A High-Throughput Flow Cytometry Screen Identifies Molecules That Inhibit Hantavirus Cell Entry

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), which infects more than 200,000 people worldwide. Sin Nombre virus (SNV) and Andes virus (ANDV) cause the most severe form of HCPS, with case fatality ratios of 30%–40%. There are no specific therapies or vaccines for SNV. Using high-throughput flow cytometry, we screened the Prestwick Chemical Library for small-molecule inhibitors of the binding interaction between UV-inactivated and fluorescently labeled SNVR18 particles, and decay-accelerating factor (DAF) expressed on Tanoue B cells. Eight confirmed hit compounds from the primary screen were investigated further in secondary screens that included infection inhibition, cytotoxicity, and probe interference. Antimycin emerged as a bona fide hit compound that inhibited cellular infection of the major HCPS (SNV)- and HCPS (Hantaan)-causing viruses. Confirming our assay’s ability to detect active compounds, orthogonal testing of the hit compound showed that antimycin binds directly to the virus particle and blocks recapitulation of physiologic integrin activation caused by SNV binding to the integrin PSI domain.

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High-Throughput Screening with HyperCyt® Flow Cytometry to Detect Small Molecule Formylpeptide Receptor Ligands

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105274532 ◽

2005 ◽

Vol 10 (4) ◽

pp. 374-382 ◽

Cited By ~ 53

Author(s):

Susan M. Young ◽

Cristian Bologa ◽

Eric R. Prossnitz ◽

Tudor I. Oprea ◽

Larry A. Sklar ◽

...

Keyword(s):

Flow Cytometry ◽

High Throughput ◽

High Throughput Screening ◽

Receptor Ligands ◽

Chemical Library ◽

Assay Sensitivity ◽

Compound Libraries ◽

Analysis System ◽

G Protein Coupled ◽

Formylpeptide Receptor

High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt® is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein–coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (~2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [Ki]=14 μM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt® system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.

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Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

Clinical and Vaccine Immunology ◽

10.1128/cvi.00546-12 ◽

2012 ◽

Vol 20 (2) ◽

pp. 218-226 ◽

Cited By ~ 22

Author(s):

R. L. Brocato ◽

M. J. Josleyn ◽

V. Wahl-Jensen ◽

C. S. Schmaljohn ◽

J. W. Hooper

Keyword(s):

Dna Vaccine ◽

Neutralizing Antibodies ◽

High Titer ◽

Disease Model ◽

Case Fatality ◽

Hemorrhagic Fever ◽

Puumala Virus ◽

M Gene ◽

Reading Frame ◽

Andes Virus

ABSTRACTPuumala virus (PUUV) is a causative agent of hemorrhagic fever with renal syndrome (HFRS). Although PUUV-associated HFRS does not result in high case-fatality rates, the social and economic impact is considerable. There is no licensed vaccine or specific therapeutic to prevent or treat HFRS. Here we report the synthesis of a codon-optimized, full-length M segment open reading frame and its cloning into a DNA vaccine vector to produce the plasmid pWRG/PUU-M(s2). pWRG/PUU-M(s2) delivered by gene gun produced high-titer neutralizing antibodies in hamsters and nonhuman primates. Vaccination with pWRG/PUU-M(s2) protected hamsters against infection with PUUV but not against infection by related HFRS-associated hantaviruses. Unexpectedly, vaccination protected hamsters in a lethal disease model of Andes virus (ANDV) in the absence of ANDV cross-neutralizing antibodies. This is the first evidence that an experimental DNA vaccine for HFRS can provide protection in a hantavirus lethal disease model.

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Neutralizing Monoclonal Antibodies against the Gn and the Gc of the Andes Virus Glycoprotein Spike Complex Protect from Virus Challenge in a Preclinical Hamster Model

mBio ◽

10.1128/mbio.00028-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 3

Author(s):

James Duehr ◽

Meagan McMahon ◽

Brandi Williamson ◽

Fatima Amanat ◽

Alan Durbin ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Treatment Options ◽

Case Fatality ◽

Hemorrhagic Fever ◽

Escape Mutant ◽

Hamster Model ◽

Virus Challenge ◽

Glycoprotein Complex ◽

The Andes ◽

Andes Virus

ABSTRACT Hantaviruses are the etiological agent of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The latter is associated with case fatality rates ranging from 30% to 50%. HCPS cases are rare, with approximately 300 recorded annually in the Americas. Recently, an HCPS outbreak of unprecedented size has been occurring in and around Epuyén, in the southwestern Argentinian state of Chubut. Since November of 2018, at least 29 cases have been laboratory confirmed, and human-to-human transmission is suspected. Despite posing a significant threat to public health, no treatment or vaccine is available for hantaviral disease. Here, we describe an effort to identify, characterize, and develop neutralizing and protective antibodies against the glycoprotein complex (Gn and Gc) of Andes virus (ANDV), the causative agent of the Epuyén outbreak. Using murine hybridoma technology, we generated 19 distinct monoclonal antibodies (MAbs) against ANDV GnGc. When tested for neutralization against a recombinant vesicular stomatitis virus expressing the Andes glycoprotein (GP) (VSV-ANDV), 12 MAbs showed potent neutralization and 8 showed activity in an antibody-dependent cellular cytotoxicity reporter assay. Escape mutant analysis revealed that neutralizing MAbs targeted both the Gn and the Gc. Four MAbs that bound different epitopes were selected for preclinical studies and were found to be 100% protective against lethality in a Syrian hamster model of ANDV infection. These data suggest the existence of a wide array of neutralizing antibody epitopes on hantavirus GnGc with unique properties and mechanisms of action. IMPORTANCE Infections with New World hantaviruses are associated with high case fatality rates, and no specific vaccine or treatment options exist. Furthermore, the biology of the hantaviral GnGc complex, its antigenicity, and its fusion machinery are poorly understood. Protective monoclonal antibodies against GnGc have the potential to be developed into therapeutics against hantaviral disease and are also great tools to elucidate the biology of the glycoprotein complex.

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High Throughput Microfluidic Electrical Impedance Flow Cytometry for Assay of Micro Particles

Micro and Nanosystems ◽

10.2174/1876402911002040298 ◽

2010 ◽

Vol 2 (4) ◽

pp. 298-308 ◽

Cited By ~ 2

Author(s):

Ashish V. Jagtiani ◽

Jiang Zhe

Keyword(s):

Flow Cytometry ◽

High Throughput ◽

Electrical Impedance ◽

Micro Particles

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Identification of a juvenile-hormone signaling inhibitor via high-throughput screening of a chemical library

Scientific Reports ◽

10.1038/s41598-020-75386-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Takumi Kayukawa ◽

Kenjiro Furuta ◽

Keisuke Nagamine ◽

Tetsuro Shinoda ◽

Kiyoaki Yonesu ◽

...

Keyword(s):

Juvenile Hormone ◽

Cell Line ◽

Signaling Pathway ◽

High Throughput ◽

High Throughput Screening ◽

Large Scale ◽

Ex Vivo ◽

Agricultural Field ◽

Chemical Library ◽

Field Development

Abstract Insecticide resistance has recently become a serious problem in the agricultural field. Development of insecticides with new mechanisms of action is essential to overcome this limitation. Juvenile hormone (JH) is an insect-specific hormone that plays key roles in maintaining the larval stage of insects. Hence, JH signaling pathway is considered a suitable target in the development of novel insecticides; however, only a few JH signaling inhibitors (JHSIs) have been reported, and no practical JHSIs have been developed. Here, we established a high-throughput screening (HTS) system for exploration of novel JHSIs using a Bombyx mori cell line (BmN_JF&AR cells) and carried out a large-scale screening in this cell line using a chemical library. The four-step HTS yielded 69 compounds as candidate JHSIs. Topical application of JHSI48 to B. mori larvae caused precocious metamorphosis. In ex vivo culture of the epidermis, JHSI48 suppressed the expression of the Krüppel homolog 1 gene, which is directly activated by JH-liganded receptor. Moreover, JHSI48 caused a parallel rightward shift in the JH response curve, suggesting that JHSI48 possesses a competitive antagonist-like activity. Thus, large-scale HTS using chemical libraries may have applications in development of future insecticides targeting the JH signaling pathway.

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Immune Serum Produced by DNA Vaccination Protects Hamsters against Lethal Respiratory Challenge with Andes Virus

Journal of Virology ◽

10.1128/jvi.01822-07 ◽

2007 ◽

Vol 82 (3) ◽

pp. 1332-1338 ◽

Cited By ~ 51

Author(s):

Jay W. Hooper ◽

Anthony M. Ferro ◽

Victoria Wahl-Jensen

Keyword(s):

Dna Vaccine ◽

Neutralizing Antibodies ◽

Lethal Dose ◽

Case Fatality ◽

Immune Serum ◽

Human Infection ◽

M Gene ◽

Hamster Model ◽

Highly Pathogenic ◽

Andes Virus

ABSTRACT Hantavirus pulmonary syndrome (HPS) is a highly pathogenic disease (40% case fatality rate) carried by rodents chronically infected with certain viruses within the genus Hantavirus of the family Bunyaviridae. The primary mode of transmission to humans is thought to be inhalation of excreta from infected rodents; however, ingestion of contaminated material and rodent bites are also possible modes of transmission. Person-to-person transmission of HPS caused by one species of hantavirus, Andes virus (ANDV), has been reported. Previously, we reported that ANDV injected intramuscularly causes a disease in Syrian hamsters that closely resembles HPS in humans. Here we tested whether ANDV was lethal in hamsters when it was administered by routes that more accurately model the most common routes of human infection, i.e., the subcutaneous, intranasal, and intragastric routes. We discovered that ANDV was lethal by all three routes. Remarkably, even at very low doses, ANDV was highly pathogenic when it was introduced by the mucosal routes (50% lethal dose [LD50], ∼100 PFU). We performed passive transfer experiments to test the capacity of neutralizing antibodies to protect against lethal intranasal challenge. The neutralizing antibodies used in these experiments were produced in rabbits vaccinated by electroporation with a previously described ANDV M gene-based DNA vaccine, pWRG/AND-M. Hamsters that were administered immune serum on days −1 and +5 relative to challenge were protected against intranasal challenge (21 LD50). These findings demonstrate the utility of using the ANDV hamster model to study transmission across mucosal barriers and provide evidence that neutralizing antibodies produced by DNA vaccine technology can be used to protect against challenge by the respiratory route.

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