The secretory IgA (sIgA) response in human milk against the SARS-CoV-2 Spike is highly durable and neutralizing for at least 1 year of lactation post-infection

Abstract

Although in the early pandemic period, COVID-19 pathology among young children and infants was typically less severe compared to that observed among adults, this has not remained entirely consistent as SARS-CoV-2 variants have emerged. There is an enormous body of evidence demonstrating the benefits of human milk antibodies (Abs) in protecting infants against a wide range of enteric and respiratory infections. It is highly plausible that the same holds true for protection against SARS-CoV-2, as this virus infects cells of the gastrointestinal and respiratory mucosae. Understanding the durability of a human milk Ab response over time after infection is critical. Previously, we examined the Abs present in milk of those recently infected with SARS-CoV-2, and concluded that the response was secretory IgA (sIgA)-dominant and that these titers were highly correlated with neutralization potency. The present study aimed to monitor the durability of the SARS-CoV-2 IgA and secretory Ab (sAb) response in milk from COVID-19-recovered lactating individuals over 12 months, in the absence of vaccination or re-infection. This analysis revealed a robust and durable Spike-specific milk sIgA response, that at 9-12 months after infection, 88% of the samples exhibited titers above the positive cutoff for IgA and 94% were above cutoff for sAb. Fifty percent of participants exhibited less than a 2-fold reduction of Spike-specific IgA through 12 months. A strong significant positive correlation between IgA and sAb against Spike persisted throughout the study period. Nucleocapsid-specific Abs were also assessed, which revealed significant background or cross reactivity of milk IgA against this immunogen, as well as limited/inconsistent durability compared to Spike titers. These data suggests that lactating individuals are likely to continue producing Spike-specific Abs in their milk for 1 year or more, which may provide critical passive immunity to infants against SARS-CoV-2 throughout the lactation period.

Figure 1.. Longitudinal human milk IgA and sAb reactivity against SARS-CoV-2 Spike demonstrates significant durability over time.

SARS-CoV-2 Ab levels were measured via optimized Luminex assay as described in methods. (A-B) Titration curves of IgA and sAb 3-8 weeks post-infection. A) IgA, B) sAb. (C-D) Individual endpoint titers over time. C) IgA, D) sAb. Dotted lines: positive cutoff value. (E-F) Grouped endpoint titers with mean values and ANOVA analysis for change over time. E) IgA, F) sAb. Dotted lines: positive cutoff value. (G-H) Relative endpoint titers over time compared to 3-8 weeks post-infection. G) IgA; H) sAb. Dotted line indicates relative titer of 1, meaning no change. PI: post-infection.

Figure 2.. Longitudinal human milk IgA and sAb against SARS-CoV-2 Nucleocapsid are maintained less consistently compared to Spike Abs and subject to significant background reactivity.

(A-C) Titration curves of IgA and sAb 3-8 weeks post-infection. A) IgA; B) sAb, C) high background IgA of COVID-19-naïve milk hinders determination of a positive cutoff value. MFI at 1/18 dilution (screening dilution) are shown. (D-E) Individual endpoint titers over time. D) IgA, E) sAb. (F-G) Grouped endpoint titers with mean values and ANOVA analysis for change over time. F) IgA, G) sAb. Relative endpoint titers over time compared to 3-8 weeks post-infection. H) IgA, I) sAb. Dotted line indicates relative titer of 1, meaning no change. PI: post-infection.

Figure 3.. Spike-specific human milk IgA and sAb remain highly correlated throughout the study period, while Nucleocapsid Abs exhibit moderate correlation and Spike vs Nucleocapsid Ab reactivities are unrelated.

A) Spike IgA vs sAb 3-8 weeks post-infection. B) Nucleocapsid IgA vs. sAb 3-8 weeks post-infection. C) Correlation between Spike IgA and sAb at the end of the study period. D) Spike vs Nucleocapsid IgA 3-8 weeks post infection. E) Spike vs Nucleocapsid sAb 3-8 weeks post infection. Endpoint titers were compared by Spearman correlation analysis.

Figure 4.. SARS-CoV-2-neutralizing Ab in human milk is maintained for at least 9 months after infection.

A proxy neutralization assay that measures Abs specific for the Spike receptor binding domain (RBD) that block the RBD-ACE2 interaction was performed using a premade kit according to instructions (Genscript). Dotted line: background neutralization level of COVID-19-naïve milk. Paired t-test was used for analysis. PI: post-infection.