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AIDS:
doi: 10.1097/QAD.0b013e3282ef1bc8
Research Letters

Virological fitness of HIV in patients with resistance to enfuvirtide

Chibo, Dorisa,b; Roth, Normanc; Roulet, Vanessad,e; Skrabal, Katharinad; Gooey, Megana; Carolan, Louisea; Nicholls, Jodiea; Papadakis, Anastasiaa; Birch, Chrisa

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Author Information

aVictorian Infectious Diseases Reference Laboratory, Melbourne, Australia

bSchool of applied Sciences, RMIT University, Melbourne, Australia

cPrahran Market Clinic, Melbourne, Australia

dEurofins Viralliance Inc., Michigan, USA

eBioalliance Pharma, Paris, France.

Received 15 April, 2007

Revised 11 June, 2007

Accepted 20 June, 2007

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Abstract

Resistance to the HIV fusion inhibitor enfuvirtide is associated with mutations in the first heptad repeat region of gp41, but little is known of their impact on replicative fitness in vivo. We followed seven patients undergoing salvage therapy that included enfuvirtide in order to document the temporal generation of genotypic and phenotypic resistance in parallel with replicative fitness. Resistance to enfuvirtide was not associated with decreased replicative fitness of HIV strains infecting these patients.

Long-term treatment and associated adherence issues with HAART have facilitated the emergence of drug resistance, especially to protease inhibitors and reverse transcriptase inhibitors, but also to enfuvirtide (Fuzeon), the only clinically available drug targeting the HIV fusion process [1]. Resistance to enfuvirtide is associated with mutations in the first heptad repeat (HR1) region of gp41 involving amino acids 36–45 [1]. Combinations of these mutations generally lead to higher levels of phenotypic resistance [2]. Mutations in the second heptad repeat (HR2) may play a compensatory role by restoring or improving the interaction between it and HR1 [3].

We followed seven patients treated with optimized HAART that included enfuvirtide, but in whom subsequent virological control was not achieved. This allowed us to study the temporal generation of mutations in HR1 and HR2 in parallel with the replicative fitness of HIV strains present before and during treatment. The patients studied had previously failed treatment with reverse transcriptase and protease inhibitors and had significant resistance to them. All had virological failure at the start of enfuvirtide treatment and six of the seven had CD4 cell counts less than 250 cells/μl. They were treated for at least 3 years with enfuvirtide on an optimized antiretroviral drug background.

HIV RNA was extracted from plasma samples using a QIAmp viral RNA purification protocol (Qiagen, Hilden, Germany). A region of env was amplified in a nested reverse transcriptase–polymerase chain reaction encompassing HR1 and HR2. The mutations in HR1 considered to indicate enfuvirtide resistance were G36D/S/V, I37V, V38A/E/M, Q39R, Q40H, N42T, N43D, L44M and L45M [3,4].

HIV strains in 27 plasma samples from seven patients were evaluated for co-receptor tropism, enfuvirtide susceptibility and replicative capacity using a previously described method (Phenoscript) [5]. The fitness of each enfuvirtide-resistant virus was expressed as a percentage compard with the pretreatment strain from that patient (defined as 100%).

The viral load and CD4 cell count were monitored over the course of the study. Before initiation of enfuvirtide therapy, six of the seven patients had CD4 cell counts less than 250 cells/μl. After approximately 3 years of treatment, three patients (1, 2 and 7) maintained CD4 cell counts higher than at the commencement of therapy, despite phenotypic resistance and detectable viral loads (Fig. 1). This observation has been noted by others, and suggests that an enfuvirtide-containing salvage regimen may have immunological benefits even in the face of poor virological response [1,6].

Fig. 1
Fig. 1
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At the time of initiating enfuvirtide treatment, three patients (1, 3 and 4) were infected with R5 tropic HIV strains and three (2, 6 and 7) were infected with mixed/dual tropic (R5/X4) virus. One patient (5) had a mixed/dual tropic strain before commencing enfuvirtide therapy, but only R5 strains were detected at subsequent timepoints.

Pretherapy IC50 susceptibilities to enfuvirtide were generally in the range of 10–75 ng/ml (Fig. 1). Patient 5 was infected with a strain that was particularly sensitive to enfuvirtide (IC50 0.002 ng/ml). Natural variation in enfuvirtide susceptibility has been reported previously [6] and may be linked to the fusion rate [7]. Slow fusion, with a relatively protracted exposure of HR1 to enfuvirtide, may facilitate reduced inhibitory concentrations (relatively low IC50) [7]. Enfuvirtide resistance mutations may reduce the exposure time of HR1 to the drug, resulting in higher concentrations needed for inhibition (increased IC50) compared with the pretreatment strain. Of note is the fact that although the virus from patient 5 had the highest pretherapy level of susceptibility (IC50 < 0.002 ng/ml), on-treatment strains had increased IC50, which remained lower than resistant virus from the other patients (Fig. 1).

All patients developed enfuvirtide-associated mutations in HR1 (Fig. 1). Mutations developed between 3 weeks and 17 months after the commencement of therapy, and were associated with high-level resistance (40-fold to 35 000-fold). Apparently random changes in HR2 were identified, but we were unable to establish whether they contributed to altered enfuvirtide susceptibility, a finding noted by others [8].

Switching from one resistant genotype to a second resistant genotype during enfuvirtide therapy was observed in patients 3, 4 and 5 (Fig. 1), a phenomenon that has been noted previously [7]. It generally resulted in an increased fold-change in resistance, suggesting an evolutionary requirement by the virus, possibly facilitated by the presence of quasi-species containing discrete resistance mutations, and in response to subtle changes in enfuvirtide concentrations in vivo.

Despite the presence of genotypic and phenotypic resistance, little or no impact was observed on HIV replicative fitness in the R5 tropic strains of five of the seven patients studied, with changes in fitness from 0.5 to 1.8-fold compared with baseline (Fig. 1). This result was supported by the persistence of resistance mutations for periods ranging from at least 4 to 14 months after the cessation of enfuvirtide treatment in three evaluable patients (1, 3 and 5), an observation recently supported by one study [5] but not by a second [8]. In contrast, the replicative fitness of viruses from patients 2 and 6 increased during enfuvirtide treatment. The increase in fitness was at least sixfold compared with viruses present before enfuvirtide treatment, and involved mixed/dual-tropic variants.

Previous studies involving the site-directed mutagenesis of HR1 have generated virus with impaired fitness [9,10]. The assay we used to determine replication fitness incorporated the entire env region from patient-derived virus. The contributions of any compensatory mutations in this region were therefore accounted for in our assay. Despite genotypic and phenotypic resistance to enfuvirtide, no obvious impact on fitness, particularly a negative impact, was observed in our patients. Our findings are in agreement with those of Labrosse and colleagues [5], who also showed improved replication fitness in two enfuvirtide-resistant viruses. Our results suggest that the improvements or stabilization of CD4 cell counts sometimes observed in patients treated with enfuvirtide are not the result of reduced virological fitness of the resistant strains present.

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Acknowledgements

The authors thank Dr Gillian Hales for her interest in the study and scientists from Eurofins VIRalliance Inc. for their help with experimental work.

Sponsorship: Part of this work was supported by a grant from Roche Products Pty Ltd. under its Fuzeon Initiatives for National Discoveries Awards programme.

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References

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© 2007 Lippincott Williams & Wilkins, Inc.

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