Biology subjects

Wells, A.

Publications and source records attributed to Wells, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Donor-derived CD8+CD122+ Tregs generated in mixed donor chimeric NOD mice delete autoreactive T cells

The establishment of mixed hematopoietic chimerism is a promising way to induce immune tolerance for islet replacement therapy and to treat the underlying autoimmunity in type 1 diabetes (T1D). Mixed chimerism not only promotes effective thymic negative selection of autoreactive cells but also restores regulatory T cell (Treg) function and peripheral tolerance. In the current study, we determined that a novel class of donor-derived CD8+CD44+CD122+ Tregs (d-CD8+CD122+ Tregs) plays a crucial role in controlling autoimmunity in non-obese diabetic (NOD) mice with induced mixed chimerism. Using adoptive T cell transfer experiments, we showed that d-CD8+CD122+ Tregs abrogate autoimmunity by selectively depleting the exogenously injected diabetogenic T cells in Recombination-Activating Gene deficient NOD mice. These d-CD8+CD122+ Tregs from NOD chimeras show upregulation of Helios, Programmed cell death protein 1, perforin, granzyme-B, CD39, Folate receptor 4, and downregulation of proinflammatory markers like Scart1 and Scart2. Using in vitro assays, we show that d-CD8+CD122+ Tregs respond specifically to a Complementarity-Determining Region-3 peptide sequence derived from T cell receptors of islet antigen-specific autoreactive T cells. Similarly, we found that individuals with T1D have a deficiency in CD8+CD122+ Tregs, suggesting a potential loss of regulatory function accompanies disease onset. Revitalizing CD8+CD122+ Tregs may offer a new therapeutic strategy of restoring immune tolerance in autoimmune diabetes.

immunology↗

bioRxiv2026

Tonsillar expression quantitative trait loci verify and expand genetic contributors to childhood atopic diseases

The spectrum of causal variants, mechanisms, and immunologic gene networks that influence pediatric atopic traits are not completely understood. Human genetic variation associated with transcript abundance (eQTLs) can help to advance our understanding, yet prior work has focused on profiling immune cell populations collected from peripheral blood primarily in adult populations, leaving uncharacterized tissue-resident lymphocytes collected from children. Here, we paired genotyping with gene expression profiling across four populations of tonsil-derived immune cell types - including previously uncharacterized germinal center B cells - collected from 103 children across development (ages 1-18). Using these data, we report cell-type specific gene networks and identify 13,393 eGenes (1,793 eGenes not previously reported in similar datasets) influenced by 27,603 eQTLs (5,199 not previously reported). We link discovered eQTLs to associations identified in pediatric and adult asthma and atopy traits, nominating 78 eGenes like TRAF3, ZBTB10 and JAZF1 in disease relevant cell types. Our resource is freely available and exemplifies the importance of discovery in native tissues and across human development.

immunology↗

bioRxiv2025

Fifteen-year microbiome survey of endangered killer whales (Orcinus orca) reveals declining diversity and population differences

The endangered Southern Resident killer whale (Orcinus orca) (SRKW) population is burdened by multiple anthropogenic stressors with limited non-invasive approaches for health surveillance. The gut microbiome is interconnected with host physiology and can be characterized using remotely collected fecal samples, creating unique opportunities to integrate environmental and individualized host-associated features of health. In this study, we used fecal samples collected over a 15-year period (2005-2019) from 77% of the living, wild SRKW population (56 individuals, 1-17 time points per individual), as well as fecal samples from the conspecific Northern and Alaska Resident killer whale populations, to characterize distal gut microbiota structure and genomic composition. SRKW microbiotas were individualized and distinct from those of the nearby Northern and Alaska Resident populations, both of which exhibit consistently higher fecundity and survivorship. During the study period, SRKW fecal microbiota species richness declined, despite stability over periods of time less than or equal to 1 year. Several potential bacterial pathogens, such as Fusobacterium spp., achieved dominance in the fecal microbiotas of SRKW individuals sampled within 6 months of death. These findings demonstrate the feasibility and value of harnessing non-invasively collected fecal samples and microbiome profiles for longitudinal killer whale health surveillance.

microbiology↗

bioRxiv2025

Modeling cetacean eDNA distribution along the Washington coast using metabarcoding from opportunistic samples and generalized additive models

Effective cetacean conservation depends on robust monitoring, yet traditional visual and passive-acoustic surveys have constraints. We evaluated environmental DNA (eDNA) metabarcoding coupled with species distribution modeling (SDM) as a tool to study habitat use of cetaceans along the Washington State coast, USA. Seawater was collected at the surface and at a 50 m depth from 43 sites (86 samples) during the 2019 U.S.-Canada Integrated Ecosystem & Acoustic-Trawl Survey. A partial section of the mitochondrial control region was amplified with cetacean - specific primers, sequenced on an Illumina MiSeq, and taxonomically assigned with a curated reference database. Nine species were detected; we modelled the three most frequent: Pacific white-sided dolphin (Lagenorhynchus obliquidens) (10 detections), humpback whale (Megaptera novaeangliae) (8 detections), and Rissos dolphin (Grampus griseus) (6 detections). Binomial generalized additive models related presence-absence to bathymetry, distance to shore, longitude, slope, and sea-surface temperature; model performance was assessed with stratified five-fold cross-validation. SDMs explained 17-51% of null deviance and presented high specificity ([≥] 0.80). The Pacific white-sided dolphin showed the highest eDNA presence probabilities offshore, beyond the shelf break. Humpback whale eDNA presence probabilities showed hotspots along the shelf break with secondary high-probability patches in near-shore waters. Rissos dolphin eDNA presence probabilities were elevated in offshore zones characterized by steep bathymetric gradients, particularly northwest of the sampled transect. These spatial patterns are consistent with historical visual-acoustic records, suggesting that eDNA-informed SDMs can capture cetacean habitat use. This proof of concept indicates that combining eDNA detections with flexible SDMs could provide a cost-effective, non-invasive complement to conventional surveys and may offer a scalable pathway for marine-mammal monitoring and spatial planning.

genomics↗

bioRxiv2025

Quantitative, Multispecies Monitoring at a Continental Scale

Molecular data from environmental samples can reflect the abundance of species DNA, an index immediately relevant to natural-resources management at a broad geographic scale. These data commonly derive from assays designed and targeted for individual species (e.g., using qPCR or ddPCR), or from metabarcoding that use more general PCR primers to amplify many species simultaneously. Multispecies analyses make efficient use of field samples and laboratory time, and speak to inherently multispecies questions of management and ecological interest. However, unlike single-species techniques, metabarcoding alone generally reflects only the environmental DNA (eDNA) proportions of target-species DNA present, not their absolute quantities. Here, we combine qPCR and metabarcoding data derived from the same samples to estimate quantities of eDNA from many fish species along the US West Coast in three dimensions, demonstrating a technique of practical relevance for both management and ecology. We derive spatially explicit maps of eDNA abundances for 12 common species of ecological and management importance and point the way to quantitative surveys of wild species using molecules alone. We find that species distribution maps derived from eDNA largely mirror known species niches and known spatial distributions. Notably, our analyses indentify biodiversity hotspots that align with previously documented regions of ecological significance, such as the Columbia River plume and the Heceta Bank.

ecology↗

bioRxiv2025

An estrogen-independent and IL-1-dependent pathway controls vulvovaginal candidiasis through combined IL-17/IL-22 signaling

Vulvovaginal candidiasis (VVC) affects >75% of women, with considerable morbidity and high medical cost burden. While Type 17 cytokines (IL-17, IL-22) are critical for oral and dermal immunity to C. albicans, their role in VVC has been less clear. Th17 gene signatures are potently upregulated in VVC, yet impairment of individual Th17 components (IL-17A, IL-17R subunits, IL-22) does not worsen disease. Rather, estrogen activity is tightly linked to VVC, leading to a paradigm that hormonal pathways rather than immune defense, dominate susceptibility. Here, we reveal a previously unappreciated role for IL-1/Type 17 in VVC that operates independently of estrogenic hormones. In contrast to mice lacking IL-17A, IL-17RA, IL-22, or IL-22R individually, mice lacking IL-17RA and IL-22RA1 together (Il17raIl22ra1-/-) exhibited high fungal loads and exacerbated tissue damage and inflammation. In human vulvar epithelial cells, IL-17 and IL-22 drive synergistic signaling. IL-1R signaling but surprisingly not IL-23 wa upstream of this response. Il17raIl22ra1-/- mice expressed high IL-1{beta} yet did not control disease, indicating that IL-1 is upstream but not downstream of Type 17 responses. Unexpectedly, Type 17-dependent control occurred in the absence of exogenous estrogen administration and persisted even when estrus was prevented by progesterone treatment. Collectively, these data indicate that susceptibility to VVC is driven not only by estrogen sensitization but through combinatorial loss of IL-17 and IL-22. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/671995v2_ufig1.gif" ALT="Figure 1000"> View larger version (52K): org.highwire.dtl.DTLVardef@19946e9org.highwire.dtl.DTLVardef@1feac71org.highwire.dtl.DTLVardef@eb433aorg.highwire.dtl.DTLVardef@18ab622_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Open-science discovery of DNDI-6510, a compound that addresses genotoxic and metabolic liabilities of the COVID Moonshot SARS-CoV-2 Mpro lead inhibitor

The 2020 SARS-CoV-2 coronavirus pandemic highlighted the urgent need for novel small molecule antiviral drugs. (S)-x38 DNDI-6510 is a non-covalent SARS-CoV-2 main protease inhibitor developed by the open science collaboration COVID Moonshot. Here, we report on the metabolic and toxicologic optimization of the lead series previously disclosed by the COVID Moonshot Initiative, leading up to the selection of (S)-x38 DNDI-6510 as the preclinical candidate. We describe the thorough profiling of the series, identifying key risks such as formation of genotoxic metabolites and high clearance, which were successfully addressed during lead optimization. In addition, we disclose the in vitro and in vivo evaluation of (S)-x38 DNDI-6510 in pharmacokinetic and pharmacodynamic models, exploring multiple approaches to ameliorate rodent-specific metabolic clearance, and show that both co-dosing of (S)-x38 DNDI-6510 with an ABT inhibitor and utilizing a metabolically humanized mouse model (8HUM) achieve significant improvements in exposure. Through comparisons of ABT co-dosing and humanized mouse models in efficacy experiments, we demonstrate that continuous exposure over cellular EC90 is required for SARS-CoV-2 antiviral efficacy in vivo in an antiviral model using a mouse-adapted SARS-CoV-2 strain. Finally, (S)-x38 DNDI-6510 was assessed in maximum tolerated dose experiments in two species, demonstrating significant in vivo PXR-linked auto-induction of metabolism, leading to the discontinuation of this compound. In summary, we report the successful effort to overcome series-specific AMES liabilities in a lead development program. Downstream optimization of existing series will require in-depth optimization of rodent-specific liabilities and metabolic induction profile.

pharmacology and toxicology↗

bioRxiv2024

Hope for Others: Research Results from the University of Pittsburgh Rapid Autopsy Program for Breast Cancer

Breast cancer affects 1/8 of women throughout their lifetimes, with over 90% of cancer deaths being caused by metastasis. However, metastasis poses unique challenges to research, as complex changes in the microenvironment in different metastatic sites and difficulty obtaining tissue for study hinder the ability to examine in depth the changes that occur during metastasis. Rapid autopsy programs thus fill a unique need in advancing metastasis research. Here, we describe our protocol and processes for establishing and improving the US-based Hope for OTHERS (Our Tissue Helping Enhance Research and Science) program for organ donation in metastatic breast cancer. As of August 2024, we consented 114 patients and performed 37 autopsies, from which we collected 551 unique metastatic frozen tumor samples, 1244 FFPE blocks, 90 longitudinal liquid biopsy samples and developed 14 patient-derived organoid and 8 patient-derived xenograft models. We report in-depth clinical and histopathological information and discuss extensive new research and novel findings in patient outcomes, metastatic phylogeny, and factors in successful living model development. Our results reveal key logistical and protocol improvements that are uniquely beneficial to certain programs based on identifiable features, such as working closely with patient advocates, methods to rescue RNA quality in cases where tissue quality may degrade due to time delays, as well as guidelines and future expansions of our program. Statement of SignificanceRapid autopsy programs are unique research settings with huge potential for studying metastatic cancer, however, they have complex research challenges. Our work provides a valuable resource in advancing this field of research.

cancer biology↗

bioRxiv2024

The diverse diet of southern Alaska resident killer whales changes across spatiotemporally distinct foraging aggregations

Top predators influence ecological communities in part through the prey they consume, which they often track through cycles of seasonal and geographic abundance. Killer whales are top predators in the marine ecosystem. In the North Pacific, they have diverged into three distinct lineages with different diets, of which the fish-eating type is most abundant. In this study, we examine the diet of the southern Alaska resident killer whale population across three major foraging aggregations. We take advantage of two unique sampling methods to reveal strong spatiotemporal patterns in diet from May through September. Chinook, chum, and coho salmon were each dominant in different locations and times, with substantial dietary contributions from Pacific halibut, arrowtooth flounder, and sablefish. The diverse, location-specific, and seasonal nature of the feeding habits of this marine top predator highlights the importance of diet sampling across broad spatiotemporal and population-level scales.

ecology↗

bioRxiv2024

APP and β-amyloid modulate protein aggregation and dissociation from recycling endosomal and exosomal membranes

Secretory proteins frequently aggregate into non-soluble dense-core granules (DCGs) in recycling endosome-like compartments prior to release. By contrast, aberrantly processed A{beta}-peptides derived from Amyloid Precursor Protein (APP) form pathological amyloidogenic aggregations in late-stage Alzheimers Disease (AD) after secretion. By examining living Drosophila prostate-like secondary cells, we show both APP and A{beta}-peptides affect normal DCG biogenesis. These cells generate DCGs and secreted nanovesicles called Rab11-exosomes within enlarged recycling endosomes. The fly APP homologue, APP-like (APPL), associates with Rab11-exosomes and the compartmental limiting membrane, from where its extracellular domain controls protein aggregation. Proteolytic release of this membrane-associated domain permits aggregates to coalesce into a large central DCG. Mutant A{beta}-peptide expression, like Appl loss-of-function, disrupts this assembly step and compartment motility, and increases lysosomal targeting, mirroring pathological events reported in early-stage AD. Our data therefore reveal a physiological role for APP in membrane-dependent protein aggregation, which when disrupted, rapidly triggers AD-relevant intracellular pathologies.

cell biology↗

bioRxiv2024

Modeling Molecular Mechanisms of Pirfenidone Interaction with Kinases

Scar formation is a process that occurs due to increased collagen deposition and uncontrolled inflammation. Previous studies have demonstrated that Pirfenidone (Pf), an FDA approved anti-inflammatory and anti-fibrotic drug can reduce inflammation in vivo as well as regulate activation of LPS-stimulated neutrophils. However, the molecular level mechanism of Pfs action is not well understood. Here, we used neural networks to identify new targets and molecular modeling methods to investigate the Pfs action pathways at the molecular level that are related to its ability to reduce both the inflammatory and remodeling phases of the wound healing process. Out of all the potential targets identified, both molecular docking and molecular dynamics results suggest that Pf has a noteworthy binding preference towards the active conformation of the p38 mitogen activated protein kinase-14 (MAPK14) and it is potentially a type I inhibitor-like molecule. In addition to p38 MAPK (MAPK14), additional potential targets of Pf include AKT1, MAP3K4, MAP2K3, MAP2K6, MSK2, MAP2K2, ERK1, ERK2, and PDK1. We conclude that several proteins/kinases, rather than a single target, are involved in Pfs wound healing ability to regulate signaling, inflammation, and proliferation.

systems biology↗

bioRxiv2023

A Rab6 to Rab11 transition is required for dense-core granule and exosome biogenesis in Drosophila secondary cells

Secretory cells in glands and the nervous system frequently package and store proteins destined for regulated secretion in dense-core granules (DCGs), which disperse when released from the cell surface. Despite the relevance of this dynamic process to diseases such as diabetes and human neurodegenerative disorders, our mechanistic understanding is relatively limited, because of the lack of good cell models to follow the nanoscale events involved. Here, we employ the prostate-like secondary cells (SCs) of the Drosophila male accessory gland to dissect the cell biology and genetics of DCG biogenesis. These cells contain unusually enlarged DCGs, which are assembled in compartments that also form secreted nanovesicles called exosomes. We demonstrate that known conserved regulators of DCG biogenesis, including the small G-protein Arf1 and the coatomer complex AP-1, play key roles in making SC DCGs. Using real-time imaging, we find that the aggregation events driving DCG biogenesis are accompanied by a change in the membrane associated small Rab GTPases which are major regulators of membrane and protein trafficking in the secretory and endosomal systems. Indeed, a transition from trans-Golgi Rab6 to recycling endosomal protein Rab11, which requires conserved DCG regulators like AP-1, is essential for DCG and exosome biogenesis. Our data allow us to develop a model for DCG biogenesis that brings together several previously disparate observations concerning this process and highlights the importance of communication between the secretory and endosomal systems in controlling regulated secretion. Author summaryCells communicate with each other by releasing signalling molecules that bind receptors on target cells and alter their behaviour. Before their release, these signals are typically stored in condensed structures called dense-core granules (DCGs). DCGs are found in many animal species and their dysregulation is linked to several major human diseases, such as diabetes and neurodegenerative disorders. However, the mechanisms controlling DCG formation and secretion are only partly understood. Here we study this process in fruit flies using a secretory cell, which contains unusually large DCGs. We show that known regulators of DCG formation in mammals also control DCG production in these fly cells and identify new DCG assembly steps by following the process in living cells. Furthermore, we show that the cells secretory and recycling endosomal compartments must interact to induce the rapid condensation of proteins into a DCG, and that known regulators of DCG formation are needed for this crucial event to take place. Our work provides a platform from which to work out the molecular mechanisms that enable this critical secretory-endosomal interaction and probe its roles in diseases of secretion.

cell biology↗

bioRxiv2022

A CXCR3-activating peptide increases Tear Break Up Time and corrects corneal haze in a rabbit model of environmental dry eye

PurposeEnvironmentally-triggered dry eye disease (DED) or keratoconjunctivitis sicca (KCS), which constitutes the majority of DED cases, currently is palliatively treated with aqueous replacement solutions that do not target the dysfunction of the mucin and lipid components of tears. Herein, we tested whether a peptide that increased goblet cell numbers in a model of scleral chemical injury would also improve tear quality in environmental DED. MethodsEnvironmental DED was established by exposing New Zealand white rabbits (8 per group, female) to 20% humidity with rapid air replacement and b.i.d. atropine sulfate eyedrops for 3 weeks prior to test article administration; this continued for the subsequent 3 weeks of testing. Animals were dosed by (A) saline, (B) b.i.d. eyedrop of peptide in saline, (C) b.i.d. eyedrop of peptide in coacervate, or (D) weekly subconjunctival injection of peptide. ResultsThe environmental DED was established with both Schirmer and TBUT being reduced by from baseline at the start of test article; these levels were maintained as low through the testing period. All three treatment regimens increased TBUT approximately 3x to levels greater than prior to desiccation, with little effect on Schirmer. Corneal haze was present in all eyes after induction, and largely cleared up by all three treatments. End of study enucleation of the eye did not show any changes in goblet cells numbers, which remained high throughout the induction and treatment. ConclusionsThe treatment of environmental DED/KCS with a peptide that activates CXCR3 improved tear quality and reversed corneal pathology by promoting tear stability, while not affecting aqueous volume of the tears.

pathology↗

bioRxiv2022

Axl contributes to efficient migration and invasion of melanoma cells

Axl, a member of the TAM receptor family has been broadly suggested to play a key role in tumor metastasis. However, the function of Axl in the invasion and metastasis of melanoma, the most lethal skin cancer, remains largely unknown. In the present study, we found that melanoma cell lines present variable protein levels of Axl and Tyro3; interestingly, MerTK is not noted at detectable levels in any of tested MGP (metastatic growth phase) cell lines. Treatment with recombinant human Gas6 significantly activates Akt in the Axl-expressing WM852 and IgR3 lines but just slightly in WM1158. IgR3, WM852 and WM1158 demonstrate different autocrine signaling. Knockdown of Axl by siRNA or the treatment with Axl-specific inhibitor R428 dramatically inhibits the migration and invasion of both IgR3 and WM852 in vitro. These findings suggest that Axl enhances the invasion of melanoma cells.

cell biology↗

bioRxiv2021

A multi-layer functional genomic analysis to understand noncoding genetic variation in lipids

A major challenge of genome-wide association studies (GWAS) is to translate phenotypic associations into biological insights. Here, we integrate a large GWAS on blood lipids involving 1.6 million individuals from five ancestries with a wide array of functional genomic datasets to discover regulatory mechanisms underlying lipid associations. We first prioritize lipid-associated genes with expression quantitative trait locus (eQTL) colocalizations, and then add chromatin interaction data to narrow the search for functional genes. Polygenic enrichment analysis across 697 annotations from a host of tissues and cell types confirms the central role of the liver in lipid levels, and highlights the selective enrichment of adipose-specific chromatin marks in high-density lipoprotein cholesterol and triglycerides. Overlapping transcription factor (TF) binding sites with lipid-associated loci identifies TFs relevant in lipid biology. In addition, we present an integrative framework to prioritize causal variants at GWAS loci, producing a comprehensive list of candidate causal genes and variants with multiple layers of functional evidence. Two prioritized genes, CREBRF and RRBP1, show convergent evidence across functional datasets supporting their roles in lipid biology.

genomics↗

bioRxiv2021

Interferon-γ Increases Sensitivity to Chemotherapy and Provides Immunotherapy Targets in Models of Metastatic Castration-Resistant Prostate Cancer

Interferon-{gamma} (IFN{gamma}) is a cytokine with limited evidence of benefit in cancer clinical trials to date. However, it could potentially play a role in potentiating anti-tumor immunity in the immunologically "cold" metastatic castration-resistant prostate cancer (mCRPC) by inducing antigen presentation pathways and concurrently providing targets for immune checkpoint blockade therapy. Moreover, it could additionally increase sensitivity to chemotherapy based on its pleiotropic effects on cell phenotype. Here, we show that IFN{gamma} treatment induced expression of major histocompatibility class-I (MHC-I) genes and PD-L1 in prostate cancer cells in vitro. Furthermore, IFN{gamma} treatment led to a decrease in E-cadherin expression with a consequent increase in sensitivity to chemotherapy in vitro. In an in vivo murine tumor model of spontaneous metastatic prostate cancer, IFN{gamma} systemic pretreatment upregulated the expression of HLA-A and decreased E-cadherin expression in the primary tumor, and more importantly in the metastatic site led to increased apoptosis and limited micrometastases in combination with paclitaxel treatment compared to diffuse metastatic disease in control and monotherapy treatment groups. These findings suggest that IFN{gamma} may be useful in combinatorial regimens to induce sensitivity to immunotherapy and chemotherapy in hepatic metastases of mCRPC.

cancer biology↗

bioRxiv2020

Human FcRn expression and Type I Interferon signaling control Echovirus 11 pathogenesis in mice

Neonatal echovirus infections are characterized by severe hepatitis and neurological complications that can be fatal. Here, we show that expression of the human homologue of the neonatal Fc receptor (hFcRn), the primary receptor for echoviruses, and ablation of type I interferon (IFN) signaling are key host determinants involved in echovirus pathogenesis. We show that expression of hFcRn alone is insufficient to confer susceptibility to echovirus infections in mice. However, expression of hFcRn in mice deficient in type I interferon (IFN) signaling, hFcRn-IFNAR-/-, recapitulate the echovirus pathogenesis observed in humans. Luminex-based multianalyte profiling from E11 infected hFcRn-IFNAR-/- mice revealed a robust systemic immune response to infection, including the induction of type I IFNs. Furthermore, similar to the severe hepatitis observed in humans, E11 infection in hFcRn-IFNAR-/- mice caused profound liver damage. Our findings define the host factors involved in echovirus pathogenesis and establish in vivo models that recapitulate echovirus disease.

microbiology↗

bioRxiv2020

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients

SARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

microbiology↗