title: Comparative immunological evaluation of recombinant Salmonella Typhimurium strains expressing model antigens as live oral vaccines.
abstract: BACKGROUND
  Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines.
  
  
  RESULTS
  To compare vaccine strains encoded with different antigen delivery and expression strategies, a series of recombinant Salmonella Typhimurium strains were constructed that expressed either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. The antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in either the cytoplasm or the outer membrane. Combinations of strategies were employed to evaluate the efficacy of combined delivery/expression approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities; the immunogenicity of the constructed recombinant Salmonella strains was evaluated in mice. Using the soluble model antigen EGFP, our results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, whilst eukaryotic expression or colonization with good construct stability was critical for T cell responses. For the insoluble model antigen HA, an outer membrane expression strategy induced better B cell and T cell responses than a cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited.
  
  
  CONCLUSIONS
  Through systematically evaluating and comparing the immunogenicity of the constructed recombinant Salmonella strains in mice, we identified their respective advantages and deleterious or synergistic effects. Different construction strategies were optimally-required for soluble versus insoluble forms of the protein antigens. If an antigen, such as EGFP, is soluble and expressed at high levels, a low-copy plasmid-cytoplasmic expression strategy is recommended; since it provokes the highest B cell responses and also induces good T cell responses. If a T cell response is preferred, a eukaryotic expression plasmid or a chromosome-based, cytoplasmic-expression strategy is more effective. For insoluble antigens such as HA, an outer membrane expression strategy is recommended.
authors.affiliation: Department of Biochemistry, the University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Song-yue
authors.initials: SY
authors.lastname: Zheng
authors.forename: Bin
authors.initials: B
authors.lastname: Yu
authors.forename: Ke
authors.initials: K
authors.lastname: Zhang
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.forename: Yan-Hong
authors.initials: YH
authors.lastname: Hua
authors.forename: Shuofeng
authors.initials: S
authors.lastname: Yuan
authors.forename: Rory M
authors.initials: RM
authors.lastname: Watt
authors.forename: Bo-Jian
authors.initials: BJ
authors.lastname: Zheng
authors.forename: Kwok-Yung
authors.initials: KY
authors.lastname: Yuen
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Huang
mesh_terms: D000284:Administration, Oral; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000942:Antigens, Bacterial / Q000276:immunology*; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006385:Hemagglutination Inhibition Tests; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology*; D056724:Immunity, Humoral / Q000187:drug effects / Q000276:immunology; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology; D007371:Interferon-gamma / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D050296:Microbial Viability / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D012481:Salmonella Infections, Animal / Q000276:immunology / Q000382:microbiology; D022562:Salmonella Vaccines / Q000008:administration & dosage / Q000276:immunology*; D012486:Salmonella typhimurium / Q000166:cytology / Q000187:drug effects / Q000254:growth & development / Q000276:immunology*
chemical_list: D000900:Anti-Bacterial Agents; D000942:Antigens, Bacterial; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D022562:Salmonella Vaccines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma