Francisella tularensis (Ft), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Although attenuated for humans, i.p. infection of mice with <10 Ft live vaccine strain (LVS) organisms causes lethal infection that resembles human tularemia, whereas the LD50 for an intradermal infection is >10(6) organisms. To examine the immunological consequences of Ft LVS infection on the innate immune response, the inflammatory responses of mice infected i.p. or intradermally were compared. Mice infected i.p. displayed greater bacterial burden and increased expression of proinflammatory genes, particularly in the liver. In contrast to most LPS, highly purified Ft LVS LPS (10 microg/ml) was found to be only minimally stimulatory in primary murine macrophages and in HEK293T cells transiently transfected with TLR4/MD-2/CD14, whereas live Ft LVS bacteria were highly stimulatory for macrophages and TLR2-expressing HEK293T cells. Despite the poor stimulatory activity of Ft LVS LPS in vitro, administration of 100 ng of Ft LVS LPS 2 days before Ft LVS challenge severely limited both bacterial burden and cytokine mRNA and protein expression in the absence of detectable Ab at the time of bacterial challenge, yet these mice developed a robust IgM Ab response within 2 days of infection and survived. These data suggest that prior administration of Ft LVS LPS protects the host by diminishing bacterial burden and blunting an otherwise overwhelming inflammatory response, while priming the adaptive immune response for development of a strong Ab response.

Periodontal disease (PD) results from the immune response to the bacterial components of the sub-gingival plaque. PD progresses from gingival inflammation, to bone resorption and ultimately tooth loss. Receptor activator of nuclear factor-kappa B ligand (RANKL) is critical for regulating inflammatory bone resorption. Studies have shown that activated T and B cells are one of the main sources of RANKL in PD. The ELISPOT assay is widely used tool that can measure antigen specific cellular immune responses. We developed a RANKL specific ELISPOT assay for quantifying the number of RANKL secreting cells in the murine Porphyromonas gingivalis induced bone loss model. Various potential positive controls were tested for their ability to induce RANKL secretion including IL-7, ionomycin, LPS, and α-CD3 combined with α-CD28. We found that the combination of α-CD3 and α-CD28 was the most effective positive control for cells collected from either murine lymph nodes or spleens. Gingipains are trypsin-like cysteine proteases and have been implicated as important virulence factors for Porphyromonas gingivalis. When we performed the RANKL ELISPOT using the purified native gingipain complex as the recall antigen, we were able to detect more RANKL secreting cells from the mandibular lymph nodes of Porphyromonas gingivalis challenged animals than in the buffer challenged control animals.

Francisella tularensis (Ft), the etiological agent of tularemia and a Tier 1 select agent, has been previously weaponized and remains a high priority for vaccine development. Ft tularensis (type A) and Ft holarctica (type B) cause most human disease. We selected six attenuating genes from the live vaccine strain (LVS; type B), F. novicida and other intracellular bacteria: FTT0507, FTT0584, FTT0742, FTT1019c (guaA), FTT1043 (mip) and FTT1317c (guaB) and created unmarked deletion mutants of each in the highly human virulent Ft strain Schu S4 (Type A) background. FTT0507, FTT0584, FTT0742 and FTT1043 Schu S4 mutants were not attenuated for virulence in vitro or in vivo. In contrast, Schu S4 gua mutants were unable to replicate in murine macrophages and were attenuated in vivo, with an i.n. LD50 > 10(5) CFU in C57BL/6 mice. However, the gua mutants failed to protect mice against lethal challenge with WT Schu S4, despite demonstrating partial protection in rabbits in a previous study. These results contrast with the highly protective capacity of LVS gua mutants against a lethal LVS challenge in mice, and underscore differences between these strains and the animal models in which they are evaluated, and therefore have important implications for vaccine development.

Abstract Francisella tularensis (Ft) is a Gram-negative, immune-evasive coccobacillus that causes tularemia for which there is no FDA-approved vaccine. We have utilized a novel vaccine approach using synthetic nanoparticles made from catanionic surfactant vesicles (V), functionalized by incorporation of either Ft type B Live Vaccine Strain (Ft LVS) or Ft type A Schu S4 strain (Ft Schu S4) components (i.e., LVS-V and Schu S4-V, respectively). Immunization of C57BL/6 mice with bare V partially protected against Ft LVS, presumably through activation of an innate immune response, yet failed to protect against Ft Schu S4. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.) Ft LVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.) Ft Schu S4 challenge. LVS-V-immunization, but not immunization with V, elicited high levels of IgG against non-LPS epitopes and these antisera conferred passive protection against challenge with Ft LVS. Our recently published and ongoing studies aim to identify the protein targets of mouse antisera, study the mechanism of non-specific protection gained by immunization with this nanoparticle vaccine platform (adjuvant effect) in Ft LVS challenge, and enhance protection in Ft Schu S4 challenge. Our data extend the utility of functionalized catanionic surfactant vesicles as a vaccine platform for pathogens.

ABSTRACTFrancisella tularensisis a Gram-negative immune-evasive coccobacillus that causes tularemia in humans and animals. A safe and efficacious vaccine that is protective against multipleF. tularensisstrains 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 eitherF. tularensistype B live vaccine strain (F. tularensisLVS [LVS-V]) orF. tularensistype A Schu S4 strain (F. tularensisSchu S4 [Schu S4-V]) components. The immunization of C57BL/6 mice with “bare” vesicles, which did not expressF. tularensiscomponents, partially protected againstF. tularensisLVS, presumably through activation of the innate immune response, and yet it failed to protect against theF. tularensisSchu S4 strain. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.)F. tularensisLVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.)F. tularensisSchu 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 afterF. tularensisLVS challenge and significantly increased early cytokine production. Antisera from LVS-V-immunized mice conferred passive protection against challenge withF. tularensisLVS. Together, these data indicate that functionalized catanionic surfactant vesicles represent an important and novel tool for the development of a safe and effectiveF. tularensissubunit vaccine and may be applicable for use with other pathogens.

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation.

In the companion article by Yang and colleagues [Yang Y, et al. (2012) Proc Natl Acad Sci USA, 109, 10.1073/pnas.1121631109], we have shown that priming with glycolipid (FtL) from Francisella tularensis live-vaccine strain (i) induces FtL-specific B-1a to produce robust primary responses (IgM >>IgG); (ii) establishes persistent long-term production of serum IgM and IgG anti-FtL at natural antibody levels; and (iii) elicits FtL-specific B-1a memory cells that arise in spleen but rapidly migrate to the peritoneal cavity, where they persist indefinitely but divide only rarely. Here, we show that FtL rechallenge alone induces these PerC B-1a memory cells to divide extensively and to express a unique activation signature. However, FtL rechallenge in the context of a Toll-like receptor 4 agonist-stimulated inflammatory response readily induces these memory cells to migrate to spleen and initiate production of dominant IgM anti-FtL secondary responses. Thus, studies here reveal unique mechanisms that govern B-1a memory development and expression, and introduce B-1a memory as an active participant in immune defenses. In addition, at a practical level, these studies suggest previously unexplored vaccination strategies for pathogen-associated antigens that target the B-1a repertoire.

B-1a cells are primarily thought of as natural antibody-producing cells. However, we now show that appropriate antigenic stimulation induces IgM and IgG B-1a antibody responses and long-lived T-independent antigen-specific B-1a memory that differs markedly from canonical B-2 humoral immunity. Thus, we show here that in the absence of inflammation, priming with glycolipid (FtL) from Francisella tularensis live vaccine strain induces splenic FtL-specific B-1a to mount dominant IgM and activation-induced cytidine deaminase-dependent IgG anti-FtL responses that occur within 3-5 d of FtL priming and fade within 1 wk to natural antibody levels that persist indefinitely in the absence of secondary FtL immunization. Equally surprising, FtL priming elicits long-term FtL-specific B-1a memory cells (IgM>>IgG) that migrate rapidly to the peritoneal cavity and persist there indefinitely, ready to respond to appropriately administrated secondary antigenic stimulation. Unlike B-2 responses, primary FtL-specific B-1a responses and establishment of persistent FtL-specific B-1a memory occur readily in the absence of adjuvants, IL-7, T cells, or germinal center support. However, in another marked departure from the mechanisms controlling B-2 memory responses, rechallenge with FtL in an inflammatory context is required to induce B-1a secondary antibody responses. These findings introduce previously unexplored vaccination strategies for pathogens that target the B-1a repertoire.

Immunization with Ft-LPS provokes an antigen-specific, B-1a cell-derived antibody response that protects WT mice against an otherwise lethal challenge with Ft LVS. However, this same regimen offers limited protection to TLR2(-/-) mice, despite production of WT levels of anti-Ft-LPS antibodies. As Ft-LPS exhibits no TLR2 agonist activity, and macrophage-induced cytokine production in response to Ft LVS is overwhelmingly TLR2-dependent, we hypothesized that treatment of TLR2(-/-) mice with an alternative, MyD88-dependent TLR agonist would compensate for reduced recognition of Ft LVS in TLR2(-/-) mice and thereby, restore Ft-LPS-mediated protection. Administration of the nontoxic TLR4 agonist, synthetic Escherichia coli MPL, at the time of Ft-LPS immunization or Ft LVS challenge, fully protected TLR2(-/-) mice, whereas treatment of WT or TLR2(-/-) mice with MPL alone conferred partial protection. The TLR5 agonist, flagellin, also synergized with Ft-LPS to protect TLR2(-/-) mice from lethal Ft LVS challenge. In contrast to Ft LVS, Ft-LPS pretreatment failed to protect mice against i.n. challenge with Ft Schu S4, whereas MPL, administered in the absence or presence of Ft-LPS, conferred significant, albeit partial, protection. MPL treatment of macrophages increased the uptake of Ft LVS and decreased intracellular bacterial survival while shifting the macrophage-differentiation phenotype from "alternatively activated" to "classically activated". Collectively, our data suggest that optimal, Ft-LPS-mediated protection against Ft LVS infection requires two discrete events, i.e., production of Ft-LPS-specific antibody, as well as TLR-mediated macrophage activation, to fully control Francisella infection. J. Leukoc. Biol. 90: 787-797; 2011.

ABSTRACTImmunization with Ft-LPS provokes an antigen-specific, B-1a cell-derived antibody response that protects WT mice against an otherwise lethal challenge with Ft LVS. However, this same regimen offers limited protection to TLR2−/− mice, despite production of WT levels of anti-Ft-LPS antibodies. As Ft-LPS exhibits no TLR2 agonist activity, and macrophage-induced cytokine production in response to Ft LVS is overwhelmingly TLR2-dependent, we hypothesized that treatment of TLR2−/− mice with an alternative, MyD88-dependent TLR agonist would compensate for reduced recognition of Ft LVS in TLR2−/− mice and thereby, restore Ft-LPS-mediated protection. Administration of the nontoxic TLR4 agonist, synthetic Escherichia coli MPL, at the time of Ft-LPS immunization or Ft LVS challenge, fully protected TLR2−/− mice, whereas treatment of WT or TLR2−/− mice with MPL alone conferred partial protection. The TLR5 agonist, flagellin, also synergized with Ft-LPS to protect TLR2−/− mice from lethal Ft LVS challenge. In contrast to Ft LVS, Ft-LPS pretreatment failed to protect mice against i.n. challenge with Ft Schu S4, whereas MPL, administered in the absence or presence of Ft-LPS, conferred significant, albeit partial, protection. MPL treatment of macrophages increased the uptake of Ft LVS and decreased intracellular bacterial survival while shifting the macrophage-differentiation phenotype from “alternatively activated” to “classically activated”. Collectively, our data suggest that optimal, Ft-LPS-mediated protection against Ft LVS infection requires two discrete events, i.e., production of Ft-LPS-specific antibody, as well as TLR-mediated macrophage activation, to fully control Francisella infection.

TLR2 plays a central role in the activation of innate immunity in response to Ft, the causative agent of tularemia. We reported previously that Ft LVS elicited strong, dose-dependent NF-kappa B reporter activity in TLR2-expressing human embryo kidney 293 T cells and that Ft LVS-induced murine macrophage proinflammatory cytokine gene and protein expression is TLR2-dependent. We demonstrated further that Ft can signal through TLR2 from within the phagosome and that phagosomal retention of Ft leads to greatly increased expression of a subset of proinflammatory genes. The two adaptor proteins associated with TLR2-mediated signaling are MyD88 and TIRAP. Although MyD88 is absolutely required for the Ft-induced macrophage cytokine response, the requirement for TIRAP can be overcome through retention of Ft within the phagosome. TIRAP-independent signaling was observed whether Ft was retained in the phagosome as a result of bacterial mutation (LVS Delta iglC) or BFA-mediated inhibition of phagosome acidification. The requirement for TIRAP in TLR2 signaling could also be overcome by increasing the concentrations of synthetic bacterial TLR2 agonists. Taken together, these data suggest that prolonging or enhancing the interaction between TLR2 and its agonist overcomes the "bridging" function ascribed previously to TIRAP. J. Leukoc. Biol. 87: 275-281; 2010.

The AIM2 inflammasome induces maturation of the proinflammatory cytokines IL-1β and IL-18. Using AIM2-deficient mice, Fitzgerald and colleagues and Alnemri and colleagues show that the AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Inflammasomes regulate the activity of caspase-1 and the maturation of interleukin 1β (IL-1β) and IL-18. AIM2 has been shown to bind DNA and engage the caspase-1-activating adaptor protein ASC to form a caspase-1-activating inflammasome. Using Aim2-deficient mice, we identify a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1β and IL-18, as well as pyroptosis, in response to synthetic double-stranded DNA. AIM2 was essential for inflammasome activation in response to Francisella tularensis, vaccinia virus and mouse cytomegalovirus and had a partial role in the sensing of Listeria monocytogenes. Moreover, production of IL-18 and natural killer cell–dependent production of interferon-γ, events critical in the early control of virus replication, were dependent on AIM2 during mouse cytomegalovirus infection in vivo. Collectively, our observations demonstrate the importance of AIM2 in the sensing of both bacterial and viral pathogens and in triggering innate immunity.

Background: It has been shown previously that administration of Francisella tularensis (Ft) Live Vaccine Strain (LVS) lipopolysaccharide (LPS) protects mice against subsequent challenge with Ft LVS and blunts the pro-inflammatory cytokine response.Methods: To further investigate the molecular mechanisms that underlie Ft LVS LPS-mediated protection, we profiled global hepatic gene expression following Ft LVS LPS or saline pre-treatment and subsequent Ft LVS challenge using Affymetrix arrays.Results: A large number of genes (> 3,000) were differentially expressed at 48 hours post-infection. The degree of modulation of inflammatory genes by infection was clearly attenuated by pre-treatment with Ft LVS LPS in the surviving mice. However, Ft LVS LPS alone had a subtle effect on the gene expression profile of the uninfected mice. By employing gene set enrichment analysis, we discovered significant up-regulation of the fatty acid metabolism pathway, which is regulated by peroxisome proliferator activated receptors (PPARs).Conclusions: We hypothesize that the LPS-induced blunting of pro-inflammatory response in mouse is, in part, mediated by PPARs (alpha and gamma).

The Category A select agent Francisella tularensis (Ft) is the causative agent of tularemia, a potentially fatal infectious disease. Intraperitoneal infection of mice with ~10 Ft Live Vaccine Strain (Ft LVS) organisms causes a lethal infection that resembles human tularemia. Immunization with 100 ng Ft LVS lipopolysaccharide (Ft-LPS) provokes an antibody response that protects wild-type (WT) C57BL/6J mice against an otherwise lethal Ft LVS challenge, but offers limited protection to TLR2-/- mice (WT, 85% survival vs. TLR2-/-, <25% survival; p < 0.0001, N = 4 separate experiments). Since macrophage recognition of Ft LVS is overwhelmingly TLR2-dependent (Cole, L. E., et al. 2007. Infect. Immun. 75:4127-37), we hypothesized that treatment of TLR2-/- mice with an alternative MyD88-dependent, TLR agonist would compensate for the failure of Ft LVS to signal in TLR2-/- mice and thereby lead to increased Ft-LPS-mediated protection. Concurrent administration of the TLR4 agonists, E. coli LPS (5μg) or monophosphoryl lipid A (MPL; 100μg), a synthetic non-toxic derivative of E. coli lipid A, with either the Ft-LPS immunization or the Ft LVS challenge, increased the survival of Ft-LPS-pretreated TLR2-/- to 100% (N=4 separate experiments). This suggests that effective Ft-LPS-mediated protection against Ft LVS challenge requires both Ft-LPS recognition and MyD88-dependent, TLR signaling. Supported by NIH AI157168.

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

Francisella tularensis (Ft), a gram-negative intracellular bacterium, is the etiologic agent of tularemia. Infection of mice with <10 Ft Live Vaccine Strain (Ft LVS) organisms i.p. causes a lethal infection that resembles human tularemia. Here, we show that immunization with as little as 0.1 ng Ft LVS lipopolysaccharide (Ft-LPS), but not Ft lipid A, generates a rapid antibody response that protects wild-type (WT) mice against lethal Ft LVS challenge. Protection is not induced in Ft-LPS-immunized B cell-deficient mice (muMT or JhD), male xid mice, or Ig transgenic mice that produce a single IgH (not reactive with Ft-LPS). Focusing on the cellular mechanisms that underlie this protective response, we show that Ft-LPS specifically stimulates proliferation of B-1a lymphocytes that bind fluorochrome-labeled Ft-LPS and the differentiation of these cells to plasma cells that secrete antibodies specific for Ft-LPS. This exclusively B-1a antibody response is equivalent in WT, T-deficient (TCRalphabeta(-/-), TCRgammadelta(-/-)), and Toll-like receptor 4 (TLR4)-deficient (TLR4(-/-)) mice and thus is not dependent on T cells or typical inflammatory processes. Serum antibody levels peak approximately 5 days after Ft-LPS immunization and persist at low levels for months. Thus, immunization with Ft-LPS activates a rare population of antigen-specific B-1a cells to produce a persistent T-independent antibody response that provides long-term protection against lethal Ft LVS infection. These data support the possibility of creating effective, minimally invasive vaccines that can provide effective protection against pathogen invasion.

Francisella tularensis ( Ft ), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Infection of mice with <10 Ft Live Vaccine Strain ( Ft LVS) organisms i.p. causes a lethal infection that resembles human tularemia. Here, we show that immunization with as little as 0.1 ng Ft LVS lipopolysaccharide ( Ft -LPS), but not Ft lipid A, generates a rapid antibody response that protects wild-type (WT) mice against lethal Ft LVS challenge. Protection is not induced in Ft -LPS-immunized B cell-deficient mice (μMT or JhD), male xid mice, or Ig transgenic mice that produce a single IgH (not reactive with Ft -LPS). Focusing on the cellular mechanisms that underlie this protective response, we show that Ft -LPS specifically stimulates proliferation of B-1a lymphocytes that bind fluorochrome-labeled Ft -LPS and the differentiation of these cells to plasma cells that secrete antibodies specific for Ft -LPS. This exclusively B-1a antibody response is equivalent in WT, T-deficient (TCRαβ −/− , TCRγδ −/− ), and Toll-like receptor 4 (TLR4)-deficient (TLR4 −/− ) mice and thus is not dependent on T cells or typical inflammatory processes. Serum antibody levels peak ≈5 days after Ft -LPS immunization and persist at low levels for months. Thus, immunization with Ft -LPS activates a rare population of antigen-specific B-1a cells to produce a persistent T-independent antibody response that provides long-term protection against lethal Ft LVS infection. These data support the possibility of creating effective, minimally invasive vaccines that can provide effective protection against pathogen invasion.

Francisella tularensis is a Category A select agent for which vaccine and countermeasure development are a priority. In the past eight years, renewed interest in this pathogen has led to the generation of an enormous amount of new data on both the pathogen itself and its interaction with host cells. This information has fostered the development of various vaccine candidates including acellular subunit, killed whole cell and live attenuated. This review summarizes the progress and promise of these various candidates.