DESIGN OF AN ORAL VACCINE AGAINST CHAGAS DISEASE COMPOSED BY A MULTICOMPONENT ANTIGEN AND A GRAS BACTERIUM

VAZQUEZ, MARIA ELISA; BARRIENTOS CONSTANZA; MASIAS RUTH EMILSE; ZABALA, BRENDA ANDREA; NATALIA CORBALAN; PIOLI MATIAS; ACUÑA, LEANDRO; CECILIA PEREZ BRANDAN

Encuentro; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Chagas disease (CD) is a neglected disease, endemic in Argentina, caused by a flagellar protozoan named Trypanosoma cruzi for which neither prevention nor treatment is available by a vaccine. One of our perspectives in this regard is the development of a multi-component vaccine. Previously, we constructed a chimeric fusion protein based on T. cruzi antigens named NTc52/TSKB20able to control parasitemia levels caused by T. cruzi in a mouse model. In this work, we present an approach that combines the adjuvant properties of a generally recognized as safe (GRAS) bacterium with the immunogenicity of NTc52/TSKB20 antigen. We developed a genetic construction between the chimeric antigen and the surface layer protein of Lactobacillus acidophilus SlpA to obtain a system that enables the antigen-self-assembly and adhesion on L. acidophilus surface. The gene encoding to SlpA was amplified by PCR from and fused to the gene encoding to NTc52/TSKB20 in an asymmetric PCR. After cloning, the recombinantplasmid derived of pRSETa containing the hybrid gene 6His-NTc52/TSKB20-SlpA was inserted into Escherichia coli BL21[DE3]. The expression was carried out by the addition of IPTG and cells were lysed by repetitive cycles of sonication, freezing and thawing. Subsequently, the lysate was centrifuged and the pellet, containing the protein in inclusion bodies, was resuspended in Urea 8M.The protein was purified through a Ni-NTA resin and dialyzed against PBS. The purified protein was added to L. acidophilus cells devoid of its S-layer by LiCl pre-treatment thus obtaining the vaccine formulation named lactos- NTc52/TSKB20-SlpA. The formulation was treated with digestive enzymes from mice to determine its endurance in a first approach to potential oral delivery.lactos-Tc52/TSKB20-SlpA diminished its ability to form colonies after treatment but chimeric antigen NTc52/TSKB20-SlpA kept up its integrity. An immunization scheme in mice was diagrammed to prove the potential activity of formulation by oral in comparison with subcutaneous administration. Animals were inoculated 3 times separated between 21 days. Blood was collectedbefore each dose and 21 days after last dose and mice were challenged with a lethal dose of T. cruzi trypomastigotes. Parasitemia were analyzed twice weekly for 25 days for tested vaccine efficiency and then animals were sacrificed. Samples were taken to assed the immune response.