Influenza A-induced cystic fibrosis transmembrane conductance regulator dysfunction increases susceptibility to Streptococcus pneumoniae.

Influenza A virus (IAV) infection is commonly complicated by secondary bacterial infections that lead to increased morbidity and mortality. Our recent work demonstrates that IAV disrupts airway homeostasis, leading to airway pathophysiology resembling cystic fibrosis disease through diminished cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we use human airway organotypic cultures to investigate how IAV alters the airway microenvironment to increase susceptibility to secondary infection with Streptococcus pneumoniae (Spn). We observed that IAV-induced CFTR dysfunction and airway surface liquid acidification is central to increasing susceptibility to Spn. Additionally, we observed that IAV induced profound transcriptional changes in the airway epithelium and proteomic changes in the airway surface liquid in both CFTR-dependent and -independent manners. These changes correspond to multiple diminished host defense pathways and altered airway epithelial function. Collectively, these findings highlight both the importance of CFTR function during infectious challenge and demonstrate a central role for the lung epithelium in secondary bacterial infections following IAV.

A single intranasal administration of AdCOVID protects against SARS-CoV-2 infection in the upper and lower respiratory tracts.

The coronavirus disease 2019 (COVID-19) pandemic has illustrated the critical need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive approach for preventing COVID-19 as the nasal mucosa is the site of initial SARS-CoV-2 entry and viral replication prior to aspiration into the lungs. We previously demonstrated that a single intranasal administration of a candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain of the SARS-CoV-2 spike protein (AdCOVID) induced robust immunity in both the airway mucosa and periphery, and completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge. Here we show that a single intranasal administration of AdCOVID limits viral replication in the nasal cavity of K18-hACE2 mice. AdCOVID also induces sterilizing immunity in the lungs of mice as reflected by the absence of infectious virus. Finally, AdCOVID prevents SARS-CoV-2 induced pathological damage in the lungs of mice. These data show that AdCOVID not only limits viral replication in the respiratory tract, but it also prevents virus-induced inflammation and immunopathology following SARS-CoV-2 infection.

Single-Dose Intranasal Administration of AdCOVID Elicits Systemic and Mucosal Immunity against SARS-CoV-2 and Fully Protects Mice from Lethal Challenge.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.

The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I.

A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid-inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function.

Influenza-mediated reduction of lung epithelial ion channel activity leads to dysregulated pulmonary fluid homeostasis.

Severe influenza (IAV) infection can develop into bronchopneumonia and edema, leading to acquired respiratory distress syndrome (ARDS) and pathophysiology. Underlying causes for pulmonary edema and aberrant fluid regulation largely remain unknown, particularly regarding the role of viral-mediated mechanisms. Herein, we show that distinct IAV strains reduced the functions of the epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane regulator (CFTR) in murine respiratory and alveolar epithelia in vivo, as assessed by measurements of nasal potential differences and single-cell electrophysiology. Reduced ion channel activity was distinctly limited to virally infected cells in vivo and not bystander uninfected lung epithelium. Multiple lines of evidence indicated ENaC and CFTR dysfunction during the acute infection period; however, only CFTR dysfunction persisted beyond the infection period. ENaC, CFTR, and Na,K-ATPase activities and protein levels were also reduced in virally infected human airway epithelial cells. Reduced ENaC and CFTR led to changes in airway surface liquid morphology of human tracheobronchial cultures and airways of IAV-infected mice. Pharmacologic correction of CFTR function ameliorated IAV-induced physiologic changes. These changes are consistent with mucous stasis and pulmonary edema; furthermore, they indicate that repurposing therapeutic interventions correcting CFTR dysfunction may be efficacious for treatment of IAV lung pathophysiology.