Background:
Clonal expansion of latently infected CD4+ T cells contributes to the latent reservoir. Disruption of this process may be needed for viral eradication to be achieved. Recent studies suggest that intact proviruses integrated in certain Zinc Finger (ZNF) genes have a survival advantage due to reduced viral expression caused by a repressive epigenetic environment. However, direct evidence of this mechanism is lacking.
Methods:
We characterized HIV integration sites and used digital PCR assays to measure total, intact, and provirus-specific HIV DNA in an elite controller (EC) before and after he received antiretroviral therapy, chemoradiation, and immunotherapy for metastatic lung cancer. CD8-depleted PBMC were stimulated with overlapping Gag peptide for 10 days. Provirus expansion and virus production were used to establish epitope recognition.
Results:
We detected a marked yet transient contraction in the number of expanded infected-clones after chemoradiation. In addition, there was a modest decline in both total and intact HIV DNA that returned to pre-treatment levels. Of note, his plasma HIV RNA remained undetectable (< 20 copies/ml) throughout the follow-up. We found that the proviral landscape at the end of treatment was dominated by two large clones with replication competent virus integrated into ZNF 721 and 470 genes. One clone, with a provirus integrated in ZNF470, was stable during treatment. In contrast the other clone, which has a provirus integrated in ZNF721 and recognizes the Gag peptide STLQEQIGWMTNNPP (241-255), underwent a 70-fold expansion after chemotherapy was completed. A third clone with a deletion in the primer binding site region and an unknown integration site recognized the overlapping Gag peptides EKAFSPEVIPMFSAL (162-176) and SPEVIPMFSALSEGA (166-180). Stimulation with Gag peptides in vitro resulted in virus production and cell proliferation. However, we observed an RNA to DNA ratio of 0.2 for the clone integrated in ZNF721, and 147 for the defective clone, suggesting that the lower inducibility of the intact provirus in the ZNF721 gene can favor its persistence.
Conclusions:
Our results suggest that chemoradiation can transiently disrupt clonal expansion and can be used in conjunction with other interventions as part of an HIV cure strategy. Despite the lower inducibility of the ZNF721 integrant, virus production can still occur, suggesting that strong CTL killing is required to maintain elite control of HIV replication.