RetroVirox is the only CRO exclusively focused on the antiviral field. RetroVirox has a large menu of validated antiviral services, including assays to evaluate small-molecule antivirals, antibodies and vaccines against many human viruses such as SARS-CoV-2, HIV, influenza, RSV, Dengue, Zika, CMV, rhinovirus, and other infectious agents. Several coronavirus related assays are offered, including antiviral and neutralization assays with live SARS-CoV-2, and also assays against seasonal coronaviruses (OC43 and 229E). RetroVirox offers also neutralization assays against highly pathogenic viruses (including Ebola, Lassa, and pandemic influenza). The company has optimized assays with heterologous pseudovirus carrying the glycoproteins of highly pathogenic viruses. The company also evaluates the titers of antisera from animals and humans against adeno-associated vectors and viruses used in gene therapy approaches.
Since 2010, RetroVirox has also provided customized services to companies worldwide, including optimization and validation of novel antiviral assays, tailored studies to understand the mechanism of action of antiviral agents, and production and characterization of viruses for use in preclinical studies. RetroVirox has broad expertise in high throughput screening (HTS) for antiviral targets, and has successfully completed the screening of thousands of small-molecules in multiple HTS campaigns utilizing live infectious viruses. The company is headquartered in San Diego, California, and has provided services to Biotechnology and Pharmaceutical companies in more than 20 countries.
Antiviral services offered at RetroVirox include a full menu of in vitro antiviral assays against several important human pathogens (SARS-CoV-2, HCoV, RSV, HIV, influenza, DENV, ZIKA, CMV, LCMV, and HRV among others). The company can also determine the neutralizing activity of antibodies and sera against highly pathogenic viruses using HIV pseudovirions carrying heterologous proteins of Ebola virus, Lassa virus, Marburg virus, SARS-CoV-2, and avian influenza. The Company also performs neutralization assays against adeno-associated vectors (AAV) used in Gene Therapy approaches. Unlike other Contract Research Organizations (CRO), RetroVirox is specialized in offering biological services exclusively in the antiviral field. We work with virtual start-up companies lacking the appropriate facilities to perform antiviral studies, and with large Biotechnology and Pharmaceutical companies seeking CROs with unique expertise in the virology field. We also offer a number of tailored antiviral assays to address the mechanism of action of novel antivirals. Development of novel antiviral assays against other viruses is also available on FTE basis and also fixed prices. With one of the largest collections of viral strains available (over 250 viral strains of 18 different viral species). RetroVirox can optimize assays and evaluate your molecules for activity against many commercially important human viruses. Evaluation of cytoxicity assays in uninfected stable cells or primary cells are also available when requested as part of antiviral services. RetroVirox is 100% "IP and ownership-friendly", and does not request any ownership of intellectual property rights of any data, discoveries, or materials generated during any of its services.
Microneutralization assays are available with several live viruses, including SARS-CoV-2, HIV, influenza and respiratory syncytial virus (RSV). Microneutralization assays utilizing pseudoviruses are available against SARS-CoV-2, Lassa, Ebola, LCMV, and avian influenza. These assays allow us to evaluate the neutralization activity of monoclonal antibodies, antisera and small-molecule entry inhibitors against highly pathogenic viruses. Pseudoviruses carrying the envelope glycoproteins of HIV or VSV (vesicular stomatitis virus) can be used as controls to determine the specificity of your entry inhibitors.
Additional pseudovirus against other pathogenic viruses can be developed and validated upon request. Pseudovirus assays are amenable for high throughput screening (HTS) of small-molecule libraries (ranging from 10,000 up to 100,000 molecules)
RetroVirox has successfully screened over 200,000 small molecules in different HTS campaigns with live viruses utilizing a variety of readouts. Several of these campaigns have resulted in the generation of validated leads against novel targets and at now at the medicinal chemistry optimization stage. RetroVirox has broad experience optimizing phenotypic cell-based assays from the scratch and validating them with pilot assays of a few thousand compounds to determine the feasibility of the campaign (signal to background, variation and Z’ values) and set the criteria for hit identification. Among the readouts typically used are high content (flow cytometry), fluorescence, luminescence , colorimetric, and cytopathic effect (CPE) readouts.
AAV Neutralization Assays
Our standard assay is performed with AAV serotypes 2 (AAV2) and 9 (AAV9) utilizing HeLa cells. The assay determines the titer of antisera in experimental animals (mice, pig, rats, non-human primates) and also in clinical samples of humans. The assay monitors the level of a reporter gene expressed (beta-galactosidase or luciferase) upon transduction of permissive cells with AAV reporter vectors. The signal is revealed using commercially available kits that detect a luminescent signal.
The assay has been optimized to generate a linear signal, and can detect small reductions in transduction of cells. This assay is useful to determine the level of immunity against specific AAV vectors. The existence of neutralizing antibodies in small animals or humans, even at low titers, can be sufficient to block in vivo transduction. Additionally, these studies can help predict the appearance of strong immune responses to the vector(s), which could result in "cytokine storms" responsible for strong adverse reactions. The standard assays are optimized with AAV2, and AAV9, but assays against other AAV serotypes may be available upon request.
Lead Identification and Validation Services
The influenza panel is comprised of 20 different influenza A seasonal strains. Strains span from 1933 to 2012 season and include the H1N1 and H3N2 serotypes. The panel includes strains resistant to several neuraminidase inhibitors and also pandemic strains from the 2009 and 201 seasons. This assay utilizes infected A549 cells (human lung epithelial carcinoma cells) to evaluate the ability of test-items to block influenza replication. The assay detects the presence of intracellular viral antigens in influenza-infected cells. Immunodetection is performed with a proprietary cocktail of monoclonal antibodies (mAbs) designed to maximize the detection of different strains of Influenza A. The assay is optimized to evaluate inhibition of influenza replication and has been validated with known inhibitors of influenza replication (baloxavir, ribavirin and pimodivir). Importantly, the cocktail excludes antibodies against the viral hemagglutinin (HA) protein, to avoid interference with test-items targeting the influenza HA protein. The assay can be used also to reveal the neutralization activity of influenza entry inhibitors, including antisera and monoclonal antibodies (mAbs)
Unlike other antiviral assays such as plaque reduction (PRA), which measures the number of plaques, or the focus forming assays (FFA), which measures foci or units of infected cells stained with fluorescent antibodies (FFU), this assay is faster, more robust and reproducible, superior in sensitivity, and also displays significantly lower variation than the more traditional assays. Additionally, many influenza strains are known to develop plaques or display foci in strain-dependent manners, with some strains known to poorly develop plaques. Our microneutralization assay has been optimized to generate a linear signal and can detect small variations in inhibition of viral replication. Another flaw observed with plaque-reduction based assays with some inhibitors is the appearance of plaques of diminished size without significantly altering the total number of plaques monitored.
TAT for the assay is 3 to 4 weeks, and up to 20 test-items can be evaluated per month (full dose response curves) with the entire panel. Test-items are assessed at 8 concentrations in duplicates.
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Cell Viability & Proliferation Assays Services
RetroVirox offers a panel of HIV clinical isolates for the evaluation of therapeutic antibodies, antisera and antivirals. The assay includes up to 30 clinical isolates covering subtypes A, B, C, D, and also some recombinants strains. The assay utilizes live infectious primary isolates. Inhibition of virus infectivity is revealed with a HeLa reporter cell line. The panel is offered for the evaluation of test-items against 10, 20 or 30 different viruses. Upon request, testing with additional viruses can be performed.
Antiviral services against human coronaviruses (alpha and betacoronaviruses) are available at RetroVirox, including assays with live SARS-CoV-2, the causative agent of COVID-19. The coronavirus assays utilize a colorimetric readout to determine the extent of prevention of the virus-induced cytopathic effect (CPE). The assay reveals activities of antivirals at most stages of the coronavirus life cycle, including entry, fusion viral replication, viral protease inhibition, and also late stages affecting virus production. Assays are performed with the live SARS-CoV-2 virus, and also with a prototypic betacoronavirus strain (HCoV-OC43). Assays are also available with a seasonal alphacoronavirus strain (HCoV-229E). Importantly, many of the inhibitors with activity against the novel coronavirus (SARS-CoV-2), also displayed activity against other "milder" human betacoronaviruses. Testing of prospective inhibitors with our CPE-based assay against the seasonal coronaviruses OC43 is also a valuable tool for preliminary screens of potential inhibitors of SARS-CoV-2.
Assays are also available to specifically evaluate inhibition of viral entry of the SARS-CoV-2 novel coronavirus utilizing pseudoviruses that are coated with the spike (S) glycoprotein of SARS-CoV-2. The assay reveals only entry inhibitors targeting either the S protein of the novel coronavirus, or its viral receptor (ACE-2). The pseudovirus entry assay does not reveal inhibitors at later stages of replication. Replication inhibitors can be assessed with the live SARS-CoV-2 virus assay.
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