Construction of a stable attenuated Shigella sonnei DeltavirG vaccine strain, WRSS1, and protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model - PubMed (original) (raw)

Construction of a stable attenuated Shigella sonnei DeltavirG vaccine strain, WRSS1, and protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model

A B Hartman et al. Infect Immun. 1998 Sep.

Abstract

Construction of a stable Shigella sonnei vaccine has been complicated by the instability of the virulence phenotype caused by the spontaneous loss of the invasion plasmid. To select a suitable candidate for vaccine construction, 16 S. sonnei strains were screened for stability of the virulence phenotype. A stable strain, S. sonnei Mosely, was selected for further work. pDeltavirG2, a deletion derivative of the virG gene in the sacB suicide vector pCVD442, was used to generate an S. sonnei virG deletion strain, WRSS1, which was invasive in HeLa cells but negative in the Sereny test. WRSS1 was found to be both immunogenic and protective in the guinea pig keratoconjunctivitis model.

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Figures

FIG. 1

FIG. 1

Construction of strain WRSS1. (A) Plasmid pΔvirG contains a virG gene with a 212-bp deletion cloned into pCVD442. The deleted version is shaded to distinguish it from the wild-type gene. Primer BA76 is located in the oriR6K portion of pCVD442. (B) The virG gene located on the invasion plasmid of S. sonnei Mosely with the positions of the primers used to monitor recombinants. BA118 is located in the 5′ noncoding region of virG. (C) The product of the first recombination event. Primers BA118 and BA76 were used to monitor insertion of the pΔvirG plasmid into virG. (D) The product of the second recombination event generating WRSS1. Sequences homologous to primers BA114 and BA117 are present in panels A, B, C, and D. Sequences homologous to primer BA118 are present only in panels B, C, and D. Sequences homologous to primers BA76 are present only in panels A and C.

FIG. 2

FIG. 2

PCR analysis of the virG gene in wild-type Shigella and vaccine strain WRSS1. PCR analysis of the size of virG was carried out with primers BA114 and BA117. Products were run on an ethidium-bromide-stained 0.8% agarose gel. Lane 1, molecular size markers; lane 2, S. flexneri 2a strain 2457T; lane 3, S. flexneri 5a strain M90T-W; lane 4, S. sonnei Mosely; lane 5, WRSS1. The sizes of the wild-type virG gene and the deleted gene found in WRSS1 are shown at the right.

FIG. 3

FIG. 3

Serum IgG and IgA titers against the S. sonnei O antigen 2 weeks after the boosting immunization of guinea pigs with WRSS1. The geometric mean titers from experiments 1 and 2 are shown. Background titers were determined from preimmunization bleeds (PB) and were <50 for both IgG and IgA. Standard errors of the mean are shown. Exp, experiment.

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