CN116942835A - Composite and use thereof - Google Patents

Composite and use thereof Download PDF

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Publication number
CN116942835A
CN116942835A CN202310517596.1A CN202310517596A CN116942835A CN 116942835 A CN116942835 A CN 116942835A CN 202310517596 A CN202310517596 A CN 202310517596A CN 116942835 A CN116942835 A CN 116942835A
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opc
acid
vaccine
complex
compound
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任晋
孙芸
邵帅
张欣明
李媛媛
韩子泊
张靖
康子瑶
李媛
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China National Biotec Research Institute Co ltd
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China National Biotec Research Institute Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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Abstract

The invention discloses a complex and application thereof, wherein the complex is obtained by connecting a polyphenol compound, a conjugate formed by metal ions and the polyphenol compound or a conjugate formed by nonmetal ions and the polyphenol compound with a protein with immunogenicity through a non-covalent bond. The novel protein delivery system can replace vaccine adjuvant, plays an equivalent or better effect with the adjuvant in vaccine preparation, particularly can activate cellular immune response, thereby further enhancing the efficacy of the vaccine, has simple preparation process, and effectively solves the inherent defects of the vaccine adjuvant in the current market.

Description

Composite and use thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a compound and application thereof.
Background
Vaccination is a major approach for humans against a wide variety of pathogenic microorganisms. The subunit vaccine is a vaccine which is prepared by utilizing a certain surface structural component (antigen) of microorganism, does not contain nucleic acid, can induce organisms to generate antibodies, has definite antigen component and good safety, but needs to be added with an adjuvant to enhance the vaccine effect. Currently, the only adjuvants approved for sale by the U.S. FDA are aluminum adjuvants, MF-59, AS03, AS04, AS01, and CPG 1018. Among them, one third of subunit vaccines are all using aluminum adjuvants, but no proprietary intellectual property adjuvants are currently applied clinically in China. Although aluminum adjuvant is the most widely used adjuvant in clinical practice at present, there are still many problems to be solved, such as the failure of aluminum adjuvant to cause Th1 and killer CD8 + T reaction, freeze thawing and the like cannot be performed in the preparation process. Therefore, searching for a novel vaccine preparation which is safe, efficient and capable of inducing strong cellular immune response is beneficial to fundamentally solving the problem of the adjuvant, namely the neck of the vaccine industry.
Disclosure of Invention
Technical problem to be solved：
One aspect of the invention is directed to the presence of vaccine adjuvants of the prior art, e.g., not capable of eliciting Th1 and killer CD8 + T reaction, can not freeze thawing in the course of preparation, has provided a kind of complex and its use.
In particular, the present inventors have unexpectedly found that a complex can replace a vaccine adjuvant, exert an effect equivalent to or better than that of an adjuvant in a vaccine preparation, and in particular, can activate a cellular immune response, thereby further enhancing the efficacy of a vaccine, and that the preparation process of the complex is simple, effectively solving inherent defects of vaccine adjuvants currently on the market.
The technical proposal provided by the invention：
A complex, characterized in that the complex is obtained by linking a polyphenol compound, a conjugate of a metal ion and a polyphenol compound, or a conjugate of a nonmetallic ion and a polyphenol compound, with a protein having immunogenicity through a non-covalent bond.
In the present invention, the assembly of the metal ion with the polyphenol compound, or the nonmetal ion with the polyphenol compound is mainly based on the pH dependence of the coordination bond between the catechol group and the metal or nonmetal ion as a crosslinking unit. The catechol or galloyl groups present in phenolic compounds provide binding sites for the chelation of metal or non-metal ions, thereby forming highly stable complexes. At a certain pH, the nature of the complex depends mainly on the concentration of the polyphenol and its ionization degree.
In certain preparation embodiments of the present invention, methods of assembling the above metal ions with a polyphenol compound, or the nonmetallic ions with a polyphenol compound, are provided. For example, the polyphenol is dissolved into a clear aqueous solution by a suitable method, and then the substance containing the metal or nonmetal is dissolved into a clear aqueous solution as well, and the two are mixed according to a molar ratio and reacted for 30 to 40 minutes at room temperature. Preferably, in certain embodiments of the present invention, the molar ratio of the metal ion to the polyphenol compound is from 0.5 to 2:1, and the molar ratio of the non-metal ion to the polyphenol compound is from 0.5 to 2:1. The molar ratio may be, for example, 0.5:1, 0.7:1, 0.9:1, 1.1:1, 1.3:1, 1.5:1, 1.7:1, 1.9:1 or 2:1. More preferably, in one embodiment of the present invention, the molar ratio of the metal ion to the polyphenol compound is 1:1, and the molar ratio of the nonmetallic ion to the polyphenol compound is 1:1.
In the present invention, the metal ion may be any suitable metal ion or ions. For example, the metal ion may be selected from Sn 2+ 、Al 3+ 、Cu 2+ 、Fe 3+ 、Mn 2+ 、Zn 2+ 、Co 2+ 、Ti 4+ 、Ag + 、Ni 2+ Or Mg (Mg) 2+ At least one of them. However, preferably, in one embodiment of the present invention, the metal ion is Mn 2+ 。
The metal ions described above may be present in any suitable manner, including, but not limited to, ionic salt forms, for example. In certain embodiments of the invention, the ionic salt may be, for example, mnSO 4 ·H 2 O。
In the present invention, the nonmetallic ion may be any suitable nonmetallic ion or ions. Preferably, however, in one embodiment of the present invention, the nonmetallic ion is a boron ion.
The nonmetallic ions described above may be present in any suitable manner. Preferably, in certain embodiments of the invention, the boron ions originate from a boric acid group. More preferably, in certain embodiments of the present invention, the boric acid group is a phenylboric acid group as shown in formula I or a pyridineboric acid group as shown in formula II:
formula I and formula II
Further preferably, in certain embodiments of the present invention, the boric acid group is a phenylboric acid group as shown in formula I.
Further preferably, in certain embodiments of the present invention, the phenylboronic acid group is derived from at least one of the phenylboronic acid groups of aliphatic chain substitution, benzene ring substitution, amide substitution, fluorine atom substitution, halogen atom substitution, amino substitution, carboxyl substitution, aldehyde substitution. More preferably, in certain embodiments of the present invention, the phenylboronic acid group is derived from at least one of 3-acrylamidophenylboronic acid, 4-bromomethylphenylboronic acid, 4-carboxy-3-fluorophenylboronic acid, 3-acyl-4-methylphenylboronic acid, 4-aminophenylboronic acid, or fluoro-substituted phenylboronic acid;
further preferably, in certain embodiments of the present invention, the phenylboronic acid groups are derived from 3-acrylamidophenylboronic acid and/or 4-bromomethylphenylboronic acid.
In the present invention, the above-mentioned polyphenols may be compounds comprising at least two phenol groups, which may be associated in a more or less complex structure, which generally have a high molecular weight. For example, but not limited to, flavonoid polyphenols such as epigallocatechin, 3-gallate, epicatechin 3-gallate, baicalin, douglas fir extract, quercetin, hesperetin, or genistein; or non-flavonoid polyphenols such as resveratrol and gossypol. In certain embodiments of the invention, the polyphenol is at least one selected from catechin, epicatechin, tannic acid, gallic acid, protocatechuic aldehyde, or tannic acid, or a polymer or derivative of the foregoing. The polymers or derivatives of the above compounds may be prepared using suitable methods, which generally have the same or similar biological functions as the above compounds. Preferably, in one embodiment of the present invention, the polyphenol is Oligomeric Procyanidin (OPC).
In certain preparation embodiments of the present invention, there is provided a method of complexing a conjugate of the above polyphenol compound, metal ion and polyphenol compound, or a conjugate of a nonmetallic ion and polyphenol compound with a protein having immunogenicity through a non-covalent bond. For example, the above-mentioned polyphenol compound, metal ion and polyphenol compound, or a reaction mixture of the above-mentioned nonmetallic ion and polyphenol compound is added with a certain proportion of the above-mentioned protein having immunogenicity, and reacted at room temperature to obtain the above-mentioned complex. Preferably, in certain embodiments of the present invention, the mass ratio of the polyphenol compound, the conjugate of a metal ion and the polyphenol compound, or the conjugate of a nonmetal ion and the polyphenol compound is 1 to 100:1 by non-covalent bond to the protein having immunogenicity. More preferably, in certain embodiments of the invention, the mass ratio is from 2 to 20:1, for example, 2:1,5:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1.
The composite obtained by the scheme of the invention forms uniform composite particles. The particle size of the composite particles is 140.9 nm-3514.7 nm.
In another aspect of the present invention, there is provided a vaccine formulation comprising the complex as described above and a pharmaceutically acceptable carrier. In the present invention, the above pharmaceutically acceptable carrier may be any pharmaceutically acceptable additive, including, but not limited to, physiological saline, solvents, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, surfactants, preservatives, isotonic agents, absorption delaying agents, and the like, which are compatible with pharmaceutical administration.
As an innovative point of the present invention, the vaccine formulation does not need to contain an adjuvant. Through certain experiments described in the invention, the vaccine preparation can achieve or be better than the equivalent vaccine preparation containing the adjuvant.
Preferably, the vaccine preparation may be any subunit vaccine. More preferably, the subunit vaccine may be a novel coronavirus subunit vaccine, an influenza subunit vaccine, a respiratory syncytial virus subunit vaccine or a rabies subunit vaccine.
The invention has the beneficial effects that:
the novel protein delivery system can replace vaccine adjuvant, plays an equivalent or better effect with the adjuvant in vaccine preparation, particularly can activate cellular immune response, thereby further enhancing the efficacy of the vaccine, has simple preparation process, and effectively solves the inherent defects of the vaccine adjuvant in the current market.
Drawings
FIG. 1 is a graph showing the results of particle size measurements of an antigen complex of an OPC-novel crown subunit vaccine with different mass ratios in the examples of the present invention;
FIG. 2 is a graph showing the results of particle size measurements of antigen complexes of OPC-Mn-novel crown subunit vaccines with different mass ratios in the examples of the present invention;
FIG. 3 is a graph showing the results of particle size measurements of different mass ratios of OPC-3-acrylamidophenylboronic acid-neocoronal subunit vaccine antigen complexes in accordance with the present invention;
FIG. 4 is a graph showing the results of particle size measurements of different mass ratios of OPC-4-bromomethylphenylboronic acid-neocoronal subunit vaccine antigen complexes in accordance with the present invention;
FIG. 5 is a graph showing the results of particle size measurements of antigen complexes of different mass ratios of OPC-influenza subunit vaccines in an embodiment of the invention;
FIG. 6 is a graph showing the results of particle size measurements of an OPC-influenza subunit vaccine antigen complex at different mass ratios (1:1-10:1) in an example of the present invention;
FIG. 7 is a graph showing the results of particle size measurements of antigen complexes of different mass ratios of OPC-Mn-influenza subunit vaccines in examples of the present invention;
FIG. 8 is a graph showing the results of particle size stability measurements of an OPC/OPC-Mn-influenza subunit vaccine antigen complex in an example of the present invention;
FIG. 9 is a graph showing the results of particle size measurements of different mass ratios of OPC-RPE protein complexes in examples of the invention;
FIG. 10 is a graph showing the results of particle size measurements of OPC-Mn-RPE protein complexes of different mass ratios in the examples of the present invention;
FIG. 11 is a graph showing the transmission electron microscope results of an OPC-3-acrylamidophenylboronic acid-novel crown vaccine composition in an embodiment of the present invention;
FIG. 12 is a graph showing the transmission electron microscope results of an OPC/OPC-Mn-influenza vaccine complex in an embodiment of the invention;
FIG. 13 is a graph showing the result of cell transfection of OPC/OPC-Mn-RPE protein complexes in examples of the present invention;
FIG. 14 is a graph showing cytotoxicity results of OPC in an embodiment of the invention;
FIG. 15 is a graph showing cytotoxicity results of OPC-Mn in examples of the present invention;
FIG. 16 is a graph showing the results of three immunization of the OPC-neocrown vaccine antigen protein complex with antibodies (left in FIG. 16) and neutralizing antibodies (right in FIG. 16) in the examples of the present invention;
FIG. 17 is a graph showing the results of antibody levels after immunization of an OPC-Mn-novel crown vaccine antigen protein complex in the examples of the present invention;
FIG. 18 is a graph showing the results of cellular immunity levels of an OPC-Mn-novel crown vaccine antigen protein complex in an embodiment of the invention;
FIG. 19 is a graph showing the results of antibody levels after immunization of an OPC-3-acrylamidophenylboronic acid-neocoronal vaccine antigen protein complex in an embodiment of the present invention;
FIG. 20 is a graph showing the results of cellular immunity levels after immunization of an OPC-3-acrylamidophenylboronic acid-neocoronal vaccine antigen protein complex in an embodiment of the present invention;
FIG. 21 is a graph showing the results of antibody levels after immunization of an OPC-4-bromomethylbenzoborate-novel crown vaccine antigen protein complex in an example of the present invention;
FIG. 22 is a graph showing the results of cellular immunity levels after immunization of an OPC-4-bromomethylphenylboronic acid-neocoronal vaccine antigen protein complex in an embodiment of the present invention;
FIG. 23 is a graph showing the results of sequential vaccination of an OPC/OPC-Mn-novel crown vaccine antigen protein complex with mouse antibody levels in an example of the present invention;
FIG. 24 is a graph showing the results of sequential vaccination of mice with OPC/OPC-Mn-novel crown vaccine antigen protein complexes in accordance with an embodiment of the invention;
FIG. 25 is a graph showing the results of weight change in mice injected with an OPC-based delivery system in accordance with an embodiment of the present invention;
FIG. 26 is a graph showing the results of measuring the levels of inflammatory factors in plasma of mice after immunization in the examples of the present invention.
Detailed Description
The invention discloses application of a compound in preparing vaccine preparations, and a person skilled in the art can properly improve process parameters by referring to the content of the compound. It is to be particularly pointed out that all similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention and that the relevant person can make modifications and appropriate alterations and combinations of what is described herein to make and use the technology without departing from the spirit and scope of the invention.
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components. The terms "such as," "for example," and the like are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
Definition:
the term "compound" refers to a product made from two or more different components and which provides a design that is superior to the properties of the starting materials.
The terms "vaccine formulation", "vaccine" refer to any formulation comprising an antigenic component. Subunit vaccine formulations typically contain an adjuvant. The vaccine formulation may also comprise excipients or vehicles suitable for pharmaceutical use, or any compound having immunomodulatory activity such as cytokines, ligand compounds for Toll receptors, and the like.
The term "adjuvant" refers to a compound that, upon administration in combination with an antigen, can augment, enhance, and/or promote an immune response to the antigen, but that does not produce an immune response to the antigen when the adjuvant compound is administered alone.
The term "procyanidins" (PC) is polymerized from different numbers of monomers (catechins or epicatechins) as shown in formulas III-VI below. According to the degree of polymerization, it is divided into Oligomeric Procyanidins (OPC) polymerized from 2-4 monomers and Polymeric Procyanidins (PPC) polymerized from more than 5 monomers. Researches show that the oligomeric proanthocyanidin has the advantages of high bioavailability, quick oral absorption, long in-vivo residence time and the like in human bodies, so that the oligomeric proanthocyanidin has strong biological activity.
Procyanidins are widely found in fruits, leaves, seeds, and peels of a variety of plants, with procyanidins content in grape seeds being as high as 95%. Procyanidins have pharmacological activities of resisting oxidation, myocardial ischemia reperfusion injury, atherosclerosis, protecting vascular endothelial cells, resisting cancer, lowering blood pressure, reducing blood lipid, lowering blood sugar and the like. At present, procyanidine is widely applied to the fields of food addition, medical health care and cosmetics due to extremely strong antioxidant activity and free radical scavenging capability, and the application in the field of vaccines is not reported yet. The procyanidine has resorcinol and phloroglucinol structural units in the structure, and can be complexed with metal ions to form a complex.
The term "pharmaceutically acceptable" refers to the non-toxic nature of a substance that does not interact with the action of the active components of the pharmaceutical composition.
The term "derivative" refers to a compound having a structure derived from the parent compound and having a structure substantially similar to those disclosed herein, and based on that similarity, one skilled in the art will expect to exhibit the same or similar activity and use as the claimed compound, or to induce the same or similar activity and use as the claimed compound as a precursor.
In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail with reference to specific embodiments.
Examples:
example 1: preparation of OPC-New coronal subvaccine antigen Complex
OPC was solublized with a trace of DMSO and diluted with sterile, sterile water to a clear solution. And (3) absorbing different volumes of antigen protein stock solutions according to the mass ratio (2:1, 5:1,10:1,20:1, 40:1) of OPC and antigen proteins, mixing with an OPC solution, and reacting for 1h at room temperature to obtain OPC-new crown vaccine complexes with different proportions.
Example 2: preparation of OPC-Mn-New crown subunit vaccine antigen Complex
Dissolving OPC with trace DMSO, and diluting with sterile and asepsis water to obtain clear solution; mnSO 4 ·H 2 O was dissolved with sterile, water. The corresponding volumes are sucked according to the molar ratio of 1:1, and the mixture is mixed and reacted for 30min. Absorbing different volumes of antigen protein stock solution according to the mass ratio of OPC to antigen protein (2:1, 5:1,10:1,20:1, 40:1), and adding OPC and MnSO 4 ·H 2 And (3) in the mixed solution of O, reacting for 1h at room temperature to obtain OPC-Mn-new crown vaccine complexes with different proportions.
Example 3: OPC-3-acrylamidophenylboronic acid/4-bromomethylphenylboronic acid-novel crown subunit vaccine antigen complex Preparation of the article
Dissolving OPC with trace DMSO, and diluting with sterile and asepsis water to obtain clear solution; 3-acrylamidophenylboronic acid/4-bromomethyl phenylboronic acid is dissolved with a little absolute ethyl alcohol, and diluted into a clear solution with sterile and enzyme-free water. The corresponding volumes are sucked according to the molar ratio of 1:1, and the mixture is mixed and reacted for 30min. Absorbing different volumes of antigen protein stock solutions according to the mass ratio (1:1, 2:1,5:1,10:1, 20:1) of OPC and antigen proteins, adding the stock solutions into the mixed solution of OPC and phenylboronic acid compounds, and reacting for 1h at room temperature to obtain OPC-phenylboronic acid-new crown vaccine complexes with different proportions.
Example 4: preparation of OPC-influenza subunit vaccine antigen complexes
OPC was solublized with a trace of DMSO and diluted with sterile, sterile water to a clear solution. And absorbing different volumes of influenza antigen protein stock solutions according to the mass ratio of OPC to antigen protein (1:1, 2:1,5:1,10:1,20:1,40:1,60:1,80:1, 100:1), mixing with an OPC solution, and reacting for 1h at room temperature to obtain OPC-influenza vaccine complexes with different proportions.
Example 5: preparation of OPC-Mn-influenza sub-vaccine antigen complex
Dissolving OPC with trace DMSO, and diluting with sterile and asepsis water to obtain clear solution; mnSO 4 ·H 2 O was dissolved with sterile, water. The corresponding volumes are sucked according to the molar ratio of 1:1, and the mixture is reacted for 30min at room temperature. Absorbing antigen protein stock solutions with different volumes according to the mass ratio (1:1, 2:1,5:1, 10:1) of OPC and antigen protein, and adding OPC and MnSO 4 ·H 2 And (3) in the mixed solution of O, reacting for 1h at room temperature to obtain the OPC-Mn-influenza vaccine compound with different proportions.
Example 6: preparation of OPC/OPC-Mn-model protein (RPE) complexes
OPC-RPE complexes were prepared using the procedure of example 1 at the mass ratio of OPC to RPE (2:1, 5:1,10:1, 20:1).
OPC-Mn-RPE composites were prepared using the method of example 2 at mass ratios of OPC to RPEOPC to RPE (2:1, 5:1,10:1, 20:1).
Experimental example 1: particle size analysis of OPC-New crown subunit vaccine antigen Complex
Particle size of the OPC-new crown subunit vaccine antigen complex prepared in example 1 was measured using a Dynamic Light Scattering (DLS) nanolaser particle sizer. The detection instrument was Zetasizer Nano ZS and the detection temperature was 25℃and each sample was repeated three times, and the detection results are shown in FIG. 1. From the results, it can be seen that different proportions of OPC adsorb the novel coronal subunit vaccine antigen proteins to form uniform complex particles. Wherein the composite particle size is the smallest and the uniformity is the best when the mass ratio is 10:1.
Experimental example 2: particle size analysis of OPC-Mn-New crown subunit vaccine antigen Complex
Particle size of the OPC-Mn-new coronal subunit vaccine antigen complex prepared in example 2 was measured using a Dynamic Light Scattering (DLS) nanolaser particle sizer. The detection instrument was Zetasizer Nano ZS and the detection temperature was 25℃and each sample was repeated three times, and the detection results are shown in FIG. 2. From the results, it can be seen that different proportions of OPC-Mn can adsorb the novel coronal subunit vaccine antigen protein to form uniform complex particles.
Experimental example 3: particle size analysis of OPC-3-acrylamidophenylboronic acid-novel crown subunit vaccine antigen complex
The particle size of the OPC-3-acrylamidophenylboronic acid-neocrown subunit vaccine antigen complex prepared in example 3 was measured using a Dynamic Light Scattering (DLS) nano laser particle sizer. The detection instrument was Zetasizer Nano ZS and the detection temperature was 25℃and each sample was repeated three times, and the detection results are shown in FIG. 3. From the results, it can be seen that different proportions of OPC-3-acrylamidophenylboronic acid can adsorb the antigen protein of the novel crown subunit vaccine to form uniform composite particles. Wherein, the particle size of the compound is reduced to about 200nm when the mass ratio is 10:1 and 20:1, and the particle size is minimum when the mass ratio is 20:1, namely 197nm.
Experimental example 4: particle size analysis of OPC-4-bromomethylphenylboronic acid-novel crown subunit vaccine antigen complex
The particle size of the OPC-4-bromomethylbenzoic acid-neocrown subunit vaccine antigen complex prepared in example 3 was measured using a Dynamic Light Scattering (DLS) nanolaser particle sizer. The detection instrument was Zetasizer Nano ZS and the detection temperature was 25℃and each sample was repeated three times, and the detection results are shown in FIG. 4. From the results, it can be seen that different proportions of OPC-4-bromomethylphenylboronic acid can adsorb the antigen protein of the novel crown subunit vaccine to form uniform composite particles. Wherein, the particle size of the compound is reduced to nanometer level when the mass ratio is 10:1 and 20:1, and the particle size is minimum when the mass ratio is 10:1, and is 252nm.
Experimental example 5: particle size analysis of OPC-influenza subunit vaccine antigen complexes
Particle size of the OPC-influenza subunit vaccine antigen complex prepared in example 4 was measured using a Dynamic Light Scattering (DLS) nanolaser particle sizer. The detection instrument was Zetasizer Nano ZS and the detection temperature was 25℃and each sample was repeated three times, and the detection results are shown in FIG. 5. When the OPC loads influenza antigen protein, the particle size of the compound is greatly increased after the decrease of the OPC mass ratio, wherein the particle size is relatively smaller at 5:1 and 10:1. Further verification of particle size results from 1:1 to 10:1 shows (as shown in FIG. 6), that the mass ratio is about 200nm when the particle size is 2:1 and 10:1, the particles are uniform, and the PDI is less than 0.3.
Experimental example 6: particle size analysis of OPC-Mn-influenza subunit vaccine antigen complexes
Particle size of the OPC-Mn-influenza subunit vaccine antigen complex prepared in example 5 was measured using a Dynamic Light Scattering (DLS) nanolaser particle sizer. The detection instrument was Zetasizer Nano ZS and the detection temperature was 25℃and each sample was repeated three times, and the detection results are shown in FIG. 7. OPC and MnSO 4 The particle size of the protein complex is improved after coordination, wherein the particle size of the OPC-Mn-influenza vaccine complex is kept within 200nm at three mass ratios of 2:1,5:1 and 10:1.
Experimental example 7: particle size stability analysis of OPC/OPC-Mn-influenza subunit vaccine antigen complexes
The OPC-based delivery system/influenza vaccine complex prepared in example 4 and example 5 was placed at 4 ℃ and its particle size was measured for three consecutive days to determine the stability of the formulation, and the measurement results are shown in fig. 8. Within three days, the particle size of the preparation is not obviously increased, which indicates that the particles are not obviously aggregated and the system is stable.
Experimental example 8: particle size analysis of OPC/OPC-Mn-model protein (RPE) complexes
The particle size of the OPC/OPC-Mn-model protein (RPE) complex prepared in example 6 was measured using a Dynamic Light Scattering (DLS) nanolaser particle sizer. The detection instrument is Zetasizer Nano ZS, the detection temperature is 25 ℃, each sample is repeated three times, and the detection results are shown in fig. 9 and 10. OPC loads RPE protein, can form stable nano particles, and has good uniformity. Adding MnSO 4 After that, OPC coordinates with Mn, and then protein is adsorbed, the particle size is changed, and the mass ratio is 2:1, the particle size was the smallest, 141nm.
Experimental example 9: morphological characterization of OPC-3-acrylamidophenylboronic acid-novel crown subunit vaccine antigen complex
OPC-3-acrylamidophenylboronic acid-neocrown vaccine complexes prepared using example 3 at 5:1,10:1,20:1 mass ratio of OPC to protein. The specific operation method is as follows: and (3) carrying out ultrafiltration concentration on the prepared compound solution, dripping 10 mu L of the compound solution on a copper mesh, sucking the solution to dryness by using filter paper after 3min, dripping 10 mu L of 2% phosphotungstic acid solution on the copper mesh, sucking the solution to dryness by using filter paper after 30s, and photographing under a Hitachi H-7650 electron microscope after the solution is dried. As shown in FIG. 11, at different mass ratios, OPC-3-acrylamidophenylboronic acid was able to form spherical nanoparticles after binding to the novel crown subunit vaccine antigen protein.
Experimental example 10: morphological characterization of OPC/OPC-Mn-influenza subunit vaccine antigen complexes
OPC-influenza vaccine complexes and OPC-Mn-influenza vaccine complexes prepared in example 4 and example 5 were used with mass ratios of OPC to antigenic protein of 2:1,5:1, 10:1. The specific operation method is as follows: and (3) carrying out ultrafiltration concentration on the prepared compound solution, dripping 10 mu L of the compound solution on a copper mesh, sucking the solution to dryness by using filter paper after 3min, dripping 10 mu L of 2% phosphotungstic acid solution on the copper mesh, sucking the solution to dryness by using filter paper after 30s, and photographing under a Hitachi H-7650 electron microscope after the solution is dried. The results are shown in FIG. 12, where OPC and OPC-Mn bind to influenza antigen proteins at different mass ratios and are capable of forming spherical nanoparticles.
Experimental example 11: determination of cell transfection efficiency of OPC/OPC-Mn-RPE protein Complex
(1) Cell transfection efficiency of OPC-RPE protein Complex
OPC was solublized with a trace of DMSO and diluted with sterile, sterile water to a clear solution. Different volumes of RPE stock solution are sucked according to different mass ratios of OPC and RPE proteins, and are uniformly mixed and reacted for 1h at room temperature. HeLa cells were plated in 24 well plates, 2.0X10 per well 5 The transfection experiment can be carried out when the cell fusion degree is 60-90% after the cells are incubated for 18-24 hours in an incubator. The original complete medium was replaced with 500. Mu.L of DMEM medium without serum and double antibodies, incubated for 2h, and the prepared OPC-RPE complex was then added to 24 well plates at 2. Mu.g of RPE per well for transfection. After culturing the transfected cells in an incubator for 4 hours, fluorescence is generatedThe results were observed under a microscope. Cells were digested and collected after PBS wash for flow detection. The results are shown in FIG. 13.
(2) Cell transfection efficiency of OPC-Mn-RPE protein Complex
Dissolving OPC with trace DMSO, and diluting with sterile and asepsis water to obtain clear solution; mnSO 4 ·H 2 O was dissolved with sterile, water. The corresponding volumes are sucked according to the molar ratio of 1:1, and the mixture is mixed and reacted for 30min. Sucking RPE stock solution with different volumes according to different mass ratios of OPC and RPE, adding OPC and MnSO 4 ·H 2 And (3) in the O mixed solution, reacting for 1h at room temperature.
HeLa cells were plated in 24 well plates, 2.0X10 per well 5 And (3) incubating the cells in an incubator for 18-24 hours, and carrying out transfection experiments until the cell fusion degree is 60-90%. The original complete medium is replaced by 500 mu L of DMEM medium replacement liquid without serum and double antibodies, and after incubation for 2 hours, the prepared OPC-Mn-RPE compound is added into a 24-well plate for transfection according to 2 mu g R-PE per well. After the transfected cells were cultured in an incubator for 4 hours, the results were observed by a fluorescence microscope. Cells were digested and collected after PBS wash for flow detection. The results are shown in FIG. 13.
From the transfection results, both OPC-RPE and OPC-Mn-RPE complexes can enter cells, wherein the OPC-wrapping complex has lower capability of entering cells, the transfection capability is improved after Mn is added, and the transfection efficiency is highest when the mass ratio is 2:1.
Experimental example 12: cytotoxicity of OPC and OPC-Mn delivery systems
(1) Cytotoxicity of OPC
OPC was reconstituted with trace DMSO, diluted with sterile, sterile water to a clear solution, and finally diluted with serum-and diabody-free DMEM basal medium. HeLa cells were plated in 96-well plates 1.0X10 s per well 5 And (4) incubating the cells in the incubator for 18-24 hours until the cell fusion degree is 90%, and performing subsequent operation.
Toxicity for 4 h: the prepared OPC solution was added to a 96-well plate with 100. Mu.L of each well, and 5 complex wells were set per proportion. A cell-free blank control group and a simple cell control group are simultaneously set. Incubation was performed for 4h. CCK-8 detection solution is prepared, the solution is changed into a 96-well plate, 100 mu L of each well is incubated for 30min-1h, and the absorbance is measured by a enzyme-labeled instrument 492nm, and the result is shown in FIG. 14.
Toxicity for 24 h: the prepared OPC solution was added to a 96-well plate with 100. Mu.L of each well, and 5 complex wells were set per proportion. A cell-free blank control group and a simple cell control group are simultaneously set. After 4h incubation, 10 μl FBS was added to each well for a further 20h incubation. CCK-8 detection solution is prepared, the solution is changed into a 96-well plate, 100 mu L of each well is incubated for 30min-1h, and the absorbance is measured by a enzyme-labeled instrument 492nm, and the result is shown in FIG. 14. As can be seen from the graph, the cell viability gradually decreased with increasing OPC ratio.
(2) Cytotoxicity of OPC-Mn delivery systems
OPC was solublized with a trace of DMSO and diluted with sterile, sterile water to a clear solution. MnSO 4 ·H 2 O sterile, aqueous enzymatic dissolution. The corresponding volumes are sucked according to the molar ratio of 1:1, and the mixture is mixed and reacted for 30min. Sucking RPE stock solution with different volumes according to different mass ratios of OPC and RPE, adding OPC and MnSO 4 ·H 2 And (3) in the O mixed solution, reacting for 1h at room temperature. Finally, the culture medium is diluted with DMEM basal medium without serum and double antibodies. Hela/Vero/293T cells were plated in 96-well plates 1.0X10 s per well 5 And (4) incubating the cells in the incubator for 18-24 hours until the cell fusion degree is 90%, and performing subsequent operation.
Toxicity for 4 h: the prepared solution was added to a 96-well plate at 100. Mu.L per well, with 5 multiplex wells per proportion. A cell-free blank control group and a simple cell control group are simultaneously set. Incubation was performed for 4h. Preparing CCK-8 detection liquid, changing the liquid into 96-well plates, and incubating for 30min-1h with a microplate reader 492nm to measure absorbance.
Toxicity for 24 h: the prepared solution was added to a 96-well plate at 100. Mu.L per well, with 5 multiplex wells per proportion. A cell-free blank control group and a simple cell control group are simultaneously set. After 4h incubation, 10 μl FBS was added to each well for a further 20h incubation. CCK-8 detection solution is prepared, the solution is changed into a 96-well plate, 100 mu L of each well is incubated for 30min-1h, and the absorbance is measured by a enzyme-labeled instrument 492nm, and the result is shown in FIG. 15. It can be seen from the figure that the OPC-Mn delivery system is less toxic to three cells at low mass ratios.
Experimental example 13: evaluation of in vivo delivery Effect of OPC-New coronal vaccine antigen protein Complex
Using the preparation method in example 1, OPC-neocrown vaccine complexes were prepared at OPC to neocrown antigen protein mass ratios of 5:1,10:1 and 20:1. Mice were given intramuscular injection of the posterior calf shank anterior muscle, 0.1 mL/mouse, immunized at 0d,21d,42d, respectively. The mice were bled from the orbit 14 days after each immunization, serum was isolated and collected, and antibody levels were determined by ELISA (results are shown in fig. 16). From the results, OPC is able to load new coronal vaccine antigen proteins to produce a stronger antibody response in vivo, resulting in no significant difference compared to the aluminum hydroxide adjuvant control.
Experimental example 14: evaluation of in vivo delivery Effect of OPC-Mn-New coronal vaccine antigen protein Complex
Using the preparation method in example 2, OPC-Mn-neocrown vaccine complexes were prepared at OPC to neocrown antigen protein mass ratios of 5:1,10:1 and 20:1. Mice were given intramuscular injection of the posterior calf shank anterior muscle, 0.1 mL/mouse, immunized at 0d,21d,42d, respectively. The mice were bled from the orbit 14 days after each immunization, serum was isolated and collected, antibody levels were determined by ELISA (results shown in FIG. 17), and cellular immunity levels were determined by ELISPOT (results shown in FIG. 18). From the results, it can be seen that different proportions of OPC-Mn-neocrown vaccine are capable of eliciting strong humoral immune responses, with no significant difference in antibody levels compared to the aluminium hydroxide adjuvant group. The results of the cellular immune level test showed that the IFN-gamma level was increased in the two proportions of OPC-Mn groups and the IL-4 level was decreased compared with the aluminum hydroxide adjuvant group, indicating that OPC-Mn was more prone to initiate Th1 type cellular immune response.
Experimental example 15: evaluation of in vivo delivery effect of OPC-3-acrylamidophenylboronic acid-novel crown vaccine antigen protein complex Price of price
Using the preparation method in example 3, OPC-3-acrylamidophenylboronic acid-neocrown vaccine complexes were prepared with OPC to neocrown antigen protein mass ratios of 5:1,10:1 and 20:1. Mice were given intramuscular injection of the posterior calf shank anterior muscle, 0.1 mL/mouse, immunized at 0d,21d,42d, respectively. The mice were bled from their orbitals 14 days after each immunization, serum was isolated and collected, and the antibody levels were determined by ELISA (results shown in fig. 19), which showed no significant difference in the OPC-3-acrylamidophenylboronic acid levels in the different proportions after three immunizations compared to the aluminum hydroxide adjuvant control. The results of the cellular immune level measurement using ELISPOT (results shown in fig. 20) showed that the OPC-3-acrylamidophenylboronic acid group had increased levels of IFN- γ and IL-2 compared to the aluminum hydroxide adjuvant control group, indicating that the OPC-3-acrylamidophenylboronic acid group was more prone to elicit a Th1 type cellular immune response.
Experimental example 16: evaluation of in vivo delivery Effect of OPC-4-bromomethylphenylboronic acid-novel crown vaccine antigen protein Complex
Using the preparation method in example 3, OPC-3-acrylamidophenylboronic acid-neocrown vaccine complexes were prepared with OPC to neocrown antigen protein mass ratios of 5:1,10:1 and 20:1. Mice were given intramuscular injection of the posterior calf shank anterior muscle, 0.1 mL/mouse, immunized at 0d,21d,42d, respectively. The mice were bled from the orbit 14 days after each immunization, serum was isolated and collected, and the antibody levels were measured by ELISA (results are shown in FIG. 21), which showed that the antibody levels tended to decrease as the proportion of OPC-4-bromomethylphenylboronic acid increased. The results of the ELISPOT assay on cellular immune levels (results are shown in FIG. 22) indicate that OPC-4-bromomethylphenylboronic acid also tends to elicit Th1 type cellular immune responses.
Experimental example 17: evaluation of immune Effect of OPC/OPC-Mn-New crown vaccine antigen protein Complex sequential vaccinated mice
The mice were sequentially vaccinated with the OPC/OPC-Mn and novel crown vaccine complexes of experimental examples 13 and 14 described above, and then serum antibody levels and cellular immune levels were measured, and as shown in fig. 23 and 24, it can be seen from the results that the total antibody levels of OPC and OPC-Mn delivery systems were less than that of aluminum hydroxide after the mice were sequentially vaccinated, but the levels of IgG1 and IgG2a were equivalent to that of aluminum hydroxide, and the cellular immune results showed that the level of Th1 type cellular immune responses was higher than that of aluminum hydroxide.
Experimental example 18: in vivo safety assessment for OPC-based delivery systems
(1) Monitoring of body weight changes in mice after immunization
After immunization of the mice, the weights of the mice were weighed at fixed times daily and changes in the weights were recorded. The results are shown in FIG. 25. Throughout the immunization period, mice had no significant weight loss, indicating that the safety of the delivery system was better.
(2) Determination of the level of inflammatory factors in the plasma of immunized mice
To assess whether OPC-like delivery systems would cause a strong inflammatory response, we measured the levels of major inflammatory factors in plasma after mice were vaccinated with OPC and OPC-Mn. The specific operation is as follows: after 6h and 24h of carrier inoculation, mice were bled from the eyeballs and plasma was isolated. Cytokine in plasma was then assayed using a Bio-Plex MAGPIX System based immunoassay. As a result, as shown in FIG. 26, the levels of several inflammatory factors did not rise severely, and although IL-6 increased after 6h of inoculation, the levels returned to normal after 24h, indicating that this type of delivery system was safer.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (12)

1. A complex, characterized in that the complex is obtained by linking a polyphenol compound, a conjugate of a metal ion and a polyphenol compound, or a conjugate of a nonmetallic ion and a polyphenol compound, with a protein having immunogenicity through a non-covalent bond.
2. The compound of claim 1, wherein the polyphenol compound is at least one selected from the group consisting of catechin, epicatechin, tannic acid, gallic acid, protocatechuic aldehyde, and tannic acid, or a polymer or derivative of the foregoing;
preferably, the polyphenol is Oligomeric Procyanidin (OPC).
3. The compound of claim 1, wherein the metal ion is selected from Sn 2+ 、Al 3+ 、Cu 2+ 、Fe 3 + 、Mn 2+ 、Zn 2+ 、Co 2+ 、Ti 4+ 、Ag + 、Ni 2+ Or Mg (Mg) 2+ At least one of (a) and (b);
preferably, the metal ion is Mn 2+ 。
4. The composite of claim 1, wherein the nonmetallic ions are boron ions and derivatives thereof.
5. The composite of claim 4, wherein the boron ions are derived from a boric acid group;
preferably, the boric acid group is a phenylboric acid group shown in a formula I or a pyridinylboric acid group shown in a formula II:
formula I and formula II
More preferably, the boric acid group is a phenylboric acid group as shown in formula I.
6. The compound according to claim 5, wherein the phenylboronic acid group is derived from at least one selected from the group consisting of aliphatic chain substitution, benzene ring substitution, amide substitution, fluorine atom substitution, halogen atom substitution, amino substitution, carboxyl substitution, and aldehyde group substitution;
preferably, the phenylboronic acid group is derived from at least one selected from the group consisting of 3-acrylamidophenylboronic acid, 4-bromomethylphenylboronic acid, 4-carboxy-3-fluorophenylboronic acid, 3-acyl-4-methylphenylboronic acid, 4-aminophenylboronic acid and fluoro-substituted phenylboronic acid;
more preferably, the phenylboronic acid group is derived from 3-acrylamidophenylboronic acid and/or 4-bromomethylphenylboronic acid.
7. The composite of claim 1, wherein the molar ratio of metal ions to polyphenol compounds is 0.5-2:1, and the molar ratio of non-metal ions to polyphenol compounds is 0.5-2:1;
preferably, the molar ratio of the metal ion to the polyphenol compound is 1:1, and the molar ratio of the nonmetallic ion to the polyphenol compound is 1:1.
8. The complex according to claim 1, wherein the mass ratio of the complex of the polyphenol compound, the metal ion and the polyphenol compound, or the complex of the nonmetal ion and the polyphenol compound to the protein having immunogenicity is 1 to 100:1;
preferably, the mass ratio is 2-20:1.
9. The composite of any one of claims 1 to 8, wherein the composite has a particle size of 140.9nm to 3514.7nm.
10. Use of a complex as claimed in any one of claims 1 to 9 in the manufacture of a vaccine formulation, wherein no adjuvant is required in the vaccine formulation.
11. A vaccine formulation, characterized in that it consists of a complex as claimed in any one of claims 1 to 9 and a pharmaceutically acceptable carrier.
12. The vaccine formulation of claim 11, wherein the vaccine formulation is a subunit vaccine;
preferably, the subunit vaccine is a novel coronavirus subunit vaccine, an influenza subunit vaccine, a respiratory syncytial virus subunit vaccine or a rabies subunit vaccine.
CN202310517596.1A 2023-05-09 2023-05-09 Composite and use thereof Pending CN116942835A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116033898A (en) * 2020-06-18 2023-04-28 贺发研究Ip(Pyc)公司 Procyanidins for the treatment of endothelial dysfunction induced by covd-19

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116033898A (en) * 2020-06-18 2023-04-28 贺发研究Ip(Pyc)公司 Procyanidins for the treatment of endothelial dysfunction induced by covd-19

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