Background: CD4 T-cells have been shown to harbor replication competent HIV (RCV) in both children and adults on HAART with prolonged undetectable plasma viremia.

Methods: We prospectively evaluated 11 HIV-infected infants and children (35 days to 9-yrs-old) and 1 teenager (16 yrs) with durable plasma viremia suppression (< 50 cp/ml) at 0.5–1 yr intervals for 4 yrs post-HAART. Peripheral blood mononuclear cells (PBMC) were separated into purified CD4 T-cells alone, CD45RA or CD45RO T-cells using magnetic beads and/or FACS. CD4 T-cells were activated with monoclonal antibody CD3, CD28 for 48 hours then co-cultured with PHA stimulated HIV-1 (-) donor’s CD4 T-cells for up to 48 days. Samples with 1x10⁶ CD4 T-cells were considered adequate. Supernatants were then assayed for P24 Antigen (Ag) as previously described.

Results: RCV was obtained from 9/11 (82%) patients (pts) post-HAART. We were unable to obtain RCV from CD4 T-cells on 4/4 attempts in 1 infant and 1 teenager.

 

Years on HAART	< 1	1.0	1.5	2	3	> 4
Positive recovery/# attempts	3/6	7/11	4/8	4/7	7/9	6/11
% recovery of RCV	50	64	50	57	78	55

 

Pts on HAART < 2 yrs had RCV detected in vitro at day 7–14 as compared to day 21–45, 3–4 yrs post-HAART. We isolated RCV from CD45RA only in 1 young infant (3 mo) and from both CD45RA and RO in 3 older children (2.5–8 years). All CD4 T-cell RCVs were R5 tropic in HOS cell lines. Comparison of in vitro replication kinetics of pre-HAART PBMC virus isolates in pooled 3 donor PBMC to later CD4 T-cell RCV showed a decrease in peak P24Ag production (mean peak P24Ag 20,000 vs10,000 pg/ml).

Conclusions: We have isolated RCV in the majority of children (82%) with durable viral suppression on HAART over a 4-yr period. At later time points, virus recovery from latent CD4 T-cells in vitro was delayed. It is of interest that one infant treated early with HAART (< 3 mos) had no RCV detected on multiple attempts and virus was isolated only from CD45RA 62L naïve cells in another infant. In addition, RCV isolates from CD4 T-cells have lower replication kinetics in vitro and may be less fit. Further studies of the establishment, persistence, and factors influencing these reservoirs will be important for future therapeutic intervention.