Identification of a live attenuated vaccine candidate for tularemia prophylaxis

Abstract

Francisella tularensis is the causative agent of a fatal human disease, tularemia. F. tularensis was used in bioweapon programs in the past and is now classified as a category A select agent owing to its possible use in bioterror attacks. Despite over a century since its discovery, an effective vaccine is yet to be developed. In this study four transposon insertion mutants of F. tularensis live vaccine strain (LVS) in Na/H antiporter (FTL_0304), aromatic amino acid transporter (FTL_0291), outer membrane protein A (OmpA)-like family protein (FTL_0325) and a conserved hypothetical membrane protein gene (FTL_0057) were evaluated for their attenuation and protective efficacy against F. tularensis SchuS4 strain. All four mutants were 100-1000 fold attenuated for virulence in mice than parental F. tularensis. Except for the FTL_0304, single intranasal immunization with the other three mutants provided 100% protection in BALB/c mice against intranasal challenge with virulent F. tularensis SchuS4. Differences in the protective ability of the FTL_0325 and FTL_0304 mutant which failed to provide protection against SchuS4 were investigated further. The results indicated that an early pro-inflammatory response and persistence in host tissues established a protective immunity against F. tularensis SchuS4 in the FTL_0325 immunized mice. No differences were observed in the levels of serum IgG antibodies amongst the two vaccinated groups. Recall response studies demonstrated that splenocytes from the FTL_0325 mutant immunized mice induced significantly higher levels of IFN-γ and IL-17 cytokines than the FTL_0304 immunized counterparts indicating development of an effective memory response. Collectively, this study demonstrates that persistence of the vaccine strain together with its ability to induce an early pro-inflammatory innate immune response and strong memory responses can discriminate between successful and failed vaccinations against tularemia. This study describes a live attenuated vaccine which may prove to be an ideal vaccine candidate for prevention of respiratory tularemia.

Figure 1. Immunization with the FTL_0291, FTL_0325 or FTL_0057 mutant protects BALB/c mice against F. tularensis SchuS4 challenge.

BALB/c mice (n = 10) were immunized i.n. with 1×10⁷ CFU of the indicated mutants, challenged i.n. with 100 CFU of F. tularensis SchuS4 30 days post-immunization, and observed for mortality for a period of 21 days. Unvaccinated mice were kept as controls. Data are represented as Kaplan-Meier survival curves and are cumulative of two independent experiments. The P values were determined using Logrank test. Survival comparisons between mice immunized with the FTL_0291, FTL_0325 or FTL_0057 mutant and unvaccinated controls or FTL_0304 immunized mice are shown in the table. Differences in survival between the experimental groups were considered statistically significant at a P < 0.005 level.

Figure 2. The FTL_0325 and FTL_0304 mutants of F. tularensis LVS exhibit no growth defect under acellular growth conditions.

Growth curves for bacteria grown in MHB were generated in a 96-well plate using 200 µl culture volumes while the growth curves for bacteria grown in BHI and CDM were generated using a 25 ml culture volume. Results shown are representative of two independent experiments.

Figure 3. The FTL_0325 mutant persists longer than the FTL_0304 mutant in infected mice.

BALB/c mice (n = 4) were infected with 1×10⁷ CFU of the F. tularensis LVS, FTL_0325 or FTL_0304 mutant. At the indicated times, homogenates of lung (A), liver (B) and spleen (C) from the infected mice were diluted 10-fold and plated on MH-chocolate agar plates for quantification of bacterial numbers. The results are expressed as Log₁₀ CFU/organ and are representative of two independent experiments conducted. The data were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and P values were recorded. *P<0.05; **P<0.01; ***P<0.001. Ψ = Mice infected with 1×10⁷ CFU of F. tularensis LVS succumbed to infection by day 7 PI and hence were unavailable for comparison. ND = Not detected.

Figure 4. Immunization with the FTL_0325 mutant induces an early pro-inflammatory cytokine response in lungs.

BALB/c mice (n = 4) were infected with 1×10⁷ CFU of the indicated mutants and wild type F. tularensis LVS. At the indicated times, the levels of pro-inflammatory cytokines were measured in lung homogenates using a Cytometric Bead Array assay. The data are representative of two independent experiments conducted and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and P values were recorded. **P<0.01; ***P<0.001. Ψ = Mice infected with 1×10⁷ CFU of F. tularensis LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.

Figure 5. Immunization with the FTL_0325 mutant induces a higher pro-inflammatory cytokine response in the spleen.

BALB/c mice (n = 4) were infected with 1×10⁷ CFU of the indicated mutants and wild type F. tularensis LVS. At the indicated times, the levels of pro-inflammatory cytokines were measured in spleen homogenates using a Cytometric Bead Array assay. The data are representative of two independent experiments conducted and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and P values were recorded. *P<0.05; **P<0.01; ***P<0.001. Ψ = Mice infected with 1×10⁷ CFU of F. tularensis LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.

Figure 6. Immunization with the FTL_0325 mutant induces an early inflammatory response.

BALB/c mice were infected with 1×10⁷ CFU of the F. tularensis LVS, FTL_0325 or FTL_0304 mutant and the lungs were harvested at the indicated times, sectioned and stained with H & E. Representatives of H & E stained lung sections are shown. Infiltration of neutrophils is shown in the inset (Magnification 10×; Inset 100×). Ψ = Mice infected with 1×10⁷ CFU of F. tularensis LVS succumbed to infection by day 7 PI and hence were unavailable for comparison. Green arrows indicate the sites of cellular infiltration. Yellow circles in inset show neutrophilic infiltration in the lungs of FTL_0325 mutant immunized mice.

Figure 7. Antibody responses in mice immunized with the FTL_0325 and FTL_0304 mutants.

BALB/c mice (n = 3) were immunized with 1×10⁷ CFU of the mutants intranasally. The mice were bled at days 45 (A) and 60 (B) post-immunization and total IgG levels were determined in the serum by ELISA.

Figure 8. Immunization with the FTL_0325 mutant results in a potent memory recall response.

Cell culture supernatants collected from BMDMs infected with either the F. tularensis LVS (A–D) or F. tularensis SchuS4 (E and F) and co-cultured with the splenocytes prepared from immunized mice at day 45 post-immunization (A and C) or at day 60 (B–F) were analyzed for IFN-γ (A, B and E) and IL-17a (C,D and F) cytokines by Cytometric Bead Flex sets. The data are representative of two independent experiments and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and P values recorded. *P<0.05; **P<0.01; ***P<0.001.

Figure 9. Recall memory response results in an enhanced killing of F. tularensis SchuS4.

The BMDMs were infected with F. tularensis SchuS4 and co-incubated with the splenocytes from naïve, FTL_0325 or FTL_0304 immunized mice. The BMDMs were lysed 72 hrs PI, diluted 10-fold and plated on MH-chocolate agar plates to determine the intracellular bacterial replication. The results were expressed as log₁₀ CFU/ml and are representative of two independent experiments. The data were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and P values were recorded. ***P<0.001.