Novel coronavirus broad-spectrum nasal mucosa vaccine based on conservative polypeptide / glycopeptide as well as preparation method and application of novel coronavirus broad-spectrum nasal mucosa vaccine

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a conservative polypeptide / glycopeptide-based novel coronavirus broad-spectrum nasal mucosa vaccine, which comprises a carrier and an antigen, the carrier is chitosan oligosaccharide, the antigen is a conjugate of an antigen fragment and an adjuvant, the antigen fragment comprises polypeptide and glycopeptide, and the adjuvant is a structural analogue of a TLR7 / 8 agonist. The invention further provides a preparation method of the novel broad-spectrum coronavirus nasal mucosa vaccine based on conservative polypeptide glycopeptide, chitosan oligosaccharide is used as a nano-carrier, and a conjugate of an antigen fragment and an adjuvant is loaded through an ionic cross-linking method. The novel broad-spectrum nasal mucosa vaccine for the coronavirus based on the conserved polypeptide glycopeptide can effectively prevent and treat the novel coronavirus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a broad-spectrum nasal mucosa vaccine against novel coronavirus based on conserved polypeptides / glycopeptides, and a preparation method and application thereof. Background Art

[0002] SARS-CoV-2 has an extremely high mutation rate, and the key antigenic sites of its spike protein (S protein) frequently mutate, resulting in the continuous emergence of multiple variants of concern (VOCs, such as Alpha, Delta, Omicron, etc.) and variants of interest (VOIs) defined by the WHO. These mutant strains have significantly reduced the neutralizing efficacy of existing diagnostic reagents, monoclonal antibody drugs, and vaccines designed based on the original strain (including mRNA vaccines, inactivated vaccines, and recombinant protein vaccines), and the virus can be transmitted through multiple routes such as human-to-human and object-to-human, exacerbating the difficulty of epidemic prevention and control.

[0003] Currently approved COVID-19 vaccines mainly induce systemic immunity (such as IgG antibodies and T cell responses) through intramuscular injection, but have limited immune protection for the local respiratory mucosa and are difficult to effectively block infection and transmission in the early stage of virus invasion. In addition, existing vaccines are mostly designed against the receptor binding domain (RBD) or full-length S protein of the S protein, and the mutation rate of the RBD region is extremely high (for example, there are 15 mutations in the RBD of Omicron BA.1), resulting in a significant decrease in the neutralizing antibody titer of the vaccine against mutant strains. Although multivalent vaccines (such as bivalent mRNA vaccines) can partially cover known mutant strains, it is still difficult to cope with immune escape mutant strains that may appear in the future, and there is an urgent need to develop a broad-spectrum vaccine based on conserved epitopes.

[0004] The core of a broad-spectrum vaccine lies in targeting highly conserved functional epitopes in virus evolution. Research shows that certain regions in the S protein of SARS-CoV-2 (such as the fusion peptide (FP), heptapeptide repeat domain (HR1 / HR2), and transmembrane domain) are highly conserved in mutant strains and are related to the key mechanisms of virus membrane fusion and invasion of host cells. However, the immunogenicity of conserved linear polypeptides used alone is weak, and it is difficult to induce highly efficient neutralizing antibodies. Therefore, it is necessary to enhance its immunogenicity through multi-epitope chimeric design, glycosylation modification, or nanoparticle delivery systems, and optimize the immune response in combination with mucosal adjuvants.

[0005] Nasal mucosal vaccination can simultaneously activate mucosal immunity (secretory IgA) and systemic immunity, forming the first line of defense in the respiratory tract locally and significantly reducing the viral load and transmission risk. However, existing mucosal vaccines face the following bottlenecks: ① Protein antigens are easily enzymatically degraded or cleared in the nasal cavity environment; ② There is a lack of highly effective and safe mucosal adjuvants; ③ The cross-protection ability against variant strains is insufficient. Therefore, developing nasal vaccines based on conserved polypeptides / glycopeptides and combining mucosal targeting delivery technologies (such as chitosan nanoparticles, liposome carriers) is an important direction to break through the current technical barriers. Summary of the Invention

[0006] To solve the above technical problems, the present invention proposes a broad-spectrum nasal mucosal vaccine for novel coronavirus based on conserved polypeptides / glycopeptides, its preparation method and application.

[0007] The broad-spectrum nasal mucosal vaccine for novel coronavirus based on conserved polypeptides / glycopeptides of the present invention comprises a carrier and an antigen. The carrier is chitosan oligosaccharide, and the antigen is a conjugate of an antigen fragment and an adjuvant, wherein the antigen fragment comprises a polypeptide and a glycopeptide, and the adjuvant is a structural analog of a TLR7 / 8 agonist.

[0008] The preparation method of the above broad-spectrum nasal mucosal vaccine for novel coronavirus based on conserved polypeptides / glycopeptides comprises the following steps:

[0009] S1. Screening of antigen fragments:

[0010] Based on virus evolution analysis, screening for polypeptide sequences that are highly conserved among multiple coronavirus variant strains; preferentially selecting polypeptides containing disulfide bonds to enhance structural stability; excluding polypeptides with glycosylation sites, or retaining glycosylation sites to prepare glycopeptide antigens;

[0011] S2. Construction of a conjugate of an antigen fragment and an adjuvant:

[0012] S2.1. Preparing a polypeptide antigen fragment by solid-phase polypeptide synthesis method;

[0013] S2.2. Preparing an adjuvant molecule conjugated with an alkynyl group by chemical synthesis method;

[0014] S2.3. Coupling the antigen fragment and the adjuvant molecule by click chemistry method;

[0015] S3. Preparing a broad-spectrum nasal mucosal vaccine for novel coronavirus:

[0016] S3.1. Dissolving the carrier chitosan oligosaccharide in an acetic acid solution with pH = 4 to obtain solution A;

[0017] S3.2. Mixing the conjugate of the antigen fragment and the adjuvant with an active agent TPP solution to obtain a mixed solution B;

[0018] S3.3. Under stirring conditions, add solution B dropwise into solution A to react and form vaccine chitosan oligosaccharide nanoparticles.

[0019] Preferably, in S2, the polypeptide is prepared by a solid-phase polypeptide synthesizer. During the preparation process, glycin is added to the synthesizer in the same way as ordinary amino acids to prepare glycopeptides. A linker with azide / alkyne groups is coupled to the polypeptide fragment. The cleavage reagent used for polypeptide cleavage is trifluoroacetic acid / thioanisole / 1,2-ethanedithiol / anisole = 90 / 5 / 3 / 2 (volume ratio) to prevent the oxidation of disulfide bonds.

[0020] Preferably, in S3, the final concentration of chitosan oligosaccharide is 3.5 mg / mL, the added volume of the TPP solution is 5% of the final volume, and the concentration is 0.8% (w / v). The concentration of the conjugate of the antigen fragment and the adjuvant is 0.5 mg / mL, and the added volume is 15% of the final volume.

[0021] Preferably, in 3.3, the stirring conditions are 400 r / min. The prepared nanoparticles have a particle size of 155.89 nm, a potential of +24.92 mV, and a PDI of 0.1996 - 0.2304.

[0022] The present invention also proposes the application of a novel coronavirus broad-spectrum nasal mucosa vaccine based on a conserved polypeptide / glycopeptide prepared according to the above method in the prevention and treatment of novel coronavirus.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) Broad-spectrum: Based on the conserved epitope screening strategy, it can cover a variety of coronavirus variants;

[0025] (2) Mucosal immune enhancement: The chitosan oligosaccharide nanocarrier promotes nasal mucosa adsorption, and the co-delivery of adjuvant-antigen enhances the immune response;

[0026] (3) Stability optimization: The antioxidant cleavage system retains the disulfide bond structure, and the nanopreparation parameters are accurately controllable;

[0027] (4) Process feasibility: The combination of click chemistry and solid-phase synthesis technology is suitable for large-scale production. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments.

[0029] Figure 1 Two novel coronavirus conserved peptide segments PY36 and IF23 screened from the RBD fragment;

[0030] Figure 2The PY36 and IF23 fragments of different mutant strains with biotin linkers (A) and the determination of the affinity between antigen peptides and patient sera by ELISA method (B);

[0031] Figure 3 Antigen peptide segments conjugated with azide groups (A); Representative high-resolution mass spectrum of PY36 (B); Conjugation of antigen and adjuvant by Click method (C);

[0032] Figure 4 Schematic diagram of preparing nanoparticles with mucosal adhesion by ionic crosslinking method (A), and the induction of specific systemic immune response after mucosal administration of nanoparticles (B). Detailed implementation manners

[0033] The following further describes the detailed implementation manners of the present invention. It should be noted here that the description of these implementation manners is used to help understand the present invention, but does not constitute a limitation to the present invention.

[0034] Example 1

[0035] Screening of antigen fragments:

[0036] Disulfide bonds play a crucial role in maintaining the spatial three-dimensional structure and biological activity of RBD; Antigen glycosylation sites promote the production of specific antibodies; There are conserved antigen fragments in the viral sequence, and the broadly neutralizing antibodies produced by the conserved antigen fragments can recognize different mutant strains. Therefore, during the screening of conserved antigen fragments in the RBD domain, the following three criteria are used for screening: the conservation of polypeptide sequences, the presence or absence of disulfide bonds, and the presence or absence of glycosylation sites. According to the above three criteria, by comparing the RBD sequences of different SARS-CoV-2 mutant strains, two conserved peptide segments PY36 and IF23 as shown in Figure 1 are screened out.

[0037] Synthesis of PY36 and IF23 peptide segments of different mutant strains and determination of serum affinity

[0038] The PY36 and IF23 fragments of different mutant strains with biotin linkers are prepared by solid-phase peptide synthesis method, and the antigen sequences are as shown in Figure 2As shown in Figure A. The specific synthesis steps are as follows: resin selection and swelling (select the resin coupled with the first amino acid and swell it in DMF for 30 minutes); washing (wash the resin with DMF 3 - 5 times to remove unreacted reagents); step - by - step coupling of amino acids (remove the Fmoc protecting group with 20% piperidine / DMF solution, repeat twice, 5 - 10 minutes each time; wash the resin with DMF 3 - 5 times; mix the next Fmoc - protected amino acid with HBTU / HOBt and DIEA, add it to the resin, and react for 1 - 2 hours); synthesize PY36 glycopeptide by coupling glycine to the polypeptide chain in the same way as ordinary amino acids; at the end of the synthesis, couple biotin with a carboxyl - terminal to the polypeptide chain in the same way; use a cleavage reagent (trifluoroacetic acid / thioanisole / 1,2 - ethanedithiol / anisole = 90 / 5 / 3 / 2, v / v / v / v) to cleave the polypeptide from the resin, react for 2 - 3 hours, and the role of 1,2 - ethanedithiol is to prevent disulfide bond oxidation. Purify the crude peptide by high - performance liquid chromatography (HPLC) on a Waters e2695 HPLC system equipped with a dual - absorbance UV detector. The chromatographic column is a C18 column (Waters SymmetryPrepTM, 19×300mm, 7μm), the flow rate is 20 mL / min, and a linear gradient of 60% - 80% acetonitrile containing 0.1% trifluoroacetic acid is run for 20 minutes.

[0039] Co - incubate the prepared antigen peptide coupled with biotin with an ELISA plate containing streptavidin, use the serum of COVID - 19 infected patients as the primary antibody, and determine the affinity between the antigen peptide and the patient serum by ELISA method. As Figure 2 As shown in Figure B, according to the ELISA test results, the binding force between PY36 of mutant strains BA.2.75 (G339H) and BA.2.76 (G339D, R346T) and antibodies is relatively strong; most mutations in the IF23 peptide segment are S477N, T478K, E484A, F486V; based on the binding experiment results and the number of infected people, select PY36 and IF23 of BQ.1 and XBB.1.5 for synthesis as antigens to construct the vaccine.

[0040] Construct the conjugate of the antigen fragment and the adjuvant:

[0041] Similarly, prepare the antigen peptide segment coupled with an azide group by SPPS method, as Figure 3 shown in Figure A. The representative high - resolution mass spectrum of PY36 is as Figure 3 shown in Figure B. Prepare the TLR7 / 8 agonist IMDQ coupled with an alkyne group as an adjuvant by chemical synthesis method, and couple the peptide segment with the adjuvant by Click reaction, as Figure 3 shown in Figure C.

[0042] Prepare a broad - spectrum nasal vaccine against SARS - CoV - 2:

[0043] Using chitosan oligosaccharide as a carrier and TPP as an anionic crosslinking agent, nanoparticles with mucosal adhesion were prepared by the ionic crosslinking method. Figure 4 A). The optimized preparation conditions for the empty chitosan oligosaccharide nanoparticles were as follows: 35 mg of chitosan oligosaccharide was dissolved in 9.5 mL of acetic acid solution with a pH of 4, and 0.5 mL of 0.8% TPP solution was slowly added dropwise under stirring at 400 r / min to finally form chitosan oligosaccharide nanoparticles. The prepared nanoparticles had a particle size of 155.89 nm, a zeta potential of +24.92 mV, and a PDI of 0.1996 - 0.2304. The polypeptide - adjuvant conjugate was dissolved in the TPP solution during the preparation of the nanoparticles. The conjugate concentration was 0.5 mg / mL, and the addition amount was 1.5 mL. The prepared nanoparticles had a particle size of 302.225 nm, a PDI of 0.2326, and a polypeptide encapsulation efficiency of 82.96%.

[0044] Evaluation of the immune effect of the nanoparticles:

[0045] CS@IF23, CS@PY36, and CS@IF23 - PY36 nanoparticles were prepared respectively and administered via nasal mucosa. The dosage was 10 μg of polypeptide per mouse. Nasal mucosal administration was performed once every 7 days, and blood was collected 7 days after the second administration. The plate was coated with IF23 / PY36 - biotin, and the serum antibody titer was measured by ELISA. The results showed that the prepared nanoparticles had a uniform particle size, a good encapsulation efficiency, and chitosan oligosaccharide had a mucosal adhesion effect, which could induce a specific systemic immune response against the antigen. Figure 4 B), which is suitable for the encapsulation of the same type of polypeptide antigen and mucosal administration.

[0046] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification.

Claims

1. A broad-spectrum nasal mucosal vaccine for novel coronavirus based on conservative polypeptides / glycopeptides, characterized in that: The invention comprises a carrier and an antigen, wherein the carrier is chitosan oligosaccharide, the antigen is a conjugate of an antigen fragment and an adjuvant, wherein the antigen fragment comprises a polypeptide and a glycopeptide, and the adjuvant is a structural analogue of a TLR7 / 8 agonist.

2. The method for preparing a novel coronavirus broad-spectrum nasal mucosal vaccine based on conserved polypeptides / glycopeptides according to claim 1, characterized in that: The following steps are involved: S1. Screening of antigen fragments: Based on virus evolution analysis, highly conserved peptide sequences in multiple coronavirus variants were screened; peptides containing disulfide bonds were given priority to enhance structural stability; peptides containing glycosylation sites were excluded, or glycosylation sites were retained to prepare glycopeptide antigens; S2. Construction of conjugates of antigen fragments and adjuvants: S2.1, preparation of polypeptide antigen fragments by solid phase polypeptide synthesis; S2.2, preparing adjuvant molecules coupled with alkyne groups by chemical synthesis; S2.3, coupling antigen fragments and adjuvant molecules by click chemistry; S3. Preparation of a broad-spectrum nasal mucosal vaccine for novel coronavirus: S3.1, dissolving the carrier chitosan oligosaccharide in an acetic acid solution at pH = 4 to obtain solution A; S3.2, mixing the conjugate of the antigen fragment and the adjuvant with the active agent TPP solution to obtain a mixed solution B; S3.

3. Add solution B dropwise into solution A under stirring to react and form vaccine chitosan oligosaccharide nanoparticles.

3. The method for preparing a novel coronavirus broad-spectrum nasal mucosal vaccine based on conserved polypeptides / glycopeptides according to claim 2, characterized in that: The polypeptide in S2 is prepared by a solid phase peptide synthesizer. During the preparation process, glucosine is added to the synthesizer in the form of common amino acids to prepare a glycopeptide. A linker with an azide / alkyne group is coupled to the polypeptide fragment. The cleavage reagent used when cleaving the polypeptide is trifluoroacetic acid / thioanisole / 1,2-ethanedithiol / anisole = 90 / 5 / 3 / 2 (volume ratio) to prevent oxidation of disulfide bonds.

4. The method for preparing a novel coronavirus broad-spectrum nasal mucosal vaccine based on conserved polypeptides / glycopeptides according to claim 2, characterized in that: The final concentration of chitosan oligosaccharide in S3 is 3.5 mg / mL, the volume of TPP solution added is 5% of the final volume, the concentration is 0.8% (w / v), the concentration of the conjugate of antigen fragment and adjuvant is 0.5 mg / mL, and the volume added is 15% of the final volume.

5. The method for preparing a novel coronavirus broad-spectrum nasal mucosal vaccine based on conserved polypeptides / glycopeptides according to claim 4, characterized in that: The stirring condition in 3.3 is 400 r / min, the particle size of the prepared nanoparticles is 155.89 nm, the potential is +24.92 mV, and the PDI is 0.1996-0.2304.

6. Use of a broad-spectrum nasal mucosal vaccine for novel coronavirus based on conserved polypeptides / glycopeptides prepared according to any one of claims 2-5 in the prevention and treatment of novel coronavirus.

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