Core-linked LPS expression of Shigella dysenteriae serotype 1 O-antigen in live Salmonella Typhi vaccine vector Ty21a: Preclinical evidence of immunogenicity and protection
Introduction
Bacillary dysentery, caused typically by the enteroinvasive bacterial genus Shigella, is characterized by severe inflammation of the colon. An estimated 165 million cases of shigellosis occur annually worldwide and 69% of episodes occur in children under 5 years of age. Moreover, approximately 1.1 million deaths are attributed to Shigella infection each year in developing countries, 60% of which occur in the under-5 age group [1]. Shigellosis is spread via the fecal–oral route and is highly transmissible due to its very low infective dose (i.e. <100 bacteria) [2]. Shigella dysenteriae 1 is the primary causative agent of epidemic outbreaks of severe bacillary dysentery which is characterized by serious complications (e.g., hemorrhagic colitis, sepsis, hemolytic uremic syndrome, purpura) and high mortality [3], [4]. The severity of this disease results from the relatively high levels of Shiga toxin produced by strains of S. dysenteriae 1 over those produced by other Shigellae [3], [5]. Another complication in the treatment of S. dysenteriae 1 is the emergence of multiply antibiotic resistant strains, particularly in developing countries. For these reasons, the World Health Organization has given high priority to the development of an effective vaccine against S. dysenteriae 1 [6], [7], [8]. Further impetus for the development of an effective S. dysenteriae 1 vaccine has come from the consideration of this highly infectious food- and water-borne pathogen as a potential bioterrorist agent.
Natural protective immunity against shigellosis is generally thought to be serotype-specific and correlates with stimulation of both systemic and local intestinal immunity against the O-specific surface lipopolysaccharide (LPS) [6], [9], [10]. Genes for S. dysenteriae 1 O-antigen biosynthesis are uniquely located in two unlinked genetic loci. One essential gene, rfp, is located on a 9 kb multicopy plasmid [11], and the remaining biosynthetic genes are clustered in the rfb chromosomal locus [12], [13].
We have previously reported on the use of the proven safe, licensed oral vaccine strain S. Typhi Ty21a as a general antigen delivery system [14], and specifically for expression of heterologous Shigella LPS [15], [16]. In prior studies directed toward the development of an S. dysenteriae 1 vaccine, the essential genes for S. dysenteriae 1 O-Ps biosynthesis were introduced together into attenuated strains of Escherichia coli or Salmonella enterica serovars Typhimurium (S. Typhimurium) or Typhi [12], [17], [18] to create live vaccine candidates for protection against this Shigella serotype. In these studies the S. dysenteriae 1 O-Ps antigen appeared to be core-linked in Shigella, E. coli, or S. Typhimurium recombinants, but was reportedly not core-linked in S. Typhi [18]. In the current studies, the genetic loci for S. dysenteriae 1 O-antigen biosynthesis were cloned, sequenced, and analyzed for their ability to direct O-Ps expression. The S. dysenteriae 1 O-antigen was found to be core-linked in both E. coli and S. Typhi Ty21a. Further, mice immunized with Ty21a expressing the heterologous S. dysenteriae 1 O-Ps were solidly protected against challenge with virulent S. dysenteriae 1.
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Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids utilized are described below and in Table 1. The wild type parent S. dysenteriae 1 strain 1617 was obtained from the Walter Reed Army Institute of Research (WRAIR) collection [19] as a lyophilized culture in vacuum-sealed glass ampules, which were prepared shortly after original isolation from an outbreak of epidemic Shiga bacillus dysentery in Guatemala, Central America in 1968 [20]. Strain 1617 is sensitive to ampicillin, spectinomycin, streptomycin,
Construction of S. dysenteriae 1 O-antigen gene expression cassette
Previous studies showed that the determinants for the production of LPS in S. dysenteriae 1 are distributed on the chromosome (i.e. rfb genes) and on a small plasmid (rfp) [11], [12], [13], [21]. These regions were PCR-amplified from S. dysenteriae 1 strain 1617 genomic DNA and cloned tandemly to create an O-Ps biosynthetic expression cassette in the low copy vector pGB2, as detailed in Section 2.2 and schematically depicted in Fig. 1A. In this pSd1 construct, the rfp gene (wbbP) encoding a
Discussion
There is renewed interest in the development of a vaccine to protect against infection due to S. dysenteriae 1, both because of its association with high mortality and its potential use as a bioterrorist agent. We have previously demonstrated that heterologous Shigella sonnei O-Ps genes are stably maintained in the low copy plasmid pGB2, which when carried by the live, attenuated typhoid vaccine strain Ty21a provides protection in animals against virulent Shigella challenge [14], [15], [16].
Acknowledgements
We thank A. O’Brien (Uniformed Univ. of the Health Sciences) for conducting Stx assays and Dr. K. Shima and Eric Patzer (Aridis Pharmaceuticals, LLD) for critical review of the manuscript. This work was supported in part by the Intramural Research Program of the NIH, NIDCR.
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