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Overview of the development of mRNA vaccines: Part Four
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Overview of the development of mRNA vaccines: Part Four.
Part Four: Application of mRNA Vaccine
mRNA vaccine against COVID-19
Like other human coronaviruses including SARS and MERS, SARS-CoV-2 is an enveloped, positive-stranded single-stranded RNA virus. Its genomic RNA encodes non-structural polyproteins and structural proteins, including thorns. Spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. As surface proteins, the S, E, and M proteins are inserted into the virus envelope, and the S glycoprotein gives the virus particle a “crown” and name. The N protein surrounds the positive-strand genomic RNA. SARS-CoV-2 uses the S protein to enter the host cell.
The S glycoprotein has a total length of 1,273 amino acids and is composed of an N-terminal signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain. It is functionally divided into S1 and S2 subunits ( Figure 1A). The receptor binding domain (RBD) is located at the C-terminus of the S1 subunit and is responsible for binding to human angiotensin-converting enzyme 2 (hACE2). S2 mediates membrane fusion. The S structure before fusion reveals the four domains of the S1 subunit, the N-terminal domain (NTD), RBD, C-terminal domain (CTD) 1 and CTD2, which surround the triple axis of the S trimer and cover the S2 below. Fragment; The S2 subunit appears to be a symmetrical trimer in which the first heptapeptide repeat (HR1) curves toward the viral membrane (Figure 1B).
The structure of the SARS-CoV-2 S trimer in the fusion conformation shows that after a large number of structural rearrangements, HR1 and the central helix (CH) form an unusually long, central, three-stranded coiled coil (Figure 1C) . In addition, it is reported that the spontaneous transition of SARS-CoV-2 S protein to the post-fusion state has nothing to do with target cells. When the full-length S-encoding plasmid is transfected into cells, S protein will be produced before and after fusion.
The high instability of S protein in the pre-fusion conformation is undoubtedly a major obstacle in the development of S-based vaccines. Fortunately, the introduction of two consecutive proline residues (2P) at the beginning of the CH is a general strategy for keeping the β-coronavirus S protein in the pre-fusion conformation. The limited torsion angle of the proline backbone may not be conducive to the refolding of the joint between CH and HR1, preventing the S protein from transforming into the fusion conformation.
In addition, the cryo-EM structure of the SARS-CoV-2 S-2P mutant showed that the 2P substitution did not change the conformation of the S protein (Figure 1D). At the same time, various structures including RBD-hACE2 and RBD-monoclonal antibody are also reported. RBD contains two structural subdomains, of which five antiparallel b-strands contain a conserved core subdomain, and the other outer subdomain is controlled by a flexible ring stabilizing the disulfide bond that recognizes hACE2 (Figure 1E). In addition, both RBD and S-2P proteins can induce effective SARS-CoV-2 neutralizing antibodies and T cell responses. Therefore, they are widely used as immunogens in the development of COVID-19 mRNA vaccines.
Figure 1 Antigen of COVID-19 mRNA vaccine
A: A schematic diagram of the full-length SARS-CoV-2S primary structure colored by domain. SS, signal peptide; NTD, N-terminal domain; RBD, receptor binding domain; CTD, C-terminal domain; FP, fusion peptide; HR1, heptapeptide repeat 1; CH, central helix; CD, connector domain; HR2 , Heptapeptide repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. B: The structure of SARS-CoV-2 wild-type S protein before fusion. C: The structure of SARS-CoV-2 wild-type S protein after fusion. D: The structure of SARS-CoV-2S-2P. E: The structure of SARS-CoV-2RBD.
The mRNA vaccine contains the S protein coding region, flanked by optimized 5′- and 3′-UTR and polyA tails, synthesized by IVT, then 5′-capped with 5′-cap mimics, and encapsulated with LNP for IM Injection (intramuscular injection) (Figure 2 Step 1). The vaccine enters muscle cells or antigen-presenting cells, such as dendritic cells or macrophages, through endocytosis (step 2). The mRNA molecule is unloaded from the LNP and translated into the S protein in the ribosome (step 3). The newly synthesized S protein is secreted into the extracellular space, is internalized into antigen-presenting cells through endocytosis, and presents the antigen to immune cells as part of the MHC class II antigen-presenting complex (steps 5b, 6b, and 7). Including T cells and B cells. The partially degraded S peptide of the proteosome is integrated into the MHC class I complex, which is then transported to the plasma membrane and presented to immune cells as an antigen (steps 4a, 4b, 5a, and 7).
Figure 2 Delivery and working mechanism of COVID-19 mRNA vaccine
With its versatility and rapid development advantages, two COVID-19 mRNA vaccines (mRNA-1273 and BNT162b2) have been approved for marketing, one drug candidate is in phase III clinical trials, and the other three drug candidates are currently in phase I or II clinical evaluation . Based on the published data of preclinical experiments or clinical trials, Table 1 lists all COVID-19 mRNA vaccines or candidate vaccines in clinical trials, and summarizes their safety, neutralizing antibody response and protective efficacy.
Table 1 Clinical application of COVID-19 mRNA vaccine
BNT162b2 is a lipid nanoparticle preparation, a nucleoside modified mRNA vaccine, used to prevent new coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The expression of the SARS-CoV-2 S protein encoded by BNT162b2 can induce the receptor’s immune response to the antigen. BNT162b2 was administered intramuscularly in a two-dose schedule. The protection rate can reach 95%.
BNT162b2 needs to be refrigerated (a logistical challenge for vaccine distribution), and must be thawed and diluted before use. After dilution, each vial contains six 30 µg doses. BNT162b2 is injected intramuscularly in two courses of 30 µg (into the deltoid muscle ideally), with a recommended dose interval of 21 days. Seven days after the second dose is administered, complete protection from COVID-19 may not be achieved. As with other vaccines, BNT162b2 may not protect every recipient.
Mechanism of action
BNT162b2 is composed of nucleoside modified mRNA encapsulated by lipid nanoparticles, which encodes a membrane-anchored full-length SARS-CoV-19 S protein and contains a mutation that stabilizes the S protein in an antigen-preferred pre-fusion conformation. Lipid nanoparticles protect non-replicating RNA from degradation and allow it to enter host cells after intramuscular injection. Once in the host cell, the mRNA is translated into the SARS-CoV-2 S protein, which is expressed on the surface of the host cell and induces neutralizing antibodies and cellular immune responses against it (Figure 3).
Figure 3 The mechanism of action of intramuscular injection of BNT162b2
Although mRNA vaccines have shown obvious advantages in the past few years, it was not until the SARS-CoV-2 pandemic significantly accelerated and accelerated clinical trials and reviews that humans approved the use of the first mRNA vaccine. This is undoubtedly a milestone in the history of vaccination. If successful, mRNA-based vaccines may become an immediate “standard” solution for future pandemics, but they may also replace some conventional protein-based live attenuated vaccines. The mRNA platform may also be superior to other platforms, and can be modified and distributed the fastest to combat new mutant virus strains that emerge during a pandemic.
Based on the major technological innovations and advancements of the mRNA vaccine platform in the past decade, an mRNA vaccine against COVID-19 has been successfully developed at an unprecedented speed. In clinical trials, compared with protein subunit vaccines and inactivated virus vaccines, COVID-19 mRNA vaccines showed a higher incidence of systemic adverse events, such as fever and fatigue. Therefore, it is necessary to monitor the safety of the COVID-19 mRNA vaccine for a long time. Most mRNA vaccines are designed to produce neutralizing IgG antibodies, and neutralizing IgG antibodies can only effectively protect the lower respiratory tract through muscle immunity. However, IgA antibodies, which are mainly responsible for protecting the upper respiratory tract, may be necessary for disinfection and immunity, and the level of IgA antibodies induced by mRNA vaccines has not been determined in current clinical trials. In addition, although the results of the mRNA-1273 study show that after the first vaccination in humans, high school and antibody titers last at least 4 months, but the time that these mRNA vaccines can protect humans against COVID-19 needs further elucidation.
On the other hand, data is still needed to evaluate whether mRNA vaccines are suitable for everyone, including children, the elderly, immunosuppressed individuals, and patients with chronic diseases such as autoimmune diseases. Compatibility with different drugs also needs to be evaluated. Will the type I IFN response induced by mRNA vaccine become a problem for patients with various underlying diseases or type I IFN treatment? Some of these types of investigations have already begun or are planned. During the distribution of the approved SARS-CoV-2 mRNA vaccine, other issues with the mRNA vaccine have caused great concern in the health care system, including cold chain and storage restrictions. Even in high-income countries, many clinics and vaccination sites cannot use low-temperature refrigerators to meet the needs of certain mRNA vaccines. This challenge will become more prominent in low-income countries. Therefore, there is an urgent need to improve and verify these issues.
Other clinical applications of mRNA vaccines
Since Wolf et al. demonstrated that proteins can be produced from in vitro transcribed mRNA in living tissues, mRNA vaccines have proven their effectiveness in many applications. The first clinical trial using mRNA technology based on RNA-pulsed DC cancer vaccine can be traced back to 2003. Today, more than 140 clinical trials can be found that use mRNA to address different situations such as cancer or infectious diseases (Figure 4).
Figure 4 The breakdown of mRNA vaccine clinical trials submitted by disease type (left) and delivery system (right) each year
Cancer is currently the preferred target of mRNA technology. More than 50% of clinical trials focus on the treatment of melanoma, prostate cancer and brain cancer (Figure 5), and most trials are still in the early stages (I and II). In addition to safety and immune response, the lack of benchmarks for cancer treatment hinders the assessment of vaccine effectiveness. However, this is not the case for infectious diseases, as many traditional vaccines can be used as benchmarks for validating new mRNA vaccines. mRNA also shows other potentials, not only for the treatment of cancer, but also as a therapeutic agent for protein expression in many other diseases (such as cardiovascular disease and type II diabetes).
Figure 5 Application of mRNA vaccine
Table 2 and Table 3 summarize the clinical application of mRNA vaccines against cancer and infectious diseases.
Table 2 Clinical trials of mRNA vaccines against cancer
Table 3 Clinical trials of mRNA vaccines against viral diseases
(source:internet, reference only)