HIV-1 integrase variability and relationship with drug resistance in antiretroviral-naive and -experienced patients with different HIV-1 subtypes

Sir, The prevalence of natural polymorphisms and mutations associated with integrase (IN) inhibitor (INI) resistance in the HIV-1 IN has already been analysed.1–5 The aim of the study was to characterize the HIV-1 IN variability in antiretroviral (ARV)-naive and -experienced patients, never treated with INIs, in a panel of different HIV-1 subtypes, and the relationship with drug resistance. This multicentre study included 590 HIV-1-infected individuals never treated with INIs (308 drug-naive and 282 ARV-experienced patients) who were enrolled in seven clinical centres in France and one centre in Switzerland. Nucleotide and amino acid sequences were compared with the HxB2 HIV-1 clade B consensus sequence (GenBank accession number K03455.1) using the Bioedit software program. The sequences of the samples have been submitted to GenBank and assigned accession numbers JX425421 to JX425885 and JX451875 to JX451963. Thirty-seven of the 590 sequences are not available in the NCBI database because we have only the description of the polymorphism for these sequences and not the nucleotide sequence. The median CD4+ T cell count at the time the samples were drawn was 280 CD4+ T cells/mm3 (range 2–1692) and the median log10 viral load was 4.43 log10 copies/mL (1.92–6.94). Drug-treated patients were exposed to an average of 1.96 ± 2.65 nucleotide reverse transcriptase inhibitors (n = 526), 0.18 ± 0.41 non-nucleoside reverse transcriptase inhibitors (n = 473) and 0.86 ± 1.59 protease inhibitors (n = 481). The entire IN protein sequences (288 amino acids) derived from 308 drug-naive and 282 experienced patients infected with HIV-1 B or non-B subtype, all INI-naive, were analysed (Figure 1). All important residues involved in catalytic activity or in binding to the human cellular cofactor LEDGF/p75 were conserved in both drug-naive and ARV-treated patients (variability <0.5%). The presence of polymorphic substitutions at codon 124 was significantly associated with previous ARV exposure (P = 0.03). Limited data are available on the prevalence of specific polymorphisms in the IN gene of HIV-1 non-B subtypes. We investigated the diversity of the IN region of different HIV-1 subtypes in INI-naive patients. Among the 590 samples, 252 corresponded to non-B subtypes. No difference in reverse transcriptase and IN sequences was observed between subtypes. Twenty-six changes were found with significantly different prevalence between B isolates and at least one non-B group (prevalence of polymorphism >50%; P < 0.05). Polymorphisms at codons D10, K14, S17, A21, S24, D25, V31, V32, S39, V72, L101, T112, T125, G134, I135, K136, D167, V201, T206, I208, K215, T218, L234, D256, D278 and S283 could be related to specific non-B subtypes. Among the 26 analysed codons, we analysed the T125A mutations. T125A (specific GCA codon) was significantly more prevalent in non-B samples (P < 0.0001). A similar result was found in a recent study on the analysis of polymorphisms in the IN gene of ART-naive patients infected with HIV-1 non-B subtypes.6 Distribution of variants among group M HIV-1 IN sequences. Amino acid polymorphism in HIV-1 IN from 308 plasma samples from drug-naive patients and 282 samples from experienced patients are reported. The consensus subtype B sequence is shown in bold at the top of each 30 amino acid section. Numbers given as superscripts below each position are the numbers of isolates with that specific polymorphism. Grey boxes signify positions associated with in vivo resistance defined according to the algorithms from the ANRS (update October 2012, v.22, http://www.hivfrenchresistance.org/2012/Algo-sep-2012.pdf). Highly conserved motifs, the HHCC motif (coordinates zinc binding), the DDE motif, catalytic core domains I–VI and the Q sequence, are indicated by boxes. Twenty-two out of 36 HIV-1 IN resistance mutations (H51Y, L68I/V, V72I, L74M, Q95K, T97A, S119G/R, A128T, T125K, V151I, M154I, K156N, E157Q, K160N, G163K/R, V165I, V201I, I203M, T206S, S230N, D232N and V249I) already associated with INI resistance were detected. Polymorphic changes included some known residues associated with INI resistance, such as V72I, L74M, T97A, V151I, E157Q and I203M, but were not statistically different between ARV-naive and -experienced patients not including INIs. In contrast, the frequency of L101I and T124A mutations, but not the M154I mutation, selected in vitro by dolutegravir was higher in naive patients. Regarding viral subtypes, mutations L101I and T124A, either alone or in combination, were significantly more prevalent in non-B than B subtypes in ARV-naive patients (65.5% versus 34.6% for L101I, 74.4% versus 25.6% for T124A and 85.7% versus 14.3% for L101I+T124A; P < 0.0001 in all cases), as recently described by Garrido et al.7 Except at position 157 (E157Q), none of the primary mutations detected in patients failing on raltegravir-containing regimens (Y143R/C, Q148H/K/R and N155H) or on elvitegravir-containing regimens (T66I, E92Q, E138K, S147G, Q148H/K/R and N155H) was detected. The E157Q mutation was observed among 2.9% (n = 17) of patient samples, including four of subtype B, one of subtype H, seven of subtype CRF02_AG, one of subtype A, two of subtype D, one of subtype CRF11_cpx and one of subtype G, without a significant difference in polymorphism between ARV-naive and -experienced patients. The primary mutations detected in patients failing on dolutegravir-containing regimens (V151L, S153Y, T66K/L74M, E92Q/N155H, E138A/K+Q148H/K/R, G140C/S+Q148H/K/R and Q148R/N155H) were completely absent. In our study, dolutegravir resistance-associated mutations, in particular R263K, were not found to be polymorphic. Only the mutations L101I and T124A, which were previously shown to be selected in vitro in the presence of dolutegravir8,9 either alone or in combination, were common in both naive and experienced patients. However, these mutations have shown little impact on virological response to dolutegravir. Recently, the HIV-1 CRF01_AE IN coding region of the pol gene was evaluated for the presence of natural polymorphisms in 87 ARV-naive individuals from Cambodia, Thailand and Vietnam.10 Amino acid substitutions occurred in 60% of the subjects and none of these substitutions have been reported to be associated with resistance to INIs. Many polymorphisms in non-B viruses are considered to be secondary resistance mutations since they emerge in B subtype viruses after drug exposure.11 Nevertheless, the selection of resistance mutations could be influenced by the naturally occurring variations between the different non-B subtypes. In conclusion, all patients in our study lacked previously described major resistance mutations to raltegravir, elvitegravir and dolutegravir. However, we found evidence of important variations regarding the IN polymorphisms according to the different HIV-1 subtypes. Further studies of INI-treated patients will be needed to fully elucidate the role of polymorphic IN mutations in the context of HIV-1 variability. C. Roussel (Amiens), C. Alloui (Avicennes), H. Leguillou-Guillemette (Angers), D. Bettinger (Besançon), C. Pallier (Bicètre), D. Descamps, F. Brun-Vezinet and G. Peytavin (Bichat, Paris), B. Masquelier, P. Pinson and S. Reigadas (Bordeaux), S. Vallet (Brest), J. D. Poveda (Cerba), A. Mirand (Clermont-Ferrand), A. Krivine (Cochin, Paris), C. Auvray and A. de Rougemont (Dijon), S. Yerly (Genève), A. Signori-Schmuck (Grenoble), L. Bocket (Lille), S. Rogez (Limoges), C. Tamalet (Marseille), V. Schneider and C. Amiel (Tenon), M. Bouvier-Alias (Mondor), B. Montes (Montpellier), E. Schvoerer (Nancy), V. Ferré (Nantes), M. L. Chaix (Necker, Paris), J. Guinard (Orleans), S. Haim-Boukobza (Paul Brousse), C. Soulié, A. G. Marcelin, P. Flandre, L. Assoumou and V. Calvez (Pitié-Salpétrière, Paris), A. Maillard (Rennes), L. Morand-Joubert (St Antoine, Paris), C. Chaplain (St Denis), C. Delaugerre (St Louis, Paris), T. Bourlet (St Etienne), S. Bertsch (Strasbourg), J. C. Plantier (Rouen), S. Raymond (Toulouse) and S. Marque-Juillet (Versailles). The research leading to these results received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under the project ‘Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)’—grant agreement no. 223131 and the Agence Nationale de Recherche sur le SIDA et les Hépatites virales (ANRS, France). None to declare. We thank all patients included in the study and the Agence Nationale de Recherche sur le SIDA et les Hépatites virales (ANRS, France). We also acknowledge Professor Ray Cooke for editing the manuscript and all the members of the ANRS AC11 Resistance Study Group.