571-T. <i>In Vitro</i> Selection of the T215Y Mutation by Stavudine (d4T) in Viruses Carrying 215D/C from Drug-Naïve Persons

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Session 75 Poster Session
Resistance to Antiretroviral Chemotherapeutic Agents
Session Time: 4:30-6:30 pm
Room 4E-F

571-T.

In Vitro Selection of the T215Y Mutation by Stavudine (d4T) in Viruses Carrying 215D/C from Drug-Naïve Persons
G. García-Lerma*, H. MacInnes, S. Nidtha, D. Bennett, H. Weinstock, and W. Heneine
CDC, Atlanta, GA

Background: The T215Y mutation has long been associated with resistance to AZT and, more recently, has been seen in patients failing d4T therapy. We have previously identified a distinct group of viruses that have a unique set of mutations at codon 215 among newly diagnosed persons. These mutations include 215D and 215C and do not confer resistance to AZT or d4T. However, HIV-1_215D/C have higher ability than WT viruses to select 215Y in vitro in the presence of AZT. In the present study, we evaluated the capacity of these viruses to acquire 215Y in the presence of d4T.
Methods: 9 recombinant viruses were studied: 6 had cloned RT sequences containing 215C or D, and 3 had control WT sequences. All viruses were cultured in the presence of increasing concentrations of d4T. Mutations at codon 215 and other positions of the RT were monitored by sequence analysis after each passage. Fold-changes in IC₅₀ values for d4T-TP and d4T were determined by an RT assay and by a replication-based assay (MT4/MTT).
Results: Of the 6 recombinant viruses carrying 215D or C, 4 acquired 215Y after a mean of 57 days (range=32-89) in culture. In contrast, the 215Y mutation was not seen in the 3 control WT viruses. Interestingly, 7 of 9 viruses acquired the K65R mutation. Among these, 4 had K65R only, 2 had K65R and 215Y, and 1 had K65R and V75A (HXB2). Reduced susceptibility to d4T-TP was seen by an RT-based assay in 7 isolates carrying either 215Y, K65R/215Y, or K65R/V75A, with a mean increase in IC₅₀ of 3.6-fold (range=2.2-6.3). However, resistance to d4T in the MT4/MTT assay was low or undetectable, with a mean increase in IC₅₀ of 1.4-fold (range=0.9-2.4), likely reflecting the inability of this assay to detect d4T resistance in these mutants.
Conclusions: Our results document for the first time the selection of 215Y by d4T in vitro. These findings suggest that 215Y confers a selective advantage in the presence of d4T, and may explain the selection of 215Y by d4T observed in vivo. The observed selection of K65R suggests that mutations at codons other than 215 and 75 may also contribute to d4T resistance. Our findings suggest that patients infected with HIV-1_215D/C and treated with antiretroviral regimens containing d4T may be at increased risk for development of 215Y.