An in situ subunit hydrogel vaccine preparation and its preparation method and application

Abstract

The present invention relates to an in situ subunit hydrogel vaccine preparation, a preparation method and application thereof, an in situ subunit hydrogel vaccine preparation, comprising a supramolecular polypeptide hydrogel carrier and a subunit vaccine component loaded on the supramolecular polypeptide hydrogel carrier; the subunit vaccine component comprises an antigen, an adjuvant and an immunomodulator. The in situ subunit hydrogel vaccine preparation provided by the present invention can control the slow release of vaccine components, continuously activate the immune response effect in the body, thereby establishing an immune barrier, timely cutting off virus transmission and blocking viral infection. The designed polypeptide sequence can self-assemble to form positively charged nanofibers, which can adsorb vaccine components and further form subunit hydrogel vaccines in situ in a physiological environment; by detecting the drug release behavior, it is shown that its subunit hydrogel vaccine can slowly release vaccine components for a long time, thereby increasing the antigen exposure time and continuously activating the immune response mechanism in the body.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an in situ subunit hydrogel vaccine preparation, a preparation method and an application thereof. Background Art

[0002] Vaccines are the most effective strategy for preventing SARS-CoV-2 infection, as our immune system is the most important line of defense against viral infection. The high mutation rate of SARS-CoV-2 variants, asymptomatic transmission, and lack of effective treatments make its spread difficult to control. Therefore, deploying a safe and effective vaccine to end SARS-CoV-2 infection and interrupt viral transmission is essential.

[0003] SARS-CoV-2 contains numerous protein targets. Among various regions of the S protein, the receptor-binding domain (RBD) protein, located in the S1 subunit, is a promising candidate for vaccine development. It plays a crucial role in viral entry by interacting with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells. Furthermore, the RBD protein is highly efficient and stable. Given its importance in virus / cell interactions, its immunogenicity at the B and T cell levels, and its ability to induce neutralizing antibodies in infected patients, it has been a primary target antigen in vaccine development. However, RBD exhibits low immunogenicity. Improving its immune response with the adjuvant CpG ODN can enhance the magnitude, quality, and durability of the immune response elicited by vaccination, even with lower antigen doses. However, both the RBD and the adjuvant CpG ODN exhibit a short half-life after administration due to rapid clearance from the body. This results in a short antigen exposure time, often insufficient to elicit a robust immune response. If the controlled and sustained delivery of RBD antigens and adjuvant CpG ODN can be achieved after a single vaccination, inducing high concentrations and long-lasting neutralizing antibodies, the efficacy and durability of subunit vaccines will be greatly improved, effectively helping to establish a population immune barrier, blocking viral mutations, and ending the epidemic as soon as possible. Therefore, the development of a delivery platform that can enhance the controlled release of subunit vaccines is currently urgently needed. Summary of the Invention

[0004] The purpose of the present invention is to provide an in situ subunit hydrogel vaccine preparation, its preparation method and application in order to overcome the defects of existing vaccine technology. The existing new coronavirus vaccine has problems such as dependence on cold chain, multiple vaccinations, and large toxic side effects. The present invention constructs a supramolecular polypeptide hydrogel for delivering subunit vaccines, and prepares a hydrogel vaccine delivery system with good safety, high stability and high drug loading, thereby achieving controllable and long-term delivery of subunit vaccine components, inducing high concentration and long-lasting neutralizing antibodies, and thus effectively preventing SARS-CoV-2 infection and spread.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] A supramolecular polypeptide hydrogel carrier is obtained by the following method: polypeptides are self-assembled in a solvent to form nanofibers, and then the supramolecular polypeptide hydrogel carrier is formed in a physiological environment;

[0007] The polypeptide molecule sequences include but are not limited to: KKFKFEFEF, AEAEAKAKAAEAEAKAK, AGEDQLKHVFS, FFFRGDR, FKFEFKFF, FEFEFKFK, FHFDFHFD, GV2Q2HKD, GGVVVRGDR, GSFSIQYTYHV, IKVAV, KFKFEFKFE, IKVKIKVKVPPTKIKVKIK, KYFIL, K2(QL)6K2, K2(SL)6K2, K(SL)6KGP RKLYDY, VKVKVPPTKVKVKVKVKVKV, KLDLPVGLIGKLDL, K2W(QL)6K2, KNEFKAAFDI, KFDLKKDLKLDL, RGDSRGDS, RADARADA RADARADA, Fmoc-FFVPGVGQGK, VLTKVKTKVPLPTKVEVKVLV, Fmoc-FFRGD, Fmoc-FRGDF, Nap-GFFYGRGD, Nap-GFFYGRGDH.

[0008] An in situ subunit hydrogel vaccine preparation comprises the supramolecular polypeptide hydrogel carrier and a subunit vaccine component loaded on the supramolecular polypeptide hydrogel carrier; the subunit vaccine component comprises an antigen, an adjuvant and an immunomodulator.

[0009] The present invention also provides a method for preparing an in situ subunit hydrogel vaccine preparation, the specific steps of which are as follows:

[0010] S1, synthesis of self-assembling peptides;

[0011] S2, dissolving the self-assembling polypeptide obtained in step S1 in a solvent to obtain a self-assembling nanofiber solution;

[0012] S3. Add the subunit vaccine components to the self-assembled nanofiber solution obtained in step S2 and incubate, and then form an in situ subunit hydrogel vaccine preparation in a physiological environment.

[0013] Furthermore, in step S1, the self-assembling polypeptide is obtained by solid phase synthesis.

[0014] Further, in step S1, the self-assembling polypeptide molecule sequence is as follows, including but not limited to: KKFKFEFEF, AEAEAKAKAAEAEAKAK, AGEDQLKHVFS, FFFRGDR, FKFEFKFF, FEFEFKFK, FHFDFHFD, GV2Q2HKD, GGVVVRGDR, GSFSIQYTYHV, IKVAV, KFKFEFKFE, IKVKIKVKVPPTKIKVKIK, KYFIL, K2(QL)6K2, K2(SL)6K2, K (SL)6KGPRKLYDY, VKVKVPPTKVKVKVKVKVKV, KLDLPVGLIGKLDL, K2W(QL)6K2, KNEFKAAFDI, KFDLKKDLKLDL, RGDSRGDS, RADA RADARADARADA, Fmoc-FFVPGVGQGK, VLTKVKTKVPLPTKVEVKVLV, Fmoc-FFRGD, Fmoc-FRGDF, Nap-GFFYGRGD, Nap-GFFYGRGDH;

[0015] Among them, A is alanine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I isoleucine, K is lysine, L is leucine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, and Y is tyrosine.

[0016] Furthermore, in step S2, the solvent is selected from water, physiological saline or phosphate buffer.

[0017] Furthermore, in step S2, the concentration of the self-assembling polypeptide in the solvent is 1-100 mM.

[0018] Furthermore, in step S3, the incubation temperature is 4-50° C., and the incubation time is 0-3 h.

[0019] Furthermore, in step S3, the subunit vaccine components include antigens, adjuvants and immunomodulators.

[0020] Furthermore, the vaccine antigens include but are not limited to RBD protein, S protein, S1 protein, S2 protein, N protein, E protein, and M protein.

[0021] Furthermore, the vaccine adjuvant includes but is not limited to CpG ODN, CpG 1018, Poly (I: C), R837, R848, MF59, AS03, AS01B, MPLA, ADU-S100, MAVU-140, GSK3745417, IMSA-101, MK-1454, E7766, SB11285, BMS-986301, SR-717lithium, MSA-2, c-di-GMP, c-di-AMP, tautomerism, 3',3'-cGAMP, 2',3'-cGAMP, HG381, alum, lipopolysaccharide, and aluminum salt.

[0022] Furthermore, the immunomodulators include but are not limited to GM-CSF, IL-2, IFN-α, IFN-γ, IFN-β, CXCL-9, CXCL-10, CXCL-11, sotigalimab, cifurtilimab, tecaginlimab, CDX-1140, YH-003, AMG-994, SL-172154, IL-12, IL-21, IL-22, CCL27, CCL28, and QS-21.

[0023] Furthermore, the vaccine component antigen is RBD protein, and the mass of RBD is 0.001-50 mg;

[0024] The vaccine adjuvant is CpG ODN, and the mass of the CpG ODN is 0.001-50 mg;

[0025] The immunomodulator is GM-CSF, and the mass of the GM-CSF is 0.001-50 mg.

[0026] In addition, the present invention also provides an application of an in situ subunit hydrogel vaccine preparation, which is used in the preparation of a vaccine for terminating SARS-CoV-2 infection and blocking virus transmission.

[0027] Furthermore, the SARS-CoV-2 includes but is not limited to wild, Alpha, Beta, Gamma, Delta, Omicron, B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.1.529, BA.2, BA.1, BA.1.1, BA.3, BA.4, and BA.5.

[0028] Hydrogels are a class of highly promising "smart" drug delivery vehicles that can achieve controlled delivery of vaccine components. They have adjustable mechanical properties and good biocompatibility. Among them, supramolecular hydrogels constructed with self-assembling polypeptides have attracted widespread attention due to their good safety, biodegradability, easy preparation and functionality. They can achieve the co-delivery of multiple components, control the release of payloads and prevent the occurrence of undesirable immune responses.

[0029] The present invention relates to an in situ subunit hydrogel vaccine, which can increase the retention of vaccine components at the vaccination site through local delivery, and control and continuously release the vaccine components. While improving drug concentration and bioavailability, it greatly reduces the exposure time of the vaccine components in the blood and reduces their toxic side effects.

[0030] The present invention is based on the fact that supramolecular polypeptide self-assembly can achieve its instant "solution-gel" transition under physiological conditions, directly subcutaneously inoculated in liquid state, and then undergoes in situ phase transition to form a hydrogel vaccine preparation.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention utilizes the "solution-gel" transition property of supramolecular polypeptides. After inoculation in solution form, the supramolecular subunit hydrogel vaccine (RCG / KKFE8) is triggered at the injection site to construct an in situ drug reservoir, control the slow and sustained release of vaccine components (RBD, CpG ODN, and GM-CSF), and prevent the occurrence of undesirable immune responses.

[0033] (2) The hydrogel vaccine prepared by the present invention can serve as an "artificial tertiary lymphatic structure" to recruit and program immune cells at the vaccination site. The activated immune cells further migrate to the lymph nodes, inducing high titers of binding and neutralizing antibodies, blocking the recognition of the virus and ACE2, thereby effectively preventing SARS-CoV-2 infection;

[0034] (3) The in situ subunit hydrogel vaccine preparation prepared by the present invention is simple to prepare, convenient to administer, and easy to transport and store, which provides favorable conditions for the transportation, storage, and vaccination of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the mass spectrum of the KFKFEFKFE polypeptide of Example 1 of the present invention;

[0036] Figure 2 This is an electron microscope characterization image of the KKFE8 nanofiber of Example 2 of the present invention;

[0037] Figure 3 This is a characterization diagram of the "solution-gel" transition of KKFE8 nanofibers according to Example 2 of the present invention;

[0038] Figure 4 This is the Zeta potential diagram of the RCG / KKFE8 solution of Example 3 of the present invention;

[0039] Figure 5 This is a diagram showing the drug release characteristics of Example 4 of the present invention;

[0040] Figure 6 This is a schematic diagram of the formation of the in situ subunit hydrogel vaccine according to Example 5 of the present invention;

[0041] Figure 7 This is a diagram of antibody production induced by the RCG / KKFE8 subunit hydrogel vaccine of Example 6 of the present invention. DETAILED DESCRIPTION

[0042] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] A supramolecular polypeptide hydrogel carrier is obtained by the following method: polypeptides are self-assembled in a solvent to form nanofibers, and then the supramolecular polypeptide hydrogel carrier is formed in a physiological environment;

[0044] The polypeptide molecule sequences include but are not limited to: KKFKFEFEF, AEAEAKAKAAEAEAKAK, AGEDQLKHVFS, FFFRGDR, FKFEFKFF, FEFEFKFK, FHFDFHFD, GV2Q2HKD, GGVVVRGDR, GSFSIQYTYHV, IKVAV, KFKFEFKFE, IKVKIKVKVPPTKIKVKIK, KYFIL, K2(QL)6K2, K2(SL)6K2, K(SL)6KGP RKLYDY, VKVKVPPTKVKVKVKVKVKV, KLDLPVGLIGKLDL, K2W(QL)6K2, KNEFKAAFDI, KFDLKKDLKLDL, RGDSRGDS, RADARADA RADARADA, Fmoc-FFVPGVGQGK, VLTKVKTKVPLPTKVEVKVLV, Fmoc-FFRGD, Fmoc-FRGDF, Nap-GFFYGRGD, Nap-GFFYGRGDH.

[0045] An in situ subunit hydrogel vaccine preparation comprises the supramolecular polypeptide hydrogel carrier and a subunit vaccine component loaded on the supramolecular polypeptide hydrogel carrier; the subunit vaccine component comprises an antigen, an adjuvant and an immunomodulator.

[0046] The present invention also provides a method for preparing an in situ subunit hydrogel vaccine preparation, the specific steps of which are as follows:

[0047] S1, synthesis of self-assembling peptides;

[0048] S2, dissolving the self-assembling polypeptide obtained in step S1 in a solvent to obtain a self-assembling nanofiber solution;

[0049] S3. Add the subunit vaccine components to the self-assembled nanofiber solution obtained in step S2 and incubate, and then form an in situ subunit hydrogel vaccine preparation in a physiological environment.

[0050] Furthermore, in step S1, the self-assembling polypeptide is obtained by solid phase synthesis.

[0051] Further, in step S1, the self-assembling polypeptide molecule sequence is as follows, including but not limited to: KKFKFEFEF, AEAEAKAKAAEAEAKAK, AGEDQLKHVFS, FFFRGDR, FKFEFKFF, FEFEFKFK, FHFDFHFD, GV2Q2HKD, GGVVVRGDR, GSFSIQYTYHV, IKVAV, KFKFEFKFE, IKVKIKVKVPPTKIKVKIK, KYFIL, K2(QL)6K2, K2(SL)6K2, K (SL)6KGPRKLYDY, VKVKVPPTKVKVKVKVKVKV, KLDLPVGLIGKLDL, K2W(QL)6K2, KNEFKAAFDI, KFDLKKDLKLDL, RGDSRGDS, RADA RADARADARADA, Fmoc-FFVPGVGQGK, VLTKVKTKVPLPTKVEVKVLV, Fmoc-FFRGD, Fmoc-FRGDF, Nap-GFFYGRGD, Nap-GFFYGRGDH;

[0052] Among them, A is alanine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I isoleucine, K is lysine, L is leucine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, and Y is tyrosine.

[0053] Furthermore, in step S2, the solvent is selected from water, physiological saline or phosphate buffer.

[0054] Furthermore, in step S2, the concentration of the self-assembling polypeptide in the solvent is 1-100 mM.

[0055] Furthermore, in step S3, the incubation temperature is 4-50° C., and the incubation time is 0-3 h.

[0056] Furthermore, in step S3, the subunit vaccine components include antigens, adjuvants and immunomodulators.

[0057] Furthermore, the vaccine antigens include but are not limited to RBD protein, S protein, S1 protein, S2 protein, N protein, E protein, and M protein.

[0058] Furthermore, the vaccine adjuvant includes but is not limited to CpG ODN, CpG 1018, Poly (I: C), R837, R848, MF59, AS03, AS01B, MPLA, ADU-S100, MAVU-140, GSK3745417, IMSA-101, MK-1454, E7766, SB11285, BMS-986301, SR-717lithium, MSA-2, c-di-GMP, c-di-AMP, tautomerism, 3',3'-cGAMP, 2',3'-cGAMP, HG381, alum, lipopolysaccharide, and aluminum salt.

[0059] Furthermore, the immunomodulators include but are not limited to GM-CSF, IL-2, IFN-α, IFN-γ, IFN-β, CXCL-9, CXCL-10, CXCL-11, sotigalimab, cifurtilimab, tecaginlimab, CDX-1140, YH-003, AMG-994, SL-172154, IL-12, IL-21, IL-22, CCL27, CCL28, and QS-21.

[0060] Furthermore, the vaccine component antigen is RBD protein, and the mass of RBD is 0.001-50 mg;

[0061] The vaccine adjuvant is CpG ODN, and the mass of the CpG ODN is 0.001-50 mg;

[0062] The immunomodulator is GM-CSF, and the mass of the GM-CSF is 0.001-50 mg.

[0063] In addition, the present invention also provides an application of an in situ subunit hydrogel vaccine preparation, which is used in the preparation of a vaccine for terminating SARS-CoV-2 infection and blocking virus transmission.

[0064] Furthermore, the SARS-CoV-2 includes but is not limited to wild, Alpha, Beta, Gamma, Delta, Omicron, B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.1.529, BA.2, BA.1, BA.1.1, BA.3, BA.4, and BA.5.

[0065] In the following examples, the required materials are as follows: RBD (Arg319-Ser591) was purchased from Suzhou Jinan Protein Technology Co., Ltd., and CpG ODN and GM-CSF were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0066] In the following examples, the sequences are as follows:

[0067] The sequence of CpG ODN is shown in SEQ ID NO. 1: 5′-TCCATGACGTTCCTGACGTT-3′;

[0068] The sequence of GM-CSF is shown in SEQ ID NO. 2:

[0069] MAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQ TRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFL TDIPFECKKPVQK;

[0070] The sequence of the polypeptide molecule KKFKFEFEF is shown in SEQ ID NO. 3: KKFKFEFEF;

[0071] The sequence of the polypeptide molecule AEAEAAKAKAEAEAKAK is shown in SEQ ID NO.4:

[0072] AEAEAKAKAEAEAKAK;

[0073] The sequence of the polypeptide molecule AGEDQLKHVFS is shown in SEQ ID NO. 5: AGEDQLKHVFS;

[0074] The sequence of the polypeptide molecule FFFRGDR is shown in SEQ ID NO. 6: FFFRGDR;

[0075] The sequence of the polypeptide molecule FKFEFKFF is shown in SEQ ID NO. 7: FKFEFKFF;

[0076] The sequence of the polypeptide molecule FEFEFKFK is shown in SEQ ID NO. 8: FEFEFKFK;

[0077] The sequence of the polypeptide molecule FHFDFHFD is shown in SEQ ID NO. 9: FHFDFHFD;

[0078] The sequence of the polypeptide molecule GV2Q2HKD is shown in SEQ ID NO. 10: GVVQQHKD;

[0079] The sequence of the polypeptide molecule GGVVVRGDR is shown in SEQ ID NO. 11: GGVVVRGDR;

[0080] The sequence of the polypeptide molecule GSFSIQYTYHV is shown in SEQ ID NO. 12: GSFSIQYTYHV;

[0081] The sequence of the polypeptide molecule IKVAV is shown in SEQ ID NO. 13: IKVAV;

[0082] The sequence of the polypeptide molecule KFKFEFKFE is shown in SEQ ID NO. 14: KFKFEFKFE;

[0083] The sequence of the polypeptide molecule IKVKIKVKVPPTKIKVKIK is shown in SEQ ID NO.15:

[0084] IKVKIKVKVPPTKIKVKIK;

[0085] The sequence of the polypeptide molecule KYFIL is shown in SEQ ID NO. 16: KYFIL;

[0086] The sequence of the polypeptide molecule K2(QL)6K2 is shown in SEQ ID NO. 17: KKQLQLQLQLQLQLKK;

[0087] The sequence of the polypeptide molecule K2(SL)6K2 is shown in SEQ ID NO.18: KKSLSLSLSLSLSLKK;

[0088] The sequence of the polypeptide molecule K(SL)6KGPRKLYDY is shown in SEQ ID NO.19:

[0089] KSLSLSLSLSLSLKGPRKLYDY;

[0090] The sequence of the polypeptide molecule VKVKVPPTKVKVKVKVKVKV is shown in SEQ ID NO. 20: VKVKVPPTKVKVKVKVKVKV;

[0091] The sequence of the polypeptide molecule KLDLPVGLIGKLDL is shown in SEQ ID NO. 21: KLDLPVGLIGKLDL;

[0092] The sequence of the polypeptide molecule K2W(QL)6K2 is shown in SEQ ID NO. 22: KKWQLQLQLQLQLQLKK;

[0093] The sequence of the polypeptide molecule KNEFKAAFDI is shown in SEQ ID NO. 23: KNEFKAAFDI;

[0094] The sequence of the polypeptide molecule KFDLKKDLKLDL is shown in SEQ ID NO. 24: KFDLKKDLKLDL;

[0095] The sequence of the polypeptide molecule RGDSRGDS is shown in SEQ ID NO. 25: RGDSRGDS;

[0096] The sequence of the polypeptide molecule RADARADARADARADA is shown in SEQ ID NO.26:

[0097] RADARADARADARADA;

[0098] The sequence of the polypeptide molecule FFVPGVGQGK is shown in SEQ ID NO. 27: FFVPGVGQGK;

[0099] The sequence of the polypeptide molecule VLTKVKTKVPLPTKVEVKVLV is shown in SEQ ID NO.28:

[0100] VLTKVKTKVPLPTKVEVKVLV;

[0101] The sequence of the polypeptide molecule FFRGD is shown in SEQ ID NO. 29: FFRGD;

[0102] The sequence of the polypeptide molecule FRGDF is shown in SEQ ID NO. 30: FRGDF;

[0103] The sequence of the polypeptide molecule GFFYGRGD is shown in SEQ ID NO. 31: GFFYGRGD;

[0104] The sequence of the polypeptide molecule GFFYGRGDH is shown in SEQ ID NO.32: GFFYGRGDH.

[0105] Unless otherwise specified, the remaining raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0106] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0107] Example 1

[0108] Synthesis of self-assembling peptides

[0109] The peptide KFKFEFKFE was synthesized by solid-phase synthesis using a Liberty Blue fully automated peptide synthesizer. The crude product was separated and purified using preparative high-performance liquid chromatography. Finally, the dried product was obtained using a freeze dryer. The chemical structure of KFKFEFKFE is as follows:

[0110]

[0111] Figure 1 This is the mass spectrum of the polypeptide KFKFEFKFE in Example 1 of the present invention. Mass spectrometry data analysis proves that the polypeptide molecule KFKFEFKFE is successfully synthesized.

[0112] Example 2

[0113] Preparation of supramolecular polypeptide hydrogel

[0114] The amphiphilic polypeptide KFKFEFKFE (13 mg) was dissolved in deionized water (1 mL), and then allowed to stand at room temperature to obtain a self-assembled nanofiber KKFE8 solution. Then, PBS (20 μL) was added to the KKFE8 solution (180 μL) to obtain the KKFE8 supramolecular hydrogel.

[0115] In this example, the inversion method was used to test the “solution-gel” transition characteristics by simulating the physiological environment in vivo in vitro.

[0116] Figure 2 This is the structure of the assembled KKFE8 according to Example 2 of the present invention. Transmission electron microscopy shows that KKFE8 is a nanofiber structure.

[0117] Figure 3 This is the "solution-gel" transition of the assembly KKFE8 of Example 2 of the present invention. The inversion method was used to determine that KKFE8 has an instant "solution-gel" transition characteristic.

[0118] Example 3

[0119] Preparation method of subunit hydrogel vaccine system

[0120] A KKFE8 solution (10 mM, 180 μL) was prepared, and RBD (10 μg), CpG ODN (100 μg), and GM-CSF (2 μg) were added. The mixture was vortexed and incubated at 37°C for 30 min to prepare the RCG / KKFE8 system. The RCG / KKFE8 solution was then pelleted by ultracentrifugation, and the drug loading efficiency was determined by measuring the RBD, CpG ODN, and GM-CSF contents in the supernatant. Changes in surface potential were measured using a zeta potential meter.

[0121] Figure 4The Zeta potential is characterized by a graph showing that the Zeta potential of the KKFE8 solution is 46.6±3.5 mV. After the KKFE8 solution adsorbs the RBD protein, the Zeta potential decreases to 39.6±2.5 mV, indicating that the RBD protein is loaded onto the surface of the KKFE8 nanofiber through electrostatic action. When CpG ODN and GM-CSF are added to the solution, the Zeta potential of the solution decreases to 26.9±0.9 mV. In addition, no free drug is detected in the supernatant. The above results indicate that the KKFE8 nanofiber solution adsorbs subunit vaccine components (RBD, CpG ODN, GM-CSF) through electrostatic action.

[0122] Example 4

[0123] Controlled drug release effect of subunit hydrogel vaccine system

[0124] To prepare the RCG / KKFE8 gel vaccine, 180 μL of RCG / KKFE8 solution (with Cy5-labeled RBD) was placed in a 0.6 mL centrifuge tube and 20 μL of PBS was added. Release medium (60 μL of PBS solution) was added to the centrifuge tube, and 40 μL of the sample was taken at the designated time points and supplemented with 40 μL of fresh PBS solution. Finally, the release of RBD, CpG ODN (Quant-iTOliGreen ssDNA Assay Kit), and GM-CSF (ELISA GM-CSF Kit) was measured using a multifunctional microplate reader. The cumulative drug release was calculated, and the drug release curve was plotted.

[0125] Figure 5 In vitro drug release results demonstrate that this subunit hydrogel vaccine system is capable of slow and sustained release of vaccine components. Over the 20-day experiment, the release amounts of RBD protein, CpG ODN, and GM-CSF were 72.8% ± 1.4%, 75.5% ± 4.25%, and 83.89% ± 4.68%, respectively. Therefore, this supramolecular hydrogel can serve as a drug reservoir, effectively controlling the slow and sustained release of subunit vaccine components.

[0126] Example 5

[0127] In situ subunit hydrogel vaccine formation

[0128] RCG / KKFE8 solution was subcutaneously inoculated into mice. Fifteen minutes later, the mice were dissected to observe the formation of in situ subunit hydrogel vaccine and take pictures.

[0129] from Figure 6 It can be seen that the RCG / KKFE8 system has a "solution-gel" transition characteristic, is inoculated in a solution state, and triggers the formation of an in situ subunit hydrogel vaccine under physiological conditions.

[0130] Example 6

[0131] In situ subunit hydrogel vaccine induces sustained antibody production

[0132] Healthy BALB / c mice (6-8 weeks) were subcutaneously injected with 100 μL of RCG / KKFE8 solution (n=3). Blood was collected from the eye sockets of mice 21, 28, 35, 42, and 49 days after vaccination, and the serum was collected and tested for IgG antibody production by ELISA.

[0133] from Figure 7 It can be seen that when the RCG / KKFE8 system was inoculated subcutaneously in mice, the secretion of IgG antibodies could be detected at 21, 28, 35, 42, and 49 days, indicating that the RCG / KKFE8 subunit hydrogel vaccine system can induce the continuous production of antibody IgG.

[0134] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Use of an in situ subunit hydrogel vaccine formulation in the preparation of a vaccine to prevent SARS-CoV-2 infection and transmission, characterized in that: The in situ subunit hydrogel vaccine preparation includes a supramolecular polypeptide hydrogel carrier and a subunit vaccine component loaded on the supramolecular polypeptide hydrogel carrier; The subunit vaccine components include antigen, adjuvant and immunomodulator, the antigen is RBD protein, the adjuvant is CpG ODN, and the immunomodulator is GM-CSF; The supramolecular polypeptide hydrogel carrier is formed by self-assembly of polypeptides in a solvent to form nanofibers, and then forms a supramolecular polypeptide hydrogel carrier in a physiological environment. The solvent is water, and the polypeptide sequence is KFKFEFKFE.

2. The use according to claim 1, characterized in that The SARS-CoV-2 includes wild, Alpha, Beta, Gamma, Delta or Omicron.

3. The use according to claim 1, characterized in that The preparation steps of the in situ subunit hydrogel vaccine formulation are as follows: S1. Synthesize a self-assembling polypeptide, wherein the polypeptide sequence is KFKFEFKFE; S2, dissolving the self-assembling polypeptide obtained in step S1 in a solvent to obtain a self-assembling nanofiber solution, wherein the solvent is water; S3, adding the subunit vaccine components to the self-assembled nanofiber solution obtained in step S2 and incubating, and then forming an in situ subunit hydrogel vaccine preparation in a physiological environment, The subunit vaccine components include antigen, adjuvant and immunomodulator, the antigen is RBD protein, the adjuvant is CpG ODN, and the immunomodulator is GM-CSF.

4. The use according to claim 3, characterized in that In step S1, the self-assembling polypeptide is obtained by solid phase synthesis.

5. The use according to claim 3, characterized in that In step S2, the concentration of the self-assembling polypeptide in the solvent is 1-100 mM.

6. The use according to claim 3, characterized in that In step S3, the incubation temperature is 4-50° C., and the incubation time is 0.5-3 h.

7. The use according to claim 3, characterized in that The RBD mass is 0.001-50 mg; The mass of the CpG ODN is 0.001-50 mg; The mass of the GM-CSF is 0.001-50 mg.

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