Abstract Purpose: Aging is the greatest risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. With advancing age, impaired immune surveillance and cellular senescence create a pro-tumorigenic microenvironment that disrupts epithelial homeostasis and promotes oncogenic remodeling. However, the molecular mechanisms by which age-related transcriptional and splicing changes in lung contribute to immune dysregulation and cancer susceptibility remain poorly understood. We performed integrated transcriptomic profiling of lung progenitor cells to identify age-associated molecular alterations predisposing to immune dysfunction and early tumorigenic changes. Methods: Primary human broncho-epithelial cells from young (age: 18y to 27y, n=5) and old (age: 42y to 67y, n=5) donors were enriched for progenitor cells in 2D culture for seven days following Fulcher et al. We performed Illumina short-read and PacBio long-read RNA-sequencing. Isoform-level analysis on long-reads was performed through IsoSeq3 and SQANTI3. Differential expression was analyzed using DESeq2 (FDR < 0.05) and alternative splicing using rMATS (>10% Delta PSI, FDR < 0.05) with a long-read derived transcriptome. Functional consequences of splicing events were predicted using SpliceDecoder. Results: We identified 47 differentially expressed genes (36 upregulated, 11 downregulated with age). Notably, IL18, MMP25, PGLYRP4, THY1, CDH2, MFAP5, and PLXNC1 were upregulated in older donors, reflecting activation of inflammatory and immune response programs alongside altered epithelial and extracellular matrix remodeling. We also detected 991 age-related differentially spliced events in 632 genes. Among these, intron retention occurred in immune genes including HLA-A, HLA-B, HLA-C, TRIM65, and FOSB, while splicing of cassette exons was observed in IFNAR2-IL10RB, DMKN, HLA-F-AS1, and NOD1. SpliceDecoder predicted that these age-related splicing alterations introduce premature stop codons, alter coding sequences, and modify protein domains, potentially leading to gain- or loss-of-function effects. Finally, long-read RNA-sequencing identified 42,206 full-length spliced isoforms, the majority of which were novel and absent from reference transcriptomes, revealing extensive isoform diversity in lung progenitor cells. Conclusions: Our integrated approach demonstrates that aging reshapes the transcriptomic landscape through changes in gene expression and splicing, that may contribute to immune dysfunction and epithelial remodeling. These changes may create a pro-inflammatory environment that enhances susceptibility to oncogenic transformation and disrupts tissue homeostasis. Together, these findings uncover molecular mechanisms linking aging to lung cancer risk and identify potential biomarkers and therapeutic targets for prevention and intervention. Citation Format: Mohammed Toufiq, Florentina Marches, Hyeon Gu Kang, Te-Chia Wu, Ryan Englander, Sanaz Keshavarz Shahbaz, Marina Yurieva, Phylip Chen, Mark E Peeples, Adolfo Garcia-Sastre, Michael Schotsaert, Damien Chaussabel, Karolina Palucka, Olga A. Anczukow-Camarda. Age-related transcriptional and alternative splicing changes in lung progenitor cells predisposing to immune dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2256.

Globally, RSV is a major contributor to severe lower respiratory tract infections among children. Despite the significant medical concern posed by RSV, efforts to develop effective vaccines and antiviral drugs have largely fallen short, with the exception of immune prophylaxis available only for specific high-risk infants. We employed a suite of computational tools to investigate the role of microRNAs in the host's response to RSV infection. miRanda and RNAHybrid were instrumental in predicting microRNA-mRNA binding sites. For a deeper structural analysis, MC-Fold and MC-Sym were used to predict the 3D structures of both the miRNAs and their target mRNAs. The interactions between these molecules were then studied through RNA-RNA docking, with the resulting poses evaluated based on binding affinities and interaction profiles. This analysis focused on twelve selected miRNAs and their binding to specific sites on RSV mRNA. Finally, molecular dynamics (MD) simulations were conducted to evaluate the stability of the docked complexes. Taken together, these results suggest that two miRNAs, hsa_miR-2278 and hsa_miR-6732-3p, could potentially regulate the transcriptional activity during RSV infection and may warrant consideration as therapeutic agents.

Abstract Introduction The interpretation of large-scale transcriptional data remains a significant challenge in functional genomics, particularly in complex biological contexts such as host-pathogen interactions. Methods We present a systematic approach combining air-liquid interface (ALI) cultures with stepwise Large Language Model (LLM) analysis to achieve deep functional interpretation of ciliary gene regulation during influenza infection. From 2,828 differentially expressed genes, initial high-throughput LLM screening identified 29 genes with high confidence scores specifically associated with ciliated cell biology. We conducted detailed functional profiling of these candidates through human-in-the-loop validation. Results Analysis revealed a coordinated program of ciliary gene dysregulation. Key genes, including DNAH5, DYNC2H1, and DNAAF4-CCPG1, showed consistent downregulation by 24-48 hours post-infection. This pattern was validated across independent datasets from Influenza, Rhinovirus, and SARS-CoV-2 infections, suggesting a conserved mechanism of mucociliary clearance impairment across respiratory viral infections. Conclusion By focusing our analysis on ciliated cell-associated genes, we uncovered specific mechanisms of viral pathogenesis while establishing a generalizable framework for context-aware interpretation of complex biological datasets. Funding Source National Institute of Allergy and Infectious Diseases (NIAID) Topic Categories Viral Immunology (VIR)

Since the COVID-19 pandemic, several reverse genetics platforms for SARS-CoV-2 have been established. In general, a plasmid-based reverse genetics system is stable and easy to manipulate, distribute, and store. However, traditional methods for the assembly of a large viral genome in a plasmid rely on natural and artificially engineered restriction sites, which are inefficient, time-consuming, labor-intensive, and frequently not successful. Here, we developed a yeast-based homologous recombination system that allows the assembly of the SARS-CoV-2 genome as a cDNA in a bacterial artificial chromosome (BAC) plasmid in a single step. The entire protocol from cDNA construction to virus rescue is simple, rapid, accurate, highly efficient, and can be completed in 2 weeks. Using this system, we have quickly generated recombinant SARS-CoV-2 (rSARS-CoV-2) WA1, Omicron BA.2.86, and Omicron JN.1 viruses expressing mCherry, green fluorescent protein (GFP), and NanoLuc luciferase (Nluc) reporters. Insertion of these reporter genes does not significantly alter the replication of SARS-CoV-2 in cell culture. We also compared the replication kinetics of rSARS-CoV-2-WA1, BA.2.86, and JN.1 reporter viruses in ex vivo primary human nasal epithelial (HNE) and human bronchial epithelial (HBE) cultures. Omicron BA.2.86 replicated and spread more efficiently than JN.1, which spread much faster than SARS-CoV-2 WA1 in these cultures. In summary, we have developed a highly efficient yeast-based recombinant system for the construction of infectious cDNA clones of SARS-CoV-2, enabling rapid genetic manipulation of SARS-CoV-2. In addition, the reporter viruses generated in this study will be useful for monitoring SARS-CoV-2 infection in vitro and in vivo.IMPORTANCEReverse genetics systems are an essential tool for probing the biology of viruses, testing antivirals, and developing live-attenuated vaccines. However, it has been a challenge to generate a rapid reverse genetics system for coronaviruses. Here, we developed a rapid, highly efficient reverse genetics system for SARS-CoV-2 that uses yeast homologous recombination. In this procedure, overlapping DNA fragments encompassing the entire SARS-CoV-2 and BAC plasmid fragments containing a yeast replication origin were mixed and transformed into yeast cells to assemble infectious cDNA clones in a single step. This system has enabled us to rapidly generate nine SARS-CoV-2 viruses: WA1, Omicron BA.2.86, and JN.1 viruses each expressing one of three reporters for tracking virus infection in vitro and in vivo. This method is easy, convenient, and highly efficient, generating infectious cDNA clones within 2 weeks. This system could readily be adapted to construct infectious cDNA clones for other large RNA viruses.

Abstract Introduction The interpretation of large-scale transcriptional data remains a significant challenge in functional genomics, particularly in complex biological contexts such as host-pathogen interactions. Methods We present a systematic approach combining air-liquid interface (ALI) cultures with stepwise Large Language Model (LLM) analysis to achieve deep functional interpretation of ciliary gene regulation during influenza infection. From 2,828 differentially expressed genes, initial high-throughput LLM screening identified 29 genes with high confidence scores specifically associated with ciliated cell biology. We conducted detailed functional profiling of these candidates through human-in-the-loop validation. Results Analysis revealed a coordinated program of ciliary gene dysregulation. Key genes, including DNAH5, DYNC2H1, and DNAAF4-CCPG1, showed consistent downregulation by 24-48 hours post-infection. This pattern was validated across independent datasets from Influenza, Rhinovirus and SARS-CoV-2 infections, suggesting a conserved mechanism of mucociliary clearance impairment across respiratory viral infections. Conclusion By focusing our analysis on ciliated cell-associated genes, we uncovered specific mechanisms of viral pathogenesis while establishing a generalizable framework for context-aware interpretation of complex biological datasets. Funding Source National Institute of Allergy and Infectious Diseases (NIAID) Topic Categories Computational and Systems Immunology (COMP)

Throughout the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown the capacity to infect a wide range of nonhuman hosts, including farmed mink. In early 2020, a mink-associated variant, termed mink cluster 5 (MC5V), emerged in Denmark and spread to mink farmers and their household contacts but failed to cause a sustained outbreak and eventually disappeared. Here, we demonstrate that the spike protein (S) of MC5V is intrinsically unstable and impaired in processing, leading to markedly attenuated infectivity and fusogenicity. Remarkably, these defects are primarily driven by a single mutation, I692V, located in the S2 subunit of S, with additional contribution from the Y453F substitution in the receptor-binding domain. Structural analyses indicate that I692V induces conformational instability in S, promoting spontaneous S1 shedding and impairing spike incorporation into virions. These findings reveal that spike instability constrains viral fitness and emphasize the importance of monitoring zoonotic SARS-CoV-2 variants and other emerging viral pathogens.

Influenza D virus (IDV), primarily found in livestock species, has demonstrated cross-species transmission potential, yet its threat to humans remains poorly understood. Here, we curated a panel of IDV isolates collected during field surveillance from 2011 to 2020 from swine and cattle to assess their ability to infect human airway cells as a proxy for zoonotic threat assessment. Using lung epithelial cell lines, primary well-differentiated airway epithelial cultures, and precision-cut lung slices, we demonstrated that IDV efficiently propagates in cells and tissues from the human respiratory tract, reaching titers comparable to human influenza A virus (IAV). Infection kinetics in primary porcine airway cultures and respiratory tissues mirrored those from humans, suggesting similar infectivity across species. To define host responses to IDV infection, we evaluated innate immune sensing and downstream interferon signaling in human respiratory cells. IDV infection resulted in markedly reduced activation of interferon regulatory factor signaling and diminished induction of interferon lambda 1 and interferon-stimulated genes compared to IAV, indicating inefficient activation of innate immune sensing pathways. However, IDV replication was potently restricted in interferon-pretreated cells, demonstrating sensitivity to interferon-mediated antiviral effector mechanisms once an antiviral state was established. Together, these findings show that IDV can efficiently infect the human airway while limiting innate immune sensing, a feature that may facilitate zoonotic spillover. Our study highlights the need for enhanced surveillance of IDV at the animal-human interface and provides a foundation for further investigation into its biology and potential for causing human infection and disease.

Physical interactions with bacterial colonizers at mucosal barrier surfaces can have beneficial or detrimental effects on viral infectivity and transmission. The oropharyngeal mucosal surface is a major portal of entry for many pathogenic respiratory viruses, such as respiratory syncytial virus (RSV), and also harbors a rich and diverse microbiome. Whether oropharyngeal bacteria directly or indirectly influence host susceptibility to respiratory viruses remains unknown. Here, we show that Porphyromonas gingivalis, an oral pathobiont, potently and uniquely suppresses airway epithelial antiviral immunity by degrading interferons (IFNs) and transcriptionally repressing multiple IFN-stimulated genes (ISGs), which are essential for providing resistance to viral infection. Despite inducing a state of IFN hyporesponsiveness, we found that P. gingivalis counterintuitively protected against severe infection by RSV and the closely related murine-specific Sendai virus (SeV) in two independent models: human airway bronchial epithelial transwell cultures and airway infection in mice. This protection was conferred by the activity of P. gingivalis cysteine proteases (gingipains) that cleaved envelope glycoproteins on RSV and SeV, thereby impairing their infectious capacity. Thus, our data show a nuanced role for P. gingivalis in modulating host susceptibility to viral infection. While P. gingivalis can significantly inhibit host IFN responses, its proteases preemptively reduce viral infectious capacity, protecting the host from severe damage associated with respiratory infections.

Negative-sense RNA viruses have been widely used as viral vectors for vaccine delivery. However, little is known about coronaviruses as vectors for delivering vaccines. Here, we have developed safe SARS-CoV-2 Omicron JN.1-based live attenuated vaccine candidates by combining a mutation (D130A) in the viral nsp16 protein, deletion of the furin cleavage site (dFCS) in the spike protein, deletion of accessory proteins, and/or modification of the transcription regulatory sequences (mTRS). Subsequently, using rJN.1, rJN.1-D130A-dFCS, and rJN.1-mTRS-D130A-dFCS as the backbones, we generated three recombinant viruses expressing a nonfunctional, soluble, and stabilized prefusion F protein of human respiratory syncytial virus (RSVF). Among them, rJN.1-D130A-dFCS-RSVF virus was sufficiently attenuated and highly immunogenic, providing complete protection against challenge with both JN.1 and RSV in hamsters. However, rJN.1-mTRS-D130A-dFCS-RSVF was poorly immunogenic. Collectively, we demonstrate that attenuated SARS-CoV-2 is an effective viral vector for delivering RSV vaccine, warranting further development as a novel intranasal bivalent vaccine for SARS-CoV-2 and RSV.

Rationale: Influenza A virus (IAV) poses a major public health threat, causing severe lung inflammation. Due to frequent mutations, IAVs often develop resistance to current antiviral treatments. Hence, to identify potential alternative therapies, it is crucial to understand how IAVs interact with host proteins. Our previous research showed that IAV induces the host cellular protein Bcl-2 interacting killer (BIK), promoting viral replication in airway epithelial cells (AECs). However, the precise mechanism remains unclear. Here, we further investigated how IAV hijacks the host cellular proteins to promote viral replication and exacerbate lung inflammation. Methods: Human precision-cut lung slices (hPCLS) were transduced with adeno-associated viral (AAV) vectors expressing EGFP or ARIH2, followed by IAV infection. To explore the translational potential, ARIH2 was delivered intranasally via AAV6.2 vector in C57BL/6 mice followed by IAV-infection, and the impact on lung inflammation and survival was evaluated. Lung histology was assessed using hematoxylin and eosin (H&E) staining. Western blot, co-immunoprecipitation, ubiquitination assays, mass spectrometry (MS), and immunofluorescence were performed to assess protein expression. Luminex multiplex assays quantified inflammatory cytokines/chemokines in lung homogenates and supernatants of IAV-infected mice and hPCLS. Results: MS analysis identified ARIH2 as the primary BIK-interacting E3 ligase suppressed by IAV to stabilize BIK. This finding was supported by the observation that decreasing ARIH2 levels increased BIK protein levels in IAV-infected AECs. IAV nucleoprotein, distinct from other viral ribonucleoprotein components, specifically inhibited ARIH2 and induced BIK. Overexpression of ARIH2 reduced BIK levels and diminished IAV yield. Conversely, ARIH2 depletion led to increased BIK protein levels. Furthermore, ARIH2 showed a robust interaction with BIK. IAV inhibited ARIH2-mediated K48-linked BIK ubiquitination. On the contrary, ARIH2 overexpression in IAV-infected cells restored BIK ubiquitination and dampened IAV-induced pro-inflammatory cytokines, affirming that IAV interferes with ARIH2-mediated BIK degradation. In hPCLS, IAV infection suppressed ARIH2, thereby increasing BIK protein levels, while AAV-ARIH2 treatment significantly reduced viral load, underscoring its significant translational implications. In vivo, intranasal delivery of AAV-ARIH2 reduced lung viral load and inflammation, thereby reducing IAV-induced morbidity and mortality. Conclusion: ARIH2 emerges as a novel host cellular protein exploited by IAV to inhibit BIK degradation, thereby promoting viral replication. Enhancing ARIH2-mediated BIK ubiquitination presents a promising novel therapeutic strategy to reduce IAV replication and lung inflammation.

In this study, we developed a 3D lung model that incorporated alveolar and vascular components, allowing for the investigation of lung physiology and responses to infection. We investigated the role of ventilation in formation of the alveolar epithelial layer and its response to viral infections. We subjected our perfused model to a continuous respiratory cycle at the air-liquid interface (ALI) for up to 10 days. The results revealed that ventilation increased tight-junction formation with better epithelial barrier function over time. Two viruses, influenza and respiratory syncytial virus (RSV), were tested, where ventilation enhanced infectivity with an increased progression of viral spread over time while sensitizing the epithelium for viral recognition. Ventilation also attenuated the production of key proinflammatory chemokines. Our findings represent a critical step forward in advancing our understanding of lung-specific viral responses and respiratory infections in response to ventilation, shedding light on vital aspects of pulmonary physiology and pathobiology.

Respiratory viral infections with negative-strand RNA viruses, like influenza A virus (IAV) and respiratory syncytial virus (RSV) cause significant morbidity and mortality. Previously we demonstrated mice treated with neuregulin-1 (NRG1) survived normally lethal infections with IAV or Sendai virus, a rodent virus related to RSV. Using well-differentiated human airway epithelial cells (hBEC) grown at air-liquid interface and infected with RSV, NRG-1 reduced viral titer and epithelial leak, which might explain increased survival following a severe respiratory viral infection. NRG1 binds three receptors of the EGFR family (HER-2 (ERbB2), ErbB3, and ErbB4) and signals through homo or heterodimers consisting of two ErbB receptors. To determine which ErbB receptors are necessary for NRG1’s function in vitro, we added mAbs against each of the ErbB receptors to the basolateral compartment of hBECs along with NRG1 (100 ng) for 5 days before gfp-RSV inoculation. Viral titer was determined by quantifying GFP by fluorescent microscopy 48h post-inoculation (PI) RSV. Epithelial leakage was determined by treating the transwell apical side with 70 KDa FITC-Dextran (25 mcg) and collecting basal media for spectrophotometric analysis at day 4 PI. Blocking HER-2 but not other ErbB receptors significantly increased viral titer and permeability, suggesting HER-2 is the primary receptor for NRG1 signaling and protection from viral insult in airway epithelium. This work was supported by NIH grant R01AI171027 (to Mitchell H Grayson) Translational and Interventional Immunology (TI)

Respiratory infections with RNA viruses such as respiratory syncytial virus (RSV) and influenza lead to significant morbidity and mortality. Using a natural rodent pathogen, Sendai virus (SeV), which is similar to RSV, mice made atopic with house dust mite survived a normally lethal SeV infection. One protein that we found markedly elevated in the lungs and bronchoalveolar lavage fluid of atopic mice was neuregulin-1 (NRG1). Administration of NRG1 protected naïve (non-atopic) mice from death with both SeV and mouse adapted influenza A virus (IAV). Survival was associated with reduced alveolar epithelium permeability and reduced phosphorylation of mixed lineage kinase domain-like (MLKL) protein indicating inhibition of necroptosis. In vitro, treatment of mouse lung epithelial cells with NRG1 inhibited SeV induced necroptosis, and NRG1 administration to differentiated human bronchial epithelial cells infected with RSV reduced transepithelial fluid leak and expression of necroptosis associated genes RIPK3 and MLKL, while regulating genes associated with homeostatic maintenance, suggesting stabilized epithelial integrity. In conclusion, our data demonstrate a unique function of NRG1 in respiratory viral infections by reducing alveolar leak, inhibiting epithelial necroptosis, and promoting homeostatic regulation of airway epithelium, all of which associate with markedly reduced mortality to the respiratory viral insult.

BACKGROUND:Maternal antibodies are critical for infant protection. We analyzed the dynamics of placental transferred antibodies generated after SARS-CoV-2 maternal infection and/or vaccination. METHODS:Prospective, multicenter, observational study of SARS-CoV-2-infected and/or vaccinated pregnant people and their infants. We collected maternal and cord blood samples at delivery and neonatal/infant samples at delivery, 1, 2, 6 and 12 months of age. Receptor Binding Domain (RBD) and Spike immunoglobulin G antibody titers were measured by Enzyme Linked Immunosorbent Assay (ELISA). Serum maternal cytokines were measured at delivery using the Olink platform. We analyzed differences in antibody transfer according to infection versus vaccination, adjusted for trimester of gestation. RESULTS:We collected blood samples from 193 pregnant people (infected = 96, vaccinated = 60 and infected and vaccinated = 37) and 154 infants (n = 76, n = 47 and n = 31, respectively). At birth, RBD median (interquartile range) log 10 ng/mL antibody titers of infants from vaccinated-only [4.28 (3.48-4.80)] and from infected-and-vaccinated mothers [4.61 (4.27-4.93)] were higher than from infected-only mothers [2.20 (0.10-3.30); P < 0.001]. Differences persisted through 6 months of age. Median (interquartile range) transplacental antibody transfer ratio was higher in vaccinated-only [2.94 (1.34-3.74)] versus infected-only pregnant people [1.19 (0.33-2.52); P < 0.01]. Spike antibodies showed similar results. Linear regression analysis showed that mean RBD and Spike antibodies transfer ratios were higher in infants from vaccinated-only versus infected-only mothers, adjusted for trimester of infection or vaccination. Maternal concentrations of CXCL10, CXCL11, IL-18 and IFNg at delivery were inversely correlated with placental antibody transfer. CONCLUSIONS:Antibodies generated by maternal vaccination were transplacentally transferred more efficiently and persisted longer in infants than those generated by SARS-CoV-2 infection alone.

BackgroundRespiratory syncytial virus (RSV) is a common cause of bronchiolitis in children under the age of five. RSV infection proceeds by fusion of the viral envelope with the target cell membrane, but it is unclear whether fusion occurs with plasma or endosomal membranes.MethodsEntry and/or infection was studied in undifferentiated primary cultures of human bronchial epithelial cells. Synchronization of viral entry or infection was achieved by attaching the virus to the plasma membrane at temperatures of 4°C or 22°C. Cells in which entry events had occurred were identified by the enzymatic action of beta-lactamase M (BlaM) fused to the RSV P protein (BlaM-P) carried by rgRSV virions. BlaM cleaves the beta-lactam ring of CCF2 loaded into the cells, disrupting FRET and allowing blue light to be emitted. Green fluorescent protein (GFP) expression, encoded by the rgRSV genome, was used to identify infected cells.ResultsWe found that adsorption of RSV at 4°C favors entry via endocytosis, whereas binding of the virus to the membrane at 22°C favors RSV entry via the plasma membrane. The induction of endocytosis by synchronization at 4°C is, therefore, an artifact. In addition, we found that all drugs that interfered with RSV infection reduced cell membrane deformations such as filopodia and lamellipodia, suggesting a mechanism by which they may interfere with RSV fusion with the cell membrane.DiscussionIn conclusion, RSV enters the cell by direct fusion of its envelope with the plasma membrane.

The addition of marker protein genes to respiratory syncytial virus (RSV) has enabled studies of the spread of RSV in different types of cell cultures and quantification of viral replication in those cultures. Genetic deletion of individual RSV genes from RSV genome has been used to determine their importance in virus infection and the differences between infection of cultured cells lines and of primary well-differentiated human bronchial epithelial (HBE) cultures. Modifications of individual viral proteins can identify the importance of a particular glycosylation, cleavage, or antigenic sites or reveal sites with these functions. However, the standard recombinant systems for the RSV genome, based on natural and inserted restriction sites, have been difficult to use, slow to accomplish, and frequently not successful. Here, we describe a yeast-based cDNA assembly system that streamlines both the construction of a cDNA clone of an RSV strain as well as any modification of an RSV cDNA, thereby enabling the rapid generation of an RSV mutant virus.

Human viruses have traditionally been studied in immortalized, usually tumor-derived, cell lines because of their availability, low cost, ease of expansion, and ease of care. Although most viruses infect, replicate, and spread in these cells, all aspects of in vivo virus-host interaction are not accurately reflected in immortalized cells. The isolation, storage, and differentiation of human airway epithelial basal cells have enabled ex vivo studies of RSV infection in a near natural setting. Here we provide our rationale for using well-differentiated primary human bronchial and nasal epithelial cultures, and a current protocol for generating these fully differentiated airway cultures from donor tissue for ex vivo studies of RSV and other respiratory virus infections.

Severe SARS-CoV-2 infection is characterized by lung hyperinflammation, impaired interferon responses, and defective T-cell activation, yet the molecular drivers of these immune dysregulations remain incompletely understood. Caspase-11 (CASP11), a key mediator of the non-canonical inflammasome, has been shown to mediate an innate hyperinflammatory response and cytokine release in a non-severe, non-lethal SARS-CoV-2 infection model. However, the role played by CASP11 in severe SARS-CoV-2 disease and how it impacts adaptive immunity is not identified. Here, we newly discover that CASP11 exacerbates severe SARS-CoV-2 pathogenesis by amplifying early innate immune responses while concurrently impairing antiviral CD8 T-cell immunity. Using global knockouts, reciprocal bone marrow chimeras, and phagocyte- monocyte system (PMS) cell-specific CASP11 deletion models, we show that CASP11 deletion in monocyte-derived cells reduces lung inflammation, enhances type I and II interferon signaling, and promotes robust virus-specific effector CD8+ T-cell response. This was associated with enhanced viral clearance and improved survival, even under lethal infection conditions. Importantly, CASP11 KO mice also exhibited faster resolution of post-viral inflammation, suggesting a role in long-term immune remodeling. These findings position CASP11 as a promising immunomodulatory target for acute and delayed manifestations of severe SARS-CoV-2.

We demonstrate that proteases produced by the oro-pharyngeal bacterial colonizer Porphyromonas gingivalis (Pg) reduce viral burden and modulate host interferon responses during respiratory syncytial virus (RSV) infection. Several oral bacteria, including Pg , have been shown to translocate to the upper airways through sub-clinical micro-aspiration. Our findings reveal that Pg , upon translocating to this new niche, significantly attenuated lung damage by reducing viral loads during respiratory viral infections in the lungs of wild-type mice. This protective effect was attributed to the activity of gingipains, cysteine endopeptidases produced by Pg , which cleaved envelope glycoproteins on RSV as well as on related murine-specific Sendai virus (SeV), thereby impairing their infectious capacity. Notably, the reduction in viral loads was independent of interferon lambda (IFN-λ) signaling, which is actively suppressed by Pg in airway epithelial cells. However, the complete absence of IFN-λ signaling resulted in a stronger inflammatory response despite a low viral load. Thus, we show a previously undescribed role for the oro-respiratory bacterial colonizer Pg in creating bottlenecks to viral infection by the activity of its proteases. SIGNIFICANCE STATEMENT:Reciprocal interactions between microbial colonizers and host epithelial cells are critical for providing initial defense against viral infections. However, our understanding of this phenomenon has been limited to microbiota-derived ligands that activate host pattern recognition receptors (PRRs), inducing basal interferon expression and downstream antiviral genes. Here, we present a novel mechanism that relies on microbial proteases to directly reduce viral load. Specifically, we discovered that the infectious capacity of the Respiratory Syncytial Virus (RSV) was significantly inhibited upon contact with the proteases (gingipains) produced by the oropharyngeal colonizer Porphyromonas gingivalis . Gingipains caused proteolytic degradation of the RSV envelope and attachment proteins, rendering them inactive. This preemptive reduction in viral infectious capacity consequently diminished the severity of respiratory viral infections in an IFN-independent manner.