Search

Background Face masks have been critical in the COVID-19 pandemic, but supplies were sometimes limited and disposable masks contribute greatly to environmental waste. Studies suggest that filtration capacity is retained with repeated use, and surveys indicate many people re-use surgical masks. However, the impact of mask re-use on the host is under-studied. Methods We applied 16S rRNA gene sequencing to investigate the bacterial microbiome of the facial skin and oropharynx of individuals randomized to wearing fresh surgical masks daily versus masks re-used for one week. Results Compared to daily fresh masks, re-use was associated with increased richness (number of taxa) of the skin microbiome and trend towards greater diversity, but no difference in the oropharyngeal microbiome. Used masks had either skin-dominant or oropharynx-dominant bacterial sequences, and re-used masks had >100-fold higher bacterial content but no change in composition compared to those used for one day. Conclusions One week of mask re-use increased the number of low-abundance taxa on the face but did not impact the upper respiratory microbiome. Thus, face mask re-use has little impact on the host microbiome, though whether minor changes to the skin microbiome might relate to reported skin sequelae of masking (“maskne”) remains to be determined.

The spread of SARS-CoV-2, like that of many other pathogens, is governed by heterogeneity. “Superspreading,” or “over-dispersion,” is an important factor in transmission, yet it is hard to quantify. Estimates from contact tracing data are prone to potential biases due to the increased likelihood of detecting large clusters of cases, and may reflect variation in contact behavior more than biological heterogeneity. In contrast, the average number of secondary infections per contact is routinely estimated from household surveys, and these studies can minimize biases by testing all members of a household. However, the models used to analyze household transmission data typically assume that infectiousness and susceptibility are the same for all individuals or vary only with predetermined traits such as age. Here we develop and apply a combined forward simulation and inference method to quantify the degree of inter-individual variation in both infectiousness and susceptibility from observations of the distribution of infections in household surveys. First, analyzing simulated data, we show our method can reliably ascertain the presence, type, and amount of these heterogeneities with data from a sufficiently large sample of households. We then analyze a collection of household studies of COVID-19 from diverse settings around the world, and find strong evidence for large heterogeneity in both the infectiousness and susceptibility of individuals. Our results also provide a framework to improve the design of studies to evaluate household interventions in the presence of realistic heterogeneity between individuals.

Redondoviruses are circular Rep-encoding single-stranded DNA (CRESS) viruses of high prevalence in healthy humans. Redondovirus abundance is increased in oro-respiratory samples from individuals with periodontitis, acute illness, and severe COVID-19. We investigated potential host cells supporting redondovirus replication in oro-respiratory samples and uncovered the oral amoeba Entamoeba gingivalis as a likely host. Redondoviruses are closely related to viruses of Entamoeba and contain reduced GC nucleotide content, consistent with Entamoeba hosts. Redondovirus and E. gingivalis co-occur in metagenomic data from oral disease and healthy human cohorts. When grown in xenic cultures with feeder bacteria, E. gingivalis was robustly positive for redondovirus RNA and DNA. A DNA proximity-ligation assay (Hi-C) on xenic culture cells showed enriched cross-linking of redondovirus and Entamoeba DNA, supporting E. gingivalis as the redondovirus host. While bacteria are established hosts for bacteriophages within the human virome, this work shows that eukaryotic commensals also contribute an abundant human-associated virus.

Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Here, we investigated the respiratory tract microbiome in coronavirus disease 2019 (COVID-19) and its relationship to disease severity, systemic immunologic features, and outcomes. We examined 507 oropharyngeal, nasopharyngeal, and endotracheal samples from 83 hospitalized COVID-19 patients as well as non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, and the commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR. We found that COVID-19 patients had upper respiratory microbiome dysbiosis and greater change over time than critically ill patients without COVID-19. Oropharyngeal microbiome diversity at the first time point correlated inversely with disease severity during hospitalization. Microbiome composition was also associated with systemic immune parameters in blood, as measured by lymphocyte/neutrophil ratios and immune profiling of peripheral blood mononuclear cells. Intubated patients showed patient-specific lung microbiome communities that were frequently highly dynamic, with prominence of Staphylococcus. Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Thus, the respiratory tract microbiome and commensal viruses are disturbed in COVID-19 and correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, its possible use as biomarkers, and the role of bacterial and viral taxa identified here in COVID-19 pathogenesis. IMPORTANCE COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized COVID-19 patients and found that each was markedly abnormal compared to that in healthy people and differed from that in critically ill patients without COVID-19. Early airway samples tracked with the level of COVID-19 illness reached during hospitalization, and the airway microbiome also correlated with immune parameters in blood. These findings raise questions about the mechanisms linking SARS-CoV-2 infection and other microbial inhabitants of the airway, including whether the microbiome might regulate severity of COVID-19 disease and/or whether early microbiome features might serve as biomarkers to discriminate disease severity.

An unexpected Diplorickettsia species closely related to the tickborne pathogen D. massieliensis was found in the microbiome of an Ixodes scapularis tick in Vermont, USA. This evidence of Diplorickettsia in North American ticks suggests a need for disease surveillance using molecular screening of ticks and serologic studies of humans.

ObjectiveThe immune response to invasive carcinoma has been the focus of published work, but little is known about the adaptive immune response to bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma. This study was designed to characterize the T cell receptor (TCR) repertoire in PMLs and its association with clinical, pathological, and molecular features.MethodsEndobronchial biopsies (n=295) and brushings (n=137) from high-risk subjects (n=50), undergoing lung cancer screening at approximately 1-year intervals via autofluorescence bronchoscopy and CT, were profiled by RNA-seq. We applied the TCR Repertoire Utilities for Solid Tissue/Tumor (TRUST) tool to the RNA-seq data to identify TCR CDR3 sequences across all samples. In the biopsies, we measured the correlation of TCR diversity with previously derived immune-associated PML transcriptional signatures and outcome. We also quantified the spatial and temporal distribution of shared and clonally expanded TCRs. Using the biopsies and brushes, the ratio of private (i.e., found in one patient only) and public (i.e., found in two or more patients) TCRs was quantified and the CDR3 sequences were compared to those found in curated databases with known antigen specificities.ResultsWe detected 39,303 unique TCR sequences across all samples. In PML biopsies, TCR diversity was negatively associated with a transcriptional signature of T-cell mediated immune activation (Spearman’s rho −0.34, p < 0.001) associated with PML outcome.Additionally, in lesions of the proliferative molecular subtype, TCR diversity was decreased in regressive versus progressive/persistent PMLs (p=0.045). Within each patient, TCRs were more likely to be shared between biopsies sampled at the same timepoint than biopsies sampled at the same anatomic location at different times. Clonally expanded TCRs, within a biopsied lesion, were more likely to be expanded at future time points than non-expanded clones. The majority of TCR sequences were found in a single sample, with only 3,396 (8.6%) found in more than one sample and 1,057 (2.7%) found in two or more patients (i.e. public), however, when compared to a public database of CDR3 sequences, 4,543 (11.6%) of TCRs were identified as public. TCRs with known antigen specificities were enriched among Public TCRs (p < 0.001).ConclusionsDecreased TCR diversity may reflect nascent immune responses that contribute to PML elimination. Further studies are needed to explore the potential for immunoprevention of PMLs.

A chemopreventive effect of aspirin (ASA) on lung cancer risk is supported by epidemiologic and preclinical studies. We conducted a randomized, double-blinded study in current heavy smokers to compare modulating effects of intermittent versus continuous low-dose ASA on nasal epithelium gene expression and arachidonic acid (ARA) metabolism. Fifty-four participants were randomized to intermittent (ASA 81 mg daily for one week/placebo for one week) or continuous (ASA 81 mg daily) for 12 weeks. Low-dose ASA suppressed urinary prostaglandin E2 metabolite (PGEM; change of −4.55 ± 11.52 from baseline 15.44 ± 13.79 ng/mg creatinine for arms combined, P = 0.02), a surrogate of COX-mediated ARA metabolism, but had minimal effects on nasal gene expression of nasal or bronchial gene-expression signatures associated with smoking, lung cancer, and chronic obstructive pulmonary disease. Suppression of urinary PGEM correlated with favorable changes in a smoking-associated gene signature (P < 0.01). Gene set enrichment analysis (GSEA) showed that ASA intervention led to 1,079 enriched gene sets from the Canonical Pathways within the Molecular Signatures Database. In conclusion, low-dose ASA had minimal effects on known carcinogenesis gene signatures in nasal epithelium of current smokers but results in wide-ranging genomic changes in the nasal epithelium, demonstrating utility of nasal brushings as a surrogate to measure gene-expression responses to chemoprevention. PGEM may serve as a marker for smoking-associated gene-expression changes and systemic inflammation. Future studies should focus on NSAIDs or agent combinations with broader inhibition of pro-inflammatory ARA metabolism to shift gene signatures in an anti-carcinogenic direction.

Introduction: Bronchial premalignant lesions (PMLs), precursors to lung squamous cell carcinoma, represent a relevant system for studying early tumor development. Some PMLs will progress through increasing grades of dysplasia to become squamous cell carcinoma, while others will regress back to normal airway without intervention. Previous work has identified molecular subtypes of PMLs and identified immune alterations associated with lesion progression/persistence and regression. Here, we used gene expression profiling to identify genes that may be responsible for immune suppression or activation in PMLs. Methods: We collected endobronchial biopsies (n=295) longitudinally from 50 high-risk smokers in approximately one-year intervals at Roswell Park Comprehensive Cancer Center. Two biopsies were collected from each lesion, one for standard pathological assessment and one for RNA sequencing. We derived eight immune-related gene expression phenotypes based on immune-oncologic gene coexpression signatures taken from literature sources. Mediation analysis was used to identify genes that may be upstream modulators or downstream effectors of the association between immune phenotypes and lesion progression/persistence. Gene-Set Enrichment Analysis (GSEA) and Virtual Inference of Protein-activity by Enriched Regulon analysis (VIPER) were used to identify specific signaling and transcription pathways that these mediators were acting through. Results: Predicted immune phenotypes related to B cell activation, interferon signaling, and antigen presentation were significantly associated with lesion progression/persistence within the Proliferative molecular subtype (FDR ≤ 0.01). Via mediation analysis, we identified 15 genes as candidate immune regulators or downstream immune effectors essential for activation/suppression of the immune response in PMLs. One of these genes, GSTP1, is up-regulated in progressing lesions and negatively correlated with several immune activation pathways, thus representing a possible mechanism for immune suppression in these lesions. Further in silico analysis suggests that GSTP1 acts as a repressor of STAT3 signaling and NF-κB pathways, and in vitro experiments are underway to determine if suppression of GSTP1 is sufficient to induce expression of these immune pathways. Conclusion: Therapies are needed to intercept lung cancer development and these results suggest a targetable pathway to activate the immune system to stimulate PML regression. Citation Format: Carter Merenstein, Julia Breda, Sarah Mazzilli, Eric Burks, Christopher Stevenson, Mary Reid, Avrum Spira, Marc Lenburg, Jennifer Beane. Mediators of early immune response in bronchial premalignant lesions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1098.

102 Background: The immune response to frank malignancy has been the focus of published work, but little is known about the adaptive immune response to bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma. This study was designed to characterize the T cell receptor (TCR) repertoire in PMLs and its association with clinical, pathological and molecular features. Methods: Endobronchial biopsies (n=294) and brushings (n=137) from high-risk subjects (n=50), undergoing lung cancer screening at approximately 1-year intervals via auto fluorescence bronchoscopy and CT, were profiled by RNA-Seq as part of the Pre-Cancer Genome Atlas (PCGA). We applied the TCR Repertoire Utilities for Solid Tissue/Tumor (TRUST) tool to identify TCR CDR3 sequences in the RNA-Seq data. We quantified the ratio of private (i.e., found in one patient only) and public (i.e. found in two or more patients) TCRs that are found in curated databases with known antigen specificities. We measured the correlation of TCR diversity with previously derived PML transcriptional signatures and molecular subtypes; and with mutational burden among a subset of biopsies (n=115) also profiled with whole exome sequencing. Results: We detected 40,421 unique TCR sequences, of which 3,396 (8.4%) were found in more than one sample and 1,057 (2.6%) were found in two or more patients (i.e. Public). TCRs with known antigen specificities were enriched among public TCRs (p < 0.001). In PMLs with a proliferative molecular subtype reflective of dysplasia (n=80), TCR diversity was decreased in PMLs that regressed versus PMLs that progressed (p=0.045). TCR diversity was negatively associated with a transcriptional signature of T-cell mediated immune activation (Spearman’s rho -0.26, p < 0.001) but was not associated with mutational burden. Conclusions: To our knowledge, this is the first characterization of the TCR repertoire associated with bronchial premalignant lesions. TCR diversity may help predict the efficacy of the host immune response to PMLs but it is not associated with mutational burden. Further studies are needed to leverage these findings and explore the potential for immunoprevention of PMLs.