Application of broad-spectrum influenza virus polypeptide vaccine in preparation of medicine for preventing influenza virus infection and controlling influenza virus reinfection

Abstract

The invention discloses application of a broad-spectrum influenza virus polypeptide vaccine in preparation of a medicine for preventing influenza virus infection and controlling influenza virus reinfection, and belongs to the technical field of biological medicine. The vaccine is designed on the basis of artificially synthesized polypeptide, a polypeptide sequence which is highly conservative and has immunocompetence is screened and optimized aiming at various subtype influenza virus proteins, a broad-spectrum polypeptide vaccine P125-H is constructed, and an Ii-Key technology and an MF59 adjuvant are applied to enhance immune response. The vaccine can effectively induce immune protection against H1N1 and H7N9 influenza A viruses, significantly reduce the virus load, reduce pathological injury to the lung and improve the survival rate. The P125-H vaccine is composed of three optimized polypeptides, has a cross protection effect on various influenza virus subtypes, has the potential of being developed into a broad-spectrum influenza vaccine, and provides a new technical scheme for coping with influenza virus variation and improving vaccine efficacy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a broad-spectrum influenza virus polypeptide vaccine in the preparation of drugs for preventing influenza virus infection and controlling influenza virus reinfection. Background Technology

[0002] Influenza viruses are among the most common and public health hazards of respiratory viruses worldwide, exhibiting extremely high transmissibility and mutation rates. According to the World Health Organization (WHO), approximately one billion people globally are infected with influenza viruses each year, with 3-5 million severe cases and about 290,000-650,000 deaths. It poses a serious threat to the elderly, children, and those with chronic diseases. Influenza viruses are mainly classified into four types: Influenza A, Influenza B, Influenza C, and Influenza D. Influenza A virus (IAV), due to its ability to spread across host species and its rapid antigenic mutation, is the primary pathogen causing seasonal epidemics and pandemics.

[0003] Currently, vaccination remains the most effective prevention and control strategy for influenza. However, due to the continuous evolution and antigenic drift of the influenza virus, existing vaccines often suffer from poor or even non-matching with circulating strains due to their screening strategies. This leads to reduced or even no protective efficacy, and existing vaccines lack cross-protective efficacy across different subtypes, making them unreliable as a means of controlling emerging pandemic influenza. Adjusting vaccine design strategies and developing broad-spectrum vaccines that protect against multiple influenza virus subtypes have become urgent problems to be solved in the field of influenza vaccine research and development. Summary of the Invention

[0004] Because multi-epitope peptide vaccines overcome the antigenic variation problem of influenza viruses by screening for highly conserved antigenic epitopes, they provide long-term and stable cross-protection. The primary objective of this invention is to provide a broad-spectrum influenza virus peptide vaccine. Based on the above, multiple computational biology tools are used to systematically screen the hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2) sequences targeting influenza viruses, identifying high-quality, highly conserved candidate peptide fragments with potential for cellular immune activity and strong immunogenicity. Using an Ii-key immune enhancement element combined with antigenicity and immunogenicity assessment, a novel broad-spectrum influenza virus peptide vaccine is prepared. This vaccine overcomes the bottleneck problems of existing influenza vaccines, such as frequent antigenic variation, limited immune protection, and insufficient cross-protection.

[0005] The second objective of this invention is to provide a broad-spectrum influenza virus peptide vaccine for use in the preparation of drugs for the prevention of influenza virus infection. This vaccine significantly improves antigen presentation efficiency and immunogenicity by tandemly integrating antigenic epitope fragments derived from the HA stalk, NA catalytic head, and M2e conserved region of the influenza virus, and introducing an Ii-Key immune-enhancing element at the carboxyl terminus of the peptide.

[0006] The third objective of this invention is to provide a broad-spectrum influenza virus polypeptide vaccine for use in the preparation of drugs to control influenza virus reinfection. This vaccine has shown strong immune response and broad-spectrum cross-protection in animal experiments. It can induce humoral and cellular immune responses to multiple subtypes of influenza virus in both healthy animals and previously infected models, and has good potential for practical application and promotion value.

[0007] This invention is achieved through the following technical solution:

[0008] The application of a broad-spectrum influenza virus polypeptide vaccine in the preparation of drugs for preventing influenza virus infection and controlling influenza virus reinfection, wherein the broad-spectrum influenza virus polypeptide vaccine is composed of three functional antigen peptides mixed in equal proportions.

[0009] The three functional antigenic peptides are as follows:

[0010] P1: M2e+Ii-Key, P2: HA-NA tandem +Ii-Key and P5: another pair of HA-NA tandem +Ii-Key;

[0011] Each of the functional antigen peptide segments is connected in series via a flexible connector to form a complete immune module, and an Ii-Key immune enhancement element is introduced at the carboxyl terminus to form a complete immune module.

[0012] The three functional antigenic peptides are derived from the hemagglutinin (HA) stem region, the conserved neuraminidase (NA) catalytic head region, and the matrix protein M2e of the influenza virus, respectively.

[0013] The amino acid sequences of the three functional antigenic peptides are shown in SEQ ID NO.1-3.

[0014] Preferably, the influenza virus includes multiple influenza virus subtypes, specifically any one of H1N1, H3N2, H5N1 and H7N9.

[0015] Preferably, the broad-spectrum influenza virus peptide vaccine further includes an adjuvant, wherein the adjuvant is MF59.

[0016] Preferably, the drug for preventing influenza virus infection is used to enhance broad-spectrum neutralizing antibody response, enhance cellular immune activation, reduce viral load in the lungs, alleviate pathological damage to lung tissue, alleviate post-infection weight loss, and improve post-infection survival rate.

[0017] Preferably, the enhancement of broad-spectrum neutralizing antibody response includes enhancing the titers of hemagglutination inhibition antibodies and neutralizing antibodies against the subtype of influenza virus;

[0018] The enhancement of cellular immune activation includes inducing mouse spleen lymphocytes to increase the secretion of interferon-γ under peptide stimulation;

[0019] The reduction of viral load in the lungs includes reducing viral titer in the lungs, inhibiting viral replication and spread in the lungs, and reducing viral load in the lungs.

[0020] The relief of lung tissue pathological damage includes reducing inflammatory cell infiltration and alleviating alveolar structural damage.

[0021] Preferably, the drug for controlling influenza virus reinfection is used to increase antibody titers, alleviate weight loss after reinfection, improve survival rate, reduce viral titers in the lungs, enhance T cell-mediated cellular immune responses, inhibit viral replication and spread, reduce the degree of inflammatory cell infiltration, and alleviate lung tissue damage.

[0022] Preferably, the enhancement of antibody titers includes enhancing the titers of hemagglutination-inhibiting antibodies and neutralizing antibodies against multiple influenza subtypes.

[0023] The various subtypes include any one of H1N1, H3N2, H5N1, H7N9, and BV.

[0024] Preferably, the enhancement of T cell-mediated cellular immune response includes promoting T cell differentiation and activating functional memory T cells.

[0025] The application of a broad-spectrum influenza virus peptide vaccine in the preparation of a broad-spectrum cross-protective agent for heterologous subtype influenza viruses, wherein the vaccine can activate the immune memory response and enhance the strength of humoral and cellular immunity in previously infected individuals with only one immunization.

[0026] Compared with the prior art, the present invention has at least the following technical effects:

[0027] (i) Because multi-epitope peptide vaccines can overcome the problem of antigenic variation of influenza virus by screening highly conserved antigenic epitopes, they can provide long-term and stable cross-protection.

[0028] This invention provides a broad-spectrum influenza virus peptide vaccine, P125-H. It utilizes multiple computational biology tools to systematically screen the sequences of hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2) targeting influenza viruses, identifying high-quality, highly conserved candidate peptide fragments with potential for cellular immune activity and strong immunogenicity. Using an Ii-key immune enhancement element combined with antigenicity and immunogenicity assessment, a novel broad-spectrum influenza virus peptide vaccine is prepared. This vaccine overcomes the bottlenecks in influenza prevention and control caused by frequent antigenic mutations, limited immune protection, and insufficient cross-protection in existing influenza vaccines.

[0029] The broad-spectrum influenza virus peptide vaccine P125-H is based on highly conserved antigenic regions of multiple influenza virus subtypes. Through immunoinformatics screening, physicochemical optimization, and peptide tandem design, it possesses excellent humoral and cellular immune activation capabilities. It can induce broad-spectrum neutralizing antibodies and provide cross-protection between different subtypes. It has also shown excellent secondary activation capabilities in previous infection models, providing a feasible strategy and key technical support for the development of novel broad-spectrum influenza vaccines.

[0030] (II) Application of this broad-spectrum influenza virus peptide vaccine in the preparation of drugs for the prevention of influenza virus infection. This vaccine significantly improves antigen presentation efficiency and immunogenicity by tandemly integrating antigenic epitope fragments derived from the HA stalk, NA catalytic head and M2e conserved region of the influenza virus, and introducing an Ii-Key immune enhancement element at the carboxyl terminus of the peptide.

[0031] (III) Application of this broad-spectrum influenza virus polypeptide vaccine in the preparation of drugs to control influenza virus reinfection. The vaccine has shown strong immune response and broad-spectrum cross-protection in animal experiments. It can induce humoral and cellular immune responses to multiple subtypes of influenza virus in both healthy animals and previously infected models, and has good potential for practical application and promotion value. Attached Figure Description

[0032] Figure 1 Flowchart for the design, screening and construction of candidate peptides for P125-H vaccine;

[0033] Figure 2 The cross-reactivity of the five candidate single peptides with different viral subtypes was determined.

[0034] Figure 3 The graph shows the changes in serum levels of anti-autopeptide IgG antibodies in mice after immunization with three combined polypeptides.

[0035] Figure 4 This is a schematic diagram illustrating the detection of hemagglutination inhibition antibody titers against different subtypes of influenza in the serum of immunized mice.

[0036] Figure 5 This is a schematic diagram illustrating the detection of neutralizing antibody titers against the above five subtypes of influenza virus in the serum of immunized mice.

[0037] Figure 6 Schematic diagram of the cellular immune activity of different combinations of polypeptides stimulating splenic lymphocytes in immunized mice;

[0038] Figure 7 The curves of body weight change and survival rate of immunized mice after receiving two doses of P125-H and being infected with H1N1-CA04 and H7N9-MA viruses;

[0039] Figure 8 This is a schematic diagram showing the viral load in the lung tissue of mice infected with the virus after immunization.

[0040] Figure 9 This is a schematic diagram of the lung-to-body weight ratio and pathological changes in lung tissue of mice after infection with P125-H immunization and adjuvant control groups.

[0041] Figure 10 The process of establishing a mouse model previously infected with H1N1;

[0042] Figure 11 This is a schematic diagram of the detection of hemagglutination inhibition antibody titers after a single P125-H immunization in previously infected mice.

[0043] Figure 12 This is a schematic diagram of the detection of neutralizing antibody titers after a single P125-H immunization in previously infected mice.

[0044] Figure 13 This is a schematic diagram comparing the changes in body weight and survival rate of mice in the P125-H immunized group after viral challenge in a previous infection model.

[0045] Figure 14 This is a schematic diagram of viral titer in lung tissue of mice on day 7 after a single P125-H immunization following a prior infection.

[0046] Figure 15 The results of lung-to-body weight ratio changes and H&E staining of lung tissue in mice immunized with P125-H in a previous infection model;

[0047] Figure 16 The distribution of CD4+ and CD8+ T cell subsets in the spleen and lung tissues of mice in the immunized and adjuvanted groups after P125-H immunization.

[0048] Figure 17 This is a flow cytometry analysis of the proportion of specific IFN-γ+ memory T cells in spleen lymphocytes and bronchoalveolar lavage fluid of mice in the immunized group and the adjuvant control group after stimulation with the P125-H polypeptide component. Detailed Implementation

[0049] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0050] One specific embodiment of the present invention is as follows:

[0051] I. Experimental Design and Expected Results:

[0052] 1.1 First, the complete sequences of HA, NA, and M2 proteins of various influenza subtypes were collected from the NCBI database. Multiple sequence alignment was performed using MEGA5 software to identify highly conserved regions. Further immunoinformatics and physicochemical characterization tools such as VaxiJen, Bepipred, NetMHCIIpan, and ProtParam were used. Based on multi-dimensional screening principles including epitope immunogenicity scoring, MHCII affinity, protective antigen potential, linear B-cell epitopes, and hydrophilicity, nine candidate antigenic peptides originating from the HA stem, NA catalytic head, and M2e region were ultimately identified. After initial screening experiments, three peptides with the best immune response were selected, and the final vaccine component was named P125-H. This vaccine, P125-H, is composed of three functional antigenic peptides mixed in equal proportions and linked by a flexible peptide (6-aminocaproic acid) and an immune-enhancing element Ii-Key to form a complete immune module. These are P1 (M2e+Ii-Key), P2 (HA-NA tandem + Ii-Key), and P5 (another pair of HA-NA tandem + Ii-Key), and their specific amino acid sequences are shown in SEQ ID NO.1-SEQ ID NO.3.

[0053] 1.2 The constructed P125-H exhibited good antigenicity and immunogenicity. Peptide ELISA assays showed that it specifically bound to the serum of mice infected with multiple influenza virus subtypes, demonstrating broad-spectrum antigen recognition. In mouse immunization experiments, two intraperitoneal injections of P125-H with MF59 adjuvant induced high titers of autospecific IgG, HI antibodies, and neutralizing antibodies (MN), exhibiting broad-spectrum cross-neutralizing ability against representative influenza subtypes such as H1N1, H3N2, H5N1, H7N9, and B / Victoria. Serum antibody levels plateaued after the second immunization, and no significant gain effect was observed after the third immunization.

[0054] 1.3 Regarding cellular immunity detection, ELISA Pot analysis revealed that P125-H significantly induced interferon-γ (IFN-γ) secretion from mouse splenic lymphocytes upon peptide stimulation, with a significantly stronger effect than other peptide combinations, indicating that this combination possesses excellent cellular immune activation capabilities. Challenge protection experiments showed that the P125-H vaccine significantly delayed weight loss and improved survival rate in mice infected with lethal H1N1-CA04 and H7N9-MA vaccines, and significantly reduced viral load in the lungs and the degree of histopathological damage. Mice in the adjuvant control group all died after infection, while 100% of the mice in the P125-H immunized group survived H1N1 infection and 50% survived H7N9 infection, demonstrating its clear cross-protective efficacy against different subtypes of influenza viruses.

[0055] 1.4 To more realistically simulate the widespread immune memory state in the human population, a mouse model based on a non-lethal dose of H1N1-CA04 primary infection was constructed. In this model, serum HI antibodies peaked at 120 days post-infection and then gradually declined, falling below the protective threshold after 180 days, forming a state of "previous infection but diminished protective antibody levels." At this point, mice were immunized with P125-H. The results showed that this vaccine significantly increased HI and MN antibody titers against multiple influenza subtypes, including H1N1, H3N2, H5N1, H7N9, and B / Victoria, with levels significantly higher than the adjuvant group and the peak levels at primary infection, demonstrating a potent immune memory activation ability.

[0056] 1.5 In the previous infection model, flow cytometry was used to detect T cell response. P125-H can significantly amplify CD4+ and CD8+ central memory T cells (TCM) and effector memory T cells (TEM) subsets, and memory T cell activation characteristics were observed in bronchoalveolar lavage fluid. In hosts with immune memory background, it can further enhance the breadth and depth of cellular immunity and expand the cross-protection spectrum.

[0057] II. Preparation of polypeptide vaccines:

[0058] A broad-spectrum influenza virus combination peptide vaccine was developed. Based on the full-length sequences of HA, NA, and M2 proteins of multiple influenza virus subtypes downloaded from GeneBank and NCBI databases, immunoinformatics tools were used to analyze their conserved regions, identifying nine peptides that possess both conservation and immunogenicity. After further screening, tandem sequencing, and the addition of Ii-Key elements, three combinations (P123, P124, and P125) were constructed. The P125 combination showed the best immunogenicity and was named P125-H.

[0059] The three component polypeptide sequences of P125-H are as follows:

[0060] SEQ ID NO.1: Ac-LRMK-ACS-MSLLTEVETPTRSEWECRCSDSS-NH2

[0061] SEQ ID NO.2: Ac-LRMK-ACS-PFQNVHPITIGKCPKYVKSTKLRLATG-ACS-CYRACFYVELIRGRPKEDKVWWTSNSIVSM-NH2

[0062] SEQ ID NO.3: Ac-LRMK-ACS-LYDKVRLQLKDNAKELGNGCFEFYHKCD-ACS-WYSFGFEIKDKKCDVPCIGIEMVHDGGK-NH2

[0063] III. Vaccine Design, Construction, and Immunogenicity Evaluation:

[0064] 3.1 Antigen Design and Screening:

[0065] This technical solution is based on highly conserved regions in the HA, NA, and M2 protein sequences of different influenza virus subtypes. Immuninformatics methods are used to screen for candidate polypeptide fragments that simultaneously possess CD4+ T cell epitopes, B cell linear epitopes, and high immunogenicity scores.

[0066] Nine candidate peptides were screened from the M2e, HA stem and NA catalytic head, respectively. They were linked together by a flexible linker peptide (6-aminocaproic acid) and fused with an Ii-Key immune enhancement element at the carboxyl terminus to enhance antigen processing and presentation efficiency and immune activation.

[0067] Three combinations were eventually constructed (P123, P124, and P125), among which the P125 combination showed the best immunogenicity and was named P125-H.

[0068] The above design and screening process is as follows: Figure 1 As shown. Figure 1 This is a flowchart illustrating the design, screening, and construction of candidate peptides for the P125-H vaccine. A represents the overall design and evaluation process for P125-H; B shows the composition of the five candidate peptide fragments; C and D respectively represent the immunoinformatics software used for peptide screening and the screening scoring results.

[0069] 3.2 Cross-reactivity of peptides with different viral subtypes:

[0070] The lyophilized peptides were dissolved in PBS buffer and diluted to a working concentration (10 μg / peptide), then thoroughly mixed with an equal volume of MF59 emulsion adjuvant. Each mouse was intraperitoneally injected with 200 μL of the vaccine mixture. Six- to eight-week-old C57BL / 6J mice weighing approximately 20 g were used for the initial immunization, followed by a booster immunization on day 14. A small blood sample was collected before each immunization to separate serum for subsequent testing.

[0071] like Figure 2 As shown, the binding ability of five candidate peptides to IgG antibodies in the serum of convalescent mice infected with different subtypes of influenza virus (H1N1, H5N1, H7N9) was detected by ELISA. Normal mouse serum served as a negative control, demonstrating the cross-reactivity of the peptides to different viral subtypes. AE corresponds to candidate peptides 1-5, respectively.

[0072] The results showed that orbital serum was collected on days 14, 28 and 42, and the level of anti-P125-H IgG antibody was detected by peptide-coated ELISA.

[0073] 3.3 Antibody level detection:

[0074] like Figure 3 The figure shows the changes in serum anti-autopeptide IgG antibody levels on days 14, 28, and 42 after mice were immunized with high-dose (10 μg / peptide, group H) and low-dose (2.5 μg / peptide, group L), respectively. In the figure, A represents the result after one immunization (day 14); B represents the result after two immunizations (day 28); and C represents the result after three immunizations (day 42). The Blank group served as the adjuvant control.

[0075] Figure 3 The results showed that antibodies were detected in the vaccine group mice after the first immunization, and the antibody titer increased significantly with the number of immunizations. It reached a plateau after the second immunization, and the level of the third immunization was similar to that of the second immunization.

[0076] 3.4 Antibody titer detection

[0077] Meanwhile, the serum of immunized mice was tested for HI and MN neutralizing antibodies.

[0078] like Figure 4 The figure shows the hemagglutination inhibition antibody (HI) GMT in the serum of immunized mice against different subtypes of influenza virus (H1N1-CA04, H3N2, H5N1, H7N9, BV). AD represents the HI antibody titers after three immunizations (A, B) or two immunizations (C, D) in different groups, respectively. The performance of P125-H / P123-H / P124-H and the adjuvant group was compared on days 14, 28, and 42, with statistical significance markers attached.

[0079] like Figure 5 The results show the detection results of neutralizing antibody (MN) titers against the above five subtypes of influenza virus in the serum of immunized mice, indicating that P125-H induces a broad-spectrum neutralizing response.

[0080] Results combined Figure 4 and Figure 5 It is known that P125-H immunization can induce the production of high-titer HI and MN antibodies, which show cross-neutralizing activity against multiple influenza virus subtypes such as H1N1, H3N2, H5N1, and H7N9, suggesting that it has broad-spectrum protective potential.

[0081] 3.5 Detection of cellular immune activity:

[0082] On day 42, the spleens of mice were collected, splenic lymphocytes were isolated, and the cells were stimulated in vitro with the three polypeptides that make up P125-H. The IFN-γ secretion level was detected by ELISpot.

[0083] like Figure 6 As shown, different combinations of peptides stimulated splenic lymphocytes of immunized mice, and the ELISpot results of IFN-γ secretion in vitro were compared. The immune level was expressed as the number of spot-forming cells (SFCs). The cellular immune activity of different peptide combinations was compared.

[0084] The results showed that P125-H significantly induced T cells to produce IFN-γ, indicating that it has good cellular immunogenicity.

[0085] 3.6 Weight change curve and survival rate:

[0086] On day 14 post-immunization, mice were anesthetized with isoflurane inhalation at a dose of 1×10 5 TCID 50 H1N1-CA04 or 2×10 2 TCID 50 (10x LD) 50 Mice were challenged with H7N9-MA virus via nasal drops, 25 μL instilled into each nostril, for a total of 50 μL. An adjuvant control group and a PBS blank group were established. Changes in mouse body weight and survival rate were recorded for 14 consecutive days post-challenge.

[0087] like Figure 7Figure A shows the weight change curves and survival rates (Kaplan-Meier survival analysis) of immunized mice after receiving two doses of P125-H and being infected with H1N1-CA04 and H7N9-MA viruses. Figure B shows the weight change curves within 14 days after immunization with H1N1-CA04 virus, Figure C shows the survival curves of mice within 14 days after immunization with H1N1-CA04 virus, and Figure D shows the survival curves of mice within 14 days after immunization with H7N9-MA virus. The control group was the group receiving MF59 adjuvant, n=10.

[0088] The results showed that the P125-H group mice experienced a significant reduction in body weight loss after infection and a significantly improved survival rate.

[0089] 3.7 Lung tissue viruses:

[0090] Lung tissue was collected from mice on day 7 of infection to detect viral load (TCID). 50 (and immunohistochemical staining (IHC) results)

[0091] like Figure 8 The figure shows the total viral load (TCID) in the lung tissue of mice infected with H1N1-CA04(A) or H7N9-MA(B) after immunization. 50 The results of immunohistochemical staining of serum IFN-γ concentration (C after H1N1-CA04 infection, D after H7N9-MA infection) and influenza virus NP protein in lung tissue (E, scale bar: 100 μm) showed that the vaccine reduced viral replication and promoted cellular immunity.

[0092] like Figure 8 Results A and B showed that the viral titer in the lung tissue of group P125-H was significantly reduced;

[0093] Figure 8 The results from the E-test show that the viral NP protein is mainly confined to the epithelial region, and viral replication is restricted.

[0094] at the same time, Figure 8 The elevated IFN-γ levels in serum C and D indicate that the vaccine activated a coordinated humoral and cellular immune response.

[0095] 3.8 Severity of lung injury:

[0096] like Figure 9 As shown in Figure C, this is a comparison of the lung-to-body weight ratio (A after infection with H1N1-CA04; B after infection with H7N9-MA) and pathological changes in lung tissue (H&E staining, scale bar: 100μm) in mice infected with P125-H immunized mice and mice in the adjuvant control group. The pathological images show inflammatory cell infiltration and alveolar damage, and quantitative analysis reflects the degree of lung injury.

[0097] In terms of lung injury assessment, the results were combined Figure 9 It can be seen that the lung-to-body weight ratio of mice in the P125-H group decreased, and H&E staining showed reduced inflammatory cell infiltration and relatively intact alveolar structure, suggesting that the vaccine can effectively reduce lung tissue damage.

[0098] IV. Immunoprotective effect of P125-H in the context of prior infection

[0099] 4.1 Model Construction:

[0100] To simulate the immune status of individuals after natural infection, a prior infection model was constructed. Mice were infected with a non-lethal dose of H1N1-CA04, and serum HI antibody levels were monitored for 7 consecutive months.

[0101] like Figure 10 The diagram shows the process of establishing a mouse model previously infected with H1N1. A represents the dynamic change curve of serum HI antibody titer; B represents the change in body weight within 14 days post-infection, used to verify the effectiveness of the model construction.

[0102] like Figure 10 The results from the Chinese model showed that the antibody titer peaked on day 120 of infection (GMT=243) and dropped below the protective threshold on day 180 (GMT=26), indicating that the model was successfully constructed. Figure 10 The B-mode indicates that weight changes are stable.

[0103] 4.2 Antibody titer:

[0104] like Figure 11 The figure shows the titers of HI antibodies against H1N1-CA04 (A), H3N2 (B), H5N1 (C), H7N9 (D), and BV (E) on day 14 after a single P125-H immunization in mice previously infected with the virus. The differences were compared among the vaccine group, the adjuvant group, and the model group with the highest titer (120d).

[0105] like Figure 12 The image shows the results of MN antibody titers against various influenza viruses in the serum of mice 14 days after a single P125-H immunization (A for H1N1-CA04; B for H3N2; C for H5N1; D for H7N9; E for B / Victoria), indicating that the vaccine can still induce a neutralizing response in mice with a history of infection.

[0106] Based on this, mice were immunized with P125-H once, and serum HI and MN antibodies were detected on day 14. Figure 11 and Figure 12The results showed that P125-H could significantly increase antibody titers against five subtypes: H1N1, H3N2, H5N1, H7N9, and BV, indicating that it still has a broad-spectrum immune-enhancing effect in the memory background.

[0107] 4.3 Weight Changes and Survival Rate:

[0108] The model mice were then challenged again with high-lethal doses of H1N1-CA04 or H7N9-MA virus via nasal drops.

[0109] like Figure 13 The figure shows a comparison of body weight changes and survival rates in mice immunized with P125-H after challenge with H1N1-CA04 or H7N9-MA in previous infection models. Specifically, A represents the body weight change curve within 14 days of H1N1-CA04 infection after immunization; B represents the body weight change curve within 14 days of H7N9-MA infection after immunization; C represents the survival curve of mice within 14 days of H1N1-CA04 infection after immunization; and D represents the survival curve of mice within 14 days of H7N9-MA infection after immunization. This indicates that the vaccine still possesses cross-protective efficacy in the context of immune memory.

[0110] The results showed that the weight loss of mice in the P125-H immunization group was significantly slowed down, and the survival rate reached 100%, which was far better than that of the control group, indicating that it has a complete protective effect.

[0111] 4.4 Viral titer in lung tissue

[0112] The viral load and IHC results in lung tissue were measured on day 7 of the infection.

[0113] like Figure 14 As shown, the viral titer (TCID) in the lung tissue of mice 7 days after a single P125-H immunization in a previously infected background is... 50 Comparison (A: H1N1-CA04; B: H7N9-MA) and influenza NP protein immunohistochemical staining (C; scale bar: 100μm) reflects the vaccine's ability to control viral replication and spread in the lungs.

[0114] like Figure 14 Results A and B showed that the viral titer in the lungs of group P125-H was significantly reduced; Figure 14 The C IHC results showed that the viral distribution in the immune group was more limited and the replication was more controlled.

[0115] 4.5 Pathological analysis of lung tissue

[0116] Lung tissue pathological analysis results as follows Figure 15As shown, the lung-to-body weight ratio changes in mice immunized with P125-H in previous infection models (A is after infection with H1N1-CA04; B is after infection with H7N9-MA) and H&E staining results of lung tissue (C, scale bar: 100 μm) are used to assess the degree of inflammatory response and tissue damage.

[0117] The results showed that the lung-to-body weight ratio of the immunized mice was significantly lower than that of the adjuvant group ( Figure 15 (A, B); H&E staining results showed that the alveolar structure was well preserved and the infiltration of inflammatory cells was significantly reduced. Figure 15 (C) suggests that the vaccine can alleviate lung tissue damage.

[0118] 4.6 Cellular immunity:

[0119] In terms of cellular immunity, cells were collected from the spleen and bronchoalveolar lavage fluid (BALF) for flow cytometry analysis. The proportions of TCM, TEM, and TEMRA subsets in CD4+ and CD8+ T cells were detected.

[0120] like Figure 16 The figures show the distribution of CD4+ and CD8+ T cell subsets (T naive, TCM, TEM, TEMRA) in the spleen and lung tissues of mice in the immunized and adjuvanted groups after P125-H immunization (A: CD4+ T cell subset in spleen; B: CD4+ T cell subset in BALF; C: CD8+ T cell subset in spleen; D: CD8+ T cell subset in BALF). The differentiation status of circulating and tissue-resident immune memory cells was also compared (E: circulating / resident CD4+ T cell subset; F: circulating / resident CD8+ T cell subset).

[0121] The results showed that P125-H significantly promoted the differentiation of CD4+ and CD8+ T cells into TCM and TEM subsets and enhanced immune memory function.

[0122] 4.7 Detection of IFN-γ+ memory T cell activation

[0123] Furthermore, spleen and BALF cells were stimulated in vitro with the three components P1, P2, and P5 of P125-H to detect the activation of IFN-γ+ memory T cells.

[0124] like Figure 17The image shows flow cytometry analysis of the proportions of specific IFN-γ+ memory T cells (TCM, TEM, TEMRA) in splenic lymphocytes and bronchoalveolar lavage fluid (BALF) of mice in the immunized group and adjuvant control group after stimulation by P125-H polypeptide components P1, P2, and P5, respectively. (A, B, and C represent the specific IFN-γ lymphocyte populations of TCM, TEMRA, and TEM in the spleen under polypeptide stimulation, respectively; C, D, and E represent the specific IFN-γ lymphocyte populations of TCM, TEMRA, and TEM in BALF under polypeptide stimulation, respectively). This demonstrates the activation effect of polypeptide stimulation on memory T cell function.

[0125] The results showed that a high proportion of IFN-γ positive cells were observed in all three subsets of the P125-H immunization group (TCM, TEM, and TEMRA), indicating that the vaccine can activate functional memory T cells and has a strong secondary immune response capability.

[0126] In summary, the P125-H peptide vaccine composed of three peptides screened and constructed in this invention has excellent humoral and cellular immunogenicity, and can provide broad-spectrum protection against multiple influenza subtypes in both primary immunization and prior infection backgrounds, showing potential as a candidate for novel influenza vaccines.

[0127] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a broad-spectrum influenza virus polypeptide vaccine in the preparation of drugs for preventing influenza virus infection and controlling influenza virus reinfection, characterized in that, The broad-spectrum influenza virus polypeptide vaccine is composed of three functional antigenic peptides mixed in equal proportions. The three functional antigenic peptides are as follows: P1: M2e+Ii-Key, P2: HA-NA tandem +Ii-Key and P5: another pair of HA-NA tandem +Ii-Key; Each of the functional antigen peptide segments is connected in series via a flexible connector to form a complete immune module, and an Ii-Key immune enhancement element is introduced at the carboxyl terminus to form a complete immune module. The three functional antigenic peptides are derived from the hemagglutinin (HA) stem region, the conserved neuraminidase (NA) catalytic head region, and the matrix protein M2e of the influenza virus, respectively. The amino acid sequences of the three functional antigenic peptides are shown in SEQ ID NO.1-3.

2. An application according to claim 1, characterized in that, The influenza virus includes multiple influenza virus subtypes, specifically any one of H1N1, H3N2, H5N1, and H7N9.

3. An application according to claim 1, characterized in that, The broad-spectrum influenza virus peptide vaccine also includes an adjuvant, wherein the adjuvant is MF59.

4. An application according to claim 1, characterized in that, The application of the drug for preventing influenza virus infection in enhancing broad-spectrum neutralizing antibody response, enhancing cellular immune activation, reducing viral load in the lungs, alleviating pathological damage to lung tissues, alleviating post-infection weight loss, and improving post-infection survival rate.

5. An application according to claim 4, characterized in that, The enhancement of broad-spectrum neutralizing antibody response includes increasing the titers of hemagglutination inhibition antibodies and neutralizing antibodies against subtype influenza viruses; The enhancement of cellular immune activation includes inducing mouse spleen lymphocytes to increase the secretion of interferon-γ under peptide stimulation; The reduction of viral load in the lungs includes reducing viral titer in the lungs, inhibiting viral replication and spread in the lungs, and reducing viral load in the lungs. The relief of lung tissue pathological damage includes reducing inflammatory cell infiltration and alleviating alveolar structural damage.

6. An application according to claim 1, characterized in that, The drug for controlling influenza virus reinfection is used to increase antibody titers, alleviate weight loss after reinfection, improve survival rate, reduce viral titers in the lungs, enhance T cell-mediated cellular immune responses, inhibit viral replication and spread, reduce the degree of inflammatory cell infiltration, and alleviate lung tissue damage.

7. An application according to claim 6, characterized in that, The enhancement of antibody titers includes enhancing the titers of hemagglutination inhibition antibodies and neutralizing antibodies against multiple influenza virus subtypes; The various subtypes include any one of H1N1, H3N2, H5N1, H7N9, and BV.

8. An application according to claim 6, characterized in that, The enhancement of T cell-mediated cellular immune responses includes promoting T cell differentiation and activating functional memory T cells.

9. The application of a broad-spectrum influenza virus polypeptide vaccine in the preparation of a broad-spectrum cross-protective agent for heterologous subtype influenza viruses, characterized in that, The vaccine requires only one immunization in previously infected individuals to activate the immune memory response and enhance the strength of humoral and cellular immunity.

Images