Paul B. McCray

University of Iowa

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Patrick L. Sinn

University of Iowa

Christine Wohlford-Lenane

University of Iowa

David K. Meyerholz

University of Iowa

Michael J. Welsh

Howard Hughes Medical Institute

Stanley Perlman

University of Iowa

Alejandro A. Pezzulo

University of Iowa

Joseph Zabner

University of Iowa

David A. Stoltz

University of Iowa

Beverly L. Davidson

Children's Hospital of Philadelphia

Jennifer A. Bartlett

University of Iowa

Cooperative Institutions

University of Iowa

475

Museum of Heilongjiang Province

179

Harvard University

113

University of Iowa Hospitals and Clinics

112

Howard Hughes Medical Institute

102

University of Pittsburgh

New York Proton Center

National Institutes of Health

Communities In Schools of Orange County

Johns Hopkins University

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Receptor Variation and Susceptibility to Middle East Respiratory Syndrome Coronavirus Infection

Journal of Virology (2014)

Arlene Barlan Jincun Zhao Mayukh K. Sarkar Kun Li Paul B. McCray

ABSTRACT The Middle East respiratory syndrome coronavirus (MERS-CoV) recently spread from an animal reservoir to infect humans, causing sporadic severe and frequently fatal respiratory disease. Appropriate public health and control measures will require discovery of the zoonotic MERS coronavirus reservoirs. The relevant animal hosts are liable to be those that offer optimal MERS virus cell entry. Cell entry begins with virus spike (S) protein binding to DPP4 receptors. We constructed chimeric DPP4 receptors that have the virus-binding domains of indigenous Middle Eastern animals and assessed the activities of these receptors in supporting S protein binding and virus entry. Human, camel, and horse receptors were potent and nearly equally effective MERS virus receptors, while goat and bat receptors were considerably less effective. These patterns reflected S protein affinities for the receptors. However, even the low-affinity receptors could hypersensitize cells to infection when an S-cleaving protease(s) was present, indicating that affinity thresholds for virus entry must be considered in the context of host-cell proteolytic environments. These findings suggest that virus receptors and S protein-cleaving proteases combine in a variety of animals to offer efficient virus entry and that several Middle Eastern animals are potential reservoirs for transmitting MERS-CoV to humans. IMPORTANCE MERS is a frequently fatal disease that is caused by a zoonotic CoV. The animals transmitting MERS-CoV to humans are not yet known. Infection by MERS-CoV requires receptors and proteases on host cells. We compared the receptors of humans and Middle Eastern animals and found that human, camel, and horse receptors sensitized cells to MERS-CoV infection more robustly than goat and bat receptors. Infection susceptibility correlated with affinities of the receptors for viral spike proteins. We also found that the presence of a cell surface lung protease greatly increases susceptibility to MERS-CoV, particularly in conjunction with low-affinity receptors. This cataloguing of human and animal host cell factors allows one to make inferences on the distribution of MERS-CoV in nature.

Coronavirus

Betacoronavirus

Coronavirus Infections

2019-20 coronavirus outbreak

10.1128/jvi.00161-14

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Citations (112)

Lentivirus Vectors Pseudotyped with Filoviral Envelope Glycoproteins Transduce Airway Epithelia from the Apical Surface Independently of Folate Receptor Alpha

Journal of Virology (2003)

Patrick L. Sinn Melissa A. Hickey Patrick D. Staber Douglas E. Dylla Scott A. Jeffers

The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.

10.1128/jvi.77.10.5902-5910.2003

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Citations (118)

A SARS-CoV-2 Infection Model in Mice Demonstrates Protection by Neutralizing Antibodies

Cell (2020)

Ahmed O. Hassan James Brett Case Emma S. Winkler Larissa B. Thackray Natasha M. Kafai

Coronavirus

Pathogenesis

Angiotensin-converting enzyme 2

10.1016/j.cell.2020.06.011

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Citations (538)

Broad-Spectrum In Vitro Activity and In Vivo Efficacy of the Antiviral Protein Griffithsin against Emerging Viruses of the Family Coronaviridae

Journal of Virology (2009)

Barry R. O’Keefe Barbara Giomarelli Dale L. Barnard Shilpa R. Shenoy Paul K.S. Chan

ABSTRACT Viruses of the family Coronaviridae have recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection both in vitro and in vivo . We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals.

Coronaviridae

Coronavirus

Antiviral protein

10.1128/jvi.02322-09

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Citations (289)

KGF and FGF-10 Stimulate Liquid Secretion in Human Fetal Lung

Pediatric Research (1999)

Ronald W. Graeff Guoshun Wang Paul B. McCray

Keratinocyte growth factor

10.1203/00006450-199911000-00006

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Citations (35)

Prostaglandins stimulate fluid secretion in human fetal lung.

PubMed (1993)

Paul B. McCray Bettencourt Jd

Fluid secretion by the fetal pulmonary epithelium is thought to be important for normal lung development yet little is known about factors regulating its production. As prostaglandins are synthesized in human fetal lung and stimulate secretion in a variety of epithelia, we investigated the effect of prostaglandins E2 and F2a (PGE2 and PGF2 alpha) on ion transport and fluid secretion in cultured first trimester human fetal lung tissue explants. We used conventional microelectrodes to continuously record the transepithelial potential difference (psi t). The addition of either PGE2 or PGF2 alpha to the bathing solution significantly hyperpolarized the lumen negative psi t and the subsequent addition of bumetanide, an inhibitor of chloride secretion in other systems, depolarized psi t by approximately 60% suggesting chloride transport contributed to the voltage. To assess whether this acute change in psi t represented stimulation of fluid secretion, we measured the change in luminal area of the explants after a 24-h exposure to prostaglandins. Both PGE2 and PGF2 alpha caused significant increases in the mean % luminal area of the explants compared with control tissues consistent with a stimulation of lung fluid secretion. Cultured lung tissue explants produced prostaglandins E2 and F2 alpha as assessed by radioimmunoassay of cell culture media samples and both prostaglandins stimulated cAMP accumulation in the explants. These findings show that lung fluid secretion in the human fetal pulmonary epithelium can be stimulated by prostaglandins. This effect may be mediated through cAMP dependent pathways. Prostaglandins may play a physiologic role in regulation of fetal lung fluid transport in vivo.

Transepithelial potential difference

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Suspected COVID-19 Reinfections at a Tertiary Care Center, Iowa 2020

Antimicrobial Stewardship & Healthcare Epidemiology (2021)

Takaaki Kobayashi Mohammed Alsuhaibani Miguel Ángel Mañez Ortiz Katherine L. Imborek Stephanie Holley

Background: Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 RNA can be detected by real-time reverse-transcription polymerase chain reaction (RT-PCR) for several weeks after infection. Discerning persistent RT-PCR positivity versus reinfection is challenging and the frequency of COVID-19 reinfections is unknown. We aimed to determine the frequency of clinically suspected reinfection in our center and confirm reinfection using viral whole-genome sequencing (WGS). Methods: The University of Iowa Hospitals and Clinics (UIHC) is an 811-bed academic medical center. Patients with respiratory complaints undergo COVID-19 RT-PCR using nasopharyngeal swabs. The RT-PCR (TaqPath COVID-19 Combo kit) uses 3 targets (ORF1ab, S gene, and N gene). We identified patients with previous laboratory-confirmed COVID-19 who sought care for new respiratory complaints and underwent a repeated SARS-CoV-2 test at least 45 days from their first positive test. We then identified patients with median RT-PCR cycle threshold (Ct) values. Results: During the study period, 13,603 patients had a SARS-CoV-2– positive RT-PCR. Of these, 296 (2.2%) had a clinical visit for new onset of symptoms and a repeated RT-PCR assay >45 days from the first test. Moreover, 29 patients (9.8%) had a positive RT-PCR assay in the repeated testing. Ct values were available for samples from 25 patients; 7 (28%) had Ct values. Conclusions: In patients with a recent history of COVID-19 infection, repeated testing for respiratory symptoms was infrequent. Some had a SARS-CoV-2–positive RT-PCR assay on repeated testing, but only 1 in 4 had Ct values suggestive of a reinfection. We confirmed 1 case of reinfection using WGS. Funding: No Disclosures: None

Coronavirus

10.1017/ash.2021.34

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Citations (0)

piggyBac-mediated phenotypic correction of factor VIII deficiency

Molecular Therapy — Methods & Clinical Development (2014)

Janice M. Staber Molly Pollpeter Angela M. Arensdorf Patrick L. Sinn D. Thomas Rutkowski

Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder. Hemophilia A is an attractive gene therapy candidate because even small increases in FVIII levels will positively alter the phenotype. While several vectors are under investigation, gene addition from an integrated transgene offers the possibility of long term expression. We engineered the DNA transposon-based vector, piggyBac (PB), to carry a codon-optimized B-domain deleted human FVIII cDNA. Evaluation of gene transfer efficiency in FVIII null mice demonstrated that PB containing the FVIII cDNA, delivered via hydrodynamic injection to immunocompetent hemophilia mice, conferred persistent gene expression, attaining mean FVIII activity of approximately 60% with 3/19 developing inhibitors. In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity. To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress. To evaluate phenotypic correction, a tail clip assay performed at the end of the study revealed reduced blood loss. These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.

10.1038/mtm.2014.42

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Increased CFTR expression and function from an optimized lentiviral vector for cystic fibrosis gene therapy

Molecular Therapy — Methods & Clinical Development (2021)

Laura Marquez‐Loza Ashley L. Cooney Qian Dong Christoph O. Randak Stefano Rivella

Despite significant advances in cystic fibrosis (CF) treatments, a one-time treatment for this life-shortening disease remains elusive. Stable complementation of the disease-causing mutation with a normal copy of the CF transmembrane conductance regulator (CFTR) gene fulfills that goal. Integrating lentiviral vectors are well suited for this purpose, but widespread airway transduction in humans is limited by achievable titers and delivery barriers. Since airway epithelial cells are interconnected through gap junctions, small numbers of cells expressing supraphysiologic levels of CFTR could support sufficient channel function to rescue CF phenotypes. Here, we investigated promoter choice and CFTR codon optimization (coCFTR) as strategies to regulate CFTR expression. We evaluated two promoters—phosphoglycerate kinase (PGK) and elongation factor 1-α (EF1α)—that have been safely used in clinical trials. We also compared the wild-type human CFTR sequence to three alternative coCFTR sequences generated by different algorithms. With the use of the CFTR-mediated anion current in primary human CF airway epithelia to quantify channel expression and function, we determined that EF1α produced greater currents than PGK and identified a coCFTR sequence that conferred significantly increased functional CFTR expression. Optimized promoter and CFTR sequences advance lentiviral vectors toward CF gene therapy clinical trials. Despite significant advances in cystic fibrosis (CF) treatments, a one-time treatment for this life-shortening disease remains elusive. Stable complementation of the disease-causing mutation with a normal copy of the CF transmembrane conductance regulator (CFTR) gene fulfills that goal. Integrating lentiviral vectors are well suited for this purpose, but widespread airway transduction in humans is limited by achievable titers and delivery barriers. Since airway epithelial cells are interconnected through gap junctions, small numbers of cells expressing supraphysiologic levels of CFTR could support sufficient channel function to rescue CF phenotypes. Here, we investigated promoter choice and CFTR codon optimization (coCFTR) as strategies to regulate CFTR expression. We evaluated two promoters—phosphoglycerate kinase (PGK) and elongation factor 1-α (EF1α)—that have been safely used in clinical trials. We also compared the wild-type human CFTR sequence to three alternative coCFTR sequences generated by different algorithms. With the use of the CFTR-mediated anion current in primary human CF airway epithelia to quantify channel expression and function, we determined that EF1α produced greater currents than PGK and identified a coCFTR sequence that conferred significantly increased functional CFTR expression. Optimized promoter and CFTR sequences advance lentiviral vectors toward CF gene therapy clinical trials.

10.1016/j.omtm.2021.02.020

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Citations (15)

Engineered Transfer RNA Suppression of CFTR Nonsense Mutations

Biophysical Journal (2018)

John D. Lueck Adam L. Mackey Daniel T. Infield Marshall R. Pope Paul B. McCray

Stop codon

Nonsense mutation

Nonsense-Mediated Decay

Loss function

10.1016/j.bpj.2017.11.2673

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Citations (1)