Biology subjects

Harrod, K. S.

Publications and source records attributed to Harrod, K. S..

10 recordsLinked to original sources

bioRxiv2026

Intranasal immunization with live-attenuated RSV-vectored SARS-CoV-2 vaccines elicits antigen-specific systemic and mucosal immunity and protects against viral challenge and natural infection

The emergence of new SARS-CoV-2 variants and breakthrough infections underscores the need for next-generation vaccines capable of protecting from natural infection and/or preventing virus transmission to others. Intranasal vaccination offers a promising approach by eliciting local immune responses in the nasal mucosa, the primary site of infection and reservoir for transmissible virus. We evaluated two live-attenuated, respiratory syncytial virus vectored vaccines in which the RSV F and G surface glycoproteins were replaced with a chimeric SARS-CoV-2 Spike protein from either the ancestral USA/WA-1/2020 strain (MV-014-212) or the Delta variant (MV-014-212-delta). A single intranasal dose of either vaccine elicited systemic and mucosal immunity in K18-hACE2 mice, including serum neutralizing antibodies, Spike-specific memory B cells and plasmablasts, and Spike-specific CD8 lung-resident memory T cells. Although MV-014-212-delta vaccination provided the best protection against Delta variant virus challenge, both vaccines decreased viral loads in nasal discharge, lung and brain, and reduced weight loss and mortality. In naturally acquired infection studies, vaccinated hamsters exposed to infected cagemates exhibited minimal weight loss, limited viral replication within the nasal mucosa, and attenuated lung pathology. Therefore, intranasal RSV-vectored vaccines can elicit broad protective respiratory immunity, suggesting that this platform could be leveraged for other respiratory pathogens.

immunology↗

bioRxiv2025

Mucosal delivery of influenza antigens using a replication deficient adenovirus supports broadly reactive antibody responses and heterologous viral immunity in the respiratory tract of animals

Systemically administered influenza vaccines provide strain-limited protection, while influenza infection of the respiratory epithelium supports development of lung resident memory B and T cells and more broadly reactive antibody responses. To test whether local antigen delivery is critical for establishing broad immunity, we directly compared respiratory tract and systemic delivery of influenza antigens to mice and hamsters using a replication-deficient adenovirus serotype 5 vector (Ad5[E1-,E2b-,E3-]). Both immunization routes elicited antibody responses in the lower respiratory tract and antigen-specific B and T cells in the draining lymph node. However, only intranasal immunization established lung-resident memory B and T cells, induced IgA responses in the upper respiratory tract directly at the site of viral entry and supported generation of IgA and IgG antibodies that bound antigenically drifted and distantly related influenza strains, including those of avian origin. Intranasal immunization accelerated viral clearance following heterologous virus challenge and was associated with limited pulmonary inflammation and fibrosis. Thus, intranasal immunization with the immunologically stealthy Ad5[E1-,E2b-,E3-] platform supports respiratory and systemic immunity to divergent influenza strains in the absence of overt lung immunopathology, suggesting that local antigen delivery may be key to development of more broadly protective "universal" flu vaccines.

immunology↗

bioRxiv2024

ACE-2 Blockade & TMPRSS2 Inhibition Mitigate SARS-CoV-2 Severity Following Cigarette Smoke Exposure in Airway Epithelial Cells In Vitro

Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking alters the expression of the SARS-CoV-2 cellular receptor and point of entry, angiotensin converting enzyme-2 (ACE-2), and its cofactors including transmembrane protease serine 2 (TMPRSS2). We investigated the impact of cigarette smoking on the expression of ACE-2, TMPRSS2, and other known cofactors of SARS-CoV-2 infection and the resultant effects on infection severity in vitro. Cigarette smoke extract (CSE) exposure increased ACE-2 and TMPRSS2 mRNA expression compared to air control in ferret airway cells, Calu-3 cells, and primary human bronchial epithelial (HBE) cells derived from normal and COPD donors. CSE-exposed ferret airway cells inoculated with SARS-CoV-2 had a significantly higher intracellular viral load versus vehicle-exposed cells. Likewise, CSE-exposure increased both SARS-CoV-2 intracellular viral load and viral replication in both normal and COPD HBE cells over vehicle control. Apoptosis was increased in CSE-exposed, SARS-CoV-2-infected HBE cells. Knockdown of ACE-2 via an antisense oligonucleotide (ASO) reduced SARS-CoV-2 viral load and infection in CSE-exposed ferret airway cells that was augmented by co-administration of camostat mesylate to block TMPRSS2 activity. Smoking increases SARS-CoV-2 infection via upregulation of ACE2 and TMPRSS2.

cell biology↗

bioRxiv2024

Pulmonary Fibrosis Ferret Model Demonstrates Sustained Fibrosis, Restrictive Physiology, and Aberrant Repair

RationaleThe role of MUC5B mucin expression in IPF pathogenesis is unknown. Bleomycin-exposed rodent models do not exhibit sustained fibrosis or airway remodeling. Unlike mice, ferrets have human-like distribution of MUC5B expressing cell types and natively express the risk-conferring variant that induces high MUC5B expression in humans. We hypothesized that ferrets would consequently exhibit aberrant repair to propagate fibrosis similar to human IPF. MethodsBleomycin (5U/kg) or saline-control was micro-sprayed intratracheally then wild-type ferrets were evaluated through 22 wks. Clinical phenotype was assessed with lung function. Fibrosis was assessed with {micro}CT imaging and comparative histology with Ashcroft scoring. Airway remodeling was assessed with histology and quantitative immunofluorescence. ResultsBleomycin ferrets exhibited sustained restrictive physiology including decreased inspiratory capacity, decreased compliance, and shifted Pressure-Volume loops through 22 wks. Volumetric {micro}CT analysis revealed increased opacification of the lung bleomycin-ferrets. Histology showed extensive fibrotic injury that matured over time and MUC5B-positive cystic structures in the distal lung suggestive of honeycombing. Bleomycin ferrets had increased proportion of small airways that were double-positive for CCSP and alpha-tubulin compared to controls, indicating an aberrant proximalization repair phenotype. Notably, this aberrant repair was associated with extent of fibrotic injury at the airway level. ConclusionsBleomycin-exposed ferrets exhibit sustained fibrosis through 22 wks and have pathologic features of IPF not found in rodents. Ferrets exhibited proximalization of the distal airways and other pathologic features characteristic of human IPF. MUC5B expression through native cell types may play a key role in promoting airway remodeling and lung injury in IPF.

physiology↗

bioRxiv2023

Mucociliary Clearance Augmenting Drugs Block SARS-Cov-2 Replication in Human Airway Epithelial Cells

The coronavirus disease (COVID-19) pandemic, caused by SARS-CoV-2 coronavirus, is devastatingly impacting human health. A prominent component of COVID-19 is the infection and destruction of the ciliated respiratory cells, which perpetuates dissemination and disrupts protective mucociliary transport (MCT) function, an innate defense of the respiratory tract. Thus, drugs that augment MCT could improve barrier function of the airway epithelium, reduce viral replication and, ultimately, COVID-19 outcomes. We tested five agents known to increase MCT through distinct mechanisms for activity against SARS-CoV-2 infection using a model of human respiratory epithelial cells terminally differentiated in an air/liquid interphase. Three of the five mucoactive compounds tested showed significant inhibitory activity against SARS-CoV-2 replication. An archetype mucoactive agent, ARINA-1, blocked viral replication and therefore epithelial cell injury, thus, it was further studied using biochemical, genetic and biophysical methods to ascertain mechanism of action via improvement of MCT. ARINA-1 antiviral activity was dependent on enhancing the MCT cellular response, since terminal differentiation, intact ciliary expression and motion was required for ARINA-1-mediated anti-SARS-CoV2 protection. Ultimately, we showed that improvement of cilia movement was caused by ARINA-1-mediated regulation of the redox state of the intracellular environment, which benefited MCT. Our study indicates that Intact MCT reduces SARS-CoV-2 infection, and its pharmacologic activation may be effective as an anti-COVID-19 treatment.

microbiology↗

bioRxiv2023

Influenza A virus modulation of Streptococcus pneumoniae infection using ex vivo transcriptomics in a human primary lung epithelial cell model reveals differential host glycoconjugate uptake and metabolism

BackgroundStreptococcus pneumoniae (Spn) is typically an asymptomatic colonizer of the nasopharynx but it also causes pneumonia and disseminated disease affecting various host anatomical sites. Transition from colonization to invasive disease is not well understood. Studies have shown that such a transition can occur as result of influenza A virus coinfection. MethodsWe investigated the pneumococcal (serotype 19F, strain EF3030) and host transcriptomes with and without influenza A virus (A/California/07 2009 pH1N1) infection at this transition. This was done using primary, differentiated Human Bronchial Epithelial Cells (nHBEC) in a transwell monolayer model at an Air-Liquid Interface (ALI), with multispecies deep RNA-seq. ResultsDistinct pneumococcal gene expression profiles were observed in the presence and absence of influenza. Influenza coinfection allowed for significantly greater pneumococcal growth and triggered the differential expression of bacterial genes corresponding to multiple metabolic pathways; in totality suggesting a fundamentally altered bacterial metabolic state and greater nutrient availability when coinfecting with influenza. Surprisingly, nHBEC transcriptomes were only modestly perturbed by infection with EF3030 alone in comparison to that resulting from Influenza A infection or coinfection, which had drastic alterations in thousands of genes. Influenza infected host transcriptomes suggest significant loss of ciliary function in host nHBEC cells. ConclusionsInfluenza A virus infection of nHBEC promotes pneumococcal infection. One reason for this is an altered metabolic state by the bacterium, presumably due to host components made available as result of viral infection. Influenza infection had a far greater impact on the host response than did bacterial infection alone, and this included down regulation of genes involved in expressing cilia. We conclude that influenza infection promotes a pneumococcal metabolic shift allowing for transition from colonization to disseminated disease. Author summarySecondary Streptococcus pneumoniae bacterial infections typically occur after influenza A virus respiratory infection. Such coinfections often lead to invasive pneumococcal disease. The mechanisms involved in this process are not well understood. Here, using an ex vivo human lung bronchial epithelial cell model, we investigated the biological processes of the host and pneumococcus occurring at this niche, during coinfection with multi-species transcriptomics techniques, and in vivo mouse model experimentation. We observed stark differences in global pneumococcal metabolism in different infection states, as well as viral induced epithelial cell changes in ciliary function, potentially aiding pneumococcal dissemination. Overall, this study identified broad and targeted biological processes involved in this host-pathogen interaction.

microbiology↗

bioRxiv2022

Mucociliary Transport Deficiency and Disease Progression in Syrian Hamsters with SARS-CoV-2 Infection

Substantial clinical evidence supports the notion that ciliary function in the airways plays an important role in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, consequent impaired mucociliary transport (MCT) remains unknown for the intact MCT apparatus from an in vivo model of disease. Using golden Syrian hamsters, a common animal model that recapitulates human COVID-19, we quantitatively followed the time course of physiological, virological, and pathological changes upon SARS-CoV-2 infection, as well as the deficiency of the MCT apparatus using micro-optical coherence tomography, a novel method to visualize and simultaneously quantitate multiple aspects of the functional microanatomy of intact airways. Corresponding to progressive weight loss up to 7 days post-infection (dpi), viral detection and histopathological analysis in both the trachea and lung revealed steadily descending infection from the upper airways, as the main target of viral invasion, to lower airways and parenchymal lung, which are likely injured through indirect mechanisms. SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 dpi, largely due to diminished motile ciliation coverage, but not airway surface liquid depth, periciliary liquid depth, or cilia beat frequency of residual motile cilia. Further analysis indicated that the fewer motile cilia combined with abnormal ciliary motion of residual cilia contributed to the delayed MCT. The time course of physiological, virological, and pathological progression suggest that functional deficits of the MCT apparatus predispose to COVID-19 pathogenesis by extending viral retention and may be a risk factor for secondary infection. As a consequence, therapies directed towards the MCT apparatus deserve further investigation as a treatment modality.

microbiology↗

bioRxiv2021

Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase

The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs1-4, the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits5. Here we show that water-soluble derivatives of -tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D--tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and {beta}-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of -tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV-2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp6. In summary, we conclude that solubilizing modifications to -tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection7-9.

microbiology↗

bioRxiv2020

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.

immunology↗

bioRxiv2020

Influenza-induced oxidative stress sensitizes lung cells to bacterial toxin-mediated necroptosis

RationalePneumonia caused by Influenza A virus (IAV) co- and secondary bacterial infections are characterized by their severity. Previously we have shown that pore-forming toxin (PFT)-mediated necroptosis is a key driver of acute lung injury during bacterial pneumonia. Here, we evaluate the impact of IAV on PFT-induced acute lung injury during co- and secondary Streptococcus pneumoniae (Spn) infection. ObjectivesDetermine the impact of IAV infection on bacterial PFT-mediated lung epithelial cell (LEC) necroptosis. Determine the molecular basis for increased sensitivity and if inhibition of necroptosis or oxidative stress blocks IAV sensitization of LEC to PFT. MethodsMice and cells were challenged with IAV followed by Spn. Necroptosis was monitored by measuring cell death at fixed time points post-infection and immunofluorescent detection of necroptosis. Wildtype mice and LEC were treated with necroptosis inhibitors. Necroptosis effector molecule MLKL deficiency was tested for infection synergy. Oxidative damage to DNA and lipids as result of infection was measured in vitro and in vivo. Necroptosis and anti-oxidant therapy efficacy to reduce disease severity was tested in vivo. Measurements and Main ResultsIAV synergistically sensitized LEC for PFT-mediated necroptosis in vitro and in murine models of Spn co-infection and secondary infection. Pharmacological induction of oxidative stress sans virus sensitized cells for PFT-mediated necroptosis. Necroptosis inhibition reduced disease severity during secondary bacterial infection. ConclusionsIAV-induced oxidative stress sensitizes LEC for PFT-mediated necroptosis. This is a new molecular explanation for severe influenza-associated bacterial infections. Necroptosis inhibitors are potential therapeutic strategies to reduce IAV-primed bacterial pneumonia severity. SummaryHere we demonstrate that Influenza A virus (IAV) infection synergistically sensitizes lung cells to bacterial pore-forming toxin (PFT)-mediated necroptosis. Moreover, this contributes to the severity of lung injury that is observed during co- and secondary infection with Streptococcus pneumoniae. IAV-induced oxidative stress was identified as a key factor contributing to cell sensitization and induction of oxidative stress sans virus was sufficient to synergistically enhance susceptibility to PFT-mediated killing. Our results advance our understanding on the molecular basis of co- and secondary bacterial infection to influenza and identifies necroptosis inhibition and antioxidant therapy as potential intervention strategies.

microbiology↗