Intranasal Administration of a Two-Dose Adjuvanted Multi-Antigen TMV-Subunit Conjugate Vaccine Fully Protects Mice Against Francisella Tularensis LVS Challenge

Tularemia is a fatal human disease caused by Francisella tularensis, a Gram-negative encapsulated coccobacillus bacterium. Due to its low infectious dose, ease of aerosolized transmission, and lethal effects, the CDC lists F. tularensis as a Category A pathogen, the highest level for a potential biothreat agent. Previous vaccine studies have been conducted with live attenuated, inactivated, and subunit vaccines, which have achieved partial or full protection from F. tularensis live vaccine strain (LVS) challenge, but no vaccine has been approved for human use. We demonstrate the improved efficacy of a multi-antigen subunit vaccine by using Tobacco Mosaic virus (TMV) as an antigen carrier for the F. tularensis SchuS4 proteins DnaK, OmpA, SucB and Tul4 (DOST). The magnitude and quality of immune responses were compared after mice were immunized by subcutaneous or intranasal routes of administration with a TMV-DOST mixture, with or without four different adjuvants. Immune responses varied in magnitude and isotype profile, by antigen, by route of administration, and by protection in an F. tularensis LVS challenge model of disease. Interestingly, our analysis demonstrates an overwhelming IgG2 response to SucB after intranasal dosing, as well as a robust cellular response, which may account for the improved two-dose survival imparted by the tetravalent vaccine, compared to a previous study that tested efficacy of TMV-DOT. Our study provides evidence that potent humoral, cellular and mucosal immunity can be achieved by optimal antigen combination, delivery, adjuvant and appropriate route of administration, to improve vaccine potency and provide protection from pathogen challenge

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

Intranasal administration of a two-dose adjuvanted multi-antigen TMV-subunit conjugate vaccine fully protects mice against Francisella tularensis LVS challenge.

Tularemia is a fatal human disease caused by Francisella tularensis, a Gram-negative encapsulated coccobacillus bacterium. Due to its low infectious dose, ease of aerosolized transmission, and lethal effects, the CDC lists F. tularensis as a Category A pathogen, the highest level for a potential biothreat agent. Previous vaccine studies have been conducted with live attenuated, inactivated, and subunit vaccines, which have achieved partial or full protection from F. tularensis live vaccine strain (LVS) challenge, but no vaccine has been approved for human use. We demonstrate the improved efficacy of a multi-antigen subunit vaccine by using Tobacco Mosaic virus (TMV) as an antigen carrier for the F. tularensis SchuS4 proteins DnaK, OmpA, SucB and Tul4 (DOST). The magnitude and quality of immune responses were compared after mice were immunized by subcutaneous or intranasal routes of administration with a TMV-DOST mixture, with or without four different adjuvants. Immune responses varied in magnitude and isotype profile, by antigen, by route of administration, and by protection in an F. tularensis LVS challenge model of disease. Interestingly, our analysis demonstrates an overwhelming IgG2 response to SucB after intranasal dosing, as well as a robust cellular response, which may account for the improved two-dose survival imparted by the tetravalent vaccine, compared to a previous study that tested efficacy of TMV-DOT. Our study provides evidence that potent humoral, cellular and mucosal immunity can be achieved by optimal antigen combination, delivery, adjuvant and appropriate route of administration, to improve vaccine potency and provide protection from pathogen challenge

IFNγ ELISpot assay on splenocytes of mice vaccinated two times with TMV-DOST vaccine.

<p>Splenocytes were isolated from 2 mice per group on D65 after intranasal vaccination and incubated on IFNγ antibody coated plates at a final concentration of 10⁵ cells/well. Whole protein antigen stimulants were added at 20 μg/mL, and plates incubated at 37°C in a 5% CO₂, 99% humidity incubator for ~36h. IFNγ secreting cells were detected and developed using Mouse IFNγELISpot Ready-SET-Go! Kit, developed with AEC, and read on an AID ELISpot plate reader. IFNγ secretion from spleen cells stimulated with DnaK (<b>A</b>.) OmpA (<b>B.</b>) SucB (<b>C.</b>) and Tul4 (<b>D</b>.) are compared to no stimulation (no stim), or PBS vaccinated mice (Norm). Values were adjusted positive IFNγ spots per 10⁶ cells, and groups that show significant augmentation with adjuvants compared to no adjuvant are shown (*p<0.05, **p<0.001). Means are presented ± standard deviation, and p values were calculated by non-parametric t-test. Results are representative of two experiments.</p

Subunit protein conjugation to the surface of TMV.

<p>A. A single TMV virion, consisting of a helical single RNA encapsidated by coat protein. B-D single TMV virion with the subunit antigen of increasingly larger sizes conjugated to the surface of the TMV rod.</p

Pathogen challenge with <i>F</i>. <i>tularensis</i> LVS.

<p>C57BL/6 mice (n = 7–10) were vaccinated 2 times, 28 days apart, with either TMV-DOST with adjuvant or with PBS + adjuvant. Mice were challenged with 10xLD₁₀₀ dose of <i>F</i>. <i>tularensis</i> LVS 21 days after the second vaccine and monitored for weight loss and survival. Mice were euthanized when weight loss exceeded 30%. (<b>A.</b>) Mice were vaccinated with TMV-DOST vaccine + CPG, or left untreated, and survival and body weights were recorded (<b>B.</b>) for 21 days. TMV-DOST was administered with di-GMP or left untreated and survival (<b>C.</b>) and body weights (<b>D.</b>) were recorded for 21 days. Statistical significance was determined via Kaplan-Meier Log-rank Test (GraphPad Prism).</p

Recall IFNγ ELISpot and Granzyme B cellular immune response on D100.

<p><b>A.</b> Splenocytes of twice-vaccinated mice were harvested at Day 100 after intranasal vaccination as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194614#pone.0194614.g007" target="_blank">Fig 7</a>. (<b>A.</b>) Splenocytes from TMV-DOST vaccine groups were tested for Granzyme B secretion after antigen stimulation by the low responder (OmpA) and (<b>B.</b>) high responder (SucB) antigens given with no adjuvant (-), dIC, CpG or diGMP adjuvant. (<b>C</b>.) IFNγ responses after TMV-DOST + diGMP are compared to no stimulation, or PBS vaccinated mice (Norm) groups (2 mice per group). Values for both Granzyme B and IFNγ were adjusted positive spots per 10⁶ cells, and groups that show significant augmentation with adjuvants compared to no adjuvant are shown (** p<0.001 and *p<0.05). Means are presented ± standard deviation, and statistical analysis was done via non-parametric t-test.</p

Systemic IgA response to TMV-DOST intranasal vaccination.

<p>Systemic serum IgA titers against each specific antigen in the pooled sera of mice immunized twice intranasally with the TMV-conjugated tetravalent vaccine as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194614#pone.0194614.g005" target="_blank">Fig 5</a>.</p

Antigen conjugation to TMV.

<p>Purified antigens were conjugated to TMV in a 1:1 molar ratio in an amide-carboxylate reaction using conditions optimized for each specific protein. M: Marker (Precision Plus Dual Color Standard- BioRad). T0: start time (mixture of TMV and antigen), R (1–4): independent reactions, T90-T180: time points (minutes) after reaction start. Successful conjugation is demonstrated by reduction in protein and an increase in conjugate product (>250kDa; arrow, or in the gel stack; arrowhead).</p

IgG isotype profile analysis of TMV-DOST (D, DnaK;O, OmpA; S, SucB; T, Tul4) vaccinated mice.

<p>Bleed 4 sera pooled from mice after the second immunization of 20 μg TMV-conjugated DOST antigen vaccine with (dIC, CpG, MF59 or diGMP) or without (neat) adjuvant was analyzed by ELISA against each antigen-using isotype specific IgG1 and IgG2b secondary antibodies to deduce type of immune response. Isotype profile is depicted as a ratio of the two, where direct addition gives total isotype content. <b>A</b>: Subcutaneous immunization profile. <b>B</b>: Intranasal immunization profile.</p