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.

FIG. 1

RTV structure and overview of drug susceptibility assay. (A) Amplified PR and RT gene segments from patient plasma samples are inserted into an indicator gene viral vector by using ApaI and PinAI restriction sites (vertical arrows). The ApaI site is located upstream of the gag polyprotein p7-p1-p6 cleavage sites. The PinAI site is located at amino acid 313 of RT. To monitor virus replication, a luciferase indicator gene cassette was inserted into a deleted region of the env gene, preventing HIV-1 envelope protein expression. (B) Pseudotyped virus particles are produced by cotransfecting cells with RTV DNA and a plasmid that expresses the envelope proteins of amphotropic murine leukemia virus (MLV). Following transfection, virus particles are harvested and are used to infect fresh target cells. The ability of virus particles to complete a single round of replication is assessed by measuring luciferase activity in target cells. The antiviral activities of PRIs and RTIs are measured by adding PRIs to transfected cells and RTIs to infected cells.

FIG. 2

Phenotypic drug susceptibility profile. Phenotypic drug susceptibility testing was performed with a plasma sample obtained from a patient receiving combination antiretroviral therapy. The viral load was 78,620 RNA copies per ml. Susceptibility to a panel of 15 antiretroviral drugs is shown. A reference virus (strain CNDO) that exhibits wild-type levels of susceptibility to all drugs was tested in parallel. Inhibition curves shifted to the right (higher drug concentration) of the reference curve for the drug-susceptible virus indicate reduced drug susceptibility. Inhibition curves shifted to the left (lower drug concentration) of the reference curve for the drug-susceptible virus indicate increased drug susceptibility. Fold differences in drug susceptibility were determined by comparing the IC₅₀ for the reference virus to the IC₅₀ for the sample virus. Drug abbreviations are defined in Materials and Methods. Dashed blue line, drug-sensitive reference virus; solid red line, patient virus. The patient virus genotype was as follows: for RT, M41L, D67N, M184V, L210W, and T215Y; for PR, L10F, D30N, L63P, V77I, N88D.

FIG. 3

Detection of drug-resistant virus subpopulations. RTV DNA representing genetically defined wild-type or drug-resistant mutant viruses were mixed at various ratios and were cotransfected into 293 cells to generate virus stocks. The drug susceptibility of each mixture was determined by the phenotypic assay. (A) Mixtures of wild-type virus and a patient virus clone containing RT mutations M41L, A62V, T69SSA, L74V, M184V, L210W, and T215Y tested against ZDV. (B) Mixtures of wild-type virus and a patient virus clone containing PR mutations K20R, L24I, G48V, I54V, V77I, and V82A tested against NFV. ◊, 0% mutant and 100% wild type; ■, 25% mutant and 75% wild type; □, 50% mutant and 50% wild type; ●, 75% mutant and 25% wild type; ⧫, 100% mutant and 0% wild type. Vertical dashed lines denote the IC₅₀.