Luisa De Sordi

Marta Lourenço

Laurent Debarbieux
email: laurent.debarbieux@pasteur.fr

"I will survive": a tale of bacteriophage-bacteria coevolution in the gut

Viruses that infect bacteria, or bacteriophages, are among the most abundant entities in the gut microbiome. However, their role and the mechanisms by which they infect bacteria in the intestinal tract remain poorly understood. We recently reported that intestinal bacteria are an evolutionary force, driving the expansion of the bacteriophage host range by boosting the genetic variability of these viruses. Here, we expand these observations by studying antagonistic bacteriophage-bacteria coevolution dynamics and revealing that bacterial genetic variability is also increased under the pressure of bacteriophage predation. We propose a model showing how the expansion of bacteriophage-bacteria infection networks is relative to the opportunities for coevolution encountered in the intestinal tract. Our data suggest that predator-prey dynamics are perpetuated and differentiated in parallel, to generate and maintain intestinal microbial diversity and equilibrium.

INTRODUCTION

The homeostasis of the intestinal microbiome is crucial to health, as shown by the evergrowing list of chronic conditions linked to microbiota dysbiosis, including obesity, diabetes, asthma, inflammatory bowel disease (IBD) and central nervous system disorders [START_REF] Tremaroli | Functional interactions between the gut microbiota and host metabolism[END_REF][START_REF] Fujimura | Microbiota in allergy and asthma and the emerging relationship with the gut microbiome[END_REF][START_REF] Gallo | Modulation of microbiota as treatment for intestinal inflammatory disorders: An uptodate[END_REF][START_REF] Sampson | Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson's Disease[END_REF] . The antagonistic coevolution between the two most abundant components of the microbiome, bacteria and their viruses, bacteriophages, is a key candidate player in the maintenance of this microbial equilibrium [START_REF] Scanlan | Bacteria-Bacteriophage Coevolution in the Human Gut: Implications for Microbial Diversity and Functionality[END_REF] .

The perpetuation of bacteriophages is intrinsically dependent on their ability to predate on the bacterial populations and experimental coevolution studies have characterised the dynamics of interactions between bacteria and bacteriophages [START_REF] Buckling | Antagonistic coevolution between a bacterium and a bacteriophage[END_REF][START_REF] Gandon | Host-parasite coevolution and patterns of adaptation across time and space[END_REF] . The development of bacterial resistance, and the consequent bacteriophage adaptation towards such resistance, have been identified as major forces driving their antagonistic coevolution in vitro and in environmental samples. This arms race necessarily results in an increase in the genomic diversity of both partners to ensure population survival [START_REF] Koskella | Bacteria-phage coevolution as a driver of ecological and evolutionary processes in microbial communities[END_REF][START_REF] Gomez | Local adaptation of a bacterium is as important as its presence in structuring a natural microbial community[END_REF] , as seen in aquatic ecosystems [START_REF] Enav | Adaptation to sub-optimal hosts is a driver of viral diversification in the ocean[END_REF][START_REF] Middelboe | Bacteriophages drive strain diversification in a marine Flavobacterium: implications for phage resistance and physiological properties[END_REF] . However, most studies of this type are limited to single pairs of bacteria and bacteriophages and are frequently performed in laboratory settings.

Metagenomic analyses of intestinal bacterial populations have revealed that these organisms are diverse and differently abundant in healthy humans and diseased patients. Fewer studies have focused on viral populations (virome), but those that have been performed have revealed an unprecedented complexity of relationships between bacteriophages, bacteria and the mammalian host [START_REF] Mirzaei | Menage a trois in the human gut: interactions between host, bacteria and phages[END_REF] . A recent comparative study showed that healthy humans share a pool of conserved intestinal bacteriophages that differs significantly from the viruses found in patients with inflammatory bowel disease (IBD) [START_REF] Manrique | Healthy human gut phageome[END_REF] . Also, in these patients, lower bacterial diversity is associated with a significantly larger number and diversity of bacteriophages [START_REF] Norman | Disease-specific alterations in the enteric virome in inflammatory bowel disease[END_REF] . Similarly, a recent microbiome study conducted on malnourished pediatric patients hospitalized with acute diarrhea showed an increase in Escherichia coli bacteriophages compared to healthy individuals, that negatively correlated with the abundance of the bacterial host [START_REF] Kieser | Antibiotic Treatment Leads to Fecal Escherichia coli and Coliphage Expansion in Severely Malnourished Diarrhea Patients[END_REF] . Other studies suggest that bacteriophages play a key role in regulating intestinal bacterial populations by showing that filtered (bacteria-free) faecal microbiota transplantation (FMT) yields curative results comparable to those obtained with traditional FMT, and that viral transfer correlates with the resolution of gut infections caused by Clostridium difficile [START_REF] Ott | Efficacy of Sterile Fecal Filtrate Transfer for Treating Patients With Clostridium difficile Infection[END_REF][START_REF] Zuo | Bacteriophage transfer during faecal microbiota transplantation in Clostridium difficile infection is associated with treatment outcome[END_REF] . Nonetheless, exploitation of this genomic information at the molecular level remains limited, because most of the sequences obtained do not match to a known function. Another major hurdle is the lack of association between bacteriophage sequences and those of their specific bacterial hosts.

There is, therefore, a considerable gap between studies of interactions between bacteriophages and bacteria in laboratory conditions and the complexity of these interactions in the gut [START_REF] Manrique | Healthy human gut phageome[END_REF][START_REF] Reyes | Gut DNA viromes of Malawian twins discordant for severe acute malnutrition[END_REF] .

In the environment, bacteria and bacteriophages coexist in intricate, structured interaction networks [START_REF] Flores | Statistical structure of host-phage interactions[END_REF][START_REF] Weitz | Phage-bacteria infection networks[END_REF] . Bacterial species are represented by distinct genetic lineages (strains) and bacteriophages are mostly strain-specific: rare are bacteriophages that infect most strains within one given species and even fewer are those infecting distinct species. Thus, little is known about the role of bacteriophage-bacteria infection networks in driving the diversification of the gut microbial ecosystem in the context of health and disease.

MICROBIOTA-DRIVEN BACTERIOPHAGE ADAPTATION

Reductionist approaches using E. coli and its bacteriophages have successfully deciphered major mechanisms of molecular biology [START_REF] Hershey | Independent functions of viral protein and nucleic acid in growth of bacteriophage[END_REF][START_REF] Brenner | An unstable intermediate carrying information from genes to ribosomes for protein synthesis[END_REF][START_REF] Cairns | Phage and the Origins of Molecular Biology[END_REF] . By lifting the reductionist approach to the next level of complexity, namely the study of the intestinal microbiota, we recently described the coevolution of one bacteriophage with multiple host strains within the mouse gut [START_REF] Sordi | The Gut Microbiota Facilitates Drifts in the Genetic Diversity and Infectivity of Bacterial Viruses[END_REF] . We studied P10, a virulent bacteriophage from the Myoviridae family, infecting the E coli strain LF82, and we assessed its ability to adapt to E. coli strain MG1655, to which it was initially unable to bind and therefore could not infect. Such host-range expansion was observed, but only occurred during coevolution in the gut of conventional mice hosting E. coli strains LF82 and MG1655 within their microbiota. In planktonic in vitro cultures or in the gut of dixenic mice colonized solely by the two E. coli strains, this event was never detected. Based on these findings, we hypothesized that the mouse microbiota played a crucial role in promoting adaptation. Indeed, we showed that this adaptation was initiated by the infection of an intermediate host, E. coli strain MEc1, which we isolated from the murine microbiota. Mixing bacteriophage P10 in vitro with the three E. coli strains also promoted viral host-range expansion. This adaptation was accompanied by genomic differentiation in the bacteriophage population: a single point mutation in a tail fibre-encoding gene was found to be sufficient to promote host adaptation, but additional mutations were required to optimise the infectious cycle.

The spatial and temporal dynamics of the acquisition of these mutations in the structured intestinal environment remain unclear. However, our data are consistent with the hypothesis that the genomic differentiation of bacteriophage subpopulations depends on the diversity of the bacteria encountered, making the microbiota an ideal site to generate viral diversity. In addition to bacterial diversity, the spatial distribution of bacterial populations along the gut may also influence the dynamics of bacteriophage evolution [START_REF] Suzuki | Spatial Heterogeneity of Gut Microbial Composition along the Gastrointestinal Tract in Natural Populations of House Mice[END_REF][START_REF] Wang | Comparison of bacterial diversity along the human intestinal tract by direct cloning and sequencing of 16S rRNA genes[END_REF] . The colonisation of macroenvironments, such as the small versus the large intestine and the their compartments (luminal and mucosal), and the occupation of specific niches within these contexts (nutrient-niche hypothesis [START_REF] Freter | Mechanisms that control bacterial populations in continuous-flow culture models of mouse large intestinal flora[END_REF] ), give rise to structured networks of single or mixed bacterial populations [START_REF] Pereira | Microbial nutrient niches in the gut[END_REF] likely to promote the diversification of bacteriophages into multiple subpopulations with diverging infectivity profiles.

GENETIC BACTERIAL RESISTANCE IN THE GUT

Here, we analyse a second source of genomic diversity, the emergence of bacterial resistance, one of the drivers of antagonistic evolution [START_REF] Scanlan | Bacteria-Bacteriophage Coevolution in the Human Gut: Implications for Microbial Diversity and Functionality[END_REF][START_REF] Koskella | Bacteria-phage coevolution as a driver of ecological and evolutionary processes in microbial communities[END_REF][START_REF] Hall | Host-parasite coevolutionary arms races give way to fluctuating selection[END_REF] . Faecal pellets of mice in which P10 adaptation had occurred, yielded five MG1655 clones displaying different degrees of resistance to adapted P10 bacteriophages (Fig. 1A). The genomes of these five strains presented different mutations in the waaZ gene, which encodes a protein involved in the biosynthetic pathway for the core lipopolysaccharide (LPS) (Fig. 1B; Table S1). We identified four convergent paths of adaptation, characterised by gene disruption by insertion sequences (ISs), IS5 and IS2, at different gene positions. We hypothesise that independent convergent events leading to modifications of the LPS core biosynthesis pathway had served as the first step towards adaptation of the newly targeted strain MG1655, under the selective pressure of bacteriophage predation.

Another gene, waaY, flanking waaZ, was also targeted by IS elements in three of the coevolved MG1655 clones. The occurrence of these mutations, coupled to the high degree of sequence identity between bacteriophage P10 and the LPS-binding WV8 and Felix-O1 bacteriophages [START_REF] Villegas | The genome and proteome of a virulent Escherichia coli O157:H7 bacteriophage closely resembling Salmonella phage Felix O1[END_REF][START_REF] Hudson | Lipopolysaccharide core defects in Salmonella typhimurium mutants which are resistant to Felix O phage but retain smooth character[END_REF] , suggests a bacterial resistance strategy based on the masking of the bacteriophage receptor. Interestingly, natural populations of Vibro cholerae isolated from patients with diarrhoea have also been shown to consist of heterogeneous mixtures of unique mutants resistant to bacteriophage predation [START_REF] Seed | Evolutionary consequences of intra-patient phage predation on microbial populations[END_REF] . However, these mutants were subject to fitness and virulence costs that might arguably affect their infection potential. Similarly, experimental phage therapy studies revealed that bacterial pathogens can develop bacteriophage resistance at the expenses of their major virulence factors, as shown in bovine enteropathogenic E. coli [START_REF] Smith | Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves, piglets and lambs[END_REF] or during experimental endocarditis due to Pseudomonas aeruginosa [START_REF] Oechslin | Synergistic Interaction Between Phage Therapy and Antibiotics Clears Pseudomonas Aeruginosa Infection in Endocarditis and Reduces Virulence[END_REF] .

Further genomic analysis of the MG1655 clones that had coevolved with P10 identified a second hotspot for mutations in the galactitol operon, which was previously shown to be pervasive in E. coli clones adapting to the gut environment [START_REF] Barroso-Batista | The first steps of adaptation of Escherichia coli to the gut are dominated by soft sweeps[END_REF][START_REF] Lourenco | A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the Gut[END_REF] . In addition, two sugar metabolism pathways (maltose and galactonate) were targeted by IS insertions in genes encoding the DNA-binding transcriptional regulators (malT, lgoR), with probable positive or negative overall effects on pathway activation.

The contextual genomic variability of bacteriophages was also analysed by sequencing five adapted bacteriophages differing in their ability to infect the five MG1655 clones considered (Fig. 1C). The only bacteriophage able to infect all the bacterial clones had the largest number of mutations (12 mutations, versus 5 to 9 in the other bacteriophages isolated; Table S1), suggesting a possible faster pace of adaptation in response to bacterial resistance. The mutations were clustered into four genomic regions. The first corresponds to the rIIA (gp37) gene, the function of which is probably related to infection fitness, as this gene was also highlighted in our population genomics study in in vitro conditions [START_REF] Sordi | The Gut Microbiota Facilitates Drifts in the Genetic Diversity and Infectivity of Bacterial Viruses[END_REF] . A second, larger region encompasses several structural genes, including the tail fibre genes. The gp55 and gp57 genes, which are predicted to encode two subunits of the class I ribonucleotide reductase, were also affected, together with gp108, the function of which is unknown.

However, the functions of the affected genes were not sufficient to associate genomic mutations with differences in bacteriophage infectivity, highlighting the versatility of bacteriophage infection. It remains to be determined which of these mutations accumulated before and after the development of bacterial resistance.

We investigated these dynamics further, by performing a time-shift interaction study. We isolated P10 clones (n=40) from three time points during coevolution: one time-point before, and two after the adaptation of P10 to strain MG1655. We characterised the ability of these clones to infect MG1655 clones (n=40) isolated at past, present and future time points in the same experiment. As expected, bacteriophages isolated before the adaptation event were unable to infect any of the contemporary MG1655 clones (present) (Fig. 1D). While adapted bacteriophages were always able to infect bacterial clones from the past time points, those isolated at the first time point after the adaptation event (day 1) showed reduced infectivity towards MG1655 bacterial clones isolated at the present and, particularly, future time points. However, all bacteriophages isolated subsequently (day 21) were able to infect past, present and future bacterial clones, overcoming the bacterial resistance that had developed and demonstrating the occurrence of continuous adaptive evolution in the mouse gut (Fig. 1D).

It could, therefore, be argued that bacteriophage adaptation in the gut led to a two-step coevolution pathway, in which the evolutionary arms race was initially characterised by the rapid development of bacterial resistance followed by a refining of bacteriophage adaptation.

The two populations subsequently continued to coexist, with no evidence of renewed bacterial resistance, suggesting that transient resistance occurred in situ, protecting the bacteria against bacteriophage predation, as discussed below.

TRANSIENT BACTERIAL RESISTANCE IN THE GUT

Bacterial resistance to bacteriophages has long been studied and characterised in vitro [START_REF] Luria | Mutations of Bacteria from Virus Sensitivity to Virus Resistance[END_REF] , and is known to involve several mechanisms. These include the prevention of adsorption, superinfection exclusion, restriction modifications, CRISPR-Cas systems, bacteriophage exclusion (BREX), and many new recently discovered systems revealing the extreme versatility of bacterial resources for defence [START_REF] Labrie | Bacteriophage resistance mechanisms[END_REF][START_REF] Goldfarb | BREX is a novel phage resistance system widespread in microbial genomes[END_REF][START_REF] Doron | Systematic discovery of antiphage defense systems in the microbial pangenome[END_REF] . Nonetheless, little is known about the mechanisms activated in vivo, and their relevance and impact in natural communities. In our study, the resistance of strain MG1655 to the newly adapted bacteriophage P10 seemed to depend on preventing adsorption by modifying the bacteriophage receptor. However, this may simply reflect part of the process of bacteriophage adaptation to a new bacterial host, as the bacteriophage could rapidly fine-tune its mechanism of infection to overcome this resistance. This hypothesis is supported by the lack of emergence of resistant clones of the original bacterial host, strain LF82, in mouse faeces (data not shown), despite the presence of large numbers of both the bacteriophage and the bacterium during the course of the experiment.

We have already reported similar observations for a different E. coli strain, 55989, coevolving in mouse gut with either a cocktail of three virulent bacteriophages or with each bacteriophage separately. No resistance was ever detected when 20 bacterial isolates were tested against the individual bacteriophages [START_REF] Maura | Virulent bacteriophages can target O104:H4 enteroaggregative Escherichia coli in the mouse intestine[END_REF][START_REF] Maura | On the interactions between virulent bacteriophages and bacteria in the gut[END_REF] . However, two to six hours of co-incubation with the same bacteriophages in vitro was sufficient to trigger the development of bacterial resistance [START_REF] Maura | Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice[END_REF] . We also previously tested the ability of each bacteriophage to replicate in the intestinal environment ex vivo, both in homogenates of the small and the large intestines and in the faeces of mice colonised with E. coli strain LF82 or strain 55989 [START_REF] Maura | Virulent bacteriophages can target O104:H4 enteroaggregative Escherichia coli in the mouse intestine[END_REF][START_REF] Galtier | Bacteriophages Targeting Adherent Invasive Escherichia coli Strains as a Promising New Treatment for Crohn's Disease[END_REF] . We found that all bacteriophages were infectious in the ileal sections, but that replication in colonic or faecal samples was significantly impaired for some of them [START_REF] Maura | Virulent bacteriophages can target O104:H4 enteroaggregative Escherichia coli in the mouse intestine[END_REF][START_REF] Galtier | Bacteriophages Targeting Adherent Invasive Escherichia coli Strains as a Promising New Treatment for Crohn's Disease[END_REF] .

These results support the hypothesis that the metabolic state of bacteria, which is not uniform throughout the gut [START_REF] Denou | Gene expression of commensal Lactobacillus johnsonii strain NCC533 during in vitro growth and in the murine gut[END_REF] , is the principal barrier to bacteriophage infection. Indeed, several factors, such as the availability of carbon sources, oxygen, and stress responses, can have a marked effect on cell surface structures, some of which are required for bacteriophage infection. This physiological and structural versatility provides bacteria with opportunities for transient resistance to bacteriophages without paying the cost or irreversible mutations, but remaining susceptible when the physiological conditions change, such as during growth in the laboratory environment. Conversely, bacteriophages can escape such resistance strategies by entering into a state of pseudolysogeny or hibernation, in which the infectious cycle is halted until better conditions for progeny production occur [START_REF] Bryan | Bacteriophage T4 Infection of Stationary Phase E. coli: Life after Log from a Phage Perspective[END_REF][START_REF] Los | Pseudolysogeny[END_REF] .

MODEL OF BACTERIOPHAGE -BACTERIA COEVOLUTION IN THE GUT

This dynamic picture of the coevolution of bacteria and bacteriophages serves as the basis of a theoretical model describing how microbiome diversity is generated and expanded via these interactions (Fig. 2). Mutations in the bacteriophage genome accumulate when they confer a fitness advantage and favour perpetuation of the infection cycle. This corresponds to adaptation to new host strains, and/or host strains that have acquired resistance. However, it remains unclear whether bacteriophage evolution discriminates between these two bacterial situations, since each adaptation event would involve specific mechanisms to overcome the obstacles to predation.

The evolution of the microbiome results in a growing number of bacteriophage populations infecting new bacterial hosts with which perpetuating the process of antagonistic coevolution. This is likely to occur at the expense of the most abundant and available bacterial populations, providing a major contribution to microbiome homeostasis and to bacterial differentiation.

Bacterial hosts also have opportunities to escape bacteriophage predation, resulting in genomic differentiation between microbial populations. In addition, some of these populations are likely to be protected against bacteriophage predation due to their physical inaccessibility in the environment, their limited density and/or the development of transient resistance due to their metabolic and phenotypic states. An example of such viral diversification in the human gut can be found with the expanding population of Crassphage [START_REF] Yutin | Discovery of an expansive bacteriophage family that includes the most abundant viruses from the human gut[END_REF] .

CONCLUDING REMARKS

The timing, frequency and conditions required for bacteriophage adaptation and bacterial resistance during coevolution in the intestinal microbiota remain largely unpredictable. Illumina technology and mutations were called using the Breseq variant report software v0.26 [START_REF] Barrick | Genome evolution and adaptation in a long-term experiment with Escherichia coli[END_REF] . Mutations (orange, red and blue triangle -IS1, IS2 and IS5 respectively, black triangle pointing down -1-5bp insertion, black triangle pointing up -1-5bp deletion, vertical black rectangle -SNP and black horizontal rectangle ->1kb deletion) are reported relative to their positions in the genome. For mutation hotspots, the relative targeted genes are reported as purple arrows. For a complete list of bacterial genomic mutations see Table S1

Figure 1 :

 1 Figure 1: Bacteriophages and bacteria coevolve in the mouse gut.

  . The corresponding sequences are deposited at ENA under project PRJEB24878. C) Bacteriophage genomic mutations accumulated during coevolution with strain MG1655 in the mouse gut. Sequences of five adapted P10 bacteriophages (ad_P10_1-3,5-6) were analysed as described for bacterial clones. Mutations are relative to their positions in the bacteriophage genome (ORFs are shown as purple arrows) and mutation hotspots are indicated (same legend as for panel B). For a complete list of viral genomic mutations, see TableS2

  . The corresponding sequences are deposited at ENA under project PRJEB18073. D) Bacteriophages overcome genetic bacterial resistance. A time-shift experiment shows the percentage infectivity of fourty P10 bacteriophages from different time points tested towards fourty MG1655 clones

Figure 1

 1 Figure 1

Acknowledgements

We thank Harald Brüssow for critically reading the manuscript and Varun Khanna for support in bioinformatics analysis. LDS is the recipient of a Roux-Cantarini fellowship from the

Disclosure of potential conflicts of interest

The authors declare no potential conflicts of interest. isolate from past, present and future time-points during coevolution in the mouse gut.

Bacterial lysis was tested by double-spot assay [START_REF] Saussereau | Effectiveness of bacteriophages in the sputum of cystic fibrosis patients[END_REF] .


Alicia Moreno-Sabater
email: alicia.moreno-sabater@upmc.fr

Jean Louis Pérignon

Dominique Mazier

Catherine Lavazec

Valerie Soulard

Humanized mouse models infected with human Plasmodium species for antimalarial drug discovery

Keywords: Antimalarial compounds, humanized mouse models, malaria eradication, preclinical pathway, P. falciparum, P. vivax, P. ovale

Introduction: Efforts on malaria drug discovery are expected to increase in the coming years to achieve malaria eradication. Owing to the increasing number of new potential candidates together with the actual limitations of the primate models, humanized mouse models infected with human Plasmodium spp. (HmHP) now appear as an alternative to the primate model. Areas covered: The authors review the progress obtained in the HmHP in the last two decades, with a special emphasis of their input on the drug discovery pathway. The authors discuss the methodologies and strategies used in these models to obtain an accurate assessment of the compound activity and a reliable prediction of the human efficacious regimen. Expert opinion: Research efforts have led us to an era in which HmHP can successfully be infected with P. falciparum, P vivax and P. ovale. Furthermore, it is now a reality that the complete human cycle of P. falciparum can be obtained in HmHP. The HmHP has shown a real input mainly in the preclinical evaluation of new compounds against the erythrocytic stages of P. falciparum. However, further technical improvements are needed before HmHP may replace the primate model.

Introduction

There has been a dramatic increase in malaria drug discovery in the last 10 years and efforts are expected to increase in the coming years, for two main reasons. The first reason is that the present armamentarium against Plasmodium falciparum (P. falciparum), the parasite responsible for the large majority of malaria mortality (ca. 429,000 in 2015, 92% in Africa, and 70% in children under 5 years old) [START_REF] Wmr | World malaria report[END_REF], may soon become insufficient. Indeed, due to worldwide resistance to chloroquine, artemisinin-based combination therapies (ACTs) are the mainstay in the current malaria therapy. ACTs are recommended in most malaria-endemic countries as first-line treatment of uncomplicated P. falciparum malaria. However, it is well known that P. falciparum can become resistant against almost any drug that is in widespread use. Unfortunately, this has been confirmed 8 years ago, when first cases of reduced sensitivity of P. falciparum against artemisinins were reported from South-East Asia, which were characterized by a prolonged clearance time of the parasite [START_REF] Dondorp | Artemisinin resistance in Plasmodium falciparum malaria[END_REF]. In addition, laboratory strains have been rendered resistant to artemisinin, allowing the identification of a genetic marker of resistance and K13 propeller mutations were found to correlate with delayed in vivo clearance times [START_REF] Ariey | that K13-propeller mutations are important determinants of artemisinin resistance[END_REF]. The emergence and spread of resistance to antimalarial drugs are of major concern and are globally monitored, e.g. by the WorldWide Antimalarial Resistance Network, in order to inform the malaria community of potential efficacy loss of antimalarials [START_REF] Verlinden | Resisting resistance: is there a solution for malaria?[END_REF]. This situation highlights the urgent need to find new drugs to treat the disease.

The second reason is that the objective of malaria drug discovery has become much more ambitious than circumventing the remarkable ability of malaria parasites to develop resistance to drugs. Indeed, 10 years ago (thus before the first report on reduced artemisinin efficacy), at the Malaria Forum in October 2007, the paradigm of malaria control and elimination has been extended to encompass an ultimate goal of malaria eradication [START_REF] Tanner | Malaria eradication back on the table[END_REF]. The objective was thus not limited to P. falciparum, but was extended to all malaria parasites including Plasmodium vivax, which is responsible for ~50% of the malaria cases outside sub-Sahara Africa. This position was rapidly supported by the World Health Organization, the Roll Back Malaria Partnership, and many other organizations and institutions. This call for malaria eradication led to a new research agenda laying out priorities with broader horizons [START_REF] Alonso | A research agenda to underpin malaria eradication[END_REF]. Ideally, the objective is now Single Encounter Radical Cure and Prophylaxis (SERCaP), corresponding to a target product profile that includes (1) radical cure, defined as elimination of all parasites (including the long-lived hypnozoites of P. vivax or Plasmodium ovale), (2) suitability for mass administration (including administration to healthy subjects and the consequent need of a very good safety profile), and (3) prophylaxis for at least 1 month after treatment, to outlast the typical development period of Plasmodium parasites in Anopheline mosquitoes.

For these two reasons, the past decade has seen an unprecedented renewed focus on the discovery of new antimalarial drugs through extraordinary collaboration between academia (parasitologists, medicinal chemists, pharmacologists, clinicians) and industrial/private partnerships (e.g. Medicines for Malaria Venture). Cell-, chemistry-, and target-based approaches have already selected tens of thousands molecules [START_REF] Flannery | Antimalarial drug discovery -approaches and progress towards new medicines[END_REF][START_REF] Flannery | Antimalarial drug discovery -approaches and progress towards new medicines[END_REF]. The big challenge is to identify among them interesting compounds, advance them through preclinical and clinical development, to finally develop them into a registered antimalarial drug [START_REF] Held | Novel approaches in antimalarial drug discovery[END_REF]. Indeed, several compounds recently entered Phase II testing or are in transition to Phase III evaluation [START_REF] Flannery | Antimalarial drug discovery -approaches and progress towards new medicines[END_REF][START_REF] Anthony | The global pipeline of new medicines for the control and elimination of malaria[END_REF][START_REF] Held | Antimalarial compounds in Phase II clinical development[END_REF]. Apart from two herbal medicines and the ozonide arterolane, preparations recently approved or in advanced stages of clinical development are mainly combinations of long known drugs. Given the high attrition rate even in advanced stages of development, only a few compounds with novel mechanisms will finally be introduced in therapy. In addition, none of the compounds corresponds to the ideal objective of SERCaP. Therefore, continued efforts are mandatory to keep the antimalarial pipeline filled, and for all the candidates that will emerge from the first screens, the major bottleneck will obviously be the translation into clinical development.

A major characteristic of research on drugs active against P. falciparum is that this parasite can be cultured, facilitating in vitro studies, whereas in vivo studies are restricted owing to strict host-parasite relationship. Indeed, promising antimalarial compounds are routinely assessed in the murine model infected with rodent Plasmodium spp. (most frequently Plasmodium berghei), with the obvious limitation of a different parasite and a different host. A next step may be provided by nonhuman primate models, which are much closer to malaria in the human host and are a valuable tool under some circumstances [START_REF] Beignon | In vivo imaging in NHP models of malaria: challenges, progress and outlooks[END_REF]. Splenectomized Aotus monkeys support growth of some P. falciparum strains and are mainly used to test novel vaccine candidates rather than drugs [START_REF] Held | Novel approaches in antimalarial drug discovery[END_REF]. Till now, primate model was the only realistic possibility that could be proposed. However, the high costs and scarcity of primates have limited cohort sizes, whichin combination with the interindividual variability of an outbred specieslimits the reproducibility of research findings. In addition, the use of primates, especially chimpanzees, has raised ethical concerns in many countries. The moratorium issued by the National Institutes of Health on 'nonessential' chimpanzee research is likely to further constrain aspects of malaria research in the future [START_REF] Wadman | Animal rights: chimpanzee research on trial[END_REF].

Humanized mouse models (Hm) were initially developed to study viral infections with strict host specificity, most notably human immunodeficiency virus/acquired immunodeficiency syndrome [START_REF] Akkina | Improvements and limitations of humanized mouse models for HIV research: NIH/NIAID "meet the experts" 2015 workshop summary[END_REF]. Since then, the number of Hm for infectious diseases has rapidly expanded over the past years [START_REF] Brehm | Generation of improved humanized mouse models for human infectious diseases[END_REF]. Applicated to human malaria, this approach led to graft immunodeficient mice with human erythrocytes or hepatocytes, thus allowing in vivo growth of P. falciparum and providing a promising tool for antimalarial drug discovery. The final goal was to obtain the full cycle of the human parasite in immunodeficient mice double grafted with both human red blood cells (HRBC) and human hepatocytes (HH); ideally, adding the graft of human lymphoid cells would provide an attractive model for vaccine research.

The purpose of this review is to present improvements recently obtained with Hm infected with human Plasmodium species (HmHP) such as P. falciparum (HmHPf), P. vivax (HmHPv), and P. ovale (HmHPo), their input in the preclinical pathway of new compounds, their ability to confirm the in vivo activity of new compounds and to help to predict human doses. We discuss to what extent these models may contribute to the general endeavor to alleviate the threat of malaria parasites resistance against existing drugs and to design drugs fulfilling the objective of SERCaP. In our opinion, further technical improvements are needed before they may replace the primate model.

HmHP for malaria research

The proof of concept

Although great efforts have been employed to develop Hm for infectious diseases, the potential of HmHP for malaria research has been undervalued for many years (Figure 1). The initial development of HmHP was mainly sustained by severe combined immunodeficiency (SCID) mice, congenitally immunedeficient bg/nu/xid mice, or diabetes-susceptible nonobese diabetic (NOD)/SCID mice [START_REF] Moreno | Plasmodium falciparuminfected mice: more than a tour de force[END_REF]. Using these precursor mouse models, several groups provided the proof of concept that P. falciparum liver stages (LS) could be obtained in immunodeficent mice grafted with HH, to set up a Hm infected with P. falciparum LS (HmHPf-LS) [START_REF] Butcher | The SCID mouse as a laboratory model for development of the exoerythrocytic stages of human and rodent malaria[END_REF][START_REF] Sacci | Mouse model for exoerythrocytic stages of Plasmodium falciparum malaria parasite[END_REF]. In parallel, P. falciparum erythrocytic stages (ES) could be obtained in immunodeficient mice grafted with HRBC to obtain a Hm infected with P. falciparum ES (HmHPf-ES) [START_REF] Moore | Maintenance of the human malarial parasite, Plasmodium falciparum, in scid mice and transmission of gametocytes to mosquitoes[END_REF][START_REF] Tsuji | Establishment of a SCID mouse model having circulating human red blood cells and a possible growth of Plasmodium falciparum in the mouse[END_REF].

Soon after, the HmHPf-ES, easier to obtain than the HmHPf-LS, proved to be a useful tool to study immunological mechanisms and the activity of classical antimalarial drugs [START_REF] Badell | Human malaria in immunocompromised mice: an in vivo model to study defense mechanisms against Plasmodium falciparum[END_REF][START_REF] Druilhe | A malaria vaccine that elicits in humans antibodies able to kill Plasmodium falciparum[END_REF][START_REF] Moreno | Human malaria in immunocompromised mice: new in vivo model for chemotherapy studies[END_REF]. However, HmHPf-ES available at this time lacked reproducibility and showed a low rate of HRBC engraftment,

Article highlights

• To face antimalarial drugs resistance and to achieve malaria eradication, continued efforts are mandatory to keep the antimalarial pipeline filled. • For all the candidates that will emerge from the first screens, the major bottleneck will obviously be the translation into clinical development.

• In this context, limitations of the primate model become more important, leading to favor the HmHP in the preclinical pathway. • Recent efforts carried out by the scientific community have permitted to proceed to an era in which different HmHP can successfully be infected with human P. falciparum, P vivax and P. ovale, allowing the in vivo study of the parasite liver development for all of them and the complete human parasite development for P. falciparum. • HmHPf-ES has shown to be an efficient tool to confirm in vivo antimalarial activity of new compounds, including against resistant parasites, and to help to predict human doses. • Further technical improvements are needed before HmHP may replace the primate model in the drug discovery preclinical pathway.

This box summarizes key points contained in the article.

leading to a disinterest of the scientific community. First reports pointed to the macrophagic system as responsible for HRBC clearance [START_REF] Sabater | Experimental infection of immunomodulated NOD/LtSz-SCID mice as a new model for Plasmodium falciparum erythrocytic stages[END_REF]. A significant improvement in the model reproducibility was obtained working with the NOD/ SCID mouse and the previously described immunomodulatory protocol [START_REF] Moreno | The course of infections and pathology in immunomodulated NOD/LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates[END_REF]. Using this combination, all the P. falciparum ES, asexual and sexual parasites, were observed in the peripheral blood of mice infected with laboratory strains or with clinical isolates.

Toward a HmHP for all parasite stages

HmHP for ES

Although the HmHPf-LS fell into desuetude due to the technical difficulties and the scarce access to HH, the interest in the HmHPf-ES was renewed thanks to the generation of new immunodeficient mouse strains (Figure 1). NOD/SCID beta2m-/- mice [START_REF] Angulo-Barturen | A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes[END_REF] or NOD.Cg-Prkdc scid Il2rg tm1WjI /SzJ (also named NSG mice), with reduced activity of natural killer cells (NK), allowed further improvement in the graft of HRBC and in the reproducibility of the HmHPf-ES [START_REF] Arnold | Further improvements of the P. falciparum humanized mouse model[END_REF][START_REF] Arnold | Analysis of innate defences against Plasmodium falciparum in immunodeficient mice[END_REF][START_REF] Chen | Human natural killer cells control Plasmodium falciparum infection by eliminating infected red blood cells[END_REF][START_REF] Jimenez-Diaz | Improved murine model of malaria using Plasmodium falciparum competent strains and nonmyelodepleted NOD-acid IL2Rgammanull mice engrafted with human erythrocytes[END_REF]. Two different protocols should to be used to maintain P. falciparum infection: (1) a protocol requiring the use of immunochemical treatments to reduce HRBC clearance by macrophages or (2) a protocol requiring daily transfusions of HRBC and infection with an adapted strain of P. falciparum (i.e. a strain that has been selected after several successful infections in the HmHPf-ES).

One characteristic of the HmHPf-ES is that all the asexual stages are circulating in the peripheral blood, in contrast with the human infection where only the ring stages are circulating, whereas the mature stages cytoadhere to the vascular endothelium of organs. This suggests that parasite cytoadherence to the mouse vascular endothelium may not occur in the HmHPf-ES. Although some studies have observed a parasite accumulation in several organs as compared with the peripheral blood [START_REF] Moreno | The course of infections and pathology in immunomodulated NOD/LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates[END_REF][START_REF] Angulo-Barturen | A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes[END_REF], this may be related to a parasitized erythrocytes retention due to their lower deformability.

Using the NSG mouse strain, the complete sexual development of P. falciparum parasites has been obtained [START_REF] Duffier | A humanized mouse model for sequestration of Plasmodium falciparum sexual stages and in vivo evaluation of gametocytidal drugs[END_REF], confirming initial results obtained in the NOD/SCID strain [START_REF] Moreno | The course of infections and pathology in immunomodulated NOD/LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates[END_REF]. Until then, the in vivo study of parasite sexual stages had been restricted to rodent Plasmodium spp., which show important biological differences with P. falciparum, or to splenectomised monkeys, precluding the use of these animals to address gametocytes' interactions with the spleen. One of the major challenges to obtain the sexual stages in the HmHPf-ES was to reduce the rapid clearance of grafted HRBC (3-5 days) as gametocyte maturation needs at least 10 days. By modifying previously described immunosuppression protocols and using the NSG strain, it could be possible to obtain high gametocytemia and further characterize P. falciparum gametocytogenesis in this model. Interestingly, an enrichment of immature gametocytes was observed in the bone marrow and the spleen, suggesting a sequestration mechanism reminiscent to that observed in humans [START_REF] Duffier | A humanized mouse model for sequestration of Plasmodium falciparum sexual stages and in vivo evaluation of gametocytidal drugs[END_REF] and confirming that this is a valuable model to investigate the biology of the sexual forms in vivo.

While waiting a new generation of immunodeficient mouse strains with a reduced macrophage population, an alternative method to humanize mice with HRBC is the engraftment of human stem cells [START_REF] Noulin | Malaria modeling: in vitro stem cells vs in vivo models[END_REF]. Recent progresses have established the proof of concept that the graft of stem cells from different sources allows the continuous production of HRBC in the peripheral blood of NSG mice. Generally, CD34+ cells mainly isolated from umbilical cord blood are injected intrahepatic or intravenously within immunodeficient mice. Recent promising improvements have shown that it is possible to generate HRBC permissive to different P. falciparum strains in NSG mice by transfecting the human stem cells with a plasmid that expresses IL-3 and erythropoietin [START_REF] Amaladoss | De novo generated human red blood cells in humanized mice support Plasmodium falciparum infection[END_REF]. Unfortunately, HRBC obtained by this procedure were only in low amounts (1.5-2.8%), with a low parasite density. Consequently, new efforts should be concentrated on overcoming the short lifespan of those cells.

Thus, the increased numbers of studies using the HmHPf-ES in the last two decades witness its usefulness and its place in malaria research. This model has proved its value in immunological studies such as evaluating the potential inhibition of antibodies directed against proteins implicated in erythrocyte invasion by the merozoite cysteine-rich protective antigen [START_REF] Dreyer | Passive immunoprotection of Plasmodium falciparum-infected mice designates the CyRPA as candidate malaria vaccine antigen[END_REF], but its major contribution has been in the evaluation of new antimalarial drugs, which will be reviewed in the following section.

HmHP for LS

The HmHPf-LS, long time forgotten, has also benefited from the generation of broader immunodeficient mice [START_REF] Good | Humanized mouse models to study cell-mediated immune responses to liver-stage malaria vaccines[END_REF] (Figure 1). Efficient liver humanization relies simultaneously on the host immunodeficiency that facilitates xenotransplantation and on the selective elimination of endogenous murine hepatocytes in order to make room for the transplanted HH to repopulate the liver, prior to inoculation of human malaria sporozoites. Hence, P. falciparum LS development was first observed in homozygous Alb-UpA SCID mice [START_REF] Morosan | Liver-stage development of Plasmodium falciparum, in a humanized mouse model[END_REF][START_REF] Sacci | Plasmodium falciparum infection and exoerythrocytic development in mice with chimeric human livers[END_REF] which express the hepatotoxic Urokinase Plasminogen Activator (UpA) transgene under the albumin (Alb) promoter leading to a constitutive loss of endogenous hepatocytes. Alb-UpASCID mice are best engrafted at very young age (3-4 weeks) but given their low level of immunodeficiency, additional treatment to deplete NK cells and macrophages is required. Mice deficient for fumarylacetoacetate hydrolase (FAH -/-) with the broader immunodeficient Rag2 -/-, IL2R gamma null -/-background (FRG) has been also used in malaria studies [START_REF] Vaughan | Complete Plasmodium falciparum liver-stage development in liver-chimeric mice[END_REF]. The FAH -/-mice suffer from an acute liver failure that can be rescued by providing NTBC (2-(2nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione) at regular intervals pre-and post-HH transplantation. The FRG mice backcrossed onto the NOD background (FRG NOD) allowed full maturation of the P. falciparum LS up to the generation of infectious hepatic merozoites [START_REF] Vaughan | Complete Plasmodium falciparum liver-stage development in liver-chimeric mice[END_REF]. Although this HmHPf-LS supported reproducible transition from LS to ES, the graft of HRBC was ephemeral and the ES were only observed when infected HRBC were cultured ex vivo. More recently, the TK-NOG mouse strain, a model of inducible liver injury, has also proved to be valuable as a HmHPf-LS [START_REF] Soulard | Plasmodium falciparum full life cycle and Plasmodium ovale liver stages in humanized mice[END_REF]. TK-NOG mice harbor the herpes simplex virus type 1 thymidine kinase (HSVtk) transgene under the albumin promoter onto the NSG background and upon a brief exposure to a nontoxic dose of gancyclovir, endogenous hepatocytes are depleted [START_REF] Hasegawa | The reconstituted 'humanized liver' in TK-NOG mice is mature and functional[END_REF]. Numerous studies have then provided the proof of concept that HmHPf-LS are robust new experimental models to evaluate passive transfer therapies [START_REF] Foquet | Vaccine-induced monoclonal antibodies targeting circumsporozoite protein prevent Plasmodium falciparum infection[END_REF][START_REF] Sack | Model for in vivo assessment of humoral protection against malaria sporozoite challenge by passive transfer of monoclonal antibodies and immune serum[END_REF], to study the identification of genetic determinants of parasite traits and adaptations [START_REF] Vaughan | Plasmodium falciparum genetic crosses in a humanized mouse model[END_REF], and to evaluate prophylaxis therapies [START_REF] Douglass | Host-based prophylaxis successfully targets liver stage malaria parasites[END_REF]. Furthermore, LS development of other human Plasmodium species has been obtained with the FRG and TK-NOG mouse strains grafted with HH and infected with P. vivax and P. ovale (HmHPv-LS and HmHPo-LS), including the generation, persistence, and activation of hypnozoites [START_REF] Soulard | Plasmodium falciparum full life cycle and Plasmodium ovale liver stages in humanized mice[END_REF][START_REF] Mikolajczak | Plasmodium vivax liver stage development and hypnozoite persistence in human liver-chimeric mice[END_REF].

HmHP for LS and ES

To obtain a Hm which harbors both the LS and ES of P. falciparum (HmHPf-LES), the scientific community has had to wait until the generation of new immunodeficient mice accept the simultaneous graft of HH and HRBCs. The proof of concept was showed up with the arrival of DR4.RagKO.IL2Rγ cKO.NOD (DRAG) mouse strain. To obtain mouse reconstitution with human liver and erythrocytes, DRAG mice were infused with HLA-matched human hematopoietic stem cells [START_REF] Wijayalath | Humanized HLA-DR4. RagKO.IL2RgammacKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria[END_REF]. Although LS and ES could be obtained in this double Hm, the level of humanization and the parasite load were very low, limiting the use of this model to malaria research.

Finally, the robust proof of concept that the whole human P. falciparum life cycle can be obtained in a double grafted model has been obtained in the TK-NOG mouse strain [START_REF] Soulard | Plasmodium falciparum full life cycle and Plasmodium ovale liver stages in humanized mice[END_REF]. HRBC graft has been maintained for 5 weeks in these Pf-LSm through daily injections. In this double grafted mouse model, following inoculation of P. falciparum sporozoites, ES parasites could be detected in the blood from day 8 postinfection (parasitemia reached up to 1.52%) and sexual stages after 21 days.

The achievement of the complete life cycle of P. ovale and P. vivax is the following step that could be obtained in a near future with progress in stem cells graft and in vivo production of human reticulocytes. So far, transition from P. vivax LS to ES has been described in the FRG mice following transient enrichment of the infected liver-humanized mice with reticulocyteenriched HRBC, but no maintenance of a parasitemia has been reported [START_REF] Mikolajczak | Plasmodium vivax liver stage development and hypnozoite persistence in human liver-chimeric mice[END_REF].

HmHP for antimalarial drug discovery

The huge investment in new drug discovery to face artemisinin resistance and to achieve malaria eradication has also transformed the classical preclinical pathway. Drug efficacy, pharmacology, and toxicity are important parameters in the screening of compounds to proceed toward the clinical phase. In order to determine these parameters, new compounds are subjected to a series of in vitro and in vivo models with a decisive stage in the primate model, which is the most representative experimental model of the human infection, to confirm the therapeutic potential of compounds. Owing to the increased number of new potential candidates together with the actual limitations of the primate models, the progress realized in the HmHP makes them now appear as an alternative to the primate model.

In vivo test used for antimalarial screening

As seen in the previous section, different HmHP are now available but mainly the HmHPf-ES have been largely used in the preclinical evaluation of different antimalarial drugs targeting ES (Table 1). The efficacy of new compounds in the HmHPf-ES has usually been analyzed by transposing the classical tests set up to evaluate in vivo activity in rodent models, such as the 4 days Peter's test [START_REF] Peters | Drug resistance in Plasmodium berghei Vincke and Lips, 1948. I. Chloroquine resistance[END_REF]. However, this test evaluates the suppressive effect of a compound, since the treatment is administered just after mouse infection with a rodent Plasmodium. The need of determining the therapeutic value of new compounds in the HmHPf-ES has led to introduce slight modifications in this test, such as administration of the therapeutic regimen once the parasitemia has developed [START_REF] Angulo-Barturen | A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes[END_REF][START_REF] Cosledan | Selection of a trioxaquine as an antimalarial drug candidate[END_REF]. In the actual context of antimalarial drug discovery and to support the global malaria eradication effort, there is a need for new medicines that can be given as a single dose to allow direct monitoring of administration and to ensure compliance. To this aim, the Thompson's test, which monitors the progress of infection following single-dose administration [START_REF] Thompson | Quinine-resistant Plasmodium berghei in mice[END_REF], has been used in the HmHPf-ES to assess the potential of radical cure of a compound after a single dose [START_REF] Nilsen | Quinolone-3-diarylethers: a new class of antimalarial drug[END_REF]. Most of the studies have also realized a 'dose-ranging test' to determine the classical parameters such as ED50 and ED90 (50% or 90% reduction in parasitemia) of these new compounds. Other parameters which are now being evaluated in the HmHPf-ES are the maximum kill rate after the first dose of treatment and the complete parasite clearance which allows to estimate the potential of parasite resistance development to a compound. Finally, some authors prefer to compare the parasite clearance rate induced by a compound with that induced by a classical antimalarial treatment [START_REF] Angulo-Barturen | A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes[END_REF][START_REF] Jimenez-Diaz | Improved murine model of malaria using Plasmodium falciparum competent strains and nonmyelodepleted NOD-acid IL2Rgammanull mice engrafted with human erythrocytes[END_REF][START_REF] Cosledan | Selection of a trioxaquine as an antimalarial drug candidate[END_REF]63].

An added value of the HmHPf-ES is that it can also give precious information about compound activity against resistant strains. Thus, new compounds such as PA1103/ SAR116242, and SC81458 and SC83288 have shown an excellent activity in mice infected with the P. falciparum 3D7 strain as well as against the chloroquine-resistant P. falciparum W2 strain [START_REF] Cosledan | Selection of a trioxaquine as an antimalarial drug candidate[END_REF]65]. Other authors have taken advantage of the characteristic of this modelin which the different stages of the parasite are free in the circulationto determine against which stage was acting the compound DDD107498, i.e. at the early ring stage or at the later mature schizont stage [59].

Antimalarial activity of new promising compounds in the HmHPf-ES

Using these tests, the HmHPf-ES has participated in the assessment of new compounds, most of them obtained from phenotypic or whole cell screenings; for some of them, the mechanism of action is known (Table 1). As the number of studies is consequent [START_REF] Angulo-Barturen | A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes[END_REF][START_REF] Jimenez-Diaz | Improved murine model of malaria using Plasmodium falciparum competent strains and nonmyelodepleted NOD-acid IL2Rgammanull mice engrafted with human erythrocytes[END_REF][START_REF] Ibrahim | Artemisinin nanoformulation suitable for intravenous injection: preparation, characterization and antimalarial activities[END_REF], in this review, we focus on the most promising compounds. Among them, some compounds target the asexual ES with the aim to develop new therapies against simple or severe malaria access, such as PA1103/SAR116242, triaminopyrimidine, or SC81458 and SC83288 [START_REF] Cosledan | Selection of a trioxaquine as an antimalarial drug candidate[END_REF]65,71]. Other compounds also target the sexual ES such as (+)-SJ733, ACT-451840, E209 [START_REF] O'neill | A tetraoxane-based antimalarial drug candidate that overcomes PfK13-C580Y dependent artemisinin resistance[END_REF]57,64], or the LS which make them potential new therapeutic tools to provide radical cure and prophylaxis, such as the ELQ-300, DSM265, DDD107498, and MMV390048 [START_REF] Nilsen | Quinolone-3-diarylethers: a new class of antimalarial drug[END_REF]52,59,63]. It is interesting to highlight that in none of these works, the primate model was used to confirm the results obtained in the HmHPf-ES, which indirectly confirms the role of the latter model in the preclinical evaluation of a compound.

Compounds targeting different pathways such as protein synthesis, membrane transport, heme polymerization, or transcription have been evaluated in the HmHPf-ES to confirm the activity observed using the classical in vitro model and in vivo rodent Plasmodium spp. models (Table 1). When comparing efficacy parameters between different experimental models, efficacies observed for PA1103/SAR116242, triaminopyrimidine, ELQ-300, and DDD107498 compounds in rodent Plasmodium model and in HmHPf-ES were similar. In contrast, (+)-SJ733, ACT-451840, or E209 showed an efficacy higher in the HmHPf-ES than in the rodent Plasmodium model. It even happened that a compound was inactive in the rodent Plasmodium model infected with P. berghei whereas an excellent activity had been determined in vitro and could be confirmed using the HmHPf-ES, such as SC81458 and SC83288 [65]. Different therapeutic efficacy between Plasmodium species has been associated with charged compounds so that it could be hypothesized that discrepancies may be explained by selective uptake of charged compounds through parasite-induced channels in the host cell plasma membrane and species-specific characteristics of these new permeation pathways. These differences can be important in the progression of a compound throughout preclinical pathway toward the clinical phase. Since P. berghei is usually used in the first step of this screening, a reduced or a lack of activity in this model can stop the further development of a compound. Thus, HmHPf-ES may help to not unduly eliminate compounds during the preclinical pathway.

Pharmacodynamic/Pharmacokinetic studies in the HmHPf-ES

The HmHPf-ES has also opened new perspectives in pharmacokinetic (PK) studies together with pharmacodynamic (PD) studies that could allow a human dose prediction. In this context, the HmHPf-ES has participated in the preclinical assessment of dose-efficacy modeling, with the remarkable example of the ACT-451840 [64]. Briefly, assessment of PD/PK parameters in rodent Plasmodium infected and noninfected mice was used to model and predict dose regimens against the rodent Plasmodium. In parallel, PD/PK parameters were also evaluated in the HmHPf-ES. Finally, combining PD/PK obtained in the rodent and human Plasmodium models, a modeling approach was achieved to predict human efficacious exposure [64]. In an attempt to obtain a more accurate human dose prediction, other authors prefer to modify the 4day therapeutic assay by taking into account the PK parameters of the compound previously evaluated in mice. For example, because the half-life of DSM265 in mice is much shorter (2-4 h) than the one predicted in humans, this compound was dosed twice daily for 4 days in the HmHPf-ES [52].

Then, blood concentrations obtained using this therapeutic regimen in infected mice were used to determine the minimum concentration required for parasite killing.

New HmHP for drug discovery

The value of the HmHPf-ES has been also explored as a platform for testing drugs against P. falciparum erythrocytic sexual forms, those responsible for malaria transmission [START_REF] Duffier | A humanized mouse model for sequestration of Plasmodium falciparum sexual stages and in vivo evaluation of gametocytidal drugs[END_REF]. As a proof of concept, the effect of primaquine, which is currently the only licensed drug that has demonstrated efficacy to eliminate P. falciparum gametocytes in humans, was evaluated. After primaquine treatment, gametocytes were eliminated from peripheral blood and from sequestration sites, proving that the HmHPf-ES can be used for testing drugs against gametocytes. Primaquine can also eradicate the LS of P. vivax and P. ovale. Its activity against different stages and different Plasmodium spp. makes primaquine an important antimalarial drug, but the risk of inducing hemolytic anemia in G6PD-deficient persons has reduced its utilization. Evaluation of drugs safer than primaquine in vivo is hampered by the fact that treatment with drugs known to cause hemolytic anemia in humans does not cause damage to mouse RBCs. To overcome this limitation, NSG mice have been humanized with G6PD-deficient HRBCs. In this new model, oral administration of primaquine induces a dose-dependent hemolytic response [START_REF] Rochford | Humanized mouse model of glucose 6-phosphate dehydrogenase deficiency for in vivo assessment of hemolytic toxicity[END_REF][START_REF] Wickham | Single-dose primaquine in a preclinical model of glucose-6-phosphate dehydrogenase deficiency: implications for use in malaria transmission-blocking programs[END_REF] and treatment with drugs known to cause hemolytic toxicity (pamaquine, sitamaquine, tafenoquine, and dapsone) resulted in loss of G6PD-deficient HRBCs comparable to primaquine. In contrast, results obtained in this model show that (-)-(R)-primaquine may have a better safety margin than the racemate in human [START_REF] Nanayakkara | Scalable preparation and differential pharmacologic and toxicologic profiles of primaquine enantiomers[END_REF].

Finally, the HmHPf-LSm has recently proved its value to investigate liver parasites and facilitate the discovery of drugs targeting this stage [START_REF] Douglass | Host-based prophylaxis successfully targets liver stage malaria parasites[END_REF]. For an easier evaluation of parasite infection in liver, mice are infected using a GFP-luc transgenic P. falciparum strain and drug efficacy can be monitored without killing mice or using complicated and timeconsuming histological methods. The accuracy of this model has been evaluated after treatment with different antimalarial drugs. Parasite clearance could be observed after atovaquone treatment or using a combination of Bcl-2 inhibitors and P53 agonists treatments [START_REF] Douglass | Host-based prophylaxis successfully targets liver stage malaria parasites[END_REF].

Conclusions

Recent efforts carried out by the scientific community have permitted to proceed to an era in which HmHP can successfully be infected with human Plasmodium spp.: P. falciparum, P vivax, and P. ovale. Furthermore, it is now a reality that the complete human cycle of P. falciparum can be obtained in HmHP, ultimately allowing to reproducibly obtain transmission to mosquitoes. The HmHPf-ES has shown a real input in the preclinical evaluation of new compounds against the ES: its efficacy is similar to that of the rodent Plasmodium model, it allows to study compound activity against P. falciparum resistant strains and to realize a prediction of the human dose by taking into account the PK parameters.

Expert opinion

The present review of HmHP suggests that, theoretically, these models may replace the rodent Plasmodium model in the preclinical pathway, thereby reducing the risk of unduly eliminate compounds inactive against rodent Plasmodium spp. However, considering 10 years of experience in the HmHPf-ES, it appears that compounds with a species-specific activity are more an exception than a rule. Furthermore, for economic and technical reasons, the use of the HmHPf-ES is reduced to a limited number of laboratories. Consequently, the rodent Plasmodium model keeps a main place in the initial step of the preclinical pathway. Nonetheless, HmHP is a valuable tool in case of discrepancies between in vitro data and those obtained in the rodent model. In addition, there is an important advantage of the HmHPf-ES, which has been largely underexploited: its ability to evaluate compound activity against strains artemisin resistant or against clinical isolates with in vivo reduced clearance. Thus, it is regrettable that most studies have evaluated compounds activity against a sensitive mouse-adapted P. falciparum strain and only a reduced number of works have explored the activity against chloroquineresistant strains.

In the present context where an unprecedented number of new compounds must be evaluated to confirm their in vivo activity against P. falciparum, the ethical, economical, and practical limitations of the primate model become more important, leading to favor the HmHPf-ES. The input of the primate model in the preclinical pathway is primary to confirm the efficacy of compounds and to predict the human dose, as primate metabolism is closer to human metabolism than rodent metabolism. Considerable efforts have been done to overcome these differences and PD/PK studies are now realized in the HmHPf-ES to obtain a more accurate prediction. Preliminary studies comparing the data obtained in the HmHP and in the human challenge model are encouraging [START_REF] Mccarthy | Linking murine and human Plasmodium falciparum challenge models in a translational path for antimalarial drug development[END_REF]. Nonetheless, the proof of concept of this modelization has not yet been demonstrated, as none of the compounds has reached the clinical phase in human. Meanwhile, several technical improvements of the HmHPf-ES should contribute to the final objective of replacing the primate model.

A technical improvement concerns the tests used to evaluate the activity of compounds. Till now, the modified 4-day test [START_REF] Peters | Drug resistance in Plasmodium berghei Vincke and Lips, 1948. I. Chloroquine resistance[END_REF] or the Thompson's test have been accommodated to the HmHPf-ES. However, these tests have been set up to evaluate the activity of compounds against the rodent Plasmodium infection. The biology between rodent and human Plasmodium spp. diverges in many aspects but especially in time that parasites need to achieve their erythrocytic cycle: P. falciparum requires 48 h whereas rodent Plasmodium spp. need only 24 h. Considering that PK parameters are faster in mouse than in humans, there is an imbalance between parasite maturation and mouse PK. This imbalance might explain that for some antimalarials with a short half-life such as artesunate, dosages of 50 mg/kg are needed in the HmHPf-ES to clear parasitemia while the human dose chosen is 4 mg/kg. Thus, the recently introduced test in which the compound is administrated twice a day could counteract rapid mouse PK. Consequently, new studies should tend to use this 'twice a day -4 days test' or 'twice a day -Thompson's test.' Another technical improvement will be to carry out PD/PK studies in mouse grafted with HH to mimic HH metabolism.

Further improvements will require the generation of new immunodeficient mice. Protocols to obtain HRBC humanization are still a constraint. It is not known whether these protocols could alter the compound evaluation. Daily transfusion of HRBC may induce a dilution factor during parasitemia assessment and the use of immunosuppressive treatments could interact with drug activity or reduce the physiological clearance of parasites. As it is now clearly demonstrated that phagocyte population plays a major role in HRBC clearance, it is mandatory to generate new immunodeficient mice with a reduced macrophagic population. In parallel, efforts should be made to improve the graft of CD34 cells and the production of HRBC in the HmHP. This methodology could allow the in vivo generation of reticulocytes and the setup of a Hm for P. vivax and P. ovale ES.

Correcting the lack of cytoadherence in the HmHPf-ES, as compared with human infection, will also require the generation of new immunodeficient mice. A first objective is to reduce the difference between the parasite biomass that a compound must face in the HmHPf-ES as compared to human infection. Indeed, the parasite biomass in the HmHPf-ES, where parasites freely circulate, may be quite less than that in human infection, where the parasites cytoadhere throughout the vascular system. Consequently, it is reasonable to wonder whether the dose determined in the HmHPf-ES could face the parasite biomass in humans. This question has further consequences when the ability of P. falciparum to develop resistances is taken into account. A second objective is to set up a new animal model for in vivo studies of mechanisms leading to the onset of severe malaria. The lack of cytoadherence is most probably due to the lack of interactions between P. falciparum antigens and mouse vascular adhesion receptors. Therefore, new immunodeficient mice transgenic for human vascular adhesion molecules could overcome this limitation.

After two decades of continuous developments and progress obtained in the HmHP, the state of the art shows that it is now a valuable and accurate model for antimalarial drug discovery. The present review shows its input in the preclinical pathway of new compounds, its ability to confirm their in vivo activity including against resistant parasites and to help to predict human doses. In our opinion, further technical improvements are needed before HmHP may replace the primate model.

Figure 1 .

 1 Figure 1. Evolution of humanized mouse models infected with human Plasmodium species (HmHP). The figure above shows the main improvements obtained during the last 20 years in the HmHP infected with P. falciparum (Pf) erythrocytic stages (HmHPf-ES), the liver stages (HmHPf-LS) or both stages (HmHPf-LES) comparing with the generation of new immunodeficient and transgenic mouse strains. The graph below shows the cumulative number of publications implicating the HmHP and it is correlated with the upper picture showing the progress in the improvements obtained during the last 20 years in the HmHP. Human hepatocytes (HH), Human red blood cells (HRBC).

Table 1 .

 1 Input of HmHPf-ES in antimalarial drug discovery.

	Mode of action	Chemical family	Bibliography
	Hemozoin metabolism	Trioxaquines (PA1103/SAR116242)	[47]
		Tetraoxane (E209)	[48]
	Mitochondrial process
	Via cytochrome bc1	Pyridone (GSK932121)	[26]
		Quinolone-3-diarylethers (ELQ-300)	[49]
	Via inhibition of dihydroorotate
	dehydrogenase

Funding

This manuscript has not been funded.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Peer reviewers on this manuscript have no relevant financial or other relationships to disclose