Development of a glycoconjugate vaccine to prevent invasive Salmonella Typhimurium infections in sub-Saharan Africa

Abstract

Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30-43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63-74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa.

Fig 1. STm COPS size characterization and the effect of pH on rhamnose and abequose O-acetylation in 1925wzzB-COPS.

(A) SDS-PAGE and Pro-Q staining for LPS from CVD 1925 (lane 1) and CVD 1925 (pSEC10-wzzB) (lane 2). (B) Chromatogram of purified D65 COPS (dashed line) and 1925wzzB-COPS (solid line) assessed by HPLC-SEC with detection by refractive index. (C) ¹H NMR analysis of 1925wzzB-COPS after exposure to different pH levels. Peaks for rhamnose C2/C3 and abequose C2 O-acetyls are indicated. (D) Hestrin analysis of residual O-acetylation of 1925wzzB-COPS after incubation at different pH levels. (E) ELISA reactivity of 1925wzzB-COPS and dOAc-1925wzzB-COPS with monoclonal antibodies against O4 (α-STm O4) and O5 (α-STm-O5) or polyclonal sera recognizing O1,12 and core epitopes (α-SE). Error bars represent s.d. and were derived from technical replicates from one experiment.

Fig 2. ¹H and ¹³C NMR analysis of native and de-O-acetylated 1925wzzB-COPS and native D65 COPS.

¹H and ¹³C spectra obtained by 800 MHz NMR are shown for 1925wzzB-COPS, dOAc-1925wzzB-COPS, and D65 COPS. NMR shifts for O-acetylated and native monosaccharides within the COPS polymer are differentiated by letter: α-D-Abequp [A], α-D-Abequp + OAc at C2 [B], α-L-Rhap [C], α-L-Rhap + OAc at C2 [D], α-L-Rhap + OAc at C3 [E].

Fig 3. COPS epitope selectivity for sera from mice immunized with a live-attenuated STm vaccine.

(A) Serum IgG titers for 1925wzzB-COPS (black), dOAc-1925wzzB-COPS (grey), and SE COPS (open) from mice immunized with CVD 1931 (n = 6). Each point represents an individual mouse. Solid bars indicate the GMT; comparisons between paired serological analyses were accomplished by two-tailed Wilcoxon signed-rank test, *P ≤ 0.05. (B) Western blot of crude LPS extracts from stationary-phase growth cultures of various Salmonella strains (detailed in S1 Table). Two volumes from each extract were separated: H, “high” 10 μL; L, “low” 2 μL. Abequose O-acetyl groups were detected with a STm O5-specific monoclonal antibody and normalized to reactivity with an anti-core polysaccharide monoclonal antibody [α-Sal core PS].

Fig 4. Immunogenicity and protection against STm infection in mice immunized with different COPS:FliC conjugates or unconjugated flagellin.

(A,B,C) Serum IgG titers for 1925wzzB-COPS (black), dOAc-1925wzzB-COPS (grey) or STm FliC (open) from mice (n = 40/group) immunized with PBS or STm-COPS^Lat:FliC (A), STm-COPS^KDO:FliC (B,C),or STm FliC (C). Each point represents an individual mouse. Red squares indicate mice that succumbed to challenge. Solid bars indicate the GMT; comparisons between groups were accomplished by either a two-tailed Mann-Whitney U test (PBS vs. conjugate) or two-tailed Wilcoxon signed-rank test (paired conjugate sera analyses). (D,E) Kaplan-Meier survival curves of mice immunized with PBS (circles, dashed lines) or conjugates of STm-COPS^Lat:FliC (D) or STm-COPS^KDO:FliC (E) (squares, solid lines) after challenge (n = 20/group) with 1x10⁵ CFU (black) or 5x10⁵ CFU (grey) of STm D65. Survival curves were compared using log rank analysis. Adjustments for multiple comparisons were not made. *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001, n.s. not-significant.

Fig 5. Immunogenicity, protective efficacy and functional analyses of vaccine-induced antibody for mice immunized with conjugates of native or dOAc-1925wzzB-COPS with CRM₁₉₇.

(A) Serum IgG titers for 1925wzzB-COPS (black), dOAc-1925wzzB-COPS (grey) or SE COPS (open) from mice (n = 40/group) immunized with PBS, STm-COPS^KDO:CRM₁₉₇ or dOAc-STm-COPS^KDO:CRM₁₉₇ as indicated. Each point represents an individual mouse. Red squares indicate mice that succumbed after challenge. Solid bars indicate the GMT; comparisons between groups were accomplished by either a two-tailed Mann-Whitney U test (PBS vs. conjugate, conjugate 1 vs. conjugate 2) or two-tailed Wilcoxon signed-rank test (paired serological analyses). (B) Kaplan-Meier survival curves of mice immunized with STm-COPS^KDO:CRM₁₉₇ (circle), dOAc-STm-COPS^KDO:CRM₁₉₇ (square) or PBS (diamond) after challenge (n = 20/group) with 1x10⁶ CFU (open symbol, dashed line) or 5x10⁶ CFU (closed symbol, solid line) of STm D65. Adjustments for multiple comparisons were not made. *P ≤ 0.0001 for indicated comparisons or for vaccinated mice relative to the respective PBS challenge group. ^#P ≤ 0.05, ^##P ≤ 0.005 for STm-COPS^KDO:CRM₁₉₇ vaccinated mice relative to the respective challenge dose group immunized with dOAc-STm-COPS^KDO:CRM₁₉₇. (C) Representative serum anti-COPS epitope profiles (identified by symbol) from mice immunized with either STm-COPS^KDO:CRM₁₉₇ or dOAc-STm-COPS^KDO:CRM₁₉₇. Selected sera were chosen to assess serum bactericidal antibodies (“SBA”, D) and opsonophagocytic antibodies (“OPA”, E) and were diluted such that each sample contained equivalent anti-1925wzzB-COPS IgG EU. Curves were fitted to each serial dilution of serum and were compared using nonlinear regression analysis. Dashed line indicates 0% killing. Results are representative of two independent assays, error bars represent s.d. and were derived from technical replicates; n.s., not significant.

Fig 6. Chemical structure of the OPS used for computational simulations and chemical models and sampled volumes of the most prevalent conformational cluster “11111111”.

(A) Structure of the base (O:4,12) 3-repeat STm polysaccharide unit used for computational analyses. Individual monosaccharide units are indicated numerically, with the reducing end rhamnose (Rha) designated as (1, blue). The representative O-acetylated polysaccharide was constructed with the hydroxyl group substituted by an acetyl at the C2 position of both α-D-Abequp and α-L-Rhap (-OH, red). (B,C) Structural models of GL cluster “11111111” displaying 90° of rotation around the carbohydrate stem for the base (B) and O-acetylated base (C) polysaccharides. The acetyl group is shown in brown, glucose in blue, mannose in green, galactose in yellow, abequose in purple, and rhamnose in cyan. For clarity, all hydrogen atoms are omitted except those on the acetyl group. (D,E,F,G) 3D spatial distributions (wire frame) displaying 90° of rotation around the carbohydrate stem for the base (D,E) and O-acetylated base (F,G) polysaccharides for all non-hydrogen atoms. The reducing end Rha (1, blue), indicated at the base, is approximated as linked to the core polysaccharide or preceding OPS repeat. Conformational flexibility increases progressively relative to the reducing end anchor point. The base polysaccharide backbone is identified by a transparent grey surface overlaid with the individual O-acetyl groups in color for monosaccharides (1) [blue], (4) [red], (5) [orange], (8) [tan], (9) [green] and (12) [purple]. Contour levels are set to 10⁻⁶ for the full polysaccharide and to 10⁻⁵ for the individual acetyl groups.