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.