Detoxified endotoxin vaccine (J5dLPS/OMP) protects mice against lethal respiratory challenge with Francisella tularensis SchuS4

Abstract

Francisella tularensis is a category A select agent. J5dLPS/OMP is a novel vaccine construct consisting of detoxified, O-polysaccharide side chain-deficient, lipopolysaccharide non-covalently complexed with the outer membrane protein of N. meningitidis group B. Immunization elicits high-titer polyclonal antibodies specific for the highly-conserved epitopes expressed within the glycolipid core that constitutes gram-negative bacteria (e.g., F. tularensis). Mice immunized intranasally with J5dLPS/OMP exhibited protective immunity to intratracheal challenge with the live vaccine strain, as well as the highly-virulent SchuS4 strain, of F. tularensis. The efficacy of J5dLPS/OMP vaccine suggests its potential utility in immunizing the general population against several different gram-negative select agents concurrently.

Fig. 1

J5 core glycolipid-specific antibodies are elevated in mice immunized with J5dLPS/OMP vaccine. Groups of mice were inoculated i.n. with J5dLPS/OMP and CpG ODN (squares); control mice received CpG ODN alone (circles). Anti-J5 core glycolipid-specific IgG (A) and IgA (B) antibodies in the sera (open symbols) and BAL fluids (closed symbols) obtained on day 28 following the last inoculation were quantified. The pooled data derived from 2 separate experiments are shown. IgG and IgA anti-J5 core glycolipid-specific antibody concentrations in the sera and BAL fluids obtained from the vaccinated animals are significantly greater (P<0.05; non-paired Student’s t test or Mann-Whitney Rank sum test).

Fig. 2

J5dLPS/OMP vaccine protects mice against F. tularensis LVS respiratory challenge. Groups of 10 control (CpG ODN alone) and 10 vaccinated (J5dLPS/OMP plus CpG ODN) mice were challenged i.t. with 9,800 CFUs F. tularensis LVS on day 28 post-vaccination. Data are derived from a single experiment representative of 4 separate experiments. J5dLPS/OMP vaccinated and CpG ODN control groups are significantly different; P = 0.0006 (LogRank test).

Fig. 3

The bacterial burden is diminished in the tissues of vaccinated mice. Mice administered J5dLPS/OMP plus CpG ODN or CpG ODN alone were challenged i.t. with 6,800 CFUs (~5 LD₅₀) F. tularensis LVS on day 35 following final vaccine delivery. The lungs (A), blood (B) and livers (C) were collected on day 4 postinfection and the bacterial burden was determined. The pooled data derived from 2 separate experiments and a total of 5-6 mice per group are shown; each symbol represents a single mouse. The bacterial burden was significantly lower in all tissues derived from vaccinated animals (p<0.05, Student’s t test).

Fig. 4

Proinflammatory cytokine/chemokine message and protein concentrations in the lungs are unchanged shortly following i.t. inoculation of F. tularensis LVS. Groups of control and vaccinated mice were challenged i.t. with 7,200 CFUs F. tularensis LVS. The lungs were dissected at 4 hours post-infection, and the expression of IL-12 (A), IFN-γ (B) and TNF-α (C) mRNAs quantified. The protein concentrations of the cytokines and chemokines listed were quantified (D). Data are the means ± SD derived from 4 mice treated similarly in each group. Second experiments yielded comparable results. ND = not detected. All differences between vaccinated and control mice were statistically insignificant; P>0.05 (Student’s t test).

Fig. 5

Proinflammatory cytokine/chemokine protein levels are diminished in the livers of vaccinated mice on day 3 post-challenged with F. tularensis LVS. Control (CpG ODN) and vaccinated (J5dLPS/OMP plus CpG ODN) mice were challenged i.t. with ~5 LD₅₀ F. tularensis LVS. On day 3 post-infection, the lungs (top panel) and livers (bottom panel) were dissected; the cytokines and chemokines listed were quantified. Data are the means ± SD derived from 4 mice treated comparably. A second experiment yielded similar results. *Vaccinated group is significantly less than control: P<0.05 (Student’s t test). ND = not detected.

Fig. 6

Vaccination suppresses the accumulation of neutrophils in the lungs subsequent to infection. Control and vaccinated groups of mice were challenged i.t. with 7,200 CFUs F. tularensis LVS. At 4, 24 and 72 hours post-infection, the lungs were dissected from four mice in each group and myeloperoxidase activity was quantified as a measure of neutrophil accumulation. *Significantly less than 72-hour group administered CpG ODN alone; P<0.01 (McNemar’s test).

Fig. 7

Vaccination with J5dLPS/OMP and CpG ODN protects mice against respiratory, F. tularensis SchuS4 challenge. Groups of mice were vaccinated with J5dLPS/OMP and CpG ODN; negative control mice received CpG ODN alone; positive control mice were immunized by infection i.t. with 100 CFUs F. tularensis LVS on day 0. All three groups were challenged i.t. with 10 CFUs F. tularensis SchuS4 on day 37 post-vaccination. Data are combined from 2 separate experiments and a total of 18-20 mice per group. J5dLPS/OMP vaccinated and CpG ODN control groups are significantly different; P = 0.0013 (LogRank test).