The characteristics and research progress of COVID-19 vaccines developed by different technical routes

Since the global pandemic, although countries or regions have adopted non-pharmaceutical interventions, it is assessed from a global perspective that the new coronavirus pneumonia epidemic has not been contained. Even in some countries where the epidemic has been initially controlled, the epidemic has also appeared Local rebound. 

People pin their hopes of controlling the epidemic on the application of the new coronavirus pneumonia vaccine. This research starts from 7 different vaccine development routes including inactivated vaccines, live attenuated vaccines, virus vector vaccines, virus-like particle vaccines, subunit vaccines, DNA vaccines and RNA vaccines. 

Introduce the advantages and limitations of vaccine research and development mechanism, similar vaccine situation, production speed, technological maturity, etc., and systematically sort out and summarize the characteristics of vaccines developed by different technologies, in order to provide reference for the development and use of vaccines.

Unless otherwise stated, all articles published in this journal do not represent the views of the Chinese Medical Association and the editorial board of this journal.

According to the statistics of Johns Hopkins University, as of August 23, 2020 Beijing time, the global pandemic of the new coronavirus (new crown virus) in 2019 has reported a total of more than 23.21 million cases and more than 800,000 deaths. [1] . Experts in the fields of medicine and biology around the world are committed to finding, developing, and preventing new coronavirus pneumonia (new coronary pneumonia for short), but the current clinical research results of hydroxychloroquine and remdesivir are not optimistic.

 There are documents suggesting that 2 months after the onset of new coronary pneumonia patients, 87% of discharged patients still have symptoms, and 44% of patients feel that their quality of life has deteriorated [2] . The treatment of the new coronavirus is still based on symptomatic treatment, and there is no effective antiviral therapy. Therefore, everyone has focused more on the research and development of vaccines for the prevention and control of the new coronavirus. 

According to statistics from the World Health Organization (WHO), the world is currently conducting research and development from 8 different types of technology platforms, namely inactivated vaccines, non-replicating vector vaccines, replicating vector vaccines, live attenuated vaccines, DNA vaccines, and RNA Vaccines, protein subunit vaccines, and virus-like particle (VLP) vaccines. 

This study combines replicating vector vaccines and non-replicating vector vaccines into viral vector vaccines, introducing 7 different vaccine development technology platforms. The new coronary pneumonia vaccine research and development technology route covers almost the vast majority of technology types for existing vaccine research and development. 

It can be divided into three categories as a whole. The first category is a classic technical route, including inactivated vaccines and live attenuated vaccines through continuous passage; the second category is protein subunit vaccines and VLP vaccines that express antigens in vitro by genetic recombination technology; The three types are viral vector vaccines (replicating, non-replicating) and nucleic acid (DNA and mRNA) vaccines that use genetic material to directly express antigens in vivo.

 As of August 20, 2020, there are a total of 168 vaccines in the research phase worldwide, of which 30 vaccines have entered clinical trials and 138 candidate vaccines are in the preclinical stage ( Table 1 ) [3] . This article introduces the mechanism of vaccines developed by different technical routes, the situation of similar vaccines, production speed, technological maturity, advantages and limitations, etc., and systematically sorts out, summarizes and compares the characteristics of vaccines developed by different technologies, hoping to be a vaccine Provide reference for the development and use of.

1. Inactivated vaccine technology

(1) Introduction to inactivated vaccine technology

The inactivated virus vaccine is to culture the virus on the cell matrix, and then use physical (heating) or chemical (usually using formaldehyde solution or β-propiolactone) to kill the intact and infectious pathogens, making them lose their pathogenicity. Disease and retain antigenicity [4] . Inactivated vaccines can be composed of whole viruses or bacteria, or they can be composed of fragments [5] .

 Currently available inactivated vaccines on the market mainly include inactivated polio vaccine, inactivated influenza vaccine, inactivated Japanese encephalitis vaccine, inactivated hepatitis A vaccine, rabies vaccine, EV71 hand-foot-mouth disease vaccine, cholera vaccine, Leptospira vaccine, epidemic hemorrhagic fever vaccine, forest encephalitis virus vaccine [6] . The key links in the development of virus inactivated vaccines mainly include the acquisition of virus strains, virus amplification, inactivation and other process determination, safety and effectiveness evaluation, etc. [7] . 

The most commonly used virus strains are human diploid cells (2BS, KMBl7, MEC-5), African monkey kidney cells (Vero), Hela cells, etc., which are cultured in cell factories or bioreactors [8] . Virus inactivation is critical to vaccine quality and safety. Formaldehyde and β-propiolactone are the most widely used in the production of human vaccines as inactivating agents [9,10,11,12,13,14] , alkylating agents (including diethyleneimine, acetylethyleneimine, etc.) and hydrogen peroxide can also be used to inactivate viruses to prepare vaccines [15,16,17,18] To ensure the complete inactivation of the virus and at the same time ensure its immunogenicity.

(2) Progress in research and development of new coronary pneumonia vaccines developed with inactivated vaccine technology

Previously, a domestic team had developed an inactivated vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV), and completed a phase I clinical trial, which proved the human safety of the obtained inactivated coronavirus vaccine, and at the same time it can be 100% Induce the production of neutralizing antibodies [19] . 

At present, there are 5 kinds of inactivated new coronary pneumonia vaccines in clinical trials in the world, of which 3 kinds of inactivated vaccines have entered phase III clinical trials. They are respectively sponsored by Beijing Kexing Biological Products Co., Ltd., Sinopharm Wuhan Institute of Biological Products and Beijing Institute of Biological Products; In addition, the inactivated vaccine developed by the Chinese Academy of Medical Sciences and India’s Bharat Biotech has also entered phase I clinical trials. 

The results of the vaccine interim analysis of the Wuhan Institute of Biological Products showed that the main adverse events were pain at the site of inoculation, followed by fever, all of which were mild and self-limiting adverse events with good safety performance. In terms of immunogenicity, in the low-dose and high-dose two groups, the vaccinators had a positive conversion rate of 100%, while the placebo positive conversion rate was 0, indicating that the vaccine has a better immunogenicity result [20] . 

According to the official news of the State-owned Assets Management Committee of the State Council of China, the inactivated vaccine of the Beijing Institute of Biological Products has a neutralizing antibody positive conversion rate of 100% in the 0, 21 day program and the 0, 28 day program group [21] , The immunogenicity is good.

(3) Advantages and limitations of inactivated vaccine development

Inactivated vaccine research and development technology is a relatively traditional and mature vaccine research and development technology. It has the advantages of simple production process, mature preparation and industrialization technology platform, good stability, and safe use. [22] . In addition, the quality control points and evaluation methods of inactivated vaccines are relatively clear, and they usually have better safety and immunogenicity [23] . Inactivated vaccines have a relatively mature production process, so R&D can use pre-existing technology and infrastructure, which can start the R&D process more quickly than new vaccine technologies.

Although inactivated vaccines have a high degree of technological maturity, the production process requires cell and virus culture, establishment of a multi-level seed bank of virus strains and cell lines as substrates, so it is difficult to achieve rapid expansion of production capacity during the production stage [24] ; In addition, because it is an inactivated vaccine, its immunogenicity is relatively weak, and it often requires multiple vaccination to produce sufficient protective antibodies, which is insufficient when used as an emergency vaccine [4] . 

In the production process of inactivated vaccines, it is necessary to maintain the integrity of the immunogenic particles to maintain the immune effect of the vaccine. The inactivation process is required to be high, and the biosafety level required during the production process is high [24] . In addition to the above limitations, the inactivation process may have potential antibody-dependent enhancement (ADE) effects. This effect is mainly manifested as the increase in the susceptibility to the pathogen or the aggravation of the disease when the body is re-infected with the virus after being vaccinated [25] . 

Previous animal experimental studies on the development of inactivated SARS-CoV vaccines suggested that after vaccination, mice suffered more severe lung damage and eosinophil infiltration in the lungs [26,27] , Suggesting that an enhanced disease may occur when re-infected after immunization [28] . Another animal experiment on Middle East Respiratory Syndrome Virus (MERS-CoV) showed that compared with the placebo group, eosinophils and related cytokines IL-5 and IL-13 appeared in mice in the inactivated vaccine group. 

Significant increase, suggesting that the vaccine has the risk of causing pathological changes in the lungs [29] . Animal experiments on inactivated vaccines against both viruses indicate the risk of ADE. SARS-CoV, MERS-CoV and the new virus belong to the genus Coronavirus, so the development of inactivated vaccines needs to be alert to the occurrence of ADE.

2. Viral vector vaccine

(1) Introduction to virus vector vaccine technology

Viral vector vaccine refers to the use of a specific virus as a carrier of genetic information, using genetic engineering technology to implant foreign antigen genes into the viral genome and transfecting cells to obtain a recombinant virus. The obtained recombinant virus can utilize the host's inheritance in the body The substance expresses the target antigen protein and induces the body to produce corresponding antibodies, so as to achieve the purpose of immunization [30] . 

According to whether the viral vector can produce live progeny viruses, it can be divided into replication-defective (ie, non-replicating) viral vectors. After the replicating virus vector enters the cells of the body, it can produce a large number of progeny viruses, and the number of foreign genes will increase with the replication of the vector genome, thereby inducing the body to produce a strong and lasting immune response. 

But at the same time, the protein of the viral vector itself will also be expressed in large quantities, which may have a toxic effect on the body, so the safety of the replicating viral vector needs to be optimized. Non-replicating viral vector vaccines can only infect cells at one time and will not produce progeny viruses. It is safe, but the expression of foreign genes is relatively low, and the immune effect may be worse than that of replicating viral vectors [31] . 

Before the emergence of the new crown pneumonia epidemic, a variety of viral vectors such as poxvirus, adenovirus, herpes virus, vesicular stomatitis virus and flavivirus 17D strain have been used in the development and research of viral vectors for non-new crown pneumonia vaccines [32] . Among them, adenovirus, poxvirus and vesicular stomatitis virus have been studied the most [33] , this article mainly introduces vaccine research and development information using adenovirus as a vector.

Adenovirus is a non-enveloped double-stranded DNA virus that can cause infections such as the respiratory tract, eyes, gastrointestinal tract, and urinary tract. It has a high level of adenovirus antibody positive rate among adults in many parts of the world [33] . The research and development of adenovirus vector vaccines is mainly based on the research of non-replicating adenovirus. At present, the recombinant Ebola virus vaccine jointly developed by the Chinese Academy of Military Medical Sciences and Tianjin Kangsino Biological Co., Ltd. based on the adenovirus vector vaccine was approved in China in 2017 [34] . The adenovirus vector Ebola vaccine developed by the American Janssen Pharmaceutical Company has also completed phase III clinical trials [35] . 

The chimpanzee adenovirus type 3 vector Ebola vaccine (ChAd3-EBO-Z) developed by the National Institute of Allergy and Infectious Diseases in the United States and GlaxoSmithKline in the United Kingdom also showed good immunity in the expanded phase II clinical trial Originality and safety [36] . In addition, the glandular carrier nasal flu vaccine inoculated by Altimmune of the United States and the gland carrier-based oral influenza vaccine (VXA-A1.1) developed by Vaxart of the United States have shown good immunogenicity and safety in clinical trials [37,38]。

(2) Advantages and limitations of adenovirus vector vaccine

The vaccine developed by adenovirus as a gene delivery vector has its unique advantages. First, the host range of adenovirus is wide, and most types of adenovirus have low pathogenicity to humans, and there is no risk of integration into the host. Insertion risk of mutagenesis [39] . In addition, adenovirus can simultaneously infect proliferating and non-proliferating cells [40] And the construction technology of adenovirus recombinant vector is relatively mature, and large-scale cultivation in suspension cells can be realized, the production cost is relatively low, and the production capacity has been improved compared with traditional vaccine research and development technology [41,42] ; Under different formulations, the storage conditions of adenovirus vector vaccines are convenient and flexible, and can be stored in a liquid buffer at 4 ℃, or in the form of freeze-dried powder for more than 1 year [43] ; In addition, adenovirus itself can be used as an antigen, or as a carrier of recombinant genes, and studies have shown that adenovirus also has the function of an immune adjuvant, which can stimulate the body's natural immune response [44] , so it makes the development of vaccines easier and helps reduce production costs; adenovirus vector vaccination is relatively simple and flexible, and there are multiple vaccination routes. Viral vector vaccines can be used for mucosal immunity through the intestinal or respiratory tract, such as oral and nasal vaccination Wait [45,46,47,48] . It can infect different types of cells and tissues, differentiated and undifferentiated cells, and even antigen-presenting cells [49] . Adenovirus as a vector has good safety and certain efficacy in clinical trials of related gene therapy [50] . In addition, the platform of the virus-based vaccine production process is highly versatile, and vaccine development against multiple different pathogens can be carried out based on the same platform.

Despite the above advantages, there are unique difficulties and challenges in the development of adenovirus vaccines. Among them, the biggest problem encountered in the process of vaccine construction is the existence of the human body's own immunity to adenovirus. The human body’s immunity to adenovirus may affect the strength of the immune response induced by the vaccine and thus affect the protective effect [51] . Severely ill patients infected by the new coronavirus are mostly elderly people or people with underlying diseases. These people have weaker immunity than healthy people. It is currently impossible to determine whether the vaccine can protect these people. [52]。

(3) Progress in research and development of new coronary pneumonia vaccines using adenovirus as a carrier

At present, there are 5 kinds of new coronary pneumonia vaccines that have entered clinical trials with adenovirus as a vector, all of which are non-replicating adenovirus vector vaccines. Among them, the vaccine jointly developed by the University of Oxford and AstraZeneca has entered phase III clinical trials; the vaccine jointly developed by the Chinese Academy of Military Medical Sciences and Cansino Bioproducts has completed phase II clinical trials, and phase III clinical trials have not yet been carried out; The vaccine developed by Janssen has launched phase I/II clinical trials; the vaccine developed by the Gamaleya Institute in Russia has launched phase I clinical trials; the vaccine developed by the three companies of Italy ReiThera, Germany LEUKOCARE, and Belgium Univercells has also launched phase I clinical trials. test. In addition, the replication-type measles virus vector vaccine led by the Pasteur Institute in France has also started phase I clinical trials. According to the results of a phase I/II study published by the University of Oxford, the safety of adenovirus vector vaccines is good. From the results of immunogenicity, the vaccine can induce both humoral and cellular immune responses [54] . The results of Phase I and Phase II clinical trials of the vaccine developed by Cansino Company showed that the positive conversion rates of serum antibodies were 96% and 97% in the two dose groups 28 days after one dose of vaccine. The incidence of adverse events in the two groups 72% and 74% respectively [55,56]。

3. Protein subunit vaccine

(1) Introduction to protein subunit vaccine technology

The research and development process of protein subunit vaccine is: based on the published viral gene sequence, the virus target antigen gene is constructed on an expression vector by recombination, and then transformed into bacteria, yeast, mammalian or insect cells to induce the expression of the antigen The protein is finally purified and made into a vaccine [53] . The vaccine research and development technology is relatively mature, and currently marketed vaccines include hepatitis B vaccine and influenza subunit vaccine.

(2) Advantages and limitations of protein subunit vaccine research and development

Compared with the traditional inactivation and attenuation routes, the biggest advantages of the protein subunit vaccine technical route are mainly reflected in: (1) The production process is highly safe, and the production and development of the vaccine does not require high-level biosafety experiments. room [57] . (2) Compared with the traditional vaccine research and development platform, the vaccine production capacity is higher, and this technology is suitable for large-scale production. After the process route of vaccine production is determined, it is easy to produce on a large scale [24] . (3) The safety of the vaccine is good. Because the vaccine only has a certain specific antigen protein of the pathogen, and not the entire pathogen, there is no risk of infection, and the vaccine is relatively well tolerated [58] . (4) The stability of the vaccine is relatively good. The main challenges of protein subunit vaccines are that because vaccines use different expression systems to express part of the pathogen’s proteins as antigens, the final expressed antigen protein may be different from the natural conformation of the viral protein [59] , this may affect the immunogenicity of the vaccine to a certain extent. In addition, because only a part of the pathogen’s protein is used as an antigen, the immunogenicity is weak, and adjuvants are generally needed to enhance it, and the immune memory effect may be weak [60]。

(3) Progress in research and development of new coronary pneumonia vaccines developed with protein subunit vaccine technology

There are currently 7 new coronavirus protein subunit vaccines in clinical trials, of which the adjuvant-added recombinant protein vaccine jointly developed by the Institute of Microbiology of the Chinese Academy of Sciences and Anhui Zhifeilongkema Biopharmaceutical Company has entered phase II clinical trials; the remaining 6 Two vaccines have started phase I clinical trials. They are jointly organized by Novavax in the United States, Kentucky Bioprocessing in the United States, Clover in China, GlaxoSmithKline in the United Kingdom, and Dynavax in the United States, Vaxine Pty Ltd in Australia, Medytox in South Korea, Queensland University in Australia, and Seqirus in South Korea. The company is jointly developed by China Kea Biotechnology Company, the National Institute of Allergy and Infectious Diseases of the United States, and Dynavax of the United States. No relevant clinical trial results have been published yet.

Four, DNA vaccine

(1) Introduction to DNA vaccine technology

DNA vaccine is a third-generation vaccine developed on the basis of the first-generation live attenuated vaccine, inactivated vaccine and the second-generation subunit vaccine. It is a kind of nucleic acid vaccine [61] . The mechanism of DNA vaccine is to recombine the gene encoding the exogenous antigen protein into the plasmid of the eukaryotic expression element, and then introduce the plasmid into the host body, use the genetic material in the host cell to express the antigen protein, and induce the body through the antigen protein. Produce the corresponding immune response, so as to achieve the purpose of preventing and treating diseases [62] . DNA vaccines have been used for the development of various human pathogens, such as HIV, influenza virus, malaria, hepatitis B virus, respiratory syncytial virus, and herpes simplex virus, and have entered the clinical trial stage [63] . At present, no DNA vaccine has been approved for marketing, but DNA vaccines have been used in the field of animal vaccines for many years, and a variety of animal DNA vaccines have been approved.

(2) Advantages and limitations of DNA vaccine research and development technology

Compared with traditional vaccine research and development technology, DNA vaccines have very obvious advantages. First of all, DNA vaccines have no risk of infection; in addition, DNA vaccines, like live attenuated vaccines and carrier live vaccines, cause cytolytic T lymphocyte responses [64] , But there is no risk of infection, and there are no virulence changes related to scattered virus, virus contamination and the sensitivity of individual sources of infection. For pathogens that are difficult to cultivate or dangerous with conventional vaccines, the construction of DNA vaccines is relatively simple. [65] , And may produce a lasting immune response. DNA vaccines can exist in the body for a long time and continue to express low-level antigen proteins, and can induce a continuous immune effect without subsequent immunization injections. Therefore, a very small amount of antigen can stimulate the body to produce a strong and long-lasting immune response [66] . Research data shows that after the DNA vaccine is injected into mice intramuscularly, the vaccine DNA can be continuously detected for 2 years [67] , suggesting that DNA vaccines may induce sustained immune effects. In addition, DNA vaccines have relatively simple production methods, high production efficiency, and low production costs. DNA vaccines are mainly produced by constructing highly efficient expression plasmids and then using bacterial clones for mass production. Compared with ordinary vaccines, DNA vaccines have no tedious and time-consuming processes such as antigen extraction and purification. DNA vaccines are used in a small amount, which is more cost-effective than other vaccines. It also avoids the advantages of reduced efficacy and high cost after long-term repeated use of biological agents [68] . In addition, compared with traditional vaccines, DNA vaccines are relatively stable at room temperature and do not require refrigeration equipment, so they are easier to store and transport.

In addition to the above advantages, DNA vaccines still have their difficulties and challenges to be solved. (1) The selection of target genes, vectors and promoters for DNA vaccines should be cautious. The construction and selection of the target gene, the vaccine DNA, is the key to determining the effect of DNA immunity [69] . (2) Potential safety risks. DNA vaccines need to integrate part or all of the plasmid sequence into the host genome, which may result in inactivation of tumor suppressor genes or activation of oncogenes, or chromosomal instability (breaks and mutations), so plasmids The persistence and stability of DNA need to be effectively evaluated before clinical use [70] . In addition, DNA vaccines may stimulate the body to produce anti-DNA antibodies associated with autoimmune diseases such as systemic lupus erythematosus. Preclinical data indicate that DNA vaccines can increase the production of autoantibodies against DNA. In normal mice, bacterial plasmid DNA induces the production of anti-double-stranded DNA autoantibodies, while in lupus mice, DNA vaccines accelerate the development of autoimmunity [71] . (3) The barrier of the DNA vaccine delivery system may affect the vaccine effect, and an appropriate vaccination route needs to be selected. Plasmid DNA vaccines need to cross several barriers to enter the nucleus. The vaccine plasmid must pass through the phospholipid cell membrane through endocytosis or pinocytosis to avoid degradation of endosomes, lysosomes and cytoplasmic nucleases, and transnuclear transport [72] . Therefore, without an effective delivery system, it is difficult to deliver DNA plasmids into host cells. The injection immunization method, including direct injection of naked DNA and injection after liposome encapsulation, is the most commonly used DNA vaccine immunization method by direct intramuscular injection of the protein expressing the encoding gene to induce the host immune response [73] . The protective effect of intramuscular injection is better than that of intranasal, intraperitoneal, intravenous and intracutaneous vaccination. Another study showed that the effect of different vaccination methods on DNA immunization was compared, and it was found that the effect of gene gun vaccination is 600~6 000 times better than direct injection of DNA vaccine [66]。

(3) Progress of new coronary pneumonia vaccines developed with DNA vaccine technology

At present, a total of 4 new coronavirus vaccines developed based on the DNA technology platform have entered the clinical trial stage. The 4 DNA vaccines are all in the phase 1/2 research. They were jointly conducted by Inovio and International Vaccine Institutions, Osaka University, Japan, and AnGes, Japan. , Japan’s Takara Bio company, three companies jointly, India’s Cadila Healthcare Limited company, South Korea’s Genexine Consortium company for research and development. At present, Inovio company has announced the results of animal experiments. When animals are challenged several months after vaccination, they can quickly produce T cell and B cell responses to the new coronavirus spike protein [74]。

Five, mRNA vaccine

(1) Introduction to mRNA vaccine technology

mRNA vaccine is a kind of nucleic acid vaccine. Its mechanism is to inoculate the host with mRNA encoding the antigen protein, and then use the host's genetic material to express and synthesize the antigen protein in the cells of the body, and induce and activate the body's immune system through the antigen protein. Produce an immune response, thereby achieving the purpose of preventing and treating diseases [75] . According to whether mRNA vaccines can self-amplify and replicate, mRNA vaccines can be divided into self-amplifying mRNA vaccines and non-replicating mRNA vaccines [76] . Self-amplified mRNA induces high-level antigen expression through self-amplification of RNA in host cells [77] . In recent years, various mRNA vaccine technology platforms have been developed, and their immunogenicity has been verified in clinical studies [78] . At present, all mRNA vaccine products are still in pre-clinical research or clinical research stage, and there is no mRNA vaccine used on the market [79] . But several of the world's major mRNA vaccine development company, in addition to crown a new virus vaccine has several other products have entered clinical studies, under detailed in Table 3 .

The mRNA vaccine development process mainly includes several key steps: target antigen selection, gene sequence acquisition, target gene plasmid construction, RNA in vitro transcription, and mRNA vaccine modification and optimization. Since different proteins are encoded by different RNA sequences, finding the best protein antigen is the key to determining the development direction of mRNA vaccines [80] . The mRNA sequence is generally obtained by in vitro transcription, but because the mRNA itself is very easy to be degraded, in order to ensure its stability, it is usually necessary to modify the nucleotides and optimize the codons of the mRNA after obtaining the mRNA in vitro. It is very important to improve the translation efficiency of mRNA vaccines and ensure their stability [81]。

(2) Advantages and limitations of mRNA vaccine research and development technology

As a third-generation vaccine, mRNA has its unique advantages compared to traditional vaccines ( Table 2 ). First of all, the acquisition of mRNA vaccines is produced through the process of enzymatic in vitro transcription. This process does not depend on cell expansion, so it is easy to realize the monitoring and quality control of all production processes [82] , and this process saves cell culture, antigen extraction and purification processes, greatly shortens the production time, can easily achieve mass production, and improves the production capacity of vaccines [75] . According to the report of the current research and development unit, the production capacity of mRNA can easily achieve an annual production capacity of hundreds of millions or even billions of doses, which is essential for rapid response to emerging infectious diseases on a global scale. The development and production cycle of mRNA is short, and the initial synthesis is relatively fast. Once the clinical data verifies its effectiveness and safety, it can promote the production of vaccines downstream in a short period of time [83] . And there are clinical data showing that mRNA vaccine may induce the body to produce humoral immunity and cellular immunity at the same time [84] , Through a variety of mechanisms to protect the body. Due to its own characteristics, mRNA can be quickly degraded in the cytoplasm of transfected cells after immunization, which can reduce the risk of its safety. And there is no risk of DNA vaccine integration into host chromatin [85]。

There are also challenges that need to be overcome during the development of mRNA vaccines ( Table 2 ). The first is the stability of RNA. The instability of RNA itself will seriously affect the effectiveness of vaccines [86] . Enhancing the stability of RNA is a systematic project. It can start with improving the stability of mRNA itself, such as optimizing the synthesis system, codon optimization, and modifying nucleosides, and it can also achieve the goal by improving the efficiency of the delivery system at the same time [87] . Naked mRNA will be degraded when it enters the body directly; at present, it is more common to use carriers to assist the delivery of mRNA. Commonly used carriers include dendritic cells, protamine, liposomes and polymer carriers, etc. [88] . Lipid nanoparticle carriers are currently one of the most commonly used carriers for mRNA vaccines [89] . In addition, the potential safety risks of mRNA as a new generation of vaccines also need to be fully evaluated. The toxicity risk of RNA vaccines mainly comes from the toxicity produced by the nucleic acid analogues in synthetic raw materials and the toxicity produced by the packaging materials used in mRNA [90] . In addition, mRNA vaccines have the risk of causing apoptosis of surrounding host cells, and a large number of apoptosis will cause a certain intensity of inflammatory response [91] . The mRNA produced by artificial in vitro transcription often cannot be consistent with the mRNA naturally transcribed from human DNA in terms of primary sequence and secondary structure, so there are differences in the process of translating proteins and activating the immune response. RNA chemical synthesis and large-scale production are more difficult [92] . In terms of quality control, how to detect residual template DNA and incompletely synthesized mRNA is also a major quality control problem facing the current research and development of mRNA vaccines [85]。

(3) Research progress of new coronary pneumonia vaccine developed by mRNA technology

Based on mRNA technology research and development, there are currently six candidate new coronavirus vaccine products entering the clinical trial stage. Among them, the fastest progress is the mRNA-1273 vaccine candidate jointly developed by Moderna of the United States/National Institute of Allergy and Infectious Diseases, which has launched phase III clinical trials, and is jointly developed by Pfizer, BioNTech, and Fosun Pharmaceutical in China. The BNT-162 vaccine candidate has also started phase III clinical trials. The mRNA vaccine jointly developed by Arcturus of Singapore and Duke National University of Singapore has started phase I/II clinical trials. The other three mRNA vaccines jointly developed by Imperial College London in the United Kingdom, Curevac in Germany, the Chinese Academy of Military Medical Sciences and Watson Biotechnology are all in the phase I clinical research phase. The mRNA-1273 vaccine candidate had adverse events in more than 1/2 of the subjects, but they were all mild or moderate, and 3 subjects had serious adverse events; in terms of immunogenicity, 2 doses of vaccination Afterwards, neutralizing antibodies were detected in all subjects, and the intensity of neutralizing antibodies was similar to that of convalescent patients [93] . The Phase I/II results of the BNT-162 vaccine jointly developed by Pfizer/German BioNTech showed that after two doses of the BNT-162b1 candidate vaccine, the neutralizing antibody titers increased compared to the serum titers of convalescent patients with new crowns It is 1.9-4.6 times higher, and the adverse events are mostly mild and moderate, suggesting that the vaccine has a good safety and immunogenicity [94]。

Six, VLP vaccine

(1) Introduction to VLP vaccine technology

Virus-like particles are highly structured hollow particles that are self-assembled by one or several capsid proteins of the virus in a heterologous system and have the same or similar structure as natural virus particles, but do not contain viral genetic material, with a diameter of about approx. It is 20~200 nm. Studies have suggested that the formation of virus-like particles may be related to the overexpression of some structural proteins of the virus [95] . According to the source of VLP, it can be divided into natural VLP and recombinant VLP. Natural VLP is mainly synthesized by self-assembly of virus mutation and modified body. In 1978, scientists discovered for the first time that the main capsule protein of polyoma virus can assemble into VLP by itself without viral nucleic acid [96] . The reorganization of VLP is completely artificially designed [97] , the components of synthetic VLP epitopes can be modified in various ways during preparation to meet different preparation requirements. Therefore, artificially synthesized VLP as a carrier platform has gradually become a research hotspot of VLP vaccines. According to different structures, VLP can be divided into two types: enveloped and non-enveloped. Non-enveloped VLPs are assembled by self-assembly of one or more components of the pathogen or fused with one or more target antigens on the surface of the VLP, and usually do not contain host components [98] . The role of VLP and the immune system is mainly related to the two factors of VLP size and surface geometry [99] . Because of its viral structure, after VLP enters the body, it can not only interact with the human innate immune response system, but also effectively cause specific humoral immune responses and specific cellular immune responses. As an exogenous antigen, VLP can be effectively presented by MCH Ⅰ and Ⅱ [100] . VLP vaccine research and development technology has been extensively researched and applied. VLP vaccines currently on the market or under development include human papillomavirus vaccine, hepatitis B vaccine, hepatitis E vaccine, norovirus vaccine, etc. [101]。

(2) Advantages and limitations of VLP vaccine research and development technology

VLP vaccine does not have the ability to infect and replicate autonomously because its structure does not contain the genetic material of the pathogen, but because it has a three-dimensional structure similar to that of natural virus particles, it exhibits a conformational epitope more similar to that of natural virus particles. It can be presented to immune cells in the same way as virus infection, effectively inducing the immune system to produce an immune protective response. It is a safer and effective antigen, and it also has an adjuvant function [102,103] . The preparation of VLP vaccine virus-like particles mainly includes the cloning and expression of virus structural genes, selection of host expression system, purification and identification. Commonly used expression systems include: Escherichia coli, yeast, insect cells, etc., through which the structural protein of the virus can be expressed. Then the expressed protein is purified and artificially assembled to form a VLP. Due to the complicated production process of the vaccine, the production speed of the vaccine is relatively slow.

At present, only the product of Medicago, a Canadian company, has entered phase I clinical trials for the new coronavirus vaccine developed based on VLP technology.

Seven, live attenuated vaccine

(1) Introduction to live attenuated vaccine technology

Live attenuated vaccines refer to pathogens undergoing various treatments to make them mutate, and to obtain pathogen variants with weakened or even non-toxic effects through subculture, but the variants still retain their immunogenicity, and the live virus Variant strains are inoculated into the body as a vaccine and will not cause the occurrence of diseases, but the pathogen can induce an immune response in the body to achieve the effect of immune protection [104] . The history of human vaccines began with live attenuated vaccines. In 1796, British Doctor Jenner used live vaccinia virus to prevent human smallpox for the first time, thus unveiling the beginning of vaccine development. Later, French doctor Pasteur made an attenuated rabies vaccine through physical and chemical methods in 1885, and Calmette and Guerin made the first live attenuated bacterial vaccine in 1921, BCG, to prevent tuberculosis. Subsequently, a variety of live attenuated vaccines have been successfully developed. The live attenuated vaccines currently on the market include monovalent live attenuated vaccines for measles, mumps, rubella, and varicella, and four combined live attenuated vaccines and rotavirus attenuated vaccines. Live attenuated polio vaccine, live attenuated polio vaccine, live attenuated influenza vaccine, live attenuated Japanese encephalitis vaccine, live attenuated hepatitis A vaccine, live attenuated dengue vaccine, live attenuated yellow fever vaccine In addition, there are still a variety of live attenuated vaccines under development, such as oral live attenuated cholera vaccine, live attenuated respiratory syncytial virus vaccine, etc. [105]。

(2) Advantages and limitations of live attenuated vaccine technology

The research and development history of human attenuated live vaccines is relatively long and the technology is relatively mature. The advantages of live attenuated vaccines are mainly manifested in: (1) Because they are live pathogens, live attenuated vaccines have very strong immunogenicity, which can stimulate the body to produce specific memory B cells and T cells, and at the same time activate humoral immunity and Cellular immunity, long-term or even life-long protection, has the advantages of strong immunity and long acting time [106] , vaccination through natural infection route can also induce mucosal immunity, allowing the body to obtain a wider range of immune protection [107] ; (2) Due to the induction of a strong immune response, live attenuated vaccines generally do not need to add adjuvants, and their vaccine doses are generally less [108] ; (3) Since living pathogens have the property of increasing value, there is the possibility of horizontal transmission, which can expand the immune effect and strengthen the herd immune barrier [107] ; (4) The production process of attenuated live vaccines generally does not require concentration and purification, and the vaccine production prices are generally relatively low [109] . In addition to the above advantages, live attenuated vaccines also present certain challenges. The first is the issue of safety. Since live pathogens are vaccinated into the human body, generally live attenuated vaccines retain a certain residual virulence, which may induce serious diseases for some individuals (such as immunodeficiency), and after the vaccine is vaccinated into the human body , Due to various reasons (such as genetic modification, etc.), live attenuated vaccines may appear "return to virulence" phenomenon [108] . In addition, because it is a live microbial preparation, pathogens excreted by the human body may cause environmental pollution and cause cross-infection [110] . Because it is a vaccine made from a live pathogen, the live attenuated vaccine has relatively high requirements for the storage and transportation of the product [111] . Moreover, the key steps in the development of live attenuated vaccines include [112] : (1) Passage screening or artificially mutated pathogenic sites to obtain attenuated strains; (2) strain safety and immunogenicity verification, both of which are very time-consuming, so the development of live attenuated vaccines Very long period [108] . In addition, there are certain biosafety risks in the isolation and cultivation of virus strains, related research and evaluation work, and subsequent production. The biosafety level of laboratories or production workshops is high, and the general requirement is biosafety level three; in addition, , For the development of a live attenuated vaccine for new coronary pneumonia, it is a unique challenge. The population is generally susceptible to the new crown virus. The elderly and those with underlying diseases are more ill after infection, children and infants also have the disease, and pregnant women are susceptible. [113,114] . Therefore, it is required that all age groups should be covered for research and development; however, children’s immune system functions are often weaker than adults, and the preventive effect of live attenuated vaccines is poor and prone to adverse reactions [115] Therefore, the adaptability of different populations needs to be fully considered in the development process of the new crown live attenuated vaccine.

With the development of genetic recombination technology, new forms of attenuated live vaccine research and development technologies are also developing. A live attenuated vaccine combining influenza virus and new coronavirus genetic recombination currently being developed by the University of Hong Kong in China is expected to enter clinical trials in the near future [116] , the design of the vaccine is based on the development of a live influenza virus strain lacking the NS1 protein, which reduces the virulence of the influenza virus while maintaining high immunogenicity, and then through genetic engineering, the expression of the new coronavirus will be spiked The gene of the receptor binding region of the protein (S protein) is integrated into the influenza virus genome to specifically express the RBD region protein of the coronavirus spike protein on the surface of the influenza virus, thereby inducing the body to produce and target at the same time after injection into the human body. The immune response of influenza virus and new crown virus achieves the effect of protecting influenza virus and new crown virus at the same time [117] . The vaccine is vaccinated by nasal spray, which is expected to produce a better immune response than wild influenza strains [118] . In addition, the research and development of attenuated vaccines is also introducing new technical means, such as "cold adaptation" virus strain cultivation, reverse genetics technology and genetic recombination classification techniques [118]。

(3) Progress of new coronary pneumonia vaccines developed with live attenuated vaccine technology

At present, the new coronary pneumonia vaccines developed with live attenuated vaccine technology are in the preclinical research stage, and no new coronavirus vaccine developed with live attenuated vaccine technology has entered clinical trials in the world.

Different types of vaccine research and development platforms have their own advantages and limitations ( Table 5 ). Under the situation of a global pandemic of new coronary pneumonia, vaccination is the most effective way to respond to and control the epidemic. The development of vaccines requires systematic clinical trial evaluation from the perspective of safety and effectiveness, but the prevention and control of the epidemic needs to fully consider the availability of vaccines (including short-term supply), affordability, and the fairness of vaccine use . No vaccine can deal with pandemic infectious diseases in a short period of time. Vaccine research and development must be based on a larger perspective, multi-technology parallel research and development, and multi-channel accelerated research and development. This is the correct way for us to develop vaccines and the key strategy to overcome the epidemic.