Circulation CD4+CD25+FoxP3+ regulatory T cells (Tregs) have been associated with the delicate balancing between control of overwhelming acute malaria infection and prevention of immune pathology due to disproportionate inflammatory responses to erythrocytic stage of the parasite. While the role of Tregs has been well-documented in murine models and P. falciparum infection, the phenotype and function of Tregs in P. vivax infection is still poorly characterized. In the current study, we demonstrated that patients with acute P. vivax infection presented a significant augmentation of circulating Tregs producing anti-inflammatory (IL-10 and TGF-β) as well as pro-inflammatory (IFN-γ, IL-17) cytokines, which was further positively correlated with parasite burden. Surface expression of GITR molecule and intracellular expression of CTLA-4 were significantly upregulated in Tregs from infected donors, presenting also a positive association between either absolute numbers of CD4+CD25+FoxP3+GITR+ or CD4+CD25+FoxP3+CTLA-4+ and parasite load. Finally, we demonstrate a suppressive effect of Treg cells in specific T cell proliferative responses of P. vivax infected subjects after antigen stimulation with Pv-AMA-1. Our findings indicate that malaria vivax infection lead to an increased number of activated Treg cells that are highly associated with parasite load, which probably exert an important contribution to the modulation of immune responses during P. vivax infection.

Abstract. The antibody response to the C-terminal 19-kD fragment of Plasmodium falciparum merozoite surface protein-1 (PfMSP1-19) was investigated in groups of subjects living in areas of Brazil with different levels of malaria transmission. The prevalence and the levels of IgG to PfMSP1-19 increased with the time of exposure and were positively correlated with the absence of clinical symptoms in parasitemic patients. The frequency of positive response and the mean level of IgG were higher in areas where malaria prevalence was more intense, especially among asymptomatic patients. The serum absorbance values of the IgG1 isotype were significantly higher among subjects with long-term exposure and in asymptomatic infections. These data suggest a protective role of IgG1 in naturally acquired immunity in spite of the unstable transmission levels in the Brazilian Amazon.

The antibody responses to the apical membrane antigen 1 of the Plasmodium vivax (PvAMA-1) were investigated in subjects living in areas of Brazil with different levels of malaria transmission. The prevalence and the levels of IgG to PvAMA-1 increased with the time of exposure. The frequency of a positive response and the mean IgG level were higher in areas where malaria prevalence was more intense, especially among non-infected subjects exposed to moderate transmission over a period of 20 years. The proportions and levels of IgG1and IgG3 isotypes were significantly higher among those subjects with long-term exposure. Antibodies, mainly IgG1, to PvAMA-1 persisted for seven years among subjects briefly exposed to malaria in an outbreak outside the Brazilian malaria-endemic area. These data show the highly immunogenic properties of PvAMA-1 and emphasize its possible use as a malaria vaccine candidate.

Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

Subclasses of antibodies to the C-terminal 19 kDa fragment of the Plasmodium vivax merozoite surface protein 1 (PvMSP-1(19)) were assessed among subjects with distinct degrees of malaria exposure in the Brazilian endemic area. The PvMSP-1(19) specific IgG1and IgG3 levels were low among subjects with long-term exposure (approximately 19 years) when compared to subjects less and sporadically exposed (<1 year). No statistically difference was observed in IgG subclass distribution of antibodies from symptomatic Plasmodium-infected patients, asymptomatic parasite carriers and non-infected subjects living in a same mesoendemic area. Subjects briefly exposed to a P. vivax outbreak living in a rural community outside the endemic area were also evaluated to measure the persistence of specific antibodies. IgG anti-PvMSP-1(19) antibodies persisted in 40% of the subjects who had had malarial symptoms 8 months before and decreased after 7 years (28%). Specific IgG1 were the predominant isotype. Our study emphasizes the highly immunogenicity of the PvMSP-1(19) and points toward its possible use as a potential malaria vaccine.