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

Figures & data

Table 1. List of Salmonella strains used in the study

Strain	Source/characteristics	Reference
S. Newport Chile 361	S. Newport Chile 361, wild-type, serogroup C2-C3, (6,8:e,h:1,2), challenge strain	Fuche et al., (2018) this issue of HVI
CVD 1962	S. Newport Chile 361 ΔguaBA ΔclpX (6,8:e,h:1,2), attenuated, source of COPS hapten	Fuche et al., (2018) this issue of HVI
CVD 1964	S. Newport Chile 361 ΔguaBA ΔclpX ΔfljB ΔfliD, attenuated, lacks phase 2 flagellin, secretes phase 1 flagellin monomers, source of FliC carrier protein	Fuche et al., (2018) this issue of HVI
S. Muenchen ATCC-8388	Wild-type, serogroup C2-C3 (6,8:d:1,2), challenge strain	ATCC
S. Enteritidis R11	Wild-type, serogroup D (1,9,12:g,m)	^Citation20
S. Typhimurium D65	Wild-type, serogroup B (1,4,5,12:i:1,2)	^Citation20
S. Virchow Q23	Wild-type, serogroup C1 (6,7,14:r:1,2)	^Citation26

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

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