Vaccinia expression of Mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of M-tuberculosis Ag85

There is increasing evidence to implicate a role for CD8(+) T cells in prot ective immunity against tuberculosis. Recombinant vaccinia (rVV) expressing Mycobacterium tuberculosis (MTB) proteins can be used both as tools to dis sect CD8(+) T-cell responses and, in attenuated form, as candidate vaccines capable of inducing a balanced CD4(+)/CD8(+) T-cell response. A panel of r VV was constructed to express four immunodominant secreted proteins of MTB: 85A, 85B and 85C and ESAT-6. A parallel group of rVV was constructed to in clude the heterologous eukaryotic tissue plasminogen activator (tPA) signal sequence to assess if this would enhance expression and immunogenicity. Cl ear expression was obtained for 85A, 85B and ESAT-6 and the addition of tPA resulted in N-glycosylation and a 4-10-fold increase in expression. Female C57BL/6 mice were immunised using the rVV-Ag85 constructs, and interleukin -2 and gamma-interferon were assayed using a co-culture of immune splenocyt es and recall antigen. There was a marked increase in cytokine production i n mice immunised with the tPA-containing constructs. We report the first da ta demonstrating enhanced immunogenicity of rVV using a tPA signal sequence , which has significant implications for future vaccine design. (C) 2000 Ed itions scientifiques et medicales Elsevier SAS.