CN118126138A

CN118126138A - Mutant of novel coronavirus RBD protein and application thereof

Info

Publication number: CN118126138A
Application number: CN202311786421.7A
Authority: CN
Inventors: 陈仙莹
Original assignee: Huzhou Weikexin Biotechnology Co ltd
Current assignee: Huzhou Weikexin Biotechnology Co ltd
Priority date: 2023-12-22
Filing date: 2023-12-22
Publication date: 2024-06-04

Abstract

The invention discloses a mutant of a novel coronavirus RBD protein and application thereof. Through scientific experimental design and a large number of detection, the invention modifies RBD region of wild S protein, provides RBD mutant with better immune effect and stronger broad spectrum aiming at SARS-CoV-2 variant strain, detects immunogenicity of the mutant at subunit protein and mRNA vaccine level, and proves that the mutant can induce broad spectrum anti-novel coronavirus neutralizing antibody in vivo and can provide strong and broad spectrum anti-novel coronavirus protection effect.

Description

Mutant of novel coronavirus RBD protein and application thereof

Technical Field

The invention relates to the fields of virology and immunology, in particular to a novel mutant of a coronavirus RBD protein and application thereof.

Background

Pneumonia (COVID-19) caused by novel coronavirus infection is an infectious acute respiratory disease caused by novel coronavirus, and mainly comprises fever, dry cough, hypodynamia and the like, wherein dyspnea occurs in severe cases, serious illness state is critical, and even death occurs.

At present, no specific treatment method is adopted for the pneumonia caused by the novel coronavirus infection, and the transmission path is cut off by a separation means, so that the disease support treatment is mainly adopted. Vaccines are the most effective means of controlling infectious diseases. The existing new crown vaccine types comprise inactivated vaccines, attenuated live vaccines, recombinant protein vaccines, viral vector vaccines, nucleic acid vaccines and the like. The current domestic approved technical routes for using the marketed vaccines relate to inactivated vaccines, recombinant protein vaccines, adenovirus vector novel crown vaccines and mRNA vaccines.

The S protein of SARS-CoV-2 effects viral invasion by binding to the ACE2 receptor on the host cell, and comprises an S1 subunit and an S2 subunit, wherein the S1 subunit comprises primarily a receptor binding domain (Recept or binding domain, RBD) responsible for recognizing the receptor of the cell. S2 contains the essential elements required for the membrane fusion process. The Receptor Binding Domain (RBD) is effective in inducing the production of nAb in vivo, and thus SARS-CoV-2RBD can be used as a good immunogen for inducing antibody production.

With the advent of new crown variants, the protective effect provided by current vaccines has been significantly reduced, as demonstrated in the several waves COVID-19 pandemic caused by Alpha, beta, iota, kappa, delta and Omicron variants. Omicron variants have replication advantages over previous variants and are able to evade humoral immunity induced by infection and vaccine to a greater extent. These SARS-CoV-2 variants continue to threaten global public health, and the development of more effective, broader-spectrum vaccines is critical to control the pneumonia pandemic caused by new coronavirus infection and to prevent breakthrough infections of post-vaccination variants. The specific vaccines developed to date against new crown variants are mostly direct combinations of variant antigens, the key sites determining immunogenicity of which are not yet clear. Thus, there is an urgent need for a new generation of broad spectrum SARS-CoV-2 vaccine with accurate and clear mutation sites against variants including Omikovia.

Disclosure of Invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a novel mutant of the receptor binding domain of the coronavirus S protein. Through scientific experimental design and a large number of detection, the invention modifies RBD region of wild S protein, provides RBD mutant with better immune effect and stronger broad spectrum aiming at SARS-CoV-2 variant strain, detects immunogenicity of the mutant at subunit protein and mRNA vaccine level, proves that the mutant can induce broad spectrum anti-novel coronavirus neutralizing antibody in vivo, and can provide strong broad spectrum anti-novel coronavirus protection effect.

In order to achieve the above purpose, the invention adopts the following technical scheme:

In a first aspect, the present invention provides a novel mutant of the receptor binding domain RBD of the S protein of coronavirus, said mutant of the receptor binding domain RBD of the S protein being mutated from 319-541aa of the sequence shown in SEQ ID NO.1, at least at amino acid residue positions 417, 452, 484 and 501 of SEQ ID NO. 1; and compared with the receptor binding domain RBD of the S protein shown in SEQ ID NO.1, the novel anti-coronavirus neutralizing antibody can induce a broad spectrum in vivo.

In certain embodiments, the mutation site further comprises amino acid residue 478.

In certain preferred embodiments, the mutation site further comprises amino acid residue 440.

According to an embodiment of the invention, the amino acid sequence of the mutant of the receptor binding domain RBD of the novel coronavirus S protein is mutated at one or more sites selected from the group consisting of the amino acid residues shown below:

417. Asn or Thr;

lys at position 440;

arg at position 452;

478 th Lys;

Lys at position 484;

501 th bit Tyr.

In a specific embodiment, the mutant of the receptor binding domain RBD of the novel coronavirus S protein is selected from any one of the following (a) - (c):

(a) The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO 5,6,8 or 9;

Or (b)

(B) The mutant of RBD has 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence of (a), and has the function of the mutant of RBD of (a), wherein the amino acid residue at position 417, 440, 452, 478, 484 or 501 corresponding to the amino acid sequence shown in SEQ ID NO. 1 is identical to that of the amino acid sequence of (a);

Or (b)

(C) The mutant of RBD consists of adding or deleting 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, most preferably 1 to 3 amino acid residues at the C-terminal and/or N-terminal of the amino acid sequence of (a), and has the function of the mutant of RBD of (a), wherein the amino acid residue at position 417, 440, 452, 478, 484 or 501 corresponding to the amino acid sequence shown in SEQ ID NO. 1 is the same as that of the amino acid sequence of (a).

In a second aspect, the present invention provides a recombinant protein comprising a mutant of the receptor binding domain RBD of the novel coronavirus S protein of the first aspect, further comprising at least one of the following modifications based on the amino acid sequence of the mutant:

Modification 1: replacing or adding protein tag, adding or replacing hFc protein tag with one of mFc tag, his6 tag, avi tag, MBP tag, etc.;

Modification 2: the RBD regions are truncated at the N-or C-terminus, respectively or simultaneously, but still contain the amino acid sequence of the respective mutation site.

In a third aspect, the present invention provides a polynucleotide encoding a mutant of the receptor binding domain RBD of the novel coronavirus S protein of the first aspect or the recombinant protein of the second aspect.

In a fourth aspect, the present invention provides an expression vector comprising a polynucleotide according to the third aspect of the present invention.

In a fifth aspect, the present invention provides a host cell comprising the expression vector according to the fourth aspect of the invention or a polynucleotide having integrated in its genome a mutant of the receptor binding domain RBD of the novel coronavirus S protein according to the third aspect of the invention.

In a sixth aspect, the present invention provides a linearized mRNA obtained by transcription of a polynucleotide according to the third aspect.

In a seventh aspect, the present invention provides a pharmaceutical composition (e.g. a vaccine) comprising a mutant of the receptor binding domain RBD of the novel coronavirus S protein of the first aspect or the recombinant protein of the second aspect or the polynucleotide of the third aspect or the expression vector of the fourth aspect or the host cell of the fifth aspect or the linearized mRNA of the sixth aspect.

In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In an eighth aspect, the present invention provides the use of a mutant of the receptor binding domain RBD of the novel coronavirus S protein according to the first aspect or the recombinant protein according to the second aspect or the polynucleotide according to the third aspect or the expression vector according to the fourth aspect or the host cell according to the fifth aspect or the linearized mRNA according to the sixth aspect or the pharmaceutical composition according to the seventh aspect for the preparation of a medicament for the prevention or treatment of a novel coronavirus infection or a disease caused by a novel coronavirus infection.

In certain embodiments, the drug is a subunit vaccine or an mRNA vaccine.

In certain embodiments, the novel coronavirus is the novel coronavirus mutant SARS-CoV-2D614G, omicron ba.1 and Omicron ba.2.

In a ninth aspect, the present invention provides a method of preventing or treating a novel coronavirus infection or a disease caused by a novel coronavirus infection in a subject, comprising administering to the subject a prophylactically or therapeutically effective amount of the mutant of the first aspect or the recombinant protein of the second aspect or the pharmaceutical composition of the seventh aspect.

In a tenth aspect, the present invention provides a method of preparing a vaccine, the method comprising: mixing the mutant of the first aspect, or the recombinant protein of the second aspect, the polynucleotide of the third aspect, or the linearized mRNA of the sixth aspect with a pharmaceutically acceptable carrier and/or excipient (e.g., aluminum adjuvant, LNP), and/or additional active ingredient.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA, etc., as used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the terms "native RBD", "wild S protein RBD", "WT-RBD" refer to the biologically active, naturally occurring receptor binding domain of S protein (Receptor binding domain, RBD), which have the same meaning and are used interchangeably. The amino acid sequences of the RBD of the native S protein or the RBD of the wild S protein can be conveniently obtained from various public databases (e.g., NCBI' S GenBank database). For example, the invention obtains SARS-CoV-2Spike protein amino acid sequence (protein_id= "YP_ 009724390.1") from NCBI database, which is described using the sequence shown in SEQ ID NO:1, the 319-541 segment of this amino acid sequence being the natural RBD protein amino acid sequence of SARS-CoV-2 described in the invention (referred to as WT-RBD in the invention), as shown in SEQ ID NO: 2.

As used herein, when referring to the amino acid sequence of a wild RBD, it uses the amino acid sequence of SEQ ID NO:2, and a sequence shown in the following. For example, the expression "amino acid residue 140 of wild-type RBD" refers to the amino acid sequence of SEQ ID NO:2 at amino acid residue 140. However, it is understood by those skilled in the art that wild RBDs may have multiple versions that have substantially the same primary structure (i.e., amino acid sequence) and higher structure (i.e., spatial structure), as well as substantially the same biological function, but that may still differ slightly in amino acid sequence from one another. Thus, in the present application, the wild RBD is not limited to SEQ ID NO:2, but is intended to cover all known wild-type RB D. Thus, in the present application, the term "wild RBD" shall include various naturally occurring, biologically functional RBDs, including, for example, the RBDs of SEQ ID NO:2 and naturally occurring variants thereof. Also, when describing the amino acid position of RBD, it includes not only SEQ ID NO:2, and further includes an amino acid position in a natural variant thereof that corresponds to the particular amino acid position. For example, the expression "amino acid residue 140 of wild-type RBD" includes SEQ ID NO:2, and the corresponding amino acid position in its natural variant. According to the application, the expression "corresponding amino acid position" refers to an amino acid position in the sequences being compared which is located at an equivalent position when optimally aligned, i.e. when the sequences are aligned to obtain the highest percentage identity. Similarly, the expression "position corresponding to position 140 of SEQ ID NO. 2" means that when optimally aligned with SEQ ID NO. 2, i.e. when aligned with SEQ ID NO. 2 to obtain the highest percent identity, the amino acid position in the sequence compared is located at the position equivalent to position 140 of SEQ ID NO. 2.

In certain preferred embodiments, the wild RBD has an amino acid sequence set forth in SEQ ID NO. 2. In certain preferred embodiments, the wild-type RBD is a naturally occurring, biologically functional receptor binding domain, and has an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO. 2. In certain preferred embodiments, the wild-type RBD is a naturally occurring, biologically functional receptor binding domain, and has an amino acid sequence that differs from SEQ ID NO. 2 by one or more (e.g., 1-10 or 1-5 or 1-3) amino acid differences (e.g., conservative amino acid substitutions).

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CA GGTT share 50% identity (3 position matches out of 6 positions total). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithm of E.Meyers and W.Miller (Comput. ApplBiosci.,4:11-17 (1988)) which has been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available on www.gcg.com) using the Blossum 62 matrix or PAM250 matrix and the GAP weights (GAP WEIGHT) of 16, 14, 12, 10, 8, 6 or 4 and the length weights of 1, 2, 3, 4, 5 or 6.

As used herein, the term "mutant" refers to a polynucleotide or polypeptide comprising an alteration (i.e., substitution, insertion, and/or deletion) at one or more (e.g., several) positions relative to a "wild-type" or "comparable" polynucleotide or polypeptide, wherein a substitution refers to a substitution of a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletions refer to the removal of a nucleotide or amino acid occupying a position. Insertion refers to the addition of a nucleotide or amino acid following the nucleotide or amino acid that abuts and immediately occupies the position. Those skilled in the art know that if a mutation is to be made to an enzyme in order to obtain a mutant with improved activity, it is critical that the site of activity be improved after the mutation is found. In the invention, mutation is carried out on a specific site of 319-541 section in the amino acid sequence of wild SARS-CoV-2Spike protein with the amino acid sequence shown as SEQ ID NO.1, thus obtaining RBD mutant with better immune effect and stronger broad spectrum aiming at SARS-CoV-2 variant strain.

In a specific embodiment, the RBD mutant obtained by the present inventors through mutation at the following multiple sites of the amino acid sequence shown in SEQ ID NO. 1 has better immune effect and stronger broad spectrum against SARS-CoV-2 variant: 417 bits, 440 bits, 452 bits, 478 bits, 484 bits, 501 bits.

In view of this, in a specific embodiment, the amino acid sequence of the RBD mutant of the invention has the amino acid residues shown below at a plurality of positions selected from the group consisting of:

417. Asn or Thr;

lys at position 440;

arg at position 452;

478 th Lys;

Lys at position 484;

501 th bit Tyr.

In a specific embodiment, the RBD mutant has an amino acid sequence set forth in any one of SEQ ID NOs 5,6,8 or 9.

In view of the teachings of the present invention and the prior art, it will also be appreciated by those skilled in the art that the "RBD of the present invention" shall also include variants thereof which have the same or similar function as the "RBD of the present invention" but which differ in amino acid sequence by a small amount from the amino acid sequence of the RBD in the examples of the present invention. These variants include (but are not limited to): deletion, insertion, and/or substitution of one or more (usually 1 to 30, preferably 1 to 10, more preferably 1 to 6, still more preferably 1 to 3, most preferably 1) amino acids, and addition of one or more (usually 30 or less, preferably 10 or less, more preferably 6 or 3 or less) amino acids at the C-terminal and/or N-terminal. For example, it is well known to those skilled in the art that substitution with amino acids having similar or similar properties, e.g., isoleucine to leucine, does not alter the function of the resulting protein. As another example, the addition of one or several amino acids at the C-terminal and/or N-terminal, e.g., a 6 XHis tag added for ease of isolation, will not typically alter the function of the resulting protein.

The term "corresponding to" as used herein has a meaning commonly understood by one of ordinary skill in the art. Specifically, "corresponding to" means that two sequences are aligned by homology or sequence identity, and that one sequence corresponds to a specified position in the other sequence. Thus, for example, in the case of "amino acid residue corresponding to position 417 of the amino acid sequence shown in sequence 1", if a 6×hi s tag is added to one end of the amino acid sequence shown in sequence 1, position 417 of the resulting mutant corresponding to the amino acid sequence shown in sequence 1 may be position 423.

As used herein, the term "protective antibody" refers to an antibody that has a protective effect against a virus. Protective antibodies include, but are not limited to, antibodies capable of neutralizing viral virulence, antibodies capable of inhibiting viral recognition and binding to host cells, and antibodies capable of inhibiting fusion of a virus with a host cell.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19^th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, e.g., pH modifiers include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, e.g., tween-80, adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvant (e.g., complete freund's adjuvant), ionic strength enhancers include, but are not limited to sodium chloride).

As used herein, the term "adjuvant" refers to a non-specific immunopotentiator that, when delivered with an antigen or pre-delivered into an organism, can enhance the organism's immune response to the antigen or alter the type of immune response. There are many adjuvants including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in current animal trials. Aluminum hydroxide adjuvants are used more in clinical trials. In the present invention, it is particularly preferred that the adjuvant is CpG+Al (OH) ₃.

As used herein, the term "effective amount" refers to an amount effective to achieve the intended purpose. For example, a prophylactically or therapeutically effective amount is an amount effective to prevent, or delay the onset of a disease (e.g., a coronavirus infection), or to alleviate, reduce, or treat the severity of an existing disease (e.g., a disease caused by a coronavirus infection). Determination of such effective amounts is within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

As used herein, the term "immunogenicity" (immunogenicity) refers to the ability of the body to be stimulated to form specific antibodies or sensitized lymphocytes. It refers to the characteristic that an antigen can stimulate a specific immune cell to activate, proliferate and differentiate the immune cell and finally produce immune effector substances such as antibodies and sensitized lymphocytes, and also refers to the characteristic that the immune system of an organism can form specific immune responses of the antibodies or sensitized T lymphocytes after the antigen stimulates the organism. Immunogenicity is the most important property of an antigen, whether an antigen can successfully induce an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the manner of immunization.

As used herein, "subject" refers to an animal, such as a vertebrate. In certain embodiments, the subject is a mammal, such as a human, bovine, equine, feline, canine, rodent, or primate. Particularly preferably, the subject is a human. The term is used interchangeably herein with "patient".

The terms "comprising," "having," "including," or "containing," as used herein, are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

As used herein, "about" means: one value includes the standard deviation of the error of the device or method used to determine the value.

As used herein, the term "or" is defined as only alternatives and "and/or" but, unless expressly indicated otherwise as only alternatives or as mutually exclusive between alternatives, the term "or" in the claims means "and/or".

Unless defined otherwise or clearly indicated by context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The invention has the beneficial effects that:

the mutant of RBD of S protein provided by the application can induce protective antibodies against different SARS-CoV-2 variants (for example, SARS-CoV-2D614G, delta, omicron BA.1 or Omicron BA.2) to realize the protection effect on different SARS-CoV-2 variants. The subunit vaccine and the mRNA vaccine prepared by the mutant can induce the mice to generate high-titer neutralizing antibodies in vivo, and show great application prospect as broad-spectrum novel coronavirus vaccine.

Drawings

FIG. 1 is a schematic representation of the mutation sites of various RBD muteins according to the present application;

FIG. 2 is a graph showing the results of the detection of neutralization of different mutant pseudoviruses of the new crown by antibodies in serum of BALB/C mice immunized with RBD mutants M2-RBD, M3-RBD, and M4-RBD of example 1; wherein A is SARS-CoV-2D614G pseudovirus; b is SARS-CoV-2Omicron BA.1 pseudovirus; c is SARS-CoV-2Omicron BA.2 pseudovirus;

FIG. 3 is a graph showing the results of the detection of neutralization of different mutant pseudoviruses of the new crown by antibodies in serum from RBD mutant M4T-RBD, M4A-RBD immunized BALB/C mice of example 2; wherein A is SARS-CoV-2D614G pseudovirus; b is SARS-CoV-2Omicron BA.1 pseudovirus; c is SARS-CoV-2Omicron BA.2 pseudovirus;

FIG. 4 is a graph showing the results of the detection of neutralization of different mutant pseudoviruses of the new crown by antibodies in serum from RBD mutant M5-RBD, M6K-RBD immunized BALB/C mice of example 3; wherein A is SARS-CoV-2D614G pseudovirus; b is SARS-CoV-2Omicron BA.1 pseudovirus; c is SARS-CoV-2Omicron BA.2 pseudovirus;

FIG. 5 is a graph showing the results of neutralizing the SARS-CoV-2Spike protein mutant M5-Spike and M5-RBD immunized BALB/C mice of example 4, and detecting antibodies in serum against different mutant pseudoviruses of the new crown; wherein A is SARS-CoV-2D614G pseudovirus; b is SARS-CoV-2Omicron BA.1 pseudovirus; c is SARS-CoV-2Omicron BA.2 pseudovirus;

FIG. 6 is a graph showing the results of the mRNA vaccines for WT-RBD, M5-RBD and M6K-RBD in example 5, immunization of BALB/C mice, and detection of neutralization of SARS-CoV-2D614G, SARS-CoV-2Omicron BA.1 and SARS-CoV-2Omicron BA.2 pseudoviruses by mouse serum.

Detailed Description

The invention is explained in detail below with reference to the drawings and the specific embodiments.

Unless specifically indicated, the following examples employ conventional methods well known in the art and described in various references.

In addition, the specific conditions not specified in the examples were either conventional conditions or conditions recommended by the manufacturer. The reagents or equipment used are conventional products available from commercial sources, and specific manufacturers are not identified.

EXAMPLE 1 preparation and immunogenicity detection of SARS-CoV-2RBD mutant recombinant protein

1. Molecular cloning design

The amino acid sequence of SARS-CoV-2Spike protein (protein_id= "YP_ 009724390.1"), i.e. the amino acid sequence of prototype Spike protein, obtained from NCBI database is shown as SEQ ID NO.1, 319-541 segment in the amino acid sequence is SARS-CoV-2RBD protein amino acid sequence in the present study (referred to as WT-RBD in the present invention), and the amino acid sequence of WT-RBD is shown as SEQ ID NO. 2.

The nucleotide sequence encoding the WT-RBD was determined by codon optimization and synthesized. After synthesizing the prototype strain sequence added with the hFc tag, designing a primer according to the target mutation site, synthesizing a required mutation combined fragment by a PCR technology, and connecting the mutation combined fragment to PCAGGS vectors by using HindIII and NotI enzyme cutting sites. The following plasmids were constructed together for a total of 4 combinations of mutations, including the prototype strain. K represents lysine, G represents glycine, L represents leucine, R represents arginine, T represents threonine, E represents glutamic acid, A represents alanine, N represents asparagine, Y represents tyrosine, and H represents histidine. Wherein, the position of the mutation site refers to the position of the site relative to the prototype Spike protein. Mutation sites are shown in FIG. 1.

(1) WT-RBD is prototype sequence, no mutation site is added, and its amino acid sequence is shown in SEQ ID NO. 2.

(2) Compared with SEQ ID NO.1, the 452 th amino acid L of the protein sequence is mutated into R, and the 478 th amino acid T is mutated into K. The mutated RBD protein is named as M2-RBD, and the amino acid sequence of the mutated RBD protein is shown as SEQ ID NO. 3.

(3) Compared with SEQ ID NO.1, the 417 th amino acid K of the protein sequence is mutated into N, the 484 th amino acid E is mutated into K, and the 501 st amino acid N is mutated into Y. The mutated RBD protein is named as M3-RBD, and the amino acid sequence of the mutated RBD protein is shown as SEQ ID NO. 4.

(4) Compared with SEQ ID NO.1, the 417 th amino acid K of the protein sequence is mutated to N, the 452 th amino acid L is mutated to R, the 484 th amino acid E is mutated to K, and the 501 th amino acid N is mutated to Y. The mutated protein is named M4-RBD, and the amino acid sequence of the mutated protein is shown as SEQ ID NO. 5.

2. Transformation amplification plasmid:

After the constructed plasmid is verified to be correct by sequencing, 100ng of the plasmid with correct sequencing is added into 100 mu LDH5 alpha competent cells (escherichia coli), and after the plasmid is uniformly mixed, the plasmid is incubated on ice for 30min, and after heat shock is carried out at 42 ℃ for 30sec, the plasmid is incubated on ice for 3min. Spread on LB plates with ampicillin resistance and incubated overnight at 37℃in an incubator. Single colonies were picked at intervals, grown in LB medium with ampicillin resistance, and endotoxin-removed plasmids were extracted for transfection.

3. Expression and purification of proteins

Mammalian 293F suspension cells were cultured in a shaker incubator at 120rpm,37℃with 5% CO ₂. When the density of the cells in the logarithmic growth phase is 2×10 ⁶ cells/mL and the activity is 98% or more. The plasmid was transfected with PEI and cultivation continued for 4 days during which time nutrient solution was supplemented daily. The cell suspension was collected on the fourth day and the supernatant was collected by centrifugation at 4000rpm at 4℃for 20 min. Filtering the supernatant with 0.22 μm, affinity purifying with Protein A filler to obtain target Protein, and purifying with gel chromatography to obtain target Protein with purity higher than 99%. The purified protein was then checked for size and purity by polyacrylamide gel electrophoresis (SDS-PAGE). The purified protein is quantified by using a BCA protein quantification kit, then split charging into 200 mu LEP tubes, quick freezing with liquid nitrogen and storing at-80 ℃.

4. Mutant subunit vaccine immunization mice experiments:

4.1. Mice immunization experiments:

Female BABL/c mice of 6-8 weeks old were purchased from Beijing Bei Fu and kept in the university of Wuhan laboratory animal center. The CpG adjuvant used in the invention is derived from CpG 6 in Table 1 of CN108728444A, the sequence is 5'-tcgcgaacgttcgccgcgtacgtacgcgg-3', and the sequence is a full thio CpG ODN sequence. The CpG adjuvant lyophilized powder is prepared into 1mg/mL solution by sterile PBS and then split-packed for freezing at-80 ℃. Groups of 12 mice were prepared for immunization, 6 each. The experimental groups were prepared with 5. Mu.g WT-RBD or RBD mutein per mouse, 20. Mu.g CpG and 0.26mg Al (OH) ₃ in amounts of the immune system, and the 100. Mu.L sterile PBS group was used as control group, while the sterile PBS group was used to make up to a volume of 100. Mu.L. Each group of mice was immunized by intramuscular injection twice, with a two week time interval. After 14 days of immunization, blood of the mice is collected through an orbital blood taking method, the blood is kept standing at 4 ℃ overnight, serum is obtained through centrifugal separation, the serum is inactivated at 56 ℃ for 30 minutes, and the serum is split-packaged and frozen at-80 ℃ for subsequent detection.

4.2. Neutralizing antibody titer detection based on pseudovirus in vitro neutralization system:

the present experiment examined the level of neutralizing antibodies to SARS-CoV-2D614G, omicr-on BA.1 and Omicron BA.2 pseudoviruses in the serum of day 28 of mice immunized with the different muteins. The mouse serum diluted in gradient is evenly mixed with SARS-CoV-2 pseudovirus (5000 TCID ₅₀) with the same volume, incubated for 30 minutes at 37 ℃, then the mixed solution is evenly mixed with BHK21-hACE2 steady transfer cell line suspended with the same volume, and then cultured overnight at 37 ℃, after 16 hours, the cell culture solution is discarded, luciferase chromogenic substrate is added, and the serum dilution with 50% neutralization activity is calculated by luciferase activity, namely pVNT ₅₀. The Geometric Mean Titer (GMT) is the geometric mean of all the tested samples pVNT ₅₀ of the group.

Preparation of SARS-CoV-2 pseudovirus is described in the following literature (Nie,J.,Li,Q.,Wu,J.,Zhao,C.,Hao,H.,Liu,H.,...Wang,Y.(2020).Establishment and validation of a ps eudovirus neutralization assay for SARS-CoV-2.Emerg Microbes Infect,9(1),680-686.doi:10.1080/22221751.2020.1743767)

As a result, as shown in FIG. 2, the RBD mutant immune groups had a level of reduced neutralization activity against SARS-CoV-2D614G pseudovirus compared to the WT-RBD group (FIG. 2A). When tested for neutralizing activity against Omicron BA.1 and Omicron BA.2 pseudoviruses, the M4-RBD immunized group had a significant increase in serum neutralizing activity relative to the WT-RBD immunized group, and the GMT had a 15.6 and 28.9 fold increase relative to the WT-RBD group, respectively (FIGS. 2B, 2C).

Example 2 optimized screening of M4-based SARS-CoV-2RBD mutant recombinant proteins

1. Optimization of immunogen:

We further optimized the sequence on the M4 sequence with better immunogenicity, replaced K417N with K417T or replaced K417N with K417T and E484K with E484A mutation site at the same time, and further screened. Mutation sites are shown in FIG. 1, and specific mutation sites are as follows:

(1) Compared with SEQ ID NO.1, the 417 th amino acid K of the protein sequence is mutated into T, the 452 th amino acid L is mutated into R, the 484 th amino acid E is mutated into K, and the 501 st amino acid N is mutated into Y. The mutated protein is named M4T-RBD, and the amino acid sequence of the mutated protein is shown as SEQ ID NO. 6.

(2) Compared with SEQ ID NO.1, the 417 th amino acid K of the protein sequence is mutated into T, the 452 th amino acid L is mutated into R, the 484 th amino acid E is mutated into A, and the 501 st amino acid N is mutated into Y. The mutated protein is named M4A-RBD, and the amino acid sequence of the mutated protein is shown as SEQ ID NO. 7.

2. After constructing the vector, expressing and purifying the protein in the same amount as in example 1, mice were immunized and the serum of the mice was taken for detection of neutralizing antibodies. The results showed that the neutralizing activity of the M4T-RBD immune group on SARS-CoV-2D614G was close to (1.1 times) that of the WT-RBD immune group, but that the neutralizing activity on Omicron BA.1 and Omicron BA.2 pseudoviruses was elevated to some extent compared to that of the WT-RBD group, and that the GMT was increased by 10.9 and 37.2 times, respectively, relative to the WT-RBD group (FIG. 3). The neutralizing activity of the M4A-RBD immune group to the pseudoviruses is not obviously improved.

Example 3 optimized screening of M4T-based SARS-CoV-2RBD mutant recombinant proteins

1. Optimization of immunogen:

We further optimized the sequence on the M4T-RBD sequence with better immunogenicity, and increased T478K or T478K and N440K mutation sites, and further screened. Mutation sites are shown in FIG. 1, and specific mutation sites are as follows:

(1) Compared with SEQ ID NO.1, the 417 th amino acid K of the protein sequence is mutated into T, the 452 th amino acid L is mutated into R, the 478 th amino acid T is mutated into K, the 484 th amino acid E is mutated into K, and the 501 st amino acid N is mutated into Y. The mutated protein is named M5-RBD, and the amino acid sequence of the mutated protein is shown as SEQ ID NO. 8.

(2) Compared with SEQ ID NO.1, the 417 th amino acid K of the protein sequence is mutated into T, the 440 th amino acid N is mutated into K, the 452 th amino acid L is mutated into R, the 478 th amino acid T is mutated into K, and the 484 th amino acid E is mutated into K and the 501 th amino acid N is mutated into Y. The mutated protein is named M6K-RBD, and the amino acid sequence of the mutated protein is shown as SEQ ID NO. 9.

2. The vector was constructed as described in example 1, the DNA nucleic acid sequences of M5-RBD and M6K-RBD were as shown in SEQ ID No.11 and SEQ ID No.12, and after expressing and purifying the proteins, mice were immunized at the same dose and the serum of the mice was taken for detection of neutralizing antibodies. The results showed that both M5-RBD and M6K-RBD immune groups had a level of enhancement in neutralizing activity against SARS-CoV-2D614G, omicron BA.1 and Omicron BA.2, with the M5-RBD immune group GMT being increased 1.2, 28.3 and 46.3 fold relative to WT-RBD groups, and the M6K-RBD immune group GMT being increased 0.8, 13.4 and 31.7 fold relative to WT-RBD groups, respectively (FIG. 4).

EXAMPLE 4 comparison of immunogenicity of M5-RBD-based SARS-CoV-2M5 RBD and SARS-CoV-2M5Spike mutant recombinant proteins

1. Optimization of immunogen:

The SARS-CoV-2Spike protein amino acid sequence (protein_id= "YP_ 009724390.1") was obtained from NCBI database, amino acid K at position 417 of the protein sequence was mutated to T, amino acid L at position 452 was mutated to R, amino acid T at position 478 was mutated to K, amino acid E at position 484 was mutated to K, amino acid N at position 501 was mutated to Y, and the amino acid sequence was regarded as SARS-CoV-2Spike protein amino acid sequence in the present invention (referred to as M5-Spike in the present invention), and the amino acid sequence was represented by SEQ ID NO. 10. The SARS2-CoV-2M5Spike base sequence with HIS6 tag was determined by codon optimization and synthesized. This fragment was ligated into PCAGGS vector by PCR technique using HindIII and NotI cleavage sites.

2. After expression and purification of the protein, mice were immunized at the same dose and the serum of the mice was taken for detection of neutralizing antibodies. The results showed that the neutralizing antibody levels were significantly lower in the SARS2-CoV-2M5-Spike immunized group than in the M5-RBD immunized group (FIG. 5).

Example 5: mRNA vaccine immunization mice experiments

Preparation of SARS-CoV-2RBD mutant mRNA vaccine

Rna transcription and purification:

DNA sequences of WT-RBD, M5-RBD and M6K-RBD were added with T7 promoter-5 'UTR-GCCACC and 3' UTR-polyadenylation (Poly A) tails at both ends, respectively, and constructed on pUC57 plasmid, and transformed into E.coli. The positive strain was cultivated by amplification, and the plasmid was extracted and purified. The plasmid is digested and linearized, the mRNA is transcribed into mRNA by using a T7 in vitro transcription system after purification, the 5 '-end of the mRNA is capped into a cap-1 structure by using vaccinia virus capping enzyme and 2' -O-methyltransferase simultaneously after purification, and the mRNA obtained by purification is used for preparing Lipid Nano Particles (LNP), wherein the sequences of the mRNA of M5-RBD and M6K-RBD are shown as SEQ ID NO.13 and 14.

RNA-LNP liposome preparation and concentration determination

The ionizable lipid, neutral lipid, cholesterol and PEG lipid in certain proportion are dissolved in absolute ethyl alcohol, and the glass bottle is gently shaken to be fully dissolved to be used as an ethanol phase. mRNA was diluted with 20mM sodium acetate buffer at ph=4 as the aqueous phase. Inserting a chip into a microfluidic instrument, respectively sucking an ethanol phase and a water phase by using an injector, and mounting the chips on an injection port of the chip; on microfluidic software, the temperature was set at room temperature, the total injection flow rate was 12mL/min, the flow rate ratio was ethanol phase: water phase=2:3, the equipment was started, and the effluent was collected. Sodium acetate buffer and ethanol were replaced with PBS (ph=7.4) using a 100kDa ultrafiltration centrifuge tube. The concentration of the actual encapsulated RNA was calculated by measuring free and total RNA with Ribogreen dye.

1. Mice immunization experiments:

Female BABL/c mice of 6-8 weeks old were purchased from Beijing Bei Fu and kept in the university of Wuhan laboratory animal center. RNA-LNP was diluted to an RNA concentration of 10. Mu.g/mL based on the actual encapsulated RNA concentration in LNP, immunized at a dose of 0.3. Mu.g per mouse, immunized every 3 weeks, and mice were orbital bled on day 35, and serum was isolated for subsequent detection of neutralizing antibody titers with pseudovirus.

2. Neutralizing antibody titer detection based on pseudovirus in vitro neutralization system:

The present experiment detects the neutralizing antibody levels of the serum of mice immunized with RNA vaccine on day 35 against SARS-CoV-2D614G, delta and Omicron BA.1 pseudovirus. The serum of the mice subjected to gradient dilution is evenly mixed with the SARS-CoV-2 pseudovirus (5000 TCID ₅₀) with the same volume and incubated for 30 minutes at 37 ℃, the mixed solution is evenly mixed with the BHK21-hACE2 steady transfer cell line with the same volume and then cultured overnight at 37 ℃, after 16 hours, the cell culture solution is discarded, a luciferase chromogenic substrate is added, and the serum dilution with 50% neutralization activity is calculated through luciferase activity, namely pVNT ₅₀.

The neutralization results showed that both the M5-RBD-mRNA and M6K-RBD-mRNA immune groups had higher serum neutralization titers GMT for D614G, delta and Omicron pseudovirus than the WT-RBD-mRNA immune group (FIG. 6).

Claims

1. A mutant of a receptor binding domain RBD of a novel coronavirus S protein, characterized in that the mutant of the receptor binding domain RBD of the S protein is mutated from 319-541aa of the sequence shown in SEQ ID No.1, at least at amino acid residues 417, 452, 484 and 501 of SEQ ID No. 1; compared with the receptor binding domain RBD of the S protein shown in SEQ ID NO.1, the anti-coronavirus neutralizing antibody can induce a broad spectrum in vivo;

preferably, the mutation site further comprises an amino acid residue at position 478;

further preferably, the mutation site further comprises amino acid residue 440;

417. Asn or Thr;

lys at position 440;

arg at position 452;

478 th Lys;

Lys at position 484;

501 th bit Tyr.

2. The mutant of the receptor binding domain RBD of the novel coronavirus S protein as recited in claim 1, wherein the mutant of the receptor binding domain RBD of the novel coronavirus S protein is selected from any one of the following (a) - (c):

Or (b)

3. A recombinant protein comprising a mutant of the receptor binding domain RBD of the novel coronavirus S protein of claim 1 or 2, further comprising at least one of the following modifications based on the amino acid sequence of the mutant:

4. A polynucleotide encoding a mutant of the receptor binding domain RBD of the novel coronavirus S protein of claim 1 or 2 or the recombinant protein of claim 3.

5. An expression vector comprising the polynucleotide of claim 4.

6. A host cell comprising the expression vector of claim 5 or a polynucleotide having integrated in its genome a mutant of the receptor binding domain RBD of the novel coronavirus S protein of claim 4.

7. A linearized mRNA obtained by in vitro transcription of the polynucleotide of claim 4.

8. A pharmaceutical composition comprising a mutant of the receptor binding domain RBD of the novel coronavirus S protein of claim 1 or 2 or the recombinant protein of claim 3 or the polynucleotide of claim 4 or the expression vector of claim 5 or the host cell of claim 6 or the linearized mRNA of claim 7;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

9. Use of a mutant of the receptor binding domain RBD of the novel coronavirus S protein of claim 1 or 2 or a recombinant protein of claim 3 or a polynucleotide of claim 4 or an expression vector of claim 5 or a host cell of claim 6 or a linearized mRNA of claim 7 or a pharmaceutical composition of claim 8 for the preparation of a medicament for the prevention or treatment of a novel coronavirus infection or a disease caused by a novel coronavirus infection;

Preferably, the drug is a subunit vaccine or an mRNA vaccine;

Further preferred, the novel coronavirus is the novel coronavirus mutant SARS-CoV-2D614G, omicronBA.1 and OmicronBA.2.

10. A method of preparing a vaccine, the method comprising: mixing the mutant of claim 1 or 2, or the recombinant protein of claim 3, the polynucleotide of claim 4, or the linearized mRNA of claim 7 with a pharmaceutically acceptable carrier and/or excipient, and/or with an additional active ingredient.