Human Milk from Previously COVID-19-Infected Mothers: The Effect of Pasteurization on Specific Antibodies and Neutralization Capacity

Abstract

Background: Since the outbreak of coronavirus disease 2019 (COVID-19), many put their hopes in the rapid availability of effective immunizations. Human milk, containing antibodies against syndrome coronavirus 2 (SARS-CoV-2), may serve as means of protection through passive immunization. We aimed to determine the presence and pseudovirus neutralization capacity of SARS-CoV-2 specific IgA in human milk of mothers who recovered from COVID-19, and the effect of pasteurization on these antibodies.

Methods: This prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Human milk and serum samples were collected. To assess the presence of SARS-CoV-2 antibodies we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein (specific for IgA and IgG), receptor binding domain (RBD) and nucleocapsid (N) protein for IgG in serum, and bridging ELISA with the SARS-CoV-2 RBD and N protein for specific Ig (IgG, IgM and IgA in human milk and serum). To assess the effect of pasteurization, human milk was exposed to Holder (HoP) and High Pressure Pasteurization (HPP).

Results: Human milk contained abundant SARS-CoV-2 antibodies in 83% of the proven cases and in 67% of the suspected cases. Unpasteurized milk with and without these antibodies was found to be capable of neutralizing a pseudovirus of SARS-CoV-2 in (97% and 85% of the samples respectively). After pasteurization, total IgA antibody levels were affected by HoP, while SARS-CoV-2 specific antibody levels were affected by HPP. Pseudovirus neutralizing capacity of the human milk samples was only retained with the HPP approach. No correlation was observed between milk antibody levels and neutralization capacity.

Conclusions: Human milk from recovered COVID-19-infected mothers contains SARS-CoV-2 specific antibodies which maintained neutralization capacity after HPP. All together this may represent a safe and effective immunization strategy after HPP.

Keywords: COVID-19; breastfeeding; immunoglobulins; pasteurization.

Figure 1

Schematic representation of SARS-CoV-2 and the different ELISA assays used to detect SARS-CoV-2-reactive antibodies. The spike (including the receptor binding domain (RBD)) and nucleocapsid proteins of SARS-CoV-2 are depicted in the context of the virus. SARS-CoV-2 specific antibodies were detected using multiple complementary ELISA assays. The indirect ELISA assays using S, RBD or N were used to detect IgG or IgA specific-antibodies (green, blue or orange, respectively) and the bridging ELISA assay was used to detect total Ig against the RBD.

Figure 2

Multiple assay assessment of SARS-CoV-2 antibody levels in human milk and serum. Colors from blue to yellow indicate increasing levels of antibodies in milk and serum, relative to the cut-off values of the respective assays, with all levels over 2 times the cut-off being bright yellow, as indicated by the color scale. For the neutralization of the pseudovirus by milk (unpasteurized (UP), high pressure pasteurized (HPP) and holder pasteurized (HoP)) or serum, colors from blue to yellow indicate increasing neutralization capacity. Ab; total Ig. * multiplication sign.

Figure 3

Assessment of human milk antibody interaction with SARS-CoV-2 proteins in unpasteurized milk from the case and control groups. All box plots depict the interquartile range (IQR) as a median value with a lower 25th and upper 75th quartile range, and lower and upper whiskers to indicate the variability outside of the IQR. All cases are depicted in red and all controls are depicted in grey. *** indicates a p-value < 0.001 (a) OD450 nm SARS-CoV-2 Spike-IgA (p < 0.001), (b) OD450 nm SARS-CoV-2 RBD-Ab (p < 0.001) and (c) neutralization titers in unpasteurized milk of the cases and controls (p < 0.001).

Figure 4

LC-MS profiles of Fab fragments originating from IgA clones present in human milk. (A) Profile of IgA clones detected in the unpasteurized milk of Patient 1; (B) SARS-CoV-2 antigen-specific affinity purification yields specific clones (orange line; right y-axis), distinct from the depleted flow-through fraction (green line, left y-axis); (C) Overlay of the three profiles reveals the high specificity of a few antigen-specific clones.

Figure 5

Detection of antibody levels in human milk and serum relative to onset of COVID-19 symptoms. The OD_{450 nm} values for human milk spike protein IgA (blue dots) and serum IgG (red squares) levels from ELISA are plotted against the sampling time point in weeks after the onset of COVID-19 symptom.

Figure 6

Assessment the effects of pasteurization on human milk IgA levels. All figures are shown as box and whisker plots as median and IQR for human milk expressed as the percentage of treated relative to untreated human milk. ◆ are values outside the IQR. The * indicates a p-value < 0.05, the ** a p-value < 0.01. (a) IgA retention according to LC-MS profiles following HoP (n = 9) and HPP (n = 9) (p = 0.020) (b) Spike IgA titers following HoP (n = 38) and HPP (n = 38) (p = 0.006).

Figure 7

SARS-CoV-2 virus neutralization in unpasteurized and pasteurized human milk. The median and interquartile ranges of neutralizing activity against the pseudovirus, expressed as ln neutralizing capacity (50% inhibitory dilution), for unpasteurized (UP) milk, high pressure pasteurized (HPP) milk and Holder pasteurized (HoP) milk of PCR positive and suspected participants (n = 38). Neutralization capacity was generally preserved after the HPP pasteurization (p = 0.906) and not after HoP (p < 0.001) ***, relative to UP milk.