Salmonella Paratyphi A Outer Membrane Vesicles Displaying Vi Polysaccharide as a Multivalent Vaccine against Enteric Fever

Abstract

Typhoid and paratyphoid fevers have a high incidence worldwide and coexist in many geographical areas, especially in low-middle-income countries (LMIC) in South and Southeast Asia. There is extensive consensus on the urgent need for better and affordable vaccines against systemic Salmonella infections. Generalized modules for membrane antigens (GMMA), outer membrane exosomes shed by Salmonella bacteria genetically manipulated to increase blebbing, resemble the bacterial surface where protective antigens are displayed in their native environment. Here, we engineered S Paratyphi A using the pDC5-viaB plasmid to generate GMMA displaying the heterologous S Typhi Vi antigen together with the homologous O:2 O antigen. The presence of both Vi and O:2 was confirmed by flow cytometry on bacterial cells, and their amount was quantified on the resulting vesicles through a panel of analytical methods. When tested in mice, such GMMA induced a strong antibody response against both Vi and O:2, and these antibodies were functional in a serum bactericidal assay. Our approach yielded a bivalent vaccine candidate able to induce immune responses against different Salmonella serovars, which could benefit LMIC residents and travelers.

Keywords: GMMA; OMV; S. Paratyphi; S. Typhi; Salmonella; Vi; enteric fever; typhoid fever; vaccine.

FIG 1

Display of polysaccharide antigens on the bacterial surface. Flow cytometry analysis of surface polysaccharides of ParA O:2 Vi⁻, ParA O:2 Vi⁺, S. Typhi O:9 Vi⁺, and S. Typhi O:9 Vi⁻. Flow cytometry was performed using rabbit anti-O:2, -O:9, and -Vi polyclonal serum, followed by Alexa Fluor 488-conjugated secondary antibodies. Bacteria stained with a rabbit anti-O:4 polyclonal serum were included as a negative control.

FIG 2

Immunogenic (ELISA) and functional (SBA) assessment of vaccines. Total anti-O:2, anti-O9, and anti-Vi ELISA IgG (top) and SBA titers (IC₅₀) against S. Paratyphi A (O:2-positive), S. Enteritidis (O:9-positive), and C. freundii s.l. (Vi-positive) strains (bottom) are shown. Unpaired, nonparametric t test (Mann-Whitney) was used to determine the statistically significant differences between groups (ns, not significant; *, P < 0.033; **, P < 0.002).