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.

Malaria absorption peaks acquired through the skin of patients with infrared light can detect patients with varying parasitemia.

To eliminate malaria, scalable tools that are rapid, affordable, and can detect patients with low parasitemia are required. Non-invasive diagnostic tools that are rapid, reagent-free, and affordable would also provide a justifiable platform for testing malaria in asymptomatic patients. However, non-invasive surveillance techniques for malaria remain a diagnostic gap. Here, we show near-infrared Plasmodium absorption peaks acquired non-invasively through the skin using a miniaturized hand-held near-infrared spectrometer. Using spectra from the ear, these absorption peaks and machine learning techniques enabled non-invasive detection of malaria-infected human subjects with varying parasitemia levels in less than 10 s.

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.

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.

Análise do polimorfismo genético da PvCelTOS (P. vivax Cell-Traversal Protein for Ookinetes and Sporozoites) em isolados de diferentes áreas endêmicas da Amazônia Brasileira

A PvCelTOS (Plasmodium vivax Cell-Traversal Protein for Ookinetes and Sporozoites) desempenha um papel importante na travessia de células hospedeiras. Mesmo sendo essencial para o progresso da PvCelTOS como uma candidata vacinal, o conhecimento sobre a sua diversidade genética permanece desconhecido. Assim, investigamos o polimorfismo genético da PvCelTOS a partir da amplificação gênica por PCR e sequenciamento de 119 amostras de isolados de P. vivax oriundos de cinco diferentes localidades da Amazônia Brasileira (Manaus, Novo Repartimento, Porto Velho, Plácido de Castro e Oiapoque) e avaliamos o potencial impacto de mutações não sinônimas na estrutura tridimensional e nos epítopos da PvCelTOS através de ferramentas de predição in silico. Nossos dados mostram que os isolados de campo apresentam alta similaridade (99,3% de pb) com a cepa de referência Sal-1, apresentando apenas quatro polimorfismos de um único nucleotídeo (SNP) nas posições 24A, 28A, 109A e 352C. A frequência da mutação sinônima C109A (82%) foi maior do que todas as outras (p<0,0001). As mutações não sinônimas G28A e G352C foram observadas em 9,2% e 11,7% dos isolados, respectivamente. A grande maioria dos isolados (79,8%) revelou homologia completa da sequência de aminoácidos com Sal-1, 10,9% apresentaram homologia completa com o genoma Brazil I e duas sequências não descritas da PvCelTOS foram observadas em 9,2% dos isolados de campo. Em relação à análise de predição, previu-se que a substituição Nterminal (Gly10Ser) estava dentro de um potencial epítopo de célula B e exposta à superfície da proteína, enquanto que a substituição Val118Leu não é um epítopo predito Por conseguinte, os nossos dados sugerem que, o SNP G28A pode interferir em epítopos potenciais de células B na região N-terminal da PvCelTOS, enquanto que não parece haver interferência nos epítopos de células TCD8+. Além disso, sua sequência genética é altamente conservada entre isolados de diferentes regiões geográficas, sendo uma característica importante em relação ao seu potencial como vacina candidata.