Avaliação do papel de genes imunorreguladores (B18R, D9R e D10R) em infecções causadas por amostras de Vaccinia virus: utilização de vírus recombinantes como ferramenta de análise

Abstract

The Orthopoxvirus (OPXV) are part of the Poxviridae family comprising large, complex viruses having double-stranded DNA genomes and with viral multiplication taking place in the cytoplasm of the host cell. In order to escape host immune responses, the OPXV have genes encoding immune evasion proteins. These viruses block or interfere with many innate immunity mediators including IFNs, TNFs, interleukins (IL), the complement system, chemokines, as well as different cell signaling pathways. The MVA virus, an OPXV, was obtained after more than 500 successive passages of the chorioalantoid virus Vaccinia Ankara (CVA) in cultures of chicken embryo fibroblasts (CEF). Following these passages the MVA lost about 15% of its parental genome and became unable to replicate productively in most mammalian cell lines. This makes MVA an excellent vaccine vector candidate. To determine and explore the immunomodulatory genes that are missing in the MVA, it is essential to completely understand the interaction between VACV and the host antiviral response. Even though the safety and efficacy of the MVA as a vaccine vector are well established, the genetic basis that defines these features are not totally known. Since the MVA was generated through an attenuation process by random deletion, studying each gene individually would allow us to better understand its role in virulence. This knowledge would enable the development of new viral vectors in a more rational way, through specific gene deletion. Thus, the aim of this work was to evaluate the role of the genes B18R (soluble type I IFN receptor), D9R and D10R (mRNA decapping enzymes) by in vitro characterization of different constructions of the Western Reserve (WR) and MVA expressing or not those genes. We also aimed to compare the immunogenicity of these viruses in a murine model of infection. We observed that the inactivation of the catalytic sites of the enzymes D9 and D10 of the MVA virus delayed the transition between the early and late stages of the viral cycle. Furthermore, the interruption of cellular protein synthesis caused a buildup of dsRNA in BS-C-1 cells infected by the double mutant virus. This build-up led to the activation of cell signaling pathways, triggering the antiviral response that primarily disrupts protein synthesis in infected cells and stimulates the production of type I IFN. It could be possible that the MVAD9muD10mu engineered here is more immunogenic than the original sample, and therefore a good vaccine vector candidate. We saw no changes in virulence or plaque phenotype between the MVA samples that carried or lacked the B18R gene. In addition, the insertion of the B18R gene did not cause significant changes in the activation of dendritic cells and lymphocytes. With respect to the virus WR, the deletion caused a significant decrease in virulence of the WRΔB18 sample when compared to the original virus. Based on the results obtained from the analyzed parameters we can conclude that the absence of the protein B18 in the WR virus leads to an increase in DC activation and a reduction in the activation of T lymphocytes and B cells.