Identification of a live attenuated vaccine candidate for tularemia prophylaxis.

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

Preclinical testing of a vaccine candidate against tularemia.

Tularemia is caused by a gram-negative, intracellular bacterial pathogen, Francisella tularensis (Ft). The history weaponization of Ft in the past has elevated concerns that it could be used as a bioweapon or an agent of bioterrorism. Since the discovery of Ft, three broad approaches adopted for tularemia vaccine development have included inactivated, live attenuated, or subunit vaccines. Shortcomings in each of these approaches have hampered the development of a suitable vaccine for prevention of tularemia. Recently, we reported an oxidant sensitive mutant of Ft LVS in putative EmrA1 (FTL_0687) secretion protein. The emrA1 mutant is highly sensitive to oxidants, attenuated for intramacrophage growth and virulence in mice. We reported that EmrA1 contributes to oxidant resistance by affecting the secretion of antioxidant enzymes SodB and KatG. This study investigated the vaccine potential of the emrA1 mutant in prevention of respiratory tularemia caused by Ft LVS and the virulent SchuS4 strain in C57BL/6 mice. We report that emrA1 mutant is safe and can be used at an intranasal (i. n.) immunization dose as high as 1x106 CFU without causing any adverse effects in immunized mice. The emrA1 mutant is cleared by vaccinated mice by day 14-21 post-immunization, induces minimal histopathological lesions in lungs, liver and spleen and a strong humoral immune response. The emrA1 mutant vaccinated mice are protected against 1000-10,000LD100 doses of i.n. Ft LVS challenge. Such a high degree of protection has not been reported earlier against respiratory challenge with Ft LVS using a single immunization dose with an attenuated mutant generated on Ft LVS background. The emrA1 mutant also provides partial protection against i.n. challenge with virulent Ft SchuS4 strain in vaccinated C57BL/6 mice. Collectively, our results further support the notion that antioxidants of Ft may serve as potential targets for development of effective vaccines for prevention of tularemia

<i>FTL_0304</i>, <i>FTL_0291</i>, <i>FTL_0325</i> and <i>FTL_0057</i> mutants are highly attenuated for virulence in BALB/c and C57BL/6 mice.

a<p>6–8 weeks old mice were infected i.n. with either <i>F. tularensis</i> LVS or the indicated mutants and monitored for mortality for 28 days. Data are shown as number of mice survived/total number of mice infected.</p

Immunization with the <i>FTL_0325</i> mutant induces a higher pro-inflammatory cytokine response in the spleen.

<p>BALB/c mice (n = 4) were infected with 1×10⁷ CFU of the indicated mutants and wild type <i>F. tularensis</i> 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 <i>P</i> values were recorded. *<i>P<</i>0.05<i>; **P</i><0.01; ***<i>P<</i>0.001. Ψ = Mice infected with 1×10⁷ CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.</p

Immunization with the <i>FTL_0325</i> mutant induces an early inflammatory response.

<p>BALB/c mice were infected with 1×10⁷ CFU of the <i>F. tularensis</i> LVS, <i>FTL_0325</i> or <i>FTL_0304</i> 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 <i>F. tularensis</i> 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 <i>FTL_0325</i> mutant immunized mice.</p

Immunization with the <i>FTL_0325</i> mutant results in a potent memory recall response.

<p>Cell culture supernatants collected from BMDMs infected with either the <i>F. tularensis</i> LVS (A–D) or <i>F. tularensis</i> 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 <i>P</i> values recorded. *<i>P<</i>0.05<i>; **P</i><0.01; ***<i>P<</i>0.001.</p

Correlation analysis of bacterial burden and pro-inflammatory cytokine levels in mice infected with <i>F. tularensis</i> LVS, the <i>FTL_0325</i> and <i>FTL_0304</i> mutants.

*<p>r² value of 0.6 and above was considered as a strong correlation. Values in bold are strongly correlated.</p>**<p>P-value.</p

Immunization with the <i>FTL_0325</i> mutant induces an early pro-inflammatory cytokine response in lungs.

<p>BALB/c mice (n = 4) were infected with 1×10⁷ CFU of the indicated mutants and wild type <i>F. tularensis</i> 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 <i>P</i> values were recorded. <i>**P</i><0.01; ***<i>P<</i>0.001. Ψ = Mice infected with 1×10⁷ CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.</p

Recall memory response results in an enhanced killing of <i>F. tularensis</i> SchuS4.

<p>The BMDMs were infected with <i>F. tularensis</i> SchuS4 and co-incubated with the splenocytes from naïve, <i>FTL_0325</i> or <i>FTL_0304</i> 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 <i>P</i> values were recorded. ***<i>P<</i>0.001.</p

The <i>FTL_0325</i> and <i>FTL_0304</i> mutants of <i>F. tularensis</i> LVS exhibit no growth defect under acellular growth conditions.

<p>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.</p