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Sars-cov2 Spike protein and derivates toxicology :fertility and teratogen evaluation. State of Evidence -Hypotesys of Work 2022-01-21-16-29

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M. Luisetto Pharm.D.Spec. PharmacologyEurop. Specialist Lab. Medicine; ADVANCED univ. course CLINICAL PHARMACIST, Execution contract DIRECTOR H. med gas at -

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Sars-cov2 Spike protein and derivates toxicology :fertility and teratogen evaluation. State of Evidence -Hypotesys of Work 2022-01-21-16-29

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Journal of Medical Research and Health Sciences
Received 20 Nov 2021 | Revised 10 Dec 2021 | Accepted 5 Jan 2022 | Published Online 21 Jan 2022
DOI: https://doi.org/10.52845/JMRHS/2022-5-1-6
JMRHS 5 (1), 1678−1752 (2002) ISSN (O) 2589-9031 | (P) 2589-9023
RESEARCH ARTICLE
Sars-cov-2 Spike-protein and derivates toxicology : fertility- and teratogen
evaluation State of Evidence – Hypotesys of Work
Mauro Luisetto 1∗
Almukthar Naseer 2
Tarro G 3
Farhan Ahmad Khan 4
Nili B.
Ahmadabadi 5
Mashori Gulam Rasool 6
Gamal A. Hamid 7
Edbey K, 8
Fiazza C. 9
ilnaf Ilman 10
Yesvi A. Rafa 11
Cabianca L. 12
latyshev Oleg yurevich 13
1
IMA academy Marijnskaya ,Natural science
branch , applied pharmacologist Professorship
toxicology italy 29121 italy
2
Professor, Department of Physiology/College of
Medicine, University of Babylon, Iraq
3
President of the T & L de Beaumont Bonelli
Foundation for Cancer Research, Naples Italy
4
Professor, Department of Pharmacology,
Jawaharlal Nehru Medical College, AMU,
Aligarh.
5
Nano Drug Delivery, (a Product Development
Firm), United States
6
Profesor, Department of Medical& Health
Sciences for Woman, Peoples University of
Medical and Health Sciences for Women,
Pakistan
7
Professor Hematology Oncology,University of
Aden, Yemen
8
Professor, Department of Chemistry, Libya
Physical Chemistry, University of Benghazi,
Libya
9
Independent researcher pharmacologist italy
29121,
10
Student, Dhaka Residential Model College,
Dhaka, Bangladesh
11University of Nebraska-Lincoln,NE , USA
Majoring in Biological sciences -Human Health
and Disease.
12
biomedical laboratory turin italy Citta' della
Salute
13
IMA Academy president RU
Abstract
The aim of this work is to analyze the pathological
and toxicological aspect of covid-19 pathology in organ
different form lungs like ovarian and placentia .
pharmaceutical technology of nano-lipids used in some
covid-19 vaccine to preserve m-rna vaccine activity
repilication activity of m-RNA vaccine toxicological
aspect involevd in this kinetics and related other kind
of vaccine like viral vector and inactivate.
With a neutral approach a review of relevat literature is
submitted to the researcher in order to produce A global
conclusion related hypotesys of effect on human
fertility produced by Spike-protein . This result are
usefull also in vaccine design strategy for next
generation of products . Great enphasys is given to
the numeric value evaluation between cellular intake
of nano-lipids m-RNA vaccine vs the naked ones .
Keywords: coronavirus, covid-19, spike-protein, sars
cov-2 m-rna vac-cine safety, fertility, toxicology,
epidemiology, evidence, pharmaceuti-cal technology,
nano drug delivery, epidemiology, literature interpreta-
tion errors, fold, self amplyfing, cellular intake.
Copyright : © 2002 The Authors. Published by
Medical Editor and Educational Research Publishers
Ltd. This is an open access article under the CC BY-
NC-ND license
(https://creativecommons.org/licenses/by-nc-nd/4.0/).
JMRHS (1), 1678−1752 MEERP LTD 1678
Open Access Journal
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
JMRHS 5 (1), 1678−1752 MEERP LTD 1678
MEERP LTD
MAURO LUISETTO ET AL.
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
MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1679
SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN
EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK
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
JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1680
MEERP LTD
MAURO LUISETTO ET AL.
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
MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1681
SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN
EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK
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
JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1682

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Sars-cov2 Spike protein and derivates toxicology :fertility and teratogen evaluation. State of Evidence -Hypotesys of Work 2022-01-21-16-29

  • 1. Journal of Medical Research and Health Sciences Received 20 Nov 2021 | Revised 10 Dec 2021 | Accepted 5 Jan 2022 | Published Online 21 Jan 2022 DOI: https://doi.org/10.52845/JMRHS/2022-5-1-6 JMRHS 5 (1), 1678−1752 (2002) ISSN (O) 2589-9031 | (P) 2589-9023 RESEARCH ARTICLE Sars-cov-2 Spike-protein and derivates toxicology : fertility- and teratogen evaluation State of Evidence – Hypotesys of Work Mauro Luisetto 1∗ Almukthar Naseer 2 Tarro G 3 Farhan Ahmad Khan 4 Nili B. Ahmadabadi 5 Mashori Gulam Rasool 6 Gamal A. Hamid 7 Edbey K, 8 Fiazza C. 9 ilnaf Ilman 10 Yesvi A. Rafa 11 Cabianca L. 12 latyshev Oleg yurevich 13 1 IMA academy Marijnskaya ,Natural science branch , applied pharmacologist Professorship toxicology italy 29121 italy 2 Professor, Department of Physiology/College of Medicine, University of Babylon, Iraq 3 President of the T & L de Beaumont Bonelli Foundation for Cancer Research, Naples Italy 4 Professor, Department of Pharmacology, Jawaharlal Nehru Medical College, AMU, Aligarh. 5 Nano Drug Delivery, (a Product Development Firm), United States 6 Profesor, Department of Medical& Health Sciences for Woman, Peoples University of Medical and Health Sciences for Women, Pakistan 7 Professor Hematology Oncology,University of Aden, Yemen 8 Professor, Department of Chemistry, Libya Physical Chemistry, University of Benghazi, Libya 9 Independent researcher pharmacologist italy 29121, 10 Student, Dhaka Residential Model College, Dhaka, Bangladesh 11University of Nebraska-Lincoln,NE , USA Majoring in Biological sciences -Human Health and Disease. 12 biomedical laboratory turin italy Citta' della Salute 13 IMA Academy president RU Abstract The aim of this work is to analyze the pathological and toxicological aspect of covid-19 pathology in organ different form lungs like ovarian and placentia . pharmaceutical technology of nano-lipids used in some covid-19 vaccine to preserve m-rna vaccine activity repilication activity of m-RNA vaccine toxicological aspect involevd in this kinetics and related other kind of vaccine like viral vector and inactivate. With a neutral approach a review of relevat literature is submitted to the researcher in order to produce A global conclusion related hypotesys of effect on human fertility produced by Spike-protein . This result are usefull also in vaccine design strategy for next generation of products . Great enphasys is given to the numeric value evaluation between cellular intake of nano-lipids m-RNA vaccine vs the naked ones . Keywords: coronavirus, covid-19, spike-protein, sars cov-2 m-rna vac-cine safety, fertility, toxicology, epidemiology, evidence, pharmaceuti-cal technology, nano drug delivery, epidemiology, literature interpreta- tion errors, fold, self amplyfing, cellular intake. Copyright : © 2002 The Authors. Published by Medical Editor and Educational Research Publishers Ltd. This is an open access article under the CC BY- NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/). JMRHS (1), 1678−1752 MEERP LTD 1678 Open Access Journal
  • 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 JMRHS 5 (1), 1678−1752 MEERP LTD 1678
  • 3. MEERP LTD MAURO LUISETTO ET AL. 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 MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1679
  • 4. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK 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 JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1680
  • 5. MEERP LTD MAURO LUISETTO ET AL. 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 MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1681
  • 6. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK 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 JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1682
  • 7. MEERP LTD MAURO LUISETTO ET AL. 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 MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1683
  • 8. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK 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” JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1684
  • 9. MEERP LTD MAURO LUISETTO ET AL. 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). MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1685
  • 10. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK 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- JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1686
  • 11. MEERP LTD MAURO LUISETTO ET AL. 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 MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1687
  • 12. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK 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 JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1688
  • 13. MEERP LTD MAURO LUISETTO ET AL. 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 MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1689
  • 14. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK 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 JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1690
  • 15. MEERP LTD MAURO LUISETTO ET AL. 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), MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1691
  • 16. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK 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 JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1694 FIGURE 4
  • 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 MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1695
  • 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- JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1696
  • 21. MEERP LTD 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 MEERP LTD JMRHS 5 (1), 1678−1752 (2002) 1697 FIGURE 5
  • 22. SARS-COV-2 SPIKE-PROTEIN AND DERIVATES TOXICOLOGY : FERTILITY- AND TERATOGEN EVALUATION STATE OF EVIDENCE – HYPOTESYS OF WORK 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 JMRHS 5 (1), 1678−1752 (2002) MEERP LTD 1698
  • 23. MEERP LTD 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