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.

The cell-traversal protein for ookinetes and sporozoites (CelTOS), a highly conserved antigen involved in sporozoite motility, plays an important role in the traversal of host cells during the preerythrocytic stage of Plasmodium species. Recently, it has been considered an alternative target when designing novel antimalarial vaccines against Plasmodium falciparum. However, the potential of Plasmodium vivax CelTOS as a vaccine target is yet to be explored. This study evaluated the naturally acquired immune response against a recombinant P. vivax CelTOS (PvCelTOS) (IgG and IgG subclass) in 528 individuals from Brazilian Amazon, as well as the screening of B-cell epitopes in silico and peptide assays to associate the breadth of antibody responses of those individuals with exposition and/or protection correlates. We show that PvCelTOS is naturally immunogenic in Amazon inhabitants with 94 individuals (17.8%) showing specific IgG antibodies against the recombinant protein. Among responders, the IgG reactivity indexes (RIs) presented a direct correlation with the number of previous malaria episodes (p = 0.003; r = 0.315) and inverse correlation with the time elapsed from the last malaria episode (p = 0.031; r = −0.258). Interestingly, high responders to PvCelTOS (RI > 2) presented higher number of previous malaria episodes, frequency of recent malaria episodes, and ratio of cytophilic/non-cytophilic antibodies than low responders (RI < 2) and non-responders (RI < 1). Moreover, a high prevalence of the cytophilic antibody IgG1 over all other IgG subclasses (p < 0.0001) was observed. B-cell epitope mapping revealed five immunogenic regions in PvCelTOS, but no associations between the specific IgG response to peptides and exposure/protection parameters were found. However, the epitope (PvCelTOSI136-E143) was validated as a main linear B-cell epitope, as 92% of IgG responders to PvCelTOS were also responders to this peptide sequence. This study describes for the first time the natural immunogenicity of PvCelTOS in Amazon individuals and identifies immunogenic regions in a full-length protein. The IgG magnitude was mainly composed of cytophilic antibodies (IgG1) and associated with recent malaria episodes. The data presented in this paper add further evidence to consider PvCelTOS as a vaccine candidate.

Background The GMZ2.6c malaria vaccine candidate is a multi-stage Plasmodium falciparum chimeric protein which contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, a fusion protein of GLURP and MSP-3, that has been shown to be well tolerated, safe and immunogenic in clinical trials performed in a malaria-endemic area of Africa. However, there is no data available on the antigenicity or immunogenicity of GMZ2.6c in humans. Considering that circulating parasites can be genetically distinct in different malaria-endemic areas and that host genetic factors can influence the immune response to vaccine antigens, it is important to verify the antigenicity, immunogenicity and the possibility of associated protection in individuals living in malaria-endemic areas with different epidemiological scenarios. Herein, the profile of antibody response against GMZ2.6c and its components (MSP-3, GLURP and Pfs48/45) in residents of the Brazilian Amazon naturally exposed to malaria, in areas with different levels of transmission, was evaluated. Methods This study was performed using serum samples from 352 individuals from Cruzeiro do Sul and Mâncio Lima, in the state of Acre, and Guajará, in the state of Amazonas. Specific IgG, IgM, IgA and IgE antibodies and IgG subclasses were detected by Enzyme-Linked Immunosorbent Assay. Results The results showed that GMZ2.6c protein was widely recognized by naturally acquired antibodies from individuals of the Brazilian endemic areas with different levels of transmission. The higher prevalence of individuals with antibodies against GMZ2.6c when compared to its individual components may suggest an additive effect of GLURP, MSP-3, and Pfs48/45 when inserted in a same construct. Furthermore, naturally malaria-exposed individuals predominantly had IgG1 and IgG3 cytophilic anti-GMZ2.6c antibodies, an important fact considering that the acquisition of anti-malaria protective immunity results from a delicate balance between cytophilic/non-cytophilic antibodies. Interestingly, anti-GMZ2.6c antibodies seem to increase with exposure to malaria infection and may contribute to parasite immunity. Conclusions The data showed that GMZ2.6c protein is widely recognized by naturally acquired antibodies from individuals living in malaria-endemic areas in Brazil and that these may contribute to parasite immunity. These data highlight the importance of GMZ2.6c as a candidate for an anti-malarial vaccine.

In the last years, the development of small molecule antagonists of VLA-4 for the treatment of diseases, where cell trafficking and activation are important, has increased considerably. Among them, the MK-0617 ligand has proven to be a highly potent and orally active α4β1 antagonist. However, the binding mode of this ligand in the integrin binding site remains unknown. Herein we report a thermodynamic analysis of the interaction between MK-0617 (and one of its isomers) and the VLA-4 protein using molecular docking and the free energy perturbation calculations, based on a comparative model of the α4β1 receptor. Initial complex coordinates were taken from molecular docking assays and submitted to alchemical transformations. Free energy of binding ΔΔG values, derived from experimental IC50 values, were taken as a parameter for determining the most likely binding mode. In addition, molecular dynamics simulations of these ligands within the α4β1 binding site were carried out to elucidate the binding energy profile and identify the most significant residues. Our results indicate that MK-0617 fits within the binding site in a stretched conformation, pointing the carboxylate group towards the MIDAS ion. We observe that, despite the fact that the main contribution to the energetic binding process is due to the electrostatic ion contribution, the nonpolar contribution is not negligible. Additionally, a network of hydrogen bonds participate in stabilizing the ligand-receptor interaction.

The Plasmodium vivax Cell-traversal protein for ookinetes and sporozoites (PvCelTOS) plays an important role in the traversal of host cells. Although essential to PvCelTOS progress as a vaccine candidate, its genetic diversity remains uncharted. Therefore, we investigated the PvCelTOS genetic polymorphism in 119 field isolates from five different regions of Brazilian Amazon (Manaus, Novo Repartimento, Porto Velho, Plácido de Castro and Oiapoque). Moreover, we also evaluated the potential impact of non-synonymous mutations found in the predicted structure and epitopes of PvCelTOS. The field isolates showed high similarity (99.3% of bp) with the reference Sal-1 strain, presenting only four Single-Nucleotide Polymorphisms (SNP) at positions 24A, 28A, 109A and 352C. The frequency of synonymous C109A (82%) was higher than all others (p<0.0001). However, the non-synonymous G28A and G352C were observed in 9.2% and 11.7% isolates. The great majority of the isolates (79.8%) revealed complete amino acid sequence homology with Sal-1, 10.9% presented complete homology with Brazil I and two undescribed PvCelTOS sequences were observed in 9.2% field isolates. Concerning the prediction analysis, the N-terminal substitution (Gly10Ser) was predicted to be within a B-cell epitope (PvCelTOS Accession Nos. AB194053.1) and exposed at the protein surface, while the Val118Leu substitution was not a predicted epitope. Therefore, our data suggest that although G28A SNP might interfere in potential B-cell epitopes at PvCelTOS N-terminal region the gene sequence is highly conserved among the isolates from different geographic regions, which is an important feature to be taken into account when evaluating its potential as a vaccine candidate.