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.

Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model.

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

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.

Avaliação da resposta imune humoral contra a proteína Trap (proteína adesiva relacionada à trombospondina) de Plasmodium vivax em populações de área endêmica brasileira / Evaluation of the humoral immune response against the Trap protein (adhesive protein related to thrombospondin) of Plasmodium vivax in populations from the Brazilian endemic area

A TRAP (Proteína Adesiva Relacionada à Trombospondina) do Plasmodium spp. é essencial para a motilidade dos esporozoítos e para a invasão da glândula salivar do mosquito e do hepatócito do vertebrado. Devido ao seu importante papel biológico, a TRAP é considerada um alvo promissor para o desenvolvimento de uma vacina pré-eritrocítica. Apesar dos poucos relatos disponíveis acerca da resposta imune naturalmente adquirida contra a TRAP do Plasmodium vivax (PvTRAP), os resultados são conflitantes e nunca foram explorados na região Amazônica. Portanto, nós visamos caracterizar a reatividade de anticorpos (IgG e subclasses de IgG) contra a PvTRAP recombinante em um estudo de corte transversal envolvendo 299 indivíduos naturalmente expostos a infecções de malária, moradores de 3 comunidades na Amazônia brasileira (Cruzeiro do Sul, Mâncio Lima e Guajará), além disso os epítopos de células B também foram mapeados na sequência completa da PvTRAP através do uso de abordagens in vitro e in silico. Primeiramente, nós confirmamos que a PvTRAP é naturalmente imunogênica pois 49% dos indivíduos foram respondedores para IgG de PvTRAP. A resposta imune observada foi conduzida principalmente pela sublasse de anticorpo citofílico IgG1. Além disso, os níveis de IgG1 apresentaram correlação com idade e tempo de residência em área endêmica (p<0,05). Entretanto, curiosamente, apenas os níveis de anti-IgG3específicos para PvTRAP parecem estar associados com proteção, visto que os indivíduos respondedores para IgG3 apresentaram um tempo decorrido desde o último episódio de malária significatimente maior do que os indivíduos não-respondedores para IgG3. Em relação ao mapeamento dos epítopos de células B, entre os 148 indivíduos respondedores para a proteína PvTRAP recombinante, quatro epítopos preditos foram reconhecidos pelos anticorpos (PvTRAPR197-H227; PvTRAPE237-T258; PvTRAPP344-G374 e PvTRAPE439-K454). Contudo, as frequências de respondedores contra os peptídeos foram baixas e não apresentaram uma clara correlação contra o antígeno recombinante. Além disso, nenhum dos epítopos confirmados foram localizados nas regiões de ligação da PvTRAP. Em conjunto, nossos dados confirmam a imunogenicidade da PvTRAP entre os habitantes da Amazônia, entretanto nós sugerimos que os principais epítopos de células B não são lineares.