Novel single-cell-level phenotypic assay for residual drug susceptibility and reduced replication capacity of drug-resistant human immunodeficiency virus type 1

Abstract

Human immunodeficiency virus type 1 (HIV-1)-infected individuals who develop drug-resistant virus during antiretroviral therapy may derive benefit from continued treatment for two reasons. First, drug-resistant viruses can retain partial susceptibility to the drug combination. Second, therapy selects for drug-resistant viruses that may have reduced replication capacities relative to archived, drug-sensitive viruses. We developed a novel single-cell-level phenotypic assay that allows these two effects to be distinguished and compared quantitatively. Patient-derived gag-pol sequences were cloned into an HIV-1 reporter virus that expresses an endoplasmic reticulum-retained Env-green fluorescent protein fusion. Flow cytometric analysis of single-round infections allowed a quantitative analysis of viral replication over a 4-log dynamic range. The assay faithfully reproduced known in vivo drug interactions occurring at the level of target cells. Simultaneous analysis of single-round infections by wild-type and resistant viruses in the presence and absence of the relevant drug combination divided the benefit of continued nonsuppressive treatment into two additive components, residual virus susceptibility to the drug combination and selection for drug-resistant variants with diminished replication capacities. In some patients with drug resistance, the dominant circulating viruses retained significant susceptibility to the combination. However, in other cases, the dominant drug-resistant viruses showed no residual susceptibility to the combination but had a reduced replication capacity relative to the wild-type virus. In this case, simplification of the regimen might still allow adequate suppression of the wild-type virus. In a third pattern, the resistant viruses had no residual susceptibility to the relevant drug regimen but nevertheless had a replication capacity equivalent to that of wild-type virus. In such cases, there is no benefit to continued treatment. Thus, the ability to simultaneously analyze residual susceptibility and reduced replication capacity of drug-resistant viruses may provide a basis for rational therapeutic decisions in the setting of treatment failure.

FIG. 1.

Single-cell phenotypic assay for drug susceptibility and replication capacity. (A) Proviral construct used to generate pseudotyped virus for infections. Patient-derived gag-pol sequences were cloned in frame into a pNL4-3 proviral clone with the coding sequence for GFP replacing a portion of the env gene. The GFP sequence was followed by a KDEL ER retention signal and a stop codon. As a result, cells transfected with this vector or infected with pseudotyped viruses generated from this vector express an Env-GFP fusion protein that is directed into the ER by the Env signal peptide and retained in the ER by the KDEL sequence. (B) Expression of GFP by infected CD4⁺ T cells. The CD4⁺-T-cell Jurkat line was infected in vitro with GFP-encoding HIV-1 pseudotyped with VSV-G. Representative dot plots of GFP expression are shown for uninfected Jurkat cells (left), Jurkat cells infected with pseudovirions carrying the reference NL4-3 gag-pol sequence (center), and Jurkat cells infected with pseudovirions carrying a patient-derived wild-type gag-pol sequence, Pt0311-5M7 (right). (C) Linear relationship between the number of infected Jurkat cells and the amount of input viral inoculum. Jurkat cells were infected under standard conditions with increasing amounts of viral supernatant equivalent to the indicated numbers of virus-producing cells. GFP expression in Jurkat cells was measured by flow cytometry on day 2 after infection. The dotted line represents a fitted linear regression.

FIG. 2.

Measurement of viral susceptibility to antiretroviral drugs using the pNL4-3-ΔE-GFP-derived pseudoviruses. The ability of pseudovirions carrying wild-type NL4-3 gag-pol sequences to infect Jurkat cells was measured in the presence of increasing concentrations of the NRTI d4T, the NNRTI EFV, and the PI APV. APV was added to cultures of virus-producing cells beginning 4 hours after transfection and was maintained throughout the course of viral assembly, release, maturation, and spin inoculation into the target cells. d4T was added to target cells beginning 16 h before infection and was maintained in all steps thereafter. EFV was added at the time of spin infection and was maintained thereafter. The IC₅₀ for each drug was calculated by fitting data to the median-effect pharmacokinetic model (6, 7).

FIG. 3.

Ratios of C_min and C_max to IC₅₀ for individual antiretroviral drugs. The in vitro IC₅₀ was measured for each drug by using pseudoviruses carrying the reference NL4-3 sequence as described in Fig. 3. The ratios between published C_max and C_min values and the IC₅₀ for each drug are plotted. ABC, abacavir; TDF, tenofovir disoproxil fumarate; RTV, ritonavir; NFV, nelfinavir; SQV, saquinavir; LPV, lopinavir; IDV, indinavir.

FIG. 4.

Simultaneous measurement of susceptibility to 3TC and replication capacity for different HIV-1 clones. Pseudovirions carrying patient-derived isolates and NL4-3 gag-pol sequences were used to infect Jurkat cells in the presence of the indicated concentrations of 3TC. Drug resistance mutations in the protease and RT of patient-derived HIV-1 clones are shown in Table 2.

FIG. 5.

Three different patterns of replication of drug-resistant HIV-1 clones reflecting differential contributions of residual drug susceptibility and reduced replication capacity. gag-pol sequences amplified from the plasma of patients failing therapy were used to generate pseudovirions that were then used to infect Jurkat cells in the absence of drugs and in the presence of failing drug combinations at the C_min and C_max of each drug. Drug resistance mutations present in each isolate are indicated in Table 2. The replication of each isolate was compared to that of the wild-type NL4-3 sequence. (A) Resistant virus with significant residual susceptibility and marginally reduced replication capacity. (B) Resistant virus with minimal residual susceptibility and high replication capacity. (C) Resistant virus with no residual susceptibility and significantly reduced replication capacity.

FIG. 6.

In vitro demonstration of selection for a drug-resistant virus with reduced replication capacity by a simplified regimen. The drug-resistant virus analyzed for Fig. 5C was tested for replication in the absence of drugs, in the presence of the failing regimen, and in the presence of a simplified regimen consisting of only 3TC. Replication of this isolate was compared to that of the wild-type NL4-3 sequence.

FIG. 7.

Schematic plot showing decomposition of the clinical benefit of nonsuppressive HAART into two additive effects, the residual suppression of viral replication and the selection for resistant virus with diminished replication capacity. The difference in viral replication in the absence of drug represents the diminished replication capacity of the selected resistant virus versus the counterselected archival wild-type virus. The different replication capacities of the resistant virus in the absence and presence of the failing drug regimen represent residual suppression of the resistant virus. The addition of these two effects on a log scale represents the total inhibition of potential viral replication by nonsuppressive HAART or the treatment benefit.