Method for enhancing the broad spectrum of new crown mutant strain vaccine and new crown broad spectrum vaccine

Abstract

The application discloses a method for enhancing the broad spectrum of a new crown mutant vaccine and a new crown broad spectrum vaccine. + Through research, six different new crown mutant RBDs and six CD8 T cell epitope peptides highly conserved on N and S proteins of Sarbecovirus are fused and expressed, respectively, and the six fusion protein antigens are displayed on the surface of the same nanoparticle to form a Mosaic new crown nanoparticle vaccine. The new crown broad spectrum vaccine scheme can overcome the problem of the vaccine caused by the immune escape and the decreased immunogenicity of the new crown mutant due to the severe mutation, the designed vaccine immunogen can significantly improve the level of specific antibodies against the virus generated by the host, thereby generating the ability to block the invasion of the virus into the body, and can induce the body to generate humoral immunity and cellular immunity responses against various new crown mutants and SARS, and has obvious broad spectrum. In addition, the new crown broad spectrum vaccine can be suitable for various inoculation modes, such as nasal spray immunization, inhalation immunization or injection immunization. Moreover, the vaccine preparation method is simple, easy to purify, safe, and the vaccine can be quickly applied to clinical trials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More specifically, it relates to a method for enhancing the broad-spectrum of new crown mutant vaccine and improving the immune protection effect of vaccine and a new crown broad-spectrum vaccine. BACKGROUND

[0002] The vaccine targets of new crown virus mainly include whole virus or its structural proteins, such as spike protein (S) and membrane protein (M) and envelope protein (E). The currently approved vaccines include inactivated vaccine, DNA vaccine, recombinant protein vaccine, mRNA vaccine, etc. Except for inactivated vaccine, all of them use S protein of virus as antigen to stimulate the immune response of human body. Since S protein is extremely variable, Omicron and original wild type have 32 Spike mutations. From the perspective of single base mutation causing amino acid mutation, this is already an extreme form of SARS-CoV-2 evolution, which has caused a great threat to the protection of existing vaccines. At the same time, the data published by us and multiple mainstream laboratories in the world show that the new mutant strains often produce serious immune escape after early virus or vaccine immunization. The plasma of convalescent patients with new crown also hardly recognizes the RBD of coronavirus other than SARS-CoV-2, so vaccine companies and research institutions are also constantly updating and iterating their own vaccines against new coronavirus variants.

[0003] Although the Chinese population has been widely vaccinated with various vaccines developed based on early strains of new crown, it has shown a very high protection rate, but the continuous emergence of new crown variants, the trend of vaccine "preventing severe cases, but not preventing infection" is very obvious. However, the strategy of developing targeted vaccines after the emergence of viruses (mutant strains) has been passive and lagging behind. The fastest mRNA vaccine from research and development to clinical use also needs nearly 8 months, and in this period of time, new variants may have emerged, and old variants may have evolved into weak strains. Therefore, based on the current situation that the mutation speed of new crown virus is faster than the development speed of vaccine, in order to get rid of the passive situation, the development of broad-spectrum / universal vaccine is particularly urgent.

[0004] There are various definitions of universal coronavirus vaccines in the academic and industrial communities. Currently, there are mainly the following definitions: ① Variant-proof COVID-19 vaccines, which aim to protect against multiple variants of COVID-19 (reduce infection and severity). ② Pan-sarbecovirus vaccines, which refer to vaccines that can protect against SARS coronavirus, COVID-19, and future sarbecovirus subgenus viruses. ③ Pan-betacoronavirus vaccines, which refer to vaccines that can protect against multiple existing and future beta coronavirus viruses, including SARS and MERS. ④ Pan-CoV vaccines, which refer to vaccines that can protect against multiple existing and future coronavirus viruses. Obviously, the fourth Pan-CoV vaccine is the most ideal and anticipated vaccine, but achieving this goal requires tremendous challenges and efforts. The first and second vaccines are the most likely to be achieved. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the problems and deficiencies of the existing novel coronavirus vaccines and to develop a new coronavirus broad-spectrum / universal vaccine based on the mosaic nanoparticle technology to cope with the current and future variants of the novel coronavirus.

[0006] The purpose of the present application is to provide a method for enhancing the broad-spectrum of the novel coronavirus mutant vaccine.

[0007] Another purpose of the present application is to provide a novel coronavirus broad-spectrum antigen.

[0008] Another purpose of the present application is to provide a recombinant plasmid for preparing the novel coronavirus broad-spectrum antigen.

[0009] Another purpose of the present application is to provide a novel coronavirus broad-spectrum vaccine.

[0010] The above purposes of the present application are achieved by the following technical solutions:

[0011] To solve the technical bottleneck of poor broad-spectrum of the new crown mutant strain vaccine, we combined two strategies together, one is to combine different antigens from multiple coronavirus subgroups and deliver them to the body to stimulate immune protection against multiple coronaviruses, and the other is to find common conserved protein structures in multiple coronaviruses and use them as antigens to develop vaccines; specifically, we express 6 different new crown mutant strain RBDs and 6 CD8 + T cell epitope peptides by fusing, and then display the six fusion-expressed protein antigens on the surface of the same nanoparticle to form our original Mosaic new crown nanoparticle vaccine. By comparing the immune responses of the Mosaic new crown nanoparticle vaccine induced by the body against various new crown mutant strains and SARS, etc., the application of the vaccine is optimized.

[0012] The research results show that the Mosaic new crown nanoparticle vaccine prepared by us can overcome the problem of immune escape caused by the severe mutation of the new crown mutant strain and the decrease of immunogenicity, and can significantly improve the level of specific antibodies produced by the host against the virus, thereby producing the ability to block the invasion of the virus into the body, and has obvious broad-spectrum.

[0013] Therefore, the present application provides a method for enhancing the broad-spectrum of the new crown mutant strain vaccine, using two or more combinations of the CD8 + T cell epitope peptides shown in any one of SEQ ID NO: 6-11 and the new crown mutant strain RBD shown in any one of SEQ ID NO: 12-17 as antigens.

[0014] Preferably, the six CD8 + T cell epitope peptides are connected to the six new crown mutant strain RBDs shown in SEQ ID NO: 12-17 respectively to obtain six fusion proteins, and the six fusion proteins are combined as antigens.

[0015] As a preferred embodiment, the six fusion proteins are:

[0016] Gv-BA.1 RBD-N395-403, the amino acid sequence is shown in SEQ ID NO: 18,

[0017] Gv-BA.2 RBD-N316-333, the amino acid sequence is shown in SEQ ID NO: 19,

[0018] Gv-BA.5 RBD-N360-369, the amino acid sequence is shown in SEQ ID NO: 20,

[0019] Gv-BA.2.75RBD-S539-546, the amino acid sequence of which is shown as SEQ ID NO: 21,

[0020] Gv-Delta RBD-S1136-1155, the amino acid sequence of which is shown as SEQ ID NO: 22,

[0021] Gv-D614G RBD-S865-873, the amino acid sequence of which is shown as SEQ ID NO: 23.

[0022] Further preferably, the connecting element used in the connection is a Gv / Sd connecting element, the amino acid sequence of which is shown as SEQ ID NO: 5.

[0023] Further preferably, the fusion proteins are co-displayed on the surface of the same nanoparticle to form a mosaic nanoparticle antigen.

[0024] The application also provides a novel coronavirus broad-spectrum antigen, which is a CD8 + T cell epitope peptide segment connected to two or more combinations of the fusion proteins of any one of SEQ ID NOs: 6-11 and the RBDs of the novel coronavirus mutant strains shown in any one of SEQ ID NOs: 12-17.

[0025] Further preferably, the connecting element used in the connection is a Gv / Sd connecting element, the amino acid sequence of which is shown as SEQ ID NO: 5.

[0026] Further preferably, the fusion proteins are co-displayed on the surface of the same nanoparticle to form a mosaic nanoparticle antigen.

[0027] Preferably, the novel coronavirus broad-spectrum antigen is six CD8 + T cell epitope peptide segments connected to the RBDs of six novel coronavirus mutant strains shown in SEQ ID NOs: 12-17, respectively, to obtain six fusion proteins, and the six fusion proteins are co-displayed on the surface of the same nanoparticle to form a mosaic nanoparticle antigen.

[0028] As a preferred embodiment, the six fusion proteins are:

[0029] Gv-BA.1RBD-N395-403, the amino acid sequence of which is shown as SEQ ID NO: 18,

[0030] Gv-BA.2RBD-N316-333, the amino acid sequence of which is shown as SEQ ID NO: 19,

[0031] Gv-BA.5 RBD-N360-369, the amino acid sequence of which is shown as SEQ ID NO: 20,

[0032] Gv-BA.2.75 RBD-S539-546, the amino acid sequence of which is shown as SEQ ID NO: 21,

[0033] Gv-Delta RBD-S1136-1155, the amino acid sequence of which is shown as SEQ ID NO: 22,

[0034] Gv-D614G RBD-S865-873, the amino acid sequence of which is shown as SEQ ID NO: 23.

[0035] As an alternative embodiment, the preparation method of the new coronavirus broad-spectrum antigen is as follows:

[0036] The six Gv-RBD-CD8 + T Peptide monomer proteins (amino acid sequences SEQ ID NO: 18-23) mixed solution (preferably mixed in pH = 7.5 20mM Tri-HCl 50mM NaCl buffer at equal proportions) and SdCatcher-HPF protein (amino acid sequence SEQ ID NO: 3) are coupled in a buffer system by room temperature shaking incubation (preferably incubated for 1 hour), to obtain the coupled Mosaic-Nanoparticle (NP), that is, the new coronavirus broad-spectrum antigen (Mosaic nanoparticle antigen).

[0037] Preferably, the buffer system is a pH = 7.5 20mM Tri-HCl 50mM NaCl buffer system.

[0038] In addition, the present application also provides a recombinant plasmid for expressing and preparing the new coronavirus broad-spectrum antigen, which comprises the coding sequence of the above-mentioned Gv-RBD-CD8 + T Peptide monomer protein.

[0039] Preferably, the recombinant plasmid for expressing and preparing Gv-RBD-CD8 + T Peptide monomer protein is as follows: the fusion protein Gv-RBD-CD8 +The nucleotide sequence of T Peptide is used as the basis to separate the Gv / Sd connecting element and the RBD in the fusion protein by a Linker GSG, and add a secretory peptide SP (amino acid sequence SEQ ID NO: 4) at the 5' end of the fusion protein, and add a 6xHis and a translation termination codon at the 3' end of the fusion protein; then cloned into the expression vector (pcDNA3.1-Intron-WPRE) added with Intron and WPRE to enhance expression between the Xho I and Xba I enzyme digestion sites, to construct the expression vector pcDNA3.1-Intron-SP-Gv-RBD-CD8 + T Peptide-His-IRES-GFP-WPRE, which is a recombinant plasmid for preparing the novel coronavirus broad-spectrum antigen.

[0040] The recombinant plasmid is transiently transfected into a eukaryotic expression system (such as 293F cells) to express the nanoantigen, and the cell supernatant is collected after expression and purified to obtain Gv-RBD-CD8 + T Peptide monomer protein (novel coronavirus 6 RBD fusion protein monomer).

[0041] The eukaryotic expression system includes but is not limited to HEK293T cells, 293F cells, CHO cells, sf9 and other cell strains and cell lines that can be used to express eukaryotic proteins. The corresponding protein introduction scheme for the eukaryotic expression system includes but is not limited to various transfection, infection, transposition schemes, etc.

[0042] As an alternative embodiment, the purification method is to filter the cell supernatant expressing the antigen to remove cell debris, and then perform preliminary purification through a 10K ultrafiltration tube (Millipore), and then capture the target protein through a HisTrap HP nickel column (GE) and a Lectin column (GE), and finally purify the target protein through molecular sieve chromatography using a Siperose6Increase10 / 300GL column (GE) to obtain a high-purity target protein (as shown in the examples herein). Figure 6

[0043] Finally, the application also provides a novel coronavirus broad-spectrum vaccine prepared from the antigen described above.

[0044] As an alternative embodiment, a nasal spray vaccine, an inhalation vaccine or an injection vaccine can be prepared as needed to achieve different immunization modes.

[0045] The application has the following beneficial effects:

[0046] The application provides a method for enhancing the broad spectrum of a novel coronavirus mutant vaccine and a novel coronavirus broad-spectrum vaccine.​

[0047] In terms of antigen selection, we selected RBDs of four subtypes of the currently globally dominant Omicron mutant strain, BA.1, BA.2, BA.5 and BA.2.75, combined with the RBDs of the previous dominant Delta strain and the original strain, and displayed the RBDs of the six coronavirus mutants on the surface of the same nanoparticle to form our original Mosaic coronavirus nanoparticle vaccine. In addition, we innovatively fused six highly conserved CD8+ T cell epitope peptides on the N and S proteins of Sarbecovirus with the above-mentioned six coronavirus subunit RBD proteins for expression, which not only can quantitatively identify different RBD proteins, but also further significantly enhances the cellular immune response of the vaccine and improves its broad-spectrum nature against coronavirus and SARS and other Sarbecoviruses.

[0048] We utilized the characteristic of Helicobacter pylori ferritin (HPF) that can spontaneously form a 24-mer, and completed the assembly of the above-mentioned six coronavirus subunit receptor binding domain (RBD) antigens through a self-developed optimized linker peptide segment, displayed the immunogen RBD on the surface of the nanoparticle, and aggregated together to form a nanoparticle, which increased the number of antigens carried by a single immunization and fully stabilized its extensive contact with immune cells in the human body to stimulate the production of high-titer neutralizing antibodies.

[0049] The research results show that the scheme of the present application can overcome the problem of vaccine design caused by the severe mutation of coronavirus mutants, which leads to immune escape and causes a decrease in immunogenicity. The optimized design of the vaccine immunogen significantly improves the level of specific antibodies produced by the host against the virus, thereby producing the ability to block the invasion of the virus into the body, and has the advantages of high titer, low side effects, and high safety. Moreover, the nanoparticle vaccine of the present application has the advantages of simple preparation method, easy purification, low production and storage cost, and the vaccine can be quickly applied to clinical trials.

[0050] In addition, the coronavirus broad-spectrum vaccine of the present application can be applied in various vaccination methods, such as nasal spray immunization, inhalation immunization or injection immunization. Among them, the nasal spray immunization and inhalation immunization methods have the advantages of more convenience, more effective prevention of infection and blocking of transmission in the work of coronavirus vaccination. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 It is a schematic diagram of self-assembly of RBD-HPF nanoparticle vaccine.

[0052] Figure 2 It is a highly conserved CD8+ T cell epitope peptide.

[0053] Figure 3 Schematic diagram of Mosaic nanoparticle vaccine for six new coronavirus mutant RBD fusion protein and nanoparticle hook connection.

[0054] Figure 4 For the expression of Gv-RBD-CD8 + Schematic diagram of plasmid structure of T Peptide.

[0055] Figure 5 Gv-RBD-CD8 + T Peptide protein and the corresponding Ferritin connection verification diagram.

[0056] Figure 6 Molecular sieve diagram for purification of Mosaic nanoparticle vaccine and mouse immunization strategy diagram.

[0057] Figure 7 For detecting the titer level of various RBD-specific IgG antibodies in mouse serum by ELISA method.

[0058] Figure 8 For the immune serum of mice immunized for 6 weeks, the titer level of anti-virus infected cells was detected by pseudovirus neutralization experiment.

[0059] Figure 9 For the spleen cells of mice immunized with Mosaic nanoparticle vaccine for 8 weeks, the IFN-γ cellular immune response stimulated by Mosaic nanoparticle vaccine CD8 + T Peptide.

[0060] Figure 10 For the immune serum of mice immunized with Mosaic nanoparticle vaccine for 6 weeks, the titer level of anti-virus infected cells was detected by pseudovirus neutralization experiment. DETAILED DESCRIPTION

[0061] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0062] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0063] The sequences involved in the present application are as follows:

[0064] SEQ ID NO: 1 (amino acid sequence of HPF)

[0065] 1 DIIKLLNEQV NKEMQSSNLY MSMSSWCYTH SLDGAGLFLF DHAAEEYEHA KKLIIFLNEN

[0066] 61 NVPVQLTSIS APEHKFEGLT QIFQKAYEHE QHISESINNI VDHAIKSKDH ATFNFLQWYV

[0067] 121 AEQHEEEVLF KDILDKIELI GNENHGLYLA DQYVKGIAKS RKS

[0068] SEQ ID NO: 2 (amino acid sequence of Sd Catcher)

[0069] 1 SGETGQSGNT TIEEDSTTHV KFSKRDINGK ELAGAMIELR NLSGQTIQSW VSDGTVKDFY

[0070] 61 LMPGTYQFVE TAAPEGYELA APITFTIDEK GQIWVDSTLI VGDDPI

[0071] SEQ ID NO: 3 (amino acid sequence of Sd Catcher-HPF)

[0072]

[0073]

[0074] SEQ ID NO: 4 (amino acid sequence of Signal Peptide)

[0075] 1 MGILPSPGMP ALLSLVSLLS VLLMGCVA

[0076] SEQ ID NO: 5 (amino acid sequence of GV)

[0077] 1 KVGNTIVMVD KLKEVPTP

[0078] SEQ ID NO: 6 (amino acid sequence of N395-403)

[0079] 1 LPAADLDDF

[0080] SEQ ID NO: 7 (amino acid sequence of N316-333)

[0081] 1 GMSRIGMEVTPSGTWLTY

[0082] SEQ ID NO: 8 (amino acid sequence of N360-369)

[0083] 1 YKTFPPTEPK

[0084] SEQ ID NO: 9 (amino acid sequence of S539-546)

[0085] 1 VNFNFNGL

[0086] SEQ ID NO: 10 (amino acid sequence of S1136-1155)

[0087] 1 TVYDPLQPELDSFKEELDKY

[0088] SEQ ID NO: 11 (amino acid sequence of S865-873)

[0089] 1 LTDEMI AQY

[0090] SEQ ID NO: 12 (amino acid sequence of BA.1 RBD)

[0091]

[0092] SEQ ID NO: 13 (amino acid sequence of BA.2 RBD)

[0093]

[0094] SEQ ID NO: 14 (amino acid sequence of BA.5 RBD)

[0095]

[0096] SEQ ID NO: 15 (amino acid sequence of BA.2.75 RBD)

[0097]

[0098] SEQ ID NO: 16 (amino acid sequence of Delta RBD)

[0099]

[0100] SEQ ID NO: 17 (amino acid sequence of D614G RBD)

[0101]

[0102] SEQ ID NO: 18 (amino acid sequence of Gv-BA.1 RBD-N395-403)

[0103]

[0104] SEQ ID NO: 19 (amino acid sequence of Gv-BA.2 RBD-N316-333)

[0105]

[0106] SEQ ID NO: 20 (amino acid sequence of Gv-BA.5 RBD-N360-369)

[0107]

[0108] SEQ ID NO: 21 (amino acid sequence of Gv-BA.2.75 RBD-S539-546)

[0109]

[0110] SEQ ID NO: 22 (amino acid sequence of Gv-Delta RBD-S1136-1155)

[0111]

[0112] SEQ ID NO: 23 (amino acid sequence of Gv-D614G RBD-S865-873)

[0113]

[0114] Example 1 Construction of six SARS-CoV-2 mutant RBD fusion protein plasmids and purification of proteins

[0115] 1. Construction of six SARS-CoV-2 mutant RBD fusion protein Gv-RBD-CD8 + T Peptide

[0116] In the selection of vaccine antigens, we targeted the main SARS-CoV-2 mutant Omicron currently prevalent in the world. We selected four Omicron RBD subtypes, including BA.1, BA.2, BA.5 and BA.2.75. We also combined the Delta and early strain (D614G) RBD as vaccine antigens. By using the self-developed optimized linker peptide Gv / Sd linker (amino acid sequence SEQ ID NO: 5) (as shown in Figure 1 , we completed the vaccine assembly by displaying the six RBDs on the same nanoparticle surface, so the conserved epitope regions of the six RBDs will be enriched and preserved, and the mutant regions will be diluted. The conserved epitope region usually induces SARS-CoV-2 3 / 4 class neutralizing antibodies with broad spectrum.

[0117] In addition, we found 6 highly conserved CD8 + T cell epitope peptides (such as Figure 2 indicated) on the N and S proteins of sarbecovirus, and tried to fuse them with the above-mentioned 6 RBDs respectively. Not only can different RBD proteins be quantitatively identified, but also the cellular immune response of the vaccine can be further significantly improved, and its broad protection against sarbecovirus viruses can be improved.

[0118] The 6 RBD amino acid sequences (the encoded amino acid sequences SEQ ID NO: 12-17) were connected with the Gv / Sd connecting element (the encoded amino acid sequence SEQ ID NO: 5) and the 6 CD8 + T cell Peptide (the encoded amino acid sequence SEQ ID NO: 6-11) according to Figure 3 The schematic diagram of Gv-RBD-CD8 + T Peptide (the encoded amino acid sequence SEQ ID NO: 18-23) fusion protein cloning.

[0119] 2. Construction of expression vector

[0120] The Gv-RBD-CD8 + T Peptide nucleotide sequence 5' is added with the coding sequence of the secretion peptide SP (the encoded amino acid sequence SEQ ID NO: 4), the Gv / Sd connecting element and the RBD are separated by the Linker GSG, and the 3' end is added with 6xHis and a translation termination codon. Then it is cloned between the Xho I and Xba I enzyme cutting sites of the expression vector (pcDNA3.1-Intron-WPRE) added with Intron and WPRE to enhance expression (as Figure 4 indicated) to construct the expression vector pcDNA3.1-Intron-SP-Gv-RBD-CD8 + T Peptide-His-IRES-GFP-WPRE (as Figure 4 indicated).

[0121] 3. Expression and purification of six new coronavirus mutant RBD fusion proteins

[0122] The prepared expression vector was transformed into DH5a competent cells, which were cultured overnight at 37°C. Positive clones were screened and identified by PCR. Endotoxin-free plasmids were extracted, and HEK293F cells were transfected by a liposome transfection protocol. After 4 days of transfection, the cell supernatant was harvested by centrifugation, and the target protein was purified. The supernatant was incubated with Ni-NTA agarose (GE Healthcare) to enrich the His-labeled target protein, and then the protein was eluted with an imidazole-containing Tris buffer. The purified protein was concentrated and replaced with a pH = 7.5 20 mM Tri-HCl 50 mM NaCl buffer. The protein concentration was determined by the BCA method, and Coomassie blue staining was used. The results are shown in Figure 5 (left).

[0123] 3. Experimental results

[0124] The results are shown in Figure 5 (left). It is shown that six Gv-RBD-CD8 + T Peptide plasmids were successfully constructed, and the corresponding mutant fusion proteins were expressed and purified.

[0125] Example 2: Preparation of Mosaic nanoparticle antigen

[0126] 1. Six Gv-RBD-CD8 + T Peptide monomer proteins were coupled to Sd Catcher-HPF proteins.

[0127] The six purified Gv-RBD-CD8 + T Peptide monomer proteins were mixed in equal proportions and diluted in a pH = 7.5 20 mM Tri-HCl 50 mM NaCl buffer to a concentration of 5 μM. 5 μM of the mixed protein solution was mixed with 5 μM of prokaryotic expression and purified Sd Catcher-HPF protein (amino acid sequence SEQ ID NO: 3, obtained by connecting Sd shown in SEQ ID NO: 2 and HPF shown in SEQ ID NO: 1 via GSG) in 1 ml of a pH = 7.5 20 mM Tri-HCl 50 mM NaCl buffer system at room temperature for 1 hour, and a coupled Mosaic-Nanoparticle (NP) was obtained.

[0128] In this scheme, the RBD can be coupled with the HPF monomer through the self-developed and optimized Gv and Sd Catcher connecting elements (i.e., Gv / Sd connecting elements, amino acid sequence SEQ ID NO: 5), and the size of the combined monomer protein is 70 KDa. As shown in Figure 5(Right) shown. Because HPF has the feature of being able to assemble into a 24-mer nanoparticle autonomously in vitro, the coupled RBD can be displayed on the surface of the 24-mer HPF nanoparticle to obtain the coupled Mosaic-Nanoparticle (NP).

[0129] 2. The coupled Mosaic-Nanoparticle (NP) was purified using a Siperose 6 Increase 10 / 300 GL column (GE) for molecular sieve chromatography. The buffer for molecular sieve chromatography was: pH = 7.5, 20 mM Tri-HCl 50 mM NaCl. The 24-mer Mosaic-NP protein (as shown in Figure 6 ) was obtained, which is the Mosaic nanoparticle antigen.

[0130] 3. Experimental results

[0131] The results are shown in Figure 5 , Figure 6 . The six Gv-RBD-CD8 + T Peptide proteins can be successfully displayed on the surface of the nanoparticle to form a macromolecular Mosaic nanoparticle antigen.

[0132] Example 3 IgG antibody titer level induced by Mosaic nanoparticle vaccine immunization of mice.

[0133] After the target protein Mosaic-NP (i.e., Mosaic nanoparticle antigen) was concentrated, 100 μl of pH = 7.5 20 mM Tri-HCl 50 mM NaCl was added, and an equal volume of Alum adjuvant was used for grouping emulsification to prepare the Mosaic nanoparticle vaccine.

[0134] Then, 6-8 week old Balb / C mice were grouped and immunized at Day 0 and Day 28. Each mouse was injected subcutaneously with 200 μl of inoculum. Every two weeks, the mice were taken blood from the eye orbit (as shown in Figure 6 ). After the mouse serum was allowed to stand for a period of time, the serum was separated by centrifugation at 4°C, 2800 rpm for 15 minutes, and the supernatant was aspirated to obtain the mouse serum, which was immediately used for Anti-RBD IgG ELISA detection experiment.

[0135] The results are shown in Figure 7 . The Mosaic nanoparticle vaccine produced very high IgG antibody titers against Omicron BA.1, BA.2, BA.5, BA.2.75, Delta and D614G RBD in the immunized mice, and the specific RBD IgG antibody titers all exceeded 10 4In addition, the Mosaic nanoparticle vaccine immunized mice not only produced high specific RBD IgG antibody titers against the 6 RBDs of the vaccine antigen, but also produced high cross-specific RBD IgG antibody titers against other sarbecovirus RBDs other than the 6 RBDs of the vaccine antigen, such as the currently most prevalent Omicron subtypes BF.7, BQ.1.1 and XBB, and SARS. It shows that the Mosaic nanoparticle vaccine of the application can overcome the problem of reduced immunogenicity caused by mutations of the new crown mutant strain in vaccine design, and significantly improve the level of specific antibodies produced by the host against the virus, and enhance the broad-spectrum of the new crown mutant strain vaccine.

[0136] Example 4 SARS-CoV-2 Pseudovirus Neutralization Experiment

[0137] 1. Preparation of Pseudovirus:

[0138] According to the sequence published by NCBI, the Spike proteins of SARS-CoV-2 Omicron BA1, BA.2, BA.5, BA.2.75, Delta, D614G, BF.7, BQ.1.1 and XBB were synthesized and inserted into pcDNA3.1 expression vector. The expression vectors of the above Spike proteins were co-transfected with pHIV-luciferase and psPAX2 plasmids into 293T cells, and 5 hours after transfection, the cells were washed twice with PBS and replaced with serum-free DMEM medium for continuous culture. After 48 hours, the supernatant was collected and centrifuged to remove cell debris. Then small volume of serum-free DMEM was used to dissolve to obtain HIV-luc / SARS-CoV-2 mutant S pseudovirus. This pseudovirus can effectively simulate the process of wild-type SARS-CoV-2 invading cells. When it infects producer cells or target cells, the expression of luciferase reporter gene carried by SARS-CoV-2 pseudovirus can accurately reflect the results of virus infection, so that the results of the experimental system can be accurately and quickly read, which can be used as an excellent antibody neutralization titer monitoring system.

[0139] 2. Pseudovirus TCID 50 Determination

[0140] The virus solution collected in the previous step was diluted by 5 times, and added to HEK293T cells in a 96-well plate. After 4 hours of infection, the virus solution was discarded, the cells were washed twice with PBS, and replaced with complete DMEM medium containing 10% serum. After 48 hours, the culture medium was discarded, washed twice with PBS, and 60ul of cell lysis solution was added for 15 minutes of shaking lysis. After freezing and thawing once at -80℃, 30ul per well was taken to detect the luciferase activity value by GloMax 96 (Promega). The TCID 50 was calculated by the Reed-Muech method.

[0141] 3. Neutralization test

[0142] The serum of the 6-week-old mice immunized with the Mosaic nanoparticle vaccine was diluted by 10 times, mixed with the TCID50 final concentration of pseudovirus at 37°C for 1 hour. The mixture was added to a 96-well plate with HEK293T cells at a density of about 70%. After 48 hours, the culture solution was discarded, the cells were washed twice with PBS, and cell lysis solution was added to detect the luciferase activity value.

[0143] 4. Result analysis

[0144] The results are shown in Figure 8 The Mosaic nanoparticle vaccine produced high neutralizing antibody titers against Omicron BA.1, BA.2, BA.5, BA.2.75, Delta, D614G RBD, BF.7, BQ.1.1 and XBB in immunized mice. It shows that the Mosaic nanoparticle vaccine of the present application can overcome the problem of vaccine design caused by the rapid mutation of the new crown mutant strain, and the optimized vaccine immunogen significantly improves the level of specific neutralizing antibodies produced by the host against the virus, and enhances the broad-spectrum nature of the new crown mutant vaccine, thereby producing the ability to block the invasion of the virus into the body.

[0145] Example 5: IFN-γ cellular immune response produced by Mosaic nanoparticle vaccine immunization

[0146] 1. The 8-week-old mice immunized with the Mosaic nanoparticle vaccine were sacrificed, the mouse spleen was taken and ground, filtered with a cell filter, then treated with red blood cell lysis solution to lyse red blood cells, then centrifuged at 500 rpm for 5 min, the supernatant was discarded, and washed once with PBS. Centrifuged again at 500 rpm for 5 min, the supernatant was discarded. Resuspend the cells with 1 ml of PBS to obtain mouse spleen lymphocytes, and count the cells with a cell counting plate.

[0147] 2. We designed and developed Mosaic CD8 +T cell peptide pool, containing 13 peptides, and commissioned GenScript to synthesize, the sequence of the 13 peptides are: N360-369 (SEQ ID NO: 8), N395-403 (SEQ ID NO: 6), S539-546 (SEQ ID NO: 9), S865-873 (SEQ ID NO: 11), and 4 peptides N316-324, N319-327, N322-330, N325-333 derived from N316-333 (SEQ ID NO: 7) and 5 peptides S1136-1144, S1139-1147, S1142-1150, S1145-1153, S1148-1155 derived from S1136-1155 (SEQ ID NO: 10).

[0148] 3, take 10 6 mouse spleen lymphocytes and Mosaic CD8 T cell peptide pool (synthesized by GenScript) and CD28 antibody (1000X) with a concentration of 2 ug / ml were co-incubated and plated in IFN-γ ELISPOT plate (Dakewe; Cat#2210005) for 18-24 hours. According to the product manual of IFN-γ ELISPOT kit (Dakewe; Cat#2210005), sample collection and detection were performed, and ImmunoSpot 5.1.34 software was used to calculate the number of IFN-γ positive T cells. +

[0149] 4, the results are shown in Figure 9 Table 1, spleen cells of mice immunized for 8 weeks can produce strong IFN-γ cellular immune response by Mosaic nanoparticle vaccine CD8 T peptide stimulation. +

[0150] Example 6 Mice immunized with Mosaic nanoparticle vaccine by nasal spray produce high titer neutralizing antibody titers.

[0151] After concentrating the Mosaic nanoparticle vaccine, 20 mM Tri-HCl 50 mM NaCl with pH = 7.5 to 30 μl, 6-8 week old Balb / C mice were immunized by nasal spray on Day 0 and Day 28. By nasal drop, each mouse was inoculated with 10 ug of Mosaic nanoparticle vaccine, and the inoculation volume was 30 μl, without the need for adjuvant. After 6 weeks, the mice were taken out of the orbit (as shown in Table 2) and the mouse serum was obtained by centrifugation at 4°C, 2800 rpm for 15 minutes. The supernatant was immediately used for pseudovirus neutralization experiment. Figure 6 ​​​

[0152] The procedure of the pseudovirus neutralization experiment is as described in Example 4. The serum of the 6-week-old mice immunized by the Mosaic nanoparticle vaccine by nasal spray was diluted by 10 times, mixed with the pseudovirus with a final concentration of TCID50, and incubated at 37°C for 1 hour. The mixture was added to a 96-well plate with HEK293T cells with a density of about 70%. After 48 hours, the culture solution was discarded, the cells were washed twice with PBS, and cell lysate was added to detect the luciferase activity value.

[0153] The results are shown in Table 2. Figure 10 The mice immunized by the Mosaic nanoparticle vaccine by nasal spray also produced high neutralizing antibody titers against the pseudovirus of Omicron BA.1, BA.2, BA.5, BA.2.75, Delta, D614G RBD, BF.7, BQ.1.1, and XBB. This shows that the inoculation method of the Mosaic nanoparticle vaccine of the present application is not only suitable for subcutaneous injection immunization, but also for non-invasive nasal spray immunization. The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method for enhancing the broad-spectrum activity of a novel coronavirus mutant vaccine, characterized in that: Six fusion proteins were used as antigens; the six fusion proteins were: Gv-BA.1RBD-N395-403, amino acid sequence as shown in SEQ ID NO: 18, Gv-BA.2RBD-N316-333, amino acid sequence as shown in SEQ ID NO: 19, Gv-BA.5RBD-N360-369, the amino acid sequence is shown in SEQ ID NO: 20, Gv-BA.2.75RBD-S539-546, the amino acid sequence is shown in SEQ ID NO: 21, Gv-Delta RBD-S1136-1155, the amino acid sequence is shown in SEQ ID NO: 22, Gv-D614G RBD-S865-873, the amino acid sequence is shown in SEQ ID NO:

23.

2. The method according to claim 1, characterized in that The fusion proteins are co-displayed on the surface of the same nanoparticle to form mosaic nanoparticle antigens.

3. A new coronavirus broad-spectrum antigen, characterized in that The novel coronavirus broad-spectrum antigen is a combination of 6 fusion proteins; the 6 fusion proteins are: Gv-BA.1RBD-N395-403, amino acid sequence as shown in SEQ ID NO: 18, Gv-BA.2RBD-N316-333, amino acid sequence as shown in SEQ ID NO: 19, Gv-BA.5RBD-N360-369, the amino acid sequence is shown in SEQ ID NO: 20, Gv-BA.2.75RBD-S539-546, the amino acid sequence is shown in SEQ ID NO: 21, Gv-Delta RBD-S1136-1155, the amino acid sequence is shown in SEQ ID NO: 22, Gv-D614G RBD-S865-873, the amino acid sequence is shown in SEQ ID NO:

23.

4. The novel coronavirus broad-spectrum antigen according to claim 3, characterized in that The fusion proteins are displayed together on the surface of the same nanoparticle to obtain mosaic nanoparticle antigens.

5. A recombinant plasmid for preparing the novel coronavirus broad-spectrum antigen, characterized in that: Contains the coding sequence of the fusion protein according to claim 3 or 4.

6. A broad-spectrum COVID-19 vaccine, characterized in that: Prepared from the antigen according to claim 3 or 4.

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