Geração de proteínas recombinantes e aplicação em testes sorológicos para detecção de anticorpos anti-Chikungunya, Zika e SARS-CoV-2.

Abstract

Serological tests, such as enzyme-linked immunosorbent assay (ELISA) and rapid tests available in Brazil are dependent on foreign technology. The main imported raw material is the antigen, which makes the diagnosis more expensive, less accessible and subject to availability fluctuation due to international demands. This study aims to generate antigens to detect the presence of specific antibodies against CHIKV, ZIKV and SARS-CoV-2 using diagnostic tools. The nucleotide coding sequences of proteins from these viruses were analyzed in silico and expressed in different expression systems (prokaryotic and eukaryotic). The ZIKV-E protein produced in E.coli was not able to differentiate sera from infected patients and healthy individuals. However, the same protein produced in a baculovirus system showed potential for ELISA applicability (100% accuracy for a preliminary experiment, using eight positive and seven negative samples). An ELISA to detect antibodies against CHIKV allowed the comparison between the E2 protein produced in two different expression systems: E.coli (procaryotic) versus HEK-293T (eucaryotic). The IgG ELISA accuracy was higher than 90% for both versions of E2 and the antigen of the prokaryotic system was considered preferable because it had a higher yield option. In contrast, for IgM, the accuracy of the E2 protein produced in E.coli was lower when compared to the same protein produced in HEK-293T (75% and 90%, respectively). Also, SARS-CoV-2 nucleocapsid (N) and a truncated Spike (S1) was produced in E.coli. The S1 protein presented lower yields, insolubility, and poorer performances to recognize SARS-CoV-2-positive sera when compared to the N protein. Seroconversion patterns against the in-house N and commercial S proteins were evaluated in COVID-19 patients. An immunoassay using N protein (EIE COVID19 IgG) was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (93%) and specificity (100%) when compared to a commercial reference kit. During the development of this work, a prototype of a rapid test and two ELISA kits using antigens produced at CT-Vacinas have been developed. The EIE COVID19 IgG technology was validated and transferred to the Bio-Manguinhos Institute and is currently under the registration process at the Brazilian Health Regulatory Agency (ANVISA)as well as the CHIKV ELISA kit.