Minimal mRNA uptake and inflammatory response to COVID-19 mRNA vaccine exposure in human placental explants

Abstract

Despite universal recommendations for COVID-19 mRNA vaccination in pregnancy, uptake has been lower than desired. There have been limited studies of the direct impact of COVID-19 mRNA vaccine exposure in human placental tissue. Using a primary human villous explant model, we investigated the uptake of two common mRNA vaccines (BNT162b2 Pfizer-BioNTech or mRNA-1273 Moderna), and whether exposure altered villous cytokine responses. Explants derived from second or third trimester chorionic villi were incubated with vaccines at supraphysiologic concentrations and analyzed at two time points. We observed minimal uptake of mRNA vaccines in placental explants by in situ hybridization and quantitative RT-PCR. No specific or global cytokine response was elicited by either of the mRNA vaccines in multiplexed immunoassays. Our results suggest that the human placenta does not readily absorb the COVID-19 mRNA vaccines nor generate a significant inflammatory response after exposure.

Figure 1.. Detection of vaccine-derived Spike protein mRNA in human placental explants and four human cell lines using qRT-PCR.

(A) Vaccine messenger RNA (mRNA) standard curves. Standard curves for mRNA-1273 and BNT162b2 vaccines were generated to calculate vaccine-derived Spike mRNA concentrations in placental explants and cells. The limit of detection (LOD) level was set according to the standard curve (0.05 pg/μL, mRNA-1237 CT = 23.2, BNT162b2 CT = 24.6). (B) Vaccine mRNA detected in placental explants (villi) and four different types of cell lines exposed to mRNA-1273 or BNT162B2 at 0.1 μg/mL or 1 μg/mL for 0.5h or 4h. Results were calculated by extrapolating CT values based on the derived standard curves for each mRNA vaccine. Black dotted line represents the LOD level of 0.05 pg/μL. Grey box represents the area under LOD. All data represent 2 independent experiments. CT = Cycle threshold.

Figure 2.. Lack of detection of COVID mRNA vaccine via in situ hybridization in placental explants.

Chorionic villi explants derived from 2nd (n=2) or 3rd trimester (n=2) human placentas were incubated with 0.1 μg/mL (not shown) or 1 μg/mL mRNA-1237 or BNT162B2 vaccines. After 0.5h or 4h, tissues were fixed, paraffin-embedded, sectioned and probed for mRNA vaccine using RNAscope in situ hybridization. (A) Positive and (B) negative controls for RNAscope detection of mRNA vaccine. Peptidylprolyl isomerase B (PPIB) was used as a positive control. Pink dots corresponding to PPIB mRNA can be observed within the chorionic villi at 20X and 40X. DapB was used as a negative control. No signal was detected at 20 or 40X. (C) In situ detection of mRNA vaccine in vaccine-exposed explants. No signal was evident in explants incubated with either vaccine at any of the two timepoints.

Figure 3.. Expression of inflammatory mediators in conditioned media of placental explants incubated with COVID mRNA vaccine.

Chorionic villi explants derived from 2nd or 3rd trimester human placenta (n=6) were incubated with 0.1 μg/mL or 1 μg/mL mRNA-1237 (n=4) or BNT162B2 (n=2) vaccines for 0.5h or 4h. Conditioned media was collected and levels of 20 unique cytokines were measured using the Luminex multiplex assay. (A) Distribution in abundance of cytokines in all samples. Black bars represent median levels in cytokine abundance. (B) Hierarchical clustering of samples by levels of cytokines. We excluded cytokines of low variance from this analysis (n=6 cytokines). In general, sample profiles clustered by culture time (0.5 vs. 4h). (C) Significance of factors in cytokine expression evaluated using multiple linear regression models. Asterisks represent significance cutoffs: p<0.05 (*); p<0.005 (**); and p<0.0005 (***). Abbreviations listed for factors assessed in our analysis: mRNA-1237 (M), BNT162B2 (P) vaccines, culture time (Time), or trimester (Age).