Human milk-derived extracellular vesicles (HMEVs) are crucial functional components in breast milk, contributing to infant health and development. Maternal conditions could affect HMEV cargos; however, the impact of SARS-CoV-2 infection on HMEVs remains unknown. Researchers at the University of Cincinnati College of Medicine evaluated the influence of SARS-CoV-2 infection during pregnancy on postpartum HMEV molecules. Milk samples (9 prenatal SARS-CoV-2 vs. 9 controls) were retrieved from the IMPRINT birth cohort. After defatting and casein micelle disaggregation, 1 mL milk was subjected to a sequential process of centrifugation, ultrafiltration, and qEV-size exclusion chromatography. Particle and protein characterizations were performed following the MISEV2018 guidelines. EV lysates were analyzed through proteomics and miRNA sequencing, while the intact EVs were biotinylated for surfaceomic analysis. Multi-Omics was employed to predict HMEV functions associated with prenatal SARS-CoV-2 infection. Demographic data between the prenatal SARS-CoV-2 and control groups were similar. The median duration from maternal SARS-CoV-2 test positivity to milk collection was 3 months (range: 1-6 months). Transmission electron microscopy showed the cup-shaped nanoparticles. Nanoparticle tracking analysis demonstrated particle diameters of <200 nm and yields of >1e11 particles from 1 mL milk. Western immunoblots detected ALIX, CD9 and HSP70, supporting the presence of HMEVs in the isolates. Thousands of HMEV cargos and hundreds of surface proteins were identified and compared. Multi-Omics predicted that mothers with prenatal SARS-CoV-2 infection produced HMEVs with enhanced functionalities involving metabolic reprogramming and mucosal tissue development, while mitigating inflammation and lower EV transmigration potential. These findings suggest that SARS-CoV-2 infection during pregnancy boosts mucosal site-specific functions of HMEVs, potentially protecting infants against viral infections. Further prospective studies should be pursued to reevaluate the short- and long-term benefits of breastfeeding in the post-COVID era.
Multi-Omic analysis revealed prenatal SARS-CoV-2 infection affected HMEV molecular cargos in human milk at 2 weeks of lactation period
A. HMEV proteomics. Of 1143 identified proteins, 91 significantly altered proteins were analyzed by heatmap with unsupervised clustering. Functional enrichment analysis with network ensembled topology of up- and down-regulated proteins identified four functional-protein clusters. Representative six relevant EV proteins were showed as boxplots (all 30 relevant EV proteins showed in Supplementary figure 1). B. HMEV surfaceomics. Of 366 surface EV proteins, top 10% increased (36) and decreased (36) proteins were analyzed by STRING protein-protein interaction with functional enrichment analysis. Color codes represent proteins involved in predicted functions. C. HMEV miR-Omics. Of 2588 identified miRs, the top 20 high abundant miRs in HMEVs were illustrated. Gene target prediction with network analysis showed 24 common gene targets shared among top 5 HMEV-miRs Analyzing HMEVs at different molecular levels provided a consistent functional prediction that prenatal SARS-CoV-2 infection altered HMEV functions to promote mucosal epithelial development, mitigate inflammation, and lower systemic bioavailability, thus boosting mucosal-site specific effects. *, p<0.05; **, p<0.01.