In the present work, the profile of acquired cellular and antibody immune responses against PvMSP-119 was evaluated in individuals naturally exposed to P. vivax and P. falciparum infections in a malaria-endemic area in the north-western Amazon region of Brazil.
It is well known that suboptimal cryopreservation could result in a significant decrease of cell number and viability, causing alterations on the cellular phenotype and on the immune response to specific antigens. To avoid troublesome events, a protocol to successfully recovery PBMC after freezing and thawing was previously standardized . Thus, in the present study, an important frequency of mononuclear cell viability after thawing was observed (around 78%), regardless the individuals being infected or not. Because 75% PBMC viability is required for lymphocyte proliferation assays, representing the ability of cells to respond to the antigenic or mitogenic stimulation, independently of the nature or intensity of the stimulus , the cryopreservation procedure seems not have been a limiting factor in the present study.
The phenotypic analysis of ex-vivo PBMC revealed that CD4+ T cells were found more frequently than CD8+ and CD20+ cells, in both malaria and control individuals. In malaria individuals, a higher prevalence of activated CD4+ than CD8+ T cells was observed, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. It can be speculated that CD4+ T cells were the most stimulated cells due the nature of antigenic stimulus because CD4+ T cells are activated by the parasite in blood stage, while CD8+ T cells are most often activated during the hepatic stage of parasite life cycle .
Low proliferative responses in the presence of MSP-119 of P. falciparum and Plasmodium chabaudi chabaudi have already been demonstrated [37, 38]. The results also showed a low proliferative response against PSS1 crude antigen or PvMSP-119 after 96 h culture. This low proliferative response may occur because the majority of activated cells undergo activated-induced cell death, an active cell suicide mechanism of widespread biological importance that constitutes the physiological response of normal cells to activation and is believed to control the number of antigen-stimulated cells during the immune response .
Anti-inflammatory cytokines are involved in a feedback mechanism to regulate the expression of pro-inflammatory cytokines, and prevent the pathological effects that may result from their continuous secretion. Previous in vitro studies have shown that IL-10 suppresses the expression of malaria parasite-induced production of TNF by PBMC . In fact, in the present study, high plasmatic levels of IL-10 and low plasmatic levels of TNF were observed in malaria patients.
Parasite clearance seems to be related to IL-10 and IFN-γ levels. Here, an intense secretion of IFN-γ and IL-10 as detected by dosage of plasmatic levels, were shown in malaria patients. However no correlation was found between parasitaemia and IFN-γ or IL-10 levels. The high levels of IL-10 and IFN-γ are in agreement with previous clinical reports that IL-10 is up-regulated in concern with IFN-γ [41, 42], suggesting that IL-10 may be up-regulated as a direct consequence of IFN-γ production as part of homeostatic feedback mechanism to limit IFN-γ-mediated pathology, as is seen in murine malaria infections .
IL-10 levels were similar in P. vivax and P. falciparum infected individuals. However, besides plasmodial species, other factors, like the number of previous malaria infections, may influence the levels of this and other cytokines. In fact, a negative correlation between number of previous malaria infections and IL-10 levels was found. In addition, previous studies have shown that an adaptive type 1 regulatory CD4+ cells have been identified as the main source of IL-10 in experimental murine infection with Plasmodium yoelii, while in P. chabaudi infections in mice, an activated effector TH1 cells were major IL-10 producers . Understanding how these regulatory cells are induced could help to explain differences in IL-10 production in human malaria.
High production of TNF in malaria individuals is related to the development of severe malaria. In the present work, lower than expected plasmatic TNF levels (lower than those observed in control individuals) were detected in malaria patients. Interestingly, PvMSP-119-stimulated PBMCs from malaria patients showed higher TNF response than those recorded in both PSS1-stimulated and non-stimulated PBMCs. The production of TNF in recall to PvMSP-119 may suggest a protective role of PvMSP-119 immune response because TNF has been related to parasite clearance.
A great majority of studied individuals presented antibodies against PvMSP-119, independently of the reported number of previous malaria attacks or the time of residence in a malaria-endemic area, indicating that this protein is immunogenic in natural conditions of exposure and seems to be independent of the time of exposure. The high immunogenicity of PvMSP-119 may be reflex of its limited polymorphism, because independent groups demonstrated that PvMSP-119 is conserved [17, 46, 47]. Another possible explanation is that PvMSP-119 is the single fragment that remains on the parasite surface during red cell invasion through glycosylphosphatidylinositol (GPI) anchor  and studies have described GPI as a potent agonists of toll-like receptors that may provide the adjuvant required for stronger immune responses .
Anti-PvMSP-119 antibodies were not detected in the sera of five individuals from malaria group (one with vivax and four with falciparum malaria). The absence of anti-PvMSP-119 antibodies could be related to immunosuppression observed in humans in malaria infection [49, 50], however, this hypothesis seems unlikely because these non-responders individuals presented antibodies against PSS1 crude antigen. Another possible explanation for the absence of these antibodies is that PvMSP-119-specific B cells were present, although circulating antibodies titers were undetectable. Alternatively, the lack of PvMSP-119 antibody response may be due the genetic restriction of immune response that has been described to several plasmodial recombinant proteins and synthetic peptides [24, 51–55]. However, in a recent study performed in the Brazilian Amazon region, no association between HLADRB1* and HLADQB1* allelic groups and the antibody response against PvMSP-119 was found .
Considering that different Plasmodium antigens in the same population as well as the same Plasmodium antigen in different populations can induce different antibody profiles, it was also evaluated the frequency and levels of IgG, IgM and IgE class and IgG subclass against PvMSP-119. IgM antibodies were the most prevalent and the one with the highest levels. PvMSP-119-induced IgG antibodies were predominantly of non-cytophilic subclasses. Different data have been reported for individuals living in other Brazilian-endemic areas with different levels of exposure where the PvMSP-119-induced antibodies were predominantly of IgG1 subclass [20, 23]. Differences in antibody profile may be due to transmission intensity. In fact, previous study has demonstrated that the levels of IgG1 and IgG3 specific antibodies were low among individuals with long-term exposure (~19 years) when compared to subjects less and sporadically exposed (< 1 year) . In this concern, one must emphasize that the individuals from malaria group claimed living in malaria endemic area for 19 years. Another additional hypothesis could be related to cytokine modulation of specific antibodies production because IL-10 has been reported to demonstrate both potentiating and inhibiting IgE and increasing IgG4 productions  as well as to be associated with IgM antibodies against P. vivax. In addition, associations between polymorphism in cytokine genes and anti-plasmodial antibody response have been reported . One other possibility could be the context of the response because co-infection with helminths is known to shift the Th1 to the Th2 pattern of immune response modulating the IgG subclass expression. Independently of its origin, the differences between antibody profile reported in the present study and the aforementioned underline the importance of conducting immunoepidemiological studies in different malaria endemic areas where transmission intensities and human genetic background are different.
Associations between antibody responses with time of residence and/or number of previous episodes have been commonly reported to several malaria antigens [52, 58, 59]. However, in the present study, no association between levels of PvMSP-119 antibodies and number of previous malaria episodes was observed. This finding may reflect that people living for a longer period of time in the region may have acquired some degree of clinical immunity after experiencing a number of infections, therefore, reporting less episodes of clinical malaria in the more recent years. Interestingly, only the levels of IgE antibodies were inversely correlated with the number of previous malaria episodes. Thus, it can be supposed that higher levels of IgE antibodies may have an important role against clinical malaria. Similar results were already reported that shown that high levels of malaria specific IgE were associated with reduced risk for subsequent clinical malaria episodes .
In this study the frequency and the levels of IgG, IgM and IgE antibodies against PvMSP-119 were higher in individuals infected with P. vivax than P. falciparum or who had reported the last malaria episode due P. vivax. Similar results were reported elsewhere where sera from subjects who had had P. vivax in their last malaria clinical episode presented higher levels of antibodies when compared with those whose last malaria episode was due to P. falciparum.
The individuals infected with P. falciparum studied in this work reported previous infection with P. vivax. This may explain the recognition of PvMSP119 antigen by antibodies from individuals infected with P. falciparum. However, the similarity between PfMSP1 and PvMSP1 antigens may also explain, at least in part, the recognition of PvMSP1 by patients infected with P. falciparum, although a recent study has shown that sera from immunized mice with PfMSP119 or PvMSP119 failed to cross-react with heterologous antigen .
In conclusion, the results presented here shows that PvMSP-119 was able to induce a high cellular activation leading to production of TNF, emphasizing the high immunogenicity of PvMSP-119 in naturally exposed individuals and therefore its potential as a malaria vaccine candidate.