3. Considerations in the Design of mRNA Vaccines
The primary goal of the developers of the SARS-CoV-2 mRNA vaccines was
to design a vaccine that could induce a robust antibody response to the
spike protein. Preexisting antibodies to spike protein should cause the
invading viruses to be quickly cleared before they could invade host
cells, thus arresting the disease process early on. As stated succinctly
by Kaczmarek et. al. (2021) [58]:
“The rationale behind vaccination is to provide every vaccinated person
with protection against the SARS‐CoV‐2 virus. This protection is
achieved by stimulating the immune system to produce antibodies against
the virus and to develop lymphocytes that will retain memory and the
ability to fight off the virus for a long time.”
Vaccines generally depend upon adjuvants such as aluminum and squalene
to provoke immune cells to migrate to the injection site immediately
after vaccination. In the history of mRNA vaccine development, it was
initially hoped that the mRNA itself could serve as its own adjuvant.
This is because human cells recognize viral RNA as foreign, and this
leads to upregulation of type I IFNs, mediated via toll like receptors
such as TLR3, TLR7 and TLR8 [59].
However, with time it became clear that there were problems with this
approach, both because the intense reaction could cause flu-like
symptoms and because IFN-α could launch a cascade response that would
lead to the breakdown of the messenger RNA before it could produce
adequate amounts of spike protein to induce an immune response [60].
A breakthrough came when it was discovered experimentally that the mRNA
coding for the spike protein could be modified in specific ways that
would essentially fool the human cells into recognizing it as harmless
human RNA. A seminal paper by Karikó et al. (2005) demonstrated through
a series of in vitro experiments that a simple modification to
the mRNA such that all uridines were replaced with pseudouridine could
dramatically reduce innate immune activation against exogenous mRNA
[59]. Andries et al. (2015) later discovered that
1-methylpseudouridine as a replacement for uridine was even more
effective than pseudouridine and could essentially abolish the TLR
response to the mRNA, preventing the activation of blood-derived
dendritic cells [61]. This modification is applied in both the mRNA
vaccines on the market [62].
For successful mRNA vaccine design, the mRNA needs to be encapsulated in
carefully constructed particles that can protect the RNA from
degradation by RNA depolymerases. The mRNA vaccines are formulated as
lipid nanoparticles containing cholesterol and phospholipids, with the
modified mRNA complexed with a highly modified polyethylene glycol (PEG)
lipid backbone to promote its early release from the endosome and to
further protect it from degradation [63]. The host cell’s existing
biological machinery is co-opted to facilitate the natural production of
protein from the mRNA through endosomal uptake of a lipid particle
[63]. A synthetic cationic lipid is added as well, since it has been
shown experimentally to work as an adjuvant to draw immune cells to the
injection site and to facilitate endosomal escape. De Beuckelaer et al.
(2016) observed that “condensing mRNA into cationic lipoplexes
increases the potency of the mRNA vaccine evoked T cell response by
several orders of magnitude.” [60] Another important modification
is that they replaced the code for two adjacent amino acids in the
genome with codes for proline, which causes the spike protein to stay in
a prefusion stabilized form [64].
The spike protein mRNA is further “humanized” with the addition of a
guanine-methylated cap, 3’ and 5’ untranslated regions (UTRs) copied
from those of human proteins, and finally a long poly(A) tail to further
stabilize the RNA [65]. In particular, researchers have cleverly
selected the 3’UTR taken from globins which are produced in large
quantities by erythrocytes, because it is very effective at protecting
the mRNA from degradation and maintaining sustained protein production
[66]. This is to be expected, since erythrocytes have no nucleus, so
they are unable to replace the mRNAs once they are destroyed. Both the
Moderna and the Pfizer vaccines adopted a 3’UTR from globins, and the
Pfizer vaccine also uses a slightly modified globin 5’UTR [67]. De
Beuckelaer et al. (2016) aptly summed up the consequences of such
modifications as follows: “Over the past years, technical improvements
in the way IVT [in vitro transcribed] mRNAs are prepared (5′
Cap modifications, optimized GC content, improved polyA tails,
stabilizing UTRs) have increased the stability of IVT mRNAs to such
extent protein expression can now be achieved for days after directin vivo administration of the mRNA.” [60]
However, the optimized analogue cap formation of synthetic mRNAs
inevitably forces the recipient cells to undergo a cap-dependent
prolonged translation, ignoring homeostatic demands of cellular
physiology [65]. The cap 2’ O methylation carried out by cap 2’ O
methyltransferase (CMTR1) serves as a motif that marks the mRNA as
“self,” to prevent recognition by IFN-induced RNA binding proteins
[68]. Thus, the mRNA in the vaccines, equipped with the cap 2’ O
methylation motif, evades detection as a viral invasion. Furthermore,
the overwhelming impetus for cells to perform a single and artificial
approach to translation according to the robust capping and synthetic
methylations of mRNAs in vaccines is fundamentally associated with
disease progression due to differential rather than normal signaling of
pattern recognition receptors (PRRs) [69].
The regulatory process controlling mRNA translation is extremely
complex, and it is highly disturbed in the context of mRNA vaccines
[65,69]. Briefly, the idea is for mRNA vaccines to achieve the
intended goal (i.e., production of the modified spike protein) through a
stealth strategy that bypasses the natural immunological response to
RNA-type viral infection. Injected lipid nanoparticles containing mRNA
are brought to the cell interior via endocytosis. The mRNA escapes its
lipid carrier and migrates to the ribosome, where it is abundantly
translated into its final protein product, following an optimized
program for producing large quantities of a specific protein over an
extended period of time. These modified spike proteins then follow one
of three primary pathways. Some are proteolytically degraded and
fragments are bound by MHC class I molecules for surface presentation to
cytotoxic T-cells. A second pathway has those same spike fragments bind
MHC class II molecules, move to the cell surface, and activate T-helper
cells. A final pathway has soluble spike proteins extruded from the cell
in exosomes, where they can be recognized by B-cell-activated
spike-specific antibodies [70].
In the end, it is through utilization of nanolipids and sophisticated
mRNA technology that the normal immune response to exogenous RNA is
evaded in order to produce a strong antibody response against an
exogenous RNA virus.