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

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

Comparative analysis of absent in melanoma 2-inflammasome activation in Francisella tularensis and Francisella novicida

Francisella tularensis is a highly virulent Gram-negative bacterium that causes the fatal zoonotic disease tularemia. The mechanisms and signaling pathways leading to the absent in melanoma 2 (Aim2) inflammasome activation have been elegantly elucidated using Francisella novicida as a model. Although not pathogenic for humans, F. novicida can cause tularemia in mice, and the inflammatory response it triggers is the polar opposite to that observed in mice infected with F. tularensis strains. This study aimed to understand the mechanisms of Aim2 inflammasome activation in F. tularensis-infected macrophages. The results reveal that macrophages infected with the F. tularensis live vaccine strain (LVS) induce lower levels of Aim2-dependent IL-1β than those infected with F. novicida. The suppression/weak activation of Aim2 in F. tularensis LVS-infected macrophages is due to the suppression of the cGAS-STING DNA-sensing pathway. Furthermore, the introduction of exogenous F. tularensis LVS DNA into the cytosol of the F. tularensis LVS-infected macrophages, alone or in conjunction with a priming signal, failed to restore IL-1β levels similar to those observed for F. novicida-infected macrophages. These results indicated that, in addition to the bacterial DNA, DNA from some other sources, specifically from the damaged mitochondria, might contribute to the robust Aim2-dependent IL-1β levels observed in F. novicida-infected macrophages. The results indicate that F. tularensis LVS induces mitophagy that may potentially prevent the leakage of mitochondrial DNA and the subsequent activation of the Aim2 inflammasome. Collectively, this study demonstrates that the mechanisms of Aim2 inflammasome activation established for F. novicida are not operative in F. tularensis

Chronically hypertensive transgenic mice expressing human AT1R haplotype-I exhibit increased susceptibility to Francisella tularensis

Age-related illnesses, including hypertension and accompanying metabolic disorders, compromise immunity and exacerbate infection-associated fatalities. Renin-angiotensin system (RAS) is the key mechanism that controls blood pressure. Upregulation of RAS through angiotensin receptor type 1 (AT1R), a G-protein coupled receptor, contributes to the pathophysiological consequences leading to vascular remodeling, hypertension, and end-organ damage. Genetic variations that increase the expression of human AT1R may cause the above pathological outcomes associated with hypertension. Previously we have shown that our chronically hypertensive transgenic (TG) mice containing the haplotype-I variant (Hap-I, hypertensive genotype) of human AT1R (hAT1R) gene are more prone to develop the metabolic syndrome-related disorders as compared to the TG mice containing the haplotype-II variant (Hap-II, normotensive genotype). Since aging and an increased risk of hypertension can impact multiple organ systems in a complex manner, including susceptibility to various infections, the current study investigated the susceptibility and potential effect of acute bacterial infection using a Gram-negative intracellular bacterial pathogen, Francisella tularensis in our hAT1R TG mice. Our results show that compared to Hap-II, F. tularensis-infected aged Hap-I TG mice have significantly higher mortality post-infection, higher bacterial load and lung pathology, elevated inflammatory cytokines and altered gene expression profile favoring hypertension and inflammation. Consistent with worsened phenotype in aged Hap-I mice post-Francisella infection, gene expression profiles from their lungs revealed significantly altered expression of more than 1,400 genes. Furthermore, bioinformatics analysis identified genes associated with RAS and IFN-γ pathways regulating blood pressure and inflammation. These studies demonstrate that haplotype-dependent over-expression of the hAT1R gene leads to enhanced susceptibility and lethality due to F. tularensis LVS infection, which gets aggravated in aged animals. Clinically, these findings will help in exploring the role of AT1R-induced hypertension and enhanced susceptibility to infection-related respiratory diseases

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

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

Superoxide Dismutase B Gene (sodB)-Deficient Mutants of Francisella tularensis Demonstrate Hypersensitivity to Oxidative Stress and Attenuated Virulence

A Francisella tularensis live vaccine strain mutant (sodB(Ft)) with reduced Fe-superoxide dismutase gene expression was generated and found to exhibit decreased sodB activity and increased sensitivity to redox cycling compounds compared to wild-type bacteria. The sodB(Ft) mutant also was significantly attenuated for virulence in mice. Thus, this study has identified sodB as an important F. tularensis virulence factor

Nlrp3 Increases the Host\u27s Susceptibility to Tularemia

Francisella tularensis (F. tularensis) is a Gram-negative, intracellular bacterium and the causative agent of a fatal human disease known as tularemia. The CDC has classified F. tularensis as a Tier 1 Category A select agent based on its ease of aerosolization, low infectious dose, past use as a bioweapon, and the potential to be used as a bioterror agent. Francisella has a unique replication cycle. Upon its uptake, Francisella remains in the phagosomes for a short period and then escapes into the cytosol, where the replication occurs. Francisella is recognized by cytosolic pattern recognition receptors, Absent In Melanoma 2 (Aim2) and Nacht LRR and PYD domains containing Protein 3 (Nlrp3). The recognition of Francisella ligands by Aim2 and Nlrp3 triggers the assembly and activation of the inflammasome. The mechanism of activation of Aim2 is well established; however, how Nlrp3 inflammasome is activated in response to F. tularensis infection is not known. Unlike Aim2, the protective role of Nlrp3 against Francisella infection is not fully established. This study investigated the role of Nlrp3 and the potential mechanisms through which Nlrp3 exerts its detrimental effects on the host in response to F. tularensis infection. The results from in vitro studies demonstrate that Nlrp3 dampens NF-κB and MAPK signaling, and pro-inflammatory cytokine production, which allows replication of F. tularensis in infected macrophages. In vivo, Nlrp3 deficiency results in differential expression of several genes required to induce a protective immune response against respiratory tularemia. Nlrp3-deficient mice mount a stronger innate immune response, clear bacteria efficiently with minimal organ damage, and are more resistant to Francisella infection than their wild-type counterparts. Together, these results demonstrate that Nlrp3 enhances the host\u27s susceptibility to F. tularensis by modulating the protective innate immune responses. Collectively, this study advances our understanding of the detrimental role of Nlrp3 in tularemia pathogenesis