Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

Prenatal SARS-CoV-2 infection is associated with higher rates of pregnancy and birth complications, despite that vertical transmission rates are thought to be low. Here, multi-omics analyses of human placental tissues, cord tissues/plasma, and amniotic fluid from 23 COVID-19 mother-infant pairs revealed robust inflammatory responses in both maternal and fetal compartments. Pronounced expression of complement proteins (C1q, C3, C3b, C4, C5) and inflammatory cytokines (TNF, IL-1α, and IL-17A/E) was detected in the fetal compartment of COVID-19-affected pregnancies. While ~26% of fetal tissues were positive for SARS-CoV-2 RNA, more than 60% of fetal tissues contained SARS-CoV-2 ORF8 proteins, suggesting transplacental transfer of this viral accessory protein. ORF8-positive fetal compartments exhibited increased inflammation and complement activation compared to ORF8-negative COVID-19 pregnancies. In human placental trophoblasts in vitro, exogenous ORF8 exposure resulted in complement activation and inflammatory responses. Co-immunoprecipitation analysis demonstrated that ORF8 binds to C1q specifically by interacting with a 15-peptide region on ORF8 (C37-A51) and the globular domain of C1q subunit A. In conclusion, an ORF8-C1q-dependent complement activation pathway was identified in COVID-19-affected pregnancies, likely contributing to fetal inflammation independently of fetal virus exposure. Synopsis COVID-19 during pregnancy is associated with higher risk of maternal disease severity and/or death, characterized by a dysregulated inflammatory response when compared to non-pregnant individuals. Therefore, this work characterizes the maternal-fetal crosstalk that occurs during COVID-19-affected pregnancies through multi-omic analysis of placental and fetal tissues. Ultrasensitive droplet digital PCR detected vertical transmission of SARS-CoV-2 viral RNA in 26% of fetal samples from a COVID-19 pregnancy cohort. COVID-19-positive pregnancies had robust complement activation in fetal compartments with increase expression of C1q, C3, C4, C5 in the amnions and amniotic fluid. In >60% of COVID19 pregnancies, SARS-CoV-2 accessory protein ORF8 was detected in fetal compartments, including amniotic fluid, cord blood, and amnion. ORF8-positive COVID-19 fetal amnion tissues are associated with higher expression of inflammatory genes and complement factors. A 15-amino acid region of ORF8 binds specifically to the globular domain of C1qA to induce complement activation in