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Environmental changes associated with global warming create new opportunities for pathogen and parasite transmission in Arctic wildlife. As an apex predator ranging over large, remote areas, changes in pathogens and parasites in polar bears are a useful indicator of changing transmission dynamics in Arctic ecosystems. We examined prevalence and risk factors associated with exposure to parasites and viral and bacterial pathogens in Chukchi Sea polar bears. Serum antibodies to six pathogens were detected and prevalence increased between 1987-1994 and 2008-2017 for five: Toxoplasma gondii, Neospora caninum, Francisella tularensis, Brucella abortus/suis, and canine distemper virus. Although bears have increased summer land use, this behavior was not associated with increased exposure. Higher prevalence of F. tularensis, Coxiella burnetii, and B. abortus/suis antibodies in females compared to males, however, could be associated with terrestrial denning. Exposure was related to diet for several pathogens indicating increased exposure in the food web. Elevated white blood cell counts suggest a possible immune response to some pathogens. Given that polar bears face multiple stressors in association with climate change and are a subsistence food, further work is warranted to screen for signs of disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Francisella tularensis is an intracellular gram-negative bacterium known as the causative agent of tularemia, which can be transmitted to humans by direct contact with wild animals or by tick bites. Although F. tularensis is highly pathogenic, its recent prevalence in Japan is underreported due to the small number of reported cases. To clarify the current situation of F. tularensis in wild animals, we conducted surveillance on various species of wild animals in Yamaguchi prefecture. In this study, we screened 809 samples collected from 90 Japanese black bears, 105 Japanese monkeys, 168 sika deer, 205 wild boars, and 84 bats. For seroprevalence analysis, we tested 177 serum samples from 75 black bears and 102 monkeys using the microagglutination test. The results showed that serums from five black bears exhibited slight agglutination. Western blot was performed as a confirmatory test on these five samples, but no positive signals were detected. Additionally, molecular surveillance was conducted using DNA extracted from 464 whole blood and 168 tissues, targeting the gene encoding 23 KDa hypothetical protein by real-time PCR and outer membrane protein A gene by conventional PCR. No positive samples of F. tularensis were detected by either real-time or conventional PCR. Although we did not detect any F. tularensis-positive samples through serological and molecular analyses, continuous surveillance studies are necessary since sporadic human cases have been reported in Japan., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Francisella tularensis can cause severe disease in humans via the respiratory or cutaneous routes and a case fatality ratio of up to 10 % is reported due to lack of proper antibiotic treatment, while F. novicida causes disease in severely immunocompromised individuals. Efforts are needed to develop effective vaccine candidates against Francisella species. Thus, in this study, a systematic computational work frame was used to deeply investigate the whole proteome of Francisella novicida containing 1728 proteins to develop vaccine against F. tularensis and related species. Whole-proteome analysis revealed that four proteins including (A0Q492) (A0Q7Y4), (A0Q4N4), and (A0Q5D9) are the suitable vaccine targets after the removal of homologous, paralogous and prediction of subcellular localization. These proteins were used to predict the T cell, B cell, and HTL epitopes which were joined together through suitable linkers to construct a multi-epitopes vaccine (MEVC). The MEVC was found to be highly immunogenic and non-allergenic while the physiochemical properties revealed the feasible expression and purification. Moreover, the molecular interaction of MEVC with TLR2, molecular simulation, and binding free energy analyses further validated the immune potential of the construct. According to Jcat analysis, the refined sequence demonstrates GC contents of 41.48 % and a CAI value of 1. The in-silico cloning and optimization process ensured compatibility with host codon usage, thereby facilitating efficient expression. Computational immune simulation studies underscored the capacity of MEVC to induce both primary and secondary immune responses. The conservation analysis further revealed that the selected epitopes exhibit 100 % conservation across different species and thus provides wider protection against Francisella., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: Tularemia, caused by the bacterium Francisella tularensis , poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of F. tularensis (FopA) for rapid and accurate diagnosis of tularemia., Methods: We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA., Result: Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 × 10-10 M and 1.32 × 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from 0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices., Discussion: Our findings demonstrate the feasibility of a novel diagnostic approach for detecting F. tularensis based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jang, Kwon, Jang, Lee, Chang, Jeon, Jeong, Song, Min, Park, Lee, Han, Yang, Lee and Lee.)

Traditionally, successful vaccines rely on specific adaptive immunity by activating lymphocytes with an attenuated pathogen, or pathogen subunit, to elicit heightened responses upon subsequent exposures. However, recent work with Mycobacterium tuberculosis and other pathogens has identified a role for "trained" monocytes in protection through memory-like but non-specific immunity. Here, we used an in vitro co-culture approach to study the potential role of trained macrophages, including lung alveolar macrophages, in immune responses to the Live Vaccine Strain (LVS) of Francisella tularensis. F. tularensis is an intracellular bacterium that replicates within mammalian macrophages and causes respiratory as well as systemic disease. We vaccinated mice with F. tularensis LVS and then obtained lung alveolar macrophages, or derived macrophages from bone marrow. LVS infected and replicated comparably in both types of macrophages, whether naïve or from LVS-vaccinated mice. LVS-infected macrophages were then co-cultured with either naïve splenocytes, splenocytes from mice vaccinated intradermally, or splenocytes from mice vaccinated intravenously. For the first time, we show that immune (but not naïve) splenocytes controlled bacterial replication within alveolar macrophages, similar to previous results using bone marrow-derived macrophage. However, no differences in control of intramacrophage bacterial replication were found between co-cultures with naïve macrophages or macrophages from LVS-vaccinated mice; furthermore, nitric oxide levels and interferon-gamma production in supernatants were largely comparable across all conditions. Thus, in the context of in vitro co-cultures, the data do not support development of trained macrophages in bone marrow or lungs of mice vaccinated with LVS intradermally or intravenously., Importance: The discovery of non-specific "trained immunity" in monocytes has generated substantial excitement. However, to date, training has been studied with relatively few microbes (e.g., Mycobacterium bovis Bacille Calmette-Guérin, a live attenuated intracellular bacterium used as a vaccine) and microbial substances (e.g., LPS), and it remains unclear whether training during infection is common. We previously demonstrated that vaccination of mice with Francisella tularensis Live Vaccine Strain (LVS), another live attenuated intracellular bacterium, protected against challenge with the unrelated bacterium Listeria monocytogenes . The present study therefore tested whether LVS vaccination engenders trained macrophages that contributed to this protection. To do so, we used a previous in vitro co-culture approach with murine bone marrow-derived macrophages to expand and study lung alveolar macrophages. We demonstrated that alveolar macrophages can be productively infected and employed to characterize interactions with LVS-immune lymphocytes. However, we find no evidence that either bone marrow-derived or alveolar macrophages are trained by LVS vaccination., Competing Interests: The authors declare no conflict of interest.

Tularemia is a zoonotic disease caused by the facultative intracellular gram-negative bacterium Francisella tularensis. F. tularensis has a very low infection dose by the aerosol route which can result in an acute, and potentially lethal, infection in humans. Consequently, it is classified as a Category A bioterrorism agent by the US Centers for Disease Control (CDC) and is a pathogen of concern for the International Biodefence community. There are currently no licenced tularemia vaccines. In this study we report on the continued assessment of a tularemia subunit vaccine utilising β-glucan particles (GPs) as a vaccine delivery platform for immunogenic F. tularensis antigens. Using a Fischer 344 rat infection model, we demonstrate that a GP based vaccine comprising the F. tularensis lipopolysaccharide antigen together with the protein antigen FTT0814 provided partial protection of F344 rats against an aerosol challenge with a high virulence strain of F. tularensis, SCHU S4. Inclusion of imiquimod as an adjuvant failed to enhance protective efficacy. Moreover, the level of protection afforded was dependant on the challenge dose. Immunological characterisation of this vaccine demonstrated that it induced strong antibody immunoglobulin responses to both polysaccharide and protein antigens. Furthermore, we demonstrate that the FTT0814 component of the GP vaccine primed CD4+ and CD8+ T-cells from immunised F344 rats to express interferon-γ, and CD4+ cells to express interleukin-17, in an antigen specific manner. These data demonstrate the development potential of this tularemia subunit vaccine and builds on a body of work highlighting GPs as a promising vaccine platform for difficult to treat pathogens including those of concern to the bio-defence community., Competing Interests: The authors have declared that no competing interest exist., (Copyright: © 2024 Crown copyright, Dstl. This material is licensed under the terms of the Open Government Licence except where otherwise stated, which permits unrestricted use, distribution, and reproduction in any medium, provided you acknowledge the source of the Information or link to any attribution statement specified in the license. To view this licence, visit http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3 or write to the Information Policy Team, The National Archives, Kew, London TW9 4DU, or email: psi@nationalarchives.gov.uk.)

Pulmonary infections elicit a combination of tissue-resident and circulating T cell responses. Understanding the contribution of these anatomically distinct cellular pools in protective immune responses is critical for vaccine development. Francisella tularensis is a highly virulent bacterium capable of causing lethal systemic disease following pulmonary infection for which there is no currently licensed vaccine. Although T cells are required for survival of F. tularensis infection, the relative contribution of tissue-resident and circulating T cells is not completely understood, hampering design of effective, long-lasting vaccines directed against this bacterium. We have previously shown that resident T cells were not sufficient to protect against F. tularensis , suggesting circulating cells may serve a critical role in host defense. To elucidate the role of circulating T cells, we used a model of vaccination and challenge of parabiotic mice. Intranasally infected naive mice conjoined to immune animals had increased numbers of circulating memory T cells and similar splenic bacterial burdens as vaccinated-vaccinated pairs. However, bacterial loads in the lungs of naive parabionts were significantly greater than those observed in vaccinated-vaccinated pairs, but despite early control of F. tularensis replication, all naive-vaccinated pairs succumbed to infection. Together, these data define the specific roles of circulating and resident T cells in defense against infection that is initiated in the pulmonary compartment but ultimately causes disseminated disease. These data also provide evidence for employing vaccination strategies that elicit both pools of T cells for immunity against F. tularensis and may be a common theme for other disseminating bacterial infections.

The essential micronutrient Selenium (Se) is co-translationally incorporated as selenocysteine into proteins. Selenoproteins contain one or more selenocysteines and are vital for optimum immunity. Interestingly, many pathogenic bacteria utilize Se for various biological processes suggesting that Se may play a role in bacterial pathogenesis. A previous study had speculated that Francisella tularensis , a facultative intracellular bacterium and the causative agent of tularemia, sequesters Se by upregulating Se-metabolism genes in type II alveolar epithelial cells. Therefore, we investigated the contribution of host vs. pathogen-associated selenoproteins in bacterial disease using F. tularensis as a model organism. We found that F. tularensis was devoid of any Se utilization traits, neither incorporated elemental Se, nor exhibited Se-dependent growth. However, 100% of Se-deficient mice (0.01 ppm Se), which express low levels of selenoproteins, succumbed to F. tularensis -live vaccine strain pulmonary challenge, whereas 50% of mice on Se-supplemented (0.4 ppm Se) and 25% of mice on Se-adequate (0.1 ppm Se) diet succumbed to infection. Median survival time for Se-deficient mice was 8 days post-infection while Se-supplemented and -adequate mice was 11.5 and >14 days post-infection, respectively. Se-deficient macrophages permitted significantly higher intracellular bacterial replication than Se-supplemented macrophages ex vivo , corroborating in vivo observations. Since Francisella replicates in alveolar macrophages during the acute phase of pneumonic infection, we hypothesized that macrophage-specific host selenoproteins may restrict replication and systemic spread of bacteria. F. tularensis infection led to an increased expression of several macrophage selenoproteins, suggesting their key role in limiting bacterial replication. Upon challenge with F. tularensis , mice lacking selenoproteins in macrophages (TrspM) displayed lower survival and increased bacterial burden in the lung and systemic tissues in comparison to WT littermate controls. Furthermore, macrophages from TrspM mice were unable to restrict bacterial replication ex vivo in comparison to macrophages from littermate controls. We herein describe a novel function of host macrophage-specific selenoproteins in restriction of intracellular bacterial replication. These data suggest that host selenoproteins may be considered as novel targets for modulating immune response to control a bacterial infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Markley, Restori, Katkere, Sumner, Nicol, Tyryshkina, Nettleford, Williamson, Place, Dewan, Shay, Carlson, Girirajan, Prabhu and Kirimanjeswara.)

Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of linear compared to conformational epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Prediction of linear epitopes through use of computational tools is fast and cost-effective, but typically of low accuracy, necessitating extensive experimentation to verify results. On the other hand, identification of linear epitopes through experimental screening has been an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B-cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. Although B-cell epitope prediction tools Bepipred 2.0 and iBCE-EL classified many of our seroreactive peptides as epitopes, they did not score them significantly higher than the non-reactive tryptic peptides in our study, nor did they assign higher scores to seroreactive peptides from known Ft or Bp antigens, highlighting the need for experimental data instead of relying on computational epitope predictions alone. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B-cell epitopes and development of vaccines to counter emerging biological threats., Competing Interests: MF, NC, and PD’h are inventors on a provisional patent application for the method for rapid detection of immunogenic epitopes, filed by Lawrence Livermore National Security, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 D’haeseleer, Collette, Lao, Segelke, Branda and Franco.)

B1 cells, a subset of B lymphocytes whose developmental origin, phenotype, and function differ from that of conventional B2 cells, are the main source of "natural" IgM but can also respond to infection by rapidly producing pathogen-specific IgM directed against T-independent antigens. Francisella tularensis (Ft) is a Gram-negative bacterium that causes tularemia. Infection with Ft Live Vaccine Strain activates B1 cells for production of IgM directed against the bacterial LPS in a process incompletely understood. Here we show that immunization with purified Ft LPS elicits production of LPS-specific IgM and IgG3 by B1 cells independently of TLR2 or MyD88. Immunization, but not infection, generated peritoneum-resident memory B1 cells that differentiated into LPS-specific antibody secreting cells (ASC) upon secondary challenge. IL-5 was rapidly induced by immunization with Ft LPS and was required for production of LPS-specific IgM. Antibody-mediated depletion of ILC2 indicated that these cells were the source of IL-5 and were required for IgM production. IL-25, an alarmin that strongly activates ILC2, was rapidly secreted in response to immunization or infection and its administration to mice significantly increased IgM production and B1 cell differentiation to ASC. Conversely, mice lacking IL-17RB, the IL-25 receptor, showed impaired IL-5 induction, IgM production, and B1 ASC differentiation in response to immunization. Administration of IL-5 to Il17rb-/- mice rescued these B1 cells-mediated responses. Il17rb-/- mice were more susceptible to infection with Ft LVS and failed to develop immunity upon secondary challenge suggesting that LPS-specific IgM is one of the protective adaptive immune mechanisms against tularemia. Our results indicated that immunization with Ft LPS triggers production of IL-25 that, through stimulation of IL-5 release by ILC2, promotes B1 cells activation and differentiation into IgM secreting cells. By revealing the existence of an IL-25-ILC2-IL-5 axis our results suggest novel strategies to improve vaccination against T-independent bacterial antigens., Competing Interests: The authors have declared that no competing interests exist.

Mucosal-associated Invariant T (MAIT) cells are recognized for their antibacterial functions. The protective capacity of MAIT cells has been demonstrated in murine models of local infection, including in the lungs. Here we show that during systemic infection of mice with Francisella tularensis live vaccine strain results in evident MAIT cell expansion in the liver, lungs, kidney and spleen and peripheral blood. The responding MAIT cells manifest a polarised Th1-like MAIT-1 phenotype, including transcription factor and cytokine profile, and confer a critical role in controlling bacterial load. Post resolution of the primary infection, the expanded MAIT cells form stable memory-like MAIT-1 cell populations, suggesting a basis for vaccination. Indeed, a systemic vaccination with synthetic antigen 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil in combination with CpG adjuvant similarly boosts MAIT cells, and results in enhanced protection against both systemic and local infections with different bacteria. Our study highlights the potential utility of targeting MAIT cells to combat a range of bacterial pathogens., (© 2021. The Author(s).)

Francisella tularensis is a facultative, intracellular, Gram-negative bacterium that causes a fatal disease known as tularemia. Due to its extremely high virulence, ease of spread by aerosolization, and potential to be used as a bioterror agent, F. tularensis is classified by the CDC as a tier 1 category A select agent. Previous studies have demonstrated the roles of the inflammasome sensors absent in melanoma 2 (AIM2) and NLRP3 in the generation of innate immune responses to F. tularensis infection. However, contributions of both the AIM2 and NLRP3 to the development of vaccine-induced adaptive immune responses against F. tularensis are not known. This study determined the contributions of Aim2 and Nlrp3 inflammasome sensors to vaccine-induced immune responses in a mouse model of respiratory tularemia. We developed a model to vaccinate Aim2- and Nlrp3-deficient ( Aim2 -/- and Nlrp3 -/- ) mice using the emrA1 mutant of the F. tularensis live vaccine strain (LVS). The results demonstrate that the innate immune responses in Aim2 -/- and Nlrp3 -/- mice vaccinated with the emrA1 mutant differ from those of their wild-type counterparts. However, despite these differences in the innate immune responses, both Aim2 -/- and Nlrp3 -/- mice are fully protected against an intranasal lethal challenge dose of F. tularensis LVS. Moreover, the lack of both Aim2 and Nlrp3 inflammasome sensors does not affect the production of vaccination-induced antibody and cell-mediated responses. Overall, this study reports a novel finding that both Aim2 and Nlrp3 are dispensable for vaccination-induced immunity against respiratory tularemia caused by F. tularensis.

CCR2 is the major chemokine receptor that regulates appropriate trafficking of inflammatory monocytes, but the role of this chemokine receptor and its ligands during primary and secondary infection with intracellular infections remains incompletely understood. Here we used murine infection with the Live Vaccine Strain (LVS) of Francisella tularensis to evaluate the role of CCR2 during primary and secondary parenteral responses to this prototype intracellular bacterium. We find that mice deficient in CCR2 are highly compromised in their ability to survive intradermal infection with LVS, indicating the importance of this receptor during primary parenteral responses. Interestingly, this defect could not be readily attributed to the activities of the known murine CCR2 ligands MCP-1/CCL2, MCP-3/CCL7, or MCP-5/CCL12. Nonetheless, CCR2 knockout mice vaccinated by infection with low doses of LVS generated optimal T cell responses that controlled the intramacrophage replication of Francisella, and LVS-immune CCR2 knockout mice survived maximal lethal Francisella challenge. Thus, fully protective adaptive immune memory responses to this intracellular bacterium can be readily generated in the absence of CCR2., Competing Interests: The authors have declared that no competing interests exist.

Tularemia is a zoonotic disease of global proportions. Francisella tularensis subspecies tularensis (type A) and holarctica (type B) cause disease in healthy humans, with type A infections resulting in higher mortality. Repeated passage of a type B strain in the mid-20th century generated the Live Vaccine Strain (LVS). LVS remains unlicensed, does not protect against high inhalational doses of type A, and its exact mechanisms of attenuation are poorly understood. Recent data suggest that live attenuated vaccines derived from type B may cross-protect against type A. However, there is a dearth of knowledge regarding virulent type B pathogenesis and its capacity to stimulate the host's innate immune response. We therefore sought to increase our understanding of virulent type B in vitro characteristics using strain OR96-0246 as a model. Adding to our knowledge of innate immune kinetics in macrophages following infection with virulent type B, we observed robust replication of strain OR96-0246 in murine and human macrophages, reduced expression of pro-inflammatory cytokine genes from "wild type" type B-infected macrophages compared to LVS, and delayed macrophage cell death suggesting that virulent type B may suppress macrophage activation. One disruption in LVS is in the gene encoding the chloride transporter ClcA. We investigated the role of ClcA in macrophage infection and observed a replication delay in a clcA mutant. Here, we propose its role in acid tolerance. A greater understanding of LVS attenuation may reveal new mechanisms of pathogenesis and inform strategies toward the development of an improved vaccine against tularemia., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Sepsis results from the dysregulated immune response to infection. While the stimulator and progression of the septic response is poorly understood, the systemic production of a storm of cytokines is common in all etiologies of sepsis. While the complexity of this uncontrolled cascade is difficult to replicate using single molecule agonist, for example, lipopolysaccharide (LPS), several whole organism models can stimulate this cytokine storm. Herein, we detail protocols developed to trigger and analyze the systemic septic response in mouse models using the bacterium Francisella tularensis.

There is a need for development of an effective vaccine against Francisella tularensis , as this potential bioweapon has a high mortality rate and low infectious dose when delivered via the aerosol route. Moreover, this Tier 1 agent has a history of weaponization. We engineered targeted mutations in the Type A strain F. tularensis subspecies tularensis Schu S4 in aro genes encoding critical enzymes in aromatic amino acid biosynthesis. F. tularensis Schu S4 ΔaroC , Schu S4Δ aroD , and Schu S4Δ aroC Δ aroD mutant strains were attenuated for intracellular growth in vitro and for virulence in vivo and, conferred protection against pulmonary wild-type (WT) F. tularensis Schu S4 challenge in the C57BL/6 mouse model. F. tularensis Schu S4Δ aroD was identified as the most promising vaccine candidate, demonstrating protection against high-dose intranasal challenge; it protected against 1,000 CFU Schu S4, the highest level of protection tested to date. It also provided complete protection against challenge with 92 CFU of a F. tularensis subspecies holarctica strain (Type B). Mice responded to vaccination with Schu S4Δ aroD with systemic IgM and IgG2c, as well as the production of a functional T cell response as measured in the splenocyte-macrophage co-culture assay. This vaccine was further characterized for dissemination, histopathology, and cytokine/chemokine gene induction at defined time points following intranasal vaccination which confirmed its attenuation compared to WT Schu S4. Cytokine, chemokine, and antibody induction patterns compared to wild-type Schu S4 distinguish protective vs . pathogenic responses to F. tularensis and elucidate correlates of protection associated with vaccination against this agent.

Tularemia is a zoonotic disease caused by the bacterium Francisella tularensis . The predominant sources, routes of infection, and clinical manifestations of human infections greatly vary according to the geographic area considered. Moreover, clinical suspicion of tularemia is often tricky because of the lack of specificity of the clinical manifestations. Because F. tularensis isolation is tedious and detection of its DNA usually requires removal of infected tissues, serological techniques are most often used for diagnostic confirmation. However, these techniques are varied and poorly standardized. The microagglutination test (MAT), the indirect immunofluorescence assay (IFA), and ELISA tests are currently the most frequently used techniques. These home-made and commercial tests are mainly used for tularemia diagnosis but also seroprevalence studies. ELISA tests detect specific antibodies within two weeks of disease evaluation, compared to 2-3 weeks for MAT and IFA. However, more false-positive results are usually reported with ELISA. The long-term persistence of anti- F. tularensis antibodies in patients with past tularemia infection hampers the diagnostic specificity of all these tests. Also, cross-reacting antibodies have been described (especially with Brucella and Yersinia species), although usually at a low level. The immunoblotting technique can highlight these serological cross-reactions. Tularemia remains an underdiagnosed disease in most endemic areas, and the clinical presentations of this disease are evolving. It is necessary to improve further speed and accuracy of tularemia diagnosis, as well as the standardization of diagnostic procedures., (Copyright © 2020 Maurin.)

Intracellular pathogens affect diverse host cellular defence and metabolic pathways. Here, we used infection with Francisella tularensis to identify SON DNA-binding protein as a central determinant of macrophage activities. RNAi knockdown of SON increases survival of human macrophages following F. tularensis infection or inflammasome stimulation. SON is required for macrophage autophagy, interferon response factor 3 expression, type I interferon response and inflammasome-associated readouts. SON knockdown has gene- and stimulus-specific effects on inflammatory gene expression. SON is required for accurate splicing and expression of GBF1, a key mediator of cis-Golgi structure and function. Chemical GBF1 inhibition has similar effects to SON knockdown, suggesting that SON controls macrophage functions at least in part by controlling Golgi-associated processes., (© 2020 Federation of European Biochemical Societies.)

Mucosa-associated invariant T (MAIT) cells are a unique T cell subset that contributes to protective immunity against microbial pathogens, but little is known about the role of chemokines in recruiting MAIT cells to the site of infection. Pulmonary infection with Francisella tularensis live vaccine strain (LVS) stimulates the accrual of large numbers of MAIT cells in the lungs of mice. Using this infection model, we find that MAIT cells are predominantly CXCR6 + but do not require CXCR6 for accumulation in the lungs. However, CXCR6 does contribute to long-term retention of MAIT cells in the airway lumen after clearance of the infection. We also find that MAIT cells are not recruited from secondary lymphoid organs and largely proliferate in situ in the lungs after infection. Nevertheless, the only known ligand for CXCR6, CXCL16, is sufficient to drive MAIT cell accumulation in the lungs in the absence of infection when administered in combination with the MAIT cell antigen 5-OP-RU. Overall, this new data advances the understanding of mechanisms that facilitate MAIT cell accumulation and retention in the lungs., (Copyright © 2020 Yu, Yang, Liu, Mak, Fairlie and Cowley.)

Production of IFN-γ is a key innate immune mechanism that limits replication of intracellular bacteria such as Francisella tularensis (Ft) until adaptive immune responses develop. Previously, we demonstrated that the host cell types responsible for IFN-γ production in response to murine Francisella infection include not only natural killer (NK) and T cells, but also a variety of myeloid cells. However, production of IFN-γ by mouse dendritic cells (DC) is controversial. Here, we directly demonstrated substantial production of IFN-γ by DC, as well as hybrid NK-DC, from LVS-infected wild type C57BL/6 or Rag1 knockout mice. We demonstrated that the numbers of conventional DC producing IFN-γ increased progressively over the course of 8 days of LVS infection. In contrast, the numbers of conventional NK cells producing IFN-γ, which represented about 40% of non-B/T IFN-γ-producing cells, peaked at day 4 after LVS infection and declined thereafter. This pattern was similar to that of hybrid NK-DC. To further confirm IFN-γ production by infected cells, DC and neutrophils were sorted from naïve and LVS-infected mice and analyzed for gene expression. Quantification of LVS by PCR revealed the presence of Ft DNA not only in macrophages, but also in highly purified, IFN-γ producing DC and neutrophils. Finally, production of IFN-γ by infected DC was confirmed by immunohistochemistry and confocal microscopy. Notably, IFN-γ production patterns similar to those in wild type mice were observed in cells derived from LVS-infected TLR2, TLR4, and TLR2xTLR9 knockout (KO) mice, but not from MyD88 KO mice. Taken together, these studies demonstrate the pivotal roles of DC and MyD88 in IFN-γ production and in initiating innate immune responses to this intracellular bacterium., Competing Interests: The authors have declared that no competing interest exist.

Francisella tularensis is a highly infectious intracellular bacterium that causes tularemia by invading and replicating in mammalian myeloid cells. Francisella primarily invades host macrophages, where it escapes phagosomes within a few hours and replicates in the cytoplasm. Less is known about how Francisella traffics within macrophages or exits into the extracellular environment for further infection. Immune T lymphocytes control the replication of Francisella within macrophages in vitro by a variety of mechanisms, but nothing is known about intracellular bacterial trafficking in the face of such immune pressure. Here we used a murine model of infection with a Francisella attenuated live vaccine strain (LVS), which is under study as a human vaccine, to evaluate the hypothesis that immune T cells control intramacrophage bacterial growth by re-directing bacteria into toxic intracellular compartments of infected macrophages. We visualized the interactions of lymphocytes and LVS-infected macrophages using confocal microscopy and characterized LVS intramacrophage trafficking when co-cultured with immune lymphocytes. We focused on the late stages of infection after bacteria escape from phagosomes, through bacterial replication and the death of macrophages. We found that the majority of LVS remained cytosolic in the absence of immune pressure, eventually resulting in macrophage death. In contrast, co-culture of LVS-infected macrophages with LVS-immune lymphocytes halted LVS replication and inhibited the spread of LVS infection between macrophages, but bacteria did not return to vacuoles such as lysosomes or autophagosomes and macrophages did not die. Therefore, immune lymphocytes directly limit intracellular bacterial replication within the cytoplasm of infected macrophages.

Francisella tularensis ( Ft ) is a highly virulent, intracellular Gram-negative bacterial pathogen. Acute Ft infection by aerosol route causes pneumonic tularemia, characterized by nodular hemorrhagic lesions, neutrophil-predominant influx, necrotic debris, fibrin deposition, and severe alveolitis. Ft suppresses activity of neutrophils by impairing their respiratory burst and phagocytic activity. However, the fate of the massive numbers of neutrophils recruited to the infection site is unclear. Here, we show that Ft infection resulted in prominent induction of neutrophil extracellular traps (NETs) within damaged lungs of mice infected with the live attenuated vaccine strain of Ft ( Ft -LVS), as well as in the lungs of domestic cats and rabbits naturally infected with Ft . Further, Ft -LVS infection increased lung myeloperoxidase (MPO) activity, which mediates histone protein degradation during NETosis and anchors chromatin scaffolds in NETs. In addition, Ft infection also induced expression of peptidylarginine deiminase 4, an enzyme that causes citrullination of histones during formation of NETs. The released NETs were found largely attached to the alveolar epithelium, and disrupted the thin alveolar epithelial barrier. Furthermore, Ft infection induced a concentration-dependent release of NETs from neutrophils in vitro. Pharmacological blocking of MPO reduced Ft -induced NETs release, whereas addition of H 2 O 2 (a substrate of MPO) significantly augmented NETs release, thus indicating a critical role of MPO in Ft- induced NETs. Although immunofluorescence and electron microscopy revealed that NETs could efficiently trap Ft bacteria, NETs failed to exert bactericidal effects. Taken together, these findings suggest that NETs exacerbate tissue damage in pulmonary Ft infection, and that targeting NETosis may offer novel therapeutic interventions in alleviating Ft -induced tissue damage., (Copyright © 2020 Pulavendran, Prasanthi, Ramachandran, Grant, Snider, Chow, Malayer and Teluguakula.)

Methods used to prepare bone marrow-derived macrophages (BMDMs) may influence the outcomes of immunological assays in which they are used. Supernatant conditioned by growth of L929 cells has often been used to generate mouse macrophages from bone marrow in vitro but is subject to lot-to-lot variability. To reduce experimental variability and to standardize techniques across laboratories, we investigated recombinant M-CSF (rM-CSF) as an alternative supplement for BMDM maturation in the context of macrophage infection, using the intracellular bacterium Live Vaccine Strain (LVS) of Francisella tularensis as a prototype. We compared rM-CSF with L929 supernatant in terms of their effects on mouse and rat macrophage growth, maturation patterns, surface marker expression, and the expression of selected genes. Further, we compared macrophage infectivity and bacterial replication using LVS. Finally, we compared the in vitro function of BMDMs co-cultured with splenocytes from vaccinated animals in terms of their control of intramacrophage bacterial replication, as well as production of cytokines and nitric oxide. We demonstrated that rM-CSF produced BMDMs with similar, or minimal, phenotypic and gene expression outcomes compared to those generated with media containing L929 supernatant. Most importantly, functional outcomes were similar. Taken together, our data support the use of the rM-CSF in cell culture media as an alternative to L929-supplemented media for functional bioassays that use C57BL/6J mouse or Fischer 344 rat BMDMs to study intracellular infections. This comparison therefore facilitates future protocol standardization., (Published by Elsevier B.V.)

Francisella tularensis, a category-A bioterrorism agent causes tularemia. F. tularensis suppresses the immune response of host cells and intracellularly proliferates. However, the detailed mechanisms of immune suppression and intracellular growth are largely unknown. Here we developed a transposon mutant library to identify novel pathogenic factors of F. tularensis. Among 750 transposon mutants of F. tularensis subsp. novicida (F. novicida), 11 were isolated as less cytotoxic strains, and the genes responsible for cytotoxicity were identified. Among them, the function of slt, which encodes soluble lytic transglycosylase (SLT) was investigated in detail. An slt deletion mutant (Δslt) was less toxic to the human monocyte cell line THP-1 vs the wild-type strain. Although the wild-type strain proliferated in THP-1 cells, the number of intracellular Δslt mutant decreased in comparison. The Δslt mutant escaped from phagosomes during the early stages of infection, but the mutant was detected within the autophagosome, followed by degradation in lysosomes. Moreover, the Δslt mutant induced host cells to produce high levels of cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and IL-1β, compared with the wild-type strain. These results suggest that the SLT of F. novicida is required for immune suppression and escape from autophagy to allow its survival in host cells., Competing Interests: The authors have declared that no competing interests exist.

Previous in vivo studies established that inactivated Francisella tularensis immune complexes (mAb-iFt) are a more protective vaccine against lethal tularemia than iFt alone. Subsequent in vitro studies revealed enhanced DC maturation marker expression with mAb-iFt stimulation. The goal of this study was to determine the mechanism of enhanced DC maturation. Multiparameter analysis of surface marker expression and cytokine secretion demonstrates a requirement for FcγR signaling in enhanced DC maturation. MyD88 was also found to be essential for heightened DC maturation, implicating MyD88-dependent TLRs in DC maturation. Upon further study, we discovered that TLRs 2 & 4 drive cytokine secretion, but surprisingly TLR9 is required for DC maturation marker upregulation. These studies reveal a separation of DC cytokine and maturation marker induction pathways and demonstrate that FcγR-TLR/MyD88 synergy underlies the enhanced dendritic cell maturation in response to the mAb-iFt vaccine., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Vaccines are considered the bedrock of preventive medicine. However, for many pathogens, it has been challenging to develop vaccines that stimulate protective, long-lasting immunity. We have developed a novel approach using β-1,3-D-glucans (BGs), natural polysaccharides abundantly present in fungal cell walls, as a biomaterial platform for vaccine delivery. BGs simultaneously provide for receptor-targeted antigen delivery to specialized antigen-presenting cells together with adjuvant properties to stimulate antigen-specific and trained non-specific immune responses. This review focuses on various approaches of using BG particles (GPs) to develop bacterial and fungal vaccine candidates. A special case history for the development of an effective GP tularaemia vaccine candidate is highlighted., (© 2019 Crown copyright. Clinical and Experimental Immunology © 2019 British Society for Immunology. This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.)

Informed management of American black bears ( Ursus americanus ) requires knowledge of the distribution and pathology of diseases affecting the species. Little information is available on pathogen prevalence from black bear populations in the Southwest, US, and it is unknown how these infections may influence black bear populations or disease transmission. We captured New Mexico black bears ( Ursus americanus amblyceps ) during 2016-17 as part of a long-term monitoring project and opportunistically collected 36 blood samples from 12 female and 17 male black bears. We wanted to determine prior exposure to canine distemper virus, canine parvovirus, Yersinia pestis , Francisella tularensis , West Nile virus, Toxoplasma gondii , and the tick-borne pathogens, Anaplasma spp., Ehrlichia spp., Borrelia burgdorferi , Rickettsia spp., and Babesia spp. Approximately half (55%, 16/29) of the individuals sampled had antibodies to Y. pestis , and 37% (10/27) had antibodies to T. gondii . Prevalence of antibodies to West Nile virus, F. tularensis , and canine parvovirus were lower (i.e., 11, 10, and 3%, respectively). We detected no antibodies to canine distemper, B. burgdorferi , Rickettsia spp., or Babesia spp. We documented changes in antibody titer levels for both sexes of several recaptured black bears. Our data will inform managers of pathogen prevalence and distribution in black bears in north-central New Mexico and provide a vital baseline dataset for future pathogen monitoring. Additionally, these data support actions to minimize exposure through handling wild individuals or through hunter harvest activities.

Francisella tularensis (Ft), the causative agent of lethal tularemia, is classified as a category A biological warfare threat agent. While Ft infection is treatable by antibiotics, many failed antibiotic treatments were reported, highlighting the need for effective new treatments. It has been demonstrated that binding of antibody-coated bacteria to the Fc receptor located on phagocytic cells is a key process needed for efficient protection against Ft. Yet, Ft utilizes the same receptor to enter the phagocytic cells in order to escape the immune system. To address the question whether an anti-Ft LPS antibody lacking the ability to bind the Fc receptor may inhibit the entry of Ft into host cells, a soluble scFv (TL1-scFv) was constructed from an anti Ft-LPS antibody (TL1) that was isolated from an immune single-chain (scFv) phage-display library. Bacterial uptake was assessed upon infection of macrophages with Ft live attenuated strain (LVS) in the presence of either TL1 or TL1-scFv. While incubation of LVS in the presence of TL1 greatly enhanced bacterial uptake, LVS uptake was significantly inhibited in the presence of TL1-scFv. These results prompt further experiments probing the therapeutic efficacy of TL1-scFv, alone or in combination with antibiotic treatment.

Inhalation of Francisella tularensis causes pneumonic tularemia in humans, a severe disease with a 30 to 60% mortality rate. The reproducible delivery of aerosolized virulent bacteria in relevant animal models is essential for evaluating medical countermeasures. Here we developed optimized protocols for infecting New Zealand White (NZW) rabbits with aerosols containing F. tularensis We evaluated the relative humidity, aerosol exposure technique, and bacterial culture conditions to optimize the spray factor (SF), a central metric of aerosolization. This optimization reduced both inter- and intraday variability and was applicable to multiple isolates of F. tularensis Further improvements in the accuracy and precision of the inhaled pathogen dose were achieved through enhanced correlation of the bacterial culture optical density and the number of CFU. Plethysmograph data collected during exposures found that respiratory function varied considerably between rabbits, was not a function of weight, and did not improve with acclimation to the system. Live vaccine strain (LVS)-vaccinated rabbits were challenged via aerosol with human-virulent F. tularensis SCHU S4 that had been cultivated in either Mueller-Hinton broth (MHB) or brain heart infusion (BHI) broth. LVS-vaccinated animals challenged with SCHU S4 that had been cultivated in MHB experienced short febrile periods (median, 3.2 days), limited weight loss (<5%), and longer median survival times (∼18 days) that were significantly different from those for unvaccinated controls. In contrast, LVS-vaccinated rabbits challenged with SCHU S4 that had been cultivated in BHI experienced longer febrile periods (median, 5.5 days) and greater weight loss (>10%) than the unvaccinated controls and median survival times that were not significantly different from those for the unvaccinated controls. These studies highlight the importance of careful characterization and optimization of protocols for aerosol challenge with pathogenic agents., (Copyright © 2019 American Society for Microbiology.)

Primary interaction of an intracellular bacterium with its host cell is initiated by activation of multiple signaling pathways in response to bacterium recognition itself or as cellular responses to stress induced by the bacterium. The leading molecules in these processes are cell surface membrane receptors as well as cytosolic pattern recognition receptors recognizing pathogen-associated molecular patterns or damage-associated molecular patterns induced by the invading bacterium. In this review, we demonstrate possible sequences of events leading to recognition of Francisella tularensis , present findings on known mechanisms for manipulating cell responses to protect Francisella from being killed, and discuss newly published data from the perspective of early stages of host-pathogen interaction.

Tularemia is a severe infectious zoonotic disease caused by Francisella tularensis. Although F. tularensis is considered to be a potential biological weapon due to its high infectivity and mortality rate, no vaccine has been currently licensed. Recently, we reported that F. tularensis SCHU P9 derived ΔpdpC strain lacking the pathogenicity determinant protein C gene conferred stable and good protection in a mouse lethal model. In this study, the protective effect of ΔpdpC was evaluated using a monkey lethal model. Two cynomolgus macaques (Macaca fascicularis) intratracheally challenged with the virulent strain SCHU P9 were euthanized on 7 and 11 days post-challenge after the development of severe clinical signs. The bacterial replication in alveolar macrophages and type II epithelial cells in the lungs would cause severe pneumonia accompanied by necrosis. Conversely, two animals subcutaneously immunized with ΔpdpC survived 3 weeks after SCHU P9 challenge. Though one of the two animals developed mild symptoms of tularemia, bacterial replication was limited in the respiratory organs, which may be due to a high level of humoral and cellular immune responses against F. tularensis. These results suggest that the ΔpdpC mutant would be a safe and promising candidate as a live attenuated tularemia vaccine.

Pulmonary immune control is crucial for protection against pathogens. Here we identify a pathway that promotes host responses during pulmonary bacterial infection; the expression of CD200 receptor (CD200R), which is known to dampen pulmonary immune responses, promotes effective clearance of the lethal intracellular bacterium Francisella tularensis. We show that depletion of CD200R in mice increases in vitro and in vivo infectious burden. In vivo, CD200R deficiency leads to enhanced bacterial burden in neutrophils, suggesting CD200R normally limits the neutrophil niche for infection. Indeed, depletion of this neutrophil niche in CD200R -/- mice restores F. tularensis infection to levels seen in wild-type mice. Mechanistically, CD200R-deficient neutrophils display significantly reduced reactive oxygen species production (ROS), suggesting that CD200R-mediated ROS production in neutrophils is necessary for limiting F. tularensis colonisation and proliferation. Overall, our data show that CD200R promotes the antimicrobial properties of neutrophils and may represent a novel antibacterial therapeutic target.

Francisella tularensis is the causative agent of tularemia, a category A bioterrorism agent. The lipopolysaccharide (LPS) O antigen (OAg) of F. tularensis has been considered for use in a glycoconjugate vaccine, but conjugate vaccines tested so far have failed to confer protection necessary against aerosolized pulmonary bacterial challenge. When F. tularensis OAg was purified under standard conditions, the antigen had a small molecular size [25 kDa, low molecular weight (LMW)]. Using milder extraction conditions, we found the native OAg had a larger molecular size [80 kDa, high molecular weight (HMW)], and in a mouse model of tularemia, a glycoconjugate vaccine made with the HMW polysaccharide coupled to tetanus toxoid (HMW-TT) conferred better protection against intranasal challenge than a conjugate made with the LMW polysaccharide (LMW-TT). To further investigate the role of OAg size in protection, we created an F. tularensis live vaccine strain (LVS) mutant with a significantly increased OAg size [220 kDa, very high molecular weight (VHMW)] by expressing in F. tularensis a heterologous chain-length regulator gene ( wzz ) from the related species Francisella novicida Immunization with VHMW-TT provided markedly increased protection over that obtained with TT glycoconjugates made using smaller OAgs. We found that protective antibodies recognize a length-dependent epitope better expressed on HMW and VHMW antigens, which bind with higher affinity to the organism., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Background: Tularaemia is a zoonotic disease caused by inoculation with the Gram-negative coccobacillus Francisella tularensis. It was first described in the United States in 1911 and is a rare disease with an annual reported incidence in France between 2002 and 2012 of 0.07 cases per 100,000 habitants. Reporting of the disease in humans has been mandatory in France since 2003., Patients and Methods: Herein we report a case of tularaemia following a tick bite in a patient in the north of France., Discussion: Tularaemia is a rare form of zoonosis that should be sought in the event of unexplained adenitis. Clinical presentations vary, and in certain cases only dermatological signs are manifest. Diagnosis is confirmed by bacterial serology. Rapid initiation of suitable antibiotics produces a favourable and benign outcome in most cases. However, the offending organism, which is potentially lethal, is classed as a potential bioterrorism agent., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Francisella tularensis (Ft) is a biothreat agent for which there is no FDA-approved human vaccine. Currently, there are substantial efforts underway to develop both vaccines and the tools to assess these vaccines. Tularemia laboratory research has historically relied primarily upon a small number of inbred mouse strains, but the utility of such findings to outbred animals may be limited. Specifically, C57BL/6 mice are more susceptible than BALB/c mice to Ft infection and less easily protected against challenge with highly virulent type A Ft. Thus, depending on the inbred mouse strain used, one could be misled as to which immunogen(s)/vaccine will ultimately be effective in an outbred human population. Accordingly, we evaluated an outbred Swiss Webster (SW) mouse model in direct comparison to a well-established, inbred C57BL/6 mouse model. Mucosal vaccination with the live, attenuated Ft LVS superoxide dismutase (sodB) mutant demonstrated significantly higher protection in outbred SW mice compared to inbred C57BL/6 mice against Ft SchuS4 respiratory challenge. The protection observed in vaccinated outbred mice correlated with lower bacterial density, reduced tissue inflammation, and reduced levels of pro-inflammatory cytokine production. This protection was CD4+ and CD8+ T cell-dependent and characterized by lower titers of serum antibody (Ab) that qualitatively differed from vaccinated inbred mice. Enhanced protection of vaccinated outbred mice correlated with early and robust production of IFN-γ and IL-17A. Neutralizing Ab administered at the time of challenge revealed that IFN-γ was central to this protection, while IL-17A neutralization did not alter bacterial burden or survival. The present study demonstrates the utility of the outbred mouse as an alternative vaccination model for testing tularemia vaccines. Given the limited MHC repertoire in inbred mice, this outbred model is more analogous to the human in terms of immunological diversity., Competing Interests: The authors have declared that no competing interests exist.

Tularemia has sustained seroprevalence in Eurasia, with estimates as high as 15% in endemic regions. The purpose of this report is to characterise the current epidemiology of Francisella tularensis subspecies holarctica in Georgia. Three surveillance activities are summarised: (1) acute infections captured in Georgia's notifiable disease surveillance system, (2) infectious disease seroprevalence study of military volunteers, and (3) a study of seroprevalence and risk factors in endemic regions. Descriptive analyses of demographic, exposure and clinical factors were conducted for the surveillance studies; bivariate analyses were computed to identify risk factors of seropositivity using likelihood ratio χ2 tests or Fisher's exact tests. Of the 19 incident cases reported between 2014 and August 2017, 10 were confirmed and nine met the presumptive definition; the estimated annual incidence was 0.12/100 000. The first cases of tularemia in Western Georgia were reported. Seroprevalences of antibodies for F. tularensis were 2.0% for military volunteers and 5.0% for residents in endemic regions. Exposures correlated with seropositivity included work with hay and contact with multiple types of animals. Seroprevalence studies conducted periodically may enhance our understanding of tularemia in countries with dramatically underestimated incidence rates.

Disease tolerance can preserve host homeostasis and limit the negative impact of infections. We report that vaccinated mice survived pulmonary challenge with the extremely virulent SchuS4 strain of Francisella tularensis for at least 100 days, despite the persistence of large numbers (~104) of organisms. Transfer of 100 of these resident bacteria to naive animals caused 100% lethality, demonstrating that virulence was maintained. Tissue damage in the lung was limited over the course of infection and was associated with increased levels of amphiregulin. Mice depleted of CD4+ cells had reduced amphiregulin and succumbed to infection. In addition, neutralization of interferon-γ or depletion of CD8+ cells resulted in increased pathogen loads, bacteremia, and death of the host. Conversely, depletion of Ly6G+ neutrophils had no effect on survival and actually resulted in reduced bacterial levels. Understanding the interplay between host resistance and disease tolerance will provide new insights into the understanding of chronic infectious diseases.

Bacteria that are highly virulent, expressing high infectivity, and able to survive nebulization, pose great risk to the human population. One of these is Francisella tularensis, the etiological agent of tularemia. F. tularensis is a subject of intense scientific interest due to the fact that vaccines for its immunoprophylaxis in humans are not yet routinely available. One of the substantial obstacles in developing such vaccines is our insufficient knowledge of processes that initiate and regulate the expression of effective protective immunity against intracellular bacteria. Here, we present data documenting the different pattern of cellular behavior occurring in an environment unaffected by microbiota using the model of germ-free mice mono-associated with F. tularensis subsp. holarctica strain LVS in comparison with a classic specific-pathogen-free murine model during early stages of infection., (Copyright © 2018. Published by Elsevier Ltd.)

Tularaemia is a rare zoonotic disease caused by Francisella tularensis in humans. In Europe infections of humans and animals are mainly caused by F. tularensis subspecies holarctica. We report the first three documented cases of tularaemia in humans in Western Austria., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Tularemia is an infectious zoonosis caused by Francisella tularensis , an aerobic, noncapsulated, Gram-negative coccobacillus. It is more common in the northern hemisphere, and there are sporadic reports in non-endemic areas. The bacterium is usually transmitted by the bite or feces of a tick or other arthropods such as mosquitoes and horseflies. We report a case of an Italian patient with tularemia after a horsefly bite.

There are no defined correlates of protection for any intracellular pathogen, including the bacterium Francisella tularensis, which causes tularemia. Evaluating vaccine efficacy against sporadic diseases like tularemia using field trials is problematic, and therefore alternative strategies to test vaccine candidates like the Francisella Live Vaccine Strain (LVS), such as testing in animals and applying correlate measurements, are needed. Recently, we described a promising correlate strategy that predicted the degree of vaccine-induced protection in mice given parenteral challenges, primarily when using an attenuated Francisella strain. Here, we demonstrate that using peripheral blood lymphocytes (PBLs) in this approach predicts LVS-mediated protection against respiratory challenge of Fischer 344 rats with fully virulent F. tularensis, with exceptional sensitivity and specificity. Rats were vaccinated with a panel of LVS-derived vaccines and subsequently given lethal respiratory challenges with Type A F. tularensis. In parallel, PBLs from vaccinated rats were evaluated for their functional ability to control intramacrophage Francisella growth in in vitro co-culture assays. PBLs recovered from co-cultures were also evaluated for relative gene expression using a large panel of genes identified in murine studies. In vitro control of LVS intramacrophage replication reflected the hierarchy of protection. Further, despite variability between individuals, 22 genes were significantly more up-regulated in PBLs from rats vaccinated with LVS compared to those from rats vaccinated with the variant LVS-R or heat-killed LVS, which were poorly protective. These genes included IFN-γ, IL-21, NOS2, LTA, T-bet, IL-12rβ2, and CCL5. Most importantly, combining quantifications of intramacrophage growth control with 5-7 gene expression levels using multivariate analyses discriminated protected from non-protected individuals with greater than 95% sensitivity and specificity. The results therefore support translation of this approach to non-human primates and people to evaluate new vaccines against Francisella and other intracellular pathogens., Competing Interests: The authors have declared that no competing interest exist.

Bacillus anthracis, Yersinia pestis, and Francisella tularensis are the causative agents of Tier 1 Select Agents anthrax, plague, and tularemia, respectively. Currently, there are no licensed vaccines against plague and tularemia and the licensed anthrax vaccine is suboptimal. Here we report F. tularensis LVS ΔcapB (Live Vaccine Strain with a deletion in capB)- and attenuated multi-deletional Listeria monocytogenes (Lm)-vectored vaccines against all three aforementioned pathogens. We show that LVS ΔcapB- and Lm-vectored vaccines express recombinant B. anthracis, Y. pestis, and F. tularensis immunoprotective antigens in broth and in macrophage-like cells and are non-toxic in mice. Homologous priming-boosting with the LVS ΔcapB-vectored vaccines induces potent antigen-specific humoral and T-cell-mediated immune responses and potent protective immunity against lethal respiratory challenge with all three pathogens. Protection against anthrax was far superior to that obtained with the licensed AVA vaccine and protection against tularemia was comparable to or greater than that obtained with the toxic and unlicensed LVS vaccine. Heterologous priming-boosting with LVS ΔcapB- and Lm-vectored B. anthracis and Y. pestis vaccines also induced potent protective immunity against lethal respiratory challenge with B. anthracis and Y. pestis. The single vaccine platform, especially the LVS ΔcapB-vectored vaccine platform, can be extended readily to other pathogens.

Mucosa-associated invariant T (MAIT) cells are a subset of innate T cells that express a semi-invariant Vα chain paired with limited Vβ chains. MAIT cells are activated by riboflavin metabolite derivatives presented by the nonpolymorphic major histocompatibility complex class I (MHC-I)-like molecule MR1. The precise mechanisms required to activate MAIT cells are an area of intense interest. Here we used two closely related intracellular pathogens with distinct inflammasome activation phenotypes to probe the role of innate cytokines in MAIT cell activation. Using an in vitro assay containing transgenic murine MAIT cells, we show that macrophages infected with Francisella novicida , a strong inflammasome activator, released high levels of interleukin-18 (IL-18) and stimulated high levels of MAIT cell gamma interferon (IFN-γ) through a partially MR1-independent pathway. In contrast, macrophages infected with Francisella tularensis live vaccine strain (LVS), a weak inflammasome activator, generated little IL-18 and stimulated low MAIT cell IFN-γ through an MR1-dependent pathway. By manipulating the quantities of IL-18 in these cultures, we show that the IL-18 concentration is sufficient to influence the magnitude of MAIT cell IFN-γ production. Correspondingly, infected IL-18-deficient macrophages failed to induce substantial MAIT cell IFN-γ. In contrast, we found that MAIT cell IFN-γ production in the lungs of IL-18-deficient mice was not significantly different from that in WT mice during F. tularensis LVS pulmonary infection. Overall, we demonstrate that while IL-18 is essential for the MAIT cell IFN-γ response in vitro , it is not essential for MAIT cell IFN-γ production during in vivo LVS pulmonary infection, suggesting that additional signals can drive MAIT cell IFN-γ production in vivo ., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Tularemia is a fatal human disease caused by Francisella tularensis, a Gram-negative encapsulated coccobacillus bacterium. Due to its low infectious dose, ease of aerosolized transmission, and lethal effects, the CDC lists F. tularensis as a Category A pathogen, the highest level for a potential biothreat agent. Previous vaccine studies have been conducted with live attenuated, inactivated, and subunit vaccines, which have achieved partial or full protection from F. tularensis live vaccine strain (LVS) challenge, but no vaccine has been approved for human use. We demonstrate the improved efficacy of a multi-antigen subunit vaccine by using Tobacco Mosaic virus (TMV) as an antigen carrier for the F. tularensis SchuS4 proteins DnaK, OmpA, SucB and Tul4 (DOST). The magnitude and quality of immune responses were compared after mice were immunized by subcutaneous or intranasal routes of administration with a TMV-DOST mixture, with or without four different adjuvants. Immune responses varied in magnitude and isotype profile, by antigen, by route of administration, and by protection in an F. tularensis LVS challenge model of disease. Interestingly, our analysis demonstrates an overwhelming IgG2 response to SucB after intranasal dosing, as well as a robust cellular response, which may account for the improved two-dose survival imparted by the tetravalent vaccine, compared to a previous study that tested efficacy of TMV-DOT. Our study provides evidence that potent humoral, cellular and mucosal immunity can be achieved by optimal antigen combination, delivery, adjuvant and appropriate route of administration, to improve vaccine potency and provide protection from pathogen challenge.

The CDC Tier 1 select agent Francisella tularensis is a small, Gram-negative bacterium and the causative agent of tularemia, a potentially life-threatening infection endemic in the United States, Europe and Asia. Currently, there is no licensed vaccine or rapid point-of-care diagnostic test for tularemia. The purpose of this research was to develop monoclonal antibodies (mAbs) specific to the F. tularensis surface-expressed lipopolysaccharide (LPS) for a potential use in a rapid diagnostic test. Our initial antigen capture ELISA was developed using murine IgG3 mAb 1A4. Due to the low sensitivity of the initial assay, IgG subclass switching, which is known to have an effect on the functional affinity of a mAb, was exploited for the purpose of enhancing assay sensitivity. The ELISA developed using the IgG1 or IgG2b mAbs from the subclass-switch family of 1A4 IgG3 yielded improved assay sensitivity. However, surface plasmon resonance (SPR) demonstrated that the functional affinity was decreased as a result of subclass switching. Further investigation using direct ELISA revealed the potential self-association of 1A4 IgG3, which could explain the higher functional affinity and higher assay background seen with this mAb. Additionally, the higher assay background was found to negatively affect assay sensitivity. Thus, enhancement of the assay sensitivity by subclass switching is likely due to the decrease in assay background, simply by avoiding the self-association of IgG3.

Francisella tularensis is an intracellular bacterium that causes the disease tularemia. There are several subspecies of F. tularensis whose ability to cause disease varies in humans. The most virulent subspecies, tularensis , is a Tier One Select Agent and a potential bioweapon. Although considerable effort has made to generate efficacious tularemia vaccines, to date none have been licensed for use in the United States. Despite the lack of a tularemia vaccine, we have learned a great deal about the adaptive immune response the underlies protective immunity. Herein, we detail the animal models commonly used to study tularemia and their recapitulation of human disease, the field's current understanding of vaccine-mediated protection, and discuss the challenges associated with new vaccine development.

The aim of this work was to ascertain the usefulness of a new commercially-available single-assay chemiluminescence test (CHT) for the diagnosis of human tularemia (Tularaemia VIRCLIA IgG + IgM monotest, Vircell, Santa Fe, Granada, Spain). A total of 773 sera from 773 patients including 364 initial sera from patients with diagnosed tularemia, patients with suspected tularemia not confirmed (100), healthy people (152), patients with serology positive to Brucella (97), patients diagnosed with other infectious diseases (30), and patients diagnosed with autoimmune diseases (30) were included. All sera were tested by CHT, "in-house" microagglutination test (MAT), immunochromatographic test (ICT) (Virapid Tularaemia, Vircell, Santa Fe Granada, Spain), and "in-house" ELISA IgG, and ELISA IgM. Of the total initial sera, 334 (sensitivity 91.8%) were positive in the CHT, 332 (sensitivity 91.2%) in the MAT, 330 (sensitivity 90.7%) in the ICT, and 328 (sensitivity 90.1%) in the ELISA IgG and ELISA IgM tests. The specificity of the CHT was 96.7%; of the MAT, 100%; of the ICT, 98.7%; and of the ELISA IgG and ELISA IgM, 97.4%. In the group of patients with serology positive to Brucella, at least 12.4% of sera were positive in tularemia tests (12.4% in ELISA IgM, 13.4% in MAT, 14.4% in ICT, and 15.5% in CHT and ELISA IgG). In conclusion, CHT presents a sensitivity and specificity in early diagnosis of human tularemia, similar to MAT, ICT, and ELISA IgG and ELISA IgM. Its single assay design allows lower costs, especially in areas of low endemicity or inter-epidemic periods.

Francisella tularensis is a remarkably infectious facultative intracellular bacterium of macrophages that causes tularemia. Early evasion of host immune responses contributes to the success of F. tularensis as a pathogen. F. tularensis entry into human monocytes and macrophages is mediated by the major phagocytic receptor, complement receptor 3 (CR3, CD11b/CD18). We recently determined that despite a significant increase in macrophage uptake following C3 opsonization of the virulent Type A F. tularensis spp. tularensis Schu S4, this phagocytic pathway results in limited pro-inflammatory cytokine production. Notably, MAP kinase/ERK activation is suppressed immediately during C3-opsonized Schu S4-CR3 phagocytosis. A mathematical model of CR3-TLR2 crosstalk predicted early involvement of Ras GTPase-activating protein (RasGAP) in immune suppression by CR3. Here, we link CR3-mediated uptake of opsonized Schu S4 by human monocytes and macrophages with inhibition of early signal 1 inflammasome activation, evidenced by limited caspase-1 cleavage and IL-18 release. This inhibition is due to increased RasGAP activity, leading to a reduction in the Ras-ERK signaling cascade upstream of the early inflammasome activation event. Thus, our data uncover a novel signaling pathway mediated by CR3 following engagement of opsonized virulent F. tularensis to limit inflammasome activation in human phagocytic cells, thereby contributing to evasion of the host innate immune system.