Integrase variability and susceptibility to HIV integrase inhibitors: impact of subtypes, antiretroviral experience and duration of HIV infection

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1 Journal of Antimicrobial Chemotherapy Advance Access published December 9, 2009 J Antimicrob Chemother doi: /jac/dkp423 Integrase variability and susceptibility to HIV integrase inhibitors: impact of subtypes, antiretroviral experience and duration of HIV infection Carolina Garrido 1, Anna Maria Geretti 2, Natalia Zahonero 1, Clare Booth 2, Angela Strang 2, Vincent Soriano 1 * and Carmen De Mendoza 1 1 Department of Infectious Diseases, Hospital Carlos III, Madrid, Spain; 2 Department of Virology, Royal Free Hampstead NHS Trust and University College London Medical School, London, UK *Corresponding author. Tel: þ ; Fax: þ ; vsoriano@dragonet.es Received 12 June 2009; returned 25 October 2009; revised 28 October 2009; accepted 29 October 2009 Background: Little is known about the extent and predictors of polymorphisms potentially influencing the susceptibility to HIV integrase inhibitors (INIs). Methods: Genetic sequences of HIV integrase were obtained from INI-naive patients at two European clinics. The 39 amino acid changes at 29 integrase positions so far associated with INI resistance were examined according to HIV clade, prior antiretroviral exposure and duration of HIV infection. Results: Integrase sequences were obtained from 418 patients, 294 (70.3%) infected with clade B and 124 (29.7%) infected with non-b variants (predominantly CRF02, A, C and D). Overall, 40% of patients were antiretroviral experienced and 32.8% were recent seroconverters. The most prevalent INI resistance-associated mutations were V72I (63.9%), V201I (54.8%), T206S (25.4%), I203M (9.8%) and K156N (7.4%). Major INI resistance mutations at positions 66, 92, 143, 148 and 155 were not detected. The mean number of polymorphic sites was greater in non-b than in B variants (2.17 versus 1.59; P, 0.001), and in antiretroviralexperienced than in drug-naive patients (1.89 versus 1.68; P¼0.034), whereas no significant differences were seen comparing recent seroconverters and chronically infected persons. Conclusions: Major INI resistance-associated mutations are very rare, if indeed ever present, in INI-naive patients. However, polymorphisms at positions which may influence the genetic barrier and/or drive the selection of specific INI resistance pathways are common, especially in HIV non-b subtypes. Keywords: polymorphisms, non-b subtypes, drug resistance, raltegravir, elvitegravir Introduction HIV replication requires three viral enzymes along with metabolites and machinery provided by the infected cell. The viral enzymes are encoded by the pol gene, and have been the main targets of antiretroviral drugs. 1 Integrase inhibitors (INIs) represent the latest approved drug family for the treatment of HIV infection. 2,3 The integrase enzyme is a 288 amino acid protein encoded by the 5 0 end of the pol gene. It consists of three different structural and functional domains: the N-terminal domain (amino acids 1 49), which contains the HHCC motif for zinc binding; the central catalytic domain (CCD; amino acids ), which contains the DDE motif; and the C-terminal domain (amino acids ), which displays DNA binding activity. 3,4 Integration is a complex process that comprises different steps: (i) assembly of the integrase enzyme at the end of the HIV long terminal repeat (LTR), forming the pre-integration complex; (ii) cleavage of the terminal GT dinucleotide from the 3 0 end of each LTR (known as 3 0 processing ); (iii) translocation of the pre-integration complex to the nucleus through the nucleopore; (iv) covalent linkage of the viral DNA into the cellular chromosome (known as strand transfer ); and (v) gap repair by cellular enzymes. 5 Although the integrase catalyses both 3 0 processing and strand transfer, the current INIs raltegravir and elvitegravir only inhibit its strand transfer activity. 3,6,7 Raltegravir (Isentress, Merck) is the first INI commercially available, and elvitegravir (GS-3197, Gilead) is currently in Phase III clinical development. 7 Other INIs are in earlier steps of clinical development, some of which (e.g. GSK or GSK ) may retain activity against viruses resistant to raltegravir or elvitegravir. Overall, all INIs display high potency against viruses resistant to other antiretroviral agents and constitute a valuable option for salvage therapy. 7 More recently # The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org 1of7

2 Garrido et al. raltegravir has also received approval to be used as part of firstline antiretroviral therapy. However, resistance to INIs rapidly emerges as a result of selection of a few resistance-associated mutations (RAMs) within the integrase gene. Several changes influencing the susceptibility to INIs have been described both in vitro 8 10 and in vivo, including mutations that play a major role in resistance (major INI RAMs) and changes that play a contributing role (minor INI RAMs). The aim of this study was to assess the prevalence of naturally occurring polymorphisms within the HIV-1 integrase gene which might influence the susceptibility to INIs, including both major and minor INI RAMs. Particular attention was devoted to recognizing the influence of HIV-1 subtype, prior exposure to antiretroviral agents other than INIs, and duration of HIV infection on INI variability. Patients and methods Study population Plasma samples from HIV-1-infected individuals on regular follow-up at Hospital Carlos III (Madrid, Spain) and Royal Free Hampstead NHS Trust 3 p15 protease p66, p51 retrotranscriptase NTD aa 50 pol & UCL University College London Medical School (London, UK) were selected. All patients were naive to INIs. Integrase amplification and sequencing RNA was extracted from plasma using the automatic extractor EasyMag (Biomerieux, Madrid, Spain), which isolates nucleic acids based on the Boom methodology. 14 The integrase gene was amplified using an in-house PCR protocol designed on the basis of a well conserved integrase region in order to detect all HIV-1 variants. Briefly, an RT PCR was carried out using as primers Forward INtegrase EXternal (FINEX) (5 0 AGT GCT GGA ATC AGG AAA GTA C3 0 ) and Reverse INtegrase EXternal (RINEX) (5 0 TAA TCC TCA TCC TGT CTA CTT GC3 0 ). The reverse transcription step was carried out at 488C for 45 min, and PCR involved 35 repeated cycles (948C for 45 s, 578C for 45 s and 728C for 1 min) followed by incubation at 728C for 7 min. Then, a nested PCR was performed using as primers Forward INtegrase INternal (FININ) (5 0 TAG ATG GAA TAG ATA AGG CCC3 0 ) and the Reverse INtegrase INternal (RININ) (5 0 ATC ACC TGC CAT CTG TTT TCC3 0 ). PCR involved 35 repeated cycles (948C for 45 s, 578C for 45 s and 728C for 1 min) followed by incubation at 728C for 7 min. After checking the presence of amplicons with electrophoresis on an agarose gel, purification using Montage w PCR Centrifugal Filter Devices (Montáge, Madrid, Spain) was carried out. The integrase gene was then p15 RNase CCD P31 integrase aa 212 CTD 5 aa 288 H H C C D D E T L L E T F E G Y S Q V S N E G S D R I/A V/I M Q A Y K/A A/S/C R/H/C G K/R/H I Y H/S Q K/R R N K Figure 1. HIV-1 integrase gene and positions associated with resistance to integrase inhibitors. aa, amino acid; NTD, N-terminal domain; CTD, C-terminal domain; CCD, catalytic core domain. 2of7

3 Integrase variability in HIV non-b subtypes JAC sequenced using a cycle sequencing reaction with the Rhodamine terminator kit (Applied Biosystems, Foster City, CA, USA), and a set of four primers was used for complete coverage of both strands of the integrase region. Primers were as follows: two forward primers FININ (5 0 TAG ATG GAA TAG ATA AGG CCC3 0 ), FINMED (5 0 TAC AAT CCC CAA AGT C3 0 ), and two reverse primers RINEX (5 0 ATC ACC TGC CAT CTG TTT TCC3 0 ) and RINMED (5 0 ACT TTG GGG ATT GTA G3 0 ). Sequencing was carried out with the automated sequencer ABI Prism 3100 and integrase sequences were analysed using SeqScape v2.5 (Applied Biosystems). HIV-1 subtyping Phylogenetic and molecular evolutionary analyses were conducted using the MEGA v4 program. 15 All integrase sequences were first aligned together with reference subtype sequences available at the Los Alamos Database using ClustalX. A total of 56 reference sequences were considered, including 9 subtypes and 42 circulating recombinant forms (CRFs) of HIV-1 group M and one from HIV-1 group O, which was considered as an outgroup. INI resistance-associated mutations According to information derived from in vitro studies and clinical trials, to date 39 mutations at 29 positions within the integrase gene may influence INI susceptibility. 16,17 They are the following: H51Y, T66I, V72I, L74M/A/I, E92Q, T97A, F121Y, T125K, A128T, E138K/A, G140S/A, Y143R/C, Q146K/R, S147G, Q148K/R/H, V151I, S153A/Y, M154I, N155H/S, K156N, E157Q, K160D, G163R, V165I, V201I, I203M, T206S, S230M and V249I The major INI RAMs 16 are highlighted in bold in Figure 1. The prevalence of these mutations was examined in the whole study population. Additionally, the variability at the DDE and HHCC motifs, and the overall heterogeneity at the CCD was investigated. Results were stratified by HIV clade, prior antiretroviral exposure and duration of HIV infection. In addition, integrase sequence variability was examined longitudinally in plasma specimens collected from a subset of HIV-1-infected patients who began antiretroviral therapy and experienced virological failure on drugs other than INIs. Statistical analysis The main characteristics of the study population were reported as numbers, percentages or mean values. Fisher s exact test was used to compare the proportion of patients with distinct integrase amino acid changes. All statistical analyses were performed using SPSS v15.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance was considered for two-tailed P values,0.05. Results Study population A total of 418 integrase sequences were obtained. Of these 294 (70.3%) were from patients infected with HIV-1 clade B and 124 (29.7%) from subjects infected with non-b variants. Seven different HIV-1 subtypes and 12 CRFs were represented in the latter group, the most prevalent being CRF02_AG, C, A, D and F. The main characteristics of the study population are recorded in Table 1. A total of 249 (60%) individuals were antiretroviral naive, including 137 who had been infected with HIV-1 within the last 12 months (recent seroconverters). Table 1. Main characteristics of the study population (n¼418) Prevalence of INI resistance-associated mutations The integrase sequences harboured between 0 and 5 INI RAMs, with a median number of 2 [interquartile range (IQR) 1 2]. A total of 33 (7.9%) integrase sequences did not show any resistance change, whereas 143 (34.2%) harboured one, 148 (35.4%) harboured two, 78 (18.7%) harboured three, 15 (3.6%) harboured four, and 1 (0.2%) harboured five INI RAMs. Of the 39 recognized INI RAMs, 22 did not appear in any sequence (Figure 2), comprising H51Y, E92Q, F121Y, T125K, A128T, E138K/A, G140S/A, Y143R/C, Q146R/K, S147G, Q148K/R/ H, S153A/Y, N155H/S and K160D, and including the major RAMs E92Q, Y143R, Q148K/H/R and N155H ,16 In contrast, the most frequent INI RAMs were V72I (63.9%), V201I (54.8%), T206S (25.4%), I203M (9.8%), K156N (7.4%) and L74M/A/I (6.9%), all of which are considered as minor (accessory or compensatory). Other mutations appeared less frequently, such as V165I (2.4%), V151I (2.2%), M154I (1%) or E157Q (1%). No. Percentage Male gender White ethnicity Risk group MSM IDU heterosexual others Antiretroviral naive Recent HIV seroconverters HIV-1 non-b subtypes A C D F G H J CRF01_AE CRF02_AG CRF CRF CRF CRF CRF12_BF CRF CRF14_BG CRF CRF CRF MSM, men who have sex with men; IDU, intravenous drug users. 3of7

4 Garrido et al. T66I V72I L74MAI T97A V151I M154I K156N E157Q G163R V165I V201I I203M T206S S230R V249I other subtypes Percentage of isolates with the change Figure 2. Prevalence of changes at the integrase gene associated with resistance to integrase inhibitors across HIV-1 subtypes. The following mutations were not found in any sample: H51Y, E92Q, F121Y, T125K, A128T, E138KA, G140SA, Y143RC, Q146KR, S147G, Q148KRH, S153AY, N155HS, K160D. CRF02 F D C A B Distribution of INI resistance-associated mutations Significant differences were found in the prevalence of INI RAMs when comparing clade B and non-b viruses, with a mean number of mutations of 1.59 and 2.17, respectively (P, 0.001). Similarly, antiretroviral-naive patients showed a significantly lower mean number of INI RAMs than antiretroviral-experienced patients (1.68 and 1.89; P¼0.034). Patients with recent infection showed fewer INI RAMs than chronically infected subjects (1.61 versus 1.86; P¼0.015). Nevertheless, the only clinically relevant difference was for HIV-1 clades (see Table 2). Several amino acid changes occurred significantly more commonly in non-b subtypes than in subtype B, including L74M/A/I (P¼0.001), T97A (P¼0.007), V165I (P, 0.001), V201I (P, 0.001) and T206S (P, 0.001). Conversely, K156N (P, 0.001) and I203M (P¼0.001) were more common in clade B than in non-b viruses. Comparing antiretroviral-naive with antiretroviral-experienced patients, the only significant differences were found for T97A (P¼0.025) and I203M (P¼0.012), which were more common in the latter. There were no significant differences in the prevalence of any INI RAMs when comparing recent seroconverters and chronically infected individuals. Variability at the integrase CCD A total of 163 amino acids (residues ) constitute the CCD of the HIV-1 integrase. A position was considered as polymorphic 4of7

5 Integrase variability in HIV non-b subtypes JAC Table 2. Rate of integrase inhibitor (INI) resistance-associated mutations in the study population, according to HIV subtype and antiretroviral experience Total B subtype Non-B subtypes Antiretroviral naive Antiretroviral experienced Patients (number) Number of INI resistance-associated mutations mean (SD) 1.76 (0.97) 1.59 (0.88) 2.17 (1.06) 1.68 (1.00) 1.89 (0.92) Number of polymorphic positions within the CCD mean (SD) 9.89 (5.05) 8.75 (4.58) (5.11) 9.82 (5.34) 9.9 (4.62) INI resistance-associated mutations T66I, G163R, S230R, V249I 1 (0.2%) V72I 267 (63.9%) 189 (64.3%) 78 (62.9%) 153 (61.4%) 114 (68.7%) L74A/M/I 29 (6.9%) 12 (4.1%) 17 (13.7%) 18 (7.2%) 10 (6%) T97A 4 (1%) 0 4 (3.2%) 0 4 (2.4%) V151I 9 (2.2%) 9 (3.1%) 0 6 (2.4%) 3 (1.8%) M154I 4 (1%) 3 (1%) 1 (0.8%) 2 (0.8%) 2 (1.2%) K156N 31 (7.4%) 31 (10.5%) 0 17 (6.8%) 13 (7.8%) E157Q 4 (1%) 3 (1%) 1 (0.8%) 4 (1.6%) 0 V165I 10 (2.4%) 1 (0.3%) 9 (7.3%) 6 (2.4%) 4 (2.4%) V201I 229 (54.8%) 130 (44.2%) 99 (78.8%) 138 (55.4%) 89 (53.6%) I203M 41 (9.8%) 36 (12.2%) 5 (4%) 17 (6.8%) 24 (14.5%) T206S 106 (25.4%) 54 (18.4%) 52 (41.9%) 57 (22.9%) 49 (29.5%) Bold text indicates significant differences between groups (P,0.05). when amino acid changes were observed in.1% of tested sequences. 17 Given that 418 sequences were analysed in our study, changes should be found in at least 5 samples. A total of 73 (44.7%) positions within the CCD were conserved, while the remaining 90 were polymorphic. The median number of polymorphic changes in the study population was 9 (IQR 7 12). DDE and HHCC motifs were relatively conserved, with polymorphic changes in 41 (9.8%) sequences, of which 31 (7.4%) occurred within the DDE triad and 10 (2.3%) within the HHCC motif. The number of integrase polymorphic sites was higher in non-b clades compared with subtype B. The mean number was 12.6 and 8.75, respectively (P, 0.001). No significant differences were found when comparing antiretroviral-experienced and drug-naive subjects (9.96 versus 9.82; P¼0.776), or recent seroconverters and chronically infected individuals (10.02 versus 9.82; P¼0.742). Moreover, the rate of polymorphisms was not influenced by plasma HIV RNA values (data not shown). Longitudinal analysis A total of 34 plasma samples were collected longitudinally from 17 patients before starting antiretroviral therapy and at subsequent virological failure. There was no evidence for selection of INI RAMs comparing paired samples in 13 out of 17 patients. However, four individuals selected one minor INI RAM upon failure under antiretroviral drugs other than INI. The selected change differed in each case (data not shown). Nine subjects showed variability within the CCD when comparing baseline and failing specimens. Differences ranged from 0.5 to 6 changes, considering 0.5 when only a mixture was seen either at baseline or at failure. The remaining eight patients did not show any change at the CCD motif. Discussion The introduction of INIs has represented a major breakthrough in the treatment of antiretroviral-experienced HIV-1 patients. In particular, raltegravir has shown high antiviral potency and good tolerability, allowing the virological rescue of many patients who had failed other antiretroviral drug families Nevertheless, as with other antiretroviral agents, selection of drug resistance represents an important drawback in patients treated with INIs ,21 Although a few key resistance mutations in the integrase have been well characterized as producing loss of susceptibility to INIs, 11,16 other multiple changes seem to modulate the degree of susceptibility to these compounds. 17 In this study, the variability within the integrase gene at positions associated with resistance to INIs was examined in a large group of HIV-1 patients, with the specific aim of analysing any relevant impact of HIV-1 subtypes, prior exposure to antiretroviral drugs other than INIs, and duration of HIV-1 infection. A total of 418 integrase sequences belonging to patients infected with 20 different HIV-1 clades were analysed. Interestingly, primary INI RAMs were not seen in INI-naive individuals, regardless of exposure to other antiretroviral agents, duration of HIV-1 disease or infection with diverse non-b subtypes. However, naturally occurring polymorphisms, which could influence INI susceptibility and the genetic barrier to resistance, were found to be relatively common, particularly in subjects infected with non-b subtypes. Similar results have recently been found by others. In contrast, the prevalence of these polymorphisms appeared to be only marginally influenced by prior antiretroviral exposure and/or the duration of HIV-1 infection. The absence of detectable major INI RAMs in our study is consistent with findings from others 22,23 and strongly argues in favour of a wide activity of INIs across HIV-1 subtypes, and 5of7

6 Garrido et al. regardless of exposure to other antiretroviral drugs or duration of HIV-1 infection. This observation is in contrast to sporadic reports of primary INI RAMs, including major (E92K) and minor (E157Q, G140S) changes, among INI-naive individuals Moreover, our findings indirectly support major INI RAMs significantly impairing viral fitness. 27,28 The recognition of a substantial variability within the integrase at secondary resistance positions which modulate the susceptibility to INIs, especially in non-b subtypes, may require further investigation. Prior studies examining in vitro the susceptibility to raltegravir and elvitegravir with large panels of clinical isolates, representing multiple HIV-1 subtypes with several polymorphisms, have concluded that fold changes in the IC 50 are almost always below the biological threshold Moreover, the more distant HIV-1 group O and HIV-2 viruses, which show 40% heterogeneity in the integrase gene compared with HIV-1 group M viruses, 32,33 have been shown to be susceptible to raltegravir inhibition. 24,34 37 It should be highlighted that the catalytic triad DDE and the HHCC and RKK motifs are fully conserved across all HIV variants. 17,38 Altogether, these data suggest that secondary INI RAMs per se do not compromise INI activity. Nevertheless, at this time it is unclear whether they may influence the genetic barrier to resistance or drive the selection of specific INI resistance pathways. In this regard, the presence of the V165I polymorphism might favour the selection of N155H upon raltegravir failure, while the presence of T97A could favour selection of Y143R. 39 Mutation E157Q was found in four sequences (1%) in our study, belonging to three subtype B viruses and one CRF06 virus. Although E157Q has been reported to cause resistance to raltegravir by itself, besides dramatically reducing the enzymatic activity of the integrase, 13 in vitro studies have demonstrated only a minimal impact of this change on phenotypic susceptibility to INIs (fold change of 1.14). 28 Finally, the results of our longitudinal study show that antiretroviral treatment with drugs other than INIs does not result in the selection of integrase changes which may significantly influence the susceptibility to INIs. Similar results have recently been found by others 27 and argue against previous reports suggesting that selection of resistance mutations at the protease and/or reverse transcriptase might condition changes at the integrase which subsequently might influence INI susceptibility. 38 In summary, major INI resistance-associated mutations for raltegravir and elvitegravir are not seen in HIV-infected persons naive for INIs, regardless of exposure to other antiretroviral agents. However, naturally occurring polymorphisms at integrase positions which may influence the genetic barrier and/or drive specific INI resistance pathways are common, especially in subjects infected with non-b subtypes. Funding This work was supported by grants from Fundación Investigación y Educación en SIDA (IES), Red de Investigación en SIDA (RIS ISCIII-RETIC RD06/006), Fondo de Investigación Sanitaria (FIS, projects: PI06/1826; CP06/0284; CP08/0214), NEAT and Agencia Laín Entralgo. 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