574-T. Fine Structure Analysis of HIV Sequence Variation <i>in Vivo</i>: Longitudinal Study of Drug Naļve Patients and Dynamics of Response to Initiating HAART

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Session 76 Poster Session
HIV Resistance and Fitness
Session Time: 4:30-6:30 pm
Room 4E-F

574-T.

Fine Structure Analysis of HIV Sequence Variation in Vivo: Longitudinal Study of Drug Naļve Patients and Dynamics of Response to Initiating HAART
F. Maldarelli*¹, M. Kearney¹, S. Palmer¹, D. Rock², J. Falloon², J. Mican², S. Liu², A. Pau³, M. VanHoutte⁴, L. Michels⁴, K. Hurtogs⁴, R. Stephens⁵, J. Mellors⁶, and J. Coffin¹
¹NCI, NIH, Frederick, MD; ²NIAID, NIH, Bethesda, MD; ³NIH, Bethesda, MD; ⁴Virco, Mechelen, Belgium; ⁵SAIC, Frederick, MD; and ⁶Univ. of Pittsburgh, PA

Background: Drug-resistant variants of HIV emerge in vivo from a heterogeneous swarm of virus. To further understand mechanisms contributing to viral diversity and the evolution of drug resistance, we initiated a clinical protocol to derive a comprehensive description of the genetic structure of HIV populations in infected individuals prior to and following administration of antiretrovirals.
Methods: HIV-infected adults with no prior antiretroviral experience and plasma viral loads (VL) >1000 copies/mL were enrolled in an intensive sampling protocol. Plasma samples were obtained daily for 10 days, weekly for 16 weeks, and monthly thereafter. Patients requiring highly active antiretroviral therapy (HAART) began therapy following the initial 10-day sampling period, and frequent samples were obtained after HAART. Individual HIV pol sequences were obtained using an endpoint dilution-PCR amplification strategy; 15-20 amplicons (PR and nt 1-1200 of RT) were analyzed from each plasma sample.
Results: 17 patients (CD4 180-840 cells/µL, VL 5000-1.5X10⁶ copies/mL) are enrolled; 4 patients initiated HAART. HIV sequences from 1 chronically infected patient (CD4 342, VL 10,500 copies/mL) over 18 months remained highly related; >85% of nucleotide positions remained invariant in 180 sequences; no drug resistant mutations were identified. No evidence of positive selection was present (ds/dn>1). Evidence suggestive of new allele acquisition was present at 1 locus, but no allele extinction was identified. Variable loci included 24 sites with significant polymorphisms (frequencies of minor allele >10%). Linkage analysis revealed evidence of frequent recombination, and unlinked sites were identified within 40-50 nt. Analyses of additional patients also detected 24-34 polymorphisms; no relationship was identified between numbers of polymorphisms and duration of infection or magnitude of viral load. Preliminary analysis of a patient initiating HAART revealed that HIV variants present prior to therapy were not reduced uniformly on HAART. Analyses of additional patients initiating HAART are under study. Analyses of a newly infected individual prior to HAART revealed a highly monomorphic virus population; only 8 nt changes were detected in 47,600 nt in 28 amplicons.
Conclusions: HIV populations undergo frequent recombination in vivo but are not significantly affected by random mutation, suggesting the effective population size is large.