Biology subjects

Harding, A. T.

Publications and source records attributed to Harding, A. T..

7 recordsLinked to original sources

bioRxiv2025

Oxidative Stress and Interferon Signaling Drive Differential Pathogenesis of Ancestral and Contemporary Zika Viruses in Human Cerebral Organoids

Neurotropic Zika viruses (ZIKV) cause serious human disease with pandemic potential. Pathogenesis severities resulting from Asian/American versus African ZIKV lineage infections range from mild to severe, respectively; however, mechanisms underlying differential ZIKV pathogenesis remain unclear, as do effective therapeutic strategies. The limitations of mechanistic understanding are due in part to the challenges of comparing data generated in disparate experimental models, as well as approaches that did not test both ancestral and contemporary ZIKV infections. The goal of this work was to define differential pathogenesis mechanisms among ancestral and contemporary ZIKVs by direct infection comparisons using a relevant human stem cell-derived cerebral organoid experimental model. While Asian/American ZIKV lineage infections enhanced antiviral and interferon gene expression responses that correlated with viral RNA clearance from organoid ventricles, ancestral African lineage ZIKV infections enhanced apoptotic and stress response signaling that correlated with diminished STAT2 signaling protein levels, ongoing ZIKV replication, and production of damaging reactive oxygen species (ROS). We discovered that, surprisingly, severe ancestral Zika virus pathogenesis was dramatically reduced by Trolox, a hydroxyl radical scavenger antioxidant, thereby confirming ROS imbalance as a major pathogenesis driver. These results demonstrate that ZIKV lineage infections and pathogenesis are differentiated by their signaling responses and suggest that preventing or controlling hydroxyl radical imbalance may offer therapeutic benefits to address microcephaly and Congenital Zika Virus Syndrome. One Sentence SummaryDifferential signal transduction responses to lineage-specific Zika virus infections cause reduction-oxidation imbalance-mediated pathogenesis that is blocked by Trolox, an antioxidant.

microbiology↗

bioRxiv2024

Single-cell analysis of human airway epithelium identifies cell type-specific responses to Aspergillus and Coccidioides

Respiratory fungal infections pose a significant threat to human health. Animal models do not fully recapitulate human disease, necessitating advanced models to study human-fungal pathogen interactions. In this study, we utilized primary human airway epithelial cells (hAECs) to recapitulate the lung environment in vitro and investigate cellular responses to two diverse, clinically significant fungal pathogens, Aspergillus fumigatus and Coccidioides posadasii. To understand the mechanisms of early pathogenesis for both fungi, we performed single-cell RNA sequencing of infected hAECs. Analysis revealed that both fungi induced cellular stress and cytokine production. However, the cell subtypes affected and specific pathways differed between fungi, with A. fumigatus and C. posadasii triggering protein-folding-related stress in ciliated cells and hypoxia responses in secretory cells, respectively. This study represents one of the first reports of single-cell transcriptional analysis of hAECs infected with either A. fumigatus or C. posadasii, providing a vital dataset to dissect the mechanism of disease and potentially identify targetable pathways. ImportanceFungal infections in the lungs are dreaded complications for those with compromised immune systems and have limited treatment strategies available. These options are restricted further by the increased prevalence of treatment-resistant fungi. Many studies focus on how our immune systems respond to these pathogens, yet airway epithelial cells remain an understudied component of fungal infections in the lungs. Here, the authors provide a transcriptional analysis of primary human airway epithelial cells stimulated by two distinct fungal pathogens, Aspergillus fumigatus and Coccidioides posadasii. These data will enable further mechanistic studies of the contribution of the airway epithelium to initial host responses and represent a powerful new resource for investigators.

immunology↗

bioRxiv2022

Single-cell genome-wide association reveals a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus

Diversity in the human genome is one factor that confers resistance and susceptibility to infectious diseases. This is observed most dramatically during pandemics, where individuals exhibit large differences in risk and clinical outcomes against a pathogen infecting large portions of the worlds populations. Here, we developed scHi-HOST (single cell High-throughput Human in vitrO Susceptibility Testing), a method for rapidly identifying genetic variants that confer resistance and susceptibility to pathogens. scHi-HOST leverages scRNA-seq (single-cell RNA-sequencing) to simultaneously assign genetic identity to individual cells in mixed infections of cell lines of European, African, and Asian origin, reveal associated genetic variants for viral entry and replication, and identify expression quantitative trait loci (eQTLs). Applying scHi-HOST to influenza A virus (IAV), we identified eQTLs at baseline and in genes that are induced by IAV infection. Integration of scHi-HOST with a human IAV challenge study (Prometheus) revealed that a missense variant in ERAP1 (Endoplasmic reticulum aminopeptidase 1; rs27895) was associated with IAV burden in cells and human volunteers. Functional studies using RNA interference, ERAP1 inhibitor, and overexpression of alternative alleles demonstrated that ERAP1 is exploited by IAV to promote infection. Specifically, the nonsynonymous substitution, which results in a glycine to aspartate substitution at ERAP1 residue 348, would disrupt the substrate binding pocket of ERAP1, likely resulting in a significantly altered preference for substrates, poorer catalytic efficiency, or both. Finally, rs27895 exhibits substantial population differentiation, with the higher frequency of the minor T allele in two African populations likely contributing to the greater permissivity of cells from these populations to IAV infection. scHi-HOST is an important resource for understanding susceptibility to influenza and is a broadly applicable method for decoding human genetics of infectious disease.

genetics↗

bioRxiv2021

Influenza viral particles harboring the SARS-CoV-2 spike RBD as a combination respiratory disease vaccine

Vaccines targeting SARS-CoV-2 have gained emergency FDA approval, however the breadth against emerging variants and the longevity of protection remains unknown. Post-immunization boosting may be required, perhaps on an annual basis if the virus becomes an endemic pathogen. Seasonal influenza virus vaccines are already developed every year, an undertaking made possible by a robust global vaccine production and distribution infrastructure. To create a seasonal combination vaccine targeting influenza viruses and SARS-CoV-2 that is also amenable to frequent reformulation, we have developed a recombinant influenza A virus (IAV) genetic platform that "reprograms" the virus to package an immunogenic domain of the SARS-CoV-2 spike (S) protein onto IAV particles. Vaccination with this combination vaccine elicits neutralizing antibodies and provides protection from lethal challenge with both pathogens. This technology may allow for leveraging of established influenza vaccine infrastructure to generate a cost-effective and scalable seasonal vaccine solution for both influenza and coronaviruses.

microbiology↗

bioRxiv2020

SRSF protein kinases 1 and 2 are essential host factors for human coronaviruses including SARS-CoV-2

While vaccines are vital for preventing COVID-19 infections, it is critical to develop new therapies to treat patients who become infected. Pharmacological targeting of a host factor required for viral replication can suppress viral spread with a low probability of viral mutation leading to resistance. In particular, host kinases are highly druggable targets and a number of conserved coronavirus proteins, notably the nucleoprotein (N), require phosphorylation for full functionality. In order to understand how targeting kinases could be used to compromise viral replication, we used a combination of phosphoproteomics and bioinformatics as well as genetic and pharmacological kinase inhibition to define the enzymes important for SARS-CoV-2 N protein phosphorylation and viral replication. From these data, we propose a model whereby SRPK1/2 initiates phosphorylation of the N protein, which primes for further phosphorylation by GSK-3/{beta} and CK1 to achieve extensive phosphorylation of the N protein SR-rich domain. Importantly, we were able to leverage our data to identify an FDA-approved kinase inhibitor, Alectinib, that suppresses N phosphorylation by SRPK1/2 and limits SARS-CoV-2 replication. Together, these data suggest that repurposing or developing novel host-kinase directed therapies may be an efficacious strategy to prevent or treat COVID-19 and other coronavirus-mediated diseases.

microbiology↗

bioRxiv2019

ETV7 represses a subset of interferon-stimulated genes that restrict influenza viruses

The type I interferon (IFN) response is an important component of the innate immune response to viral infection. Precise control of interferon responses is critical; insufficient levels of interferon-stimulated genes (ISGs) can lead to a failure to restrict viral spread while excessive ISG activation can result in interferon-related pathologies. While both positive and negative regulatory factors control the magnitude and duration of IFN signaling, it is also appreciated that a number of ISGs regulate aspects of the interferon response themselves. However, the mechanisms underlying these ISG regulatory networks remain incompletely defined. In this study, we performed a CRISPR activation screen to identify new regulators of the type I IFN response. We identified ETS variant transcription factor 7 (ETV7), a strongly induced ISG, as a protein that acts as a negative regulator of the type I IFN response; however, ETV7 did not uniformly suppress ISG transcription. Instead, ETV7 preferentially targeted a subset of known antiviral ISGs. Further, we showed the subset of ETV7-modulated ISGs was particularly important for IFN-mediated control of some viruses including influenza viruses and SARS-CoV-2. Together, our data assign a function for ETV7 as an IFN response regulator and also identify ETV7 as a therapeutic target to increase innate responses and potentiate the efficacy of interferon-based antiviral therapies. One Sentence SummaryETV7 is an interferon-induced, repressive transcription factor that negatively regulates antiviral interferon-stimulated genes essential for controlling influenza virus and SARS-CoV-2 infections.

microbiology↗

bioRxiv2019

Replication competent, 10-segmented influenza viruses as antiviral therapeutics

Influenza A viruses (IAVs) encode their genome as eight negative sense RNA segments. During viral assembly, the failure to package all eight segments, or packaging of a mutated segment, renders the resultant virion incompletely infectious. It is known that the accumulation of these defective particles can limit viral disease by interfering with the spread of fully infectious particles. In order to harness this phenomenon therapeutically, we defined which viral packaging signals were amenable to duplication and developed a viral genetic platform which allowed the production of replication competent IAVs that package up to two additional artificial genome segments for a total of 10 segments. These artificial genome segments are capable of acting as "decoy" segments that, when packaged by wild-type (WT) viruses, lead to the production of non-infectious viral particles. Despite 10-segmented viruses being able to replicate and spread in vivo, these genomic modifications render the viruses avirulent. Excitingly, administration of 10-segmented viruses, both prophylactically and therapeutically, was able to rescue animals from normally lethally influenza virus infections. Thus, 10-segmented influenza viruses represent a potent anti-influenza biological therapy that targets the strain-independent process of viral assembly to slow the kinetics of productive viral spread and therefore limit viral disease. Author SummarySeasonal influenza infections are best prevented using vaccination. Vaccination, however, is not capable of completely preventing influenza infection, necessitating the use of anti-influenza therapeutics. To date, several different classes of anti-influenza therapeutics have been developed and used in order to combat these infections. Unfortunately, the incidence of influenza resistance to many of these therapeutics has begun to rise, necessitating the development of new strategies. One such strategy is to mimic the activity of naturally occurring viral particles that harbor defective genomes. These defective interfering particles have the ability to interfere with productive viral assembly, preventing the spread of influenza viruses across the respiratory tract. Furthermore, given the manner in which they target influenza segment packaging, a conserved feature of all influenza A viruses, resistance to this therapeutic strategy is unlikely. Here, we report the development of a genetic platform that allows the production of replication competent, 10-segmented influenza viruses. These viruses are capable of amplifying themselves in isolation, but co-infection with a wild-type virus leads to segment exchange and compromises the spread of both viruses. This interference, while mechanistically distinct from naturally occurring defective particles, was able to target the same viral process and rescue animals exposed to an otherwise lethal viral infection. This viral-based approach may represent a cost effective and scalable method to generate effective anti-influenza therapeutics when vaccines or anti-viral drugs become ineffective due to acquisition of viral resistance mutations.

microbiology↗