HIV Drug Susceptibility Testing

AIDS and Other Manifestations of HIV Infection Fourth Edition, edited by Gary P. Wormser Copyright © 2004, Elsevier (USA). All rights reserved

Chapter 34

HIV Drug Susceptibility Testing Joseph K. Wong, Davey Smith and Douglas Richman

Resistance of clinical HIV isolates to the first antiretroviral drug, the nucleoside analogue azidothymidine (zidovudine, ZDV), was identified in 1989 soon after its introduction into clinical trials (1–3). These studies utilized standard methods of virus isolation and in vitro assays for susceptibility based on measurement of inhibition of viral replication by varying concentrations of drug. The following year, the genotypic basis for ZDV resistance was defined (4) and opened the field of HIV drug susceptibility testing based on viral genetic sequence determination. Since that time, the introduction of each new antiretroviral agent has been quickly followed by the recognition of resistance. HIV drug resistance is both a cause and an effect of incomplete viral suppression, but it is not the only impediment to effective, long-term antiretroviral therapy. A number of techniques have been developed to diagnose and study HIV drug resistance, each with particular strengths and weaknesses, but the testing strategies that may be most clinically effective still need to be determined. Based on current data: circumstances that merit drug resistance testing include patients who need to switch therapy due to drug failure, patients with primary HIV infection, patients with established infection from regions where the prevalence of primary resistance is known to be high who are about to initiate therapy and pregnant patients starting long term treatment or perinatal prophylaxis. (5,6). The apparent ease with which HIV evades both pharmacologic inhibition and immune control through sequence change is explained by the massive scale of HIV replication (7–9) and by the high mutational rates characteristic of replication of RNA viruses, which lack proof-reading mechanisms (10). These circumstances result in the random generation on a daily basis of viral Joseph K. Wong, Davey Smith and Douglas Richman: Division of Infectious Diseases, UC San Diego School of Medicine and VA San Diego Healthcare System, La Jolla, CA 92130.

variants bearing every single mutation (and many double mutations) along the 9kb HIV genome (11,12). Therefore, the genetically complex population (termed quasispecies) at any given time has a high likelihood of containing viral variants that harbor mutations that can contribute to resistance. Incomplete suppression of viral replication in the presence of antiviral drug(s) results in the selective outgrowth of these variants with reduced susceptibility. For some drugs, single mutations can render mutants fully resistant (13,14), while for other drugs and for combinations of drugs the evolution of high level drug resistance requires iterative events during successive viral generations that result in the accumulation of multiple mutations (15,16). Genetic recombination among viral quasispecies may facilitate the emergence of multi-drug resistant virus (17–19). OVERVIEW OF RESISTANCE TESTING Most clinical resistance testing is now performed on HIV RNA in plasma (or serum). This has the advantage of sampling contemporaneous virus because the HIV population in plasma turns over rapidly (8,9). Additionally, the typically large concentrations of free virus in plasma from untreated patients or from those failing treatment with high level drug resistance increases the likelihood that the sampling will be more representative and less susceptible to sampling artifact (8,20,21). Alternatively, peripheral blood mononuclear cells (PBMC) can be used for virus isolation as well as direct molecular studies. However, this viral compartment turns over relatively slowly and may not provide an accurate picture of the most recent evolutionary events (22,23). Drug resistance can be detected in cell free virion RNA weeks and even months before their appearance in PBMC DNA (8,24–27). In addition, direct molecular assays targeting HIV DNA in PBMC risk over sampling the relatively large numbers of “replication defective” genomes known to exist in PBMC (28,29), which may not accurately reflect the replicating virus pool.

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Two categories of resistance testing are available. Phenotypic assays determine drug susceptibility by directly measuring viral growth rates or biochemical activity of the viral molecular target in the presence of varying concentrations of a particular inhibitor. Genotypic assays assess resistance based on the absence or presence of mutations or combinations of mutations known to confer reduced susceptibility to a particular drug or class of drugs. Some considerations and caveats are generic to all resistance testing methods. Plasma collected for resistance testing should be stored at  70°C to minimize degradation of viral RNA prior to processing. Methods that include polymerase chain reaction (PCR) amplification are very susceptible to contamination because of the exquisite sensitivity of these amplification procedures. Where these methods are used, active measures to prevent and survey for cross contamination should be employed. This includes separate dedicated areas for specimen processing, PCR set up and post PCR processing, the inclusion of negative controls with all assays, and adjunctive measures to limit aerosol formation during processing such as positive displacement or plugged pipet tips. Additionally, when sequences are generated, each new sequence should be compared to known laboratory strains and previously generated sequences for unexpected similarities or differences. Another generic issue is the timing of resistance testing. For patients who undergo resistance testing because of a failing drug regimen, sampling should ideally be performed while the patient is still on treatment or as soon after stopping therapy as possible. Most drug resistance mutations result in selective advantage for the virus only in the presence of drug and stopping treatment will result in their replacement by wildtype, drug susceptible virus. However, this does not indicate that all traces of the drug resistant variants will have been eliminated. On the contrary, resumption of therapy results in the rapid reemergence of drug resistant virus that lingers in latently infected cells (30). This highlights another potential weakness in drug resistance testing as resistance may have developed in response to treatment received in the remote past and is unlikely to be detected with standard assays. An exception may be certain “reversion” mutations that are seen when patients have previously had resistance to ZDV (31). Because of this, drug selection should not be based solely on the most recent drug resistance data but should take into account treatment history and prior resistance test data when available.

GENOTYPIC ASSAYS Cycle Sequencing The most broadly available resistance testing strategy is based on sequencing the viral genes that are targeted by

individual inhibitor drugs. The utility of genotypic testing is dependent on the accuracy and completeness of existing inventories of resistance associated mutations (32,33). Such inventories are constantly updated, but many of the key (major) and secondary resistance associated mutations are well described for most of the available antiretroviral drugs. The identification of individual mutations as possibly conferring drug resistance is made when mutations reproducibly arise during the use of a drug in vivo or when virus is passaged in the presence of the drug in vitro. Establishing the relationship of mutations or groups of mutations conclusively to drug resistance depends on the demonstration that the viral isolate has a reduced susceptibility to the drug in phenotype assays in vitro and that site directed mutation(s) can confer reduced susceptibility. Genetic sequencing for resistance testing begins with isolation of RNA from cell free virus in plasma which is used to generate complementary DNA (cDNA) by in vitro reverse transcription (RT) or by isolation of total DNA from cells, which contain viral DNA (see earlier discussion of comparative relevance of RNA and DNA sequences). Next, the target sequence is amplified by polymerase chain reaction (PCR), a repetitive procedure of annealing, extension and denaturation, which approximately doubles the amount of the sequence target with every cycle. Both the RT and PCR steps require the presence of primers which have high homology to the targeted genes. The amplified products are separated from unincorporated primers and deoxynucleotides, and can be cloned to generate individual clonal sequences that can be individually sequenced or, more typically, the entire mixture of products can be sequenced as a “population”. The most common sequencing methods are based on the Sanger chain termination strategy (34). Denatured amplicons are annealed to sequencing primers in the presence of one or more DNA dependent DNA polymerases and both unlabelled dNTP and individual 2,3-dideoxy, chain terminating nucleotides. The iterative steps of annealing, extention and denaturation results in generation of chain terminated products of varying lengths in process known as “cycle sequencing.” The use of fluorescently (“dye”) labeled dideoxy chain terminators (or in some cases of dye labeled primers) and automated genetic analyzers has greatly simplified this method of nucleotide sequencing (Fig. 34.1). The labeled single stranded DNA fragments are separated by electrophoresis and the individual fragments detected as they migrate past a laser source. The raw data from “automated sequencing” are interpreted by base calling software. A chromatogram consisting of a series of peaks representing the pattern of individual fluorescently labeled fragments is generated (Fig. 34.2a). This approach is the basis of both commercial genotyping kits (TrueGene™, Bayer Diagnotics; Viroseq™, Celera Diagnostics) as well as many “in-house” genotypic assays. Because HIV exists in vivo as a complex genetic mix, more than one base may be present at each position when

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FIG. 34.1. Overview of drug resistance genotyping. RNA or DNA is extracted from a clinical sample and undergoes PCR amplification with or without reverse transcription (not shown). The amplified target to be sequenced is mixed with conserved sequencing primers and deoxy-nucleotide triphosphate as well as dideoxy nucleotide triphosphate that carry a different fluorescent label for each of the 4 bases (Top left). Following multiple cycles of primer annealing, extension and denaturation, fragments of different length are generated, each labeled by one of the four fluorescent dyes corresponding to the last incorporation of the “chain terminating” base. The products are resolved electrophoretically and analyzed by fluorescence emission following laser excitation in an automated instrument (Bottom left). The emission pattern is translated by software into a base sequence which is then compared to a prototype “wildtype” sequence. Typically, a manual review of the virtual representation of the electropherogram (also called chromatogram) is performed and any necessary manual alignments and edits are made before a final report is generated (Top right).

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FIG. 34.2. Electropherogram demonstrating pure or mixed sequences at a single position. (A) arrow denotes 1st base position of codon 184 of the HIV-1 RT where 100% of the composition is Adenine. (B) 75% Adenine, 25% Guanine. (C) 50% Adenine, 50% Guanine.

used to fix arrays of probe oligonucleotides based on wildtype and mutant HIV pol sequences to a silicon wafer (37–39). These oligonucleotides include various permutations of wildtype and mutant codons together with polymorphisms in the flanking sequences. The arrays are designed with many redundant probes for each position interrogated. RT PCR is performed to generate complementary DNA (c-DNA) templates, and the inclusion of RNA polymerase promoter sequences in the amplification primers permits the generation of complementary RNA (cRNA). These labeled c-RNAs are partially fragmented to generate short oligomers of varying lengths and hybridized to the “GeneChip” (39). These arrays contain many more probes than can be tested using conventional southern blotting or even the line probe approach. In general, the agreement of this method of resistance testing with cycle sequencing approaches is high ( > 96% concordance); however, even this method may not be able to detect novel (previously unrecognized) resistance mutations or clusters of mutations and sequence regions with extensive polymorphisms. An example of this limitation are the recently identified insertion mutants between codon 69 and 70 (40,41).

Oligonucleotide Hybridization

bulk sequencing is performed. This is observed as multiple peaks at a single location on the chromatogram (Fig. 34.2b and 34.2c). The threshold for identifying a base position as mutant is set with the base-calling software of the sequencing system (typically > 20–30%). Most sequencing protocols involve sequencing both strands of DNA and comparing these results for consistency. In many sequencing laboratories, some manual inspection and editing is also done. In order to screen for sequences that may be the result of contamination by exogenous sequences during PCR amplification, many laboratories perform additional quality assurance procedures by comparing every newly generated sample sequence with laboratory strain HIV sequences and sequences generated from other patient samples by phylogenetic reconstruction and BLAST searching against sequence databases (35,36). The sample sequence is then compared with a reference “wild-type” sequence to generate a summary of mutations which can be annotated with information on whether the position is associated with resistance to one or more drugs. (Fig. 34.3).

An alternatives to long range sequencing are hybridization based assays that inspect only select positions known to confer drug resistance. These assays detect the presence or absence of mutations in the viral genome by the use of individual oligonucleotides that are either complementary to the wildtype (drug sensitive) or the mutant (drug resistant) sequence. The oligonucleotides are used to directly probe for the presence of mutant or wildtype sequences by hybridization under stringent conditions (temperature and salt conditions that prevent annealing unless the probe and template are a perfect match) or to prime ligation or extention reactions that are dependent on annealing of the oligonucleotide to the viral target “template”. Hybridization assays function best to detect well characterized and predictable mutation patterns, but they may yield an indeterminate result when there are flanking sequence polymorphisms around the interrogated site which affect probe or primer binding. These assays are generally easier to perform with less instrumentation than required for the long range sequencing methods, but they are not designed for discovery of new resistance associated mutations.

Hybridization Sequencing: GeneChip

Line Probe Assay (LiPA)

Sequencing the entire length of HIV protease and the relevant 5 portion of the HIV RT can be performed by large scale hybridization to gene arrays. In one such approach, phosphoramidite chemistry and lithography are

The line probe assay (LiPA, Innogenetics) uses oligonucleotide probes that are fixed onto membrane strips (42). Biotinylated primers are used in RT-PCR amplification on RNA extracted from plasma and the amplified products are

FIG. 34.3. Example of genotypic drug resistance report on patient sample. Drugs corresponding to FDA licensed agents as of February 2003 are shown grouped according to drug class. Mutations at codons predicted to confer resistance to individual drugs are shown individually. Here, mutations conferring high level resistance are marked in red, mutations which alone confer low level resistance are in yellow, mutations which contribute to resistance in the presence of other mutations are in blue and mutations that increase susceptibility to a drug are shown as bracketed and in green. Note that a single mutation can confer resistance to multiple drugs or confer resistance to some drugs while simultaneously sensitizing virus to other drugs (for example M184V). (Reproduced with permission.)

888 Chapter 34 allowed to anneal to the probe strips, washed and treated with streptavidin-alkaline phosphatase conjugate and nitroblue tetrazolium substrate to detect hybridization. Like other hybridization based assays, the applicability of the system depends on well defined resistance mutations hence only known resistance mutations can be detected. In addition, binding of the amplified products to the probe strips is also affected by sequences flanking the codon of interest therefore highly polymorphic regions can be problematic (43). Where high-throughput examination of one or a few resistance codons in well-conserved regions is the principal goal, the time savings with LiPA is appealing. An important feature of the LiPA system is its relative independence from sophisticated instrumentation following the PCR step. Thus, this system may have advantages when a fully equipped molecular diagnostic laboratory is unavailable. The LiPA also appears capable of detecting minor resistant mutant populations that make up as little as 5–10% of the total population. In addition to HIV resistance testing, it has been adapted for hepatitis B resistance testing and hepatitis C genotyping (44,45).

Primer Extension and Primer Ligase Assays The ability of primers, that match either wild-type or resistant sequence, to anneal and prime a PCR amplification reaction has also been used for mutation detection either with conventional detection of product following electrophoresis (46,47) or for use in real-time PCR assays (48,49). These latter assays can be exquisitely sensitive and can be used to detect minority populations < 0.1%. However, each assay is directed at a single codon and not all positions and mutational patterns may be amenable to this approach because of polymorphisms flanking the positions of interest. These assays are currently limited to research applications. Other assays have been described that use primers whose 3 ends terminate at the site of the position to be studied. This is the basis of both the Point-mutation Assay (50) and the Oligo-ligase assay (51). In the former, RTPCR generated template are tested in duplicate polymerase extension assays with either a wild-type or mutant primer and labeled dNTPs. The incorporation of label (originally described with radioactive labeling) in each reaction is proportional to the match between templates to each primer. Minority populations as low as 10% can be detected with this assay but only assays for NRTIs have been described. In the Oligo-ligase assay, mutant is distinguished from wildtype sequence by the ability of respective primers that are differentially modified to anneal to template and become ligated to a third common oligonucleotide (51,52). This system has been used to study NRTI, NNRTI and PI resistance. Sensitivity for detecting minority species approaches 5% which is important because a resistant variant may exist as a minority species, especially when certain drug pressure is

not present, but then become the major variant when this drug pressure is reinstated.

Interpretation of Resistance Data Mutations associated with HIV drug resistance have been classified as primary or secondary. Typical characteristics of primary resistance associated mutations include their ability to alter susceptibility of the virus to the antiviral compound, their early appearance and their relative specificity for one or a class of drugs. Secondary mutations typically accumulate in virus that has already developed primary mutations. By definition, secondary mutations confer little or no reduction in drug susceptibility. However, secondary mutations can increase the level of resistance of virus that already possess primary resistance associated mutations and can also restore or augment the replication capacity of such virus. Secondary mutations that have this latter effect are also called compensatory mutations. The identification of a mutation as important for drug resistance is usually supported by one or more lines of evidence. First, before drugs enter human trials, cell culture passage of virus in the presence of escalating concentrations of drugs can select for mutations in vitro that can be detected by sequencing. Virus isolates can then be tested in growth assays (as described in the section Phenotypic Assays) for their drug susceptibility in comparison with the parental virus. Further confirmation that particular mutations confer the resistance phenotype can be obtained by creating the individual or groups of mutations in molecular viral clone and testing these “site directed mutants.” Second, virus can be recovered from patients on a particular drug and these viruses can be similarly tested. The resistance mutations selected in vivo do not always correlate with resistance patterns selected in vitro. Finally, some mutations may be consistently observed in the setting of treatment failure with or without the simultaneous development of known drug resistance conferring mutations. The secondary role that such mutations play in enhancing resistance or in restoring replication fitness can be established in some but not all cases. While a comprehensive inventory of all primary and secondary RAM is beyond the scope of this chapter, a summary of important resistance associated mutations is provided in Figs. 34.4a–c and a discussion of mechanisms of resistance relating to some key mutations concludes this chapter. Comprehensive, updated databases describing known drug resistance genotypic patterns are accessible on the Web at http://resdb.lanl.gov/Resist_DB and http:/ /hivdb.stanford.edu. Additional information can be obtained through the International AIDS Society (http:/ /www.ias.se/). The standard nomenclature to describe mutations is to use the single letter abbreviation for the wild-type or reference strain amino acid, according to the International

HIV Drug Susceptibility Testing 889 Union of Pure and Applied Chemistry (IUPAC) followed by the location of the codon in question followed by the single letter amino acid abbreviation of the new amino acid of the mutant. For example, K103N represents a mutation at the 103rd codon of the RT gene from a Lysine (K) to an Asparagine (N). It is commonly associated with high-grade resistance to NNRTIs (53).

PHENOTYPIC ASSAYS Inhibition of Virus Replication in PBMC and Cell Lines A consensus PBMC assay has been described that assesses the phenotypic drug susceptibility of clinical

FIG. 34.4. Summary of mutations conferring resistance to NRTI, NNRTI and Protease inhibitors according to drug. The wildtype amino acid is shown above each relevant codon position while resistance conferring mutations are shown below. The single letter amino acid codes are according to the IUPAC convention: Alanine, A; Cysteine, C; Aspartate, D; Glutamate, E; Phenelalanine, F; Glycine, G; Histidine, H; Isoleucine, I; Lysine, K; Leucine, L; Methionine, M; Asparagine, N; Proline, P; Glutamine, Q; Arginine, R; Serine, S; Threonine, T; Valine, V; Tryptophan, W; Tyrosine, Y.

FIG. 34.4. Continued.

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FIG. 34.4. Continued.

isolates of HIV to AZT by determining the drug concentration necessary to inhibit virus replication in vitro (54). The assay uses virus isolated from co-cultivation of stimulated patient PBMC with sero-negative donor PBMC. The titered virus is then used to infect seronegative donor cells in the presence of varying concentrations of drug. The concentration of drug that reduces the amount of viral p24 production is called the inhibitory concentration 50 or IC50. The fold change in drug concentration for the sample virus to a “wildtype” drug sensitive control virus provides a measure of susceptibility. Adaptations of this assay have been used to assess the susceptibility to other antiretroviral drugs as well (55–60). Because of the requirement for virus isolation and titration of the viral stock prior to performance of the actual drug susceptibility tests, this approach is too time consuming and labor intensive for routine clinical monitoring. Furthermore, there are concerns that the process of virus isolation itself can alter the makeup of the viral population and that virus recovered from PBMC may overly represent archival viral forms and therefore not reflect the contemporaneous replicating viral population in plasma (25,61). However, some data suggest that this may not be major problem in practice (53). A related assay utilizes HeLa cells, which have been engineered to express CD4, to test drug susceptibility (1,62). It scores the amount of HIV infection of HeLa cells by counting syncitial plaques formed by infected cells. Like the PBMC assay, a titered viral isolate must be prepared. However, the readout for infection is relatively simple and less expensive than quantifying p24 production. Because HeLa cells are more homogeneous than

donor PBMC, the reproducibility of the assay tends to be better. However, this assay can only be performed with syncitia inducing virus (i.e. those that use the CXCR4 coreceptor) and hence has limited utility for primary virus isolates, which more often do not induce syncitia. Recombinant Virus Assays The large time requirement of conventional phenotypic drug susceptibility testing has led to the development of “recombinant-virus” assays that measure in vitro phenotypic susceptibility but can be performed rapidly enough to have clinical utility (63–67). These assays utilize virus chimeras that are created from the target gene(s) of interest from clinical specimens using molecular techniques. These assays have advantages over conventional phenotypic assays since they do not require time consuming viral isolation and they minimize the potential for alteration of viral characteristics during in vitro cell culturing that can occur during virus isolation (25,61). Several commercial assays have been developed using this strategy. In one recombinant virus assay, the Phenosense™ (Virologic, South San Francisco) the HIV gene(s) of interest (in current application, the HIV protease and first 340 bases of the RT) are cloned into a vector which encodes an indicator gene, the firefly enzyme, luciferase, under the transcriptional control of the HIV-1 LTR. This chimeric construct and a separate vector that provides the murine leukemia virus envelope (a-MLV) are co-transfected into a cell line, which permits production of

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FIG. 34.5. Phenosense™ Phenotypic resistance testing. (A) Schematic of procedures used in one recombinant virus phenotypic resistance assay. Vector expressing the pol gene from the clinical isolate and a second vector lacking pol but containing components for virion encapsidation, envelope and the luciferace gene are introduced into a producer cell line with or without protease inhibitors at varying concentrations, permitting production of virions capable of only a single round of infection. These virions are then used to infect a target cell line with or without reverse transcriptase inhibitors at varying concentrations and luciferase activity is observed.

pseudotyped virions (Figs. 34.5a–c). These virions are either produced in the presence of drugs (protease inhibitors) or are exposed to inhibitors during the infection step (reverse transcriptase inhibitors). Successful infection of a second cell line is quantitatively assayed by detection of luciferase activity in cell lysates. These are compared with no-drug controls and are used to calculate drug concentrations where 50% or 90% of virus replication is inhibited, the inhibitory concentration50 (I.C.50) or inhibitory concentration90 (I.C.95). Comparisons with values for wildtype virus are used for calculation of a fold change over wildtype. (Figs. 34.6a and b showing readout from Phenosense assay and inhibition curves) (67). The two other commercial phenotypic assays, the Antivirogram™ (Tibotec-Virco, Belgium) (63,65,68) and the Phenoscript™ (Bioalliance, Paris) (68,69) differ from the Phenosense™ in that they use homologous recombination rather than cloning to construct the recombinant viruses. cDNA corresponding to the viral protease and first 3 to 400 codons of RT derived from the patient sample is cotransfected into a cell line along with a vector containing a laboratory clone of HIV missing the corresponding region in the HIV pol. A complete viral genome is reconstituted by homologous recombination within the target cell by the cellular machinery and results in virus production. The virus is harvested and used in assays of infectivity and virus growth at varying concentrations of drugs. Calculation of I.C.50 and I.C.95 and fold-change in drug susceptibility are similarly calculated (Fig. 34.6). Interpretation of these phenotypic assays depends on three assay characteristics: assay reliability, biologic

FIG. 34.5. Continued. (B) Inhibition curves for patient virus without evidence of resistance to a test drug overlies curve for reference virus (upper graph) while the displaced inhibition curve for resistant test virus is shifted to the right of the reference (lower graph).

measures and clinical measures. Cutoffs for reliability are determined by observations on assay to assay variability (i.e. consistency of replicate assays on the same sample). Biologic cutoffs are based on the range of results on samples from patients not receiving therapy. Clinical cutoffs are based on relationship between change in I.C.50 for a drug and the likelihood of a clinical response. This last and arguably most important cutoff has been determined for some but not all drugs. The automation of these assays now permits their rapid and reproducible performance and simultaneous testing against all 16 of the licensed antiretroviral agents available in the U.S. Such studies using conventional approaches for phenotypic testing would be hard to perform in a timely manner. However, commercial phenotypic assays are expensive compared to genotypic assays with costs ranging from three to four times more. It remains to be determined when phenotypic testing carries sufficient advantages over genotyping to be indicated. Scenarios where phenotypic testing may provide potentially useful

FIG. 34.5. Continued. (C) Inhibition curves with various antiretrovirals for a multi-drug resistant virus. Note with 3TC, no inhibition is noted even with the highest drug concentrations tested. Broken line, reference; solid line, patient.

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FIG. 34.6. Examples of two phenotypic resistance reports: Phenosense™ (Virologic) and Antivirogram™ (Virco).

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FIG. 34.6. Continued.

HIV Drug Susceptibility Testing 895 information for clinical management include cases of highly experienced patients whose viral genotypes indicate no available therapeutic alternatives. In such cases, phenotypic testing with precise endpoints may be able to distinguish drugs with little or no effect from drugs that retain some activity. Furthermore, subtle increases in I.C.50 for some drugs such as didanosine, stavudine and possibly tenofovir appear to correlate with loss of in vivo inhibitory activity even when genotypic resistance is not apparent (70,71). In other cases, mutations to one drug may enhance susceptibility to another rendering genotypically resistant virus phenotypically susceptible. Finally, phenotypic assays can indicate loss of susceptibility to drugs due to novel mutations or combinations of resistance mutations not yet characterized.

Biochemical Assays Other phenotypic assays, which do not depend on measures of viral infection, assess the biochemical activity of RT in the presence of varying concentrations of inhibitor (72–74). In the system described by Heinene and colleagues, HIV RT present in virions from patient plasma are used to reverse transcribe a heterologous RNA template belonging to the encephalomyocarditis virus (ECMV) in the presence of varying concentrations of RT inhibitors (73). Like genotypic assays and recombinant phenotype assays, the biochemical assays have the advantage that virus isolation is unnecessary, saving time. This assay has an additional advantage in that minor populations of resistant virus in frequencies as low as 10% can be detected. Furthermore, the assay is independent of sequence type so it is theoretically adaptable to divergent viral forms such as non-B subtype and O group viruses. However, there are a limited number of drugs for which this test has been validated (Nevirapine and 3TC) and some drugs for which this approach does not work (ZDV). Finally, the levels of viremia required are typically higher than for assays that include a genetic amplification step.

Virtual Phenotype The difficulties with interpretation of genotypic data and the cost and time requirement of many phenotypic assays have led to the examination of large databases of simultaneous genotypes and phenotypes to generate algorithms for predicting phenotype from genetic sequence (Vircogen Virtual Phenotype; Virco) (75,76). In a study of clinical specimens, virtual phenotype and actual phenotype have been found to be in agreement in most but not all cases (77). Prospective comparisons between virtual and actual phenotype are needed to determine clinical utility. Further refinement of the prediction algorithms may be possible by the accumulation of additional “learning” data. Finally, the updating of databases with actual phenotypes

for virus with new genotypic resistance patterns developing in response to new and new combinations of antiretroviral drugs will need to be continued.

ROLE OF RESISTANCE TESTING IN CLINICAL PRACTICE Predictive Value and Clinical Utility Support for resistance testing in clinical practice is mounting although precise guidelines for which assays to use and how best to use them are not yet available. Abundant retrospective data clearly show the predictive value of the presence of individual and categories of resistance mutations to NRTI (78–85), NNRTI (83,86–88) and PI (76,85,89) on treatment responses and outcomes. These observations suggest the utility of resistance testing in choosing antiretroviral drugs when switching therapy. Evidence from prospective studies is less clear. Some but not all prospective studies performed to date provide support for the short term benefits of resistance testing. The VIRADAPT study randomly assigned 108 treatment experienced patients either to receive treatment based on genotypic resistance testing or to standard of care (90). By 12 weeks, mean change in plasma RNA was significantly greater in the genotyping arm (–1.04log vs. –0.46log) as was the proportion of patients achieving viral loads < 200 copies/ml (29% vs. 14%). Similar results were maintained to the conclusion of the study at six months. In the CPCRA 046 study, genotypic testing along with expert interpretation and recommendations was compared to standard of care in 153 patients failing dual NRTI plus PI therapy (91). Reduction of plasma RNA was greater in the genotype arm than in the standard of care arm (–1.19 log vs. –0.61 log) and the proportion with plasma VL < 500 copies/ml was also greater (55% vs. 25%) (at week 8). However, the difference was no longer statistically significant at week 12 (34% vs. 22%). Similar results studying phenotypic assays have been reported. In a study of 273 ARV experienced patients randomized to treatment based on a recombinant phenotypic assay (Antivirogram, Virco) or to standard of care, viral load decrease (–1.23 log vs. –0.89 log) and proportion of patients with undetectable viral load ( < 400 copies/ml) (59% vs. 42%) were significantly better with phenotyping at week 16 (92). However, another study using the same assay reported benefits at week 4 that were lost by week 16 (93). Meynard and colleagues found no overall benefit to either phenotypic or genotypic testing in a study of 541 patients randomized to standard of care, genotyping or phenotyping (in-house assay) at week 12 but in a subset of patients experiencing failure on their first PI regimen, genotyping appeared superior (94). In another study using the Phenosense™ assay, Haubrich and colleagues noted that baseline phenotype was indeed strongly predictive of outcomes however, at months 6 and 12, viral

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suppression was similar for those randomized to phenotype and those randomized to standard of care (95). These investigators stressed the need to refine the cutoff values for some drugs such as stavudine and didanosine. Several important conclusions can be drawn from these prospective studies. First is the need for expert interpretation of resistance data for optimal benefit from resistance testing. In the one study where expert interpretation of resistance data and recommendations were not provided to clinicians, neither genotype nor phenotype provided convincing benefit (94). Computerized algorthims have also been advocated for the interpretation of resistance data, but it remains to be seen how these compare with results based on the recommendations of experienced clinicians and virologists. Another factor that appears to affect the utility of resistance testing is the range of treatment options available to patients failing therapy. Patients with many treatment options based on treatment history and those with no options may receive less benefit from resistance testing than would patients who are in between these extremes (6,94,95). The development of appropriate clinical cutoffs for each drug should also enhance the benefits from resistance testing.

IAS Consensus Recommendations The International AIDS Society expert panel has recently recommended the use of resistance testing to help guide the selection of new drugs following treatment failure and for the selection of drugs for HIV infected, pregnant women. The panel also recommends consideration of resistance testing of drug naïve chronically infected patients prior to initiation of therapy in areas where the prevalence of drug resistance is high. Finally, testing is strongly advocated in patients with primary HIV-1 infection who are about to undergo treatment because of the increasing incidence of primary drug resistance (96).

Viral Resistance in Tissues Drug resistance testing of virus in blood is currently the only commonly employed clinical strategy. Because the vast majority of virus in blood arises from active viral replication and production lymphoid tissue, it is not surprising that drug susceptibility of virus in blood and lymphoid tissues correlate quite well (97,98). However, virus in other anatomic compartments such as the CNS and genital tract has been shown in small studies to comprise distinct genetic sub-populations (99–102). In the absence of treatment, this may reflect the selective effects of differences in target cell availability and host immune response (reviewed in Blankson) (103). With therapy, variation in drug penetration into different anatomical sites may result in suboptimal drug levels, promoting the evolution of resistance (104), or when no significant drug

penetration occurs, give rise to sanctuary sites that permit the replication of drug susceptible virus (25,105–110). The discordance of resistance genotypes between virus in plasma and CSF has been noted in two recent studies (106 107). The clinical significance of these observations has not been determined.

EPIDEMIOLOGY OF DRUG RESISTANCE Through analysis of samples derived from clinical trials and population-based surveys of resistance in treated patients, the risks of emergence of resistance in those receiving specific drug combinations have been well documented. The largest such database to date has been presented by the Virco group, who assessed the prevalence of specific mutations and phenotypic resistance in over 11,000 samples submitted for routine clinical testing in the U.S. (111). These data demonstrate that less than 25% of patients had a wild type phenotype and over 20% had reduced susceptibility to drugs within all three classes of currently available antiretroviral drugs. In nearly 50% of patients, plasma virus carrying M184V in reverse transcriptase could be detected, representing high level resistance to lamivudine, and a large proportion also had the zidovudine resistance-associated mutations at positions 215, 70, 67 and 41 of reverse transcriptase (Fig. 34.7). Multinucleoside resistance mutations (Q151M and 69 insertions) were only rarely detected, although this may rise as patients become more treatment-experienced. Of note, around 30% of patients also had at least one of the nonnucleoside analog and/or one of the key protease inhibitor resistance mutations. In the U.K., the Public Health Laboratory Service started monitoring the prevalence of HIV drug resistance in 1998, recruiting patients on a random basis, who were receiving antiretroviral drugs with a viral load > 5,000 copies/mL. Results from the first year identified nearly 50% of 100 patients with resistance to drugs within at least one class of drugs, and approximately 20% having demonstrable resistance to drugs within all three classes. The most common mutations were associated with reduced susceptibility to the nucleoside analogs. These results, from a much smaller data set, are remarkably similar to those from the U.S. discussed above, and demonstrate the problems faced in choosing effective antiretroviral therapies for patients failing treatment. There is also increasing concern about the possible transmission of drug resistant HIV, so called “primary drug resistance” (112–115). In many developed countries, the incidence of HIV/AIDS related mortality fell dramatically since the mid-1990s, coincident with the introduction of HAART while the number of new HIV diagnoses has remained constant. It is therefore unsurprising that some of these individuals have been infected with resistant virus, with prevalences of 5–20% being reported from Europe, Australia, and the U.S. (112–116).

HIV Drug Susceptibility Testing 897 MECHANSIMS OF ANTIRETROVIRAL RESISTANCE Antiretroviral resistance develops as viral replication is allowed to continue in the presence of drug selective pressure. For some agents (such as lamivudine and the non-nucleoside agents), a single point mutation induces high-grade phenotypic resistance in a predictable manner. For others (such as zidovudine, abacavir and most of the protease inhibitors), high-grade phenotypic resistance requires the serial accumulation of multiple mutations, and is thus much slower to emerge. A final group of drugs (including didanosine and stavudine) is only associated with low-grade phenotypic resistance, despite the presence of one or more key mutations. Although it was originally thought that this could be associated with persistent in vivo efficacy of these agents, clinical trial data now show that this is not the case, and the cut-off for phenotypic resistance to didanosine and stavudine has been lowered to reflect this.

Despite the fact that many antiretroviral agents are available for use, the phenomenon of cross-resistance among drugs in a same class limits the options for second and third-line therapy. For protease inhibitors, it appears that a primary resistance mutation develops, which leads to an increase in drug resistance at a cost of decreased viral replicative capacity or “fitness”. As an example, isolates carrying the D30N or M46I mutation in the protease gene may be as much as 20% less fit. The majority of isolates resistant to PIs demonstrate alterations in substrate specificity, which in theory, could be exploited to design novel protease inhibitors (117) that would retain activity against these less fit isolates. However, the subsequent accumulation of secondary mutations (such as at codons 46 and 63) may restore fitness and allow the strain to replicate and persist. Recent data show that these strains (with multiple primary and secondary mutations in the protease and reverse transcriptase genes) can be transmitted, and persist for many years in the absence of drug pressure. It is likely that this phenomenon is not fully

FIG. 34.7. Frequency of NRTI resistance associated mutations in the USA among patients on drug treatment range from > 40% with codon 184 mutation conferring resistance to 3TC (lamivudine) to < 1% for mutations at codon 75 conferring multinucleoside resistance. (From ref. 111, Bloor et al.)

898 Chapter 34 explained by changes in the target genes themselves, as interesting experiments have shown that if multi-resistant protease genes are cloned into recombinant backgrounds, they appear much less fit than when wild-type protease genes are cloned into recombinant backgrounds (118). However, the fitness of resistant viruses may not parallel that of wild-type isolates, as the persistence of this resistance tended to be associated with more favorable virologic and immunologic set points (30). The sequential use of PIs is based on the fact that a number of agents in this class have unique primary resistance mutations. This is particularly true of nelfinavir and amprenavir. Indeed, the I50V amprenavir resistance mutation alters the hydrophobic interaction with the substrate without altering the binding of other drugs in this class (119). This advantage may be lost if the isolates are allowed to continue to replicate under drug pressure, and develop secondary mutations. These would, at the very least, hasten the development of the primary mutations to other agents in the class. One agent that seems to retain activity in this setting is lopinavir/ritonavir, due to the high circulating drug concentrations that are achieved. It may thus retain activity in a situation where other protease inhibitors are no longer effective. Finally, protease cleavage site mutations may be more important than previously appreciated. In one study of drug naïve patients such mutations were present in 77/473 individuals and were associated with a poorer therapeutic response to protease inhibitor-based HAART (120). Although most of the mutations associated with NRTI resistance are not at the active site of the enzyme, they do lead to conformational changes that propagate to the active site (121). The fact that the mutations occur in two separate domains leads to two specific mechanisms for resistance: decreased substrate binding and increases pyrophosphorolysis, leading to a net decrease in chain termination (122). Resistance to another agent, phospohonoformic acid, reduces pyrophosphorolysis, and partially restores zidovudine susceptibility of resistant isolates, confirming the importance of both mechanisms in highgrade zidovudine resistance. For multi-drug resistance three patterns have been identified. Two of these are associated with an inability of the reverse transcriptase enzyme to incorporate ATP and the third (the Q151M complex) leads to substrate-independent inhibition of enzyme function (123). Further, the insertion of a dipeptide between codons 69 and 70 has been shown to enhance the removal of the terminal nucleotide and enhance extension of the unblocked primer (124). These data provide a biochemical explanation as to why the latter pattern is most readily associated with multi-drug resistance within the class. It is worrisome that these isolates may be more fit than corresponding wild type strains in vivo. This is also true of certain strains carrying other multi-drug resistant mutations in the reverse transcriptase genes, suggesting the need to intervene in the course of therapy before such progressive genetic changes occur. A

new nucleotide agent, tenofovir, has preserved activity against many isolates carrying multi-nucleoside resistance mutations, but seems less active against those carrying K65R, the T69S insertion or four or more resistance associated mutations (79). The small size of the molecule and its ability to interact with its substrate in multiple conformations likely explain its retained activity against most resistant isolates (125). The K103N mutation confers resistance to all currently available NNRTIs, presumably by stabilizing the closed pocket form of the enzyme, thus inhibiting the binding of the drug to its substrate (126). The fact that all agents in this class bind to the same area explains the broad pattern of cross-resistance, and should prompt the development of new agents that would interact with this side chain more favorably. Although mutations at codons 181 and 188 confer high-grade resistance to certain drugs in this class such as nevirapine, this is not generalized to newer drugs such as efavirenz, which is a more compact molecule, and these changes do not alter the binding to its substrate (127). Therefore, cross-resistance across this class is not absolute. In practice, as many as 20% or more of patients developing nevirapine resistance will still have isolates that are sensitive to efavirenz (53,128). Although it may be that subsequent exposure to efavirenz will lead to a more rapid development of resistance, than if the baseline isolate were wild-type, which limits the possibility of sequencing of drugs within this class. Certain mutations may confer resistance to one drug and enhanced susceptibility to another, such as M184V (129,130) and L74V (131), associated with resistance to lamivudine and didanosine respectively, which leads to enhanced sensitivity to zidovudine. M184V may also restore susceptibility to stavudine and adefovir (132,133). Isolates carrying these single changes have been consistently shown to be less fit. It has been postulated that isolates with M184V mutations have increased fidelity, thereby decreasing their ability to accumulate new resistance mutations (134). They may also lead to a decrease in pyrophosphorolysis, further enhancing zidovudine susceptibility (135). Indeed in early studies of the combination of zidovudine and lamivudine as double therapy, the development of zidovudine resistance was considerably delayed (129). Based on these data, it has been suggested that the use of lamivudine in salvage therapy settings could help reduce the development of resistance to other agents and enhance the efficacy of the overall regimen, a fact that remains to be proven in clinical trials. Enhanced susceptibility to NNRTIs has been described in association with multiple mutations conferring broad cross-resistance to nucleoside analogues. It now appears that this phenomenon has biological significance, since its presence enhances the response to efavirenz-based therapy in both uncontrolled (136) and controlled (137) trials. However, the presence of hyper-susceptibility did not appear to delay the emergence of delavirdine resistance in

HIV Drug Susceptibility Testing 899 one controlled salvage therapy study (138). This phenomenon may also extend to protease inhibitors, with mutations at codons 30 and 88 possibly conferring hyper susceptibility to amprenavir and ritonavir (139). Some preliminary data suggest that non-B HIV isolates may have a reduced response to HAART. This may be due to natural polymorphisms in the protease gene, such as M36I, with or without additional changes at codons 71 and 77 (140,141). Secondary mutations that are more frequent in subtype C isolates exposed to protease inhibitors appear to be unique, a finding of unclear clinical significance (142). Additionally, it appears that subtype G isolates develop nelfinavir resistance through the L90M rather than the D30N pathway, a difference that would confer different patterns of cross-resistance to other agents in this class than we are used to seeing (143). In vitro data exist to suggest that subtype C isolates develop resistance to NNRTIs more rapidly (144,145). FUTURE DIRECTIONS Clinical Outcomes with Resistance Testing While available data strongly suggest a role for resistance testing in clinical practice, they also suggest that we currently underestimate the degree of cross-resistance (in particular with NRTIs) and that more accurate clinical cutoffs need to be established for all drugs. Additionally, the most cost effective approach to resistance testing remains to be defined and given the rapid pace of new drug development and the ever growing complexity of drug resistance is an issue that will require continued review and revision. Early Detection of Resistance In research applications, sequences of individual viral RNA molecules or proviral DNA sequences can be obtained by performing a cloning step (105,146) Such procedures can permit detection of minor populations of drug resistant virus when the bulk sequences only reflect the major variant. How important this might be to clinical practice is not known. Hypersusceptibility The precision of some of the automated phenotypic assays have permitted the identification of genetic polymorphisms of HIV from some patients that exhibit greater susceptibility to drugs than the control susceptible or “wild-type” strains (136,147–150). Some of these polymorphisms represent resistance mutations to other drugs or classes of drugs and are elicited by prior treatment while others appear to be naturally occurring. Whether treatment with drugs for which a patient’s virus is hypersusceptible translates to a better clinical outcome remains to be determined but these observations suggest an additional potential benefit of drug susceptibility assessment to guide drug selection.

Viral Fitness The recent observation that some drug resistant virus may exhibit attenuated virulence properties (30,151–153) adds additional complexity to therapeutic decisions based on drug resistance testing. Continued treatment with drugs against which resistance has developed can still provide some residual viral inhibition and also serves to maintain drug resistant viral populations that may be associated with lower viral replication fitness and virulence compared to “wild-type” virus. In net, continued treatment can be associated with prolonged immunologic benefit. Under circumstances where therapeutic options are limited, the potential benefits of continued treatment need to be weighed against the possibility that continued treatment will result in selection of viral variants with greater resistance and replication fitness (154,155). Resistance to New Drug Classes Inhibitors of the HIV-1 integrase are now in clinical trials and resistance to these agents will doubtless be recognized in the near future. The investigational fusion inhibitor enfuviritide (156,157) is already under review by the FDA and resistance to this drug appears to develop in the gp41 coding region (20, 158). This and future progress in drug development against new viral targets will necessitate the development of new tests for resistance and the adaptation of existing resistance testing methods. Non-Clade B Resistance While the field of HIV drug resistance testing has largely been restricted to the study of subtype-B virus, three issues make increased attention to drug resistance in non-B viruses important. First, drug therapy is being introduced into areas of the world where the predominant subtypes are non-B (159,160). While there remain serious financial and other logistical barriers to antiretroviral therapy in the developing world (160,161), the need to begin to build the infrastructure to support treatment in these areas of the world is acknowledged to be great and is a major goal of many governmental and non-governmental agencies. Second, some non-B viruses exhibit natural polymorphisms that appear to confer intrinsic resistance to certain drugs. The need for pre-treatment screening may therefore be greater under these circumstances. Third, the genetic background of these viruses may theoretically result in mutational patterns not previously encountered with clade B virus (162). Because many of the existing assays were developed and refined specifically for type B virus, their performance for the testing of non-B virus is only recently being explored (163,164). CONCLUSION HIV drug resistance testing is a rapidly evolving field. Technical advances and refinement of testing modalities

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are matched by the growing complexity of therapeutic options and the complex resistance patterns that result. A further challenge will be the application of resistance testing to non-subtype B virus as therapy is introduced into areas outside North America and Europe. Ultimately, analyses of large data bases relating genetic resistance patterns to phenotypic susceptibility, drug treatment and clinical outcomes will be needed to fully optimize and define the use of resistance testing in clinical care. In the meantime, the standard of care for HIV infected patients includes the judicious use of resistance testing along with expert interpretation. Resistance testing has been advocated when switching patients from a failing drug regimen, for choosing drugs for pregnant patients and should be considered in all patients with primary HIV infection or any patient about to embark on therapy in areas where the prevalence of drug resistance is high.

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