To investigate the longitudinal antibody response against SARS-CoV-2 in human milk after COVID-19 booster vaccination, we conducted a prospective cohort follow-up study of previously fully vaccinated individuals who participated in our original vaccination study10.
To assess whether the human milk antibody response against COVID-19 differs after homologous and heterologous booster vaccination, participants were divided into two cohorts based on the type of vaccine they received for their primary vaccination series, and human milk samples from both groups were collected longitudinally following booster vaccination.
This study was conducted in accordance with the Declaration of Helsinki and the ICH GCP Guidelines and complied with the Regulation on Medical Research Involving Human Subjects. The study protocol was approved by the Ethics Committee of the Amsterdam University Medical Center. We have obtained written informed consent from all participants.
Population and setting
The current study is a follow-up of our previous vaccination study10. All lactating individuals in the Netherlands receiving a vaccination against COVID-19 were eligible to participate. Participants of the initial vaccination study who were still lactating and were going to receive a booster dose of a COVID-19 vaccine could register to participate in the current prospective follow-up study.
Recruitment of participants for the original study was done through several social media platforms and registration for participation in the current follow-up study was requested from participants who had consented to be contacted for possible further research.
The first study group consisted of participants who received an mRNA-based vaccine, namely BNT162b2 or mRNA-1273, both for their primary vaccination series and for their booster vaccination (homologous booster vaccination group). The second study group consisted of lactating women who completed a primary series of an adenoviral vector-based COVID-19 vaccine which included either two doses of the AZD1222/Vaxzevria vaccine or a single dose of the Ad26.COV2.S vaccine, and for their booster vaccination, they received an mRNA-based vaccine (heterologous booster vaccination group).
Over a period of 16 days, participants collected eight human milk samples: one sample before and one sample on days 3, 5, 7, 9, 11, 13, and 15 after booster vaccination. All samples were collected in a standardized way. Participants were instructed to empty one breast completely before the first feeding moment, either manually or with an electric breast pump, mix the milk (so that fore- and hindmilk were mixed), and subsequently store 5 ml in the freezer until collection by one of the researchers during the home visit at the end of the follow-up period. Human milk samples were collected in sterile polypropylene tubes. During transport to the study site, milk samples were placed on dry ice and at the study site, samples were stored at −80 °C until analysis.
Antibody analysis in human milk
SARS-CoV-2-specific IgA and IgG antibodies in human milk were determined using an enzyme-linked immunosorbent assay with the SARS-CoV-2 spike protein. A more detailed description was also reported in our previous study19 as follows:
Soluble prefusion-stabilized S-protein of SARS-CoV-2 was generated. This protein was immobilized on a 96-well plate (Greiner, Kremsmünster, Austria) at 5 µg/mL in 0.1 M NaHCO3 overnight, followed by a 1-h blocking step with 1% casein PBS (Thermo Scientific, Waltham, MA, USA). Human milk was diluted 1:5 in 1% casein PBS and incubated on the S-protein coated plates for 2 h to allow binding. Antibody binding was measured in the human milk samples using 1:3000 diluted HRP-labeled goat anti-human IgG (Jackson Immunoresearch, West Grove, PA, USA) and 1:3000 diluted HRP-labeled goat anti-human IgA (Biolegend, San Diego, CA, USA) in casein. Healthy controls were used (of our previous study) to determine cutoff values defined as the mean plus two times the standard deviation. Sensitivity was 68% for IgA and 96% for IgG in human milk. Specificity was 99% for both IgA and IgG in human milk. All samples of human milk were analyzed as one batch at the same time. Milk samples were considered positive at an optical density 450-nm cutoff value of 0.5 for IgA in human milk (sensitivity, 68%; specificity, 99%) and 0.2 for IgG in human milk (sensitivity, 96%; specificity, 99%) . All biologically independent samples were assayed in duplicate.
Statistics and reproducibility
Characteristics of participants and their infants were compared between study groups, IBM SPSS Statistics for macOS (Version 29) was used. Discrete variables are denoted by the observed number and percentages within a study group, and continuous variables are presented by mean and standard deviation after testing for normality.
Human milk antibody dynamics following COVID-19 vaccination are displayed using Graphpad Prism 9.1.0 for macOS. To assess the total antibody response after booster vaccination, we determined the area under the curve with respect to ground (AUCG), with respect to increase from mean pre-booster level (AUCI), and the area under the curve with respect to the cutoff: both the area of positive peaks (AUCPP) and the net area which subtracts the area of peaks below the cutoff (AUCNet), as described in ref. 20. The AUCs were calculated for the human milk antibody response up to 15 days after the administration of the booster dose. The mean AUCs with standard errors of the mean (SEM) and degrees of freedom (df) for both study groups were compared using an unpaired-sampled t-test, with a significance level set at a p value <0.05. GraphPad Prism version 9.1.0 for macOS was used to determine and compare the AUCs.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.