1. CXCR4-tropism and the preferential establishment of an HIV-reservoir in naïve CD4+ T-cells among HIV-infected Ugandan children Theodore Ruel 1, Jane Achan 2, Wei Huang 3, Maudi Killian 1, Elizabeth Sinclair 1, Edwin Charlebois 1, Peilin Li 1, Huyen Cao 1, Leigh Anne Eller 4, Moses Kamya 2, Diane Havlir 1, Joseph Wong 1 1 University of California, San Francisco, U.S.A.; 2 Makerere University, Kampala, Uganda; 3 Monogram Biosciences, South San Francisco, CA, U.S.A.; 4 US Military HIV Research Program, Bethesda, MD, U.S.A. THPDA0205

2. CXCR4-tropism and the preferential establishment of an HIV-reservoir in naïve CD4+ T-cells among HIV-infected Ugandan children Table 1. Subject Characteristics* DMR5 Number48 Age (years)4.9 ( 4.1-6.2)5.1 (3.7-7.9) CD4%23 (21-24)28 (23-30) CD4 (cells/μl)655 (581–837)834 (555-1049) HIV RNA Levelall <400 c/ml Duration of ART (days) 98 (96-113)99 (95-150) HIV subtype2 A, 2 D4 A, 4 D * Values are number or median (interquartile range) Background: Children have large populations of naive CD4+ T-cells that express high levels of CXCR4 HYPOTHESIS: HIV-infected Ugandan children infected with CXCR4-tropic virus would exhibit larger HIV- DNA reservoirs in naïve CD4+ T-cells, compared to children infected with CCR5-tropic virus. HIV-DNA: real time Taqman PCR assay using a lock nucleotide (LNA™) - based probe in HIV gag p24 region (HXB2, 1400–1412). Lymphocyte DNA: commercial TERT-assay. Calculated number of HIV copies per 10 6 copies of cellular DNA in the naïve and memory lymphocyte subsets for each child. Non-parametric Kruskal-wallis testing Methods: Banked specimens from a cohort of 300 HIV-infected Ugandan children. Selected 4 dual-mixed (CXCR4- and CCR5-tropic, DM) and 8 pure CCR5-tropic (R5) virus infected children (Trofile Assay); all children on antiretroviral therapy (ART). Naïve and memory CD4+ T-cell separation: Cryopreserved peripheral blood mononuclear cells were sorted into naïve (CD27+CD45RA+) and memory (CD27-CD45+ and CD45-CD27±) CD3+CD4+ lymphocytes. Ruel et. al., THPDA0205

3. CXCR4-tropism and the preferential establishment of an HIV-reservoir in naïve CD4+ T-cells among HIV-infected Ugandan children Ratio of HIV-infection frequency in Naïve : Memory CD4+ T-Cells* DM8.9 (6.6-13.0) R50.7 (0.2-4.3) *Median (IQR); p=0.04 Proportion of circulating HIV-infected CD4+ T-cells that are naive Conclusions In contrast to adults, a significant portion of the HIV- reservoir is located among naïve CD4+ T lymphocytes among ART-treated children. Infection with DM virus was associated with preferential infection of naïve CD4+ T-cells In developing strategies to eradicate HIV, it will be important to take into account the high levels of naïve T- cell infection in children, particularly among those with with DM virus. Bars represent medians. P-values are for Kruskal- Wallis Test. DM92% (88%-95%) R553% (25%-80%) *Median (IQR); P=0.07