Maternal-Fetal Immune Responses in Pregnant Women Infected with SARS-CoV-2

Pregnant women are a high-risk population for severe/critical COVID-19 and mortality. However, the maternal-fetal immune responses initiated by SARS-CoV-2 infection, and whether this virus is detectable in the placenta, are still under investigation. Herein, we report that SARS-CoV-2 infection during pregnancy primarily induced specific maternal inflammatory responses in the circulation and at the maternal-fetal interface, the latter being governed by T cells and macrophages. SARS-CoV-2 infection during pregnancy was also associated with a cytokine response in the fetal circulation (i.e. umbilical cord blood) without compromising the cellular immune repertoire. Moreover, SARS-CoV-2 infection neither altered fetal cellular immune responses in the placenta nor induced elevated cord blood levels of IgM. Importantly, SARS-CoV-2 was not detected in the placental tissues, nor was the sterility of the placenta compromised by maternal viral infection. This study provides insight into the maternal-fetal immune responses triggered by SARS-CoV-2 and further emphasizes the rarity of placental infection.


INTRODUCTION
To date, over 65,000 pregnant women in the United States have been infected with 54 SARS-CoV-2 1 , the virus responsible for the coronavirus disease 2019 . During 55 pregnancy, SARS-CoV-2 infection can lead to variable outcomes, which range from 56 experiencing no symptoms to developing severe/critical disease 2,3 . Most pregnant women with 57 SARS-CoV-2 infection are asymptomatic or only experience mild symptoms 4,5 . Regardless, in 58 the first six months of the COVID-19 pandemic, it was documented that pregnant women with 59 SARS-CoV-2 were at an increased risk for hospitalization, mechanical ventilation, intensive care 60 unit admission, and preterm birth 2,3,6-8 , but rates of maternal mortality were reported to be similar 61 between pregnant and non-pregnant women 6 . More recently, it has been clearly shown that 62 pregnant women are at a high risk for severe/critical disease and mortality as well as preterm 63 birth 9-12 . Therefore, investigating host immune responses in pregnant women infected with 64 SARS-CoV-2, even if they are asymptomatic, is timely. 8 VEGF (1.56-log2 fold change), IL-5 (1.23-log2 fold change), and IL-8 (0.99-log2 fold change) 145 than those born to control mothers (Fig. 1C, Supplementary Fig. 2, Supplementary Table 2). 146 Such inflammatory changes in the neonates were not solely driven by the severe COVID-19 147 case. Based on an unsupervised analysis, the primary source of variability in the maternal and  between SARS-CoV-2 cases and controls. In general, fetal cell types were minimally altered by 195 the presence of SARS-CoV-2 infection in the mother (Fig. 4D&E). 196 The effects of SARS-CoV-2 on gene expression in maternal T cells from the CAM and 197 PVBP were compared to those from peripheral T cells from hospitalized COVID-19 patients 40 , 198 which we will refer to as the reference database hereafter. Maternal T-cell gene expression  with SARS-CoV-2 41 ) but not in the placental tissues from women with SARS-CoV-2 infection 238 ( Supplementary Fig. 6B&C). 239 Subsequently, we investigated the presence of viral RNA in the CAM, basal plate (BP), 240 and placental villi (PV) using RT-qPCR for the N1 and N2 viral genes ( Supplementary Fig. 7A).

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SARS-CoV-2 N1 and N2 proteins were not detected in any of the placental samples from women 242 with SARS-CoV-2 infection or healthy controls ( Supplementary Fig. 7B). Yet, in the spike-in 243 positive control, N1 and N2 RNA was detected in the CAM, BP, and PV. A sensitivity assay 244 revealed that 10 is the minimum confident copy number of viral particles detectable in the 245 placental villi using RT-qPCR ( Supplementary Fig. 7C).

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Next, we determined whether the spike and nucleocapsid proteins were detected in the 247 placental tissues of women with SARS-CoV-2 infection using immunohistochemistry (Fig. 6A).

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Several histological slides from the CAM, BP, and PV were included in our evaluation, 249 including negative and spike-in positive controls (Supplementary Table 4 were also included. None of the placentas from women with SARS-CoV-2 infection or controls 260 had detectable levels of N1 and N2 RNA viral genes; yet, the spike-in positive controls were 261 detected (Fig. 6F).

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Collectively, these data show that SARS-CoV-2 is not detected in the placental tissues, 263 including the chorioamniotic membranes, of women infected with SARS-CoV-2.  profiles of the placental tissues, we did not find evidence that the same is true for maternal 282 SARS-CoV-2 infection.

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This study provides evidence that, in a largely asymptomatic population, SARS-CoV-2 285 infection in pregnancy is primarily associated with maternal inflammatory responses in the 286 circulation and at the maternal-fetal interface. First, we showed that pregnant women with IgM cannot cross the placenta due to its large molecular weight, and thus the detection of this 298 immunoglobulin in the cord blood represents an acute fetal response in the clinical setting 47,48 .

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Therefore, the absence of detectable IgM in the cord blood suggests that vertical transmission in 300 utero of SARS-CoV-2 was unlikely to occur in our study population.

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In the current study, we report that pregnant women mount a mild systemic inflammatory 302 response to SARS-CoV-2, which is consistent with observations in asymptomatic non-pregnant 303 individuals with SARS-CoV-2 infection 49 . Interestingly, we found that neonates born to SARS-      de-multiplexing. The fastq files were then aligned using kallisto 122 , and bustools 123 summarized 563 the cell/gene transcript counts in a matrix for each sample using the "lamanno" workflow for 564 scRNAseq. Each library was then processed using DIEM 124 to eliminate debris and empty 565 droplets. In parallel, "cellranger counts" was also used to align the scRNAseq reads using the 566 STAR 125 aligner to produce the bam files necessary for demultiplexing the individual of origin, 567 28 based on the genotype information using souporcell 126 and demuxlet 127 . We removed any 568 droplet/GEM barcode that was assigned to doublet or ambiguous cells in demuxlet or souporcell, 569 and only those cells that could be assigned a pregnancy case and maternal/fetal origin were kept.

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All count data matrices were then normalized and combined using the "NormalizeData,"  the FDR method 135 . The functions "enrichPathway", "enrichKEGG", and "gseGO" from 616 "clusterProfiler" were used to perform the ORA and GSEA analyses separately for each list of 617 genes obtained as differentially expressed for each cell type, placental compartment, and 618 maternal/fetal origin. Only results that were significant after correction were reported with q < 619 0.05 being considered statistically significant.  Reactions were incubated at 25°C for 2 min followed by 50°C for 15 mins. Initial denaturation 657 was set for 2 min at 95°C followed by 45 amplification cycles at 95°C for 3 sec and 55°C for 30         were both calculated from the control group. The Z-scores were visualized as a heat map and 820 compared between SARS-CoV-2 (+) and control groups using two-sample t-tests. P-values were 821 adjusted for multiple comparisons using the false discovery rate method to obtain q-values. A q-822 value < 0.1 was considered statistically significant. The principal components (PC) of the flow 823 cytometry data were also determined, and PC1, PC2, and PC3 were plotted on a 3D scatter plot.