A Novel Phenotypic Drug Susceptibility Assay for Human Immunodeficiency Virus Type 1

Abstract

Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.

Human immunodeficiency virus type 1: drug resistance in treated and untreated Brazilian children

“…By itself, it causes low-level resistance to NFV and IDV. However, in the presence of D30N or M46I it causes high-level NFV resistance (Petropoulos et al 2000) as reported in 1 child. The M46L was reported in 2 children; it contributes resistance to each of the PIs except possibly SQV and has been commonly reported during therapy with IDV, RTV, APV, and NFV (Condra et al 2000b).…”

“…The Y181C and G190S mutations were reported in 1 child and the other one presented additional resistance mutations at sites A98G and V108I. The Y181C mutation causes a more than 30-fold resistance to NVP and DLV and 2 to 3-fold resistance to EFV (Petropoulos et al 2000). The G190S mutation causes high-level resistance to both NVP and EFV .…”

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

“…The only commercially available fitness assay is a parallel-infection, recombinant-virus, single-cycle RC assay developed by Monogram Biosciences, Inc. (formerly ViroLogic) (15). This assay, which is a variation on Monogram's phenotypic drug susceptibility assay (133), compares test and reference strains in the absence of drug (Fig. 2).…”

“…2). An amplicon spanning the p7-p1-p6 cleavage sites in Gag, the entire protease, and part of the reverse transcriptase (codons 1 to 313) is obtained from patient plasma and cloned in bulk into an HIV-1 vector derived from the pNL4-3 infectious molecular clone containing a luciferase reporter gene that replaces part of env (133). Luciferase is expressed upon establishment of infection and is a measure of viral infection (133).…”

“…Examples of reporter genes used to detect viral replication are luciferase (133), green fluorescent protein (GFP) (184), hisD from Salmonella enterica serovar Typhimurium, human placental alkaline phosphatase (PLAP) (103), and the mouse Thy1.1 and Thy1.2 alleles (57). A major advantage of the enzymatic assay to quantify luciferase is its high sensitivity and wide dynamic range of quantitation; a disadvantage is that different variants cannot be distinguished, and therefore, a growth competition assay is not feasible.…”