A GMMA-CPS-Based Vaccine for Non-Typhoidal Salmonella

Abstract

Non-typhoidal Salmonella are a major cause of gastroenteritis worldwide, as well as causing bloodstream infections in sub-Saharan Africa with a high fatality rate. No vaccine is currently available for human use. Current vaccine development strategies are focused on capsular polysaccharides (CPS) present on the surface of non-typhoidal Salmonella. This study aimed to boost the amount of CPS purified from S. Typhimurium for immunization trials. Random mutagenesis with Tn10 transposon increased the production of CPS colanic acid, by 10-fold compared to wildtype. Immunization with colanic acid or colanic acid conjugated to truncated glycoprotein D or inactivated diphtheria toxin did not induce a protective immune response in mice. However, immunization with Generalized Modules for Membrane Antigens (GMMAs) isolated from colanic acid overproducing isolates reduced Salmonella colonization in mice. Our results support the development of a GMMA-CPS-based vaccine against non-typhoidal Salmonella.

Keywords: GMMAs; Salmonella; capsular polysaccharide; colanic acid; vaccines.

Figure 1

Evaluating the role of yih operon in the virulence of S. Typhimurium. A competitive index experiment was performed with C57BL/6 mice orally infected with wildtype and S. Typhimurium 14028 Δyih. At 4–7 days post-infection, the CFU levels were enumerated from the liver, spleen, cecum and MLN. Each dot represents the CFU counts (per organ) from the designated organ from a single mouse. Competitive index values were calculated from each organ as follows: (CFU yih mutant/ wt)_output/(CFU yih/wt)_input. A CI value of 1, which represents a situation where both strains are equally virulent, is represented by the horizontal dotted line. Red circles represent CI values where the S. Typhimurium 14028 Δyih strain won the competition. Statistical differences between groups of mice were noted as ns: p > 0.05.

Figure 2

Expression of yihUTSRQPO and yihVW operons in S. Typhimurium. Promoter luciferase fusions for (A) yihUTSRQPO and (B) yihVW were used to measure expression in S. Typhimurium (ST, wildtype), S. Typhimurium ΔbcsA (ΔbcsA), S. Typhimurium ΔbcsA pBR322-yihVW (pyihVW), S. Typhimurium ΔbcsA ΔyihVW (ΔyihVW), S. Typhimurium ΔbcsA ΔyihW (ΔyihW) and S. Typhimurium ΔbcsA ΔyihW Tn10 (Tn10C). Cultures were grown in 1% tryptone at 28 °C with agitation, and luminescence (in counts per second (cps)) was recorded every 30 min for 48 hours. The log maximum CPS value recorded over 48 hours is shown. Statistical differences were noted as **** p < 0.0001, ns: p > 0.05.

Figure 3

Effect of different precursor sugars on the expression of yihUTSRQPO. The S. Typhimurium ΔbcsA (A) and S. Typhimurium ΔbcsA ΔyihW (B) strains were grown in 1% tryptone media supplemented with or without different precursor sugars (glucose, galactose, rhamnose, mannose, or all four sugars), at 28 °C. Luminescence (in counts per second (cps)) was recorded every 30 min for 48 hours. The log maximum CPS value recorded over 48 hours is shown. Statistical differences were noted as ** p < 0.01, *** p < 0.001, or ns: p > 0.05.

Figure 4

Immune response to colanic acid, truncated glycoprotein D and inactivated diphtheria toxin. ELISA was performed with serum collected from CB7BL/6 mice (A,C) or BALB/c mice (B,D), immunized with phosphate-buffered saline (PBS), colanic acid (CA), CA conjugated to inactivated diphtheria toxin (CA-CRM197) or CA conjugated to truncated glycoprotein D (CA-tgD).

Figure 5

Amounts of S. Typhimurium recovered from mice previously immunized with colanic acid. (A) C57BL/6 mice were immunized with phosphate buffered saline (PBS), colanic acid (CA) or CA conjugated to inactivated diphtheria toxin (CA-CRM197). (B) BALB/c mice were immunized with PBS, CA or CA conjugated to truncated glycoprotein D (CA-tgD). All immunized mice were orally challenged with 10⁷ CFU of Salmonella, and 4–7 days post-infection, the liver, spleen, cecum, mesenteric lymph node (MLN) and blood (C57BL/6) were harvested. Organs were homogenized before plating on LB agar supplemented with kanamycin. The log₁₀ CFU values of Salmonella recovered from individual organs from each mouse are shown. The dashed line represents the limit of detection of 100 CFU. For each group of mice, the black line represents the median values. Statistical significance: Not significant (ns): p > 0.05.

Figure 6

Immune response to GMMAs and colanic acid in immunized mice. Mice were immunized with GMMAs purified from S. Typhimurium 14028 ∆tolR (wildtype), S. Typhimurium 14028 ∆tolR ∆lon (Lon), Tn10C ∆tolR (Tn10C) or PBS. ELISA was performed with sera collected on days 0, 21, 42 and 63 and used to detect Anti-CPS IgG (A), Anti- ST GMMAs IgG (wildtype) (B), Anti-Lon GMMAs IgG (C) and Anti-Tn10C GMMAs IgG (D). Each point represents the value from an individual mouse, the horizontal line represents the median from each group of mice. Statistical significance: ns: p > 0.05.

Figure 7

Ability of GMMAs to protect mice against lethal challenge of S. Typhimurium. C57BL/6 mice were immunized with PBS and GMMAs purified from S. Typhimurium ΔtolR (wildtype), S. Typhimurium ΔtolR Δlon (Lon) and S. Typhimurium ΔtolR ΔbcsA ΔyihW Tn10dtet (Tn10C). Immunized mice were orally challenged with 10⁷ CFU of S. Typhimurium 14028, and 4–7 days post-infection, mice were euthanized, and liver, spleen, cecum, MLN and blood were collected for bacterial enumeration. The log₁₀ CFU of Salmonella recovered from each mouse is shown. The dotted line represents the limit of detection of 100 CFU. The black line represents the median log CFU values determined from each group of mice.