Estudios sobre la inmunogenicidad y los mecanismos fisiopatológicos de la proteína NSP4 de rotavirus.

Abstract

The baculovirus expression system in insect cells was utilized to produce and purify the biologically active form of the NSP4 protein from rotavirus strains isolated from different animal and human strains. After the immunization of mice with the different ourified NSP4 proteins the super-immune sera obtained from NSP4 immunized mice, strongly recognize the carboxi-end of the protein (aminoacids 114 to 175), being indicative of the immunodominance of this region. One of the objebtives of this thesis was to obtain monoclonal antibodies, using the phage display technique that showed to be a useful tool in producing antibodies that recognize different epitopes in the NSP4 protein, thus being able to conduct inhibitory studies to identify biological activity. One interesting result when using this technique was that the phage display technique allowed us for the isolation of single chain monoclonal antibodies against non-immunodominant regions of NSP4. We also present data on the immunogenicity of the NSP4 protein in natural rotavirus infections in humans. Our results show that the NSP4 protein elicits a humoral immune response in children that had been naturally infected by rotavirus. The IgG serical antibodies developed against the NSP4 in children suffering acute rotavirus gastroenteritis, present cross reactivity against more than one genotype of the protein in 40% of the studied cases, indicating that the recognition of A and B NSP4 genotypes is not always heterotypic. Finally we studied the possible role of the nitric oxide (NO) in rotavirus pathophysiology. The NSP4 protein produced an increase in NO secretion in cultured HT-29 human epithelial cells. This increase occurred in parallel to the intracellular calcium increase previously observed in the same cell line in response to the NSP4. The rotaviral infections in Blab/c mice produced an increase in the concentration of the nitric oxide derived products in the urine of the infected animals, through the induction of the inducible nitric oxide enzyme (iNOS) in the murine model. This increase was also observed in the urine of children suffering acute rotaviral gastroenteritis.