DEVELOPMENT OF A NEW VACCINE CANDIDATE AGAINST YELLOW FEVER BASED ON BACULOVIRUS-SURFACE DISPLAY

ARRIAS PN; THOMAS PT; BACIGALUPO L; CALDERONE M; PIDRE ML; ROMANOWSKI V; CARRERA SILVA EA; GOMEZ RM; FERRER MF

Otro; Reunión Conjunta SAIC. SAI. AAFE. NANOMED-AR; 2021

SAIC. SAI. AAFE. NANOMED-AR

Yellow Fever (YF) is a disease caused by the homonymous flavivirus. There is an excellent attenuated virus vaccine for the prevention of this disease. However, this vaccine cannot be administered to immunocompromised and pregnant individuals. For this reason, development of new alternatives is of the utmost importance. The aim of this work was to generate a new generation vaccine candidate based on a recombinant baculovirus (rBV) that expresses YFV protein E and NS1 on its surface capable of inducing both humoral (protein E) and cellular (NS1) immune responses. To this end, a pBacPak9 vector containing the BV GP64 signal peptide, YFV?s prM, E, NS1 open reading frames and the ectodomain of VSV-G was synthetically constructed. This plasmid and the bApGOZA bacmid were co-transfected in High Five cells. Between 5 to 7 days post-transfection, characteristic BV infection signs (polyhedra and cytopathic effect) were observed. Culture supernatant was harvested and used to infect new cultures. When the aforementioned infection signs firstly appeared in the subsequent infection, the supernatant was again collected. This procedure was repeated 3 times. The rBV was recovered from the supernants and used to make a viral stock. Viral genome DNA extraction from the stock was subjected to PCR with specific primers to confirm the incorporation of the target genes in the rBV genome. Protein expression was confirmed by western blot using an anti-E polyclonal antibody (GTX134024, Genetex) using another rBV carrying YFV prM-E genes as a positive control. The stock was then escalated and concentrated by ultracentrifugation at 80.000g for 1:15 hs in a 25%w/w sucrose cushion and the pellet was resuspended in PBS and filtered using a 0.2µm filter. This stock was titrated using the Sf9-GFP transgenic reporter line reaching 105.5 virus/ml. In conclusion, we have generated a valid YFV vaccinate candidate to be tested in a murine model.