In the history of human prevention of infectious diseases, the invention of vaccines is a milestone, saving countless lives in a century. The essence of preventive vaccines is "attack with poison", that is, artificially introduce viral antigens and use the body's own immune system to perform epitope recognition, immune amplification and memory of target antigens, and then produce durable and specific humoral and cellular immunity. According to the epitope sequence carried by the target antigen, antigen-presenting cells present epitopes to different T cell subgroups, and further activate them into helper T cells (Th cells) and cytotoxic T cells (Tc cells). Th cells help B cells recognize antigens and produce humoral immunity represented by antibodies, while Tc cells can directly recognize antigens and lyse infected host cells to achieve cellular immunity.

The new crown pneumonia is following the severe acute respiratory syndrome coronavirus (SARS-CoV) discovered and circulating in China from 2002 to 2003, and the Middle East respiratory syndrome that was circulating in Saudi Arabia and South Korea in 2012 and 2015 respectively. After the coronavirus (middle east respiratory syndrome coronavirus, MERS-CoV), one of the third serious public health emergencies caused by the coronavirus. At present, vaccination is one of the most effective measures to overcome the new coronavirus pneumonia epidemic. It can stimulate the body to produce an immune response, produce specific antibodies against the virus and immune memory, and thus resist the invasion of the virus.

The main transmission routes of SARS-CoV-2 are respiratory droplet transmission, aerosol transmission and contact transmission. After SARS-CoV-2 infects the human body, the most common symptoms are cough, fever, shortness of breath, and difficulty breathing. Severe infections may lead to severe acute respiratory syndrome, renal failure and even death. Studies have found that SARS, MERS, SARS-CoV-2 and other coronavirus infections can cause an imbalance in the body’s immune regulatory network, trigger cytokine storm syndrome (CSS), and cause diffuse damage to target cells such as alveolar epithelial cells. As a result, the human body suffers from acute respiratory distress syndrome (ARDS), septic shock, multiple organ dysfunction, and even death. CSS is an excessive immune response that is not controlled by the immune system and is dysfunctional by the body to antigens such as viruses. The main cells involved are endothelial cells, dendritic cells, epithelial cells, macrophages, lymphocytes and other immune cells. This reaction will cause the immune cells to continuously activate and expand, producing a large number of cytokines. The clinical features of CSS are systemic inflammation, hemodynamic instability, multiple organ failure, and even death. SARS-CoV-2 mainly attacks the human lungs, presenting diffuse alveolar damage and the formation of hyaline membrane, which is consistent with ARDS performance. In addition, the number of CD4+ and CD8+ T cells in peripheral blood decreased significantly, but they were over-activated, manifested by an increase in highly irritating CCR4+CCR6+Th17 cells and high cytotoxicity of CD8+ T cells, which partially explained the severe immune system damage, indicating The number of lymphocytes is closely related to disease severity and mortality.

The particle diameter of coronaviruses ranges from 70 to 120 nm, and the length of the single-stranded non-segmental RNA genome ranges from 26 to 32 kb. It is a type of positive-stranded single-stranded RNA virus with envelope. It is also the RNA virus with the largest known genome. Coronaviruses belong to the Orthocoronavirinae subfamily (Coronaviridae). This subfamily includes 4 genera (α, β, γ, and δ), and usually only α and β genera can cause disease in humans. The virus structure of SARS-CoV-2 is very similar to SARS-CoV and MERS-CoV. It is a β-coronavirus and has a genome homology of up to 82% with SARS-CoV. It is the seventh discrete coronavirus that can cause human diseases. Virus species. The spike (S) protein of SARS-CoV-2 is a trimeric structure, and each monomer has a cell receptor binding site. When the virus is combined with the host cell, the S protein will undergo structural changes, which will cause the host cell membrane to fuse with the virus plasma membrane, and the virus will invade the host cell and multiply.

The pathogenic mechanisms of the same viruses often have similarities, and they are often used for reference and comparison. Regrettably, they belong to the same family of coronaviruses. For severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), humans have not yet been able to develop effective vaccines. Similarly, the RNA inside SARS-CoV-2 carries genetic information and is responsible for virus replication, while the protein body is composed of four parts, including S protein (Spike protein), E protein (Envelope protein), and M protein (Membrane protein) And N protein (Nucleocapsid protein). Among them, the S protein on the surface of SARS-CoV-2 can mediate the virus invading host cells. The mechanism is that the receptor-binding domain (RBD) on the S protein can recognize the angiotensin converting enzyme 2 (ACE2).

ACE2 is a carboxypeptidase and a protein receptor, widely distributed in various organs of the human body. The receptor ACE2 of SARS-CoV-2 is mainly expressed in type II alveolar cells, and many genes related to the pathogenic process of the virus are also overexpressed in this type of cell. Studies on the infection of SARS-CoV-2 in the digestive system show that ACE2 can not only be expressed in large quantities in type II alveolar cells, esophageal epithelial cells and stratified epithelial cells, but also in ileum and colon absorptive intestinal epithelial cells, indicating The digestive system and respiratory system are potential routes of SARS-CoV-2 infection. The study found that the use of small molecule inhibitors and patient serum can block the recognition of S protein and ACE2 and cell invasion. Moreover, the neutralizing antibody induced by SARS-CoV-2 S protein can significantly block the recognition pathway and reduce the virus' infectivity. These studies fully illustrate the important role of S protein neutralizing antibodies in preventing virus invasion, and provide important guidance for vaccine design based on B cell humoral immunity. At the same time, T cell immunity is essential to eliminate infected cells and viruses. Because once a small number of viruses escape the entrapment of neutralizing antibodies, the infected host cell will become a hotbed for virus replication and even mutation, and at this time, antigen-specific Tc cells can recognize the infected host cell and further eliminate the virus. Studies have found that severe COVID-19 patients have experienced T cell exhaustion and decreased functional diversity, which reflects the importance of T cell immunity for disease control.

Due to the specificity of the immune response, the target antigen epitope sequence (including B cell epitope and T cell epitope) directly determines the type of immune response. Given that both B cell immunity and T cell immunity are indispensable in the elimination of SARS-CoV-2, selecting the appropriate immunogenic fragments as much as possible is a key step in the design of the new crown vaccine. Since it contains multiple B cell epitopes and T cell epitopes, the full-length S protein is still the most important target antigen choice in the current new crown vaccine design. However, after evaluating the types of antibodies in patients and mice after SARS-CoV-2 and SARS-CoV infections, it was found that conserved epitopes in the S protein can induce the production of non-neutralizing antibodies. According to previous studies on SARS-CoV candidate vaccines, this type of antibody does not provide effective protective immunity. On the contrary, it may aggravate the antibody-dependent enhancement (ADE) and cause the virus to expand infection. Studies have found that the RBD fragment of S protein contains multiple B cell and T cell epitopes and can trigger strong protective antiviral immunity; N protein also has high immunogenicity and is one of the potential targets of vaccines. The discovery provides strong support for RBD as an important immunogenic region of S protein, and provides ideas for subsequent subunit vaccine design.

Vaccine design steps include the rational selection of target antigens, the design of immune-enhancing adjuvants, the regulation of humoral and cellular immune types, and the clinical optimization of immune procedures. Based on the previous safety and effectiveness verification on MERS-CoV and SARS-CoV vaccines, the new crown vaccine has expanded new platform technologies. Specific development strategies include inactivated and attenuated virus vaccines, adenovirus vector vaccines, and recombinant proteins Vaccines, nucleic acid vaccines and other vaccines.

1 Inactivated and attenuated vaccines

During the development of inactivated virus vaccines, it is necessary to ensure that the virus is infectious without losing its immunogenicity. This technology is easy to implement and has a good humoral immune response effect, so it has become the preferred form of vaccine for emerging infectious diseases. The principle of its preparation is to inactivate pathogenic microorganisms and their metabolites by various physical and chemical methods to gradually lose infectivity, but maintain the immunogenicity of the virus, and then prepare candidate vaccines through purification and other steps. The candidate vaccines can stimulate body produces antibodies to protect the body against viruses. Live attenuated vaccine is a type of pathogen that is artificially processed to make the virus lose its pathogenicity, but retain its original proliferation ability and immunogenicity. It has a long immunity time in the body and can induce systemic immunity and mucosal membranes. Immune response. The SARS-CoV E protein plays an important role in the formation of the virus envelope. Viruses with mutations in the E protein gene can produce temperature-sensitive mutations and morphological changes. Preliminary studies have shown that the lack of SARS-CoV E protein in live attenuated vaccines can induce humoral and cellular immunity in mice, and can achieve partial protection from challenge.

Inactivated and attenuated vaccines essentially eliminate or reduce the toxicity of the virus while maintaining a certain degree of immunogenicity, so as to simulate the complete virus structure as much as possible to achieve immune protection. In terms of preparation methods, the production process of inactivated virus vaccines roughly includes: (1) appropriate strain selection and cell culture, (2) chemical or physical method inactivation and purification process exploration, (3) adjuvant addition. The preparation of attenuated vaccines includes: (1) passage screening or artificially mutated pathogenic sites to obtain attenuated strains, (2) strain safety and immunogenicity verification. Both have their own advantages and disadvantages: inactivated vaccines have a short preparation cycle and high safety. They are the main force in the development of traditional antiviral vaccines. However, their immunogenicity is low, and the main induced humoral immunity; while the attenuated vaccine has stronger immunogenicity, needn’t to add additional adjuvants, it can induce humoral immunity and cellular immunity at the same time, but it is more time-consuming to screen attenuated strains. In addition, because the virus is still viable, it may return to strong virulence in the body, so Its safety assessment is extremely important.

As two classic R&D platforms, inactivated and attenuated vaccines against COVID-19 are also under preparation and expansion. From the perspective of vaccine development timeline, inactivated vaccines are in a leading position in this new crown vaccine research and are the most in-depth clinical research.

2. Recombinant virus vector vaccine

Recombinant virus vector vaccine is a vaccine made by using virus as a carrier to recombine antigen genes into the viral genome and using recombinant viruses that can express antigen genes. Currently, commonly used viral vectors include DNA viruses such as poxvirus, herpes virus, and adenovirus, and RNA viruses such as attenuated influenza virus and flavivirus. Based on the successful research and development of the Ebola vaccine, the team of Academician Chen Wei from the Institute of Bioengineering of the Academy of Military Medical Sciences of the Academy of Military Sciences and CanSino Biotech Co., Ltd. used adenovirus as a carrier to rapidly develop the pharmaceutical Research on pharmacodynamics, pharmacology and toxicology, etc., quickly completed the design of the SARS-CoV-2 vaccine, construction of recombinant virus species, and the safety, effectiveness, broad-spectrum evaluation and quality review of the vaccine, on March 16, 2020 A phase I clinical trial was launched on Japan. The vaccine is a recombinant viral vector vaccine based on the type 5 replication-deficient adenovirus as a vector to express the SARS-CoV-2 S protein. The focus of the Phase I clinical research trial is to determine the human tolerance of different doses of vaccines by observing the safety of vaccine use. On April 12, 2020, the vaccine entered phase II clinical trials, and it is the world's only SARS-CoV-2 vaccine that entered human phase II clinical trials. In the research and development of Ebola virus vaccines and MERS-CoV vaccines, recombinant viral vectors are the most commonly used method, especially Ebola vaccines. Both types of approved vaccines are vaccines that use adenovirus as a carrier. This technology has also been widely used in the development of HIV vaccines and respiratory syncytial virus vaccines.

3 Recombinant protein vaccine

Recombinant protein vaccine is considered to be the safest vaccine. It integrates the target antigen gene of the virus into an expression vector, and then transforms the expression vector into bacteria, yeast, mammalian or insect cells, and induces the expression of a large number of antigen proteins. The vaccine obtained by purification. For this new crown pneumonia epidemic, the development of recombinant protein vaccines is also an important technical route. Because recombinant protein vaccines are exogenous antigens, MHC-I molecules are not the main way of presentation and cannot produce effective sensitized cytotoxic T lymphocytes (CTL). However, in the process of clearing coronavirus infection, cell-mediated immune effects play a key role. Therefore, it is recommended to use recombinant protein vaccines in combination with other forms of vaccines. In addition, designing a recombinant protein vaccine into a polymer or virus like particle (VLP) structure and choosing a suitable vaccine adjuvant can make up for its weak immunogenicity. Experiments have shown that adjuvants with squalene components such as MF59, AS03, AF03, etc., can induce humoral and cellular immunity of recombinant protein vaccines in a more balanced manner, and can induce a wider range of cross-reactions, so they may be more suitable for SARS -CoV-2 recombinant protein vaccine.

4 Nucleic acid vaccine

Nucleic acid vaccines are also called genetic vaccines, including DNA vaccines and mRNA vaccines. The principle is to introduce the DNA or mRNA genes of a certain antigen into the host by intramuscular injection or microprojectile bombardment, and express the antigen protein in the host to induce the host cell produces an immune effect on the antigen protein to achieve the purpose of preventing and treating diseases. In the vaccine development of this epidemic, nucleic acid vaccines are also an important technical route. Such vaccines are easy to operate, low in production costs, and have a short development and production cycle. They can quickly respond to the epidemic and enter the evaluation stage.

DNA vaccine is to introduce the foreign gene of the target antigen into the cells of the animal so that the foreign gene is expressed in the living body, and the generated antigen induces the body to produce specific humoral and cellular immunity. Previous studies have found that DNA vaccine against SARS coronavirus can induce the production of protective neutralizing antibodies in a mouse model. The researchers tested the vaccine on mice and non-human primates. By measuring the content of INF-γ, they found that the vaccine can induce a strong immune response from the body's T cells. This type of vaccine can cause the body to produce an immune effect and can play a role in this new coronary pneumonia epidemic. On April 6, 2020, INOVIO Pharmaceuticals announced that the U.S. Food and Drug Administration (FDA) has accepted the company's research new drug application for INO-4800 and started a phase I clinical trial. This is the world's third new coronavirus vaccine candidate to start clinical trials, and the first new coronavirus DNA vaccine to enter phase I human clinical trials.

The mRNA vaccine is to synthesize in vitro the mRNA sequence that contains the specific antigen, deliver it to the body and express it into the antigen protein by the cell, induce the body to produce a specific immune effect, and then achieve the effect of immune protection. mRNA vaccines can express intracellular antigens like DNA vaccines, but they solve the problems of low immunogenicity of DNA vaccines and generate non-specific immunity against vectors, and have the advantage of short development and production time. The American pharmaceutical company Moderna developed the world’s first SARS-CoV-2 vaccine (mRNA-1273) based on the previous mRNA vaccine research and development and the National Institutes of Health (NIH), which will be released on March 16, 2020. Japan officially started human clinical trials at the Washington Institute of Health of the Caesars Medical Group in the United States. The vaccine is currently in phase I human clinical trials, and further clinical trials are needed to evaluate its safety.

In addition to the many technical platforms mentioned above, virus-like particle (VLP) vaccines and peptide vaccines have also become preclinical research hotspots. VLP is a viral protein body without nucleic acid. It has many advantages such as safety, stability, structural order, suitable size, and surface modification. It is a very promising platform and carrier in vaccine development. Due to the maintenance of the assembly properties of viral proteins, VLP vaccines can achieve high-efficiency vaccine delivery while fully retaining target antigen information, which helps the immune system to recognize. Compared with recombinant protein vaccines, peptide vaccines have a smaller scale, and their essence is the identified B epitope or T epitope sequence. Polypeptide vaccines are prepared quickly and have high safety, making them a promising candidate platform for research. Moreover, the backbone of the polypeptide is composed of amide bonds, which is more conducive to the uptake by cells than nucleic acid vaccines. Considering the high specificity of polypeptide epitopes, reasonable target sequence selection is the top priority. At the same time, in order to improve the immunogenicity of polypeptides, the selection of adjuvants and delivery systems is essential in the design of polypeptide vaccines.

Preliminary studies have shown that patients who have been infected with SARS-CoV lack immune memory response, and the level of neutralizing antibodies can only be maintained for a short period of time. Therefore, the method of obtaining herd immunity through population infection is not feasible, and safe and effective vaccines are the most effective measure to stop the spread of the virus.

References

[1] ADAMS M J, LEFKOWITZ E J, KING A M Q, et al. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2014) [J]. Arch Virol, 2014, 159(10):2831-2841. 0

[2] XU X, CHEN P, WANG J, et al. Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission[J]. Sci China Life Sci, 2020, 63(3): 457 -60. 0

[3] CHAN FW, KOK KH, ZHU Z, et al. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan[J]. Emerg Microbes Infect, 2020, 9(1): 221-236. 0

[4] WRAPP D, WANG N, CORBETT K S, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation [EB/OL]. [2020-02-25] (2020-05-17).