Compositions and uses thereof

Abstract

The application discloses a kind of complex and purposes, the complex is obtained by non-covalent bond with the protein with immunogenicity by the conjugate of polyphenol compound, metal ion and polyphenol compound, or the conjugate of non-metal ion and polyphenol compound connection.New protein delivery system in the application can replace vaccine adjuvant, and play the effect comparable to or better than adjuvant in vaccine preparation, especially can activate cellular immune response, thereby further enhancing the efficacy of vaccine, and the complex preparation process is simple, effectively solves the inherent defects of vaccine adjuvant existing in current market.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to a complex and its uses. Background Technology

[0002] Vaccination is the primary means by which humans combat various pathogenic microorganisms. Subunit vaccines are vaccines made using a specific surface structural component (antigen) of a microorganism, containing no nucleic acid, and capable of inducing the body to produce antibodies. Their antigenic components are clearly defined, and their safety profile is good, but adjuvants are needed to enhance vaccine efficacy. Currently, the US FDA has approved only aluminum adjuvants, MF-59, AS03, AS04, AS01, and CPG 1018. One-third of these subunit vaccines use aluminum adjuvants, while my country currently lacks adjuvants with independent intellectual property rights used clinically. Although aluminum adjuvants are currently the most widely used adjuvants in clinical practice, many problems still need to be solved, such as their inability to induce Th1 and cytotoxic CD8 antibodies. + The T-reaction cannot be frozen or thawed during its preparation. Therefore, finding safe, efficient, and potent novel vaccine formulations that can elicit strong cellular immune responses is beneficial for fundamentally solving the bottleneck problem of adjuvants in the vaccine industry. Summary of the Invention

[0003] Technical problems to be solved :

[0004] One aspect of the present invention addresses vaccine adjuvants in the prior art that, for example, fail to induce Th1 and cytotoxic CD8 responses. + The T-reaction method addresses issues such as the inability to freeze-thaw during preparation, providing a composite material and its applications.

[0005] Specifically, the inventors unexpectedly discovered through research that a complex can replace vaccine adjuvants and exert effects in vaccine formulations that are comparable to or better than adjuvants. In particular, it can activate cellular immune responses, thereby further enhancing the efficacy of vaccines. Moreover, the preparation process of this complex is simple and effectively solves the inherent defects of vaccine adjuvants currently on the market.

[0006] The technical solution provided by this invention :

[0007] The complex is characterized in that it is obtained by non-covalently linking a polyphenolic compound, a metal ion and a polyphenolic compound conjugate, or a non-metal ion and a polyphenolic compound conjugate to an immunogenic protein.

[0008] In this invention, the assembly of the metal ions with polyphenolic compounds, or the non-metal ions with polyphenolic compounds, is primarily based on the pH dependence of the coordination bonds between the catechol groups and the metal or non-metal ions that serve as crosslinking units. The catechol or galloyl groups present in the phenolic compounds provide binding sites for the chelation of metal or non-metal ions, thereby forming highly stable complexes. At a constant pH, the properties of the complexes depend mainly on the concentration and degree of ionization of the polyphenols.

[0009] In some preparation embodiments of the present invention, a method for assembling the aforementioned metal ions with polyphenolic compounds, or the aforementioned non-metal ions with polyphenolic compounds, is provided. For example, the polyphenol is dissolved into a clear aqueous solution using a suitable method, and then the substance containing the metal or non-metal is similarly dissolved into a clear aqueous solution. The two are then mixed in a molar ratio and reacted at room temperature for 30–40 minutes. Preferably, in some embodiments of the present invention, the molar ratio of the metal ions to the polyphenolic compounds is 0.5–2:1, and the molar ratio of the non-metal ions to the polyphenolic compounds is 0.5–2:1. The aforementioned molar ratio of 0.5–2:1 can 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 ions to the polyphenolic compounds is 1:1, and the molar ratio of the non-metal ions to the polyphenolic compounds is 1:1.

[0010] In this invention, the metal ion can be any suitable one or more metal ions. For example, the metal ion can be selected from Sn. 2+ Al 3+ Cu 2+ Fe 3+ Mn 2+ Zn 2+ Co 2+ Ti 4+ Ag + Ni 2+ or Mg 2+ At least one of the following. However, preferably, in one embodiment of the present invention, the metal ion is Mn. 2+ .

[0011] The aforementioned metal ions can exist in any suitable manner, including but not limited to, in the form of ionic salts. In some embodiments of the present invention, the ionic salt may be, for example, MnSO4·H2O.

[0012] In this invention, the non-metallic ion can be any suitable one or more non-metallic ions. However, as a preferred embodiment of the invention, the non-metallic ion is a boron ion.

[0013] The aforementioned nonmetallic ions can exist in any suitable manner. Preferably, in some embodiments of the invention, the boron ions are derived from borate groups. More preferably, in some embodiments of the invention, the borate group is a phenylboronic acid group as shown in Formula I or a pyridineboronic acid group as shown in Formula II.

[0014]

[0015] Formula I Formula II

[0016] More preferably, in some embodiments of the present invention, the borate group is a phenylboronic acid group as shown in Formula I.

[0017] Further preferably, in certain embodiments of the present invention, the phenylboronic acid group is derived from at least one of the following phenylboronic acid groups: aliphatic chain substitution, benzene ring substitution, amide substitution, fluorine atom substitution, halogen atom substitution, amino substitution, carboxyl substitution, and 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 fluorinated phenylboronic acid.

[0018] More preferably, in some embodiments of the present invention, the phenylboronic acid group is derived from 3-acrylamidophenylboronic acid and / or 4-bromomethylphenylboronic acid.

[0019] In this invention, the polyphenols can be compounds containing at least two phenolic groups, which can be associated in more or less complex structures and typically have high molecular weights. Examples include, but are not limited to, flavonoid polyphenols such as epigallocatechin, epigallocatechin 3-gallate, epicatechin 3-gallate, baicalin, piperidin, quercetin, hesperidin, or genistein; or non-flavonoid polyphenols such as resveratrol and gossypol. In some embodiments of this 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 above compounds. The polymers or derivatives of the above compounds can be prepared using suitable methods and typically have the same or similar biological functions as the above compounds. Preferably, in one embodiment of this invention, the polyphenol is oligomeric proanthocyanidins (OPCs).

[0020] In some embodiments of the present invention, a method is provided for complexing the above-mentioned polyphenolic compound, a conjugate composed of a metal ion and a polyphenolic compound, or a conjugate composed of a non-metal ion and a polyphenolic compound with an immunogenic protein via non-covalent bonds. For example, a certain proportion of the immunogenic protein is added to a reaction mixture of the above-mentioned polyphenolic compound, a metal ion and a polyphenolic compound, or the non-metal ion and a polyphenolic compound, and the reaction is carried out at room temperature to obtain the complex. Preferably, in some embodiments of the present invention, the mass ratio of the conjugate composed of a polyphenolic compound, a metal ion and a polyphenolic compound, or the conjugate composed of a non-metal ion and a polyphenolic compound to the immunogenic protein via non-covalent bonds is 1 to 100:1. More preferably, in some embodiments of the present invention, the mass ratio is 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.

[0021] The composite obtained by the method of the present invention forms uniform composite particles. The particle size of the composite particles is 140.9 nm to 3514.7 nm.

[0022] Another aspect of the present invention is to provide a vaccine formulation comprising the aforementioned complex and a pharmaceutically acceptable carrier. In this invention, the pharmaceutically acceptable carrier may be any pharmaceutically permissible additive, including but not limited to physiological saline, solvents, glucose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, surfactants, preservatives, isotonic agents, absorption delay agents, etc., compatible with drug administration.

[0023] As an innovative aspect of this invention, the above-mentioned vaccine formulation does not require the presence of an adjuvant. Through certain experiments described in this invention, the vaccine formulation of this invention can achieve or surpass the effects of equivalent vaccine formulations containing adjuvants.

[0024] Preferably, the aforementioned vaccine formulation can be any subunit vaccine. More preferably, the aforementioned subunit vaccine can be a novel coronavirus subunit vaccine, an influenza subunit vaccine, a respiratory syncytial virus subunit vaccine, or a rabies subunit vaccine.

[0025] The beneficial effects of this invention are:

[0026] The novel protein delivery system of this invention can replace vaccine adjuvants and exert effects comparable to or better than adjuvants in vaccine formulations. In particular, it can activate cellular immune responses, thereby further enhancing the efficacy of vaccines. Moreover, the preparation process of this complex is simple and effectively solves the inherent defects of vaccine adjuvants currently on the market. Attached Figure Description

[0027] Figure 1 This is a graph showing the particle size determination results of OPC-COVID-19 subunit vaccine antigen complexes with different mass ratios in embodiments of the present invention;

[0028] Figure 2 This is a graph showing the particle size determination results of OPC-Mn-COVID-19 subunit vaccine antigen complexes with different mass ratios in embodiments of the present invention;

[0029] Figure 3 This is a graph showing the particle size determination results of OPC-3-acrylamidophenylboronic acid-COVID-19 subunit vaccine antigen complexes with different mass ratios in embodiments of the present invention;

[0030] Figure 4 This is a graph showing the particle size determination results of OPC-4-bromomethylphenylboronic acid-COVID-19 subunit vaccine antigen complexes with different mass ratios in embodiments of the present invention;

[0031] Figure 5 This is a graph showing the particle size determination results of OPC-influenza subunit vaccine antigen complexes with different mass ratios in embodiments of the present invention;

[0032] Figure 6 This is a graph showing the particle size determination results of OPC-influenza subunit vaccine antigen complexes with different mass ratios (1:1-10:1) in embodiments of the present invention;

[0033] Figure 7 This is a graph showing the particle size determination results of OPC-Mn-influenza subunit vaccine antigen complexes with different mass ratios in embodiments of the present invention;

[0034] Figure 8 This is a graph showing the particle size stability determination results of the OPC / OPC-Mn-influenza subunit vaccine antigen complex in an embodiment of the present invention.

[0035] Figure 9 The figure shows the particle size determination results of OPC-RPE protein complexes with different mass ratios in the embodiments of the present invention;

[0036] Figure 10 The figure shows the particle size determination results of OPC-Mn-RPE protein complexes with different mass ratios in the embodiments of the present invention.

[0037] Figure 11 This is a transmission electron microscope (TEM) image of the OPC-3-acrylamidophenylboronic acid-COVID-19 vaccine complex in an embodiment of the present invention.

[0038] Figure 12 This is a transmission electron microscope (TEM) image of the OPC / OPC-Mn-influenza vaccine complex in an embodiment of the present invention.

[0039] Figure 13This is a diagram showing the cell transfection results of the OPC / OPC-Mn-RPE protein complex in an embodiment of the present invention;

[0040] Figure 14 This is a graph showing the cytotoxicity results of OPC in an embodiment of the present invention;

[0041] Figure 15 This is a graph showing the cytotoxicity results of OPC-Mn in an embodiment of the present invention;

[0042] Figure 16 The antibody (antibody) formed after three immunizations with the OPC-COVID-19 vaccine antigen-protein complex in this embodiment of the invention. Figure 16 (left) and neutralizing antibodies ( Figure 16 (Right) Horizontal result diagram;

[0043] Figure 17 This is a graph showing the antibody level results after immunization with the OPC-Mn-COVID-19 vaccine antigen-protein complex in an embodiment of the present invention.

[0044] Figure 18 This is a graph showing the cellular immune level results of the OPC-Mn-COVID-19 vaccine antigen protein complex in an embodiment of the present invention;

[0045] Figure 19 This is a graph showing the antibody level results after immunization with the OPC-3-acrylamidophenylboronic acid-COVID-19 vaccine antigen-protein complex in an embodiment of the present invention.

[0046] Figure 20 This is a graph showing the results of cellular immunity levels after immunization with the OPC-3-acrylamidophenylboronic acid-COVID-19 vaccine antigen protein complex in an embodiment of the present invention.

[0047] Figure 21 This is a graph showing the antibody level results after immunization with the OPC-4-bromomethylphenylboronic acid-COVID-19 vaccine antigen protein complex in an embodiment of the present invention;

[0048] Figure 22 This is a graph showing the results of cellular immunity levels after immunization with the OPC-4-bromomethylphenylboronic acid-COVID-19 vaccine antigen protein complex in an embodiment of the present invention.

[0049] Figure 23 This is a graph showing the antibody level results in mice after sequential inoculation with the OPC / OPC-Mn-COVID-19 vaccine antigen protein complex in an embodiment of the present invention.

[0050] Figure 24 This is a graph showing the results of sequential inoculation of mice with the OPC / OPC-Mn-COVID-19 vaccine antigen protein complex in an embodiment of the present invention, indicating the level of cellular immunity.

[0051] Figure 25 This is a graph showing the changes in body weight of mice after injection using the OPC delivery system in an embodiment of the present invention.

[0052] Figure 26 This is a graph showing the results of measuring the levels of inflammatory factors in the plasma of immunized mice in an embodiment of the present invention. Detailed Implementation

[0053] This invention discloses the use of a complex in the preparation of vaccine formulations. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately alter and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0054] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or variations thereof such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components. The terms "such as," "for example," etc., are intended to refer to exemplary embodiments and are not intended to limit the scope of this disclosure. Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural indicators.

[0055] definition:

[0056] The term "complex" refers to a product made of two or more different components that offers superior performance compared to the starting materials.

[0057] The terms "vaccine formulation" and "vaccine" refer to any formulation containing an antigenic component. Subunit vaccine formulations typically contain adjuvants. Vaccine formulations may also contain excipients or mediators suitable for pharmaceutical application, or any compound with immunomodulatory activity such as cytokines, Toll receptor ligands, etc.

[0058] The term "adjuvant" refers to a compound that, when administered in combination with an antigen, can increase, enhance, and / or promote an immune response to the antigen, but does not produce an immune response to the antigen when the adjuvant compound is administered alone.

[0059] The term "proanthocyanidins" (PC) refers to proanthocyanidins polymerized from varying numbers of monomers (catechins or epicatechins), as shown in formulas III to VI below. Based on the degree of polymerization, they are classified into oligomeric proanthocyanidins (OPC), composed of 2-4 monomers, and polymeric proanthocyanidins (PPC), composed of 5 or more monomers. Studies have shown that oligomeric proanthocyanidins possess advantages such as high bioavailability, rapid oral absorption, and long retention time in the body, thus exhibiting strong biological activity.

[0060]

[0061]

[0062] Proanthocyanidins are widely found in the fruits, leaves, seeds, and skins of various plants, with grape seeds containing up to 95% proanthocyanidins. Proanthocyanidins possess a variety of pharmacological activities, including antioxidant activity, protection against myocardial ischemia-reperfusion injury, anti-atherosclerosis, protection of vascular endothelial cells, anti-cancer activity, and the ability to lower blood pressure, blood lipids, and blood sugar. Currently, due to their extremely strong antioxidant activity and free radical scavenging ability, proanthocyanidins are widely used in food additives, pharmaceuticals, and cosmetics; however, their application in the vaccine field has not yet been reported. The structure of proanthocyanidins contains resorcinol and phloroglucinol structural units, which can form complexes with metal ions.

[0063] The term "pharmaceutical acceptable" means that a substance is non-toxic and does not interact with the active component of a pharmaceutical composition.

[0064] The term "derivative" refers to a compound having a structure derived from a parent compound and whose structure is sufficiently similar to those of the compounds disclosed in this invention, and based on this similarity, those skilled in the art would expect it to exhibit the same or similar activities and uses as the claimed compound, or to induce the same or similar activities and uses as a precursor as the claimed compound.

[0065] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0066] Example:

[0067] Example 1: Preparation of OPC-COVID-19 sub-vaccine antigen complex

[0068] OPC was dissolved with a trace amount of DMSO and diluted with sterile, enzyme-free water to obtain a clear solution. Different volumes of antigen protein stock solution were taken according to the mass ratio of OPC to antigen protein (2:1, 5:1, 10:1, 20:1, 40:1), mixed with OPC solution, and reacted at room temperature for 1 hour to obtain OPC-COVID-19 vaccine complexes with different ratios.

[0069] Example 2: Preparation of OPC-Mn-COVID-19 subunit vaccine antigen complex

[0070] OPC was dissolved with a trace amount of DMSO and diluted with sterile, enzyme-free water to obtain a clear solution; MnSO4·H2O was dissolved with sterile, enzyme-free water. The corresponding volumes were taken at a molar ratio of 1:1 and reacted for 30 min. Different volumes of antigen protein stock solution were taken according to the mass ratio of OPC to antigen protein (2:1, 5:1, 10:1, 20:1, 40:1) and added to the OPC and MnSO4·H2O mixture. The mixture was reacted at room temperature for 1 h to obtain OPC-Mn-COVID-19 vaccine complexes with different ratios.

[0071] Example 3: OPC-3-acrylamidophenylboronic acid / 4-bromomethylphenylboronic acid-COVID-19 subunit vaccine antigen complex Preparation of substances

[0072] OPC was dissolved with a trace amount of DMSO and diluted with sterile, enzyme-free water to obtain a clear solution; 3-acrylamidophenylboronic acid / 4-bromomethylphenylboronic acid was dissolved with a trace amount of anhydrous ethanol and diluted with sterile, enzyme-free water to obtain a clear solution. The corresponding volumes were pipetted at a molar ratio of 1:1 and reacted for 30 min. Different volumes of antigen protein stock solution were pipetted according to the mass ratio of OPC to antigen protein (1:1, 2:1, 5:1, 10:1, 20:1) and added to the OPC and phenylboronic acid compound mixture. The mixture was reacted at room temperature for 1 h to obtain OPC-phenylboronic acid-COVID-19 vaccine complexes with different proportions.

[0073] Example 4: Preparation of OPC-Influenza Subunit Vaccine Antigen Complex

[0074] OPC was dissolved with a trace amount of DMSO and diluted with sterile, enzyme-free water to obtain a clear solution. Different volumes of influenza antigen protein stock solution were taken 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), mixed with OPC solution, and reacted at room temperature for 1 hour to obtain OPC-influenza vaccine complexes with different ratios.

[0075] Example 5: Preparation of OPC-Mn-influenza subvaccine antigen complex

[0076] OPC was dissolved with a trace amount of DMSO and diluted with sterile, enzyme-free water to obtain a clear solution; MnSO4·H2O was dissolved with sterile, enzyme-free water. The corresponding volumes were pipetted at a molar ratio of 1:1 and reacted at room temperature for 30 min. Different volumes of antigen protein stock solution were pipetted according to the mass ratio of OPC to antigen protein (1:1, 2:1, 5:1, 10:1) and added to the OPC and MnSO4·H2O mixture, reacting at room temperature for 1 h to obtain OPC-Mn-influenza vaccine complexes with different ratios.

[0077] Example 6: Preparation of OPC / OPC-Mn-model protein (RPE) complex

[0078] OPC-RPE composites were prepared using the method described in Example 1 at mass ratios of OPC to RPE (2:1, 5:1, 10:1, 20:1).

[0079] The OPC-Mn-RPE complex was prepared using the method in Example 2 according to the mass ratio of OPC to RPE (2:1, 5:1, 10:1, 20:1).

[0080] Experimental Example 1: Particle Size Analysis of OPC-COVID-19 Subunit Vaccine Antigen Complex

[0081] The particle size of the OPC-COVID-19 subunit vaccine antigen complex prepared in Example 1 was determined using a dynamic light scattering (DLS) nanolaser particle size analyzer. The instrument used was a Zetasizer Nano ZS90, the detection temperature was 25°C, and each sample was tested three times. The results are shown below. Figure 1 As shown in the results, different proportions of OPC can adsorb the COVID-19 subunit vaccine antigen protein, forming uniform complex particles. The complex with the mass ratio of 10:1 has the smallest particle size and the best uniformity.

[0082] Experimental Example 2: Particle Size Analysis of OPC-Mn-COVID-19 Subunit Vaccine Antigen Complex

[0083] The particle size of the OPC-Mn-COVID-19 subunit vaccine antigen complex prepared in Example 2 was determined using a dynamic light scattering (DLS) nanolaser particle size analyzer. The instrument used was a Zetasizer Nano ZS90, the detection temperature was 25°C, and each sample was tested three times. The results are shown below. Figure 2 As shown in the results, different proportions of OPC-Mn can adsorb the COVID-19 subunit vaccine antigen protein, forming uniform complex particles.

[0084] Experimental Example 3: Particle Size Analysis of OPC-3-acrylamidophenylboronic Acid-COVID-19 Subunit Vaccine Antigen Complex

[0085] The particle size of the OPC-3-acrylamidophenylboronic acid-COVID-19 subunit vaccine antigen complex prepared in Example 3 was determined using a dynamic light scattering (DLS) nanolaser particle size analyzer. The instrument used was a Zetasizer Nano ZS90, the detection temperature was 25°C, and each sample was tested three times. The results are shown below. Figure 3 As shown in the results, different proportions of OPC-3-acrylamidophenylboronic acid can adsorb the COVID-19 subunit vaccine antigen protein, forming uniform complex particles. Specifically, at mass ratios of 10:1 and 20:1, the particle size of the complex shrinks to approximately 200 nm, with the smallest particle size (197 nm) observed at a ratio of 20:1.

[0086] Experimental Example 4: Particle Size Analysis of OPC-4-bromomethylphenylboronic acid-COVID-19 subunit vaccine antigen complex

[0087] The particle size of the OPC-4-bromomethylphenylboronic acid-COVID-19 subunit vaccine antigen complex prepared in Example 3 was determined using a dynamic light scattering (DLS) nanolaser particle size analyzer. The instrument used was a Zetasizer Nano ZS90, the detection temperature was 25°C, and each sample was tested three times. The results are shown below. Figure 4 As shown in the results, different proportions of OPC-4-bromomethylphenylboronic acid can adsorb the COVID-19 subunit vaccine antigen protein, forming uniform complex particles. Specifically, at mass ratios of 10:1 and 20:1, the particle size of the complex shrinks to the nanometer scale, with the smallest particle size (252 nm) observed at a ratio of 10:1.

[0088] Experimental Example 5: Particle Size Analysis of OPC-Influenza Subunit Vaccine Antigen Complex

[0089] The particle size of the OPC-influenza subunit vaccine antigen complex prepared in Example 4 was determined using a dynamic light scattering (DLS) nanolaser particle size analyzer. The instrument used was a Zetasizer Nano ZS90, the detection temperature was 25°C, and each sample was tested three times. The results are shown below. Figure 5 As shown, when OPC is loaded with influenza antigen protein, the particle size of the complex first decreases and then increases significantly with the increase of OPC mass ratio, with relatively smaller particle sizes at 5:1 and 10:1. Further verification of particle size results from mass ratios of 1:1 to 10:1 shows (e.g.) Figure 6 As shown in the figure, when the mass ratio is 2:1 and 10:1, the particle size is around 200nm, the particles are uniform, and the PDI is less than 0.3.

[0090] Experimental Example 6: Particle Size Analysis of OPC-Mn-Influenza Subunit Vaccine Antigen Complex

[0091] The particle size of the OPC-Mn-influenza subunit vaccine antigen complex prepared in Example 5 was determined using a dynamic light scattering (DLS) nanolaser particle size analyzer. The instrument used was a Zetasizer Nano ZS90, the detection temperature was 25°C, and each sample was tested three times. The results are shown below. Figure 7 As shown, coordination between OPC and MnSO4 improved the particle size of the protein complex. The particle size of the OPC-Mn-influenza vaccine complex remained within 200 nm at three mass ratios of 2:1, 5:1, and 10:1.

[0092] Experimental Example 7: Particle size stability analysis of OPC / OPC-Mn-influenza subunit vaccine antigen complex

[0093] The OPC-based delivery system / influenza vaccine complexes prepared using Examples 4 and 5 were placed at 4°C and their particle size was measured for three consecutive days to determine the stability of the formulation. The test results are as follows: Figure 8 As shown, the particle size of the formulation did not increase significantly within three days, indicating that there was no significant particle aggregation and the system was stable.

[0094] Experimental Example 8: Particle Size Analysis of the OPC / OPC-Mn-Model Protein (RPE) Complex

[0095] The particle size of the OPC / OPC-Mn-model protein (RPE) complex prepared in Example 6 was determined using a dynamic light scattering (DLS) nanolaser particle size analyzer. The instrument used was a Zetasizer Nano ZS90, the detection temperature was 25°C, and each sample was tested three times. The results are shown below. Figure 9 , Figure 10 As shown, OPC loaded with RPE protein can form stable nanoparticles with good uniformity. After adding MnSO4, OPC coordinates with Mn and then adsorbs the protein, resulting in a change in particle size. The smallest particle size of 141 nm is obtained when the mass ratio is 2:1.

[0096] Experimental Example 9: Morphological characterization of the OPC-3-acrylamidophenylboronic acid-COVID-19 subunit vaccine antigen complex

[0097] OPC-3-acrylamidophenylboronic acid-COVID-19 vaccine complexes with OPC to protein ratios of 5:1, 10:1, and 20:1 were prepared in Example 3. The specific procedure was as follows: The prepared complex solution was concentrated by ultrafiltration. 10 μL was added dropwise onto a copper grid, and after 3 minutes, it was blotted dry with filter paper. Then, 10 μL of a 2% phosphotungstic acid solution was added dropwise onto the copper grid, and after 30 seconds, it was blotted dry with filter paper. After drying, the images were taken under a Hitachi H-7650 electron microscope. The results are as follows: Figure 11 As shown, under different mass ratios, OPC-3-acrylamidophenylboronic acid can form spherical nanoparticles after binding with the COVID-19 subunit vaccine antigen protein.

[0098] Experimental Example 10: Morphological characterization of OPC / OPC-Mn-influenza subunit vaccine antigen complex

[0099] The OPC-influenza vaccine complex and OPC-Mn-influenza vaccine complex, prepared in Examples 4 and 5 with OPC to antigen protein ratios of 2:1, 5:1, and 10:1, were used. The specific procedures were as follows: The prepared complex solution was concentrated by ultrafiltration. 10 μL was added dropwise onto a copper grid, and after 3 minutes, it was blotted dry with filter paper. Then, 10 μL of 2% phosphotungstic acid solution was added dropwise onto the copper grid, and after 30 seconds, it was blotted dry with filter paper. After drying, the images were taken under a Hitachi H-7650 electron microscope. The results are as follows: Figure 12 As shown, OPC and OPC-Mn can form spherical nanoparticles after binding with influenza antigen proteins at different mass ratios.

[0100] Experimental Example 11: Determination of cell transfection efficiency of OPC / OPC-Mn-RPE protein complex

[0101] (1) Cell transfection efficiency of OPC-RPE protein complex

[0102] OPC was dissolved with a trace amount of DMSO and diluted with sterile, enzyme-free water to a clear solution. Different volumes of RPE stock solution were pipetted according to different mass ratios of OPC to RPE protein, mixed thoroughly, and incubated at room temperature for 1 hour. HeLa cells were seeded into 24-well plates, 2.0 × 10⁶ cells per well. 5 Cells were incubated in an incubator for 18-24 hours until cell confluence reached 60-90%, at which point transfection could be performed. The original complete culture medium was replaced with 500 μL of serum- and antibiotic-free DMEM medium. After incubation for 2 hours, the prepared OPC-RPE complex was added to each well of a 24-well plate at a rate of 2 μg RPE. After transfection, cells were cultured in an incubator for 4 hours, and the results were observed under a fluorescence microscope. Cells were washed with PBS, digested, and collected for flow cytometry analysis. Results are as follows: Figure 13 As shown.

[0103] (2) Cell transfection efficiency of OPC-Mn-RPE protein complex

[0104] OPC was dissolved with a trace amount of DMSO and diluted with sterile, enzyme-free water to a clear solution; MnSO4·H2O was dissolved with sterile, enzyme-free water. The corresponding volumes were taken at a molar ratio of 1:1 and reacted for 30 min. Different volumes of RPE stock solution were taken according to different mass ratios of OPC to RPE and added to the OPC and MnSO4·H2O mixture, and reacted at room temperature for 1 h.

[0105] HeLa cells were seeded in 24-well plates, with 2.0 × 10⁶ cells per well. 5Cells were incubated in a culture incubator for 18-24 hours until cell confluence reached 60-90%, at which point transfection could be performed. The original complete culture medium was replaced with 500 μL of serum- and antibiotic-free DMEM medium. After incubation for 2 hours, the prepared OPC-Mn-RPE complex was added to each well of a 24-well plate at a rate of 2 μg R-PE. After transfection, cells were cultured in an incubator for 4 hours, and the results were observed under a fluorescence microscope. Cells were washed with PBS, digested, and collected for flow cytometry analysis. Results are as follows: Figure 13 As shown.

[0106] The transfection results showed that both OPC-RPE and OPC-Mn-RPE complexes could enter the cells. However, the OPC-encapsulated complex had a lower ability to enter the cells. The transfection ability was improved after the addition of Mn, and the transfection efficiency was the highest when the mass ratio was 2:1.

[0107] Experimental Example 12: Cytotoxicity of OPC and OPC-Mn Delivery Systems

[0108] (1) Cytotoxicity of OPC

[0109] OPC was dissolved with a trace amount of DMSO, diluted with sterile, enzyme-free water to a clear solution, and finally diluted with DMEM basal medium free of serum and antibiotics. HeLa cells were seeded in 96-well plates at 1.0 × 10⁶ cells per well. 5 When the cells are incubated in an incubator for 18-24 hours and the cell confluence reaches 90%, subsequent operations can be performed.

[0110] 4-hour toxicity test: Add 100 μL of the prepared OPC solution to each well of a 96-well plate, with 5 replicates per well. A cell-free control group and a cell-only control group were also included. Incubate for 4 hours. Prepare CCK-8 assay solution, add 100 μL to each well of the 96-well plate, and incubate for 30 min–1 hour. Measure the absorbance at 492 nm using a microplate reader. Results are as follows: Figure 14 As shown.

[0111] 24-hour toxicity: The prepared OPC solution was added to 96-well plates at 100 μL per well, with 5 replicates per well. A cell-free control group and a cell-only control group were also included. After 4 hours of incubation, 10 μL of FBS was added to each well, and incubation continued for another 20 hours. CCK-8 assay solution was prepared, and 100 μL was added to each well of the 96-well plate. Incubation was performed for 30 minutes followed by 1 hour, and absorbance was measured at 492 nm using a microplate reader. The results are as follows: Figure 14 As shown in the figure, cell viability gradually decreases as the proportion of OPC increases.

[0112] (2) Cytotoxicity of OPC-Mn delivery system

[0113] OPC was dissolved with a trace amount of DMSO and diluted to a clear solution with sterile, enzyme-free water. MnSO4·H2O was dissolved in sterile, enzyme-free water. The appropriate volumes were pipetted at a 1:1 molar ratio and reacted for 30 min. Different volumes of RPE stock solution were added to the OPC and MnSO4·H2O mixture according to different mass ratios of OPC to RPE, and reacted at room temperature for 1 h. Finally, the solution was diluted with DMEM basal medium without serum and antibiotics. HeLa / Vero / 293T cells were seeded in 96-well plates at 1.0 × 10⁶ cells per well. 5 When the cells are incubated in an incubator for 18-24 hours and the cell confluence reaches 90%, subsequent operations can be performed.

[0114] 4-hour toxicity test: Add 100 μL of the prepared solution to each well of a 96-well plate, with 5 replicates for each ratio. A cell-free control group and a cell-only control group were also included. Incubate for 4 hours. Prepare CCK-8 assay solution, add 100 μL to each well of the 96-well plate, and incubate for 30 min–1 hour. Measure the absorbance at 492 nm using a microplate reader.

[0115] 24-hour toxicity: The prepared solution was added to 96-well plates at 100 μL per well, with 5 replicates per ratio. A cell-free control group and a cell-only control group were also included. After 4 hours of incubation, 10 μL of FBS was added to each well, and incubation continued for another 20 hours. CCK-8 assay solution was prepared, and 100 μL was added to each well of the 96-well plate. Incubation was performed for 30 minutes followed by 1 hour, and absorbance was measured at 492 nm using a microplate reader. The results are as follows: Figure 15 As shown in the figure, the OPC-Mn delivery system exhibits lower toxicity to the three cell types at low mass ratios.

[0116] Experiment Example 13: Evaluation of the in vivo delivery efficiency of OPC-COVID-19 vaccine antigen-protein complex

[0117] Using the preparation method in Example 1, OPC-COVID-19 vaccine complexes were prepared at OPC to SARS-CoV-2 antigen protein ratios of 5:1, 10:1, and 20:1. Mice were immunized via intramuscular injection into the tibialis anterior muscle of the hind leg, 0.1 mL per mouse, on days 0, 21, and 42. Blood was collected from the orbital sinus 14 days after each immunization, and serum was separated and collected. Antibody levels were measured using ELISA (results are shown below). Figure 16 (As shown in the figure). The results show that OPC can load the COVID-19 vaccine antigen protein and produce a strong antibody response in vivo. The antibodies produced are not significantly different from those in the aluminum hydroxide adjuvant control group.

[0118] Experimental Example 14: Evaluation of the in vivo delivery efficiency of the OPC-Mn-COVID-19 vaccine antigen-protein complex

[0119] Using the preparation method in Example 2, OPC-Mn-COVID-19 vaccine complexes were prepared at OPC to SARS-CoV-2 antigen protein ratios of 5:1, 10:1, and 20:1. Mice were immunized via intramuscular injection into the tibialis anterior muscle of the hind leg, 0.1 mL per mouse, on days 0, 21, and 42. Blood was collected from the orbital sinus 14 days after each immunization, and serum was separated and collected. Antibody levels were measured using ELISA (results are shown below). Figure 17 As shown), cellular immunity levels were measured using ELISPOT (results are shown below). Figure 18 (As shown in the image). The results show that both ratios of OPC-Mn COVID-19 vaccine can induce a strong humoral immune response, with no significant difference in antibody levels compared to the aluminum hydroxide adjuvant group. Cellular immunity level detection results show that, compared to the aluminum hydroxide adjuvant group, both ratios of OPC-Mn groups showed increased IFN-γ levels and decreased IL-4 levels, indicating that OPC-Mn is more likely to elicit a Th1-type cellular immune response.

[0120] Experimental Example 15: Evaluation of the in vivo delivery efficacy of OPC-3-acrylamidophenylboronic acid-COVID-19 vaccine antigen-protein complex price

[0121] Using the preparation method in Example 3, OPC-3-acrylamidophenylboronic acid-COVID-19 vaccine complexes were prepared at OPC to SARS-CoV-2 antigen protein ratios of 5:1, 10:1, and 20:1. Mice were immunized via intramuscular injection into the tibialis anterior muscle of the hind leg, 0.1 mL per mouse, on days 0, 21, and 42. Blood was collected from the orbital sinus 14 days after each immunization, and serum was separated and collected. Antibody levels were measured using ELISA (results are shown in Figure 1). Figure 19 As shown in the figure), the results indicated that after three immunizations, there was no significant difference in antibody levels between the OPC-3-acrylamidophenylboronic acid groups and the aluminum hydroxide adjuvant control group. Cellular immunity levels were measured using ELISPOT (results are shown in the figure). Figure 20 As shown in the figure, the results showed that compared with the aluminum hydroxide adjuvant control group, the levels of IFN-γ and IL-2 in the OPC-3-acrylamidophenylboronic acid group were increased, indicating that the OPC-3-acrylamidophenylboronic acid group was more likely to induce Th1-type cellular immune responses.

[0122] Experimental Example 16: Evaluation of the in vivo delivery efficacy of OPC-4-bromomethylphenylboronic acid-COVID-19 vaccine antigen protein complex

[0123] Using the preparation method in Example 3, OPC-3-acrylamidophenylboronic acid-COVID-19 vaccine complexes were prepared at OPC to SARS-CoV-2 antigen protein ratios of 5:1, 10:1, and 20:1. Mice were immunized via intramuscular injection into the tibialis anterior muscle of the hind leg, 0.1 mL per mouse, on days 0, 21, and 42. Blood was collected from the orbital sinus 14 days after each immunization, and serum was separated and collected. Antibody levels were measured using ELISA (results are shown in Figure 1). Figure 21As shown in the figure), the results indicated that antibody levels decreased with increasing proportion of OPC-4-bromomethylphenylboronic acid. ELISPOT assays of cellular immunity levels (results shown in the figure) Figure 22 The results (as shown) indicate that OPC-4-bromomethylphenylboronic acid also tends to induce Th1-type cellular immune responses.

[0124] Experiment Example 17: Evaluation of the Immunization Efficacy of Sequential Inoculation of Mice with OPC / OPC-Mn-COVID-19 Vaccine Antigen Protein Complex

[0125] Mice were sequentially inoculated with the OPC / OPC-Mn and COVID-19 vaccine complex from Experiments 13 and 14 above, and then serum antibody levels and cellular immunity levels were measured. The results are as follows: Figure 23 and Figure 24 As shown in the results, the total antibody levels of OPC and OPC-Mn delivery systems were lower than those of aluminum hydroxide after sequential inoculation of mice, but the levels of IgG1 and IgG2a were comparable to those of aluminum hydroxide. Cellular immunization results showed that the level of Th1-type cellular immune response was higher than that of aluminum hydroxide.

[0126] Experiment Example 18: In vivo safety evaluation of OPC-type delivery systems

[0127] (1) Monitoring of weight changes in mice after immunization

[0128] After immunization, the mice were weighed at fixed times daily, and weight changes were recorded. The results are as follows: Figure 25 As shown, there was no significant decrease in mouse weight throughout the immunization cycle, indicating that the delivery system has good safety.

[0129] (2) Determination of the level of inflammatory factors in the plasma of mice after immunization

[0130] To evaluate whether OPC delivery systems induce a strong inflammatory response, we measured the levels of major inflammatory factors in the plasma of mice after inoculation with OPC and OPC-Mn. The specific procedure was as follows: Blood was collected from the eyeballs of mice 6 h and 24 h after inoculation, and plasma was separated. Then, an immunoassay based on the Bio-Plex MAGPIX System was used to measure cytokines in the plasma. The results are as follows: Figure 26 As shown, the levels of several inflammatory factors did not increase significantly. Although IL-6 increased 6 hours after vaccination, its level returned to normal after 24 hours, indicating that this type of delivery system is relatively safe.

[0131] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A complex, characterized in that, The complex is obtained by non-covalently linking a polyphenolic compound, a conjugate of a metal ion and a polyphenolic compound, or a conjugate of a non-metal ion and a polyphenolic compound to an immunogenic protein. The polyphenolic compound is oligomeric proanthocyanidins (OPC), and the metal ion is Mn. 2+ The non-metallic ion is 3-acrylamidophenylboronic acid or 4-bromomethylphenylboronic acid.

2. The complex according to claim 1, characterized in that, The molar ratio of the metal ion to the polyphenol compound is 0.5 to 2:1, and the molar ratio of the non-metal ion to the polyphenol compound is 0.5 to 2:

1.

3. The complex according to claim 2, characterized in that, The molar ratio of the metal ion to the polyphenol compound is 1:1, and the molar ratio of the non-metal ion to the polyphenol compound is 1:

1.

4. The complex according to claim 1, characterized in that, The mass ratio of the polyphenol compound, the conjugate of the metal ion and the polyphenol compound, or the conjugate of the non-metal ion and the polyphenol compound to the immunogenic protein is 1 to 100:

1.

5. The complex according to claim 4, characterized in that, The mass ratio is 2 to 20:

1.

6. The complex according to any one of claims 1 to 5, characterized in that, The particle size of the composite is 140.9 nm to 3514.7 nm.

7. The use of the complex according to any one of claims 1 to 6 in the preparation of vaccine formulations, characterized in that, No adjuvants are required in the vaccine formulation.

8. A vaccine formulation, characterized in that, The vaccine formulation comprises the complex as described in any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

9. The vaccine formulation according to claim 8, characterized in that, The vaccine formulation is a subunit vaccine.

10. The vaccine formulation according to claim 9, characterized in that, The subunit vaccine mentioned is a novel coronavirus subunit vaccine, an influenza subunit vaccine, a respiratory syncytial virus subunit vaccine, or a rabies subunit vaccine.

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