A genotypic assay for the amplification and sequencing of integrase from diverse HIV-1 group M subtypes

Journal of Virological Methods 153 (2008) 176–181

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A genotypic assay for the amplification and sequencing of integrase from diverse HIV-1 group M subtypes Kristel Van Laethem a,b,∗ , Yoeri Schrooten a,b , Kris Covens a , Nathalie Dekeersmaeker a , Paul De Munter c , Eric Van Wijngaerden c , Marc Van Ranst a,b , Anne-Mieke Vandamme a a b c

Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium Aids Reference Laboratory, University Hospitals Leuven, Leuven, Belgium Internal Medicine, University Hospitals Leuven, Leuven, Belgium

a b s t r a c t Article history: Received 6 March 2008 Received in revised form 14 July 2008 Accepted 17 July 2008 Available online 2 September 2008 Keywords: PCR Sequencing HIV-1 subtypes Resistance

Recently, the Food and Drug Administration (FDA) of the USA approved the first integrase inhibitor for inclusion in treatment regimens of HIV-1 patients failing their current regimens with multi-drug resistant strains. However, treatment failure has been observed during integrase inhibitor-containing therapy. Several mutational pathways have been described with signature mutations at integrase positions 66, 92, 148 and 155. Therefore, a genotypic assay for the amplification and sequencing of HIV-1 integrase was developed. The assay displayed a detection limit of 10 HIV-1 IIIB RNA copies/ml plasma. As the HIV-1 pandemic is characterised by a large genetic diversity, the new assay was evaluated on a panel of 74 genetically divergent samples belonging to the following genetic forms A, B, C, D, F, G, J, CRF01-AE, CRF02-AG, CRFF03-AB, CRF12-BF and CRF13-cpx. Their viral load ranged from 178 until >500,000 RNA copies/ml. The amplification and sequencing was successful for 70 samples (a success rate of 95%). The four failures were most probably due to low viral load or poor quality of RNA and not to subtype issues. Some of the sequences obtained from integrase inhibitor-naïve patients displayed polymorphisms at integrase positions associated with resistance: 74IV, 138D, 151I, 157Q and 163AE. The relevance of these polymorphisms in the absence of the signature mutations remains unclear. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Antiretroviral drug combinations for the treatment of patients infected with HIV-1 generally comprise three drugs that target mostly the viral enzymes reverse transcriptase and protease (DHHS Guidelines, 2007). Recently, the Food and Drug Administration (FDA) approved the first integrase (IN) inhibitor (INI) for use in combination with other antiretroviral drugs for the treatment of therapy-experienced HIV-1 patients failing their current regimens with multi-drug resistant strains. This decision was based upon the efficacy by which raltegravir in combination with an optimized background regimen could improve the viral load and CD4+ count at week 24 in phase III clinical trials (Cooper et al., 2007; Steigbigel et al., 2007; Grinsztejn et al., 2007). However, treatment failure, whether caused by lack of adherence, insufficient

∗ Corresponding author at: Rega Institute for Medical Research and University Hospitals Leuven, Microbiology and Immunology, Clinical and Epidemiological Virology, AIDS Reference Laboratory, Minderbroedersstraat 10, 3000 Leuven, Belgium. Tel.: +32 16 332177; fax: +32 16 332131. E-mail address: [email protected] (K. Van Laethem). 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.07.008

potency of the regimen or pre-existing antiviral drug resistance, has been observed during raltegravir-containing therapy. Several mutational pathways have been described, mainly within subtype B strains, with signature mutations at integrase positions 148 and 155 (Hazuda et al., 2007). The majority of HIV-1 infections worldwide, and also in Belgium, are caused by non-B subtypes (Snoeck et al., 2004; Vercauteren et al., 2008). Therefore, an IN genotypic resistance assay performing well for all HIV-1 subtypes was developed. 2. Materials and methods 2.1. Samples Dilution series of HIV-1 (IIIB ) virus (subtype B) were used (kindly provided by R.C. Gallo when at the National Institutes of Health, Bethesda, MD) to fine-tune conditions for RNA extraction, cDNA synthesis and PCR. In addition to this laboratory strain, 74 plasma samples were included from patients who had attended the University Hospitals in Leuven and for whom a genotypic resistance analysis towards protease and reverse transcriptase inhibitors had been performed as part of their routine management. The sam-

K. Van Laethem et al. / Journal of Virological Methods 153 (2008) 176–181 Table 1 Characteristics of the patient samples tested with the IN assay

Table 1 (Continued ) Genetic forma

Genetic forma

Sample

A

AR07-2743 AR02-689 AR04-1158 AR04-1145 AR02-852 AR04-320 AR05-073 P00-235 AR02-025

479 1,521 6,908 13,204 17,390 18,226 56,855 77,055 94,885

AR07-1935 AR07-2127 AR02-781 AR06-1245 AR07-1965 AR00-175 AR03-127 AR01-751 AR01-273 P00-470 P00-200 AR02-092 H97-3457 AR02-884 AR02-471

398 447 526 575 1,479 3,009 4,172 4,273 12,492 18,644 41,032 44,411 61,722 254,839 >500,000

1(+) 1(+) 1(−)/2(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+)

AR07-955 AR05-124 AR07-050 AR07-1554 AR05-390 AR05-1284 AR04-354 AR05-756 AR06-505 AR01-760 AR01-491

562 569 1,096 1,380 2,649 4,778 7,324 11,378 23,988 50,118 138,739

1(+) 1(−)/2(−) 1(−)/2(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+)

CRF01

AR02-766 AR02-969 AR04-1031 AR03-1252 AR03-1146 AR05-781 AR05-673 P98-015

2,017 3,629 9,765 10,554 11,400 59,544 141,551 145,465

1(+) 1(+) 1(+) 1(+) 1(−) 1(+) 1(+) 1(+)

CRF02

AR06-602 AR07-1637 AR06-309 AR05-569 AR05-1056 AR03-228 AR05-747 AR03-1196 AR05-1089

269 2,042 2,750 4,883 5,446 7,124 14,523 45,293 318,615

1(+) 1(−)/2(+) 1(−) 1(+) 1(+) 1(−)/2(+) 1(+) 1(+) 1(+)

B

C

Viral loadb

177

Successc 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+) 1(+)

CRF03

AR03-506

2,000

1(−)

CRF12

P99-538

CRF13

AR03-754 P00-530 AR06-225

80,577 767 29,979 102,329

1(+) 1(−)/2(+) 1(+) 1(+)

D

AR02-071 AR01-109 AR04-342 AR01-699 AR06-2632 AR04-1171

7,943 12,589 14,921 23,000 25,704 100,670

1(+) 1(+) 1(+) 1(+) 1(+) 1(+)

F

AR06-659 AR04-538 AR05-661

178 9,422 13,714

G

AR06-1056 AR01-066 AR06-1186

1,318 2,268 2,818

1(−)/2(+) 1(+) 1(+) 1(+) 1(+) 1(+)

J

Sample

Viral loadb

Successc

AR06-009 AR06-2205

25,349 27,542

1(+) 1(+)

AR00-287 AR03-1144 AR01-485

2,219 7,939 419,945

1(+) 1(+) 1(+)

a Genetic forms as obtained by submitting the PR-RT-IN sequences to the Rega HIV-1 subtyping tool (de Oliveira et al., 2005), except for samples that failed, for which only the PR-RT sequence was used. b RNA copies/ml. c 1(+) and 1(−)/2(+): successful amplification and sequencing in the first, respectively, second round. 1(−)/2(−): unsuccessful amplification and sequencing in both rounds. 1(−): unsuccessful amplification and sequencing in the first round and for which no samples were available to perform a second round.

ples were selected based on the subtype that was determined by submitting the pol sequences to the Rega HIV-1 subtyping tool (de Oliveira et al., 2005) (Table 1). The plasma viral load was determined using the VERSANT HIV-1 RNA 3.0 Assay (bDNA, Bayer HealthCare, Brussel, Belgium) and Abbott RealTime HIV-1 (Abbott Molecular, Louvain-La-Neuve, Belgium). 2.2. Primer development and synthesis The PCR and sequencing primers were designed using the polvif gene alignment of several HIV-1 group M strains (Leitner et al., 2005). The primer sequences are listed in Table 2. Primers were developed and analysed using the Oligo software (Medprobe, Oslo, Norway). The primers were synthesised by Invitrogen (Merelbeke, Belgium). 2.3. Extraction For HIV-1 viral RNA detection, 1 ml plasma samples or 1 ml virus IIIB supernatant were ultracentrifuged at 37,100 × g for 1 h to pellet the virus and were extracted subsequently using the extraction procedure of the Viroseq HIV-1 Genotyping System (Abbott Molecular, Louvain-La-Neuve, Belgium). 2.4. cDNA synthesis and amplification of the IN region Ten microliters out of 50 ␮l RNA extract was reverse transcribed and amplified in a one-step RT-PCR using the SuperScriptTM III One-Step RT-PCR System with Platinum® Taq High Fidelity (Invitrogen, Merelbeke, Belgium) and the following conditions: 1× Reaction Mix, 2.25 mM MgSO4 , 0.2 ␮M primer KVL068, 0.2 ␮M primer KVL069, 10 U Protector RNase Inhibitor (Roche Diagnostics, Vilvoorde, Belgium) and 1 ␮l SuperScriptTM III RT/Platinum® Taq High Fidelity Enzyme Mix. Cycling conditions were: a cDNAsynthesis step of 30 min at 55 ◦ C, a denaturation step of 2 min at 94 ◦ C, 40 cycles of 15 s at 94 ◦ C, 30 s at 53 ◦ C, 2 min 30 s at 68 ◦ C and a final extension step of 5 min at 68 ◦ C. A 2128-bp nucleotide fragment, encompassing the integrase region, was herewith obtained. A 1254-bp nucleotide fragment of integrase was amplified by adding 1 ␮l outer PCR product into in an inner PCR using the Expand High Fidelity PCR System and the following conditions: 1× Expand HF Buffer, 2 mM MgCl2 , 200 ␮M dNTPs, 0.4 ␮M primer KVL070, 0.4 ␮M primer KVL084 and 2.625 U Expand High Fidelity PCR System enzyme mix. Cycling conditions were: a denaturation step of 2 min at 94 ◦ C, 10 cycles of 15 s at 94 ◦ C, 30 s at 53 ◦ C, 1 min 30 s at 72 ◦ C, 30 cycles of 15 s at 94 ◦ C, 30 s at 53 ◦ C, 1 min 30 s at 72 ◦ C + cycle elongation of 5 s for each cycle and a final extension step of 7 min at 72 ◦ C. Amplification products were separated on a 1% agarose gel and visualized by ethidium bromide staining.

178

K. Van Laethem et al. / Journal of Virological Methods 153 (2008) 176–181

Table 2 Primers for the amplification and sequencing of the HIV-1 integrase region Primers

Sequence 5 –3

Positiona

Description

KVL068 KVL069 KVL070 KVL084 KVL076 KVL082 KVL083

AGGAGCAGAAACTTWCTATGTAGATGG TTCTTCCTGCCATAGGARATGCCTAAG TTCRGGATYAGAAGTAAAYATAGTAACAG TCCTGTATGCARACCCCAATATG GCACAYAAAGGRATTGGAGGAAATGAAC GGVATTCCCTACAATCCCCAAAG GAATACTGCCATTTGTACTGCTG

3854–3880 5955–5981 4013–4042 5243–5266 4161–4188 4647–4669 4750–4772

Sense outer primer for amplification Antisense outer primer for amplification Sense inner primer for amplification Antisense inner primer for amplification and sequencing Sense primer for sequencing Sense primer for sequencing Antisense primer for sequencing

a

Positions according to pNL4.3 (AF324493).

The images were processed on a videoimager (ImageMaster VDS, Amersham Biosciences, Roosendaal, The Netherlands). 2.5. Sequencing and data analysis PCR products for population sequencing were purified with Microspin S-400 (Amersham Biosciences, Roosendaal, The Netherlands). Sequencing was performed using the ABI PRISM BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit and the primers KVL084, KVL076, KVL082 and KVL083 (Table 2). The sequencing reactions were run on an ABI3100 Genetic Analyzer (Applera, Nieuwerkerk a/d Ijssel, The Netherlands). The sequences were analysed using Sequence Analysis version 3.7 and SeqScape version 2.0 (Applera, Nieuwerkerk a/d Ijssel, The Netherlands). The genetic form of the newly obtained IN fragments was obtained by submitting the sequence to the Rega HIV-1 subtyping tool (de Oliveira et al., 2005). The variability within the IN was defined as the number of observed amino acids divided by the frequency of the most prevalent amino acid at a certain position. The variability can range theoretically between 1 (1/1; completely conserved) and 400 (20/0.05). The accession numbers of the sequences obtained in this manuscript are EU882742–EU882811. 3. Results 3.1. Performance of the assay Experimental conditions were optimized starting from dilution series in PBS of HIV-1 IIIB, ranging from 1,000,000 until 10 RNA copies/ml (final conditions as described in Section 2). The optimized nested PCR procedure was specific, generating only a single amplification band, and it was very sensitive (10 RNA copies/ml). Subsequently, it was validated on a set of 74 clinical samples from INI-naïve patients, belonging to a broad range of HIV-1 group M subtypes (A, B, C, D, F, G, J, CRF01, CRF02, CRF03, CRF12, CRF13) and displaying a viral load range between 178 and >500,000 RNA copies/ml (Table 1). At first, the amplification was successful for 64 samples (64/74; 86% success rate). The negative samples were of subtype B, C, F, CRF01, CRF02, CRF03 and CRF13, and half had a viral load below 1000 RNA copies/ml. As the IN amplification was performed on RNA extracts, obtained previously for routine resistance testing purposes and stored for many years at −80 ◦ C, 7 of the 10 negative samples were re-extracted. Plasma was not available for the three remaining negative samples. In this run, only one sample remained unamplifiable (AR05-124, 569 RNA copies/ml), giving a total success rate of 95% (70/74). Full-length sequences were obtained for all 70 amplified samples (70/74; 95%). Ninety-four percent of the IN sequences were covered fully in both directions (66 samples sequenced in sense and antisense direction). The four other IN sequences were partly unidirectional. When amplification was successful, primers KVL083 and KVL084 were always able to generate a good quality sequence, whereas primers KVL076 and KVL082 failed in, respectively, three

and one instance. Two failures were due to a shift in the fluorograms after 160, respectively, 220 covered nucleotides (KVL082 on AR07-2743 and KVL076 on AR06-1186). The primer KVL076 was unsuccessful for two samples that displayed a deletion of nine nucleotides at the primer-binding site (nucleotide string GAGGAAATG) (samples AR07-050 and AR07-1554). 3.2. Variability of the HIV-1 IN region The analysis of the 70 full-length IN sequences revealed that 110 positions of IN were polymorphic (110/288; 38%) (Table 3). The variability per site ranged from 1 until 12.7. The most variable positions were 136 (12.7 variability), 125 (9.8), 112 (9.5), 124 (8.8) and 119 (8.4). None of these high polymorphic positions are associated with INI resistance (Hazuda et al., 2007; McColl et al., 2007). Of the secondary resistance mutations against raltegravir and elvitegravir, only 151I and 157Q were observed in, respectively, four and five of our INI-naïve patients. In addition, other polymorphic changes at positions associated with resistance were observed, i.e. 74IV, 138D and 163AE. 4. Discussion The approval of raltegravir provides clinicians with the means of designing sufficiently potent regimens to attain long-term success for HIV-1 patients failing their current therapy with multi-drug resistant strains (Cooper et al., 2007; Steigbigel et al., 2007; Grinsztejn et al., 2007). Nevertheless, development of drug resistance against integrase inhibitors is expected, especially in patients in whom these new drugs cannot be supported by a potent backbone regimen due to broad cross-resistance in existing drug classes (Hazuda et al., 2007). The aim of this study was to develop a genotypic drug resistance test applicable to all HIV-1 group M subtypes. Therefore, amplification and sequencing primers were chosen at the most conserved sites surrounding and within the IN region. Within the primers, ambiguity positions were also included to take into account the genetic diversity at the primer binding sites. This approach resulted in a successful amplification of 70 genetically divergent samples belonging to the genetic forms A, B, C, D, F, G, J, CRF01-AE, CRF02-AG, CRF12-BF and CRF13-cpx. Their viral load ranged from 178 to >500,000 RNA copies/ml. However, for 4 samples (1 subtype C, 1 CRF01, 1 CRF02 and 1 CRF03) no amplification product could be obtained. These negative results could be due to the genetic differences between target and primer sequence, low viral load or the quality of the RNA extracts. Indeed, in the first attempt, when using old RNA extracts, only 64 samples could be amplified, while when using freshly extracted RNA, all but one of the failures could be resolved, indicating that the quality of the RNA is probably one of the most important determinants of success. For three of the remaining failures, a sample was not available to carry out a fresh extraction, and the fourth repeated failing sample had a low viral load (569 RNA copies/ml). The most likely rea-

Table 3 Natural polymorphisms within the HIV-1 integrase region Genetic form

Position in NTD of IN K7

Variability Genetic form

E10 D2

E11 D2 ; ED1 D4 ; ED1 D1 D2

E13

K14

S17

R20

A21

N1 C3 ; SN1 N6

K1 K1

T3 ; AT1 T1

D3

R5 R1 ; KR1 R2 ; KR1 R7 R5 ; KR1

KR1 D1 D1 D4

Q1

R1 Q1 3.2

S24

V2 ; AV1

H2 G1 ; N1 D1 ; G1 ; N1

D25 E1 ; DE1 E5

SN1

E1

N27

V1

P30

V31

H1

SN1

V32

I9 I1 I9 I7 I7

I4 ; L1

K34

V37

A38

KR1

I1 ; VI1 I2

D1

S39

D41

L45

N1 C1 ; SC1 C1 ; N1 N1 ; SN1 N2

N1

I1 V1 ; LV1 Q3

I1 R2

D1 2.1

T5

A23

R4 R3

2.8

2.1

3.7

N2 ; ST1 C1 ; N1 ; SN1 N3

K1

5.7

2.1

2.4

L74

V77

AT1

N2

G1

VI1

N1 2.1

6.1

2.3

3.1

S1

I1 I3

2.0

3.8

3.5

K103

A105

X1 T1

2.0

2.1

2.0

4.8

2.0

4.4

Position in CCD of IN M50

A9 B15 C10 CRF017 CRF028 CRF121 CRF133 D6 F3 G5 J3

I1 ; MI1 I4 ; T1 I2 ; T1 MI2 ; MIT2

Variability

5.2

S57 SG1

G59 GE1

I60

L63

V72

V1

I3 I6 ; VI2 I8 VI1 I8

M1 ; L1

I73

LV1 I1

I2

3.2

3.8

I1

2.0

2.0

3.1

IV1

A91

E96

F100

VI1

I1 I3

M5 ; MI1 ; ML1 M1 ; V1 IM1 L1

I1

IM1

Y5

AG1

D1

I1 I2

2.0

Y99

M1

L1 I3 I2 I2 I4 I1

I84

F2

3.5

2.0

4.9

2.0

2.0

2.1

2.1

L101 I1 I5 ; LI1 I9 I3 ; V1 I6 ; LI1 I1 I2 ; V1 I2 I2 I3 ; V1 I1 ; IV2

6.0

2.1

T122

T124

T125

V126

G134

I135

K136

E138

I141

I151

A7 ; TA1 ; GS1 A2 ; N3 ; TA2 ; DN1 N3 ; A2 ; S3 ; GS1 A7 A6 ; N2 A1 A3 A4 ; TA1 ; GS1 A2 A3 ; N2 A3

A7 ; S1 A1 ; MV1 A8 A7 A8

F1

N3 ; D2 ; S1

V4

Q2 ; R2 ; HQ1

ED1

V1

V9 V13 ; IV1 V10 V7 V8 V1 V1 V6 V3 V5 V3

Variability

2.4

8.8

9.8

A1 ; TA1 A1 ; S1 ; TA1 A1 ; V1 ; TA1 A5 A3

N2 ; X1 F1

2.1

ED1

IV1 D1 N5 D2

V3 V3

8.5

2.9

H1 ; Q2 T3 ; Q1 ; RT1 12.7

2.1

2.0

2.1

S119 P2 P4 ; T1 ; SG1 P1

GA1

R1 R3 ; KT1

3.8

8.4

V1

I1 I4 ; TI1

V4 V7

V113 I7 I15 I10 I6 ; VI1 I7 I1 I3 VI1 I2 ; L1 I4 ; L1 I1 ; VI2

R2

A9 B15 C10 CRF017 CRF028 CRF121 CRF133 D6 F3 G5 J3

N6 N5 ; DN1

T112 V7 ; A1 ; IV1 A1 ; TI1 V10 V7 V8

R1

Position in CCD of IN

TI1 I1 I1 I1

K111 T1 ; KR1 Q1

KR1

Genetic form

Q8 ; X1 Q4 ; R2 ; KQ1 T5 Q1 T2

G106 A1

2.0

K156 R1

2.1

E157

4.6

K160

Q1

E1

Q2

Q1

Q2

N2 ; KQ1

V1 V4 ; I1 ; M1 R1 ; IL1 V5 V2

9.5

SG1 P1 P1 ; X1 A1 ; R1 P1 P2

G163

V165

D167

E1

I1 I1

E7

I1

E6

K. Van Laethem et al. / Journal of Virological Methods 153 (2008) 176–181

A9 B15 C10 CRF017 CRF028 CRF121 CRF133 D6 F3 G5 J3

A1 E1 ; DE1 E2 I1 E1

2.0

2.2

4.3

2.9

E1 2.1

2.7

179

Table 3 (Continued ) 180

Genetic form

Position in CCD of IN H171

9

Q1

Variability

2.0

R R2 R3 ; KR1

G193

E198

I200

V201

V1

GE1 1

KR

3.2

1

X

M

KR1

2.1

A205

T206

L2 2.0

2.1

2.0

3.1

2.0

K219

I220

N222

3.2

D207

I208

T210

K211

1

I I4 I9 ; VI1 I7 I8 I1 I2 I6 I3 I5 I3

D1

1

IV1

2.3

I203

8

S S4 S2

1

M

DE1

TI1

S8 S1

S1 AS1

2.5

2.0

2.1

S1 S2 S5 S3

E1

3.2

2.1

L3 IM1

TA1

L1 3.2

KR1 KR1 R1 KR1 Q1 ; R1

R1 3.1

3.3

Position in CTD of IN K215 2

Q216

I217

1

T218 1

R ;N N2 KN2

1

1

S ; TI S1 I3 ; TI1

L L1 V1

N1

R224

1

Y227

S230

2

K K1

X1 QR1 A1 A1 3.1

3.4

2.0

2.0

I3 ; TI3 ; IL1 I1 I1 M1 ; TI1 ; TM1 I1 I2 ; A1 ; S1 I1

Q1

F F1

I L12 ; I3 I10 I7 I8

N1

7.8

4.3

K2 ; NH1

KQ1

L1

I3 I5 ; T1 I1 ; T1 I5 I3

Q1 YF1 F2

K1

E1 Q1 3.2

3.3

2.0

V234 8

IV1

2.2

2.0

5.3

K240

D253

1

N254 1

R

K K1 Q1

G2 ; N1 G1

ND1

N3

E1

G1 N1 N3 ; G1

KR1 2.1

S255

2.0

4.3

3.7

Position in CTD of IN

9

Variability

G189

R L1 ; Y1

R1

255-ins A B15 C10 CRF017 CRF028 CRF121 CRF133 D6 F3 G5 J3

K188

1

D256

V259

2

E E3 E3

A265 1

I1

AV V1 V4 ; AV2

I268

R269

D270

1

1

L

K273

M275

D278

1

A282

1

H ; DN 6

V281

8

M

2

8

K ; RK

1

A

P VM1

E1 E1 E2

I1

G1

V2 AT1

E3 E4 E3 –

2.9

V2

K2

V1 IL2

H3

Q1

N1 DH1

3.7

3.2

1.7

3.4

2.0

2.0

G G1 G3 ; SG1 G7 G7 G1 G2 ; SG1 G3 G3 G3

V1 2.1

S283

2.3

2.1

2.0

3.7

R284

D286

E287

D288

1

G

N1 G2 ; RG1

DN1

RG1 G1

N1

Q1

2.2

2.1

2.0

2.0

Wild-type amino acids and positions according to pNL4.3 (AF324493). The number of sequences belonging to a certain genetic form (subtype or CRF) or displaying a certain amino acid at a particular position is displayed in superscript. A mixture is indicated by a 2-letter code listing both possible amino acids, when containing more than 2 amino acids it is annotated as X. Amino acid changes and positions associated with raltegravir or elvitegravir resistance are in bold or underlined, respectively. Variability is defined as the number of observed amino acids at a certain position divided by the frequency of the most prevalent amino acid. 255-ins: insertion between amino acids 255 and 256. IN: integrase; NTD: amino-terminal domain; CCD: catalytic core domain; CTD: carboxyl-terminal domain.

K. Van Laethem et al. / Journal of Virological Methods 153 (2008) 176–181

Genetic form

K186

D1

9

Variability

I182

L1

E212 A B15 C10 CRF017 CRF028 CRF121 CRF133 D6 F3 G5 J3

F181

1

A B15 C10 CRF017 CRF028 CRF121 CRF133 D6 F3 G5 J3

Genetic form

K173

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son for failure is thus probably not related to divergence of the subtype. Full-length sequences covered in both directions were obtained for almost all sequences. Intriguingly, one of the sequencing primers failed in two epidemiologically linked sequences due to a deletion of 9 nucleotides at the primer-binding site. This would result into a yet unreported deletion of three amino acids at the 3 prime end of RNase H (amino acids GGN at position 543–545). In the study of Hazuda et al. (2007) in which they investigated drug resistance development in patients failing raltegravir, the majority of patients failed with either 155H or 148HKR within IN. Each of these signature mutations were associated often with secondary resistance mutations, i.e. 74M, 92Q, 97A, 143HR, 151I, 163KR or 232N for 155H and 74M, 138AK or 140AS for 148HKR. In a phase II clinical trial of elvitegravir, another integrase inhibitor, the most common mutations were 66AIK, 92Q, 138K, 148HKR and 155H, suggesting cross-resistance between the first generation integrase inhibitors (McColl et al., 2007). In vitro selection experiments with elvitegravir revealed two distinct resistance pathways, i.e. 66I with 95K, 138K, 146P and 147G; and 92Q with 51Y, 147Q and 157Q (Shimura et al., 2008). In general, the signature mutations lead to a decrease in susceptibility towards the respective integrase inhibitors, whereas secondary resistance mutations augment the resistance level (Hazuda et al., 2007; Malet et al., 2008; Shimura et al., 2008). Within this study of IN sequences from INI-naïve patients, the majority of the positions associated with IN resistance were conserved. Only positions 74, 138, 151, 157 and 163 displayed some low-level variability (range 2.1–3.5). With the exception of 151I in four and 157Q in five patients, they all showed other amino acid changes than those reported previously (Hazuda et al., 2007; McColl et al., 2007; Shimura et al., 2008). The relevance of these secondary mutations in absence of signature mutations remains unclear. However, the single mutants 151I and 157Q revealed a rather highly reduced susceptibility towards raltegravir and elvitegravir in one particular in vitro study (Shimura et al., 2008). Additionally, from studies investigating resistance development against protease and reverse transcriptase inhibitors, it is known that the genetic background and the presence of secondary mutations can influence drug susceptibility, resistance development and clinical response (Perno et al., 2001; Abecasis et al., 2005, 2006; Deforche et al., 2006, 2007). In conclusion, this integrase assay displayed a high amplification and sequencing success rate and was shown to be a useful tool for the genotypic characterisation of diverse HIV-1 group M subtypes. Acknowledgements This work was supported by the AIDS Reference Laboratory of Leuven that receives support from the Belgian Ministry of Social Affairs through a fund within the Health Insurance System, by the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (grant G.0266.04) and by the Centers of Excellence of the Katholieke Universiteit Leuven (krediet EF/05/015). Kris Covens was funded by a PhD grant of the Institute for the Promotion of Innovation through Sciences and Technology in Flanders (IWT). References Abecasis, A., Deforche, K., Snoeck, J., Bachelor, L.T., McKenna, P., Carvalho, A.P., Gomes, P., Camacho, R., Vandamme, A.-M., 2005. Protease mutation M89I/V is linked to therapy failure in patients infected with the HIV-1 non-B subtypes C, F or G. AIDS 19, 1799–1806.

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