Introduction

Brief history of vaccination

Infectious diseases have been an important cause of mortality throughout the history of mankind and have always played a key role in regulating the numerousness of humans. Vaccination is considered one of the most powerful means to save lives and to increase the level of health of humans.

1 Vaccination is the active immunization with an immunogen (the vaccine) administered so that the host develops specific antibodies and B and T memory cells that can act against the natural immunogen. These cells stand ready to be activated should the host later be exposed to the pathogen bearing the natural immunogen (Figure 1). Vaccines are prophylactic in the sense that they are administered to healthy individuals to prevent a disease. Nevertheless, there is a growing trend to use vaccines to alleviate the suffering of those already with disease. Preventive vaccines carry immunologic specificity for individual infective disease agents and provoke an immune response against them. Prophylactic vaccines represent the possibility to prevent global epidemics of infectious diseases. Historically vaccination was born with the work of Edward Jenner when, in 1796, he inoculated an eight years old boy, the son of his gardener, with pus scraped from cowpox blisters. The young boy was then protected from smallpox when he was challenged with variolous material. After subsequent experimentation vaccination was eventually accepted by British government and offered to the population in 1840 free of charge replacing variolation, an older practice, introduced in England by Lady Mary Wortley Montagu on her return from Costantinople, based on immunization of patients by infecting them with substance from the pustules of patients with a mild form of the smallpox. The worldwide implementation of vaccination campaigns against smallpox allowed the World Health Organization (WHO) to declare its eradication in 1980. During the 1900s many successes and discoveries have been registered on vaccine development and the effectiveness of vaccination has been proved on human health. Figure 2 shows how the incidence of serogroup C meningococcal disease decreased after the introduction of the glycoconjugate vaccine.

As reported in Table 1, different types of approaches have been studied so far for vaccine development and some of them became a reality such as: traditional whole pathogens live attenuated (polio oral vaccine), killed or inactivated (inactivate polio, inactivated whole virus influenza), toxins isolated from the pathogen and subsequently inactivated (diphtheria and tetanus vaccines), other subunit vaccines such as proteins produced by recombinant DNA technologies or from genetically detoxified microorganisms and bacterial polysaccharides (CPS) and glycoconjugates .

Type of vaccine Description

Live, attenuated Whole pathogen (polio) Table 1: Types of vaccines.

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Polysaccharide Vaccines

Polysaccharides are important virulence factor especially for encapsulated bacteria that present on their surface complex carbohydrate structures. Surface polysaccharides have several functions: they protect microorganisms from desiccation when they are exposed to the external environment, in some cases, for example the hyaluronic capsule of group A Streptococcus, their adhesive properties help the pathogen to invade the host; other capsular polysaccharides prevent the activation of the alternate complement pathway; sometimes they mimic molecules produced by human cells so that the pathogen is not recognized as foreign by our immune system (serogroup B meningococcal capsular polysaccharide, hyaluronic acid). 4 Around 1930s the protective role of antibodies (Abs) induced by pneumococcal polysaccharide started to be investigated and in 1945 the first vaccine composed by purified polysaccharide from selected pneumococcal serotypes was tested in man. 5 6 The research on vaccines development was subsequently slowed down by the introduction of antibiotics, however with the emergence of drug resistant strains the development of polysaccharide vaccines started again and a number of them have been studied in large clinical studies. 7 8 Polysaccharide vaccines against meningococcus serogroup ACWY, Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) were licensed between the seventies and eighties.

Polysaccharide vaccines however did not completely solve the problem of bacterial diseases caused by encapsulated microorganisms. Bacterial polysaccharides are made by repeated units constituted of monosaccharides linked together by glycosidic linkages, and one of their main feature emerged from clinical trials, is that they are poorly immunogenic in children less than two years of age. 9 10 11 In addition, the antibody response to bacterial polysaccharides is weakly affected by adjuvants, IgM represents the major class of antibodies induced and since their immune response does not induce memory, it is not boosted by subsequent immunizations.

These characteristics are due to the fact that, unlike proteins that are T-cell dependent (TD)

antigens, polysaccharides are T-cell independent (TI) antigens. Polysaccharide antigens directly activate polysaccharide-specific B cells which differentiate then into plasma cells to produce antibodies, but memory B cells are not formed.

Proteins instead are TD antigens; following interaction with antigen-presenting cells (APC) like dendritic cells, macrophages and B-cells, protein antigens are internalized and processed into small peptides which are then re-exposed and presented to T lymphocytes in association with the major histocompatibility complex (MHC) class II molecules. Interaction with T cells induces B cells to differentiate into plasma cells and memory B cells. Unlike TI antigens, TD antigens are immunogenic early in infancy, the immune response induced can be boosted, enhanced by Page 10

Glycoconjugate vaccines

The limitation of polysaccharides vaccines has been overcome by covalent conjugation to a carrier protein as source of T-cell epitopes. Since 1929 Avery and Goebel have demonstrated that nonimmunogenic sugars after conjugation to a carrier protein become able to induce antibodies in the animal model. 15 However the first application of this concept to a vaccine for human use started only in 1980 with the development of the first conjugate vaccine against Haemophilus influenzae type b (Hib) that was later on licensed vaccine between 1987 and 1990. 16 17 18 19 Many other glycoconjugate vaccines have been developed against bacterial pathogens such as Neisseriae meningitidis, Streptococcus pneumoniae and group B Streptococcus. 20 21 22 23 24 25 26 27 Today glycoconjugate vaccines are among the safest and most efficacious vaccines developed during the last 30 years and they are currently used in the immunization schedules of different countries like for example the United States (US) as reported in Table 2. Table 2: Schedules of immunization currently used in US. [START_REF] Bardotti | Physicochemical characterisation of glycoconjugate vaccines for prevention of meningococcal diseases[END_REF] Currently licensed glycoconjugate vaccines are reported on the Table 3. Many studies have been performed to understand the mechanisms of internalization and processing of the glycoconjugate molecules by the immune system and many data have been published. 14 29 30 31 32 33 Chemical conjugation of polysaccharides to protein carriers allows processing of the protein carrier by polysaccharide-specific B cells and presentation, on their surface, of the resulting peptides or glycopeptides in association with MHC class II. Further interaction with carrier-specific T cells then induces polysaccharide-specific B cells differentiation (Figure 4). As a consequence, a conjugate vaccine induces a T-cell-dependent response already early in life which leads to immunological memory and boosting of the response by further doses of the vaccine.

Vaccine

Vaccine indication Type of conjugate Manufacturer

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Figure 4: Schematic diagram of mechanism of action for conjugate vaccines. 34 Recently a new working model for glycoconjugate vaccines has been proposed where T-cell populations that recognize carbohydrate epitopes derived by APC processing of conjugate vaccines and that, when presented by MHCII, these epitopes recruit T-cell help for the induction of adaptive immune responses to these vaccines. [START_REF] Costantino | The design of semi-synthetic and synthetic glycoconjugate vaccines[END_REF] To prepare glycoconjugate vaccines, two main approaches based on different chemistry of conjugation, have been used so far. One is based on the random chemical activation of the saccharide chain followed by covalent binding with the protein carrier obtaining a cross-linked structure between the polysaccharide and the protein. A second approach is based on the generation by controlled fragmentation of the native polysaccharide, of appropriately sized oligosaccharides which are then activated at their terminal groups, usually with a linker molecule, and subsequently conjugated to the carrier protein obtaining a radial structure (Figure 5).

Depending on the conjugation chemistry employed, a chemical spacer can be used in order to facilitate the coupling of the protein to the saccharide antigens and, in some cases, conjugation chemistry also requires prior derivatization of the protein carriers.

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Figure 5: Structures of glycoconjugates.

The synthetic approach which, starting from suitable monosaccharides, can provide pure and welldefined oligosaccharides for coupling to protein carriers, is increasingly attractive. 36 37 38 39 40 A new approach called bioconjugation is based on glyco-engineering the bacterial N-glycosylation pathway in bacteria such as Escherichia coli. The polysaccharide, encoded by the inserted genes, is produced on a polyisoprenoid carrier and then is transferred to an asparagine residue of the carrier protein which has to contain at least one (native or engineered) N-glycosylation site. 41 42 43 44 A chemistry which favors the formation of cross-linked structures was used for Hib conjugate vaccines by Sanofi-Pasteur, Merck and Pfizer (Table 3).

Novartis Vaccine has instead developed a conjugation chemistry which results in conjugates having oligosaccharides oriented radially from the carrier protein. 34 45 In particular Hib and meningococcal Novartis Vaccines conjugate vaccines are made starting from purified capsular polysaccharides which have been size reduced by acid hydrolysis and fractionated to select an intermediate chain length population. The oligosaccharides were then derivatized at their reducing termini introducing first a primary amino group by reductive amination followed by a 6-carbon atom flexible hydrocarbon spacer having a terminal active ester ready for conjugation. 20 23 46 The same technology was used by Novartis Vaccine to develop its quadrivalent A, C, W135, Y anti-meningococcal conjugate vaccine. [START_REF] Matjila | Safety and immunogenicity of two Haemophilus influenzae type b conjugate vaccines[END_REF] Page 14

Variables influencing the immunogenicity and physicochemical properties of glycoconjugate vaccines

Many aspects can influence the immunogenicity of conjugate vaccines and the main variables investigated so far are the size of the saccharide moiety, the saccharide: protein ratio in the purified conjugate, the conjugation strategy, the nature of the spacer and the protein carrier.

The size of the saccharide moiety and saccharide/protein ratio were investigated in different works such as Seppälä and Mäkelä in one of the first studies on the effect of size and chemistry on the immunogenicity of dextrans-protein conjugates found that dextrans of low molecular weight conjugated to chicken serum albumin, induced strong anti-dextran responses in mice, while increasing the dextrans' size resulted in reduced immunogenicity. [START_REF] Broker | Chemistry of a new investigational quadrivalent meningococcal conjugate vaccine that is immunogenic at all ages[END_REF] LPS oligosaccharides conjugated to human serum albumin (HSA). The authors found that octa-, dodeca-, and hexadecasaccharide fragments induced high levels of lipopolysaccharide binding IgG antibodies in mice after three injections and were superior to a tetrasaccharide conjugate. The influence of the carbohydrate/protein ratio was different for the three conjugates. The octasaccharide-HSA conjugate with the highest density evoked a good immune response, while in the case of dodeca-and hexadecasaccharide conjugates, the median density was optimal. 51 These studies suggest that oligosaccharide chain length and hapten loading might be interconnected in determining the immunogenicity of glycoconjugate vaccines.

The spacer is a short linear molecule that is generally linked to the polysaccharide chain or to the protein or to both moieties, depending on the chemistry, used to facilitate the coupling between the protein and sugar. There are evidences in the literature which suggest that rigid, constrained spacers like cyclohexyl maleimide, elicit a significant amount of undesirable antibodies, with the risk of driving the immune response away from the targeted epitope on the hapten. 52 53 The use of a flexible alkyl type maleimido spacer has been reported as a way to overcome the previous observed immunogenicity of cyclic maleimide linkers. [START_REF] Buskas | The immunogenicity of the tumor-associated antigen Lewis(y) may be suppressed by a bifunctional cross-linker required for coupling to a carrier protein[END_REF] A number of protein carriers have been used so far in preclinical and clinical evaluation of conjugate vaccines. 55 56 57 58 59 60 61 Page 15

Proteins such as diphtheria and tetanus toxoids, which derive from the respective toxins after chemical detoxification with formaldehyde, were initially selected as carriers because of the safety track record accumulated with tetanus and diphtheria vaccination. CRM197, a non-toxic mutant of diphtheria toxin [START_REF] Micoli | Vi-CRM 197 as a new conjugate vaccine against Salmonella Typhi[END_REF] which instead does not need chemical detoxification, has been extensively used as carrier for licensed Hib, pneumococcal, meningococcal conjugate vaccines and for other vaccines being developed. An outer membrane protein complex of serogroup B meningococcus has been used by Merck as carrier for their Hib conjugate vaccine. [START_REF] Giannini | The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197[END_REF] GSK in their multivalent pneumococcal conjugate vaccine introduced the use of the Hib-related protein D as carrier for most of the polysaccharides included into the vaccine. 64 65 The team of John Robbins made extensive use of the recombinant non toxic form of Pseudomonas aeruginosa exo-toxin as carrier for Staphylococcus aureus type 5 and 8 as well as for Salmonella typhi Vi conjugates. 66 67 A number of clinical trials have been conducted to compare the immunogenicity of different conjugate vaccines with different carrier proteins. 68 69 70 71 72 It is however very difficult a direct comparison of the effect of different protein carriers, due to the coexistence of other variables as conjugation chemistry, saccharide chain length, adjuvant, formulation technology, and previous or concomitant vaccination with other antigens.

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Aim of the thesis

The aim of my PhD thesis is to investigate the role that different variables have on the immunogenicity and physicochemical properties of glycoconjugate vaccines.

In particular in this work three main aspects have been investigated: the carrier protein, the sugar moiety and the linker region.

Concerning the protein, a screening of new candidate carrier proteins were investigated and a comparative study on three common carriers, CRM197, TT and DT, has been done.

The role of the sugar moiety has been investigated through two different studies: the first was conducted on glycoconjugate of the biologically derived capsular polysaccharide from meningococcus serogroup A. Different chain length oligosaccharides and glycosylation degrees were tested. The second study was about synthetic antigens and their ability to represent the native epitope.

The influence of the linker on the immunogenicity of glycoconjugate vaccines in animal model was studied using four different linker molecules.

Moreover studies on the mechanism of antigen uptake by antigen presenting cells have been conducted.

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2 Results

The influence of the carrier protein

In this part of my PhD project I have carried out two investigations, one directed to discover new proteins that could be used as carriers in future glycoconjugate vaccines, and another focused on the immunological and physicochemical comparison of glycoconjugates made with three commonly used protein carriers.

As model carbohydrate antigens I have used Laminarin (Lam) and the serogroups A, C, W 135 and Y meningococcal oligosaccharides (MenA, MenC, MenW and MenY).

Laminarin (Lam) is a β-(1, 3) glucan with sporadic β-(1, 6) branches (Figure 6), extracted from the brown alga Laminaria digitata that has been studied as vaccine candidate against infections induced by C. albicans. 39 73 74 Figure 6: Laminarin structure.

Meningococcal saccharides from serogroups A, C, W 135 and Y have been used as conjugate vaccines against Neisseriae meningitidis by different companies. The chemical structure of the different capsular polysaccharides is reported in the Figure 7.

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MenC →9)--D-Neu5Ac(7/8OAc)-(2→ MenY →6)--D-Glcp-(1→4)--D-Neu5Ac(7/9OAc)-(2→ MenW →6)--D-Galp-(1→4)--D-Neu5Ac(7/9OAc)-(2→ MenA

Conjugates preparation and characterization

The different Lam conjugates have been obtained by chemical derivatization of Lam at the reducing end with an adipic acid linker having a terminal succinimido ester.

The activated oligosaccharide was then reacted with the different protein carriers using the same active ester/protein molar ratio.

A conjugate between Lam and CRM197 was also prepared to be used as control during the immunological studies in animals.

To obtain the glycoconjugates, Laminarin has been activated as reported in Mat&Met

Page 21

A schematic description of the chemical reactions to obtain Lam-CRM conjugate is reported on the Figure 8.

Figure 8: scheme of reaction to obtain Laminarin conjugate.

Laminarin conjugates have been characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-page) to asses that conjugation took place. Figure 9 shows the electrophoretic profiles of the selected lam conjugates. After conjugation the typical smear of a glycoconjugates could be observed on the SDS-page, the smear is due to the variable number of sugar chains bound to the protein and to the variable chain lenght of the sugar. The glycosylation degree of the different glycoconjugates varies in a range from 0.1 and 1.7 w/w. This difference is in accordance with the different molecular weight of the proteins since for example PNEU19 of 27kDa had reached a saccharide/protein w/w ratio of 1.2 while PNEU7, PNEU8 and PNEU9 of 115 and 194kDa have reached a ratio of 0.1. Moreover the different glycosylation degree could be due also to a different number of lysine on each protein and/or to differences in the conjugation efficiencies.

Evaluation of Laminarin glycoconjugates in mice

The Lam conjugates with the different candidate protein carriers have been tested in mice and the anti laminarin response compared to that provided by lam-CRM conjugates used as control. Ten different studies without adjuvant have been performed with an immunization dose of 5µg in term of saccharide. The anti Laminarin antibodies response after the third immunization has been evaluated

and among the proteins tested, eight of them, namely Ex 1, Ex 2, MB 2, MB 3, GAS 1, PNEU 8, PNEU 11, PNEU 20, were selected as new candidate carriers since they were able to induce a response against the saccharide antigen comparable to that induced by CRM197 (Figure 10). In particular the conjugate with the PNEU 20 protein induced the highest anti laminarin antibodies titer.

MenC conjugation with selected new candidate carrier proteins: conjugates preparation and characterization

Among the new candidate carrier proteins, seven have been conjugated to the serogroup C meningococcal oligosaccharide (MenC) to study their carrier functionality with a second saccharide antigen. PNEU 19 has been used as negative control since it didn't work as carrier for Laminarin, while as positive control MenC-CRM conjugate was used.

The preparation of the MenC conjugates was performed according to the scheme reported in Figure 11. Activated MenC oligosaccharides were obtained from Novartis V&D manufacturing department.

Figure 11: Scheme of reactions to obtain the MenC conjugates.

After purification conjugates have been characterized by SDS-page, as reported in the Figure 12, to asses that conjugation took place. For protein Ex2 we observed a double band profile corresponding to two protein isoforms always present on the purified batch. The two isoforms are equally involved in the conjugation process. Following purification, MenC conjugates were characterized for their saccharide and protein content.

The calculated carbohydrate to protein ratios is reported in

Evaluation of MenC glycoconjugates in mouse animal model

MenC glycoconjugates with the selected carrier proteins have been tested in mice without adjuvant with an immunization dose of 1µg in term of saccharide. The antibody response against MenC was evaluated on post third dosage immunization sera. The conjugates of four protein carriers elicited an antibody response comparable to that induced by MenC-CRM197: PNEU20, EX1, EX2 and GAS1 (Figure 13).

PNEU 20 evaluation in MenAWY glycoconjugate: conjugation as well as products characterization

Since PNEU 20 induced the best antibody response against Laminarin and a good response against MenC, this protein was considered for future experiment and tested also in MenAWY glycoconjugates.

The conjugation of PNEU20 to the meningococcal oligosaccharides has been performed according to the scheme reported below (Figure 14). The activated oligosaccharides were provided by Novartis V&D manufacturing department. In brief the individual oligosaccharides were first reacted with an excess of ammonium acetate in the presence of sodium cyanoborohydride to provided amino-oligosaccharides which were subsequently derivatized to active esters by reaction with an excess of disuccinimidyl adipate. The resulting activated oligosaccharides, after purification, were then reacted with PNEU20 using an active ester/protein (mol/mol) ration of 12:1 in 10-100mM

NaH 2 PO 4 pH 7, as described in Materials and Methods.

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Figure 14: Scheme of the reaction to obtain MenAWY conjugates with PNEU20.

After purification, the conjugates were characterized by SDS-page, as reported in the Figure 15, to confirm that conjugation took place. The purified conjugates have been characterized by their saccharide and protein content. The glycosylation degree (w/w) of each conjugate was then calculated. Glycosylation degrees are between 0.3 and 0.7 w/w (Table 7). Despite the fact that the same stechiometry was used, a different glycocylation degree was obtained for MenW. In particular as compared with MenY which has a similar structure, the conjugation reaction appears to proceed with higher yelds. This is consistent also with previous observation. Table 7: MenAWY-PNEU20 glycoconjugates characterization by saccharide/protein ratio w/w.

Evaluation of PNEU20 glycoconjugates in mouse animal model

The individual meningoccus A, C, W, and Y conjugates with PNEU20 have been evaluated in mouse animal model without adjuvant with an immunization dose of 2 µg for MenA and 1 µg for MenCWY in term of saccharide. After the third immunization anti polysaccharides antibody response was evaluated by coating the different CPS on the ELISA plates.

The protein PNEU20 protein was able to induce a level of anti-saccharide antibody comparable with that of CRM197 when conjugated with the different meningococcal oligosaccharides (Figure 16). To better investigate if some differences were present between the response induced by CRM197 and PNEU20 as carriers, sera after the 2 nd immunization were analyzed by complement mediated bactericidal activity using rabbit complement (rSBA). The antibodies elicited by PNEU20

glycoconjugates have shown functional activities against each meningococcal bacterial strain (Table 8), confirming the capability of this protein to work as carrier for different glycoconjugate vaccines.

rSBA titers post 2 nd dose

Conjugates

CRM197 PNEU20

MenA

Comparison study of three common carrier proteins in MenACWY glycoconjugates

In this section of my PhD a comparative study of glycoconjugates obtained with three different carrier proteins currently used in vaccines has been performed. The carrier proteins taken into consideration were: tetanus toxoid (TT), diphtheria toxoid (DT) and CRM197.

The proteins TT and DT derive from their homologous toxins after chemical detoxification with formaldehyde. [START_REF] Torosantucci | A novel glycoconjugate vaccine against fungal pathogens[END_REF] CRM197 is a non-toxic mutant of diphtheria toxin and does not need detoxification treatment. In CRM197 the glycine residue of diphtheria toxin in position 52 it is substituted by a glutamic acid residue (Figure 17). [START_REF] Neumuller | Detoxification of Diphtheria Toxin with Formaldehyde mixed with an Amino-Acid[END_REF] Lysine residues

Diphtheria toxoid (DT) CRM 197

Gly 52

Glu 52

Figure 17: CRM197 and Diphtheria toxoid structures comparison. [START_REF] Schneerson | Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates[END_REF] Recently the crystal structure of CRM197 has been solved investigating the structural basis for lack of toxicity of the protein. [START_REF] Giannini | The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197[END_REF] The proteins have been used to conjugate with MenACWY oligosaccharides and products have been compared in their physicochemical characteristics and in their immunogenicity.

Serogroup A, C, W, Y meningococcal oligosaccharides conjugation with CRM197, TT and DT as carrier proteins: conjugates preparation and characterization

Conjugates of CRM, TT and DT with the meningococcal A, C, W, Y oligosaccharides were prepared following the same chemistry described in paragraphs 2.2.3 and 2.2.5 (Figure 11 Each protein has been conjugated with the oligosaccharides of the four serogroups using the chemistry of conjugation previously reported before for MenACWY (paragraphs 2.2.3 and 2.2.5).

Conjugates have been characterized by SDS-page, as shown in Figure 19, to confirm that conjugation took place. Depending on the type of saccharide, conjugates with different electrophoretic profile were observed. The tendency of MenW to give higher glycosylated products was confirmed.

Page 34 Purified conjugates have been also characterized for their saccharide content, protein content and residual unconjugated saccharide (free saccharide %). The saccharide/protein ratio w/w and the free saccharide % for each conjugate are reported in Table 9. Glycosylation degrees obtained Page 35 ranged between 0.1 and 0.7 w/w depending on the saccharide protein combination. The free saccharide % varied from 3.7 to 22. This difference on the free saccharide content could derive from a different behaviour of the conjugates, depending on the protein/saccharide combination, during the ammonium sulphate precipitation applied for the purificationIn any case the 0.4 w/w ratio of MenA-TT conjugate will become 0.31 considering the 22% of free saccharide and doesn't affect considerably the immunization dosage content in term of conjugated saccharide.

Conjugates Sacch/Prot (w/w) Free Sacch (%)

MenA-CRM 0,5 16,3

MenC-CRM 0,6 17,7

MenW-CRM 0,7 10,7

MenY-CRM 0,4

MenA-TT 0,4

MenC-TT 0,2 14,1

MenW-TT 0,2 7,9

MenY-TT 0,1 16,2

MenA-DT 0,2 13,7

MenC-DT 0,1 10,3

MenW-DT 0,3 6,2

MenY-DT 0,1 11,8

Table 9: MenACWY glycoconjugates with CRM, TT and DT characterization by saccharide/protein ratio w/w.

Evaluation of glycoconjugates in mouse animal model

The meningococcal oligosaccharides conjugates with the different carrier proteins have been evaluated for their immunogenicity in Balb/c mice, with and without alum phosphate (AlPO 4 ) as adjuvant.

The adjuvanted formulations have been characterized concerning the absorption degree to AlPO 4 in PBS buffer pH 7-7.5. After centrifugation and supernatant separation from the pellet containing the AlPO 4 , the protein content was determined in the supernatant and compared to that of a control constituted by the formulation not exposed to the adjuvant.

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Conjugates % absorption

MenA-CRM 23

MenC-CRM 13

MenW-CRM 19

MenY-CRM 14

MenA-TT 12

MenC-TT 11

MenW-TT 0

MenY-TT 0

MenA-DT 10

MenC-DT 18

MenW-DT 6

MenY-DT 7

MenACWY-CRM 21

MenACWY-TT 0

MenACWY-DT 14

Table 10: % of absorption on AlPO 4 adjuvant of MenACWY-CRM, TT and DT vaccine formulations.

As shown in the Table 10, with these buffer and pH conditions used, none of the conjugates significantly absorbed to AlPO 4 .

All immunizations were done with the same schedule of injection and bleedings as reported on the

MenA-CRM 2 4 MenC-CRM 1 1,6 MenW-CRM 1 1,4 MenY-CRM 1 2,5 ACWY-CRM 2,1,1,1 9,5
MenA-TT 2 5

MenC-TT 1 5

MenW-TT 1 5

MenY-TT 1 10

ACWY-TT 2,1,1,1 25
MenA-DT 2 10

MenC-DT 1 10

MenW-DT 1 3,3 MenY-DT 1 10 ACWY-DT 2,1,1, 1 33,3
Table 11: Conjugates immunization dosages.

Initially we tested in Balb/c mice formulations without adjuvant of individual meningococcal conjugates with CRM, TT and DT. Sera post 2 nd and 3 rd immunization were analyzed by ELISA and complement mediated bactericidal activity using rabbit complement (rSBA). In all cases after the 2 nd immunization the antibodies response against the individual unconjugated polysaccharides was low and increased with the 3 rd immunization. However the high variability of the response did not allow to measuring any statistical difference between the different conjugates (Figure 22). In term of bactericidal activity all the conjugates elicited low levels of rSBA titer after the second dose which increased after the third (Table 12). Due to the high variability of the response without adjuvant we performed the next studies using AlPO 4 as adjuvant. Two different studies with the individual meningococcal conjugates have been conducted with AlPO 4 as adjuvant and the same trend of response has been observed inside the immunization groups, for this reason the titers have been grouped to calculate the GMT (CI 95%) on 16 mice. Only one study was performed comparing three tetravalent formulations containing the conjugates of the four meningococcal oligosaccharides with CRM197, DT and TT respectively and in this case the immunization groups were composed of eight mice.

Each carrier protein was able to induce a specific response against each saccharide antigen, vaccination induced a significant level of antibodies in comparison with PBS alone (Table 13).

All groups showed a significant booster effect following the third dose.

To compare the different vaccines a statistical analysis using a non parametric t test has been done on ELISA titers and highly significant differences have been obtained between MenA-CRM vs MenA-TT (*** 3 P= 0.0002) at post 3 and MenA-DT vs MenA-TT at post 3 (** The analysis of immunological response was completed by analyzing the functional activity of the induced antibodies. This was achieved by performing the rSBA assay on pools of mice sera immunized with the different conjugates. In general a similar trend of response was observed when comparing rSBA and ELISA titers (Table 14). However, interestingly, rSBA revealed a decreased of bactericidal titer against serogroup A meningococcus when the MenA conjugates were tested in tetravalent formulation. This difference was not evident with ELISA titers and might be related to the maturation of anti-meningococcal antibodies affinity which might proceed in a different way when MenA conjugates are in tetravalent formulation. TT and DT conjugates seem to have a similar behavior in inducing high levels of anti-carrier antibodies already after the second dose. A similar trend has been observed also for the tetravalent formulations (Figure 23 A andB). Due to the lower glycosylation degree of the TT and DT conjugates, the amount of protein per dose that mice received is generally higher for these two types of conjugates (Table 11) which could explain the higher anti carrier antibody titer. However

MenA-CRM and MenA-TT have a similar carbohydrate to protein ratio indicating that CRM197 has the tendency to drive the response towards the carbohydrate moiety which is the role of the protein carrier in a conjugate.

Characterization of the influence of the sugar moiety on glycoconjugate immunogenicity

To investigate how the saccharide moiety could influence the glycoconjugate immunogenicity two different studies have been performed. In the first study a natural antigen, MenA oligosaccharide, has been taken as model to study how the saccharide chain length and glycosylation degree can influence the immunogenicity of a glycoconjugate in mouse animal model.

A second investigation has been carried out on a synthetic antigen, the synthetic -glucan oligosaccharides, to evaluate if this antigen could be able to represent the epitope of the natural sugar.

Saccharide chain length and protein loading of a natural carbohydrate antigen

This investigation has been performed using serogroup A meningococcus oligosaccharides as thne concentrated on a 30kDa membrane and after diafiltration the retentate was recovered and used for conjugation with CRM197.

AvDP15 and avDP6/10 oligosaccharides were obtained firstly processing the hydrolyzed material trough UF30K or 2K respectively; subsequently the two permeate were further purified by anionic exchange chromatography column which was eluted with a sodium chloride gradient. Fractions containing the desired chain length oligosaccharides were identified by HPAEC-PAD analysis and pooled (Figure 25). All oligosaccharides have been chemically activated according to the following scheme of reaction (Figure 28) and using the experimental conditions reported in Mat&Met for oligosaccharides activation with succinimidyl diester of adipic acid (SIDEA).

Conjugates preparation and characterization

Using different conjugation conditions conjugates with different saccharide/protein ration have been obtained which have been characterized by SDS-page, as reported on the Figure 29, to confirm that conjugation took place. The excess of oligosaccharide used was sufficient to conjugate all the CRM197 protein in reation. The different glycoconjugates were purified by gel filtration chromatography in order to remove the unreacted oligosaccharides. After purification the conjugates were characterized for their protein and saccharide content. As described before the conjugation conditions were varied in order to obtain products with different glycosylation degree which are reported in the following table (Table 16).

Evaluation of conjugates in mice

Both monovalent (MenA-CRM) and tetravalent (ACWY-CRM) formulations were evaluated using AlPO 4 as adjuvant. As far as the tetravalent formulation is concerned the immunization dosage was 2µg for MenA conjugates and 1µg for the other conjugates expressed on saccharide basis.

The immunological response against serogroup A meningococcal polysaccharide was evaluated by ELISA, the functional activity of the induced antibodies was evaluated by rSBA. The results are reported in Figure 30 (A,B,C) and Table 17 (A1, B1, C1).

Within the three categories of avDP we did not observe significant immunogenicity differences in the range of glycosylation degree studied. However both ELISA titers and rSBA titers showed a trend of higher response induced by the conjugates with lower glycosylation degree. This behavior is particularly evident in tetravalent formulations. The reasons for this trend are not clear; however we should note that conjugates with low glycosylation degree corresponded to a higher amount of molecules (moles) injected into the animals per dose.

MenA-CRM LL

MenA-CRM LL

MenA-CRM ML +CWY

MenA-CRM LL +CWY

MenA-CRM LL +CWY

MenA

Epitope mapping of a synthetic carbohydrate antigen

Another aspect that we wanted to investigate, concerning the influence of the carbohydrate moiety on the immunogenicity of glycoconjugate vaccines, is the ability of synthetic oligosaccharides to represent the corresponding epitope of the native polysaccharide structure. We focused our attention on synthetic -glucan oligosaccharides.

The synthetic -glucans represent the cell wall -glucans produced by pathogenic fungi like C. albicans. It has been already shown that Laminarin a -glucans derived from the alga Laminaria digitata, when conjugated with CRM197 and injected in mice, induce antibodies that react with the cell wall -glucans of C. albicans and protect mice from the challenge with this fungus. It has also been shown that conjugates with synthetic -glucans made of 15 repeating unit can also induce Page 51 protecting antibodies. We wanted therefore perform additional studies to understand the minimal chain length that could induce antibodies against Laminarin and therefore against C.albicans cell wall; moreover we designed experiments to understand the role of branching on the immunogenicity and specificity of the induced antibodies. With this goal in mind our carbohydrate chemistry team synthesized five -glucan oligosaccharides whose structure is reported in Figure 31. From this competitive inhibition study we obtained the information that the linear -glucans better represent the immunogenic epitope located on Laminarin and that a chain length of at least six sugars would be needed in order to efficiently fill the combining site of anti Laminarin antibodies.

Accordingly we conjugated the hexasaccharide 6 to CRM197 and we evaluated its immunogenicity in mice. The β-(1,3) glucan hexasaccharide-CRM197 conjugate was tested in Balb/c mice for its ability to induce anti laminarin antibodies. For this purpose groups of 8 mice were immunized with 5 μg carbohydrate based dose of Lam-and Hexa-CRM197 conjugates, respectively. For Hexa-CRM197 a 1 μg dose was used as well, with the aim of studying the dose effect. Vaccines were formulated with the adjuvant MF59, an oil in water emulsion, which was shown to be effective in boosting both cellular and humoral immune response and is commonly used for seasonal flu vaccination. [START_REF] Malito | Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197[END_REF] Adjuvant alone in phosphate buffer (PBS) was used as negative controls.

Sera were analyzed by ELISA for their content of anti laminarin IgG. After three injections, the hexasaccharide conjugate, at both 1 and 5 μg dosages, induced titers of specific anti laminarin IgG antibody significantly higher as compared to Lam-CRM197 (geometric mean titers 18101 vs 5191; p< 0.05). Interestingly, as shown in Figure 33, the immunological response of the hexasaccharide conjugate appeared to be more homogeneous in comparison with Lam-CRM.

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The nature of the spacer

In the manufacturing process of glycoconjugate vaccines a spacer molecule is frequently used in order to facilitate the coupling of the protein to the saccharide antigen. Usually the spacer, also called linker, is installed on the saccharide structure (Figure 34) but sometimes also on the protein or both moieties as was applied for PedvaxHIB vaccine production. Accordingly four activated MenA oligosaccharides have been prepared with the different linker molecules, using reaction conditions which allowed the retention of a reducing group on one end of the linker thus enabling the subsequent coupling with the carrier protein CRM197.

The conjugates with the different linkers were then purified, characterized and tested in Balb/c mice for their immunogenicity.

Preparation and characterization of conjugates with different linkers

Serogroup A meningococcal oligosaccharide avDP15 was obtained as described in Mat&Met and briefly summarized in paragraph 2.5. The MenA-CRM conjugate with adipic acid residue spacer was obtained from the Manufacturing Department of Novartis V&D. MenA-CRM conjugate with "penta ethylene glycol" residue as spacer was prepared following the scheme reported in Figure 35A with a procedure very similar to that conventional used for MenA-CRM with adipic acid spacer in Novartis V&D.

Page 56

MenA-CRM conjugate with SMPB as spacer has been prepared according to the Figure 35C. In this case the MenA has been firstly derivatized at its reducing ends with cystamine following by reduction with TCEP to provide a thiol group. The thiolated oligosaccharide was then allowed to react with the maleimido group of SMPB according to the conditions described in Mat&Met. The derived oligosaccharide was then purified by acetone precipitation, dried and tested for its content of active ester group.

The same procedure was adopted for MenA derivatization with SMCC spacer (Figure 35B).

In addition in the case of SMCC we have prepared also a conjugate where the protein was first derivatized by reaction of its lysine amino group with the active ester moiety of SMCC spacer (Figure 35D). The activation degree of CRM197 with SMCC spacer was determined by MALDI-TOF mass spectrometry and turned out to be an average 8 mol of linker /mol CRM197.

The three activated oligosaccharides were than conjugated with CRM197 in 50-100mM NaH 2 PO 4 pH 7 using a molar ration 13:1 (active ester/protein).

CRM SMCC was reacted with thiolated MenA oligosaccharides using a molar ratio of 10:1 SH:linker (mol/mol).

Conjugates were purified by size exclusion chromatography as described in Mat&Met. CRM and MenA SMPB -CRM as compared to MenA SIDEA -CRM and MenA SMCC -CRM, supporting a higher carbohydrate/protein ratio for the last two. This was also confirmed by the chemical analysis of carbohydrate and protein content of the conjugate as reported in Table 19. The higher glycosylation degree obtained for MenA SIDEA -CRM as compare to MenA SMCC -CRM implies that a lower number of conjugate moles are administered to mice during immunization.

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MenA-CRM SMCC MenA PEG -CRM MenA SMCC -CRM MenA SMPB -CRM MenA SIDEA -CRM

Evaluation of conjugates in mouse animal model

Conjugates were evaluated in mice using the immunization schedule reported before (Figure 21, monovalent formulation with AlPO 4 as adjuvant).

Vaccines were used at 2µg saccharide/dose.

Anti-MenA and anti-protein response have been evaluated by ELISA and the functionality of anti-MenA antibody has been evaluated by rSBA.

Conjugates obtained with different chemistry of conjugation were able to induce a specific antibody response against the saccharide antigen MenA (Figure 38A) that was comparable between the different molecules, differences have been observed between MenA SMPB -CRM that has induced the lower response and the two conjugates MenA SIDEA -CRM and MenA SMCC -CRM that induced the higher response, these differences after the third dose were also confirmed by statistical analysis using a non-parametric t test (P= 0.0041; P= 0.0034). For all vaccines a booster effect is present between the second and the third dose. The anti-MenA antibodies induced were bactericidal and while high comparability was observed at post 3, post 2 data shown that only MenA SIDEA -CRM and MenA SMCC -CRM were able to induce functional Ab after two doses (Table 20). Similar anti-carrier response (Figure 38B) was observed after three doses, instead at post 2 MenA SIDEA -CRM and MenA SMCC -CRM have induced a lower level of anti-CRM abs. MenA SMPB -CRM that have induced the lowest anti-MenA response have induced an higher level of anti-protein Ab.

P B S + A l u m P h o s p h a t e P B S + A l u m P h o s p h a t e M e n A S I D E

PBS + AlPO4

MenA

SIDEA -CRM

MenA

PEG -CRM

MenA

SMPB -CRM

MenA

SMCC -CRM

MenA-CRM

SMCC

Labelling of glycoconjugate

Glycoconjugates are among the safest and most efficacious vaccines developed in the last 30 years. Many papers have been published concerning their mechanism of action; however a complete understanding of the uptake and processing by the immune system has not been reached yet. We decide therefore to perform some experiments in order to study some still poorly investigated aspects of their mechanism of action. In particular we focused our attention on: cell recruitment after glycoconjugate administration; type of cells involved in the uptake of the glycoconjugate antigen; intracellular localization of the antigen after uptake.

In order to carry out these interrogations we needed to track the antigen during uptake and internalization/processing events. Therefore we needed to label the conjugate antigen with a fluorescent dye suitable for fluorescence activated cell sorting analysis (FACS) and confocal microscopy. MenC-CRM was selected as glycoconjugate model and this molecule was double labeled in order to be able to track both the protein as well as the carbohydrate moiety.

To obtain the double labelled glycoconjugate, MenC oligosaccharides were activated and labelled with A488, as reported in Mat&Met and subsequently were conjugated with CRM197 as reported.

After conjugation the second labelling with A647 has been done on the protein moiety. The scheme of reaction to obtain MenC-CRM with double labelling (MenC-CRM dl) is reported in Figure 39.

Page 64 Labelled conjugates have been characterized by spectrophotometric analysis between 200 and 800 nm and two peaks have been determined at 495 and 650 nm that are the typical wave lengths of the dyes A488 and A647 (Figure 41). The double labelled conjugate was characterized after purification for the saccharide and protein content and degree of labelling as reported on the Table 21. A glycosylation degree of 0.4 w/w has been obtained, a degree of labelling (DOL) of 0.5 on the sugar moiety with A488 and of 7 on the protein moiety with A647. DOL has been calculated as reported in Mat&Met applying the equation reported below DOL= (Amax * MW)/[sample] * ε dey.

Conjugate

Sacch/prot w/w DOL488 DOL647

MenC/A488-CRM/A647 0.4 0.5 7

Table 21: MenC-CRM dl characterization by saccharide/protein ratio w/w and DOL.

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In-and ex-vivo antigen uptake characterization

We took advantage of the MenC-CRM dl glycoconjugate to investigate the internalization process in vitro and ex vivo, mainly focusing on the characterization of cell types involved in antigen uptake and internalization.

Briefly, Balb/c mice were treated intraperitoneally with MenC-CRM dl and peritoneal wash samples were collected at different time point post injection (0, 30 min, 6 h and 24 h). The cells in these samples were fixed and stained for the presence of specific cellular markers, enabling the identification of macrophages, B cells, monocytes, neutrophils and eosinophils.

In Figure 41 we show the cell recruitment into the peritoneum after GC injection. The cell type that is recruited at highest numbers are B cells, showing a massive influx immediately at 30 minutes p.i.

This increase is maintained during the course of the analysis. Neutrophils show a peak of recruitment at 6 h and then decrease rapidly back to basal level. Macrophages slightly increase at 30 min p.i and then are maintained for up to 24 h. The same samples were also analyzed for antigen uptake and the results are reported in Figure 42 A and B. Professional antigen presenting cells (APC) like macrophages and B cells rapidly take up the labeled glycoconjugate. Uptake by macrophages is generally fast, non-specific and antigen will most likely be processed and presented on MHC-II molecules to T cells. The fast uptake was also observed in our setting: macrophages were found to be Ag-positive at already 30 min post injection and remain positive up to 24 h. In contrast, antigen uptake by B cells is slower and generally mediated by ligation of the specific B cell receptor. Accordingly, only a minor cell fraction of B cells stains positive for the glycoconjugate at any time point. Also monocytes are recruited and become Ag positive between 30 min and 6 h post injection, but their number is quite low and does not contribute in a major way to antigen uptake.

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Besides professional APC, some cells of the innate arm of the immune system like eosinophils or neutrophils can bind antigen on their surface and act as antigen transporters. Eosinophils are recruited and most cells become antigen positive, instead neutrophils were recruited but most cells did not become antigen positive.

In conclusion, the major contribution to antigen uptake in our system comes from macrophages. Notably, in both cell types, we can observe a complete co-localization of protein with the sugar moieties, indicating that during the GC processing the two components, the protein and the sugar moiety, tend to stay in the same cellular compartments.

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Discussion

Glycoconjugate vaccines are among the safest and more efficacious vaccines developed during the last 30 years. They have been developed to improve the immunogenicity of polysaccharide vaccines which are T-cell independent, poorly immunogenic in infants and young children < 2 years of age 56 80 and do not induce immunological memory. [START_REF] Granoff | Reconsideration of the use of meningococcal polysaccharide vaccine[END_REF] There is no other example where the introduction of a new approach to vaccine development has had such a rapid and positive effect in preventing infections by a series of human pathogens. [START_REF] Pollard | Maintaining protection against invasive bacteria with protein-polysaccharide conjugate vaccines[END_REF] The Hib conjugate vaccines, the prototypes of successful conjugate vaccines, have resulted in the virtual eradication of disease due to Hib in the United States and much of the developed world. [START_REF] Pace | MenACWY-CRM a novel quadrivalent glycoconjugate vaccine against N.meningitidis for the prevention of meningococcal infection[END_REF] Given the success of Hib vaccine other glycoconjugate vaccines have been successfully developed against other pathogens such as Neisseriae meningitidis, Streptococcus pneumoniae and they are currently present on WHO schedule of immunization.

The introduction of anti-pneumococcal glycoconjugate vaccines has demonstrated the utility of these vaccines in pediatric and other high-risk populations, in reducing the carriage rates of pneumococci, and the prevalence of antimicrobial-resistant organisms. 83 84 85 86 87 The introduction of a monovalent, group C meningococcal polysaccharide-protein conjugate vaccine in England in late 1999 led to an initial dramatic decrease in the incidence and mortality of meningococcal disease in targeted pediatric populations. Similar results were demonstrated elsewhere in Europe over the last decade. 88 89 90 The development of glycoconjugate vaccines has allowed the investigation of their immunogenicity in preclinical and clinical studies in relation with their chemical physical properties. In addition glycoconjugates have been introduced in vaccines combinations that are now part of human immunization schedules. 34 91 92 Moreover the success of glycoconjugates vaccines has triggered studies on new possible application such as therapeutic vaccines for cancer, Alzheimer and conjugates vaccines for drug abuse, which are currently under development. 93 94 Ideally, knowing the immunogenic properties of many conjugates vaccines that have been developed and the understanding of their mechanism of action, a rational design of a glycoconjugate could be possible. On the other hand data obtained until now show that many variables can affect the immunogenicity of a conjugate vaccine and they have to be tested and optimized whenever a new vaccine will be developed.

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Glycoconjugate vaccines developed so far have different characteristics depending on their carbohydrate antigen, the carrier protein and the conjugation chemistry. All these aspects confer to the glycoconjugate different physic-chemical characteristics that may result in different immunological properties.

This PhD work is fitting into this framework by proposing an investigation of the three main moieties that could influence the immunogenicity of a glycoconjugate vaccine: the protein carrier, the sugar moiety and the chemistry of conjugation in particular studying the spacer moiety.

The influence of the carrier protein

A number of protein carriers have been used so far in preclinical and clinical evaluation of conjugate vaccines. Diphtheria and tetanus toxoids (DT and TT), CRM197, the outer membrane protein complex of serogroup B meningococcus (OMPC) and Hib-related protein D (PD) are the proteins used so far in licensed conjugate vaccines. OMPC has been used as carrier for Haemophilus influenzae (Hib) capsular polysaccharide (PRP) which is immunogenic in children as young as 2 month of age. DT has been used as carrier for the first Hib vaccine and subsequently to develop a quadrivalent meningococcal ACWY vaccine which was proven to be safe and immunogenic in children and adolescents. CRM197 has been successfully used as carrier for many conjugate vaccines against Hib, Pneumococcus (7 and 13 valent) and recently for a tetravalent (ACWY) meningococcal conjugate vaccine which prove to be immunogenic in all classes of age. TT, besides Hib and MenC, has been used also as carrier for a tetravalent meningococcal vaccine that induces equal or higher SBA persistence in adolescents when compared with the PS vaccine. PD has been used as carrier to develop the 10-valent pneumococcal nontypeable Heamophilus influenzae (PHiD-CV) and the usage of this protein as carrier might provide additional protection against acute otitis media due to pneumococcus in infants and children. 55 95 96 97 98 Studies to obtain an optimized carrier protein which contains selected human T cell epitopes have been published. A rationally designed recombinant protein containing strings of promiscuous human CD4+ T-cell epitopes derived from various pathogens including tetanus, influenza virus, Plasmodium falciparum and hepatitis B virus, proved to be a very good carrier for Hib and meningococcal oligosaccharides. 99 100 Other carrier proteins such as keyhole limpet hemocyanin (KLH) and the virus like particle Qb are taken into consideration in developing conjugate vaccines for non-infectious diseases. 101 We have conducted a screening study on different bacterial proteins to investigate their ability to behave as carrier for different saccharide antigens. Data obtained have shown that only 8 among 31

proteins tested were able to induce a specific IgG response against the model carbohydrate antigen

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Laminarin. These proteins were additionally tested with a different saccharide antigen, the meningococcal serogroup C oligosaccharide, and four of them, PNEU20, EX1, EX2 and GAS1, confirmed their functionality as carrier. In particular the protein PNEU20 turned out to be the best carrier and when it was conjugated to meningococcal serogroups ACWY oligosaccharides, the resulting conjugates induced, in mice, a response comparable with that of CRM197 based meningococcal ACWY oligosaccharides. The antibodies elicited by PNEU20-MenACWY conjugates were also functional when tested by rSBA.

We were therefore able to identify a new candidate carrier protein, PNEU20, which works as carrier for different carbohydrate antigens, inducing high titers of anti-carbohydrate specific and functional antibodies.

A number of clinical trials have been conducted to compare the immunogenicity of different conjugate with different carrier proteins. Comparison of four Hib conjugate vaccines developed with four different carrier proteins resulted in differences of antibody response generated in infants: DT conjugate (PRP-D) was the less immunogenic when compared with TT and CRM197 PRP conjugates. PRP-CRM and PRP-TT were the most immunogenic and have induced a similar level of anti-PRP antibody. PRP-OMP was the only able to produce a significant immune response after one injection, it also performed well after two injection but a similar antibody levels was induced after three injection, with final levels one/third of those achieved by PRP-CRM and PRP-TT. Additional studies in infants residing in different geographic areas, confirmed that OMP conjugate induces significant antibody levels after a single injection at 2 months of age. Studies published to compare meningococcal tetravalent vaccines obtained using CRM or DT as carrier protein showed that in adolescents MenACWY-CRM generates a robust immune response agains all serogroups, with a response noninferior for serogroup C and a statistical significant superiority for serogroups AWY, compared with MenACWY-DT vaccine. Comparing the two vaccines also in children aged 2-10 years, the MenACWY-CRM vaccine was non inferior to MenCAWY-DT for all four serogroups, and met statistical superiority for groups CW and Y. 67 68 69 71 But a direct comparison of the influence of different protein carriers on glycoconjugates immunogenicity is difficult due to the coexistence of other variables such as conjugation chemistry, saccharide chain length, adjuvant, formulation technology, and previous or concomitant vaccination with other antigens. 34 We have therefore performed a preclinical comparison between the three main carrier proteins, CRM197, TT and DT which have been conjugated with the same conjugation chemistry to serogroups A, C, W135 and Y meningococcal oligosaccharides.

This study allowed to do a comparison of the physico-chemical characteristics between the conjugates obtained using the three different proteins firstly. Despite applying the same chemistry of conjugation and stoichiometry, we observed that using CRM197 we were able to obtain conjugates with higher glycosylation degree. This could be due to differences between the proteins derived from the manipulation that DT and TT undergo during chemical detoxification. In fact diphtheria and tetanus toxins are detoxified often directly in culture medium by treatment with formaldehyde and lysine. This complex reaction involves also the lysine residues of the toxins through the formation of Schiff bases which are further stabilized by the reaction with other residues like imidazole and tyrosine rings. The products are cross-linked and heterogeneous species which might be less reactive and contain reduced number of of lysines available for conjugation. 102 Since CRM197 is a non-toxic protein and does not require the chemical detoxification, the protein has a well defined physico-chemical profile. Consequently CRM197 allows the preparation of defined glycoconjugates.

The immunogenicity evaluation in mice of the different conjugates has shown that all carriers were able to induce a specific antibodies response against each saccharide antigens. When tested without the adjuvant all responses were affected by high variability.

Immunogenicity studies with adjuvant showed that the proteins induced a comparable response against each saccharide antigen and for all vaccines we observed a significant booster effect after the third dose of immunization. No significant differences have been observed between the responses of the different vaccines except that for serogroup A meningococcus conjugates: in fact, when tested as monovalent vaccines, MenA-CRM conjugate induced, after the third dose, an antibody titer significantly higher than that of MenA-TT (*** 3 P= 0.0002) and also MenA-DT (** 3 P< 0.008). Tetravalent formulations confirmed the significant difference between MenA-CRM and MenA-TT after three doses (** 3 P< 0.008). The elicited antibodies were also functional when tested by rSBA. When anti carrier antibodies were determined, CRM197 appeared to be the less immunogenic.

This study demonstrates that the three common carriers CRM197, DT and TT are comparable in inducing a specific response against four different saccharide antigens, in mice. At the same time CRM197 was confirmed an excellent carrier also considering its physico-chemical characteristics of high pure protein with non-cross linked structure and consequently amenable to better characterization.

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The influence of the sugar moiety

The sugar moiety of glycoconjugate vaccines is key to the induction of pathogen specific functional antibodies.

Many studies have been published about the influence of the carbohydrate on the immunogenicity of glycoconjugate vaccines, 49 50 103 104 however it has not been possible until now to identify predetermined characteristics for their optimal immunogenicity. Data published suggest that saccharide chain length and saccharide loading onto the protein could influence the glycoconjugate immunogenicity.

To further investigate these parameters we used meningococcal serogroup A saccharide, as model.

Three different saccharide average chain lengths (avDP6/10, 15 and 30/40) were obtained applying ad hoc purification of the oligosaccharides. The oligosaccharides were activated and then conjugated to CRM197 using reaction stoichiometries which allowed to obtain products at average glycosylation degree varying from 9 to 3 moles of saccharide per mole of protein. Depending on the oligosaccharides length some limitations in the protein loading were found, for example loadings higher than 5 (mol/mol) could not be obtained when using the longer saccharides, while on the other hand, loadings lower than 4 (mol/mol) were not achievable with the shorter oligosaccharides. This behavior probably reflects a better reactivity of the activated short oligosaccharides which can be explained by a reduced steric hindrance and/or a higher diffusion rate as compared to the longer saccharide chains.

The immunological evaluation in mice of these conjugate in both, monovalent and tetravalent formulations, has showed that in all cases we have induced the specific response against the serogroup A meningococcus capsular polysaccharide, and the elicited antibodies were functional when tested by rSBA.

The immunogenic properties of the different vaccines seem not having been affected by the saccharide average chain length, although is difficult to draw conclusions due to the overlapping of the sugar population. Considering the carbohydrate loading, a trend to a lower response could be observed for the conjugate with high glycosylation degree and this might be related to the lower number of conjugate moles administered to mice using constant saccharide based dosages.

In this part of the PhD work we showed that in the case of serogroup A meningococcus, as already reported for other carbohydrate antigens 102 , short chain length oligosaccharides (avDP6/10) are sufficient to induce high antibody titres, with bactericidal activity against the serogroup A meningococcus, in mice.

Short oligosaccharides antigens could therefore be attractive as candidate vaccines considering the possibility to obtain them via chemical synthesis.

Currently, most of the conjugate vaccines are produced from poly-or oligosaccharides extracted and purified from microbial cultures. They can be obtained with very high purity, although heterogeneity and traces of bacterial contaminants (endotoxins, variants containing non-protective epitopes, etc.) cannot be avoided. The chemical synthesis of oligosaccharides instead provides homogeneous, well defined and characterized molecules with built-in chemical terminal functionalities suitable for conjugation to carrier proteins. 34 An attractive concept for future conjugate vaccines, although challenging, is based on fully synthetic glycopeptide vaccines where not only the hapten moiety is obtained by chemical synthesis but also the carrier is constituted by synthetic peptides representing CD4+ T-cell epitopes. 105 106 We investigated five synthetic -glucan oligosaccharides for their ability to represent the natural epitope of Laminarin antigen. By competitive studies we showed that linear -glucans better represent the immunogenic epitope located on Laminarin and that a chain length of at least six sugars would be needed in order to efficiently fill the combining site of anti Laminarin antibodies.

Our observation was than confirmed by immunogenic study in mice were the hexasaccharide antigen conjugated with CRM197 have induced a comparable response with that induced by the native conjugate with Laminarin. Synthetic -glucan hexasaccharide could be then considered as a new candidate vaccine for C.albicans.

The influence of the spacer molecule

Data present in the literature have shown that also the spacer molecule could have a role in driving the immunogenicity of the glycoconjugate vaccine. 51 52 53 We studied therefore four different linker molecules having different chemical physical aspects, maintaining the strategy of conjugation and the carbohydrate antigen, the serogroup A meningococcus.

Conjugation strategy for each spacer molecule involved the saccharide end reducing group and conjugates with similar glycosylation degree have been obtained except that for SMCC when used to activate the sugar moiety and SIDEA that allow to obtain glycoconjugate with higher saccharide/protein ratio.

Immunogenicity data have shown that all conjugates were able to induce an anti-MenA polysaccharide response and the higher response has been obtained with MenA SIDEA -CRM and MenA SMCC -CRM that have induced bactericidal antibodies also after the second dose of vaccine. On Page 77

the contrary significant lower response has been induced by MenA SMPB -CRM. Otherwise evaluating the anti-carrier antibodies induced by MenA SMPB -CRM we can see that are comparable with those induced by the other conjugates after three doses. This observation could allow us to conclude that rigid molecules containing aromatic rings might not be the optimal linkers for conjugate vaccines. It has been already shown that the rigid linker containing the aromatic ring induces very high anti linker antibodies 51 , this could cause a reduction on the anti-carbohydrate antibodies induction.

To study more in deep this aspect, future work should be focused on determination of anti-spacer antibodies induced by each type of conjugate to find whether a correlation exists between the anticarbohydrate response and the intrinsic immunogenicity of the spacer molecule.

Investigation on antigen uptake

In another context of this thesis we have investigated the immune cells recruitment and the processing and re-exposure of glycoconjugate antigens in order to get information about the mechanism of action of this class of vaccines. For this purpose, CRM-MenC has been used as molecular model, and particularly it has been double labeled in order to track both protein and carbohydrate moiety during glycoconjugate processing.

Data obtained with cell recruitment experiments shows that professional antigen presenting cells (APC) like macrophages and B cells rapidly take up the labeled glycoconjugate. Uptake by macrophages is fast, non-specific and antigen will be processed and presented on MHC-II molecules to T cells. In contrast, antigen uptake by B cells is slower and only a minor cell fraction stains positive for the glycoconjugate. Also some cells of the innate arm of the immune system like eosinophils or neutrophils can bind antigen on their surface and act as antigen transporters. Yet, the major contribution to antigen uptake in our system comes from macrophages.

Intraperitoneally treatment with double labeled glycoconjugate has induced immune cells activation in mice as expected for a general immunity mechanism consequent to the antigen. Macrophages occupy a position of central importance in the immune response, since they are key effectors in both the innate and adaptive systems. They play a role in the inflammatory response during the early stages of host innate defense, phagocytosing foreign entities and secreting chemotactic factors and cytokines, after they function as accessory cells to the lymphocytes of the adaptive immune response, secreting pro-inflammatory cytokines and also processing and presenting antigen to T cells. Macrophages act also as effectors in the humoral immune response and in cell-mediated immunity. 107 Also with confocal microscopy experiments we were able to distinguish two different types of cells, those with higher amount of antigen could be still referred to macrophages due to their prolonged Page 78

role on the antigen uptake; in these cells the antigens co-localized with Lamp-1, a lysosomeassociated membrane proteins demonstrated to be present on the phagosomal membrane. 108 Instead in the cells with lower antigen content we identified some region where both the peptide and the carbohydrate moieties are co-localized with the MHCII. Likely these are B cells where antigens are loaded on the MHCII for further interaction with T cells. Similar results have been obtained also by other research teams. 109 The mechanism of action of glycoconjugate vaccines on the immune system, in particular about the function of the sugar moiety during antigen presentation in association with MHCII complex and subsequent interaction with T cell receptor, is still unclear. Knowing that CPSs are T independent antigens 13 and that most of the carbohydrate antigens are not able to bind MCHII after APC processing 110 , the elicitation of T cell help by glycoconjugates has been attributed to the peptide moiety derived from protein processing. This attractive model has been derived with GBS type III glycoconjugates and needs further investigations to understand if it is a general mechanism. Our results show that the carbohydrate portion of MenC glycoconjugate is exposed on the surface of B-cells in association with a peptide moiety and the MHCII; we cannot exclude that the carbohydrate further interacts with specific T-cell receptors, however additional experiments, for example isolation of a MenC specific T cell clone, are needed in order to confirm this.

Suggestions for the design of future glycoconjugate vaccines

Based on the data obtained in this work, the commonly used carriers protein (CRM197, DT and TT) appear suitable to develop also new glycoconjugate vaccines. In fact their clinical development will take advantages in using well known carriers with a well demonstrated history of safety and efficacy.

However if a new carrier would be needed, in order for example to reduce the amount of the same protein administered in human vaccines, our work identified other potential candidates able to work as carrier with a performance comparable to CRM197. Our method of investigation could also be used to select additional proteins as new carriers.

In the design of new glycoconjugate vaccines particular attention should be devoted to answer the question whether there is an optimal chain length to maximize the immune response in animal Page 79 models, and later on in humans. This information will be essential also to asses the feasibility of the synthetic approach for vaccine manufacturing, considering that the production of saccharides with short chain length will be easier to scale up.

Since to a selected saccharide chain length, probably corresponds an optimal loading onto the carrier protein, this need to be defined by comparing in animal model the immunogenicity of conjugates at different glycosylation degree and fixed oligosaccharide chain length.

In order to obtain vaccines that can be deeply characterized, it is recommended the use of conjugation chemistries which involve the reducing end terminus to couple the sugar moiety to the protein. This will results in conjugates with a well defined radial structure, without cross-linking and easier to characterize.

Our data on different linker molecules suggest that the presence of an aromatic structure might jeopardize immune response against the sugar antigen.

Finally additional studies to better understand the mechanism of action of glycoconjugates vaccines will inform the development of future glycoconjugate vaccines.

This PhD work contributes to obtain more information on the properties that determine the optimal immunogenicity of the vaccine and leads to improve the design of glycoconjugate vaccines.

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Size Exclusion High Performance Liquid Chromatography (SEC-HPLC)

SEC-HPLC has been performed on UltiMate® 3000 HPLC system (Dionex part of Thermo Fisher Scientific) equipped with a PDA, RF2000 Fluorescence Detector and RI-101 Shodex Detector.

Chromatography has been performed in 0.1M NaSO 4 0.1M NaPi 5% CH 3 CN pH 7.2 on TSK 3000-4000 Gel SW (Tosoh Bioscience) analytical column at flow rate of 0.5 ml/min, with 50 µl of injection volume loading 15-25 µg of sample in protein content. The resulting chromatographic data processed using Chromeleon® 6.7 software.

Colorimetric analyses

Protein content on the conjugates has been determined by MicroBCA (Thermo) colorimetric assay. 112 Amino groups were determined by colorimetric assay. 113 Active ester groups introduced in the oligosaccharides were determined by analysis of released Nhydroxy-succinimido groups as previously published. 114 Sulphydryl groups (SH) groups content on oligosaccharides have been determined by colorimetric assay. Where MW=molecular weight of the sample, ε dey = extinction coefficient of the dye at its absorbance maximum, sample concentration was in mg/ml. as an external standard (0 ppm). All the NMR spectra were obtained in quantitative manner using a total recycle time to ensure a full recovery of each signal (5 x Longitudinal Relaxation Time T 1 ).

Dye

The avDP of MenA oligosaccharides were calculated by the integration of 31 P NMR peaks and expressed as [(P de /P me ) +1], where P de is molar concentration of the phosphodiester in chain groups and P me the molar concentration of phosphomonoester end groups.

Profiling by use of ion chromatography

The chain length distribution of MenA oligosaccharides preparation has been controlled by profiling analysis. Analysis of the hydrolyzed products has been performed by HPAEC-PAD with a Dionex ICS3000 system, using a CarboPac PA200 column and a NaNO 3 gradient in 100mM NaOH. The resulting chromatographic data have been processed using Chromeleon ® 6.8 software.

MALDI analysis

MALDI-TOF mass spectra linker-derivatized CRM 197 was recorded by UltraFlex III MALDI-TOF/TOF instrument (Bruker Daltonics) in linear mode and with positive ion detection. The samples for analysis were prepared by mixing 2.5 μL of product and 2.5 μL of Super DHB matrix. 2.5 μL of each mixture were deposited on samples plate, dried at room temperature for 10 min and subjected to the spectrometer.

Determination of total saccharide in the conjugates preparations

Total saccharide was determined by high performance anionic exchange chromatography-pulsed amperometric detection (HPAEC-PAD) using different sample pre-treatment and chromatographic conditions depending on the saccharide antigens.

Laminarin conjugates

Total saccharide content of laminarin conjugates was determined by HPAEC-PAD 72 using a CarboPac PA1 column (50mm×250mm) coupled to a CarboPac PA1 guard column and connected to a Dionex ICS3000 system. Samples, diluted at 5 µg/mL of saccharide were treated with trifluoroacetic acid (TFA) at a final concentration of 2M, heated at 100 •C for 2 h in a closed screwcap test tube, dried on a Speed Vac concentrator fitted with a refrigerated condensation trap (Savant SC110) under vacuum, dissolved in 0.5mL of distilled water and filtered 0.45µm before the analysis. The separation was performed with a flow rate of 1mL/min using isocratic elution of 40mM

NaOH for 12 min, followed by a washing step with 500mM NaOH for 5min. The chromatography was monitored using the pulsed amperometric mode with a gold working electrode and an Ag/AgCl reference electrode, a quadruple-potential waveform for carbohydrates was applied. The chromatographic data were processed using Dionex Chromeleon TM software. Calibration curve, treated as samples, was set up with glucose (Fluka) in the range 0.5-10.0µg/mL.

Meningococcal A,C,W ,Y conjugates

In MenACWY conjugates total saccharide was determined by HPAEC-PAD 116 Saccharide quantification for MenA conjugate has been performed by incubation in TFA with a final concentration of 2 M at 100 °C for 2 h in a closed screw-cap test tube, then samples has been chilled and centrifuged to combine the condensate with the bulk liquid before to be neutralized with NaOH 2M. Analysis of the hydrolyzed products has been performed by HPAEC-PAD with a Dionex ICS3000 system fitted with DP, DC and AS modules, using a CarboPac PA1 column and a

Introduction of primary amino groups or cystamine at the reducing end of meningococcal serogroup A oligosaccharides by reductive amination

MenA has been treated by reductive amination to introduce a NH 2 group on the reducing end of the sugar using these conditions: 4 mg/ml of polysaccharides were subjected to reductive amination by incubation for 5 days at 37°C in 300 mg/ml ammonium acetate (Sigma-Aldrich) and 0.2 M sodium cyanoborohydride (Sigma-Aldrich) at pH 6.5-7.

Reductive amination with cystamine has been performed using these conditions: 4 mg/ml of polysaccharides were subjected to amination with cystamine (cyst) by incubation for 5 days at 37°C in 1:23 mol/mol MenA/cyst and 1:40 mol/mol MenA/sodium cyanoborohydride at pH 6.5-7.

In both cases the modified MenA oligosaccharides, Oligo MenA-NH 2 and OligoMenA-Cystamine, have been purified by tangential flow filtration with 2kDa cut-off membrane to remove the excess of low molecular weight reagents and the retentate was collected. Amino group content has been determined by colorimetric assay as reported in 4.2.

Preparation of MenA oligosaccharides of different average degree of polymerization (avDP)

MenA CPS hydrolysis has been conducted in 50mM sodium acetate pH4.73 at saccharide concentration of 2.5mg/ml, 73°C for 30min-1h to obtain avDP 30/40, for 4-4.30h for avDP15 and for 7-7.30h to obtain avDP 6/10.

After hydrolysis the chain length selection has been performed applying different purification techniques. To select the avDP 30/40 after hydrolysis a tangential flow filtration has been done with 100kDa cut-off membrane (Sartorius) and the retentate containing the avDP saccharides higher than 30/40 have been eliminated. Instead the permeate has been treated with a second tangential flow filtration with 30kDa cut-off membrane that allow to eliminate in permeate the avDP saccharide lower than 30/40. The material in the 30kDa retentate contains the avDP 30/40 oligosaccharides of our interest.

To select the avDP6/10, the material has been treated with a tangential flow filtration with 2kDa cutoff membrane to eliminate in the retentate the saccharides at high avDP. The permeate has been further purified by anion exchange chromatography using a Q Sepharose resin (GEHealthcare): the sample has been charged on the column in 50mM NaCl and after a gradient to 500mM NaCl has been applied on 30 column volumes. After profiling analysis of the single fractions the pool containing the avDP oligosaccharides 6/10 has been collected. The oligosaccharides avDP 30/40 and 6/10 have been treated by reductive amination and activated with SIDEA as reported before.

Derivatization of OligoMenA-NH 2 with Succinimidyl diester of adipic acid (SIDEA)

This reaction as carried as described before for the laminarin oligosaccharides. After, acetone precipitation has been done and on lyophilized pellet active ester groups have been determined.

Derivatization of serogroup C menigococcal oligosaccharide with fluorescent labels

To obtain a glycoconjugate selectively labelled on the protein and on the saccharide moiety, MenC oligosaccharides avDP15 have been labelled with Alexa488 (A488, Invitrogen). At first MenC was aminated by reductive amination using the same conditions described for MenA. After, the hydrazide groups on A488 have reacted with the carboxylic groups on the sialic acid of MenC sugar:

10% mol of A488 respect to mol of MenC-COOH were put in reaction with 50% mol of EDC (Sigma)

Vaccines formulation and immunological studies

Preparation of glycoconjugates formulations

Antigens formulations have been done under sterile woods using sterile instrumentation and solutions.

All formulations have been done using PBS pH 7.2 as buffer where the vaccines have been diluted

to obtain the require dosage of saccharide per mice in a total volume of 200µl.

Aluminium phosphate (AlPO 4 ) has been used as adjuvant and has been prepared as 2x solution to be mixed 1:1 to the total volume of PBS. Each dose contained 0.12 mg (expressed as Al 3+ ) of aluminium phosphate.

To study the absorption of vaccines on AlPO 4 , formulated vaccines were incubated 1h at R.T.

measuring the pH and after centrifuged at 13000g, 4°C for 40 min. Protein dosage has been done as reported on the supernatant. The % of absorption has been calculated relatively to the formulation without adjuvant.

MenA vaccines have been administered to mice in 2µg per dose in saccharide content; MenCWY vaccines have been administered to mice in 1µg per dose in saccharide content; Laminarin vaccines have been administered to mice in 5µg per dose in saccharide content.

Mice have been immunized subcutaneously at day 1, 14 and 28. Bleedings were performed at day 0 (pre immune), day 28 (post 2) and day 42 (post 3). Control groups received PBS. Animal experimental guidelines set forth by the Novartis Animal Care Department were followed in the conduct of all animal studies. Page 91

Immunochemical evaluation of response

The antibody response induced by the glycoconjugates against the homologous polysaccharide and carrier protein has been measured by ELISA. Plates have been coated with the different meningococcal polysaccharides or carrier proteins by adding 100 µl/well of a 5 µg/ml polysaccharide solution in PBS buffer at pH 8.2 or 100 µl/well of a 1-2 µg/ml carrier protein solution in PBS buffer at pH 7.2, followed by incubation overnight (o.n.) at 4°C. Instead Laminarin coating has been done at 5 µg/ml of polysaccharide solution in 0.05M Na 2 CO 3 -NaHCO 3 buffer at pH 9.6.

Coating solutions were removed from the plates by washing tree times per well with PBS buffer with 0.05% of Tween 20 (Sigma) (TPBS). A blocking step has been then performed by adding 100 µl of BSA solution at 3% in TPBS and incubating the plates 1 h at 37°C. Blocking solution has been removed from the plates by washing three times per well with TPBS. Two hundred µl of pre-diluted serum (1:25 for pre immune, 1:100-1:200 for a reference serum from 1:50 to 1:1000 for test sera)

was added in the first well of each column of the plate, while on the other wells 100 µl of TPBS has been dispensed. Eight twofold serial dilutions along each column were then performed by transferring from well to well 100 µl of sera solutions. After primary Abs dilution, plates have been incubated for 2h at 37°C. After three washes with TPBS, 100 µl TPBS solutions of secondary antibody alkaline phosphates conjugates (anti mouse IgG 1:10000 or anti mouse IgG 1, 2a, 2b, 3 , Sigma-Aldrich, diluted 1:2000 or anti mouse IgM 1:5000) were added and the plates incubated 1h at 37°C. After three more washes with TPBS, 100 µl/well of a 1mg/ml of p-NPP (Sigma) in a 0.5 M diethanolammine buffer pH 9.6 was added. After 30min of incubation at room temperature, plates were read at 405nm using a Biorad plate reader. Raw data acquisition has been performed by Microplate Manager Software (Biorad). Sera titers were expressed as the reciprocal of sera dilution corresponding to a cut-off OD = 1 or to a cut-off OD=0.2. Each immunization group has been represented as the geometrical mean (GMT) of the single mouse titers. The statistical and graphical analysis has been done by GraphPad 5.0 software.

Competitive ELISA

The protocol described above was followed to prepare laminarin-coated plates. For confocal microscopy analysis, fixed cells collected from peritoneal wash samples were permeabilized with PBS, 0.1% saponin, 1% BSA (permeabilization buffer, PB) fro 30 minutes. Then cells were stained with anti MHCII or with anti Lamp1 antibodies, followed by secondary AF568 conjugated anti mouse IgG antibodies. Cells were washed twice with PB and then resuspended in 100 ul of PBS and centrifuged onto a slide by cytospin (300 rpm, 4 min). Samples were treated with

ProLong antifade medium and analyzed by laser scanning microscopy (LSM700, Zeiss).

4. 3 . 1

 31 Introduction of primary amino groups at the reducing end of laminarin oligosaccharide by reductive amination ................................................................................................................................................................. 4.3.2 Derivatization of the laminarin amino oligosaccharides to active esters ................................................... 4.3.3 Introduction of primary amino groups or cystamine at the reducing end of meningococcal serogroup A oligosaccharides by reductive amination ................................................................................................................. 4.3.4 Preparation of MenA oligosaccharides of different average degree of polymerization (avDP) ................. 4.3.5 Derivatization of OligoMenA-NH 2 with Succinimidyl diester of adipic acid (SIDEA) ................................... 4.3.6 Derivatization of OligoMenA-NH 2 with Bis succinimidyl penta ethylene glycol (Bis(NHS)PEG5)................ 4.3.7 Derivatization of OligoMenA-Cystamine with Succinimidyl4 p-maleimidophenyl butyrate (SMPB) ..........

Figure 1 :

 1 Figure 1: Active immunization after vaccination. Vaccination induces activation by cytokines, B cells differentiate into memory B cells (long-lived antigen-specific B cells) or plasma cells (effector B cells that secrete large quantities of antibodies). Most antigens activate B cells using activated T helper (Th) cells, primarily Th1 and Th2 cells. The Th1 response leads mainly to a cell-mediated immunity (cellular response) instead Th2 cells generally induce a humoral response (antibody) 2 .

Figure 2 :

 2 Figure 2: Example of the Effectiveness of Vaccine Coverage. The graph shows how disease incidence, in this case serogroup C meningococcal disease, can fall dramatically with increasing vaccine coverage.*Laboratory-confirmed cases.

Figure 7 :

 7 Figure 7: Repeating unit structure of Neisseriae meningitidis serogroups A, C, W 135 and Y saccharides.

8 Figure 9 :

 89 Figure 9: SDS-page of Laminarin glycoconjugates with new candidate carrier proteins

Figure 10 :

 10 Figure 10: anti-Laminarin Abs level induced by new candidate carrier proteins in comparison with the positive control Lam-CRM. Each spot indicate a single mouse ELISA titer, the horizontal bar refers to the geometric mean of the group, while the vertical bar shows the 95% CI.

8 Figure 12 :

 812 Figure 12: SDS-page of MenC glycoconjugates with new candidate carrier proteins.

Figure 13 :

 13 Figure 13: Anti-MenC Abs levels induced by new candidate carrier proteins in comparison with the positive control MenC-CRM. Each spot indicate single mouse ELISA titer, the horizontal bar was the geometric mean of the group, instead the vertical bar shows the statistical 95% CI.

6 .

 6 Figure 15: SDS-page of MenACWY glycoconjugates with PNEU20.

27

 27

Figure 16 :

 16 Figure 16: Anti-MenACWY Abs levels induced by PNEU20 glycoconjugates in comparison with the positive control MenACWY-CRM conjugates. Each spot indicate a single mouse ELISA titer, the horizontal bar was the geometric mean of the group, instead the vertical bar shows the statistical 95% CI.

33 Figure 18 :

 3318 Figure 18: A) Chromatographic profile of TT protein obtained by gel filtration on Sephacryl S300.

Figure 19 :Figure20:

 19 Figure 19: Electrophoretic profiles of glycoconjugates with different carrier proteins: CRM197 panel A, DT panel B and TT panel C. Lanes 1 to 4 have been loaded with MenA, MenC, MenW and MenY conjugates respectively.

Figure 21 :

 21 Figure 21: Mice immunization schedule.

Figure 22 :

 22 Figure 22: Antibody response induced in mice by the individual meningococcal conjugates formulated without adjuvant. Each spot indicate single mouse ELISA titer, the horizontal bar was the geometric mean of the group, while the vertical bar shows the statistical 95% CI.

Figure 23 :

 23 Figure 23: Anti-protein IgG detection on sera post 2 nd and post 3 rd immunization from the study with adjuvant. A) monovalent formulations; B) tetravalent formulations. Data have been grouped for type of protein.

Figure 24 :

 24 Figure 24: Purification steps applied to hydrolyzed MenA to select avDP 6/10, 15 and 30/40.

Figure 25 :

 25 Figure 25: Chromatogram obtained by anionic exchange chromatography column to select avDP 6/10 and 15. Blue box indicates the fractions collected.

Figure 26 :Figure 27

 2627 Figure 26: HPAEC-PAD profiles obtained for each avDP oligosaccharides.

Figure 28 :Figure 29 :

 2829 Figure 28: MenA-CRM conjugation scheme after amination and activation of the oligosaccairde.

Figure 30 :

 30 Figure 30: Antibody (ELISA) response of monovalent and tetravalent MenA-CRM formulations. A) avDP 30/40 MenA conjugates; B) avDP15 MenA conjaugates; C) avDP6/10 MenA conjugates.

Figure 31 :

 31 Figure 31: Chemical structure of Laminarin and synthetic fragments.

Figure 32 :

 32 Figure 32: Competitive ELISA of synthetic glucans 1-4 and 6. Meningococcal C polysaccharide was used as negative control (neg).

Fiure 33 :

 33 Antibodies titers induced in mice by the synthetic β-(1, 3) glucan hexasaccharide-CRM197 conjugate formulated with adjuvant MF59. Horizontal and vertical bars refer to the geometric means and 95% confidence interval, respectively. Page 54

79

 79

Figure 34 : 18 :

 3418 Figure 34: Schematic conjugation process with linker activation of the sugar moiety.

Figure 35 :

 35 Figure 35: Scheme of reactions to obtain MenA-CRM conjugates with different linkers. A) MenA PEG -CRM; B) MenA SMCC -CRM; C) MenA SMPB -CRM; D) MenA-CRM SMCC

Figure 36 :

 36 Figure 36: SDS-page of MenA-CRM conjugates obtained with different linkers.

Figure 38 :

 38 Figure 38: Antibodies response induced by MenA-CRM conjugates obtained with different linkers after the 2 nd and the 3 rd dose of vaccines. A) Anti-Serogroup A meningoccocus CPS IgG response; B) Anti-CRM IgG response.

Figure 39 : 3 Figure 40 :

 39340 Figure 39: Scheme of reaction to obtain MenC-CRM conjugates with double labeling with A488 and A647.

Figure 41 :

 41 Figure 41: Spectrophotometric analysis of the labelled conjugates.

Figure 41 :

 41 Figure 41: Graphical representation of cell recruitment after intraperitoneally mice treatment with glycoconjugate (GC).

Figure 42 A 69 Figure 43B :

 426943B Figure 42 A and B: antigen uptake experiment by FACS. A) FACS analysis conducted at different time. B) Graphical representation of antigen uptake experiment, data were expressed both as % of total cell subset and as absolute cell number at different time.

Figure 43C :

 43C Figure 43C: Confocal microscopy experiment. Cells with lower antigen content where the antigen localizes on the cell surface with some regions of co-localization with MHCII.

  Recently a different mechanism has been proposed by Kasper et al. In their model there are T-cell populations that recognize carbohydrate epitopes derived by APC processing of conjugate vaccines. When presented in association with MHCII, these epitopes recruit T-cell help for the induction of specific anti carbohydrate antibodies. 111

  115

4. 2 . 4

 24 Spectrophotometric analysis The label degree (DOL) of oligosaccharides labelled with fluorescent dyes has been determined by spectrophotometer. DOL calculation has been done by measuring the absorbance of the sample at the λ max (Amax) of the dye and applying the Lambert-Beer law to calculate the number of dye molecule per molecule of sample knowing the sample concentration. DOL= (Amax * MW)/[sample] * ε dey

  using different sample pre-treatment and chromatographic conditions for the different saccharides. MenY and MenW conjugates were hydrolyzed by incubation in trifluoroacetic acid (TFA) with a final concentration of 2 M at 100 °C for 2 h in a closed screw-cap test tube, then centrifuged to combine the condensate with the bulk liquid and dried on a Speed Vac concentrator fitted with a refrigerated condensation trap (Savant SC110) under vacuum. Samples have been first dissolved in distilled and degassed water and then filtered (0.45 μm). Analysis of the hydrolyzed products has been performed by HPAEC-PAD with a Dionex ICS3000 system fitted with DP, DC and AS modules, using a CarboPac PA1 column and a NaOH/Na acetate step elution. The resulting chromatographic data have been integrated and processed using Chromeleon® 6.8 software. Quantification has been performed with calibration curves, treated as samples, set up with galactose and glucose, for MenW and MenY respectively, in the range of 0.5-4.0 μg/ml. Saccharide quantification for MenC conjugate has been performed by incubation in trifluoroacetic acid (TFA) with a final concentration of 1 M at 90 °C for 2 h in a closed screw-cap test tube, then samples have been chilled and centrifuged to combine the condensate with the bulk liquid before neutralization with sodium hydroxide (NaOH) 4M. Analysis of the hydrolyzed products has been performed by HPAEC-PAD with a Dionex ICS3000 system fitted with DP, DC and AS modules, using a CarboPac PA1 column and a NaOH/Na acetate elution gradient. The resulting chromatographic data have been integrated and processed using Chromeleon® 6.8 software. Quantification has been performed with a calibration curve, treated as samples, set up with sialic acid in the range of 0.5-5.0 μg/ml.

2 , 3 , 4 , 92 37C.

 23492 The plate was designed to contain (a) a blank column with TPBS alone, without serum and inhibitors, and (b) a column with serum alone, without inhibitors (b0); the other columns contained both, the serum and the inhibitors, which included also laminarin and the not correlated polysaccharide MenC as positive and negative controls, respectively. The different competitors (compounds 1, and 6) were prediluted to obtain the starting concentration of 4 mM and eight 10-fold dilutions were performed on the plate. Meningococcal C polysaccharide was used as negative control.The competitors at different concentrations starting from 10 mg/mL concentration were mixed with an equal volume of a fixed dilution of antilaminarin immune serum, followed by 2 h incubation at Page After primary antibody incubation the general protocol described in the ELISA Analysis section was followed. All OD lectures were subtracted from the mean value of the blank column (b). The inhibition percentage was expressed as follows: % inhibition = [(B0 ODx)/B0] x100, where B0 is the mean values of the b0 column (serum without inhibitor) and ODx is the optical density corresponding to each inhibitor concentration. IC50 was defined as the inhibitor concentration resulting in 50% inhibition of the main reaction. Fitting of inhibition curves and calculation of IC50 values were performed on the Graphpad Prism software using the variable slope model (Graphpad Prism Inc.).Native MenA CPS and oligosaccharides were provided by Novartis Vaccines and Diagnostics, Siena, Italy. Polyclonal serum derived from immunization of mice with a native meningococcal A oligosaccharide-protein conjugate was provided by Preclinical Serology Laboratory in Novartis Vaccines and Diagnostics, Siena, Italy. 96-Well Maxisorp plates (Nunc, Thermo Fisher scientific)were coated overnight at +4 •C with Meningococcal A native capsular polysaccharide (MenA CPS), 5 µg/well in Phosphate Saline Buffer (PBS) pH 8.2. After coating, the plates were washed three times with 300 µl per well of PBS with 0,05% Tween 20 (TPBS) at pH 7.4. Plates were blocked with 3% bovine serum albumin (Fraction V, Sigma-Aldrich) in TPBS for 1 h at 37°C and then washed again. 50 µl of polyclonal immune mouse sera pre-diluted in TPBS were put on the plate and mixed with 50 µl of inhibitor previously diluted with tenfold serial dilution on another plate. On the column without inhibitor 50 µl of immune mouse sera pre-diluted in TPBS were mixed with 50 µl of TPBS.Polyclonal serum obtained from native Meningococcal A immunization was used at the final dilution of 1:1600. After 2h of incubation at 37°C and washing with TPBS, 100 µl/well of 1:10,000 of antimouse IgG alkaline phosphatase conjugated (Sigma-Aldrich) were added and plates were incubated for 1 h at 37°C. After, plates were developed for 30 min at room temperature with 100 µl/well of 1 mg/ml p-nitrophenyl phosphate disodium (Sigma-Aldrich) in 1 M diethanolamine (pH 9.8) and red at 405 nm with a microplate spectrophotometer (Biorad). The plate was designed to contain: a) blank column with TPBS alone, without serum and inhibitors, b) column with serum alone, without inhibitors (b0); the other columns contained both, the serum and the inhibitors which included also MenA CPS, native MenA oligosaccharides and the not correlated Laminarin polysaccharide as positive and negative controls respectively.The different competitors (compounds 1 = monosaccaride, 2 = disaccharide, 3 = trisaccharide and nativeavDP3 = native MenA oligosaccharide average polymerization degree 3 ) were pre-diluted to obtain the starting concentration of 0.5 mM and ten fold dilutions were performed eight times on the plate. Native MenA CPS and avDP15 oligosaccharide used as positive control were pre-diluted to obtain a starting concentration of 0.05mM, instead Laminarin of 1mM and was used as negative control. All OD lectures were subtracted of the mean value of the blank column (b). The inhibition percentage was expressed as follows: % inhibition= [(B0 -ODx)/B0] x 100. Where B0 is the mean values of the b0 column (serum without inhibitor) and ODx is the optical density corresponding to each inhibitor concentration. IC50 was defined as the inhibitor concentration resulting in 50% inhibition of the main reaction. Fitting of inhibition curves and calculation of IC 50 values was performed on the Graphpad Prism software using variable slope model (Graphpad Prism Inc.).

4. 5 . 4

 54 Serum bactericidal assay (rSBA)Functional antibodies induced by vaccine immunization were analysed by measuring the complement-mediated lysis of Neisseria meningitidis with an in vitro bactericidal assay.118 A commercial lot of baby rabbit complement was used as source of active complement. Briefly, N. meningitidis strains were grown overnight on chocolate agar plates at 37°C in 5% CO 2 . Colonies were inoculated in Mueller-Hinton broth, containing 0.25% glucose to reach an OD600 of 0.05-0.08 and incubated at 37°C with shaking. When bacterial suspensions reached OD600 of 0.25-0.27, bacteria were diluted in the assay buffer (Gey's balanced salt solution with 1% BSA) at the working dilution (ca. 104 CFU/mL). The total volume in each well was 50µL with 25µL of serial two-fold dilutions of the test serum, 12.5µL of bacteria at the working dilution and 12.5µL of baby rabbit complement. All sera to test were heat-inactivated for 30 minutes at 56°C. Negative controls included bacteria incubated, separately, with the complement serum without the test serum and with test sera and the heat-inactivated complement. Immediately after the addition of the baby rabbit complement, negative controls were plated on Mueller-Hinton agar plates, using the tilt method (time 0). Plate were incubated for 1 hour at 37°C, then each sample was spotted in duplicate on Mueller-Hinton agar plates while the controls were plated using the tilt method (time 1). Agar plates were incubated overnight at 37°C and the colonies corresponding to time 0 and time 1 (surviving bacteria) were counted. The serum bactericidal titre was defined as the serum dilution resulting in 50% decrease in colony forming units (CFU) per mL, after 60 min incubation of bacteria in the reaction mixture, compared to control CFU per mL at time 0. Typically, bacteria incubated without the test serum in the presence of complement (negative control) showed a 150 to 200% increase in CFU/mL, during the 60 min incubation time. The reference strains for each meningococcal serotype used were: F8238 (MenA); C11 (MenC); 240070 (MenW135); 860800 (MenY).

4. 5 . 5

 55 In vivo and ex vivo cell uptake experimentsBalb/c mice were treated IP with double labeled CRM-MenC antigen containing an equivalent of 1 mcg of MenC/ mouse in a volume of 200 l. Peritoneal wash samples were collected at time 0, 30 min, 6 h and 24 h post treatment. Cells were harvested by centrifugation at 1300 rpm, washed 2 times with PBS and then fixed with 2% of formaldehyde solution for 20 min at RT. Cells were then washed and stained in 2% rabbit serum with the following antibody cocktail: CD19-PE; MHCII-A700; F4/80-Pacific Blue; Gr1-PerCP Cy5.5; CD11c-APC e700 CD11b PECy7. Samples were acquired using a FACS LSRII (BectonDickinson, special order system) and analyzed using BD-DIVA software (BectonDickinson).

Table 3 :

 3 Licensed glycoconjugate vaccine.

2.2 Screening of new carrier proteins

  In this study 31 proteins derived from different bacteria have been studied for their capacity to work as carrier for different saccharide antigens. The proteins used derived from different bacteria like The proteins were selected by five main criteria: molecular weigh between 40 and 100kDa, non-enzymatic activity, non-toxicity, water solubility, availability by recombinant expression and appropriate number of lysine residues for conjugation reaction. All the proteins tested as new carrier candidate are reported in the following Table4.

	PNEU 6	Streptococcus pneumoniae	49,1
	PNEU 7	Streptococcus pneumoniae	115,4
	PNEU 8	Streptococcus pneumoniae	194
	PNEU 9	Streptococcus pneumoniae	194
	PNEU 10	Streptococcus pneumoniae	35,5
	PNEU 11	Streptococcus pneumoniae	92
	Protein PNEU 12	Micro organism Streptococcus pneumoniae	MW (kDa) 38,9
	Ex 1 PNEU 13	Extraintestinal Pathogenic Escherichia coli Streptococcus pneumoniae	54 36,5
	Ex 2 PNEU 14	Extraintestinal Pathogenic Escherichia coli Streptococcus pneumoniae	165 70,4
	MB 1 PNEU 15	N. Meningitidis serogroup B Streptococcus pneumoniae	27 52
	MB 2 PNEU 16	N. Meningitidis serogroup B Streptococcus pneumoniae	47,4 38
	MB 3 PNEU 17	N. Meningitidis serogroup B Streptococcus pneumoniae	65 122
	MB 4 PNEU 18	N. Meningitidis serogroup B Streptococcus pneumoniae	34,5 15
	MB 5 PNEU 19	N. Meningitidis serogroup B Streptococcus pneumoniae	68 27
	GAS 1 PNEU 20	Group A streptococcus Streptococcus pneumoniae	60 61
	GAS 2	Group A streptococcus	89,4
	GBS 3	Group B streptococcus	30
	PNEU 1	Streptococcus pneumoniae	48,3
	PNEU 2	Streptococcus pneumoniae	41,8
	PNEU 3	Streptococcus pneumoniae	72,5
	PNEU 4	Streptococcus pneumoniae	72,6
	PNEU 5	Streptococcus pneumoniae	46,7
		Page 20

Extraintestinal Pathogenic Escherichia coli (ExPEC), N. Meningitidis serogroup B, Group A streptococcus (GAS), Group B streptococcus (GBS) and Streptococcus pneumoniae.

Table 4 : Bacterial protein selected to be studied as new candidate carrier proteins for glycoconjugate vaccines

 4

Table 5 . Laminarin conjugates Saccharide/protein w/w

 5

	Ex 1	0.4
	Ex 2	0.3
	MB 1	0.5
	MB 2	0.4
	MB 3	0.6
	MB 4	1.7
	MB 5	0.6
	MB 6	0.6
	GAS 1	0.9
	GAS 2	0.2
	GBS 3	0.7
	PNEU 1	0.6
	PNEU 2	0.4
	PNEU 3	0.6
	PNEU 4	0.4
	PNEU 5	0.6
	PNEU 6	0.7
	PNEU 7	0.1
	PNEU 8	0.1
	PNEU 9	0.1
	PNEU 10	0.6

Table 5 :

 5 Laminarin glycoconjugates characterization by saccharide/protein ratio w/w.

Table 6

 6

	MenC Conjugates	Saccharide/protein w/w
	Ex 1	0.3
	Ex 2	0.1
	MB 2	0.5
	GAS 1	0.3
	PNEU 8	0.1
	PNEU 11	0.1
	PNEU 19	0.6
	PNEU 20	0.3
	CRM197	0.6

and varied between 0.1 and 0.6 w/w for the different conjugates.

Table 6 :

 6 MenC glycoconjugates characterization by saccharide/protein ratio w/w

Table 8 :

 8

	(Men A -F8238)	2048	4096
	MenC (Men C -60E)	1024	2048
	MenW (Men W -240070)	128	512
	MenY (Men Y -860800)	2048	1024

rSBA titers induced by sera post 2 nd dose of immunization with MenACWY-CRM and MenACWY-PNEU20 glycoconjugates. Page 32

rd 2 nd 3 rd 2 nd 3 rd 2 nd 3 rd

	Bacteria strain	A		C		W		Y
	Dose	2 nd 3 Monovalent formulation
	CRM conjugates 64 2048 8	32	8 128 64 2048
	TT conjugates 64 4096 32 128	8 128 128 512
	DT conjugates 32 128 128 2048 64 256 128 2048
	PBS	8	8	8	8	8	8	8	8

Table 12 :

 12

Rabbit complement SBA titers determined in pool sera from mice immunized with individual meningococcal conjugates without adjuvant.

Table 13 :

 13

	Page 40

3 P< 0.008) for monovalent vaccines. In tetravalent formulation the difference between MenA-CRM and MenA-TT is maintained at post 3 (** 3 P< 0.008). Antibody response induced in mice by the individual meningococcal conjugates and their combinations formulated with AlPO 4 . (GMT 95%CI) ** 1 P< 0.0014; * 2 P< 0.05; *** 3 P= 0.0002; ** 3 P< 0.008.

Table 14 :

 14 Rabbit complement SBA titers determined in pool of sera from mice immunized with individual meningococcal conjugates and their combinations formulated with AlPO 4 . Values for monovalent formulations are the average of rSBA titers obtained in two different studies.

	Page 41

*Average of three replicates.

Additional information on the immunological response of the meningococcal conjugates with the three different carrier proteins was obtained by analyzing the level of anti-carrier antibody. Each conjugate induced a specific antibody response against the protein moiety. As opposed to CRM197,

Table 15 .

 15 The 31 P NMR minor peak observed in the P de region (up-field) was assigned to the de-O-acetylated repeating units, which don't contain OAc groups both at C 3 and C 4 .

	Chain length oligosaccharide	31 P NMR avDP
	6/10	9
	15	12
	30/40	36

Table 15

 15

: avDP calculation by

[START_REF] Pollard | Maintaining protection against invasive bacteria with protein-polysaccharide conjugate vaccines[END_REF]

P NMR.

Table 16 :

 16

	Conjugate Sacch/Prot (w/w) Sacch/Prot (mol/mol)
	avDP6/10
	ML (M13)	0.15	4
	HL (M15)	0.34	8
	avDP15
	LL (M10)	0.23	3
	ML (GMP)	0.42	5
	HL (M5)	0.8	9
	avDP30/40
	LL (MT2)	0.46	3
	LL (M8)	0.56	3
	ML (M11)	0.88	5

Saccharide/protein ratio of MenA-CRM conjugates obtained with saccharide antigen at different avDP. Page 48

Table 17 :

 17 rSBA titers of monovalent and tetravalent MenA-CRM formulations. A1) avDP 30/40 MenA conjugates; B1) avDP15 MenA conjugates; C1) avDP6/10 MenA conjugates.

MenA SIDEA -CRM MenA PEG -CRM MenA SMPB -CRM MenA SMCC -CRM MenA-CRM SMCC

	Conjugates	Sacc/Prot (w/w)	Sacc/Prot (mol/mol)
	MenA SIDEA -CRM	0.42	4.9
	MenA PEG -CRM	0.16	1.93
	MenA SMPB -CRM	0.15	1.84
	MenA SMCC -CRM	0.39	4.71
	MenA-CRM SMCC	0.16	1.94

Figure 37: HPLC profile of MenA-CRM conjugates obtained with different linkers.

Table 19 :

 19 Chemical comparison of MenA-CRM conjugates obtained with different linkers.

	Page 59

Table 20 :

 20 rSBA titers of the elicited antibodies with MenA-CRM conjugates with different linkers, post the 2 nd and the 3 rd dose of vaccines.

	rSBA (MenA)
	Conjugates	Post 2	Post 3
	MenA SIDEA -CRM	1024	8192
	MenA PEG -CRM	8	4096
	MenA SMPB -CRM	8	4096
	MenA SMCC -CRM	512	4096
	MenA-CRM SMCC	1024	4096
	PBS\AlPO 4	8	8

λ max Em ε dey CF 280

  NMR experiments were recorded at 25  0.1°C on Bruker Avance III 400 MHz spectrometer, equipped with a high precision temperature controller, and using 5-mm broadband probe (Bruker). All the samples were dissolved in 0.75 mL of deuterium oxide (D 2 O, 99.9% atom D,

	4.2.5 NMR analyses
	1 H and 31 P Aldrich) and inserted in 5-mm NMR tube (Wilmad). For data acquisition and processing, TopSpin
	version 2.6 software (Bruker) was used.
	1 H NMR spectra were collected at 400 MHz over a 10 ppm spectral width, accumulating
	approximately 128 scans. The transmitter was set at the HDO frequency which was used as the
	reference signal (4.79 ppm). 31 P NMR spectra were recorded at 161.9 MHz over 20 ppm spectral
	width, accumulating approximately 1k of scans. 75% phosphoric acid in deuterium oxide was used
	Alexa Fluor® 488	495	519	71.000	0.11
	Alexa Fluor® 647	650	665	239.000	0.03
	Em= fluorescence excitation and emission maxima in nm; CF 280 = correction factor at 280nm (A 280
	free dye/A max free dye)
			Page 82

  After 3hthe reaction has been dialyzed on 3.5kDa cut-off membrane against H 2 O. After DOL determination (DOL=0.484), MenC/A488 has been activated with SIDEA as reported before and active ester group determination has been done on the lyophilized sample. All reactions have been done in the dark as required by the dye usage instructions.Laminarin conjugations have been carried out in 10-100mM NaH 2 PO 4 pH 7 or in phosphate buffer saline pH7 (PBS, 150mM NaCl, 10mM Na 2 HPO 4 , 2mM KCl, 2mM KH 2 PO 4 ) depending on the protein buffer, using an active ester(AE):protein ratio mol:mol of 30:1. The reaction has been carried over night (o.n.) at RT with gentle stirring.The conjugates obtained have been purified from the unreacted sugar by affinity chromatography with His MultiTrap 96-Well Filter Plates (GEHealthcare) using the His tag portion on the recombinant protein to bind the conjugate to the resin. Conjugates have been charged on the resin 250µg per well on volume of 200µl in the same buffer of conjugation and incubate for 15min after mixing on the well, other 15min of incubation has been done. After, the flow through have been taken by using the vacuum air and two wash of the resin have been done with 200 µl of PBS buffer pH 7.2. Conjugates Conjugates preparation with CRM197, TT and DT as carrier proteinsProtein CRM197 was produced internally by Novartis V&D, Siena, Italy. TT and DT were produced by Novartis V&D, Marburg, Germany. For TT protein, the monomer portion has been isolated by gel filtration using a Sephacryl S300 resin and 0.15M NaCl, 0.01M NaPi pH7.2 as running buffer and collecting the fractions at lower molecular weight.Meningococcal ACWY oligosaccharides avDP15 after SIDEA activation have been conjugated to the three different carrier with an AE:protein ratio mol:mol of 12:1, in 100mM NaH 2 PO 4 pH 7 and incubated o.n. at RT in gentle stirring. CRM197, TT and DT conjugates have been purified by (NH 4 ) 2 SO 4 precipitation to remove the unreacted oligosaccharides. Precipitation has been carried out adding 500g/l of solid ammonium sulphate to the conjugate solution; the suspension has been incubated on ice for 15 min and then centrifuged at 4°C for 30 min. After centrifugation, the pellet has been dissolved in 0.01M NaH 2 PO 4 solution pH 7 and the treatment has been repeated for 3 times. Then, the pellet has been washed 3 times with an ammonium sulphate solution 500g/l. Finally pellets have been dissolved in 0.01M NaH 2 PO 4 pH 7. 1M NaHCO 3 buffer pH8.2 for 6h at RT. After, the sample has been diluted 1:2 with 10mM NaH 2 PO 4 and purified by (NH 4 ) 2 SO 4 precipitation to remove the unreacted sugar. Precipitation has been carried out adding 500g/l of solid ammonium sulphate to the conjugate solution; the suspension has been incubated on ice for 15 min and then centrifuged at 4°C for 30 min. After centrifugation, the pellet has been dissolved in 0.01M NaH 2 PO 4 solution pH 7 and the treatment has been repeated for 3 times. Then, the pellet has been washed 3 times with an ammonium sulphate solution 500g/l. Finally pellets have been dissolved in 0.01M NaH 2 PO 4 pH 7. After purification by precipitation, the sample has been dialyzed to remove the unreacted dye, dialysis has been done on 3-8kDa cut-off membrane on 0.01M NaH 2 PO 4 pH7 for 24h at 4°C, changing the buffer for 5 times.

	4.4 Conjugates preparation and purification 4.4.1 Conjugates with new carrier candidate proteins have been eluted from the resin by adding 150 µl of elution buffer 0.5M NaCl, 0.5M NaH 2 PO 4 , 0.5M unconjugate protein; M11 by gel filtration using a Sephacryl S300 resin and 0.01M NaCl, 0.01M NaPi pH 7.2 as running buffer to eliminate the unreacted sugar. 4.4.4 Conjugates with different linkers MenA oligosaccharides avDP 15 have been activated as reported above with different linker molecules. Activated oligosaccharides have been conjugated with CRM197 using these conditions of reaction: MenASIDEA: AE:protein ratio mol:mol 12:1, in 50-100mM NaH 2 PO 4 pH 7 and incubated o.n. at RT in gentle stirring. MenAPEG: AE:protein ratio mol:mol 13:1, in 50-100mM NaH 2 PO 4 pH 7 and incubated o.n. at RT in gentle stirring. MenASMPB: AE:protein ratio mol:mol 13:1, in 50-100mM NaH 2 PO 4 pH 7 and incubated 48h at RT in gentle stirring. MenASMCC: AE:protein ratio mol:mol 13:1, in 50-100mM NaH 2 PO 4 pH 7 and incubated 48h at RT in gentle stirring. With SMCC a second type of conjugate has been obtained activating the protein moiety with the linker molecule and after conjugating with the saccharide: CRM197 has been taken in reaction with 4.4.2 Conjugation conditions for avDP6-11 oligosaccharides: two different conjugates have been using 3M NaCl as sample loading buffer and stripping with a gradient of H 2 O to eliminate the 0.
	Imidazole pH 6.3, incubating for 15min and collecting the flow through; the elution has been prepared (M13 13:1 prot 40mg/ml, M15 13:1 prot 40mg/ml) with AE:protein ratio mol:mol of 13:1, in 30% of mol of SMCC linker considering the total mol of lysine in the CRM197 (39 lysine present in
	repeated two times. Eluted conjugates have been dialyzed with 3-8kDa membrane vs PBS buffer 100mM NaH 2 PO 4 pH 7 and incubated o.n. at RT in gentle stirring. Conjugates have been purified by CRM197), that are the NH 2 groups free to react. Reaction has been carried in 50mM NaH 2 PO 4 pH
	pH 7.2 to eliminate the elution buffer, dialysis have been carried out one day at 4°C changing the gel filtration using a Sephacryl S300 resin and 0.01M NaCl, 0.01M NaPi pH 7.2 as running buffer to 7 for 3h at RT. After, CRM197-SMCC has been purified in size exclusion chromatography using a
	buffer for four times. eliminate the unreacted sugar. G25 resin (GE Healthcare) and 10mM NaH 2 PO 4 pH 7 with 10% of saccharose as running buffer.
	Purified CRM197-SMCC has been conjugated with MenA oligosaccharides after cystamine
	If the protein used in the conjugate was expressed without the His tag, conjugates have been Conjugation conditions for avDP15 oligosaccharides: two different conjugates have been prepared activation and reduction. Conjugation has been carried SH:SMCC ratio mol:mol of 10:1, in 50-
	purified by size exclusion chromatography or by ultrafiltration with Vivaspin system (Sartorius) using (M5 40:1, M10 13:1) with AE:protein ration mol:mol of 40:1-13:1, in 100mM NaH 2 PO 4 pH 7 and 100mM NaH 2 PO 4 pH 7 and incubated 48h at RT in gentle stirring. Conjugate has been purified by
	a 30kDa cut-off membrane. incubated o.n. at RT in gentle stirring. Conjugates have been purified by by gel filtration using a size exclusion chromatography using a S300 resin (GEHealthcare) and 10mM NaH 2 PO 4 10mM
	MenC conjugations reaction have been carried with an AE:protein mol:mol of 12:1, in 10-100mM Sephacryl S300 resin and 0.01M NaCl, 0.01M NaPi pH 7.2 as running buffer to eliminate the NaCl pH 7 with as running buffer.
	unreacted sugar. NaH 2 PO 4 pH 7 or in PBS pH 7 depending on the protein buffer, and incubated o.n. at RT with gentle 4.4.5 Labeling of glycoconjugate
	stirring. Purification has been done or by His MultiTrap 96-Well Filter Plates (GEHealthcare) or by Conjugation conditions for avDP30-40 oligosaccharides: tree different conjugates have been MenC oligosaccharide avDP15 labelled with A488 as reported and after SIDEA activation, have
	gel filtration or by ultrafiltration as reported above. prepared (M8, M11, MT2) with AE:protein ration mol:mol of 25:1-13:1-8:1, in 100mM NaH 2 PO 4 pH 7 been conjugated with CRM197 with an AE:protein ratio mol:mol 13:1, in 50mM NaH 2 PO 4 pH 7 and
	and incubated o.n. at RT in gentle stirring. Conjugates have been purified by: M8 ultrafiltration with
	100kDa cut-off membrane (0.1m 2 Hydrosart Sartorius) against 10 volumes of 10mM NaH 2 PO 4 pH 7;
	MT2 by gel filtration using a Sephacryl S300 resin and 0.15M NaCl, 0.01M NaPi pH 7.2 as running
	buffer to eliminate the unreacted sugar and by hydrophobic resin Phenyl Sepharose (GEHealthcare)
	Page 88 Page 90

in Mes buffer (2-(N-morpholino) ethanesulfonic acid) 30mM pH 5.6 for 3h in gentle stirring. PNEU20 conjugations with MenAWY oligosaccharides have been done using the same conditions described above for MenC.

4.4.3 Conjugates with different saccharide chain length and different glycosylation degree

Activated oligosaccharides at different avDP (6/10, 15, 30/40) have been conjugated with CRM197 after SIDEA activation and using different reaction conditions conjugates with different glycosylation degree have been obtained. incubated o.n. at RT in gentle stirring. After conjugation a second labelling has been done selectively on the protein moiety with Alexa Fluor® 647 (A647, Invitrogen) using the active ester group on the dye to react with the free amino group on the protein after conjugation. A647 reaction with MenC/A488-CRM197 conjugate has been carried with CRM197:A647 ratio mol:mol 1:30 in

adjuvants, and is characterized by antibody class switch and production of antigen-specific IgG.[START_REF] Makela | Polysaccharide vaccines of group A Neisseria meningtitidis and Haemophilus influenzae type b: a field trial in Finland[END_REF]

Page 68

To characterize the intracellular localization of the antigen at the cellular level, cells from IP wash samples at 24 h post injection. were analyzed by confocal microscopy. Briefly, cells were fixed, permeabilized and stained with specific cellular marker, MHCII and Lamp1. We were able to identify at least 2 distinct populations (Figure 43 A, B andC):

1. Cells with high antigen content, but poorly expressing MHCII on their surface (A). In these cells MenC-CRM dl is intracellular and co-localizes with Lamp1, a marker for the late endosomal compartment (B). These cells are generally larger than other Agpositive cells and we speculate that they could represent macrophages that, referring to general mechanism of antigen uptake, they remain longer antigen positive and are very active in the uptake mechanism. NaOH/Na acetate elution gradient. The resulting chromatographic data have been integrated and processed using Chromeleon® 6.8 software. Quantification has been performed with a calibration curve, treated as samples, set up with MenA polysaccharide in the range of 0.5-8.0 μg/ml.

Determination of unconjugated (free) saccharide content in glycoconjugates preparations

Unconjugated saccharide content (free saccharide %) was determined after separation of the free from the conjugated saccharide as follows. Conjugate vaccines (500 μl at saccharide concentration of 300µg/mL) have been applied to Microcon-30 filters previously washed with water, centrifuged at 10 000×g for 15 min at r.t. and washed twice with 500µl of water followed by centrifugation.

Saccharide quantification on filtrates has been then performed as described above.

Preparation of oligosaccharides

Introduction of primary amino groups at the reducing end of laminarin oligosaccharide by reductive amination

Laminarin has been treated by reductive amination to introduce a NH 2 group on the reducing end of the sugar using these conditions: 2 mg/ml of polysaccharides were subjected to reductive amination by incubation for 5 days at 50°C in 300 mg/ml ammonium acetate (Sigma-Aldrich) and 0.2 M sodium cyanoborohydride pH 7.5 (Sigma-Aldrich). Aminated laminarin was purified from the reaction mixture by diafiltration on regenerated cellulose membrane (cut-off 1kDa; Millipore) and the amount of primary amino groups introduced was determined as reported in paragraph 3.2.

Derivatization of the laminarin amino oligosaccharides to active esters

The aminated oligosaccharides were vacuum dried, solubilized in 1:9 H 2 O:DMSO solution with a final [NH 2 ]=40µmol/ml, and then reacted with the succinimido diester of adipic acid (SIDEA) in a 12fold molar excess as compared with primary amino groups in the presence of 5-fold molar excess triethylamine as compared with primary amino groups. 72 117 The reaction was kept under gentle stirring at room temperature for 2 h. The activated oligosaccharides were then separated from the reagents by precipitation with 1:4 of acetone, followed by washing of the precipitate with 1:4 acetone and dried under vacuum.

Content of N-hydroxysuccinimide ester groups introduced in the activated oligosaccharide was determined as reported in paragraph 3.1.