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This study presents the engineering of a less endotoxic Salmonella Typhimurium strain by manipulating the lipid-A structure of the lipopolysaccharide (LPS) component. Salmonella lipid A was dephosphorylated by using lpxE from Francisella tularensis. The 1-phosphate group from lipid-A was removed selectively , resulting in a close analog of monophosphoryl lipid A. We observed a significant impact of ∆ pagL on major virulence factors such as biofilm formation, motility, persistency, and immune evasion. In correlation with biofilm and motility retardation, adhesion and invasion were elevated but with reduced intracellular survival, a favorable phenotype prospect of a vaccine strain. Western blotting and silver staining confirmed the absence of the O-antigen and truncated lipid-A core in the detoxified Salmonella mutant. In vitro and in vivo studies demonstrated that the dephosphorylated Salmonella mutant mediated lower pro-inflammatory cytokine secretion than the wild-type strain. The vaccine strains were present in the spleen and liver for five days and were cleared from the organs by day seven. However, the wild-type strain persisted in the spleen, liver, and brain, leading to sepsis-induced death. Histological evaluations of tissue samples further confirmed the reduced endotoxic activity of the detoxified Salmonella mutant. The detoxification strategy did not compromise the level of protective immunity, as the vaccine strain could enhance humoral and cellular immune responses and protect against the wild-type challenge in immunized mice. • Engineering a live attenuated Salmonella Typhimurium (SG) strain with reduced endotoxicity. • Knocking out of pagL hindered the motility and biofilm formation ability of Salmonella. • Lipid A modified Salmonella strain generated significantly lower levels of pro-inflammatory cytokines. • Monophosphoryl lipid A created by insertion of lpxE showed improved adjuvanticity. • Detoxified Salmonella mutant strain showed enhanced protective immune responses against a virulent wild-type challenge. [ABSTRACT FROM AUTHOR]

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

Respiratory infection of mice with Francisella novicida has recently been used as a model for the highly virulent human pathogen Francisella tularensis . Similar to F. tularensis , even small doses of F. novicida administered by respiratory routes are lethal for inbred laboratory mice. This feature obviously limits study of infection-induced immunity. Parenteral sublethal infections of mice with F. novicida are feasible, but the resulting immune responses are incompletely characterized. Here we use parenteral intradermal (i.d.) and intraperitoneal (i.p.) F. novicida infections of C57BL/6J mice to determine the role of B cells in controlling primary and secondary F. novicida infections. Despite developing comparable levels of F. novicida -primed T cells, B cell knockout mice were much more susceptible to both primary i.d. infection and secondary i.p. challenge than wild type (normal) C57BL/6J mice. Transfer of F. novicida -immune sera to either wild type C57BL/6J mice or to B cell knockout mice did not appreciably impact survival of subsequent lethal F. novicida challenge. However, F. novicida -immune mice that were depleted of T cells after priming but just before challenge survived and cleared secondary i.p. F. novicida challenge. Collectively these results indicate that B cells, if not serum antibodies, play a major role in controlling F. novicida infections in mice. [ABSTRACT FROM AUTHOR]

Francisella tularensis inhalation results in bacterial interaction with numerous lung cell types, including those of the epithelium. This work investigates a three-dimensional cell-culture system to characterise the epithelial response to F. tularensis . Immortalised human pneumocytes (A549s) grown in rotating-wall vessel (RWV) bioreactors display an in vivo-like phenotype, which has been confirmed to be driven by specific transcriptional events (8454 genes, p ≤ 0.05). These data support the RWV model as a more in vivo-like culture system to investigate the lung epithelium, compared to monolayer counterparts. RWV-cultured A549s were infected with F. tularensis SchuS4 and LVS and intracellular replication mapped over 22 h compared to monolayer cells. The RWV-cultured A549s were more resistant to SchuS4 and LVS infection ( p ≤ 0.05). Transcriptomics identified 2086 genes ( p ≤ 0.05) as candidates for host–pathogen interactions which result in the observed increase in resistance of the RWV-cultured A549 cells. Gene and pathway analysis identified processes involved in MMP modulation, endocytosis, mucin production and the complement pathway amongst others. The role of these pathways during infection was further characterised using chemical inhibitors. This work has revealed several new hypotheses worthy of further testing in order to understand the epithelial host response to F. tularensis infection. [ABSTRACT FROM AUTHOR]

Activation of the FcγR via antigen containing immune complexes can lead to the generation of reactive oxygen species, which are potent signal transducing molecules. However, whether ROS contribute to FcγR signaling has not been studied extensively. We set out to elucidate the role of NADPH oxidase-generated ROS in macrophage activation following FcγR engagement using antigen-containing immune complexes. We hypothesized that NOX2 generated ROS is necessary for propagation of downstream FcγR signaling and initiation of the innate immune response. Following exposure of murine bone marrow-derived macrophages (BMDMs) to inactivated Francisella tularensis (iFt)-containing immune complexes, we observed a significant increase in the innate inflammatory cytokine IL-6 at 24 h compared with macrophages treated with Ft LVS-containing immune complexes. Ligation of the FcγR by opsonized Ft also results in significant ROS production. Macrophages lacking the gp91phox subunit of NOX2 fail to produce ROS upon FcγR ligation, resulting in decreased Akt phosphorylation and a reduction in the levels of IL-6 compared with wild typemacrophages. Similar results were seen following infection of BMDMs with catalase deficient Ft that fail to scavenge hydrogen peroxide. In conclusion, our findings demonstrate that ROS participate in elicitation of an effective innate immune in response to antigen-containing immune complexes through FcγR. [ABSTRACT FROM AUTHOR]

Abstract: Francisella tularensis subspecies tularensis is an extremely virulent facultative intracellular bacterial pathogen capable of causing significant mortality in humans when inhaled. Consequently, subspecies tularensis was developed as a biological weapon more than 50 years ago. To counter this threat the US Army empirically developed a live vaccine strain, F. tularensis LVS, from the less virulent holarctica subspecies. In human experiments LVS afforded substantial protection against transdermal challenge with clinical subspecies tularensis strain, SCHU S4, but lesser protection against infection initiated by inhalation of the pathogen. Several regulatory and clinical issues remain unresolved for this vaccine, including the absence of a robust correlate of protection. To try to address this, we have developed several defined gene deletion mutants of SCHU S4 that elicit varying degrees of protection in a mouse dermal or respiratory challenge model. In the present study, we have examined whether host immune responses to immunization with such live vaccine candidates can serve as correlates of protection. Antibody responses were unable to distinguish between effective and ineffective vaccine strains. However, several cytokine responses to vaccination showed some promise. Especially, serum levels of TNFα, IFNγ, and MCP-1 between days 4 and 7 after vaccination appear to correlate with protection against respiratory challenge. [Copyright &y& Elsevier]

Abstract: Pneumonic tularemia is a potentially fatal disease caused by the Category A bioterrorism agent Francisella tularensis. Understanding the pulmonary immune response to this bacterium is necessary for developing effective vaccines and therapeutics. In this study, characterization of immune cell populations in the lungs of mice infected with the type A strain Schu S4 revealed a significant loss in natural killer (NK) cells over time. Since this decline in NK cells correlated with morbidity and mortality, we hypothesized these cells contribute to host defense against Schu S4 infection. Depletion of NK cells prior to Schu S4 challenge significantly reduced IFN-γ and granzyme B in the lung but had no effect on bacterial burden or disease progression. Conversely, increasing NK cell numbers with the anti-apoptotic cytokine IL-15 and soluble receptor IL-15Rα had no significant impact on Schu S4 growth in vivo. A modest decrease in median time to death, however, was observed in live vaccine strain (LVS)-vaccinated mice depleted of NK1.1+ cells and challenged with Schu S4. Therefore, NK cells do not appear to contribute to host defense against acute respiratory infection with type A F. tularensis in vivo, but they play a minor role in protection elicited by LVS vaccination. [Copyright &y& Elsevier]

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpAlike protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium. [ABSTRACT FROM AUTHOR]

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

Abstract: Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in infection remain poorly understood. Murine pulmonary models of infection were therefore employed to assess and compare dissemination and pathology and to elucidate the host immune response to infection with the highly virulent Type A F. tularensis strain Schu4 versus the less virulent Type B live vaccine strain (LVS). We found that dissemination and pathology in the spleen was significantly greater in mice infected with F. tularensis Schu4 compared to mice infected with F. tularensis LVS. Using gene expression profiling to compare the response to infection with the two F. tularensis strains, we found that there were significant differences in the expression of genes involved in the apoptosis pathway, antigen processing and presentation pathways, and inflammatory response pathways in mice infected with Schu4 when compared to LVS. These transcriptional differences coincided with marked differences in dissemination and severity of organ lesions in mice infected with the Schu4 and LVS strains. Therefore, these findings indicate that altered apoptosis, antigen presentation and production of inflammatory mediators explain the differences in pathogenicity of F. tularensis Schu4 and LVS. [Copyright &y& Elsevier]

Abstract: Although survival of primary infection with the live vaccine strain (LVS) of Francisella tularensis depends on interferon gamma (IFN-γ), the relative importance of IFN-γ to secondary protective immunity in vivo has not been clearly established. Here we examine the role of IFN-γ in T cell priming and expression of vaccine-induced protection against lethal intraperitoneal challenge of mice. Large amounts of IFN-γ were detected between days 3 and 7 in the sera of LVS-immunized mice, while relatively small amounts were found transiently after secondary LVS challenge. Consistent with the production of this cytokine, mice lacking IFN-γ (gamma interferon knockout, GKO, mice) could not be successfully vaccinated with LVS or an attenuated mglA mutant of F. novicida to withstand secondary Francisella LVS challenge. Further, splenocytes from such primed mice did not adoptively transfer protection to naive GKO recipient mice in vivo, nor control the intramacrophage growth of LVS in vitro. Finally, LVS-immune WT mice depleted of IFN-γ prior to intraperitoneal challenge survived only the lowest doses of challenge. Thus successful priming of protective LVS-immune T cells, as well as complete expression of protection against Francisella during secondary challenge, depends heavily on IFN-γ. [Copyright &y& Elsevier]

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

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

Abstract: The remarkable infectiousness of Francisella tularensis suggests that the bacterium efficiently evades innate immune responses that typically protect the host during its continuous exposure to environmental and commensal microbes. In our studies of the innate immune response to F. tularensis, we have observed that, unlike the live vaccine strain (LVS) of F. tularensis subsp. holarctica, F. tularensis subsp. novicida U112 opsonized in pooled human serum activated the NADPH oxidase when incubated with human neutrophils. Given previous observations that F. tularensis fixes relatively small quantities of complement component C3 during incubation in human serum and the importance of C3 to neutrophil phagocytosis, we hypothesized that F. tularensis subsp. novicida may fix C3 in human serum more readily than would LVS. We now report that F. tularensis subsp. novicida fixed approximately six-fold more C3 than did LVS when incubated in 50% pooled human serum and that this complement opsonization was antibody-mediated. Furthermore, antibody-mediated C3 deposition enhanced bacterial uptake and was indispensable for the neutrophil oxidative response to F. tularensis subsp. novicida. Taken together, our results reveal important differences between these two strains of F. tularensis and may, in part, explain the low virulence of F. tularensis subsp. novicida for humans. [Copyright &y& Elsevier]

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

Abstract: The live vaccine strain (LVS) of Francisella tularensis is the only vaccine against tularemia available for humans, yet its mechanism of protection remains unclear. We probed human immunological responses to LVS vaccination with transcriptome analysis using PBMC samples from volunteers at time points pre- and post-vaccination. Gene modulation was highly uniform across all time points, implying commonality of vaccine responses. Principal components analysis revealed three highly distinct principal groupings: pre-vaccination (−144h), early (+18 and +48h), and late post-vaccination (+192 and +336h). The most significant changes in gene expression occurred at early post-vaccination time points (≤48h), specifically in the induction of pro-inflammatory and innate immunity-related genes. Evidence supporting modulation of innate effector function, specifically antigen processing and presentation by dendritic cells, was especially apparent. Our data indicate that the LVS strain of F. tularensis invokes a strong early response upon vaccination. This pattern of gene regulation may provide insightful information regarding both vaccine efficacy and immunopathogenesis that may provide insight into infection with virulent strains of F. tularensis. Additionally, we obtained valuable information that should prove useful in evaluation of vaccine lots as well as efficacy testing of new anti-F. tularensis vaccines. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a category A biothreat agent, and as a result, it has recently generated much research interest. F. tularensis live vaccine strain (LVS) is an attenuated form of the virulent F. tularensis organism and has previously been used as a vaccine. However, because of safety concerns, it is no longer approved for this purpose. Thus, the use of inactivated organisms is preferable for vaccine purposes. Although many studies have been performed that examine human peripheral blood mononuclear cells (PBMC), and in particular CD4 T cells, responses to inactivated F. tularensis, there has been no study identifying the individual human cell populations within a mixed PBMC population that respond to this organism. We sought to address this deficit. Our results indicate that natural killer and CD8 T cells comprise the majority of cells responding to F. tularensis LVS. In addition, data suggest CD8 T cell responses are maximal when antibiotic-treated organisms are used and are minimal when formaldehyde-fixed organisms are used. Given the belief that CD8 T cells can play an important role in protection against F. tularensis infection, these studies have direct relevance to the development of F. tularensis vaccines that use inactivated organisms. In addition, important new knowledge is added to our understanding of the human immune response to F. tularensis LVS. [Copyright &y& Elsevier]