Construction, Expression, and Evaluation of the Naturally Acquired Humoral Immune Response against Plasmodium vivax RMC-1, a Multistage Chimeric Protein.

The PvCelTOS, PvCyRPA, and Pvs25 proteins play important roles during the three stages of the P. vivax lifecycle. In this study, we designed and expressed a P. vivax recombinant modular chimeric protein (PvRMC-1) composed of the main antigenic regions of these vaccine candidates. After structure modelling by prediction, the chimeric protein was expressed, and the antigenicity was assessed by IgM and IgG (total and subclass) ELISA in 301 naturally exposed individuals from the Brazilian Amazon. The recombinant protein was recognized by IgG (54%) and IgM (40%) antibodies in the studied individuals, confirming the natural immunogenicity of the epitopes that composed PvRMC-1 as its maintenance in the chimeric structure. Among responders, a predominant cytophilic response mediated by IgG1 (70%) and IgG3 (69%) was observed. IgM levels were inversely correlated with age and time of residence in endemic areas (p < 0.01). By contrast, the IgG and IgM reactivity indexes were positively correlated with each other, and both were inversely correlated with the time of the last malaria episode. Conclusions: The study demonstrates that PvRMC-1 was successfully expressed and targeted by natural antibodies, providing important insights into the construction of a multistage chimeric recombinant protein and the use of naturally acquired antibodies to validate the construction.

Sph2(176-191) and Sph2(446-459): Identification of B-Cell Linear Epitopes in Sphingomyelinase 2 (Sph2), Naturally Recognized by Patients Infected by Pathogenic Leptospires.

Sphingomyelin is a major constituent of eukaryotic cell membranes, and if degraded by bacteria sphingomyelinases may contribute to the pathogenesis of infection. Among Leptospira spp., there are five sphingomyelinases exclusively expressed by pathogenic leptospires, in which Sph2 is expressed during natural infections, cytotoxic, and implicated in the leptospirosis hemorrhagic complications. Considering this and the lack of information about associations between Sph2 and leptospirosis severity, we use a combination of immunoinformatics approaches to identify its B-cell epitopes, evaluate their reactivity against samples from leptospirosis patients, and investigate the role of antibodies anti-Sph2 in protection against severe leptospirosis. Two B-cell epitopes, Sph2(176-191) and Sph2(446-459), were predicted in Sph2 from L. interrogans serovar Lai, presenting different levels of identity when compared with other pathogenic leptospires. These epitopes were recognized by about 40% of studied patients with a prevalence of IgG antibodies against both Sph2(176-191) and Sph2(446-459). Remarkably, just individuals with low reactivity to Sph2(176-191) presented clinical complications, while high responders had only mild symptoms. Therefore, we identified two B-cell linear epitopes, recognized by antibodies of patients with leptospirosis, that could be further explored in the development of multi-epitope vaccines against leptospirosis.

B-Cell Epitope Mapping of the Plasmodium falciparum Malaria Vaccine Candidate GMZ2.6c in a Naturally Exposed Population of the Brazilian Amazon.

The GMZ2.6c malaria vaccine candidate is a multi-stage P. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, an asexual-stage vaccine construction consisting of the N-terminal region of the glutamate-rich protein (GLURP) and the C-terminal region of the merozoite surface protein-3 (MSP-3). Previous studies showed that GMZ2.6c is widely recognized by antibodies from Brazilian exposed individuals and that its components are immunogenic in natural infection by P. falciparum. In addition, anti-GMZ2.6c antibodies increase with exposure to infection and may contribute to parasite immunity. Therefore, identifying epitopes of proteins recognized by antibodies may be an important tool for understanding protective immunity. Herein, we identify and validate the B-cell epitopes of GMZ2.6c as immunogenic and immunodominant in individuals exposed to malaria living in endemic areas of the Brazilian Amazon. Specific IgG antibodies and subclasses against MSP-3, GLURP, and Pfs48/45 epitopes were detected by ELISA using synthetic peptides corresponding to B-cell epitopes previously described for MSP-3 and GLURP or identified by BepiPred for Pfs48/45. The results showed that the immunodominant epitopes were P11 from GLURP and MSP-3c and DG210 from MSP-3. The IgG1 and IgG3 subclasses were preferentially induced against these epitopes, supporting previous studies that these proteins are targets for cytophilic antibodies, important for the acquisition of protective immunity. Most individuals presented detectable IgG antibodies against Pfs48/45a and/or Pfs48/45b, validating the prediction of linear B-cell epitopes. The higher frequency and antibody levels against different epitopes from GLURP, MSP-3, and Pfs48/45 provide additional information that may suggest the relevance of GMZ2.6c as a multi-stage malaria vaccine candidate.

Leptolysin, a Leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity.

Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.

Genetic Diversity of Plasmodium vivax Cysteine-Rich Protective Antigen (PvCyRPA) in Field Isolates from Five Different Areas of the Brazilian Amazon.

The Plasmodium vivax Cysteine-Rich Protective Antigen (PvCyRPA) has an important role in erythrocyte invasion and has been considered a target for vivax malaria vaccine development. Nonetheless, its genetic diversity remains uncharted in Brazilian malaria-endemic areas. Therefore, we investigated the pvcyrpa genetic polymorphism in 98 field isolates from the Brazilian Amazon and its impact on the antigenicity of predicted B-cell epitopes. Genetic diversity parameters, population genetic analysis, neutrality test and the median-joining network were analyzed, and the potential amino acid polymorphism participation in B-cell epitopes was investigated. One synonymous and 26 non-synonymous substitutions defined fifty haplotypes. The nucleotide diversity and Tajima's D values varied across the coding gene. The exon-1 sequence had greater diversity than those of exon-2. Concerning the prediction analysis, seven sequences were predicted as linear B cell epitopes, the majority contained in conformational epitopes. Moreover, important amino acid polymorphism was detected in regions predicted to contain residues participating in B-cell epitopes. Our data suggest that the pvcyrpa gene presents a moderate polymorphism in the studied isolates and such polymorphisms alter amino acid sequences contained in potential B cell epitopes, an important observation considering the antigen potentiality as a vaccine candidate to cover distinct P. vivax endemic areas worldwide.

Identification of Immunogenic Linear B-Cell Epitopes in C. burnetii Outer Membrane Proteins Using Immunoinformatics Approaches Reveals Potential Targets of Persistent Infections.

Coxiella burnetii is a global, highly infectious intracellular bacterium, able to infect a wide range of hosts and to persist for months in the environment. It is the etiological agent of Q fever-a zoonosis of global priority. Currently, there are no national surveillance data on C. burnetii's seroprevalence for any South American country, reinforcing the necessity of developing novel and inexpensive serological tools to monitor the prevalence of infections among humans and animals-especially cattle, goats, and sheep. In this study, we used immunoinformatics and computational biology tools to predict specific linear B-cell epitopes in three C. burnetii outer membrane proteins: OMP-H (CBU_0612), Com-1 (CBU_1910), and OMP-P1 (CBU_0311). Furthermore, predicted epitopes were tested by ELISA, as synthetic peptides, against samples of patients reactive to C. burnetii in indirect immunofluorescence assay, in order to evaluate their natural immunogenicity. In this way, two linear B-cell epitopes were identified in each studied protein (OMP-H(51-59), OMP-H(91-106), Com-1(57-76), Com-1(191-206), OMP-P1(197-209), and OMP-P1(215-227)); all of them were confirmed as naturally immunogenic by the presence of specific antibodies in 77% of studied patients against at least one of the identified epitopes. Remarkably, a higher frequency of endocarditis cases was observed among patients who presented an intense humoral response to OMP-H and Com-1 epitopes. These data confirm that immunoinformatics applied to the identification of specific B-cell epitopes can be an effective strategy to improve and accelerate the development of surveillance tools against neglected diseases.

Changes in B Cell Pool of Patients With Multibacillary Leprosy: Diminished Memory B Cell and Enhanced Mature B in Peripheral Blood.

Despite being treatable, leprosy still represents a major public health problem, and many mechanisms that drive leprosy immunopathogenesis still need to be elucidated. B cells play important roles in immune defense, being classified in different subgroups that present distinct roles in the immune response. Here, the profile of B cell subpopulations in peripheral blood of patients with paucibacillary (TT/BT), multibacillary (LL/BL) and erythema nodosum leprosum was analyzed. B cell subpopulations (memory, transition, plasmablasts, and mature B cells) and levels of IgG were analyzed by flow cytometry and ELISA, respectively. It was observed that Mycobacterium leprae infection can alter the proportions of B cell subpopulations (increase of mature and decrease of memory B cells) in patients affected by leprosy. This modulation is associated with an increase in total IgG and the patient's clinical condition. Circulating B cells may be acting in the modulation of the immune response in patients with various forms of leprosy, which may reflect the patient's ability to respond to M. leprae.

The Serological Cross-Detection of Bat-Borne Hantaviruses: A Valid Strategy or Taking Chances?

Bats are hosts of a range of viruses, and their great diversity and unique characteristics that distinguish them from all other mammals have been related to the maintenance, evolution, and dissemination of these pathogens. Recently, very divergent hantaviruses have been discovered in distinct species of bats worldwide, but their association with human disease remains unclear. Considering the low success rates of detecting hantavirus RNA in bat tissues and that to date no hantaviruses have been isolated from bat samples, immunodiagnostic tools could be very helpful to understand pathogenesis, epidemiology, and geographic range of bat-borne hantaviruses. In this sense, we aimed to identify in silico immunogenic B-cell epitopes present on bat-borne hantaviruses nucleoprotein (NP) and verify if they are conserved among them and other selected members of Mammantavirinae, using a combination of (the three most used) different prediction algorithms, ELLIPRO, Discotope 2.0, and PEPITO server. To support our data, we in silico modeled 3D structures of NPs from representative members of bat-borne hantaviruses, using comparative and ab initio methods due to the absence of crystallographic structures of studied proteins or similar models in the Protein Data Bank. Our analysis demonstrated the antigenic complexity of the bat-borne hantaviruses group, showing a low sequence conservation of epitopes among members of its own group and a minor conservation degree in comparison to Orthohantavirus, with a recognized importance to public health. Our data suggest that the use of recombinant rodent-borne hantavirus NPs to cross-detect antibodies against bat- or shrew-borne viruses could underestimate the real impact of this virus in nature.

Recombinant Plasmodium vivax circumsporozoite surface protein allelic variants: antibody recognition by individuals from three communities in the Brazilian Amazon.

Circumsporozoite protein (CSP) variants of P. vivax, besides having variations in the protein repetitive portion, can differ from each other in aspects such as geographical distribution, intensity of transmission, vectorial competence and immune response. Such aspects must be considered to P. vivax vaccine development. Therefore, we evaluated the immunogenicity of novel recombinant proteins corresponding to each of the three P. vivax allelic variants (VK210, VK247 and P. vivax-like) and of the C-terminal region (shared by all PvCSP variants) in naturally malaria-exposed populations of Brazilian Amazon. Our results demonstrated that PvCSP-VK210 was the major target of humoral immune response in studied population, presenting higher frequency and magnitude of IgG response. The IgG subclass profile showed a prevalence of cytophilic antibodies (IgG1 and IgG3), that seem to have an essential role in protective immune response. Differently of PvCSP allelic variants, antibodies elicited against C-terminal region of protein did not correlate with epidemiological parameters, bringing additional evidence that humoral response against this protein region is not essential to protective immunity. Taken together, these findings increase the knowledge on serological response to distinct PvCSP allelic variants and may contribute to the development of a global and effective P. vivax vaccine.

Main B-cell epitopes of PvAMA-1 and PvMSP-9 are targeted by naturally acquired antibodies and epitope-specific memory cells in acute and convalescent phases of vivax malaria.

Although antibodies are considered critical for malaria protection, little is known about the mechanisms/factors that maintain humoral immunity, especially regarding the induction and maintenance of memory B cells over time. In Brazilian endemic areas, this is the first time that the profile of antibody responses and the occurrence of antigen-specific memory B cells (MBC) against P vivax were investigated during acute malaria and up to six months after parasite clearance. For this, we selected two peptides, PvAMA-1(S290-K307) and PvMSP-9(E795-A808) , which represent the apical membrane antigen-1 and merozoite surface protein-9 of P vivax, respectively. Both peptides were previously described as containing linear B-cell epitopes. Our findings were as follows: 1-both peptides were recognized by IgG antibodies at a high frequency (between 24% and 81%) in all study groups; 2-in the absence of infection, the IgG levels remained stable throughout 6 months of follow-up; and 3-PvAMA-1(S290-K307) and PvMSP-9(E795-A808) -specific MBCs were detected in all individual groups in the absence of reinfection throughout the follow-up period, suggesting long-lived MBC. However, no positive association was observed between malaria-specific antibody levels and frequency of MBCs over time. Taken together, these results suggest that peptides can be, in the future, an alternative strategy to polypeptidic vaccine formulation.

Identification and validation of specific B-cell epitopes of hantaviruses associated to hemorrhagic fever and renal syndrome.

BACKGROUND: Orthohantavirus infection is a neglected global health problem affecting approximately 200,000 people/year, spread by rodent hosts and associated to fatal human diseases, such as hemorrhagic fever with renal syndrome (HFRS) and orthohantavirus cardiopulmonary syndrome (HCPS). Circulation of HFRS-associated orthohantaviruses, such as Seoul, Gou, Amur, Dobrava and Hantaan, are supposed to be restricted to Eurasian countries even though their hosts can be a worldwide distribution. Few confirmed HFRS orthohantavirus infections in humans have been reported in American countries, but due to lower medical awareness of the symptoms of this zoonosis, it could be associated to viral underreporting or to misdiagnosis with several tropical hemorrhagic diseases. Serological evidence of orthohantavirus infections, using enzyme-linked immunosorbent assay for the presence of immunoglobulin M and G against recombinant nucleoprotein protein, remains as an essential assay for viral surveillance. In this study, we aimed to identify in silico immunogenic B-cell linear epitopes present on orthohantavirus nucleoprotein that are exclusive to HFRS-related species. METHODOLOGY/PRINCIPAL FINDINGS: In silico analysis were performed using Seoul orthohantavirus nucleoprotein (SHNP) sequence as a model. Linear B-cell-epitopes on SHNP and its immunogenicity were predicted by BepiPred-2.0 and Vaxijen algorithms, respectively. The conservancy of predicted epitopes was compared with the most clinically relevant HFRS or HCPS-associated orthohantavirus, aiming to identify specific sequences from HFRS-orthohantavirus. Peptide validation was carried out by ELISA using Balb/c mice sera immunized with purified recombinant rSHNP. Peptides cross-reactivity against HCPS orthohantavirus were evaluated using immunized sera from mice injected with recombinant Juquitiba orthohantavirus nucleoprotein (rJHNP). CONCLUSION/SIGNIFICANCE: In silico analysis revealed nine potential immunogenic linear B-cell epitopes from SHNP; among them, SHNP(G72-D110) and SHNP(P251-D264) showed a high degree of sequence conservation among HFRS-related orthohantavirus and were experimentally validated against rSHNP-IMS and negatively validated against rJHNP-IMS. Taken together, we identified and validated two potential antigenic B-cell epitopes on SHNP, which were conserved among HFRS-associated orthohantavirus and could be applied to the development of novel immunodiagnostic tools for orthohantavirus surveillance.

Antibody Responses Against Plasmodium vivax TRAP Recombinant and Synthetic Antigens in Naturally Exposed Individuals From the Brazilian Amazon.

Thrombospondin-related adhesive protein (TRAP) is essential for sporozoite motility and the invasion of mosquitoes' salivary gland and vertebrate's hepatocyte and is, thus, considered a promising pre-erythrocytic vaccine candidate. Despite the existence of a few reports on naturally acquired immune response against Plasmodium vivax TRAP (PvTRAP), it has never been explored so far in the Amazon region, so results are conflicting. Here, we characterized the (IgG and IgG subclass) antibody reactivity against recombinant PvTRAP in a cross-sectional study of 299 individuals exposed to malaria infection in three municipalities (Cruzeiro do Sul, Mâncio Lima and Guajará) from the Acre state of the Brazilian Amazon. In addition, the full PvTRAP sequence was screened for B-cell epitopes using in silico and in vitro approaches. Firstly, we confirmed that PvTRAP is naturally immunogenic in the cohort population since 49% of the individuals were IgG-responders to it. The observed immune responses were mainly driven by cytophilic IgG1 over all other sublcasses and the IgG levels that was corelated with age and time of residence in the studied area (p < 0.05). Interestingly, only the levels of specific anti-TRAP IgG3 seemed to be associated with protection, as IgG3 responders presented a significantly higher time elapse since the last malaria episode than those recorded for IgG3 non-responders. Regarding the B-cell epitope mapping, among the 148 responders to PvTRAP, four predicted epitopes were confirmed by recognition of antibodies (PvTRAPR197-H227; PvTRAPE237-T258; PvTRAPP344-G374; and PvTRAPE439-K454). Nevertheless, the frequency of responders against these peptides were low and did not show a clear correlation with the antibody response against the corresponding antigen. Moreover, none of the linear confirmed epitopes were located in the binding regions of PvTRAP in respect to the host cell ligand. Collectively, our data confirm the PvTRAP immunogenicity among Amazon inhabitants, while suggesting that the main important B-cell epitopes are not linear.

Apical membrane protein 1-specific antibody profile and temporal changes in peripheral blood B-cell populations in Plasmodium vivax malaria.

Plasmodium falciparum-specific antibodies tend to be short-lived, but their cognate memory B cells (MBCs) circulate in the peripheral blood of exposed subjects for several months or years after the last infection. However, the time course of antigen-specific antibodies and B-cell responses to the relatively neglected parasite Plasmodium vivax remains largely unexplored. Here, we showed that uncomplicated vivax malaria elicits short-lived antibodies but long-lived MBC responses to a major blood-stage P vivax antigen, apical membrane protein 1 (PvAMA-1), in subjects exposed to declining malaria transmission in the Amazon Basin of Brazil. We found that atypical (CD19+ CD10- CD21- CD27- ) MBCs, which appear to share a common precursor with classical MBCs but are unable to differentiate into antibody-secreting cells, significantly outnumbered classical MBCs by 5:1 in the peripheral blood of adult subjects currently or recently infected with P vivax and by 3:1 in healthy residents in the same endemic communities. We concluded that malaria can drive classical MBCs to differentiate into functionally impaired MBCs not only in subjects repeatedly exposed to P falciparum, but also in subjects living in areas with low levels of P vivax transmission in the Amazon, leading to an impaired B-cell memory that may affect both naturally acquired and vaccine-induced immunity.

Plasmodium vivax ookinete surface protein (Pvs25) is highly conserved among field isolates from five different regions of the Brazilian Amazon.

The Plasmodium vivax Ookinete Surface Protein (Pvs25) is one of the leading malaria Transmission-Blocking Vaccine candidates based on its high immunogenicity in animal models, transmission-blocking activity of antibodies elicited in clinical trials and high conservation among P. vivax isolates from endemic areas. However, the polymorphism in gene encoding Pvs25 in endemic areas from South America has been poorly studied so far. Here, we investigated the genetic polymorphism of pvs25 in P. vivax isolates from five different regions of the Brazilian Amazon (Cruzeiro do Sul, Mâncio Lima, Guajará, Manaus and Oiapoque) and its impact on antigenicity of predicted B-cell epitopes using gene sequencing and epitope prediction tools. Firstly, only a non-synonymous substitution was found in the 657 bp amplified fragment in all sequenced samples, which represented an exchange of Gln by Lys at position 87 (Q87K) of protein amino acid sequence (domain II EGF-like). Q87K substitution was also present in all studied sites with a total frequency of 37.8%. Cruzeiro do Sul presented Q87K substitution in almost half of the isolates (48.4%), and an expressive frequency (40.5%) was also found in Manaus, while in Mâncio Lima, Guajará and Oiapoque, the frequencies were low (23.5%, 25% and 22.2% respectively). We also observed the Q87K mutation in a predicted B-cell epitope of pvs25, with no significant changes on its putative antigenicity. Our data suggest that the pvs25 gene is conserved among isolates from different Brazilian Amazon geographic regions, an important observation considering the antigen potentiality as a vaccine candidate to cover distinct P. vivax endemic areas worldwide.

Immunogenicity of synthetic peptide constructs based on PvMSP9E795-A808, a linear B-cell epitope of the P. vivax Merozoite Surface Protein-9.

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.

Corrigendum: Leishmania infantum Virulence Factor A2 Protein: Linear B-Cell Epitope Mapping and Identification of Three Main Linear B-Cell Epitopes in Vaccinated and Naturally Infected Dogs.

[This corrects the article DOI: 10.3389/fimmu.2018.01690.].

Leishmania infantum Virulence Factor A2 Protein: Linear B-Cell Epitope Mapping and Identification of Three Main Linear B-Cell Epitopes in Vaccinated and Naturally Infected Dogs.

In Brazil, canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, presenting a broad spectrum of clinical manifestations. Dogs are the main parasite reservoir in urban areas and canine cases precede human infection. Currently, A2 protein based Leish-Tec® vaccine is the only vaccine commercially available against CVL in Brazil. Considering that the main screening and confirmatory tests of canine infection are serological, it is possible that the antibody response elicited after vaccination interfere with diagnosis, leading to the inability to distinguish between vaccinated and infected animals. In order to identify the specific B-cell response induced after vaccination, A2 protein sequence was screened for main linear B-cell epitopes using in silico prediction (Bepipred) and immunological confirmation by ELISA. Three amino acid sequences were described as potential B-cell epitopes (SV11-SAEPHKAAVDV, PP16-PQSVGPLSVGPQSVGP, and VQ34-VGPLSVGPQSVGPLSVGPLSVGPQAVGPLSVGPQ). Specific IgG ELISAs were performed in sera of 12 immunized dogs living in non-endemic areas, followed for up to 1 year after immunization. The results were compared with those obtained in a group of 10 symptomatic and 10 asymptomatic CVL dogs. All predicted epitopes were confirmed as linear B-cell epitopes broadly recognized by sera from studied dogs. Total IgG ELISAs demonstrated distinct patterns of response between peptides in the immunized and CVL groups. VQ34 peptide was recognized by the majority of sera from vaccinated and symptomatic dogs, and increases after vaccination. PP16 induced low levels of specific IgG that increased 1 year after immunization. Interestingly, a low frequency of reactivity was found against SV11 in naturally infected dogs (symptomatic and asymptomatic), while 83.3% of vaccinated dogs presented positive responses 1 year after immunization. The two animals in the vaccinated group that did not respond to SV11 1 year after immunization presented positive serology both 30 days and 6 months after immunization. In summary, we identified three main linear B-cell epitopes in A2 based vaccine. Moreover, the humoral response against SV11 presented marked differences between infected and Leish-Tec vaccinated dogs, and should be further investigated, in large trials, to confirm its potential as a serological marker able to distinguish between infected and vaccinated dogs.

Plasmodium vivax Cell Traversal Protein for Ookinetes and Sporozoites (PvCelTOS) gene sequence and potential epitopes are highly conserved among isolates from different regions of Brazilian Amazon.

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.

Plasmodium vivax Cell-Traversal Protein for Ookinetes and Sporozoites: Naturally Acquired Humoral Immune Response and B-Cell Epitope Mapping in Brazilian Amazon Inhabitants.

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.

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.