Caracterização de antígenos e produção de anticorpos anti-peptídeos dos vírus dengue, Zika e chikungunya

Abstract

Dengue, Zika and chikungunya viruses are responsible for outbreaks in several countries. In Brazil, the population has already faced simultaneous epidemics of these diseases. Diagnosis type are similar between diseases and may range between serological tests, RT-PCR and antigen tests. Currently, part of the efforts are focused on the development of low-cost tests, with quick results and that not require a laboratory structure, thus facilitating their execution in regions with few resources. Therefore, the antigen test is a great candidate, appearing as an alternative to established tests. DENV NS1, ZIKV NS1 and CHIKV E2 are viral proteins that are the focus of studies for the development of antigen detection kits, as they are more easily detected in infected samples compared to other proteins. Thus, in silico predictions were performed using bioinformatics tools in order to know which regions of these proteins were antigenic, corresponding to an epitope, were accessible, hydrophilic and were not present in a β-turn. The 2 predicted peptides of each protein that achieved the greatest number of desired characteristics were synthesized and characterized by HPLC and mass spectrometry. Then, they were inoculated into rabbits to produce antibodies against each antigen. Sera were collected, and Dot blots and Western blots were performed. The predicted peptides were 103SLRPQPTELKY113 (DENV-2 NS1), 111YKYSWKSWGKA121 (DENV-1 NS1), 238ESDLIIPKSLAGPLSH253 and 83GVQLTVVVGS92 (ZIKV NS1), 24EGHSCHSPVAL34 and 147HGKELPCSTYVQS159 (CHIKV E2). From Dot blot results it could be seen that all sera produced presented antipeptide antibodies, seeing that all showed interaction with the peptides. As the Western blot used full length proteins instead of peptides, some results diverged from the previous one. Of all produced sera, anti-SLR, antiYKY and anti-ESD showed interaction with full length proteins expressed in Escherichia coli and HEK293 cells, with the exception of sera against CHIKV E2 peptides, which were tested only with the expressed protein in insect cells containing recombinant baculovirus, and the anti-EGH serum reacted to protein.