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 Vaccination with an Iron-Superoxide Dismutase (FeSOD) Deficient Mutant of Francisella tularensis LVS Protects Mice against Lethal Challenge with F. tularensis SchuS4 (47.29)

Abstract Francisella tularensis, the causative agent of tularemia is a potential bioweapon because of ease of its dissemination, multiple routes of infection, high infectivity and lethality. We have previously reported a mutant of F. tularensis LVS deficient in iron superoxide dismutase (sodBFt) and have demonstrated that this mutant is hypersensitive to oxidative stress and attenuated for virulence in mice. Herein, we evaluated further the efficacy of this mutant as a vaccine candidate against respiratory tularemia. We observed that immunization with sodBFt mutant offered 100% protection against 10–100LD100 doses of the highly virulent F. tularensis SchuS4 strain in BALB/c mice and a consistent 40% protection in C57BL/6 mice with a significantly extended median time to death as compared to naïve or LVS vaccinated mice. The sodBFt vaccinated mice exhibited significantly lowered bacterial burden, less severe histopathological lesions in the liver and spleen and a regulated production of proinflammatory cytokines following SchuS4 challenge as compared to the LVS vaccinated mice. We observed increased levels of heat shock protein GroEL, peroxidase/catalase (KatG) and bacteroferritin (Bfr) in the cell lysates and culture filtrate of sodBFt mutant as compared to F. tularensis LVS. Studies on the role of these proteins in the generation of a strong protective immunity in the sodBFt vaccinated mice are currently underway. To conclude, our results demonstrate that sodBFt mutant is a better vaccine candidate than F. tularensis LVS and this study is a major step forward towards the development of a live attenuated vaccine for the prevention of respiratory tularemia.</jats:p

Identification of live attenuated vaccine candidates for tularemia prophylaxis (166.20)

Abstract Francisella tularensis (Ft) is the causative agent of a fatal human disease, tularemia. Ft was used in bioweapon programs in the past and is now classified as a category A agent owing to its possible use as a bioterror agent. Despite over a century since its discovery, an effective vaccine is yet to be developed. A live attenuated vaccine strain (LVS) exists; however, an unknown cause of attenuation and adverse effects in immunized individuals eluded its licensing as a vaccine. This study identified potential vaccine candidates for prophylaxis of respiratory tularemia. Five mutants of Ft LVS [FTL_0057, FTL_0291, FTL_0304, FTL_0325 and superoxide dismutase B (SodB)] were tested for their protective efficacy against Ft SchuS4 challenge. BALB/c mice were immunized intranasally (i.n.) with these mutants. Doses as high as 5x10(3) CFU (for SodB) or 1x10(7) CFU (for other mutants) did not cause any adverse reaction in immunized mice. A kinetic experiment revealed that the immunized mice cleared the mutants by days 14-21 post-immunization (PI). I.n. challenge with 100 CFU of Ft SchuS4 30 days PI protected 100% of mice immunized with SodB, FTL_0057, FTL_0291 and FTL_0325 mutants, while all the mice immunized with FTL_0304 mutant succumbed to infection. Mice that survived the challenge cleared Ft SchuS4 by day 45 post-challenge. This study has identified superior, safer and genetically defined live attenuated vaccine candidates for prevention of respiratory tularemia.</jats:p

ThioredoxinA1 Controls the Oxidative Stress Response of Francisella tularensis Live Vaccine Strain (LVS)

The role of thioredoxins in the oxidative stress response of F. tularensis is not known. This study demonstrates that of the two thioredoxins, TrxA1 is vital to counter the oxidative stress in F. tularensis live vaccine strain (LVS). </jats:p

Aim2 and Nlrp3 Are Dispensable for Vaccine-Induced Immunity against Francisella tularensis Live Vaccine Strain

Francisella tularensis is a facultative, intracellular, Gram-negative bacterium that causes a fatal disease known as tularemia. Due to its extremely high virulence, ease of spread by aerosolization, and potential to be used as a bioterror agent, F. tularensis is classified by the CDC as a tier 1 category A select agent. </jats:p

The Impact of Primary Immunization Route on the Outcome of Infection With SARS-Cov-2 in a Hamster Model of COVID-19

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has resulted in over 6.7 million deaths worldwide. COVID-19 vaccines administered parenterally via intramuscular or subcutaneous (SC) routes have reduced the severity of respiratory infections, hospitalization rates, and overall mortality. However, there is a growing interest in developing mucosally delivered vaccines to further enhance the ease and durability of vaccination. This study compared the immune response in hamsters immunized with live SARS-CoV-2 virus via SC or intranasal (IN) routes and assessed the outcome of a subsequent IN SARS-CoV-2 challenge. Results showed that SC-immunized hamsters elicited a dose-dependent neutralizing antibody response but of a significantly lower magnitude than that observed in IN-immunized hamsters. The IN challenge with SARS-CoV-2 in SC-immunized hamsters resulted in body weight loss, increased viral load, and lung pathology than that observed in IN-immunized and IN-challenged counterparts. These results demonstrate that while SC immunization renders some degree of protection, IN immunization induces a stronger immune response and better protection against respiratory SARS-CoV-2 infection. Overall, this study provides evidence that the route of primary immunization plays a critical role in determining the severity of a subsequent respiratory infection caused by SARS-CoV-2. Furthermore, the findings suggest that IN route of immunization may be a more effective option for COVID-19 vaccines than the currently used parenteral routes. Understanding the immune response to SARS-CoV-2 elicited via different immunization routes may help guide more effective and long-lasting vaccination strategies

Prophylactic and Therapeutic Use of Antibodies for Protection against Respiratory Infection with <i>Francisella tularensis</i>

Abstract The role of Abs in protection against respiratory infection with the intracellular bacterium Francisella tularensis is not clear. To investigate the ability of Abs to clear bacteria from the lungs and prevent systemic spread, immune serum was passively administered i.p. to naive mice before intranasal F. tularensis live vaccine strain infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcγR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro experiments demonstrated that macrophages were more readily infected by Ab-opsonized bacteria but became highly efficient in killing upon activation by IFN-γ. Consistent with this finding, in vivo Ab-mediated protection was found to be dependent upon IFN-γ. SCID mice were not protected by passive Ab transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-γ. These data suggest that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis. Of considerable interest was the finding that serum Abs were capable of conferring protection against lethal respiratory tularemia when given 24–48 h postexposure. Thus, this study provides the first evidence for the therapeutic use of Abs in Francisella-infected individuals.</jats:p