Chutipongtanate Somchai, Cetinkaya Hatice, Zhang Xiang, Kuhnell Damaris, Benefield Desirée, Haffey Wendy, Wyder Michael, Patel Richa, Conrey Shannon C, Burrell Allison R, Langevin Scott, Nommsen-Rivers Laurie, Newburg David S, Greis Kenneth D, Staat Mary A, Morrow Ardythe L
bioRxiv. 2023 Jun 1:2023.06.01.543234. doi: 10.1101/2023.06.01.543234.
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. This study 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. Our 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.
人乳源细胞外囊泡（HMEVs）是母乳中的关键功能成分，对婴儿的健康和发育至关重要。母亲的身体状况可能会影响HMEV的货物成分；然而，新型冠状病毒2（SARS-CoV-2）感染对HMEVs的影响仍不清楚。本研究评估了孕期SARS-CoV-2感染对产后HMEV分子的影响。从IMPRINT出生队列中获取了牛奶样本（9例产前SARS-CoV-2感染产妇与9例对照产妇）。在进行脱脂和酪蛋白胶粒分解后，取1 mL牛奶依次进行离心、超滤和qEV尺寸排阻色谱法处理。按照MISEV2018指南进行颗粒和蛋白质表征。通过蛋白质组学和miRNA测序分析细胞外囊泡裂解物，同时将完整的细胞外囊泡进行生物素化用于表面组学分析。采用多组学方法预测与产前SARS-CoV-2感染相关的HMEV功能。产前SARS-CoV-2感染组和对照组之间的人口统计学数据相似。从母亲SARS-CoV-2检测呈阳性到采集乳汁的中位持续时间为3个月（范围：1 - 6个月）。透射电子显微镜显示为杯状纳米颗粒。纳米颗粒跟踪分析表明颗粒直径<200 nm，且每1 mL牛奶产生的颗粒产量>1×10¹¹个。蛋白质免疫印迹检测到了ALIX、CD9和HSP70，证实了分离物中存在HMEVs。鉴定并比较了数千种HMEV货物成分和数百种表面蛋白。多组学预测，产前感染SARS-CoV-2的母亲产生的HMEVs具有增强的功能，涉及代谢重编程和黏膜组织发育，同时减轻炎症并降低细胞外囊泡的迁移潜力。我们的研究结果表明，孕期SARS-CoV-2感染可增强HMEVs的黏膜部位特异性功能，可能保护婴儿免受病毒感染。应进一步开展前瞻性研究，以重新评估新冠疫情后母乳喂养的短期和长期益处。
