Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

Transcription

1 CLINICAL MICROBIOLOGY REVIEWS, Oct. 2007, p Vol. 20, No /07/$ doi: /cmr Copyright 2007, American Society for Microbiology. All Rights Reserved. Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness Carrie Dykes and Lisa M. Demeter* Infectious Diseases Division, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York INTRODUCTION ASSAYS TO MEASURE HIV-1 REPLICATION FITNESS Cell Culture Assays General features of cell culture assays to measure HIV-1 replication fitness Growth competition assays versus parallel infections Single-cycle versus multiple-cycle assays Whole-virus versus recombinant-virus assays Direct measure of virus replication versus use of a reporter gene Use of cell lines versus primary human cells Other differences among fitness assays Examples of specific assays Other Assays To Measure Fitness MATHEMATICAL APPROACHES TO QUANTIFYING REPLICATION FITNESS Multiple-Cycle Assays Single-Cycle Assays EFFECTS OF SPECIFIC DRUG RESISTANCE MUTATIONS ON FITNESS Mutations Conferring Resistance to Reverse Transcriptase Inhibitors nrtis (i) M184V (ii) TAMs (iii) L74V (iv) K65R (v) Q151M complex NNRTIs Mutations Conferring Resistance to Protease Inhibitors Major protease inhibitor resistance mutations Minor protease inhibitor resistance mutations Mutations Conferring Resistance to Entry Inhibitors MUTATIONAL INTERACTIONS THAT AFFECT FITNESS Intragenic Interactions Reverse transcriptase (i) Interactions among nrti resistance mutations (ii) Interactions between NNRTI and nrti resistance mutations (iii) Effects of codons in reverse transcriptase not associated with drug resistance on fitness of nrti-resistant mutants Protease Envelope Extragenic Interactions Protease cleavage site mutations in gag Insertions in gag Other mutations in gag UTR GENETIC DETERMINANTS OF FITNESS CORRELATION OF HIV-1 REPLICATION FITNESS WITH CLINICAL OUTCOMES Rationale Transmission Efficiency Outcomes in Primary HIV-1 Infection Evolution of fitness during primary infection Impact of HIV-1 replication fitness on CD4 T-cell count and viral load * Corresponding author. Mailing address: University of Rochester, School of Medicine and Dentistry, Infectious Diseases Unit, 601 Elmwood Ave., Box 689, Rochester, NY Phone: (585) Fax: (585) lisa_demeter@urmc.rochester.edu. 550

2 VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 551 Outcomes in Chronic HIV-1 Infection Correlation of HIV-1 replication fitness with clinical outcome in untreated patients Correlation between fitness and outcome of antiretroviral treatment interruptions Association of HIV-1 replication fitness with antiretroviral treatment responses Clinical Predictive Potential of Specific HIV-1 Replication Fitness Assays CONCLUSIONS ACKNOWLEDGMENTS REFERENCES INTRODUCTION Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that infects primarily CD4-expressing T cells. Primary HIV-1 infection occurs with a single variant initially. However, the rapid replication and turnover of HIV-1 (79, 177) and the high mutation rate of the viral reverse transcriptase (110, 147) result in high mutation frequencies that quickly lead to the production of a population of genetically distinct but related variants called a quasispecies (53). If the effective size of the replicating HIV-1 population is large, the variant that predominates in an HIV-1 quasispecies at any one point in time should be the variant that is most fit under the selective pressures that exist (31). According to population genetics, fitness is defined as a variant s ability to contribute to successive generations (reviewed in reference 54). By this definition, HIV-1 variants with high levels of fitness should have a selective advantage over other less-fit variants in clinical infections. It has also been postulated that HIV-1 variants with reduced fitness may be less pathogenic, leading to improved clinical outcomes. HIV-1 fitness has been studied primarily using in vitro cell culture model systems in which the rate of replication is measured, and it is not known how closely this measure of fitness correlates with viral fitness in patients or clinical outcomes. For the sake of simplicity, we will refer to all such assays as replication fitness assays, recognizing that these assays are varied in their design and do not fully reflect the selective forces impacting viral fitness during clinical HIV-1 infection. The degree to which HIV-1 replication fitness correlates with clinical outcome continues to be a controversial issue. We believe that this controversy stems in part from a lack of consensus on what the best approaches are to measure fitness and which clinical outcomes are most impacted by fitness. In addition, the labor-intensive nature of most assays used to quantify fitness limits the sample sizes of studies correlating fitness with clinical outcomes. This review will summarize and put into perspective the types of assays used to measure HIV-1 replication fitness, the effects of specific drug resistance mutations on fitness, and the studies done to date evaluating the correlation between in vitro measures of HIV-1 replication fitness and clinical outcome. ASSAYS TO MEASURE HIV-1 REPLICATION FITNESS Cell Culture Assays General features of cell culture assays to measure HIV-1 replication fitness. The published literature contains data on a multitude of cell culture assays that have been used to measure HIV-1 replication fitness. This variety makes it difficult to comprehensively list the different approaches to measuring fitness and limits the comparisons that can be made between studies from different groups of investigators. One common feature of all assays is that they compare the replication of a test variant to that of a reference strain. The test variant is either a site-directed mutant of a laboratory strain of HIV-1 or an isolate derived from a patient sample, usually peripheral blood (Fig. 1a). We have proposed further categorizing cell culture fitness assays on the basis of five major features: whether replication of the test strains and replication of the reference strains are compared in parallel infections or directly in a single culture (the latter is referred to as a growth competition assay); whether replication is measured over a single virus life cycle, using pseudotyped virus, or over multiple cycles; whether the test virus is an isolate obtained from a clinical specimen ( whole virus ) versus a recombinant virus containing only a portion of the clinical viral sequence; whether virus is detected directly by assaying a viral gene or protein or indirectly through the use of a reporter gene; and whether the assay utilizes cell lines versus primary human cells (Table 1). These features of fitness assays are summarized in more detail below. Growth competition assays versus parallel infections. Differences between growth competition assays and parallel infections are illustrated in Fig. 1b. Growth competition assays are generally preferable to parallel infections for measuring replication fitness since the replication of the test strain and replication of the reference strain are compared in the same culture, eliminating potential artifacts resulting from differences in culture conditions of the test and reference strains. As the total virus population expands, the prevalence of the test variant in a growth competition assay should decrease over time relative to the reference strain if the test variant has lower fitness. If the relative proportions of the test and reference strains are compared at more than one time point after infection, growth competition assays are also relatively insensitive to the specific method used to determine virus inoculum, which is another significant advantage over parallel infections (more detail on methods to quantify virus inoculum is provided below). A third advantage of growth competition assays is that they can detect small differences in replication fitness that are not identified in parallel infections (33, 137). An important potential disadvantage of growth competition assays is that a quantitative assay that can distinguish the test and reference strains must be available (Fig. 1b). Quantitation of the relative amounts of test and reference strains usually adds significantly to the complexity and cost of the assay. In addition, the question of viral recombination must be considered, since the production of recombinant progeny viruses could alter the apparent replication fitness of the test strain if it differs genetically from the reference strain at more than one nucleotide position. In order to minimize recombination be

3 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 552

4 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. 553

5 554 DYKES AND DEMETER CLIN. MICROBIOL. REV. TABLE 1. General features of cell culture assays to measure HIV-1 replication fitness Feature Description Parallel infection vs growth competition assay Parallel infection...test and reference viruses are grown in separate cultures Growth competition assay...test and reference viruses are grown in the same culture Single cycle vs multiple cycles of replication Single cycle...assays are conducted using an env-deleted viral vector that completes only one replication cycle Multiple cycles of replication...env is not deleted, and the virus completes several rounds of replication Whole virus vs recombinant virus Whole virus...assays are performed using intact isolates cultured from patient PBMCs Recombinant virus...assays are performed using a laboratory strain that contains a portion of the patient s viral genome previously PCR amplified from a clinical sample Direct measure of virus replication vs use of a reporter gene Direct measure of virus replication...virus replication is measured by quantifying a viral protein such as p24 Use of a reporter gene...virus replication is measured by quantifying a reporter gene that is expressed by a recombinant virus such as luciferase Use of cell lines vs primary human cells Use of cell lines...assays are performed in a transformed T-cell line Primary human cells...assays are performed in primary human cells (e.g., PBMCs or macrophages) tween the test and reference strains in a growth competition assay, the experimental design should minimize dual infection of cells, which is a prerequisite for retroviral recombination (80), by using a low multiplicity of infection initially and limiting the duration of infection. Single-cycle versus multiple-cycle assays. Single-cycle assays are typically conducted by deleting the envelope gene from an HIV-1 vector and then producing pseudotyped virus by transfecting an appropriate cell line with the env-deleted HIV vector together with a plasmid expressing a gene product that can serve as an envelope (see Fig. 2 for a depiction of the Monogram Biosciences replication capacity [RC] assay, which is a single-cycle assay utilizing murine leukemia virus [MLV] envpseudotyped virus). The MLV envelope and vesicular stomatitis virus G protein have each been used to pseudotype HIV-1 variants in single-cycle replication fitness assays (15, 184). Such pseudotyped virions can infect susceptible cells but cannot produce infectious progeny due to the fact that the genome still carries the env deletion. Infection with pseudotyped virus is thus limited to a single round of replication. Single-cycle infections have the advantage of a shorter time frame, typically 24 to 72 h, compared to several days to weeks for a multiplecycle assay. Multiple-cycle assays have the theoretical advantage of greater sensitivity because differences between the two variants can be amplified over many life cycles, although the two types of assays have not been extensively compared. Whole-virus versus recombinant-virus assays. Whole-virus assays are performed using intact isolates cultured directly from patient samples. In contrast, recombinant-virus assays require the amplification of a region of interest by PCR, for example, protease and reverse transcriptase, and the subsequent cloning of that region into an HIV-1 vector encoding the remaining viral genome from a laboratory strain (Fig. 1a). The recombinant virus may be derived from a single clone of the PCR amplicon, or a pool of recombinant viruses can be produced from the bulk cloning of all amplicons and transfection of the pooled viral vectors. The major advantage of the recombinant-virus assay is that it does not require the isolation of infectious HIV-1 from the patient, which adds significantly to the time and cost of the assay. In addition, viral vectors used in the recombinant-virus assay can be modified to express a reporter gene, such as luciferase, which can be used to detect viral infection. Recombinant assays allow one to look at the effects of a particular gene segment on fitness but have the disadvantage of not taking into account the possible modulating effects of other gene segments located outside the region of amplification. Whole-virus assays have the advantage of looking at the replication of the complete viral population present in peripheral blood and thus are likely to be a more accurate measure of viral fitness in a patient. However, whole-virus assays are more difficult in that they require target cells that can propagate all variant types contained within the isolate, usually primary human peripheral blood mononuclear cells (PBMCs); in addition, the methods to detect virus growth are limited to those that directly assay viral genes or gene products. Studies have compared whole-virus and recombinant-virus assays in relatively small numbers of clinical samples in order to determine the relative impact of different gene segments on overall replication. Using virus isolates without drug resistance mutations in pol, overall fitness in a wholevirus assay correlated most closely with that of a recombinant-virus assay in which the env gene was amplified from the patient specimen (142). In contrast, in one isolate with

6 VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 555 Downloaded from 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 15 with permission of the publisher.) on April 8, 2019 by guest drug resistance mutations, the pol gene appeared to make a major contribution to viral fitness (142). In addition, studies have demonstrated interactions among gag, protease, and reverse transcriptase that affect viral fitness (19, 156). Thus, the correlation between the two assay types will likely vary and will depend on the gene segment included in the recombinant-virus assay, whether the patient population is treatment-naive or -experienced, and, if treatment failure is occurring, the duration of virologic failure. Direct measure of virus replication versus use of a reporter gene. Fitness assays can also vary in the methodologies used to detect and quantify the test and reference strains (Fig. 1b). The major distinguishing feature is whether an assay measures a viral gene or gene product directly or whether a reporter gene is used as a surrogate measure of viral replication. 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);

7 556 DYKES AND DEMETER CLIN. MICROBIOL. REV. 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. The most common method to directly quantify virus growth is p24 antigen concentration in the culture supernatant. This approach has been used to quantify the relative replication rates of different variants in parallel infections (27, 163). p24 antigen concentration can also be used to evaluate the relative expansion of virus during a growth competition experiment in which the relative proportions of test and reference strains are measured by a PCR-based method that does not directly measure virus replication (174). A number of different methods have been used to quantify the relative proportion of test and reference variants in a growth competition assay. 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. The mobility of the heteroduplex is affected more by insertions and deletions than by base substitutions. Therefore, if the test and reference strains are sufficiently divergent in their sequences, HTAs can be used to quantify their relative proportions in a growth competition assay over time. The env region is particularly amenable to being assayed by HTA because it often exhibits substantial sequence divergence and insertions/deletions among HIV strains. This method is less labor-intensive than other methods that rely on PCR amplification of the test and reference strains but requires that the two variants have substantial divergence in their nucleotide sequences. Sequence analysis has also been used to quantify the relative amounts of the two variants in a growth competition assay either by direct sequence analysis of the bulk PCR product (65, 69, 78, 86, 117, 154, 155, 174) or by analysis of individual clones derived from the PCR amplicon (119). One disadvantage of bulk sequencing is that differences in the surrounding sequence may influence the assay s sensitivity for detecting a minority variant at a given codon. This is less of a concern when site-directed mutants that differ by only one or a few codons are compared but may impact reproducibility when clinical isolates are studied. In addition, quantitation of the relative amounts of mutants by analysis of the sequencing electropherogram from the bulk amplicon has a limited linear range. Clonal sequence analysis does not have these limitations if large numbers of clones are assayed, but this approach is significantly more labor-intensive and costly. Real-time PCR has also been used to quantify the relative amounts of test and reference strains in a growth competition assay by allele-specific amplification of viral nucleic acid at a codon where the two variants differ (33, 142, 176). Real-time PCR has an advantage over other molecular assays used to quantify the relative proportion of the test and reference strains because of its high throughput and wide linear range of detection. However, surrounding genetic variation, which can influence the efficiency of primer or probe hybridization to the target sequence, can significantly influence the performance characteristics of allelespecific real-time PCR. Therefore, if real-time PCR is used to detect a specific viral mutation, the assay must be optimized and validated for each codon of interest. Examples of reporter genes used to detect viral replication are luciferase (133), green fluorescent protein (GFP) (184), hisd from Salmonella enterica serovar Typhimurium, human placental alkaline phosphatase (PLAP) (103), and the mouse Thy1.1 and Thy1.2 alleles (57). A major advantage of the enzymatic assay to quantify luciferase is its high sensitivity and wide dynamic range of quantitation; a disadvantage is that different variants cannot be distinguished, and therefore, a growth competition assay is not feasible. The advantage of the paired reporter genes hisd/plap and Thy1.1/Thy1.2, which are detected by real-time PCR and flow cytometry, respectively, is that each gene in the pair can be cloned into a different vector, allowing the test and reference strains to be distinguished in growth competition assays (57, 103). A further potential advantage of the Thy1.1/Thy1.2 flow cytometry-based assay is that the number of cells infected by each variant is quantified, allowing an assessment of both virus growth and the relative proportions of test and reference strains (57). However, reporter genes are surrogate measures of viral replication, and linkage of the HIV-1 genome and the reporter gene is necessary for an accurate interpretation of the data; this linkage could potentially be disrupted if recombination between the test and reference strains that express different reporter genes were to occur. This theoretical limitation has not posed a problem under the conditions reported in previously published assays, which utilized a low virus inoculum to initiate infection (57, 103). Use of cell lines versus primary human cells. The specific target cells used in fitness assays may also affect the apparent fitness of a mutant. The best evidence for an impact of cell type on fitness has been obtained for HIV-1 mutants that are resistant to nucleoside analog inhibitors of the viral reverse transcriptase. For example, the replication fitness of M184V, which is resistant to lamivudine, was reduced in primary human PBMCs but was indistinguishable from that of wild-type HIV-1 in a lymphoid cell line (10). Purified reverse transcriptase with the M184V mutation showed reduced processivity of polymerization that was accentuated at low nucleotide concentrations (10). Since PBMCs are known to have lower concentrations of nucleotides than T-cell lines, this finding offers a potential biochemical mechanism for the differences in fitness of this mutant in primary cells versus cell lines. A systematic study of HIV-1 with K65R or different numbers of thymidine analog mutations (TAMs) showed that the replication of each of these variants was less efficient in primary human macrophages than in T-cell lines (129). It is interesting that a mutant virus with the four TAMs D67N, K70R, T215Y, and K219Q actually had a replication advantage compared to wildtype virus in unstimulated PBMCs; this difference was not observed when the PBMCs were stimulated before infection (26). Similar to the M184V mutant, the differences in replication fitness observed under these conditions were associated with differences in processivity under limiting nucleotide concentrations. Thus, it seems likely that differences in the replication fitness of nucleoside resistance mutants are more likely to be observed in cell types in which nucleotide pools are limited. In contrast, no such impact of cell type was observed when the replication fitness of HIV-1 mutants that are resistant to

8 VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 557 nonnucleoside reverse transcriptase inhibitors (NNRTIs) was studied (4, 69, 94, 174). These findings are consistent with the fact that these mutants do not appear to affect the processivity of polymerization or nucleotide affinity (51, 69, 174). We are not aware of published studies comparing the replication fitness of mutants that are resistant to protease inhibitors in different cell types. However, based on the known mechanisms for drug resistance of this class of mutant, one would not expect to see the pronounced effects of cell type on replication fitness that are seen with nucleoside-resistant mutants. Because of the difficulty in maintaining primary human PBMCs in culture for extended periods of time, most assays of replication fitness utilize T-cell lines rather than primary cells. Thus, these assays may overestimate the replication efficiencies of isolates containing nucleoside resistance mutations. Other differences among fitness assays. In addition to these major differences in assay design, methods to determine the amount of virus used to infect a culture-based fitness assay may differ. The method used to determine virus inoculum can have a significant impact on the apparent fitness of a strain in parallel infections, single-cycle assays, or growth competition assays if the relative proportions of test and reference variants at a single time point after infection are compared to the proportions in the original inoculum. One study rigorously evaluated the correlation between different measures of virus input using a number of different clinical isolates and found that the reverse transcriptase activity of the virus stock correlated better with infectious titer than p24 antigen or quantitative viral RNA assays (115). It is not clear whether this analysis included strains containing drug resistance mutations in reverse transcriptase, which might alter the relationship between reverse transcriptase activity and virus infectivity, so these findings may not necessarily apply to drug-resistant strains of HIV-1. Examples of specific assays. The only commercially available fitness assay is a parallel-infection, recombinant-virus, single-cycle RC assay developed by Monogram Biosciences, Inc. (formerly ViroLogic) (15). This assay, which is a variation on Monogram s phenotypic drug susceptibility assay (133), compares test and reference strains in the absence of drug (Fig. 2). An amplicon spanning the p7-p1-p6 cleavage sites in Gag, the entire protease, and part of the reverse transcriptase (codons 1 to 313) is obtained from patient plasma and cloned in bulk into an HIV-1 vector derived from the pnl4-3 infectious molecular clone containing a luciferase reporter gene that replaces part of env (133). Luciferase is expressed upon establishment of infection and is a measure of viral infection (133). MLV envelope is supplied in trans during the production of the virus stock, yielding pseudotyped virus. Since the RC assay is a single-cycle assay and since luciferase detection can be automated, it has a high throughput. Luciferase quantitation also has a wide linear range. The potential disadvantages of this assay are that it measures the fitness contributions of the 3 end of gag, protease, and part of reverse transcriptase only and uses parallel infections, which may be less sensitive and more susceptible to variations in virus input than growth competition assays. The use of a murine retroviral envelope instead of HIV-1 could also possibly affect the relative fitness of mutants that are resistant to protease and/or reverse transcriptase inhibitors, although this seems unlikely. The Monogram RC assay has been the most widely used fitness assay in clinical cohorts, and results correlating RC with clinical outcome will be summarized later in this review. There are several other types of assays developed by individual research laboratories that have been used to quantify the relative fitness of site-directed mutants or clinical isolates. Here, we will briefly describe several different assays to illustrate the types of approaches that have been taken, recognizing that this is not an exhaustive list. An assay used by several laboratories is a multiple-cycle recombinant-virus growth competition assay in which the relative proportion of test and reference strains is measured using either bulk or clonal sequence analysis (4, 65, 69, 78, 86, 117, 119, 154, 155, 174). This type of assay is labor-intensive and difficult to apply to large numbers of clinical samples but has been commonly used to characterize the relative fitness of site-directed drug-resistant mutants of HIV-1 in protease and reverse transcriptase. Depending on the laboratory and the specific mutants studied, either T-lymphocyte lines or primary human PBMCs have been used as target cells. A recombinantvirus growth competition assay in which test and reference strains are quantified using real-time PCR of the hisd and PLAP reporter genes has been used to study the relative fitnesses of mutants that are resistant to the nucleoside analog zidovudine (81, 105) and the fusion inhibitor enfuvirtide (104). A potential advantage of this assay is the greater dynamic range and reduced labor of real-time PCR. 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. Some laboratories have utilized multiple-cycle, whole-virus, parallel infections to measure growth kinetics in primary human PBMCs (27, 163). This type of assay offers the advantages of using primary cells and an intact virus isolate as well as improved throughput compared to growth competition assays. If replication fitness is estimated using the slope of virus expansion after initial infection, this will markedly reduce the influence of virus inoculum on apparent replication fitness. However, a theoretical concern is assay-to-assay variation, since there is no internal control as in a growth competition assay. These assays have demonstrated associations of replication fitness with viral load and will be discussed in more detail later in this review. Another recently reported assay developed by Tibotec and Virco is a high-throughput assay to measure replication rates in parallel infections. Eight serial dilutions of virus are made, with six time points tested per sample, and the measurements are normalized for viral input by multiplying by the dilution. Viral replication is measured using a cell line containing an enhanced GFP (EGFP) reporter gene under the control of an HIV long terminal repeat (LTR). Infection with HIV results in the activation of LTR and EGFP expression. The log 10 of the

9 558 DYKES AND DEMETER CLIN. MICROBIOL. REV. product of fluorescence and viral dilution is linear with respect to time, and the slope of this line is the replication rate, which is independent of virus inoculum (146a). The very high throughput of this assay would make it very amenable to use in a clinical setting, although this use of the assay has not yet been reported. Some recent publications have described fitness assays that utilize flow cytometry as a method to determine virus growth and variant proportions. The potential advantages of such assays are higher throughput and the ability to monitor the spread of infection at a cellular level. Zhang and coworkers designed a recombinant-virus single-cycle assay in which the GFP gene replaces env in the infectious HIV-1 molecular clone pnl4-3; test and reference strains were then compared in parallel infections (184). The use of red fluorescent proteins (DsRed2) and EGFPs as markers in a multiple-cycle recombinant-virus growth competition assay in primary human PBMCs has also been described and was used to characterize the relative fitness of mutants that are resistant to the fusion inhibitor enfuvirtide (125). A flow cytometry-based recombinant-virus multiple-cycle growth competition assay has also been developed in which the test and reference viral vectors contain the mouse Thy1.1 or Thy1.2 allele in place of nef. The Thy gene products are expressed on the surface of infected cells early during viral infection and can be detected by commercially available fluorescence-labeled monoclonal antibodies (57). There was an excellent correlation of relative fitness of different drug-resistant mutants, as measured by flow cytometry, compared to direct and clonal sequence analysis, indicating that the linkage between the resistance mutations and the reporter gene was not disrupted by recombination (57). Such dual-marker flow cytometry-based assays are exciting in that they have the potential to allow the use of growth competition assays to measure replication fitness in larger-scale clinical trials. Other Assays To Measure Fitness An interesting modification to the cell culture growth competition assays described above is the rapid cell turnover assay (172). In contrast to standard cell culture assays in which half the cultured cells are typically replaced every 4 to 7 days, 90% of the cultured cells are replaced with fresh uninfected cells every 2 days, a time interval similar to the life span of T cells in patients. Under these conditions, it has been shown that the fitness of the highly pathogenic simian immunodeficiency virus clone Mne170 was higher than that of its parental clone CL8, whereas under normal culture conditions, they were indistinguishable. The result obtained under rapid cell turnover conditions is more consistent with the relative pathogenicity of the two clones in animals, suggesting that rapid cell turnover conditions may be a better predictor of viral pathogenicity in clinical infection. This interesting finding needs to be confirmed with more extensive studies of HIV-1 isolates. Although more labor-intensive and much less commonly used, HIV-1 replication fitness has also been measured in SCID-hu mice that are reconstituted with human peripheral blood leukocytes and then infected with patient isolates. This methodology has been used to look at the properties of isolates with resistance mutations in protease and reverse transcriptase (91a, 135, 159). An interesting feature of this assay is that CD4 T-cell depletion can be evaluated, suggesting that this assay may be able to evaluate some aspects of virus pathogenicity that are not captured in traditional cell culture-based assays of replication fitness. The disadvantages are that it is much more labor-intensive and costly than cell culture assays, and therefore, the ability to correlate viral replication fitness with clinical outcomes in significant numbers of clinical samples is extremely limited. Nonetheless, this assay has the potential to be a very interesting research tool to better understand the relationship between HIV-1 replication efficiency and pathogenicity. Measurements of viral fitness have also been made by assaying the prevalence of TAMs in viral quasispecies present in plasma from patients not receiving antiretroviral therapy (71, 72). Allelespecific PCR assays or clonal sequence analysis was used to quantify the relative proportions of the different drug-resistant variants over time, and mathematical modeling was used to determine the fitness gain of the revertant virus over the mutant. An important advantage of this approach is that it takes into account selective pressures present in patients. However, this approach assumes that the specific mutation being assayed has the same effect in each viral genome and does not take into account the potential modulating effects of other mutations on viral fitness. For example, if the prevalence of T215Y in a patient did not decrease after the discontinuation of the drug due to a selective advantage conferred by a nonresistance mutation at another codon, this fitness value could erroneously be attributed to T215Y. In addition, the replication properties of the wild-type reference strain that overgrows the mutant will differ from patient to patient. Therefore, one must use caution in attributing fitness values obtained from such studies solely to the resistance mutation(s) being assayed. MATHEMATICAL APPROACHES TO QUANTIFYING REPLICATION FITNESS Although cell culture-based fitness assays can provide qualitative information regarding the replication fitness of one variant relative to that of another, determining whether such in vitro measures of fitness correlate with clinical outcome requires reliable methods to quantitate the degree to which fitness is altered relative to the reference strain. Reliable quantitation will also be required if fitness assays are to be used in clinical practice. Unfortunately, there has been no clear consensus on how to quantify relative fitness, and publications differ in their mathematical definitions of a fitness coefficient. This lack of consensus has led to confusion in the literature and has further limited the ability to compare results from different studies. We will discuss methods to quantify fitness for multiple-cycle and singlecycle assays separately, since investigators have taken different approaches for these two types of assays. Multiple-Cycle Assays According to population genetics, the relative fitness of a variant, defined as 1 s, is the relative contribution of that variant to the next generation; s is defined as the selection

10 VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 559 TABLE 2. Different approaches to calculating fitness in multiple-cycle assays Measure of fitness a Mathematical definition b Formula b Production p k m /k w ln T m (t 2 )/T m (t 1 ) m t ln2 rate ratio ln T w (t 2 )/T w (t 1 ) w t ln2 Log fitness r g m /g w ln T m (t 2 )/T m (t 1 ) ln2 ratio ln T w (t 2 )/T w (t 1 ) ln2 Log relative d g m g w 1/ t ln T m (t 2 ) T w (t 1 )/T w (t 2 ) fitness T m (t 1 ) Relative fitness 1 s exp(d) exp{1/ t ln T m (t 2 ) T w (t 1 )/T w (t 2 ) T m (t 1 ) } a Nomenclature proposed by Wu et al. (181). Computational tools are available at the following website: /virusfitness.htm. b Definition of terms used in these formulas is as follows: T m is the number of cells infected by infectious mutant virus, T w is the number of cells infected by infectious wild-type virus, m and w are death rates of infected mutant and wild-type cells, respectively, k m and k w are infection rates of mutant and wildtype virus, respectively, g m and g w are net growth rates of mutant- and wild-typeinfected cells, respectively, and s is the selection coefficient, as defined by population genetics theory. coefficient. If a mutant is more fit than the reference strain, 1 s will be greater than 1, and successive generations will have an increasing proportion of progeny derived from that mutant. If a mutant is less fit than the reference strain, 1 s will be less than 1, and the prevalence of that mutant will decline over time relative to the reference strain. A confusing aspect of the literature on fitness is that the value of 1 s has sometimes been defined differently (reviewed in more detail in reference 181). Using viral dynamic models, Wu and coworkers attempted to clarify the different definitions of fitness, proposing standard nomenclature that distinguishes different approaches to measuring fitness and providing approaches to perform statistical evaluations (181). In these viral dynamic models, it is assumed that only two variants are present (i.e., no recombinants) and that there is a constant number of target cells over time (i.e., a large excess of susceptible target cells). Three parameters were defined, each of which takes a different approach to quantifying fitness: log relative fitness, log fitness ratio, and production rate ratio. The log relative fitness value, d, is equal to the natural log of 1 s, where s is the selection coefficient defined by population genetics theory (Table 2). d is equivalent to the difference between the net growth rates of the mutant and reference strains (Table 2). In contrast, the log fitness ratio, r, is the ratio of the net growth rates of the mutant and reference strains. The production rate ratio, p, differs from the two previous parameters in that it is the ratio of the production rates of the mutant and reference strains and assumes that the life span of infected cells does not contribute to fitness. p is equivalent to 1 plus the selection coefficient defined by Maree and coworkers (112), which differs from the selection coefficient defined by population genetics theory. An important point is that d and r compare the growth rates of the mutant and the wild type at the same time point and therefore do not need to be corrected if the culture is diluted during the course of the growth competition assay. In addition, p must incorporate the total expansion of the two viral variants; use of relative proportions of variants without accounting for the degree of viral expansion will give incorrect values. A publicly available website provides calculators and guidance on the use and statistical analysis of these different fitness parameters (http: // Single-Cycle Assays The Monogram Biosciences assay expresses RC of a test strain as a percentage of the wild-type reference virus (14, 15, 41). All values are normalized for the efficiency of the transfections used to produce the virus stocks. For example, if infection with a recombinant virus derived from a clinical sample leads to half of the luciferase activity of the wild-type reference strain, and if the test and reference virus stocks had equal transfection efficiencies and were used in equal volumes in the infection, the RC value of the clinical isolate would be reported as 50%. There are no published data on how this method of normalizing virus inoculum compares to other methods. EFFECTS OF SPECIFIC DRUG RESISTANCE MUTATIONS ON FITNESS A major focus of early literature on HIV-1 replication fitness was an evaluation of the impact of specific drug resistance mutations on viral fitness. The majority of these studies utilized site-directed mutants of laboratory strains, although some studies also evaluated clinical isolates (either whole virus or recombinant viruses containing a genome segment of the clinical isolate). This continues to be an important area of study as new drugs and combination therapies are introduced into clinical practice. In general, nearly all the drug resistance mutations studied adversely impact HIV-1 replication fitness to some extent, although different mutations vary in the magnitudes of their effect. Since replication fitness is proposed to influence the prevalence of an HIV-1 variant in patients and may also impact clinical outcome, an understanding of the relative impact of different drug-resistant mutations may provide important, clinically relevant information. The following section will describe what is known about the replication fitness of specific drug resistance HIV-1 variants conferring resistance to drugs that are currently FDA approved, organized according to the drug class that is affected. Because of the variations in assay design and approaches to calculating fitness values summarized above, much of this discussion will be qualitative, with comparisons among mutants limited primarily to those that were studied with the same methods (see Table 3 for a summary of the effects of the single drug resistance mutations discussed below on fitness). Mutations Conferring Resistance to Reverse Transcriptase Inhibitors nrtis. Nucleoside and nucleotide reverse transcriptase inhibitors (nrtis) are competitive inhibitors of nucleotides that are normally incorporated during the synthesis of the viral genome. nrtis bind to the polymerase active site of reverse transcriptase and competitively inhibit the synthesis of proviral DNA by acting as chain terminators during synthesis (for a review of this class of drugs, see reference 171). Several nrti resistance mutations can confer cross-resistance to more than one nrti.

11 560 DYKES AND DEMETER CLIN. MICROBIOL. REV. TABLE 3. Effects of drug resistance mutations on HIV-1 replication fitness Gene (drug) Resistance mutation Fitness assay used (cell type) a Fitness relative to wild type (reference s ) Reverse transcriptase K65R Multiple cycle, parallel cultures (cell line) Slight reduction (48) (nrtis) Single cycle, parallel cultures (cell line, primary cells PBMCs ) Similar (129) Multiple cycle, growth competition b (cell line) Reduced (35) Single cycle, parallel cultures (primary cells macrophages ) Reduced (129) Multiple cycle, growth competition (primary cells PBMCs ) c Reduced (175) Single cycle, parallel cultures (cell line) d Reduced (179) L74V Multiple cycle, parallel cultures (cell line) Similar (48) Multiple cycle, parallel cultures (primary cells [PBMCs]) Reduced (154) Multiple cycle, growth competition b (primary cells [PBMCs]) Reduced (154) Multiple cycle, growth competition b (cell line) Reduced (35) M184V Multiple cycle, parallel cultures (cell line) Similar (10) Single cycle, parallel cultures (cell line, primary cells PBMCs ) Similar (129) Multiple cycle, parallel cultures (primary cells PBMCs ) Reduced (10) Single cycle, parallel cultures (primary cells macrophages ) Reduced (129) Single cycle, parallel culture (cell line) d Reduced (48) Multiple cycle, growth competition b (cell line) Reduced (35) T215Y Multiple cycle, growth competition b (cell line) Reduced (78) Multiple cycle, growth competition b (cell line) Reduced (35) Multiple cycle, parallel infections (cell line) Slight reduction (106) Q151M Multiple cycle, parallel infections (cell lines, primary cells PBMCs ) Similar (106) Multiple cycle, growth competition b (cell line) Improved (93) Reverse transcriptase (NNRTIs) K103N Multiple cycle, growth competition b,e (cell line) Similar (33, 57, 94) Y181C Multiple cycle, growth competition f (cell line) Increased (84) Multiple cycle, growth competition b,g (cell line) Reduced (4, 33) G190S Multiple cycle, growth competition b,g (cell line) Reduced (57, 82, 174) Single cycle, parallel infections d (cell line) Reduced (82) Protease (protease L90M Multiple cycle, growth competition b,e ; single cycle, parallel Similar (57, 119, 130) inhibitors) infections (cell line) D30N Multiple cycle, growth competition b,e ; single cycle, parallel infections Reduced (57, 119, 130) (cell line) SCID-hu mouse model Reduced (91a) V82A Single cycle, parallel infections Similar (109) SCID-hu mouse model h Reduced (135) Env (enfuvirtide) I37T Multiple cycle, growth competition i (cell line, primary cells Reduced (104) PBMCs ) V38M or A Multiple cycle, growth competition i (cell line, primary cells Reduced (104) PBMCs ) N42T Multiple cycle, growth competition i (cell line, primary cells PBMCs ) Reduced (104) a All assays tested site-directed mutants of laboratory strains of HIV-1, unless stated otherwise. b Relative proportions of the mutant and wild type were measured by sequence analysis. c Intact clinical isolates were tested. Relative proportions of the mutant and wild type were measured by real-time PCR of env. d Monogram Biosciences RC assay using patient-derived recombinant viruses. e Relative proportions of the mutant and wild type were measured using flow cytometry to detect Thy1.1 and Thy1.2 reporter genes. f Relative proportions of wild-type and mutant strains, which were selected for in the presence of nevirapine, were determined by direct sequence analysis. Relative proportions of the wild type and a site-directed mutant of Y181C were determined by HTA. g Relative proportions of the mutant and wild type were measured using an allele-specific real-time PCR assay. h Clinical isolates from patients whose virus had acquired the V82A mutation were studied. i Relative proportions of the mutant and wild type were measured using real-time PCR to detect hisd and PLAP reporter genes. (i) M184V. The replication fitness deficit conferred by the M184V mutation in reverse transcriptase, which causes highlevel resistance to the cytosine analogs lamivudine and emtricitabine, has been the subject of extensive study (reviewed in reference 132). M184V occurs frequently in viral isolates from patients failing lamivudine or emtricitabine therapy (63, 153). M184V has a fitness similar to that of the wild type in most T-cell lines where nucleotide concentrations are high (10) but reduced fitness in primary cells that have limited nucleotide pools, such as PBMCs and macrophages (3, 10, 129). Of note is that there are some more-recent studies that have found reduced fitness of this mutant in cell lines (35, 48). A multitude of biochemical abnormalities have been proposed to explain the reduced fitness conferred by the M184V mutation, including decreased processivity of polymerization that is aggravated by low nucleotide concentrations, decreased initiation of