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HIV-1 PHENOSCRIPT ENV: A Sensitive Assay for the Detection of HIV X4 Minority Species and Determination of Non-B Subtype Viral Tropism
Vanessa Roulet*1,2,3, Vanessa Roulet*1,2,3, Vanessa Roulet*1,2,3, S Rochas1,2, S Rochas1,2, J L Labernardière1,3, J L Labernardière1,3, F Mammano3, J L Faudon2, N Raja2, S Lebel-Binay1, K Skrabal1,2, and K Skrabal1,2
1BioAlliance Pharma, Paris, France; 2Eurofins Viralliance Inc, Kalamazoo, MI, US; and 3INSERM U552, Paris, France
Background: Entry inhibitors that block the attachment of
HIV gp120 to CCR5 or CXCR4 co-receptors are currently in clinical development
and represent a new generation of antivirals for the treatment of HIV
infection. PHENOSCRIPT ENV is a sensitive phenotypic test for the evaluation of
viral tropism. Its sensitivity has been established by detecting X4-tropic
minority species; evaluating the success rate for plasma samples with different
viral loads; and determining viral tropism of the most prevalent European HIV-1
non-B subtypes.
Methods: To measure the sensitivity of the assay for
detecting X4-tropic viruses, different ratios of proviral plasmid carrying X4-
and R5-tropic envelopes from both reference and primary isolates were mixed.
The gp120-gp41extracellular region was polymerase chain reaction (PCR) -amplified
and co-transfected into producer cells along with pNL4-3-deleted of the
corresponding region. Supernatants containing the recombinant virus were used
to infect U373MG-CD4 indicator cells, expressing CCR5 or CXCR4, in the presence
or absence of CXCR4-antagonist AMD-3100. To determine the sensitivity of the
assay towards plasma samples with viral load between 1000 and 10,000 copies/mL,
4 characterized samples with known viral load were diluted and viral RNA was
extracted before PCR-amplification of env
and analyzed in PHENOSCRIPT.
In
addition, to establish the success rate of the assay in determining the tropism
of HIV non-B subtypes, we tested a panel of 400 plasma samples representing the
most prevalent European non-B subtypes.
Results: PHENOSCRIPT ENV allowed the detection of as few
as 5 to 10% X4 variants in mixtures of viruses with similar infectivity. For
plasma samples with viral load between 1000 and 10,000 copies/mL, the success
rate of PHENOSCRIPT was 100%, whereas the success rate of the assay is mainly
dependent on the PCR-success rate when the viral load was below 1000 copies/mL.
The PCR success rate for non-B subtype samples ranged from 69 to 92%, depending
on the subtype. The overall recombinant virus assay success rate for all non-B
subtypes was 71% (range 36 to 95%). The prevalence of viruses using CXCR4
exclusively or in addition to CCR5 ranged from 0 to 25%, for different
subtypes.
Conclusions: Given the efficiency of
our assay, our test will contribute to the optimization of drug therapy
and the follow-up for HIV-infected individuals non-B subtype samples are an
appropriate source for the determination of baseline susceptibility to enfuvirtide
(T-20).
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