Construção de um vírus recombinante Vaccínia Ankara Modificado (MVA) expressando a Enzima Conversora de Angiotensina 2 (ACE2) humana

Abstract

In December 2019, a new member of the Coronaviridae family, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China, and triggered a new type of viral pneumonia called coronavirus disease 2019 (COVID-19). The disease is currently classified as a pandemic and is the sixth public health emergency of international concern ever declared by the World Health Organization. Thus, herculean efforts have been undertaken by researchers around the world to elucidate the infection and pathogenesis of SARS-CoV-2 and to outline effective vaccination strategies and antiviral therapies. Modified Vaccinia Ankara virus (MVA) is one of the best-studied poxvirus vectors used worldwide for expression of heterologous genes. In view of the above, the present work aimed at the construction of a recombinant MVA (rMVA) expressing the human angiotensin II converting enzyme (hACE2), a protein used by SARS-CoV-2 to infect host cells, for the creation of a model of experimental virus infection. In summary, cultures of BHK-21 cells were infected with wild-type MVA (MVA-WT) and transfected with the transfer plasmid containing the human ACE2 cDNA, pLW44-ACE2, generating through homologous recombination, the recombinant MVA virus expressing hACE2 (rMVA-hACE2). Recombinant viruses underwent rounds of plaque purification to choose the most robust rMVA-hACE2 clones and eliminate MVA-WT; for further gradual amplification of the virus. Parallel to the selection technique in semi-solid medium, the use of the Cell sorting technique was proposed. The use of the new technique was effective in obtaining clones, in addition to allowing a more efficient and faster amplification. The constructed recombinant virus had protein expression confirmed by flow cytometry and subsequently purified and titrated. The data obtained suggest a reformulation of the technique for producing recombinant MVAs and confirm the success of the rMVA-hACE2 construct, making it eligible for the proposed construction of an experimental infection model for SARS-CoV-2.