key: cord-0969938-0cihei1f authors: Selma-Royo, M.; Bauerl, C.; Mena-Tudela, D.; Aguilar-Camprubi, L.; Perez-Cano, F. J.; Parra-Llorca, A.; Lerin, C.; Martinez-Costa, C.; COLLADO, M. C. title: Anti-Sars-Cov-2 IgA And IgG In Human Milk After Vaccination Is Dependent On Vaccine Type And Previous Sars-Cov-2 Exposure: A Longitudinal Study. date: 2021-05-23 journal: nan DOI: 10.1101/2021.05.20.21257512 sha: 2e9606b1d5ae0277fe2f9b3bc43174e54b5ce07b doc_id: 969938 cord_uid: 0cihei1f Importance: Limited data are available on COVID-19 vaccine impact in lactating women. Objective: To evaluate the impact of different COVID-19 vaccines on specific anti-SARS-CoV-2 IgA and IgG levels in human milk. Design, Settings and Participants: In this prospective observational study in Spain, 75 lactating women from priority groups receiving vaccination against SARS-CoV-2 were included (January to April 2021). Human milk samples were collected at seven-time points. A group with confirmed SARS-CoV-2 infection (n=19) and a group of women from prepandemic time (n=13) were included. Exposure: mRNA vaccines (BNT162b2 and mRNA-1273) and adenovirus-vectored vaccine (ChAdOx1 nCoV-19). Main Outcome(s) and Measure(s): Presence of IgA and IgG against RBD SARS-CoV-2 in breast milk. Results: Seventy-five vaccinated lactating women [mean age, 34.9{+/-}3.7 years] of whom 96% were Caucasic and 92% were health care workers. A total of 417 milk samples were included and vaccine distribution was BioNTech/Pfizer (BNT162b2, n=30), Moderna (mRNA-1273, n=21), and AstraZeneca (ChAdOx1 nCoV-19, n=24). For each vaccine, 7 time points were collected from baseline up to 25 days after the 1st dose and same points were collected for mRNA vaccines 30 days after 2nd dose. A strong reactivity was observed for IgG and IgA after vaccination mainly after the 2nd dose. Presence and the persistence of specific SARS-CoV-2 antibodies in breast milk were dependent on the vaccine-type and, on previous virus exposure. High inter-variability was observed, being relevant for IgA antibodies. IgG levels were significantly higher than those observed in milk from COVID-19 women while IgA levels were lower. Women with previous COVID-19 increased the IgG levels after the 1st dose to a similar level observed in vaccinated women after the 2nd dose. Conclusions and Relevance: Breast milk from vaccinated women contains anti-SARS-CoV-2 IgA and IgG, with highest after the 2nd dose. Levels were dependent on vaccine type and previous exposure to SARS-CoV-2. Previous COVID-19 influenced the vaccine effect after a single dose, which could be especially relevant in the design of vaccination protocols . Further studies are warranted to demonstrate the potential protective role of these antibodies against COVID-19 in infants from vaccinated and infected mothers through breastfeeding. 1 Importance: Limited data are available on COVID-19 vaccine impact in lactating women. Presence and the persistence of specific SARS-CoV-2 antibodies in breast milk were 21 dependent on the vaccine-type and, on previous virus exposure. High inter-variability was 22 observed, being relevant for IgA antibodies. IgG levels were significantly higher than 23 those observed in milk from COVID-19 women while IgA levels were lower. Women 24 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2021. ; https://doi.org/10.1101/2021.05.20.21257512 doi: medRxiv preprint with previous COVID-19 increased the IgG levels after the 1 st dose to a similar level 1 observed in vaccinated women after the 2 nd dose. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Introduction 1 Breastfeeding is the most important postnatal link between mothers and infants, being the 2 best source of nutrition with effects for infant health and development 1 including the 3 maturation of the neonatal immune system, which is especially relevant in the context of CoV-2 infection. 23 We investigated whether maternal immunization with the available vaccines resulted in 24 secretion of antibodies directed against SARS-CoV-2 into breast milk and evaluated any 25 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 23, 2021. ; https://doi.org/10.1101/2021.05.20.21257512 doi: medRxiv preprint potential adverse events among women and their infants. Furthermore, we compared milk 1 antibody levels from vaccinated women to those found in naturally immunized women. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 23, 2021. ; https://doi.org/10.1101/2021.05.20.21257512 doi: medRxiv preprint issues, it is being evaluated for other target-population groups and is not provided 1 systematically to all people vaccinated with a first dose. 2 Furthermore, a group with confirmed SARS-CoV-2 infection (n=19) (ClinicalTrials.gov 3 Identifier: NCT04768244) from a previous study 14 and a control group of women not 4 exposed to SARS-CoV-2 from prepandemic time 15 (n=13) (ClinicalTrials.gov Identifier: 5 NCT03552939) were included. 17 Antibodies against the receptor-binding domain (RBD) SARS-CoV-2 S protein were 18 determined as previously described 14 . 1:4 diluted samples were incubated for 2 h in 1 % 19 (w/v) milk powder and SARS-Cov-2 antibodies were detected using anti-human IgA (α-20 chain-specific) HRP antibody (Thermo-Fisher Scientific; A18781; 1:6.000) and anti- CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2021. ; https://doi.org/10.1101/2021.05.20.21257512 doi: medRxiv preprint IgA, respectively. An arbitrary antibody concentration unit of 3000 was assigned to the 1 highest OD value corresponding to the undiluted sample. The standard curve corresponds 2 to eight 3-fold serial dilution from 3000 to 1.38 arbitrary units. In the case of IgG levels, 3 an additional standard curve using a monoclonal anti-SARS-CoV-2 IgG (Clone CR3022 4 produced in Nicotiana benthamiana, BEI Resources, NR-53876) was obtained to assess 5 the equivalence between the arbitrary units and IgG quantification (eFigure 1). Study population characteristics 20 The study included seventy-five lactating women receiving COVID-19 vaccination 21 (Table 1) , with age of 34.9±3.9 years (mean±SD) and infant age of 13.4±7.3 months 22 (mean±SD). No significant differences were observed for the vaccine-type groups in 23 clinical variables (Table 1) . Mothers vaccinated with the adenoviral-vectored vaccine 24 reported more side effects after vaccination compared to those reported with the mRNA-25 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2021. ; https://doi.org/10.1101/2021.05.20.21257512 doi: medRxiv preprint based vaccines including fever (p=0.0005) and headache (p<0.0001) (eTable 1). Four 1 infants developed fever after maternal vaccination. There were no serious adverse events 2 during the study period. Anti-SARS-CoV-2 antibody levels in milk according to vaccine type 23 We next compared the immunogenic response for each vaccine type. While all three 24 vaccines increased anti-SARS-CoV-2 IgG (Figure 2A) and IgA levels (Figure 2B) , CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Figures 2C and 2D) . Moreover, after the 2 nd dose, IgG levels reached higher 19 levels compared to the 1 st dose while IgA levels did not further increase. The subset of mothers with previous SARS-CoV-2 infection (n=8) showed higher IgG 23 and IgA levels at baseline when compared to the prepandemic group and participants 24 without infection (Figure 3) . Baseline anti-SARS-CoV-2 IgG levels in milk were similar 25 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2021. than those observed in non-vaccinated and infected women (SARS-CoV-2) and higher 1 than prepandemic group. IgG levels increased after the 1 st dose reaching values similar to 2 those observed after the 2 nd dose in participants without previous exposure to SARS-CoV-3 2 ( Figure 3A) . Baseline anti-SARS-CoV-2 IgA levels were lower compared to the We observed a robust secretion of SARS-CoV-2 specific IgGs in breast milk after the 2 nd 16 dose of vaccination, reaching higher levels than in naturally SARS-CoV-2 infected 17 women, suggesting a powerful effect of vaccination on SARS-CoV-2 IgG production. In 18 agreement with our data, a 2 nd mRNA vaccine dose increased the levels of anti-SARS- CoV-2 IgG but not IgA in both maternal blood and breast milk 10 . IgA secretion was evident as early as 2 weeks after vaccination followed by a peak after 21 4 weeks (a week after the 2 nd vaccine). Afterwards, levels of specific IgA against RBD CoV-2 IgG levels were higher than those found in the SARS-CoV-2 group, IgA was lower 24 compared to naturally infected women. Further studies with longer follow-up are needed 25 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2021. to track persistence of IgA specific antibodies. Indeed, we reported higher baseline signal 1 for anti-SARS-CoV-2 IgA than those found for IgG, which agrees with previous studies 2 14 . In this sense, a recent study reported higher S1 + S2-reactive IgA in breast milk from 3 women that had viral respiratory infection symptoms before the pandemic than in milk 4 from women without symptoms 21 . These data would partially explain the potential signal 5 or cross-reactivity in breast milk samples before 2020 21 . 6 While different studies have reported presence of specific IgA antibodies against SARS- Furthermore, although it has been suggested that antibodies found in breast milk would 23 exert strong neutralizing effects, no functional assays were performed. In addition, the 24 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2021. potential impact on neonatal growth as well as the potential protective effect against 1 infection in the infant remains elusive. infants from vaccinated and/or infected mothers and the differences in the vaccine-type. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 23, 2021. ; https://doi.org/10.1101/2021.05.20.21257512 doi: medRxiv preprint Padró-Arocas 2 A. 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