A self-adjuvanting viral-derived glycopeptide and its use in the preparation of vaccines

Abstract

The application relates to a self-adjuvant based on a virus-derived glycopeptide and application thereof in preparation of a vaccine, the self-adjuvant being a varicella-zoster virus (VZV)-derived glycopeptide fragment, which is a B cell epitope domain V50 glycopeptide (from a valine at 50 to an isoleucine at 135, V50-I135) in a glycoprotein E. After fusion expression of the V50 glycopeptide and a novel coronavirus RBD, coupling on the surface of a ferritin-based nanoparticle, a self-adjuvant vaccine is formed, which can significantly enhance the RBD-specific antibody and pseudovirus neutralizing antibody levels induced by the RBD nanoparticle vaccine, and also has a synergistic effect in B cell response and T cell response of systemic and mucosal immunity. The immunoprotective effect of the novel coronavirus RBD nanoparticle vaccine is significantly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology and vaccine technology, in particular to a self-adjuvanting based on virus-derived glycopeptide and its use in the preparation of vaccines. BACKGROUND

[0002] Adjuvant refers to a substance that is injected into the body at the same time or in advance of an antigen, which can non-specifically enhance the immune response of the body to the antigen, and is an immune enhancer. In the past few decades, adjuvants of different modes of action have been developed to enhance immunogenicity, including aluminum salts, Toll-like receptor (TLR) agonists, organic extracts, oil-in-water reagents, etc. However, these types of adjuvants will cause varying degrees of inflammatory response and cytotoxicity, and the vaccine field urgently needs to develop safer and more efficient adjuvant systems. Self-adjuvanting vaccines are vaccines in which antigens are covalently coupled with adjuvants that stimulate immune responses, and do not require additional adjuvant systems to assist. Self-adjuvanting vaccines use components of pathogens or synthetic analogs to activate the immune system, and exhibit higher antigenicity than simple mixtures of antigens and adjuvants. The ligands that activate the innate immune system conjugated to the antigens bind to pattern recognition receptors to promote better uptake of the conjugated antigens into APCs. In addition, the conjugated adjuvants simultaneously induce cytokine production by APCs, promoting the maturation of APCs and thereby enhancing innate immunity and adaptive immunity. Self-adjuvanting vaccines have stronger and more durable immune responses than traditional vaccines, but reduce the risk of inflammatory response and cytotoxicity caused by additional adjuvant systems.

[0003] Ferritin has physicochemical properties such as monodispersity, uniform size at the nanometer level, biocompatibility, and biodegradability, can self-assemble into nanocages, and can be surface conjugated with antigens through chemical or genetic methods, and has become a promising antigen display platform in the field of vaccine research. Using the Gv / Sd system independently developed, RBD proteins of various new coronavirus variants and conserved T cell epitope polypeptides were conjugated to ferritin to form Mosaic nanoparticle vaccines, which produced strong humoral and mucosal immune responses through muscle injection or nasal immunization, showed broad-spectrum protection against major epidemic mutant strains of new coronaviruses, and could block the invasion of new coronavirus mutant viruses from the upper respiratory tract into the host.

[0004] Varicella-zoster virus (VZV) belongs to the Varicella virus genus of the Herpesviridae family, and is a double-stranded DNA virus transmitted by respiratory tract and mucosal contact. VZV is closely related to Herpes Simplex Virus (HSV) and has many genomic homologues. In addition to envelope glycoprotein gD, VZV virus has envelope glycoproteins gB, gC, gE, gH, gI, gK and gL corresponding to Herpes Simplex Virus. Chinese patent 202310168957.6 A varicella-zoster virus vaccine and a preparation method thereof discloses a multivalent VZV protein subunit vaccine. The mice subcutaneously receiving the multivalent vaccine containing gE protein component have a virus-specific IgG endpoint titer as high as 10 4 two weeks after immunization, and significantly improve germinal center response and cellular immune level. In mice immunized with a multivalent vaccine without gE, the immune response level is reduced, suggesting that certain domains in the gE protein have extremely high immunogenicity and enhance immune response. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art and provide a self-adjuvant based on virus-derived glycopeptide and its application in preparing a vaccine.

[0006] The first purpose of the present application is to provide a peptide segment.

[0007] The second purpose of the present application is to provide a nucleic acid molecule.

[0008] The third purpose of the present application is to provide a biological material.

[0009] The fourth purpose of the present application is to provide the application of the peptide segment, the nucleic acid molecule or the biological material in preparing a self-adjuvant, an adjuvant or a vaccine.

[0010] The fifth purpose of the present application is to provide the application of the peptide segment, the nucleic acid molecule or the biological material in improving the immunogenicity of an antigen.

[0011] The sixth purpose of the present application is to provide a protein composition.

[0012] The seventh purpose of the present application is to provide a nucleic acid composition.

[0013] The eighth purpose of the present application is to provide another biological material.

[0014] The ninth purpose of the present application is to provide a nanoparticle with enhanced immunogenicity.

[0015] The tenth object of the present application is to provide the use of the protein composition, the nucleic acid composition, the biological material or the nanoparticle in preparing a vaccine.

[0016] In order to achieve the above object, the present application is implemented by the following technical scheme:

[0017] The present application analyzes the domain and the distribution of O-glycosylation sites of the gE protein of VZV, obtains the B cell epitope domain V50 (V50-I135), and finds that V50 can significantly enhance the RBD-specific antibodies and pseudovirus antibodies induced by the RBD nanoparticle vaccine after being displayed on Ferritin by using the Sd / Gv coupling system, and has a good protective effect in the application of the novel coronavirus.

[0018] The present application claims a peptide segment V50, and the amino acid sequence is shown in SEQ ID NO: 1.

[0019] The present application also claims a nucleic acid molecule, and the coding gene of the peptide segment.

[0020] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2.

[0021] The present application also claims a biological material, which is any one of the following:

[0022] (1) an expression cassette containing the nucleic acid molecule;

[0023] (2) a recombinant vector containing the nucleic acid molecule or the expression cassette in (1);

[0024] (3) a recombinant microorganism containing the nucleic acid molecule, the expression cassette in (1) or the recombinant vector in (2);

[0025] (4) a cell line containing the nucleic acid molecule, the expression cassette in (1), the recombinant vector in (2) or the recombinant microorganism in (3).

[0026] The present application also claims the use of the peptide segment, the nucleic acid molecule or the biological material in preparing a self-adjuvant, an adjuvant or a vaccine.

[0027] The present application also claims the use of the peptide segment, the nucleic acid molecule or the biological material in improving the immunogenicity of an antigen.

[0028] The present application also claims a protein composition, which comprises component 1 and component 2: the component 1 is a fusion protein of an SD protein, the peptide segment and an immunogen from N-terminal to C-terminal in sequence;

[0029] Component 2 is a fusion protein of GV peptide segment and HPF protein (ferritin) immunogen from N-terminal to C-terminal.

[0030] The SD protein and the GV peptide segment form a GvTagOpti / Sdcatcher (Gv / Sd) system based on isopeptide bond, see Chinese patent CN113621031A A peptide linker for covalent self-assembly of proteins using spontaneous isopeptide bond.

[0031] As a specific embodiment, the immunogen shown is RBD.

[0032] As a specific embodiment, the immunogen shown is novel coronavirus RBD.

[0033] As a specific embodiment, the immunogen shown is novel coronavirus RBD, and the amino acid sequence of the novel coronavirus RBD is shown in SEQ ID NO: 5.

[0034] As a specific embodiment, the amino acid sequence "GSG" is connected between the SD protein, the peptide segment and the immunogen.

[0035] As a specific embodiment, the amino acid sequence of the SD protein is shown in SEQ ID NO: 13; and the amino acid sequence of the Gv protein is shown in SEQ ID NO: 19.

[0036] As a specific embodiment, the amino acid sequence of component 1 is shown in SEQ ID NO: 15.

[0037] As a specific embodiment, the amino acid sequence of component 2 is shown in SEQ ID NO: 23.

[0038] The present application also claims to protect a nucleic acid composition comprising component A and component B, which are the coding genes of component 1 and component 2 of the protein composition, respectively.

[0039] As a specific embodiment, the amino acid sequence of component A is shown in SEQ ID NO: 16.

[0040] As a specific embodiment, the amino acid sequence of component B is shown in SEQ ID NO: 24.

[0041] The present application also claims to protect a biological material, characterized in that it is any one of the following:

[0042] (a) an expression cassette containing the nucleic acid composition;

[0043] (b) a recombinant vector containing the nucleic acid composition or the expression cassette in (a);

[0044] (c) A recombinant microorganism containing the nucleic acid composition described in (a) or the expression cassette described in (b);

[0045] (d) A cell line containing the nucleic acid composition described in (a), the expression cassette described in (b), or the recombinant vector described in (c).

[0046] The present invention also claims protection for an immunogenicity-enhancing nanoparticle obtained by self-assembly of the components of a protein composition.

[0047] Preferably, the preparation method is as follows: the protein composition, after component 1 is self-assembled to obtain nanoparticles, is mixed with component 2 for self-assembly.

[0048] The present invention also claims protection for the use of the said protein composition, the said nucleic acid composition, the said biomaterial, or the said nanoparticle in the preparation of vaccines.

[0049] In one specific implementation, the vaccine is a novel coronavirus vaccine.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] This invention discloses a glycopeptide fragment derived from varicella-zoster virus (VZV), specifically the V50 glycopeptide (from valine at position 50 to isoleucine at position 135, V50-I135) of the B cell epitope domain in glycoprotein E. After fusing the V50 glycopeptide with the novel coronavirus RBD and conjugating it to the surface of ferritin-based nanoparticles, a self-adjuvanted vaccine is formed. This vaccine significantly enhances the levels of RBD-specific antibodies and pseudovirus neutralizing antibodies induced by the RBD nanoparticle vaccine, and also exhibits synergistic effects in systemic immunity and mucosal immunity, particularly in B cell and T cell responses. This significantly improves the immunoprotective efficacy of the novel coronavirus RBD nanoparticle vaccine. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the fusion expression protein construction in Example 1; SP: secretion signal peptide; His: 6×His purification tag.

[0053] Figure 2 The diagram shows the expression and purification of RBD, MLD-RBD, and V50-RBD proteins; A is: the protein sample after purification by SDS-PAGE electrophoresis and Coomassie brilliant blue staining; B is: the Western blot result of the purified R protein sample after SDS-PAGE electrophoresis, rapid semi-dry transfer to cellulose acetate membrane (NC membrane), and labeling with Anti-His mouse monoclonal antibody.

[0054] Figure 3 Figure 15. RBD-specific IgG and IgA titers in mouse sera at different time points. RBD-specific IgG antibody titers of sera collected at weeks 2, 4, 6 (n=4) and IgA titers of sera at week 6 (n=3) were determined by gradient dilution method and expressed as the inverse of the endpoint serum dilution. A: x-axis represents serum samples at 2, 4, 6 weeks post-immunization, y-axis represents the titers of specific antibodies IgG against RBD antigen of XBB virus. B: x-axis represents different immunogen immunized mice, y-axis represents the titers of specific antibodies IgA against RBD antigen of XBB virus in sera of mice at week 6.

[0055] Figure 4 Figure 16. SARS-CoV-2 pseudovirus neutralizing antibody titers in sera of ALB / c mice at week 4 post-first immunization.

[0056] Figure 5 Figure 17. Schematic diagram of fusion expression proteins of Example 2. SP, secretion signal peptide; Sd, SdCatcher; His, 6xHis purification tag.

[0057] Figure 6 Figure 18. Schematic diagram of protein expression and purification of Sd-RBD and Sd-V50-RBD. A: Coomassie blue staining of protein samples after purification by SDS-PAGE electrophoresis. B: Western Blot results of protein samples after SDS-PAGE electrophoresis and rapid semi-dry transfer to NC membrane, labeled with Anti-Sd mouse monoclonal antibody.

[0058] Figure 7 Figure 19. Purification and characterization of RBD-HPF and V50-HPF nanoparticles. A: SEC chart, x-axis represents liquid retention volume, y-axis represents 280 nm absorbance value. B: Coomassie blue staining chart. C: Scanning transmission electron microscopy image after negative staining, the white scale in the figure represents 200 nm.

[0059] Figure 8 Figure 20. RBD-specific IgG titers in mouse sera at different time points. A: RBD-specific IgG detection of serum diluted 6 times at day 7, y-axis represents OD 450 absorbance reading of RBD-specific binding IgG; B: RBD-specific IgG titers at weeks 2, 4, 6, x-axis represents serum samples at 2, 4, 6 weeks post-immunization, y-axis represents the titers of specific antibodies IgG against RBD antigen of XBB virus.

[0060] Figure 9SARS-CoV-2 pseudovirus neutralizing antibody titers in BALB / c mice serum at the fourth, sixth week: A is SARS-CoV-2 pseudovirus neutralizing antibody titers at the fourth week; B is SARS-CoV-2 pseudovirus neutralizing antibody titers at the sixth week; x axis represents different pseudovirus neutralization experiments, y axis represents the antibody level with 50% inhibition rate of neutralization activity to the pseudovirus.

[0061] Figure 10 RBD-specific GC B cell proportion in the spleen of mice at the sixth week; x axis represents different nanoparticle vaccine immunization conditions of mice, y axis represents the proportion of germinal center B (GCB) cells in the spleen cells of mice; n=4.

[0062] Figure 11 RBD-specific memory B cell proportion in the spleen of mice at the sixth week; n=4; A is IgA + and IgG + plasma cell proportion, y axis represents the percentage of IgA positive cells (left) and IgG positive cells (right) in plasma cells; B is RBD-specific IgA+ and IgG+ memory B cell proportion, y axis represents the percentage of IgA positive cells (left) and IgG positive cells (right) in memory B cells.

[0063] Figure 12 CD4 + TCM proportion in the spleen of mice at the sixth week; x axis represents different nanoparticle vaccine immunization conditions of mice, y axis represents the proportion of central memory T (TCM) cells in the spleen cells of mice; n=4.

[0064] Figure 13 T cell proportion secreting different types of cytokines in the spleen of mice at the sixth week; A is CD4 + T cell proportion secreting IFN-γ; B is CD4 + T cell proportion secreting IL-2; C is CD4 + T cell proportion secreting TNF-α; D is CD8 + T cell proportion secreting IFN-γ; E is CD8 + T cell proportion secreting IL-2; F is CD8 + T cell proportion secreting TNF-α.

[0065] Figure 14 RBD-specific IgA titer in the lung alveolar lavage fluid of mice at the sixth week; x axis represents the gradient dilution condition of the lung alveolar lavage fluid, y axis represents the absorbance value of RBD-specific IgG at 450 nm; n=5.

[0066] Figure 15 SARS-CoV-2 JN.1 pseudovirus neutralizing antibody titers in the bronchoalveolar lavage fluid of C57BL / 6 mice at the sixth week; n=5.

[0067] Figure 16 Proportion of tissue-resident memory B cells in different types of tissues in the lungs of C57BL / 6 mice at the sixth week; n=5; A: IgA + Proportion of tissue-resident memory B cells; B: IgG + Proportion of tissue-resident memory B cells; C: proportion of RBD-specific tissue-resident memory B cells in the lungs.

[0068] Data are expressed as: mean ± SD; adjusted p value was calculated by data using Student's t-test; ns, no statistical difference, . DETAILED DESCRIPTION

[0069] In order to make the purposes, technical solutions and advantages of the present application clearer, further detailed description of the present application will be made in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0070] The GvTag Opti / Sd catcher (Gv / Sd) system based on isopeptide bond is disclosed in Chinese Patent CN113621031A, a combination of peptide linkers for covalent self-assembly of proteins using spontaneous isopeptide bond.

[0071] Example 1 Enhancement of RBD immunogenicity by candidate self-adjuvanting proteins from gE protein of VZV

[0072] I. Expression and purification of fusion protein V50-RBD and fusion protein MLD-RBD

[0073] 1. Experimental methods

[0074] Based on the analysis of the distribution of functional domains and O-linked glycosylation sites of the gE protein of VZV, the B cell epitope V50 (V50-I135) and the mucin-like domain MLD (P510-A546) on the gE protein were selected as candidate self-adjuvanting proteins, which were respectively fused and expressed with the RBD protein of the new coronavirus Omicron XBB variant to obtain the fusion protein V50-RBD and the fusion protein MLD-RBD.

[0075] The specific method for expressing and purifying is as follows:

[0076] As shown in Figure 1 , the fusion protein V50-RBD, the fusion protein MLD-RBD and the N-terminal fusion secretion peptide (SP, amino acid sequence as shown) of RBD are fused with His tag at the C-terminal to obtain SP-V50-RBD-His, SP-MLD-RBD-His and SP-RBD-His. The encoding genes thereof are codon-optimized after mammalian cells, and are cloned into pcDNA3.1-IRES-GFP vector (reference: https: / / doi.org / 10.1073 / pnas.2024202118) to obtain three eukaryotic expression plasmids pcDNA3.1-SP-V50-RBD-His-IRES-GFP, pcDNA3.1-SP-MLD-RBD-His-IRES-GFP and pcDNA3.1-SP-RBD-His-IRES-GFP. The correct sequence of the expression plasmid is obtained by colony PCR and Sanger sequencing.

[0077] The specific information of the above-mentioned sequences is shown in Table 1.

[0078] Table 1:

[0079]

[0080] The three plasmids are respectively transfected into Expi293F cells using polyethyleneimine (PEI-MAX), and the culture supernatant containing the target protein is centrifuged 5 days after transfection. The target protein with His tag in the supernatant is purified by nickel column to obtain V50-RBD protein, MLD-RBD protein and RBD protein. After protein electrophoresis of the obtained target protein, the protein is identified by Coomassie brilliant blue staining and Western Blot identification with Anti-His mouse monoclonal antibody.

[0081] The specific method for Western Blot identification is as follows:

[0082] Take the prepared recombinant protein sample, 5 μg per well, mix with the loading buffer, denature at 95 ℃ for 5 min. Use SDS-PAGE separation (12% separation gel), voltage 120 V. The protein is transferred to PVDF membrane by wet transfer method (100 V, 4 ℃, 60 min). The membrane is blocked with 5% skim milk / TBST for 1 h, and the primary antibody (anti-His monoclonal antibody) (1:2000 dilution) is added and incubated at 4 ℃ overnight. After washing the membrane with TBST for 3 times, the fluorescently labeled secondary antibody (1:10 000 dilution) is added and incubated at room temperature for 1 h. After washing the membrane again, the LI-COR imager is used for detection.

[0083] 2. Experimental results

[0084] The results of Coomassie blue staining identification are shown in A of Figure 2 The results show that the main component of each sample is the target protein of its own size, and the purity is reliable.

[0085] The Western Blot identification is shown in B of Figure 2 The results show that the target protein can be specifically recognized by anti-His, and the size is consistent with the expected and Coomassie blue staining size, verifying that the protein is complete and correct.

[0086] II. Immunization of mice

[0087] The fusion protein V50-RBD, the fusion protein MLD-RBD and the protein RBD were mixed with aluminum adjuvant in equal volume, and the "prime-boost" strategy, i.e. the "primary immunization + booster immunization" strategy was used for 2 times of immunization. Specifically, BALB / c mice were subcutaneously immunized with 10 μg of protein per mouse per dose of vaccine, and booster immunization was performed at the fourth week after immunization, and orbital blood sampling was performed every two weeks during the period.

[0088] III. ELISA detection of IgG and IgA antibody levels in serum

[0089] 1. Experimental method

[0090] The titers of RBD-specific IgG at weeks 2, 4 and 6 after the first immunization were determined, and the titers of RBD-specific IgA at week 6 were determined. The specific method is as follows:

[0091] The serum samples collected at weeks 2, 4 and 6 after the first immunization were diluted 1:50 with blocking solution (5% skim milk-PBS), and then 10-fold gradient dilution was performed.

[0092] Coating: Dilute RBD antigen with coating buffer (0.05M carbonate buffer) to 5μg / mL, add 100μL per well, incubate at 4℃ overnight, discard the liquid in the well, then wash the plate 3 times with washing buffer and pat dry;

[0093] Blocking: Add 200μL blocking solution per well, incubate at 37℃ for 1 hour, discard the liquid and pat dry directly;

[0094] Sample incubation: Add the 2nd, 4th, 6th week gradient-diluted serum to the wells respectively, incubate at 37℃ for 2 hours, then wash the plate 3 times and pat dry;

[0095] Secondary antibody incubation: Dilute the HRP-labeled secondary antibody according to the instructions (use anti-human IgG secondary antibody for IgG detection, use anti-human IgA secondary antibody for IgA detection), add 100μL per well, incubate at 37℃ for 1 hour, wash the plate 5 times and pat dry; then develop the color, add 100μL TMB solution per well, incubate at 37℃ in the dark for 15 minutes; add 50μL stop solution (2M H2SO4), shake for 10-15 seconds, measure the OD value within 10 minutes using an enzyme-labeled instrument (reference wavelength 630nm, detection wavelength 450nm), and fit the absorbance curve.

[0096] 2、Experimental results

[0097] The V50-RBD group can produce higher RBD-specific IgG titers at the fourth week of immunization (A in Figure 3 , and can stimulate higher IgA titers after two weeks of the second immunization compared to the other two groups (B in Figure 3 ).

[0098] III. Pseudovirus antibody neutralization experiment

[0099] 1、Experimental method

[0100] At the fourth week of immunization (without booster immunization), SARS-CoV-2 pseudovirus neutralization experiments of different variants were performed to measure the pVNT50 of nAb of serum of each group of BALB / c mice. The specific method is as follows:

[0101] Peripheral blood serum samples were prepared after immunizing mice, and SARS-CoV-2 pseudovirus neutralization experiments of different variants were used to detect the neutralizing antibody titers in the serum.

[0102] The shell protein of the new coronavirus pseudovirus is composed of the spike protein (S protein) of SARS-CoV-2, which is responsible for binding to the ACE2 receptor of host cells, simulating the infection characteristics of the real virus, while the core genome is derived from other safe viruses and is modified to have no replication ability, and contains a reporter gene luciferase for quantifying infection events. The preparation of the pseudovirus is by co-transfecting plasmids expressing the spike protein, psPAX2, and plasmids expressing luciferase into 293T cells, and after 48 hours of transfection, the culture supernatant is collected and the virus content is titrated by detecting luciferase activity.

[0103] The specific method is as follows: collect mouse serum, treat at 56 °C for 30 min to inactivate complement, and dilute according to the gradient (5-fold serial dilution). Mix the diluted serum with an equal volume of SARS-CoV-2 pseudovirus, incubate at 37 °C for 1 h to allow the neutralization reaction. Then add the mixture to pre-seeded HEK293T-hACE2 cells and continue to culture for 48 h. Use luciferase detection reagent to determine the luciferase signal in the cells. The signal intensity is positively correlated with the degree of virus infection, and a decrease in signal indicates an increase in serum neutralization activity.

[0104] Nonlinear fitting analysis of neutralizing antibody titer is performed by Prism software, and the neutralizing antibody titer corresponding to half the inhibition rate of pseudovirus (pVNT50) is calculated.

[0105] 2、Experimental results

[0106] The results are shown in Figure 4 At four weeks after the initial immunization of BALB / c mice with RBD monomer protein, the RBD and MLD-RBD groups produced almost no antibodies with neutralizing activity against XBB, XBB1.15 and XBB1.16 pseudoviruses, while the V50-RBD group produced about 10 2 times more neutralizing antibodies against the three XBB lineage pseudoviruses, with a statistically significant difference from the other two groups. Therefore, the V50 peptide segment helps to stimulate the humoral immune response more quickly and produce higher titers of neutralizing antibodies.

[0107] Example 2 Preparation of nanoparticles

[0108] I. Protein expression and purification

[0109] 1、Experimental method

[0110] As Figure 5As shown, V50-RBD and RBD are fused with N-terminal secretion peptide (SP) and Sd sequence, and His tag at C-terminal, each element is connected by amino acid sequence "GSG", obtaining SP-Sd-V50-RBD-His and SP-Sd-RBD-His, and the encoding genes are codon-optimized by mammalian cells, then cloned into pcDNA3.1-IRES-GFP vector, obtaining pcDNA3.1-Sd-V50-RBD-His-IRES-GFP and pcDNA3.1-Sd-RBD-His-IRES-GFP plasmids, and Sd-V50-RBD-His and Sd-RBD-His expression plasmids with correct sequences are obtained by colony PCR and Sanger sequencing.

[0111] The specific information of the above-mentioned sequences is shown in Table 2.

[0112] Table 2

[0113]

[0114] The three plasmids are transfected into Expi293F cells using polyethyleneimine (PEIMAX) respectively, and the culture supernatant containing the target protein is harvested by centrifugation 5 days after transfection, and the target protein with His tag in the supernatant is purified by nickel column to obtain Sd-V50-RBD and Sd-RBD proteins, and the purified target proteins are identified by protein electrophoresis, Coomassie brilliant blue staining and Western Blot with Anti-His mouse monoclonal antibody.

[0115] Gv is fused with ferritin for prokaryotic expression (reference: PMID: 33275896), forming Gv-ferritin, in which ferritin can self-assemble into 24-mer nanoparticles, and Gv can self-catalyze with Sd to form isopeptide bond.

[0116] 2、Experimental results

[0117] The Coomassie brilliant blue staining identification results are shown in A of FIG. 6, and the results show that the main components of each sample of Sd-V50-RBD and Sd-RBD proteins are the respective target protein sizes, and the purity is reliable. Figure 6

[0118] The Western Blot identification is shown in B of FIG. 6, and the results show that the target protein can be specifically recognized by anti-His, and the size is consistent with the expected and Coomassie brilliant blue staining size, verifying that the protein is complete and correct. Figure 6

[0119] II. Preparation of fusion nanoparticles

[0120] 1、Experimental method​​

[0121] Sd-V50-RBD and Sd-RBD proteins were incubated with Gv-ferritin protein at equimolar ratio at 37 ℃ for 6 hours, and the nanoparticles were separated using size-exclusion chromatography (SEC), and 11-12 mL of peak effluent was collected to obtain V50-RBD-HPF and RBD-HPF nanoparticles.

[0122] The two kinds of nanoparticles were subjected to Coomassie brilliant blue staining and negative staining electron microscopy sample preparation.

[0123] 2. Experimental results

[0124] The SEC peak chart is shown in FIG. 1A, which shows a single smooth polymer peak, indicating that the prepared V50-RBD-HPF and RBD-HPF nanoparticles have good purity; Figure 7 The Coomassie brilliant blue staining identification result is shown in FIG. 1B, and the negative staining electron microscopy result is shown in FIG. 1C, which shows that the V50-RBD-HPF and RBD-HPF nanoparticle vaccines have good uniformity.

[0125] Figure 7 The Coomassie brilliant blue staining identification result is shown in FIG. 1B, and the negative staining electron microscopy result is shown in FIG. 1C, which shows that the V50-RBD-HPF and RBD-HPF nanoparticle vaccines have good uniformity. Figure 7 Example 3: Injection of mice with nanoparticle vaccines

[0126] I. Immunization of mice

[0127] The V50-RBD-HPF and RBD-HPF nanoparticles prepared in Example 2 were mixed with aluminum adjuvant at the same volume, and a “prime-boost” strategy was used, then BALB / c mice were subcutaneously immunized with 10 μg of protein / dose / mouse dose of vaccine, and a booster needle immunization was performed at the fourth week after immunization, and the humoral immune effect was evaluated.

[0128] II. ELISA detection of IgG antibody levels in serum

[0129] 1. Experimental method

[0130] The initial dilution was 6 times, the RBD-specific IgG antibody titer of the serum collected on the 7th day without booster immunization was detected, and the OD 450 reading was used; the RBD-specific IgG antibody titer of the serum collected at weeks 2, 4, and 6 was determined by gradient dilution method (n=5), and was expressed as the reciprocal of the end-point serum dilution.

[0131] The specific steps are the same as in Example 1.

[0132] 2. Experimental results

[0133] The results are shown in FIG. 2.

[0134] Figure 8 ​​On day 7 and weeks 2 and 4 after initial subcutaneous immunization of BALB / c mice, the V50-RBD-HPF group showed earlier and higher levels of stimulated anti-RBD-specific IgG antibody titers compared to the RBD-HPF group. This indicates that the V50 fragment can promote a highly efficient immune response to the RBD nanoparticle vaccine, producing more RBD-specific IgG in a shorter time.

[0135] III. Detection of pseudovirus neutralizing antibody levels

[0136] 1. Experimental Methods

[0137] pVNT50 was used at half maximum inhibitory concentration (IC50). 50 This indicates that it is the reciprocal of the semi-maximum neutralizing dilution (n = 4).

[0138] The specific testing method is the same as in Example 1.

[0139] 2. Experimental Results

[0140] Four weeks after the initial immunization of BALB / c mice (before booster immunization), the V50-RBD-HPF group showed an average increase of approximately 10% in neutralizing antibody titers against the three XBB lineages of pseudoviruses compared to the RBD-HPF group. 0.5 The level of neutralizing antibodies against JN.1 pseudovirus increased by approximately 10%. 0.2 ( Figure 9 (A) After two booster immunizations (6 weeks after the initial immunization), the V50-RBD-HPF group had a 10-fold higher titer of neutralizing antibodies against the above pseudoviruses than the RBD-HPF group. 0.8 -10 1 Magnitude ( Figure 9 (B in the text). Therefore, the V50-RBD-HPF group produced a higher titer of pseudovirus neutralizing antibodies and had a stronger neutralizing titer for different pseudovirus lineages, indicating that its neutralizing ability is more broad-spectrum.

[0141] IV. Effects on immune cells

[0142] 1. Experimental Methods

[0143] Two weeks after booster immunization of C57BL / 6 mice (6 weeks after the first immunization), the mice were euthanized, and lymphocytes were isolated from the spleen and analyzed by flow cytometry to detect RBD-specific GC B cells, RBD-specific IgA+ and IgG+ memory B cells, and the proportion of IgA+ and IgG+ plasma cells.

[0144] Among them, the gate-gathering strategies are: ① FSC / SSC gates mononuclear cells and removes debris and aggregates; ② FVD-APC-Cy7 - Screening for live cells; ③ Enclosing CD19-Amcyano in live cells+ B cells; IV Circled CD95-PE-Cy7 in B cells + GL7-FITC + GCB cells; V RBD-BV421 in GCB cells + GCB cells, i.e. RBD-specific GCB cells.

[0145] 2. Experimental results

[0146] Results as shown in Figure 10 showed that the V50-RBD-HPF group induced a higher proportion of RBD-specific GC B cells compared with the RBD-HPF control group, indicating that the V50 self-adjuvanting peptide fragment can to some extent improve the proportion of clonal expansion and affinity maturation of RBD-specific B cells.

[0147] Results as shown in Figure 11 showed that the V50-RBD-HPF group induced a higher proportion of IgA+ and IgG+ plasma cells (A in Figure 11 ) and a higher proportion of RBD-specific IgA+ and IgG+ memory B cells (B in Figure 11 ), indicating that the V50 peptide fragment can to some extent improve the proportion of plasma cells and RBD-specific memory B cells induced by the RBD nanoparticle vaccine, and can play a self-adjuvanting function. That is, the V50 peptide fragment can enhance the immune response of different types of B cells induced by the RBD-HPF vaccine.

[0148] Five. Immune response of RBD-specific T cells induced in vivo in mice

[0149] 1. Experimental method

[0150] Using the SARS-CoV-2 S protein peptide library synthesized by Kings River as a stimulant, the proportion of cytokine RBD-specific T cells of CD4 + and CD8 + T cells secreting TNF-α, IL-2 and IFN-γ in the spleen was analyzed by intracellular cytokine staining (ICCS) experiment.

[0151] The specific method is as follows:

[0152] After collecting the spleen of the mouse after immunization to prepare a single cell suspension, removing red blood cells by treating with red blood cell lysis solution, resuspending using RPMI-1640 complete culture medium. Take 1×10 6SARS-CoV-2 S protein peptide library (purchased from Genecript, Cat. No. RP30223, final concentration 2 pg / peptide / mL) was added, and the cells were stimulated for 6 h at 37 °C and 5% CO2, while Brefeldin A (final concentration 10 pg / mL) was added to block cytokine secretion.

[0153] After stimulation, the cells were washed with PBS, labeled with Fixable Live / Dead stain to mark live and dead cells, and then stained with anti-CD3, CD4, CD8, and CD44 surface antibodies in sequence. After fixation and permeabilization, intracellular staining was performed using fluorescently labeled antibodies against TNF-a, IL-2, and IFN-g. After staining, the cells were detected by flow cytometry, and the proportions of CD4 + and CD8 + T cells secreting TNF-a, IL-2, and IFN-g were analyzed to evaluate the level of RBD-specific T cell response.

[0154] 2. Experimental results

[0155] The results are shown in Figure 12 Compared with the RBD-HPF group, the V50-RBD-HPF induced a higher proportion of central memory (CM) CD4+ TCM cells. Figure 13 The results showed that, compared with the RBD-HPF group, the proportion of RBD-reactive CD4+ and CD8+ cells secreting IL-2-specific response after stimulation with viral antigen peptides was significantly increased in the V50-RBD-HPF group. This indicates that after immunization with a vaccine prepared using V50 peptides as a self-adjuvant, the cell-mediated immune response is more likely to be activated to protect against pathogen invasion.

[0156] Example 4. Intranasal immunization of mice with nanoparticle vaccines

[0157] I. Immunization of mice

[0158] The V50-RBD-HPF and RBD-HPF nanoparticles prepared in Example 2 were used to intranasally immunize BALB / c mice twice using a “prime-boost” strategy, with a dose of 10 pg per mouse per immunization.

[0159] Six weeks after the first immunization (i.e., two weeks after the booster intranasal immunization), the mice were sacrificed, and their lung tissues and bronchoalveolar lavage fluid were collected.

[0160] II. ELISA detection of RBD-specific IgA antibody levels in bronchoalveolar lavage fluid

[0161] 1. Experimental method

[0162] Dilution 3, 9, 27, 81 times to measure the RBD-specific IgA antibody titer of the alveolar lavage fluid collected at the 6th week (n = 5), OD 450 The reading represents the titer of RBD-specific IgA, and the specific method is the same as that of Example 1.

[0163] 2. Experimental results

[0164] The ELISA results are shown in Figure 14 At the 6th week of twice nasal immunization of C57BL / 6 mice, the alveolar lavage fluid of the V50-RBD-HPF group has a higher RBD-specific IgA titer, indicating that the V50 fragment can induce high-titer IgA type antibodies in the lungs.

[0165] III. Pseudovirus antibody neutralization experiment

[0166] 1. Experimental method

[0167] SARS-CoV-2 pseudovirus neutralization experiment of alveolar lavage fluid (BALF) of different variants was carried out, and the specific method was: referring to the serum neutralization experiment method, the neutralization activity of antibodies in the alveolar lavage fluid was detected. The alveolar lavage fluid of BALB / c mice was collected, centrifuged at 400 x g at 4 °C for 10 min to remove cells and impurities, and the supernatant was filtered and stored as needed. The sample was diluted by 2 times in series with 1:5 as the starting point, mixed with an equal volume of SARS-CoV-2 pseudovirus of each variant (pseudovirus preparation is the same as Example 1), and incubated at 37 °C for 1 h. The mixed solution was added to 293T-ACE2 cells for culture for 48 h, and the luciferase signal was detected. The lower the signal, the stronger the neutralization activity of BALF.

[0168] Nonlinear fitting analysis of neutralizing antibody titer was performed by Prism software, and the neutralizing antibody titer corresponding to half the inhibition rate of pseudovirus (pVNT50) was calculated.

[0169] 2. Experimental results

[0170] The results of the neutralizing antibody experiment are shown in Figure 15 The neutralizing antibody titer of the V50-RBD-HPF group against the JN.1 variant pseudovirus is 10 1 orders of magnitude higher than that of the RBD-HPF group, indicating that the V50 peptide segment helps the RBD nanoparticle vaccine to induce neutralizing antibodies against other novel coronavirus immune escape variants in mucosal immunization.

[0171] IV. Detection of lung immune response

[0172] 1. Experimental method

[0173] Lung-resident IgA+and IgG+memory B cells and lung tissue-resident RBD antigen-specific memory B cells were isolated from the lung and analyzed by flow cytometry. The specific method is as follows:

[0174] The lung tissue of BALB / c mice after immunization was cut into pieces in a culture dish containing RPMI-1640 complete medium, and then digested with a mixed solution of collagenase D (1 mg / mL) and DNase I (50 μg / mL) at 37°C for 30 min. After digestion, the single cell suspension was collected by filtering with a 70 μm filter. After centrifugation at 400 x g for 10 min, the obtained lymphocytes were washed with PBS and stained with live / dead dye to distinguish live cells, and then surface markers were detected. Fluorescently labeled antibodies were used to detect B220, CD19, CD38, IgA, IgG, CD27, etc. to identify lung-resident IgA + and IgG + memory B cell populations.

[0175] To detect RBD antigen-specific memory B cells, biotinylated RBD protein was used to form a probe by binding with fluorescently labeled streptavidin (BV421). The probe was incubated with isolated lung lymphocytes for 30 min (4°C, dark), and then washed and co-stained with surface marker antibodies. The sample was detected by flow cytometry, and the proportion of RBD probe positive, B220 + , CD38 + , IgA + or IgG + cells was analyzed by FlowJo software to evaluate the immune response level of lung tissue-resident RBD-specific memory B cells.

[0176] Among them, the gate strategy is: ①FSC / SSC circle single nucleus cells, remove debris and agglomerates; ②FVD-APC-Cy7 - screen live cells; ③IgD-PE - CD19-Amcyan + antigen experienced B cells; ④CD95-PE-Cy7 - CD38-APC - memory B cells; ⑤RBD-BV421 + cells in memory B cells, i.e. RBD-specific memory B cells.

[0177] 2. Experimental results

[0178] The results showed that the V50-RBD-HPF group induced a higher proportion of IgA+ tissue-resident memory B cells in the lungs of mice. Figure 16 A) IgG+ tissue-resident memory B cells ( Figure 16 B cells in the brain) and RBD-specific tissue-resident memory B cells ( Figure 16 The C in the text indicates that the V50 peptide helps induce a higher proportion of tissue-resident memory B cells of various types in the mouse lungs.

Claims

1. The application of a peptide in the preparation of adjuvants or self-adjuvants, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The application of a nucleic acid molecule in the preparation of adjuvants or self-adjuvants, characterized in that, The gene encoding the peptide described in claim 1.

3. The application of a biomaterial in the preparation of adjuvants or self-adjuvants, characterized in that, It is any one of the following: (1) An expression cassette containing the nucleic acid molecule of claim 2; (2) A recombinant vector containing the nucleic acid molecule of claim 2 or the expression cassette of (1); (3) A recombinant microorganism containing the nucleic acid molecule of claim 2, the expression cassette of (1), or the recombinant vector of (2); (4) A cell line containing the nucleic acid molecule of claim 2, the expression cassette of (1), the recombinant vector of (2), or the recombinant microorganism of (3).

4. A protein composition, characterized in that, It comprises component 1 and component 2: component 1 is a fusion protein consisting of SD protein, the peptide described in claim 1, and an immunogen, in sequence from the N-terminus to the C-terminus; Component 2 is a fusion protein consisting of a GV peptide and an HPF protein immunogen, arranged sequentially from the N-terminus to the C-terminus.

5. A nucleic acid composition, characterized in that, It contains component A and component B, which are the encoding genes of component 1 and component 2 of the protein combination described in claim 4, respectively.

6. A biomaterial, characterized in that, It is any one of the following: (a) An expression cassette containing the nucleic acid composition of claim 5; (b) A recombinant vector containing the nucleic acid composition of claim 5 or the expression cassette of (a); (c) A recombinant microorganism comprising the nucleic acid composition of claim 5, the expression cassette of (a), or the recombinant vector of (b); (d) A cell line containing the nucleic acid composition of claim 5, the expression cassette of (a), the recombinant vector of (b), or the recombinant microorganism of (c).

7. An immunogenicity-enhanced nanoparticle, characterized in that, It is obtained by self-assembly of the components of the protein composition according to claim 6.

8. The use of the protein composition of claim 4, the nucleic acid composition of claim 5, the biomaterial of claim 6, or the nanoparticles of claim 7 in the preparation of vaccines.

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