A polysaccharide conjugate vaccine and its preparation method and application

Abstract

The present invention discloses a polysaccharide conjugate vaccine and a preparation method and application thereof. The polysaccharide conjugate vaccine comprises (a) a polysaccharide; (b) a polypeptide that can bind to a major histocompatibility complex class II molecule; and (c) a plant virus or a bacteriophage; wherein the polysaccharide and the polypeptide are covalently linked to form a polysaccharide-polypeptide conjugate, and the polysaccharide-polypeptide conjugate is covalently bound to the outer surface of the plant virus or the bacteriophage. The polysaccharide conjugate vaccine of the present invention can effectively improve the defect of low immunogenicity of the antigen polysaccharide, and can produce the effect of preventing fungal or bacterial infection after immunizing the body.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a polysaccharide conjugate vaccine and a preparation method and application thereof. Background Art

[0002] Antibiotic resistance (AMR) has become a serious global health problem, which has had a serious impact on society and the economy, and is particularly evident in low-income and lower-middle-income countries and regions. Therefore, how to reduce the use of antibiotics is an urgent issue.

[0003] Vaccination is an important means to reduce the threat of AMR. Vaccination can reduce the spread of microorganisms and infections, which can directly reduce the use of antibiotics; specific vaccines can even accurately prevent bacteria or fungi that have already developed drug resistance, and are not prone to drug resistance, which further reduces the increase of AMR.

[0004] However, as a typical T cell-independent antigen, bacterial and fungal antigen polysaccharides (such as capsular polysaccharides and lipopolysaccharides) cannot induce effective and long-term immune responses, and even have little immunogenicity in infants. This is because polysaccharides cannot bind to the major histocompatibility complex (MHC) class II molecules in antigen presenting cells (APCs) and be presented to CD4 + The T cell receptor (TCR) on the surface of T cells then activates the interaction between T cells and B cells, which makes it impossible to further induce B cells to proliferate and differentiate into plasma cells and memory B cells through the Th2 cytokines (such as IL-4) released by T cells. It can neither form high-affinity IgG antibodies nor quickly respond to subsequent foreign antigens. Ultimately, it can only induce the production of low-affinity IgM antibodies. The lack of proliferation and differentiation process even reduces the number of B cells, leading to a decrease in immune ability. The polysaccharide-protein conjugate vaccine can better overcome this problem. Therefore, the polysaccharide conjugate vaccine is the focus of the next vaccine research and development. Summary of the invention

[0005] In order to solve the problem of low immunogenicity of antigen polysaccharide, the first objective of the present invention is to construct a combined vaccine by using virus-polypeptide-polysaccharide conjugates to improve its immunogenicity.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned combined vaccine.

[0007] The third object of the present invention is to provide a use of the above-mentioned combined vaccine in the preparation of antifungal or antibacterial immune preparations.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a polysaccharide conjugate vaccine, the polysaccharide conjugate vaccine comprising:

[0010] (a) polysaccharides;

[0011] (b) a polypeptide that binds to a major histocompatibility complex class II molecule; and

[0012] (c) plant viruses or bacteriophages;

[0013] The polysaccharide and the polypeptide are covalently linked to form a polysaccharide-polypeptide conjugate, and the polysaccharide-polypeptide conjugate is covalently bound to the outer surface of the plant virus or bacteriophage.

[0014] The polysaccharide conjugate vaccine of the present invention connects polysaccharides, especially antigenic polysaccharides, to the outer surface of viruses (bacteriophages or plant viruses) through polypeptides, thereby improving their immunogenicity. The characteristic of the polypeptide is that it can bind well to MHC class II molecules, thereby ensuring that the polysaccharides connected thereto can be presented to T cells by antigen presenting cells. As nanoparticles, bacteriophages and plant viruses are more likely to enter lymph nodes and be taken up by antigen presenting cells, which can enhance the immunogenicity of polysaccharides.

[0015] In the present invention, optionally, the polysaccharide is an antigenic polysaccharide of fungi or bacteria, and the molecular weight can range from several hundred to several million, including but not limited to antigenic polysaccharides derived from yeast, antigenic polysaccharides derived from cryptococcus, antigenic polysaccharides derived from Staphylococcus aureus, antigenic polysaccharides derived from Streptococcus pneumoniae, or antigenic polysaccharides derived from meningococci, such as β-glucan, β-mannan, glucuronic acid chloromannan, Staphylococcus aureus type 5 capsule polysaccharide, Staphylococcus aureus type 8 capsule polysaccharide, Streptococcus pneumoniae C polysaccharide, meningococcal A polysaccharide, meningococcal C polysaccharide, etc. These antigenic polysaccharides can be extracted from the above-mentioned fungi or bacteria, or synthesized based on the reported structures.

[0016] Optionally, the polypeptide is a polypeptide that can bind well to major histocompatibility complex (MHC) class II molecules, including but not limited to polypeptides having the following amino acid sequences: YGKDVKDLFDYAQE, VFDSFNSLTWSNAA, CKALNPKEIE or FAQLLNKNNEVNSEPE.

[0017] Optionally, the polysaccharide conjugate vaccine design of the present invention also utilizes bacteriophages or plant viruses that are not infectious to animal cells, wherein the plant viruses include but are not limited to tobacco mosaic virus, cowpea mosaic virus, papaya mosaic virus or potato virus; the bacteriophages include but are not limited to T4, M13 or f1 phages.

[0018] In a second aspect, the present invention provides a method for preparing the above-mentioned polysaccharide conjugate vaccine, which comprises: covalently linking a polysaccharide and a polypeptide that can bind to a major histocompatibility complex class II molecule to form a polysaccharide-polypeptide conjugate, and then covalently binding the polysaccharide-polypeptide conjugate to the outer surface of a plant virus or a bacteriophage to form a polysaccharide conjugate vaccine.

[0019] Optionally, in the above preparation method, the covalent connection method for forming the polysaccharide-polypeptide conjugate can use the connection method of polysaccharides and polypeptides known in the prior art, including but not limited to, esterification reaction, Schiff base reaction, amidation reaction, click chemistry reaction or NCA ring-opening reaction, etc.; for example, the hydroxyl group of the polysaccharide and the carboxyl group of the polypeptide are coupled through an EDC / NHS activation reaction; or the aldehyde group of the polysaccharide and the α-amino group of the polypeptide form a CN double bond.

[0020] Optionally, in the above preparation method, the method for covalently binding the polysaccharide-polypeptide conjugate to the outer surface of the plant virus or bacteriophage can also use the method of connecting polypeptides and viruses known in the prior art, including but not limited to esterification reaction, Schiff base reaction, amidation reaction or click chemistry reaction, that is, the polysaccharide-polypeptide conjugate is covalently bound to the outer surface of the plant virus or bacteriophage through esterification reaction, Schiff base reaction, amidation reaction or click chemistry reaction.

[0021] Exemplarily, when the polysaccharide-polypeptide conjugate is covalently bound to the outer surface of the virus through a click chemistry reaction, the outer surface of the plant virus or bacteriophage can be modified with an alkynyl group, an azide group, a maleimide group, a double bond group, a sulfhydryl group or a DBCO group, and one end of the polypeptide can be modified with an alkynyl group, an azide group, a maleimide group, a double bond group, a sulfhydryl group or a DBCO group, and the two are covalently bound through a click chemistry reaction.

[0022] In a third aspect, the present invention also claims to protect the use of the above-mentioned polysaccharide conjugate vaccine in the preparation of antifungal or antibacterial immune preparations.

[0023] Optionally, the immune preparation may or may not contain an adjuvant.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention provides a novel polysaccharide conjugate vaccine, which can effectively improve the defect of low immunogenicity of antigen polysaccharide. The virus polypeptide polysaccharide conjugate vaccine prepared by the present invention can produce IgG antibodies after immunizing the body, producing the effect of preventing fungal or bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0027] Figure 1a A schematic diagram showing the synthesis process of the tobacco mosaic virus polypeptide-polysaccharide conjugate in Example 1.

[0028] Figure 1b The SDS-PAGE gel electrophoresis result image of the tobacco mosaic virus polypeptide-polysaccharide conjugate in Example 1 is shown; the samples from left to right are TMV, TMV-Fba, and TMV-Fba-G.

[0029] Figure 1c The MALDI-TOF-MS mass spectrum of the tobacco mosaic virus polypeptide-polysaccharide conjugate in Example 1 is shown.

[0030] Figure 2a The experimental process of immunizing mice with the samples in Example 2 is shown.

[0031] Figure 2b The figure shows the comparison of IgG levels produced by three blood draws from mice in Example 2, and the dilution ratio during detection was 1:200.

[0032] Figure 2c The figure shows the comparison of IgG levels produced by three blood draws from mice in Example 2, and the dilution ratio during detection was 1:200-1:25600. DETAILED DESCRIPTION

[0033] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments and drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0034] Example 1: Preparation and characterization of tobacco mosaic virus polypeptide polysaccharide (TMV-Fba-G) conjugate vaccine

[0035] (1) Preparation of tobacco mosaic virus (TMV) with alkyne modification on the outer surface. This step was based on the previous literature report (SMITH ML, FITZMAURICE WP, TURPEN TH, et al. Display of Peptides on the Surface of Tobacco Mosaic Virus Particles [M]. Current Topics in Microbiology and Immunology. Springer Berlin Heidelberg. 2009: 13-31.) with slight modifications. Add 381.37 mg of borax and 233.76 mg of sodium chloride to 20 mL of pure water to prepare a 0.05 M borax solution for use; add 247.32 mg of boric acid and 233.76 mg of sodium chloride to 20 mL of pure water to prepare a 0.2 M boric acid solution for use; mix 16 mL of borax solution and 4 mL of boric acid solution, adjust the pH to 8.8-9.0, and prepare a borax boric acid buffer solution for use; prepare p-toluenesulfonic acid in pure water to make a 57 mg / mL solution for use; prepare acetylene aniline in acetonitrile to make a 79 mg / mL solution for use; prepare sodium nitrite to make a 207 mg / mL solution for use; then gently mix 800 μL of p-toluenesulfonic acid solution, 150 μL of acetylene aniline solution and 50 μL of sodium nitrite solution to prepare a pre-synthesized salt; 2.696 mL of H2O, 4 mL of borax boric acid buffer solution, 1 mL TMV (16 mg / mL) solution and 0.304 mL of pre-synthesized salt (35 equivalents in TMV) were mixed and stirred at 4°C for 50 min, and then dialyzed with pure water (the water was changed every 1 h at the beginning of dialysis, and the water was changed 4 times at intervals of 5 min, 10 min, 15 min and 30 min), and the obtained product was TMV modified with alkyne on the outer surface;

[0036] (2) The hydroxyl group of the antigen polysaccharide was coupled to the carboxyl group of the N-terminal azidated Fba polypeptide (sequence: YGKDVKDLFDYAQE) through an EDC / NHS activation reaction. Specifically, 6 mg of yeast-derived laminarin (purchased from Shanghai McLean, catalog number G856759-500 mg-) was dissolved in 0.3 mL of pure water to form a laminarin solution. At the same time, in another reaction bottle, 132 μL of Fba polypeptide aqueous solution (30 mg / mL) (0.33 equivalent to the hydroxyl group of laminarin) was added to 204 μL of pure water, and 8 μL of MES buffer (0.5 M, pH = 5.0) was added. Further, 6.2 mg of EDC (15 times equivalent to the carboxyl group of the Fba polypeptide) and 3.3 mg of NHS (15 times equivalent to the carboxyl group of the Fba polypeptide) were added to activate the carboxyl group. The activated reaction solution was stirred at room temperature for 20 minutes, then added into the laminarin solution, stirred at room temperature, and reacted for another 24 hours. When the reaction was carried out for 6 hours and 12 hours, 6.2 mg EDC and 3.3 mg NHS were added respectively.

[0037] (3) The polysaccharide-polypeptide conjugate is covalently bound to the alkyne group of TMV by click chemistry. The click chemistry of the azide group and the alkyne group is based on previous literature reports (SMITH ML, FITZMAURICE WP, TURPEN TH, et al. Display of Peptides on the Surface of Tobacco Mosaic Virus Particles [M]. Current Topics in Microbiology and Immunology. Springer Berlin Heidelberg. 2009: 13-31.) and slightly modified. The product obtained in step (1) is recorded as solution 1, and the solution obtained in step (2) is recorded as solution 2. 195 μL of water, 100 μL of PBS (100 mM, pH range 7.2-7.4), 455 μL of solution 1 and 200 μL of solution 2 were mixed, and then 10 μL of sodium ascorbate aqueous solution (1 M), 10 μL of aminoguanidine aqueous solution (1 M), and 10 μL of CuSO4 aqueous solution (0.5 M) were added and the mixture was stirred at 4°C for 30 min. After the reaction was completed, 10 μL of ETA / Na (pH ≈ 8, 100 mM) was added and stirred for 1 hour. The obtained TMV-Fba-G conjugate was purified by dialyzing in deionized water through a dialysis bag (MWCO = 1,000,000 Da) in deionized water to obtain.

[0038] Take 20 μL of the conjugate vaccine sample prepared above, add 5 μL of protein loading buffer (5×), boil for 10 minutes, load the sample on the gel, and then stain with Coomassie Brilliant Blue. Since the molecular weight of the polysaccharide used is not single, the observation that the sample band of the TMV-linked polysaccharide-peptide conjugate is wider than the band of the TMV-linked polypeptide sample can prove that the connection is successful, such as Figure 1b .

[0039] Take 10 μL of sample and perform MALDI-TOF-MS detection. It is also found that the molecular peak becomes wider and shifts to the right, which proves that the connection is successful. Figure 1c .

[0040] Example 2: Vaccination of mice and detection of antibody levels

[0041] Three balb / c female mice aged 6-8 weeks were purchased. They were kept for at least one week after purchase and observed to be in good condition before immunization. During immunization, 50 μL (0.9 mg / mL) of the TMV-Fba-G combined vaccine solution prepared in Example 1 was injected subcutaneously (the site was the skin depression that appeared after the ears of the mice were lifted). The second and third immunizations were performed 14 days and 29 days after the first immunization, respectively. 100 μL of blood was collected 1 day before the first immunization, 7 days after the second immunization, and 7 days after the third immunization. Figure 2a After blood was collected, it was left to stand at room temperature for 1-2 hours, then centrifuged at 4000r for 10 minutes, and serum was collected and frozen at -80℃. After the serum was diluted in a ratio of 1:200-1:25600, ELISA was performed, and it was found that the mice produced IgG antibodies corresponding to the antigen polysaccharide, such as Figure 2b and 2c .

[0042] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A polysaccharide conjugate vaccine, characterized in that: The polysaccharide conjugate vaccine comprises: (a) Polysaccharides; (b) a polypeptide that binds to a major histocompatibility complex class II molecule; and (c) plant viruses; The polysaccharide and the polypeptide are covalently linked to form a polysaccharide-polypeptide conjugate, and the polysaccharide-polypeptide conjugate is covalently bound to the outer surface of the plant virus; The polysaccharide is laminarin derived from yeast; The amino acid sequence of the polypeptide is YGKDVKDLFDYAQE; The plant virus is tobacco mosaic virus.

2. The method for preparing the polysaccharide conjugate vaccine according to claim 1, characterized in that: The preparation method comprises: covalently linking a polysaccharide and a polypeptide that can bind to a major histocompatibility complex class II molecule to form a polysaccharide-polypeptide conjugate, and then covalently binding the polysaccharide-polypeptide conjugate to the outer surface of a plant virus to form a polysaccharide conjugate vaccine.

3. The preparation method according to claim 2, characterized in that: The covalent linking method for forming the polysaccharide-polypeptide conjugate is selected from esterification reaction, Schiff base reaction, amidation reaction, click chemistry reaction or NCA ring-opening reaction.

4. The preparation method according to claim 2, characterized in that: The method for covalently binding the polysaccharide-polypeptide conjugate to the outer surface of the plant virus is selected from esterification reaction, Schiff base reaction, amidation reaction or click chemistry reaction.

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