Introduction
The ongoing global pandemic of coronavirus disease 2019 (COVID-19) raised the questions about newborn infants’ protection against respiratory infection caused by severe acute respiratory coronavirus 2 (SARS-CoV-2). Due to the immaturity of the immune system, infants represent a unique at-risk population for which SARS-CoV-2 vaccines are not intended [
1]. The main source of newborns’ passive immunity is maternal milk [
2]. The benefits of breastfeeding are well established, therefore even in case of COVID-19 disease, the World Health Organization (WHO, 2020) strongly recommends that women should be encouraged and supported to breastfeed [
3]. Transmission of SARS-CoV-2 via breast milk is rare. A meta-analysis in 2020 showed that SARS-CoV-2 genome is generally not found in human milk of COVID-19 infected lactating women [
4]. Human milk, as previous studies reported, is a source of maternal SARS-CoV-2 antibodies, which may serve as protection against SARS-CoV-2 [
5‐
7]. However, research on immunoglobulins in human milk derived from women affected by COVID-19 during pregnancy are mostly limited due to small group size or insufficient information about milk collection and its analysis. The purpose of this study was to investigate the concentrations of SARS-CoV-2 IgG and IgA in human milk and serum and factors that may affect their levels. We performed Enzyme-Linked ImmunoSorbent Assay of blood serum and human milk from maternity patients who had recovered from COVID-19 at different trimesters of pregnancy and from mothers who had COVID-19 during the time of delivery.
Materials and methods
Study population
The study involved 72 women of which 54 had a history of COVID-19 at various trimesters of their pregnancy (11 1st trimester, 8 2nd trimester, 35 3rd trimester) and 18 women with an active COVID-19 during delivery. Women were patients at the Wroclaw Medical Teaching Hospital or were recruited via social media during the 3rd wave of coronavirus pandemic between 15 February to 1 May 2021. The inclusion criteria were as follows: PCR-confirmed coronavirus infection during pregnancy or delivery and lactation in the postpartum period. Seventeen women with no history of COVID-19 and any recent symptoms consistent with the infection constituted a control group. All women were seronegative for anti-SARS-CoV-2 antibodies. None of the study participants nor controls has been anti-SARS-CoV-2 vaccinated at the time of the study. All women gave written informed consent before study procedures were performed. Data regarding mothers and their infants were collected at the time of enrollment by filling the personal questionnaire.
Serum and human milk collection
Maternal blood (5 ml) and breast milk (10 – 15 ml) samples were collected on the same day. Whole blood samples were left to clot at room temperature (15 min.) and then centrifuged at 1000 x g for 15 minutes. Subsequent serum was aliquoted and frozen at − 80 °C. Human milk samples were collected:
(I)
at home and transported in insulated boxes up to 4 h to hospital (recruitment via social media)
(II)
or in the hospital (recruitment in hospital).
Serum and breast milk samples were collected 1-229 (median 68) days after the patients tested positive for COVID-19. All mothers were instructed to express milk with clean electric breast pumps into sterile plastic containers after feeding the baby. Then, received milk samples were separated into aliquots, frozen and stored at − 80 °C until the ELISA measurements.
Measurement of SARS-CoV-2-specific antibodies
The anti-SARS-CoV-2 immune response was assessed by determining the presence of anti-SARS-CoV-2 IgG and IgA antibodies in the maternal serum and breastmilk samples. The detection of antibodies against SARS-CoV-2 was based on the S1 domain of the spike protein including the immunologically relevant receptor binding domain (RBD) as an antigen. RBD represents important target antigen for virus neutralizing antibodies. The concentration of serum anti-SARS-CoV-2 IgG was measured using semi-quantitative enzyme linked immunosorbent assay (anti-SARS-CoV-2 IgG ELISA, #EI 2606-9601 G, Euroimmun, Germany). The concentration of breastmilk anti-SARS-CoV-2 IgG were measured using semi-quantitative enzyme linked immunosorbent assay (Euroimmun) and quantitative enzyme linked immunosorbent assay (anti-SARS-CoV-2 QuantiVac ELISA, #EI 2606-9601-10 G, Euroimmun, Germany). Serum samples were diluted 101 times as suggested by the manufacturer, while breastmilk samples were diluted 5 times. The breastmilk samples were vortexed for 3 minutes immediately before sample dilution. The presence of serum and breastmilk anti-SARS-CoV-2 IgA was measured using semi-quantitative ELISA (anti-SARS-CoV-2 IgA ELISA, #EI 2606-9601 A, Euroimmun, Germany). Serum samples were diluted 101 times as suggested by the manufacturer, while breastmilk samples were diluted 5 times. The breastmilk samples were vortexed for 3 minutes immediately before sample dilution. The quantitative test results of the measurement were converted to standardized binding antibody units (BAU/ml), which are in agreement with the First WHO International Standard Anti-SARS-CoV-2 Immunoglobulin (NIBSC 20/136). According to manufacturer guidelines, the results of semi-quantitative tests are presented as a ratio to the cut-off value, with the following interpretation of the results for the serum samples: < 0.8 interpreted as negative, ≥0.8 and < 1.1 borderline, and ≥ 1.1 positive. No cut-off values for breastmilk samples are available in the manufacturer guidelines. The measurements were performed on the coded samples, by researchers blinded to the information of the participant status. The participants were called seropositive, if they presented anti-SARS-CoV-2 IgG or IgA level defined positive according to manufacturer guidelines. If serum levels of both anti-SARS-CoV-2 IgG and IgA antibodies were below detection limit a participant was called seronegative.
Statistical analysis
The descriptive statistics of variables is presented as mean with standard deviation (SD) and median with interquartile range (IQR). The normality of the distribution was checked using the Shapiro-Wilk test. All presented comparisons and correlations included not normally distributed variables. Intergroup comparisons of continuous data was assessed using the non-parametric Mann-Whitney U test or Wilcoxon signed-rank test for dependent variables. The correlations were performed with using rang correlation (Spearman). Receiver operating characteristic (ROC) curve was used to define the cut-off value for breastmilk IgG and IgA level related to former COVID-19. The results with p < 0.05 were considered significant. Statistical analysis was performed using the STATISTICA 13 statistical package (TIBCO Software Inc. Palo Alto, CA, USA) and MedCalc (version 20, MedCalc Software).
Discussion
The paper describes a group of 72 mothers recovered from COVID-19 during the 3rd wave of pandemic. The study included women with a different time window between the COVID-19 infection and time of sampling. Therefore investigation over the antibody levels dynamics over time was possible. Human milk is a source of antigen-specific antibodies. IgA is the predominant antibody isotype in human milk (approximately 90%) nearly all in secretory (s) form (sIgA). These antibodies are responsible for conferring mucosal immunity and passive transfer during breastfeeding protecting infants from potential pathogens [
8,
9].
We have detected the presence of IgA antibodies against the virus in 62 out 72 patients (86.1%) in the breast milk samples. Low et al. in a systematic review from 2021 gathered the data of 161 lactating individuals with COVID-19 infection. According to this study human milk from 69 of 86 individuals (80.2%) contained SARS-CoV-2-specific IgA [
10]. Specific IgA to SARS-CoV-2 was found in 80% of samples in the study of Fox et al. (mostly being secretory IgA) [
5] in 97% of samples in the study of Demers-Mathieu et al .[
11] and in 76% of samples in Pace et al. study [
6]. In our study all sera samples (
n = 18) collected up to 7 days postpartum already contained specific IgA antibodies. Lebrão et al. demonstrate positive IgA at 3 and 6 days after delivery in one mother [
12]. In our study we also found level of antibodies in one individual persisting up to 8 months after the infection (229 days). However, median durability of human milk antibody production could not be reported due to heterogeneity of reporting. Based on systematic review by Low et al. [
10] the longest duration of antibody persistence in human milk reported from onset of COVID-19 infection until end of study was 195 days in a single individual [
13]. Duncombe et al. reported durable anti-RBD IgA antibodies in breast milk persisting over 6 months [
14] and recent study from Juncker et al. reported that IgA antibodies in human milk remain present at least 10 months after PCR confirmed infection [
15].
In our study anti-SARS-CoV-2 IgG antibodies were detected in 61 out of 72 (84.7%) breastmilk samples. The breastmilk samples obtained from women with formerly confirmed COVID-19 presented higher concentrations of SARS-CoV-2 IgG and IgA compared to controls (
p < 0.001). Moreover, the breastmilk samples obtained from seropositive study participants presented higher (
p < 0.001) concentration of anti-SARS-CoV-2 IgG and IgA compared to seronegative. In our study the concentrations of anti-SARS-CoV-2 IgA antibodies were consistently higher than those of IgG antibodies. Our findings are consistent with Pace et al. study where all samples of recently diagnosed women comprised IgA and IgG antibodies against the virus and their levels were higher than in milk collected from women before pandemic. Observed anti-RBD-IgA levels were also higher than IgG antibodies [
6]. In fact, it has been demonstrated that for SARS-CoV- 2, the majority of antibodies (60%) are IgA or secretory IgA, which may explain higher IgA levels [
16]. Moreover, only a small fraction of milk antibodies originates from serum- IgG comprise only 2% of milk Ab [
8].
Besides milk samples we managed to collect additionally 55 serum samples. Anti-SARS-CoV-2 IgG antibodies were detected in the sera of 81.1% mothers with former COVID-19 and anti-SARS-CoV-2 IgA antibodies in 75.7% samples. The serum of women with COVID-19 was characterized by a higher concentration of anti-RBD IgA and IgG than the serum from the control group without COVID-19 with statistical significance of
p < 0.001 for both antibodies. A statistically significant positive correlation was found between the values of IgG in milk and maternal serum (rs = 0.92,
p < 0.001) and of IgA in milk and maternal serum (rs = 0.67,
p < 0.001). Levels of SARS-CoV-2 IgA were higher in human milk than in serum samples, regarding SARS-CoV-2 IgG- their levels in serum were higher than in human milk. Our findings are in accordance with Dong et al. study, which showed that the serum SARS-CoV-2- specific IgG level was higher (292 to 2093 fold) than human milk SARS-CoV-2-specific IgG from one mother with confirmed SARS-CoV-2 infection [
17]. Demers-Mathieu et al. demonstrated differences between human milk and serum samples from COVID-19-recovered women. When the AUCs were not divided by the antibody concentration, SARS-CoV-2 RBD-specific IgA and IgG levels were higher in serum than the human milk. However, diving AUC by antibody concentrations demonstrated SARS-CoV-2 RBD-specific IgG (AUC/lgG) higher in human milk than in serum (
p < 0.05), whereas, SARS-CoV-2 RBD-specific IgA (AUC/mg of IgA) was higher in the serum than the human milk (
p < 0.01) [
16].
Neutralization capacity tends to correlate with RBD-specific antibody levels in various serological studies [
18]. Although our study did not evaluate the neutralization capacity of IgG and IgA, previous studies showed that human milk can neutralize SARS-CoV-2 in vitro [
6,
13,
19]. Samples from 20 of 48 individuals (41.7%) – data from systematic review were found to neutralize SARS-CoV-2 infectivity in vitro [
10]. Further investigations are warranted to evaluate protective capacity of human milk and whether lactating mothers who have recovered from COVID-19 transfer protective antibodies to their nursing infants. Regarding human milk banking neutralizing capacity of human milk was also evaluated after Holder pasteurization and high-pressure pasteurization. After Holder pasteurization SARS-CoV-2 IgA levels remained unchanged and the neutralizing function was significantly reduced or lost. Regarding high-pressure pasteurized-treated milk antibodies remain effective at neutralizing the virus [
20].
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