One hundred malaria attacks since birth. A longitudinal study of African children and young adults exposed to high malaria transmission.

Background: Despite significant progress in malaria control over the past twenty years, malaria remains a leading cause of child morbidity and mortality in Tropical Africa. As most patients do not consult any health facility much uncertainty persists about the true burden of the disease and the range of individual differences in susceptibility to malaria. Methods: Over a 25-years period, from 1990 to 2015, the inhabitants of Dielmo village, Senegal, an area of intense malaria transmission, have been monitored daily for their presence in the village and the occurrence of diseases. In case of fever thick blood films were systematically examined through microscopy for malaria parasites and patients received prompt diagnosis and treatment. Findings: We analysed data collected in 111 children and young adults monitored for at least 10 years (mean 17.3 years, maximum 25 years) enrolled either at birth (95 persons) or during the two first years of life. A total of 11,599 episodes of fever were documented, including 5268 malaria attacks. The maximum number of malaria attacks in a single person was 112. Three other persons suffered one hundred or more malaria attacks during follow-up. The minimum number of malaria attacks in a single person was 11. The mean numbers of malaria attacks in children reaching their 4th, 7th, and 10th birthdays were 23.0, 37.7, and 43.6 attacks since birth, respectively. Sixteen children (14.4%) suffered ten or more malaria attacks each year at ages 1-3 years, and six children (5.4%) each year at age 4-6 years. Interpretation: Long-term close monitoring shows that in highly endemic areas the malaria burden is higher than expected. Susceptibility to the disease may vary up to 10-fold, and for most children childhood is an endless history of malaria fever episodes. No other parasitic, bacterial or viral infection in human populations has such an impact on health. Funding: The Pasteur Institutes of Dakar and Paris, the Institut de Recherche pour le Développement, and the French Ministry of Cooperation provided funding.

PvDBPII elicits multiple antibody-mediated mechanisms that reduce growth in a Plasmodium vivax challenge trial.

The receptor-binding domain, region II, of the Plasmodium vivax Duffy binding protein (PvDBPII) binds the Duffy antigen on the reticulocyte surface to mediate invasion. A heterologous vaccine challenge trial recently showed that a delayed dosing regimen with recombinant PvDBPII SalI variant formulated with adjuvant Matrix-MTM reduced the in vivo parasite multiplication rate (PMR) in immunized volunteers challenged with the Thai P. vivax isolate PvW1. Here, we describe extensive analysis of the polyfunctional antibody responses elicited by PvDBPII immunization and identify immune correlates for PMR reduction. A classification algorithm identified antibody features that significantly contribute to PMR reduction. These included antibody titre, receptor-binding inhibitory titre, dissociation constant of the PvDBPII-antibody interaction, complement C1q and Fc gamma receptor binding and specific IgG subclasses. These data suggest that multiple immune mechanisms elicited by PvDBPII immunization are likely to be associated with protection and the immune correlates identified could guide the development of an effective vaccine for P. vivax malaria. Importantly, all the polyfunctional antibody features that correlated with protection cross-reacted with both PvDBPII SalI and PvW1 variants, suggesting that immunization with PvDBPII should protect against diverse P. vivax isolates.

Unveiling P. vivax invasion pathways in Duffy-negative individuals.

Vivax malaria has long been thought to be absent from sub-Saharan Africa owing to the high proportion of individuals lacking the Duffy antigen receptor for chemokines (DARC) in their erythrocytes. The interaction between P. vivax Duffy-binding protein (PvDBP) and DARC is assumed to be the main pathway used by merozoites to invade reticulocytes. However, the increasing number of reports of vivax malaria cases in genotypically Duffy-negative (DN) individuals has raised questions regarding the P. vivax invasion pathway(s). Here, we show that a subset of DN erythroblasts transiently express DARC during terminal erythroid differentiation and that P. vivax merozoites, irrespective of their origin, can invade DARC+ DN erythroblasts. These findings reveal that a large number of DN individuals may represent a silent reservoir of deep P. vivax infections at the sites of active erythropoiesis with low or no parasitemia, and it may represent an underestimated biological problem with potential clinical consequences in sub-Saharan Africa.

Immunogenicity of a Plasmodium vivax vaccine based on the duffy binding protein formulated using adjuvants compatible for use in humans.

The invasion of reticulocytes by Plasmodium vivax merozoites is dependent on the interaction of the Plasmodium vivax Duffy Binding Protein (PvDBP) with the Duffy antigen receptor for chemokines (DARC). The N-terminal cysteine-rich region II of PvDBP (PvDBPII), which binds DARC, is a leading P. vivax malaria vaccine candidate. Here, we have evaluated the immunogenicity of recombinant PvDBPII formulated with the adjuvants Matrix-M and GLA-SE in mice. Analysis of the antibody responses revealed comparable ELISA recognition titres as well as similar recognition of native PvDBP in P. vivax schizonts by immunofluorescence assay. Moreover, antibodies elicited by the two adjuvant formulations had similar functional properties such as avidity, isotype profile and inhibition of PvDBPII-DARC binding. Furthermore, the anti-PvDBPII antibodies were able to block the interaction of DARC with the homologous PvDBPII SalI allele as well as the heterologous PvDBPII PvW1 allele from a Thai clinical isolate that is used for controlled human malaria infections (CHMI). The cross-reactivity of these antibodies with PvW1 suggest that immunization with the PvDBPII SalI strain should neutralize reticulocyte invasion by the challenge P. vivax strain PvW1.

Vaccination with Plasmodium vivax Duffy-binding protein inhibits parasite growth during controlled human malaria infection.

There are no licensed vaccines against Plasmodium vivax. We conducted two phase 1/2a clinical trials to assess two vaccines targeting P. vivax Duffy-binding protein region II (PvDBPII). Recombinant viral vaccines using chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) vectors as well as a protein and adjuvant formulation (PvDBPII/Matrix-M) were tested in both a standard and a delayed dosing regimen. Volunteers underwent controlled human malaria infection (CHMI) after their last vaccination, alongside unvaccinated controls. Efficacy was assessed by comparisons of parasite multiplication rates in the blood. PvDBPII/Matrix-M, given in a delayed dosing regimen, elicited the highest antibody responses and reduced the mean parasite multiplication rate after CHMI by 51% (n = 6) compared with unvaccinated controls (n = 13), whereas no other vaccine or regimen affected parasite growth. Both viral-vectored and protein vaccines were well tolerated and elicited expected, short-lived adverse events. Together, these results support further clinical evaluation of the PvDBPII/Matrix-M P. vivax vaccine.

Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission.

Transmission of Plasmodium falciparum involves a complex process that starts with the ingestion of gametocytes by female Anopheles mosquitoes during a blood meal. Activation of gametocytes in the mosquito midgut triggers "rounding up" followed by egress of both male and female gametes. Egress requires secretion of a perforin-like protein, PfPLP2, from intracellular vesicles to the periphery, which leads to destabilization of peripheral membranes. Male gametes also develop flagella, which assist in binding female gametes for fertilization. This process of gametogenesis, which is key to malaria transmission, involves extensive membrane remodeling as well as vesicular discharge. Phospholipase A2 enzymes (PLA2) are known to mediate membrane remodeling and vesicle secretion in diverse organisms. Here, we show that a P. falciparum patatin-like phospholipase (PfPATPL1) with PLA2 activity plays a key role in gametogenesis. Conditional deletion of the gene encoding PfPATPL1 does not affect P. falciparum blood stage growth or gametocyte development but reduces efficiency of rounding up, egress, and exflagellation of gametocytes following activation. Interestingly, deletion of the PfPATPL1 gene inhibits secretion of PfPLP2, reducing the efficiency of gamete egress. Deletion of PfPATPL1 also reduces the efficiency of oocyst formation in mosquitoes. These studies demonstrate that PfPATPL1 plays a role in gametogenesis, thereby identifying PLA2 phospholipases such as PfPATPL1 as potential targets for the development of drugs to block malaria transmission.

Antibody responses to Plasmodium vivax Duffy binding and Erythrocyte binding proteins predict risk of infection and are associated with protection from clinical Malaria.

BACKGROUND: The Plasmodium vivax Duffy Binding Protein (PvDBP) is a key target of naturally acquired immunity. However, region II of PvDBP, which contains the receptor-binding site, is highly polymorphic. The natural acquisition of antibodies to different variants of PvDBP region II (PvDBPII), including the AH, O, P and Sal1 alleles, the central region III-V (PvDBPIII-V), and P. vivax Erythrocyte Binding Protein region II (PvEBPII) and their associations with risk of clinical P. vivax malaria are not well understood. METHODOLOGY: Total IgG and IgG subclasses 1, 2, and 3 that recognize four alleles of PvDBPII (AH, O, P, and Sal1), PvDBPIII-V and PvEBPII were measured in samples collected from a cohort of 1 to 3 year old Papua New Guinean (PNG) children living in a highly endemic area of PNG. The levels of binding inhibitory antibodies (BIAbs) to PvDBPII (AH, O, and Sal1) were also tested in a subset of children. The association of presence of IgG with age, cumulative exposure (measured as the product of age and malaria infections during follow-up) and prospective risk of clinical malaria were evaluated. RESULTS: The increase in antigen-specific total IgG, IgG1, and IgG3 with age and cumulative exposure was only observed for PvDBPII AH and PvEBPII. High levels of total IgG and predominant subclass IgG3 specific for PvDBPII AH were associated with decreased incidence of clinical P. vivax episodes (aIRR = 0.56-0.68, P≤0.001-0.021). High levels of total IgG and IgG1 to PvEBPII correlated strongly with protection against clinical vivax malaria compared with IgGs against all PvDBPII variants (aIRR = 0.38, P<0.001). Antibodies to PvDBPII AH and PvEBPII showed evidence of an additive effect, with a joint protective association of 70%. CONCLUSION: Antibodies to the key parasite invasion ligands PvDBPII and PvEBPII are good correlates of protection against P. vivax malaria in PNG. This further strengthens the rationale for inclusion of PvDBPII in a recombinant subunit vaccine for P. vivax malaria and highlights the need for further functional studies to determine the potential of PvEBPII as a component of a subunit vaccine for P. vivax malaria.

Genetic diversity in two Plasmodium vivax protein ligands for reticulocyte invasion.

The interaction between Plasmodium vivax Duffy binding protein (PvDBP) and Duffy antigen receptor for chemokines (DARC) has been described as critical for the invasion of human reticulocytes, although increasing reports of P. vivax infections in Duffy-negative individuals questions its unique role. To investigate the genetic diversity of the two main protein ligands for reticulocyte invasion, PvDBP and P. vivax Erythrocyte Binding Protein (PvEBP), we analyzed 458 isolates collected in Cambodia and Madagascar from individuals genotyped as Duffy-positive. First, we observed a high proportion of isolates with multiple copies PvEBP from Madagascar (56%) where Duffy negative and positive individuals coexist compared to Cambodia (19%) where Duffy-negative population is virtually absent. Whether the gene amplification observed is responsible for alternate invasion pathways remains to be tested. Second, we found that the PvEBP gene was less diverse than PvDBP gene (12 vs. 33 alleles) but provided evidence for an excess of nonsynonymous mutations with the complete absence of synonymous mutations. This finding reveals that PvEBP is under strong diversifying selection, and confirms the importance of this protein ligand in the invasion process of the human reticulocytes and as a target of acquired immunity. These observations highlight how genomic changes in parasite ligands improve the fitness of P. vivax isolates in the face of immune pressure and receptor polymorphisms.

Targeting a Reticulocyte Binding Protein and Duffy Binding Protein to Inhibit Reticulocyte Invasion by Plasmodium vivax.

Plasmodium vivax merozoite invasion is restricted to Duffy positive reticulocytes. Merozoite interaction with the Duffy antigen is mediated by the P. vivax Duffy binding protein (PvDBP). The receptor-binding domain of PvDBP maps to an N-terminal cysteine-rich region referred to as region II (PvDBPII). In addition, a family of P. vivax reticulocyte binding proteins (PvRBPs) mediates interactions with reticulocyte receptors. The receptor binding domain of P. vivax reticulocyte binding protein 1a (PvRBP1a) maps to a 30 kD region (PvRBP1a30). Antibodies raised against recombinant PvRBP1a30 and PvDBPII recognize the native P. vivax antigens and inhibit their binding to host receptors. Rabbit IgG purified from sera raised against PvRBP1a30 and PvDBPII were tested individually and in combination for inhibition of reticulocyte invasion by P. vivax field isolates. While anti-PvDBPII rabbit IgG inhibits invasion, anti-PvRBP1a30 rabbit IgG does not show significant invasion inhibitory activity. Combining antibodies against PvDBPII and PvRBP1a30 also does not increase invasion inhibitory activity. These studies suggest that although PvRBP1a mediates reticulocyte invasion by P. vivax merozoites, it may not be useful to include PvRBP1a30 in a blood stage vaccine for P. vivax malaria. In contrast, these studies validate PvDBPII as a promising blood stage vaccine candidate for P. vivax malaria.

Artesunate tolerance in transgenic Plasmodium falciparum parasites overexpressing a tryptophan-rich protein.

Due to their rapid, potent action on young and mature intraerythrocytic stages, artemisinin derivatives are central to drug combination therapies for Plasmodium falciparum malaria. However, the evidence for emerging parasite resistance/tolerance to artemisinins in southeast Asia is of great concern. A better understanding of artemisinin-related drug activity and resistance mechanisms is urgently needed. A recent transcriptome study of parasites exposed to artesunate led us to identify a series of genes with modified levels of expression in the presence of the drug. The gene presenting the largest mRNA level increase, Pf10_0026 (PArt), encoding a hypothetical protein of unknown function, was chosen for further study. Immunodetection with PArt-specific sera showed that artesunate induced a dose-dependent increase of the protein level. Bioinformatic analysis showed that PArt belongs to a Plasmodium-specific gene family characterized by the presence of a tryptophan-rich domain with a novel hidden Markov model (HMM) profile. Gene disruption could not be achieved, suggesting an essential function. Transgenic parasites overexpressing PArt protein were generated and exhibited tolerance to a spike exposure to high doses of artesunate, with increased survival and reduced growth retardation compared to that of wild-type-treated controls. These data indicate the involvement of PArt in parasite defense mechanisms against artesunate. This is the first report of genetically manipulated parasites displaying a stable and reproducible decreased susceptibility to artesunate, providing new possibilities to investigate the parasite response to artemisinins.