Antigen-specific B-1a antibodies induced by Francisella tularensis LPS provide long-term protection against F. tularensis LVS challenge

Abstract

Francisella tularensis (Ft), a gram-negative intracellular bacterium, is the etiologic agent of tularemia. Infection of mice with <10 Ft Live Vaccine Strain (Ft LVS) organisms i.p. causes a lethal infection that resembles human tularemia. Here, we show that immunization with as little as 0.1 ng Ft LVS lipopolysaccharide (Ft-LPS), but not Ft lipid A, generates a rapid antibody response that protects wild-type (WT) mice against lethal Ft LVS challenge. Protection is not induced in Ft-LPS-immunized B cell-deficient mice (muMT or JhD), male xid mice, or Ig transgenic mice that produce a single IgH (not reactive with Ft-LPS). Focusing on the cellular mechanisms that underlie this protective response, we show that Ft-LPS specifically stimulates proliferation of B-1a lymphocytes that bind fluorochrome-labeled Ft-LPS and the differentiation of these cells to plasma cells that secrete antibodies specific for Ft-LPS. This exclusively B-1a antibody response is equivalent in WT, T-deficient (TCRalphabeta(-/-), TCRgammadelta(-/-)), and Toll-like receptor 4 (TLR4)-deficient (TLR4(-/-)) mice and thus is not dependent on T cells or typical inflammatory processes. Serum antibody levels peak approximately 5 days after Ft-LPS immunization and persist at low levels for months. Thus, immunization with Ft-LPS activates a rare population of antigen-specific B-1a cells to produce a persistent T-independent antibody response that provides long-term protection against lethal Ft LVS infection. These data support the possibility of creating effective, minimally invasive vaccines that can provide effective protection against pathogen invasion.

Fig. 1.

Survival of saline- or Ft-LPS-pretreated mice after challenge with Ft LVS. (A) WT C57BL/6J mice were injected i.p. with saline or 0.1 - 100 ng Ft-LPS 2 d before challenge with ≈10³ CFU Ft LVS i.p. Data shown are from 1 experiment, 5 mice per treatment. (B) 5 WT mice per treatment/experiment were injected i.p. with saline, 100 ng Ft-LPS, 100 ng Ft LVS lipid A, or 1000 ng Ft LVS lipid A 2 d before challenge with ≈10⁴ CFU Ft LVS. Combined results are shown for 2 experiments. (C) 5 WT or μMT mice were injected i.p. with saline or 100 ng Ft-LPS 2 d before challenge with ≈10⁴ CFU Ft LVS. Data in the experiment shown are representative of 7 with similar design and outcome.

Fig. 2.

Ft-LPS induces the appearance and proliferation of splenic Ft-LPS-binding (Ft-LPS⁺) B cells and plasma cells of the B-1a subset/lineage. (A) (Upper) Spleen cells from C57BL/6J mice immunized i.p. with Ft-LPS (100 ng) were stained with fluorochrome-labeled Ft-LPS and fluorescent antibodies recognizing a panel of B cell surface antigens. The total live cell population was further gated to display splenic B cells (IgM⁺B220⁺), for which κ light chain expression and Ft-LPS binding are shown in the figure. The circled cell population contains B cells that bind Ft-LPS; the proportion of the total B cell population represented within this gate is indicated for each day. (Lower) Ft-LPS⁺ B cells were further gated to distinguish Ft-LPS⁺ plasma cells, which express CD138. The proportion of cells expressing the plasma cell phenotype is shown. Note: on days 1 and 2, spleens from Ft-LPS immunized animals are equivalent to spleens in control (PBS) animals; i.e., few cells are within the gated region for Ft-LPS⁺Igκ⁺. (B) Surface marker expression on live Igκ⁺ Ft-LPS⁺ CD138⁻ B cells (black histogram) and Igκ⁺ Ft-LPS⁺ CD138⁺ plasma cells (gray histogram) in spleen 4 days after i.p. injection of 100 ng Ft-LPS. Reference histograms show surface marker expression for the relevant B cell population as indicated. B-2 cells were gated as IgD^hi IgM^lo CD23^hi B cells and Marginal Zone (MZ) B cells were gated as IgD^lo IgM^hi CD21^hi B cells. The reference histogram for the CD5⁺ cells shows data for the FMO staining control, in which cells were stained with the full reagent mixture from which anti-CD5 antibody has been omitted to reveal the appropriate threshold for identifying CD5⁺ cells. Similar data were obtained on day 3, day 5 and day 7 (data not shown).

Fig. 3.

Absolute numbers of Ft-LPS⁺ B-1a cells and plasma cells (CD138⁺) in spleen peak on day 5 after immunization, whereas anti-Ft LVS IgM titers in the sera peak at day 5 and are sustained. C57BL/6J mice were killed 1–7 days after i.p. injection of 100 ng Ft-LPS. Mice killed on day 0 were injected with saline only. Tissues and sera were collected from all mice at the time of killing and analyzed by Hi-D FACS (A) and ELISA (B) (n = 3). (A) Each triangle represents Hi-D FACS data from an individual mouse. Horizontal lines in the “quartile box plots” indicate the 25th, 50th and 75th percentile values. (B) ELISAs using Ft-LPS-coated plates were performed on serum from collected from the mice analyzed in A. Each symbol (squares, circles, and crosses) represents the anti-Ft-LPS titer from an individual mouse.