Novel Two-Round Phenotypic Assay for Protease Inhibitor Susceptibility Testing of Recombinant and Primary HIV-1 Isolates

Abstract

Antiretroviral drug susceptibility tests facilitate therapeutic management of HIV-1-infected patients. Although genotyping systems are affordable, inaccuracy in the interpretation of complex mutational patterns may limit their usefulness. Currently available HIV-1 phenotypic assays are based on the generation of recombinant viruses in which the specific viral gene of interest, derived from a patient plasma sample, is cloned into a susceptible genetic viral backbone prior to in vitro drug susceptibility evaluation. Nevertheless, in the case of protease inhibitors, not only are mutations in the HIV-1 protease-coding region involved in resistance, but the role of Gag in drug susceptibility has also recently been reported. In order to avoid the inherent limitations resulting from partial cloning of the viral genome, we designed and evaluated a new experimental strategy to test the in vitro susceptibility of primary viral isolates to protease inhibitors. Our protocol, which is based on a two-round infection protocol using the reporter TZM-bl cell line, showed a good correlation with genotypic resistance prediction and with the Antivirogram phenotypic assay, in both protease-recombinant viruses and primary viral isolates. The protocol is suitable for any HIV-1 subtype and enables rapid in-house measurement of protease inhibitor susceptibility, thus making it possible to evaluate the concomitant effects of both patient-derived gag and protease-coding regions.

Agreement between an in‐house replication competent and a reference replication defective recombinant virus assay for measuring phenotypic resistance to HIV‐1 protease, reverse transcriptase, and integrase inhibitors

“…Previously published academic small‐scale methods for phenotypic resistance testing have been not usually tested in comparison with reference systems. One exception is the system published by Puertas et al . for measuring resistance to PIs, which also includes partial cloning of the Gag region.…”

System-oriented optimization of multi-target 2,6-diaminopurine derivatives: Easily accessible broad-spectrum antivirals active against flaviviruses, influenza virus and SARS-CoV-2

“…As previously reported, comparison between SYRA and direct yield reduction assay (DYRA) data can be used to obtain preliminary information about early versus late antiviral activity of drug candidates [ [24] , [25] , [26] ]. Compound 6i was thus analyzed through DYRA against ZIKV and DENV replication.…”

Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication

“…For example, a similar two-step system is adopted to measure the anti-HIV activity of drugs acting at different steps of virus replication. 40,41 Thus, the combined use of the direct and secondary YRA can not only measure antiviral activity but also help characterize the mechanism of action. As proof of concept, we tested celgosivir, an inhibitor of endoplasmic reticulum (ER) α-glycosidases, found to be active against DENV both in vitro, with IC 50 values ranging from the sub- (0.2 µM) to low- (5.7 µM) micromolar range, 30,42 and in vivo in a mouse model, demonstrating the reduction of viremia and inducement of protection against virus-induced mortality.…”