Biodistribution of mRNA COVID-19 vaccines in human breast milk

Abstract

Background: COVID-19 mRNA vaccines play a vital role in the fight against SARS-CoV-2 infection. However, lactating women have been largely excluded from most vaccine clinical trials. As a result, limited research has been conducted on the systemic distribution of vaccine mRNA during lactation and whether it is excreted in human breast milk (BM). Here, we evaluated if COVID-19 vaccine mRNA is detectable in BM after maternal vaccination and determined its potential translational activity.

Methods: We collected BM samples from 13 lactating, healthy, post-partum women before and after COVID-19 mRNA vaccination. Vaccine mRNA in whole BM and BM extracellular vesicles (EVs) was assayed using quantitative Droplet Digital PCR, and its integrity and translational activity were evaluated.

Findings: Of 13 lactating women receiving the vaccine (20 exposures), trace mRNA amounts were detected in 10 exposures up to 45 h post-vaccination. The mRNA was concentrated in the BM EVs; however, these EVs neither expressed SARS-COV-2 spike protein nor induced its expression in the HT-29 cell line. Linkage analysis suggests vaccine mRNA integrity was reduced to 12-25% in BM.

Interpretation: Our findings demonstrate that the COVID-19 vaccine mRNA is not confined to the injection site but spreads systemically and is packaged into BM EVs. However, as only trace quantities are present and a clear translational activity is absent, we believe breastfeeding post-vaccination is safe, especially 48 h after vaccination. Nevertheless, since the minimum mRNA vaccine dose to elicit an immune reaction in infants <6 months is unknown, a dialogue between a breastfeeding mother and her healthcare provider should address the benefit/risk considerations of breastfeeding in the first two days after maternal vaccination.

Funding: This study was supported by the Department of Pediatrics, NYU-Grossman Long Island School of Medicine.

Keywords: Biodistribution; Breast milk; COVID-19; Extracellular vesicles; Lipid nanoparticles; Vaccine mRNA.

Fig. 1

COVID Vaccine mRNA detected in breast milk (BM) measured by ddPCR. The heat map represents the vaccine mRNA concentrations in the BM. The X-axis represents the time points between vaccination and sample collection (hours/weeks). The Y axis represents different individual exposures (as detailed in Table 1) to BNT162b2 (upper panel) and mRNA1273 (lower panel). White cells indicate there were no samples collected in that time interval. Gray cells indicate vaccine mRNA was not detected. The amount of mRNA (copies/mL BM) in the sample is indicated by the color gradient.

Fig. 2

Vaccine mRNA-positive BM EVS did not induce spike protein expression when incubated with intestinal HT-29 cells for 24 h. HT-29 cells treated with vaccine mRNA1273 at different dilutions were used as positive controls (1:10⁴, 1:10⁶, and 1:10⁷, lanes 1–3, respectively). Lane 4 represents EVs from pre-vaccination BM. Lanes 5–7 represent EVs positive for the vaccine mRNA (lane 5, E5; lane 6, E7; lane 7, E17, respectively). Cells were lysed, and the expression of spike protein was assayed by automated capillary western blot (WES). S: Full-length spike protein. No S protein was detected in any of the BM EV samples tested. However, positive control samples in concentrations similar to those of BM EVs (lane 3) also failed to induce S protein expression. The only positive control sample that induced spike protein was the HT-29 cells treated with a higher concentration of stock mRNA vaccine (1:10⁴, lane 1).

Fig. 3

Integrity of vaccine mRNA in breast milk from vaccinated women.(a) Vaccine mRNA integrity was assayed in a duplex ddPCR assay using a two-primer set targeting 1602 nt of the mRNA1273 sequence. (b) Representative dot plot profiles of FAM-labeled mRNA1273-1 primer and probe (channel 1, amplitude) and HEX-labeled mRNA1273-2 primer and probe (channel 2, amplitude). Droplets emitting 2D signals were separated into four groups (Gray, double negative for mRNA1273-1 and mRNA1273-2; Blue, positive for mRNA1273-1, negative for mRNA1273-2; Green, positive for mRNA1273-2, negative for mRNA1273-1; Orange, double positive for both mRNA1273-1 and mRNA1273-2). Left panel, No template control; middle panel, RNA isolated from vaccine mRNA1273 stock (positive control); right panel, a representative BM sample from a vaccinated woman. (c) The number of droplets in each single or double positive group was derived by QX Manager Software. Percent linkage of each sample was expressed as the percentage of linked molecules in relation to the total molecules detected, normalized to the original vaccine stock solution.

Fig. 4

Cytokine levels in COVID-19 mRNA vaccines stimulated CBMCs and HT-29 cells. (a) Following 24 h stimulation with 1–10³ and 1–10⁶ diluted reconstituted vaccine product, cytokine concentrations in supernatant from CBMC (5 biological replicates) and HT-29 cells (n = 5) were measured by ELISA as described in Material and Methods. TLR agonists LPS, Poly IC, and R848 were used as positive control. Cytokine concentrations of CBMC (Upper panel) and HT-29 cells (Lower panel) are presented as box-and-whisker plot showing the median and IQR with minimum and maximum whiskers. p values were computed using repeated measure one-way ANOVA with posttest Holm-Šídák's multiple comparisons test (CBMC: TNFa, IFNa and HT-29: IL6, IFNa) or Friedman with posttest Dunn’s multiple comparisons (CBMC: IL6 and HT-29: TNFa). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (b) Representative sample of S protein expression in vaccine BNT162b2 treated CBMCs. 1) CBMCs received no treatment, 2) CBMCs treated with 1:10³ diluted BNT162b2, and 3) treated with 1:10⁶ diluted BNT162b2. S: full-length spike protein.

Fig. 5

Proposed model of biodistribution of vaccine mRNA to breast milk (BM). Following intramuscular administration, the vaccine mRNA enclosed in lipid nanoparticles (LNPs) is transported to the mammary glands through either hematogenous or lymphatic pathways. Within the mammary cell cytosol, a portion of the released vaccine mRNA is recruited and packaged into developing extracellular vesicles (EVs). The vaccine mRNA can be packaged into multivesicular bodies as intraluminal vesicles that will fuse with the mammary cell's plasma membrane, resulting in the release of mRNA-containing exosomes/EVs into breast milk. Some vaccine mRNA can also be packaged into microvesicles (MVs) formed by the outward budding of the mammary cell's plasma membrane and released into BM. This illustration was created with BioRender.com