A multiomic investigation of human milk-derived extracellular vesicles in perinatal HIV and antiretroviral therapy exposure

BACKGROUND: Human milk (HM) is beneficial for early infant development, and breastfeeding is recommended globally for women with HIV (WHIV) on antiretroviral therapy (ART). HM has a high extracellular vesicle (EV) content, and distinct HM fractions may be enriched for unique small metabolites, lipids, oxylipins, and microRNAs, which may differ by HIV/ART exposure. Our objective was to characterize the metabolomic and miRNA differences between milk fractions and in relation to HIV/ART. METHODS: HM was collected at 6 weeks postpartum from WHIV receiving ART and HIV-seronegative women in South Africa (N = 20/group). Milk was fractionated into whole milk, skim milk, and EVs, which we isolated from skim milk using size-exclusion chromatography. Widely targeted small metabolite (~ 550 metabolites) and lipid profiling (~ 1500 lipid species in 24 + lipid classes) were measured with Ultra Performance Liquid Chromatography-Triple quadrapole Mass Spectrometry on whole milk, skim milk, and EVs. EV-miRNAs were sequenced on an Illumina NovaSeq. We used linear models to compare levels of lipids, metabolites, and miRNAs in HM EVs of WHIV versus HIV-seronegative women adjusting for covariates. RESULTS: There were 658 lipids, 130 oxylipins, and 274 small metabolites detected across all HM fractions, with fewer compounds detected in HM EVs compared to whole and skim milk. HM EVs were enriched for fatty acid metabolism and metabolic products of linoleic acid (false discovery rate [FDR] p < 0.05). Triacylglycerols, TAG47:0 and TAG49:0, were lower in whole and skim milk of WHIV participants. Conversely, 18 small metabolites in HM EVs were associated with HIV status, but not in whole or skim milk. Of 187 detected EV-miRNAs, miR-148b-5p was significantly lower in WHIV, which was also validated by qPCR. CONCLUSIONS: These preliminary results suggest that EVs may better represent differences in milk metabolites from HIV/ART exposure than whole milk or skim milk. EV contents altered in WHIV may be relevant for lipid, vitamin and energy metabolism, and metabolic health. Future research is warranted to assess the relationship between HM EV contents and child health. TRIAL REGISTRATION: ClinicalTrials.gov-ID: NCT04991402.