Elsevier

Vaccine

Volume 25, Issue 33, 14 August 2007, Pages 6167-6175
Vaccine

Core-linked LPS expression of Shigella dysenteriae serotype 1 O-antigen in live Salmonella Typhi vaccine vector Ty21a: Preclinical evidence of immunogenicity and protection

https://doi.org/10.1016/j.vaccine.2007.06.003Get rights and content

Abstract

Shigella dysenteriae serotype 1 (S. dysenteriae 1) causes severe shigellosis that is typically associated with high mortality. Antibodies against Shigella serotype-specific O-polysaccharide (O-Ps) have been shown to be host protective. In this study, the rfb locus and the rfp gene with their cognate promoter regions were PCR-amplified from S. dysenteriae 1, cloned, and sequenced. Deletion analysis showed that eight rfb ORFs plus rfp are necessary for biosynthesis of this O-Ps. A tandemly-linked rfb–rfp gene cassette was cloned into low copy plasmid pGB2 to create pSd1. Avirulent Salmonella enterica serovar Typhi (S. Typhi) Ty21a harboring pSd1 synthesized S. Typhi 9, 12 LPS as well as typical core-linked S. dysenteriae 1 LPS. Animal immunization studies showed that Ty21a (pSd1) induces protective immunity against high stringency challenge with virulent S. dysenteriae 1 strain 1617. These data further demonstrate the utility of S. Typhi Ty21a as a live, bacterial vaccine delivery system for heterologous O-antigens, supporting the promise of a bifunctional oral vaccine for prevention of shigellosis and typhoid fever.

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.

Section snippets

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.

References (45)

  • H.L. DuPont et al.

    Inoculum size in shigellosis and implications for expected mode of transmission

    J Infect Dis

    (1989)
  • G. Keusch et al.

    Shigellosis

    Harrison's Principles of Internal Medicine

    (2005)
  • P.J. Sansonetti

    The bacterial weaponry: lessons from Shigella

    Ann NY Acad Sci

    (2006)
  • R.A. Oberhelman et al.

    Prospective study of systemic and mucosal immune responses in dysenteric patients to specific Shigella invasion plasmid antigens and lipopolysaccharides

    Infect Immun

    (1991)
  • World Health Organization

    New strategies for accelerating Shigella vaccine development

    Wkly Epidemiol Rec

    (1997)
  • World Health Organization, Division of diarrhoeal and acute respiratory disease control. Interim report 1994. Document...
  • R. Raqib et al.

    Innate immune responses in children and adults with shigellosis

    Infect Immun

    (2000)
  • D.K. Windsor et al.

    Comparison of the serum and fecal antibody responses of patients with naturally acquired Shigella sonnei infection

    J Infect Dis

    (1988)
  • H. Watanabe et al.

    Small plasmid in Shigella dysenteriae 1 specifies one or more functions essential for O antigen production and bacterial virulence

    Infect Immun

    (1984)
  • T.L. Hale et al.

    Expression of lipopolysaccharide O antigen in Escherichia coli K-12 hybrids containing plasmid and chromosomal genes from Shigella dysenteriae 1

    Infect Immun

    (1984)
  • D.J. Kopecko et al.

    Oral anti-Shigella vaccines—a model system

    Infectious diarrhea of the young

    (1985)
  • S.B. Formal et al.

    Construction of a potential bivalent vaccine strain: introduction of Shigella sonnei form I antigen genes into the galE Salmonella typhi Ty21a typhoid vaccine strain

    Infect Immun

    (1981)

    • Cited by (27)

    • Oral shigella vaccines

      2019, Mucosal Vaccines: Innovation for Preventing Infectious Diseases

    • A bivalent vaccine derived from attenuated Salmonella expressing O-antigen polysaccharide provides protection against avian pathogenic Escherichia coli O1 and O2 infection

      2018, Vaccine
      Citation Excerpt :

      Prior studies have shown that recombinant Salmonella is capable biosynthesizing O-antigen polysaccharides of many bacterial species including Pseudomonas aeruginosa, Burkholderia mallei, Shigella dysenteriae, and Escherichia coli. Moreover, immunization with live-attenuated Salmonella vaccines recombinantly expressing O-antigen polysaccharides elicits robust protective immune responses [13,20–23]. In our previous study, we have utilized an innovative yeast/bacterial system to express the 10.8 kb operon synthesizing the APEC O1 O-antigen polysaccharide in attenuated Salmonella strain S740 [14].

    • Pushing the Bacterial Envelope: Strategies for Re-Engineering Bacterial Surfaces with Heterologous Proteins and Sugars

      2014, Micro-and Nanoengineering of the Cell Surface

    • Stable expression of Shigella sonnei form I O-polysaccharide genes recombineered into the chromosome of live Salmonella oral vaccine vector Ty21a

      2013, International Journal of Medical Microbiology
      Citation Excerpt :

      Unfortunately, but not totally unexpected, the considerable protection afforded volunteers receiving the first two lots of vaccine was not observed with subsequent vaccine lots, due to loss of the form I gene region from the genetically unstable, large cointegrate plasmid in 5076-1C (Formal et al., 1981). In subsequent studies, the minimal size gene regions needed for stable expression of S. sonnei or S. dysenteriae serotype 1 O-antigens were determined to be between 11 and 15 kb (Xu et al., 2002, 2007). These studies also revealed that the S. sonnei O-antigen could be expressed as core-linked LPS or as a Group 4 O-antigen capsule in either Ty21a or in Shigella, as are LPS's of certain other Shigella serotypes and of S. Typhi, but that either form of O-antigen expression appears to be equally immunogenic (Xu et al., 2007).

    • Oral immunization using HgbA in a recombinant chancroid vaccine delivered by attenuated Salmonella typhimurium SL3261 in the temperature-dependent rabbit model

      2012, Journal of Immunological Methods

    • Shigellosis

      2011, Tropical Infectious Diseases: Principles, Pathogens and Practice
    View all citing articles on Scopus
    View full text
    Published by Elsevier Ltd.