Vaccine compositions and vaccine formulations based on protein nanotube hydrogels

Abstract

The application provides a vaccine composition and vaccine preparation based on a protein nanotube hydrogel, and belongs to the technical field of biological medicine and biomaterials. The immunization sample prepared by simply mixing a vaccine antigen prepared from a fusion glycoprotein F and an attachment glycoprotein G of a Nipah virus (NiV) is used as an immunization object. The antigen clearance is delayed and strong humoral immunity is excited through subcutaneous immunization. The humoral immunity and cellular immunity are synergistically excited through nasal immunization, and the adjuvant blank of a nasal spray vaccine is filled. Moreover, the protein nanotube hydrogel preparation has high safety, and the subcutaneous and nasal immunization has no adverse effect on mice under a specified dose. The protein nanotube hydrogel provides a new solution for a viral subunit vaccine efficiency and delivery system, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of interdisciplinary technology of biomedicine and biomaterials, and in particular to the application of a protein nanotube hydrogel in the preparation of vaccine compositions or vaccine formulations, especially suitable for a subunit vaccine of Nipah virus prepared based on protein nanotube hydrogel. Background Technology

[0002] Nipah virus (NiV) belongs to the genus Hennipahvirus of the family Paramyxoviridae. It is a single-stranded negative-sense RNA virus whose natural host is the fruit bat (Pteropodidae family). Nipah virus can cause serious zoonotic diseases and is highly pathogenic. It can be transmitted from animals to humans, from humans to humans, and through the consumption of contaminated food. The case fatality rate of Nipah virus is as high as 40% to 75%. Due to its high mortality rate, human-to-human transmission, and lack of specific treatments, it is listed as a Biosafety Level 4 (BSL-4) pathogen and is included in the World Health Organization's (WHO) list of priority emerging infectious disease pathogens for development. Currently, there are no approved Nipah virus vaccines or specific antiviral drugs. Existing vaccine development focuses on the viral surface G (attached glycoprotein) and F (fusion glycoprotein) as antigens or drug targets, including multiple technical routes such as adenovirus vectors, mRNA, and subunits. The vaccine research process also faces technical difficulties such as high dependence on BSL-4 laboratories, high development costs, and long development cycles.

[0003] Viral subunit vaccines are composed of specific proteins or polysaccharides of the pathogen. Because they do not contain the complete pathogen and have clearly defined components, they offer relatively high safety and have become an important direction in Nipah virus vaccine development. The starting point for Nipah virus subunit vaccine development is the screening of target antigens, often using G and F alone or in combination. Technical challenges include: primarily in delivery system design, maintenance of antigen conformation, and optimization of immunogenicity. These issues severely restrict the immunogenicity of Nipah virus subunit vaccines: 1) Antigen conformation maintenance: G and F glycoproteins have complex structures and are prone to misfolding after leaving the lipid environment, making it difficult to maintain a stable and correct conformation, resulting in weak immunogenicity and difficulty in effectively activating innate immunity; 2) Limitations in immune response: Compared with live attenuated vaccines, subunit vaccines are less likely to induce a sustained, higher level of antibody response and are almost ineffective in stimulating cytotoxic T lymphocyte (CTL) immune responses. A: This is crucial for clearing Nipah virus (NiV) infection within cells; 3) Adjuvant system challenges: Aluminum adjuvants (such as alum) are widely used, but mainly favor Th2 humoral immunity, with poor efficacy against Th1 immunity and CTL responses; novel adjuvants (such as AS01, Spy) can partially enhance immunity, but face limitations such as high cost, demanding storage conditions, and strong potential side effects; 4) Mucosal immunity bottleneck: The natural route of Nipah virus infection is mainly through the respiratory tract. Mucosal immune routes such as nasal sprays or drops can establish "local blockage" at the first line of defense against viral invasion, but there are currently no approved dedicated mucosal adjuvants, and only a few (such as polyphosphazene nanocarriers) are still in the preclinical stage. Therefore, developing efficient delivery and adjuvant systems to enhance the immunogenicity of Nipah virus subunit vaccines, prolong response time, and expand immune pathways are currently key research directions.

[0004] Virus-like particles (VLPs) are nanoparticles formed by the self-assembly of one or more structural proteins of a virus, lacking viral genetic material. Their size and morphology make them easily uptaken by antigen-presenting cells, exhibiting excellent adjuvant properties. MS2 bacteriophage, as a model vector for virus-like particles (VLPs), has been applied as a platform for vaccine development and drug delivery. MS2 VLPs have a region called the "AB loop" on their surface, which acts like a "slot" to accommodate exogenous antigen fragments, displaying them at high density on the particle surface, thereby eliciting a strong immune response. For example, it has been used in vaccines against pathogens such as human papillomavirus (HPV), hand-foot-and-mouth disease virus, malaria parasites, and chlamydia trachomatis. As a display platform with an adjuvant effect, it has successfully induced specific immune responses in mouse models. The internal cavity of MS2 VLPs can encapsulate exogenous substances. For instance, preclinical experiments have shown that exogenous RNA sequences (such as RNA used as internal controls or mRNA with therapeutic potential) can be encapsulated inside MS2 VLPs, protecting the RNA from degradation. However, traditional MS2 VLPs, used as vaccine adjuvant platforms to display antigens or as delivery vector platforms to package RNA, are typically in their natural icosahedral form. Existing research (“Transformation of a Viral Capsid from Nanocages to Nanotubes and Then to Hydrogels Redirected Self-Assembly and Effects on Immunogenicity”, Mengsi Yang et al., ACS Nano 2024, 18, 13755−13767) provides a programmable, tunable, and reversible protein nanomaterial platform: a two-site mutant (CP) created by site-directed mutation of sites 46 and 70 of the MS2 phage capsid protein (CP) to alanine and cysteine, respectively. C46A / Q70C This allows them to transform from spherical VLPs in a nanocage-like structure to nanotubes with a diameter of approximately 7.2 nm and a length of approximately 1 µm; furthermore, a three-point mutant (CP) with a site-directed mutation at position 75 to cysteine ​​is created. C46A / Q70C / V75CThe nanotubes can cross-link through disulfide bonds to form a viscoelastic solid nanotube hydrogel. Molecular dynamics analysis shows that it exhibits typical hydrogel rheological properties, and immunogenicity results indicate that this three-point mutant is more likely to elicit a cellular immune response. This study reveals that a multi-level, deformable (nanocages, nanotubes, nanotube hydrogels) self-assembly system of MS2 CP can be obtained through site-directed mutagenesis in genetic engineering, and different morphologies impart significantly different immunogenicity. Therefore, compared to the single morphology of traditional MS2 VLPs, using morphology-controllable mutants as a delivery or adjuvant platform for next-generation protein subunit vaccines (such as Nipah virus subunit vaccines) is a highly promising research direction. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a vaccine adjuvant or delivery system in the form of protein nanotube hydrogel and its application in Nipah virus vaccines.

[0006] To achieve the above objectives, the present invention also provides a method for preparing protein nanotube hydrogels and Nipah virus subunit vaccines.

[0007] In a first aspect, the present invention provides a vaccine composition based on a protein nanotube hydrogel, the vaccine composition comprising a protein nanotube hydrogel and a specific antigen, wherein the protein nanotube hydrogel is a three-point mutant CP of the MS2 phage capsid protein. C46A / Q70C / V75C A three-dimensional network hydrogel formed by the self-assembly of disulfide bonds between nanotubes, and a three-point mutant CP. C46A / Q70C / V75C It has an amino acid sequence as shown in SEQ ID NO:1; the specific antigen is an antigen in the form of a subunit vaccine; the protein nanotube hydrogel and the specific antigen are physically mixed to form a complex.

[0008] According to a preferred embodiment of the present invention, the protein nanotube hydrogel is abbreviated as SS3 (S-Scrosslinked triple mutant).

[0009] According to a preferred embodiment of the present invention, in the vaccine composition based on protein nanotube hydrogel, the specific antigen is an antigen against Nipah virus, and the physical mixing is a simple mixing of protein nanotube hydrogel and specific antigen. The purification and preparation process of protein nanotube hydrogel does not require the addition of chemical cross-linking agents.

[0010] According to a preferred embodiment of the present invention, in the vaccine composition based on protein nanotube hydrogel, the specific antigen is a dual antigen complex against Nipah virus, and the concentration of protein nanotube hydrogel added is 0.3 to 0.5 mg / mL.

[0011] Preferably, in the vaccine composition based on protein nanotube hydrogel, the dual antigen complex is selected from the epitope enrichment segment of Nipah virus fusion glycoprotein F and the extracellular domain of attachment glycoprotein G, wherein the epitope enrichment segment of fusion glycoprotein F has the amino acid sequence shown in SEQ ID NO:2, and the extracellular domain of attachment glycoprotein G has the amino acid sequence shown in SEQ ID NO:3.

[0012] In a second aspect, the present invention provides a vaccine formulation based on protein nanotube hydrogel, comprising the vaccine composition based on protein nanotube hydrogel described in the first aspect, but excluding other vaccine adjuvants.

[0013] According to a preferred embodiment of the present invention, the vaccine formulation based on protein nanotube hydrogel is an injection, nasal drops, inhalation solution, or spray.

[0014] According to a preferred embodiment of the present invention, the immunization route of the vaccine formulation based on protein nanotube hydrogel is subcutaneous injection. After subcutaneous injection, the protein nanotube hydrogel forms an antigen library at the injection site, extending the in vivo clearance half-life of the specific antigen to no less than 4 days.

[0015] Preferably, when the immunization route is subcutaneous injection, the dose of the vaccine preparation administered subcutaneously is 50 µL per mouse.

[0016] Preferably, when the immunization route is subcutaneous injection, the content of protein nanotube hydrogel does not exceed 25 µg / mouse.

[0017] Preferably, when the immunization route is subcutaneous injection, a single immunization can produce a higher concentration and longer-lasting effect of specific IgG antibodies, and a booster immunization can produce an even higher concentration of specific IgG antibodies.

[0018] According to a preferred embodiment of the present invention, when the immunization route of the vaccine formulation based on protein nanotube hydrogel is mucosal immunity, mucosal immunity combined with booster immunization simultaneously induces and stimulates humoral immunity and cellular immunity.

[0019] Preferably, the immunization route is mucosal immunization, specifically nasal instillation, with the dose of vaccine preparation administered via nasal drops being 10 µL per mouse.

[0020] Preferably, when the immunization route is mucosal immunization, the content of protein nanotube hydrogel does not exceed 5 µg / mouse.

[0021] Preferably, when the immune route is mucosal immunity, it can synergistically generate serum-specific IgG antibodies, serum IgA antibodies, and Th1-type cell immune responses.

[0022] Thirdly, the present invention provides a vaccine delivery platform based on the vaccine composition based on protein nanotube hydrogel as described in the first aspect, or the vaccine formulation based on protein nanotube hydrogel as described in the second aspect.

[0023] Fourthly, the present invention provides a vaccine composition based on protein nanotube hydrogel as described in the first aspect, or a vaccine formulation based on protein nanotube hydrogel as described in the second aspect, or the application of the vaccine delivery platform as described in the third aspect in viral subunit vaccines. This application is applicable not only to Nipah virus antigens, but also to other infectious disease antigens and tumor antigens.

[0024] The beneficial effects of this invention are: This invention relates to a vaccine composition based on protein nanotube hydrogel (SS3) and its application in Nipah virus subunit vaccines. Firstly, protein nanotube hydrogel (SS3) can be prepared using conventional prokaryotic or eukaryotic expression systems. The purification method is simple and yields products with high purity. By simply physically mixing the protein nanotube hydrogel SS3 with viral antigens, a viral subunit vaccine with immunomodulatory effects can be obtained. This invention aims to develop a non-chemically synthesized protein nanotube hydrogel (SS3) that possesses both good biocompatibility and highly efficient immune activation capabilities. This has the potential to be an important research and application direction in the development of vaccine adjuvants with sustained-release delivery effects for viral subunit vaccines.

[0025] To address the problems of toxic residues in the preparation materials, poor biocompatibility, and uncontrollable delivery and release processes after mixing with antigens, the protein nanotube hydrogel (SS3) provided by this invention is a hydrogel system entirely composed of bio-derived proteins. It forms a unique three-dimensional network structure through disulfide bond cross-linking between nanotubes. Its essential component is protein, exhibiting excellent biocompatibility and biodegradability. Furthermore, because the protein nanotube hydrogel (SS3) is entirely derived from a three-point mutant (CP) of the bacteriophage MS2 capsid protein... C46A / Q70C / V75C It not only has good biocompatibility, but also naturally has the advantage of stimulating the immune system, fundamentally avoiding the safety hazards that may exist in traditional synthetic adjuvants or materials.

[0026] Regarding safety, when protein nanotube hydrogel (SS3) was used as an adjuvant for Nipah virus subunit vaccines, the maximum injection dose for subcutaneous immunization was 25 µg, and the maximum dose for intranasal immunization was 5 µg. The mental state, weight, and body size of the experimental mice under both immunization methods were not significantly different from those of the negative control group, demonstrating good safety.

[0027] Regarding the advantages of subcutaneous immunization, protein nanotube hydrogel (SS3) forms a unique three-dimensional network structure through disulfide bond cross-linking between nanotubes. After subcutaneous injection, it can efficiently physically encapsulate antigens, forming a local "antigen reservoir," and can be considered as a vaccine delivery platform. Experiments have shown that adding protein nanotube hydrogel (SS3) not only significantly delays antigen clearance in vivo, but also simultaneously induces higher antigen-specific antibodies in mice after a single subcutaneous immunization. Booster immunization yields even higher serum-specific antibody titers. In vivo imaging experiments have confirmed that, compared with free antigens, fluorescently labeled antigens with added protein nanotube hydrogel (SS3) have an average extended fluorescence signal half-life of at least 2 days at the injection site (from 2 days to 4.3 days), while simultaneously stimulating strong humoral immunity, producing specific antibody titers similar to or even slightly higher than those of traditional adjuvants, without any side effects. The continuous antigen release pattern of protein nanotube hydrogel (SS3) provides long-lasting stimulation for the continuous activation of the immune system, which is the physical basis for inducing a strong and durable immune response.

[0028] Regarding the advantages of the mucosal immune pathway, the application of protein nanotube hydrogel (SS3) in nasal spray immunization can simultaneously stimulate strong humoral and cellular immunity, filling the gap of no approved adjuvants for nasal spray vaccines. Viral subunit vaccines mixed with this protein nanotube hydrogel (SS3), administered via nasal spray immunization with booster immunization, can induce significantly higher titers of serum Nipah virus antigen-specific IgG and IgA antibodies in terms of humoral immunity; and in terms of cellular immunity, ELISA shows efficient activation of Th1 T cell responses.

[0029] The vaccine delivery platform containing protein nanotube hydrogel (SS3) provided by this invention has broad versatility. The protein nanotube hydrogel (SS3) itself can achieve potent immunomodulation without the need for co-administration with other adjuvants, simplifying vaccine formulation, improving process stability, and reducing the complexity and uncontrollable risks associated with the coexistence of multiple components. Therefore, the vaccine delivery platform containing protein nanotube hydrogel (SS3) is not only suitable for dual-antigen complexes such as Nipah virus fusion glycoprotein F and attachment glycoprotein G, but is also expected to be applicable to other infectious disease antigens, tumor antigens, etc., providing a novel technological tool for developing new and highly effective preventive and therapeutic vaccines.

[0030] The advantages of the protein nanotube hydrogel (SS3) system provided by this invention are as follows: The protein nanotube hydrogel (SS3) system simultaneously possesses vaccine sustained-release and immunomodulatory effects. The protein nanotube hydrogel (SS3) is entirely composed of biologically derived proteins, is easily biodegradable, and has good biocompatibility. Because it is derived naturally from bacteriophages, it has the advantage of stimulating the immune system, avoiding the safety hazards of traditional synthetic adjuvants or materials. The protein nanotube hydrogel (SS3) can achieve potent immunomodulatory enhancement without the need for combination with other adjuvants, simplifying vaccine formulations, improving process stability, and reducing complexity and uncontrollable risks. This provides a novel technological tool for developing new and highly effective "therapeutic vaccines" and "preventive vaccines." Attached Figure Description

[0031] Figure 1 The image shows the purification results of the protein nanotube hydrogel (SS3) of this invention; A is an SDS-PAGE image, B is a scanning electron microscope image at different magnifications at high concentration, and C is a scanning electron microscope image at different magnifications at low concentration. Figure 2 ; This is a diagram showing the purification results of the Nipah virus fusion glycoprotein F and attachment glycoprotein G of the present invention; A is an SDS-PAGE image of F and G after purification respectively, and B is an SDS-PAGE image of the mixture of F and G after coupling with the fluorescent dye Alexa Fluor 750 Dye. Figure 3 Immunoassays prepared with fluorescently labeled Nipah virus dual antigen complex (F / G) and different amounts of protein nanotube hydrogel (SS3) were used to immunize mice via subcutaneous route. The fluorescence trend and fluorescence half-life were displayed by in vivo imaging. A is the fluorescence trend; B is the fluorescence half-life. Figure 4 The image shows the ELISA IgG antibody titer determination results of the immunogenicity of SS3-F / G mice subcutaneously immunized according to the present invention.

[0032] Figure 5 The graph shows the results of immunogenicity assays in SS3-F / G nasally immunized mice according to the present invention. A is the result of ELISA IgG antibody titer assay, B is the result of ELISA IgA antibody titer assay, and C is the result of Elispot quantitative detection of IFN-γ secretion. Figures 3-5 In the table, "ns" indicates no significant difference, "*" represents P < 0.05 (significant difference), "**" represents P < 0.01 (very significant difference), "***" represents P < 0.001 (extremely significant difference), and "****" represents P < 0.0001 (extremely significant difference). Detailed Implementation

[0033] The exemplary embodiments of the present invention are described in detail below with reference to practical applications. For ease of understanding, many specific details are set forth to provide a comprehensive understanding of the present invention. In this context, "same" or "similar" refers to the same concepts, such as protein nanotube hydrogel = SS3, Nipah virus = NiV, attached glycoprotein G extracellular domain = glycoprotein G = G protein = G, fusion glycoprotein F epitope enrichment segment = glycoprotein F = F protein = F, enzyme-linked immunosorbent assay = ELISA, enzyme-linked immunospot = Elispot.

[0034] The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods, and the materials and reagents used are commercially available. Scanning Electron Microscope (SEM, Hitachi SU8010) purchased from Hitachi Corporation; high-purity single-crystal SEM silicon wafer purchased from Wuhan Xuanshilin Biotechnology Co., Ltd.; Alexa Fluor 750 Dye purchased from Thermo Fisher Scientific; SPF-grade BALB / c mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; In Vivo Imaging System (IVIS) purchased from PerkinElmer (now Revvity); HRP-labeled goat anti-mouse IgG (heavy and light chains) (Goat Anti-Mouse IgG (H+L), HRP Conjugate) purchased from Wuhan Boster Biological Engineering Co., Ltd.; HRP-labeled goat anti-mouse IgA (HRP-conjugated goat anti-mouse IgA (α), HRP-goat anti-mouse IgA) purchased from Wuhan Kaishengxiang Biotechnology Co., Ltd.; TMB chromogenic solution and stop solution (ELISA) HRP staining solution was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; MONTANIDE ISA 201 VG adjuvant (ISA 201) was purchased from Sepico (Shanghai) Specialty Chemicals Co., Ltd.; mouse lymphocyte separation medium, Mouse IFN-γ Precoated ELISpot Kit, and cell sieves were all purchased from Dakota Biotechnology Co., Ltd. However, it is obvious that one or more examples can be performed without these specific details, and conditions not specifically specified in the examples should be performed under standard conditions or the manufacturer's recommended conditions. Immunobiological experimental methods not specifically described in the following examples were performed according to the specific methods listed in the textbook (Medical Immunology Experimental Tutorial, edited by Zhuang Guohong and Gao Fengguang, Higher Education Press, September 27, 2021), or according to the product instructions of the kit.

[0035] In the embodiments of this invention, the detection or determination targets are all in vitro expressed proteins, which are isolated, non-living, artificially synthesized samples. The direct purpose of the detection or determination is solely to perform relevant biological or immunological laboratory tests or analyses, and is unrelated to disease diagnosis or treatment. Therefore, the detection or determination described in this invention does not constitute a disease diagnosis method and meets the basic requirements for patentable subject matter under the Patent Law. This invention primarily deals with in vitro expressed proteins (G, F), and therefore does not involve direct manipulation of live Nipah virus. Specific experimental operations involving gene cloning and protein expression are all performed in a BSL-2 laboratory and comply with relevant regulations.

[0036] Example 1: Purification and preparation method of protein nanotube hydrogels The expression, purification, and preparation process of protein nanotube hydrogels includes culture and induction of expression, collection and disruption of bacterial culture, purification by ammonium sulfate precipitation, and identification of purified samples. The specific steps are as follows.

[0037] 1.1 Cultivation and Induction of Expression The existing study (“Transformation of a Viral Capsid from Nanocages to Nanotubes and Then to Hydrogels Redirected Self-Assembly and Effects on Immunogenicity”, Mengsi Yang et al., ACS Nano 2024, 18, 13755−13767) provided a three-point mutant (CP) C46A / Q70C / V75C Expression plasmid pET32a-CP C46A / Q70C / V75C The BL21(DE3) strain was removed from -80 °C and rapidly thawed in a 37 °C water bath. After thawing, it was spread onto LB agar plates containing ampicillin (final concentration 100 μg / mL) and incubated upside down at 37 °C for 12 hours. Among them, the three-point mutant (CP) C46A / Q70C / V75C The coding sequence of the bacteria is shown in SEQ ID NO:1. A single colony was picked from an LB plate and inoculated into a test tube containing 5 mL of LB liquid medium containing ampicillin (final concentration 100 μg / mL), and cultured overnight at 37°C with shaking at 200 rpm. The entire 5 mL of bacterial culture from the test tube was then inoculated into a shake flask containing 500 mL of LB liquid medium containing ampicillin, and cultured at 37°C with shaking at 200 rpm until OD (dose elapsed). 600 =0.4-0.6, and the shaking culture time is about 3 hours. Add IPTG (isopropyl β-D-thiogalactoside) to the bacterial culture with a final concentration of 1 mM, and incubate at 25℃ and 160 rpm for 10-12 hours.

[0038] Table 1 Summary of Sequence Lists

[0039] 1.2 Collection and disruption of bacterial culture After induction, the bacterial culture was transferred to centrifuge tubes and centrifuged at 4°C and 5000 rpm for 15 minutes. The supernatant was discarded. The bacterial pellet was washed once with PBS buffer (8 g NaCl, 0.2 g KCl, 3.58 g Na2HPO4·12H2O, 0.24 g KH2PO4, and ultrapure water to a final volume of 1 L, pH=7.4). The pellet was then centrifuged again at 4°C and 5000 rpm for 15 minutes, and the supernatant was discarded. The washed bacterial pellet was resuspended in the same PBS buffer and subjected to high-pressure disruption at 850 psi for 5 minutes each time, repeated 3 times.

[0040] 1.3 Ammonium sulfate precipitation purification The disrupted bacterial culture was centrifuged at 10,000 rpm for 30 minutes at 4°C. The supernatant was subjected to ammonium sulfate fractionation and ultracentrifugation purification. The ammonium sulfate precipitation and ultracentrifugation purification process mainly included: transferring the supernatant after disruption and centrifugation to a new tube, placing it on ice, and gradually adding ammonium sulfate solids to 15% saturation (generally about 9.3 g of ammonium sulfate per 100 mL of supernatant) while slowly stirring, and continuing to stir for at least 30 minutes. Then, centrifuged at 10,000 rpm for 30 minutes at 4°C, discarding the supernatant, resuspending the precipitate in 50 mL of pre-chilled PBS buffer, and stirring slowly overnight at 4°C. The resuspended solution was aliquoted into Beckman Type 90 Ti centrifuge tubes, balanced, and centrifuged at 30,000 rpm for 1 hour at 4°C using a Beckman Type 90 Ti rotor, discarding the supernatant. The precipitate was resuspended in pre-chilled PBS and stirred overnight at 4°C. This PBS resuspended solution was the target protein (three-point mutant CP). C46A / Q70C / V75C The protein nanotube hydrogel solution formed by this process is based on the known three-point mutant (MS2 CP with site-directed mutations at three sites). C46A / Q70C / V75C The protein nanotube hydrogel is formed by introducing two cysteine ​​residues to form a strongly interacting disulfide bond (–SS–). Therefore, the protein nanotube hydrogel can be simply referred to as SS3 (SS crosslinked triple mutant).

[0041] 1.4 Identification of purified samples The purified protein nanotube hydrogel (SS3) solution sample was identified, including SDS-PAGE analysis and scanning electron microscopy (SEM) observation. SDS-PAGE analysis: Samples were treated with a reducing agent (e.g., DTT) before loading, electrophoresis, and then stained with Coomassie brilliant blue. The results showed the purity and size of the SS3 protein as follows: Figure 1 As shown in Figure A, the main band of the SS3 protein is clearly visible, and its size (approximately 14 kDa) is correct. Protein concentration was semi-quantitatively determined using SDS-PAGE grayscale comparison. Scanning electron microscopy (SEM) observation: A suitable amount of SS3 solution sample was dropped onto a silicon wafer and allowed to stand for 10 minutes to allow adsorption. After removing excess droplets from the wafer, it was placed in a dust-free environment to air dry naturally. A layer of gold film was uniformly deposited on the sample surface using an ion sputtering coating instrument to improve conductivity. The sample preparation was then complete. Finally, the sample was placed in the SEM chamber, the accelerating voltage was set to 10 kV, and the working distance and focusing parameters were adjusted. The microstructure of the sample was observed, and images were acquired as shown below. Figure 1 As shown in Figures B and C, the results indicate that the purified protein nanotube hydrogels (SS3) at different concentrations and magnifications all formed the expected nanotube network structure with uniform morphology. Under high concentration conditions, SS3 exhibited the following characteristics: Figure 1 The dense network structure shown in Figure B indicates that even at low concentrations, the nanotubes of SS3 can still exhibit similar characteristics. Figure 1 The cross-linked network structure shown in C is shown in the figure.

[0042] Example 2: Preparation of Nipah virus fusion glycoprotein F as an antigen complex stock solution 2.1 Purification and preparation method of Nipah virus fusion glycoprotein F.

[0043] The expression and purification process of Nipah virus fusion glycoprotein F is as follows. The sequence of Nipah virus fusion glycoprotein F is obtained from published literature. 1–5Based on literature, three bases were mutated to make it a pre-F stable conformation (L104C disulfide bond, S191P helical proline substitution and L172F cavity filling mutation). The sequence was codon optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. Culture and induction: The BL21 strain carrying the expression plasmid pET32a-F encoding the Nipah virus fusion glycoprotein F epitope enrichment segment as shown in SEQ ID NO:2 was removed from -80 ℃ and rapidly thawed in a 37 ℃ water bath. After thawing, it was spread onto LB agar plates containing ampicillin (final concentration 100 μg / mL) and incubated upside down at 37 ℃ for 12 hours. A single colony was picked from the LB plate and inoculated into a test tube containing 5 mL of LB liquid medium containing ampicillin (final concentration 100 μg / mL) and incubated overnight at 37 ℃ with shaking at 200 rpm. The entire 5 mL of bacterial culture from the test tube was inoculated into a shake flask containing 500 mL of LB liquid medium containing ampicillin and incubated at 37 ℃ with shaking at 200 rpm until OD600 = 0.4-0.6, with shaking incubation time of approximately 3 hours. Add IPTG, the inducer, to the bacterial culture at a final concentration of 1 mM, and incubate at 25°C and 160 rpm for 10-12 hours.

[0044] Cell collection and disruption: After induction, the bacterial culture was transferred to centrifuge tubes and centrifuged at 4°C and 5000 rpm for 15 minutes. The supernatant was discarded, and the cell pellet was washed once with PBS buffer. The pellet was then centrifuged again at 4°C and 5000 rpm for 15 minutes, and the supernatant was discarded. The washed cell pellet was resuspended in PBS buffer and subjected to autoclaving at 850 psi for 5 minutes each time, repeated three times. The disrupted bacterial culture was centrifuged at 4°C and 10000 rpm for 30 minutes. The disrupted cell pellet contained Nipah virus fusion glycoprotein F. This pellet was purified by refolding inclusion bodies of protein F.

[0045] The F protein inclusion body refolding and purification process includes: 1) Resuspension of inclusion body precipitate: Resuspend the broken bacterial cell precipitate in 1 mL of inclusion body washing buffer (50 mM Tris-HCl, 100 mM NaCl, 3 mM β-mercaptoethanol, 0.3 mM EDTA, 0.5% Triton X-100, 1 M urea, and ultrapure water to a final volume of 1 L, pH 7.4), and stir at room temperature for 20 minutes; 2) Inclusion body washing: After stirring, centrifuge at 10,000 rpm for 20 minutes and discard the supernatant. Repeat this washing process 3 times, or repeat the washing until the precipitate turns white; 3) Inclusion body denaturation and dissolution: Add 15 mL of denaturing buffer (50 mM Tris-HCl, 6 M urea, 100 mM NaCl, 10 mM β-mercaptoethanol, pH 8.0) to resuspend the precipitate, stir and dissolve overnight at 4°C, or stir until the solution is clear and then dissolve at 10,000 rpm. 4) Dialysis refolding: Transfer the supernatant to a dialysis bag with a molecular weight cutoff of 14 kDa, and place the dialysis bag in a 1 L beaker containing 200 mL of denaturing solution. Incubate the beaker at 4°C with stirring overnight. Simultaneously, slowly pump refolding solution (50 mM Tris-HCl, 100 mM NaCl, 5% glycerol, pH 7.4) into the beaker using a peristaltic pump. The peristaltic pump reading should be 4 (flow rate of 0.5-1 mL / min). Pump the refolding solution to a volume of 1 L. 5) Dialysis after refolding: Transfer the refolded target protein solution to a new dialysis bag. Place the new dialysis bag in a new beaker containing PBS buffer (pH 7.4, containing 5% glycerol) and dialyze at 4°C for 10-12 h. Change the PBS buffer 2-3 times during dialysis. After dialysis, take the target protein F solution from the dialysis bag and perform SDS-PAGE. The concentration of protein F was semi-quantitatively determined using the grayscale comparison method. If necessary, the concentration could be determined by concentrating the F solution through ultrafiltration and centrifugation, and stored at 4°C for later use. The dialysate was the purified solution of the obtained Nipah virus fusion glycoprotein F, and was stored at 4°C for later use.

[0046] Purified fusion glycoprotein F was identified by SDS-PAGE: The purified fusion glycoprotein F was subjected to SDS-PAGE electrophoresis, followed by Coomassie brilliant blue staining. The position, brightness, and presence of contaminants in the target band were observed. Protein purity and size were confirmed by protein analysis and Coomassie brilliant blue staining. The results are as follows: Figure 2 As shown in the left band of A, the main band of the unconcentrated and purified fusion glycoprotein F is clearly visible, and its size (49 kDa) is correct.

[0047] 2.2 Preparation of Nipah virus fusion glycoprotein F as an antigen complex stock solution.

[0048] The purified and quantitatively determined protein nanotube hydrogel (SS3) and Nipah virus fusion glycoprotein F were added to the same EP tube at a 1:1 ratio and simply mixed with a pipette or shaker to obtain the F antigen complex stock solution for animal immunization. It can be seen that the monoantigen fusion glycoprotein F can be prepared into a mixture with protein nanotube hydrogel (SS3) by simple mixing.

[0049] Example 3: Preparation of Nipah virus attachment glycoprotein G as an antigen complex stock solution 3.1 Purification and preparation method of Nipah virus attachment glycoprotein G.

[0050] The expression and purification process of Nipah virus attachment glycoprotein G is as follows. The Nipah virus fusion glycoprotein G sequence was obtained from published literature. 2–4,6 The sequence was codon optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. Cultivation and Induction: The BL21 strain carrying the expression plasmid pET32a-F encoding the extracellular domain of the Nipah virus attachment glycoprotein G as shown in SEQ ID NO:3 was removed from -80 ℃ and rapidly thawed in a 37 ℃ water bath. After thawing, it was spread onto LB agar plates containing ampicillin (final concentration 100 μg / mL) and incubated upside down at 37 ℃ for 12 hours. A single colony was picked from the LB plate and inoculated into a test tube containing 5 mL of LB liquid medium containing ampicillin (final concentration 100 μg / mL), and incubated overnight at 37 ℃ with shaking at 200 rpm. The entire 5 mL of bacterial culture from the test tube was inoculated into a shake flask containing 500 mL of LB liquid medium containing ampicillin and incubated at 37 ℃ with shaking at 200 rpm until OD600 = 0.4-0.6, with shaking incubation time approximately 3 hours. Add IPTG, the inducer, to the bacterial culture at a final concentration of 1 mM, and incubate at 25°C and 160 rpm for 10-12 hours.

[0051] Cell collection and disruption: After induction, the bacterial culture was transferred to centrifuge tubes and centrifuged at 4°C and 5000 rpm for 15 minutes. The supernatant was discarded, and the bacterial pellet was washed once with PBS buffer. The pellet was then centrifuged again at 4°C and 5000 rpm for 15 minutes, and the supernatant was discarded. The washed bacterial pellet was resuspended in PBS buffer and subjected to high-pressure disruption at 850 psi for 5 minutes each time, repeated three times. The disrupted bacterial culture was centrifuged at 4°C and 10000 rpm for 30 minutes. The disrupted bacterial pellet contained Nipah virus attachment glycoprotein G. Following the F protein inclusion body refolding and purification process described in Example 1, the G protein inclusion bodies were refolded and purified. After dialysis, the target protein G solution was taken from the dialysis bag, and the G protein concentration was semi-quantitatively determined using SDS-PAGE grayscale comparison. If necessary, the G solution could be concentrated by ultrafiltration centrifugation before concentration determination. The solution was stored at 4°C for later use. The dialysis buffer was the purified solution of Nipah virus attachment glycoprotein G, and was stored at 4°C for later use.

[0052] Purified attached glycoprotein G was identified by SDS-PAGE: The purified attached glycoprotein G was subjected to SDS-PAGE electrophoresis, followed by Coomassie brilliant blue staining. The position, brightness, and presence of contaminants in the target band were observed. Protein purity and size were confirmed by protein analysis and Coomassie brilliant blue staining. The results are as follows: Figure 2 As shown in the right band of A, the main band of the purified attachment glycoprotein G is clearly visible, and its size (greater than 60 kDa) is correct.

[0053] 3.2 Preparation of Nipah virus attachment glycoprotein G as an antigen complex stock solution.

[0054] The purified and quantitatively determined protein nanotube hydrogel (SS3) from Example 1 and Nipah virus attachment glycoprotein G were added to the same EP tube at a 1:1 ratio and simply mixed with a pipette or shaker to obtain the F antigen complex stock solution for animal immunization. It can be seen that the single antigen attachment glycoprotein G can also be prepared into a mixture with the protein nanotube hydrogel (SS3) by simple mixing.

[0055] Example 4: The sustained-release effect of the Nipah virus dual antigen complex 4.1 Fluorescent labeling of Nipah virus dual antigen complex The preparation and validation of fluorescently labeled dual antigen complexes require strict adherence to aseptic and light-protected operating procedures in a clean bench. The specific steps are as follows: Take the purified solution of the fusion glycoprotein F (hereinafter referred to as F) and the purified solution of the attached glycoprotein G (hereinafter referred to as G) prepared above, and accurately measure F and G respectively according to a molar ratio of 1:1. Mix them thoroughly to obtain the dual antigen complex (denoted as F / G). Subsequently, under strict light-protected conditions, slowly add fluorescent dye (Alexa Fluor 750 Dye) to the complex, precisely controlling the molar ratio (F / G total protein: fluorescent dye = 1:2). After gently mixing by pipetting, allow it to stand at room temperature in the dark for 1 hour to obtain the fluorescently dye-conjugated dual antigen complex (hereinafter referred to as the conjugated complex stock solution, F / G-Alexa750). Dialysis purification: The fluorescently labeled coupling complex (F / G-Alexa750) stock solution was placed in a dialysis bag with a molecular weight cutoff of 14 kDa. Dialysis was performed overnight at 4°C in the dark using PBS buffer (pH 7.4) to remove unbound free fluorescent dye. The PBS buffer was changed 2-3 times during dialysis to ensure complete removal of free dye. The next day, the dialysis bag contained the purified coupling complex (F / G-Alexa750) solution. Identification of the purified solution: An appropriate amount of the F / G-Alexa750 purified solution was separated by SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue G-250. The results are as follows. Figure 2 As shown in Figure B, the protein band size and protein concentration were confirmed to meet the requirements by comparing with the standard protein molecular weight marker (M). It can be seen in the figure that the F / G-Alexa750 purified solution sample has two obvious main bands with the correct size (60kDa, 50kDa).

[0056] 4.2 Animal Immunization The immunized animals were 6-week-old female SPF-grade BALB / c mice, which were divided into 3 groups according to the amount of protein nanotube hydrogel (SS3) added: 0 mg / mL, 0.3 mg / mL, and 0.5 mg / mL, with 3 mice in each group; the immunization route was subcutaneous injection in the neck, and the immunization dose was 50 µL / mouse.

[0057] Immune sample preparation: Prepare immune samples according to the number of mice to be immunized. According to different SS3 concentrations (0 mg / mL, 0.3 mg / mL, 0.5 mg / mL) added by groups, take the protein nanotube hydrogel (SS3) solution prepared in Example 1 and add it into the purified solution of the coupling complex (F / G-Alexa750) prepared above, then add PBS buffer to 50 µL per mouse. Meanwhile, ensure that each experimental mouse receives the double-antigen complex (F / G) at the same dose and concentration, wherein in the coupling complex (F / G-Alexa750) received by each mouse, G and F are ensured to be in equal amounts, 5 µg each per mouse. The negative control is the coupling complex without SS3 added, that is, the SS3 concentration is 0 mg / mL. Perform grouped immunization and labeling on the experimental mice according to the immunization schedule, and the day when the immunization is performed is counted as Day 0.

[0058] 4.3 In vivo imaging Imaging is performed every day for 0-5 days after immunization, and then performed once every other day thereafter. Before the first imaging, the experimental mice are first anesthetized with isoflurane gas, then the hair at the immunization injection site is shaved, and finally depilatory cream is applied to completely remove the hair. During imaging, each experimental mouse to be observed is placed in the in vivo imaging system (IVIS) for small animals for imaging in sequence. The set imaging conditions are: select the Alexa Fluor 750 program, the imaging stage height is 13.3 cm, and automatic exposure is adopted. After the imaging is completed, the Living Image software matched with the in vivo imaging system (IVIS) for small animals is used for fluorescence intensity analysis, the average value of the fluorescence value imaging results of each group of experimental mice is calculated, and the result graph is drawn as Figure 3As shown in the figure, A is the trend of the ratio of the average fluorescence value to the initial fluorescence value of each group over time, and B is the half-life of fluorescence value disappearance. It is clear from the figures that: regarding the trend of fluorescence value change, compared to the negative control (SS3 = 0 mg / mL), the rate at which the fluorescence value of the corresponding conjugate complex was reduced by metabolism was significantly slower in the subcutaneously injected immune samples containing different concentrations (SS3 = 0.3 mg / mL, SS3 = 0.5 mg / mL) of protein nanotube hydrogel (SS3), and there was no significant difference between different concentrations. This indicates that protein nanotube hydrogel (SS3) can significantly slow down the metabolic efficiency of antigen in vivo, that is, SS3 has a sustained-release effect on antigen immunization in vivo; regarding the half-life of fluorescence value disappearance, compared to the negative control (half-life = 2 days), the subcutaneous injection of samples containing different concentrations (SS3 = 0.3 mg / mL, SS3 = 0.5 mg / mL) of protein nanotube hydrogel (SS3 = 0.5 mg / mL) showed a significant reduction in fluorescence value over time. The (mg / mL) protein nanotube hydrogel (SS3) significantly prolonged the half-life of the conjugate complex in mice to 4.5 days, indicating that SS3 can increase the in vivo half-life of the antigen to 225% of the original value, a 1.25-fold increase. Therefore, SS3 is beneficial for forming an "antigen library" to achieve a sustained-release effect, thereby not only significantly prolonging the exposure time of the antigen in vivo but also enhancing and maintaining the level of the immune response. This beneficial effect makes it highly suitable for widespread application as an adjuvant in viral subunit vaccines. Example 5: Humoral immune effects of dual antigen complex (F / G) via subcutaneous immunization.

[0059] 5.1 Preparation of immunization samples in the subcutaneous immunization route experimental group Preparation of immunization samples for the experimental group: The immunized animals and groups continued to use the same three groups as in Example 4 (0 mg / mL, 0.3 mg / mL, 0.5 mg / mL; 3 mice per group). The number of experimental mice in Example 4 was used to prepare the dual antigen complex for booster immunization. The preparation process of the dual antigen complex was the same as the steps before adding the fluorescent dye in Example 4: In a clean bench, the F purification solution and G purification solution prepared in Example 2 and Example 3 were accurately measured in a molar ratio of 1:1. The dual antigen complex (F / G) obtained after mixing the two was used as the immunization antigen. According to the number and grouping of experimental mice in Example 4, different concentrations of SS3 were added to the dual antigen complex (F / G), and then PBS buffer was added to 50 µL / mouse. The three groups of experimental mice continued to receive immune samples with final SS3 concentrations of 0 mg / mL, 0.3 mg / mL, and 0.5 mg / mL (F / G-SS3-0, F / G-SS3-0.3, F / G-SS3-0.5), and it was ensured that the amount of G and F in the dual antigen complex (F / G) received by each mouse was equal, 5 µg / mouse.

[0060] 5.2 Preparation of subcutaneous immunization route control group and antigen-free negative control immunization samples Two control groups were set up. Immunogens were prepared into immunization samples for animal immunization by mixing with traditional adjuvants ISA201 and rHF, respectively. The two control groups were the ISA201 group and the rHF group.

[0061] Immunization samples for ISA201 group: Aseptic operation was performed according to the product instructions of adjuvant ISA201. The prepared dual antigen complex (F / G) solution and adjuvant ISA201 were taken in advance and left to stand at room temperature (about 25 ℃) for about 10 minutes to allow the solution temperature to equilibrate to room temperature. Immunization samples for ISA201 group were prepared according to the number of mice 3, and emulsification was performed by manual push-pull double syringe mutual push method. Take two 1 mL sterile syringes (Luer connectors): draw 75 μL of the dual antigen complex (F / G) into one syringe, with 15 µg of G and 15 µg of F; draw an equal volume (75 μL) of adjuvant ISA 201 into the other syringe; alternately and slowly push the plungers of the two syringes, repeatedly injecting about 40-60 times, so that the dual antigen complex (F / G) and adjuvant ISA 201 repeatedly pass through and mix in the narrow Luer connector channel. Repeatedly inject until the mixture forms a homogeneous, viscous, and opaque milky white emulsion, which is the ISA201 group of immunization samples prepared by emulsification, referred to as F / G-ISA201.

[0062] rHF group immunized samples: First, the fusion glycoprotein F-SpyTag and attachment glycoprotein G-SpyTag gene clones were constructed, expressed, and purified: The coding sequences of glycoproteins F and G shown in SEQ ID NO:2-3 of Nipah virus were respectively fused to the C-terminus of the coding sequences of SpyTag with flexible linkers (GGGGS). The amino acid sequence of SpyTag is shown in SEQ ID NO:4. The expression vector plasmids pET32a-F-SpyTag and pET32a-G-SpyTag containing the coding sequences of F-SpyTag and G-SpyTag were used to express the fusion proteins G-SpyTag and F-SpyTag, respectively. After purification and confirmation, they were ready for use. Secondly, SpyCatcher modification and assembly of rHF nanoadjuvant were carried out: the 92-amino acid SpyCatcher gene was cloned and fused into the N-terminus of the recombinant human heavy chain ferritin (rHF) subunit to construct the pET32a-rHF-SpyCatcher recombinant plasmid; the recombinant plasmid was transformed into Escherichia coli BL21(DE3), and the fusion protein rHF-Spy was obtained by induction expression. The assembled 24-mer rHF nanoparticles were then purified by nickel column affinity chromatography. Third, the conjugation of antigen and adjuvant: First, the purified two antigens (fusion proteins F-SpyTag and G-SpyTag) were mixed evenly at a molar ratio of 1:1 to obtain a dual antigen mixture. Then, the dual antigen mixture was added to PBS buffer (pH 7.4) containing rHF-SpyCatcher at a molar ratio of 5:1 (total fusion protein antigen to adjuvant rHF-SpyCatcher). The mixture was incubated at 4°C for 12 hours to allow SpyTag on the fusion protein to be directionally conjugated to SpyCatcher on the rHF nanoparticles through covalent amide bonds. After the incubation reaction, without removing unbound antigens, this conjugated mixture was directly used as an immunization sample for subcutaneous immunization in experimental mice, namely the F / G-SpyTag-rHF dual antigen complex (abbreviated as F / G-rHF).

[0063] Blank control: The blank control is an immune sample without antigen. PBS buffer without the addition of dual antigen complex (F / G) or any single antigen (F or G) is selected as the negative control immune sample. The immune sample F / G-SS3-0 in the experimental group does not contain any immune-enhancing auxiliary components and is actually used as a negative control to enhance the effect of auxiliary components.

[0064] On the same day as the subcutaneous injection in the neck in Example 4, the two control group immunization samples prepared above (the ISA201 group was the F / G-ISA201 prepared above, and the rHF group was the F / G-rHF prepared above) and PBS buffer without antigen blank control were taken and the first immunization on day 0 was carried out by subcutaneous injection in the neck and the immunization dose was 50 µL / mouse. At the same time, in the three experimental groups and two control groups, it was ensured that the amount of G and F in the immunogen received by each experimental mouse was equal, and each was 5 µg / mouse.

[0065] 5.3 Strengthen immunity Booster immunizations: Mice in the three experimental groups (different concentrations of SS3 dual antigen complexes: F / G-SS3-0.5, F / G-SS3-0.3, F / G-SS3-0), two control groups (F / G-ISA201, F / G-rHF), and a control group without antigen (PBS) were given a second and third booster immunization at weeks 2-3 (e.g., day 21) and weeks 5-6 (e.g., day 42) following the initial immunization. The booster immunizations were administered subcutaneously in the neck, with a dose of 50 µL per mouse.

[0066] Sample collection: Blood samples were collected from mice via the orbital cavity before the second and third immunizations (1st and 2nd) and 3 weeks after the third immunization (3rd or 63rd day). The serum samples were centrifuged after being placed at 37°C for 1 hour to retain the serum samples. The processed serum samples were either frozen at -20°C for later use or directly subjected to ELISA to determine the F / G specific IgG antibody titer to verify the humoral immune effect of different immunization samples.

[0067] 5.4 Humoral immune effect (IgG antibody titer) Serum samples collected and processed three times (1st, 2nd, and 3rd) were used for F / G specific IgG antibody titer determination (ELISA method). 1) Antigen coating: The purified solution (F) of fusion glycoprotein F prepared in Example 2 and the purified solution (G) of attachment glycoprotein G prepared in Example 3 were mixed at a molar ratio of 1:1 with ELISA coating buffer (such as carbonate-bicarbonate buffer, pH 9.6) and diluted to 5 µg / mL. 100 µL / well was added to a 96-well ELISA plate and incubated overnight at 4°C (or incubated at 37°C for 2 hours). The coating solution was discarded, and the plate was washed three times with PBST (PBS containing 0.05% Tween-20), 300 µL / well each time, and then patted dry. 2) Blocking: 200 µL of blocking solution (PBST containing 1% sheep serum) was added to each well and the plate was incubated overnight at 4°C (or incubated at 37°C for 2 hours) for blocking. The blocking solution was discarded, and the plate was washed three times and then patted dry. 3) Add serum samples: Take serum samples from the above 3 experimental groups, 2 control groups, and 1 blank control group collected and processed 3 times, and perform serial dilutions of 2-fold using diluent (PBST containing 1% sheep serum), with 11 consecutive dilutions. Add samples sequentially according to the initial dilution factor of 1:16, adding 100 µL of diluted serum sample to each well; the blank control well contains mouse serum samples immunized with PBS (without specific immune antigens); after adding the serially diluted serum samples, incubate the 96-well ELISA plate at 37℃ for 1-2 hours; discard the solution, wash the plate 3 times, and pat dry. 4) Add enzyme-labeled secondary antibody: Dilute HRP-sheep anti-mouse IgG with PBST at a ratio of 1:10000, add 100 µL to each well, and incubate at 37℃ for 1-2 hours; discard the solution, wash the plate 3-5 times, and pat dry. 5) Color development and detection: Add 100 µL of TMB chromogenic solution to each well and incubate at room temperature in the dark for about 2-5 minutes. When the positive wells show obvious color development (blue) while the negative wells remain colorless or very pale blue, add 100 µL of stop solution to each well; immediately place the well under a microplate reader and read the absorbance value (OD) at a wavelength of 450 nm. 450 6) Result Judgment and Analysis: The judgment criterion is based on the detection of OD using a blank control sample. 450 The cut-off value was 2.1 times the average value, and OD was measured on positive serum samples. 450 The highest dilution factor with a value greater than the cut-off value is taken as the antibody titer of the serum sample to be tested. The results of the F / G specific IgG antibody titer (ELISA method) determination are plotted as follows: Figure 4 .

[0068] Figure 4It is evident that: First, compared to the blank control group (PBS), serum samples containing the dual antigen complex (F / G) as the immunogen could elicit significantly different F / G-specific IgG antibodies in experimental mice. This indicates that the dual antigen complex (F / G), as the immunogen of the viral subunit vaccine, can elicit a specific humoral immune response, and the difference is extremely significant (P < 0.0001). Second, compared to the serum sample corresponding to (F / G-SS3-0) which uses only the dual antigen complex as the immunogen (the concentration of the auxiliary component SS3 is 0, i.e., a pure dual antigen complex without any auxiliary components), other immunized samples containing blood samples with different auxiliary components (F / G-SS3-0.3, F / G-SS3-0.5, F / G-ISA201, F / G-rHF) immunized mice, all of which could synergistically elicit higher F / G-specific IgG antibody titers. This indicates that different auxiliary components (or different concentrations) have varying degrees of synergistic effects on eliciting humoral immunity. Third, in terms of absolute IgG antibody titers, ISA201 showed the strongest synergistic effect, rHF the weakest, and protein nanotube hydrogel (SS3) had a synergistic effect in between; that is, ISA201 > SS3 > rHF. However, from a statistical analysis of absolute values, comparing ISA201 and rHF, only after the second immunization did ISA201 show a significantly stronger synergistic effect than rHF (P < 0.01, the difference was very significant), while there was no significant difference (ns) between the synergistic effects of ISA201 and rHF after the first immunization (1st) and the third immunization (3rd). Similarly, comparing ISA201 and SS3, there was no significant difference (ns) between different concentrations of SS3 (0.3 mg / mL, 0.5 mg / mL) and the synergistic effect of ISA201. Fourth, considering the absolute values ​​of IgG antibody titers induced by different concentrations of SS3, after the first and second immunizations, the higher concentration of SS3 (SS3=0.5 mg / mL) had a relatively stronger synergistic effect on humoral immunity. However, after the third immunization, the synergistic effect of the lower concentration of SS3 (SS3=0.3 mg / mL) on humoral immunity showed a reversal and enhancement. It is evident that protein nanotube hydrogel (SS3), as a potential adjuvant for viral subunit vaccines, still needs further optimization in terms of optimal concentration. However, the optimal addition concentration is generally between 0.3 mg / mL and 0.5 mg / mL.

[0069] The potential application of protein nanotube hydrogel (SS3) as an adjuvant for viral subunit vaccines: Using SS3 as an auxiliary component of the immunogen (dual antigen complex F / G), it was found that on day 21 after the first immunization (1st), it could activate humoral immune enhancement with extremely significant differences (SS3 & PBS: P < 0.0001). On day 63 after the third immunization (3rd), immunization samples with different concentrations of SS3 all produced immune enhancement effects that were non-inferior to commercial ISA201 adjuvant. This demonstrates that protein nanotube hydrogel (SS3) as a potential adjuvant for viral subunit vaccines can have a significant adjuvant enhancement effect in subcutaneous immunization.

[0070] Example 6: Immunological effects of dual antigen complex (F / G) via the mucosal immune pathway

[0071] 6.1 Methods for preparing immunosampling samples via the mucosal immunoassay route Preparation of immunization samples for the experimental group: The immunization route was similar to that for subcutaneous immunization, using 6-week-old female SPF-grade BALB / c mice, with 3 mice in each group; the immunization protocol was 3 times, including the first immunization (day 0), the second immunization (day 21), and the third immunization (day 42); the immunization groups included the 3 experimental groups from Example 4 (SS3=0 mg / mL, SS3=0.3 mg / mL, SS3=0.5 mg / mL), with the SS3=0 mg / mL dual antigen complex immunization sample group serving as a negative control; according to the number of mice in the above 3 experimental groups, immunization samples containing dual antigen complexes were prepared in the doses required for the 3 immunizations: F / G-SS3-0, F / G-SS3-0.3, and F / G-SS3-0.5. The preparation process of the immunization samples was the same as that of the three experimental groups in Example 5. The specific preparation steps are as follows: 1) In a clean bench, the F purification solution and G purification solution prepared in Example 2 and Example 3 were accurately measured according to a molar ratio of 1:1. The two were mixed evenly to obtain the dual antigen complex (F / G) as the immunization antigen; 2) Different concentrations of SS3 were added to the dual antigen complex (F / G), and then PBS buffer was added to 10 µL / mouse. The three groups of experimental mice continued to receive immunization samples with SS3 final concentrations of 0 mg / mL, 0.3 mg / mL, and 0.5 mg / mL, respectively. Under the premise that the dose of immunization sample received by each mouse was 10 µL / mouse, the G and F in the dual antigen complex (F / G) were equal in amount, and each was 5 µg / mouse.

[0072] Design of control group immunization samples. Since the ISA201 adjuvant, which was used as one of the control groups in the subcutaneous immunization route mentioned above, is a water / oil / water (W / O / W) double emulsion adjuvant, its design purpose is for subcutaneous or intramuscular injection immunization routes, and it is not suitable for mucosal immunization routes. Therefore, in this mucosal immunization route, only rHF adjuvant was selected as the control group for comparative analysis.

[0073] Regarding the preparation of immunization samples for the control group (rHF group), the pure F / G-rHF obtained by directly using the expression plasmids pET32a-F-SpyTag and pET32a-G-SpyTag from Example 5, expressing the fusion proteins G-SpyTag and F-SpyTag, purifying them, and conjugating them with rHF nanoparticles, was directly diluted to an immunization dose of 10 µL / mouse. It was ensured that under the immunization dose of 10 µL / mouse per mouse, the G and F in the dual antigen complex (F / G) were equal in amount, each being 5 µg / mouse.

[0074] 6.2 Animal Immunization The immunization route was mucosal immunization, and the immunization method was nasal instillation immunization. The specific procedure for nasal instillation immunization was as follows: using a 10 µL pipette, 10 µL of immunization sample was instilled into the nasal cavity of the mouse. After instillation, the nostrils were kept facing upward for 1 minute to ensure that all immunization sample was aspirated into the nasal cavity by the mouse.

[0075] Sample collection: Blood samples were collected from mice via the orbital cavity before the second and third immunizations (1st and 2nd) and 3 weeks after the third immunization (3rd or 63rd day). The serum samples were centrifuged after being placed at 37°C for 1 hour and the serum samples were retained. The serum samples were used to determine the F / G specific IgG antibody titer (ELISA method). After blood collection via the orbital cavity on the 63rd day of immunization, the experimental animals were euthanized by cervical dislocation, and the spleen samples were aseptically collected and processed for Elispot detection of T cell immune response.

[0076] The steps for separating and processing spleen samples are as follows: 1) Add 4 mL of mouse lymphocyte separation medium to a cell culture dish with a diameter of approximately 3.5 cm. Place a cell sieve above the culture dish, place the collected fresh spleen on the cell sieve, and gently grind the spleen with the end of a 5 mL sterile syringe plunger. During grinding, keep the cell sieve suspended to avoid the bottom of the sieve contacting the surface of the separation medium or pressing on the bottom of the culture dish, in order to reduce cell death; 2) Gently mix the spleen cell suspension that has passed through the cell sieve into the culture dish after grinding, and then transfer it to a 15 mL centrifuge tube. Then, slowly add 500–1000 µL of serum-free RPMI above the liquid surface. 1) Centrifuge the RPMI 1640 medium, ensuring a clear separation of the liquid surface and avoiding mixing by pipetting; 2) Centrifuge the centrifuge tube at room temperature with a horizontal centrifugal force of 800×g for 30 minutes, adjusting both the ascending and descending speeds to the lowest to maintain stable stratification of the liquid surface. After centrifugation, the tube will separate into four layers: the top layer is a plasma layer diluted with the medium, the middle layer is a cloud-like lymphocyte layer (i.e., a mononuclear cell layer), below which is a clear separation liquid layer, and the bottom layer is a red blood cell sediment; 3) Discard the upper plasma layer and most of the medium, and gently aspirate the middle cloud-like lymphocyte layer with a Pasteur pipette or a 200 μL pipette, transferring the lymphocytes to a new 15 mL centrifuge tube, taking care to avoid aspirating the separation liquid or supernatant; 4) Add 10 mL of serum-free RPMI 1640 medium to the collected lymphocytes, gently invert or slowly pipette to wash the cells, and then centrifuge at room temperature with a horizontal centrifuge force of 250×g for 10 minutes. 7) After centrifugation, discard the supernatant and resuspend the lymphocyte pellet in RPMI 1640 complete medium containing 10% FBS. Take a small amount of the lymphocyte suspension and count the viable cells using a cell counter. Based on the counting results, adjust the cell concentration to 5 × 10⁶ cells / mL using complete medium. 6 Cells / mL can be used for subsequent ELISpot assays.

[0077] 6.3 Humoral immune effect (IgG antibody titer) Referring to the determination procedure for F / G specific IgG antibody titer (ELISA method) in Example 5, the IgG antibody titer of serum samples collected and processed after three immunizations (1st, 2nd, and 3rd) was determined. The operation steps were similar, mainly as follows: 1) Antigen coating: The purified solution of fusion glycoprotein F (F) and the purified solution of attached glycoprotein G (G) were mixed evenly at a molar ratio of 1:1 and diluted to 5 µg / mL. 100 µL was added to 96-well ELISA plates for coating and the plates were washed. 2) Blocking solution (PBST containing 1% sheep serum) was used for blocking. 3) Serum sample incubation: Serum samples collected after three immunizations of the three experimental groups (F / G-SS3-0.3, F / G-SS3-0.5, F / G-ISA201) and one control group (F / G-rHF) were added sequentially according to an initial dilution of 1:16 and 11 dilution gradients of 2:16. Blank control wells contained serum samples from unimmunized mice. 4) Adding enzyme-labeled secondary antibodies: Two secondary antibodies were prepared in parallel: HRP-goat anti-mouse IgG and HRP-goat anti-mouse IgA. HRP-goat anti-mouse IgG was diluted 1:10000 with PBST, and HRP-goat anti-mouse IgA was diluted 1:2000 with PBST. 100 µL / well of each secondary antibody was added to each well. Parallel detection plates for both secondary antibodies were incubated at 37°C for 1-2 hours. The solutions were discarded, the plates were washed 3-5 times, and then patted dry. 5) Color development and detection: After incubating 100 µL / well of heated TMB colorimetric solution at room temperature in the dark, add 100 µL / well of stop solution and immediately read the absorbance value (OD450) using a microplate reader. 6) Result interpretation and analysis: The OD values ​​of the blank control samples were also determined according to the same procedure. 450 Use 2.1 times the average value as the cut-off value to detect OD. 450 Samples with a value greater than the cut-off value are considered positive, and the highest dilution of the positive sample is taken as the antibody titer of the serum sample to be tested. The antibody titers (ELISA method) of F / G specific IgG and F / G specific IgA are plotted as follows: Figure 5 As shown in A and B in the diagram.

[0078] Regarding the results of F / G specific IgG in the nasal immunization route, the absolute values ​​of the detection results in Example 5 (subcutaneous immunization route) showed a similar trend. Statistical analysis showed that the immune synergistic effect of different concentrations of SS3 and rHF control groups after the first and second immunizations was not significant, and even the absolute value of the immune synergistic effect of rHF as an adjuvant showed a negative effect of decreasing. On day 63 after the third immunization (3rd), although there was no significant difference in the synergistic effect between the immunization sample groups with different concentrations of SS3 and the rHF control group, the synergistic effect of SS3 was stronger than that of rHF (P < 0.05, significant difference). Regarding the results of F / G specific IgA via nasal drop immunization, although the synergistic effect of different concentrations of SS3 and rHF in the control group on the titer of stimulating IgA specific antibodies was not significant after the first immunization, the two experimental groups with added SS3 significantly increased the titer of F / G specific IgA antibodies after the second immunization booster, indicating that SS3 had a significantly different synergistic effect on stimulating IgA antibodies (P < 0.05, significant difference); especially after the third immunization booster, the synergistic effect of SS3 on stimulating IgA antibodies was greatly enhanced, showing an extremely significant synergistic effect (P < 0.0001). It is evident that although the addition of SS3 did not significantly enhance the production of F / G-specific IgG compared to the rHF control group, it significantly increased the titer of F / G-specific IgA antibodies in the stimulation serum after a third booster immunization. This demonstrates that SS3 represents a significant advancement as a viral subunit vaccine adjuvant specifically for mucosal immunization, and further optimization and expansion of its application as a vaccine adjuvant are warranted.

[0079] 6.4 Cellular Immunotherapy Effect (Elispot Detection of IFN-γ Antibody) Lymphocyte suspension (5×10⁻⁶) was obtained by separating and processing a spleen sample collected on day 63 post-immunization. 6 Cells / mL), directly perform Elispot detection of IFN-γ secreted by T cells. Refer to the instructions of the Mouse IFN-γ Precoated ELISPOT Kit for Elispot detection. The stimulant is a synthetic immunoepitope peptide fragment of 15 Nipah virus dual antigens (F, G). The specific steps are as follows: 1) Pre-coated plate activation: Take an ELISPOT detection plate pre-coated with anti-IFN-γ antibody on a PVDF membrane, add 200 µL of RPMI 1640 medium containing serum (10% FBS) to each well, incubate at room temperature for 5-10 minutes, then remove the medium liquid; the purpose is to activate the coated antibody and block non-specific binding sites on the PVDF membrane. 2) Cell seeding and stimulation incubation: Take the above-mentioned cells, adjusted to an appropriate concentration (5 × 10⁻⁶ cells / mL), and incubate. 6A suspension of mouse spleen cells (cells / mL) was added to the wells of an ELISPOT assay plate at a volume of 100 µL / well until the final cell concentration was 5 × 10⁻⁶ cells / mL. 5Cells / wells; For each mouse, spleen cell samples were prepared in parallel with 3 replicates, 1 positive control well, and 1 negative control well; the spleen cell samples were obtained from 3 experimental group immunologic samples (F / G-SS3-0, F / G-SS3-0.3, F / G-SS3-0.5) and 1 control group immunologic sample (F / G-rHF). Stimulants (a mixture of 15 F and G immunoepitope peptide fragments) were added to the wells of each sample, with 2 µL of peptide stimulant added to each well to achieve a final concentration of 20 µg / mL for each peptide; 10 µL of positive stimulant (PMA / ionomycin or Con A) was added to the positive control well; 10 µL of RPMI 1640 medium containing 10% FBS for cell resuspending was added to the negative control well; after sample addition, the ELISPOT assay plate was incubated at 37°C in a 5% CO2 incubator for 18-20 hours. 3) Cell lysis: Discard the liquid from the wells, add 200 µL of pre-chilled sterile distilled water (ddH2O) to each well, and incubate at 4°C for 10 minutes to lyse the cells under hypotonic conditions. The purpose is to remove cells and culture supernatant, leaving only the cytokine spots captured on the PVDF membrane. 4) Washing: Discard the liquid from the wells, add 1×Washing Buffer at a dose of 260 µL / well, let stand for 1 minute, then discard the liquid from the wells and blot dry on absorbent paper. Repeat this washing process 6 times. 5) Antibody incubation: Add diluted biotinylated antibody working solution at a dose of 100 µL / well and incubate at 37°C for 1 hour. After incubation, repeat step 4) 6 times. 6) Incubation and washing of enzyme-labeled avidin: Add diluted enzyme-labeled avidin (Streptavidin-HRP) working solution at a dose of 100 µL / well and incubate at 37°C for 1 hour; after incubation, repeat the washing process in step 4) above 5 times; after washing, carefully peel off the base plate, wash the bottom of the PVDF membrane and the ELISpot plate base with sterile distilled water (ddH2O), blot dry the base and the bottom of the PVDF membrane with absorbent paper, and then close the base. 7) Color Development, Termination, and Counting: Add freshly prepared AEC chromogenic solution at a dose of 100 µL / well and incubate at 37°C in the dark for 5-30 minutes. During the color development process, check the spot formation every 5 minutes. When dense red spots appear in the positive control wells, terminate the color development immediately. When terminating the color development, pour out the liquid in the wells, remove the base plate, and wash the front and back of the PVDF membrane and the base 3-5 times with sterile distilled water. After the ELISPOT detection plate has air-dried naturally in a cool, shady place at room temperature, close the base and use an enzyme-linked immunospot analyzer (CTL ImmunoSpot) for automatic counting. 8) Result Interpretation: The count results of the experimental wells are reliable only if the positive control wells have more than 500 spots / well and the negative control wells have less than 10 spots / well.The average count of wells that were deemed positive for IFN-γ secretion (ELISPOT method) is plotted as shown below. Figure 5 The results are shown.

[0080] Analysis of the specific counting results of the ELISPOT assay for nasal immunoassay IFN-γ is as follows: Figure 5 As shown in Figure C, nasal immunization via the mucosal immune route can increase the amount of IFN-γ secreted. Although there was no statistically significant difference between the two experimental groups of immune samples (F / G-SS3-0.3 and F / G-SS3-0.5) with added protein nanotube hydrogel (SS3) and the control group (F / G-rHF), numerically, the preferred concentration group (F / G-SS3-0.3) showed a 50% increase (from 40 to 60). A 50% increase is generally considered a biologically significant synergistic trend in cellular immune responses, and this level of improvement is noteworthy. Note: On the one hand, the added protein nanotube hydrogel (SS3) may enhance the clonal expansion of antigen-specific T cells through a mechanism of action different from rHF (such as stronger antigen-presenting cell activation or better lymph node targeting); on the other hand, when antigen production is limited (such as high production costs), candidate adjuvants can elicit higher levels of T cell responses with the same amount of antigen, and can achieve better effects than rHF with less protein nanotube hydrogel (SS3) added. Therefore, the addition of protein nanotube hydrogel (SS3) has the advantages of reducing the effective immunization dose and cost-effectiveness. Therefore, protein nanotube hydrogel (SS3) can not only activate Th1-type cellular immunity, but also, with the addition of SS3 at a concentration of 0.3 mg / mL, an equivalent dose of antigen (dual antigen complex F / G) can activate a relatively higher IFN-γ level on day 63 post-immunization. Moreover, under experimental conditions with limited statistical power, protein nanotube hydrogel (SS3), as an adjuvant for viral subunit vaccines, still showed a superior trend of enhanced cellular immunity (50% increase) compared to the current reference group (rHF). This numerical difference is sufficient to support its inclusion as a potential candidate adjuvant in further optimization and validation studies (such as increasing animal sample size, exploring higher doses or different immunization routes) to confirm its statistical significance and explore its antigen-saving value in practical applications. In the future, it may have unique application value in the development of nasal spray vaccine adjuvants via mucosal immunization, tumor immunotherapy, and chronic infection control.

[0081] Example 7: Safety of using protein nanotube hydrogel (SS3) as an adjuvant for viral subunit vaccines. To preliminarily assess the safety of protein nanotube hydrogel (SS3) as an adjuvant for viral subunit vaccines, this study conducted a maximum immunization dose experiment. The general physiological state and weight changes of experimental animals (mice) were observed after mice were immunized once via two routes of immunization: subcutaneous injection and intranasal instillation, in order to determine the preliminary safety and maximum tolerated dose range of SS3.

[0082] Maximum dose animal immunization experiment: Mice were randomly divided into three groups: subcutaneous immunization group, intranasal immunization group, and negative control group, with two mice in each group. Group 1 received the protein nanotube hydrogel (SS3) prepared according to Example 1, and mice in the subcutaneous immunization group were injected subcutaneously into the neck at the maximum dose of 25 µg / mouse, with an immunization dose of 50 µL / mouse. Group 2 received the intranasal immunization group at the maximum dose of 5 µg / mouse, with an immunization dose of 10 µL / mouse. Group 3 was the control group, and mice in the control group were injected subcutaneously into the neck with phosphate-buffered saline (PBS), with an immunization dose of 50 µL / mouse. From the day of immunization (day 0) of the three groups, the general signs of the experimental mice, such as mental state, activity level, coat luster, and body shape, were observed daily for 30 consecutive days. The weight of the mice was weighed and recorded every 3 days, and the differences between the experimental group and the control group were compared.

[0083] Results of the highest-dose animal immunization experiment: Throughout the 30-day observation period, mice in the subcutaneous immunization group (25 µg / mouse) and the intranasal immunization group (5 µg / mouse) did not exhibit any abnormal behaviors or stress responses such as lethargy, piloerection, or arched back. Furthermore, the mice in both experimental groups had smooth fur, normal activity, good mental state, and stable weight gain. All observed indicators showed no significant differences compared to the negative control group. In particular, weight monitoring data showed no statistically significant difference in weight gain trends between the subcutaneous and intranasal immunization groups and the negative control group (PBS group).

[0084] The results of the highest-dose animal immunization experiments show that the protein nanotube hydrogel (SS3) exhibits good local tolerability and systemic safety in mouse models. Regardless of the subcutaneous injection dose as high as 25 µg or the nasal drop dose as high as 5 µg, SS3 did not produce any visually perceptible acute toxicity or adverse physiological effects on the growth of mice. These results provide important evidence for the efficacy and safety studies and dosage selection of SS3 as an adjuvant for viral subunit vaccines.

[0085] Specific embodiments of the present invention have been described in detail, making them readily understandable to those skilled in the art. However, based on all the disclosed descriptions, various modifications or substitutions can be made to those details, and all such changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

[0086] References (1) ADDIN ZOTERO_BIBL {"uncited":[],"omitted":[],"custom":[]} CSL_BIBLIOGRAPHY (1) Loomis, R. J.; Stewart-Jones, G. B. E.; Tsybovsky, Y.;Caringal, R. T.; Morabito, K. M.; McLellan, J. S.; Chamberlain, A. L.;Nugent, S. T.; Hutchinson, G. B.; Kueltzo, L. A.; Mascola, J. R.; Graham, B.S. Structure-Based Design of Nipah Virus Vaccines: A Generalizable Approachto Paramyxovirus Immunogen Development. Front. Immunol. 2020, 11, 842.https: / / doi.org / 10.3389 / fimmu.2020.00842. (2) Loomis, R. J.; DiPiazza, A. T.; Falcone, S.; Ruckwardt, T. J.;Morabito, K. M.; Abiona, O. M.; Chang, L. A.; Caringal, R. T.; Presnyak, V.;Narayanan, E.; Tsybovsky, Y.; Nair, D.; Hutchinson, G. B.; Stewart-Jones, G.B. E.; Kueltzo, L. A.; Himansu, S.; Mascola, J. R.; Carfi, A.; Graham, B. S.Chimeric Fusion (F) and Attachment (G) Glycoprotein Antigen Delivery by mRNAas a Candidate Nipah Vaccine. Front. Immunol. 2021, 12, 772864. https: / / doi.org / 10.3389 / fimmu.2021.772864. (3) Kalodimou, G.; Veit, S.; Jany , S. ; Kalinke, U.; Broder , CC ;Sutter , G. ; Volz, A. A Soluble Version of Nipah Virus Glycoprotein GDelivered by Vaccinia Virus MVA Activates Specific CD8 and CD4 T Cells inMice. Viruses 2019, 12(1), 26. https: / / doi.org / 10.3390 / v12010026. (4) Avanzato, VA; Oguntuyo, KY; Ladder-Zamudio , M. ;Gutierrez , B. ; Golden , M. ; Kosakovsky Pond, SL; Pryce , R. ; Walter , TS ;Seow , J. ; Doores , KJ ; Pybus, OG; Munster, VJ; Lee, B.; Bowden, TAA Structural Basis for Antibody-Mediated Neutralization of Nipah VirusReveals a Site of Vulnerability at the Fusion Glycoprotein Apex. Proc. Natl.Acad. Sci. Rev. 2019, 116(50), 25057–25067. https: / / doi.org / 10.1073 / pnas. (5) Eshaghi, M.; Tan , WS ; Yusoff, K. Identification of Epitopes inthe Nucleocapsid Protein of Nipah Virus Using a Linear Phage‐displayed RandomPeptide Library. J. Med. Virol. 2005, 75(1), 147–152. https: / / doi.org / 10.1002 / jmv.20249. (6) Yin, J.; Yao, YF; Yang, P.; Liu, H.; Gao, G.; Peng, Y.; Chen,M.; Lu, M.; Zhang, X.; Guo, W.; Zhang, Z.; Hu, X.; Yuan, Z.; Shan, C. AHighly Effective Ferritin-Based Divalent Nanoparticle Vaccine Shields SyrianHamsters against Lethal Nipah Virus. Front. Immunol. 2024, 15, 1387811.https: / / doi.org / 10.3389 / fimmu.2024.1387811.

Claims

1. A vaccine composition based on protein nanotube hydrogel, characterized in that, The vaccine composition comprises a protein nanotube hydrogel and a specific antigen, wherein the protein nanotube hydrogel is a three-point mutant CP of the MS2 phage capsid protein. C46A / Q70C / V75C A three-dimensional network structure hydrogel formed by self-assembly of disulfide bonds between nanotubes, wherein the three-point mutant CP C46A / Q70C / V75C It has an amino acid sequence as shown in SEQ ID NO:1; the specific antigen is an antigen in the form of a subunit vaccine; the protein nanotube hydrogel and the specific antigen are physically mixed to form a complex.

2. The vaccine composition based on protein nanotube hydrogel according to claim 1, characterized in that, The specific antigen is an antigen against Nipah virus, and the physical mixing is a simple mixing of protein nanotube hydrogel and specific antigen. The purification and preparation process of the protein nanotube hydrogel does not require the addition of chemical cross-linking agents.

3. The vaccine composition based on protein nanotube hydrogel according to claim 1, characterized in that, The specific antigen is a dual antigen complex against Nipah virus, and the concentration of the protein nanotube hydrogel is 0.3–0.5 mg / mL.

4. The vaccine composition based on protein nanotube hydrogel according to claim 3, characterized in that, The dual antigen complex is selected from the epitope enrichment segment of Nipah virus fusion glycoprotein F and the extracellular domain of attachment glycoprotein G, wherein the epitope enrichment segment of fusion glycoprotein F has the amino acid sequence shown in SEQ ID NO:2 and the extracellular domain of attachment glycoprotein G has the amino acid sequence shown in SEQ ID NO:

3.

5. A vaccine formulation based on protein nanotube hydrogel, characterized in that, The vaccine composition includes the protein nanotube hydrogel-based composition of claim 1, but excludes other vaccine adjuvants.

6. The vaccine formulation based on protein nanotube hydrogel according to claim 5, characterized in that, The vaccine preparation is an injection, nasal drop, inhaler, or spray.

7. The vaccine formulation based on protein nanotube hydrogel according to claim 5, characterized in that, The vaccine formulation is administered via subcutaneous injection. After subcutaneous injection, the protein nanotube hydrogel forms an antigen library at the injection site, extending the in vivo clearance half-life of the specific antigen to no less than 4 days.

8. The vaccine formulation based on protein nanotube hydrogel according to claim 5, characterized in that, The vaccine formulation provides immunization via mucosal immunity, and this mucosal immunity, when combined with booster immunization, simultaneously induces and stimulates humoral and cellular immunity.

9. A vaccine delivery platform, characterized in that, The vaccine is constructed based on the protein nanotube hydrogel-based vaccine composition as described in any one of claims 1-4, or the protein nanotube hydrogel-based vaccine formulation as described in any one of claims 5-8.

10. The vaccine composition based on protein nanotube hydrogel according to any one of claims 1-4, or the vaccine formulation based on protein nanotube hydrogel according to any one of claims 5-8, or the application of the vaccine delivery platform according to claim 9 in a viral subunit vaccine, characterized in that, The application is applicable not only to Nipah virus antigen, but also to other infectious disease antigens and tumor antigens.

Images