Genetic mechanisms of Coxiella burnetii lipopolysaccharide phase variation.

Coxiella burnetii is an intracellular pathogen that causes human Q fever, a disease that normally presents as a severe flu-like illness. Due to high infectivity and disease severity, the pathogen is considered a risk group 3 organism. Full-length lipopolysaccharide (LPS) is required for full virulence and disease by C. burnetii and is the only virulence factor currently defined by infection of an immunocompetent animal. Transition of virulent phase I bacteria with smooth LPS, to avirulent phase II bacteria with rough LPS, occurs during in vitro passage. Semi-rough intermediate forms are also observed. Here, the genetic basis of LPS phase conversion was investigated to obtain a more complete understanding of C. burnetii pathogenesis. Whole genome sequencing of strains producing intermediate and/or phase II LPS identified several common mutations in predicted LPS biosynthesis genes. After passage in broth culture for 30 weeks, phase I strains from different genomic groups exhibited similar phase transition kinetics and elevation of mutations in LPS biosynthesis genes. Targeted mutagenesis and genetic complementation using a new C. burnetii nutritional selection system based on lysine auxotrophy confirmed that six of the mutated genes were necessary for production of phase I LPS. Disruption of two of these genes in a C. burnetii phase I strain resulted in production of phase II LPS, suggesting inhibition of the encoded enzymes could represent a new therapeutic strategy for treatment of Q fever. Additionally, targeted mutagenesis of genes encoding LPS biosynthesis enzymes can now be used to construct new phase II strains from different genomic groups for use in pathogen-host studies at a risk group 2 level.

Investigations into the Immunotoxicity and Allergic Potential Induced by Topical Application of N-Butylbenzenesulfonamide (NBBS) in a Murine Model.

N-Butylbenzene sulfonamide (NBBS) is a commonly used plasticizer found in numerous products. Due to its extensive use, lack of adequate toxicological data, and suspicion of toxicity based on the presence of structural alerts, it was nominated to the National Toxicology Program for comprehensive toxicological testing. The purpose of this study was to evaluate the potential for hypersensitivity and immune suppression following dermal exposure to NBBS using a murine model. NBBS tested negative in a combined irritancy/local lymph node assay (LLNA), classifying it as nonirritating and nonsensitizing. To estimate the immunosuppressive potential of NBBS, assays that assessed immunotoxicity were performed, including the immumnoglobulin (Ig) M response to T-cell-dependent antigen sheep red blood cells (SRBC), using the plaque-forming cell (PFC) assay and immune cell phenotyping. After a 28-d treatment with NBBS, mice exposed to the lowest concentration (25% NBBS) showed a significant increase in IgM-producing B cells in the spleen. No marked changes were identified in immune cell markers in the lymph node. In contrast to body weight, a significant elevation in kidney and liver weight was observed following dermal exposure to all concentrations of NBBS. These results demonstrate that dermal exposure to NBBS, other than liver and kidney toxicity, did not apparently induce immunotoxicity in a murine model.

Natural reversion promotes LPS elongation in an attenuated Coxiella burnetii strain.

Lipopolysaccharide (LPS) phase variation is a critical aspect of virulence in many Gram-negative bacteria. It is of particular importance to Coxiella burnetii, the biothreat pathogen that causes Q fever, as in vitro propagation of this organism leads to LPS truncation, which is associated with an attenuated and exempted from select agent status (Nine Mile II, NMII). Here, we demonstrate that NMII was recovered from the spleens of infected guinea pigs. Moreover, these strains exhibit a previously unrecognized form of elongated LPS and display increased virulence in comparison with the initial NMII strain. The reversion of a 3-bp mutation in the gene cbu0533 directly leads to LPS elongation. To address potential safety concerns, we introduce a modified NMII strain unable to produce elongated LPS.

A simple method for enrichment of phase I Coxiella burnetii.

Coxiella burnetii is the bacterial causative agent of the zoonosis Q fever. This bacterium undergoes lipopolysaccharide (LPS) phase transition similar to Enterobacteriaciae upon in vitro passage. Full-length, phase I C. burnetii LPS is a critical virulence factor and profoundly impacts vaccine-induced immunogenicity; thus, LPS phase is an important consideration in C. burnetii experimentation and Q fever vaccine design. Typically, phase I LPS-expressing organisms are obtained from the tissues of infected experimental animals. In this process, residual phase II LPS-expressing organisms are thought to be cleared by the host immune system. Here, we propose an efficient and non-animal-based method for the enrichment of C. burnetii phase I LPS-expressing bacteria in vitro. We utilize both Vero cell culture to selectively enrich solutions with phase I and intermediate phase LPS-expressing bacteria. This simple and quick method decreases reliance on experimental animals and is a sustainable solution for Q fever diagnostic and vaccine development hurdles.

Biodefence research two decades on: worth the investment?

For the past 20 years, the notion of bioterror has been a source of considerable fear and panic worldwide. In response to the terror attacks of 2001 in the USA, extensive research funding was awarded to investigate bioterror-related pathogens. The global scientific legacy of this funding has extended into the present day, highlighted by the ongoing COVID-19 pandemic. Unsurprisingly, the surge in biodefence-related research and preparedness has been met with considerable apprehension and opposition. Here, we briefly outline the history of modern bioterror threats and biodefence research, describe the scientific legacy of biodefence research by highlighting advances pertaining to specific bacterial and viral pathogens, and summarise the future of biodefence research and its relevance today. We sought to address the sizeable question: have the past 20 years of investment into biodefence research and preparedness been worth it? The legacy of modern biodefence funding includes advancements in biosecurity, biosurveillence, diagnostics, medical countermeasures, and vaccines. In summary, we feel that these advances justify the substantial biodefence funding trend of the past two decades and set a precedent for future funding.

Contributions of lipopolysaccharide and the type IVB secretion system to Coxiella burnetii vaccine efficacy and reactogenicity.

Coxiella burnetii is the bacterial causative agent of the zoonosis Q fever. The current human Q fever vaccine, Q-VAX®, is a fixed, whole cell vaccine (WCV) licensed solely for use in Australia. C. burnetii WCV administration is associated with a dermal hypersensitivity reaction in people with pre-existing immunity to C. burnetii, limiting wider use. Consequently, a less reactogenic vaccine is needed. Here, we investigated contributions of the C. burnetii Dot/Icm type IVB secretion system (T4BSS) and lipopolysaccharide (LPS) in protection and reactogenicity of fixed WCVs. A 32.5 kb region containing 23 dot/icm genes was deleted in the virulent Nine Mile phase I (NMI) strain and the resulting mutant was evaluated in guinea pig models of C. burnetii infection, vaccination-challenge, and post-vaccination hypersensitivity. The NMI ∆dot/icm strain was avirulent, protective as a WCV against a robust C. burnetii challenge, and displayed potentially altered reactogenicity compared to NMI. Nine Mile phase II (NMII) strains of C. burnetii that produce rough LPS, were similarly tested. NMI was significantly more protective than NMII as a WCV; however, both vaccines exhibited similar reactogenicity. Collectively, our results indicate that, like phase I LPS, the T4BSS is required for full virulence by C. burnetii. Conversely, unlike phase I LPS, the T4BSS is not required for vaccine-induced protection. LPS length does not appear to contribute to reactogenicity while the T4BSS may contribute to this response. NMI ∆dot/icm represents an avirulent phase I strain with full vaccine efficacy, illustrating the potential of genetically modified C. burnetii as improved WCVs.

Optimization of Single-Dose VSV-Based COVID-19 Vaccination in Hamsters.

The ongoing COVID-19 pandemic has resulted in global effects on human health, economic stability, and social norms. The emergence of viral variants raises concerns about the efficacy of existing vaccines and highlights the continued need for the development of efficient, fast-acting, and cost-effective vaccines. Here, we demonstrate the immunogenicity and protective efficacy of two vesicular stomatitis virus (VSV)-based vaccines encoding the SARS-CoV-2 spike protein either alone (VSV-SARS2) or in combination with the Ebola virus glycoprotein (VSV-SARS2-EBOV). Intranasally vaccinated hamsters showed an early CD8+ T cell response in the lungs and a greater antigen-specific IgG response, while intramuscularly vaccinated hamsters had an early CD4+ T cell and NK cell response. Intranasal vaccination resulted in protection within 10 days with hamsters not showing clinical signs of pneumonia when challenged with three different SARS-CoV-2 variants. This data demonstrates that VSV-based vaccines are viable single-dose, fast-acting vaccine candidates that are protective from COVID-19.

Optimization of single dose VSV-based COVID-19 vaccination in hamsters.

The ongoing COVID-19 pandemic has resulted in global effects on human health, economic stability, and social norms. The emergence of viral variants raises concerns about the efficacy of existing vaccines and highlights the continued need the for the development of efficient, fast-acting, and cost-effective vaccines. Here, we demonstrate the immunogenicity and protective efficacy of two vesicular stomatitis virus (VSV)-based vaccines encoding the SARS-CoV-2 spike protein either alone (VSV-SARS2) or in combination with the Ebola virus glycoprotein (VSV-SARS2-EBOV). Intranasally vaccinated hamsters showed an early CD8 + T cell response in the lungs and a greater antigen-specific IgG response, while intramuscularly vaccinated hamsters had an early CD4 + T cell and NK cell response. Intranasal vaccination resulted in protection within 10 days with hamsters not showing clinical signs of pneumonia when challenged with three different SARS-CoV-2 variants. This data demonstrates that VSV-based vaccines are viable single-dose, fast-acting vaccine candidates that are protective from COVID-19.

Novel cutaneous mediators of chemical allergy.

Chemical allergy can manifest into allergic contact dermatitis and asthma and the importance of skin sensitization in both of these diseases is increasingly being recognized. Given the unique characteristics of chemical allergy, coupled with the distinct immunological microenvironment of the skin research is still unraveling the mechanisms through which sensitization and elicitation occur. This review first describes the features of chemical sensitization and the known steps that must occur to develop a chemical allergy. Next, the unique immunological properties of the skin - which may influence chemical sensitization - are highlighted. Additionally, mediators involved with the development of allergy are reviewed, starting with early ones - including the properties of haptens, skin integrity, the microbiome, the inflammasome, and toll-like receptors (TLR). Novel cellular mediators of chemical sensitization are highlighted, including innate lymphoid cells, mast cells, T-helper (TH) cell subsets, and skin intrinsic populations including γδ T-cells and resident memory T-cells. Finally, this review discusses two epigenetic mechanisms that can influence chemical sensitization, microRNAs and DNA methylation. Overall, this review highlights recent research investigating novel mediators of chemical allergy that are present in the skin. It also emphasizes the need to further explore these mediators to gain a better understanding of what makes a chemical an allergen, and how best to prevent the development of chemical-induced allergic diseases.

Comparative virulence of diverse Coxiella burnetii strains.

Coxiella burnetii is an intracellular, gram-negative bacterium that causes the zoonosis Q fever. This disease typically presents as an acute flu-like illness with persistent, focalized infections occurring less frequently. Clinical outcomes of Q fever have been associated with distinct genomic groups of C. burnetii, suggesting that gene content is responsible for virulence potential. To investigate this hypothesis, the virulence of thirteen C. burnetii strains (representing genomic groups I-VI) was evaluated in a guinea pig infection model by intraperitoneal injection. Seven strains caused a sustained fever (at least two days ≥39.5°C) in at least half of the animals within each experimental group. At fourteen days post infection, animals were euthanized and additional endpoints were evaluated, including splenomegaly and serology. The magnitude of these endpoints roughly correlated with the onset, duration, and severity of fever. The most severe disease was caused by group I strains. Intermediate and no virulence were evidenced following infection with group II-V and group VI strains, respectively. Flow cytometric analysis of the mesenteric lymph nodes revealed decreased CD4+ T cell frequency following infection with highly virulent group I strains. These findings buttress the hypothesis that the pathogenic potential of C. burnetii strains correlates with genomic grouping. These data, combined with comparative genomics and genetic manipulation, will improve our understanding of C. burnetii virulence determinants.

Topical application of the anti-microbial chemical triclosan induces immunomodulatory responses through the S100A8/A9-TLR4 pathway.

The anti-microbial compound triclosan is incorporated into numerous consumer products and is detectable in the urine of 75% of the general United States population. Recent epidemiological studies report positive associations with urinary triclosan levels and allergic disease. Although not sensitizing, earlier studies previously found that repeated topical application of triclosan augments the allergic response to ovalbumin (OVA) though a thymic stromal lymphopoietin (TSLP) pathway in mice. In the present study, early immunological effects following triclosan exposure were further evaluated following topical application in a murine model. These investigations revealed abundant expression of S100A8/A9, which reportedly acts as an endogenous ligand for Toll-like Receptor 4 (TLR4), in skin tissues and in infiltrating leukocytes during topical application of 0.75-3.0% triclosan. Expression of Tlr4 along with Tlr1, Tlr2 and Tlr6 increased in skin tissues over time with triclosan exposure; high levels of TLR4 were expressed on skin-infiltrating leukocytes. In vivo antibody blockade of the TLR4/MD-2 receptor complex impaired local inflammatory responses after four days, as evidenced by decreased Il6, Tnfα, S100a8, S100a9, Tlr1, Tlr2, Tlr4 and Tlr6 expression in the skin and decreased lymph node cellularity and production of IL-4 and IL-13 by lymph node T-cells. After nine days of triclosan exposure with TLR4/MD-2 blockade, impaired T-helper cell type 2 (TH2) cytokine responses were sustained, but other early effects on skin and lymph node cellularity were lost; this suggested alternative ligands/receptors compensated for the loss of TLR4 signaling. Taken together, these data suggest the S100A8/A9-TLR4 pathway plays an early role in augmenting immunomodulatory responses with triclosan exposure and support a role for the innate immune system in chemical adjuvancy.

Robust growth of avirulent phase II Coxiella burnetii in bone marrow-derived murine macrophages.

Published data show that murine bone marrow-derived macrophages (BMDM) restrict growth of avirulent phase II, but not virulent phase I, Coxiella burnetii. Growth restriction of phase II bacteria is thought to result from potentiated recognition of pathogen-associated molecular patterns, which leads to production of inhibitory effector molecules. Past studies have used conditioned medium from L-929 murine fibroblasts as a source of macrophage-colony stimulating factor (M-CSF) to promote differentiation of bone marrow-derived myeloid precursors into macrophages. However, uncharacterized components of conditioned medium, such as variable amounts of type I interferons, can affect macrophage activation status and their permissiveness for infection. In the current study, we show that the C. burnetii Nine Mile phase II (NMII) strain grows robustly in primary macrophages from C57BL/6J mice when bone marrow cells are differentiated with recombinant murine M-CSF (rmM-CSF). Bacteria were readily internalized by BMDM, and replicated within degradative, LAMP1-positive vacuoles to achieve roughly 3 logs of growth over 6 days. Uninfected BMDM did not appreciably express CD38 or Egr2, markers of classically (M1) and alternatively (M2) activated macrophages, respectively, nor did infection change the lack of polarization. In accordance with an M0 phenotype, infected BMDM produced moderate amounts of TNF and nitric oxide. Similar NMII growth results were obtained using C57BL/6J myeloid progenitors immortalized with an estrogen-regulated Hoxb8 (ER-Hoxb8) oncogene. To demonstrate the utility of the ER-Hoxb8 system, myeloid progenitors from natural resistance-associated macrophage protein 1 (Nramp1) C57BL/6J knock-in mice were transduced with ER-Hoxb8, and macrophages were derived from immortalized progenitors using rmM-CSF and infected with NMII. No difference in growth was observed when compared to macrophages from wild type mice, indicating depletion of metal ions by the Nramp1 transporter does not negatively impact NMII growth. Results with NMII were recapitulated in primary macrophages where C57BL/6J Nramp1+ BMDM efficiently killed Salmonella enterica serovar Typhimurium. M-CSF differentiated murine macrophages from bone marrow and conditional ER-Hoxb8 myeloid progenitors will be useful ex vivo models for studying Coxiella-macrophage interactions.

Investigations of immunotoxicity and allergic potential induced by topical application of triclosan in mice.

Triclosan is an antimicrobial chemical commonly used occupationally and by the general public. Using select immune function assays, the purpose of these studies was to evaluate the immunotoxicity of triclosan following dermal exposure using a murine model. Triclosan was not identified to be a sensitizer in the murine local lymph node assay (LLNA) when tested at concentrations ranging from 0.75-3.0%. Following a 28-day exposure, triclosan produced a significant increase in liver weight at concentrations of ≥ 1.5%. Exposure to the high dose (3.0%) also produced a significant increase in spleen weights and number of platelets. The absolute number of B-cells, T-cells, dendritic cells and NK cells were significantly increased in the skin draining lymph node, but not the spleen. An increase in the frequency of dendritic cells was also observed in the lymph node following exposure to 3.0% triclosan. The IgM antibody response to sheep red blood cells (SRBC) was significantly increased at 0.75% - but not at the higher concentrations - in the spleen and serum. These results demonstrate that dermal exposure to triclosan induces stimulation of the immune system in a murine model and raise concerns about potential human exposure.

Triclosan Induces Thymic Stromal Lymphopoietin in Skin Promoting Th2 Allergic Responses.

Triclosan is an antimicrobial chemical incorporated into many personal, medical and household products. Approximately, 75% of the U.S. population has detectable levels of triclosan in their urine, and although it is not typically considered a contact sensitizer, recent studies have begun to link triclosan exposure with augmented allergic disease. We examined the effects of dermal triclosan exposure on the skin and lymph nodes of mice and in a human skin model to identify mechanisms for augmenting allergic responses. Triclosan (0%-3%) was applied topically at 24-h intervals to the ear pinnae of OVA-sensitized BALB/c mice. Skin and draining lymph nodes were evaluated for cellular responses and cytokine expression over time. The effects of triclosan (0%-0.75%) on cytokine expression in a human skin tissue model were also examined. Exposure to triclosan increased the expression of TSLP, IL-1ß, and TNF-α in the skin with concomitant decreases in IL-25, IL-33, and IL-1α. Similar changes in TSLP, IL1B, and IL33 expression occurred in human skin. Topical application of triclosan also increased draining lymph node cellularity consisting of activated CD86(+)GL-7(+) B cells, CD80(+)CD86(+) dendritic cells, GATA-3(+)OX-40(+)IL-4(+)IL-13(+) Th2 cells and IL-17 A(+) CD4 T cells. In vivo antibody blockade of TSLP reduced skin irritation, IL-1ß expression, lymph node cellularity, and Th2 responses augmented by triclosan. Repeated dermal exposure to triclosan induces TSLP expression in skin tissue as a potential mechanism for augmenting allergic responses.

Toluene diisocyanate (TDI) disposition and co-localization of immune cells in hair follicles.

Diisocyanates (dNCOs) are potent chemical allergens utilized in various industries. It has been proposed that skin exposure to dNCOs produces immune sensitization leading to work-related asthma and allergic disease. We examined dNCOs sensitization by using a dermal murine model of toluene diisocyanate (TDI) exposure to characterize the disposition of TDI in the skin, identify the predominant haptenated proteins, and discern the associated antigen uptake by dendritic cells. Ears of BALB/c mice were dosed once with TDI (0.1% or 4% v/v acetone). Ears and draining lymph nodes (DLNs) were excised at selected time points between 1 h and 15 days post-exposure and were processed for histological, immunohistochemical, and proteomic analyses. Monoclonal antibodies specific for TDI-haptenated protein (TDI-hp) and antibodies to various cell markers were utilized with confocal microscopy to determine co-localization patterns. Histopathological changes were observed following exposure in ear tissue of mice dosed with 4% TDI/acetone. Immunohistochemical staining demonstrated TDI-hp localization in the stratum corneum, hair follicles, and sebaceous glands. TDI-hp were co-localized with CD11b(+) (integrin αM/Mac-1), CD207(+) (langerin), and CD103(+) (integrin αE) cells in the hair follicles and in sebaceous glands. TDI-hp were also identified in the DLN 1 h post-exposure. Cytoskeletal and cuticular keratins along with mouse serum albumin were identified as major haptenated species in the skin. The results of this study demonstrate that the stratum corneum, hair follicles, and associated sebaceous glands in mice are dendritic cell accessible reservoirs for TDI-hp and thus identify a mechanism for immune recognition following epicutaneous exposure to TDI.