Evaluation of recombinant BCG expressing rotavirus VP6 as an anti-rotavirus vaccine

Abstract

Recombinant BCG expressing rotavirus VP6 was explored as an anti-rotavirus vaccine in a mouse model. Three promoters and five ribosome-binding sites were used in episomal and integrative E. coli-mycobacterium shuttle vectors to express VP6 in BCG. The VP6 gene was configured for accumulation within the BCG cytoplasm, secretion from the BCG cell or targeting to the BCG cell membrane. Vectors were assessed in terms of stability, levels of antigen production, immunogenicity and protection in mice. Gross instability occurred in episomal vectors utilizing the hsp60 promoter. However, three integrative vectors using the same expression system and two episomal vectors using inducible promoters were successfully recovered from BCG. Growth rates of the former were not detectably reduced. Growth rates of the latter were considerably reduced, implying the existence of a significant metabolic load. In the absence of selection, loss rate of these plasmids was high. VP6 production levels (0.04-1.78% of total cytoplasmic protein) were on the lower end of the range reported for other rBCG. One episomal and one integrated vaccine reduced viral shedding in intraperitoneally vaccinated mice challenged with rotavirus. Compared to controls, infection-associated faecal shedding of virus was reduced by 66% and 62%, respectively. These protective vectors differ in promoter, ribosome-binding site and antigen production level, but both link the VP6 protein to the 19kDa lipoprotein signal sequence, suggesting that transport of VP6 to the BCG membrane is important for induction of a protective immune response. Protection occurred in the absence of detectable anti-rotavirus antibody in serum or faeces, implicating cellular immunity in protection.