Characterization of SARS-CoV-2 antibodies in human milk from 21 women with confirmed COVID-19 infection

Abstract

Background: One potential mechanism for protection from SARS-CoV-2 in children is through passive immunity via breast milk from a mother infected with the novel coronavirus. The primary objectives of this study were to establish the presence of SARS-CoV-2-specific IgA and IgG and to characterize the antigenic regions of SARS-CoV-2 proteins that were reactive with antibodies in breast milk.

Methods: Between March 2020 and September 2020, 21 women with confirmed SARS-CoV-2 infection were enrolled in Mommy's Milk. Participants donated serial breast milk samples around their time of illness. Breast milk samples were used to probe a multi-coronavirus protein microarray containing full-length and variable-length overlapping fragments of SARS-CoV-2 proteins. Samples were also tested against S and N proteins by electrochemiluminescence assay.

Results: The breast milk samples contained IgA reactive with a variety of SARS-CoV-2 antigens. The most IgA-reactive SARS-CoV-2 proteins were N (42.9% of women responded to ≥1 N fragment) and S proteins (23.9% responded to ≥1 fragment of S1 or S2). IgG responses were similar. A striking observation was the dissimilarity between mothers in antibody recognition, giving distinct antibody reactivity and kinetic profiles.

Conclusions: Individual COVID-19 cases had diverse and unique milk IgA profiles following the onset of symptoms.

Impact: In this observational longitudinal case series of 21 women with confirmed SARS-CoV-2 infection, IgA binding to SARS-CoV-2 proteins detected by orthologous proteome microarray and electrochemiluminescence assays was observed in >75% of women, but there was heterogeneity in which antigens and how many were reactive between women. Immunological profiles of protein regions recognized by each woman were distinct. Diverse repertoires of mucosal breast milk antibody to SARS-CoV-2 reflect heterogeneous passive transfer of maternal antibody to exposed breastfeeding infants.

Fig. 1. Reactivity of individual COVID-19 patient breast milk IgA to SARS-CoV-2 proteins.

a The circular graphic maps the amino acid (aa) position of SARS-CoV-2 fragments, showing a heatmap of antibody levels for each individual mother for overlapping regions of different aa length. Proteins are indicated outside the circle plot above an axis that shows aa positions from the N-terminus to C-terminus of each protein. The following line graph shows the sequence homology of other HCoVs with SARS-CoV-2 for each gene. The inner circular heatmap shows proteins and protein fragments produced in cell-free E. coli in vitro transcription and translation reactions by bars that represent length and position of each fragment in each protein. Full-length, 100 aa and 50 aa fragments are shown. Fragments of 30 aa size were mostly non-reactive and are not shown but are included in the full data sets (see Supplementary Data). Each fragment is drawn 21 times, once for each COVID-19 maternal patient ordered by subject ID number, and the colored bars show the normalized signal intensity (SI) of antibody binding to each fragment. Only one data point per subject, per fragment is shown, representing the maximum SI measured among all of their respective breast milk samples taken after onset of symptoms. IgG signal intensity is shown by color gradient (gray to red). Seroreactive regions of the proteins are highlighted by magenta outlines. The inner circle bands represent the responses to full-length purified recombinant S protein (shown crossing both S1 and S2 regions) and the receptor-binding domain (RBD) of S protein from the array. This is followed by responses acquired in the electrochemiluminescence assay (ECLIA) to the full-length S and N proteins, the N-terminal domain (NTD) of S protein and the RBD of S protein. b The zoomed cutout of the circle graphic includes additional labeling for clarity, including fragment length labels on the left of the heatmap and subject ID labels to the right.

Fig. 2. Heatmap depicting relative IgA antibody responses to SARS-CoV-2 as compared to other HCoVs and clinical data.

The heatmap presents the signals of antibody binding to individual proteins and protein fragments within the antigenic regions of SARS-CoV-2, as well as the full-length structural proteins of MERS-CoV, HCoV-NL63 and HCoV-OC43, for individual samples. Columns represent breast milk samples, and rows represent proteins or protein fragments: 26 SARS-CoV-2 proteins or fragments filtered for having a maximal normalized log2 signal intensity of at least 0.5 in one or more mother’s samples, and five proteins each of MERS-CoV, HCoV-OC43 and HCoV-NL63. Antibody signal intensity is shown on a color scale from gray to red. Log2 signal intensities from recombinant purified proteins on the array and log2 signal intensity from proteins assayed on the ECLIA platform are overlaid above the array cell-free expressed proteins and shown with independent gray-to-red color scales. Sample clinical information is overlaid above the heatmaps and includes categories at time of sampling for COVID-19 symptoms, number of symptoms, number of days symptomatic, baby’s symptoms (presence or absence of respiratory symptoms, or asymptomatic if testing positive for SARS-CoV-2 “+/Asymptomatic”), maternal age and baby age. Protein/fragment information is annotated to the left of the heatmaps and includes the virus, full-length protein name and the amino acid length of the protein fragments (“AA Length”, as full length, 100, 50, or 30 aa).

Fig. 3. Unique longitudinal profiles of mothers’ breast milk IgA response to SARS-CoV-2 show heterogeneity in antibody recognition.

a The line plots show breast milk IgA responses to SARS-CoV-2 and human common cold coronavirus selected full-length and protein fragments produced by the cell-free E. coli in vitro transcription and translation system. Antigens were selected to illustrate differences in reactivity profiles of four unique responders to SARS-CoV-2. The timing of breast milk sampling in days since onset of symptoms is shown on a free x-axis, and the normalized IgA signal intensity is shown in the y-axis. Each colored line represents an antigen’s IgA response measured for each of the longitudinal samples of one of the four mothers (each displayed in separate panels). aa amino acid, FL full-length, SCoV2 SARS-CoV-2, HCoV common cold human coronavirus. b The line plots show the log2 signal intensity of IgA responses to SARS-CoV-2 proteins assayed on the ECLIA platform (only SARS-CoV-2 proteins were assayed by ECLIA), where each panel is a unique mother’s responses to the four antigens. The number of days since onset of symptoms when samples were taken is shown on a free x-axis.