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Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, California
SUMMARY INTRODUCTION RATIONALE FOR HIV-1 DRUG RESISTANCE TESTING EVOLUTION OF HIV-1 DRUG RESISTANCE IDENTIFYING AND CHARACTERIZING DRUG RESISTANCE MUTATIONS PI RESISTANCE HIV-1 Protease PIs Protease Substrate Cleft Mutations Protease Flap Mutations Protease Mutations at Other Conserved Residues Polymorphic Sites Contributing to Resistance PI Cross-Resistance Patterns Investigational PIs NRTI RESISTANCE HIV-1 RT NRTIs NEMs M184V Mutations at Codons 65, 69, 74, and 75 Multinucleoside Resistance Due to Q151M Other NRTI Resistance Mutations NRTI Cross-Resistance Patterns NNRTI RESISTANCE MUTATIONS NNRTIs NNRTI Mutations between Codons 98 and 108 NNRTI Mutations between Codons 179 and 190 NNRTI Mutations between Codons 225 and 236 Other NNRTI Resistance Mutations NNRTI Mutation Interactions HIV-1 FUSION INHIBITORS TECHNICAL ASPECTS OF HIV-1 GENOTYPIC TESTING IN CLINICAL SETTINGS Source of Virus and Initial Sample Processing Population-Based versus Clonal Sequencing Dideoxynucleotide Sequencing Hybridization Methods Sequence Quality Control Global HIV-1 Isolates GENOTYPIC INTERPRETATION General Principles VirtualPhenotype HIV RT and Protease Sequence Database LIMITATIONS OF HIV-1 DRUG RESISTANCE TESTING Complex Relationship between Drug Resistance and Disease Progression Quasispecies Nature of HIV-1 Cross-Resistance ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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| RATIONALE FOR HIV-1 DRUG RESISTANCE TESTING |
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Genotypic testing has been shown to be clinically useful in four of five prospective randomized studies (19, 47a, 92, 380a; Meynard, J. L., M. Vray, L. Monard-Joubert, S. Matheron, G. Peytavin, F. Clavel, F. Brun-Vezinet, and P. M. Girard, 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, abstract 698, p. 294, 2000); in contrast, phenotypic testing has been shown to be clinically useful in just one of four prospective randomized studies (50a, 139, 256; Melnick, D., J. Rosenthal, M. Cameron, M. Snyder, S. Griffith-Howard, K. Hertogs, W. Verbiest, N. Graham, and S. Pham, Abstract 786, 7th Conference on Retroviruses and Opportunistic Infections, San Francisco, Calif., 2000).
Several reviews on the genetic basis of HIV-1 drug resistance have recently been published (69, 127, 128, 235, 260, 337). This review will focus on how knowledge of the genetic basis of HIV-1 drug resistance can be exploited to test for HIV drug resistance in clinical settings. HIV drug resistance is an interdisciplinary field; important data have been derived from structural biology, biochemistry, virology, and clinical studies. This review will integrate data from these different disciplines that are relevant to the development of new HIV drugs and to the optimal use of those HIV drugs that are already available.
| EVOLUTION OF HIV-1 DRUG RESISTANCE |
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The HIV-1 quasispecies within an individual undergo continuous genetic variation, competition, and selection. Development of drug resistance depends on the size and heterogeneity of the HIV-1 population within an individual, the extent to which virus replication continues during drug therapy, the ease of acquisition of a particular mutation (or set of mutations), and the effect of drug resistance mutations on drug susceptibility and virus fitness. Some mutations selected during drug therapy confer measurable phenotypic resistance by themselves, whereas other mutations increase resistance only when present with other mutations or compensate for the diminished replicative activity that can be associated with drug resistance.
It has been estimated that every possible single point mutation occurs between 104 and 105 times per day in an untreated HIV-1-infected individual and that double mutants also occur commonly (50). It is not known, however, whether multidrug-resistant viruses already exist at low frequencies in untreated persons or if they are generated by residual viral replication during therapy (304). Answers to this question depend on the effective population number of HIV-1 in vivo. Some authors have argued in favor of a high effective population number and a deterministic model of HIV-1 evolution in which chance effects play a small role (313); others have argued in favor of a lower effective population number and a stochastic model of HIV-1 evolution (31, 34, 107).
Resistant virus strains can also be transmitted between individuals. In the United States and Europe, about 10% of new infections are with HIV-1 strains harboring resistance to at least one of three classes of anti-HIV drugs (16, 23, 30, 93, 144, 233, 234, 317, 357, 372, 384, 416; Grant, R. M., F. Hecht, C. Petropoulos, N. Hellmann, M. Warmerdam, N. I. Bandrapalli, T. Gittens, M. Chesney, and J. Kahn, abstract 142, Antivir. Ther. 4[Suppl. 1]:98-99, 1999; Harzic, M., C. Deveau, I. Pellegrin, b. Dubeaux, P. Sageat, N. Ngo, H. Fleury, B. Hoen, D. Sereni, and J. F. Delfraissy, abstract 91, Antivir. Ther. 4[Suppl. 1]:61-62, 1999) (Table 3).
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| IDENTIFYING AND CHARACTERIZING DRUG RESISTANCE MUTATIONS |
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Drug susceptibility testing involves culturing a fixed inoculum of HIV-1 in the presence of serial dilutions of an inhibitory drug. The concentration of drug required to inhibit virus replication by 50% (IC50) or 90% (IC90) is the most commonly used measure of drug susceptibility. Drug susceptibility assays are not designed to determine the amount of drug required to inhibit virus replication in vivo, but rather to compare the drug concentration required to inhibit a fixed inoculum of the isolated virus with the concentration required to inhibit the same inoculum of wild-type virus.
Drug susceptibility results depend on multiple unstandardized factors including the inoculum of virus tested, the cells used for virus replication, and the means of assessing virus replication. Susceptibility testing of NRTIs is further complicated by the fact that NRTIs are triphosphorylated to their active form at different rates in different cell lines. The dynamic susceptibility range between wild-type and the most drug-resistant isolates depends on the drug tested and the susceptibility assay used. It is as low as 10-fold for some drugs and as high as 1,000-fold for others. The dynamic susceptibility range does not necessarily correlate with the potency of a drug; rather it provides a useful context for interpreting an individual susceptibility result. For example, a 10-fold reduction in susceptibility to a drug would be considered high-level resistance if the dynamic susceptibility range for that drug is 10-fold but not if it is 1,000-fold.
The process of identifying drug resistance mutations using virus passage studies and characterizing their impact by testing the susceptibility of site-directed mutants containing the same amino acid changes is highly rigorous but has several limitations. First, the spectrum of mutations developing during in vitro passage experiments is narrower than in isolates from treated patients. This is particularly true for patients receiving combinations of drugs targeting the same enzyme. Second, site-directed mutagenesis studies cannot capture the complicated patterns of mutations observed in clinical isolates and cannot account for the impact of background polymorphisms that may influence the viability and extent of resistance in isolates containing known drug resistance mutations. Finally, clinical data often provide additional insight into which mutations are the most significant in vivo.
To characterize the mutations responsible for drug resistance, it is therefore necessary to also study HIV-1 isolates from patients receiving treatment. Specifically, three additional types of data must be collected: correlations between mutations and drug susceptibility in clinical HIV isolates, correlations between mutations and the drug treatment histories of persons from whom the sequenced isolates have been obtained, and correlations between mutations and the virologic response to a new HIV drug regimen. HIV-1 isolates from persons failing drug therapy are crucial observations of HIV-1 evolution that show which mutations the virus uses to escape from drug suppression in vivo. Such data are particularly important for elucidating the genetic mechanisms of resistance to drugs that are difficult to test in vitro susceptibility tests.
Correlations between genotype and virologic response to a new regimen are essential for demonstrating the clinical significance of drug resistance mutations. Because drug resistance mutations arise in the enzymes targeted by therapy, many of these mutations compromise enzymatic function. Although, the fitness of these variants can be tested in vitro, such fitness tests are not standardized and are unable to detect subtle changes in replication or the likelihood that certain defects in fitness may be readily compensated for by other genetic changes in the virus while other defects may be more crippling. How a mutant virus responds to a new drug regimen in vivo, therefore provides the most meaningful test of virus fitness.
Because insertions and deletions are uncommon in HIV-1 RT and protease, researchers have been able to develop a standardized numbering system for HIV-1 drug resistance mutations. The most commonly used wild type reference sequence is the subtype B consensus sequence. This sequence was originally derived from alignments in the HIV Sequence Database at Los Alamos (205) and can now be found on the HIV RT and Protease Sequence Database (339). The standardized numbering system and reference sequence have led to the development of a shorthand for mutations in which a letter indicating the consensus B wild-type amino acid is followed by the amino acid residue number, followed by a letter indicating the mutation (e.g., T215Y).
So many mutations in both the protease and RT have been associated with drug resistance that it has become customary to label some mutations either primary (or less commonly major) and other mutations secondary (or minor). The term primary is used to indicate mutations that reduce drug susceptibility by themselves whereas the term secondary is used to indicate mutations that reduce drug susceptibility or improve the replicative fitness of isolates with a primary mutation. However, the labels primary and secondary are not strictly defined. For example, some mutations might be considered to be primary for one drug but secondary for another drug. Moreover, secondary mutations commonly arise before primary mutations.
| PI RESISTANCE |
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PI resistance usually develops gradually from the accumulation of multiple primary and secondary mutations. Most primary mutations, by themselves, cause a two- to fivefold reduction in susceptibility to one or more PIs. However, this level of resistance is often insufficient to interfere with the antiviral activity of these drugs. Higher levels of resistance are resulting from the accumulation of additional primary and secondary mutations are often required for clinically significant reductions in drug susceptibility. This requirement for multiple mutations to overcome the activity of PI inhibitors has been referred to as a "genetic barrier" to drug resistance (54, 188, 265).
Sequence analyses of drug resistant isolates has shown that mutations at several of the protease cleavage sites are also selected during treatment with protease inhibitors (60, 87, 180, 187, 237, 240, 420). Growth kinetic studies have shown that cleavage site mutations in some circumstances improve the kinetics of protease enzymes containing drug resistance mutations and that the cleavage site mutations are compensatory rather than primary. Moreover, there have been no reports that changes at cleavage sites alone can cause PI resistance.
Pharmacologic factors influence the clinical efficacy of PIs more than that of the other two classes of HIV drugs. Virologic response is highly correlated with the ratio of the trough drug concentration divided by the inhibitory concentration of the drug (e.g., the IC50 in a standardized assay), a ratio that is commonly referred to as the inhibitory quotient (IQ) (153). Drug levels achieved during PI monotherapy can vary greatly among individuals, often resulting in low IQs. This has led to the practice of administering subtherapeutic doses of ritonavir (a P450 enzyme inhibitor) in combination with other PIs to increase their drug levels—a practice known as PI boosting. Lopinavir is formulated in a fixed combination with ritonavir; and saquinavir, indinavir, and amprenavir are also increasingly likely to be administered with low-dose ritonavir (162). Boosted PIs require higher levels of resistance than PIs given as monotherapy before significant loss of antiviral activity and virologic rebound occur.
I84V has been reported in patients receiving indinavir, ritonavir, saquinavir, and amprenavir 54, 61, 148, 237a, 265, 330) and causes phenotypic 42, 54, 188, 277, 280, 282, 378 387) and clinical (278, 423; Kempf, D., S. Brun, R. Rode, J. Isaacson, M. King, Y. Xu, K. Real, A. Hsu, R. Granneman, Y. Lie, N. Hellmann, B. Bernstein, and E. Sun, abstract 89, Antivir. Ther. 5[Suppl. 3]:70-71, 2000) resistance to each PI. I84V tends to develop in isolates that already have the mutation L90M and is rarely the first major mutation to develop in patients receiving a PI (178).
G48V occurs primarily in patients receiving saquinavir and rarely in patients receiving indinavir. This mutation causes 10-fold resistance to saquinavir and about 3-fold resistance to indinavir, ritonavir, and nelfinavir (149, 172, 282, 409). Isolates with a combination of mutations at codons 48, 54, and 82 have been tested against each of the PIs except lopinavir and found to have high-level resistance to each (277, 343).
D30N occurs solely in patients receiving nelfinavir and confers no in vitro or clinical cross-resistance to the other PIs (243, 282, 409). Cross-resistance to indinavir, ritonavir, and saquinavir has been observed in isolates that have D30N along with mutations at positions 88 and 90 (279).
I50V has been reported only in patients receiving amprenavir as their first PI (237a). In addition to causing reduced amprenavir susceptibility, it has been shown to increased ki values to ritonavir, indinavir, and nelfinavir in biochemical studies (Xu, R., W. Andrews, A. Spaltenstein, D. Danger, W. Dallas, L. Carter, M. Hanlon, L. Wright, and E. Furfine, abstract 54, Antivir. Ther. 6[Suppl. 1]:43, 2001) and to cause in vitro cross-resistance to ritonavir and lopinavir (279, 280, 378). Possibly because of the rarity of this mutation, there have been few reports of multidrug-resistant isolates containing this mutation.
V32I occurs in patients receiving indinavir, ritonavir, and amprenavir. It usually occurs only in association with other PI resistance mutations in the substrate cleft or flap and by itself appears to cause minimal resistance to any one drug. R8K and R8Q are substrate cleft mutation that cause high-level resistance to one of the precursors of ritonavir (A-77003) (124, 152), but they have not been reported with the current PIs.
Mutations at position 46 contribute to resistance to each of the PIs except saquinavir and have been commonly reported during therapy with indinavir, ritonavir, amprenavir, and nelfinavir (55, 237a, 265, 281, 320). Mutations at codon 47 have been reported in patients receiving amprenavir, indinavir, and ritonavir, and often occur in conjunction with the nearby substrate cleft mutation, V32I. F53L has been reported rarely in patients receiving PI monotherapy, but it occurs in more than 10% of patients treated with multiple PIs (178). It has most recently come to attention as one of the mutations associated with phenotypic resistance to lopinavir in multivariate analyses (188).
Mutations at codon 73, particularly G73S, have been reported in 10% of patients receiving indinavir and saquinavir monotherapy and occasionally during nelfinavir monotherapy (178, 334). However, this mutation occurs most commonly in patients failing multiple PIs, usually in conjunction with L90M. Mutations at position 88 (N88D and N88S) commonly occur in patients receiving nelfinavir and occasionally in patients receiving indinavir. By itself, a mutation at this position causes low-level nelfinavir resistance. However, a mutation at this position causes high-level nelfinavir resistance in the presence of D30N or M46I (290, 421). N88S (but not N88D) has been shown to hypersensitize isolates to amprenavir (421), but the clinical significance of this finding is not known. L24I has been reported only in HIV-1 isolates from patients receiving indinavir (55) and has not been shown to confer cross-resistance to other PIs, except possibly lopinavir (188).
Mutations at codons 10, 20, 36, and 71 occur in up to 5 to 10% of untreated persons infected with subtype B viruses. However, in heavily treated patients harboring isolates with multiple mutations in the substrate cleft, flap, or at codon 90, the prevalence of mutations at these positions increases dramatically. Mutations at codon 10 and 71 increase to 60 to 80%, whereas mutations at codons 20 and 36 increase to 30 to 40% (148, 177). Codon 63 is the most polymorphic protease position. In untreated persons about 45% of isolates have 63L (considered the subtype B consensus), about 45% have 63P, and about 10% have other residues at this position. However, the prevalence of amino acids other than L increases to 90% in heavily treated patients (177, 413). Mutations at codons 77 and 93 double in prevalence from 15 to 20% in untreated persons to 30 to 40% in heavily treated persons (177).
In some HIV-1 subtypes, mutations at codons 10, 20, and 36 occur at higher rates than they do in subtype B isolates (58, 114, 293, 331). It has been hypothesized that individuals harboring isolates containing multiple accessory mutations may be at a greater risk of virologic failure during PI therapy (288, 289). However, most studies to date have not supported this hypothesis (3a, 25, 106, 208, 288, 289, 329).
Patients in whom nelfinavir-resistant isolates arise after nelfinavir treatment often respond to a regimen containing a different PI because D30N and N88D/S confer little cross-resistance to other PIs (185, 423). But because as many as 15% of nelfinavir failures may be associated with mutations at codons 46 and/or 90, virologic failure while receiving nelfinavir does not guarantee susceptibility to other PIs (9, 281). Nelfinavir is usually unsuccessful as salvage therapy because most of the mutations that confer resistance to other PIs confer cross-resistance to nelfinavir (148, 227, 399).
In vitro drug susceptibility studies suggest that patients failing other PIs often have isolates that retain susceptibility to amprenavir and saquinavir (300, 321). But neither drug has demonstrated usefulness when administered as salvage therapy without ritonavir boosting (82, 92a, 102). In a study of ritonavir/saquinavir salvage therapy, the number of mutations at positions 46, 48, 54, 82, 84, and 90 predicted the virologic response at 4, 12, and 24 weeks. Patients with three or more of these mutations had no response to salvage (423). Data on salvage therapy with ritonavir-boosted amprenavir are not yet available.
In a study of salvage therapy with a regimen containing lopinavir and efavirenz, the number of mutations at positions 10, 20, 24, 46, 53, 54, 63, 71, 82, 84, and 90 predicted the level of phenotypic resistance and the virologic response after 24 weeks of therapy (188; Kempf, D., S. Brun, R. Rode, J. Isaacson, M. King, Y. Xu, K. Real, A. Hsu, R. Granneman, Y. Lie, N. Hellmann, B. Bernstein, and E. Sun, abstract 89, Antivir. Ther. 5[Suppl. 3]:70-71, 2000). A decreased response to therapy was observed only in those patients that had 
6 of the listed mutations. Subsequent analyses have suggested that mutations at positions 10, 20, 46, 54, and 82 may be more predictive than the others listed (40, 264) and that other mutations, including I50V and G73S may contribute to resistance in different patient cohorts (135, 279). Nonetheless, the large number of mutations required to interfere with a clinical response to therapy demonstrates the high genetic barrier to resistance associated with a drug that achieves high levels in vivo.
In summary, clinical studies have shown that most patients developing virologic failure during treatment with one PI have a diminished virologic response to treatment with a second PI (Table 4). Indeed, most of the successful cases of salvage therapy in patients failing a PI regimen have included regimens with dual PIs or a change to a new PI in combination with an NNRTI (20a, 92a, 294, 423; Kempf, D., S. Brun, R. Rode, J. Isaacson, M. King, Y. Xu, K. Real, A. Hsu, R. Granneman, Y. Lie, N. Hellmann, B. Bernstein, and E. Sun, abstract 89, Antivir. Ther. 5[Suppl. 3]:70-71, 2000). There continues to be great interest in discovering ways to use genotypic data to help switch from one PI to another, although the second PI is increasingly being given as part of a boosted regimen.
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Tipranavir is a nonpeptidomimetic PI with greater flexibility in conforming to enzyme variants with PI resistance mutations (219, 260, 383). However, tipranavir is less potent than other PIs both in vitro and in vivo, and has a narrower dynamic susceptibility range compared with other PIs (14, 219, 316). The narrow dynamic susceptibility range makes it difficult to assess the clinical significance of the decreased cross-resistance between tipranavir and the currently approved PIs. Data are pending on its activity when used in combination with ritonavir.
| NRTI RESISTANCE |
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Most RT inhibitor resistance mutations are in the 5' polymerase coding regions, particularly in the "fingers" and "palm" subdomains (Fig. 2). Structural information for RT is available from X-ray crystallographic studies of RT bound to an NNRTI (198), unliganded RT (309), and RT bound to double-stranded DNA (158, 171). However, only one structure exists that enables visualization of the interaction between the catalytic complex and the incoming deoxynucleoside triphosphate (dNTP) (158). There have been fewer structural determinations of mutant RT enzymes than of mutant protease enzymes (302, 318).
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There are two biochemical mechanisms of NRTI drug resistance. The first mechanism is mediated by mutations that allow the RT enzyme to discriminate against NRTIs during synthesis, thereby preventing their addition to the growing DNA chain (158, 226, 319). The second mechanism is mediated by nucleotide excision mutations (NEMs) that increase the rate of hydrolytic removal of the chain-terminating NRTI and enable continued DNA synthesis (6, 7, 252, 254).
In most drug susceptibility assays, the dynamic susceptibility range is >100-fold for zidovudine and lamivudine and 15- to 20-fold for didanosine, stavudine, zalcitibine, abacavir, and tenofovir (393). Mutant isolates from patients failing therapy with zidovudine, lamivudine, and abacavir usually have measurable phenotypic drug resistance. In contrast, mutant isolates from patients failing therapy with stavudine or didanosine are often found to be drug susceptible in phenotypic assays. Because tenofovir has only recently been approved, there are fewer data on the genotypic correlates of drug resistance and on how well these changes can be detected in phenotypic assays. The difficulty in detecting didanosine resistance is thought to be an artifact of susceptibility testing caused by the inefficient conversion of didanosine to the active compound ddATP when stimulated lymphocytes are used for susceptibility testing (111). The difficulty in detecting stavudine resistance may also be an artifact of the current susceptibility tests that rely on stimulated lymphocytes (230, 253).
In a ddNTP-terminated primer, the presence of the dNTP that would have been incorporated next, had the primer been free for elongation, results in the formation of a stable "dead-end" catalytic complex between RT, primer, template, and dNTP (29, 230, 262, 379). The formation of such a dead-end complex may interfere with the ability of NEMs to facilitate the resumption of virus DNA chain elongation. Biochemical and structural modeling studies have suggested that the bulky azido group of zidovudine may interfere with the formation of a dead-end catalytic complex by sterically preventing the addition of the next dNTP (29, 230). This observation may explain, at least in part, why the NEMs cause the highest levels of phenotypic resistance to zidovudine, despite the fact that biochemical studies have shown that some combinations of NEMs elevate ATP-dependent removal of blocked stavudine-monophosphate (MP) to the same degree as zidovudine-MP (230, 253).
The structural basis underlying the NEMs mechanism of action is not yet understood. Two crystallographic studies have described possibly different roles for the NEMs depending on the particular mutant enzyme studied. One study suggested that positions 215 and 219 give rise to changes that propagate to the active site residues via adjacent residues in the enzyme (302). Whereas the second study suggested that in some mutant structures, T215Y may make direct contact with the dNTP substrate (Stammers, D. K., J. Ren, C. Nichols, P. Chamberlain, L. Douglas, J. Lennerstrand, B. Larder, and D. I. Stuart, abstract 72, Antivir. Ther. 6[Suppl. 1]:54-55, 2001).
During the past few years, several studies have shown that the NEMs are associated with resistance not just to zidovudine, but also to stavudine, abacavir, and to a lesser extent, to didanosine, zalcitibine, and tenofovir (259, 262, 398). The NEMs are selected primarily in patients treated with zidovudine or stavudine alone or in combination with other NRTIs (27, 49, 169, 203, 232, 266, 285, 291, 305, 311, 335, 353). They occur in about 10% of patients treated with didanosine monotherapy (77, 410; Winters, M. A., M. Hughes, S. Lustgarten, and D. A. Katzenstein, abstract 131, Antivir. Ther. 6[Suppl. 1]:96-97, 2001) but do not appear to occur during abacavir monotherapy (261). There are few data on the development of NEMs in patients receiving zalcitibine or tenofovir without other NRTIs.
K70R causes low-level (four- to eightfold) zidovudine resistance and is usually the first drug resistance mutation to develop in patients receiving zidovudine monotherapy (27, 64). T215Y/F results from a two base-pair mutation and causes intermediate (10- to 20-fold) zidovudine resistance. It arises in patients receiving dual NRTI therapy, as well as, in those receiving zidovudine monotherapy (207, 224, 335). T215S/C/D are transitional mutations between wild-type and Y or F that do not cause reduced drug susceptibility but rather indicate the presence of previous selective drug pressure (67, 221, 417). Mutations at positions 70 and 215 are antagonistic in their effect on zidovudine resistance and these two mutations rarely occur together unless additional NEMs are also present (27).
Mutations at positions 41 and 210 usually occur with mutations at position 215 (133, 156, 414). Mutations at positions 67 and 219 may occur with mutations at position 70 or with mutations at position 215. T215Y and K219Q are associated with increased processivity. L210W is strongly associated with M41L and T215F/Y and decreases the susceptibilities of isolates with these mutations by several fold. L210W may stabilize the interaction of 215YF with the dNTP binding pocket (262, 414). In patients failing multiple dual nucleoside therapy it is not unusual for isolates to have four, five, or even all six NEMs.
Clinical studies have shown that the NEMs, particularly mutations at position 215 interfere with the clinical response to zidovudine (203, 303), stavudine (353), abacavir (102, 191, 214), didanosine (155, 173), and most dual NRTI combinations (169, 266; Costagliola, D., D. Descamps, V. Calvez, B. Masquelier, A. Ruffault, F. Telles, J. L. Meynard, and F. Brun-Vizinet, abstract 7, Antivir. Ther. 6:S8, 2001; Mayers, D., T. Merigan, and P. Gilbert, abstract 129, 6th Conference on Retroviruses and Opportunistic Infections, Chicago, Ill., 1999) (Table 5). Complete loss of response to abacavir appears to require the combination of three or more NEMs together with the mutation M184V (214; Costagliola, D., D. Descamps, V. Calvez, B. Masquelier, A. Ruffault, F. Telles, J. L. Meynard, and F. Brun-Vizinet, abstract 7, Antivir. Ther. 6:S8, 2001). The extent to which NEMs interfere with response to tenofovir is not known; however, preliminary data presented to the FDA have shown that tenofovir usually retains antiviral activity even in patients with extensive previous NRTI therapy.
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The NEMs cause minimal lamivudine resistance and do not greatly compromise lamivudine activity (3) except to the extent that they interfere with the synergism between lamivudine and zidovudine and lamivudine and stavudine. One abstract that correlated the presence of NEMs with low-level lamivudine resistance (Skowron, G., J. Whitcomb, M. Wesley, C. Petropoulos, N. Hellmann, M. Holodniy, J. Kolberg, J. Detmer, M. T. Wrin, and K. Frost, abstract 81, Antivir. Ther. 4[Suppl. 1]:55, 1999) relied on a point mutation assay and did not account for other RT mutations which were likely to have explained the results (e.g., codons 44 and 118 [147]).
M184I results from a G to A mutation (ATG to ATA) and usually develops before M184V in patients receiving lamivudine because HIV-1 RT is more prone to G to A mutations than to A to G mutations (ATG to GTA) (174, 189). Although M184I also causes high-level resistance to lamivudine, the enzymatic efficiency of M184I is less than that of M184V and nearly all patients with mutations at this position eventually also develop M184V (107).
M184V alone renders lamivudine ineffective but may not significantly compromise virologic response to treatment with abacavir (145, 179, 391, 399a). However, M184V in combination with multiple zidovudine resistance or in combination with mutations at positions 65, 74, or 115 leads to both in vitro and in vivo abacavir resistance (134, 179, 277, 343; Lanier, R., J. Scott, H. Steel, B. Hetherington, M. Ait-Khaled, G. Pearce, W. Spreen, and S. Lafon, abstract 82, Antivir. Ther. 4[Suppl. 1]:56, 1999). The effect of M184V on the virologic response to didanosine-containing regimens has been less well studied though in one small observational study showed that in heavily treated patients infected with isolates containing multiple NEMs and M184V, a change from lamivudine to didanosine was usually associated with an RNA decrease of 0.5 log10 RNA (315).
Position 184 is in a conserved part of the RT close to the active site. M184V sterically hinders certain NRTIs, particularly lamivudine, while still allowing the enzyme to function (318). The possibility that isolates containing M184V are compromised was suggested by the initial lamivudine monotherapy studies which showed that RNA levels remained about 0.5 log copies below their starting value in patients receiving lamivudine for 6 to 12 months despite the presence of lamivudine-resistant isolates containing M184V (98, 167, 296). Several studies have shown that in vitro RT enzymes with M184V displayed increased fidelity (89, 275, 396) and others decreased processivity (12, 13, 28, 348). The clinical significance of these biochemical studies is not known and the increased fidelity does not appear to limit the ability of HIV to develop new mutations under continued selective drug pressure (176, 190).
M184V reverses T215Y-mediated zidovudine resistance (26, 223, 377); in plaque-forming assays, HIV-1 isolates containing M41L/T215Y displayed 64-fold resistance, while isolates containing M41L/T215Y and M184V were just 4-fold resistant. Resensitization may be due to the ability of M184V to impair the rescue of chain-terminated DNA synthesis (115) and does not appear to apply to zidovudine resistance caused by Q151M (342). This resensitization is probably clinically significant and explains the slow evolution of phenotypic zidovudine resistance in patients receiving zidovudine plus lamivudine (209, 223, 247). Resensitization, however, can be overcome by the presence of four or more zidovudine resistance mutations (343, 377). M184V also appears to reverse the effect of the classical zidovudine mutations on resistance to stavudine and tenofovir, but not abacavir (89, 257, 270, 277).
T69D was initially identified as causing resistance to zalcitibine (104) but substitutions at this position have since been reported after treatment with each of the available NRTIs. In site-directed mutagenesis studies, other mutations at this position including T69N, T69S, and T69A have been shown to confer resistance to zidovudine, didanosine, zalcitibine, and stavudine (404a). It also appears likely that mutations at position 69 may contribute to resistance to each of the NRTIs when they occur together with NEMs (149, 257, 397, 404a).
Insertions at position 69 occur in about 2% of heavily treated HIV-1-infected patients (390). By themselves, these insertions cause low-level resistance to each of the NRTIs, but isolates containing insertions together with T215Y/F and other zidovudine-resistance mutations have high-level resistance to each of the NRTIs (63, 218, 248, 373, 406). Insertions at this position are associated with about 20-fold resistance to tenofovir, which is the highest reported level of resistance to this drug (258). The precise mechanism by which this mutation causes resistance is not known with certainty but one paper suggests that higher levels of resistance occur in the presence of ATP suggesting that this mutation may act in a manner similar to the NEMs by causing ATP-mediated primer unblocking (230). Single amino acid deletions between codons 67 to 70 occur in <1% of heavily treated patients (164 to 166, 405). These deletions contributes to resistance to each of the NRTIs in patients with viruses containing multiple NRTI mutations.
L74V occurs commonly during didanosine and abacavir monotherapy (202, 261, 338, 410) and confers two- to fivefold resistance to didanosine and zalcitibine (368, 410) and two- to threefold resistance to abacavir (376). L74V is sufficient to cause virologic failure in patients receiving didanosine monotherapy (202) but additional mutations may be required to cause virologic failure to abacavir monotherapy. L74V causes hypersensitivity to zidovudine and possibly also to stavudine (368) and is consequently rarely observed in patients receiving dual nucleoside therapy with didanosine/zidovudine or didanosine/stavudine (49, 200, 285, 335, 338). L74V has also been shown to be cause decreased RT processivity in enzymatic studies and decreased replication in cell culture (347, 348).
K65R confers intermediate levels of resistance to didanosine, abacavir, zalcitibine, lamivudine, and tenofovir (120, 121, 259, 261, 290, 359, 367, 376, 397, 419). This mutation has been shown to increase the replication fidelity of HIV-1 RT in vitro and to cause increased enzymatic processivity mediated by a decrease in the rate of template-primer dissociation (5, 344). K65R occurs rarely in vivo (404, 413), and the biological and clinical significance of these biochemical findings are not known.
V75T develops in isolates cultured in the presence of increasing concentrations of stavudine and causes about fivefold resistance to stavudine, didanosine, and zalcitibine (212). Biochemical data and modeling data suggest that mutations at this position cause drug resistance through nucleotide discrimination and possibly also through a non-ATP-mediated mechanism of primer unblocking (230, 327). V75T occurs rarely even in patients receiving stavudine. V75I generally occurs in isolates that also have the multinucleoside resistance mutation, Q151M. The phenotypic effects of other mutations at this position including V75 M/A have not been well-characterized.
G333E is a polymorphism that has been reported in 4 of 70 (6%) untreated persons and 26 of 212 (12%) of persons receiving NRTIs (109). G333E has been reported to facilitate zidovudine resistance in isolates from patients receiving zidovudine and lamivudine who also have multiple NEMs (184). However, dual resistance to these drugs usually emerges without this change (247, 343). There are no data suggesting that this mutation by itself reduces zidovudine susceptibility. Two abstracts have suggested that in some isolates the common polymorphisms R211K and L214F also facilitate dual zidovudine and lamivudine resistance in the presence of mutations at positions 41, 184, and 215 (262, 380). P157A/S is a rare mutation associated with lamivudine resistance. This mutation was first identified in a feline immunodeficiency virus isolate cultured in the presence of lamivudine and has subsequently shown to be associated with high-level lamivudine resistance even in isolates lacking M184V (291, 362, 363).
High-level resistance to both drugs in a dual NRTI combination usually requires multiple NRTI resistance mutations. Two genetic mechanisms of multidrug resistance have received much attention: (i) Q151M usually together with V75I, F77L, and F116Y; and (ii) a double amino acid insertion at position 69 in combination with T215Y/F and other NEMs. These two mutational patterns, however, are responsible for only a minority of multidrug resistant isolates. Multidrug resistance more commonly results from a combination of 4 NEMs, M184V, and 1 to 2 mutations in the ß2-ß3 loop, particularly at position 69.
The extent of cross-resistance between one dual NRTI combination and a second dual NRTI combination is currently being evaluated in clinical trials (360). Preliminary data suggest that patients switching from one dual NRTI combination to a second dual NRTI combination will generally have some response as long as high-level resistance to the first combination has not yet emerged. Because of the high-level of cross-resistance between zidovudine and stavudine, it is unlikely that substituting one drug for the other is likely to be highly effective. There appears to be less clinical cross-resistance between lamivudine and didanosine and a salvage regimen that substitutes one of these drugs for the other is likely to have some activity.
The optimal uses of abacavir and tenofovir, the two most recently approved NRTIs have not yet been defined. In previously untreated patients, abacavir is highly potent, reducing plasma HIV-1 RNA levels by 1.5 log10 copies/ml. Its activity in treated patients, however, is compromised by the fact that a combination of M184V together with 3 NEMs appear to prevent a clinical response to the addition of this drug (214). This would suggest that its main role should be as part of an initial treatment regimen and not for salvage therapy. Preliminary data presented to the FDA from phase III trials in which tenofovir was added to a failing treatment regimen suggest that this drug may be uniquely effective (reducing plasma HIV-1 RNA levels by 0.7 log10 copies/ml) in heavily treated patients harboring viruses resistant to most other NRTIs. The usefulness of tenofovir in salvage therapy should not necessarily preclude a possible role in initial therapy.
| NNRTI RESISTANCE MUTATIONS |
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V106A causes >30-fold resistance to nevirapine, intermediate resistance to delavirdine, and low-level resistance to efavirenz (18, 38, 95, 108, 220, 284, 290, 418). L100I causes intermediate resistance to efavirenz and delavirdine and low-level resistance to nevirapine (37, 38, 108, 290, 403, 418). L100I usually occurs with K103N in patients receiving efavirenz and significantly increases efavirenz resistance in these isolates (11). A98G, K101E, and V108I each cause low-level resistance to each of the NNRTIs (11a, 37, 290, 418).
Y188C/L/H causes high-level resistance to nevirapine and efavirenz and intermediate resistance to delavirdine (38, 108, 290, 418). G190A/S causes high-level resistance to nevirapine and efavirenz but do not cause in vitro resistance to delavirdine (11a, 108, 290). There are no clinical data, however, on the usefulness of delavirdine in patients harboring isolates with these mutations. V179D causes low-level (about twofold) resistance to each of the NNRTIs (38, 195, 403, 418).
