Durable antibody and effector memory T cell responses in breastmilk from women with SARS-CoV-2.

Background: Given that only 25% of pregnant women elect to receive a COVID-19 vaccine, maternal SARS-CoV-2 infection remains an important route of conferring protective passive immunity to breastfed infants of mothers who are not vaccinated. Methods: We enrolled 30 lactating participants between December 2020 and March 2021 who had a positive PCR-test and their first COVID-19 symptoms within the previous 21 days. Participants were asked to provide serial bilateral milk samples at 12 timepoints (~ every 3 days) over a period of 35 days. A second set of samples was collected at least four months after the beginning of the first set. Participants also were asked to provide their dried blood spots and infant stool samples. All samples were tested for receptor-binding domain (RBD)-specific immunoglobulin (Ig)A, IgG, and IgM. Milk samples were assessed for neutralizing ability against the spike protein and four SARS-CoV-2 variants: D614G, Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Permeability of the breast epithelium was assessed by measuring the sodium to potassium ions (Na:K) in milk. Using flow cytometry, memory CD4 and CD8 T cells (CD45RO+ and CCR7+/-) and mucosal-homing CD4 and CD8 T cells (CD103+) were determined in cells from milk expressed at 35 days and at least 4 months after their first milk donation. Results: Milk antibodies from SARS-CoV-2 positive participants neutralized the spike complex. Milk from 73, 90, and 53% of participants had binding reactivities to RBD-specific IgA, IgG, and IgM, respectively. In contrast to blood spots, which showed increased levels of IgG, but not IgA or IgM, the COVID-19 response in milk was associated with a robust IgA response. Twenty-seven percent of participants had increased breast-epithelium permeability, as indicated by Na:K ≥ 0.6. The percentage of CD45RO+CCR7- effector-memory T cells in the day ≥120 milk samples was significantly higher than day 35 samples (P< 0.05). Conclusions: Antibodies in milk from participants with recent SARS-CoV-2 infection and those who recovered can neutralize the spike complex. For the first time we show that breastmilk T cells are enriched for mucosal memory T cells, further emphasizing the passive protection against SARS-CoV-2 conferred to infants via breastmilk.

Neutralizing Antibodies and Cytokines in Breast Milk After Coronavirus Disease 2019 (COVID-19) mRNA Vaccination.

OBJECTIVE: To evaluate immune responses to coronavirus disease 2019 (COVID-19) mRNA-based vaccines present in breast milk and transfer of the immune responses to breastfeeding infants. METHODS: We enrolled 30 lactating women who received mRNA-based COVID-19 vaccines from January through April 2021 in this cohort study. Women provided serial milk samples, including milk expressed before vaccination, across 2-3 weeks after the first dose, and across 3 weeks after the second dose. Women provided their blood, spotted on cards (dried blood spots), 19 days after the first dose and 21 days after the second dose. Stool samples from the breastfed infants were collected 21 days after mothers' second vaccination. Prepandemic samples of milk, dried blood spots, and infant stool were used as controls. Milk, dried blood spots, and infant stool were tested by enzyme-linked immunosorbent assay for receptor-binding domain (RBD)-specific immunoglobulin (Ig)A and IgG. Milk samples were tested for the presence of neutralizing antibodies against the spike and four variants of concern: D614G, Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Levels of 10 cytokines were measured in milk samples. RESULTS: Milk from COVID-19-immunized women neutralized the spike and four variants of concern, primarily driven by anti-RBD IgG. The immune response in milk also included significant elevation of interferon-γ. The immune response to maternal vaccination was reflected in breastfed infants: anti-RBD IgG and anti-RBD IgA were detected in 33% and 30% of infant stool samples, respectively. Levels of anti-RBD antibodies in infant stool correlated with maternal vaccine side effects. Median antibody levels against RBD were below the positive cutoffs in prepandemic milk and infant stool samples. CONCLUSION: Humoral and cellular immune responses to mRNA-based COVID-19 vaccination are present in most women's breast milk. The milk anti-RBD antibodies can neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike and variants of concern. Anti-RBD antibodies are transferred to breastfed infants, with the potential to confer passive immunity against SARS-CoV-2.

Humoral and Cell-Mediated Immune Response in Colostrum from Women Diagnosed Positive for SARS-CoV-2.

Objective: To evaluate the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in colostrum from women who tested positive for the virus. Methods: Between March and September 2020 we obtained bilateral colostrum samples collected on spot cards within 48 hours of delivery from 15 new mothers who had previously tested positive for SARS-CoV-2. Four of 15 women provided liquid colostrum, which was used for validating results obtained from spot cards. Archived bilateral colostrum samples collected from 8 women during 2011-2013 were used as pre-coronavirus disease 2019 (COVID-19) controls. All samples were tested for reactivity to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein using an enzyme-linked immunosorbent assay that measures SARS-CoV-2 RBD-specific IgA, IgG, and IgM and for levels of 10 inflammatory cytokines (interferon-gamma [IFN-γ], tumor necrosis factor-alpha, interleukin [IL]-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13) using a multiplex electrochemiluminescent sandwich assay. Results: Our validation studies indicate that the levels of SARS-CoV-2-specific antibodies and the associated cytokines measured in liquid colostrum are comparable to levels eluted from spot cards. Bilateral colostrum samples from 73%, 73%, and 33% of the 15 COVID-19 mothers exhibited IgA, IgG, and IgM reactivity to RBD, respectively. In addition, symptomatic COVID-19 mothers had statistically significant elevated levels of 4 of the 10 inflammatory markers (IFN-γ, IL-4, IL-6, and IL-12) compared to asymptomatic COVID-19 mothers. Conclusions: A strong humoral immune response is present in the colostrum of women who were infected with SARS-CoV-2 before delivering. The evolution and duration of the antibody response, as well as dynamics of the cytokine response, remain to be determined. Our results also indicate that future large-scale studies can be conducted with milk easily collected on paper spot cards.