key: cord-0078841-ltulgbx8 authors: Esteve-Palau, Erika; Gonzalez-Cuevas, Araceli; Eugenia Guerrero, M.; Garcia-Terol, Clara; Carmen Alvarez, M.; Garcia, Geneva; Moreno, Encarna; Medina, Francisco; Casadevall, David; Diaz-Brito, Vicens title: Quantification and progress over time of specific antibodies against SARS-CoV-2 in breast milk of lactating women vaccinated with BNT162b2 Pfizer-BioNTech COVID-19 vaccine (LacCOVID) date: 2022-05-11 journal: Open Forum Infect Dis DOI: 10.1093/ofid/ofac239 sha: 572b9d92e24da930cb18798725ec18d6cac79cf9 doc_id: 78841 cord_uid: ltulgbx8 BACKGROUND: Transfer of passive and active immunity through human milk is a key aspect in infant protection against infections. Several observational studies demonstrated the passage of postvaccine antibodies through breast milk in women vaccinated against COVID-19, mostly with mRNA-based vaccines, but lacked long-term data. METHODS: A six-month prospective cohort study was performed to determine SARS-CoV-2 vaccine-induced antibody levels in the breast milk of 33 lactating healthcare workers at different time-points after mRNA BNT162b2 Pfizer-BioNTech COVID-19 vaccination. Moreover, we examined the correlation of SARS-CoV-2 antibody levels between serum and breast milk, adverse events related to vaccination (AErV) and rate of SARS-CoV-2 infections. RESULTS: Mothers’ median (IQR) age was 38(36-39) years and 15(10-22) months for infants. SARS-CoV-2 IgG-S1 vaccine-induced levels at different time-points for serum–milk pairs, median (IQR), were: 519(234-937) to 1(0-2.9) arbitrary units (AU)/mL at 2w after first dose, 18,644(9,923-29,264) to 78(33.7-128) AU/mL at 2w, 12,478(6,870-20,801) to 50.4(24.3-104) AU/mL at 4w, 4,094(2,413-8,480) to 19.9(10.8-51.9) AU/mL at 12w, and 1,350(831-2,298) to 8.9(7.8-31.5) at 24w after second dose. We observed a positive correlation of antibody levels between serum and breast milk (Pearson correlation coefficient 0.68), no serious AErV and 2(6%) COVID-19 vaccine breakthrough infections. CONCLUSIONS: Women vaccinated with Pfizer-BioNTech transmit antibodies into breast milk with a positive correlation with serum levels. Both decreased over time in a 6-month follow-up. Finally, Infants of breastfeeding vaccinated women could be acquiring vaccine antibodies for at least six months after vaccination and serum determination of SARS-CoV-2 IgG-S1 could indicate breastmilk antibody levels. Breastfeeding is one of the most efficacious means of preventing diseases and promoting 3 health in both mothers and children 1 . Transfer of passive and active immunity through human 4 milk is a key element in infant protection against infections 2, 3 . In the neonatal period, newborns 5 are exposed to a myriad of microorganisms, whose main entry point is through mucosal 6 barriers, and infants initially have an immune system which is too immature to cope with 7 pathogens. Breast milk contributes to a significant reduction in infant morbidity and mortality when 9 breastfeeding is performed exclusively in the first six months of life [4] [5] [6] [7] [8] [9] . Apart from its nutritional 10 richness, both colostrum and mature milk have a high content of immunoglobulins, proteins, 11 lactoferrin and leukocytes, among other immunomodulatory factors, which makes it the first 12 immunological contribute that the infant can receive in the first weeks and months of life 10, 11 . In The COVID-19 pandemic has raised many questions among people who are breastfeeding, both because of the possibility of viral transmission to infants during breastfeeding and, with the 23 approval of the SARS-CoV-2 vaccines, of the potential risks and benefits of vaccination in this 24 specific population. Pregnant and breastfeeding women were excluded from all pre-marketing 25 trials of anti-SARS-CoV-2 vaccines, so some doubts exist regarding its compatibility. In this 26 regard, a meta-analysis of 48 studies with 183 infected unvaccinated women analyzed the rate 27 of SARS-CoV-2 genome identification in breast milk, concluding that this was found in 5% of 28 cases, associated mainly with mild cases of COVID-19 in breastfed infants 15 . However, other 29 studies have observed that although SARS-CoV-2 RNA was found through PCR in the milk of pose a risk of infection for the infant 16, 17 . Different studies during the pandemic suggest that, far 2 from posing a risk of infection to the infant, breast milk from infected mothers may be protective Antibodies and Ag-RDT determination 13 When blood samples were obtained, they were centrifuged for 15 minutes at 3,500 rpm and 14 processed to determine levels of immunoglobulin (Ig) G antibodies against the spike protein S1 15 subunit (IgG-S1) and against the nucleocapsid (IgG-NC) of SARS-CoV-2 (Architect, Abbott®). As vaccination does not induce nucleocapsid antibody response, any IgG-NC positive result 17 was considered a prior infection. The milk samples were centrifuged for 15 minutes at 3,500 rpm and after removing the fat layer 19 with a pipette, the liquid layer of the milk was collected. Subsequently, we repeated the same 20 process once more to determine IgG-S1 (Architect, Abbott®). The determination was performed 21 in milk only when obtaining positive IgG-NC in serum.. Nasopharyngeal swabs were analyzed with Panbio® Abbot COVID-19 (Ag-RDT) to determine 23 SARS-CoV-2 infection. No tests were performed on the infants. In cases of suspicion of SARS-CoV-2 infection or close 25 contact, the infants were referred to pediatrics to perform a PCR SARS-CoV-2 assay to exclude Numeric variables were summarized using median and IQR (Q1-Q3). Association between 29 serum-and milk SARS-CoV-2 antibodies was analyzed using repeated measures (rm). and figures created using ggplot2 R package. Characteristics of participants and samples 4 A total of 33 volunteers were included in the study. The median (IQR) age of mothers was 38 5 (36-39) years and 15 (10-22) months for the infants at the time of the vaccination. All mother 6 and infant characteristics are described in Table 1 . We were able to recruit and obtain samples to determine the primary end-point, antibody titers 8 at 4 weeks after complete vaccination, in all participants (n=33). However, we only achieved 9 samples after the first dose of vaccine in 28 participants, and in 32 at two weeks after the 10 second dose. In the three-month follow-up, two participants were lost to follow-up (one due to 11 not being able to express milk, the other due to having weaned) and at six-month follow-up a 12 total of 8 participants were lost (4 were unable to express milk, 2 weaned, and 2 failed to visit). Results of all time-points after vaccination are summarized in Table 2 . Although maximum levels of IgG-S1, in both serum and milk, were observed two weeks after The main AErV (mean of 2 doses) in our cohort were: pain at the injection site (95%), feverish 8 feeling and/or confirmed fever (15%), general malaise (15%), headache (11%), arthromyalgia 9 (9%), asthenia (6%), axillary adenopathy (5%), and other (<3%). All AErV are summarized in 10 Therefore, it is reasonable to hypothesize that serum determination of SARS-CoV-2 IgG-S1 The current COVID-19 pandemic has raised multiple concerns for breastfeeding mothers. At We have also observed how, in cases in which a participant became infected by SARS-CoV-2, 28 the levels of IgG-S1 showed a new increase in both serum and breast milk. Not so in the case 29 of IgG-NC which, despite having been positive in serum, did not have its presence in milk 1 of detection could be higher than IG-S1 quantitative antibody determination. On the basis of the substantial increase in IgG-S1 in serum and milk observed after SARS-CoV-3 2 infection in 2 volunteers, it may be reasonable to consider that a COVID-19 vaccine booster 4 shot could increase serum and milk levels in the same way as a natural infection. The main limitation of this study is its small sample size. In our sample, there was only one 8 infant of exclusive breastfeeding age (<6 months) so it was not possible to analyze whether 9 there is a greater passage of antibodies to milk in this period compared to a longer postpartum 10 period. Moreover, with a larger sample, we could confirm the correlation between antibody 11 levels in serum and breast milk that we found and predict the levels in milk from a serum sample 12 determination. Another limitation is the lack of pre-vaccine antibody levels. However, we carried out serial 27 Acquisition, analysis, or interpretation of data: All authors. Drafting of the manuscript: Esteve-Palau, Casadevall, Diaz-Brito. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: Esteve-Palau, Casadevall. Supervision: Diaz-Brito. IgG(S1): immunoglobulin G-S1 subunit; AU/mL; arbitrary units/mL. 6 Time point (TP) 1: 2 weeks after first dose, TP2: 2 weeks after second dose, TP3: 4 weeks 7 after second dose, TP4: 12 weeks after second dose, TP4: 24 weeks after second dose. 8 Two patients were excluded for the 6-month analysis (time-point 5) due to they suffered a 9 SARS-CoV-2 Infection. Their values are highlighted with an asterisk. 10 11 Figure 3 . Correlation between immunoglobulin G-S1 subunit levels in serum and breast 12 milk of vaccinated participants. 13 IgG(S1): immunoglobulin G-S1 subunit; AU/mL; arbitrary units/mL. 15 Correlation between serum and breast milk SARS-CoV2 IgG-S1 levels overlaying patient-16 level regression lines. It can be seen that inter-patient variability, shown by the red 17 regression line, is parallel to intra-patient variability (dashed patient-level regression lines). 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