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

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

Vaccination with virosomally formulated recombinant CyRPA elicits protective anti against Plasmodium falciparum parasites in preclinical in vitro and in vivo modelsbodies

The; Plasmodium falciparum; (; Pf; ) cysteine-rich protective antigen (; Pf; CyRPA) has emerged as a promising blood-stage candidate antigen for inclusion into a broadly cross-reactive malaria vaccine. This highly conserved protein among various geographical strains plays a key role in the red blood cell invasion process by; P. falciparum; merozoites, and antibodies against; Pf; CyRPA can efficiently prevent the entry of the malaria parasites into red blood cells. The aim of the present study was to develop a human-compatible formulation of the; Pf; CyRPA vaccine candidate and confirming its activity in preclinical studies. Recombinant; Pf; CyRPA expressed in HEK 293 cells was chemically coupled to phosphoethanolamine and then incorporated into the membrane of unadjuvanted influenza virosomes approved as antigen delivery system for humans. Laboratory animals were immunised with the virosome-based; Pf; CyRPA vaccine to determine its immunogenic properties and in particular, its capacity to elicit parasite binding and growth-inhibitory antibodies. The vaccine elicited in mice and rabbits high titers of; Pf; CyRPA-specific antibodies that bound to the blood-stage parasites. At a concentration of 10 mg/mL, purified total serum IgG from immunised rabbits inhibited parasite growth in vitro by about 80%. Furthermore, in a; P. falciparum; infection mouse model, passive transfer of 10 mg of purified total IgG from; Pf; CyRPA vaccinated rabbits reduced the in vivo parasite load by 77%. Influenza virosomes thus represent a suitable antigen delivery system for the induction of protective antibodies against the recombinant; Pf; CyRPA, designating it as a highly suitable component for inclusion into a multivalent and multi-stage virosomal malaria vaccine

Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization

Due to their rising incidence and progressive geographical spread, infections with mosquito-borne viruses, such as dengue (DENV), chikungunya and zika virus, have developed into major public health challenges. Since all of these viruses may cause similar symptoms and can occur in concurrent epidemics, tools for their differential diagnosis and epidemiological monitoring are of urgent need.; Here we report the application of a novel strategy to rapidly generate monoclonal antibodies (mAbs) against native viral antigens, exemplified for the DENV nonstructural glycoprotein 1 (NS1). The described system is based on the immunization of mice with transfected mammalian cells expressing the target antigens in multiple displays on their cell surface and thereby presenting them efficiently to the host immune system in their native conformation. By applying this cell-based approach to the DENV NS1 protein of serotypes 1 (D1NS1) and 4 (D4NS1), we were able to rapidly generate panels of DENV NS1 serotype cross-reactive, as well as D1NS1- and D4NS1 serotype-specific mAbs. Our data show that the generated mAbs were capable of recognizing the endogenous NS1 protein in DENV-containing biological samples.; The use of this novel immunization strategy, allows for a fast and efficient generation of hybridoma cell lines, producing mAbs against native viral antigens. Envisaged applications of the mAbs include the development of test platforms enabling a differentiation of the DENV serotypes and high resolution immunotyping for epidemiological studies

Passive immunoprotection of Plasmodium falciparum-infected mice designates the CyRPA as candidate malaria vaccine antigen

An effective malaria vaccine could prove to be the most cost-effective and efficacious means of preventing severe disease and death from malaria. In an endeavor to identify novel vaccine targets, we tested predicted Plasmodium falciparum open reading frames for proteins that elicit parasite-inhibitory Abs. This has led to the identification of the cysteine-rich protective Ag (CyRPA). CyRPA is a cysteine-rich protein harboring a predicted signal sequence. The stage-specific expression of CyRPA in late schizonts resembles that of proteins known to be involved in merozoite invasion. Immunofluorescence staining localized CyRPA at the apex of merozoites. The entire protein is conserved as shown by sequencing of the CyRPA encoding gene from a diverse range of P. falciparum isolates. CyRPA-specific mAbs substantially inhibited parasite growth in vitro as well as in a P. falciparum animal model based on NOD-scid IL2Rgamma(null) mice engrafted with human erythrocytes. In contrast to other P. falciparum mouse models, this system generated very consistent results and evinced a dose-response relationship and therefore represents an unprecedented in vivo model for quantitative comparison of the functional potencies of malaria-specific Abs. Our data suggest a role for CyRPA in erythrocyte invasion by the merozoite. Inhibition of merozoite invasion by CyRPA-specific mAbs in vitro and in vivo renders this protein a promising malaria asexual blood-stage vaccine candidate A

Structure of the malaria vaccine candidate antigen CyRPA and its complex with a parasite invasion inhibitory antibody

Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. 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 vaccines

Additional file 1: Figure S1. of Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization

Dengue virus NS1 protein sequences. Sequence alignment of the D1NS1 - D4NS1 protein sequences expressed by the transfected HEK cells showing 30 % sequence variability. Identical amino acid positions among the four serotypes are highlighted in grey. (TIF 2689 kb

7-N-Substituted-3-oxadiazole Quinolones with Potent Antimalarial Activity Target the Cytochrome bc1 Complex.

The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class