Malaria caused by the Plasmodium vivax parasite is a major global health burden. Immunity against blood-stage infection reduces parasitemia and disease severity. Duffy-binding protein (DBP) is the primary parasite protein responsible for the invasion of red blood cells and it is a leading subunit vaccine candidate. An effective vaccine, however, is still lacking despite decades of interest in DBP as a vaccine candidate. This review discusses the reasons for targeting DBP, the challenges associated with developing a vaccine, and modern structural vaccinology methods that could be used to create an effective DBP vaccine. Next-generation DBP vaccines have the potential to elicit a broadly protective immune response and provide durable and potent protection from P. vivax malaria.

Genetically-growth-attenuated blood-stage parasites were generated inPlasmodium falciparumby targeted deletion of NT1 (Nucleoside Transporter-1) gene, and Pfnt1(-) parasites only grew after providing the culture with supra-physiological concentrations of purines. Genetically-attenuatedP. yoeliint1(-)parasites induced sterile-protection against homologous blood-stage infectious challenge after immunization with single subpatent doses, which remained subpatent even in immune-compromised mice. Here, we showed that immunizations with frozen-stocks of equally-mixedP. bergheiandP. yoelii nt1(-)parasites in single subcutaneous doses, which did not lead to patent blood-stage infection, conferred sterile protection against intravenous infectious blood-stage challenge with wild-type parasites ofP. bergheiANKA andP. yoelii17X-NL strains. This data highlights the possibility that a single subcutaneous sub-patent dose of two species of genetically-growth-attenuated parasites, which can protect humans against twoPlasmodiumspp. infections, could be developed in cultures provided with supra-physiological concentrations of purines, and shipped to endemic areas as frozen-stock doses.

The highly conserved Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is a key target for next-generation vaccines against blood-stage malaria. PfCyRPA constitute the core of a ternary complex, including the reticulocyte binding-like homologous protein 5 (PfRh5) and the Rh5-interacting protein (PfRipr), and is fundamental for merozoite invasion of erythrocytes. In this study, we show that monoclonal antibodies (mAbs) specific to PfCyRPA neutralize the in vitro growth of Ghanaian field isolates as well as numerous laboratory-adapted parasite lines. We identified subsets of mAbs with neutralizing activity that bind to distinct sites on PfCyRPA and that in combination potentiate the neutralizing effect. As antibody responses against multiple merozoite invasion proteins are thought to improve the efficacy of blood-stage vaccines, we also demonstrated that combinations of PfCyRPA- and PfRh5 specific mAbs act synergistically to neutralize parasite growth. Yet, we identified prominent strain-dependent neutralization potencies, which our results suggest is independent of PfCyRPA expression level and polymorphism, demonstrating the importance of addressing functional converseness when evaluating blood-stage vaccine candidates. Finally, our results suggest that blood-stage vaccine efficacy can be improved by directing the antibody response towards defined protective epitopes on multiple parasite antigens.

During intraerythrocytic development Plasmodium falciparum deploys numerous proteins to support erythrocyte invasion, intracellular growth and development, as well as host immune evasion. Since these proteins are key for parasite intraerythrocytic survival and propagation, they represent attractive targets for antimalarial vaccines. In this study we sought to characterize a member of the PHISTc family of proteins, PF3D7_0801000, as a potential vaccine target. Using the wheat germ cell-free system we expressed the N-terminal region of PF3D7_0801000 (G93-L494, PF3D7_0801000N) and generated specific immune sera. We observed that PF3D7_0801000 localizes in merozoites, and antibodies against PF3D7_0801000N modestly inhibit P. falciparum parasite growth in in vitro culture. Sliding window analysis of the coding sequence revealed that pf3d7_0801000n is relatively conserved among African parasite isolates. Antibody profiles in a malaria-exposed Ugandan population revealed that PF3D7_0801000N is strongly immunoreactive with antibody acquisition increasing with age. Taken together, these findings suggest the need for further evaluation of PF3D7_0801000 for its role in merozoite invasion and utility as an asexual blood-stage vaccine candidate antigen.

Clinical manifestation of malaria is mainly due to intra-erythrocytic development of Plasmodium parasites. Plasmodium falciparum merozoites, the invasive form of the blood-stage parasite, invade human erythrocytes in a complex but rapid process. This multi-step progression involves interactions between parasite and human host proteins. Here we show that antibodies against a vaccine antigen, PfGAMA, co-immunoprecipitate with PfMSP10. This interaction was validated as direct by surface plasmon resonance analysis. We then demonstrate that antibodies against PfMSP10 have growth inhibitory activity against cultured parasites, with the region PfMSP10 R1 that is critical for its interaction with PfGAMA being the key target. We also observe that the PfMSP10 R1 region is highly conserved among African field isolates. Lastly, we show that high levels of antibodies against PfMSP10 R1 associate with reduced risk to clinical malaria in children resident in a malaria endemic region in northern Uganda. Put together, these findings provide for the first time the functional context of the important role of PfGAMA/PfMSP10 interaction in erythrocyte invasion and unveil a novel asexual blood-stage malaria vaccine target for attenuating P. falciparum merozoite invasion.

Merozoite surface proteins (MSPs) are considered as promising blood-stage malaria vaccine candidates. MSP3 has long been evaluated for its vaccine candidacy, however, the candidacy of other members of MSP3 family is insufficiently characterized. Here, we investigated Plasmodium falciparum MSP11 (PF3D7_1036000), a member of the MSP3 family, for its potential as a blood-stage vaccine candidate. The full-length protein (MSP11-FL) as well as the N-terminal half-MSP11 (MSP11-N), known to be unique among the MSP3 family members, were expressed by wheat germ cell-free system, and used to raise antibodies in rabbit. Immunoblot analysis of schizont lysates probed with anti-MSP11-N antibodies detected double bands at approximately 40 and 60 kDa, consistent with the previous report thus confirming antibodies specificity. However, inconsistent with previously reported merozoite\'s surface localization, immunofluorescence assay (IFA) revealed that MSP11 likely localizes to rhoptry neck of merozoites in mature schizonts. After invasion, MSP11 localized to parasitophorous vacuole and thereafter in Maurer\'s clefts in trophozoites. Anti-MSP11-FL antibody levels were significantly higher in asymptomatic than symptomatic P. falciparum cases in malaria low endemic Thailand. This reconfirmed that anti-MSP11 antibodies play an important role in protection against clinical malaria, as previously reported. Furthermore, in vitro growth inhibition assay revealed that anti-MSP11-FL rabbit antibodies biologically function by inhibiting merozoite invasion of erythrocytes. These findings further support the vaccine candidacy of MSP11.

Proteins coating Plasmodium merozoite surface and secreted from its apical organelles are considered as promising vaccine candidates for blood-stage malaria. The rhoptry neck protein 12 of Plasmodium falciparum (PfRON12) was recently reported as a protein specifically expressed in schizonts and localized to the rhoptry neck of merozoites. Here, we assessed its potential as a vaccine candidate. We expressed a recombinant PfRON12 protein by a wheat germ cell-free system to obtain anti-PfRON12 antibody. Immunoblot analysis of schizont lysates detected a single band at approximately 40 kDa under reducing conditions, consistent with the predicted molecular weight. Additionally, anti-PfRON12 antibody recognized a single band around 80 kDa under non-reducing conditions, suggesting native PfRON12 forms a disulfide-bond-mediated multimer. Immunofluorescence assay and immunoelectron microscopy revealed that PfRON12 localized to the rhoptry neck of merozoites in schizonts and to the surface of free merozoites. The biological activity of anti-PfRON12 antibody was tested by in vitro growth inhibition assay (GIA), and the rabbit antibodies significantly inhibited merozoite invasion of erythrocytes. We then investigated whether PfRON12 is immunogenic in P. falciparum-infected individuals. The sera from P. falciparum infected individuals in Thailand and Mali reacted with the recombinant PfRON12. Furthermore, human anti-PfRON12 antibodies affinity-purified from Malian serum samples inhibited merozoite invasion of erythrocytes in vitro. Moreover, pfron12 is highly conserved with only 4 non-synonymous mutations in the coding sequence from approximately 200 isolates deposited in PlasmoDB. These results suggest that PfRON12 might be a potential blood-stage vaccine candidate antigen against P. falciparum.

The target molecules of antibodies against falciparum malaria remain largely unknown. Recently we have identified multiple proteins as targets of immunity against Plasmodium falciparum using African serum samples. To investigate whether potential targets of clinical immunity differ with transmission intensity, we assessed immune responses in residents of low malaria transmission region in Thailand. Malaria asymptomatic volunteers (Asy: n=19) and symptomatic patients (Sym: n=21) were enrolled into the study. Serum immunoreactivity to 186 wheat germ cell-free system (WGCFS)-synthesized recombinant P. falciparum asexual-blood stage proteins were determined by AlphaScreen, and subsequently compared between the study groups. Forty proteins were determined as immunoreactive with antibody responses to 35 proteins being higher in Asy group than in Sym group. Among the 35 proteins, antibodies to MSP3, MSPDBL1, RH2b, and MSP7 were significantly higher in Asy than Sym (unadjusted p<0.005) suggesting these antigens may have a protective role in clinical malaria. MSP3 reactivity remained significantly different between Asy and Sym groups even after multiple comparison adjustments (adjusted p=0.033). Interestingly, while our two preceding studies using African sera were conducted differently (e.g., cross-sectional vs. longitudinal design, observed clinical manifestation vs. functional activity), those studies similarly identified MSP3 and MSPDBL1 as potential targets of protective immunity. This study further provides a strong rationale for the application of WGCFS-based immunoprofiling to malaria vaccine candidate and biomarker discovery even in low or reduced malaria transmission settings.

Having the ability to rapidly, accurately, and robustly measure Plasmodium falciparum merozoite invasion is a critical component in effective assessment of a blood stage vaccine\'s mechanism of action. Being able to measure invasion of erythrocytes accurately, objectively and in a high throughput fashion is of critical importance. Here, we describe a simple and robust flow cytometry method that allows for the measurement of the key invasion parameters of parasite multiplication rate and erythrocyte selectivity-both important determinants of disease severity-from the schizont to the ring stage of the parasite\'s life-cycle, thus separating invasion from growth of the parasite. Importantly, this method is able to accurately detect low levels of parasitemia and heterogeneity within the population that can be missed by enzymatic methods. Lastly, this method has been successfully adapted and employed in field based research settings for parasitemia measurements in vivo, ex vivo, and in vitro and to measure invasion inhibition by antibodies and the use of alternative pathways for invasion.