Clinical significance of human immunodeficiency virus type 1 replication fitness

Abstract

The relative fitness of a variant, according to population genetics theory, is that variant's relative contribution to successive generations. Most drug-resistant human immunodeficiency virus type 1 (HIV-1) variants have reduced replication fitness, but at least some of these deficits can be compensated for by the accumulation of second-site mutations. HIV-1 replication fitness also appears to influence the likelihood of a drug-resistant mutant emerging during treatment failure and is postulated to influence clinical outcomes. A variety of assays are available to measure HIV-1 replication fitness in cell culture; however, there is no agreement regarding which assays best correlate with clinical outcomes. A major limitation is that there is no high-throughput assay that incorporates an internal reference strain as a control and utilizes intact virus isolates. Some retrospective studies have demonstrated statistically significant correlations between HIV-1 replication fitness and clinical outcomes in some patient populations. However, different studies disagree as to which clinical outcomes are most closely associated with fitness. This may be in part due to assay design, sample size limitations, and differences in patient populations. In addition, the strength of the correlations between fitness and clinical outcomes is modest, suggesting that, at present, it would be difficult to utilize these assays for clinical management.

FIG. 1.

Approaches to measuring HIV-1 replication fitness in cell culture. (a) Production of a virus stock from the peripheral blood of an HIV-infected patient. In this example, peripheral blood is obtained from the patient by venipuncture and separated into its component parts by density gradient centrifugation. A whole-virus isolate is obtained by coculturing the patient's PBMCs with a susceptible cell, either PBMCs from an HIV-negative human donor or an appropriate cell line (left-hand side). The clinical isolate is illustrated as red hexagons and can be harvested by separating the culture supernatant from the cells in culture. A recombinant virus derived from the patient's HIV-1 strain can also be obtained by purifying plasma and amplifying a specific region of the viral genome using reverse transcription followed by PCR (RT-PCR) (right-hand side). The PCR product can then be cloned into a viral vector containing the remainder of the HIV genome. Recombinant virus (illustrated by red striped hexagons) can then be produced by transfecting an appropriate cell line with the recombinant HIV vector. Either of these methods can be used to generate the clinical test strain for a fitness assay, as illustrated in b. RBC, red blood cells. (b) Methods used to carry out growth competition assays versus parallel infections. In this example, the test strain has reduced replication fitness relative to the reference strain. Virions are illustrated by hexagons, infected cells are illustrated by rectangles containing a circle, and nucleic acid is illustrated by curled lines. The reference virus, the cells that it infects, and the nucleic acid derived from it are colored blue; the analogous illustrations for the test virus are colored red. Uninfected cells are white. In parallel infections (left-hand side of the illustration), the reference and test viruses are used to separately infect different flasks containing susceptible cells. Virus replication for each culture is usually measured by quantitating p24 capsid antigen or a reporter gene such as luciferase. In a growth competition assay, the reference and test viruses infect the same culture; therefore, additional methods are needed to quantify the relative replication rates of the two variants. Viral or proviral nucleic acids can be purified from the culture (A), and the relative amounts of each variant can be quantitated using real-time PCR, a heteroduplex tracking assay, or sequence analysis. More recently, assays that utilize flow cytometry to detect reporter genes expressed by the viruses in infected cells have been developed (B). ELISA, enzyme-linked immunosorbent assay.

FIG. 1.

Approaches to measuring HIV-1 replication fitness in cell culture. (a) Production of a virus stock from the peripheral blood of an HIV-infected patient. In this example, peripheral blood is obtained from the patient by venipuncture and separated into its component parts by density gradient centrifugation. A whole-virus isolate is obtained by coculturing the patient's PBMCs with a susceptible cell, either PBMCs from an HIV-negative human donor or an appropriate cell line (left-hand side). The clinical isolate is illustrated as red hexagons and can be harvested by separating the culture supernatant from the cells in culture. A recombinant virus derived from the patient's HIV-1 strain can also be obtained by purifying plasma and amplifying a specific region of the viral genome using reverse transcription followed by PCR (RT-PCR) (right-hand side). The PCR product can then be cloned into a viral vector containing the remainder of the HIV genome. Recombinant virus (illustrated by red striped hexagons) can then be produced by transfecting an appropriate cell line with the recombinant HIV vector. Either of these methods can be used to generate the clinical test strain for a fitness assay, as illustrated in b. RBC, red blood cells. (b) Methods used to carry out growth competition assays versus parallel infections. In this example, the test strain has reduced replication fitness relative to the reference strain. Virions are illustrated by hexagons, infected cells are illustrated by rectangles containing a circle, and nucleic acid is illustrated by curled lines. The reference virus, the cells that it infects, and the nucleic acid derived from it are colored blue; the analogous illustrations for the test virus are colored red. Uninfected cells are white. In parallel infections (left-hand side of the illustration), the reference and test viruses are used to separately infect different flasks containing susceptible cells. Virus replication for each culture is usually measured by quantitating p24 capsid antigen or a reporter gene such as luciferase. In a growth competition assay, the reference and test viruses infect the same culture; therefore, additional methods are needed to quantify the relative replication rates of the two variants. Viral or proviral nucleic acids can be purified from the culture (A), and the relative amounts of each variant can be quantitated using real-time PCR, a heteroduplex tracking assay, or sequence analysis. More recently, assays that utilize flow cytometry to detect reporter genes expressed by the viruses in infected cells have been developed (B). ELISA, enzyme-linked immunosorbent assay.

FIG. 2.

Design of the Monogram Biosciences RC assay. (a) Production of patient-derived recombinant viruses. HIV-1 genomic RNA is purified from patient plasma. Reverse transcriptase PCR (RT-PCR) is used to amplify a region of the viral genome spanning the 3′ end of gag, protease (PR), and the first 313 codons of reverse transcriptase (RT). The pooled PCR amplicons are cloned into an HIV-1 vector containing a luciferase reporter gene. The resulting recombinant HIV-1 clones are cotransfected together into a mammalian cell line with a plasmid that allows the expression of an amphotropic MLV (A-MLV) envelope. The resultant pool of recombinant pseudotyped viruses will utilize the A-MLV envelope to infect susceptible cells and will express patient-derived protease and reverse transcriptase as well as luciferase. The A-MLV envelope allows the infection of CD4-negative cells. (b) Determination of the replication capacity of patient-derived recombinant viruses. The pool of recombinant viruses is used to infect a cell line; virus replication is quantified by measuring luciferase activity at a single time point. Because the patient-derived recombinant viruses do not encode an envelope protein, progeny viruses will not be infectious, i.e., only a single round of virus replication will occur. (Reproduced from reference with permission of the publisher.)

FIG. 3.

Correlation of HIV-1 replication fitness, using the Monogram Biosciences RC assay, with the presence of drug resistance mutations in HIV-1 obtained from antiretroviral-naive patients with early infection. The y axis represents RC, expressed as a percentage of the wild-type reference strain. Isolates from 191 patients were placed into mutually exclusive categories (x axis), depending on whether mutations conferring resistance to protease inhibitors (PI) with or without other resistance mutations (PI + any), nucleoside analogs (nRTI [NRTI]) only, NNRTIs only, or nRTIs plus NNRTIs were present. All isolates in the “PI + any” category contained primary (or major) protease inhibitor resistance mutations. Numbers beneath the graph refer to the numbers of samples in each category. Only the presence of protease inhibitor resistance mutations was statistically significantly associated with RC (P = 0.01). (Reprinted from reference with permission. © 2004 by the Infectious Diseases Society of America. All rights reserved.)

FIG. 4.

Correlation of HIV-1 replication fitness, using the Monogram Biosciences RC assay, with baseline CD4⁺ T-cell count in antiretroviral-naive patients with early infection. The y axis represents the CD4⁺ T-cell count at baseline, expressed as cells/μl (the y axis label in the figure is in error); the x axis represents the RC thresholds for each quartile. There was a statistically significant correlation between reduced CD4⁺ T-cell count and RC, which was driven primarily by the higher CD4⁺ T-cell values in the lowest quartile (Spearman's ρ = −0.29; P < 0.0001). (Reprinted from reference with permission. © 2004 by the Infectious Diseases Society of America. All rights reserved.)

FIG. 5.

Correlation of HIV-1 replication fitness, using a whole-virus parallel infection assay, with baseline plasma HIV-1 RNA concentration in chronically infected untreated patients (A) and RC, as measured by the Monogram Biosciences assay (B). Correlation coefficients (r²) were 0.71 (P < 0.001) (A) and 0.53 (P = 0.007) (B). The solid line indicates the fit of data by linear regression; the dotted lines indicate the 95% confidence interval for the linear regression. (Reprinted from reference with permission.)

FIG. 6.

Correlation of HIV-1 replication fitness, as measured by the Monogram Biosciences RC assay, with baseline CD4⁺ T-cell count (a) and baseline plasma HIV-1 RNA concentration (b) in a cohort of chronically infected children and adults with hemophilia. (a) y axis, square root-transformed baseline CD4⁺ T-cell count; x axis, RC expressed as a percentage of the wild-type reference strain. The solid line indicates fit of data by linear regression (r² = −0.199; P = 0.02). (b) y axis, log₁₀-transformed baseline plasma HIV-1 RNA concentration; x axis, RC expressed as a percentage of the wild-type reference strain. The solid line indicates the fit of data by linear regression (r² = +0.189; P = 0.03). (Reprinted from reference with permission of the publisher.)

FIG. 7.

Kaplan-Meier curves for HIV-1 disease progression in chronically infected children and adults with hemophilia according to baseline HIV-1 replication fitness as measured by the Monogram Biosciences RC assay. The y axis indicates the proportion of patients with AIDS-free survival; the x axis indicates years after baseline sample. Solid and dashed lines represent the first (RC, <69%), second (RC, 69 to 94%), third (RC, 95 to 118%), and fourth (RC, >118%) quartiles of RC values, as labeled in the graph. (Reprinted from reference with permission of the publisher.)

FIG. 8.

Association of HIV-1 replication fitness, as measured in a whole-virus parallel infection assay, with ability to control viremia after treatment interruption. The x axis indicates the slope of the change in culture p24 antigen between days 0 and 6 after infection; the y axis indicates the log₁₀ of patient's viral load. Panels depict the correlation between HIV-1 replication fitness using this assay and preantiretroviral therapy (ART) viral load (a), post-structured treatment interruption (STI) viral load (b), and viral load change during STI (c). (Modified from reference with permission.)