The global burden of SARS-CoV-2 variants, particularly affecting unvaccinated children, necessitates vaccination of breastfeeding women to protect both mother and infant. While SARS-CoV-2-specific antibodies are transferred via breast milk, concerns remain regarding potential adverse effects on infants and vaccine-induced changes to the breast milk microbiome. The early-life microbiome significantly impacts long-term health and immune development. Previous research demonstrated the gut microbiota's role in modulating immune response to SARS-CoV-2 vaccines. However, the effects of mRNA COVID-19 vaccination on breast milk microbiota composition and its influence on antibody responses remain unclear. This study aimed to investigate the changes in breast milk microbiome composition before and after mRNA COVID-19 vaccination and its impact on anti-SARS-CoV-2 antibody kinetics in breast milk.

Several studies have shown that SARS-CoV-2 specific antibodies are present in breast milk and infant stool post-vaccination, conferring potential protection to the infant. However, there is a relatively low vaccination rate among lactating mothers due to fears of unpredictable adverse effects on their infants. These fears are significant because breast milk antibodies not only protect against pathogens but also shape the infant's immune system development and gut microbiome. Prior research has also shown that the gut microbiota modulates the immune response to SARS-CoV-2 vaccines, with specific species potentially influencing both immunogenicity and adverse events. This study builds upon this foundation by focusing specifically on the breast milk microbiome and its relationship to vaccine-induced antibody responses.

This prospective longitudinal study recruited 49 lactating mothers in Hong Kong who received two doses of the BNT162b2 mRNA vaccine between June and December 2021. Breast milk samples were self-collected at four time points: pre-vaccination, one week post-first dose, one week post-second dose, and one month post-second dose. SARS-CoV-2 spike protein-specific IgA and IgG levels were measured using ELISA. 16S rRNA amplicon sequencing was performed to analyze the breast milk microbiota composition and diversity. Statistical analyses included paired Wilcoxon rank-sum tests, linear mixed modeling, PERMANOVA, LEfSe, and random forest analysis to assess the relationships between antibody levels, microbiota composition, and various demographic factors. An external unvaccinated cohort was used for comparison.

Levels of SARS-CoV-2 spike protein-specific IgA and IgG in breast milk significantly increased one week post-second dose. IgA levels returned to baseline one month later, while IgG levels remained elevated. Breast milk microbiota richness and composition changed dynamically throughout the vaccination regimen, but the abundances of beneficial microbes such as Bifidobacterium species did not significantly change. Baseline breast milk bacterial composition effectively predicted spike-specific IgA levels one week post-second dose (AUC 0.72, 95% CI: 0.58–0.85). Specifically, unclassified Neisseria and Neisseria elongata were enriched in participants with higher IgA levels. Analysis of microbial functional pathways revealed enrichment of nine pathways in high-IgA subjects, including fucose degradation and arginine and polyamine biosynthesis. While the abundance of Lactobacillus and Bifidobacteria genera generally remained unchanged following vaccination, some species showed altered abundance.

This study provides the first longitudinal evidence in humans that baseline breast milk microbiota composition and its post-vaccination changes reflect vaccine-conferred anti-SARS-CoV-2 antibody levels. The observed changes in bacterial richness differ from those in the unvaccinated cohort, suggesting a vaccine-specific effect. The lack of significant impact on beneficial Bifidobacteria suggests the vaccine's safety profile. The identified differentially abundant bacterial species at baseline, particularly unclassified Neisseria and Neisseria elongata, might enhance antibody levels through immunomodulatory actions and/or by acting as adjuvants. The influence of microbiota on immunomodulatory metabolic pathways, particularly fucose degradation, further highlights the complex interaction between the microbiome and immune response. The increased abundance of Bifidobacterium species in high-IgA subjects points towards their potential role in boosting immunity.

This study demonstrates the dynamic interplay between BNT162b2 vaccination, breast milk microbiota, and anti-SARS-CoV-2 antibody levels. Specific baseline microbial markers are associated with higher antibody responses, while the overall abundance of beneficial probiotic species remains unaffected by vaccination. This suggests that vaccination of lactating mothers provides immunological protection to infants via both antibody and microbiota transmission. Future studies should investigate the mechanistic details and validate these findings in larger, more diverse populations, including analyses of infant immune responses.

The study had a relatively small sample size, and the lack of a contemporaneous unvaccinated control group limits the strength of conclusions regarding vaccine-specific effects. The use of an external, pre-pandemic control cohort, while valuable, introduces potential biases due to differences in collection time points and potential variations in microbial composition. Further research is needed to fully understand the long-term consequences and detailed mechanisms of the observed effects.