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Recombinant attenuated Salmonella Typhimurium with heterologous expression of the Salmonella Choleraesuis O-polysaccharide: high immunogenicity and protection.

ABSTRACT: Non-typhoidal Salmonella are associated with gastrointestinal disease worldwide and invasive disease in Africa. We constructed novel bivalent vaccines through the recombinant expression of heterologous O-antigens from Salmonella Choleraesuis in Salmonella Typhimurium. A recombinant Asd⁺ plasmid pCZ1 with the cloned Salmonella Choleraesuis O-antigen gene cluster was introduced into three constructed Salmonella Typhimurium ?asd mutants: SLT11 (?rfbP), SLT12 (?rmlB-rfbP) and SLT16 (?rfbP ?pagL::TT araCP_BAD rfbP). Immunoblotting demonstrated that SLT11 (pCZ1) and SLT12 (pCZ1) efficiently expressed the heterologous O-antigen. In the presence of arabinose, SLT16 (pCZ1) expressed both the homologous and heterologous O-antigens, whereas in the absence of arabinose, SLT16 (pCZ1) mainly expressed the heterologous O-antigen. We deleted the crp/cya genes in SLT12 (pCZ1) and SLT16 (pCZ1) for attenuation purposes, generating the recombinant vaccine strains SLT17 (pCZ1) and SLT18 (pCZ1). Immunization with either SLT17 (pCZ1) or SLT18 (pCZ1) induced specific IgG against the heterologous O-antigen, which mediated significant killing of Salmonella Choleraesuis and provided full protection against a lethal homologous challenge in mice. Furthermore, SLT17 (pCZ1) or SLT18 (pCZ1) immunization resulted in 83% or 50% heterologous protection against Salmonella Choleraesuis challenge, respectively. Our study demonstrates that heterologous O-antigen expression is a promising strategy for the development of multivalent Salmonella vaccines.

SUBMITTER: Zhao X

PROVIDER: S-EPMC5533773 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Recombinant attenuated Salmonella Typhimurium with heterologous expression of the Salmonella Choleraesuis O-polysaccharide: high immunogenicity and protection.

Zhao Xinxin X Dai Qinlong Q Zhu Dekang D Liu Mafeng M Chen Shun S Sun Kunfeng K Yang Qiao Q Wu Ying Y Kong Qingke Q Jia Renyong R

Scientific reports 20170728 1

Non-typhoidal Salmonella are associated with gastrointestinal disease worldwide and invasive disease in Africa. We constructed novel bivalent vaccines through the recombinant expression of heterologous O-antigens from Salmonella Choleraesuis in Salmonella Typhimurium. A recombinant Asd<sup>+</sup> plasmid pCZ1 with the cloned Salmonella Choleraesuis O-antigen gene cluster was introduced into three constructed Salmonella Typhimurium Δasd mutants: SLT11 (ΔrfbP), SLT12 (ΔrmlB-rfbP) and SLT16 (ΔrfbP ...[more]

PMID: 28754982

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Project description:Attenuated Salmonella enterica serovar Typhimurium expressing recombinant antigens from other pathogens elicits primarily a Th1-type dominant immune response to both recombinant and Salmonella antigens. The immunogenicity and appropriate subcellular location of the recombinant antigen in the Salmonella vaccine strain may contribute to augmenting immune responses by facilitating adequate exposure of recombinant antigen to antigen-presenting cells for processing. To allow for secretion from gram-negative bacteria and overexpression of antigen, a DNA fragment encoding a highly antigenic alpha-helical region of PspA (pneumococcal surface protein A) was subcloned downstream from the beta-lactamase signal sequence in the multicopy Asd(+) pYA3493 vector to create pYA3494. pYA3493 was derived from a class of Asd(+) vectors with reduced expression of Asd to minimize selective disadvantage and enhance immunization of expressed recombinant antigens. The S. enterica serovar Typhimurium vaccine strain was constructed by the introduction of deletion mutations Delta crp-28 and Delta asdA16. Approximately 50% of the recombinant PspA (rPspA) expressed in a Salmonella strain harboring pYA3494 was detected in the combined supernatant and periplasmic fractions of broth-grown recombinant Salmonella. After a single oral immunization in BALB/c mice with 10(9) CFU of the recombinant Salmonella vaccine strain carrying pYA3494, immunoglobulin G (IgG) antibody responses were stimulated to both the heterologous antigen rPspA and Salmonella lipopolysaccharide (LPS) and outer membrane proteins (OMPs). About half, and even more at later times after immunization, of the antibodies induced to rPspA were IgG1 (indicating a Th2-type response), whereas 60 to 70% of the antibodies to LPS and 80 to 90% of those to OMPs were IgG2a (indicating a Th1-type response). A sublethal infection with Streptococcus pneumoniae WU2 boosted PspA antibody levels and maintained IgG2a/IgG1 ratios similar to those seen before the challenge. Oral immunization with Salmonella-PspA vaccine protected 60% of immunized mice from death after intraperitoneal challenge with 50 times the 50% lethal dose of virulent S. pneumoniae WU2.

Project description:Salmonella enterica serovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesized in vivo We used an in vivo expression technology (IVET) strategy to identify four fimbrial operons, agf, saf, sti, and stc that are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 × 105 CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 × 109 CFU of wild-type S Typhimurium. We also examined the possible effect of these fimbriae on the ability of a Salmonella vaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, χ9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express the Streptococcus pneumoniae antigen PspA. Strains that constitutively expressed saf or stc elicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carrying saf or stc deletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulent S. pneumoniae Our results indicate that these fimbriae play important roles, as yet not understood, in Salmonella virulence and immunogenicity.IMPORTANCESalmonella enterica is the leading cause of bacterial food-borne infection in the United States. S. Typhimurium is capable of producing up to 13 distinct surface structures called fimbriae that presumably mediate its adherence to surfaces. The roles of most of these fimbriae in disease are unknown. Identifying fimbriae produced during infection will provide important insights into how these bacterial structures contribute to disease and potentially induce protective immunity to Salmonella infection. We identified four fimbriae that are produced during infection. Deletion of all four of these fimbriae results in a significant reduction in virulence. We explored ways in which the expression of these fimbriae may be exploited for use in recombinant Salmonella vaccine strains and found that production of Saf and Stc fimbriae are important for generating a strong immune response against a vectored antigen. This work provides new insight into the role of fimbriae in disease and their potential for improving the efficacy of Salmonella-based vaccines.

Dataset Information

Recombinant attenuated Salmonella Typhimurium with heterologous expression of the Salmonella Choleraesuis O-polysaccharide: high immunogenicity and protection.

Publications

Recombinant attenuated Salmonella Typhimurium with heterologous expression of the Salmonella Choleraesuis O-polysaccharide: high immunogenicity and protection.

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