CN115708869A - Polypeptide vaccine microneedle patch and preparation method thereof - Google Patents

Abstract

The invention provides a polypeptide vaccine microneedle patch and a preparation method thereof, belonging to the technical field of biology. The polypeptide vaccine microneedle patch comprises a base and microneedles positioned on the base, wherein the microneedles are made of a mixture of a polypeptide vaccine and a soluble matrix, the material of the soluble matrix comprises hyaluronic acid and low-molecular-weight polyvinylpyrrolidone, and the mass ratio of the hyaluronic acid to the low-molecular-weight polyvinylpyrrolidone in the soluble matrix is (1-6): 20. according to the invention, low molecular weight polyvinylpyrrolidone is selected as a framework material, hyaluronic acid rich in hydroxyl and carboxyl is introduced, and a complex three-stage net structure is formed by utilizing the hydrogen bond effect between the low molecular weight polyvinylpyrrolidone and the hyaluronic acid and the crosslinking winding effect of a macromolecular chain, so that the microneedle which is easy to hydrolyze and diffuse and has high strength and hardness is obtained. The polypeptide vaccine is delivered to the epidermal layer and the dermal layer by using the microneedle, so that the painless delivery of the polypeptide vaccine is realized, and the curative effects of low dose and high immunity can be realized.

Description

Polypeptide vaccine microneedle patch and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a polypeptide vaccine microneedle patch and a preparation method thereof.

Background

The epitope peptide vaccine is a novel vaccine developed in recent years, also called polypeptide vaccine, and is a vaccine prepared by a chemical synthesis technology according to the known or predicted amino acid sequence of a certain section of antigen epitope in a pathogen antigen gene. The polypeptide vaccine has no problem of virulence reversion or incomplete inactivation, and the synthesis method is simple and accords with the development direction of safe vaccines. Prophylactic or therapeutic polypeptide vaccines designed against 2019 novel coronaviruses (2019-nCoV) can generate antigen-specific immune responses. However, polypeptide vaccines often have molecular weights of thousands to hundreds of thousands and cannot penetrate into the subcutaneous tissue by diffusion through the stratum corneum as do small molecule drugs. And due to the instability of the polypeptide vaccine in vitro and in vivo, the main clinical dosage form of the polypeptide vaccine is injection, namely the vaccine is delivered into the body by a syringe injection mode. The injection by syringe is divided into intravenous injection, intramuscular injection, subcutaneous injection and intradermal injection. The concentrated novel coronavirus vaccines currently on the market are all injected intramuscularly. Such vaccine delivery methods are associated with a great pain sensation, poor patient compliance, risk of infection, and also generate more medical waste, and the vaccines do not exert their immunogenicity well. Therefore, the development of novel polypeptide vaccine administration technology is very important.

In recent years, the transdermal drug delivery technology of the micro-needle is rapidly developed, and the micro-needle can pierce through the stratum corneum to create a plurality of micron-level channels in the epidermis so as to enable macromolecular drugs to enter subcutaneous tissues. Due to the elasticity of the skin and the tiny size of the microneedle, the depth of the microneedle entering the skin usually cannot reach the dermis, obvious pain is not caused, and the microneedle is a painless drug delivery technology. At present, the materials used for preparing microneedles are mainly metals, silicon, and polymers. The micro-needle prepared in the early stage mainly adopts monocrystalline silicon or silicon dioxide and other semiconductor materials, the semiconductor micro-needle has good biocompatibility and high hardness, is easy to pierce skin, but has large brittleness, cannot be degraded if being retained in a body after being broken, and cannot store medicines, and a complex medicine storage and slow release system is needed when the corresponding micro-needle transdermal administration patch is prepared, so that the process is complex, the cost is high, and the clinical application of the patch is limited. The metal micro-needle appears later than the semiconductor micro-needle, generally adopts titanium and nickel alloy, and the biological safety of the metal micro-needle is good, and the needle point is easy to pierce the skin without breaking, but the metal micro-needle can not store the medicine, and a medicine storage and slow release system is also needed to be arranged, so that the metal micro-needle can not be widely applied clinically. The polymer micro-needle appears in 2004 or so, can be degraded in vivo due to good biocompatibility, has high safety, can coat the drug on the needle body, has relatively simple preparation process, and can be developed rapidly. At present, the polymer micro-needle mainly adopts materials such as polymethyl methacrylate, polylactic acid, polyglycolic acid, vinyl pyrrolidone, polydioxanone and copolymers thereof, and has the defects that the mechanical strength is generally not enough to pierce the skin, and most of polymer materials for preparing the micro-needle cannot be dissolved in water and need to be cast and molded in a molten state, but the high-temperature treatment easily makes high-temperature sensitive medicines such as polypeptide vaccines lose activity.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a polypeptide vaccine microneedle patch and a preparation method thereof.

In order to achieve the purpose, the invention is realized by the following technology:

a polypeptide vaccine microneedle patch comprises a base and microneedles positioned on the base, wherein the microneedles are made of a mixture of a polypeptide vaccine and a soluble matrix, the material of the soluble matrix comprises hyaluronic acid and low molecular weight polyvinylpyrrolidone, and the mass ratio of the hyaluronic acid to the low molecular weight polyvinylpyrrolidone in the soluble matrix is 1-6:20.

further, the molecular weight of the hyaluronic acid is 3-5WDa, and the molecular weight of the low molecular weight polyvinylpyrrolidone is 4-6WDa.

Further, the polypeptide vaccine comprises a B cell epitope peptide and an adjuvant, wherein the B cell epitope peptide comprises at least one selected from the following a) to c):

a) The amino acid sequence is one or more of polypeptides shown in SEQ ID NO.1 to SEQ ID NO. 4;

b) The derivative polypeptide is formed by inserting, substituting or deleting one or more amino acids into the polypeptide shown in the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.4, and has the same or basically the same function as the polypeptide shown in the amino acid sequences of SEQ ID NO.1 to SEQ ID NO. 4;

c) And a multimeric polypeptide formed from one or more of the polypeptides having amino acid sequences represented by SEQ ID No.1 to SEQ ID No. 4.

Further, the B cell epitope peptide comprises a polypeptide with an amino acid sequence shown as SEQ ID NO. 4. Furthermore, the B cell epitope peptide also comprises one or more polypeptides with amino acid sequences shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

Further, the B cell epitope peptide comprises a polymer polypeptide formed by the polypeptide with an amino acid sequence shown as SEQ ID NO.1, and the polymer polypeptide is a homotetramer formed by connecting 4 polypeptides with amino acid sequences shown as SEQ ID NO.1 through 3 lysines. Still further, the B cell epitope peptide also includes a multimeric polypeptide formed by one or more of the polypeptides shown in SEQ ID NO.2 to SEQ ID NO.4 in amino acid sequence.

Further, the mass ratio of the polypeptide vaccine to the soluble matrix in the microneedle is 1:100-1000.

Further, the material of the base comprises high molecular weight polyvinylpyrrolidone, and the molecular weight of the high molecular weight polyvinylpyrrolidone is 32-36WDa.

Further, the whole height of the micro-needle is 500-1500 μm, the diameter of the tip head is 5-20 μm, and the bottom end size of the needle body is (100-500) × (100-500) & ltmu & gt ² ；

The size of the base is (1-30) x (1-30) mm ² The thickness is 0.5-3mm.

Furthermore, the microneedles are distributed on the base in an array shape, the array specification is 1 x 1-20 x 20, and the needle axis distance between every two adjacent microneedles is 100-1000 mu m.

In addition, another embodiment of the present invention provides a method for preparing the polypeptide vaccine microneedle patch, which comprises the following steps:

s1, dissolving hyaluronic acid and polyvinylpyrrolidone in ultrapure water to obtain a soluble matrix solution;

s2, adding the polypeptide vaccine into the soluble matrix solution and uniformly mixing to obtain a microneedle solution;

s3, sucking the microneedle solution, placing the microneedle solution in a microneedle mould, performing vacuum decompression treatment, filling the microneedle solution in pinholes of the microneedle mould, removing redundant microneedle solution, and drying at low temperature to obtain a microneedle structure;

and S4, adding a base solution on the surface of the microneedle structure, curing, forming and demolding to obtain the polypeptide vaccine microneedle patch.

Further, in step S3, the vacuum pressure reduction process specifically includes: the microneedle mould containing the microneedle solution was placed under-100 kPa vacuum for 15min and bubbles were removed by allowing the bubbles to escape by depressurization.

Further, in step S4, the curing and forming specifically includes: and (3) placing the microneedle mould containing the base solution at the temperature of 4 ℃ for 48 hours or at room temperature for 24 hours.

Has the beneficial effects that:

1. the invention selects low molecular weight polyvinylpyrrolidone as a framework material, introduces hyaluronic acid rich in hydroxyl and carboxyl, and forms a complex three-level network structure by utilizing the hydrogen bonding effect between the hyaluronic acid and the cross-linking and winding effect of a macromolecular chain to obtain the microneedle which is easy to hydrolyze and diffuse and has higher strength and hardness. The microneedle is used for delivering the vaccine to the epidermal layer and the dermal layer, thereby realizing the painless delivery of the polypeptide vaccine, reducing the risk of the inactivation of the polypeptide vaccine and realizing the curative effects of low dosage and high immunity.

2. The soluble matrix of the microneedle is formed by hyaluronic acid and low molecular weight polyvinylpyrrolidone which are easily soluble in water, so that the coating of the polypeptide vaccine can be realized at normal temperature, and the activity of the polypeptide vaccine can be maintained to the maximum extent. And because the water solubility is good, the soluble matrix can be quickly dispersed when meeting tissue fluid, the release speed of the polypeptide vaccine is accelerated, the immunogenicity of the polypeptide vaccine is better exerted, the response intensity of immune response is improved, the irritation of long-time medicine absorption to skin is reduced, and the biocompatibility and the safety are good.

3. The polypeptide vaccine microneedle patch has the advantages of simple preparation process and low cost, reduces the generation of medical waste, and has a higher application prospect.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

Fig. 1 is a flowchart illustrating a process for preparing a polypeptide vaccine microneedle patch according to an embodiment of the present invention;

FIG. 2 is a scanning electron microscope image of the polypeptide vaccine microneedle patch of example 1 of the present invention;

FIG. 3 is a fluorescent inverted microscope image of a polypeptide vaccine microneedle patch according to example 1 of the present invention; wherein, the picture a is a fluorescence inverted microscope picture of the head part of the needle tip, the picture b is a fluorescence inverted microscope picture of the bottom end of the needle body, and the picture c is a fluorescence inverted microscope picture of the side surface of the micro needle;

FIG. 4 is a stress-strain curve of the polypeptide vaccine microneedle patch of example 1;

FIG. 5 is a fluorescence inverted microscope image of the depth of the polypeptide vaccine microneedle patch penetrating the pigskin in example 1 of the present invention;

FIG. 6 is a diagram of the recovery of a pinhole at different times after a polypeptide vaccine microneedle patch punctures the skin of a mouse in example 1 of the present invention;

FIG. 7 is a diagram showing the release and diffusion properties of the active ingredients of the polypeptide vaccine of example 1 after the microneedle punctures the agarose hydrogel;

FIG. 8 is a graph showing the results of mass spectrometric identification of the B-cell epitope peptide Pep-916 of example 3 of the present invention;

FIG. 9 is a graph showing the result of mass spectrometric identification of the B-cell epitope peptide Pep-1510 in example 3 of the present invention;

FIG. 10 is a graph showing the results of mass spectrometric identification of the B-cell epitope peptide Pep-233 of example 3 of the present invention;

FIG. 11 is a graph showing the result of mass spectrometric identification of the B-cell epitope peptide Pep-252 of example 3 of the present invention;

FIG. 12 shows the specific antibody titer of the B-cell epitope peptide Pep-252 in positive and negative horse sera in example 4 of the present invention;

FIG. 13 shows the specific antibody titers of the B cell epitope peptides Pep-252 and Pep-control in the serum of positive horses in example 4 of the present invention;

FIG. 14 shows the specific antibody titer of B-cell epitope peptide Pep-233 in positive and negative horse sera of example 4 of the present invention;

FIG. 15 shows the specific antibody titers of the B cell epitope peptides Pep-233 and Pep-control in the serum of positive horse of example 4 of the present invention;

FIG. 16 shows the specific antibody titer of B-cell epitope peptide Pep-1510 in positive and negative horse sera of example 4 of the present invention;

FIG. 17 shows the specific antibody titers of the B-cell epitope peptide Pep-1510 and Pep-control in the sera of positive horses in example 4 of the present invention;

FIG. 18 shows the specific antibody titer of the B-cell epitope peptide Pep-1510 in the serum of positive mice and the serum of negative mice in example 4 of the present invention;

FIG. 19 shows the specific antibody titers of the B-cell epitope peptide Pep-1510 and Pep-control in the serum of positive mice in example 4 of the present invention;

FIG. 20 shows the specific antibody titer of the B cell epitope peptide Pep-916 in the serum of positive and negative mice in example 4 of the present invention;

FIG. 21 shows the specific antibody titers of the B-cell epitope peptides Pep-916 and Pep-control in the serum of positive mice in example 4 of the present invention;

FIG. 22 shows the specific antibody titers of B cell epitope peptide Pep-916 in the sera of positive monkeys and the sera of negative monkeys in example 4 of the present invention;

FIG. 23 shows the specific antibody titers of the B-cell epitope peptides Pep-916 and Pep-control in the serum of the positive monkeys of example 4 of the present invention;

FIG. 24 shows the specific antibody titer of the B-cell epitope peptide Pep-233 in the serum of positive monkey and the serum of negative monkey in example 4 of the present invention;

FIG. 25 shows the specific antibody titers of the B-cell epitope peptides Pep-233 and Pep-control in the sera of positive monkeys of example 4 of the present invention;

FIG. 26 shows the specific antibody titers of B cell epitope peptide Pep-916 in convalescent patient positive serum and normal human negative serum of example 4 of the present invention;

FIG. 27 shows the specific antibody titers of the B cell epitope peptides Pep-916 and Pep-control in convalescent patient positive sera of example 4 of the present invention;

FIG. 28 is a graph showing the result of the neutralization experiment of SARS-CoV-2 pseudovirus in example 6 according to the present invention;

FIG. 29 is a graph showing the results of the SARS-CoV-2 virus microneutralization experiment in example 7 of the present invention;

FIG. 30 is a structural formula of multimeric polypeptide BH1 of example 8 of the present invention;

FIG. 31 is a structural formula of multimeric polypeptide BH2 of example 8 of the present invention;

FIG. 32 is a chart showing the results of a SARS-CoV-2 true virus neutralization assay using the multimeric polypeptide of example 8 of the present invention;

FIG. 33 is a graph showing the results of the SARS-CoV-2 true virus neutralization assay performed by the polypeptide vaccine microneedle patch of example 9 of the present invention.

Detailed Description

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. In addition, the terms "comprising", "containing", "having" and "having" are intended to be non-limiting, i.e., that other steps and other ingredients can be added which do not affect the results. Materials, equipment and reagents are commercially available unless otherwise specified.

For a better understanding of the invention, without limiting its scope, all numbers expressing quantities, volume fractions, and other numerical values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.

The embodiment of the invention provides a polypeptide vaccine microneedle patch, which comprises a base and microneedles positioned on the base, wherein the microneedles are made of a mixture of a polypeptide vaccine and a soluble matrix, the material of the soluble matrix comprises Hyaluronic Acid (HA) and Polyvinylpyrrolidone (PVP), and the mass ratio of the Hyaluronic acid to the Polyvinylpyrrolidone in the soluble matrix is (1-6): 20.

in the invention, PVP is selected as a framework material, HA rich in hydroxyl and carboxyl is introduced, and a complex three-stage net structure is formed by utilizing the hydrogen bond effect between the PVP and the HA, and the macromolecular chain is crosslinked and wound, so that the microneedle which is easy to hydrolyze and diffuse and HAs higher strength and hardness is obtained. When the micro needle is inoculated with the polypeptide vaccine, the base can be tightly adhered to the skin, the micro needle penetrates into the surface skin, and the soluble matrix can be quickly dissolved under the action of the tissue fluid, so that the effect of releasing the polypeptide vaccine is achieved. Since the microneedles deliver the vaccine to the epidermal and dermal layers, with a large number of antigen presenting cells therebetween, a low vaccine dose with a high immune response can be achieved.

Because the polypeptide vaccine is unstable in vivo and is easily degraded by relevant enzymes in blood, the immunogenicity of the polypeptide vaccine is reduced and even lost. The microneedles deliver the vaccine to the epidermal and dermal layers without direct contact with enzymes in the body, and thus avoid degradation and loss of immunogenicity. Therefore, compared with the traditional injection, the delivery mode is improved, and the painless and high-efficiency delivery of the polypeptide vaccine is realized.

In addition, HA and PVP are very easy to dissolve in water, operations such as high-temperature dissolution and high-temperature pouring are not needed, the polypeptide vaccine can be coated at normal temperature, and the activity of the polypeptide vaccine can be kept to the maximum extent. And the hyaluronic acid endogenous substances do not cause inflammatory reaction, do not harm human bodies and have high biological safety. Because the water solubility is good, the three-level net structure of the soluble matrix can be quickly dispersed when meeting tissue fluid, the release speed of the polypeptide vaccine is accelerated, and the polypeptide vaccine can be quickly absorbed by a human body, thereby being beneficial to better exerting the immunogenicity of the polypeptide vaccine, improving the response strength of immune response, reducing the irritation of long-time drug absorption to skin, and having good biocompatibility and safety.

In the soluble matrix, HA content is higher, and the dissolving speed of micropin is faster, and the release rate of polypeptide vaccine active ingredient is also faster, does benefit to more and is absorbed by the human body fast, also helps reducing the irritability of micropin to skin, but HA content is too high can lead to the viscosity height to be unfavorable for the micropin shaping, and the micropin HAs the hardness not enough, mechanical strength defect such as not good enough, when using the micropin, easily leads to the phenomenon of being difficult for puncturing in the skin. Preferably, the mass ratio of the hyaluronic acid to the low molecular weight polyvinylpyrrolidone in the soluble matrix is 2-4:20, more preferably, 3:20. at this mass ratio, good moldability of the microneedle is ensured and it is made to have sufficient mechanical strength and hardness to be able to pierce the skin while being unlikely to break.

The molecular weight of the hyaluronic acid is 3-5WDa, and the molecular weight of the low molecular weight polyvinylpyrrolidone is 4-6WDa. The HA and PVP with the molecular weight have proper viscosity and solid content after being dissolved in water, and are beneficial to forming to obtain the microneedle with better hardness and strength. HA and PVP are linear macromolecules, when the HA and PVP with lower concentration are dissolved in water, the viscosity of an aqueous solution is increased sharply and is increased along with the increase of molecular weight, the viscosity is too high, the microneedle patch is not easy to produce, the solid content of the manufactured needle body is insufficient, the needle body is not easy to break, and the hardness is insufficient.

Optionally, the polypeptide vaccine includes a B cell epitope peptide and an adjuvant. Meanwhile, the immunogenicity of the polypeptide vaccine can be further enhanced by loading the polypeptide vaccine and the adjuvant.

In the context of the present invention, the term "vaccine" refers to an agent useful for eliciting, stimulating or enhancing the immune system of a cell or animal or human against a pathogen. The term "adjuvant" refers to a specific immunopotentiator which, when injected or previously injected into a body together with the B cell epitope peptide of the present invention, can enhance the body's immune response to the B cell epitope peptide or change the type of immune response, such as Al (OH) ₃ Adjuvants, the use of adjuvants for vaccine actives is well known in the art and will not be described in detail herein. In some preferred embodiments of the present invention, the mass ratio of the B cell epitope peptide to the adjuvant in the polypeptide vaccine is 4:3.

the above-mentioned B-cell epitope peptide includes at least one selected from the following a) to c):

a) The amino acid sequence is one or more of polypeptides shown in SEQ ID NO.1 to SEQ ID NO. 4; specifically, the amino acid sequences of the polypeptides are respectively:

SEQ ID NO.1 (hereinafter referred to as Pep-916): CVNFNFNFNGL

SEQ ID NO.2 (hereinafter referred to as Pep-1510): YQPYRVVLVFLSFELLH

SEQ ID NO.3 (hereinafter referred to as Pep-233): GDEVRQIAPGQTGKIADYNYKLP

SEQ ID NO.4 (hereinafter referred to as Pep-252): YNSASFSFKCYGVSPTKLDLCFT

b) The derivative polypeptide is formed by inserting, substituting or deleting one or more amino acids into the polypeptide shown in the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.4, and has the same or basically the same function as the polypeptide shown in the amino acid sequences of SEQ ID NO.1 to SEQ ID NO. 4;

c) And a multimeric polypeptide formed from one or more of the polypeptides having amino acid sequences represented by SEQ ID No.1 to SEQ ID No. 4.

In the context of the present invention, the term "derivative polypeptide" refers to variants having the same or similar function as the polypeptide of the present invention, but with a minor difference in amino acid sequence to that shown in any of SEQ ID No.1-4, including but not limited to: deletion, insertion and/or substitution of one or more (usually 1 to 5, preferably 1 to 4, more preferably 1 to 3, most preferably 1 to 2) amino acids. It is well known to those skilled in the art that substitutions with amino acids that are close or similar in properties, such as substitutions between polar amino acids (between glutamine and asparagine), hydrophobic amino acids (such as between leucine and isoleucine), and similarly charged amino acids (such as between arginine, lysine and histidine, or between glutamic acid and aspartic acid), will not alter the function of the resulting polypeptide. As another example, the addition of one or several amino acids at the C-terminus and/or N-terminus, such as a tag added for ease of isolation, does not generally alter the function of the resulting polypeptide, nor does it have any adverse effect on its performance.

The term "multimer" refers to a substance comprising two or more polypeptides, and is divided into homomultimers and heteromultimers. The "multimeric" polypeptide comprises a plurality of polypeptides which are linked to each other covalently or via a connecting chain, wherein the covalent linkage can be the linkage of the plurality of polypeptides via ester bonds, ether bonds, phosphate bonds, amide bonds, peptide bonds, imide bonds, carbon-sulfur bonds, or carbon-phosphorus bonds; the linking chain connection can be a structural unit that connects adjacent polypeptides by one or more amino acids, the introduction of which does not compromise the immunogenicity of the multimeric polypeptide. For example, in some embodiments, 2 Pep-252 polypeptides are linked by 1 lysine to form a homodimeric polypeptide, and 4 Pep-916 polypeptides are linked by 3 lysines to form a homotetrameric polypeptide; in other embodiments, pep-916 and Pep-252 can be linked by other amino acids to form heterodimeric polypeptides, and so on, and will not be described further.

The present invention illustratively provides several multimeric polypeptides, which should not be construed as limiting the scope of the invention, and are exemplified by:

BH1, the amino acid sequence of which is: (YNSASFSFKCYGVSSPTKLNDLCFT) 2-K, wherein the multimeric polypeptide is formed by connecting 2 Pep-252 through 1 lysine to form homodimer, the molecular weight is 5699.12, and the structural formula is shown in figure 30;

BH2, the amino acid sequence of which is: [ (CVNFNFNFNGL) 2-K ]2-K, the multimeric polypeptide is 4 Pep-916 connected by 3 lysines to form homotetramer, the molecular weight is 4440.84, the structural formula is shown in figure 31.

The Pep-916, pep-1510, pep-233, and Pep-252 polypeptides of the present invention may be used alone or in any combination. Wherein, for a) 15 cases of B cell epitope peptides, specifically, there are 4 cases of using Pep-916, pep-1510, pep-233 or Pep-252 polypeptides alone, and 11 cases of using Pep-916, pep-1510, pep-233 and Pep-252 polypeptides in combination, including: the total of 2 polypeptide combinations is 6 cases, the total of 3 polypeptide combinations is 4 cases, and the total of 4 polypeptide combinations is 1 case. For B) and c), the same includes B cell epitope peptides of different species used alone and in combination, and the polypeptides contained in a), B) and c) may also be used in combination.

The polypeptide vaccine of the invention takes specific B cell epitope peptides (Pep-916, pep-1510, pep-233 and Pep-252) or derivative polypeptides or polymer polypeptides thereof as active ingredients, the B cell epitope peptides have good intermiscibility with PVP and HA, can improve the skin permeability of the active ingredients, improve the administration efficiency, ensure the full play of the efficacy of the polypeptide vaccine, further efficiently stimulate the human body to generate immune response against SARS-CoV-2, and provide powerful immune protection effect.

The invention deeply excavates 2019S protein sequence, S protein 3D crystal structure and 3D crystal structure of interaction of S protein RBD region and human receptor ACE-2 by using the method of immunoinformatics, then selects candidate linear B cell epitope peptide, uses horse antibody, mouse antibody, monkey antibody and disease convalescent phase antibody for further selection to obtain the B cell epitope peptide, and further verifies the immunogenicity and functionality of the B cell epitope peptide obtained by the invention through animal experiments, and then selects and obtains B cell epitope peptides Pep-916, pep-1510, pep-233 and Pep-252, wherein the B cell epitope peptide has strong immunogenicity and immune persistence, can induce organism to generate corresponding immune protection reaction, obviously improves the titer of total antibody and neutralizing antibody aiming at S protein, effectively blocks the novel coronavirus S protein from being combined with host cell receptor ACE2, therefore, the novel coronavirus S protein is developed into SARS-CoV-2 neutralizing vaccine with excellent virus effect. In addition, the B cell epitope peptide uses the polypeptide sequence as immunogen, does not use inactivated virus or attenuated virus, has no side effect of infecting host with virus, and has higher safety.

A large number of experiments show that the Pep-252 polypeptide has the highest immunogenicity in four polypeptides, can generate stronger protective neutralizing antibodies, and better protects a matrix from being attacked by a novel coronavirus. Therefore, preferably, the B cell epitope peptide comprises a polypeptide having an amino acid sequence shown in SEQ ID No. 4. More preferably, the polypeptide also comprises one or more of the polypeptides with the amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3. Generally, a single epitope has weak immunogenicity, and the combination of multiple epitopes can enhance immunogenicity, induce stronger immune response and stimulate the body to generate strong antibody immune response with virus neutralization activity.

The polymer polypeptide of Pep-916 has higher immunogenicity compared with the Pep-252 polypeptide, and preferably, the B cell epitope peptide comprises a polymer polypeptide formed by a polypeptide with an amino acid sequence of SEQ ID NO.1, and the polymer polypeptide is a homotetramer formed by connecting 4 polypeptides with the amino acid sequence of SEQ ID NO.1 through 3 lysines; more preferably, the polypeptide also comprises a polymer polypeptide formed by one or more of the polypeptides shown in SEQ ID NO.2 to SEQ ID NO. 4.

The B cell epitope peptide is synthesized by adopting an Fmoc solid-phase polypeptide synthesis method, and comprises the following steps:

1) Connecting the first Fmoc-amino acid carboxyl at the C terminal of the B cell epitope peptide to be synthesized to Rink resin in a covalent bond mode, and then removing the protecting group at the N terminal of the first Fmoc-amino acid; taking the N end of the first Fmoc-amino acid as the synthesis starting point of the B cell epitope peptide, and carrying out dehydration condensation reaction with the carboxyl end of the next amino acid to form a peptide bond;

2) Adding a second Fmoc-amino acid, performing dehydration condensation reaction of the second Fmoc-amino acid and the first Fmoc-amino acid with the N-terminal of the protecting group removed to bond the second Fmoc-amino acid to the Rink resin, and then removing the protecting group from the N-terminal of the second Fmoc-amino acid; the N-terminus of the second amino acid is reacted with the carboxyl group of the subsequent Fmoc-amino acid.

3) Repeating the step 3) until the synthesis of the B cell epitope peptide is finished;

4) Cutting the synthesized B cell epitope peptide from Rink resin, and precipitating and washing by using diethyl ether to obtain a primary peptide;

5) And separating and purifying the primary peptide to obtain the B cell epitope peptide with the purity of more than 95 percent.

Optionally, the mass ratio of the polypeptide vaccine to the soluble matrix in the microneedle is 1:100-1000. The polypeptide vaccine provided by the invention has high immunogenicity and high release speed of the soluble matrix, can reduce the dosage of the polypeptide vaccine, realizes the effects of low dosage and high immunity, and reduces the cost.

Optionally, the material of the base comprises high molecular weight polyvinylpyrrolidone, the high molecular weight polyvinylpyrrolidone having a molecular weight of 32-36WDa. The high molecular weight PVP is adopted as a base material, so that the base layer has good flexibility and toughness, has excellent fit with skin irregularities such as joints and the like, and is favorable for transdermal drug delivery of microneedles.

The overall height of the microneedles is preferably not too high, or else they penetrate the skin easily and develop a sensation of pain, preferably 500-1500 μm, more preferably 500-1000 μm, with a tip-head diameter of 5-20 μm. The bottom of the needle body is not suitable to be too small, which easily causes small supporting area and reduces the mechanical property, or too large, which causes larger wound, is not easy to repair in time and may cause infection, and the bottom of the needle body is preferably (100-500) × (100-500) μm ² More preferably (200-400). Times. (200-400). Mu.m ² The needle body is in the shape of a quadrangular pyramid, a cone or other shapes suitable for penetrating into the skin, and is not limited herein.

Optionally, a plurality of microneedles are distributed on the base in an array shape to form a microneedle array, the array specification is 1 × 1-20 × 20, in order to ensure that each microneedle patch carries a certain drug amount, the microneedle patch array is not too small, preferably 5 × 5-20 × 20, and the needle axis distance between two adjacent microneedles is 100-1000 μm.

Optionally, the base has dimensions of (1-30) × (1-30) mm ² The thickness is 0.5-3mm.

Another embodiment of the present invention provides a method for preparing a polypeptide vaccine microneedle patch as described above, referring to fig. 1, including the following steps:

s1, dissolving hyaluronic acid and polyvinylpyrrolidone in ultrapure water to obtain a soluble matrix solution;

s2, adding the polypeptide vaccine into the soluble matrix solution and uniformly mixing to obtain a microneedle solution;

s3, sucking the microneedle solution, placing the microneedle solution in a microneedle mould, performing vacuum decompression treatment, filling the microneedle solution in pinholes of the microneedle mould, removing redundant microneedle solution, and drying at low temperature to obtain a microneedle structure;

and S4, adding a base solution on the surface of the microneedle structure, curing, forming and demolding to obtain the polypeptide vaccine microneedle patch.

In the step S1, the mass concentration ratio of the hyaluronic acid to the low molecular weight polyvinylpyrrolidone in the matrix solution is 1 to 6:20.

in step S2, the mass concentration ratio of the polypeptide vaccine to the soluble matrix in the microneedle solution is 1:100-1000.

In step S3, the vacuum pressure reduction treatment specifically includes: the microneedle mould containing the microneedle solution was placed under-100 kPa vacuum for 15min, and the air bubbles were removed by allowing the air bubbles to escape by depressurization.

In step S4, the curing and forming operations include: and (3) placing the microneedle mould containing the base solution at the temperature of 4 ℃ for 48 hours or at room temperature for 24 hours.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are examples of experimental procedures not specified under specific conditions, generally according to the conditions recommended by the manufacturer.

Example 1 polypeptide vaccine microneedle patch and evaluation of Performance thereof

A polypeptide vaccine microneedle patch comprises a base and microneedles positioned on the base, wherein the microneedles are prepared from the following components in a mass ratio of 1:230 and a soluble matrix, the soluble matrix being prepared from a mixture of the polypeptide vaccine of mass ratio 3:20 Hyaluronic Acid (HA) and low molecular weight polyvinylpyrrolidone (PVP), the molecular weight of hyaluronic acid is 3-5WDa, and the molecular weight of low molecular weight polyvinylpyrrolidone is 4-6WDa; the base is made of high molecular weight polyvinylpyrrolidone having a molecular weight of 32-36WDa.

The polypeptide vaccine comprises B cell epitope peptide and Al (OH) ₃ The mass ratio of the adjuvant, the B cell epitope peptide and the adjuvant is 4:3.

in this embodiment, the microneedle has an overall height of 670 μm, a tip diameter of 5-20 μm, and a base size of 300 × 300 μm ² The shape of the needle body is quadrangular pyramid, the micro-needles are distributed on the base in an array shape, and the array specification is10 x 10, and the needle axis distance between two adjacent microneedles is 500 mu m; the size of the base is 9.8 multiplied by 9.8mm ² And the thickness is 1.5mm.

The preparation method of the polypeptide vaccine micro-needle patch comprises the following steps:

s1, dissolving HA and low molecular weight PVP in ultrapure water to obtain a soluble matrix solution, wherein the final concentration of the HA in the soluble matrix solution is 150mg/mL, and the final concentration of the PVP in the soluble matrix solution is 1000mg/mL;

s2, polypeptide vaccine (wherein, B cell epitope peptide and Al (OH) ₃ Adding the adjuvant into the soluble matrix solution according to the mass ratio of 4);

s3, sucking 20 mu L of microneedle solution, placing the microneedle solution in a microneedle mould, carrying out vacuum decompression treatment, filling the microneedle solution in pinholes of the microneedle mould, removing the redundant microneedle solution, and drying at low temperature to obtain a microneedle structure;

s4, adding a base solution on the surface of the microneedle structure, then placing the microneedle structure at room temperature for 24 hours for curing and molding, and demolding to obtain the polypeptide vaccine microneedle patch; wherein the base solution is 600mg/mL aqueous solution of high molecular weight PVP.

The polypeptide vaccine microneedle patch of the present example was observed by a scanning electron microscope, and the result is shown in fig. 2. In the same way, the polypeptide vaccine was replaced by Alexa

488 dye, the prepared micro-needle is attached under a fluorescence inverted microscope for observation, and a-c in fig. 3 are pictures of the head part of the needle point, the bottom end of the needle body and the side surface of the micro-needle respectively. As can be seen from fig. 3, the dye is uniformly distributed in the microneedles, which indicates that the polypeptide vaccine in the polypeptide vaccine microneedle patch prepared by the invention has good dispersibility and uniform distribution in the microneedles.

The mechanical properties of the polypeptide vaccine microneedle patch were tested on a miniforce tester, and a set of stress-strain data was obtained by compressing the microneedles at a constant speed, the results being shown in fig. 4. As can be seen from fig. 4, the polypeptide vaccine microneedle patch of the present invention can withstand a high load.

Taking fresh dehaired and cleaned pigskin, loading Alexa

The 488-dye microneedle patch is perpendicularly pierced aiming at the surface layer of the pigskin until the soluble matrix is completely dissolved, the base layer is uncovered after 10min, the pigskin applied with the microneedle is observed under an optical microscope, the part of the pigskin applied with the microneedle can be seen to have clear holes, and the piercing rate reaches 100 percent, which indicates that the microneedle of the embodiment has enough mechanical strength and can effectively form the skin layer. The pig skin was also longitudinally sectioned at different depths (40 μm, 140 μm, 260 μm and 380 μm) from the surface layer and then viewed under a fluorescence inverted microscope image, which showed that the microneedles penetrated the skin to a depth of approximately 400 μm, indicating that they were effective in delivering the vaccine into the dermal layer.

And puncturing the skin of a mouse by taking a microneedle to observe the recovery condition of the pinhole, puncturing the microneedle into the skin of the anesthetized mouse, uncovering the back lining after 10min, and observing the healing condition of the pinhole on the skin, wherein the result is shown in figure 6. It can be seen that the pinholes on the skin of the mice gradually shrink and heal within 5min, indicating that the microneedles applied on the skin of the living animals will not cause damage to the skin, and the formed pinholes will recover quickly after the microneedle patch is uncovered.

The agarose hydrogel is used for simulating human skin, researching the release and diffusion performance of active ingredients of the polypeptide vaccine microneedle patch, and encapsulating pigment and Al (OH) ₃ The microneedles of the adjuvant were inserted into the hydrogel, and the diffusion diameters at different times were measured with the center of the hydrogel as the center of the circle, and the results are shown in fig. 7. The upper graph in fig. 7 shows the measurement process of the diffusion diameter, and the lower graph is a graph of the diffusion diameter with time. As can be seen from FIG. 7, the pigment and Al (OH) encapsulated in the microneedle ₃ The adjuvant can be quickly released after the microneedle is dissolved, and most of active ingredients can be released within about 20 min.

Example 2 screening of B cell epitope peptides in polypeptide vaccines

In the embodiment, a 2019 novel coronavirus S protein sequence is subjected to immune informatics analysis, a linear B cell epitope peptide with strong immunogenicity is predicted, the variation frequency of amino acid sites is counted, and a polypeptide without variation and with little variation is selected; meanwhile, the 3D crystal structure combined with the S protein and the 3D crystal structure combined with the human receptor ACE-2 protein position out the polypeptide which is positioned on the RBD area of the S protein and on the interaction surface with the ACE-2, and the possible linear B cell epitope peptide with strong immunogenicity is chemically synthesized. Then, the polypeptides are screened by in vitro experiments to obtain the polypeptides capable of binding to horse antibody, mouse antibody, monkey antibody and patient convalescent antibody. Finally, the immunogenicity is verified through further animal experiments, and the neutralizing antibody titer of the immunized mice is functionally measured to obtain the B cell epitope peptide, which is used as a synthetic peptide antigen, has stronger immunogenicity and good immune effect.

The present example provides a group of B cell epitope peptides for preparing a 2019 novel coronavirus vaccine for prevention and/or treatment, wherein the B cell epitope peptides of the present invention are derived from a 2019 novel coronavirus S protein sequence, and the amino acid sequences thereof are respectively:

pep-916: CVNFNFNGL (see SEQ ID NO. 1), its three-letter abbreviated form: cys-Val-Asn-Phe-Asn-Phe-Asn-Gly-Leu;

pep-1510: YQPYRVVLVFLSFELLH (see SEQ ID NO. 2), a three-letter abbreviated form thereof: tyr-Gln-Pro-Tyr-Arg-Val-Val-Val-Leu-Ser-Phe-Glu-Leu-Leu-His;

pep-233: GDEVRQIAPGQTGKIADYNYKLP (see SEQ ID NO. 3), the three-letter abbreviated form of which: gly-Asp-Glu-Val-Arg-Gln-Ile-Ala-Pro-Gly-Gln-Thr-Gly-Lys-Ile-Ala-Asp-Tyr-Asn-Tyr-Lys-Leu-Pro;

pep-252: YNSASFSFKCYGVSPSTKLNDLCFT (see SEQ ID NO. 4), a three-letter abbreviated form: tyr-Asn-Ser-Ala-Ser-Phe-Ser-Thr-Phe-Lys-Cys-Tyr-Gly-Val-Ser-Pro-Thr-Lys-Leu-Asn-Asp-Leu-Cys-Phe-Thr.

Example 3 Synthesis of B cell epitope peptide Using Fmoc solid phase polypeptide Synthesis method

The B cell epitope peptides Pep-916, pep-1510, pep-233 and Pep-252 of example 1 were synthesized by Fmoc solid phase polypeptide synthesis, comprising the following steps:

s1, connecting a first Fmoc-amino acid carboxyl group at the C end of a B cell epitope peptide to be synthesized to Rink resin through a covalent bond, then putting the Rink resin into N-methylpyrrolidone (NMP) solution of hexahydropyridine with the volume percentage of 15-30%, reacting for 25-40 minutes at the temperature of 20-28 ℃, thereby removing a protecting group at the N end of the first Fmoc-amino acid, then blowing the Rink resin dry by using nitrogen, and washing the Rink resin by using NMP;

s2, adding a second Fmoc-amino acid and 1-Hydroxybenzotriazole (HOBT), N' -Diisopropylcarbodiimide (DIC), performing condensation reaction at 20-28 ℃ for 30-90 minutes, performing dehydration condensation reaction on the second Fmoc-amino acid and the first Fmoc-amino acid of which the N terminal is protected, so as to bind the second Fmoc-amino acid to Rink resin, putting the Rink resin into an N-methylpyrrolidone (NMP) solution of hexahydropyridine with the volume percentage of 15-30%, performing reaction at 20-28 ℃ for 25-40 minutes, so as to remove the protecting group at the N terminal of the second Fmoc-amino acid, blow-drying the Rink resin by using nitrogen, and washing the Rink resin by using NMP;

s3, repeating the step S2 until the synthesis of the B cell epitope peptide is finished, washing Rink resin with the B cell epitope peptide for 3 times by using 100% methanol, drying the Rink resin in a fume hood, transferring the Rink resin into a brown bottle, and placing the bottle into a refrigerator at the temperature of-20 ℃ for later use;

s4, according to the volume ratio of trifluoroacetic acid (TFA), triisopropylsilane (TIS), phenol and water of 85:8:6:1, mixing Rink resin with B cell epitope peptide and the prepared lysate in a fume hood, stirring for 60 minutes by a magnetic stirrer at room temperature until the reaction is complete, cutting the synthesized B cell epitope peptide from the Rink resin, continuously evaporating for 30-120 minutes by using a cold trap rotary evaporator, removing TFA in crude peptide, and then precipitating and washing by using diethyl ether to obtain primary peptide;

and S5, washing the primary peptide for multiple times by using Dimethylformamide (DMF) to obtain the B cell epitope peptide with the purity of more than 95%.

The B cell epitope peptide prepared by the embodiment is freeze-dried to obtain solid powder, so that the B cell epitope peptide is convenient to transport and store for a long time.

And carrying out qualitative and quantitative analysis on the synthesized B cell epitope peptide. Qualitative and quantitative analysis was performed using matrix assisted laser desorption time of flight mass spectrometry (MALLDL-TOF) and reversed phase high pressure liquid chromatography (RP-HPLC). The mass spectrometric identification results of the prepared B-cell epitope peptides Pep-916, pep-1510, pep-233, and Pep-252 are shown in FIG. 8 (Pep-916), FIG. 9 (Pep-1510), FIG. 10 (Pep-233), and FIG. 11 (Pep-252), respectively. In fig. 8 to 11, the abscissa indicates the value of the mass-to-charge ratio (m/z) of the ion, the value of the mass-to-charge ratio increases from left to right, and the ordinate indicates the intensity (intensity) of the ion current.

As can be seen from FIGS. 8 to 11, the molecular weight of the synthesized B cell epitope peptide was in accordance with the theoretical value, indicating that the expected B cell epitope peptide was successfully obtained by the above-mentioned method.

Example 4 determination of IgG titres specific for B-cell epitope peptides in the serum of immunized animals by Indirect ELISA

In this example, SARS-CoV-2 virus S1 antigen (i.e., the B cell epitope peptide of the present invention) was purchased from Biotech, ohio, chinesian, oenoki, inc., and a 96-well ELISA plate pre-coated with SARS-CoV-2 virus S1 antigen and a washing buffer were purchased from Beijing medical research institute, oriental sea, at the time of pre-coating, the concentration of S1 antigen was 2. Mu.g/mL, and the coating amount was 50. Mu.L/well. HRP-labeled coat Anti-Mouse IgG antibody (Goat Anti-Mouse IgG antibody) was purchased from Southern Biotech and used at a concentration of 1:5000 preparation; HRP-labeled coat Anti-monkey IgG antibodies (Goat Anti-monkey IgG antibodies) were purchased from beijing solibao biotechnology ltd, and were used at a concentration of 1:3000, the medicines are allocated and are ready to use. Sample dilution buffer: bovine serum albumin and PBS solution were purchased from beijing solibao biotechnology ltd, and prepared into 3% bovine serum albumin PBS solution for use. The TMB single component color developing solution was purchased from biotechnology limited, solebao, beijing. ELISA stop solutions were purchased from Biotech, inc. of Solebao, beijing.

An indirect ELISA method for determining the specific IgG titer of the B cell epitope peptide in the serum of an immunized animal comprises the following steps:

s41, animal immunization and sample collection: based on the novel coronavirus immunization scheme of horses, mice and monkeys, blood is collected from tail veins at specified time points, and serum is separated for standby;

s42, collecting patient samples in a recovery period: blood samples are extracted from the veins of convalescent patients, and serum is separated for standby;

s43, performing an ELISA experiment:

(1) Adding a horse, mouse, monkey or human serum sample diluted by a multiple ratio in each reaction hole of a 96-hole enzyme label plate precoated with SARS-CoV-2 virus S1 antigen by 50 mu L/hole, and setting a negative control without serum;

(2) Placing the 96-hole enzyme label plate in an incubator at 37 ℃ for incubation for 45 minutes, then washing the enzyme label plate for 3 times by using a plate washing machine, and completely beating residual moisture;

(3) Adding a fresh diluted enzyme-labeled antibody into each reaction hole in an amount of 50 mu L/hole, incubating for 30 minutes at 37 ℃, washing the enzyme-labeled plate for 3 times by using a plate washing machine, and completely beating residual water;

(4) Adding TMB single-component color developing solution into each reaction hole in an amount of 50 mu L/hole, and incubating for 10 minutes at 37 ℃;

(5) Adding ELISA stop solution into each reaction hole in an amount of 50 mu L/hole;

(6) OD reading using microplate reader ₄₅₀ And (4) measuring the absorbance value.

The determination of antibody titer is noted as: mean OD of individual serum samples before immunization ₄₅₀ The value is the background value, if the background is lower than 0.05, the value is calculated according to 0.05, and 2.1 times of the background value is the titer judgment value. The data were analyzed by using statistical software Graphpad Prism 8, each group was repeated 3 times, and the results are shown in fig. 12-27, and fig. 12-27 are the serum of horse, mouse, monkey immunized with the novel coronavirus and the specific IgG titer of the B-cell epitope peptide in the serum of convalescent patients, respectively.

FIGS. 12-13 show the specific antibody titer of Pep-252 in positive horse serum and the comparison result of the binding force of the horse serum antibody and Pep-252 and Pep-control (the amino acid sequence is RRRRRRRRRRRRRRRRRRRR, see SEQ ID No. 5) after horse serum is immunized by the novel coronavirus respectively, and it can be known from FIGS. 12-13 that the B cell epitope peptide Pep-252 can be specifically bound with horse antibody.

FIGS. 14 to 15 show the specific antibody titer of Pep-233 in positive horse serum and the comparative results of binding force of horse serum antibody with Pep-233 and Pep-control, respectively, after horse serum is immunized with the novel coronavirus, and it can be seen from FIGS. 14 to 15 that B cell epitope peptide Pep-233 can be specifically bound with horse antibody.

FIGS. 16-17 show the specific antibody titer of Pep-1510 in positive horse serum and the binding strength of horse serum antibodies to Pep-1510 and Pep-control, respectively, after immunization of horse serum with the novel coronavirus, and it can be seen from FIGS. 16-17 that the B-cell epitope peptide Pep-1510 can specifically bind to horse antibody.

FIGS. 18 to 19 show the specific antibody titer of Pep-1510 in the serum of positive mice and the comparison of the binding force of the antibodies in the serum of the mice with Pep-1510 and Pep-control, respectively, after the mice were immunized with the novel coronavirus, and it can be seen from FIGS. 18 to 19 that the B cell epitope peptide Pep-1510 can specifically bind to the mouse antibody.

FIGS. 20-21 show the specific antibody titer of Pep-916 in the serum of positive mice and the comparative results of the binding force of the antibodies in the serum of mice with Pep-916 and Pep-control, respectively, after the sera of mice are immunized with the novel coronavirus, and it can be seen from FIGS. 20-21 that the B-cell epitope peptide Pep-916 can be specifically bound with the mouse antibody.

FIGS. 22 to 23 show the results of comparison of specific antibody titer of Pep-916 in the serum of positive monkeys and binding force of antibody in the serum of monkeys to Pep-916 and Pep-control, respectively, after immunization of monkey serum with the novel coronavirus, and it can be seen from FIGS. 22 to 23 that the B-cell epitope peptide Pep-916 can bind specifically to monkey antibody.

FIGS. 24 to 25 show the specific antibody titer of Pep-233 in the serum of positive monkeys and the binding force of antibody in the serum of monkeys against Pep-233 and Pep-control, respectively, after immunization of the sera with the novel coronavirus, and it can be seen from FIGS. 24 to 25 that the B-cell epitope peptide Pep-233 can bind specifically to monkey antibodies.

FIGS. 26 to 27 are the results of comparing the specific antibody titer of Pep-916 in serum of convalescent patients and the binding force between serum antibodies of convalescent patients and Pep-916 and Pep-control, respectively, and it can be seen from FIGS. 26 to 27 that B-cell epitope peptide Pep-916 can be specifically bound to anti-convalescent patients.

In conclusion, pep-916 can be specifically combined with the anti-specificity of mouse antibody, monkey antibody and convalescent patient, and the specific binding force of Pep-916 and convalescent patient antibody is much larger than that of other antibodies. The Pep-233 can be specifically combined with the horse antibody and the monkey antibody, and the combination force of the Pep-233 and the horse antibody is greater than that of the monkey antibody. The Pep-1510 can be specifically combined with horse antibody and mouse antibody, and the combination force of the Pep-1510 and the horse antibody is greater than that of the mouse antibody. The results show that the B cell epitope peptides Pep-916, pep-1510, pep-233 and Pep-252 screened by the invention have the capability of being specifically combined with 2019 novel coronavirus antibodies, and the affinity of different polypeptides for different antibodies is different, so that the B cell epitope peptides can be used for specifically detecting IgG.

Example 5 immunization of mice with B cell epitope peptides

Mice of BALB/c strain were used 20, divided into 4 groups of 5 mice each; each group of mice was immunized by intravenous injection of the B-cell epitope peptide of the present invention (Pep-916, pep-1510, pep-233, and Pep-252) at a dose of 5. Mu.g/mouse, respectively. Mice were immunized once a week for a total of 5 immunizations. The peripheral blood of the mice after 5 times of immunization is collected, and serum is separated for standby.

In order to explore the tolerance and safety of the B cell epitope peptide, the activity, body temperature and the like of 20 mice are continuously observed, and the results show that all the mice are normal in ingestion, free of adverse reaction and free of death after being injected with the B cell epitope peptide, and suggest that the B cell epitope peptide has high tolerance and biological safety when being used as a vaccine.

EXAMPLE 6 SARS-CoV-2 pseudovirus neutralization assay

S61, cloning an S protein gene from a Wuhan-Hu-1 strain (GenBank: MN 908947) into a plasmid pcDNA3.1 to obtain a recombinant plasmid pcDNA3.1.S2, wherein the recombinant plasmid pcDNA3.1.S2 can form S protein in host expression;

s62, transfecting pcDNA3.1.S2 plasmid (30. Mu.g/T75 flask) by adopting 293T cells;

s63, 293T cells after transfection of pcDNA3.1.S2 plasmid, within 24 hours, four times of infection with G delta G-VSV, every 2 hours of infection, using PBS solution to wash cells, then adding new cell culture solution;

s64, after 24 hours of infection with G.times.. DELTA.G-VSV virus, SARS-CoV-2 pseudovirus containing cell supernatant was collected, the main framework of the SARS-CoV-2 pseudovirus system was constituted by G.times.. DELTA.G-VSV, and cell debris was removed by filtration using a 0.45-. Mu.m pore size (Millipore, SLHP033 RB), and the cells were frozen in a freezer at-70 ℃ for use;

s65, diluting the immune mouse serum collected in the example 5 in a mode of 3 times each time, diluting the immune mouse serum for 6 times in total, mixing the immune mouse serum with the prepared SARS-CoV-2 pseudovirus, and incubating the immune mouse serum for 60 minutes at the temperature of 37 ℃;

s66, adding the cells treated with trypsin to the reaction solution, and adding CO ₂ 5% of content and incubation for 24 hours at 37 ℃;

s67, calculating the EC50 value neutralization titer of each mouse serum by using a Reed-Muench method.

The experimental result is shown in FIG. 28, and it can be seen from the figure that the antibody generated by the mice immunized with the B cell epitope peptides Pep-916, pep-1510, pep-233 and Pep-252 has very high neutralizing titer, and can rapidly identify and bind to the S protein, thereby neutralizing the pseudovirus, and the B cell epitope peptide obtained by screening has very good immune effect, and the antibody obtained by immunization can well neutralize the SARS-CoV-2 pseudovirus.

EXAMPLE 7 SARS-CoV-2 Virus microneutralization experiment

In this example, SARS-CoV-2 novel coronavirus was obtained from a virus strain isolated from the institute of virology of the institute of microbiology, military medical institute of microbiology, and stored in a virus library, betaCov/Beijing/IMEBJ01/2020 (abbreviated as 131, CPE/PFU, for cross-neutralization titer measurement), and its genome sequence numbers were GWHACAX01000000 and virus stock titer was 5X 10 ⁵ PFU/mL。

The main experimental materials and equipment include: DMEM medium, purchased from Gibco, usa; fetal Bovine Serum (FBS) purchased from Hyclone, usa; the antibiotic Penicilin-Streptomyces solution was purchased from Hyclone, USA; vero cells were purchased from ATCC, USA and stored in the institute for epidemic diseases of microorganisms. The cell culture solution contains 10% fetal calf serum, 100U/mL penicillin and 100ug/mL streptomycin; the cell maintenance solution is 2% fetal calf serum, 100U/mL penicillin and 100ug/mL streptomycin; 96-well cell culture plates, purchased from Corning biotechnology limited (Corning-Costar, usa).

Serum samples and controls: the sample was the serum collected from the immunized mouse in example 5, and the standard positive serum was the positive serum obtained by subcutaneously injecting a subunit vaccine (purchased from Cassia, cat.: 40592-V05H) in an amount of 5. Mu.g. The standard negative serum is the negative serum obtained by adjuvant subcutaneous injection, namely blank control.

Serum anti-SARS-CoV-2 neutralizing antibody titers were evaluated by microneutralization experiments after immunization of mice with B-cell epitope peptides (Pep-916, pep-1510, pep-233, and Pep-252). The experimental principle is that SARS-CoV-2 virus can cause sensitive target cell (Vero) to generate specific lesion, and the specific neutralizing antibody can prevent the virus-caused specific cell lesion, so that the neutralizing antibody titer is calculated by measuring the inhibition rate of the neutralizing antibody on the cell lesion. The experimental steps include:

and S71, conventionally culturing and passaging the Vero cells by using a DMEM complete culture medium containing 10% fetal calf serum, wherein the number of the passages of the Vero cells is 145-155. Immediately before use, cell growth medium was used to dilute the cell concentration to 1X 10 ⁵ Per mL, the cell suspension was added to the cell culture plate at 100 uL/well and mixed well in CO ₂ Culturing in an incubator for 18-24 hours;

s72, diluting the serum sample with cell maintenance liquid (different original dilution concentrations of different samples) to an initial concentration 4-8 hours before measurement, and then diluting by 2 times, wherein each sample is diluted by 46 dilutions, and each dilution is subjected to 2-4 repeated holes;

s73, diluting a subpackaged virus strain 131 stored in a virus Stock (Stock) to 100CCID50/0.05mL by using a cell maintenance solution 1-2 hours before measurement to obtain a virus solution;

s74, in the sample well with the serial dilution, 50 mu L of virus liquid (except for a sample toxicity test control and a normal cell control) with the concentration of 100CCID50/0.05mL is added in a hanging drop mode in a vertical mode in CO ₂ Incubating in the incubator for 1-1.5 hours, and simultaneously adding a virus control (50 uL containing 100TCID 50) and a normal cell control without adding virus into each cell culture plate in a hole for culturing Vero cells;standard positive serum, standard negative serum and virus back drop are set as inter-batch difference internal references in each batch of Neutralization Titer (NT) experiment.

S75, inoculated cell culture plates at 37 ℃ and 5% CO ₂ Culturing in an incubator, observing cytopathic effect (CPE) by using an inverted microscope, recording the cytopathic effect, taking the highest dilution of serum inhibiting 50% of cytopathic effect as an end point titer, judging the final result within 72-96 hours, and calculating the titer of neutralizing antibodies (NT 50) by using a Karber method or a Reed-Muench method.

The results of the experiments are shown in FIG. 29, from which it can be seen that the antibodies produced by mice immunized with the B-cell epitope peptides Pep-916, pep-1510, pep-233, and Pep-252 have high neutralizing titer (NT 50); pep-252 has the highest neutralization potency; NT50 average value is up to 78.2; the average NT50 value of Pep-1510 reaches 47.4; pep-233 and Pep-916 also have higher neutralization potency, NT50 is 18.6 and 15.2, respectively. The B cell epitope peptide screened by the invention has good immune effect and strong neutralizing capacity to SARS-CoV-2 virus.

EXAMPLE 8 SARS-CoV-2 Euvirus neutralization assay of multimeric Polypeptides

The present example provides 2B cell epitope peptides in the form of multimeric polypeptides for the preparation of a novel 2019 coronavirus vaccine for prevention and/or treatment, the amino acid sequences of which are:

BH1: the amino acid sequence is: (YNSASFSFKCYGVSSPTKLNDLCFT) 2-K, its three-letter abbreviated form: (Tyr-Asn-Ser-Ala-Ser-Phe-Ser-Thr-Phe-Lys-Cys-Tyr-Gly-Val-Ser-Prp-Thr-Lys-Leu-Asn-Asp-Leu-Cys-Phe-Thr) 2-Lys, wherein the polymer polypeptide is 2 Pep-252 which is connected by 1 lysine to form a homodimer, the molecular weight is 5699.12, and the structural formula is shown in figure 30;

BH2: the amino acid sequence is: [ (CVNFNFNFNGL) 2-K ]2-K, its three-letter abbreviated form: [ (Cys-Val-Asn-Phe-Asn-Phe-Asn-Gly-Leu) 2-Lys ]2-Lys, the polymer polypeptide is 4 Pep-916 which is connected by 3 lysines to form a homotetramer, the molecular weight is 4440.84, and the structural formula is shown in figure 31.

The BH1 and BH2 are synthesized by Fmoc solid-phase polypeptide synthesis, and the specific synthesis process is similar to that of example 3 and is not described herein again.

10 mice of BALB/c strain were used, divided into 2 groups of 5 mice each; each group of mice was immunized by subcutaneous injection of the B cell epitope peptides (BH 1 and BH 2) of the present invention at a dose of 5. Mu.g/mouse, respectively. Mice were immunized once a week for a total of 5 immunizations. The peripheral blood of the mice after 5 times of immunization is collected, and serum is separated for standby. Serum samples and controls: the sample was the serum collected from the immunized mouse in example 5, and the standard positive serum was the positive serum obtained by subcutaneous injection of subunit vaccine (purchased from Yi Qiao Shen, cat # 40592-V05H) in an amount of 5. Mu.g. The standard negative serum was a blank (blank) with adjuvant injected subcutaneously as negative serum.

The microclimation experiment of example 7 was used to evaluate serum anti-SARS-CoV-2 neutralizing antibody titers after immunization of mice with the multimeric polypeptides BH1 and BH2, with the confidence limit of the assay being the serum initial dilution factor (1. The measurement results are shown in FIG. 32.

As can be seen from FIG. 32, the NT50 value of BH2 is over 200, which is much higher than that of Pep-916 single peptide, and is also higher than that of the positive control. The NT50 value of BH1 is close to 100, and is very close to that of a positive control; proves that the antibodies generated by the two polymers have higher virus neutralization activity and can efficiently induce the neutralizing antibody against SARS-CoV-2.

The results show that the B cell epitope peptide has strong SARS-CoV-2 virus resistance as a vaccine, and has very good popularization prospect and market value.

EXAMPLE 9 neutralization assay of SARS-CoV-2 true virus by polypeptide vaccine microneedle patch

25 mice of BALB/c strain were used, divided into 5 groups of 5 mice each; the experimental groups were as follows: in the present embodiment, pep-916 is taken as an example, the dosage of the administered polypeptide is 5 μ g, the polypeptide-916 is taken as a positive control 1 (subcutaneously injected Pep 16), the subunit vaccine (purchased from yokewa, cat # 40592-V05H, intramuscular injection is taken as a positive control 2 (intramuscularly injected subunit recombinant vaccine), the microneedle patch encapsulated with the subunit recombinant vaccine is taken as a positive control 3 (MN + subunit recombinant vaccine), the dosage of the administered subunit vaccine is 5 μ g, each group of mice is immunized, the second immunization is performed on the 21 st day after the first immunization, the third immunization is performed on the 35 th day, the peripheral blood of the mice after the 3 immunizations is collected, and the neutralizing antibody using the transdermal administration mode of the microneedle patch is evaluated by the micro-neutralizing experiment of example 7, and the longitudinal coordinate is the real virus neutralizing antibody titer, see fig. 33.

As can be seen from fig. 33, the immunization effect can be improved by transdermal administration of the microneedle, and compared with subcutaneous injection, the real virus neutralization capacity of the B cell epitope peptide Pep16 is greatly improved, which is not much different from that of subunit recombinant vaccines, and thus the microneedle patch of the present invention has important significance for stably and efficiently delivering polypeptide vaccines and improving vaccine titer. The remaining B cell epitope peptides have similar effects and conclusions as Pep16 and are not repeated here.

Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Various changes and modifications may be effected therein by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present disclosure, and these changes and modifications are intended to be within the scope of the present disclosure.

Claims (10)

1. The polypeptide vaccine microneedle patch comprises a base and microneedles positioned on the base, and is characterized in that the microneedles are made of a mixture of a polypeptide vaccine and a soluble matrix, the material of the soluble matrix comprises hyaluronic acid and low-molecular-weight polyvinylpyrrolidone, and the mass ratio of the hyaluronic acid to the low-molecular-weight polyvinylpyrrolidone in the soluble matrix is 1-6:20.

2. the polypeptide vaccine microneedle patch according to claim 1, wherein the molecular weight of hyaluronic acid is 3-5WDa, and the molecular weight of low molecular weight polyvinylpyrrolidone is 4-6WDa.

3. The polypeptide vaccine microneedle patch according to claim 1, wherein the polypeptide vaccine comprises a B cell epitope peptide comprising at least one selected from the following a) to c) and an adjuvant:

a) The amino acid sequence is one or more of polypeptides shown in SEQ ID NO.1 to SEQ ID NO. 4;

b) The derivative polypeptide is formed by inserting, substituting or deleting one or more amino acids into the polypeptide shown in the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.4, and has the same or basically the same function as the polypeptide shown in the amino acid sequences of SEQ ID NO.1 to SEQ ID NO. 4;

c) And a multimeric polypeptide formed from one or more of the polypeptides shown in SEQ ID NO.1 to SEQ ID NO. 4.

4. The polypeptide vaccine microneedle patch according to claim 3, wherein the B cell epitope peptide comprises a polypeptide having an amino acid sequence shown in SEQ ID No. 4.

5. The polypeptide vaccine microneedle patch according to claim 3, wherein the B cell epitope peptide comprises a multimeric polypeptide formed by a polypeptide having an amino acid sequence shown in SEQ ID No.1, and the multimeric polypeptide is a homotetramer formed by connecting 4 polypeptides having an amino acid sequence shown in SEQ ID No.1 through 3 lysines.

6. The polypeptide vaccine microneedle patch according to any one of claims 1 to 5, wherein the mass ratio of the polypeptide vaccine to the soluble matrix in the microneedle is 1:100-1000.

7. The polypeptide vaccine microneedle patch according to claim 1, wherein a material of the base includes high molecular weight polyvinylpyrrolidone having a molecular weight of 32-36WDa.

8. The polypeptide vaccine microneedle patch of claim 1The microneedle is characterized in that the whole height of the microneedle is 500-1500 mu m, the diameter of the tip head is 5-20 mu m, and the bottom end size of the needle body is (100-500) x (100-500) mu m ² ；

The size of the base is (1-30) x (1-30) mm ² The thickness is 0.5-3mm.

9. The polypeptide vaccine microneedle patch according to claim 1, wherein a plurality of microneedles are distributed on the base in an array form, the array specification is 1 x 1-20 x 20, and the needle axis distance between two adjacent microneedles is 100-1000 μm.

10. A method for preparing a polypeptide vaccine microneedle patch, which is used for preparing the polypeptide vaccine microneedle patch as claimed in any one of claims 1 to 9, comprising the steps of:

s1, dissolving hyaluronic acid and polyvinylpyrrolidone in ultrapure water to obtain a soluble matrix solution;

s2, adding the polypeptide vaccine into the soluble matrix solution and uniformly mixing to obtain a microneedle solution;

s3, sucking the microneedle solution, placing the microneedle solution in a microneedle mould, performing vacuum decompression treatment, filling the microneedle solution in pinholes of the microneedle mould, removing redundant microneedle solution, and drying at low temperature to obtain a microneedle structure;

and S4, adding a base solution on the surface of the microneedle structure, curing, forming and demolding to obtain the polypeptide vaccine microneedle patch.

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