Construction and expression of F. tularensis antigens by attenuated recombinant L. monocytogenes vaccines in broth culture and in human macrophages. A. Construction of attenuated recombinant L. monocytogenes expressing F. tularensis antigens. The F. tularensis nucleotide sequence encoding each antigen was PCR amplified from F. tularensis RCI genomic DNA and cloned into the vector pKB199 downstream of the promoter (Phly) and signal sequence (hly S.S.) of L. monocytogenes hly to generate pKB/Ft. The expression cassette of the fusion protein in pKB/Ft was excised by NotI and subcloned into the NotI site of the integration plasmid pDP4189 to generate pDP/Ft. The pDP/Ft plasmid was then integrated into the 3′ end of an arginine tRNA gene in the attenuated L. monocytogenes strain rLmΔactA through conjugation [60]. The resulting recombinant L. monocytogenes vaccines carried one single copy of the F. tularensis gene. A portion of the Figure was modified from Bruhn et al [60]. B and C. Protein expression in the culture supernatant of recombinant L. monocytogenes. Culture filtrates of each recombinant L. monocytogenes strain were concentrated and a volume of filtrate equivalent to 1 ml of the bacterial culture was analyzed by Western blotting using rabbit polyclonal antibody to AcpA (B, top panel), Bfr (B, middle panel), DnaK (B, bottom panel), GroEL and KatG (C, top panel), or IglC (C, bottom panel). D and E. Protein expression in human macrophages. THP-1 cells were mock infected or infected with the parental rLmΔactA strain, or the recombinant L. monocytogenes vaccines expressing F. tularensis IglC (rLm/iglC) or KatG (rLm/katG) at an MOI of 50. At 24 h post-infection, the infected cells were lysed and subjected to Western blotting using polyclonal antibody to IglC (D) or KatG (E). On the right border of each blot are listed the proteins of interest and their predicted mass. On the left border are listed the sizes of the molecular mass standards. M, Molecular mass standards.

Bacterial burden in the host tissues of mice after intranasal challenge with F. tularensis LVS. Forty BALB/c mice were anesthetized with Ketamine/Xylazine and then infected i.n. with 4400 CFU of LVS. At the indicated times post-infection, 8 mice per group were euthanized and the liver, spleen and lung were removed and homogenized in 2 ml of sterile PBS for 15 seconds in a Pro200 homogenizer. CFU of F. tularensis in each organ was determined by plating serial dilutions of the homogenates on Chocolate agar containing sulfamethoxazole (40 mg/ml), trimethoprim (8 mg/ml) and erythromycin (50 mg/ml) and culturing for 3 days at 37°C. Values represent the mean Log CFU per organ ± SE.

Intradermal immunization with attenuated recombinant L. monocytogenes vaccines expressing IglC protects mice against i.n. challenge with lethal dose of LVS. A. Intranasal challenge with 6 × LD₅₀ of LVS. This challenge study was performed simultaneously with the one shown in Fig. 4. Six groups of 8 mice were sham-immunized or immunized with LVS or rLm vaccines at weeks 0 and 4. At week 8, mice were anesthetized with Ketamine/Xylazine, challenged i.n. with 4400 CFU of LVS, and monitored for survival for up to 3 weeks after challenge. B. Intranasal challenge with 65 × LD₅₀ of LVS. Groups of mice were sham-immunized or immunized i.d. twice at weeks 0 and 4 with 5 × 10⁴ CFU LVS or 1 × 10⁶ CFU of rLmΔactA (vector), rLm/iglC, rLm/katG or rLm/iglC plus rLm/katG. At 4 – 6 weeks after the last immunization, mice were anesthetized with Ketamine/Xylazine, challenged i.n. with 4.5 × 10⁴ CFU F. tularensis LVS (65 LD₅₀s), and monitored for 3 weeks. Data shown in Fig. 5B are combined from three independent experiments totaling 20 mice per group. Survival curves among mice in the sham or vector-immunized group and mice in the LVS or rLm vaccine-immunized groups were compared by logrank analysis. ns, not significant (P > 0.05). Mean survival time (MST) was calculated by dividing the sum of the survival times of all mice in a group by the total number of mice challenged, with surviving animals given a survival time of 21 days, when the experiment was terminated.

Antigen-specific intracellular IFNγ secretion after intradermal immunization with recombinant L. monocytogenes vaccines expressing F. tularensis proteins. Groups of 3 BALB/c mice were sham-immunized or immunized i.d. twice at weeks 0 and 3 with 5 × 10⁴ CFU LVS, with 1×10⁶ CFU rLm/iglC, rLm/katG, or the same dose of both vaccines. Six days after the second immunization, mice were euthanized and their splenocytes were incubated with or without the relevant F. tularensis protein (10 μg/ml): IglC (A and B), KatG (C and D), or IglC plus KatG (E and F). The cells were stained with FITC-conjugated rat anti-mouse CD4 (A, C and E) or PE-Cy5-conjugated rat anti-mouse CD8 monoclonal antibody (B, D, and F). Cells were then washed and fixed per protocol and stained for intracellular IFNγ with PE-conjugated rat anti-mouse IFNγ antibody. Stained cells were analyzed on a FACSCalibur cytometer using CellQuest software. Each individual symbol represents result from an individual mouse. The bars represent the mean value for three animals. Asterisks indicate that the difference in the cell numbers between the mice in the indicated group and that of mice in the sham- or vector-immunized group was significant. *, P<0.05; **, P<0.01; ***, P<0.001 by one-way ANOVA with Bonferroni’s post test.

Intradermal immunization with recombinant L. monocytogenes vaccines protects mice against aerosol challenge with F. tularensis Type A SchuS4 strain. Ten groups of eight BALB/c mice were sham-immunized, or immunized i.d. twice at weeks 0 and 4 with 1×10⁷ CFU rLmΔactA, rLm/iglC or rLm/katG. Mice immunized twice with 1 × 10⁴ CFU LVS served as a positive control. Six weeks after the second immunization (week 10), mice were challenged by aerosol with F. tularensis SchuS4 with a dose estimated to be either 1 × LD₁₀₀ (A) or 10 × LD₁₀₀ (B). [(The effective dose actually delivered in (A) was less than 1 × LD₁₀₀ as 50% of the animals survived, i.e. the dose was effectively 1 × LD₅₀. The animals in (B) received 10 times the dose of the animals in (A), as detailed in Methods]. The difference in survival between the mice in a vaccinated group and mice in a sham- or vector-vaccinated group was evaluated using a logrank analysis. ns, not significant (P > 0.05).

Immunogenicity and protective immunity induced by attenuated recombinant L. monocytogenes vaccines. A. Immunogenicity. Nine groups of 3 BALB/c mice were sham-vaccinated, or vaccinated twice at weeks 0 and 5 with 1 × 10⁶ CFU of the parental recombinant L. monocytogenes vector (rLmΔactA) or the rLm vaccines expressing various F. tularensis antigens as indicated on the horizontal scale of the graph. Mice vaccinated twice with 5 × 10⁴ CFU LVS served as positive controls. At week 9, mice were euthanized, a single cell suspension of splenocytes was prepared and incubated with the F. tularensis antigen (10 μg/ml) indicated on the horizontal scale of the graphs, and the culture then pulsed with 0.25 μCi of [³H]thymidine/ml for 2 h. The mean CPM varied from group to group, ranging from 90 to 380 in the absence of the F. tularensis protein and from 350 to 1500 in the presence of F. tularensis protein. The Stimulation Index (SI) was determined by dividing the mean CPM in the presence of the F. tularensis protein by the mean CPM in the absence of the F. tularensis protein. Values are the mean ± SE of the SI. The asterisks indicate that the difference in SI between the mice in a control group and the mice in the rLm/iglC-vaccinated group was significant. *, P < 0.05 and **, P < 0.01 by student t-test. B. Protective Immunity. Ten groups of 8 mice were immunized as described in A. At week 9, mice were anesthetized with Isoflurane, challenged i.n. with 1 × 10⁶ CFU LVS (30 × LD₅₀s), and monitored for illness and survival for three weeks. The differences in survival between the mice in the vaccinated groups and mice in the sham-vaccinated group were evaluated using a logrank analysis. ns, not significant (P > 0.05). Mean survival time (MST) was calculated by dividing the sum of the survival times of all mice in a group by the total number of mice challenged, with surviving animals given a survival time of 21 days, when the experiment was terminated.

Intradermal immunization with attenuated recombinant L. monocytogenes vaccines expressing IglC reduces the bacterial organ burden after i.n. challenge with LVS. Six groups of 8 BALB/c mice were sham-immunized, immunized once with LVS [LVS(1)], or twice with LVS [(LVS(2)], rLmΔactA, rLm/iglC or rLm/katG at an interval of 4 weeks. At week 8, mice were anesthetized with Ketamine/Xylazine and challenged i.n. with 4400 CFU LVS. At day 5 post-challenge, mice were euthanized, and the liver, spleen and lung were removed and assayed for CFU of F. tularensis as described in Figure 3B. Values represent the mean Log CFU per organ ± SE. The asterisks indicate that the differences in bacterial burden among the animals in the indicated paired groups are statistically significant. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by one-way ANOVA with Bonferroni’s post test.

Lymphocyte proliferation induced by intradermal immunization with attenuated L. monocytogenes vaccines expressing F. tularensis IglC or KatG. Groups of 3 BALB/c mice were sham-immunized, immunized i.d. twice with 1 × 10⁴ CFU LVS, or 1×10⁶ CFU recombinant L. monocytogenes vaccines expressing IglC (rLm/iglC), KatG (rLm/katG), or the combination of both at weeks 0 and 3. Six days after the second immunization, mice were euthanized and their splenocytes isolated and assayed for lymphocyte proliferation in response to recombinant IglC (A), KatG (B) or the IglC plus KatG (C) proteins. The mean CPM varied from group to group, ranging from 355 to 564 in the absence of the F. tularensis protein and from 493 to 4840 in the presence of the F. tularensis protein. The Stimulation Index (SI) was determined by dividing the mean CPM obtained in the presence of the F. tularensis protein by the mean CPM obtained in the absence of F. tularensis protein. Standard errors of the means of triplicate samples from three mice are shown. Asterisks indicate that the differences in SIs between the indicated groups are significant. *, P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA with Bonferroni’s post test.

Antibody response induced by intradermal immunization with recombinant L. monocytogenes. Groups of 5 mice were sham-immunized or immunized i.d. twice at weeks 0 and 4 with LVS, vector control (rLmΔactA), or recombinant L. monocytogenes vaccines expressing KatG (rLm/katG), IglC (rLm/iglC), or the combination of both vaccines. At week 8, mice were euthanized and sera were collected to measure the antibody (IgG) response by ELISA. For measuring antibody response to KatG (A), sera were diluted 1:50. For measuring antibody response to IglC (B), sera were diluted 1:25. The antibody responses are presented as the Absorbance at 414 nm (A_414nm) in the presence of antigen divided by the A_414nm in the absence of antigen for each individual mouse. Bars represent the mean of the antibody titer from the 5 mice per group. Asterisks indicate that the difference between the antibody response of mice in the indicated vaccinated group and mice in the sham-vaccinated group was significant. *, P<0.05; **, P<0.01; ***, P<0.001 by one-way ANOVA with Bonferroni’s post test.