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Journal of Clinical Microbiology, September 2006, p. 3384-3387, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00666-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Performance of Human Immunodeficiency Virus Type 1 gp41 Assays for Detecting Enfuvirtide (T-20) Resistance Mutations

Michelle Marcial,1 Jing Lu,1 Steven G. Deeks,2 Rainer Ziermann,3,{dagger} and Daniel R. Kuritzkes1*

Section of Retroviral Therapeutics, Brigham and Women's Hospital and Division of AIDS, Harvard Medical School, Boston, Massachusetts,1 Department of Medicine, University of California, San Francisco, and San Francisco General Hospital, San Francisco, California,2 Diagnostics Division, Bayer HealthCare LLC, Berkeley, California3

Received 28 March 2006/ Returned for modification 16 May 2006/ Accepted 5 July 2006


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ABSTRACT
 
The performance of a gp41 assay, created using reagents designed for use with the OpenGene DNA Sequencing System, was evaluated using a panel of plasma samples obtained from enfuvirtide-naive and -experienced human immunodeficiency virus type 1-infected subjects. Resulting sequence data were highly accurate compared to a "home brew" assay and clonal sequence analysis.


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TEXT
 
The role of genotypic drug resistance testing in guiding the choice of antiretroviral therapy for treatment of human immunodeficiency virus type 1 (HIV-1) is well established (4). Routine use of genotypic resistance testing requires convenient assays with high reproducibility that can be performed by laboratories skilled in molecular diagnostic techniques. Genotyping systems approved for clinical use have focused on the PR- and reverse transcriptase (RT)-encoding segments of HIV-1 pol (1, 2, 6). With the advent of novel classes of antiretroviral drugs that target other viral gene products, additional sequencing approaches are needed.

Enfuvirtide (ENF) is a synthetic oligopeptide that inhibits fusion of HIV-1 to CD4+ cells by binding to the first heptad repeat (HR-1) of gp41, the transmembrane subunit of the viral envelope glycoprotein (5, 13). Resistance to enfuvirtide is mediated by substitutions within HR-1 at amino acids 36 to 45 of gp41 (11, 12). Substitutions most frequently associated with enfuvirtide resistance include G36D, -S, -V, or -E; V38A, -E, or -M; Q40H; N42T; and N43D (9, 10, 14). These mutations confer significantly reduced binding of enfuvirtide to HR-1 and a substantial decrease in antiviral activity in vitro (10). Viruses carrying enfuvirtide resistance mutations show reduced viral fitness in growth competition assays performed in the absence of enfuvirtide (7).

An assay utilizing Bayer primers and reagents (Bayer HealthCare, Berkeley, Calif.) (referred to here as the "gp41 assay") was designed for use together with the OpenGene DNA Sequencing System to detect enfuvirtide resistance mutations in the gp41-encoding segment of env. In this study, we evaluated performance of the gp41 assay using a panel of plasma samples obtained from enfuvirtide-naive and -experienced HIV-1-infected subjects.

(These data were presented, in part, at the following conferences: (i) Targeting Viral Entry—1st International Workshop, 2 to 3 December 2005, Bethesda, MD [abstract 31], and (ii) 13th Conference on Retroviruses and Opportunistic Infections, 5 to 8 February 2006, Boston, MA [abstract 659].).

Patient plasma samples. Fifty-one plasma samples were obtained from 26 subjects (20 prior to ENF exposure and 31 during or after ENF exposure) with HIV-1 RNA levels in plasma ranging from 3.12 to 6.30 log10 copies/ml. All subjects provided written informed consent, and all aspects of this study were conducted according to institutional guidelines for research with human subjects.

Sequence analysis of gp41. HIV-1 RNA was extracted using a QIAGEN viral RNA extraction minikit (QIAGEN, Valencia, CA). Samples were then subjected to an RT-PCR, and the HR-1-encoding region of env was sequenced using the gp41 assay. The reagents were used with the Bayer HealthCare OpenGene Sequencing System and were implemented as a laboratory-developed assay. Sequence data were acquired and analyzed using the OpenGene DNA Sequencing System. Alternatively, a 650-bp fragment of gp41 that included the HR-1 coding region (corresponding to nucleotides 7660 to 8310 of the Hxb2 sequence [http://hiv-web.lanl.gov]) was amplified by a nested RT-PCR and sequenced using Taq DyeDeoxy Terminator cycle sequencing (Applied Biosystems, Foster City, CA) on an ABI 3700 PRISM automated sequencer. For 30 of the plasma samples, independent clones of gp41 were obtained for sequencing by cloning the amplicon into plasmid vector pCR-2.1 (Stratagene, La Jolla, CA) using the TOPO TA cloning system (Invitrogen, California), as described previously (8). The sequences obtained were then manually aligned with each other and with a subtype B reference sequence (NL4-3), and differences between the two test sequences were scored to determine the percent agreement. Mutations in HR-1 at codons 36, 38, 40, and 42 to 45 were scored as possible ENF resistance mutations.

Analysis. Comparisons of results generated by the gp41 assay and "home brew" sequencing methods included identification of bases, codons, and resistance mutations in a sequence. Samples were considered to contain a mixture at a given position when multiple peaks were observed on the chromatogram. For each test sequence, the proportion of agreement of the entire base sequence between the gp41 assay-generated sequence and the "home brew" sequence was determined by comparing the extent of agreement at nucleotides 43 to 273 of gp41 and at amino acids 15 to 91. The proportion of agreement was defined as the number of exactly matching bases or codons between the two methods divided by the total number of bases or codons, respectively, in the consensus sequence (this analysis was performed only for samples that generated results with both assays). Percent agreement of the number and identity of resistance-associated mutations at codons 36 to 45 was calculated for the sequences generated by the gp41 assay relative to the "home brew" technology from each sample using the following proportion: [(total number of resistance codons) – (number of resistance codons on which the two sequences disagree)]/(total number of resistance codons). For the purpose of this analysis, the N42S substitution was not counted as a resistance mutation because previous data show that this polymorphism does not affect susceptibility to enfuvirtide (12).

Concordance between the gp41 assay and "home brew" sequencing. Sequence data were obtained from 50 of 51 samples (98%) with the HIV-1 gp41 assay and in 44 (86%) with the "home brew" method; 43 samples (84%) yielded sequence data by both methods. The mean number of enfuvirtide resistance mutations per sample was 1.0 (range, 0 to 2). Mean percent agreement across a shared 231-nucleotide region encompassing HR-1 was 99.4% (range, 97.4 to 100%) at the nucleotide level and 99.1% (range, 96.1 to 100%) at the codon level (Table 1). Mean percent agreement at codons 36 to 45 was 97.7% (range, 80 to 100%). Discordances were explained by the detection of mixtures by one method but not the other (Table 2).


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  		TABLE 1. Concordance between gp41 assay and "home brew" sequencing methods


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  		TABLE 2. Discordant predicted amino acid sequences at enfuvirtide resistance-associated mutations (gp41 codons 36 to 45)a

Clonal sequence analysis. Sequence data from multiple independent clones (n = 7 to 14) were available for 30 samples (Table 3). There was good agreement overall between the clonal sequences and those generated by the gp41 assay. However, in three samples (3151, 3174, and 3506) the gp41 assay detected additional mutations not detected by clonal analysis. Conversely, clonal analysis detected additional mutations in six samples that were not detected by the gp41 assay. In nearly all cases, these discrepancies arose because of detection of mixtures by the gp41 assay at codons that were wild type by clonal analysis or because a minor variant was detected by clonal analysis that was not detected by the gp41 assay.


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  		TABLE 3. Comparison of HR-1 mutations detected by gp41 assay and clonal analysis

Although it is possible that mixtures might have been overestimated from the chromatograms in the gp41 assay, examination of relative peak heights suggested that typically the minor variants constituted approximately 20% of the population, suggesting that a sequencing artifact is an unlikely explanation. Given that in most cases only eight clones were analyzed per sample, the clonal analysis would not be expected to detect consistently minor variants present at less than 10 to 15%.

In summary, these results show excellent agreement between population-based sequencing of HIV-1 gp41 using the gp41 assay and "home brew" methods. The percent agreement between the two methods was comparable to the high level of agreement observed between various methods for genotyping of the HIV-1 pol gene (3). Use of the gp41 assay resulted in a higher success rate for generating gp41 sequences from test samples with low copy number and from clinical samples. These results suggest that the gp41 assay may be a useful approach to detecting enfuvirtide-associated resistance mutations.


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ACKNOWLEDGMENTS
 
This work was funded by Bayer and received partial support from a Virology Specialty Laboratory contract from the AIDS Clinical Trials Group (NIH grant U01AI-38858).


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FOOTNOTES
 
* Corresponding author. Mailing address: Brigham and Women's Hospital, 65 Landsdowne Street, Room 449, Cambridge, MA 02139. Phone: (617) 768-8371. Fax: (617) 768-8738. E-mail: dkuritzkes{at}partners.org. Back

{dagger} Present address: Novartis Vaccines and Diagnostics, Chiron, Inc., Emeryville, Calif. Back


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Journal of Clinical Microbiology, September 2006, p. 3384-3387, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00666-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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