In less than one year since the outbreak of the COVID-19 pandemic, two mRNA-based vaccines, BNT162b2 and mRNA-1273, were granted the first historic authorization for emergency use, while another mRNA vaccine, CVnCoV, progressed to phase 3 clinical testing. The COVID-19 mRNA vaccines represent a new class of vaccine products, which consist of synthetic mRNA strands encoding the SARS-CoV-2 Spike glycoprotein, packaged in lipid nanoparticles to deliver mRNA to cells. This review digs deeper into the scientific breakthroughs of the last decades that laid the foundations for the rapid rise of mRNA vaccines during the COVID-19 pandemic. As well as providing momentum for mRNA vaccines, SARS-CoV-2 represents an ideal case study allowing to compare design-activity differences between the different mRNA vaccine candidates. Therefore, a detailed overview of the composition and (pre)clinical performance of the three most advanced mRNA vaccines is provided and the influence of choices in their structural design on to their immunogenicity and reactogenicity profile is discussed in depth. In addition to the new fundamental insights in the mRNA vaccines’ mode of action highlighted here, we also point out which unknowns remain that require further investigation and possibly, optimization in future mRNA vaccine development.

All mRNA vaccines target the same SARS-CoV-2 antigen and incor porate mRNA encoding the full-length, transmembrane anchored S protein. The genetic sequence is slightly altered to stabilize the prefusion conformation of the glycoprotein using two proline (2P) substitutions (K986P and V987P mutations) (Fig. 2a) [21]. A major advantage of the mRNA approach, is that the proteins are produced by the host cells as they would be in case of a natural infection with the virus. As a result, the produced proteins will undergo the same post-translational pro cessing, including glycosylation, subunit cleavage and proper protein folding. As such, the S glycoprotein is eventually incorporated as a trimer in the membrane of mRNA-transfected cells, allowing it to be efficiently exposed in its antigenically native prefusion conformation to B cells. Precedent work by Moderna and the NIH National Institute for Allergy and Infectious Diseases (NIAID) demonstrated that the membrane-bound MERS-CoV 2P S mRNA elicited more potent neutral izing antibody responses as compared to secreted MERS-CoV 2P S, or wild-type S mRNA; fundamental insights that could immediately be transferred to the mRNA vaccine design for the SARS-CoV-2 pandemic [27]. Moreover, isolation of neutralizing antibodies from the serum of COVID-19 patients confirmed the strong immunogenicity of the S pro tein, but showed an equal immunogenicity of the RBD and the N-ter minal domain (NTD). This implies that vaccinating against the entire S protein, rather than only one of its structural components, is expected to result in an improved response which will be less affected when the virus undergoes genetic drift [28].

Several optimizations to the mRNA structure can drastically improve the final outcome. The design of (non-coding) structural elements of the mRNA such as the CAP structure, poly(A) tail and untranslated regions (UTRs) all have a major impact on the mRNA stability and translation capacity [31,32]. Codon optimization in the mRNA sequence to e.g., match host transfer (t)RNA abundances, or as a determinant of intro ducing secondary structures, can drastically impact the protein synthesis rate and ribosome dwell time (i.e. mRNA functional half-life) [33,34]. In this context, 1m. nucleotide-modifications were shown to provide additional base pair stability, giving rise to a high degree of secondary structure which significantly improves the mRNA translation [35]. Furthermore, the secondary structure design of mRNA can be optimized in order to improve mRNA stability against cleavage by endonucleases and chemical degradation processes, including hydrolysis [36]. BNT162b2 and mRNA-1273 implement a combination of modifie nucleotide 1m. replacement and removal of dsRNA fragments in the mRNA production process, which strongly reduces the innate immune signaling in response to mRNA through decreased activation of TLR signaling and cytosolic RNA sensors. Moderna claimed that with such an approach, both local and systemic innate immune effects upon mRNA (vaccine) administration can be limited to a bare minimum in mice [37]. In contrast to BNT162b2 and mRNA-1273, the CVnCoV vaccine candi date contains an “unmodified” mRNA, which employs sequence engi “ neering (e.g., reduction in uridine content), selected UTRs, and a stringent purification protocol to remove dsRNA fragments [11].

Moreover, BioNTech’s and Moderna’s LNPs also contain (more or less) the same helper lipids; 1,2-dis tearoyl- snglycero-3-phosphocholine (DSPC), cholesterol and a diffusible PEG-lipid (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, PEG2000-DMA in BNT162b2 or 1,2-dimyristoyl-rac-glycero3-methoxypo lyethylene glycol-2000, PEG2000-DMG in mRNA-1273) [23,24].

Fig. 2. COVID-19 mRNA vaccine design. (a) The COVID-19 mRNA vaccines contain a mRNA sequence encoding the full length S protein with two proline substitutions (K986P and V987P). The S protein’s genetic code is flanked by structural elements to produce a mature mRNA. Each of these elements can be optimized in order to modulate mRNA stability, translation capacity and innate immune activity. (b) While the CVnCoV vaccine candidate make use of unmodified uridines, BNT162b2 and mRNA-1273 are nucleoside-modified with a substitution of N1-methylpseudouridine (1mψ) for uridine (U). (c) Chemical structures of the ionizable cationic lipids ALC-0315 (((4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) and SM-102 (Heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)- 8-oxooctyl)amino)octanoate) used in the LNP formulation of BNT162b2 and mRNA-1273, respectively. The ionizable cationic lipid used in CVnCoV has not (yet) been disclosed. Abbreviations: SS; Signal sequence, NTD; N-terminal domain, S1/S2; native furine cleavage site, TM; transmembrane domain.

Fig. 3. The mode of action of mRNA vaccines. (a –at the injection site) Upon endocytosis by muscle-resident cells, mRNA LNPs trigger a transient inflammatory response recruiting neutrophils, monocytes and DCs to the injection site. Local and recruited APC subsets transiently express the S protein mRNA and undergo maturation in response to innate immune sensing of the mRNA. The migration of targeted/activated APCs and direct lymphatic transport of mRNA LNPs and cell debris containing S proteins, brings the S antigen to B cells and T cells in draining lymph nodes. (b – at the cellular level) To avoid lysosomal degradation, mRNA must escape the endosomes and binds to ribosomes, known as a complex and rate-limiting process, which is facilitated by the ionizable LNP carrier. After translation and transport of S proteins through the endoplasmatic reticulum and Golgi apparatus, S proteins are exposed as prefusion-stabilized trimer constructs at the cell surface. This membrane-bound S antigen can efficiently be recognized and internalized by B cells, which leads to a series of events activating B cells responses towards neutralizing antibody generation against the S protein. Moreover, the expressed S antigens can gain access to the MHC class I antigen presentation pathway to prime CD8+ T cells that can eliminate infected cells, while recycling mechanisms allow the presentation of antigenic epitopes in MHC-II complexes to CD4+ helper T cells, especially needed to promote the antibody production by providing B cell help. Abbreviations: APC; Antigen presenting cell, RBD; Receptor binding domain, MHC; major histocompatibility complex.

Fig 4. Proposed innate immune signaling in response to mRNA vaccination. The internalization of mRNA LNPs can be detected by innate immune sensors that are localized in the endosomes and cytosol. The detection of mRNA, by the endosomal TLR/8, recruits the MYD88 signal transduction adaptor and leads to the expression of type I IFNs (IFN-α and IFN-β) through IFN regulatory factor 7, and to the secretion of other proinflammatory cytokines through nuclear factor κB (NF-κB). In addition, dsRNA contaminants and/or secondary structures in the mRNA product can interact with TLR3 in the endosomes, recruiting TRIF, as well as upon their arrival in the cytosol be detected by RIG-I and MDA5, binding MAVS. The activation of TRIF and MAVS is followed by molecular cascades that results in the expression of type I IFNs in control of IRF3 and IRF7. In turn, the type I IFN cytokines bind autocrine or paracrine receptors, which eventually regulates the gene expression of hundreds of proteins involved in antiviral immunity. This includes the expression of MHC-I and co-stimulatory molecules, needed for T cell responses, as well as antiviral proteins involved with undesirable anti-RNA responses. Methods such as the introduction of modified nucleotides, the removal of dsRNA fragments, and sequence-engineering, can be utilized to minimize or control the type I IFN activity of mRNA. However, it remains unclear how to strike the perfect balance between obtaining sufficient mRNA-encoded antigen expression and adequate immunostimulation in order to support adaptive immunity. In addition, more research is needed to investigate whether and how the recognition of lipid components in the LNP vehicle might contribute to the innate immune response to mRNA vaccines. Abbreviations: IRF; interferon regulatory factor, ISG; interferon-stimulated gene, NF-κB; nuclear factor-κB, MAVS; mitochondrial antiviral signaling protein, MDA5; melanoma differentiation-associated protein 5, MYD88; myeloid differentiation primary response protein 88, TRIF, Toll-IL-1 receptor domain-containing adapter protein inducing IFNβ.

DOI. 10.1016/j.jconrel.2021.03.043

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