The altered TBI fecal microbiome is stable and functionally distinct.

Introduction: Patients who suffer a traumatic brain injury (TBI) often experience chronic and sometimes debilitating sequelae. Recent reports have illustrated both acute and long-term dysbiosis of the gastrointestinal microbiome with significant alterations in composition and predicted functional consequences. Methods: Working with participants from past research, metagenomic stability of the TBI- associated fecal microbiome (FMB) was evaluated by custom qPCR array comparing a fecal sample from 2015 to one collected in 2020. Metatranscriptomics identified differently expressed bacterial genes and biochemical pathways in the TBI FMB. Microbiota that contributed the largest RNA amounts identified a set of core bacteria most responsible for functional consequences of the TBI FMB. Results: A remarkably stable FMB metagenome with significant similarity (two-tail Spearman nonparametric correlation p < 0.001) was observed between 2015 and 2020 fecal samples from subjects with TBI. Comparing the 2020 TBI FMB metagenome to FMBs from healthy controls confirmed and extended the dysbiotic genera and species. Abundance differences between average TBI and healthy FMBs revealed Bacteroides caccae, B. uniformis, Blautia spp., Collinsella spp., Dialister spp., and Ordoribacter spp. were significantly different. Functionally, the Parabacteroides genus contributed the highest percentage of RNA sequences in control FMBs followed by the Bacteroides genus as the second highest contributor. In the TBI FMB, the Corynebacterium genus contributed the most RNA followed by the Alistipes genus. Corynebacterium and Pseudomonas were distinct in the top 10 contributing genera in the TBI FMB while Parabacteroides and Ruminococcus were unique to the top 10 in controls. Comparing RNA profiles, TBI samples had ∼1.5 fold more expressed genes with almost 700 differently expressed genes (DEGs) mapped to over 100 bacterial species. Bioinformatic analysis associated DEGs with pathways led identifying 311 functions in the average TBI FMB profile and 264 in the controls. By average profile comparison, 30 pathways had significantly different abundance (p < 0.05, t-test) or were detected in >80% of the samples in only one of the cohorts (binary distinction). Discussion: Functional differences between TBI and healthy control FMBs included amino acid metabolism, energy and carbon source usage, fatty acid metabolism, bacterial cell wall component production and nucleic acid synthesis and processing pathways. Together these data shed light on the functional consequences of the dysbiotic TBI FMB decades after injury.

Boosting of vaginal HSV-2-specific B and T cell responses by intravaginal therapeutic immunization results in diminished recurrent HSV-2 disease.

Boosting herpes simplex virus (HSV)-specific immunity in the genital tissues of HSV-positive individuals to increase control of HSV-2 recurrent disease and virus shedding is an important goal of therapeutic immunization and would impact HSV-2 transmission. Experimental therapeutic HSV-2 vaccines delivered by a parenteral route have resulted in decreased recurrent disease in experimental animals. We used a guinea pig model of HSV-2 infection to test if HSV-specific antibody and cell-mediated responses in the vaginal mucosa would be more effectively increased by intravaginal (Ivag) therapeutic immunization compared to parenteral immunization. Therapeutic immunization with HSV glycoproteins and CpG adjuvant increased glycoprotein-specific IgG titers in vaginal secretions and serum to comparable levels in Ivag- and intramuscular (IM)-immunized animals. However, the mean numbers of HSV glycoprotein-specific antibody secreting cells (ASCs) and IFN-γ SCs were greater in Ivag-immunized animals demonstrating superior boosting of immunity in the vaginal mucosa compared to parenteral immunization. Therapeutic Ivag immunization also resulted in a significant decrease in the cumulative mean lesion days compared to IM immunization. There was no difference in the incidence or magnitude of HSV-2 shedding in either therapeutic immunization group compared to control-treated animals. Collectively, these data demonstrated that Ivag therapeutic immunization was superior compared to parenteral immunization to boost HSV-2 antigen-specific ASC and IFN-γ SC responses in the vagina and control recurrent HSV-2 disease. These results suggest that novel antigen delivery methods providing controlled release of optimized antigen/adjuvant combinations in the vaginal mucosa would be an effective approach for therapeutic HSV vaccines. IMPORTANCE HSV-2 replicates in skin cells before it infects sensory nerve cells where it establishes a lifelong but mostly silent infection. HSV-2 occasionally reactivates, producing new virus which is released back at the skin surface and may be transmitted to new individuals. Some HSV-specific immune cells reside at the skin site of the HSV-2 infection that can quickly activate and clear new virus. Immunizing people already infected with HSV-2 to boost their skin-resident immune cells and rapidly control the new HSV-2 infection is logical, but we do not know the best way to administer the vaccine to achieve this goal. In this study, a therapeutic vaccine given intravaginally resulted in significantly better protection against HSV-2 disease than immunization with the same vaccine by a conventional route. Immunization by the intravaginal route resulted in greater stimulation of vaginal-resident, virus-specific cells that produced antibody and produced immune molecules to rapidly clear virus.

Microbiota Associated With Cholesteatoma Tissue in Chronic Suppurative Otitis Media.

Otitis media (OM), defined as infection or inflammation of the middle ear (ME), remains a major public health problem worldwide. Cholesteatoma is a non-cancerous, cyst-like lesion in the ME that may be acquired due to chronic OM and cause disabling complications. Surgery is required for treatment, with high rates of recurrence. Current antibiotic treatments have been largely targeted to previous culturable bacteria, which may lead to antibiotic resistance or treatment failures. For this study, our goal was to determine the microbiota of cholesteatoma tissue in comparison with other ME tissues in patients with long-standing chronic OM. ME samples including cholesteatoma, granulation tissue, ME mucosa and discharge were collected from patients undergoing tympanomastoidectomy surgery for chronic OM. Bacteria were profiled by 16S rRNA gene sequencing in 103 ME samples from 53 patients. Respiratory viruses were also screened in 115 specimens from 45 patients. Differences in bacterial profiles (beta-diversity) and the relative abundances of individual taxa were observed between cholesteatoma and ME sample-types. Additionally, patient age was associated with differences in overall microbiota composition while numerous individual taxa were differentially abundant across age quartiles. No viruses were identified in screened ME samples. Biodiversity was moderately lower in cholesteatoma and ME discharge compared to ME mucosal tissues. We also present overall bacterial profiles of ME tissues by sample-type, age, cholesteatoma diagnosis and quinolone use, including prevalent bacterial taxa. Our findings will be useful for fine-tuning treatment protocols for cholesteatoma and chronic OM in settings with limited health care resources.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Case Report: Post-Partum Complications of NFκB1 Deficiency Underscore a Need to Better Understand Primary Immunodeficiency Management During Pregnancy.

Nuclear factor κappa-B (NFκB) is a family of transcription factors involved in regulating inflammation and immunity. Mutations in the NFκB1 pathway are associated with primary immune defects and underlie the most common monogenic etiology of common variable immunodeficiency (CVID). However, little is known about how NFκB1 defects or primary immunodeficiency (PID) complicate pregnancy. We present a previously healthy 34-year-old patient who suffered from poor wound healing and sterile sepsis during the post-partum period of each of her three pregnancies. She was otherwise asymptomatic, but her daughter developed Evans Syndrome (ES) with hypogammaglobulinemia prompting expanded genetic testing which revealed a novel monoallelic variant in NFκB1. This case highlights that pregnancy-related complications of PID can be difficult to recognize and may portend adverse patient outcomes. For these reasons, guidance regarding diagnosis and management of women of childbearing age with PID is warranted.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Microbiota Detection Patterns Correlate With Presence and Severity of Barrett's Esophagus.

Background: The microbiome has been increasingly associated with different disease processes, but its role in esophagus is largely unknown. Our goal was to determine the associations of the esophageal microbiota with Barrett's esophagus. Methods: A total of 74 patients were included in this prospective study, including 34 patients with Barrett's esophagus and 40 patients without Barrett's esophagus. Esophageal swabs were obtained from the uvula, and mucosal biopsies were obtained from the proximal esophagus and distal esophagus in each patient. The microbiome of each sample was assessed using a customized Esophageal Microbiome qPCR array (EMB). For each clinical sample, we completed a detection/non-detection analysis for each organism in the EMB. The limit of detection (LOD) for each target was established by analysis of plasmid dilutions. Results: Average age was 60.2 years. There were significantly different microbial detection patterns in patients with Barrett's esophagus compared to the control population. There were a greater number of organisms which had different likelihoods of detection in the distal esophagus, compared to the proximal esophagus or uvula. In addition, as the length of the Barrett's column increased, multiple organisms were less likely to be detected. This decreased likelihood occurred only in the distal esophagus. Beside Barrett's esophagus, no other demographic factors were associated with differences in detection patterns. Conclusions: Microbial community structures differ between patients with and without Barrett's esophagus. Certain organisms are less likely to be detected as the severity of Barrett's esophagus worsens. These results suggest that particular organisms may have a protective effect against the development of Barrett's esophagus.

Characterization of the Ovine Vaginal Microbiome and Inflammation Patterns as an Improved Testing Model of Human Vaginal Irritation.

The development of therapies targeted to improve the health of women has utilized direct vaginal delivery as a more effective and less toxic method of protection from HIV and other pathogens. Vaginal applicants and delivery devices that provide sustained effects have been met with increasing acceptability, but the efficacy and toxicity outcomes have not been successfully predicted by preclinical in vitro studies and animal modeling. We have explored the utilization of sheep as a model for testing the safety of vaginal applicants and devices based on spatial and structural similarities to the human vagina. As recently noted by the FDA, an additional safety measure is an impact on the vaginal microbiome (VMB) that is known to contribute to vaginal health and influence pathogen susceptibility and drug metabolism. To advance the utility of the sheep vaginal model, we completed a thorough molecular characterization of the ovine VMB utilizing both next-generation sequencing (NGS) and PCR methods. The process also created a custom PCR array to quantify ovine VMB community profiles in an affordable, higher throughput fashion. The results from vaginal swabs (>475 samples) collected from non-pregnant crossbred Dorset and Merino ewes treated with selected vaginal applicants or collected as sham samples established 16 VMB community types (VMB CTs). To associate VMB CTs with eubiosis or dysbiosis, we also completed custom ELISAs for six cytokines identifying IL1B, IL8, TNFa, and CXCL10 as useful markers to support the characterization of ovine vaginal inflammation. The results indicated that Pasteurella, Actinobacillus, Pseudomonas, Bacteroides, Leptotrichia, and E. coli were common markers of eubiosis (low inflammatory marker expression), and that Haemophilus, Ureaplasma, and Corynebacterium were associated with dysbiosis (high cytokine levels). Utilizing the optimized workflow, we also confirmed the utility of three commonly used vaginal applicants for impact on the VMB and inflammatory state, producing a dataset that supports the recommendation for the use of sheep for testing of vaginal applicants and devices as part of preclinical pipelines.

Development of Gram Stain Scoring System Based on Pro-Inflammatory Cytokines in the Sheep Model for Testing Toxicity of Vaginal Products.

Background: Development of safe, effective products to prevent the sexual transmission of HIV remains a priority. Prior to clinical testing, the products must undergo strict safety evaluations to avoid mucosal drug toxicity, inflammation, and vaginal microbiome (VMB) shifts. Based on the Food and Drug Administration (FDA) guidance, we designed a study to measure the inflammatory markers and VMB changes after intravaginal treatment with products that have been associated with toxicity, with the objective to develop a Gram stain slide scoring system, similar to Nugent scoring, correlated with the proinflammatory cytokines in sheep. Methods: Non-pregnant Dorset ewes (n = 34) were randomized to receive 5 ml intravaginal 4% nonoxynol-9 (N9) contraceptive gel, positive control (0.2% benzalkonium chloride), placebo control [hydroxethyl cellulose (HEC)], or no application daily for 10 days, with 11-day post-treatment follow-up. The vaginal swabs were collected for the cytokines, VMB, and Gram-stained slides. An enzyme-linked immunosorbent assay (ELISA) analysis of cytokines interleukin (IL)-1ß, IL-8, CXCL10, and tumor necrosis factor-α (TNF-α) was used to determine inflammatory state of the sample. Vaginal microbiome community types (CT) were utilized to create five equivalent slide subsets for iterative development of a Gram-stained slide scoring system with comparisons with inflammatory state based on the cytokine levels. Results: Digital images of the Gram-stained slides were scored based on Gram staining and morphology of bacteria, presence of sheep epithelial cells, and immune cells. The scoring system was modified in an iterative fashion with weighting based on cytokine categorization of inflamed samples, with three of four cytokine values above the mean indicating that the sample was inflamed. The parameters in the final version of the scoring system included mature epithelial cells, Gram-negative rods, and Gram-positive diplococci indicating normal and immune cells indicating inflammation. The area under the receiver operator characteristic curve (ROC AUC) was 0.725 (ROC AUCs range between 0.5 and 1.0) with a greater area indicating higher diagnostic ability of a test with a binary outcome: inflamed or normal. Conclusion: The scoring system, derived from the advanced VMB and cytokine analyses, provides a validated, practical method for quantification of Gram-stained slides that can be performed in most laboratories, increasing the potential for standardization. The training plan can assist laboratories to determine the safety of intravaginal products in their sheep studies or the methodological approach can be applied to other animal models where such data are also needed.

Effects of proton pump inhibitor use on the esophageal microbial community.

BACKGROUND: Changes in the esophageal microbiome correlate with esophageal disease, but the effects of proton pump inhibitor (PPI) drugs are incompletely characterized. Our objective was to identify the effects of PPI use on the microbial community of the esophagus. METHODS: Mucosal biopsies of the distal esophagus were analyzed using a customized esophageal microbiome qPCR panel array (EMB). Patient demographics, use of PPIs, duration of use and dose were recorded. RESULTS: Fifty-eight patients were included. Mean age was 60.5 years. Ninety percent (52/58) of patients were on PPIs. Mean dose was 42.7 mg. Mean duration of use was 2.5 years. The use of PPIs led to a significant difference in absolute levels of only one organism, Actinomyces, in the entire array (p < 0.01). Among patients who used proton pump inhibitors, there was no significant association between dose and absolute levels of any organism. Similarly, there was no association between duration of use and absolute levels of any organism. CONCLUSIONS: PPI use does not seem to cause significant changes in the distal esophageal microbial community. Future studies with larger sample sizes and esophageal pH testing should be performed to determine the level of acidity and its relationship to the microbial community.

Methylation Patterns of the HTR2A Associate With Relapse-Related Behaviors in Cocaine-Dependent Participants.

Relapse during abstinence in cocaine use disorder (CUD) is often hastened by high impulsivity (predisposition toward rapid unplanned reactions to stimuli without regard to negative consequences) and high cue reactivity (e.g., attentional bias towards drug reward stimuli). A deeper understanding of the degree to which individual biological differences predict or promote problematic behaviors may afford opportunities for clinical refinement and optimization of CUD diagnostics and/or therapies. Preclinical evidence implicates serotonin (5-HT) neurotransmission through the 5-HT2A receptor (5-HT2AR) as a driver of individual differences in these relapse-related behaviors. Regulation of 5-HT2AR function occurs through many mechanisms, including DNA methylation of the HTR2A gene, an epigenetic modification linked with the memory of gene-environment interactions. In the present study, we tested the hypothesis that methylation of the HTR2A may associate with relapse-related behavioral vulnerability in cocaine-dependent participants versus healthy controls. Impulsivity was assessed by self-report (Barratt Impulsiveness Scale; BIS-11) and the delay discounting task, while levels of cue reactivity were determined by performance in the cocaine-word Stroop task. Genomic DNA was extracted from lymphocytes and the bisulfite-treated DNA was subjected to pyrosequencing to determine degree of methylation at four cytosine residues of the HTR2A promoter (-1439, -1420, -1224, -253). We found that the percent methylation at site -1224 after correction for age trended towards a positive correlation with total BIS-11 scores in cocaine users, but not healthy controls. Percent methylation at site -1420 negatively correlated with rates of delay discounting in healthy controls, but not cocaine users. Lastly, the percent methylation at site -253 positively correlated with attentional bias toward cocaine-associated cues. DNA methylation at these cytosine residues of the HTR2A promoter may be differentially associated with impulsivity or cocaine-associated environmental cues. Taken together, these data suggest that methylation of the HTR2A may contribute to individual differences in relapse-related behaviors in CUD.

"Submergence" of Western equine encephalitis virus: Evidence of positive selection argues against genetic drift and fitness reductions.

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century.

Altered Fecal Microbiome Years after Traumatic Brain Injury.

Patients with chronic traumatic brain injury (TBI) requiring long-term, permanent care suffer a myriad of clinical symptoms (i.e., impaired cognition, fatigue, and other conditions) that persist for years beyond the acute brain injury. In addition to these comorbid clinical symptoms, chronic TBI patients exhibit altered amino acid and hormonal profiles with distinct cytokine patterns suggesting chronic inflammation. This metabolic link suggests a role of the gut-brain axis in chronic TBI. Thus, we utilized a two-site trial to investigate the role of the gut-brain axis in comorbidities of chronic TBI. The fecal microbiome profile of 22 moderate/severe TBI patients residing in permanent care facilities in Texas and California was compared to 18 healthy age-matched control subjects working within the participating facilities. Each fecal microbiome was characterized by 16S(V4) ribosomal RNA (rRNA) gene sequencing and metagenomic genome sequencing approaches followed by confirmatory full 16S rRNA gene sequencing or focused tuf gene speciation and specific quantitative polymerase chain reaction evaluation of selected genera or species. The average chronic TBI patient fecal microbiome structure was significantly different compared to the control cohort, and these differences persisted after group stratification analysis to identify any unexpected confounders. Notably, the fecal microbiome of the chronic TBI cohort had absent or reduced Prevotella spp. and Bacteroidies spp. Conversely, bacteria in the Ruminococcaceae family were higher in abundance in TBI compared to control profiles. Previously reported hypoaminoacidemia, including significantly reduced levels of l-tryptophan, l-sarcosine, ß-alanine, and alanine, positively correlated with the reduced levels of Prevotella spp. in the TBI cohort samples compared to controls. Although the sequelae of gut-brain axis disruption after TBI is not fully understood, characterizing TBI-related alterations in the fecal microbiome may provide biomarkers and therapeutic targets to address patient morbidity.

Microbiota of the Oropharynx and Endoscope Compared to the Esophagus.

The role of the microflora in the development of esophageal disease is still largely unknown and is being investigated in more detail. Our goal was to determine how the microbiota levels of endoscope and uvular swabs compared to the levels of tissue biopsies along various points of the esophagus. 17 patients with Barrett's esophagus agreed to participate in the study. Biopsies of esophageal mucosa were taken from the (1) proximal esophagus, (2) mid-esophagus, (3) distal esophagus, and (4) Barrett's esophagus. Swabs were also taken from the uvula and the endoscope. Throughout the esophagus, 17 bacterial genera were detected from the samples. The microflora pattern obtained from the uvula and endoscopic swabs did not correlate well with mucosal biopsies along any aspect of the esophagus. There were statistically significant differences in the levels and proportions of bacteria found when comparing the uvula swab to the esophageal biopsies and when comparing the endoscope swab to the esophageal biopsies. Obtaining a simple swab of the uvula or endoscope itself appears to be a poor substitute for tissue biopsy of esophageal mucosa when evaluating microflora patterns. When performing microflora studies of the esophagus, mucosal biopsies should be used for analysis.

Development of a Novel ex vivo Nasal Epithelial Cell Model Supporting Colonization With Human Nasal Microbiota.

The nasal mucosa provides first line defense against inhaled pathogens while creating a unique microenvironment for bacterial communities. Studying the impact of microbiota in the nasal cavity has been difficult due to limitations with current models including explant cultures, primary cells, or neoplastic cell lines. Most notably, none have been shown to support reproducible colonization by bacterial communities from human donors. Therefore, to conduct controlled studies of the human nasal ecosystem, we have developed a novel ex vivo mucosal model that supports bacterial colonization of a cultured host mucosa created by immortalized human nasal epithelial cells (NEC). For this model, immortalized NEC established from 5 male and 5 female donors were cultured with an air-interfaced, apical surface on a porous transwell membrane. NEC were grown from nasal turbinate tissues harvested from willed bodies or from discarded tissue collected during sinonasal procedures. Immortalized cells were evaluated through molecular verification of cell type, histological confirmation of tissue differentiation including formation of tight junctions, NEC multilayer viability, metabolism, physiology and imaging of the luminal surface by scanning electron microscopy. Results showed proper differentiation and multilayer formation at seven to 10 days after air interface that was maintained for up to 3 weeks. The optimized mucosal cultures created an environment necessary to sustain colonization by nasal microbiomes (NMBs) that were collected from healthy volunteers, cryogenically preserved and characterized with customized quantitative polymerase chain reaction (qPCR) arrays. Polymicrobial communities of nasal bacteria associated with healthy and inflamed states were consistently reproduced in matured NEC co-cultures by transplant of NMBs from multiple community types. The cultured NMBs were stable after an initial period of bacterial replication and equilibration. This novel ex vivo culture system is the first model that supports controlled cultivation of NMBs, allowing for lab-based causation studies and further experimentation to explore the complexities of host-microbe and microbe-microbe interactions.

Development of disease and immunity at the genital epithelium following intrarectal inoculation of male guinea pigs with herpes simplex virus type 2.

Most analyses of genital immunity to herpes simplex virus type 2 (HSV-2) have been performed in females, consequently immune protection of the male genital epithelium is incompletely understood. We developed a model of male genital HSV-2 infection resulting from intrarectal inoculation of guinea pigs. Vesicular lesions developed transiently on the perineum and foreskin concurrent with acute virus shedding. Virus shedding and recurrent genital lesions were also detected after establishment of a latent infection. Analysis of perineum and foreskin RNA detected transcripts for IFNγ, proinflammatory and regulatory cytokines, and for genes involved in migration and regulation of leukocytes. HSV-specific T cells were detected in lymphoid and genital tissues after resolution of the primary infection whereas virus-specific antibody secreting cells were detected only in lymphoid tissue. Taken together, the ability to quantify pathogenesis and local immunity in this guinea pig model represent an important advance towards understanding immunity to HSV-2 in males.

Increased Frequency of Virus Shedding by Herpes Simplex Virus 2-Infected Guinea Pigs in the Absence of CD4+ T Lymphocytes.

Reactivation of herpes simplex virus 2 (HSV-2) results in infection of epithelial cells at the neuro-epithelial junction and shedding of virus at the epithelial surface. Virus shedding can occur in either the presence or absence of clinical disease and is usually of short duration, although the shedding frequency varies among individuals. The basis for host control of virus shedding is not well understood, although adaptive immune mechanisms are thought to play a central role. To determine the importance of CD4+ T cells in control of HSV-2 shedding, this subset of immune cells was depleted from HSV-2-infected guinea pigs by injection of an anti-CD4 monoclonal antibody (MAb). Guinea pigs were treated with the depleting MAb after establishment of a latent infection, and vaginal swabs were taken daily to monitor shedding by quantitative PCR. The cumulative number of HSV-2 shedding days and the mean number of days virus was shed were significantly increased in CD4-depleted compared to control-treated animals. However, there was no difference in the incidence of recurrent disease between the two treatment groups. Serum antibody levels and the number of HSV-specific antibody-secreting cells in secondary lymphoid tissues were unaffected by depletion of CD4+ T cells; however, the frequency of functional HSV-specific, CD8+ gamma interferon-secreting cells was significantly decreased. Together, these results demonstrate an important role for CD4+ T lymphocytes in control of virus shedding that may be mediated in part by maintenance of HSV-specific CD8+ T cell populations. These results have important implications for development of therapeutic vaccines designed to control HSV-2 shedding.IMPORTANCE Sexual transmission of HSV-2 results from viral shedding following reactivation from latency. The immune cell populations and mechanisms that control HSV-2 shedding are not well understood. This study examined the role of CD4+ T cells in control of virus shedding using a guinea pig model of genital HSV-2 infection that recapitulates the shedding of virus experienced by humans. We found that the frequency of virus-shedding episodes, but not the incidence of clinical disease, was increased by depletion of CD4+ T cells. The HSV-specific antibody response was not diminished, but frequency of functional HSV-reactive CD8+ T cells was significantly diminished by CD4 depletion. These results confirm the role of cell-mediated immunity and highlight the importance of CD4+ T cells in controlling HSV shedding, suggesting that therapeutic vaccines designed to reduce transmission by controlling HSV shedding should include specific enhancement of HSV-specific CD4+ T cell responses.

Transcriptional Analysis of the Guinea Pig Mucosal Immune Response to Intravaginal Infection with Herpes Simplex Virus Type 2.

Genital herpes infection in guinea pigs closely models human infection but tools for immune characterization are limited. Immunity to HSV infection at the vaginal epithelial surface was characterized in guinea pigs using PCR-based array analysis of vaginal swab samples. IFNγ was one of the most significantly upregulated genes throughout the infection and over 40% of genes with significantly altered expression were linked to IFNγ based on INTERFEROME analysis. IFNγ transcripts and biologically active IFNγ at the genital mucosa were confirmed by RTPCR and IFNγ reporter cells. Gene ontology analysis revealed activation of many biological processes related to genital immunity shared by humans and mice demonstrating the similarities of the local immune response to primary genital HSV-2 infection in guinea pigs and other established models. This transcription-based array will be useful for dissection of immunity during reactivation from latency, an infection outcome that is not well recapitulated by other animal models.

Cultivated Human Vaginal Microbiome Communities Impact Zika and Herpes Simplex Virus Replication in ex vivo Vaginal Mucosal Cultures.

The human vaginal microbiome (VMB) is a complex bacterial community that interacts closely with vaginal epithelial cells (VECs) impacting the mucosal phenotype and its responses to pathogenic insults. The VMB and VEC relationship includes nutrient exchange and regulation of signaling molecules that controls numerous host functions and defends against invading pathogens. To better understand infection and replication of sexually transmitted viral pathogens in the human vaginal mucosa we used our ex vivo VEC multilayer culture system. We tested the hypothesis that selected VMB communities could be identified that alter the replication of sexually transmitted viruses consistent with reported clinical associations. Sterile VEC multilayer cultures or those colonized with VMB dominated by specific Lactobacillus spp., or VMB lacking lactobacilli, were infected with Zika virus, (ZIKV) a single stranded RNA virus, or Herpes Simplex Virus type 2 (HSV-2), a double stranded DNA virus. The virus was added to the apical surface of the cultured VEC multilayer to model transmission during vaginal intercourse. Viral replication was measured 48 h later by qPCR. The results indicated that VEC cultures colonized by VMB containing Staphylococcus spp., previously reported as inflammatory, significantly reduced the quantity of viral genomes produced by ZIKV. HSV-2 titers were decreased by nearly every VMB tested relative to the sterile control, although Lactobacillus spp.-dominated VMBs caused the greatest reduction in HSV-2 titer consistent with clinical observations. To explore the mechanism for reduced ZIKV titers, we investigated inflammation created by ZIKV infection, VMB colonization or pre-exposure to selected TLR agonists. Finally, expression levels of human beta defensins 1-3 were quantified in cultures infected by ZIKV and those colonized by VMBs that impacted ZIKV titers. Human beta defensins 1-3 produced by the VEC showed no association with ZIKV titers. The data presented expands the utility of this ex vivo model system providing controlled and reproducible methods to study the VMB impact on STIs and indicated an association between viral replication and specific bacterial species within the VMB.