Inhibit Merozoite Invasion Research Articles

Inhibition of Plasmodium knowlesi merozoite invasion into human erythrocytes by antibodies raised against the parasite's secreted protein with altered thrombospondin repeat (SPATR).

The Plasmodium secreted protein with an altered thrombospondin repeat (SPATR) has been known to play an important role in the malaria parasite's invasion into host erythrocytes. This protein is immunogenic and has been considered as one of the potential vaccine candidates against malaria parasite infection. Thus far, only a handful immunological studies have been carried out on P. knowlesi SPATR (PkSPATR), and none of these studies investigated the immunoprotective properties of the protein. In the present study, the ability of anti-PkSPATR antibodies to inhibit invasion of human erythrocytes was assessed in an in vitro merozoite invasion inhibition assay. The antibodies were harvested from the serum of a rabbit which was immunised with recombinat PkSPATR. Results from the merozoite invasion inhibition assay revealed significant antibody invasion inhibitory activity in a concentration dependent manner (concentration range: 0.375 - 3.00 mg/ml) with inhibition rate ranging from 20% to 32%. Future studies, such as anti-PkSPATR antibodies inhibitory effect on sporozoite invasion of human liver cells, need to be carried out to assess the potential of PkSPATR as a knowlesi malaria vaccine candidate.

Characterisation of PfCZIF1 and PfCZIF2 in Plasmodium falciparum asexual stages

Plasmodium falciparum exerts strong temporal control of gene expression across its lifecycle. Proteins expressed exclusively during late schizogony of blood stages, for example, often have a role in facilitating merozoite invasion of the host red blood cell (RBC), through merozoite development, egress, invasion or early establishment of infection in the RBC. Here, we characterise P. falciparum C3H1 zinc finger 1 (PfCZIF1, Pf3D7_1468400) and P. falciparum C3H1 zinc finger 2 (PfCZIF2, Pf3D7_0818100) which we identified as the only C3H1-type zinc finger proteins with peak expression at schizogony. Previous studies reported that antibodies against PfCZIF1 inhibit merozoite invasion, suggesting this protein may have a potential role during RBC invasion. We show using C-terminal truncations and gene knockouts of each of Pfczif1 and Pfczif2 that neither are essential for blood stage growth. However, they could not both be knocked out simultaneously, suggesting that at least one is needed for parasite growth in vitro. Immunofluorescence localisation of PfCZIF1 and PfCZIF2 indicated that both proteins occur in discrete foci on the periphery of the parasite’s cytosol and biochemical assays suggest they are peripherally associated to a membrane. Transcriptomic analyses for the C-terminal truncation mutants reveal no significant expression perturbations with PfCZIF1 truncation. However, modification of PfCZIF2 appears to modify the expression for some exported proteins including PfKAHRP. This study does not support a role for PfCZIF1 or PfCZIF2 in merozoite invasion of the RBC and suggests that these proteins may help regulate the expression of proteins exported into the RBC cytosol after merozoite invasion.

Plasmodium knowlesi Duffy binding protein alpha region II (PkDBPαII) in clinical isolates from Peninsular Malaysia and Malaysian Borneo exhibit different immune responses in animal models

Plasmodium knowlesi utilizes the Duffy binding protein alpha (PkDBPα) to facilitate its invasion into human erythrocytes. PkDBPα region II (PkDBPαII) from Peninsular Malaysia and Malaysian Borneo has been shown to occur as distinct haplotypes, and the predominant haplotypes from these geographical areas demonstrated differences in binding activity to human erythrocytes in erythrocyte binding assays. This study aimed to determine the effects of genetic polymorphisms in PkDBPαII to immune responses in animal models. The recombinant PkDBPαII (~ 45kDa) of Peninsular Malaysia (PkDBPαII-H) and Malaysian Borneo (PkDBPαII-S) were expressed in a bacterial expression system, purified, and used in mice and rabbit immunization. The profile of cytokines IL-1ra, IL-2, IL-6, IL-10, TNF-α, and IFN-γ in immunized mice spleen was determined via ELISA. The titer and IgG subtype distribution of raised antibodies was characterized. Immunized rabbit sera were purified and used to perform an in vitro merozoite invasion inhibition assay. The PkDBPαII-immunized mice sera of both groups showed high antibody titer and a similar IgG subtype distribution pattern: IgG2b > IgG1 > IgG2a > IgG3. The PkDBPαII-H group was shown to have higher IL-1ra (P = 0.141) and IL-6 (P = 0.049) concentrations, with IL-6 levels significantly higher than that of the PkDBPαII-S group (P ≤ 0.05). Merozoite invasion inhibition assay using purified anti-PkDBPαII antibodies showed a significantly higher inhibition rate in the PkDBPαII-H group than the PkDBPαII-S group (P ≤ 0.05). Besides, anti-PkDBPαII-H antibodies were able to exhibit inhibition activity at a lower concentration than anti-PkDBPαII-S antibodies. PkDBPαII was shown to be immunogenic, and the PkDBPαII haplotype from Peninsular Malaysia exhibited higher responses in cytokines IL-1ra and IL-6, antibody IgM level, and merozoite invasion inhibition assay than the Malaysian Borneo haplotype. This suggests that polymorphisms in the PkDBPαII affect the level of immune responses in the host.

Dynamic Palmitoylation of Red Cell Membrane Proteins Governs Susceptibility to Invasion by the Malaria Parasite, Plasmodium falciparum.

Phosphorylation and other post-translational modifications of red blood cell (RBC) proteins govern membrane function and have a role in the invasion of RBCs by the malaria parasite, Plasmodium falciparum. Furthermore, a percentage of RBC proteins are palmitoylated, although the functional consequences are unknown. We establish dynamic palmitoylation of 118 RBC membrane proteins using click chemistry and acyl biotin exchange (ABE)-coupled LC-MS/MS and characterize their involvement in controlling membrane organization and parasite invasion. RBCs were treated with a generic palmitoylation inhibitor, 2-bromopalmitate (2-BMP), and then analyzed using ABE-coupled LC-MS/MS. Only 42 of the 118 palmitoylated proteins detected were palmitoylated in the 2-BMP-treated sample, indicating that palmitoylation is dynamically regulated. Interestingly, membrane receptors such as semaphorin 7A, CR1, and ABCB6, which are known to be involved in merozoite interaction with RBCs and parasite invasion, were found to be dynamically palmitoylated, including the blood group antigen, Kell, whose antigenic abundance was significantly reduced following 2-BMP treatment. To investigate the involvement of Kell in merozoite invasion of RBCs, a specific antibody to its extracellular domain was used. The antibody targeting Kell inhibited merozoite invasion of RBCs by 50%, implying a role of Kell, a dynamically palmitoylated potent host-derived receptor, in parasite invasion. Furthermore, a significant reduction in merozoite contact with the RBC membrane and a consequent decrease in parasite invasion following 2-BMP treatment demonstrated that palmitoylation does indeed regulate RBC susceptibility to parasite invasion. Taken together, our findings revealed the dynamic palmitoylome of RBC membrane proteins and its role in P. falciparum invasion.

Potent Virustatic Polymer-Lipid Nanomimics Block Viral Entry and Inhibit Malaria Parasites In Vivo.

Infectious diseases continue to pose a substantial burden on global populations, requiring innovative broad-spectrum prophylactic and treatment alternatives. Here, we have designed modular synthetic polymer nanoparticles that mimic functional components of host cell membranes, yielding multivalent nanomimics that act by directly binding to varied pathogens. Nanomimic blood circulation time was prolonged by reformulating polymer–lipid hybrids. Femtomolar concentrations of the polymer nanomimics were sufficient to inhibit herpes simplex virus type 2 (HSV-2) entry into epithelial cells, while higher doses were needed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given their observed virustatic mode of action, the nanomimics were also tested with malaria parasite blood-stage merozoites, which lose their invasive capacity after a few minutes. Efficient inhibition of merozoite invasion of red blood cells was demonstrated both in vitro and in vivo using a preclinical rodent malaria model. We envision these nanomimics forming an adaptable platform for developing pathogen entry inhibitors and as immunomodulators, wherein nanomimic-inhibited pathogens can be secondarily targeted to sites of immune recognition.

Enhancing neutralization of Plasmodium falciparum using a novel monoclonal antibody against the rhoptry-associated membrane antigen

The pathogenesis of malaria is associated with blood-stage infection and there is strong evidence that antibodies specific to parasite blood-stage antigens can control parasitemia. This provides a strong rational for applying blood-stage antigen components in a multivalent vaccine, as the induced antibodies in combination can enhance protection. The Plasmodium falciparum rhoptry-associated membrane antigen (PfRAMA) is a promising vaccine target, due to its fundamental role in merozoite invasion and low level of polymorphism. Polyclonal antibodies against PfRAMA are able to inhibit P. falciparum growth and interact synergistically when combined with antibodies against P. falciparum reticulocyte-binding protein 5 (PfRh5) or cysteine-rich protective antigen (PfCyRPA). In this study, we identified a novel PfRAMA-specific mAb with neutralizing activity, which in combination with PfRh5- or PfCyRPA-specific mAbs potentiated the neutralizing effect. By applying phage display technology, we mapped the protective epitope to be in the C-terminal region of PfRAMA. Our results confirmed previous finding of synergy between PfRAMA-, PfRh5- and PfCyRPA-specific antibodies, thereby paving the way of testing these antigens (or fragments of these antigens) in combination to improve the efficacy of blood-stage malaria vaccines. The results emphasize the importance of directing antibody responses towards protective epitopes, as the majority of anti-PfRAMA mAbs were unable to inhibit merozoite invasion of erythrocytes.

Evaluation of X-ray ionizing radiation on Plasmodium berghei invasion of erythrocytes

Developing new strategies for designing effective vaccines has become a priority for parasitologists worldwide. There is high interest in designing a vaccine against malaria since it is considered one of the most prevalent infectious diseases in the tropics. We evaluated the effects of X-rays irradiation on the erythrocytic stage of Plasmodium berghei ANKA merozoites and schizonts using doses of ionizing radiation ranging between 10 and 300 Gy on parasitized red blood cells (PRBC) to study the attenuating effects of radiation on the merozoites. Parasitic activity diminution was observed starting at 50 Gy, and the dose for complete attenuation was established at 200 Gy, corresponding with a 100% survival rate of mice. In vivo invasion experiments and immunofluorescence assays (IFA) showed inhibition of merozoite invasion of the host red blood cells (RBC). Nonetheless, immunization with irradiated parasitized red blood cells (IPRBC) was ineffective in protective assays. We perform cytoadherence and inhibition of cytoadhesion assays on irradiated merozoites. The results showed that high irradiation doses caused an unspecific cellular adhesion phenomenon independent of the ICAM-1 and CD36 interaction, which was determined by Cytoadhesion assays. Our results show that, even though X-ray irradiation is an effective method to induce complete parasite attenuation, it might affect the parasite's membrane surface structures triggering unspecific adhesion.

Genetic Diversity Analysis of Surface-Related Antigen (SRA) in Plasmodium falciparum Imported From Africa to China

Plasmodium falciparum surface-related antigen (SRA) is located on the surfaces of gametocyte and merozoite and has the structural and functional characteristics of potential targets for multistage vaccine development. However, little information is available regarding the genetic polymorphism of pfsra. To determine the extent of genetic variation about P. falciparum by characterizing the sra sequence, 74 P. falciparum samples were collected from migrant workers who returned to China from 12 countries of Africa between 2015 and 2019. The full length of the sra gene was amplified and sequenced. The average pairwise nucleotide diversities (π) of P. falciparum sra gene was 0.00132, and the haplotype diversity (Hd) was 0.770. The average number of nucleotide differences (k) for pfsra was 3.049. The ratio of non-synonymous (dN) to synonymous (dS) substitutions across sites (dN/dS) was 1.365. Amino acid substitutions of P. falciparum SRA could be categorized into 35 unique amino acid variants. Neutrality tests showed that the polymorphism of PfSRA was maintained by positive diversifying selection, which indicated its role as a potential target of protective immune responses and a vaccine candidate. Overall, the ability of the N-terminal of PfSRA antibodies to evoke inhibition of merozoite invasion of erythrocytes and conserved amino acid at low genetic diversity suggest that the N-terminal of PfSRA could be evaluated as a vaccine candidate against P. falciparum infection.

Localization and function of a Plasmodium falciparum protein (PF3D7_1459400) during erythrocyte invasion

Plasmodium falciparum malaria is a global health problem. Erythrocyte invasion by P. falciparum merozoites appears to be a promising target to curb malaria. We have identified and characterized a novel protein that is involved in erythrocyte invasion. Our data on protein subcellular localization, stage-specific protein expression pattern, and merozoite invasion inhibition by α-peptide antibodies suggest a role for PF3D7_1459400 protein during P. falciparum erythrocyte invasion. Even more, the human immunoepidemiology data present PF3D7_1459400 protein as an immunogenic antigen which could be further exploited for the development of new anti-infective therapy against malaria.

Antibodies against a short region of PfRipr inhibit Plasmodium falciparum merozoite invasion and PfRipr interaction with Rh5 and SEMA7A

Plasmodium falciparum merozoite invasion into erythrocytes is an essential step of the blood-stage cycle, survival of parasites, and malaria pathogenesis. P. falciparum merozoite Rh5 interacting protein (PfRipr) forms a complex with Rh5 and CyRPA in sequential molecular events leading to erythrocyte invasion. Recently we described PfRipr as a conserved protein that induces strain-transcending growth inhibitory antibodies in in vitro assays. However, being a large and complex protein of 1086 amino acids (aa) with 87 cysteine residues, PfRipr is difficult to express in conventional expression systems towards vaccine development. In this study we sought to identify the most potent region of PfRipr that could be developed to overcome difficulties related to protein expression, as well as to elucidate the invasion inhibitory mechanism of anti-PfRipr antibodies. Using the wheat germ cell-free system, Ecto- PfRipr and truncates of approximately 200 aa were expressed as soluble proteins. We demonstrate that antibodies against PfRipr truncate 5 (PfRipr_5: C720-D934), a region within the PfRipr C-terminal EGF-like domains, potently inhibit merozoite invasion. Furthermore, the antibodies strongly block PfRipr/Rh5 interaction, as well as that between PfRipr and its erythrocyte-surface receptor, SEMA7A. Taken together, PfRipr_5 is a potential candidate for further development as a blood-stage malaria vaccine.

Preliminary Evaluation of the Safety and Immunogenicity of an Antimalarial Vaccine Candidate Modified Peptide (IMPIPS) Mixture in a Murine Model.

Malaria continues being a high-impact disease regarding public health worldwide; the WHO report for malaria in 2018 estimated that ~219 million cases occurred in 2017, mostly caused by the parasite Plasmodium falciparum. The disease cost the lives of more than 400,000 people, mainly in Africa. In spite of great efforts aimed at developing better prevention (i.e., a highly effective vaccine), diagnosis, and treatment methods for malaria, no efficient solution to this disease has been advanced to date. The Fundación Instituto de Inmunología de Colombia (FIDIC) has been developing studies aimed at furthering the search for vaccine candidates for controlling P. falciparum malaria. However, vaccine development involves safety and immunogenicity studies regarding their formulation in animal models before proceeding to clinical studies. The present work has thus been aimed at evaluating the safety and immunogenicity of a mixture of 23 chemically synthesised, modified peptides (immune protection-inducing protein structure (IMPIPS)) derived from different P. falciparum proteins. Single and repeat dose assays were thus used with male and female BALB/c mice which were immunised with the IMPIPS mixture. It was found that single and repeat dose immunisation with the IMPIPS mixture was safe, both locally and systemically. It was observed that the antibodies so stimulated recognised the parasite's native proteins and inhibited merozoite invasion of red blood cells in vitro when evaluating the humoral immune response induced by the IMPIPS mixture. Such results suggested that the IMPIPS peptide mixture could be a safe candidate to be tested during the next stage involved in developing an antimalarial vaccine, evaluating local safety, immunogenicity, and protection in a nonhuman primate model.

Evaluation of Plasmodium falciparum MSP10 and its development as a serological tool for the Peruvian Amazon region

BackgroundDifferent antigens are needed to characterize Plasmodium falciparum infection in terms of seroreactivity and targets for invasion inhibition, in order to guide and identify the proper use of such proteins as tools for the development of serological markers and/or as vaccine candidates.MethodsIgG responses in 84 serum samples from individuals with P. falciparum infection [classified as symptomatic (Sym) or asymptomatic (Asym)], or acute Plasmodium vivax infection, from the Peruvian Amazon region, were evaluated by enzyme-linked immunosorbent assays specific for a baculovirus-produced recombinant protein P. falciparum Merozoite Surface Protein 10 (rMSP10) and for non-EGF region selected peptides of PfMSP10 selected by a bioinformatics tool (PfMSP10-1, PfMSP10-2 and PfMSP10-3). Monoclonal antibodies against the selected peptides were evaluated by western blotting, confocal microscopy and inhibition invasion assays.ResultsSeroreactivity analysis of the P. falciparum Sym- and Asym-infected individuals against rMSP10 showed a higher response as compared to the individuals with P. vivax acute infection. IgG responses against peptide PfMSP10-1 were weak. Interestingly high IgG response was found against peptide PfMSP10-2 and the combination of peptides PfMSP10-1 + PfMSP10-2. Monoclonal antibodies were capable of detecting native PfMSP10 on purified schizonts by western blot and confocal microscopy. A low percentage of inhibition of merozoite invasion of erythrocytes in vitro was observed when the monoclonal antibodies were compared with the control antibody against AMA-1 antigen. Further studies are needed to evaluate the role of PfMSP10 in the merozoite invasion.ConclusionsThe rMSP10 and the PfMSP10-2 peptide synthesized for this study may be useful antigens for evaluation of P. falciparum malaria exposure in Sym and Asym individuals from the Peruvian Amazon region. Moreover, these antigens can be used for further investigation of the role of this protein in other malaria-endemic areas.

RON2, a novel gene in Babesia bigemina, contains conserved, immunodominant B-cell epitopes that induce antibodies that block merozoite invasion

Bovine babesiosis is the most important protozoan disease transmitted by ticks. In Plasmodium falciparum, another Apicomplexa protozoan, the interaction of rhoptry neck protein 2 (RON2) with apical membrane antigen-1 (AMA-1) has been described to have a key role in the invasion process. To date, RON2 has not been described in Babesia bigemina, the causal agent of bovine babesiosis in the Americas. In this work, we found a ron2 gene in the B. bigemina genome. RON2 encodes a protein that is 1351 amino acids long, has an identity of 64% (98% coverage) with RON2 of B. bovis and contains the CLAG domain, a conserved domain in Apicomplexa. B. bigemina ron2 is a single copy gene and it is transcribed and expressed in blood stages as determined by RT-PCR, Western blot, and confocal microscopy. Serum samples from B. bigemina-infected bovines were screened for the presence of RON2-specific antibodies, showing the recognition of conserved B-cell epitopes. Importantly, in vitro neutralization assays showed an inhibitory effect of RON2-specific antibodies on the red blood cell invasion by B. bigemina. Therefore, RON2 is a novel antigen in B. bigemina and contains conserved B-cell epitopes, which induce antibodies that inhibit merozoite invasion.

Anti-MSP11 IgG inhibits Plasmodium falciparum merozoite invasion into erythrocytes in vitro

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.

Antibodies against a Plasmodium falciparum RON12 inhibit merozoite invasion into erythrocytes

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.

SURGE complex of Plasmodium falciparum in the rhoptry-neck (SURFIN4.2-RON4-GLURP) contributes to merozoite invasion.

Plasmodium falciparum invasion into red blood cells (RBCs) is a complex process engaging proteins on the merozoite surface and those contained and sequentially released from the apical organelles (micronemes and rhoptries). Fundamental to invasion is the formation of a moving junction (MJ), a region of close apposition of the merozoite and the RBC plasma membranes, through which the merozoite draws itself before settling into a newly formed parasitophorous vacuole (PV). SURFIN4.2 was identified at the surface of the parasitized RBCs (pRBCs) but was also found apically associated with the merozoite. Using antibodies against the N-terminus of the protein we show the presence of SURFIN4.2 in the neck of the rhoptries, its secretion into the PV and shedding into the culture supernatant upon schizont rupture. Using immunoprecipitation followed by mass spectrometry we describe here a novel protein complex we have named SURGE where SURFIN4.2 forms interacts with the rhoptry neck protein 4 (RON4) and the Glutamate Rich Protein (GLURP). The N-terminal cysteine-rich–domain (CRD) of SURFIN4.2 mediates binding to the RBC membrane and its interaction with RON4 suggests its involvement in the contact between the merozoite apex and the RBC at the MJ. Supporting this suggestion, we also found that polyclonal antibodies to the extracellular domain (including the CRD) of SURFIN4.2 partially inhibit merozoite invasion. We propose that the formation of the SURGE complex participates in the establishment of parasite infection within the PV and the RBCs.

Protein-protein interaction studies reveal the Plasmodium falciparum merozoite surface protein-1 region involved in a complex formation that binds to human erythrocytes.

Plasmodium falciparum merozoite surface protein (PfMSP) 1 has been studied extensively as a vaccine candidate antigen. PfMSP-1 undergoes proteolytic processing into four major products, such as p83, p30, p38, and p42, that are associated in the form of non-covalent complex(s) with other MSPs. To delineate MSP1 regions involved in the interaction with other MSPs, here we expressed recombinant proteins (PfMSP-165) encompassing part of p38 and p42 regions and PfMSP-119 PfMSP-165 interacted strongly with PfMSP-3, PfMSP-6, PfMSP-7, and PfMSP-9, whereas PfMSP-119 did not interact with any of these proteins. Since MSP-1 complex binds human erythrocytes, we examined the ability of these proteins to bind human erythrocyte. Among the proteins of MSP-1 complex, PfMSP-6 and PfMSP-9 bound to human erythrocytes. Serological studies showed that PfMSP-165 was frequently recognized by sera from malaria endemic regions, whereas this was not the case for PfMSP-119 In contrast, antibodies against PfMSP-119 showed much higher inhibition of merozoite invasion compared with antibodies against the larger PfMSP-165 fragment. Importantly, anti-PfMSP-119 antibodies recognized both recombinant proteins, PfMSP-119 and PfMSP-165; however, anti-PfMSP-165 antibody failed to recognize the PfMSP-119 protein. Taken together, these results demonstrate that PfMSP-1 sequences upstream of the 19 kDa C-terminal region are involved in molecular interactions with other MSPs, and these sequences may probably serve as a smoke screen to evade antibody response to the membrane-bound C-terminal 19 kDa region.

Plasmodium falciparum ligand binding to erythrocytes induce alterations in deformability essential for invasion.

The most lethal form of malaria in humans is caused by Plasmodium falciparum. These parasites invade erythrocytes, a complex process involving multiple ligand-receptor interactions. The parasite makes initial contact with the erythrocyte followed by dramatic deformations linked to the function of the Erythrocyte binding antigen family and P. falciparum reticulocyte binding-like families. We show EBA-175 mediates substantial changes in the deformability of erythrocytes by binding to glycophorin A and activating a phosphorylation cascade that includes erythrocyte cytoskeletal proteins resulting in changes in the viscoelastic properties of the host cell. TRPM7 kinase inhibitors FTY720 and waixenicin A block the changes in the deformability of erythrocytes and inhibit merozoite invasion by directly inhibiting the phosphorylation cascade. Therefore, binding of P. falciparum parasites to the erythrocyte directly activate a signaling pathway through a phosphorylation cascade and this alters the viscoelastic properties of the host membrane conditioning it for successful invasion.

Identification of Plasmodium falciparum reticulocyte binding protein homologue 5-interacting protein, PfRipr, as a highly conserved blood-stage malaria vaccine candidate

Genetic variability in Plasmodium falciparum malaria parasites hampers current malaria vaccine development efforts. Here, we hypothesize that to address the impact of genetic variability on vaccine efficacy in clinical trials, conserved antigen targets should be selected to achieve robust host immunity across multiple falciparum strains. Therefore, suitable vaccine antigens should be assessed for levels of polymorphism and genetic diversity. Using a total of one hundred and two clinical isolates from a region of high malaria transmission in Uganda, we analyzed extent of polymorphism and genetic diversity in four recently reported novel blood-stage malaria vaccine candidate proteins: Rh5 interacting protein (PfRipr), GPI anchored micronemal antigen (PfGAMA), rhoptry-associated leucine zipper-like protein 1 (PfRALP1) and Duffy binding-like merozoite surface protein 1 (PfMSPDBL1). In addition, utilizing the wheat germ cell-free system, we expressed recombinant proteins for the four candidates based on P. falciparum laboratory strain 3D7 sequences, immunized rabbits to obtain specific antibodies (Abs) and performed functional growth inhibition assay (GIA). The GIA activity of the raised Abs was demonstrated using both homologous 3D7 and heterologous FVO strains in vitro. Both pfripr and pfralp1 are less polymorphic but the latter is comparatively more diverse, with varied number of regions having insertions and deletions, asparagine and 6-mer repeats in the coding sequences. Pfgama and pfmspdbl1 are polymorphic and genetically diverse among the isolates with antibodies against the 3D7-based recombinant PfGAMA and PfMSPDBL1 inhibiting merozoite invasion only in the 3D7 but not FVO strain. Moreover, although Abs against the 3D7-based recombinant PfRipr and PfRALP1 proteins potently inhibited merozoite invasion of both 3D7 and FVO, the GIA activity of anti-PfRipr was much higher than that of anti-PfRALP1. Thus, PfRipr is regarded as a promising blood-stage vaccine candidate for next-generation vaccines against P. falciparum.

In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9.

Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with the linear epitope, since the IgG1 subclass against PvMSP9E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and experimentally confirmed the potential of PvMSP9E795-A808 as an immunogenic linear B cell epitope within the P. vivax malaria vaccine candidate PvMSP9 and support its inclusion in future subunit vaccines.