Daily Clout 4/20/22 Report: Pfizer mRNA Construct
Researcher/Author: Daniel B. Demers, PhD
Reviewed by: Team 5
Team 5 Leader: Linnea Wahl
Introduction
In the first paragraph of Pfizer document 2.4 NONCLINICAL
OVERVIEW, Pfizer states that “BNT162b2 is a nucleoside modified
mRNA (modRNA) expressing full-length S [spike] with two proline
mutations (P2) to lock the transmembrane protein in an antigenically
optimal prefusion conformation” (p. 6, https://phmpt.org/wp-content/
uploads2022/03/125742_S1_M2_24_nonclinical-overview.pdf).
They list two references (Pallesen et al., 2017; Wrapp et al., 2020) as
their justification for this design. That is the end of Pfizer’s discussion
on why that particular design was selected, and it appears that Pfizer
conducted no further research before selecting this design (or
construct) and proceeding with vaccine development. This, as it turns
out, is quite important.
Concerns Regarding the Pfizer mRNA Construct
There are three primary concerns regarding the Pfizer approach used
to design their mRNA vaccine.
1. The basic Pfizer construct utilizing two proline substitutions
to stabilize the spike protein molecule is flawed, and the protein
molecule as well as the mRNA itself, remain unstable.
2. The spike protein has been shown to cause disease;
therefore, a vaccine based on the spike protein will promote
pathogenesis, not prevent it.
3. The S1 subunit of the spike protein has been shown to shed
into the circulatory system, thereby furthering disease.
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The following discussion expands on these three concerns.
Concern 1: Pfizer selected the Pallesen et al. (2017) construct as
the basis for the Pfizer vaccine.
The work described by Pallesen et al. (2017) was performed on the
MERS-CoV virus. Pallesen selected proline substitutions based on
the work of others (Qiao et al., 1998; Sanders et al., 2002; Krarup, et
al., 2015).
Pfizer also references a paper in the journal Science authored by
Daniel Wrapp (Wrapp et al., 2020). Wrapp cites Pallesen et al. (2017)
and the work of Robert Kirchdoerfer et al. (2018) who evaluated the
Pallesen-style double proline substitutions (S2P) in the spike protein
of SARS-CoV. Wrapp et al. (March 2020) assessed the 2P
substitution in the spike protein of SARS-CoV-2, evaluating the
construct for its affinity for the host cell receptor ACE2. Wrapp did not
evaluate the SARS-CoV-2 S2P antigenicity nor the fate of the S1
subunit that is shed when the spike protein binds to the cell.
Wrapp et al. (March 2020) states that “Knowing the atomic level
structure of the SARS-CoV-2 spike will allow for additional protein
engineering efforts that could [emphasis added] improve antigenicity
and protein expression for vaccine development.” It appears that
Pfizer took this article and used it as is to create a vaccine without
“additional protein engineering efforts” as suggested by Wrapp et al.
(2020).
Moreover, the purpose of introducing two proline substitutions into the
spike protein as described by Pallesen and Wrapp (Pallesen et al.,
2017; Wrapp et al., 2020; Pfizer, p. 6, https://phmpt.org/wp-content/
uploads2022/03/125742_S1_M2_24_nonclinical-overview.pdf) was to
stabilize the spike protein to improve its thermal stability,
conformation and antigenicity. But, as stated by Hsieh et al. (2020)
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with co-author Daniel Wrapp, “even with these (2P) substitutions the
SARS-CoV-2 S-protein remains unstable and difficult to produce
reliably in mammalian cells, hampering R&D of subunit vaccines.”
Hsieh and Wrapp (Hsieh et al., July 2020) found that 26 of 100
variants that they created and tested had higher expression than the
S-2P substitution that Pfizer selected. One of their variants, labeled
Hexa-Pro, contained four proline substitutions in addition to the S-2P
substitutions and had nearly 10X greater expression, had improved
thermal stability and retained the desired conformation.
Numerous articles since then state that the 2P substitution used by
Pallesen/Pfizer is unstable (McCallum et al., 2020, posted on-line Aug.
2020; Xiong et al., 2020; Brun et al., 2020; Juraszek et al., 2021).
Brun et al. (posted November 2020) even made suggestions for
improving the Pfizer BNT162b2 vaccine after describing why it was a
suboptimal design.
Why did Pfizer select the Pallesen construct requiring storage in
ultra-low-temperature freezers when the HexaPro construct is
more stable, can be stored at room temperature and has much
greater expression?
Concern 2: Pfizer did not address the well-documented
pathogenesis caused by the coronavirus spike protein before
release of their vaccine and before FDA approval.
In a 2005 article, Kuba demonstrated that SARS-CoV spike protein
injected into mice worsened their lung disease (Kuba, 2005).
In 2008, Wang et al. demonstrated that the receptor binding domain
(RBD) of the spike protein of SARS-CoV leads to internalization of
ACE2, resulting in downregulation and subsequent lung injury (Wang
et al., 2008). The authors concluded that “because the RBD spike
binding to ACE2 contributes to SARS pathogenesis, the use of subunit
vaccines based on RBD spike should be considered carefully.”
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Wang et al. (2020) and Semimukai et al. (2020) noted that
recombinant spike protein induced antibodies in mice and protected
against SARS-CoV infection, but lung eosinophilic immunopathology
was observed in the immunized mice after SARS infection.
Elizabeth M. Rhea and her co-authors reported on-line in December
2020 and published in March 2021 (Rhea et al., 2021) that S1 subunit
labeled with radioiodine (I-S1) readily crosses the mouse blood-brainbarrier (BBB) and could explain the adverse effects of S1 and/or
SARS-CoV-2 such as encephalitis, respiratory difficulties and reduced
ability to smell. I-S1 was also detected in kidney, liver and spleen.
In January 2021, Letarov et al. published an article in the journal
Biochemistry (Moscow), titled Free Sars-CoV-2 Spike Protein S1
Particles May Play a Role in the Pathogenesis of COVID-19 Infection
(Letarov et al., 2021). They noted that the upregulation of cell surface
expression of ACE1 and/or downregulation of ACE2 can lead to
pulmonary damage. This occurs during SARS infection and by
recombinant SARS-CoV spike protein. They hypothesize that S1
molecules carry intact RBDs, and their binding to ACE2 may induce
ACE2 downregulation and deleterious downstream effects such as
increased inflammation, thrombosis, and pulmonary damage.
Letarov et al. (2021) also reference the work of Zhang et al. (2020)
who elucidated a spike protein mutation in SARS-CoV-2 (the D614G
variant) that is associated with increased infectivity but reduced S1
shedding and mild symptoms. This is further evidence that the spike
protein is responsible for pathogenesis.
Nuovo et al. (2021, posted on-line Dec. 2020) reported on the
endothelial cell damage caused by the S1 subunit of the spike protein.
They reported two main findings: 1) Human COVID-19 cases
demonstrated microvessel endothelial damage in the brain and other
organs, including the skin, due to circulating spike protein that induces
cytokine production resulting in microencephalopathy; and 2) injection
of the S1 full-length spike subunit into mice (but not the S2 subunit)
induced an equivalent microvascular encephalopathy as seen in
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human COVID-19 cases. The authors further note that although their
study “focused on the brain, it should be stressed that there are other
sites where there is a rich bed of microvessels with the ACE2 receptor,
including skin/subcutaneous fat and the liver. As has been
documented in human patients, microvessels at these sites can also
display an endothelialitis that, in the skin/fat can induce complement
activation/hypercoagulable state and the so called cytokine storm
typical of fatal COVID-19.”
“In sum, the data presented indicates that the full length S1 subunit of
the spike protein of SARS-CoV-2 alone is capable, without the
infectious virus, of inducing systemic microendothelial cell damage in
mice with a cognate pattern of complement activation and increased
cytokine expression and the concomitant thrombosis/hypercoagulable
state. This disease pattern strongly parallels the extra-pulmonary
manifestation of severe human COVID-19 and suggests that the latter
may not represent systemic infectious virus. Thus, prevention of the
CNS disease so common in severe COVID-19 may require
neutralization/removal of the circulating pseudovirus.”
Lei et al. (April 2021) created a pseudovirus exhibiting spike protein
but containing no virus inside and concluded that the spike protein
alone is sufficient to cause damage to the vascular endothelial cells.
With so much evidence demonstrating a direct link between the
presence of the spike protein S1 subunit in the circulatory
system and pathogenesis, why would Pfizer create a vaccine that
not only injects spike protein into the patient, but converts the
cells of the patient into “spike protein factories” that turn out the
spike protein S1 subunit, the very molecule that causes illness?
Concern 3: Pfizer did not address the well-documented shedding
of the coronavirus spike protein into the circulatory system,
where it crosses over to multiple organ systems to cause
pathogenesis, before release of their vaccine.
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It was shown as early as 1994 (Bullough et al., 1994) that the surface
spike protein of an enveloped virus (Influenza) would release a
subunit after proteolytic cleavage of the structure upon binding to the
host cell surface. Work by Alexandra Walls (2017) demonstrated that
the proteolytic processing of coronavirus spike proteins allows
shedding of the S1 subunit.
Brun et al. (posted on-line November 2020) reported the process by
which spike protein is processed within the host cell and soluble S1
subunit was secreted into the extracellular space via lysosomes.
Their work indicated that the production of spike vaccine antigen
protein without a virus to incorporate the protein into the viral envelope
created an overexpression system and secretion of the protein by the
cell (shedding). They suggest that the secreted spike proteins do not
mimic the spike glycoproteins as they are presented on the actual
virus and may effectively act as a decoy, eliciting more of the
unwanted sub-optimal, non-neutralizing antibodies that are incapable
of neutralizing the virus.
The authors state that the Pfizer BNT162b vaccines (and other similar
type vaccines) rely on the supplied RNA sequence to use the host cell
machinery to faithfully produce the spike protein in its fully folded,
glycosylated and assembled state, resembling a natural infection, and
they trigger a robust innate and humoral response; however, this does
not happen. They go on to suggest a better vaccine design, one that
abolishes the furin cleavage site (which is intact in the Pfizer
construct) and introduces mutations that lock the spike protein in the
prefusion conformation to prevent shedding and elicit a more potent
antibody response.
Rhea et al. (2021, posted on-line December 2020) noted that
coronavirus spike proteins are often cleaved; therefore, S1 could be
shed and shed S1 may cross the BBB. Shedding of the S1 subunit of
the spike protein was also noted by Liu et al. (2020), Letarov et al.
(2021), Rhea et al. (2020), Zhang et al. (2020) and Henderson et al.
(2020).
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Given that the Pfizer mRNA construct design is sub-optimal;
given that it has been well established (since 2005 to 2008) that
spike proteins cause disease; and given that the spike protein S1
subunit is shed during binding of the virus or pseudovirus with
the host cell, as well as secreted by host cells producing spike
protein following injection with an mRNA-derived spike protein
vaccine, why would Pfizer develop and release an mRNA vaccine
that demonstrates all three of these deleterious qualities? Why
would Pfizer develop and release an mRNA vaccine that
demonstrates poor design with limited immunogenicity, requires
storage at very low temperatures, and results in the production
of a spike protein that readily sheds into the circulatory system
to cause pathogenesis in multiple organ systems? And why
would the FDA approve it?
References:
Brun, J., et al., bioRxiv, https://doi.org/10.1101/2020.11.16.384594,
Analysis of SARS-CoV-2 spike glycosylation reveals shedding of a vaccine
candidate.
Bullough, P., et al., Nature, 1994; 371:37-43. Structure of Influenza
haemagglutinin at the pH of membrane fusion.
Henderson, R., et al, Nat Struct Mol Biol, 2020; 27(10):925-933.
Controlling the SARS-CoV-2 spike glycoprotein conformation.
Hsieh, C., et al., (co-author Wrapp), Science, 2020; DOI:10.1126/
science.abd0826. Structure-based design of profusion-stabilized SARSCoV-2 spikes.
Juraszek, J., et al., Nature Communications, 2021; 12, Article number 244.
Stabilizing the closed SARS-CoV-2 spike trimer.
Kirchdoerfer, R., et al., Scientific Reports, 2018; 8:15701; DOI:10.1038/
s41598-018-34171-7. Stabilized coronavirus spikes are resistant to
conformational changes induced by receptor recognition or proteolysis.
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Krarup, A., et al., Nat Commun, 2015; 6:8143. A highly stable prefusion
RSV F vaccine derived from structural analysis of the fusion mechanism.
Kuba, K., et al., Nature Medicine, 2005; 11(8):875-879. A crucial role of
angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced
lung injury.
Lei, Y., et al., Circulation Research. 2021; 128:1323-1326. SARS-CoV-2
Spike Protein Impairs Endothelial Function via Downregulation of ACE 2.
Letarov, A., et al., Biochemistry (Moscow), 2021; 86(3):257-261. Free
SARS-CoV-2 Spike Protein S1 Particles May Play a Role in the
Pathogenesis of COVID-19 Infection.
Liu, C., et al., Structure, 2020; 28(11):1218-1224. The Architecture of
Inactivated SARS-CoV-2 with Postfusion Spikes Revealed by Cryo-EM
and Cryo-ET.
McCallum, M., et al., Nature Structural & Molecular Biology, October 2020;
27:942–949. Structure-guided covalent stabilization of coronavirus spike
glycoprotein trimers in the closed conformation.
Nuovo, G., et al. Annals of Diagnostic Pathology 51, 2021; 151682.
Endothelial cell damage is the central part of COVID-19 and a mouse
model induced by injection of the S1 subunit of the spike protein.
Pallesen, J., et al., Proc Natl Acad Sci, 2017; 114:E7348-E7357.
Immunogenicity and structures of a rationally designed prefusion MERSCoV spike antigen.
Qiao, H., et al., Journal Cell Biol, 1998; 141:1335-1347. Specific single or
double proline substitutions in the “spring-loaded” coiled-coil region of the
influenza hemagglutinin.
Rhea, E., et al., Nature Neuroscience, March 2021; 24:368-378. The S1
protein of SARS-CoV-2 crosses the blood–brain barrier in mice.
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Sanders, R., et al., Journal Virology, 2002; 76:8875-8889. Stabilization
of the soluble, cleaved, trimeric form of the envelope glycoprotein
complex of human immunodeficiency virus type1.
Semimukai et al., Microbiol Immunol, 2020; 64:33-51. Gold
nanoparticle-adjuvanted S protein induces a strong antigen-specific
IgG response against severe acute respiratory syndrome-related
coronavirus infection, but fails to induce protective antibodies and limit
eosinophilic infiltration in lungs.
Walls, A., et al., PNAS, |October 17, 2017; 114(42):11157-11162. Tectonic
conformational changes of a coronavirus spike glycoprotein promote
membrane fusion.
Wang, S., et al., Virus Research, 2008; 136:8-15. Endocytosis of the
receptor-binding domain of SARS-CoV spike protein together with virus
receptor ACE2.
Wang, Y., et al., J Med Virol, 2020; 93:892-898. SARS‐CoV‐2 S1 is
superior to the RBD as a COVID‐19 subunit vaccine antigen.
Wrapp, D., et al., Science, 2020; 367:1260-1263. Cryo-EM structure of the
2019-nCoV spike in the prefusion conformation.
Xiong, X., et al., Nat Struct Mol Biol, October 1, 2020; 27(10):934-941. A
thermostable, closed SARS-CoV-2 spike protein trimer.
Zhang, L., et al., bioRxiv. Preprint. June 12, 2020. The D614G mutation in
the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity.
Source: Report: Pfizer mRNA Construct – DailyClout