The porin OmpD from nontyphoidal Salmonella is a key target for a protective B1b cell antibody response

Abstract

Invasive nontyphoidal Salmonella (NTS), including Salmonella typhimurium (STm), are major yet poorly-recognized killers of infants in sub-Saharan Africa. Death in these children is usually associated with bacteremia, commonly in the absence of gastrointestinal symptoms. Evidence from humans and animal studies suggest that severe infection and bacteremia occur when specific Ab is lacking. Understanding how Ab responses to Salmonella are regulated will help develop vaccines against these devastating infections. STm induces atypical Ab responses characterized by prominent, accelerated, extrafollicular T-independent (TI) Ab against a range of surface antigens. These responses develop without concomitant germinal centers, which only appear as infection resolves. Here, we show STm rapidly induces a population of TI B220(+)CD5(-) B1b cells during infection and TI Ab from B1b cells targets the outer membrane protein (Omp) porins OmpC, OmpD and OmpF but not flagellin. When porins are used as immunogens they can ablate bacteremia and provide equivalent protection against STm as killed bacterial vaccine and this is wholly B cell-dependent. Furthermore Ab from porin-immunized chimeras, that have B1b cells, is sufficient to impair infection. Infecting with porin-deficient bacteria identifies OmpD, a protein absent from Salmonella Typhi, as a key target of Ab in these infections. This work broadens the recognized repertoire of TI protein antigens and highlights the importance of Ab from different B cell subsets in controlling STm infection. OmpD is a strong candidate vaccine target and may, in part, explain the lack of cross-protection between Salmonella Typhi and STm infections.

Fig. 1.

Porins from STm induce a T-independent Ab response. (A) T cell-deficient TCRβδ^−/− mice were infected i.p. with 5 × 10⁵ attenuated STm for 4 days, and splenic CD138⁺ plasmacytoid cells per square millimeter (Left) were assessed by immunohistology. Anti-STm, anti-LPS, anti-flagellin (FliC), and anti-total Omp serum IgM was assessed by ELISA (Right). Data are representative of 2 experiments. (B) Analysis of porin purity and structure. Electrophoretic separation of porins showing their purity (Left) reveals 2 major species OmpC and OmpD with trace amounts of OmpF (see Materials and Methods). (Center) A spectrum of porins, using CD [millidegrees (mdeg)], indicating they are β-stranded. The boxed gel (Right) shows the electrophoretic pattern of porins under denaturing (100 °C) or seminative (25 °C) conditions. (Lower Left) s distributions (c(s)) for porins after AUC, indicating that the porins had a peak coefficient indicative of oligomeric structures with Mr of 120–240 kDa. (C) ELISA assessment of porin-specific IgM from nonimmunized and 7-day (d) porin-immunized TCRβδ^−/− mice. Immunoblot of IgM binding to Omp from naïve and porin-immunized mice. Porins separate to the boxed area. Binding of sera to other proteins (such as 27 kDa) reflects the presence of natural Ab and is independent of porin immunization. ELISA data representative of 3 experiments; immunoblots of 5 naïve and immune sera. The Mann–Whitney test was applied to compare the statistical significance between groups linked by bars. NI, noninfected. *, P < 0.05.

Fig. 2.

Immunization with porins impairs STm infection in a B cell-dependent manner. (A) Nonimmunized (NI), porin-immunized or HK STm immunized WT mice were infected with 5 × 10⁶ STm cells for 5 days and splenic bacterial numbers enumerated. (B) NI or porin immunized WT (filled circles) or B cell-deficient (open circles) (IgH^−/−) mice were infected with 5 × 10⁶ STm cells for 5 days and spleen and liver bacterial numbers enumerated (Left) (representative of 3 repeats). B cell-deficient mice were infected with STm opsonized with sera from NI (filled circles) or porin-immunized (open circles) mice (Right) (representative of 2 repeats). (C) NI or porin-immunized WT, CD28^−/− or TCRβδ^−/− mice were infected with 5 × 10⁶ STm for 5 days and splenic bacterial numbers enumerated (Left) (representative of 3 and 2 experiments). (Right) Serum IgM titers from 5-day STm-infected NI and porin-immunized WT and TCRβδ^−/− mice (representative of 2 experiments). (D) Blood bacterial counts from NI and porin-immunized WT mice infected with 5 × 10⁶ STm for 5 days (representative of 2 repeats). (E) Spleen and liver bacterial counts from WT mice immunized once (Left) or twice (Right) with porins and infected with 3 × 10³ STm strain SL1344 cells for 4 days (representative of 2 experiments). (F) Splenic bacterial counts from NI WT mice or WT mice immunized with STm porins, ST porins or LPS and infected with 5 × 10⁶ STm for 5 days (representative of 3 experiments). The Mann–Whitney test was applied to compare the statistical significance between groups linked by bars. *, P < 0.05; **, P < 0.01; NS, not significant.

Fig. 3.

OmpD is an important target of the humoral immune response to STm. (A) Nonimmunized (NI) or porin-immunized WT mice were infected with 5 × 10⁶ STm (closed circles) or 5 × 10⁶ STm lacking OmpD (STmOmpD⁻ strain RAK126; open circles) for 5 days and splenic, liver and blood bacterial numbers enumerated (representative of 3 experiments for spleen and liver). Immunoblot analysis of IgM from NI or porin-immunized TCRβδ^−/− mice against cell walls from STm or STm lacking OmpD. The boxed region shows the approximate Mr of the porins OmpC and F. (B) NI or porin-immunized WT mice were infected with 5 × 10⁶ STm 5 × 10⁶ STm cells (filled circles) or 5 × 10⁶ STm cells lacking OmpR (STmOmpR⁻ strain RAK83) (open circles) for 5 days and splenic, liver, and blood bacterial numbers enumerated (representative of 2 experiments). Immunoblot analysis of IgM from NI or porin-immunized TCRβδ^−/− mice against cell walls from STm or STm lacking OmpR. The boxed region shows the approximate Mr of the porin OmpD. The Mann–Whitney test was applied to compare the statistical significance between groups linked by bars. *, P < 0.05; NS, not significant; d, day.

Fig. 4.

Porins induce B1b cells. (A) Peritoneal cells from nonimmunized (NI) WT mice and WT mice immunized with the described antigens for 4 days were stained using 7 color FACS. IgM⁺CD19⁺ cells were gated from the lymphocyte population. Cells with low CD21 and CD23 were gated from the IgM⁺CD19⁺ population, and B1b cells were IgM⁺CD19⁺CD21^loCD23^lo cells that were CD5⁻B220⁺. The final FACS gate shows the proportion of B1b cells that express CD11b. Graphs show the proportion of B1 cells that are B1b and the proportion of B1b cells that are CD11b⁺. Data representative of ≥2 repeat experiments. (B) B1b cells from TCRβδ^−/− mice before and after immunization with porins and STm for 4 days were identified as in A. Graphs show the proportion of B1 cells that are B1b and the proportion of B1b cells that are CD11b⁺. Numbers refer to the percentages of cells in the gated boxes and are representative of 2 experiments. The Mann–Whitney test was applied to compare the statistical significance between NI and immunized groups linked by bars. *, P < 0.05; **, P < 0.01. Approximately 5 × 10³ events are shown in each CD21/CD23 gate. EU, endotoxin units.

Fig. 5.

Ab derived from B1b cells against porins is sufficient to impair STm infection. (A) (Upper) FACS plot showing the B220⁺CD5⁻ cells (boxed) sorted for transfer into B cell-deficient IgH^−/− mice. (Lower) Plot showing that the purity of the sorted cells was >95% and were CD5⁻. Graph shows serum IgM Ab titers from B1b chimeras 20 days after transfer in the absence of porin immunization. (B) Splenic bacterial burden (Left) and IgM titer (Right) of B cell-deficient mice that have either only been infected or been infected after porin immunization or have been infected after B1b transfer and porin immunization. FACS plots show the absence of B cells in the peritoneal cavity of B cell-deficient mice (Left) but their presence in B1b chimeras (Center). (Right) Plot showing CD11b expression on chimera B cells. The Mann–Whitney test was applied to compare the statistical significance between groups linked by bars. *, P < 0.05. NS, not significant.