Vector vaccine based on human-derived horseshoe-shaped protein skeleton

Abstract

The invention discloses a vector vaccine based on a human-derived horseshoe-shaped protein skeleton. The vector vaccine is formed by displacing neutralizing epitope peptide into an alpha helical structural domain of a human-derived horseshoe-shaped protein. The vector vaccine can induce efficient, specific, lasting and broad-spectrum neutralizing antibody reaction, has the characteristics of low autoimmunogenicity, high safety and the like, and is expected to be used as a safe, stable and efficient novel epitope vaccine.

Description

Technical Field

[0001] The invention relates to a carrier vaccine based on a human horseshoe protein skeleton and application thereof, belonging to the technical field of vaccines. Background Art

[0002] Epitope vaccines are a new type of vaccine designed based on specific antigenic epitopes. They can induce specific immune responses by accurately selecting and presenting key antigenic epitopes of pathogens (Bagwe, Bagwe et al. 2022, Int J PeptRes Ther 28 (3): 94.). Epitopes are the parts of antigens that can be recognized and bound by the immune system, and epitope vaccines stimulate the body's highly specific immune response by simulating these epitopes, reducing the potential side effects caused by nonspecific reactions. Through bioinformatics and structural biology techniques, different epitope combinations can be designed and optimized to improve the immunogenicity and coverage of epitope vaccines. Epitope vaccines can also be quickly adjusted and redesigned for different pathogens and variants, and have strong adaptability.

[0003] In epitope vaccines, single or multiple epitopes usually need to be combined with carrier proteins to enhance their immunogenicity. Commonly used carrier proteins include KLH (Keyhole Limpet Hemocyanin), CRM197 (Cross-Reactive-Material-197), TT (Tetanus Toxoid), etc. (Broker, Costantino et al. 2011, Biologicals 39(4): 195-204., Pichichero 2013, Hum Vaccin Immunother 9(12):2505-2523., Swaminathan, Lucas et al. 2014, Br J Clin Pharmacol 78(5): 1135-1142.). KLH is a ceruloplasmin from giant marine gastropod mollusk. Its advantages are strong immunogenicity and easy to bind antigens, but its disadvantage is that it may cause carrier-specific immune response. CRM197 is a non-toxic mutant of diphtheria toxin. Its advantages are high safety and has been verified in many vaccines. Its disadvantage is high production cost. TT is a tetanus toxoid, which is widely used as a carrier for many vaccines. Its advantages are high immunogenicity and long-term use have proven its safety. Its disadvantage is that it may induce carrier-related immune response.

[0004] In summary, an ideal carrier protein should effectively enhance the immune response of the epitope and have good safety and low toxicity. At the same time, the production process should be mature, cost-controllable, and suitable for large-scale production. The carrier protein should minimize its own immunogenicity and cross-reaction with other vaccine components to avoid triggering nonspecific immune responses.

[0005] It is of great value to discover new carrier protein skeletons from natural proteins to better display epitopes. Human ribonuclease inhibitor protein (Ribonuclease / angiogenin 1, RNH1) is a 50kDa protein present in most human tissues and has been widely studied as a ribonuclease inhibitor (Shapiro and Vallee 1987, Proc Natl Acad Sci USA 84(8): 2238-2241.). RNH1 has a unique horseshoe-shaped structure, so it is also called horseshoe protein. Its inner and outer walls are composed of parallel β-strands and α-helices. It belongs to the protein family containing leucine-rich repeat sequences (LRR) (Bella, Hindle et al. 2008, Cell Mol Life Sci 65(15): 2307-2333.). These characteristics make it an ideal backbone carrier for enhancing the immunogenicity of α-helical epitopes, and the target antigens of many important pathogens including the new coronavirus, influenza virus, respiratory syncytial virus, HIV, etc. contain key α-helical neutralizing epitopes.

[0006] The purpose of the present invention is to provide a vector vaccine based on a human horseshoe protein skeleton. By replacing multiple outer α-helical regions with target epitopes, multiple displays of target epitopes can be achieved, efficient epitope-specific immune responses can be stimulated, and it has the characteristics of low autoimmunogenicity and high safety. It is expected to be a safe and efficient new epitope vaccine. Summary of the invention

[0007] Based on the above purpose, the present invention provides a vector vaccine based on a human horseshoe protein skeleton, wherein the vector vaccine based on a human horseshoe protein skeleton is composed of replacing the α-helical domain in the human horseshoe protein with a neutralizing epitope peptide.

[0008] The human horseshoe skeleton described in the present invention is composed of parallel β-strands and α-helices as its inner and outer walls, and belongs to the protein family containing leucine-rich repeat sequences (LRR); the vector vaccine based on the human horseshoe protein skeleton is composed of the α-helical domain of the human horseshoe protein shown in the amino acid sequence of SEQ ID NO.1 replaced with a neutralizing epitope peptide, and can evenly and multiplexly display the conformation of the neutralizing epitope peptide on the outside of the protein skeleton, thereby stimulating a highly efficient and specific immune response.

[0009] In the vector vaccine with a human horseshoe protein backbone, one or more of the alpha helical domains composed of amino acid residues 15-25, 40-50, 68-78, 97-107, 125-135, 154-164, 182-192, 211-221, 239-249, 268-278, 303-306, 325-335, 353-363, 382-392, 410-420 and 439-449 in the human horseshoe protein whose amino acid sequence is shown in SEQ ID NO.1 are replaced with a neutralizing epitope peptide.

[0010] In a preferred embodiment, the neutralizing epitope peptide is 10-50 amino acid residues in length.

[0011] In a preferred embodiment, the vector vaccine with a human horseshoe protein backbone also includes adding 1-3 copies of the β-sheet domain in the human horseshoe protein in a tandem form. The tandem copies of the β-sheet domain are intended to expand the backbone size and accommodate longer neutralizing epitope peptides in the α-helix.

[0012] Particularly preferably, the neutralizing epitope peptide is a neutralizing epitope peptide whose amino acid sequence in the neck helix of the S2 protein of SARS-CoV-2 is shown in SEQ ID NO.2.

[0013] In a specific embodiment of the present invention, the amino acid sequence of the vector vaccine based on the human horseshoe protein skeleton is as shown in SEQ ID NO.3, and the vector vaccine is designed to replace the α-helical domain composed of amino acid residues at positions 15-25, 40-50, 68-78, 97-107, 125-135, 154-164, 182-192, 211-221, 239-249, 268-278, 303-306, 325-335, 353-363, 382-392, 410-420, and 439-449 in the amino acid sequence shown in SEQ ID NO.1 with the S2 protein neutralizing epitope peptide of SARS-CoV-2 as shown in SEQ ID NO.2. In order to ensure that the neutralizing epitope peptide is fully displayed on the carrier skeleton, the present invention adjusts the β-sheet domain of the human horseshoe protein skeleton, specifically, the β-sheet in the original structure is copied once in series to expand the size of the skeleton.

[0014] In another preferred embodiment of the present invention, the neutralizing epitope peptide is a neutralizing epitope peptide whose amino acid sequence in the RSV F protein is shown as SEQ ID NO.4.

[0015] In another specific embodiment of the present invention, the amino acid sequence of the vector vaccine based on the human horseshoe protein backbone is shown in SEQ ID NO. 5. The vector vaccine is designed to replace the α-helical domains composed of amino acid residues at positions 15-25, 40-50, 68-78, 97-107, 125-135, 154-164, 182-192, 211-221, 239-249, 268-278, 303-306, 325-335, 353-363, 382-392, 410-420, and 439-449 in the amino acid sequence shown in SEQ ID NO. 1 with the RSV F protein neutralizing epitope peptide with the amino acid sequence shown in SEQ ID NO. 4.

[0016] Secondly, the present invention provides a polynucleotide of the above-mentioned vector vaccine based on the human horseshoe protein skeleton. When the neutralizing epitope peptide is a neutralizing epitope peptide with an amino acid sequence in the neck spiral of the S2 protein of SARS-CoV-2 as shown in SEQ ID NO.2, the sequence of the polynucleotide of the vector vaccine is as shown in SEQ ID NO.6.

[0017] When the neutralizing epitope peptide is a neutralizing epitope peptide in the RSV F protein whose amino acid sequence is shown in SEQ ID NO.4, the sequence of the polynucleotide of the vector vaccine is shown in SEQ ID NO.7.

[0018] Third, the present invention provides an expression vector containing the above polynucleotide.

[0019] Finally, the present invention provides a host cell containing the above expression vector.

[0020] The present invention replaces multiple α-helical regions on the outside of human horseshoe protein with target epitopes to form a carrier vaccine that can realize multiple displays of target epitopes and stimulate efficient epitope-specific immune responses. In a specific embodiment of the present invention, RNH1-S1139 exhibits strong antigenicity and can bind to the neutralizing antibody B6 specific for the S2 epitope with high affinity, which is two orders of magnitude higher than the affinity of the S protein trimer with B6. By multivalently displaying epitopes, RNH1-S1139 significantly enhances immune recognition ability, and can stimulate the production of high titers of binding antibodies 14 days after the first immunization. 14 days after the second immunization, the titers of specific binding antibodies against the S1139 epitope in the serum of the RNH1-S1139 immune group are much higher than those of the single S1139 peptide, S1139-KLH, S1139 3-string peptide, 12-string peptide and 24-string peptide, indicating the great potential of RNH1 as an epitope vaccine skeleton carrier. Further in vivo experiments showed that RNH1-S1139 can induce a long-lasting immune response, and the binding antibody titer remained at a high level 180 days after the third immunization. In addition, RNH1-S1139 can also induce a broad-spectrum neutralizing antibody response and effectively inhibit the occurrence of membrane fusion, which is better than a single epitope. Preliminary safety evaluation showed that RNH1-S1139 antigen is safe after immunization.

[0021] In another specific embodiment of the present invention, RNH1-RSV-F also exhibits strong antigenicity and can bind to RSV-F-specific neutralizing antibody D25 with high affinity, which is 10 times the affinity of RSV pre-fusion conformation F protein and D25. RNH1-RSV-F vaccine can effectively induce the production of high-titer specific IgG antibodies against RSV-F peptides in mice. The antibody titer is 100 times that of the RSV-F protein immunization group 14 days after the three immunizations, and can still maintain a high titer after 14 days.

[0022] Based on the above technical effects, it is shown that the vector vaccine based on the human horseshoe protein skeleton provided by the present invention has the characteristics of low autoimmunogenicity and high safety, and is expected to be used as a safe, stable and efficient new epitope vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 . Schematic diagram of the structural design of RNH1-S1139 (A) and SDS-PAGE electrophoresis verification of the recombinant product (B); Figure 2 . Circular dichroism characterization of RNH1-S1139 (A) and affinity curve with neutralizing antibody B6 (B); Figure 3 . Immunological evaluation of RNH1-S1139: binding antibody titer (A) and duration (B); Figure 4 . Broadly neutralizing antibody response induced by RNH1-S1139: results of pseudovirus neutralization assay (A) and membrane fusion inhibition assay (B); Figure 5 . Safety evaluation results of RNH1-S1139; Figure 6 . Schematic diagram of the structural design of RNH1-RSV-F (A) and SDS-PAGE electrophoresis verification of the recombinant product (B); Figure 7 . Circular dichroism characterization of RNH1-RSV-F (A) and affinity curve with neutralizing antibody D25 (B); Figure 8 . Immunological evaluation of RNH1-RSV-F. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described in detail below in conjunction with two specific embodiments. Through these embodiments, the advantages and features of the present invention will be more clearly demonstrated. However, these embodiments are only exemplary and do not constitute any limitation to the scope of protection defined by the claims of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are all conventional reagents, methods and equipment used in the art.

[0025] Example 1: Design, preparation and immune evaluation of novel coronavirus RNH1-S1139 The rapid mutation of the virus, such as the Omicron series variants, significantly reduces the effectiveness of the vaccine. To address these challenges, this example designs a horseshoe protein backbone vector based on RNH1 for multivalent display of the conserved neutralizing epitope S1139 of the S2 neck spiral of the new coronavirus, aiming to induce broad-spectrum neutralizing antibodies and improve resistance to mutant strains.

[0026] 1.1 Design ideas Select target epitope: Select the conserved neutralizing epitope S1139 in the SARS-CoV-2 S2 neck spiral sequence as described in SEQ ID NO.2 ( 1139 DPLQPELDSFKEELDKYFKNHTSP 1162 ). S1139 is a highly conserved sequence that remains unchanged in multiple variants and is an ideal candidate neutralizing epitope.

[0027] Genetic engineering: The skeleton of human horseshoe protein is composed of parallel β-chains and α-helices, which form its inner and outer walls. It belongs to the protein family containing leucine-rich repeat sequences (LRR). Each LRR is composed of approximately 20-25 amino acids, and multiple LRRs form an adaptive horseshoe protein scaffold. Human horseshoe proteins represented by RNH1 can stably and multiplexly display inserted α-helical epitopes due to their rich leucine repeat sequences. They can evenly and multiplexly display neutralizing epitope peptide conformations on the outside of the protein skeleton, stimulate efficient specific immune responses and enhance immunogenicity (SEQ ID NO.1). Through genetic engineering technology, 16 S1139 epitopes were replaced to the 16 α-helical regions on the outside of the RNH1 protein. Since the S1139 epitope is 24 amino acids, which is longer than the α-helical fragment of the original RNH1, in order to maintain the regular horseshoe-shaped conformation, the β-sheet sequence in the original structure of the RNH1 protein was copied in tandem to expand the skeleton size and accommodate the longer α-helical epitope, and the RNH1-S1139 horseshoe-shaped protein (SEQ ID NO.3) was designed.

[0028] 1.2 Antigen preparation Vector construction: The RNH1-S1139 protein sequence was codon-optimized and synthesized into a gene (SEQ ID NO.6), which was cloned into the prokaryotic expression vector pTig through restriction sites (EcoRI and NotI) and then transformed into Escherichia coli ( E. coli Origami). E. coli The Origami strain, due to its special redox environment, helps to form the correct disulfide bonds and ensure the correct folding of proteins.

[0029] Optimization of expression conditions: The protein expression and solubility were maximized by optimizing the final concentration of the inducer IPTG (0.5 mM, OD = 0.6-0.8), induction temperature (37 ° C) and time (4 hours).

[0030] Protein purification: The recombinant RNH1-S1139 protein containing the His tag was purified by nickel column affinity chromatography and the protein purity was improved by molecular sieve Superdex75. This method is simple and efficient, and can quickly obtain high-purity and high-concentration target proteins. Figure 1 Figure B is the SDS-PAGE electrophoresis of the recombinant RNH1-S1139 protein, and its molecular weight is about 90 kDa, which is in line with expectations. The sample after affinity purification showed a single peak when the volume of the molecular sieve solution was 65 mL, indicating that the protein properties were relatively uniform.

[0031] 1.3 Immune evaluation Vaccine preparation and immunization: The purified RNH1-S1139 protein was mixed with aluminum salt adjuvant to prepare a vaccine; the vaccine was immunized to mice by intramuscular injection, 100 μL per mouse each time. Blood was collected from the retro-orbital venous plexus of each mouse on days 0, 14, and 28 to separate serum. The specific immunization plan is as follows: Table 1. Types of experimental SARS-CoV-2 vaccines and immunization regimens

[0032] Among them, S Trimer is S protein trimer, RBD is RBD monomer, and S1139-KLH is S1139 epitope peptide coupled to KLH carrier protein through cysteine.

[0033] ELISA test: S1139 epitope peptide was used as coating antigen, gradiently diluted mouse immune serum was used as primary antibody, goat anti-mouse IgG Fc was used as secondary antibody, and the S1139-specific IgG antibody titer in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA) to evaluate the binding antibody response of mouse serum in each group.

[0034] Neutralization experiment: Pseudovirus neutralization experiment and membrane fusion inhibition experiment were used to evaluate the neutralization ability of mouse immune serum against SARS-CoV-2 pseudovirus and the ability to inhibit S protein-mediated membrane fusion, and to determine the protective effect of the vaccine. For the pseudovirus neutralization experiment, the mouse immune serum was first incubated with the gradient diluted new crown pseudovirus with lentivirus as the vector at 37°C for one hour, and then mixed with HEK293T cells that stably expressed ACE2 receptors on the cell surface, and the intensity of the luciferase reporter gene was measured two days later. For the qualitative membrane fusion inhibition experiment, the immune serum was incubated with HEK293T cells transiently transfected with the new crown S protein-EGFP plasmid at 37°C for one hour, and then mixed with HEK293T cells that stably expressed ACE2 receptors on the cell surface, and the phenomenon of cell membrane fusion was observed under a microscope. For the quantitative membrane fusion inhibition experiment, the immune serum was incubated with HEK293T cells stably expressing the SARS-CoV-2 S protein-β-galactosidase subunit α plasmid at 37°C for one hour, and then mixed with HEK293T cells stably expressing the ACE2 receptor-β-galactosidase subunit ω. The fluorescence signal intensity was detected two days later (Anne, Carsten et al. 2004, Virology 319(2):343-352).

[0035] 1.4 Safety Assessment Toxicity test: Blood was collected 3 days after the first immunization and 28 days after the third immunization to observe the blood biochemical and routine physiological indicators of mice after vaccination and to preliminarily evaluate the acute and chronic toxicity of the vaccine.

[0036] Histopathological examination: Histopathological examination (H&E staining and Sirius red staining) was used to preliminarily evaluate the pathological changes in the main organs of the vaccinated mice (such as heart, liver, spleen, lung, kidney, injection site and brain) to ensure the safety of the vaccine.

[0037] 1.5 Experimental Results IPTG was used at a final concentration of 0.5 mM at 37 °C to adjust the OD 600 The expression was induced in the bacterial solution with a concentration of 0.6-0.8, and the bacteria were harvested after four hours. The results showed that RNH1-S1139 was expressed in E. coli ( E. coli Origami) with high yield and low cost ( Figure 1 ). The secondary structure was analyzed by circular dichroism (CD). The results showed that the secondary structure of RNH1-S1139 maintained a certain proportion of α-helical conformation, similar to RNH1, and maintained the expected conformational stability ( Figure 2 A).

[0038] RNH1-S1139 exhibits strong antigenicity and can bind with high affinity to the neutralizing antibody B6 specific for the S2 epitope. K D The value is 0.175 nM, which is two orders of magnitude higher than the affinity of S protein trimer for B6 ( Figure 2 B). By displaying epitopes in multiple ways, RNH1-S1139 significantly enhanced immune recognition. ELISA results showed that RNH1-S1139 could stimulate the production of high titers of binding antibodies 14 days after the first immunization. 14 days after the second immunization, the titers of specific binding antibodies against the S1139 epitope in the serum of the RNH1-S1139 immunization group were much higher than those of the single S1139 peptide, the conventional KLH carrier protein coupled peptide to improve immunogenicity S1139-KLH, S1139's 3-string peptide (S1139×3), 12-string peptide (S1139×12) and 24-string peptide (S1139×24). The binding antibodies stimulated by RNH1-S1139 are still one order of magnitude higher than those stimulated by the most immunogenic S1139-KLH (10 3.39 VS 10 4.74 ), indicating the great potential of RNH1 as an epitope vaccine backbone carrier ( Figure 3 A). Further in vivo experiments showed that RNH1-S1139 could induce a long-lasting immune response, and the binding antibody titer remained at a high level 180 days after the third immunization, with the endpoint rarefaction factor approaching 10 5 ( Figure 3In addition, RNH1-S1139 can also induce a broad spectrum of neutralizing antibody responses and effectively inhibit the occurrence of membrane fusion, which is better than a single epitope, once again confirming the potential of RNH1 as an epitope vaccine backbone carrier.

[0039] RNH1-S1139 rabbit immune serum not only showed certain neutralizing activity against the new coronavirus pseudovirus, but also inhibited the occurrence of S protein-mediated membrane fusion. RNH1-S1139 rabbit immune serum can neutralize the new coronavirus original strain and variant strains (including BQ.1, XBB, BF.7, XBB.1.5) pseudovirus to a certain extent and prevent them from infecting cells that stably express ACE2-293T. Figure 4 As shown in A, the serum dilution multiple gradually increases from left to right. When the serum dilution multiple is low, the serum of the S Trimer immune group can 100% inhibit the invasion of pseudovirus, and the RNH1-S1139 immune group also has a certain inhibitory effect. As the serum dilution multiple increases, the inhibitory effect gradually weakens. At the same time, the qualitative and quantitative experiments of membrane fusion also show that the addition of immune serum diluted 10 times can prevent the occurrence of cell membrane fusion expressing S protein and ACE2 receptor on the cell membrane surface, and when the serum is diluted 500 times, the incidence of membrane fusion increases. The serum of the control group injected with phosphate buffer does not show the ability to inhibit membrane fusion.

[0040] Physiological and biochemical tests (see Table 2) and histopathological evaluation showed that at different time points after RNH1-S1139 injection, the physiological and biochemical indicators of the test animals were normal, and no fibrosis or other pathological changes were found in the heart, liver, spleen, lung, kidney, brain, and muscle at the injection site ( Figure 5 These data preliminarily indicate that the S1139 epitope displayed by the RNH1 backbone is safe.

[0041] Table 2. Physiological and biochemical indicators of the test animals at different time points after injection of RNH1-S1139

[0042] This example reports the design, preparation and preliminary immunological evaluation of an epitope-focused immunogen RNH1-S1139 based on the RNH1 backbone. The results show that RNH1-S1139 not only has high immunogenicity, but also can induce a long-lasting and broad-spectrum immune response, and has good safety.

[0043] Example 2: Design, preparation and immunological evaluation of RSV virus RNH1-RSV-F Respiratory syncytial virus (RSV) is an important pathogen that causes severe respiratory infections in infants, young children and the elderly, resulting in a large number of hospitalizations and deaths worldwide each year. In order to develop an efficient and broad-spectrum vaccine against RSV, this example designs a horseshoe-shaped protein skeleton based on human ribonuclease inhibitor (RNH1) for multivalent display of conserved neutralizing epitopes in RSV-F protein, aiming to induce broad-spectrum neutralizing antibodies and improve resistance to RSV infection.

[0044] 2.1 Design Idea Select target epitope: Select the conserved neutralizing epitope peptide S-193 (S-193) in RSV-F protein that binds to the monoclonal antibody D25 (Mclellan, Chen et al. 2013 Science 340(6136): 1113-111) 193 LDLKNYIDKQLLPIVNKQSCS 213 ). F protein is an important fusion protein of RSV, and its structure is highly conserved. The D25 binding epitope is an ideal candidate epitope.

[0045] Genetic engineering: Through genetic engineering technology, 16 conserved epitopes of RSV-F protein were inserted into the outer α-helical region of RNH1 protein, and the inner side adopted the same sequence as RNH1-S1139 backbone to form RNH1-RSV-F horseshoe protein backbone (such as Figure 6 (as shown in A).

[0046] 2.2 Antigen preparation Vector construction: The RNH1-RSV-F protein sequence was codon-optimized and the gene SEQ ID NO.7 was synthesized. It was cloned into the prokaryotic expression vector pTig through restriction sites (EcoRI and NotI) and then transformed into Escherichia coli ( E. coli Origami). E. coli The Origami strain contains mutated sulfhydryl reductase and glutathione reductase genes, which help form correct disulfide-bonded proteins and enhance protein solubility.

[0047] Optimization of expression conditions: The protein expression and solubility were maximized by optimizing the final concentration of the inducer IPTG (0.5 mM, OD = 0.6-0.8), induction temperature (37 ° C) and time (four hours).

[0048] Protein purification: After the target protein in the inclusion body was denatured with urea, the recombinant RNH1-RSV-F protein containing the His tag was purified by nickel column affinity chromatography and renatured, and finally the target protein was purified by combining with molecular sieve Superdex75. Figure 6Figure B is the SDS-PAGE electrophoresis of the recombinant RNH1-RSV-F protein, which has a molecular weight of approximately 77 kDa and is in expected size. The affinity-purified sample showed a single peak when the volume of the molecular sieve solution was 75 mL, indicating that the method is simple and efficient and can quickly obtain a high-purity target protein.

[0049] 2.3 Immune evaluation Vaccine preparation: The purified RNH1-RSV-F protein was mixed with aluminum salt adjuvant to prepare the vaccine; the vaccine was injected intramuscularly into mice on days 0, 14, and 28, with 100 μL per mouse each time. The specific immunization scheme is as follows: Table 3. Types of experimental RSV vaccines and immunization regimens

[0050] RSV-F is the RSV pre-fusion F protein, and RNH1-RSV-F is the protein design involved in the present invention.

[0051] ELISA test: RSV-F peptide was used as the coating antigen, gradiently diluted mouse immune serum was used as the primary antibody, and goat anti-mouse antibody was used as the secondary antibody. The RSV-F specific IgG antibody titer in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA) to evaluate the antibody response of the mouse serum in each group.

[0052] 2.4 Experimental Results and Discussion The expression was induced with IPTG at a final concentration of 0.5 mM at 37°C. The bacteria were harvested four hours later. The protein in the inclusion body was purified through a series of denaturation-affinity column-renaturation-molecular sieve purification to obtain the target protein with high purity. The experimental results showed that RNH1-RSV-F was expressed in E. coli ( E. coli Origami). RNH1-RSV-F showed strong antigenicity and was able to bind with high affinity to RSV-F-specific neutralizing antibody D25. K D Values ​​as low as 5.9X10 -11 M, which is 10 times the affinity of RSV pre-fusion conformation F protein for D25 ( Figure 7 The RNH1-RSV-F vaccine can effectively induce the production of high-titer specific IgG antibodies against RSV-F peptides in mice. The antibody titer was 100 times that of the RSV-F protein immunization group 14 days after the third immunization, and it can still maintain a titer nearly two orders of magnitude higher than the RSV-F group 28 days after the third immunization ( Figure 8 ).

[0053] The RNH1-RSV-F vaccine designed in this example successfully inserted the conserved neutralizing epitope of RSV-F protein into the RNH1 protein backbone through genetic engineering technology, forming a stable and efficient immunogen. The results of animal experiments showed that the RNH1-RSV vaccine has good immunogenicity, confirming the potential of RNH1 as a universal epitope vaccine backbone carrier.

[0054] The above embodiments are preferred implementations of the present invention, but are not intended to limit the present invention. Any other changes and modifications that do not violate the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A vector vaccine based on a human horseshoe protein skeleton, characterized in that: The vector vaccine based on the human horseshoe protein skeleton is formed by replacing the α-helical domain in the human horseshoe protein with a neutralizing epitope peptide.

2. The vector vaccine based on human horseshoe protein skeleton according to claim 1, characterized in that: In the vector vaccine with a human horseshoe protein backbone, one or more of the alpha helical domains composed of amino acid residues 15-25, 40-50, 68-78, 97-107, 125-135, 154-164, 182-192, 211-221, 239-249, 268-278, 303-306, 325-335, 353-363, 382-392, 410-420 and 439-449 in the human horseshoe protein whose amino acid sequence is shown in SEQ ID NO.1 are replaced with a neutralizing epitope peptide.

3. The vector vaccine based on human horseshoe protein skeleton according to claim 2, characterized in that: In the vector vaccine with human horseshoe protein skeleton, 1-3 copies of the β-sheet domain in the human horseshoe protein are also added in series.

4. The vector vaccine based on human horseshoe protein skeleton according to claim 3, characterized in that: The neutralizing epitope peptide is 10-50 amino acid residues in length.

5. The vector vaccine based on human horseshoe protein skeleton according to claim 4, characterized in that: The neutralizing epitope peptide is a neutralizing epitope peptide whose amino acid sequence in the neck helix of the S2 protein of SARS-CoV-2 is shown in SEQ ID NO.

2.

6. The vector vaccine based on human horseshoe protein skeleton according to claim 5, characterized in that: The amino acid sequence of the vector vaccine based on the human horseshoe protein skeleton is shown in SEQ ID NO.

3.

7. The vector vaccine based on human horseshoe protein skeleton according to claim 4, characterized in that: The neutralizing epitope peptide is a neutralizing epitope peptide whose amino acid sequence in the RSV F protein is shown as SEQ ID NO.

4.

8. The vector vaccine based on human horseshoe protein skeleton according to claim 7, characterized in that: The amino acid sequence of the vector vaccine based on the human horseshoe protein skeleton is shown in SEQ ID NO.

5.

9. A polynucleotide encoding the vector vaccine based on the human horseshoe protein skeleton according to claim 6, characterized in that: The sequence of the polynucleotide is shown as SEQ ID NO.

6.

10. A polynucleotide encoding the vector vaccine based on the human horseshoe protein skeleton according to claim 8, characterized in that: The sequence of the polynucleotide is shown in SEQ ID NO.

7.

11. An expression vector comprising the polynucleotide according to claim 9 or 10.

12. A host cell containing the expression vector according to claim 11.

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