Report on the XVII International HIV Drug Resistance Workshop

Antiviral Therapy 13:1097–1113

Workshop report Broad advances in understanding HIV resistance to antiretrovirals: report on the XVII International HIV Drug Resistance Workshop

Mark Mascolini1, Brendan A Larder 2, Charles AB Boucher 3, Douglas D Richman4 and John W Mellors5*

1Allentown, PA, USA 2RDI, London, UK 3Erasmus Medical Center Rotterdam, Rotterdam, the Netherland and University Hospital Utrecht, Utrecht, the Netherlands 4University of California San Diego, VA San Diego Healthcare System, San Diego, CA, USA 5University of Pittsburgh, Pittsburgh, PA, USA

*Corresponding author: E-mail: [email protected]

The 2008 International HIV Drug Resistance Workshop resistance in antiretroviral-experienced patients and a explored six topics on viral resistance: new antiretro- heightened resistance risk in injecting drug users regard- virals; clinical implications; epidemiology; new tech- less of adherence. New research on resistance technolo- nologies and interpretations; HIV pathogenesis, fit- gies involved an enhanced assay for HIV-1 coreceptor ness, and resistance; and mechanisms of resistance. The determination and improved gene-based tools for pre- last of these topics provided a forum for new work on dicting coreceptor use. In the pathogenesis arena, a small resistance of hepatitis B and C viruses, which were also study of intensification shed light on the likely source explored in two poster sessions. Much work focused on of residual viraemia in patients on successful antiretro- resistance to the two most recent antiretroviral classes viral therapy. A large study in Mozambique correlated (integrase inhibitors and CCR5 antagonists), a new set of the timing of infant infection with selection, transmis- entry inhibitor candidates and one new class represented sion and persistence of nevirapine resistance mutations. by the maturation inhibitor bevirimat. Other research Mechanistic research explored resistance to the integrase explored two novel non-nucleoside reverse transcriptase inhibitor raltegravir, K65R-mediated resistance to teno- inhibitors, etravirine and IDX899. Epidemiological work fovir and the role of connection domain mutations in analysed rates of transmitted resistant virus, multiclass resistance to zidovudine.

Introduction

Resistance to antiretroviral drugs can usually be traced countries where HIV-infected pregnant women take to intermittent adherence caused by drug intolerance single-dose nevirapine, with or without short courses and behavioural factors, or to prescription of too few of other antiretrovirals, to prevent vertical transmis- active drugs after virological failure of one or more reg- sion, the prophylactic regimen often engenders resist- imens. Although treatment regimen may fail to control ant virus that can imperil treatment for both mother HIV replication because a patient finds it too difficult to and infant. Infected sexually active adults or injecting take regularly, many recently licensed agents have good drug users anywhere may transmit resistant virus to sex safety and tolerability profiles. or needle-sharing partners, especially if their own viral Treatment failure with drug-resistant virus poses replication is not well controlled. an especially grave threat in low- and middle-income Research presented at the XVII International HIV countries with fledgling antiretroviral programmes Drug Resistance Workshop addressed these critical that use suboptimal initial regimens and often provide issues as well as much of the basic and applied science enough drugs for only a first and perhaps a second regi- essential to inform clinical understanding of HIV resist- men. In such countries, failure may leave patients with ance. This report summarizes all oral presentations and few or no effective options for subsequent therapy. In selected posters from the Workshop.

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Resistance to new antiretroviral agents et al. cautioned that this finding was preliminary and must be confirmed in other patient populations. They Diverse pathways to raltegravir resistance suggested that, although greater replication capacity of Separate analyses of resistance to raltegravir among Q148 mutations may confer an evolutionary advan- patients in whom that integrase inhibitor failed in clini- tage over N155H, the advantage is probably better cal trials identified diverse and non-overlapping evolu- explained by heightened resistance with Q148 changes tionary pathways to resistance [1–3]. Fransen et al. ana- than with N155H. lysed susceptibility to raltegravir in 69 patients enrolled Hatano et al. studied long-term virological and in the Phase III BENCHMRK trials of this agent. The immunological outcomes after integrase inhibitor fail- authors used cloned integrase sequences from 11 of these ure among 18 patients who had taken a median of 14.5 patients with matched pretreatment and failure samples, antiretrovirals from a median of four antiretroviral and site-directed mutants bearing the mutations N155H, classes before starting a rescue regimen containing an Q148R/H/K, E92Q and G140S/A alone or in combi- integrase inhibitor [3]. Median plasma viraemia before nation [1]. Earlier work identified N155H and Q148 beginning an integrase inhibitor was 4.92 HIV-1 RNA + changes as primary raltegravir resistance mutations and log10 copies/ml. Most patients continued to gain CD4 E92Q and G140S/A as secondary mutations [4]. T-cells after documented virological failure: median Evaluation of patient-derived isolates and clonal CD4+ T-cell counts were 42 cells/mm3 higher than base- analysis confirmed that raltegravir selects N155H and line values 3 months after integrase inhibitor failure mutations at Q148 independently, that is, on different and 50 cells/mm3 higher 6 months after failure. viral genomes. These primary mutations alone, and on The investigators measured evolution of integrase occasion secondary mutations alone, conferred reduced inhibitor resistance both genotypically and phenotypi- susceptibility to raltegravir. Analysis of cloned sequences cally. Most patients had evidence of resistance at the demonstrated that E92Q evolved with N155H, but earliest follow-up point. However, 4 of 18 patients with never with Q148 substitutions. G140A/S, on the other poor regimen adherence had no genotypical or pheno- hand, arose with Q148 mutations, but never with typical evidence of resistance to integrase inhibitors. N155H. Secondary mutations generally further reduced Mutations at position Q148 or the G140S mutation susceptibility to raltegravir. Clones carrying E92Q and evolved, usually together, in 11 patients (61% of those neither N155H nor Q148 mutations were rare, but they with detectable mutations). N155H emerged upon fail- did have reduced susceptibility to raltegravir. ure in four patients (22%). Y143R/C evolved in three Analysis of site-directed mutants showed that N155H patients (17%), never with mutations at positions 140, and E92Q together decreased susceptibility to raltegra- 148 or 155. Mutations at position 143 tended to emerge vir and decreased replication capacity more than either later than other mutations. High-level resistance (>400-

of these mutations alone. G140A or G140S generally fold increases in 50% inhibitory concentration [IC50]) enhanced resistance of Q148 site-directed mutants, often took several months to develop. with one exception: G140S plus Q148K was more sus- One patient in whom the N155H mutation emerged ceptible to raltegravir than G148K alone. G140S plus stopped raltegravir and continued the other drugs in the Q148H or G148K had increased replication capacity regimen. Plasma viraemia remained relatively stable for compared with Q148H or Q148K alone. G140S did 1 week after raltegravir stopped, a finding suggesting not alter replication capacity of Q148R alone. that raltegravir had some residual activity after failure in Longitudinal genotypical and clonal analysis of virus this patient. HIV-1 RNA in plasma rose approximately from 35 Phase II trial patients in whom raltegravir 10-fold over the following month, as evidence of resist- failed revealed a preference for Q148 pathways over ance to raltegravir waned. That finding, the investigators N155H [2]. Miller et al. reported that initial genotypes proposed, suggests N155H has a negative fitness effect. at virological failure identified Q148H/R/K in 20 iso- Hatano et al. noted that scientists who developed ralte- lates, N155H in 14 and Y143R in one. As in the work gravir reported nearly identical findings in macaques by Fransen et al. [1], mutations at positions 148 and infected with simian immunodeficiency virus [5]. 155 never occurred on the same genome. Virus from seven patients harbouring N155H and Q148Q/X mixes Correlates of non-response to an HIV-1 maturation eventually evolved to a pure Q148 mutant population. inhibitor Viral populations in five patients evolved from N155H Bevirimat inhibits formation of mature HIV-1 virions without Q148 substitutions to populations dominated by blocking cleavage of the Gag capsid (CA) precur- by Q148 mutations. sor, CA-SP1, to mature CA protein. Although patients Time to loss of virological response was longer in in a Phase II trial of bevirimat attained optimal con- patients whose virus bore N155H without Q148 muta- centrations of the drug, some did not have virologi- tions than in patients with other mutations. Miller cal responses [6]. To elucidate bevirimat response

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correlates, McCallister et al. analysed pretreatment it has an intracellular half-life of approximately 9 h,

and on-treatment variables in 20 responders (>0.5 log10 compared with a zidovudine triphosphate intracellular

copies/ml decrease) and 24 non-responders (<0.5 log10 half-life of 3–4 h. copies/ml decrease) on day 14. All trial participants had The investigators passaged wild-type HIV-1 against extensive antiretroviral experience [7] increasing concentrations of AZG in MT-2 cells and After 14 days, reduction in plasma HIV RNA levels human peripheral blood mononuclear cells (PBMCs),

averaged 1.26 log10 copies/ml in responders and 0.05 then assayed passaged virus for susceptibility to AZG

log10 copies/ml in non-responders. Of 20 responders, and other NRTI in P4/R5 cells. A total of 88 passages in 19 (95%) had at least a 20 µg/ml bevirimat trough MT-2 cells yielded virus 7.3-fold resistant to AZG com- concentration, compared with 19 of 24 non-respond- pared with wild-type virus (50% effective concentra-

ers (79%). Although non-responders had pretreat- tion [EC50] 10.3 versus 1.40 µM). AZG-resistant virus ment polymorphisms in HIV-1 gag more frequently had reduced susceptibility to lamivudine (14.3-fold), than responders (7.3 versus 5.9), the difference did not zidovudine (5.6-fold), didanosine (4.3-fold), stavudine reach statistical significance. (3.5-fold) and tenofovir (1.8-fold). Substitutions in gag at Q369, V370 and T371 cor- Population sequencing of virus at passage 88 disclosed related with mean plasma HIV-1 RNA reductions of the mutations L74V, F77L, V106I, L214F and K476N,

0.16, 0.24 and 0.32 log10 copies/ml, whereas patients the last of which lies in the RNase H primer grip region with consensus sequence at those positions had 0.69, of RT. None of these mutations are TAMs. After 45

0.79 and 0.73 log10 copies/ml reductions in HIV-1 weeks of serial passage in PBMCs, AZG-selected virus RNA, respectively. Twelve of 13 patients (92%) with harboured V75I, F77L and H221Y, none of which are a bevirimat trough concentration >20 µg/ml and with TAMs. A site-directed mutant incorporating these latter consensus sequences at positions 369, 370 and 371 had three mutations had a 1.5-fold decrease in susceptibil-

HIV-1 RNA decreases >0.5 log10 copies/ml. ity to AZG. L74V confers resistance to abacavir and In a separate data set of 567 antiretroviral-naive didanosine [10], whereas V75I and F77L are part of HIV-infected individuals, 60% had HIV-1 subtype B the multi-NRTI Q151M complex [10]. Meteer et al. consensus sequences at Gag amino acid positions 369, proposed that these findings are consistent with inef- 370 and 371, predicting poor antiviral activity of bev- ficient excision of AZG-5′-monophosphate by RT with irimat in 40% of individuals with subtype B infection TAMs and thus the selection of resistance mutations [8]. Of particular note, Van Baelen et al. showed that other than TAMs. these polymorphisms that predict non-response repre- sent consensus sequences in most non-B subtypes of Tenofovir-related compound potent against HIV-1, a concern that merits further evaluation. tenofovir-resistant virus CMX157 (hexadecyloxypropyl tenofovir) proved more Experimental nucleoside selects novel mutations potent than tenofovir in cell studies and active against In serial passage studies, the nucleoside reverse tran- virus resistant to tenofovir [11]. Lanier et al. observed scriptase inhibitor (NRTI) 3′-azido-2′,3′-dideoxy no toxicity, including nephrotoxicity, in rats given guanosine (AZG) selected mutations in the polymerase CMX157 for 28 days at a dose of 50 or 200 mg/kg/ and RNase H domains of reverse transcriptase (RT) that day. Because CMX157 is not cleaved to free tenofo- do not include classic thymidine analogue mutations vir in plasma in vivo, the investigators propose that the (TAMs) selected by zidovudine or stavudine [9]. How- new compound will result in higher levels of active ten- ever, AZG-resistant virus had moderate cross-resistance ofovir diphosphate in target cells. The lower doses and to zidovudine and stavudine, as well as to the non-thy- decreased cleavage of prodrug in blood could decrease midine NRTIs lamivudine and didanosine and to the tenofovir levels in the kidney. nucleotide reverse transcriptase inhibitor tenofovir. Testing CMX157 against a panel of 30 isolates with Zidovudine (3′-azidothymidine) selects TAMs that one or more NRTI resistance mutations including K65R, promote high-level resistance to zidovudine, but low- multiple TAMs and multi-NRTI resistant virus – all of level cross-resistance to other NRTIs. To determine which render virus resistant to tenofovir – Lanier et al.

whether this relative specificity of resistance can be recorded IC50s ranging from 0.66 nM for L74V/M184V attributed to the 3′-azido moiety, Meteer et al. evaluated to 57 nM for A62V/T69S/V/G/V75I/T215I. Respective

the resistance profile of AZG, another ′3 -azido NRTI. IC50s for tenofovir were 227 nM and 16,959 nM. For the AZG has several potential safety and pharmacological multiple-TAM mutation M41L/L210W/T215Y with-

advantages: it does not exhibit significant cytotoxicity out M184V, CMX157 IC50 measured 6.3 nM; for the at concentrations <100 µM in four cell lines, it does same multiple-TAM mutation with M184V, CMX157

not exhibit mitochondrial toxicity, it is taken up rapidly IC50 was 2.2 nM. Tenofovir IC50s for these two mutant in cells and converted to the active triphosphate, and viruses were 2,240 nM and 770 nM, respectively.

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Against a panel of 14 NRTI-susceptible wild-type The average count fell by approximately 80 cells/mm3 isolates representing HIV-2, HIV-1 subtypes A–G, and from a baseline of 654 cells/mm3 in the placebo group.

HIV-1 group O, CMX157 IC50s ranged from 0.2 nM The investigators recorded no treatment-emergent against B to 7.2 nM against the rare O group. Tenofovir serious adverse events or premature discontinuations,

IC50s for those subtypes range from 1,600 to 4,900 nM, no grade 3 or 4 laboratory abnormalities, and similar according to the package insert. laboratory profiles with IDX899 and placebo. In PBMCs stimulated by phytohaemagglutinin and Serial passage studies with IDX899 selected the follow- interleukin 2, tenofovir diphosphate levels measured ing RT mutations: V90I, E138K, Y181C/I, S134I, I135R, 1.65 pM/106 cells after 24 h of exposure to 1,000 nM G190E and M230L [12]. The resistance pathways started of CMX157 versus 0.05 pM/106 cells after 24 h of with either E138K or Y181C. Y181C and Y181I also exposure to 1,000 nM of tenofovir. The investigators confer resistance to the currently licensed NNRTIs nevi- plan to explore pharmacokinetics of CMX157 further rapine, efavirenz and etravirine [10] and to the experi- to see if once weekly dosing may be feasible. mental NNRTI rilpivirine (TMC278) [14]. V90I is also an etravirine-related mutation [10]. Serial passage and Phase I studies of new NNRTI In vitro selection experiments found that an experi- Novel entry inhibitor with potential resistance mental non-nucleoside reverse transcriptase inhibitor advantages (NNRTI), IDX899, required more passages than efa- A novel trimeric d-peptide fusion inhibitor showed potent virenz for evolution of resistance-conferring mutations activity against diverse HIV-1 subtypes and a resistance [12]. An 8 day, single-centre, double-blind, placebo- profile apparently superior to that of enfuvirtide [15]. d controlled Phase I trial recorded a 1.8 log10 copies/ml Investigators at the University of Utah designed -peptide reduction in plasma viraemia with this investigational inhibitors for testing because, unlike natural peptides, agent alone [13]. d-peptides are insensitive to degradation by proteases. Murphy et al. randomized 30 antiretroviral-naive They also might be orally bioavailable and have proper- patients to 200, 400, or 800 mg of IDX899 daily, or to ties that make them good microbicide candidates, such placebo for 7 days [13]. After stopping IDX899, study as long shelf-life and resistance to breakdown in mucosa. participants took lopinavir/ritonavir monotherapy for Welch et al. focused on trimeric d-peptides that simulta- 28 days to prevent evolution of virus resistant to the neously bind to all three pockets of the HIV-1 gp41 HR1 NNRTI. All patients had ≥5,000 HIV-1 RNA copies/ region trimer via flexible polyethylene glycol linkers. ml, ≥200 CD4+ T-cells/mm3, no history of AIDS and no The lead compound, PIE12-trimer, inhibited a panel NNRTI resistance mutations detectable at screening. of primary isolates representing HIV-1 subtypes A–G at

On study day 8, mean change in HIV-1 RNA, deter- an average IC50 >60-fold lower than that of enfuvirtide, mined by using the COBAS Amplicor HIV-1 RNA the only licensed HIV-1 entry inhibitor that disrupts

assay, exceeded 1.7 log10 copies/ml in all IDX899 arms fusion mediated by gp41. Of seven enfuvirtide-resistant

while rising marginally in the placebo group (Table 1). strains tested (IC50>100 nM), six were sensitive to

The trial also showed consistent underestimation of PIE12-trimer (IC50<10 nM), the lone exception being a plasma HIV-1 RNA with the COBAS TaqMan assay group O isolate. Two group O strains were resistant to (Table 1). Measuring IDX899 minimum concentration PIE12-trimer and one of these was susceptible to enfu- before the final dose revealed no clear pharmacokinetic– virtide. Group O viruses account for a tiny fraction of pharmacodynamic relationship, probably because drug worldwide HIV-1 infections. d plasma levels far exceeded the EC90 for wild-type HIV-1 Antiviral potency of several trimeric -peptide candi- at all doses. Mean CD4+ T-cell counts rose approxi- dates with sub-pM affinity appeared to be determined by mately 60 cells/mm3 from baseline means ranging from association rate rather than by binding affinity because 432 to 466 cells/mm3 in each of the three IDX899 arms. of limited time-window of exposure to the target during

Table 1. Mean change in plasma viraemia with IDX899 monotherapy for 7 days

COBAS Amplicor COBAS TaqMan Baseline, Day-8 change, Baseline, Day-8 change

log10 copies/ml log10 copies/ml log10 copies/ml log10 copies/ml

IDX899 (800 mg, n=8) 4.36 -1.78 4.31 -1.78 IDX899 (400 mg, n=8) 5.09 -1.78 4.64 -1.65 IDX899 (200 mg, n=8) 4.74 -1.83 4.38 -1.61 Placebo (n=6) 4.45 +0.05 4.18 -0.02

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viral entry. By exploiting this property, the investigators Exposure to single-dose nevirapine frequently selects sought to engineer inhibitors with a reserve of binding K103N, which renders HIV-1 resistant to nevirapine energy against mutations that disrupt affinity. Doing so, and efavirenz. Long-term follow-up of women with they propose, will also block HIV from stepwise accu- single-dose nevirapine-induced resistance mutations mulation of resistance mutations by dissociating affinity shows that these mutations fade over time to levels and inhibitory potency. undetectable by standard population sequencing [19] and do not affect overall response to NNRTI regimens Binding changes in virus resistant to CCR5 begun 6 months or more after single-dose nevirapine monoclonal antibody [20]. To measure the impact of low-frequency K103N Resistance to a humanized anti-CCR5 monoclonal on later response to nevirapine or efavirenz plus zido- antibody, RoAb3952, involved a change in the viral vudine/lamivudine, Hunt et al. used highly sensitive binding site from the extracellular loop 2 (ECL2) AS-PCR to search for K103N in pretreatment samples region of CCR5 to the N-terminal loop of CCR5 [16]. of 94 women exposed to single-dose nevirapine 18–36 In the presence of RoAb13 (an antibody that binds months before beginning NNRTI-based therapy and in to the N-terminal domain), RoAb3952-sensitive virus 60 previously pregnant women beginning an NNRTI required ECL2 for viral entry, whereas RoAb3952- regimen, but never exposed to single-dose nevirapine. resistant virus efficiently entered target cells after The single-dose nevirapine-exposed and -unexposed binding to the N-terminal loop. groups did not differ in rates of attaining an HIV-1 Jekle et al. passaged two CCR5-using HIV-1 isolates, RNA <50 copies/ml through 24 weeks of follow-up or Bal and CC1/85, in the presence of increasing concen- in viral frequency of viral rebound. AS-PCR detected trations of RoAb3952, which is a humanized version K103N in 11% of nevirapine-exposed women and of a mouse anti-CCR5 monoclonal antibody, RoAb14 in 13% of women who reported never receiving sin-

[17]. IC50s of RoAB3952 and RoAb14 against the JRCSF gle-dose nevirapine or other antiretrovirals. Among strain of HIV were 0.06 ±0.03 µg/ml and 0.03 ±0.02 women with pretreatment K103N, 11 of 19 (57.8%) µg/ml in PBMC assays. The investigators used CD8+ either did not reach an HIV-1 RNA level <50 copies/ T-cell-depleted PBMCs and high titres of virus to speed ml with triple therapy or reached that mark and failed emergence of resistant virus. to sustain suppression for 78 weeks. However, AS-PCR Two highly resistant viruses evolved. Both the Bal- detected K103N in pretreatment isolates of only 11 of and CC1/85-resistant viruses were more susceptible to 30 women (36.7%) with virological failure, a finding RoAb13, the N-terminal-recognizing CCR5 monoclonal suggesting other minor resistant virus populations or antibody, than to no-drug control viruses. Jekle et al. other factors affected response in these women. proposed this finding suggests a shift in binding abil- The investigators did not attempt a multivariate ity of the resistant virus from ECL2 to the N-terminal analysis to determine what other variables – such as domain. RoAb3952-resistant HIV continued to use the pretreatment plasma viraemia or adherence to therapy CCR5 coreceptor. – may explain poor virological response. They sug- Bal and CC1/85 resistance to RoAb3952 was gested that some reportedly unexposed women with conferred by mutations throughout gp120, includ- K103N may have forgotten taking single-dose nevi- ing mutations in C1, C2, V2 and gp41. Although rapine, may not have been told they were getting it or Jekle et al. could not identify a resistance ‘hot spot’, may have been infected with the mutant virus. RoAb3952-resistant Bal and CC1/85 shared two muta- Standard genotyping of the first virological failure sam- tions: L85M in C1 and S535A in gp41. RoAb3952- ple in these women revealed differing resistance patterns resistant strains were cross-resistant to 2D7, another in women who did or did not receive nevirapine: K103N CCR5 monoclonal antibody. evolved in 9 of 18 (50%) women exposed to nevirapine, but in only 1 of 10 (10%) not exposed to nevirapine. The Clinical implications of resistance lamivudine-induced M184V/I mutations arose in 7 of 10 (70%) women not exposed to nevirapine, but in 6 of Pretreatment K103N mutation affects response in 18 (33%) exposed women. Most women in both groups African women had at least one major NNRTI mutation upon failure, Allele-specific (AS)-PCR detected low-frequency K103N including 13 of 18 (72%) in the nevirapine-exposed mutations in 11–13% of South African women, regard- group and 8 of 10 (80%) in the unexposed group. less of a history of exposure to single-dose nevirapine [18]. Women with pretreatment K103N had a higher rate of poor Clinical cutoff and weighted mutation scores virological response to triple therapy including an NNRTI, proposed for etravirine but K103N did not entirely explain virological response in Analysing samples from patients enrolled in the this observational study in Johannesburg, South Africa. DUET trials, researchers from Tibotec and Monogram

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Table 2. Baseline fold-change in susceptibility to etravirine and 24-week response in DUET 1 and 2

Baseline fold change in susceptibility to etravirine ≤3 3–13 >13

Patients at baseline, % 67 18 15 Week-24 HIV-1 RNA <50 copies/ml, % 71 50 37

Week-24 change in HIV-1 RNA, log10 copies/ml -2.67 -2.39 -1.51

Biosciences independently set a low-end cutoff for 199 samples from DUET trial participants, excluding maximal response to the NNRTI etravirine at a three- those taking enfuvirtide. An analysis not adjusted for

fold or lower increase in EC50 compared with wild- activity of the background regimen found only a mod- type virus [21,22]. Tibotec investigators proposed an est correlation between fold change in susceptibility intermediate cutoff for response at ≤13-fold change in to etravirine and week 4 change in plasma viraemia susceptibility; however, the data used to make this esti- (r2=0.05, P=0.02). Analyses adjusted for background mate are sparse. Furthermore, the analysis of cutoffs regimen activity correlated reduced response to etra- for treatment response with the more recently approved virine with a fold change >2.9, a finding indicating drugs is confounded by the activity of the other drugs that 2.9 is the clinical cutoff for maximal response. used as part of an optimized background regimen. Among the 199 samples phenotyped, the median fold The Tibotec study [21] involved 403 patients taking change in susceptibility to etravirine stood at 0.75 and etravirine plus darunavir/ritonavir and other antiretro- ranged from 0.06 to 200. Sixty-seven samples (33.7%) virals in the DUET 1 and DUET 2 trials, which enrolled were hypersusceptible to etravirine, defined as a fold patients with mutations conferring resistance to change <0.4. Only 23 baseline samples (11.5%) had NNRTI, with three or more primary protease inhibi- reduced susceptibility to both etravirine (fold change tor (PI) mutations and with >5,000 HIV-1 RNA copies/ >2.9) and darunavir (fold change >10). In a subset of ml [23,24]. The analysis excluded anyone taking enfu- samples with reduced susceptibility to darunavir (fold virtide for the first time and anyone who discontinued change >30), regression analyses demonstrated a gradu- treatment before 24 weeks for reasons other than viro- ated reduction in etravirine activity as etravirine fold

logical failure. Baseline fold change in etravirine EC50 change increased. However, the number of samples in predicted HIV-1 RNA <50 copies/ml at study week this subgroup (46) limited precision in defining an upper 24. An analysis of covariance model including baseline clinical cutoff for etravirine, above which response was plasma viraemia, CD4+ T-cell count, darunavir fold unlikely. Testing of further subsets of samples is under change in susceptibility, etravirine fold change in sus- way to define an upper clinical cutoff for etravirine ceptibility and NRTI susceptibility, set an initial clinical In another study, Monogram investigators established

cutoff of ≤13-fold change in etravirine EC50. a weighted genotypical score to predict response to etra- Because 22 of 60 patients (37%) with an etravirine virine [25]. International AIDS Society-USA (IAS-USA) fold change >13 reached undetectable plasma viraemia panellists designate 13 mutations at eight positions by week 24 (Table 2), Peters et al. proposed 13-fold as as etravirine-specific mutations: V90I, A98G, L100I, an intermediate response cutoff. Among 269 patients K101E/P, V106I, V179D/F, Y181C/I/V and G190A/S with a less than threefold change in susceptibility to [10]. Virus with three or more mutations from this list is etravirine at baseline, 190 (71%) had a week-24 HIV-1 considered resistant to etravirine. Monogram analysed RNA <50 copies/ml and a mean 24-week plasma virae- 2,756 viral isolates bearing one or more recognized

mia decrease of 2.67 log10 copies/ml, compared with NNRTI-related mutations, excluding samples with mix-

1.79 log10 copies/ml among patients with a baseline tures at positions previously associated with resistance fold change >13 (Table 2). Tibotec selected threefold to etravirine. Within this dataset, the investigators also as the lower clinical cutoff for etravirine, meaning that considered a subset of 1,006 isolates that did not har- patients with this level of susceptibility to etravirine bour NRTI mutations that may enhance susceptibility have the best chance of responding to etravirine in a to etravirine [26]. regimen that includes other active drugs. Tibotec could With the 2.9-fold lower clinical cutoff established not set an upper clinical cutoff because of the relatively in the phenotypical study [22], Monogram found an small number of DUET participants with a baseline overall 16.8% discordance between the three-mutation fold change >13. rule and susceptibility to etravirine. After elimination In a separate analysis, Monogram investigators of samples with mixtures of NRTI mutations, discord- confirmed a lower clinical cutoff for etravirine at 2.9- ance between the three-mutation rule and phenotypical fold [22]. Coakley et al. phenotyped and genotyped resistance remained at 15.7%.

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Using a stepwise, iterative algorithm considering Table 3. Proposed weighting factors for etravirine-related mutations mutations in the order in which they affect etravirine susceptibility, Monogram assigned weighting factors Weighting factor Mutations to mutations that furthered resistance to etravirine in 4 L100I, K101P, Y181C and Y181I this analysis. Four mutations (L100I, K101P, Y181C 3 E138A/G, V179E, G190Q, M230L and K238N and Y181I) earned a weighting factor of four, indi- 2 K101E, V106A, E138K, V179Land Y188L cating the greatest association with resistance to etra- 1 V90I, K101H, V106M, E138Q, V179D/F/M, virine (Table 3). Four mutations in the original list Y181F, V189I, G190E/T, H221Y, P225H of etravirine-specific mutations (A98G, V106I and and K238T G190A/S) were not observed to affect etravirine susceptibility and thus were assigned no weight in the final algorithm. frequently monitored groups. Rates of K65R were <5% A total weighted mutation score of four correlated in both groups. with phenotypical resistance to etravirine. Apply- Excluding data from one cohort that had only week ing this score to the original dataset, the investiga- 80 resistance data rendered the difference in TAM prev- tors determined that the weighted scoring system had alence as non-significant. Analysing data by 3 monthly an overall genotype–phenotype discordance of only monitoring, 6 monthly monitoring and no monitor- 7.6% (excluding mixtures). Among the 1,006 samples ing did not change results except that the difference in without NRTI mutations, discordance was only 3.2% TAM prevalence became non-significant. (excluding mixtures). These weighting factors remain Gupta et al. suggested that higher resistance mutation to be validated in separate antiretroviral-experienced detection rates in less frequently monitored populations populations starting an etravirine regimen. could point to more extensive viral evolution during peri- ods of uncontrolled viraemia. They proposed that lower Resistance rates higher with less frequent HIV-1 resistance rates in populations with more frequent moni- RNA monitoring toring suggest that more frequent monitoring detects NRTI and NNRTI mutation rates were much higher in poor adherence early. patient groups with less frequent plasma viraemia monitoring, according to results of a systematic review involv- Epidemiology of HIV resistance ing 526 patients in Uganda, 1,700 in Thailand, 389 in Malawi, 300 in Zimbabwe, 60 in Cameroon, 1,352 in Increasing prevalence of resistance mutations in naive the United Kingdom and 236 in Switzerland [27]. US trial recruits The analysis focused on adult trial and cohort popu- Although a recent multinational European study found lations with 36–96 weeks of follow-up after starting a a declining rate of resistant virus in antiretroviral-naive first-line regimen including an NNRTI (efavirenz or nev- people with HIV infection from 2003 through to 2005 irapine) plus lamivudine and one thymidine nucleoside [28], a prospective study of 3,542 US clinical trial recruits analogue (zidovudine or stavudine). All patients started charted an increasing prevalence of resistance mutations therapy at a CD4+ T-cell count <200 cells/mm3. Gupta in untreated people over the past 8 years [29]. et al. considered major NNRTI mutations, TAMs, Using the IAS-USA resistance mutation list [10] and M184V and K65R. They defined frequent monitoring the World Health Organization (WHO) 2008 Surveil- as every 12 weeks or less and infrequent monitoring as lance mutation list [30], Ross et al. analysed mutation any greater interval. prevalence in HIV-1-infected, antiretroviral-naive Pretreatment parameters including age, CD4+ T-cell adults seeking enrolment in GlaxoSmithKline trials count and plasma viraemia were similar across the from 2000 through 2007 [29]. Median HIV-1 RNA + monitoring groups. The resistance analysis considered stood at 4.884 log10 copies/ml and median CD4 T-cell 261 genotypes from patients with virological failure. In count at 226 cells/mm3; 83% of trial participants countries with virological monitoring every 3 months or were men. The respective overall prevalence of NRTI, less, 89.3% of virological failure samples had resistance NNRTI and PI mutations for the 8 years measured to an NNRTI, compared with 62.2% of failure samples 4%, 13% and 3% with the IAS-USA list and 5%, 7% from countries with more frequent virological moni- and 3% with the WHO Surveillance list. According toring. Evidence of resistance to lamivudine could be to the IAS-USA list, 15% of trial recruits had resist- detected in 84.5% of failure samples in less frequently ant virus in 2007. Detection of NNRTI mutations monitored populations and in 30.8% of samples from increased significantly from 2000 through to 2007 more frequently monitored populations. Prevalence of with either list (odds ratio [OR] 1.08, standard error at least one thymidine analogue mutation was 28.9% [s e ] 0.07 for IAS-USA [P=0.003]; OR 1.08, s e 0.01 for in less frequently monitored groups and 14.9% in more WHO [P=0.004]). With the IAS-USA list, prevalence

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of primary resistance mutations for all three anti- type virus (P<0.001). Those differences remained sig- retroviral classes also rose 8% annually (OR 1.08, s e nificant after adjustment for other variables including 0.08, P<0.001). older age (P=0.04), injecting drug use transmission According to the IAS-USA mutation list, 63 of 3,542 (P<0.001), lower CD4+ T-cell count (P<0.0001) and (1.8%) trial participants had dual-class resistance, higher plasma viraemia (P<0.01). Deeks et al. sug- compared with 50 of 3,542 (1.4%) by the WHO sur- gested that higher mortality with wild-type virus than veillance list. Less than 1% had triple-class resistance with NRTI/PI resistance reflects poor adherence in by either method. The IAS-USA list found the high- patients with wild-type virus. A 5 year study in Brit- est resistance mutation prevalence in the west (20%), ish Columbia found a 75% higher risk of death in followed by the south (17%), northeast (15%), and antiretroviral-treated patients with resistance to any midwest (15%) of the USA. Prevalence ranking was class and a tripled risk of death in those with resist- the same with the WHO surveillance list. ance to NNRTI [32]. Multivariate analysis determined a significantly lower risk of any WHO surveillance list mutation in Higher risk of resistance in IDUs regardless of Blacks than in Whites and others (P<0.001). Individu- adherence als who reported heterosexual contact as their trans- Injecting drug users (IDUs) in British Columbia ran a mission risk were significantly more likely than those 70% higher risk of resistance to antiretrovirals than who did not indicate heterosexual contact as a trans- other transmission risk groups regardless of adher- mission risk to have a major IAS-USA PI mutation ence, according to results of an observational study of (P<0.001). Every additional 10 years of age decreased 2,350 individuals by Gill et al. [33]. Earlier research the risk of having an IAS-USA PI mutation (P=0.06) by these investigators identified a higher risk of resist- in all races and increased the likelihood of having an ance during 30 months of follow-up in IDUs than in NNRTI WHO Surveillance list mutation (P=0.03). others starting their first antiretrovirals [34]. The new analysis involved antiretroviral-naive Multiclass resistance decreases in treated North adults beginning treatment between August 1996 and American cohort November 2004. Gill et al. examined 6,066 genotypes Rates of mutations conferring resistance to multiple from viral samples collected when plasma viraemia antiretroviral classes decreased in a US–Canadian was >1,000 copies/ml after treatment began. The study from 2000 through to 2005 [31]; however, the investigators estimated adherence by prescription study correlated class-wide NNRTI resistance with a refill and, in a subset of 751 patients who began treat- higher risk of death within 8 years of a first genotype. ment between August 1996 and September 1999, by The North American AIDS Cohort Collaboration on untimed plasma drug concentrations of prescribed PIs Research and Design (NA-ACCORD) studied antiret- and NNRTIs measured in the first two plasma samples roviral-treated patients from eight clinics. All patients collected for genotyping. had plasma viraemia >1,000 HIV-1 RNA copies/ml, Of the 2,350 cohort members, 1,919 (82%) were and most had genotypical test results. The analysis men, 645 (27%) were IDUs, 1,210 (51%) had a CD4+ included 1,591 patients in 2000 (52% with genotypical T-cell count <200 cells/mm3, 1,360 (58%) had a pre- results), 1,597 in 2001 (58%), 1,499 in 2002 (60%), treatment HIV-1 RNA <10,000 copies/ml, 991 (42%) 1,396 in 2003 (61%), 1,290 in 2004 (66%) and 1,004 began treatment with a PI not boosted by ritonavir in 2005 (71%). Deeks et al. used the Stanford HIV and 1,272 (54%) had ≥95% adherence. Database to identify resistance mutations and defined In multivariate analysis adjusted for pretreatment antiretroviral class resistance as pan-class resistance to CD4+ T-cell count and HIV RNA in plasma, age, type all available drugs in a class or within-class resistance of initial regimen, year of first therapy and adherence, to any available drug in a class. IDUs had a 1.7-fold higher risk of resistance, defined Within-class resistance to two classes fell from as detection of a major NRTI, NNRTI or PI muta- 50% in 2000 to 33% in 2005 (P<0.001). Over the tion (adjusted OR 1.71, 95% confidence interval [CI] same period, within-class resistance to three classes 1.39–2.11). The higher risk of resistance held true for decreased from 24% to 16% (P<0.001). Frequency comparisons of IDUs with non-IDUs across the same of pan-class resistance to all three classes did not adherence brackets (0–<40%, 40–<80%, 80–95% and change significantly from 2000 to 2005 (9–8.5%, ≥95%). Higher resistance risk among IDUs also held P=0.32). Kaplan–Meier survival estimates determined true for both men and women. For all antiretroviral that patients with pan-class resistance to PI/NRTI class categories (except NRTI excluding lamivudine had significantly longer survival through 8 years of and emtricitabine), the probability of treatment- follow-up than patients with pan-class NRTI/NNRTI emergent resistant virus was consistently 1.5–1.6-fold resistance, pan-class PI/NNRTI resistance or wild- higher among IDUs than others.

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New resistance technologies and Enhanced Trofile assay improves screening for CCR5 interpretations antagonist Using the enhanced Trofile coreceptor tropism assay in the Minor variants emerge during vicriviroc failure in ACTG 5211 trial of vicriviroc [36] would have improved Phase II trial response rates to the CCR5 antagonist by eliminating Viral variants representing <1% of a person’s HIV-1 patients with previously undetectable levels of CXCR4- quasispecies before treatment with the CCR5 antago- using virus at trial screening or entry [37]. Reeves et al. nist vicriviroc emerged during failure of rescue regimens compared the newly validated enhanced assay with the including that investigational agent [35]. Tsibris et al. former assay in determining coreceptor use in stored reached that conclusion by using the high-throughput samples of 118 ACTG 5211 participants randomized to pyrosequencing platform from 454 Life Science to a rescue regimen including 5, 10 or 15 mg of vicriviroc or analyse sequences encoding the gp120 V3 loop in one placebo. The enhanced assay, which can detect a minority pretreatment and two on-treatment samples from four population of CXCR4 viruses of 0.3%, determined that patients enrolled in ACTG study 5211 [36]. All patients 25 of these patients had virus that could use either CCR5 were taking vicriviroc at failure. or CXCR4 (dual/mixed or DM virus) before beginning Producing 25,000–140,000 sequences per patient their trial regimen. Fifteen of these 25 received vicriviroc, at each time point, 454 sequencing revealed far more which cannot control replication of DM virus. The previ- V3 loop heterogeneity than previously recognized with ous Trofile assay initially classified virus from all 15 as standard sequencing. All patients had CCR5-using CCR5 using, then detected emerging CXCR4-using virus virus at trial entry. One of them also had CCR5-using during treatment with vicriviroc in 12 of these 15. HIV-1 at virological failure. In the other three patients, Reclassification of virus as CXCR4 using would not CXCR4-using variants emerged during vicriviroc fail- have changed virological response rates of patients ran- ure. In two of these three, CXCR4-using virus emerged domized to placebo. However, an intention-to-treat 2 weeks after vicriviroc therapy began. Deep sequenc- analysis determined that more accurate coreceptor clas- ing revealed that the CXCR4-using majority population sification with the enhanced Trofile assay would have at failure evolved from sequences representing <1% of improved 14-day and 24-week response rates among pretreatment sequences. patients randomized to each dose of vicriviroc (Table 4). In the third individual whose vicriviroc regimen failed The 64 patients with CCR5-using virus at trial with CXCR4-using virus, those variants emerged later screening and entry according to the enhanced Trofile in the course of treatment. When resistance to vicrivi- assay would have had significantly greater reductions in roc developed at treatment week 19, the predominant plasma viraemia at day 14 than the 15 people reclassified viral population at that point matched sequences repre- as having DM virus by the enhanced assay (-1.15 versus

senting 0.2–3.0% of pretreatment sequences. -0.09 log10 copies/ml at day 14, P<0.0001). Reeves et al. In the patient whose vicriviroc regimen failed with also observed a significantly greater 24-week response CCR5-using virus, the pretreatment CCR5-using viral when comparing these two groups (-1.95 versus -0.57

population was replaced by different CCR5-using vari- log10 copies/ml, P=0.0003). Thirty-six of 66 patients ants that made up a minority of the pretreatment viral (55%) who could have taken vicriviroc if screened by quasispecies. No change in susceptibility to vicriviroc the enhanced assay would have had HIV-1 RNA <400 could be detected in this patient at failure. copies/ml at week 24, compared with 40 of 83 (48%) Tsibris et al. concluded that minor variants repre- screened with the less sensitive Trofile assay. senting <1% of a pretreatment population can emerge rapidly during vicriviroc therapy and contribute to Improving genotypical approaches to coreceptor failure. They proposed that high-throughput pyrose- classification quencing platforms can comprehensively assess viral Because the HIV-1 envelope sequence determines diversity and can quantify minor sequence variants. coreceptor use, researchers are trying to develop

Table 4. Differences in response to vicriviroc with enhanced versus standard Trofile coreceptor assay

Mean change in HIV-1 RNA, log10 copies/ml Enhanced assay, Standard assay, Enhanced assay, Standard assay, Vicriviroc dose, mg 14 days 14 days 24 weeks 24 weeks

5 -1.10 -0.87 -1.85 -1.51 10 -1.31 -1.15 -2.09 -1.86 15 -0.93 -0.92 -1.75 -1.68

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sequencing and interpretation tools that will permit (area under the receiver operating characteristic curve faster and less expensive coreceptor classification than [AUC] 0.933 for V2 plus V3 versus 0.914 for V3 alone, is possible with phenotypical assays. A study first pre- P=0.0019). At 90% specificity and the default 10% sented at the 2007 Resistance Workshop found that false-positive rate of geno2pheno, sensitivity for pre- genotypical methods lacked adequate sensitivity in dicting CXCR4-using virus was 89.8% for V2 plus V3 predicting coreceptor use compared with the phe- versus 85.3% for V3 alone. At a specificity of 96.1% for notypical Trofile test [38]. Three reports at the 2008 the 11/25 rule coreceptor prediction algorithm, sensitiv- Resistance Workshop presented improved genotype- ity improved from 78.5% with analysis of V3 sequences based coreceptor-classifying techniques. to 83.5% with analysis of V2 plus V3 sequences. Moores et al. developed a method that relies on three At 90% specificity, sensitivity in predicting pheno- independent genotypes of the V3 loop-encoding region typically determined coreceptor use in the 268 sequence of HIV-1 gp120 and two algorithms, position-specific validation set was 52.4% when analysing only V3 scoring matrices (PSSM) and geno2pheno, to interpret sequences. With V2 plus V3 sequencing, sensitivity the genotypes [39]. Testing 63 viral samples for which improved to 62.8%. AUC was 0.778 with V3 alone Trofile had already determined coreceptor use, these and 0.841 with V2 plus V3. investigators calculated that geno2pheno had 61.2% Däumer et al. used 454 sequencing to analyse V3 sensitivity and 93% specificity for producing the same sequences and then predicted coreceptor use with the coreceptor result as Trofile, whereas PSSM had 75% geno2pheno tool [41]. The study began with 55 sam- sensitivity and 83% specificity. Sensitivity was 75.8% ples from antiretroviral-treated patients sequenced by and specificity 91.1% when the investigators combined standard population-based methods and interpreted by geno2pheno and PSSM. Moores et al. then validated geno2pheno. The phenotypical Trofile assay determined this combined approach in an independent set of 278 coreceptor use for all these samples. The investigators samples from the Phase III MOTIVATE trials of the chose 14 of these isolates for 454 sequencing coupled CCR5 antagonist maraviroc. In the validation set, sen- with geno2pheno coreceptor prediction. sitivity was 72% and specificity 88% with the com- Compared with Trofile coreceptor determinations, bined method. Sequencing methods to detect minor specificity was 90.9% and sensitivity 59.1% for gen- envelope variants yielded further improvements. o2pheno predictions based on the 55 bulk-sequenced These investigators also analysed V3 sequences in samples. Among the 14 samples submitted to 454 proviral HIV-1 DNA sampled from 26 patients with sequencing, geno2pheno made one incorrect CCR5 call virus that could use either CCR5 or CXCR4 and 14 and four incorrect CXCR4 calls when relying on pop- with strictly CCR5-using virus. All patients had unde- ulation-based sequencing. The geno2pheno algorithm tectable plasma viraemia as a result of antiretroviral made only one coreceptor assignment discordant with therapy, so adequate HIV-1 RNA sampling was not Trofile results when interpreting genotypes provided by possible. Yet such patients may need to take a CCR5 454 sequencing. antagonist because of intolerance to other drugs in With an average 10,000 reads for every V3 sequence their regimen. Trofile had determined coreceptor use in analysed, 454 sequencing detected at least a few these patients before they began treatment. Compared sequences indicating CXCR4 use in all 14 samples with Trofile results, PSSM had 76% sensitivity and analysed, regardless of whether Trofile designated that 71% specificity in predicting coreceptor use from DNA sample as using CXCR4 or CCR5. The predictive value sequences, whereas sensitivity and specificity were 77% of these analyses for treatment responses to CCR5 and 93% with geno2pheno. inhibitors needs to be determined. Thielen et al. used a test set of 916 Los Alamos Database sequences from 312 people and a valida- HIV pathogenesis, fitness and resistance tion set of 268 bulk-sequenced isolates from antiretroviral-naive patients in the British Columbia HOMER Intensification study points to source of residual cohort to test the value of adding V2 sequencing to V3 viraemia sequencing in predicting coreceptor use [40]. Repeat- A study of antiretroviral regimen intensification with ing analyses 10 times for each sequence, they corre- efavirenz or lopinavir/ritonavir found no virological ben- lated seven V2 mutations with CXCR4 use: S164V, efit with the strategy and produced evidence suggesting R166I, V169T, Y173H, I182E, K192I and S195H. that long-lived chronically infected cells are the probable Mutations at five other V2 positions correlated with source of persistent low-level viraemia in patients with CCR5 use. viraemia undetected by standard assays [42]. The results Prediction models trained on V2 plus V3 sequences contradict findings in an earlier intensification study significantly outperformed V3-only models in predict- whose authors concluded that ongoing complete cycles ing the same coreceptor determined by phenotyping of viral replication explain low-level viraemia [43].

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Maldarelli et al. used a single-copy assay to quantify (PCR positive at birth) and 24 had acute peripartum HIV RNA plasma levels in six patients whose standard infection (PCR positive between 2–8 weeks of age). triple regimen kept plasma viraemia <50 HIV-1 RNA Using an oligonucleotide ligation assay to search copies/ml for >1 year [42]. Three patients used a PI not for four critical NNRTI-selected mutations (K103N, boosted by ritonavir (atazanavir or nelfinavir), one used V106M, Y181C and G190A), the investigators deter- ritonavir-boosted lopinavir and two used the NNRTI mined that all infants with in utero infection had wild-type efavirenz. Everyone was taking two or three nucleo- virus at birth. Micek et al. focused on 29 infants infected sides with their PI or NNRTI. HIV-1 infection duration in utero, dividing them into two groups: 23 infants with averaged 9 years (range 4–16 years) and duration of the established in utero infection who had high HIV-1 DNA current regimen averaged 4 years (range 1–10 years). levels at birth and 6 infants with acute in utero infection Before intensification, average HIV RNA levels in these had who low HIV-1 DNA at birth. Nevirapine resistance patients measured 4.5 HIV-1 RNA copies/ml, a value mutations emerged in 20 of 23 infants (87%) with estab- similar to those measured in an earlier study by these lished in utero infection and in 2 of 6 (33%) infants with investigators [44]. The four patients already taking a acute in utero infection (P=0.013). Mutations arose in PI added efavirenz for 30 days and the two taking an all 17 infants who received single-dose nevirapine alone NNRTI added lopinavir/ritonavir for 30 days. Average and in 3 of 6 who received single-dose nevirapine plus HIV-1 RNA levels rose non-significantly to 5.3 HIV-1 zidovudine (P=0.013). RNA copies/ml during intensification, then decreased Of the 24 infants with peripartum infection, nevirap- marginally to 5.2 HIV-1 RNA copies/ml after intensi- ine resistance mutations evolved in nine (38%; P=0.001 fication. CD4+ T-cell count did not change significantly versus infants with established in utero transmission). during or after intensification. Among the nine infants with resistance mutations, only Maldarelli et al. argued that their findings are two (22%) initially had exclusively wild-type virus inconsistent with the hypothesis that complete cycles (P<0.001 versus infants with established in utero infec- of viral replication in sanctuary sites contribute sub- tion). Among five infants of mothers who did not receive stantially to residual plasma viraemia during other- single-dose nevirapine prophylaxis, none had nevirap- wise well-controlled replication. One would expect ine resistance mutations, compared with 9 of 19 whose residual HIV-1 RNA plasma levels to decrease fur- mothers did take single-dose nevirapine (P=0.12). ther with intensification if that hypothesis were true. Nevirapine resistance mutations decayed more rap- Rather, the investigators suggested that long-lived idly in infants with established in utero infection than chronically infected cells are the most likely source of in infants with peripartum infection. Resistance muta- persistent residual viraemia. tions in infants with acute in utero infection decayed In the earlier study, Havlir et al. found that intensifying more slowly than in infants with established in utero a suppressive regimen with the NRTI abacavir correlated infection. Decay of resistance mutations was slowest in with a further decrease in already low HIV-1 RNA lev- infants with peripartum infection. els in four of five patients [43]. This finding led them to In infants with established in utero infection, Micek propose that ongoing replication contributes to residual et al. proposed that viral replication in utero promotes viraemia. Maldarelli suggested that patients in the earlier emergence of mutations that can later be selected by study had not yet controlled replication as tightly as they single-dose nevirapine. Adding zidovudine to single- might have with a standard triple regimen because three dose nevirapine for these infants reduces selection of of the four responders had episodes of transient virae- resistance mutations, they suggested, by raising the mia >50 HIV-1 RNA copies/ml. None of the six patients genetic barrier to resistance and slowing viral replica- studied by Maldarelli et al. had such episodes. tion. Rapid decay of resistance mutations in these infants suggests that antiretroviral therapy may be effective if Timing of infant infection affects selection, begun at some still-undefined interval after birth. transmission and persistence of In infants with acute in utero infection, the investiga- nevirapine-resistant virus tors suggested resistance rates are probably lower than A prospective cohort study of 741 Mozambican infants in established in utero infection because there is too lit- prophylaxed with single-dose nevirapine found differ- tle viral diversity and replication in utero to generate ences in rates of selection, transmission and persistence mutations for selection by nevirapine. However, when of nevirapine resistance mutations when comparing nevirapine mutations are selected, they decay slowly, infants infected in utero with those infected after birth perhaps because they populate long-lived reservoirs. [45]. Estimating the timing of transmission by nested In infants with peripartum infection, Micek et al. pro- PCR for HIV-1 pol in dried blood spots collected at posed that 100% infection with mutant virus suggests birth and periodically in the first weeks of life, Micek transmission of nevirapine resistance mutations from the et al. determined that 44 infants were infected in utero mother. Persistence of mutations in these infants suggests

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the resistant variants populate long-lived reservoirs and Using SplitTree, Simplot and Hightighter programs to that nevirapine-based antiretroviral therapy may fail. measure recombination frequency in vivo, the investigators found more frequent recombination in gag–pro–pol Cell-mediated immunity may play role in evolution of than in env and more frequent recombination in acute resistance mutations than in chronic infection. Initial recombination fre- In the RESINA cohort of 1,343 antiretroviral-naive quency in gag–pro–pol during acute infection measured individuals with HIV infection, 10% have mutations 0.29. Most of these recombinants did not persist over that confer resistance to antiretrovirals [46]. Schweitzer time and recombination frequency decreased to 0.12 in et al. hypothesized that some of these transmitted muta- chronic infection. Recombination in env was infrequent tions disappear and others persist not only because of during both acute and chronic infection, averaging 0.05 differences in viral replicative capacity, but also because overall. In one patient a gag–pro–pol variant disap- of interpatient differences in human leukocyte antigens peared over time, whereas two env variants persisted. (HLA) among infected individuals. To test the impact Kearney et al. suggested the findings in this patient indi- of HLA class 1 alleles on resistance mutations, they cate that recombination between gag–pro–pol and env analysed correlations between the two in 73 untreated contributed to viral evolution. members of the RESINA cohort. Fisher’s exact prob- The investigators concluded that recombination is ability test made the following mutation–allele correla- frequent in early HIV infection. They suggested that tions: L33F and HLA-A*01 (P=0.0440, risk ratio [RR] frequent recombination means many cells are dually or 9.1667), M46I/L and HLA-A*03 (P=0.0181, RR 10.6), multiply infected and recombination contributes to HIV M46I/L and HLA-B*35 (P=0.1070, RR 4.2632), V75I evolution by allowing different regions of the genome and HLA-A*11 (P=0.0251, RR cannot be calculated), to evolve independently. K103R and HLA-B*44 (P=0.00046, RR 23.0769), V118I and HLA-B*07 (P=0.0146, RR 2.95), and Limited potential of HIV recombination in PBMCs L210F and HLA-B*44 (P=0.0366, RR 6.9231). Analysis of HIV-1 DNA copies in PBMCs of five Epitope mapping showed that 4 of 28 patients (14%) chronically infected individuals found that 80–90% of with HLA-A*01 recognized peptides comprising the cells harbour only one copy of DNA, a result implying amino acid at protease position 33, 17 of 28 (61%) with a low chance of viral recombination in this compart- HLA-A*03 recognized peptides at protease position 46, ment [48]. To study DNA in infected cells, Palmer et al. 4 of 10 (40%) with HLA-B*35 recognized peptides at developed a technique that measures DNA in PBMCs protease position 46, 4 of 9 (44%) with HLA-A*11 rec- diluted in a microtitre plate to much less than 1 infected ognized peptides at RT position 75, 6 of 22 (27%) with cell per well. They sampled plasma and PBMCs from HLA-B*44 recognized peptides at RT position 103, 7 five patients. of 17 (41%) with HLA-B*07 recognized peptides at RT The first patient was infected for >15 years and had position 118 and 8 of 11 (73%) with HLA-B*44 rec- 1,801,380 HIV-1 RNA copies/ml in plasma and 111 ognized peptides at RT position 210. Further analysis CD4+ T-cells/mm3. The investigators calculated a pro- of cytotoxic T-lymphocyte cell recognition at RT posi- virus rate of 0.464 proviruses per well per 333 cells in tion 210 showed that L210F is an escape mutation for each well, or 1 HIV-1 DNA molecule per 717 cells. The patients with HLA-B*44, so patients with this allele second patient, infected for at least 3 years and with may select L210F. 12,482 HIV-1 RNA copies/ml in plasma, had 1 HIV-1 Schweitzer et al. concluded that resistance-associated DNA molecule per 14,018 cells. Of the remaining three mutations can be assigned to specific HLA alleles. They patients, one was infected for 1.5 years, one for 9.5 proposed that the HLA system may play roles in the years and one for an unknown period. Plasma virae- evolution and persistence of transmitted resistance mia in these patients ranged from 29,700 to 339,000 mutations in antiretroviral-naive individuals. HIV-1 RNA copies/ml and CD4+ T-cell counts from 133 to 512 cells/mm3. All had <1 HIV-1 DNA copy per Recombination contributes to viral diversity in early infected cell in most cells. infection Phylogenetic analysis showed that intracellular DNA Single-genome sequencing of HIV-1 env and the gag– sequences were indistinguishable from single genome pro–pol region in 15 patients with acute or early infec- sequences derived from plasma RNA, a result suggesting tion found evidence of frequent recombination, espe- ongoing viral exchange between PBMCs and plasma. In cially in gag–pro–pol [47]. Kearney et al. analysed one patient who began antiretrovirals, nearly 2 years longitudinal samples from these patients because recom- of suppressive therapy had no significant impact on bination is difficult to assess in chronic HIV infection. genetic similarity in plasma and PBMCs. Four of the 15 patients had viral populations reflecting The finding of infrequent multiplying HIV- infection with multiple HIV variants. infected cells appears to contradict frequent in vivo

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recombination observed by Kearney et al. [47]; how- These structures revealed that K65R does not signifi- ever, cells from lymph nodes and other lymphocyte- cantly alter interactions of the 65 residue with deoxyribo- dense compartments where multiple infection and nucleotide triphosphate (dNTP). They showed, however, recombination may occur were not examined. that K65R forms a molecular platform by enabling an interaction between R65 and the conserved amino acid Mechanisms of HIV drug resistance residue R72. This platform apparently restricts R65 and R72 in a way that hinders DNA polymerisation by N155H in integrase confers resistance to raltegravir RT. Das et al. hypothesized that the restriction on con- The N155H mutation in HIV-1 integrase confers formational adaptability of K65R and R72 explains the resistance to the integrase inhibitors raltegravir and lower rate of nucleotide incorporation by K65R and the elvitegravir [4]. Although research presented at this decreased excision rate. workshop indicates that mutations at position Q148 This mechanism differs from steric hindrance, by may be the preferred raltegravir resistance pathway which M184V confers resistance to lamivudine, and [2], analysing the mechanism of N155H-mediated from ATP-mediated excision, the mechanism behind resistance is critical in understanding how HIV-1 resistance to thymidine nucleoside analogues. The evolves under pressure from these inhibitors. K65R–R72 platform interfaces with L74V, M184 and Raltegravir inhibits integrase function by interacting TAMs at discrete sites. In virus bearing both K65R with magnesium ions in the integrase binding pocket. and M184V, side chains of R72 and V184 restrict the Interactions with the metals and direct contact with pocket conformation. Das et al. suggested this restric- integrase mediate raltegravir binding. To determine the tion could explain decreased dNTP incorporation and precise mechanism by which N155H disrupts raltegra- enhanced resistance to lamivudine by K65R/M184V, vir activity, Grobler et al. used a radioligand binding which would lead to partial tenofovir resensitization assay to measure magnesium-dependent binding of with this mutation combination. this antiretroviral to wild-type integrase and N155H If virus bears both K65R and the TAM K70R, mutant integrase [49]. further restrictions to the K65R–R72 platform may Magnesium ion concentration was a determining impair dNTP binding, ATP binding (essential for factor in raltegravir binding affinity to both wild-type excision) or nucleotide incorporation. These effects, and mutant integrase. In the absence of magnesium, Das et al. proposed, may explain the rare co-evo- wild-type and mutant integrase had similar binding lution of K65R and K70R. TAMs, such as T215Y, affinities for magnesium and for raltegravir. Although may be antagonistic to K65R because the K65R–R72 at saturating magnesium concentrations, raltegravir platform probably hinders proper positioning of ATP bound to N155H mutant integrase less efficiently than phosphates and thus decreases excision. to wild-type integrase. Those findings led Grobleret al. to conclude that N155H confers resistance to raltegra- Role of connection domain mutations in resistance vir mainly by disturbing the magnesium ion arrange- to zidovudine ments in the integrase active site. N155H appears not Mutations in the RT connection domain appeared to to mediate resistance by affecting the affinity of inte- enhance TAM-induced resistance to zidovudine by grase for magnesium or by affecting direct contacts compromising RNase H cleavage and thus allowing between raltegravir and viral integrase. more time for excision of chain-terminating zidovudine monophosphate (AZT-MP) from the RNA/DNA tem- Crystallographic view of K65R-mediated resistance plate/primer [51]. To clarify the mechanism underlying to tenofovir this interaction, Beilhartz et al. used biochemical tools to X-ray crystallography elucidated the mechanism by analyse the molecular impact of A360V and N348I con- which mutant virus with K65R in RT escapes inhibi- nection domain mutations in combination with TAMs tion by tenofovir, which appeared to differ from the [52]. A360V appears to evolve with TAMs, whereas mechanisms by which other RT mutations confer N348I may or may not be associated with TAMs. resistance to lamivudine and the thymidine nucleoside These experiments determined that these two con- analogues zidovudine and stavudine [50]. Tenofovir, nection domain mutations, separately or together, cause abacavir, didanosine and, occasionally, stavudine can reduced binding to substrate in the RNase H-compe- select K65R, which confers resistance to all NRTIs tent complex with TAMs. In the polymerase-competent except zidovudine. Das et al. determined the crystal complex, A360V plus TAMs result in improved sub- structure of K65R mutant RT with double-stranded strate binding, but N348I does not. Compared with DNA in complexes with tenofovir diphosphate (the TAMs, both N348I and A360V increase processive active metabolite) and deoxyadenosine triphosphate DNA synthesis. Pyrophosphate-mediated excision of (dATP). AZT-MP occurred with A360V and/or N348I plus

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TAMs. N348I alone increased pyrophosphate-mediated in isolates from experienced versus naive patients. excision of AZT-MP, but A360V did not. A360V and The most frequent of these mutations were A371V in N348I increased AZT-MP excision independently of 41.5%, A376S in 20.0% and N348I in 19.1%. N348I RNase H activity. or E399D correlated with earlier virological failure Beilhartz et al. concluded that RNHase H-dependent during treatment with lamivudine, but not with earlier and -independent mechanisms contribute to enhanced failure of thymidine analogues or NNRTI. resistance to zidovudine. A360V, which emerges after Dendrograms revealed no clustering of connection the evolution of TAMs, appears to compensate for domain mutations with TAMs, but N348I always TAM-mediated deficits. N348I, which emerges early occurred with the lamivudine mutation M184V. and independently of TAMs, can recruit pyrophosphate Descriptive analysis of sampling times since antiretro- as a substrate for the excision reaction. viral therapy initiation suggested that M184V precedes The investigators proposed that diminished RNase H emergence of N348I. Because of these findings, Von Wyl cleavage plus enhanced DNA synthesis seen with con- et al. concluded that N348I alone probably does not nection domain mutations and TAMs take advantage of cause virological failure. Sampling time analysis also both pyrophosphate- and ATP-dependent mechanisms indicated that genotypes with both TAMs and N348I in engendering resistance. TAMs alone, on the other were sampled later during treatment than genotypes hand, exploit only the ATP-dependent mechanism. that showed M348I without TAMs. Earlier work by Brehm et al. showed that the connection domain mutation A371V and the RNase H Differing PI resistance pathway with HIV-1B and domain mutation Q509L plus the TAMs D67N, K70R CRF01_AE and T215F increase resistance to zidovudine 50-fold Weaker affinity of HIV-1 circulating recombinant form compared with the TAMs alone [53]. This work dem- (CRF)01_AE than HIV-1 clade B for PIs may lie behind onstrated increased AZT-MP excision as a result of differing protease resistance pathways with the two decreased RNase H cleavage. To further elucidate this HIV-1 clades, according to results of crystal structure mechanism, Brehm et al. determined rates of RNase H and calorimetry experiments by Bandaranayake et al. cleavage by transient or steady-state kinetic approaches [56]. CRF01_AE, seen predominantly in Southeast and assessed the ability of wild-type or mutant RTs to Asia, differs about 10% from clade B in its protease- bind template/primers with a duplex length <18 nucle- encoding sequence and a novel protease mutation out- otides in a DNA polymerase-competent or RNase side the active site, N88S, evolves in CRF01_AE in H-competent mode [54]. response to nelfinavir. Because initial RNase H cleavage with mutant and Bandaranayake et al. determined the crystal structure wild-type RTs were similar, the investigators suggested of CRF01_AE N88S protease in complex with the PI that A37IV and Q509L have no direct effect on catalytic darunavir to examine the impact of protease sequence activity of the RNase H active site. Q509L decreased polymorphisms on inhibitor binding. Comparing 10-nucleotide RNA/DNA duplex formation that occurs binding constants and thermodynamic parameters of after template/primer dissociation. AZT-MP excision CRF01_AE and clade B by calorimetry, they found that was more efficient on RNA/DNA duplexes >10 nucle- CRF01_AE had approximately eightfold weaker bind- otides. After template/primer dissociation and rebind- ing to nelfinavir and twofold weaker binding to daru- ing, RNase H cleavage decreased approximately two- navir. Binding to amprenavir did not differ between the fold with Q509L plus TAMs. two HIV-1 clades. Q509L increased ATP-mediated AZT-MP excision Crystal structure analysis revealed a difference in the on RNA/DNA template/primers, but not on DNA/ flap hinge region of protease between CRF01_AE and DNA template/primers. The mutation did not increase clade B. Specifically, the CRF01_AE flap hinges packed initial rates of polymerase-directed RNase H activity. against the protease core region via hydrogen bonds Q509L decreased secondary cleavages requiring tem- not seen with clade B. The Ser88 side chain directly plate/primer dissociation and it increased dissocia- interacted with Asp30 and caused its side chain to tion of RT from template/primer bound in the RNase flip away from the protease active site. Bandaranay- H-competent mode. ake et al. hypothesized that this interaction disrupts a Von Wyl et al. analysed the evolution of connection hydrogen bond essential for nelfinavir binding to pro- domain mutations E312Q, G333D, G335C/D, N348I, tease and favours evolution of N88S under pressure A360I/V, A365I, T369I, A371V, A376S and E399D, from nelfinavir. Clade B virus protease has a relatively together with TAMs and M184V, in sequences from higher affinity for nelfinavir and thus requires a com- 445 antiretroviral-naive patients and from 797 with bination of active site mutations and non-active site antiretroviral experience [55]. Most connection mutations (D30N plus N88D) to develop resistance to domain mutations appeared significantly more often this PI. CRF01_AE protease has a weaker affinity for

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nelfinavir and thus requires only a non-active site muta- substitution in subtype 1a. Because two successive tion to become resistant. Furthermore, the N88S path- substitutions are needed to render HCV-1b resistant way allows CRF01_AE protease to preserve Asp30, to PIs, resistance is less likely to emerge in 1b viruses which is important for substrate recognition. than in 1a viruses. Inability of VX950 or ITMN191 to select resistant virus in vitro could reflect use of HCV Differing resistance profiles of three HCV PIs replicon 1b laboratory strains. Courcambeck et al. The hepatitis C virus (HCV) NS3/4A protease mutations concluded that subtype 1a HCV resistance selection R155K/T differ in their impact on the HCV PIs VX950, assays are needed. ITMN191 and SCH503034 [57]. Although R155K and R155T are not selected in vitro in subtype 1b replicon Entecavir mechanism underpins rationale for novel assays, VX-950 monotherapy selects these mutations in nucleoside development HCV genotype 1a patients. R155K confers an 89-fold Although entecavir potently inhibits replication of decrease in susceptibility to ITMN191 in vitro in subtype hepatitis B virus (HBV), discovery that it selects the 1b replicon assays. SCH503034 does not select R155K/T M184V mutation in HIV-1 RT [58] prevents its use mutations in patients infected with HCV genotype 1. without antiretrovirals in HBV–HIV-coinfected peo- Courcambeck et al. sought to clarify these findings ple. Entecavir is a guanosine nucleoside analogue, by analysing the complexes formed between HCV but chain termination after incorporation of entecavir NS3-4A R155K/T and each of these agents. In R155K monophosphate (ETV-MP) into the template/primer complexes, SCH503034 and VX950 can partially van does not explain its antiviral activity. To elucidate der Waals interactions with the K155 side chain. Cour- the mechanisms of that activity, Tchesnokov et al. cambeck et al. suggested that HCV NS3-4A R155K/T conducted biochemical studies that disclosed several induce loss of a key salt bridge with D168, and this resi- mechanistic findings [59]. due stays in the hydrophobic S4 binding pocket. There- RT pausing at positions n and n+3 following fore, D168, a polar residue, is in front of the bulky and ETV-MP incorporation can be overcome by increas- hydrophobic VX950 P4 cyclohexyl moiety and causes ing natural dNTP concentrations at n+1 and n+4, repulsive interactions. Thus, discrimination of VX950 but incorporation of the natural nucleotide at n+4 by R155K/T is indirectly transferred through D168 to becomes severely limited. Nucleotide incorporation the P4–S4 interaction domain. at n+1 decreased 7.5-fold with entecavir-terminated The investigators found that, compared with VX950, primers versus natural counterparts, whereas incorpo- SCH503034 undergoes minor perturbations with ration at n+4 decreased 1,233-fold. ETV-MP forced R155K/T mutations and can compensate for these RT to slide away from the 3′-end of the primer at n+3, mutations through its overall interactions with the an effect Tchesnokov et al. proposed as a plausible HCV NS3-4A protease catalytic domain. However, cause of delayed chain termination. Delayed chain SCH503034 appears to have more difficulty compen- termination at position n+3 appears to be the major sating for the P2–S2 interactions lost with the R155T mechanism of action of entecavir. mutant. Indeed, the R155T strain increases the dis- Demonstration that RT bearing TAMs efficiently crimination capacity of HCV NS3-4A compared with excises ETV-MP at position n reflects the same finding R155K, by loss of hydrophobic interactions in the P2–S2 with other NRTIs. Nevertheless, entecavir remained interactions domain. R155K affects the recognition and fully sensitive to virus harbouring TAMs. Thus, Tch- covalent steps of VX950. Selection of a second mutation esnokov et al. concluded, the additional nucleotides at affecting the capacity of VX950 to be covalently linked n+1 to n+3 protect ETV-MP from excision. That ability to HCV NS3-4A protease, such as V36M, will have a to circumvent TAM-mediated excision prompted these synergistic effect on R155K-mediated resistance. investigators to propose the development of entecavir- Courcambeck et al. described HCV NS3-4A R155K like delayed chain terminators as antiretrovirals. resistance to ITMN191 by a direct resistance mechanism in the P2–S2 interactions area. The R155K muta- References tion replaces the planar R155 guanidinium π system by + 1. Fransen S, Gupta S, Danovich R, et al. Loss of raltegravir a formal charge –NH3 in front of the hydrophobic P2 susceptibility in treated patients is conferred by multiple isoindoline moiety of ITMN191. Therefore, ITMN191 non-overlapping genetic pathways. Antivir Ther 2008; undergoes strong repulsive interactions in the P2–S2 13 Suppl 3:A9. interactions domain. An indirect mechanism transferred 2. Miller MD, Danovich RM, Ke Y, et al. Longitudinal analysis of resistance to the HIV-1 integrase inhibitor by D168 to the P4–S4 interactions area also contributes raltegravir: results from P005, a phase II study in treatment- to resistance to ITMN191. experienced patients. Antivir Ther 2008; 13 Suppl 3:A8. The R155K/T mutations require two nucleotide 3. Hatano H, Lampiris H, Huang W, et al. Virological and immunological outcomes in a cohort of patients failing substitutions in HCV subtype 1b, but only one integrase inhibitors. Antivir Ther 2008; 13 Suppl 3:A12.

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4. Hazuda DJ, Miller MD, Nguyen BY, Zhao J. Resistance to 23. Madruga JV, Cahn P, Grinsztejn B, et al. Efficacy and the HIV-integrase inhibitor raltegravir: analysis of protocol safety of TMC125 (etravirine) in treatment-experienced 005, a Phase II study in patients with triple-class resistant HIV-1-infected patients in DUET-1: 24-week results from a HIV-1 infection. Antivir Ther 2007; 12:S10. randomised, double-blind, placebo-controlled trial. Lancet 5. Hazuda DJ, Young SD, Guare JP, et al. Integrase inhibitors 2007; 370:29–38. and cellular immunity suppress retroviral replication in 24. Lazzarin A, Campbell T, Clotet B, et al. Efficacy and safety rhesus macaques. Science 2004; 305:528–532. of TMC125 (etravirine) in treatment-experienced HIV- 6. Smith PF, Ogundele A, Forrest A, et al. Phase I and II 1-infected patients in DUET-2: 24-week results from a study of the safety, virologic effect, and pharmacokinetics/ randomised, double-blind, placebo-controlled trial. Lancet pharmacodynamics of single-dose 3-o-(3′,3′- 2007; 370:39–48. dimethylsuccinyl)betulinic acid (bevirimat) against human 25. Benhamida J, Chappey C, Coakley E, Parkin NT. immunodeficiency virus infection. Antimicrob Agents HIV-1 genotype algorithms for prediction of etravirine Chemother 2007; 51:3574–3581. susceptibility: novel mutations and weighting factors 7. McCallister S, Lalezari J, Richmond G, et al. HIV-1 gag identified through correlations to phenotype. Antivir Ther polymorphisms determine treatment response to bevirimat 2008; 13 Suppl 3:A142. (PA-457). Antivir Ther 2008; 13 Suppl 3:A10. 26. Benhamida J, Coakley E, Parkin NT, Chappey C. Increased 8. Van Baelen K, Salzwedel K, De Wolf H, et al. HIV-1 phenotypic susceptibility to etravirine in HIV-1 with susceptibility to the maturation inhibitor bevirimat is nucleoside reverse transcriptase inhibitor resistance. 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Hexadecyloxypropyl Dec 2005). 6th European HIV Drug Resistance Workshop tenofovir (CMX157) has enhanced potency in vitro against 26–28 March 2008, Budapest. Poster 5. NRTI-resistant HIV relative to tenofovir and a favorable preclinical profile. Antivir Ther 2008; 13 Suppl 3:A6. 29. Ross LL, Dix l, Wine B, et al. Change in regional prevalence, clade and epidemiology of HIV-1 drug 12. Murphy R, Zala C, Jakubik JJ, et al. In vitro cross- resistance mutations and clade among antiviral therapy- resistance profile, antiviral activity, safety and naive patients in the United States from 2000 to 2007. pharmacokinetics in HIV-1 infected patients of IDX899, a Antivir Ther 2008; 13 Suppl 3:A160. novel HIV-1 NNRTI with high barrier to resistance. Antivir Ther 2008; 13 Suppl 3:A7. 30. The World Health Organization 2008 List of Mutations for Surveillance of Transmitted Drug Resistant HIV Strains. 13. Murphy R, Zala C, Zhou XJ, et al. Antiviral activity, safety http://hivdb.stanford.edu/pages/WHOResistanceList.html and pharmacokinetics of IDX899, a novel HIV-1 non- nucleoside reverse transcriptase inhibitor with high barrier 31. Deeks SG, the North American AIDS Cohort Collaboration to resistance, in treatment-naive HIV-1-infected patients. on Research and Design (NA-ACCORD) of the IeDEA. The Antivir Ther 2008; 13 Suppl 3:A27. prevalence of multidrug resistance among treated patients 14. de Bethune MP, Andries K, Azijn H, et al. TMC278, a new in North America and its impact on mortality. Antivir Ther potent NNRTI, with an increased barrier to resistance and 2008; 13 Suppl 3:A153. favourable pharmacokinetic profile. 12th Conference on 32. Hogg RS, Bangsberg DR, Lima VD, et al. Emergence of drug Retroviruses and Opportunistic Infections. February 22–25, resistance is associated with an increased risk of death among 2005. Boston. Poster 556. patients first starting HAART. PLoS Med 2006; 3:e356. 15. Welch BD, Redman JP, Paul S, et al. Potent HIV-1 inhibitors 33. Gill VS, Lima VD, Fernandes KA, Harrigan PR. Injection with a reserve of binding energy against resistance drug users have a higher probability of developing mutations. Antivir Ther 2008; 13 Suppl 3:A3. resistance than non-injection drug users regardless of 16. Jekle A, Chhabra M, Chou E, et al. Resistance to adherence or initial treatment regimen. Antivir Ther 2008; CCR5mAb RoAb3952 is associated with a shift in binding 13 Suppl 3:A157. from the extracellular domain 2 to the N terminus of CCR5. Antivir Ther 2008; 13 Suppl 3:A4. 34. Harrigan PR, Hogg RS, Dong WWY, et al. Predictors of HIV drug-resistance mutations in a large antiretroviral- 17. Ji C, Brandt M, Dioszegi M, et al. Novel CCR5 monoclonal naive cohort initiating triple antiretroviral therapy. J Infect antibodies with potent and broad-spectrum anti-HIV Dis 2005; 191:339–347. activities. Antiviral Res 2007; 74:125–137. 35. Tsibris AMN, Arnaout R, Korber B, et al. Dynamic HIV-1 18. Hunt G, Coovadia A, Abrams JE, et al. Low frequency escape from vicriviroc therapy in vivo. Antivir Ther 2008; K103N mutations are strongly associated with inadequate 13 Suppl 3:A97. virological responses to non-nucleoside reverse transcriptase inhibitor based therapy. Antivir Ther 2008; 13 Suppl 3:A132. 36. Gulick RM, Su Z, Flexner C, et al. Phase II study of the safety and efficacy of vicriviroc, a CCR5 inhibitor, in HIV- 19. Flys TS, Donnell D, Mwatha A, et al. Persistence of K103N- 1-Infected, treatment-experienced patients: AIDS clinical containing HIV-1 variants after single-dose nevirapine for trials group 5211. J Infect Dis 2007; 196:304–312. prevention of HIV-1 mother-to-child transmission. J Infect Dis 2007; 195:711–715. 37. Su Z, Reeves JD, Krambrink A, et al. Response to vicriviroc in HIV-infected, treatment-experienced individuals using 20. Lockman S, Shapiro RL, Smeaton LM, et al. Response to antiretroviral therapy after a single, peripartum dose of an enhanced version of the Trofile HIV co-receptor tropism nevirapine. N Engl J Med 2007; 356:135–147. assay [Trofile (ES)]: reanalysis of ACTG 5211 results. Antivir Ther 2008; 13 Suppl 3:A98. 21. Peeters M, Nijs S, Vingerhoets J, et al. Determination of phenotypic clinical cut-offs for etravirine: pooled week 24 38. Low AJ, Dong W, Chan D, et al. Current V3 genotyping results of the DUET-1 and DUET-2 trial. Antivir Ther 2008; algorithms are inadequate for predicting X4 co-receptor 13 Suppl 3:A133. usage in clinical isolates. AIDS 2007; 21:F17–F24. 22. Coakley E, Chappey C, Benhamida J, et al. Biological and 39. Moores A, Thielen A, Dong W, et al. Improved detection of clinical cut-off analyses for etravirine in the PhenoSense X4 virus by V3 genotyping: application to plasma RNA and proviral DNA. Antivir Ther 2008; 13 Suppl 3:A99. HIV assay. Antivir Ther 2008; 13 Suppl 3:A134.

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40. Thielen A, Harrigan PR, Lou AJ, et al. Improved genotypic 50. Das K, Bandwar R, White KL, et al. Structural basis for K65R prediction of HIV-1 coreceptor usage by incorporating V2 function: tenofovir resistance, reduced nucleotide incorporate loop sequence variation. Antivir Ther 2008; 13 Suppl 3:A100. and excision antagonism. Antivir Ther 2008; 13 Suppl 3:A44. 41. Däumer MP, Kaiser R, Klein R, et al. Inferring viral 51. Nikolenko GN, Delviks-Frankenberry KA, Palmer S, tropism from genotype with massively parallel sequencing: et al. Mutations in the connection domain of HIV-1 qualitative and quantitative analysis. Antivir Ther 2008; reverse transcriptase increase 3′-azido-3′-deoxythymidine 13 Suppl 3:A101. resistance. Proc Natl Acad Sci U S A 2007; 104:317–322. 42. Maldarelli F, Wiegand A, Palmer S, et al. Intensification 52. Beilhartz GL, Ehteshami M, Scarth B, et al. Zidovudine with efavirenz or lopinavir/ritonavir does not reduce resistance related connection mutations in HIV-1 reverse residual HIV-1 viraemia in patients on standard transcriptase cause selective dissociation from RNase H antiretroviral therapy. Antivir Ther 2008; 13 Suppl 3:A79. competent complexes. Antivir Ther 2008; 13 Suppl 3:A45. 43. Havlir DV, Strain MC, Clerici M, et al. Productive infection 53. Brehm JH, Koontz D, Meteer JD, et al. Selection of maintains a dynamic steady state of residual viremia in mutations in the connection and RNase H domains of human immunodeficiency virus type 1-infected persons human immunodeficiency virus type 1 reverse transcriptase treated with suppressive antiretroviral therapy for five years. that increase resistance to 3′-azido-3′-dideoxythymidine. J Virol 2003; 77:11212–11219. J Virol 2007; 81:7852–7859. 44. Maldarelli F, Palmer S, King MS, et al. ART suppresses 54. Brehm J, Mellors J, Sluis-Cremer N. Q509L in HIV-1 plasma HIV-1 RNA to a stable set point predicted by reverse transcriptase increases zidovudine resistance pretherapy viremia. PLoS Pathog 2007; 3:e46. by promoting polymerase-competent versus RNase H-competent binding on RNA/DNA template/primers with 45. Micek MA, Blanco AJ, Beck IA, et al. The selection, short duplex lengths. Antivir Ther 2008; 13 Suppl 3:A46. transmission and persistence of drug-resistant HIV-1 in 55. von Wyl V, Bürgisser P, Yerly S, et al. Evolution of reverse infants prophylaxed with single-dose nevirapine varies by transcriptase connection domain mutations in patients on timing of infection. Antivir Ther 2008; 13 Suppl 3:A78. antiretroviral therapy. Antivir Ther 2008; 13 Suppl 3:A47. 46. Schweitzer F, Mueller SM, Daeumer M, et al. Correlations 56. Bandaranayake RM, Ng C, Kolli M, et al. Structural between transmitted HIV-1 drug resistance mutations and explanations to altered drug resistance pathways in HIV-1 the human leukocyte antigen alleles of therapy-naive HIV non-clade B proteases. Antivir Ther 2008; 13 Suppl 3:A42. patients. Antivir Ther 2008; 13 Suppl 3:A76. 57. Courcambeck J, Bouzidi M, Roche G, et al. How hepatitis 47. Kearney M, Shao W, Maldarelli F, et al. HIV-1 recombination C virus NS3-4A protease R155K/T strains can discriminate in patients infected with multiple HIV-1 variants from the VX-950 and ITMN-191 but affect differentially SCH- same donor. Antivir Ther 2008; 13 Suppl 3:A77. 503034. Antivir Ther 2008; 13 Suppl 3:A43. 48. Palmer S, Maldarelli F, Kearney M, et al. Single cell 58. McMahon MA, Jilek BL, Brennan TP, et al. The HBV drug analysis of HIV DNA from infected patients. Antivir Ther entecavir—effects on HIV-1 replication and resistance. N 2008; 13 Suppl 3:A75. Engl J Med 2007; 356:2614–2621. 49. Grobler JA, Stillmock KA, Miller MD, Hazuda DJ. 59. Tchesnokov E, Obikhod A, Schinazi RF, Götte M. Delayed Mechanism by which the HIV integrase active-site mutation chain-termination protects the hepatitis B virus drug N155H confers resistance to raltegravir. Antivir Ther 2008; entecavir from excision by HIV-1 reverse transcriptase. 13 Suppl 3:A41. Antivir Ther 2008; 13 Suppl 3:A48.

Accepted for publication 31 October 2008

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