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. 2017 Dec 21;48(1):89.
doi: 10.1186/s13567-017-0494-6.

Salmonella enterica serovar Choleraesuis vector delivering SaoA antigen confers protection against Streptococcus suis serotypes 2 and 7 in mice and pigs

Yu-An Li  1   2 , Zhenying Ji  1   2 , Xiaobo Wang  1   2 , Shifeng Wang  3 , Huoying Shi  4   5
Affiliations

Salmonella enterica serovar Choleraesuis vector delivering SaoA antigen confers protection against Streptococcus suis serotypes 2 and 7 in mice and pigs

Yu-An Li et al. Vet Res. .

Abstract

Streptococcus suis is one of the major pathogens that cause economic losses in the swine industry worldwide. However, current bacterins only provide limited prophylactic protection in the field. An ideal vaccine against S. suis should protect pigs against the clinical diseases caused by multiple serotypes, or at least protect against the dominant serotype in a given geographic region. A new recombinant Salmonella enterica serotype Choleraesuis vaccine vector, rSC0011, that is based on the regulated delayed attenuation system and regulated delayed antigen synthesis system, was developed recently. In this study, an improved recombinant attenuated Salmonella Choleraesuis vector, rSC0016, was developed by incorporating a sopB mutation to ensure adequate safety and maximal immunogenicity. In the spleens of mice, rSC0016 colonized less than rSC0011. rSC0016 and rSC0011 colonized similarly in Peyer's patches of mice. The recombinant vaccine rSC0016(pS-SaoA) induced stronger cellular, humoral, and mucosal immune responses in mice and swine against SaoA, a conserved surface protein that is present in many S. suis serotypes, than did rSC0011(pS-SaoA) without sopB or rSC0018(pS-SaoA), which is an avirulent, chemically attenuated vaccine strain. rSC0016(pS-SaoA) provided 100% protection against S. suis serotype 2 in mice and pigs, and full cross-protection against SS7 in pigs. This new vaccine vector provides a foundation for the development of a universal vaccine against multiple serotypes of S. suis in pigs.

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Figures

Figure 1
Figure 1
Diagrams of chromosomal mutation, plasmid maps and phenotypic feature of the sopB mutant in Salmonella Choleraesuis strains. A Chromosomal map of ΔsopB deletion mutation. B Empty plasmid vector pYA3493 and expression vector pS-SaoA. C Histopathological observations of rabbit ileal loops injected with different Salmonella Choleraesuis strains or LB control for 8 h. Bar: 50 μm. The small square showed the neutrophils in the submucosa of rabbit ileal injected with wild-type C78-3 strain. D Fluid secretion induced by Salmonella Choleraesuis strains in rabbit ileal loops. a, P < 0.05, wildtype Salmonella Choleraesuis C78-3 compared with LB, rSC0017, rSC0018(pS-SaoA), rSC0016(pS-SaoA) and rSC0011(pS-SaoA); b, P < 0.05, Salmonella Choleraesuis with ΔsopB mutation rS0017 compared to LB, rSC0018(pS-SaoA), rSC0011(pS-SaoA) and rSC0016(pS-SaoA); *P < 0.05, for the indicated strains were compared each other.
Figure 2
Figure 2
Phenotypic characterization of Salmonella Choleraesuis strains rSC0016. A Phenotype of ΔPcrp527::TT araC PBAD crp. Strain rSC0016 with the ΔPcrp527::TT araC PBAD crp mutation were grown on MacConkey maltose agar with and without 0.2% arabinose. B Phenotype of Δpmi mutation. LPS profiles of Δpmi mutant strains rSC0016 in NB grown with or without 0.2% mannose. Lanes: 1, wild-type C78-3; 2, rSC0016 with mannose; 3, rSC0016 without mannose. C Regulated decreased synthesis of LacI and regulated delayed synthesis of SaoA proteins in rSC0016(pS-SaoA) containing ΔrelA::araC PBAD lacI TT mutation. Strain rSC0016(pS-SaoA) were grown in NB with arabinose and mannose (Lane 1) and then diluted 1:10 into fresh NB without arabinose and mannose until OD600 to 0.8. The process was repeated for four times (Lane 2–5); each lane was loaded around 2.5 × 107 CFU bacteria. Synthesis of LacI and SaoA were detected by Western blot using correspondent antiserum. M: protein marker. D Phenotype of ΔasdA mutation. Growth curves of rSC0016 in LB with and without DAP. Growth was monitored by measuring OD600 at the indicated time intervals. E Synthesis of SaoA in Salmonella Choleraesuis vector rSC0011, rSC0016 and rSC0018. An equal amount of cells was subjected to SDS-PAGE analysis. Immunoblot was detected with SaoA—specific polyclonal antibody. Densitometry ratio was quantified using Image J software. M: protein Marker; 1: rSC0011(pS-SaoA); 2: rSC0016(pS-SaoA); 3: rSC0018(pS-SaoA); 4: rSC0011(pYA3493); 5: rSC0016(pYA3493); 6: rSC0018(pYA3493); GroEL was used as a control.
Figure 3
Figure 3
Colonization of Salmonella Choleraesuis rSC0016 in BALB/c mice at different time points. The numbers of Salmonella Choleraesuis C78-3, rSC0018(pYA3493), rSC0018(pS-SaoA), SC0016(pYA3493), rSC0016(pS-SaoA), rSC0011(pYA3493), and SC0011(pS-SaoA) in Peyer’s patches (A), spleen (B) and liver (C), of mice at 3, 7, 14, and 21 days after oral inoculation with 1.0 ± 0.3 × 109 CFU of the indicated strains were plotted. Bars represent the arithmetic mean ± standard deviations from ten mice per group. **P < 0.01, for rSC0018 compared to rSC0016 or to rSC0011 with either pYA3493 or pS-SaoA, respectively; # P < 0.05, ## P < 0.01, for rSC0011 compared to rSC0016 carrying either pYA3493 or pS-SaoA, respectively; $$ P < 0.01, for C78-3 compared to rSC0018, rSC0016 and rSC0011 carrying either pYA3493 or pS-SaoA, as indicated. The data were collected from two independent experiments.
Figure 4
Figure 4
Antibody responses in mice. A Serum IgG responses to SaoA. B Vaginal IgA responses to SaoA and C Serum IgG to Salmonella Choleraesuis OMPs were measured by ELISA. The data represent reciprocal antibody titers in sera from ten mice orally immunized with attenuated Salmonella carrying either pS-SaoA or pYA3493 (empty vector) and BSG at indicated weeks after immunization. Serum and vaginal wash obtained from individual mice were serially diluted to obtain titers, starting from either 1:50 or 1:10. Error bars represent variation between mice. Significant differences were indicated. **P < 0.01, for Salmonella Choleraesuis with pS-SaoA compared to Salmonella Choleraesuis with pYA3493 in A and C; # P < 0.05; ## P < 0.01, for Salmonella carrying either pS-SaoA or pYA3493 compared each other. No antibody responses were detected to antigen tested in mice immunized with the only BSG or in pre-immune sera from vaccinated mice. ELISA was performed twice with identical results.
Figure 5
Figure 5
Cytokines levels in ten mice immunized with Salmonella Choleraesuis vaccines. IFN-γ (A), IL-4 (B) or IL-17A (C) in sera at 0.5, 3, and 5 days, in the spleen at 7 days after the boost were assayed with ELISA kit. BSG control was also included. *P < 0.05; **P < 0.01, for the cytokines levels induced by strains rSC0011, rSC0016 and rSC0018 containing pS-SaoA compared with that containing the empty vector pYA3493; # P < 0.05, ## P < 0.01 for the significant differences between groups were indicated. The assay was performed in triplicate. The data were collected from two experiments and analyzed.
Figure 6
Figure 6
Serum IgG responses to SaoA (A), nasal IgA responses to SaoA (B), serum IgG responses to Salmonella Choleraesuis OMPs (C) in ten pigs were measured by ELISA. The data represent reciprocal anti-IgG antibody titers from the piglets orally immunized with attenuated Salmonella carrying either pS-SaoA and pYA3493 at the indicated weeks after immunization. Serum and nasal cavity wash obtained from individual pig were serially diluted to obtain titers, starting from either 1:50 or 1:10. Error bars represent variation between different pigs. Significant differences were indicated. **P < 0.01, for Salmonella Choleraesuis with pS-SaoA compare to Salmonella Choleraesuis with pYA3493 in A and B; # P < 0.05; ## P < 0.01, for Salmonella carrying either pS-SaoA or pYA3493 compared each other. No responses were detected to antigen tested in pigs immunized with BSG or in pre-immune sera from vaccinated piglets. ELISA was performed twice with identical results.
Figure 7
Figure 7
Cytokine levels in ten pigs. IFN-γ (A), IL-4 (B) or IL-17A (C) in sera from 0.5, 3, and 5 days, in the spleen at 7 days after the booster from single piglet were assayed with ELISA. BSG controls were also included. *P < 0.05; **P < 0.01, for the cytokines levels induced by strains rSC0011, rSC0016 and rSC0018 containing pS-SaoA compared with that containing the empty vector pYA3493; # P < 0.05, ## P < 0.01 for significant differences between groups were indicated. The assay was performed in triplicate. The data were collected from two experiments and analyzed.
Figure 8
Figure 8
Protection in pigs. A Survival curve of swine after challenge with SS2. Groups of ten pigs were orally immunized twice at 3-week intervals with indicated strains and challenged intravenously with 108 CFU of S. suis 2 at 2 weeks after the 2nd immunization. (B) Clinical symptom of leg and histopathology in the brain of immunized pigs challenged with SS2. (C) Western blots analysis the ability of sera from the pigs vaccinated with rSC0016 or rSC0018 containing pS-SaoA plasmid to recognize SaoA protein of SS2 and SS7. PB: Post-boost serum of pigs. (D) Rectal temperature of immunized piglets after challenged intravenously with 1010 CFU of SS7. The rectal temperatures were measured at 0, 1, 2, 3, 4, 5, 6, and 7 days after challenge. The experiment was performed twice.
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