From reverse to structural vaccinology : profiling of CyRPA as new Plasmodium falciparum malaria vaccine candidate antigen

Abstract

Malaria is one of the most important and life-threatening infectious diseases worldwide. In 2015 malaria claimed about 429 000 lives, mostly among children below five year of age in sub‐Saharan Africa, and caused 212 million clinical episodes in a population of approximately 3.3 billion people living in regions at risk of infection. The development of an effective malaria vaccine is recognized as one of the most promising approaches for preventing infections and reducing transmission. To date, there is no vaccine on the market for prevention of malaria and only a few candidate vaccines were able to induce some protective efficacy. Thus, there is an urgent need to accelerate the pace of design and development of new malaria vaccine candidates that induce broad and long-lasting protective immunity. Reverse vaccinology and structural vaccinology are two complementary techniques that hold much promise in this regard. The pathogenesis of malaria is primarily associated with blood-stage infection and there is strong evidence that antibodies specific for parasite blood-stage antigens can control parasitemia. This provides a strong rationale for incorporation of asexual blood-stage antigen components into an effective multivalent malaria subunit vaccine. In this thesis, we exploited the great potential of the ‘omics’ sciences for the selection of hypothetical surface-exposed protein and the evaluation of their potential as vaccine candidate antigens. For the characterization of selected antigens we have exploited an entirely cell-based, rapid and reliable approach for the generation of antigen-specific and parasite cross-reactive monoclonal antibodies (mAbs): (I) generation of mammalian cell lines expressing high levels of the selected predicted malaria antigens as transmembrane proteins; (II) living­-cell immunization of mice; (III) generation of hybridoma cell lines producing mAbs capable of recognizing the endogenous antigen in its native context. This strategy has led us to the identification of the Plasmodium falciparum Cysteine-­Rich Protective Antigen (PfCyRPA) as promising blood­-stage malaria vaccine candidate: (I) PfCyRPA has limited natural immunogenicity, (II) is highly conserved among P. falciparum isolates and (III) forms together with the Reticulocyte-binding Homolog 5 (PfRH5) and the PfRH5-interacting Protein (PfRipr) a multiprotein complex crucial for P. falciparum erythrocyte invasion; (IV) PfCyRPA-specific mAbs showed parasite in vitro growth­-inhibitory activity due to inhibition of merozoite invasion; (V) passive immunization experiments in P. falciparum infected NOD­scid IL2Rγnull mice engrafted with human erythrocytes demonstrated in vivo growth-­inhibitory activity of PfCyRPA specific mAbs. To investigate whether growth inhibitory anti­PfCyRPA and anti­PfRH5 Abs can be induced by active immunization with the adjuvanted recombinant proteins, PfCyRPA and PfRH5 were recombinantly expressed as soluble protein in mammalian and insect cells respectively, purified from culture supernatant and employed for immunization of mice. mAbs raised against recombinant PfCyRPA and PfRH5 proteins showed potent parasite growth-­inhibitory activity both in vitro and in vivo. Furthermore, both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in a vaccine. To examine the 3D structure of PfCyRPA and to explore the dynamics of its surface loops, we generated co-crystals of it in complex with an inhibitory mAb and elucidated the 3D structure of PfCyRPA and of the epitope–paratope interface by X-ray crystallography. Elucidation of the structure of the epitope recognized by the protective mAb will strongly facilitate design of peptidomimetics in a structural vaccinology approach. The overall structure of PfCyRPA is a six-bladed β-propeller with each blade of the propeller being a four-stranded anti-parallel β-sheet. The five disulfide bonds of the protein are located within blades 1-5, stabilizing each individual blade. Since the 6th blade is composed of β-strands both from the N- and the C-terminus and has no disulfide bond, PfCyRPA has the potential to undergo large conformational changes by disassembly of blade 6. Among additional hypothetical antigens investigated in the framework of this thesis, PF14_0044 showed interesting features: while none of the generated PF14_0044-specific mAbs significantly inhibited parasite growth, a synergistic in vitro inhibitory activity was observed when anti-PF14_0044 mAbs were combined with anti-PfCyRPA mAbs. Applying the principle of reverse vaccinology, we thus identified PfCyRPA and PF14_0044 as targets of merozoite invasion‐inhibitory antibodies. Taken together results show how a combination of reverse and structural vaccinology approaches can enable the identification of new target antigens for incorporation into subunit vaccines.