A Dual Infection/Competition Assay Shows a Correlation between Ex Vivo Human Immunodeficiency Virus Type 1 Fitness and Disease Progression

Abstract

This study was designed to examine the impact of human immunodeficiency virus type 1 (HIV-1) fitness on disease progression through the use of a dual competition/heteroduplex tracking assay (HTA). Despite numerous studies on the impact of HIV-1 diversity and HIV-specific immune response on disease progression, we still do not have a firm understanding of the long-term pathogenesis of this virus. Strong and early CD8-positive cytotoxic T-cell and CD4-positive T-helper cell responses directed toward HIV-infected cells appear to curb HIV pathogenesis. However, the rate at which the virus infects the CD4 ؉ T-cell population and possibly destroys the HIV-specific immune response may also alter the rate of disease progression. For HIV-1 fitness studies, we established conditions for dual HIV-1 infections of peripheral blood mononuclear cells (PBMC) and a sensitive HTA to measure relative virus production. A pairwise comparison was then performed to estimate the relative fitness of various non-syncytium-inducing/CCR5-tropic (NSI/R5) and syncytium-inducing/CXCR4-tropic (SI/X4) HIV-1 isolates. Four HIV-1 strains (two NSI/R5 and two SI/X4) with moderate ex vivo fitness were then selected as controls and competed against primary HIV-1 isolates from an HIV-infected Belgian cohort. HIV-1 isolates from long-term survivors (LTS) were outcompeted by control strains and were significantly less fit than HIV-1 isolates from patients with accelerated progression to AIDS (PRO). In addition, NSI/R5 HIV-1 isolates from PRO overgrew control SI/X4 strains, suggesting that not all SI/X4 HIV-1 isolates replicate more efficiently than all NSI/R5 isolates. Finally, there were strong, independent correlations between viral load and the total relative fitness values of HIV-1 isolates from PRO (r ‫؍‬ 0.84, P ‫؍‬ 0.033) and LTS (r ‫؍‬ 0.86, P ‫؍‬ 0.028). Separation of the PRO and LTS plots suggest that HIV-1 fitness together with viral load may be a strong predictor for the rate of disease progression.

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

“…Another study, which used a multiple-cycle whole-virus growth competition assay in PBMCs also found a correlation between replication fitness and disease progression in a small number of patients (140). Two other studies demonstrated good correlations between HIV-1 replication fitness, as measured in multiple-cycle whole-virus parallel infections in PBMCs, and plasma HIV-1 RNA concentration before the initiation of antiretroviral therapy (27,163).…”

“…Some examples of measuring virus directly include quantitation of a viral protein, such as p24, by enzyme-linked immunosorbent assay (106,156,163); measurement of reverse transcriptase activity (164, 175); and quantitation of proviral DNA (116). In addition, in growth competition assays, the relative amounts of test and reference strains can be quantified by sequence analysis of proviral DNA or viral RNA (65,69,78,86,117,154,155,169,174), heteroduplex tracking assays (HTAs) (140), or allele-specific real-time PCR (33,142,176). It should be noted that the latter methods do not provide information on the extent of viral replication.…”

“…HTAs of the env gene have been used in whole-virus growth competition assays (7,116,140,164). Hybridization of a PCR-amplified product to a labeled nucleic acid probe results in a heteroduplex that has a different electrophoretic mobility from that of the homoduplex if there are sufficient sequence differences between the amplicon and the probe.…”

“…A whole-virus, multiple-cycle growth competition assay in primary human PBMCs in which the relative proportion of test and reference viruses is measured using an HTA has been used to study the effects of env on fitness and disease progression and to evaluate the relative fitnesses of different subtypes of HIV-1 (6,11,140,164). Because this growth competition assay utilizes an intact viral isolate and primary human PBMCs, it likely provides the closest approximation to HIV-1 replication fitness in patients compared to other currently available assays, but its labor-intensive nature limits its use for large-scale studies correlating fitness with clinical outcomes.…”

A new strategy based on recombinant viruses for assessing the replication capacity of HIV‐1

“…Competition experiments, in which the proportions of competing viruses are carefully monitored over time by measuring small differences in replication rates, provide a more accurate measurement of viral replicative fitness. Different techniques have been developed to measure dual virus production in competitive culture assays: sequencing assays [23,24], heteroduplex tracking assays [25,26], real-time polymerase chain reaction (PCR) [27,28] and reporter gene RVs [29][30][31]. Although it has been shown that sensitivity is higher in competitive culture assays than in replication kinetics assays, competitive culture assays are timeconsuming and laborious, making them impractical for general clinical use.…”