CN117357645A

CN117357645A - Novel universal Mosaic-RBD nanoparticle vaccine for coronaviruses and preparation method and application thereof

Info

Publication number: CN117357645A
Application number: CN202311258618.3A
Authority: CN
Inventors: 施一; 高福; 景佳美; 王敏; 杨金月; 彭齐; 赵学金; 高思维
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2023-09-27
Filing date: 2023-09-27
Publication date: 2024-01-09

Abstract

The invention discloses a novel universal Mosaic-RBD nanoparticle vaccine for coronaviruses, and a preparation method and application thereof. The invention provides a novel coronavirus Mosaic-RBD nanoparticle vaccine, which is obtained by embedding RBD proteins of three SARS-CoV-2variants of Delta, BA.5 and XBB.1.5 on 24-polymer ferritin nanoparticles together through a spatag 003-spatcher 003 covalent connection system. The Mosaic-RBD nanoparticle vaccine provided by the invention can stimulate mice to generate strong antibody response, has a strong neutralization effect on prototype strains, delta and a series of Omikovia strains, and is an ideal novel coronavirus universal vaccine.

Description

Novel universal Mosaic-RBD nanoparticle vaccine for coronaviruses and preparation method and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a novel universal Mosaic-RBD nanoparticle vaccine for coronaviruses, a preparation method and application thereof.

Background

Covd-19 is an acute respiratory infectious disease caused by infection with a novel coronavirus (also known as the new coronavirus, SARS-CoV-2). The novel coronavirus belongs to the genus beta-coronavirus of the family coronaviridae, has a envelope and has a genome of positive strand RNA. The new coronavirus is a novel coronavirus with high infectivity, and poses serious threat to human health and public safety. Although there are many new coronavirus vaccines on the market, new coronavirus variants continue to appear and become popular, some of which escape the immune response of the existing vaccine, causing breakthrough infections. The Omicron variant has severe immune escape to both new coronavirus neutralizing antibody drugs and vaccine-activated humoral immune responses. However, it has been reported that vaccine-activated immune responses developed with omacron sequences are strong against omacron variants, but cross-reactions against prototype strains and other strains are weak, and do not adapt to coexistence of the current prototype strain and various variants. Therefore, it is necessary to develop a vaccine which has a strong protective effect against the current epidemic strains and can induce a broad-spectrum immune response.

The spike protein (S) on the surface of the novel coronavirus is responsible for the recognition and membrane fusion of the virus and host cell receptor, and the Receptor Binding Domain (RBD) at the C-terminal end of the S protein is considered to be a main area for inducing the organism to produce neutralizing antibodies, and is also an important vaccine target.

Disclosure of Invention

The invention aims to provide a novel universal Mosaic-RBD nanoparticle vaccine for coronaviruses, a preparation method and application thereof.

In a first aspect, the invention claims a novel coronavirus Mosaic-RBD nanoparticle vaccine.

The novel coronavirus Mosaic-RBD nanoparticle vaccine provided by the invention is obtained by embedding RBD proteins of three SARS-CoV-2variants of Delta, BA.5 and XBB.1.5 on 24-mer ferritin nanoparticles together through a spatag 003-spatcher 003 covalent connection system.

Further, the novel coronavirus Mosaic-RBD nanoparticle vaccine can be prepared by the following steps: incubating the spatcher 003-ferrotin protein and the RBD-spattag 003 protein in a buffer system according to a molar ratio of 1:5 to realize covalent connection of the spatcher 003-ferrotin protein and the RBD-spattag 003 protein, and naming the obtained connection product as Mosaic 3_RBD-ferrotin, namely the novel coronavirus Mosaic-RBD nanoparticle vaccine.

Wherein the sportsher 003-ferritin protein is formed by fusing the sportsher 003 protein and the ferritin protein.

The RBD-spytag003 protein is formed by mixing three proteins of Delta_RBD-spytag003 protein, BA.5_RBD-spytag003 protein and XBB.1.5_RBD-spytag003 protein in an equimolar ratio. The number of moles of RBD-spytag003 protein is the sum of the numbers of the three hatching eggs Bai Maer.

The Delta-RBD-spytag 003 protein is formed by fusion of an RBD sequence of a Delta strain of SARS-CoV-2and the spytag003 protein.

The BA.5_RBD-spytag003 protein is formed by fusing an RBD sequence of a BA.5 strain of SARS-CoV-2 with the spytag003 protein.

The XBB.1.5_RBD-spytag003 protein is formed by fusing an RBD sequence of an XBB.1.5 strain of SARS-CoV-2 with the spytag003 protein.

Further, the amino acid sequence of the spycatcher003 protein is shown in positions 2-119 of SEQ ID No. 1.

Further, the amino acid sequence of the ferritin protein is shown in the 123 th to 295 th positions of SEQ ID No. 1.

Further, the RBD sequence of the Delta strain of SARS-CoV-2 is shown at positions 19-237 of SEQ ID No. 2.

Further, RBD sequence of BA.5 strain of SARS-CoV-2 is shown in 19-237 of SEQ ID No. 3.

Further, the RBD sequence of XBB.1.5 strain of SARS-CoV-2 is shown at positions 19-237 of SEQ ID No. 4.

Further, the amino acid sequence of the spytag003 protein is shown in positions 238-253 of SEQ ID No.2 (or SEQ ID No.3 or SEQ ID No. 4).

In a specific embodiment of the invention, the amino acid sequence of the spycatcher003-ferritin protein is shown as SEQ ID No.1 or as positions 1-295 of SEQ ID No. 1.

Further, the Delta_RBD-spytag003 protein, the BA.5_RBD-spytag003 protein and the XBB.1.5_RBD-spytag003 protein also contain signal peptide sequences; the signal peptide sequence is shown in positions 1-18 of SEQ ID No.2 (or SEQ ID No.3 or SEQ ID No. 4).

More specifically, in a specific embodiment of the present invention, the amino acid sequence of the Delta_RBD-spytag003 protein is shown in SEQ ID No.2 or as positions 1-253 of SEQ ID No. 2.

More specifically, in a specific embodiment of the present invention, the amino acid sequence of the BA.5_RBD-spytag003 protein is shown in SEQ ID No.3 or as positions 1-253 of SEQ ID No. 3.

More specifically, in a specific embodiment of the present invention, the amino acid sequence of the XBB.1.5_RBD-spytag003 protein is shown in SEQ ID No.4 or as positions 1-253 of SEQ ID No. 4.

In a specific embodiment of the present invention, the incubation in the buffer system is specifically: incubate in buffer A at 25℃for 8h. Wherein the composition of the buffer A is as follows: 25mM Tris,150mM NaCl,pH 8.0.

In a second aspect, the invention claims a method of preparing a novel coronavirus Mosaic-RBD nanoparticle vaccine.

The method of preparing a novel coronavirus Mosaic-RBD nanoparticle vaccine as claimed in the present invention may comprise the steps as described in the first aspect hereinbefore.

In a third aspect, the invention claims a kit for preparing the novel coronavirus Mosaic-RBD nanoparticle vaccine as described in the first aspect above.

The kit for preparing the novel coronavirus Mosaic-RBD nanoparticle vaccine as claimed in the first aspect of the present invention may comprise: the spycatcher 003-ferrotin protein described in the first aspect above and the RBD-spytag003 protein.

Further, the kit may also comprise a buffer A as described in the first aspect above.

In a fourth aspect, the invention claims the use of a novel coronavirus Mosaic-RBD nanoparticle vaccine as described in the first aspect hereinbefore or a kit as described in the third aspect hereinbefore for any of the following:

(A1) Preparing a product capable of neutralizing SARS-CoV-2;

(A2) Preparing a product for treating and/or preventing diseases caused by SARS-CoV-2 infection;

(A3) A product for improving symptoms due to SARS-CoV-2 infection is prepared.

Further, SARS-CoV-2 is a protogenic strain or a variant strain.

Still further, the variant strain may be a Delta strain, an Omicron BA.1 strain, an Omicron BA.2 strain, an Omicron BA.5 strain, an Omicron BF.7 strain, an Omicron BQ.1 strain, an Omicron BQ.1.1 strain, an Omicron XBB strain, an Omicron XBB.1.5 strain, an Omicron XBB.1.16 strain or an Omicron CH.1.1 strain.

The invention designs a novel universal Mosaic-RBD nanoparticle vaccine for coronaviruses, which can stimulate mice to generate strong antibody reaction and has stronger neutralization effect on prototype strains, delta strains and a series of Omiko-curt strains by a spatag 003-spyccher 003 covalent connection system and embedding RBD proteins of three variant strains of Delta, BA.5 and XBB.1.5 on 24-polymer ferritin nanoparticles.

Drawings

FIG. 1 shows gel filtration chromatography and gel electrophoresis analysis of the spatcher 003-ferritin.

FIG. 2 shows gel filtration chromatography and gel electrophoresis analysis of Delta RBD-spytag 003.

FIG. 3 shows the gel filtration chromatography and gel electrophoresis analysis of BA.5_RBD-spytag 003.

FIG. 4 shows the gel filtration chromatography and gel electrophoresis analysis of XBB.1.5_RBD-spytag 003.

FIG. 5 shows the gel filtration chromatography curve of the Delta_RBD-ferritin protein and the SDS-PAGE identification of the elution peak.

FIG. 6 is a negative electron microscopic view of the Delta RBD-ferritin nanoparticle.

FIG. 7 shows the gel filtration chromatography curve of BA.5-RBD-ferritin and the SDS-PAGE identification of the elution peak.

FIG. 8 is a negative electron microscope observation of BA.5-RBD-ferritin nanoparticles.

FIG. 9 shows the gel filtration chromatography curve of XBB.1.5_RBD-ferritin and the SDS-PAGE identification of the elution peak.

FIG. 10 is a negative electron microscopic view of XBB.1.5-RBD-ferritin nanoparticles.

FIG. 11 shows the gel filtration chromatography curve of Mosaic3_RBD-ferritin and the SDS-PAGE identification of the elution peak.

FIG. 12 is a negative electron microscope observation of the Mosaic3_RBD-ferritin nanoparticle.

Fig. 13 is a mouse immunization protocol.

FIG. 14 is a serum-free RBD specific antibody titer assay.

FIG. 15 is a diagram of a di-immune serum RBD specific antibody titer assay.

FIG. 16 is a trisimmune serum RBD specific antibody titer assay.

FIG. 17 shows the neutralizing antibody titers of the di-immune serum pseudoviruses.

FIG. 18 is a graph showing neutralizing antibody titers against the three-way serum pseudovirus.

FIG. 19 is a titer of a di-immune serum against 12 pseudovirus neutralizing antibodies.

FIG. 20 is a three-way serum neutralizing antibody titer assay against 12 pseudoviruses.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 construction, expression and purification of the SPycatcher003-ferritin 24 mer nanoparticle protein plasmid construction

The N-terminal of the helicobacter pylori ferritin sequence (shown as 123-295 of SEQ ID No. 1) is connected with the spycatter 003 sequence (shown as 2-119 of SEQ ID No. 1) and a start codon is added, and the C-terminal of the spycatter 003 sequence is added with a Thrombin cleavage site sequence (LVPRGS), 8 histidines (HHHHHH) and a stop codon in sequence to obtain the spycatter 003-ferritin construct (the amino acid sequence of which is shown as SEQ ID No. 1). The amino acid sequence of the construct was optimized using the Escherichia coli codon, and the DNA sequence (shown as SEQ ID No. 5) was synthesized by Souzhou Jin Weizhi Biotechnology Co., ltd, and the synthesized DNA sequence was cloned into vector pET28a (+) by 5'NcoI and 3' XhoI to obtain a plasmid expressing spycatcher 003-ferritin.

The structure of the recombinant plasmid is described as follows: the recombinant plasmid is obtained by inserting the DNA fragment shown in 7 th to 392 th positions of SEQ ID No.5 between the cleavage sites NcoI and XhoI of the pET28a (+) vector.

The 3 rd to 932 rd positions of SEQ ID No.5 encode the amino acid sequence shown as SEQ ID No.1 (spycatcher 003-ferritin protein). The 2-119 th site of SEQ ID No.1 is spycatcher003 sequence, the 120-122 th site is linker, the 123-295 th site is ferritin sequence, the 296-301 th site is Thrombin cleavage site sequence, and the 302-309 th site is 8 His.

2. Protein expression and purification

The plasmid expressing the spycatcher003-ferritin constructed in the step one is introduced into E.coli BL21 competent strain by a heat shock transformation method, and then coated on a kanamycin-resistant LB plate at 37 DEG CAfter 12h of inversion culture, several colonies were randomly selected for small amounts of test expression of the target protein to determine the optimal expression conditions. Single colonies were picked and transferred to 6mL LB medium, cultured to OD600 of about 0.6-0.8, induced by adding 0.5mM IPTG, and expressed at 37℃for 5h. The cells were collected by centrifugation, the culture supernatant was discarded, and about 100mL of buffer (25 mmol/L Tris,150mmol/L NaCl, pH 8) was added to each 2L of the cultured cells for resuspension, the bacterial suspension was sonicated, the supernatant was collected by centrifugation, and the supernatant was filtered with a 0.22 μm microporous membrane and subjected to His affinity chromatography and ion exchange chromatography. Concentrating the target protein eluent collected by ion exchange chromatography with a 100kD concentration tube to a final volume of less than 1ml; then pass through Superose ^TM Gel filtration chromatography was performed on a 6 incoase 10/300GL column (GE Healthcare) to further purify the target protein, and during the chromatography, the eluate at the elution peak was collected for SDS-PAGE identification of the target protein.

The SDS-PAGE identification of gel filtration chromatography curve and elution peak of the spycatcher003-ferritin protein is shown in FIG. 1. The SDS-PAGE gel electrophoresis analysis result of the elution peak shows that the molecular weight of the eluted protein is correct, the purified spycatcher003-ferritin protein is proved to be obtained, and the electrophoresis band shows that the purified target protein has higher purity and yield.

EXAMPLE 2 construction, expression and purification of RBD-spytag003 proteins of three variants of Delta, BA.5 and XBB.1.5

Constructs expressing Delta_RBD-spytag003, BA.5_RBD-spytag 003H and XBB.1.5_RBD-spytag003 were designed as follows:

(1) The N end of RBD sequence (19-237 th site of SEQ ID No. 2) of new coronavirus Delta strain is connected with signal peptide (MHSSALLCCLVLLTGVRA), and the C end of RBD sequence is added with spatag 003 sequence (RGVPHIVMVDAYKRYK), thrombin enzyme cutting site sequence (LVPRGS), 6 histidines (HHHHH) and stop codon in turn to obtain Delta_RBD-spatag 003 construct (its amino acid sequence is shown in SEQ ID No. 2).

(2) A signal peptide (MHSSALLCCLVLLTGVRA) is connected to the N end of RBD sequence (shown as 19-237 of SEQ ID No. 3) of a new coronavirus strain, and a spatag 003 sequence (RGVPHIVMVDAYKRYK), a Thrombin cleavage site sequence (LVPRGS), 6 histidines (HHHHH) and a stop codon are sequentially added to the C end of the RBD sequence to obtain a BA.5_RBD-spatag 003 construct (the amino acid sequence of which is shown as SEQ ID No. 3).

(3) A signal peptide (MHSSALLCCLVLLTGVRA) is connected to the N end of RBD sequence (shown as 19-237 th site of SEQ ID No. 4) of a novel coronavirus XBB.1.5 strain, and a spytag003 sequence (RGVPHIVMVDAYKRYK), a Thrombin cleavage site sequence (LVPRGS), 6 histidines (HHHHH) and a stop codon are sequentially added to the C end of the RBD sequence to obtain an XBB.1.5_RBD-spytag003 construct (the amino acid sequence of which is shown as SEQ ID No. 4).

1. Plasmid construction

The amino acid sequences of the above three constructs were optimized using human codons, and a Kozak sequence gccac was added upstream of these DNA coding sequences, respectively, and DNA sequences (SEQ ID No.6, SEQ ID No.7 and SEQ ID No. 8) were synthesized by su Jin Weizhi biotechnology limited; the synthesized 3-segment DNA sequence was cloned into pCAGGS plasmid vector (supplied by SU Jin Weizhi) through EcoRI and XhoI cleavage sites, and expression plasmids for expressing the prototypes Delta_RBD-spytag003, BA.5_RBD-spytag003 and XBB.1.5_RBD-spytag003 were obtained, respectively.

The structure of the Delta_RBD-spytag003 expression plasmid is described as: the recombinant plasmid was obtained by inserting the DNA fragment shown in positions 7-810 of SEQ ID No.6 between EcoRI and XhoI of the pCAGGS vector.

The structure of the BA.5_RBD-spytag003 expression plasmid is described as: the recombinant plasmid was obtained by inserting the DNA fragment shown in positions 7-810 of SEQ ID No.7 between EcoRI and XhoI of the pCAGGS vector.

The structure of the XBB.1.5_RBD-spytag003 expression plasmid is described as: the recombinant plasmid was obtained by inserting the DNA fragment shown in positions 7-810 of SEQ ID No.8 between EcoRI and XhoI of the pCAGGS vector.

Positions 13-810 of SEQ ID No.6 encode the amino acid sequence shown as SEQ ID No.2 (Delta_RBD-spatag 003 protein). The 1 st to 18 th positions of SEQ ID No.2 are signal peptide sequences, the 19 th to 237 th positions are Delta_RBD, the 238 th to 253 th positions are spytag003 sequence, the 254 th to 259 th positions are Thrombin cleavage site sequences, and the 260 th to 265 th positions are 6His.

Positions 13-810 of SEQ ID No.7 encode the amino acid sequence shown in SEQ ID No.3 (BA.5-RBD-spytag 003 protein). The 1 st to 18 th positions of SEQ ID No.3 are signal peptide sequences, the 19 th to 237 th positions are BA.5_RBD, the 238 th to 253 th positions are spytag003 sequence, the 254 th to 259 th positions are Thrombin cleavage site sequences, and the 260 th to 265 th positions are 6His.

Positions 13-810 of SEQ ID No.8 encode the amino acid sequence shown in SEQ ID No.4 (XBB.1.5_RBD-spytag 003 protein). The 1 st to 18 th positions of SEQ ID No.4 are signal peptide sequences, the 19 th to 237 th positions are XBB.1.5_RBD, the 238 th to 253 th positions are spytag003 sequence, the 254 th to 259 th positions are Thrombin enzyme cleavage site sequences, and the 260 th to 265 th positions are 6His.

2. Protein expression and purification

Transfection of the plasmid expressing the Delta_RBD-spytag003 protein constructed in step one into an Expi293F ^TM Cells were collected by centrifugation after 5 days, the supernatant was filtered through a 0.22 μm filter, the resulting cell supernatant was adsorbed on a nickel affinity column (His Trap, GE Healthcare) at 4℃and washed with buffer A (25mM Tris,150mM NaCl,pH 8.0) to remove non-specific binding proteins, then the target protein was eluted from the His Trap with buffer B (25mM Tris,150mM NaCl,pH 8.0,300mM imidazole), and the eluate at the elution peak was collected for SDS-PAGE identification and subsequent gel filtration chromatography of the target protein. Concentrating the eluent at the collected eluting peak by using a 10kD concentration tube to a final volume of less than 1ml; then pass through Superdex ^TM 75 The Increase 10/300GL column (GE Healthcare) is subjected to gel filtration chromatography to further purify the target protein, and during the chromatography, the eluent at the eluting peak is collected for SDS-PAGE identification of the target protein.

The SDS-PAGE identification of the gel filtration chromatography curve and elution peak of the Delta_RBD-spytag003 protein is shown in FIG. 2. The SDS-PAGE gel electrophoresis analysis result of the elution peak shows that the molecular weight of the elution protein is correct, the Delta-RBD-spytag 003 protein is obtained through purification, and the electrophoresis band shows that the purified target protein has higher purity and yield.

The same method is used for expressing and purifying the BA.5_RBD-sptag 003 and XBB.1.5_RBD-sptag 003 proteins, gel filtration chromatography curves of the BA.5_RBD-sptag 003 and XBB.1.5_RBD-sptag 003 and SDS-PAGE identification results of elution peaks thereof are respectively shown in fig. 3 and 4, on each gel filtration chromatography curve, an arrow indicates an elution peak corresponding to the target protein, the eluate at the elution peak is subjected to SDS-PAGE gel electrophoresis analysis, and the SDS-PAGE gel electrophoresis analysis results of the elution peaks show that the molecular weight of the eluted proteins is correct, so that the purified target proteins are obtained, namely the BA.5_RBD-sptag 003 and XBB.1.5_RBD-sptag 003 proteins, and the purified target proteins have higher purity and yield.

EXAMPLE 3 preparation and purification of RBD-ferritin homonanoparticles of three variants of Delta, BA.5 and XBB.1.5 and preparation, purification and characterization of chimeric Mosaic3_RBD-ferritin nanoparticles

The purified spatcher 003-ferritin protein (SEQ ID No. 1) and Delta_RBD-spytag003 protein (SEQ ID No. 2) were incubated in buffer A (25mM Tris,150mM NaCl,pH 8.0) at 25℃overnight in a molar ratio of 1:5 for 8h, then gel filtration chromatography was performed through Superdex 75 Increase 10/300GL column (GE Healthcare), the covalently attached Delta_RBD-ferritin protein and excess Delta_RBD-spytag003 protein were purified, and during chromatography, the eluate at the elution peak was collected for SDS-PAGE identification of the protein of interest.

The gel filtration chromatography curve of the Delta_RBD-ferritin protein and the SDS-PAGE identification result of the elution peak thereof are shown in FIG. 5, wherein peak 1 is the Delta_RBD-ferritin nanoparticle protein which is successfully covalently connected, and peak 2 is the excess Delta_RBD-spytag003 protein. SDS-PAGE gel electrophoresis analysis results of the elution peak show that the molecular weight of the eluted protein is correct, and the covalent linkage efficiency is close to 100%. The Delta_RBD-ferritin protein was collected to prepare a negative dye sample and observed by an electron microscope, and the result is shown in FIG. 6, the uniformity of the prepared purified Delta_RBD-ferritin nanoparticle protein is better, and the particle size is about 18nm.

The same procedure was used to prepare, purify and characterize the BA.5-RBD-ferritin and XBB.1.5-RBD-ferritin nanoparticle proteins. The gel filtration chromatography curve of BA.5_RBD-ferritin and the SDS-PAGE identification result of the elution peak are shown in FIG. 7, wherein peak 1 is the BA.5_RBD-ferritin nanoparticle protein which is successfully covalently linked, and peak 2 is the excessive BA.5_RBD-spytag003 protein. SDS-PAGE gel electrophoresis analysis results of the elution peak show that the molecular weight of the eluted protein is correct, and the covalent linkage efficiency is close to 100%. The BA.5_RBD-ferritin protein was collected to prepare a negative-stain sample, which was observed by an electron microscope, and the result was shown in FIG. 8, and the prepared purified BA.5_RBD-ferritin nanoparticle protein was excellent in uniformity and had a particle size of about 18nm. The gel filtration chromatography curve of XBB.1.5_RBD-ferritin and the SDS-PAGE identification result of the elution peak thereof are shown in FIG. 9, wherein peak 1 is the XBB.1.5_RBD-ferritin nanoparticle protein which is successfully covalently connected, and peak 2 is the excessive BA.5_RBD-spytag003 protein. SDS-PAGE gel electrophoresis analysis results of the elution peak show that the molecular weight of the eluted protein is correct, and the covalent linkage efficiency is close to 100%. The XBB.1.5_RBD-ferritin protein is collected to prepare a negative dyeing sample, and the negative dyeing sample is observed by an electron microscope, and the result is shown in figure 10, and the prepared purified XBB.1.5_RBD-ferritin nanoparticle protein has good uniformity and a particle size of about 18nm.

As can be seen from the above experimental results, the covalent linking efficiencies of the preparation of Delta RBD-spytag003, BA.5_RBD-ferritin and XBB.1.5_RBD-ferritin were substantially identical, and the Mosaic3 RBD-ferritin nanoparticle protein was prepared, purified and characterized in the same manner by incubating the spycatcher003-ferritin protein and RBD-spytag003 protein in buffer A (formulation above) at a molar ratio of 1:5 for 8h overnight at 25℃wherein RBD-spytag003 was mixed by the three proteins Delta RBD-spytag003, BA.5_RBD-spytag003 and XBB.1.5_RBD-spytag003 in the same molar ratio. The gel filtration chromatography curve of the mosaic3_rbd-ferritin and the SDS-PAGE identification result of the elution peak thereof are shown in fig. 11, wherein peak 1 is the mosaic3_rbd-ferritin nanoparticle protein which is successfully covalently linked, and peak 2 is the three RBD-spytag003 proteins which are excessive. SDS-PAGE gel electrophoresis analysis results of the elution peak show that the molecular weight of the eluted protein is correct, and the covalent linkage efficiency is close to 100%. The negative staining sample prepared by collecting the Mosaic3_RBD-ferritin protein is observed by an electron microscope, and the result is shown in figure 12, and the prepared purified Mosaic3_RBD-ferritin nanoparticle protein has good uniformity and a particle size of about 18nm.

EXAMPLE 4 experiments on three homotype RBD-ferritin nanoparticle proteins and on Mosaic3_RBD-ferritin nanoparticle protein immunized mice

To examine the immunogenicity of the prepared nanoparticle proteins, we immunized BALB/c mice with purified three homotype RBD-ferritin nanoparticle proteins and Mosaic3_RBD-ferritin protein. BALB/c mice used were purchased from Vetong Lihua, inc., and were female, 6-8 weeks old. The immunization groups set up for the mouse immunization experiments used Mosaic3_RBD-ferritin, delta _RBD-ferritin, BA.5_RBD-ferritin, and XBB.1.5_RBD-ferritin as immunogens. The 3 kings RBD-ferritin immune group was mixed with three nanoparticles of Delta RBD-ferritin, BA.5 RBD-ferritin, XBB.1.5 RBD-ferritin in equimolar ratio, the spycatcher003-ferritin protein as the experimental control group, PBS as the negative control group, the mouse groups and vaccine doses as shown in Table 1, the immune doses were calculated as RBD molecular weight (the spycatcher003-ferritin group was calculated as the whole protein molecular weight). The immunogen is diluted to 100 mug/mL by PBS, and then mixed and emulsified with an adjuvant AddaVax according to the volume ratio of 1:1 to prepare the vaccine. The mixed vaccine was immunized against BALB/c mice, 6 per group. The experimental procedure of mice is shown in figure 13, all mice were vaccinated 3 times by intramuscular injection at weeks 0,3 and 6, respectively, with an inoculation volume of 100 μl each. The mice were collected by centrifugation at two weeks after each immunization, and stored in a-80 ℃ refrigerator, and then used to determine antigen-specific antibody titers and pseudovirus neutralization titers.

Table 1, mosaic-RBD nanoparticle vaccine immunized mice group and dose

Group of	Immunogens	Immunization dose	Adjuvant	Quantity of
					1	Mosaic3_RBD-ferritin	5μg	AddaVax	6
2	3kinds RBD-ferritin	5μg	AddaVax	6
					3	Delta_RBD-ferritin	5μg	AddaVax	6
4	BA.5_RBD-ferritin	5μg	AddaVax	6
					5	XBB.1.5_RBD-ferritin	5μg	AddaVax	6
6	spycatcher003-ferritin	5μg	AddaVax	6
					7	PBS	-	AddaVax	6

Example 5 detection of antigen-specific antibody titres produced by vaccine by enzyme-linked immunosorbent assay (ELISA)

Delta_RBD (positions 19-237 of SEQ ID No. 2), BA.5_RBD (positions 19-237 of SEQ ID No. 3) or XBB.1.5_RBD (positions 19-237 of SEQ ID No. 4) monomeric proteins were diluted to 3. Mu.g/mL with ELISA coating solution, 100. Mu.l per well of a 96-well ELISA plate (Coring, 3590) were coated overnight at 4 ℃. The coating liquid was discarded, washed once with PBS, and blocked for 1 hour at room temperature with 5% skimmed milk prepared with PBS as blocking liquid. After blocking, the cells were washed once with PBS. The serum of the mice is diluted during the blocking period, the serum samples are sequentially diluted according to a gradient of 4 times from 20 times, then are added into a 96-well plate, and are incubated for 1h at room temperature, and negative control is blocking solution. Then, the goat anti-mouse secondary antibody coupled with HRP diluted 1:2000 in blocking solution was added to the mixture after washing 4 times with PBST, and incubated at room temperature for 1h, followed by washing 4 times with PBST. After a proper reaction time, 50. Mu.L of 2M hydrochloric acid was added to stop the reaction, and the OD450 reading was measured on a microplate reader. Antibody titer values were defined as the highest dilution of serum with a response value greater than 2.5 times the negative control value, and the titer of the sample was defined as half of the lowest dilution when the response value for the lowest dilution (limit of detection) was still less than 2.5 times the background value.

The experimental results of RBD specific antibody titer of serum after three immunizations are shown in FIG. 14, FIG. 15 and FIG. 16, the mouse serum of each group of immunized RBD-Ferritin nano-particles can generate antibodies with Delta_RBD, BA.5_RBD and XBB.1.5_RBD specificities, and the titer of the antibodies after the second immunization is obviously increased compared with that of the first immunization and can basically reach 10 ⁶ And controlNo specific antibody is produced in the group of the sportsher 003-ferritin and the PBS group, which indicates that the three homotype RBD-ferritin nanoparticle proteins and the Mosaic3_RBD-ferritin nanoparticle proteins prepared by the invention have better immunogenicity.

EXAMPLE 6 pseudo-Virus neutralization assay detection of broad-spectrum neutralization by vaccine

The 50% pseudovirus neutralization titers (pVNT 50) of the pooled immune mouse serum against the pseudoviruses of SARS-CoV-2 prototype strain (PT) and variant strains, including Delta (B.1.617.2), omicron BA.1, BA.2, BA.5, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.16, CH.1.1, were measured, respectively, using the novel coronavirus pseudoviruses. The novel coronavirus pseudoviruses used in this example are pseudoviruses prepared based on the Vesicular Stomatitis Virus (VSV) backbone, which exhibit the novel coronavirus S protein, for preparation as described in the methods section of the published papers of the subject group (Effects of a Prolonged Booster Interval on Neutralization of Omicron Variant, N Engl J Med,2022, PMID: 35081296). The method comprises the following steps:

pseudoviruses of the SARS-CoV-2 prototype strain (SARS-CoV-2-PT) and the variant strain Delta (B.1.617.2) are described in "Zhao X, zheng A, li D, zhang R, sun H, wang Q, gao GF, han P, dai L.neutral of ZF2001-elicited antisera to SARS-CoV-2variants.Lancet Microbe.2021Oct;2 (10) e494.doi. 10.1016/S2666-5247 (21) 00217-2.Epub 2021 Aug 20.PMID:34458880; PMCID PMC8378832", wherein the SARS-CoV-2 prototype strain is referred to herein as" SARS-CoV-2wild type "is publicly available from the applicant and is not available for use as far as possible in repeating the experiments of the present invention.

Pseudoviruses of SARS-CoV-2variant BA.1 are described in "Huang M, wu L, zheng A, xie Y, he Q, rong X, han P, du P, han P, zhang Z, zhao R, jia Y, li L, bai B, hu Z, hu S, niu S, hu Y, liu H, liu B, cui K, li W, zhao X, liu K, qi J, wang Q, gao GF.atlas of currently available human neutralizing antibodies against SARS-CoV-2and escape by Omicron sub-varians BA.1/BA.2/BA.3. Immunity.2022Aug 9;55 (8) 1501-1514.e3.doi:10.1016/j.immini.2022.06.005.Epub 2022Jun 15.PMID:35777362; PMCID PMC9197780", available to the public from the applicant, is used as far as possible for repeated experiments of the invention, and cannot be used as far as it is.

Pseudoviruses of SARS-CoV-2variant strains BA.2, BA.5, BQ.1.1, XBB are described in "He Q, wu L, xu Z, wang X, xie Y, chai Y, zheng A, zhou J, qiao S, huang M, shang G, zhao X, feng Y, qi J, gao GF, wang Q.an updated atlas of antibody evasion by SARS-CoV-2Omicron sub-variants including BQ.1.1and XBB.cell Rep Med.2023 Apr 18;4 (4) 100991.doi:10.1016/j.xcrm.2023.100991.epub 2023 Mar 21.PMID:37019110; PMCID PMC10027947", available to the public from the applicant, is used as far as possible for repeated experiments of the invention, and cannot be used as far as it is.

The rest pseudoviruses of SARS-CoV-2variant strains are prepared by the following method:

the nucleotide sequences BF.7-S-del18 (SEQ ID No. 9), BQ.1-S-del18 (SEQ ID No. 10), XBB.1.5-S-del18 (SEQ ID No. 11), CH.1.1-S-del18 (SEQ ID No. 12), XBB.1.16-S-del18 (SEQ ID No. 13) were obtained by removing the 18-amino acid nucleotide after the S protein encoding the variant strains Omicron (BF.7), omicron (BQ.1), omicron (XBB.1.5), omicron (CH.1.1) and Omicron (XBB.1.16) and were synthesized by GENEWIZ. The nucleotide sequences obtained above were cloned between EcoRI and XholI of pCAGGS expression vector (supplied by SU Jin Weizhi company) through EcoRI and XholI cleavage sites, respectively, and the recombinant expression plasmids were obtained after sequencing verification to be correct, and named pCAGGS-BF.7-S-del18, pCAGGS-BQ.1-S-del18, pCAGGS-XBB.1.5-S-del18, pCAGGS-CH.1.1-S-del18, pCAGGS-XBB.1.16-S-del18, respectively, depending on the difference of the insertion sequences.

Each of the above-constructed expression plasmids of S protein was transfected at 30. Mu.g in 10cm two plates (cell amount 80% -90%) from ATCC cell bank, after 4 hours, the supernatant was changed to DMEM (10% FBS), after 24 hours, VSV-. DELTA.G-GFP pseudovirus (Wohyokokumi brain science, inc., V03001) was added at 5ml, after 2 hours, DMEM (containing 10% FBS and VSV-G antibody, diluted at 1:1000 ratio, expressed by I1Hybridoma ATCC CRL2700 cells, final concentration 10. Mu.g/ml, the objective of adding VSV-G antibody was to neutralize the original VSV pseudovirus, after 20 hours of pseudovirus addition, supernatant was collected at 3000rpm,10min was centrifuged, and 0.45 μm was filtered. Sub-packaging and freezing to-80 ℃. Cells not transfected with S protein expression plasmid were subsequently also added with the group of VSV-. DELTA.G-GFP pseudovirus and antibody as a pseudovirus packaging control.

Through the above steps, pseudoviruses of Omicron (bf.7), omicron (bq.1), omicron (xbb.1.5), omicron (ch.1.1) and Omicron (xbb.1.16) were obtained, respectively.

Pseudovirus titer assay:

the day before the experiment, the Vero cells in logarithmic growth phase (from ATCC cell bank) were harvested by digestion with pancreatin, counted and re-inoculated in 96-well plates, and used for the experiment when cell density reached 80-100% for 18-24 hours; pseudoviruses were diluted 100 μl/well in DMEM (containing 10% FBS) and added to 96-well plates, three wells were made per sample in parallel, and CQ1 was read after 15h to calculate titers.

Pseudovirus neutralization assay:

the method for detecting neutralizing antibody titer of novel coronavirus pseudovirus (hereinafter referred to as pseudovirus) is as follows: in 96-well plates, immunized mouse serum was diluted 2-fold in gradient ratio, then mixed with pseudovirus separately, and blank medium was also mixed with pseudovirus as a control, and incubated at 37℃for 1 hour. The immune serum-pseudovirus mixture was transferred to 96-well plates that were confluent with Vero cells. After 15 hours, positive cell values were calculated by a CQ1 confocal cell imager (Yokogawa), then fitted curves were drawn in GraphPad Prism software, and the reciprocal of the corresponding serum dilution at 50% neutralization was calculated as 50% pseudovirus neutralization titer.

The results of the neutralizing antibody titer assays for three pseudoviruses Delta, ba.5 and xbb.1.5 in post-di-and tri-exempt mouse sera are shown in fig. 17 and 18, respectively: the mosaic3_RBD-ferritin group generates higher neutralizing antibody titer to three pseudoviruses, and the titer is obviously increased after the three-immunity and the two-immunity, and basically reaches 10 ⁵ The method comprises the steps of carrying out a first treatment on the surface of the None of the control spycatcher003-ferritin and PBS had associated pseudovirus neutralizing antibodies produced. Neutralizing antibody titre measurement of immune serum against 12 pseudoviruses of group 5 Experimental groups, namely PT, delta, omicron BA.1, BA.2, BA.5, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.16 and CH.1.1The determination results are shown in fig. 19 and 20, respectively: the three homotype RBD-ferritin nanoparticle immune groups have higher neutralizing antibody titer to corresponding pseudovirus variant strains, but have different degrees of reduction to other variant strains; the mosaic3_rbd-ferritin group produced higher neutralizing antibody titers against 12 pseudoviruses, and the 3 kings RBD-ferritin immune group also showed the advantage of higher neutralizing antibody titers over a broader spectrum.

In conclusion, the novel coronavirus Mosaic3_RBD-ferritin nanoparticle prepared by the method can stimulate mice to generate strong antibody response, has strong neutralization effect on prototype strains, delta and a series of Omikovia strains, and is an ideal novel coronavirus universal vaccine.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims

1. A novel coronavirus Mosaic-RBD nanoparticle vaccine is obtained by embedding RBD proteins of three SARS-CoV-2variants of Delta, BA.5 and XBB.1.5 on ferritin nanoparticles together through a spag 003-spatcher 003 covalent connection system.

2. The novel coronavirus Mosaic-RBD nanoparticle vaccine of claim 1, wherein: the novel coronavirus Mosaic-RBD nanoparticle vaccine is prepared by the method comprising the following steps: incubating the sportsher 003-ferrotin protein and the RBD-sportsag 003 protein in a buffer system according to a molar ratio of 1:5 to realize covalent connection of the sportsher 003-ferrotin protein and the RBD-sportsag 003 protein, and naming the obtained connection product as a Mosaic 3_RBD-ferrotin, namely the novel coronavirus Mosaic-RBD nanoparticle vaccine;

wherein the sportscher 003-ferritin protein is formed by fusing sportscher 003 protein and ferritin protein;

the RBD-spytag003 protein is formed by mixing three proteins of Delta_RBD-spytag003 protein, BA.5_RBD-spytag003 protein and XBB.1.5_RBD-spytag003 protein in an equimolar ratio; the Delta_RBD-spytag003 protein is formed by fusion of an RBD sequence of a Delta strain of SARS-CoV-2and the spytag003 protein; the BA.5_RBD-spytag003 protein is formed by fusing an RBD sequence of a BA.5 strain of SARS-CoV-2 with the spytag003 protein; the XBB.1.5_RBD-spytag003 protein is formed by fusing an RBD sequence of an XBB.1.5 strain of SARS-CoV-2 with the spytag003 protein.

3. The novel coronavirus Mosaic-RBD nanoparticle vaccine of claim 2, wherein: the amino acid sequence of the spycatcher003 protein is shown in positions 2-119 of SEQ ID No. 1; and/or

The amino acid sequence of the ferritin protein is shown in the 123 th-295 th positions of SEQ ID No. 1;

and/or

The RBD sequence of the Delta strain of SARS-CoV-2 is shown in the 19 th-237 th position of SEQ ID No. 2; and/or

The RBD sequence of the BA.5 strain of SARS-CoV-2 is shown as 19-237 of SEQ ID No. 3; and/or

The RBD sequence of XBB.1.5 strain of SARS-CoV-2 is shown in 19 th-237 th position of SEQ ID No. 4; and/or

The amino acid sequence of the spatag 003 protein is shown in positions 238-253 of SEQ ID No. 2.

4. The novel coronavirus Mosaic-RBD nanoparticle vaccine of claim 3, wherein: the amino acid sequence of the spycatcher003-ferritin protein is shown as SEQ ID No.1 or as 1 st to 295 th positions of SEQ ID No. 1.

5. The novel coronavirus Mosaic-RBD nanoparticle vaccine of claim 3 or 4, wherein: the Delta_RBD-spytag003 protein, the BA.5_RBD-spytag003 protein and the XBB.1.5_RBD-spytag003 protein also contain signal peptide sequences; the signal peptide sequence is shown in positions 1-18 of SEQ ID No. 2;

further, the amino acid sequence of the Delta_RBD-spytag003 protein is shown as SEQ ID No.2 or as 1 st-253 rd position of SEQ ID No. 2; and/or

Further, the amino acid sequence of the BA.5_RBD-spytag003 protein is shown as SEQ ID No.3 or as 1 st-253 rd position of SEQ ID No. 3; the method comprises the steps of carrying out a first treatment on the surface of the And/or

Further, the amino acid sequence of the XBB.1.5_RBD-spytag003 protein is shown as SEQ ID No.4 or as 1 st to 253 rd positions of the SEQ ID No. 4; .

6. The novel coronavirus Mosaic-RBD nanoparticle vaccine of any of claims 2-5, wherein: the incubation in the buffer system is as follows: incubating in buffer a at 25 ℃ for 8h;

the composition of the buffer A is as follows: 25mM Tris,150mM NaCl,pH 8.0.

7. A method for preparing a novel coronavirus Mosaic-RBD nanoparticle vaccine comprising the steps of any of claims 2-6.

8. A kit for preparing the novel coronavirus Mosaic-RBD nanoparticle vaccine of any of claims 1-6, comprising: the spycatcher003-ferritin protein according to any one of claims 1 to 6 and said RBD-spytag003 protein.

9. The kit of claim 8, wherein: also included in the kit is buffer a as described in claim 6.

10. Use of a novel coronavirus Mosaic-RBD nanoparticle vaccine as claimed in any of claims 1-6 or a kit as claimed in claim 8 or 9 in any of the following:

(A1) Preparing a product capable of neutralizing SARS-CoV-2;

(A3) Preparing a product for ameliorating symptoms caused by SARS-CoV-2 infection;

further, SARS-CoV-2 is a prototype strain or a variant strain;

further, the variant strain is Delta strain, omicron BA.1 strain, omicron BA.2 strain, omicron BA.5 strain, omicron BF.7 strain, omicron BQ.1.1 strain, omicron BB strain, omicron XB.1.5 strain, omicron XB.1.16 strain or Omicron CH.1.1 strain.