Background:
We studied the proviral landscape of neonates with in utero HIV-1 who initiated antiretroviral treatment (ART) within 48 hours of birth in the IMPAACT P1115 study.
Methods:
21 neonates with in utero HIV-1 in IMPAACT P1115 met criteria for near full-length single genome sequencing (nFLSGS) at either Step 1 (within 48 hours post-birth; N=16), Step 2 (7-18 days post-birth when infection was confirmed; N=17) or both (N=12). Of the 13 neonates excluded at either timepoint, 11 had low proviral DNA loads (<20 copies/10^6 PBMCs) and 2 had low sample volumes (<5 uL) insufficient for nFLSGS amplification. nFLSGS was performed using a standard input of HIV-1 DNA per neonate to ensure single proviral genome amplification. nFLSGS data were classified with HIVSeqinR as intact, defective, or hypermutated, and copy numbers were standardized and efficiency adjusted to c/10^6 PBMCs. The limit of detection was set to 0.5 copies per number of cells assayed. ART drug resistance was evaluated using the Stanford HIV Drug Resistance Database.
Results:
In the 21 neonates analyzed, median log10 HIV-1 DNA load was 2.8 at Step 1 and 2.9 at Step 2 (median 15 days). nFLSGS examined 93 genomes at Step 1 and 111 genomes at Step 2 (median 6 genomes per neonate in median 50000 cells). At our sampling depth, we detected intact proviruses in 13/16 (81%) neonates at Step 1 and 14/17 (82%) at Step 2. Intact proviruses comprised a median of 50% of each neonate’s observed genomes at Step 1 and 33% at Step 2. Median log10 intact proviral load was 2.0 at Step 1. Among the 12 neonates with data at both timepoints (median 14 days apart), intact proviruses shared >99.8% sequence identity. Defective and hypermutated proviruses were detected at either timepoint in 20/21 and 9/21 neonates. The K103N resistance mutation was detected in all intact proviruses at both timepoints for one neonate, confirmed with maternal genotyping. No other major resistance mutations were identified in the 21 neonates.
Conclusions:
The proviral landscape at birth of neonates with in utero HIV-1 and quantifiable HIV-1 DNA includes a mix of intact, defective, and hypermutated proviruses, indicating in utero HIV-1 replication. Intact proviruses share >99.8% sequence identity through Step 2. These findings suggest early formation of abundant intact HIV-1 DNA populations, which could establish reservoirs that pose challenges to HIV-1 remission.