The Coronavirus disease-19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus -2 (SARS-CoV-2), has impacted human lives in the most profound ways with millions of infections and deaths. Scientists and pharmaceutical companies ...
mRNA vaccine development for COVID-19
Optimization of mRNA vaccine design
Typical vaccine development using live-attenuated or inactivated virus or a pseudovirus system involves tedious and time-consuming steps and has become a bottleneck for responding to an epidemic or pandemic caused by newly emerging viruses. As described above, mRNA vaccines possess distinctive advantages of rapid development and versatility as exemplified by the swift development of multiple COVID-19 mRNA vaccines. More importantly, recent preliminary data from clinical trials have shown that two licensed mRNA vaccines, mRNA-1273 and BNT162b, have higher protective efficacy than ChAdOx1 vaccine developed using a chimpanzee adenovirus (~95% vs. ~70%) 4, 41.
However, some intrinsic features of mRNA molecules demand special strategies to guarantee the stability, efficacy and safety of mRNA vaccines. First, mRNA are intrinsically unstable and prone to degradation due to the omnipresence of RNases in the serum and plasma 42. Second, the cellular machinery recognizes exogenous RNA molecules as immunological mimic of viral infection, which results in an immediate immune response 43. Thus, it is a prerequisite for the design of mRNA vaccines to maximize the stability of RNA and translation efficiency and avoid the innate immune response by host cells 44, 45. Below we will discuss the major strategies used in designing mRNA vaccines, including 5'-capping, nucleoside modification, codon optimization, and efficient delivery of mRNA molecules with nanoparticles (Table (Table22).
5'-capping of mRNA vaccines
Endogenous mRNA molecules undergo post-transcriptional modifications, including 5'-capping and polyadenylation, for the stability of mRNA and efficient translation. Naturally, 7-methylguanosine cap (m7G) is added to the first nucleotide of a mRNA chain via 5' to 5' linkage. The 2'-OH of the ribose of the first nucleotide is further methylated to form m7GpppNm, also known as cap1. 5'-capping is critical for protecting mRNA from exonuclease activity, facilitating pre-mRNA splicing, and serving as the binding site for eIF4F, the heterodimeric translation initiation complex 46-49. Recent studies have indicated the 5'-cap structure as a major determinant by which the host can discriminate between self vs. non-self mRNA molecules 50-53. A m7GpppNm cap was added to the 5'-end of the majority of the mRNA vaccines reported thus far during their IVT 54-57.
Optimization of 5'- and 3'-untranslated regions and the length of polyadenylation tail
Regulatory elements in the 5′-untranslated region (UTR) 58 and the length of 3′-UTR 59 increase protein translation. In addition, the polyadenylation (polyA) tail stabilizes mRNA and increases protein translation. Several recent reports have shown that the length of polyA tail is closely associated with the translation efficiency 60. However, the information on 5'- and 3'-UTRs and the nature of polyA signal sequence remains proprietary and undisclosed for the seven reported mRNA vaccines.
Nucleoside modification during IVT
Kariko, et al., demonstrated that RNA recognition by Toll-like receptors (TLRs) is suppressed via modification of the nucleosides in mRNA molecules 61, 62. Incorporating m5C, m6A, m5U, s2U, or pseudouridine into mRNA molecules abrogates the immune response by evading the activation of TLR-3, -7, and -8 61. For all the seven reported vaccines, pseudouridine was incorporated into the mRNA vaccines in the place of uridine. In addition, the substitution with pseudouridine, m6A, and s2U in RNA molecules suppresses the degradation of RNA by RNase L 63. Thus, the nucleoside modifications not only enhance the stability of RNA but also reduce the innate immune response.
Purification of IVT
The contaminating impurities during IVT can massively affect the safety of mRNA vaccines once they are introduced to human cells. Even residual amounts of double-stranded RNA and DNA-RNA hybrid molecules can trigger the innate immune response as they can be recognized by the cellular sensors pattern recognition receptors. Various purification techniques have been used to remove residual impurities from IVT reactions for all the seven mRNA vaccines currently on clinical trials. A previous study indicates that the purification of mRNA reduces the expression of type I interferon and increases the protein translation 64. As summarized in Table Table2,2, various purification techniques such as Oligo dT column, LiCl precipitation, and silicone column have been employed to remove contaminants from in vitro synthesized mRNA 45.
Codon optimization
Several parameters have been considered for the codon optimization, which affects the translation efficiency, protein folding, and mRNA abundance. One example is that the GC content in the sequence. Although GC-rich sequences may be problematic for the secondary structure formation of mRNA, the translation efficiency of a GC-rich sequence can be 100-fold higher than that of a GC-poor sequence 65. The translation elongation rate highly depends on the availability of the cognate tRNA species and the optimization of the codon usage to avoid sequences that match rare tRNA species and incorporate sequences that match more abundant tRNA species 66. Moreover, the codon optimization is essential for the mRNA stability as the codon-dependent translation elongation rate has been implicated as a major determinant of the mRNA stability 67. Mechanistically, reduced translational elongation of mRNA with suboptimal codons results in the recruitment of the DEAD-box RNA helicase, Dhh1p, which triggers mRNA decay 68. Two additional codon optimization methods involve the use of the codons with human bias and the maximum adaptation index 69, 70. Other bioinformatics approaches can be explored to further enhance the stability of mRNA, e.g., via design of the secondary structures and prediction of the expression level based on deep learning 71, 72.
Designing platform and target immunogen for the seven mRNA candidate vaccines
Each of the seven mRNA candidate vaccines was synthesized in vitro from a DNA template encoding either the full-length S protein or RBD of SARS-CoV-2 using bacteriophage T7 RNA polymerase. mRNA-1273, CVnCoV, LUNAR-CoV19, and LNP-nCoVsaRNA mRNA vaccines used the template encoding the full-length S protein with 2P substitutions at K986 and V987 positions to produce the stable pre-fusion form of S protein 73. Pfizer/BioNTech have developed two immunogens, the RBD (BNT162b1) and the full-length S protein (BNT162b2). BNT162b2 has been shown to be safer than BNT162b1, especially in older adults in a preliminary clinical trial, and thus was chosen for a phase 3 clinical trial 74. ARCoV vaccines are based on the RBD of SARS-CoV-2. Whereas the sequences of the 5'- and 3'-UTR of the mRNA templates were not revealed in the literature, the 3'-UTR of BNT162b mRNA vaccine derived empirically by screening naturally occurring 3'-UTRs for the highest RNA stability 75. On the other hand, CVnCoV and LNP-nCoVsaRNA were built on the saRNA platform containing a self-replicating replicon of Trinidad donkey Venezuelan equine encephalitis virus (VEEV). The viral protein-encoding gene of the replicon is replaced with a modified S protein-encoding gene of SARS-CoV-2 with two proline mutations in the S2 subunit, K986P and V987P 54, 55. Consistent with the notion that saRNA vaccines can self-amplify after delivery into host cells, the dose used for vaccination was one to two magnitude lower than conventional mRNA vaccines. As shown in Table Table2,2, the dosage range for CVnCoV and LNP-nCoVsaRNA was 2-12 μg and 0.01-10 μg, respectively. In comparison, the typical dose range for the conventional mRNA vaccines was 30-100 μg.
Packaging mRNA vaccines with lipid nanoparticle (LNPs)
An early study has shown that the transfection efficiency of naked mRNAs is nearly two orders of magnitude lower than that of mRNA bound to lipofectin formulation 27. The lipofectin-based carriers effectively help mRNA delivery into target cells and protect mRNA from RNase 36, 76. The formulation of liposome-based transfection reagents containing cationic lipids has remarkably been improved in recent years 77. In particular, LNPs, composed of proprietary components including positively charged lipids, cationic polypeptides, polymers, micelles or dendrimers, have been widely used for in vivo RNA delivery 78, 79. LNPs encapsulate mRNA and assemble it into the stable lipid bilayers, which are then ingested by cells through a variety of endocytosis pathways. Below is the information for packaging of mRNA vaccines with various LNPs.
1. mRNA-1273: It was loaded into two proprietary cationic LNPs, WO2017070626 and WO2018115527. Although the exact formulation is not known, the composition of the LNPs was described as follows, SM-102, polyethylene glycol-2000-dimyristoyl glycerol (PEG2000-DMG), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 80.
2. BNT162b mRNA: It was encapsulated by patented LNPs with improved efficiency of the mRNA delivery according to its clinical trial report (#NCT04368728) 81, 82. The LNPs are composed of ionizable amino lipid, phospholipid, cholesterol and a PEGylated lipid prepared at a ratio of 50:10:38.5:1.5 mol/mol 82, 83. It is interesting to note that BTN162b and mRNA-1273 vaccines are suggested to be shipped and stored at -80˚C and -20°C, respectively 80, 82.
3. CVnCoV: It was formulated with a proprietary LNP, referred to as RNActive® technology platform. The LNP consists of four lipid components: cholesterol, DSPC, PEGylated lipid, and a cationic lipid, however the detailed formulation information was not disclosed. CVnCoV remains stable for at least three months when it is stored at 5℃ as suggested by its manufacturer. Moreover, CVnCoV can be stored at room temperature as a ready-to-use the vaccine for up to 24 hours 84, 85.
4. ARCoV: It was encapsulated in LNPs of a proprietary composition using a preformed vesicle method and found thermostable at different temperatures, including 4°C, 25°C, and 37°C for up to one week 86.
5. ARCT-021: Currently undergoing phase 1/2 clinical trials, it combines two technologies, i.e., saRNA STARR™ and LUNAR® lipid-mediated delivery method. It was designed to enhance and extend antigen expression, enabling vaccination at lower doses 87. In addition, LUNAR® lipids are pH-sensitive and biodegradable, causing minimal lipid accumulation in cells after multiple dosing 87
6. LNP-nCoVsaRNA: Developed by Imperial College London using cationic liposome as the carrier, it has just entered phase 1 clinical trial 55.
4. ARCoV: It was encapsulated in LNPs of a proprietary composition using a preformed vesicle method and found thermostable at different temperatures, including 4°C, 25°C, and 37°C for up to one week 86.
5. ARCT-021: Currently undergoing phase 1/2 clinical trials, it combines two technologies, i.e., saRNA STARR™ and LUNAR® lipid-mediated delivery method. It was designed to enhance and extend antigen expression, enabling vaccination at lower doses 87. In addition, LUNAR® lipids are pH-sensitive and biodegradable, causing minimal lipid accumulation in cells after multiple dosing 87
6. LNP-nCoVsaRNA: Developed by Imperial College London using cationic liposome as the carrier, it has just entered phase 1 clinical trial 55.
