title: Vaccinia expression of Mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of M. tuberculosis Ag85.
abstract: There is increasing evidence to implicate a role for CD8(+) T cells in protective immunity against tuberculosis. Recombinant vaccinia (rVV) expressing Mycobacterium tuberculosis (MTB) proteins can be used both as tools to dissect CD8(+) T-cell responses and, in attenuated form, as candidate vaccines capable of inducing a balanced CD4(+)/CD8(+) T-cell response. A panel of rVV 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 include the heterologous eukaryotic tissue plasminogen activator (tPA) signal sequence to assess if this would enhance expression and immunogenicity. Clear 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 splenocytes and recall antigen. There was a marked increase in cytokine production in mice immunised with the tPA-containing constructs. We report the first data demonstrating enhanced immunogenicity of rVV using a tPA signal sequence, which has significant implications for future vaccine design.
authors.affiliation: Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, WC1E 7HT, London, UK. adam.malin@bigfoot.com
authors.forename: A S
authors.initials: AS
authors.lastname: Malin
authors.forename: K
authors.initials: K
authors.lastname: Huygen
authors.forename: J
authors.initials: J
authors.lastname: Content
authors.forename: M
authors.initials: M
authors.lastname: Mackett
authors.forename: L
authors.initials: L
authors.lastname: Brandt
authors.forename: P
authors.initials: P
authors.lastname: Andersen
authors.forename: S M
authors.initials: SM
authors.lastname: Smith
authors.forename: H M
authors.initials: HM
authors.lastname: Dockrell
mesh_terms: D000217:Acyltransferases; D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000096:biosynthesis*; D001428:Bacterial Vaccines / Q000276:immunology*; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018920:Coculture Techniques; D005260:Female; D005822:Genetic Vectors; D006031:Glycosylation; D007371:Interferon-gamma / Q000032:analysis; D007376:Interleukin-2 / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D017510:Protein Folding; D018271:Signal Recognition Particle; D010959:Tissue Plasminogen Activator / Q000737:chemistry*; D014616:Vaccinia virus / Q000378:metabolism*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D007376:Interleukin-2; D018271:Signal Recognition Particle; C503637:Srp72 protein, mouse; D007371:Interferon-gamma; D000217:Acyltransferases; C112580:antigen 85A, Mycobacterium tuberculosis; D010959:Tissue Plasminogen Activator