Advances in COVID-19 mRNA vaccine development

Abstract

To date, the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has determined 399,600,607 cases and 5,757,562 deaths worldwide. COVID-19 is a serious threat to human health globally. The World Health Organization (WHO) has declared COVID-19 pandemic a major public health emergency. Vaccination is the most effective and economical intervention for controlling the spread of epidemics, and consequently saving lives and protecting the health of the population. Various techniques have been employed in the development of COVID-19 vaccines. Among these, the COVID-19 messenger RNA (mRNA) vaccine has been drawing increasing attention owing to its great application prospects and advantages, which include short development cycle, easy industrialization, simple production process, flexibility to respond to new variants, and the capacity to induce better immune response. This review summarizes current knowledge on the structural characteristics, antigen design strategies, delivery systems, industrialization potential, quality control, latest clinical trials and real-world data of COVID-19 mRNA vaccines as well as mRNA technology. Current challenges and future directions in the development of preventive mRNA vaccines for major infectious diseases are also discussed.

Fig. 1. Cellular and humoral immune responses induced by messenger RNA (mRNA) vaccine.

mRNA delivered in an mRNA vaccine enters cells by endocytosis and, after release from the endosome, is translated into protein by ribosomes. Translated proteins can then activate the immune system primarily in two ways: i) proteins are degraded by the proteasome into peptides subsequently presented as antigens on the cell surface by major histocompatibility complex (MHC) class I molecules which bind to the T cell receptor (TCR) to activate CD8⁺ T cells to kill infected cells thorugh the secretion of perforin and granzyme; ii) proteins secreted extracellularly are engulfed by antigen-presenting cells (APCs) and degraded into peptides subsequently presented on the cell surface by MHC class II molecules for recognition by CD4⁺ T cells, which can activate both the cellular immune responses by secreting cytokines and the humoral immune responses by co-activating B cells. In addition, single-stranded RNA and double-stranded RNA delivered in mRNA vaccines bind to Toll-like receptor (TLR) in the endosome to activate the antiviral innate immune responses via the production of type-I interferon (IFN-I) which results in the induction of several IFN-1-stimulated genes involved in antiviral innate immunity, in a mechanism known as the self-adjuvant effect of a sequence-engineered mRNA. This figure is created with BioRender.com

Fig. 2. Proposed mechanism of endosomal escape of delivered mRNA.

Endosomal escape of delivered mRNA is largely dependable on interactions between ionizable lipids and naturally occurring anionic phospholipids in the endosomal membrane. Prior to membrane fusion, ionizable lipids in lipid nanoparticles (LNPs) and anionic lipids in the endosomal membrane adopt a cylindrical conformation which is compatible with molecular packing in a bilayer phase. The acidic environment in endosomes facilitates protonation of ionizable lipids into cationic lipids. Cationic and anionic lipids generate ion pairs whose combined cross-sectional headgroup area is smaller than the total of individual headgroup areas before membrane fusion. Consequently, the ion pair adopts a conical shape which promotes the formation of inverted, non-bilayer phases, such as the hexagonal shape illustrated above. Thus, the formation of ion pairs between lipids promotes membrane fusion and disruption, allowing mRNA to escape from endosomes. This figure is created with BioRender.com

Fig. 3. Antigen expression in different types of mRNA vaccines.

A The vaccine immunogen is encoded by a non-replicating RNA flanked by 5′ and 3′ UTRs (S protein). B Self-amplifying RNA (saRNA) encodes four nonstructural proteins (nsp 1–4) and a subgenomic promoter derived from the alphavirus genome. saRNA encodes a replicase and amplifies vaccine-encoding transcripts. C Trans-amplifying RNA (taRNA) uses two transcripts to enable self-amplification of replicase and the immunogen. D Circular RNA (circRNA) is circularized by the autocatalytic Group I ribozyme. The exon 2 is ligated upstream to exon 1, and a coding region is inserted between the exon-exon junction. During splicing, the 3′-OH of a guanosine nucleotide engages in a transesterification reaction at the 5′ splice site. The 5′ intron is excised, and the 3′-OH at the end of the intermediate engages in a second transesterification reaction at the 3′ splice site, resulting in the circularization of the immunogen mRNA. Upon entering the cell, the internal ribosome entry site (IRES) of circRNA initiates protein translation. The figures are created with BioRender.com

Fig. 4. Structure of mRNA and nucleotide modifications.

mRNA molecules are synthesized in vitro with a 5′-cap 1 structure and chemically modified nucleotides as substitutes for natural nucleotides, which enhances stability and translation efficiency of mRNA as well as reduces innate immune response. This figure is created with BioRender.com

Fig. 5. mRNA capping procedure using capping enzymes or cap analogs.

A Production of post-transcriptional modifications of mRNA with cap0 requires three enzymes: triphosphatase, guanylyltransferase, and N7-methyltransferase with S-adenosylmethionine (SAM) as the methyl donor. Subsequently, the cap0 is modified with 2′-O-ribose methyltransferase to generate the cap1 structure. B Cap analogs commonly used for in vitro transcription of mRNA are CleanCap® Reagent AG (TriLink) and CleanCap® Reagent AU (TriLink). The proposed mechanism of CleanCap co-transcriptional initiation involves the docking of AmG or AmU dimers onto the +1 and +2 positions in template nucleotides. Initiation occurs upon coupling of CleanCap with an nucleoside triphosphate (NTP) occupying the +3 position.

Fig. 6. Rationale underlying the design strategy of COVID-19 mRNA vaccine.

Representation of the SARS-CoV-2 reference genome showing structural, nonstructural, and accessory proteins, consisting of ORF1a, ORF1b, Spike protein (S), ORF3a, ORF3b, Envelope (E), Membrane (M), ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF14, Nucleocapsid (N) and ORF10. Spike and receptor-binding domain (RBD) proteins are mainly used as target antigens for the design and optimization of COVID-19 mRNA vaccines. This figure is created with BioRender.com

Fig. 7. Structure of lipid nanoparticles (LNPs) and lipid components employed in currently available COVID-19 mRNA vaccines.

LNPs are composed of four components: ionizable lipid, helper lipid, cholesterol, and PEGylated lipid. Binding with mRNA occurs by the ionizable lipid that occupies the central core of the LNP. PEGylated lipid is found on the surface of LNPs along with helper lipid forming the bilayer. Cholesterol, charged ionizable lipids, and neutral ionizable lipids are distributed throughout LNPs. The confirmed or the most likely chemical structure of ionizable lipids employed in COVID-19 mRNA vaccines developed by Moderna, BioNTech, CureVac, Arcturus, Imperial College London, and Chulalongkorn University. *Molar lipid ratio (%) of ionizable lipid: helper lipid: cholesterol: PEGylated lipid; **NA: Not applicable

Fig. 8. Production process of mRNA vaccines.

The design of an mRNA vaccine is conditioned to the definition of the antigen sequence of the target pathogen. By determining the target antigen and optimizing its coding sequence, the mRNA can be transcribed in vitro by RNA polymerase. The synthesized mRNA is purified by different processes and then mixed with a lipid phase using microfluidics and encapsulated into an mRNA-lipid nanoparticle (mRNA-LNP) complex. Subsequently, self-assembly of LNPs is completed by dilution and concentration by ultrafiltration. Finally, after sterile filtration, filling, and capping, the mRNA vaccine is obtained