An adjuvant and its application in preparing vaccines

Abstract

The invention discloses an adjuvant and its application in the preparation of a vaccine, wherein the adjuvant amino acid sequence is shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 23, 27, 31, 35 or 39. It can improve the immune protection effect of nanoparticle vaccines by activating TLR2, and show good vaccine adjuvant activity in general immunogen SD or new coronavirus RBD immunogen models. Further, the assembly and construction method of the self-adjuvant-nanoparticle vaccine is optimized, and the self-adjuvant-nanoparticle vaccine is prepared to produce a significant effect of inducing antibody production in animals. The new adjuvant provided by the present invention can be widely used in vaccine research and development, and the assembled nanoparticle self-adjuvant vaccine can also become a new vaccine.

Description

Technical Field

[0001] The present invention relates to the field of immunity and vaccines, and more specifically, to an adjuvant and application thereof in preparing vaccines. Background Art

[0002] Vaccines are preventive biological products that stimulate the body's immune system to produce an immune response against a specific pathogen (such as a virus or bacteria), thereby providing protection against the disease caused by the pathogen. In order to enhance the immune effect, vaccines are often added with adjuvants, which are an indispensable component of vaccines and can induce stronger and more lasting immune responses by enhancing the immunogenicity of antigens.

[0003] In the field of vaccine development, the role of adjuvants is not limited to improving the effectiveness of vaccines, but also plays a key role in reducing antigen doses, optimizing the direction of immune responses, and improving protective efficacy. Traditional adjuvants such as aluminum salt adjuvants have been widely used in a variety of vaccines, but their limitations in inducing cellular immunity and complex pathogen protection have promoted the development of new adjuvants. In recent years, with the advancement of immunology and biotechnology, innovative adjuvants based on molecular mechanisms have gradually become a research hotspot, including Toll-like receptor (TLR)-based immunomodulators, nanoparticle adjuvants, and new adjuvants targeting inflammasome pathways.

[0004] Toll-like receptor 2 (TLR2) is an important member of the pattern recognition receptor family and plays a key role in recognizing pathogen-associated molecular patterns (PAMPs) such as bacterial lipid molecules such as lipoylated peptides and lipoteichoic acid. Activation of TLR2 can initiate innate and adaptive immune responses, induce the secretion of inflammatory factors and regulate the activity of immune cells. TLR2 can sense a variety of pathogenic molecules through heterodimerization with TLR1 or TLR6. This diversity enables adjuvants targeting TLR2 to flexibly activate multiple immune pathways, which can not only induce effective humoral immunity to produce high-titer neutralizing antibodies, but also stimulate cellular immunity and enhance T cell responses, thereby providing greater flexibility for the design of vaccines against complex pathogens. Compared with some strongly stimulating TLRs (such as TLR4), TLR2 activation can usually induce a mild and controllable immune response with a lower risk of systemic inflammatory response. This feature makes adjuvants targeting TLR2 more suitable for children, the elderly and other immune-vulnerable populations. Therefore, in recent years, adjuvants targeting TLR2 have gradually attracted attention due to their unique immunomodulatory properties and have shown significant research and development advantages.

[0005] At present, studies have found that some TLR2 adjuvants based on lipopeptides or lipopolysaccharides have shown significant immune enhancement effects, especially in viral diseases and bacterial vaccines. TLR2 agonists are generally mild, but the immune responses of different populations may vary significantly, and further exploration of ligand selection and dosage regulation of targets is needed.

[0006] Ferritin is a nanoparticle with self-assembly ability. It is composed of 24 subunits and can spontaneously form a hollow spherical structure with a diameter of about 12 nanometers under physiological conditions. Ferritin nanoparticles are used as delivery carriers, and a series of new crown RBD vaccines have been developed. They induce broad-spectrum and highly effective neutralizing antibodies in mice and rhesus monkeys and produce antiviral infection protection effects. In recent years, the development of various antiviral vaccines based on ferritin nanoparticles has attracted widespread attention. It can not only serve as an antigen delivery platform, but also enhance the immune response through self-adjuvant effect by coupling other substances. Self-adjuvant refers to certain substances or components that have the ability to enhance the immune response without the addition of traditional adjuvants. This type of substance can activate and regulate the immune system through its own characteristics without relying on external auxiliary substances, thereby improving the effectiveness of vaccines or other immunotherapies.

[0007] The development of self-adjuvant ferritin nanoparticle vaccine has many significant advantages. Ferritin is a protein naturally present in mammals, has good biocompatibility and safety, and is not prone to allergic or toxic reactions. Its degradation mechanism based on the iron metabolic pathway can further reduce the risk of immune side effects, making it particularly suitable for vaccination of the elderly, children or immune-vulnerable people. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide an adjuvant and its application in the preparation of vaccines.

[0009] The first object of the present invention is to provide a peptide segment.

[0010] The second object of the present invention is to provide a nucleic acid molecule.

[0011] The third object of the present invention is to provide a biomaterial.

[0012] The fourth object of the present invention is to provide the use of the peptide segment, the nucleic acid molecule or the biological material in the preparation of a sub-adjuvant, an adjuvant or a vaccine.

[0013] The fifth object of the present invention is to provide the use of the peptide segment, the nucleic acid molecule or the biological material in improving the immunogenicity of an antigen.

[0014] A sixth object of the present invention is to provide a protein composition.

[0015] The seventh object of the present invention is to provide another protein composition.

[0016] The eighth object of the present invention is to provide a nucleic acid composition.

[0017] The ninth object of the present invention is to provide another nucleic acid composition.

[0018] The tenth object of the present invention is to provide another biomaterial.

[0019] The eleventh object of the present invention is to provide nanoparticles with enhanced immunogenicity.

[0020] The twelfth object of the present invention is to provide another nanoparticle with enhanced immunogenicity.

[0021] The thirteenth object of the present invention is to provide the use of the protein composition, the nucleic acid composition, the biomaterial or the nanoparticle in the preparation of vaccines.

[0022] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0023] A peptide segment, whose amino acid sequence is shown as SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35 or SEQ ID NO:39.

[0024] A nucleic acid molecule encodes the coding gene of the sub-peptide segment, or is the reverse complementary sequence of the coding gene.

[0025] The amino acid sequence of peptide 2SBspA is shown in SEQ ID NO:23, and the nucleotide sequence of the gene encoding the SBspA is shown in SEQ ID NO:24.

[0026] The nucleotide sequence of the gene encoding the peptide 2SMVH whose amino acid sequence is shown in SEQ ID NO:27 is shown in SEQ ID NO:28.

[0027] The nucleotide sequence of the gene encoding the peptide 4SMVH whose amino acid sequence is shown in SEQ ID NO:31 is shown in SEQ ID NO:32.

[0028] The amino acid sequence of peptide 1BmpA is shown in SEQ ID NO:33, and the nucleotide sequence of the gene encoding the peptide 1BmpA is shown in SEQ ID NO:34.

[0029] A biological material, which is any of the following:

[0030] (1) an expression cassette containing the nucleic acid molecule;

[0031] (2) A recombinant vector containing the nucleic acid molecule described above or the expression cassette described above in (1);

[0032] (3) A recombinant microorganism containing the nucleic acid molecule described above, the expression cassette described above in (1) or the recombinant vector described above in (2);

[0033] (4) A cell line containing the nucleic acid molecule described above, the expression cassette described above in (1), the recombinant vector described above in (2), or the recombinant microorganism described above in (3).

[0034] The use of the peptide segment, the nucleic acid molecule or the biological material in the preparation of a sub-adjuvant, an adjuvant or a vaccine.

[0035] The use of the peptide segment, the nucleic acid molecule or the biological material in improving the immunogenicity of an antigen.

[0036] A protein composition 1 comprises component 1 and component 2: component 1 is a fusion protein which comprises SD protein, the peptide segment and HPF protein from N-terminus to C-terminus;

[0037] Component 2 is a fusion protein consisting of a GV peptide segment and an immunogen from the N-terminus to the C-terminus.

[0038] The amino acid sequence of the SD protein is shown in SEQ ID NO: 1; the amino acid sequence of the GV peptide segment is shown in SEQ ID NO: 3.

[0039] The amino acid sequence of the HPF protein is shown in SEQ ID NO:67.

[0040] As a specific embodiment, the peptide segment is 1BmpA, the amino acid sequence of component 1 is shown in SEQ ID NO:53, and the amino acid sequence of component 2 is shown in SEQ ID NO:59.

[0041] A protein composition 2, comprising component 1, component 2 and component 3:

[0042] Component 1 is a fusion protein comprising SD protein and the peptide segment of claim 1 from N-terminus to C-terminus;

[0043] Component 2 is a fusion protein containing SD protein and immunogen from N-terminus to C-terminus.

[0044] Component 3 is a fusion protein consisting of GV peptide and HPF protein from N-terminus to C-terminus.

[0045] As a specific embodiment, the peptide segment is 1BmpA, the amino acid sequence of component 1 is shown in SEQ ID NO:61, the amino acid sequence of component 2 is shown in SEQ ID NO:63; and the amino acid sequence of component 3 is shown in SEQ ID NO:67.

[0046] A nucleic acid composition comprises component 1 and component 2, which are respectively the coding genes of component 1 and component 2 of the protein combination or the complementary sequences of the coding genes.

[0047] A nucleic acid composition comprises component 1, component 2 and component 3, which are coding genes of component 1, component 2 and component 3 of protein combination 2 or complementary sequences of coding genes.

[0048] A biological material, which is any of the following:

[0049] (1) an expression cassette containing the nucleic acid composition;

[0050] (2) A recombinant vector containing the nucleic acid composition described above or the expression cassette described above in (1);

[0051] (3) A recombinant microorganism containing the nucleic acid composition described above, the expression cassette described above in (1) or the recombinant vector described above in (2);

[0052] (4) A cell line containing the nucleic acid composition described above, the expression cassette described above in (1), the recombinant vector described above in (2), or the recombinant microorganism described above in (3).

[0053] A nanoparticle with enhanced immunogenicity, wherein the nanoparticle is a nanoparticle obtained by self-assembly using HPF protein:

[0054] Wherein, the N-terminus of the HPF protein is linked to the peptide segment and the immunogen;

[0055] Or the N-terminus of the HPF protein is linked to the peptide segment, and the N-terminus of the peptide segment is linked to the immunogen.

[0056] As a specific embodiment, the immunogen is RBD, and its amino acid sequence is shown in SEQ ID NO:41; the amino acid sequence of HPF ferritin is shown in SEQ ID NO:67.

[0057] Preferably, the connection is through or not through a GV peptide segment-SD protein self-assembly system, wherein the amino acid sequence of the GV peptide segment is as shown in SEQ ID NO: 4, and the amino acid sequence of the SD protein is as shown in SEQ ID NO: 2.

[0058] A nanoparticle with enhanced immunogenicity, wherein the protein composition is obtained by self-assembly of various components.

[0059] Use of the protein composition 1 or 2, the nucleic acid composition, the biomaterial or the nanoparticle in preparing a vaccine.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The present invention discloses a peptide segment with adjuvant activity, which can improve the immune protection effect of nanoparticle vaccines by activating TLR2, and show good vaccine adjuvant activity in general immunogen SD or new coronavirus RBD immunogen models. Further, the assembly and construction method of the self-adjuvant-nanoparticle vaccine is optimized to prepare a self-adjuvant-nanoparticle vaccine that produces a significant effect of inducing antibody production in animals. The new adjuvant provided by the present invention can be widely used in vaccine research and development, and the assembled nanoparticle self-adjuvant vaccine can also become a new vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The SD protein and SD-potential self-adjuvant fusion protein obtained by prokaryotic expression and purification by nickel column affinity chromatography were stained with Coomassie Brilliant Blue.

[0063] Figure 2 Figure 2 ELISA test of serum SD-specific IgG antibody titers in Balb / c mice after intranasal immunization; A is the test results in the second week after the primary immunization: n=4 for SD group, SD-BspA group, SD-truMVH-2 group, and SD-SC2-EP-CTD group, and n=3 for the other groups; B is the test results in the fifth week after the booster immunization in the third week after the primary immunization: n=4 for SD group, SD-BspA group, SD-truMVH-2 group, and SD-SC2-EP-CTD group, and n=3 for the other groups; Data are expressed as mean ± standard deviation, and the adjusted p value was calculated using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test.

[0064] Figure 3 are the truncated sequences of BspA, truMVH-2, and BmpA; A is a schematic diagram of the truncation of BspA, truMVH-2, and BmpA, the rectangular diagram indicates the protein length, and the numbers indicate the number of amino acids; B is the SD protein and SD-potential self-adjuvant truncated sequence protein obtained by prokaryotic expression and purification by nickel column affinity chromatography stained with Coomassie Brilliant Blue.

[0065] Figure 4The ELISA test was used to detect the serum SD-specific IgG antibody titer of Balb / c mice after intranasal immunization; A is the test result in the second week after the primary immunization; B is the test result in the fifth week after the booster immunization in the third week after the primary immunization: n=3 mice in each group, expressed as mean ± standard deviation, and the adjusted p value was calculated using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test.

[0066] Figure 5 The RBD-self-adjuvant fusion protein was obtained by prokaryotic expression stained with Coomassie Brilliant Blue and purified by nickel column affinity chromatography.

[0067] Figure 6 The serum SD-specific IgG antibody titers of Balb / c mice were detected by ELISA at the eighth week after the first immunization after intranasal immunization; n=8 for RBD group and RBD-4SMVH group, n=7 for RBD-1BmpA group, and n=6 for RBD-2SBspA group. The values ​​are expressed as mean ± standard deviation. The adjusted p-values ​​were calculated using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test.

[0068] Figure 7 A is a self-adjuvant nanoparticle; A is a schematic diagram of the self-adjuvant nanoparticle vaccine assembly; B is a protein gel image detection of the nanoparticle vaccine; C is a molecular sieve (SEM) detection image of the SD-1BmpA-HPF nanoparticle vaccine.

[0069] Figure 8 A is the preparation of self-adjuvant-nanoparticle RBD-1BmpA HPF NP; A is the strategy for preparing nanoparticle self-adjuvant vaccine; B is the result of Coomassie Brilliant Blue stained protein electrophoresis.

[0070] Fig. 9 A is the preparation of self-adjuvant-nanoparticle 1BmpA-HPF RBD NP; A is the strategy for preparing nanoparticle self-adjuvant vaccine; B is the result of Coomassie Brilliant Blue stained protein electrophoresis.

[0071] Fig.10 A is the self-adjuvant nanoparticle Mosaic NP; A is the preparation strategy of nanoparticle self-adjuvant vaccine; B is the result of Coomassie Brilliant Blue stained protein electrophoresis.

[0072] Fig.11The effects of different new coronavirus vaccines on immunity; A is the ELISA test of serum SD-specific IgG antibody titer of Balb / c mice at the seventh week after the initial immunization with intranasal drops; B is the pseudovirus neutralization experiment to detect the level of new coronavirus neutralizing antibodies in mouse serum at the fifth week; C is the ELISA test of RBD-specific IgG antibody titer in bronchoalveolar lavage fluid of Balb / c mice at the seventh week after the initial immunization with intranasal drops; D is the ELISA test of RBD-specific IgA antibody titer in bronchoalveolar lavage fluid of Balb / c mice at the seventh week after the initial immunization with intranasal drops; n=5 mice in each group, expressed as mean±standard deviation, and adjusted p-values ​​were calculated using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test.

[0073] Fig.12 Activation of hTLR1 / 2-293T cells, NF-κB activation fold = NF-κB-activated firefly fluorescence intensity / internal reference Renilla fluorescence intensity; n = 3 per group, expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and Tukey's multiple comparison test were used to calculate the adjusted p value. DETAILED DESCRIPTION

[0074] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0075] GvTagOpti / Sdcatcher (Gv / Sd) system based on isopeptide bonds, see Chinese patent CN113621031A, a combination of peptide-linked adapters that utilize spontaneous isopeptide bonds for protein covalent self-assembly.

[0076] The autocatalytic isopeptide bond system composed of the SD protein and the GV peptide segment used in the following examples, wherein the amino acid sequence of the SD protein is shown in SEQ ID NO:1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:2; the amino acid sequence of the GV peptide segment is shown in SEQ ID NO:3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:4.

[0077] The amino acid sequence of HPF ferritin is shown in SEQ ID NO:67, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:68.

[0078] Example 1: Improvement of the immunogenicity of pathogen-related proteins with the potential to activate TLR2 against SD

[0079] 1. Expression of SD-potential self-adjuvant fusion protein

[0080] 1. Experimental methods

[0081] For pathogen-related proteins with the potential to activate TLR2, sequences that may have immune activation effects were obtained based on the National Center for Biotechnology Information (NCBI) database, as shown in Table 1 (tru, truncated, truncated).

[0082] Table 1:

[0083]

[0084] In order to utilize the autocatalytic isopeptide bond system composed of SD protein and GV peptide to couple it to the surface of ferritin nanoparticles in the later stage, SD protein was coupled to the N-terminus of the sequence in Table 1, and prokaryotic expression and purification were performed to facilitate purification and immunogenicity detection.

[0085] The specific steps of prokaryotic expression are as follows: First, the coding sequences of the sequences in Table 1 are inserted into the expression vector pET28a by molecular cloning technology, wherein the C-terminus of the expression vector pET28a contains a 6×His tag.

[0086] After sequencing detection, the correctly constructed recombinant vector was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and positive clones were screened on LB solid medium containing kan (50 μg / mL).

[0087] The positive colonies were cultured in shake flasks and inoculated into LB liquid medium containing kan (50 μg / mL) at 37°C until OD 600 When the value reaches 0.6-0.8, add IPTG to 0.5 mM to induce expression and culture at 16°C for 12-16 hours to reduce the risk of inclusion body formation and improve protein solubility.

[0088] After induction, the cells were collected by centrifugation (4°C, 8000×g, 10 minutes) and resuspended in pre-cooled lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl). The resuspended cells were lysed by high-pressure crusher and then centrifuged (4°C, 12000×g, 30 minutes) to obtain the supernatant.

[0089] The supernatant contains the target protein and is directly loaded onto a pre-equilibrated nickel column for affinity chromatography purification. During the purification process, different concentrations of imidazole were used for elution, and 1 mL of elution buffer (containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 250 mM imidazole) was used for elution.

[0090] The elution peak of the target protein was verified by SDS-PAGE for purity, and the imidazole was removed by dialysis against 50 mM Tris-HCl (pH 8.0) buffer containing 300 mM NaCl. Finally, a highly pure target fusion protein (SD-potential self-adjuvant) was obtained for subsequent experiments. The SD protein was also expressed in the same prokaryotic manner.

[0091] 2. Experimental results

[0092] The results are as follows Figure 1 As shown, the results showed that SD protein, SD-BspA protein (sequence source: surface protein A of Tannerella forsythia), SD- BCSP31 protein (sequence source: surface protein 31 of Brucella abortus), SD-LipL32 protein (corresponding to the outer membrane protein of Leptospira), SD-BmpA protein (sequence source: surface A protein of Helicobacter burgdorferi), SD-HCV-truCP protein (sequence source: core protein of hepatitis C virus), SD- HCV-truNS3 protein (sequence source: non-structural protein 3 of hepatitis C virus), SD-truMVH-2 protein (sequence source: hemagglutinin protein of measles virus), and SD-SC2-EP-CTD protein (sequence source: the extracellular segment of the envelope protein of the new coronavirus) were purified, and protein electrophoresis and Coomassie blue staining were used for identification and analysis. The main bands were clear, indicating that the purity was consistent.

[0093] 2. Immunological activity of fusion protein

[0094] 1. Experimental methods

[0095] The prepared SD-potential self-adjuvant and SD protein were used to immunize Balb / c mice by nasal drops, respectively. The mass of SD contained in each immunization of each group of mice was 10 μg, and the second dose of nasal drops was performed in the third week after the initial immunization.

[0096] The SD-specific IgG antibody titer in the mouse serum at the second and fifth weeks after the initial immunization was detected by ELISA. The specific ELISA detection method is as follows:

[0097] Coating: Dilute SD protein to 2 μg / mL with carbonate buffer as coating solution, add 100 μL per well of 96-well ELISA plate, and incubate overnight at 4°C for coating.

[0098] Blocking: The next day, discard the coating solution and wash the plate three times with PBST, soaking for 5 minutes each time; then, add 5% bovine serum albumin (BSA) or skimmed milk powder in PBST to block the plate, 100 μL per well, and incubate at 37°C for 1 hour.

[0099] Sample addition: After blocking, wash the plate again 3 times with PBST; then, dilute the serum sample to be tested in a gradient manner (starting from 1:50, 3-fold gradient dilution), add 500 μL to each well, and incubate at 37°C for 1 hour.

[0100] Add secondary antibody: After incubation, wash the plate 5 times with PBST, soak for 2 minutes each time. Add HRP-labeled anti-species secondary antibody (anti-mouse IgG, 1:10000 dilution), 100 μL per well, and incubate at 37°C for 1 hour.

[0101] Color development: After washing the plate 5 times with PBST, add TMB color development solution, 100 μL per well, react at room temperature in the dark for 10-15 minutes, then add 50 μL 2M sulfuric acid to terminate the reaction.

[0102] Detection: Read the OD value at 450 nm on a microplate reader.

[0103] The difference in OD value between samples and controls was used to evaluate the level of SD-specific IgG antibodies.

[0104] 2. Experimental results

[0105] The results are as follows Figure 2 As shown, by fusing SD protein with pathogen-related proteins with the potential to activate TLR2, the pathogen-related proteins with the potential to activate TLR2 can improve the immunogenicity of SD protein. Among them, after immunizing mice with SD-BspA, SD-BmpA, and SD-truMVH-2 obtained by fusion expression of BspA, BmpA, and truMVH-2 with SD protein, it was found that the effect of increasing the antibody level against SD in immunized mice was the most prominent.

[0106] Example 2 Improvement of SD immunogenicity by truncated peptides of proteins that increase SD antibody levels

[0107] 1. Protein truncation to improve SD antibody levels

[0108] 1. Experimental methods

[0109] In order to further clarify the key active regions in BspA, BspA and truMVH-2 that promote antigen immunogenicity, BspA, BspA and truMVH-2 were further truncated to obtain self-adjuvant truncated sequences).

[0110] See the truncation strategy Figure 3A in:

[0111] BspA was truncated into 1SBspA (three short fragments were connected by a fusion protein linker with the amino acid sequence GSG) and 2SBspA;

[0112] truMVH-2 is truncated to 1SMVH, 2SMVH, 3SMVH, 4SMVH, and 5SMVH;

[0113] BmpA is truncated into 1BmpA, 2BmpA, 3BmpA and 4BmpA.

[0114] The specific sequences are shown in Table 2.

[0115] Table 2:

[0116]

[0117] The N-terminus of each adjuvant truncated sequence was coupled with SD protein to obtain a fusion protein (SB-self-adjuvant truncated sequence), and the coding sequence of the fusion protein was cloned into the pET28a vector to obtain a recombinant vector, which was expressed in prokaryotic cells according to the method of Example 1 to obtain the SD-self-adjuvant truncated sequence. The SD protein was also expressed in the same prokaryotic cell manner, and the purified target protein was identified by protein electrophoresis and Coomassie blue staining.

[0118] 2. Experimental results

[0119] The results are as follows Figure 3 As shown in B, the results showed that except for SD-4BmpA, other proteins were successfully expressed, and each purified protein showed a main target band, and the purity was sufficient for subsequent experiments.

[0120] 2. Immunological activity of truncated sequences of SD-self-adjuvant

[0121] 1. Experimental methods

[0122] The prepared SD-self-adjuvant truncated sequence was used to immunize Balb / c mice by intranasal drops, wherein the mass of SD contained in each group of mice per immunization was 10 μg, and the second dose of intranasal drops was performed for re-immunization in the third week after the initial immunization.

[0123] The SD-specific IgG antibody titers in the sera of mice at the second and fifth weeks were detected by ELISA.

[0124] The specific ELISA detection method is the same as in Example 1.

[0125] 2. Experimental results

[0126] The results are as follows Figure 4As shown in the figure, the fusion proteins SD-2SBspA, SD-4SMVH and SD-1BmpA can significantly increase the SD-specific IgG antibody titer in mice after immunization. Therefore, the fusion proteins 2SBspA, 4SMVH and 1BmpA were selected for further experiments.

[0127] Example 3: Improvement of RBD antigenicity by truncated sequences of proteins that increase antibody levels against SD

[0128] 1. Protein truncation to improve SD antibody levels

[0129] 1. Experimental methods

[0130] 2SBspA, 4SMVH and 1BmpA were designed respectively, and fused with the novel coronavirus receptor binding domain protein (RBD, nucleotide sequence is SEQ ID NO: 41, amino acid sequence is SEQ ID NO: 42) for expression, and the RBD sequence was coupled to the N-terminus of the self-adjuvant truncated sequences 2SBspA, 4SMVH and 1BmpA, and the amino acid sequence was codon-optimized for eukaryotic expression according to the preference of 293F cells to obtain the coding sequence of the RBD-self-adjuvant truncated sequence, the sequence of which is shown in Table 3 below, and the coding sequence of the 6×His tag was further added to the 3' end of the coding sequence to obtain the coding sequence of the RBD-self-adjuvant truncated sequence-6×His:

[0131] Table 3

[0132]

[0133] The optimized RBD-self-adjuvant truncated sequence-6×His coding sequence was cloned into the pcDNA3.1 vector to obtain a recombinant vector. The recombinant plasmid was transfected into 293F cells using PEI max reagent, and the cells secreted the synthesized target protein into the culture supernatant. The supernatant was collected on the fourth day after transfection, and the target protein was purified by nickel column affinity chromatography, and then identified by protein electrophoresis and Coomassie blue staining.

[0134] 2. Experimental results

[0135] The results are as follows Figure 5 As shown, the main target bands of RBD-2SBspA, RBD-4SMVH and RBD-1BmpA with 6×His tags were consistent with expectations, and the fusion proteins RBD-2SBspA, RBD-4SMVH and RBD-1BmpA were successfully prepared.

[0136] 2. The truncated sequence of the protein that increases the antibody level of SD enhances the antigenicity of RBD

[0137] 1. Experimental methods

[0138] Balb / c mice were immunized with RBD protein, fusion protein RBD-2SBspA, fusion protein RBD-4SMVH and fusion protein RBD-1BmpA by intranasal drops. The mass of RBD contained in each group of mice was 20 μg each time. The mice were immunized again by intranasal drops in the third and sixth weeks after the initial immunization, for a total of three doses of immunization.

[0139] The RBD protein-specific IgG antibody titer in the mouse serum at the eighth week after the initial immunization was detected by ELISA. The specific detection method was the same as that in the example, except that the SD protein in the coating solution was replaced by the RBD protein.

[0140] 2. Experimental results

[0141] The results are as follows Figure 6 The results showed that compared with the mice in the RBD group, the mice in the RBD-2SBspA, RBD-4SMVH, and RBD-1BmpA groups were able to produce higher levels of RBD-specific antibodies, indicating that for eukaryotic expressed antigen proteins, 2SBspA, 4SMVH, and 1BmpA have adjuvant effects, can improve the immunogenicity of antigens, and can serve as self-adjuvant proteins.

[0142] Example 4 Preparation of a self-adjuvant-nanoparticle

[0143] 1. Preparation of Self-adjuvant Nanoparticles

[0144] like Figure 7 As shown in A in FIG, SD sequence is added to the N-terminus of the self-adjuvant truncated sequence 2SBspA, 4SMVH and 1BmpA, and HPF sequence is added to the C-terminus of the self-adjuvant protein to obtain SD-self-adjuvant truncated sequence-HPF. The sequence information is shown in Table 4.

[0145] Table 4:

[0146]

[0147] The coding sequence of SD-self-adjuvant truncated sequence-HPF was cloned into the pET28a vector, and prokaryotic expression was performed using BL21 (DE3) according to the method of Example 1, and purification was performed by nickel column affinity chromatography to obtain each fusion protein (SD-self-adjuvant truncated sequence-HPF). Since HPF molecules can spontaneously assemble to form 24-mers, the purified protein exists in the form of nanoparticles. The purified target protein was identified by protein electrophoresis and Coomassie blue staining.

[0148] 2. Protein Electrophoresis

[0149] The molecular weight and purity of the obtained SD-self-adjuvant truncated sequence-HPF were analyzed by polyacrylamide gel electrophoresis and Coomassie blue staining.

[0150] 3. Molecular sieve

[0151] The spontaneously formed "SD-self-adjuvant protein-HPF" twenty-tetrameric nanoparticle protein was purified by molecular sieve chromatography using a Siperose6Increase10 / 300 GL column (GE). The molecular sieve chromatography buffer was PBS, pH 7.4.

[0152] The molecular sieve peak is single, indicating that the present invention has obtained a twenty-tetrameric protein with a purity higher than 99%.

[0153] IV. Experimental Results

[0154] Protein electrophoresis results Figure 7 As shown in B, each SD-self-adjuvant protein-HPF can be successfully expressed and purified. The molecular screening results are as follows Figure 7 As shown in C, protein electrophoresis showed that SD-2SBspA-HPF could not form ferritin 24-mers, SD-4SMVH-HPF formed super-polymers, and only SD-1BmpA-HPF could form relatively homogeneous ferritin 24-mers, showing a main peak of 24-mers in the molecular sieve experiment.

[0155] Comparative Example 1 Preparation of a novel coronavirus vaccine based on nanoparticles RBD NP

[0156] 1. Experimental Methods

[0157] Preparation of a nanoparticle RBD NP coronavirus vaccine that does not contain a self-adjuvant peptide and is not modified: SD and RBD protein are fused and expressed (from N-terminus to C-terminus) to obtain the fusion protein SD-RBD (nucleotide sequence is SEQ ID NO: 64, amino acid sequence is SEQ ID NO: 63), Gv and HPF are fused and expressed (from N-terminus to C-terminus) to obtain the fusion protein Gv-HPF (nucleotide sequence is SEQID NO: 58, amino acid sequence is SEQ ID NO: 57), Gv-HPF self-assembles to obtain HPF nanoparticles coupled with Gv, SD-RBD and HPF nanoparticles coupled with Gv are connected using the isopeptide bond-based GvTagOpti / Sdcatcher (Gv / Sd) system, RBD-1BmpA is connected to the surface of HPF nanoparticles to construct RBD NP, which is then analyzed by protein electrophoresis and Coomassie blue staining.

[0158] Among them, the expression and purification methods of the fusion protein SD-RBD and the fusion protein Gv-HPF are the same as those in Example 4; the specific method of connection based on the isopeptide bond GvTagOpti / Sdcatcher (Gv / Sd) system and HPF self-assembly is as follows: after the fusion protein SD-RBD and the fusion protein Gv-HPF are mixed in a molar ratio of 1:1, they are incubated overnight at 25°C to obtain.

[0159] 2. Experimental Results

[0160] The results showed (electrophoresis diagram see Figure 8 (B) SD-HPF+Gv-RBD lane results: SD-RBD was successfully connected to Gv-HPF using the Gv / Sd system, and successfully self-assembled to obtain twenty-tetrameric RBD NP, i.e., nanoparticle vaccine.

[0161] Example 5 Preparation of a novel coronavirus vaccine based on self-adjuvant-nanoparticles

[0162] 1. Experimental Methods

[0163] 1. Preparation of self-adjuvant nanoparticles RBD-1BmpA HPF NP

[0164] SD, 1BmpA and RBD protein were fused and expressed (from N-terminus to C-terminus) to obtain the fusion protein SD-RBD-1BmpA (nucleotide sequence of SEQ ID NO:56, amino acid sequence of SEQ ID NO:55), Gv and HPF were fused and expressed (from N-terminus to C-terminus) to obtain the fusion protein Gv-HPF (nucleotide sequence of SEQ ID NO:58, amino acid sequence of SEQ ID NO:57), Gv-HPF self-assembled to obtain HPF nanoparticles coupled with Gv, SD-RBD-1BmpA and HPF nanoparticles coupled with Gv were connected using the GvTagOpti / Sdcatcher (Gv / Sd) system based on isopeptide bonds, and RBD-1BmpA was connected to the surface of HPF nanoparticles to construct self-adjuvant-nanoparticle RBD-1BmpA HPF NP. The construction strategy is shown in Figure 8 A in the sample was then analyzed by protein electrophoresis and Coomassie blue staining. The specific preparation method is as follows:

[0165] The expression and purification methods of the fusion protein SD-RBD-1BmpA and the fusion protein Gv-HPF are the same as those in Example 4;

[0166] The specific method of connection based on the isopeptide bond GvTagOpti / Sdcatcher (Gv / Sd) system and HPF self-assembly is as follows: after the fusion protein SD-RBD-1BmpA and the fusion protein Gv-HPF are mixed in a molar ratio of 1:1, they are incubated overnight at 25°C to obtain the product.

[0167] 2. Experimental Results

[0168] The results are as follows Figure 8 As shown in B, the results show that in the SD-RBD-1BmpA+Gv-HPF lane, SD-RBD-1BmpA was successfully connected to Gv-HPF using the Gv / Sd system, and successfully self-assembled to obtain twenty-tetrameric RBD-1BmpA HPFNP, i.e., nanoparticle vaccine.

[0169] Example 6 Preparation of a novel coronavirus vaccine based on self-adjuvant-nanoparticles

[0170] 1. Experimental Methods

[0171] 1. Preparation of self-adjuvant nanoparticles 1BmpA-HPF RBD NP

[0172] SD, 1BmpA and HPF were fused and expressed (from N-terminus to C-terminus) to obtain the fusion protein SD-1BmpA-HPF (nucleotide sequence is SEQ ID NO: 54, amino acid sequence is SEQ ID NO: 53), Gv and RBD were fused and expressed (from N-terminus to C-terminus) to obtain the fusion protein Gv-RBD (nucleotide sequence is SEQ ID NO: 60, amino acid sequence is SEQ ID NO: 59), SD-1BmpA-HPF self-assembled to form HPF nanoparticles coupled with SD-self-adjuvant (SD-1BmpA), and then the RBD protein was connected to the surface of HPF nanoparticles coupled with SD-1BmpA using the isopeptide bond-based GvTagOpti / Sdcatcher (Gv / Sd) system to construct the self-adjuvant-nanoparticle 1BmpA-HPF RBD NP. The construction strategy is shown in Fig. 9 In A, the specific preparation method refers to Example 5, and then analyzed by protein electrophoresis and Coomassie blue staining.

[0173] 2. Experimental Results

[0174] The results are as follows Fig. 9 As shown in B, the results of the SD-1BmpA-HPF+Gv-RBD lane showed that Gv-RBD was successfully connected to SD-1BmpA-HPF using the Gv / Sd system, and twenty-tetrameric 1BmpA-HPF RBD NP, i.e., nanoparticle vaccine, was successfully self-assembled.

[0175] Example 7 Preparation of a novel coronavirus vaccine based on self-adjuvant-nanoparticles

[0176] 1. Experimental Methods

[0177] 1. Preparation of self-adjuvant-nanoparticle Mosaic NP

[0178] SD and 1BmpA were fused and expressed (from N-terminus to C-terminus) to obtain the fusion protein SD-1BmpA (nucleotide sequence of SEQ ID NO: 62, amino acid sequence of SEQ ID NO: 61), SD and RBD were fused and expressed (from N-terminus to C-terminus) to obtain the fusion protein SD-RBD (nucleotide sequence of SEQ ID NO: 64, amino acid sequence of SEQ ID NO: 63), SD-1BmpA and SD-RBD were mixed at a molar ratio of 2:3 to obtain a SD-1BmpA+SD-RBD mixture, Gv and HPF were fused and expressed (from N-terminus to C-terminus) to obtain the fusion protein Gv-HPF (nucleotide sequence of SEQ ID NO: 66, amino acid sequence of SEQ ID NO:65), Gv-HPF self-assembled to obtain HPF nanoparticles coupled with Gv, SD-1BmpA+SD-RBD mixture and HPF nanoparticles coupled with Gv were connected using the GvTagOpti / Sdcatcher (Gv / Sd) system based on isopeptide bonds, 1BmpA and RBD were connected to the surface of HPF nanoparticles to construct self-adjuvant-nanoparticle Mosaic NP. The construction strategy is shown in Fig.10 In A, the specific preparation method refers to Example 5, and then analyzed by protein electrophoresis and Coomassie blue staining.

[0179] 2. Experimental Results

[0180] The results are as follows Fig.10 As shown in B, the results showed that SD-1BmpA and SD-RBD were successfully connected to Gv-HPF using the Gv / Sd system, and successfully self-assembled to obtain twenty-tetramers, namely nanoparticle vaccines.

[0181] Example 8 Effects of different novel coronavirus vaccines on serum RBD-specific IgG titers

[0182] 1. Experimental Methods

[0183] The RBD-1BmpA HPF NP prepared in Example 5, the 1BmpA-HPF RBDNP prepared in Example 6, the Mosaic NP prepared in Example 7 and the RBD NP prepared in Comparative Example 1 were used as vaccines to immunize Balb / c mice by nasal drops.

[0184] Each dose of vaccine for each group of mice contained 10 μg of RBD per dose, and booster immunizations were performed in the third and sixth weeks after the initial immunization, and serum was collected from the mice in the seventh week.

[0185] The RBD protein was coated on the ELISA plate to detect the serum RBD-specific IgG titer of each group, and the detection method was the same as that in Example 3.

[0186] 2. Experimental Results

[0187] The results are as follows Fig.11 As shown in A in the figure, the results show that, when comparing the immune effects of different nanoparticles, the serum RBD-specific IgG titer of the RBD-1BmpA HPF NP prepared in Example 5 was not significantly improved compared with the RBD NP prepared in Comparative Example 1; the 1BmpA-HPF RBD NP prepared in Example 6 and the Mosaic NP prepared in Example 7 were both significantly improved.

[0188] Example 9 Effects of different novel coronavirus vaccines on serum neutralizing antibody titers

[0189] 1. Experimental Methods

[0190] 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 the host cell and simulating the infection characteristics of the real virus. The core genome is derived from other safe viruses and is modified to be non-replicative. It also contains the reporter gene luciferase for quantifying infection events. For the preparation method and neutralization test of the new coronavirus pseudovirus, please refer to Example 4 of the Chinese patent "CN115043915A A method for enhancing the immunogenicity of new coronavirus variants and its application"

[0191] That is, the method for preparing SARS-CoV-2 pseudovirus is: by co-transfecting a plasmid expressing the spike protein Spike, a psPAX2 packaging plasmid, and a plasmid expressing luciferase into 293T cells, 48 ​​hours after transfection, collecting the culture supernatant to obtain the SARS-CoV-2 pseudovirus.

[0192] The serum of each group of mice in the fifth week after immunization in Example 8 was gradiently diluted and pre-incubated with the SARS-CoV-2 pseudovirus at 37°C for 1 hour, and hACE2-293T cells (HEK293T expressing hACE2, which can be recognized and invaded by the pseudovirus Spike, and the amount of luciferase expressed in hACE2-293T is positively proportional to the amount of virus that can be infected) were added. After culturing for 48 hours, the luciferase activity of the cells was lysed. The lower the luciferase activity, the lower the amount of infected virus, the stronger the ability of the serum to neutralize the virus, and the higher the neutralizing antibody titer.

[0193] 2. Experimental Results

[0194] Experimental Fig.11 As shown in B, the results show that the neutralizing antibody titer of the RBD-1BmpA HPF NP prepared in Example 5 was not significantly increased compared with the RBD NP without self-adjuvant protein in the mouse serum at the fifth week after immunization in Example 8, while the 1BmpA-HPF RBD NP prepared in Example 6 and the Mosaic NP prepared in Example 7 could significantly increase the neutralizing antibody level.

[0195] Example 10 Effects of different novel coronavirus vaccines on RBD-specific IgG and IgA in mouse bronchoalveolar lavage fluid

[0196] 1. Experimental Methods

[0197] The mice immunized in Example 8 were euthanized 6 weeks after immunization, and the alveolar lavage fluid was taken out to analyze the antibodies contained therein. The IgG detection method was the same as in Example 8; the HRP-labeled IgG detection antibody in the IgA detection method was replaced with the HRP-labeled IgA detection antibody.

[0198] 2. Experimental Results

[0199] Experimental Fig.11 As shown in C and D in the figure, the results show that, similar to the results in serum, the RBD-1BmpA HPF NP prepared in Example 5 did not significantly increase the RBD-specific IgA level or IgG level in the alveolar lavage fluid compared with the RBD NP prepared in Comparative Example 1; while the 1BmpA-HPF RBD NP prepared in Example 6 and the Mosaic NP prepared in Example 7 were significantly improved.

[0200] Example 11 Activation of TLR2 by BmpA

[0201] 1. Experimental Methods

[0202] Referring to the prior art PMID: 23765228, PMID: 30478312 and PMID: 28606988, the activation effect of 1BmpA on TLR2 was investigated, and the specific method is as follows:

[0203] 293T cells were transfected with plasmids expressing TLR1 / TLR2 receptors, firefly luciferase plasmids with NF-κB binding element activation, and Renilla luciferase plasmids as internal reference. After 24 hours, PBS (negative control group), Pam2CSK4 (positive control group), gradient concentrations of RBD-1BmpA protein, and RBD protein (to exclude the effect of RBD on NF-κB activation) were added respectively. After 6 hours of activation, the cells were lysed, and the fluorescence intensity of firefly luciferase activated by NF-κB and the internal reference Renilla luciferase were detected to calculate the hTLR1 / 2 activation level.

[0204] 2. Experimental Results

[0205] The results are as follows Fig.12 , as the concentration of RBD-1BmpA fusion protein increases, its activation level of NF-κB pathway increases, while RBD has no effect on NF-κB activation, indicating that 1BmpA can activate TLR2 and cause downstream signaling pathway responses ( Fig.12 ).

Claims

1. A fusion protein, characterized in that From N-terminus to C-terminus, there are SD protein and peptide segment. The amino acid sequence of the SD protein is shown in SEQ ID NO: 1, The peptide segment has an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35 or SEQ ID NO:

39.

2. A nucleic acid molecule, characterized in that A gene encoding the fusion protein of claim 1.

3. A biomaterial, characterized in that: It is any 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. Use of the fusion protein according to claim 1, the nucleic acid molecule according to claim 2 or the biological material according to claim 3 in the preparation of an adjuvant or a vaccine.

5. A protein composition, characterized in that It comprises component 1 and component 2: the amino acid sequence of component 1 is shown in SEQ ID NO:53, and the amino acid sequence of component 2 is shown in SEQ ID NO:

59.

6. A protein composition, characterized in that Contains component 1, component 2 and component 3: The amino acid sequence of component 1 is shown in SEQ ID NO:61, the amino acid sequence of component 2 is shown in SEQ ID NO:63; and the amino acid sequence of component 3 is shown in SEQ ID NO:

67.

7. A nucleic acid composition, characterized in that It comprises component 1 and component 2, which are respectively the coding genes of component 1 and component 2 of the protein combination according to claim 5.

8. A nucleic acid composition, characterized in that It comprises component 1, component 2 and component 3, which are respectively the coding genes of component 1, component 2 and component 3 of the protein combination according to claim 6.

9. A biomaterial, characterized in that: It is any of the following: (1) An expression cassette containing the nucleic acid composition of claim 7 or 8; (2) A recombinant vector containing the nucleic acid composition of claim 7 or 8 or the expression cassette of (1); (3) A recombinant microorganism comprising the nucleic acid composition of claim 7 or 8, the expression cassette of (1) or the recombinant vector of (2); (4) A cell line comprising the nucleic acid composition of claim 7 or 8, the expression cassette of (1), the recombinant vector of (2), or the recombinant microorganism of (3).

10. A nanoparticle with enhanced immunogenicity, characterized in that: The nanoparticles are fusion proteins with an amino acid sequence as shown in SEQ ID NO: 53, and are self-assembled using HPF proteins.

11. A nanoparticle with enhanced immunogenicity, characterized in that: The protein composition of claim 5 or 6, wherein each component is obtained by self-assembly.

12. The nanoparticle according to claim 11, characterized in that The preparation method thereof is as follows: the protein composition according to claim 5, component 1 self-assembles to obtain nanoparticles and then mixes with component 2 for self-assembly.

13. The nanoparticle according to claim 11, characterized in that The preparation method is the protein composition according to claim 6, wherein component 3 is self-assembled to obtain nanoparticles, which are then mixed with component 1 and component 2 for self-assembly.

14. Use of the protein composition of claim 5 or 6, the nucleic acid composition of claim 7 or 8, the biomaterial of claim 9 or the nanoparticle of any one of claims 10 to 13 in the preparation of a vaccine.

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