Dolutegravir is a new HIV integrase inhibitor showing a potent antiviral activity with 88% of the patients receiving this once daily unboosted integrase inhibitor (INI) having a viral load below 50 copies/ml at W48 .
This compound is expected to display an improved resistance profile with a limited cross-resistance to raltegravir and elvitegravir [2,3], as none of the key resistance mutations previously described for these drugs (Y143C/H/R, Q148H/R/K, and N155H) were selected during the in-vitro serial passage experiments of dolutegravir. These latter experiments identified seven, single or combined, substitutions in integrase generated by passage of wild-type virus in the presence of dolutegravir: E92Q, T124A, S153Y, G193E, T124A/S153F, T124A/S153Y and L101I/T124A/S153Y [4,5]. Although the substitutions T124A and L101I are polymorphic and do not impact viral susceptibility level to dolutegravir, the substitutions E92Q, S153F/Y and G193E exhibit an approximate two- to three-fold increase in phenotypic susceptibility [4,5].
A study, based on 650 INI-naive, subtype B-infected, patients reported the prevalence of the L101I and T124A polymorphic substitutions of 45.8% and 24.5%, respectively . The double-mutant profile L101I/T124A was found in 10% of cases in INI-naive patients . Thus, although integrase gene sequences were found conserved among diverse HIV-1 subtypes , some substitutions observed during dolutegravir passage in vitro can be detected in virus isolated from INI-naive patients. Recently, a clinical trial showed that, in vivo, the integrase mutation R263K with a dolutegravir fold change below two was found to be selected in viruses from two INI-naive patients in virological failure with dolutegravir-based treatment .
Limited data are available as regards HIV-1 ‘non-B’ subtypes and dolutegravir phenotypic susceptibility. Indeed, most of the data are obtained from genotypic and phenotypic recombinant virus assays testing with HIV-1 subtype B viruses. Thus, considering the increasing frequency of HIV-1 ‘non-B’ subtypes in France and in Europe, dolutegravir phenotypic susceptibility data in INI-naive patients infected with ‘non-B’ subtypes are needed.
The aim of this study was to assess phenotypic susceptibility to dolutegravir and raltegravir in a large variety of HIV-1 ‘non-B’ subtypes in order to assess the impact of polymorphisms on drug susceptibility.
Phenotypic susceptibility to dolutegravir and raltegravir was determined using the Agence Nationale de Recherches sur le SIDA et les hépatites virales (ANRS) peripheral blood mononuclear cells (PBMC) method, as previously described . Phenotypic assay was performed with both dolutegravir and raltegravir drugs on: (i) BRU HIV-1 reference strain and (ii) co-cultivated HIV-1 clinical isolates obtained from 72 INI-naive ‘non-B’-infected patients. Briefly, after HIV-1 isolation from PBMC, the cell-free HIV-1 positive supernatant was serially diluted (100–10−2) and incubated with fresh normal phytohaemagglutinin-stimulated PBMC. After being washed, the cells were placed in 96-well plates containing six serial dilutions of the antiretroviral drug. Each dilution was tested in quadruplicate. On day 3, the supernatant was collected and the 50% tissue culture-infective dose (TCID50) was assessed by measuring the number of HIV-1 RNA copies in the supernatant with a real-time quantitative reverse transcription-PCR assay . Drug concentrations inhibiting the replication of 100 TCID50 by 50% were calculated (IC50). Integrase gene direct sequencing was performed in all specimens both on plasma samples and on supernatant at time of phenotypic assay. In this study, we assessed the mutations previously described in the development of resistance to raltegravir and elvitegravir, as primary or secondary mutations; and substitutions observed during the in-vitro serial passage experiments of dolutegravir. Taken together, the mutations list was: H51Y; T66A/I/K; V72I; L74A/I/M; E92Q; T97A; L101I; T112I; G118R, F121Y; T124A, T125K; A128T; E138A/D/K; G140A/C/H/R/S; Y143C/H/R; Q146P/K; S147G; Q148H/K/R; V151I; S153A/F/Y; M154I; N155H/S; K156N; E157Q; K160D/N; G163R/K; V165I; G193E; V201I; I203 M; T206S; S230N/R; V249I; R263K; and C280Y [4,5,7].
Among the 72 HIV-1 clinical isolates tested, recombinant CRF02_AG was the most prevalent virus (n = 16, 22%). The distribution of HIV-1 subtypes was: A (n = 12, 17%), C (n = 8, 11%), D (n = 8, 11%), F (n = 5, 7%), G (n = 4, 6%), CRF01_AE (n = 4, 6%), CRF06_cpx (n = 4, 6%), H (n = 2, 3%), CRF11_cpx (n = 2, 3%), CRF12_BF (n = 1, 1%), CRF14_BG (n = 1, 1%), CRF18_cpx (n = 1, 1%), CRF25_cpx (n = 1, 1%), CRF45_cpx (n = 1, 1%), and two undetermined subtypes (3%). No changes were observed in integrase gene for all of the HIV-1 clinical isolates between day 0 and day 3 of the PBMC phenotypic assay.
No major key INI resistance mutations were identified by direct sequencing. The most prevalent integrase substitutions detected were: V201I (n = 66, 84%), T124A (n = 56, 78%), L101I (n = 53, 74%); V72I (n = 49, 68%), and T206S (n = 37, 51%). All CRF02_AG viruses displayed both L101I and T124A. Additionally, the E157Q, G193E, and R263K mutations were detected in two, six, and one samples, respectively.
Phenotypic data showed that all the clinical isolates were susceptible to both dolutegravir and raltegravir with median IC50 values of 1.22 nmol/l (range = 0.08–3.72) for dolutegravir and of 1.53 nmol/l (range = 0.03–5.95) for raltegravir (Fig. 1). The control HIV-1 subtype B BRU reference strain showed an IC50 of 1.86 and 2.17 nmol/l for dolutegravir and raltegravir, respectively. The highest IC50 fold-changes observed were 2.0 for dolutegravir and 2.7 for raltegravir.
Focusing on the virus with the R263K mutation, this was a CRF01_AE virus showing an IC50 value of 1.39 nmol/l. The two E157Q-mutated viruses were CRF01_AE and CRF02_AG, showing susceptibility to raltegravir (IC50 = 0.16 and 1.09 nmol/l).
We assessed the in-vitro susceptibility of 72 HIV-1 ‘non-B’ clinical isolates, representing a large variety of viral subtypes, to the new INI compound dolutegravir and to raltegravir. In this study, despite the high prevalence of polymorphic substitutions in integrase gene in ‘non-B’ clinical isolates, IC50 values to dolutegravir and raltegravir remained similar to that observed for the B subtype. Indeed, fold-changes less than 2.7 obtained with the PBMC phenotypic assay do not induce a significant decrease in viral susceptibility to these drugs. The highest IC50 observed was not found to be associated with specific subtype/CRF or polymorphic substitutions.
The recent study by Vavro et al.  showed that the most prevalent integrase polymorphic substitutions did not impact phenotypic susceptibility to dolutegravir in the context of B subtype site-directed mutants; we confirmed these data in ‘non-B’ clinical isolates. Indeed, in our series, all CRF02_AG samples exhibited both L101I and T124A, added to the G193E for two of them, with no impact on dolutegravir susceptibility.
In conclusion, our findings did not identify a higher risk of decreased susceptibility to dolutegravir for ‘non-B’ subtypes, despite the genotypic variability.
C.C., F.B.V., J.C.P., and D.D. contributed to the study concept. M.B., B.V., J.L. and L.L. performed the phenotypic assays. C.C., G.P., G.C., T.M. and D.D. contributed to the analysis and interpretation of the data. C.C., F.B.V., J.C.P., and D.D. contributed to the writing of the article. All authors contributed to the critical reviewing of the article.
Conflicts of interest
There are no conflicts of interest.
Financial support: French National Agency for Research on AIDS and viral hepatitis [Agence Nationale de Recherches sur le SIDA et les hépatites virales (ANRS)] and from the European Community's Seventh Framework Programme (FP7/2007–2013) under the project ‘Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)’ (grant no. 223131). We also thank Shionogi-ViiV Healthcare LLC for financial support.
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