The main phylogenetic group of HIV-1 variants (group M) is responsible for the majority of HIV infections worldwide; this group is subdivided into 10 subtypes or clades designated A through K. Although subtype B is the most prevalent form in the western world, approximately 90% of infections worldwide are caused by non-B subtypes, with subtype C accounting for almost 50% . Genetic diversity among HIV-1 subtypes could affect the development of resistance mutations; several reverse transcriptase and protease mutations appear to occur more frequently in non-B subtypes .
Raltegravir is an HIV-1 integrase strand transfer inhibitor that is active against multidrug-resistant HIV-1 and both CCR5-tropic and CXCR4-tropic HIV-1 in vitro[3,4]. The in-vitro potency of raltegravir is similar against subtype B and non-B isolates . The clinical efficacy of raltegravir as part of a combination regimen has been demonstrated in double-blind, randomized, phase III studies of treatment-naive patients (STARTMRK) [6,7] and patients with triple-class resistant virus who have failed prior therapy (BENCHMRK-1 and 2) [8,9]. Using data from these three studies, we examined the long-term efficacy of raltegravir in patients infected with non-B subtype HIV-1, as compared with those with subtype B.
STARTMRK (MK-0518 Protocol 021; NCT00369941) is a randomized, double-blind, active-controlled, noninferiority study conducted at 67 centers in Australia, Europe, North and South America, India, and Thailand. Treatment-naive patients were randomly assigned in a 1 : 1 ratio to receive raltegravir 400 mg b.i.d. or efavirenz 600 mg q.h.s., both in combination with tenofovir/emtricitabine [6,7]. BENCHMRK-1 (MK-0518 Protocol 018; NCT00293267) and BENCHMRK-2 (MK-0518 Protocol 019; NCT00293254) are randomized, double-blind, placebo-controlled, comparative studies conducted at 114 centers in Australia, Europe, North and South America, Taiwan, and Thailand. Highly treatment-experienced patients with multidrug-resistant virus and prior treatment failures were randomly assigned in a 2 : 1 ratio to receive raltegravir 400 mg b.i.d. or placebo, both in combination with optimized background therapy (OBT) [8,9]. All studies were approved by the Institutional Review Boards or Ethical Review Committees at each site, and all participants provided written informed consent. Combined analysis of the BENCHMRK studies was prespecified because of their identical study design; homogeneity testing confirmed the consistency of treatment effects between the studies (P > 0.10) . In the current study, data from STARTMRK and BENCHMRK-1 and 2 are presented separately due to the different study designs and treatment populations.
Viral subtypes were determined from patients’ baseline specimens using genotypic data of the PhenoSense GT test (Monogram Biosciences, South San Francisco, California, USA). Non-B subtypes were combined for the current analysis due to small numbers of each specific subtype. In order to focus on virologic responses, the observed failure approach was used for the subgroup efficacy analysis (only discontinuations due to lack of efficacy were treated as failures). A post-hoc stratified categorical analysis of a 2-by-2 contingency table was used to compare the treatment difference between strata (B-subtype vs. non-B subtype); P values were calculated using the Breslow–Day homogeneity test. Data through 96 weeks of treatment were included; this time point represents the last prespecified efficacy analysis for STARTMRK and the latest common time point available for all three studies.
Resistance to raltegravir was investigated in patients with virologic failure by genotyping the integrase coding sequence according to the following method: the entire integrase coding region was amplified from viral RNA by nested reverse transcriptase PCR (RT-PCR) using methods developed at Merck. Primers for reverse transcriptase and first-round PCR were INREV1 (5′-TCTCCTGTATGCAGACCCCAATAT-3′) and INFOR1 (5′-GGAATCATTCAAGCACAACCAGA-3′). Second-round primers were HIV + 4141 (5′-TCTACCTGGCATGGGTACCA-3′) and INREVII (5′-CCTAGTGGGATGTGTACTTCTGA-3′). PCR products were treated with shrimp alkaline phosphatase and exonuclease I and then sequenced on both strands using BigDye 3.1 sequencing reagents (Applied Biosystems by Life Technologies, Carlsbad, California, USA) and the following primers: INREVII, HIV + 4620 (5′-AARGCMGCCTGTTGGTGGGC-3′), HIV-4760 (5′-AGATTCYAYTACTCCTTGACTTTGG-3′), IN4 (5′-GGGCGGGGATCAAGCAGGAA-3′), and IN1REV (5′-TGCTGTCCCTGTAATAAACCC-3′); and RT9 (5′-GGAGGAAATGAACAAGTAGATA-3′) or HIV + 4141. Sequences were determined using an ABI3100 capillary sequencer (Applied Biosystems) and assembled and analyzed using SeqScape software (Applied Biosystems). For each patient, the inferred integrase amino acid sequences determined after virologic failure were compared with the baseline (pretreatment) sequence and treatment-emergent changes noted. The following mutations were considered raltegravir resistance mutations: L74M, E92Q, T97A, E138A/K, G140A/S, Y143C/H/R, Q148H/K/R, V151I, N155H, G163R, and S230R.
A total of 743 patients received raltegravir and 519 received the comparator agent, both in combination regimens, across the three studies. Non-B subtype virus isolated at baseline included subtypes A, A/C, A/D, A/G, A1, AE, AG, B/G, BF, C, D, D/F, F, F1, G, and complex. A total of 98 patients (13%) receiving raltegravir and 62 patients (12%) receiving comparator therapy were infected with HIV-1 non-B subtype virus; the most common non-B subtypes overall were AE and C, isolated in 41 and 43 patients, respectively. Non-B subtype infection was more common than B subtype among black patients and Asian patients, whereas B subtype infection was more common than non-B subtype among white patients and Hispanic patients. Baseline HIV-1 RNA levels and CD4 cell counts were generally similar between patients with B subtype and those with non-B subtypes (Table 1).
Among patients who received raltegravir for up to 96 weeks, virologic responses in patients with non-B subtype HIV-1 were similar to those in patients with B-subtype HIV-1 infection (Table 2). In treatment-naive patients, HIV RNA below 50 copies/ml was achieved in 89% of raltegravir recipients with B subtype and 95% of those with non-B subtype. In treatment-experienced patients, 61% of raltegravir recipients with B subtype and 67% with non-B subtype achieved HIV RNA below 50 copies/ml. Virologic response rates by specific non-B subtype are shown in Table 3. Across both patient populations, the most common non-B subtypes in the raltegravir group were AE (n = 25) and C (n = 25); at week 96, HIV-1 RNA below 50 copies/ml was achieved in 91% of patients with these HIV-1 subtypes.
Immunologic responses after 96 weeks of treatment also were similar in patients with non-B subtype as compared with those with B subtype infection (Table 2). The mean change from baseline in CD4 cell count for B and non-B subtype, respectively, was 243 vs. 221 cells/μl among treatment-naive patients and 121 vs. 144 cells/μl among treatment-experienced patients.
Among patients with non-B subtype infection who received raltegravir, virologic failure occurred in two patients from STARTMRK and seven patients from BENCHMRK-1 and 2. At time of failure, resistance-associated mutations were found in viral isolates from one of the STARTMRK patients and four of the BENCHMRK patients (Table 4). Viruses isolated from the four BENCHMRK patients also displayed phenotypic resistance to raltegravir, consistent with the observation that signature raltegravir resistance mutations (N155H or Y143H/C/R) were present in these viruses. In contrast, the resistance-associated mutation observed in the one STARTMRK patient, L74L/I/M, did not confer phenotypic resistance and may represent the emergence of a polymorphism. All of the phenotypic raltegravir resistance could be explained by the presence of known resistance mutations, and there was no evidence of previously unknown resistance mutations.
We examined the virologic and immunologic response to raltegravir of patients with non-B subtype HIV-1 infection, as compared with patients with B subtype HIV-1, in three phase III clinical trials. In these studies, raltegravir exhibited comparable antiviral clinical activity against B subtype and non-B subtype HIV-1 infection for up to 96 weeks in both treatment-naive and treatment-experienced populations. Although limited data preclude conclusions on individual non-B subtypes, all subtypes studied to date appeared to show favorable responses. The most common individual non-B subtypes were the AE and C subtypes, which also showed virologic responses comparable to those in B subtype infection. Clinically this is of great importance since non-B subtypes (and in particular subtype C) account for the majority of HIV-1 infections worldwide .
The HIV-1 integrase region is highly conserved across all HIV-1 subtypes , thus it was anticipated that raltegravir, as an HIV integrase inhibitor, would be active against all HIV-1 subtypes. This has been previously demonstrated in vitro and is now documented in clinical studies. Currently available protease and reverse transcriptase inhibitors also appear to be equally active against all HIV-1 subtypes . The effect of viral subtype on the development of resistance to antiviral therapy is of potential concern. However, in our study, phenotypic raltegravir resistance in non-B virus was associated with integrase mutations that have been observed previously in B subtype infection. Similar results have been reported for protease and reverse transcriptase inhibitors, although several mutations have been found to occur more frequently in non-B subtypes . The low number of mutations observed in our patient population limits the overall conclusions and underlines the need for more data to understand resistance development in non-B subtypes.
With the increasing availability of antiretroviral drugs and also the emergence of relevant HIV drug resistance, the availability of new drug classes with a different mode of action and preserved activity across all subtypes is desirable. Clearly more data for the various non-B subtypes are needed in order to obtain larger sample sizes and to further increase the level of confidence when using raltegravir in patients with non-B subtype HIV-1 infection.
All authors contributed to the conception and design of the studies and/or the analysis and interpretation of the data. The manuscript was drafted by J.K.R., K.M.S., and B-Y.T.N. and was critically reviewed and subsequently approved by all authors. Merck, Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., provided financial support for the clinical studies, which contributed data to these analyses. H.T., J.Z., P.S., M.D.M., C.M.H., K.M.S., R.Y.L., and B-Y.T.N. are employees of Merck, Sharp & Dohme Corp. and may own stock and/or stock options in the company.
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Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
HIV-1 subtype; integrase inhibitor; raltegravir; resistance mutations