BACULOVIRUS WITH A DELETION IN THE AC109 GENE PRODUCES HIGH LEVELS OF HETEROLOGOUS PROTEIN AND MINIMAL IMPACT ON THE ANTIVIRAL RESPONSE IN MAMMALS

AMALFI SABRINA; MATTERA RAMIRO EZEQUIEL; PLASTINE MARÍA DEL PILAR; LOPEZ MARÍA GABRIELA; OSCAR TABOGA; ALFONSO VICTORIA

Congreso; LVII SAIB Meeting - XVI SAMIGE Meeting; 2021

Baculoviruses are enveloped viruses, with double-stranded DNA genomes, pathogens of insects. The Baculovirus Expression Vector System (BEVS) is a widely used eukaryotic expression system for the production of recombinant proteins in insect cells to be used as supplies in human and veterinary medicine. In particular, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is one of the most used and studied members of the Baculoviridae family. In a previous study, we described ac109 of AcMNPV as an essential gene that codifies a structural protein required for the production of infective viral progeny. Thus, this work aimed to employ an ac109 knockout virus (Ac109KO) to obtain recombinant proteins with minimal levels of co-produced virions. First, to determine the best strategy to produce infectious Ac109KO viruses, co-transfections of an Ac109KO bacmid that expressed a reporter gene with plasmids containing ac109 gene under the regulation of different promoters were performed. As a result, we observed that the use of a constitutive promoter did not produce any virions. On the contrary, the ac109 promoter and the very late polyhedrin promoter boosted by upstream regulatory sequences (XXLPolh) produced high levels of complemented viruses. Next, to determine the presence and percentage of recombination between Ac109KO bacmid and each of the plasmids, co-transfection supernatants were evaluated by a viral plaque assay that allowed the differentiation of plaques from single infected cells. Due to the regeneration of wild-type viruses with frequencies between 4 and 10%, Ac109 expression vectors with a modified codon usage were constructed and evaluated. Transient expression assays showed that the recombination rates decreased by half. Next, we constructed a transgenic Sf9 cell line containing the modified ac109 sequence regulated by XXLPolh, and observed that the recombination rate drastically decreased below 1%. This strategy also rendered higher titers of complemented virus, although approximately 9-fold lower than the obtained from control virus in Sf9 cells. Finally, to analyze protein production in this system, the reporter gene expression was evaluated by fluorescence microscopy and flow cytometry. Sf9 cells were infected with supernatants from transgenic Sf9 cell line transfected with the Ac109KO bacmid or from Sf9 cells transfected with control bacmid. At 48 hours post-infection, the trans-complemented Ac109KO virus showed similar levels of protein expression as the control virus. In addition, supernatants of infection with complemented Ac109KO in NIH/3T3 cells induced approximately 5-fold less IFN β mRNA production than the control virus. In conclusion, we describe a recombinant protein system based on defective Ac109KO baculovirus that enables the production of proteins at levels comparable to the control virus and the induction of a minimal impact on the mammalian antiviral response.