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Shedding Study Spike & mRNA


Infectious Diseases Research 2022;3(4):22.

Submit a manuscript: 1

Current state of knowledge on the excretion of mRNA and spike produced by

anti-COVID-19 mRNA vaccines; possibility of contamination of the entourage of

those vaccinated by these products

Helene Banoun1*

1Pharmacist biologist, Former Inserm researcher, Member of the Independent Scientific Council, Marseille 13000, France.

*Corresponding to: Helene Banoun, Pharmacist biologist, Former Inserm researcher, Member of the Independent Scientific Council, Marseille 13000, France.


Competing interests

The author declares no conflicts of interest.


This research received no specific grant from any funding

agency in the public, commercial, or not-for-profit sectors.


LNPs, lipid nanoparticles; MMR, measles/mumps/rubella; EVs,

extracellular vesicles; VEGF, vascular endothelial growth

factor; pDNA, plasmid DNA; IM, intramuscular; VLP, virus like

particles; RBD, receptor binding domain (spike).


Banoun H. Current state of knowledge on the excretion of

mRNA and spike produced by anti-COVID-19 mRNA vaccines;

possibility of contamination of the entourage of those

vaccinated by these products. Infect Dis Res. 2022;3(4):22. doi:


Executive editor: Na Liu.

Received: 11 October 2022; Accepted: 07 November 2022;

Available online: 14 November 2022.

© 2022 By Author(s). Published by TMR Publishing Group

Limited. This is an open access article under the CC-BY license.



The massive COVID-19 vaccination campaign is the first time that mRNA vaccines have

been used on a global scale. The mRNA vaccines correspond exactly to the definition of gene

therapy of the American and European regulatory agencies. The regulations require

excretion studies of these drugs and their products (the translated proteins). These studies

have not been done for mRNA vaccines (nor for adenovirus vaccines). There are numerous

reports of symptoms and pathologies identical to the adverse effects of mRNA vaccines in

unvaccinated persons in contact with freshly vaccinated persons. It is therefore important to

review the state of knowledge on the possible excretion of vaccine nanoparticles as well as

mRNA and its product, the spike protein.

Vaccine mRNA-carrying lipid nanoparticles spread after injection throughout the body

according to available animal studies and vaccine mRNA (naked or in nanoparticles or in

natural exosomes) is found in the bloodstream as well as vaccine spike in free form or

encapsulated in exosomes (shown in human studies). Lipid nanoparticles (or their natural

equivalent, exosomes or extracellular vesicles (EVs)) have been shown to be able to be

excreted through body fluids (sweat, sputum, breast milk) and to pass the transplacental

barrier. These EVs are also able to penetrate by inhalation and through the skin (healthy or

injured) as well as orally through breast milk (and why not during sexual intercourse

through semen, as this has not been studied). It is urgent to enforce the legislation on gene

therapy that applies to mRNA vaccines and to carry out studies on this subject while the

generalization of mRNA vaccines is being considered.

Keywords: COVID-19 vaccine; vaccine shedding; COVID vaccine adverse effects; Lipid

nanoparticles; LNPs; mRNA vaccine; exosome; exosome excretion route; gene therapy; spike

protein; LNPs excretion routes; exosomes penetration


Infectious Diseases Research 2022;3(4):22.

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Why are we interested in this hypothesis, which may seem


The expression “vaccine shedding” classically refers to the possible

excretion of a virus by a person who has been freshly vaccinated

against that virus; this is valid only for live attenuated virus vaccines

(measles/mumps/rubella (MMR), chickenpox, rotavirus, nasal spray


No COVID-19 vaccine uses this formula. Therefore, there is no risk

that a vaccine recipient will transmit a vaccine virus. However,

mRNA-based COVID-19 vaccines are the first to be used commercially

in humans on a global scale and no studies have been conducted

regarding the possible excretion of the vaccine itself (lipid

nanoparticles containing mRNA) of the vaccine mRNA or of the

vaccine product, the spike protein translated by the cells of the


The COVID vaccination started in December 2020. The first

published testimony of vaccine shedding that I saw dates from

December 2021 and is that of Dr Ray Sahelian [1]: he reported cases

of medical or scientific colleagues who had observed symptoms close

to those of the adverse effects of the vaccine after having been in

contact with freshly vaccinated persons; he proposed an excretion of

the products of the vaccine by the skin and the respiratory tract and

asked for complementary studies.

At the beginning, this type of testimony did not seem very credible

to me, but they accumulated and in October 2021, I received a

testimony from a group of French caregivers: they observed a stroke in

a 7-year-old child with no risk factors and whose parents had been

freshly vaccinated. There are Telegram groups listing testimonies from

patients and doctors. All of these testimonials report symptoms or

conditions reported in the COVD-19 vaccine adverse event databases :

the adverse effects of mRNA vaccines against COVID-19 are now

recognized by regulatory agencies (see VAERS and Eudravigilance

databases, as well as the ANSM, France).

The vaccines are all based on the spike protein, which has since

been recognized as the main responsible for the pathogenicity of

SARS-CoV-2 [2-6]. Therefore, in the event that the vaccine or its

product (the spike) passes from vaccinated to unvaccinated, the

adverse effects of the vaccine should be found in some unvaccinated

people in contact with vaccinated people. The exploration of

vaccine-related pathologies in non-vaccinated age groups in contact

with vaccinated people could give indications in the sense of vaccine

shedding, but it does not give significant results (unpublished). As

there are more than 400 pathologies related to adverse vaccine

reactions in the pharmacovigilance reporting databases (see for

example, the UK data, spontaneous notification data for Pfizer vaccine

in May 2021 [7]), this large number dilutes the signals that could

appear in non-vaccinated age groups.

On the other hand, an analysis of European, Israeli and US data

shows that for the non-vaccinated 0-14 age group, most of the

associations between mortality and vaccination in adults are positive:

the excess mortality in non-vaccinated age groups when vaccination

campaigns begin could be explained by a transmission phenomenon of

the vaccine or its products. This pattern of positive correlations

increases from the week of vaccination to week 18 after vaccination

and then disappears. It indicates indirect negative effects of adult

vaccination on mortality in children aged 0-14 years during the first

18 weeks after vaccination [8].

What is the biological plausibility of transmission of the vaccine

or its products from vaccinated to unvaccinated?

To answer this question, we need to explore the possibility and routes

of excretion of the vaccine or its products and the routes of their

possible penetration.

Concerning the vaccine and its products, it may be the transmission

of the circulating spike in the vaccinated (in free form or included in

exosomes or EVs), the transmission of circulating naked mRNA or

complete lipid nanoparticles (LNPs).

Therefore, the ability of LNPs, mRNA and vaccine spike to be

excreted by different possible routes and then the ability of the same

products to enter by different routes into the body of unvaccinated in

close contact with vaccinated should be explored.

The excretion of mRNA-containing LNPs, the excretion of modified

spike-encoding mRNA, and the excretion of spike produced by

vaccinees have not been studied in the trial phase of the vaccines,

contrary to the recommendations of the regulators concerning gene

therapies. Pharmacokinetic studies of nanoparticles in general have

not explored the excretion of the transporters or the transported

molecules. This area should be explored.

Pfizer documents obtained by FOIA [9] show that only the excretion

of some components of the LNPs (ALC-0315 and ALC-0159) was

studied in the urine and feces of IM injected rats.

Regulations regarding the excretion of gene therapies by regulatory


There was no regulation of mRNA clinical trials prior to RNA vaccines,

yet there is strict regulation of gene therapy products. It is difficult to

justify that mRNA vaccines are not considered in the same way as

gene therapies regarding this regulation; indeed the only difference is

that they are supposed to protect against a disease and not cure it.

Gene therapies are intended for a small number of people in poor

health, whereas vaccines are used on a large scale on healthy people:

it would therefore be wise to apply stricter rules to them. However,

the description of gene therapy products provided by the regulatory

agencies does include mRNA and adenovirus vaccines.

The 2015 FDA document on Gene Product Shedding Studies [10]

concerns gene therapies, which are defined as “all products that exert

their effects by transcription and/or translation of transferred genetic

material and/or by integration into the host genome and that are

administered in the form of nucleic acids, viruses or genetically

modified microorganisms”. In this sense mRNA vaccines are indeed

gene therapy products and should have been submitted to these

excretion studies.

Excretion studies must be conducted for each VBGT (virus or

bacteria-based gene therapy products), first in animals but also in

humans, especially when there is a risk of transmission to untreated

individuals. According to this document, clinical excretion studies are

not stand-alone studies but are integrated into the design of a safety or

efficacy trial. The term “shedding” refers to the release of VBGT

products from the patient by any or all of the following routes: feces

(feces); secretions (urine, saliva, nasopharyngeal fluids, etc.); or

through the skin (pustules, lesions, sores).

The NIH guidelines [11] provide biosafety principles specifically for

“synthetic nucleic acid molecules, including those that are chemically

or otherwise modified but can pair with naturally occurring nucleic

acid molecules”; these are molecules of more than 100 nucleotides

with the potential to be transcribed or translated. This April 2019

document is about modified and unmodified synthetic nucleic acids.

Any experiment involving the deliberate transfer of a nucleic acid to a

human must be preceded by Institutional Biosafety Committee

approval (which is confirmed here [12]), but approval was not given

because of the emergency clearance given to mRNA vaccines.

Based on an EMA document on excretion of gene products [13],

mRNA vaccines meet the definition of GMTPs (gene therapy medicinal

products), however their designation as a “vaccine” has allowed them

to escape the clinical trial requirements for gene products that relate

in particular to excretion potential, biodistribution,

pharmacodynamics, genotoxicity, insertional mutagenesis (page 36 :

Pharmacokinetic studies should be performed when a protein is

excreted into the bloodstream). The expression of the nucleic acid

sequence (its translation into protein) should also be studied (page

37). Excretion is defined as the dissemination of the vector through

secretions and/or feces and should be addressed in animal models

(page 30).

Therefore, according to the regulations of the American and

European agencies, mRNA vaccines correspond to the definition of


Infectious Diseases Research 2022;3(4):22.

Submit a manuscript: 3

gene therapy products and should have been subjected to excretion

studies by all secreted fluids (urine, saliva, sputum, nasopharyngeal

fluids, semen, breast milk), feces and skin (healthy or injured). These

studies should have concerned the nanoparticles containing the

mRNA, the naked mRNA and the product of the vaccine after

translation (the spike protein).

An example of an excretion study corresponding to this regulation

of gene products can be found in a report submitted to the EMA to

authorize a drug intended to treat an orphan disease; it is a product

based on LNPs with a composition close to that of mRNA vaccines.

Here the LNPs contain siRNA. The regulations require extensive

studies for this gene therapy, unlike those for mRNA vaccines, which

are similar. However, studies on the excretion of these LNPs give little

information. In animals, the radioactivity of LNPs is found in the urine

(50%) and in the feces (between 10% and 24%). In humans, no study

with radioactive LNPs has been performed, but the components of

LNPs are found in the urine for less than 1% of the plasma

concentrations. It is assumed that elimination is via the feces but this

has not been proven. There have been no studies on excretion in milk

or other body fluids [14].

Reference to possible vaccine shedding in Pfizer documents

The protocol for the Pfizer Phase I/II/III trial of COVID-19 mRNA

vaccines (which began in May 2020) mentions the possibility of

passage of the study product through inhalation or skin contact and

passage through semen from a man exposed through inhalation or

skin contact and passage through breast milk; the possibility of an

adverse vaccine reaction from these exposures is also mentioned [15].

Pfizer's data clearly indicate that a pregnant woman may be exposed

to “the intervention studied due to environmental exposure.”

Environmental exposure can occur through “inhalation or skin

contact.” Examples of environmental exposure during pregnancy

include: -A female family member or health care provider reports that

she is pregnant after being exposed to the study intervention through

inhalation or skin contact. -A male family member or health care

provider who was exposed to the study intervention by inhalation or

skin contact subsequently exposes his female partner before or around

the time of conception. This clearly means that any contact, including

sexual contact with someone who has received the vaccines, exposes

those who have not received the vaccines to the “intervention”, i.e.

mRNA. Exposure during breastfeeding had also to be immediately

notified during the trial: it is assumed that the investigator is

concerned that a breastfeeding mother could transmit the

experimental mRNA to her baby if she received the vaccines directly

or if she is “exposed to the study intervention by inhalation or skin


Structure and function of extracellular vesicles (EVs) or exosomes

and lipid nanoparticles (LNPs)

Natural extracellular vesicles (EVs or exosomes) are generated by most

living cells, they are spherical bilayer proteolipids ranging in size from

20 to 4,000 nm and they can contain various molecules (lipids,

proteins and nucleic acids, like signaling RNAs). EVs are natural

carriers in the human body and are involved in intercellular

communications, they can serve as transporters for different molecules

that can thus pass from cell to cell, resulting in a marked response

from the target cell [16]. Synthetic mRNA vaccine LNPs have the same

structure as the natural exosomes they seek to mimic [17, 18].

Naturally produced exosomes can carry spike or vaccine mRNA as

discussed below. LNPs have the ability (like natural exosomes) to fuse

with cell membranes and release their cargo into the cytosol.

LNPs used for mRNA vaccines are nanosized (less than 1

micrometer) lipid systems made of 2 or more (usually 4) lipids at

varying ratios. The most typical lipid composition used for mRNA-LNP

systems consists of a cationic/ionizable lipid, a phospholipid “helper

lipid”, cholesterol and/or a poly(ethylene glycol) (PEG) associated

lipid. LNPs can be administered IM, subcutaneously, intradermally,

intratracheally, orally, ophthalmically and even topically. LNP

injected by all of these routes is capable of driving translation of

mRNA to protein for several days [19]. The size of LNPs in COVID-19

mRNA vaccines is reported to be between 60 and 100 nm [20].

This trafficking of EVs is bidirectional during pregnancy (EVs cross

the fetomaternal barrier and uterine cells constantly secrete

exosomes) and EVs can be used to deliver drugs to the fetus during

pregnancy [21].

EVs have a potential advantage for use in vaccine therapies because

they are the body's natural antigen carriers and can circulate in body

fluids to distribute antigens even to distal organs [16].

Little is known about the pharmacokinetics of mRNA vaccines

Nanoparticles in animals

According to a study by researchers independent of mRNA vaccine

manufacturers, in mice, mRNA-carrying LNPs injected IM pass from

the injection site into the lymph nodes and then into the systemic

circulation, accumulating primarily in the liver and spleen. LNPs pass

first into the lymphatic circulation and then into the bloodstream

(LNPs smaller than 200 nm pass directly into the lymph, while those

between 200 and 500 nm are transported into the lymph by dendritic

cells). Unintentional direct injection into a blood vessel may also

occur during intramuscular (IM) injection [22].

Nanoparticles in humans

Exposure of the human body to nanoparticles can occur accidentally

through inhalation, skin contact, or ingestion. In the case of

inhalation, the possible routes of transfer of nanoparticles are the

bloodstream (systemic), the lymphatic vessels, the gastrointestinal

tract and the central and/or peripheral nervous system [23].

Excretion of PEG-coated LNPs is primarily through feces and urine

and primarily through feces when they are > 80 nm in diameter.

LNPs can be excreted through saliva, sweat, and breast milk [24].

LNPs of size < 5 nm are rapidly excreted by the kidney.

Nanoparticles that are between 5 and 200 nm tend to have extensive

blood circulation. Larger LNPs have prolonged blood circulation and

little renal excretion. Because of the size of LNPs, inhalation is the

most direct route of entry into the pulmonary system. Exposure can be

intentional, as in the case of targeting or therapeutic nanoparticles, or

unintentional, through inhalation or dermal exposure, due to the

increasing number of industrial applications of nanoparticles [25].

The mRNA

Persistence of viral mRNA after viral infections. The viral RNA of

some viruses persists for a long time in the brain, the eyes, the

testicles: this has ben showed for the measles virus, the Ebola virus,

Zika and Marburg. SARS-CoV-2 persists in the respiratory tract and

intestine. Viral RNAs are also detected in secretions, blood, or tissue.

Prolonged shedding of these RNAs in the respiratory tract, feces,

sweat, conjunctival fluid, and urine is common. Studies have shown

that full-length viral RNA can persist over the long term. This

persistent RNA can be translated into protein even if no viable virus

can be assembled.

In patients who later develop long COVID, viral RNA is found in the

blood in the acute phase of the disease [26].

Fate of vaccine mRNA. Huge amounts of mRNA are injected

compared to the circulation of a virus during a natural infection: up to

10 to 7 times more, according to Professor Jean-Michel Claverie [27].

Vaccine mRNA is present from day one and persists in the

bloodstream for at least 2 weeks after injection; its concentration

starts to decrease after 4 days. This lifetime is much longer than was

claimed by the manufacturers on the basis of brief studies in rats. The

transported mRNA is encapsulated in LNPs but is found in plasma (i.e.

not associated with white blood cells). This mRNA is capable of being

translated into spike protein in susceptible cells and tissues [28].

mRNA packaged in LNPs is able to escape from LNPs and form

extracellular vesicles that transport it to other cells: these vesicles are

secreted after endocytosis of mRNA-loaded LNPs. These EVs protect


Infectious Diseases Research 2022;3(4):22.

Submit a manuscript: 4

the mRNA during transport and distribute it intact to the recipient

cells, the mRNA is functional and can then be translated into the

protein of interest. The inflammatory response is lower after

transfection with EVs than with LNPs. The uptake pathways of EVs

differ from those of LNPs and are not likely to trigger the

autophagic-lysosomal pathway, as they release their contents into the

cytoplasm presumably without undergoing lysosomal trapping.

Moreover, because of their small size, EVs can escape rapid

phagocytosis and routinely transport and deliver RNA into the

circulation, crossing the vascular endothelium to target cells [29].

The presence of extracellular vesicles in all biofluids is attested.

They can contain nucleic acids. In sweat, we find EVs containing

nucleic acids from bacteria, viruses, skin fungi but also from human

cells. These EVs can also contain viruses (hepatitis C, for example).

Small mRNAs (20 to 200 bp) are found in these sweat EVs; they are

functional (can be translated), RNAs are protected from skin nucleases

in the EVs [30].

Note that the vaccine RNA comprises 4,284 nucleotides (Pfizer)

[31]. Thus, the possibility of RNAs of this size being excreted through

sweat should be explored.

EVs may contain “signal” molecules such as miRNAs. It is possible

that EVs contain full-length mRNAs, which are key mediators of

intracellular communication. Blood and sweat RNA analyses are

correlated: the EVs found in sweat reflect the circulation of EVs in

plasma. Bare RNAs are also found in sweat (not encapsulated in EVs).

MiRNAs are selectively selected and enriched in sweat EVs from blood

and do not passively circulate in any blood or sweat fractions [32].

An increase in sweating after the COVID vaccine has been noted

[33] and people who have received the vaccine have complained of

increased sweating, particularly at night [34].

The possibility of exudation of extracellular vesicles from the skin

has been shown: keratinocytes are able to exude extracellular vesicles

capable of carrying miRNAs. In psoriasis, EVs excreted by

keratinocytes pass from cell to cell: from keratinocyte to neighboring

keratinocyte. In patients with lichen planus (inflammatory rash)

extracellular vesicles carrying miRNAs are excreted in saliva [35].

Nanoparticles are naturally present in sputum [36]: RNA-containing

exosomes were isolated from sputum of mild asthmatic patients [37].

Passage of vaccine mRNA into milk. Vaccine mRNA was found in

the milk of 1/10 women studied (4/40) in the first week after

vaccination with mRNA vaccine (either after dose 1 or dose 2).

Amounts can reach 2 ng/mL of milk [38]. This amount may seem

small compared to the 30 micrograms of mRNA injected with the

vaccine, but it can be enough to produce a significant amount of spike.

Indeed, an infant makes several feedings per day, for approximately

240 to 360 mL per day and a total over a week of 1680 to 2,520 mL in

the first week. The newborn, weighing between 2 and 5 kg, could

therefore be exposed to a dose of 5 μg of mRNA in its first week. This

seems disproportionate compared to the 10 μg injected into children

aged 5 to 11 years who weigh approximately 18 to 35 kg respectively

[39]. The method used in the latter study is more sensitive than that of

Golan et al. who did not find mRNA in milk [40]. This same team had

also explored the passage of vaccine mRNA into milk by indirectly

searching for PEG contained in LNPs. PEG was searched for in the milk

of 13 women at varying times after vaccination: Figure 1 of the article

shows the detection of vaccine PEG in milk between 24 hours and one

week after injection. However, the authors concluded without

specifying that these quantities were not significant [41].

Another study investigated whether COVID-19 vaccine mRNA can

be detected in the expressed breast milk of breastfeeding individuals

who received vaccination within 6 months of delivery. The presence

of mRNA was investigated in free form and encapsulated in

extracellular vesicles. EVs were isolated by centrifugation of milk.

Vaccine RNA was found within 48 h of vaccination and in higher

concentrations in EVs than in whole milk. The highest concentration

found was 17 pg/mL in EVs and the lowest was 1.3 pg/mL in whole

milk. The priority presence of mRNA in EVs and not in whole milk

may explain why Golan et al. did not find it [42].

It has been known for some years that mRNA encapsulated in

extracellular vesicles is protected from gastric juices and can transfect

intestinal cells [43, 44]. A recent review by Melnik and Schmitz

confirms that milk EVs survive the extreme conditions of the

gastrointestinal tract, are internalized by endocytosis, are

bioavailable, reach the bloodstream, and penetrate peripheral tissue

cells [45].

Transplacental passage of nanoparticles? In mice, LNPs of the same

type as those used in COVID-19 mRNA vaccines have shown their

ability to transfect mRNA after injection into a fetal vein or in utero


In an attempt to immunize fetuses against neonatal herpes in

pregnant mice by injection of mRNA-loaded LNPs into the mother, the

possibility that transplacental passage of LNPs would explain both

fetal immunization and maternal passage of induced Ig is not

discussed [47].

Studies have shown that it is very possible that nanoparticles of

comparable size to those used for mRNA vaccines are capable of

transplacental passage in humans [48, 49].

The delivery of LNP-based therapies during pregnancy poses risks

that should be investigated. Detection of transplacental passage

depends on the sensitivity of the detection methods: for some types of

nanoparticles embryotoxicity has been observed while no absorption

by the fetus was observed; this absorption does not seem to correlate

with the type, size or surface electric charge of the nanoparticles.

Translocation of nanoparticles is likely to depend on the different

stages of pregnancy. During the first trimester, the placental barrier is

very thick to protect the developing embryo and becomes thin at term

when large amounts of nutrients are needed to support fetal growth.

However, in animals, placental transfer appears to be higher in early

pregnancy. There is a need to develop human models for NP transfer

studies in early pregnancy. Comparison with animal studies is

essential, as the placenta is the most species-specific organ [50, 51].

240 nm nanoparticles are able to cross the human placental barrier


All these publications underline the difficulty of extrapolating

animal studies to humans concerning the transplacental passage of

nanoparticles. From a 2022 review [53], nanoparticles can transit

through ordinary placental transcellular transport mechanisms such as

pinocytosis, active transport, facilitated diffusion and passive

diffusion. RNA cargo exosomes are also able to cross the human

placental barrier. PEG-coated LNPs are reported to have less diffusion

across the placental barrier than liposome-based formulations, but are

able to deliver some of their cargo to the fetus [54].

All of these data cannot rule out that LNPs from mRNA vaccines are

capable of reaching the fetus of a vaccinated mother during


Excretion of LNPs into the sperm? I have not found any studies

regarding the possibility of LNPs passing into the sperm; however, the

effect of nanoparticles on fertility and sperm quality has been widely

studied in animals [55]. The toxicity of nanoparticles on male

reproductive function is well established, gold nanoparticles have

been shown to act only by interacting with the surface of sperm cells

but not penetrating them. No data is available on the possible

penetration of LNPs into the sperm.

According to a confidential Pfizer document obtained through the

FOIA [56] concerning pharmacokinetic studies in rats, LNPs

concentrate in the ovaries and to a lesser extent in the testes.


Infectious Diseases Research 2022;3(4):22.

Submit a manuscript: 5

Figure 1 State of knowledge on excretion of mRNA vaccines

Fate of spike protein after mRNA translation

A CDC-sponsored news site accessed on July 21, 2021 notes that the

lifespan of spike in the bloodstream is “unknown and may be a few

weeks.” [57]. Injection of LNPs containing pseudouridine-modified

mRNA by IM, subcutaneously and intradermally results in protein

production at the site of injection, the duration of active translation is

6 to 10 days in mice. Intradermal injection produces a lower initial

amount of protein but over a longer period of time than the IM route.

By the intradermal route, the half-life of protein production is the

longest compared with other injection routes (IM, subcutaneous, IV,

Iperitoneal, intra-tracheal). By IM delivery, the majority of translation

ceased in the liver at day 2 post-injection but lasted for up to 8 days in

muscles [58].

In humans, the spike protein could persist for a long time in

vaccinees, monitoring of vaccine adverse effects should therefore be

extended [59]. Comparison of spike concentrations achieved during

disease and after vaccination shows that during severe COVID-19 the

median concentration observed is 50 pg/mL with maximums at 1

ng/mL. During severe Covid infection, levels of up to 135 pg/mL of S1

spike can be detected, most commonly between 6 and 50 pg/mL. After

vaccination with mRNA vaccine concentrations up to 150 pg/mL are

commonly observed but may reach 10 ng/mL in individuals with

vaccine-induced thrombocytopenia [60].

The same team [61] also shows that spike protein persists for a long

time in free form: vaccine-induced spike mRNA circulates in plasma as

early as D1 after vaccination and up to 14 days, with the peak

occurring at D5 with 68 pg/mL of S1 sub-unity detected; full-length

spike is detected up to D15, with a peak at 62 pg/mL. After the 2nd

dose, free spike is no longer detected as it would be bound to

antibodies; the study does not detect antibody-spike immune


Another team also showed that, after vaccination with mRNA, spike

protein enters the bloodstream, persists for more than a week and is

completely eliminated within 1 month. The increase in blood spike

concentration after vaccination is rapid (1 to 3 days) [62].

According to an autopsy, vaccine spike is found up to three weeks

after injection in different organs (heart, brain, muscles, germinal

centers, etc.) and particularly in the endothelium of capillaries [63].

Circulating spike-containing exosomes

After COVID-19 infection, spike circulates as exosomes. Exosomes are

released from cells into the extracellular environment under normal

and pathological conditions. Exosomes are an important tool for

intercellular communication, as they serve as shuttles for the transfer

of biologically active proteins, lipids, and RNA. EVs can incorporate

pathogenic proteins and/or viral RNA fragments from infected cells to

mRNA Vaccine

= mRNA in LNPs




CNS, Testes,

Concentration in

liver, spleen, ovaries


naked or encapsulated

in natural EVs

2 weeks in bloodstream


mRNA > spike protein

at the injection site in

mice: lasts 6 to 10 days

Naked spike circulates 1

month, present in heart, brain,

muscles, germinal centers at

least 3 weeks post injection

included in exosomes

circulates 4 months


Feces, urine, saliva,

sweat, maternal milk,

unexplored in semen

mRNA naked

and in EVs in

human maternal


Spike in EVs in

keratinocytes 3

months post


Penetration of

vaccine products

mRNA and spike

circulate in LNPs or in natural EVs

that have been shown to penetrate

transdermally and by inhalation,

orally (breast milk) or by trans

placental route



Infectious Diseases Research 2022;3(4):22.

Submit a manuscript: 6

transport material to target cells, an event that plays an important role

in responses to viral infections. SARS-CoV-2-S protein or derived

fragments were clearly present in the exosomes of COVID-19 patients.

SARS-CoV-2-S-derived fragments are present in exosomes from all

COVID-19 patients [64].

Spike also circulates in exosomes after mRNA vaccination in

humans. Authors have proposed that after LNP internalization and

mRNA release, antigen sorting and trafficking can induce the release

of S protein-containing exosomes. The events presented would occur

in the apical and/or basolateral surfaces of polarized (e.g. epithelial)

cells [65]. Indeed, the vaccine spike is spontaneously enveloped in

exosomes or extra-cellular vesicles: Vaccination with mRNA and

translation of the mRNA induces the production of exosomes carrying

the spike protein and circulating in the blood 14 days after injection

and up to 4 months after vaccination. Injection of these exosmoes into

mice induces the synthesis of anti-spike antibodies [66].

Vaccine spike was found in keratinocyte vesicles of dermal

endothelial cells from a patient with skin lesions 3 months after

vaccination with Pfizer-BioNTech vaccine. This patient had

varicella-zoster virus infection. A plausible hypothesis was that RNA

stabilization by methyl-pseudouridine substitution at all uridine

nucleotides for BNT162b2 could result in long-term production of the

encoded spike from any cell, persistently affecting the

microenvironment of the protective immune system, including the

skin [67].

All these data indicate that vaccine LNPs or exosomes naturally

formed after vaccination could contain mRNA or spike and could be

present in body fluids. Are these nanoparticles capable of entering

from these fluids into the bodies of unvaccinated individuals in

contact with freshly vaccinated individuals?

Ability of LNPs or natural extracellular vesicles (EVs or exosomes)

and mRNA to enter through different pathways

Use of nanoparticles for therapeutic purposes by inhalation,

transdermal, in utero, conjunctival route

In a review dedicated to the safety of nanoparticles in biomedical

applications, we learned that exposure to LNPs can occur through

ingestion, injection, inhalation and skin contact. Some exposures are

unintentional, such as pulmonary inhalation of NPs in the

environment or at manufacturing sites [68].

Nanosystems are increasingly being exploited for topical and dermal

delivery, including therapeutic peptides, proteins, vaccines, gene

fragments, or drug carrier particles [69]. Intradermal administration

of mRNA encoding vascular endothelial growth factor (VEGF) has

been shown to result in functional protein expression in the skin even

in the absence of lipid nanoparticles [70]. According to Palmer et al

[71], in a lipid nanoparticle formulation, liposomes increase the

transdermal passage of molecules used to treat skin diseases. Skin

penetration of siRNAs has been demonstrated in the form of

nano-carriers, these siRANs transfect cells and express the targeted

gene of interest. Nanocarriers have been tested for use in transdermal

vaccination [72].

Extracellular vesicles are used to deliver therapies other than

vaccines: clinical studies are underway by local route (periondontitis,

ulcers, epidermolysis bullosa) and by inhalation (ongoing trial against

Alzheimer's disease) [73]. Lipid nanoparticles with a lipid bilayer are

able to pass the skin barrier and carry genetic material. These particles

can penetrate the skin through hair follicles or directly into

keratinocytes due to their similarity to cell membranes [74].

Intranasal, oral, and intraocular and subconjunctival administration

of extracellular vesicles capable of carrying drugs has been

successfully tested.

Intranasal administration represents the second most frequently

reported route. It is effective in transporting drugs into the CNS, into

the lungs. Most of the protective effects were obtained in a similar way

for intravenous and intranasal administration. Oral administration has

been described for EVs from bovine milk in a mouse model. Six hours

after administration, vesicles were localized in the liver, heart, spleen,

lungs, and kidneys. Intraocular and subconjunctival injection of

MSC-derived EVs (stem cells) delivered vesicles to the retina in a

rabbit model of diabetes-induced retinopathy [75].

Nanovesicles naturally produced by plants are morphologically and

functionally identical to their mammalian analogues. A review on

plant nanovesicles brings together knowledge on the transdermal,

transmembrane, and targeting mechanisms of these vesicles.

Experiments on mice have shown that it is possible to deliver RNA

into a brain tumor via these intranasally introduced nanovesicles.

These nanovesicles would also be able to efficiently transport their

cargo through the skin and into the skin cells [76].

Lipid nanoparticles are a potential carrier for delivering molecules

to the posterior chamber of the eye: they have demonstrated excellent

ocular permeation characteristics and penetration-enhancing

capabilities, while exhibiting high drug loading and entrapment

efficiencies [77].

Nanoparticles in vaccination and gene therapy trials (LNPs

containing nucleic acids) via the respiratory route

Nucleic acid cargo nanoparticles are capable of transfecting airway

cells in animals and humans by local administration (instillation or

nebulization). The DEFUSE project [78], submitted by Eco Health

Alliance in response to a DARPA call for proposals, deals with the

transcutaneous administration of vaccines in animals using

nanoparticles. For therapeutic purposes, the LNPs formulation was

optimized for lung penetration by inhalation and it was verified that

mRNA is efficiently translated in the lung after nebulization (mouse

assay) [79].

The intranasal route has also been studied for vaccination with

mRNA cargo LNPs as well as for gene therapy in cystosis fibrosis with

mRNAs encapsulated in LNPs by the intranasal route by instillation in

the nostrils of mice: the mRNA transfects the nasal cells and expresses

the protein of interest in cells that did not express it because of a

genetic defect [80].

In humans, liposomal DNA-containing nanoparticles administered

locally by nebulization have transfected airway cells. A recent phase

2b trial of cystic fibrosis transmembrane conductance regulator

(CFTR) DNA delivery using a liposomal delivery system showed that

after repeated monthly nebulizations for one year, the cystic fibrosis

patient groups experienced a stabilization of lung function, while the

placebo group experienced a decline [81].

Clinical trials for influenza prevention have shown the efficacy and

safety of inhaled mRNA vaccines: Bare mRNA or mRNA enveloped in

lipid particles (especially PEG-based as in the anti-COVID mRNA

vaccines), is able to be inhaled in an aerosol and transfect lung

epithelial cells [82]. In utero administration of lipid nanoparticle

formulations containing mRNA can be applied to deliver mRNA to

mouse fetuses, resulting in protein expression in the fetal liver, lungs,

and intestines [70].

Testing LNPs for transcutaneous vaccination

In a review [72] on the possibility of transcutaneous vaccination with

LNPs, we learn that undamaged human skin is impermeable to microand

nanoparticles but there is evidence of some dermal penetration

into viable tissues (mainly in the stratum spinosum of the epidermal

layer, but also possibly in the dermis) for very small particles (less

than 10 nm). When using intact skin penetration protocols, there is no

conclusive evidence of skin penetration into viable tissues for particles

with a primary size of about 20 nm and larger. But there is no

information appropriate for skin with impaired barrier function, e.g.,

atopic skin or sunburned skin. Some data are available on psoriatic

skin. There is evidence that some mechanical effects (e.g., bending) on

the skin may affect nanoparticle penetration. But it has been shown

that nanoparticles accumulate in follicular openings, sebaceous glands

or skin folds. An aqueous suspension of nanoparticles as well as a

hydrogel formulation of these particles, applied to pig ear skin in

vitro, penetrated deep into the hair follicles. These particles can

release various encapsulated compounds that then penetrate the skin.

There is evidence in the literature that the trans-follicular route can be


Infectious Diseases Research 2022;3(4):22.

Submit a manuscript: 7

used: topical application of naked plasmid expression vectors to intact

mouse skin induced antigen-specific immune responses.

HBsAg-specific cellular and antibody responses were induced in the

same order of magnitude as those produced by intramuscular (IM)

injection of recombinant HBsAg polypeptide vaccine. In contrast, no

immune response could be induced in nude mice: the presence of

normal hair follicles was a prerequisite for the induction of a response.

The particles were approximately 150 nm in size. LNPs in mRNA

vaccines are between 100 and 400 nm [22].

One system that has been clinically tested transdermally is the

DermaVir HIV-1/AIDS patch. It contains a plasmid DNA (pDNA)

vaccine encoding all major HIV-1 antigens and viral-like particle

formation.) The pDNA is formulated as mannosylated

polyethyleneimine nanoparticles (80-400 nm) similar to those of

pathogens. This study involved 12 people immunized with the

vaccine: they developed higher and broader levels of CD8+ T cells

compared to placebo, although it had no effect on CD4+ T cell

numbers [72].

Naked RNA could also be used via skin passage and inhalation

RNA oligonucleotides can penetrate intact skin and retain their

biological activity, penetration through the skin does not depend on

the size of the molecule under study (12.5 to 29.3 kDA) [83].

The feasibility of inhaled RNA for passive transfection has also been

demonstrated in a number of studies. Mechanistically, inhaled RNA

can lead to passive synthesis of non-infectious spike proteins using cell

transfection machines, thus leading to immunization of the individual


Therapeutic and vaccine LNPs in COVID-19

Given that vaccine LNPs are synthetic exosomes it is not surprising

that COVID therapeutics and vaccines with natural exosomes as

vectors are being tested. Nebulization of exosomes for inhalation

therapy has been tested against COVID-19. Clinical trials are

underway to deliver aerosolized anti-viral therapies in EVs in

COVID-19. Currently, over sixty clinical trials are underway to study

the effects of MSCs (mesenchymal stem cells) and EVs (containing

these MSCs) in COVID-19 patients. A phase 1 clinical trial to evaluate

the safety and efficacy of inhaled exosomes derived from allogeneic

adipose MSCs for the treatment of COVID-19 pneumonia has been

completed. 3 clinical trials used aerosol as the route of administration

[16]. In 2022, MSCs exosomes showed efficacy for nebulization

therapy in COVID-19 patients [85].

Natural exosome vaccines against SARS-CoV-2: plantar or

inhalation route

Exosome vaccines carrying mRNA have been considered against

SARS-CoV-2 [86]. Vaccine trials injected as exosomes into the footpad

of mice showed induction of antibodies against spike [87]. Spike RBD

exosomes (nanoparticles) are capable of nebulizing and inhaling

antigen into mouse lung cells and inducing an immune response. They

are virus-like particles (virus like particles (VLP)) naturally obtained

from lung cells and carry RNA from their parent cell as well as various

proteins expressed on their surface [88]. Also by inhalation, exosomes

containing mRNA or spike protein are able to immunize mice or

non-human primates against SARS-CoV-2 and natural EVs are more

effective than synthetic EVs [89].

The possible reinterpretation of a study may support vaccine


Scientists compared unvaccinated children living with unvaccinated

parents with children who were also unvaccinated but living with

vaccinated parents [90]. The children of vaccinated parents have

anti-COVID IgG in their nose and the difference with the children of

unvaccinated parents is significant. The authors think that this is due

to antibody shedding by droplets: what is transferred would be the IgG

antibody itself in the saliva droplets. But it is possible that children

develop intranasal IgG because other vaccine byproducts or exosomes

are excreted by their vaccinated parents. This could be due to lipid

nanoparticles of mRNA that could be excreted and transferred through

saliva, sputum or skin. Children would develop an immune response

to the nanoparticles (or vaccine by-products) instead of IgG being

transferred directly from parents to children. The antibodies sought

are IgG and IgA against the RBD of the spike and not against the

nucleocapsid of the virus, which is a pity because the authors have

developed both types of tests [91]: this does not allow to distinguish

children who would have been naturally infected by the virus (and

would have anti-RBD and anti-N antibodies) from children who would

have developed antibodies following their parents' vaccination (and

would only have anti-RBD and no anti-N because not induced by the



There are many testimonies of non-vaccinated persons who

experienced symptoms identical to the adverse effects of the vaccine

after having been in contact with freshly vaccinated persons. A study

shows an excess of mortality in the non-vaccinated age groups when

vaccination campaigns begin, which could be explained by a

phenomenon of transmission of the vaccine or its products.

It is important not to neglect these testimonies because the required

studies of pharmacokinetics and in particular of excretion of the

vaccine and its products have not been carried out in spite of the

regulations in force for gene therapies, which include mRNA vaccines

according to the definition of these gene products. Moreover, the

doubt about the possible transmission of the vaccine creates an

unhealthy climate of suspicion of the non-vaccinated towards the

vaccinated: a clarification would therefore be welcome.

The vaccines are all based on the spike protein, which has since

been recognized as the main responsible for the pathogenicity of the

SARS-CoV-2 virus: if transmission of the vaccine or of the spike is

possible, it is logical to find the adverse effects of the vaccine in

non-vaccinated people in contact with vaccinated people.

Little is known about the pharmacokinetics of the vaccine. Vaccine

LNPs are very similar to natural EVs or exosomes, whose structure and

function scientists have tried to mimic as closely as possible.

According to the few studies conducted by manufacturers and

independent researchers, mRNA vaccine LNPs circulate in the blood

and accumulate in the spleen and liver of mice (and to a lesser extent

in many organs including ovaries and testes, bone marrow,..).

Translation into spike protein persists 6 to 10 days in mice at the

injection site and 8 days in the muscles.

The route of excretion of LNPs varies according to their size, in the

case of LNPs of mRNA vaccines excretion should be mainly by the

feces but also by the urine. The quantitative results of these studies

suggest that other routes of excretion than feces and urine should be

explored. Studies prior to mRNA vaccines suggest that EV excretion is

possible through saliva, sweat, and breast milk.

Studies have shown that it is very possible that nanoparticles of

comparable size to those used for mRNA vaccines are capable of

transplacental passage in humans. Natural nanoparticles (EVs) are

naturally present in all body fluids (including sputum, saliva, and

sweat) and in keratinocytes and can carry nucleic acids that are thus

protected from nucleases. Certain types of RNA (miRNAs) are

selectively selected and enriched in sweat EVs from blood. No studies

have been found regarding the possibility of passage of LNPs into

semen; given the biodistribution in all organs and fluids, such passage

is a priori possible and should be explored.

Viral RNA of many viruses is found in blood, secretions and tissues.

Vaccine mRNA is injected in quantities orders of magnitude greater

than the viral RNA produced during natural infection. This mRNA is

found in the blood as early as the first day after injection and persists

for up to 15 days. It is able to escape from LNPs and to be

encapsulated in EVs, it is functional and can be translated into protein.

Vaccine mRNA naked or encapsulated in EVs is found in breast milk

within the first week after injection; it is protected from gastric juices

and can transfect neonatal cells.


Infectious Diseases Research 2022;3(4):22.

Submit a manuscript: 8

RNA embedded in EVs or even naked is capable of transfecting cells

by inhalation or transdermal passage. Intranasal, oral, transdermal

intraocular and subconjunctival administration of extracellular

drug-carrying vesicles has been tested: LNPs can be administered

through the skin, intranasally, intraconjunctivally and by inhalation;

experiments have shown that mRNA included in these LNPs is capable

of transfecting cells Vaccination trials against COVID by inhalation of

EVs containing mRNA or spike protein have shown positive results in

mice and nonhuman primates. Natural EVs are more effective than

synthetic EVs.

Spike protein translated from vaccine mRNA persists for months in

large quantities in vaccinees; it is found in free form in plasma and

encapsulated in EVs that form spontaneously from the cells where

spike was produced. These EVs can deliver their cargo to different cell

types, in particular to fetal cells of vaccinated mothers. Spike can be

found in keratinocytes of the skin.

Specifically against coronaviruses, gene therapy and vaccination

trials (especially with mRNA) have shown the possibility of

transfecting cells transcutaneously, nasally and by nebulization from

LNPs and even from naked mRNA. Spike or mRNA RBD vector

exosomes have been tested by inhalation in animals for anti-COVID-19


All these studies show that EVs carrying mRNA and spike could

therefore be excreted by different body fluids and could enter by

transcutaneous or inhalation route in unvaccinated individuals (as

well as by breast milk in infants and by transplacental passage in

fetuses and why not by semen). Naked mRNA could also be excreted

and entered.

The mRNA (and adenovirus) vaccines correspond exactly to the

definition of gene therapy given by the health agencies (FDA, NIH and

EMA). According to the regulations of these agencies, these products

should be subject to additional pharmacokinetic studies (in particular

excretion studies) as a matter of urgency as the widespread use of

mRNA technology becomes apparent. Indeed, Sanofi launched clinical

trial of the first mRNA-based seasonal flu vaccine candidate [92],

Moderna launched phase 3 trial of mRNA influenza vaccine [93]. For

these flu vaccines, emergency approval should not be applied and the

requirement for these additional studies should not be exceeded.


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