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, 10 (1), 82-96

Attenuated Salmonella Typhimurium Expressing Salmonella Paratyphoid A O-antigen Induces Protective Immune Responses Against Two Salmonella Strains

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Attenuated Salmonella Typhimurium Expressing Salmonella Paratyphoid A O-antigen Induces Protective Immune Responses Against Two Salmonella Strains

Qing Liu et al. Virulence.

Abstract

Salmonella enterica serovar Paratyphi A is the main causative agent of paratyphoid fever in many Asian countries. As paratyphoid is spread by the fecal-oral route, the most effective means of controlling S. Paratyphi A infection is through the availability of clean water supplies and working sanitation services. Because sanitation facilities improve slowly in these poor areas and antibiotic resistance is severe, the development of a safe and effective vaccine remains a priority for controlling the spread of paratyphoid disease. In this study, we investigated the strategy of heterologous O-antigenic O2 serotype (S. Paratyphi A characterized) conversion in S. Typhimurium to prevent paratyphoid infections. A series of S. Typhimurium mutants were constructed with replacement of abe, wzxB1 and wbaVB1 genes with respective prt-tyvA1, wzxA1 and wbaVA1, and the results showed that only three genes including prt, wbaVA1 and wzxA1 from S. Paratyphi A presence enable S. Typhimurium to sufficiently express O2 antigen polysaccharide. We also constructed a series of live attenuated S. Typhimurium vaccine candidates expressing heterologous O2 O-antigens, and a mouse model was used to evaluate the immunogenicity of live vaccines. ELISA data showed that vaccine candidates could induce a comparatively high level of S. Paratyphi A and/or S. Typhimurium LPS-specific IgG and IgA responses in murine model, and IgG2a levels were consistently higher than IgG1 levels. Moreover, the functional properties of serum antibodies were evaluated using in vitro C3 complement deposition and opsonophagocytic assays. Our work highlights the potential for developing S. Typhimurium live vaccines against S. Paratyphi A.

Keywords: O-antigen; Paratyphoid fever; Recombinant attenuated Salmonella vaccine (RASV); S. Paratyphi A; S. Typhimurium.

Figures

Figure 1.
Figure 1.
LPS profile of O2 serotype-converted mutants in S. Typhimurium. (a) The LPS profiles of the wild-type S. Paratyphi A and S. Typhimurium and mutants S1049 (Δabe::prt-tyvA1), S1071 [Δ(abe-wzxB1)::(prt-wzxA1)], S1072 (Δabe::prt-tyvA1 ΔwbaVB1:: wbaVA1) and S1074 [Δ(abe-wbaVB1)::(prt-wbaVA1)] were visualized by silver staining. (b and c) Western blot analysis confirmed the O-serotype conversion from O4 to O2. Salmonella O4 and O2 single-factor rabbit antisera were used as primary antibodies in the Western blot assay. Samples derived from the same experiment and gel/blots were processed in parallel.
Figure 2.
Figure 2.
LPS profile of the live attenuated vaccine candidates S1151 (pSS978) and S1166 (pSS978). (a) The LPS profiles of the live attenuated vaccine candidates S1151 (Δabe ΔpagL::araC PBAD abe TT ΔrelA::araC PBAD lacI TT Δcrp Δcya Δasd) harboring pSS978 and S1166 (Δcrp Δcya ΔrelA ΔpagL Δasd) harboring pSS978 were visualized by silver staining. Vaccine candidates were grown in medium with (+) or without (-) 0.2% arabinose, as indicated. (b and c) Western blot analysis of the LPS profile from panel A probed with anti-Salmonella serogroup A single-factor O2 (anti-O2) or serogroup B single-factor O4 (anti-O4) antibodies, as indicated. Samples derived from the same experiment and gel/blots were processed in parallel.
Figure 3.
Figure 3.
Serum IgG and mucosal IgA responses against S. Paratyphi A and S. Typhimurium LPS. The LPS of S. Paratyphi A and S. Typhimurium were used to coat ELISA plates. After a booster immunization, the serum IgG and vaginal wash IgA responses were measured by ELISA. (a) The levels of anti-S. Paratyphi A LPS IgG induced by S1112, S1151 (pSS978) and S1166 (pSS978) were significantly higher than those induced by S738 (***, P < 0.001). (b) The level of anti-S. Typhimurium LPS IgG induced by S738 was significantly higher than that induced by S1112 (***, P < 0.01), while there were no significant differences among the others. (c) Vaginal wash anti-S. Paratyphi A LPS IgA levels induced by S1112, S1151 (pSS978) and S1166 (pSS978) were significantly higher than those induced by S738 (***, P < 0.01). (D) Vaginal wash anti-S. Typhimurium LPS IgA induced by S738 was significantly higher than that induced by S1112 (***, P < 0.01). The negative control groups (BSG) did not mount a detectable immune response in any test. The concentration of antibodies was calculated using a standard curve. All concentrations of the measured samples were within the range of the standard curve. Error bars represent the standard errors of the means.
Figure 4.
Figure 4.
Flow cytometry histograms of C3 complement deposition. The percentage of FITC-positive bacteria was determined by flow cytometry. The wild-type S. Paratyphi A was incubated with the sera from mice that had been vaccinated with S738 (a), S1112 (b), S1151 (pSS978) (c) and S1166 (pSS978) (d). Accordingly, approximately 69.6%, 85.4%, 94.8% and 89.1% S. Paratyphi A were detected positive, respectively. The wild-type S. Typhimurium was incubated with sera from mice that had been vaccinated with S738 (e), S1112 (f), S1151 (pSS978) (g) and S1166 (pSS978) (h). Accordingly, approximately 94.0%, 86.6%, 87.7%, 90.9% of S. Typhimurium were detected positive, respectively. The blue line indicated the test groups mentioned above. The negative control (black line) was the wild-type S. Paratyphi A or S. Typhimurium incubated with naïve mice serum, and the positive control (red line) was the wild-type S. Paratyphi A or S. Typhimurium incubated with O2 or O4 single-factor rabbit antisera.
Figure 5.
Figure 5.
The differential uptake of S. Paratyphi A and S. Typhimurium by RAW264.7 cells after serum opsonization. Opsonophagocytic uptake of the wild-type S. Paratyphi A and S. Typhimurium by RAW264.7 macrophages was evaluated after treatment with the sera from mice that had been immunized with live attenuated vaccine candidates. (A) In comparison to the control (S. Paratyphi A opsonized with naïve serum), the uptake of S. Paratyphi A opsonized with sera from mice primed with S1112, S1151 (pSS978) and 1066 (pSS978) was significantly higher (**, P < 0.01). (B) The uptake of S. Typhimurium opsonized with sera from mice primed with S738, S1112, S1151 (pSS978) and S1166 (pSS978) was significantly higher than those opsonized with naïve mice serum (**, P < 0.01; ***, P < 0.001). The number of enumerated cells was normalized to 100% as the maximal value. The error bars represented the standard errors of the means.

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Grant support

This work was supported by grants 31570928 and 31472179 from the National Natural Science Foundation of China, and R01 AI112680 from the National Institutes of Health.

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