Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria: Effects of host background and route of immunization

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.     

Cellular and humoral immunity are synergistic in protection against types A and B Francisella tularensis

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.     

Characterization of rationally attenuated Francisella tularensis vaccine strains that harbor deletions in the guaA and guaB genes

Francisella tularensis, the etiologic agent of tularemia, can cause severe and fatal infection after inhalation of as few as 10–100 CFU. 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.     

Recombinant attenuated Listeria monocytogenes vaccine expressing Francisella tularensis IglC induces protection in mice against aerosolized Type A F. tularensis

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.     

Vaccination of Fischer 344 rats against pulmonary infections by Francisella tularensis type A strains

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.     

Protection of vaccinated mice against pneumonic tularemia is associated with an early memory sentinel-response in the lung

Francisella tularensis is the intracellular bacterial pathogen causing the respiratory life-threatening disease tularemia. Development of tularemia vaccines has been hampered by an incomplete understanding of the correlates of immunity. Moreover, the importance of lung cellular immunity in vaccine-mediated protection against tularemia is a controversial matter. Live attenuated vaccine strains of F. tularensis such as LVS (Live Vaccine Strain), elicit an immune response protecting mice against subsequent challenge with the virulent SchuS4 strain, yet the protective immunity against pulmonary challenge is limited in its efficacy and longevity. We established a murine intra-nasal immunization model which distinguishes between animals fully protected, challenged at 4 weeks post double-vaccination (200 inhalation Lethal Dose 50%, LD₅₀, of SchuS4), and those which do not survive the lethal SchuS4 infection, challenged at 8 weeks post double vaccination. Early in the recall immune response in the lung (before day 3), disease progression and bacterial dissemination differed considerably between protected and non-protected immunized mice. Pre-challenge analysis, revealed that protected mice, exhibited significantly higher numbers of lung Ft-specific memory T cells compared to non-protected mice. Quantitative PCR analysis established that a higher magnitude, lung T cells response was activated in the lungs of the protected mice already at 24 h post-challenge. The data imply that an early memory response within the lung is strongly associated with protection against the lethal SchuS4 bacteria presumably by restricting the dissemination of the bacteria to internal organs. Thus, future prophylactic strategies to countermeasure F. tularensis infection may require modulation of the immune response within the lung.     

Francisella tularensis Live Vaccine Strain deficient in capB and overexpressing the fusion protein of IglA,IglB, and IglC from the bfr promoter induces improved protection against F. tularensis respiratory challenge

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 ΔcapB 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 ΔcapB, we developed recombinant LVS ΔcapB (rLVS ΔcapB) 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 ΔcapB/bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS ΔcapB 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 ΔcapB/bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS ΔcapB, comparable to those from LVS-immunized mice. Mice immunized with rLVS ΔcapB/bfr-iglA, iglB, iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS ΔcapB/bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS ΔcapB-immunized mice. Mice immunized intranasally with rLVS ΔcapB/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 ΔcapB/bfr-iglABC shows improved immunogenicity and protective efficacy compared with parental LVS ΔcapB and, in contrast to LVS, has partial efficacy as immediate pre- and post-exposure prophylaxis.     

Expansion and retention of pulmonary CD4+ T cells after prime boost vaccination correlates with improved longevity and strength of immunity against tularemia

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.     

Aerosol-, but not intradermal-immunization with the live vaccine strain of Francisella tularensis protects mice against subsequent aerosol challenge with a highly virulent type A strain of the pathogen by an alphabeta T cell- and interferon gamma- dependent mechanism

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 (2000 cfu) intradermal (i.d.) challenge, but not against low dose (approximately 10 cfu) 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 (IFNgamma) 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.     

Long lived protection against pneumonic tularemia is correlated with cellular immunity in peripheral,not pulmonary,organs

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.     

Mucosal immunotherapy for protection from pneumonic infection with Francisella tularensis

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 24 h 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.     

Susceptibility of various mouse strains to systemically- or aerosol-initiated tularemia by virulent type A Francisella tularensis before and after immunization with the attenuated live vaccine strain of the pathogen

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 10(5)cfu of LVS, except for A/J mice the majority of which died following exposure to 10(4)cfu (colony forming units). Most LVS-immunized mouse strains survived subsequent i.d. challenge with >50 LD(50) of a virulent type A strain of the pathogen. However, all mouse strains tested remained susceptible to lethal infection by a low dose ( approximately 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.     

Vaccination with a defined Francisella tularensis subsp. novicida pathogenicity island mutant (ΔiglB) induces protective immunity against homotypic and heterotypic challenge

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 <10 CFU) 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.     

Vaccination evokes gender-dependent protection against tularemia infection in C57BL/6Tac mice

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 (iFt) 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 iFt-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.     

Role of antibody to lipopolysaccharide in protection against low- and high-virulence strains of Francisella tularensis

Mice immunised with lipopolysaccharide (LPS) from Francisella tularensis were protected against challenge with the live vaccine strain (LVS). However, when similarly immunised mice were challenged using the fully virulent F. tularensis strain Schu4, only an increase in the time to death was observed. Passive transfer of serum from LPS-immunised mice to naive mice afforded protection against F. tularensis LVS. LPS-immunised mice depleted of either CD4+ or CD8+ T-cells survived a F. tularensis LVS challenge although the rate of clearance of bacteria from the spleen was significantly reduced in the CD8+ depleted group. LPS-immunised mice boosted with F. tularensis LVS were re-challenged with F. tularensis Schu4. This cohort was significantly protected (LD(50) increased from <1 to >1000 CFU). However, passive transfer of serum did not confer protection and mice depleted of CD4+ or CD8+ T-cells did not survive.     

Oral immunization of mice with the live vaccine strain (LVS) of Francisella tularensis protects mice against respiratory challenge with virulent type A F. tularensis

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-gamma 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.     

Efficacy of the live attenuated Francisella tularensis vaccine (LVS) in a murine model of disease

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 4 cfu and 2.24 x 10(4) cfu, respectively, in BALB/c mice and less than 1 cfu and 1.29 x 10(4) cfu, respectively, in C57BL/6 mice. When delivered subcutaneously, the MLD for F. tularensis LVS was greater then 1 x 10(8) cfu 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.     

Intradermal vaccination with a Pseudomonas aeruginosa vaccine adjuvanted with a mutant bacterial ADP-ribosylating enterotoxin protects against acute pneumonia

Respiratory infections are a leading cause of morbidity and mortality globally. This is partially due to a lack of effective vaccines and a clear understanding of how vaccination route and formulation influence protective immunity in mucosal tissues such as the lung. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing acute pulmonary infections and is a leading cause of hospital-acquired and ventilator-associated pneumonia. With multidrug-resistant P. aeruginosa infections on the rise, the need for a vaccine against this pathogen is critical. Growing evidence suggests that a successful P. aeruginosa vaccine may require mucosal antibody and Th1- and Th17-type CD4⁺ T cells to prevent pulmonary infection. Intradermal immunization with adjuvants, such as the bacterial ADP-Ribosylating Enterotoxin Adjuvant (BARE) double mutant of E. coli heat-labile toxin (dmLT), can direct protective immune responses to mucosal tissues, including the lungs. We reasoned that intradermal immunization with P. aeruginosa outer membrane proteins (OMPs) adjuvanted with dmLT could drive neutralizing antibodies and migration of CD4⁺ T cells to the lungs and protect against P. aeruginosa pneumonia in a murine model. Here we show that mice immunized with OMPs and dmLT had significantly more antigen-specific IgG and Th1- and Th17-type CD4⁺ memory T cells in the pulmonary environment compared to control groups of mice. Furthermore, OMPs and dmLT immunized mice were significantly protected against an otherwise lethal lung infection. Protection was associated with early IFN-γ and IL-17 production in the lungs of immunized mice. These results indicate that intradermal immunization with dmLT can drive protective immunity to the lung mucosa and may be a viable vaccination strategy for a multitude of respiratory pathogens.     

An improved vaccine for prevention of respiratory tularemia caused by Francisella tularensis SchuS4 strain

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.     

Epitope-based vaccination against pneumonic tularemia

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.