Patient subtyping analysis of baseline multi-omic data reveals distinct pre-immune states associated with antibody response to seasonal influenza vaccination.

Understanding the molecular mechanisms underpinning diverse vaccination responses is critical for developing efficient vaccines. Molecular subtyping can offer insights into heterogeneous nature of responses and aid in vaccine design. We analyzed multi-omic data from 62 haemagglutinin seasonal influenza vaccine recipients (2019-2020), including transcriptomics, proteomics, glycomics, and metabolomics data collected pre-vaccination. We performed a subtyping analysis on the integrated data revealing five subtypes with distinct molecular signatures. These subtypes differed in the expression of pre-existing adaptive or innate immunity signatures, which were linked to significant variation in baseline immunoglobulin A (IgA) and hemagglutination inhibition (HAI) titer levels. It is worth noting that these differences persisted through day 28 post-vaccination, indicating the effect of initial immune state on vaccination response. These findings highlight the significance of interpersonal variation in baseline immune status as a crucial factor in determining the effectiveness of seasonal vaccines. Ultimately, incorporating molecular profiling could enable personalized vaccine optimization.

CD4+ and CD8+ T cells are required to prevent SARS-CoV-2 persistence in the nasal compartment.

SARS-CoV-2 infection induces the generation of virus-specific CD4+ and CD8+ effector and memory T cells. However, the contribution of T cells in controlling SARS-CoV-2 during infection is not well understood. Following infection of C57BL/6 mice, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract, and a vast proportion secrete the cytotoxic molecule granzyme B. Using depleting antibodies, we found that T cells within the lungs play a minimal role in viral control, and viral clearance occurs in the absence of both CD4+ and CD8+ T cells through 28 days postinfection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent, culturable virus replicating in the nasal epithelial layer through 28 days postinfection. Viral sequencing analysis revealed adapted mutations across the SARS-CoV-2 genome, including a large deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

Emergence of transmissible SARS-CoV-2 variants with decreased sensitivity to antivirals in immunocompromised patients with persistent infections.

We investigated the impact of antiviral treatment on the emergence of SARS-CoV-2 resistance during persistent infections in immunocompromised patients (n=15). All patients received remdesivir and some also received nirmatrelvir-ritonavir or monoclonal antibodies. Sequence analysis showed that nine patients carried viruses with mutations in the nsp12 (RNA dependent RNA polymerase), while four had viruses with nsp5 (3C protease) mutations. Infectious SARS-CoV-2 with a double mutation in nsp5 (T169I) and nsp12 (V792I) was recovered from respiratory secretions 77 days after initial COVID-19 diagnosis from a patient treated with remdesivir and nirmatrelvir-ritonavir. In vitro characterization confirmed its decreased sensitivity to remdesivir and nirmatrelvir, which was overcome by combined antiviral treatment. Studies in golden Syrian hamsters demonstrated efficient transmission to contact animals. This study documents the isolation of SARS-CoV-2 carrying resistance mutations to both nirmatrelvir and remdesivir from a patient and demonstrates its transmissibility in vivo.

Impaired immune responses in the airways are associated with poor outcome in critically ill COVID-19 patients.

Introduction: Mounting evidence indicates that an individual's humoral adaptive immune response plays a critical role in the setting of SARS-CoV-2 infection, and that the efficiency of the response correlates with disease severity. The relationship between the adaptive immune dynamics in the lower airways with those in the systemic circulation, and how these relate to an individual's clinical response to SARS-CoV-2 infection, are less understood and are the focus of this study. Material and methods: We investigated the adaptive immune response to SARS-CoV-2 in paired samples from the lower airways and blood from 27 critically ill patients during the first wave of the pandemic (median time from symptom onset to intubation 11 days). Measurements included clinical outcomes (mortality), bronchoalveolar lavage fluid (BALF) and blood specimen antibody levels, and BALF viral load. Results: While there was heterogeneity in the levels of the SARS-CoV-2-specific antibodies, we unexpectedly found that some BALF specimens displayed higher levels than the paired concurrent plasma samples, despite the known dilutional effects common in BALF samples. We found that survivors had higher levels of anti-spike, anti-spike-N-terminal domain and anti-spike-receptor-binding domain IgG antibodies in their BALF (p<0.05), while there was no such association with antibody levels in the systemic circulation. Discussion: Our data highlight the critical role of local adaptive immunity in the airways as a key defence mechanism against primary SARS-CoV-2 infection.

Host obesity impacts genetic variation in influenza A viral populations.

Obesity is well established as a risk factor for many noncommunicable diseases; however, its consequences for infectious disease are poorly understood. Here, we investigated the impact of host obesity on influenza A virus (IAV) genetic variation using a diet-induced obesity ferret model and the A/Hong Kong/1073/1999 (H9N2) strain. Using a co-caging study design, we investigated the maintenance, generation, and transmission of intrahost IAV genetic variation by sequencing viral genomic RNA obtained from nasal wash samples over multiple days of infection. We found evidence for an enhanced role of positive selection acting on de novo mutations in obese hosts that led to nonsynonymous changes that rose to high frequency. In addition, we identified numerous cases of mutations throughout the genome that were specific to obese hosts and that were preserved during transmission between hosts. Despite detection of obese-specific variants, the overall viral genetic diversity did not differ significantly between obese and lean hosts. This is likely due to the high supply rate of de novo variation and common evolutionary adaptations to the ferret host regardless of obesity status, which we show are mediated by variation in the hemagglutinin and polymerase genes (PB2 and PB1). We also identified defective viral genomes (DVGs) that were found uniquely in either obese or lean hosts, but the overall DVG diversity and dynamics did not differ between the two groups. Our study suggests that obesity may result in a unique selective environment impacting intrahost IAV evolution, highlighting the need for additional genetic and functional studies to confirm these effects.IMPORTANCEObesity is a chronic health condition characterized by excess adiposity leading to a systemic increase in inflammation and dysregulation of metabolic hormones and immune cell populations. Influenza A virus (IAV) is a highly infectious pathogen responsible for seasonal and pandemic influenza. Host risk factors, including compromised immunity and pre-existing health conditions, can contribute to increased infection susceptibility and disease severity. During viral replication in a host, the negative-sense single-stranded RNA genome of IAV accumulates genetic diversity that may have important consequences for viral evolution and transmission. Our study provides the first insight into the consequences of host obesity on viral genetic diversity and adaptation, suggesting that host factors associated with obesity alter the selective environment experienced by a viral population, thereby impacting the spectrum of genetic variation.

CD4+ and CD8+ T cells are required to prevent SARS-CoV-2 persistence in the nasal compartment.

SARS-CoV-2 is the causative agent of COVID-19 and continues to pose a significant public health threat throughout the world. Following SARS-CoV-2 infection, virus-specific CD4+ and CD8+ T cells are rapidly generated to form effector and memory cells and persist in the blood for several months. However, the contribution of T cells in controlling SARS-CoV-2 infection within the respiratory tract are not well understood. Using C57BL/6 mice infected with a naturally occurring SARS-CoV-2 variant (B.1.351), we evaluated the role of T cells in the upper and lower respiratory tract. Following infection, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract and a vast proportion secrete the cytotoxic molecule Granzyme B. Using antibodies to deplete T cells prior to infection, we found that CD4+ and CD8+ T cells play distinct roles in the upper and lower respiratory tract. In the lungs, T cells play a minimal role in viral control with viral clearance occurring in the absence of both CD4+ and CD8+ T cells through 28 days post-infection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent and culturable virus replicating in the nasal compartment through 28 days post-infection. Using in situ hybridization, we found that SARS-CoV-2 infection persisted in the nasal epithelial layer of tandem CD4+ and CD8+ T cell-depleted mice. Sequence analysis of virus isolates from persistently infected mice revealed mutations spanning across the genome, including a deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

Patient Subtyping Analysis of Baseline Multi-omic Data Reveals Distinct Pre-immune States Predictive of Vaccination Responses.

Understanding the molecular mechanisms that underpin diverse vaccination responses is a critical step toward developing efficient vaccines. Molecular subtyping approaches can offer valuable insights into the heterogeneous nature of responses and aid in the design of more effective vaccines. In order to explore the molecular signatures associated with the vaccine response, we analyzed baseline transcriptomics data from paired samples of whole blood, proteomics and glycomics data from serum, and metabolomics data from urine, obtained from influenza vaccine recipients (2019-2020 season) prior to vaccination. After integrating the data using a network-based model, we performed a subtyping analysis. The integration of multiple data modalities from 62 samples resulted in five baseline molecular subtypes with distinct molecular signatures. These baseline subtypes differed in the expression of pre-existing adaptive or innate immunity signatures, which were linked to significant variation across subtypes in baseline immunoglobulin A (IgA) and hemagglutination inhibition (HAI) titer levels. It is worth noting that these significant differences persisted through day 28 post-vaccination, indicating the effect of initial immune state on vaccination response. These findings highlight the significance of interpersonal variation in baseline immune status as a crucial factor in determining vaccine response and efficacy. Ultimately, incorporating molecular profiling could enable personalized vaccine optimization.

Repurposed Drugs That Activate Autophagy in Filarial Worms Act as Effective Macrofilaricides.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases caused by filarial nematodes that utilize insect vectors for transmission to their human hosts. Current control strategies are based on annual or biannual mass drug administration (MDA) of the drugs Ivermectin or Ivermectin plus Albendazole, respectively. These drug regimens kill the first-stage larvae of filarial worms (i.e., microfilariae) and interrupt the transmission of infections. MDA programs for these microfilaricidal drugs must be given over the lifetime of the filarial adult worms, which can reach 15 years in the case of Onchocerca volvulus. This is problematic because of suboptimal responses to ivermectin in various endemic regions and inefficient reduction of transmission even after decades of MDA. There is an urgent need for the development of novel alternative treatments to support the 2030 elimination goals of onchocerciasis and lymphatic filariasis. One successful approach has been to target Wolbachia, obligatory endosymbiotic bacteria on which filarial worms are dependent for their survival and reproduction within the human host. A 4-6-week antibiotic therapy with doxycycline, for example, resulted in the loss of Wolbachia that subsequently led to extensive apoptosis of somatic cells, germline, embryos, and microfilariae, as well as inhibition of fourth-stage larval development. However, this long-course regimen has limited use in MDA programs. As an alternative approach to the use of bacteriostatic antibiotics, in this study, we focused on autophagy-inducing compounds, which we hypothesized could disturb various pathways involved in the interdependency between Wolbachia and filarial worms. We demonstrated that several such compounds, including Niclosamide, an FDA-approved drug, Niclosamide ethanolamine (NEN), and Rottlerin, a natural product derived from Kamala trees, significantly reduced the levels of Wolbachia in vitro. Moreover, when these compounds were used in vivo to treat Brugia pahangi-infected gerbils, Niclosamide and NEN significantly decreased adult worm survival, reduced the release of microfilariae, and decreased embryonic development depending on the regimen and dose used. All three drugs given orally significantly reduced Wolbachia loads and induced an increase in levels of lysosome-associated membrane protein in worms from treated animals, suggesting that Niclosamide, NEN, and Rottlerin were effective in causing drug-induced autophagy in these filarial worms. These repurposed drugs provide a new avenue for the clearance of adult worms in filarial infections.

Miniature spatial transcriptomics for studying parasite-endosymbiont relationships at the micro scale.

Several important human infectious diseases are caused by microscale-sized parasitic nematodes like filarial worms. Filarial worms have their own spatial tissue organization; to uncover this tissue structure, we need methods that can spatially resolve these miniature specimens. Most filarial worms evolved a mutualistic association with endosymbiotic bacteria Wolbachia. However, the mechanisms underlying the dependency of filarial worms on the fitness of these bacteria remain unknown. As Wolbachia is essential for the development, reproduction, and survival of filarial worms, we spatially explored how Wolbachia interacts with the worm's reproductive system by performing a spatial characterization using Spatial Transcriptomics (ST) across a posterior region containing reproductive tissue and developing embryos of adult female Brugia malayi worms. We provide a proof-of-concept for miniature-ST to explore spatial gene expression patterns in small sample types, demonstrating the method's ability to uncover nuanced tissue region expression patterns, observe the spatial localization of key B. malayi - Wolbachia pathway genes, and co-localize the B. malayi spatial transcriptome in Wolbachia tissue regions, also under antibiotic treatment. We envision our approach will open up new avenues for the study of infectious diseases caused by micro-scale parasitic worms.

Wolbachia interferes with Zika virus replication by hijacking cholesterol metabolism in mosquito cells.

IMPORTANCE: Arthropod-borne viruses are emerging pathogens that are spread widely by mosquitos. Zika virus is an arbovirus that can infect humans and be transmitted from an infected mother to the fetus, potentially leading to microcephaly in infants. One promising strategy to prevent disease caused by arboviruses is to target the insect vector population. Recent field studies have shown that mosquito populations infected with Wolbachia bacteria suppress arbovirus replication and transmission. Here, we describe how intracellular bacteria redirect resources within their host cells and suppress Zika virus replication at the cellular level. Understanding the mechanism behind Wolbachia-induced interference of arbovirus replication could help advance strategies to control arbovirus pathogens in insect vectors and human populations.

Lower Airway Dysbiosis Augments Lung Inflammatory Injury in Mild-to-Moderate Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and healthcare costs. Cigarette smoke is a causative factor; however, not all heavy smokers develop COPD. Microbial colonization and infections are contributing factors to disease progression in advanced stages. Objectives: We investigated whether lower airway dysbiosis occurs in mild-to-moderate COPD and analyzed possible mechanistic contributions to COPD pathogenesis. Methods: We recruited 57 patients with a >10 pack-year smoking history: 26 had physiological evidence of COPD, and 31 had normal lung function (smoker control subjects). Bronchoscopy sampled the upper airways, lower airways, and environmental background. Samples were analyzed by 16S rRNA gene sequencing, whole genome, RNA metatranscriptome, and host RNA transcriptome. A preclinical mouse model was used to evaluate the contributions of cigarette smoke and dysbiosis on lower airway inflammatory injury. Measurements and Main Results: Compared with smoker control subjects, microbiome analyses showed that the lower airways of subjects with COPD were enriched with common oral commensals. The lower airway host transcriptomics demonstrated differences in markers of inflammation and tumorigenesis, such as upregulation of IL-17, IL-6, ERK/MAPK, PI3K, MUC1, and MUC4 in mild-to-moderate COPD. Finally, in a preclinical murine model exposed to cigarette smoke, lower airway dysbiosis with common oral commensals augments the inflammatory injury, revealing transcriptomic signatures similar to those observed in human subjects with COPD. Conclusions: Lower airway dysbiosis in the setting of smoke exposure contributes to inflammatory injury early in COPD. Targeting the lower airway microbiome in combination with smoking cessation may be of potential therapeutic relevance.

Host obesity impacts genetic variation in influenza A viral populations.

Obesity is a chronic health condition characterized by excess adiposity leading to a systemic increase in inflammation and dysregulation of metabolic hormones and immune cell populations. Obesity is well established as a risk factor for many noncommunicable diseases; however, its consequences for infectious disease are poorly understood. Influenza A virus (IAV) is a highly infectious pathogen responsible for seasonal and pandemic influenza. Host risk factors, including compromised immunity and pre-existing health conditions, can contribute to increased infection susceptibility and disease severity. During viral replication in a host, the negative sense single stranded RNA genome of IAV accumulates genetic diversity that may have important consequences for viral evolution and transmission. Here, we investigated the impact of host obesity on IAV genetic variation using a diet induced obesity ferret model. We infected obese and lean male ferrets with the A/Hong Kong/1073/1999 (H9N2) IAV strain. Using a co-caging study design, we investigated the maintenance, generation, and transmission of intrahost IAV genetic variation by sequencing viral genomic RNA obtained from nasal wash samples over multiple days of infection. We found evidence for an enhanced role of positive selection acting on de novo mutations in obese hosts that led to nonsynonymous changes that rose to high frequency. In addition, we identified numerous cases of recurrent low-frequency mutations throughout the genome that were specific to obese hosts. Despite these obese-specific variants, overall viral genetic diversity did not differ significantly between obese and lean hosts. This is likely due to the high supply rate of de novo variation and common evolutionary adaptations to the ferret host regardless of obesity status, which we show are mediated by variation in the hemagglutinin (HA) and polymerase genes (PB2 and PB1). As with single nucleotide variants, we identified a class of defective viral genomes (DVGs) that were found uniquely in either obese or lean hosts, but overall DVG diversity and dynamics did not differ between the two groups. Our study provides the first insight into the consequences of host obesity on viral genetic diversity and adaptation, suggesting that host factors associated with obesity alter the selective environment experienced by a viral population, thereby impacting the spectrum of genetic variation.

CRISPR arrays as high-resolution markers to track microbial transmission during influenza infection.

BACKGROUND: Disruption of the microbial community in the respiratory tract due to infections, like influenza, could impact transmission of bacterial pathogens. Using samples from a household study, we determined whether metagenomic-type analyses of the microbiome provide the resolution necessary to track transmission of airway bacteria. Microbiome studies have shown that the microbial community across various body sites tends to be more similar between individuals who cohabit in the same household than between individuals from different households. We tested whether there was increased sharing of bacteria from the airways within households with influenza infections as compared to control households with no influenza. RESULTS: We obtained 221 respiratory samples that were collected from 54 individuals at 4 to 5 time points across 10 households, with and without influenza infection, in Managua, Nicaragua. From these samples, we generated metagenomic (whole genome shotgun sequencing) datasets to profile microbial taxonomy. Overall, specific bacteria and phages were differentially abundant between influenza positive households and control (no influenza infection) households, with bacteria like Rothia, and phages like Staphylococcus P68virus that were significantly enriched in the influenza-positive households. We identified CRISPR spacers detected in the metagenomic sequence reads and used these to track bacteria transmission within and across households. We observed a clear sharing of bacterial commensals and pathobionts, such as Rothia, Neisseria, and Prevotella, within and between households. However, due to the relatively small number of households in our study, we could not determine if there was a correlation between increased bacterial transmission and influenza infection. CONCLUSION: We observed that airway microbial composition differences across households were associated with what appeared to be different susceptibility to influenza infection. We also demonstrate that CRISPR spacers from the whole microbial community can be used as markers to study bacterial transmission between individuals. Although additional evidence is needed to study transmission of specific bacterial strains, we observed sharing of respiratory commensals and pathobionts within and across households. Video Abstract.

Tracking the emergence of antigenic variants in influenza A virus epidemics in Brazil.

Influenza A virus (IAV) circulation patterns differ in North America and South America, with influenza seasons often characterized by different subtypes and strains. However, South America is relatively undersampled considering the size of its population. To address this gap, we sequenced the complete genomes of 220 IAVs collected between 2009 and 2016 from hospitalized patients in southern Brazil. New genetic drift variants were introduced into southern Brazil each season from a global gene pool, including four H3N2 clades (3c, 3c2, 3c3, and 3c2a) and five H1N1pdm clades (clades 6, 7, 6b, 6c, and 6b1). In 2016, H1N1pdm viruses belonging to a new 6b1 clade caused a severe influenza epidemic in southern Brazil that arrived early and spread rapidly, peaking mid-autumn. Inhibition assays showed that the A/California/07/2009(H1N1) vaccine strain did not protect well against 6b1 viruses. Phylogenetically, most 6b1 sequences that circulated in southern Brazil belong to a single transmission cluster that rapidly diffused across susceptible populations, leading to the highest levels of influenza hospitalization and mortality seen since the 2009 pandemic. Continuous genomic surveillance is needed to monitor rapidly evolving IAVs for vaccine strain selection and understand their epidemiological impact in understudied regions.

Influenza Defective Interfering Virus Promotes Multiciliated Cell Differentiation and Reduces the Inflammatory Response in Mice.

Influenza defective interfering (DI) viruses have long been considered promising antiviral candidates because of their ability to interfere with replication-competent viruses and induce antiviral immunity. However, the mechanisms underlying DI-mediated antiviral immunity have not been extensively explored. Here, we demonstrated the interferon (IFN)-independent protection conferred by the influenza DI virus against homologous virus infection in mice deficient in type I and III IFN signaling. We identified unique host signatures responding to DI coinfection by integrating transcriptional and posttranscriptional regulatory data. DI-treated mice exhibited reduced viral transcription, less intense inflammatory and innate immune responses, and primed multiciliated cell differentiation in their lungs at an early stage of infection, even in the absence of type I or III IFNs. This increased multiciliogenesis could also be detected at the protein level via the immunofluorescence staining of lung tissue from DI-treated mice. Overall, our study provides mechanistic insight into the protection mediated by DIs, implying a unifying theme involving inflammation and multiciliogenesis in maintaining respiratory homeostasis and revealing their IFN-independent antiviral activity. IMPORTANCE During replication, the influenza virus generates genetically defective viruses. These are found in natural infections as part of the virus population within the infected host. Some versions of these defective viruses are thought to have protective effects through their interference with replication-competent viruses and induction of antiviral immunity. To better determine the mechanisms underlying the protective effects of these defective interfering (DI) viruses, we tested a DI that we previously identified in vitro with mice. Mice that were infected with a mix of wild-type influenza and DI viruses had less intense inflammatory and innate immune responses than did mice that were infected with the wild-type virus only, even when type I or III interferons, which are cytokines that play a prominent role in defending the respiratory epithelial barrier, were absent. More interestingly, the DI-infected mice had primed multiciliated cell differentiation in their lungs, indicating the potential promotion of epithelial repair by DIs.

US National Institutes of Health Prioritization of SARS-CoV-2 Variants.

Since late 2020, SARS-CoV-2 variants have regularly emerged with competitive and phenotypic differences from previously circulating strains, sometimes with the potential to escape from immunity produced by prior exposure and infection. The Early Detection group is one of the constituent groups of the US National Institutes of Health National Institute of Allergy and Infectious Diseases SARS-CoV-2 Assessment of Viral Evolution program. The group uses bioinformatic methods to monitor the emergence, spread, and potential phenotypic properties of emerging and circulating strains to identify the most relevant variants for experimental groups within the program to phenotypically characterize. Since April 2021, the group has prioritized variants monthly. Prioritization successes include rapidly identifying most major variants of SARS-CoV-2 and providing experimental groups within the National Institutes of Health program easy access to regularly updated information on the recent evolution and epidemiology of SARS-CoV-2 that can be used to guide phenotypic investigations.

Influenza A virus reassortment is strain dependent.

RNA viruses can exchange genetic material during coinfection, an interaction that creates novel strains with implications for viral evolution and public health. Influenza A viral genetic exchange can occur when genome segments from distinct strains reassort in coinfected cells. Predicting potential genomic reassortment between influenza strains has been a long-standing goal. Experimental coinfection studies have shed light on factors that limit or promote reassortment. However, determining the reassortment potential between diverse Influenza A strains has remained elusive. To address this challenge, we developed a high throughput genotyping approach to quantify reassortment among a diverse panel of human influenza virus strains encompassing two pandemics (swine and avian origin), three specific epidemics, and both circulating human subtypes A/H1N1 and A/H3N2. We found that reassortment frequency (the proportion of reassortants generated) is an emergent property of specific pairs of strains where strain identity is a predictor of reassortment frequency. We detect little evidence that antigenic subtype drives reassortment as intersubtype (H1N1xH3N2) and intrasubtype reassortment frequencies were, on average, similar. Instead, our data suggest that certain strains bias the reassortment frequency up or down, independently of the coinfecting partner. We observe that viral productivity is also an emergent property of coinfections, but uncorrelated to reassortment frequency; thus viral productivity is a separate factor affecting the total number of reassortants produced. Assortment of individual segments among progeny and pairwise segment combinations within progeny generally favored homologous combinations. These outcomes were not related to strain similarity or shared subtype but reassortment frequency was closely correlated to the proportion of both unique genotypes and of progeny with heterologous pairwise segment combinations. We provide experimental evidence that viral genetic exchange is potentially an individual social trait subject to natural selection, which implies the propensity for reassortment is not evenly shared among strains. This study highlights the need for research incorporating diverse strains to discover the traits that shift the reassortment potential to realize the goal of predicting influenza virus evolution resulting from segment exchange.

Bordetella bronchiseptica-Mediated Interference Prevents Influenza A Virus Replication in the Murine Nasal Cavity.

Colonization resistance, also known as pathogen interference, describes the ability of a colonizing microbe to interfere with the ability of an incoming microbe to establish infection, and in the case of pathogenic organisms, cause disease in a susceptible host. Furthermore, colonization-associated dysbiosis of the commensal microbiota can alter host immunocompetence and infection outcomes. Here, we investigated the role of Bordetella bronchiseptica nasal colonization and associated disruption of the nasal microbiota on the ability of influenza A virus to establish infection in the murine upper respiratory tract. Targeted sequencing of the microbial 16S rRNA gene revealed that B. bronchiseptica colonization of the nasal cavity efficiently displaced the resident commensal microbiota-the peak of this effect occurring 7 days postcolonization-and was associated with reduced influenza associated-morbidity and enhanced recovery from influenza-associated clinical disease. Anti-influenza A virus hemagglutinin-specific humoral immune responses were not affected by B. bronchiseptica colonization, although the cellular influenza PA-specific CD8+ immune responses were dampened. Notably, influenza A virus replication in the nasal cavity was negated in B. bronchiseptica-colonized mice. Collectively, this work demonstrates that B. bronchiseptica-mediated pathogen interference prevents influenza A virus replication in the murine nasal cavity. This may have direct implications for controlling influenza A virus replication in, and transmission events originating from, the upper respiratory tract. IMPORTANCE The interplay of microbial species in the upper respiratory tract is important for the ability of an incoming pathogen to establish and, in the case of pathogenic organisms, cause disease in a host. Here, we demonstrate that B. bronchiseptica efficiently colonizes and concurrently displaces the commensal nasal cavity microbiota, negating the ability of influenza A virus to establish infection. Furthermore, B. bronchiseptica colonization also reduced influenza-associated morbidity and enhanced recovery from influenza-associated disease. Collectively, this study indicates that B. bronchiseptica-mediated interference prevents influenza A virus replication in the upper respiratory tract. This result demonstrates the potential for respiratory pathogen-mediated interference to control replication and transmission dynamics of a clinically important respiratory pathogen like influenza A virus.