Purified lipopolysaccharide from Francisella tularensis live vaccine strain (LVS) induces protective immunity against LVS infection that requires B cells and gamma interferon

Abstract

Previous results have demonstrated that nonspecific protective immunity against lethal Francisella tularensis live vaccine strain (LVS) or Listeria monocytogenes infection can be stimulated either by sublethal infection with bacteria or by treatment with bacterial DNA given 3 days before lethal challenge. Here we characterize the ability of purified lipopolysaccharide (LPS) from F. tularensis LVS to stimulate similar early protective immunity. Treatment of mice with surprisingly small amounts of LVS LPS resulted in very strong and long-lived protection against lethal LVS challenge within 2 to 3 days. Despite this strong protective response, LPS purified from F. tularensis LVS did not activate murine B cells for proliferation or polyclonal immunoglobulin secretion, nor did it activate murine splenocytes for secretion of interleukin-4 (IL-4), IL-6, IL-12, or gamma interferon (IFN-gamma). Immunization of mice with purified LVS LPS induced a weak specific anti-LPS immunoglobulin M (IgM) response and very little IgG; however, infection of mice with LVS bacteria resulted in vigorous IgM and IgG, particularly IgG2a, anti-LPS antibody responses. Studies using various immunodeficient mouse strains, including LPS-hyporesponsive C3H/HeJ mice, muMT(-) (B-cell-deficient) knockout mice, and IFN-gamma-deficient mice, demonstrated that the mechanism of protection does not involve recognition through the Lps(n) gene product; nonetheless, protection was dependent on B cells as well as IFN-gamma.

FIG. 1

Dose response of protection against lethal LVS challenge stimulated by immunization with LVS LPS. Groups of five BALB/cByJ mice were given the indicated dose of LVS LPS (or PBS) i.d. Three days later all mice were challenged with either 10³ LVS bacteria i.p. (black bars) or 10⁴ LVS bacteria i.p. (open bars). Percent survival is shown for a single experiment. This experiment is representative of four experiments of similar design.

FIG. 2

Strength of protection against lethal LVS challenge stimulated by immunization with LVS LPS. Groups of five BALB/cByJ mice were given 100 ng of LVS LPS (or PBS) i.d. Three days later all mice were challenged with the indicated number of LVS bacteria i.p. Percent survival is shown for a single experiment. This experiment is representative of three experiments of similar design.

FIG. 3

Time course of development of protection against lethal LVS challenge stimulated by immunization with LVS LPS. Groups of five BALB/cByJ mice were given 100 ng of LVS LPS (or PBS) i.d. on the indicated day before all mice were challenged with 10⁴ LVS bacteria i.p. Percent survival is shown for a single experiment. This experiment is representative of two experiments of similar design.

FIG. 4

Ability of various types of LPS to stimulate protection against lethal LVS challenge. Groups of 5 BALB/cByJ mice were given 100 ng of the indicated LPS (or PBS) i.d.; three days later, all mice were challenged with 10⁴ LVS bacteria i.p. Percent survival is shown for a single experiment. This experiment is representative of four experiments of similar design.

FIG. 5

Time course of specific antibody responses in BALB/cByJ mice immunized with LVS LPS compared to infection with LVS. Serum samples were obtained from five normal BALB/cByJ mice (prebleeds), and approximately equal amounts of blood from each mouse were pooled. Mice were then given 100 ng of LPS i.d. (A and B) or 10⁴ LVS bacteria i.d. (C and D); actual bacterial doses were confirmed by plate count at the time of inoculation. On the indicated days after immunization or infection, pooled serum samples were obtained and tested by ELISA using plates coated with either purified LPS (A and C) or whole LVS bacteria (B and D). Antibodies were detected by the use of either horseradish peroxidase-labeled goat anti-mouse IgM (●) or anti-mouse IgG (▿) antibodies. Endpoint titers from a single representative experiment using pooled serum samples are shown; titers are defined in relationship to the binding of matched prebleed normal mouse serum, which was uniformly low and similar to that of germfree mouse serum (see Materials and Methods). This experiment is representative of three experiments of similar design.

FIG. 6

Induction of proliferation of BALB/cByJ spleen cells by LVS LPS. Spleen cells obtained from normal BALB/cByJ mice were cultured at 2 × 10⁵/well in complete medium with the indicated amounts of E. coli O111 or LVS LPS. Proliferation was assessed by adding [³H]thymidine overnight on day 2 before harvesting and counting. Mean uptake of ³H per well for triplicate wells is shown; error bars indicate standard deviation of the mean. The response to 1 μg of concanavalin A per ml was 138,244 ± 16,481 in this experiment. This experiment is representative of four experiments of similar design.