Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System

Francisella tularensisis a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensisSchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD100) doses of F. tularensisLVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensisantigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensisproteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens

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

Expression and Purification of Recombinant DnaK, OmpA and Tul4 Proteins of <i>F</i>. <i>tularensis</i> SchuS4.

<p>Purification of recombinant OmpA, DnaK and Tul4 proteins of <i>F</i>. <i>tularensis</i> SchuS4 proteins was confirmed by SDS-PAGE and western blot analysis using anti-His antibodies.</p

OmpA, DnaK and Tul4 Specific Antibody Responses in Mice Immunized with TMV-Multiconjugate Vaccines using Schedule I and II of Immunization.

<p><i>F</i>. <i>tularensis</i> SchuS4 recombinant proteins OmpA, DnaK and Tul4 specific IgG, antibody levels on day 28 in serum samples of C57BL/6 mice immunized with TMV-multiconjugate vaccine using Schedule II were determined by ELISA. The plates were coated with recombinant <i>F</i>. <i>tularensis</i> SchuS4 proteins. Serum samples obtained from naïve mice or those inoculated with TMV alone were used as controls. The data are represented as Mean ±S.D. of absorbance values measured at 450nm. The comparisons are shown with the data obtained from mice immunized with TMV-multiconjugate vaccine using schedule I (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130858#pone.0130858.g007" target="_blank">Fig 7</a>). Table shows comparison of antibody titers between groups of mice vaccinated with Schedule I and II vaccination regimens.</p

OmpA, DnaK and Tul4 Specific Antibody Responses in Mice Immunized with TMV-Monoconjugate and TMV-Multiconjugate Vaccines using Schedule I of Immunization.

<p><i>F</i>. <i>tularensis</i> SchuS4 recombinant proteins OmpA, DnaK and Tul4 specific IgG antibody levels on day 28 in serum samples of C57BL/6 mice immunized with TMV-monoconjugate and TMV-multiconjugate vaccine using Schedule I were determined by ELISA. Serum samples obtained from naïve mice or those inoculated with TMV alone were used as controls. The data are represented as Mean ±S.D. of absorbance values measured at 450nm. Table shows comparison of antibody titers between groups of mice vaccinated with these vaccine formulations.</p

Vaccine Formulations.

<p>Two different vaccine formulations were used. In the first vaccine formulation all three recombinant proteins OmpA, DnaK and Tul4 were conjugated to a single TMV virion (TMV-monoconjugate vaccine). The second vaccine formulation contained each recombinant protein of <i>F</i>. <i>tularensis</i> conjugated individually to TMV and then mixed in equal concentrations to generate a TMV-multiconjugate vaccine.</p

Conjugation of DnaK, OmpA and Tul4 Proteins of <i>F</i>. <i>tularensis</i> SchuS4 to TMV.

<p>Purified OmpA, DnaK and Tul4 proteins were combined with purified TMV and incubated with EDC and NHS for 0, 30 min, 1, or 2 hours as described in Methods section. Two μg of TMV or recombinant proteins DnaK, OmpA, Tul4 or 4 μg of the TMV-protein mixtures were resolved on an 8–16% SDS-PAGE gel to observe conjugation products indicated by changes in the molecular masses of the starting materials. <b>(A)</b> Conjugation of DnaK, OmpA and Tul4 to a single TMV virion to generate TMV-monoconjugate vaccine. The progress of conjugation process was observed over a period of time: Lane M = Precision Plus Dual Color standard (BioRad) Marker; Lane 1 = TMV-protein mix, 0 min; Lane 2 = TMV-protein mix, 30 min; Lane 3 = TMV-protein mix,1 hour; Lane 4 = TMV-protein mix, 2 hours. <b>(B, C, D)</b> Kinetics of DnaK, OmpA and Tul4 TMV-protein conjugations over a two hour incubation period to generate TMV-protein conjugates. The individual TMV-protein conjugates were then admixed to generate TMV-multiconjugate vaccine. Lane M = Precision Plus Dual Color standard (BioRad) Marker; Lane 1 = TMV; Lane 2 = Recombinant protein; Lane 3 = TMV-protein mix, 0 hour; Lane 4 = TMV-protein mix, 1 hour; Lane 5 = TMV-protein mix, 2 hours. In all cases, 2 hour time points were used for scale-up and vaccine preparation. Solid arrows indicate TMV-protein conjugate(s), dashed arrows indicate free TMV or free proteins.</p

Protective Efficacy of TMV-Conjugate Vaccine.

<p><b>(A)</b> C57BL/6 mice (N = 8 per group) immunized with TMV-monoconjugate vaccine; <b>(C)</b> with TMV-multiconjugate vaccine (schedule I) or <b>(E)</b> with TMV-multiconjugate vaccine (Schedule II) were challenged i.n. with 10xLD₁₀₀ of <i>F</i>. <i>tularensis</i> LVS on day 28 post-immunization. Mice vaccinated with TMV alone were used as controls. Challenged mice were observed for morbidity and mortality for a period of 21 days post-challenge. The survival results are expressed as Kaplan-Meier survival curves and statistical analysis was performed using Log-rank test. <b>(B, D and F)</b> Body weight of the challenged mice at the indicate time points. The data are represented as Mean ± S.D.</p

Immunization with the <i>emrA1</i> mutant results in minimal weight loss, rapid bacterial clearance, and histopathological lesions in lung, liver and spleen.

<p>C57BL/6 mice were immunized i.n. with 1×10⁶ CFU of the <i>emrA1</i> mutant. Mice infected with equal numbers of wild type <i>Ft</i> LVS were used as controls. <b>(A)</b> The immunized mice were weighed at the indicated times post-immunization to track the progress of infection. <b>(B)</b> On days 1, 5, 7, 14 and 21 post-immunization, mice (n = 4 per group/time point) were euthanized and bacterial burdens were quantified in their lung, liver and spleen. Bacterial counts in organs are expressed as Log₁₀CFU/mL. The <i>P</i> values were determined using one way ANOVA. *<i>P<0</i>.<i>05; **P<0</i>.<i>01; ***P<0</i>.<i>001</i>. <b>(C)</b> Excised lungs, livers and spleens were preserved in 10% formalin, paraffin embedded, sliced into 5 μM thin sections and stained with Hematoxylene & Eosin. Stained sections were observed for histopathological lesions under a light microscope (Magnification 100×). # = <i>Ft</i> LVS infected mice succumbed to infection.</p

The <i>emrA1</i> mutant vaccinated mice induce sustained production of pro-inflammatory cytokines and a potent antibody response following lethal <i>Ft</i> LVS challenge.

<p>C57BL/6 mice immunized i.n. with 1×10⁶ CFU of the <i>emrA1</i> mutant were challenged i.n. with 1×10⁷ CFU of wild type <i>Ft</i> LVS 42 days post-immunization. <b>(A-D)</b> On days 5, 7 and 14 post-challenge, mice (n = 3 per group/time point) were euthanized and their excised lungs were homogenized. Clear lung homogenates were used for quantification of indicated pro-inflammatory cytokines using flow cytometric analysis. The data are represented as Mean ± S.D. <b>(E)</b> On day 21 post-challenge, mice (n = 3 per group) were anesthetized and bled retroorbitally to obtain serum. <i>Ft</i> specific total IgG, IgG2a, IgG2b, IgG1 and IgA levels in serum samples were determined by ELISA. The data are represented as Mean ± S.D. of absorbance values measured at 450 nm. Red arrows indicate antibody titers. ND = Not detected.</p