Background. Maternal breastmilk is a source of pre- and pro-biotics that impact neonatal gut microbiota colonisation. Since oral rotavirus vaccines (ORVs) are administered at a time when infants are often breastfed, breastmilk microbiota composition may have a direct or indirect influence on vaccine take and immunogenicity. Methods. Using standardised methods across sites, we compared breastmilk microbiota composition in relation to geographic location and ORV response in cohorts prospectively

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... ollowed up from birth to 18 weeks of age in India (n = 307), Malawi (n = 119), and the UK (n = 60). Results. Breastmilk microbiota diversity was higher in India and Malawi than the UK across three longitudinal samples spanning weeks of life 1 to 13. Dominant taxa such as Streptococcus and Staphylococcus were consistent across cohorts; however, significant geographic differences were observed in the prevalence and abundance of common and rare genera throughout follow-up. No significant associations were identified between breastmilk microbiota composition and ORV outcomes including seroconversion, post-dose 1 vaccine shedding, and/or post-vaccination rotavirus-specific IgA level. Conclusions. Our findings suggest that breastmilk microbiota composition may not be a key factor in shaping trends in ORV response within or between countries.