Immune and pathophysiologic profiling of antenatal coronavirus disease 2019 in the GIFT cohort: A Singaporean case-control study

Abstract

COVID-19 can be severe in pregnant women, and have adverse consequences for the subsequent infant. We profiled the post-infectious immune responses in maternal and child blood as well as breast milk in terms of antibody and cytokine expression and performed histopathological studies on placentae obtained from mothers convalescent from antenatal COVID-19. Seventeen mother-child dyads (8 cases of antenatal COVID-19 and 9 healthy unrelated controls; 34 individuals in total) were recruited to the Gestational Immunity For Transfer (GIFT) study. Maternal and infant blood, and breast milk samples were collected over the first year of life. All samples were analyzed for IgG and IgA against whole SARS-CoV-2 spike protein, the spike receptor-binding domain (RBD), and previously reported immunodominant epitopes, as well as cytokine levels. The placentae were examined microscopically. The study is registered at clinicaltrials.gov under the identifier NCT04802278. We found high levels of virus-specific IgG in convalescent mothers and similarly elevated titers in newborn children. Thus, antenatal SARS-CoV-2 infection led to high plasma titers of virus-specific antibodies in infants postnatally. However, this waned within 3-6 months of life. Virus neutralization by plasma was not uniformly achieved, and the presence of antibodies targeting known immunodominant epitopes did not assure neutralization. Virus-specific IgA levels were variable among convalescent individuals' sera and breast milk. Antibody transfer ratios and the decay of transplacentally transferred virus-specific antibodies in neonatal circulation resembled that for other pathogens. Convalescent mothers showed signs of chronic inflammation marked by persistently elevated IL17RA levels in their blood. Four placentae presented signs of acute inflammation, particularly in the subchorionic region, marked by neutrophil infiltration even though > 50 days had elapsed between virus clearance and delivery. Administration of a single dose of BNT162b2 mRNA vaccine to mothers convalescent from antenatal COVID-19 increased virus-specific IgG and IgA titers in breast milk, highlighting the importance of receiving the vaccine even after natural infection with the added benefit of enhanced passive immunity.

Keywords: GIFT; SARS-CoV-2; antenatal COVID-19; breast milk antibodies; placental inflammation; transplacental antibody transfer.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Gu, Low, Tan, Ng, Ng, Shunmuganathan, Gupta, MacAry, Amin, Lee, Lian, Shek, Zhong and Wang.

Figures

FIGURE 1

Neutrophilic infiltration present in the placentae of convalescent mothers. Microscopic analysis shows a neutrophilic infiltrate in the subchorionic region of the placentae of three patients—(A) patient CS2, H&E × 100, (B) patient CS3, H&E ×100, (C) patient CS7, H&E × 100. (D) Higher power view of the inflammatory process (patient CS3, H&E × 200).

FIGURE 2

Spike and RBD-specific IgG and IgA present in maternal plasma of convalescent mothers. Using a protein-based ELISA, maternal plasma from control (CT01- CT10) and convalescent (CS01-CS07) mothers obtained 1-month post-partum were used for the detection of spike (A,C) and RBD (B,D) specific IgG and IgA antibodies. The bar graphs represent the normalized average signals of antibodies binding to the respective proteins. Convalescent mothers (CS samples) are arranged in ascending order according to their time from COVID diagnosis to delivery.

FIGURE 3

Neutralization of SARS-CoV-2 pseudovirus using maternal plasma. Plasma from control (n = 10) and convalescent mothers (n = 7). Bar graphs represent the average percentage neutralization. Convalescent mothers (CS samples) are arranged in ascending order according to their time from COVID diagnosis to delivery.

FIGURE 4

Spike and RBD-specific IgG are passively transferred from convalescent mothers to infants. (A) Spike and (B) RBD-specific IgG from maternal plasma 1 month post-partum and at three time points from babies’ plasma was determined using ELISA. Transfer ratio at the 1 month timepoint of (C) spike and (D) RBD-specific IgG from mother to infant was calculated by taking the averaged IgG signal of baby’s over the mother’s IgG. Convalescent mothers (CS samples) are arranged in ascending order according to their time from COVID diagnosis to delivery.

FIGURE 5

Spike and RBD-specific IgA present in maternal breast milk of convalescent mothers. Using a protein-based ELISA, maternal breast milk from control (CT01- CT10) and convalescent (CS01-CS08) mothers obtained at two time points 0–1 months and 2–3 months post-partum were used for the detection of spike and RBD-specific IgG and IgA antibodies. Breast milk was screened for (A) spike-specific IgG, (B) RBD-specific IgG, (C) spike-specific IgA, and (D) RBD-specific IgA. The bar graphs represent the normalized average signals of antibodies binding to the respective proteins. Convalescent mothers (CS samples) are arranged in ascending order according to their time from COVID diagnosis to delivery.

FIGURE 6

Cytokine profile differences in healthy and convalescent mothers’ plasma. Using 1-month post-partum plasma (n = 6 for controls and n = 7 for cases), cytokines were evaluated using the 41-plex Luminex™ assay. (A) A representation of all cytokines that were detected, (B) IL-17RA levels and (C) MCP-1. Convalescent mothers are represented in red while healthy controls are represented in black. Bar graphs represent the average log(concentration) from the samples from the respective groups in A while the measured concentrations were used for (B,C). *p < 0.05.

FIGURE 7

Increased secretion and production of spike-specific IgA and IgG antibodies post-vaccination in convalescent mothers. Breast milk from three nursing convalescent mothers were collected at 4 time points, prior to vaccination (0), 3 days post-vaccination, 7 days post-vaccination, and 30 days post-vaccination for a (A,C) spike and (B,D) RBD protein-based IgA and IgG ELISA assay. The x-axis refers to the number of days post-vaccination.