Immunogenic evaluation of recombinant proteins expressed in Pichia pastoris based on Plasmodium vivax antigens from different parasite stages

Plasmodium vivax is the species of malaria more widely distributed worldwide and with higher prevalence in the Americas. The complexity of the parasite life cycle and its extensive antigenic diversity have hampered the achievement of an effective vaccine and infer that it is unlikely that this goal will be achieved using a single antigen. In this context, the combination of immunodominant regions of antigens of one or more stages of the Plasmodium life cycle can be a strategy with better prognosis at inducing protective and durable immune responses against this parasite. Our study assessed the immunogenicity of vaccine formulations consisting of mixture of antigens CSP, pre-erythrocytic and AMA-1, which is expressed in the both stages, pre-erythrocytic and erythrocytic asexual, in mice. The chimeric protein yPvCSAllFL, which contains B-cell epitopes of the central region (repeats) of the 3 allelic variants PvCSP-VK210, PvCSP-VK247 and PvCSP-P. vivax-like fused, and the yPvAMA-1 were successfully expressed in the yeast Pichia pastoris and purified by chromatographic methods for immunization of BALB/c and C57BL/6 mice in the presence of the adjuvant Poly(I:C), a TLR3 agonist. By ELISA, we determined the titles, IgG subclasses and the avidity of the antibodies to these proteins, administered alone or in combination. The immune response to yPvCSAllFL proved to be dependent on mouse strain, having been observed high titers of IgG antibodies (106) in C57BL/6, which remained high for up to 6 months after the last dose. Anti-yPvCSAllFL antibodies, predominantly IgG1, were able to recognize proteins representing the 3 allelic variants. In general, the co-administration of yPvCSAllFL and yPvAMA-1 antigens did not compromise the individual antibodies response. Using this vaccination protocol, we could not detect cell specific proliferative responses of TCD3+TCD4+ or TCD3+TCD8+ after stimulation with yPvCSAllFL. The proliferation (8.31%) and the pattern of secretion of cytokines IFN-γ, IL-2, TNF-α and IL-10, associated with the yPvAMA-1, were reduced during the co-administration (6.33%) and compensated by the elevation of IL-2. The data generated on the study of vaccine formulations presented in this thesis may be useful for the development of a vaccine anti-P. vivax, mainly by exploiting strategies of combination and fusion of antigens. (AU)