Phenotypic analysis of HIV-1 E157Q integrase polymorphism and impact on virological outcome in patients initiating an integrase inhibitor-based regimen

Phenotypic analysis of HIV-1 E157Q integrase polymorphism and impact on virological outcome in... Abstract Objectives To assess the phenotypic susceptibility of the E157Q polymorphism in HIV-1 integrase (IN) and the virological outcome of patients infected with E157Q-mutated virus initiating an IN inhibitor (INI)-based regimen. Methods This was a multicentre study assessing IN sequences from INI-naive patients among 17 French HIV clinical centres. E157Q site-directed mutants in pNL4.3 and pCRF02_AG contexts were assessed in a recombinant phenotypic assay. Results Prevalence of the E157Q polymorphism was 2.7% among 8528 IN sequences from INI-naive patients and its distribution was 1.7%, 5.6% and 2.2% in B, CRF02_AG and various non-B subtypes, respectively. Thirty-nine INI-naive patients with E157Q-mutated virus initiated an INI-based regimen. Among them, 15 had a viral load (VL) <50 copies/mL at initiation and virological suppression was maintained during the first year of follow-up in all but two exhibiting a viral blip. Twenty-four patients had a VL > 50 copies/mL at the time of INI-based regimen initiation. Among them eight were receiving a first-line regimen and the only two patients who did not reach VL < 50 copies/mL at week 24 were receiving elvitegravir. The 16 remaining patients were ART experienced in virological failure with drug-resistant viruses displaying several virological outcomes independently of the genotypic susceptibility score. Phenotypic analyses showed a fold change in EC50 of 0.6, 0.9 and 1.9 for raltegravir, dolutegravir and elvitegravir, respectively, in a subtype B context, and 1.1, 1.9 and 2.4 for raltegravir, dolutegravir and elvitegravir, respectively, in a CRF02_AG context. Conclusions Assessment of virological response in 39 patients initiating an INI-based regimen with E157Q-mutated virus, in combination with phenotypic analysis, suggests that particular attention should be paid to antiretroviral-naive patients and dolutegravir should be preferentially used in these patients. Introduction Integrase (IN) inhibitors (INIs) are now recommended by multiple international guidelines in first-line regimens of HIV-1 infection treatment and their use is increasing. The major INI resistance mutations, resulting in a high level of phenotypic resistance, are well described for first-generation INIs (raltegravir and elvitegravir), but remain not fully elucidated for dolutegravir. The IN E157Q substitution has been previously described as a polymorphism present in around 3%–5% of viral sequences obtained from ART-naive patients.1,2 However, the E157Q change has also been observed in cases of virological failure under a raltegravir-based treatment in two case reports.3,4 More recently, a case report of a non-virological response to a dolutegravir-based regimen has been described in a patient infected with an E157Q-mutated virus.5 Thus, there are few data on the prevalence of the E157Q substitution in a large dataset of IN sequences representing the genetic diversity of HIV-1. Regarding the virological response to an INI-based regimen for patients displaying E157Q mutation, only case reports are available.6,7 In the study of Ambrosioni et al.,6 only two ART-naive patients initiated a dolutegravir-based regimen with a very short follow-up of 12 weeks. In the study by Pavkovich et al.,7 only 6 of the 15 patients initiating an INI-based regimen achieved virological success at month 12; however, heterogeneous profiles of ART-naive and ART-experienced patients were not distinguished in the analysis. The aim of this study was to assess the prevalence of E157Q polymorphism in IN sequences obtained from INI-naive patients across various HIV-1 subtypes and the virological outcome at week 24 (W24) and week 48 (W48) of patients infected with an E157Q-mutated virus initiating an INI-based regimen. Methods IN sequences This was a national multicentre study assessing all available IN sequences from INI-naive patients among 17 French HIV clinical centres included in the Agence Nationale de Recherche sur le SIDA et les hépatites virales (ANRS) virology network. Ethics Participating laboratories belong to ANRS and participate in the ANRS quality control assessment of HIV-1 drug resistance sequencing. The study was approved by the scientific committee of Action Coordonnée n° 11 of ANRS. pol sequencing The pol gene was sequenced by standard Sanger technology using ANRS procedures (www.hivfrenchresistance.org). The genotypic susceptibility score (GSS) of the ART was calculated using the ANRS algorithm (September 2016, version 26), translating the interpretations ‘susceptible’, ‘possible resistance’ and ‘resistance’ into scores of 1, 0.5 and 0, respectively. HIV-1 subtyping Phylogenetic relationships of reverse transcriptase (RT) were estimated from sequence comparisons with previously reported representatives of HIV-1 group M subtypes and circulating recombinant forms (CRFs) for which sequences are available in the Los Alamos HIV database (https://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html). Phenotypic assays The E157Q substitution was introduced into the pNL4-3 sequence (subtype B) and the pCRF02_AG plasmid (kindly provided by M. Wainberg and T. Mesplede) by site-directed mutagenesis using the Quickchange II XL Site-Directed Mutagenesis® Kit (Agilent), according to the manufacturer’s instructions. HIV-1 stocks were prepared by transfecting 293-T cells using Lipofectamine reagent (Thermo Fisher) with the various HIV-1 molecular clones. The titre of the supernatant was determined by quantifying HIV-1 p24 antigen using the VIDAS® instrument (bioMérieux). Briefly, HeLa-P4 cells were infected in triplicate in 96-well plates with WT virus or mutants containing the E157Q IN mutation (equivalent of 10 ng of p24 antigen), with increasing concentrations of raltegravir or dolutegravir (from 0 to 200 nM). The viral titre was determined 48 h post-infection by quantifying β-galactosidase activity in HeLa-P4 lysates in a colorimetric assay based on the cleavage of chlorophenol red-β-d-galactopyranoside by β-galactosidase. The EC50 of each drug was defined as the drug concentration resulting in β-galactosidase levels that were 50% lower than those in infected cells without the drug. Results Prevalence of the E157Q natural polymorphism among various HIV-1 subtypes A total of 8528 IN sequences obtained between 2010 and 2016 from INI-naive patients in routine clinical care were analysed; 4759 (56%) of the sequences were subtype B, 1945 (23%) were CRF02_AG and 1824 (21%) were various non-B subtypes other than CRF02_AG. Overall, prevalence of E157Q polymorphism was 2.7% (95% CI 2.4%–3.1%). Distribution of E157Q polymorphism among HIV-1 subtypes was 1.7% (95% CI 1.3%–2.1%), 5.6% (95% CI 4.6%–6.6%) and 2.2% (95% CI 1.6%–2.9%) in B, CRF02_AG and various non-B subtypes, respectively. The distribution of E157Q polymorphism was as follows among the various non-B subtypes: 16.7% in subtype H (n = 4/24), 11.8% in CRF09_cpx (n = 2/17), 7.0% in CRF06_cpx (n = 3/43), 5.6% in subtype D (n = 7/124), 3.1% in subtype G (n = 4/127), 2.1% in CRF01_AE (n = 3/140), 2.0% in subtype C (n = 4/199), 1.6% in subtype A (n = 5/320), 1.1% in subtype F (n = 2/189) and 1.5% in complex recombinants (n = 7/452) (Figure 1). Figure 1. View largeDownload slide Prevalence of the E157Q IN polymorphism among various HIV-1 subtypes from INI-naive patients. *Non-B subtypes other than CRF02_AG. **Complex recombinants not identified. Figure 1. View largeDownload slide Prevalence of the E157Q IN polymorphism among various HIV-1 subtypes from INI-naive patients. *Non-B subtypes other than CRF02_AG. **Complex recombinants not identified. Virological outcome of patients with an E157Q-mutated virus initiating an INI-based regimen Thirty-nine INI-naive patients with an E157Q-mutated virus initiated an INI-based regimen, including raltegravir in 19 cases, elvitegravir in 10 cases and dolutegravir in 10 cases. IN sequences showed that E157Q was the only INI resistance mutation in all 39 viruses, except in two cases in which it was associated with the E92Q or T97A mutation. Among these 39 patients, 15 patients were in virological success [i.e. plasma viral load (VL) <50 copies/mL] under their ongoing regimen at the time of INI-based regimen initiation. Virological suppression was maintained during the first year of the follow-up in 13 patients (87%). The two remaining patients exhibited a VL >50 copies/mL at W48 (53 and 267 copies/mL), which became undetectable again at the VL control performed within 4 weeks, thus corresponding to a viral blip. Twenty-four patients initiated an INI-based regimen with a VL >50 copies/mL (median 4.9 log10 copies/mL, IQR 4.3–5.1): 8 were receiving a first-line regimen (Table 1) and 16 were ART-experienced patients in virological failure with viruses harbouring drug resistance mutations (Table 2). INI-based regimen discontinuation occurred between W24 and W48 in 2/8 patients on a first-line regimen and in 5/16 antiretroviral-experienced patients. Table 1. Description and virological outcome of antiretroviral-naive patients initiating a first-line INI-based regimen INI  ARV associated with INI  GSS of associated ARV  HIV-1 subtype  VL at baseline of INI treatment (copies/mL)  VL at W24 of INI treatment (copies/mL)  VL decrease between day 0 and W24 (log10 copies/mL)  VL at W48 of INI treatment (copies/mL)  EVG  TDF/FTC/c  2  B  109 483  <50  −3.4  <50  EVG  TDF/FTC/c  2  CRF02_AG  3061  <50  −1.8  <50  EVG  TDF/FTC/c  2  B  425 900  <50  −4.0  STOP  EVG  TDF/FTC/c  2  B  99 750  151  −2.8  STOP  EVG  TDF/FTC/c  2  B  27 352  236  −2.0  NA  RAL  TDF/FTC  2  CRF02_AG  90 746  <50  −3.4  <50  RAL  DRV/r  1  B  168 255  <50  −3.6  <50  DTG  TDF/FTC  2.5  CRF02_AG  405 790  <50  −4.0  <50  INI  ARV associated with INI  GSS of associated ARV  HIV-1 subtype  VL at baseline of INI treatment (copies/mL)  VL at W24 of INI treatment (copies/mL)  VL decrease between day 0 and W24 (log10 copies/mL)  VL at W48 of INI treatment (copies/mL)  EVG  TDF/FTC/c  2  B  109 483  <50  −3.4  <50  EVG  TDF/FTC/c  2  CRF02_AG  3061  <50  −1.8  <50  EVG  TDF/FTC/c  2  B  425 900  <50  −4.0  STOP  EVG  TDF/FTC/c  2  B  99 750  151  −2.8  STOP  EVG  TDF/FTC/c  2  B  27 352  236  −2.0  NA  RAL  TDF/FTC  2  CRF02_AG  90 746  <50  −3.4  <50  RAL  DRV/r  1  B  168 255  <50  −3.6  <50  DTG  TDF/FTC  2.5  CRF02_AG  405 790  <50  −4.0  <50  /c, boosted with cobicistat; ARV, antiretroviral, DRV/r, darunavir boosted with ritonavir; DTG, dolutegravir; EVG, elvitegravir; FTC, emtricitabine; NA, not available; RAL, raltegravir; TDF, tenofovir disoproxil fumarate. Table 2. Description and virological outcome of pretreated patients initiating an INI-based regimen with VL > 50 copies/mL INI  ARV associated with INI  GSS of associated ARV  HIV-1 subtype  VL at baseline of INI treatment (copies/mL)  VL at W24 of INI treatment (copies/mL)  VL decrease between day 0 and W24 (log10 copies/mL)  VL at W48 of INI treatment (copies/mL)  RAL  ETR/DRV/r  1  D  59 900  <50  −3.1  <50  RAL  DRV/r  1  CRF02_AG  45 574  <50  −3.1  <50  RAL  ZDV/ABC/3TC/DRV/ATV/r  0.5  non-B  66 200  <50  −3.1  <50  RAL  ABC/3TC/DRV/r  3  B  336 211  <50  −3.8  63  RAL  DRV/r  1  CRF02_AG  139  <50  −0.5  97  RAL  DRV/r  1  CRF02_AG  472  <50  −1.1  STOP  RAL  ABC/3TC/TDF/EFV  2  CRF02_AG  1423  454  −0.5  STOP  RAL  DRV/r  1  B  128 000  128  −3.0  STOP  RAL  NVP  1  CRF02_AG  2200  NA  NA  2852  RAL  ABC/3TC/ETR/DRV/r/ENF  1.5  CRF02_AG  179 594  102  −3.2  131  RAL  TDF/FTC  2  B  136  343  +0.4  STOP  EVG  TDF/FTC/c  2  CRF02_AG  81 989  121  −2.8  182  EVG  TDF/FTC/c  2  CRF02_AG  1 094 101  23 434  −1.7  6660  DTG  TDF/FTC  2.5  non-B  65  <50  −0.5  <50  DTGa  ABC/3TC/TDF/DRV/r  1  G  75 700  105  −2.9  145  DTGb  TDF/ETR/DRV/r/ENF  3  B  118 000  <50  −3.5  STOP  INI  ARV associated with INI  GSS of associated ARV  HIV-1 subtype  VL at baseline of INI treatment (copies/mL)  VL at W24 of INI treatment (copies/mL)  VL decrease between day 0 and W24 (log10 copies/mL)  VL at W48 of INI treatment (copies/mL)  RAL  ETR/DRV/r  1  D  59 900  <50  −3.1  <50  RAL  DRV/r  1  CRF02_AG  45 574  <50  −3.1  <50  RAL  ZDV/ABC/3TC/DRV/ATV/r  0.5  non-B  66 200  <50  −3.1  <50  RAL  ABC/3TC/DRV/r  3  B  336 211  <50  −3.8  63  RAL  DRV/r  1  CRF02_AG  139  <50  −0.5  97  RAL  DRV/r  1  CRF02_AG  472  <50  −1.1  STOP  RAL  ABC/3TC/TDF/EFV  2  CRF02_AG  1423  454  −0.5  STOP  RAL  DRV/r  1  B  128 000  128  −3.0  STOP  RAL  NVP  1  CRF02_AG  2200  NA  NA  2852  RAL  ABC/3TC/ETR/DRV/r/ENF  1.5  CRF02_AG  179 594  102  −3.2  131  RAL  TDF/FTC  2  B  136  343  +0.4  STOP  EVG  TDF/FTC/c  2  CRF02_AG  81 989  121  −2.8  182  EVG  TDF/FTC/c  2  CRF02_AG  1 094 101  23 434  −1.7  6660  DTG  TDF/FTC  2.5  non-B  65  <50  −0.5  <50  DTGa  ABC/3TC/TDF/DRV/r  1  G  75 700  105  −2.9  145  DTGb  TDF/ETR/DRV/r/ENF  3  B  118 000  <50  −3.5  STOP  /c, boosted with cobicistat; /r, boosted with ritonavir; 3TC, lamivudine; ABC, abacavir; ARV, antiretroviral; ATV, atazanavir; DRV, darunavir; DTG, dolutegravir; ENF, enfuvirtide; EFV, efavirenz; ETR, etravirine; EVG, elvitegravir; FTC, emtricitabine; NA, not available; NVP, nevirapine; RAL, raltegravir; TDF, tenofovir disoproxil fumarate; ZDV, zidovudine. a Double mutant E92Q-E157Q. b Double mutant T97A-E157Q. Among the eight patients receiving a first-line regimen, five received a tenofovir/emtricitabine/elvitegravir/cobicistat single-tablet regimen (STR), one received tenofovir/emtricitabine and raltegravir, one received tenofovir/emtricitabine and dolutegravir and one received raltegravir plus darunavir/ritonavir. Five patients were infected with subtype B and three with CRF02_AG. Six of the eight patients reached VL <50 copies/mL at W24 and virological suppression was maintained at W48 in five of these patients still receiving an INI-based regimen at W48 (Table 1). Regarding the two patients who had not reached VL <50 copies/mL at W24, VL was 236 and 151 copies/mL after a significant VL decrease between INI-based regimen initiation and W24 of −2.0 and −2.8 log10 copies/mL, respectively. Both patients were receiving an elvitegravir-based STR and were infected with subtype B. Their follow-up between W24 and W48 showed that one discontinued the INI-based regimen after retrospective evidence of the E157Q polymorphism in the pre-therapeutic genotype and one died (not related to HIV infection). Regarding the 16 ART-experienced patients in virological failure harbouring viruses with drug resistance mutations, 4 were in virological success with VL <50 copies/mL at W24 and at W48 (Table 2). Four additional patients had reached VL <50 copies/mL at W24, including one displaying the T97A–E157Q double mutant at baseline. Among these four patients, two exhibited a viral blip at W48 (63 and 97 copies/mL) and two discontinued INI after W24: one was lost to follow-up and in the other there was retrospective detection of the E157Q polymorphism in the pre-therapeutic genotype. Four other patients showed an initial significant VL decrease >2.5 log10 copies/mL between INI-based regimen initiation and W24, but without reaching 50 copies/mL at W24 (102, 105, 121 and 128 copies/mL), including one patient displaying the E92Q–E157Q double mutation at baseline. At W48, three of these patients were still receiving an INI-based regimen and all displayed low-level viraemia between 100 and 200 copies/mL. Four other patients had no virological response, with a VL decrease <2 log10 copies/mL at W24. The virological outcome of the 16 antiretroviral-experienced patients was not associated with the number of active antiretroviral drugs associated with the INI. Indeed, 8 of the 16 patients had a GSS ≤1 and they were equally distributed among the different profiles of virological outcome described (Table 2). Phenotypic susceptibility of the E157Q mutant to raltegravir, elvitegravir and dolutegravir In cells carrying E157Q site-directed mutations, the fold change in EC50 of raltegravir, elvitegravir and dolutegravir was 0.6, 1.9 and 0.9, respectively, in the subtype B context and 1.1, 2.4 and 1.9, respectively, in the CRF02_AG context (Table 3). Table 3. Effects of the E157Q mutation on the EC50 and standard deviation for raltegravir, elvitegravir and dolutegravir in HeLa-P4 cells Backbone  Genotype  Raltegravir   Elvitegravir   Dolutegravir   EC50 (nM)  standard deviation (nM)  fold change  EC50 (nM)  standard deviation (nM)  fold change  EC50 (nM)  standard deviation (nM)  fold change  pNL43 (B)  WT  20.3  7.8  –  20.1  5.5  –  7.7  2.1  –  E157Q  12.2  5.2  0.6  39.1  2.4  1.9  6.9  1.5  0.9  pCRF02_AG  WT  26  7.1  –  39.8  15.6  –  7.1  3.4  –  E157Q  29.3  0.9  1.1  95.5  31  2.4  13.5  1.0  1.9  Backbone  Genotype  Raltegravir   Elvitegravir   Dolutegravir   EC50 (nM)  standard deviation (nM)  fold change  EC50 (nM)  standard deviation (nM)  fold change  EC50 (nM)  standard deviation (nM)  fold change  pNL43 (B)  WT  20.3  7.8  –  20.1  5.5  –  7.7  2.1  –  E157Q  12.2  5.2  0.6  39.1  2.4  1.9  6.9  1.5  0.9  pCRF02_AG  WT  26  7.1  –  39.8  15.6  –  7.1  3.4  –  E157Q  29.3  0.9  1.1  95.5  31  2.4  13.5  1.0  1.9  Discussion In this study, based on a large dataset of HIV-1 IN sequences, with >8500 viral sequences obtained from INI-naive patients infected with a large variety of HIV-1 subtypes, we described an overall prevalence of E157Q polymorphism of 2.7%. Virological response of patients displaying an E157Q-mutated virus and initiating an INI-based regimen was assessed in 39 patients showing different profiles of virological outcome. The prevalence of the E157Q IN polymorphism in viral sequences obtained from antiretroviral-naive patients was 2.4% in the Italian database and 5.0% in the last French transmitted drug resistance survey study,1,2 and so was in a similar range to the prevalence observed in the present study. Thus, this polymorphism is not so rare. The high HIV-1 genetic diversity observed in the present study evidenced a differential distribution of the E157Q polymorphism among the different HIV-1 subtypes. Thus, the prevalence of the E157Q polymorphism was >3-fold higher in CRF02_AG than in B subtype sequences (5.6% versus 1.7%, P < 0.0001). This is a key point for the epidemiology of HIV infection in France because 59% of new infections are due to a non-B virus and half of them are CRF02_AG.2 Regarding non-B subtypes other than CRF02_AG, there is also a differential distribution of the E157Q polymorphism among them. The highest prevalences of E157Q were observed in H, CRF09_cpx, CRF06_cpx, D and G subtypes, and the lowest ones in CRF01_AE, C, A and F. Prevalence differences across various HIV-1 subtypes have also been described for other polymorphisms. For example, E138A in RT, impacting susceptibility to the NNRTIs rilpivirine and etravirine, was found to be twice as high in CRF02_AG than in B subtype sequences (4.1% versus 2.0%) in antiretroviral-naive patients.8 In this study, we assessed the virological outcome of 39 patients infected with an E157Q-mutated virus and receiving an INI-based regimen. Different clinical situations were found among the following groups: (i) virologically suppressed patients; (ii) antiretroviral-experienced patients in virological failure; and (iii) patients initiating their first-line antiretroviral regimen. Regarding virologically suppressed patients who switched to an INI-based regimen (n = 15), we observed a high level of virological suppression: 87% (n = 13/15) during the first year following the switch. Thus, the presence of E157Q does not seem to have a deleterious impact on the virological outcome in this specific clinical situation. Similar observations of virological suppression maintenance were described in very few cases of patients infected with an E138A-mutated virus who were switched to a rilpivirine-based STR.9,10 Regarding antiretroviral-experienced patients in virological failure, this is a more heterogeneous clinical situation with different therapeutic histories across the patients. In most cases, only one active antiretroviral drug was associated with the INI. However, as could be expected, the virological outcome was not associated with the GSS. Eight of the 16 patients in this clinical situation in our study did not reach VL <50 copies/mL. Among them, four still showed a significant VL decrease after INI-based initiation, but the remaining four represent a profile of non-virological response with no change in VL in three cases and only a slight VL decrease in the remaining case. In our study, the eight patients with a GSS ≤1 showed different virological responses, making it difficult to interpret INI intrinsic activity. Regarding antiretroviral-naive patients, most of them initiated an elvitegravir-based STR (n = 5/8), and two did not reach VL <50 copies/mL at W24 (i.e. 236 and 151 copies/mL) after a significant VL decrease following INI-based regimen initiation. These findings are unexpected since baseline plasma VL was <5 log10 copies/mL in both patients and since the INI drug class is characterized by a very rapid VL decrease after initiation. Unfortunately, no plasma drug concentrations were available for these patients. Obviously, observations in only two patients must be interpreted with caution. However, this could constitute a warning signal and emphasizes the need to pay particular attention to antiretroviral-naive patients infected with an E157Q-mutated virus who initiate an INI-based regimen. In vitro phenotypic assays were performed here in context B, as usual, but also in context CRF02_AG as half of the patients initiating a first-line INI-based regimen were infected with CRF02_AG. The E157Q site-directed mutants did not show an increased phenotypic resistance level to dolutegravir or raltegravir in either the B or the CRF02_AG subtype. These in vitro phenotypic data are in accordance with recent findings conducted in six clinical isolates with the E157Q polymorphism using similar phenotypic assays. They showed that all six clinical samples were susceptible to dolutegravir and to raltegravir, except one at the limit of the biological cut-off for raltegravir.1 The E157Q mutant did not result in an increased fold change in resistance to raltegravir and dolutegravir in our study or in the Italian study. However, in both studies, only the effect of the single E157Q mutant was characterized. Interestingly, we also evaluated phenotypic susceptibility to elvitegravir and observed a slight increase in fold change of 1.9 and 2.4 in the presence of E157Q in context B and context CRF02_AG, respectively. Similar results with E157Q tested in the presence of elvitegravir have been previously published showing an increased fold change of between 2 and 10.11 In addition, these assays did not assess the whole IN of the virus, still less the whole viral genome. Indeed, we can hypothesize the possible impact of polymorphic IN residues associated with the E157Q mutation in clinical isolates or the impact of mutations located outside the IN region, as recently shown with mutations selected in vitro in the nef gene.12 Furthermore, previous studies have shown the importance of the IN genetic context for INI phenotypic susceptibility.13 These findings showed that the prevalence of E157Q polymorphism was not negligible, particularly in CRF02_AG, which is of interest in European and French HIV epidemiology since recent data showed that 59% of new infections in France are with non-B subtypes.2 In addition, among INI-naive patients two did not reach VL <50 copies/mL while receiving elvitegravir. These findings support the recommendation of IN pre-therapeutic genotypic resistance tests in France and indicate the need to pay particular attention to ART-naive patients with an E157Q-mutated virus who initiate an INI-based regimen. Based on the present data and on previous in vitro findings, in the case of E157Q polymorphism the most preferable INI might be dolutegravir in such patients; elvitegravir should not be considered owing to potential low-level resistance, especially in the context of the CRF02_AG recombinant, and raltegravir seems adequate, with IC50 not increased, but with the caveat that selection of E157Q has been described at virological failure in two case reports. Acknowledgements We would like to thank all of the virological and clinical investigators involved in the ANRS AC11 virology network. Funding The research leading to these results has received funding from the French National Agency for Research on AIDS and Viral Hepatitis (ANRS). Transparency declarations C. C., E. A.-G., V. C., A.-G. M. and D. D. have received honoraria and travel grants from ViiV Healthcare, Janssen-Cilag, Gilead Sciences and MSD. All other authors: none to declare. References 1 Saladini F, Giannini A, Boccuto A et al.   The HIV-1 integrase E157Q polymorphism per se does not alter susceptibility to raltegravir and dolutegravir in vitro. AIDS  2017; 31: 2307– 9. Google Scholar CrossRef Search ADS PubMed  2 Assoumou L, Bocket L, Pallier C et al.   Stable prevalence of transmitted drug resistance mutations and increase circulation of non-B subtype in antiretroviral-naive chronically HIV-infected patients in 2015/2016 in France: the ANRS Odyssee study. In: Abstracts of the Ninth IAS Conference on HIV Science, Paris, France, 2017. Abstract TUPEC0851. 3 Malet I, Delelis O, Valantin M-A et al.   Mutations associated with failure of raltegravir treatment affect integrase sensitivity to the inhibitor in vitro. Antimicrob Agents Chemother  2008; 52: 1351– 8. Google Scholar CrossRef Search ADS PubMed  4 Ghosn J, Mazet A-A, Avettand-Fenoel V et al.   Rapid selection and archiving of mutation E157Q in HIV-1 DNA during short-term low-level replication on a raltegravir-containing regimen. J Antimicrob Chemother  2009; 64: 433– 4. Google Scholar CrossRef Search ADS PubMed  5 Danion F, Belissa E, Peytavin G et al.   Non-virological response to a dolutegravir-containing regimen in a patient harbouring a E157Q-mutated virus in the integrase region. J Antimicrob Chemother  2015; 70: 1921– 3. Google Scholar PubMed  6 Ambrosioni J, Nicolás D, Manzardo C et al.   Integrase strand-transfer inhibitor polymorphic and accessory resistance substitutions in patients with acute/recent HIV infection. J Antimicrob Chemother  2017; 72: 205– 9. Google Scholar CrossRef Search ADS PubMed  7 Pavkovich D, Richmond D, Veltman F. Reviewing clinical outcomes of patients with the E157Q mutation in Detroit. In: Abstracts of IDWeek 2017, San Diego, CA, USA. Abstract 1374. 8 Lambert-Niclot S, Charpentier C, Storto A et al.   Prevalence of pre-existing resistance-associated mutations to rilpivirine, emtricitabine and tenofovir in antiretroviral-naive patients infected with B and non-B subtype HIV-1 viruses. J Antimicrob Chemother  2013; 68: 1237– 42. Google Scholar CrossRef Search ADS PubMed  9 Porter DP, Toma J, Tan Y et al.   Clinical outcomes of virologically-suppressed patients with pre-existing HIV-1 drug resistance mutations switching to rilpivirine/emtricitabine/tenofovir disoproxil fumarate in the SPIRIT study. HIV Clin Trials  2016; 17: 29– 37. Google Scholar CrossRef Search ADS PubMed  10 Amiel C, Schneider V, Guessant S et al.   Initiation of rilpivirine, tenofovir and emtricitabine (RPV/TDF/FTC) regimen in 363 patients with virological vigilance assessment in ‘real life’. J Antimicrob Chemother  2014; 69: 3335– 9. Google Scholar CrossRef Search ADS PubMed  11 Serrao E, Odde S, Ramkumar K et al.   Raltegravir, elvitegravir, and metoogravir: the birth of ‘me-too’ HIV-1 integrase inhibitors. Retrovirology  2009; 6: 25. Google Scholar CrossRef Search ADS PubMed  12 Malet I, Subra F, Charpentier C et al.   Mutations located outside the integrase gene can confer resistance to HIV-1 integrase strand transfer inhibitors. MBio  2017; 8: e00922– 17. Google Scholar CrossRef Search ADS PubMed  13 Munir S, Thierry E, Malet I et al.   G118R and F121Y mutations identified in patients failing raltegravir treatment confer dolutegravir resistance. J Antimicrob Chemother  2015; 70: 739– 49. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

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Oxford University Press
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0305-7453
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1460-2091
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10.1093/jac/dkx511
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Abstract

Abstract Objectives To assess the phenotypic susceptibility of the E157Q polymorphism in HIV-1 integrase (IN) and the virological outcome of patients infected with E157Q-mutated virus initiating an IN inhibitor (INI)-based regimen. Methods This was a multicentre study assessing IN sequences from INI-naive patients among 17 French HIV clinical centres. E157Q site-directed mutants in pNL4.3 and pCRF02_AG contexts were assessed in a recombinant phenotypic assay. Results Prevalence of the E157Q polymorphism was 2.7% among 8528 IN sequences from INI-naive patients and its distribution was 1.7%, 5.6% and 2.2% in B, CRF02_AG and various non-B subtypes, respectively. Thirty-nine INI-naive patients with E157Q-mutated virus initiated an INI-based regimen. Among them, 15 had a viral load (VL) <50 copies/mL at initiation and virological suppression was maintained during the first year of follow-up in all but two exhibiting a viral blip. Twenty-four patients had a VL > 50 copies/mL at the time of INI-based regimen initiation. Among them eight were receiving a first-line regimen and the only two patients who did not reach VL < 50 copies/mL at week 24 were receiving elvitegravir. The 16 remaining patients were ART experienced in virological failure with drug-resistant viruses displaying several virological outcomes independently of the genotypic susceptibility score. Phenotypic analyses showed a fold change in EC50 of 0.6, 0.9 and 1.9 for raltegravir, dolutegravir and elvitegravir, respectively, in a subtype B context, and 1.1, 1.9 and 2.4 for raltegravir, dolutegravir and elvitegravir, respectively, in a CRF02_AG context. Conclusions Assessment of virological response in 39 patients initiating an INI-based regimen with E157Q-mutated virus, in combination with phenotypic analysis, suggests that particular attention should be paid to antiretroviral-naive patients and dolutegravir should be preferentially used in these patients. Introduction Integrase (IN) inhibitors (INIs) are now recommended by multiple international guidelines in first-line regimens of HIV-1 infection treatment and their use is increasing. The major INI resistance mutations, resulting in a high level of phenotypic resistance, are well described for first-generation INIs (raltegravir and elvitegravir), but remain not fully elucidated for dolutegravir. The IN E157Q substitution has been previously described as a polymorphism present in around 3%–5% of viral sequences obtained from ART-naive patients.1,2 However, the E157Q change has also been observed in cases of virological failure under a raltegravir-based treatment in two case reports.3,4 More recently, a case report of a non-virological response to a dolutegravir-based regimen has been described in a patient infected with an E157Q-mutated virus.5 Thus, there are few data on the prevalence of the E157Q substitution in a large dataset of IN sequences representing the genetic diversity of HIV-1. Regarding the virological response to an INI-based regimen for patients displaying E157Q mutation, only case reports are available.6,7 In the study of Ambrosioni et al.,6 only two ART-naive patients initiated a dolutegravir-based regimen with a very short follow-up of 12 weeks. In the study by Pavkovich et al.,7 only 6 of the 15 patients initiating an INI-based regimen achieved virological success at month 12; however, heterogeneous profiles of ART-naive and ART-experienced patients were not distinguished in the analysis. The aim of this study was to assess the prevalence of E157Q polymorphism in IN sequences obtained from INI-naive patients across various HIV-1 subtypes and the virological outcome at week 24 (W24) and week 48 (W48) of patients infected with an E157Q-mutated virus initiating an INI-based regimen. Methods IN sequences This was a national multicentre study assessing all available IN sequences from INI-naive patients among 17 French HIV clinical centres included in the Agence Nationale de Recherche sur le SIDA et les hépatites virales (ANRS) virology network. Ethics Participating laboratories belong to ANRS and participate in the ANRS quality control assessment of HIV-1 drug resistance sequencing. The study was approved by the scientific committee of Action Coordonnée n° 11 of ANRS. pol sequencing The pol gene was sequenced by standard Sanger technology using ANRS procedures (www.hivfrenchresistance.org). The genotypic susceptibility score (GSS) of the ART was calculated using the ANRS algorithm (September 2016, version 26), translating the interpretations ‘susceptible’, ‘possible resistance’ and ‘resistance’ into scores of 1, 0.5 and 0, respectively. HIV-1 subtyping Phylogenetic relationships of reverse transcriptase (RT) were estimated from sequence comparisons with previously reported representatives of HIV-1 group M subtypes and circulating recombinant forms (CRFs) for which sequences are available in the Los Alamos HIV database (https://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html). Phenotypic assays The E157Q substitution was introduced into the pNL4-3 sequence (subtype B) and the pCRF02_AG plasmid (kindly provided by M. Wainberg and T. Mesplede) by site-directed mutagenesis using the Quickchange II XL Site-Directed Mutagenesis® Kit (Agilent), according to the manufacturer’s instructions. HIV-1 stocks were prepared by transfecting 293-T cells using Lipofectamine reagent (Thermo Fisher) with the various HIV-1 molecular clones. The titre of the supernatant was determined by quantifying HIV-1 p24 antigen using the VIDAS® instrument (bioMérieux). Briefly, HeLa-P4 cells were infected in triplicate in 96-well plates with WT virus or mutants containing the E157Q IN mutation (equivalent of 10 ng of p24 antigen), with increasing concentrations of raltegravir or dolutegravir (from 0 to 200 nM). The viral titre was determined 48 h post-infection by quantifying β-galactosidase activity in HeLa-P4 lysates in a colorimetric assay based on the cleavage of chlorophenol red-β-d-galactopyranoside by β-galactosidase. The EC50 of each drug was defined as the drug concentration resulting in β-galactosidase levels that were 50% lower than those in infected cells without the drug. Results Prevalence of the E157Q natural polymorphism among various HIV-1 subtypes A total of 8528 IN sequences obtained between 2010 and 2016 from INI-naive patients in routine clinical care were analysed; 4759 (56%) of the sequences were subtype B, 1945 (23%) were CRF02_AG and 1824 (21%) were various non-B subtypes other than CRF02_AG. Overall, prevalence of E157Q polymorphism was 2.7% (95% CI 2.4%–3.1%). Distribution of E157Q polymorphism among HIV-1 subtypes was 1.7% (95% CI 1.3%–2.1%), 5.6% (95% CI 4.6%–6.6%) and 2.2% (95% CI 1.6%–2.9%) in B, CRF02_AG and various non-B subtypes, respectively. The distribution of E157Q polymorphism was as follows among the various non-B subtypes: 16.7% in subtype H (n = 4/24), 11.8% in CRF09_cpx (n = 2/17), 7.0% in CRF06_cpx (n = 3/43), 5.6% in subtype D (n = 7/124), 3.1% in subtype G (n = 4/127), 2.1% in CRF01_AE (n = 3/140), 2.0% in subtype C (n = 4/199), 1.6% in subtype A (n = 5/320), 1.1% in subtype F (n = 2/189) and 1.5% in complex recombinants (n = 7/452) (Figure 1). Figure 1. View largeDownload slide Prevalence of the E157Q IN polymorphism among various HIV-1 subtypes from INI-naive patients. *Non-B subtypes other than CRF02_AG. **Complex recombinants not identified. Figure 1. View largeDownload slide Prevalence of the E157Q IN polymorphism among various HIV-1 subtypes from INI-naive patients. *Non-B subtypes other than CRF02_AG. **Complex recombinants not identified. Virological outcome of patients with an E157Q-mutated virus initiating an INI-based regimen Thirty-nine INI-naive patients with an E157Q-mutated virus initiated an INI-based regimen, including raltegravir in 19 cases, elvitegravir in 10 cases and dolutegravir in 10 cases. IN sequences showed that E157Q was the only INI resistance mutation in all 39 viruses, except in two cases in which it was associated with the E92Q or T97A mutation. Among these 39 patients, 15 patients were in virological success [i.e. plasma viral load (VL) <50 copies/mL] under their ongoing regimen at the time of INI-based regimen initiation. Virological suppression was maintained during the first year of the follow-up in 13 patients (87%). The two remaining patients exhibited a VL >50 copies/mL at W48 (53 and 267 copies/mL), which became undetectable again at the VL control performed within 4 weeks, thus corresponding to a viral blip. Twenty-four patients initiated an INI-based regimen with a VL >50 copies/mL (median 4.9 log10 copies/mL, IQR 4.3–5.1): 8 were receiving a first-line regimen (Table 1) and 16 were ART-experienced patients in virological failure with viruses harbouring drug resistance mutations (Table 2). INI-based regimen discontinuation occurred between W24 and W48 in 2/8 patients on a first-line regimen and in 5/16 antiretroviral-experienced patients. Table 1. Description and virological outcome of antiretroviral-naive patients initiating a first-line INI-based regimen INI  ARV associated with INI  GSS of associated ARV  HIV-1 subtype  VL at baseline of INI treatment (copies/mL)  VL at W24 of INI treatment (copies/mL)  VL decrease between day 0 and W24 (log10 copies/mL)  VL at W48 of INI treatment (copies/mL)  EVG  TDF/FTC/c  2  B  109 483  <50  −3.4  <50  EVG  TDF/FTC/c  2  CRF02_AG  3061  <50  −1.8  <50  EVG  TDF/FTC/c  2  B  425 900  <50  −4.0  STOP  EVG  TDF/FTC/c  2  B  99 750  151  −2.8  STOP  EVG  TDF/FTC/c  2  B  27 352  236  −2.0  NA  RAL  TDF/FTC  2  CRF02_AG  90 746  <50  −3.4  <50  RAL  DRV/r  1  B  168 255  <50  −3.6  <50  DTG  TDF/FTC  2.5  CRF02_AG  405 790  <50  −4.0  <50  INI  ARV associated with INI  GSS of associated ARV  HIV-1 subtype  VL at baseline of INI treatment (copies/mL)  VL at W24 of INI treatment (copies/mL)  VL decrease between day 0 and W24 (log10 copies/mL)  VL at W48 of INI treatment (copies/mL)  EVG  TDF/FTC/c  2  B  109 483  <50  −3.4  <50  EVG  TDF/FTC/c  2  CRF02_AG  3061  <50  −1.8  <50  EVG  TDF/FTC/c  2  B  425 900  <50  −4.0  STOP  EVG  TDF/FTC/c  2  B  99 750  151  −2.8  STOP  EVG  TDF/FTC/c  2  B  27 352  236  −2.0  NA  RAL  TDF/FTC  2  CRF02_AG  90 746  <50  −3.4  <50  RAL  DRV/r  1  B  168 255  <50  −3.6  <50  DTG  TDF/FTC  2.5  CRF02_AG  405 790  <50  −4.0  <50  /c, boosted with cobicistat; ARV, antiretroviral, DRV/r, darunavir boosted with ritonavir; DTG, dolutegravir; EVG, elvitegravir; FTC, emtricitabine; NA, not available; RAL, raltegravir; TDF, tenofovir disoproxil fumarate. Table 2. Description and virological outcome of pretreated patients initiating an INI-based regimen with VL > 50 copies/mL INI  ARV associated with INI  GSS of associated ARV  HIV-1 subtype  VL at baseline of INI treatment (copies/mL)  VL at W24 of INI treatment (copies/mL)  VL decrease between day 0 and W24 (log10 copies/mL)  VL at W48 of INI treatment (copies/mL)  RAL  ETR/DRV/r  1  D  59 900  <50  −3.1  <50  RAL  DRV/r  1  CRF02_AG  45 574  <50  −3.1  <50  RAL  ZDV/ABC/3TC/DRV/ATV/r  0.5  non-B  66 200  <50  −3.1  <50  RAL  ABC/3TC/DRV/r  3  B  336 211  <50  −3.8  63  RAL  DRV/r  1  CRF02_AG  139  <50  −0.5  97  RAL  DRV/r  1  CRF02_AG  472  <50  −1.1  STOP  RAL  ABC/3TC/TDF/EFV  2  CRF02_AG  1423  454  −0.5  STOP  RAL  DRV/r  1  B  128 000  128  −3.0  STOP  RAL  NVP  1  CRF02_AG  2200  NA  NA  2852  RAL  ABC/3TC/ETR/DRV/r/ENF  1.5  CRF02_AG  179 594  102  −3.2  131  RAL  TDF/FTC  2  B  136  343  +0.4  STOP  EVG  TDF/FTC/c  2  CRF02_AG  81 989  121  −2.8  182  EVG  TDF/FTC/c  2  CRF02_AG  1 094 101  23 434  −1.7  6660  DTG  TDF/FTC  2.5  non-B  65  <50  −0.5  <50  DTGa  ABC/3TC/TDF/DRV/r  1  G  75 700  105  −2.9  145  DTGb  TDF/ETR/DRV/r/ENF  3  B  118 000  <50  −3.5  STOP  INI  ARV associated with INI  GSS of associated ARV  HIV-1 subtype  VL at baseline of INI treatment (copies/mL)  VL at W24 of INI treatment (copies/mL)  VL decrease between day 0 and W24 (log10 copies/mL)  VL at W48 of INI treatment (copies/mL)  RAL  ETR/DRV/r  1  D  59 900  <50  −3.1  <50  RAL  DRV/r  1  CRF02_AG  45 574  <50  −3.1  <50  RAL  ZDV/ABC/3TC/DRV/ATV/r  0.5  non-B  66 200  <50  −3.1  <50  RAL  ABC/3TC/DRV/r  3  B  336 211  <50  −3.8  63  RAL  DRV/r  1  CRF02_AG  139  <50  −0.5  97  RAL  DRV/r  1  CRF02_AG  472  <50  −1.1  STOP  RAL  ABC/3TC/TDF/EFV  2  CRF02_AG  1423  454  −0.5  STOP  RAL  DRV/r  1  B  128 000  128  −3.0  STOP  RAL  NVP  1  CRF02_AG  2200  NA  NA  2852  RAL  ABC/3TC/ETR/DRV/r/ENF  1.5  CRF02_AG  179 594  102  −3.2  131  RAL  TDF/FTC  2  B  136  343  +0.4  STOP  EVG  TDF/FTC/c  2  CRF02_AG  81 989  121  −2.8  182  EVG  TDF/FTC/c  2  CRF02_AG  1 094 101  23 434  −1.7  6660  DTG  TDF/FTC  2.5  non-B  65  <50  −0.5  <50  DTGa  ABC/3TC/TDF/DRV/r  1  G  75 700  105  −2.9  145  DTGb  TDF/ETR/DRV/r/ENF  3  B  118 000  <50  −3.5  STOP  /c, boosted with cobicistat; /r, boosted with ritonavir; 3TC, lamivudine; ABC, abacavir; ARV, antiretroviral; ATV, atazanavir; DRV, darunavir; DTG, dolutegravir; ENF, enfuvirtide; EFV, efavirenz; ETR, etravirine; EVG, elvitegravir; FTC, emtricitabine; NA, not available; NVP, nevirapine; RAL, raltegravir; TDF, tenofovir disoproxil fumarate; ZDV, zidovudine. a Double mutant E92Q-E157Q. b Double mutant T97A-E157Q. Among the eight patients receiving a first-line regimen, five received a tenofovir/emtricitabine/elvitegravir/cobicistat single-tablet regimen (STR), one received tenofovir/emtricitabine and raltegravir, one received tenofovir/emtricitabine and dolutegravir and one received raltegravir plus darunavir/ritonavir. Five patients were infected with subtype B and three with CRF02_AG. Six of the eight patients reached VL <50 copies/mL at W24 and virological suppression was maintained at W48 in five of these patients still receiving an INI-based regimen at W48 (Table 1). Regarding the two patients who had not reached VL <50 copies/mL at W24, VL was 236 and 151 copies/mL after a significant VL decrease between INI-based regimen initiation and W24 of −2.0 and −2.8 log10 copies/mL, respectively. Both patients were receiving an elvitegravir-based STR and were infected with subtype B. Their follow-up between W24 and W48 showed that one discontinued the INI-based regimen after retrospective evidence of the E157Q polymorphism in the pre-therapeutic genotype and one died (not related to HIV infection). Regarding the 16 ART-experienced patients in virological failure harbouring viruses with drug resistance mutations, 4 were in virological success with VL <50 copies/mL at W24 and at W48 (Table 2). Four additional patients had reached VL <50 copies/mL at W24, including one displaying the T97A–E157Q double mutant at baseline. Among these four patients, two exhibited a viral blip at W48 (63 and 97 copies/mL) and two discontinued INI after W24: one was lost to follow-up and in the other there was retrospective detection of the E157Q polymorphism in the pre-therapeutic genotype. Four other patients showed an initial significant VL decrease >2.5 log10 copies/mL between INI-based regimen initiation and W24, but without reaching 50 copies/mL at W24 (102, 105, 121 and 128 copies/mL), including one patient displaying the E92Q–E157Q double mutation at baseline. At W48, three of these patients were still receiving an INI-based regimen and all displayed low-level viraemia between 100 and 200 copies/mL. Four other patients had no virological response, with a VL decrease <2 log10 copies/mL at W24. The virological outcome of the 16 antiretroviral-experienced patients was not associated with the number of active antiretroviral drugs associated with the INI. Indeed, 8 of the 16 patients had a GSS ≤1 and they were equally distributed among the different profiles of virological outcome described (Table 2). Phenotypic susceptibility of the E157Q mutant to raltegravir, elvitegravir and dolutegravir In cells carrying E157Q site-directed mutations, the fold change in EC50 of raltegravir, elvitegravir and dolutegravir was 0.6, 1.9 and 0.9, respectively, in the subtype B context and 1.1, 2.4 and 1.9, respectively, in the CRF02_AG context (Table 3). Table 3. Effects of the E157Q mutation on the EC50 and standard deviation for raltegravir, elvitegravir and dolutegravir in HeLa-P4 cells Backbone  Genotype  Raltegravir   Elvitegravir   Dolutegravir   EC50 (nM)  standard deviation (nM)  fold change  EC50 (nM)  standard deviation (nM)  fold change  EC50 (nM)  standard deviation (nM)  fold change  pNL43 (B)  WT  20.3  7.8  –  20.1  5.5  –  7.7  2.1  –  E157Q  12.2  5.2  0.6  39.1  2.4  1.9  6.9  1.5  0.9  pCRF02_AG  WT  26  7.1  –  39.8  15.6  –  7.1  3.4  –  E157Q  29.3  0.9  1.1  95.5  31  2.4  13.5  1.0  1.9  Backbone  Genotype  Raltegravir   Elvitegravir   Dolutegravir   EC50 (nM)  standard deviation (nM)  fold change  EC50 (nM)  standard deviation (nM)  fold change  EC50 (nM)  standard deviation (nM)  fold change  pNL43 (B)  WT  20.3  7.8  –  20.1  5.5  –  7.7  2.1  –  E157Q  12.2  5.2  0.6  39.1  2.4  1.9  6.9  1.5  0.9  pCRF02_AG  WT  26  7.1  –  39.8  15.6  –  7.1  3.4  –  E157Q  29.3  0.9  1.1  95.5  31  2.4  13.5  1.0  1.9  Discussion In this study, based on a large dataset of HIV-1 IN sequences, with >8500 viral sequences obtained from INI-naive patients infected with a large variety of HIV-1 subtypes, we described an overall prevalence of E157Q polymorphism of 2.7%. Virological response of patients displaying an E157Q-mutated virus and initiating an INI-based regimen was assessed in 39 patients showing different profiles of virological outcome. The prevalence of the E157Q IN polymorphism in viral sequences obtained from antiretroviral-naive patients was 2.4% in the Italian database and 5.0% in the last French transmitted drug resistance survey study,1,2 and so was in a similar range to the prevalence observed in the present study. Thus, this polymorphism is not so rare. The high HIV-1 genetic diversity observed in the present study evidenced a differential distribution of the E157Q polymorphism among the different HIV-1 subtypes. Thus, the prevalence of the E157Q polymorphism was >3-fold higher in CRF02_AG than in B subtype sequences (5.6% versus 1.7%, P < 0.0001). This is a key point for the epidemiology of HIV infection in France because 59% of new infections are due to a non-B virus and half of them are CRF02_AG.2 Regarding non-B subtypes other than CRF02_AG, there is also a differential distribution of the E157Q polymorphism among them. The highest prevalences of E157Q were observed in H, CRF09_cpx, CRF06_cpx, D and G subtypes, and the lowest ones in CRF01_AE, C, A and F. Prevalence differences across various HIV-1 subtypes have also been described for other polymorphisms. For example, E138A in RT, impacting susceptibility to the NNRTIs rilpivirine and etravirine, was found to be twice as high in CRF02_AG than in B subtype sequences (4.1% versus 2.0%) in antiretroviral-naive patients.8 In this study, we assessed the virological outcome of 39 patients infected with an E157Q-mutated virus and receiving an INI-based regimen. Different clinical situations were found among the following groups: (i) virologically suppressed patients; (ii) antiretroviral-experienced patients in virological failure; and (iii) patients initiating their first-line antiretroviral regimen. Regarding virologically suppressed patients who switched to an INI-based regimen (n = 15), we observed a high level of virological suppression: 87% (n = 13/15) during the first year following the switch. Thus, the presence of E157Q does not seem to have a deleterious impact on the virological outcome in this specific clinical situation. Similar observations of virological suppression maintenance were described in very few cases of patients infected with an E138A-mutated virus who were switched to a rilpivirine-based STR.9,10 Regarding antiretroviral-experienced patients in virological failure, this is a more heterogeneous clinical situation with different therapeutic histories across the patients. In most cases, only one active antiretroviral drug was associated with the INI. However, as could be expected, the virological outcome was not associated with the GSS. Eight of the 16 patients in this clinical situation in our study did not reach VL <50 copies/mL. Among them, four still showed a significant VL decrease after INI-based initiation, but the remaining four represent a profile of non-virological response with no change in VL in three cases and only a slight VL decrease in the remaining case. In our study, the eight patients with a GSS ≤1 showed different virological responses, making it difficult to interpret INI intrinsic activity. Regarding antiretroviral-naive patients, most of them initiated an elvitegravir-based STR (n = 5/8), and two did not reach VL <50 copies/mL at W24 (i.e. 236 and 151 copies/mL) after a significant VL decrease following INI-based regimen initiation. These findings are unexpected since baseline plasma VL was <5 log10 copies/mL in both patients and since the INI drug class is characterized by a very rapid VL decrease after initiation. Unfortunately, no plasma drug concentrations were available for these patients. Obviously, observations in only two patients must be interpreted with caution. However, this could constitute a warning signal and emphasizes the need to pay particular attention to antiretroviral-naive patients infected with an E157Q-mutated virus who initiate an INI-based regimen. In vitro phenotypic assays were performed here in context B, as usual, but also in context CRF02_AG as half of the patients initiating a first-line INI-based regimen were infected with CRF02_AG. The E157Q site-directed mutants did not show an increased phenotypic resistance level to dolutegravir or raltegravir in either the B or the CRF02_AG subtype. These in vitro phenotypic data are in accordance with recent findings conducted in six clinical isolates with the E157Q polymorphism using similar phenotypic assays. They showed that all six clinical samples were susceptible to dolutegravir and to raltegravir, except one at the limit of the biological cut-off for raltegravir.1 The E157Q mutant did not result in an increased fold change in resistance to raltegravir and dolutegravir in our study or in the Italian study. However, in both studies, only the effect of the single E157Q mutant was characterized. Interestingly, we also evaluated phenotypic susceptibility to elvitegravir and observed a slight increase in fold change of 1.9 and 2.4 in the presence of E157Q in context B and context CRF02_AG, respectively. Similar results with E157Q tested in the presence of elvitegravir have been previously published showing an increased fold change of between 2 and 10.11 In addition, these assays did not assess the whole IN of the virus, still less the whole viral genome. Indeed, we can hypothesize the possible impact of polymorphic IN residues associated with the E157Q mutation in clinical isolates or the impact of mutations located outside the IN region, as recently shown with mutations selected in vitro in the nef gene.12 Furthermore, previous studies have shown the importance of the IN genetic context for INI phenotypic susceptibility.13 These findings showed that the prevalence of E157Q polymorphism was not negligible, particularly in CRF02_AG, which is of interest in European and French HIV epidemiology since recent data showed that 59% of new infections in France are with non-B subtypes.2 In addition, among INI-naive patients two did not reach VL <50 copies/mL while receiving elvitegravir. These findings support the recommendation of IN pre-therapeutic genotypic resistance tests in France and indicate the need to pay particular attention to ART-naive patients with an E157Q-mutated virus who initiate an INI-based regimen. Based on the present data and on previous in vitro findings, in the case of E157Q polymorphism the most preferable INI might be dolutegravir in such patients; elvitegravir should not be considered owing to potential low-level resistance, especially in the context of the CRF02_AG recombinant, and raltegravir seems adequate, with IC50 not increased, but with the caveat that selection of E157Q has been described at virological failure in two case reports. Acknowledgements We would like to thank all of the virological and clinical investigators involved in the ANRS AC11 virology network. Funding The research leading to these results has received funding from the French National Agency for Research on AIDS and Viral Hepatitis (ANRS). Transparency declarations C. C., E. A.-G., V. C., A.-G. M. and D. D. have received honoraria and travel grants from ViiV Healthcare, Janssen-Cilag, Gilead Sciences and MSD. All other authors: none to declare. References 1 Saladini F, Giannini A, Boccuto A et al.   The HIV-1 integrase E157Q polymorphism per se does not alter susceptibility to raltegravir and dolutegravir in vitro. AIDS  2017; 31: 2307– 9. Google Scholar CrossRef Search ADS PubMed  2 Assoumou L, Bocket L, Pallier C et al.   Stable prevalence of transmitted drug resistance mutations and increase circulation of non-B subtype in antiretroviral-naive chronically HIV-infected patients in 2015/2016 in France: the ANRS Odyssee study. In: Abstracts of the Ninth IAS Conference on HIV Science, Paris, France, 2017. Abstract TUPEC0851. 3 Malet I, Delelis O, Valantin M-A et al.   Mutations associated with failure of raltegravir treatment affect integrase sensitivity to the inhibitor in vitro. Antimicrob Agents Chemother  2008; 52: 1351– 8. Google Scholar CrossRef Search ADS PubMed  4 Ghosn J, Mazet A-A, Avettand-Fenoel V et al.   Rapid selection and archiving of mutation E157Q in HIV-1 DNA during short-term low-level replication on a raltegravir-containing regimen. J Antimicrob Chemother  2009; 64: 433– 4. Google Scholar CrossRef Search ADS PubMed  5 Danion F, Belissa E, Peytavin G et al.   Non-virological response to a dolutegravir-containing regimen in a patient harbouring a E157Q-mutated virus in the integrase region. J Antimicrob Chemother  2015; 70: 1921– 3. Google Scholar PubMed  6 Ambrosioni J, Nicolás D, Manzardo C et al.   Integrase strand-transfer inhibitor polymorphic and accessory resistance substitutions in patients with acute/recent HIV infection. J Antimicrob Chemother  2017; 72: 205– 9. Google Scholar CrossRef Search ADS PubMed  7 Pavkovich D, Richmond D, Veltman F. Reviewing clinical outcomes of patients with the E157Q mutation in Detroit. In: Abstracts of IDWeek 2017, San Diego, CA, USA. Abstract 1374. 8 Lambert-Niclot S, Charpentier C, Storto A et al.   Prevalence of pre-existing resistance-associated mutations to rilpivirine, emtricitabine and tenofovir in antiretroviral-naive patients infected with B and non-B subtype HIV-1 viruses. J Antimicrob Chemother  2013; 68: 1237– 42. Google Scholar CrossRef Search ADS PubMed  9 Porter DP, Toma J, Tan Y et al.   Clinical outcomes of virologically-suppressed patients with pre-existing HIV-1 drug resistance mutations switching to rilpivirine/emtricitabine/tenofovir disoproxil fumarate in the SPIRIT study. HIV Clin Trials  2016; 17: 29– 37. Google Scholar CrossRef Search ADS PubMed  10 Amiel C, Schneider V, Guessant S et al.   Initiation of rilpivirine, tenofovir and emtricitabine (RPV/TDF/FTC) regimen in 363 patients with virological vigilance assessment in ‘real life’. J Antimicrob Chemother  2014; 69: 3335– 9. Google Scholar CrossRef Search ADS PubMed  11 Serrao E, Odde S, Ramkumar K et al.   Raltegravir, elvitegravir, and metoogravir: the birth of ‘me-too’ HIV-1 integrase inhibitors. Retrovirology  2009; 6: 25. Google Scholar CrossRef Search ADS PubMed  12 Malet I, Subra F, Charpentier C et al.   Mutations located outside the integrase gene can confer resistance to HIV-1 integrase strand transfer inhibitors. MBio  2017; 8: e00922– 17. Google Scholar CrossRef Search ADS PubMed  13 Munir S, Thierry E, Malet I et al.   G118R and F121Y mutations identified in patients failing raltegravir treatment confer dolutegravir resistance. J Antimicrob Chemother  2015; 70: 739– 49. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Apr 1, 2018

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