Novel recombinant virus assay for measuring susceptibility of human immunodeficiency virus type 1 group M subtypes to clinically approved drugs

Abstract

Combination therapy can successfully suppress human immunodeficiency virus (HIV) replication in patients but selects for drug resistance, requiring subsequent resistance-guided therapeutic changes. This report describes the development and validation of a novel assay that offers a uniform method to measure susceptibility to all clinically approved HIV type 1 (HIV-1) drugs targeting reverse transcriptase (RT), protease (PR), integrase (IN), and viral entry. It is an assay in which the antiviral effect on infection within a single replication cycle is measured in triply transfected U87.CD4.CXCR4.CCR5 cells, based on homologous recombination between patient-derived amplicons and molecular proviral clones tagged with the enhanced green fluorescent protein (EGFP) reporter gene and from which certain viral genomic regions are removed. The deletions stretch from p17 codon 7 to PR codon 98 in pNL4.3-DeltagagPR-EGFP, from PR codons 1 to 99 in pNL4.3-DeltaPR-EGFP, from RT codons 1 to 560 in pNL4.3-DeltaRT-EGFP, from IN codons 1 to 288 in pNL4.3-DeltaIN-EGFP, and from gp120 codon 34 to gp41 codon 237 in pNL4.3-Deltaenv-EGFP. The optimized experimental conditions enable the investigation of patient samples regardless of viral subtype or coreceptor use. The extraction and amplification success rate for a set of clinical samples belonging to a broad range of HIV-1 group M genetic forms (A-J, CRF01-03, CRF05, and CRF12-13) and displaying a viral load range of 200 to >500,000 RNA copies/ml was 97%. The drug susceptibility measurements, based on discrimination between infected and noninfected cells on a single-cell level by flow cytometry, were reproducible, with coefficients of variation for resistance ranging from 7% to 31%, and were consistent with scientific literature in terms of magnitude and specificity.

FIG. 1.

Construction of molecular proviral clones containing deletions and the EGFP reporter gene. ΔX indicates a deletion of gag-PR, PR, RT, or IN. The deletions were generated in the 5′-hemigenomic molecular clone p83-2 by inverse PCR and self-ligation (A), followed by subcloning into pNL4.3-EGFP (B). (C) Schematic representation of generated vectors. (Adapted from reference with permission of the publisher.)

FIG. 2.

Discrimination between infected and noninfected cells. U87.CD4.CCR5.CXCR4 cells were infected with HIV-1-NL4.3-EGFP (right) or were mock infected (left). Twenty-four hours later, cells were washed with PBS, trypsinized, and fixed in 2% paraformaldehyde for 10 min. (A) Afterwards, nuclei were stained for 5 min with DAPI (4′,6-diamidino-2-phenylindole). Subsequently, cells were washed three times with PBS, resuspended in PBS, and visualized by fluorescence microscopy. (B) After fixation, cells were analyzed by flow cytometry.

FIG. 3.

Representative examples of susceptibility curves for different inhibitors and recombinant viruses. Means and standard deviations of percentages of infection relative to the positive control are shown (data for triplicate experiments are shown). Susceptibility curves are shown for wild-type virus (open squares with dashed line) and RT recombinant virus with A62V, S68G, V75I, F77L, F116Y, and Q151M mutations (filled squares with dashed line) or K65R mutation (filled circles with dashed line) against TDF; for wild-type virus (open squares with solid line) and RT recombinant virus with A62V, S68G, V75I, F77L, F116Y, and Q151M mutations (filled squares with solid line) or K65R mutation (filled circles with sold line) against abacavir; for wild-type virus (× with dashed line) and IN recombinant virus with T66I mutation (open circles with dashed line) against EVG; and for wild-type virus (× with dotted and dashed line) and IN recombinant virus with T66I mutation (filled circles with dotted and dashed line) against RAL.

FIG. 4.

Schematic overview of recombinant virus assay. *, for testing of susceptibility to PIs, the titration procedure as well as the IC₅₀ determination should be modified as indicated in the text. FACS, fluorescence-activated cell sorting; ARV, antiretroviral.