Protein synthesis in HeLa or Henle 407 cells infected with Shigella dysenteriae 1, Shigella flexneri 2a, or Salmonella typhimurium W118.

ABSTRACT We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S. Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S. Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever.

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Characterization and immunogenicity of a Shigella flexneri 2a O-antigen bioconjugate vaccine candidate

Glycobiology ◽

10.1093/glycob/cwz044 ◽

2019 ◽

Vol 29 (9) ◽

pp. 669-680 ◽

Cited By ~ 5

Author(s):

Neil Ravenscroft ◽

Martin Braun ◽

Joerg Schneider ◽

Anita M Dreyer ◽

Michael Wetter ◽

...

Keyword(s):

Diarrheal Disease ◽

Vaccine Candidate ◽

Shigella Flexneri ◽

Preventive Measure ◽

Shigella Dysenteriae ◽

O Antigen ◽

Shigella Flexneri 2A ◽

Single Production ◽

Functional Antibodies ◽

Efficacious Vaccine

AbstractShigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Vaccination represents a promising preventive measure to fight the burden of the disease, but despite enormous efforts, an efficacious vaccine is not available to date. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of a multivalent conjugate vaccine to prevent shigellosis. This bioconjugation approach has been used to produce the Shigella dysenteriae type O1 conjugate that has been successfully tested in a phase I clinical study in humans. In this report, we describe a similar approach for the production of an additional serotype required for a broadly protective shigellosis vaccine candidate. The Shigella flexneri 2a O-polysaccharide is conjugated to introduced asparagine residues of the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa by co-expression with the PglB oligosaccharyltransferase. The bioconjugate was purified, characterized using physicochemical methods and subjected to preclinical evaluation in rats. The bioconjugate elicited functional antibodies as shown by a bactericidal assay for S. flexneri 2a. This study confirms the applicability of bioconjugation for the S. flexneri 2a O-antigen, which provides an intrinsic advantage over chemical conjugates due to the simplicity of a single production step and ease of characterization of the homogenous monomeric conjugate formed. In addition, it shows that bioconjugates are able to raise functional antibodies against the polysaccharide antigen.

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