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HIV Medicine 2006 825 pages Download PDF, 5.3 MB
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HIV Drug Resistance by Eva Wolf
Table 1. Mutations leading to RTI resistance
The development of resistant viral strains is one of the main reasons for failure of antiretroviral therapy. If there is resistance to several drug classes, the number of alternative treatment regimens is limited and the virological success of subsequent therapies, or so-called salvage regimens, may be short-lived. The rapid development of resistant variants is due to the high turnover of HIV - approximately 10 million new viral particles are produced every day (Perelson 1996) - and the exceptionally high error rate of HIV reverse transcriptase. This leads to a high mutation rate and constant production of new viral strains, even in the absence of treatment. In the presence of antiretroviral drugs, resistant strains are selected for as the dominant species (Drake 1993). There are two established assays for measuring resistance or sensitivity of HIV to specific antiretroviral drugs - the genotypic and the phenotypic resistance tests (Wilson 2003). Both assays are commercially available. Examples of commercially available genotypic resistance tests are: HIV-1 TrueGene™, Bayer Healthcare Diagnostics; or ViroSeq™, Celera Diagnostics/Abbott Laboratories. Other genotypic resistance assays such as Virco™TYPE HIV-1, Virco, GenoSure (Plus), LabCorp, or GeneSeq, Monogram Biosciences (formerly Virologic) are established in the laboratories of the respective manufacturers and are used in clinical trials. Phenotypic resistance tests include: Antivirogram™, Virco; PhenoSense™, Monogram Biosciences (formerly ViroLogic); and Phenoscript™, Viralliance. Disadvantages of phenotypic testing include the lengthy procedure and high expense of the assay. The cost of genotyping ranges from 350 to 500 Euro, depending on the assay and laboratory used. It is approximately twice as much for phenotyping. The drawback with both methods is that a minimum amount of virus is necessary in order to perform the test. A viral load below 500-1,000 copies/ml often does not allow any detection of resistance. Phenotyping Phenotypic resistance tests involve direct quantification of drug sensitivity. Viral replication is measured in cell cultures under the selective pressure of increasing concentrations of antiretroviral drugs and is compared to viral replication of wild-type virus. Drug concentrations are expressed as IC50 values (50 % inhibitory concentration). The IC50 is the concentration of drug required to inhibit viral replication by 50 %. The sensitivity of the virus is expressed as the IC50 divided by the IC50 of a wild type reference virus (fold-change value) and compared to the so-called cut-off value. The cut-off value indicates by which factor the IC50 of an HIV isolate can be increased in comparison to that of the wild type, whilst still being classified as sensitive. Determination of the cut-off is crucial for the interpretation of the results! Cut-off definitions Three different cut-offs are currently used. The technical cut-off is a measure of the methodological variability of the assay. The biological cut-off involves the inter-individual variability of wild type virus isolates from ART-naïve HIV patients. If the IC50 is below the biological cut-off, virological success is very likely. However, an IC50 above the biological cut-off does not allow prediction of the virological response to a drug. In contrast, the clinical cut-off indicates up to which levels of IC50 virological success can still be expected. Clinical cut-offs for boosted protease inhibitors (PIs) are higher than cut-offs for unboosted PIs. But, through boosting with ritonavir, drug levels may overcome certain levels of resistance. The VircoType and PhenoSense reports have included lower and upper clinical cut-offs (Bacheler 2004). The lower clinical cut-off is the fold-change in IC50 and indicates a slightly reduced virological response. A fold-change above the upper clinical cut-off indicates resistance, and a fold-change between the two cut-offs indicates partial resistance.
Genotyping Genotypic assays are based on the analysis of mutations associated with resistance. These are determined by the direct sequencing of the amplified HIV genome or by specific hybridization techniques with wild-type or mutant oligonucleotides. Genotype tests only detect viral mutants comprising at least 20 to 30 % of the total population and provide an indirect measurement of drug resistance. Mutations that are associated with reduced sensitivity have been well described for most HIV drugs, but the high number of different resistance patterns, which may also contain compensatory mutations, makes the determination of the degree of resistance to particular drugs difficult. The interpretation of genotypic resistance patterns is based on the correlation between the geno- and the phenotype. Data is available from in vitro studies, clinical observations and duplicate testing, in which genotypically localized mutations were investigated for phenotypic resistance. Ruled-based interpretation systems For the phenotypic interpretation of genotypic mutation patterns, rule-based interpretation systems are commonly available. Expert panels (e.g. the French ANRS AC11 Resistance group) have developed algorithms based on literature and clinical outcomes. Virtual phenotype One further approach to predict phenotype from genotype is the so-called "virtual" phenotype: a genotypic mutation pattern is interpreted with the aid of a large database of samples of paired genotypic and phenotypic data (Winters 2004). The genotypic interpretation systems vircoTYPE and geno2pheno are both based on a virtual phenotype. For the VircoType interpretation, genotypes matching the patient's virus were identified through a database search. The IC50 results of each of the matching viruses were averaged, thus producing the probable phenotype of the patient's virus. In the updated version of VircoTYPE, all mutations and mutation pairs of the patient's virus that contribute to specific drug resistance according to the new multiple linear regression modeling are identified. They are then included in the respective linear regression model using the drug specific resistance weight factors of the observed mutations and mutation pairs. The outcome variable of the regression model is the predicted fold-change comparing the IC50 of the patient's virus to the IC50 of the wild type reference virus. In addition, machine learning approaches such as decision trees and support vector machines (as implemented by the geno2pheno system) can be applied to predict phenotypic drug resistance (Beerenwinkel 2003, Larder 2005). Some of the most important databases for resistance profiles and interpretational systems are available free of charge on the following websites:
Some commercial suppliers of resistance tests also provide interpretation guidelines for their systems (e.g. TruGene™, GeneSeq™, Retrogram™). The discussion about genotypic resistance in this chapter focuses on the sequencing of the reverse transcriptase, the protease and the env (gp41) gene and on the respective resistance patterns that emerge with treatment. Most data are derived from patients with subtype B viruses (representing only 12 % of the worldwide HIV-infected population). However, by now, non-subtype B viruses have also been investigated for the development of resistance (van de Vijver 2004). Resistance pathways and patterns may differ in the various subtypes. Within the nucleotide sequences of the HIV genome, a group of three nucleotides, called a codon, defines a particular amino acid in the protein sequence. Resistance mutations are described using a number, which shows the position of the relevant codon, and two letters: the letter preceding the number corresponds to the amino acid specified by the codon at this position in the wild-type virus; the letter after the number describes the amino acid that is produced from the mutated codon. M184V indicates a mutation in codon 184 of the reverse transcriptase gene leading to a valine for methionine substitution in the reverse transcriptase enzyme. Mechanisms of resistance Nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs) are prodrugs that only become effective after being converted to triphosphates. Nucleotide analogs require only two instead of three phosphorylation steps. Phosphorylated NRTIs compete with naturally occurring dNTPs (deoxynucleotide triphosphates). The incorporation of a phosphorylated NRTI into the proviral DNA blocks further elongation of the proviral DNA and leads to interruption of the chain. There are two main biochemical mechanisms that lead to NRTI resistance (De Mendoza 2002). Sterical inhibition is caused by mutations enabling the reverse transcriptase to recognize structural differences between NRTIs and dNTPs. Incorporation of NRTIs is then prevented in favor of dNTPs (e.g. in the presence of the mutations M184V, Q151M, L74V, or K65R; Naeger 2001, Clavel 2004). Phosphorylysis via ATP (adenosine triphosphate) or pyrophosphate leads to the excision of the NRTIs already incorporated in the growing DNA chain. This is the case with the following mutations: M41L, D67N, K70R, L210W, T215Y and K219Q (Meyer 2000). Phosphorylysis leads to cross-resistance between NRTIs, the degree of which may differ between substances (AZT, d4T > ABC > ddC, ddI > 3TC). Contrary to the excision mutations, K65R leads to a decreased excision of all NRTIs when compared to the wild type, resulting in a greater stability once incorporated. For K65R, the combined effect of its opposing mechanisms - on the one hand decreased incorporation and on the other, decreased excision - results in a decreased susceptibility to most NRTIs but an increased susceptibility to AZT (White 2005). Non-nucleoside RT inhibitors (NNRTIs) also inhibit the viral enzyme reverse transcriptase (RT). NNRTIs are small molecules that bind to the hydrophobic pocket close to the catalytic domain of the RT. Mutations at the NNRTI binding site reduce the affinity of the NNRTI to the RT and thus lead to loss of antiviral activity of NNRTI and treatment failure. Protease inhibitors (PIs) hinder the cleavage of viral precursor gal-pol-polyprotein by the enzyme protease, thereby producing immature, non-infectious viral particles. PI resistance usually develops slowly, as several mutations must first accumulate. This is also referred to as the genetic barrier. For PIs, a distinction is made between major (or primary) and minor (or secondary) mutations. Major mutations are responsible for phenotypic resistance. They are selected for early on in the process of resistance to one drug, and are located within the active site of the target enzyme, the HIV protease. They reduce the ability of the protease inhibitor to bind to the enzyme. Major or primary mutations may also lead to a reduced activity of the protease. Minor mutations (often referred to as secondary mutations) are located outside the active site and usually occur after major mutations. Minor mutations can be particularly found at polymorphic sites of non-B subtypes. Minor mutations compensate for the reduction in viral fitness caused by major mutations (Johnson 2004). However, the differentiation of major and minor mutations can only provide an approximate estimation of the degree of resistance. Fusion inhibitors differ from NRTIs, NNRTIs and PIs, which block the replication of HIV in the infected cell. Instead, fusion inhibitors prevent HIV from entering its target cells. The first step in cell entry occurs when the HIV envelope glycoprotein, gp120, binds to the CD4 receptor and the chemokine coreceptors, CCR5 or CXCR4, of the target cell. Interactions between the two heptad repeat regions HR1 and HR2 within the transmembrane glycoprotein subunit gp41 lead to a conformational change in gp41, enable fusion of the viral and cellular membranes and thereby entry of HIV into the host cell. The fusion inhibitor T-20 (enfuvirtide), a synthetic peptide consisting of 36 amino acids, mimics the C-terminal HR2 domain of gp41 and competitively binds to HR1. Thus, interactions between HR1 and HR2 are blocked and the conformational change of gp41 that is necessary for fusion of virions to host cells is inhibited. A single amino acid substitution in gp41 can reduce the efficacy of T-20.
Transmission of resistant HIV strains The prevalence of mutations already present in treatment-naïve patients differs among demographic regions. High prevalences of more than 20 % were observed in big US cities with large populations of homosexual men and a long period of access to antiretroviral treatment. In San Francisco, the resistance prevalence among patients with acute or recent infections was between 18 and 27 % during the period 1996-2002. Comparably high rates of resistance transmission were observed in Madrid from 1997 to 1999 and in 2002 (Grant 2003, Wensing 2003a, De Mendoza 2003). In a multicentric evaluation in 40 US cities, 14 % of 371 isolates from treatment-naïve patients had at least one resistance mutation (Ross 2004). In 2003, the first results of the CATCH-Study (which later transferred into the European SPREAD study) were published. Data from more than 1,600 newly diagnosed HIV patients from 17 European countries were evaluated. From 1996 until 2002, the prevalence of primary mutations was 10 % (Wensing 2003b). These data were confirmed by the SPREAD study, which gathered data from 2,008 patients (Strategy to Control Spread of HIV Drug Resistance). The goal of the SPREAD study is to monitor primary resistances in newly infected and ART-naïve HIV patients and their clinical implications. Whereas the proportion of NRTI mutations, which was 13 % at the start of the observation, decreased by half over time, the frequency of NNRTI resistance mutations increased from 2.3 to 9.8 %. The frequency of PI resistance remained stable at 3-4 %. From 1996 to 2002, primary resistance was mainly observed in subtype B infections. Resistance mutations were present in 12.9 % of patients with subtype B infection compared to only 4.8 % in non-B subtypes. However, an increase over time was also observed in non-B subtypes (from 2.0 % in 1996-1998 to 8.2 % in 2000-2001). Transmission rates of resistant virus are possibly underestimated in the different regions. Minority viral populations below 25 % are not detected by standard sequencing techniques. Forty-nine virus isolates of acute seroconverters were tested for the presence of L90M, K103N and M184V by quantitative real-time polymerase chain reaction using specific oligonucleotides for the three key resistance mutations. In 10 out of 49 patients these mutants were detected. In 5 of these 10 patients the detected population represented a minor viral quasi-species and was not detected by direct sequencing (Metzner 2005). Transmitted primary resistance can persist for a long time. In a Spanish seroconverters study, 10 patients with primary resistance mutations were followed over a median time of 41 months. The following mutations were detected: T215Y in three isolates, T215N/S/C in four, M41L in six, L74V in one, I54V in one, V82S/A in two, and L90M in two isolates. In only three of 10 cases (partial) reversion (of T215Y) was observed: T215Y revertants (T215S) were detected in two patients, and wild type virus was detected in one patient after 7 years (De Mendoza 2005). The clinical relevance of primary resistance has been shown in several studies. Transmitted resistance mutations can limit further treatment options and reduce treatment response rates (Harzic 2002, Little 2002, Riva 2002, Hanna 2001, Balotta 2000). A retrospective study with 202 patients showed that, when initiating treatment without information on pre-existing resistance, patients with pre-existing mutations had a slower treatment response and a higher risk of treatment failure (Little 2002). However, on careful consideration of any pre-existing resistance, primary treatment success is often possible (Oette 2005, Little 2002, Hanna 2001). In early 2005, a patient from New York caused a sensation. He was infected with a multidrug resistant virus harboring 7 relevant NRTI mutations, 2 NNRTI mutations and 12 PI mutations. After 4 to 20 months (the exact time of infection is unknown), the patient's CD4 count had decreased to 80 cells/µl. The replication capacity of this resistant virus was comparable to that of wild type virus. Only two available antivirals, T-20 and efavirenz were still active. Even though the transmission of multidrug resistant virus and rapid clinical progression are rare events, this case report demonstrates the possible clinical consequences of primary drug resistance (Markowitz 2005).
The clinical importance of performing resistance testing before making changes to the therapy, has been demonstrated in several prospective, controlled studies, both for genotypic (Durant 1999, Baxter 1999, Tural 2001) and phenotypic resistance testing (Cohen 2000). Patients whose physicians had access to information about any existing mutations before the therapy was changed usually had more significant decreases in the viral load than patients in whom treatment was changed without knowledge of the resistance profile. With regard to the ongoing development of new antivirals with different resistance profiles, the clinical relevance of resistance testing might be even higher than that shown in studies several years ago. Interpretation of genotypic resistance profiles For several NRTIs, such as lamivudine, and for NNRTIs, a high degree of resistance can develop following only a single mutation (Havlir 1996, Schuurman 1995). For this reason, such drugs should only be used in highly effective regimens. However, the lamivudine-specific mutation, M184V, also reduces viral replication capacity (often referred to as reduced viral fitness) by 40-60 % (Sharma 1999, Miller 2003). After 52 weeks on lamivudine monotherapy, the viral load remained 0.5 logs below the initial levels, despite early development of the M184V mutation (Eron 1995). When compared to treatment interruptions, continuous monotherapy with 3TC delays virological and immunological deterioration (Castagna 2005). FTC (emtricitabine) has the same resistance pattern as 3TC. Treatment failure is associated with the M184V mutation (van der Horst 2003). Thymidine analog mutations, mostly referred to as "TAMs", include the mutations M41L, D67N, K70R, L210W, T215Y and K219Q, which were initially observed on zidovudine therapy (Larder 1989). It is now known that these mutations can also be selected for by stavudine (Loveday 1999). Three or more TAMs are associated with a relevant reduction in the sensitivity to stavudine (Shulman 2001, Calvez 2002, Lafeuillade 2003). The term "NAMs" (nucleoside analog mutations) is also used instead of TAMs, as these mutations are associated with cross-resistance to all other nucleoside analogs, with the exception of 3TC and FTC. Viral mutants, isolated from patients in whom treatment on AZT, 3TC or abacavir has failed, usually have a measurable phenotypic resistance. Two TAMs result in a 5.5-fold, three TAMs in a 29-fold and four TAMs or more in a > 100-fold reduced sensitivity to zidovudine. The use of abacavir in cases where there is a more than 7-fold reduction in sensitivity no longer promises success. This usually requires at least 3 TAMs in addition to the M184V mutation (Harrigan 2000). A score, which has been developed in the context of the Narval study (ANRS 088), seems to have a good predictive value concerning virological response to abacavir. Virological response is poor if 5 mutations out of M41L, D67N, L74V, M184V, L210W, and T215Y/F are present (Brun-Vézinet 2003). The virological response to ddI depends on the number of specific TAMs. In the Jaguar study, using treatment-experienced patients, T215Y/F, M41L and L210W - to a lesser extent also D67N und K219Q - were associated with a reduced efficacy (Marcelin 2005). The virological response was not dependent on the presence of the mutations M184V and K70R. The development of a measurable phenotypic resistance to d4T or ddI has been observed less frequently, and has been more moderate in character (Larder 2001). The clinical cut-off for stavudine lies below the biological cut-off of 1.8. Presumably, this is also the case for ddI (Shulman 2004). Since most interpretation systems still use biological cut-offs, phenotypic resistance might be underestimated. Clinical data indicates that tenofovir is effective even in the presence of NAMs such as D67, K70R, T215Y/F or K219Q/E. However, if three or more NAMs include M41L or L210W, a reduced virological response can be expected (Antinou 2003). The lamivudine-associated mutation, M184V, as well as the L74V mutation, observed on didanosine treatment, and the NNRTI-specific mutations, L100I and Y181C, may have an antagonistic effect on the development of resistance (Vandamme 1999). M184V induces re-sensitization to AZT, resulting in a 50-60 % reduction of IC50. Re-sensitization to stavudine results in a 30 % reduction of IC50. However, re-sensitization is of clinical relevance only if there are no more than three other AZT- or d4T-associated mutations present (Shafer 1995, Naeger 2001, Underwood 2005). In one genotypic and phenotypic resistance study consisting of 9,000 samples, a combination of M41L, L210W and T215Y decreased the susceptibility to AZT by more than 10-fold in 79 % of cases. If the M184V mutation was also present, only 52 % had a more than 10-fold decreased susceptibility to AZT (Larder 1999a). The M184V mutation also increases the sensitivity to tenofovir (Miller 2001, Miller 2004a). In contrast, the presence of M184V plus multiple NAMs or mutations at positions 65, 74 or 115 increased the resistance to ddI, ddC and abacavir (Harrigan 2000, Lanier 2001). So-called multidrug resistance (MDR) to all nucleoside analogs - except lamivudine - is established if one of the following combinations occurs: T69SSX, i.e. the T69S mutation plus an insertion of 2 amino acids (SS, SG or SA) between positions 69 and 70, plus a AZT-associated mutation or Q151M, plus a further MDR mutation (V75I, F77L or F116Y; Masquelier 2001). The MDR mutation, Q151M, alone leads to intermediate resistance to AZT, d4T, ddI, ddC and abacavir (Shafer 2002a). It is relatively uncommon, with a prevalence of less than 5 %. In contrast, Q151M does not lead to the loss of activity of tenofovir. Instead, the T69S insertion induces an approximately 20-fold increase in the resistance to tenofovir (Miller 2001, Miller 2004a). The insertion T69SSX together with the mutation M184V, as well as the mutation Q151M together with M184V, leads to a 70 % reduction in the viral replication capacity (Miller 2003). The L74V mutation emerges on ddI or abacavir and leads to a 2- to 5-fold increase in the resistance to ddI (Winters 1997). The loss of efficacy by a factor of around 2-3 for abacavir is not considered clinically relevant and requires further mutations (Tisdale 1997, Brun-Vézinet 2003). L74V/I with or without M184V leads to a reduction in IC50 of about 70 %; phenotypic susceptibility increases by a factor of 3 (Underwood 2005). The K65R mutation can emerge while on tenofovir, abacavir or ddI and leads to an intermediate resistance to tenofovir, abacavir, ddI, 3TC, FTC, and possibly d4T (Shafer 2002a, Garcia-Lerma 2003). There is no cross-resistance with AZT (Miller 2004b). In antiretroviral combinations containing AZT, the incidence of the K65R mutation is lower. K65R emerges very rarely together with TAMs on the same genome. K65R and TAMs represent two antagonistic resistance pathways. Genotypes harboring K65R and L74V are also very unlikely (Wirden 2005). Since abacavir was mostly used as part of the combination AZT+3TC+abacavir or in the presence of multiple TAMs, K65R was rare prior to the use of tenofovir. Similar to large clinical trials using tenofovir within divergent (PI- or NNRTI-containing) treatment regimens, the incidence of K65R stabilized at ≤ 5 %. However, virological failure of triple NRTI combinations such as Tenofovir+3TC+ABC or Tenofovir+3TC+ddI was often associated with the development of K65R (Farthing 2003, Gallant 2003, Landman 2003, Jemsek 2004). The main reason for the high failure rate seems to be the low genetic barrier of these regimens: the emergence of K65R induces a loss of sensitivity to all three drugs. K65R increases the sensitivity to AZT and induces a resensitization to zidovudine in the presence of (few) TAMs. K65R alone increases sensitivity to AZT by a factor of 2, together with M184V/I by a factor of 2.5 (White 2005, Underwood 2005). Vice versa, TAMs reduce the K65R-associated resistance to TDF, abacavir, ddI and ddC (Parikh 2004). As with M184V, the mutation K65R leads to a reduction in the viral replication capacity. This is not the case with TAMs or L74V/I. The median replication capacities for viruses with M184V/I (n=792), K65R (n=72) or L74V/I (n=15) alone were 68 % (P < 0.0001), 72 % (p < 0.0001) and 88 % (p=0.16), respectively. With the exception of M184V, NAMs did not change the replication capacities of viruses containing K65R or L74V/I (McColl 2005). If both mutations, K65R and M184V, were present, a replication of only 29 % was observed (Miller 2003). The V75T mutation, which is associated with an approximately 5-fold increase in the resistance to d4T, ddI and ddC, is only rarely observed (Lacey 1994). In large patient cohorts, quantitative measurements of sensitivity have shown that up to 29 % of NRTI-experienced patients have a hypersusceptibility to NNRTIs (i.e. a reduction in the inhibitory concentration by a factor of 0.3 - 0.6). A reduction in the AZT or 3TC sensitivity correlated with an increased NNRTI susceptibility. Shulman et al. pheno- and genotyped 444 virus isolates from NRTI-experienced patients. Mainly the reverse transcriptase mutations T215Y, H208Y and V118I were predictive for efavirenz hypersusceptibility. A database analysis of pair wise geno- and phenotypes showed NNRTI hypersusceptibility for TAMs and for non-thymidine analog-associated NAMs. Hypersusceptibility for efavirenz was detected for 1-2 TAMs, multiple TAMs plus M184V and for non-thymidine analog-associated NAMs such as K65R, T69X, M184V and in particular for K65R+M184V (Whitcomb 2000, Shulman 2004b, Coakley 2005a). However, these results have not influenced treatment strategies so far. A single mutation can confer a high degree of resistance to one or more NNRTIs. The relatively frequent K103N mutation leads to a 20- to 30-fold increase in resistance to all available NNRTIs (Petropolus 2000). Further use of the approved first generation NNRTIs in the presence of this mutation is therefore not recommended. V106A leads to a 30-fold increase in nevirapine resistance and intermediate efavirenz resistance. In contrast to subtype B viruses, the mutation V106M is more frequent in subtype C viruses. V106M is associated with high-level resistance not only to nevirapine but also to efavirenz (Grossman 2004). A98G (which occurs more frequently in subtype C viruses), K101E and V108 lead to low-grade resistance to all available NNRTIs. Intermediate resistance to efavirenz and delavirdine and low-grade resistance to nevirapine result from the L101I mutation. Y181C/I causes a 30-fold increase in nevirapine resistance, and response to efavirenz is only temporary. G190A is associated with a high degree of nevirapine resistance and an intermediate resistance to efavirenz and delavirdine. G190S and Y188C/L/H are mutations that result in a high degree of nevirapine and efavirenz resistance (Shafer 2002b, De Mendoza 2002). The spectrum of PI mutations is very large. Although there is a moderate to high degree of cross-resistance between PIs, the primary mutations are relatively specific for the individual drugs. If treatment is changed early on to another PI combination, i.e. before the accumulation of several mutations, the subsequent regimen may still be successful. Most data on primary mutations selected for first in the presence of a PI, are derived from studies using unboosted PIs. In studies evaluating first-line triple therapy with boosted lopinavir, fosamprenavir or saquinavir, no patient with virological failure developed detectable major PI mutations, and the incidence of minor mutations was low (Gulick 2004, DeJesus 2004, Anaworanich 2005). Development of primary PI resistance in patients failing boosted PI therapy is rare (Conradie 2004, Friend 2004, Lanier 2003, Coakley 2005b). Polymorphisms at positions 10, 20, 36, 63, 71, 77 and 93 do not lead to resistance per se, but compensate for the reduced protease activity caused by primary mutations (Nijhuis 1999). The typical nelfinavir-specific resistance profile, with the D30N primary mutation and further secondary mutations, results in only a low degree of cross-resistance to other PIs (Larder 1999a). Virological failure on nelfinavir can also be associated with the emergence of L90M (Craig 1999). In subtype B viruses, treatment with nelfinavir generally leads to the emergence of D30N or M46I plus N88S. In subtype C, G and AE viruses, however, the mutations L90M and I84V occur more frequently. One reason for these different resistance pathways is the prevalence of natural polymorphisms: whereas the polymorphism M36I is present in only 30 % of subtype B viruses, M36I is present in 70 - 100 % of non-B subtypes (Gomes 2002, Gonzales 2004, Grossman 2004, Sugiura 2002, Hackett 2003). A comparison between the replicative capacities of a virus with a single protease mutation (D30N or L90M) and that of the wild-type virus, demonstrated a significant loss of viral fitness in the presence of the D30N mutation selected by nelfinavir. In contrast, the L90M mutation only leads to a moderate reduction in the replicative capacity, which can be compensated for by the frequently occurring L63P polymorphism. Conversely, the L63P mutation hardly influences the reduced replicative capacity of D30N mutants (Martines 1999). G48V mainly emerges on unboosted saquinavir and leads to a 10-fold decrease in the susceptibility to saquinavir - in combination with L90M it results in a high degree (over 100-fold) of decreased susceptibility to saquinavir (Jakobson 1995). Yet generally, any 4 mutations out of L10I/R/V, G48V, I54V/L, A71V/T, V77A, V82A, I84V and L90M, are required to reduce the efficacy of RTV-boosted saquinavir (Valer 2002). Marcelin et al. (2005) re-evaluated the genotypic interpretation of saquinavir resistance in a retrospective analysis of 138 PI-experienced patients. In this study, the mutations 10F/I/M/R/V, 15A/V, 20I/M/R/T, 24I, 62V, 73ST, 82A/F/S/T, 84V, and 90M were identified as those most strongly associated with virological response. The presence of 3 to 4 mutations was associated with a reduced response to boosted saquinavir. Unboosted indinavir and/or ritonavir mainly selected for the major mutation V82A(T/F/S), which in combination with other mutations led to cross-resistance to other PIs (Shafer 2002c). Mutants that frequently developed on indinavir, harboring M46I, L63P, V82T, I84V or L10R, M46I, L63P, V82T, I84V, were just as fit as the wild-type virus. The resistance pattern of amprenavir and fosamprenavir is somewhat different to that of other first generation PIs. In the course of failing treatment with unboosted amprenavir or fosamprenavir, the following mutations have been selected: I54L/M, I50V or V32I plus I47V - often together with the mutation M46I. In a small study, the corresponding virus isolates showed full susceptibility to saquinavir and lopinavir (Chapman 2004, Ross 2003). A loss of sensitivity to (fos-) amprenavir and all other approved PIs can be anticipated if the mutation I84V (together with other mutations) is present (Snowden 2000, Schmidt 2000, Kempf 2001, Maguire 2002, MacManus 2003). Researchers on a small study, with 49 PI-experienced patients who were switched to boosted amprenavir, developed an algorithm that also included resistance mutations at positions 35, 41, 63 and 82 (Marcelin 2003). Several mutations are required to confer resistance to boosted (fos-)amprenavir (Table 3). The response to lopinavir in PI-experienced patients correlates with the number of any of the following mutations: L10F/I/R/V, K20M/R, L24I, M46I/L, F53L, I54L/T/V, L63P, A71I/L/T/V, V82A/F/T, I84V, and L90M (Kempf 2000, Kempf 2001). Five mutations or less result in an increase in the IC50 by a median factor of 2.7, with 6-7 mutations this factor is 13.5, and with at least 8 mutations it is 44. The good efficacy, even with several mutations, is due to the high plasma levels of boosted lopinavir, which - for the wild-type virus - are > 30-fold above the EC50 concentration during the entire dose interval (Prado 2002). In studies where boosted lopinavir is part of a first-line regimen, no primary PI-mutations have been observed to date. Very few case reports of primary lopinavir resistance have been published. In one patient, virological failure was associated with the occurrence of V82A followed by the mutations V32I, M46M/I and I47A. Phenotyping resulted in high-grade lopinavir resistance. Susceptibility to other PIs, especially saquinavir, was not affected (Friend 2004, Parkin 2004). In a second case, with some pre-existing polymorphisms (M36I, L63P and I93L), the mutations 54V and V82A, followed by L33F, were selected (Conradie 2004). A different algorithm to predict lopinavir resistance also includes mutations at novel amino acid positions. Viruses with any 7 mutations out of L10F/I, K20I/M, M46I/L, G48V, I50V, I54A/M/S/T/V, L63T, V82A/F/S, G16E, V32I, L33F, E34Q, K43T, I47V, G48M/V, Q58E, G73T, T74S, and L89I/M display approximately a 10-fold increase in IC50. Mutations at positions 50, 54 and 82 particularly affect the phenotypic resistance (Parkin 2003, Jimenez 2005). In-vivo selection of lopinavir resistance was described in 54 PI-experienced patients failing treatment with boosted lopinavir. Mutations at positions 82, 54 and 46 frequently emerged. Mutations such as L33F, I50V or V32I together with I47V/I were selected less frequently. New mutations at positions 84, 90 and 71 were not observed (Mo 2005). Recently, the mutation I47A, which has rarely been observed since lopinavir has become available, has been associated with lopinavir resistance. I47A reduces the binding affinity to lopinavir and results in an 86- to > 110-fold loss in sensitivity. In contrast, I47A leads to saquinavir hypersusceptibility due to an enhanced binding affinity to saquinavir (Kagan 2005). A German team reported that even with 5 - 10 PI-mutations, which normally confer broad PI-cross-resistance, resensitization is possible. The mutation L76V, which is primarily selected for by lopinavir and rarely by amprenavir, is associated with high-grade resistance to lopinavir and (fos-) amprenavir, but can lead to resensitization to atazanavir and saquinavir (Müller 2004). The resistance profile of atazanavir, an aza-peptidomimetic PI, partly differs to that of other PIs. In patients, in whom first-line treatment with atazanavir failed, the mutation I50L - often combined with A71V - was primarily observed. On the one hand, I50L leads to a loss of sensitivity to atazanavir; on the other hand, I50L leads to an increased susceptibility to other currently approved PIs. Mutants harboring I50L plus A71V showed a 2- to 9-fold increase in the binding affinity to the HIV protease. Even in the presence of other major and minor PI mutations, I50L can increase susceptibility to other PIs (Colonno 2002, Colonno 2003, Weinheimer 2005, Yanchunas 2005). In PI-experienced patients, the I50L mutation was selected for in only one third of patients failing atazanavir (Colonno 2004). In PI-experienced patients, at least partial cross-resistance to atazanavir is probable (Snell 2003). The accumulation of PI-mutations such as L10I/V/F, K20R/M/I, L24I, L33I/F/V, M36I/L/V, M46I/L, M48V, I54V/L, L63P, A71V/T/I, G73C/S/T/A, V82A/F/S/T, L90M, and, in particular, I84V, leads to a loss of sensitivity to atazanavir. In the expanded access program using unboosted atazanavir, the number of the respective PI mutations correlated with the change in viral load. For unboosted atazanavir, the threshold for resistance is generally met if 3 or 4 PI mutations are present; for boosted atazanavir, resistance is likely with 6 or more mutations (Colonno 2004, Johnson 2004, Gianotti 2005). Tipranavir, the first non-peptidic protease inhibitor, shows good efficacy against viruses with multiple PI mutations. In phenotypic resistance testing, 90 % of isolates with a high degree of resistance to ritonavir, saquinavir, indinavir and nelfinavir were still sensitive to tipranavir (Larder 2000). Although tipranavir has shown activity against viruses with up to 20 - 25 PI mutations, a reduced sensitivity can be anticipated if three or more PRAMs (protease inhibitor-resistance associated mutations) - also referred to as UPAMs (universal PI-associated mutations) - are present (Cooper 2003). PRAMs include the following mutations: L33I/V/F, V82A/F/L/T, I84V and L90M. On the other hand, a sufficient short term reduction in the viral load of 1.2 logs was seen after two weeks on treatment with boosted tipranavir plus an optimized backbone in patients with at least three PRAMs, compared to only 0.2-0.4 logs with boosted amprenavir, saquinavir or lopinavir plus an optimized backbone (Mayers 2004). In a pooled analysis of 291 patients in three Phase II trials, the mutations, V82T, V82F and V82L, but not L90M or V82A, were associated with tipranavir-resistance. The mutations, D30N, I50V and N88D, were associated with an increased susceptibility for tipranavir (Kohlbrenner 2004). In pooled data analyses of Phase II and III studies, the mutations I10V, I13V, K20M/R/V, L33F, E35G, M36I, N43T, I47V, I54A/M/V, Q58E, H69K, T74P, V82L/T, N83D and I84V were identified as being associated with a virological response to tipranavir (Schapiro 2005). The presence of 4 to 7 mutations leads to a reduced tipranavir response. The accumulation of 8 or more mutations is predictive for tipranavir failure. In vitro, L33F and I84V were the first mutations that were selected for by tipranavir, but the respective loss in sensitivity was only two-fold. At the end of the selection experiments, virus isolates with 10 mutations (L10F, I13V, V32I, L33F, M36I, K45I, I54V, A71V, V82L, I84V) and sensitivity reduced by 87-fold, were observed (Doyon 2005). Similar resistance mutations were also found in clinical isolates of tipranavir-treated patients (L10F, I13V, K20M/R/V, L33F, E35G, M36I, K43T, M46L, I47V, I54A/M/V, Q58E, H69K, T74P, V82L/T, N83D, I84V) (Croom 2005). Fusion inhibitors This section focuses on enfuvirtide (T-20) resistance. The gp41 genome has positions of high variability and highly conserved regions. There seems to be no differences between B and non-B subtypes. Polymorphic sites are observed in all regions of gp41. The heptad repeat 2 (HR2) region has the highest variability. Primary T-20 resistance is a rare phenomenon (Wiese 2005). Loss of efficacy is generally accompanied by the appearance of mutations at the T-20 binding site, which is the heptad repeat 1 (HR1) region of gp41. In particular, mutations at positions 36 to 45 emerge, most frequently with substitutions at positions 36, 38, 40, 42, 43 and 45 (e.g. G36D/E/S, 38A/M/E, Q40H/K/P/R/T, N42T/D/S, N43D/K, or L45M/L). The IC50fold change, which ranges from £ 10 to several hundred, depends on the position of the mutation and the substitution of the amino acid. The decrease in susceptibility is higher for double mutations than for a single mutation. For double mutations such as G36S+L44M, N42T+N43K, N42T+N43S or Q40H+L45M, a fold-change of > 250 has been observed. Additional mutations in HR2 and envelope regions also contribute to T-20 resistance (Sista 2004, Mink 2005). In clinical isolates with G36D as a single mutation a 4- to 450-fold decrease in susceptibility was found. In the isolate showing a 450-fold decrease in susceptibility, a heterozygote change at position 126 in HR2 was observed (N/K). In a small study, 6 out of 17 patients with virological failure developed the mutation S138A in the HR2 region of gp41 in addition - mostly combined with a mutation at position 43 in the HR1 region and a range of HR2 sequence changes at polymorphic sites (Xu 2004). The replication capacity (RC) in the presence of HR1 mutations is markedly reduced when compared to wild type virus with a relative order of RC wild type > N42T > V38A > N42T, N43K » N42T, N43S > V38A, N42D » V38A, N42T (Lu 2004). Viral fitness und T-20 susceptibility are inversely correlated (r=0.99, p < 0.001) (Lu 2004). The following chapter describes the resistance profiles of several newly developed antiretroviral drugs.
In a study on 25 virus isolates with one or two NNRTI-associated mutations, etravirine was still active in 18 isolates with only a small change in IC50 (less than 4-fold). A more than 10-fold increase in IC50 was observed in only 3 virus isolates. The corresponding resistance profile noted in one case was the combination L100I+K103N, and in the two other cases the single mutations Y181I and F227C. However, the prevalence of these mutations is small (3 % for L100I+K103N and ≤ 0.5 % for Y181I and F227C; Andries 2004). Etravirine has a higher genetic barrier than other NNRTIs due to its flexible binding to the reverse transcriptase. High-grade resistance is observed only with multiple mutations. After several in-vitro passages, the dominant virus population showed the RT mutation V179F (a new variant at this position) and Y181C. Further mutations that were selected for in vitro were L100I, E138K, Y188H, G190E, M230L, M230L and V179I (Brillant 2004, Vingerhoets 2005).
Pooled data analyses of the clinical studies Power 1, 2 and 3 showed that the presence of specific baseline mutations was associated with reduced virological response (i.e. V11I, V32I, L33F, I47V, I50V, I54L/M, G73S, L76V, I84V, and L89V). The mutations V32I, L33F, I47V, I54L or L89V developed in ≥ 10 % of virological failures (De Béthune 2006). A preceding failure on lopinavir was not predictive for virological outcome on TMC 114 (Koh 2003, Peters 2004). Out of 447 PI-experienced patients with a median number of 8 PI mutations and a median of 3 major PI mutations, 30 to 47 % of patients in the different TMC114 study arms had a viral load of < 50 copies/ml compared to only 10 % in the control PI arm (Katlama 2005). With the aid of HIV resistance tests, antiretroviral treatment strategies can be improved. Pharmaco-economic studies have shown that these tests are also cost-effective both in treatment-experienced and in ART-naïve patients (Sax 2005, Corzillius 2004, Weinstein 2001). For several years, national and international HIV treatment guidelines have recommended the use of resistance testing (Salzberger 2004, US DHHS 2005, BHIVA 2005). With some delay, resistance tests are now covered by public health insurances in several countries. Currently, both genotypic and phenotypic tests show good intra- and inter-assay reliability. However, the interpretation of genotypic resistance profiles has become very complex and requires constant updating of the guidelines. The determination of the thresholds associated with clinically relevant phenotypic drug resistance is crucial for the effective use of (virtual) phenotypic testing. Even if treatment failure requires the consideration of other causal factors, such as compliance of the patient, metabolism of drugs and drug levels, resistance testing is of great importance in antiretroviral therapy. Finally, it needs to be emphasized that - even with the benefit of well-interpreted resistance tests - only experienced HIV practitioners should start, stop or change antiretroviral therapy with respect to the clinical situation and the psychosocial context of the patient.
Table 1. Mutations leading to RTI resistance Table 2. Mutations leading to NNRTI resistance Table 3. Mutations leading to PI resistance Table 4. Mutations leading to PI resistance
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