Background
The persistent HIV-1 reservoir is comprised of individual proviral clones with a corresponding integration site. The proviral landscape in the blood of long-term ART-suppressed persons with HIV-1(PWH) is often dominated by identical sequences, signifying antigen-driven proliferation. The clonal structure of the SIV reservoir during ART suppression is less well-characterized, particularly in the first 2 years of ART, which is commonly when HIV-1 cure strategies undergo pre-clinical testing.
Methods
We employed a linker-mediated PCR-based assay coupled with next-generation sequencing to longitudinally assess proviral integration sites in the blood of SIVmac239M-infected macaques (n=3) at 3 and 10 months of ART.
Results
We sampled a total of 357 SIV integration sites during both ART time points. Of these, we encountered predominantly single unique proviral sites (Fig. A). 39 (10.9%) proviral clones were found to be identical, with no statistical difference in the number of identical integration sites between the two timepoints (p = 0.9934). We also did not observe clonality increase from 3 to 10 months of ART, a phenomenon known to occur in PWH over long-term ART intervals. We observed a very low frequency of SIV integration sites shared between 3- and 10-month ART timepoints (1.1%) (Fig. B). Shared clones however had a high likelihood to be clonal at 3 or 10 months, confirming cellular proliferation as a driver of reservoir persistence. One animal was found to have 7 clones at 3 months, ~10% of integration sites sampled. This clone decreased to 2 clones (4.9%) at 10 months. This integration site was within the DDX49 gene; an RNA Helicase that has previously been associated with cellular proliferation.
Conclusions
Our data indicate an absence of dominating SIV proviral clones up to 10 months of ART. Suggesting the SIV reservoir during periods of pre-clinical therapeutic testing is polyclonal, and predominantly maintained by homeostatic rather than antigenic proliferation. At 3 months of ART, one animal displayed a highly expanded clone integrated into DDX49. Given that early ART reservoirs are orders of magnitude larger than those of long-term suppression, this clone is likely to be massive. The frequency of this clone contracted at 10 months, suggesting that large clones within genic regions may be under negative selection pressure during reservoir decay. Overall, the data suggest that the SIV proviral landscape is markedly distinct from that of long-term suppressed PWH.