CN117486980A - Coronavirus VLP (virus-like particle) and preparation method and application thereof - Google Patents

Abstract

The present invention relates to the field of virology, in particular, the invention relates to the engineering of the N protein, M protein, E protein and/or part of the ORF1ab genomic RNA of SARS-CoV-2 and provides a method for preparing coronavirus VLPs based on said engineering. Furthermore, the present invention provides coronavirus VLPs prepared based on said method, and uses of said VLPs.

Description

Coronavirus VLP (virus-like particle) and preparation method and application thereof

Technical Field

The present invention relates to the field of virology, in particular, the invention relates to the engineering of the N protein, M protein, E protein and/or part of the ORF1ab genomic RNA of SARS-CoV-2 and provides a method for preparing coronavirus VLPs based on said engineering. Furthermore, the present invention provides coronavirus VLPs prepared based on said method, and uses of said VLPs.

Background

Replication-defective SARS-CoV-2 virus-like particle (VLP) pseudoviruses are infectious virus-like particles similar to true viruses, which contain 4 structural proteins of a novel coronavirus (S protein, spike protein; M protein, membrane glycoprotein; E protein, envelope protein; N protein, nucleocapsid phosphoprotein) and bring foreign genes into the virus particles by self-assembly through the combination of a segment of RNA of ORF1ab and the N protein, and are safe to operate because the ORF1ab has only a partial segment of RNA so that the virus-like particles do not have replication capacity, so that the virus-like particles have only one round of infectivity; and the virus-like particle contains structural proteins of all new coronaviruses, and compared with pseudo viruses of a VSV system only containing S proteins, the virus-like particle is more similar to the state of a living virus, and the structure is more stable.

Replication-defective SARS-CoV-2 virus-like particle pseudoviruses are mainly formed by cotransfecting 293T cells with plasmids, self-expressing and assembling in the cells, and are obtained by a culture medium supernatant collecting method. The virus-like particle pseudovirus not only has the same infectivity as the live virus, but also has no capacity of replicating to generate virus after entering the cell, so that the virus-like particle pseudovirus is harmless, and can be more safely used for detecting neutralizing antibodies and antiviral drugs and researching the influence of new coronavirus structural gene changes on infectivity.

The SARS-CoV-2 has strong infectivity, strong pathogenicity, extremely high risk for living virus research, limited quantity, high safety of virus-like particle pseudoviruses, and can be prepared in large quantity by transfection technology, thereby providing convenience for SARS-CoV-2 virus research.

The SARS-CoV-2 virus-like particle pseudovirus features that it overcomes the defect of traditional method, and makes the detection technique safer, simpler, faster and higher in flux, and makes some viruses which are difficult to culture themselves also can be detected by culture method. However, when 4 structural proteins of SARS-CoV-2 virus are generally used directly for construction of virus-like particle pseudoviruses, the viral titer of the VLPs prepared therefrom tends to be low, which limits the application of the virus-like particle pseudoviruses. Thus, there is a need to further increase the viral titer of virus-like particle pseudoviruses.

Disclosure of Invention

The inventors of the present application have studied, through extensive experimentation and repeated investigation, a number of methods that can significantly increase VLP titers:

(i) The inventor discovers key mutation sites capable of improving the virus titer by single-point mutation experiments on SARS-CoV-2N protein, and further performs joint mutation and codon optimization on N protein, so that SARS-CoV-2 virus-like particle pseudovirus with further improved titer is obtained.

(ii) After the inventors performed replacement and transformation of the M & E gene, it was unexpectedly found that the substitution of the SARS-CoV-2M & E gene with the M & E gene of SARS-CoV-1 could also increase the titer of the pseudovirus of the virus-like particle.

(iii) The inventors truncate and remodel part of RNA of SARS-CoV-2ORF1ab and use the modified RNA in VLP preparation, and find that specific truncations can significantly improve the titer of VLP.

On this basis, the inventors carried out the preparation of VLPs in combination with (i) to (iii) above, and the overall titer of the VLPs obtained could be improved by more than about 200 times compared with the conventional methods.

In addition, the inventors replaced the S gene of the novel coronavirus mutant with the S genes of the other 3 human coronaviruses 229E, NL, SARS and the other 21 coronaviruses Sarbecovirus clade a and Sarbecovirus clade b, all of which could be packaged into virus-like particle pseudoviruses using the above method.

Exemplary SARS-CoV-2 that can be used to make the corresponding VLPs using the methods of the present application include, but are not limited to: wuhan Hu-1 strain, b.1 strain, b.1.1.7 strain, b.1.351 strain, p.1 strain, b.1.671.2 strain, ba.1 strain, ba.2 strain, ba.3 strain, ba.4/5 strain, ba.2.12.1 strain.

Exemplary coronaviruses other than SARS-CoV-2 that can be used to prepare the corresponding VLPs using the methods of the present application include, but are not limited to: 229E, NL, SARS, sin852, GZ-C, sino1-11, urbani, HGZ8L1-A, GD01, PC4-127, PC4-13, PC4-137, GD03T0013, GZ0402, SZ1, LYRa11, WIV1, rs7327, rs4231, rsSHC014, rs4084, raTG13, pandolin_GD (1/2019), pandolin_GX (P5L).

Thus, in one aspect, the present application provides a coronavirus virus-like particle (VLP) comprising a mutant of an N protein derived from SARS-CoV-2, wherein said N protein mutant comprises one or more mutations selected from the group consisting of:

(i) A residue at a position corresponding to amino acid residue 199 of SEQ ID NO. 1 (e.g., a P residue) is replaced with an L residue;

(ii) A residue at a position corresponding to amino acid residue 202 of SEQ ID NO. 1 (e.g., an S residue) is substituted with an R residue;

(iii) The residue at the position corresponding to amino acid residue 203 of SEQ ID NO. 1 (e.g., R residue) is replaced with an M residue.

In certain embodiments, the N protein mutant comprises mutations (i) and (ii), or the N protein mutant comprises mutations (i), (ii), and (iii).

In certain embodiments, the VLP is not capable of autonomous replication.

In certain embodiments, the wild-type N protein derived from SARS-CoV-2 has an amino acid sequence as set forth in SEQ ID NO. 1.

In certain embodiments, the N protein mutant has an amino acid sequence selected from any one of SEQ ID NOs 2-7.

In certain embodiments, the VLP further comprises: an RNA molecule comprising a packaging signal sequence; wherein the packaging signal sequence is selected from the PS9 region derived from SARS-CoV-2 or a truncation thereof, wherein the truncation truncates 110-140 (e.g., 110-130, 110-126, 120-140, 126-130, 120-130, 126) nucleotide residues at the 3' end of the PS9 region as compared to the PS9 region.

In certain embodiments, the packaging signal sequence is selected from the PS9 region truncations.

In certain embodiments, the truncations are truncated by 126 nucleotide residues at the 3' end of the PS9 region as compared to the PS9 region.

In certain embodiments, the PS9 region has the nucleotide sequence set forth in SEQ ID NO. 12.

In certain embodiments, the truncations of the PS9 region have the nucleotide sequence set forth in SEQ ID NO. 13.

In certain embodiments, the VLP further comprises: m and E proteins; wherein the M and E proteins are selected from the group consisting of SARS-CoV-2 or SARS-CoV-1 derived M and E proteins.

In certain embodiments, the M and E proteins are selected from the group consisting of SARS-CoV-1 derived M and E proteins.

In certain embodiments, the M protein derived from SARS-CoV-2 has an amino acid sequence as set forth in SEQ ID NO. 8.

In certain embodiments, the SARS-CoV-2 derived E protein has an amino acid sequence as set forth in SEQ ID NO. 10.

In certain embodiments, the M protein derived from SARS-CoV-1 has the amino acid sequence as set forth in SEQ ID NO. 9.

In certain embodiments, the E protein derived from SARS-CoV-1 has the amino acid sequence as set forth in SEQ ID NO. 11.

In certain embodiments, the VLP further comprises: s protein; wherein the S protein is selected from the group consisting of coronavirus-derived S proteins.

In certain embodiments, the coronavirus is selected from the group consisting of human coronaviruses (e.g., SARS-CoV-2, 229E, NL63, SARS-CoV-1), sarbecovirus clade a (SARS-CoV-1-like coronavirus) and Sarbecovirus clade 1b (SARS-CoV-2-like coronavirus).

In certain embodiments, the coronavirus is selected from the group consisting of SARS-CoV-2 (e.g., omicron series strain), 229E, NL63, SARS-CoV-1, sin852, GZ-C, sino1-11, urbani, HGZ8L1-A, GD01, PC4-127, PC4-13, PC4-137, GD03T0013, GZ0402, SZ1, LYRa11, WIV1, rs7327, rs4231, rsSHC014, rs4084, raTG13, panglin_GD (1/2019), panglin_GX (P5L).

In certain embodiments, the SARS-CoV-2 is selected from the group consisting of Wuhan Hu-1 strain, B.1 strain, B.1.1.7 strain, B.1.351 strain, P.1 strain, B.1.671.2 strain, BA.1 strain, BA.2 strain, BA.3 strain, BA.4/5 strain, BA.2.12.1 strain.

In certain embodiments, the S protein derived from coronavirus has an amino acid sequence selected from the group consisting of SEQ ID NOS.14-45.

In certain embodiments, the VLP comprises or consists of:

(1) An N protein mutant as defined hereinabove;

(2) S protein as defined hereinabove;

(3) An RNA molecule comprising a PS9 region truncate as defined hereinabove;

(4) The M and E proteins derived from SARS-CoV-1 as defined hereinabove.

In certain embodiments, the RNA molecule comprising a packaging signal sequence further comprises a coding sequence for a reporter protein; in certain embodiments, the reporter protein is selected from the group consisting of luciferase, fluorescent proteins.

In certain embodiments, the RNA molecule further comprises a coding sequence for a luciferase. In certain embodiments, the amino acid sequence of the luciferase is shown in SEQ ID NO. 46.

In another aspect, the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an N protein mutant, wherein the N protein mutant is as defined above.

In certain embodiments, the isolated nucleic acid molecule further comprises: a coding sequence for an S protein as defined hereinabove; a coding sequence of an RNA molecule comprising a packaging signal sequence as defined hereinabove; and/or coding sequences for M protein and E protein as defined hereinabove.

In certain embodiments, the isolated nucleic acid molecule comprises: a coding sequence of an N protein mutant as defined hereinabove; a coding sequence for an S protein as defined hereinabove; a coding sequence of an RNA molecule comprising a PS9 region truncations as defined hereinabove; and coding sequences for the M and E proteins derived from SARS-CoV-1 as defined hereinabove.

In another aspect, the present application also provides a vector comprising an isolated nucleic acid molecule as described above.

In another aspect, the present application also provides a carrier system comprising one or more carriers, wherein the one or more carriers comprise:

(1) A coding sequence for an N protein mutant, the N protein mutant being as defined above;

(2) A coding sequence for an S protein, the S protein being as defined above;

(3) A coding sequence for an RNA molecule comprising a packaging signal sequence, said RNA molecule being as defined above;

(4) Coding sequences for M and E proteins as defined hereinabove.

In certain embodiments, the coding sequence of (3) is a DNA representation of the RNA molecule.

In certain embodiments, the sequence of (3) is the coding sequence of an RNA molecule comprising the PS9 region. In certain embodiments, the DNA sequence of the PS9 region is as set forth in SEQ ID NO. 52.

In certain embodiments, the sequence of (3) is a coding sequence for an RNA molecule comprising a PS9 region truncate. In certain embodiments, the DNA sequence of the PS9 domain truncations is set forth in SEQ ID NO. 53.

In certain embodiments, the sequences of (4) are the coding sequences for the M and E proteins derived from SARS-CoV-1.

In certain embodiments, the sequences recited in (3) are located in a first vector and the sequences recited in (1), (2) and (4) are located in one or more additional vectors.

In certain embodiments, the sequence of (3) is located in a first vector, the sequences of (1) and (2) are located in a second vector and a third vector, respectively, and the M protein coding sequence and the E protein coding sequence of (4) are co-located in a fourth vector.

In certain embodiments, the M protein coding sequence and the E protein coding sequence of (4) are linked by an IRES coding sequence.

In certain embodiments, the IRES has a coding sequence as set forth in SEQ ID NO. 47.

In certain embodiments, the RNA molecule comprising a packaging signal sequence further comprises a coding sequence for a reporter protein; in certain embodiments, the reporter protein is selected from the group consisting of luciferase, fluorescent proteins.

In certain embodiments, the RNA molecule further comprises a coding sequence for a luciferase. In certain embodiments, the amino acid sequence of the luciferase is shown in SEQ ID NO. 46.

In another aspect, the present application also provides a host cell comprising a vector system as described above.

In another aspect, the present application also provides a method of preparing a coronavirus VLP comprising: culturing a host cell as described above under conditions allowing expression of said VLP; and recovering the expressed VLPs.

In certain embodiments, the method comprises: introducing the vector system as described above into a host cell and culturing, recovering VLPs in the culture supernatant of said host cell.

In another aspect, the present application also provides the use of a vector system or host cell as described above for the preparation of coronavirus VLPs.

In another aspect, the present application also provides the use of a coronavirus VLP as described above for screening a drug or detecting antibody activity (e.g., binding activity and/or neutralizing activity).

In certain embodiments, the agent is an agent that inhibits coronavirus infection.

In certain embodiments, the antibody is an antibody against a coronavirus.

In another aspect, the present application also provides a method of detecting neutralizing activity of an antibody comprising:

(1) Contacting a sample comprising an antibody to be tested with VLPs as described above;

(2) Contacting the product of step (1) with a cell, said cell being capable of being infected with a coronavirus; and, a step of, in the first embodiment,

(3) Detecting the infection rate of the cells, thereby evaluating the neutralizing activity of the antibody to be tested.

In certain embodiments, the antibody is an antibody against a coronavirus.

In certain embodiments, the VLP comprises a coding sequence for a reporter protein.

In certain embodiments, in step (3), the infection rate of the cell is detected by expression of the reporter protein in the cell.

In certain embodiments, the reporter protein is selected from the group consisting of luciferase, fluorescent proteins.

In certain embodiments, the method is provided with one or more selected from the group consisting of:

(a) The cells are cells expressing ACE2 and Furin; in certain embodiments, the cell is a 293T cell expressing ACE2 and Furin;

(b) In step (2), the product of step (1) is contacted with the cells for a period of 15-22 hours (e.g., 15-20 hours, 15-18 hours, 18-22 hours, 18-20 hours, 18 hours);

(c) In step (1), the VLP is vaccinated in an amount of 512-65610TCID50.

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. Moreover, the virology, biochemistry, immunology laboratory procedures 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.

When used herein, the terms "for example," such as, "" including, "" comprising, "or variations thereof, are not to be construed as limiting terms, but rather as meaning" but not limited to "or" not limited to.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "virus-like particle" or "VLP" refers to a non-replicating self-assembled biomolecule. VLPs are typically composed of one or more viral proteins, such as, but not limited to, proteins of the classes known as capsid proteins, coat proteins, capsid proteins, surface proteins and/or envelope proteins, or polypeptides derived from the same. Typically, VLPs are infectious but do not contain intact viral nucleic acids and thus do not have complete replication capacity.

It will be appreciated by those skilled in the art that mutations or variations (including, but not limited to, substitutions, deletions and/or additions, e.g., N proteins of different types of coronaviruses) can be naturally occurring or artificially introduced in the amino acid sequence of the N protein without affecting its biological function. For example, the amino acid sequence of the N protein may naturally vary (mutation or variation) among different coronavirus strains without affecting its biological function. Thus, in the present invention, the term "wild-type N protein" shall include all such sequences, including, for example, SEQ ID NO:1 and natural or artificial variants thereof. Also, when describing the sequence fragment or amino acid position of the wild-type N protein, it includes not only the sequence of SEQ ID NO:1, and also includes the corresponding sequence fragment or amino acid position in a natural or artificial variant thereof.

According to the invention, for example, when the expression "at a position corresponding to amino acid residue 199 of SEQ ID NO. 1" is intended to mean that the sequence is optimally aligned with SEQ ID NO. 1, i.e. when the sequence is aligned with SEQ ID NO. 1 to obtain the highest percentage identity, the amino acid residue of the sequence compared with amino acid residue 199 of SEQ ID NO. 1 is at an equivalent position. For example, when the expression "at the position corresponding to amino acid residue 202 of SEQ ID NO. 1" means that when optimally aligned with SEQ ID NO. 1, i.e. when aligned with SEQ ID NO. 1 to obtain the highest percent identity, the amino acid residue of the compared sequence that is at the equivalent position to amino acid residue 202 of SEQ ID NO. 1. For example, when the expression "at the position corresponding to amino acid residue 203 of SEQ ID NO. 1" means that when optimally aligned with SEQ ID NO. 1, i.e. when the sequences are aligned with SEQ ID NO. 1 to obtain the highest percent identity, the amino acid residue in the sequence compared with amino acid residue 203 of SEQ ID NO. 1 is at the equivalent position. The rest of the amino acid residues defined in a similar expression are determined in a similar manner.

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 CAGGTT share 50% identity (3 out of 6 positions in total are matched). 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 algorithms of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which have 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)) algorithm that has 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 GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

Those skilled in the art will readily appreciate that coronaviruses from which the "S proteins" described herein are derived are not limited. For example, the S protein may be derived from a naturally occurring coronavirus, or the S protein may be an S protein obtained by artificially introducing mutation or variation on the basis of an S protein derived from a natural coronavirus. Coronaviruses from which the S protein is derived include, but are not limited to: SARS-CoV-2 (Wuhan Hu-1 strain, B.1 strain, B.1.1.7 strain, B.1.351 strain, P.1 strain, B.1.671.2 strain, BA.1 strain, BA.2 strain, BA.3 strain, BA.4/5 strain, BA.2.12.1 strain), 229E, NL63, SARS, sin852 (e.g., as GenBank: sin852_ay 559082), GZ-C (e.g., as GenBank: GZ-c_ay 394979), sino1-11 (e.g., as GenBank: sino1-11_AY485277), urbani (e.g., such as GenBank: urbani_AY 278741), HGZ L1-A (e.g., such as GenBank: HGZ L1-A_AY 394981), GD01 (e.g., such as GenBank: GD01_AY 278489), PC4-127 (e.g., such as GenBank: PC4-127_AY 613951), PC4-13 (e.g., such as GenBank: PC 4-13_AY613948), PC4-137 (e.g., such as GenBank: PC 4-137_AY627045), GD03T0013 (e.g., such as GenBank: GD03T0013_AY 525636), GZ0402 (e.g., such as GenBank: GZ0402_AY 613947), SZ1 (e.g., such as GenBank: SZ1_AY 304489), LYRa11 (e.g., such as GenBank: LY11_89), ra (e.g., such as GenBank: LY11_89), such as Pak: pad3_AY 4575), GZ 03 (e.g., such as GZ 3_AY 4576), GZ0402 (e.g., such as GenBank: GZ 3_AY 394979), GZ 03 (e.g., such as GZ 3) and GZ 03 (e.g., such as GYwork 35), GY35), GK 03 (e.g., such as GK 3_SrK 2_SrK 2, srK 5, srK_SrK 2, srK 5, srK 1_SrK 3, sr3 1L 13, sr1 3, sr3 3, S1 3, S1 3 G3, such (e G3 GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG,.

The amino acid sequences of the N, S, M, E proteins shown herein comprise an amino acid (e.g., methionine (Met)) encoded by an initiation codon (e.g., ATG) at the N-terminus. In certain embodiments, the N, S, M, E proteins of the invention each independently encompass not only an amino acid sequence comprising an amino acid encoded by a start codon (e.g., met) at their N-terminus, but also an amino acid sequence comprising no amino acid encoded by a start codon (e.g., met) at their N-terminus. Thus, sequences that do not include the amino acid encoded by the start codon (e.g., met) at the N-terminus of the amino acid sequence are also within the scope of the invention.

As used herein, the term "PS9 region" has the meaning commonly understood by those skilled in the art to be a cis-acting element of the RNA package that triggers SARS-CoV-2. See, e.g., syled AM, et al rapid assessment of SARS-CoV-2-evolved variants using virus-like parts science 2021;374 (6575):1626-1632. In general, the PS9 region refers to the nucleic acid fragment of SARS-CoV-2 genome at a position corresponding to the Wuhan Hu-1 strain genome (GenBank: NC-045512.2) 20080-21171.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., immunology-a Synthesis (2nd Edition,E.S.Golub and D.R.Gren,Eds, sinauer Associates, sundland, mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

Advantageous effects of the invention

The methods of making coronavirus VLPs provided herein are capable of significantly increasing viral titers of VLPs compared to methods disclosed in the prior art, and the methods are generally applicable to the production of a variety of coronavirus VLPs, e.g., are generally applicable to human coronaviruses (e.g., SARS-CoV-2, 229E, NL, SARS-CoV-1), sarbecovirus clade a (SARS-CoV-1-like coronavirus) and Sarbecovirus clade 1b (SARS-CoV-2-like coronavirus). Furthermore, omacron-series VLPs, which are more difficult to obtain by means of the prior art, can also be prepared and obtained using the methods provided herein.

The coronavirus VLP prepared by the method has high titer and strong infectivity, and can be advantageously applied to screening of coronavirus infection inhibition drugs and detection of antibody activity (such as binding activity and/or neutralizing activity).

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

Fig. 1: n protein mutant expression plasmid.

Fig. 2: titers of wild-type N protein and P199L, S202R, R M based N protein mutants correspond to VLPs.

Fig. 3: titers of wild-type N protein and P13L, G204R-based N protein mutants correspond to VLPs.

Fig. 4: SARS-COV-1-M & E protein expression plasmid.

Fig. 5: titer of M & E protein derived from SARS-COV-2 or SARS corresponds to VLP.

Fig. 6: PS966 expression plasmid.

Fig. 7: PS9 and each truncate corresponds to the titer of VLPs.

Fig. 8: titer of the omacron series SARS-CoV-2 VLPs before and after optimization.

Fig. 9: titers of each coronavirus VLP in example 5.

Fig. 10: titers of VLPs after repeated freeze thawing.

Fig. 11: titers of VLPs after reconstitution after lyophilization.

Fig. 12: titer of VLPs after 37 degrees celsius treatment.

Fig. 13: luminescence values in example 7 as a function of inoculation time.

Fig. 14: the luminescence values detected for the different cell types in example 7.

Fig. 15: the luminescence values detected for the different cell inoculum sizes in example 7.

Fig. 16: an antibody concentration-inhibition ratio curve corresponding to each virus inoculum size in example 7.

Fig. 17: inhibition versus antibody dilution curve in example 7.

Fig. 18: serum dilution-inhibition curves corresponding to each serum sample in example 7.

Fig. 19: correlation of live virus and VLP detection results in example 7.

Fig. 20: results of neutralizing antibody titer assays for live virus and VLP pseudoviruses in example 7. Fig. 21: an antibody concentration-inhibition ratio curve corresponding to each virus inoculum size in example 7.

Sequence information

A description of the sequences to which the present application relates is provided in the following table.

Table 1: sequence information

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Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, molecular biology experimental methods and immunoassays used in the present invention are basically described in j.sambrook et al, molecular cloning: laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-compiled guidelines for molecular biology experiments, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the manufacturer of the product. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: optimizing the production of replication defective SARS-CoV-2 VLPs by mutation of the N protein

In this example, the inventors studied their effect on VLP titers by mutating the N protein.

The preparation method comprises the following steps:

1. plasmid construction

Plasmid construction of PS 9:

coding sequences of reporter genes luciferases (the amino acid sequence of which is shown as SEQ ID NO: 46) and PS9 (the nucleotide sequence of which is shown as SEQ ID NO: 12) are cloned into a PCNDA3.1 vector, and synthesis and connection of the target genes are completed by general biological companies.

Construction of N protein mutant expression plasmid:

first, a wild-type N-WT (amino acid sequence shown as SEQ ID NO: 1) gene was cloned into a PCDNA3.1 vector, and then P199L, S R and/or R203M was introduced into the vector by point mutation (expression vector of the combined mutation of P199L, S202R and R203M is shown in FIG. 1), and synthesis and ligation of the target gene and point mutation were all accomplished by general biological company. The amino acid sequences of the N protein mutant P199L, S202R, R M, P199L & S202R, S R & R203M, P199L & S202R & R203M are shown in SEQ ID NO. 2-7 respectively.

Construction of SARS-CoV-2-M & E protein expression plasmid:

the M protein (amino acid sequence shown as SEQ ID NO: 8) gene and E protein (amino acid sequence shown as SEQ ID NO: 10) gene derived from SARS-CoV-2 are linked together by the coding DNA sequence (SEQ ID NO: 47) of IRES sequence and then are linked in PCDNA3.1 vector, and the synthesis and linkage of the target gene are completed by general biological company.

Construction of S protein expression plasmid:

the coding sequence of S protein (amino acid sequence shown as SEQ ID NO: 14) from SARS-CoV-2 is synthesized into PCDNA3.1 vector by general biology, the obtained plasmid is transformed into colibacillus, single colony is picked up, inoculated into LB liquid culture medium (containing ampicillin resistance), a large amount of bacterial liquid is obtained, plasmid extraction is carried out, and the sequence is sent to sequence, and the sequence is correctly used for subsequent virus packaging.

Preparation of VLP pseudoviruses

1) 293T cells as long as 90% -100% were passaged 1 to 2.5 a day in T75 or T150 cell flasks for plasmid transfection.

2) When the cells grow to a density of more than 65%, the method can be used for transfection, and the transfection reagent lipo3000 has toxicity and has great damage to the cells.

3) First, two 4mL EP tubes were prepared, which were divided into tube 1 and tube 2, 937.5. Mu.L opti-MEM was added to each of the two tubes, and the S protein plasmid, N protein plasmid, M & E protein plasmid and PS9 plasmid prepared as described above were added to tube 1 in the following proportions: a total of 39ug of plasmid 1:10:5:10 was added to 30. Mu.L of p3000.

4) 30. Mu.L of lipo3000 was added to opti-MEM in tube 2 and the two tubes were thoroughly mixed; (note that opti-MEM is left at room temperature as much as possible, making it easier to form liposomes).

5) After 5min of standing, pouring the solution in the pipe 2 into the pipe 1, fully and uniformly mixing, and standing at room temperature for 20min; tube 2, which does not contain plasmids, is poured into tube 1 to reduce wastage and wastage of plasmids during transfer.

6) The mixed liposome is added into a cell culture flask, and the mixture is gently mixed (the culture medium can be properly sucked out by 30% to increase the plasmid concentration, so that the toxicity-producing efficiency is higher).

7)5％CO ₂ After 12 hours of incubation in a 37℃incubator, the medium from the flask was aspirated, and 15mL of fresh DMEM complete medium was added to continue the incubation for 48 hours.

8) The supernatant was aspirated, centrifuged at 4000rpm for 10min, and filtered with a 0.45 μm filter to harvest the prepared VLPs, split 1 mL/tube and frozen at-80 ℃.

VLP titer detection

1) 100. Mu.L of medium was added to the middle 60 wells of the 96 well plate except for the edge 36 wells.

2) 50. Mu.L of VLP pseudovirus solution prepared in step 2 above was added to 6 wells of wells B2-G2.

3) Then, from wells B2 to G2, 3-fold ratio dilutions were performed in sequence to wells B3 to G3 and thereafter to wells, and finally 50. Mu.L of the mixed liquid taken out from wells B10 to G10 was discarded.

4) 100 μl of 293T cells overexpressing ACE2 and Furin were added to 60 wells (1 ten thousand cells/well) in the middle of a 96-well plate and the edge wells were sealed with 260 μl of sterile water.

5) Dilute 96 wells were placed in 5% co ₂ Incubation in incubator at 37℃for 18 hours.

6) 100 mu L of liquid is sucked and discarded from each hole, 100 mu L of Bright-GloTM fluorescent detection reagent is added, the mixture is placed for 2 minutes at normal temperature in a dark place, then the mixture is blown for 4 to 6 times, and 150 mu L of liquid is transferred to a white board or a blackboard.

7) And reading the luminescence value of each hole by using a microplate spectrophotometer.

8) VLP pseudovirus titers were calculated using the Reed-Muench method with 10-fold cut off value for the cell control and the results are shown in FIG. 2.

VLP dilution factor settings for specific wells are shown in table 2, column 10 dilution factor 390625 ×5:

TABLE 2 dilution of VLPs from wells

The results of FIG. 2 show that the titers of VLPs constructed based on the N protein mutant P199L, S202R, R203M, P L & S202R, S R & R203M, P199L & S202R & R203M are increased by about 4, 12, 8, 26, 13, 28 fold, respectively, compared to VLPs constructed based on the wild-type N protein of the SARS-CoV-2Wuhan Hu-1 original strain. The N protein mutation can obviously improve the packaging efficiency of VLPs with primary infection capacity, and the mutation P199L & S202R, P199L & S202R & R203M has obvious synergistic effect.

Furthermore, to further highlight the synergistic effect of the mutations, the inventors further provided titer data for VLPs constructed based on the N protein mutant P13L, G R, P L & S202R 203M & P13L, P199L & S202R & R203M & G204R (shown in fig. 3), which showed that although the titers of VLPs constructed based on the N protein single point mutations N-P13L and G204R were respectively enhanced compared to VLPs constructed based on the wild-type N protein, the titers of VLPs did not continue to increase upon subsequent superposition of the mutations P199L, S202R, R203M, indicating that there was not necessarily a synergistic effect between the N protein single point mutations that were able to increase the titers of VLPs.

Example 2: preparation of replication defective SARS-CoV-2VLP by replacement of M & E protein

In this example, the inventors further studied their effect on VLP titers by substituting M & E proteins on the basis of N protein mutational engineering.

The preparation method comprises the following steps:

1. plasmid construction

Construction of SARS-CoV-1-M & E protein expression plasmid (schematic view shown in FIG. 4):

the M protein (amino acid sequence shown as SEQ ID NO: 9) gene and E protein (amino acid sequence shown as SEQ ID NO: 11) gene from SARS-CoV-1 are connected together by IRES coding DNA sequence and then connected in PCDNA3.1 vector, and the synthesis and connection of target gene are completed by general biological company.

Construction of N protein mutant expression plasmid:

an expression plasmid of the N protein mutant P199L & S202R & R203M (amino acid sequence shown as SEQ ID NO: 7) was constructed, and the construction method was as described in example 1.

The construction methods of the PS9 expression plasmid and the S protein expression plasmid are described in example 1.

Preparation of VLP pseudoviruses

Reference is made to example 1.

Titer detection of VLPs

Reference is made to example 1.

The results of the VLP titer test are shown in FIG. 5, and the results show that the titer of the packaged VLP can be further improved by about 3.71 times after the replacement of the M & E of the packaged VLP by SARS-COV-2-M & E, and compared with the VLP constructed based on the original wild-type N protein and SARS-COV-2-M & E, the titer of the packaged VLP is improved by about 100 times.

Furthermore, the inventors have also found that substitution of SARS-COV-2-M & E with the M & E protein of the remaining coronavirus (229E-M & E, NL63-M & E, OC-M & E, HKU1-M & E, MERS-M & E) results in decreased VLP titers or unsuccessful packaging of the virus.

Example 3: preparation of replication defective SARS-CoV-2VLP by truncation of PS9

In this example, the inventors further studied their effect on VLP titers by truncating PS9, based on N protein mutation engineering and M & E protein substitution engineering.

The preparation method comprises the following steps:

1. plasmid construction

Plasmid construction of PS966 (schematic diagram as in fig. 6):

the coding sequences of the reporter genes luciferases and PS9 truncated PS966 (a segment of RNA sequence of non-coding new coronavirus NSP15-NSP16, and a nucleotide sequence shown as SEQ ID NO: 13) are cloned into a PCNDA3.1 vector, and the synthesis and connection of the target genes are completed by general biological companies.

Construction of N protein mutant expression plasmid:

an expression plasmid of the N protein mutant P199L & S202R & R203M (amino acid sequence shown as SEQ ID NO: 7) was constructed, and the construction method was as described in example 1.

Construction of SARS-CoV-1-M & E protein expression plasmid reference example 2, and construction method of S protein expression plasmid reference example 1.

Preparation of VLP pseudoviruses

Reference is made to example 1.

Titer detection of VLPs

Reference is made to example 1.

As shown in fig. 7, the results of the VLP titer detection showed that VLP constructed from PS966 was increased by about 1.91 times compared to VLP constructed from PS9 (also called PS 2) by the VLP constructed from PS966 by the 3 'end truncations of PS9 (882 to PS9, PS882 to PS903 to PS9, 924 to PS945 to PS9, 966 to PS9, PS966 to PS987, 1008 to PS9, PS1008 to PS9, 1029 to PS1029, 1050 to PS1050, 1071 to PS 1091 to PS 1050) by the 987 to PS987, indicating that the VLP constructed from PS966 by 126 nucleotides to the 3' end of PS9 further increased the packaging efficiency of VLP with primary infectious capability.

Example 4: preparation of replication-defective Omacron series SARS-CoV-2VLP

In this example, the inventors tried to combine the above-described N protein mutation engineering, M & E protein substitution engineering, and PS9 truncation engineering to prepare omacron series SARS-CoV-2 VLPs, and studied their titer changes relative to control VLPs prepared from an unoptimized system (i.e., VLPs prepared from wild-type N protein, untruncated PS9, M & E protein derived from SARS-CoV-2).

The experimental method is as follows:

1. plasmid construction

Construction of N protein mutant expression plasmid:

an N protein mutant P199L & S202R & R203M (amino acid sequence shown as SEQ ID NO: 7) expression plasmid was constructed according to example 1.

Construction of S protein expression plasmid:

expression plasmids derived from Omicron strain BA.1, BA.2, BA.3, BA.4/5 and BA.2.12.1 were constructed in accordance with example 1, and the amino acid sequences thereof were shown in SEQ ID NOS: 17-21, respectively.

Construction of the PS966 expression plasmid is described in example 3, and construction of the SARS-CoV-1-M & E protein expression plasmid is described in example 2.

Preparation of VLP pseudoviruses

Reference is made to example 1.

Titer detection of VLPs

Reference is made to example 1.

The results of the VLP titer assays are shown in fig. 8, and demonstrate that each omacron-series VLP pseudovirus can be packaged out of the virus by the methods optimized herein and that each strain has a significant increase in VLP virus titer compared to the control VLP prepared from the pre-optimization system (i.e., VLP constructed based on the untruncated PS9, wild-type N protein, and SARS-CoV-2m & e proteins), wherein ba.1 is increased by about 4.93-fold, ba.2 is increased by about 8.3-fold, ba.3 is increased by about 14.67-fold, ba.4/5 is increased by about 15.05-fold, and ba.2.12.1 is increased by about 41.65-fold.

Furthermore, the inventors have noted that the luminescent value of Omicron-series VLPs when subjected to titer detection was lower than that of other SARS-CoV-2 strains, probably due to the low Omicron infectivity (studies have shown that the use efficiency of Furin enzyme and TMPRSS2 by Omicron-series new coronavirus was reduced, resulting in reduced cleavage effect and reduced Omicron-series virus infectivity), resulting in lower detected titers than other strains.

Example 5: preparation of other coronavirus replication defective VLPs

In this example, the inventors tried to make VLPs of other coronaviruses by combining the above-described N protein mutation engineering, M & E protein substitution engineering, and PS9 truncation engineering.

The experimental method is as follows:

1. plasmid construction

Construction of N protein expression plasmid:

an N protein mutant P199L & S202R & R203M (amino acid sequence shown as SEQ ID NO: 7) expression plasmid was constructed according to example 1.

Construction of S protein expression plasmid:

reference example 1 constructs expression plasmids of S proteins (amino acid sequences of which are shown in SEQ ID NOs: 22-45) derived from respective coronaviruses (229E, NL, SARS, sin852, GZ-C, sino1-11, urbani, HGZ8L1-A, GD01, PC4-127, PC4-13, PC4-137, GD03T0013, GZ0402, SZ1, LYRa11, WIV1, rs7327, rs4231, rsSHC014, rs4084, raTG13, panglin_GD (1/2019), panglin_GX (P5L)).

Construction of the PS966 expression plasmid is described in example 3, and construction of the SARS-CoV-1-M & E protein expression plasmid is described in example 2.

Preparation of VLP pseudoviruses

Reference is made to example 1.

Titer detection of VLPs

Reference is made to example 1.

The results of the VLP titer assays are shown in FIG. 9, which shows that the optimized methods of the present application can successfully package 24 coronavirus VLP pseudoviruses other than SARS-CoV-2.

Example 6: stability test of VLPs prepared herein

In this example, the inventors repeatedly freeze-thaw, freeze-dry re-thaw or 37 degrees celsius treatments were performed on VLPs prepared by the optimization method of the present application, and the virus titer of the treated VLPs was monitored to test the stability thereof.

Repeated freezing and thawing:

the VLPs were freeze-thawed once and then a portion was aspirated for storage, and the remaining freeze-thawed portions were again aspirated and then freeze-thawed, and after 10 times of freeze-thawing, the VLP samples aspirated after each freeze-thawing were subjected to titer detection together.

Freeze-drying and re-melting:

after freeze-drying the VLPs by conventional freeze-drying techniques, the titer detection was performed simultaneously on the multiplex and non-freeze-dried samples, and the luminescence values were compared.

Stability at 37 degrees celsius:

VLPs of the same titer were simultaneously placed at 37 degrees celsius and after a period of time were recovered for cryopreservation until final titer monitoring was performed together and luminescence values were compared.

The results are shown in figures 10-12, and the results show that the VLP has good stability after repeated freezing and thawing for 10 times without reducing the virus titer; the freeze-dried and re-melted titer of the VLP is unchanged, and the stability is good; VLPs were treated at 37 degrees celsius for less than 48 hours with essentially unchanged viral titers, and the VLP viral titers remained high when treated at 37 degrees celsius for up to 168 hours.

Example 7: antibody neutralization activity assay using VLPs

In this example, the inventors used VLPs prepared by the optimization method of the present application to detect the neutralizing activity of antibodies, and optimized parameters such as the cell type, the cell addition amount, the virus inoculation amount, the detection time of the neutralization test, and established a stable and reliable neutralizing antibody detection method.

The experimental method comprises the following steps:

1) Sample preparation: inactivating guinea pig serum in 56 ℃ water bath for 0.5-1h, and adjusting the initial concentration of the antibody (m 9A8, baioesegram) to be detected to 30 mug/mL;

2) Taking a 96-well plate, adding 150 mu L/well of DMEM complete medium into the 2 nd column, adding 100 mu L/well of DMEM complete medium into the 3 rd-11 th column, and adding 42.5 mu L/well of DMEM medium into the B4-B11 wells;

3) 7.5 mu L of sample to be tested is added into the holes B4-B11, and two to three compound holes are arranged in each sample;

4) Repeatedly blowing and sucking the liquid in the holes B4-B11 for 6-8 times, and sequentially transferring 50 mu L of liquid to the corresponding holes C4-C11, wherein the liquid is diluted by 3 times;

5) Diluting 2050TCID50/mL pseudovirus with DMEM complete medium, and adding 50 mu L of pseudovirus to each well of columns 3-11;

6) The diluted 96-well plate was placed in a cell incubator (37 ℃,5% co) ₂ ) Incubating for 1h;

7) Digesting the cells and diluting the cell concentration to 1X 10 ⁵ mu.L of cells per well was added to each well of a 96-well plate at a rate of 1X 10 cells per well ⁴ A plurality of;

8) Adding 260 mu L of sterile water into 36 holes around the 96-well plate for sealing; placing 5% CO ₂ Culturing in a cell incubator at 37 ℃ for 18h;

9) After the culture is completed, absorbing and discarding 150 mu L of supernatant, then adding 100 mu L of Bright-gloTM luciferase detection reagent, reacting for 2min at normal temperature in a dark place, blowing for 4-6 times, and transferring 150 mu L of liquid into a white board or a blackboard;

10 Reading the luminescence value of each hole by using a microplate spectrophotometer;

11 Calculating the inhibition ratio:

inhibition ratio = (1- (luminescence intensity mean of sample group-blank CC mean)/(VC mean-CC mean)) ×100%;

wherein "CC" represents a cell control; "VC" means a virus control;

neutralizing antibody titer was expressed as the corresponding antibody concentration at 50% inhibition or the corresponding fold of serum dilution at 50% inhibition;

The sample dilution factor settings for each well are shown in table 3:

TABLE 3 dilution factor settings for each well sample

1. Determination of time for detection of neutralizing Activity of antibodies

After the VLP pseudovirus is used for infecting cells, the luminescence value is detected at 15h, 18h and 24h, and the result is shown in figure 13, and the luminescence value reaches the maximum value from 18h and tends to be stable, which is far lower than that of the conventional method at 24-48h. The VLPs of the present application are shown to be effective for antibody neutralization activity detection and to be able to effectively shorten detection times.

2. Selection of neutralizing antibody detection sensitive cells

VLP-sensitive cells suitable for detection of neutralizing antibody activity were screened by infection experiments on Vero, calu3,293T-ACE2 (293T expressing ACE 2), 293T-ACE2+Furin (293T expressing ACE2 and Furin), 293T-ACE2+TMPRSS2 (293T expressing ACE2 and TMPRSS 2), 293T-ACE2+cathepsin (293T expressing ACE2 and Cathepsin), 293T-ACE2+NRP1 (293T expressing ACE2 and NRP 1), respectively.

The results (FIG. 14) show that the best sensitive cell is 293T-ACE2+Furin.

3. Optimization of cell inoculum size in antibody detection

The amount of infected cells affects the expression level of luciferase, frozen VLP pseudovirus is diluted 3 times and added into 96-well plate, the cells are digested and counted, and 5×10 cells are added into each well ³ 、1×10 ⁴ 、3×10 ⁴ 、5×10 ⁴ 、8×10 ⁴ HEK293T cells overexpressing ACE2 and Furin enzymes were examined for luminescence after 18h infection.

The results (FIG. 15) show that at 3X 10 per well ⁴ Higher relative fluorescence intensities can be obtained with more than one cell.

To further determine the effect of cell number on antibody detection sensitivity, the antibody was first diluted 30-fold and then serially diluted 3-fold, frozen Wuhan Hu-1 strain VLP pseudovirus was taken and 20-fold diluted and then added to 96-well platesIncubation at 37℃for 1h, digestion of cells, counting and dilution to 5X 10 ⁴ /mL、1×10 ⁵ /mL、3×10 ⁵ /mL、5×10 ⁵ /mL，8×10 ⁵ Per mL, 100 μl per well was added, and after 18h of infection, luminescence was detected, inhibition was calculated, and IC50 was calculated using graphpad software. The results (Table 4) show that at 5X 10 ³ ～8×10 ⁴ The sensitivity of detecting the antibody was high in the cell/well range, in which the number of cells was 1X 10 ⁴ The linearity was best (r2=0.9165), and the cell number was finally determined to be 1×10 ⁵ Neutralization experiments were performed on cells/well.

TABLE 4 Effect of different cell inoculum sizes on antibody detection

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4. Optimization of VLP pseudovirus inoculum size in antibody detection

On the premise of fixing the number of infected cells, the test detects several different pseudovirus inoculum sizes, and uses 256, 512, 1025, 2050, 4100, 8200, 16400, 32800 and 65600TCID50 pseudoviruses in each hole to perform neutralization test, firstly, diluting the antibody by 30 times, then serially diluting the antibody by 3 times, then adding the pseudoviruses with different TCID50, incubating at 37 ℃ for 1h, digesting the cells, and adding 1X 10 in each hole ⁴ After 24h of infection, the cells were examined for luminescence, the inhibition was calculated, and IC50 was calculated using GraphPad.

The results (fig. 16) show: the TCID50 of each hole 2050 has higher sensitivity, and the neutralization experiment is carried out by selecting the TCID50 of about 2050 as the most virus amount, so that the error caused by dilution can be prevented, the neutralization experiment result is influenced, the error of the titer of the neutralizing antibody of the TCID50 from 512 to 65610 is not more than twice from the virus amount, and the influence of the virus amount on the neutralization result is very small, which is very important for the stability of the neutralization experiment.

5. Determination of neutralizing antibody sensitivity for detection of novel coronaVLP pseudoviruses

Diluting WHO new crown neutralizing antibody standard (NIBSC code:20/136, middle inspection yard) by 2 times, and diluting the standard with original and 2 timesSerial dilutions were performed 3-fold, 10 dilutions each, incubated with 2050TCID50 pseudovirus at 37 ℃ for 1h, 1 x 10 additions per well ⁴ Cells were detected after 18 h.

The result of the detection of the neutralizing antibody (figure 17) is an S-shaped curve, and the inhibition rate is higher than 98% when the dilution is within 810 times (12.35 IU/mL); the dilution rate is lower than 10% when the dilution is above 2187 times (< 0.457 IU/mL); the inhibition rate was about 50% at a dilution around 700-fold (. Apprxeq.1.42 IU/mL). The lowest detection limit of the S-shaped curve was 0.15IU/mL by linear analysis.

6. Method for detecting neutralizing antibody specificity analysis of novel coronaVLP pseudovirus

To verify the specificity of the novel coronaVLP pseudovirus detection neutralizing antibody method, 1X 10 was used ⁴ The amount of cell inoculum, amount of pseudovirus inoculum per well 2050TCID50, was measured on 10 samples of different sources of neocoronal negative serum and 8 samples of antibody positive serum. Serum was serially diluted 3-fold after the initial 30-fold dilution, incubated with pseudovirus for 1h at 37℃and HEK293T-ACE2+ Furin cells were added. After 18h, the results (FIG. 18) showed that none of the 10 antibody negative sera had a neutralization reaction, while 8 antibody positive sera showed a significant inhibitory effect. The method has good specificity.

7. Method for detecting neutralizing antibody by using novel coronaVLP pseudovirus and repeatedly analyzing

Cell inoculum size was 1X 10 ⁴ The inoculum size of the pseudovirus is 2050TCID50 per well, and the WHO new corona neutralizing antibody standard (NIBSC code:20/136, middle school of care; sample corresponding to number 11 of Table 5), national standard for neutralizing antibodies for new crowns (generation 1) (cat No. 280034-202001, middle inspection; sample corresponding to number 22 of Table 5), human immunoglobulin for WT new crowns (candidate standard of generation 2) (middle inspection; sample corresponding to number 5 of Table 5), plasma mix for WT healers (candidate standard of generation 2) (middle inspection; sample corresponding to number 66 of Table 5), plasma (high titer) of WT healers (middle inspection; sample corresponding to number 77 of Table 5), plasma mix for Delta healers (middle inspection; sample corresponding to number 88 of Table 5), plasma mix for healers (middle inspection; sample corresponding to number 99 of Table 5) were entered Antibody titer assays were performed and each sample was repeated three times.

The results are shown in Table 5, wherein the WHO new crown neutralizing antibody standard has a variation coefficient of 2.10%, the variation coefficient of six sera is lower than 10%, and the variation coefficient of 1 serum is 10.28%. From the results, the novel coronaVLP pseudovirus has better stability in detecting the titer of the neutralizing antibody.

TABLE 5 repeatability analysis of neutralizing antibodies for pseudovirus detection

8. Method for detecting neutralizing antibody by using novel coronaVLP pseudovirus and analyzing accuracy and precision

The established method for detecting neutralizing antibodies by using the novel crown VLP pseudovirus is used for detecting the national standard (generation 1) of the novel crown neutralizing antibodies with three concentrations of 1000U/mL (high) 500U/mL (medium) and 250U/mL (low). The concentration standards were tested in eight replicates in one experiment, and the coefficient of variation and recovery were calculated and the results are shown in table 6. The detection result shows that the variation coefficient of repeated detection is lower than 12%, the recovery rate is between 90.43% and 101.84%, and the method is good in precision and accuracy.

TABLE 6 precision and accuracy analysis of neutralizing antibodies for pseudovirus detection

9. Method correlation analysis of neutralizing antibody detection method for novel coronaVLP pseudovirus

The consistency of VLPs prepared by the optimization method of the application with live viruses is analyzed by comparing the detection results of antibody neutralization activities of clinical serum live viruses and VLP pseudoviruses.

The results showed that P values were less than 0.001, the correlation coefficient was equal to 0.7125, and the live and VLP pseudotoxic results had strong correlation (fig. 19); the GMT values of the results of neutralizing antibody titer assays for live and VLP pseudoviruses were substantially identical with no significant differences (fig. 20).

In addition, a neutralizing antibody experiment was performed on VLP pseudoviruses by means of mAb-9A8, with a neutralizing antibody titer of IC50=0.16. Mu.g/ml (FIG. 21), consistent with the live-virus results reported in the paper, also 0.16. Mu.g/ml (see Liu, S., et al, A broader neutralizing antibody against all the current VOCs and VOIs targets unique epitope of SARS-CoV-2RBD.Cell Discov,2022.8 (1): p.81.).

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims (19)

1. A coronavirus virus-like particle (VLP) comprising a mutant of an N protein derived from SARS-CoV-2, wherein said N protein mutant comprises one or more mutations compared to a corresponding wild-type N protein selected from the group consisting of:

(i) A residue at a position corresponding to amino acid residue 199 of SEQ ID NO. 1 (e.g., a P residue) is replaced with an L residue;

(ii) A residue at a position corresponding to amino acid residue 202 of SEQ ID NO. 1 (e.g., an S residue) is substituted with an R residue;

(iii) A residue at a position corresponding to amino acid residue 203 of SEQ ID NO. 1 (e.g., R residue) is replaced with an M residue;

preferably, the N protein mutant comprises mutations (i) and (ii), or the N protein mutant comprises mutations (i), (ii) and (iii);

preferably, the VLP is not capable of autonomous replication;

preferably, the wild type N protein derived from SARS-CoV-2 has the amino acid sequence as shown in SEQ ID NO. 1;

preferably, the N protein mutant has an amino acid sequence selected from any one of SEQ ID NOs 2 to 7.

2. The VLP of claim 1, further comprising: an RNA molecule comprising a packaging signal sequence; wherein the packaging signal sequence is selected from the PS9 region derived from SARS-CoV-2 or a truncation thereof, wherein the truncation is truncated at the 3' end of the PS9 region by 110-140 (e.g., 110-130, 110-126, 120-140, 126-130, 120-130, 126) nucleotide residues compared to the PS9 region;

Preferably, the packaging signal sequence is selected from the PS9 region truncations;

preferably, the truncate is truncated by 126 nucleotide residues at the 3' end of the PS9 region compared to the PS9 region;

preferably, the PS9 region has the nucleotide sequence shown as SEQ ID NO. 12;

preferably, the truncations of the PS9 region have the nucleotide sequence shown as SEQ ID NO. 13.

3. The VLP of claim 1 or 2, further comprising: m and E proteins; wherein the M and E proteins are selected from the group consisting of SARS-CoV-2 or SARS-CoV-1 derived M and E proteins;

preferably, the M and E proteins are selected from the group consisting of SARS-CoV-1 derived M and E proteins;

preferably, the M protein derived from SARS-CoV-2 has the amino acid sequence as shown in SEQ ID NO. 8;

preferably, the E protein derived from SARS-CoV-2 has the amino acid sequence as shown in SEQ ID NO. 10;

preferably, the M protein derived from SARS-CoV-1 has the amino acid sequence as shown in SEQ ID NO. 9;

preferably, the E protein derived from SARS-CoV-1 has the amino acid sequence as shown in SEQ ID NO. 11.

4. The VLP of any one of claims 1-3, further comprising: s protein; wherein the S protein is selected from the group consisting of coronavirus-derived S proteins;

Preferably, the coronavirus is selected from the group consisting of human coronaviruses (e.g., SARS-CoV-2, 229E, NL, SARS-CoV-1), sarbecovirus clade a (SARS-CoV-1-like coronavirus) and Sarbecovirus clade 1b (SARS-CoV-2-like coronavirus);

preferably, the coronavirus is selected from SARS-CoV-2 (e.g., omicron series strain), 229E, NL, SARS-CoV-1, sin852, GZ-C, sino1-11, urbani, HGZ8L1-A, GD01, PC4-127, PC4-13, PC4-137, GD03T0013, GZ0402, SZ1, LYRa11, WIV1, rs7327, rs4231, rsSHC014, rs4084, raTG13, pangolin_GD (1/2019), pangolin_GX (P5L);

preferably, the SARS-CoV-2 is selected from the group consisting of Wuhan Hu-1 strain, B.1 strain, B.1.1.7 strain, B.1.351 strain, P.1 strain, B.1.671.2 strain, BA.1 strain, BA.2 strain, BA.3 strain, BA.4/5 strain, BA.2.12.1 strain;

preferably, the S protein derived from coronavirus has an amino acid sequence selected from the group consisting of SEQ ID NOS.14-45.

5. The VLP of any one of claims 1-4, comprising or consisting of:

(1) An N protein mutant as defined in claim 1;

(2) An S protein as defined in claim 4;

(3) An RNA molecule comprising a PS9 region truncate as defined in claim 2;

(4) The M and E proteins derived from SARS-CoV-1 as defined in claim 3.

6. The VLP of any one of claims 1-5, wherein said RNA molecule comprising a packaging signal sequence further comprises a coding sequence for a reporter protein; preferably, the reporter protein is selected from the group consisting of luciferase and fluorescent proteins;

preferably, the RNA molecule further comprises a coding sequence for a luciferase; preferably, the amino acid sequence of the luciferase is shown in SEQ ID NO. 46.

7. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an N protein mutant, wherein the N protein mutant is as defined in claim 1.

8. The isolated nucleic acid molecule of claim 7, further comprising: a coding sequence for an S protein as defined in claim 4; a coding sequence of an RNA molecule comprising a packaging signal sequence as defined in claim 2; and/or the coding sequences for the M protein and the E protein as defined in claim 3;

preferably, the isolated nucleic acid molecule comprises: a coding sequence for an N protein mutant as defined in claim 1; a coding sequence for an S protein as defined in claim 4; a coding sequence of an RNA molecule comprising a PS9 region truncations as defined in claim 2; and the coding sequences of the M and E proteins derived from SARS-CoV-1 as defined in claim 3.

9. A vector comprising the isolated nucleic acid molecule of claim 7 or 8.

10. A carrier system comprising one or more carriers, wherein the one or more carriers comprise:

(1) A coding sequence for an N protein mutant, said N protein mutant being as defined in claim 1;

(2) A coding sequence for an S protein as defined in claim 4;

(3) A coding sequence of an RNA molecule comprising a packaging signal sequence, said RNA molecule being as defined in claim 2;

(4) Coding sequences for M and E proteins as defined in claim 3;

preferably, the sequence of (3) is the coding sequence of an RNA molecule comprising a PS9 region truncate;

preferably, the sequences of (4) are the coding sequences for the M and E proteins derived from SARS-CoV-1.

11. The vector system of claim 10, wherein the sequence in (3) is located in a first vector and the sequences in (1), (2) and (4) are located in one or more additional vectors;

preferably, the sequence of (3) is located in a first vector, the sequences of (1) and (2) are located in a second vector and a third vector, respectively, and the M protein coding sequence and the E protein coding sequence of (4) are co-located in a fourth vector;

Preferably, the M protein coding sequence and the E protein coding sequence of (4) are linked by an IRES coding sequence;

preferably, the IRES has a coding sequence as shown in SEQ ID NO. 47.

12. The vector system of any one of claims 10-11, wherein the RNA molecule comprising a packaging signal sequence further comprises a coding sequence for a reporter protein; preferably, the reporter protein is selected from the group consisting of luciferase and fluorescent proteins;

preferably, the RNA molecule further comprises a coding sequence for a luciferase; preferably, the amino acid sequence of the luciferase is shown in SEQ ID NO. 46.

13. A host cell comprising the vector system of any one of claims 10-12.

14. A method of making a coronavirus VLP comprising: culturing the host cell of claim 13 under conditions that allow expression of the VLP; and, recovering the expressed VLP;

preferably, the method comprises: introducing and culturing the vector system of any one of claims 10-12 into a host cell and recovering VLPs in the culture supernatant of said host cell.

15. Use of the vector system of any one of claims 10-12 or the host cell of claim 13 for the preparation of coronavirus VLPs.

16. Use of the coronavirus VLP of any one of claims 1-6 for screening a drug or detecting antibody activity (e.g., binding activity and/or neutralizing activity);

preferably, the drug is a drug that inhibits coronavirus infection;

preferably, the antibody is an antibody against coronavirus.

17. A method of detecting neutralizing activity of an antibody comprising:

(1) Contacting a sample comprising an antibody to be tested with the VLP of any one of claims 1-6;

(2) Contacting the product of step (1) with a cell, said cell being capable of being infected with a coronavirus; and, a step of, in the first embodiment,

(3) Detecting the infection rate of the cells, thereby evaluating the neutralizing activity of the antibody to be tested;

preferably, the antibody is an antibody against coronavirus.

18. The method of claim 17, wherein the VLP comprises a coding sequence for a reporter protein;

preferably, in step (3), the infection rate of the cell is detected by expression of the reporter protein in the cell;

preferably, the reporter protein is selected from the group consisting of luciferase and fluorescent proteins.

19. The method of claim 17 or 18, provided with one or more selected from the group consisting of:

(a) The cells are cells expressing ACE2 and Furin; preferably, the cell is a 293T cell expressing ACE2 and Furin;

(b) In step (2), the product of step (1) is contacted with the cells for a period of 15-22 hours (e.g., 15-20 hours, 15-18 hours, 18-22 hours, 18-20 hours, 18 hours);

(c) In step (1), the VLP is vaccinated in an amount of 512-65610TCID50.