Technological breakthroughs and advancements in the application of mRNA vaccines: a comprehensive exploration and future prospects

Abstract

mRNA vaccines utilize single-stranded linear DNA as a template for in vitro transcription. The mRNA is introduced into the cytoplasm via the corresponding delivery system to express the target protein, which then performs its relevant biological function. mRNA vaccines are beneficial in various fields, including cancer vaccines, infectious disease vaccines, protein replacement therapy, and treatment of rare diseases. They offer advantages such as a simple manufacturing process, a quick development cycle, and ease of industrialization. Additionally, mRNA vaccines afford flexibility in adjusting antigen designs and combining sequences of multiple variants, thereby addressing the issue of frequent mutations in pathogenic microorganisms. This paper aims to provide an extensive review of the global development and current research status of mRNA vaccines, with a focus on immunogenicity, classification, design, delivery vector development, stability, and biomedical application. Moreover, the study highlights current challenges and offers insights into future directions for development.

Keywords: biomedical application; classification; delivery vector development; design; immunogenicity; mRNA vaccines; stability.

Figure 1

mRNA vaccines immunogenicity. The mRNA delivered in mRNA vaccines enters cells through endocytosis and is subsequently released from endosome into the cytoplasm, where ribosomes translate it into proteins. The proteins are degraded into peptides by proteasomes and presented on the cell surface as antigens by MHC-I. These antigens bind to T-cell receptors (TCRs), activating CD8⁺ T cells, which secrete perforins and granzymes to kill infected cells. Proteins secreted extracellularly are phagocytosed by APCs and degraded into peptides within lysosomes. These peptides are then presented on the cell surface by MHC-II for recognition by CD4⁺ T cells. CD4⁺ T cells can not only activate cellular immune responses by secreting cytokines but also induce humoral immune responses by activating B cells. ssRNA in mRNA vaccines binds to Toll-like receptor (TLR) 7/8 within endosomes, while dsRNA interacts with TLR3, Retinoic acid-inducible gene I (RIG-I), and Melanoma Differentiation-Associated protein 5 (MDA-5). These interactions induce the production of pro-inflammatory cytokines and type I IFN (IFN-I), thereby activating antiviral innate immune responses, through which mRNA exhibits a self-adjuvant effect. Additionally, dsRNA also can activate PKR and OAS. IFN-I binds to Interferon-α/β Receptor (IFNAR) and trigger the JAK-STAT signaling pathway, inducing the expression of PKR and OAS, thereby exerting a negative regulatory effect on the immune response. Green arrow: cellular immunity, black arrow: homoral immunity, red arrow: self-adjuvant effect, blue arrow: negative immune effect. TCR, T-cell receptor; BCR, B-cell receptor; MHC, Major Histocompatibility Complex; APC, antigen-presenting cell; ADCC, antibody-dependent cellular cytotoxicity; RNase L, Ribonuclease L; eIF2, eukaryotic translation initiation factor 2; dsRNA, Double-stranded RNA; ssRNA, Single-stranded RNA; TBK-1, TANK-binding kinase 1; IKKϵ, IκB kinase ϵ; IFN, interferon; IRF, IFN regulatory factor; MyD88, Myeloid Differentiation Primary Response 88; ER, Endoplasmic Reticulum.

Figure 2

Different types of mRNA vaccines. (A) Non-replicating mRNA (nrmRNA) vaccines. (B) Self-amplifying RNA (saRNA) vaccines. (C) Trans-amplifying RNA (taRNA) vaccines. (D) Circular RNA (circRNA) vaccines. UTR, untranslated region; IRES, internal ribosome entry site.

Figure 3

Enzyme capping method. RNA triphosphatase (RTPase) hydrolyzes the 5’γ-phosphate of messenger RNA (mRNA), resulting in a β-phosphate group. Under the action of guanylyltransferase (GTase), the β-phosphate at the 5’ end is linked to guanosine monophosphate (GMP) via a 5’-to-5’ triphosphate bridge. RNA guanine-N7 methyltransferase (G-N7 MTase) uses S-adenosyl-L-methionine (AdoMet) as a substrate to methylate the guanine base at the N7 position, forming Cap 0. Subsequently, 2’-O-methyltransferase methylates the R1 group on Cap 0 to produce Cap 1. Further methylation at the R2 position results in the formation of Cap 2.

Figure 4

mRNA circular translation complex. 5’ cap binds to eIF4E, which, in turn, associates with eIF4G and eIF4A to form the cap-binding initiation complex eIF4F. The eIF4F complex recruits the 40S ribosomal small subunit, while the poly(A)-binding protein (PABP) interacts with eIF4G. Acting as an anchor, eIF4G directly links the 5’ cap and the poly(A) tail of mRNA, forming a “ closed-loop “ structure.