COVID-19 Vaccine mRNA Biodistribution: Maternal and Fetal Exposure Risks

Abstract

SARS-CoV-2 infection during pregnancy has severe consequences on maternal and neonatal health. Presently, vaccination stands as a critical preventive measure for mitigating infection-related risks. Although the initial clinical trials for the COVID-19 vaccines excluded pregnant women, subsequent investigations have indicated mRNA vaccinations’ effectiveness and short-term safety during pregnancy. However, there is a lack of information regarding the potential biodistribution of the vaccine mRNA during pregnancy and lactation. Recent findings indicate that COVID-19 vaccine mRNA has been detected in breast milk, suggesting that its presence is not confined to the injection site and raises the possibility of similar distribution to the placenta and the fetus. Furthermore, the potential effects and responses of the placenta and fetus to the vaccine mRNA are still unknown. While potential risks might exist with the exposure of the placenta and fetus to the COVID-19 mRNA vaccine, the application of mRNA therapies for maternal and fetal conditions offers a groundbreaking prospect. Future research should leverage the unique opportunity provided by the first-ever application of mRNA vaccines in humans to understand their biodistribution and impact on the placenta and fetus in pregnant women. Such insights could substantially advance the development of safer and more effective future mRNA-based therapies during pregnancy.

1 |. Introduction

Messenger RNA (mRNA) has revolutionized the field of therapeutics with applications in vaccines, cancer immunotherapies, and genome editing. Since their debut in 1995, mRNA vaccines have undergone rapid development, presenting a novel approach to immunization [1]. Following mRNA vaccine administration, the engineered mRNA strands are transported to cells for translation into a target protein. The proteasome subsequently degrades the protein into peptides presented on the cell surface, initiating an immune response that allows the body to develop immunity against the antigen [1].

As COVID-19 remains a substantial worldwide concern for public health, it is crucial to understand the safety and efficacy of vaccines for all populations, including pregnant women [2]. The Pfizer and Moderna COVID-19 mRNA vaccines, which contain mRNA encoding the full-length SARS-CoV-2 spike protein encapsulated in lipid nanoparticles (LNPs), have been widely adopted to reduce the morbidity and mortality of SARS-CoV-2 infection [3]. However, pregnant women were excluded from the initial clinical trials [4]. Currently, the Center for Disease Control and Prevention (CDC) recommends mRNA-based COVID-19 vaccination for pregnant or to be pregnant women [5]. However, many pregnant mothers are hesitant to receive the COVID-19 vaccine, citing long-term safety concerns for their newborns [6].

This review aims to highlight the current knowledge regarding the potential biodistribution of mRNA vaccines in pregnant mothers, examining whether vaccine mRNA can traverse the placenta and cross the placental barrier to reach the fetus. Furthermore, we explore the potential implications of the transplacental passage of vaccine mRNA to the fetus. This review will highlight knowledge gaps and future research directions to guide the development of future mRNA therapies administered during pregnancy.

2 |. Biodistribution of COVID-19 mRNA Vaccines

The efficacy of the mRNA vaccine depends on the delivery system since naked mRNA is degraded in the systemic circulation [7]. Nanoparticles (NPs) are nonviral delivery systems that prevent mRNA degradation and facilitate cellular uptake [7]. LNPs and other NPs differ in composition.

LNPs consist mainly of lipids, while other NPs can be composed of various materials such as metals, polymers, and ceramics, which influences their degradation and biodistribution.

Currently, the two major COVID-19 vaccines, Pfizer BNT162b2 and Moderna mRNA-1273 are both LNP-encapsulated mRNA vaccines that encode the SARS-CoV-2 spike glycoprotein [8, 9]. LNPs’ in vivo distribution and potency depend on characteristics such as size, shape, charge, chemical composition, and targeting ligands [10, 11]. Furthermore, when NPs interact with physiologic environments, they are rapidly coated with a layer of biomolecules, mainly proteins adsorbing to the surfaces. This protein-rich layer formed around NPs is called the “protein corona,” which can significantly influence the NPs’ biodistribution, mRNA release, and potential toxicity [12–15]. Identifying key parameters that control the NPs’ transfer to a target organ will significantly reduce their off-target effects [11], which are of particular concern during pregnancy. Few studies have explored the biodistribution of mRNA vaccines in humans [16–18] and examined the impact of the LNP characteristics and protein corona on their biodistribution and mRNA release [12–15].

At present, relatively little has been reported on the tissue localization of the LNPs after intramuscular (IM) administration of the mRNA vaccine [19]. Upon IM injection, the LNP-encapsulated mRNA vaccine causes local inflammation that recruits neutrophils and antigen-presenting cells, which have the ability to take up the LNP and migrate to local draining lymph nodes [18]. However, the assumption that the mRNA vaccine is rapidly broken down at its administration site with no biodistribution to other organs has been challenged [19–21]. In mice, the biodistribution of the LNPs after IM administration demonstrated transportation and active translation of the vaccine mRNA in the liver, heart, kidney, lung, and spleen for up to 96 h post-IM administration [22, 23]. The Pfizer Assessment Report provided to the European Medicines Agency (EMA) [18] acknowledged that no secondary pharmacodynamics studies had been conducted with the BNT162b2 vaccine in humans. The report showed that in rats, a fraction of the administered mRNA vaccine was distributed to the liver, adrenal glands, spleen, and ovaries. The Moderna Assessment Report provided to the EMA showed similar results [17]. However, in both reports, the biodistribution to the placenta was not included in these experiments.

In humans, vaccine mRNAs have been detected in various sites other than the inoculation location. Krauson et al. reported that vaccine mRNA was present in axillary lymph node and myocardium in autopsies samples 30 days post-vaccination [24]. Further, the vaccine mRNA has been found in germinal centers in lymph nodes for up to 8 weeks postimmunization [25]. It was also demonstrated that the vaccine mRNA can remain in circulation for at least 2 weeks following injection, with most mRNA found in the plasma, which implies free circulation rather than transportation by neutrophils or other immune cells [26]. Since free mRNA cannot escape degradation by serum proteins, it is likely that the free mRNA is traveling within the LNP capsule [26], suggesting that unprocessed LNP-mRNA can circulate freely in the bloodstream. Vaccine mRNA may also be transported via extracellular vesicles (EVs), including exosomes. Circulating exosomes have been shown to carry the SARS-CoV-2 spike protein 14 days post-vaccination [27]. It has also been found that mice immunized with exosomes carrying spike proteins from vaccinated individuals also developed SARS-CoV-2 spike protein-specific antibodies [27]. Exosomes may also have the potential to carry vaccine mRNA encoding the spike protein, which allows the vaccine mRNA to circulate in the bloodstream [28]. Along this line, our published studies demonstrated that the COVID-19 vaccine mRNA is detected in human breast milk EVs in the first 48 h after maternal vaccination, suggesting biodistribution from the injection site to distant organs [21]. The ability of vaccine mRNA to travel to different areas of the body may have significant implications for pregnant mothers.

3 |. Potential Biodistribution of the Vaccine mRNA to the Placenta

It has been previously shown in animal studies that the IM administration of the vaccine can be detected in many organs, including the liver. Since the placenta shares many features with the liver, such as high blood flow and fenestrated endothelia [29], it is likely that the placenta may also be a major target of LNP-mRNA delivery. In a mouse study, intravenously injected inorganic NPs were accumulated in the placenta and mice embryos [30], demonstrating that NPs can travel to the placenta. These results suggest that LNP-mRNAs have the potential to reach the placenta, given their similar sizes to inorganic NPs. The transfer of NPs across the placenta was dependent on their size, shape, surface composition, and charge [11]. Fluorescent polystyrene NPs up to a size of 240 nm can be taken up by the placenta and cross the placental barrier [31]. However, coating NPs with polyethylene glycol (PEG) decreases the transfer of the NPs across the placenta [30]. PEGylated gold NPs of 10–30 nm can be endocytosed into trophoblastic cells of the placenta, but the NPs cannot cross the placenta into the fetal circulation [32]. This suggests that PEGylation of LNPs may prevent transfer across the placenta. However, the composition of LNPs consists of ionizable lipids, phospholipid distearoylphosphatidylcholine, and cholesterol in addition to PEG [33], making them more lipophilic than inorganic NPs. This suggests that despite PEGylation, LNPs could potentially cross the placenta. Recent research into how LNPs are delivered to the placenta indicates that the delivery efficiency is determined by the molar ratios of each lipid component within the LNPs rather than their size [34]. However, the biodistribution of LNPs to the human placenta is uncertain due to limited human data and the selective nature of the placental barrier. Additional research is necessary to confirm the systemic distribution of LNPs to the placenta.

Another potential means of vaccine mRNA traveling to and crossing the placental barrier is via EVs, as previously demonstrated in breast milk EVs [21, 35]. Once LNPs are endocytosed by cells, they are subsequently acidified when the endosome fuses with the lysosome [36]. Acidification of LNPs allows for the release of mRNA into the cytoplasm [36]. Once released into the cytoplasm, the mRNA can be translated into the target protein (e.g., SARS-CoV-2 spike protein) and subsequently displayed on the surface of APCs, triggering an immune response. However, it has also been found that the mRNA and other components of LNPs can be secreted in EVs [28]. When EVs are secreted from the cell of origin and enter the extracellular space, they can influence neighboring cells or travel in body fluids to modulate distal cells [37]. In pregnancy, EVs facilitate bidirectional intercellular communication between the fetus and mother [38], regulating maternal physiology and fetal growth. Maternal uterine cells also have the ability to secrete EVs that maintain homeostasis during pregnancy [39]. Given that vaccine mRNA can travel in EVs, it is possible that maternal-fetal transfer of vaccine mRNA may occur across the placenta through EVs.

Three studies with human cohorts have explored the presence of the COVID-19 mRNA vaccine in the placenta after maternal vaccination, yielding mixed results, possibly due to methodological limitations. One study failed to detect vaccine mRNA in the placenta using in situ hybridization (ISH) [40], likely because it used a probe designed for SARS-CoV-2 RNA, not the specific vaccine sequence (which are not identical), making it less likely to detect vaccine mRNA. Another study using qRT-PCR did not detect the vaccine mRNA in maternal blood, cord blood, or placental tissue [41]. However, in most samples, there was a long interval between vaccination and birth, along with the use of a mismatched primer set [41]. A follow-up study by the same group also failed to detect the vaccine mRNA using RNAscope-based ISH assay [42]. However, a mismatch probe targeting the SARS-CoV-2 S gene was used rather than the vaccine mRNA sequence. In our recent publications [21, 35, 43], we employed sensitive techniques to detect the vaccine mRNA, including using two primer sets covering ~1.5 kb of the full-length mRNA vaccine to enhance detection sensitivity. Furthermore, we utilized ddPCR for more precise quantification, offering superior accuracy and sensitivity over qRT-PCR in detecting SARS-CoV-2 [44]. For the RNAscope-based ISH assay, we used a probe tailored explicitly for the vaccine mRNA, thus ensuring more reliable detection. Using these improved methodologies, we demonstrated vaccine mRNA biodistribution to the placenta in two mothers who received the vaccine days before birth [43]. Our findings indicate that the vaccine mRNA can distribute systemically to the placenta and umbilical cord blood. Additionally, our study demonstrated the presence of vaccine mRNA in the placental villus stroma and the decidua using ISH, and the spike protein was detected in placental tissue using Western blot analysis [43]. The detection of spike protein in placental tissue indicates the bioactivity of the vaccine mRNA that reached the placenta. However, the degree of biological activity and possible transcriptomic effect(s) of the vaccine mRNA in the placenta needs further research.

4 |. Potential Vaccine mRNA Crossing the Placental-Fetal Barrier

Although the systemic distribution of LNPs after IM administration is likely, including to the placenta, the transfer of LNPs across the placental-fetal barrier is unclear. It is possible that the mRNA vaccine can be transferred to the fetus via the vaccine LNPs-mRNA crossing the placental-fetal barrier or via vaccine mRNA packaged in placental EVs. To our knowledge, this has not been explored in the human placenta. Furthermore, whether the mRNA biodistributed to the placenta/fetus is intact and biologically active is unclear. One animal study injected LNP-mRNA with a similar composition to the COVID-19 mRNA vaccines into the vitelline vein. Functional mRNA was demonstrated to be delivered to the fetal liver, lung, and intestines [45]. Similarly, mRNA was shown to be functional after being delivered to target cells carried in exosomes [46]. Thus, vaccine mRNA may have a biological effect on the fetus if transferred to the fetus via either LNP-mRNA crossing the placental barrier or vaccine mRNA-carrying exosomes. In murine models, exosomes injected intraperitoneally into pregnant females led to functional changes in the fetus, demonstrating that maternal EVs can be transported across the placenta to the fetus and exert a biological effect [38]. In humans, evidence suggests that the placental EVs can cross the placental-fetal barrier, enter fetal circulation, and induce the cord blood immune cells [47–49]. In a significant study [28], mice treated with LNPs containing hEPO mRNA showed that part of the hEPO mRNA escaped into the cytoplasm, was packed into exosomes, and secreted to distant cells. We observed similar mechanisms with COVID-19 vaccine mRNA crossing into human breast milk [21, 35], suggesting the possibility of placental EVs carrying vaccine mRNA to the fetus. It was also demonstrated that LNPs are processed via the endosomal pathway and subsequently secreted in exosomes rather than directly fusing with exosomes extracellularly [28]. These secreted exosomes protected the exogenous mRNA during in vivo delivery to produce hEPO protein in other organs [28]. Of note, embryotoxicity can be observed for certain nanoparticles despite no detected fetal absorption [50, 51]. Several studies have reported fetotoxicity despite undetectable nanomaterial translocation across the placenta, which prompted the hypothesis that indirect pathways may play a role in embryotoxicity [51]. Data from a recent human study evaluating the effects of maternal COVID-19 vaccination on the hematopoietic progenitor cells in the umbilical cord blood suggested that LNPs/mRNA vaccines may be reaching the fetus of a vaccinated mother [52]. In our recent publication [43], a mother who received the vaccine 2 days before delivery had the vaccine mRNA detected in the cord blood in significant concentrations. However, it was highly fragmented, which might explain the lack of spike IgM antibody detection in cord blood after maternal vaccination in several studies [53, 54].

Understanding the mechanisms through which mRNA vaccines might cross the placental-fetal barrier is essential for evaluating the potential impact of future mRNA therapies on the fetus. Further research is needed to elucidate the specific pathways and factors that may play a role in the transplacental passage of vaccine mRNA. Furthermore, understanding the role of gestational age is critical for evaluating the safety of mRNA vaccines during pregnancy. Early gestation poses unique vulnerabilities due to organogenesis, while mid to late gestation offers a more permeable placental barrier that facilitates antibody transfer but also raises concerns about potential fetal exposure to vaccine components. Future research should continue to investigate the biodistribution and effects of mRNA vaccines at different stages of pregnancy to optimize vaccination strategies and ensure the safety of both the mother and the fetus. By considering the gestational age in vaccine administration, we can better tailor vaccination recommendations and improve maternal and fetal health outcomes during the ongoing pandemic and beyond.

5 |. The Potential Implications of the mRNA Vaccine Crossing the Placental Barrier

Although many studies demonstrated the efficacy and short-term safety of the mRNA vaccination during pregnancy, to our knowledge, few studies investigated the placental potential transcriptomic responses to the vaccine, including expression of the Spike (S) protein. One study demonstrated that the mRNA vaccines promote the sustained synthesis of the S protein, suppressing type I interferon responses and impairing innate immunity [55]. S protein has been hypothesized to have a role in the pathogenesis of Long-COVID [56–59] and long-term post-COVID vaccine injury [60, 61]. Biodistribution studies have found significant expression of S protein in tissues and organs up to 2 months post-vaccine administration [25, 27, 62]. In addition, several open reading frames (ORFs) overlap the SARS-CoV-2 spike protein sequence [63]. The presence of multiple ORFs in a single mRNA vaccine molecule can lead to the translation of different proteins from a single gene (alternative translation). Consequently, the existence of these ORFs within SARS-CoV-2 mRNA vaccines might result in the synthesis of unwanted proteins during the vaccination process. This could potentially trigger alterations in the placental transcriptome, leading to possible unintended clinical outcomes [64]. Although the clinical significance of these potential proteins is unclear, this could explain some of the observed reactions seen with vaccine administration in some cases.

6 |. The Risks and Benefits of Immunization in Comparison to Consequences of Infection

In order to assess the safety of the vaccine during pregnancy, it is essential to compare the potential risks and benefits of vaccination to the risks associated with maternal SARS-COV-2 infection during pregnancy. COVID-19 during pregnancy is linked to significant adverse outcomes such as preterm birth, low birth weight, and stillbirth [65]. Additionally, the virus’s primary receptor, ACE2, is present in many cells at the maternal-fetal interface, raising concerns about the possibility of the virus transmitting from mother to fetus [66]. The long-term effects of maternal COVID-19 disease on fetal growth and development are still uncertain, necessitating ongoing research to fully understand the impact on the child’s health and overall pregnancy outcomes.

COVID-19 vaccination during pregnancy provides numerous benefits, as evidenced by scientific and medical research. The administration of mRNA-based COVID-19 vaccines induces a robust immune response in the mother, producing specific antibodies. Vaccinated pregnant women show decreased rates of preterm births, stillbirths, and the need for neonatal intensive care [67, 68]. These antibodies not only protect the mother but also cross the placental barrier, offering passive immunity to the fetus against COVID-19 [69]. Importantly, no increased risks of neonatal complications have been associated with these vaccines [70], highlighting their role in protecting both maternal and fetal health during pregnancy.

While considering COVID-19 vaccination during pregnancy, it is crucial to acknowledge potential risks based on scientific observations. Emerging evidence highlights the occurrence of post-COVID-19-vaccine syndrome in specific vaccinated individuals. This syndrome could involve various complications, such as damage to the endothelial lining of blood vessels, cardiovascular issues (like microthrombus formation due to ACE2 receptor activation on platelets and myocarditis), and neurological symptoms (including small fiber neuropathy) [61]. However, current data suggest that these adverse effects are rare and generally outweighed by the benefits of vaccination. Furthermore, studies indicate that pregnant women do not face higher risks of complications from the COVID-19 vaccine compared to non-pregnant individuals [2, 71, 72]. While mRNA vaccines have not been shown to cause teratogenic effects [4], the lack of long-term data on mRNA vaccines in pregnancy makes it difficult to predict the potential consequences, especially the potential impact on the fetal immune cells if, indeed, it can cross the placental fetal barrier. Overall, while there may be some potential risks associated with the transplacental passage of vaccine mRNA, the current knowledge suggests that maternal vaccination’s benefits outweigh these speculative risks.

7 |. Addressing the Knowledge Gaps

Despite the increasing evidence supporting the safety and efficacy of COVID-19 mRNA vaccines for pregnant women and their fetuses, several limitations and knowledge gaps persist in our current understanding. While understanding the implications of the COVID-19 mRNA vaccine in pregnant women provides valuable foundational insights, we acknowledge that future advancements in mRNA delivery vehicles and specific mRNA construction may influence outcomes. However, the fundamental principles of mRNA biodistribution, safety, and immunogenicity established through research will remain relevant. A multifaceted research approach is essential to gain comprehensive insights into the effects of COVID-19 mRNA vaccination during pregnancy. Longitudinal studies involving pregnant women who have received mRNA vaccines and subsequently tracking the health of both the mothers and their offspring over an extended period hold paramount importance. These rigorous investigations serve as a means to uncover any potential long-term consequences of maternal mRNA vaccination. In parallel, integrating animal models into the research framework is an indispensable strategy. By leveraging these models, the complex mechanisms underlying mRNA vaccine biodistribution and action during pregnancy can be dissected and comprehended. Moreover, a pressing need for exploration lies in further probing the dynamics of placental transfer and the possible fetal exposure to extra-genomic mRNA. Addressing these gaps in knowledge is instrumental in evaluating the potential hazards and benefits of maternal vaccination, facilitating informed decision-making by healthcare professionals and expectant mothers.

The significance of this knowledge extends beyond the scope of COVID-19 mRNA vaccines. mRNA therapies are an emerging technology with broad application not only to vaccines but a host of pathologies amenable to gene therapy, including cancer immunotherapies and genome editing. Given that the COVID-19 mRNA vaccine is the only mRNA vaccine that has been administered to humans thus far, the biodistribution of the COVID-19 mRNA vaccine given during pregnancy can be used as a model to provide valuable insights into the transport and potential biological effects of vaccine mRNA on the intrauterine environment. This can be relevant for assessing the safety and efficacy of future mRNA-based treatments administered to pregnant women.

8 |. Conclusions

COVID-19 immunization has proven to be an effective tool in combating the SARS-CoV-2 pandemic. Numerous studies indicate that vaccination not only protects the mother but also the fetus through passive immunity [69, 73]. However, it is critical to understand the biodistribution of mRNA vaccines and their potential effect on gestational tissues, as well as the possible introduction of extra-genomic mRNA during fetal growth and development. Although data indicates that mRNA vaccination during pregnancy is safe, it is crucial for pregnant women to engage in informed discussions with their healthcare providers regarding the benefits and potential risks associated with maternal mRNA vaccination.

The global acceptance and usage of mRNA vaccines have not only helped in controlling COVID-19 disease but also opened avenues for advancing other mRNA-based treatments [74–77]. Although the perinatal application of gene therapy shows promise, our understanding of mRNA delivery during pregnancy is still developing [45]. Despite potential unknown risks associated with mRNA exposure during pregnancy, the opportunity to treat both maternal and fetal conditions with mRNA therapies represents a significant breakthrough. Further research is necessary to enhance our understanding of how LNPs are distributed, modified, and interact at the cellular level within the body. This knowledge will be crucial in refining the development of LNPs to ensure they are safe and effective for use in pregnant women.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.