Analysis of the diverse antigenic landscape of the malaria protein RH5 identifies a potent vaccine-induced human public antibody clonotype.

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.

Why Plasmodium vivax and Plasmodium falciparum are so different? A tale of two clades and their species diversities.

The global malaria burden sometimes obscures that the genus Plasmodium comprises diverse clades with lineages that independently gave origin to the extant human parasites. Indeed, the differences between the human malaria parasites were highlighted in the classical taxonomy by dividing them into two subgenera, the subgenus Plasmodium, which included all the human parasites but Plasmodium falciparum that was placed in its separate subgenus, Laverania. Here, the evolution of Plasmodium in primates will be discussed in terms of their species diversity and some of their distinct phenotypes, putative molecular adaptations, and host-parasite biocenosis. Thus, in addition to a current phylogeny using genome-level data, some specific molecular features will be discussed as examples of how these parasites have diverged. The two subgenera of malaria parasites found in primates, Plasmodium and Laverania, reflect extant monophyletic groups that originated in Africa. However, the subgenus Plasmodium involves species in Southeast Asia that were likely the result of adaptive radiation. Such events led to the Plasmodium vivax lineage. Although the Laverania species, including P. falciparum, has been considered to share "avian characteristics," molecular traits that were likely in the common ancestor of primate and avian parasites are sometimes kept in the Plasmodium subgenus while being lost in Laverania. Assessing how molecular traits in the primate malaria clades originated is a fundamental science problem that will likely provide new targets for interventions. However, given that the genus Plasmodium is paraphyletic (some descendant groups are in other genera), understanding the evolution of malaria parasites will benefit from studying "non-Plasmodium" Haemosporida.

Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion.

Erythrocyte invasion by Plasmodium falciparum merozoites is a highly intricate process in which Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an indispensable parasite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is a leading blood-stage vaccine candidate because it exhibits limited polymorphisms and elicits potent strain-transcending parasite neutralizing antibodies. However, the mechanism by which it is anchored to the merozoite surface remains unknown because both PfRH5 and the PfRH5-interacting protein (PfRipr) lack transmembrane domains and GPI anchors. Here we have identified a conserved GPI-linked parasite protein, Cysteine-rich protective antigen (CyRPA) as an interacting partner of PfRH5-PfRipr that tethers the PfRH5/PfRipr/CyRPA multiprotein complex on the merozoite surface. CyRPA was demonstrated to be GPI-linked, localized in the micronemes, and essential for erythrocyte invasion. Specific antibodies against the three proteins successfully detected the intact complex in the parasite and coimmunoprecipitated the three interacting partners. Importantly, full-length CyRPA antibodies displayed potent strain-transcending invasion inhibition, as observed for PfRH5. CyRPA does not bind with erythrocytes, suggesting that its parasite neutralizing antibodies likely block its critical interaction with PfRH5-PfRipr, leading to a blockade of erythrocyte invasion. Further, CyRPA and PfRH5 antibody combinations produced synergistic invasion inhibition, suggesting that simultaneous blockade of the PfRH5-Basigin and PfRH5/PfRipr/CyRPA interactions produced an enhanced inhibitory effect. Our discovery of the critical interactions between PfRH5, PfRipr, and the GPI-anchored CyRPA clearly defines the components of the essential PfRH5 adhesion complex for P. falciparum erythrocyte invasion and offers it as a previously unidentified potent target for antimalarial strategies that could abrogate formation of the crucial multiprotein complex.

Rational structure-guided design of a blood stage malaria vaccine immunogen presenting a single epitope from PfRH5.

There is an urgent need for improved malaria vaccine immunogens. Invasion of erythrocytes by Plasmodium falciparum is essential for its life cycle, preceding symptoms of disease and parasite transmission. Antibodies which target PfRH5 are highly effective at preventing erythrocyte invasion and the most potent growth-inhibitory antibodies bind a single epitope. Here we use structure-guided approaches to design a small synthetic immunogen, RH5-34EM which recapitulates this epitope. Structural biology and biophysics demonstrate that RH5-34EM is correctly folded and binds neutralising monoclonal antibodies with nanomolar affinity. In immunised rats, RH5-34EM induces PfRH5-targeting antibodies that inhibit parasite growth. While PfRH5-specific antibodies were induced at a lower concentration by RH5-34EM than by PfRH5, RH5-34EM induced antibodies that were a thousand-fold more growth-inhibitory as a factor of PfRH5-specific antibody concentration. Finally, we show that priming with RH5-34EM and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens.

A complex equation - adding to Plasmodium falciparum invasion.

The Plasmodium falciparum invasion complex - consisting of the prime blood-stage vaccine candidates PfRH5, PfCyRPA and PfRipr - is essential and conserved. New data from Scally et al. reveal that the complex consists of two additional proteins, adding important knowledge to the current understanding of the biology behind the invasion process.

Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes.

Basigin is an essential host receptor for invasion of Plasmodium falciparum into human erythrocytes, interacting with parasite surface protein PfRH5. PfRH5 is a leading blood-stage malaria vaccine candidate and a target of growth-inhibitory antibodies. Here, we show that erythrocyte basigin is exclusively found in one of two macromolecular complexes, bound either to plasma membrane Ca2+-ATPase 1/4 (PMCA1/4) or to monocarboxylate transporter 1 (MCT1). PfRH5 binds to each of these complexes with a higher affinity than to isolated basigin ectodomain, making it likely that these are the physiological targets of PfRH5. PMCA-mediated Ca2+ export is not affected by PfRH5, making it unlikely that this is the mechanism underlying changes in calcium flux at the interface between an erythrocyte and the invading parasite. However, our studies rationalise the function of the most effective growth-inhibitory antibodies targeting PfRH5. While these antibodies do not reduce the binding of PfRH5 to monomeric basigin, they do reduce its binding to basigin-PMCA and basigin-MCT complexes. This indicates that the most effective PfRH5-targeting antibodies inhibit growth by sterically blocking the essential interaction of PfRH5 with basigin in its physiological context.

Establishment of an Antiplasmodial Vaccine Based on PfRH5-Encoding RNA Replicons Stabilized by Cationic Liposomes.

BACKGROUND: Nucleic acid-based vaccines have been studied for the past four decades, but the approval of the first messenger RNA (mRNA) vaccines during the COVID-19 pandemic opened renewed perspectives for the development of similar vaccines against different infectious diseases. Presently available mRNA vaccines are based on non-replicative mRNA, which contains modified nucleosides encased in lipid vesicles, allowing for entry into the host cell cytoplasm, and reducing inflammatory reactions. An alternative immunization strategy employs self-amplifying mRNA (samRNA) derived from alphaviruses, but lacks viral structural genes. Once incorporated into ionizable lipid shells, these vaccines lead to enhanced gene expression, and lower mRNA doses are required to induce protective immune responses. In the present study, we tested a samRNA vaccine formulation based on the SP6 Venezuelan equine encephalitis (VEE) vector incorporated into cationic liposomes (dimethyldioctadecyl ammonium bromide and a cholesterol derivative). Three vaccines were generated that encoded two reporter genes (GFP and nanoLuc) and the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5). METHODS: Transfection assays were performed using Vero and HEK293T cells, and the mice were immunized via the intradermal route using a tattooing device. RESULTS: The liposome-replicon complexes showed high transfection efficiencies with in vitro cultured cells, whereas tattooing immunization with GFP-encoding replicons demonstrated gene expression in mouse skin up to 48 h after immunization. Mice immunized with liposomal PfRH5-encoding RNA replicons elicited antibodies that recognized the native protein expressed in P. falciparum schizont extracts, and inhibited the growth of the parasite in vitro. CONCLUSION: Intradermal delivery of cationic lipid-encapsulated samRNA constructs is a feasible approach for developing future malaria vaccines.

High genetic and haplotype diversity in vaccine candidate Pfceltos but not Pfrh5 among malaria-infected children in Ibadan, Nigeria.

Malaria remains a global public health challenge. The disease has a great impact in sub-Saharan Africa among children under five years of age and pregnant women. Malaria control programs targeting the parasite and mosquitoes vectors with combinational therapy and insecticide-treated bednets are becoming obsolete due to the phenomenon of resistance, which is a challenge for reducing morbidity and mortality. Malaria vaccines would be effective alternative to the problem of parasite and insecticide resistance, but focal reports of polymorphisms in malaria candidate antigens have made it difficult to design an effective malaria vaccine. Therefore, studies geared towards elucidating the polymorphic pattern and how genes targeted for vaccine design evolve are imperative. We have carried out molecular and genetic analysis of two genes encoding vaccine candidates-the Plasmodium falciparum cell traversal ookinetes and sporozoites (Pfceltos) and P. falciparum reticulocyte binding protein 5 (Pfrh5) in parasite isolates from malaria-infected children in Ibadan, Nigeria to evaluate their genetic diversity, relatedness and pattern of molecular evolution. Pfceltos and Pfrh5 genes were amplified from P. falciparum positive samples. Amplified fragments were purified and sequenced using the chain termination method. Post-sequence edit of fragments and application of various population genetic analyses was done. We observed a higher number of segregating sites and haplotypes in the Pfceltos than in Pfrh5 gene, the former also presenting higher haplotype (0.942) and nucleotide diversity (θ = 0.01219 and π = 0.01148). In contrast, a lower haplotype (0.426) and nucleotide diversity (θ = 0.00125; π = 0.00095) was observed in the Pfrh5 gene. Neutrality tests do not show deviation from neutral expectations for Pfceltos, with the circulation of multiple low frequency haplotypes (Tajima's D = -0.21637; Fu and Li's D = -0.08164; Fu and Li's F = -0.14051). Strong linkage disequilibrium was observed between variable sites, in each of the genes studied. We postulate that the high diversity and circulation of multiple haplotypes has the potential of making a Pfceltos-subunit vaccine ineffective, while the low genetic diversity of Pfrh5 gene substantiates its evolutionary conservation and potential as a malaria vaccine candidate.

Expanding the Malaria Antibody Toolkit: Development and Characterisation of Plasmodium falciparum RH5, CyRPA, and CSP Recombinant Human Monoclonal Antibodies.

Malaria, an infection caused by apicomplexan parasites of the genus Plasmodium, continues to exact a significant toll on public health with over 200 million cases world-wide, and annual deaths in excess of 600,000. Considerable progress has been made to reduce malaria burden in endemic countries in the last two decades. However, parasite and mosquito resistance to frontline chemotherapies and insecticides, respectively, highlights the continuing need for the development of safe and effective vaccines. Here we describe the development of recombinant human antibodies to three target proteins from Plasmodium falciparum: reticulocyte binding protein homologue 5 (PfRH5), cysteine-rich protective antigen (PfCyRPA), and circumsporozoite protein (PfCSP). All three proteins are key targets in the development of vaccines for blood-stage or pre-erythrocytic stage infections. We have developed potent anti-PfRH5, PfCyRPA and PfCSP monoclonal antibodies that will prove useful tools for the standardisation of assays in preclinical research and the assessment of these antigens in clinical trials. We have generated some very potent anti-PfRH5 and anti-PfCyRPA antibodies with some clones >200 times more potent than the polyclonal anti-AMA-1 antibodies used for the evaluation of blood stage antigens. While the monoclonal and polyclonal antibodies are not directly comparable, the data provide evidence that these new antibodies are very good at blocking invasion. These antibodies will therefore provide a valuable resource and have potential as biological standards to help harmonise pre-clinical malaria research.

APC-Targeted DNA Vaccination Against Reticulocyte-Binding Protein Homolog 5 Induces Plasmodium falciparum-Specific Neutralizing Antibodies and T Cell Responses.

Targeted delivery of antigen to antigen presenting cells (APCs) is an efficient way to induce robust antigen-specific immune responses. Here, we present a novel DNA vaccine that targets the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5), a leading blood-stage antigen of the human malaria pathogen, to APCs. The vaccine is designed as bivalent homodimers where each chain is composed of an amino-terminal single chain fragment variable (scFv) targeting unit specific for major histocompatibility complex class II (MHCII) expressed on APCs, and a carboxyl-terminal antigenic unit genetically linked by the dimerization unit. This vaccine format, named "Vaccibody", has previously been successfully applied for antigens from other infectious diseases including influenza and HIV, as well as for tumor antigens. Recently, the crystal structure and key functional antibody epitopes for the truncated version of PfRH5 (PfRH5ΔNL) were characterized, suggesting PfRH5ΔNL to be a promising candidate for next-generation PfRH5 vaccine design. In this study, we explored the APC-targeting strategy for a PfRH5ΔNL-containing DNA vaccine. BALB/c mice immunized with the targeted vaccine induced higher PfRH5-specific IgG1 antibody responses than those vaccinated with a non-targeted vaccine or antigen alone. The APC-targeted vaccine also efficiently induced rapid IFN-γ and IL-4 T cell responses. Furthermore, the vaccine-induced PfRH5-specific IgG showed inhibition of growth of the P. falciparum 3D7 clone parasite in vitro. Finally, sera obtained after vaccination with this targeted vaccine competed for the same epitopes as PfRH5-specific mAbs from vaccinated humans. Robust humoral responses were also induced by a similar P. vivax Duffy-binding protein (PvDBP)-containing targeted DNA vaccine. Our data highlight a novel targeted vaccine platform for the development of vaccines against blood-stage malaria.

Development of a process for large scale production of PfRH5 in E. coli expression system.

The Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) has recently shown great promise to be developed as a vaccine candidate to prevent blood-stage malaria. However, because of its molecular complexity, most previous efforts were focused on expressing PfRH5 in its native and soluble form. Here, we describe the E. coli expression of full-length PfRH5 as inclusion bodies (IBs), followed by its high cell density fermentation at 1, 5 and 30 L scale. Denatured full-length PfRH5 was purified using a two-step chromatography process before being refolded using design of experiments (DoE). Refolded PfRH5 was further purified using size exclusion chromatography (SEC), recovering high purity antigen with an overall yield of 102 mg/L from fermentation cell harvest. Purified PfRH5 was further characterized using orthogonal analytical methods, and a short-term stability study revealed -80 °C as an optimum storage temperature. Moreover, refolded, and purified PfRH5, when formulated with adjuvant Glucopyranosyl A lipid stable emulsion (GLA-SE), elicited high antibody titers in BALB/c mice, proving its potential to neutralize the blood-stage malarial parasite. Here, we establish an E. coli-based process platform for the large-scale cGMP production of full-length PfRH5, enabling global malaria vaccine development efforts.

High Level of Specific Anti-Plasmodium Falciparum Merozoite IgG1 Antibodies in Rural Asymptomatic Individuals of Dienga, South-Eastern Gabon.

Plasmodium falciparum merozoite antigens (PfMAgs) play an essential role in the development of immunity to malaria. Currently, P. falciparum: protein 113 (Pf 113), apical membrane antigen 1 (AMA1), erythrocyte binding antigens (EBA175), and reticulocyte binding protein homologue 5 (RH5) are among the most PfMAgs studied. A comparative analysis of naturally acquired antibodies against these antigens in children would increase our knowledge about the development of protective immunity. Analysis of antibodies to Pf113, PfAMA1, PfEBA175, and PfRH5 was conducted in rural population during 2013 and 2014. Both prevalence and levels of total IgG anti-PfAMA1 were higher than that of IgG anti-PfEBA175, anti-PfRH5, and anti-Pf113. Seroconversion to PfAMA1 and PfEBA175 occurred moderately in young children and reached to the maximum in adolescent and in adults. High prevalence of IgG anti-Pf113 was observed in young children of 3 to 6 years old in 2013. The four antigens were recognized by IgG 1, 2, 3, and 4 antibodies from a large proportion of the subjects, and all of them induced high levels of specific IgG1 against PfAMA1, PfEBA175, fewer by Pf113 and PfRH5. Many asymptomatic children had specific IgG1 recognizing multiple antigens, and these IgG1 antibodies could be associated with a reduced risk of developing malaria symptoms.

Comparative Antibody Responses Against three Antimalarial Vaccine Candidate Antigens from Urban and Rural Exposed Individuals in Gabon.

The analysis of immune responses in diverse malaria endemic regions provides more information to understand the host's immune response to Plasmodium falciparum. Several plasmodial antigens have been reported as targets of human immunity. PfAMA1 is one of most studied vaccine candidates; PfRH5 and Pf113 are new promising vaccine candidates. The aim of this study was to evaluate humoral response against these three antigens among children of Lastourville (rural area) and Franceville (urban area). Malaria was diagnosed using rapid diagnosis tests. Plasma samples were tested against these antigens by enzyme-linked immunosorbent assay (ELISA). We found that malaria prevalence was five times higher in the rural area than in the urban area (p < 0.0001). The anti-PfAMA1 and PfRh5 response levels were significantly higher in Lastourville than in Franceville (p < 0.0001; p = 0.005). The anti-AMA1 response was higher than the anti-Pf113 response, which in turn was higher than the anti-PfRh5 response in both sites. Anti-PfAMA1 levels were significantly higher in infected children than those in uninfected children (p = 0.001) in Franceville. Anti-Pf113 and anti-PfRh5 antibody levels were lowest in children presenting severe malarial anemia. These three antigens are targets of immunity in Gabon. Further studies on the role of Pf113 in antimalarial protection against severe anemia are needed.

PfRH5 as a candidate vaccine for Plasmodium falciparum malaria.

PfRH5 has recently emerged as a strong vaccine candidate for Plasmodium falciparum malaria. Antibodies inhibit invasion of erythrocytes by merozoites and blood-stage replication, and PfRH5 is a significant target of acquired human immunity. Recent studies have shown protective efficacy of PfRH5 vaccines in a non-human primate model of malaria.

Liposomes loaded with P. falciparum merozoite-derived proteins are highly immunogenic and produce invasion-inhibiting and anti-toxin antibodies.

The formulation of an effective vaccine against malaria is still a significant challenge and the induction of high anti-parasite antibody titers plus a sustained T cell response is mandatory for the success of such a vaccine. We have developed a nanoliposome-based structure which contains plasma membrane-associated proteins (PfMNP) of Plasmodium falciparum merozoites on its surface. Incorporation of parasite-derived proteins led to a significant increase in the size and dispersity of particles. Immunization of particles in BalbC and C57BL/6 mice led to high anti-MSP119 IgG titers (10(4)) after the first dose and reached a plateau (>10(6)) after the third dose. While very high titers were observed against the C-terminal domain of the vaccine candidate MSP1, only modest titers (≤10(3)) were detected against MSP2. The induced antibodies showed also a strong growth-inhibiting effect in reinvasion assays. In addition, PfMNP immunization generated antibodies which partially blocked the inflammatory response, probably by blocking TLR-induced activation of macrophages by malarial toxins such as GPI anchors. The results underline the potential of nanoliposome-based formulations as anti-malarial vaccines.

Erratum: Dangerous liaisons: molecular basis for a syndemic relationship between Kaposi's sarcoma and P. falciparum malaria.

[This corrects the article on p. 35 in vol. 4, PMID: 23487416.].

Dangerous liaisons: molecular basis for a syndemic relationship between Kaposi's sarcoma and P. falciparum malaria.

The most severe manifestations of malaria (caused by Plasmodium falciparum) occur as a direct result of parasitemia following invasion of erythrocytes by post-liver blood-stage merozoites, and during subsequent cyto-adherence of infected erythrocytes to the vascular endothelium. However, the disproportionate epidemiologic clustering of severe malaria with aggressive forms of endemic diseases such as Kaposi's sarcoma (KS), a neoplasm that is etiologically linked to infection with KS-associated herpesvirus (KSHV), underscores the significance of previously unexplored co-pathogenetic interactions that have the potential to modify the overall disease burden in co-infected individuals. Based on recent studies of the mechanisms that P. falciparum and KSHV have evolved to interact with their mutual human host, several new perspectives are emerging that highlight a surprising convergence of biological themes potentially underlying their associated co-morbidities. Against this background, ongoing studies are rapidly constructing a fascinating new paradigm in which the major host receptors that control parasite invasion (Basigin/CD147) and cyto-adherence (CD36) are, surprisingly, also important targets for exploitation by KSHV. In this article, we consider the major pathobiological implications of the co-option of Basigin/CD147 and CD36 signaling pathways by both P. falciparum and KSHV, not only as essential host factors for parasite persistence but also as important mediators of the pro-angiogenic phenotype within the virus-infected endothelial microenvironment. Consequently, the triangulation of interactions between P. falciparum, KSHV, and their mutual human host articulates a syndemic relationship that points to a conceptual framework for prevalence of aggressive forms of KS in malaria-endemic areas, with implications for the possibility of dual-use therapies against these debilitating infections in resource-limited parts of the world.