Fig 1.
Analysis of recombinant PrgI and SipD proteins.
(A) SDS-PAGE / Coomassie blue staining (reducing conditions) of purified recombinant proteins. polyHis-SipD (38.2 kDa, lane 2) and polyHis-PrgI (9.9 kDa, lane 3) are shown with molecular mass markers in kilodaltons (kDa) (lane 1). (B) Far-UV Circular-Dichroism spectroscopy of recombinant proteins were recorded at 20°C in phosphate buffer at pH 7.4.
Fig 2.
Serum Ig(G+M) concentrations of mice immunized with PrgI or SipD (A) and PrgI/SipD (B).
Serum Ig(G+M) antibodies specific for PrgI (left) and SipD (right) were quantified by sandwich ELISA 2 weeks after the last immunization as described in Materials and Methods. Data represent mean concentrations (ng/mL) and the standard errors (SEM) from 14–16 individual mice per group. Asterisks *** indicate P value< 0.001, comparing the antibody responses using different routes versus control mice. No cross-reactions were observed between PrgI and SipD (data not shown). [°: indicates injected immunogen; *: indicates biotinylated recombinant protein].
Table 1.
Summary of the antibody responses (IgG and IgA) after the last immunization with PrgI or SipD by the SC, IN and OG routes.
Fig 3.
IgA titers of mice immunized with PrgI or SipD (A) or PrgI/SipD (B).
Serum IgA antibodies specific for PrgI (left) and SipD (right) were measured by sandwich ELISA 2 weeks after the last immunization as described in Materials and Methods. Data represent mean titers and the standard errors (SEM) from 14–16 individual mice per group. Asterisks indicate P values: *** p < 0.001, ** 0.001<p <0.01 and * p< 0.05 when comparing mice immunized by the IN or OG route versus mice immunized by the SC route and control mice. No cross-reactions were observed between PrgI and SipD (data not shown). [°: indicates injected immunogen; *: indicates biotinylated recombinant protein].
Table 2.
Summary of the antibody responses (IgG and IgA) after the last immunization with both proteins (PrgI and SipD) by the SC, IN and OG routes.
Fig 4.
IgG (2a +2b) / IgG 1 ratio after PrgI (left) and SipD (right) immunizations.
Mice immunized with PrgI or SipD separately are represented on panel A and those receiving both PrgI and SipD on panel B. Data represent mean and the standard errors (SEM) from 14–16 mice per group. [°: indicates immunogen injected; *: indicates biotinylated recombinant protein].
Fig 5.
Protective efficacy of PrgI and SipD (A-D).
Mice (N = 14–16) were immunized at days 0, 21 and 42 or 0, 21, 42 and 63 by the indicated routes. Six weeks after the last immunization: at day 84 for the SC (A), IN (B) and OG 3I (C) routes; at day 105 for the OG 4I (D) route, 106 CFU/mL (100 LD 50) of S. Typhimurium were administered orally to immunized and control mice. Survival was monitored for 21 days. Statistical significance was determined using a 2-tailed Fisher's exact test. Statistically significant differences are indicated by *** p < 0.001, ** 0.001<p <0.01 and * p< 0.05 compared to PBS groups.
Table 3.
Protection efficacy of PrgI and SipD T3SS proteins in mice from lethal challenge with S. Typhimurium.
Table 4.
SipD protein identity sequences for the 4 major Salmonella pathogens of humans.