AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 23, Number 3, 2007, pp. 381–390 © Mary Ann Liebert, Inc. DOI: 10.1089/aid.2006.0175 Slow Human Immunodeficiency Virus Type 1 Evolution in Viral Reservoirs in Infants Treated with Effective Antiretroviral Therapy DEBORAH PERSAUD, 1 STUART C. RAY, 2 JOLEEN KAJDAS, 1 AIMA AHONKHAI, 2 GEORGE K. SIBERRY, 1 KIMBERLY FERGUSON, 1 CARRIE ZIEMNIAK, 1 THOMAS C. QUINN, 2,3 JOSEPH P. CASAZZA, 4 STEVEN ZEICHNER, 5 STEPHEN J. GANGE, 6 and DOUGLAS C. WATSON 7 ABSTRACT A longitudinal study of viral reservoirs in children initiating highly active antiretroviral therapy (HAART) in early infancy was undertaken to test the hypothesis that early effective treatment affects the persistence of replication-competent viral latency and the evolution of HIV-1 in resting CD4  T cells. An end point dilution culture assay was used to measure the frequencies of latently-infected resting CD4  T cells harboring repli- cation-competent virus in early and late treated children. Gag, pol, and env also were sequenced and com- pared to pretreatment sequences. HIV-1-specific humoral and cellular immune responses were also assessed. Blood samples were obtained from 12 HIV-1-infected children who started HAART at a median of 1.9 months of age and who maintained suppression of HIV-1 replication for up to 5.5 years. Replication-competent HIV- 1 was recovered from 10/12 (84%) subjects. Evolution in gag, pol, and env was restricted for years in early- treated children. HAART initiated from early infancy does not prevent the establishment of a reservoir of la- tent provirus, but does significantly limit the evolution of HIV-1 in viral reservoirs. The effect of early therapy on HIV-1 evolution may have implications for long-term pharmacologic control of HIV-1. 381 INTRODUCTION O VER TWO MILLION CHILDREN are infected with human im- munodeficiency virus type 1 (HIV-1) worldwide. 1 Al- though antiretroviral drugs can reduce perinatal transmission, 1400 infants are infected daily, the majority of whom reside in developing countries. 1 For persons with access to antiretroviral therapy, treatment can inhibit virus replication to levels that are undetectable by ultrasensitive clinical assays (below 50 copies/ml). 2–5 This suppression can be sustained for years, but reservoirs of HIV-1 persist in long-lived cells, preventing viral clearance. 6–12 Because viral reservoirs prevent HIV-1 eradication, and pro- longed therapy is associated with toxicities, recommendations for initiating highly-active antiretroviral therapy (HAART) in older children and adults are based on the risk of disease pro- gression. 13,14 HAART is recommended for infants diagnosed before 1 year of age who are symptomatic (clinical category A, B, or C) or whose CD4 + T cells are less than 25% of periph- eral lymphocytes. 13 However, many experts recommend start- ing HAART in all infected infants because of high rates of dis- ease progression or death in the first year of life. 13 Therefore, most children diagnosed with HIV-1 infection during infancy in the United States are started on HAART once infection is confirmed. The impact of early treatment on the persistence of HIV-1 and evolution of replication-competent virus has not been studied due to the difficulty of performing such studies in children. 1 Department of Pediatrics and 2 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. 3 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. 4 Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, Maryland. 5 Children’s Reseach Institute, Children’s National Medical Center and Departments of Pediatrics and Microbiology, Immunology, and Trop- ical Medicine, George Washington University School of Medicine, Washington, DC. 6 Department of Epidemiology, Johns Hopkins University Bloomberg School of Public Health, Baltimore Maryland 21205. 7 Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland.