Biology subjects

Kell, A. M.

Publications and source records attributed to Kell, A. M..

7 recordsLinked to original sources

bioRxiv2026

Discovering Novel Therapeutic VHHs for Emerging Viruses: Perspectives from VEEV Selection Strategies

Evolution or emergence of a new viral variant is a significant public health concern. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), are mosquito-borne viruses which are becoming more prevalent due to expansion of vector habitats. The increased prevalence of such viruses provides opportunities for novel variants to evolve. Key therapeutic molecules that could be developed against viral pathogens are recombinant antibodies or antibody fragments, such as the variable heavy domain of heavy chain antibodies (VHHs). These proteins can neutralize or sequester viral particles, preventing or reducing infection. However, due to the evolution of viruses, there is a need to isolate new antibodies and direct their binding to particular epitopes on the virus. In vitro selections offer a promising pathway for the selection of therapeutic antibodies, but as we demonstrate, the choice of a target for these selections is key to obtaining the desired viral binding characteristics. Here we report four novel "human" VHHs which bind to the VEEV E2 protein selected using different strategies that include both computational and biochemical design of suitable antigens and whole virus selections. These VHHs have distinct complementarity-determining regions (CDRs). Multiple VHHs bind to the VEEV viral particles in ELISAs, and we report the peptide epitope recognized by these VHHs. Though non-neutralizing, when immobilized, these VHHs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these VHHs to perform viral "sponging". The selection strategies we report may have applications to further antibody developments against other viruses. Significance/ImportanceAlphaviruses, and in particular Venezuelan equine encephalitis virus (VEEV), are recognized for their ability to cause severe disease and for their potential to be used as a biothreat. Despite this, there are currently no antiviral therapies or FDA-approved vaccines available to treat or prevent VEEV infection. This study reports on a novel antibody selection pipeline to produce antibody fragments against VEEV. Antibodies produced via this method showed strong affinity and high specificity to the VEEV E2 glycoprotein in multiple conformations. Additionally, while not neutralizing, the antibody fragments described were shown to be effective as "viral sponges", having the ability to bind, sequester, and remove VEEV virions from solution, which represents a novel therapeutic approach.

immunology↗

bioRxiv2025

Specific and versatile monoclonal antibodies for hantavirus research

Rodent-borne hantaviruses pose a continual public health threat to humans through zoonotic transmission, with case fatality rates of up to 50% in some cases. Human infections can lead to hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardio-pulmonary syndrome (HCPS), depending on the viral species. Despite the morbidity and mortality associated with this family of viruses, no anti-viral therapeutics or vaccines are available to treat and prevent hantavirus disease. The relative shortage of commercially available reagents to study hantavirus infections in vitro and in vivo likely contributes to the challenges in developing viral countermeasures. This report describes the generation of a panel of mouse monoclonal antibodies that collectively recognize the four viral proteins of Seoul virus (Orthohantavirus seoulense), an Old World hantavirus with worldwide distribution and the causative agent of HFRS. We have validated the specificity and versatility of these antibodies against a subset of Old World and New World hantaviruses in assays relying on antigen recognition in denatured or native conformations. We present several antibodies that specifically recognize the Seoul virus nucleoprotein and polymerase protein in Western blotting and immunostaining assays. We also identified three novel antibodies directed against the glycoprotein complex that are capable of binding to the N-terminal glycoprotein of all hantaviruses tested. These antibodies are freely available to all hantavirus researchers to add to the small, but growing, collection of reliable and available reagents to be used to study hantavirus biology, identify novel antiviral compounds, and measure viral prevalence in the laboratory and the field. ImportancePathogenic hantaviruses cause severe hemorrhagic disease and pose a significant public health threat world-wide. Insufficient research into the biology of these viruses has slowed the development of effective direct-acting antivirals and vaccines. Here, we describe the generation and validation of novel, specific monoclonal antibodies for the detection of Seoul virus proteins in vitro. These reagents can be used to fill in critical gaps in knowledge regarding hantavirus entry, protein expression, and particle generation.

microbiology↗

bioRxiv2025

Impact of Species and Cell-type on the RNA Replication Kinetics of Seoul Virus

Hantaviruses are zoonotic, tri-segmented, negative-sense RNA viruses and a significant public health threat. Viral pathogenesis varies between host species, with rodent reservoir infection being asymptomatic and human infection resulting in severe, immune-mediated disease. Viral pathogenesis is highly dependent on virus replication efficiency since it affects the viruss ability to evade detection and determines the magnitude of the host immune response. However, the molecular replication kinetics for hantaviruses remain poorly defined. Therefore, we developed a sense- and segment-specific quantitative real-time PCR (ssqRT-PCR) assay and a SYBR-based qRT-PCR (Sb-qRT-PCR) assay, allowing us to quantify both negative-sense genome levels and total viral RNA synthesis of the small (S), medium (M), and large (L) segments of Seoul virus (SEOV). We then measured total viral RNA and genome accumulation in reservoir rat endothelial cells (RLMVEC), non-reservoir human endothelial cells (HUVEC-C), and Vero E6 epithelial cells. We also measured the ratio of each segment released into the culture supernatant, approximating the relative packaging efficiency. We found that, while the magnitude of viral RNA differed, RNA replication kinetics were largely similar between reservoir and non-reservoir endothelial cells. However, replication and release kinetics differed between infection of endothelial and Vero cells. We also found that the S, M, and L segments were not equally abundant during viral infection or release, but instead followed a trend of M>L>S. Overall, this study validates two qRT-PCR assays to measure SEOV RNA, details the accumulation and release of each viral segment, and demonstrates the impact of host cell type on hantavirus replication. Impact StatementHantavirus infections in humans lead to significant immune-mediated morbidity and mortality around the world and pose a threat to public health. Zoonotic transmission occurs from persistently infected rodents which show no overt signs of disease. Understanding the molecular interactions that drive divergent infection outcomes is incomplete in the field of hantavirus research due to limited tools and reagents. This novel strand- and segment-specific quantitative RT-PCR assay uncovers the unique kinetics of hantavirus genome replication in reservoir and non-reservoir cells. Further, this assay will serve as a valuable tool to investigate antiviral therapies and virus-host interactions critical for polymerase activity.

microbiology↗

bioRxiv2024

Viral susceptibility and innate immune competency of Carollia perspicillata bat cells produced for virological studies

Multiple viruses that are highly pathogenic in humans are known to have evolved in bats. How bats tolerate infection with these viruses, however, is poorly understood. As viruses engage in a wide range of interactions with their hosts, it is essential to study bat viruses in a system that resembles their natural environment like bat-derived in vitro cellular models. However, stable and accessible bat cell lines are not widely available for the broader scientific community. Here, we generated in vitro reagents for the Sebas short-tailed bat (Carollia perspicillata), tested multiple methods of immortalization, and characterized their susceptibility to virus infection and response to immune stimulation. Using a pseudotyped virus library and authentic virus infections, we show that these C. perspicillata cell lines derived from a diverse array of tissues are susceptible to viruses bearing the glycoprotein of numerous orthohantaviruses, including Andes and Hantaan virus and are also susceptible to live hantavirus infection. Furthermore, stimulation with synthetic double-stranded RNA prior to infection with VSV and MERS-CoV induced a protective antiviral response, demonstrating the suitability of our cell lines to study the bat antiviral immune response. Taken together, the approaches outlined here will inform future efforts to develop in vitro tools for virology from non-model organisms and these C. perspicillata cell lines will enable studies on virus-host interactions in bats.

microbiology↗

bioRxiv2024

Manipulation of Host Cholesterol by SARS-CoV-2

Cholesterol homeostasis relies on lysosomes, which release free cholesterol from degraded lipids. We show that SARS-CoV-2 blocks lysosomal cholesterol export through its protein ORF3a. ORF3a binds the HOPS subunit VPS39, and disrupting this interaction restores cholesterol trafficking. Two mechanisms underlie this defect. First, ORF3a-VPS39 interaction traps the sorting receptor CI-MPR and the retrieval complex retromer in endosomes/lysosomes, impairing trafficking of the cholesterol transporter NPC2. Retromer deletion reproduced these defects. Second, ORF3a reduces bis(monoacylglycerol)phosphates (BMPs), lysosomal lipids required for cholesterol export. Lipidomics and proteomics revealed altered metabolism of BMP precursors, mitochondrial phosphatidylglycerols (PGs), and reduced mitochondrial proteins at lysosomes. ORF3a-VPS39 interaction decreased lysosome-mitochondrion membrane contact sites (MCS), excluding autophagy or mitochondrion-derived vesicles as routes for PG transfer. VPS39 deletion decreased the MCS and BMPs. These findings identify VPS39 as a regulator of NPC2 trafficking and BMP biosynthesis and reveal that ORF3a disrupts both pathways to block cholesterol egress.

cell biology↗

bioRxiv2024

Seoul orthohantavirus evades innate immune activation inreservoir endothelial cells

Pathogenic hantaviruses are maintained world-wide within wild, asymptomatic rodent reservoir hosts, with increasingly frequent human spillover infections resulting in severe hemorrhagic fever disease. With no approved therapeutics or vaccines, research has, until recently, focused on understanding the drivers of immune-mediated pathogenesis. An emerging body of work is now investigating the mechanisms that allow for asymptomatic, persistent infections of mammalian reservoir hosts with highly pathogenic RNA viruses. Despite limited experimental data, several hypotheses have arisen to explain limited or absent disease pathology in reservoir hosts. In this study, we directly tested two leading hypotheses: 1) that reservoir host cells induce a generally muted response to viral insults, and 2) that these viruses employ host-specific mechanisms of innate antiviral antagonism to limit immune activation in reservoir cells. We demonstrate that, in contrast to human endothelial cells which mount a robust antiviral and inflammatory response to pathogenic hantaviruses, primary Norway rat endothelial cells do not induce antiviral gene expression in response to infection with their endemic hantavirus, Seoul orthohantavirus (SEOV). Reservoir rat cells do, however, induce strong innate immune responses to exogenous stimulatory RNAs, type I interferon, and infection with Hantaan virus, a closely related hantavirus for which the rat is not a natural reservoir. We also find that SEOV-infected rat endothelial cells remain competent for immune activation induced by exogenous stimuli or subsequent viral infection. Importantly, these findings support an alternative model for asymptomatic persistence within hantavirus reservoir hosts: that efficient viral replication within reservoir host cells prevents the exposure of critical motifs for cellular antiviral recognition and thus limits immune activation that would otherwise result in viral clearance and/or immune-mediated disease. Defining the mechanisms that allow for infection tolerance and persistence within reservoir hosts will reveal novel strategies for viral countermeasures and inform rational surveillance programs. Author SummaryDespite the significant, and continual, threat to human health, limited experimental evidence explains the mechanisms that underly asymptomatic zoonotic RNA virus persistence within natural, mammalian reservoir hosts. Here, we investigated whether reservoir host target cells for hantavirus infection are competent for antiviral activation and tested the hypothesis that, through long-term co-evolution, Seoul orthohantavirus actively antagonizes innate antiviral signaling pathways to limit immune induction and prevent viral clearance in primary reservoir cells. While we find no evidence to support these hypotheses, our findings do support an alternative hypothesis that viral replication within the natural reservoir cells may not result in the production of immune-stimulating by-products. The factors that determine viral persistence within the reservoirs may include efficient use of host co-factors for efficient genome replication and/or packaging and warrant further investigation.

microbiology↗

bioRxiv2021

Endomembrane targeting of human OAS1 p46 augments antiviral activity

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from host cell innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense viral RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flavivirus, picornavirus, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform strongly associates with COVID-19 severity. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests early control of SARS-CoV-2 replication through OAS1-p46 is an important determinant of COVID-19 severity.

immunology↗