Failure of Initial Therapy with Two Nucleosides and Efavirenz is Not Associated with Early Emergence of Mutations in the C-Terminus of HIV-1 Reverse Transcriptase

Abstract

It is uncertain how often mutations in the connection or RNase H domains of HIV-1 reverse transcriptase (RT) emerge with failure of first-line antiretroviral therapy. Full-length RT sequences in plasma obtained pre-therapy and at virologic failure were compared in 53 patients on first-line efavirenz-containing regimens from AIDS Clinical Trials Group study A5142. HIV-1 was mostly subtype B (48/53). Mutations in the polymerase but not in connection or RNase H domains of RT increased in frequency between pre-therapy and failure (K103N, p=0.001; M184I/V, p=0.016). Selection of mutations in C-terminal domains of RT is not common with early failure of efavirenz-containing regimens.

INTRODUCTION

Currently recommended antiretroviral therapy (ART) for human immunodeficiency virus type 1 (HIV-1) infection consists of combinations of two nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) with a non-nucleoside 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 three 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 non-functional 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 to ART-naïve 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 (TAMs),^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 pre-therapy and virologic failure plasma samples from the 2 NRTI plus EFV arm of AIDS Clinical Trials Group (ACTG) study A5142.¹⁸ Sequences from pre-therapy 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, pre-therapy sequences from patients who did not experience virologic failure were obtained and compared to pre-therapy sequences from patients with virologic failure to assess if polymorphisms in RT predispose to virologic failure of the 2 NRTI plus EFV regimen.

METHODS

Study Design

ACTG A5142 was a phase III, multicenter, randomized, open-label trial among ART-naïve patients consisting of three 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 i) confirmed HIV-1 RNA <1.0 log₁₀ copies/mL reduction from pre-therapy and ≥ 200 copies/mL at/or after week 8 and prior to week 32, ii) lack of suppression to <200 copies/mL by week 32, iii) confirmed rebound >1000 copies/mL after confirmed suppression to <200 copies/mL before week 32, iv) confirmed rebound >1.0 log₁₀ copies/mL from nadir and >1000 copies/mL without confirmed suppression to <200 copies/mL before week 32, or v) confirmed rebound ≥200 copies/mL after confirmed suppression to <200 copies/mL at/or after week 32. Sixty patients reached protocol-defined virologic failure among 250 patients randomized to 2 NRTI plus EFV in ACTG A5142; 53/60 had stored plasma samples and available sequences from pre-therapy and failure time points with HIV-1 RNA >450 copies/mL.

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 pre-therapy sequence.

Amplification and Sequencing

Viral RNA was extracted from paired pre-therapy/failure plasma samples from 53 patients (QIAamp^® Viral RNA Mini-kit, Qiagen, Valencia, CA) and converted to cDNA using SuperScript™ III One-Step RT-PCR System with Platinum^® Taq High Fidelity (Invitrogen, Carlsbad, CA) and template specific primers. Full-length RT (codons 1–560) was amplified by nested PCR, products were purified with ExoSAP-IT^® (USB, Cleveland, OH) and bulk sequenced with six 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 pre-therapy 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 pre-therapy polymorphisms and virologic failure.

Statistical Analysis

Emergence of Mutations at Virologic Failure

Full-length RT sequences at time of failure and pre-therapy 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 two-sided exact McNemar’s test. The same comparison was performed within two subgroups: patients experiencing failure with at least one IAS-USA RT mutation (n=26) and those without any such mutations at failure (n=27). P-values were unadjusted for multiple comparisons.

Associations between Pre-therapy Mutations and Virologic Failure

Pre-therapy sequences from patients who did not experience virologic failure (non-failures, N=144) were compared to 53 pre-therapy sequences from patients who experienced failure for each mutation occurring more than once, using Fisher’s exact test (two-sided). P-values were adjusted for multiple comparisons using the Bonferroni correction.

All sequences were compared to the appropriate reference subtype (B, C, D or circulating recombinant form (CRF) AB or AE).

RESULTS

Emergence of RT Mutations at Virologic Failure

HIV-1 subtypes in the 53 patients experiencing virologic failure were predominantly B (48/53); 4/53 were C and 1/53 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 pre-therapy. Only K103N and M184I/V were significantly more frequent at failure than pre-therapy (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 pre-therapy, but none of the changes in frequency were statistically significant (unadjusted p-values ≥0.25). All of the pre-therapy mutations listed in Table 1 were also identified in the failure sample from the same patient except for one patient who did not have R358K in the failure sample.

Table 1.

RT Mutations More Frequent at Virologic Failure than at Pre-therapy (N=53 pairs)^a

Domain	Mutation	% Pre-therapy(N)b	% Failure(N)b	p-valuec
Polymerase	K65R	0(0)	7.6(4)	0.13
	V90I	0(0)	3.8(2)	0.50
	K101E	0(0)	5.7(3)	0.25
	K103N	1.9(1)	23(12)	0.001
	V106I/M	1.9(1)	9.4(5)	0.13
	V179D	3.8(2)	7.6(4)	0.50
	M184I/V	1.9(1)	15(8)	0.016
	Y188H	0(0)	3.8(2)	0.50
	G190S	0(0)	5.7(3)	0.25
Connection	R358K	7.6(4)	11(6)	0.63
	A376S	1.9(1)	5.7(3)	0.50
	M/T377L	13(7)	17(9)	0.50
RNase H	V467I	17(9)	23(12)	0.25
	K530R	4.1(2)	8.2(4)	0.50

^aMutations ≥3% more frequent at virologic failure compared to pre-therapy.

^bTotal N=53 except K530R (N=49, 4 subtype C sequences were excluded from analysis because they differed from subtype B consensus).

^cExact McNemar’s test (two-sided).

Comparing pre-therapy and failure sequences within the two 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 pre-therapy.

Associations between Pre-therapy Mutations and Virologic Failure

Of the 144 pre-therapy sequences from non-failures, 139 were subtype B, 3 were subtype C, 1 was subtype D and 1 was CRF AB. Pre-therapy 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.

Table 2.

Associations of Pre-Therapy RT Mutations with Virologic Failure^a

		Controlsb	Failures
Domain	Mutation	N=144c	N=53d	p-valuee
		% (N)	% (N)
Polymerase	E6D	3 (4)	12 (6)f	0.023
	K103R	1 (2)	8 (4)	0.046
	Q174K	1 (2)	10 (5)	0.015
Connection	Q334H	6 (9)	16 (8)	0.045

^aMutations with p < 0.05 listed; not corrected for multiple comparisons.

^bControls did not reach protocol-defined virologic failure.

^cFor the control group, subtype C sequences (N=3) were excluded for analyses of codon 334 because they differed from subtype B consensus.

^dFor the failure group, subtype C sequences (N=4) were excluded from analysis at codon 334 because they differed from subtype B consensus. One CRF AE sequence was excluded at codon 174 for the same reason.

^eFisher’s exact test (two-sided) not corrected for multiple comparisons. No significant associations were found after correction for multiple comparisons (Bonferroni).

^fTotal N=52, missing first 16 amino acids for one sample.

DISCUSSION

This study is the first to compare full-length HIV-1 RT sequences in paired plasma samples obtained pre-therapy and at first virologic failure in predominantly subtype B infected patients. The only mutations that were significantly more frequent at virologic failure than pre-therapy 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 pre-therapy (p ≥0.25), and thus did not contribute to the virologic failure observed.

This study is the largest to date comparing pre-therapy and failure sequences from the same individuals (N=53). The only other similar study compared full-length RT sequences in one patient over three years⁴. 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 and virologic failure was detected early using a sensitive definition of failure.

Other prior analyses of RT connection or RNase H domain mutations have compared sequences from unrelated ART-naïve 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 to ART-naïve 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 (see 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 pre-therapy-failure comparison of RT sequences, the sample size (N=53 pairs) had 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 in the study population. Larger sample sizes are needed to exclude connection and RNase H domain mutations that emerge infrequently.

Secondary analyses were performed to assess if pre-therapy 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 (e.g. 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, i.e. 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.

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; 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); G. Smith, I. Ofotokun (Emory University).