Inefficient Placental Virus Replication and Absence of Neonatal Cell-Specific Immunity Upon Sars-CoV-2 Infection During Pregnancy

Abstract

Pregnant women have been carefully observed during the COVID-19 pandemic, as the pregnancy-specific immune adaptation is known to increase the risk for infections. Recent evidence indicates that even though most pregnant have a mild or asymptomatic course, a severe course of COVID-19 and a higher risk of progression to diseases have also been described, along with a heightened risk for pregnancy complications. Yet, vertical transmission of the virus is rare and the possibility of placental SARS-CoV-2 infection as a prerequisite for vertical transmission requires further studies. We here assessed the severity of COVID-19 and onset of neonatal infections in an observational study of women infected with SARS-CoV-2 during pregnancy. Our placental analyses showed a paucity of SARS-CoV-2 viral expression ex vivo in term placentae under acute infection. No viral placental expression was detectable in convalescent pregnant women. Inoculation of placental explants generated from placentas of non-infected women at birth with SARS-CoV-2 in vitro revealed inefficient SARS-CoV-2 replication in different types of placental tissues, which provides a rationale for the low ex vivo viral expression. We further detected specific SARS-CoV-2 T cell responses in pregnant women within a few days upon infection, which was undetectable in cord blood. Our present findings confirm that vertical transmission of SARS-CoV-2 is rare, likely due to the inefficient virus replication in placental tissues. Despite the predominantly benign course of infection in most mothers and negligible risk of vertical transmission, continuous vigilance on the consequences of COVID-19 during pregnancy is required, since the maternal immune activation in response to the SARS-CoV2 infection may have long-term consequences for children's health.

Keywords: SARS-CoV; T cell response; human trial; prenatal infection; variants of concern; vertical transfer.

Figure 1

Placental SARS-CoV-2 expression during or upon SARS-CoV-2 infection during pregnancy. SARS-CoV-2 SP and NP expression was assessed by IHC on placental specimen taken at delivery. Time point of infection and delivery is listed at the left side. The antigen targets of the antibodies used for respective IHC, SARS-CoV-2 specific SP or NP, are provided in the top row. Four different antibodies were used to stain for NP, which have been labelled as NP1 to NP4 in top row. One placenta from a non-infected woman was included as control. The third line from the top shows photomicrographs from a positive staining of the syncytiotrophoblast, detected in a placental specimen taken during acute infection (gestation week at infection is 39 + 1 and delivery occurred at 39 + 2). We here deliberately highlight the only positive staining of the sample, additional photomicrographs taken at lower magnification provide a more representative overview of positive staining of the same slide are provided in Supplementary Figure 1 . Bar inserted at bottom left represents 20 µm.

Figure 2

Viral expression in placental explants upon inoculation with human SARS-CoV-2 or CMV isolate. Photomicrographs of placental explants generated from the decidua basalis, the chorionic villi and the amniochorionic membranes were stained for SARS-CoV-2 or CMV expression respectively by IHC, as indicated on left. Inoculation of explants from different anatomical regions of the placenta with a human SARS-CoV-2 isolate did not yield to viral expression, whereas inoculation with CMV confirmed CMV-specific immediate-early antigen expression by IHC predominately in placental villi (bottom left). Bar represents 100 µm.

Figure 3

Low levels of viral RNA and viral load in explant supernatants of SARS-CoV-2 inoculated explants. (A) Levels of viral RNA, as indicated by Ct-values, was measured by PCR in explant supernatants of SARS-CoV-2 inoculated explants. (B) Detection of infectious virus, derived from SARS-CoV-2 plaque test using supernatants of SARS-CoV-2 infected placental explants harvested at 1, 3 and 5 days post infection (d.p.i.). Data are provided as plaque forming units (pfu). (C) Infectious virus assessment in supernatants of JEG-3 and BeWo placental cells at 24, 48, 72 and 96 hours post infection (h.p.i.). Lines in scatter plots show median and standard deviation.

Figure 4

SARS-CoV-2 cell-mediated immunity (CMI) in mother and neonate. Maternal and cord (CB) blood samples of six mother-baby pairs were analyzed for SARS-CoV-2-specific CMI applying an IFN-γ release assay. Blood samples were treated with stimulants as indicated and IFN-ɣ was measured in supernatants. (A) Absolute measurements (upper row) and calculated ratios to the negative control (lower row) are depicted as indicated. Red dots refer to the mother-baby pair from case #3, where CMI was tested two days after a positive viral RNA swab. (B) Cumulative data interpretation of all measurements applying criteria as outlined in the Material and Methods section. *, this individual’s positive control was negative after stimulation with PMA + ionomycin but positive after stimulation with SEB. Clinical details of the participants are listed in Supplementary Table 1 , please see the case IDs on left to idenitfy the cases in the table.