Background:
SIV infection recapitulates many aspects of HIV-1 infection. However, the SIV reservoir at the single-cell resolution remains to be defined. Here, we adapted our single-cell viral Assay for Transposase Accessible Chromatin with Select Antigen Profiling by sequencing (viral ASAPseq) to rhesus macaque SIV infection model, to characterize SIV/SHIV reservoir epigenetics and phenotype.
Methods:
To adapt viral ASAPseq to the nonhuman primate model, we compiled, titrated, and characterized a rhesus cross-reactive antibody panel consisting of 62 immune-related markers using custom and commercially available oligo-tagged antibodies from Biolegend. Using this cocktail, we performed viral ASAPseq on purified lymph node CD4+ T cells at day 13 post-SIVmac251 infection. Finally, we developed a customized bioinformatics analysis pipeline to assess infected cells after viral alignments were made against the SIVmac251 sequence.
Results:
From this pilot test, we identified 81 SIV+ cells out of 15,334 cells (0.5%); of these, 42 cells had 2 or more reads aligned to multiple regions of the provirus. Using both the epigenetic and surface antigen profiles, we found that 67% of infected cells had characteristics of T follicular helper, T follicular regulatory, or T regulatory cells. The remaining infected cells appeared to have effector, activated, and/or resident memory characteristics.
Conclusions:
These initial findings demonstrate that NHP viral single-cell ASAPseq can be used to identify SIV+ cells and determine the unperturbed epigenetics and phenotype of these cells. Notably, this assay allows an unprecedented view into the SIV/SHIV reservoir to understand the perturbations associated with various cure strategies.