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An improved tobacco mosaic virus (TMV)-conjugated multiantigen subunit vaccine against respiratory tularemia

Frontiers in Microbiology (2018) 9(JUN)
DOI: 10.3389/fmicb.2018.01195
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Abstract

Francisella tularensis, the causative agent of the fatal human disease known as tularemia is classified as a Category A Select Agent by the Centers for Disease Control. No licensed vaccine is currently available for prevention of tularemia in the United States. Previously, we published that a tri-antigen tobacco mosaic virus (TMV) vaccine confers 50% protection in immunized mice against respiratory tularemia caused by F. tularensis. In this study, we refined the TMV-vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetra-antigen vaccine by conjugating OmpA, DnaK, Tul4, and SucB proteins of Francisella to TMV. CpG was also included in the vaccine formulation as an adjuvant. Primary intranasal (i.n.) immunization followed by two booster immunizations with the tetra-antigen TMV vaccine protected 100% mice against i.n. 10LD100 challenges dose of F. tularensis live vaccine strain (LVS). Mice receiving three immunization doses of tetra-antigen TMV vaccine showed only transient body weight loss, cleared the infection rapidly, and showed minimal histopathological lesions in lungs, liver, and spleen following a lethal respiratory challenge with F. tularensis LVS. Mice immunized with the tetra-antigen TMV vaccine also induced strong ex vivo recall responses and were protected against a lethal challenge as late as 163 days post-primary immunization. Three immunization with the tetra-antigen TMV vaccine also induced a stronger humoral immune response predominated by IgG1, IgG2b, and IgG2c antibodies than mice receiving only a single or two immunizations. Remarkably, a single dose protected 40% of mice, while two doses protected 80% of mice from lethal pathogen challenge. Immunization of Interferon-gamma (IFN-γ)-deficient mice with the tetra-antigen TMV vaccine demonstrated an absolute requirement of IFN-γ for the generation of protective immune response against a lethal respiratory challenge with F. tularensis LVS. Collectively, this study further demonstrates the feasibility of TMV as an efficient platform for the delivery of multiple F. tularensis antigens and that tetra-antigen TMV vaccine formulation provides complete protection, and induces long-lasting protective and memory immune responses against respiratory tularemia caused by F. tularensis LVS.

Figures

  • FIGURE 1 | Design and formulation of the tetra-antigen TMV vaccine. (A) To the tri-antigen TMV vaccine consisting of OmpA, DnaK, and Tul4, a fourth protein antigen, dihydrolipoamide succinyl transferase (SucB), was added to prepare a tetra-antigen TMV vaccine. Each recombinant protein antigen was conjugated to TMV separately and then mixed in equal concentrations to generate the tetra-antigen vaccine. The CpG 1826 adjuvant was also included in the vaccine formulation. (B) Conjugation of F. tularensis SchuS4 recombinant proteins to TMV. Recombinant proteins purified by Nickel affinity chromatography were conjugated to TMV as described in the section “Materials and Methods.” The protein–TMV conjugation was visualized by the disappearance of free antigen protein and the appearance of higher molecular weight complexes. M, protein molecular weight marker; 1, TMV; 2, recombinant protein; 3, protein+TMV mixture; 4–7, protein–TMV conjugate replicates from several conjugation reactions.
  • FIGURE 2 | Immunization with tetra-antigen TMV vaccine provides 100% protection and the immunized mice clear infection rapidly following a lethal F. tularensis LVS challenge. (A) C57BL/6 mice were immunized i.n. on days 0, 14, and 28 with tetra-antigen TMV vaccine formulation as described in the section “Materials and Methods” and challenged i.n. with 1 × 105 CFU (10LD100) of F. tularensis LVS on day 49 post-primary immunization. (B) Mice were observed for mortality and morbidity by monitoring survival and body weights. The survival results are expressed as Kaplan–Meier survival curves and the statistical analysis was performed by log-rank test. The body weight data are represented as mean ± SD (n = 10–12 mice/group). The results are representative of two independent experiments conducted. (C) Kinetics of bacterial clearance in mice immunized and challenged as shown. The organs were harvested on days 7, 14, and 21 post-challenge and the bacterial burden in the lung, liver, and spleen of vaccinated and control mice was determined. The results represent the means ± standard errors of CFU counts (n = 3–4 mice/group/time point) and are from a single experiment conducted. The data were analyzed by one-way ANOVA followed by Bonferroni’s correction and P-values were determined. The dotted line represents the limit of detection. 9, all control mice succumbed to infection by day 14 post-challenge and hence were not available for comparisons; ND, not detected.
  • FIGURE 3 | Immunization with tetra-antigen TMV vaccine results in minimal pathology in lung following a lethal challenge with F. tularensis LVS. (A) C57BL/6 mice were immunized and challenged as shown. Lungs were collected from unvaccinated controls as well as vaccinated mice on days 7, 14, and 21 post-challenge. Hematoxylin and eosin stained sections of lung (B) were evaluated for histopathological lesions at the indicated times post-challenge. The experiments were conducted with 3–4 mice/group/time point and the representative images are shown. The sections were observed and images were taken on a Nikon Eclipse microscope using VIS-Elements AR Version 5.02 software. Small green boxes represent the area shown at 40× magnification in insets (magnification 10×; insets 40×; bar in 10× = 200 µM; bar in 40× = 50 µM). 9, all unvaccinated mice succumbed to infection by day 14 post-challenge and hence were not available for comparisons.
  • FIGURE 4 | Immunization with tetra-antigen TMV vaccine results in minimal pathology in liver and spleen following a lethal challenge with F. tularensis LVS. C57BL/6 mice were immunized and challenged as shown in Figure 3A. Liver and spleens were collected from unvaccinated controls as well as vaccinated mice on days 7, 14, and 21 post-challenge. Hematoxylin and eosin-stained sections of liver (A) were evaluated for histopathological lesions at the indicated times post-challenge. The arrows and arrow heads indicate granulomas in livers of unvaccinated controls and mice immunized with tetra-antigen TMV vaccine. (B) Number of granulomas were quantitated in uninfected and unvaccinated controls on day 7 post-challenge by counting the number of granulomas in 10 random fields under 10× magnification by two independent investigators in a blinded fashion (n = 3 mice/group). The data were analyzed by unpaired t-test and the P-values were determined. (C) Hematoxylin and eosin stained sections of spleens were evaluated for histopathological lesions at the indicated times post-challenge. The experiments were conducted with 3–4 mice/group/time point and the representative images are shown. The sections were observed and images were taken on a Nikon Eclipse microscope using VIS-Elements AR Version 5.02 software (magnification 10×; bar = 200 µM). 9, all unvaccinated mice succumbed to infection by day 14 post-challenge and hence were not available for comparisons.
  • FIGURE 5 | Total IgG antibody responses in immunized mice pre- and post-challenge against each individual protein of tetra-antigen TMV vaccine. (A) C57BL/6 mice were vaccinated with tetra-antigen TMV vaccine, TMV alone, or left unvaccinated. Mice were challenged on day 49 post-primary immunization. (B) Antigen-specific total IgG antibody titers were determined at the indicated times in pooled serum samples from immunized mice (n = 3 mice/group) by ELISA. Each pooled sample was run in duplicate. The results are expressed as absorbance at 450 nm (mean ± standard deviation). The cut-off absorbance values used to determine the end-point titers are indicated by dashed lines and the actual values are shown in the box (top panels). The end-point titers are represented as Log10 values (bottom panel).
  • TABLE 1 | Antibody responses in mice immunized with tetra-antigen TMV vaccine receiving none, one, or two booster immunizations.
  • FIGURE 6 | Tetra-antigen TMV vaccine induces potent recall response in immunized mice. (A) C57BL/6 mice (n = 3–4) were immunized as shown. Mice were sacrificed on day 84 post-primary immunization and single cell spleen suspensions were prepared. BMDMs isolated from syngeneic C57BL/6 mice were infected either with F. tularensis LVS (B) or F. tularensis SchuS4 (C) at an MOI of 100 (100:1 bacteria:cell ratio), for 2 h followed by gentamicin treatment for 2 h. The infected BMDMs were then overlaid with splenocytes isolated from immunized mice at 1:1 ratio. Splenocytes isolated from naïve mice (n = 3) were used as controls. The culture supernatants from co-culture were analyzed for IFN-γ and IL-17 levels 24, 48, and 120 h later by CBA. The limits of detection for IL-17 and IFN-γ by CBA are 18.9 and 3.7 pg/ml, respectively. The results are representative of two independent experiments. The data were analyzed by one-way ANOVA followed by Bonferroni’s correction and P-values were determined.
  • FIGURE 7 | Tetra-antigen TMV vaccine induces long-lasting immunity against lethal F. tularensis LVS challenge. (A) C57BL/6 mice were immunized i.n. on days 0, 14, and 28 with tetra-antigen TMV vaccine. These mice received an additional booster with half the dose on day 91 post-primary immunization and were challenged i.n. with 1 × 105 CFU (10LD100) of F. tularensis LVS on day 163 post-primary immunization. (B) Mice (n = 7/group) were observed for mortality and morbidity by monitoring survival and body weights. The results shown are from a single experiment conducted. The data are represented as mean ± SD. The survival results are expressed as Kaplan–Meier survival curves and the statistical analysis was performed by log-rank test.

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Mansour, A. A., Banik, S., Suresh, R. V., Kaur, H., Malik, M., McCormick, A. A., & Bakshi, C. S. (2018). An improved tobacco mosaic virus (TMV)-conjugated multiantigen subunit vaccine against respiratory tularemia. Frontiers in Microbiology, 9(JUN). https://doi.org/10.3389/fmicb.2018.01195

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