A nano-vaccine delivery carrier GALA-CS, a preparation method and application thereof, and a nano-vaccine

Abstract

This invention belongs to the field of nanovaccine technology, and discloses a nanovaccine delivery carrier GALA-CS, its preparation method and application, and a nanovaccine. The nanovaccine of this invention has stable physicochemical properties and good biocompatibility. GALA peptide modification significantly enhances its cellular uptake and endosome escape capabilities. G-CP-NPs can effectively activate antigen-presenting cells in vitro, significantly promote chBMDC maturation and upregulate CD11c. + and MHC II + Compared with traditional inactivated vaccines, G-CP-NPs exhibit a faster immune activation capacity; G-CP-NPs can simultaneously and efficiently induce mucosal immunity and cellular immunity, making up for the core defects of traditional inactivated vaccines; in addition, G-CP-NPs can more efficiently clear the virus in heterologous virus challenge experiments, significantly shorten the viral shedding cycle, reduce viral load in target organs, and alleviate lung pathological damage, providing new strategies and experimental evidence for the development of novel vaccines for H9N2 subtype avian influenza.

Description

Technical Field

[0001] This invention relates to the field of nanovaccine technology, and in particular to a nanovaccine delivery carrier GALA-CS, its preparation method and application, and nanovaccines. Background Technology

[0002] Influenza A virus (IAV) belongs to the Orthomyxoviridae family and is an enveloped, single-stranded, negative-sense, segmented RNA virus. Based on antigenic differences in its internal conserved nucleoprotein (NP) and matrix protein (M), influenza viruses are classified into four types: A, B, C, and D. Type A influenza virus has the widest host range and poses the greatest threat to human health and livestock safety; avian influenza virus (AIV) is an important member of this group. In recent years, the risk of cross-species transmission of AIV has increased significantly, posing a serious challenge to global public health security. HPAIV, represented by the H5 genotype AIV (clade 2.3.4.4b), has caused large-scale outbreaks in birds and mammals. These viruses spread widely through wild bird migration, leading to mass mortality in poultry and wild animals. Furthermore, human infections are mainly caused by H7N9 and H5N1, indicating that avian influenza viruses have the evolutionary potential to play a role in zoonotic disease transmission chains.

[0003] The H9N2 subtype of avian influenza virus is the most widespread AIV in poultry worldwide, causing continuous and severe economic losses to the poultry industry. Since its first report, the prevalence of H9N2 AIV has been steadily increasing, and it has now replaced H5N6 and H7N9 as the most common AIV subtype. It possesses both endemic and zoonotic characteristics, posing a dual and ongoing threat to the global poultry industry and public health. Although classified as a low-pathogenic avian influenza, H9N2 infection severely damages the reproductive system of laying hens. H9N2 can replicate efficiently in the oviduct tissue of laying hens, causing severe pathological damage, leading to a sharp decline in egg production, often accompanied by cessation of egg production or frequent production of deformed, thin-shelled, and soft-shelled eggs. H9N2 infection often induces significant immunosuppression in the host. Viral infection can impair both humoral and cellular immunity in chickens, significantly weakening the body's barrier function against other pathogens. In intensive poultry farming, this immunosuppression easily leads to severe viral or bacterial co-infections and secondary infections. The superposition of H9N2 with other common avian viruses significantly exacerbates damage. For example, if a flock is first infected with infectious bronchitis virus and then with H9N2, it will cause more severe respiratory clinical symptoms, extremely high local viral load, and severe tracheal inflammation. When H9N2 is co-infected with Newcastle disease virus (NDV), it not only further aggravates pathological damage to immune organs such as the bursa of Fabricius, spleen, and thymus, directly leading to decreased feed intake and significant weight loss, but also severely interferes with the immunizing effect of routine vaccines, exacerbating the shedding of H9N2 virus in the trachea and cloaca. In bacterial infections, H9N2 also exhibits strong synergistic pathogenicity. When H9N2 is co-infected with Salmonella, it not only significantly exacerbates clinical symptoms and mortality in chickens, but also promotes Salmonella colonization in organs and fecal shedding, while prolonging the H9N2 virus shedding cycle itself. When co-infected with avian pathogenic Escherichia coli, it triggers a more intense inflammatory response, further aggravating tissue damage to target organs such as the oviduct. This network effect of multi-pathogen synergistic pathogenicity transforms H9N2, which originally had a low single-infection mortality rate, into a complex disease syndrome leading to high culling and mortality rates in chicken flocks.

[0004] Given the harm caused by H9N2 AIV in poultry and its potential threat to human public health, the development of effective vaccines is of paramount importance. Currently, its control still mainly relies on traditional inactivated vaccines (InV). However, these vaccines are mostly administered via a single intramuscular injection route, which is insufficient to effectively induce local mucosal immunity at the natural entry points of viruses, such as the respiratory tract, resulting in unsatisfactory efficacy and leaving immunized flocks still at risk of viral shedding and transmission. Therefore, exploring novel vaccines that can synergistically stimulate systemic antibodies and local mucosal defenses has become crucial to overcoming the current immunization bottlenecks of avian influenza. Summary of the Invention

[0005] The purpose of this invention is to provide a nano-vaccine delivery carrier GALA-CS, which co-encapsulates H9N2 AIV inactivated antigen and CpG ODN molecular adjuvant to optimize the preparation of nano-vaccines G-CP-NPs. G-CP-NPs have a faster immune activation ability and can simultaneously and efficiently induce mucosal immunity and cellular immunity. In heterologous virus challenge experiments, it can more efficiently clear viruses, significantly shorten the virus shedding cycle, reduce viral load in target organs, and alleviate lung pathological damage.

[0006] Another objective of this invention is to provide a method for preparing a nano-vaccine delivery carrier GALA-CS.

[0007] Another object of the present invention is to provide an application of the nano-vaccine delivery carrier GALA-CS.

[0008] Another object of the present invention is to provide a nano-vaccine for the prevention of avian influenza virus.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A nanovaccine delivery carrier GALA-CS, comprising a cell-penetrating peptide and chitosan, wherein the cell-penetrating peptide is a GALA peptide; the molar ratio of the GALA peptide to the free amino group of chitosan is 1:5~10.

[0011] Furthermore, this invention discloses a method for preparing the GALA-CS nanovaccine delivery carrier as described above, comprising the following steps:

[0012] Step 1: Prepare a solution A of GALA peptide with MES buffer at a concentration of 8-12 mg / mL; prepare a solution B of carbodiimide hydrochloride with MES buffer at a concentration of 60-68 mg / mL; prepare a solution C of N-hydroxysuccinimide with MES buffer at a concentration of 15-25 mg / mL.

[0013] Step 2: After mixing solutions B and C, immediately add the mixture to solution A, and then shake at 2-6℃ for 15-25 minutes.

[0014] Step 3: Dissolve chitosan completely in 1 wt% glacial acetic acid, and add an appropriate amount of 0.1 M MES buffer to adjust the pH of the chitosan solution to 6.0~6.5;

[0015] Step 4: Add the solution obtained in Step 2 dropwise to the chitosan solution in Step 3, controlling the molar ratio of GALA peptide to chitosan free amino group to be 1:5~10, and then stir continuously at room temperature in the dark for 24 hours. After the reaction is completed, add 50 mM β-mercaptoethanol to the system, and then dialyze the mixture using a Zeba™ desalting column. After the dialysate is pre-frozen at -80℃, it is then freeze-dried under vacuum to obtain the GALA-CS lyophilized nanovaccine delivery carrier.

[0016] Furthermore, this invention discloses the application of the nanovaccine delivery carrier GALA-CS prepared by the above-described method or by the above-described preparation method in the preparation of nanovaccines.

[0017] Preferably, the nano-vaccine is a nano-vaccine for the prevention of avian influenza virus.

[0018] Finally, this invention discloses a nano-vaccine for the prevention of avian influenza virus, wherein the nano-vaccine comprises the nano-vaccine delivery carrier GALA-CS as described in claim 1, H9N2 inactivated whole virus antigen, and molecular adjuvant CpG-ODN.

[0019] Preferably, the molecular adjuvant CpG-ODN includes CpG-ODNs-1 and CpG-ODNs-2.

[0020] More preferably, the nucleotide sequences of the molecular adjuvants CpG-ODNs-1 and CpG-ODNs-2 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] (1) This invention successfully constructed GALA-CS, a chitosan nanovaccine delivery carrier modified with GALA peptide, and G-CP-NPs, a nanovaccine. The G-CP-NPs nanovaccine has stable physicochemical properties and good biocompatibility. GALA peptide modification significantly enhances its cellular uptake and endosome escape capabilities. G-CP-NPs can effectively activate antigen-presenting cells in vitro, significantly promote chBMDC maturation, and upregulate CD11c. + and MHC II + Express.

[0023] (2) Compared with traditional inactivated vaccines, the G-CP-NPs provided by this invention exhibit a faster immune activation ability; G-CP-NPs can simultaneously and efficiently induce mucosal immunity and cellular immunity, making up for the core defects of traditional inactivated vaccines; in addition, G-CP-NPs can clear the virus more efficiently in heterologous virus challenge experiments, significantly shorten the virus shedding cycle, reduce the viral load in target organs, and alleviate lung pathological damage, providing a new strategy and experimental basis for the development of novel vaccines for H9N2 subtype avian influenza. Attached Figure Description

[0024] Figure 1 The diagram shows the synthetic route for GALA-CS. In the diagram, A represents the chemical structure of chitosan; B represents the amino acid sequence and structural schematic of GALA peptide; and C represents the synthetic reaction mechanism of GALA-CS conjugate.

[0025] Figure 2 FT-IR analysis results for CS and GALA-CS;

[0026] Figure 3 For the GALA-CS theory based on ChemDraw simulation prediction 1 H NMR spectrum;

[0027] Figure 4 The actual measured values ​​for the product GALA-CS 1 H NMR identification results;

[0028] Figure 5 This study evaluates the uptake behavior of G-CP-NPs nanoparticles in HD11 cells and characterizes them physicochemically. Figure a shows the uptake and co-localization of different formulations and inactivated virus (WIV) in HD11 cells observed using laser confocal microscopy. The right side shows the corresponding fluorescence co-localization intensity analysis. Green represents FITC-labeled carriers, red represents AIV antigens, and blue represents DAPI-stained cell nuclei. The scale bar is 20 μm. The right side also shows the fluorescence intensity line scan analysis of the corresponding field of view, characterizing the intracellular fluorescence co-localization trend of the carrier and antigen. Figure b shows the quantitative analysis of the intracellular Pearson correlation coefficient (Rr) between different groups of nanocarriers and AIV NPs.

[0029] Figure 6 To compare the particle size distribution of optimized G-CP-NPs with that of the untreated group using dynamic light scattering (DLS) instrumentation;

[0030] Figure 7The results show the effect of GALA peptide modification on the antigen delivery efficiency of chitosan nanoparticles in HD11 cells. In the figure, a shows the uptake and co-localization of G-CP-NPs (GALA peptide modified group) and CP-NPs (unmodified control group) in HD11 cells; the blue channel represents DAPI-labeled cell nuclei, the red channel represents AIV NP antigen, and the green channel represents FITC-labeled chitosan nanocarriers. Merge is a multi-channel overlay image with a scale bar of 20 μm; b shows the quantitative analysis of intracellular CS-FITC mean fluorescence intensity (MFI); c shows the quantitative analysis of intracellular Pearson correlation coefficient (Rr) between nanocarriers and AIV antigens.

[0031] Figure 8 The in vitro safety evaluation of G-CP-NPs on different poultry cell lines is shown in the figure. a) Effect of G-CP-NPs on DEF cell viability; b) Effect of G-CP-NPs on CEF cell viability; c) Effect of G-CP-NPs on DF-1 cell viability.

[0032] Figure 9 This image shows the in vitro evaluation of G-CP-NPs-induced maturation and activation of bone marrow-derived dendritic cells (BMDCs). Figure a, from left to right, shows BMDC morphology on days 3, 5, and 7 of in vitro culture, morphology after LPS-induced maturation, and a magnified BMDC microscope image (400×). Figure b shows the flow cytometry gating strategy for BMDCs. Figure c shows the BMDC surface maturation marker CD11c. + A quantitative statistical graph of the proportion of positive cells; d represents the MHC II maturation marker on the surface of BMDCs. + A quantitative statistical graph of the proportion of positive cells;

[0033] Figure 10 The graph shows the HI antibody titer results in different groups of chicken serum.

[0034] Figure 11 Figure 1 shows the results of the detection of micro-neutralizing antibody titers in chicken serum from different groups.

[0035] Figure 12 The graph shows the detection results of specific antibody content in chicken serum from different groups; in the graph, a represents the detection result of IgM antibody content; b represents the detection result of IgY antibody content; and c represents the detection result of IgA antibody content.

[0036] Figure 13 To enhance the levels of sIgA antibodies in bronchoalveolar lavage fluid and tracheal mucosa after immunization; in the figure, a represents the sIgA content in tracheal lavage fluid; b represents the sIgA content in bronchoalveolar lavage fluid;

[0037] Figure 14The figure shows the dynamic changes in the proportion of B cells in chicken peripheral blood; in the figure, a is the gating strategy for B cell flow cytometry staining; b is the proportion of B cells in PBMCs.

[0038] Figure 15 The figure shows the dynamic changes in the proportion of T lymphocyte subsets in chicken peripheral blood; in the figure, a represents the gating strategy of T cell subset flow cytometry staining; b~e represent CD4+ in PBMCs at 7, 14, 21, and 28 DPV after immunization, respectively. + CD8 + and CD4 + CD8 + The proportion of T cells;

[0039] Figure 16 The figure shows the expression of immune-related genes in chicken PBMCs after immunization. In the figure, a represents the expression of CTL-related genes; b represents the expression of inflammation and chemokine-related genes; c represents the expression of Th2-related genes; the horizontal dashed line with y=1 represents the relative expression baseline of the non-immunized group (PBS control group); the asterisks above the bars represent the significant differences between the immunized group and the PBS control group, and the asterisks with lines represent the significant differences between the G-CP-NPs group and the InV group in pairwise comparisons.

[0040] Figure 17 The secretion level of IFN-γ by chicken spleen lymphocytes;

[0041] Figure 18 The images show the detoxification process of the throat and cloaca; in the diagram, a represents the detoxification process of a throat swab, and b represents the detoxification process of anal swabs.

[0042] Figure 19 The viral load in the organ is 3 DPI; in the figure, 3 / 3, 2 / 3, 1 / 3 and 0 / 3 represent the ratio of the number of animals with positive virus detection in the corresponding tissue to the total number of animals tested in the corresponding group;

[0043] Figure 20 HE staining pathological observation of major organs of chickens in each group after challenge (3 DPI); in the figure, Blank is the blank control group (no immunization, no challenge); PBS is the challenge model group (PBS + H9N2 AIV); InV is the traditional inactivated vaccine group; G-CP-NPs is the nanovaccine group; red arrows in the figure indicate multifocal capillary congestion, and blue arrows indicate interstitial lymphocyte infiltration; the scale bar is 100 μm. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It should be noted that, where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0045] Information on materials, reagents, and instruments:

[0046] The main sources of biological materials are shown in Table 1.

[0047] Table 1 Main Materials

[0048] H9N2 AIV challenge strain (A / Chicken / Kunming / 23540 / 2023) South China Agricultural University National-Local Joint Engineering Laboratory for Zoonotic Disease Control Preparations H9N2 AIV vaccine strain (A / Chicken / Hunan / HN / 2015) South China Agricultural University National-Local Joint Engineering Laboratory for Zoonotic Disease Control Preparations MDCK cell line South China Agricultural University National-Local Joint Engineering Laboratory for Zoonotic Disease Control Preparations DF-1 cell line South China Agricultural University National-Local Joint Engineering Laboratory for Zoonotic Disease Control Preparations HD11 cell line South China Agricultural University National-Local Joint Engineering Laboratory for Zoonotic Disease Control Preparations Duck embryo fibroblasts (DEF) South China Agricultural University National-Local Joint Engineering Laboratory for Zoonotic Disease Control Preparations Chicken embryo fibroblasts (CEF) Prepared using conventional methods 2-week-old SPF chickens Guangdong Xinxing Dahua Agricultural Poultry and Egg Co., Ltd. 3-week-old SPF chickens Guangdong Xinxing Dahua Agricultural Poultry and Egg Co., Ltd. 9-11 day old SPF chicken embryos Guangdong Xinxing Dahua Agricultural Poultry and Egg Co., Ltd. SS strain H9N2 inactivated vaccine Guangdong Wenshi Dahua Agricultural Biotechnology Co., Ltd.

[0049] The main reagents are shown in Table 2:

[0050] Table 2 Main Reagents

[0051] RPMI-1640 culture medium Gibco Fetal bovine serum (FBS) Gibco 10000UI dual resistance Gibco 0.25% Trypsin-EDTA pancreatic enzyme Gibco Chitosan (degree of deacetylation ≥ 90%) Shanghai Aladdin Company Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride Shanghai Aladdin Company β-Mercaptoethanol Shanghai Aladdin Company glacial acetic acid Shanghai Aladdin Company GALA peptide MCE Company 2-Morpholine ethanesulfonic acid (MES) MCE Company N-hydroxysuccinimide (NHS) Thermo Fisher Scientific N-Hydroxysulfosuccinimide (Sulfo-NHS) Thermo Fisher Scientific Zeba™ dye and biotin removal centrifuge columns and filter plates Thermo Fisher Scientific β-propiolactone (BPL) Shanghai Biyuntian Company BCA Protein Concentration Assay Kit Shanghai Biyuntian Company RNA extraction kit Nanjing Novozymes Biotechnology Co., Ltd. Mouse Anti-Chicken CD3-APC Southern Biotech Mouse Anti-Chicken CD4-FITC Southern Biotech Mouse Anti-Chicken CD8a-PE Southern Biotech Mouse Anti-Chicken BU 1-FITC Southern Biotech Influenza A virus Nucleoprotein antibody GeneTex SYRBR Green Premix Pro Taq HS qPCR Kit Hunan Aikerui Biotechnology Co., Ltd. Recombinant Human IL-4 PeproTechg Company Recombinant Human GM-CSF PeproTechg Company Chicken peripheral blood lymphocyte isolation kit Tianjin Haoyang Biological Products Technology Co., Ltd. Chicken spleen lymphocyte isolation kit Tianjin Haoyang Biological Products Technology Co., Ltd. Chicken bone marrow-derived cell isolation kit Tianjin Haoyang Biological Products Technology Co., Ltd. Red blood cell lysis fluid Tianjin Haoyang Biological Products Technology Co., Ltd. Chicken Immunoglobulin M (IgM) Detection Kit Xiamen Lunchangshuo Biotechnology Co., Ltd. Chicken Immunoglobulin G (IgG) Detection Kit Xiamen Lunchangshuo Biotechnology Co., Ltd. Chicken Immunoglobulin A (IgA) Detection Kit Xiamen Lunchangshuo Biotechnology Co., Ltd. Chicken secretory immunoglobulin A (sIgA) detection kit Xiamen Lunchangshuo Biotechnology Co., Ltd.

[0052] The main instruments and equipment are shown in Table 3:

[0053] Table 3 Main Instruments and Equipment

[0054] PCR instrument Bio-Rad, Inc. (USA) Real-time PCR instrument Jena, Germany Laser particle size analyzer Malvern Instruments Ltd., UK Fourier transform infrared spectrometer Thermo Fisher Scientific, USA high-speed centrifuge Thermo Fisher Scientific, USA <![CDATA[CO2 Incubator]]> Thermo Fisher Scientific, USA Vortex oscillator Thermo Fisher Scientific, USA Flow Cytometry Beckman Coulter, Inc. Laser confocal microscope German company Zeiss Nuclear magnetic resonance spectrometer Bruker Biospin, Switzerland Electronic balance BioTek, Inc. 7500 Real-Time PCR system Applied Biosystems, Inc. (USA) Refrigerated centrifuge Eppendorf -80℃ and -20℃ ultra-low temperature freezers Haier Company Biosafety cabinet Thermo Fisher Scientific, USA constant temperature water bath Qixin Technology Co., Ltd. Ultraviolet spectrophotometer Thermo Fisher Scientific, USA Freeze-drying machine Hangzhou Furuijie Technology Co., Ltd. pH meter Shanghai Leici Instrument Co., Ltd.

[0055] The main reagents are prepared as follows:

[0056] Flow Cytometry Buffer: Place 49 mL of PBS in a centrifuge tube, add 1 mL of inactivated FBS, mix well, and store at 4°C.

[0057] RPMI-1640 complete medium: Take 45 mL of RPMI-1640 medium and place it in a centrifuge tube. Add 5 mL of inactivated FBS and 500 μL of double antibiotics, mix well, and store at 4℃.

[0058] 1% chicken red blood cells: Draw 0.5 mL of chicken red blood cells into a syringe and slowly add them into 49.5 mL of sterile PBS. Gently shake to mix and store at 4°C for later use.

[0059] Cell cryopreservation solution: Place 45 mL of inactivated FBS in a centrifuge tube, add 5 mL of DMSO, mix well, and store at 4°C.

[0060] 0.1 M NaHCO3 sodium bicarbonate buffer (pH 8.3-8.5): Weigh 0.84 g of sodium bicarbonate (NaHCO3, MW=84.01) and dissolve it in 80 mL of ultrapure water. Stir magnetically until completely dissolved. Use a pH meter to monitor the pH and adjust the solution to 8.3 using 1 M NaOH or 1 M HCl.

[0061] 0.1M MES buffer: Accurately weigh 0.1952g of MES free acid powder, add the powder to a beaker, add approximately 8mL of ultrapure water, and stir with a magnetic stirrer until completely dissolved. Using a pH meter, slowly add 1M NaOH solution dropwise to adjust the pH to 6.0. Transfer the pH-adjusted solution to a volumetric flask and bring the volume to 10mL. Filter the resulting buffer solution through a 0.22μm syringe filter for sterilization and store at 4°C protected from light for later use.

[0062] The CpG ODN sequences used in the embodiments are shown in Table 4;

[0063] Table 4 CpG ODN Sequences

[0064] CpG ODNs-1 5′-GCTAGTAGGTGT-3′ SEQ ID NO: 1 CpG ODNs-2 5′-TCAACGTGT-3′ SEQ ID NO: 2

[0065] This study used GraphPad Prism8 software to perform statistical analysis on all experimental data. Here, ns indicates P > 0.05, meaning no significant difference; * indicates P < 0.05, meaning a significant difference; ** indicates P < 0.01; *** indicates P < 0.001; and **** indicates P < 0.0001, meaning an extremely significant difference.

[0066] Example 1

[0067] 1.1 Propagation of H9N2 AIV

[0068] Two H9N2 AIV strains were used: vaccine strain AA / Chicken / Hunan / HN / 2015 and challenge strain A / Chicken / Kunming / 23540 / 2023. Both strains were propagated using the following methods:

[0069] Thaw the frozen virus solution from the laboratory and dilute it 1000-fold with sterile PBS containing 10% penicillin-streptomycin. Inoculate 0.2 mL / embryo into 9-11 day old SPF chicken embryos and incubate at 37°C. Candling of the embryos every 12 hours starting 24 hours after inoculation was performed, and chicken embryos that died within 24 hours were discarded. After 72 hours of incubation, the chicken embryos were cooled at 4°C overnight, and the allantoic fluid was collected aseptically. The collected allantoic fluid was centrifuged at 2000 rpm for 10 min at 4°C to remove cell debris. The supernatant was filtered through a 0.22 μm filter for sterilization, aliquoted, and stored at -80°C for later use.

[0070] 1.2 Blood coagulation titer determination

[0071] The hemagglutination titer of the virus was determined using a standard microhemagglutination assay. In a 96-well V-type microplate, 25 µL of PBS was added to each well. 25 µL of the virus solution to be tested was added to well 1, and after mixing by pipetting, 25 µL was transferred to well 2. This process was repeated serially up to well 11, and the 25 µL solution from well 11 was discarded. 50 µL of PBS was added to well 12 as a red blood cell control. 25 µL of 1% chicken red blood cell suspension was added to each well, and after vortexing, the mixture was incubated at room temperature for 30 min. The results were then observed. The highest dilution of the virus that caused complete agglutination of red blood cells was determined as the hemagglutination titer of the virus.

[0072] 1.3 Chicken embryo median infection dose (EID) 50 Measurement

[0073] After thawing H9N2 AIV (A / Chicken / Kunming / 23540 / 2023), perform a 10-fold serial dilution with PBS containing 10% penicillin antibody. Take 10... -5 ~10 -9 Virus solution at the same dilution was inoculated into 9-day-old chicken embryos, with 0.2 mL per embryo for every 5 embryos. After inoculation, the embryos were incubated at 37°C for 72 h. Allantoic fluid was collected, and viral infection was assessed using a hemagglutination assay (HA). The 50% Embryo infectious dose (EID) was calculated using the Reed-Muench method. 50 ).

[0074] 1.4 Inactivation of H9N2 AIV and Antigen Preparation

[0075] The allantoic fluid of the H9N2 AIV vaccine strain (A / Chicken / Hunan / HN / 2015) harvested after propagation in section 1.1 was centrifuged at 8000 xg for 30 min at 4°C to remove cell debris, and the supernatant was collected. Inactivation was performed using β-propiolactone (BPL): BPL was added at a volume ratio of 1:4000 (final concentration 0.1%), thoroughly mixed, and then inactivated by rotation in a 4°C freezer for 24 h with intermittent shaking. After inactivation, the virus solution was transferred to a 37°C water bath and shaken for 2 h to ensure complete hydrolysis of BPL. 0.2 mL of the inactivated virus solution was inoculated into 9-day-old SPF chicken embryos and incubated at 37°C for 72 h. Allantoic fluid was collected after incubation, and the virus was passaged blindly for two consecutive generations. The inactivation effect was assessed by a hemagglutination test. Complete inactivation was considered achieved if all chicken embryo allantoic fluid hemagglutination tests were negative.

[0076] The completely inactivated virus solution was concentrated by ultracentrifugation (100,000 xg, 2 h, 4℃), and the precipitate was resuspended in sterile PBS. The total protein concentration was determined using a BCA protein concentration assay kit, and the solution was aliquoted and stored at -80℃ for later use as the "H9N2 inactivated whole virus antigen solution" for subsequent nanoparticle preparation.

[0077] 1.5 Synthesis of GALA peptide-modified chitosan (GALA-CS)

[0078] GALA-CS was prepared using the EDC / NHS chemical cross-linking method. The synthetic route is as follows: Figure 1 As shown, 10 mg of GALA peptide was prepared into a 10 mg / mL solution in MES buffer on ice. Based on the average molecular weight of GALA peptide (3000 Da), precise stoichiometry was performed. To achieve efficient activation of the terminal carboxyl group of the peptide, approximately 6.4 mg of carbodiimide hydrochloride and 1.9 mg of N-hydroxysuccinimide were precisely weighed into a sterile centrifuge tube at strict molar excess ratios of 10 and 5 times, respectively. These were then added to 100 μL of cold MES buffer and gently pipetted to dissolve. The two were then rapidly dissolved in an appropriate amount of ice-cold buffer and immediately added to the peptide solution. This activation system was subjected to gentle shaking at 4°C for 20 minutes to fully activate the free carboxyl group of the peptide molecule and convert it into a succinimide ester intermediate with extremely high nucleophilic reactivity.

[0079] Accurately weigh 5 mg of chitosan (CS) and dissolve it in a 1% (w / w) aqueous acetic acid solution. Stir magnetically at room temperature until completely dissolved to form a homogeneous and transparent solution. Then add an appropriate amount of 0.1 M MES buffer to the system and carefully titrate with a low concentration of NaOH solution to adjust the pH of the system to 6.0-6.5 for later use.

[0080] The activated GALA peptide solution was added dropwise to the aforementioned pH 6.0 CS solution, controlling the molar ratio of peptide to CS free amino groups to be between 1:5 and 10. The reaction system was continuously stirred at room temperature in the dark for 24 h. After the reaction was completed, 50 mM β-mercaptoethanol was added to the system to quench unreacted crosslinking agents. The mixture was dialyzed using a Zeba™ desalting column to remove unreacted peptides, reagents, and byproducts. Finally, the dialysate was pre-frozen at -80°C and then freeze-dried under vacuum to collect the GALA-CS composite lyophilized powder, which was then stored at -20°C.

[0081] 1.6 Fourier Transform Infrared Spectroscopy (FT-IR) Measurement

[0082] The internal structure and surface functional groups of the synthesized material (GALA-CS) were qualitatively analyzed using a Nicolet iS50 Fourier transform infrared spectrometer manufactured by Thermo Fisher Scientific, USA. Before testing, the CS raw material and the dialysis coupling product (GALA-CS) were freeze-dried for 4 hours to remove moisture interference. The potassium bromide (KBr) pelleting method was used for sample preparation, as follows: approximately 1–2 mg of dried sample was accurately weighed and thoroughly ground and mixed with 100–200 mg of dried KBr powder in an agate mortar. The mixture was then pressed into uniform, transparent pellets using a pellet press. The detection range was 4000–400 cm⁻¹. -1 The scanning resolution is 4 cm. -1 A single test was performed with 32 cumulative scans, and the infrared spectrum was recorded using the spectrum of pure KBr pellets as the background baseline.

[0083] The infrared absorption spectrum of GALA-CS is as follows Figure 2 As shown: 3440 cm -1 The broad peaks on the left and right belong to the stretching vibrations of -OH and -NH; 2915 cm⁻¹ -1 The specific absorption peak at 1683 cm⁻¹ is attributed to the CH stretching vibration; -1 The presence of specific strong absorption peaks on the left and right sides is attributed to the C=O stretching vibration characteristic of newly formed α,β-unsaturated esters (or amides); 1559 cm⁻¹ -1 and 1508 cm -1 The specific absorption peaks appearing on the left and right are attributed to the NH bending vibration and CN stretching vibration of the amide II band of the polypeptide amide bond. (1077 cm⁻¹) -1 The specific absorption peaks appearing on the left and right are attributed to the stretching vibration characteristics of the COC skeleton of chitosan pyranose rings.

[0084] 1.7 Proton nuclear magnetic resonance (NMR) spectrum 1 H NMR test

[0085] The chemical structure of the modified material was characterized using a Bruker AVANCE III superconducting nuclear magnetic resonance spectrometer (400 MHz or 600 MHz). The polymer sample (approximately 1–5 mg), purified by dialysis and freeze-dried, was dissolved in 0.5 mL of heavy water (D₂O). The solution was transferred to a standard 5 mm NMR tube and tested at 25 °C. A standard proton NMR pulse sequence was used, with 64 or 128 scans to obtain high signal-to-noise ratio spectral data. The obtained data were imported into MestReNova software for baseline correction, phase adjustment, and peak area integration.

[0086] ChemDraw software simulation predicts the theoretical value of monomers. 1 H NMR image as follows Figure 3 As shown. (Through) 1 ¹H NMR analysis was used to further identify the chemical structure of GALA-CS. For example... Figure 4 As shown, a specific proton peak was observed at δ 6.04 ppm, attributed to the proton signal of the GALA peptide double bond; a specific broad proton peak was observed at δ 3.74 ppm, attributed to the proton signal of the chitosan pyranose ring backbone; and specific broad proton peaks were observed at δ 2.99 ppm and δ 2.01 ppm, attributed to the proton signals of the chitosan amino and N-acetyl protons, respectively. These results indicate that the GALA peptide was successfully covalently grafted onto the CS backbone to form the GALA-CS polymer.

[0087] Example 2

[0088] 2.1 Preparation of nano-sized vaccine particles

[0089] Nanoparticles for vaccine preparation were prepared using the ionogel method. Lyophilized GALA-CS powder was weighed and fully dissolved in a 1% (v / v) aqueous acetic acid solution. The pH of the system was adjusted to 5.5 using a low-concentration sodium hydroxide solution, and the solution was filtered through a 0.22 μm filter to prepare GALA-CS solutions with mass concentrations of 0.2%, 0.1%, and 0.05%, respectively. 5 mL of pre-purified H9N2 inactivated whole virus antigen solution, along with 25 μg of molecular adjuvants CpG-ODNs-1 and CpG ODNs-2 per 200 mL of system, were mixed together and added to the above GALA-CS solutions. The mixture was thoroughly mixed to allow for preliminary electrostatic complexation. TPP powder was weighed and dissolved in ultrapure water to prepare cross-linking agent aqueous solutions with mass concentrations of 0.2%, 0.1%, and 0.05%, respectively. At room temperature, different volumes of TPP solution were slowly added dropwise to a GALA-CS solution containing antigen and adjuvant using a micro-pump at a constant flow rate of 10 mL / h and high-speed magnetic stirring at 1000 rpm. To screen for the optimal preparation conditions for nanoparticles, ionic cross-linking was performed using different GALA-CS concentrations, TPP concentrations, and TPP volumes.

[0090] The size of the samples (G-CP-NPs) was accurately measured using a Zetasizer Nano series nanoparticle size analyzer from Malvern Panalytical, UK. Before testing, the nanoparticles were appropriately diluted with ultrapure water to avoid multiple light scattering interference caused by excessive sample concentration. After dilution, the samples were placed in a dedicated sample cell and tested at a constant temperature of 20°C. Each sample was measured in triplicate to ensure data reproducibility. The test data were automatically recorded and fitted using the instrument's built-in Malvern software, and the final results were presented as volume distribution. Simultaneously, the software output the average hydrodynamic particle size (Z-average size) of the samples to evaluate the size characteristics of the prepared nanovaccines. The results are shown in Table 5.

[0091] Table 5. Effects of different crosslinking formulations on the physicochemical properties of nanoparticles

[0092] 1 0.2 0.2 1 600±18.2 +21.8±0.4 2 0.2 0.1 1 570.9±14.5 +5.08±0.3977 3 0.1 0.1 1 608.7±17.3 +22.9±1.794 4 0.1 0.1 2 786.9±22.8 +11.5±0.904 5 0.05 0.05 2.5 581.3±16.1 +18.2±1.429 6 0.05 0.1 1 532.3±13.7 +25.9±2.03 7 0.05 0.1 2.5 794±25.4 +25.6±2.004 8 0.05 0.1 1.5 499.3±12.6 +25.8±2.022 9 0.05 0.1 2 549.1±15.8 +28.7±2.248

[0093] Note: The nanoparticle size and Zeta potential data in the table are expressed as mean ± standard deviation (Mean ± SD); Groups 1 to 9 in the table are formulation screening data (n=3).

[0094] 2.3 FITC-labeled CS and GALA-CS

[0095] FITC was used to covalently fluorescently label CS and GALA-CS for subsequent cellular uptake studies. The method was as follows: 10 mg of CS or GALA-CS was dissolved in 1 mL of 0.1 M acetic acid solution to prepare a 1% (w / w) solution. Simultaneously, 10 mg of FITC was dissolved in 5 mL of dehydrated methanol. Under light-protected conditions, the FITC methanol solution was slowly added to the polymer solution. The mixture was magnetically stirred at room temperature for 3 h in the dark. After the reaction, the mixture was centrifuged at 4°C and 1000 xg for 10 min to collect the precipitate. A washing solution of methanol and water in a 70:30 ratio was prepared, and the precipitate was repeatedly washed and centrifuged until no fluorescence signal was detected in the supernatant to completely remove free FITC. The washed label was redissolved in 1% acetic acid solution, and the solution was placed in a 3500 Da dialysis bag and dialyzed in 5 L of ultrapure water in the dark for 3 days, with the ultrapure water changed every 6 h. After dialysis, the labeled product was freeze-dried, and the resulting powder was stored at -20°C in the dark for later use.

[0096] 2.4 In vitro uptake and qualitative analysis of nanoparticles by HD11 cells

[0097] To further investigate the effects of different formulations on the delivery efficiency of nanocarriers, co-localization experiments of GALA-CS with antigens were conducted using laser confocal microscopy. First, 1×10 6 Chicken macrophage cell line HD11 cells were evenly seeded in 35 mm laser confocal microplates and cultured in a 37°C, 5% CO2 incubator. When the cell density reached about 70%, the original culture medium was aspirated and the cells were washed with sterile phosphate buffer.

[0098] To evaluate the effects of different formulations on antigen loading and delivery, the formulation ratios of groups 2, 3, 5, 6, 8, and 9 in Table 5 were selected. FITC-GALA-CS was used to replace GALA-CS in the 2.1-nanometer vaccine particles, resulting in nano-vaccines with green fluorescence, which were named Group 2, Group 3, Group 5, Group 6, Group 8, and Group 9, respectively. Inactivated avian influenza virus solution was set up as a parallel control group, with 100 μL / plate added to the corresponding small plates and incubated for 1 h.

[0099] After incubation, the supernatant was aspirated, and the cells were washed three times with 500 μL of cold PBS. Then, 4% paraformaldehyde solution was added to the culture dishes for fixation at room temperature for 20 min, followed by three washes with pre-cooled PBS. After washing, 0.1% Triton X-100 solution was added for permeabilization at room temperature for 20 min, the Triton X-100 and permeabilization solution were discarded, and the cells were washed three times again. Subsequently, the cells were blocked with 5% Tween bovine serum albumin solution at room temperature for 1 hour. After washing, 50 μL of mouse influenza virus nucleoprotein primary antibody diluted 1:1000 was added to each culture dish, and the cells were incubated at 4°C in the dark for 12 h. After primary antibody incubation, the cells were washed three times, and 50 μL of rabbit anti-mouse red fluorescent secondary antibody diluted 1:500 was added to each dish, and the cells were incubated at room temperature in the dark for 1 hour. Finally, DAPI staining solution was added to induce blue fluorescence in the cell nuclei for five minutes. After three final washes, 100 μL of PBS was retained in the culture dish to prevent the cells from drying out, and then observed and photographed using a laser confocal microscope.

[0100] The results are as follows Figure 5 As shown, by Figure 5 As shown in Figure 'a', GALA-CS nanoparticles exhibit green fluorescence, AIV antigen displays red fluorescence, and cell nuclei stained with DAPI show blue fluorescence. In the Merge plots of each experimental group, the green and red fluorescence signals show significant overlap, forming a yellow co-localization signal. Fluorescence intensity analysis results show that the two fluorescence signals have a high degree of spatial overlap, while no obvious co-localization signal was observed in the WIV control group. The co-localization efficiency of the nanocomposite within cells is as follows: Figure 5As shown in b, Pearson correlation coefficient (Rr) was used to quantitatively analyze each group. Statistical analysis showed that the co-localization efficiency of Group 8 was significantly higher than that of Group 2, Group 3, Group 5, and Group 9 (P < 0.0001), while there was no significant difference between Group 6 and Group 8 (P > 0.05). This quantitative co-localization analysis result is highly consistent with the qualitative observation of laser confocal microscopy, further confirming that the formulation using Group 8 can achieve optimal binding between the GALA-CS nanocarrier and the AIV antigen.

[0101] The optimal ratio of physicochemical properties to antigen loading was selected through comprehensive screening (group 8), and the process was optimized. A suspension prepared at a constant flow rate of 10 mL / h under high-speed magnetic stirring at 1000 rpm was centrifuged at 12000 xg for 10 minutes at 4°C to collect the precipitate, which was then resuspended in ultrapure water. To eliminate soft agglomeration of particles during centrifugation and resuspension, the resuspension was briefly subjected to transient ultrasonic dispersion in an ice bath at 1000 W for 30 seconds. The resulting nanovaccine was formally named G-CP-NPs, stored in the dark for later use, and its particle size distribution and surface potential were further characterized.

[0102] The results are shown in Table 6 and Figure 6 As shown in the figure, the surface potential of G-CP-NPs particles is approximately +30 mV, and the repeated measurements are stable, indicating that the particle surface carries a strong positive charge and the system has good dispersion stability. The particle size of G-CP-NPs is mainly concentrated in the range of approximately 200–350 nm, and the distribution is relatively concentrated, which is significantly smaller than that of the untreated group.

[0103] Table 6. Physicochemical properties before and after optimization

[0104] Group 8 0.05 0.1 1.5 499.3±12.6 +25.8±2.022 G-CP-NPs 0.05 0.1 1.5 250.6±12.4 +31.3±1.7

[0105] Note: The nanoparticle size and zeta potential data in the table are expressed as mean ± standard deviation (Mean ± SD). The Untreated group is the formulation of group 8, and G-CP-NPs are obtained by repeated measurements after the preparation of the optimized process based on the formulation of group 8 (n=5).

[0106] To evaluate the role of GALA peptide in promoting delivery, two different nanoparticle suspensions were prepared using FITC-CS and FITC-GALA-CS in the optimal ratio group 8, respectively, and named CP-NPs and G-CP-NPs. 100 μL of each suspension was added to the corresponding culture dish and incubated in an incubator for 1 hour.

[0107] After incubation, the supernatant was aspirated, and the cells were washed three times with 500 μL of cold PBS. Then, 4% paraformaldehyde solution was added to the culture dishes for fixation at room temperature for 20 min, followed by three washes with pre-cooled PBS. After washing, 0.1% Triton X-100 solution was added for permeabilization at room temperature for 20 min, the Triton X-100 and permeabilization solution were discarded, and the cells were washed three times again. Subsequently, the cells were blocked with 5% Tween bovine serum albumin solution at room temperature for 1 hour. After washing, 50 μL of mouse influenza virus nucleoprotein primary antibody diluted 1:1000 was added to each culture dish, and the cells were incubated at 4°C in the dark for 12 h. After primary antibody incubation, the cells were washed three times, and 50 μL of rabbit anti-mouse red fluorescent secondary antibody diluted 1:500 was added to each dish, and the cells were incubated at room temperature in the dark for 1 hour. Finally, DAPI staining solution was added to induce blue fluorescence in the cell nuclei for five minutes. After three final washes, 100 μL of PBS was retained in the culture dish to prevent the cells from drying out, and then observed and photographed using a laser confocal microscope.

[0108] The results are as follows Figure 7 As shown in Figure a, after co-incubation of the particles with HD11 cells, FITC-labeled chitosan nanoparticles (green fluorescence) and AIV NP antigen (red fluorescence) were observed intracellularly in both groups, indicating that both nanocarriers could be effectively taken up by HD11 cells. To quantify the difference in intracellular uptake between the two groups of nanocarriers, statistical analysis was performed on the mean fluorescence intensity (MFI) of intracellular FITC at the single-cell level. The results are shown in Figure a. Figure 7 As shown in b, the mean intracellular FITC fluorescence intensity in the G-CP-NPs group was significantly higher than that in the CP-NPs group (P < 0.05). The quantitative analysis results of the Pearson correlation coefficient (Rr) between intracellular GALA-CS, CS, and AIVNP are shown in Figure 1. Figure 7 As shown in c, the G-CP-NPs group was significantly higher than the CP-NPs group (P < 0.001), indicating that the G-CP-NPs entering the cell can maintain a highly stable co-localization state with the AIV antigen, thus realizing the delivery of the vector and the antigen.

[0109] 2.5 Preparation of chicken embryo fibroblasts

[0110] Specific pathogen-free (SPF) chicken embryos incubated for 9-11 days were used as material for the isolation and culture of primary chicken embryo fibroblasts (CEF). The procedure is as follows:

[0111] (1) Surface disinfection and embryo extraction: Take a well-developed chicken embryo with the air cell end facing upwards, and wipe the surface with iodine and 75% ethanol cotton balls for disinfection and deiodination. In a clean bench, use sterile forceps to gently break the eggshell at the air cell end and peel off the inner shell membrane. Then, use brand new sterile instruments to carefully remove the main body of the chicken embryo, transfer it into a sterile petri dish, and wash it repeatedly with pre-cooled PBS solution until the allantoic fluid, egg white and blood residue attached to the surface are washed away;

[0112] (2) Target tissue removal: Remove the head, limbs, skeletal structure and all internal organs of the chicken embryo in a petri dish, and remove only the pure muscle tissue of the trunk. Transfer the remaining muscle mass to a clean petri dish, add PBS again and rinse several times to thoroughly remove free red blood cells and other residual tissue debris;

[0113] (3) Mechanical pulverization and enzymatic digestion: The muscle tissue was thoroughly minced into a paste using sterile ophthalmic scissors and then collected into a 15 mL sterile centrifuge tube. 5 mL of 0.25% trypsin solution was added to the tube, and it was incubated at 37°C for approximately 10 minutes with intermittent shaking to accelerate tissue dissociation. Once the tissue mass exhibited flocculent turbidity, an equal volume of DMEM complete medium containing 10% FBS was quickly added to neutralize and terminate the digestion. After gently pipetting the suspension, it was filtered through a 70 µm pore size cell sieve at a uniform speed to remove incompletely digested tissue clumps.

[0114] (4) Cell pelleting and culture: Collect the filtered cell suspension, centrifuge at 440 xg for 5 minutes. After the operation, carefully aspirate the supernatant, add an appropriate amount of DMEM medium containing 10% FBS and 1% antibiotics along the tube wall, and gently resuspend the cell pellet. After viable cell counting, seed the cells into T75 cell culture flasks and place them in a constant temperature incubator at 37°C with 5% CO2 for static culture. Usually, after about 24 hours of incubation, the primary CEF cells will adhere and grow into a dense monolayer. After routine digestion and passage, the secondary CEF cells can be harvested for subsequent experiments.

[0115] 2.6 In vitro cytotoxicity evaluation of G-CP-NPs

[0116] To evaluate the biosafety of the optimized nanovaccine at the in vitro level, its toxicity to three avian target cell lines was detected using the CCK-8 assay. First, primary duck embryo fibroblasts (DEF), primary chicken embryo fibroblasts (CEF), and chicken embryo fibroblast line DF-1, all in logarithmic growth phase, were cultured at approximately 5 × 10⁶ cells per well. 5Cells were evenly seeded at a density in 48-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. Once the cell density reached 80%, the culture medium was aspirated from the wells, and the cells were washed three times with PBS. Then, 300 μL of serum-free medium containing different concentrations of G-CP-NPs nanoparticle vaccine was added to each well, resulting in final concentrations of 100 μg / mL, 200 μg / mL, 500 μg / mL, and 1000 μg / mL, respectively. A negative control group was set up with an equal volume of pure serum-free medium containing no nanoparticles. Three replicates were prepared for each concentration gradient and the control group.

[0117] The treated cell culture plates were returned to the incubator for another 24 hours of incubation. After incubation, 30 μL of CCK-8 detection solution was added to each well in the dark and gently shaken to mix. The culture plates were then returned to the incubator for another one to two hours in the dark to observe the color development. Finally, the absorbance of the supernatant in each well was measured at 450 nm using a reading instrument. The relative cell viability under different concentrations was obtained by subtracting the absorbance of the cell-free blank zeroing well from the absorbance of each concentration of nanovaccine experimental well as the absorbance of the negative control well from the absorbance of the same blank zeroing well, dividing the two values, and multiplying by 100%.

[0118] The cytotoxicity of the optimized G-CP-NPs nanovaccine against DF-1, CEF, and DEF was evaluated using the CCK-8 assay. Results are as follows: Figure 8 As shown, after co-incubation with different concentrations of G-CP-NPs for 24 hours, the viability of DF-1, CEF, and DEF cells was not significantly different from that of the control group (P > 0.05), indicating that the nanoparticles did not produce significant cytotoxicity to these cells. G-CP-NPs exhibited high safety at the in vitro cellular level, and its good biocompatibility ensures a safe basis for subsequent in vivo application as a poultry vaccine vector.

[0119] 2.7 Preparation of Chicken Bone Marrow-Derived Dendritic Cells (chBMDC)

[0120] Density gradient centrifugation was used to efficiently separate chBMDCs from SPF chickens using a chicken bone marrow mononuclear cell isolation kit manufactured by Tianjin TED Company. The specific procedures were as follows: Healthy 3-week-old SPF chickens were euthanized by cervical dislocation and then immersed in 75% ethanol for 10 minutes for sterilization. The femurs and tibias were aseptically separated in a laminar flow hood, and attached muscle and connective tissue were removed. The epiphyses at both ends of the bones were cut off, and the bone marrow cavity was repeatedly flushed with a sterile syringe containing pre-chilled PBS until the bone turned white. The bone marrow flushing fluid was collected, and the cell clumps were gently dispersed by pipetting and then filtered through a 200-mesh sterile cell sieve at a uniform speed. The cell filtrate was slowly spread evenly on the surface of an equal volume of chicken lymphocyte separation medium and centrifuged at 2500 rpm for 30 minutes at room temperature. After centrifugation, the white, cloudy mononuclear cell layer at the interface was carefully aspirated and washed twice with pre-chilled PBS at 1500 rpm.

[0121] After washing, viable cell counts were performed using 0.4% trypan blue staining. When cell viability was greater than 95%, the cells were resuspended in RPMI-1640 complete medium containing 10% FBS and 1% penicillin antibiotics, and the concentration was adjusted to 1×10⁻⁶. 6 Cells / mL. The cell suspension was seeded into 6-well culture plates, and chicken recombinant granulocyte-macrophage colony-stimulating factor (rcGM-CSF) and chicken recombinant interleukin-4 (rcIL-4) were immediately added to a final concentration of 20 ng / mL for both. The plates were incubated at 37°C in a 5% CO2 incubator, with medium changes every 3 days. By day 6-7, the cells mostly exhibited semi-adherent colony growth with short pseudopodia on the surface; these were immature chBMDCs.

[0122] 2.8 In vitro induction and identification of chBMDC and the maturation-promoting effect of G-CP-NPs on it

[0123] Following the isolation and culture method described in section 2.7 above, mononuclear cells were isolated from SPF chicken bone marrow and induced to grow in complete RPMI-1640 medium containing GM-CSF (20 ng / mL) and IL-4 (20 ng / mL) at 37°C in a 5% CO2 incubator. On days 3, 5, and 7 of culture, the morphological evolution, colony formation, and dendritic development of the cells were observed and recorded under an inverted phase-contrast microscope.

[0124] Immature chBMDCs induced in vitro to day 6 were collected, and the cell concentration was adjusted to 1×10⁶ cells / day using complete culture medium. 6Cells were seeded at a density of 1 / mL into 6-well or 12-well cell culture plates. Four treatment groups were established, with equal volumes of different stimulants added to each well: PBS group: added an appropriate amount of sterile PBS; LPS group: added LPS to a final concentration of 200 ng / mL; WIV group: added avian influenza inactivated virus solution (final antigen concentration of 10 μg / mL); G-CP-NPs group: added G-CP-NPs nanodelivery solution coated with an equal volume of H9N2 virus antigen. After gentle mixing, the culture plates were returned to a 37°C, 5% CO2 incubator for 24 hours to induce dendritic cell activation and maturation. After incubation, the cells were washed three times with pre-cooled PBS. Cells from each group were collected and transferred to flow cytometry tubes, washed with 1 mL of flow cytometry buffer, centrifuged at 440 xg for 5 min, and the supernatant was discarded. CD11c and MHC II antibodies were diluted with flow cytometry buffer at the recommended concentrations in the dark according to the manufacturer's instructions. Add 100 μL of diluted antibody working solution to each sample tube, gently pipette to resuspend the cells, and incubate at 4°C in the dark for 30 min.

[0125] Add 800 μL of flow cytometry buffer to each tube for washing, centrifuge at 440 xg for 5 min, and discard the supernatant to remove unbound free antibodies. Finally, resuspend the cell pellet in 100 μL of flow cytometry buffer. Data are acquired using a flow cytometer, and the results are analyzed using FlowJo software.

[0126] The process of in vitro induction culture of BMDCs, such as Figure 9 As shown in a, the cell morphology gradually changes on Day 3, Day 5, and Day 7, with the cell volume gradually increasing and the number of dendritic protrusions gradually increasing. When it reaches the mature stage (Mature BMDC), it presents a typical dendritic cell morphology. Under high magnification (400×), the surface of mature BMDC can be seen to have obvious dendritic protrusions, which is consistent with the morphological characteristics of DC.

[0127] To evaluate the ability of G-CP-NPs to induce BMDC maturation and activation in vitro, flow cytometry was used to identify BMDCs treated with different groups. Gating strategies were employed, such as... Figure 9 As shown in b in the figure. CD11c of different processing BMDC + and MHC II + The cell proportions are as follows: Figure 9 As shown in c and d, the results show that CD11c in the LPS group, G-CP-NPs group, and WIV group... + and MHC II + The proportion of cells in the LPS-positive control group was significantly higher than that in the PBS control group. + and MHC II +The most significant upregulation was observed in the cell ratio. Compared to the WIV (fully inactivated virus) group, the CD11c level in the G-CP-NPs group was significantly higher. + and MHC II + The proportion of cells was significantly upregulated (CD11c). + P < 0.05, MHC II + (P < 0.01). This indicates that, compared to inactivated viral antigens, G-CP-NPs nanovaccines can more efficiently promote the in vitro maturation of BMDCs and significantly upregulate the expression ratio of antigen-presenting surface molecules, confirming that this delivery strategy has excellent antigen-presenting cell activation capabilities.

[0128] Example 3

[0129] 3.1 A three-group animal experimental design was adopted, with 15 SPF chickens in each group, as follows.

[0130] Group 1 (PBS control group): 14-day-old SPF chickens were injected intramuscularly with 400 μL of PBS, and two weeks later were injected with the same amount of PBS.

[0131] Group 2 (InV control group): 14-day-old SPF chickens were injected intramuscularly with 400 μL of H9N2 inactivated vaccine (ss strain) according to the instructions, and an equal amount of H9N2 inactivated vaccine (ss strain) was injected intramuscularly 14 days later.

[0132] Group 3 (G-CP-NPs group): 14-day-old SPF chickens were injected intramuscularly with 200 μL of G-CP-NPs vaccine and administered 200 μL of G-CP-NPs vaccine via eye drops or nasal drops. A booster immunization was repeated 14 days later.

[0133] 3.2 Detection of HI antibodies in chicken serum

[0134] Non-anticoagulated blood was collected from experimental animals on days 7, 14, 21, 28, and 35 post-vaccination (DPV). After allowing the blood to clot naturally at room temperature, it was centrifuged at 5000 xg for 5 min at 4°C. The supernatant serum was collected for HI antibody level detection, and the remainder was stored at -80°C. In a 96-well V-bottom microplate, 25 µL of the serum to be tested was added to well 1, 25 µL of PBS buffer to wells 1-11, and 50 µL of PBS as a red blood cell control to well 12. After mixing by pipetting from well 1, 25 µL was transferred to well 2, and this serial dilution was repeated up to well 10. Finally, 25 µL was discarded from well 10 to ensure that the reaction volume in each well was 25 µL. Add 25 µL of four units of H9N2AIV (A / Chicken / Hunan / HN / 2015 strain) to wells 1-11 and incubate at room temperature for 30 min. Then, add 25 µL of 1% chicken erythrocytes to all wells and vortex to mix. Incubate at room temperature for 30 min and observe the results. The HI titer of the serum sample is the highest dilution that completely inhibits erythrocyte aggregation. The results are statistically analyzed after log2 transformation.

[0135] To investigate the ability of the G-CP-NPs nanovaccine to induce a specific humoral immune response to H9N2 avian influenza virus in chickens, the titer of HI antibodies in chicken serum at different time points after immunization was measured. Results Figure 10 As shown, the HI antibody titers in the G-CP-NPs immunization group and the InV fully inactivated vaccine immunization group were significantly higher than those in the PBS negative control group. At 7 DPV, the G-CP-NPs group induced a highly significant upregulation of HI antibody titers compared to the InV group (P < 0.0001). However, at 14 DPV and 21 DPV, there was no significant difference in HI antibody titers between the G-CP-NPs group and the InV group (P < 0.05). At 28 DPV and 35 DPV, the HI antibody titers in the InV group were significantly higher than those in the G-CP-NPs group (P < 0.05).

[0136] 3.3 Detection of chicken serum-specific neutralizing antibodies

[0137] The titers of specific neutralizing antibodies in the serum of chickens in the DPV groups at 7, 14, and 21 days post-immunization were detected using a micro-neutralization test.

[0138] (1) Cell and virus preparation: MDCK cells were used for the neutralization test. The cells were cultured in DMEM medium containing 10% FBS and 1% double antibiotics and placed in a 37℃, 5% CO2 incubator until a dense monolayer was formed. The virus used in the experiment was the H9N2 subtype avian influenza virus (A / Chicken / Hunan / HN / 2015 strain) harvested from chicken embryos. Using MDCK cells cultured into a monolayer, the Reed-Muench method was used to determine the half-maximal tissue culture infectious dose (TCID) of the virus. 50 ), and adjusted the concentration of the virus working solution to 100 TCID. 50 / 50 µL;

[0139] (2) Serum serial dilution and virus neutralization: In a 96-well cell culture plate, add 50 µL of inactivated serum to well 1, and 50 µL of PBS buffer to wells 2-11 for serial dilution. Discard the 50 µL solution after well 10. Add 50 µL of 100 TCID to each dilution well. 50 The virus working solution was prepared, and virus control (virus and PBS only), cell control (cells and PBS only), and serum control (serum and PBS only) were also prepared. After thorough mixing, the mixture was incubated in a 37°C, 5% CO2 incubator for 1 h.

[0140] (3) Cell seeding and culture: Add 100 µL of the solution at a density of 1×10⁻⁶ cells to each well. 5 The MDCK cell suspension at 1000 cells / mL was incubated at 37°C in a 5% CO2 incubator for 48 h.

[0141] (4) Indirect immunofluorescence (IFA) identification and result determination: The procedures for IFA fixation, permeabilization, blocking, incubation with primary and secondary antibodies, and nuclear staining are as described in section 2.4 of the indirect immunofluorescence staining procedure. The highest serum dilution with the lowest fluorescence signal intensity or no specific red fluorescence signal is used as the neutralizing antibody titer of the sample.

[0142] Chicken serum neutralizing antibody titer detection Figure 11As shown in the figure, at 7 DPV, almost no neutralizing antibodies were detected in the PBS and InV groups, while the G-CP-NPs group rapidly reached its peak, significantly higher than the PBS group (P < 0.001) and the InV group (P < 0.01). At 14 DPV, the titer in the InV group increased significantly, while the titer in the G-CP-NPs group decreased slightly, with no significant difference between the two groups (P > 0.05). At 21 DPV, the neutralizing titers in the InV and G-CP-NPs groups remained stable, with no statistically significant difference between the two groups (P > 0.05), but significantly higher than the PBS group (P < 0.0001). This demonstrates that while traditional oil-emulsion inactivated vaccines can induce high levels of circulating antibodies, they exhibit significant immune lag, typically requiring two weeks after immunization to achieve effective antibody seroconversion, thus failing to provide early immune protection for chicks. In contrast, the G-CP-NPs nanovaccine of this invention enables early and rapid initiation of humoral immunity, inducing significantly higher levels of HI and neutralizing antibodies at 7 DPV compared to the traditional inactivated vaccine (InV) group, with serum IgM, IgY, and IgA levels remaining at high levels throughout the entire immunization cycle.

[0143] 3.4 Detection of IgG, IgM, and IgA antibody levels in chicken serum

[0144] The levels of IgG, IgM, and IgA antibodies in the chicken serum collected in section 3.2 were detected according to the ELISA kit instructions.

[0145] The results are as follows Figure 12 As shown, the levels of IgM, IgY, and IgA in the G-CP-NPs group were significantly higher than those in the PBS control group throughout the entire testing cycle. Further comparisons revealed that at 7 DPV, the IgM and IgY levels in the G-CP-NPs group were significantly upregulated compared to the InV group (P < 0.0001 and P < 0.05), and this significant advantage in IgY persisted until 14 DPV (P < 0.0001). The IgA level in the G-CP-NPs group was significantly higher than that in the InV group throughout the entire testing cycle (P < 0.05 at 7 DPV, and P < 0.0001 from 14 to 28 DPV). There was no significant difference in IgM levels between the G-CP-NPs and InV groups after 14 DPV (P > 0.05), while the IgY level stimulated by the InV group surpassed that of the G-CP-NPs group at 28 DPV (P < 0.05).

[0146] 3.5 Detection of sIgA antibody content in chicken lung and tracheal mucosa

[0147] On day 14 after booster immunization, three experimental chickens were randomly selected from each experimental group. Lung tissue and tracheal mucosa samples were aseptically collected. A mixture of protease inhibitor and PBS was added to a 2 mL centrifuge tube. The samples were weighed, chopped, and added to the centrifuge tube. The mixture was then ground for 5 min, and the tissue homogenate was centrifuged at 2000 × g for 5 min at 4°C. The supernatant was collected. The sIgA content in the tissue supernatant was strictly controlled according to the instructions of the commercial chicken sIgA ELISA kit.

[0148] The results are as follows Figure 13 As shown, the sIgA levels in the G-CP-NPs immunization group in the bronchoalveolar lavage fluid and tracheal mucosa were significantly higher than those in the PBS control group and the traditional InV immunization group (P < 0.0001); while there was no significant difference in sIgA content between the InV immunization group and the PBS group (P > 0.05).

[0149] 3.6 Detection of T lymphocyte subtypes and B lymphocytes in chicken peripheral blood

[0150] Every 7 days after the initial immunization, 2 mL of anticoagulated blood was collected. Peripheral blood lymphocytes were isolated according to the instructions of the chicken peripheral blood mononuclear cell isolation kit. The blood in the anticoagulated tube was mixed with the sample diluent at a 1:1 volume ratio and then pipetted to mix. The suspension was slowly added dropwise along the tube wall to a centrifuge containing an equal volume of separation solution. The mixture was centrifuged at 400 ×g for 15 min, and the white membrane layer was aspirated into a centrifuge tube containing washing solution. After centrifugation for 5 min, the cell supernatant was discarded. If red blood cells were present in the precipitate, an appropriate amount of red blood cell lysis buffer was added, and the mixture was lysed at room temperature. PBS was added to stop the lysis, and the mixture was centrifuged for 5 min before discarding the solution. Finally, the precipitated cells were resuspended in 1640 complete culture medium for cell counting.

[0151] Add the required number of cells to a flow cytometry tube, wash with 1 mL of flow cytometry buffer, centrifuge at 440 × g for 5 min, and discard the liquid. Dilute CD3, CD4, and CD8α antibodies to the recommended concentrations using flow cytometry buffer in the dark, adding 100 μL of antibody to each sample. Resuspend the cells and incubate at 4°C in the dark for 30 min. Then wash with 800 μL of flow cytometry buffer, centrifuge at 440 × g for 5 min, and discard the liquid. Finally, resuspend the pellet with 100 μL of flow cytometry buffer and collect data using a flow cytometer. Similarly, take the required number of cells, wash with flow cytometry buffer, centrifuge, dilute Bu-1-FITC antibody with flow cytometry buffer in the dark, stain, and then analyze the results using FlowJo software.

[0152] The proportion of B lymphocytes in PBMCs was measured at days 7, 14, 21, and 28 post-immunization, using a gating strategy as follows: Figure 14 As shown in 'a' in the diagram. The flow cytometry results are as follows: Figure 14Figure b shows that, compared with the PBS control group, the G-CP-NPs group showed a significant increase from 7 DPV (P < 0.01) and maintained a significant difference at subsequent time points (P < 0.0001 at 14, 21, and 28 DPV); while the InV group was significantly higher than the PBS group only at 14 DPV (P < 0.001) and 28 DPV (P < 0.01). The G-CP-NPs group was significantly better than the InV group at 7 DPV (P < 0.01), and this advantage continued at 14 and 28 DPV (P < 0.01 at 14 DPV; P < 0.001 at 28 DPV), but no statistically significant difference was observed between the two groups at 21 DPV (P > 0.05).

[0153] To assess the ability of G-CP-NPs to induce cellular immune responses in vivo, the dynamic changes in the proportion of T lymphocyte subsets in chicken peripheral blood mononuclear cells were examined at different time points after immunization. The gating strategy for T cell flow cytometry staining was as follows: Figure 15 As shown in a, CD4 + CD8 + and CD4 + CD8 + The T cell proportion results are as follows: Figure 15 b~e are displayed in CD8. + Regarding the proportion of T cells, the G-CP-NPs group was significantly higher than the PBS group throughout the entire testing period (P < 0.001 at 7 DPV, P < 0.05 at all other time points), and significantly higher than the traditional InV group at all time points (P < 0.001 at 7 DPV, P < 0.01 at 21 DPV, P < 0.05 at 14 and 28 DPV); CD4 + CD8 + The T cell G-CP-NPs group maintained a significant upregulation advantage throughout the entire cell cycle, with its proportion consistently higher than that of the InV group (P < 0.01 at 7 DPV, P < 0.05 for all other DPVs); in CD4 + Regarding the proportion of T cells, the activation rate in the G-CP-NPs group was significantly lower than that in the InV group at 7 and 14 DPV (P < 0.001 and P < 0.01, respectively). However, at 21 and 28 DPV, the activation level in the G-CP-NPs group steadily increased, converging with that of the InV group (P > 0.05). This indicates that G-CP-NPs successfully broke the traditional inactivated vaccine's early unidirectional bias towards CD4 activation. + The inherent limitations of T cells significantly compensate for the shortcomings of CD8 cells. + The core weakness lies in cellular immunity.

[0154] 3.7 Detection of Immune-Related Gene Expression in PBMCs

[0155] Using the Novizan RNA extraction kit, PBMCs with 28 DPVs were isolated from 3.6. Total RNA was extracted and reverse transcribed into cDNA. The primer sequences for qRT-PCR were referenced in the following references (Dai M, Li S, Keyi Shi, et al. Comparative analysis of key immune protection factors in H9N2 avian influenza viruses infected and immunized specific pathogen–free chicken[J]. Poultry Science, 2021, 100(1):39-46; Liu Q, Yang J, Huang X, et al. Global gene expression analysis data of chicken dendritic cells infected with H9N2 avian influenza virus[J]. Data in Brief, 2020, 30:105430.). The GAPDH gene was used as an internal control, and the experimental results were quantitatively analyzed using the ΔΔCt method.

[0156] CTL-related gene analysis, such as Figure 16 As shown in a, the expression levels of TNF-α, IL-2, and IFN-γ induced by the G-CP-NPs group were significantly higher than those in the PBS group (P < 0.05 or higher), with TNF-α and IL-2 being significantly higher than those in the InV group (P < 0.01). However, there was no significant difference in IFN-γ expression between the two immunization groups (P > 0.05).

[0157] Analysis of inflammation and chemokine-related genes, such as Figure 16 As shown in b, the expression levels of CXCLi1 and CXCLi2 in both the G-CP-NPs and InV groups were significantly higher than those in the PBS group (P < 0.05 or higher), but there was no significant difference between the two immunization groups. Notably, the G-CP-NPs group showed a particularly strong induction of IL-6, with its expression level being significantly higher than both the PBS group (P < 0.01) and the InV group (P < 0.01). Furthermore, the expression level of IL-1β did not change significantly among the groups.

[0158] Th2-related gene analysis, such as Figure 16As shown in Figure c, the expression levels of IL-10 and MHC II in the G-CP-NPs group were significantly higher than those in the PBS group (P < 0.001) and also significantly higher than those in the InV group (P < 0.01 and above). In stark contrast, the InV group elicited extremely high levels of the typical Th2 cytokine IL-4 (P < 0.001), while the G-CP-NPs group did not induce a significant upregulation of IL-4, and its expression level was significantly lower than that in the InV group (P < 0.001).

[0159] 3.8 Detection of T lymphocyte response in the spleen of chickens after H9N2 stimulation immunization

[0160] On day 14 after booster immunization, three chickens were randomly selected from each experimental group. Spleens were aseptically collected, and splenic lymphocytes were isolated according to the instructions for an ELI-Spot (Enzyme Linked Immunospot Assay, ELISpot) experiment to detect the secretion of IFN-γ by splenic T cells stimulated by H9N2 immunization. The steps are as follows:

[0161] (1) Pretreatment of PVDF membrane plate: Take a PVDF 96-well plate, add 15 μL of 35% ethanol solution to each well for membrane activation, and strictly control the standing time to within 1 minute. Then quickly aspirate the liquid in the well and wash with sterile deionized water to remove residual ethanol;

[0162] (2) Coating antibody: The working concentration of anti-chicken IFN-γ antibody was adjusted to 15 μg / mL with PBS buffer, and 100 μL of each well was coated into the activation plate. After sealing with sealing film, the plate was incubated at 4°C overnight.

[0163] (3) Blocking: Discard the coating solution, wash repeatedly with PBS 5 times, add 200 μL of RPMI 1640 complete medium containing 10% FBS to each reaction well, and block at room temperature for 1 hour.

[0164] (4) H9N2 AIV infection of APC: Take 6×10⁻⁶ samples per well. 5 One splenic lymphocyte was placed as an APC in a biochemical reaction tube and infected with H9N2 AIV at an MOI of 2. The virus solution and cell suspension were mixed 1:1 and incubated at 39°C for 1 h. Then, RP-10 medium with 0.25 μg / mL TPCK-trypsin was added, and the cells were incubated again for 4-6 h. After APC incubation, the cells were centrifuged at 440 ×g for 5 min and resuspended in 100 μL of medium per well.

[0165] (5) APC-stimulated effector T lymphocytes: Discard the blocking solution from the PVDF 96-well plate. Add the prepared chicken spleen lymphocyte suspension at a ratio of 3 × 10⁶ cells per well. 6 APC cells were seeded at a density of 100 cells / well in each well to serve as effector T lymphocytes. APC cells were added at a ratio of 5:1. The PVDF 96-well plate was then incubated at 37°C in a 5% CO2 cell culture incubator for 24-48 hours.

[0166] (6) Biotinylation detection antibody incubation: Discard the cell suspension in the wells and wash the plate five times with PBS containing 0.5% FBS. Then, add 100 μL of biotin-conjugated secondary antibody diluted to 1 μg / mL with the same washing buffer to each well and incubate at room temperature in the dark for 2 hours;

[0167] (7) Enzyme-linked complex binding: Discard the secondary antibody incubation solution and wash five times. Add streptavidin-labeled horseradish peroxidase (HRP) to the reaction wells and continue the reaction at room temperature for 1 hour;

[0168] (8) Spot development and image analysis: Discard the liquid in the wells and wash five times. Quickly add 100 μL of TMB substrate solution to each well and develop the color at room temperature in the dark for about 15 to 30 minutes. Clear blue-purple spots are visible to the naked eye in the positive control wells. Discard the chromogenic solution and rinse with sterile water to terminate the enzymatic reaction. After the plate is air-dried in the dark, use a fully automated ELI-Spot plate reader to scan and count the specific spots in each well, and perform statistical analysis on the number of spots.

[0169] The results are as follows Figure 17 As shown, the number of specific T cell spots secreting IFN-γ in the G-CP-NPs group was significantly higher than that in the InV and PBS groups (P < 0.01), while there was no significant difference between the InV and PBS groups (P > 0.05). These results indicate that, compared to the InV group, G-CP-NPs can effectively activate chicken spleen T cells and induce a stronger antigen-specific cellular immune response.

[0170] 3.9 Detection of viral shedding in infected chickens

[0171] The H9N2 AIV challenge strain (A / Chicken / Kunming / 23540 / 2023) was diluted to 10 μL with PBS containing 10% penicillin and antibiotic antibodies. 7 EID 50SPF chickens in each group (PBS control group, InV control group, and G-CP-NPs group) were infected via nasal or ocular drops at a dose of 200 μL / bird. Throat and cloacal swabs were collected from each group of chickens at 3, 5, and 7 DPIs post-infection and stored in PBS containing 10% penicillin-drug antibodies and 20% glycerol at -80°C for later use.

[0172] Nine chickens were tested in each group at each time point. A positive result for viral shedding was determined by a viral load greater than the detection limit, and the positive rate for viral shedding was calculated.

[0173] Throat detoxification results ( Figure 18 a) shows that at 3 DPI, the viral load in the throat of both the InV group and the G-CP-NPs group was significantly lower than that of the unimmunized PBS group (P < 0.001 and P < 0.0001, respectively). The positive rate of viral shedding in the G-CP-NPs group was 88.89% (8 / 9), while the positive rates in the InV and PBS groups remained at 100% (9 / 9). At 5 DPI, the positive rate of viral shedding in the G-CP-NPs group (55.56%, 5 / 9) was lower than that in the InV group (77.78%, 7 / 9) and the PBS group (88.89%, 8 / 9). The viral load in the G-CP-NPs group and the InV group was significantly lower (P < 0.001) and significantly lower (P < 0.05) than that in the PBS group, respectively. At 7 DPI... At DPI, throat swabs from the G-CP-NPs and InV groups showed no detectable viral shedding, while 4 chickens in the PBS group continued to shed the virus (44.44%, 4 / 9).

[0174] Cloacal detoxification results ( Figure 18 b) shows that at 3 DPI, the cloacal viral shedding positivity rates of the G-CP-NPs group (66.67%, 6 / 9) and the InV group (77.78%, 7 / 9) were lower than those of the PBS group (100%, 9 / 9), with the G-CP-NPs group having the lowest viral shedding positivity rate. The viral load of the G-CP-NPs group and the InV group was significantly lower than that of the PBS group (P < 0.001 and P < 0.05, respectively). At 5 DPI, the viral shedding rates of all groups decreased over time, but the viral shedding positivity rate of the G-CP-NPs group (44.44%, 4 / 9) remained lower than that of the InV group (55.56%, 5 / 9) and the PBS group (66.67%, 6 / 9). By 7 DPI, the cloaca of the G-CP-NPs group and the InV group had completely stopped shedding (0%, 0 / 9), while the PBS group still continued to shed the virus (22.22%, 2 / 9).

[0175] 3.10 Detection of viral replication capacity in chicken tissues and organs

[0176] At 3 DPI, three chickens were randomly euthanized from each immunized group and the negative control group, and tissue samples from the trachea, lungs, ileum, liver, and spleen were collected. Each tissue sample was accurately weighed and placed in a sterile homogenization tube. Pre-cooled sterile PBS buffer was added at a 1:9 (mass-to-volume ratio), and the samples were thoroughly homogenized under ice bath conditions to prepare a tissue homogenate. The homogenate was then centrifuged at 3000 ×g for 5 min at 4°C, and the clear supernatant was carefully aspirated, aliquoted, and stored at -80°C. The EID was calculated. 50 Detect the viral load in the tissues and organs of SPF chickens infected with H9N2 AIV.

[0177] The results are as follows Figure 19 As shown, at 3 DPI, only in the trachea and lung tissues, the viral load in the InV group (P < 0.01 and P < 0.001, respectively) and the G-CP-NPs group (both P < 0.0001) was significantly lower than that in the PBS group; in the trachea tissue, the viral load in the G-CP-NPs group was significantly lower than that in the InV group (P < 0.01); while in the ileum, liver, and spleen, there was no significant difference in viral load among the groups (P > 0.05).

[0178] 3.11 HE staining

[0179] On day 14 after booster immunization, three experimental chickens were randomly selected from each group. Lung, liver, kidney, heart, and skeletal muscle tissue were collected, trimmed, and immediately fixed in 4% paraformaldehyde for 24–48 hours. After fixation, the samples were sent to Wuhan Sewell Biotechnology Co., Ltd. for paraffin section preparation and HE staining. The stained sections were observed under an optical microscope to examine the pathological changes in each tissue, focusing on assessing tissue integrity, inflammatory cell infiltration, and pathological damage, and images were acquired.

[0180] The tissue sections of chickens in each of the 3 DPI groups after viral challenge were observed by HE staining. Figure 20 The study evaluated the protective efficacy of different vaccine strategies and the biosafety of delivery systems.

[0181] Evaluation of lung protective efficacy: The PBS group showed obvious characteristics of viral pneumonia, with multifocal capillary congestion and interstitial lymphocyte infiltration. Although the lungs of the InV group were relieved, a small amount of perivascular lymphocyte infiltration was still visible. In contrast, the lung tissue structure of the G-CP-NPs group was basically intact, with only occasional weak hemorrhage and a small amount of respiratory capillary dilation, and no obvious inflammatory cell infiltration. Its pathological damage was significantly less than that of the PBS group and the InV group.

[0182] Local muscle safety evaluation: In muscle sections at the injection site, the InV group showed a small amount of muscle fiber necrosis and cytoplasmic disintegration, with multifocal fibrous connective tissue proliferation in the interstitium, accompanied by focal infiltration of numerous lymphocytes and granulocytes. In contrast, the G-CP-NPs group showed neatly arranged muscle fibers and no obvious tissue necrosis or inflammatory reaction, demonstrating that this nanodelivery system has superior local biocompatibility compared to traditional oil emulsion adjuvants.

[0183] Systemic biocompatibility evaluation: No significant organic damage or inflammatory cell infiltration was observed in the liver, kidneys, or heart tissues of any group. In the Blank and G-CP-NPs groups, very minor mild edema of renal tubular epithelial cells was observed, which was not considered severe pathological damage. In conclusion, the G-CP-NPs nanovaccine not only effectively alleviates lung pathological damage caused by H9N2 AIV, but also demonstrates good biocompatibility at the injection site and in all solid organs.

[0184] In summary:

[0185] (1) This invention successfully constructed chitosan nanoparticles modified with GALA peptides (G-CP-NPs), which have stable physicochemical properties and good biocompatibility. GALA peptide modification significantly enhances their cellular uptake and endosome escape capabilities. G-CP-NPs can effectively activate antigen-presenting cells in vitro, significantly promote chBMDC maturation and upregulate CD11c. + and MHC II + Express.

[0186] (2) This invention elucidates the differences in immune response characteristics and protective efficacy between G-CP-NPs and traditional inactivated vaccines. Both can induce humoral immunity, but G-CP-NPs exhibit a faster immune activation capacity; G-CP-NPs can simultaneously and efficiently induce mucosal immunity and cellular immunity, making up for the core defects of traditional inactivated vaccines; in addition, G-CP-NPs can clear the virus more efficiently in heterologous virus challenge experiments, significantly shorten the viral shedding cycle, reduce the viral load in target organs, and alleviate lung pathological damage, providing new strategies and experimental evidence for the development of novel vaccines for H9N2 subtype avian influenza.

[0187] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A nano-vaccine delivery carrier GALA-CS, characterized in that, The nano-vaccine delivery carrier comprises a cell-penetrating peptide and chitosan, wherein the cell-penetrating peptide is a GALA peptide; the molar ratio of the GALA peptide to the free amino group of chitosan is 1:5~10.

2. A method for preparing the GALA-CS nanovaccine delivery carrier as described in claim 1, characterized in that, Includes the following steps: Step 1: Prepare a solution A with GALA peptide in MES buffer at a concentration of 8-12 mg / mL; prepare a solution B with carbodiimide hydrochloride in MES buffer at a concentration of 60-68 mg / mL; prepare a solution C with N-hydroxysuccinimide in MES buffer at a concentration of 15-25 mg / mL. Step 2: After mixing solutions B and C, immediately add the mixture to solution A, and then shake at 2-6℃ for 15-25 minutes. Step 3: Dissolve chitosan completely in 1 wt% glacial acetic acid, and add an appropriate amount of 0.1 M MES buffer to adjust the pH of the chitosan solution to 6.0~6.5; Step 4: Add the solution obtained in Step 2 dropwise to the chitosan solution in Step 3, controlling the molar ratio of GALA peptide to chitosan free amino group to be 1:5~10, and then stir continuously at room temperature in the dark for 24 hours. After the reaction is completed, add 50 mM β-mercaptoethanol to the system, and then dialyze the mixture using a Zeba™ desalting column. After the dialysate is pre-frozen at -80℃, it is then freeze-dried under vacuum to obtain the GALA-CS lyophilized nanovaccine delivery carrier.

3. The application of the nanovaccine delivery carrier GALA-CS as described in claim 1 or the preparation method described in claim 2 in the preparation of nanovaccines.

4. The application according to claim 3, characterized in that, The aforementioned nano-vaccine is a nano-vaccine for the prevention of avian influenza virus.

5. A nano-vaccine for preventing avian influenza virus, characterized in that, The nanovaccine comprises the nanovaccine delivery carrier GALA-CS as described in claim 1, H9N2 inactivated whole virus antigen, and molecular adjuvant CpG-ODN.

6. The nano-vaccine according to claim 5, characterized in that, The molecular adjuvant CpG-ODN includes CpG-ODNs-1 and CpG-ODNs-2.

7. The nanovaccine according to claim 6, characterized in that, The nucleotide sequences of the molecular adjuvants CpG-ODNs-1 and CpG-ODNs-2 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

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