title: Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA.
abstract: Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.
authors.affiliation: Center for Vaccine Development, Separtmrnt of Medicine, School of Medicine, University of Maryland, 685 W. Baltimore St., Baltimore, MD 21201, USA. jgalen@medicine.umaryland.edu
authors.forename: James E
authors.initials: JE
authors.lastname: Galen
authors.forename: Licheng
authors.initials: L
authors.lastname: Zhao
authors.forename: Magaly
authors.initials: M
authors.lastname: Chinchilla
authors.forename: Jin Yuan
authors.initials: JY
authors.lastname: Wang
authors.forename: Marcela F
authors.initials: MF
authors.lastname: Pasetti
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
authors.forename: Myron M
authors.initials: MM
authors.lastname: Levine
mesh_terms: D000818:Animals; D022122:Anthrax Vaccines / Q000276:immunology*; D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D006460:Hemolysin Proteins; D007371:Interferon-gamma / Q000096:biosynthesis; D015848:Interleukin-5 / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012485:Salmonella typhi / Q000235:genetics*; D014613:Vaccines, Attenuated / Q000276:immunology; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D022122:Anthrax Vaccines; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D015848:Interleukin-5; D011993:Recombinant Fusion Proteins; D014613:Vaccines, Attenuated; D014614:Vaccines, Synthetic; C030325:anthrax toxin; C087744:ApxI toxin, Bacteria; D007371:Interferon-gamma