broad phenotypic cross-resistance to elvitegravir in hiv-infected

1

Broad Phenotypic Cross-Resistance to Elvitegravir in HIV-Infected 1 Patients Failing on Raltegravir-Containing Regimens 2

3 Carolina Garrido1, Jorge Villacian2, Natalia Zahonero1, Theresa Pattery2, Federico Garcia3, 4

Felix Gutierrez4, Estrella Caballero5, Margriet Van Houtte2, Vincent Soriano#1 and Carmen de 5 Mendoza1; on behalf of the SINRES Group 6

7 1Hospital Carlos III, Madrid, Spain; 2Virco BVBA, Mechelen, Belgium; 3Hospital Clínico 8

Universitario San Cecilio, Granada, Spain; 4Hospital General Universitario, Elche, Spain; 9 5Hospital Vall d’Hebrón, Barcelona, Spain 10

11 12 13

Key words: HIV, elvitegravir, raltegravir, drug resistance. 14 15 Running title: Elvitegravir and Raltegravir cross-resistance 16 17 18 Corresponding author: 19 20 Dr. Vincent Soriano 21 Department of Infectious Diseases 22 Hospital Carlos III 23 Calle Sinesio Delgado 10 24 28029 Madrid, Spain 25 Phone: 34 91 4532500 26 Fax: 34 91 7336614 27 e-mail: [email protected] 28 29 Alternative corresponding author: 30 Dr. Carolina Garrido 31 Department of Infectious Diseases 32 Hospital Carlos III 33 Calle Sinesio Delgado 10 34 28029 Madrid, Spain 35 Phone: 34 91 4532500 36 Fax: 34 91 7336614 37 e-mail: [email protected] 38

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.06170-11 AAC Accepts, published online ahead of print on 26 March 2012

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Spanish INtegrase RESistance (SINRES) Group 39 J Pedreira (Hospital Juan Canalejo, La Coruña); JA Iribarren (Hospital de Donostia, San 40 Sebastián); J Solá (Hospital de Navarra, Pamplona); MJ Téllez, V Estrada (Hospital Clínico San 41 Carlos, Madrid); S García, JR Arribas (Hospital La Paz, Madrid); l Santos (Hospital de la 42 Princesa, Madrid); P Miralles (Hospital Gregorio Marañón, Madrid); JE Losa (Hospital 43 Universitario Fundación de Alcorcón, Madrid); M Cervero (Hospital Severo Ochoa, Madrid); J 44 Sanz (Príncipe de Asturias, Madrid); C Barros (Hospital de Móstoles, Madrid); M de Górgolas 45 (Fundación Jiménez Díaz, Madrid); E Ribera, E Caballero (Hospital Vall d’Hebrón, Barcelona); 46 C Vidal, M Riera (Hospital Son Dureta, Palma de Mallorca); C Gómez (Complejo Hospitalario 47 de Toledo); A Chocarro (Virgen de la Concha, Zamora); MJ Galindo (Hospital Clínico 48 Universitario, Valencia); F Gutiérrez (Hospital General Universitario, Elche); I Viciana, J. 49 Santos (Hospital Virgen de la Victoria, Málaga); M Omar (Hospital Ciudad de Jaén); A 50 Collado, C Gálvez (Hospital Torrecardenas, Almeria); AB Lozano, JM Fernández (Hospital 51 Poniente, Almería); V Gutiérrez-Ravé (Hospital de Motril, Granada); F García, MA López Ruz, 52 C Hidalgo, J Pasquau (Hospital Universitario Virgen de las Nieves, Granada); F García, V 53 Guillot, J Hernández-Quero (Hospital Universitario San Cecilio Granada); JA Pineda (Hospital 54 de Valme, Sevilla); C Garrido, L Anta, C de Mendoza, V Soriano (Hospital Carlos III, Madrid). 55

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ABSTRACT 57

58

Background: Failure of raltegravir (RAL) is generally associated with selection of mutations 59

at integrase positions Y143, Q148 or N155. However, a relatively high proportion of failures 60

occur in the absence of these changes. Herein, we report the phenotypic susceptibility to 61

RAL and elvitegravir (EVG) in a large group of HIV-infected patients failing on RAL. 62

Methods: Plasma from HIV-infected individuals failing on RAL-containing regimens 63

underwent genotypic and phenotypic resistance testing (Antivirogram v2.5.01, Virco). A 64

control group of patients failing on other regimens were similarly tested. 65

Results: Sixty-one samples were analysed, 40 of which belonged to patients failing on RAL. 66

Full RAL susceptibility was found in 20/21 controls, while susceptibility to EVG was 67

diminished in 8, with a median fold-change (FC) of 2.5 [2.1-3.1]. Fourteen samples from RAL 68

failures showed diminished RAL susceptibility, with a median FC of 38.5 [10.8-103.2]. 69

Primary integrase resistance mutations were found in 11 of these samples, displaying a 70

median FC of 68.5 [23.5-134.3]. The remaining 3 samples showed a median FC of 2.5 [2-2.7]. 71

EVG susceptibility was diminished in 19/40 samples from RAL failures (median FC=7.71 72

[2.48-99.93]). Cross-resistance between RAL and EVG was high (R2=0.8, p<0.001), being drug 73

susceptibility more frequently reduced for EVG than for RAL (44.3% vs 24.6%, p=0.035). 74

Conclusions: The susceptibility to RAL and EVG is rarely affected in the absence of primary 75

integrase resistance mutations. There is broad cross-resistance between RAL and EVG, 76

which should preclude their sequential use. Resistance to EVG seems to be more frequent 77

and might be more influenced by integrase variability. 78

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INTRODUCTION 80

81

The introduction of highly active antiretroviral therapy (HAART) has transformed the 82

prognosis of HIV-positive patients, with dramatic improvements in survival. However, 83

selection of drug resistance still represents a major challenge, and promotes switches in 84

therapeutic regimens in a substantial proportion of patients (3). Virologic failure under 85

some antiretroviral drugs is associated with cross-resistance to agents within the same 86

family, limiting rescue therapeutic options (16). Nevirapine and efavirenz are good examples 87

of cross-resistance between members of the same drug family, sharing almost completely 88

resistance patterns. To a lesser exent, it also occurs with failures under some protease 89

inhibitors (12) and nucleoside analogues. 90

91

Integrase inhibitors (INI) are a new and promising drug family for the treatment of HIV 92

infection. Raltegravir (RAL) is the first-in-class approved drug for clinical use in both 93

treatment-naïve (11) and treatment-experienced individuals (19). Elvitegravir (EVG) will 94

most likely be the second INI compound to be marketed (4). Resistance to INIs has shown to 95

be driven by changes mainly located in the central domain of the viral integrase. The results 96

of clinical trials and clinical experience have highlighted that failure on RAL-containing 97

regimens is generally driven by selection of mutations Y143RHC, Q148HRK and/or N155H, 98

often accompanied by secondary changes (G140SA, E138K, L74M, etc.) (2). However, a 99

relatively high proportion of patients do not show such changes upon virologic failure on 100

RAL. Although unidentified resistance mutations might exist, a recent report has highlighted 101

that the absence of INI resistance mutations in these individuals is mainly explained by poor 102

drug compliance, given that RAL plasma levels were undetectable in most of them (6). 103


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104

Information about resistance to EVG in vivo is scarce. In the phase II GS-US-183–105 study 105

(13), 28 individuals failed EVG after 24 weeks of treatment. Overall, 39% of them selected 106

the mutation E92Q, 32% selected Q148HRK and 25% selected N155H. Accordingly, broad 107

cross-resistance between RAL and EVG should be expected. The aim of our study was to 108

characterize RAL phenotypic susceptibility in samples from a group of patients experiencing 109

RAL failure, exploring whether mutations not previously involved in RAL resistance might be 110

recognised and the extent of phenotypic cross-resistance to EVG. 111

112

PATIENTS AND METHODS 113

114

Study population 115

HIV-1-infected individuals treated at several clinics in Spain who experienced virological 116

failure under a RAL-containing antiretroviral regimen, were identified during 2009. 117

Virological failure was defined as the first sample with plasma HIV-RNA >50 copies/mL, 118

confirmed in a second specimen collected within a month. Then, plasma specimens 119

collected at the time of first RAL failure were sent to Hospital Carlos III along with 120

information on viral load, CD4 counts and antiretroviral combinations. In addition, samples 121

from INI naïve patients were also sent to be used as controls. Most participating centres 122

belonged to RIS (Red de Investigación en SIDA), the government funded Spanish AIDS 123

research network, which involves around twenty-five HIV clinics across the country and 124

whose main characteristics have already been described elsewhere (17). All specimens that 125

were examined in the current study were part of the repository collected for the SinRes 126


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study, a previous survey that characterized virologically RAL failures in a total of 106 127

patients (6). Signed informed consent was obtained from each individual. 128

129

Genotypic resistance analysis 130

The analysis of the HIV integrase gene was performed by bulk sequencing using an in-house 131

PCR protocol, with primers and conditions previously described (7). Primary integrase 132

resistance mutations were defined as those changes that cause significant loss of drug 133

susceptibility by themselves. This is the case for Y143RHC, Q148HRK and N155H. 134

135

Phenotypic resistance analysis 136

Viral RNA was isolated from all plasma samples using NucliSENS EasyMAG (Biomerieux). One 137

amplicon per sample containing the reverse transcriptase – integrase (RT-IN) regions was 138

sequenced and cloned into an HXB2 backbone. Recombinant viruses were titrated and 139

subjected to an antiviral experiment in MT4-LTR eGFP cells, as previously described (20). 140

Briefly, MT4-LTR-eGFP cells were inoculated with a titrated amount of virus in the presence 141

of 3-fold dilutions of the compound tested. After 3 days of incubation at 37°C, infection was 142

quantified by means of fluorescent microscopy measuring HIV Tat-induced enhanced green 143

fluorescent protein (eGFP) expression. Using the HIV-1 wild-type strain IIIB as reference, fold 144

change (FC) values were calculated by dividing the mean 50% effective concentration (EC50) 145

for a recombinant virus stock by that for the reference strain. A biological cutoff (BCO) for 146

RAL and EVG was calculated on clonal database (991 clones from 153 clinical isolates) as the 147

97.5 percentile of the log FC phenotypes of patient-derived clonal viruses not containing any 148

of the mutations listed in the RAL package insert label: 74M, 92Q, 97A, 138A/K, 140A/S, 149

143C/H/R, 148H/K/R, 151I, 155H, 163R, 183P, 226C/D/F/H, 230R and 232N, and/or not 150

containing mutations with score >0 for RAL/EVG in the Stanford algorithm 6.0.11. Before 151

calculating the BCO, outliers (log FC > mean log FC +3 standard deviations) were removed 152

(22). Thus, biological cut-offs were preliminarly established as FC=2 for RAL and FC=1.9 for 153

EVG. 154


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155

Statistical analysis 156

Results are expressed as medians [IQR] and percentages. Comparisons were performed with 157

non-parametric tests using Mann-Whitney or Chi-square tests. FC values were log 158

transformed for analysis of the phenotypic resistance correlation using bivariate analysis. All 159

statistical analyses were carried out using the SPSS v15 software (SPSS Inc., Chicago, IL). 160

161

RESULTS 162

163

Study population 164

A total of 61 samples were examined, 21 belonging to INI-naïve patients and 40 obtained 165

from patients experiencing early virological failure on RAL-containing regimens. All INI-naïve 166

individuals were antiretroviral-experienced. The main characteristics of this population are 167

summarized in Table 1. 168

169

Resistance testing in INI-naïve patients 170

Phenotypic results in specimens collected from the 21 INI-naïve individuals, showed that all 171

but one (95.2%) were susceptible to RAL. The single sample displaying a FC of 2.4 to RAL did 172

not harbor any primary nor secondary known integrase resistance mutation. However, 173

compared to reference sequences the following changes at the integrase gene were 174

present: E11D, S24G, D25E, S39C, M50IM, I72V, L101I, V201I and S283G. In contrast, up to 175

8/21 (38%) of the INI-naïve samples displayed FC above 1.9 for EVG. Primary or secondary 176

resistance changes were neither found in these specimens, as shown in Table 2. 177

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Resistance testing in RAL failures 179

Overall, 11 out of 40 (27.5%) samples collected from patients experiencing early RAL failure 180

harbored primary INI resistance mutations, distributed as follows: Q148H/R (n=5), N155H 181

(n=3), Y143R (n=2) and a complex pattern including Y143HY+N155H (n=1). All sequences 182

examined in this study have been submitted to Genbank, with accession numbers 183

JQ716857-JQ716918. 184

185

Fourteen (35%) samples displayed phenotypic FC above 2 for RAL, including the 11 186

specimens with primary INI resistance mutations. Three samples, which did not contain any 187

primary INI resistance change, displayed low FC values for RAL (2, 2.5 and 2.7, respectively). 188

As a comparison, median FC was of 58.9 [26.5-125.3] for the eleven patients with INI 189

resistance mutations. The main characteristics of these patients are recorded in Table 3, 190

including information on RAL plasma levels as an indirect marker of drug adherence. 191

192

When testing EVG, 19 (47.5%) of RAL failures displayed impaired EVG sensitivity. Overall, 193

median FC was 7.71 [2.48-99.93], but it was much greater in the 11 samples harboring 194

primary INI resistance mutations than in the 8 samples without major integrase changes 195

(99.93 [46.92-99.93] vs 2.45 [2.36-2.83], p<0.001). Table 3 records the genotypic and 196

phenotypic data for EVG in these patients. 197

198

Primary INI resistance mutations led to high-level resistance to both RAL and EVG, although 199

with slight differences. Mutation Y143R conferred greater level of resistance to RAL than 200

EVG, with FC ranging from 26.5 to 58.9 vs 3.92 to 7.68, respectively. However, it is 201

noteworthy that this change, which is not considered a primary resistance mutation for 202


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EVG, significantly compromised EVG susceptibility in the presence of secondary resistance 203

mutations (T97A+V151I for one specimen and L74M for another). 204

205

Impact of minor integrase changes on RAL and EVG susceptibility 206

Comparisons of RAL and EVG FC in the presence/absence of changes at single integrase 207

positions were performed using non-parametric tests. Only samples without primary INI 208

resistance mutations were considered for this purpose, and therefore the analysis was 209

carried out in a subset of 50 specimens. Changes at 5 positions were positively associated 210

with increased FC for RAL compared to the wild type. They were as follows: S24G/N, D25E, 211

L74I, K215N and Q221H/S. On the contrary, the K156N change reduced the FC to RAL 212

compared to the wild type. Of note, mutations L74I and K215N appeared together in the 213

same specimen, as well as most S24G/N and D25E. All this information is summarised in 214

Table 4. With respect to EVG, mutations L45Q/V, V72I and R263K were associated to an 215

increased FC, while S119G/P/S/T, V126M/L and Q216H seemed to enhance EVG 216

susceptibility in comparison with the wild type. 217

218

Cross-resistance between RAL and EVG 219

Bivariate associations between FC to RAL and EVG expressed as log values resulted in a 220

significant positive correlation (r2=0.8, p<0.001), supporting that there was a high degree of 221

cross-resistance between both drugs. However, reduced susceptibility to EVG was more 222

common than for RAL, as 27/61 (44.3%) samples displayed EVG resistance in comparison 223

with only 15/61 (24.6%) of them (p=0.002). 224

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DISCUSSION 226

227

Resistance to both RAL and EVG has been described to be mainly associated with changes at 228

integrase positions 148 and 155. Our phenotypic results confirm the impact of these 229

changes, since they conferred high FC to both drugs. Mutation Y143R leads to the third RAL 230

resistance pattern, and again its impact on RAL susceptibility was confirmed in our study, 231

showing high FC values. Interestingly, this mutation also impaired EVG susceptibility, 232

showing FC of 4 to 8 when Y143R was present along with other secondary changes. This is 233

somewhat unexpected since prior reports had claimed that Y143R did not influence EVG 234

susceptibility (5,14,18). Altogether, our results suggest that EVG activity is compromised in 235

the presence of any of the RAL resistance patterns, including Y143R. Therefore, the 236

sequential use of these drugs is not advisable upon failure with either of them. 237

238

A few samples in our study displayed small, but potentially clinically significant, reductions 239

in the susceptibility to integrase inhibitors, based upon the established biological cut-offs of 240

2.0 and 1.9 for RAL and EVG, respectively. The reduced sensitivity was more prominent for 241

EVG in the absence of primary INI resistance mutations. One specimen from a subject failing 242

on RAL did not show any known INI resistance mutation but displayed a 2.5 FC for RAL and 243

7.71 for EVG. This sample only harboured the change R263K as potentially responsible for 244

this effect on INI susceptibility. Interestingly, R263K has previously been shown to be 245

selected in vitro by EVG conferring a 5.2 FC to the drug (15). In the present study, it also led 246

to a slight but significant decrease in RAL susceptibility. Another patient displayed a FC of 247

2.7 to RAL despite lacking primary INI resistance mutations. However, change L74I had been 248

selected in this patient failing on RAL. Given that mutation L74M is a secondary resistance 249


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mutation generally selected along with N155H in patients failing on RAL (8,15), we suggest 250

that change L74I might equally compromise RAL susceptibility. 251

252

Overall, testing of samples collected from RAL failures in the absence of primary INI 253

resistance mutations allowed the recognition of certain minor changes that might impact INI 254

susceptibility in a clinically relevant manner. This was the case for changes S24G/N, D25E, 255

L74I, K215N and Q221H/S for RAL and L45Q/V, V72I and R263K for EVG. Additional 256

resistance studies are being performed by Virco to clear up the phenotypic effect of each of 257

these changes. However, their clinical impact should be further investigated in larger groups 258

of patients experiencing INI failure. 259

260

Most patients displaying resistance to RAL in our study also exhibited a reduced EVG 261

susceptibility. The extent of cross-resistance was high for all different resistance patterns 262

and may have important clinical implications regarding the consideration of sequential use 263

of RAL and EVG. Moreover, it was intriguing that EVG showed more often increased FC than 264

RAL, given that confronting integrase changes, the FC was almost uniformly more influenced 265

for EVG than for RAL. Hypothetically, our findings could be explained by the faster 266

dissociation time from the viral integrase for EVG than RAL, with a half-life of 2.4 and 9 267

hours, respectively (9). 268

269

The variability at the viral integrase has been shown to be greater in antiretroviral-270

experienced compared to drug-naïve patients (1,7,21). All our patients, regardless of RAL 271

exposure, were antiretroviral-experienced, which might have led to a higher rate of 272


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polymorphisms, contributing to explain the relatively high proportion of subjects with slight 273

reductions in RAL and especially EVG susceptibility in INI-naïve patients. 274

275

In summary, our results testing phenotypic and genotypic samples from RAL failures and 276

controls support that the susceptibility to RAL and EVG is mainly impaired by resistance 277

mutations Q148H/R, N155H and/or Y143R. Slight reductions in susceptibility to any of these 278

drugs can occur in the presence of other integrase changes. The wide extent of cross-279

resistance between RAL and EVG should preclude their sequential use. 280

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22. Verlinden Y, Vermeiren H, Lecocq P, Bacheler L, McKenna P, Vanpachtenbeke M, 373

Horvat LI, Van Houtte M, Stuyver LJ. 2005. Assessment of the Antivirogram® 374

performance over time including a revised definition of biological test cut-off values. 375

Antivir Ther., 10 (suppl):S51 376

377

378


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Table 1. Main characteristics of the study population. 1

2

Samples examined (n) 61INI naïve (%) RAL failures (%)

21 (34.4%) 40 (65.6%)

Median Plasma HIV-RNA (log copies/mL) 4.13 [3.38-4.82] Median CD4 count (cells/mm3) 242 [88-392] HIV-1 subtype (%)B 93.4 F 3.3G 1.6 CRF02_AG 1.6Time under RAL therapy*(weeks) 44.71 [22.57-70.57] 3

* Only for the 40 samples belonging to patients failing on raltegravir. 4

INI, integrase inhibitor; RAL, raltegravir 5


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Table 2. Genotypic and phenotypic results in patients naïve to HIV integrase inhibitors. 1

2

Sample ID ARV regimen Subtype FC

RALFC

EVGMinor

mutationsOther changes

N_1 ABC, AZT, TDF, DRV/r B 1,10 2,08

Q44K, I72V, T112IT, I113V, S119P, T122IT, E152G, T206S, T218S

N_2 FTC, ddI, ATV B 1,30 2,08 D6E, E11D, V32I, V37I, L101I, T125A,

I135V, V201I, S283G

N_3 NA B 1,30 2,19 I203M E11D, E13D, V31I, V32I, S39C, M50I, I60V, L101I, I113V, T122I, T124N, I208L

N_4 AZT, 3TC, NVP B 1,60 2,53 E138D V32I, D41N, I72V, L101I, T122I, T124A,

V201I

N_5 TDF, AZT, 3TC, ABC,

fAPV/r B 0,80 2,56 S230NS D6E, E11D, L28I, K34R, V37I, V201I,

L234IL, D253DE, A265AV

N_6 AZT, 3TC, fAPV/r B 1,80 2,63 T97A

E11D, S24G, D25E, L45V, M50I, L101I, T125A, F181L, T206S, D256E, S283G,

D288N

N_7 FTC, TDF, fAPV/r G 1,90 3,33

K14R, L101I, T112I, T124A, T125A, G134N, K136T, D167E, V201I, T206S, Q221H, D232E, L234I, S255N, D256E, S283G

N_8 FTC, TDF, ATV/r B 1,20 3,35

M50I, I113V, T124A, F181L, V201I, N222H, D256E

N_9 FTC, TDF, LPV/r B 2,40 1,68

E11D, S24G, D25E, S39C, M50IM, I72V, L101I, V201I, S283GS

3

FC, fold change; RAL, raltegravir; EVG, elvitegravir 4

ABC, abacavir; 3TC, lamivudine; FTC, emtricitabine; TDF, tenofovir; fAPV, fosamprenavir, 5

ATV, atazanavir; LPV, lopinavir. 6


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Table 3. Genotypic and phenotypic results in patients failing on raltegravir. 1

2

Sample ID Subtype ARV regimen Months on RAL

Detectable RAL plasma levels

FCRAL

FCEVG

Majormutations

Minormutations

Other changes

F_1 B ETR, DRV/r, RAL 8.9 - 2 1.1 D6T, K7E, E13D, V31I, V79I,

L101I, V201I

F_2 B AZT, 3TC, ATV,RAL 7.6 - 2.7 1.2 I203M E11D, V32I, S39C, L74I,

V201I, I208L, K215N

F_3 B MVC, ATV, RAL NA + 2.5 7.7 R263K M50I, I72V,S119P, T124A, V201I, I220L

F_4 B 3TC, MVC, RAL 9.6 + 78 99.9 Q148HQ G140GS E10D, V31I, I72V,

L101I, S119G, T122I, T125A, K156N, V201I, T206S, Q216H, Q221R, D256E

F_5 B FTC, TDF, ETR, DRV/r, RAL 5.3 + 161.2 99.9 Q148H G140S E11D, S24N, V31I,

T112IM, T206S, I208L, D253DE

F_6 B FTC, TDF, RAL 7 NA 125.3 99.9 Q148R G140A V31I, D41N,L101I, V201I, T206S, I268L, R269K

7 B FTC, TDF, RAL 7.6 + 161.2 99.9 Q148H G140S,A128AV

D6E, K14R, S17NS, V31I,L101I, K111EK, I135V, G163Q, G193E, V201I, D256E

F_8 B TDF, MVC, RAL NA + 95.9 99.9 Q148R G140A, E157Q

K14R, S24G, D25E, L45I,I113V, T124N, T125A, K160S, V165IV,

I220L, Y227F, L234V, D256E

F_9 B NA NA + 13.5 48.0 N155H S17N, L101I, T112I, V165I, H171L,

T206S, D256E

F_10 B 3TC, ABC, RAL 6.7 + 14.6 99.9 N155H L74LM E11D, K14R, K34R, D41N, P90S, L101I, K111KQ, S119RS, K156N, I208L, D256E

F_11 B AZT,

3TC,ETR,DRV/r, T20, RAL

5.2 + 27.6 46.9 N155H T97AT, E92EQ

K7R, S17N, R20K,L101I, T112V, S119P, T124A, I135V,

I220L, D232DN, D253DN

F_12 B d4T, TDF, ETR, DRV/r, RAL 21.7 + 26.5 3.9 Y143R T97AT,

V151IVS17N, V31I, M50T, I72V, V75IV,

T124A, K156N, T206S, I220IV, D232DN, D256E


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F_13 B FTC, TDF, RAL 12.8 NA 58.9 7.6 Y143R L74M K7R, V31I,L101I, T112I, T125V, K219N, N222K

F_14 B FTC, TDF, RAL 10.4 + 49.3 99.9 Y143HY,N155H

S17N, E96N, L101I, S119P, T122I, T124A, K156N,

V201I, T206S, I208L, Y227F, D253Y, D256E

F_15 B FTC, TDF, ETR, RAL 11.8 - 1.3 2.3

D6E, L28I, V37I, S39C, I84IM, L101I, T124A, T125A, I135V

F_16 B AZT, 3TC, TDF, T20, DRV/r, RAL 5.6 NA 1.5 2.4

K7KR, S17N, L101I, K111T, S119T, T124NS, I208IL, I220L, Q221S, D253E, D256E

F_17 B FTC, TDF, DRV/r, RAL 31.7 + 1.1 2.4 S230N E10D, V32I, S39C, L101I, T218S, D253E

F_18 B DRV/r, RAL NA - 0.7 2.4 D6E, S17N, V32I, L45V, T124N, I203M, D253E

F_19 B FTC, TDF, ETR, RAL 16.5 - 1.3 2.5

K7R, S17N, V31I, S39C, L101I, G163E, K173R, V201I, D232E

F_20 B 3TC, LPV/r,RAL NA NA 0.6 2.8 V31I, L45Q, L101I, T206S, N254Q

F_21 B FTC, TDF, RAL 18 + 0.6 2.8 S230N K7R, K14R, L101I, T112I 3

FC, fold change; RAL, raltegravir; EVG, elvitegravir; -, undetectable RAL plasma levels; +, detectable RAL plasma levels 4

DRV, darunavir; ETV, etravirine; MVC, maraviroc; ABC, abacavir; 3TC, lamivudine; FTC, emtricitabine; TDF, tenofovir; fAPV, fosamprenavir, ATV, 5

atazanavir; LPV, lopinavir; d4T, stavudine; T20, enfuvirtide 6


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Table 4. Phenotypic impact of minor changes at the HIV integrase gene in the absence of 1

primary resistance mutations on susceptibility to raltegravir and elvitegravir. 2

3 Integrase Inhibitor Position

FC [median, IQR] WT Mutation p

RAL

S24G/N 1.05 [0.70-1.30](n=46)

1.65 [1.27-2.25](n=4)

0.021

D25E 1.10 [0.70-1.30](n=47)

1.80 [1.20-2.40](n=3)

0.052

L74IK215N

1.10 [0.75-1.3](n=49)

2.70(n=1)

0.040

Q221H/S 1.05 [0.7-1.3](n=46)

1.4 [1.3-1.8](n=4)

0.044

K156N 1.10 [0.8-1.3](n=47)

0.7 [0.6-0.8](n=3)

0.047

EVG

L45Q/V 1.19 [0.9-2.08](n=47)

2.63 [2.43-2.80](n=3)

0.018

V72I 1.14 [0.76-1.44](n=18)

1.48 [1-2.42](n=32)

0.051

R263K 1.2 [0.92-2.22](n=49)

7.71(n=1)

0.040

S119G/P/S/T1.48 [1.09-2.45]

(n=33)1 [0.70-1.39]

(n=17)0.006

V126M/L 1.3 [0.98-2.33](n=48)

0.76 [0.70-0.76](n=2)

0.041

Q216H 1.3 [0.98-2.33](n=48)

0.62 [0.48-0.76](n=2)

0.015

4

FC, fold change; RAL, raltegravir; EVG, elvitegravir 5


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broad phenotypic cross-resistance to elvitegravir in hiv-infected

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