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The 145 individuals exposed to P. vivax malaria included: (i) 18 healthy blood donors from areas with P. vivax endemicity: 11 adults from the municipalities of São Luís, São José do Cabo and São Francisco de Paula, all in the State of Maranhão, and seven from the municipality of Rio Branco in the State of Acre; (ii) 35 patients with a recognized clinical episode of P. vivax malaria caused by antibodies passively acquired during the first year of life and ten months later active infection (age range, 7 to 19 years; mean, 14.5 years) and spontaneously resolved without drugs. None of these patients presented any clinical evidence of peripheral blood involvement or complex neurological manifestations during the first day of follow-up. They were admitted to the hospital between 2 and 12 days after fever onset with malaria symptoms ranging from mild to severe. They used no drugs to treat the episodes. All the antigens were used as antigenic extracts from rings of third-generation infections (mature forms with a DBL2-variant NANANA composed of the binding sequences RIIIB and RIVMIIIA). The extracted antigens were used at different dilutions: 1:100, 1:1000, and 1:5000. The positive control was the antigenic extract of P. falciparum, the negative control was the antigenic extract of a parasite strain with absence of DBL2-variant NANANA. All the samples were processed by ELISA as described before. Individuals in group A were born and raised in the malaria transmission areas and had no previous contact with P. vivax. They were contacted by the Laboratório de Microparasitologia do Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, and had no antimalarial prophylaxis in their lives. For the lymphoproliferative responses (LRP), the individuals with the highest antibody responses were selected for analysis of cytokine production by antigen-specific polyclonal T-lymphocyte assays. The studies were performed at the Laboratory of Leishmania Immunobiology at the Oswaldo Cruz Foundation. All the experimental protocols were approved by the Local Committee for the Use of Animals of the Oswaldo Cruz Foundation (CEUA no. 218/13) and the Brazilian Institute of Environmental and Renewable Natural Resources (IBAMA no. 0001.02.00.00). Peripheral blood mononuclear cells from healthy individuals were isolated using Ficoll gradient centrifugation. After the removal of red blood cells, the peripheral blood mononuclear cells were washed and resuspended in RPMI 1640 medium supplemented with 2% heat-inactivated human fetal bovine serum, 100 μg/mL streptomycin, 100 U/mL penicillin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol. The non-exposed individuals were selected on the basis of age, sex, and living area, and all of them were negative for malaria infection, as detected by microscopy or by the rapid test (BKDT). The individuals with P. vivax malaria were selected, considering the quantity of parasites in the blood, the age of the individuals, and the existence of the genetic polymorphism in the sequences of the DBL2-variant NANANA gene of the infecting strain. 3d9ccd7d82
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