Proteomic profiling of single extracellular vesicles reveals association of CD31+ EV subpopulation with immune dysregulation in people living with HIV.

BACKGROUND

HIV remains globally prevalent, and the long-term complications experienced by people living with HIV (PLWH) have increased interest in the functional heterogeneity of extracellular vesicle (EV) subpopulations. Defining these EV subsets may help clarify HIV-associated immune dysregulation and inform future diagnostic and therapeutic strategies.

METHODS

Plasma samples were obtained from three PLWH (HIV+ group) and three healthy controls (HC group). Single-EV surface membrane proteins were profiled using the proximity barcoding assay (PBA), and EV subpopulations were identified by FlowSOM clustering with t-SNE visualization. Functional enrichment analysis, protein-protein interaction (PPI) network analysis, and database-based annotation were used to infer putative subpopulation functions and cellular origins. Differentially expressed EV proteins (DEPs) were screened using limma, and ligand-receptor interaction networks were constructed with CellPhoneDB and cross-referenced with publicly available single-cell RNA sequencing data.

RESULTS

HIV infection did not appear to alter overall EV abundance but was associated with selective remodeling of EV heterogeneity and expansion of distinct EV subpopulations. CD31+ EVs (cluster 2), inferred to be endothelial-derived, were markedly enriched in PLWH. Ligand-receptor analysis suggested that CD31+ EVs may communicate with CD16+ monocytes through F11R-ITGAL/ITGB2 and with plasmablasts through CD31-CD38 interactions; these interactions were associated with inflammatory, leukocyte transendothelial migration, and metabolic pathways. In addition, HIV-enriched B2M+ EVs (clusters 3) and MUC16+ EVs (clusters 9) showed predicted interactions with CD4+ T cells and with CD8+ effector memory T and NK cells, respectively, suggesting potential effects on reservoir-related and cytotoxicity-related programs.

CONCLUSIONS

These exploratory findings suggest that EV heterogeneity may encode cell-type-specific immune regulatory information in HIV infection and highlight CD31+ EVs as a candidate EV subset associated with HIV-related immune dysregulation. Larger cohorts and functional validation are required before these EV populations can be considered therapeutic targets for HIV cure strategies.