The success of mRNA vaccines ushered in a new era of vaccinology
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Nature Reviews: The success of mRNA vaccines ushered in a new era of vaccinology
The success of mRNA vaccines ushered in a new era of vaccinology. On January 12, 2018, Professor Drew Weissman published a review  in the journal Nature Reviews Drug Discovery, describing the future of a promising mRNA vaccine. At this time, he might not have thought that a quarrel with Katalin Karikó in front of the laboratory copier in 1997 would pave the way for saving the world more than 20 years later.
Drew Weissman (left), Katalin Karikó (right)
At that time, the two had a dispute over grabbing a photocopier, and they got acquainted. In the subsequent exchanges, they found that each other had similar views on mRNA and began to collaborate on research. On August 1, 2005, the two people’s paper was published in the journal Immunity.  They modified the mRNA by pseudouracil, which greatly enhanced the stability of the mRNA in the body.
We all know what happened later, the COVID-19 epidemic broke out, and the mRNA vaccine turned out.
This review was published in January 2018. At this time, the theoretical research on mRNA has been perfected, the instability of mRNA in the body and the low efficiency of delivery have been basically solved, and clinical trials of mRNA vaccines for several infectious diseases and cancers have also been completed. Shows an encouraging effect.
Professor Drew Weissman gave a detailed overview of mRNA vaccines and elaborated on the future direction and challenges of pushing this promising vaccine platform to a wide range of preventive and therapeutic uses.
Vaccines can prevent millions of diseases and save countless lives every year. Due to the widespread use of vaccines, the smallpox virus has been completely eradicated, and the incidence of polio, measles and other childhood diseases has been drastically reduced worldwide. Traditional vaccine methods, such as live attenuated and inactivated pathogens and subunit vaccines, can provide durable protection against various dangerous infectious diseases. Despite many successes, there are still major obstacles to the development of vaccines against various infectious pathogens, especially those that can better evade adaptive immune responses. In addition, for most emerging virus vaccines, the main obstacle is not the effectiveness of traditional methods, but the need for faster development and large-scale deployment. Moreover, traditional vaccine methods may not be suitable for non-communicable diseases, such as cancer. Therefore, there is an urgent need to develop a more effective and universal vaccine platform.
As early as 1990, studies have shown that injecting reporter gene mRNA into mice can detect protein production. A follow-up study in 1992 showed that injecting mRNA encoding vasopressin into the rat hypothalamus can cause physiological responses in rats. However, these studies have not ignited an upsurge in the development of mRNA therapies. This is mainly due to concerns about mRNA instability, high innate immunogenicity and low delivery efficiency in vivo. At this time, the scientific research community is pursuing treatment methods based on DNA and protein.
In the following decades, many major technological innovations and capital investment have made mRNA vaccines gradually become a promising vaccine platform. Compared with subunit vaccines, inactivated virus vaccines, live attenuated virus vaccines, and DNA-based vaccines, mRNA vaccines have several significant advantages:
1. Safety: Since mRNA is a non-infectious, non-integrative platform, there is no potential risk of infection or insertion mutation. In addition, mRNA is degraded by normal cellular processes, and its half-life in vivo can be adjusted by using various modifications and delivery methods. The inherent immunogenicity of mRNA can also be down-regulated to further improve safety.
2. Effectiveness: Various modifications make the mRNA more stable and highly translatable. By constructing the mRNA on the carrier molecule, it can be quickly taken up and expressed in the cytoplasm, thereby achieving effective in vivo delivery. mRNA is the smallest genetic carrier, therefore, anti-carrier immune responses are avoided and can be administered repeatedly.
3. High production efficiency: mRNA vaccines have the potential for rapid, cheap and scalable manufacturing, mainly due to the high yield of in vitro transcription reactions.
mRNA vaccines represent a promising alternative to traditional vaccine methods because of their high efficiency, rapid development capabilities, and the potential for low-cost manufacturing and safe management.
Basic mRNA vaccine pharmacology
mRNA, also called messenger RNA, is responsible for transmitting the genetic information stored in DNA and directing the synthesis of proteins in cells. The mRNA vaccine is to use mRNA to guide cells to produce the corresponding protein, so as to prevent and treat diseases.
There are currently two main types of RNA used as vaccines: non-replicating mRNA and self-amplified RNA from viral sources.
Traditional mRNA-based vaccines encode the antigen of interest and contain 5’and 3’untranslated regions, while self-amplified RNA not only encodes the antigen, but also encodes genes related to viral replication, thereby achieving intracellular RNA amplification and richer proteins Express.
Naked mRNA will be rapidly degraded by RNase outside the cell, making it difficult to effectively enter the cell. A variety of in vitro and in vivo transfection reagents, as well as RNA modifications have been developed to promote the uptake of mRNA by cells and prevent its degradation.
Once the mRNA is transferred to the cytoplasm, the cellular translation machinery will produce a post-translationally modified protein, resulting in a fully functional protein that is correctly folded. This feature of mRNA pharmacology is particularly advantageous for vaccines and protein replacement therapies that require the delivery of cytoplasmic or transmembrane proteins to the correct cell compartment for proper presentation or function. The mRNA transcribed in vitro will eventually be degraded by normal physiological processes, thereby reducing the risk of metabolite toxicity.
mRNA vaccine technology
mRNA translation and stability optimization
The 5’and 3’UTR elements flanking the coding sequence profoundly affect the stability and translation of mRNA, both of which are key issues for vaccines. These regulatory sequences can be derived from viral or eukaryotic genes, and greatly increase the half-life and expression level of therapeutic mRNA.
Efficient protein production from mRNA requires a 5’cap. Various versions of 5’end caps or synthetic caps or the like can be added during or after the transcription reaction using the vaccinia virus capping enzyme. The poly(A) tail also plays an important regulatory role in mRNA translation and stability. Therefore, it is necessary to synthesize directly from the encoding DNA template or use poly(A) polymerase to add the optimal length of poly(A) to the mRNA. The use of codons has an impact on protein translation. Replacing rare codons with commonly used synonymous codons is a common practice to increase the level of mRNA translated protein.
Although the level of mRNA translation protein can be increased by changing the codon composition or introducing modified nucleosides, this approach may also affect mRNA secondary structure, translation kinetics and accuracy, protein folding, and so on. All these factors may affect the strength or specificity of the immune response.
Strategies for optimizing mRNA pharmacology
Modulation of immunogenicity
Exogenous mRNA has inherent immunostimulatory properties because it can be recognized by various cell surface, endosome and cytoplasmic innate immune receptors.
Depending on the treatment application, this feature of mRNA may be beneficial or harmful. It has potential advantages for vaccination, because in some cases, it can provide adjuvant activity to drive the maturation of dendritic cells, thereby triggering a powerful T cell immune response and B cell immune response. However, the innate immune induction of mRNA is also related to the suppression of antigen expression, and may have a negative impact on the immune response.
Delivery of mRNA vaccines
Effective in vivo mRNA delivery is critical to effectiveness. Exogenous mRNA must penetrate the lipid membrane barrier to reach the cytoplasm and be translated into functional proteins. The mRNA uptake mechanism seems to depend on the cell type, and the properties of mRNA complexes can profoundly affect cell delivery and organ distribution.
There are currently two broad categories of basic methods used to deliver mRNA vaccines. One is to transfect mRNA into dendritic cells (DCs) in vitro, and then transfer the transfected dendritic cells into the body. The other is to inject mRNA directly with or without a vector. The former can precisely control conditions such as cell targets and transfection efficiency, but as a cell therapy, it is an expensive and labor-intensive vaccination method. The latter method of directly injecting mRNA is faster and more economical.
Common delivery methods and carriers of mRNA vaccines: a. naked mRNA, b. electroporation, c. protamine, d. cationic nanoemulsion, e. modified dendritic nanoparticles, f. protamine liposomes, g . Cationic polymers, h. Cationic polymer liposomes, i. Polysaccharide particles, j. Cationic lipid nanoparticles, k. Cationic lipid cholesterol nanoparticles, 1. Cationic lipid cholesterol PEG nanoparticles.
In vitro transfection of dendritic cells (DCs):
Dendritic cells are the most effective antigen-presenting cells in the immune system, which can cause adaptive immune responses, and may also present complete antigens to B cells to trigger antibody responses. And dendritic cells are also very suitable for mRNA transfection. For these reasons, dendritic cells have become attractive targets for in vivo and in vitro mRNA vaccine transfection.
Although naked mRNA can enter dendritic cells through endocytosis, electroporation technology can improve transfection efficiency. The mRNA delivery method of electroporation is favored because of its ability to produce high transfection efficiency without the need for carrier molecules. Then the dendritic cells that have been transfected with mRNA are re-infused into the body to initiate an immune response. Most dendritic cell vaccines transfected with mRNA in vitro mainly cause cell-mediated immune responses, so they are mainly used to treat cancer.
In vivo injection of naked mRNA:
Naked mRNA has been successfully used for in vivo immunization, especially in forms that preferentially target antigen-presenting cells, such as intradermal and intranodal injections. Studies have shown that repeated intranodal immunization with naked, unmodified mRNA encoding tumor-associated neoantigens can generate a strong T cell response and increase progression-free survival.
Physical delivery in vivo:
In order to improve the efficiency of uptake of mRNA in the body, studies have used physical methods to penetrate cell membranes. For example, using the gene gun method, gold nanoparticles are compounded with mRNA and then injected. In recent years, the field has become more inclined to use lipid nanoparticles or polymer nanoparticles for delivery.
Cationic lipid and polymer-based delivery:
In the past few years, cationic lipids and polymers have become widely used mRNA delivery tools. This benefits from research on the delivery of small interfering RNA (siRNA) in vivo. Lipid nanoparticles (LNP) have become one of the most attractive and commonly used mRNA delivery tools. LNP usually consists of four components: an ionizable cationic lipid, which promotes self-assembly into virus-sized particles (about 100 nanometers in diameter), and allows endosomes to release mRNA into the cytoplasm; lipid-linked polyethylene glycol (PEG), which can extend the half-life of the preparation; cholesterol, as a stabilizer; and naturally occurring phospholipids, to support the lipid bilayer structure. Both of the two mRNA vaccines currently on the market use lipid nanoparticles (LNP) as delivery vehicles.
MRNA vaccines against infectious diseases
The development of preventive or therapeutic vaccines against infectious pathogens is the most effective means to control and prevent epidemics. However, traditional vaccine methods have largely failed to produce effective vaccines against challenging viruses that cause chronic or recurrent infections, such as HIV-1, herpes simplex virus, and respiratory syncytial virus (RSV).
In addition, as demonstrated by the 2014-2016 Ebola and Zika virus outbreaks, the slow pace of development and approval of commercial vaccines based on traditional vaccines is not sufficient to deal with rapidly emerging acute viral diseases. Therefore, it is essential to develop a more effective, faster and more versatile vaccine platform.
mRNA can be divided into two main types: non-replicating mRNA vaccines and self-amplifying mRNA vaccines. Among them, non-replicating mRNA vaccines can also be divided into two types according to the delivery vector: the type of dendritic cells transfected in vitro and the type of direct injection in vivo.
Direct injection of non-replicating mRNA vaccine
Direct injection, non-replicating mRNA vaccines are an attractive form of vaccine because they are simple and economical, especially in resource-limited environments. At present, two mRNA vaccines have been approved by the FDA for marketing, from Moderna and BioNTech, both of which are used to prevent new coronavirus (SARS-CoV-2) infection. In addition, human clinical trials have also been carried out for mRNA for HIV-1, Zika virus, influenza virus, and respiratory syncytial virus (RSV). These are direct injections of non-replicating mRNA vaccines.
Self-amplified mRNA vaccine
Compared with direct injection of non-replicating mRNA vaccines, self-amplified mRNA vaccines add replicable sequences to the mRNA sequence. After entering the cell, it can be like a virus and can use host cells for self-replication. In this case, the high expression of antigen is achieved, and the ability to generate a strong immune response is achieved. The self-amplifying mRNA vaccine released the results of a phase 1 clinical trial against the new coronavirus in July 2021. The vaccine can produce a protective immune response in 87% of vaccinators, even if the injection dose is much lower than the non-replicating mRNA vaccine. Moreover, no short-term safety issues have been observed.
Comparison of non-replicating mRNA vaccines and self-amplifying mRNA vaccines
Dendritic cell mRNA vaccine
This mRNA vaccine transfects mRNA into dendritic cells in vitro. The infectious disease vaccines developed using this method are mainly limited to HIV-1 therapeutic vaccines: HIV-1 infected persons who receive high-efficiency antiretroviral therapy use autologous Dendritic cell therapy. These dendritic cells are electroporated with mRNA encoding various HIV-1 antigens. Clinical trials have shown that this vaccine is safe and triggers antigen-specific CD4+ and CD8+ T cell responses. But no clinical benefit was observed.
mRNA vaccine for cancers
Cancer vaccines and other immunotherapies represent promising new strategies for the treatment of malignant tumors. Cancer vaccines use tumor antigens to induce the body’s own immune response to specifically kill tumor cells. Because the body’s immune response is systemic and systemic, this therapy can not only specifically kill the remaining tumor lesions after surgery, but also effectively act on distantly metastatic cells, compared to other treatment methods. More specific and extensive.
Dendritic cell mRNA cancer vaccine
Since it was first reported in 1996 that dendritic cells transfected with mRNA by electroporation can trigger an effective immune response against tumor antigens, many studies and clinical trials have confirmed the feasibility and effectiveness of this method.
At present, the potential of this vaccine has been verified in a variety of cancers such as metastatic prostate cancer, metastatic lung cancer, renal cell carcinoma, brain cancer, melanoma, acute myeloid leukemia, and pancreatic cancer. In addition, a number of clinical trials have shown that the combination of dendritic cell mRNA cancer vaccines and traditional chemotherapy drugs or immune checkpoint inhibitors has a promising improvement effect.
Directly injected mRNA cancer vaccine
The route of administration and delivery of the mRNA vaccine can greatly affect the results. At present, a variety of mRNA cancer vaccine forms have been developed using common delivery routes (intradermal, intramuscular, subcutaneous, or intranasal) and some unconventional vaccination routes (intranodal, intravenous, intrasplenic or intratumoral).
Some preclinical studies have shown that combining mRNA cancer with adjuvant therapies (such as traditional chemotherapy, radiotherapy, and immune checkpoint inhibitors) increases the beneficial results of mRNA cancer vaccination.
In general, mRNA cancer vaccines have been shown to be immunogenic to humans, and further improvements in vaccination methods may require greater clinical benefits.
Thoughts on the Effectiveness of Direct Injection of mRNA Cancer Vaccine
Challenges of mRNA vaccines
With the approval of the marketing of mRNA vaccines, the mRNA field has begun to heat up, but the two currently marketed mRNA vaccines need to be stored at -20°C or even -70°C, which is much lower than the storage temperature of traditional vaccines. Therefore, there is a need to continue efforts to develop formulations that are stable at higher temperatures and more suitable for vaccine distribution.
Modern preventive vaccines have extremely strict safety requirements because these vaccines are targeted at healthy individuals. With the widespread vaccination of the two COVID-19 mRNA vaccines, it has shown that mRNA is a relatively safe form of vaccine, but some rare side effects such as myocarditis and facial paralysis should also cause concern and further research.
The future of mRNA vaccines
The mRNA vaccine has many significant advantages. The mRNA vaccine can simulate the natural infection process of the virus to activate the immune system and stimulate a potentially stronger immune response; multiple mRNAs can be packaged in the same vaccine to improve the applicability of the vaccine; mRNA The discovery and production of vaccines is faster than protein vaccines, and can respond more quickly to sudden epidemic infections; different mRNA vaccines can use the same production steps and facilities.
These unique advantages have allowed the rapid development of mRNA vaccines at an extremely alarming rate. At present, there are not only a variety of COVID-19 mRNA vaccines, but also mRNA vaccines for rabies, Zika virus and other infectious diseases, as well as cancer, autoimmune diseases, and rare genetic diseases. Under study.
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