Interleukin-17 Protects against the Francisella tularensis Live Vaccine Strain but Not against a Virulent F. tularensis Type A Strain

Francisella tularensis is a highly infectious intracellular bacterium that causes the zoonotic infection tularemia. While much literature exists on the host response to F. tularensis infection, the vast majority of work has been conducted using attenuated strains of Francisella that do not cause disease in humans. However, emerging data indicate that the protective immune response against attenuated F. tularensis versus F. tularensis type A differs. Several groups have recently reported that interleukin-17 (IL-17) confers protection against the live vaccine strain (LVS) of Francisella. While we too have found that IL-17Rα^−/− mice are more susceptible to F. tularensis LVS infection, our studies, using a virulent type A strain of F. tularensis (SchuS4), indicate that IL-17Rα^−/− mice display organ burdens and pulmonary gamma interferon (IFN-γ) responses similar to those of wild-type mice following infection. In addition, oral LVS vaccination conferred equivalent protection against pulmonary challenge with SchuS4 in both IL-17Rα^−/− and wild-type mice. While IFN-γ was found to be critically important for survival in a convalescent model of SchuS4 infection, IL-17 neutralization from either wild-type or IFN-γ^−/− mice had no effect on morbidity or mortality in this model. IL-17 protein levels were also higher in the lungs of mice infected with the LVS rather than F. tularensis type A, while IL-23p19 mRNA expression was found to be caspase-1 dependent in macrophages infected with LVS but not SchuS4. Collectively, these results demonstrate that IL-17 is dispensable for host immunity to type A F. tularensis infection, and that induced and protective immunity differs between attenuated and virulent strains of F. tularensis.     

Native outer membrane proteins protect mice against pulmonary challenge with virulent type A Francisella tularensis

Francisella tularensis is a gram-negative intracellular bacterium and the causative agent of the zoonotic disease tularemia. F. tularensis is a category A select agent and thus a potential agent of bioterrorism. Whereas an F. tularensis live, attenuated vaccine strain (LVS) is the basis of an investigational vaccine, this vaccine is not licensed for human use because of efficacy and safety concerns. In the present study, we immunized mice with isolated native outer membrane proteins (OMPs), ethanol-inactivated LVS (iLVS), or purified LVS lipopolysaccharide (LPS) and assessed the ability of each vaccine preparation to protect mice against pulmonary challenge with the virulent type A F. tularensis strain SchuS4. Antibody isotyping indicated that both Th1 and Th2 antibody responses were generated in mice after immunization with OMPs or iLVS, whereas LPS immunization resulted in only immunoglobulin A production. In survival studies, OMP immunization provided the greatest level of protection (50% survival at 20 days after infection with SchuS4), and there were associated 3-log reductions in the spleen and liver bacterial burdens (compared to nonvaccinated mice). Cytokine quantitation for the sera of SchuS4-challenged mice indicated that OMP and iLVS immunizations induced high levels of tumor necrosis factor alpha and interleukin-2 (IL-2) production, whereas only OMP immunization induced high levels of IL-10 production. By comparison, high levels of proinflammatory cytokines, including RANTES, granulocyte colony-stimulating factor, IL-6, IL-1α, IL-12p40, and KC, in nonvaccinated mice indicated that these cytokines may facilitate disease progression. Taken together, the results of this study demonstrate the potential utility of an OMP subunit (acellular) vaccine for protecting mammals against type A F. tularensis.     

Novel Catanionic Surfactant Vesicle Vaccines Protect against Francisella tularensis LVS and Confer Significant Partial Protection against F. tularensis Schu S4 Strain

Francisella tularensis is a Gram-negative immune-evasive coccobacillus that causes tularemia in humans and animals. A safe and efficacious vaccine that is protective against multiple F. tularensis strains has yet to be developed. In this study, we tested a novel vaccine approach using artificial pathogens, synthetic nanoparticles made from catanionic surfactant vesicles that are functionalized by the incorporation of either F. tularensis type B live vaccine strain (F. tularensis LVS [LVS-V]) or F. tularensis type A Schu S4 strain (F. tularensis Schu S4 [Schu S4-V]) components. The immunization of C57BL/6 mice with “bare” vesicles, which did not express F. tularensis components, partially protected against F. tularensis LVS, presumably through activation of the innate immune response, and yet it failed to protect against the F. tularensis Schu S4 strain. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.) F. tularensis LVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.) F. tularensis Schu S4 challenge and significantly increased the mean time to death for nonsurvivors, particularly following the i.n. and heterologous (i.e., i.p./i.n.) routes of immunization. LVS-V immunization, but not immunization with empty vesicles, elicited high levels of IgG against nonlipopolysaccharide (non-LPS) epitopes that were increased after F. tularensis LVS challenge and significantly increased early cytokine production. Antisera from LVS-V-immunized mice conferred passive protection against challenge with F. tularensis LVS. Together, these data indicate that functionalized catanionic surfactant vesicles represent an important and novel tool for the development of a safe and effective F. tularensis subunit vaccine and may be applicable for use with other pathogens.     

The Presence of CD14 Overcomes Evasion of Innate Immune Responses by Virulent Francisella tularensis in Human Dendritic Cells In Vitro and Pulmonary Cells In Vivo

Francisella tularensis is a Gram-negative bacterium that causes acute, lethal disease following inhalation. We have previously shown that viable F. tularensis fails to stimulate secretion of proinflammatory cytokines following infection of human dendritic cells (hDC) in vitro and pulmonary cells in vivo. Here we demonstrate that the presence of the CD14 receptor is critical for detection of virulent F. tularensis strain SchuS4 by dendritic cells, monocytes, and pulmonary cells. Addition of soluble CD14 (sCD14) to hDC restored cytokine production following infection with strain SchuS4. In contrast, addition of anti-CD14 to monocyte cultures inhibited the ability of these cells to respond to strain SchuS4. Addition of CD14 or blocking CD14 following SchuS4 infection in dendritic cells and monocytes, respectively, was not due to alterations in phagocytosis or replication of the bacterium in these cells. Administration of sCD14 in vivo also restored cytokine production following infection with strain SchuS4, as assessed by increased concentrations of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-12p70, and IL-6 in the lungs of mice receiving sCD14 compared to mock-treated controls. In contrast to homogenous cultures of monocytes or dendritic cells infected in vitro, mice treated with sCD14 in vivo also exhibited controlled bacterial replication and dissemination compared to mock-treated controls. Interestingly, animals that lacked CD14 were not more susceptible or resistant to pulmonary infection with SchuS4. Together, these data support the hypothesis that the absence or low abundance of CD14 on hDC and in the lung contributes to evasion of innate immunity by virulent F. tularensis. However, CD14 is not required for development of inflammation during the last 24 to 48 h of SchuS4 infection. Thus, the presence of this receptor may aid in control of virulent F. tularensis infections at early, but not late, stages of infection.Daily insults of inhaled particulate and foreign antigens into the lungs could result in devastating inflammation. However, the lung counters these attacks by tightly regulating inflammatory responses. This regulation occurs both in the form of inhibitory molecules, such as surfactants that dampen macrophage and dendritic cell (DC) responsiveness, as well as production of immunosuppressive cytokines, such as transforming growth factor β (TGF-β) (1, 10, 15, 33, 34). Given the immunosuppressive nature of the lung environment, it is not surprising that pathogens capable of causing lethal disease following inhalation, such as Francisella tularensis, take advantage of this property for rapid replication while evading detection by the host immune response.Francisella tularensis is a Gram-negative, facultative intracellular bacterium and is the causative agent of tularemia. Pneumonic tularemia is an acute, lethal disease mediated by F. tularensis, following inhalation of as few as 10 to 15 bacteria in mice and humans (20, 49). Surprisingly, despite the rapidity by which pulmonary F. tularensis infections progress, there is little to no evidence of inflammation in the lung until the very end stage of infection (11, 20, 54). The mechanisms by which F. tularensis replicates within the lung while evading detection by the host are not well understood and represent an important hurdle for the development of novel therapeutics and vaccines directed against tularemia.Recently we have demonstrated that, similar to pulmonary cells, conventional human dendritic cells (hDC) derived from peripheral blood fail to secrete proinflammatory cytokines following infection with virulent F. tularensis strain SchuS4 (17). The absence of production of proinflammatory cytokines was not due to the inability of the cells to become infected or support replication of strain SchuS4, nor was it due to induction of apoptosis among infected hDC. There are multiple explanations for the lack of cytokine production in hDC and pulmonary cells following SchuS4 infection. One possibility is that these cells fail to detect F. tularensis during the initial phases of infection.CD14 is a glycosylphosphatidylinositol receptor that exists in both a membrane-bound form and a soluble form in vivo. CD14 is present on monocytes, most macrophages, fibroblasts, and neutrophils (39). Conventional human DC lose surface expression of CD14 following their differentiation from blood monocytes, primarily due to exposure to interleukin-4 (IL-4) (40). Similar to hDC, alveolar macrophages have been reported to have little to no CD14 present on their surface (9, 45). Soluble CD14 (sCD14) is abundant in serum. However, it is present at very low levels to nearly undetectable concentrations in the airways of mammals (47). CD14 (both in its soluble and membrane-bound forms) is best known as a coreceptor for lipopolysaccharide (LPS), facilitating optimal delivery of LPS to the Toll-like receptor 4 (TLR4)/MD-2 complex on the cell surface (35, 64). In addition to delivery of LPS to TLR4, CD14 has been described as an important coreceptor for delivery of other microbial antigens, including polyuronic acids from Pseudomonas, lipoteichoic acid (LTA) from Staphylococcus aureus, outer surface protein of Borrelia burgdorferi and WI-1 antigen of Blastomyces dermatitidis to TLR2 (reviewed in reference 58). Thus, CD14 is an important coreceptor for initiating inflammatory responses via TLR2 and TLR4 against a wide variety of bacterial and fungal diseases.In this report, we demonstrate that CD14 serves as a critical coreceptor for detection of F. tularensis SchuS4 during the initial stage of in vitro infection in hDC and primary human monocytes. Further, CD14 was also found to have an important role in the early in vivo detection and control of pulmonary infections with strain SchuS4. However, development of inflammatory responses associated with bacterial sepsis observed at late stages of SchuS4 infection were not dependent on the presence of CD14. Thus, in contrast to infections with other microbial pathogens where the presence of CD14 in the lungs is detrimental to control of infection, CD14 represents an important sensor to initiate protective host immune responses during pulmonary infections with strain SchuS4, but it is not required to elicit responses at the end of the disease process.     

Activities of Murine Peripheral Blood Lymphocytes Provide Immune Correlates That Predict Francisella tularensis Vaccine Efficacy

We previously identified potential correlates of vaccine-induced protection against Francisella tularensis using murine splenocytes and further demonstrated that the relative levels of gene expression varied significantly between tissues. In contrast to splenocytes, peripheral blood leukocytes (PBLs) represent a means to bridge vaccine efficacy in animal models to that in humans. Here we take advantage of this easily accessible source of immune cells to investigate cell-mediated immune responses against tularemia, whose sporadic incidence makes clinical trials of vaccines difficult. Using PBLs from mice vaccinated with F. tularensis Live Vaccine Strain (LVS) and related attenuated strains, we combined the control of in vitro Francisella replication within macrophages with gene expression analyses. The in vitro functions of PBLs, particularly the control of intramacrophage LVS replication, reflected the hierarchy of in vivo protection conferred by LVS-derived vaccines. Moreover, several genes previously identified by the evaluation of splenocytes were also found to be differentially expressed in immune PBLs. In addition, more extensive screening identified additional potential correlates of protection. Finally, expression of selected genes in mouse PBLs obtained shortly after vaccination, without ex vivo restimulation, was different among vaccine groups, suggesting a potential tool to monitor efficacious vaccine-induced immune responses against F. tularensis. Our studies demonstrate that murine PBLs can be used productively to identify potential correlates of protection against F. tularensis and to expand and refine a comprehensive set of protective correlates.     

A Francisella tularensis Live Vaccine Strain (LVS) Mutant with a Deletion in capB,Encoding a Putative Capsular Biosynthesis Protein,Is Significantly More Attenuated than LVS yet Induces Potent Protective Immunity in Mice against F. tularensis Challenge

Francisella tularensis, the causative agent of tularemia, is in the top category (category A) of potential agents of bioterrorism. The F. tularensis live vaccine strain (LVS) is the only vaccine currently available to protect against tularemia; however, this unlicensed vaccine is relatively toxic and provides incomplete protection against aerosolized F. tularensis, the most dangerous mode of transmission. Hence, a safer and more potent vaccine is needed. As a first step toward addressing this need, we have constructed and characterized an attenuated version of LVS, LVS ΔcapB, both as a safer vaccine and as a vector for the expression of recombinant F. tularensis proteins. LVS ΔcapB, with a targeted deletion in a putative capsule synthesis gene (capB), is antibiotic resistance marker free. LVS ΔcapB retains the immunoprotective O antigen, is serum resistant, and is outgrown by parental LVS in human macrophage-like THP-1 cells in a competition assay. LVS ΔcapB is significantly attenuated in mice; the 50% lethal dose (LD₅₀) intranasally (i.n.) is >10,000-fold that of LVS. Providing CapB in trans to LVS ΔcapB partially restores its virulence in mice. Mice immunized with LVS ΔcapB i.n. or intradermally (i.d.) developed humoral and cellular immune responses comparable to those of mice immunized with LVS, and when challenged 4 or 8 weeks later with a lethal dose of LVS i.n., they were 100% protected from illness and death and had significantly lower levels (3 to 5 logs) of LVS in the lung, liver, and spleen than sham-immunized mice. Most importantly, mice immunized with LVS ΔcapB i.n. or i.d. and then challenged 6 weeks later by aerosol with 10× the LD₅₀ of the highly virulent type A F. tularensis strain SchuS4 were significantly protected (100% survival after i.n. immunization). These results show that LVS ΔcapB is significantly safer than LVS and yet provides potent protective immunity against virulent F. tularensis SchuS4 challenge.Francisella tularensis is a Gram-negative coccobacillus that causes tularemia, a zoonotic disease spread among small animals such as rabbits and rodents by blood-sucking insects. Humans typically acquire tularemia by handling infected animals or from the bite of infected insects. There are four subspecies of F. tularensis: F. tularensis subsp. tularensis, holarctica, mediasiatica, and novicida (41); of these, F. tularensis subsp. tularensis, found in North America and also known as type A, causes the most severe disease. Following cutaneous exposure, tularemia typically presents as an ulceronodular disease with painful, ulcerated skin lesions and swollen lymph nodes. Following inhalation exposure, tularemia presents with acute flu-like symptoms followed by pleuropneumonic and typhoidal illness. The pneumonic form of tularemia has a high fatality rate (11).Because of its high pathogenicity in humans, especially after respiratory exposure, its low infectious dose, and the relative ease with which it can be cultured and disseminated, F. tularensis is classified as a category A agent of bioterrorism, i.e., among bioterrorist agents thought to pose the greatest risk to the public. Indeed, F. tularensis was previously developed as a bioweapon and stockpiled by Japan during World War II (16) and by the United States and the Soviet Union during the Cold War (1, 6). Although tularemia can be treated with available antibiotics, F. tularensis can be genetically engineered to be antibiotic resistant (30). Moreover, pneumonic tularemia frequently requires hospitalization and intensive care, and even when an infected individual is treated with antibiotics to which the organism is sensitive, the disease may resolve slowly (12); even a moderately sized outbreak could rapidly overwhelm medical facilities (11). Hence, relying on antibiotics to protect against a bioterrorist attack with F. tularensis is not a practical public health approach. A safe and potent vaccine, on the other hand, would appear to offer a much more reliable approach.An unlicensed vaccine known as the live vaccine strain (LVS), an attenuated mutant of F. tularensis subsp. holarctica, was developed in the mid-1900s and is the only vaccine currently available in the United States. The underlying mechanism of attenuation is not fully characterized genetically, although recently, the reintroduction of deleted genes pilA and FTT0918 was shown to restore virulence to the level of virulent type B strains (35). The LVS vaccine has several drawbacks. The vaccine, which retains considerable virulence in animals, shows significant toxicity in humans after both intradermal (i.d.) and aerosol administration (19, 37). Moreover, it provides incomplete protection to humans challenged with type A F. tularensis by aerosol, the route of transmission of greatest concern in a bioterrorist attack (19, 29, 37).In a search for a vaccine that is safer and more potent than LVS, we sought to rationally attenuate LVS and to use the attenuated LVS as both a vaccine and a vector to overexpress immunogenic F. tularensis proteins. We hypothesized that we would render LVS safer by further attenuating it and that we would render it more potent by overexpressing key immunoprotective antigens. This overall strategy mirrors that used successfully to develop the first vaccine against tuberculosis that is more potent than the current Mycobacterium bovis BCG vaccine, rBCG30, a recombinant BCG vaccine overexpressing the Mycobacterium tuberculosis 30-kDa major secretory protein, and to develop the first vaccine both safer and more potent than BCG, rBCG(mbtB)30, an attenuated version of rBCG30 that is engineered and propagated such that it can multiply only a few times in the host (20, 21, 45).In attenuating LVS, we sought a mutation that would greatly reduce virulence but have a minimal impact on immunogenicity and protective efficacy. Transposon mutagenesis studies of F. tularensis subsp. novicida and holarctica (LVS) have shown that mutants with transposon insertions in genes (FTT0806, FTT0805, and FTT0798) encoding proteins putatively involved in capsular biosynthesis, on the basis of partial amino acid sequence homology with capsular biosynthesis proteins of Bacillus anthracis, are highly attenuated (∼100- to 1,000-fold) in mice (43, 47). Consequently, we decided to evaluate the vaccine potential of an LVS mutant with a deletion in one of these genes.In this study, we describe the construction of an antibiotic resistance marker-free FTL_1416/FTT0805 (capB) deletion mutant of F. tularensis LVS (LVS ΔcapB) and show that LVS ΔcapB is resistant to serum killing, outgrown by its parental LVS in human macrophage-like THP-1 cells, and highly attenuated in mice. We demonstrate further that this vaccine, after both i.d. and intranasal (i.n.) administration, induces potent cellular and humoral immune responses and significant protective immunity against respiratory challenge with virulent F. tularensis.     

Humoral and cell-mediated immunity to the intracellular pathogen Francisella tularensis

Summary: Francisella tularensis can cause fatal respiratory tularemia in humans and animals and is increasingly being isolated in the United States and several European countries. The correlates of protective immunity against this intracellular bacterium are not known, and currently there are no licensed vaccines available for human use. Cell-mediated immunity has long been believed to be critical for protection, and the importance of humoral immunity is also now recognized. Furthermore, synergy between antibodies, T cell-derived cytokines, and phagocytes appears to be critical to achieve sterilizing immunity against F. tularensis. Thus, novel vaccine approaches should be designed to induce robust antibody and cell-mediated immune responses to this pathogen.     

Features of sepsis caused by pulmonary infection with Francisella tularensis Type A strain

The virulence mechanisms of Francisella tularensis, the causative agent of severe pneumonia in humans and a CDC category A bioterrorism agent, are not fully defined. As sepsis is the leading cause of mortality associated with respiratory infections, we determined whether, in the absence of any known bacterial toxins, a deregulated host response resulting in sepsis syndrome is associated with lethality of respiratory infection with the virulent human Type A strain SchuS4 of F.?tularensis. The C57BL/6 mice infected intranasally with a lethal dose of SchuS4 exhibited high bacterial burden in systemic organs and blood indicative of bacteremia. In correlation, infected mice displayed severe tissue pathology and associated cell death in lungs, liver and spleen. Consistent with our studies with murine model strain Francisella novicida, infection with SchuS4 caused an initial delay in upregulation of inflammatory mediators followed by development of severe sepsis characterized by exaggerated cytokine release, upregulation of cardiovascular injury markers and sepsis mediator alarmins S100A9 and HMGB1. This study shows that pulmonary tularemia caused by the Type A strain of F.?tularensis results in a deregulated host response leading to severe sepsis and likely represents the major cause of mortality associated with this virulent pathogen.     

Toll-Like Receptor 3 Agonist Protection against Experimental Francisella tularensis Respiratory Tract Infection

We investigated whether Toll-like receptor 3 (TLR3) stimulation would protect the host from inhaled Francisella tularensis. TLR3 is expressed by respiratory epithelial cells and macrophages and can be activated by a synthetic double-stranded RNA ligand called polyinosine-polycytosine [poly(I:C)]. Thus, we evaluated poly(I:C) as a novel treatment against inhaled F. tularensis. In vivo, BALB/c mice intranasally (i.n.) treated with poly(I:C) (100 μg/mouse) 1 h before or after Schu 4 or LVS (100 CFU) i.n. challenge showed that poly(I:C) treatment significantly reduced bacterial load in the lungs (P < 0.05). Bronchoalveolar lavage from poly(I:C)-treated mice alone or combined with F. tularensis infection significantly increased cytokine secretion and enhanced neutrophil influx to lung tissues. Poly(I:C) responses were transient but significantly prolonged the survival of treated mice after i.n. F. tularensis challenge relative to mock treated animals. This prolonged survival providing a longer window for initiation of levofloxacin (LEVO) treatment (40 mg/kg). Animals treated with poly(I:C), challenged with F. tularensis, and then treated with LEVO 5 days later had 100% survival relative to 0% survival in animals receiving LEVO alone. Mechanistically, poly(I:C) given to human monocyte-derived macrophages before or after Schu 4 or LVS challenge (multiplicity of infection, 20:1) had significantly reduced intracellular bacterial replication (P < 0.05). These data suggest that poly(I:C) may represent a potential therapeutic agent against inhaled F. tularensis that prolongs survival and the opportunity to initiate standard antibiotic therapy (i.e., LEVO).Inhalation is likely to be one of the primary routes by which a bioweapon will be delivered to a target population. Francisella tularensis is a potential bioweapon because it can be aerosolized due to its inherently hardy nature, and less than 20 inhaled organisms can be detrimental to the host (8, 10, 28). F. tularensis, the etiologic agent of tularemia, is a small, Gram-negative nonmotile coccus and a facultative intracellular bacterium (16, 36). There are two major subspecies of F. tularensis; one is designated type A and includes strains that induce aggressive pathologies in the host and can result in pulmonary tularemia causing death if not treated (18, 37). The commonly studied virulent type A strain, Schu 4, was isolated originally from a human case of tularemia (22, 31, 38). The type B subspecies of F. tularensis causes a milder disease in humans than do type A strains. The only vaccine against tularemia known at this time was derived from subspecies type B and is called the live vaccine strain (LVS) (3, 8, 19, 31). Interestingly, most animal modeling of F. tularensis infection has used LVS-infected mice because it mimics the human disease caused by type A strains (6, 7, 9, 12). The immunological efficacy of LVS in humans is not known; vaccination with LVS does not provide complete protection against the virulent type A strains of F. tularensis (7, 19). As a result, alternative intervention strategies and vaccines need to be developed.Ideally, vaccines against bioweapons will be established to protect the general population limiting the impact of such terroristic acts. Until such vaccines are available and widely distributed, alternate methods of broad range protection must be investigated. One interesting strategy is to engender innate immune resistance against mucosal pathogens (13, 17, 24). We have investigated the potential of Toll-like receptor (TLR) agonists recognized by TLRs highly expressed by respiratory epithelial cells. Specifically, polyinosine-polycytosine [poly(I:C)] is a synthetic double-stranded RNA analog that stimulates TLR3 triggering the induction of the host innate immune response including as RANTES, gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-6 (11, 17, 23, 26).Poly(I:C) can be delivered easily by a nose spray, is cheap to manufacture, and could be offered as an over-the-counter product unlike antibiotics. Importantly, the kinetics of cytokine secretion after poly(I:C) administration showed a transient response and offered no indication of toxicity, even with repeated use in our previous study (17). We therefore examined poly(I:C) as a topical treatment for a potential F. tularensis aerosol release. Theoretically, intranasal (i.n.) poly(I:C) could engender an innate immune response against F. tularensis, providing an extended period of resistance before an antibiotic, such as levofloxacin (LEVO), can be administered. LEVO belongs to the group of antibiotics known as fluoroquinolones and has been used to treat respiratory infections such as tularemia (1, 21, 25).Recently, we established that genital application of poly(I:C) protected against lethal HSV-2 challenge in mice (17). We have extended these findings by applying poly(I:C) to the respiratory mucosa testing the hypothesis that nucleic acid-based TLR agonists may prove to be useful prophylactic and possibly therapeutic measures against select agent respiratory infections including F. tularensis. Because F. tularensis suppresses the innate immune response (2, 4, 29, 39), the host does not detect and/or respond to the organism for approximately 48 to 72 h after F. tularensis infection (2; T. D. Eaves-Pyles, unpublished data). This large gap between the time of infection and host detection of the organism limits the development of an adequate immune response against F. tularensis. As such, we hypothesized that poly(I:C) would enhance the host''s response prior to or soon after F. tularensis exposure. Our in vivo and in vitro studies show that mice treated 1 h before or 1 h after the administration of poly(I:C) had significantly less bacteria in their lungs, increased neutrophil infiltration to the lung, and extended survival after LVS or Schu 4 infection. Moreover, mice treated with poly(I:C) (1 h after Schu 4 infection), followed by LEVO administration 5 days later, were fully protected from lethal outcomes. Corresponding to these in vivo studies, we show that poly(I:C)-treated human monocyte-derived macrophages (MDM) secreted high levels of specific cytokines and engendered enhanced intracellular bacterial killing after LVS and Schu 4 exposure compared to untreated animals.     

Infection with Francisella tularensis LVS clpB Leads to an Altered yet Protective Immune Response

Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo. We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-γ) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB, can serve as a model for the rational design of attenuated vaccines.     

Differing Effects of Interleukin-10 on Cutaneous and Pulmonary Francisella tularensis Live Vaccine Strain Infection

We investigated the role of interleukin-10 (IL-10) in cutaneous and pulmonary infection with Francisella tularensis. We found that after intradermal challenge of mice with the live vaccine strain (LVS) of F. tularensis, splenic IL-10 levels increased rapidly and reached a peak 5 days after infection. However, IL-10 expression after infection was detrimental, since IL-10^−/− mice showed increased bacterial clearance and were resistant to an infectious dose (>10⁶ CFU/mouse) that was uniformly lethal for IL-10^+/+ mice. Furthermore, IL-10^+/+ mice treated with neutralizing anti-IL-10R monoclonal antibody were able to survive lethal cutaneous LVS challenge. The presence of IL-10 appeared to restrain the expression of IL-17, since high levels of splenic IL-17 were observed after intradermal LVS infection only in IL-10^−/− mice. Furthermore, treatment with neutralizing anti-IL-17R antibody ablated the enhanced survival observed in IL-10^−/− mice. However, neutralization of IL-10 activity in IL-17R^−/− mice failed to provide protection. Thus, IL-10 suppresses a protective IL-17 response that is necessary for resistance to cutaneous LVS infection. Surprisingly, however, IL-10^−/− mice were significantly more susceptible to pulmonary infection with LVS. Finally, although IL-10 is a critical and novel regulator of immunity to F. tularensis LVS infection, its effects were masked during infection with the highly virulent SchuS4 strain. Taken together, these findings suggest that differentially regulating expression of the IL-10 pathway in various tissues could ultimately have prophylactic and therapeutic benefits for protection against tularemia.     

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study

Background

Burkholderia pseudomalleiis the causative agent for melioidosis. For many bacterial infections, cytokine dysregulation is one of the contributing factors to the severe clinical outcomes in the susceptible hosts. The C57BL/6 and BALB/c mice have been established as a differential model of susceptibility in murine melioidosis. In this study, we compared the innate IFN-γ response toB. pseudomalleibetween the C57BL/6 and BALB/c splenocytes and characterized the hyperproduction of IFN-γ in the relatively susceptible BALB/c micein vitro.

Results

Naïve BALB/c splenocytes were found to produce more IFN-γ in response to live bacterial infection compared to C57BL/6 splenocytes. Natural killer cells were found to be the major producers of IFN-γ, while T cells and Gr-1^intermediatecells also contributed to the IFN-γ response. Although anti-Gr-1 depletion substantially reduced the IFN-γ response, this was not due to the contribution of Gr-1^high, Ly-6G expressing neutrophils. We found no differences in the cell types making IFN-γ between BALB/c and C57BL/6 splenocytes. Although IL-12 is essential for the IFN-γ response, BALB/c and C57BL/6 splenocytes made similar amounts of IL-12 after infection. However, BALB/c splenocytes produced higher proinflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-18 than C57BL/6 splenocytes after infection withB. pseudomallei.

Conclusion

Higher percentages of Gr-1 expressing NK and T cells, poorer ability in controlling bacteria growth, and higher IL-18 could be the factors contributing to IFN-γ hyperproduction in BALB/c mice.     

TolC-Dependent Modulation of Host Cell Death by the Francisella tularensis Live Vaccine Strain

Francisella tularensis is a facultative intracellular, Gram-negative pathogen and the causative agent of tularemia. We previously identified TolC as a virulence factor of the F. tularensis live vaccine strain (LVS) and demonstrated that a ΔtolC mutant exhibits increased cytotoxicity toward host cells and elicits increased proinflammatory responses compared to those of the wild-type (WT) strain. TolC is the outer membrane channel component used by the type I secretion pathway to export toxins and other bacterial virulence factors. Here, we show that the LVS delays activation of the intrinsic apoptotic pathway in a TolC-dependent manner, both during infection of primary macrophages and during organ colonization in mice. The TolC-dependent delay in host cell death is required for F. tularensis to preserve its intracellular replicative niche. We demonstrate that TolC-mediated inhibition of apoptosis is an active process and not due to defects in the structural integrity of the ΔtolC mutant. These findings support a model wherein the immunomodulatory capacity of F. tularensis relies, at least in part, on TolC-secreted effectors. Finally, mice vaccinated with the ΔtolC LVS are protected from lethal challenge and clear challenge doses faster than WT-vaccinated mice, demonstrating that the altered host responses to primary infection with the ΔtolC mutant led to altered adaptive immune responses. Taken together, our data demonstrate that TolC is required for temporal modulation of host cell death during infection by F. tularensis and highlight how shifts in the magnitude and timing of host innate immune responses may lead to dramatic changes in the outcome of infection.     

Live Attenuated Mutants of Francisella tularensis Protect Rabbits against Aerosol Challenge with a Virulent Type A Strain

Francisella tularensis, a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 ΔguaBA, ΔaroD, and ΔfipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD₅₀). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The ΔguaBA and ΔaroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the ΔfipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The ΔguaBA and ΔaroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection.     

Francisella tularensis T-Cell Antigen Identification Using Humanized HLA-DR4 Transgenic Mice

There is no licensed vaccine against the intracellular pathogen Francisella tularensis. The use of conventional mouse strains to screen protective vaccine antigens may be problematic, given the differences in the major histocompatibility complex (MHC) binding properties between murine and human antigen-presenting cells. We used engineered humanized mice that lack endogenous MHC class II alleles but that express a human HLA allele (HLA-DR4 transgenic [tg] mice) to identify potential subunit vaccine candidates. Specifically, we applied a biochemical and immunological screening approach with bioinformatics to select putative F. tularensis subsp. novicida T-cell-reactive antigens using humanized HLA-DR4 tg mice. Cell wall- and membrane-associated proteins were extracted with Triton X-114 detergent and were separated by fractionation with a Rotofor apparatus and whole-gel elution. A series of proteins were identified from fractions that stimulated antigen-specific gamma interferon (IFN-γ) production, and these were further downselected by the use of bioinformatics and HLA-DR4 binding algorithms. We further examined the validity of this combinatorial approach with one of the identified proteins, a 19-kDa Francisella tularensis outer membrane protein (designated Francisella outer membrane protein B [FopB]; FTN_0119). FopB was shown to be a T-cell antigen by a specific IFN-γ recall assay with purified CD4⁺ T cells from F. tularensis subsp. novicida ΔiglC-primed HLA-DR4 tg mice and cells of a human B-cell line expressing HLA-DR4 (DRB1*0401) functioning as antigen-presenting cells. Intranasal immunization of HLA-DR4 tg mice with the single antigen FopB conferred significant protection against lethal pulmonary challenge with an F. tularensis subsp. holarctica live vaccine strain. These results demonstrate the value of combining functional biochemical and immunological screening with humanized HLA-DR4 tg mice to map HLA-DR4-restricted Francisella CD4⁺ T-cell epitopes.Francisella tularensis is a Gram-negative bacterium and the etiological agent of the zoonotic disease tularemia. F. tularensis is classified into four subspecies, namely, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica, and F. tularensis subsp. novicida (F. novicida) on the basis of their biochemical and genetic profiles, virulence properties, and geographical origins (51). To this end, F. tularensis subsp. tularensis (type A) is the most virulent subspecies, with the inhalation of as few as 10 organisms causing disease and mortality rates of between 30 and 60% in untreated cases of pneumonic tularemia (53). The live vaccine strain (LVS) derived from F. tularensis subsp. holarctica has been used as a prophylactic vaccine against tularemia (48). Millions of individuals in the Soviet Union were immunized with live vaccine strains between 1946 and 1960 (52). However, LVS has not been licensed for use in the United States due to a lack of understanding of the genetic mutations that are responsible for attenuation of this strain, although it is used as an investigational new drug (IND) to immunize at-risk workers, primarily tularemia researchers. F. novicida, which causes disease only in immunocompromised humans but which is highly virulent for mice, has been used as a comparative model organism due to the high degree of genetic similarity with type A strains (98.1% homology between sequences common to strains U112 and SCHU S4 [45]). We recently reported that a defined vaccine strain (ΔiglB) generated in strain U112 was effective in inducing heterologous protection against various Francisella strains in a mouse model of pulmonary tularemia, suggesting the conservation of protective antigens (12).Cell-mediated immunity has been documented to play an important role in protection against tularemia (2, 18, 19, 49, 56). The role of antibodies, via neutralization and Fc receptor-mediated clearance (43, 44) in response to infection, has also gained significant attention. Therefore, the availability of a combination of multiple Francisella antigens containing T-cell and/or B-cell epitopes would be desirable for formulating an effective multivalent vaccine against this organism. However, the use of conventional mouse strains to identify protective antigens may not be feasible, given the differences in the major histocompatibility complex (MHC) binding properties between murine and human MHCs. These constraints can be overcome with the use of engineered humanized mice, such as the HLA-DR4 transgenic (tg) mouse. This mouse was generated to express the extracellular human α1 and β1 domains of the HLA-DRA and HLA-DRB1*0401 haplotypes, which form the peptide binding sites for antigen presentation, in conjunction with the murine α2 and β2 domains (29). These chimeric molecules have been shown to exhibit the same antigen-binding specificity as HLA-DRB1*0401 and to be functional in presenting antigens to T cells (29). The frequency of the HLA-DR allele in humans is 29% in Caucasian individuals, 10% in African American individuals, and 34% in other individuals (38), underscoring the translational value of the epitopes identified in these mice for humans. We recently demonstrated the feasibility of using the HLA-DR4 tg mouse for the identification of vaccine antigens against genital Chlamydia infection (39), demonstrating the value of the use of these animals in the rational selection of vaccine candidates.In this study, we utilized a robust biochemical membrane protein fractionation method, cytokine recall assays, and humanized HLA-DR4 tg mice to identify putative CD4⁺ T-cell-reactive antigens from U112. Moreover, using bioinformatics tools, we further validated one of the identified antigens (FTN_0119), designated Francisella outer membrane protein B (FopB), as a potential subunit vaccine candidate against pneumonic tularemia in HLA-DR4 tg mice.     

The ability of CpG oligonucleotides to protect mice against Francisella tularensis live vaccine strain but not fully virulent F. tularensis subspecies holarctica is reflected in cell-based assays

CpG DNA is a potent activator of the innate immune system. Here the protective effects of CpG DNA are assessed against the facultative intracellular pathogen Francisella tularensis. Dosing of mice with CpG DNA provided protection against disease caused by F. tularensis subsp. holarctica live vaccine strain (LVS) but did not protect against the fully virulent F. tularensis subsp holarctica strain HN63. Similarly, in vitro studies in J774A murine macrophage-like cells demonstrated that stimulation with CpG DNA enables control of intracellular replication of LVS but not HN63. These data confirm findings that CpG DNA may have limited efficacy in providing protection against fully virulent strains of F. tularensis and also suggest that in vitro assays may be useful for the evaluation of novel treatments for virulent F. tularensis.     

Protection afforded against aerosol challenge by systemic immunisation with inactivated Francisella tularensis live vaccine strain (LVS)

BALB/c mice were immunised with inactivated Francisella tularensis live vaccine strain (LVS) and the level of protection afforded against aerosol challenge with virulent strains of F. tularensis ascertained. Intramuscular (IM) injection of inactivated LVS with an aluminium-hydroxide-based adjuvant-stimulated IgG1-biased LVS-specific antibody responses and afforded no protection against aerosol challenge with subspecies holarctica (strain HN63). Conversely, IM injection of inactivated LVS adjuvanted with preformed immune-stimulating complexes (ISCOMS) admixed with immunostimulatory CpG oligonucleotides afforded robust protection against aerosol-initiated infection with HN63. However, despite a significantly extended time-to-death relative to naïve controls, the majority of mice immunised with the most potent vaccine formulation were not protected against a low-dose aerosol challenge with subspecies tularensis (strain Schu S4). These data indicate that parenterally administered non-living vaccines can be used for effective immunisation against aerosol challenges with subspecies holarctica, although not high virulence strains of F. tularensis.     

RipA, a cytoplasmic membrane protein conserved among Francisella species, is required for intracellular survival

Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS ΔripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS ΔripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS ΔripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.     

Disruption of Francisella tularensis Schu S4 iglI,iglJ, and pdpC Genes Results in Attenuation for Growth in Human Macrophages and In Vivo Virulence in Mice and Reveals a Unique Phenotype for pdpC

Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI, iglJ, or pdpC, three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10⁸ CFU iglJ- or pdpC-deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype.     

Vaccination with an Attenuated Strain of Francisella novicida Prevents T-Cell Depletion and Protects Mice Infected with the Wild-Type Strain from Severe Sepsis

Francisella tularensis is the causative agent of zoonotic tularemia, a severe pneumonia in humans, and Francisella novicida causes a similarly severe tularemia in mice upon inhalation. The correlates of protective immunity, as well as the virulence mechanisms of this deadly pathogen, are not well understood. In the present study, we compared the host immune responses of lethally infected and vaccinated mice to highlight the host determinants of protection from this disease. Intranasal infection with an attenuated mutant (Mut) of F. novicida lacking a 58-kDa hypothetical protein protected C57BL/6 mice from a subsequent challenge with the fully virulent wild-type strain U112 via the same route. The protection conferred by Mut vaccination was associated with reduced bacterial burdens in systemic organs, as well as the absence of bacteremia. Also, there was reduced lung pathology and associated cell death in the lungs of vaccinated mice. Both vaccinated and nonvaccinated mice displayed an initial 2-day delay in upregulation of signature inflammatory mediators after challenge. Whereas the nonvaccinated mice developed severe sepsis characterized by hypercytokinemia and T-cell depletion, the vaccinated mice displayed moderated cytokine induction and contained increased numbers of αβ T cells. The recall response in vaccinated mice consisted of a characteristic Th1-type response in terms of cytokines, as well as antibody isotypes. Our results show that a regulated Th1 type of cell-mediated and humoral immunity in the absence of severe sepsis is associated with protection from respiratory tularemia, whereas a deregulated host response leading to severe sepsis contributes to mortality.The causative agent of respiratory tularemia, Francisella tularensis, is a gram-negative intracellular bacterium. There are four closely related subspecies of F. tularensis, F. tularensis subsp. tularensis (type A), F. tularensis subsp. holarctica (type B), F. tularensis subsp. mediasiatica, and “F. tularensis subsp. novicida,” and type A is the most virulent subspecies in humans (20). This pathogen is capable of causing acute respiratory infection following inhalation of as few as 10 organisms (10, 48). This extremely low infectious dose, the ease of transmission via the aerosol route, and the wide host range have led the CDC to recognize this pathogen as a potential bioweapon (56). Since the fully virulent strains of F. tularensis are highly infectious, much of our knowledge about Francisella pathogenesis has been obtained by using the attenuated live vaccine strain (LVS) derived from a type B strain of F. tularensis or Francisella novicida. Although attenuated for humans, F. novicida is virulent in mice and results in a disease that closely resembles human tularemia. Despite continuous efforts, an effective vaccine for tularemia has not been developed yet. This highlights the need for understanding the virulence mechanisms of Francisella, as well as the correlates of protective immunity, in order to devise effective therapeutics for use against tularemia.Primary respiratory infections with Francisella cause a delay in the initial innate immune response. This initial delay has been postulated to be an important virulence mechanism of the organism (2, 3, 39, 40). An absence of this initial immune response is thought to aid rapid multiplication of bacteria, followed by dissemination of the bacteria to systemic organs, resulting in bacteremia. This causes widespread upregulation of multiple cytokines and chemokines that reflects contributions from both the host and the pathogen to an inappropriate inflammatory response (40, 59, 64). This kind of unbridled host response to a pathogen is now broadly accepted as the cause of host death in infectious diseases like malaria, influenza, and sepsis (6). In light of the absence of any known endo- or exotoxin activity of any virulence factor of Francisella, this hyperimmune response seems to be the cause of the mortality associated with respiratory tularemia (54).Adaptive immune responses following vaccination, as well as during sublethal infections, have highlighted the contributions of both B and T lymphocytes (8, 16, 44, 53). Most of the studies have been carried out with type B-infected humans, as well as mice (65). Both humans and mice develop antigen-specific antibodies, as well as CD4⁺ and CD8⁺ T cells, during sublethal infections (15, 17, 26, 57). The effector T-cell mechanisms that control the infections involve mainly gamma interferon (IFN-γ) and/or tumor necrosis factor alpha (TNF-α) (9, 66), but bacterial killing is partially mediated by NO produced by IFN-γ-activated macrophages (4, 14). However, a comprehensive study of the mechanisms triggering rapid death following systemic dissemination of bacteria before the onset of acquired immunity and the factors involved in bacterial clearance and host protection from lethal respiratory infection in the same experimental setting has not been done.Analysis of the genome sequence of Francisella revealed a family of five hypothetical proteins unique to this organism (38). One of these factors, a protein encoded by the FTT_0918 gene, has been shown to be a virulence factor, as mutants of type A strains lacking this gene are attenuated for infection in vitro and in vivo. In addition, intradermal inoculation with this mutant protects mice from intranasal challenge with virulent type A strains (63, 65). Our in vivo studies with the murine model organism F. novicida have shown that a transposon mutant (Mut) lacking a homolog of this 58-kDa protein is equally attenuated (54). In the current study we tested this mutant to determine whether it protects against murine respiratory tularemia and determined the host immune responses associated with protection. Intranasal immunization of C57BL/6 mice with Mut protected the mice from a subsequent challenge with an otherwise lethal dose of the wild-type (WT) bacteria. Importantly, the severe sepsis characterized by hypercytokinemia and bacteremia observed in nonvaccinated mice was not present in lungs of mice vaccinated with the mutant. Instead, a protective Th1 type of cytokine and antibody response was upregulated. Our results show that in the apparent absence of any endotoxins or exotoxins that could account for the lethality associated with respiratory tularensis, severe sepsis coupled with a lack of adaptive responses due to T-cell depletion is likely the major contributor to the severity of the disease and associated mortality, and an effective Th1 type of response coupled with the absence of severe sepsis and bacteremia is key for protection against this deadly infection.