Semelhante a Sars-cov2 Spike protein and derivates toxicology :fertility and teratogen evaluation. State of Evidence -Hypotesys of Work 2022-01-21-16-29
Semelhante a Sars-cov2 Spike protein and derivates toxicology :fertility and teratogen evaluation. State of Evidence -Hypotesys of Work 2022-01-21-16-29 (20)
2. 1 INTRODUCTION
A
ccording an JAMA article November 4,
2021 Association Between m-RNA Vacci-
nation and COVID-19 Hospitalization and
Disease Severity Mark W. Tenforde et al :
“Vaccination with an m-RNA COVID-19 vaccine
was significantly
less likely among patients with COVID-19 hospi-
talization and disease progression to death or me-
chanical -ventilation. These findings are consistent
with the risk reduction among vaccine break-through
infections compared with absence of the vaccination
“.
But also if this kind of data and results are pub-
lished it is interesting to observe the public debate
around The VARES database (USA) related death
and covid-19 vaccine :
Clin Exp Vaccine Res.
2021 Sep 30
Post-vaccination COVID-19 deaths: a review of
available evidence and recommendations for the
global population
Emmanuel Lamptey
“the Vaccine Adverse Event Reporting -System
(VAERS) is the established database set up to mon-
itor adverse –events AE following the vaccination,
including deaths not necessarily caused by a vac-
cine or related health- problems. VAERS cannot
determine whether a vaccine is responsible for the
adverse -event, but rather the AE occurred sometime
after the vaccination . While essential to monitor
vaccine- safety, the VAERS alone can not be used as
proof for adverse -events. It is a passive- surveillance
system and reports may contain information that are
incomplete, coincidental, inaccurate and unverified
.”
MMWR Surveill Summ. 2003 Jan
Surveillance for safety after immunization: Vaccine
Adverse Event Reporting System -United States,
1991-2001
Weigong Zhou et al :
“VAERS was established in 1990 under the joint
of CDC and FDA to accept reports of suspected
adverse- events AE after administration of any vac-
cine licensed in the US . VAERS is a passive -
surveillance system: reports of events are voluntarily
submitted by those who experience them, their care-
givers, or by others. Passive -surveillance systems
are subject to multiple -limitations, including under-
reporting, reporting of temporal -associations or un-
confirmed diagnoses, and lack of denominator data
and unbiased comparison groups. Due to these kind
of limitations, determining causal associations be-
tween vaccines and adverse events from VAERS re-
ports is usually not possible. Vaccine safety concerns
identified through AE monitoring nearly always re-
quire confirmation using an epidemiologic or other
( laboratory) study. Reports may be submitted by
any-one suspecting that an adverse event AE might
have been caused by vaccination and are usually
submitted by mail or fax. The objectives of VAERS
are to A) detect new, un-usual, or rare vaccine AE;
B) monitor increases in known AE; C) determine
patient risk factors for particular types of AE ; D)
identify vaccine lots with increased numbers or types
of reported AE; E) assess the safety of newly licensed
vaccines.
During 1991-2001, VAERS received 128,717 re-
ports, whereas >1.9 billion net doses of human vac-
cines were distributed. The overall dose-based re-
porting rate for the 27 frequently reported vaccine-
types was 11.4 reports per 100,000 net doses dis-
tributed. The proportions of reports in the age -
groups <1 year, 1-6 years, 7-17 years, 18-64 years,
and >/= years were 18.1%, 26.7%, 8.0%, 32.6%, and
4.9%, respectively. In all of the adult -age groups,
a predominance among the number of women re-
porting was observed, but the difference in sex was
minimal among the children. A total of 14.2% of
all reports described serious AE , which by regula-
tory definition include death, life-threatening illness,
hospitalization or prolongation of hospitalization, or
permanent- disability.”
Supplementary information The online version of
this article (https://doi.org/10.52845/JMRHS/2022-
5-1-6) contains supplementary material, which is
available to authorized users.
Corresponding Author: Mauro Luisetto
IMA academy Marijnskaya , Natural science branch
, applied pharmacologist Professorship toxicology
italy 29121 italy
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MMWR Morb Mortal Wkly Rep . 2021 Feb 26
First Month of COVID-19 Vaccine Safety Monitor-
ing - United States, December 14, 2020-Jan 13, 2021
Julianne Gee et al :
“ 2 COVID-19 vaccines are currently authorized for
use in the US. The FDA issued Emergency- Use Au-
thorization for the Pfizer-BioNTech COVID-19 vac-
cine on Dec 11, 2020, and for the Moderna COVID-
19 vaccine on Dec 18, 2020; Administered as a 2-
dose series. The Advisory Committee on Immuniza-
tion -Practices issued interim recommendations for
Pfizer-BioNTech and Moderna COVID-19 vaccines
on Dec 12, 2020 (1), and Dec 19, 2020 (2), respec-
tively; initial doses were recommended for health-
care personnel and long-term care facility residents
(3). Safety monitoring for these vaccines has been
the most intense and comprehensive in U.S. history,
using the VAERS, a spontaneous -reporting system,
and v-safe,* an active- surveillance system, during
the initial implementation phases of the COVID-19
national vaccination program (4). CDC conducted
descriptive- analyses of safety data from the first
month of vaccination (Dec 14, 2020-Jan13, 2021).
During this period, 13,794,904 vaccine doses were
administered, and VAERS received and processed
6,994 reports of adverse -events after vaccination,
including 6,354 (90.8%) that were classified as non-
serious and 640 (9.2%) as serious.The symptoms
most frequently reported to VAERS were headache
(22.4%), fatigue (16.5%), and dizziness (16.5%). A
total of 113 deaths were reported to VAERS, in-
cluding 78 (65%) among LTCF residents; available
information from death- certificates, autopsy reports,
medical- records, and clinical descriptions from
VAERS reports and health care providers did not
suggest any causal -relationship between COVID-
19 vaccination and death. Rare cases of anaphy-
laxis after receipt of both the vaccines were reported
(4.5 reported cases per million- doses administered).
Among persons who received the Pfizer-BioNTech
vaccine, reactions reported to the v-safe system were
more frequent after receipt of the second- dose than
after the first. The initial post-authorization safety-
profiles of the 2 COVID-19 vaccines in current use
did not indicate evidence of un-expected serious
adverse- events AE. These data provide reassurance
and helpful information regarding what health -care
providers and vaccine recipients might expect after
the vaccination.”
Rapid response to: Covid-19: Norway investigates
23 deaths in frail elderly patients after vaccination
BMJ 2021;372:n149 30 April 2021 J. Stone
“Rapid Response:
Agency failure? (27% of all fatal reports on VAERS,
started in 1990, are for Covid vaccines in the last 4
months)
Dear Editor
I respond to the letters of Doshi, Arby , Fujito .
In order to give an idea of the magnitude of the
current problem I made a search on VAERS us-
ing the National Vaccine Information Center web-
site, Medalerts . The search found 11,895 adverse
events “where patient died” for all vaccines since
the database opened in 1990 to 16 April 2021, of
which 3,186 were from Covid vaccines, or 27% of all
cases in the space of just 4 months from these novel
products . This is quite likely the tip of the iceberg: a
report on the database in 2010 stated “fewer than 1%
of vaccine adverse events are reported”
The failure of US- government agencies to pick up a
signal in the face of an unprecedented level of fatal
reports is disturbing”.
Editorial Sep. 3, 2021
Safety Surveillance of COVID-19 m-RNA Vaccines
Through the Vaccine Safety Datalink
Kimberly G. Blumenthal et al :
JAMA. COVID-19 Resource Center
“People in the US have received more than 342
million -doses of COVID-19 vaccines, with the vast
majority being m-RNA vaccines from Pfizer-BioN-
Tech or Moderna. In the research study by Klein
et al, the m-RNA COVID-19 vaccines were safe
for the population overall ( there was no difference
for any of the serious- outcomes assessed), but an
excess risk of myocarditis/pericarditis was identified
for vaccinees aged from 12 to 39 years”.
Deaths post COVID-19 Vaccination: An analysis
per VAERS
Wong, J. et al :
JACCP Journal of the American College of Clinical
Pharmacy, 2021.
“Using VAERS, data from Dec 2020 to Feb 2021 on
patients that died after receiving either the Moderna
or Pfizer-BioNtech vaccine. Information collected
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included vaccine- manufacturer, patient’s age, sex,
state of vaccine- administration, dose received,
date vaccinated, date of symptoms -onset, date of
death, symptoms, adverse drug -event description,
medication -history, allergies, current medications,
and illnesses. The patients were then categorized per
ADEs that possibly related to death.
568 patients were included ranging from 23 to 108
years old, with a median age of 78.5 and mean
age of 77.3. The top 3 adverse -effect categories
were respiratory (23.8%), cardiac (21.3%), and
fatigue (10.9%). The reported- deaths were similar
after receiving either the Moderna (51.1%) or the
Pfizer-Bio-Ntech vaccine (48.9%). Eighty percent
of patients died after receiving the first dose, and
20% died after receiving the second ”.
And form https://www.cdc.gov/coronavirus/2019-n
cov/vaccines/safety/adverse-events.html
Selected Adverse Events AE Reported after COVID-
19 Vaccination Nov. 30, 2021
Safety of COVID-19 Vaccines
“Reports of death after COVID-19 vaccination are
rare. More than 459 million doses of COVID-19
vaccines were administered in the US from Dec14,
2020, through Nov 29, 2021. During this time,
VAERS received 10,128 reports of death (0.0022%)
among people who received a COVID-19 vaccine.
FDA requires healthcare providers to report any
death after the COVID-19 vaccination to VAERS,
even if it’s unclear whether the vaccine was
the cause. Reports of AE to VAERS following
vaccination, including deaths, do not necessarily
mean that a vaccine caused a health- problem.
CDC -clinicians review- reports of death to VAERS
including death- certificates, autopsy, and medical
-records. A review of reports indicates a causal -
relationship between the J&J/Janssen COVID-19
Vaccine and TTS, a rare and serious adverse event
AE —that causes blood -clots with low platelets—
which has caused or directly contributed to six
confirmed -death “
The rapid need of introduction in healthcare of covid-
19 vaccine for people safety
To prevent this severe coronavirus disease needed
an emergencial authorization in the first period of
commercialization.
2 different technologies , and 2 different veicle :
nano-lipids or viral vector.
From website https://portal.ct.gov/vaccine-portal/V
accine-Knowledge-Base/Articles/m-RNA-vs-Viral-
Vector:
“The instructions in the m-RNA vaccines are
messenger RNA (m-RNA), the genetic material that
tells your cells how to make the proteins. The m-
RNA is surrounded by tiny -lipids (fatty molecules)
which help m-RNA enter directly into your cells.
Once your cells create the spike-proteins, your body
breaks down the m-RNA.
In viral-vector vaccines, spike-protein DNA is
placed inside a modified version of a different virus
that doesn’t cause illness. This non-harmful virus
delivers the DNA- instructions to your cells .”
But analizing the pkarmaco-kinetics PK of this
products it must not to be considered only the
the codifing molecule ( to produce a spike-protein
modified or not ) but also the carrier-vectors and its
influence in increace cell- intake.
Of interest also data from inactivated vaccine .
From Int.J. of Molecular Sciences Review
m-RNA Vaccine Era—Mechanisms, Drug Platform
and Clinical Prospection
Shuqin Xu et al :
“in 1989, Malone et al. demonstrated that m-RNA
could be successfully transfected and expressed
in various of eukaryotic- cells under the package
of a cationic- lipid (N-[1-(2,3-dioleyloxy) propyl]-
N,N,N-trimethyl-ammonium chloride (DOTMA)) .
In 1990, in vitro-transcribed m-RNA was sufficiently
expressed in mouse skeletal -muscle cells through di-
rect -injection, which became the first successful at-
tempt on m-RNA in vivo expression and thus proved
the feasibility of the m-RNA vaccine development
. Since then, m-RNA structure researches and other
related technologies have been rapidly developed.
Under this research condition, several development
restrictions stemmed from m-RNA instability, high
innate immune-genicity, and inefficient in vivo de-
livery have been mitigated, and now m-RNA vac-
cines have been widely studied in different kinds of
pathology .
By modifying the m-RNA sequence and delivery
system,
the expression- activity and in vivo half-life of m-
RNA can be effectively regulated
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Kuhn et al. showed that m27,20-OGppSpG (β-S-
ARCA) could significantly enhance the stability and
translation -efficiency of m-RNA in immature den-
dritic -cells (DCs)
UTR optimization is to increase the in vivo m-RNA
expression- level.
Orlandini ,von Niessen et al. once connected 2 ran-
dom 30 UTRs which contained stable elements in
series, and successfully improved the translation ef-
ficiency of m-RNA .
As the coding region of m-RNA, the translatable rate
of ORF- region is definitely crucial.
Choosing the appropriate codons in this region -
can optimize the overall -translation efficiency of m-
RNA.
Poly (A) tail and the 50 Cap structures are both
crucial elements during m-RNA translation. Poly
(A) sequence can slow down the degradation- pro-
cess of RNA -exonuclease, which increases stability,
extends in vivo half-life, and enhances translation
efficiency of m-RNA
m-RNA has self-adjuvanting -properties which ac-
tivate strong and Long lasting adaptive immune-
responses through the TNF-α,IFN-α and other cy-
tokines secretion by immune cells , while polypep-
tide and protein based vaccines need extra -adjuvants
to achieve a similar goal ”.
From Nature cell research letter to the editor article
Letter to the Editor August 2020 A COVID-19
m-RNA vaccine encoding SARS-CoV-2 virus-like
particles induces a strong antiviral-like immune re-
sponse in mice
Jing Lu et al : Cell Research
“ we designed 3 m-RNA vaccine candidates for
COVID-19, and they encode various forms of AGs
in vaccinated -hosts . RQ3011-RBD encodes the
receptor-binding domain of the S spike glycol-
protein (residues 331–524) of SARS-CoV-2 with
an N-terminal signal peptide and a C-terminal
membrane-anchoring helix. Vaccine RQ3012-Spike
encodes the full-length wild-type S, while RQ3013-
VLP is formulated from a cocktail of m-RNAs en-
coding 3 structural- proteins: S, M (membrane), and
E (envelope) to form SARS-CoV-2 virus-like par-
ticles (VLPs). To increase the expression capacity
of m-RNA vaccines, all m-RNAs were subjected to
an in-depth sequence optimization procedure of 2
parameters: codons in the DNA template and mod-
ified nucleotides incorporated into m-RNA. We de-
signed ten coding sequences of the S- gene (3822 nu-
cleotides in length) with varying GC-content, main-
taining the maximum codon adaptation -index. For
each DNA template, we tested six m-RNA species
with various modified nucleotides. The m-RNA can-
didates (total of 60) displayed a considerable vari-
ation in their abilities to express S in HEK 293A-
cells”.
JUNE 02, 2021 A single dose of self-transcribing
and replicating RNA-based SARS-CoV-2 vaccine
produces protective adaptive immunity in mice
Ruklanthi de Alwis et al
FIGURE 1: Design and expression of a
SARS-COV-2vaccine with conven onal m-RNA and
self-transcribing andreplica ng RNA (STARR)
pla orms
“A) diagram of the SARS-CoV-2 self-replicating
STARR -RNA (LUNAR-COV19) and conventional
m-RNA vaccine constructs. The STARR construct
encodes for the 4 non-structural proteins, ns1–ns4,
from Venezuelan equine- encephalitis virus (VEEV)
and the un-modified full-length pre-fusion spike-
protein of SARS-CoV-2. The m-RNA construct
also codes for the same SARS-CoV-2 full-length
spike-protein. (B) Physical characteristics and RNA
trapping efficiency of the LNPs encapsulating con-
ventional m-RNA and LUNAR-COV19 vaccines.
(C) Western- blot detection of SARS-CoV-2 Spike-
protein following transfection of Hep3b cells with
LUNAR-COV19 and conventional m-RNA. (D) In
vivo comparison of protein expression following
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i.m. administration of LNPs containing luciferase-
expressing STARR -RNA or conventional m-RNA.
BALB/c mice (n = 3/group) were injected i.m.
with 0.2 µg, 2.0 µg, or 10.0 µg of STARR RNA
or conventional m-RNA formulated with the same
LNPs. Luciferase- expression was measured by in
vivo bio-luminescence on days 1, 3, and 7 post-
i.m. administration. Results are shown as mean
with standard deviation error- bars. S1, S domain
1; S2, S domain 2; TM, trans-membrane domain;
CP, cytoplasmic domain; aka.”
Form AZ covid19 vaccine technical sheet :”
“QUALITATIVE AND QUANTITATIVE COM-
POSITION
These are multidose vials which contain 8 doses or
10 doses of 0.5 mL per vial (see section 6.5).One
dose (0.5 mL) contains:Chimpanzee- Adenovirus
encoding the SARS-CoV-2 Spike glycoprotein
(ChAdOx1-S)
*, not less than 2.5 × 108 infectious -units
(Inf.U)*Produced in genetically modified human
embryonic- kidney (HEK) 293 cells and by recom-
binant DNA technology.”
For the Pfizer one:
“QUALITATIVE AND QUANTITATIVE COM-
POSITION
This is a multi-dose vial and must be diluted before
the use. One vial (0.45 mL) contains 6 doses of 0.3
mL after dilution, see sections 4.2 and 6.6.
One dose (0.3 mL) contains 30 micrograms of
tozinameran, a COVID-19 m-RNA Vaccine (em-
beddedin lipid nano-particles).
Tozinameran is a single-stranded, 5’-capped
messenger- RNA (m-RNA) produced using a cell-
free
in vitro transcription from the corresponding DNA
-templates, encoding the viral spike (S) protein of
SARS-CoV-2.”
Various relavant literature was and is produced to
clarify aspect like this.
The same aim of this work is to observe some aspect
related m-RNA vaccine
Like pharmaco-kinetics , encoding profile, toxico-
logical implication in covid-19 disease
And effect played by coronavirus spike -protein .
But before to start this work it is interesting to
consider some relevant literature involved in the
topics under investigation:
in publication : FOCUS ON COVID-19 Vaccines:
m-RNA Vaccines
5th revision: July 2021 Public helath Ontario
“m-RNA vaccines work by delivering instructions
to human cells to produce a viral -protein, which
is then recognized by the body as foreign. These
proteins, AGs, use the body’s normal processes to
safely produce immune -response : Non-replicating
( non-amplifying) RNA -vaccines are the simplest
type and consist of m-RNA coding for the viral
AG. Our cell- machinery is used to make a specific
viral- AG and once this is accomplished, the m-
RNA is cleared.9,10 COVID-19 m-RNA vaccines
are non-replicating RNA vaccines. Self-replicating (
or self-amplifying) RNA vaccines consist of an RNA
coding for the viral- AG and the virus’ replication
machinery, allowing for abundant production of
viral -AG. m-RNA vaccines are encapsulated in a
lipid- coat, commonly referred to as a lipid nano-
particle (LNP), which allows them to easily cross
cell -membranes into our cells. Once inside in
our cells, m-RNA is released into the cytoplasm
where the body’s cell machinery makes copies of
the SARS-CoV-2 spike glycoprotein- AG. The m-
RNA instructions are then rapidly broken down and
disposed of by our cells. Next, the SARS-CoV-2
spike -glycoprotein AG is temporarily displayed
on the surface of our cells, where it is recognized
as foreign and activates B (AB-mediated) and T
(cell-mediated) cells . Activation of cell-mediated
immune- responses are expected to play a central-
role in providing us with long-term protection.AB-
mediated responses directed against the SARS-CoV-
2 spike –glyco-protein are believed to be important
for blocking the virus from entering into our cells. ”
In article Toxicology Vol.185 April 2003 Reproduc-
tive toxicity testing of vaccines
François Verdiera et al
“Vaccines play a relevant role in the prevention of
human -birth defects by protecting the pregnant
woman from teratogenic or vs harmful infections.
Until now, it has not been common -practice to
perform pre-clinical developmental toxicity tests for
new vaccines. Despite the excellent safety record of
vaccines, increased attention is now being given
to the feasibility of screening new- vaccines for
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developmental hazards in animals before their use
in the humans. Contrary to previous assumptions,
many vaccines are now given to
potentially pregnant- women. Any new components
of the vaccine -formulation (adjuvants, excipients,
stabilisers ,preservatives) could also be tested for
influences on development, although based on past
experience the risks are limited by the very low -
dosages used. The conferred immunity following
vaccination lasts for several years.The developing
conceptus may theoretically be exposed to the
induced AB and/or sensitised T-cells, even if
the pregnant -woman was last vaccinated during
childhood (particularly if she encounters the AG
during pregnancy through exposure to infection).
Non-clinical safety studies may be employed as
an aid for hazard identification. In these work
studies interactions of the vaccine with the maternal
immune- system IS or with the developmental
systems of the offspring are considered. Post-natal
examinations are necessary to detect all possible
manifestations of developmental -toxicity, such as
effects on the immune -system. Species selection for
the pre-clinical studies is based on immune-genicity
to the vaccine and the relative timing and rate of
transfer of maternal AB to the offspring. A single
study design is proposed for the pre- and post-natal
developmental- assessments of vaccines in rodents
and rabbits”
And in website https://www.technologyne2rks.com
/biopharma/articles/regulatory-requirements-for-tox
icity-studies-to-support-vaccine-clinical-developme
nt-and-348662
Toxicity -Studies in Vaccine Clinical Development
and Registration
May 19 2021 Lawrence Segal PhD ERT
“Before the safety and efficacy of a new vaccine can
be evaluated in a Phase I clinical trial with human
subjects, it must first undergo non-clinical safety
evaluation in animal models. Regulatory Agencies
such as EMA, FDA, PMDA, MHRA, Health Canada
and others require that safety- studies performed
prior to Phase 1 must be conducted under GLP
standards.Additional non-clinical safety studies are
required during the clinical development process and
are normally submitted as part of the registration
dossier for the vaccine. Specific additional non-
clinical safety -studies are needed for adjuvants and
immune-stimulants”.
Kim, E.S. et al
Spike-proteins of SARS-CoV-2 InducePathological
Changes in Molecular
Delivery and Metabolic Function in the Brain
Endothelial Cells. Viruses
2021, MDPI
“Brain endothelial -cells (ECs), one of the compo-
nents of the blood–brain barrier BBB, are a major
hurdle for the entry of pathogenic or infectious
agents into the brain. They strongly express ACE2
for its normal physiological- function, which is
also well-known to be an opportunistic receptor for
SARS-CoV-2 spike-protein, facilitating their entry
into host -cells.We identified rapid internalization of
the RBD S1 domain (S1) and active -trimer (Trimer)
of SARS-CoV-2 spike-protein through ACE2 in
brain ECs. Internalized S1 increased Rab5, an early
endosomal marker while Trimer decreased Rab5 in
the brain ECs. The permeability of transferrin and
dextran was increased in S1 treatment but decreased
in Trimer, respectively. S1 and Trimer both induced
mitochondrial -damage including functional deficits
in mitochondrial -respiration. this study shows
that SARS-CoV-2 itself has toxic effects on the
brain ECs including defective molecular delivery
and metabolic- function, suggesting a potential
pathological -mechanism to induce neurological -
signs in the brain.”
In : A Comprehensive Guide to Toxicology in
Nonclinical Drug Development.
2016 Nov Preclinical Toxicology of Vaccines M.D.
Green et al :
“To ensure the safety of new- vaccines, preclin-
ical toxicology studies are conducted prior to
the initiation of, and concurrently with, clinical-
studies. There are 5 different types of preclinical
toxicology- study in the evaluation of vaccine -
safety: single and/or repeat dose, reproductive and
developmental, mutagenicity, carcino-genicity, and
safety pharmacology. If any adverse effects are
observed in the course of these studies, they should
be fully evaluated and a final safety decision made
accordingly. Successful pre-clinical toxicology
studies depend on multiple factors including using
the appropriate study- designs, using the right animal
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model, and evoking an effective immune- response.
Additional in vivo and in vitro assays that establish
the identity, purity, safety, and potency of the
vaccine play a significant role in assessing critical-
characteristics of vaccine- safety
Reproductive - developmental toxicology -studies
should be included in the IND package if the
vaccine under study is intended to be administered
to women of childbearing potential.Certain vaccines
may automatically be contra-indicated for pregnant
women or to those planning to become pregnant .
the small-pox vaccine is contra-indicated for women
who are pregnant, and women who plan to become
pregnant within 4 weeks of vaccination. pregnant
women are advised to avoid close contact with
persons recently vaccinated, as in the case of rubella
.
Studying the potential effects of the vaccine on
fertility, fetal development, and postnatal develop-
ment of the offspring is critical . Sexual- organs and
their functions, endocrine regulation, fertilization,
transport of the fertilized -ovum, implantation, and
development could all be affected by toxic effects of
the vaccine .
Abnormal development of the fertilized -egg through
the embryo, fetus, and the offspring all the way to
maturity, due to test-vaccine exposure, is a subset
of reproductive- toxicology called developmental
toxicology. Developmental -studies include the
studies of the prenatal (embryonic and fetal) and
post-natal (development following birth until the
end differentiation of organs is achieved) events. It
has been confirmed through the development of this
vaccine that studies in animals remain relevant to the
control of infectious- diseases in humans.
Animal models in human vaccine development have
different applications, such as:
1.Route of infection, transmission of disease, and
analysis of disease -pathogenesis
2.Host immune responses to natural -infection and
vaccination characterization
3.Onset and duration of vaccine-induced immunity
assessment
4.Mucosal versus systemic -immunity induction
5.Novel strategies for vaccine -delivery and formu-
lation development.
6.Clinical symptoms and disease transmission
following infection- reduction
7.Novel vaccination concepts (such as in utero or
maternal -immunization) development
Vaccine parameters requiring the consideration of
animal -models are :
1.Vaccine -safety
2.Duration and onset of immunity
3.Mucosal, maternal, and neonatal- vaccination
4.Novel vaccine technologies
5.Vaccination of the elderly
6.Therapeutic vaccines for non-infectious diseases
On 23 august 2021 FDA completely approved Pfizer
SARS-COV-2 vaccine. :
“This is a great news for the world health -systems ,
nations, government and patients”.
But even this fact and the literature show no global
evicence in affecting fertility by covid-19 vaccine It
is the same interesting to verify some aspect involved
in covid-19 disease in pregnancy and in the before
stages.
What is obviously is that there is a great difference
in the spike -protein level during
covid-19 disease vs after a vaccine procedure .
The pathological effect played by this disease can
be of interest to better understand also the Vaccine-
profile.
And finally what is the global effect in using nano-
lipide to carry the m-Rna ?
Not only to avoid innate immune- sensor and to
protect it by degradation but also to increase cell
intake of this M-rna “.
According EMA News 03/09/2021 : COVID-19
vaccines: EMA reviewing cases of multisystem
inflammatory syndrome
“EMA’s safety committee (PRAC) is assessing
whether there is a risk of multi-system inflammatory
syndrome (MIS) with COVID-19 vaccines following
a report of MIS with Comirnaty.”
Ema REPORT : 14 July 2021 COVID-19 vaccine
safety update
COMIRNATY
“BioNTech Manufacturing Gmb “HPRAC con-
cluded that myo-carditis and pericarditis can occur
in very rare- cases following vaccination with
Comirnaty and should be added in
the product information as new side -effects, together
with a warning to raise aware”
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And FROM TECHNICAL SHEET Vaxzevria
suspension for injection
COVID-19 Vaccine (ChAdOx1-S [recombinant])
Fertility, pregnancy and lactation Pregnancy
“There is limited experience with use of Vaxzevria
in pregnant- women.
Animal studies do not indicate direct or indirect
harmful -effects with respect to pregnancy,
embryo/foetal development, parturition or post-natal
development .
Administration of Vaxzevria during pregnancy
should only be considered when the potential
benefits outweigh any potential- risks for the mother
and fetus”
Fertility :Animal -studies do not indicate direct or
indirect harmful effects with respect to fertility
AND “Thrombosis with thrombo-cytopenia syn-
drome and coagulation disorders
Thrombosis with thrombo-cytopenia -syndrome
(TTS), in some cases accompanied by bleeding,
has been observed very rarely following vaccination
with Vaxzevria”.
And for Cominarty : “Fertility, pregnancy and
lactation
Pregnancy There is limited experience with use of
Comirnaty in pregnant women. Animal studies do
not indicate direct or indirect harmful effects with
respect to pregnancy, embryo/foetal development,
parturition or post-natal development . Adminis-
tration of Comirnaty in pregnancy should only be
considered when the potential- benefits outweigh
any potential risks for the mother and foetus.”
Related ADR reported in technical sheet “Blood and
lymphatic system disorders : frequency “ Common”
for “Thrombo-cytopeniaa”
The overall safety of Vaxzevria is based on an
analysis of pooled data from 4 clinical trials
conducted in the UK, Brazil, and South Africa.
At the time of analysis, 24,244 participants ≥18
years old had been randomised and received either
Vaxzevria or control. Out of these, 12,282 received
at least one dose of Vaxzevria and 10,448 received
2 doses. The median duration of follow-up was 81
days post-dose 2, with 7,158 participants completing
>2 months followup post-dose 2.
And in contra-indicationsis reported:
“Hyper-sensitivity to the active substance or to any
of the excipients listed in section 6.1.
Individuals who have experienced thrombosis with
thrombocytopenia -syndrome (TTS) following
vaccination with Vaxzevria .
Individuals who have previously experienced
episodes of capillary leak- syndrome “
“Thrombosis with thrombo-cytopenia syndrome and
coagulation -disorders
Thrombosis with thrombo-cytopenia syndrome
(TTS), in some cases accompanied by bleeding, has
been observed very -rarely following vaccination
with Vaxzevria. This includes severe cases pre-
senting as venous -thrombosis, including unusual
sites such as cerebral venous sinus thrombosis,
splanchnic vein thrombosis, as well as arterial
thrombosis, concomitant with thrombo-cytopenia.
Some cases had a fatal- outcome. The majority
of these cases occurred within the first 3 weeks
following vaccination and occurred mostly in
women under 60 years of age”.
“Capillary leak syndrome Very rare cases of
capillary leak- syndrome (CLS) have been reported
in the first days after vaccination with Vaxzevria. A
history of CLS was apparent in some of the cases.
Fatal -outcome has been reported.
CLS is a rare disorder characterised by acute
episodes of oedema mainly affecting the limbs,
hypotension, haemo-concentration and hypo-
albuminaemia. Patients with an acute episode of CLS
following vaccination require prompt recognition
and treatment. Intensive supportive- therapy is
usually warranted. Individuals with a known history
of CLS should not be vaccinated with this vaccine”
Neurological events
Guillain-Barré Syndrome has been reported very
rarely following vaccination with Vaxzevria”
JAMA Neurol . 2021 Sep 28
Characteristics and Outcomes of Patients With
Cerebral Venous Sinus Thrombosis in SARS-CoV-
2 Vaccine-Induced Immune Thrombotic Thrombo-
cytopenia
Mayte Sánchez van Kammen et al :
“ Importance: Thrombosis with thrombo-cytopenia
-syndrome has been reported after vaccination with
the SARS-CoV-2 vaccines ChAdOx1 nCov-19 and
Ad26.COV2.S (Janssen/Johnson & Johnson).
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To describe the clinical characteristics and outcome
of patients with cerebral venous- sinus thrombosis
(CVST) after SARS-CoV-2 vaccination with and
without TTS.
This cohort study used data from an international
registry of consecutive patients with CVST within 28
days of SARS-CoV-2 vaccination included between
March 29 and June 18, 2021, from 81 hospitals in 19
countries. data from patients with CVST between
2015 and 2018 were derived from an existing
international registry. Clinical characteristics and
mortality -rate were described for adults with (1)
CVST in the setting of SARS-CoV-2 vaccine-
induced immune thrombotic- Thrombo-cytopenia,
(2) CVST after SARS-CoV-2 vaccination not
fulling criteria for TTS, and (3) CVST unrelated to
SARS-CoV-2 vaccination. Patients were classified
as having TTS if they had new-onset thrombo-
cytopenia without recent exposure to heparin, in
accordance with the Brighton Collaboration interim-
criteria.
Clinical characteristics and mortality -rate.
Of 116 patients with post-vaccination CVST, 78
(67.2%) had TTS, of whom 76 had been vaccinated
with ChAdOx1 nCov-19; 38 (32.8%) had no
indication of TTS. The control group included 207
patients with CVST before the COVID-19 pandemic.
A total of 63 of 78 (81%), 30 of 38 (79%), and 145
of 207 (70.0%) patients, respectively, were female,
and the mean (SD) age was 45 (14), 55 (20), and
42 (16) years, respectively. Concomitant thrombo-
embolism occurred in 25 of 70 patients (36%) in the
TTS group, 2 of 35 (6%) in the no TTS group, and 10
of 206 (4.9%) in the control group, and in-hospital
mortality rates were 47% (36 of 76; 95% CI, 37-58),
5% (2 of 37;
95% CI, 1-18), and 3.9% (8 of 207; 95% CI, 2.0-7.4),
respectively. The mortality- rate was 61% (14 of 23)
among patients in the TTS -group diagnosed before
the condition garnered attention in the scientific
community and 42% (22 of 53) among patients
diagnosed later.
In this cohort study of patients with CVST, a distinct
clinical profile and high mortality- rate was observed
in patients meeting criteria for TTS after SARS-
CoV-2 vaccination.”
HEALTH AND SCIENCE Nordic countries are
restricting the use of Moderna’s Covid vaccine.
Here’s why OCT 8 2021 Chloe Taylor
“Finland and Sweden are limiting the use of
Moderna’s Covid-19 vaccine in young people over
concerns around rare cardio-vascular side effects.
Finland’s national health authority, THL, announced
Thursday that it would pause the use of Moderna’s
Covid vaccine in young men. All males aged 30
or younger would be offered the Pfizer-BioNTech
vaccine instead, THL said.”
According a “The scientist “article :
Moderna Vaccine Paused for Young People in 2
European Countries
Health- authorities in both countries announced
that people under certain age cutoffs are now
ineligible for the Spikevax COVID-19 shot due to its
association with heart inflammation. young woman
smiling Chloe Tenn Oct 6, 2021
“Update (Oct 15, 2021): According to Reuters, some
countries are only administering one dose of the
Pfizer/BioNTech vaccine in children and teen-agers
out of concern over possible rare cardio-vascular side
effects, which are more common after the second
shot than the first. included Norway, South Africa,
UK.
Update (Oct 11): According to Reuters, the Danish
-Health Agency has retracted its statement about
pausing the Moderna vaccine for those under 18
years old, and is in fact still using both Moderna’s
and Pfizer’s vaccines in this age group.
Update (Oct 8, 2021): Finland’s health agency
announced yesterday that it is suspending use of
Moderna’s Spikevax shot in males under 30 years
of age, who will be offered the Pfizer/BioNTech
vaccine instead, the Associated -Press reports.
“The Danish- Health Agency is suspending adminis-
tration of Moderna’s COVID-19 vaccine for people
under 18 years of age, while Sweden announced
today that it is doing the same for people under 30 (
Reuters) .
Both countries cited data showing a potential
increased risk of rare inflammatory -heart conditions
known as myo-carditis and peri-carditis among
younger people who receive the Moderna shot, and
recommended that individuals in those age groups
get the Pfizer/BioNTech vaccine instead. Canadian
-vaccine data showed increased cases of heart-
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inflammation particularly in adolescents and young
adults after receiving the Moderna OVID-19 vaccine
Spikevax, according to Reuters. Health experts from
both Sweden and Denmark maintain that the risk of
either myo-carditis or peri-carditis after receiving
Spikevax remains small.
The US Centers for Disease -Control and Prevention
notes that young males seem to be more at risk of the
inflammatory conditions after receiving Moderna’s
or Pfizer’s m-RNA vaccines, and that patients
who develop these conditions following COVID-
19 vaccination tend to recover quickly. The agency
continues to recommend that everyone 12 and older
receive a COVID-19 vaccine, as the risks posed
by COVID-19 far outweigh those associated with
vaccination.”
Nature cell discovery articles article 26 October 2021
Comprehensive investigations revealed consistent
patho-physiological alterations after vaccination
with COVID-19 vaccines
Jiping Liu et al :
“articipants, clinical data collection, and procedures
Healthy adult -volunteers were recruited to the pro-
gram. All subjects underwent a physical examination
and completed a questionnaire by trained doctors.
Healthy adult aged 18–60 years, with axillary
temperature ≤ 37.0 ◦
C, negative for SARS-CoV-
2 nucleic acid test, and
willing to complete all scheduled study processes
were enrolled in the study. People with epilepsy,
brain or mental- diseases, history of allergies, un-
controlled major chronic illnesses, and clinically
significant abnormal findings on bi-ochemistry,
hematology tests were excluded. Pregnant or breast-
feeding women were also excluded.
A total of 11 participants were enrolled and vacci-
nated to evaluate the clinical- safety and dynamic
changes in the immune -system. Among these, five
participants (cohort A) were vaccinated with 4 µg
dose of inactivated SARS-CoV-2 Vaccine (Vero
Cell) on days 1 and 14, and six participants (cohort
B) received a 4 µg dose of the vaccine on days 1 and
28. Inactivated SARS-CoV-2 Vaccine (Vero- Cell)
(China Biotechnology Group -Corporation) was
administered intra-muscularly into the deltoid. All
vaccines were approved by the National Institutes
for Food and Drug Control of China.
Lab. safety tests including infection-related indices
(C-reactive protein, serum amyloid A protein),
hematologic- parameters (white blood cell counts,
neutrophil counts, lymphocyte counts, monocyte-
counts, red blood cell counts, hemoglobin, platelet
counts), coagulation function-related indices (PT,
APTT, fibrinogen, prothrombin activity, INR), blood
glucose-related parameters (fasting plasma glucose,
HbA1c), serum lipid (CLT, triglyceride, HDL-C,
LDL-C), cardiac function-related enzymes (CK,
CK-MB), electrolytes (potassium, sodium, chloride,
bicarbonate, total calcium, MG), liver function-
related bio-markers
( albumin, ALT, AST, total bilirubin), renal
function-related markers (creatinine, uric acid, BUN,
estimated glomerular filtration -rate) were measured.
We report, besides generation of neutralizing
AB, consistent alterations in hemoglobin- A1c,
serum sodium and potassium levels, coagulation
profiles, and renal functions in healthy volunteers
after vaccination with an inactivated SARS-
CoV-2 vaccine. Similar changes had also been
reported in COVID-19 patients, suggesting that
vaccination mimicked an infection. Single-cell m-
RNA sequencing (scRNA-seq) of peripheral blood
mononuclear -cells (PBMCs) before and 28 days
after the first inoculation also revealed consistent
alterations in gene expression of many different
immune- cell types. Reduction of CD8+ T cells
and increase in classic monocyte contents were
exemplary. scRNA-seq revealed increased NF-κB
signaling and reduced type I interferon -responses,
which were confirmed by biological assays and
also had been reported to occur after SARS-CoV-
2 infection with aggravating symptoms. Our study
recommends additional- caution when vaccinat-
ing people with pre-existing clinical conditions,
including diabetes, electrolyte imbalances, renal
dysfunction, coagulation -disorders. Coagulo-pathy
is another COVID-19-induced clinical condition. We
found that coagulation profiles changed significantly
after vaccination, in the short-term (7 days) after the
1st inoculation, coagulation profiles were leaning
toward shorter PT, whereas the long-term (28 and
42 days) effect was toward activated APTTand PT
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prolongation . By day 90, the profiles returned back
to those before vaccination . To study detailed gene
-expression changes induced by vaccination, we
merged individual samples into pseudo-bulk samples
and used paired sample test to identify differentially
expressed -genes (DEGs) .
Significantly upregulated- genes were involved
in “TNFα signaling via NF-κB”, “inflammatory-
responses”, and “cytokine-cytokine receptor in-
teraction”, “IL6-JAK STAT3 signaling”, “coag-
ulation”, “hypoxia”, which had been reported
for COVID-19, while cell cycle-related pathways
were down-regulated . These results supported the
notion that vaccination mimicked an infection.
Echoing the clinical measurement results, genes
related to “cholesterol homeostasis”, “coagulation”,
and “inflammatory- response” (CXCL8, CD14,
IL6, and TNFRSF1B), “TNFα signaling via NF-
κB” (NFKB1, NFKB2, NFKBIE, TNFAIP3,
and TNFSF9) and “hypoxia” (HIF1A) were up-
regulated. “TGFβ signaling”, “IL2-STAT5 signal-
ing” (IFNGR1, MAPKAPK2, and CASP3), and
“IL6-JAK-STAT3 signaling”-related genes were
also up-regulated.
Vaccination-induced inflammatory- responses in
monocytes
Recent reports have described conserved host
immune -response signatures to respiratory viral
infections, namely the Meta-Virus Signature (MVS),
which is also conserved in SARS-CoV-2 infection.
Higher MVS scores are associated with infection.
In all, 380 (158 positively- and 222 negatively
contributed to MVS scores) out of 396 (161
positively- and 235 negatively contributed) genes
selected for MVS measurement were detected in
our dataset. To investigate host immune- responses
after vaccination with inactivated SARS-CoV-2, we
separated the positive and negative gene sets and
calculated MVS- scores . The MVS -scores were
substantially higher after vaccination , suggesting
that vaccination mimicked an infection. the positive
MVS gene set was predominantly expressed in
monocytes, while the negative set in lymphocytes,
indicating different cell-type-specific immune -
responses would take place after vaccination . To
investigate which pathways were associated with
MVS-positive gene set and MVS-negative gene set,
we calculated Spearman- correlation among MVS
gene sets scores and previously identified differen-
tially enriched pathways using our scRNA-seq data .
The most highly correlated pathway with MVS score
and MVS-positive set was “Inflammatory response-
signaling”, which was strikingly up-regulated in
monocyte after the vaccination, together with CD14,
FPR1, C5AR1, NAMPT, NLRP3, CDKN1A, and
IFNGR2. MVS-negative set correlated well with
“Cyto-toxicity -signature”, represented by NKG7,
CCL4, CST7, PRF1, GZMA, GZMB, IFNG, and
CCL3 expression, significantly decreased in many T-
cell subtypes but not NK -cells after the vaccination
.”
February 2021
m-RNA vaccine-elicited AB to SARS-CoV-2 and
circulating variants
Zijun Wang et al : Nature volume 592
“ we report on the AB and memory B cell responses
of a cohort of 20 volunteers who received the
Moderna (m-RNA-1273) or BNT162b2 vaccine
against the SARS-CoV-2 . Eight weeks after the
second injection of vaccine, volunteers showed high
levels of IgM and IgG anti-SARS-CoV-2 spike-
protein (S) and receptor-binding-domain (RBD)
binding titre. The plasma neutralizing activity and
relative numbers of RBD-specific memory B- cells
of vaccinated volunteers were equivalent to those
of individuals who had recovered from natural -
infection “ .
From : FDA FACT SHEET FOR HEALTHCARE
PROVIDERS ADMINISTERING VACCINE
EMERGENCY USE AUTHORIZATION (EUA)
OF
THE PFIZER-BIONTECH COVID-19 VACCINE
22 spet. 2021
“USE IN SPECIFIC POPULATIONS
Pregnancy Risk Summary
All pregnancies have a risk of birth defect, loss,
or other adverse outcomes. In the US general
population, the
estimated background risk of major birth defects and
miscarriage in clinically recognized pregnancies is
2% to 4% and 15% to 20%, respectively. Available
data on Pfizer-BioNTech COVID-19 Vaccine
administered to pregnant -women are insufficient
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to inform vaccine-associated risks in pregnancy.
In a reproductive -developmental -toxicity study,
0.06 mL of a vaccine formulation containing the
same quantity of nucleoside-modified messenger
ribonucleic acid (m-RNA) (30 mcg) and other ingre-
dients included in a single human- dose of Pfizer-
BioNTech COVID-19 Vaccine was administered to
female rats by the
Intra-muscular -route on 4 occasions: 21 and 14 days
prior to mating, and on gestation days 9 and 20. No
vaccine-related adverse effects on female- fertility,
fetal development, or postnatal development were
reported in the study. Myocarditis and Pericarditis
Post-marketing data demonstrate increased risks of
myocarditis and pericarditis, particularly within 7
days following the second- dose. The observed risk
is higher among males under 40 years of age than
among females and older males. The observed risk
is highest in males 12 through 17 years of age.
Although some cases required intensive care support,
available data from short-term follow-up suggest that
most individuals
have had resolution of symptoms with conservative
-management. Information is not yet available
about potential long-term sequelae. The CDC has
published considerations related to myo-carditis
and pericarditis after vaccination, including for
vaccination of individuals with a history of myo-
carditis or pericarditis
(https://www.cdc.gov/vaccines/covid-19/clinical-co
nsiderations/myo-carditis.html).’’
Until present, the FDA has not issued any explicit
guidelines regarding the use of COVID-19 vaccines
in pregnant -women. the FDA refers to pregnant
women in the EUA letters and factsheets provided
to healthcare providers for the individual vaccines.
Review May 2021
The safety of Covid-19 m-RNA vaccines: a review
Pratibha Anand & Vincent P. Stahel Patient Safety
in Surgery volume 15
“EUA- letters for both the Moderna and the
Pfizer/BioNTech vaccines contain provisions oblig-
ing post-authorization observational studies and
label pregnant- women as a “population of interest”
for these studies, citing a lack of data regarding
vaccine risk in pregnancy. The Moderna vaccine
fact sheet specifically alludes to a reproductive-
toxicity study in female rats; adverse -effects
on fetal development, female -fertility, and early
offspring development were assessed, with no
adverse outcomes observed. The Moderna vaccine
also has a pregnancy exposure registry intended
to monitor pregnancy outcomes in women who
received the vaccine during the pregnancy. To date,
neither Moderna nor Pfizer have issued guidelines
or guidance regarding their vaccines and pregnancy
. The afore mentioned concerns not with standing,
recent data indicate “no difference in the composite
primary- outcome of pre-term birth, pre-eclampsia
with severe features, and cesarean delivery for fetal
indication among women with and without SARS-
CoV-2 infection diagnosed during pregnancy” (52
women [21 %] vs. 684 women [23 %]; relative risk,
0.94; 95 % CI, 0.73–1.21; P = .64). There were also
no still births among women with SARS-CoV-2
during pregnancy ”.
Front. Immunol., July 2021
From COVID-19 to Cancer m-RNA Vaccines:
Moving From Bench to Clinic in the Vaccine
Landscape Chiranjib Chakraborty et al:
“m-RNA Vaccine and the Innate Immune- Response
A self-adjuvant effect has also been noted for
m-RNA vaccines. In this effect, APCs recognize
m-RNA, subsequently triggering PRRs (pattern
recognition receptors). Pattern recognition receptor
members include TLR -family members, such as
TLR3, TLR7 and TLR8 , which are localized in
the endolysosomal area of the cell. Receptors in the
cytosol can detect nucleic- acids in the cytoplasm.
ssRNA molecules are recognized by 2 TLRs: TLR7
and TLR8 receptors. Auridine-rich tetramers are
a minimum requirement for both receptors for
activation. TLR7-mediated down-stream pathway
activation aids in type I IFN production . AU-
rich sequences induce TLR8-mediated down-stream
pathway activation, leading to a TNF response .
dsRNA triggers immune -system activation through
TLR3 recognition . Binding with the TLR3 receptor
requires a minimum length of 45 bp dsRNA . m-
RNA Vaccines for Cancer and Their Pre-Clinical
and Clinical Update
Different m-RNA-based cancer vaccines were
designed to target tumor-associated AGs. These
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AGs are more prevalent in cancerous -cells. The
majority of cancer vaccines are therapeutic rather
than prophylactic . These vaccines may stimulate
cell-mediated immune responses. 2 decades ago,
the proposal of RNA-based cancer vaccines was
published . Recently, several m-RNA-based cancer
vaccines have been developed that are registered
for different phases of clinical -trials . Few trials
were terminated due to lack of efficiency, immuno-
genicity, toxicity, and other side effects. Due to
lack of efficiency, one clinical trial (clinical trial no.
NCT01582672) was terminated. The trial was con-
ducted using an m-RNA vaccine against carcinoma.
Another clinical trial, an m-RNA-based prostate
cancer vaccine (clinical trial no. NCT01817738)
was terminated as the study’s outcome was not as
impactful as expected.“.
Table A : to be noted the number of TERMI-
NATED study vs COMPLETED
Article August 2020 Phase I/II study of COVID-19
RNA vaccine BNT162b1 in adults
Mark J. Mulligan et al Nature vol. 586
“after the first- dose, fever (defined as ≥38.0 ◦
C)
was reported by 8.3% (1 out of 12) of participants
who received 10 µg and 30 µg BNT162b1 and by
50.0% (6 out of 12) of individuals who received 100
µg BNT162b1. After the second- dose, 8.3% (1 out
of 12) of participants who received 10 µg BNT162b1
and 75.0% (9 out of 12) of participants who received
30 µg BNT162b1 reported fever of ≥38.0 ◦
C. On
the basis of the reacto-genicity reported after the
first dose of 100 µg and the second- dose of 30 µg,
participants who received an initial 100-µg dose did
not receive a second 100-µg dose. Fevers generally
resolved within 1 day of onset. No grade 4 systemic -
events or fever were reported (Fig. 3a, b). Most local
reactions and systemic events peaked by day 2 after
vaccination and esolved by day 7.”
Fig. n. 2 a, Systemic events and medication use
reported within 7 days after vaccination 1 for all
dose levels. b, Systemic events and medication use
reported within 7 days after vaccination 2 for the 10-
µg and 30-µg dose levels. Solicited systemic events
were: fatigue, headache, chills, new or worsened
muscle- pain, new or worsened joint pain (mild, does
not interfere with activity; moderate, some interfer-
ence with activity; severe, prevents daily activity),
vomiting (mild, 1–2 times in 24 h; moderate, >2
times in 24 h; severe, requires IV hydration), diar-
rhoea (mild, 2–3 loose stools in 24 h; moderate, 4–5
loose stools in 24 h; severe: 6 or more loose stools in
24 h); grade 4 for all events: emergency room- visit
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or hospitalization; and fever (mild, 38.0–38.4 ◦
C;
moderate, 38.5–38.9 ◦
C; severe, 39.0–40.0 ◦
C; grade
4, >40.0 ◦
C). Medication indicates the proportion of
participants who reported the use of antipyretic or
pain medication. Data were collected with the use of
electronic diaries for 7 days after each vaccination.
Fig n. 3 Major death causes for the 26 autopsy
COVID-19 cases. The major death- causes were se-
vere pulmonary injuries (n = 23), including COVID-
19-related respiratory failure without (n = 21) or with
(n = 2) pulmonary fungal -infection. The major death
causes for other 3 cases were pulmonary thromboem-
bolism, dissecting aneurysm rupture, and cardiovas-
cular disorders, respectively. b Schematic model for
SARS-CoV-2 organ- tropism. LNs, Lymph nodes. c
Heatmap showing SARS-CoV-2 distribution groups
and viral RNA (Log2) in postmortem organs in 26
autopsy- cases with COVID-19. LU, left upper; LL,
left lower; RU, right upper; RM; right middle; RL,
right lower. d Percentage of COVID-19 autopsy
cases in 3 groups of SARS-CoV-2 distribution. e
The correlation between SARS-CoV-2 viral RNA in
the lungs and the number of SARS-CoV-2-positive
organs. f Comparison of viral infection rate between
SARS-CoV-2 based on the current autopsy study and
SARS-CoV in the literature in post-mortem organs
from patients with COVID-19 and SARS. From June
2021 A cohort autopsy -study defines COVID-19
systemic patho-genesis Xiao-Hong Yao et al Cell
Research volume 31, (2021)
J clinical pathology review
Histopathological observations in COVID-19: a
systematic review FREE
Vishwajit Deshmukh,Rohini Motwani,Ashutosh
Kumar, Chiman Kumari, Khursheed Raza
“Although COVID-19 mainly affects respiratory and
immune -systems, but other systems like cardiovas-
cular, urinary,GI tract, reproductive system, NS and
integumentary system are not spared, especially in
elderly cases and those with co-morbidity”.
Xiao -Hong Yao et al :
“We found that the cellular components of alveolar
exudate were mainly CD68+ macrophages positive
for SARS-CoV-2 spike-protein ( IHC staining using
serial sections also identified the presence of SARS-
CoV-2 spike-protein in monocytes and macro-
phages in lymph
nodes and the spleen , as well as peripheral blood -
mononuclear cells in the postmortem lungs, kidneys,
lymph- nodes, spleen, intestines . Single-cell RNA-
sequencing (scRNA-seq) of lung tissues from a
COVID-19 autopsy- case (Case 17) within 2 h after
death revealed the presence of CD14+ monocytes
(CD14+ Mono-1, -2), monocyte-derived alveolar
macro-phages (MoAM-1, -2), and other cell- types
. CD14+ monocytes were characterized by VCAN
expression and MoAMs were positive for C1QA
and C1QC. We detected SARS-CoV-2 transcripts of
open reading frame 10 (ORF_10) and nucleocapsid
in alveolar CD14+ monocyte , confirming the
presence of SARS-CoV-2 in lung- monocytes.
We demonstrated that the majority of autopsy -cases
with systemic- virus distribution showed multiple
organ failures, supporting the systemic nature of the
disease. ur study identified SARS-CoV-2 presence in
endothelia located at several physiological- barriers
(blood–air, filtration, and blood–testis barriers),
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raising the possibility that the virus might invade
these barriers for dissemination. The virus was also
found in vascular endothelia of multiple organs,
which may cause vasculitis.The injured endothelia
may initiate vascular dysfunction and subsequently
a pro-coagulant state to induce thrombosis, as well
as ischemic or hemorrhagic- changes frequently
observed in fatal COVID-19 patients”.
25 February 2021
Comorbidities and inflammation associated with
ovarian cancer and its influence on SARS-CoV-2
infection Sima Chaudhari et al :
j.of Ovarian Research
“The ne2rk of inflammation-related genes modu-
lated in SARS-CoV-2 infection and the underlying
co-morbid conditions may promote alterations in
signaling pathways that could consequently lead to
severe inflammation-induced cancer pathogenesis
and/or impart undesirable- outcomes in OC patients”
Li Fet al. Impact of COVID-19 on
female fertility: a systematic review and meta-
analysis protocol. BMJ Open 2021
“SARS-CoV-2 appears to have shown adverse
effects on the reproductive- system. Some studies
have shown that SARS-CoV-2 might affect female
fertility and disturb female reproductive -functions.
SARS-CoV-2 may invade target cells by binding
to ACE2, thereby affecting female fertility. ACE2,
which is widely expressed in ovaries, uterus, vagina
and placenta, regulates the levels of angiotensin II
(Ang II) and Ang-(1–7) to exert its physiological
-functions.
ACE2, Ang II and Ang-(1–7) could regulate
follicular -development and ovulation, regulate
corpus luteum angiogenesis and degeneration, and
affect endometrial tissue- growth. Ovarian reserve
is a key determinant of female- fertility. Diminished
ovarian reserve could affect fecundity by reducing
egg quality. ACE2 is highly expressed in the ovaries.
The ovarian reserve function should be the primary
observation indicator for the impact of COVID-19
on female- fertility”.
Mol Hum Reprod . 2020 Jun
Potential influence of COVID-19/ACE2 on the
female reproductive system
Yan Jing et al
“The available evidence suggests that ACE2 is
widely expressed in the ovary, uterus, vagina and
placenta. We believe that apart from droplets and
contact- transmission, the possibility of mother-to-
child and sexual transmission also exists. Ang II,
ACE2 and Ang-(1-7) regulate follicle development
and ovulation, modulate luteal angiogenesis and
degeneration, and also influence the regular changes
in endometrial- tissue and embryo development.
Taking these functions into account, 2019-nCoV
may disturb the female reproductive- functions
through regulating ACE2”.
Am J Obstet Gynecol MFM. 2021 Nov
COVID-19 vaccination in pregnancy: early experi-
ence from a single institution
Megan E. Trostle et al :
“We identified 424 pregnant women who received an
m-RNA vaccination. Of those, 348 (82.1%) received
both doses and 76 (17.9%) received only 1 dose
This series describes our experience with women
who received an m-RNA COVID-19 vaccine
during the pregnancy. In line with other published
findings,2 we observed no concerning trends. There
were no stillbirths. Our 6.5% rate of spontaneous
abortion is within the expected rate of 10%,3 and
our preterm birth- rate of 5.9% is below the national
average of 9.5%.4 Our rate of pregnancy-related
hypertensive disorders is higher than our baseline
institutional rate of 9.5%, this may be because of the
under-lying characteristics of our study population
or skewing of our small- sample size”
N Engl J Med 2021 Preliminary Findings of m-RNA
Covid-19 Vaccine Safety in Pregnant Persons Tom
T. Shimabukuro et al
“A total of 35,691 v-safe participants 16 to 54 years
of age identified as pregnant.
Among 827 participants who had a completed
pregnancy, the pregnancy resulted in a live- birth
in 712 (86.1%), in a spontaneous- abortion in 104
(12.6%)”
Review Prim Care. 1993 Sep Spontaneous abortion
B S Apgar 1, C A Churgay
“Spontaneous abortion- rates vary with maternal age,
but the overall incidence is approximately 2% of
clinically recognized pregnancies. The incidence of
clinically unrecognized loss is approximately 20%”
According to : Stroobandt, S.; Stroobandt,R.
JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1692
17. MEERP LTD
MAURO LUISETTO ET AL.
Data of the COVID-19 m-RNAVaccine V-Safe
Surveillance System and Pregnancy Registry Re-
veals Poor Embryonic and Second Trimester Fetal
Survival Rate. Comment on Stuckelberger et al.
SARS-CoV-2 Vaccine Willingness among Pregnant
and Breastfeeding Women during the First Pandemic
Wave: A CrossSectional Study in Switzerland.
Viruses 2021 “we would like to advise readers
that the referenced article contains a serious error
regarding the interpretation of the data presented in
Table 4 ( of DOI: 10.1056/NEJMoa2104983.
Prospective cohort -studies are routinely performed
to establish the safety of novel obstetric interven-
tions. Such studies typically compare, at the same
gestational age, the well-being of a cohort that under-
went the intervention to that of a comparable control
cohort (or, in the absence of this, the pertaining
population) without the intervention. The
cited study compared the control population’s
incidence rate of spontaneous- abortions
of 10 to 26% prior to week 20 to the incidence among
the 827 study participants of which
700 received their first dose only in the third
trimester, after week 26. a
correct comparison with the remaining 127 partic-
ipants sets the 104 spontaneous abortions recorded
prior to week 20 at an alarming incidence of 82%, 3
to 8 times higher than in the control population. This
observation suggests that obstetric vaccine -safety
is severely compromised during pregnancy and
may lead to decreased willingness among pregnant-
women to be vaccinated”.
Stuckelberger, S. et al
Data on COVID-19 m-RNA-Vaccine Safety during
Pregnancy Might Be Subject to Selection Bias.
Reply to Stroobandt, S.; Stroobandt, R. Data of the
COVID-19 m-RNA-Vaccine V-Safe
Surveillance System and Pregnancy Registry Re-
veals Poor Embryonic and
Second Trimester Fetal Survival Rate.
Comment on “Stuckelberger et al.SARS-CoV-2
Vaccine Willingness among Pregnant and Breast-
feeding
Women during the First Pandemic Wave: A Cross-
Sectional Study in Switzerland. Viruses 2021, 1199”.
“Stroobandt, S. and Stroobandt, R.’s interpretation
leads to an 82% risk
of spontaneous- abortion (104 spontaneous abortions
for 127 participants exposed to the
first dose of the COVID-19 vaccine during the first-
trimester and who have completed their
pregnancy) instead of the 12.6% calculated by
Shimabukuro and coll (104 spontaneous abortions
for 807 participants with a pregnancy outcome at the
time of analysis).
As we totally disagree with their interpretation of
these data, we would like to take the
opportunity to respond to their letter.
Spontaneous abortion incidence- rates are sensitive
to gestational age at enrollment as the risk decreases
over gestation, with later enrollees carrying a lower
risk, or no risk of the outcome as mentioned by
Stroobandt, S. and Stroobandt, R. Thus, a dedicated
analysis estimating the rate of spontaneous- abortion
by considering all women vaccinated during the first-
trimester who either had or were at risk of having
a spontaneous -abortion ( beyond 20 weeks of a-
menorrhea at the time of the analysis), even if still
with an ongoing pregnancy, would probably have
provided a fairer estimate. With the information
available in the paper by Shimabukuro et al., the
spontaneous -abortion risk would have probably
been around 10% (104 spontaneous abortion for
1132 women vaccinated during the first trimester),
which is even lower than the previously published
estimate.”
Fig. 4 a Schematic diagram of the m-RNA-based
vaccine targeted to the spike-protein (S protein) of
severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2). The m-RNA-based vaccine targeted
to the S protein of SARS-CoV-2 works by active -
immunization. This technique will not use part of the
virus but only recombine m-RNA of the S protein in
vitro according to the gene sequence, which is coated
with lipid nano-particles for effective delivery. Once
injected into the muscle, the myo-cytes take up the
lipid nano-particle (LNPs) and then release the m-
RNAs into the cytoplasm for translation into the S
proteins. These endogenously synthesized S- pro-
teins will be secreted to activate both humoral and
cellular immune responses. S protein – spike-protein;
IM – IM, LNP – lipid nano-particle; DC – dendritic
cell; MHC – major histo-compatibility complex; Ag
MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1693
18. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN
EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK
– AG. From Med Sci Monit. 2020; 26: e924700-1–
e924700-8.
2020 May 5. 2020 Apr 21
An Evidence Based Perspective on m-RNA-SARS-
CoV-2 Vaccine Development
Fuzhou Wang et al :
Drug delivery Without these lipid shells, there would
be no m-RNA vaccines for COVID-19
Fragile m-RNA molecules used in COVID-19 vac-
cines can’t get into cells on their own. They owe their
success to lipid nano-particles that took decades to
refin
Ryan Cross March 6, 2021
“Modern LNPs can be traced back to work on sim-
pler systems called liposomes, hollow lipid spheres
often made of just 2 or 3 kinds of lipids. In the early
1980s, Cullis found that cancer drugs could diffuse
into these liposomes and get trapped in the hollow
core. When injected into animals with cancer, the
liposomes would slip through the leaky vasculature
of tumors, enter cells, and unleash a drug. Cullis, and
several others, started companies with the hope that
liposomes could safely deliver otherwise toxic drugs
into tumors in humans. Pegylated- lipids, in which
poly-ethylene glycol strands are attached to lipid
heads, have several functions in a nano-particle. PEG
helps control the particle size during formulation,
prevents the particles from aggregating in storage,
and initially shields the particles from being de-
tected by immune- system proteins in the body LNPs
take advantage of a natural process called receptor-
mediated endocytosis to get into cells, Madden ex-
plains. Upon binding to a cell, the nano-particle be-
comes encapsulated in an even bigger lipid bubble—
an organelle called an endosome. The endosome’s
acidic interior protonates the heads of the ioniz-
able -lipids, making them positively charged. That
positive- charge triggers a change in the shape of the
nano-particle, which scientists think helps it break
free from the endosome and ultimately release its
RNA cargo into the cell’s cytoplasm. Once released,
the RNA is free to do its job.
LNPs used in the COVID-19 vaccines contain just 4
ingredients: ionizable lipids whose positive charges
bind to the negatively charged backbone of m-RNA,
pegylated lipids that help stabilize the particle, and
phospho-lipids and cholesterol molecules that con-
tribute to the particle’s structure.
Thousands of these 4 components encapsulate m-
RNA, shield it from destructive enzymes, and shuttle
it into cells, where the m-RNA is unloaded and used
to make proteins”
Adv Drug Deliv Rev. 2021 Mar
Self-assembled m-RNA vaccines Jeonghwan Kim et
al
“Once saRNA is delivered to the cytosol, it goes
through the endogenous translation machinery in
ribosomes. The translation of the nsP series leads
to the production of precursor poly-proteins, form-
ing the early replication complex that synthesizes
the negative strand of RNA. Further cleavages of
poly-proteins yield the late replication complex that
synthesizes the positive strand of genomic and sub-
genomic RNA by using the negative strand as a
template. As a result, 1 copy of the saRNA produces
multiple copies of RNA transcripts , overcoming
the limited cytosolic- delivery of in vivo m-RNA
therapeutics.
AG expression of saRNA increases gradually ini-
tially and lasts for an extended period of time ,
which prolongs the stimulation of AG-presenting
cells (APCs)”.
Mol Ther. 2018 Feb
Self-Amplifying RNA Vaccines Give Equivalent
Protection against Influenza to m-RNA Vaccines but
at Much Lower- Doses Annette B. Vogel et al
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19. MEERP LTD
MAURO LUISETTO ET AL.
“New vaccine platforms are needed to address the
time gap between pathogen emergence and vaccine
licensure. RNA-based vaccines are an attractive can-
didate for this role: they are safe, are produced cell
free, and can be rapidly generated in response to
pathogen
emergence. 2 RNA vaccine platforms are available:
synthetic m-RNA molecules encoding only the AG
of interest and self-amplifying RNA (sa-RNA).
sa-RNA is virally derived and encodes both the AG
of interest and proteins enabling RNA vaccine repli-
cation. Both platforms have been shown to induce an
immune- response, but it is not clear which approach
is optimal. We compared synthetic m-RNA and
sa-RNA expressing influenza virus hemagglutinin.
Both platforms were protective, but equivalent levels
of protection were achieved using 1.25 µg sa-RNA
compared to 80 µg m-RNA (64-fold less material).
Having determined that sa-RNA was more effec-
tive than m-RNA, we tested hemagglutinin from 3
strains of influenza H1N1, H3N2 (X31), and B (Mas-
sachusetts) as sa-RNA vaccines, and all protected
against challenge infection. When sa-RNA was com-
bined in a trivalent formulation, it protected against
sequential H1N1 and H3N2 challenges. From this we
conclude that sa-RNA is a promising platform for
vaccines against viral -diseases.”
Ther Deliv. 2016 May
m-RNA vaccine delivery using lipid nano-particles
Andreas M Reichmuth et al
“Geall et al. showed that self-amplifying m-RNA en-
capsulated in LNPs exhibits overall higher immune-
genicity than the non-encapsulated variant
the 2 most important parameters for LN accumula-
tion are LNP size and surface composition. reports
indicate decreasing lymphatic uptake with increasing
LNP size. Only small LNPs with a diameter smaller
than about 150 nm appear to enter the lymphatic
capillaries, and are subsequently drained to the pe-
ripheral -lymphatics . larger LNPs are retained at
the injection site . Larger LNPs are believed to be
recognized and cleared more rapidly by the comple-
ment system because they present a larger number of
recognition sites on their surface . Coating the parti-
cles with a PEG-containing lipid can reduce comple-
ment activation. The right amount of PEG coating
on the LNPs is critical. Carstens et al. showed that
PEG- coating clearly improves lymphatic- drainage.
It is well known that enhanced PEGylation of LNPs
leads to longer blood- circulation times”.
Molecular therapy 2021
A single dose of self-transcribing and replicating
RNA-based SARS-CoV-2 vaccine produces protec-
tive adaptive immunity in mice
Ruklanthi de Alwis et al
“Both candidates, one from Moderna and the other
from Pfizer-BioNTech, mediate immunity by a
prime and boost regimen through i.m. injection of
a messenger RNA (m-RNA) encoding the SARS-
CoV-2 S AG encased by a lipid nano-particle (LNP).
The use of adjuvants is obviated by the ability of the
LNP itself to activate an innate immune- response.
“ direct comparison of assay results are difficult
because of differences in S glycoprotein quaternary-
structure, LNP formulation composition, and virus
neutralization assays. trends in results can be com-
pared. One study in which the furin cleavage site was
inactivated produced similar anti-S glycoprotein IgG
titers at 4 and 9 weeks post-vaccination and neutral-
izing AB titers 9 weeks post-vaccination. this was
achieved at RNA doses at least 10-fold greater than
the lowest effective LUNAR-COV19 RNA dose (2
µg), and no virus challenge study was conducted to
demonstrate protective- immunity. These preclinical
results confirmed that LUNAR-COV19 vaccination
elicited innate immune -responses, in both the blood
and draining lymph- nodes of mice, similar to those
elicited by a m-RNA vaccine lacking the VEEV
replicase but at doses that were 5–50 times lower.”
From article on NATURE : sep 2021
THE TANGLED HISTORYOF M-RNA VAC-
CINES
“The fatty nano-particle around the m-RNA is made
of 4 types of lipid molecule. One of these is ‘ioniz-
able’:in the vaccine, many of these molecules have a
positive -charge and cling to negatively charged m-
RNA,but they lose that charge in the more alkaline
conditions of the blood-stream, reducing toxicity in
the body”
Med Sci Monit. 2020 May
An Evidence Based Perspective on m-RNA-SARS-
CoV-2 Vaccine Development
Fuzhou Wang et al
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20. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN
EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK
“The penetration of the lipid membrane- barrier is
the first step for exogenous m-RNA to reach the
cytoplasm before the translation of functional protein
happen . Also, the uptake mechanisms of m-RNA
vaccines show cell specificity , and the physico-
chemical properties of the m-RNA may significantly
influence its cellular delivery and organ distribution .
All these factors must be considered when designing
an effective m-RNA-based vaccine. Even so, an m-
RNA vaccine is still considered the most promising
candidate because it can be scaled rapidly, which
can save time when the rapidly spreading COVID-
19 emerged and started to infect millions of people
world-wide .
As a (+)ss-RNA virus, SARS-CoV-2 possesses self-
amplifying RNA that can realize extreme RNA repli-
cation in the cytosol . This finding supports the role
of m-RNA-based vaccine development. the safety
- efficacy of m-RNA vaccines for use in humans
remain unknown. The hypothetical benefits of m-
RNA vaccine seem strong, whereas limitations such
as the delivery and stability issues related to RNA
degradation, and the safety concerns due to immune-
genicity hinder its development . The results from the
phase I trial of the m-RNA-1273 vaccine are awaited
The m-RNA-based vaccines actively induce activa-
tion of both B cell responses and T cell cyto-toxicity.
The m-RNA vaccines use the m-RNA sequence of
the target protein that recombine in vitro, rather than
the sequence of the target AB.
the recombinant target protein m-RNA sequence is
carried by LNPs and enters the somatic cytoplasm
to achieve direct translation and encoding the target
protein. When the target- protein is released from
the host- cell, the AG-presenting cells will quickly
capture and process the heterologous- protein. Then,
the presentation of MHC I and MHC II on the surface
of the AG-presenting cell membrane . This step is
important for the subsequent activation of B -cells
and T -cells and is also the key to the humoral
and cytotoxic response. There are 4 main safety
and efficacy advantages of the use of m-RNA-based
antiviral vaccines over traditional approaches. M-
RNA-based antiviral vaccines minimize the potential
risk of infection and insertion-induced mutagenesis
due to natural degradation of m-RNA in the cellular
micro-environment . the immunogen’s high efficacy
due to engineered m-RNA structural modifications
improves its stability and translation efficacy. The
high potency of m-RNA-based vaccines capable
of generating potent antiviral neutralizing immune-
globulins with only one or 2 low-dose immunizations
may induce strong immune -responses by activating
both CD8+ and CD4+ T cells . 4th, engineered m-
RNA production facilitates large-scale production of
sufficient vaccine doses required to treat mass popu-
lations . All these factors make the m-RNA vaccine
more suitable for a rapid response to the emerging
COVID-19 pandemic. It is important to clearly un-
derstand the potential- risks of this type of m-RNA-
based vaccine, which include local and systemic
inflammatory- responses, the bio-distribution and
persistence of the induced immunogen -expression,
possible development of autoreactive AB and toxic
effects of any non-native nucleotides and delivery -
system components .”
Engineering precision nano-particles for drug deliv-
ery
Michael J. Mitchell et al Nature Reviews Drug Dis-
covery (2021)
“nano-particles (NPs) constitute a significant portion
of reported research and advancement.
NPs have the potential to improve the stability and
solubility of en-capsulated- cargos, promote trans-
port across membranes and prolong circulation times
to increase safety and efficacy.
Many early NP iterations were unable to over-come
these biological -barriers to delivery, but more recent
NP designs have utilized advancements in controlled
synthesis strategies to incorporate complex architec-
tures, bio-responsive moieties and targeting agents
to enhance delivery. These NPs can be utilized as
more complex systems — including in nano-carrier-
mediated combination therapies — to alter multiple
pathways, maximize the therapeutic efficacy against
specific macro-molecules, target particular phases of
the cell cycle or over-come mechanisms of drug-
resistance. NPs have been extensively explored in
vaccines against SARS-CoV-2 , with multiple suc-
cessful late-stage clinical trials. Moderna and BioN-
Tech use LNPs to encapsulate m-RNA that encodes
for a COVID-19 AG. As of 30 Nov 2020, Mod-
erna and BioNTech/Pfizer have met their primary
efficacy end points in phase III trials and have ap-
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MAURO LUISETTO ET AL.
plied for Emergency Use Authorization. As with
other applications, NP architecture, material proper-
ties and active targeting can affect cellular uptake,
AG presentation and the strength of the immune-
response.Macrophages, B- cells and dendritic cells
are all APCs and can be targeted by NPs to im-
prove the specificity of immune- activation. Passive
targeting includes optimizing size, shape ratios and
using positively -charged particles to interact with
the negatively charged cell membranes. APCs also
express numerous carbohydrate-
recognizing lectin receptors for endocytosis, and
these have been exploited for cell-specific active
-targeting. Some of these lectin receptors are ex-
pressed at high levels in certain APCs, such as the
C-type lectin receptors lymphocyte- AG 75 ( DEC-
205) and C-type lectin domain family 9 member A
(CLEC9A), which can be used to target dendritic-
cells. Mannose is commonly used to target macro-
phages and tumour-associated macro-phages, but
can target dendritic cells as well. Particles coated
with galactose, dextran or sialo-adhesin can deliver
to macro-phages. CD19-targeting NPs can be used to
actively target B cells, and NPs with lipoprotein- sur-
faces can activate the scavenger receptor class- B1
(SRB1) receptor on dendritic -cells. NP properties
can be optimized for accumulation at tolerogenic or-
gans, such as the liver and spleen, where immunolog-
ical AGs are naturally produced. Immune-recruiting
systems, like as polymeric- hydrogels and scaffolds,
could also be used to optimize interactions with
APCs. These systems work with APC-targeted NPs,
allowing them to recruit and re-programme- APCs.
All of these methods aim to increase the likelihood
that an AG will interact with an APC, improving
the efficacy of AG-based therapies and lowering the
dosage needed to reach therapeutic- levels.”
From Manufacturing Strategies for m-RNA Vac-
cines and Therapeutics
Laurens Vergauwen et al :
“LNPs have a very good stability, structural plastic-
ity and enhanced gene delivery compared to other
delivery -systems. They increase the transfection -
rate compared to naked m-RNA, allow for intra-
venous injection without the risk of being degraded
by RNases present in the blood-stream and enable
active targeting if specific ligands are incorporated.
The m-RNA construct is designed to ensure efficient
expression of the gene of interest.
Stability, gene expression and efficient protein trans-
lation depend upon several structural elements :
m-RNA structure:
The cap region at the 5’ end of the sequence is
essential for m-RNA maturation and allows the ri-
bosome to recognize the m-RNA for the efficient
protein- translation. The cap also stabilizes m-RNA
by protecting it from nuclease digestion.
The un-translated regions (UTRs) located at the up-
stream and down-stream domains of the m-RNA
coding region are affecting translation efficiency,
localization and stability and can be utilized for
efficient protein- expression.
The open reading frame or coding sequence regions
contains the gene of interest (GOI).
The poly-(A) tail is crucial for protein translation and
m-RNA stability by preventing digestion by 3’ exo-
nuclease.”.
Review Trends Biotechnol. 1997 Jun
Biomedical applications of nano-technology-
implications for drug targeting and gene therapy
S S Davis 1
“Colloidal particles in the nano-metre size range
(less than 1 micron in diameter) can be engineered
to provide opportunities for the site-specific delivery
of drugs after injection into the general circulation
or lymphatic- systems. Targets include the liver
(both Kupffer- cells and hepatocytes), endothelial
cells, sites of inflammation and lymph- nodes. The
size and surface of the particle are crucial factors in
targeting, and the attachment of cell-specific ligands
can lead to increased selectivity. The applications of
such particle engineering are discussed in relation to
conventional -drugs as well as the emerging area of
gene therapy.”
Circulating SARS-CoV-2 Vaccine AG Detected in
the Plasma of m-RNA-1273 Vaccine Recipients
Alana F Ogata et al Clinical Infectious Diseases May
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2021
“ 11 participants exhibit S1 AG in plasma after the
first injection, whereas nucleo-capsid concentrations
are insignificant in all participants, confirming that
the detected S1 originates from vaccination and not
natural- infection. The presence of S1 is likely due
to the nature of the encoded m-RNA-1273 spike-
protein, which contains a cleavable S1–S2 site and
enables release of S1 from the spike- trimer . We
hypothesize that release of S1 protein could result
from cleavage via mammalian cell -proteases or
circulating proteases. We observe an increase in
S1 over an initial period of 1–5 days, suggesting
that m-RNA translation begins immediately after
vaccine -inoculation. spike -protein appears in 3
of 13 participants on average 8 days after S1 is
produced. The Simoa AG assays for the full spike-
protein are designed to require AB binding to both
the S1 and S2- sub-units for detection, resulting in a
cleaved spike-protein to be un-detectable”
ACS Nano. 2020 Oct
COVID-19 Vaccine Frontrunners and Their Nano-
technology Design
Young Hun Chung et al
“m-RNA vaccines are generally en-capsulated
within nan-oparticles. Both BioNTech/Pfizer and
Moderna en-capsulate their RNA vaccines within
LNPs, which may enable cytoplasmic delivery
via fusogenic mechanisms. neither Moderna nor
BioNTech/Pfizer specifically mention the use of
fusogenic- LNPs although BioNTech/Pfizer does
mention using cationic lipids. Cytoplasmic -delivery
may improve translation efficiency, but it may also
decrease RNA immune-stimulation. RNA stimulates
the immune -system, and acts as an adjuvant,
by activating specific toll- like receptors (TLRs),
mainly TLRs 3, 7, and 8, which are all located
within the cell’s endosomes. TLRs 7 and 8 are
especially important for m-RNA vaccines as they
recognize single stranded RNA and engage in virus-
recognition. En-capsulation within nano-particles
improves RNA phagocytosis by APCs with subse-
quent localization within the endosomes. Failure of
the RNA to be endocytosed can lead to nuclease
degradation and weak immune -stimulation. Lastly,
due to the “nano” scale of nanomaterials as well as
their composition, they can traffic in vivo differently
from other materials. The lymphatic- system is
critical in initiating immune- responses as APCs,
and other lymphocytes travel from peripheral organs
to nearby lymph- nodes using the lymphatic -system.
Accessing the lymphatic- system can be challenging,
but nano-materials can traverse the interstitial spaces
and access nearby lymph -nodes. are all located
within the cell’s endosomes. Once inside the cell, the
m-RNA is translated directly within the cytoplasm;
in contrast, DNA plasmids from the viral vectors
need to be translocated into the nucleus, transcribed,
and exported back to the cytoplasm. This means that
m-RNA vaccines may produce greater amounts of
AG from smaller doses, but a caveat is that DNA
tends to be more stable than m-RNA meaning m-
RNA expression is generally shorter lived. the m-
RNA utilized by BioNTech/Pfizer encodes for the
RBD.Named BNT162b1, the m-RNA is modified
with single nucleoside incorporations of 1-methyl-
pseudouridine , which not only reduces the immune-
genicity of the m-RNA in vivo but also increases
its translation. The exact mechanism for increased
translation has not been entirely elucidated, but
one hypothesis is that the nucleoside- modification
improves RNA stability by decreasing rates of
hydrolysis by phosphor-diesterases”
Preprint : Biorxiv oct. 2020
Zebrafish studies on the vaccine candidate to
COVID-19, the Spike-protein: Production of AB
and adverse reaction
Bianca H Ventura Fernandes et al
“Zebrafish have been used as a model to study the
safety of vaccines and to assess toxicology that could
be correlated to human- health. Although zebrafish
do not have lungs as humans do, the present study
shows similar inflammatory- responses observed in
severe cases of COVID-19 patients that could be
considered when investigating human- responses to
the virus.
Female zebrafish individuals injected with a N-
terminal fraction of SARS-CoV-2 Spike recombi-
nant protein (residues 16-165) produced specific
AB, and presented suggestive adverse reactions and
inflammatory- responses resembling the severe cases
of COVID-19 human patients.
r-Spike-protein immunization of zebrafish had an
impact on the survival rate
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MAURO LUISETTO ET AL.
2 bioassays were carried out to analyze the toxicity
of the rSpike.
The immunization administration
We performed 2 intra-peritoneal inoculations of a
solution containing 1 µg purified rSpike diluted in 10
µL of inoculation buffer (7M urea, 50 mM Tris-HCl
pH 7.5, 200 mM NaCl, and 1mM EDTA). A group
of control- animals received injections containing
only the dilution buffer. Another control -group was
challenged by a lysate of bacterial fragment of E.
coli BL21(DE3) extract. rSpike was injected into 2
immunization sections in 20 zebrafish females (pre-
viously anesthetized with tricaine methane-sulfonate
(Sigma) - at a dose of 150 mg/L) at an interval of 7
days, with the aim of producing plasma- AB. Passive
AB transfer to zebrafish eggs occurs naturally as
described by Wang and collaborators
Although the immunized- fish produced AB, the first
injection of the r-Spike generated high toxicity to
the fish . the assay was repeated by adding different
control groups to confirm that the toxicity findings
were specific to the r-Spike . In the first bio-assay,
after fish immunization with r-Spike, the survival
rate was 78.6% during the first seven days It was
significant when compared to naive control and fish
injected with protein buffer (control 1), where the
survival rate was 100% and 90%, respectively . After
a second immunization, the r-Spike immunized -
group maintained the plateau survival rate, with no
statistical significance between the groups for the
relative risk of death” .
With respect to the reproductive- tissue, female ze-
brafish injected with rSpike displayed severe damage
in the ovary (follicular atresia, cellular -infiltration,
and disorganized extracellular matrix) after 7 days of
protein -inoculation.
It is remarkable that ovarian damage was reversed
after 14 days, when zebrafish received a second
injection of rSpike). t was recovered through the
protein-protein interaction with rSpike, the Toll-like
receptor path-way (dre:04620 and hsa:04620). It can
allow interaction with the Toll-like receptors TLR1,
TLR2, TLR4, and TLR5 and the interferon-α/β re-
ceptor (IFNαβR), possibly triggering the activation
of various signaling pathways . In this pathway, we
observed a possible interaction of the r-Spike with
the signal transducer and activator of transcription
1-alpha/beta (STAT1) protein in the cytoplasmic-
region. the signal molecules and interaction path-
way (zebrafish and human) showed the possibility
of rSpike interacting with a considerable number
of cell receptors related to the neuroactive ligand-
receptor (KEGG:4080) and a cytokine-cytokine re-
ceptor ( KEGG:4060) and triggering diverse cellular
signaling such as the TGF beta signaling family,
class I and II helical cytokines, IL and TNF family.
proteins related to the extracellular- matrix, cellular
communication and motility, formation of vesicles,
transport and catabolism, VEGF signaling -pathway,
and AGE-RAGE signaling pathway in diabetic com-
plications, among others, were identified
Inflammatory- infiltrates in different systems of
zebrafish injected with rSpike-protein.
a: longitudinal- section of the whole female
zebrafish for morphological analyses of the main
organs affected. All sections were stained with
Hematoxylin -Eosin. Brain: (b)-histology of control,
(c)-brain histology after 7 days of first immunization
presenting macrophages, and (d) 14 days after first
MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1699
FIGURE 6
24. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN
EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK
immunization with a burst after 7 days from the
first immunization presenting intense mononuclear
-infiltrate. (e) The same image as panel d but at
a higher magnification. Ovary: Ovarian histology
from zebrafish control (f), after 7 (g - h) and 14 days
(i). (f-i) Follicular development was classified as
primary growth oocyte (PG), cortical alveolus (CA),
and vitellogenic (V) stages. Asterisks in panel g
indicate an abundant and disorganized extra-cellular
matrix in the ovarian stroma. (h) Inset shows a higher
magnification of the cellular- infiltration and arrows
show dense, eosinophilic inflammatory infiltrates.
(i) The histology of ovaries after 14 days is similar
to the control. Scale bars: 1000 µm (g) and 200 µm
(f, h, and i). Liver: Histology of the liver from control
(j), after 7 days from rSpike immunization (l), and
after 14 days from the first immunization with a
burst at 7 days (m). Kidney: Histology of kidney
from zebrafish -control (n), after 7 days from the
first immunization (o), and after 14 days from the
first immunization with a second immunization after
7 days (p). Scale bars: 1,000 µm (n) and 200 µm (o
- p).
Blood clearance and organ- deposition of IVly
administered colloidal particles. The effects of
particle size, nature and shape
Lisbeth Iliuma S.S. et al :
“The blood clearance and organ deposition of poly-
styrene and cellulose particles have been studied in
the rabbit using labelled material and the technique
of gamma scintigraphy in order to investigate the
importance of particle- size, shape and nature. Small
(1.27 µm diameter) poly-styrene -microspheres were
taken up by the reticuloendothelial system of the
liver, while large poly-styrene particles (15.8 µm
diameter) were mechanically filtered by capillary-
beds of the lungs. Cellulose micro-spheres and fibres
were also taken up into lung tissue. Large cellulose
fibres, 30 µm diameter, proved to be toxic whereas
large cellulose micro-spheres were well tolerated”
Molecules 2020, Therapeutic Nano-particles and
Their Targeted
Delivery Applications Abuzer Alp Yetisgin et al
“Association of therapeutic agents with nano-
particles exhibiting unique physico-chemical and
biological properties and designing their pathways
for suitable targeting is a promising approach in
delivering a wide range of molecules to certain
locations in the body . This targeted strategy
enhances the concentration of therapeutic- agent
in cells/tissues; thereby, low doses can be used,
particularly if there is a contradiction between the
therapeutic- activity and the toxic effects of the
agent. Increasing concentration of therapeutic agents
in-target location also improves their therapeutic -
index by enhancing the efficacy and/or increasing the
tolerability in biological- systems. Water-insoluble
therapeutic agents can also be combined with nano-
particles, which can protect them from physiological
barriers and improve their bio-availability.
Association of therapeutic nano-particles with
contrast agents provides a way of tracking their
pathway and imaging their delivery location in in
vivo- systems”
FIGURE 7: From PHARMACEUTICAL
MANUFACTURING HANDBOOK SHAYNE COX GAD,
PH.D., D.A.B.T. Gad Consul ng Services Cary,
North Carolina ISBN: 978--47-25958-
Review Therapeutic efficacy of nano-particles and
routes of administration
Dhrisya Chenthamara et al : Biomaterials Research
volume 23 (2019)
“Targeted drug delivery methods
Passive targeting
Drug -targeting is defined as the selective drug
release at a specific physiological destination organ
or tissue or cell in which specific pharmacological
impact is required. Nanocarrier mediated cell
targeting includes active and passive- mechanisms.
In passive targeting, the drugs can be delivered to the
target -organ passively based on the longevity of the
pharmaceutical -carrier in the blood and preferential
JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1700
