Abstract
The dynamic nature of the immune response during acute viral infection involves a complex orchestration of diverse cellular subsets. Traditional bulk proteomic approaches often obscure the functional heterogeneity inherent in these populations, failing to capture the nuanced transitions between effector, memory, and exhausted states. In this study, we employed high-dimensional single-cell proteomic profiling to characterize the immune landscape in a cohort of patients during the acute phase of viral infection. Using a combination of single-cell network profiling (SCNP) and mass spectrometry-based workflows, we identified distinct proteomic signatures within CD4+ and CD8+ T-cell subpopulations, as well as B-cell lineages. Our results reveal a significant upregulation of immune checkpoints, including PD-1 and CTLA-4, concurrent with a sharp decline in homeostatic regulators such as E2F1 during the peak of infection. Furthermore, we observed a unique enrichment of Tcf1+ memory precursors, suggesting an early commitment to long-term immunity despite the inflammatory milieu. Proteomic interrogation of secretory granules provided insights into the cytotoxic potential of specific CD8+ clusters, while analysis of extracellular vesicle-associated proteins highlighted the role of systemic signaling in modulating the immune response. These findings provide a comprehensive map of the proteomic alterations driving immune activation and regulation in acute viral contexts, offering potential biomarkers for predicting clinical outcomes and informing the design of next-generation vaccines. The integration of single-cell proteomics with clinical data underscores the importance of cellular resolution in understanding the molecular determinants of viral clearance and immune homeostasis.