mRNA-1273 is placenta-permeable and immunogenic in the fetus

Abstract

COVID-19 mRNA vaccines are generally recognized as safe for gestational administration. However, their transplacental pharmacokinetics remain obscure. In this study, mRNA-1273 intramuscularly given to pregnant mice rapidly circulated in maternal blood and crossed the placenta within 1 h to spread in the fetal circulation. Although spike mRNA in fetal circulation faded away within 4-6 h, it could accumulate in fetal tissues, mainly the liver and get translated into spike protein. Transplacental mRNA-1273 proved immunogenic in the fetuses, as postnatally equipped with anti-spike immunoglobulin (Ig)M, paternal allotypic anti-spike IgG_2a, and heightened anti-spike cellular immunity. Gestationally administered, mRNA-1273 had a dose-dependent effect on its transplacental transfer and immunogenicity in the fetuses, with higher mRNA-1273 doses leading to increased transplacental mRNA-1273 passage and greater serum titers of endogenous anti-spike IgM/IgG generated by the fetuses. Thus, gestationally maternal mRNA-1273 vaccination might endow the newborns with not only passive but also active anti-spike immunity. Our results pose new insights into transplacental capacity of mRNA vaccines and their immunogenic potential in the fetuses, advancing our knowledge of mRNA medicine to protect the unborns against pathogens in perinatal life and broaden our horizons of prenatal mRNA molecular therapy.

Keywords: IgG allotype; MT: Oligonucleotides: Therapies and Applications; fetus; in utero immunization; lipid nanoparticle; mRNA vaccine; maternal vaccination; placenta; transplacental transfer.

Graphical abstract

Figure 1

Transplacental mRNA-1273 transfer after maternal mRNA-1273 vaccination during pregnancy (A) GD14 FVB/N mothers, intramuscularly (IM) vaccinated with a single-dose mRNA-1273 of 4.0 μg, were subjected to serum collection before vaccination (Pre), and at indicated time points of 0.5–3 h and 1–7 days after injection. Their pups were delivered for serum sampling at the same time points. Immunodot blot assay demonstrated transplacental PEGylated LNP transfer. (B) ELISA disclosed that fetal sera contained significantly higher PEG levels at the time points of 1 h, 3 h, and 6 h after maternal mRNA-1273 vaccination than those with maternal saline injection (control, ANOVA with least significant difference (LSD) multiple comparison). A significant decrease of serum PEG levels occurred between 3 h and 6 h. Although PEG remained measurable in certain pups of groups 1–3 d and 7–11 d, their mean levels did not differ from that of saline controls. On days 14–18, PEG was completely absent in all neonatal sera, identical to saline controls. (C) At the time points of 1 h and 3 h following maternal vaccination, 4.0 μg mRNA-1273 led to higher PEG levels in fetal sera than a dose of 0.2 μg. (D) Spike mRNA in fetal placenta, liver, and soft tissue was quantified by RT-PCR after maternal 4 μg mRNA-1273 vaccination (dams 234, 235, and 236 in Table S1). Spike mRNA levels of “(−)” and “< 0.021” were input as “0” and “0.021,” respectively in building this chart. Spike mRNA significantly dominated in fetal liver of groups 1, 4, and 6 h (ANOVA with LSD multiple comparison). (E) Immunostaining disclosed intracellular PEGylated LNPs and spike protein in fetal liver 6 h after maternal 4.0 μg mRNA-1273 vaccination. DIC: differential interference contrast. ZI: zoom-in. (F) At the time points of 1, 4, and 6 h after maternal mRNA-1273 vaccination, levels of spike mRNA in fetal placentas did not differ between 4.0 and 0.2 μg mRNA-1273 used to vaccinate the dams (Tables S1 and S2), whereas 4.0 μg mRNA-1273 led to significantly greater spike mRNA accumulation in fetal livers than 0.2 μg mRNA-1273. Error bar charts display the boxed areas of 95% confidence intervals for the means as box-crossing horizontal lines.

Figure 2

Anti-spike IgG₁/IgG_2a with virus-blocking efficacy in dams and their offspring after gestational mRNA-1273 administration Pregnant FVB/N mice were intramuscularly vaccinated by the same doses of mRNA-1273 (0.2, 1, 2, or 4 μg) on GD14 and GD17. (A) One month after delivery (M1), all four mRNA-1273 doses elicited significant levels of serum anti-spike IgG₁/IgG_2a in dams and (B) pups (p < 0.001, ANOVA), as compared with their saline controls. There were dose-responsive anti-spike IgG₁/IgG_2a levels in pups rather than dams (multiple comparisons by Fisher’s LSD post hoc test). The dams (n = 6) kept steady anti-spike IgG₁/IgG_2a titers in sera within postnatal 3 months (M1–M3) except for an initial drop of anti-spike IgG_2a levels (p = 0.010, pairwise comparison) at M2, whereas anti-spike IgG₁/IgG_2a in pups’ sera (n = 18) gradually faded away by M3–M4. The interconnected circles at different time points were the data of IgG₁/IgG_2a levels collected from an individual mouse. (C) Virus-blocking efficacy of maternal and offspring sera was evaluated by pseudovirus neutralization assays and shown in a representative mother and its offspring. Postnatal 2- and 3.5-month maternal sera had the neutralization titers of 2048- and 1024-fold dilutions, respectively, whereas neutralization activity of offspring sera was 64-fold at 2 months old but vanished by 3.5 months old. Error bar charts display the boxed areas of 95% confidence intervals for the means as box-crossing horizontal lines.

Figure 3

Analyses of anti-spike IgG_2a allotypes and anti-spike IgM in offspring born to the dams with gestational mRNA-1273 vaccination (A) After mRNA-1273 vaccination, BALB/c (Igh-1a) × C57BL/6 (Igh-1b) F1 mice (n = 9) significantly secreted anti-spike IgG_2a (Igh-1a/b) in sera within 2–4 weeks (pairwise comparison). Igh-1a haplotype dominated the allotypes of anti-spike IgG_2a. (B) C57BL/6 females (F) mated to BALB/c males (M) were vaccinated with 4 μg mRNA-1273 twice on GD14 and GD17. Both paternal Igh-1a and maternal Igh-1b allotypic anti-spike IgG_2a significantly showed up in BALB/c (M) × C57BL/6 (F) F1 mice at 4 weeks old (p < 0.001) despite undetectable paternal Igh-1a allotype by 8 weeks old (p = 0.508). (C) In the case of 0.2 μg mRNA-1273 vaccination in C57BL/6 pregnant mice, BALB/c × C57BL/6 F1 offspring (n = 17) did not compare favorably in serum anti-spike IgG_2a of Igh-1a (p = 0.418) with their saline controls (n = 5) but owned significantly higher levels of Igh-1b (p < 0.001) allotype than the controls by their age of 4 weeks. (D) After maternal vaccination with either 0.2 or 4.0 μg mRNA-1273 twice, offspring showed significantly heightened levels of serum anti-spike IgM by their age of 4 weeks, as compared with the controls with maternal saline injection. Besides, 4.0 μg mRNA-1273 given to the dams elicited higher serum titers of anti-spike IgM in offspring than 0.2 μg mRNA-1273 (p < 0.001). OD: optic density at 450 nm. Error bar charts display the boxed areas of 95% confidence intervals for the means as box-crossing horizontal lines.

Figure 4

Anti-spike cellular immunity in dams and their pups after maternal mRNA-1273 vaccination during pregnancy (A and B) After maternal vaccination with 4 μg mRNA-1273 twice, the dams and pups were examined for spike-specific lymphocyte proliferation by the readout of incorporated tritium in vitro. Splenic lymphocytes of both dams (p = 0.001) and pups (p < 0.001) proliferated specifically in response to spike, as opposed to those with maternal saline injection. Besides, the dams (p = 0.004) and pups (p < 0.001) with maternal mRNA-1273 vaccination were superior in spike-specific lymphocyte proliferation to their respective saline controls. Bovine serum albumin (BSA) was the third-party stimulators, and Con-A was a mitogen to non-specifically stimulate T cells. (C and D) IFN-γ- and IL-2 ELISpot images in triplicate shown were from a representative dam and pup with maternal mRNA-1273 (4 μg) or saline vaccination during pregnancy. Both groups exhibited heightened frequencies of IFN-γ- and IL-2-secreting T cells, as compared with their respective control counterparts. Error bar charts display the boxed areas of 95% confidence intervals for the means as box-crossing horizontal lines.

Figure 5

Immunological consequences of in utero mRNA-1273 injection GD14 FVB/N fetuses were subjected to intraperitoneal injection of mRNA-1273 (IU mRNA-1273, n = 19). (A and B) Postnatally, serum anti-spike IgG₁/IgG_2a was examined at the age of 1 month. IU mRNA-1273 led to significantly higher titers of anti-spike IgG₁/IgG_2a, as compared with in utero saline injection (IU saline, n = 9). Serum anti-spike IgG₁/IgG_2a gradually decreased within postnatal 3 months. Circles interconnected by a line represent IgG₁/IgG_2a levels measured at 1 (M1), 2 (M2), and 3 (M3) months old from an individual mouse (n = 11). (C) Lymphocyte proliferation in response to spike protein was measured by the readout of incorporated tritium (n = 4) as counts per minute (cpm). Medium only was used as background controls, BSA as third-party stimulators, and Con-A as a mitogen to stimulate the T cell population. IU mRNA-1273 significantly proliferated specifically in response to spike protein (p < 0.027), whereas IU saline (n = 4) failed to show lymphocyte proliferation under spike protein stimulation. There was a significant difference in lymphocyte proliferation under spike protein stimulation between IU mRNA-1273 and IU saline (p < 0.006). Rectangles within a dataset represent 95% confidence intervals for the means, which are shown as transverse lines crossing the rectangles. (D) Spike-reactive IFN-γ- and IL-2-secreting cells of splenic lymphocytes were enumerated by ELISpot. Figures showed the spots with their counts from the representative mice of IU mRNA-1273 and IU saline. The frequency of spike-reactive IFN-γ- and IL-2-secreting T cells was calculated by the mean of ELISpot readouts (triplicates) divided by the CD3⁺ cell ratio of splenic lymphocytes in each individual mouse.