Determining optimal conditions for growing recombinant vectors to be used in developing a bovine tuberculosis vaccine

Abstract

Two recombinant influenza A virus vectors expressing the ESAT 6 and TB10.4 mycobacterial proteins from the nonstructural (NS) gene were constructed via reverse genetics technique to develop a specific means of prophylaxis for bovine tuberculosis. We experimented to determine optimal conditions for growing recombinant vectors in Vero cell culture and chick embryos. This study established that the maximum amount of virus builds up in a Vero cell culture with the Dulbecco′s Modified Eagle′s Medium (DMEM) serum-free medium. However, using cell culture to produce vector vaccines is labourintensive and inefficient. An alternative way, a traditional, time-tested technique, is provided by growing samples in chick embryos. One of the advantages of this technique is its affordability and availability, enabling easy scale-up of vaccine production. In the optimization experiments, the FLU-ΔNS_ESAT 6 and FLU-ΔNS_ТВ10.4 viruses constructed were inoculated into 10-day-old chick embryos. It was determined that the optimal incubation temperature that led to the highest virus build-up was 37 ± 0.5 °С. And the infectious activity level of the FLU-ΔNS_ESAT 6 recombinant vector was at 8.95 ± 0.07 log10EID50 0.2 cm-3, while that of the FLU-ΔNS_ТВ10.4 was at 9.20 ± 0.07 log10EID50 0.2 cm-3, what was provided by infectious doses of 1000–10000 EID50, which makes it possible to create a virus-containing material with a hemagglutination activity level of 1:64. The size of recombinant vector amplicons expressing proteins ТВ10.4 and ESAT 6 was 1170 bp and 1175 bp, respectively. Electron microscopy images confirm that the developed virions are morphologically similar to the avian influenza virus.