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Tularemia is caused by subspecies of Francisella tularensis and can manifest in a variety of disease states, with the pneumonic presentation resulting in the greatest mortality. Despite decades of research, there are no approved vaccines against F. tularensis in the United States. Traditional vaccination strategies, such as live-attenuated or subunit vaccines, are not favorable due to inadequate protection or safety concerns. Because of this, novel vaccination strategies are needed to combat tularemia. Here we discuss the current state of and challenges to the tularemia vaccine field and suggest novel vaccine approaches going forward that might be better suited for protecting against F. tularensis infection. [ABSTRACT FROM AUTHOR]

Background Tularemia is caused by Francisella tularensis , a gram-negative bacterium that has been weaponized as an aerosol. For protection of personnel conducting biodefense research, the United States Army required clinical evaluation of a new lot of tularemia live vaccine strain manufactured in accordance with Current Good Manufacturing Practices. Methods A phase 2 randomized clinical trial compared the new lot (DVC-LVS) to the existing vaccine that has been in use for decades (USAMRIID-LVS). The vaccines were delivered by scarification to 228 participants. Safety, reactogenicity, take and/or antibody levels were assessed on days 0, 1, 2, 8, 14, 28, 56, and 180. Principal Results Both vaccines were safe and had acceptable reactogenicity profiles during six months of follow-up. There were no serious or grade 3 and 4 laboratory adverse events. Moderate systemic reactogenicity (mostly headache or feeling tired) was reported by ∼23% of participants receiving either vaccine. Injection site reactogenicity was mostly mild itchiness and pain. The frequencies of vaccine take skin reactions were 73% (95% CI, 64, 81) for DVC-LVS and 80% (95% CI, 71, 87) for USAMRIID-LVS. The 90% CI for the difference in proportions was −6.9% (−16.4, 2.6). The rates of seroconversion measured by microagglutination assay on days 28 or 56 were 94% (95% CI, 88, 98; n = 98/104) for DVC-LVS and 94% (95% CI, 87, 97; n = 103/110) for USAMRIID-LVS (p = 1.00). Day 14 sera revealed more rapid seroconversion for DVC-LVS relative to USAMRIID-LVS: 82% (95% CI, 73, 89) versus 55% (95% CI, 45, 65), respectively (p < 0.0001). Major conclusions The DVC-LVS vaccine had similar safety, reactogenicity, take and antibody responses compared to the older USAMRIID vaccine, and was superior for early (day 14) antibody production. Vaccination take was not a sensitive surrogate for seroconversion in a multi-center study where personnel at five research clinics performed assessments. ClinicalTrials.gov identifier NCT01150695 [ABSTRACT FROM AUTHOR]

Francisella tularensis subsp. tularensis strain SchuS4 (Ftt) is a highly virulent intracellular bacterium. Inhalation of 10 or fewer organisms results in an acute and potentially lethal disease called pneumonic tularemia. Ftt infections occur naturally in the U.S. and Ftt was developed as a bioweapon. Thus, there is a need for vaccines that protect against this deadly pathogen. Although a live vaccine strain of Francisella tularensis (LVS) exists, LVS fails to generate long-lived protective immunity against modest challenge doses of Ftt. We recently identified an important role for high avidity CD4 + T cells in short-term protection and hypothesized that expanding this pool of cells would improve overall vaccine efficacy with regard to longevity and challenge dose. In support of our hypothesis, application of a prime/boost vaccination strategy increased the pool of high avidity CD4 + T cells which correlated with improved survival following challenge with either increased doses of virulent Ftt or at late time points after vaccination. In summary, we demonstrate that both epitope selection and vaccination strategies that expand antigen-specific T cells correlate with superior immunity to Ftt as measured by survival. [ABSTRACT FROM AUTHOR]

Numerous studies have demonstrated that administration of antigen (Ag)-pulsed dendritic cells (DCs) is an effective strategy for enhancing immunity to tumors and infectious disease organisms. However, the generation and/or isolation of DCs can require substantial time and expense. Therefore, using inactivated F. tularensis (i Ft ) Ag as a model immunogen, we first sought to determine if DCs could be replaced with peripheral blood mononuclear cells (PBMCs) during the ex-vivo pulse phase and still provide protection against Ft infection. Follow up studies were then conducted using the S. pneumoniae ( Sp ) vaccine Prevnar ®13 as the Ag in the pulse phase followed by immunization and Sp challenge. In both cases, we demonstrate that PBMCs can be used in place of DCs when pulsing with i Ft and/or Prevnar ®13 ex vivo and re-administering the Ag-pulsed PBMCs as a vaccine. In addition, utilization of the i.n. route for Ag-pulsed PBMC administration is superior to use of the i.v. route in the case of Sp immunization, as well as when compared to direct injection of Prevnar ®13 vaccine i.m. or i.n. Furthermore, this PBMC-based vaccine strategy provides a more marked and enduring protective immune response and is also capable of serving as a multi-organism vaccine platform. The potential for this ex-vivo vaccine strategy to provide a simpler, less time consuming, and less expensive approach to DC-based vaccines and vaccination in general is also discussed. [ABSTRACT FROM AUTHOR]

A safer and more effective vaccine than the unlicensed Francisella tularensis Live Vaccine Strain (LVS) is needed to protect against the biowarfare agent F. tularensis . Previously, we developed an LVS Δ capB mutant that is significantly safer than LVS and provides potent protective immunity against F. tularensis respiratory challenge when administered intranasally but limited protection when administered intradermally unless as part of a prime-boost vaccination strategy. To improve the immunogenicity and efficacy of LVS Δ capB , we developed recombinant LVS Δ capB (rLVS Δ capB ) strains overexpressing various F. tularensis Francisella Pathogenicity Island (FPI) proteins - IglA, IglB and IglC, and a fusion protein (IglABC) comprising immunodominant epitopes of IglA, IglB, and IglC downstream of different Francisella promoters, including the bacterioferritin ( bfr ) promoter. We show that rLVS Δ capB / bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS Δ capB in broth and in human macrophages, and stably express FPI proteins in macrophages and mice absent antibiotic selection. In response to IglC and heat-inactivated LVS, spleen cells from mice immunized intradermally with rLVS Δ capB / bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS Δ capB , comparable to those from LVS-immunized mice. Mice immunized with rLVS Δ capB / bfr-iglA , iglB , iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS Δ capB / bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS Δ capB -immunized mice. Mice immunized intranasally with rLVS Δ capB / bfr-iglABC - but not with LVS - just before or after respiratory challenge with F. tularensis Schu S4 are partially protected; protection is correlated with induction of a strong innate immune response. Thus, rLVS Δ capB / bfr-iglABC shows improved immunogenicity and protective efficacy compared with parental LVS Δ capB and, in contrast to LVS, has partial efficacy as immediate pre- and post-exposure prophylaxis. [ABSTRACT FROM AUTHOR]

Francisella tularensis ( Ft ) is a Category A biothreat agent for which there currently is no FDA-approved vaccine. Thus, there is a substantial effort underway to develop an effective tularemia vaccine. While it is well established that gender can significantly impact susceptibility to primary infection, the impact of gender on vaccine efficacy is not well established. Thus, development of a successful vaccine against tularemia will require an understanding of the impact gender has on vaccine-induced protection against this organism. In this study, a role for gender in vaccine-induced protection following Ft challenge is identified for the first time. In the present study, mucosal vaccination with inactivated Ft (i Ft ) LVS elicited gender-based protection in C57BL/6Tac mice against respiratory challenge with Ft LVS. Specifically, vaccinated male mice were more susceptible to subsequent Ft LVS challenge. This increased susceptibility in male mice correlated with increased bacterial burden, increased tissue inflammation, and increased proinflammatory cytokine production late in post-challenge infection. In contrast, improved survival of i Ft -vaccinated female mice correlated with reduced bacterial burden and enhanced levels of Ft -specific Abs in serum and broncho-alveolar lavage (BAL) fluid post-challenge. Furthermore, vaccination with a live attenuated vaccine consisting of an Ft LVS superoxide dismutase (SodB) mutant, which has proven efficacious against the highly virulent Ft SchuS4 strain, demonstrated similar gender bias in protection post- Ft SchuS4 challenge. Of particular significance is the fact that these are the first studies to demonstrate that gender differences impact disease outcome in the case of lethal respiratory tularemia following mucosal vaccination. In addition, these studies further emphasize the fact that gender differences must be a serious consideration in any future tularemia vaccine development studies. [ABSTRACT FROM AUTHOR]

Oral vaccination with the defined live attenuated Francisella novicida vaccine strain U112ΔiglB has been demonstrated to induce protective immunity against pulmonary challenge with the highly human virulent Francisella tularensis strain SCHU S4. However, this vaccination regimen requires a booster dose in mice and Exhibits 50% protective efficacy in the Fischer 344 rat model. To enhance the efficacy of this vaccine strain, we engineered U112ΔiglB to express the Salmonella typhimurium FljB flagellin D1 domain, a TLR5 agonist. The U112ΔiglB::fljB strain was highly attenuated for intracellular macrophage replication, and although the FljB protein was expressed within the cytosol, it exhibited TLR5 activation in a TLR5-expressing HEK cell line. Additionally, infection of splenocytes and lymphocytes with U112ΔiglB::fljB induced significantly greater TNF-α production than infection with U112ΔiglB. Oral vaccination with U112ΔiglB::fljB also induced significantly greater protection than U112ΔiglB against pulmonary SCHU S4 challenge in rats. The enhanced protection was accompanied by higher IgG2a production and serum-mediated reduction of Francisella infectivity. Thus, the U112ΔiglB::fljB strain may serve as a potential vaccine candidate against pneumonic tularemia. [ABSTRACT FROM AUTHOR]

Highlights: [•] IFN-? is required for survival during secondary pneumonic tularemia. [•] IL-17A is dispensable for survival during secondary pneumonic tularemia. [•] Vaccination with LVS clpB elicits the same memory T cell response as LVS. [ABSTRACT FROM AUTHOR]

Highlights: [•] Interest in vaccine development against bioterror agents has increased since 2001. [•] One such agent of interest is Francisella tularensis. [•] Live attenuated strains have shown promise as vaccine candidates in mouse models. [•] Survival and protection data from recent examples is reviewed in this article. [Copyright &y& Elsevier]

Abstract: Successful treatment of pneumonic infection with Francisella tularensis, the causative agent of tularemia, requires rapid initiation of antibiotic therapy, yet even then treatment failures may occur. Consequently, new treatments are needed to enhance the effectiveness of antimicrobial therapy for acute pneumonic tularemia. In a prior study, immunization with F. tularensis membrane protein fraction (MPF) antigens 3 days prior to challenge was reported to induce significant protection from inhalational challenge. We therefore hypothesized that MPF immunization might also be effective in enhancing infection control if combined with antibiotic therapy and administered after infection as post-exposure immunotherapy. To address this question, a 24h post-exposure treatment model of acute pulmonary Schu S4 strain of F. tularensis infection in BALB/c mice was used. Following exposure, mice were immunized with MPF and treated with low-dose gentamicin, alone or in combination and the effects on survival, bacterial burden and dissemination were assessed. We found that immunization with MPF significantly increased the effectiveness of subtherapeutic gentamicin for post-exposure treatment of pneumonic tularemia, with 100% of combination-treated mice surviving long-term. Bacterial burdens in the liver and spleen were significantly reduced in combination MPF-gentamicin treated mice at 7 days after challenge. Passively transferred antibodies against MPF antigens also increased the effectiveness of gentamicin therapy. Thus, we concluded that post-exposure immunization with MPF antigens was an effective means of enhancing conventional antimicrobial therapy for pneumonic tularemia. [Copyright &y& Elsevier]

Abstract: Francisella tularensis subspecies tularensis is highly virulent for humans especially when it is inhaled. Therefore, it has the potential to be used as a biothreat agent. Vaccines against F. tularensis will need to be approved in accordance with the FDA Animal Rule. This will require identification of robust correlates of protection in experimental animals and the demonstration that similar immune responses are generated in vaccinated humans. Towards this goal, we have developed an experimental live vaccine strain by deleting the gene, clpB, encoding a heat shock protein from virulent subsp. tularensis strain, SCHU S4. SCHU S4ΔclpB administered intradermally protects BALB/c, but not C57BL/6 mice from subsequent respiratory challenge with wildtype SCHU S4. A comparison of post-vaccination and post-challenge immune responses in these two mouse strains shows an association between several antibody and cytokine responses and protection. In particular, elevated IFNγ levels in the skin 2 days after vaccination, sero-conversion to hypothetical membrane protein FTT_1778c, and to 30S ribosomal protein S1 (FTT_0183c) of F. tularensis after 30 days of vaccination, and elevated levels of pulmonary IL-17 on day 7 after respiratory challenge with SCHU S4 were all associated with protection. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a highly pathogenic gram negative bacterium that infects multiple sites in a host, including the skin and the respiratory tract, which can lead to the onset of a deadly disease with a 50% mortality rate. The live vaccine strain (LVS) of F. tularensis, while attenuated in humans but still virulent in mice, is not an option for vaccine use in the United States due to safety concerns, and currently no FDA approved vaccine exists. The purpose of the present work was to assess the ability of recombinant Francisella outer membrane protein A (FopA) to induce a protective response in mice. The gene encoding FopA from F. tularensis LVS was cloned and expressed in Escherichia coli. The resulting recombinant protein was affinity-purified from the E. coli outer membrane, incorporated into liposomes and administered to mice via multiple routes. FopA-immunized mice produced FopA-specific antibodies and were protected against both lethal intradermal and intranasal challenges with F. tularensis LVS. The vaccinated mice had reduced bacterial numbers in their lungs, livers and spleens during infection, and complete bacterial clearance was observed by day 28 post infection. Passive transfer of FopA-immune serum protected naïve mice against lethal F. tularensis LVS challenge, showing that humoral immunity played an important role in vaccine efficacy. FopA-immunization was unable to protect against challenge with the fully virulent SchuS4 strain of F. tularensis; however, the findings demonstrate proof of principle that an immune response generated against a component of a subunit vaccine is protective against lethal respiratory and intradermal tularemia. [Copyright &y& Elsevier]

Abstract: Protection against the intracellular bacterium Francisella tularensis within weeks of vaccination is thought to involve both cellular and humoral immune responses. However, the relative roles for cellular and humoral immunity in long lived protection against virulent F. tularensis are not well established. Here, we dissected the correlates of immunity to pulmonary infection with virulent F. tularensis strain SchuS4 in mice challenged 30 and 90 days after subcutaneous vaccination with LVS. Regardless of the time of challenge, LVS vaccination protected approximately 90% of SchuS4 infected animals. Surprisingly, control of bacterial replication in the lung during the first 7 days of infection was not required for survival of SchuS4 infection in vaccinated mice. Control and survival of virulent F. tularensis strain SchuS4 infection within 30 days of vaccination was associated with high titers of SchuS4 agglutinating antibodies, and IFN-γ production by multiple cell types in both the lung and spleen. In contrast, survival of SchuS4 infection 90 days after vaccination was correlated only with IFN-γ producing splenocytes and activated T cells in the spleen. Together these data demonstrate that functional agglutinating antibodies and strong mucosal immunity are correlated with early control of pulmonary infections with virulent F. tularensis. However, early mucosal immunity may not be required to survive F. tularensis infection. Instead, survival of SchuS4 infection at extended time points after immunization was only associated with production of IFN-γ and activation of T cells in peripheral organs. [ABSTRACT FROM AUTHOR]

Abstract: Francisella tularensis is a category A select agent. J5dLPS/OMP is a novel vaccine construct consisting of detoxified, O-polysaccharide side chain-deficient, lipopolysaccharide non-covalently complexed with the outer membrane protein of N. meningitidis group B. Immunization elicits high-titer polyclonal antibodies specific for the highly-conserved epitopes expressed within the glycolipid core that constitutes gram-negative bacteria (e.g., F. tularensis). Mice immunized intranasally with J5dLPS/OMP exhibited protective immunity to intratracheal challenge with the live vaccine strain, as well as the highly-virulent SchuS4 strain, of F. tularensis. The efficacy of J5dLPS/OMP vaccine suggests its potential utility in immunizing the general population against several different gram-negative select agents concurrently. [Copyright &y& Elsevier]

Abstract: Francisella tularensis subspecies tularensis is a highly virulent facultative intracellular pathogen of humans and a potential biological weapon. A live vaccine strain, F. tularensis LVS, was developed more than 50 years ago by pragmatic attenuation of a strain of the less virulent holarctica subspecies. LVS was demonstrated to be highly effective in human volunteers who were exposed to intradermal challenge with fully virulent subsp. tularensis, but was less effective against aerosol exposure. LVS faces regulatory hurdles that to date have prevented its licensure for general use. Therefore, a better defined and more effective vaccine is being sought. To this end we have created gene deletion mutants in the virulent subsp. tularensis strain and tested them for their ability to elicit a protective immune response against systemic or aerosol challenge with the highly virulent wild-type subsp. tularensis strain, SCHU S4. Both oral and intradermal (ID) primary vaccination routes were assessed in BALB/c and C3H/HeN mice as was oral boosting. One SCHU S4 mutant missing the heat shock gene, clpB, was significantly more attenuated than LVS whereas a double deletion mutant missing genes FTT0918 and capB was as attenuated as LVS. In general mice immunized with SCHU S4ΔclpB were significantly better protected against aerosol challenge than mice immunized with LVS. A single ID immunization of BALB/c mice with SCHU S4ΔclpB was at least as effective as any other regimen examined. Mice immunized with SCHU S4Δ0918ΔcapB were generally protected to a similar degree as mice immunized with LVS. A preliminary examination of immune responses to vaccination with LVS, SCHU S4ΔclpB, or SCHU S4Δ0918ΔcapB provided no obvious correlate to their relative efficacies. [Copyright &y& Elsevier]

Abstract: There is very limited evidence concerning the phenotype, function, and homing characteristics of memory T (TM) cells elicited by vaccination against intracellular bacteria in humans. Here we studied TM subsets elicited by exposure to Francisella tularensis in humans as a model of immunity to intracellular bacteria. To this end, TM cells were evaluated in two groups: (1) subjects immunized with live attenuated tularemia vaccine by skin scarification and (2) tularemia naturally infected subjects. In both groups the immune responses were mediated by CD4+ and CD8+ effector TM cells, mostly CD45RA−CD62L− and CD45RA+CD62L−. Based on the expression of CD27, integrins α4/β7, and α4/β1, it is likely that some of these TM cells have lytic potential and the ability to enter both mucosal and non-mucosal sites. Thus, regardless of whether by immunization or natural exposure, tularemia antigens elicited a broad spectrum of specific TM subsets with diverse homing characteristics. [Copyright &y& Elsevier]

Abstract: Francisella tularensis, an intracellular Gram-negative bacterium, is the causative agent of tularemia and a potential bioweapon. Currently, there is no licensed vaccine against this organism. We have characterized the efficacy of a defined F. tularensis subsp. novicida mutant (ΔiglB) as a live attenuated vaccine against pneumonic tularemia. Replication of the iglB mutant (KKF235) in murine macrophages was significantly lower than the wild type novicida strain U112, and exhibited an LD50 greater than 106-fold (>107 CFU vs <10CFU) in an intranasal challenge model. Mice immunized with KKF235 intranasally or orally induced robust antigen-specific splenic IFN-γ recall responses, as well as the production of systemic and mucosal antibodies. Intranasal vaccination with KKF235 protected mice from subsequent homotypic challenge with U112 as well as heterotypic challenge with F. tularensis subsp. holarctica (LVS). Moreover, protected animals also exhibited minimal pathological changes compared with mock-vaccinated and challenged animals. The protection conferred by KKF235 vaccination was shown to be highly dependent on endogenous IFN-γ production. Most significantly, oral immunization with KKF235 protected mice from a highly lethal subsp. tularensis (SCHU S4) pulmonary challenge. Collectively, these results further suggest the feasibility of using defined pathogenicity island mutants as live vaccine candidates against pneumonic tularemia. [Copyright &y& Elsevier]

Abstract: Francisella tularensis, the etiological agent of tularemia, is one of the most infectious bacterial pathogens known. No vaccine is currently approved for public use. Previously, we identified epitopes recognized specifically by T cells obtained from individuals following infection with F. tularensis. Here, we report that a subunit vaccine constructed based upon these epitopes elicited protective immunity in “humanized” HLA class II (DRB1*0401) transgenic mice. Vaccinated mice challenged intratracheally with a lethal dose of F. tularensis (Live Vaccine Strain) exhibited a rapid increase in pro-inflammatory cytokine production and diminished number of organisms in the lungs, and a concurrent increased rate of survival. These results demonstrate the efficacy of an epitope-based tularemia vaccine and suggest that such an approach might be widely applicable to the development of vaccines specific for intracellular bacterial pathogens. [Copyright &y& Elsevier]

Abstract: We evaluated the safety, reactogenicity and immunogenicity of escalating doses of a new Francisella tularensis Live Vaccine Strain (LVS) lot by scarification (SCAR) or subcutaneously (SQ) in humans. Subjects (N =10/group) received one dose of LVS via SCAR at 105,107 or 109 cfu/ml or SQ at 102, 103,104 or 105 cfu/ml; 14 subjects received placebo. All doses/routes were well tolerated. When compared to placebo, vaccination with 107 SCAR and 109 SCAR resulted in significantly higher serologic response frequencies, as measured by ELISA for IgG, IgM, IgA and microagglutination; whereas vaccination with 105 SCAR, 107 SCAR 109 SCAR and 105 SQ elicited a significantly higher interferon-γ response frequency. [Copyright &y& Elsevier]

Abstract: Pneumonic tularemia caused by inhalation of the type A strains of Francisella tularensis is associated with high morbidity and mortality in humans. The only vaccine known to protect humans against this disease is the attenuated live vaccine strain (LVS), but it is not currently registered for human use. To develop a new generation of vaccines, multiple animal models are needed that reproduce the human response to F. tularensis infection and vaccination. We examined the potential use of Fischer 344 rat as such a model. Fischer 344 rats were very sensitive to intratracheal infection with the virulent type A strain SCHU S4 and generally succumbed less than 2 weeks after infection. Similar to humans and non-human primates, Fischer 344 rats vaccinated with LVS by subcutaneous or intradermal routes were protected against a greater range of respiratory SCHU S4 challenge doses than has been reported for LVS vaccinated mice. Intratracheal LVS vaccination also induced effective immunity, but it was less protective when the challenge dose exceeded 105 SCHU S4. LVS vaccination did not prevent SCHU S4 infection but rather controlled bacterial growth and pathology, leading to the eventual clearance of the bacteria. Our results suggest that the Fischer 344 rat may be a good model for studying pneumonic tularemia and evaluating potential vaccine candidates. [Copyright &y& Elsevier]

Abstract: Previous studies have demonstrated that systemically administered immunotherapy can protect mice from systemic challenge with the bacterial pathogen Francisella tularensis. However, for protection from inhalational challenge with this bacterium, we wondered if mucosally administered immunotherapy might be more effective. Therefore, we administered cationic liposome–DNA complexes (CLDC), which are potent activators of innate immunity, intranasally (i.n.) and assessed the effectiveness of protection from lethal inhalational challenge with F. tularensis. We found that pretreatment by i.n. administration of CLDC 24h prior to bacterial challenge elicited nearly complete protection of BALB/c mice from lethal challenge with F. tularensis LVS strain. We also observed that mucosal CLDC immunotherapy provided a statistically significant increase in survival time in mice challenged with the highly virulent F. tularensis Schu4 strain. Protection was associated with a significant reduction in bacterial burden in the lungs, liver, and spleen. Mucosal administration of CLDC elicited significantly increased expression of IL-12, IFN-γ, TNF-α, IFN-β and IFN-α genes in the lung as detected by real-time quantitative PCR. In vitro treatment of F. tularensis infected macrophages with CLDC-elicited cytokines also significantly suppressed intracellular replication of F. tularensis in infected macrophages. In vivo, depletion of NK cells prior to administration of CLDC completely abolished the protective effects of CLDC immunotherapy. CLDC-elicited protection was also dependent on induction of IFN-γ production in vivo. We conclude therefore that activation of local pulmonary innate immune responses is capable of eliciting significant protection from inhalational exposure to a virulent bacterial pathogen. [Copyright &y& Elsevier]

Abstract: Francisella tularensis, the etiologic agent of tularemia, can cause severe and fatal infection after inhalation of as few as 10–100CFU. F. tularensis is a potential bioterrorism agent and, therefore, a priority for countermeasure development. Vaccination with the live vaccine strain (LVS), developed from a Type B strain, confers partial protection against aerosal exposure to the more virulent Type A strains and provides proof of principle that a live attenuated vaccine strain may be efficacious. However LVS suffers from several notable drawbacks that have prevented its licensure and widespread use. To address the specific deficiencies that render LVS a sub-optimal tularemia vaccine, we engineered F. tularensis LVS strains with targeted deletions in the guaA or guaB genes that encode critical enzymes in the guanine nucleotide biosynthetic pathway. F. tularensis LVSΔguaA and LVSΔguaB mutants were guanine auxotrophs and were highly attenuated in a mouse model of infection. While the mutants failed to replicate in macrophages, a robust proinflammatory cytokine response, equivalent to that of the parental LVS, was elicited. Mice vaccinated with a single dose of the F. tularensis LVSΔguaA or LVSΔguaB mutant were fully protected against subsequent lethal challenge with the LVS parental strain. These findings suggest the specific deletion of these target genes could generate a safe and efficacious live attenuated vaccine. [Copyright &y& Elsevier]

Abstract: Fransicella tularensis, the causative agent of tularemia, is in the top category (Category A) of potential agents of bioterrorism. To develop a safer vaccine against aerosolized F. tularensis, we have employed an attenuated Listeria monocytogenes, which shares with F. tularensis an intracellular and extraphagosomal lifestyle, as a delivery vehicle for F. tularensis antigens. We constructed recombinant L. monocytogenes (rLm) vaccines stably expressing seven F. tularensis proteins including IglC (rLm/iglC), and tested their immunogenicity and protective efficacy against lethal F. tularensis challenge in mice. Mice immunized intradermally with rLm/iglC developed significant cellular immune responses to F. tularensis IglC as evidenced by lymphocyte proliferation and CD4+ and CD8+ T-cell intracellular expression of interferon gamma. Moreover, mice immunized with rLm/iglC were protected against lethal challenge with F. tularensis LVS administered by the intranasal route, a route chosen to mimic airborne infection, and, most importantly, against aerosol challenge with the highly virulent Type A F. tularensis SchuS4 strain. [Copyright &y& Elsevier]

Abstract: Herein we report studies with a novel combination vaccine that, when administered to mice, conferred protection against highly virulent strains of Francisella tularensis by stimulating both arms of the immune system. Our earlier studies with Ft.LVS::wbtA, an O-polysaccharide (OPS)-negative mutant derived from the available live vaccine strain of F. tularensis (Ft.LVS), elucidated the role of antibodies to the OPS – a key virulence determinant – in protection against virulent type A organisms. However, when expressed on the organism, the OPS enhances virulence. In contrast, in purified form, the OPS is completely benign. We hypothesized that a novel combination vaccine containing both a component that induces humoral immunity and a component that induces cellular immunity to this intracellular microbe would have an enhanced protective capacity over either component alone and would be much safer than the LVS vaccine. Thus we developed a combination vaccine containing both OPS (supplied in an OPS–tetanus toxoid glycoconjugate) to induce a humoral antibody response and strain Ft.LVS::wbtA (which is markedly attenuated by its lack of OPS) to induce a cell-mediated protective response. This vaccine protected mice against otherwise-lethal intranasal and intradermal challenge with wild-type F. tularensis strains Schu S4 (type A) and FSC 108 (type B). These results represent a significant advance in our understanding of immunity to F. tularensis and provide important insight into the development of a safer vaccine effective against infections caused by clinical type A and B strains of F. tularensis. [Copyright &y& Elsevier]

Abstract: The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with F. tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72h post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to re-challenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4-specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis. [Copyright &y& Elsevier]

Abstract: Vaccination of mice with Francisella tularensis live vaccine strain (LVS) mutants described so far have failed to induce protection in C57BL/6 mice against challenge with the virulent strain F. tularensis SchuS4. We have previously reported that a mutant of F. tularensis LVS deficient in iron superoxide dismutase (sodB Ft ) is hypersensitive to oxidative stress and attenuated for virulence in mice. Herein, we evaluated the efficacy of this mutant as a vaccine candidate against respiratory tularemia caused by F. tularensis SchuS4. C57BL/6 mice were vaccinated intranasally (i.n.) with the sodB Ft mutant and challenged i.n. with lethal doses of F. tularensis SchuS4. The level of protection against SchuS4 challenge was higher in sodB Ft vaccinated group as compared to the LVS vaccinated mice. sodB Ft vaccinated mice following SchuS4 challenge exhibited significantly reduced bacterial burden in lungs, liver and spleen, regulated production of pro-inflammatory cytokines and less severe histopathological lesions compared to the LVS vaccinated mice. The sodB Ft vaccination induced a potent humoral immune response and protection against SchuS4 required both CD4 and CD8 T cells in the vaccinated mice. sodB Ft mutants revealed upregulated levels of chaperonine proteins DnaK, GroEL and Bfr that have been shown to be important for generation of a potent immune response against Francisella infection. Collectively, this study describes an improved live vaccine candidate against respiratory tularemia that has an attenuated virulence and enhanced protective efficacy than the LVS. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a Gram-negative intracellular bacterium, and the causative agent of tularemia. The infection can be initiated by various routes and can manifest itself in several clinical forms with the disseminated typhoidal form initiated by inhalation being most fatal. The attenuated live vaccine strain (LVS), developed almost 50 years ago, remains the sole effective tularemia vaccine, which is still only available as an investigational new drug for at-risk individuals. This vaccine, when given by scarification, appears to provide solid protection against subsequent systemic infection with clinical strains of F. tularensis, but its efficacy against respiratory infection is less satisfactory. In this study, we evaluated the potential of oral immunization with LVS for eliciting protection against systemic and respiratory infection with virulent F. tularensis strains in a mouse model of tularemia. Oral LVS immunization was highly effective at protecting Balb/c mice against lethal systemic or respiratory challenges with type A and type B F. tularensis. Compared to sham-immunized mice, oral LVS-immunized mice showed significant reductions in burdens of virulent F. tularensis in the lung and spleen and milder tissue damage and inflammation in the liver. The immunization induced F. tularensis-specific antibody responses in the serum and bronchoalveolar lavage fluids, as well as antigen-specific splenocyte proliferation and IFN-γ and IL-2 production. The protective efficacy was related to the size of the immunizing dose but not the number of doses administered. Like other routes of LVS immunization in mice, the protective immunity induced by oral immunization was relatively short-lived. These results suggest that oral immunization should be explored further as an alternative vaccination strategy to combat tularemia. [Copyright &y& Elsevier]

Abstract: The T lymphocyte antigens, which may have a role in protection against tularemia, were predicted by immunoinformatics analysis of Francisella tularensis Schu4. Twenty-seven class II putative promiscuous epitopes and 125 putative class I supertype epitopes were chosen for synthesis; peptides were tested in vitro for their ability to bind HLA and to induce immune responses from PBMCs of 23 previously infected subjects. While the immune responses of individual subjects showed heterogeneity, 95% of the subjects responded strongly to a pool of 27 promiscuous peptides; 25%, 33%, and 44% of subjects responded to pools of 25 A2, A24, and B7 peptides, respectively. These data can aid in the development of novel epitope-based and subunit tularemia vaccines. [Copyright &y& Elsevier]

Abstract: Francisella tularensis subspecies novicida mutants have been made with deletions introduced into the purA or purF genes. These mutants demonstrated the expected growth requirement for purines and complementation with the wild type genes restored the ability to grow on purine deficient media. The mutants were at least 10,000-fold attenuated by the ip challenge route in Balb/C mice and defective for survival in J774A.1 mouse macrophages. Immunisation with the purA mutant did not provide protection against a subsequent challenge with 100 median lethal doses of F. tularensis subspecies novicida. Immunisation of mice with the purF mutant provided protection against a subsequent challenge with F. tularensis subspecies novicida but not against a subspecies tularensis challenge. These findings suggest that purine auxotrophs of F. tularensis should be further evaluated as live attenuated vaccines against tularemia, but that differential effects are seen depending on which step in the biosynthetic pathway is inactivated. [Copyright &y& Elsevier]

Abstract: A live attenuated vaccine Francisella tularensis live vaccine strain (LVS), that confers protection against tularemia infection in a number of animal models including man was developed during the 1960s in the US. In this study, we have established the median lethal dose (MLD) after intraperitoneal (i.p.) or intravenous (i.v.) delivery of NDBR Lot 4 F. tularensis LVS to be 4cfu and 2.24 × 104cfu, respectively, in BALB/c mice and less than 1cfu and 1.29 × 104cfu, respectively, in C57BL/6 mice. When delivered subcutaneously, the MLD for F. tularensis LVS was greater then 1 × 108cfu in both strains of mouse. Using mouse models of systemic tularemia infection it was demonstrated that F. tularensis LVS immunised BALB/c mice were fully protected after challenge with approximately 1000 MLD of a strain of F. tularensis subsp. tularensis or a strain of F. tularensis subsp. holarctica. Under similar challenge conditions, protection in C57BL/6 mice was only evident against a subsp. holarctica strain. In BALB/c mice, protection against a subsp. holarctica strain was achieved 4 days after F. tularensis LVS immunisation whereas protection against a subsp. tularensis strain was only evident 14 days after F. tularensis LVS immunisation. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is an extremely virulent facultative intracellular bacterial pathogen of many mammalian species including mice and humans in which it causes a spectrum of disease collectively called tularemia. In humans, intradermal or inhaled inocula of 10cfu or less of the most virulent strains of the pathogen are sufficient to cause severe infection and possible death; in mice similar inocula are routinely lethal. An attenuated live vaccine strain, F. tularensis LVS, was developed almost 50 years ago, and remains the sole prophylactic against virulent strains of the pathogen. Using F. tularensis LVS as a model vaccine, we recently showed that it was possible to systemically immunize various mouse strains and protect them against subsequent massive (2000cfu) intradermal (i.d.) challenge, but not against low dose (∼10cfu) aerosol challenge, with virulent strains of the pathogen. This is troubling because the latter route is considered an important means of deliberately disseminating F. tularensis in a bioterrorist attack. Others have previously shown that administering LVS to humans, guinea pigs and monkeys as an aerosol enhanced protection against subsequent aerosol challenge with virulent F. tularensis. In the present study, we show the same phenomenon in BALB/c and C3H/HeN mice. In this model, interferon gamma (IFNγ) and CD4+ and CD8+ T cells are essential for the expression of anti-Francisella immunity in the lungs. Combined this immune response operates by limiting dissemination of the pathogen to susceptible internal organs. Further, understanding of how inhaled LVS elicits local cell-mediated protective immunity will be critical for devising improved vaccines against pulmonary tularemia. [Copyright &y& Elsevier]

The following study determined whether commonly available mouse strains could be used to reveal vaccines capable of protecting against aerosol exposure to virulent type A strains of Francisella tularensis. The attenuated live vaccine strain (LVS) of the pathogen, F. tularensis LVS, was used as the model vaccine for these studies. The results showed that most mouse strains survived intradermal immunization with 105 cfu of LVS, except for A/J mice the majority of which died following exposure to 104 cfu (colony forming units). Most LVS-immunized mouse strains survived subsequent i.d. challenge with >50 LD50 of a virulent type A strain of the pathogen. However, all mouse strains tested remained susceptible to lethal infection by a low dose (∼20 cfu) aerosol challenge with virulent F. tularensis even after vaccination with LVS, though immunized C3H/HeN mice appeared to be slightly more resistant than the rest to such an assault. [Copyright &y& Elsevier]

In order to begin understanding the immunological basis for immunity to tularemia, and to establish a baseline for judging the efficacy of potential novel vaccines, the present study examined the ability of the live vaccine strain of Francisella tularensis (F. tularensis) LVS, to elicit immunity in mice against subsequent systemic and aerosol challenge with highly virulent strains of the pathogen. The results show, that infection with LVS protects BALB/c mice against systemic challenge with virulent Types A and B F. tularensis. In contrast, C57BL/6 mice vaccinated with LVS were only rendered immune to systemic challenge with Type B F. tularensis. Neither mouse strain immunized with LVS was able to resist aerosol challenge with Type A F. tularensis, and only immunized BALB/c mice withstood exposure to aerosols of Type B F. tularensis. [Copyright &y& Elsevier]

The purpose of this study was to evaluate the efficacy of a vaccine consisting of the O-polysaccharide of the lipopolysaccharide (LPS) of Francisella tularensis chemically conjugated to bovine serum albumin. The results show that conjugation preserved both the antigenicity and immunogenicity of the polysaccharide moiety. Mice vaccinated with the glyco-conjugate, but not with BSA alone, were completely protected against an intradermal challenge with a highly virulent type B strain of F. tularensis, and partially protected against an aerosol challenge with the same strain. However, such vaccination failed to protect against an aerosol challenge with a virulent type A strain of the pathogen. The results suggest that the O-antigen of F. tularensis could be considered as a potential component of a subunit vaccine against type B, but not type A strains of F. tularensis. [Copyright &y& Elsevier]

Abstract: Francisella tularensis has attracted attention historically as a biological weapon, due to its high infectivity in aerosols, and the severity of disease in humans. There is no licensed vaccine currently available, although an attenuated live vaccine strain (LVS) was identified in the middle of the last century and has been successfully used to protect humans. Efforts are underway to determine the basis of attenuation of LVS, and to understand the immunity required for protection. Alternative approaches to produce subunit vaccines and defined attenuated strains are also in progress. However, the limitations of animal models may make licensing a candidate vaccine challenging. [Copyright &y& Elsevier]