Novel coronavirus immunogenic substance, preparation method and application thereof

Abstract

The present invention provides a novel coronavirus immunogenic substance, comprising a first antigen derived from an immunodominant strain and a second antigen derived from an epidemic dominant strain, wherein the antigens respectively comprise a receptor binding region of an S protein or a portion of a receptor binding region, wherein the immunodominant strain is selected from at least one of a novel coronavirus WH01 strain and a Beta strain, and the epidemic dominant strain is selected from at least one of a novel coronavirus Delta strain and an Omicron strain. The novel coronavirus immunogenic substance of the present invention has high immunogenicity and can show significantly improved immune effects for different strains.

Description

Field of the Invention

[0001] The present invention belongs to the field of biomedical engineering technology and relates to novel coronavirus immunogenic substances, preparation methods and applications thereof. Background Art

[0002] Infection with the novel coronavirus (SARS-CoV-2) can cause coronavirus disease (COVID-19), with common symptoms such as fever, cough, sore throat, etc. In more severe cases, infection can lead to dyspnea, hypoxemia, acute respiratory distress syndrome, and even death. The novel coronavirus can be transmitted from person to person through the respiratory tract and droplet routes, and there is also the possibility of transmission through the air and digestive tract.

[0003] SARS-CoV-2 virus particles contain four structural proteins, namely spike protein (S), nucleocapsid protein (N), membrane protein (M) and envelope protein (E). Studies have found that only antibodies against the S protein have neutralizing activity, so the vaccines currently under development all contain the S protein or its components. Among them, the receptor binding region of the S protein is considered to be the most important antigenic target region for inducing the body to produce neutralizing antibodies. As a vaccine, the receptor binding region can focus the neutralizing antibodies stimulated by the body on the receptor binding against the virus, which can improve the immunogenicity and immune efficiency of the vaccine. SARS-CoV-2 enters the cell by binding to the host cell receptor hACE2 through its receptor binding region.

[0004] The new coronavirus continues to evolve during the transmission process, and multiple representative mutant strains have been detected. Most of the new coronavirus antigens currently developed or under development can only target one strain and cannot produce neutralizing antibodies against different strains. CN114369172A designed a prototype strain RBD dimer vaccine, a Beta strain RBD dimer vaccine, and a prototype strain + Beta strain chimeric RBD dimer vaccine. The results showed that the prototype strain + Beta strain chimeric RBD dimer vaccine induced a more balanced antibody response compared to the prototype strain RBD dimer vaccine and the Beta strain RBD dimer vaccine. However, from the results, compared with the prototype strain and other variants, the prototype strain + Beta strain chimeric RBD dimer vaccine had a significantly decreased neutralizing antibody titer against the Omicron variant S protein pseudovirus, which shows that the vaccine has poor protection against the currently popular Omicron variant. Therefore, it is necessary to develop a vaccine with a more balanced protective effect against different strains. Summary of the invention

[0005] The purpose of the present invention is to provide a novel coronavirus immunogenic substance, which contains the receptor binding region of the S1 subunit of the S protein of different strains. The novel coronavirus immunogenic substance has higher immunogenicity and can stimulate the production of neutralizing antibodies against different strains, thereby significantly improving the immune effect.

[0006] To achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0007] A novel coronavirus immunogenic substance, comprising a first antigen derived from an immunodominant strain and a second antigen derived from an epidemic dominant strain, wherein each antigen comprises a receptor binding region or a portion of a receptor binding region of an S protein.

[0008] In some embodiments, the immunodominant strain is selected from at least one of the novel coronavirus WH01 strain and the Beta strain.

[0009] In some embodiments, the prevalent dominant strain is selected from at least one of the Delta strain and the Omicron strain of the new coronavirus.

[0010] In some embodiments, the Omicron strain includes BA.1, BA.2, BA.3, BA.4 and BA.5 variants.

[0011] In some embodiments, the novel coronavirus immunogenic material further comprises a third antigen derived from a strain other than the immunodominant strain and the epidemic dominant strain.

[0012] In some embodiments, the novel coronavirus immunogenic material further comprises a fourth antigen derived from a strain other than the immunodominant strain and the epidemic dominant strain.

[0013] In some embodiments, the strains other than the immunodominant strain and the epidemic dominant strain are selected from the following strains: Alpha strain, Gamma strain, Epsilon strain, Zeta strain, Eta strain, Theta strain, Iota strain, Kappa strain, Lambda strain, Mu strain, etc.

[0014] In some embodiments, the antigens constitute a composition, or the antigens are directly connected or connected through an amino acid linker, for example, the amino acid linker can be GGS or multiple tandem GGSs (G and S represent glycine and serine, respectively).

[0015] For example, in some embodiments, the novel coronavirus immunogenic material comprises a first antigen and a second antigen, and the first antigen is directly connected to the second antigen or connected by an amino acid linker. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen and a second antigen, and the first antigen and the second antigen are mixed to form a composition. In some embodiments, the novel coronavirus immunogenic material is formed by directly connecting the receptor binding region of the novel coronavirus WH01 strain S protein and the receptor binding region of the Delta strain; in some embodiments, the novel coronavirus immunogenic material is formed by directly connecting the receptor binding region of the novel coronavirus WH01 strain S protein and the receptor binding region of the Omicron strain; in some embodiments, the novel coronavirus immunogenic material is formed by directly connecting the receptor binding region of the novel coronavirus Beta strain S protein and the receptor binding region of the Delta strain; in some embodiments, the novel coronavirus immunogenic material is formed by directly connecting the receptor binding region of the novel coronavirus Beta strain S protein and the receptor binding region of the Omicron strain.

[0016] In some embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, and a third antigen, which are directly connected or connected through an amino acid linker. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, and a third antigen, which are directly connected to the second antigen or connected through an amino acid linker to form a fusion antigen, which is mixed with the third antigen to form a composition. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, and a third antigen, which are mixed with the first antigen, the second antigen, and the third antigen to form a composition.

[0017] In some embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, a third antigen, and a fourth antigen, and the first antigen, the second antigen, the third antigen, and the fourth antigen are directly connected or connected through an amino acid linker to form a fusion antigen. In some embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, a third antigen, and a fourth antigen, and the first antigen is directly connected to the second antigen or connected through an amino acid linker to form a fusion antigen, and the third antigen is directly connected to the fourth antigen or connected through an amino acid linker to form a fusion antigen, and the two fusion antigens are mixed to form a composition. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, a third antigen, and a fourth antigen, and the first antigen is directly connected to the second antigen or connected through an amino acid linker to form a fusion antigen, and the fusion antigen is mixed with the fourth antigen to form a composition. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, a third antigen, and a fourth antigen, and the first antigen is directly connected to the second antigen or connected through an amino acid linker to form a fusion antigen, and the fusion antigen is mixed with the third antigen and the fourth antigen to form a composition. In other embodiments, the novel coronavirus immunogenic substance comprises a first antigen, a second antigen, a third antigen and a fourth antigen, and the first antigen, the second antigen, the third antigen and the fourth antigen are mixed to form a composition.

[0018] In the present invention, the first, second, third and fourth are only used to indicate different types of antigens, and do not indicate any order between the antigens. The types and quantities of antigens in the present invention are also not limited, and those skilled in the art can determine the appropriate types and quantities of antigens based on widely spread strains and cross-reactions between antigens and antibodies.

[0019] In some embodiments, each of the antigens comprises at least 8 cysteines, and the number of cysteines is an even number.

[0020] In some embodiments, the receptor binding region of the S protein derived from the WH01 strain comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0021] In some embodiments, the receptor binding region of the S protein derived from the Beta strain comprises the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0022] In some embodiments, the receptor binding region of the S protein derived from the Delta strain comprises the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

[0023] In some embodiments, the receptor binding region of the S protein derived from the Omicron BA.1 variant comprises the amino acid sequence shown in SEQ ID NO:42, the receptor binding region of the S protein derived from the BA.2 variant comprises the amino acid sequence shown in SEQ ID NO:62, the receptor binding region of the S protein derived from the Omicron BA.3 variant comprises the amino acid sequence shown in SEQ ID NO:63, and the receptor binding region of the S protein derived from the Omicron BA.4 and BA.5 variants comprises the amino acid sequence shown in SEQ ID NO:64.

[0024] In some embodiments, the novel coronavirus immunogenic substance comprises an amino acid sequence shown in any one of SEQ ID NOs: 19, 20, 28, 29, 47, 48, 51-55.

[0025] In some embodiments, the antigen further comprises the N-terminal domain (NTD) in the S1 subunit of the S protein.

[0026] In some embodiments, the N-terminal domain is located at the N-terminus of the amino acid sequence of the first antigen and / or the second antigen.

[0027] In some embodiments, the N-terminal domain of the S protein derived from the WH01 strain comprises the amino acid sequence shown in SEQ ID NO:13.

[0028] In some embodiments, the N-terminal domain of the S protein derived from the Beta strain comprises the amino acid sequence shown in SEQ ID NO:14.

[0029] In some embodiments, the N-terminal domain of the S protein derived from the Delta strain comprises the amino acid sequence shown in SEQ ID NO:15.

[0030] In some embodiments, the N-terminal domain of the S protein derived from the Omicron BA.1 variant comprises the amino acid sequence shown in SEQ ID NO:43, the N-terminal domain of the S protein derived from the Omicron BA.2 variant comprises the amino acid sequence shown in SEQ ID NO:65, the N-terminal domain of the S protein derived from the Omicron BA.3 variant comprises the amino acid sequence shown in SEQ ID NO:66, and the N-terminal domain of the S protein derived from the Omicron BA.4 and BA.5 variants comprises the amino acid sequence shown in SEQ ID NO:67.

[0031] In some embodiments, the novel coronavirus immunogenic substance comprises an amino acid sequence shown in any one of SEQ ID NOs: 22, 23, 30, 49, 50, 56-59.

[0032] In some embodiments, the novel coronavirus immunogenic substance further comprises the Fc domain of an immunoglobulin, and preferably, the immunoglobulin is human IgG.

[0033] In some embodiments, the Fc domain is located at the C-terminus of the amino acid sequence of the novel coronavirus immunogenic substance. Preferably, the human IgG Fc domain comprises the amino acid sequence shown in SEQ ID NO:16.

[0034] In some embodiments, the novel coronavirus immunogenic substance comprises the amino acid sequence shown in SEQ ID NO:24 or SEQ ID NO:25.

[0035] In some embodiments, the novel coronavirus immunogenic substance further comprises a Foldon domain. The Foldon domain / protein is derived from the C-terminus of the T4 bacteriophage fiber protein, is composed of 27 amino acids, and has the function of promoting non-covalent oligomerization of the target protein to form a trimer.

[0036] In some embodiments, the Foldon domain is located at the C-terminus of the amino acid sequence of the novel coronavirus immunogenic substance. Preferably, the Foldon domain comprises the amino acid sequence shown in SEQ ID NO:17.

[0037] In some embodiments, the novel coronavirus immunogenic substance comprises the amino acid sequence shown in any one of SEQ ID NO: 26, 27, 60, 61.

[0038] The present invention also provides a method for preparing the novel coronavirus immunogenic substance, comprising the following steps:

[0039] Constructing a recombinant expression plasmid using the nucleotide sequence encoding the novel coronavirus immunogenic substance;

[0040] Transform the constructed recombinant expression plasmid into the host bacteria and screen the correct recombinant expression plasmid;

[0041] The screened recombinant expression plasmid is used to transfect the cells of the expression system. After expression, the supernatant is collected and purified to obtain the new coronavirus immunogenic substance.

[0042] In some embodiments, the cells of the expression system include mammalian cells, insect cells, yeast cells or bacterial cells. Optionally, the mammalian cells include 293T cells or CHO cells, and the bacterial cells include Escherichia coli cells.

[0043] The present invention also provides a nucleotide sequence encoding the novel coronavirus immunogenic substance, a recombinant vector comprising the above nucleotide sequence, and an expression system cell carrying the above recombinant vector.

[0044] The present invention also provides the use of the novel coronavirus immunogenic substance, a nucleotide sequence encoding the novel coronavirus immunogenic substance, a recombinant vector containing the nucleotide sequence, and an expression system cell carrying the recombinant vector in the preparation of a novel coronavirus vaccine.

[0045] The present invention also provides a novel coronavirus protein vaccine, comprising the novel coronavirus immunogenic substance and an adjuvant.

[0046] In some embodiments, the adjuvant is selected from one or more of aluminum adjuvant, MF59 adjuvant, MPL adjuvant, QS-21, GLA, CpG, AS01, AS02, AS03, and AS04 adjuvants, preferably AS03 adjuvant or MF59 adjuvant.

[0047] The present invention also provides a new coronavirus DNA vaccine, which comprises a DNA sequence encoding the immunogenic substance of the new coronavirus.

[0048] The present invention also provides a new coronavirus mRNA vaccine, which comprises an mRNA sequence encoding the new coronavirus immunogenic substance.

[0049] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0050] The novel coronavirus immunogenic substance of the present invention comprises at least two antigens from an immunodominant strain and an epidemic dominant strain, and can induce the production of neutralizing antibodies against different strains. Experiments have shown that the novel coronavirus immunogenic substance of the present invention has high immunogenicity and good cross-protection effects, and can induce the production of balanced neutralizing antibody levels against different strains.

[0051] The inventors were surprised to find that the immunodominant strain based on the WH01 strain or Beta strain of the new coronavirus, combined with the currently popular mutant strains, can produce outstanding immune effects. The WH01 strain or Beta strain is the cornerstone of the immune combination, and can produce antibodies against different mutant strains to ensure the stability of immunity. In particular, the recombinant protein constructed by combining the WH01 strain and the Omicron strain, the Beta strain and the Delta strain in the embodiment can maintain a balanced neutralizing antibody GMT level for different mutant strains, while overcoming the immune escape of the mutant strains, and producing outstanding immune effects. The research of the present invention also shows that the mRNA vaccine prepared using the mRNA encoding the immunogenic substance of the present invention also has a good immune effect on different strains.

[0052] The present invention also studies the combined immune effects of different adjuvants and immunogenic substances. The results show that when an oil-in-water emulsion adjuvant (especially AS03 adjuvant) is used, a higher neutralizing antibody titer can be generated. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of the S protein domain.

[0054] Figure 2 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:18.

[0055] Figure 3 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:19.

[0056] Figure 4 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:20.

[0057] Figure 5 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:21.

[0058] Figure 6 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:22.

[0059] Figure 7 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:23.

[0060] Figure 8 The SDS-PAGE and Western Blot results of antigen SEQ ID NO:26.

[0061] Fig. 9 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:27.

[0062] Fig.10 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:28.

[0063] Fig.11 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:30.

[0064] Fig.12 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:44.

[0065] Fig.13 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:60.

[0066] Fig.14 Shown are the SDS-PAGE and Western Blot results of antigen SEQ ID NO:61. DETAILED DESCRIPTION

[0067] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] The "immunodominant strain" in the present invention refers to a variant strain whose antigen has higher immunogenicity than that of a non-immunodominant variant strain and can provide better cross-protection to other variant strains.

[0069] The "prevalent dominant strain" in the present invention refers to the main variant strain currently prevalent, which can usually be understood as the currently prevalent "variants of concern" (VOC). The definition of VOC can refer to the working definition of the World Health Organization (WHO), that is, SARS-CoV-2 variants that meet the definition of "variants of interest (VOI)" and have been proven to be associated with one or more of the following changes with certain global public health significance through comparative evaluation:

[0070] Increased transmissibility or deleterious changes in the epidemiology of COVID-19; or

[0071] Increased toxicity or changes in clinical disease presentation; or

[0072] Reduced effectiveness of public health and social measures or available diagnostics, vaccines and treatments.

[0073] The definition of "Variants of interest (VOI)" in the present invention can refer to the working definition of the World Health Organization (WHO), namely: SARS-CoV-2 variants with the following characteristics:

[0074] Have genetic changes predicted or known to affect viral characteristics, such as transmissibility, disease severity, immune escape, diagnostic or therapeutic escape; and

[0075] Confirmed as resulting in significant community transmission or multiple clusters of COVID-19 cases in multiple countries, with increasing relative prevalence, increasing case numbers, or other clear epidemiological impacts indicating an emerging risk to global public health.

[0076] The structure of SARS-CoV-2 S protein is as follows Figure 1 As shown, 1-13 is a signal peptide, 14-685 is an S1 subunit, and 686-1273 is an S2 subunit. The S1 subunit can be divided into NTD (14-303) and CTD (334-527). 319-541 is a receptor binding region, 788-806 is a fusion protein. 13-1213 is an extracellular domain, 1214-1234 is a transmembrane domain, and 1235-1273 is an intracellular domain.

[0077] Example 1 Amino Acid Sequence of Novel Coronavirus Immunogenic Substance

[0078] In an embodiment of the present invention, the inventors designed a variety of novel coronavirus immunogenic substances using the receptor binding regions of the novel coronavirus WH01 strain, Beta strain, Delta strain and Omicron variant strain.

[0079] The receptor binding region of the S protein derived from the WH01 strain comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. SEQ ID NO: 1 contains 8 cysteines, SEQ ID NO: 2 contains an additional sequence extending outward from the N-terminus of SEQ ID NO: 1, containing 10 cysteines, SEQ ID NO: 3 contains an additional sequence extending outward from the C-terminus of SEQ ID NO: 1, containing 10 cysteines, and SEQ ID NO: 4 contains additional sequences extending outward from the N-terminus and C-terminus of SEQ ID NO: 1, respectively, containing 12 cysteines.

[0080] The receptor binding region of the S protein derived from the Beta strain comprises an amino acid sequence as shown in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Among them, SEQ ID NO: 5 contains 8 cysteines, SEQ ID NO: 6 contains an additional sequence extending outward from the N-terminus of SEQ ID NO: 5, containing 10 cysteines, SEQ ID NO: 7 contains an additional sequence extending outward from the C-terminus of SEQ ID NO: 5, containing 10 cysteines, and SEQ ID NO: 8 contains additional sequences extending outward from the N-terminus and C-terminus of SEQ ID NO: 5, respectively, containing 12 cysteines.

[0081] The receptor binding region of the S protein derived from the Delta strain comprises an amino acid sequence as shown in any one of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. Among them, SEQ ID NO: 9 contains 8 cysteines, SEQ ID NO: 10 contains an additional sequence extending outward from the N-terminus of SEQ ID NO: 9, containing 10 cysteines, SEQ ID NO: 11 contains an additional sequence extending outward from the C-terminus of SEQ ID NO: 9, containing 10 cysteines, and SEQ ID NO: 12 contains additional sequences extending outward from the N-terminus and C-terminus of SEQ ID NO: 9, respectively, containing 12 cysteines.

[0082] The receptor binding region of the S protein derived from the Omicron BA.1 variant comprises the amino acid sequence shown in SEQ ID NO:42, the receptor binding region of the S protein derived from the BA.2 variant comprises the amino acid sequence shown in SEQ ID NO:62, the receptor binding region of the S protein derived from the BA.3 variant comprises the amino acid sequence shown in SEQ ID NO:63, and the receptor binding region of the S protein derived from the BA.4 and BA.5 variants comprises the amino acid sequence shown in SEQ ID NO:64.

[0083] In this embodiment, some immunogenic substances also include the N-terminal domain (NTD) in the S1 subunit of the S protein. Among them, the N-terminal domain in the S1 subunit of the WH01 strain S protein comprises the amino acid sequence shown in SEQ ID NO:13, the N-terminal domain in the S1 subunit of the Beta strain S protein comprises the amino acid sequence shown in SEQ ID NO:14, the N-terminal domain in the S1 subunit of the Delta strain comprises the amino acid sequence shown in SEQ ID NO:15, the N-terminal domain in the S1 subunit of the Omicron BA.1 variant comprises the amino acid sequence shown in SEQ ID NO:43, the N-terminal domain of the S protein of the Omicron BA.2 variant comprises the amino acid sequence shown in SEQ ID NO:65, the N-terminal domain of the S protein of the Omicron BA.3 variant comprises the amino acid sequence shown in SEQ ID NO:66, and the N-terminal domain of the S protein of the Omicron BA.4 and BA.5 variants comprises the amino acid sequence shown in SEQ ID NO:67.

[0084] In this embodiment, some immunogenic substances also include a human IgG Fc domain to form a dimer structure, or some immunogenic substances also include a Foldon domain to form a trimer structure, the human IgG Fc domain comprises the amino acid sequence shown in SEQ ID NO:16, and the Foldon domain comprises the amino acid sequence shown in SEQ ID NO:17.

[0085] Based on the above sequence, the inventors designed the amino acid sequence of the following antigen and obtained the corresponding DNA sequence by codon optimization according to the host CHO cells:

[0086] (1) The antigen derived from the WH01 strain is composed of two SEQ ID NO:1s in series, and its amino acid sequence is SEQ ID NO:18 (with a signal peptide SEQ ID NO:41 added to the 5' end and six histidines added to the 3' end), and the corresponding DNA sequence is SEQ ID NO:31;

[0087] (2) The antigen derived from the WH01 strain and the Delta strain is composed of SEQ ID NO: 1 and SEQ ID NO: 9 in series, and its amino acid sequence is SEQ ID NO: 19 (a signal peptide is added to the 5' end and six histidines are added to the 3' end), and the corresponding DNA sequence is SEQ ID NO: 32;

[0088] (3) An antigen derived from the Beta and Delta strains, which is composed of SEQ ID NO:5 and SEQ ID NO:9 in series, and its amino acid sequence is SEQ ID NO:20 (with a signal peptide added to the 5' end and six histidines added to the 3' end), and the corresponding DNA sequence is SEQ ID NO:33;

[0089] (4) The antigen derived from the Delta strain is composed of two SEQ ID NO:9 in series, and its amino acid sequence is SEQ ID NO:21 (a signal peptide is added to the 5' end and six histidines are added to the 3' end), and the corresponding DNA sequence is SEQ ID NO:34;

[0090] (5) An antigen derived from WH01 and Delta strains, comprising the N-terminal domain of WH01 strain, which is composed of SEQ ID NO: 13, SEQ ID NO: 1 and SEQ ID NO: 9 in series, and its amino acid sequence is SEQ ID NO: 22 (with a signal peptide added to the 5' end and 6 histidines added to the 3' end), and the corresponding DNA sequence is SEQ ID NO: 35;

[0091] (6) An antigen derived from Beta and Delta strains, comprising the N-terminal domain of Beta strain, which is composed of SEQ ID NO:14, SEQ ID NO:5 and SEQ ID NO:9 in series, and its amino acid sequence is SEQ ID NO:23 (with a signal peptide added at the 5' end and 6 histidines added at the 3' end), and the corresponding DNA sequence is SEQ ID NO:36;

[0092] (7) Fc dimer of antigens derived from WH01 strain and Delta strain, composed of SEQ ID NO: 1, SEQ ID NO: 9 and SEQ ID NO: 16 in series, and its amino acid sequence is SEQ ID NO: 24 (signal peptide added to the 5' end and 6 histidines added to the 3' end);

[0093] (8) an Fc dimer of an antigen derived from WH01 strain and Delta strain, comprising the N-terminal domain of WH01 strain, which is composed of SEQ ID NO: 13, SEQ ID NO: 1, SEQ ID NO: 9 and SEQ ID NO: 16 in series, and whose amino acid sequence is SEQ ID NO: 25 (a signal peptide is added to the 5' end and six histidines are added to the 3' end);

[0094] (9) Foldon trimer of antigens derived from WH01 and Delta strains, composed of SEQ ID NO: 1, SEQ ID NO: 9 and SEQ ID NO: 17 in series, with an amino acid sequence of SEQ ID NO: 26 (with a signal peptide added to the 5' end and six histidines added to the 3' end), and the corresponding DNA sequence is SEQ ID NO: 37;

[0095] (10) A Foldon trimer of antigens derived from WH01 and Delta strains, comprising the N-terminal domain of WH01 strain, which is composed of SEQ ID NO:13, SEQ ID NO:1, SEQ ID NO:9 and SEQ ID NO:17 in series, and whose amino acid sequence is SEQ ID NO:27 (with a signal peptide added to the 5' end and six histidines added to the 3' end), and the corresponding DNA sequence is SEQ ID NO:38;

[0096] (11) The antigen derived from the WH01 strain and the Delta strain is composed of SEQ ID NO: 2 and SEQ ID NO: 10 in series, and its amino acid sequence is SEQ ID NO: 28 (a signal peptide is added to the 5' end and six histidines are added to the 3' end), and the corresponding DNA sequence is SEQ ID NO: 39;

[0097] (12) An antigen derived from the WH01 strain and the Delta strain, which is composed of SEQ ID NO: 4 and SEQ ID NO: 12 in series, and its amino acid sequence is SEQ ID NO: 29 (a signal peptide is added to the 5' end and six histidines are added to the 3' end);

[0098] (13) The antigen derived from WH01 strain and Delta strain is composed of SEQ ID NO:13, SEQ ID NO:1, SEQ ID NO:15 and SEQ ID NO:9 in series, and its amino acid sequence is SEQ ID NO:30 (a signal peptide is added to the 5' end and 6 histidines are added to the 3' end), and the corresponding DNA sequence is SEQ ID NO:40.

[0099] (14) an antigen derived from the WH01 strain, which is composed of two SEQ ID NO: 2 in series, and whose amino acid sequence is SEQ ID NO: 44 (a signal peptide is added to the 5' end and six histidines are added to the 3' end);

[0100] (15) an antigen derived from the WH01 strain, comprising the N-terminal domain of the WH01 strain, which is composed of SEQ ID NO: 13 and two SEQ ID NO: 1s in series, and whose amino acid sequence is SEQ ID NO: 45;

[0101] (16) an antigen derived from the Delta strain, comprising the N-terminal domain of the Delta strain, consisting of SEQ ID NO: 15 and two SEQ ID NO: 9 in series, and having an amino acid sequence of SEQ ID NO: 46;

[0102] (17) An antigen derived from the WH01 strain and the Omicron BA.1 variant, which is composed of SEQ ID NO: 1 and SEQ ID NO: 42 in series, and its amino acid sequence is SEQ ID NO: 47;

[0103] (18) An antigen derived from the Beta strain and the Omicron BA.1 variant, which is composed of SEQ ID NO: 5 and SEQ ID NO: 42 in series, and its amino acid sequence is SEQ ID NO: 48;

[0104] (19) An antigen derived from the WH01 strain and the Omicron BA.1 variant, comprising the N-terminal domain of the WH01 strain, which is composed of SEQ ID NO: 13, SEQ ID NO: 1 and SEQ ID NO: 42 in series, and whose amino acid sequence is SEQ ID NO: 49;

[0105] (20) An antigen derived from the Beta strain and the Omicron BA.1 variant, comprising the N-terminal domain of the Beta strain, which is composed of SEQ ID NO: 14, SEQ ID NO: 5 and SEQ ID NO: 42 in series, and whose amino acid sequence is SEQ ID NO: 50;

[0106] (21) Based on the major variants, antigens derived from 2-4 variants were further designed, and their amino acid sequences are shown in SEQ ID NOs: 51-61.

[0107] The above antigens are summarized in Table 1:

[0108] Table 1

[0109]

[0110]

[0111] Example 2 Construction of expression vector

[0112] The DNA sequences encoding the above antigens were codon-optimized according to the host CHO cells and then synthesized exogenously and cloned into the expression vector pWX039 to obtain the expression vector pWX039-PR-Z, which was delivered after verification by target gene sequencing.

[0113] Using pWX039-PR-Z vector as template, the target gene DNA fragment was obtained by PCR amplification and gel purification. The purified target gene DNA fragment was cloned into the vector pWX4.1 through the SalI and NotI sites to obtain the transient expression vector pWX4.1-PR. The expression vector pWX4.1-PR was verified by Sanger sequencing, and the sequence was 100% correct.

[0114] Transform the expression vector pWX4.1-PR into competent E. coli Top10 cells. Pick the transformants for liquid culture and then streak them on LB agar plates (containing 100 μg / mL ampicillin). Pick a single clone from the plate and inoculate it into 300 ml LB medium for expansion culture. Use the NucleoBond Xtra Maxi EF kit to prepare the plasmid in large quantities and sequence and verify the target gene. The pWX4.1-PR plasmid DNA can be used for transfection after being verified by sequencing.

[0115] Example 3 Protein expression and purification

[0116] Protein expression

[0117] During all cell manipulations, mix cells by gentle swirling; avoid vigorous mixing / pipetting. Cell health is critical to achieving maximum performance. CHO cells were subcultured and expanded using ExpiCHO Expression Medium (EXPICHO from Thermo) to a cell density of 4 × 10 6 – 6×10 6 Cells were passaged at 100 μg / mL.

[0118] Day -1: Cell expansion, expand the culture cells to 3×10 6 – 4×10 6 Day 0: Transfect cells and measure viable cell density and viability. The cell density should reach 7 × 10 6 – 10×10 6 / mL, the survival rate reaches 95-99% for transfection, fresh cell expression medium, preheated to 37 ° C, the cells are diluted to a final density of 5 × 10 6 / mL, culture at 37℃ and 8% CO2 in a 50mm amplitude incubator at 90rpm. Use OPti-PRO SFM medium to prepare transfection reagent and plasmid DNA complex (4℃). For example: prepare 40μl OPti-PRO SFM for 1ml of cells, add 1ug of plasmid, mix and let stand for 5min; prepare 40μl OPti-PRO SFM, add 6μg PEI reagent, mix and let stand for 5min. Mix equal volumes of plasmid and transfection reagent, incubate at room temperature for 1-5 minutes, then slowly transfer the solution to a shake flask and shake, gently shake the shaker during the addition process. Culture the cells in a 50mm amplitude incubator at 90rpm and 37℃ and 8% CO2. 18-22 hours after transfection, add feed and perform the standard experimental plan. Example: Add 0.2ml to 1ml of cells Advanced CHO Feed 1, cells were cultured in a 50 mm amplitude incubator at 90 rpm at 37°C, 8% CO2. Six days after transfection, cells were harvested for subsequent purification.

[0119] Protein purification

[0120] The culture medium was centrifuged, the supernatant was added to the Ni column, incubated with shaking for 2 hours, and then purified by affinity chromatography through a gravity empty column. Equilibration buffer: "PBS", pH7.4, wash 10CV; wash buffer: "PBS", pH7.4, containing 20mM imidazole, wash 10CV; elution buffer: "PBS", pH7.4, containing 500mM imidazole, elution 1CV, repeated 5 times. SDS-PAGE & Western Blot profiles of different candidate antigens are shown below. Figure 2-14 shown.

[0121] Example 4 Preparation of recombinant protein vaccine

[0122] Each recombinant protein obtained in Example 3 was diluted to 80 μg / ml with 1×PBS buffer and fully mixed with an equal volume of AS03 adjuvant to prepare a vaccine product, wherein the AS03 adjuvant component includes 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of polysorbate 80, 3.53 mg of sodium chloride, 0.09 mg of potassium chloride, 0.51 mg of disodium hydrogen phosphate, 0.09 mg of potassium dihydrogen phosphate and water for injection per 0.5 ml.

[0123] Example 5 Preparation of recombinant protein vaccine

[0124] Each recombinant protein obtained in Example 3 was diluted to 80 μg / ml with 1×PBS buffer and fully mixed with an equal volume of MF59 adjuvant to prepare a vaccine product, wherein the MF59 adjuvant is a citric acid buffer solution containing 1% squalene, 0.5% Tween 80 and 0.5% Span 85.

[0125] Example 6 Preparation of recombinant protein vaccine

[0126] Each recombinant protein obtained in Example 3 was diluted to 80 μg / ml with 1×PBS buffer and fully mixed with an equal volume of aluminum adjuvant to prepare a vaccine product, wherein the aluminum adjuvant was 2 mg / ml Al(OH)3.

[0127] Example 7 Preparation of recombinant protein vaccine

[0128] Each recombinant protein obtained in Example 3 was diluted to 40 μg / ml with 1×PBS buffer and fully mixed with an equal volume of AS01 adjuvant to prepare a vaccine product, wherein the AS01 adjuvant component includes 50 μg of MPL, 50 μg of QS21, 1 mg of DOPC and 0.25 mg of cholesterol per 0.5 ml.

[0129] Example 8 Recombinant protein vaccine mouse immunization experiment

[0130] 8-10 week old BALB / c mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were immunized in groups using the vaccine prepared in Example 4-7, with 5 mice in each group. Mice were immunized on days 0 and 21, with 100 μl of immune sample (containing 8 μg antigen) injected intramuscularly each time, and blood was collected on days 0, 21, and 14 days after the second immunization. The collected blood samples were placed at 37°C for 1 hour, 4°C for 1 hour, centrifuged at 8000 r / min for 10 minutes, and serum was collected and stored at -20°C for pseudovirus neutralization detection.

[0131] Example 9 Neutralization experiment of the same strain RBD combination against different types of pseudoviruses

[0132] A pseudovirus was constructed using the SARS-CoV-2 S protein containing the mutation sites of each strain, and the serum isolated after immunization was detected using the SARS-CoV-2 pseudovirus neutralizing antibody detection method (chemiluminescence method) based on the VSV system.

[0133] In terms of adjuvants, the pseudovirus neutralizing antibody titers measured when mice were immunized with RBD(Beta)-RBD(Beta) as antigen and combined with different adjuvants are shown in Table 2.

[0134] Table 2

[0135]

[0136] The results showed that when AS03 adjuvant was used, the neutralizing antibody titers against pseudoviruses of D614G, Beta and Omicron BA.1 strains were significantly higher than those when Al(OH)3 adjuvant was used. Other experiments showed that when MF59 adjuvant was used, the antibody titers were similar to those when AS03 adjuvant was used. In subsequent experiments, the present invention used AS03 adjuvant.

[0137] In terms of antigens, the present invention first investigated the neutralizing antibody titers of candidate antigens composed of the same strain RBD against different strains. The neutralizing antibody titers and geometric mean titers (GMT) of the five serum samples in each group are shown in Table 3.

[0138] Table 3

[0139]

[0140] The results showed that the candidate antigen (SEQ ID NO: 18) formed by the combination of RBDs of the WH01 strain had a high geometric mean titer against the WH01 strain and D614G strain pseudoviruses, but the geometric mean titer against the Delta strain decreased, and the geometric mean titer against the OmicronBA.1 strain pseudovirus decreased significantly. In contrast, the candidate antigen (SEQ ID NO: 21) formed by the combination of RBDs of the Delta strain had a high geometric mean titer only against the Delta strain pseudovirus, and the geometric mean titer against the WH01 strain, D614G strain and Omicron BA.1 strain pseudovirus decreased significantly. This shows that when the RBD comes from the same strain, the candidate antigen has poor cross-protection ability against different strains, which is consistent with the published research.

[0141] In further research, the present invention screened the RBD of a specific strain as a preferred antigen component, which can increase the antibody titer against non-self strains to obtain a more balanced protection effect. This strain is the immunodominant strain described in the present invention.

[0142] Example 10 Neutralization experiment of different strains of RBD combinations against different types of pseudoviruses

[0143] In this example, the method of Example 9 was used to detect the neutralizing antibody titers of different strains of RBD combinations against different types of pseudoviruses.

[0144] (1) RBD screening of immunodominant strains

[0145] When one of the RBDs derived from the Delta variant in RBD(Delta)-RBD(Delta) (SEQ ID NO:21) was replaced with an RBD derived from the Beta variant or the WH01 strain, the changes in the neutralizing antibody titer and geometric mean titer GMT of the 5 serum samples in each group are shown in Table 4.

[0146] Table 4

[0147]

[0148] It can be seen that the average geometric mean titer of SEQ ID NO: 21 against the four pseudoviruses reached 46948, and the geometric mean titer against the Delta pseudovirus was the largest, reaching 101399, which was about 2.2 times the average value. The geometric mean titer against the Omicron BA.1 pseudovirus was only 22267, which was about 47% of the average value.

[0149] Compared with SEQ ID NO:21, the geometric mean titer of the candidate antigen (SEQ ID NO:20) formed by the combination of the RBDs of the Beta strain and the Delta strain against the Delta strain pseudovirus was slightly decreased, but the geometric mean titers against the D614G strain and the Omicron BA.1 strain pseudovirus were increased by 2 times and 1 times, respectively. The average geometric mean titer against the four pseudoviruses reached 65462, and the geometric mean titer against the D614G strain pseudovirus was the largest, reaching 92502, which was about 1.4 times the average value. The geometric mean titer against the Omicron BA.1 strain pseudovirus reached 50785, which was about 78% of the average value.

[0150] Compared with SEQ ID NO:21, the geometric mean titer of the candidate antigen (SEQ ID NO:19) formed by the combination of the RBD of the WH01 strain and the Delta strain against the WH01 and D614G strain pseudoviruses did not change much, but the geometric mean titer against the Delta strain and the Omicron BA.1 strain pseudoviruses was reduced by more than 50%. The average geometric mean titer against the four pseudoviruses was only 29505, and the geometric mean titer against the Delta strain pseudovirus was the largest, reaching 42932, which was about 1.5 times the average value. The geometric mean titer against the Omicron BA.1 strain pseudovirus was only 10520, which was about 36% of the average value.

[0151] Therefore, in general, the geometric mean titer of SEQ ID NO: 20 against WH01, D614G, Delta and Omicron BA.1 pseudoviruses is more balanced, and the RBD of the Beta strain can increase the neutralizing antibody titer of the antigen against non-Beta strain variants, so the Beta strain can be considered as an immunodominant strain.

[0152] The present invention further studies the effect of the combination of different strains of RBD on the neutralizing antibody titer when forming Foldon trimers. The neutralizing antibody titers and geometric mean titers GMT of 5 serum samples of NTD-RBD (BA.2) -RBD (BA.1) -Foldon (SEQ ID NO: 60) and NTD-RBD (BA.2) -RBD (WH01) -Foldon (SEQ ID NO: 61) against different pseudoviruses are shown in Table 5.

[0153] Table 5

[0154]

[0155] It can be seen that NTD-RBD (BA.2) -RBD (BA.1) -Foldon has a very high neutralization titer against BA.2 pseudovirus, reaching 19802, and also has a certain antibody titer against BA.4 pseudovirus, but the antibody titer against D614G and BA.1 is very low.

[0156] When the RBD from the Omicron-BA.1 variant was replaced with the RBD from the WH01 strain, not only the neutralization titer against the BA.2 pseudovirus increased, but also the antibody titer against the D614G and BA.4 pseudoviruses increased significantly. However, the antibody titer against BA.1 was lower, which may be due to the large difference between BA.1 and BA.2 and BA.4. Published literature has also shown that the antigenicity of BA.1 and BA.2 is significantly different (see Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1 and BA.2 are antigenically distinct, ANNA Z.MYKYTY Net al. SCIENCE IMMUNOLOGY, 23Jun2022).

[0157] The above results indicate that, when forming Foldon trimers, the RBD of the WH01 strain can increase the neutralizing antibody titer of the antigen against different variants. Therefore, in this case, the WH01 strain can also be considered an immunodominant strain.

[0158] Different from this, further research of the present invention shows that when antigen components are selected from the receptor binding regions of the Alpha strain, Gamma strain, Epsilon, Zeta strain, Eta strain, Theta strain, Iota strain, Kappa strain, Lambda strain, and Mu strain, the obtained immunogenic substances only improve the titer of the corresponding strain pseudovirus, but have no obvious effect on the titer of other mutant strain pseudoviruses, which indicates that the receptor binding regions of the above strains are weak in inducing antibody responses to other strains and are not the immunodominant strains described in the present invention. However, these can be used as additional components of antigens to further improve the balance of the protective effect of the vaccine against different strains.

[0159] Existing studies have shown that when the receptor binding region of a certain strain is present in the immunogenic substance, the titer of the immunogenic substance against the pseudovirus of the strain will be significantly improved. In combination with the epidemic trend of the new coronavirus, the present invention uses the Delta strain or the Omicron strain as the epidemic dominant strain. The experimental study of the present invention shows that when the immunogenic substance contains the receptor binding region derived from the immunodominant strain and the epidemic dominant strain at the same time, it can show a higher titer for the pseudovirus of different strains, indicating that it can produce excellent immune effects on different strains.

[0160] (2) Effect of adding NTD

[0161] The present invention further investigates the effect of adding NTD to the candidate antigen. After adding NTD, the neutralizing antibody titers and geometric mean titers (GMT) of the five serum samples in each group against different pseudoviruses are shown in Table 6.

[0162] Table 6

[0163]

[0164]

[0165] As shown in Table 6, when the RBD is from the same strain, compared with SEQ ID NO:18 and SEQ ID NO:21, the geometric mean titers of the candidate antigen SEQ ID NO:45 formed by the combination of NTD and RBD of the WH01 strain and the candidate antigen SEQ ID NO:46 formed by the combination of NTD and RBD of the Delta strain against different pseudoviruses were improved to varying degrees, and the average values ​​of the geometric mean titers against the four pseudoviruses increased from 38492 and 46948 to 53563 and 55471, respectively.

[0166] When the RBD comes from different strains, compared with SEQ ID NO:19, the candidate antigen SEQ ID NO:22 formed by the combination of the NTD and RBD of the WH01 strain and the RBD of the Delta strain has significantly improved geometric mean titers against different pseudoviruses, and the average geometric mean titers against four pseudoviruses increased from 29505 to 67293; compared with SEQ ID NO:20, the candidate antigen SEQ ID NO:23 formed by the combination of the NTD and RBD of the Beta strain and the RBD of the Delta strain has reduced geometric mean titers against the D614G strain and the Delta strain pseudovirus, but still maintained at a high level, while the geometric mean titers against the WH01 strain and the Omicron BA.1 strain pseudovirus have increased, especially the geometric mean titer against the Omicron BA.1 strain pseudovirus has reached the highest value among these combinations. Therefore, in general, the addition of NTD can improve the cross-protection ability.

[0167] (3) Effect of adding Foldon domain

[0168] The present invention further investigates the effect of adding Foldon domain to the candidate protein to form a trimer. After adding the Foldon domain, the neutralizing antibody titers and geometric mean titers GMT of 5 serum samples in each group against different pseudoviruses are shown in Table 7.

[0169] Table 7

[0170]

[0171] As shown in Table 7, compared with SEQ ID NO: 19, after the Foldon domain (SEQ ID NO: 26) or the NTD and Foldon domain (SEQ ID NO: 27) were further added to the RBD combination of the WH01 strain and the Delta strain to form a trimer, the geometric mean titers against different pseudoviruses were significantly improved, and the average geometric mean titers against the four pseudoviruses increased from 29505 to 63178 and 53995, respectively, indicating that the formation of a trimer can improve the cross-protection ability.

[0172] It can be seen that the antigen formed by the combination of RBDs of the immune dominant strain and the epidemic dominant strain described in the present invention has a more balanced immune effect on different strains, and the immune effect can be further improved after further adding NTD or Foldon domain.

[0173] Example 11 Preparation of mRNA

[0174] In this example, mRNAs of the antigens shown in Table 8 were prepared.

[0175] Table 8

[0176]

[0177] A tag DYKDDDDKHHHHHHHH was added to the C-terminus of the above antigen, the DNA sequence encoding the antigen was codon-optimized according to humans as the host, and a T7 RNA polymerase binding sequence, a 5'UTR (5'UTR of human β-globin), a kozak sequence and a signal peptide were added in sequence at the 5' end, and a 3'UTR (5'UTR of human β-globin) and PloyA (120) and a restriction site were added in sequence at the 3' end to obtain a DNA sequence.

[0178] The DNA sequence was synthesized and cloned into the expression vector pUC57-kan, and the target gene was verified by sequencing.

[0179] The expression vector was transformed into E. coli competent cells, and the plasmid was extracted after expansion culture. The linearized plasmid sample was obtained by enzyme digestion, and in vitro transcription was performed using T7 High Yield RNA Transcription Kit (Novoprotein, CAT: E131-01A) according to the instructions. mRNA capping was performed using Cap1 Capping System (Novoprotein, CAT: M082) according to the instructions. Finally, mRNA was purified by lithium chloride precipitation purification method.

[0180] The prepared mRNA sequences are SEQ ID NOs: 68-73, wherein,

[0181] Positions 123-2750 of SEQ ID NO:68 are the antigen coding region,

[0182] Positions 123-1436 of SEQ ID NO:69 are the antigen coding region,

[0183] Positions 123-1436 of SEQ ID NO:70 are the antigen coding region,

[0184] Positions 123-1436 of SEQ ID NO:71 are the antigen coding region,

[0185] Positions 123-2093 of SEQ ID NO:72 are the antigen coding region,

[0186] Positions 123-1436 of SEQ ID NO:73 are the antigen coding region.

[0187] Example 12 Preparation of mRNA vaccine

[0188] The mRNAs obtained in Example 11 were mixed with lipid nanoparticles to obtain nucleic acid-lipid nanoparticle complexes, wherein the lipid nanoparticles contained DOTMA and DOPE in a molar ratio of 1:1; the content of mRNA in the complex was 100 μg / ml, and the mass ratio of lipid nanoparticles to mRNA was 10:1.

[0189] Example 13 mRNA vaccine mouse immunization experiment

[0190] 8-10 week old BALB / c mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were immunized in groups using each mRNA vaccine obtained in Example 12, with 5 mice in each group. Mice were immunized on days 0 and 21, with 50 μl of immune samples (containing 5 μg mRNA) injected intramuscularly each time, and blood was collected on days 0, 21, and 14 days after the second immunization. The collected blood samples were placed at 37 ° C for 1 hour, 4 ° C for 1 hour, centrifuged at 8000r / min for 10 minutes, serum was collected, and stored at -20 ° C for pseudovirus neutralization detection. The neutralizing antibody titers and geometric mean titer GMT results of 5 serum samples in each group for different pseudoviruses are shown in Table 9.

[0191] Table 9

[0192]

[0193] It can be seen that the average GMT value of the neutralizing antibody titer against different pseudoviruses of the vaccine prepared by mRNA encoding RBD(Beta)-RBD(Beta) (SEQ ID NO:73) is 31133, and the neutralizing antibody titer against the Omicron strain is significantly lower than that of other strains. In comparison, the average GMT value of the neutralizing antibody titer against different pseudoviruses of the vaccine prepared by mRNA encoding RBD(WH01)-RBD(Beta)-RBD(Delta)-RBD(BA.1) (SEQ ID NO:68) reaches 99781, the average GMT value of the neutralizing antibody titer against different pseudoviruses of the vaccine prepared by mRNA encoding RBD(WH01)-RBD(BA.1) (SEQ ID NO:69) and mRNA encoding RBD(Beta)-RBD(Delta) (SEQID NO:70) reaches 58513, and the average GMT value of the neutralizing antibody titer against different pseudoviruses of the vaccine prepared by mRNA encoding RBD(Beta)-RBD(BA.1) (SEQ ID The average GMT of the neutralizing antibody titer of the vaccine prepared by mRNA encoding RBD(Beta)-RBD(WH01)-RBD(Delta) (SEQ ID NO:71) against different pseudoviruses reached 44561, and the average GMT of the neutralizing antibody titer of the vaccine prepared by mRNA encoding RBD(Beta)-RBD(WH01)-RBD(Delta) (SEQ ID NO:72) against different pseudoviruses reached 67155, and the neutralizing antibody titers of these vaccines against the Omicron strain increased several times.

[0194] It can be seen that the mRNA vaccine prepared by using the mRNA encoding the immunogenic substance of the present invention also has a good immune effect on different strains. It can be expected that the vaccine prepared by the immunogenic substance of the present invention and the nucleic acid encoding the immunogenic substance will still have a good preventive effect against mutant strains of the new coronavirus that may appear in the future. Therefore, in the case of continuous mutations of the new coronavirus, the present invention has important guiding significance.

[0195] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A novel coronavirus immunogenic substance, characterized in that: The novel coronavirus immunogenic substance comprises a first antigen derived from an immunodominant strain and a second antigen derived from a prevalent dominant strain, each antigen comprising a receptor binding region of an S protein, wherein the immunodominant strain is selected from at least one of the novel coronavirus WH01 strain or the Beta strain, and the prevalent dominant strain is selected from at least one of the novel coronavirus Delta strain, Omicron BA.1, BA.2, BA.3, BA.4 and BA.5 variants, and the amino acid sequence of the novel coronavirus immunogenic substance is as shown in any one of SEQ ID NO: 19, 20, 47, 48, 51, 55.

2. A novel coronavirus immunogenic substance, characterized in that: The novel coronavirus immunogenic substance comprises a first antigen derived from an immunodominant strain and a second antigen derived from a prevalent dominant strain, each antigen comprising a receptor binding region of an S protein, wherein the immunodominant strain is selected from at least one of the novel coronavirus WH01 strain or the Beta strain, and the prevalent dominant strain is selected from at least one of the novel coronavirus Delta strain, Omicron BA.1, BA.2, BA.3, BA.4 and BA.5 variants, and the novel coronavirus immunogenic substance further comprises an N-terminal domain (NTD) and / or a foldon domain in the S1 subunit of the S protein, and the amino acid sequence of the novel coronavirus immunogenic substance is as shown in any one of SEQ ID NOs: 22, 23, 26, 27, and 61.

3. The novel coronavirus immunogenic substance according to claim 1 or 2, characterized in that: The novel coronavirus immunogenic material also contains one or more antigens derived from strains other than the immunodominant strain and the epidemic dominant strain.

4. The novel coronavirus immunogenic substance according to claim 3, characterized in that: The strains other than the immunodominant strains and the epidemic dominant strains are selected from the following strains: Alpha strain, Gamma strain, Epsilon strain, Zeta strain, Eta strain, Theta strain, Iota strain, Kappa strain, Lambda strain and Mu strain.

5. The novel coronavirus immunogenic substance according to claim 4, characterized in that: The antigens constitute a composite, or the antigens are directly linked or linked via an amino acid linker.

6. A method for preparing the novel coronavirus immunogenic substance according to any one of claims 1 to 5, characterized in that: The following steps are involved: Constructing a recombinant expression plasmid using the nucleotide sequence encoding the novel coronavirus immunogenic substance; The constructed recombinant expression plasmid was transformed into the host bacteria, and the correct recombinant expression plasmid was screened. The screened recombinant expression plasmid is used to transfect the cells of the expression system. After expression, the supernatant is collected and purified to obtain the new coronavirus immunogenic substance.

7. The method according to claim 6, characterized in that The cells of the expression system include mammalian cells, insect cells, yeast cells or bacterial cells.

8. The method according to claim 7, characterized in that The mammalian cells include 293T cells or CHO cells, and the bacterial cells include Escherichia coli cells.

9. A nucleotide encoding the novel coronavirus immunogenic substance according to any one of claims 1 to 5.

10. A recombinant vector comprising the nucleotide sequence of claim 9.

11. An expression system cell carrying the recombinant vector according to claim 10.

12. Use of the novel coronavirus immunogenic substance according to any one of claims 1 to 5, the nucleotide according to claim 9, the recombinant vector according to claim 10, or the expression system cell according to claim 11 in the preparation of a novel coronavirus vaccine.

13. A novel coronavirus protein vaccine, characterized in that: It comprises the novel coronavirus immunogenic substance as described in any one of claims 1 to 5 and an adjuvant, wherein the adjuvant is selected from one or more of aluminum adjuvant, MF59 adjuvant, MPL adjuvant, QS-21, GLA, CpG, AS01, AS02, AS03, and AS04 adjuvant.

14. The novel coronavirus protein vaccine according to claim 13, characterized in that: The adjuvant is AS03 or MF59 adjuvant.

15. A novel coronavirus DNA vaccine, characterized in that: The DNA vaccine comprises a DNA sequence encoding the immunogenic substance of the new coronavirus described in any one of claims 1-5.

16. A novel coronavirus mRNA vaccine, characterized in that: The mRNA vaccine comprises an mRNA sequence encoding the new coronavirus immunogenic substance described in any one of claims 1-5.

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