Cell-associated flagella enhance the protection conferred by mucosally-administered attenuated Salmonella Paratyphi A vaccines

Abstract

Background: Antibiotic-resistant Salmonella enterica serovar Paratyphi A, the agent of paratyphoid A fever, poses an emerging public health dilemma in endemic areas of Asia and among travelers, as there is no licensed vaccine. Integral to our efforts to develop a S. Paratyphi A vaccine, we addressed the role of flagella as a potential protective antigen by comparing cell-associated flagella with exported flagellin subunits expressed by attenuated strains.

Methodology: S. Paratyphi A strain ATCC 9150 was first deleted for the chromosomal guaBA locus, creating CVD 1901. Further chromosomal deletions in fliD (CVD 1901D) or flgK (CVD 1901K) were then engineered, resulting in the export of unpolymerized FliC, without impairing its overall expression. The virulence of the resulting isogenic strains was examined using a novel mouse LD(50) model to accommodate the human-host restricted S. Paratyphi A. The immunogenicity of the attenuated strains was then tested using a mouse intranasal model, followed by intraperitoneal challenge with wildtype ATCC 9150.

Results: Mucosal (intranasal) immunization of mice with strain CVD 1901 expressing cell-associated flagella conferred superior protection (vaccine efficacy [VE], 90%) against a lethal intraperitoneal challenge, compared with the flagellin monomer-exporting mutants CVD 1901K (30% VE) or CVD 1901D (47% VE). The superior protection induced by CVD 1901 with its cell-attached flagella was associated with an increased IgG2a:IgG1 ratio of FliC-specific antibodies with enhanced opsonophagocytic capacity.

Conclusions: Our results clearly suggest that enhanced anti-FliC antibody-mediated clearance of S. Paratyphi A by phagocytic cells, induced by vaccines expressing cell-associated rather than exported FliC, might be contributing to the vaccine-induced protection from S. Paratyphi A challenge in vivo. We speculate that an excess of IgG1 anti-FliC antibodies induced by the exported FliC may compete with the IgG2a subtype and block binding to specific phagocyte Fc receptors that are critical for clearing an S. Paratyphi A infection.

Figure 1. ATCC 9150 is highly culturable, expresses flagella moderately, and is highly virulent.

(A) Growth curves of four S. Paratyphi A isolates cultured on Lennox medium at 37°C under high aeration (250 rpm, 1∶10 medium∶flask volume). (B) Final cell yields were recorded at time point of 8 h (t = 8) by performing live counts. Average yields from two independent experiments are shown. (C) Equivalent cell pellets and supernatants from t = 8 were analysed by SDS-PAGE and stained with Coomassie (upper panels) or subjected to Western blot analysis with anti-FliC antibody (middle), along with S. Paratyphi A FliC standard (100 µg). Estimation of FliC quantity was performed on the blots with Bio-Rad QuantityOne (lower). (D) Swim (0.3% agar) and swarm (0.7%) plates, following 8 h incubation at 37°C. (E) BALB/c mice were injected i.p. with 4 S. Paratyphi A isolates. Bacteria were collected from Lennox plates incubated at 37°C over-night, diluted in PBS, and mixed with hog gastric mucin. Groups of naïve BALB/c mice, 6 mice per group were challenged and monitored twice daily for three days. Data represent percent cumulative survival curves.

Figure 2. S. Paratyphi A ΔfliD and ΔflgK mutants shade FliC to culture supernatant.

ATCC 9150 and five derivative mutants were grown on Lennox medium under high aeration. (A) Cell yields (upper panel) and Coomassie-stained SDS-PAGE (lower) of culture supernatants following 8 h of growth. (B) Cell yields and SDS-PAGE of supernatants from cultures grown over-night under low-aeration. (C) SDS-PAGE (upper), anti-FliC western blot (middle) and FliC quantitation (lower) of cell pellets from cultures grown over-night under low-aeration.

Figure 3. ΔfliD and ΔflgK mutants differ in their ability to export non-assembled flagellin.

(A) Separation between monomeric and filamentous FliC using serial UF membranes. Supernatants were brought to equivalent FliC concentration using 30-kDa Amicon UF units and compared by a Coomassie-stained SDS-PAGE (upper gel). The concentrated samples, with or without heat treatment (70°C, 30 min), were then run through 100-kDa units and compared again (middle). Filtrates from the previous step were passed through 30-kDa units, and the final concentrates were detected (lower). (B) Swim (0.3% agar) and swarm (0.7, 0.6 and 0.5%) plates were scanned following 8 h incubation, 37°C. (C) EM images of negatively-stained bacteria, collected from swarm plates. For CVD1901D, cells carrying two and a single filament are shown. Numbers indicate the extent of magnification.

Figure 4. ΔfliD and ΔflgK mutations do not impair virulence of S. Paratyphi A in mice.

Survival of naïve BALB/c mice infected with different strains of S. Paratyphi A at the indicated doses (cfu/mouse). Bacteria were collected from plates, diluted in PBS and mixed with hog gastric mucin. Groups of 6 mice were injected i.p. and monitored twice daily.

Figure 5. Serum IgG against S. Paratyphi A FliC and LPS do not correlate with protection.

(A) Total serum anti-FliC and anti-LPS ELISA antibody titers (geometric means ± standard error of the mean) from days −2 (prior to vaccination), 27 (two weeks following first boost) and 42 (two weeks following second boost). S. Paratyphi A FliC was extracted from strain CVD 1902. LPS was extracted from strain CVD 1901. (B) Comparison of anti-FliC and anti-LPS total serum IgG titers at day 42 from individual mice. Closed shapes represent mice that succumbed to the challenge.

Figure 6. Immunization with CVD 1901D and CVD 1901K but not CVD 1901, induces high anti-FliC IgG1.

Subclass distribution of IgG antibodies against S. Paratyphi A flagella. (A) Anti-FliC IgG1 and IgG2a titers detected in day 42 sera. (B) The ratio between the antibody subclasses. For (A) and (B), closed shapes represent mice that succumbed to the challenge.

Figure 7. CVD 1901 cell-associated flagella elicits antibodies with opsonophagocytic rather than complement-mediated bactericidal activity.

(A) Serum-induced killing of ATCC 9150 (flagellated) and 9150K (non-flagellated) bacteria mediated by complement. Sera are from day 42 of the experiment described in Fig. 5. (B) Opsonophagocytic activity of sera withdrawn at day 27 in J774A.1 macrophage against the wild-type ATCC 9150 strain. For (A) and (B), closed shapes represent mice that succumbed to the challenge.