Zinc ion polypeptide compound immunologic adjuvant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a zinc ion polypeptide compound immunologic adjuvant as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing polypeptide, a PBS buffer solution, new coronavirus S protein, a CpG ODN immune activator and zinc ions, and standing to obtain the zinc ion polypeptide compound immunologic adjuvant. A dual-adjuvant system mixed with CpG ODN shows excellent performance in activation of lung tissue immunity, and in the cellular immunity level, the response intensity of CD4 + T and CD8 + T cells is enhanced, and formation of related tissue resident memory T cell phenotypes is promoted; on the humoral immunity level, a Gel (Zn < 2 + >) and CpG ODN dual-adjuvant system obviously stimulates the increase of the number of B cells and the activation, and can effectively stimulate the secretion of a main antibody sIgA for mucosal immunity. On the whole, the dual adjuvants realize the synergistic enhancement of T cell (containing TRM) and B cell immunity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a zinc ion polypeptide complex immune adjuvant, its preparation method, and its application. Background Technology

[0002] Clinical and epidemiological studies suggest that persistent infections and immune imbalances make a more durable, broader-spectrum, and especially mucosal, immune barrier even more crucial. Vaccination is the most effective way to protect the body from pathogens. Compared to traditional attenuated / inactivated vaccines that contain all pathogens, subunit vaccines have been widely developed due to their high safety and stability. However, they also face core bottlenecks: low protein antigen delivery efficiency, difficulty in crossing mucosal barriers, and weak immunogenicity. Therefore, adjuvants are needed to enhance immunity. Among existing adjuvants, aluminum adjuvant is the most classic, but it only induces humoral immunity. Therefore, there is an urgent need to develop novel adjuvant molecules that can induce potent cellular and humoral immunity. Summary of the Invention

[0003] To address the aforementioned issues, this invention provides a zinc ion-peptide complex immunoadjuvant, its preparation method, and its applications. The coordination of zinc ions with functional peptides drives self-assembly to form a hydrogel structure, which can effectively encapsulate the S protein antigen, achieving effective loading and controlled release. When combined with a CpG ODN immunoactivator, it synergistically achieves adjuvant-antigen co-delivery. This system can mimic the multi-pathway immune activation process of natural infection, significantly enhancing innate immune signal amplification, thereby inducing stronger and more balanced mucosal, humoral, and cellular immune responses, particularly excelling in enhancing cellular immunity. This work demonstrates the prospects and potential of zinc ion-peptide complexes as immunoadjuvants.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing a zinc ion polypeptide complex immune adjuvant, comprising the following steps: The peptide, PBS buffer, SARS-CoV-2 S protein, CpG ODN immune activator and zinc ions were mixed, the pH was adjusted to 7.4 and then allowed to stand to obtain zinc ion peptide complex immune adjuvant. The polypeptide is Nap-GFFYGGGHRGD, and all amino acids are L-shaped.

[0005] Preferably, the polypeptides are mixed in the form of a polypeptide solution, wherein the polypeptide content in the polypeptide solution is 2% by mass.

[0006] Preferably, the SARS-CoV-2 S protein is mixed in the form of a SARS-CoV-2 S protein solution, and the concentration of SARS-CoV-2 S protein in the SARS-CoV-2 S protein solution is 1 mg / mL.

[0007] Preferably, the CpG ODN immune activator is mixed in the form of an immune activator solution, wherein the concentration of the CpG ODN immune activator in the immune activator solution is 2 µg / µL.

[0008] Preferably, the zinc ions are mixed in the form of a zinc chloride solution, wherein the concentration of zinc chloride in the zinc chloride solution is 100 mM.

[0009] Preferably, the volume ratio of the polypeptide solution, PBS buffer, SARS-CoV-2 S protein solution, immune activator solution, and zinc chloride solution is 50:19.98:5:20:5.02. The pH value of the PBS buffer is 7.2~7.4.

[0010] Preferably, the settling time is 10-30 minutes and the temperature is 20-30°C.

[0011] The present invention also provides an immune adjuvant of zinc ion polypeptide complex prepared by the preparation method described in the above technical solution.

[0012] This invention also provides the application of the zinc ion polypeptide complex immune adjuvant described in the above technical solution in the preparation of drugs for immune protection against SARS-CoV-2 infection.

[0013] Preferably, the zinc ion polypeptide complex immune adjuvant enhances mucosal immunity, humoral immunity, and cellular immune responses.

[0014] The beneficial effects of this invention are: This invention reports a novel zinc ion-peptide adjuvant by coordinating metal ions with self-assembled peptides. Further, it combines this adjuvant with CpG ODN, an immune activator, to construct a composite adjuvant system. Using the S protein as an antigen model, this system synergistically achieves adjuvant-antigen co-delivery, enhancing immune control against SARS-CoV-2. Zinc ions mediate the potent self-assembly of peptides to form a hydrogel with improved mechanical properties, allowing for unimpeded antigen encapsulation and enhancing antigen stability and delivery efficiency. The dual adjuvant system, combined with CpG ODN, demonstrates excellent performance in activating lung tissue immunity. At the cellular level, it not only enhances CD4... + T and CD8 + The strength of the T cell response further promotes the formation of related tissue-resident memory T cell phenotypes; at the humoral immunity level, Gel (Zn 2+ The dual adjuvant system of T cells and CpGODN significantly stimulated the increase and activation of B cells and effectively stimulated the secretion of sIgA, the main antibody of mucosal immunity. Overall, the dual adjuvant achieved synergistic enhancement of T cell (including TRM) and B cell immunity. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 Here is the structural diagram of the Nap-GFFYGGGHRGD polypeptide; Figure 2 MS spectrum of Nap-GFFYGGGHRGD; Figure 3 This is a schematic diagram of the Fmoc peptide solid-phase synthesis method (SPPS). Figure 4 For Gel (Zn) 2+ Electron microscopy morphology, circular dichroism (CD) and rheological characterization of ) Figure 5 The activation effect of the compound adjuvant on the CD4+ T cell population in lung tissue; Figure 6 The activation effect of the compound adjuvant on the CD8+ T cell population in lung tissue; Figure 7 The activation effect of compound adjuvants on lung tissue B cell population; Figure 8 HE sections of major organs and CCK-8 cell viability curves. Detailed Implementation

[0017] This invention provides a method for preparing a zinc ion-peptide complex immune adjuvant, comprising the following steps: mixing a peptide, PBS buffer, SARS-CoV-2 S protein, CpG ODN immune activator, and zinc ions, adjusting the pH to 7.4, and allowing it to stand to obtain the zinc ion-peptide complex immune adjuvant; wherein the peptide is Nap-GFFYGGGHRGD, and the amino acid configuration is L-type.

[0018] The present invention does not impose any particular limitation on the preparation method of the polypeptide; those skilled in the art can use conventional preparation methods.

[0019] In this invention, the polypeptides are preferably mixed in the form of a polypeptide solution, and the polypeptide content in the polypeptide solution is preferably 1% by mass. In this invention, the SARS-CoV-2 S protein, i.e., the spike glycoprotein on the surface of the SARS-CoV-2 viral capsid, is provided by the project collaborator, Chengdu MaxVax Biotech. The original wild-type S protein has the Uniprot accession number P0DTC2 and the NCBI accession number YP_009724390.1. The SARS-CoV-2 S protein is preferably mixed in the form of a SARS-CoV-2 S protein solution, and the concentration of the SARS-CoV-2 S protein in the solution is preferably 1 mg / mL. In this invention, the CpG ODN immune activator is preferably mixed in the form of an immune activator solution, and the concentration of the CpG ODN immune activator in the immune activator solution is preferably 2 µg / µL. In this invention, the CpG ODN immune activator is derived from Suzhou Hongxun Biotechnology Co., Ltd., specifically CpG ODN 1826. In this invention, the zinc ions are preferably mixed in the form of a zinc chloride solution, and the concentration of the zinc chloride in the zinc chloride solution is preferably 10 mM. In this invention, the preferred volume ratio of the polypeptide solution, PBS buffer, SARS-CoV-2 S protein solution, immune activator solution, and zinc chloride solution is 50:19.98:5:20:5.02. The preferred pH value of the PBS buffer is 7.2-7.4. The preferred standing time is 10-30 minutes, and the preferred temperature is 20-30°C.

[0020] The present invention also provides an immune adjuvant of zinc ion polypeptide complex prepared by the preparation method described in the above technical solution.

[0021] This invention also provides the application of the zinc ion polypeptide complex immune adjuvant described in the above-mentioned technical solution in the preparation of drugs for immune protection against SARS-CoV-2 infection. In this invention, the zinc ion polypeptide complex immune adjuvant enhances mucosal immunity, humoral immunity, and cellular immune responses.

[0022] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention. Example 1

[0023] 1. The designed polypeptide molecule is named Nap-GFFYGGGHRGD (all amino acids are L-configured), hereinafter referred to as Gel, and its specific structure is as follows: Figure 1 As shown.

[0024] Molecular design: (hydrophobic end capping - assembly unit - zinc ion chelating fragment - cell adhesion module). Hydrophobic end capping: Nap is responsible for inducing hydrophobic stacking between molecules; Assembly unit: GFFY provides π–π stacking; Metal chelate segment: GGGH separates the hydrophobic segment from the chelate segment, providing multiple sites for His and Zn²⁺ coordination; Cell adhesion module: RGD mucosal targeting promotes lung targeting and endocytosis.

[0025] The designed polypeptide LC-MS spectrum is as follows: Figure 2 As shown.

[0026] The specific preparation method is as follows: The peptide Nap-GFFYGGGHRGD (e.g., 100mg / L) was synthesized using the standard Fmoc SPPS method. Figure 3 (As shown). The specific steps are as follows: After swelling the CTC resin with DCM for 10 min, Fmoc-Asp(Otbu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr(Otbu)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, and Fmoc-Gly-OH are sequentially coupled to the CTC resin using HBTU / DIEA. The resin is then protected with DMF containing 20% ​​piperidine. Nap is then added to the N-terminus of Gly to cap the peptides. After synthesis, a mixture of TFA / TIS / H2O (95% / 2.5% / 2.5%) is added to cleave the peptides from the resin. The reaction solution is purified by filtration using a preparative reversed-phase high-performance liquid chromatography column. 2. The designed compound adjuvant Gel (Zn 2+ ), Gel (Zn 2+ )+S, CpG ODN+Gel+S, CpG ODN+Gel(Zn 2+ The assembly parts are shown in the table below: Table 1 Components (% is volume percentage)

[0027] I. General Preparation 1. Use sterile PBS (pH 7.2–7.4) as the solvent.

[0028] 2. Prepare the following stock solutions: peptide solution (2wt%), protein antigen (SARS-CoV-2 S protein) solution (1 mg / mL), CpG ODN (CpG ODN immune activator) solution (2 µg / µL), and ZnCl2 solution (100mM).

[0029] 3. All components should be handled at room temperature or 4°C. When mixing, use gentle blowing to avoid vigorous swirling and foaming.

[0030] II. Preparation process of four formulas 1) Gel (Zn²⁺): Gel(Zn²⁺): Take 22.49 µL of PBS, add 25.00 µL of 2 wt% peptide solution and mix gently. Finally, add 2.51 µL of 100 mM ZnCl₂ solution (corresponding to Zn²⁺ being 0.7 eq of the peptide) and mix gently by pipetting. Let stand at room temperature for 10–30 min to form Gel(Zn²⁺).

[0031] 2) Gel(Zn²⁺) + antigen: Gel(Zn²⁺)+ antigen: Take 19.99 µL of PBS, add 25.00 µL of 2 wt% peptide solution and mix gently, add 2.50 µL (2.5 µg) of SARS-CoV-2 S protein solution (1 mg / mL) and mix gently, and finally add 2.51 µL (0.7 eq) of 100 mM ZnCl2 solution to induce assembly and mix gently. Let stand at room temperature for 10–30 min to obtain Gel(Zn²⁺)+ antigen composite gel.

[0032] 3) CpG ODN + Gel + Antigen (Zinc-free): Take 12.50 µL of PBS, add 25.00 µL of 2 wt% peptide solution and mix gently. Then add 2.50 µL (2.5 µg) of SARS-CoV-2 S protein solution (1 mg / mL) and 10.00 µL (20 µg) of CpG ODN solution (2 µg / µL) and mix gently. Let stand at room temperature for 10–60 min to obtain the CpG ODN + Gel + antigen complex system.

[0033] 4) CpG ODN + Gel(Zn²⁺) + Antigen: CpG ODN + Gel(Zn²⁺) + Antigen: Take 9.99 µL of PBS, add 25.00 µL of 2 wt% peptide solution and mix gently. Then add 2.50 µL (2.5 µg) of SARS-CoV-2 S protein solution (1 mg / mL) and 10.00 µL (20 µg) of CpG ODN solution (2 µg / µL) and mix gently. Finally, add 2.51 µL (0.7 eq) of 100 mM ZnCl2 solution to induce assembly and mix gently. Let stand at room temperature for 10–30 min to obtain CpG ODN + Gel(Zn²⁺) + antigen composite gel. Application Example 1

[0034] 1. Nap-GFFYGGGHRGD can be strongly assembled by zinc ions and has improved mechanical properties. The Nap-GFFYGGGHRGD peptide exhibits weak self-assembly under heating and cooling conditions, forming very sparse short fibers, named Gel. Under different zinc ion equivalents, the assembly behavior shows a transformation from nanoparticle to nanofiber morphology. Furthermore, at a zinc ion equivalent of 0.7 eq, the densest and most uniform nanofibers are formed, named Gel(Zn) 2+ Add Zn 2+ Subsequently, the CD spectrum changed, proving that Zn 2+ Successful coordination of Zn; and rheological analysis shows that, with the elastic modulus remaining constant, Zn 2+ The coordination enhances the overall mechanical properties of the hydrogel.

[0035] 2. Gel (Zn) 2+ CD4 enhancement with CpG ODN dual adjuvant system + T cell response and induction of related TRM phenotype Mice were randomly assigned to groups for inhalation administration. The KW-DM-YWH rat and mouse nebulizer (from Calvin Biotechnology Co., Ltd.) was used for nebulized inhalation drug delivery: the prepared drug solution was added to the nebulizer cup, and the nebulizer was connected to the sealed drug delivery chamber via a tubing. The nebulization time and output intensity were set, and nebulization began, allowing the mice to inhale the aerosol within the sealed chamber to complete the drug delivery. The nominal dose per mouse was 50 µL / dose, for a total of 3 doses, administered at preset time points within a 21-day dosing cycle. Dosing conditions were consistent across groups. After each administration, the mice's condition was observed, and the nebulizer cup and tubing were cleaned to avoid cross-contamination. Mice were sacrificed on day 21, and lung tissue was harvested. The lung tissue was minced and ground using a grinder. After terminating the reaction, the mixture was filtered through a cell sieve to obtain a cell suspension; erythrocyte lysis was performed if necessary. After centrifugation and washing, the cells were resuspended, counted, and their concentration adjusted for subsequent flow cytometry staining.

[0036] CpG ODN+Gel (Zn 2+ Dual adjuvants at CD45 + CD4 levels were significantly increased in immune cells. + The proportion of T cells indicates more efficient local T cell recruitment / expansion; and in CD4 + In the context of T cells, upregulation of CD69 indicates enhanced tissue retention and early activation characteristics; while further upregulation of CD103... + CD69 +The increased proportion of these cells indicates that this population better conforms to the classic tissue-resident memory T cell (TRM) phenotype, reflecting that the dual adjuvant system not only "increased the number of T cells" but also promoted their differentiation towards "tissue-resident memory." This is particularly crucial for respiratory viruses: TRMs can react rapidly at the entry point of infection, expand locally, and produce effector factors, achieving faster early control.

[0037] 3. Gel (Zn) 2+ CD8 enhancement with CpG ODN dual adjuvant system + T cell responses induce the relevant TRM phenotype (this step is consistent with the previous heading). With CD4 + The T cell population results were consistent, CpG ODN+Gel (Zn) 2+ Dual adjuvants at CD45 + The level of CD8 in lung tissue was significantly increased in immune cells. + The proportion of T cells was increased, and CD103 was further enhanced. + CD69 + The presence of a TRM-like CD8 cell population indicates that the dual adjuvant system not only initiates an immune response against the S protein but also enables its immune clearance. In summary, Gel (Zn 2+ In synergy with CpG ODN, it not only enhances CD4 in lung tissue + T and CD8 + The strength of the T-cell response also promotes the formation of the TRM phenotype, thus bringing it closer to the immunological goal of "long-term frontline protection" for mucosal vaccines.

[0038] 4. Gel (Zn) 2+ The dual adjuvant system of CpG and ODN enhances B cell population response and sIgA secretion. Mice were randomly assigned to groups for inhalation administration. The KW-DM-YWH rat and mouse nebulizer (from Calvin Biotechnology Co., Ltd.) was used for nebulized inhalation drug delivery: the prepared drug solution was added to the nebulizer cup, and the nebulizer was connected to the sealed drug delivery chamber via a tubing. The nebulization time and output intensity were set, and nebulization began, allowing the mice to inhale the aerosol within the sealed chamber to complete the drug delivery. The nominal dose per mouse was 50 µL / dose, for a total of 3 doses, administered at preset time points within a 21-day dosing cycle. Dosing conditions were consistent across groups. After each administration, the mice's condition was observed, and the nebulizer cup and tubing were cleaned to avoid cross-contamination. On day 21, the mice were dissected, blood was collected from the eyeballs, and serum was collected. Left lung tissue was harvested, homogenized using a grinder, and then filtered through a cell sieve, followed by erythrocyte lysis, washing, and resuspending to obtain a single-cell suspension. After cell counting and concentration adjustment, lung cells were labeled with antibodies and then analyzed by flow cytometry. The serum was diluted 5000-fold with PBS for sIgA ELISA testing.

[0039] Further evaluation was conducted on B-cell responses in lung tissue 21 days after administration. CpG ODN+Gel (Zn 2+ Dual adjuvants at CD45 + The level of CD19 in lung tissue was significantly increased in immune cells. + Based on the proportion of B cells, compared with other adjuvant-only groups, CpG ODN+Gel (Zn 2+ Dual adjuvants have CD38 + The highest proportion of activated B cells suggests that dual adjuvants more effectively promote B cell activation and effector differentiation in local lymphoid / lung tissues, providing an immune basis for sustained antibody production and mucosal protection. Furthermore, it is noteworthy that within the flow cytometry range, the proportions of IgA and IgG in the CD38-positive population were similar, with no significant differences between groups. Since sIgA is the main antibody in the mucosal immune response and to further refine the humoral immune evidence chain, serum sIgA levels were further measured to comprehensively assess systemic and mucosal immune responses. Results showed that CpG ODN+Gel (Zn 2 + The dual adjuvant significantly increased sIgA secretion, indicating that CpG ODN+Gel (Zn) 2+ In addition to increasing the number of B cells and activated B cells, dual adjuvants can also successfully stimulate mucosal immunity, providing more comprehensive immune protection against COVID-19 infection.

[0040] 5. The compound adjuvant system exhibits good biocompatibility. Mice were randomly assigned to groups for inhalation administration. The KW-DM-YWH rat and mouse nebulizer (from Calvin Biotechnology Co., Ltd.) was used for nebulized inhalation drug delivery: the prepared drug solution was added to the nebulizer cup, and the nebulizer was connected to the sealed drug delivery chamber via a tubing. The nebulization time and output intensity were set before nebulization began, allowing the mice to inhale the aerosol within the sealed chamber to complete the drug delivery. The nominal dose per mouse was 50 µL / dose, for a total of 3 doses, administered at preset time points within a 21-day dosing cycle. Dosing conditions were consistent across groups. After each administration, the mice's condition was observed, and the nebulizer cup and tubing were cleaned to avoid cross-contamination. On day 21, the mice were dissected, and the heart, liver, spleen, lung (right lung), and kidney were harvested. After fixation in paraformaldehyde for 24 hours, the tissues were dehydrated and cleared, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

[0041] Histological sections were taken from the heart, liver, spleen, lung, and kidney, the main organs involved. The left side of the HE test showed that the heart, liver, spleen, lung, and kidney tissues in each treatment group were structurally intact, with no obvious inflammatory infiltration or necrosis or other pathological damage. The right side of the CCK-8 assay showed that the cell viability curve remained close to the control level within the range of 0.1-1.0 eq zinc ion equivalents, indicating that the 0.7 eq zinc ion used was non-cytotoxic and had good safety.

[0042] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a zinc ion polypeptide complex immunoadjuvant, characterized by, Includes the following steps: The peptide, PBS buffer, SARS-CoV-2 S protein, CpG ODN immune activator and zinc ions were mixed, the pH was adjusted to 7.4 and then allowed to stand to obtain zinc ion peptide complex immune adjuvant. The polypeptide is Nap-GFFYGGGHRGD, and all amino acids are L-shaped.

2. The production method according to claim 1, characterized by, The polypeptides are mixed in the form of a polypeptide solution, wherein the polypeptide content in the polypeptide solution is 2% by mass.

3. The preparation method according to claim 1, characterized in that, The SARS-CoV-2 S protein is mixed in the form of a SARS-CoV-2 S protein solution, and the concentration of SARS-CoV-2 S protein in the SARS-CoV-2 S protein solution is 1 mg / mL.

4. The production method according to claim 1, characterized by, The CpG ODN immune activator is mixed in the form of an immune activator solution, wherein the concentration of CpG ODN immune activator in the immune activator solution is 2 µg / µL.

5. The preparation method according to claim 1, characterized in that, The zinc ions are mixed in the form of a zinc chloride solution, wherein the concentration of zinc chloride in the zinc chloride solution is 100 mM.

6. The method of any one of claims 1 to 5, wherein the method further comprises the step of: The volume ratio of the polypeptide solution, PBS buffer, SARS-CoV-2 S protein solution, immune activator solution, and zinc chloride solution is 50:19.98:5:20:5.02, and the pH value of the PBS buffer is 7.2~7.

4.

7. The preparation method according to claim 1, characterized in that, The settling time is 10-30 minutes, and the temperature is 20-30℃.

8. An immune adjuvant consisting of a zinc ion polypeptide complex prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the zinc ion polypeptide complex immune adjuvant according to claim 8 in the preparation of drugs for immune protection against SARS-CoV-2 infection.

10. The application according to claim 9, characterized in that, The zinc ion polypeptide complex immune adjuvant enhances mucosal immunity, humoral immunity, and cellular immune responses.

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