I would like to thank the thesis committee and all the PhD defense jury members. A special thanks to Dr. Rachel Bras-Gonçalves and Dr Phillippe Holzmuller for their insightful comments and expertise, but also for the fruitful discussions and their contribution to this work. I would like to thank Dr. Amel Garnaoui for the scientific collaboration, her support and assistance of my thesis work during my stays in Tunis.

My sincere thanks also goes to Dr. Bernard Maillere who provided me an opportunity to visit the laboratory and research facilities. His precious support was invaluable for this research project and my PhD thesis.

I would like to thank Dr. Etienne Loire and Dr. Vincent Bonhomme for their significant aid and support, determinant to this thesis work. My thanks also go out to the support I received from the collaborative work I undertook with the Plateforme de Protéomique Fonctionnelle de Montpellier. I would also like to thank Elodie Petitdidier and Julie Pagniez, great labmates who have helped and motivated me, and made the lab a friendly place. I am grateful to my sister, mother and father for the support they provided me my entire life.

A special and heartfelt thanks to Nuno. I am also grateful to my other family members and friends who have supported me along the way.

ii ABSTRACT Leishmaniasis is a vector-borne neglected tropical disease endemic to 98 countries worldwide. Twenty Leishmania species are capable of establishing intracellular infection within human macrophages, causing different clinical presentations. Vaccine development against leishmaniases is supported by evidence of natural immunity against infection, mediated by a dominant cellular Th1 response and production of IFN-γ, IL-2 and TNF-α by polyfunctional TCD4+ and TCD8+ cells, ultimately leading to macrophage activation and parasite killing.

Excreted-secreted proteins are important virulence factors present throughout Leishmania life stages and are able to induce durable protection in dogs, a good model for human infection. We aim to develop a second generation vaccine from the Leishmania secretome, with the potential for large scale dissemination in a cost-effective, reproducible approach.

The secretome of six main pathogenic species (plus L. tarentolae) was analysed by Mass-Spectrometry and conserved candidate antigens were searched in the complete dataset. A total of 52 vaccine antigen candidates were selected, including 28 previously described vaccine candidates, and an additional 24 new candidates discovered through a reverse vaccinology approach.

In silico HLA-I and -II epitope binding prediction analysis was performed on all selected vaccine antigens, with world coverage regarding HLA restriction. To select the best epitopes, an automated R script was developed in-house, according to strict rational criteria. From thousands of potential epitopes, the automated script, in combination with optimal IC50, homology to host and solubility properties, allowed us to select 50 class I and 24 class II epitopes, synthesized as individual peptides. In vitro toxicity assays showed these selected peptides are non-toxic to cells.

The peptides' immunogenicity was evaluated using immunoscreening assays with immune cells from human donors, allowing for the validation of in silico epitope predictions and selection, and the assessment of the peptide's immunogenicity and prophylactic potential. Healed individuals, which had active infection and received treatment, possess Leishmania-specific memory responses and are resistant to reinfection, being considered the gold standard of protective immunity. On the other hand, the naive population is extremely important to include in the experimental validation step since it is the target population to vaccinate with a prophylactic vaccine. Importantly, a minimum specific T-cell precursor frequency is needed to induce long-lasting memory protective responses. Furthermore, there is also a positive correlation between immunodominant epitopes and a high frequency of specific T-cell precursors. Peptides able to induce Th1 and/or cytotoxic immune responses in both background are promising candidates for a vaccine formulation. Altogether, iii experimental validation exclusively in human samples will provide us a very strong base for a vaccine formulation and allow to accelerate translation to the field. Results show Leishmania-specific peptides successfully induce IFN-γ production by total PBMC from healed donors, and by specific T cells amplified from the naïve repertoire. Preliminary evidence exists for peptides which are immunogenic in both immune backgrounds (eight HLA-class I 9-mer peptides and five class II 15-mer peptides) which are, for now, the most promising candidates to advance for the multi-epitope peptide design.

Through the combination of proteomic analysis and in silico tools, promising peptide candidates were swiftly identified and the secretome was further established as an optimal starting point for vaccine development. The proposed vaccine preclinical development pipeline delivered a rapid selection of immunogenic peptides, providing a powerful approach to fast-track the deployment of an effective pan-specific vaccine against leishmaniases.

RESUMÉ

La leishmaniose est une maladie tropicale négligée à transmission vectorielle qui est endémique dans 98 pays dont les plus pauvres. Vingt espèces de Leishmania sont capables d'établir une infection intracellulaire au sein des macrophages humains, provoquant différentes manifestations cliniques. Le développement d'un vaccin contre les leishmanioses est étayé par des preuves d'immunité naturelle contre l'infection, induite par une réponse à médiation cellulaire de type Th1 dominante associée à la production d'IFN-γ, d'IL-2 et de TNF-α par des cellules T polyfonctionnelles TCD4 + et TCD8 + , conduisant à l'activation classique des macrophages entrainant la destruction des parasites. Induire une protection robuste et durable et déterminer les épitopes immunodominants responsables de la protection naturelle représente un véritable défi.

Les protéines sécrétées sont des facteurs de virulence jouant un rôle important dans le cycle de vie des leishmanies et sont capables d'induire une protection durable chez le chien, un bon modèle pour l'infection humaine. Notre objectif est de développer, à partir du sécrétome de Leishmania, un vaccin de seconde génération reproductible et facile à produire à bas prix dans les zones d'endémie, avec des rendements de production rendant possible son utilisation à grande échelle.

Les sécrétomes des six espèces les plus pathogènes de leishmanie (plus L. tarentolae) ont été analysés et comparées par spectrométrie de masse. Les antigènes candidats ont été recherchés dans l'ensemble des données protéomiques disponibles. 52 antigènes candidats vaccin ont ainsi été sélectionnés, dont 28 avaient déjà été décrits dans la littérature et 24 sont nouveaux et découverts grâce à une approche de vaccinologie réverse.

Une analyse de la prédiction de liaison des épitopes in silico HLA-I et -II a été réalisée sur tous les antigènes candidats vaccin, prenant ainsi en compte le polymorphisme HLA de la population mondiale. Pour sélectionner les meilleurs épitopes parmi des milliers d'épitopes potentiels, un script R automatisé a été développé en interne, selon des critères rationnels stricts. Ainsi, 50 épitopes de classe I et 24 épitopes de classe II ont été sélectionnés et synthétisés sous forme de peptides individuels. Des essais de toxicité in vitro ont montré l'absence de toxicité cellulaire de ces peptides.

Les individus guéris par chimiothérapie généralement développent des réponses immunitaires protectrices à Leishmania. Des tests de stimulation des PBMC ont donc été réalisés avec des échantillons biologiques provenant de donneurs guéris de Tunisie et la production d'IFN-γ a été évaluée par ELISpot. De plus, il était important d'inclure dans l'étape de validation expérimentale des peptides des échantillons provenant d'individus naïfs, population cible à vacciner avec un vaccin prophylactique. Les résultats montrent que des peptides spécifiques de Leishmania induisent avec v succès la production d'IFN-γ par les PBMC totaux provenant de donneurs guéris et par les lymphocytes T spécifiques amplifiés à partir du répertoire naïf.

Globalement, la validation expérimentale des peptides réalisée exclusivement sur des échantillons humains nous fournira une base préclinique très solide pour développer un vaccin efficace capable de protéger les populations touchées par ces maladies. Elle constituera un moyen sûr et rentable de mieux sélectionner les candidats retenus pour le vaccin et d'éliminer ceux qui présentent un risque d'échec élevé au tout début du processus de développement du vaccin.

Grâce à la combinaison de l'analyse protéomique et d'outils in silico, des candidats peptidiques prometteurs ont été rapidement identifiés pour le développement d'un vaccin. Le « pipeline » de développement préclinique du vaccin proposé fournit une sélection rapide de peptides immunogènes, offrant une approche puissante pour accélérer le déploiement d'un vaccin panspécifique efficace contre les leishmanioses.

vi

RESUMÉ DETAILLÉ

Les leishmanioses sont des maladies parasitaires à transmission vectorielle liées à l'infection par plus de 20 espèces de parasites protozoaires flagellés appartenant au genre Leishmania. Parmi les parasitoses, la leishmaniose est le deuxième plus grand tueur dans le monde après le paludisme, avec 50 000 décès estimés par an. Signalées dans 98 pays (dont 72 sont des pays en voie de développement), elles exposent 1 milliard de personnes au risque d'être infecté et de développer une des 4 formes cliniques de la maladie : la leishmaniose viscérale (LV), forme la plus sévère aussi connue sous le nom de kala-azar ; la leishmaniose cutanée (LC), forme la plus fréquente ; la leishmaniose cutanéo-muqueuse (LCM), forme la plus mutilante et défigurante; ou la leishmaniose cutanée postkala-azar (LDPKA), pouvant présenter des complications graves après une LV.

Le contrôle de la leishmaniose repose principalement sur la lutte anti-vectorielle et le traitement, qui présentent plusieurs inconvénients, notamment la toxicité, le prix et le manque d'efficacité. A l'heure actuelle, aucun vaccin contre les leishmanioses humaines n'est disponible sur le marché. La vaccination est pourtant le moyen le plus adapté pour interrompre la transmission des leishmanies et contribuer à l'élimination des leishmanioses.

Des recherches menées par mon laboratoire d'acceuil sur les antigènes d'excrétion-sécrétion (AES) purifiés de leishmanies, est né CaniLeish® : le premier vaccin antiparasitaire Européen contre la leishmaniose viscérale canine, commercialisé par la société Virbac depuis 2011. C'est une innovation majeure en immunologie parasitaire et un atout essentiel dans la prévention de la leishmaniose canine mais aussi humaine, le chien étant le principal réservoir de parasites potentiellement transmissibles à l'homme. CaniLeish® est capable de déclencher une réponse à médiation cellulaire protectrice de type Th1 par immunisation avec des AES purifiés de cultures de promastigotes de Leishmania infantum. La résistance à Leishmania chez l'Homme est également basée sur une réponse Th1 et des réponses cytotoxiques (production d'IFN-γ, d'IL-2 et de TNF-α par les lymphocytes T CD4 + et CD8 + polyfonctionnels), conduisant à l'activation des macrophages et à la destruction des parasites intracellulaires. Par contre, la progression de la maladie est associée à des réponses cellulaires de type Th2 (prédominance d'IL-10 et d'IL-4).

L'objectif principal de mon projet de thèse est de développer un vaccin de deuxième génération contre les leishmanioses humaines. Fort du succès du vaccin CaniLeish®, les AES de leishmanies ont été choisis comme source d'antigènes pour le développement d'une stratégie de vaccination prophylactique à visée humaine, à base de peptides multi-epitopiques, réunissant "les meilleures parties des meilleurs antigènes" en un seul candidat vaccin polyvalent. Notre objectif est vii de concevoir et de synthétiser des peptides avec des épitopes immunodominants multiples (polyépitopiques) et appropriés dérivés de protéines excrétées / sécrétées identifiées à partir de données protéomiques provenant de 6 espèces pathogènes de leishmanies: L. infantum, L. major, L. tropica, L. amazonensis, L. braziliensis et L. donovani). Des études de phase préclinique ont été réalisées exclusivement sur des cellules humaines afin d'évaluer l'immunogénicité des peptides sélectionnés.

La validation expérimentale des peptides synthétiques a consisté à évaluer les profils immunitaires après stimulation peptidique de cellules d'individus exposés ayant développé une immunité à l'infection par Leishmania (individus guéris) par rapport à des sujets naïfs. D'autre part, il est extrêmement important d'inclure dans l'étape de validation expérimentale de l'immunogénicité des peptides des échantillons provenant d'individus naïfs car il s'agit de la population cible à vacciner avec un vaccin prophylactique. De manière importante, une fréquence minimale de précurseur spécifique de cellules T est nécessaire pour induire des réponses protectrices mémoire. D'autres résultats étaient attendus des données protéomiques comme la caractérisation détaillée du sécrétome et la découverte d'éventuelles corrélations entre la distribution géographique, les manifestations cliniques, l'immunomodulation et la pathogenèse. Nous avons également inclus dans notre étude les données du sécrétome d'une espèce de Leishmania non pathogène : L. tarentolae.

Nous espérons aussi contribuer à accroître nos connaissances sur la variabilité interspécifique des leishmanies et éventuellement à identifier et caractériser de nouveaux facteurs de virulence pouvant contribuer au diagnostic de ces maladies et/ou à la mise au point de médicaments contre les leishmanioses.

Les tâches accomplies dans ce projet de recherche comprennent :

• l'identification exhaustive et la caractérisation des AES présents dans le sécrétome de six espèces pathogènes de Leishmania par spectrométrie de masse (L. infantum, l'espèce utilisée pour la production de CaniLeish ®, et aussi L. major, L. tropica, L. amazonensis, L. braziliensis et L. donovani) ainsi qu'une espèce non pathogène pour l'homme, L. tarentolae;

• la sélection de 52 antigènes vaccinaux les plus pertinents sur l'ensemble des données protéomiques générées. Ceux-ci comprennent 28 protéines déjà décrites dans la littérature scientifique comme des candidats vaccins (Set A), et 24 nouvelles protéines sélectionnées, en utilisant une approche de vaccinologie reverse (Set B) selon un critère de « non-homologie de séquences protéiques avec celles de l'hôte » ;

• l'identification d'épitopes T (séquences peptidiques qui se lient spécifiquement aux molécules HLA-I et -II, et qui activent le système immunitaire adaptatif) les plus affins pour l'ensemble des molécules HLA majoritairement représentées dans les populations humaines (36 allèles viii HLA-I, correspondant à 11 supertypes et 98% de la population mondiale, et 21 allèles HLA-II, correspondant à 95% de la population mondiale), à l'aide de plusieurs serveurs de prédiction d'épitopes hautement performants;

• la seléction des épitopes le plus immunogènes, avec l'aide d'un script R automatisé, que j'ai développé pour ce projet (Pissarra J et al, publication en cours de révision), permettant de sélectionner facilement les épitopes les plus pertinents en intégrant différents critères prédéfinis. Les critères utilisés pour la hiérarchisation des épitopes sont : la forte affinité de liaison, la conservation entre les espèces, la prédiction par au moins 2 algorithmes différents, et la faible homologie des séquences peptidiques avec celles de l'hôte. Le script R applique ces critères aux données brutes collectées à partir des algorithmes de prédiction pour filtrer rapidement un nombre extrêmement important d'épitopes potentiels (stratégie "best of"). Le script est très polyvalent et applicable à d'autres prédicteurs, à d'autres protéines, mais aussi à d'autres pathogènes. Ainsi, nous avons sélectionné 50 épitopes HLA-I (9-mer) et 24 épitopes HLA-II (15-mer). Les peptides HLA-I proviennent de 23 protéines différentes (11 du Set A et12 du Set B), et les peptides HLA-II proviennent de 15 protéines (7 du Set A et 8 du Set B).

L'originalité et la pertinence de notre stratégie vaccinale réside aussi dans l'utilisation de tests fonctionnels précliniques comme voie d'exploration de l'efficacité de nos candidats vaccins : l'utilisation ex vivo de cellules humaines pour développer un vaccin humain. En effet, les modèles murins sont inappropriés pour étudier les réponses immunitaires humaines contre Leishmania. Il est difficile et dangereux d'extrapoler des résultats obtenus chez la souris à des hôtes naturels de l'infection. Globalement, la validation expérimentale des peptides réalisée exclusivement sur des cellules humaines devait fournir une base préclinique très solide pour développer un vaccin humain efficace capable de protéger les populations touchées par ces maladies. Elle constituait un moyen sûr et rentable de mieux sélectionner les candidats retenus pour le vaccin et d'éliminer ceux qui présentaient un risque d'échec élevé au tout début du processus de développement du vaccin.

Grâce à la combinaison de l'analyse protéomique et d'outils in silico, des candidats peptidiques prometteurs ont été rapidement identifiés pour le développement d'un vaccin. Le « pipeline » de développement préclinique du vaccin proposé fournit une sélection rapide de peptides immunogènes, offrant une approche puissante pour accélérer le déploiement d'un vaccin panspécifique efficace contre les leishmanioses. Les individus guéris d'une infection leishmanienne possèdent des réponses immunitaires mémoires contre les parasites qui les rendent résistants à la réinfection, et sont considérés comme le gold standard de l'immunité protectrice. La validation ix expérimentale des peptides doit également être effectuée sur des échantillons provenant de sujets ayant d'autres statuts immunitaires : individus asymptomatiques et naïfs. Les individus asymptomatiques sont infectés par le parasite mais ne développent pas la maladie, ce qui signifie que leur système immunitaire parvient à contrôler l'infection sans pour autant éliminer le parasite. De plus, la population naïve est extrêmement importante à inclure dans la validation expérimentale, car elle représente la population cible pour un vaccin prophylactique. En effet, une fréquence minimale de précurseurs de cellules T spécifiques est nécessaire pour induire une réponse protectrice de longue durée. Il existe également une corrélation positive entre les épitopes immunodominants et la fréquence élevée de précurseurs de cellules T spécifiques, ce que nous chercherons dans notre étude.

Les peptides sont validés pour leur capacité à induire des réponses mémoires préexistantes spécifiques à Leishmania sur des échantillons provenant d'individus guéris (en collaboration avec l'Institut Pasteur de Tunis). L'efficacité des peptides à stimuler ex vivo des cellules mononuclées du sang périphérique humain (PBMC) et à produire des cytokines de type Th1 comme l'IFN-γ (cytokine associée à la protection) a été évaluée.

Pour tester l'efficacité des peptides à activer le répertoire naïf d'individus sains, des essais de co-culture de cellules T sont réalisés avec plusieurs cycles d'amplification cellulaire (en raison de la rareté des cellules spécifiques), et la production d'IFN-y spécifique est recherchée par la technique ELISpot. Le typage HLA des donneurs naïfs permet une sélection « sur mesure » pour l'optimisation des immuno-essais, et aussi la conclusion effective sur les résultats de prédiction d'épitopes et la restriction HLA associée.

Ce projet aura aussi des répercussions importantes sur la connaissance de la biologie du parasite, grâce notamment au jeu de données protéomiques et à l'identification des régions d'immunogénicité des antigènes choisis (epitope-mapping). Ainsi, cela permettra d'augmenter nos connaissances sur la variabilité inter-espèces, et de révéler potentiellement de nouveaux facteurs de virulence importants et utiles pour le diagnostic de la leishmaniose ou le développement de nouveaux médicaments.

Nous pensons que ce projet contribuera à la découverte de peptides immunogènes pouvant, sous forme de peptides multi-épitopes, entrer dans la composition d'un vaccin efficace contre les leishmanioses humaines. Notre stratégie devrait concourir à minimiser les risques d'échec à un stade précoce du développement du vaccin et lors de la réalisation de futurs essais cliniques. Enfin, elle offre aussi les bases méthodologiques nécessaires aux suivis immunologiques des individus vaccinés lors d'essais cliniques de vaccination en zone d'endémie.

x

THESIS OUTLINE

The present thesis describes the preclinical development of a peptide-based vaccine against human leishmaniasis and preliminary experimental validation on the proposed peptide candidates.

Chapter I provides a contextualization of Leishmania biology, the problematic of leishmaniasis control and the importance of vaccine development. Particularly, it reviews current epidemiologic information on Leishmania spp. parasites, host-pathogen interactions and host immune responses against the parasite, as well as current vaccine pipeline and peptide-based vaccines.

Chapter II describes the proteomic analysis of the secretome of seven Leishmania species, responsible for the main clinical forms of leishmaniasis, and characterization of the secretome as an important source of virulence factors and of vaccine antigen candidates. Also, the results of this section provide the dataset used for vaccine antigen selection.

Chapter III exposes the strategies selected for vaccine antigen selection. A total of 52 protein candidates were selected from the secretome proteomic datasets through two parallel approaches: searching peptide candidates previously described in the literature (set A) and through a reverse vaccinology approach (set B).

Chapter IV describes the immuno-informatic tools available and used in this study, in silico epitope human leukocyte antigen (HLA)-binding predictions and epitope selection, including the development of a semi-automated R script to select the best epitopes from the vast HLA-binding prediction data corresponding to the selected 52 protein antigens. The selected HLA class-I and -II epitopes were synthesized as 9-and 15-mer peptides, respectively, for experimental validation.

In Chapter V, current methods to assess T-cell mediated peptide immunogenicity are reviewed, and cellular immune responses against the peptide candidates are evaluated through immunoassays with samples from humans with different immune status regarding Leishmania infection (naive and healed individuals).

Finally, Chapter VI comprehends a synthesis of main findings of this thesis work, general discussion and conclusions, as well as future perspectives. Leishmania parasites and major clinical syndromes.

7

Table I.2

Ratio between number of asymptomatic and active disease cases, according to geographical region. 9

Table I.3

Toll-like receptor agonists and Th1-inducing adjuvants for use in vaccine formulations.

52

Table I.4

Leishmania-specific peptide vaccine candidates validated using human samples.

56

CHAPTER II -THE LEISHMANIA SECRETOME

Page

Table II.1

Leishmania spp. strains cultured for the generation of ESAp. 90

Table II.2

Ordered species and group-species list established to define a "leading" protein based on the taxonomy criteria. 93

Table II.3

Leishmania reference proteomes, respective protein counts, and proteins found in the secretome. 98

Table II.4

List of Leishmania-secreted proteins associated with exosome-like and glycosomal vesicles found in the secretome datasets. 101

Table II.5

List of exosome markers and genes found in the secretome. 102

Table II.6

Leishmania proteins with moonlighting functions in the MoonProt database. 105

CHAPTER III -VACCINE ANTIGEN SELECTION

Page

Table III.1

Initial list of vaccine antigen candidates found in the literature. 120

Table III.2

A total of 72 protein antigens listed from the selected publications was searched in the secretome proteomic datasets. 124

Table III.3

Protein description of the 28 selected antigen candidates (Set A). 128

Table III.4

Deconvolution of peroxidoxin sequences annotated in the UniProt and Genbank databases. 130

Table III.5

Protein set A includes 28 antigenic proteins (A1 to A32) described in the literature as potential vaccine antigens against Leishmania spp. 131

Table III.6

ClustalOmega alignment results (percent identity matrix) for 33 uncharacterized proteins identified with the reverse vaccinology approach 134

Table III.7

Protein set B includes 14 annotated proteins and 10 uncharacterised proteins (B1 to B24).

Table IV.1

Determinants of immunodominance

Table IV.2

Available T-cell epitope databases

Table IV.3

The most commonly used sequence-based algorithms for T cell epitope prediction

Table V.4

Populations with HLA allele frequency data from four world regions most affected by leishmaniases

Table V.5

Naive donor bank description

Table V.6

HLA-typing results for donors MPL3 to MP21.

Table V.7

Personal information and medical history from recruited healed donors (TUN1-TUN20)

LIST OF PUBLICATIONS AND PRESENTATIONS

Peer-reviewed articles:

•

• Pissarra et al (under preparation)

Provisional title: Proteomic analysis of the total secretome of seven Leishmania species.

Poster presentations:

• Oral presentations:

CHAPTER I

GENERAL INTRODUCTION

Leishmaniasis is a Neglected Tropical Disease

Leishmaniasis is a term that refers to any form of a complex group of diseases caused by protozoan parasites of the genus Leishmania (belonging to the Trypanosomatida order: Leishmaniasis is one of the most neglected tropical diseases as diagnostics and treatment tools are often ineffective or toxic, and improvement and development are impaired by the lack of funding and R&D (4)(5)(6). Leishmaniasis is endemic to the poorest countries in the world, and evidence shows that epidemics (or increases in incidence) are closely associated with socio-economic conditions, war and conflicts, malnutrition and food insecurity, and access to healthcare (7-10).

Leishmaniasis surveillance is seldom based on passive case detection which further contributes to the underestimation of its burden and impairment of control efforts, whilst contributing to active parasite transmission (11,12).

In the last decades, efforts towards controlling NIDs have increased, notably since the World Health Organization's 2012 Roadmap on NTDs, which calls for enhanced control, prevention, elimination and eradication of NTDs, namely the regional elimination of VL in the Indian subcontinent by 2020 (13). Shortly after, the London Declaration on NTDs was signed (www.who.int/neglected_diseases/London_Declaration_NTDs.pdf), wherein several pharmaceutical companies, donors, endemic countries and non-government organisations declare their commitment to contribute to NIDs elimination through R&D and control programme implementation. The latest Report of the WHO's Strategic and Technical Advisory Group for Neglected Tropical Diseases (www.who.int/neglected_diseases/NTD_STAG_report_2017.pdf) further cements the commitment towards NIDs elimination and shows the progress achieved so far. Regarding leishmaniasis specifically, the Resolution WHA60.13 was adopted by the 60th World Health Assembly in 2007 to promote the awareness of the burden of leishmaniasis, and to monitor of progress of leishmaniasis control programmes (11,14).

Leishmaniasis prevention needs an integrated approach targeting both human and animal hosts (One Health approach). Measures that aim at reducing the incidence of leishmaniasis are directed: i) to people, e.g. diagnosis and treatment of cases (15); ii) to the reservoir, e.g. applying protective insecticide treatment to dogs (16); and iii) to vector, e.g. insecticide spraying. Since no effective vaccine against human leishmaniasis exists, the most effective method of controlling Leishmania transmission to date is vector control (17,18).

A large VL elimination campaign was launched in 2005 in South Asia, relying heavily on indoor residual spraying, long-lasting insecticidal bed nets, and environment management, has contributed to the reduction of reported cases (12,19,20). However, success longevity depends on continuous application of control measures, which may not be assured once targets have been achieved, and/or given the cyclical and geographical shifts in leishmaniasis transmission (12,20). As most sand fly species bite mostly outdoors, there is no strong argument for insecticide sprayingin this scenario, and for L. infantum-and L. major-endemic areas, reservoir control may prove to be a more useful tool.

The main milestones concerning leishmaniasis, since the implementation of the objectives set in 2012's Roadmap for NTDs, are i) the improvement of surveillance and case management ('District Health Information System' platforms), and ii) the introduction of standardized tools for the collection of indicators from all member states, some accessible through the Global Health

Observatory website (http://apps.who.int/gho/data/node.main.NTDLEISH), and others limited to high-burden countries (11,12). Regarding reservoir control, three canine vaccines are licensed and currently available in Europe and Brazil, Canileish ® and Letifend ® , and Leish-Tec ® , respectively.

Overall, current existing tools can greatly contribute to decrease Leishmania transmission, however, the need for investment in new diagnostics, treatment and prevention tools still stands (21)(22)(23). Alternative approaches such as immunochemotherapy or immunotherapy should also be further explored (12,21), and beyond innovation, product accessibility must also be taken into account (24,25).

Particularly, the introduction of vaccines in endemic areas remains a primary objective in the context of leishmaniasis control (26)(27)(28), either a prophylactic vaccine preventing infection, disease progression and transmission, and/or an immunotherapeutic vaccine (12).

A few cost-effectiveness studies on the impact of the introduction of human leishmaniasis vaccines were performed, and these have demonstrated that vaccines remain the most cost-effective tool for leishmaniasis control programmes. A vaccine against VL in the endemic Bihar state in India with conferring only 50% protection during 5 years is highly cost-effective compared with current treatments, as well as a vaccine against CL deployed in American countries with providing 70% protection during 10 years (29,30).

Leishmania parasites and leishmaniases

Leishmania spp. life cycle

The general life cycle is common to all Leishmania species (Figure I.1 panel A), and the vertebrate host stage begins when an infected female sand fly takes a blood meal from a naive host.

The main reservoirs for Leishmania spp. are dogs, rodents and humans.

Sand flies from the genus Phlebotomus (Old World) or from the genus Lutzomyia (New World) are modified pool feeders, meaning they bite superficially multiple times and feed on pooled blood.

Less competent flyers than mosquitoes, sand flies breed in walls, rubbish or rubble, or rodent burrows (31)(32)(33). Other transmission routes, which remain exceptional, include congenital transmission, blood transfusion, sharing of infected needles, or (rarely) sexual transmission (31,(34)(35)(36). The infected sandfly takes a bloodmeal from a naive host, injecting metacyclic promastigotes that invade phagocytes (mostly macrophages and neutrophils), where they differentiate into intracellular amastigotes and establish infection (Figure I.1 panel C). The sand fly saliva enhances promastigote infectivity as it contains vasodilator and immunomodulatory molecules (38,39).

Once in the vertebrate host, these parasites have developed key strategies that allow them to thrive in drastic conditions destined to kill them, inside acidic phagolysosomes. Phagolysosomes are cytoplasmic organelles formed after the fusion of the phagosome with one or more lysosomes, that become acidic and contain antimicrobial peptides and hydrolytic enzymes, killing intracellular pathogens (40). The complex Leishmania lifecycle is highly adapted to the host's immune system, which is actively manipulated to the parasite's benefit.

Leishmaniasis distribution

Leishmaniases are distributed worldwide across the tropical, subtropical, and temperate regions in 98 countries, 72 of which are in developing areas of the world (Figure I.2) (12). 350 million people are at risk worlwide and an estimated 12 million people suffer from leishmaniasis (11). There are an estimated half a million new VL cases per year, and 1 to 1.5 million new CL cases per year, with 2.4 million disability-adjusted life-years (DALYs), but these numbers are likely underestimated. Over 90% of all cases of VL are found in seven countries (Brazil, Ethiopia, India, Kenya, Somalia, South Sudan, Sudan) (11,12). Approximately 90% of all CL cases occur in Afghanistan in Central Asia; Iran, Saudi Arabia, and Syria in the Middle East; and in Brazil and Peru in Latin America. CL is also a substantial issue for travellers, and military personnel visiting endemic areas (31). Finally, 90% of the cases of MCL occur in three South American countries: Bolivia, Brazil, and Peru.

Co-infection with HIV has emerged as an important public health threat in areas in southern Europe and other regions where the two diseases coexist, as well as for immunocompromised individuals, as is the case of organ transplants and other conditions affecting cell-mediated immunity (41). (31,42). A) World distribution of CL-causing Leishmania species. B) World distribution of VL-causing Leishmania species.

Clinical syndromes

The main disease-causing Leishmania species and respective clinical syndromes are described in Table I.1. Depending on the infecting species and host immunity, different clinical syndromes develop.

However, most infected individuals remain asymptomatic, indicative of the development of an effective immune response, even if incomplete. Evidently, the ratio between asymptomatic:active infection varies widely according to the infecting species and endemic region (Table I.2).

India and Nepal 8,9:1 (51,52) In patients where immune responses are inadequate to control parasite proliferation, the infection progresses to active disease. Leishmaniasis encompasses a spectrum of clinical syndromes (Table I.1 and Figure I.3), from self-healing CL, to chronic, or disseminated visceral disease, indicative of both parasite diversity and variable host responses (31,53,54). (31). (56). Recovered and self-resolving individuals present long-term protection against disease, in the absence of immunosuppression (57,58). Despite no technique is really adapted and developed for asymptomatic case detection, it is common to use two or more markers for asymptomatic infection detection, seldom serology and PCR (59).

Figure I. 3 Spectrum of Leishmania infection and disease

It is still not possible to known which patients will progress to active disease, remain asymptomatic carriers or achieve parasite clearance (44,60). a) First obstacles: the complement system

The complement system comprises a large number of plasma proteins which interact among themselves and react with other components of the immune system, enhancing antibody and phagocyte function and inducing inflammatory responses against infection (65). There are three complement activation routesalternative, lectin, or classical pathways (65). All pathways culminate with the activation of C3 convertase which generates C3b and C3a from C3 (complement component 3). C3b is deposited on the surface of the pathogens allowing for parasite killing through opsonisation and subsequent phagocytosis by neutrophils and macrophages, or through the formation of the membrane attack complex (MAC) complex and cell lysis (66) (Figure I.6).

All three pathways seem to be important for Leishmania clearance, and undoubtedly the alternative complement activation is critical for parasite elimination (67). Between 85% to all promastigotes are killed by complement within a few minutes in human blood (L. donovani, L. infantum, L. major, and L. amazonensis), so parasites must quickly establish infection inside phagocytes, namely inside macrophages, their definitive host cell (67,68).

Leishmania parasites found elegant solutions to not only overcome targeting by the complement system, but also to exploit host opsonins to invade host cells and modulate their response in its favour, mostly through the interaction with glycocalyx molecules and other virulence factors (62,69,[START_REF] Naouar | Prediction of T Cell Epitopes from Leishmania major Potentially Excreted/Secreted Proteins Inducing Granzyme B Production[END_REF]. The major parasite surface proteins involved in initial first host-pathogen molecular interactions are: lipophosphoglycan (LPG), a glycoconjugate; the GP63 metalloprotease, also known as promastigote surface protease (PSP) or leishmanolysin, present only in promastigotes;

and glycosyl-inositol phospholipids (GPI), present in large numbers in both promastigotes and amastigotes (63,69).

Both LPG and GIPLs bind to the mannan-binding serum protein (MBP), which can activate the complement system in an antibody-dependent manner through the lectin activation pathway (63).

LPG is the major acceptor of C3b. Simultaneously, LPG and GP63 are involved in promastigote complement resistance. The LPG on the parasite surface prevents the complement membrane attack complex insertion: when the C5b-MAC complex binds, it does not affect the parasite membrane, and is released as soluble C5b-9 complex (63,71). GP63 can inactivate C3b and totally inhibit the formation of the C5b convertase complex (63,68). In addition, Leishmania promastigote membrane kinases (LPK-1 and potentially others) phosphorylate the C3, C5 and C9 components of the complement, inhibiting complement activation (63,72)

(Figure I.6).
Moreover, GP63 cleaves C3, and resulting iC3b opsonises promastigotes promoting macrophage through the interaction with surface C3 receptor, also known as CR3 or MAC-1 (68,73,74). The CR3-dependent entry route is important for establishing infection inside macrophages, favouring parasite development and persistence (73,75) due to associated IL-12 downregulation (63,76). GP63 also cleaves host tyrosine phosphatase SHP1 preventing IFN-γ-

Figure I. 6 Receptor-mediated phagocytosis of Leishmania parasites (73). A) GP63-mediated entry via interaction with CR3 receptor. CR3 may also mediate direct binding to promastigotes via a yet unknown surface epitope on promastigotes. GP63 also binds fibronectin, which then bridges the parasite to fibronectin receptors (FnRs). B) LPG expression on amastigotes is absent, possibly allowing the low levels of GP63 to become opsonized with iC3b protein and subsequently ligate CR3. Antibody and fibronectin detection of amastigotes leads to ligation of Fc gamma receptors (FcγRs) and FnRs, respectively. B) Immediately following inoculation by the sand fly, promastigotes parasitize predominantly Polymorphonuclear Neutrophils (PMN). Promastigotes enter PMN via CR3 and then enter macrophages

or DC while contained in the short-lived granulocyte. Promastigotes and amastigotes may also directly enter DC via DC-specific DC-SIGN.

b) Neutrophils and macrophages, evasion and exploitation of host innate immune responses

Shortly after parasites are injected in the skin, neutrophils are the first cells to be recruited to the inflammation site (79). Because of the sand fly's natural pool feeding behaviour, the tissue damage caused and the injection of saliva proteins together with the promastigote gel and Leishmaniaproduced exosomes, create a highly inflammatory environment through the release of alarmins (signal for tissue damage), cytokines, and chemokines (63,80). Neutrophils can act against the intracellular Leishmania parasites through reactive oxygen species (ROS), neutrophil elastase (NE), and neutrophil extracellular traps (NETs) (79,(81)[START_REF] Sousa | Neutrophils have a protective role during early stages of Leishmania amazonensis infection in BALB/c mice[END_REF][START_REF] Chang | Leishmanicidal mechanisms of human polymorphonuclear phagocytes[END_REF][START_REF] Pearson | Phagocytosis and killing of the protozoan Leishmania donovani by human polymorphonuclear leukocytes[END_REF][START_REF] Guimarães-Costa | Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps[END_REF][START_REF] Morgado | Are neutrophil extracellular traps playing a role in the parasite control in active American tegumentary leishmaniasis lesions?[END_REF][START_REF] De Souza Carmo | Neutrophils reduce the parasite burden in Leishmania (Leishmania) amazonensis-infected macrophages[END_REF]. Nevertheless, neutrophils become infected by promastigotes during the first 18 hours post-infection, providing a transient shelter allowing for parasite development and immunomodulation of subsequent response cascades (79,[START_REF] Carlsen | Permissive and protective roles for neutrophils in leishmaniasis[END_REF].

The parasite has evolved to both evade and exploit host innate immune responses, reviewed in Geiger et al 2016 (1). There is evidence that Leishmania infection prolongs neutrophil survival in vitro and in vivo, as parasites delay the onset of apoptosis in infected neutrophils (68,[START_REF] Aga | Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major[END_REF]. This delay provides the time needed for the recruitment of antigen-presenting cells (macrophages and DC) to the infection site. As neutrophils undergo apoptosis, the parasites take advantage of a Trojan horse strategy, where phagocytosis of apoptotic neutrophils by dermal macrophages or dendritic cells triggers anti-inflammatory signal pathways (high TGF-β, IL-10 and low IL-12), providing an optimal route for intracellular development, while simultaneously impairing subsequent adaptive immune responses [START_REF] Filardy | Proinflammatory clearance of apoptotic neutrophils induces an IL-12(low)IL-10(high) regulatory phenotype in macrophages[END_REF][START_REF] John | Neutrophil Soldiers or Trojan Horses?[END_REF][START_REF] Ribeiro-Gomes | Efficient Capture of Infected Neutrophils by Dendritic Cells in the Skin Inhibits the Early Anti-Leishmania Response[END_REF][START_REF] Ritter | Are neutrophils important host cells for Leishmania parasites?[END_REF].

Furthermore, Leishmania disease development depends on the presence of apoptotic promastigotes in the virulent inoculum, which expose phosphatidylserine (PS) also enabling a silent cell invasion and inducing the production of anti-inflammatory cytokine TGF-β (68,[START_REF] Wenzel | Leishmania major parasite stage-dependent host cell invasion and immune evasion[END_REF][START_REF] Van Zandbergen | Leishmania disease development depends on the presence of apoptotic promastigotes in the virulent inoculum[END_REF]. It is interesting to note that neutrophils readily phagocytized promastigotes, but amastigotes are not uptaken by these cells, rather by macrophages through endocytosis (CR3-or clathrin-and caveolaemediated) (63,[START_REF] Wenzel | Leishmania major parasite stage-dependent host cell invasion and immune evasion[END_REF]. 48 hours after infection, the majority of infected cells are macrophages [START_REF] Peters | In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies[END_REF].

Parasite are also capable of escaping pre-apoptotic macrophages in membrane blebs that are uptaken by bystander macrophages (68).

During promastigote internalization, the parasite transforms the phagolysosomal microenvironment and survives inside an adapted parasitophorous vacuole (PV) allowing a successful differentiation to amastigote form, by either delaying phagolysosomal fusion (L. major, L. infantum, L. donovani, with or without LPG), or producing large vacuoles that dilute and impair hydrolytic enzymes (L. mexicana and L. amazonensis) [START_REF] Michael | The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research[END_REF][START_REF] Sundar | Identifying vaccine targets for anti-leishmanial vaccine development[END_REF].

The production of reactive nitrogen and oxygen species (RNS, ROS) by macrophages is tightly controlled by the balance between energy metabolism and cell redox state (2). The enzyme arginase produces either ornithine, used for polyamine synthesis, or it produces RNS and ROS (namely, O2

Bridging the gapinnate and adaptive immune response coordination

The early innate immune effector mechanisms described above, namely phagocytosis, cytokine and chemokine production, expression of co-stimulatory molecules on APC, are essential for T cell differentiation. Early Leishmania infection is marked by a 'silent phase' during which parasites

Figure I. 7 Crosstalk between innate and adaptive immune responses (119).

Macrophages, neutrophils and NK cells are attracted to the site of pathogen entry. Dendritic cells take up antigen and migrate to regional lymph nodes.

Here antigen presentation to T cells takes place, and these differentiate into helper T cells (Th1 or Th2 T CD4+ cells) and cytotoxic T cells (T CD8+ cells). Activation of B cells and immunoglobulin production are also initiated.

Skin-resident DC, Langerhans cells and dermal DC can efficiently uptake and present parasite antigens in a pro-inflammatory environment, migrate to the lymph nodes where they can activate specific naive T cells (120). Most DC become infected after contacting with infected neutrophils.

However, some weeks post-infection, the number of DC in the lesion site is higher due to increased recruitment, and these infected DC can prime naive T cells locally, an effector function essential for Immunological synapse between APC and T cells).

Figure I. 8 Naive T cell priming. Interaction between antigen-presenting cells and naive T cells (represented here:

TCR/pMHC interaction and costimulatory molecules) leading to the differentiation into effector cells (122).

a) Antigen presentation and the Major Histocompatibility Complex (MHC)

Two classes of MHC molecules, membrane-associated glycoproteins, present antigens to TCD4 + and TCD8 + cells:

-MHC-class I exist on the surface of all nucleated cells, and present 8-12 amino-acid peptide epitopes to TCD8 + cells, acting against intracellular pathogens. Briefly, short peptides from intracellular antigens are processed in the proteasome, transported to the endoplasmatic reticulum by the transporter associated with antigen processing (TAP protein), where they are trimmed and loaded onto nascent MHC-class I molecules (123,124).

- The HLA molecules are encoded in a cluster of closely linked genes at chromosome 6 corresponds to: "HLA" prefix for human genes; "A" genomic locus; "02" allele group; "02:101" is the encoded protein, the specific HLA protein numbered by order of discovery; "01" is the allele variant, a silent/synonymous polymorphism within the coding region; "02" is a SNP number, denotes differences in a non-coding region; "N" is a suffix to denote additional information. For example, alleles that have been shown not to be expressed -'Null' alleles -have been given the suffix 'N'. Alleles The peptide-binding cleft or groove is the longitudinal cleft in the top surface of an MHC molecule into which the antigenic peptide is bound (55). Each allele has a unique binding groove that defines its specificity, and many of the MHC polymorphisms change the pockets affecting peptide binding specificities (123). The bonds between MHC molecules and the peptidic epitope are determined by their molecular structure and main anchor binding positions, respectively. The peptides insert their amino-acid side chains (anchor residues) into pockets in the peptide binding groove. For MHC-class I molecules, the peptide binding groove is formed by the α1 and α2 domains, HLA supertypes are allele groups with shared binding specificities (supermotifs), meaning that if a peptide is able to bind to an allele within a supertype, it will bind to other alleles in the same supertype (132,133). From binding data, structural analysis and motif identification, nine HLA-class I supertypes were described -HLA-A1, -A2, -A3, -A24, -B7, -B27, -B44, -B58, -B62 (134). i) stable interaction between the pMHC and the TCR and CD4 or CD8 (activation signal);

and
ii) the interaction between costimulatory molecules CD28 on the T cell with B7.1/B7.2 (also known as CD80/CD86), and between CD40L on T cells and CD40 on the APC surface (survival or costimulatory signal); the lack of costimulary molecules inhibits cell maturation and activation;

iii) cytokine signalling, namely IL-2, IL-6, IL-12, IL-4, TGF-β (differentiation signal).

Once cells are in contact, when T-cell receptors bind to an antigen and cell-adhesion molecules bind to their counterparts on the two cells, the immunological synapse is formed, a highly organised interface between a T-cell and the target cell, and the adaptive response ensues.

Figure I. 13 Interactions between T cells and antigen-presenting cells. A) TCD4 cell interaction with MHC-class II molecules on the surface of a dendritic cells and intracellular signalling. B) TCD8 cell interaction with MHC-class I on the target cell surface (122).

After cell proliferation, the interaction with infected macrophages or dendritic cells, results in iii) anergy induction, in active VL TCD8+ cells typically do not produce IFN-γ, and express high levels of CTLA-4 and PD-1, negative regulators of T cell activity associated with T cell anergy and exhaustion.

Adaptive immunity mechanisms a) Primary and secondary immune responses

The human adaptive immune system includes both naive T cells (with randomly assigned TCR specificity by genetic recombination during thymic development) and memory T cells (selected and differentiated from effector cells after initial antigen encounter into long-lasting specific T cells).

Each TCR is uniquely arranged and randomly selected from over 10 10 possible combinations.

b) T cell activation -from peptide presentation to T cell effector functions

Besides peptide-and MHC-related features, T-cell activation depends TCR signalling and TCR-pMHC binding parameters, namely binding affinity and avidity (144).

The TCR-peptide-loaded MHC (pMHC) binding strength is referred to as affinity, the sharing of a monovalent interaction, or, the binding strength of one molecule to its ligand at a single site (55,125). Antigen-presentation dictates the fate of developing T cells. In the periphery, unlike in the thymus or bone marrow, T cells are activated if they recognise antigens with high affinity. The T cells recognising antigens with low affinity generally become nonresponsive or anergic (145). Chronic infections, such as leishmaniasis, can induce high or chronic doses of antigen, in which case, T cells can become anergic, exhausted or apoptotic.

Affinity by itself cannot directly describe the activation extent of a given TCR-pMHC interaction. A high TCR-pMHC dissociation time and serial TCR triggering are more stimulatory than a strong affinity (125,144).

Binding avidity refers to all multivalent interactions involved in cell-cell interaction, i.e. the sum of the strength of binding of two molecules or cells to another at multiple sites (55). Aviditybased mechanisms mediate antigen sensitivity in T cells, which results in different binding and activation parameters in naive versus memory cells with the same TCR specificity (146). Throughout the immune response, primed T cells acquire greater sensitivity to pMHC signalling than naive T cells, undergoing avidity maturation (147). Avidity maturation may be related to decreasing antigen levels during the resolution phase, in preparation for the generation of specific memory T cells, more effective against secondary infection (148).

Although TCD8 + cells are undoubtedly important for host immune responses against Leishmania parasites (see below), some controversial results were observed in animal models, namely, CD8-knockout mice which are resistant to infection (149). These differences may be attributed to the antigen dose, as evidenced by the fact that TCD8 + cells are effective against low parasite doses, but not against high parasite loads (150). In turn, these observations further strengthen the need for optimal T cell priming for the generation of long-lasting T-cell memory.

Requirements for the successful induction of an effective adaptive immune response:

-Magnitude of response, a sufficient number of specific T cells is needed;

-Breadth of response, broader diversity of T cell specificity is more effective than responses targeting one epitope;

-Avidity, infection control mediated by high avidity T cells is more effective;

-Cell function, the induction of adequate effector phenotypes is needed (e.g. Th1 and cytotoxic responses)

c) Adaptive immunity against Leishmania spp. infectionlessons from mice

Two pivotal aspects regarding adaptive immunity make it an intrinsic mechanism for immunizationresponse specificity, through the use of highly variable and specific receptors, and memory generation, allowing for a stronger secondary immune response (151).

Generally, protective immunity against leishmaniasis is associated with a predominant inflammatory Th1 response, while disease is associated with a predominant anti-inflammatory Th2 response (63,152). During the initial 'silent phase', immune cells in the infection site secrete IL-4, promoting a Th2 response and thereby an environment that favours parasite development (116).

Interestingly, the Th1/Th2 dichotomy observed in L. major mouse infection was incorporated in the first description of Th1 and Th2 helper T cell populations by Mosmann TR et al 1988 (153).

The correlation of Th1 responses and protection against Leishmania originates from landmark discoveries from experimental infection studies in mice:

Generation of immunological memory is essential for vaccine development a) Human immunological memory

Immunological memory is defined as the ability of the immune system to respond more rapidly and more effectively on a second encounter with an antigen; immunological memory is specific for a particular pathogen and is long-lived (55 The distribution is an important aspect regarding memory T cell populations, as most subsets were described in peripheral blood but there is evidence for memory T cell presence in tissues in mice and humans (191,192). Tissue-resident memory T cells (TRM), described initially in mice, have been shown to be present throughout the human body, with TCD4 + subsets persisting in either the mucosa (CCR7 -) or lymph nodes (CCR7 + ), and TCD8 + cells persisting mostly as CCR7 -cells in peripheral tissue.

Most of these cells express a putative marker CD69, absent in circulating memory T cells. Tissuespecific properties have also been describedskin TRM express cutaneous lymphocyte antigen (CLA) and skin-associated homing chemokine receptors CCR4 and CCR10; TRM in the intestinal mucosa express gut-homing chemokines CCR9 and integrin α4β7 -suggesting tissue-specific mechanisms for memory generation (191,192). Most human TRM cells (bone-marrow, lung, mucosal) are polyfunctional for effector and cytolytic cytokines and produce IL-17, whereas skin-specific TRM produce IL-22.

Immunological memory responses against Leishmania after drug treatment or self-healing involves central and effector memory TCD4 + and TCD8 + cells (57,80,(198)(199)(200).

b) Cell populations involved in immune responses against Leishmania parasites

The Leishmanin skin test (LST), or Montenegro test, evaluates delayed-type hypersensitivity Nevertheless, LST is rarely performed nowadays due to supply issues and lack of standardization in antigen production (12,201).

In individuals with history of CL (L. major or L. tropica), studies have shown the presence of both TEM cells (CD4 + CD45RO + CD45RA -CCR7 -) that produce IFN-γ, and TCM cells (CD4 + CD45RO + CD45RA -CCR7 + ) that produce IL-2, and their role in protective recall responses (202).

An important role was also found for memory CD8+ TEM cell subsets (CD8 + CD45RO + CD45RA -CCR7 -) in recall responses in healed CL individuals after L. major or L. tropica infection (200).

Similarly, it was shown that individuals who recovered from L. infantum infection, or who are asymptomatic, possess effective and specific memory responses against Leishmania (57). Stimulated PBMC from healed individuals showed increased CD69 + expression, a T cell activation marker, in both TCD4 + and TCD8 + cells, increased CD25 expressing in TCD8 + cells, and increased percentage of TCD4 + cells expressing memory marker CD45RO when compared with controls and individuals with symptomatic disease (57). Asymptomatic individuals can control L. infantum infection through the presence of IFN-γ producing TCD8 + cells (53,203).

Furthermore, studies with cured individuals after L. braziliensis infection have shown that while both CD8 + and CD4 + specific T cells are present, with the number of TCD8 + cells increasing after cure, CD4 + TEM cells are the main source of IFN-γ produced after antigenic stimulation (204,205). Also, cured CL patients show increased lymphoproliferative potential and higher IFN-γ production, while asymptomatic individuals infected with L. braziliensis have higher levels of IL-10, probably involved in the balance between immunoregulatory and effector responses responsible for parasite control without associated tissue damage (205).

After therapy, parasite burden decreases along with Leishmania-specific immune responses, an equilibrium that appears to be essential for long-term protectionconstant antigenic stimuli (due to reinfection or parasite persistence) can induce and maintain specific memory T cells but also contribute to a chronic activation of effector cells and T cell exhaustion.

There is evidence that time after cure may influence host responses since a reduction in TCD4 + and TCD8 + cell activation is observed two years after the initial L. braziliensis infection and cure, and this is again consistent with increased regulatory responses (206). Differences between early lesions and late lesions (L. braziliensis) positively correlate the duration of illness with increased CD4 + CD69 + T cells, and negatively with CD4 + CD25 + T cells (207). Furthermore, again during active CL caused by L. braziliensis, both CD4 + and CD8 + TEM cells (CD45RO + CCR7 -) are present in lesion sites, in much higher frequency than TCM cells, and this enrichment in effector cells can be associated with immunopathology and tissue damage (207). Nevertheless, the recall responses present in healed individuals persist, and CD4 + and CD8 + TEM cells proliferate and produce IFN-γ after secondary stimulation, whilst having regulatory mechanisms that prevent immunopathology without the loss of protective immunity (206).

Humans immunized with a whole crude vaccine (prepared from promastigote antigens from New World Leishmania species) are resistant to reinfection and mount similar responses to those found in recovered patients after L. braziliensis infection, showing IFN-γ production without IL-4 as well as a higher proportion of TCD8+ cells, consistent with a long-lasting protective role of TCD8+ cells (53,175,208,209). IL-4 production is only detected during active MCL infection (175).

It is important to note, most human studies are performed with peripheral blood, and additional tissue-specific memory responses cannot be excluded.

c) Memory maintenance and concomitant immunity

Memory T cell maintenance mechanisms remain unclear, and no successful human vaccine targeting cellular-mediated immunity exists, but there is evidence of long-lasting memory T cells.

Even for antibody-based vaccines, as is the case of individuals vaccinated against the vaccinia virus, there are specific memory T cells in circulation 25 to 70 years after immunization (191,210).

Moreover, a study has found a direct correlation between influenza-specific memory TCD4 + cells and reduced disease severity after challenge (211), and circulating memory TCD4 + cells are associated with non-progression of HIV infection (194).

In mice, the cytokines IL-17 and IL-15 have been implicated in the maintenance of TCD8 + memory T cells, whereas TCD4 + memory cells need TCR and/or MHC-II signalling for their functional maintenance (191,192). In humans, these differences may be associated with tissue distribution where memory cells in the bone marrow, lymphoid tissue, spleen and blood can be maintained by IL-17 and IL-15, and memory T cells in the mucosa are maintained by TCR cross-reactions due to the high antigen density (191,212-214).

Interestingly, healed and asymptomatic individuals continue to carry viable parasites, in roughly constant numbers throughout their life (215,216). This is a very important aspect for infection transmission (217) and for potential relapse in immunosuppressive conditions (44), but also for memory generation and maintenance. Persistent parasites may be necessary for memory maintenance, as revealed by the fact that sterile cure of persistently infected mice makes them susceptible to secondary infection, and the prominent role of TEM cells in anti-Leishmania recall responses (218). As previously referred in 2.3, asymptomatic infection is much more common than active disease and even more than sterile cure, and these infections show low parasite levels, no pathology, and protective immunity against reinfection and/or disease severity. T cells, and agrees with the loss of immune protection after sterile cure (57,198,215,223,224).

Persistent parasites appear

This process of memory maintenance is named concomitant immunity. Immune memory against Leishmania is mediated through continuous cell priming that maintains memory cells in -Early immune responses greatly impact subsequent adaptive response;

-Both TCD8+ and TCD4+ cells mediate protection or susceptibility to Leishmania infection;

-IFN-γ is absolutely required to control the parasites sufficiently and control the disease;

-IL-4 is associated with the development of a non-protective Th2 phenotype;

-Human memory subsets involved in long-lasting protection are not very well described, but an important role for TEM and TCM was described;

-Memory maintenance against Leishmania is associated with parasite/antigen persistence.

Leishmaniasis is a vaccine-preventable disease

Overall, the main observations supporting vaccine feasibility are: i) protection against reinfection following spontaneous or drug-induced recovery; ii) protection can be induced through leishmanization; iii) Th1 cells are essential for resistance; iv) TCD4 + and TCD8 + cells are involved in immune memory.

The strongest evidence supporting the idea of inducing protection against Leishmania ii) The preferential host target cell is the macrophage, and current tools allow for modulation of macrophage intracellular killing and functional enhancement;

iii) High level of antigenic conservation among pathogenic species (detailed in chapter II); iv) New suitable Th1-inducing adjuvants are today under development (see Table I.3); v) Parasiteand vector-associated antigens can be used to induce protection.

The three available canine vaccines (CaniLeish ® , LeishTec ® and Letifend ® ) provide additional evidence that it is possible to induce protection against leishmaniasis through immunisation in the dog model. Several inactivated/killed and subunit vaccines have all shown to be quite ineffective in human hosts, despite promising results in animal studies (242). Also, vaccines tested in endemic regions have not performed as well as when tested in healthy volunteers (226,242). There seem to be several underlying issues at play: the use of mouse models to validate antigen candidates (golden hamsters as promising alternative as an experimental VL model); intravenous challenges instead of natural challenge used in the experimental designs; the ability of the immune system to effectively recognize live, but not killed, parasites (242); and the methods to test and measure protective immune responses (226).

Vaccine feasibility:

-Natural protective immunity after recovery from infection;

-Leishmanization; -Cost-effectiveness studies;

-Parasite-related features.

Main challenges for leishmaniasis vaccine development:

-No cellular immunity-inducing vaccine available;

-Lack of approved Th1-inducing adjuvants for human vaccine development;

-Lack of reliable correlates with immune protection;

-Poor translation from animal models;

-Undefined optimal administration route.

Immune correlates of protection against leishmaniasis

Specific immune correlates of protection, defined as measurable immune responses that are responsible for and statistically interrelated with protection (243), are largely unknown and nonstandardised in vaccine development against Leishmania. The mechanisms behind protection, and their evaluation, must be understood so that an effective vaccine is developed.

As detailed above, the resolution of human CL depends on Th1 cells that secrete IFN-γ and activate macrophages for intracellular parasite killing. Similarly for VL, if Th1 cells are absent, or if Th2 cells are predominant, patients develop chronic progressive VL (160,244).

The major role attributed to TCD8 + cells during anti-Leishmania immune responses is via IFNγ secretion that, in turn, will induce nitric oxide production and resistance against reinfection, as well as cytotoxic activity towards infected macrophages (244-246). TCD8 + cell-produced IFN-γ has been implicated in both resistance and immunopathology (247).

The known role of TCM and TEM populations in long-lasting protective responses should be explored to assess the quality of induced responses after immunisation and evaluate protection throughout time.

Antibody production, used as a correlate of protection for currently available antibody-based vaccines, does not provide quality evidence of protective responses against Leishmania. . VL and DCL are associated with high parasite burdens and, as infection progresses, DTH responses become poor and total antibody titres rise (248). The anti-Leishmania antibodies detected by the diagnostic tests rK39 (India) or rK28 (Africa) persist many years after cure, which can be due to repeated exposure or parasite persistence, and cannot distinguish between asymptomatic, previous or current infection (248). The presence of IgG2 antibodies is detected in healed patients, however, they are not actively functional in infection control (60). On the contrary, there are reports showing a deleterious effect by IgG antibodies in animal models, through the production of IL-10 by macrophages after binding to the Fcγ receptor (249).

Da-Cruz et al performed studies with healed patients infected by L. braziliensis, and suggested two markers for protective immunity: i) cure is correlated with high, but decreasing, numbers of Leishmania-specific TCD4 + cells, in the absence of IL-4; and ii) decreasing levels IL-5 are associated with long-term cure (175). These data are confirmed by studies with healed patients after L. major infection (250). In MCL long-term healing, 1 to 17 years after treatment, there is a slight increase in TCD8 + subpopulation for a final ratio TCD4 + /TCD8 + of around 1 (175). After treatment and parasite elimination, it is expected that the population of effector TCD8+ specific cells will retract. The maintenance of specific TCD8+ cells in circulation, and that can re-expand and differentiate upon restimulation, is probably mediated by the TCD4+ cell population. Interestingly, although MCL healed patients showed increasing IFN-γ levels, these were variable, with high and low responders. Probably, persistent Th1 CD4 + cells, together with decreasing proportions TCD4 + and TCD8 + specific populations in the absence of IL-4 production, are important for infection control and prevention of relapse (175).

Polyfunctional T cells produce several effector cytokines simultaneously and often in higher amounts, particularly pro-inflammatory cytokines IFN-γ, TNF-α, and IL-2. Polyfunctionality broadens the range of effector functions, and has been implicated in the development of vaccines against intracellular pathogens, i.e. targeting cellular-mediated immunity (251-253). Polyfunctionality correlates with T cell efficacy and can be used during vaccine development, for the design of candidates that target such responses and to analyse vaccine-induced responses (254).

The magnitude of response, as in the number of antigen-specific effector cells, is used for humoral responses as a correlate of protection (253). However, the quality of a given immune response, determined by the effector functions (proliferation, cytotoxicity, cytokine and chemokine production) and associated with polyfunctionality, is a better determinant for protection than the magnitude of response (253). Considering this, a high-quality response is one that specifically balances different effector functions to produce an effective and long-lasting protective immune response. Also, the diversity of antigens targeted by specific T cells (breadth of response) can be used as a correlate of protectionin studies with HIV patients, broader antigenic diversity correlates with protective immune responses (255).

Leishmania-specific Th1 cells that produce IL-2 and/or TNF-α are mostly CCR7+ TCM cells;

(256). It is possible that polyfunctional cells differentiate into IFN-γ-producing (only) T cells due to chronic antigen exposure (257), so vaccines must induce adequate Th1 responses, neither too low nor too high, to assure both effector functions and memory maintenance. These responses depend of antigen dose, stimulation duration, antigen-presenting cells targeted, and the early cytokine environment induced by the vaccine formulation (253,258).

Second and third generation vaccines typically do not induce antigen persistence, which is probably required to maintain anti-Leishmania immunity (157,218), as it occurs in natural infection or whole-organism vaccines. In highly endemic areas, natural boosts may occur, nevertheless, this is an important aspect to consider and include during vaccine design and preclinical development, and, for example, adapt vaccination schedules to include boost immunizations.

Finally, it has been demonstrated that memory responses in vaccinees correlate with the number of precursor cells in the naïve repertoire (259-261). This crucial feature should be exploited during the preclinical development of vaccine candidates through the comparison of immunogenicity profiles induced by the antigens in different immune backgroundsnaïve, healed VL, healed CL, etc.

Antigens that correlate both with the presence of memory responses and the existence of naïve precursor cells are strong candidates for a successful vaccine formulation, able to induce memory in naive individuals (prophylactic vaccine) or boost existing responses to help control infection (therapeutic vaccine).

In summary, the most important markers of immunity against leishmaniasis are: a predominant Th1 response and with the presence of polyfunctional TCD4+ cells (IFN-γ, IL-2, TNF-α),

and with low IL-10 (80). This immune environment will ultimately favour the production of effector anti-leishmanial molecules such as reactive nitrogen and oxygen intermediates for parasite control (63,68,141). Also, a relevant aspect for the induction of immunity against Leishmania seems to be the need for a balanced ratio between IFN-γ/IL-12 and IL-10/IL-4 (159), which should be preserved to prevent immunopathology and assure memory generation and maintenance.

The lack of well-established correlates of protection hinders vaccine development against leishmaniasis:

-Mouse models are of limited extrapolation to human disease and should not be used to

Vaccines against human leishmaniasis

Since the end of leishmanization, several attempts were made to develop other first generation vaccines (killed or live attenuated Leishmania parasites), as well as second generation (recombinant proteins or peptide-based vaccines) or third generation vaccines (DNA-based vaccines) (106,262,263).

Several first generation vaccines using crude antigens from Leishmania promastigotes, prepared with or without BCG as the adjuvant, were tested in human clinical trials. These candidates were found to be safe but generally ineffective, as is the case of autoclaved L. major (ALM) vaccine which showed results comparable to BCG injection alone (169). Highly variable efficacy levels were reported against CL (0-75%) and very low efficacy against VL (169). Three other first generation vaccine candidates have been tested: 'Mayrink vaccine'; 'Convit vaccine' and 'Razi Institute vaccine' with inconclusive or negative results for prophylaxis potential, but somewhat encouraging for therapeutic indications (264). These studies, although unsuccessful in launching a vaccine against leishmaniasis, further established the vaccine feasibility. Moreover, they paved the way for the development of second and third generation vaccines. Second and third generation vaccines need to be formulated with an adjuvant to increase immunogenicity and response specificity.

Current vaccine pipeline

In the last decades, several antigens have been explored as candidate antigens for both prophylactic and therapeutic second-generation vaccines against leishmaniasis. These antigens will be further characterised in chapter II.

The most advanced vaccine candidates are described below.

The vaccine candidate Leish-111f (also named LEISH-F1 or MML) was the first subunit The LEISH-F1+MPL-SE vaccine was partially protective in experimental VL, but ineffective against canine leishmaniasis in phase III field trials (266). In human trials, this formulation was found to be safe and well tolerated, and immunogenic against VL and CL (230). Furthermore, LEISH-F1

showed therapeutic efficacy against MCL, in terms of accelerated time to cure, when used in combination with chemotherapy (267). Later, the LEISH-F3 candidate was slightly modified with the addition of the cysteine protease B (CPB) to increase antigen recognition and extend the geographical affectation of the vaccine (271).

Researchers at the

This new candidate was formulated with GLA-SE and is called LEISH-F3+. This vaccine has evaluated in experimental infection models (hamsters) and has shown to provide robust immunity, similar to LEISH-F3 (271). The LEISH-F3+GLA-SE vaccine candidate was also proven to be safe, and induced A recombinant chimeric protein composed by T cell epitopes specific to human and mice MHC alleles and derived from 4 Leishmania infantum proteins (LiHyp1, LiHyp6, LiHyV and HRF proteins)

was formulated with saponin and tested in BALB/c mice. Vaccinated mice and challenged with L.

amazonensis were protected against infection. The candidate induced a specific IFN-γ, IL-12 and GM-CSF production, lower IL-4 and IL-10 levels, as well as high IgG2a isotype antibody levels. In addition, antileishmanial nitrite production by splenocytes was detected. Interestingly, the recombinant protein administered alone induced a partial protection against challenge (278). In the last decades, particularly after the completion of genome and proteome annotation of several pathogenic Leishmania species, these excreted-secreted proteins were further characterised. The canine vaccine is well-tolerated and safety studies did not show any significant adverse effects (286,287). However, a study in Spain has revealed a high rate of adverse yet mild reactions in vaccine users included in the survey (82%), the most frequent being local reactions, apathy, fever and gastroenteritis (288). Still, one often overlooked aspect is that each vaccine dose contains a fixed amount of QA-21 adjuvant, which is not adapted to the dog's weight. This highlights the need for optimal selection and dosage of the adjuvant used human vaccine formulations. An epitope-based approach and new Th1-inducing adjuvants will greatly mitigate these concerns.

Leishmania Excreted-Secreted Antigens as promising vaccine candidates and the successful canine vaccine CaniLeish®

Some attempts were made to identify the protective antigens contained in total ESP preparations. From Leishmania excreted/secreted proteins, research developed at the Lemesre Lab showed that soluble Promastigote Surface Antigens (PSA) is an immunodominant component of L.

amazonensis and L. infantum secretome, and highly conserved among Leishmania species (289).

Moreover, the vaccinated dogs with recombinant PSA (rPSA) of L. amazonensis or its carboxyterminal part, both combined with QA-21 as adjuvant, were protected at 78.8% and 80%, respectively (290). This cross-protection was associated with hallmarks of a dominant Th1-type immune response. In L. infantum and L. major-protected humans (healed individuals), it was also clearly demonstrated that the rPSA induced a dominant Th1 response associated to cytotoxicity in vitro.

During the FP7-funded RAPSODI project (2009-2012), the Lemesre Lab and collaborators developed a vaccine candidate containing the Promastigote Surface Antigen (PSA) from L. amazonensis, whose immunogenicity was validated on human cells (282). However, the very low productivity of recombinant LaPSA would result in an expensive vaccine, which is unfeasible for human mass vaccination. As peptide production is far less expensive, the adopted approach was to select only peptides derived from proteins of interest for vaccine development.

Peptide-based vaccines

The final formulation is of extreme importance for the development of an effective vaccine to be deployed in low-resource settings. The ideal target product profile (TPP) for a vaccine against leishmaniasis englobes: i) good safety profile;

ii) a minimum number of immunizations and boosts needed;

iii) the absence of animal products and impurities; iv) cost-effectiveness; v) a prophylactic and therapeutic effectiveness; vi) optimal delivery; vii) preferably, the lack of cold-chain supply (169,270).

A peptide-based vaccine formulation would answer many of these requirements (291).

Peptide-based vaccines explore the minimal immunogenic units necessary to induce protective immune responses (epitopes) and have become an increasingly important tendency in vaccine development (291,292). Peptide-based approaches allow for a highly specific stimulation of host immune responses, while decreasing the risk of unwanted cross-reactions and other adverse reactions (292).

Several epitope discovery strategies have been exploited in peptide-based vaccine design (293,294), particularly: i) in silico epitope mapping through HLA-binding epitope prediction, selection of conserved antigens by phylogenetic analysis, protein localization prediction or through reverse vaccinology approaches;

ii) phage display assays;

iii) immunodominance assays and peptide competition assays.

An epitope-based approach, in contrast to subunit recombinant protein or inactivated live vaccines, offers several advantages, namely: i) easiness of production, ii) superior stability and no need for cold-chain supply; iii) higher purity and absence of potentially harmful substances; iv) lower antigen complexity; v) low scaling-up costs; vi) high response specificity; and vii) ability to combine epitopes to design multi-epitopic and/or multi-specific vaccines (292,295).

From the CaniLeish® and recombinant PSA development studies, our lab has identified the main immunogenic portion from carboxy-terminal region of rPSA, representing 19.4% of the rPSA sequence. Vaccinated dogs with three peptides derived from this region, combined with QA-21 as adjuvant, were protected at 60% (Petitdidier E. et al. submitted). This protection level is significant given the small size of the immunogenic peptides used in this vaccine formulation, in comparison to total rPSA (78.8% of protection) or PSA carboxy-terminal region (80% of protection). This study clearly demonstrates peptide-based vaccines are a promising approach for the design of vaccines against leishmaniases.

In human leishmaniases, we need to consider the diversity of Leishmania species and HLA diversity due to population variability from numerous endemic areas.

Altogether, peptide-based vaccines are a promising approach for the design of vaccines against leishmaniases, through the ability to include multiple immunogenic and conserved epitopes from various protein antigens, increasing the chances of immunogenicity and both parasite strain and target population coverage.

However, short peptides are known poor immunogens when administered alone. The main associated challenges are the avoidance of inactivation or degradation by the host immune system, and the enhancement of peptide immunogenicity. This low immunogenicity can be overcome with the addition of adjuvants in the final vaccine formulation that direct and boost the induced responses.

There is the need to improve adjuvants for the final vaccine formulations, adjuvants that are more efficacious, less toxic, and approved for human use (226). Some new promising adjuvants are being developed, particularly Th1-inducing adjuvants and TLR agonists, which can be used in anti-Leishmania vaccine formulations (Table I.3.) BLAST was then used to reject peptides that were 100% identical with mice and human proteins. These studies are further detailed in Table I.4.

Leishmania-specific peptides tested in human cells

Project objectives and approach

The present doctoral project was part of the Marie Sklodowska-Curie Innovative Training The major aims are:

• To identify the antigenic proteins present in the Leishmania secretome;

• To design and elaborate species-conserved peptides with multiple and appropriate immunogenic epitopes to be used as vaccine antigens;

• To perform preclinical phase studies in human cells to evaluate the predictive peptide's immunogenic and immunoprotective properties; The term "co-evolution" was actually used for the first time to describe the relationship between Leishmania parasites and host sand fly species (5). Environmental cues lead to the differentiation from procyclic promastigote to metacyclic promastigote inside the sand fly, and to intracellular amastigote in the vertebrate host (temperature, pH, exposure to reactive oxygen and reactive nitrogen species, extracellular proteolytic activity, or nutritional requirements) (6,7).

•
Leishmania parasites have co-evolved with vertebrate hosts since the Jurassic period, and have adapted to diverse epidemiological scenarios divergently for 20-100 million years, and yet still share very high levels of genomic homology and synteny (6).

The first full genome sequence of a Leishmania parasite was published in 2005 (8) marking the beginning of the post-genomic era for these parasites. L. major parasites have a 32,8 megabase haploid genome, containing over 8200 protein-coding genes, and over 94% of all mRNA molecules are constitutively expressed between promastigotes and amastigotes (9,10). Since then, three other Leishmania species genomes have been sequenced and annotated -L. (L.) infantum (11), L. (V.) braziliensis (11), and L. (L.) mexicana (12). Comparative analysis showed a high level of genetic conservation among species (7392 common genes), but quite remarkable large-scale genetic differences in terms of ploidy and gene copy number (12).

The high level of conservancy observed among Leishmania genomes, with minor changes in mRNA levels between promastigotes and amastigotes, suggests that differentiation, virulence and pathogenesis depend on post-transcriptional and post-translational expression control mechanisms (6,9,(13)(14)(15)(16)(17)(18). Predicted genes are expressed as polycistronic units present on the same DNA strand, much alike bacterial operons. However, genes grouped in a common orientation are not regulated by common mechanisms nor are they functionally related (19,20). Regulation of single gene expression does not rely in RNA polymerase (RNApol) promoter controlcontrol of gene expression is performed at multiple levels (trans-splicing, mRNA polyadenylation, mRNA stability, transcript elongation, and protein stability) (19)(20)(21). Accordingly, very few RNApol promoters were identified, none for RNApol II, except for the splice leader RNA promoter and RNApol I promoter sites in the rDNA locus (10).

Post-translational modifications (PTM) are proposed to be a major mechanism in Leishmania control of gene expression (6,22,23). Over 200 different types of PTM have been generally described,

and Leishmania proteins can be cleaved eliminating signal sequences, pro-peptides and initiator methionines, or cleaved by specific proteases to generate final functional protein forms (10).

Leishmania protein modifications include methylation, acetylation, phosphorylation, hexosylation, fucosylation; the addition of complex molecules such as lipids, sugars or glycolipids; ubiquination, nytrosylation and hyperoxidation of sulfhydryl group (6,10,22).

Proteomic studies are therefore optimal tools to study Leishmania pathogenesis since gene expression is for the most part regulated post-transcriptionally (6). The first proteomic studies compared promastigote and amastigote forms and were performed for L. (L.) infantum (24), L. (L.) donovani (25), L. (L.) mexicana (26), and L. (V.) panamensis (27).

The main pathways upregulated in amastigotes are fatty acid oxidation and gluconeogenesis, mitochondrial respiration, proteins with basic pH, and some specific proteins such as tryparedoxin peroxidase, methylthioadenosine phosphatase, required for survival under oxidative conditions in the host cell (6,25,26,28). By contrast, Leishmania promastigotes rely mostly on glycolysis and aminoacid metabolism for energy generation and are also capable of hydrolysing dissacharides. In promastigotes, the glycolytic pathway is upregulated, and in metacyclic promastigotes the proteins PFR1D, alpha-and beta-tubulin, cysteine protease B, trypanoredoxin, GP63, and GP46 show higher expression levels (6,25,26). Other differentially expressed proteins are associated with diverse metabolic functionscytoskeleton components, stress responses, amino acid and carbohydrate metabolism, detoxification and proteolysis (6,25,26). Likewise, some modest differences were found between cutaneous-and visceral-adapted L. donovani strains, the latter having increased translation, biosynthetic processes, antioxidant protection and signalling functions (29).

The specific activities of some glycolytic enzymes are slightly decreased (hexokinase, phosphofructokinase and glucose-6-phophate dehydrogenase) or greatly reduced (pyruvate kinase) in amastigotes. In the amastigote form, parasites use mostly fatty acids and amino-acid metabolism for energy generation, as access to glucose and other sugars is limited (6,25,30). The capacity for sugar uptake is also reduced in amastigotes in comparison to promastigote forms, so glycolytic and pentose-phosphate pathways are present and functional but with lower activity levels. Also, glucose degradation results in succinate production (instead of pyruvate) through the action of phosphoenolpyruvate carboxykinase/malate dehydrogenase enzymes (6,25,30).

Sugar residues required by Leishmania amastigotes are generated de novo through gluconeogenesis (6,30). Both metacyclic promastigotes and amastigotes accumulate β-mannan, a complex polyssacharide, as intracellular energy storage. However, contrary to promastigotes, this molecule is essential for amastigote survival and replication and is dependent on hexose uptake (6,30).

The term 'secretome' was firstly defined by Tjalsma et al 2000 as a subset of the proteome comprising both all secreted proteins and the pathways and machinery required for protein transport/secretion (31). There is increasing interest in the secretome and excreted-secreted proteins as they play a pivotal role in virulence and interaction with the host, in this case, for Leishmania establishment of infection and immunomodulation (32)(33)(34)(35)(36)(37)(38)(39)(40). These early secreted proteins allow Leishmania parasites to survive in an otherwise lethal environment, inside the phagolysosome, as well as to modulate host response by suppressing host cell signalling and macrophage activation, while deviating the host's immune response towards a permissive antiinflammatory phenotype (32)(33)(34)(35)(36)(37)(38)(39)(40).

Proteomic analysis of the secretome of L. (L.) donovani promastigotes (41), L. (L.) mexicana promastigotes (42), L. (V.) braziliensis promastigotes (43), and L. (L.) major promastigotes inside the sand fly midgut (44) were performed, contributing to an increased knowledge about the function of these proteins. These studies show the main secretion pathway in Leishmania parasites is through exosome/micro-vesicle production, which are crucial for parasite virulence (37,40,45). Only a small proportion of proteins possesses a N-terminal secretion signal peptide or is non-classically (ER/Golgi-independently) secreted (46). Interestingly, amastigotes continue to secrete exosomes to the cytoplasm of infected and neighbouring cells (46). Exosomes are increasingly implicated in hostpathogen interactions and are determinant for the promotion or inhibition of host immunity (36,47).

A plethora of candidate virulence factors has been identified in the Leishmania secretome, meaning functional characterization is extremely difficult. It is generally accepted that the Leishmania pathogenesis depends on parasite-mediated early immune priming and continuous immunomodulation, as well as maintenance of the parasitophorous vacuole conditions (30,32,48,49).

Molecules found in the secretome can be associated to any of these essential functions. The present chapter describes the identification of the total secretome of six Leishmania species, exclusive human pathogens, as well as L. tarentolae, non-pathogenic to humans. The secretome was prepared according to the method used to produce LiESAp from promastigote cultures growing in aseric medium, used in the CaniLeish ® vaccine.

Material and Methods

Leishmania parasite cultures in aseric medium, generation and purification of total excreted-secreted proteins

The in vitro culture of Leishmania promastigotes (Table II

Protein separation

Proteins were separated on 1D SDS-PAGE gels (12 % polyacrylamide, Mini-PROTEAN® TGX™ Precast Gels, Bio-Rad, Hercules USA). Gels were stained with PageBlue Protein Staining Solution (Fermentas) and scanned using a computer-assisted densitometer (Epson Perfection V750 PRO). Gel lanes were cut into 12 gel pieces (11 bands plus the well fraction) and destained with three washes in 50% acetonitrile and 50 mM triethylammonium bicarbonate buffer (TEABC). After protein reduction (with 10 mM dithiothreitol in 50mM TEABC at 56 °C for 45 min) and alkylation (55 mM iodoacetamide TEABC at room temperature for 30 min) proteins were digested in-gel using trypsin (800 ng/band, Gold, Promega, Madison USA) as previously described (1). Desalting and preconcentration of samples were performed with a ZipTip C18. Digest products were dehydrated in a vacuum centrifuge and reduced to 3 µL.

High-performance liquid chromatography and MS measurements

Bioinformatics analysis

Raw MS spectra were processed using the MaxQuant environment (56) and Andromeda for database search with iBAQ algorithm enabled (57). The MS/MS spectra were matched against the Leishmania entries of UniProtKB SwissProt and TrEMBL sections (release 2017_01; http://www.uniprot.org) and 250 frequently observed contaminants (MaxQuant contaminants database) as well as reversed sequences of all entries. The following settings were applied for database interrogation: mass tolerance of 7 ppm (MS) and 0.5 Th (MS/MS), trypsin/P enzyme specificity, up to two missed cleavages allowed for protease digestion, minimal peptide length at 7, cysteine carbamidomethylation as fixed modification and oxidation of methionine as variable modification. FDR was set at 0.01 for peptides and proteins.

A representative protein ID in each protein group was automatically selected using in-house bioinformatics tools (leading v2.2 and multi-species script), developed by Oana Vigy (Plateforme de Proteomique Fonctionnelle de Montpellier), and the Perseus software (version 1.5.3.0). First, proteins with the most numerous identified peptides are isolated in a "match group" (proteins from the "Protein IDs" column with the maximum number of "peptides counts (all)"). For the match groups where more than one protein ID is present after filtering (no specific tryptic peptides), the "leading"

protein is firstly chosen as the best annotated protein in UniProtKB (reviewed SwissProt entries rather than automatic TrEMBL entries) corresponding to the 'Leading_AUTO' identification status. In case of ambiguous peptide identifications, proteins are identified by a given species and group-species order preference, corresponding to a 'Leading_SPECIES' identification status (Table II.2). If ambiguous identifications cannot be distinguished with specific peptides or taxonomy, these remain identified as a match group ('MultiSpecies' identification status).

Secretion pathway analysis (SecretomeP)

SecretomeP is a sequence-based method for the prediction of eukaryotic secreted proteins targeted to a non-classical secretory pathway, i.e. proteins without an N-terminal signal peptide (58).

SecretomeP queries other feature prediction servers to obtain information on various posttranslational and localisational aspects of the protein, which are integrated into the final secretion prediction. This method is also capable of predicting (signal peptide-containing) secretory proteins where only the mature part of the protein has been annotated, or cases where the signal peptide remains uncleaved.

The SecretomeP 1.0 standalone version was downloaded (http://www.cbs.dtu.dk/cgibin/nph-sw_request?secretomep) and fasta files with protein sequences corresponding to total identifications per sample were retrieved from UniProtKB. Each sample list was analysed with SecretomeP 1.0 (Signal-P and non-classical eukaryotic secretion predictions were performed).

Proteins with NNscore > 0.5 were considered non-classically secreted.

Exosome marker analysis

The secretome datasets were screened for exosome cargo protein markers, from two different sources: i) ExoCarta, an exosome database, which includes the contents that were identified in exosome cargo in multiple organisms (59); and ii) Silverman et al 2008 (41) describes 26 Leishmaniasecreted proteins associated with exosome-like and glycosomal vesicles.

To find the Leishmania genes ortholog to the most common exosome markers described by the ExoCarta database, a search strategy in OrthoMCL database was performed (Figure II.3). Briefly, 100 exosome markers (GenBank gene accessions) were downloaded from the Exocarta database (59).

Firstly, a text search ('Identify Sequences based on text terms') was performed at OrthoMCL database web server with the 100 Gene Symbols. This search retrieved 3631 sequences (step 1), 83 of which correspond to human-specific proteins (step 2). These sequences were transformed to groups (step 3) to compare with four Leishmania phyletic ortholog groups in the database (step 4). These Leishmania phyletic groups were then retransformed into sequences (step 5), and 72 sequences from

Figure II. 3 Exosome marker analysis of 100 exosome protein markers from ExoCarta. A) OrthoMCL search strategy for Leishmania ortholog genes of exosome cargo markers from ExoCarta. B) Intersection between 72

Leishmania ortholog genes and 1981 GeneDB IDs identified in the secretome proteomic analysis.

Results

Proteins found in the secretome correspond to 12-17% of total Leishmania proteins

The method used for the preparation of the secretome is analogous to the large-scale production of LiESAps for the CaniLeish ® vaccine, which allows the production and analysis of naturally excreted-secreted proteins. Leishmania promastigotes are cultured in a completely defined aseric medium until the growth stationary phase (day 6).

To identify the proteins present in the Leishmania secretome, 11 bands (plus the well fraction) from the 1D gel were carefully isolated and analysed per sample, covering all proteins per lane (Figure Considering the reference proteomes available in UniprotKB, an average total of 8034 proteins are encoded in the Leishmania genome. The proteins identified in the secretome correspond to 12% up to 17% of total proteins (Table II.3).

Figure II. 5 Comparison of protein identification lists. A) Venn diagram comparing proteins identified in six human pathogenic species. B) Intersection lists between all Leishmania species, individual unique lists, and relevant groups: human pathogens (all except L. tarentolae), VL-causing species (LEIIN+LEIDB), Old World CL-causing species (LEIMA+LEITR), Mediterranean species (LEITR+LEIIN+LEIMA), New World CL-causing species (LEIAM+LEIBR), CLcausing species (LEIAM+LEITR+LEIBR+LEIMA).

Table II. 3 Leishmania reference proteomes, respective protein counts, and proteins found in the secretome.

Proteome

The majority of Leishmania excreted-secreted proteins are non-classically secreted

A bioinformatics analysis of the secretome protein lists was performed with SecretomeP to identify proteins containing secretion signal peptides and proteins which are non-classically secreted (also known as leaderless secretion, unconventional or non-conventional secretory pathway) (58).

The consensus regarding protein secretion in eukaryotes is that membrane-associated or secreted proteins reach their final localisation via the classical secretory pathway. Proteins contain a signal peptide and/or transmembrane domain which targets them to the ER, from where they exit in vesicles, reaching the Golgi and eventually the membrane. Yet, there are extracellularly active proteins which do not enter the ER/Golgi pathway (60).

Results show that only a few proteins identified in the Leishmania secretome contain a classical secretion signal peptide (from 4.8% up to 12.9%). These results agree with previous proteomic studies which have shown as well that classical secretion is not the main secretion pathway in Leishmania parasites (33,46). although not yet well-studied, has been demonstrated for Leishmania protein HASPB (61).

Over
Importantly, the proteins secreted via non-classical secretion are predicted to be 3 to 9 times more frequent than proteins containing signal peptides for classical secretion. As indicated by the SecretomeP results, and as previous secretome studies have shown,

Leishmania-secreted proteins are associated with exosome-like vesicles as well as glycosomal vesicles. In the latter case, either Leishmania parasites secrete whole glycosomes or glycosomal cargo into the extracellular moiety (41). Exosomes are 30-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro (47).

Interestingly, in the present study we find 8 proteins associated with vesicle-based secretion, from the 26 proteins described by Silverman et al 2008 (Table II.4). Furthermore, several Leishmania proteins ortholog to exosome protein markers from other eukaryotic cell types were identified in the secretome datasets (Table II.5). The ExoCarta database describes the 100 most common proteins often associated with exosome cargo from different tissues and cell types, and across 10 different species, including humans (59). Remarkably, a high number of ortholog genes were found in Leishmania, 18 orthologs per species for a total of 72 genes identified. These correspond to 16 different proteins, 9 of which are found in the secretome datasets (Table II.5).

Table II. 4 List of Leishmania-secreted proteins associated with exosome-like and glycosomal vesicles found in the secretome datasets (Silverman et al 2008). AP, adipocyte exosome (adiposome); GLY, Leishmania glycosome; BC, B-cell lymphocyte exosome; DC, dendritic cell exosome

GeneDB accession number

Protein

Functionality of excreted-secreted proteins is conserved among

Leishmania species

To better elucidate the specialised functions performed by the Leishmania-secreted proteins, a Gene Ontology (GO) annotation analysis was performed. Gene Ontology provides a representation or categorisation of the properties assigned to different gene products, and covers

Figure II. 7 Gene Ontology (GO) terms annotation as percentage of total identified proteins. A) Molecular function GO terms. B) Cellular component GO terms. C) Biological process GO terms.

Additionally, multifunctional proteins have been described, particularly, moonlighting proteins that perform multiple independent, and often unrelated, functions, in the absence of gene fusion, splicing variants or different catalytic domains (63). Several moonlighting proteins have been described in both eukaryotes and prokaryotes which were incorporated in the MoonProt Database, a Database for Moonlighting Proteins (64). This database includes four Leishmaniaspecific proteins with described moonlighting functions (Table II.6). These four proteins were found in secretome datasets from all analysed Leishmania species. Among the most abundant proteins, we wish to highlight HSP70, a protein chaperone with key function for parasite's adaptation to higher temperatures in vertebrate hosts; and ubiquitin-60S ribosomal protein L40 involved in gene regulation. Also, the promastigote surface antigen is a major surface glycoprotein, related to glycoprotein 46 (gp46), whose functions are not fully understood, but is associated to the evasion of complement lysis (66) and parasite virulence, as evidenced by its upregulation in PKDL-causing Leishmania parasites (67). Furthermore, the elongation factor 1-alpha contributes to parasite persistence by inhibiting macrophage function through SHP-1-mediated disruption of JAK1, JAK2, STAT1 phosphorylation (68). Additionally, EF1alpha interacts with ribosomal subunits, for example, the 60S subunit also identified here, associated with protein translation (65). In turn, ribosomal subunits also interact with Sadenosylhomocysteine hydrolase, also very abundant in the secretome (65). The infective insect stage-specific protein expressed by gene META1 is specific to infective metacyclic promastigotes (69).

Several of these proteins have also been described as important antigens, namely, the protein PSA-38S (51) and elongation factor 1-alpha [START_REF] Naouar | Prediction of T Cell Epitopes from Leishmania major Potentially Excreted/Secreted Proteins Inducing Granzyme B Production[END_REF]. Interestingly, the enzyme adenosylhomocysteinase, involved in amino-acid biosynthesis (synthesis of L-homocysteine from S-adenosyl-L-homocysteine) was found to be highly abundant, which is expected for

Leishmania promastigotes, and it has also been shown to be immunogenic in hamsters and human PBMC stimulation assays (71). The uncharacterised protein (A4H3U0) identified among the most abundant proteins is actually the most abundant in the LEIBR sample. This protein is predicted to have a metalloendopeptidase activity with cell adhesion functions and could be analysed further as a potential antigen candidate.

Moreover, heat-shock proteins, other cytoplasmic and cytoskeleton proteins, such as tubulin and actin, as well as membrane-associated proteins are ubiquitous proteins identified as The putative histidine secretory acid phosphatase is found on the parasite's surface and is continuously secreted by all species analysed to date and, although its role in pathogenesis is for the most part unknown, it is implicated in nutrient scavenging as well as modulation of host environment (72).

The identification of histone proteins in stationary phase samples can be associated with the release of apoptotic vesicles. Indeed, several histone proteins are found in all datasets -Histone H4 and Histone H2B are the most abundant (normalised iBAQ values between 1.37 and 1.38).

Furthermore, we also found Histone H3 (1.29), Histone H2A (1.22), and Histone H2A.1 (1.18). The histones are core components of the nucleosome, the DNA packaging unit in eukaryotes consisting of histones bound to the DNA strands (chromatin). In the MoonProt database, the only histone described to have a moonlighting function is mouse histone H1 which acts as a thyroglobulin receptor.

Discussion

To our knowledge, this is the first proteomic analysis of the secretome comparing multiple Leishmania species, pathogenic and non-pathogenic to humans (publication in preparation).

Furthermore, the method used to produce naturally secreted proteins using aseric in vitro growth conditions is recommended to analyse such proteins. Seldom the secretome analysed in proteomic studies is prepared from cultured parasites with medium containing serum, which As expected, classical secretion mediated by N-terminal signal peptides is the least important secretion mechanism in Leishmania parasites and most proteins are predicted to be secreted through non-conventional pathways. Still, a large portion of the proteins are not predicted to be secreted despite being identified in the secretome. Despite its usefulness in deciphering the protein secretion pathways, it has been demonstrated that the SecretomeP prediction algorithm presents some limitations in predicting non-classical secretion for plant proteins and other eukaryotic organisms (76,77). These limitations may also apply to Leishmania proteins because of the algorithm training data. Possibly, Leishmania proteins possess unexpected features or share features with proteins from both eukaryotes and prokaryotes which are not considered by the algorithm. To evaluate this possibility, an additional bioinformatics analysis with SecretomeP predictions for gram-negative/gram-positive bacteria non-classical secretion will be performed and included in the publication.

Nevertheless, the results presented here are completely aligned with previous studies on Leishmania secreted proteins and the importance of vesicle-based secretion in Leishmania parasites (33,37,40,45). Exosome cargo is increasingly studied and implicated in host-host and host-pathogen interactions (35,47,78), and in this database we find proteins that are identified often in exosomes. We found many exosome marker proteins in the Leishmania secretome (9 different proteins out of 16 Leishmania orthologs to genes described in the Exocarta). This suggests a highly conserved cell communication mechanism across eukaryotic organisms, to which the parasites perfectly adapted to interrupt and manipulate so to enable their replication and continued transmission.

Several important and well-described virulence factors are among the most abundant proteins (PSA, leishmanolysin, HSP70, and others). Some of these proteins have not only been described to be key players in early establishment of infection but also as potential vaccine antigen candidates (79)(80)(81).

The biological reason for the presence of certain proteins or protein families remains elusive. We expect to find a mix of virulence factors, immunomodulators, and excretion products.

Interestingly, Leishmania proteins with moonlighting functions have been described, all of which are found in the secretome. Considering the existence of these moonlighting functions, the remaining proteins and virulence factors identified in the secretome are probably performing additional functions which remain unidentified.

Importantly, the functions attributed to the secreted proteins are highly conserved among Leishmania species. L. braziliensis is the most divergent species among the human infective species analysed and L. tarentolae which is non-infective to human. Still, they share gene product functions across all Gene Ontology domains. The secretome data generated deserves further indepth analysis to evaluate if certain GO terms are enriched in this biological compartment. This analysis will be performed and discussed in the manuscript in preparation, aiming at unravelling the essentiality of excreted-secreted proteins in host-pathogen interactions. Nevertheless, we provide evidence of the high degree of Leishmania species conservation on the proteomic level (active end products of genome expression) which further supports the discovery of conserved virulence factors and/or antigens.

The datasets generated with the proteomic analysis constitute an ancillary expected outcome of the present project. Hopefully, the detailed characterization of the secretome will contribute to novel discoveries on potential connections between geographical distribution, clinical presentation, immunomodulation and pathogenesis. Also, by including a non-pathogenic Leishmania species (L. tarentolae), we hope to contribute to increase our knowledge about interspecies variability and possibly reveal novel important virulence factors that can contribute to leishmaniasis diagnosis or drug development.

The high conservation of the proteins identified in the Leishmania secretome datasets further corroborates the proposed strategy of using promastigotes secreted proteins as a source for antigen discovery and pan-Leishmania vaccine development. The following chapter will explore these datasets to highlight relevant vaccine antigen candidates.

Objective: to search for Leishmania-specific vaccine antigens that will advance for epitope prediction analysis for the development of a human vaccine formulation against human leishmaniases.

Aims:

-To screen a maximum number of antigens, so to fully explore the potential of the secretome proteomic datasets and increase the chances of finding strong binding epitopes;

-To list and search previously described antigens in Leishmania secretome, which will allow the identification of known vaccine candidates, according to the literature;

-To perform a reverse vaccinology (RV) approach which will allow the identification of novel protein antigen candidates based on (low) homology with human host proteins.

Introduction

An antigen is a molecule that can bind specifically to an antibody or generate peptide fragments that are recognised by a T cell receptor, both produced by adaptive immune responses (1). Immunogens are molecules that, on their own, can elicit an adaptive immune response on injection into a person or animal (1). All antigens have the potential of binding specific antibodies or TCR, however, not all antigens are immunogenic, as they can be recognised by specific antibodies or TCR without inducing an immune response. In this sense, all immunogens are antigens, but not all antigens are immunogens, and vaccine candidates must be immunogenic.

As mentioned in the previous chapter, there is a high level of antigenic conservation among pathogenic species, as all species share high genome homology and synteny. Also, there is not a significant antigenic variation among Leishmania parasite forms (promastigote and amastigote).

Over the last decades, Leishmania vaccine antigens have been increasingly detailed, from non-defined crude antigen mixtures to the generation of recombinant proteins. Several factors have greatly contributed to these advances: i) the availability of pathogen-and host-specific genome and proteome information; ii) increased knowledge on mechanisms behind antigen recognition, presentation and subsequent immune activation cascades; and iii) the improvement of computational tools for epitope mapping and prediction.

Methods

Bioinformatic analysis of proteomic datasets

Raw MS spectra (AESLMTS_110414, AESLMTS_151026, AESLMTS_160115) were processed using the MaxQuant environment (version 1.5. amazonensis and L. tropica proteins in the UniProtKB were included (Uniprot_LEIAM-all_2016_01.fasta; Uniprot_LEITR-all_2016_01.fasta).

Protein set Apreviously described vaccine antigen candidates

The complete list of previously known antigen candidates (Table III vi) The final list for protein Set A contains 4 to 6 species-specific sequences per protein antigen, meaning only proteins present in all studied species were selected;

Protein set B -reverse vaccinology approach for antigen selection

A total of 618 accessions were identified in all 6 proteomic datasets from pathogenic The alignment results were retrieved using the tabular output format is a ".csv" file All accessions with "no hits found" were included in the protein set B.

Sequences were retrieved from UniProtKB and aligned to identify protein groups. In order to complete the protein groups with species-specific sequences, accessions per protein group were 'blasted' in UniProtKB. Species-specific sequences with higher homology levels and containing the highest number of identified peptides were included, resulting in 4 to 6 speciesspecific sequences per protein.

Evaluation of protein relative abundance with iBAQ

To rank the relative abundance of different proteins, an intensity-based absolute quantification (iBAQ) algorithm was used. Raw data files were analysed with the MaxQuant software package (version 1.5.5.0). As detailed in the previous chapter, the iBAQ value is obtained by dividing peptide intensities by the number of theoretically observable peptides of the protein (all fully tryptic peptides with 6 to 30 amino acids calculated by in silico protein digestion).

The iBAQ values corresponding to 2333 LEADING_CHECK accessions were retrieved. In order to estimate relative antigen abundance of protein sets A and B, absolute iBAQ values (2333 total LEADING_CHECK accessions, 88 set A accessions and 68 set B accessions) were log-transformed (log10) and normalised to overall iBAQ average (average iBAQ value 2,4x10 7 ; log10 average iBAQ value = 6,3302).

Results

3.1. Around 40% of previously described Leishmania vaccine antigens are found in the secretome (protein set A)

All previously described vaccine antigens were manually searched in the secretome datasets, either by protein accession or protein name (Table III.1). Only proteins present in all studied species were included in protein set A.

An initial list of 72 protein antigens was retrieved from the literature (Table III.2). These candidates include kinases, translation initiation factors, several proteases and other virulence factors.

The 72 proteins listed from the database (Table III.2) were manually searched in the proteomic datasets. From these, 36 were found in the secretome, and 28 are present in all tested species which corresponds roughly to 40% of the initial list (Table III.3).

Table III. 2 A total of 72 protein antigens listed from the selected publications was searched in the secretome proteomic datasets (2-7).

Antigen category

Individual proteins to search in proteomic datasets Description

Heat-shock proteins KMP11 is a widely known amastigote and promastigote antigen, noncovalently bound to LPG, and conserved in trypanosomatids. B and T cell immunogen. There is evidence it is processed by DCs and presented by MHC-II molecules, activating specific T cells. The IFN-γ produced is enough to foster TNF-α and NO production resulting in parasite death inside macrophages. In susceptible individuals, KMP11 induces IL-4, IL-10 and IL-13 production perpetuating macrophage infection.

LACK (Leishmania analogue of the kinase receptor C)

LACK is present in both amastigotes and promastigotes, and is highly conserved among Leishmania species. It is homologue of mammalian RACK1. LACK belongs to the WD repeat protein family. It is important for DNA replication, RNA synthesis, signal transduction and cell cycle, and also in the differentiation from metacyclic promastigote to amastigote, as there is a minimum threshold of this protein to establish infection. LACK is known to induce Th2 responses (IL-4 and IL-10 production).

Elongation factor 2 EF-2 is recognised by human T cells and provides protection in hamsters.

Ldp23 or 23 kDa cell surface protei (probable 60S ribosomal protein)

Ldp23 is present on the surface of L.donovani and L. major amastigotes and promastigotes, it accelerates IFN-y production and inhibits IL-4 production.

A-2 A2 is the major antigen in the Leishmune ® canine vaccine, it is only present in amastigotes. It contains multiple copies of a 10 amino-acid repeat, and is known to induce Th1 responses and provide partial protection against Leishmania species.

HASPB and HASPA (hydrophylic acylated surface proteins A and B)

Leishmania HASPB is a lipoprotein that is exported via an unconventional secretory pathway. Recombinant HASPB1 confers protection against experimental challenge in mice. At times, because proteins were manually searched by protein name, redundant proteins and isoform sequences are found, as for the case of peroxidoxins. Through sequence alignment and gene IDs three groups for peroxidoxins were generated according to their function in the antioxidation pathwaytryparedoxin peroxidase, peroxidoxin and tryparedoxin (Table III.4). Each protein antigen in set A includes 4 to 6 species-specific sequences (Table III.5). The proteins with 5 and/or 6 species-specific sequences include proteins specific to L. tropica and/or L. amazonensis samples, which are automatically identified as one of the reference proteome species by the bioinformatics analysis. The gene search in TriTrypDB and respective translated proteins allowed the inclusion of species-specific sequences.

Table III. 3 Protein description of the 28 selected antigen candidates (Set A). In yellow, the antigens used in current vaccine formulation. Significant differential expression information from

Finally, the total 28 protein antigens from protein set A include 3 protein antigens with 4 species-specific sequences, 14 protein antigens with 5 species-specific sequences, and 11 protein antigens with 6 species-specific sequences (Table III.5).

In summary, the protein set A includes i) potential vaccine antigens; ii) antigens present in the secretome; and iii) antigens common to six pathogenic Leishmania species.

Table III. 5 Protein Set A includes 28 antigenic proteins (A1 to A32) described in the literature as potential vaccine antigens against Leishmania spp. Each protein antigen includes 4 to 6 species-specific sequences. The peptide counts allowing the identification of each sequence in the several samples are shown, as well as the protein length, percentage of identity among sequences and the identification (ID) status according the bioinformatic script used (see chapter II). L. braziliensis (LEIBR), L. donovani (LEIDB), L. infantum (LEIIN), L. amazonensis (LEIAM),

L. major (LEIMA) and L. tropica (LEITR).

A reverse vaccinology approach allowed the identification of 24 novel antigen candidates, including 3 antigens previously described in the literature

The BLASTp analysis of the common 618 accessions generated a total of 15720 alignment results for 542 unique protein sequences. Proteins with no significant homology with human proteins were selecteda total of 76 accessions with "no hits found". The 76 accessions include 43 annotated sequences, corresponding to 14 proteins, and 33 accessions corresponding to uncharacterised proteins (UP). These 33 UP sequences were aligned with the ClustalOmega alignment tool, and 10 clusters were identified (Table III.6).

Considering 14 proteins and 10 UP, protein set B contains 24 protein antigen candidates.

The protein set B includes 7 protein antigens with 4 species-specific sequences, 16 proteins antigens with 5, and one protein antigen with 6 species-specific sequences (Table III.7).

Among the proteins selected through this approach, 4 accessions were already included in the protein set A. These accessions correspond to 3 different proteins: i) L. infantum nucleoside hydrolase-like protein and L. major putative inosine-guanine nucleoside hydrolase; ii) L. infantum paraflagellar rod protein 2C; and iii) L. infantum kinetoplastid membrane protein 11C.

Table III. 7 Protein set B includes 14 annotated proteins and 10 uncharacterised proteins (B1 to B24). Each protein antigen includes 4 to 6 species-specific sequences. The peptide counts allowing the identification of each sequence in the several samples are shown, as well as the protein length, percentage of identity among sequences and the identification (ID) status according the bioinformatic script used (see chapter II). L. braziliensis (LEIBR), L.

donovani (LEIDB), L. infantum (LEIIN), L. amazonensis (LEIAM), L. major (LEIMA) and L. tropica (LEITR).

Most protein antigens selected are well represented in the secretome

Normalisation of iBAQ values allowed the determination of the relative abundance of a given protein, providing a rough estimation of overall antigen abundance. The absolute iBAQ values for the 2333 LEADING accessions identified with the proteomic analysis range from 2524,8

Figure III. 2 Normalised iBAQ values show most of the selected protein antigens are more abundant than average. Absolute iBAQ values were log-transformed (log10) and normalized to overall log10-iBAQ average value. Total identified proteins (yellow), 88 set A protein antigen sequences (purple), and 68 set B protein antigen sequences (green).

From 147 unique protein accessions in the protein set A, 88 iBAQ values are available for the LEADING_CHECK accessions, with absolute values ranging from 32613 (10 4 ) to 3526300000 (10 9 ). The normalised iBAQ values for protein set A range from 0,713 to 1,508, including one highly abundant protein with a normalised iBAQ value above 1,5

Discussion

A potential limitation of the overall analysis of the promastigote secretome is to overlook amastigote-specific proteins, potentially very important in a vaccine formulation. Some amastigote-specific proteins have been described and are discussed in the selected publications from which antigens were listed, notably the A2 protein. These proteins evidently were not found present in the promastigote secretome. However, there is evidence that antigens present in the promastigote secretome can provide immunity against intracellular forms and that many secreted antigens are continuously secreted during intracellular life stages.

The simultaneous high of conservation between Leishmania species, long-term coevolution and selective pressure by the immune system suggests the parasite relies on early immune responses to establish infection. The vertebrate hosts are exposed to metacyclic promastigote forms in early infection, when the immune response cascade is activated. This provides a unique opportunity for antigen discovery since early immune responses against promastigotes will dictate the fate of the adaptive immune response. Moreover, gene expression remains stable after parasite differentiation into intracellular amastigote forms, providing the advantage of targeting the parasites in early stages of infection as well as intracellular replicative forms (9)(10)(11)(12). Additionally, it is established from the dog infection model that the Leishmania promastigote secretome is a great antigenic source for Leishmania recognition and induction of protective immune responses (13) (see Chapter I).

In this light, it is not surprising that multiple known candidates are found in the secretome.

Precisely, 36 proteins out of 72 potential antigen candidates are excreted-secreted proteins. These include several protein antigens currently in use by the most advanced vaccine candidates against leishmaniasisinitiation factors, cysteine proteases; TSA (thiol-specific antigen); LACK; protein disulfide isomerase-2; Putative glycosomal phosphoenolpyruvate carboxykinase; nucleoside hydrolase; GP63; and KMP11, among others.

The second-generation vaccine LEISH-F3, the most advanced human vaccine in clinical trial, is based on a fusion recombinant protein combining the Nucleoside hydrolase from L.

donovani, Sterol-24-c-methyl-transferase from L. infantum; delta cysteine protease "B" from L.

infantum. Interestingly, both nucleoside hydrolase and cysteine protease B antigens are found in the Leishmania secretome. Furthermore, the other antigen candidates included in vaccines LEISH-F1 and -F2, the Leishmania elongation initiation factor (LeIF) and thiol-specific antioxidant (TSA) are also present in the Leishmania secretome.

In eukaryotic organisms, glutathione (GSH) is an antioxidant that works as a redox buffer during the glutathione redox cycle, protecting the cells from reactive oxygen species. GSH reduces disulfide bonds of cytoplasmic proteins becoming oxidized (GSSG) and the ratio between GSH and GSSG is a measure of cell toxicity. Instead, Leishmania parasites have trypanothione (TSH), trypanothione reductase (TryR), tryparedoxin, tryparedoxin peroxidase also known as TSA (thiolspecific antigen), peroxidoxin, and mitochondrial peroxiredoxin. These proteins make-up their anti-oxidant system for protection against reactive oxygen and nitrogen species, and are therefore essential for parasite survival inside the phagolysosomal vesicles. Two of these were found in the secretome of all 6 speciestrypanothione reductase and tryparedoxin peroxidase, also known as TSA (thiol-specific antigen). These proteins are important virulence factors and targets particularly for drug development, but little information exists on protein immunogenicity and sequence annotation is still incomplete for the many existing isoforms.

Proteases are ubiquitous enzymes that catalyse the hydrolysis of peptide bonds and are important in several biological activities. There are at least 6 classes of proteases classified according to the nucleophilic group responsible for the first step in the proteolysis: serine, cysteine, metallo, aspartate, glutamate, and threonine proteases. The two major types in eukaryotes are cysteine (papain-like) proteases and serine (trypsin-like) proteases. Cysteine proteases are categorized into 72 families, but not all are represented in protozoan parasites. The most abundant and well-characterized cysteine proteases are the clan CA papain-family enzymes.

Cysteine proteases are vital virulence factors that ensure parasite survival and establishment of infection. Moreover, some proteases were described as potential vaccine antigen candidates, namely GP63, methionine aminopeptidase p45, and cathepsin-like proteases.

Although not specifically described as potential candidates, other Leishmania proteases were also searched (aminopeptidases and metallo-proteases) to expand the protease antigens included in the protein set A.

The antigens trypanothione reductase and glycosomal phosphoenolpyruvate carboxykinase (gPEPCK), although not used in vaccine formulation, are among the most studied vaccine antigens against Leishmania, showing very promising pre-clinical results (7). gPEPCK is upregulated in stationary phase promastigotes and L. donovani amastigotes probably due to gluconeogenesis activation (14).

The recombinant fusion protein Q is the main component of the latest canine vaccine approved in Europe (Letifend ® ), shown to be effective in pre-clinical trials in dogs. This fusion protein contains portions of L. infantum histone H2A and ribosomal proteins (p2a, p2b, and p0).

Again, these proteins are found in the Leishmania secretome.

To expand the antigen port-folio to proceed for in silico epitope prediction, we adopted a reverse vaccinology approach, using low human host homology as selection criterium. This approach allows the comprehensive exploration of the secretome proteomic analysis data thus allowing the screening of numerous protein antigen candidates.

Results show there were 618 accessions common to six Leishmania species (UniProtKB release 2016_05). These 618 common protein accessions correspond to about a third of total identifications, indicative of the high conservation among pathogenic species, and in agreement with previous studies studying excreted-secreted proteins (see chapter II). Common identified accessions between the tested samples also imply that peptides that lead to each protein identification are found in all samples (peptide counts) and are from conserved regions. The total number of species-specific sequences per protein (4 to 6) takes into account sequence variability for all given antigens.

The BLASTp alignments are a powerful tool to find interesting protein candidates which share little homology with host proteins. Several cut-off values have been suggested for selection, according to Evalue, Bit score, minimum alignment length, and/or minimum sequence identity (15,16). In the present analysis we selected only the proteins with no significant homology found, so no cutoff values were applied. The generated proteomic datasets could be further explored by including proteins with a related host protein but still with significant partial sequence divergence.

Some candidates found with the RV approach were already described as Leishmania antigen candidates -KMP11, nucleoside hydrolase, paraflagellar rod proteins and calpain-like cysteine peptidase. We believe the identification of these proteins through the RV approach further supports the validity of the proposed approach.

Some uncharacterised and hypothetical proteins have been proposed as Leishmania

antigen candidates, such as the hypothetical protein LiHyR (XP_001568689.1), which corresponds to the protein A4HNR3 in the UniProtKB database (17). LiHyR induced Th1 responses and reduced parasite burden in mice. More importantly, it is also immunogenic in human PBMC from healthy donors and cured VL patients. This protein not found in the secretome, nor any of its related proteins (A4ICT2, E9ASH2, E9BTB7, A0A088S1U1, Q4Q223). The high number of hypothetical proteins in the databases is a result of the lack of protein annotation and functional characterisation of Leishmania proteins. However, this should not deter the study of their immunogenic properties. Accordingly, the 10 uncharacterised proteins identified with the reverse vaccinology approach were included in the protein set B.

Antigen abundance is a pre-requisite for the induction of immune responses. the secretome as an antigen source contains over 1000 proteins, around 15% of total Leishmania proteins. The non-quantitative approach used does not allow to ascertain in absolute terms the amount of each identified protein. However, relative protein abundance across all tested species can be used as an indicator of overall abundance. We find most protein antigens are well represented in the secretome, 76% for set A and 74% for set B proteins. This analysis is merely descriptive, as antigen abundance was not used as an inclusion or exclusion criterion. However, this information may be useful in later stages, to favour or eliminate epitopes from more or less abundant proteins, respectively. Also, these proteomic datasets can be further explored to find new and highly abundant antigens (e.g. protein set C).

The total number of protein antigens to proceed for in silico prediction analysis is limited by the lack of high-throughput epitope selection analysis tools. Still, a total of 52 protein antigen candidates from the Leishmania secretome were selected and will proceed for epitope prediction Immunodominant epitopes present in an antigen are preferentially recognised by T cells, such that T cells specific for those epitopes come to dominate the immune response (1).

Immunodominance depends both on antigen-related factors and on T cell-related factors (Table IV.1).

Discovering immunodominant epitopes is desirable in vaccine development, as these will elicit the strongest immune responses. However, in the case of mutation, a vaccine based only on immunodominant epitopes would be rendered ineffective. Moreover, while the observed responses against dominant epitopes will be the strongest, they are more likely to present high response variability among individuals and, therefore, potentially generate an uneven vaccine performance. HLA types (4). In summary, while immunodominant epitopes will be recognised more vigorously, immunoprevalent epitopes will be recognised more frequently. Epitope-based vaccines should privilege immunoprevalent epitopes, particularly vaccines targeting large populations or against pathogens with multiple antigenic sources.

Antigen-related factors T cell-related factors

Immunodominance and immunoprevalence are relative terms rather than absolute, as they always depend on the total composition of antigenic molecules and on competitive high-affinity binding to MHC molecules. Both should be considered when designing peptide-based vaccines.

T cell epitope prediction and immunoinformatics

The knowledge of immunogenicity determinants and immunogenic epitopes increased greatly in recent years, demonstrated by available databases such as IEDB (Immune Epitope DataBase) (5-8).

Table IV. 2 Available T-cell epitope databases. General epitope databases, and pathogen-and tumor-specific databases of T-cell epitopes (9).

General Database Description

from 99% of all publications describing immune epitopes, all except HIV-and cancer-specific epitopes (10,11). The IEDB contains both epitope and assay information regarding epitopes from infectious diseases, autoimmune and allergic diseases, and alloantigens for humans, primates, mice and other host species (12).

The IPD-IMGT/HLA (or IPD-IMGT/MH-DB) database is one of the seven databases from the the Immuno Polymorphism Database (IPD) (13). The IPD-IMGT/HLA database is part of the International Immunogenetics Information System ® (www.imgt.org) and is the main specialised repository for the sequence data of polymorphic gene sequences (14). It contains allelic sequences of The MHCPEP database contains both naturally processed and synthetic peptides (over 4000 binder peptides) (15), whereas SYFPEITHI is a manually curated database from the published literature and exclusively contains naturally processed peptides (over 7000 MHC ligands, motifs and epitopes), but both these databases are no longer updated (16). The MHCBN is a curated database containing binder and non-binder epitopes (over 25800 peptide entries) with information about TAP interactions and MHC-linked autoimmune diseases, last updated in 2006 (17).

Parallel to databases, analysis resources allow the prediction of T cell epitopes. Several sophisticated immunoinformatics tools, of variable performance, are available to evaluate some of the characteristics associated with immunogenic epitopes (7,8,18,19). A determinant aspect for algorithm performance is the amount and quality of the training datasets. As epitope data increases and improves, so will the performance of epitope prediction algorithms.

T-cell epitopes prediction can be performed through direct (predicting T-cell receptor, TCR recognition) or indirect methods (predicting epitope binding to MHC/HLA molecules), the latter extensively more accurate than the former (20).

HLA-binding affinity has become the first criterion when trying to predict if a given peptide sequence constitutes an epitope, since it is the first requirement for T-cell activation and it correlates with peptide linear sequences (7,20). The first algorithms developed used basic motif listings to prediction T-cell epitopes. Subsequently, HLA-binding predictions based on machine-learning algorithms were developed, based on Artificial Neural Networks (ANNs) and Support Vector Machines (SVMs), which display better predictive performance (4,21-23) (Table IV.3). HLA-binding predictions are affected by two main issuesprediction accuracy and sensitivity, and the MHC-class I and -class II alleles for which predictions are available (10).

The performance of the several predictors was performed through benchmark studies, using curated datasets different from the algorithm's training datasets (24)(25)(26)(27). The evaluated predictors show in general somewhat similar performance, but ANN-based and consensus predictions have the best overall performance (10,27). The IEDB analysis resource centre has the highest number of predictors, including consensus predictions, the most effective prediction algorithm (23,25,28). (29).

Table IV. 3 The most commonly used sequence-based algorithms for T cell epitope prediction

In silico epitope predictions and implications for vaccine development

MHC-class II binding predictions must take into account the high peptide length variability due to the conformation of the binding groove, and are currently slightly less accurate than class I binding predictions (25,30). Due to the longer length of class II epitopes (around 15 to 25-mer), several binding registers or cores may be present in the same peptide (29,31).

Proteasomal cleavage analysis and TAP-transport prediction, although very informative in theory, do not improve MHC-binding predictions (10,32). These algorithms often display low prediction efficiency, and there are still significant knowledge gaps regarding protein intracellular processing.

Also, for the particular case of parasite-delivered antigens, the mechanisms responsible for crosspresentation remain poorly understood (32,33). Professional APC, namely DC are the main cells capable of cross-presenting antigens. However, due to the extensive modulation by Leishmania parasites and preference for macrophage infection these mechanisms remain largely unknown (33).

In this case, the use of experimental data detailing peptide-specific immune responses is indispensable to ascertain which peptides are indeed associated with natural protection and can be used in a vaccine formulation.

Additional aspects related with immunogenicity assist in epitope prediction and selection.

Analysis of subcellular localization, protein abundance and good expression dynamics are the filters with the highest selective power (32). Highly conserved epitopes are ideal for vaccine development because of pan-specific protection across multiple strains of a given pathogen (34). Furthermore, combinatorial approaches that use multiple predictors are beneficial since they increase the confidence level in the peptides' predicted binding affinity/HLA restriction (31,35).

Using homology to host proteins as rejection criterion is an unreliable filter. Self-recognition depends on the TCR-pMHC interaction which allows a reasonable amount of molecular mimicry, and therefore difficult to predict (36)(37)(38). However, potential interferences resulting in auto-immunity are correlated with epitope conservancy. BLASTp alignments can be used to compare and describe similarities pathogen-and host-derived peptide sequences.

The ability to analyse the vast amount of data generated by immunoinformatic algorithms is an additional challenge researchers must overcome (39)(40)(41).

Ultimately, in silico immunogenicity predictions on the epitope level should privilege: i) antigen abundance, subcellular localization and expression dynamics, as abundant and early expressed pathogen-specific epitopes have increased chances of being processed and presented (32,42); ii) peptide-MHC complex (pMHC) binding affinity and stability (2,43,44);

iii) homology, either as positive selection criterium of conserved sequences among pathogenic species, or as negative selection criterium of sequences homologous to host proteins (20);

Other biochemical properties can also be taken into account for optimal formulation and handling, such as solubility, since peptides should be soluble in aqueous solution (2,21).

We propose an epitope selection pipeline that starts with HLA-binding affinity prediction analysis, by at least two different algorithms, of a strong antigen pool of conserved, exposed and accessible proteins, expressed from early infection (antigens present in the Leishmania secretome).

Additional adjustable filters are homology to host proteins, promiscuity, binding affinity, and solubility, with which we can rank epitopes.

Methods

Selection of HLA allele lists

The allele lists to include in the epitope prediction algorithms were defined from the IEDB reference sets with maximal population coverage.

a) HLA-class I alleles

The HLA-class I reference allele list includes 28 alleles from HLA-A and HLA-B genes corresponding to >97% world population coverage (file from IEDB: hla_ref_set.class_i.txt) (45,46). IV.4).

b) HLA-class II alleles

The HLA-class II reference allele list includes 27 alleles from HLA-DRB, and -DQ and -DP (alpha and beta chain combinations) genes corresponding to >99% world population coverage (file from IEDB: hla_ref_set.class_ii.txt) (46).

The HLA-DQ alleles were excluded from this dataset due to their association with recognition of human epitopes and the induction of immune tolerance, to prevent a bias toward cross-reacting epitopes (47). The removal of these alleles does not greatly reduce population coverage, as only the The HLA-class II alleles selected: IEDB MHC-II binding predictions for 21 alleles, and

NetMHCIIpan predictions for 21 alleles (same allele lists) (Table IV.4).

Table IV. 5 HLA-class II alleles included in T-cell epitope prediction (46).

HLA-class II alleles

Selection and usage of in silico HLA-binding prediction algorithms

The following publications were reviewed for algorithm selection: To improve prediction sensitivity, the main criteria used for the epitope prediction and selection were i) to include at least 2 different epitope databases; ii) to include at least 2 nonredundant algorithms; iii) to use ANN-and SVM-based algorithms (9,20). Some analysis tools were explored but not selected (data not shown

Integration of HLA-binding prediction data

To merge and analyse protein-specific epitope prediction data, a selection script was developed in R (

Figure IV. 1 T-cell epitope selection pipeline. A) Epitopes were selecting through the application of relevant filters by a selection script in R. B) The result table containing conserved peptides was further analysed with spreadsheet software

Excel®.

The result table from the selection script contains the columns: peptide (epitope sequence), blastp results (middle; middle2; subject); position-specific mismatches (m_1 up to m_9), and protein supertype. Then, using Excel ® , relevant physical-chemical properties were added (MW, hydrophobicity, pI) to the result table from the selection script after analysis of the peptide list with the ProPAS software (52). Moreover, epitope promiscuity and HLA restriction were calculated (Excel

SILVIan open-source pipeline for T-cell epitope selection

The final selection steps performed in the spreadsheet analysis software were integrated in the R selection script for publication purposes. The package Peptides was used for the addition of physical-chemical properties (MW, pI and hydrophobicity). Also, promiscuity and HLA restriction are now automatically calculated by the script. The final development version of the SILVI epitope selection pipeline will be published in Pissarra J et al 2019 (under review for publication in PLoS One).

Peptide toxicity assays

Cytotoxicity assays were performed with the PrestoBlue® viability dye. Briefly, 2x10 5

Results

The T-cell epitope selection pipeline greatly reduced initial epitope lists

A T-cell epitope selection pipeline was developed in-house to optimise the process of epitope selection from vast amount of data produced by available algorithms, and to add extra relevant information, thus helping to refine the search of the most relevant epitopes through the application of filtering criteria. The R script reads epitope binding prediction data from different predictors, processes and compares data, assimilates BLASTp alignment results (53). The selection script feeds a final table with all relevant information to perform the desired selections. Additional epitopespecific information was added afterwards, in the spreadsheet analysis software Excel ® (molecular weight, pI, hydrophobicity, NetMHCpan score).

The script successfully integrated epitope prediction information from all 52 proteins.

Considering only one species-specific sequence per protein, these 52 proteins comprise 20712 9-mer overlapping peptides, and 20412 15-mer overlapping peptides (Figure IV.3). Peptides that are 100% identical in all species-specific sequences per protein were selected in the first selection step, so each peptide in the "blast_me.fasta" file are proteinand Leishmania-specific.

The result file "3_blast_mismatches_I.csv", after the second R script selection step, includes HLA-class I binding predictions for 1048 unique peptides from Set A proteins and 1069 unique peptides from Set B proteins. The total 2117 peptides correspond to 2277 predictions, after considering promiscuous peptides. These predictions are common between at least two algorithms (I+S, I+N, I+N+S).

The result file "3_blast_mismatches_II.csv", after the second R script selection step, includes HLA-class II binding predictions for 847 unique cores. These epitope core predictions are common among IEDB MHC-II binding and NetMHCIIpan (I+N).

CHAPTER IV -IN SILICO EPITOPE DISCOVERY AND SELECTION

Figure IV. 4 Stepwise HLA-class I binding prediction results per protein antigen and protein conservation. The first selection step (in blue) includes epitopes under allele-specific IC50 cut-off values, 100% conserved among species and predicted by at least 2 algorithms. These epitope lists were reduced in a second selection step (in green) with the application of filtering criteria (promiscuity>0; prediction by all 3 used algorithms; hydrophobicity<0,5; total mismatches > 1; anchor mismatches > 0). Epitopes were selected according to best predicted IC50 values per supertype (orange).

The 50 HLA-class I restricted peptides come from 23 different protein antigens (11 from Set A; 12 from Set B). 20 epitopes are from Set A antigens and 30 are from Set B antigens.

All peptides are predicted to be strong binders, with average predicted IC50 of 40 nM (minimum 3,5 nM; maximum 111,5 nM). Most peptides are not predicted to be promiscuous, 6

epitopes are predicted to be promiscuous to 2 supertypes, and 2 epitopes are promiscuous to 3 allele supertypes.

CHAPTER IV -IN SILICO EPITOPE DISCOVERY AND SELECTION 164

The final HLA-class II epitope list containing 121 unique cores was ranked according predicted promiscuity, and selections were performed per allele. The most promiscuous epitopes were selected (20), and 4 other epitopes were selected according to best predicted IC50 for remaining alleles (not represented in the overall promiscuity prediction) (Figure IV.5).

Figure IV. 5 Stepwise HLA-class II binding prediction results per protein antigen and protein conservation.

The first selection step (in blue) includes the top 10% of predicted epitopes, 100% conserved among species and predicted by at least 2 algorithms. These epitope lists were reduced in a second selection step (in green) with the application of filtering criteria (promiscuity>0; prediction by 2 algorithms; hydrophobicity<0,5; total mismatches > 3). Epitopes were selected according to predicted promiscuity (orange).

The 24 HLA-class II-restricted epitopes come from 15 different protein antigens (7 from Set A; 8 from Set B). 12 epitopes originate from Set A antigens and 12 from Set B antigens. The average prediction percentile is 1,49% (minimum 0.01%; maximum 7.32%; median 0.53). These epitopes are predicted to be promiscuous, from 3 to 13 alleles. Between 4 to 18 epitopes per allele were selected.

For HLA-class II binding predictions, the total epitope size is 15-mer, however, the determining motif for HLA binding is the epitope core or register. The predictive power of different algorithms correlates better with the ability for core prediction (4). By comparing core predictions,

Discussion

Experimental validation steps limit the number of peptide candidates to test, meaning that these candidates must be carefully selected to increase the chances of selecting immunogenic peptides. The addition of an initial in silico screening in the development pipeline greatly diminishes costs associated with epitope mapping experiments by decreasing the number of peptides to test.

Nevertheless, current in silico epitope prediction tools still present some shortcomings (Figure IV.8).

The lack of gene and protein annotations as well as an underrepresentation of protozoan-derived epitopes in the databases renders sequence-based prediction less accurate (35). Similarly, protozoan proteins with different physicochemical composition are not represented in the databases (35).

Leishmania-specific epitopes are underrepresented in epitope databases. To date, the IEDB database The term reverse vaccinology was first described by Rappuoli R. (31) to designate approaches that screen entire genomes or proteomes to filter out proteins of interest (antigens) using bioinformatic tools. Subsequently, particular features associated with antigenicity are searched, namely, subcellular localisation or expression timing.

At the time of the present analysis, only four (true) reverse vaccinology studies had been performed for vaccine development against Leishmania, with modest success in identifying strong epitopes (54,55). Briefly, most studies used BLASTp alignment to eliminate peptides or proteins with significant homology to host proteins (human or mouse). Also, most studies used sequence-based HLA-binding predictions for T-cell epitope discovery.

In Predep, a structure-based algorithm). The last analysis step involved a BLASTp analysis against human and mouse proteins, wherein epitopes showing over 80% of sequence identity were discarded, and epitopes conserved among kinetoplastids were prioritised. However, predictions were only performed for mouse alleles (H-2K d andH-2D d ). Also, proteasomal cleavage filters were applied, potentially introducing a bias in epitope prediction. Experimental validation was performed in mice.

This approach eliminated the known candidates GP63, LACK, histone 2B, LmSTI1, TSA, CPb, NH36 or beta-tubulin in the first selection step.

Similarly, Guerfali FZ et al also used the complete L. major proteome for epitope prediction.

Again, predictions were performed only for mouse alleles (BALB/c and C57BL/6 alleles) and with redundant PSSM-based algorithms (SYFPEITHI, BIMAS, RANKPEP). BLASTp analysis was also used to exclude epitopes with high levels of similarity with host proteins, but only 100% identical sequences were excluded. Moreover, SignalP predictions were used to select secreted protein antigens for epitope prediction, which is extremely unsensitive for Leishmania excreted-secreted proteins since, as shown by the literature and in chapter II of the present thesis, only up to 10% of proteins in the secretome are classically secreted.

In 2012, John L. et al retrieved total protein sequences from L. major and L. infantum and 8122 common proteins were identified through BLASTp analysis. Subcellular localisation analysis was performed with PSORT and TMHMM to select cell surface and secreted proteins (cytoplasmic nM has been extensively used in T-cell epitope selection. Allele-specific binding information allows the establishment of specific thresholds which correlate well with epitope prediction (61).

Homology was used firstly as a positive selection filter for highly conserved epitopes among Leishmania species. Logically, epitopes from proteins that show higher homology are more likely to be 100% conserved and, therefore, more likely to be selected. However, the 52 antigens processed are generally conserved, with between 60% up to 99% homology levels. Still, because selections were performed according to predicted IC50, there are conserved epitopes selected from less conserved proteinse.g. B3, A28 proteins for HLA class-I epitopes, and A1, A10 for class-II epitopes.

Homology to host was then used as negative selection filter, not to exclude prediction data, but for epitope ranking and description. In this case, epitopes were aligned with human RefSeq proteome, and position-specific mismatches were counted. This allowed us to establish a low stringency threshold for homology with human proteins, (at least 2 amino-acids in 9-mer peptides or at least 3 amino-acid mismatches for 15-mer peptides). Although the mechanisms eliciting selfrecognition and autoimmune responses are not fully understood, and therefore difficult to predict, the selection of epitopes with lower homology to host proteins is likely to decrease the chances of unwanted cross-reactivity.

The hydrophobicity filter, although not sensitive to immunogenicity, allows the discovery of soluble epitopes, which can be easily produced, solubilised and tested in vitro. After peptide synthesis, a vast majority of class I peptides was found to be water soluble, which facilitates handling. Solubility assays were not performed for class II peptides because of reduced peptide availability -HLA-class II peptides were directly solubilised in 10% DMSO to improve peptide stability upon freezing.

After peptide selection, the selection script was adapted to accommodate the steps performed in the spreadsheet analysis software and will be published under the title "Exploring -omics datasets for epitope-based development of vaccines and therapeutics -SILVI, an open-source pipeline for Tcell epitope selection". This publication aims to make SILVI available to the community and, hopefully, assist future studies in epitope selection analysis. In the manuscript, an example protein was included (Hepatitis C Virus Genome polyprotein P26664) to illustrate the workflow and SILVI's potential in assisting epitope selection. When compared to validated epitopes in the IEDB, the application of low stringency filters (promiscuity > 0 and scoreN under 1000 nM) to class I epitope predictions results in 81% sensitivity and 82% specificity (out of 614 peptides selected, 100 are P26664 validated epitopes in the IEDB). Notably, these results are not improved after proteasomal cleavage analysis.

Finally, the final epitope selections include proteins from both antigen datasets (Sets A and B). For the HLA-class I epitope list, Set A proteins correspond to 40%, with 20 epitopes compared to 30 from Set B proteins. For HLA-class II epitope lists, both datasets are equally represented (12 each).

It is noteworthy that the new antigen candidates in Set B are equally or even potentially more immunogenic than known protein candidates.

We successfully selected 50 HLA-class I-and 24 HLA-class II-restricted epitopes, through an epitope selection pipeline using high-performing HLA-binding predictions (27), and homology alignments (53). Based on this information, together with conservation among species, physicochemical properties and target population HLA restriction, prediction data was filtered, thereby reducing the number of peptides to test experimentally while increasing the chances of identifying immunogenic peptides.

The final peptide list includes high affinity binders with high immunogenicity potential according to in silico predictions. The synthetic peptides, and respective predicted immunogenicity, will be validated with experimental assays, detailed in Chapter V.

CHAPTER V

EXPERIMENTAL VALIDATION OF SYNTHETIC PEPTIDES

Introduction

Immunogenicity testing is a key aspect in the development of vaccines, immunotherapies or biological therapeutics, from both a regulatory standpoint and a preclinical development perspective

Figure V. 1 Summary of the current toolbox of cell-based assays to assess T-cell-dependent immunogenicity, adapted from (2). A) In vitro T cell assay methods and respective application and readout. B) Advantages and disadvantages regarding the different methods.

The CEF peptide pools are commonly used as positive control for peptide T cell activation in assays using total PBMC, both for HLA class I and class II peptide presentation (3). These pools contain well-defined epitopes capable of inducing memory cellular responses, from Cytomegalovirus (CMV), Epstein-Barr Virus (EBV) and Influenza virus (Flu) (CEF), which cause chronic infection with multiple reactivations or repeated acute infections.

There is, however, a lack of standardisation in immunogenicity testing protocols, specifically regarding the methods used, and the evidence needed regarding type, quantity and quality of the observed immune responses. For example, to address these issues in clinical studies using flow cytometry-based immunophenotyping assays, the Human Immune Phenotyping Consortium (HIPC) promoted standardisation guidelines, so that data could be compared across sites and studies (4).

For all mentioned techniques, the number of available cells is a limiting factor. Moreover, the time, cost and workload needed to perform the assays can be restrictive ( 5 On the other hand, naive T cells clones are rare, which constitutes a limiting issue for detection sensitivity. A method based on peptide:MHC tetramers uses four specific peptide:MHC complexes bound to a single molecule of fluorescently labelled streptavidin (13). It has proven extremely useful to identify populations of antigen-specific T cells in the naive repertoire, for example, describing the number of T cell precursors specific to ovalbumin and viral-specific epitopes (14). However, this technique can only evaluate one epitope per experiment, it is not applicable to large numbers of MHC alleles and requires previous knowledge about the peptide sequences. Also, it is very expensive and, therefore, unsuited for epitope mapping or peptide screening experiments (12).

A suitable method to prime naive T cells in vitro is to use antigen-pulsed autologous DC, and several rounds of stimulation inducing T cell activation, proliferation and effector phenotypes (7)(8)(9)(10)(11).

Cellular amplification allows the analysis and measurement of antigen-specific responses mediated by a single precursor cell or population (12). Also, the use of purified cell populations, enriched in lymphocytes, helps increase the level of detection of such responses (2).

Figure V. 2 Specific cell frequency in different immune repertoires (5).

Precursor frequency experiments use limiting dilution conditions to determine the relative frequency of antigen-reactive cells in each population. Cell suspensions are distributed with the goal of isolating one antigen-specific cell in each well of a 96-well plate. Specific T cells are then amplified through re-stimulations with peptide-pulsed antigen-presenting cells. Due to the low frequency of specific cells in circulation, not all wells seeded will contain specific T cells that will proliferate in response to antigen recognition. Therefore, a minimum of 2 million cells should be tested to maximize the chances of detecting specific naive T cells. Wells or cell lines with one or more specific T cell will be considered positive, whereas negative wells never contained antigen-specific T cells. Using this assay, it is possible to estimate the specific cell frequency through applying the Poisson distribution formula to the number of total positive and negative wells. This approach also reduces inter-assay variability. Yet, if there is a high number of peptides to test, this approach can be difficult to perform as it is extremely laborious. Methods using batch cell stimulation can provide a viable alternative, so long as response specificity is confirmed.

Experimental strategy:

Immunoassays will be performed exclusively in human cells and using two immune

Figure V. 3 Proposed in vitro T-cell assays to assess immunogenicity of synthetic peptides containing HLAclass-I and class-II Leishmania-specific epitopes. A) General view of in vitro assays with purified T cells from naive donors (naive repertoire). B) General view of the protocol used to assess immunogenicity in cells from healed individuals

(memory repertoire).

Methods

Ethics statement

The recruitment and sampling collection of different groups of volunteers were done in accordance with Good Clinical Practice (GCP).

In France, the Etablissement Français du Sang (EFS) is an established scientific partner of the Institut de Recherche pour le Développement (IRD), and performed the recruitment of blood donors and sample collection at the human blood bank of Toulouse.

In Tunisia, the recruitment of healed donors was based on the recommendations and approval of the local ethical committee from the Institut Pasteur de Tunis (IPT), the Comité d'éthique de l'Institut Pasteur de Tunis (convention de collaboration N°305256/00). A written informed consent was obtained from all subjects involved in this study.

Preparation of total soluble Leishmania promastigote antigens (TSLA)

All antigen extracts were prepared from promastigote stationary phase parasite cultures of L.

infantum. Briefly, TSLA were obtained from washed parasites in 1x phosphate-buffered saline (PBS), centrifuged at 1000×g/10 min at 4°C and supernatants were removed. The pellets were resuspended in lysis buffer (50 mM Tris/5 mM EDTA/HCl, pH7. 1 mL/1×10 9 parasites), subjected to three rapid freeze/thaw cycles followed and to three sonication pulses of 20 seconds/40W. Samples were centrifuged at 5000×g for 20 min at 4°C, and supernatants were collected, aliquoted and stored at -80°C until use. Protein quantification was performed using Bradford method. The CEF-I control peptide pool is comprised of 23 well-defined peptides derived from Cytomegalovirus (CMV), Epstein Bar virus (EBV) and Influenza viruses and is widely used as a positive control for CD8 T cell activation, designed to stimulate T cells with a broad array of HLA types.

Synthetic Peptides

CEF-I stock solution (Mabtec) was diluted 1:100 in culture medium for a final well concentration of 2 µg/mL.

Matrix-based peptide pools

A matrix-based pool testing strategy was chosen to validate and select the most immunogenic

b) Generation of monocyte-derived dendritic cells

Monocyte-derived dendritic cells (MoDC) were generated from plastic-adherent PBMCs (from 500 million to 1 billion total PBMC), after 5-day culture in AIM-V medium (Invitrogen) supplemented with 1000 U/mL of interleukin-4 (rhIL-4, Miltenyi Biotec) and 1000 U/mL of granulocyte macrophage colony-stimulating factor (rhGM-CSF, Miltenyi Biotec).

To evaluate the in vitro generation of monocyte-derived immature DC, the differentiation

c) Cell counting

Total PBMC were counted manually using a KOVA TM Glasstic TM slide, after Trypan Blue 0,4% staining (cell suspensions diluted in PBS1x).

Purified T cells were counted using the LUNA-FL TM Automated Cell Counter (Logos Biosystems, Annandale, VA), after Trypan Blue 0,4% staining (cell suspension diluted in PBS1X, 10 µL are added to the LUNA slide, focus is manually adjusted, and cells/mL are counted in the range 5x10 4 to 1x10 7 cells/mL). peptide), medium alone or CEF peptide control pool (2 µg/mL) and incubated for 4 hours at 37°C/5% CO2. Pulsed PBMC were washed and irradiated 6 minutes under 50 Hz, 312 nm UV light (VL-6.M lamp, Fischer BioBlock Scientific). Naive T CD8 + cells were added to pulsed PBMC and cultured in complete CellGro medium supplemented with human recombinant (rh) IL-7 (500 U/mL, Miltenyi Biotec) and rhIL-15 (75 U/mL, Miltenyi Biotec). Medium was changed after 3 days. Naive T CD8+ cells were restimulated on day 7 and day 14, for a total of 3 co-cultures in the same conditions (d0, d7, d14).

The protocol recommended by Wolfl et al was adapted to use total PBMC as APC. The protocol compares several cell types as APC, including total PBMC, and suggests the use of monocyte-derived dendritic cells. Monocyte-derived DCs were not generated for donor MPL3 so the adapted protocol uses total PBMC, pulsed with peptides for 4 hours, without any differentiation or maturation factors.

IFN-γ production was assessed on day 21 by IFN-γ enzyme-linked immunospot assay (ELISpot). All cells from culture wells were seeded in the ELISpot plate and compared with unstimulated cells from the same individual. Naive TCD8 + cell lines were considered positive when a spot count was twofold higher in the presence of the peptides than in their absence, with a minimal difference of 44 spots, the highest NS spot count (STIM spot count > 2xNS spot count average+44 spots).

• T cell amplification protocol_v1 (MN02):

The T cell amplification protocol using total TCD8 + cells from the naive repertoire was adapted from the protocol for TCD4 cells developed by the Bernard Maillere Lab (Laboratoire d'immunochimie de la réponse immune cellulaire, Institut de Biologie et technologies, CEA -Saclay) (8,10,17). This protocol uses magnetically purified total TCD8 cells which are co-cultured with autologous peptide-pulsed mature DC.

Immune cell populations (monocytes, TCD8 + and TCD4 + cells) from MPL9 buffy coat sample were purified differently from other donors. This sample was used to test a semi-automatic cell separator and sequential cell isolation and assess the protocol feasibility for the generation of the naive donor bank. Monocytes, TCD8 + and TCD4 + cell populations were magnetically isolated with the use of a MultiMACS™ Cell24 Separator Plus (Miltenyi Biotec). Briefly, the buffy coat sample was diluted in PBS1x 2mM EDTA and the distinct cell populations were sequentially purified: i) CD14 + Autologous DCs (20000 per well) were seeded in 96-well plates and pulsed with the different stimuli using the short peptide protocolimmature DCs are incubated in AIMV medium (Invitrogen) and matured with lipopolyssacharide (LPS, 1 µg/mL) and Resiquimod (R848, 10 µg/mL) for 16 hours, and then pulsed with either medium alone, peptide pools or positive controls for 4 hours at 37°C/5% CO2.

After peptide stimulation, APC were washed and irradiated 6 minutes under 50 Hz, 312 nm UV light (VL-6.M lamp, Fischer BioBlock Scientific).

TCD8 cell lines were generated by adding total TCD8+ cells to the pre-pulsed DC wells and cultured in IMDMc supplemented with rhIL-21 (30 U/mL, Miltenyi Biotec) for cell priming. Medium was changed after 3 days to IMDMc supplemented with rhIL-7 (500 U/mL, Miltenyi Biotec) and rhIL-15 (75 U/mL, Miltenyi Biotec). Cells were stimulated on day 0 and 7, for a total of 2 co-cultures, plus the ELISpot stimulation.

The specificity of the TCD8 + cell lines was analysed with ELISpot assays: 20.000 T cells from each cell line were incubated with autologous iDCs alone (cNS control) or with iDCs previously loaded with peptide pools (1 culture well = 2 ELISpot wells with 20.000 cells each). TCD8 cell lines were considered specific when a spot count was twofold higher in the presence of the protein than in its absence, with a minimal difference of 25 spots (STIM spot count > 2xNS spot count average+25 spots).

• T cell amplification protocol_v2 (MN03):

Three cryovials containing total TCD8+ cells from donor MPL9 were gently thawed (vials with 50 milion, 25 million and 12,5 million cells each). Total TCD8 cells were counted and cultured in IMDMc medium in: i) 96-well plates, 200.000 TCD8 cells per well and 10 cell lines per condition, or ii) 48-well plates, 1 million TCD8 cells per well and 2 wells per condition. In both formats, TCD8 cells were co-cultured at 1:10 ratio with DC cells stimulated using the short peptide protocol with mature monocyte-derived DCs, and a total of 3 co-cultures were performed (d0, d7, d14) plus the ELISpot stimulation. Different peptide concentrations were tested -1, 2,5 and 5 µM per peptide in each pool.

After peptide stimulation, APC were washed and irradiated 6 minutes under 50 Hz, 312 nm UV light (VL-6.M lamp, Fischer BioBlock Scientific).

For the IFN-γ ELISpot, and for all peptide concentrations, total TCD8 cells were incubated with autologous DCs alone (control) or with DCs previously loaded with peptide pools at a ratio of 1:10 (APC:TCD8). For cell lines cultured in 96-well plates, 20000 TCD8 cells were seeded in the ELISpot plate. For cells cultured in 48-well plates, 50.000, 100.000 or 150.000 TCD8 cells were seeded in the ELISpot plate. Responses were considered specific when a spot count was twofold higher in the presence of the protein than in its absence, with a minimal difference of 25 spots (STIM spot count > 2xNS spot count average+25 spots).

• T cell amplification protocol_v3 (MN04):

Total TCD4 and TCD8 cells were isolated using magnetic microbeads according to the manufacturer's instructions. Briefly, cryopreserved non-adherent PBMCs (PBMC NA) from healthy donor MPL10 were gently thawed. Total TCD4 + lymphocytes were isolated by positive selection using an anti-CD4 monoclonal antibody coupled to magnetic microbeads (Miltenyi Biotec, 'CD4 Microbeads kit' ref 130-045-101), as recommended by the manufacturer, and cryopreserved at -150°C for future use. Total TCD8 + cells were isolated from the flow through by negative selection using magnetic microbeads (Miltenyi Biotec, 'CD8 + T cell isolation kit' ref 130-096-495) according to manufacturer instructions.

In this experiment, only the 48-well format (batch stimulation) was used, 1 million TCD8 cells were seeded per well and 2 wells per condition (2 million TCD8 tested). A total of 3 co-cultures were performed (d0, d7, d14) and monocyte-derived DCs stimulated with the i) short peptide protocol (see above), and ii) long peptide protocol (simultaneous maturation with LPS/R848 and peptide stimulation during 16h at 37°C). After peptide stimulation, APC were washed and irradiated 6 minutes under 50 Hz, 312 nm UV light (VL-6.M lamp, Fischer BioBlock Scientific).

The selected donor was MPL10, and a tailored peptide pool was designed according to the HLA-typing results. The peptide pool consists of 3 peptides per supertype (best predicted IC50), for a total of 12 peptides (poolGOOD). Autologous monocyte-derived DCs were stimulated with 2,5 µM per peptide.

TCD8 cells were considered specific when a spot count was twofold higher in the presence of the protein than in its absence, with a minimal difference of 25 spots (STIM spot count > 2xNS spot count average+25 spots).

Immunoassays with samples from Healed Donors a) Healed donor samples from a Leishmania-endemic area in Tunisia

Human donor groups (cured individuals and healthy individuals with no history of leishmaniasis) were recruited from endemic areas for CL, based on the following defined inclusion criteria: i) individuals living in the Gadarif Region in Tunisia, endemic foci to L. major transmission, and who have not moved away in the last 10 years; and ii) the presence of typical scars for cured CL group. A complete medical questionnaire was completed during examination. Adult healthy individuals recruited in low or non-endemic areas (Tunis), and no or low IFN-γ response to SLA (<100 pg/ml) will be considered as non-immune/naive. Exclusion criteria were immunosuppressive diseases other than leishmaniasis, long term treatment and pregnancy. Each donor was assigned an internal sample identification code -TUNn.

Heparinized blood was collected from a total of 20 healed donors and 10 healthy controls (1 st healed series n=10, 2 nd healed series n=10, naive series n=10).

b) Total PBMC stimulation assays

Total PBMC were isolated from blood by density centrifugation through Ficoll-Hypaque (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Whole blood samples were transported at room temperature, in a cooler to keep temperature stability during the car trip from Gafsa Hospital to the Institut Pasteur of Tunis. Upon arrival in the lab, the cells were cultured in RPMI 1640 supplemented with 10% heat inactivated Human AB serum (SAB, Lonza), 100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamin, 1 mM sodium pyruvate and 1X non-essential amino acids. Briefly, cells were plated in 96-well plates (TPP, Switzerland), 200.000 PBMC/well, and were kept with media alone (NS) or stimulated with: i) Phytohemagglutinin 10 µg/mL (PHA, Sigma-Aldrich); ii) TSLA 10 µg/mL; iii) CaniLeish ® Antigen 10 µg/mL (GMP-produced LiESAp lyophilised without adjuvant) as positive controls; and iv) peptide pools (5 µM per peptide). Cells were cultured at 37°C, 5% CO2 for 10 days. On day 1, 4 and 6, human recombinant IL-2 (100 U/mL, R&D systems) was added to the wells.

The peptide pool conditions tested were: i) one pool containing class I 9-mer peptides (pool

ALL_I

Interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT)

Briefly, 96-well polyvinylidene difluoride (PVDF) plates (MSIP, Millipore) were coated overnight at 4°C with 2,5 ug/mL anti-human IFN-γ monoclonal antibody (mAb 1-D1K; Mabtech). Cells were cultured overnight (16-20h) with positive controls, peptide pools, or with culture medium as negative control, in triplicate. IFN-γ secretion was detected by addition of biotinylated anti-human IFN-γ mAb (7-B6-1; Mabtech) in PBS 1% bovine serum albumin (BSA) for 2 hours at 37°C. After washing, extravidin-phosphatase solution (dilution 1:3000 in PBS 1% BSA; Sigma-Aldrich) was added to the plate for 1 hour at room temperature, and spots were revealed with BCIP substate solution (tablets, Sigma-Aldrich) for 10 min (maximum revelation time of 20 min). IFN-γ spots were quantified using an AID Immunospot analyzer (C.T.L.).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 6 software (GraphPad). For healed experiment series, the non-parametric Mann-Whitney test (one-tailed with confidence interval 95%) was applied to compare independent sample groups (STIM vs NS). A p-value ≤ 0.05 was considered statistically significant.

HLA-typing

All HLA-typing services were provided by DKMS Life Science Lab, an affiliated company with DKMS German Bone Marrow Donor Centre. High resolution HLA typing (99%) is performed by Next-Generation Sequencing (Long Range Sequencing Service). Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, ref 51104) and shipped to DKMS Lab in Germany. Sequencing was performed for exons 2 and 6 for 6 HLA loci (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1) leading to the 6-digit identification of twelve HLA alleles per donor.

In order to compare allele frequencies between the recruited donors and country-specific populations, allele frequency data was retrieved from the Allele Frequency Net Database (AFND). The AFND provides a central source, freely available to all, for the storage of allele frequencies from different polymorphic areas in the Human Genome. Currently, collected data are in allele, haplotype and genotype format. In the present analysis, only HLA studies were considered (18). Allele frequency information with two-digit level of resolution for the French Population was retrieved from the AFND (Table V.2). Allele frequency information with two-digit level of resolution for the Tunisian Population was retrieved from the AFND (Table V.3).

Allele frequency refers to the total number of copies of the allele in the population sample (Alleles/2n) in decimal format. Some studies present results as percentage of individuals that have the allele (% ind), and it refers to the percentage of individuals who have the allele in the population (heterozygous allele count / n). Since Tunisian studies use mostly allele frequency to present results, for consistency purposes, only studies with results expressed as allele frequency were included. For the analysis of HLA allele frequency in different world populations affected by leishmaniasis, data was collected from the AFND for the following world regions: South and Central America, North Africa, Western Asia, South Asia (Appendix V.1). Data were retrieved from AFND through a 'HLA allele freq (Classical)' search with the following filters: i) region name; ii) level of resolution=2-digits; iii) Population standard=Gold and Silver (19); iv) show frequencies=only positives (Table V.4).

Table V. 2 Description and size of 17 French population studies from AFND with HLA data considered for HLA allele frequency analysis

Results -Immunoscreenings with samples from naive donors

The naive donor bank and respective HLA-typing results

A total of 21 buffy coat samples from naive individuals were processed (MPL1-21) (Table V.9).

HLA-typing was performed for donors MPL3-21 included in the naive donor bank (Table V.10).

Individual HLA-class I alleles sharing peptide binding properties were grouped in allele groups

HLA allele frequency of the naive donor bank includes the most prevalent alleles in the populations affected by leishmaniasis

The regions included in the AFND that match the populations most at risk for Leishmania infection are South and Central America, North Africa, Western Asia, South Asia. All positive HLA frequency studies (with Gold and Silver population standards) were included, which present results either as percentage of individuals expressing a given allele and/or HLA frequency in decimals. Typing of the HLA-DQB1 loci was performed for the individuals in the naive donor bank (Table V.10), and data exists for some of the selected populations, however, these were not included in the analysis since epitope prediction was not performed to HLA-DQ alleles.

HLA allele frequencies for these four world regions match the most prevalent alleles in the naive donor bank, suggesting the allele frequencies of naive donors are representative of these human populations.

Leishmania-specific TCD8 + cells are present in the naive repertoire

The first peptide immunoscreening performed tested all 49 HLA-class I peptides in matrixbased pools using the protocol adapted from Wolfl et al, with magnetically isolated naive TCD8+ cells from donor MPL3 (experiment MN01).

Figure V. 12 IFN-γ ELISpot results for the MN01 experiment: T cell amplification assay with naive TCD8 cells from naive donor MPL3. A) number of positive cell lines per condition (14 class I peptide pools). B) control results (TCD8 NS, PBMC NS, CEF and PHA). C) Spot counts per cell line and the threshold mark for well positivity. D) ELISpot well pictures and respective spot counts.

In experiment MN01, we successfully generated and detected specific T cell lines against In this experiment, the CEF-stimulated naive TCD8 cells underwent three co-cultures plus the ELISpot stimulation. The spot counts of the CEF peptide pool are quite variable with some low values (47,79,30,3), which is expected since this assay uses a purified population of naive T cells, and very few memory cells are present (Figure V.12 panel B). Memory T cells are responsible for long-term responses against the CEF peptide pool (CMV-, EBV-, influenza-specific epitopes), so low spot counts can be expected when using purified naive T cell populations. CD45RA + cell enrichment after magnetic purification was not assessed due to technical problems with the flow cytometer, even though staining was performed. Although there are specific naive T cells in naive individuals against the CEF peptides which can be stimulated an amplified in vitro, only 600.000 naive TCD8+ cells (triplicate wells) were tested. Still, it is noteworthy that some Leishmania-specific peptide pools induced much higher number of IFN-γ-producing cells than the CEF control pool. Considering this information, only one peptide (A24_3) corresponds to the donor's HLA typing, implying peptides are more promiscuous than predicted by in silico algorithms.

If we consider all peptide pools with at least 1 positive well, there are 9 positive matrix-based peptide pools leading to the identification of 20 double-positive peptides. However, from these 20 double positive peptides, only 2 correspond to the donor's HLA-typing. The number of restricted peptides is not significantly increased as these do not include more donor-specific supertypes.

Interestingly, peptide pool E which generated one cell line with high spot count (274) contains both A24-and B44-restricted peptides.

This assay constitutes preliminary evidence of the presence of T-cell precursors specific to

Leishmania epitopes in naive individuals' immune repertoire. Although the magnitude of response was assessed for in vitro stimulated cell lines, specificity was not absolutely confirmed because spot counts were compared to NS wells (never stimulated autologous TCD8 + cells). An ELISpot well containing culture-stimulated cells but unstimulated during the ELISpot (cNS) will demonstrate that IFN-γ production is dependent on peptide-stimulus and, therefore, specific.

The remaining issues with this experiment are i) that no cell counts were performed before seeding in ELISpot plate (in this experiment 1 culture well corresponds to 1 ELISpot well) which may introduce bias when comparing wells; ii) the use of an empirical rule for well comparison, because there are no triplicates no statistical test can be applied; iii) LiESAp or CaniLeish ® antigen, the original antigenic mixture, were not included as positive controls; iv) finally, the high number of purified naive TCD8 cells needed per experiment and per individual (MINIMUM 200.000*10 wells/condition) is logistically unviable.

In this experiment total PBMC were used as APC, without supplementation with differentiation or maturation factors. We expect the antigen presentation protocol can be further optimised using professional APC, such as monocyte-derived DCs for short peptide presentation in HLA class-I molecules.

Total TCD8 + cell in vitro amplification needs at least three stimulation rounds

In experiment MN02, total TCD8 cells from donor MPL9 were used, and the same matrixbased peptide pools were tested at 5 µM concentration per peptide. Cell populations from MPL9 were isolated differently from other donors using the MultiMACS™ Cell24 Separator Plus (see Material and Methods). Surface marker phenotyping shows that CD8 + CD45 + cells were present at 88,2% purity (66,4% of total events excluding debris) (data not shown). Also, CD14 + CD45 + monocytes were isolated with 93,9% purity (89,4% of total event excluding debris) and CD4 + CD45 + cells were isolated with 96% purity (84,7% of total events excluding debris) (data not shown).

The protocol used was adapted from analogous TCD4 + cell amplification assays (8,21), and uses the total TCD8 + cell population and mature monocyte-derived DCs (mDC) as APC. Since the class I peptides are only 9-mer in length, and do not necessarily need processing, a short peptide stimulation protocol was adopted, where monocyte-derived immatured DCs are firstly matured with LPS and TLR-7/8 agonist Resiquimod/R848, and only after stimulated with peptides for 4 hours.

In this experiment, no specific cell lines were generated against any of the pools at 5 µM peptide concentration (Figure V.14 panel A) after two co-cultures.

Negative control background levels from never stimulated TCD8 + cells remained low (NS spot counts 15/6/20/20/17, average 16). As expected, since in this experiment total TCD8 cells were used, the CEF control pool results were higher than MN01, even with just one co-culture plus the ELISpot stimulation (CEF spot counts 138/49/153/51, CEF MN02 Elispot2: 34, 238 (Too Numerous To Count), 39, 186 (TNTC), with only 20.000 TCD8+ cells seeded in the ELISpot wells.

PHA stimulation results (spot counts 366, TNTC, 404, TNTC) and average cell viability of 77% (± 9.9%) assessed before ELISpot seeding, confirm cells were viable and responsive (Figure V.13 panel B). We estimate viability is higher than calculated by LUNA reader, for there is some cellular debris from DC cells and SAB used for the co-cultures, considered as dead cells by the automatic counter (a cell size filter was not applied).

The positive controls LiESAp and CaniLeish ® antigen were tested in this experiment, however, the observed negative results remain inconclusive since the DC stimulation protocol was not adapted for protein processing. The short peptide stimulation protocol was used for all conditions and we In this experiment, response specificity per cell line was confirmed with culture-stimulated cells but unstimulated during the ELISpot (cNS). Overall, observed spot count values in stimulated wells (STIM) are sometimes higher than cNS, but not enough to consider as positive responses (Figure The use of statistical analysis to assess positive responses is more reliable and recommended as they are based on a theoretical background, have a universal application across experiments, and even more suitable to detect weaker responses. Furthermore, the primary objective of the peptide immunoscreenings is to exclude non-immunogenic and unstable peptides, before inquiring about precursor frequency. Therefore, the following adaptations were made to the protocol: i) an additional total TCD8:DC co-culture (3 instead of 2); ii) testing different peptide concentrations and fresh aliquots diluted in 10% DMSO; and iii) testing a different assay format that allow the application of statistical tests to determine well positivity, the counting of all culture wells and show results as SFC/million, a widely accepted way to present ELISpot results.

In vitro amplification of total TCD8 + cells depends on the antigenpresenting cells and respective stimulation protocol

In experiment MN03, total TCD8 cells from donor MPL9 were used, and all peptides were tested in two pools at different peptide concentrations. At the time of this experiment, HLA-typing results of MPL9 donor were not yet available to design one personalised peptide pool, so all class I peptides were tested in two pools, containing 22 peptides with predicted restriction to HLA-A genes (pool22), and 27 peptides with predicted restriction to HLA-B genes (pool27). Furthermore, two assay formats were tested: 96-well plate format, establishment of cell lines and empirical rule to determine positive responses, and the 48-well plate format (batch stimulation), that allows for statistical determination of well positivity and cell counting before seeding in the ELISpot plates, while still testing the same number of cells.

Discussion -Immunoscreenings with naive donor samples

The immunoassays with naive donor samples currently still present several challenges. The main limitation is the number of cells needed per condition, since specific cells are rare. These experiments are also extremely time-consuming (with over 20-day cultures), and resourceconsuming due to reagents' costs. Also, in vitro cellular amplification occurs under non-physiological conditions, so analysis should be performed with care. Finally, a high number of individuals should be tested to assess the peptides' overall immunogenicity in the naive repertoire. Specific approaches were adopted to mitigate some of these issues, namely: i) the generation of a naive donor bank and respective HLA-typing; and ii) the use of matrix-based peptide pools.

Peptide immunogenicity testing through immunoassays with samples from naive donors is rather innovative in the field of peptide-based vaccine development. Several laboratories, namely the Bernard Maillere Lab (CEA -Saclay), have developed techniques of in vitro cellular amplification that allow the analysis of specific T cell precursors in the naive repertoire (8)(9)(10)17,(22)(23)(24).

Immunogenicity testing is often focused on samples from exposed/healed individuals or animal models. We believe immunogenicity testing in the naive repertoire is essential to assess the prophylactic potential of peptide vaccine candidates, since there is a minimum threshold of specific naive T cells in circulation needed to generate specific effector and memory T cells. Also, the TCRspecificity of responding naive T cells correlates with the specific memory T cells present in immune individuals. We propose to assess and compare the peptide immunogenicity profiles in the naive and memory repertoires to select immunogenic peptide candidates to use in a final vaccine formulation against leishmaniasis. Overall, this approach allows for the fast validation of peptide vaccine candidates with increased confidence in late-stage human in vivo immunogenicity and vaccine effectiveness.

Naive donor bank

The Surprisingly, when the total TCD8 stimulation assay protocol was adopted, the use of mature DCs with the short peptide protocol no longer stimulated T cell proliferation and IFN-γ production.

The number of co-cultures performed (MN02: 2 co-cultures, MN03: 3 co-cultures), the peptide concentration (1, 2,5 or 5 µM), or the assay format do not seem to be as important as the DC stimulation protocol, since IFN-γ responses were not detected in both MN02 and MN03 experiments.

Many factors may contribute to this and apparently these Leishmania-specific peptides, even if short (9-mer) may need intracellular processing. Furthermore, we estimate that, even for short peptides, optimal DC stimulation requires peptide processing and presentation with simultaneous DC maturation through TLR activation, due to peptides' intrinsic weak immunogenicity.

In experiment MN04, when immature DCs were again used as APC, and stimulated during 16 Notably, both immature and mature DC populations generated from donors MPL9 and MPL10 include cells with intermediary phenotypes, not expressing any of the stained markers (CD14 -CD209 - CD83 -). To describe these populations other markers should be included in the analysis. Also, some contaminant cells (probably non-adherent PBMC) and debris due to cell thawing prior to staining are observed (18 to 47% of total events). Nevertheless, the differentiation of monocytes into DCs is successful as CD14 -cells correspond to over 98% of all cells.

For MPL9 over 40% of total CD14 -cells are immature DCs (CD14 -CD209 + CD83 -). The percentage of immature DCs after differentiation for MPL10 is much lower (18% of total CD14 -cells).

These results may result from a shorter differentiation time for monocytes from MPL10 (four-day stimulation instead of five). However, overnight maturation with LPS and Resiquimod greatly increased the number of mature DCs (from 2,3% to 28,6% of CD14 -CD209 + CD83 + cells).

The experiments MN02-04 used monocyte-derived DC as antigen-presenting cells. These were co-cultured with TCD8 + cells at a 1:10 ratio. Although no optimisation experiments were performed to improve this ratio, there is evidence that optimal APC:T-cell ratio can go up to 1:160, or be as low as 1:2, with lower ratios having potentially negative impact on T cell stimulation (28). The intermediary 1:10 ratio was adopted, same as in the total TCD4 + cell amplification protocol.

After experiment MN01, a total TCD8 amplification assay protocol was adopted due to the high number of conditions to test and to decrease the experiment-associated costs. Even though naive donors are expected to have never been exposed to leishmaniasis, there is an increased risk of triggering cross-reactive responses, potential cross-specific memory T cells in circulation against other pathogens or antigens. To assure no recall responses are being detected, short term total PBMC stimulation experiments will be performed with both TSLA and peptide pools to confirm, respectively, the naive donor status (TSLA-negative) and IFN-γ production by amplified specific naive T cells and not cross-specific memory cells.

Peptide concentrations in the peptide pools tested ranged from 1 to 5 μM. While the nature of the induced response to change remains unchanged regardless of the peptide concentration, a positive effect on T cell activation is expected in the range of 1-10 µg/mL of a 9-mer peptide (29).

Nevertheless, the peptide pool composition may also influence the observed responses. Response specificity against a single peptide may be decreased if pools contain many peptides, and likewise, pool sensitivity may be increased if there are multiple positive peptides. On the other hand, peptide competition and low frequency responses may hinder response detection. We expect that the peptides tested present binding competition since they are all predicted to be strong binders. In experiment MN03, peptide pools with over 20 peptides each were used and stimulated cells at 5 µM show decreased spot counts when compared with the other tested concentrations. Therefore, it is possible that increased peptide numbers and/or increased peptide concentration inhibit T cell activation, either through peptide competition or due to the absence of high avidity specific T cells.

We believe peptide competition is higher in the tested peptide pools than expected for traditional epitope mapping experiments, where only a few immunogenic regions are found within a full protein, and many negative peptides are included in the peptide pools. The matrix-based pools contain 7 peptides, and the poolGOOD used in MN04 contains 12 peptides at 2,5 µM each. Peptide pools containing up to 12 peptides at 2,5 µM do not seem to inhibit T cell activation, so this peptide concentration will be adopted for future experiments.

Peptide pools are an efficient and sensitive way to optimise epitope mapping experiments and even to detect low level responses while decreasing experiment-associated costs and sample size. The use of matrix-based pools provides additional advantages regarding the number of assays performed and cryovials needed (compared with single peptide testing or mini-pool peptide testing). However, some unclear results regarding double positive peptides argue for the need of a second ELISpot to confirm peptide specificity (two-stage matrix-based pool approach).

The CEF control peptide pool provides a good control for peptide processing and presentation to total TCD8+ cells. Although results were quite variable (from 16 spot counts up to TNTC), they were consistently positive and with large spots. Evidently, the observed responses are mediated by memory T cells instead of naive T cells. While CEF-induced responses do not necessarily provide evidence for amplification of specific T cell populations through clonal expansion, they still provide evidence that antigen presentation mechanisms by peptide-pulsed APC are effective in priming specific T cells in vitro. True positive control peptides for the naive T cell repertoire should be included, however, universal control peptides for naive T cell stimulation do not exist. A few peptides were so far described to have specific naive T cells in the naive repertoire. The most studied are peptides from the Melan-A/MART-1 antigen (Melanoma-Associated Antigen) (30,31). Still, these are restricted to HLA-A*02 alleles and have an unusually high precursor frequency. We propose and expect to find the most immunogenic peptides with specific naive T cells through the optimisation of T cell amplification assays, which have been shown to be effective in detecting specific T cells in the naive repertoire.

In experiment MN04, which used the long peptide stimulation and a tailored peptide pool We aim to use the 48-well batch stimulation assay with a higher number of donor samples (n=10) to screen the synthetic peptides and select the most immunogenic ones. Possibly, the number of total TCD8 cells tested per condition can be increased (3 or 4 million total TCD8 per condition).

However, the 48-well batch stimulation assay format eliminates the application of the Poisson distribution formula for the calculation of the specific T cell precursor frequency. Hence, we propose to use the batch stimulation assays to pre-select the (most) immunogenic peptides and, subsequently, to perform precursor frequency analysis experiments with a fewer number of pre-validated immunogenic peptides, and possibly bypassing polyclonal activation with peptide pools but performing co-cultures with DC pulsed with single peptides.

Because naive T cell amplification assays were performed in a limited number of individuals with all peptides, comparison is very limited. However, MN01 and MN04 clearly evidence the existence of a minimum number of naive T cells specific to Leishmania peptides in naive donors, hence, validating their predicted immunogenicity and the feasibility of using such peptides in an effective vaccine formulation. The naive donor bank, which will be augmented with additional naive donor samples, and the in vitro stimulation protocol, which is now optimised, will allow to screen all peptides and select the most immunogenic by serial routine experiments.

Results -Immunoscreenings in healed individuals from endemic areas (Tunisia)

The first peptide immunoscreenings performed with healed donors' samples tested all 49 class I peptides and 24 HLA-class I peptides in matrix-based pools, using a short term total PBMC stimulation protocol.

Two series of blood samples were received, both including 10 samples from healed donorsfirst series TUN1 to TUN10, and second series TUN11 to TUN20. In the first series (TUN1 to 10), one blood sample was hemolysed (TUN2) so no PBMC were purified. IFN-γ ELISpot was performed for both series and culture supernatants were collected and stored at -80°C. The series of blood samples with naive donors was performed in the Institut Pasteur Tunis, no IFN-γ ELISpot was performed for these samples.

Peptide immunogenicity or the capacity to prime in vitro specific recall responses was successfully assessed for 9 healed donors (see Appendix V.2 for individual data).

Healed donors' personal information and HLA-typing results

The average age for the first healed donor series was 43 years-old, ranging from 26 to 64 years-old (Table V.7). Male healed donors are much more frequent than female donors in this series, with only one woman in nine donors (TUN4). The presence of CL lesions was inquired in the medical questionnaire. On average, healed donors in the first series presented 2 cutaneous lesions, between one up to five lesions per donor. Most cutaneous lesions were localised to the legs or arms, only a few CL lesions were localised in the face (two donors, 11%) (Table V.7).

HLA-typing results for the first healed donor series was performed (Table V.8)

The HLA-typing results for the healed donors, although the sample size is quite small, mostly because only two ELISpot wells can be analysed due to an error during seeding (CaniLeish ®stimulated cells were stimulated with TSLA), but 192,5 spot count average is considerably higher than autologous NS spot counts (28,83 ±22,07). Two other donors (TUN9, TUN10) were considered TSLApositive since ELISpot results show IFN-γ production during culture stimulation, evidenced by dark, highly saturated wells, even though spot counts are low or innumerable. In addition to TSLA, CaniLeish ® was also used as positive control. Interestingly, CaniLeish ® antigens induced vigorous IFN-γ responses in >75% of individuals (7 in 9 donors). Moreover, donors TUN1, TUN7, TUN8 and TUN9 had significantly higher responses against CaniLeish ® but not TSLA (

IFN-γ responses against Leishmania-specific peptides

To evaluate recall responses against Leishmania-specific peptides, total PBMC were tropica transmission. Historically, the Gafsa region was always an endemic area for CL, with typically sporadic and occasionally epidemic transmission (32). The Gafsa region in Tunisia is estimated to include a population of almost 324000 people, of which almost 147000 are at risk of developing leishmaniasis (33). Although parasitological confirmation was not performed for all donors, we expect L. major to be the species responsible for the primary infection.

The ELISpot results for the first series of healed donors are available (n=9) and presented here. The conclusion of the CBA analysis of these samples will allow the definite confirmation of the ELISpot responses and identified immunogenic epitopes, and more importantly, characterise in more detail the induced immune response by including a complete Th1/Th2/Th17 cytokine panel. The assessment of the production of the immunoregulatory cytokine IL-10 will be particularly The first immunoscreening assays performed with healed donor samples successfully identified IFN-γ responses specific to Leishmania peptides and have led to the identification of 25 HLA-class I and 14 HLA-class II immunogenic peptides. Overall, HLA-class II peptides appear to be more immunogenic as they induced higher IFN-γ production and in more individuals. An 'ELISA' effect in the ELISpot plates is observed at timesa dark background and high well saturation due to leftover IFN-γ from the culture wells. Yet, the AID reader successfully read and count all plates (Appendix V.2).

Four donors had significantly higher IFN-γ production after TSLA stimulation (TUN3, TUN4, TUN5, TUN10). Some individuals (n=5) did not have significantly increased responses after TSLA stimulation by ELISpot. However, for two of these donors (TUN9, TUN10), although spot count may be low or innumerable, an 'ELISA' effect is observed, where secreted IFN-γ diffuses into the supernatant and is absorbed on the ELISpot plate membrane producing a color carpet. This effect was observed probably because cells were not washed before seeding the ELISpot plate (plate centrifuge unavailable in the laboratory), a simple media change was performed. These individuals were considered as TSLA-positive since there is evidence of IFN-γ production during culture stimulation.

Donor TUN1, who appears to not respond to TSLA stimulation, however, responds strongly to CaniLeish ® . Curiously, the four TSLA-positive donors received different treatment regimens (respectively, cryotherapy plus antibiotherapy, arab traditional treatment, 15-day hospitalization with administration of intramuscular Glucantime ® 60mg/Kg, or cryotherapy only). The time since diagnosis also differed greatly between donors (6 donors with date of parasitological confirmation), ranging from 1 to 12 years, and of 10 years for TUN4 and 2 years for TUN5. There appears to be no correlation with type of treatment or healing time and development of TSLA-specific immune responses in this healed donor sample series.

Two individuals did not respond to any HLA-class I peptide pool, and although there are peptides specific to all possible alleles present in the tested individuals. This may due to reduced peptide stability which prevents peptide processing or presentation and T cell activation, as class I peptides were solubilized in water. Peptide stability assays will be performed with pre-selected peptides.

Only one peptide concentration was tested for total PBMC stimulation in these experiments.

A possible inhibitory effect on induced IFN-γ responses is observed when peptide pools contain 24 or 49 peptides (peptide pools ALL_I, ALL_II, and ALL_I+II). The 5 µM concentration per peptide in these pools may inhibit T cell responses or induce apoptosis or anergy. This effect may also be due to a high dilution of culture medium, since stock peptide solutions were at 200 mM, depriving the cells from essential nutrients. For this reason, new peptide stocks were prepared at 500 mM concentration.

To establish if the peptide pools significantly increased IFN-γ production in total PBMCs from healed donors, the Mann-Whitney non-parametric statistical test compared the triplicate STIM wells and the 6 autologous NS wells. A high variability in the ELISpot background is observed, as well as some discrepancies in the ELISpot triplicates, possibly because red blood cell (RBC) lysis was not performed before seeding (reagents unavailable in the laboratory), and the number of contaminant RBC can affect successful T cell activation in a given well. Furthermore, dividing each culture well in two ELISpot seeding wells, may affect the minimum number of 100.000 PBMC per well resulting in failure to detect the specific T cells. Cell viability was also not assessed prior to ELISpot seeding, however, PHA responses were extremely high indicating the cells were viable and responsive.

The unstimulated ELISpot wells containing cells that were stimulated in the culture (cNS)

have often an intermediary spot count value between the individuals' NS and the STIM wells. This may be because in a stimulated culture well there are cells in different activation stages, especially since culture is supplemented with rhIL-2, and some cells are still producing IFN-γ from the culture stimulation. In future experiments, cells should rested for at least 3 days after the last media change, washed and checked for viability before seeding in the ELISpot plate, and a minimum number of 200.000 PBMC seeded per well.

There does not seem to be a clear correlation between in silico-predicted HLA-class I restriction and individuals' HLA typing. There is increasing evidence of extensive promiscuity in HLAclass I antigen presentation which the in silico HLA-binding prediction algorithms do not account for (34,35). It is possible that in silico HLA-binding algorithms can predict immunogenic epitopes from antigen sequence data but perform less well in predicting HLA-restriction for promiscuous peptides.

This argues for the selection of epitopes predicted to be promiscuous by the immunoinformatic tools available but, likewise, to expect unpredicted peptide HLA restriction.

There is, however, a good overlap regarding the most frequent alleles between the Tunisian

Conclusions

T cell activation in vitro occurs under non-physiological conditions, and factors influencing T cell activation should be controlled whenever possible, namely antigen-presentation, peptide dose, and stimulation conditions. Nevertheless, in vitro assays with human immune cells are still more conclusive on peptide immunogenicity than animal models. Cell-based immunoscreening assays to assess peptide immunogenicity in vitro are suitable and informative because they recreate the T cell responses induced after in vivo peptide administration, and allow the analysis of T cell populations involved. Furthermore, immunoscreening assays can be performed with cells from human donors of different immune status regarding leishmaniasis, allowing the evaluation of both correlations with protective responses and prophylactic potential of the peptide candidates. We expect to find peptides that are associated with protective T cell responses against Leishmania present in healed patients, as well as to find corresponding peptide-specific T cell precursors in the naive repertoire.

Although immunoscreenings in the naive repertoire were mostly optimisation experiments and only a few individuals were tested, preliminary evidence exists for peptides which are immunogenic in both immune backgrounds. Notably, the four double positive peptides identified in experiment MN01 from pool stimulations with more than one positive cell line (B7_3, A24_3, B8_3 and B58_3) were immunogenic in one healed donor sample (TUN10). The peptide B7_3 is also included in the poolGOOD (experiment MN04) which induced specific IFN-γ production by T cells from the naive repertoire (Table V.9).

Other peptides included in the poolGOOD induced both one positive naive T cell line (experiment MN01) and higher IFN-γ responses in healed donors: peptides A3_1, A3_3, B7_2 were immunogenic in donor TUN7, and the peptide A3_2 was immunogenic in donor TUN10. The peptide A2_1, which induced one positive naive T cell line (experiment MN01) was included in the poolGOOD (Table V.9). Some of the peptides which induced one positive cell line in experiment MN01 were also immunogenic in healed donor samples. Namely, the peptide B8_1 was immunogenic in donor TUN7, also the peptides A3_2 and B7_4 were immunogenic for donor TUN10. The double-positive peptide B8_4 identified in experiment MN01 (one positive cell line) induced significant IFN-γ responses in TSLA-positive donors TUN5 and TUN10. Similarly, peptide B62_2 which induced one positive cell line in experiment MN01 was also identified in TSLA-positive donor TUN5 (Table V.9). Some double-positive peptides inducing one positive T cell line in experiment MN01 did not induce significantly higher responses in healed donors (peptides A2_1, A2_2, A2_3, A2_5, B27_3, A26_1). Still, peptides B27_2, A2_1, and A2_2 were included in the poolGOOD that induced positive IFN-γ responses in naive donor MPL10 (Table V.9).

Table V. 9 Double-positive immunogenic peptides identified in the naive and memory repertoires.

HLA-class I peptides

Assays

were not yet performed with TCD4 + cells from the naive repertoire, so no conclusion can be made on corresponding patterns for HLA-class II peptides. However, more healed individuals responded to the HLA-class II peptides, and 14 immunogenic HLA-class II peptides were identified. Some immunoprevalent peptides were identified which induced higher IFN-γ responses in over half the individuals tested (peptide 13_b12), and in three donors (22_b4 and 4_a11). Remarkably, two of these peptides come from new protein antigen candidates discovered through the reverse vaccinology approach. Due to intellectual property issues, peptide sequences and antigen names must remain confidential and cannot be divulged.

Overall, twelve HLA-class I 9-mer peptides (A24_3, A3_1, A3_2, A3_3, B58_3, B62_2, B7_2, B7_3, B7_4, B8_1, B8_3, B8_4) and five HLA-class II 15-mer peptides (13_b12; 22_b4; 17_b15; 4_a11; 14_b12) are, for now, the most promising candidates to advance for the multi-epitope peptide design.

CHAPTER V -EXPERIMENTAL VALIDATION OF SYNTHETIC PEPTIDES 243

We aim to re-test all the synthetic peptides in more immunoscreenings with samples from both naive and healed donors. Possibly, re-test the matrix-based pools and these pre-selected peptides, with some small protocol improvements. Also, we seek to include healed donors from more endemic areas, namely L. braziliensis, L. infantum and L. donovani endemic areas.

The present results provide evidence that the proposed peptide-based vaccine development Furthermore, peptide-based vaccines and therapeutics are a promising way of reducing production costs, rather than other expensive biologicals (purified recombinant proteins or antibodies). Additionally, peptide-based products do not need cold chain transport conditions, posing a great advantage particularly for low-resource settings.

A vaccine as the most promising tool for leishmaniasis control

The vaccine development pipeline proposed in this project helps mitigate some of the

Interest and limitations of using proteomics in a vaccine development pipeline

Proteomics is the most suitable tool for the analysis of Leishmania antigens as these parasites regulate gene expression mostly at the post-translational level, even if databases still need further improvement.

It is important to note that new protein annotations in the UniProtKB Database changed the proteomic analysis results due to a database update. The same raw data were used and the same bioinformatic analysis was performed, but the database was updated between June 2016 (analysis used for antigen selection, Chapter III) and March 2017 (analysis used for the proteomic analysis, Chapter II). The database annotation updates increased the number of species-specific proteins, as a higher number of unique proteins are identified. This affects protein identification and the number of common accessions among species. Briefly, if the Reverse Vaccinology approach for antigen discovery would be applied to the datasets generated with the new analysis (March 2017), only 326 accessions would be included, instead of 618. After BLASTp against the human proteome, only 33 common accessions are identified as non-homologous to human proteins. This corresponds to a loss of selected protein antigens (33 accessions/11 proteins) from the initial 24 protein antigens (76 accessions). Overall, 9 in 50 selected HLA-class I epitopes, and 7 in 24 selected HLA-class II epitopes are no longer identified. These peptides were already synthesised when the new analysis was performed, and they were included in subsequent experimental validation.

Nevertheless, database updates are frequent in -omics analysis, so these changes are fairly expected. In our case, the decrease in the total number of protein antigens analysed is actually undesirable for a comprehensive vaccine antigen selection. The core objective of the proposed peptide-based vaccine development pipeline was to screen the highest number possible of secreted proteins as antigen candidates, in order to maximise the chances of discovering immunogenic epitopes. Nevertheless, the epitope conservation filter included in the epitope selection step ensures that the peptides are identical among Leishmania target species even if the species-specific protein sequences are different. Finally, the database updates improved the molecular analysis of the secretome of the different Leishmania species (Chapter II), but did not interfere with the overarching goal of epitope-based vaccine development (Chapter III).

Lessons from other fields (cancer immunotherapy)

Importantly, although a prophylactic vaccine remains the gold-standard, a therapeutic vaccine (immunotherapeutic formulation) would also greatly advance leishmaniasis control. The proposed multiepitope peptides should also be tested for treatment, in combination and/or comparison with current chemotherapy. A peptide candidate that enhances treatment would be a major tool to help control leishmaniasis in endemic settings.

Currently, cancer immunotherapy is harnessing patients' immune system to recognize and kill cancer cells (1). Leishmaniasis chronic infection shares some immunoregulatory features with cancer, so it is possible to incorporate inhibitors of specific immune checkpoints into vaccine or immunotherapeutic formulations which can transiently reduce immunosuppression to allow the generation of robust vaccine-mediated anti-parasitic immunity. In this context, the CTLA-4/PD-1 (Cytotoxic T-lymphocyte-associated-protein 4/Programmed cell death-1) blockade could be explored to improve drug treatment and enhance vaccine efficacy against leishmaniasis. There is preliminary evidence that CTLA-4/PD-1 blockade promotes parasite clearance and treatment efficiency in animal models (2)(3)(4). Much like cancer blockade therapies, it is expected that specific types and combinations of immune checkpoint blockade will work differently for different types of leishmaniasis (VL/CL/MCL/PKDL/DCL). Further research into Leishmania immune responses and immunoregulation must be performed.

Considerations for the final peptide vaccine formulation

The peptide vaccine candidate against leishmaniases that will stem from this research will consist of synthetic peptides which include multiple Leishmania-specific immunogenic epitopes with worldwide HLA coverage. Evidently, the assembly design of the multi-epitope peptides will be carefully performed to maximise antigen uptake and presentation without affecting the induced immune response. Altogether, a successful vaccine must integrate the safety, efficacy and cost requirements to be deployed in the field.

Also, peptide dose and the administration regimen must be optimised, in order to ensure both large number of responding T cells and an appropriate quality of the induced response (5).

Specific targeting of tissue-resident dendritic cells for the induction of protective responses and the presence of adjuvants to enhance memory generation should be included in vaccine design for improved in situ T cell memory generation (6). Dermal DC are the optimal target for the peptide vaccine formulations since they then migrate to the lymph nodes and provide the ideal antigen presentation to specific T cells inducing the desired immune response. Besides adequate adjuvantation, another important aspect to consider is the administration route, and how it affects product dispersion in the dermis and the number of targeted dermal DC (7). The anatomic location for vaccine administration is essential for optimal efficacy (6). For example, the administration in the skin or nasal mucosa could boost the generation of TRM cells in vivo. Early life vaccination is also preferential since memory T cells generated early in life have been shown to be maintained in peripheral blood, and there are fewer memory T cell clones to occupy pre-existent tissue niche environments where they were shown to persist until adulthood (6). Another key aspect of the successful assessment of vaccine immunogenicity and safety is the inclusion of sand fly challenges instead of intravenous infection in the design of clinical trials.

In this view, we believe the most well-suited final vaccine formulation against leishmaniases will include appropriate Th1-inducing and/or TLR agonist as adjuvant(s), and with optimised administration, possibly intradermally, with multiple or large injection sites, different prime-boost regimens or even novel administration devices, in order to maximise antigen uptake by DCs and ensure the ideal quality and quantity of the induced immune response.

  innovation programme under the Marie Sklodowska-Curie International Training Network grant agreement No 642609. I gratefully acknowledge the additional funding received towards my PhD from Fondation des Treilles and SATT AxLR. I would like to express my sincere gratitude to my supervisor Dr. Jean-Loup Lemesre for his guidance and help throughout my PhD. I am grateful for his continuous support, encouragement and optimism which have made my PhD experience productive and stimulating.

1 . 2 . 4 . 7 . 5 6. 1 . 6 . 2 .Figure I. 1 Leishmania parasites and lifecycle 4 Figure I. 2 Leishmania world distribution 6 Figure I. 3 Figure I. 4 Figure I. 5 Figure I. 6 Figure I. 7 Figure I. 8 Naive T cell primingFigure I. 9 HLAFigure I. 13 Figure I. 16 Figure I. 18 Figure I. 19 Figure II. 1 Figure II. 2 Figure II. 3 Figure II. 4 Leishmania secretome protein identificationsFigure II. 5 Comparison of protein identification listsFigure II. 6 Secretion pathway analysis 100 Figure II. 7 GeneFigure II. 8 122 Figure III. 2 137 Figure III. 3 Figure III. 4 Figure IV. 2 Figure IV. 3 Figure IV. 4 Figure IV. 5 Figure IV. 6 Figure IV. 7 Figure V. 1 Figure V. 2 Specific cell frequency in different immune repertoires 181 Figure V. 3 Figure V. 4 Matrix-based pool design 184 Figure V. 5 Experimental planning for immunoassays with samples from healed donors 191 Figure V. 6 Figure V. 7 HLAFigure V. 8 HLAFigure V. 9 AverageFigure V. 11 Figure V. 13 Figure V. 18 Figure V. 22 Figure V. 24 Figure VI. 1

 1247516214263456789131618191234561007812221373423456712181341845191678911131822241 Figure I.1

Geiger et al 2016 ( 1 ) 21 ••

 121 Geiger A, Bossard G, Sereno D, Pissarra J, Lemesre J-L, Vincendeau P, et al. Escaping Deleterious Immune Response in Their Hosts: Lessons from Trypanosomatids. Front Immunol. 2016;7(May):1-Holzmuller et al 2018 (2) Holzmuller P, Geiger A, Nzoumbou-Boko R, Pissarra J, Hamrouni S, Rodrigues V, et al. Trypanosomatid Infections: How Do Parasites and Their Excreted-Secreted Factors Modulate the Inducible Metabolism of l-Arginine in Macrophages? Front Immunol. Frontiers; 2018 Apr 20;9:778 Pissarra et al (under review) Pissarra J, Bonhomme V, Loire E, Lemesre J-L, Holzmuller P. Exploring -omics datasets for epitopebased development of vaccines and therapeutics -SILVI, an open-source pipeline script for T-cell epitope selection. (submitted to Plos One). • Petitdidier E et al (submitted to npj Vaccines) Petitdidier E, Pagniez J, Pissarra J, Holzmuller P, Papierok G, Vincendeau P, Lemesre J-L, Bras-Goncalves R. Peptide-based vaccine successfully induced protective immunity against canine visceral leishmaniasis.

  11th ENII EFIS EJI Summer School on Advanced Immunology, Sardinia, Italy (May 7-14 th 2016) Pissarra J, Holzmuller P, Guizani-Tabbane L, Lemesre J-L. "Development of a multiepitope peptidebased vaccine against human leishmaniasis" xxiii • 2nd International ParaFrap Conference (French Parasitology Alliance for Health Care, ParaFrap), Ile des Embiez, France (October 2-5th 2016) Pissarra J, Holzmuller P, Bras-Gonçalves R, Guizani-Tabbane L, Lemesre J-L. " Development of a multiepitope peptide vaccine against human leishmaniasis" • CBS2 day 2016 (Journée de l'Ecole Doctorale CBS2), Montpellier, France (May 26th 2016) Pissarra J, Holzmuller P, Bras-Gonçalves R, Garnaoui A, Lemesre J-L. "A new peptide-based vaccine against human leishmaniasisoptimised preclinical development to prevent late-stage failure". • 10th Meeting Club de Vaccinologie, Lyon, France (March 20-21st 2017) Pissarra J, Holzmuller P, Guizani-Tabbane L, Bras-Gonçalves R, Lemesre J-L. "A new peptide-based vaccine against human leishmaniasesoptimised preclinical development to prevent late-stage failure". • 6th World Congress on Leishmaniasis (Worldleish), Toledo, Spain (May 16-20th 2017) Pissarra J, Holzmuller P, Bras-Gonçalves R, Garnaoui A, Lemesre J-L. "A new peptide-based vaccine against human leishmaniasisoptimised preclinical development to prevent late-stage failure". • TwinntoInfect Spring School in Infection and Immunity 2018 by the TwinnToInfect Network (Instituto de Medicina Molecular, Francis Crick Institute, Institut Pasteur Paris) Pissarra J, Holzmuller P, Bras-Gonçalves R, Garnaoui A, Lemesre J-L. "Preclinical development of a new peptide-based vaccine against human leishmaniases".

Figure I. 1

 1 Figure I. 1 Leishmania parasites and lifecycle. A) Leishmania parasites general lifecycle (37). B) Microscopy images of Leishmania promastigotes cultured in vitro. Left image, contrast microscopy (© IRD Lemesre, Jean-Loup); right image, parasite DNA stained with red fluorescence dye (© IRD Vergnes, Baptiste). C) Microscopy images of Leishmania amastigotes inside human macrophages (© IRD Vergnes, Baptiste). Top image, overlap between green-labeled parasites and DNA stained with red fluorescence dye.

Figure I. 2

 2 Figure I. 2 Leishmania world distribution(31,42). A) World distribution of CL-causing Leishmania species. B) World distribution of VL-causing Leishmania species.

  L. (L.) donovani causes classic VL in Asia; L. (L.) infantum causes infantile VL in the Old World. L. (L.) chagasi=L. (L.) infantum causes VL in the Americas; L. (L.) donovani and L. (L.) infantum in East Africa (Ethiopia, Kenya, Somalia, Sudan, Uganda); L. (L.) amazonensis is an uncommon cause of atypical VL in the Americas; L. (L.) tropica is rarely associated with VL syndrome, often atypical. L.) donovani (Indian subcontinent) L. (L.) donovani, L. (L.) infantum (East Africa) PKDL develops in apparently cured VL individuals (5-10% in India, 50-60% in Sudan); PKDL lesions self-heal in up to 85% of cases in Africa but rarely lesions L. (L.) major (also known as moist or rural oriental sore) L. (L.) tropica (also known as dry or urban oriental sore) L. (L.) aethiopica L. (L.) infantum=L. (L.) chagasi (rare) L. (L.) donovani, L. (L.) infantum Self-healing in over 50% of cases within 8 or 12 months (different ulcer morphology and scarring/species); L. (L.) infantum healed lesions L.) mexicana (chiclero's ulcer) (Central and South America) L. (L.) amazonensis (Brazil and Amazon Basin) L. (V.) braziliensis (Central and South America) L. (V.) guyanensis (Guyana, Surinam, Amazon basin) L. (V.) peruviana (uta) (Western Andes) L. (V.) panamensis (Central America) L. (V.) colombiensis (Central America) L. (L.) infantum/L. (L.) chagasi (Central and South America) L.) amazonensis (Brazil, Amazon basin) L. (L.) mexicana (Central and South America) L. (L.) aethiopica (East Africa) Slow to heal lepra-like lesions (within 2 to 5 years); DCL and HIV co-V.) braziliensis (espundia) (Central and South America) Other Leishmania (V.) spp. (guyanensis, panamensis) are rare Ulcerating lesions (palpable lymph nodes before and early on in the onset of lesions; Possible self-healing) 2-5% of patients infected by these species develop MCL (31) CHAPTER I -GENERAL INTRODUCTION 9

  Factors that contribute to the spectrum of Leishmania infection and disease are associated to complex interactions between environmental factors, parasite and host-related factors (Figure I.4). The main risk factors for developing human leishmaniasis include environmental or behavioural risks (migration, urbanisation, deforestation, irrigation, lack of bed nets, open houses, house dampness), vector-associated factors (proximity to sand fly breeding sites, proportion of infected vectors, preferred sand fly feeding behaviour), host factors (malnutrition, immune status including HIV infection, other co-infections, age, genetic background), and parasite-associated factors (infecting species, tropism, virulence, co-infections with Leishmania RNA virus) (23,41).

Figure I. 4 3 . 3 . 1 .

 4331 Figure I. 4 Risk factors for the development of active or asymptomatic leishmaniasis(44). DAT, direct agglutination test; SLA, soluble leishmania antigen; ELISA, enzyme-linked immunosorbent assay; CMI, cellularmediated immune response; LST, leishmanin skin test; IGRA, Interferon-gamma release assay.

Figure I. 5

 5 Figure I. 5 Overview of the acute phase of inflammation (64). Tissue damage causes mast cell degranulation in the tissues thereby releasing histamine and chemotactic factors. This increases the expression of adhesion molecules, enabling phagocytic neutrophils to adhere and cross into the tissue. Innate immune responses are triggered first. Neutrophils, guided by chemotactic factors, ingest microorganisms by phagocytosis. Increased permeability allows complement components to enter, generating a variety of antimicrobial and pro-inflammatory molecules. Meanwhile, tissue macrophages ingest any microorganisms, releasing simultaneously inflammatory cytokines. These cause vasodilation, increased permeability and expression of adhesion molecules. Inflammatory cytokines also cause Langerhans cells (not represented) to migrate to draining lymph nodes where they activate T lymphocytes to initiate an adaptive immune response.

  mediated classical macrophage activation (63). LPG interacts with the early inflammatory C-reactive protein, triggering phagocytosis (77) (Figure I. 6). C3b-coated parasites can be internalized by macrophages via the CR1 receptor (complement receptor 1) (73). Promastigotes can also invade macrophages through interactions with host macrophage Fc-γ, mannose or fibronectin receptors (68,74). FcγR-dependent Leishmania phagocytosis is required for clearance and protection from disease (78) (Figure I. 6).

(

  ILC1s) that predominantly express interferon-γ (IFN-γ) and depend on the transcription factor Tbet, associated with Th1 T-cell development (111). NK cells are an important source of IFN-γ in early host parasite interactions, driving TCD4 + cell differentiation into a Th1 phenotype. This secretion can be dependent on IL-2 produced by specific TCD4 + cells and IL-12 secretion by DC, as in the case of L. infantum infection, where NK cell activation depends on IL-12 production and TLR9 expression by myeloid DC, emphasising the complex interactions that will eventually lead to either infection control or progression (61,112). LPG interacts with TLR2 and activates NK cells, triggering IFN-γ and TNF-α production, contributing for host protection in the mouse model, possibly through TLR9 activation (108,113). In L. major infection, NK cells produce IFN-γ, which can amplify IL-12 production by DC and induce Th1 differentiation (protective role). Pathology-associated functions have also been described -NK cells (NKp46+ CD49b+) are recruited to the spleen and hepatic granulomas where they secrete IL-10 and impair protective immunity in experimental VL models (114,115). During L. donovani infection, extensive activation of NK cells induces IL-10 secretion (negative immunoregulatory role of cell-mediated immunity) (114).

  replicate inside host cells without any associated symptoms for about 4 to 5 weeks (116,117). The development of a protective immune response requires the coordinated action of cells of the innate and adaptive immune response (63,117,118) (Figure I.7). The complex early innate immune responses and induced adaptive immune responses are summarised in Figure I.7.

  acquired resistance against Leishmania (120) (Figure I.7). After antigen recognition, naive T cell precursors differentiate into effector cells (priming) (Figure I. 8). Naive TCD8 + cells recognise specific peptides presented by major histocompatibility complex (MHC)-class I molecules on the surface of nucleated cells, and differentiate into cytotoxic CD8 + T lymphocytes which kill infected cells. On the other hand, CD4 + T lymphocytes are activated by APC and differentiate into different effector subsets -T helper 1 (Th1), Th2, Th17 or follicular TFH which activate the target cells; or regulatory T cells (Tregs), which inhibit cell activation (121). Naive T cell activation depends on i) antigen recognition of specific pMHC by the T-cell receptor (TCR); ii) expression and interaction of co-stimulatory molecules and, iii) the cytokine environment which determines the differentiation pathway (121). T cell activation will be further discussed below (3.2 b

  MHC-class II exist only on the surface of APC (B cells, macrophages, DC, epithelial thymus cells), and they present 13-25 amino-acid epitopes to TCD4 + cells, acting against extracellular pathogens as well as inducing differentiation of other T helper phenotypes thereby regulating the immune response. Briefly, endocytosed extracellular antigens or antigens from intravesicular pathogens inside macrophages are degraded in endocytic vesicles and presented by MHC class II molecules to TCD4 + cells resulting in APC activation (123). Soluble antigenic peptides of the correct length and sequence can directly bind to MHC-class I molecules. Although artificial, this direct binding can be exploited in the context of immunoassays, when peptides cannot be presented via endogenous class I presentation (123-125). Exogenous peptides can be presented by MHC class I molecules through cross-presentation (126), and it has been described to occur during Leishmania infection (127,128). In humans, MHC molecules are named Human Leukocyte Antigen (HLA) (Figure I.9).

Figure I. 9

 9 Figure I. 9 HLA class I and class II molecules (©STEMCELL Technologies Inc. 2017).

Figure I. 10

 10 Figure I. 10 Genetic loci in chromosome 6 encoding for all HLA proteins (©STEMCELL Technologies Inc. 2017).

  that have been shown to be alternatively expressed may have the suffix 'L', 'S', or 'Q'. (Figure I.11) (129).

Figure I. 11

 11 Figure I. 11 HLA gene nomenclature (130).

the 9 -

 9 mer peptide anchor residues are often determined by the B and F pockets (peptide's P2 and P9 residues) (124) (Figure I.12). On the other hand, the peptide binding groove of MHC-class II molecules is formed by the α1 and β1 domains and, contrarily to MHC-class I, they are open at both ends, presenting longer peptides of variable length (123). Also, MHC-class II molecules present higher binding pocket variability, which hinders the identification of anchor residues, although four main pockets were identified which interact with peptide's P1, P4, P6 and P9 residues (131) (Figure I.12). The MHC-class II binding pockets accommodate a 9-mer core region which determines binding affinity and specificity (131) (Figure I.12).

Figure I. 12

 12 Figure I. 12 MHC binding cleft and interacting residues along the peptide sequence (124). A) MHC-class I binding groove. B) MHC-class II binding groove.

b)

  Immunological synapse between APC and T cells Naive T cells recognise specific peptide:MHC complexes (pMHC) in the APC surface, in combination with co-stimulatory signals, which triggers T cell proliferation (IL-2 mediated) and differentiation (122). Briefly, naive T cell priming depends on 3 consecutive signals (Figure I.13):

Specifically, macrophages and

  dendritic cells need two signals for activation and differentiation from Th1 cells -IFN-γ signalling, and CD40 ligand (CD40L) interaction on the T cell surface. These signals further increase expression of CD40 and TNF-α receptors, produced by APC themselves, functioning in synergy with IFN-γ to raise and maintain activation (Figure I.13). Secreted or receptor-mediated costimulatory signals are essential for cell activation and for the generation of protective immunity (Figure I.13).

  the differentiation and acquisition of Th1 or Th2 effector functions: naive TCD4+ cells activated in presence of IL-12 and IFN-γ commit to differentiate into Th1 cells, whereas naive TCD4+ cells activated in the presence of IL-4 commit to a Th2 differentiation (Figure I.14). The main factors that influence the differentiation fate are the cytokine environment, the type of APC, the abundance of pMHC and the binding affinity with the TCR receptor. Molecules produced by activated macrophages are toxic to the host as well, so Th1 responses are tightly controlled by Th2 responses (IL-4, TGF-β, IL-10, IL-13) and regulatory T cells (122) (Figure I.14).

Figure I. 14

 14 Figure I. 14 Naive T cell differentiation in Th1 or Th2 effector cells and cross-regulation, adapted from (121). A) Th0 cells differentiate into Th1 or Th2 effector cells according to cytokine environment. B) Th1/Th2/Treg crossregulation.

  TCR expression is clonal, meaning each mature lymphocyte has a single type of receptor of unique specificity (Figure I.15). The overall diversity of T cell clones in an individual comprises at least 10 8 different lymphocyte specificities, providing the ability to recognise virtually all possible epitopes from a given pathogen and the basis for the complexity of the adaptive immune system. Upon antigen recognition, specific T cells become activated, undergo clonal expansion and become effector cells (Figure I.15). However, lymphocyte diversity and the unique TCR specificity per clone also imply that few antigen-specific T cell clones for a given epitope are present, which poses a problem for response detection.

Figure I. 15 T

 15 Figure I. 15 T cell clonal selection model (142). 1) during development, thousands of cell clones bearing unique TCR are generated in central lymphoid organs. Clones that bind self-antigens with high affinity are deleted and the remainder colonise the secondary lymphoid tissues (primary immune repertoire). 2) The introduction of foreign antigen induces activation, proliferation and differentiation of the specific T cell clones.3) a proportion of these antigen-specific T cells differentiates into memory T cells responsible for stronger and faster responses upon re-exposure.

Figure I. 16

 16 Figure I. 16 Characteristics of primary and secondary cellular responses (143). The figure represents the cellular responses (TCD8 lymphocytes) in mice against anInfluenza A virus-specific peptide. Mice were immunized twice intranasally, 8 months apart; rechallenge 8 months after initial challenge induces a faster and greater cytotoxic TCD8 response in the lungs.

  i) L. major infection in mice produces different outcome according to genetic background -BALB/c mice develop a Th2 immune response (IL-4-mediated) and are susceptible; C57BL/6 mice develop a Th1 response (with IFN-γ and IL-12 production) and are resistant (121,154); ii) Leishmania spp. immunomodulation of IL-12 productionthe decreased IL-12 production by infected macrophages, prevents IFN-γ production by NK cells, and Th1 differentiation and function favouring parasite development (121,155,156); iii) Leishmania spp. induces IL-10 production by regulatory T cells, preventing parasite clearance the increased IL-10 downregulates MHC-II expression, TNF-α and NO production, leading to reduced parasite clearance and suppressed activation of Th1 cells (157-159); iv) Passive T cell transfer from resistant C57BL/6 mice to irradiated BALB/c susceptible mice generates protection against L. major infection (160).

  TEM, hence the designation. Current consensus lies in a differentiation lineage between the different T cell subsets, based on signal strength and degree of activation, from precursor to effector functions. TCM, TEM and TSCM all produce IFN-γ, TNF-α and IL-2 but vary in the proportion of cells producing these effector cytokines (191,192) (Figure I.17).

Figure I. 17 T

 17 Figure I. 17 T cell subset populations and differentiation (193). A) Memory TCD8+ cell differentiation. B) Memory TCD4+ cell differentiation.Overall, memory TCD4 + or TCD8 + memory cells in blood circulation rapidly produce IFNγ and IL-2 upon non-specific stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin and can produce multiple cytokines in effector responses. The presence of polyfunctional memory T cells is associated to strong recall and protective responses(191,194).Initial T cell recruitment site following an infection dictates the generation of memory cellsand even a compartmentalization of pathogen-specific memory T cell responses, as observed by studies with canine leishmaniasis models (195), other viral infections(196,197), or Mycobacterium tuberculosis PPD intradermal administration (191) .

(

  DTH) reactions, a form of cell-mediated immunity elicited by an antigen in the skin stimulating sensitized Th1 CD4 and CD8 lymphocytes are methods used to evaluate infected individuals' immune response to Leishmania(55). To detect Leishmania-specific responses, Leishmania antigen (Leishmanin) is injected intradermally in the skin, and induration is measured 48 to 72 hours after (generally indurations>5 mm are considered positive). While active VL patients are generally LSTnegative due to cell anergy, six months after successful chemotherapy, around 80% of patients become LST-positive. Hence, while the LST has little value for diagnostic purposes, it may prove useful to detect asymptomatic infections and/or previous exposure in epidemiological studies.

  to include two subpopulations: i) quiescent non-replicative parasites which can resist host immune responses; and ii) persistent replicative parasites, similar to acute infection parasites, maintained through a numbers game between active parasite replication and immune clearance (215,218) (Figure I.18). HIV-Leishmania co-infection case studies, wherein an underlying Leishmania infection becomes active upon immunosuppression (219-221), and the reactivation of latent infection after administering iNOS or IFN-γ inhibitors in mice (222), further support this model. Also, constant parasite replication and killing help maintain the effector memory

  circulation and in the skin, allowing for a small number of parasites to develop and replicate inside APC and maintain the memory cell populations responsible for protective immunity (53,215) (Figure I. 18). In mouse models, protection against L. major reinfection is associated to parasite persistence mediated by CD4 + CD25 + Treg cells that produce IL-10 (218,225,226).

Figure I. 18

 18 Figure I. 18 Model for the maintenance of concomitant immunity against Leishmania parasites (227).

  infection comes from leishmanization (LZ), the deliberate inoculation with live, virulent L major parasites(229,230). The intradermal inoculation of naive individuals with live promastigotes mimics a natural cutaneous infection, inducing self-resolving lesions, typically smaller than by natural infection, which in turn protect the individual from secondary infection (229,230). The outcomes of LZ reflect the natural host response diversity observed in natural infection, with most of the individuals developing a self-healing lesion and some being able to control infection and never develop a lesion. LZ was used in large scale control programs in endemic countries in Asia and Middle East (namely in Uzbekistan, Iran, Iraq) until the 80's, and has proven to be efficacious against Old World CL (231).LZ-induced protection is associated with parasite persistence and development of concomitant immunity (see 3.4.c). This protection is T cell-mediated: IFN-γ-producing TCD4 + cells are recruited to dermal site of infection, where they perform effector functions including activating microbicidal mechanisms in infected macrophages (232-234).This protection depends on high parasite virulence and infectivity, as low virulence parasites stimulate DTH responses but do not provide protection (174,232). LZ has also been proposed as an assay to measure vaccine candidates' efficacy, as it stands as the gold-standard regarding correlates of protection (231). LZ was abandoned due to logistical and safety issues related to dose control, strain and virulence standardization, and also the occasional development of persistent lesions and associated ethical concerns(229,230). LZ can provide cross-protection against VL and there is further evidence for cross-species protection: i) L. major-infected CL patients seem to be protected against L. infantum infection (235,236); ii) longitudinal studies have shown that humans in L. major endemic areas in Sudan are protected against L. donovani VL (237); iii) a CL-causing L. donovani clinical isolate from Sri Lanka protects mice against visceral disease (238); iv) in experimental VL, C57BL/B6 mice infected with L. major are protected from L. infantum VL, associated with the recruitment of IFN-γ producing Ly6C+ CD4+ cells to skin and visceral organs (162). Also, several parasite-related factors support the development of a vaccine against leishmaniases (239,240): i) No significant antigenic variation is observed between Leishmania spp. life stage forms (detailed in chapter II);

  Thus far, no major breakthroughs in the development of a second-generation vaccine were observed. There are many gaps in the knowledge about immune responses against Leishmania, correlates of protection and even knowledge on the number of natural challenges after the first inoculation. From 11 published studies testing first generation vaccines (fractions of the parasite or whole killed Leishmania with or without adjuvants), only 4 showed a decrease in human infection, measured by LST or Montenegro skin test (MST) seroconversion, highlighting the need for better methods to assess of T-cell mediated responses and correlates of protection (241).

  screen potential candidates designed for human vaccine formulations; -Protective responses against Leishmania are characterised by mixed Th1/Th2 responses with predominant IFN-γ and low IL-10; -Memory T cells (CD45RO+) present in healed individuals are expected to proliferate and acquire effector functions after vaccine antigen stimulation; -Vaccine candidates should induce Th1 cellular responses, and respective associated cytokines, maintaining the balance high IFN-γ/IL-12 and low IL-10/IL-4; -Cell surface phenotype should be performed to identify the cell populations involved in the induced responses; -It is advisable to include a complete panel of induced cytokines (Th1, Th2, Th17 and Treg) to assess polyfunctionality and to better elucidate the immune mechanisms induced by the vaccine candidates.

  vaccine to progress to human phase I and II clinical trials, safety and immunogenicity testing in healthy subjects (265). Leish-111f consists of a chimeric recombinant protein containing three antigens in tandem (Leishmania elongation initiation factor, LeIF; L. major stress-inducible protein, LmSTI1; and thiol-specific antioxidant, TSA), combined with monophosphoryl lipid A-stable emulsion (MPL-SE), and induces a potent Th1-type immune response (265). This formulation was tested in five clinical trials: i) Phase I trials in 2005, "Study to Evaluate the Leish-111f + MPL-SE Vaccine in Healthy Adults Not Previously Exposed to Leishmania Parasite" and "Safety Study to Evaluate the Leish-111f + MPL-SE Vaccine in the Prevention of Cutaneous Leishmaniasis in Healthy Subjects Previously Exposed to the Leishmania Parasite" in Colombia (ClinicalTrials.gov Identifiers: NCT00121862 and NCT00121849, respectively); ii) Phase I trials in 2005-2007, "Study to Evaluate the Leish-111F + MPL-SE Vaccine in the Treatment of Mucosal Leishmaniasis" in Peru, and "Study to Evaluate the Leish-111F + MPL-SE Vaccine in the Treatment of Cutaneous Leishmaniasis" in Brazil (ClinicalTrials.gov Identifiers: NCT00111514 and NCT00111553, respectively); iii) Phase I trial in 2005-2006, "Safety Study to Evaluate the Leish-111f + MPL-SE Vaccine in the Prevention of Cutaneous Leishmaniasis in Healthy Subjects Previously Exposed to the Leishmania Parasite" (ClinicalTrials.gov Identifier: NCT00121849); iv) Phase I trial in 2007-2015, "Open-Label Safety Study of Three-Antigen Leishmania Polyprotein with Adjuvant MPL-SE in Healthy Adults in India" (ClinicalTrials.gov Identifier NCT00486382).

  Infectious Disease Research Institute (IDRI) redesigned Leish-111f into a new construct Leish-110f (also named LEISH-F2), to better comply with regulatory and manufacture concernsthe 6-Histidine tag near the amino terminus was removed, and the residue Lys274 was replaced by glutamine, so to eliminate an apparent proteolytic site (230,268). The safety, immunogenicity and efficacy of the LEISH-F2+MPL-SE candidate were tested in a phase I clinical trial with promising results, and in a phase II trial to assess its immunotherapeutic potential against CL in comparison with standard chemotherapy with SSG (showing very limited success): i) Interventional Phase I trial in 2009-2011, "Safety and Immunogenicity of the LEISH-F2 + MPL-SE Vaccine with sodium stibogluconate (SSG) for Patients with PKDL" in Sudan (ClinicalTrials.gov Identifier NCT00982774); ii) Interventional Phase II trials in 2009-2013, "A Study of the Efficacy and Safety of the LEISH-F2 + MPL-SE Vaccine for Treatment of Cutaneous Leishmaniasis" in Peru (ClinicalTrials.gov Identifier NCT01011309). A third vaccine candidate was developed by IDRI, LEISH-F3. From previously identified 43 antigen candidates recognised by VL patients' serum from Sudan, researchers applied rational criteria (sequence conservation among species, low homology with human proteins, and proved efficacy in animal models) and selected two proteins -L. infantum/donovani nonspecific nucleoside hydrolase (NH) and sterol 24-c-methyltransferase (SMT). These proteins were combined to form the recombinant protein LEISH-F3 and adjuvanted with either MPL-SE, glucopyranosyl lipid A formulated as stable emulsion (GLA-SE), or second generation lipid adjuvant stable emulsion (SLA-SE) (269). The LEISH-F3 vaccine candidate was tested in three clinical trials to assess safety and immunogenicity of the different formulations (270): i) Interventional Phase I trial in 2011-2013, "Phase 1 LEISH-F3 Vaccine Trial in Healthy Adult Volunteers" in USA (ClinicalTrials.gov Identifier NCT01484548); ii) Interventional Phase I trial in 2012-2016, "LEISH-F3 + GLA-SE and the LEISH-F3 + MPL-SE Vaccine" in USA (ClinicalTrials.gov Identifier NCT01751048); iii) Interventional Phase I trial in 2014-2016, "Phase 1 LEISH-F3 + SLA-SE Vaccine Trial in Healthy Adult Volunteers" in USA (ClinicalTrials.gov Identifier NCT02071758).

  strong and specific immune responses, measured by cytokine production and immunoglobulin subclass information. This candidate should, therefore, be tested in additional clinical trials in endemic countries, in populations susceptible to Leishmania infection (269). Another promising candidate in the pipeline is the ChAd63-KH vaccine candidate, also named Leish2a, developed by researchers at York University. It consists of a chimpanzee adenovirus-based vaccine (ChAd63) carrying a synthetic HASPB gene linked to a KMP11 gene with a viral 2A sequence (272). This candidate progressed to a Phase I clinical trial where the safety profile was confirmed, and strong TCD8 + cellular responses were observed (273). The Leish2a vaccine was also assessed in a Phase II clinical trial in Sudan to test safety and induced immune responses in individuals with persistent PKDL infection (ClinicalTrials.gov Identifier: NCT02894008, "A Study of a New Leishmania Vaccine Candidate ChAd63-KH (Leish2a)"). In earlier stages of development, there is also a new candidate developed by the European FP7 network MuLeVaClin, which is based on defined Leishmania antigens KMP11 and LeishF3+, and sand fly saliva antigen LJL143, formulated into Influenza virus-like particles (VLP) and adjuvanted with GLA-SE, a TLR-4 agonist (274). Pre-clinical studies in the mouse model show this candidate is safe and immunogenic, particularly against the vector-derived antigen (274). Furthermore, other polypeptidic/chimeric vaccines against leishmaniasis are in pre-clinical stages of development, and were validated in human samples. The protein Nucleoside Hydrolase NH36 is the main component of the Leishmune® vaccine, once commercialised in Brazil. The immunogenicity of three NH36 polypeptides (N-terminal, central, C-terminal domains) was tested pre-clinically with samples from cured CL and VL patients, from areas endemic to L. infantum in Spain (275). Recombinant NH36 and the N-terminal domain (F1) induced lymphoproliferation in samples from cured and asymptomatic individuals, accompanied by increased IFN-γ, TNF-α and granzymeB production. Also, F1 induced IL-17 production cured CL patients and asymptomatic patients. Additional studies were performed with samples from healed and asymptomatic individuals, from areas endemic to L. braziliensis in Brazil (276). Here, the F1 domain induced Th1 and Th17 responses in cured/exposed patients infected with L. (L.) infantum (chagasi). The polyprotein KSAC includes fragments of the proteins KMP11, SMT (24-cmethyltransferase), A2 (A2 amastigote-specific protein), and CPB (cysteine protease B) (277). KSAC formulated with monophosphoryl lipid A (MPL-SE) induced antigen-specific multifunctional Th1 cells and conferred protection against L. infantum and L. major challenge in mice. Post-challenge responses in the KSAC/MPL-SE-vaccinated mice show higher IFN-γ/IL-4 ratios and decreased IgG1 responses to SLA (268,277).

Leishmania

  excreted-secreted proteins (ESP or ESA) are important virulence factors, and are implicated in early host-parasite interactions, establishment of infection and in the regulation of host immune responses (279-281). Their role in the modulation and evasion of the host immune responses is reviewed in Holzmuller et al 2018 (2).

  Additionally, ESP have been shown to be processed and presented by antigen-presenting cells (127), as well as, to trigger protective cellular immune responses (282-285). Gour et al described the fractions F1 (11, 13 and 16 KDa) and F3 (26, 29 and 33 KDa) from Leishmania ESA, which contained Th1-inducing proteins (283). Interestingly, several of the antigens used in the most advanced vaccine candidates are found in the Leishmania secretome, such as KMP11, nucleoside hydrolase, thiolspecific antioxidant (TSA) or translation initiation factors (detailed in chapter III -Vaccine Antigen Selection).The Lemesre lab (UMR177, IRD, France) developed a method for the production of naturally excreted-secreted antigens from culture supernatants of L. infantum promastigotes, and evaluated their potential use in a vaccine formulation. Notably, LiESAp were shown to provide significant protection in dogs by inducing a strong and long-lasting Th1 response, leading to the development of the european canine vaccine Canileish ® (284,286). The adaptation and scaling-up of this vaccine for human use is impossible due to elevated costs, and because the active principle remains undefined, although likely to contain a plethora of different protein antigens, which may induce non-specific responses and/or response variability. Also, this vaccine is formulated with QA-21 (purified extract of Quillaja saponaria) as adjuvant, which is currently not approved for human use. However, QA-21 is derived from the QS-21 adjuvant which has been approved for human use by the FDA.

  Potential peptides are tested for immunogenicity in mice or human PBMC. Most immunogenicity testing of candidate proteins and peptides in leishmaniasis vaccine development have been performed in mouse models. Although these models provided important insights into Leishmania pathogenesis and host immunity, they are inconclusive in terms of vaccine antigen design.Many vaccine candidates under development fail in early stages of clinical development probably dueto weak antigenicity and lack of predictive animal models. Promising candidates against leishmaniasis developed with animal studies often were unsuccessful in human trials (297,298). The failure of protective vaccines in human trials may be because of differences in peptide processing (TAP binding) and presentation between murine H-2 and human HLA systems. In which case, humanized animal models may provide a useful tool for vaccine preclinical development.Still, some peptide vaccine candidates against Leishmania were tested in human PBMC. These studies used mostly samples from LST+ or healed patients, and lymphoproliferation and cytokine production by ELISA as readout. Briefly, studies testing Leishmania-specific peptide immunogenicity in human immune cells are: i) Russo 1993 (299): peptides from L.major GP63 (13 peptides with 14 a.a.) (300), three of which increased PBMC proliferation, and IFN-γ production. Epitope prediction was performed with an algorithm developed by Rothbard and Taylor (301) based on sequence motifs based on known human, mouse and guinea pig epitopes. ii) Delgado G 2003 (302): peptides from L. panamensis KMP11 (6 peptides with 20 a.a. spanning the whole protein sequence) induced lymphoproliferation and IFN-γ production by T-cells. Authors assess peptide binding to HLA molecules with binding assays performed with four purified HLA-DR molecules (DRB1* 0101, 0401, 0701 and 1101). Donors were HLA-typed (PCR to amplify the exon 2 of the DRB gene) and the correlation between in vitro immune response and peptide binding assay predictions was ascertained. iii) Basu R 2007 (303): peptides from L. donovani KMP11 (84 octamers spanning the whole protein sequence), some of which induced IFN-γ, IL-10 and IL-17 production by TCD8 + cells and increased splenocyte proliferation. Authors propose potential HLA restriction according to the experimental data and donor HLA typing (HLA-A, -B and -C alleles). iv) Seyed N 2011 (304): peptide pools from L. major antigens (18 nonamers from CPB, CPC, TSA, LeIF, LmSTI, LPG) increased IFN-γ production by TCD8 + cells. The six protein antigens were examined for epitopes restricted to the HLA-A*0201 allele using in silico HLA-binding prediction algorithms (see Chapter IV). Authors used a 2-step epitope selection pipeline using 2 prediction algorithms (SYFPEITHI and BIMAS) and 5 five other algorithms (EpiJen, Rankpep, nHLApred, NetCTL and Multipred) which further filtered the peptide list and, finally, analysed with NetMHCpan1.1 to check for the possibility of binding to different alleles of HLA-A2 supertype (HLA-A*0202-A*0206 and A*0209).

  v)Elfaki ME 2012 (305): peptides from L. donovani GP63 (4 peptides with 15-21 a.a.), two of which increased IL-10 production, while another peptide and peptide pools decreased IL-10 production, with no significant IL-4 production. Epitope prediction was performed with EpiMatrix (www.immunome.org/iVAX/) for alleles DRB1*1101 and DRB1*0804 and a panel of 8 HLA-DR "supertype" alleles (> 90% of human populations worldwide).Peptide hydrophobicity analysis and BLAST against the human genome were performed. vi) Naouar I 2016 (306): peptides from L. major antigens (78 nonamers from 33 proteins), six of which induced cytotoxicity, assessed by granzyme B production, but low IFN-γ and IL-10 levels were found in PBMC culture supernatants. Epitope prediction was performed for the HLA-A*0201 allele and proteasomal cleavage prediction (RANKPEP algorithm, imed.med.ucm.es/Tools/rankpep.HTML). Affinity to HLA-0201 alleles was assessed with stabilisation assays. vii) Freitas e Silva R 2016 (307): ten peptides (15-mer) conserved in L. braziliensis, L. major and L. infantum discovered with a reverse vaccinology approach using proteomic datasets, five of which induced PBMC proliferation. Linear epitope prediction was performed for 9 HLA-class I supertypes (HLA-A1, -A2, -A3, -A24, -A26, -B7, -B27, -B44 and -B58) and for 4 HLA-class II supertypes (HLA-DPA, HLA-DPB, HLA-DQA, HLA-DRB). The algorithms NETMHC and NETCTL were used to predict HLA-class I-binding epitopes, and NETMHC Class II was used for a MHC II prediction. Additionally, the linear peptides were structurally modelled to MHC molecules to find the most stable in silico epitope + allele complexes. Structures from 33 different alleles of MHC I (21) and MHC II (12) were used. viii) Mahantesh V 2017 (308): peptides from L. donovani 3'-nucleotidase (5 nonamers) induced IFN-γ and IL-2 production, increased T-cell proliferation in PBMC culture and CTL activity. Epitope prediction was performed for HLA-A*02 and HLA-B40 alleles with six HLA-binding prediction algorithms (SYFPEITHI, BIMAS, RankPep, NetCTL1.2, IEDB tools.iedb.org, and ProPredI). In the last epitope selection step, the selected peptides were analysed by NetMHCpan3.4 to check the binding affinity to other HLA class I alleles, and promiscuous epitopes that bind to at least 10 HLA-A*02 or 5 HLA-B40 were selected.

  Network EUROLEISH.net (project 2.1 -Development of a multiepitope peptide-based vaccine against human leishmaniasis). Project 2.1 is integrated within the 15 EUROLEISH.net projects as a basic sciences project focused on leishmaniasis prevention. The project's main objective is the development of a human-compatible second-generation peptide-based vaccine against leishmaniasis, with global coverage, and based on immunogenic epitopes conserved among several pathogenic Leishmania species.

  In summary, the principal Leishmania species causing cutaneous leishmaniasis (CL) in the Mediterranean Basin, Africa, Middle East, and India are L. (L.) major (zoonotic CL, ZCL), L. (L.) tropica (anthroponotic CL); whereas in Central and South America, the main CL-causing species are L. (L.) mexicana, L. (L.) amazonensis, and L. (V.) braziliensis (1). VL is caused by L. (L.) donovani parasites in the Indian and African regions, and L. (L.) infantum parasites in the Mediterranean Basin, Central and South America and China (1-3). Other Leishmania species infect non-human vertebrate hosts, as is the case of L. tarentolae whose natural host is the lizard (4). For pathogenesis and epidemiological aspects see Chapter I -General Introduction.

Furthermore, other than

  virulence factors necessary for parasite survival, many interesting molecules have been identified in the secretome, namely drug and vaccine candidates. As referred in Chapter I -General Introduction, Leishmania secreted antigens have been shown to trigger Th1 responses in several disease models (39,50-52) and several antigen candidates in the human vaccine CHAPTER II -THE LEISHMANIA SECRETOME 89 development pipeline are known to be secreted. However, the total content of the naturally secreted LiESAp, the active principle of the CaniLeish® canine vaccine, remains largely undefined. The development of a second-generation vaccine based on antigens present in the Leishmania secretome requires the comprehensive characterisation of the antigenic proteins responsible for the observed immune responses. The present analysis aims to fill this knowledge gap, to identify all the proteins present the L. infantum secretome produced in aseric conditions. Furthermore, we provide the first simultaneous analysis of five other major pathogenic Leishmania.

  Figure II. 2 General workflow for the mass spectrometry analysis of the Leishmania promastigote secretome.

  L. infantum, L. mexicana, L. major and L. braziliensis were retrieved (step 6) (Figure II.3 panel A). In parallel, 2140 accessions identified in the secretome proteomic datasets were converted to GeneDB IDs in the UNIProtKB database web server. 1860 out of 2140 identifiers from UniProtKB accessions identified in the secretome were successfully mapped to 1988 GeneDB Ids. This list was then compared (list intersection) with the 72 Leishmania ortholog exosome marker genes in TriTrypDB (Figure II.3 panel B).

  II.4 panel A). A total of2140 unique proteins were identified (UniProtKB database release 2017_01), between 909 and 1357 total accessions per sample (Figure II.4 Panel B), considering all identification categories defined by the bioinformatics identification script. Amongst the identified proteins, only a portion correspond to characterised proteins with SwissProt annotation (2.8%). Remaining proteins correspond to transcripts with an in silico characterisation in the TrEMBL database.

Figure II. 4

 4 Figure II. 4 Leishmania secretome protein identifications. A) Synthetic images of one-dimensional gel electrophoresis of total secretome of different Leishmania species. B) Total number of protein accessions identified.Total number of accessions identified per sample, according to the bioinformatic script: 'Leading_AUTO' (accessions unique to the tested species), 'Leading_SPECIES' (ambiguous identifications assigned according to tested species), and 'Multi_species' (ambiguous identifications, impossible to distinguish between species).

  All six human pathogenic species analysed share a high number of common accessions (326), corresponding to 24% up to35.9% of total identifications per sample (Figure II.5 panel A). The number of commonly identified proteins decreases to 306 accessions when considering all seven Leishmania species, meaning 20 proteins were not identified in the L. tarentolae sample. The 20 accessions identified only in human pathogenic species are: beta-fructofuranosidaselike protein (EC 3.2.1.26); chaperonin HSP60, mitochondrial; cystathionine beta-synthase (EC 4.2.1.22); putative cytochrome c; GDP-mannose pyrophosphorylase (EC 2.7.7.22); GDPMP protein (EC 2.7.7.13); mannose-1-phosphate guanyltransferase (EC 2.7.7.13); paraflagellar_rod_component_-_putative; putative 2-hydroxy-3-oxopropionate reductase (EC 1.1.1.60); putative ribosomal protein S6; putative small GTP-binding protein Rab1; S-methyl-5'-thioadenosine phosphorylase (EC 2.4.2.28) (MTA phosphorylase); superoxide dismutase (EC 1.15.1.1); three uncharacterised proteins (A4HIL9; E9AZC3; E9B159) and one hypothetical conserved protein (A4I5W4). As expected, the L. braziliensis and L. major samples, two species with reference proteomes in UniProtKb (LEIBR and LEIMA), have the highest number of specific proteins (Figure II.2 panel B). Interestingly, LEIAM and LEITA, although underrepresented in the databases, show a high number of specific proteins, respectively, 66 and 63 accessions. Also, shared proteins between LEIIN and LEIDO samples are more numerous than LEIIN-specific proteins (Figure II.2 panel B).

  42% of proteins are predicted to be secreted through non-classical pathways for all Leishmania species analysed. 'Leaderless' proteins are extracellularly active despite not having a signal peptide or a transmembrane domain. So far, four non-conventional mechanisms have been described in eukaryotes: i) pore-mediated translocation (type I); ii) ABC transporter-based secretion (type II or membrane flip-flop) of acylated proteins; iii) organelle-based translocation or autophagosome/endosome-based secretion (type III) by diverting endosomal and other membranebound compartments from their normal function to become secretory (60). The fourth unconventional protein secretion mechanism occurs when proteins containing signal peptides or transmembrane domains bypass the Golgi and are still secreted (type IV) (60). Interestingly, the type II membrane flip-flop mechanism (membrane translocation through N-terminus dual acylation)

Figure II. 6

 6 Figure II. 6 Secretion pathway analysis. Total accessions identified in each sample were analysed with SecretomeP 1.0 (mammalian proteins). Percentage of proteins with NN score > 0,5 and no predicted signal peptide are predicted to be non-classically secreted (blue bars). SignalP analysis predicts the presence of signal peptides which target proteins for classical secretion (percentage of total proteins, red bars).

  three main domains which represent how individual genes contribute to organism biology: Molecular function, the molecular activities of individual gene products; Cellular component, where the gene products are active; and Biological process, the pathways to which the gene product's activities contribute(62). Results show secreted proteins by all seven Leishmania species analysed have conserved functions, across all GO domains (Figure II.7).In all analysed Leishmania species, the vast majority of secreted Leishmania proteins possess, regarding molecular function pathways, either binding (GO:0005488, refers to the selective, non-covalent, often stoichiometric, interaction of a molecule with one or more specific sites on another molecule) or catalytic activity functions (GO:0003824, refers to the catalysis of a biochemical reaction at physiological temperatures; gene products possess specific binding sites for substrates, and are usually composed wholly or largely of protein). The remaining molecular functions identified are structural molecule activity (GO:0005198, refers to the action of a molecule that contributes to the structural integrity of a complex or its assembly within or outside a cell) or antioxidant activity (GO:0016209, refers to the inhibition of the reactions brought about by dioxygen (O2) or peroxides; usually the antioxidant is effective because it can itself be more easily oxidized than the substance protected) (Figure II.7 panel A). These results agree entirely with previous proteomic analysis of L. donovani and L. braziliensis secretomes(41,43).Regarding the biological process domain, a high number of proteins are involved in cellular (GO:0009987, refers to any process that is carried out at the cellular level, but not necessarily restricted to a single cell, for example, cell communication) or metabolic processes (GO:0008152, refers to the chemical reactions and pathways, including anabolism and catabolism, by which living organisms transform chemical substances), regulation (GO:0065007, refers to any process that modulates a measurable attribute of any biological process, quality or function.) and response to stimulus (GO:0050896, refers to Any process that results in a change in state or activity of a cell or an organism as a result of a stimulus in terms of movement, secretion, enzyme production, gene expression, etc.) (Figure II.7 panel B). Cellular compartment GO annotations are more disperse, but still conserved among Leishmania species (Figure II.7 panel C). Most gene products are described to be active in the cytoplasm (GO:0005737, refers to all of the contents of a cell excluding the plasma membrane and nucleus, but including other subcellular structures) and nucleus (GO:0005634, refers to membrane-bounded organelle of eukaryotic cells in which chromosomes are housed and replicated). The main gene products which possess these functions are: tubulin, elongation factors, proteasome-associated proteins and histones.

3. 4 .

 4 Several important virulent factors are found among the most abundant proteins (iBAQ analysis)The analysis of the proteomic data (UniProtKB database release 2017_01) resulted in a total of 2140 accessions identified, from all identification status (Leading_AUTO, Leading_SPECIES, Multispecies = LEADING_CHECK). A normalised iBAQ value of 1 corresponds to the overall iBAQ average (absolute iBAQ average 1,81x10 7 ; Log10 iBAQ average 6,195). 582 accessions with normalised iBAQ values over 1,1 were considered as abundant (see Appendix II.1). The most abundant protein overall is nucleoside diphosphate kinase, the only identified accession with a normalised iBAQ value over 1,5 (Figure II.8). This enzyme is required for the synthesis of nucleoside triphosphates (other than ATP) and catalyses the reaction transforming ATP and nucleoside diphosphate in ADP and nucleoside triphosphate. The generated nucleoside triphosphates are then used for nucleic acid, lipid or polyssacharide synthesis, protein elongation, signal transduction and microtubule polymerisation. It is likely this protein possesses additional moonlighting functions such as protein binding, similarly to other kinases described in the MoonProt database. Several important well-documented virulence factors were identified in the Leishmania promastigote secretome. Leishmania parasites secrete virulence factors to the host cell cytoplasm where they actively modulate host signalling molecules and immune responses (33,65). There are fourteen highly abundant proteins in the secretome, with normalised iBAQ values over 1,4, including well-known virulence factors (Figure II.8, dark orange). By descending order: soluble promastigote surface antigen PSA-38S, tubulin beta chain (2 accessions), tubulin alpha chain, putative small myristoylated protein-3, ubiquitin-60S ribosomal protein L40, an uncharacterized protein, infective insect stage-specific protein, peroxidoxin 2, soluble promastigote surface antigen PSA-34S (Fragment), adenosylhomocysteinase (2 accessions), putative heat-shock protein hsp70, and elongation factor 1-alpha (Appendix II.1).

  exosome cargo. Indeed, among the most abundant proteins identified (normalised iBAQ value over 1.4) the proteins tubulin (alpha and beta chains), heat-shock protein 70 and surface antigens are found. The high abundance of these proteins further supports the importance of exosomebased secretion in Leishmania parasites.

Figure II. 8

 8 Figure II. 8 Log-transformed iBAQ values normalised to overall average. The iBAQ values from 2140 accessions identified in the Leishmania secretome were analysed. A normalised iBAQ value superior to 1,1 is associated to abundant proteins.

  negatively affects protein secretion. The lack of duplicates for each sample was due to cost constraints and it can pose some limitations to the present study. However, previous experiments from the development of the canine vaccine show limited variability between secretome batches (vaccine quality control, unpublished data).The present study focuses on the Leishmania promastigote secretome, a critical player in early stage host-pathogen interactions. Leishmania parasites do undergo differentiation to amastigotes inside the vertebrate host, however, previous studies show low levels of differential gene expression, with a big role for post-translational regulation of the stage-specific gene expression and survival in the intracellular environment. As gene and protein annotation improve, so will our understanding of all players involved in host-parasite interactions. The key to potentially discover targets or markers and develop the much-needed improved treatments and diagnostics.

  Leishmania species (UniProtKB release 2016_05) (Figure III.1 panel A).All 618 protein sequences were submitted to BLASTp analysis against the human .nlm.nih.gov/genomes/H_sapiens/protein/). This analysis was performed using the standalone BLAST tool for Windows™, with imported search parameters from the online version, to keep standard alignment parameters (Figure III.1 panel B). Briefly, to import the default BLASTp search strategy, one random accession was run on the BLASTp online tool, against Human RefSeq proteins, with standard algorithm parameters. The search strategy was saved to a local directory as an ".asn" file and used in the standalone BLAST analysis (Figure III.1 panel B).

  outfmt=6), containing the columns (Figure III.1 panel B): query sequence name (qseqid), length (qlen), sequence (qseq); subject sequence name (sseqid), alignment length (length), start of alignment in query (qstart), end of alignment in query (qend), expect value (evalue), bit score (bitscore), percentage of identical matches (pident), number of identical matches (nident), percentage of positive-scoring matches (ppos), number of positive-scoring matches (positive).

Figure III. 1 A

 1 Figure III. 1 A reverse vaccinology approach to find novel excreted-secreted Leishmania antigen candidates. A) Venn diagram comparing total identified proteins from 6 Leishmania species -L. infantum (LEIIN), L. major (LEIMA), L. donovani (LEIDB), L. braziliensis (LEIBR), L. tropica (LEITR) and L. amazonensis (LEIAM) showing 618 accessions were identified in all samples. B) Standalone blastp code (MS-DOS) used to blast 618 common accessions against the human proteome (RefSeq proteome) using default parameters from the online BLASTp server.

HSP70Heat-

  shock proteins (HSP) were one of the first proposed antigen candidates. These proteins are encoded by a single copy gene, and the temperature shift the parasites undergo increases HSP expression, to adapt to survival in the vertebrate host. HSP90 is the most abundant form, with 17 tandem gene copies. HSP70 has been identified has the immunodominant antigen in antibody responses against Leishmania and has shown to stimulate DC maturation and cytokine production. HSP90 and HSP70 are B-

  Alcolea P. et al 2010. Transcriptomics throughout the life cycle of Leishmania infantum: high down-regulation rate in the amastigote stage. Int J Parasitol. 40(13):1497-516 (8). associated GO terms) Flagellar glycoprotein-like protein (FPG) 1, 3 integral component of membrane [GO:0016021]; motile cilium [GO:0031514] Trypanothione reductase 1 -25,7 +-1,4 cell [GO:0005623]; disulfide oxidoreductase activity [GO:0015036]; flavin adenine dinucleotide binding [GO:0050660]; trypanothione-disulfide reductase activity [GO:0015042]; cell redox homeostasis [GO:0045454] GO:0004601]; peroxiredoxin activity [GO:0051920] LACK (leishmania analogue of the kinase receptor C) 1 -5 -ribosome [GO:0005840]; kinase activity [GO:0016301]; regulation of cytokinesis [GO:0032465]

(10 3 )

 3 to 3526300000(10 9 ). Nine protein accessions have iBAQ values of 0, including 4 uncharacterized proteins, Calpain-like cysteine peptidase (Fragment), Putative ubiquitin conjugation factor E4 B; Tubulin binding cofactor A-like protein; Serine/threonine-protein phosphatase and DNA-directed RNA polymerase subunit beta. The null iBAQ values imply the detection of specific tryptic peptides but with very low intensity, still, these values were included in the analysis. After normalisation, iBAQ values range from 0,537 to 1,508, and proteins with normalised iBAQ values over 1 can be considered abundant in the secretome.

  The most abundant proteins in set B have normalised iBAQ values above 1,35 (putative aldolase; putative beta-fructofuranosidase; putative small myristoylated protein-1). The least abundant proteins in the set B, with normalised iBAQ values under 0,8, are a putative eukaryotic translation initiation factor 3 and glutamate dehydrogenase.

Figure III. 3

 3 Figure III. 3 Estimation of selected protein antigens' abundance with normalised iBAQ values.

  analysis (Figure III.4).

Figure III. 4

 4 Figure III. 4 Overall protein antigen selection results from the secretome proteomic data.

  Kinetics of transcription and translation; Timing of CTL clonal expansion; Antigen processing and transport; T cell precursor frequency; Antigen abundance; TCR repertoire; Affinity to MHC molecules; Signal strength; Stability and turnover of pMHC complexes; TCR affinity and avidity; Type of APCs Proliferative capacity; Intrinsic ability to respond; Pre-conditioning or priming; Dwell time on APCs; Elimination of APCs; Competition for resources (cytokines, antigens or physical niche). On the other hand, immunoprevalent T-cell epitopes are frequently immunogenic in the context of multiple MHC alleles. These epitopes can induce specific IFN-γ responses with high responding T-cell frequency within the repertoire and are common across individuals with different

HLA genes and official

  sequences named by the WHO Nomenclature Committee for Factors of the HLA System (as of April 2019, release 3.36.0 contains 16,200 HLA Class I Alleles, 6,162 HLA Class II Alleles and 186 other non-HLA Alleles).

To the 28

 28 alleles included in the IEDB HLA-class I reference list, 8 more were included, for a total of 36 alleles included in the HLA-binding prediction analysis. The IEDB reference stipulates epitopes with variable length, with 8 up to 11 amino-acid long epitopes. However, in the present study only 9-mer epitopes were searched. The HLA-class I alleles selected are: IEDB MHC-I binding predictions for 36 alleles (11 supertypes), SYFPEITHI predictions for 22 alleles (11 supertypes); and NetMHCpan predictions for 11 alleles (11 supertype representatives) (Table

  eight common DR alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501) cover around 97% of human populations worldwide (48).

--

  Tung CW. Chapter 6 -Databases for T-Cell Epitopes. Immunoinformatics 2014, 2nd Ed (9); -Desai SV, Kulkarni-Kale. Chapter 19 -T-Cell Epitope Prediction Methods: An Overview. Immunoinformatics 2014, 2nd Ed (20); -Wang M, Mogens MH. Chapter 17 -Classification of Human Leukocyte Antigen (HLA) Supertypes; Algorithm benchmarking studies -Lin HH et al 2008 (24), Wang P et al 2008 (25), Peters B et al 2006 (26), Trolle T et al 2015 (27); -Castelli M et al 2013 Clin Transl Immunology ID:521231 (29).

  Figure IV.1 panel A). Briefly, the selection pipeline allows the integration of HLAbinding prediction data from multiple algorithms, and a first selection of conserved peptides (100% conserved among the six Leishmania species tested) and predicted by at least 2 non-redundant algorithms. After this first selection step, short-BLASTp results are added (position-specific total mismatches and/or anchor position mismatches compared to host proteins). Briefly, in the BLASTp online server, the human RefSeq proteins were used as the host proteome (Homo sapiens Taxid:9606). The corresponding .txt result file was downloaded to the working directory and renamed "alignment_I.txt" or "alignment_II.txt" (Figure IV.1 panel A). The result file generated by the selection script after these 2 selection steps was further analysed with the help of spreadsheet analysis software Excel ® (Figure IV.1 panel B).

  function 'UniqueFromCell' -Figure IV.2 panel A) and used to duplicate peptide information (Excel macro 'Splt' -Figure IV.2 panel B), so to generate a filterable table per supertype.

Figure IV. 2

 2 Figure IV. 2 Excel ® software code (VBA code) for analysis of final peptide list, from the selection script in R. A)VBA code for the creation of the 'UniqueFromCell' function, that identifies common HLA restriction between IEDB and NetMHCpan predictions. B) VBA code for the 'Splt' macro that duplicates peptide rows according to predicted (consensual) promiscuity.

  human total PBMC were seeded per well in 96-well plates and incubated overnight at 37°C, 5% CO2. Individual peptides were added at 3 different concentrations in triplicate wells (1 or 5, 10 and 25 µM). After 4 hours, 10% final PrestoBlue® (Life Technologies, Switzerland) was added to each well and the plates were returned to the incubator for 16 hours. The fluorescence intensity (bottom-read) was measured using a multiwell plate reader (EnVision 2105 Multimode Plate Reader, Perkin Elmer) excitation 560 CHAPTER IV -IN SILICO EPITOPE DISCOVERY AND SELECTION 161 nm, emission 595 nm. 560 nm absorbance values were normalized to the 595 nm values for the experimental wells.

Figure IV. 3 3 . 2 .

 332 Figure IV. 3 Summary of selection filters and list reduction for HLA-class I and -class II binding predictions.

  contains a total of 538,374 peptidic epitopes, which include only 965 Leishmania epitopes (IEDB search: any epitopes + Leishmania ID 5658, February 2019 annotation). Of these, only 555 are T-cell epitopes, and if we consider positive assays only this number decreases to 379, corresponding to less than 1% of total 448402 T-cell epitopes with positive results in the database.

Figure IV. 7

 7 Figure IV. 7 Current challenges affecting reverse vaccinology approaches for vaccine design.

  the first study, in 2009, Herrera-Najera et al analysed the complete L. major proteome (8272 annotated proteins) and performed class I epitope predictions for mouse alleles. The first epitope prediction round used the algorithm RANKPEP based on position-specific score matrices (PSSM), subsequently, proteins with top scoring peptides were re-analysed using multiple HLAbinding prediction algorithms to generate consensus predictions (SYFPEITHI, BIMAS, ProPred-I, and MAPPP which are PSSM-based; ANNPred; SVMHC; ComPred which is combines ANN and QM; and

( 1 )

 1 . Researchers may seek unwanted immunogenic regions within therapeutic molecules, for instance, therapeutic antibodies, or, on the other hand, immunogenic epitopes to induce specific protective immune responses through vaccination or immunotherapy. In either case, immunoassays must be performed, during clinical development and post-marketing surveillance, to identify and assess the ability of a given molecule to induce humoral or cellular immune responses in exposed or naive individuals. The mechanisms of immunogenicity include T cell-dependent and T cell-independent responses. In the context of T-cell dependent immune responses, T-cell epitope mapping can be performed initially through in silico methods, subsequently validated with in vitro and in vivo assays, to describe Th1, Th2, cytotoxic, or regulatory epitopes. The most common marker associated with Th1-cell activation is IFN-γ, however, a maximum number of parameters should be included in the characterisation of induced responses. There are currently several methods available to assess immunogenicity and T-cell responses in vitro, summarised in Figure V.1. Importantly, several tools exist to assess the magnitude and breadth of specific responses, as well as to describe cell function according to cytokine production or cell phenotype.

  ) (Figure V.1). High-or medium-throughput approaches allow for the reduction of the number of cells needed and overall experiment costs(6).Although in vitro lymphocyte cultures do not allow assessment of cell migration and systemic interactions, they provide extremely useful information on antigen recognition and cellular activation. Still, the cell frequency in peripheral blood differs greatly between naive and memory T cell populations.Naive T cells specific to any given antigen have been shown to constitute only 0.01 to 1% of total T cells -1 in 10 5 up to 10 6 T cells (Figure V.2). Memory cells, on the other hand, constitute more than 1% of total T cells in peripheral blood (Figure V.2). These differences in cell frequency imply that detection of induced responses in both repertoires must be performed differently.Recall responses in immunized or protected individuals can be readily detected using total PBMC since specific memory cells are present at high frequencies in circulation (Figure V.2). Usually, short-term cultures (5 to 10 days) are performed in triplicates of 2.10 5 total PBMC, for a total of 6.10 5 cells tested per condition.

  backgrounds or status relative to Leishmania infectionnaive and healed individuals (Figure V.3). Most healed individuals after Leishmania infection possess specific memory T cells, responsible for immunity against reinfection. Importantly, peptides that successfully induce T cell activation in both backgrounds are promising vaccine candidates (see Chapter I). These candidates are associated with protective recall responses and will be able to induce long-lasting memory responses in naive individuals, the objective of prophylactic vaccines. The primary focus is the experimental validation of Leishmania-specific peptides, in order to validate in silico predictions and pre-select the most immunogenic peptides (Figure V.3). To this end, the in vitro immunoassays with naive human samples were optimised, healed donor samples were collected from endemic areas in Tunisia, and the 49 HLA-class I-and 24 HLA-class II-restricted synthetic peptides were screened in both immune backgrounds. The main experimental readout for the proposed immunoassay screenings will be IFN-γ production, assessed by IFN-γ ELISpot assays.

HLA-class I 9 -

 9 mer peptides were synthesized by the Peptide Synthesis Platform from the Institut de Biologie Paris-Seine (UMPC-Sorbonne Universités, Paris, France), and HLA-class II 15-mer peptides by Proteogenix (Schiltigheim, France). The first peptide stocks were stored as 200 µM solutions in sterile MilliQ water, or Sodium Bicarbonate 0.2 mM (used in Tunisia and MN01-02), and the second peptide stocks were stored as 500 µM solutions in 10% DMSO and sterile MilliQ water (MN03-04).

  epitopes among the initial 48 class I and 24 class II peptides (Figure V.4). Briefly, peptides are attributed a random peptide number (pi1 to pi48 for HLA-class-I peptides, and pii1 to pii24 for HLAclass II peptides) and arranged in a matrix. Each peptide is included in two independent pools (1 vertical and 1 horizontal), and double-positive peptides are considered immunogenic.

Figure V. 4 3 . 5 .

 435 Figure V. 4 Matrix-based pool design. A) 49 peptides restricted to HLA-class I alleles (pi) were randomly distributed in 14 pools, pools 1 to 7 and pools A to G. B) 24 peptides restricted to HLA-class II alleles (pii) were randomly distributed in 10 pools, pools 1 to 5 and pools A to E(15). Pools highlighted in yellow are example positive pools, leading to the identification of 2 immunogenic peptides.

  status of MPL9 and MPL10's immature DC was assessed with the Mo-DC Differentiation Inspector human (Miltenyi Biotec, ref. 130-093-567), according to manufacturer's instructions. It comprises monoclonal antibodies recognizing CD14, CD83, and CD209 and corresponding isotype controls. After staining, samples were acquired by flow cytometry (FACSCanto, Becton Dickinson) and analysed with the BD FACSDiva TM software (version 6.2).

  monocytes through positive selection with the StraightFrom ® Buffy Coat CD14 MicroBead Kit, human (Miltenyi Biotec, ref 130-114-976); ii) total TCD8 + cells through positive selection with the StraightFrom® Buffy Coat CD8 MicroBead Kit, human (Miltenyi Biotec, ref. 130-114-978); and, finally, iii) total TCD4 + cells through positive selection with the StraightFrom® Buffy Coat CD4 MicroBead Kit,human (Miltenyi Biotec, ref. 130-114-980). Monocytes were cultured in T175 flasks for the generation of monocyte-derived DC (see 3.5.b) and, subsequently, immature DCs were cryopreserved. All T cell populations were cryopreserved and kept at -150°C until use. Cell purity was assessed by surface marker staining with the following antibodies: mouse anti-CD8-PE antibody (BD Biosciences); mouse anti-CD4-PeCy7 antibody (BD Biosciences); mouse anti-CD14-FITC antibody (BD Biosciences); and mouse anti-CD45-PerCPCy5 (BD Biosciences).Three cryovials containing total TCD8 + cells from donor MPL9 were gently thawed (vials with 50 million, 25 million and 12.5 million TCD8 + cells). Total TCD8 + cells were seeded in 96-well plate, 200.000 cells per well in Iscove Modified Dulbecco medium (IMDM) supplemented with 10% human AB serum, 1x non-essential amino-acids (Invitrogen), 50 U/mL of penicillin and 50 μg/mL of streptomycin (Invitrogen) (=IMDMc), for a total of 10 cell lines (2 million TCD8 + cells) per condition.

  with 48 peptides); ii) fourteen matrix-based class I peptide pools 1 to 7 and pools A to G (containing 6 or 7 9-mer peptides each); iii) one pool containing class II 15-mer peptides (pool ALL_II with 24 peptides); iv) and ten matrix-based class II peptide pools 1 to 5 and pools A to E (containing 4 or 5 15-mer peptides each).

Figure V. 5

 5 Figure V. 5 Experimental planning for immunoassays with samples from healed donors.

(

  supertypes) according to the classification by Sidney J et al 2008supertypes A01, A02, A03, A24, A26, B07, B08, B27, B44, B58, B62 (cf chapter IV). Whenever specific alleles possess B and F pocket specificities that are shared by different allele groups they can be classified, for example, as A01A24 or remain unclassified. Table V. 5 Naive donor bank description. Buffy coat samples from 21 individuals from the French blood bank EFS (Établissement Français du Sang), in Toulouse, France. EFS code = collection site code/internal sample ID. Total PBMC were recovered using Ficoll density gradient. CHAPTER V -EXPERIMENTAL VALIDATION OF SYNTHETIC PEPTIDES 196 Table V. 6 HLA-typing results for donors MPL3 to MP21. A) Six loci corresponding to HLA-class I and class II polymorphic regions (HLA-A, -B, -C, -DRB1, -DPB1, -DQB1) were sequenced by NGS (exons 2 and 3), leading to the 6-digit identification of the alleles expressed by each donor. Allele supertype classification by Sidney et al 2008 (20). B) Ambiguous identifications are given a multiple allele code (MAC, https://hml.nmdp.org/MacUI/). HLA typing results for the 19 individuals describe the diploid loci for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 genes corresponding to 12 alleles per donor. The only HLA class I genes analysed were the HLA-A and -B genes (4 gene loci per donor), and homozygous alleles were excluded from allele counts. The only HLA class II genes analysed were the HLA-DRB1 and -DPB1 genes (4 loci) and homozygous alleles were excluded from allele counts. HLA-typing results for the 19 naive donors included in the naive donor bank show the most frequent HLA-A allele is HLA-A*03, with 42,1% of individuals expressing an A*03 allele (8 allele counts) (Figure V.6). Almost 80% of individuals express an allele from the A03 supertype (15 in 19 donors), which also includes the alleles HLA-A*11:01:01G, A*31:01:02G, A*33:03:01G, A*66:01:01G (Table V.10). The most frequent HLA-B allele expressed by the naive donors is HLA-B*07, with 36,8% of individuals expressing HLA-B*07 alleles (7 allele counts) (Figure V.6). Almost 75% of individuals express an allele from the B07 supertype (14 in 19 donors), which also includes the B*35 alleles, namely HLA-B*35:01:01G (Table V.10). The individuals expressing alleles that belong to the B07 supertype include 36,8% and 31,6% of individuals who express HLA-B*07 and -B*35 alleles, respectively. Even if these alleles share peptide binding specificities, the high frequency of B*35 alleles in the human populations could justify a separate allele group to minimise bias in epitope selection. Another common HLA class I allele expressed by naive donors is the HLA-B*44, with 26,3% individuals expressing HLA-B*44 alleles (5 allele counts) and 63,2% of individuals expressing an allele from the B44 supertype (12 in 19 donors) (Figure V.6). The other main alleles belong to supertypes A01 and A02, with 8 and 6 individuals expressing alleles from these supertypes, respectively (Figure V.6). Three of the donors are homozygous for one of the HLA class I loci: donor MPL3 (HLA-A*23:01:01G, A24), donor MPL07 (HLA-B*07:02:01G, B07), and donor MPL14 (HLA-B*44:03:01G, B44). The HLA-class I supertype B62, with B*15:01 as the representative allele, is not represented in the donor bank, and the supertypes B58 (B*58:01) and A01A24 are only represented once. The only B*15 allele expressed is B*15:03:01G (donor MPL10) which is classified in the B27 supertype. The supertype A01A24, according to Sidney J et al 2008, includes alleles with specificities that are compatible with multiple supertypes (i.e HLA-A*29:02) -B pocket specificity for small, aliphatic and aromatic residues, and F pocket specificity for aromatic and large hydrophobic residues. In this case, these features are compatible with both the A1 supertype (B pocket specificity for small and aliphatic residues, and F pocket specificity for aromatic and large hydrophobic residues), and the A24 of individuals), HLA-B*44 alleles (0,1316 frequency / 26,3% of individuals), HLA-A*01 alleles (0,1316 frequency / 26,3% of individuals), and HLA-B*08 alleles (0,079 frequency / 15,8% of individuals) (Figure V.7).

Figure V. 7

 7 Figure V. 7 HLA class I allele frequency in the French population and in the naive donor bank. Allele frequency for HLA-A and -B genes (allele / 2n).

Figure V. 8

 8 Figure V. 8 HLA class II allele frequency in the French population and in the naive donor bank. Allele frequency for HLA-DRB1 and -DPB1 alleles (allele / 2n).

Figure V. 9

 9 Figure V. 9 Average HLA-class I allele frequencies according to world region. Averaged results from the population studies considered (%individuals and allele frequency) for HLA-class II alleles HLA-A and HLA-B. A) South and Central America populations. B) North Africa population. C) Western Asia populations. D) South Asia populations.

Figure V. 10

 10 Figure V. 10 Average HLA-class II allele frequencies according to world region. Averaged results from the population studies considered (%individuals and allele frequency) for HLA-class II alleles HLA-DRB1 and HLA-DPB1. A) South and Central America populations. B) North African populations. C) Western Asian populations. D) South Asia populations.

Figure V. 11

 11 Figure V. 11 Monocyte-derived DC phenotype analysis for donors MPL9 and MPL10. DC were stained in independent experiments with Mo-DC Inspector kit, human (Miltenyi) that detects CD14, CD209 (DC-SIGN), and CD83 expression. A) immature DC from donor MPL9. B) immature DC from donor MPL10. C) mature DC from donor MPL10.

Leishmania-specific peptide

  pools (Figure V.12 panel A). A total of 17 positive cell lines (out of 140) specific to 9 different peptide pools were generated from donor MPL3. Results show that 5 (out of 14) peptide pools generated 1 positive cell line, and 4 pools generated between 2 and 5 specific cell lines. ELISpot results for negative controls (never stimulated TCD8 cells) showed overall low background levels (NS spot counts: 25, 20, 44, 13, 39, 3) for an average of 24 spots (±15,5). The highest NS value (44) was included in the empirical test to determine well positivity (2*NS average+44 spots). The PHA solution used in this experiment is thought to have been from on old stock, probably degraded, as only 2 wells showed high spot counts, and still much lower than expected (PHA spot counts plate A: 222, 196, 60, 56; plate B: 46, 36) (Figure V.12 panel B).

Figure V. 13

 13 Figure V. 13 Four peptide pools successfully generated over 2 positive cell lines. A) Cross-matching results with matrix peptide pool constitution leads to the identification of 4 double-positive, hence immunogenic peptides. B) Naive donor MPL3's HLA-typing results for 6 HLA loci (HLA-A, -B, -C, -DRB, -DP, -DQ)

V. 14

 14 panel C). Otherwise, spot counts are similar to respective cNS unstimulated wells. Also, STIM and cNS are mostly similar to background levels for negative controls (never stimulated TCD8 cells -NS) but much more variable. All conditions and all cell lines show some variability in background levels (cNS spot counts), and even some cell lines present high background levels (e.g. pools 1, C, E, F and G)(Figure V.14 panel C). For peptide pool C, one cell line has cNS spot count of 93 and STIM spot count of 142, which if we compared with the never stimulated TCD8+ negative control would be considered a positive cell line (Figure V.13 panel C). This can be due to approximation errors when averaging cell counts to calculate 20.000 seeded cells in the ELISpot (3 in 10 wells measured per condition). Still, pools that successfully generated more than one cell line in MN01pools B, D, 2 and 6did not induce IFN-γ production/positive cell lines (maximum spot counts were 41, 43, 39 and 25, respectively).

Figure V. 15

 15 Figure V. 15 IFN-γ ELISpot results for the MN03 experiment (96-well format), T cell amplification assay with total TCD8 cells from donor MPL9. A) Number of specific cell lines and IFN-γ ELISpot spot counts for 96-well plate format control conditions; B. all results per cell line, 7 seven lines per condition (1, 2.5 and 5µM peptide concentrations).

Figure V. 16

 16 Figure V. 16 IFN-γ ELISpot results for the MN03 experiment (48-well format), T cell amplification assay with total TCD8 cells from donor MPL9. A) IFN-γ ELISpot results for control conditions. B) Pool22 IFN-γ ELISpot results for allconditions tested: 1, 2.5 and 5µM peptide concentrations and 50.000 (50k), 100.00 (100k), or 150.000 (150k) TCD8 cells per ELISpot well. C) Pool27 IFN-γ ELISpot results for all conditions tested: 1, 2.5 and 5µM peptide concentrations and 50.000 (50k), 100.00 (100k), or 150.000 (150k) TCD8 cells per ELISpot well.

Figure V. 17

 17 Figure V. 17 MPL10 HLA-typing for HLA-class I loci and peptides selected to compose the peptide pool.

Figure V. 18

 18 Figure V. 18 IFN-γ ELISpot results for experiment MN04, T cell amplification assays with total TCD8 T cells from donor MPL10. A) TCD8+ amplified with the short peptide protocol (mDC stimulation). B) TCD8+ cells amplified with the long peptide protocol (iDC stimulation). C) ELISpot well images and respective spot counts for CEF and positive well.

  generation of a naive donor bank and respective HLA-typing results allows the optimization of samples to use in the immunoscreening experiments. Donors can be selected according to HLA-typing results to maximise HLA coverage while testing a minimal number of individuals. The donor MPL3 is homozygous for the HLA-A loci (HLA-A*23:01:01G alleles classified as A24 supertype) and expresses HLA-B*44:03:01G (B44 supertype) and HLA-B*49:01:01G, an unclassified allele. Considering four double-positive immunogenic peptides that generated more than 2 specific cell lines, only one (A24_3) matches MPL3's HLA typing results. Interestingly, the allele B*49:01 has ambiguous binding pocket specificities. This allele has a B pocket specificity matching to the B44 supertype, and F pocket binding like the B*38:01 allele (hydrophobic), so it likely this allele shares binding specificities with other alleles in the B44 supertype, and perhaps even other supertypes.

  hours with both peptides and maturation factors, specific IFN-γ production was induced and detected. These results suggest APC's immature state and the simultaneous peptide/TLR stimulation are key for proper peptide presentation and T cell activation.During in vivo responses, immature DC uptake antigen in the periphery and migrate to the lymph nodes as mature cells where they prime responding T cells. Immature DCs are phagocytic and process and present exogenous antigens. TLR stimulation of immature DCs induces cell maturation process consisting of the upregulation of MHC and costimulatory molecules (CD40, CD80 and CD86) and migration to the lymph nodes. Mature DC become powerful APC, able to prime naive T cells and influence T-cell differentiation. TLRs are therefore a unique link between pathogen recognition and induction of T cell responses. The maturation conditions and stimulation by different pattern recognition receptors (PRR) influence DC's costimulatory molecules and the ability to produce inflammatory cytokines, therefore impacting the observed T cell responses(26,27).

  containing 12 peptides (2,5 µM each), we successfully amplified and detected Leishmania-specific T cells from the naive repertoire. Only one culture well was positive (1 million TCD8 cells seeded) among two stimulated wells (2 million TCD8 cells tested), as expected for low frequency specific cells. The 48-well batch stimulation assay format allows for the successful amplification of specific T cells present in the naive repertoire, as well as practical advantages that permit cell counting, ELISpot well triplicates, and therefore the application of statistical tests for well positivity determination. Although, in this experiment, only two wells per condition were seeded in the ELISpot plate due to the optimisation of peptide concentration and cell density.

  match the most frequent alleles in the Tunisian population (Figure V.19 and V.20). The HLA-class I alleles HLA-A*02 are the most frequent. HLA-A*24 alleles, although very frequent in the Tunisian population, were not represented in the healed donor series. On the other hand, other alleles such as HLA-B*44, -B*35 and -A*01 were more frequent in the healed donor series (Figure V.19).

Figure V. 19 6 . 2 .

 1962 Figure V. 19 HLA class I allele frequency in the Tunisian populations and in the healed donor series. Allele frequency for HLA-A and -B alleles (allele / 2n).

  Figure V.21). IFN-γ ELISpot results show donors have quite different background levels, from an average NS spot count of 14,5 (TUN7) up to an average spot count of 161,5 (TUN1) (Figure V.21).

Figure V. 21

 21 Figure V. 21 IFN-γ ELISpot results for controls for 1st healed series (n=9). A) Spot counts for NS (never stimulated PBMC), for positive controls PHA, TSLA and CaniLeish®, and pools ALL_I (49 class I peptides), ALL_II (24 class II peptides) and ALL_I+II (all 73 Leishmania-specific peptides). B) Table with averaged spot counts for all controls (* significantly different from autologous NS with the Mann-Whitney statistical test, p-value ≤ 0.05).

stimulated

  in vitro in the absence or presence of peptide pools containing HLA-class I or -class II peptides. Positive IFN-γ responses were induced by multiple peptide pools and by most donorsseven responder donors for HLA-class I peptide pools, and nine responder donors for HLA-class II peptide pools (Figure V.22). The number of responders and the number of peptide-specific responses induced for each donor were different according to the peptide pool (Figure V.22). Overall, all HLA-class II pools induced IFN-γ production in at least one individual (II_pool5 and B), up to 7 individuals (II_pool4) (Figure V.22 panel B). On the other hand, three HLA-class I peptide pools did not elicit IFN-γ production in any of individuals tested (I_pool4, A and C), and the remaining 11 pools induced IFN-γ production in at least one donor (I_pool3 and 7), up to 4 donors (I_pool1 and E) (Figure V.22 panel A). Individualised ELISpot results are included in Appendix V.2 (donors TUN1, TUN3-10). For HLA-class II peptide pools, two donors had only one positive peptide pool so single immunogenic peptides were not identified (TUN1, II_poolE and TUN6, II_pool1). Likewise, for HLAclass I peptide pools, TUN6 had only one positive peptide pool (I_poolE) (Figure V.22). Results regarding classI peptide pools I_pool5 and I_pool6 are inconclusive for donor TUN7 since results are not available (no revelation antibody added to row C during ELISpot revelation).

Figure V. 22

 22 Figure V. 22 Responder frequency per peptide pool for the 1st healed series (n=9). Number of responders, individuals with significantly increased IFN-γ production (responders) per peptide pool. A) Responder donors to class I peptide pools. B) Responder donors to class II peptide pools.

Figure V. 23

 23 Figure V. 23 IFN-γ ELISpot results for TSLA-positive donors TUN3 (A), TUN4 (B), TUN5 (C), and TUN10 (D).Highlighted pools (*) induced significantly higher IFN-γ production when compared with autologous NS (Mann-Whitney test, p-value < 0.5).

Figure V. 23

 23 Figure V.23 (continued)

Figure V. 24

 24 Figure V. 24 Magnitude of response against matrix-based peptide pools for all healed donors. Fold-change differences in IFN-γ production after peptide stimulation. A) Pools containing HLA-class I peptides. B) Pools containing HLA-class II peptides.

  informative. It has been described that Tunisian LST-positive individuals produce IL-10, also, IL-10 mRNA is detected in CL lesions caused by L. major. These observations further support the analysis of balanced Th1/Th2 responses in individuals with protective immune responses against Leishmania.The second series of samples yielded no ELISpot results (data not shown), with few and faint spots in the control wells indicating very low cell viability before seeding in the ELISpot plates, although ELISpot revelation was successful after 20-minute incubation with substrate solution. These results are probably due to the change, at culture day 5, from commercial human AB serum to homemade serum. This change probably caused cell death, and cells were mostly unviable when seeded in the ELISpot plate. Still, the CBA analysis on the culture supernatants will still be performed to confirm this hypothesis.

  population and the healed donors. Furthermore, if we consider the HLA coverage of the 25 immunogenic HLA-class I peptides, the most frequent alleles in the Tunisian and world population are covered (A*01, A*02, A*03, A*24, B*07, B*08, B*44), although only one peptide is restricted to A*02 alleles.The cellular source of IFN-γ production was not assessed. TCD4 + cells are the most common origin for IFN-γ production in response to TSLA, and probably to HLA-class II peptide pools, but this remains to be confirmed.Finally, positive immune responses are observed against several Leishmania-specific peptide pools, as PBMC from healed donors produced IFN-γ in response to in vitro stimulation. Also, it is the first time IFN-γ responses from human PBMC after CaniLeish ® stimulation are observed. This preliminary observation needs further characterisation. As expected from the dog immune responses and observed here through in vitro human PBMC stimulation, the Leishmania promastigote secretome contains promising vaccine antigen candidates, which are also immunogenic in humans and associated with memory responses.

  bottlenecks associated with product development for NTDs(Figure VI.1). Briefly, the exploitation of large proteomic datasets, comprising the most important pathogenic Leishmania species, allowed the selection of 52 relevant vaccine antigens from the Leishmania secretome, a known immunogenic antigen pool. Moreover, this screening approach was coupled to an HLA-based in silico epitope prediction and selection workflow leading to the synthesis of over 70 promising short peptide vaccine candidates. Then, these peptide candidates were tested exclusively in human samples, a key aspect that ensures peptide immunogenicity in human hosts and vaccine population coverage. Experimental validation with samples from both healed individuals from endemic areas and naive individuals is extremely important to effectively assess the vaccine candidate's feasibility (Figure VI.1).

Figure VI. 1

 1 Figure VI. 1 Summary of major project outcomes.

M

  well not stimulated in ELISPOT NeverStimulated average in the legend OVERALL RESULTS -HLA-CLASS I Pools 1-7 cNS = culture well not stimulated in ELISPOT NeverStimulated average in the legend OVERALL RESULTS -HLA-CLASS I Pools A-B cNS = culture well not stimulated in ELISPOT NeverStimulated average in the legend OVERALL RESULTS -HLA-CLASS II Pools 1-5 cNS = culture well not stimulated in ELISPOT NeverStimulated average in the legend OVERALL RESULTS -HLA-CLASS II Pools A-E cNS = culture well not stimulated in ELISPOT NeverStimulated average in the legend TUN1

Table I . 1

 I1

Table IV . 4

 IV4

HLA-class I alleles included in T-cell epitope prediction.

Table IV . 5

 IV5

HLA-class II alleles included in T-cell epitope prediction.

Table IV . 6

 IV6 Selected databases and HLA-class I binding prediction algorithms

Table IV . 7

 IV7 Selected databases and HLA-class II binding prediction algorithms

CHAPTER V -EXPERIMENTAL VALIDATION OF SYNTHETIC PEPTIDES

Page

Table V.1

T cell amplification assays performed

Table V . 2

 V2

Description and size of 17 French population studies from AFND with HLA data considered for HLA allele frequency analysis

Table V . 3

 V3

Description and size of 8 Tunisian population studies from AFND with HLA data considered for HLA frequency analysis

Table V . 8

 V8

HLA-typing results for the first healed donor series (TUN1-TUN3 to TUN10)

Table V .9

 V

		ABBREVIATIONS
	a.a.	Amino-acid	MHC	Major histocompatibility complex
	Abs	Antibodies	MoDC	Monocyte-derived dendritic cells
	APC	Antigen-Presenting Cells	MPL-	monophosphoryl lipid A
	CCL	Chemokine (C-C motif) ligand	SE
	CCR	C-C chemokine receptor type	NF-κB	Nuclear factor-kappa B
	CD40L	ligand of CD154	NK	Natural killer cells
	CL	Cutaneous leishmaniasis	cells
	CMI	cellular-mediated immune	NO	Nitric Oxide
		response	NTD	Neglected Tropical Disease
	CTL	Cytotoxic T lymphocytes	PAMP	Pathogen-Associated Molecular
	DAMP	Damage-Associated Molecular		pattern
		Pattern	PBMC	Peripheral Blood Mononuclear
	DAT	Direct Agglutination Test		cells
	DC	Dendritic cells	PBS	Phosphate-buffered saline
	DCL	Diffuse Cutaneous	PHA	Phytohemagglutinin-L
		Leishmaniasis	PKDL	Post Kala-Azar Dermal
	DMSO	Dimethyl sulfoxide		Leishmaniasis
	ELISpot Enzyme-linked Immunospot	pMHC	peptide:MHC complex
		assay	PMN	Polymorphonuclear Neutrophils
	ELISA	enzyme-linked immunosorbent	PRR	Pattern Recognition Receptor
		assay	PSA	Promastigote Surface Antigen
	ESA	Excreted-secreted antigens	R&D	Research and Development
	ESP	Excreted-secreted proteins	SLA	Soluble Leishmania Antigen
	FcR	Fc receptor	SSG	Sodium StiboGluconate
	GM-CSF	Granulocyte-macrophage Double-positive immunogenic peptides identified in the naive and memory repertoires TCM Central Memory T cell colony-stimulating factor TCR T-cell receptor
	HLA	Human Leukocyte Antigen	TEM	Effector Memory T cell
	IDRI	Infectious Disease Research	TFH	Follicular T Helper
		Institute	TGF	Transforming Growth Factor
	IL	Interleukin	Th1	Type 1 T helper
	ILC	Innate Lymphoid Cell	Th2	Type 2 T helper
	IgG	Immunoglobulin G	Th17	Type 17 T helper
	IFN-	Interferon	TLR	Toll-Like Receptor
	L.	Leishmania	TNF	Tumor Necrosis Factor
	LCL	Local cutaneous leishmaniasis	Tregs	Regulatory T cells
	LPS	Lipopolysaccharide	TRM	Resident memory T cell
	LST	Leishmanin Skin Test	TSCM	Stem cell memory T cell
	LZ	Leishmanization	VL	Visceral leishmaniasis
	MCL	Mucocutaneous leishmaniasis
				xxi xxii

European Congress on Tropical Medicine and International Health Institute of

  The abstracts of the 10 th ECTMIH were published at Oral Presentation Sessions. Tropical Medicine and International Health 2017 Oct;22:2-114 (3).

	• 1st International Caparica Congress on Leishmaniasis 2018, Lisbon, Portugal (October 29-
	31 st 2018)
	Shotgun Presentation (awarded prize for best shotgun presentation): Pissarra J, Holzmuller P, Bras-
	Gonçalves R, Garnaoui A, Lemesre J-L. "A new epitope-based peptide vaccine against human
	leishmaniasis".
	Tropical Medicine (ECTMIH) during Session 1S5 -Vaccinology (October 16-20 th 2018)
	Oral presentation: Pissarra J, Holzmuller P, Bras-Gonçalves R, Garnaoui A, Lemesre J-L. "Preclinical
	development of a new peptide-based vaccine against human leishmaniasis".
	xxiv

• CBS2 day 2017 (Journée de l'Ecole Doctorale CBS2), Montpellier, France (May 24th 2017)

Oral presentation (awarded prize for best oral presentation): Pissarra J, Holzmuller P, Bras-Goncalves R, Garnaoui A, Lemesre JL. "A new peptide-based vaccine against human leishmaniasisoptimized preclinical development to prevent late-stage failure".

• 10th

• Final EUROLEISH.net meeting, Barcelona, Spain (November 12-13 th 2018) Oral presentation (awarded prize for best oral presentation): Pissarra J. "Preclinical development of a new peptide-based vaccine against human leishmaniasis" .

  Trypanosomatidae family) and are transmitted by sand flies, phlebotomine vectors. Over 20 different Leishmania (L.) species are known to cause disease in humans and other mammals. Despite being closely related and sharing a common lifecycle and an invertebrate host, different Leishmania species are transmitted by different vector species, have different epidemiological features, namely, zoonotic or anthroponotic transmission, and cause quite different clinical presentations. The main four clinical

forms of leishmaniasis are: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), visceral leishmaniasis (VL) also known as kala-azar, and post-kala-azar dermal leishmaniasis (PKDL).

Neglected Tropical Diseases (NTDs), also known as Neglected Infectious Diseases (NIDs), englobe several communicable diseases caused by diverse infectious organisms. They prevail in 149 tropical and subtropical countries causing a massive economic and development burden to the affected societies (http://www.who.int/neglected_diseases/diseases/en/). Poor and/or rural populations are the most vulnerable to these infections, and the least likely to have access to healthcare services. NIDs share features that advocate both for Public Health initiatives that successfully detect, prevent disease and treat patients, but also for the development of new and more effective control tools.

Table I . 1 Leishmania parasites and major clinical syndromes

 I

	Clinical	Leishmania spp. and location	Natural Progression
	Syndromes
	Visceral
	leishmaniasis (VL),
	also known as
	kala-azar:
	generalized
	involvement of
	the
	reticuloendothelial
	system (spleen,
	bone marrow, liver,
	lymph nodes)

. Adapted from Burza S. et al 2018 and Magill A. 2015

(12,31)

. ©WHO, World Health Organization campaign photos.

Table I . 2 Ratio between number of asymptomatic and active disease cases, according to geographical region.

 I Adapted from Singh OP et al 2014(44).

	Country	Ratio (asymptomatic:active infection)	Reference
	Sudan	1:2,4	(45)
	Kenya	4:1	(46)
	Ethiopia	5,6:1	(47)
	Brazil	18:1	(48)
	Spain	50:1

c) Sensing danger -TLR signalling

	are shown to induce IL-12 and NO production, with low concomitant IL-10 production, suggesting a
	protective effect by the TLR4/MyD88/IL-12 pathway (108,109).
	-, TLRs are crucial for the coordination of the type and magnitude of the innate response, with
	NO, H2O2, and peroxynitrite ONOO -), responsible for intracellular parasite killing. Leishmania a dual role in protection against Leishmania parasites. Albeit a non-decisive role in infection outcome,
	parasites actively modulate this balance, skewing arginase function towards glycolysis, and ensuring TLRs contribute towards an early Th2 bias, promoting parasite survival (108). TLR signalling and
	parasite intracellular survival. These mechanisms are reviewed in Holzmuller et al 2018 (2). control of early innate responses becomes particularly relevant for Leishmania immunity since
	Neutrophils and macrophages have concerted activities: neutrophils augment macrophage PAMPs are conserved and expressed constitutively across pathogenic species, and they are key
	function to quickly neutralise early inflammatory stimulus, and apoptotic neutrophils signal virulence factors, suggesting their inclusion when designing vaccine formulations against Leishmania
	macrophages to decrease inflammation and promote healing (99,100). In the context of non-healing (108).
	lesions, neutrophils contribute to the development of chronic infection through the impairment of the
	recruitment of inflammatory monocytes and monocyte-derived DC and of subsequent Th1 response d)
	(92,99,101-104).
	In addition to receptor-mediated phagocytosis, macrophages and other antigen-presenting
	cells (APC) possess receptors responsible for sensing danger and reacting accordingly through the
	production of cytokines and chemokines. In this context, danger is perceived as any molecule able to
	harm the host, from pathogens (Pathogen-Associated Molecular patterns, PAMPs) or from cell stress
	and injury (Damage-Associated Molecular Patterns, DAMPs). The main PAMPs are microbial nucleic
	acid, lipoproteins, surface glycoproteins, and other membrane components, and are recognised by
	Pattern Recognition Receptors (PRRs), including toll-like receptors (TLRs) (66).
	Humans have ten different membrane-bound TLRs, most use MyD88 for intracellular signal
	transduction, except TLR3 that uses MyD88-independent mechanisms (TRIF, TIR-domain-containing
	adapter-inducing interferon-β; TIRAP, TIR domain containing adaptor protein; or TRAM, TRIF-
	related adaptor molecule), and TLR4 that uses both. Among them, TLR2 and TLR4 can recognize
	extracellular promastigotes, TLR3 and TLR9 can recognize parasites in intracellular vacuoles
	(66,105).
	Studies have shown a major role for TLR activation in anti-Leishmania immunity, namely
	TLR4 and TLR9 (106). TLR9 activation has been linked to protective adaptive responses (107). TLR
	recognition results in the activation of transcription factors (NF-κb and IRFs) and the production of
	inflammatory cytokines and promotion of NO production (68). TLR4 inhibition results in a M2b
	macrophage phenotype, high IL-10 production contributing to Th2 responses, whereas TLR4 ligands

Innate lymphoid cells -NK cells

	Natural killer (NK) cells belong to the group of Innate lymphoid cells (ILCs), immune cells that
	mirror T cell phenotypes and functions, yet ILCs do not express antigen receptors not do they undergo
	clonal selection and expansion upon activation (110). According to cytokine production patterns and
	associated transcription factors, ILCs can be divided in 3 groups. NK cells belong to group 1 ILCs

DM, DO, oligomorphic genes encoding proteins involved in the loading of other class II molecules; DR, DQ, DP, highly

	comprising 6 class I loci and 13 class II loci (Figure I.10). There are two components to HLA diversity:
	i) gene families, encoding HLA-class I heavy chains and -class II alpha and beta chains; and ii) genetic
	polymorphism, responsible for multiple alternative forms of a given gene (alleles), and respective
	encoded protein (allotype). HLA gene polymorphism corresponds to variability at a gene locus in
	which all variants occur at a frequency greater than 1% (55). HLA gene expression is codominant, the
	two alleles are expressed in roughly equal amounts in the heterozygote, and each individual expresses
	a unique set of HLA alleles, specific to a unique set of antigenic peptides (123,124).
	A given isoform is the product of an individual HLA allele (123). There are monomorphic
	genes and oligomorphic genes, for which different alleles have been described (Figure I.10). Six HLA-
	class I isotypes: A, B, C, highly polymorphic genes, and with a role in antigen presentation to TCD8 +

cells; F, monomorphic gene, intracellular; E and G genes, oligomorphic and important for NKmediated responses. Also, there are five HLA-class II isotypes: polymorphic genes, encoding proteins responsible for presentation to TCD4 + cells (123).

d) Adaptive immunity -pathogenesis and human-specific mechanisms against Leishmania spp. infection

	and enhanced Th1 responses (170,173). IL-10 immunomodulation may also involve the induction of MCL is characterised by the chronic inflammation of the nasal mucosa and a hyperactive T
	T cell exhaustion and anergy (174). It is interesting to note that while in VL patients IL-10 is detected cell response (high levels of IFN-γ and TNF-α, decreased levels of IL-10 and TGF-β). In this case,
	Human leishmaniases often exhibit a mixed type 1 and type 2 cytokine profiles, so the in the plasma (170), this is not the case for patients infected with L. major and L. aethiopica (175). pathogenesis is caused by a poorly regulated T cell response (174,185). In human MCL patients, the
	However, IL-10 mRNA is detected in lesions caused by L. major and L. tropica (176). This is most likely number of TCD8 + cells in the lesion site increases as disease progresses. These cells express high Th1/Th2 dichotomy observed in mouse models does not apply (163-165). Although mouse models due to the immune response compartmentalisation, and possibly due to different sources of IL-10 levels of granzymes and perforin (cytolytic activity), contributing to inflammation and disease are extremely useful to detail the molecular mechanisms involved in immunopathology, these only offer an incomplete assessment human-specific mechanisms (163,166,167). Notwithstanding, the production. pathology via perforin-mediated cytotoxicity (186). Th17 cells is also associated with pathogenesis
	complex immune responses observed in dogs against canine leishmaniases bear a much closer The mechanisms behind Th1/immunosuppression regulation during Leishmania infection in MCL patients: MCL lesions have increased IL-17A mRNA levels, and TGF-β, RORyT, IL-23
	resemblance to human responses (168). are not fully understood, however, the cytokine IL-10 has a crucial role in these processes. T cell-(associated with Th17 differentiation), and IL-17 is produced by not only TCD4 + but also TCD8 + ,
	derived IL-10 is mostly produced by IFN-γ-producing TCD4 + cells, Type 1 regulatory cells (Tr1, CD14 + and CCR6 + cells (187). High IL-17 increases neutrophil recruitment, suggesting it promotes
	dysfunctional, and in human CL and MCL immune-mediated tissue pathology is observed. Active VL derived Tregs (CD4 + CD25 + FoxP3 + ) (177). The Treg cell subpopulation has an important dual role in During active VL and DCL (diffuse cutaneous leishmaniasis), the host immune response is conventional T cells that convert to FoxP3+ regulatory cells) and, to a lesser extent, by thymus-inflammatory response making it a potential target for immunotherapy (187).
	patients possess weak T-cell mediated responses unable to control infection -Th1 responses are immunosuppression and promotion of concomitant immunity in CL. Treg-produced IL-10 modulates DCL is a severe manifestation of CL, characterised by high antibody titers, TGF-β in
	balanced with immunosuppressive mechanisms, i.e. specific Th1 cells are activated but not enough to APC functions and suppresses TCD4+ effector functions against L. major and L. braziliensis and helps circulation, and a defective cellular immune response against Leishmania antigens which is restricted
	maintain a stable infected macrophage reservoir. Tregs bind IL-2 (high levels of CD25 = IL2R) and to anti-parasitic responses, as responses to unrelated antigens rest intact (188). Human DCL patients prevent disease development. deprive other cells from this growth factor, causing apoptosis (176,178). Ambiguous and little have increased parasite numbers in skin lesions, as well as low levels IFN-γ, IL-2 mRNA, and high In human CL, the cell-mediated responses at the lesion site are crucial for disease outcome. Human CL caused by L. major induces high levels of IFN-γ, IL-10, IL-12 mRNA in lesions, indicative of evidence for Treg involvement in human or experimental VL needs further development levels of IL-10, IL-4, IL-5. Therapeutic cure enhances IFN-y production with low IL-10, further
	mixed TCD4+ responses. IFN-γ-producing cells are definitely predominant in healing cutaneous (173,179,180). suggesting the need for Th1 responses. In DCL, high antigen exposure may be responsible for T cell
	lesions, but in chronic cutaneous lesions and mucocutaneous lesions we observe both Th1 and Th2 unresponsiveness, alternatively, T cell responses may promote localized parasite growth in the skin
	PKDL pathogenesis remains mostly unknown, there is consensus that immune suppression (189). DCL patients respond poorly to drug treatment and better results are obtained in combination cytokines, with high levels of IL-4 and IL-10 (169). been implicated with the development of active disease in infected patients (169,170). In active VL, characterised by increased IFN-γ and TNF-α, and IL-10 and TGF-β at lesion sites (181). In Indian In human VL, no clear correlation exists with increased IL-4 levels (169). However, IL-10 has allows the multiplication of latent parasites from the viscera or residing in the skin. PKDL is with immunomodulation (IFN-γ and BCG plus antimonial treatment) (189).
	high levels of IL-4 and IFN-γ are detected in the spleen, but these levels decline after cure, and the PKDL, a low expression of receptors is observed, a high number of TCD8 + cells in circulation and in
	same profile is observed upon antigenic in vitro PBMC stimulation (165,169). IL-10 is produced by lesions, as well as increased antigen-induced IL-10 production by TCD8+ cells, impaired antigen-
	induced proliferation and increased Th17 responses (IL-17A, IL-23, RORyT) (182). The presence of different cell types (Tregs, Th2, Th1 and other cells) and differences between CL and VL are still not Tregs at the skin (FoxP3 + CD25 + CTLA4 + ) is correlated with parasite burden in Indian PKDL (183). In clear. The anti-inflammatory cytokine IL-10 plays a key role in the regulation of host immune Sudanese PKDL, some susceptibility-associated polymorphisms in the IFN-γ receptor have been More recent studies in L. major mouse infection (in resistant C57BL/6), CCL2/MCP-1 responses (121). There is a consensus regarding the suppressive effect of IL-10 on the immune described, and immune responses similar to cured VL patients (184). PBMC from Sudanese PKDL production resulted in the recruitment of Ly6C + inflammatory monocytes, capable of capturing and response in VL and its association with disease severity, since this cytokine is an important patients proliferate in response to Leishmania antigens and TCD4 + cells secrete IL-10 and IFN-γ, killing parasites by oxidative burst and migration to lymph nodes where they differentiate into specialized DC subsets (161). These CCR2 + monocytes capture L major parasites, produce IL-12 and differentiate into iNOS-expressing DC in the lymph nodes, promoting Th1-mediated protection, making them an interesting target cell population for vaccine formulations (161). Also, studies have similarly to responses observed in cured VL patients. Th17 cells may have a role in parasite clearance immunosuppressant and inhibitor of macrophage microbicidal activity in both mice and humans with in PKDL: PBMC from Indian PKDL patients stimulated with Leishmania antigens produce IL-17A and VL (63,152,171). In human VL, IL-10 is increased in plasma, as well as increased mRNA levels in the spleen, bone marrow and lymph nodes (170). Total PBMC from healed VL patients stimulated with IL-23, and PBMC stimulated with IL-17A show enhanced production of TNF-α and NO (182).
	found that in experimental VL and CL (mice), resistance needs anti-parasitic Tbet+ IFN-γ producing Leishmania soluble antigens co-produce IFN-γ and IL-10 (172). On the other hand, IL-10 blockade in
	TCD4+ cells (161,162). VL patient sera or splenic aspirate cultures results in suppressed parasite replication in macrophages

  ).

	(191). Both subsets produce effector cytokines in response to infection and other stimuli, yet TCM cells
	produce more IL-2, and TEM have higher proliferative capacity (191). Stem cell memory T cells (TSCM)
	are CD45RO -CD45RA + , similarly to naïve T cells, they also express co-receptors CD27 and CD28, IL-
	7Rα, CD62L and CCR7, and most importantly, can differentiate into other subsets, including TCM and
	Memory T cells are identified by the expression of CD45RO isoform, and by the lack of CD45RA
	isoform expression (CD45RO + CD45RA -) (190) (Figure I.17). CD45RA is considered a marker for naive
	T cells (CD45RO -CD45RA + ). They are also categorized according to the expression of lymph node-
	homing molecules, such as CCR7 -central memory T cells (TCM) express CCR7 and traffic to lymphoid
	tissues, and effector memory T cells (TEM) do not express CCR7 and migrate to peripheral tissues

Table I . 3 Toll-like receptor agonists and Th1-inducing adjuvants for use in vaccine formulations, adapted from

 I

	In summary, the most important bottlenecks in peptide-based vaccine design are the epitope
	choice, the adjuvant selection and availability; and vaccine administration. Despite vaccines targeting
	cellular immunity are not yet available, recent advancements human Th1 inducing adjuvants make
	this objective are a closer reality.
	Moreover, synthetic peptides present great versatility for adaptation to innovative delivery
	systems (292,295). The exploitation of innovative approaches should also be considered for anti-
	Leishmania vaccine development, such novel devices for optimal intradermal delivery (i.e. dermal
	patches, nano-delivery systems, etc) that ensure proper immune response priming throughout time
	(296). Also, chimeric proteins combining peptides, polyproteins and delivery vectors are promising
	formulations.
	Adjuvant	Class	Mechanism of action Type of immune response
	Lipid A analogues (e.g., MPL, GLA)	Immunomodulatory molecule	Toll-like receptor 4	Antibody, Th1
	Imidazoquinolines (e.g.,Imiquimod, R848)	Immunomodulatory molecule	Toll-like receptor 7 / 8	Antibody, Th1
	CpG ODN	Immunomodulatory molecule	Toll-like receptor 9	Antibody, Th1, Th2, TCD8+ cells
	Saponins (e.g., QS21)	Immunomodulatory molecule	Unknown	Antibody and cell-mediated immune responses
	Virosomes	Particulate formulation	Antigen delivery	Antibody, Th1, Th2
	GLA-SE	Combination	Toll-like receptor 4	Antibody, Th1

(239)

.

  correlates with pathology in human Leishmania donovani infections. J Clin Invest.

	American Society for Clinical Investigation; 1993 Jul 1;92(1):324-9.
	171. Cummings HE, Tuladhar R, Satoskar AR. Cytokines and their STATs in cutaneous and visceral 2005 Nov 1;23(45):5245-51.
	leishmaniasis. J Biomed Biotechnol. Hindawi; 2010 Mar 15;2010:294389. 267. Nascimento E, Fernandes DF, Vieira EP, Campos-Neto A, Ashman JA, Alves FP, et al. A clinical
	172. Kumar R, Goto Y, Gidwani K, Cowgill KD, Sundar S, Reed SG. Evaluation of ex vivo human trial to evaluate the safety and immunogenicity of the LEISH-F1 + MPL-SE vaccine when used
	immune response against candidate antigens for a visceral leishmaniasis vaccine. Am J Trop in combination with meglumine antimoniate for the treatment of cutaneous leishmaniasis.
	Med Hyg. The American Society of Tropical Medicine and Hygiene; 2010 May;82(5):808-13. Vaccine. Elsevier; 2010 Sep 14;28(40):6581-7.
	173. Nylén S, Sacks D. Interleukin-10 and the pathogenesis of human visceral leishmaniasis.pdf. 268. Bertholet S, Goto Y, Carter L, Bhatia A, Howard RF, Carter D, et al. Optimized subunit vaccine
	Trends Immunol. 2007;28(9):378-84. protects against experimental leishmaniasis. Vaccine. Elsevier; 2009 Nov 23;27(50):7036-45.
	174. Kumar R, Chauhan SB, Ng SS, Sundar S, Engwerda CR. Immune checkpoint targets for host-269. Coler RN, Duthie MS, Hofmeyer KA, Guderian J, Jayashankar L, Vergara J, et al. From mouse to
	directed therapy to prevent and treat leishmaniasis. Front Immunol. Frontiers; 2017 Nov
	8;8(NOV):1492.
	Louzir H, editor. PLoS Negl Trop Dis. Public Library of Science; 2017 Nov
	27;11(11):e0005951.
	275. Carrillo E, Fernandez L, Ibarra-Meneses AV, Santos MLB, Nico D, de Luca PM, et al. F1 Domain
	of the Leishmania (Leishmania) donovani Nucleoside Hydrolase Promotes a Th1 Response in
	in mediating protection againstLeishmania donovani and the expansion of IL-10+ CD4+ T Leishmania (Leishmania) infantum Cured Patients and in Asymptomatic Individuals Living in
	cells. Eur J Immunol. John Wiley & Sons, Ltd; 2006 Jul 1;36(7):1764-71. an Endemic Area of Leishmaniasis. Front Immunol. 2017;8:750.
	181. Zijlstra EE. The immunology of post-kala-azar dermal leishmaniasis (PKDL). Parasit Vectors. 276. Santos MLB, Nico D, de Oliveira FA, Barreto AS, Palatnik-de-Sousa I, Carrillo E, et al.
	BioMed Central; 2016;9(1):464. Leishmania donovani nucleoside Hydrolase (NH36) domains induce T-cell cytokine

to evaluate the immunoprotective profiles of exposed individuals who have developed immunity to Leishmania infection. Considering the limitations of previous candidates and restrictive budgets associated with NTD research, the proposed strategy aims combine several data and methods to increase cost-efficiency and shorten development time to maximise late-stage success of a vaccine against human leishmaniases (Figure I.19). production 175. Da-Cruz AM, Bittar R, Mattos M, Oliveira-Neto MP, Nogueira R, Pinho-Ribeiro V, et al. T-cellmediated immune responses in patients with cutaneous or mucosal leishmaniasis: long-term evaluation after therapy. Clin Diagn Lab Immunol. American Society for Microbiology; 2002 Mar 1;9(2):251-6. 176. Katara GK, Raj A, Kumar R, Avishek K, Kaushal H, Ansari NA, et al. Analysis of localized immune responses reveals presence of Th17 and Treg cells in cutaneous leishmaniasis due to Leishmania tropica. BMC Immunol. 2013;14(Il):52. 177. Belkaid Y. Regulatory T cells and infection: a dangerous necessity. Nat Rev Immunol. Nature Publishing Group; 2007 Nov 1;7(11):875-88. 178. Costa DL, Guimarães LH, Cardoso TM, Queiroz A, Lago E, Roselino AM, et al. Characterization of regulatory T cell (Treg) function in patients infected with Leishmania braziliensis. Hum Immunol. 2013;72(12). 179. Maurya R, Kumar R, Prajapati VK, Manandhar KD, Sacks D, Sundar S, et al. Human visceral leishmaniasis is not associated with expansion or accumulation of Foxp3+ CD4 cells in blood or spleen. Parasite Immunol. John Wiley & Sons, Ltd (10.1111); 2010 Mar 19;32(7):479-83. 180. Stäger S, Maroof A, Zubairi S, Sanos SL, Kopf M, Kaye PM. Distinct roles for IL-6 and IL-12p40 182. Katara GK, Ansari NA, Singh A, Ramesh V, Salotra P. Evidence for Involvement of Th17 Type a multi-subunit recombinant leishmanial vaccine (MML) to protect dogs from Leishmania infantum infection and to prevent disease progression in infected animals. Vaccine. Elsevier; man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE. Clin Transl Immunol. Wiley-Blackwell; 2015 Apr;4(4):e35. 270. Didwania N, Shadab M, Sabur A, Ali N. Alternative to Chemotherapy-The Unmet Demand against Leishmaniasis. Front Immunol. Frontiers; 2017 Dec 21;8:1779. 271. Duthie MS, Pereira L, Favila M, Hofmeyer KA, Reed SJ, Metangmo S, et al. A defined subunit vaccine that protects against vector-borne visceral leishmaniasis. npj Vaccines. 2017;2(1):23. 272. Kreutzer S. Nursing Body and Soul in the Parish: Lutheran Deaconess Motherhouses in Germany and the United States. Nurs Hist Rev. 2010;18(1):134-50. 273. Osman M, Mistry A, Keding A, Gabe R, Cook E, Forrester S, et al. A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis: First-in-human trial of ChAd63-KH. McDowell MA, editor. PLoS Negl Trop Dis. Public Library of Science; 2017 May 12;11(5):e0005527. 274. Cecílio P, Pérez-Cabezas B, Fernández L, Moreno J, Carrillo E, Requena JM, et al. Pre-clinical antigenicity studies of an innovative multivalent vaccine for human visceral leishmaniasis. responses in human visceral leishmaniasis. Front Immunol. Frontiers; 2017 Mar

Table II . 1 Leishmania spp. strains cultured for the generation of ESAp. Leishmania spp. strains cultured for the generation of ESAp

 II

Leishmania infantum MHOM/MA/67/ITMAP-263 Leishmania major MHOM/SY/91/LEM-2420 Leishmania tropica MHOM/SY/90/LEM-2067 Leishmania donovani MHOM/IN/80/DD8 Leishmania braziliensis MHOM/BR/75/M-2904 Leishmania amazonensis MHOM/BR/76/LTB-012 Leishmania tarentolae WT commercial strain Figure II. 1 General workflow for the preparation of the Leishmania promastigote secretome.

Table II . 2 Ordered species and group-species list established to define a "leading" protein based on the taxonomy criteria (only for match groups cases where more than one protein ID are present after filtering).

 II

	Taxonomy	Group Species Order
	Leishmania infantum	1
	Leishmania chagasi	2
	Leishmania donovani (strain BPK282A1)	4
	Leishmania donovani	3
	Leishmania donovani donovani	4
	Leishmania donovani archibaldi	4
	Leishmania donovani complex sp. CR-2013	4
	Leishmania major	5
	Leishmania gerbilli	6
	Leishmania turanica	6
	Leishmania arabica	6
	Leishmania tropica	7
	Leishmania tropica complex sp. CR-2013	8
	Leishmania aethiopica	8
	Leishmania killicki	8
	Leishmania mexicana (strain MHOM/GT/2001/U1103)
	Leishmania mexicana
	Leishmania amazonensis	9
	Leishmania mexicana mexicana
	Leishmania mexicana venezuelensis
	Leishmania pifanoi
	Leishmania braziliensis
	Leishmania braziliensis complex EV-2015
	Leishmania guyanensis
	Leishmania peruviana
	Leishmania panamensis
	Leishmania shawi
	Leishmania lainsoni
	Leishmania naiffi
	Leishmania utingensis
	Leishmania lindenbergi
	Leishmania sp. MHOM/BR/2002/NMT-RBO004
	Leishmania sp. CR-2014
	Leishmania sp.

ID Organism Organism ID Protein count Secretome protein counts % of total proteins UP000000542 Leishmania

		major (Strain: MHOM/IL/81/Friedlin)	5664	8038	1290	16,1
	UP000008153 Leishmania infantum (Strain: JPCM5)	5671	8045	1241	15,4
	UP000007258	Leishmania braziliensis (Strain: MHOM/BR/75/M2904)	5660	8084	981	12,1
	UP000008980 Leishmania donovani (strain BPK282A1)	981087	7960	1357	17,1

Table II . 5 List of exosome markers and genes found in the secretome

 II

	GeneIDs	Found in Secretome	[Gene Product]	[PFam Domains]	Domain name
	LbrM.15.1040; LbrM.33.0760;	LinJ.15.1060;
	LinJ.15.1060; LinJ.33.0770; LmjF.15.1000; LmjF.33.0720;	LinJ.33.0770; LmjF.15.1000;	60S ribosomal protein L6, putative	PF01159	Ribosomal protein L6e
	LmxM.15.1000; LmxM.32.0720	LmjF.33.0720
					tRNA synthetases class II
	LbrM.30.0540; LinJ.30.0470;	LinJ.30.0470;	aspartyl-tRNA	PF00152,	(D, K and N), OB-fold
	LmjF.30.0460; LmxM.29.0460;	LmxM.29.0460	synthetase, putative	PF01336	nucleic acid binding
					domain
	LbrM.32.0470; LinJ.32.0410; LmjF.32.0400; LmxM.31.0400; LinJ.15.1060	LinJ.32.0410; LmxM.31.0400	ATP-dependent RNA helicase, putative	PF00270, PF00271	DEAD/DEAH box helicase, Helicase conserved C-terminal domain
	LbrM.34.3140; LinJ.35.3280; LmjF.35.3230; LmxM.34.3230; LmxM.15.1000	LbrM.34.3140; LinJ.35.3280	cystathione gamma lyase, putative	PF01053	Cys/Met metabolism PLP-dependent enzyme
	LbrM.29.2180; LinJ.29.2310; LmjF.29.2200; LmxM.08_29.2200; LmjF.35.3230	LbrM.29.2180; LinJ.29.2310; LmjF.29.2200; LmxM.08_29.2200	GTP-binding protein, putative	PF00350, PF01031 PF02212,	Biotin-requiring enzyme
	LbrM.32.4110; LmxM.31.3870; LinJ.32.4020; LmjF.32.3870; LmjF.36.2660	LinJ.32.4020; LmxM.31.3870	myosin XXI	PF00063	Myosin head (motor domain)
	LbrM.11.0920; LinJ.11.1140; LmjF.11.1160; LmxM.11.1160; LinJ.32.4020	LmjF.11.1160	protein transport protein Sec31, putative	PF00400	WD domain, G-beta repeat
	LbrM.19.0760; LinJ.19.0440; LmjF.19.0440; LmxM.19.0440; LmjF.36.3660	LbrM.19.0760	nucleosome assembly protein, putative	PF00956	Nucleosome assembly protein (NAP)
	LbrM.31.1980; LinJ.31.1770; LmjF.31.1750; LmxM.30.1750; LmjF.11.1160	LinJ.31.1770; LmjF.11.1160	nucleosome assembly protein-like protein	PF00956	Nucleosome assembly protein (NAP)
	LbrM.14.0470; LinJ.14.0470; LmjF.14.0460; LmxM.14.0460; LmjF.30.0460	None found	cystathionine beta-lyase-like protein	PF01053	Cys/Met metabolism PLP-dependent enzyme
	LbrM.24.1520; LinJ.24.1400; LmjF.24.1360; LmxM.24.1360; LmjF.14.0460	None found	hypothetical protein, conserved	null
	LbrM.20.2180; LinJ.34.2450; LmjF.34.2620; LmxM.33.2620; LmjF.29.2200	None found	RNA helicase, putative,mitochondrial, putative	PF00271	Helicase conserved C-terminal domain
	LbrM.35.3890; LinJ.36.3840; LmjF.36.3660; LmxM.36.3660; LmjF.24.1360	None found	nudix hydrolase-like protein	PF00293	NUDIX domain
	LbrM.29.2720; LinJ.13.0260; LmjF.13.0260; LmxM.13.0260; LmjF.34.2620	None found	hypothetical protein, conserved	PF00583	Acetyltransferase (GNAT) family
	LbrM.31.2920; LinJ.31.2650; LmjF.31.2580; LmxM.30.2580; LmjF.13.0260	None found	ubiquinol-cytochrome-c reductase-like protein	PF02320	Ubiquinol-cytochrome C reductase hinge protein
	LbrM.20.0631; LinJ.34.0740; LmjF.34.0705; LmxM.33.0705; LmjF.19.0440	None found	hypothetical protein, conserved	PF09799	Predicted membrane protein

. From the 100 most abundant proteins associated with exosome cargo (Exocarta database) (59), 72 Leishmania ortholog genes were found (18 per species), and 20 of these were found in this dataset through OrthoMCL and TriTrypDB searches, corresponding to 9 different proteins. Yellow = proteins with signal peptide prediction (SignalP).

Table II . 6 Leishmania proteins with moonlighting functions in the MoonProt database

 II (64).

	UniProt ID	Protein Name	Function 1 (primary function)	Function 2 (moonlighting function)	Species Name
	Q3HL75 Enolase	enolase, enzyme reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate	binds plasminogen	Leishmania mexicana
	E9BTJ1	fructose-1,6-bisphosphate aldolase	aldolase, cleavage of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, in glycolysis	Activation of mouse (host) macrophage protein tyrosine phosphatase-1 (SHP-1), causes macrophage disfunction	Leishmania donovani
				binds to and activates Src homology 2
	Q95VF2	elongation factor 1-alpha	translation elongation factor, the rate and fidelity of protein translation	domain containing tyrosine phosphatase-1 (SHP-1) in host macrophages, inhibits	Leishmania donovani
				activity of infected macrophages
	Q95U89	mitochondrial 2-cysteine peroxiredoxin	peroxidase activity, detoxification of reactive oxygen species (ROS), removal of peroxide, use redox Cys) to reduce substrates like H2O2 active cysteine residue (peroxidatic	chaperone and activators of signal transduction cascades, prevents thermal aggregation of citrate synthase in vitro, mammalian host (37°C) more sensitive to temperature in the lack of expression makes promastigoes	Leishmania infantum

  5.0), as in Chapter II. The MS/MS spectra were matched against the Leishmania entries of UniProtKB SwissProt and TrEMBL sections (release 2016_05: 50997 entries with 229 SwissProt + 50768 TrEMBL). Again, a representative protein ID in each protein group was automatically selected using in-house bioinformatics tools (leading v2.2 and multi-species scripts) developed by Oana Vigy (Plateforme de Proteomique Fonctionnelle de Montpellier), and the Perseus software (version 1.5.3.0).

All Leishmania entries available in UniProtKB (UniProtKB release 2016_05) were used for tryptic peptide identification: four reference proteomes from Leishmania infantum strain JPCM5 (RefProteome_LEIIN-all_2016_01.fasta), Leishmania major strain MHOM/IL/81/Friedlin (RefProteome_LEIMA-all_2016_01.fasta), Leishmania braziliensis strain MHOM/BR/75/M2904 (RefProteome_LEIBR-all_2016_01.fasta), Leishmania mexicana strain MHOM/GT/2001/U1103, Leishmania donovani strain BPK282A1 (Proteome_LEIDB-all_2016_01.fasta). Also, all L.

Table III .2 (continued) Antigen category Individual proteins to search in proteomic datasets Description Histones

 III

			H2B
			H3
			H4
			LeIF (Leishmania elongation
			initiation factor)
			Putative eukaryotic translation initiation factor 2 subunit	donovani trypanothione reductase (LdTPR) induces lymphoproliferation and NO production in vitro, and protects hamsters
	Translation initiation factors		Putative eukaryotic translation initiation factor 3 subunit 8 Putative eukaryotic translation initiation factor 3 subunit 7	against challenge, but also, in human samples from active and cured VL LeIF protein belongs to the DEAD box protein family, it is homologous to patients. LdTPR induces PBMC lymphoproliferation, even if slightly milder. In eIF4A. There is evidence LeIF functions as a Th1-type natural adjuvant, cured VL samples, LdTPR also induced IL-12 and IFN-γ production, accompanied by low IL-10 levels, contrary to soluble L. donovani antigens, which induce a mixed Th1/Th2 response with higher IL-10 levels and low mimicking IL-12-mediated downregulation of IL-4 production in lymph node cultures.
			Probable eukaryotic initiation	IFN-γ.
			factor 4A; Short=eIF-4A;	Pxn1 and Pxn2 tested in mice exhibit differences in the elicited response.
		EC=3.6.4.13 Peroxidoxin 1 and 2 Eukaryotic initiation factor 5a Hexokinase (EC=2.7.1.1) Glucose-6-phosphate isomerase	Both are immunogenic, however Pxn1 induced a predominant Th2 response and high IgG1 whereas Pxn2 induced a mixed Th1/Th2 response with high IgG2a antibody levels. 3 forms in L. infantum with different immunological properties: cytosolic tryparedoxin peroxidase (LicTXNPx), mitochondrial tryparedoxin peroxidase
			Phosphofructokinase	(LimTXNPx), and tryparedoxin (LiTXN1). LiTXNPx elicits strong humoral
		Tryparedoxin peroxidase (also Fructose-biphosphate aldolase	response and has no influence on cytokine production. LmTXNPx, non-
	Glycolytic enzymes	known as TSA, thiol-specific antioxidant antigen) and mitochondrial tryparedoxin Triosephosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase peroxidase Phosphoglycerate kinase and	Leishmania glycolytic enzymes are compartmentalized in glycosomes, unique trypanosomatid organelles, and are phylogenetically distant from mammalian host enzymes, making them useful vaccine antigen candidates.
			mutase
			Phosphopyrvate hydratase
			(Enolase)	GP46, also known as M-2, is a promastigote surface membrane glycoprotein.
		GP46 Pyruvate kinase (EC=2.7.1.40)	When administered with adjuvant, it induces protective cellular immune
			mitogen activated protein (MAP)	responses.
	Surface glycoprotein	Promastigote surface antigen-2 kinase (Putative MAP kinase) CRK (creatine kinase) Putative surface antigen protein 2 Putative fucose kinase;	Promastigote Surface Antigen (PSA) exists in soluble and membrane-anchored form, and contains a leucine rich repeat (LRR) region which is an immunogenic epitope after protein fragmentation, inducing IFN-γ production and Th1 responses. Sequence analysis between PSA molecules from 9
	Other kinases	Putative surface antigen protein EC=2.7.1.52; Glycosomal phosphoenolpyruvate	different Leishmania species revealed highly conserved segments in the N-terminal region, while the central LRR domain and C-terminal regions are more divergent
		PFR-2C (paraflagellar rod carboxykinase (EC=4.1.1.-)
	Flagellar and flagelle-	protein) Putative nima-related protein kinase; EC=2.7.11.1; PFR-1D (paraflagellar rod Nucleoside diphosphate kinase; protein) EC=2.7.4.6;	Flagelar proteins are upregulated in promastigote forms. PFR-2 is a highly conserved immunogen inducing a Th1 immune response in dogs and mice. FPG was identified in a high-throughput in vitro immunological screening Leishmania parasites depend upon the purine salvage system to utilize purine bases from their mammalian hosts; for this purpose they utilize a
	associated proteins Enzymes involved in the purine salvage pathway	Flagellar glycoprotein-like protein (FPG) Lcr1 peptide (T-cell proliferation stimulating peptide) Putative adenosine kinase; EC=2.7.1.20; Nucleotide hydrolase	(with mouse splenocytes) and induced a predominant Th1 response (IL-12 and IFN-γ). Lcr1 is present in L. infantum amastigotes, it stimulates Th1 cytokines (IFN-γ) and antibodies, partially protecting BALB/c mice. variety of nucleoside transporters that can be the potent targets for vaccine development. During parasite-delayed macrophage apoptosis, Leishmania nucleoside diphosphate kinase can inhibit ATP binding to P2X receptors thereby preventing apoptosis. The nucleoside hydrolase (NH) is a glycoprotein, part of the Leishmania fucose-mannose ligand (FML) complex, and essential for early infection
				establishment. NH is a known vaccine antigen, known to induce protective
				immune responses in mice and dogs against experimental infection.
				DNA topoisomerases in the kinetoplastid parasites are mainly involved in
	Topoisomerases and other	DNA topoisomerase	kDNA replication, essential for the survival of parasite. These are, however, distinct from other eukaryotic counterparts, making them interesting antigen candidates.
	isomerases

secreted, decreases IL-4 secretion both in vitro and in vivo. LiTXN1 is poorly immunogenic and promotes induces IL-10 secretion both in vitro and in vivo favouring parasite internalization and survival. TSA elicits strong Th1 responses in infected BALB/c mice infected by L. major, and there is evidence for its immunogenicity in humans. TSA is abundantly present and homogeneously distributed on the promastigote surface. TSA-based DNA vaccine induced cytotoxicity and increased levels of IgG1 and IgG2a.

H2A

Histones are evolutionary conserved DNA-binding nuclear proteins. In Leishmania, these present relatively low homology with human proteins and are stably expressed by both amastigotes and promastigotes. H2B has been shown to be a promising candidate, inducing PBMC proliferation, IFN-γ production, promoting a Th1 protective immune response. H1 from L. infantum and L. braziliensis induce humoral responses Protein disulfide isomerase (PDI) PDI catalyses thiol-disulfide interchange to prevent cell toxicity associated with ER stress and protein misfolding.

Table III .2 (continued) Antigen category Individual proteins to search in proteomic datasets Description

 III

		Peptidyl-prolyl cis-trans
	Cyclophilins	isomerase; EC=5.2.1.8;
	(peptidyl-prolyl-isomerases)	Peptidyl-prolyl cis-trans isomerase; EC=5.2.1.8;
		(Cyclophilin-40)
		SMT (sterol 24-c-
		methyltransferase)
	Sterol 24-c	arginine methyltransferase
	methyltransferase	Putative carnitine/choline	SMT is involved in the final step of the sterol biosynthetic pathway in
	and other	acetyltransferase; EC=2.3.1.-	Leishmania, leading to the production of ergosterol.
	transferases	Aspartate aminotransferase
		serine hydroxy methyl
		transferase
		Cysteine protease A and B
		GP63 (surface metalloprotease) /
		MSP (major surface protease) /
		leishmanolysin
		Cathepsin B-like (cpB) and
		cathepsin L-like (cpL) cysteine
		proteases
		Ldp45 (Methionine
		aminopeptidase 45)
		Putative aminopeptidase
		(Metallo-peptidase, clan mf,
		family m17; EC=3.4.11.)
		Putative aminopeptidase;
		(Metallo-peptidase, clan ma(E),
	Proteases	family m1; EC=3.4.-.-) Putative aminopeptidase P
		(Metallo-peptidase, clan mg,
		family m24)
		Putative carboxypeptidase
		(Metallo-peptidase, clan ma(E),
		family 32)
		Putative thimet oligopeptidase
		(Metallo-peptidase, clan ma(E),
		family m3; EC=3.4.24.15)
		Putative dipeptidyl-peptidase III
		(Metallo-peptidase, clan m-,
		family m49; EC=3.4.14.4)
		Putative peptidyl dipeptidase
		(Metallo-peptidase, Clan MA(E),
		Family M3,putative, partial)

Cyclophilins possess peptidyl-prolyl cis-trans isomerase activity, can perform different functions and are known receptors for cyclosporine, an immunosuppressive drug. Leishmania presents different cyclophilin isoforms, not fully characterized. Immunization with the L. infantum recombinant cyclophilin protein-1 confers partial protection in mice and generates specific memory T cells.

Leishmania cysteine protease is the major protease involved in survival and adaptation to the host cell. Cysteine proteases have been shown to stimulate Th2 responses (IL-4 and IL-1). However, L. mexicana cysteine protease is a T-cell immunogen causes the development of protective Th1 responses (IL-12 and IFN-γ). GP63 or major surface protease (MSP) is a zinc protease and a key virulence factor responsible for evasion of the immune system, and degradation of fibronectin enhancing movement within the connective tissue (see chapter I). The cpL and cpB cysteine proteases are responsible for the lack of MHC-II invariant chain, therefore, modulating the cytokine production (increased IL-4 and IL-1) to the parasite's benefit. Also, the human host's papain-like cysteine proteases cathepsin A-like and B-like proteases, are targets of Leishmania immunomodulation and induction of Th2 responses. Ldp45 is involved in protein maturation. It is recognized by human T-cells and protects hamsters when formulated with BCG.

Table III .2 (continued) Antigen category Individual proteins to search in proteomic datasets Description

 III

	KMP11 (kinetoplastid membrane
	protein-11)
	Other antigens

Table III .3 (continued) Protein name REFS differential expression in stat.promast vs amastigotes? [Alcolea PJ 2010] Protein function (associated GO terms)

 III

	Putative eukaryotic translation initiation factor 5a	1, 2	N.D.
	Histone2A	1, 4	-	nucleosome [GO:0000786]; nucleus [GO:0005634]; DNA binding [GO:0003677]
	Histone H2B	1, 4	2,6 +-0,2	nucleosome [GO:0000786]; nucleus [GO:0005634]; DNA binding [GO:0003677]
	Protein disulfide isomerase-2 Histone H4 OTHER KINASES Nucleoside diphosphate kinase; EC=2.7.4.6; (Purine salvage system) Triosephosphate isomerase Putative adenosine kinase; EC=2.7.1.20; (Purine metabolism) Nucleoside hydrolase PFR-2C Pyruvate kinase; EC=2.7.1.40; (glycolytic enzyme) HSP60 Putative glycosomal phosphoenolpyruvate carboxykinase; EC=4.1.1.-; GP63 Metallo-peptidase, clan mf, family m17); EC=3.4.11.-; Putative hexokinase; EC=2.7.1.1; (glycolytic enzyme) Putative aminopeptidase (Metallo-peptidase, clan ma(E), family m1); EC=3.4.-.-; INITIATION FACTORS Putative eukaryotic translation initiation factor 2 subunit Putative dipeptidyl-peptidase III (Metallo-peptidase, clan m-, family m49); EC=3.4.14.4; Putative thimet oligopeptidase (Metallo-peptidase, clan ma(E), family m3); EC=3.4.24.15;	4, 5 1, 4 1 1 1 1, 2, 5 1 1 6 5 1 1, 2	--14+-0 ------N.D. ---N.D. -	endoplasmic reticulum [GO:0005783]; protein disulfide isomerase activity nucleosome [GO:0000786]; nucleus [GO:0005634]; DNA binding [GO:0003677] [GO:0003756]; cell redox homeostasis [GO:0045454] triose-phosphate isomerase activity [GO:0004807]; gluconeogenesis ATP binding [GO:0005524]; nucleoside diphosphate kinase activity [GO:0006094]; glycolytic process [GO:0006096]; pentose-phosphate shunt [GO:0004550]; CTP biosynthetic process [GO:0006241]; GTP biosynthetic [GO:0006098] process [GO:0006183]; UTP biosynthetic process [GO:0006228] hydrolase activity [GO:0016787] adenosine kinase activity [GO:0004001]; purine ribonucleoside salvage motile cilium [GO:0031514] [GO:0006166] kinase activity [GO:0016301]; magnesium ion binding [GO:0000287]; cytoplasm [GO:0005737]; ATP binding [GO:0005524]; protein refolding [GO:0042026] potassium ion binding [GO:0030955]; pyruvate kinase activity [GO:0004743] membrane [GO:0016020]; metalloendopeptidase activity [GO:0004222]; cell adhesion [GO:0007155] ATP binding [GO:0005524]; kinase activity [GO:0016301]; cytoplasm [GO:0005737]; aminopeptidase activity [GO:0004177]; phosphoenolpyruvate carboxykinase (ATP) activity [GO:0004612]; manganese ion binding [GO:0030145]; metalloexopeptidase activity gluconeogenesis [GO:0006094] [GO:0008235] cell [GO:0005623]; ATP binding [GO:0005524]; glucose binding [GO:0005536]; hexokinase activity [GO:0004396]; cellular glucose aminopeptidase activity [GO:0004177]; metallopeptidase activity [GO:0008237]; zinc ion binding [GO:0008270] homeostasis [GO:0001678]; glycolytic process [GO:0006096] cytoplasm [GO:0005737]; dipeptidyl-peptidase activity [GO:0008239]; metal ion binding [GO:0046872] GTP binding [GO:0005525]; GTPase activity [GO:0003924]; translation metal ion binding [GO:0046872]; metalloendopeptidase activity initiation factor activity [GO:0003743] [GO:0004222]
	Elongation factor 2	1	-	GTP binding [GO:0005525]; GTPase activity [GO:0003924]; translation elongation factor activity [GO:0003746]
	KMP11	1 -5
	Calpain-like cysteine protease	1

ribosome binding [GO:0043022]; translation elongation factor activity [GO:0003746]; translation initiation factor activity [GO:0003743]; positive regulation of translational elongation [GO:0045901]; positive regulation of translational termination [GO:0045905]; translational frameshifting [GO:0006452] eIF4A 1, 2, 5 -ATP binding [GO:0005524]; helicase activity [GO:0004386]; translation initiation factor activity [GO:0003743] HISTONES

Table III . 4 Deconvolution of peroxidoxin sequences annotated in the UniProt and Genbank databases.

 III

	Underlined sequences were selected.
	Species	Name	UniProt Acc Nr	UniProt GeneID	Genbank ID
		thiol-specific antioxidant antigen	A9LJZ6_LEIMA	TSA	ABX11567.1.
		thiol-specific antioxidant antigen	Q4QF68_LEIMA	TRYP7	AAC31146.1
		tryparedoxin peroxidase	Q4QF74_LEIMA	TRYP4	CAJ03332.1
		tryparedoxin peroxidase	Q4QF80_LEIMA	TRYP1	CAJ03330.1
	L. major	tryparedoxin peroxidase	Q4QF76_LEIMA	TRYP3	CAJ03334.1
		tryparedoxin peroxidase	A9LNR9_LEIMA	TRYP6	ABX26130.1
		peroxidoxin	Q9TZS4_LEIMA	N/A	AAC79432.1
		peroxidoxin	Q4QBH2_LEIMA	LMJF_23_0040	CAJ03825.1
		Tryparedoxin	E9ADX3_LEIMA	TXN2 / LMJF_29_1150	CBZ12452.1
		thiol-specific antioxidant antigen	Q8MU50_LEIIN	N/A	AAK58478.1
		tryparedoxin peroxidase	A4HWK3_LEIIN	TRYP / LINJ_15_1120	CAM66832.1
		tryparedoxin peroxidase	A4HWK2_LEIIN	TRYP / LINJ_15_1100	CAM66830.1
	L.	cytosolic peroxiredoxin	Q95NF5_LEIIN	TRYP / LINJ_15_1140	AAL25847.1
	infantum	putative mitochondrial peroxiredoxin	Q95U89_LEIIN	mTXNPx / LINJ_23_0050	AAL25846.1
		Tryparedoxin	Q6RYT3_LEIIN	TXN1 / LINJ_29_1250	AAS48350.1./ CAM69684.1.
	L.	peroxidoxin 1	Q07DU6_LEITR	Pxn1	AAZ23600.1
	tropica	peroxidoxin 2	Q07DU5_LEITR	Pxn2	AAZ23601.1

Table IV . 1 Determinants of immunodominance

 IV

. Adapted from (3).

Table IV . 4 HLA-class I alleles included in T-cell epitope prediction (45).

 IV

	Supertype group	Allele	Predictor (I = IEDB/ N=NetMHCpan / S=SYFPEITHI)	Population frequency of allele	Allele specific affinity cut-off (IC50 nM)
	A1	A*0101	I + N + S	16.2
		A*3002	I	5
		A*3201	I	5.7
	A2	A*0201	I + N + S	25.2
		A*0203	I	3.3	92
		A*0206	I	4.9	60
		A*0211	I
	A3	A*0301	I + N + S	15.4
		A*1101	I + S	12.9
		A*3001	I	5.1
		A*3101	I	4.7
		A*3301	I	3.2
		A*6601	I
		A*6801	I + S	4.6
	A24	A*2301	I	6.4
		A*2402	I + N + S	16.8
	A26	A*2601	I + N + S	4.7
	B7	B*0702	I + N + S	13.3
		B*3501	I + S	6.5
		B*3503	I	1.2
		B*5101	I + S	5.5
		B*5301	I + S	5.4
	B8	B*0801	I + N + S	11.5
	B27	B*1402	I + S	2.8
		B*2705	I + N + S	2
		B*3801	I + S	2
		B*4801	I	1.8
	B44	B*1801	I + S	4.4
		B*4001	I + N + S	10.3
		B*4402	I + S	9.2
		B*4403	I	7.6
		B*4501	I + S
	B58	B*5801	I + N + S	3.6
		B*5701	I + S	3.2
		B*1517	I
	B62	B*1501	I + N + S	5.2

  pipeline, from the in silico epitope selection to the immunoscreenings exclusively in human samples, swiftly delivered some potentially interesting vaccine candidates.Although still presenting some limitations, experimental validation with human samples from naive and healed individuals is conclusive regarding future in vivo immunogenicity of vaccine candidates, and it may accelerate clinical trial testing while preventing late-stage failure. This approach particularly benefits vaccine research against leishmaniases, since experience shows a development of new or more effective tools for leishmaniasis control is challenging and is often not a priority for standard product development pipelines, as the market does not allow the recovery of development costs, similarly to other NTDs. Furthermore, leishmaniasis is associated with poverty, which implies that costs are determinant for the feasibility of any tool or

	product (diagnosis, treatment, prevention). Also, when new products are available, these will
	likely need government and/or philanthropic subsidisation, and access in endemic areas must be
	facilitated.
	Nevertheless, improving NTD control, including vaccine development against
	leishmaniasis, is part of the Millennium Development Goal 3, a United Nations initiative (target
	3.3: by 2030, end the epidemics of AIDS, tuberculosis, malaria and neglected tropical diseases and
	combat hepatitis, water-borne diseases and other communicable diseases), and has received
	increasing focus and resources by pharmaceutical companies and other stake-holders.
	CHAPTER VI
	GENERAL DISCUSSION AND
	FUTURE PERSPECTIVES

development gap between promising vaccine candidates tested in animal challenge experiments and de facto candidate immunogenicity in humans living in endemic areas, This evaluation model for preclinical development of peptide-based vaccines, especially in the context of NTDs, provides a powerful approach to fast-track the development and deployment of effective tools that will assist leishmaniasis control and improve Public Health programmes in affected areas.

The

Table 1 Population studies from AFND included in the HLA allele frequency analysis of the South and Central America World region. World region: SOUTH AND CENTRAL AMERICA

 1

						NORMALISED
	LEADING_CHECK	CASE_CHECK	DESC_CHECK	iBAQ	LOG10	TO LOG10
						AVERAGE
		LEADING_SPECIES
	Q9GP00	-DO NOT	Nucleoside diphosphate kinase	2552100000 9,4069	1,5186
		CHANGE!
	D1GJ50	LEADING_AUTO -DO NOT CHANGE!	Soluble promastigote surface antigen PSA-38S	1908300000 9,2806	1,4982
	A4I8U5	Multi SPECIES	Tubulin beta chain	1456100000 9,1632	1,4792
	A4HTR1	Multi SPECIES	Tubulin beta chain	1456100000 9,1632	1,4792
	A4H727	LEADING_SPECIES -DO NOT CHANGE!	Tubulin alpha chain	1090500000 9,0376	1,4590
	Q4QFP8	LEADING_AUTO -DO NOT CHANGE!	Putative small myristoylated protein-3		8,8223	1,4242
	P69201	LEADING_AUTO -DO NOT CHANGE!	Ubiquitin-60S ribosomal protein L40		8,7815	1,4176
	A4H3U0	LEADING_AUTO -DO NOT CHANGE!	Uncharacterized protein		8,7652	1,4150
	Q25307	LEADING_AUTO -DO NOT CHANGE!	Infective insect stage-specific protein		8,7630	1,4146
	Q07DU4	LEADING_AUTO -DO NOT CHANGE!	Peroxidoxin 2		8,7620	1,4145
	D1GJ46	LEADING_AUTO -DO NOT CHANGE!	Soluble promastigote surface antigen PSA-34S (Fragment)		8,7261	1,4087
	A4HPQ9	Multi SPECIES	Adenosylhomocysteinase		8,6863	1,4023
	A4HPQ8	Multi SPECIES	Adenosylhomocysteinase		8,6863	1,4023
	Q4Q7Y4	LEADING_AUTO -DO NOT CHANGE!	Putative heat-shock protein hsp70		8,6839	1,4019
	E9ARD0	LEADING_AUTO -DO NOT CHANGE!	Elongation factor 1-alpha		8,6828	1,4017
	O02614	LEADING_AUTO -DO NOT CHANGE!	Peptidyl-prolyl cis-trans isomerase		8,6613	1,3982
	A4H3V1	LEADING_AUTO -DO NOT CHANGE!	Putative beta-fructofuranosidase		8,6552	1,3972
	A4I058	LEADING_SPECIES -DO NOT CHANGE!	Putative endoribonuclease L-PSP (Pb5)		8,6306	1,3933
	O43990	LEADING_SPECIES -DO NOT CHANGE!	Infective insect stage-specific protein		8,5945	1,3874
	P15706	LEADING_AUTO -DO NOT CHANGE!	Leishmanolysin		8,5636	1,3824
	A4I7Z7	LEADING_SPECIES -DO NOT CHANGE!	Superoxide dismutase		8,5399	1,3786
	A4HYX4	LEADING_AUTO -DO NOT CHANGE!	Putative small myristoylated protein-1		8,5391	1,3785
	Q4QJ67	LEADING_AUTO -DO NOT CHANGE!	Peptidyl-prolyl cis-trans isomerase		8,5183	1,3751
	A4HW98	LEADING_SPECIES -DO NOT CHANGE!	Histone H4		8,5146	1,3745
	Q27673	LEADING_AUTO -DO NOT CHANGE!	Leishmanolysin		8,5036	1,3728
	Q4QDL6	Multi SPECIES	Histone H2B		8,4884	1,3703
	Q9BJC7	Multi SPECIES	Histone H2B		8,4884	1,3703
	Q4QHP1	Multi SPECIES	Histone H2B		8,4884	1,3703

Table 1 (continued)

 1

	Population	Ethnicity	Study	Sample Size	HLA Loci
	Brazil Guarani Nandeva	Amerindian HLA	86	DQA1, DQB1, DRB1
	Brazil Kaingang	Amerindian HLA	235	DQA1, DQB1, DRB1
	Brazil Mato Grosso do Sul	Mixed	HLA	203	DRB1
	Brazil Minas Gerais State Mixed	Mixed	HLA	1	A, B, DRB1
	Brazil North East Mixed	Mixed	HLA	205	DQA1, DQB1, DRB1
	Brazil Parana Afro Brazilian	Black	HLA	77	A, B, DRB1
	Brazil Parana Cafuzo	Mixed	HLA	319	A, B, DRB1
	Brazil Parana Caucasian	Caucasoid	HLA	2775	A, B, DRB1
	Brazil Parana Japanese		HLA	192	A B C
	Brazil Parana Mulatto	Mulatto	HLA	186	A, B, DRB1
	Brazil Parana Oriental	Oriental	HLA	33	A, B, DRB1
	Brazil Pernambuco Mixed	Mixed	HLA	101	A, B, C
	Brazil Piaui Mixed	Mixed	HLA	21943	A, B, DRB1
	Brazil REDOME Acre	Mixed	HLA	859	A, B, DRB1
	Brazil REDOME Alagoas	Mixed	HLA	25349	A, B, DRB1
	Brazil REDOME Amapa	Mixed	HLA	17,864 A, B, DRB1
	Brazil REDOME Amazonas	Mixed	HLA	24,129 A, B, DRB1
	Brazil REDOME Bahia	Mixed	HLA	47399	A, B, DRB1
	Brazil REDOME Ceara	Mixed	HLA	101217 A, B, DRB1
	Brazil REDOME Espirito Santo	Mixed	HLA	88,485 A, B, DRB1
	Brazil REDOME Federal District	Mixed	HLA	29549	A, B, DRB1
	Brazil REDOME Goias	Mixed	HLA	88,574 A, B, DRB1
	Brazil REDOME Maranhao	Mixed	HLA	10180	A, B, DRB1
	Brazil REDOME Mato Grosso	Mixed	HLA	34649	A, B, DRB1
	Brazil REDOME Mato Grosso do Sul Mixed	HLA	95667	A, B, DRB1
	Brazil REDOME Minas Gerais	Mixed	HLA	211275 A, B, DRB1
	Brazil REDOME Para	Mixed	HLA	72637	A, B, DRB1
	Brazil REDOME Paraiba	Mixed	HLA	43868	A, B, DRB1
	Brazil REDOME Parana	Mixed	HLA	341639 A, B, DRB1
	Brazil REDOME Pernambuco	Mixed	HLA	92332	A, B, DRB1
	Brazil REDOME Piaui	Mixed	HLA	46140	A, B, DRB1
	Brazil REDOME Rio de Janeiro	Mixed	HLA	139322 A, B, DRB1
	Brazil REDOME Rio Grande do Norte	Mixed	HLA	46603	A, B, DRB1
	Brazil REDOME Rio Grande do Sul	Mixed	HLA	241329 A, B, DRB1
	Brazil REDOME Rondonia	Mixed	HLA	54396	A, B, DRB1
	Brazil REDOME Roraima	Mixed	HLA	4140	A, B, DRB1
	Brazil REDOME Santa Catarina	Mixed	HLA	106673 A, B, DRB1
	Brazil REDOME Sao Paulo	Mixed	HLA	800809 A, B, DRB1

Table 1 (continued)

 1

	Population	Ethnicity	Study	Sample Size	HLA Loci
	Brazil REDOME Sergipe	Mixed	HLA	7321	A, B, DRB1
	Brazil REDOME Tocantins	Mixed	HLA	20692	A, B, DRB1
	Brazil Rio Grande do Norte Mestizo	Mestizo	HLA	12973	A, B, DRB1
	Brazil Rio Grande do Sul Black	Black	HLA	248	A, B, DRB1
	Brazil Rio Grande do Sul Caucasoid Caucasoid	HLA	4428	A, B, DRB1
	Brazil Rio Grande do Sul Mixed	Mixed	HLA	324	A, B, DRB1
	Brazil Sao Paulo Bauru Mixed	Mixed	HLA	3542	A, B, DRB1
	Brazil Sao Paulo Mixed	Mixed	HLA	239	A, B, DQA1, DQB1, DRB1
	Brazil South East Cord Bllod	Unknown	HLA	11409	A B DRB1
	Brazil South Ribeirao Preto	Mixed	HLA	184	A, B, DRB1
	Brazil Southeast Campinas Mixed	Mixed	HLA	99	DRB1
	Chile Easter Island	Amerindian HLA	21	A, B, C, DRB1
	Chile Huilliche	Amerindian HLA	40	DRB1
	Chile Santiago		HLA	920	A, B, DRB1
	Chile Santiago Mixed	Mixed	HLA	70	A, B, C, DQB1, DRB1
	Colombia Antioquia Paisa	Amerindian HLA	100	DQB1, DRB1
	Colombia Barranquilla		HLA	188	A, B, DRB1
	Colombia Bogota and Medellin Mestizo	Mestizo	HLA	65	DQB1, DRB1
	Colombia Guajira Peninsula Wayuu	Amerindian HLA	88	DQA1, DQB1, DRB1
	Colombia Jaidukama		HLA	39	A, B, DQB1, DRB1
	Colombia Northwest Tule	Amerindian HLA	29	DPB1, DQA1, DQB1, DRB1
	Colombia San Basilio de Palenque		HLA	42	A B DQB1 DRB1
	Colombia Sierra Nevada de Santa Marta Arhuaco	Amerindian HLA	107	DQA1, DQB1, DRB1
	Colombia Sierra Nevada de Santa Marta Arsario	Amerindian HLA	18	DQA1, DQB1, DRB1
	Colombia Sierra Nevada de Santa Marta Kogi	Amerindian HLA	42	DQA1, DQB1, DRB1
	Colombia Wayu from Guajira Peninsula	Wayu	HLA	48	A B DQB1 DRB1
	Costa Rica Central Valley Mestizo	Mestizo	HLA	130	A, B, C, DQB1, DRB1, DRB3 DRB4 DRB5
	Cuba Caucasian	Caucasoid	HLA	70	A, B
	Cuba Mixed	Mixed	HLA	78	A, B, DQA1, DQB1, DRB1
	Cuba Mixed pop 2	Mixed	HLA	189	A, B, C, DRB1
	Cuba Mulatto	Mulatto	HLA	42	A, B
	Ecuador African	Black	HLA	58	DPA1, DPB1, DQA1, DQB1
	Ecuador Amazonia Mixed Ancestry		HLA	39	A, B, DQB1, DRB1
	Ecuador Andes Mixed Ancestry		HLA	824	A, B, DQB1, DRB1
	Ecuador Cayapa	Amerindian HLA	183	A, DPB1, DQA1, DQB1, DRB1
	Ecuador Coast Mixed Ancestry		HLA	238	A, B, DQB1, DRB1
	Ecuador Mixed Ancestry		HLA	1173	A, B, DQB1, DRB1
	Guatemala Mayan	Amerindian HLA	132	A, B, DQB1, DRB1
	Jamaica	Black	HLA	132	DQA1, DQB1, DRB1

Table 2 Population studies from AFND included in the HLA allele frequency analysis for the North Africa World region. World region: NORTH AFRICA

 2

	Population	Ethnicity	Study	Sample Size	HLA Loci
	Algeria pop 2	Arab	HLA	106	A, B, C, DQA1, DQB1, DRB1
	Ethiopia Amhara	Black	HLA	98	DQA1, DQB1, DRB1
	Ethiopia Oromo	Black	HLA	83	DQA1, DQB1, DRB1
	Morocco	Arab	HLA	96	A, B, DQA1, DQB1, DRB1
	Morocco Casablanca	Arab	HLA	100	A, B
	Morocco pop 2	Mixed	HLA	110	A, B, DQB1, DRB1
	Morocco Souss Region	Arab	HLA	98	DQA1, DQB1, DRB1
	Sudan Central Shaigiya Mixed Mixed	HLA	36	A, B, C, DRB1
	Sudan East Rashaida	Arab	HLA	27	A, B, C, DRB1
	Sudan Mixed	Mixed	HLA	200	A, B, C, DPB1, DQB1, DRB1
	Sudan South Nuba	Black	HLA	46	A, B, C, DRB1
	Tunisia	Arab	HLA	100	A, B, C, DPB1, DQA1, DQB1, DRB1
	Tunisia Ghannouch	Arab	HLA	82	A, B, DQB1, DRB1
	Tunisia pop 3	Arab	HLA	104	A, B, DQB1, DRB1

Table 3 Population studies from AFND included in the HLA allele frequency analysis for the Western Asia World region. World region: WESTERN ASIA

 3

	Population	Ethnicity	Study	Sample Size	HLA Loci
	Armenia combined Regions Arab	HLA	100	A, B, DRB1
	Armenia Ghegharkunik	Arab	HLA	242	A, B, DRB1
	Armenia living in Iran	Arab	HLA	85	A, B, DRB1
	Armenia living in Karabakh	Arab	HLA	445	A, B, DRB1
	Armenia living in Lebanon	Arab	HLA	368	A, B, DRB1
	Armenia living in USA	Arab	HLA	233	A, B, DRB1
	Armenia Lori	Arab	HLA	102	A, B, DRB1
	Armenia Shirak	Arab	HLA	76	A, B, DRB1
	Armenia Syunik	Arab	HLA	117	A, B, DRB1
	Armenia Yerevan	Arab	HLA	445	A, B, DRB1
	Gaza Palestinians	Arab	HLA	165	A, B, DQB1, DRB1
	Georgia Kurds	Kurds	HLA	30	A, B, DQB1, DRB1
	Georgia Svaneti Region Svan	Caucasoid HLA	80	A, B, DPB1, DQA1, DQB1, DRB1
	Iran	Persian	HLA	58	DQA1, DQB1, DRB1
	Iran Baloch	Persian	HLA	100	A, B, C, DQA1, DQB1, DRB1
	Iran pop 1	Arab	HLA	64	A, B
	Iran pop 3	Arab	HLA	100	DQB1, DRB1

Table 3 (continued)

 3

	Population	Ethnicity	Study	Sample Size	HLA Loci
	Iran Royan Cord Blood Bank	Persian	HLA	15600	A, B, DRB1
	Iran Tehran	Persian	HLA	120	C
	Iraq Erbil		HLA	372	A B C DQB1 DRB1
	Israel Arab	Arab	HLA	109	DQA1, DQB1, DRB1
	Israel Arab		HLA	12301	DRB1, A, B
	Israel Ashkenazi Jews	Jew	HLA	80	DQA1, DQB1, DRB1
	Israel Ashkenazi Jews		HLA	4625	DRB1, A, B
	Israel Ashkenazi Jews pop 2	Jew	HLA	132	A, B, DQA1, DQB1, DRB1
	Israel Ethiopian Jews	Jew	HLA	122	DQA1, DQB1, DRB1
	Israel Iranian Jews	Jew	HLA	101	DQA1, DQB1, DRB1
	Israel Jews pop 2	Jew	HLA	23000	A, B, DRB1
	Israel Libyan Jews	Jew	HLA	119	DQA1, DQB1, DRB1
	Israel Moroccan Jews	Jew	HLA	113	A, B, DQA1, DQB1, DRB1
	Israel Non Ashkenazi Jews	Jew	HLA	80	DQA1, DQB1, DRB1
	Israel Yemenite Jews	Jew	HLA	76	DQA1, DQB1, DRB1
	Jordan	Arab	HLA	15141	A, B, DRB1
	Jordan Amman	Arab	HLA	146	A, B, C, DQA1, DQB1, DRB1
	Iraq Kurdistan Region	Kurd	HLA	209	A, B, C, DQB1, DRB1
	Lebanon Kafar Zubian	Arab	HLA	94	DPB1, DQB1, DRB1
	Lebanon Niha el Shouff	Arab	HLA	61	DPB1, DQB1, DRB1
	Lebanon Yuhmur	Arab	HLA	82	DPB1, DQB1, DRB1
	Oman	Arab	HLA	118	A, B
	Saudi Arabia	Arab	HLA	18	A, B, DPB1, DQB1, DRB1
	Saudi Arabia pop 2	Arab	HLA	383	A, B, DQB1, DRB1
	Saudi Arabia pop 4	Arab	HLA	499	A, B, C, DQB1, DRB1
	Turkey Ankara	Caucasoid HLA	50	DQA1, DQB1, DRB1
	Turkey Istanbul	Caucasoid HLA	250	DRB1
	Turkey pop 1	Caucasoid HLA	250	DQA1, DQB1, DRB1
	Turkey pop 2	Caucasoid HLA	228	A, B, DQB1, DRB1
	Turkey pop 3	Caucasoid HLA	50	DQA1, DQB1
	Turkey pop 5	Caucasoid HLA	142	A, B, C
	United Arab Emirates pop 2	Arab	HLA	373	A, B, DRB1

Table 4 Population studies from AFND included in the HLA allele frequency analysis for the South Asia World region. World Region: SOUTH ASIA

 4

	Population	Ethnicity	Study	Sample Size	HLA Loci
	Bangladesh Dhaka Bangalee	Asian	HLA	141	A, B, DRB1
	India Andhra Pradesh Brahmin	Asian	HLA	98	DRB1
	India Andhra Pradesh Sunni	Asian	HLA	100	DRB1
	India Bombay	Asian	HLA	59	DPA1, DPB1, DQA1, DQB1
	India Delhi pop 2	Asian	HLA	90	A, B, C
	India Jalpaiguri Toto	Asian	HLA	40	A, B
	India Kerala Adiya	Asian	HLA	21	B, C
	India Kerala Hindu Ezhava	Asian	HLA	24	A, B, C
	India Kerala Hindu Nair	Asian	HLA	41	A, B, C
	India Kerala Hindu Namboothiri	Asian	HLA	40	A, B, C
	India Kerala Hindu Pulaya	Asian	HLA	16	A, B, C
	India Kerala Kanikkar	Asian	HLA	22	B, C
	India Kerala Kattunaikka	Asian	HLA	17	B, C
	India Kerala Kurichiya	Asian	HLA	10	B, C
	India Kerala Kuruma	Asian	HLA	15	B, C
	India Kerala Malabar Muslim	Asian	HLA	34	A, B, C
	India Kerala Malapandaram	Asian	HLA	10	B, C
	India Kerala Paniya	Asian	HLA	10	B, C
	India Kerala Syrian Christian	Asian	HLA	31	A, B, C
	India Lucknow	Asian	HLA	123	DPB1, DQA1, DQB1, DRB1
	India New Delhi pop 2	Asian	HLA	102	DQB1, DRB1
	India North	Asian	HLA	85	DRB1
	India North Gujarat	Asian	HLA	338	A, B, C, DQB1, DRB1
	India North pop 2	Asian	HLA	72	A, B, C, DQA1, DQB1, DRB1
	India North pop 3	Asian	HLA	587	A, B, DRB1
	India Northeast Kayastha	Asian	HLA	190	DQA1, DQB1, DRB1
	India Northeast Lachung	Asian	HLA	58	DQA1, DQB1, DRB1
	India Northeast Mathur	Asian	HLA	155	DQA1, DQB1, DRB1
	India Northeast Mech	Asian	HLA	63	DQA1, DQB1, DRB1
	India Northeast Rajbanshi	Asian	HLA	98	DQA1, DQB1, DRB1
	India Northeast Rastogi	Asian	HLA	196	DQA1, DQB1, DRB1
	India Northeast Shia	Asian	HLA	190	DQA1, DQB1, DRB1
	India Northeast Sunni	Asian	HLA	188	DQA1, DQB1, DRB1
	India Northeast Vaish	Asian	HLA	198	DQA1, DQB1, DRB1
	India Tamil Nadu Chennai	Asian	HLA	137	DRB1
	India Tamil Nadu Dravidian	Asian	HLA	156	DRB1
	India Uttar Pradesh	Asian	HLA	202	DQA1, DQB1, DRB1

Table 4 (continued)

 4

	Population	Ethnicity	Study	Sample Size	HLA Loci
	Pakistan Baloch	Asian	HLA	66	A,

Table I. 4 Leishmania-specific peptide vaccine candidates validated using human samples. 'Ref', bibliographical references.

Table I.4 (continued)

Figure I. 19 Overview of the project's work plan, the key challenges or bottlenecks and how these will be addressed.

Trop Dis. Public Library of Science; 2016 Jun 28;10(6):e0004811.

Table III.5 (continued)

Table III. 6 ClustalOmega alignment results (percent identity matrix) for 33 uncharacterized proteins identified with the reverse vaccinology approach

Table III.7 (continued)

Table IV. 3 (continued)

Figure IV. 6 Peptide toxicity assay results. Peptide toxicity was assessed for 49 9-mer peptides in human total PBMC at three concentrations (1 or 5 µM, 10 µM and 25 µM) with PrestoBlue ® Viability dye after 20-hour incubation.

Table V. 7 Personal information and medical history from recruited healed donors (TUN1-TUN20).

Table V.7 (continued)

Table V. 8 HLA-typing results for the first healed donor series (TUN1-TUN3 to TUN10). A) Six loci corresponding to HLA-class I and class II polymorphic regions (HLA-A, -B, -C, -DRB1, -DPB1, -DQB1) were sequenced by NGS (exons 2 and 3), leading to the 6-digit identification of the alleles expressed by each donor. Allele supertype classification bySidney et al 2008 (20). B) Ambiguous identifications are given a multiple allele code (MAC, https://hml.nmdp.org/MacUI/)

Figure V. 25 Double-positive immunogenic peptides (peptides present in two positive matrix-based pools). A) 25 class I immunogenic peptides identified in 6 healed donors. B) 14 class II immunogenic peptides identified in 7 healed donors. C) Selected class I and class II peptides per protein antigen and peptides found to be immunogenic in samples from healed donors.

ACKNOWLEDGEMENTS

Undertaking this PhD has been a eventful and exciting experience and it would not have been possible without the support and guidance that I received. This project has received funding from the European Union's Horizon 2020 research and

Objective: to discover T-cell epitopes present in antigens from the Leishmania secretome capable of inducing cellular immune responses mediated by TCD8 + and TCD4 + cells.

Aims:

-To select the best performing immunoinformatic tools; -To perform epitope prediction targeting world HLA coverage; -To select immunogenic epitopes according to in silico prediction data. Availability MHCPEP Objectives: to assess T-cell-dependent peptide immunogenicity and discover Leishmania-specific epitopes restricted to HLA-class I and -class II alleles for vaccine development, using immunoassays exclusively with human samples.

Aims:

to evaluate peptide in vitro immunogenicity in both naive and healed immune backgrounds;

to validate the in silico epitope predictions;

to select the most immunogenic peptides that will proceed for multi-epitope peptide design HLA-class II-restricted peptide pii19 (13_b12) was immunogenic for 6 donors, becoming undoubtedly the most promising peptide candidate so far (Figure V.25 panel B). HLA-class II peptides pii4 (22_b4) and pii16 (4_a11) were immunogenic for 3 donors. Two HLA-class II peptides (pii14/17_b15 and pii1/14_b12) and seven HLA-class I peptides (pi8/B27_1, pi12/B58_4, pi22/A1_1, pi29/A24_1, pi36/A2_4, pi37/b4_B8, pi43/B58_5) were immunogenic for two donors. The remaining nine double-positive HLA-class II peptides and eighteen HLA-class I peptides were immunogenic only for one donor (Figure V.25).

antigen and its responding CD4 + T cells. Sci. Transl. Med. 7, 310ra167 (7). After listing all vaccine antigen candidates cited in the selected publications, a manual search was performed in the secretome datasets, either by protein accession or by protein name, as follows:

Table III. 1 Initial list of vaccine antigen candidates found in the

i) A word search by protein name was performed on the 3 already available secretome proteomic datasets from experiment AESLMTS_110414 (L. infantum, L. major and L.

tropica datasets analysed with the UniProtKB database release 2011_04 (SwissProt_TrEMBL_2011_04: 15,082,690 sequences; 4,872,194,356 residues / Leishmania 42,035 sequences). All associated identifiers (protein entry numbers) were retrieved from all match groups and respective identified tryptic peptides, so that each protein contains at least 3 species-specific sequences (protein x = ID1 + ID2 + ID3 + …).

ii) The identified tryptic peptides were aligned with the UniProtKB protein sequences (through ClustalOmega alignments) to check if all peptides are found and to check for unique residues or peptides.

iii) For L. tropica, with less annotated proteins in UniProtKB, proteins are often identified as one of the species with complete or reference proteome in UniProtKB. To include the maximal number of species-specific sequences and include L. tropica-specific sequences, the coding genes were searched in the TriTrypDB. The corresponding translated proteins were retrieved and aligned with identified tryptic peptidesif the sequences contained the tryptic peptides, they were included as L. tropica species-specific sequence. The sequences of all 52 protein antigens, including 4 to 6 species-specific sequences, were retrieved from UniProtKB in fasta format. HLA-binding predictions were performed separately for each protein from Sets A and B.

CHAPTER IV

IN SILICO EPITOPE DISCOVERY AND SELECTION

HLA-binding predictions were performed through the online servers, where the fasta files were uploaded per protein. -HLA-class I predictions files contain 4 columns (allele|peptide|seq_num|score), and are named as 'proteincode_predictorcode.csv', e.g. "a1_iedb.csv".

-HLA-class II prediction files contain 6 columns (allele|seq_num|full_peptide|rank|core|ic50)

and are named as 'proteincode_predictorcode.csv', e.g. "a1_iedbii.csv".

The data generated by each algorithm for each protein antigen were saved as .csv files and integrated in the epitope selection script developed in R.

we compare different predictors and consider all potential cores within a 15-mer peptide, selecting the best core/full_peptide combination according to predicted IC50.

HLA-class I peptides are mostly water-soluble

From the 50 HLA-class I peptides ordered, one was never successfully synthesized (B44_2): against the human and mouse proteomes, 19 peptides showed no similarity and were selected but not validated experimentally.

Overall, these RV studies focus on mouse alleles instead of human alleles (except John L et al), so extrapolation for human immune responses is very limited. Also, the filtering criteria used (proteasomal cleavage, SignalP and PSORT predictions) are likely to reject important Leishmaniaspecific proteins and/or epitopes. Discovered peptides were either validated in the mouse model or not at all.

In 2016, Freitas e Silva et al performed a reverse vaccinology approach followed by peptide validation in human samples. This study is detailed in chapter I -Table I.4 (Leishmania-specific peptide vaccine candidates validated using human samples) and describes the most complete RV approach. It includes the proteomes of three Leishmania species (L. braziliensis, L. major, and L. infantum), it uses one of the best available HLA-binding prediction algorithms (NetMHC), BLASTp analysis and structure-based epitope prediction. However, the proposed pipeline uses intensive computational analysis, namely structural modelling to HLA molecules, which is difficult to reproduce due to its extensive computational resource requirements.

We believe the successful identification of immunogenic epitopes depends on both the quality of used immunoinformatic algorithms and on the rationality of epitope selection criteria. These criteria should be permissive enough not to falsely reject immunogenic epitopes, and restrictive enough to effectively filter HLA-binding prediction data. Hence, there is an optimal balance between the used tools and the criteria chosen to filter their results, which sought to optimise. Overall, our proposed reverse vaccinology pipeline uses proteomic information from six Leishmania species, the most clinically relevant ones (L. braziliensis, L. major, L. infantum, L. donovani, L.amazonensis and L. tropica), it makes use of BLASTp analysis and the best available HLA-binding prediction algorithms.

Because our starting sample is the Leishmania secretome, an optimal antigenic source, no subcellular localization filters need to be applied.

The best sequence-based HLA-binding prediction tools currently available were used in this study (24)(25)(26)(27). Structure-based epitope prediction tools are also available, which combine binding data, sequence information, known crystallographic data of pMHC complexes, and computational modelling. However, structure-based prediction tools only apply to the few alleles with crystallographic data and PDB files (Protein Data Bank), thereby excluding alleles and affecting vaccine coverage or introducing bias in predictions for other alleles (56). Moreover, these tools require great computational processing power and time. Altogether, it is more practical and accurate to use sequence-based pan-specific methods and based on machine-learning algorithms for epitope prediction.

Some previously described Leishmania protein antigens were excluded from epitope prediction analysis. The antigen KMP11 was removed due to ambiguous results showing evidence of induced Th2 responses (57). Also, the LACK antigen was excluded since it may have an important role in parasite immunomodulation of host immune responses. Interestingly, studies have shown that LACK-specific TCD4 + cells are present in individuals never exposed to Leishmania (4,5). These cells are primed in gut-associated lymphoid tissues by cross-reactive microbial antigens and are able to quickly secrete IL-4 (6). Finally, the promastigote surface antigen was not included in the present epitope prediction analysis because this analysis previously performed by our team, both soluble and membrane-associated isoforms (58)(59)(60).

Furthermore, after performing in silico epitope prediction, the ability to compare different data sources and to synergistically combine diverse algorithms in the context of epitope prediction remains challenging. To this end, the selection script developed in R greatly assisted in data integration and epitope selection.

The binding affinity and peptide conservation filters applied to the result table allowed a 90% reduction of the initial epitope list. Despite this broad selection, testing over 2100 9-mer peptides or over 840 15-mer peptides is still an issue for the experimental validation assays and experiment costs.

To further reduce this list, peptides were selected by relative comparison according to the predicted HLA restriction, promiscuity and binding affinity to select the best epitopes per supertype or allele.

Binding affinity is a key characteristic of peptide immunogenicity and the general cut-off value of 500

d) Immunoassays with samples from naive donors (MN01-MN04)

A total of four optimisation experiments with naive donor samples were performed, summarised in Table V.8.

Table V. 1 T cell amplification assays performed.

Four different protocols were tested and optimised: in experiment MN01, the Wolfl protocol described in (16) was used. In experiment MN02, the T cell amplification assay was adapted from the protocol for TCD4 cells described in (8,9). In experiment MN03, this protocol was further optimised through the addition of a third co-culture and different peptide concentrations, tested in two different formats (96- supertype (B pocket specificity for aromatic and aliphatic residues, and a F pocket specificity for aromatic, aliphatic and hydrophobic residues).

Figure V. 14 IFN-γ ELISpot results for the MN02 experiment (96-well format), T cell amplification assay with total TCD8+ cells from donor MPL9. A) Number of specific T cell lines generated per condition. B) Spot counts for control conditions. C) Spot counts per condition showing individual cell lines; cNS, culture-stimulated cells but unstimulated in the ELISpot plate.

Likewise, in the 48-well batch stimulation format, no differences were found in IFN-γ productions were observed between unstimulated (cNS) and stimulated cells (STIM). For some conditions, in the 48-well assay format, stimulated conditions had fewer spot counts than unstimulated cNS wells (Figure V.16 panels B and C). Also, there appears to be a dose-dependent effect between peptide concentration and decreased spot counts in stimulated wells, very noticeable at 5 µM per peptide (Figure V.15 panels B and C). This effect is probably not due to peptide toxicity since cell viability was assessed, but probably to peptide binding competition, as each pool contains a large number of peptides (22 and 27 peptides).

From this experiment, we realized that stimulation of monocyte-derived matured DCs is not optimal and should be further optimised. Even if class I peptides are 9-mer in length, apparently these were not successfully presented by MHC molecules and were unable to prime specific T cells in vitro under these conditions. Furthermore, the cells used in the MN01 experiment were highly immature and were successful for T cell activation and cell line generation. Therefore, two different protocols for monocyte-derived DC stimulation were adopted: i) short peptide protocol, immature DC are matured (with LPS and Resiquimod/R848) for 16 hours, and then pulsed with peptides for 4 hours at 37°C/5% CO2; and ii) long peptide protocol, immature DC are matured (with LPS and Resiquimod/R848), and simultaneously pulsed with peptides for 16 hours at 37°C/5% CO2.

The naive donor bank was generated with samples from the French Blood Bank in Toulouse.

The HLA-typing results for HLA-A, -B, -DRB1, and -DPB1 alleles expressed by the naive donors match that of the French population. The HLA diversity represented in the naive donor bank also corresponds to the most common alleles expressed by populations in South and Central America, North Africa, Western Asia and South Asia. These regions include the populations most at risk of developing leishmaniasis and are the target populations for an anti-Leishmania vaccine.

Unquestionably, the naive donor bank is an extremely small population compared with the populations considered for the allele frequency studies, but the most frequent alleles are nonetheless represented.

Ultimately, we aim at increasing the sample size of the naive donor bank and increase the number of represented alleles, particularly those which are currently underrepresented. However, the most prevalent HLA alleles in the world population, and particularly in populations in endemic leishmaniasis areas, already represented in the naive donor bank will provide a great HLA coverage for future experiments.

Peptide immunogenicity testing with samples from naive donors

The naive T cell assay (experiment MN01) confirmed the presence of Leishmania-specific TCD8 cells in the naive repertoire, with 4 peptide pools generating at least 2 positive cell lines (in 10 tested per condition, i.e. 2 million T CD8+ cells). This assay shall be repeated in the future, after preselecting peptides, with fewer peptides further along the peptide validation pipeline and with some protocol changes (i.e. cell counting before ELISpot seeding, ELISpot triplicates and higher number of cell lines).

The protocol used in experiment MN01 was adapted from Wolfl et al, to a simplified version using total PBMC as APC. While the authors recommend the use of monocyte-derived dendritic cells, at the time of this experiment it was not possible to induce DC differentiation in vitro, and the use of autologous total PBMC as APC is commonly done as well (25). The B cells and monocytes present in this population are for the most part in an immature state. expected to be promiscuous. This observation agrees with the presence of unpredicted epitope promiscuity, the ability of peptides to bind to two or more different HLA alleles.

The analysis of cytokine production in response to Leishmania-specific peptides will help not only confirm, but also better characterise the immune responses and cellular functional activity induced by the peptide pools. Cytokine analysis of culture supernatants will be performed by Cytokine-Bead assays (CBA) for healed donors and the naive control donors, for which no results

were presented here.

Appendix II.1

Proteins identified in the secretome datasets ranked according to abundance (normalised iBAQ values) -582 protein accessions with normalised iBAQ values over 1,10.

Appendix V.1

Data was collected from the Allele Frequency Net Database for the following world regions: South and Central America (Table 1), North Africa (Table 2), Western Asia (Table 3), South Asia (Table 4).

Data were retrieved from AFND through a 'HLA allele freq (Classical)' search with the following filters: i) region name; ii) level of resolution=2-digits; iii) Population standard=Gold and Silver (8); iv) show frequencies=only positives