WO2021203236A1

WO2021203236A1 - Bat-derived coronavirus vaccine for prevention of covid-19

Info

Publication number: WO2021203236A1
Application number: PCT/CN2020/083487
Authority: WO
Inventors: 余力; 曾蓁; 曾莲
Original assignee: 四川骋誉生物制品有限公司
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2021-10-14

Abstract

A bat-derived coronavirus vaccine for prevention of COVID-19, which uses bat-derived coronavirus Bat/CovRATG13 to produce a vaccine to control and prevent the ongoing COVID-19 pandemic and future epidemics of the disease. Bat/CovRaTG13 has the closest genetic fingerprint to the 2019 novel coronavirus (SARS-CoV-2). Homology of full amino acid sequences determines not only relatedness of amino acids, but also the similarity of biological properties of the amino acids, with antigenicity being one of the main factors determining vaccine specificity and effectiveness. The vaccine strain is derived from a Bat/CovRaTG13 family member and has sequence homology. The bat-derived coronavirus vaccine can be prepared, by means of corresponding construction and manufacturing method procedures, into three types of vaccines: a live vaccine, an inactivated vaccine and/or a recombinant vaccine to prevent a COVID-19 pandemic.

Description

Bat-derived coronavirus vaccine to prevent COVID-19

Technical field

The present invention is in the field of biomedicine, and relates to a bat-derived coronavirus vaccine for preventing COVID-19.

Background technique

[Corrected according to Rule 26 23.06.2020]
Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although most cases cause mild symptoms, COVID-19 can quickly develop into acute respiratory distress syndrome, causing respiratory failure, septic shock, or multiple organ failure. The World Health Organization (WHO) declared the 2019-2020 coronavirus outbreak as a Public Health Emergency of International Concern (PHEIC) on January 30, 2020, and declared a COVID-19 pandemic on March 11, 2020. Many countries in all six WHO regions have found evidence of local spread of the disease. To date, the coronavirus pandemic has caused more than 1 million infections, more than 40,000 deaths, and led to a global economic recession.

Coronavirus (CoV) is a positive-stranded RNA virus. Under an electron microscope, due to the presence of spinous glycoprotein on the envelope, it has the appearance of a tree crown. The orthocoronavirus subfamily (Nidovirales order) of the Coronavirus family is divided into four genera: alphacoronavirus (alphaCoV), Betacoronavirus (betaCoV), Deltacoronavirus (deltaCoV) and Gammacoronavirus (gammaCoV). In addition, the betaCoV genus is further divided into 5 subgenres or lineages. The genomic characteristics indicate that bats and rodents may be the source of alphaCoVs and betaCoV genes. (Chan JF et al., "Interspecies Transmission and the Emergence of New Viruses: Lessons from Bats and Birds", Trend Microbiology, 2013, 21(10):544-55).

In general, it is estimated that 2% of the population are healthy carriers of CoV, and these viruses are responsible for approximately 5% to 10% of acute respiratory infections (Chen Y et al., Emerging Coronavirus: Genome Structure, Replication and Pathogenesis [J] , Medical Journal, Verol, 2020, 92(4): 418-423). This includes common human coronaviruses: HCoV-OC43 and HCoV-HKU1 (betaCoVs lineage); HCoV-229E and HCoV-NL63 (alphaCoVs). They can cause the common cold and self-limiting upper respiratory tract infections in people with strong immunity. In immunocompromised subjects and the elderly, lower respiratory tract infections may occur. Other human coronaviruses: SARS-CoV, SARS-CoV-2 and MERS-CoV (betaCoVs belonging to the B and C lines respectively) can cause epidemics of different clinical severity with respiratory and extra-respiratory manifestations. The mortality of SARS coronavirus and MERS coronavirus can reach 10% and 35%, respectively.

Chan et al. have demonstrated that the genome of a new human coronavirus isolated from a group of patients with atypical pneumonia has 89% nucleotide homology with bat SARS-CoVZXC21, which is the same as that of human SARS-CoV. The source is 82% (Chan JF, et al. Analysis of the genome characteristics of the 2019 new human pathogenic coronavirus in a patient with atypical pneumonia in Wuhan[J], Microbial Infection, 2020, 9(1):221-236 ). The pangolin coronaviruses discovered so far share only 92% of their entire genome with SARS-CoV-2, which makes them 96% less similar than another bat coronavirus, Bat/CovRaTG13. Therefore, this new virus is called SARS-CoV-2 and is classified as BetaCoVs.

Vaccination is one of the most effective public health interventions. In the past 100 years, it has played an important role in saving lives and greatly changing the burden of many infectious diseases. Vaccines can be divided into two categories: replicating vaccines (ie live vaccines, attenuated vaccines or recombinant vaccines) and non-replicating vaccines (such as inactivated vaccines or subunit vaccines). It has been confirmed that live attenuated vaccines are superior to non-replicating vaccines in terms of the quality of antiviral immune response, the level of protective immunity, and the duration of protective immunity. However, safety is still a major issue for live attenuated vaccines, some of which are eventually replaced by non-replicating or inactivated vaccines. For example, from 1961 to 1989, the routine vaccination of live oral polio vaccine (OPV) in the United States resulted in an average of 9 cases of vaccine-associated paralytic polio (VAPP) each year. Recently, oral polio vaccination in Nigeria has also resulted in at least 69 cases of VAPP. For these safety considerations, the United States completely replaced the live polio vaccine with inactivated polio vaccine (IPV) in 2000, which led to the complete elimination of VAPP. Although live attenuated vaccines can produce a stronger protective immune response in healthy people than inactivated vaccines, inactivated vaccines still play an important role in public health, especially in the absence of live vaccines. Controlling the epidemic of deadly infectious diseases. Inactivated vaccines do not contain any live pathogens; they cannot cause the diseases they protect, even in people with severely weakened immune systems.

As the genetic mutations that may cause human infection and the pathogenesis of COVID-19 are not fully understood, there is an urgent need to develop an anti-SARS-CoV-2 vaccine to control the ongoing pandemic and prevent future outbreaks.

Summary of the invention

In order to solve the above technical problems, the present invention discloses a bat-derived coronavirus vaccine for preventing COVID-19. The prototype or paternal strain that is biologically homologous to the bat-derived coronavirus of COVID-19 is used to prepare the vaccine as a vaccine. Virus strain.

In order to achieve the above invention objectives, the present invention provides the following technical solutions:

A bat-derived coronavirus vaccine for the prevention of COVID-19. The vaccine is prepared using a weakly virulent prototype or paternal strain of SARS-CoV-2 which has biological homology with SARS-CoV-2 virus.

Further, the weakly virulent prototype or paternal strain of SARS-CoV-2 has at least 96% homology in RNA sequence and at least 97% homology in protein amino acid sequence with SARS-CoV-2 virus.

Further, the attenuated prototype or paternal strain of SARS-CoV-2 is Bat/CovRaTG13 strain of coronavirus.

Further, the attenuated prototype or paternal strain of SARS-CoV-2 is any subgenus virus strain with homologous sequence to the Bat/CovRaTG13 strain.

Further, the vaccine is a live vaccine, an inactivated vaccine or a recombinant vaccine prepared by the attenuated prototype or paternal strain of SARS-CoV-2.

Further, the live vaccine is prepared by cell culture, purification, and preparation of the weakly toxic prototype or paternal strain of SARS-CoV-2, and does not undergo genetic modification in vitro.

Further, the inactivated vaccine is prepared by cell culture, purification, inactivation, and preparation of the weakly toxic prototype or paternal strain of SARS-CoV-2, without genetic modification in vitro.

Further, the inactivated vaccine is inactivated by formalin; the inactivated vaccine also includes an aluminum adjuvant.

Further, the recombinant vaccine is a genetically modified replicable vaccine produced by artificial point mutation and/or insertion of gene fragments into any gene of Bat/CovRaTG13.

The present invention prepares a vaccine as a vaccine virus strain by using a weakly virulent prototype or paternal strain that has biological homology with the bat-derived coronavirus of COVID-19, which has better homology with the bat-derived coronavirus of COVID-19. It has higher homology in the protein sequence and RNA sequence, and the antibody obtained by the vaccine prepared with the virus strain is better.

The bat-derived coronavirus vaccine for COVID-19 disclosed in the present invention is based on the parental relationship between SARS-CoV-2 and Bat/CovRaTG13. The parental relationship between Bat/CovRaTG13 and SARS-CoV-2 is proved by the homologous amino acid sequence in each viral protein. Except for the 97.4% homology of the S protein, all other proteins have 98.45% homology and significant similarity, which strongly proves that these two viruses are genetically evolved through a common ancestor, and their variation of less than 1.6% of the amino acid sequence still shows their Genetic relatedness. The alignment of multiple amino acid sequences showed that, except for the RBD and furin signal regions in the S protein, all regions of each protein are homologous between the two viruses. The sequence homology of the S protein shows similar biological phenotypes, including antigenicity, and the variation or mutation in the RBD and furine motifs may imply genetic transfer of non-human pathogens to human infections. For these reasons, Bat/CovRaTG13 can be the (non-pathogenic) attenuated prototype of SARS-CoV-2.

The present invention discloses the use of Bat/CovRaTG13 strain of CoV derived from bats to develop an active and/or inactive vaccine against COVID-19. The resulting vaccine organism maintains the ability to replicate (grow) and induce immunity, but usually does not cause disease. So far, the Bat/CoVRaTG13 strain has been found to be the only bat coronavirus that has a common ancestor with SARS-CoV-2. Therefore, Bat/CovRaTG13, which is not pathogenic to humans, can be used as a live vaccine against COVID-19.

Description of the drawings

Figure 1 is a multiple comparison between the Bat/CovRaTG13 RNA sequence of the present invention and the most relevant coronavirus;

Figure 2 is a multiple alignment analysis of the Bat/CovRaTG13 polyprotein amino acid sequence of the present invention;

Figure 3 is a multiple alignment analysis of the Bat/CovRaTG13 S protein amino acid sequence of the present invention;

Figure 4 is a comparison of homology between the RBD region (in frame) of the S protein of the Bat/CovRaTG13 sequence of the present invention and the self-pair;

Figure 5 is a comparison of the homology of the S protein RBD region (in frame) of the Bat/CovRaTG13 sequence of the present invention with another coronavirus of the same bat subspecies;

Figure 6 is the homology comparison between the S protein RBD region (in frame) of the Bat/CovRaTG13 sequence of the present invention and ARS-CoV-2;

Figure 7 is a comparison of homology between the RBD region (in frame) of the S protein of the Bat/CovRaTG13 sequence of the present invention and the SARS-like coronavirus;

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings.

In order to make the objectives, technical solutions, and advantages of the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but not used to limit the present invention.

The source of SARS-CoV-2 is believed to be natural and derived from animals. Through research on the natural reservoir of virus strains, many bat coronaviruses similar to SARS have been found, which are 80% similar to SARS-CoV-2 virus. However, the viral nucleic acid sequences of almost all samples of COVID-19 patients are 96% similar to the Bat/CovRaTG13, a bat (Rhinolophus affinis) coronavirus collected from Yunnan Province in 2013. The SARS-CoV-2 and Bat/CovRaTG13 strains have 1141 nucleotide variations (substitutions) (genetic distance 0.04 and 48 INDELs). And with other bat-SL-CoVZC45/2017 viruses and SARS human-BJ182-12/2008 viruses with 88% and 80% sequence homology (distance 0.23 and 0.46), 3542 and 5879 nucleotide substitutions ( 137 and 535 INDELs).

Simplot analysis and comparison or non-comparison analysis show that SARS-CoV-2 is highly similar to Bat/CovRaTG13 in the entire genome, with the whole genome RNA sequence homology greater than 96.2%, and the entire amino acid sequence reaching 97.95% homology. Among them, the homology of S protein is 97.41%; and the homology of the remaining 87% of total structural and non-structural protein amino acids is 98.45%, as shown in Figure 1. Systematic genetic analysis also confirmed that SARS-CoV-2 is a close relative of Bat/CovRaTG13, and forms different pedigrees with other SARSr-CoV.

Coronavirus particles contain four structural proteins, including spike protein (S), envelope protein, membrane protein and nucleocapsid protein. Among them, the S protein plays the most important role in virus adhesion, fusion and cell entry, and is the target for the development of antibodies, inhibitors and vaccines. The S protein mediates the virus into the host cell, firstly binds to the host receptor through the receptor binding domain (RBD) of the S1 subunit, and then fuses the viral membrane and the host membrane through the S2 subunit. Similar to SARS-CoV, SARS-CoV-2s protein also recognizes ACE2 as its host receptor. The S protein gene containing the RBD sequence is highly different from other SARSr-Cov. The nucleotide sequence homology with all previously described SARSr-Cov is less than 75%, but the nucleotide homology with Bat/CovRaTG13 is 93.1%. . The comparative analysis of the amino acid composition of the S protein showed that between SARS-CoV-2 and Bat/CovRaTG13, the N-terminal 400 amino acids (S1 subunit) are 98% identical, which is higher than all other coronaviruses, SARS-CoV or Similar to SARS-CoV, as shown in Figure 3. However, the remaining C-terminal residues in SARS-CoV-2 and Bat/CovRaTG13S1 protein have 94.4% similarity, and their homology is higher than other bat-like coronaviruses and SARS-like coronaviruses. Most of the amino acid variation in the SARS-CoV-2 S protein is concentrated in the RBD region. Four of the five key residues in the RBD motif of SARS-CoV-2 and Bat/CovRaTG13 are different from other coronaviruses. However, the variation of the RBD region does not seem to affect the binding of the SARS-CoV-2s protein to the ACE2 receptor. In addition, there are four amino acids inserted into the S protein, and they only exist in the S protein of SARS-CoV-2, not in other coronaviruses. The inserted four amino acids PRRA are located at the junction of S1 and S2 subunits and are considered to be furin cleavage signals. However, it is not clear whether the inserted PRRA amino acid has an effect on the host range, infectivity, and increased infectivity, as shown in Figures 4-7.

Compared with Bat/CovRaTG13, the mutations of SARS-CoV-2 nucleotides are mostly silent mutations. Less than 1% of point mutations in the whole genome cause amino acid changes. As shown in Figure 3, the aggregation and variation of the 17 amino acids of the S protein are mainly concentrated in the RBD region. In conclusion, genetic evidence indicates that SARS-CoV-2 is closely related to Bat/CovRaTG13. As shown in Figure 2, the amino acid alignment between Bat/CovRaTG13 and 57 sequenced SARS-CoV-2 (except S protein) is 98.45% homology. Therefore, their sequence homology proves that the latter is the paternal or parental strain of SARS-CoV-2. Bat/CovRaTG13 (GenBank: MN996532.1) was originally isolated from a species of bat (Rhinolophus affinis) in Yunnan Province, China in 2013. The ancestors of the currently circulating SARS-CoV-2 may have undergone significant evolution between 2013 and 2019, resulting in a genetic shift from bat species to humans, although the intermediate host has not yet been identified.

As mentioned above, although the genetic mutations that cause human infections and the pathogenesis of COVID-19 are not fully understood, because the genetic relationship between SARS-CoV-2 and Bat/CovRaTG13 is the closest, the homology of its S protein is 97.4 %, with the same antigenicity, so the parental Bat/CovRaTG13 as an attenuated strain of SARS-CoV-2 is a good candidate for COVID-19 vaccine. The parent type of Bat/CovRaTG13, as the source of bat-CoV, has no or low pathogenicity to humans, making it a good candidate vaccine. Due to the biological homology of the two viruses, including antigenicity, Bat/CovRaTG13 vaccination can induce a specific immune response to SARS-CoV-2 and protect healthy people from SARS-CoV-2 infection.

In order to effectively use Bat/CovRaTG13 as a preventive vaccine, the present invention provides alternative solutions, including: Bat/CovRaTG13 can be used as a live vaccine, inactivated vaccine or recombinant vaccine to prevent COVID-19. Due to its non (or low) pathogenicity to humans, the Bat/CovRaTG13 vaccine can be produced in low-level safety facilities.

The specific preparation method of the vaccine is as follows:

The live vaccine is prepared by cell culture, purification, and preparation of the weakly toxic prototype or paternal strain of SARS-CoV-2, and does not undergo genetic modification in vitro. The resulting vaccine organism maintains the ability to replicate (grow) and induce immunity, but usually does not cause disease. It specifically includes the following steps: cell culture to amplify the Bat/CovRaTG13 virus; collecting virus particles from the culture solution; purifying and concentrating virus particles by methods such as culture solution filtration, chromatography or ultracentrifugation; sterile ultrafiltration, and obtaining it before its production Live vaccines.

The inactivated vaccine is prepared by cell culture, purification, inactivation, and preparation of the weakly toxic prototype or paternal strain of SARS-CoV-2, and does not undergo genetic modification in vitro. It is specifically: the inactivated vaccine is made through the process of inactivation or "killing" of chemical substances. Inactivated vaccines are composed of whole viruses (molecules), but have lost the ability to replicate. However, inactivated vaccines are safe and can be used with adjuvants to enhance immunogenicity. The chemical substance may be formalin, and the adjuvant may be an aluminum adjuvant.

It includes the following steps: cell culture to amplify the Bat/CovRaTG13 virus; collecting virus particles from the culture medium; purifying and concentrating the virus particles by methods such as culture fluid filtration, chromatography or ultracentrifugation; using formalin to "kill" the virus particles ; Filter and mix with adjuvant; finally, the product is formulated into a product vaccine with a buffer or protective agent and a predetermined dose, and then packaged.

The recombinant vaccine is a genetically modified replicable vaccine produced by artificial point mutation and/or insertion of gene fragments into any gene of Bat/CovRaTG13. Specifically, it is prepared by artificial amino acid mutation or recombination in Bat/CovRaTG13 S protein on a molecular basis, without changing the antigenicity of S protein and not causing COVID-19 related diseases.

The present invention is not limited to the foregoing specific embodiments. The present invention extends to any new feature or any new combination disclosed in this specification, and any new method or process step or any new combination disclosed.

Claims

A bat-derived coronavirus vaccine for preventing COVID-19, which is characterized in that the vaccine is prepared using an attenuated prototype or paternal strain of SARS-CoV-2 which has biological homology with SARS-CoV-2 virus.
The bat-derived coronavirus vaccine for preventing COVID-19 according to claim 1, characterized in that: the attenuated prototype or paternal strain of SARS-CoV-2 is in RNA sequence with SARS-CoV-2 virus It has at least 96% homology in the protein amino acid sequence and at least 97% homology in the protein amino acid sequence.
The bat-derived coronavirus vaccine for preventing COVID-19 according to claim 2, wherein the attenuated prototype or paternal strain of SARS-CoV-2 is the Bat/CovRaTG13 strain of SARS-CoV-2.
The bat-derived coronavirus vaccine for the prevention of COVID-19 according to claim 2, wherein the attenuated prototype or paternal strain of SARS-CoV-2 is the same as that of the Bat/CovRaTG13 strain of SARS-CoV-2 Any subgenus virus strain with homologous sequence.
The bat-derived coronavirus vaccine used to prevent COVID-19 according to any one of claims 1 to 4, characterized in that: the vaccine is a live vaccine prepared by the attenuated prototype or male parent strain of SARS-CoV-2 Vaccines, inactivated vaccines or recombinant vaccines.
The bat-derived coronavirus vaccine for preventing COVID-19 according to claim 5, characterized in that: the live vaccine is cultured and purified by the attenuated prototype of SARS-CoV-2 or the male parent strain. , The preparation is prepared without genetic modification in vitro.
The bat-derived coronavirus vaccine for the prevention of COVID-19 according to claim 5, characterized in that: the inactivated vaccine is cultured by the attenuated prototype or the paternal strain of SARS-CoV-2. Purification, inactivation, preparation of preparations, no genetic modification in vitro.
The bat-derived coronavirus vaccine for preventing COVID-19 according to claim 7, wherein the inactivated vaccine is inactivated by formalin; the inactivated vaccine also includes an aluminum adjuvant .
The bat-derived coronavirus vaccine for preventing COVID-19 according to claim 5, characterized in that: the recombinant vaccine is made by artificial point mutation and/or insertion of gene fragments into any gene of Bat/CovRaTG13 Generated genetically modified replicable vaccines.