Immunogenicity and protective efficacy against Salmonella C2-C3 infection in mice immunized with a glycoconjugate of S. Newport Core-O polysaccharide linked to the homologous serovar FliC protein

Abstract

Nontyphoidal Salmonella (NTS) are important human enteric pathogens globally. Among the different serovars associated with human NTS disease, S. Newport (a serogroup C₂-C₃Salmonella) accounts for a measurable proportion of cases. However, to date there are no licensed human NTS vaccines. NTS lipopolysaccharide-associated O polysaccharides are virulence factors and protective antigens in animal models. As isolated molecules, bacterial polysaccharides are generally poorly immunogenic, a limitation overcome by conjugation to a protein carrier. We report herein the development of a candidate serogroup C₂-C₃ glycoconjugate vaccine based on S. Newport Core-O polysaccharide (COPS) and phase 1 flagellin (FliC). S. Newport COPS and FliC were purified from genetically engineered reagent strains, and conjugated at the polysaccharide reducing end to FliC protein lysines with thioether chemistry. S. Newport COPS:FliC immunization in mice improved anti-polysaccharide immune responses, generated high anti-FliC IgG titers, and mediated robust protection against challenge with both the homologous serovar as well another serogroup C₂-C₃ serovar (S. Muenchen). Analyses of S. Newport COPS:FliC induced sera found that the anti-COPS IgG antibodies were specific for serogroup C₂-C₃ lipopolysaccharide, and could promote bactericidal killing by complement and uptake into phagocytes. These preclinical studies establish the protective capacity of serogroup C₂-C₃ OPS glycoconjugates, and provide a path forward for the development of a multivalent Salmonella vaccine for humans that includes serogroup C₂-C₃.

Keywords: Group C; O polysaccharide; conjugate; flagellin; vaccine.

Figure 1.

Biochemical and biophysical characterization of purified S. Newport COPS, FliC and end-linked glycoconjugate. (A) 5 μg of CVD 1964 FliC alone [F] and conjugated to CVD 1962 COPS [C] were assessed by SDS-PAGE with Coomassie staining. M = molecular weight marker. (B) 2 μg CVD 1964 FliC [F] detected by western blot using pan-Salmonella flagellin monoclonal IgG CB7IH2. M = molecular weight marker. (C) HPLC-SEC chromatogram with RI detection for CVD 1962 COPS (dash trace), CVD 1964 FliC (dot trace), and COPS:FliC conjugate (solid trace). (D) Dionex HPAEC-PAD analysis of depolymerized CVD 1962 COPS with the published Salmonella serogroup C₂-C₃ OPS structure¹ inlaid.

Figure 2.

Immunogenicity of S. Newport COPS:FliC in mice. Serum IgG titers for anti-S. Newport COPS (A) or FliC (B) from mice (n = 20/group) immunized with PBS, HK S. Newport or COPS:FliC conjugate, with or without aluminum hydroxide as an adjuvant. Each point represents an individual mouse. Solid bars indicate the GMT; comparisons between groups were accomplished by unpaired two-tailed Mann-Whitney. *P < 0.001, **P < 0.0001, N.S. – not significant.

Figure 3.

OPS antigen specificity of IgG antibodies in conjugate-immunized mice sera. (A) LPS extracts from various non-typhoidal Salmonella serovars described in Table 1 (Np – Newport Chile 361, Mu – Muenchen ATCC 8388, Vir – Virchow Q23, Ent – Enteritidis R11, Tm – Typhimurium D65) were transferred to PVDF membranes and probed with S. Newport COPS:FliC immune antisera. M – Molecular weight marker. (B) SDS-PAGE with Pro-Q staining for LPS from S. Newport Chile 361 (Np) or S. Muenchen ATCC 8388 (Mu).

Figure 4.

Protection against S. Newport infection in mice immunized with S. Newport COPS:FliC conjugate with or without adjuvant. BALB/c mice (n = 14/group) as described in Figure 2, were immunized with heat-killed S. Newport Chile 361 (HK), PBS or S. Newport COPS:FliC conjugate alone or with formulated with alum. Kaplan-Meier survival curves after challenge with 3 × 10⁷ CFU (6xLD₅₀) were compared using log rank analysis. *P <0.0001, N.S. – not significant.

Figure 5.

Functional activity of immune sera. Serially diluted pre-immune, heat-killed immune and COPS:FliC immune sera were incubated with S. Newport Chile 361 and assessed for (A) SBA, or (B) OPA. Data represent the mean +/- standard deviation (SD) from three independent experiments.

Figure 6.

Intra-serogroup protection after S. Newport COPS:FliC immunization. Protection against challenge with 3 × 10⁷ CFU S. Newport Chile 361 (triangles) or S. Muenchen ATCC 8388 (squares) in mice (n = 20/group) immunized with homologous heat-killed strain (solid lines, black shapes), PBS (dotted lines, open shapes) or S. Newport COPS:FliC (dashed lines, grey shapes). Kaplan-Meier survival curves were compared using log rank analysis. *P < 0.0001.