Background
Despite effective antiretroviral therapy (ART), HIV-1 persistence is the major obstacle to a functional cure. Thus, understanding the tissue microenvironment during ART of the reservoirs that lead to a rapid viral rebound after treatment failure or analytical treatment interruption (ATI) is key.
Methods
We have developed immunoPET/CT-guided spatial transcriptomics, combined with immunofluorescence detection of viral proteins and viral sequencing using the SIV/rhesus macaque model. With this approach we can find and study foci of viral replication in tissues of all animals. Here we compare the local neighborhood of the rebound eclipse-phase foci (4-6 days post-ATI) from animals initiating ART 4-days (early-seeded reservoir with short lifespan) or 10-weeks (well-seeded reservoir) after high-dose challenge, as well as early-seeded tissues from animals on ART for 3-or-7-weeks. Additionally, we performed viral long-read deep sequencing and population dynamics analysis in the same tissue sections.
Results
Overall, SIV presence in every condition was associated with higher transcriptional levels and up-regulation of genes related to SIV infection. Activation of innate immune responses was observed only in the eclipse-phase of the rebound for both types of reservoirs and not during therapy indicating the specificity of the analysis. Notably, we also detected significant differences between early- and well-seeded reservoirs. Translation activation was associated with SIV presence in early-seeded but downregulated in well-seeded reservoirs, while mitochondrial translation was activated in all cases. This is consistent with integrated stress response (ISR) induced decreased protein synthesis in well-seeded reservoirs. Inference of cell type frequency per foci indicated that SIV presence was always associated with epithelial cells. SIV in well-seeded reservoirs was distinctly associated with IgA plasma cells, monocytes, and cycling gamma-delta T-cells. Analysis of intra-host viral population dynamics from the same “hot” tissues indicates that the highest viral diversity levels are consistently detected in GALT reservoirs where our spatial transcriptomics show activation of stress responses.
Conclusions
Our results indicate that persistent reservoirs are associated with gut tertiary lymphoid structures and might be characterized by a status of low translation consistent with ISR which could be favoring long-term viral persistence during ART and rapid rebound of robust viral populations post-ATI.