HIV-1 V3 envelope deep sequencing for clinical plasma specimens failing in phenotypic tropism assays

Ina Vandenbroucke,Dolores Vaira,Linos Vandekerckhove,Kim Thys,Wendy Mostmans,Stephane De Wit,Veerle Van Eygen,Eric Florence,Kurt Van Baelen,Katrien Fransen,Lieven J Stuyver,Kabamba Kabeya,Michel Moutschen,Chris Verhofstede,Herwig Van Marck,Evelien Rondelez

doi:10.1186/1742-6405-7-4

Abstract

BackgroundHIV-1 infected patients for whom standard gp160 phenotypic tropism testing failed are currently excluded from co-receptor antagonist treatment. To provide patients with maximal treatment options, massively parallel sequencing of the envelope V3 domain, in combination with tropism prediction tools, was evaluated as an alternative tropism determination strategy. Plasma samples from twelve HIV-1 infected individuals with failing phenotyping results were available. The samples were submitted to massive parallel sequencing and to confirmatory recombinant phenotyping using a fraction of the gp120 domain.ResultsA cut-off for sequence reads interpretation of 5 to10 times the sequencing error rate (0.2%) was implemented. On average, each sample contained 7 different V3 haplotypes. V3 haplotypes were submitted to tropism prediction algorithms, and 4/14 samples returned with presence of a dual/mixed (D/M) tropic virus, respectively at 3%, 10%, 11%, and 95% of the viral quasispecies. V3 tropism prediction was confirmed by gp120 phenotyping, except for two out of 4 D/M predicted viruses (with 3 and 95%) which were phenotypically R5-tropic. In the first case, the result was discordant due to the limit of detection for the phenotyping technology, while in the latter case the prediction algorithms were not computing the viral tropism correctly.ConclusionsAlthough only demonstrated on a limited set of samples, the potential of the combined use of "deep sequencing + prediction algorithms" in cases where routine gp160 phenotype testing cannot be employed was illustrated. While good concordance was observed between gp120 phenotyping and prediction of R5-tropic virus, the results suggest that accurate prediction of X4-tropic virus would require further algorithm development.

Highlights

HIV-1 infected patients for whom standard gp160 phenotypic tropism testing failed are currently excluded from co-receptor antagonist treatment
Since the drug is only effective in individuals exclusively harboring CCR5-tropic (R5) virus, viral tropism has to be determined before the initiation of maraviroc treatment
There is an ongoing search for alternatives [4,5,6], most commonly relying on the amplification of the V3 domain of gp120, which is the major determinant for viral tropism [7,8]

Summary

Introduction

HIV-1 infected patients for whom standard gp160 phenotypic tropism testing failed are currently excluded from co-receptor antagonist treatment. To provide patients with maximal treatment options, massively parallel sequencing of the envelope V3 domain, in combination with tropism prediction tools, was evaluated as an alternative tropism determination strategy. Maraviroc (Selzentry/Celsentri, Pfizer, NY) is a chemokine co-receptor antagonist, designed to prevent HIV-1 infection of CD4+ T-cells by blocking the CCR5 co-receptor. The only clinically validated tropism test is the Trofile assay (Monogram Biosciences, CA). Prediction of co-receptor usage based on V3 sequences using bioinformatics tools could be a good alternative for phenotypic tropism testing in routine clinical practice [9,10,11]. When combined with genotypic prediction tools, they could become a sensitive alternative to phenotypic assays

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Results

Discussion

Conclusion