Modified vaccinia virus Ankara (MVA) is a highly attenuated, replication defective strain of Vaccinia virus with the attributes of an optimal viral vaccine delivery system. An improved vaccine for the prevention of tuberculosis (TB) is urgently required and until recently, the most advanced clinical candidate under development for use against TB was based on MVA vectoring a single mycobacterial antigen (MVA85A). Unfortunately, pre-clinical efficacy and potent immunogenicity in humans did not lead to improved protection in BCG vaccinated infants. There are a number of design features to consider in the development of a recombinant MVA (rMVA)-based vaccine. The current study undertook to assess how these might impact upon the immunological potency of MVA vectoring TB antigens. Specifically, the influence of vaccinia promoter selection and leader sequences was assessed. Vaccinia promoter selection influences antigen transcription and thereby expression. The relationship between antigen production, murine immunogenicity and protective efficacy was evaluated. The results support the conclusion that vaccinia promoter optimisation can increase immunological potency and efficacy. Virally vectored antigens can be fused to sorting signals for optimised intracellular processing. The immunomodulatory effects of two leader sequences were compared. The results suggest that the sorting signal currently incorporated in virally vectored TB candidates, such as MVA85A, might be subject to further optimisation for the induction of enhanced T cell responses. Overall, the results demonstrate that rMVA immunogenicity is intrinsically linked to the precise nature of transgene insertion. The value of an optimised vaccinia promoter might be to reduce the dose of rMVA required. Leader sequences represent an opportunity to enhance the quality of the antigen-specific response for improved protection. It is concluded that both vaccinia promoter selection and leader sequence optimisation should be a feature of future rMVA TB vaccine development
