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.     

Fc receptor-targeting of immunogen as a strategy for enhanced antigen loading,vaccination, and protection using intranasally administered antigen-pulsed dendritic cells

Dendritic cells (DCs) play a critical role in the generation of adaptive immunity via the efficient capture, processing, and presentation of antigen (Ag) to naïve T cells. Administration of Ag-pulsed DCs is also an effective strategy for enhancing immunity to tumors and infectious disease organisms. Studies have also demonstrated that targeting Ags to Fcγ receptors (FcγR) on Ag presenting cells can enhance humoral and cellular immunity in vitro and in vivo. Specifically, our studies using a Francisella tularensis (Ft) infectious disease vaccine model have demonstrated that targeting immunogens to FcγR via intranasal (i.n.) administration of monoclonal antibody (mAb)-inactivated Ft (iFt) immune complexes (ICs) enhances protection against Ft challenge. Ft is the causative agent of tularemia, a debilitating disease of humans and other mammals and a category A biothreat agent for which there is no approved vaccine. Therefore, using iFt Ag as a model immunogen, we sought to determine if ex vivo targeting of iFt to FcγR on DCs would enhance the potency of i.n. administered iFt-pulsed DCs. In this study, bone marrow-derived DCs (BMDCs) were pulsed ex vivo with iFt or mAb-iFt ICs. Intranasal administration of mAb-iFt-pulsed BMDCs enhanced humoral and cellular immune responses, as well as protection against Ft live vaccine strain (LVS) challenge. Increased protection correlated with increased iFt loading on the BMDC surface as a consequence of FcγR-targeting. However, the inhibitory FcγRIIB had no impact on this enhancement. In conclusion, targeting Ag ex vivo to FcγR on DCs provides a method for enhanced Ag loading of DCs ex vivo, thereby reducing the amount of Ag required, while also avoiding the inhibitory impact of FcγRIIB. Thus, this represents a simple and less invasive strategy for increasing the potency of ex vivo-pulsed DC vaccines against chronic infectious diseases and cancer.     

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.     

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.     

Identification of Francisella tularensis outer membrane protein A (FopA) as a protective antigen for tularemia

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.     

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.     

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.     

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.     

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.     

Ex vivo antigen-pulsed PBMCs generate potent and long lasting immunity to infection when administered as a vaccine

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 (iFt) 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 iFt 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.     

Lipopolysaccharide-specific memory B cell responses to an attenuated live cholera vaccine are associated with protection against Vibrio cholerae infection

BackgroundThe single-dose live attenuated vaccine CVD 103-HgR protects against experimental Vibrio cholerae infection in cholera-naïve adults for at least 6 months after vaccination. While vaccine-induced vibriocidal seroconversion is associated with protection, vibriocidal titers decline rapidly from their peak 1–2 weeks after vaccination. Although vaccine-induced memory B cells (MBCs) might mediate sustained protection in individuals without detectable circulating antibodies, it is unknown whether oral cholera vaccination induces a MBC response.MethodsIn a study that enrolled North American adults, we measured lipopolysaccharide (LPS)- and cholera toxin (CtxB)-specific MBC responses to PXVX0200 (derived from the CVD 103-HgR strain) and assessed stool volumes following experimental Vibrio cholerae infection. We then evaluated the association between vaccine-induced MBC responses and protection against cholera.ResultsThere was a significant increase in % CT-specific IgG, % LPS-specific IgG, and % LPS-specific IgA MBCs which persisted 180 days after vaccination as well as a significant association between vaccine-induced increase in % LPS-specific IgA MBCs and lower post-challenge stool volume (r = −0.56, p < 0.001).DiscussionOral cholera vaccination induces antigen-specific MBC responses, and the anamnestic LPS-specific responses may contribute to long-term protection and provide correlates of the duration of vaccine-induced protection.Clinical trials registration: NCT01895855.     

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.     

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.     

Protection against H5N1 by multiple immunizations with seasonal influenza vaccine in mice is correlated with H5 cross-reactive antibodies

BackgroundCurrent seasonal influenza vaccines are believed to confer protection against a narrow range of virus strains. However, their protective ability is commonly estimated based on an in vitro correlate of protection that only considers a subset of anti-influenza antibodies that are typically strain specific, i.e., hemagglutination inhibiting antibodies. Here, we evaluate the breadth of protection induced with a seasonal trivalent influenza vaccine (composition H1N1 A/California/07/09, H3N2 A/Victoria/210/08, B/Brisbane/60/08) against influenza challenge in mice.MethodsBalb/c mice were immunized once, twice, or three times with seasonal influenza vaccine to assess protection against heterosubtypic H5N1 influenza challenge, or homologous H1N1 influenza virus as a control. Passive transfer of immune serum was used to determine the contribution of humoral immunity to protection.ResultsMultiple immunizations with seasonal influenza vaccine induced up to 80% protection against heterosubtypic H5N1 influenza challenge in mice without eliciting detectable H5N1 neutralizing antibodies. Comparable levels of protection were reached by passive transfer of immune serum, and protection was correlated with the titer of vaccine-induced, H5 cross-reactive, non-neutralizing antibodies that are at least in part directed against conserved HA epitopes.ConclusionsHere, we demonstrate that seasonal vaccine has the ability to induce broad serum-mediated protection, and that the mechanism of this protection is different from the vaccine-induced homologous protection.     

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.     

Reciprocal interference of maternal and infant immunization in protection against pertussis

BackgroundBecause of the current re-emergence of pertussis, vaccination during the 3rd trimester of pregnancy is recommended in several countries in order to protect neonates by placental transfer of maternal antibodies. Here, we examined the potential reciprocal interference of mother and infant vaccination in protection against pertussis in mice.MethodsFemale mice were vaccinated with acellular pertussis vaccines and protection against Bordetella pertussis challenge, as well as functional antibodies were measured in their offspring with or without re-vaccination.ResultsMaternal immunization protected the offspring against B. pertussis challenge, but protection waned quickly and was lost after vaccination of the infant mice with the same vaccine. Without affecting antibody titers, infant vaccination reduced the protective functions of maternally-derived antibodies, evidenced both in vitro and in vivo. Protection induced by infant vaccination was also affected by maternal antibodies. However, when mothers and infants were immunized with two different vaccines, no interference of infant vaccination on the protective effects of maternal antibodies was noted.ConclusionIt may be important to determine the functionality of antibodies to evaluate potential interference of maternal and infant vaccination in protection against pertussis.     

Protection by novel vaccine candidates,Mycobacterium tuberculosis ΔmosR and ΔechA7, against challenge with a Mycobacterium tuberculosis Beijing strain

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion people, claiming around 1.5 million lives annually. The only vaccine approved for clinical use against this disease is the Bacillus Calmette–Guérin (BCG) vaccine. Unfortunately, BCG has limited efficacy against the adult, pulmonary form of tuberculosis. This vaccine was developed from M. bovis with antigen expression and host specificity that differ from M. tuberculosis. To address these problems, we have designed two novel, live attenuated vaccine (LAV) candidates on an M. tuberculosis background: ΔmosR and ΔechA7. These targeted genes are important to M. tuberculosis pathogenicity during infection. To examine the efficacy of these strains, C57BL/6 mice were vaccinated subcutaneously with either LAV, BCG, or PBS. Both LAV strains persisted up to 16 weeks in the spleens or lungs of vaccinated mice, while eliciting minimal pathology prior to challenge. Following challenge with a selected, high virulence M. tuberculosis Beijing strain, protection was notably greater for both groups of LAV vaccinated animals as compared to BCG at both 30 and 60 days post-challenge. Additionally, vaccination with either ΔmosR or ΔechA7 elicited an immune response similar to BCG. Although these strains require further development to meet safety standards, this first evidence of protection by these two new, live attenuated vaccine candidates shows promise.     

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.     

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.