Comprehensive immune profiling of dengue and chikungunya viral responses using a novel miniaturized automated whole blood cellular analysis system and mass cytometry in a pediatric cohort in Msambweni, Kenya

Abstract Chikungunya (CHIKV) and dengue (DENV) are mosquito-borne viruses that cause severe epidemics, often in remote regions. A limitation to our understanding of these pathogens is the difficulty of performing assays of the cellular immune response. To fill this gap, we developed a novel miniaturized automated system capable of processing 250 μl of whole blood for high-throughput cellular analysis. In a field study with a pediatric cohort in Msambweni, Kenya, known for previous exposure to CHIKV and/or DENV, we processed 133 whole blood samples using our system under three conditions: no stimulation, and stimulation with CHIKV or DENV peptide pools. These samples underwent CyTOF or flow cytometry analysis to evaluate virus-specific memory T cell responses and phenotypes. CyTOF analysis of 81 participant samples revealed significant cytokine responses to CHIKV and DENV, particularly IFNγ (P < 0.01 and P < 0.0001, respectively) and TNF-α (P < 0.0001) by γδ T cells. Additionally, a significant TNF-α response was observed in the CD8+ TEMRA memory subset to DENV, albeit to a lesser degree than in γδ T cells. To confirm our CyTOF findings, we employed flow cytometry on the remaining 40 samples using a targeted panel, validating significant TNF-α (P < 0.0001 and P < 0.01) and IFN-γ (P < 0.05) responses by γδ T cells to CHIKV and DENV, respectively. Our study demonstrates that our innovative automated system enables detailed assessment of immune function, particularly beneficial in pediatric populations and resource-limited settings with limited sample volumes. This approach holds promise for advancing our understanding of cellular immune responses to various viral and infectious diseases.