Construction of an unmarked recombinant BCG expressing a pertussis antigen by auxotrophic complementation: protection against Bordetella pertussis challenge in neonates

Mycobacterium bovis BCG has long been investigated as a candidate for heterologous antigen presentation. We have previously described an rBCG-Pertussis that confers protection against challenge with Bordetella pertussis in neonate and adult mice. In order to obtain stable expression in vivo , we constructed an unmarked BCG lysine auxotrophic and a complementation vector containing the lysine and the genetically detoxified S1 pertussis toxin genes, both under control of the same promoter. Complemented BCG-Δlysine growth and expression of the pertussis antigen were stable, without the use of an antibiotic marker. Our results show that the complemented rBCG-ΔlysA-S1PT-lysA + (kan − ), which is now suitable to be evaluated in clinical trials, maintains similar characteristics of the original rBCG-pNL71S1PT strain, such as the antigen expression level, cellular immune response and protection against the same model challenge in neonatal-immunized mice.