Currently recommended antiretroviral therapy (ART) for HIV-1 infection consists of combinations of 2 nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) with a nonnucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor. NRTI and NNRTI target HIV-1 reverse transcriptase (RT), an essential enzyme that catalyzes the synthesis of double-stranded HIV-1 DNA from single-stranded genomic RNA. HIV-1 RT is a heterodimeric protein composed of a catalytically active 66 kDa subunit with 3 domains: polymerase (residues 1-319), connection (residues 320-440) and RNase H (residues 441-560); and a catalytically inactive 51 kDa subunit that contains only nonfunctional polymerase and connection domains.1
Mutations in the polymerase domain of RT are known to confer HIV-1 resistance to NRTI and NNRTI.2 Recently, studies have described RT connection and RNase H domain mutations that are more frequent in ART-experienced patients compared with ART-naive patients.3-13 For example, connection domain mutation N348I has been observed to emerge early with failure of zidovudine (ZDV)/lamivudine (3TC)/nevirapine (NVP) and reduces sensitivity to ZDV, didanosine, NVP, efavirenz (EFV), delavirdine, tenofovir, and etravirine, either as a single mutation or in the context of polymerase domain resistance mutations.4,11,12,14,15 Other connection domain mutations, including G333D/E, G335C/D, A360I/V, A365I, and A376S have been shown to decrease ZDV susceptibility in the presence of thymidine analog mutations,5-7 and mutation T369I increases resistance to NNRTI in the presence of NNRTI resistance mutations.14
Importantly, genotype-based HIV-1 resistance tests available for clinical use only identify RT mutations in the polymerase domain and some portions of the connection domain. This restricted approach has raised concern that clinically important resistance mutations in RT are being missed.13,16,17 To address this concern, we sequenced full-length RT in pretherapy and virologic failure plasma samples from the 2 NRTI plus EFV arm of AIDS Clinical Trials Group (ACTG) study A5142.18 Sequences from pretherapy and time of confirmed virologic failure were compared to assess if mutations in the RT polymerase, connection, and RNase H domains emerged at virologic failure. In addition, pretherapy sequences from patients who did not experience virologic failure were obtained and compared with pretherapy sequences from patients with virologic failure to assess if polymorphisms in RT predispose to virologic failure of the 2 NRTI plus EFV regimen.
ACTG A5142 was a phase III, multicenter, randomized, open-label trial among ART-naive patients consisting of 3 arms: lopinavir/ritonavir (LPV/r) (Abbott, Abbott Park, IL) plus EFV (Bristol-Myers Squibb, Princeton, NJ), 2 NRTI plus LPV/r, and 2 NRTI plus EFV (ClinicalTrials.gov number, NCT00050895).18 NRTI administered were 3TC plus ZDV (GlaxoSmithKline, Research Triangle Park, NC), stavudine (Bristol-Myers Squibb, Princeton, NJ), or tenofovir disoproxil fumarate (Gilead Sciences, Foster City, CA). Patients' treatment interruptions were recorded. All patients provided written informed consent, and the study was approved at each site by an institutional review board or ethics committee.18
Virologic failure was defined as (1) confirmed HIV-1 RNA <1.0 log10 copies per milliliter reduction from pretherapy and ≥200 copies per milliliter at/or after week 8 and before week 32, (2) lack of suppression to <200 copies per milliliter by week 32, (3) confirmed rebound >1000 copies per milliliter after confirmed suppression to <200 copies per milliliter before week 32, (4) confirmed rebound >1.0 log10 copies per milliliter from nadir and >1000 copies per milliliter without confirmed suppression to <200 copies per milliliter before week 32, or (5) confirmed rebound ≥200 copies per milliliter after confirmed suppression to <200 copies per milliliter at/or after week 32. Sixty patients reached protocol-defined virologic failure among 250 patients randomized to 2 NRTI plus EFV in ACTG A5142; 53 of 60 had stored plasma samples from pretherapy and failure time points with HIV-1 RNA >450 copies per milliliter.
One hundred forty-four patients among 190 patients in the 2 NRTI plus EFV arm of A5142 who did not experience protocol-defined virologic failure had an available full-length RT pretherapy sequence.
Amplification and Sequencing
Viral RNA was extracted from paired pretherapy/failure plasma samples from 53 patients (QIAamp Viral RNA Mini-kit, Qiagen, Valencia, CA) and converted to cDNA using SuperScript III One-Step reverse transcriptase-polymerase chain reaction System with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA) and template-specific primers. Full-length RT (codons 1-560) was amplified by nested polymerase chain reaction, products were purified with ExoSAP-IT (USB, Cleveland, OH) and bulk sequenced with 6 bidirectional primers using Big Dye terminator (v.3.1) on an ABI 3100 automated DNA sequencer (Applied Biosystems, Foster City, CA). Sequences were assembled and analyzed using Sequencher 4.9 software (Gene Codes Corporation, Ann Arbor, MI).
Sequences from patients who experienced virologic failure were examined at failure for known NRTI and NNRTI resistance mutations in the polymerase domain using the International AIDS Society-USA (IAS-USA) resistance table2 and for novel mutations in the polymerase, connection, and the RNase H domains. Sequences from pretherapy plasma samples among patients who did not experience virologic failure were generated and obtained from Mina John and Simon Mallal.19 These sequences were analyzed to identify associations between pretherapy polymorphisms and virologic failure.
Emergence of Mutations at Virologic Failure
Full-length RT sequences at time of failure and pretherapy from 53 patients who experienced virologic failure were compared for IAS-USA polymerase domain mutations and mutations that occurred more than once in the connection and RNase H domains, using 2-sided exact McNemar test. The same comparison was performed within 2 subgroups: patients experiencing failure with at least 1 IAS-USA RT mutation (n = 26) and those without any such mutations at failure (n = 27). The P values were unadjusted for multiple comparisons.
Associations between Pre-therapy Mutations and Virologic Failure
Pretherapy sequences from patients who did not experience virologic failure (nonfailures, n = 144) were compared with 53 pretherapy sequences from patients who experienced failure for each mutation occurring more than once, using Fisher exact test (2-sided). The P values were adjusted for multiple comparisons using the Bonferroni correction.
All sequences were compared with the appropriate reference subtype [B, C, D or circulating recombinant form (CRF) AB or AE].
Emergence of RT Mutations at Virologic Failure
HIV-1 subtypes in the 53 patients experiencing virologic failure were predominantly B (48 of 53); 4 of 53 were C and 1 of 53 was CRF AE. RT inhibitor resistance mutations in the polymerase domain were identified in 26 of 53 (49%) failure samples. Of the 27 patients without resistance mutations, 15 (56%) had interrupted therapy at the time of virologic failure.
Table 1 shows polymerase domain mutations that were detected at a higher frequency (3% or greater) at failure than pretherapy. Only K103N and M184I/V were significantly more frequent at failure than pretherapy (unadjusted P = 0.001 and P = 0.016, respectively). Table 1 also shows connection and RNase H domain mutations that were more frequent at virologic failure than pretherapy, but none of the changes in frequency were statistically significant (unadjusted P values ≥0.25). All of the pretherapy mutations listed in Table 1 were also identified in the failure sample from the same patient except for 1 patient who did not have R358K in the failure sample.
Comparing pretherapy and failure sequences within the 2 patient subgroups with (n = 26) and without (n = 27) IAS-USA resistance mutations at failure did not identify additional mutations in RT that were significantly more frequent at failure than at pretherapy.
Associations Between Pretherapy Mutations and Virologic Failure
Of the 144 pretherapy sequences from nonfailures, 139 were subtype B, 3 were subtype C, 1 was subtype D, and 1 was CRF AB. Pretherapy mutations associated with virologic failure, unadjusted for multiple comparisons, were E6D (P = 0.023), K103R (P = 0.046), and Q174K (P = 0.015) in the polymerase domain and Q334H in the connection domain (P = 0.045) (Table 2). However, these associations were not significant after correction for multiple comparisons.
This study is the first to compare full-length HIV-1 RT sequences in paired plasma samples obtained pretherapy and at first virologic failure in predominantly subtype B-infected patients. The only mutations that were significantly more frequent at virologic failure than pretherapy were K103N (P = 0.001) and M184I/V (P = 0.016) in the polymerase domain, which confer resistance to the study drugs EFV and 3TC, respectively. Other known polymerase domain mutations were not significantly associated with failure, although there were possible trends for K65R (P = 0.13) and V106I/M (P = 0.13). Mutations in the RT connection and RNase H domains were not significantly more frequent at virologic failure than at pretherapy (P ≥ 0.25), and thus did not contribute to the virologic failure observed.
This study is the largest to date comparing pretherapy and failure sequences from the same individuals (n = 53). The only other similar study compared full-length RT sequences in 1 patient over 3 years.4 In this patient, the N348I connection domain mutation was selected by ZDV and/or didanosine therapy4 and confers resistance to ZDV, didanosine, NVP, EFV, delavirdine, tenofovir, and etravirine.4,11,12,14,15 N348I was probably not identified in our study because efavirenz, not NVP, was the NNRTI used for initial randomized therapy in ACTG A5142.
Other prior analyses of RT connection or RNase H domain mutations have compared sequences from unrelated ART-naive patients and ART-experienced patients.3-13 These studies have identified a number of mutations in the C-terminus of RT that are more frequent in ART-experienced patients compared with ART naive including E312Q, G333D/E, G335C/D, N348I, R356K, R358K, A360I/V, A365I, T369I, A371V, A376S, and K451R. Our study did not identify these mutations at virologic failure. This may be due, in part, to the strict definition of virologic failure used in ACTG A5142 (Methods). As a consequence, the failure samples analyzed were early in the course of virologic breakthrough with limited time on failing therapy for drug-resistant variants to emerge. Nevertheless, our study shows that the first mutations to arise in association with virologic failure in HIV-1 subtype B-infected patients are in the polymerase domain and not frequently in the connection or RNase H domains.
Although our study is the largest pretherapy failure comparison of RT sequences, the sample size (n = 53 pairs) provided limited power to detect mutations that emerge at low frequency. Specifically, the upper bound of the 95% confidence interval for the probability of a mutation that is not detected in 53 patients is 6.7%. Therefore, at alleles where no mutation was observed, there may have been up to 7% of emergent resistance that was not detected. Larger sample sizes are needed to exclude connection and RNase H domain mutations that emerge infrequently.
Secondary analyses were performed to assess if pretherapy RT polymorphisms predispose to virologic failure (Table 2). Polymerase, connection, and RNase H domain mutations were not significantly associated with failure after correction for multiple comparisons. To achieve 80% power to detect such an association, assuming a low proportion (eg, 2%) of the 144 patients that did not experience virologic failure had a specific polymorphism, would require that 14% of the 53 patients that experienced virologic failure have the polymorphism, that is, a difference in proportion of 12%. As a consequence, our study had power to detect relatively large differences (>10%) in polymorphism frequency between patients experiencing virologic failure and those who did not. Such differences were not detected in our study.
In summary, this study of full-length RT did not identify mutations in the connection or RNase H domains associated with virologic failure in predominantly HIV-1 subtype B infection. These findings suggest that full-length RT sequencing is not essential for management of failure first-line efavirenz-containing regimens when failure is detected early, although analyses of larger datasets are needed before firm conclusions can be drawn.
We thank Mina John and Simon Millal (Royal Perth Hospital-Perth, Australia) for providing pretherapy RT sequences, the study sites and their personnel, and the patients for their participation in the study.
1. Wang J, Smerdon SJ, Jager J, et al. Structural basis of asymmetry in the human immunodeficiency virus type 1 reverse transcriptase heterodimer. Proc. Natl. Acad. Sci. USA
2. Johnson VA, Brun-Vezinet F, Clotet B, et al. Update of the Drug Resistance Mutations in HIV-1. Top HIV Med
3. Cane PA, Green H, Fearnhill E, et al. Identification of accessory mutations associated with high-level resistance in HIV-1 reverse transcriptase. AIDS
4. Hachiya A, Kodama EN, Sarafianos SG, et al. Amino acid mutation N348I in the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase confers multiclass resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors. J Virol
5. Hachiya A, Shimane K, Sarafianos SG, et al. Clinical relevance of substitutions in the connection subdomain and RNase H domain of HIV-1 reverse transcriptase from a cohort of antiretroviral treatment-naive patients. Antiviral Res
6. Kemp SD, Shi C, Bloor S, et al. A novel polymorphism at codon 333 of human immunodeficiency virus type 1 reverse transcriptase can facilitate dual resistance to zidovudine and L-2',3'-dideoxy-3'-thiacytidine. J Virol
7. Nikolenko GN, Delviks-Frankenberry KA, Palmer S, et al. Mutations in the connection domain of HIV-1 reverse transcriptase increase 3'-azido-3'-deoxythymidine resistance. Proc Natl Acad Sci U S A
8. Ntemgwa M, Wainberg MA, Oliveira M, et al. Variations in reverse transcriptase and RNase H domain mutations in human immunodeficiency virus type 1 clinical isolates are associated with divergent phenotypic resistance to zidovudine. Antimicrob Agents Chemother
9. Roquebert B, Wirden M, Simon A, et al. Relationship between mutations in HIV-1 RNase H domain and nucleoside reverse transcriptase inhibitors resistance mutations in naive and pre-treated HIV infected patients. J Med Virol
10. Santos AF, Lengruber RB, Soares EA, et al. Conservation patterns of HIV-1 RT connection and RNase H domains: identification of new mutations in NRTI-treated patients. PLoS ONE
11. Yap SH, Sheen CW, Fahey J, et al. N348I in the connection domain of HIV-1 reverse transcriptase confers zidovudine and nevirapine resistance. PLoS Med
12. Waters JM, O'Neal W, White KL, et al. Mutations in the thumb-connection and RNase H domain of HIV type-1 reverse transcriptase of antiretroviral treatment-experienced patients. Antivir Ther
13. von Wyl V, Ehteshami M, Demeter LM, et al. HIV-1 reverse transcriptase connection domain mutations: dynamics of emergence and implications for success of combination antiretroviral therapy. Clin Infect Dis
14. Gupta S, Fransen S, Paxinos EE, et al. Combinations of mutations in the connection domain of human immunodeficiency virus type 1 reverse transcriptase: assessing the impact on nucleoside and nonnucleoside reverse transcriptase inhibitor resistance. Antimicrob Agents Chemother
15. Sluis-Cremer N, Moore K, Radzio J, et al. N348I in HIV-1 reverse transcriptase decreases susceptibility to tenofovir and etravirine in combination with other resistance mutations. AIDS
16. Gotte M. Should we include connection domain mutations of HIV-1 reverse transcriptase in HIV resistance testing. PLoS Med
17. Roquebert B, Marcelin AG. The involvement of HIV-1 RNAse H in resistance to nucleoside analogues. J Antimicrob Chemother
18. Riddler SA, Haubrich R, DiRienzo AG, et al. Class-sparing regimens for initial treatment of HIV-1 infection. N Engl J Med
19. John M, Heckerman D, James I, et al. Adaptive Interactions between HLA and HIV-1: highly divergent selection imposed by HLA class I molecules with common supertype motifs. J Immunol
APPENDIX I: INVESTIGATORS IN THE AIDS CLINICAL TRIALS GROUP STUDY A5142 TEAM
In addition to the authors, the study team included the following members: D. Havlir (University of California, San Francisco), protocol Vice Chair; W. G. Powderly (University College Dublin), co-investigator; K. L. Klingman (Division of AIDS, National Institute of Allergy and Infectious Diseases), co-investigator; U. G. Lalloo (University of KwaZulu Natal), co-investigator; R. L. Murphy (Northwestern University), co-investigator; S. Swindells (University of Nebraska Medical Center), co-investigator; B. Brizz (Social and Scientific Systems), clinical trials specialist; A. G. DiRienzo, L. Peeple (Harvard School of Public Health), statisticians; M. Wantman (Harvard School of Public Health), data analysis; P. Cain (Stanford University Medical Center), protocol field representative; S. Hart, H. Sprenger, M. Cooper, M. Dobson (Frontier Science and Technology Research Foundation), laboratory data coordinators; A. Manzella, D. Rusin (Frontier Science and Technology Research Foundation), data manager; M. Dorosh (University of Colorado Health Sciences Center), community representative; P. Kondo (University of Hawaii), protocol laboratory technologist; K. Squires (Thomas Jefferson University), co-investigator; and P. Tran (Division of AIDS, National Institute of Allergy and Infectious Diseases), protocol pharmacist.
The following were pharmaceutical representatives: T. George (Bristol-Myers Squibb); S. Brun, K. W. Garren, R. Rode (Abbott Laboratories); J. F. Rooney, M. Poblenz, M. Hitchcock (Gilead Sciences).
Other investigators included the following: K. Coleman (Northwestern University); B. Sha (Rush University); O. Adeyemi (Cook County CORE Center); W.K. Henry, W. Calvert (University of Minnesota); M. Morgan, B. Jackson (Vanderbilt University); M. Goldman, J. Hernandez (Indiana University); H.H. Bolivar, M. A. Fischl (University of Miami School of Medicine); C. J. Fichtenbaum, J. Baer (University of Cincinnati); S. Byars, M. Stewart (University of Alabama); H. Edmondson-Melancon, C.A. Funk (University of Southern California); J. N. Connor, M. Torres (Columbia Collaborative HIV/AIDS Clinical Trials Unit); W. E. Maher, L. Laughlin (Ohio State University); M. Adams, C. Hurley (University of Rochester); C. Zelasky, D. Wohl (University of North Carolina-Chapel Hill); T. Sheen, D. McMahon, B. Rutecki (University of Pittsburgh); P. Kumar, I. Vvedenskaya (Georgetown University); G. M. Cox, D. Wade (Duke University Medical Center); P. Sax, J. Gothing (Harvard-Boston Medical Center AIDS Clinical Trials Unit); A. A. Amod (Durban International Clinical Trials Unit); B. Rodriguez, B. Philpotts (Case Western Reserve University); H. Friedman, A. Thomas (University of Pennsylvania, Philadelphia); B. Putnam, C. Basler (Colorado AIDS Clinical Trials Unit); W. A. O'Brien, G. Casey (University of Texas Medical Branch-Galveston); I. Wiggins, G. Casey (Johns Hopkins University); M. Carlson, E. Daar (University of California, Los Angeles); A. Olusanya, M. Schreiber (University of California, Davis, Medical Center); C. Davis, B. Boyce (University of Maryland); G.-Y. Kim, K. Gray (Washington University, St. Louis); J. Volinski (University of California, San Francisco); J. Norris, S. Valle (Stanford University); J. Hoffman, S. Cahill (University of California, San Diego); D. Garmon, D. Mildvan (Beth Israel Medical Center); J. Forcht, C. Gonzalez (New York University); K. Tashima, D. Perez (Miriam Hospital); P. Keiser, T. Petersen (University of Texas-Southwestern Medical Center at Dallas); N. Hanks, S. Souza (University of Hawaii at Manoa and Queen's Medical Center); A. C. Collier, S. Storey (University of Washington, Seattle); V. Hughes, T. Stroberg (Cornell University); and G. Smith, I. Ofotokun (Emory University).