Cellular and humoral immune responses against the Plasmodium vivax MSP-1₁₉ malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract

Background: Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil.

Methods: The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-1₁₉) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay.

Results: The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-1₁₉ and PSS1 antigen. A low proliferative response against PvMSP-1₁₉ and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-1₁₉ stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient's cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-1₁₉ was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses.

Conclusions: The results presented here shows that PvMSP-1₁₉ was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-1₁₉ in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Figure 1

Ex-vivo analysis of the expression of IL-2R in PBMC by cytometry flow. (A) PBMC from malaria patients (n = 34) and control individuals (n = 16) and (B) PBMC from individuals infected with P. falciparum, P. vivax and control individuals. *p = 0.01, malaria versus control individuals; ** p = 0.009 P vivax versus control individuals. Lines represent geometric mean.

Figure 2

Ex-vivo analysis of the expression of IL-2R by cytometry flow in T cells subpopulations. CD4⁺/IL2R + and CD8⁺/IL2R + T cells from (A) malaria patients (n = 34), (B) individuals infected with P. falciparum and (C) individuals infected with P. vivax. CD4⁺versus CD8⁺: *p < 0.0001, ** p = 0.0006, *** p < 0.0001. Lines represent geometric mean.

Figure 3

Analysis of cellular activation by the expression of IL-2R after 96 h culture in presence of PvMSP-1₁₉ and PSS1 crude antigen by cytometry flow. (A) PBMC from malaria patients (n = 35) and control individuals (n = 17) and (B) PBMC from individuals infected with P. falciparum, P. vivax and control individuals. *p = 0.001 for PvMSP1₁₉ and p = 0.0003 for PSS1, malaria versus control individuals; ** p = 0.009 for P. falciparum versus controls and p = 0.003 for P. vivax versus controls; *** p = 0.004 for P. falciparum versus controls and p = 0.001 for P. vivax versus controls.

Figure 4

Cytometry flow analysis of T cells activation (IL-2R⁺) after 96 h of culture in the presence of PvMSP-1₁₉ and PSS1 crude antigen. CD4⁺/IL2R + and CD8⁺/IL2R + T cells from (A) malaria patients (n = 35); individuals infected with P. falciparum and P. vivax in presence of (B) PvMSP1₁₉ and (C) PSS1. *p = 0.0002 for PvMSP1₁₉ and p = 0.003 for PSS1, CD4⁺versus CD8⁺; **p = 0.02 for P. falciparum and p = 0.003 for P.vivax CD4⁺ versus CD8+; ***p = 0.02 for P. vivax.

Figure 5

Analysis of cellular proliferation after 96 hour culture by cytometry flow using a CFSE vital staining. PBMC from malaria patients and control individuals in the absence or presence of plasmodial antigens (PvMSP-1₁₉ and PSS1 crude antigen) or PHA mitogen. *p = 0.02 malaria versus control individuals

Figure 6

Plasmatic concentrations of cytokines evaluated by ELISA assays. Levels of TNF, IFN-γ and IL-10 in plasma samples from control individuals, malaria patients, individuals infected with P. falciparum and individuals infected with P. vivax. *p = 0.01 control versus malaria individuals and control versus P. vivax; ** p = 0.001 for controls versus malaria; p = 0.0007 control versus P. vivax; *** p < 0.0001 controls versus malaria and controls versus P. vivax; p = 0.001 controls versus P. falciparum.

Figure 7

Plasmatic concentrations of IFN-γ. Levels of IFN-γ in plasma samples from P. falciparum and P. vivax malaria patients. *p = 0.03, P. falciparum versus P. vivax.

Figure 8

Analysis of cytokines concentrations in plasma and in supernatant of the cultures measured by ELISA. Levels of TNF, IFN-γ and IL-10 in plasma samples and in supernatant of the PBMC cultures from (A) control individuals (n = 17) and (B) malaria patients (n = 35). *p = 0.03, PvMSP-1₁₉versus PSS1; p = 0.0006, PvMSP-1₁₉versus without stimulus.

Figure 9

Analysis of antibodies in sera from malaria patients. Frequency of malaria patients (n = 35) with IgG, IgM and IgE antibodies against PvMSP-1₁₉. *p = 0.02, IgM versus IgE.

Figure 10

Analysis of IgG, IgM and IgE antibodies levels against PvMSP-1₁₉ in malaria patients.*p = 0.005, IgM versus IgG. Lines represent mean with standard deviation. Continuous line represents cut off.

Figure 11

Prevalence of antibody responses against PvMSP-1₁₉ in malaria infected individuals. Frequency of IgG, IgM and IgE antibodies in individuals with P. vivax or P. falciparum malaria. * p = 0.02 for IgE, p = 0.03 for IgM and IgG, P. vivax versus P. falciparum.

Figure 12

Levels of IgG, IgM and IgE antibodies. Analysis of antibodies levels against PvMSP-1₁₉ in P. vivax- or P. falciparum- infected individuals. * p = 0.01 for IgG, p = 0.04 for IgM, p = 0.003 for IgE, P. vivax versus P. falciparum. Lines represent mean with standard deviation.

Figure 13

Frequency of IgG subclasses. Prevalence of IgG1, IgG2, IgG3 and IgG4 antibodies against PvMSP-1₁₉ in IgG-positive malaria patients (n = 35). * p = 0.001, IgG3 versus IgG1; p < 0.0001, IgG3 versus IgG2 and IgG4.

Figure 14

Levels of IgG1, IgG2, IgG3 and IgG4 antibodies against PvMSP-1₁₉ in malaria patients. * p = 0.04, IgG4 versus IgG1; p = 0.03, IgG4 versus IgG2; p = 0.01, IgG4 versus IgG3. Lines represent mean with standard deviation. Dotted line represents cut off.