In vivo and in vitro immune responses against Francisella tularensis vaccines are comparable among Fischer 344 rat substrains.

Identifying suitable animal models and standardizing preclinical methods are important for the generation, characterization, and development of new vaccines, including those against Francisella tularensis. Non-human primates represent an important animal model to evaluate tularemia vaccine efficacy, and the use of correlates of vaccine-induced protection may facilitate bridging immune responses from non-human primates to people. However, among small animals, Fischer 344 rats represent a valuable resource for initial studies to evaluate immune responses, to identify correlates of protection, and to screen novel vaccines. In this study, we performed a comparative analysis of three Fischer rat substrains to determine potential differences in immune responses, to evaluate methods used to quantify potential correlates of protection, and to evaluate protection after vaccination. To this end, we took advantage of data previously generated using one of the rat substrains by evaluating two live vaccines, LVS and F. tularensis SchuS4-ΔclpB (ΔclpB). We compared immune responses after primary vaccination, adaptive immune responses upon re-stimulation of leukocytes in vitro, and sensitivity to aerosol challenge. Despite some detectable differences, the results highlight the similarity of immune responses to tularemia vaccines and challenge outcomes between the three substrains, indicating that all offer acceptable and comparable approaches as animal models to study Francisella infection and immunity.

Mice with lung airway ciliopathy develop persistent Mycobacterium abscessus lung infection and have a proinflammatory lung phenotype associated with decreased T regulatory cells.

Introduction: Human pulmonary infection with non-tuberculous mycobacteria (NTM) such as Mycobacterium abscessus (Mabs) occurs in seemingly immunocompetent patients with underlying structural lung disease such as bronchiectasis in which normal ciliary function is perturbed. In addition to alterations in mucociliary clearance, the local immunologic milieu may be altered in patients with structural lung disease, but the nature of these changes and how they relate to NTM persistence remain unclear. Methods: We used a mouse strain containing a conditional floxed allele of the gene IFT88, which encodes for the protein Polaris. Deletion of this gene in adult mice reportedly leads to loss of cilia on lung airway epithelium and to the development of bronchiectasis. In a series of experiments, IFT88 control mice and IFT88 KO mice received different preparations of Mabs lung inocula with lung CFU assessed out to approximately 8 weeks post-infection. In addition, cytokine levels in bronchoalveolar lavage (BAL) fluid, lung T cell subset analysis, and lung histopathology and morphometry were performed at various time points. Results: Mabs embedded in agarose beads persisted in the lungs of IFT88 KO mice out to approximately 8 weeks (54 days), while Mabs agarose beads in the lungs of IFT88 control mice was cleared from the lungs of all mice at this time point. T cells subset analysis showed a decrease in the percentage of CD4+FoxP3+ T cells in the total lymphocyte population in the lungs of IFT88 KO mice relative to IFT88 control mice. Proinflammatory cytokines were elevated in the BAL fluid from infected IFT88 KO mice compared to infected IFT88 control mice, and histopathology showed an increased inflammatory response and greater numbers of granulomas in the lungs of infected IFT88 KO mice compared to the lungs of infected IFT88 control mice. Scanning lung morphometry did not show a significant difference comparing lung airway area and lung airway perimeter between IFT88 KO mice and IFT88 control mice. Discussion: Persistent lung infection in our model was established using Mabs embedded in agarose beads. The utility of using IFT88 mice is that a significant difference in Mabs lung CFU is observed comparing IFT88 KO mice to IFT88 control mice thus allowing for studies assessing the mechanism(s) of Mabs lung persistence. Our finding of minimal differences in lung airway area and lung airway diameter comparing IFT88 KO mice to IFT88 control mice suggests that the development of a proinflammatory lung phenotype in IFT88 KO mice contributes to Mabs lung persistence independent of bronchiectasis. The contribution of cilia to immune regulation is increasingly recognized, and our results suggest that ciliopathy associated with structural lung disease may play a role in NTM pulmonary infection via alteration of the local immunologic lung milieu.

Working correlates of protection predict SchuS4-derived-vaccine candidates with improved efficacy against an intracellular bacterium, Francisella tularensis.

Francisella tularensis, the causative agent of tularemia, is classified as Tier 1 Select Agent with bioterrorism potential. The efficacy of the only available vaccine, LVS, is uncertain and it is not licensed in the U.S. Previously, by using an approach generally applicable to intracellular pathogens, we identified working correlates that predict successful vaccination in rodents. Here, we applied these correlates to evaluate a panel of SchuS4-derived live attenuated vaccines, namely SchuS4-ΔclpB, ΔclpB-ΔfupA, ΔclpB-ΔcapB, and ΔclpB-ΔwbtC. We combined in vitro co-cultures to quantify rodent T-cell functions and multivariate regression analyses to predict relative vaccine strength. The predictions were tested by rat vaccination and challenge studies, which demonstrated a clear relationship between the hierarchy of in vitro measurements and in vivo vaccine protection. Thus, these studies demonstrated the potential power a panel of correlates to screen and predict the efficacy of Francisella vaccine candidates, and in vivo studies in Fischer 344 rats confirmed that SchuS4-ΔclpB and ΔclpB-ΔcapB may be better vaccine candidates than LVS.

Identification of an Attenuated Substrain of Francisella tularensis SCHU S4 by Phenotypic and Genotypic Analyses.

Pneumonic tularemia is a highly debilitating and potentially fatal disease caused by inhalation of Francisella tularensis. Most of our current understanding of its pathogenesis is based on the highly virulent F. tularensis subsp. tularensis strain SCHU S4. However, multiple sources of SCHU S4 have been maintained and propagated independently over the years, potentially generating genetic variants with altered virulence. In this study, the virulence of four SCHU S4 stocks (NR-10492, NR-28534, NR-643 from BEI Resources and FTS-635 from Battelle Memorial Institute) along with another virulent subsp. tularensis strain, MA00-2987, were assessed in parallel. In the Fischer 344 rat model of pneumonic tularemia, NR-643 and FTS-635 were found to be highly attenuated compared to NR-10492, NR-28534, and MA00-2987. In the NZW rabbit model of pneumonic tularemia, NR-643 caused morbidity but not mortality even at a dose equivalent to 500x the LD50 for NR-10492. Genetic analyses revealed that NR-10492 and NR-28534 were identical to each other, and nearly identical to the reference SCHU S4 sequence. NR-643 and FTS-635 were identical to each other but were found to have nine regions of difference in the genomic sequence when compared to the published reference SCHU S4 sequence. Given the genetic differences and decreased virulence, NR-643/FTS-635 should be clearly designated as a separate SCHU S4 substrain and no longer utilized in efficacy studies to evaluate potential vaccines and therapeutics against tularemia.

COVID-19 global pandemic planning: Decontamination and reuse processes for N95 respirators.

IMPACT STATEMENT: There is a critical shortage of personal protective equipment (PPE) around the globe. This article describes the safe collection, storage, and decontamination of N95 respirators using hydrogen peroxide vapor (HPV). This article is unique because it describes the HPV process in an operating room, and is therefore, a deployable method for many healthcare settings. Results presented here offer creative solutions to the current PPE shortage.

Increased Mortality in Mice following Immunoprophylaxis Therapy with High Dosage of Nicotinamide in Burkholderia Persistent Infections.

Bacterial persistence, known as noninherited antibacterial resistance, is a factor contributing to the establishment of long-lasting chronic bacterial infections. In this study, we examined the ability of nicotinamide (NA) to potentiate the activity of different classes of antibiotics against Burkholderia thailandensis persister cells. Here we demonstrate that addition of NA in in vitro models of B. thailandensis infection resulted in a significant depletion of the persister population in response to various classes of antibiotics. We applied microfluidic bioreactors with a continuous medium flow to study the effect of supplementation with an NA gradient on the recovery of B. thailandensis persister populations. A coculture of human neutrophils preactivated with 50 µM NA and B. thailandensis resulted in the most efficient reduction in the persister population. Applying single-cell RNA fluorescence in situ hybridization analysis and quantitative PCR, we found that NA inhibited gene expression of the stringent response regulator relA, implicated in the regulation of the persister metabolic state. We also demonstrate that a therapeutic dose of NA (250 mg/kg of body weight), previously applied as immunoprophylaxis against antibiotic-resistant bacterial species, produced adverse effects in an in vivo murine model of infection with the highly pathogenic bacterium Burkholderia pseudomallei, indicating that therapeutic dose and metabolite effects have to be carefully evaluated and tailored for every case of potential clinical application.

A panel of correlates predicts vaccine-induced protection of rats against respiratory challenge with virulent Francisella tularensis.

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-12rß2, 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.

High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems.

The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis, we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host-targeted therapies against infectious disease caused by intracellular pathogens.

The Natural History of Pneumonic Tularemia in Female Fischer 344 Rats after Inhalational Exposure to Aerosolized Francisella tularensis Subspecies tularensis Strain SCHU S4.

The inbred Fischer 344 rat is being evaluated for testing novel vaccines and therapeutics against pneumonic tularemia. Although primary pneumonic tularemia in humans typically occurs by inhalation of aerosolized bacteria, the rat model has relied on intratracheal inoculation of organisms because of safety and equipment issues. We now report the natural history of pneumonic tularemia in female Fischer 344 rats after nose-only inhalational exposure to lethal doses of aerosolized Francisella tularensis subspecies tularensis, strain SCHU S4. Our results are consistent with initial uptake of aerosolized SCHU S4 from the nasal cavity, lungs, and possibly the gastrointestinal tract. Bacteremia with hematogenous dissemination was first detected 2 days after exposure. Shortly thereafter, the infected rats exhibited fever, tachypnea, and hypertension that persisted for 24 to 36 hours and then rapidly decreased as animals succumbed to infection between days 5 and 8 after exposure. Tachycardia was observed briefly, but only after the core body temperature and blood pressure began to decrease as the animals were near death. Initial neutrophilic and histiocytic inflammation in affected tissues became progressively more fibrinous and necrotizing over time. At death, as many as 1010 colony-forming units were found in the lungs, spleen, and liver. Death was attributed to sepsis and disseminated intravascular coagulation. Overall, the pathogenesis of pneumonic tularemia in the female F344 rat model appears to replicate the disease in humans.

A stable live bacterial vaccine.

Formulating vaccines into a dry form enhances its thermal stability. This is critical to prevent administering damaged and ineffective vaccines, and to reduce its final cost. A number of vaccines in the market as well as those being evaluated in the clinical setting are in a dry solid state; yet none of these vaccines have achieved long-term stability at high temperatures. We used spray-drying to formulate a recombinant live attenuated Listeria monocytogenes (Lm; expressing Francisella tularensis immune protective antigen pathogenicity island protein IglC) bacterial vaccine into a thermostable dry powder using various sugars and an amino acid. Lm powder vaccine showed minimal loss in viability when stored for more than a year at ambient room temperature (∼23°C) or for 180days at 40°C. High temperature viability was achieved by maintaining an inert atmosphere in the storage container and removing oxygen free radicals that damage bacterial membranes. Further, in vitro antigenicity was confirmed by infecting a dendritic cell line with cultures derived from spray dried Lm and detection of an intracellularly expressed protective antigen. A combination of stabilizing excipients, a cost effective one-step drying process, and appropriate storage conditions could provide a viable option for producing, storing and transporting heat-sensitive vaccines, especially in regions of the world that require them the most.

Progression of cancer from indolent to aggressive despite antigen retention and increased expression of interferon-gamma inducible genes.

Many cancers escape host immunity without losing tumor-specific rejection antigens or MHC class I expression. This study tracks the evolution of one such cancer that developed in a mouse following exposure to ultraviolet light. The primary autochthonous tumor was not highly malignant and was rejected when transplanted into naïve immunocompetent mice. Neoplastic cells isolated from the primary tumor were susceptible to the growth-inhibitory effects of IFNγ in vitro, but expressed very low levels of MHC I antigen and were resistant to tumor-specific T cells unless they were first exposed to IFNγ. Serial passage of the primary tumor cells in vivo led to a highly aggressive variant that caused fast-growing tumors in normal mice. In vitro, the variant tumor cells showed increased resistance to the growth-inhibitory effects of IFNγ but expressed high levels of immunoproteasomes and MHC I molecules and were susceptible to tumor-specific T cells even without prior exposure to IFNγ.

Antibodies contribute to effective vaccination against respiratory infection by type A Francisella tularensis strains.

Pneumonic tularemia is a life-threatening disease caused by inhalation of the highly infectious intracellular bacterium Francisella tularensis. The most serious form of the disease associated with the type A strains can be prevented in experimental animals through vaccination with the attenuated live vaccine strain (LVS). The protection is largely cell mediated, but the contribution of antibodies remains controversial. We addressed this issue in a series of passive immunization studies in Fischer 344 (F344) rats. Subcutaneous LVS vaccination induced a robust serum antibody response dominated by IgM, IgG2a, and IgG2b antibodies. Prophylactic administration of LVS immune serum or purified immune IgG reduced the severity and duration of disease in naïve rats challenged intratracheally with a lethal dose of the virulent type A strain SCHU S4. The level of resistance increased with the volume of immune serum given, but the maximum survivable SCHU S4 challenge dose was at least 100-fold lower than that shown for LVS-vaccinated rats. Protection correlated with reduced systemic bacterial growth, less severe histopathology in the liver and spleen during the early phase of infection, and bacterial clearance by a T cell-dependent mechanism. Our results suggest that treatment with immune serum limited the sequelae associated with infection, thereby enabling a sterilizing T cell response to develop and resolve the infection. Thus, antibodies induced by LVS vaccination may contribute to the defense of F344 rats against respiratory infection by type A strains of F. tularensis.

The Fischer 344 rat reflects human susceptibility to francisella pulmonary challenge and provides a new platform for virulence and protection studies.

BACKGROUND: The pathogenesis of Francisella tularensis, the causative agent of tularemia, has been primarily characterized in mice. However, the high degree of sensitivity of mice to bacterial challenge, especially with the human virulent strains of F. tularensis, limits this animal model for screening of defined attenuated vaccine candidates for protection studies. METHODS AND FINDINGS: We analyzed the susceptibility of the Fischer 344 rat to pulmonary (intratracheal) challenge with three different subspecies (subsp) of F. tularensis that reflect different levels of virulence in humans, and characterized the bacterial replication profile in rat bone marrow-derived macrophages (BMDM). In contrast to the mouse, Fischer 344 rats exhibit a broader range of sensitivity to pulmonary challenge with the human virulent subsp. tularensis and holarctica. Unlike mice, Fischer rats exhibited a high degree of resistance to pulmonary challenge with LVS (an attenuated derivative of subsp. holarctica) and subsp. novicida. Within BMDM, subsp. tularensis and LVS showed minimal replication, subsp. novicida showed marginal replication, and subsp. holartica replicated robustly. The limited intramacrophage replication of subsp. tularensis and novicida strains was correlated with the induction of nitric oxide production. Importantly, Fischer 344 rats that survived pulmonary infection with subsp. novicida were markedly protected against subsequent pulmonary challenge with subsp. tularensis, suggesting that subsp. novicida may be a useful platform for the development of vaccines against subsp. tularensis. CONCLUSIONS: The Fischer 344 rat exhibits similar sensitivity to F. tularensis strains as that reported for humans, and thus the Fischer 344 ray may serve as a better animal model for tularemia vaccine development.

Vaccination of Fischer 344 rats against pulmonary infections by Francisella tularensis type A strains.

Pneumonic tularemia caused by inhalation of the type A strains of Francisella tularensis is associated with high morbidity and mortality in humans. The only vaccine known to protect humans against this disease is the attenuated live vaccine strain (LVS), but it is not currently registered for human use. To develop a new generation of vaccines, multiple animal models are needed that reproduce the human response to F. tularensis infection and vaccination. We examined the potential use of Fischer 344 rat as such a model. Fischer 344 rats were very sensitive to intratracheal infection with the virulent type A strain SCHU S4 and generally succumbed less than 2 weeks after infection. Similar to humans and non-human primates, Fischer 344 rats vaccinated with LVS by subcutaneous or intradermal routes were protected against a greater range of respiratory SCHU S4 challenge doses than has been reported for LVS vaccinated mice. Intratracheal LVS vaccination also induced effective immunity, but it was less protective when the challenge dose exceeded 10(5) SCHU S4. LVS vaccination did not prevent SCHU S4 infection but rather controlled bacterial growth and pathology, leading to the eventual clearance of the bacteria. Our results suggest that the Fischer 344 rat may be a good model for studying pneumonic tularemia and evaluating potential vaccine candidates.

T cells from lungs and livers of Francisella tularensis-immune mice control the growth of intracellular bacteria.

Parenteral and respiratory vaccinations with the intracellular bacterium Francisella tularensis have been studied using the live vaccine strain (LVS) in a mouse model, and spleen cells from immune mice are often used for immunological studies. However, mechanisms of host immunological responses may be different in nonlymphoid organs that are important sites of infection, such as lung and liver. Using parenteral (intradermal) or respiratory (cloud aerosol) vaccination, here we examine the functions of resulting LVS-immune liver or lung cells, respectively. Surprisingly, LVS was considerably more virulent when administered by cloud aerosol than by intranasal instillation, suggesting method-dependent differences in initial localization and/or dissemination patterns. Only low doses were sublethal, and resolution of sublethal cloud aerosol infection was dependent on gamma interferon (IFN-gamma), tumor necrosis factor alpha, and inducible nitric oxide synthase. Nonetheless, survival of cloud aerosol or parenteral infection resulted in the development of a protective immune response against lethal LVS intraperitoneal or aerosol challenge, reflecting development of systemic secondary immunity in both cases. Such immunity was further detected by directly examining the functions of LVS-immune lung or liver lymphocytes in vitro. Lung lymphocytes primed by respiratory infection, as well as liver lymphocytes primed by parenteral infection, clearly controlled in vitro intracellular bacterial growth primarily via mechanisms that were not dependent on IFN-gamma activity. Thus, our results indicate functional similarities between immune T cells residing in spleens, livers, and lungs of LVS-immune mice.

Animal models of Francisella tularensis infection.

The increased incidence of emerging infections has caused a resurgence in the development of animal models in order to study their pathophysiology and develop therapeutics against them. Optimizing these models and improving our ability to extrapolate information from animals to humans is critical because in many cases the animal model will represent the only modality for efficacy testing. Francisella tularensis (F. tularensis) is an emerging pathogen that fits this category. While there is a significant body of literature that has examined infections with F. tularensis in a variety of species, the optimal small animal model has yet to be defined. A vast majority of studies have used two strains of F. tularensis, the more virulent type A strain commonly found in North America and the less virulent type B strain common to Europe. None of the small animal models described in the literature thus far behave in a fashion identical to humans with respect to their sensitivity to SCHU S4 (type A) or live vaccine strains (LVS) (attenuated type B) and an ability of LVS vaccination to consistently protect against a SCHU S4 aerosol challenge, suggesting that significant work on animal model development still remains. This report briefly describes the parameters important for animal model development and reviews the literature related to animal models of F. tularensis, including the human model, and the characterization performed for those models.

Intranasal vaccination induces protective immunity against intranasal infection with virulent Francisella tularensis biovar A.

The inhalation of Francisella tularensis biovar A causes pneumonic tularemia associated with high morbidity and mortality rates in humans. Exposure to F. tularensis usually occurs by accident, but there is increasing awareness that F. tularensis may be deliberately released in an act of bioterrorism or war. The development of a vaccine against pneumonic tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. Vaccine models for F. tularensis in inbred mice would facilitate investigations of the protective mechanisms and significantly enhance vaccine development. Intranasal vaccination with the attenuated live vaccine strain (LVS) of F. tularensis reproducibly protected BALB/c mice, but not C57BL/6 mice, against intranasal and subcutaneous challenges with a virulent clinical isolate of F. tularensis biovar A (NMFTA1). The resistance of LVS-vaccinated BALB/c mice to intranasal NMFTA1 challenge was increased 100-fold by boosting with live NMFTA1 but not with LVS. The protective response was specific for F. tularensis and required both CD4 and CD8 T cells. The vaccinated mice appeared outwardly healthy for more than 2 months after NMFTA1 challenge, even though NMFTA1 was recovered from more than half of the vaccinated mice. These results show that intranasal vaccination induces immunity that protects BALB/c mice from intranasal infection by F. tularensis biovar A.

Long-term suppression of tumor growth by TNF requires a Stat1- and IFN regulatory factor 1-dependent IFN-gamma pathway but not IL-12 or IL-18.

Tumor cells engineered to secrete TNF were used as a model to examine how persistently high local concentrations of TNF suppress tumor growth. TNF secretion had no effect on tumor cell proliferation in vitro but caused a very impressive growth arrest in vivo that was dependent on both bone marrow- and non-bone marrow-derived host cells expressing TNFR. Suppression also required an endogenous IFN-gamma pathway consisting minimally of IFN-gamma, IFN-gamma receptor, Stat1, and IFN regulatory factor 1 since mice with targeted disruption of any of the four genes failed to arrest tumor growth. The ability of these mice to suppress tumor growth was restored after they were reconstituted with bone marrow cells from Wt mice. Interestingly, mice lacking the major IFN-gamma-inducing cytokines IL-12 and IL-18 or T cells, B cells, and the majority of NK cells that are potential sources of IFN-gamma nevertheless inhibited tumor development. Moreover, multiple lines of evidence indicated that local release of IFN-gamma was not required to inhibit tumor formation. These results strongly suggest a novel function for the endogenous IFN-gamma pathway that without measurable IFN-gamma production or activity affects the ability of TNF to suppress tumor development.