Chronic Infection Model Research Articles

Identification of a nitrite reductase in Pseudomonas aeruginosa as a potential antimicrobial target

The opportunistic pathogen Pseudomonas aeruginosa utilizes a wide range of virulence factors to adapt to the host environment. With the antimicrobial pipeline drying up, understanding and targeting virulence factors for therapeutic development is an exciting alternative for the discovery of novel disease inhibitors. An integrated genome-wide transposon mutagenesis screening approach was performed in P. aeruginosa using multiple in vivo disease models with the aim to identify new virulence factors required for infection. A mutant attenuated in the production of multiple virulence determinants using in vitro assays was identified. This mutant also showed severe attenuation using in vivo models with up to an 80 % increased survival in murine chronic and acute lung infection models. The predicted protein coded by the mutated gene showed homology to nitrite and sulphite reductases. Using a methyl viologen reduction assay, we have shown that this gene encondes a nitrite reductase, operating in a siroheme and 4Fe-4S dependant manner. The preference for nitrite and the requirement of siroheme revealed that product of this gene is an assimilatory nitrite reductase and hence we propose it to be named as NirA. Work is now on-going to understand how NirA contributes to virulence and determine the crystal structure of this protein with a view to screen for novel inhibitors of this enzyme using a drug discovery platform available in our laboratories.

Identification of Potential Candidate Genes of Oral Cancer in Response to Chronic Infection With Porphyromonas gingivalis Using Bioinformatical Analyses.

Recent investigations revealed the relationship between chronic periodontitis, Porphyromonas gingivalis and cancer. However, host genes that change in response to chronic infection with P. gingivalis and may contribute to oral cancer have remained largely unknown. In the present study, we aimed to comprehensively analyze microarray data obtained from the chronic infection model of immortalized oral epithelial cells that were persistently exposed to P. gingivalis for 15 weeks. Using protein-protein interaction (PPI) networks and Ingenuity Pathway Analysis (IPA), we identified hub genes, major biological processes, upstream regulators and genes potentially involved in tumor initiation and progression. We also validated gene expression and demonstrated genetic alteration of hub genes from clinical samples of head and neck cancer. Overall, we utilized bioinformatical methods to identify IL6, STAT1, LYN, BDNF, C3, CD274, PDCD1LG2, and CXCL10 as potential candidate genes that might facilitate the prevention and treatment of oral squamous cell carcinoma (OSCC), the most common type of head and neck squamous cell carcinoma (HNSCC).

The Transcription Factor TCF1 Preserves the Effector Function of Exhausted CD8 T Cells During Chronic Viral Infection.

The long-term persistence of viral antigens drives virus-specific CD8 T cell exhaustion during chronic viral infection. Yet exhausted, CD8 T cells are still endowed with certain levels of effector function, by which they can keep viral replication in check in chronic infection. However, the regulatory factors involved in regulating the effector function of exhausted CD8 T cell are largely unknown. Using mouse model of chronic LCMV infection, we found that the deletion of transcription factor TCF-1 in LCMV-specific exhausted CD8 T cells led to the profound reduction in cytokine production and degranulation. Conversely, ectopic expression of TCF-1 or using agonist to activate TCF-1 activities promotes the effector function of exhausted CD8 T cells. Mechanistically, TCF-1 fuels the functionalities of exhausted CD8 T cells by promoting the expression of an array of key effector function-associated transcription regulators, including Foxo1, Zeb2, Id3, and Eomes. These results collectively indicate that targeting TCF-1 mediated transcriptional pathway may represent a promising immunotherapy strategy against chronic viral infections by reinvigorating the effector function of exhausted virus-specific CD8 T cells.

TLR4 signaling improves PD-1 blockade therapy during chronic viral infection.

CD8 T cells are necessary for the elimination of intracellular pathogens, but during chronic viral infections, CD8 T cells become exhausted and unable to control the persistent infection. Programmed cell death-1 (PD-1) blockade therapies have been shown to improve CD8 T cell responses during chronic viral infections. These therapies have been licensed to treat cancers in humans, but they have not yet been licensed to treat chronic viral infections because limited benefit is seen in pre-clinical animal models of chronic infection. In the present study, we investigated whether TLR4 triggering could improve PD-1 therapy during a chronic viral infection. Using the model of chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, we show that TLR4 triggering with sublethal doses of lipopolysaccharide (LPS) followed by PD-1 blockade results in superior improvement in circulating virus-specific CD8 T cell responses, relative to PD-1 blockade alone. Moreover, we show that the synergy between LPS and PD-1 blockade is dependent on B7 costimulation and mediated by a dendritic cell (DC) intrinsic mechanism. Systemic LPS administration may have safety concerns, motivating us to devise a safer regimen. We show that ex vivo activation of DCs with LPS, followed by adoptive DC transfer, results in a similar potentiation of PD-1 therapy without inducing wasting disease. In summary, our data demonstrate a previously unidentified role for LPS/TLR4 signaling in modulating the host response to PD-1 therapy. These findings may be important for developing novel checkpoint therapies against chronic viral infection.

Abstract B038: A unified genome-wide analysis of dysfunctional T-cell states in cancer and chronic viral infection

Abstract Tumor-specific T-cells that have differentiated into a terminal dysfunctional state exist in the tumor microenvironment. A systematic understanding of the requirements of immunotherapeutic rescue of these cells is critically needed to improve clinical results in patients. Mouse models of chronic infection and cancer have been studied to elucidate biologic mechanisms of persistent antigen stimulation resulting in T-cell dysfunction, or “exhaustion.” Recently, chromatin accessibility imprinting has been associated with T-cells falling back into the dysfunctional state after temporary rescue by checkpoint blockade, suggesting epigenetic mechanisms in control of T-cell dysfunction. However, comprehensive characterization of T-cell dysfunction across models based on their epigenetic and transcriptional profiles is lacking.We collected 106 chromatin accessibility (ATAC-seq) samples and 87 gene expression (RNA-seq) samples from seven recent publications. We analyzed these data by first applying batch effect correction using generalized linear modeling. This enabled mapping profiles of chromatin accessibility peaks in gene promoters and enhancers from different studies into the same space. We observed that epigenetic profiles of dysfunctional tumor-infiltrating T-cells and dysfunctional T-cells in chronic viral infection were, surprisingly, extremely similar. Furthermore, a recently characterized discrete distinction between epigenetic profiles of early (day 7-8) and late (day 28-35) dysfunction in the tumor was recapitulated in the model of chronic infection. Overall we observed across mouse models that T-cells committed to becoming dysfunctional early after an immune challenge, rather than first mounting and then loosing an effector response. These observations were also largely recapitulated in gene expression analysis. Differentially expressed genes with massive differential accessibility of their promoter and enhancer peaks during development of dysfunction, observed consistently across models, including transcription factors (TF) well studied in immunity such as Tcf7, Lef1, Satb1, Ikzf2, Tox, are good candidates for further targeted analysis.We then turned to TF binding analysis. We associated absolute levels of chromatin accessibility in peaks of each sample with TF binding (predicted by motif analysis) using regularized negative binomial regression with cross-validation. We estimated the effect of each TF in each sample, which allowed us to map chromatin accessibility profiles into the TF activity space of much lower dimensionality. This mapping largely preserved the hierarchy of relative similarities between samples. We identified key TFs whose binding was associated with open or closed chromatin in functional and dysfunctional cell states. For example, not surprisingly, binding of well known effector factors Eomes and Batf was associated with closed chromatin in naive cells and open chromatin in effector cells. Strikingly, the strongest association with closing chromatin in dysfunction, consistently across mouse models, was observed for Tcf7/Lef1 binding, further suggesting the role of these TFs in establishing the terminal CD8 T-cell dysfunctional state. Notably, we found two large groups of TFs anti-correlated to each other whose predicted binding sites had higher accessibility in either functional or dysfunctional cells. This suggested that coordinated activity of a broad range of TFs (not necessarily binding at the same sites) might be responsible for establishing and maintaining T-cell functional states, and focusing on one or a handful of TFs is not sufficient to explain them. This analysis provides a better systematic understanding of cell-intrinsic mechanisms driving different functional states of CD8 T-cells, and the developed computational methods are broadly applicable in other experimental setups where diverse cell states are profiled by high-throughput genomic assays. Citation Format: Yuri Pritykin, Christina Leslie. A unified genome-wide analysis of dysfunctional T-cell states in cancer and chronic viral infection [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B038.

Abstract B012: Characterizing tumor-induced exhaustion in melanoma patients treated with neoadjuvant pembrolizumab

Abstract Immune checkpoint blockade, including anti-PD-1 therapies, has had unprecedented success in treating various forms of cancer, including melanoma. Yet the majority of patients do not achieve durable clinical remission. The limited understanding of how these therapies work at the cellular and molecular level prevents the optimization required to improve patient outcomes. We performed RNA-seq and ATAC-seq on four sorted T-cell subpopulations (naïve CD8 T-cells, non-naïve CD8, non-naïve CD4 T-cells, T regulatory cells) from patients with metastatic melanoma before and after pembrolizumab treatment. While there were no significant changes before and after treatment in the sorted peripheral blood populations, we identified many alterations in the tumor compared to the blood following treatment. These differences may represent impediments to fully reinvigorating exhausted T-cells and be potential targets for achieving more durable clinical responses. We identified a zinc finger gene, ZC3H12C, as not only one of the most highly upregulated in non-naïve CD8 T-cells in the tumor, but it also had the highest number of associated altered open chromatin regions. This gene and its locus are also significantly different in exhausted T-cells compared to effector and memory cells in a mouse model of chronic viral infection using Lymphocytic choriomeningitis (LCMV). This conservation between species suggest an important role in T-cell exhaustion and provided an avenue to test its function in vivo, so we generated ZC3H12C knockout mice using CRISPR-Cas9. Following C13 (chronic) LCMV infection, virus-specific P14 CD8 T-cells lacking ZC3H12C are unable to persist compared to wild type P14 cells. These results suggest ZC3H12C may be critical to the survival of exhausted T-cells in the tumor and their ability to persist after anti-PD1 treatment. Here, we used transcriptomic and epigenetic analysis of a clinical human cancer data set to identify a novel target and used reverse translation in a mouse model to validate its importance in T-cell exhaustion. This gene and more broadly this strategy have the potential to provide immediately useful targets for improving immunotherapies. Citation Format: Josephine R. Giles, Alexander C. Huang, Saskinath Manne, Jorge Henao-Meji, E. John Wherry. Characterizing tumor-induced exhaustion in melanoma patients treated with neoadjuvant pembrolizumab [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B012.

Macrophage infection with combinations of BCG mutants reduces induction of TNF-α, IL-6, IL-1β and increases IL-4

Tuberculosis (TB) is the most prevalent infectious disease worldwide, with no fully effective vaccine yet available. Considering that BCG strains devoid of the BCG1416c or BCG1419c genes afforded protection in mice versus highly virulent M. tuberculosis challenge, or in chronic infection models compared to BCG, respectively, we hypothesized that a synergistic effect of these strains might occur and provide enhanced protection against TB. Herein, we evaluated this hypothesis throughout an experimental design approach, where different combinations of these strains were tested for their capacity to induce cytokines in vitro, compared to individual strains. Our results show that mixed-infection of murine macrophages using these strains significantly decreases induction of TNF-α, IL-1β, IL-6 but increases IL-4 induction compared with individual strains. These results suggest the existence of interaction effects during infection, which reduce induction of pro-inflammatory cytokines, even though individual intracellular replication is not altered when strains are combined. This is the first report of the evaluation of a potential whole-live combined vaccine against tuberculosis, which paradoxically seems to reduce production of pro-inflammatory cytokines while induces IL-4, leading us to further hypothesize that this combination might contribute as a therapeutic vaccine to reduce inflammation in severe TB cases.

Risk Assessment of Etanercept in Mice Chronically Infected With Toxoplasma gondii.

Toxoplasma gondii (T. gondii) is a zoonotic parasite that severely harms the health of the host. The cysts of T. gondii can reactivate from bradyzoites to tachyzoites, if the individual develops low or defective immunity, causing lethal toxoplasmosis. The host resists T. gondii infection by mediating Th1-type cellular immunity to generate pro-inflammatory cytokines. Tumor necrosis factor (TNF) is an important pro-inflammatory cytokine, which can induce lysosomal fusion of parasitophorous vacuole (PV) to kill parasites. Etanercept is a soluble TNF receptor fusion protein, which is widely used clinically to cure autoimmune diseases. The effects and specific molecular mechanisms of etanercept treatment on patients co-infected with autoimmune diseases and chronic toxoplasmosis are rarely reported. In our study, a mouse model of chronic infection with T. gondii and murine macrophages RAW264.7 cells infected with T. gondii were employed to investigate the impact of etanercept on the status of chronic infection. The cytokines levels and a series of phenotypic experiments in vivo and in vitro were measured. In the present study, the expression levels of TNF, IL-1β, and IL-6 were decreased and the brain cysts number was increased in mice chronically infected with T. gondii after being treated with etanercept. In vivo experiments confirmed that etanercept caused a decrease in the immune levels of the mice and activated the brain cysts, which would lead to conversion from chronic infection to acute infection, causing severe clinical and pathological symptoms. Murine macrophages RAW264.7 cells were pretreated with etanercept, and then infected with T. gondii. In vitro experiments, the expression levels of cytokines were decreased, indicating that etanercept could also reduce the cells’ immunity and promote the transformation of bradyzoites to tachyzoites, but did not affect the intracellular replication of tachyzoites. In summary, etanercept treatment could activate the conversion of bradyzoites to tachyzoites through reducing host immunity in vivo and in vitro. The results obtained from this study suggest that the use of etanercept in patients co-infected with autoimmune diseases and chronic toxoplasmosis may lead to the risk of activation of chronic infection, resulting in severe acute toxoplasmosis.

Efficient and reproducible experimental infections of rats with Blastocystis spp.

Although Blastocystis spp. infect probably more than 1 billion people worldwide, their clinical significance is still controversial and their pathophysiology remains poorly understood. In this study, we describe a protocol for an efficient and reproducible model of chronic infection in rats, laying the groundwork for future work to evaluate the pathogenic potential of this parasite. In our experimental conditions, we were unable to infect rats using vacuolar forms of an axenically cultivated ST4 isolate, but we successfully established chronic infections of 4 week-old rats after oral administration of both ST3 and ST4 purified cysts isolated from human stool samples. The infection protocol was also applied to 4 week-old C57BL/9, BALB/C and C3H mice, but any mouse was found to be infected by Blastocystis. Minimal cyst inoculum required for rat infection was higher with ST3 (105) than with ST4 (102). These results were confirmed by co-housing experiments highlighting a higher contagious potential of ST4 in rats compared to ST3. Finally, experiments mimicking fecal microbiota transfer from infected to healthy animals showed that Blastocystis spp. could easily infect a new host, even though its intestinal microbiota is not disturbed. In conclusion, our results provide a well-documented and robust rat model of Blastocystis chronic infection, reproducing “natural” infection. This model will be of great interest to study host parasite interactions and to better evaluate clinical significance of Blastocystis.

Exposure to Freeze-Thaw Conditions Increases Virulence of Pseudomonas aeruginosa to Drosophila melanogaster.

Groundwater contamination by pathogenic bacteria present in land-applied manure poses a threat to public health. In cold climate regions, surface soil layers experience repeated temperature fluctuations around the freezing point known as freeze-thaw (FT) cycles. With global climate change, annual soil FT cycles have increased, and this trend is expected to continue. It is therefore of interest to understand how FT cycles impact soil microbial communities. This study investigates the influence of FT cycles on the growth, culturability, biofilm formation, and virulence of the bacterial opportunistic pathogen Pseudomonas aeruginosa, a ubiquitous bacterium found in soil and water, responsible for infections in immunocompromised hosts. Our findings demonstrate that exposure to FT had no significant effect on growth or culturability of the bacteria. However, FT treatment significantly increased biofilm formation and delayed the onset of swimming motility, factors that are important for the pathogenicity of P. aeruginosa. An in vivo study using a chronic infection model revealed an increase in the virulence of P. aeruginosa after FT exposure. These results suggest that the impact of climate change on natural FT cycles may be affecting the ecology of soil-borne pathogens and host-pathogen interactions in unexpected ways.

Intrahepatic Cross-Presentation and Hepatocellular Antigen Presentation Play Distinct Roles in the Induction of Hepatitis B Virus-Specific CD8+ T Cell Responses.

CD8+ T cells are the key cellular effectors mediating the clearance of hepatitis B virus (HBV) infections. However, early immunological events surrounding the priming of HBV-specific CD8+ T cell responses remain poorly understood. This study examined the importance of priming location and the relative contribution of endogenous antigen presentation by hepatocytes versus cross-presentation by bone marrow-derived cells to the induction of functional HBV-specific CD8+ T cell responses using the animal models of acute and chronic HBV infection. Functional HBV-specific CD8+ T cell responses could be induced to intrahepatically expressed HBV even when T cell homing to the lymphoid tissues was severely suppressed, suggesting that functional priming could occur in the liver. The expansion of HBV-specific CD8+ T cells was significantly reduced in the mice whose major histocompatibility complex (MHC) class I expression was mostly restricted to nonhematopoietic cells, suggesting the importance of cross-presentation by hematopoietic cells in the induction of HBV-specific CD8+ T cells. Strikingly, the expansion and cytolytic differentiation of HBV-specific CD8+ T cells were reduced even more severely in the mice whose MHC class I expression was restricted to hematopoietic cells. Collectively, these results indicate that cross-presentation is required but relatively inefficient in terms of inducing the cytolytic differentiation of HBV-specific CD8+ T cells by itself. Instead, the expansion and functional differentiation of HBV-specific CD8+ T cells are primarily dependent on hepatocellular antigen presentation.IMPORTANCE Hepatitis B virus (HBV) causes acute and chronic hepatitis. Approximately 260 million people are chronically infected with HBV and under an increased risk of developing cirrhosis and hepatocellular carcinoma. Host immune responses, particularly HBV-specific CD8+ T cell responses, largely determine the outcome of HBV infection. It is widely accepted that antigen inexperienced CD8+ T cells should be initially activated by professional antigen-presenting cells (pAPCs) in lymphoid tissues to differentiate into effector CD8+ T cells. However, this notion has not been tested for HBV-specific CD8+ T cells. In this study, we show that HBV-specific CD8+ T cell responses can be induced in the liver. Surprisingly, antigen presentation by hepatocytes is more important than cross-presentation by hematopoietic cells for the induction of HBV-specific CD8+ T cell responses. These results revealed a previously unappreciated role of antigen presentation by hepatocytes in the induction of HBV-specific CD8+ T cell responses.

Human Lymph Nodes Maintain TCF-1hi Memory T Cells with High Functional Potential and Clonal Diversity throughout Life.

Translating studies on T cell function and modulation from mouse models to humans requires extrapolating in vivo results on mouse T cell responses in lymphoid organs (spleen and lymph nodes [LN]) to human peripheral blood T cells. However, our understanding of T cell responses in human lymphoid sites and their relation to peripheral blood remains sparse. In this study, we used a unique human tissue resource to study human T cells in different anatomical compartments within individual donors and identify a subset of memory CD8+ T cells in LN, which maintain a distinct differentiation and functional profile compared with memory CD8+ T cells in blood, spleen, bone marrow, and lungs. Whole-transcriptome and high-dimensional cytometry by time-of-flight profiling reveals that LN memory CD8+ T cells express signatures of quiescence and self-renewal compared with corresponding populations in blood, spleen, bone marrow, and lung. LN memory T cells exhibit a distinct transcriptional signature, including expression of stem cell-associated transcription factors TCF-1 and LEF-1, T follicular helper cell markers CXCR5 and CXCR4, and reduced expression of effector molecules. LN memory T cells display high homology to a subset of mouse CD8+ T cells identified in chronic infection models that respond to checkpoint blockade immunotherapy. Functionally, human LN memory T cells exhibit increased proliferation to TCR-mediated stimulation and maintain higher TCR clonal diversity compared with memory T cells from blood and other sites. These findings establish human LN as reservoirs for memory T cells with high capacities for expansion and diverse recognition and important targets for immunotherapies.

Giving antibodies a boost

Antibodies Persistent immune activation during chronic infections is often associated with increased generation and deposition of immune complexes. The actions of antibody-based drugs can thus be severely impaired in individuals with chronic infections. Using lymphocytic choriomeningitis virus (LCMV) as a model of chronic infection, Wieland et al. examined how to enhance antibody functions in this setting. The ability of antibodies to deplete target cells was dependent on antigen expression levels. Furthermore, afucosylation of antibodies directed against CD4 and CD8α enhanced their ability to deplete CD4+ and CD8+ T cells in mice persistently infected with LCMV. Whether afucosylation can be universally used to enhance antibody functions during chronic infections remains to be seen. Sci. Immunol. 3 , eaao3125 (2018).

Optimization and Lead Selection of Benzothiazole Amide Analogs Toward a Novel Antimycobacterial Agent.

Mycobacteria remain an important problem worldwide, especially drug resistant human pathogens. Novel therapeutics are urgently needed to tackle both drug-resistant tuberculosis (TB) and difficult-to-treat infections with nontuberculous mycobacteria (NTM). Benzothiazole adamantyl amide had previously emerged as a high throughput screening hit against M. tuberculosis (Mtb) and was subsequently found to be active against NTM as well. For lead optimization, we applied an iterative process of design, synthesis and screening of several 100 analogs to improve antibacterial potency as well as physicochemical and pharmacological properties to ultimately achieve efficacy. Replacement of the adamantyl group with cyclohexyl derivatives, including bicyclic moieties, resulted in advanced lead compounds that showed excellent potency and a mycobacteria-specific spectrum of activity. MIC values ranged from 0.03 to 0.12 μg/mL against M. abscessus (Mabs) and other rapid- growing NTM, 1–2 μg/mL against M. avium complex (MAC), and 0.12–0.5 μg/mL against Mtb. No pre-existing resistance was found in a collection of n = 54 clinical isolates of rapid-growing NTM. Unlike many antibacterial agents commonly used to treat mycobacterial infections, benzothiazole amides demonstrated bactericidal effects against both Mtb and Mabs. Metabolic labeling provided evidence that the compounds affect the transfer of mycolic acids to their cell envelope acceptors in mycobacteria. Mapping of resistance mutations pointed to the trehalose monomycolate transporter (MmpL3) as the most likely target. In vivo efficacy and tolerability of a benzothiazole amide was demonstrated in a mouse model of chronic NTM lung infection with Mabs. Once daily dosing over 4 weeks by intrapulmonary microspray administration as 5% corn oil/saline emulsion achieved statistically significant CFU reductions compared to vehicle control and non-inferiority compared to azithromycin. The benzothiazole amides hold promise for development of a novel therapeutic agent with broad antimycobacterial activity, though further work is needed to develop drug formulations for direct intrapulmonary delivery via aerosol.

Inhibition of HBV Expression in HBV Transgenic Mice Using AAV-Delivered CRISPR-SaCas9

The chronic production of hepatitis B viral (HBV) antigens could cause inflammation and necrosis, leading to elevation of liver enzymes from necrotic hepatocytes, hepatitis, cirrhosis, hepatocellular carcinoma, and liver failure. However, no current treatment is capable of significantly reducing HBsAg expression in patients. Our previous studies had confirmed the ability of CRISPR-Cas9 in disrupting HBV cccDNA. Here, to inhibit HBV expression efficiently in the mouse model of chronic HBV infection, the miniaturized CRISPR-SaCas9 system compatible with a HBV core region derived guide-RNA had been packaged in recombinant adeno-associated virus (AAV) type 8, which lowered the levels of serum HBsAg, HBeAg, and HBV DNA efficiently in HBV transgenic mice during 58 days continuous observation after vein injection. It further confirms the potential of the CRISPR-Cas9 technique for use in hepatitis B gene therapy.

Phage therapy for respiratory infections.

A respiratory infection caused by antibiotic-resistant bacteria can be life-threatening. In recent years, there has been tremendous effort put towards therapeutic application of bacteriophages (phages) as an alternative or supplementary treatment option over conventional antibiotics. Phages are natural parasitic viruses of bacteria that can kill the bacterial host, including antibiotic-resistant bacteria. Inhaled phage therapy involves the development of stable phage formulations suitable for inhalation delivery followed by preclinical and clinical studies for assessment of efficacy, pharmacokinetics and safety. We presented an overview of recent advances in phage formulation for inhalation delivery and their efficacy in acute and chronic rodent respiratory infection models. We have reviewed and presented on the prospects of inhaled phage therapy as a complementary treatment option with current antibiotics and as a preventative means. Inhaled phage therapy has the potential to transform the prevention and treatment of bacterial respiratory infections, including those caused by antibiotic-resistant bacteria.

A novel small RNA Bmsr1 enhances virulence in Brucella melitensis M28

Brucellosis, caused by Brucella spp., is one of the most serious zoonotic bacterial diseases. Small RNAs (sRNAs) are recognized as a key player in bacterial post-transcription regulation, since they participate in many biological processes with high efficiency and may govern the intracellular biochemistry and virulence of some pathogenic bacteria. Here, a novel small regulatory RNA, Bmsr1 (Brucella melitensis M28 small RNA 1), was identified in a virulent Brucella melitensis M28 strain based on bioinformatic analysis, reverse transcription PCR (RT-PCR), and Northern blot. The Bmsr1 expression level was highly induced after infection of macrophage cells RAW264.7 at 48 h, suggesting a role for Bmsr1 during in vitro infection. Indeed, bmsr1 deletion mutant of M28 attenuated its intracellular survival in RAW264.7 at 24 h and 48 h post-infection. In a mouse model of chronic infection, bmsr1 deletion strain displayed decreased colonization in the spleen while Bmsr1-overexpressed strain showed higher colonization levels than wild type pathogen. Isobaric tags for relative and absolute quantification (iTRAQ) revealed that 314 proteins were differentially expressed in M28Δbmsr1 compared with wild type. Functional annotation analysis demonstrated that most of those proteins are involved in biological processes and those proteins in the ribosome and nitrogen metabolism pathways were enriched. iTRAQ results combined with target prediction identified several potential target genes related to virulence, including virB2, virB9, virB10, virB11, and vjbR and many metabolism genes. Taken together, this study revealed the contribution of a novel sRNA Bmsr1 to virulence of B. melitensis M28, probably by influencing genes involved in T4SS, virulence regulator VjbR and other metabolism genes.

Characterizing interactions of Leptospira interrogans with proximal renal tubule epithelial cells

BackgroundLeptospira interrogans is a pathogenic, spirochetal bacterium that is responsible for leptospirosis, an emerging worldwide zoonosis. Leptospires colonize the renal proximal tubules and chronically infect the kidney. Live bacteria are excreted into urine, contaminating the environment. While it is well known that leptospires can persist in the kidneys without signs of disease for several months, the interactions of leptospires with the proximal renal epithelial tubule cells that allow the chronic renal colonization have not been elucidated yet. In the present study, we compared the interactions between a virulent, low passage (LP) strain and a cultured-attenuated, high passage (HP) strain with renal proximal tubule epithelial cells (RPTECs) to elucidate the strategies used by Leptospira to colonize the kidney.ResultsKinetics analysis of kidney colonization in a mouse model of chronic infection performed by quantitative real-time PCR and immunofluorescence, showed that the LP strain reached the kidney by 3 days post infection (pi) and attached to the basal membrane side of the renal epithelial cells. At 10 days pi, some leptospires were attached to the luminal side of the tubular epithelia and the number of colonizing leptospires gradually increased. On the other hand, the HP strain was cleared during hematogenous dissemination and did not colonize the kidney. Transmission electron microscopy analysis of LP-infected kidneys at 25 days pi showed aggregated leptospires and membrane vesicles attached to the epithelial brush border. Leptospiral kidney colonization altered the organization of the RPTEC brush border. An in vitro model of infection using TCMK-1 cells, showed that leptospiral infection induced a host stress response, which is delayed in LP-infected cells.ConclusionsAfter hematogenous dissemination, leptospires create protective and replicative niches in the base membrane and luminal sides of the RPTECs. During the long-term colonization, leptospires attached to the RPTEC brush borders and membrane vesicles might be involved in the formation of a biofilm-like structure in vivo. Our results also suggested that the virulent strain is able to manipulate host cell stress responses to promote renal colonization.

Abstract SY07-02: Epigenetic regulation of T-cell exhaustion: Implications for cancer immunotherapy

Abstract Immune checkpoint blockade (ICB)-mediated rejuvenation of CD8 T-cell effector functions has emerged as one of the most promising frontiers for treating cancer and chronic infections. However, antigen-specific T cells that have experienced prolonged antigen exposure are often terminally differentiated, and have a limited capacity to mount an effector response during ICB treatment. Such exhaustion of effector potential is a major impediment of current T cell-based immunotherapy efforts. Using in vivo mouse models of tumor and chronic viral infection, we assessed the role of de novo epigenetic programming in establishing ICB-refractory exhausted T cells. We observed that genetic deletion of the de novo DNA methyltransferase, Dnmt3a, in T cells at the effector stage of an immune response to chronic lymphocytic choriomeningitis virus (LCMV) infection allowed antigen-specific T cells to remain highly functional despite expressing high levels of PD-1 and having prolonged exposure to antigen. Quite strikingly, PD-1 blockade treatment of chronically infected animals resulted in massive expansion of PD-1+ Dnmt3a-deficient antigen-specific T cells. Whole-genome methylation profiling of WT and Dnmt3a-deficient LCMV-specific CD8 T cells identified de novo DNA methylation programs that are coupled to development of ICB-nonresponsive virus and tumor-specific T cells. Building upon these findings, we have identified de novo epigenetic programs acquired in human tumor-associated PD-1hi CD8 T cells. Collectively, these data establish Dnmt3a-mediated de novo DNA methylation programming as a key regulator in establishing ICB-refractory exhausted CD8 T cells and highlight epigenetic reprogramming of T cells as a novel approach to enhance T cell-based cancer therapies. Citation Format: Benjamin A. Youngblood, Hazem Ghoniem, Ardiana Moustaki, Hossam Abdelsamed, Yiping Fan, Paul Thomas, Elizabeth Stewart, Sara Federico. Epigenetic regulation of T-cell exhaustion: Implications for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY07-02.

CD4 T Cell Affinity Diversity Is Equally Maintained during Acute and Chronic Infection.

TCR affinity for peptide MHC dictates the functional efficiency of T cells and their propensity to differentiate into effectors and form memory. However, in the context of chronic infections, it is unclear what the overall profile of TCR affinity for Ag is and if it differs from acute infections. Using the comprehensive affinity analysis provided by the two-dimensional micropipette adhesion frequency assay and the common indirect affinity evaluation methods of MHC class II tetramer and functional avidity, we tracked IAb GP61-80-specific cells in the mouse model of acute (Armstrong) and chronic (clone 13) lymphocytic choriomeningitis virus infection. In each response, we show CD4 T cell population affinity peaks at the effector phase and declines with memory. Of interest, the range and average relative two-dimensional affinity was equivalent between acute and chronic infection, indicating chronic Ag exposure did not skew TCR affinity. In contrast, functional and tetramer avidity measurements revealed divergent results and lacked a consistent correlation with TCR affinity. Our findings highlight that the immune system maintains a diverse range in TCR affinity even under the pressures of chronic Ag stimulation.