Are pvcrt-o and pvmdr1 Gene Mutations Associated with Plasmodium vivax Chloroquine-Resistant Parasites?

(1) Background: Malaria remains a significant global public health issue. Since parasites quickly became resistant to most of the available antimalarial drugs, treatment effectiveness must be constantly monitored. In Brazil, up to 10% of cases of vivax malaria resistant to chloroquine (CQ) have been registered. Unlike P. falciparum, there are no definitive molecular markers for the chemoresistance of P. vivax to CQ. This work aimed to investigate whether polymorphisms in the pvcrt-o and pvmdr1 genes could be used as markers for assessing its resistance to CQ. (2) Methods: A total of 130 samples from P. vivax malaria cases with no clinical and/or parasitological evidence of CQ resistance were studied through polymerase chain reaction for gene amplification followed by target DNA sequencing. (3) Results: In the pvcrt-o exons, the K10 insert was present in 14% of the isolates. Regarding pvmdr1, T958M and F1076L haplotypes showed frequencies of 95% and 3%, respectively, while the SNP Y976F was not detected. (4) Conclusions: Since K10-pvcrt-o and F1076L/T958M-pvmdr1 polymorphisms were detected in samples from patients who responded well to CQ treatment, it can be concluded that mutations in these genes do not seem to have a potential for association with the phenotype of CQ resistance.

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.

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.

Plasmodium vivax metacaspase 1 (PvMCA1) catalytic domain is conserved in field isolates from Brazilian Amazon.

In the present study, we investigated the genetic diversity of Plasmodium vivax metacaspase 1 (PvMCA1) catalytic domain in two municipalities of the main malaria hotspot in Brazil, i.e., the Juruá Valley, and observed complete sequence identity among all P. vivax field isolates and the Sal-1 reference strain. Analysis of PvMCA1 catalytic domain in different P. vivax genomic sequences publicly available also revealed a high degree of conservation worldwide, with very few amino acid substitutions that were not related to putative histidine and cysteine catalytic residues, whose involvement with the active site of protease was herein predicted by molecular modeling. The genetic conservation presented by PvMCA1 may contribute to its eligibility as a druggable target candidate in vivax malaria.

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.

Chloroquine and mefloquine resistance profiles are not related to the circumsporozoite protein (CSP) VK210 subtypes in field isolates of Plasmodium vivax from Manaus, Brazilian Amazon.

BACKGROUND: The central repetitive region (CRR) of the Plasmodium vivax circumsporozoite surface protein (CSP) is composed of a repetitive sequence that is characterised by three variants: VK210, VK247 and P. vivax-like. The most important challenge in the treatment of P. vivax infection is the possibility of differential response based on the parasite genotype. OBJECTIVES: To characterise the CSP variants in P. vivax isolates from individuals residing in a malaria-endemic region in Brazil and to profile these variants based on sensitivity to chloroquine and mefloquine. METHODS: The CSP variants were determined by sequencing and the sensitivity of the P. vivax isolates to chloroquine and mefloquine was determined by Deli-test. FINDINGS: Although five different allele sizes were amplified, the sequencing results showed that all of the isolates belonged to the VK210 variant. However, we observed substantial genetic diversity in the CRR, resulting in the identification of 10 different VK210 subtypes. The frequency of isolates that were resistant to chloroquine and mefloquine was 11.8 and 23.8%, respectively. However, we did not observe any difference in the frequency of the resistant isolates belonging to the VK210 subtypes. MAIN CONCLUSION: The VK210 variant is the most frequently observed in the studied region and there is significant genetic variability in the CRR of the P. vivax CSP. Moreover, the antimalarial drug sensitivity profiles of the isolates does not seem to be related to the VK210 subtypes.

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.

Chloroquine and mefloquine resistance profiles are not related to the circumsporozoite protein (CSP) VK210 subtypes in field isolates of Plasmodium vivax from Manaus, Brazilian Amazon

BACKGROUND The central repetitive region (CRR) of the Plasmodium vivax circumsporozoite surface protein (CSP) is composed of a repetitive sequence that is characterised by three variants: VK210, VK247 and P. vivax-like. The most important challenge in the treatment of P. vivax infection is the possibility of differential response based on the parasite genotype. OBJECTIVES To characterise the CSP variants in P. vivax isolates from individuals residing in a malaria-endemic region in Brazil and to profile these variants based on sensitivity to chloroquine and mefloquine. METHODS The CSP variants were determined by sequencing and the sensitivity of the P. vivax isolates to chloroquine and mefloquine was determined by Deli-test. FINDINGS Although five different allele sizes were amplified, the sequencing results showed that all of the isolates belonged to the VK210 variant. However, we observed substantial genetic diversity in the CRR, resulting in the identification of 10 different VK210 subtypes. The frequency of isolates that were resistant to chloroquine and mefloquine was 11.8 and 23.8%, respectively. However, we did not observe any difference in the frequency of the resistant isolates belonging to the VK210 subtypes. MAIN CONCLUSION The VK210 variant is the most frequently observed in the studied region and there is significant genetic variability in the CRR of the P. vivax CSP. Moreover, the antimalarial drug sensitivity profiles of the isolates does not seem to be related to the VK210 subtypes.