Adjuvanted influenza vaccine and use thereof

Abstract

Provided are an adjuvanted influenza vaccine and use thereof, relating to the technical field of biological vaccines. The adjuvanted influenza vaccine comprises a CpG adjuvant. The CpG adjuvant is selected from at least one of CpG HP007, CpG PV001, CpG 7909, and CpG 1018. The adjuvanted influenza vaccine may further comprise an aluminum adjuvant. Compared with conventional vaccines, the provided adjuvanted influenza vaccine can increase antibody levels by several times, induce stronger humoral immune responses and cellular immune responses, and effectively reduce antigen dosage, significantly improving the immunogenic efficacy of influenza vaccines in healthy populations.

Description

An adjuvanted influenza vaccine and its application

[0001] This application claims priority to the invention application filed on January 24, 2025, with application number "202510118949.X" and patent title "An Adjuvant Influenza Vaccine and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of biological vaccine technology, and more specifically, to an adjuvanted influenza vaccine and its application. Background Technology

[0003] Influenza is an acute respiratory infectious disease caused by the influenza virus, seriously endangering public health. Influenza typically presents with acute onset, fever (some cases may experience high fever, reaching 39-40℃), accompanied by chills, headache, muscle and joint pain, extreme fatigue, loss of appetite, and other systemic symptoms. Sore throat and cough are common, and nasal congestion, runny nose, retrosternal discomfort, facial flushing, and mild conjunctival congestion may also occur. Vomiting and diarrhea may also be present. Mild influenza often resembles the common cold, but its fever and systemic symptoms are more pronounced. Severe cases can lead to viral pneumonia, secondary bacterial pneumonia, acute respiratory distress syndrome, shock, disseminated intravascular coagulation, extrapulmonary manifestations such as cardiovascular and neurological issues, and various complications.

[0004] Influenza viruses are antigenically variable and spread rapidly, causing seasonal epidemics annually. Outbreaks can occur in crowded places such as schools, childcare facilities, and nursing homes. Globally, seasonal influenza epidemics result in 3-5 million severe cases and 290,000-650,000 respiratory disease-related deaths each year. High-risk groups, such as pregnant women, infants, the elderly, and those with chronic underlying diseases, have a higher risk of severe illness and death after contracting influenza. Furthermore, influenza not only imposes a heavy economic burden but also severely impacts the health-related quality of life for individuals with underlying conditions. Influenza infection can also significantly affect patients' quality of life; over 60% of outpatient and inpatient cases of influenza report pain, malaise, anxiety, and depression. Influenza also leads to decreased productivity, such as absenteeism.

[0005] Annual influenza vaccination is the most effective way to prevent influenza, significantly reducing the risk of contracting the flu and developing serious complications. Neuraminidase inhibitors such as oseltamivir, zanamivir, and peramivir are effective treatments for influenza A and B. Early use of antiviral drugs, especially within 48 hours of symptom onset, can significantly reduce the incidence of severe influenza and death. Antiviral drugs should be used under the guidance of a doctor. Drug prophylaxis cannot replace vaccination and should only be used as an emergency temporary preventative measure for high-risk groups of severe influenza who have not been vaccinated or have not yet developed immunity after vaccination. Oseltamivir, zanamivir, etc., can be used in these cases.

[0006] In July 2019, the Healthy China Action Promotion Committee formulated and issued the "Healthy China Action (2019-2030)," which clearly stated that children, the elderly, and patients with chronic diseases have low immunity and weak resistance, making them high-risk groups for influenza. It recommended that they get vaccinated against influenza under the guidance of a doctor before each influenza season, and encouraged areas with the necessary conditions to provide free influenza vaccinations to people aged 60 and above, children in childcare institutions, primary and secondary school students, and students in secondary vocational schools. At the same time, it required that the supply of influenza vaccines be guaranteed.

[0007] Getting a flu vaccine is the most effective way to prevent influenza, reducing the harm caused by flu-related diseases and the burden on medical resources.

[0008] Given the widespread susceptibility to influenza in the general population and the need for annual influenza vaccination, the demand for influenza vaccination is extensive. Furthermore, in seasonal influenza environments, there is a need to further enhance the immunogenicity of influenza vaccines in specific populations (such as the elderly). Therefore, there remains a need to develop novel adjuvant vaccines with high immunogenicity to meet this demand.

[0009] Application content

[0010] In view of this, the main objective of the present invention is to provide a novel adjuvanted influenza vaccine. The adjuvanted influenza vaccine provided by the present invention can significantly enhance humoral and cellular immune responses, while effectively reducing the amount of antigen used and improving the immune efficacy of the influenza vaccine in healthy individuals.

[0011] To achieve the above objectives, the technical solution of the present invention is as follows:

[0012] An adjuvanted influenza vaccine comprising a CpG adjuvant selected from at least one of CpG HP007, CpG PV001, CpG 7909, and CpG 1018. CpG HP007 has the nucleotide sequence shown in SEQ ID NO: 1; CpG PV001 has the sequence shown in SEQ ID NO: 2; CpG 7909 has the sequence shown in SEQ ID NO: 3; and CpG 1018 has the sequence shown in SEQ ID NO: 4.

[0013] In this invention, different types of CpG adjuvants can significantly increase the level of influenza antibody production in the body, including H1N1 antibodies, H3N2 antibodies, and BV antibodies, and can also stimulate the production of higher levels of cytokines, including IFN-γ and IL-2. In other words, CpG adjuvants can significantly enhance the immunogenicity of adjuvanted influenza vaccines.

[0014] Furthermore, the vaccine is a single-adjuvant vaccine, and the content of the CpG adjuvant is 200-3000 μg / ml, preferably 200-1000 μg / ml, and more preferably 300-400 μg / ml.

[0015] Furthermore, the vaccine is a dual-adjuvant vaccine containing both CpG adjuvant and aluminum adjuvant, wherein the content of CpG adjuvant is 20-1000 μg / ml, preferably 20-200 μg / ml, and more preferably 160 μg / ml.

[0016] An appropriate amount of CpG adjuvant can significantly enhance the immunogenicity of influenza vaccines, while an excessive amount of CpG adjuvant may overactivate the immune system, leading to overactivation and depletion of immune cells, thus reducing immunogenicity. Conversely, an insufficient amount of CpG adjuvant may fail to effectively activate the immune system, resulting in an inadequate immune response and reduced vaccine immunogenicity. In this invention, in the case of a single adjuvant, when the CpG adjuvant content in the adjuvanted influenza vaccine is 300-400 μg / ml, more preferably 320 μg / ml (i.e., 160 μg per dose), it can stimulate the strongest cellular and humoral immune responses.

[0017] Because the synthesis process of CpG adjuvant is complex, requires high-purity raw materials and strict quality control, and is relatively expensive, adding aluminum adjuvant to adjuvanted influenza vaccines can appropriately reduce the amount of CpG adjuvant used, thereby reducing the production cost of the vaccine and further improving the immunogenicity of the vaccine.

[0018] Furthermore, the aluminum adjuvant is selected from one or more of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, and amorphous aluminum hydroxyphosphate adjuvant.

[0019] Furthermore, the content of the aluminum adjuvant is 0.6-1.0 mg / ml, preferably 0.8 mg / ml.

[0020] Further, the mass ratio of the CpG adjuvant to the aluminum adjuvant is (10-500):(300-500).

[0021] Furthermore, the influenza vaccine includes an immunogenic substance, wherein the immunogenic substance is an immunogenic substance of influenza A virus and / or influenza B virus.

[0022] Furthermore, the immunogenic substances of the influenza A virus and / or influenza B virus include influenza virus split vaccine stock solution, including at least one of a monovalent stock solution containing influenza A virus hemagglutinin and a monovalent stock solution containing influenza B virus hemagglutinin; in particular, the monovalent stock solution containing influenza A virus hemagglutinin includes at least one of a monovalent stock solution containing H1N1 influenza A virus hemagglutinin and a monovalent stock solution containing H3N2 influenza A virus hemagglutinin; the monovalent stock solution containing influenza B virus hemagglutinin includes at least one of a monovalent stock solution containing BV influenza B virus hemagglutinin and a monovalent stock solution containing BY influenza B virus hemagglutinin.

[0023] Optionally, the immunogenic substance can also be a subunit antigen, a whole virus vaccine, an attenuated antigen, etc. In this invention, influenza virus split vaccine stock solution is used as the immunogenic substance, which retains key antigenic components such as hemagglutinin (HA) and neuraminidase (NA) of the influenza virus, resulting in strong immunogenicity. Furthermore, during the preparation process, non-antigenic components such as viral nucleic acids and lipids are removed, reducing unnecessary impurities, resulting in high purity and a lower incidence of adverse reactions. This makes it suitable for most healthy individuals, including the elderly and patients with chronic diseases.

[0024] The above viruses often cause seasonal epidemics in the population. Among them, the trivalent vaccine contains A(H3N2) subtype, A(H1N1) subtype and one lineage of type B virus, while the quadrivalent vaccine contains A(H3N2) subtype, A(H1N1) subtype and type B Victoria lineage and Yamagata lineage, and has a wide market.

[0025] Further, each immunogenic substance is present at a concentration of 7.5-30 μg / ml; preferably, at 15-30 μg / ml.

[0026] Similarly, immunogenic substances, or antigens, are components in vaccines that stimulate the body to produce a specific immune response, and their content directly affects the protective effect of the vaccine. An appropriate amount of antigen can effectively activate the immune system and generate a sufficient immune response, including humoral and cellular immunity. However, excessive antigen may over-activate the immune system, leading to over-activation and depletion of immune cells, thus reducing immunogenicity. Furthermore, excessive antigen can cause more severe local reactions (such as injection site pain, swelling, induration, and rash) and systemic symptoms (such as flu-like symptoms), which may affect the overall effectiveness of the vaccine and patient tolerability. Insufficient antigen may fail to effectively activate the immune system, resulting in an insufficient immune response and reducing the immunogenicity of the vaccine. In this invention, for adjuvanted influenza vaccines, the optimal content was screened and determined to stimulate the production of higher levels of antibodies and cytokines while ensuring vaccine safety.

[0027] Furthermore, in the single-adjuvant vaccine, the mass ratio of the CpG adjuvant to the monovalent influenza virus antigen is (100-1500):(3.75-15); in the dual-adjuvant vaccine, the mass ratio of the CpG adjuvant to the monovalent influenza virus antigen is (10-500):(3.75-15).

[0028] In this invention, CpG adjuvant is mixed with the monovalent stock solution of influenza virus hemagglutinin in a special ratio to ensure the immune activity of the vaccine.

[0029] Furthermore, the adjuvanted influenza vaccine is a multivalent influenza vaccine.

[0030] Furthermore, the multivalent influenza vaccine includes an immunogenic substance selected from at least one of H1N1 influenza A virus immunogenic substance, H3N2 influenza A virus immunogenic substance, BV influenza B virus immunogenic substance, and BY influenza virus.

[0031] Furthermore, the subjects of the adjuvanted influenza vaccine were humans.

[0032] It should be noted that, regarding vaccine dosage, each dose is sufficient to induce an immune protective response in the human body without significant toxic side effects. In this invention, for the human influenza vaccine, the dosage for one dose is 0.5 ml.

[0033] Furthermore, the vaccine is a single-adjuvant vaccine, and the content of CpG adjuvant in each dose of the vaccine is 100-1500 μg, preferably 100-500 μg, and more preferably 160 μg.

[0034] Furthermore, the vaccine is a dual-adjuvant vaccine containing both CpG adjuvant and aluminum adjuvant, wherein the content of CpG adjuvant in each dose of vaccine is 10-500 μg, preferably 10-100 μg, more preferably 80 μg; and the content of aluminum adjuvant is 0.3-0.5 mg, preferably 0.4 mg.

[0035] Adding an appropriate amount of aluminum adjuvant to adjuvanted influenza vaccines can further reduce the amount of CpG adjuvant required.

[0036] In one dose of adjuvanted influenza vaccine, the content of each monovalent stock solution, i.e. each immunogenic substance, is 3.75-15 μg, preferably 7.5-15 μg.

[0037] Furthermore, the vaccination dose per person is 1 to 3 doses of the adjuvanted vaccine.

[0038] Of course, in animal experiments, such as mouse experiments, the mouse dose is generally one-tenth of the human dose (or it can be converted according to other methods such as body surface ratio data). According to this standard, the dosage for each mouse experiment is 0.05 ml. The content of each monovalent stock solution, the content of CpG adjuvant, and the content of aluminum adjuvant can be converted accordingly.

[0039] Furthermore, the CpG adjuvant is CpG HP007.

[0040] Another object of the present invention is to provide the use of any of the above-described adjuvant influenza vaccines in any of the following:

[0041] 1) Use in the preparation of products for the treatment and / or prevention of diseases caused by influenza pathogens;

[0042] 2) Application in the preparation of products that can enhance cellular immune activity against influenza pathogens;

[0043] 3) Application in the preparation of products that can enhance humoral immune activity against influenza pathogens.

[0044] Furthermore, the influenza pathogen is an influenza A virus or an influenza B virus; specifically, it is an influenza A1 virus, an influenza A3 virus, an influenza Bv virus, or an influenza By virus.

[0045] Furthermore, the symptoms caused by the influenza pathogen include fever (some cases may have high fever, reaching 39-40℃), chills, headache, muscle and joint pain, extreme fatigue, loss of appetite and other systemic symptoms, often accompanied by sore throat, cough, nasal congestion, runny nose, retrosternal discomfort, facial flushing, mild conjunctival congestion, vomiting and diarrhea.

[0046] Compared with the prior art, the present invention has the following advantages:

[0047] The influenza vaccine provided by this invention can increase antibody levels several times compared to traditional vaccines, induce higher humoral and cellular immune responses, and effectively reduce the amount of antigen used, thus significantly improving the immune effect of the influenza vaccine on healthy individuals. Attached Figure Description

[0048] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0049] Figure 1 shows the results of the detection of the effects of different types and doses of CpG ODN on the immunogenicity of influenza vaccine in Example 2 (humoral immunity results against H1N1); the horizontal axis is divided into multiple groups, each group has four pillars, and each group corresponds to the detection results on days 14, 28, 35 and 56 in sequence.

[0050] Figure 2 shows the results of the detection of the effects of different types and doses of CpG ODN on the immunogenicity of influenza vaccine in Example 2 (humoral immunity results against H3N2); the horizontal axis is divided into multiple groups, each group has four pillars, and each group corresponds to the detection results on days 14, 28, 35 and 56 in sequence.

[0051] Figure 3 shows the results of testing the effects of different types and doses of CpG ODN on the immunogenicity of influenza vaccine in Example 2 (humoral immunity results against BV); the horizontal axis is divided into multiple groups, each group has four pillars, and each group corresponds to the test results on days 14, 28, 35 and 56 in sequence.

[0052] Figure 4 shows the results of the detection of the effects of different types and doses of CpG ODN on the immunogenicity of influenza vaccine in Example 2 (cell-mediated immunity results against H1N1).

[0053] Figure 5 shows the results of the detection of the effects of different types and doses of CpG ODN on the immunogenicity of influenza vaccine in Example 2 (cell immunization results against H3N2).

[0054] Figure 6 shows the results of the detection of the effects of different types and doses of CpG ODN on the immunogenicity of influenza vaccine in Example 2 (cell-mediated immunity results against BV).

[0055] Figure 7 shows the results of the detection of the effects of different antigen dosages and different CpG ODN dosages on the immunogenicity of influenza vaccine in Example 3 (humoral immunity results against H1N1); the horizontal axis is divided into multiple groups, each group has three pillars, and each group corresponds to the detection results on days 14, 21, and 28 in sequence.

[0056] Figure 8 shows the results of the detection of the effects of different antigen dosages and different CpG ODN dosages on the immunogenicity of influenza vaccine in Example 3 (humoral immunity results against H3N2); the horizontal axis is divided into multiple groups, each group has three pillars, and each group corresponds to the detection results on days 14, 21, and 28 in sequence.

[0057] Figure 9 shows the results of the detection of the effects of different antigen dosages and different CpG ODN dosages on the immunogenicity of influenza vaccine in Example 3 (humoral immunity results against BV); the horizontal axis is divided into multiple groups, each group has three pillars, and each group corresponds to the detection results on days 14, 21, and 28 in sequence.

[0058] Figure 10 shows the results of the detection of the effects of different antigen dosages and different CpG ODN dosages on the immunogenicity of influenza vaccine in Example 3 (IFN-γELISPOT cell immunization results); the horizontal axis is divided into multiple groups, each group has three pillars, and each group corresponds to the detection results of BV, H1N1, and H3N2 in sequence.

[0059] Figure 11 shows the results of the detection of the effects of different antigen dosages and different CpG ODN dosages on the immunogenicity of influenza vaccine in Example 3 (IL-2ELISPOT cell immunization results); the horizontal axis is divided into multiple groups, each group has three pillars, and each group corresponds to the detection results of BV, H1N1, and H3N2 in sequence.

[0060] Figure 12 shows the results of the detection of the effect of different CpG ODN dosages combined with aluminum adjuvant on the immunogenicity of influenza vaccine in Example 4 (humoral immunity results against H1N1); the horizontal axis is divided into multiple groups, each group has three pillars, and each group corresponds to the detection results on days 14, 28 and 35 in sequence.

[0061] Figure 13 shows the results of the detection of the effect of different CpG ODN dosages combined with aluminum adjuvant on the immunogenicity of influenza vaccine in Example 4 (humoral immunity results against H3N2); the horizontal axis is divided into multiple groups, each group has three pillars, and each group corresponds to the detection results on days 14, 28 and 35 in sequence.

[0062] Figure 14 shows the results of testing the effect of different CpG ODN dosages combined with aluminum adjuvant on the immunogenicity of influenza vaccine in Example 4 (humoral immunity results against BV); the horizontal axis is divided into multiple groups, each group has three pillars, and each group corresponds to the test results on days 14, 28 and 35 in sequence.

[0063] Figure 15 shows the results of the detection of the effect of different CpG ODN dosages combined with aluminum adjuvant on the immunogenicity of influenza vaccine in Example 4 (IFN-γELISPOT cell immunoassay results); the horizontal axis is divided into multiple groups, each group has three pillars, and each group corresponds to the detection results of BV, H1N1, and H3N2 in sequence.

[0064] Figure 16 shows the results of the detection of the effect of different CpG ODN dosages combined with aluminum adjuvant on the immunogenicity of influenza vaccine in Example 4 (IL-2ELISPOT cell immunization results); the horizontal axis is divided into multiple groups, each group has three pillars, and each group corresponds to the detection results of BV, H1N1, and H3N2 in sequence.

[0065] Figure 17 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. Peripheral blood was collected on Day 21 (before the second immunization) and Day 42 (before challenge), serum was separated, and the specific total IgG against H1N1 in the ferret serum was detected by indirect ELISA.

[0066] Figure 18 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. Peripheral blood was collected on Day 21 (before the second immunization) and Day 42 (before challenge), serum was separated, and the specific total IgG against H3N2 in the ferret serum was detected by indirect ELISA.

[0067] Figure 19 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. Peripheral blood was collected on Day 21 (before the second immunization) and Day 42 (before challenge), serum was separated, and the specific total IgG against BV in the ferret serum was detected by indirect ELISA.

[0068] Figure 20 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. Peripheral blood was collected on Day 21 (before the second immunization) and Day 42 (before challenge), serum was separated, and the hemagglutination inhibition titer of the ferret serum against the H1N1 A / Victoria / 4897 / 2022 strain was tested.

[0069] Figure 21 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. Peripheral blood was collected on Day 21 (before the second immunization) and Day 42 (before challenge), serum was separated, and the hemagglutination inhibition titer against the H3N2 IVR-228 strain in the ferret serum was detected.

[0070] Figure 22 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. Peripheral blood was collected on Day 21 (before the second immunization) and Day 42 (before challenge), serum was separated, and the hemagglutination inhibition titer of the ferret serum against the Influenza B / Austria / 1359417 / 2021 strain was detected.

[0071] Figure 23 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with H1N1 A / Victoria / 4897 / 2022 virus via nasal drops (1 mL / ferret). Nasal wash fluid was collected on Day 1, Day 3, and Day 5, and the viral load of H1N1 A / Victoria / 4897 / 2022 in the nasal wash fluid was detected using the TCID50 assay.

[0072] Figure 24 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with H3N2A / Hong Kong / 4801 / 2014 virus via nasal drops (1 mL / ferret). Nasal wash fluid was collected on Day 1, Day 3, and Day 5, and the viral load of H3N2A / Hong Kong / 4801 / 2014 in the nasal wash fluid was detected using the TCID50 assay.

[0073] Figure 25 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with Influenza B / Austria / 1359417 / 2021 via nasal drops (1 mL / ferret). Nasal wash fluid was collected on Day 1, Day 3, and Day 5, and the viral load of Influenza B / Austria / 1359417 / 2021 in the nasal wash fluid was detected using the TCID50 assay.

[0074] Figure 26 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with Influenza B / Austria / 1359417 / 2021 via nasal drops (1 mL / ferret). On the 6th day after the challenge, the right lung of the ferret was harvested, the tissue was homogenized, centrifuged, and the supernatant was collected. The viral load of Influenza B / Austria / 1359417 / 2021 in the lung was detected using the TCID50 method.

[0075] Figure 27 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with H1N1 A / Victoria / 4897 / 2022 virus via nasal drops (1 mL / ferret). On day 6 after the challenge, the right lung of the ferret was harvested, the tissue was homogenized, and mRNA was extracted from the lung tissue. The viral load of H1N1 A / Victoria / 4897 / 2022 in the lung was detected by qPCR.

[0076] Figure 28 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with H3N2 A / Hong Kong / 4801 / 2014 virus via nasal drops (1 mL / ferret). On day 6 after challenge, the right lung of the ferret was harvested, the tissue was homogenized, and mRNA was extracted from the lung tissue. The viral load of H3N2 A / Hong Kong / 4801 / 2014 virus in the lung was detected by qPCR.

[0077] Figure 29 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with Influenza B / Austria / 1359417 / 2021 via nasal drops (1 mL / ferret). On day 6, the right lung of the ferret was harvested, the tissue was homogenized, and mRNA was extracted from the lung tissue. The viral load of Influenza B / Austria / 1359417 / 2021 in the lung was detected by qPCR.

[0078] Figure 30 shows the results of the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with H1N1 A / Victoria / 4897 / 2022 via intranasal drops (1 mL / ferret). The left lung of the ferret was taken on the 6th day after the challenge, fixed with 10% formalin, and the tissue sections were stained with HE for pathological analysis.

[0079] Figure 31 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with H1N1 A / Victoria / 4897 / 2022 via intranasal instillation (1 mL / ferret). On the 6th day after the challenge, the left lung of the ferret was harvested, fixed with 10% formalin, and the tissue sections were subjected to HE staining for pathological analysis and pulmonary lesion extent score.

[0080] Figure 32 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with H1N1 A / Victoria / 4897 / 2022 via intranasal instillation (1 mL / ferret). On the 6th day after the challenge, the left lung of the ferret was harvested, fixed with 10% formalin, and the tissue sections were subjected to HE staining for pathological analysis. The histopathological score was obtained.

[0081] Figure 33 shows the results of the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with H3N2 A / Hong Kong / 4801 / 2014 via intranasal drops. The left lung of the ferret was taken on the 6th day after the challenge, fixed with 10% formalin, and the tissue sections were stained with HE for pathological analysis.

[0082] Figure 34 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with H3N2 A / Hong Kong / 4801 / 2014 via nasal drops (1 mL / ferret). On the 6th day after the challenge, the left lung of the ferret was harvested, fixed with 10% formalin, and the tissue sections were subjected to HE staining for pathological analysis and pulmonary lesion extent score.

[0083] Figure 35 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with H3N2 A / Hong Kong / 4801 / 2014 via intranasal drops (1 mL / ferret). On the 6th day after the challenge, the left lung of the ferret was harvested, fixed with 10% formalin, and the tissue sections were subjected to HE staining for pathological analysis. The histopathological score was obtained.

[0084] Figure 36 shows the results of the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with Influenza B / Austria / 1359417 / 2021 via intranasal drops. The left lung of the ferret was taken on the 6th day after the challenge, fixed with 10% formalin, and the tissue sections were stained with HE for pathological analysis.

[0085] Figure 37 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with Influenza B / Austria / 1359417 / 2021 via nasal drops. On the 6th day after the challenge, the left lung of the ferret was harvested, fixed in 10% formalin, and the tissue sections were subjected to HE staining for pathological analysis and pulmonary lesion extent score.

[0086] Figure 38 shows the initial immunization of ferrets on Day 0 and the booster immunization on Day 21. On Day 43, ferrets were challenged with Influenza B / Austria / 1359417 / 2021 via intranasal drops. On the 6th day after the challenge, the left lung of the ferret was harvested, fixed with 10% formalin, and the tissue sections were subjected to HE staining for pathological analysis. The histopathological score was obtained. Detailed Implementation

[0087] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0089] Unless otherwise specified, all materials and reagents used in this invention are available from commercially available products in the field.

[0090] Terminology Description

[0091] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0092] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0093] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0094] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM may fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0095] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0096] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0097] As used herein, the terms “patient,” “subject,” or “subject” are used to refer to any animal, particularly a mammal, and the methods disclosed may be used to treat any type of bird, mammal, or aquatic species, including, in particular, human and mammalian veterinary patients such as cattle, sheep, goats, horses, dogs, pigs, cats, giant pandas, elephants, rabbits, rats, and mice.

[0098] As used in this invention, the term "vaccine" refers to any biological product that, when administered via injection or mucosal route, can induce the body to produce specific antibodies and / or cellular immunity against a specific pathogen, thereby providing the body with the ability to protect against or eliminate the pathogen. Vaccines include proteins, polysaccharides, nucleic acids, live vectors, or infectious agents. Vaccines are active immunizing agents used to prevent infectious diseases, prepared by artificial attenuation, inactivation, or genetic engineering of pathogenic microorganisms (such as bacteria, rickettsiae, viruses, etc.) and their metabolites. Vaccines retain the characteristic of pathogens stimulating the animal's immune system. When an animal comes into contact with this harmless pathogen, the immune system produces certain protective substances, such as immune hormones, active physiological substances, and specific antibodies. When the animal is exposed to the same pathogen again, its immune system, following its previous memory, produces more protective substances to prevent harm from the pathogen.

[0099] Globally available influenza vaccines are categorized into inactivated influenza vaccines (IIV), live attenuated influenza vaccines (LAIV), and recombinant influenza vaccines (RIV). Based on their components, influenza vaccines are available in trivalent and quadrivalent forms. Trivalent vaccines contain one lineage of A(H3N2), A(H1N1), and type B strains, while quadrivalent vaccines contain one lineage of A(H3N2), A(H1N1), and type B (Victoria and Yamagata lineages). Based on manufacturing processes, they can be further classified as chicken embryo-based, cell culture-based, and recombinant influenza vaccines. High-antigen-content inactivated influenza vaccines, adjuvanted vaccines, and intradermal vaccines targeting specific populations are also available internationally.

[0100] Immunogenicity refers to the ability of an antigen to stimulate the body to form specific antibodies or sensitized lymphocytes. The main evaluation indicators are the level of virus strain-specific HI antibodies and the seroconversion rate of serum antibodies. The evaluation results are affected by the age of the recipient, immune function, and antibody level before vaccination.

[0101] Vaccine efficacy typically refers to its effectiveness under ideal conditions in a pre-market randomized controlled trial (RCT). Vaccine effectiveness, on the other hand, refers to its effectiveness in actual population use. Outcome indicators for evaluating influenza vaccine efficacy and effectiveness mainly include seroprevalence and seroconversion rate, laboratory-confirmed influenza, presentation of acute respiratory illness or influenza-like illness, and influenza-related hospitalizations or deaths.

[0102] As used in this article, "antigen" (Ag) refers to any substance that can induce an immune response in the body. Specifically, it refers to substances that can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, producing immune response products (sensitized lymphocytes or antibodies), and that can specifically bind to these products in vivo and in vitro. This includes, but is not limited to, heteroantigens, such as pathogenic microorganisms, viruses, toxoids, and other antigens between different species; allogeneic antigens, autoantigens, and heterophilic antigens.

[0103] The WHO-recommended trivalent influenza vaccine components for the Northern Hemisphere based on chicken embryos for the 2020-2021 season consist of: A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm09 similar strain, A / Hong Kong / 2671 / 2019(H3N2) similar strain, and B / Washington / 02 / 2019 (Victoria lineage) similar strain. The quadrivalent influenza vaccine components include two lineages of type B virus, namely the above three strains and B / Phuket / 3073 / 2013 (Yamagata lineage) similar strain. Compared to the previous year, the strains of type A H1N1, A(H3N2), and type B (Victoria lineage) viruses have changed.

[0104] As used in this article, "influenza virus" belongs to the Orthomyxoviridae family and is a single-stranded, negative-sense, segmented RNA virus. Influenza A virus can be divided into several subtypes based on the protein structure and genetic characteristics of its surface hemagglutinin (HA) and neuraminidase (NA). Currently, 18 HA subtypes (H1-18) and 11 NA subtypes (N1-11) have been identified. In addition to infecting humans, influenza A virus is widely present in animals such as birds, pigs, horses, seals, whales, and mink. Influenza B virus is divided into the Victoria and Yamagata lineages and can circulate in humans; recent data show that seals can also be infected. Influenza C virus infects humans, dogs, and pigs, causing only sporadic cases of upper respiratory tract infection. Influenza D virus mainly infects pigs and cattle; no human infections have been found. Currently, the viruses that cause seasonal influenza outbreaks are the H1N1 and H3N2 subtypes of influenza A and the Victoria and Yamagata lineages of influenza B.

[0105] "Adjuvant" refers to a substance that, when added to a composition containing an antigen, enhances or strengthens the immune response of a mammalian recipient to the antigen after exposure. Examples include, but are not limited to, alum (aluminum salts), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles such as poly(lactide-co-glycolic acid) microparticles.

[0106] As used herein, the term "CpG ODN" refers to CpG oligodeoxynucleotides, which are synthetically produced oligonucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine dinucleotides (CpG). They are at least about ten nucleotides in length, including one unmethylated CpG. CpG ODNs are single-stranded. The entire CpG ODN can be unmethylated or partially unmethylated. CpG ODNs include D-type (also known as A-type), K-type (also known as B-type), C-type, and P-type ODNs. CpG ODNs are TLR9 agonists, inducing cellular and humoral immunity and enhancing the body's immune response by specifically stimulating TLR9.

[0107] As used herein, the term "aluminum adjuvant" refers to adjuvants that include aluminum. Aluminum adjuvants include, but are not limited to, aluminum hydroxide, aluminum hydroxide gel, aluminum phosphate, aluminum phosphate gel, aluminum hydroxyphosphate, aluminum hydroxyphosphate sulfate, amorphous aluminum hydroxyphosphate sulfate, potassium aluminum sulfate, aluminum monostearate, or combinations of the above aluminum adjuvants. Currently, aluminum hydroxide and aluminum phosphate adjuvants are commonly used in vaccine production. Aluminum adjuvants have a "reservoir effect" and an "immunostimulatory effect," and can also promote the activation and maturation of antigen-presenting cells, enhance their ability to take up antigens, and activate inflammasomes to participate in the immune response.

[0108] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, the referenced documents involved in this invention are incorporated in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When references are made in this invention, examples and preferred embodiments of the relevant technical features cited may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptively based on the description in this application.

[0109] The present invention will now be described in detail through specific embodiments.

[0110] The materials used in the following embodiments are from the following sources:

[0111] Clean-grade embryos were purchased from Tianjin Longwei Poultry Co., Ltd.

[0112] SPF hatching eggs were purchased from Jinan Spafars Poultry Co., Ltd.

[0113] Disodium hydrogen phosphate (pharmaceutical grade) was purchased from Sichuan Jinshan Pharmaceutical.

[0114] Potassium dihydrogen phosphate (pharmaceutical grade) was purchased from Sichuan Jinshan Pharmaceutical.

[0115] Sodium chloride (pharmaceutical grade) was purchased from Hebei Huachen Pharmaceutical Co., Ltd.

[0116] Potassium chloride (pharmaceutical grade) was purchased from Sichuan Jinshan Pharmaceutical.

[0117] Sodium hydroxide (pharmaceutical grade) was purchased from Hunan Ercon Pharmaceutical Co., Ltd.

[0118] Sucrose (pharmaceutical grade), purchased from Hunan Ercon Pharmaceutical Co., Ltd.

[0119] Triton X-100, purchased from Merck, Germany.

[0120] Formaldehyde (pharmaceutical grade), purchased from Jiangsu Baoyi Pharmaceutical Co., Ltd.

[0121] PBS phosphate buffer is prepared using the above-mentioned disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride.

[0122] HP007, PV001, CpG7909, and CpG1018 were provided by Huapu Biotechnology (Hebei) Co., Ltd.

[0123] The nucleotide sequence of CpG HP007 is shown in SEQ ID NO: 1, specifically: 5'-tcgcgaacgttcgccgcgtacgtacgcgg-3';

[0124] The nucleotide sequence of CpG PV001 is shown in SEQ ID NO: 2, specifically: 5'-tcgacgttcgttcgtcgttcgttc-3';

[0125] The nucleotide sequence of CpG 7909 is shown in SEQ ID NO: 3, specifically: 5'-tcgtcgttttgtcgttttgtcgtt-3';

[0126] The nucleotide sequence of CpG 1018 is shown in SEQ ID NO: 4, specifically: 5'-tgactgtgaacgttcgagatga-3'.

[0127] Aluminum hydroxide adjuvant, purchased from Thermo;

[0128] The influenza virus strains H1N1 (A / Victoria / 4897 / 2022(H1N1)pdm09), H3N2 (IVR-228reassortant derived from A / Darwin / 9 / 2021), and influenza B (B / Austria / 1359417 / 2021(BV type), B / Phuket / 3073 / 2013(BY type)) all originated from the National Institute for Biological Standards and Control (NIBSC) in the United Kingdom.

[0129] The quadrivalent influenza virus split vaccine was purchased from Hualan Biological Engineering Co., Ltd. Female BALB / c mice aged 6-8 weeks were purchased from Vital River Laboratory Animal Technology Co., Ltd.

[0130] Example 1: Preparation of a novel adjuvant influenza vaccine

[0131] To investigate the technical effects of the novel adjuvanted influenza vaccine provided by this invention, this embodiment prepared several novel adjuvanted influenza vaccines containing influenza antigen, CpG ODN adjuvant, and aluminum adjuvant (optional). The specific preparation methods are as follows:

[0132] The preparation method of the novel adjuvanted influenza vaccine includes the steps of preparing the stock solution and preparing the finished product, as detailed below:

[0133] 1. Virus inoculation and culture: Chicken embryos that pass the light inspection are inoculated after being disinfected with 75% alcohol. They are then transferred to an incubator for 48-72 hours of culture.

[0134] 2. Virus harvesting: Use an inoculation and harvesting machine to harvest chicken embryos and extract allantoic fluid.

[0135] 3. Virus clarification and concentration.

[0136] 4. Purification.

[0137] 5. Pyrolysis and inactivation.

[0138] 6. Sterilize and filter to obtain the single-valent stock solution.

[0139] 7. Prepare H1N1 influenza A virus hemagglutinin, H3N2 influenza A virus hemagglutinin, BV influenza B virus hemagglutinin and / or BY influenza B virus hemagglutinin according to the above steps.

[0140] 8. Preparation of adjuvanted influenza vaccine composition: Dilute the antigen obtained in step 7, mix it thoroughly with adjuvant (CpG ODN or CpG ODN and aluminum adjuvant) and pharmaceutical excipients, and then filter and package it to obtain the novel adjuvanted influenza vaccine.

[0141] Example 2: Effects of different types and doses of CpG ODN on the immunogenicity of influenza vaccine

[0142] To evaluate the immunogenicity of different types and doses of CpG ODN influenza vaccines, this example uses BALB / c mice as an animal model to conduct immunogenicity studies. The specific methods for evaluating immunogenicity are as follows:

[0143] 1. Immunosample preparation:

[0144] The trivalent influenza virus split vaccine formulations (groups A-F) shown in Table 1 were prepared according to the method of Example 1. Trivalent influenza virus split vaccine without adjuvant (group G), commercially available quadrivalent influenza virus split vaccine (group H), and PBS blank (group I) were used as controls.

[0145] Table 1: Immunosamples Note: This vaccine is a quadrivalent influenza virus split vaccine, manufactured by Hualan Biological Engineering Co., Ltd.

[0146] 2. Mouse immunization:

[0147] BALB / c mice were randomly divided into nine groups of six or nine mice each. Mice were immunized on days 0 and 28 (groups J, K, and L were immunized only once on day 0) via intramuscular injection of different samples, with an injection volume of 50 μL. Blood was collected from the orbital rim on days 14, 28, 35, and 56; spleens were collected from three mice on days 28, 35, and 56. If mice were immunized twice, blood or spleen collection on day 28 was performed before the second immunization. Antibodies were detected using a hemagglutination inhibition assay, and the number of IL-2-secreting cells in the spleen was determined using the ELISPOT assay. The mouse immunization groupings in this example are shown in Table 2.

[0148] Table 2: Mouse Immunization Groups Note: HD refers to human dose.

[0149] 3. Humoral immune response detection

[0150] The intensity of humoral immune response can be assessed by detecting hemagglutination inhibition antibodies.

[0151] The principle of hemagglutination inhibition antibody detection: The hemagglutinin (HA) protein (i.e., antigen) on the surface of the influenza virus can cause agglutination of red blood cells in humans or certain animals (chickens, guinea pigs, etc.), a phenomenon known as hemagglutination. After the influenza virus binds to specific antibodies, the HA antigen undergoes a neutralization reaction with the antibody. When the amount of antigen is higher than that of the antibody, the unneutralized antigen agglutinates with red blood cells. When the amount of antigen is lower than that of the antibody, the antigen is neutralized by the antibody and cannot agglutinate with red blood cells. At this time, the hemagglutination phenomenon is inhibited. This method can be used to identify different types of influenza viruses and to test the antibody titer in influenza vaccine-immunized serum.

[0152] The detection process for hemagglutination inhibition antibodies includes the following steps:

[0153] (1) Preparation of solution

[0154] Chicken / Guinea Pig Hematocrit Red Blood Cells: Take freshly collected chicken / guinea pig blood, centrifuge at 800g for 10 minutes, discard the supernatant, add 0.9% sodium chloride injection solution, mix well, centrifuge again, discard the supernatant, and store at 2-8℃.

[0155] 1% Chicken / Guinea Pig Red Blood Cell Suspension: Prepared by mixing hematocrit chicken / guinea pig red blood cells with 0.9% sodium chloride injection at a ratio of 1:99. Store at 2-8℃.

[0156] Receptor-destroying enzyme (RDE): Carefully open the stopper of the receptor-destroying enzyme dry powder bottle, add 20 ml of 0.9% sodium chloride injection, stopper the bottle, mix well, and let stand until completely dissolved.

[0157] (2) Serum treatment

[0158] Mix the sample with 1 volume of the corresponding type of hemagglutinin standard antiserum (positive control) and 4 volumes of receptor-destroying enzyme, incubate at 37°C for 16-18 hours; incubate at 56°C for 50 minutes; cool to room temperature, add the hematocrit red blood cells, mix by pipetting, and incubate at 2-8°C for 2 hours or overnight for testing.

[0159] (3) Preparation and labeling of tetravalent antigens

[0160] Reconstitute the hemagglutinin standard antigen, test the hemagglutination titer, and dilute the hemagglutinin standard antigen with physiological saline to a hemagglutination titer of 1:4 to label the tetravalent antigen.

[0161] (4) Blood coagulation inhibition test

[0162] Add 25 μl of 0.9% sodium chloride injection (physiological saline) to each well of a 96-well U-shaped plate; 25 μl of treated serum supernatant; serum control; serial dilution; add 25 μl of the corresponding tetravalent antigen to each well except the last well, mix with a vortex mixer and incubate at room temperature for 45 min; add 25 μl of 1% erythrocyte suspension to each well, mix with a vortex mixer and incubate at room temperature for 30 or 60 min before observing the results.

[0163] (5) Result determination

[0164] (1) Tilt the blood coagulation plate. The red blood cells precipitated at the bottom of the serum control well will flow downward along the collar bevel in a linear pattern, i.e., "teardrop". Using the serum control well as the standard, if the test sample well shows the same "teardrop" length as the serum control well, it is judged as negative (-), indicating 100% inhibition of agglutination; if the test sample well is shorter than the serum control well, it is judged as (±), indicating partial inhibition of agglutination; if the red blood cells in the test sample well are completely agglutinated (without "teardrop"), it is positive (+), indicating no inhibition of agglutination; the blood coagulation titer of the highest dilution inhibition well (- / ±) is the result of the blood coagulation inhibition test.

[0165] (2) The remaining tetravalent antigen is subjected to a hemagglutination titer back titer test. The back titer result should be consistent with the initial calibration result of the tetravalent antigen or within the same dilution (i.e., the back titer result should be between 1:3.5 and 1:5). Otherwise, the test is invalid and the test should be repeated.

[0166] 4. Detection of cellular immune response

[0167] The intensity of cellular immune responses was assessed by measuring antigen-specific T cells in the mouse spleen using the ELISPOT method. Specifically, the number of mouse spleen cells secreting IFN-γ and IL-2 was measured using IFN-γ ELISPOT (IFN-γ ELISPOT kit, BD Bioscience) and IL-2 ELISPOT (IL-2 ELISPOT kit, BD Bioscience), respectively.

[0168] Detection principle: After the target cells are activated by the stimulant and effectively secrete the target protein (cytokine), they are captured by the antibody pre-coated on the solid-phase carrier; after cell degradation, the captured cytokines bind to the biotin-labeled secondary antibody; after incubation with the chromogenic substrate, spots will appear on the solid-phase carrier.

[0169] Assay method: Spleen cells from immunized mice (2 × 10⁵ cells / well) were seeded in 96-well plates. Hemagglutinin from H1N1 influenza A virus, H3N2 influenza A virus, or BV influenza B virus was added to a final concentration of 10 μg / mL, with culture medium used as a negative control. Cells were then incubated at 37°C and 5% CO₂ for 18–48 hours. The incubation medium was discarded, and after washing, the detection antibody was added and incubated at room temperature for 2 hours. After washing, Streptavidin-HRP was added and incubated at room temperature for 1 hour. After washing, the substrate was added for color development. The reaction was terminated with deionized water and the cells were dried. The spots were counted using an ELISPOT reader.

[0170] 5. Experimental Results:

[0171] Figures 1-3 show the results of the humoral immune response. Figure 1 shows the humoral immune response against H1N1; Figure 2 shows the humoral immune response against H3N2; and Figure 3 shows the humoral immune response against BV.

[0172] Figures 4-6 show the results of the cellular immune responses. Figure 4 shows the results of cellular immunity against H1N1; Figure 5 shows the results of cellular immunity against H3N2; and Figure 6 shows the results of cellular immunity against BV. In these figures, Ag represents the antigen.

[0173] The experimental results show that, compared with PV001, 1018, and 7909, HP007 can significantly increase antibody levels in mice. Results of a single dose of humoral immunization 28 days later: At an adjuvant dose of 160 μg / dose, HP007 showed superior humoral and cellular immunity compared to commercially available vaccines, and antibody levels for different antibody types were significantly higher than the commercially available control, with BV antibody levels increasing nearly 20-fold.

[0174] Example 3: Effects of different antigen dosages and different CpG ODN dosages on the immunogenicity of influenza vaccines

[0175] To evaluate the effects of different antigen dosages and different CpG ODN dosages on the immunogenicity of influenza vaccines, this example uses BALB / c mice as an animal model to conduct immunogenicity studies. The specific methods for evaluating immunogenicity are as follows:

[0176] 1. Immunosample preparation:

[0177] The trivalent influenza virus split vaccine formulations (groups A-G) shown in Table 3 were prepared according to the method of Example 1, with PBS (group H) as a control.

[0178] Table 3: Immunosamples

[0179] 2. Mouse immunization:

[0180] BALB / c mice were randomly divided into 8 groups of 12 mice each. Mice were immunized on days 0 and 28 by intramuscular injection of different samples (50 μL each). Blood was collected from the orbital sinus on days 14, 21, 28, and 56. Spleens were collected from 6 mice on days 28 and 56. Blood collection or spleen collection on day 28 was performed before the second immunization. Antibodies were detected by hemagglutination inhibition assay, and the number of IFN-γ and IL-2 secreting cells in the spleen was determined by the ELISPOT assay. The mouse immunization grouping in this example is shown in Table 4.

[0181] Table 4: Mouse Immunization Groups

[0182] 3. Humoral immune response detection

[0183] The experimental procedure is the same as in Example 2.

[0184] 4. Detection of cellular immune response

[0185] The experimental procedure is the same as in Example 2.

[0186] 5. Experimental Results:

[0187] Figures 7-9 show the results of the humoral immune response. Figure 7 shows the humoral immune response against H1N1; Figure 8 shows the humoral immune response against H3N2; and Figure 9 shows the humoral immune response against BV.

[0188] Figures 10 and 11 show the results of the cellular immune response. Figure 10 shows the IFN-γ ELISPOT results for D56; Figure 11 shows the IL-2 ELISPOT results for D56.

[0189] The results of the humoral immunity experiment showed that there was no significant difference between HP007 (400 μg) and HP007 (160 μg); there was no significant difference in antibody levels between 15 μg HA and different adjuvant ratios; when the antigen decreased by 1 / 2, there was no significant difference in hemagglutination inhibition antibody titer compared with the original amount; when the antigen amount was reduced to 1 / 4, the hemagglutination inhibition antibody titer also decreased with the decrease in antigen content.

[0190] The results of the cellular immunity experiments showed that a strong cellular immune response was produced when 15 μg HA was mixed with 400 μg HP007 or 160 μg HP007; the strongest cellular immune response was produced when 7.5 μg HA was mixed with 160 μg HP007. This demonstrates that CpG can still induce high humoral and cellular immune responses in influenza vaccines even with reduced antigen dosage.

[0191] Example 4: Effect of different amounts of CpG ODN combined with aluminum adjuvant on the immunogenicity of influenza vaccine

[0192] To evaluate the effect of different CpG ODN dosages combined with aluminum adjuvant on the immunogenicity of influenza vaccine, this example uses BALB / c mice as an animal model to conduct an immunogenicity study. The specific evaluation methods for immunogenicity are as follows:

[0193] 1. Immunosample preparation:

[0194] The quadrivalent influenza virus split vaccine formulations (groups A-G) shown in Table 5 were prepared according to the method in Example 1, with PBS (group H) as a control.

[0195] Table 5: Immunosamples

[0196] 2. Mouse immunization:

[0197] BALB / c mice were randomly divided into 8 groups of 12 mice each. Mice were immunized on days 0 and 28 by intramuscular injection of different samples (50 μL each). Blood was collected from the orbital sinus on days 14, 28 (before the second immunization), and 35. Spleens were collected from 6 mice on day 35. Antibody levels were detected by hemagglutination inhibition assay, and the number of IFN-γ and IL-2 secreting cells in the spleen was determined by the ELISPOT assay. The immunization grouping of mice in this example is shown in Table 6.

[0198] Table 6: Mouse Immunization Grouping

[0199] 3. Humoral immune response detection

[0200] The experimental procedure is the same as in Example 2.

[0201] 4. Detection of cellular immune response

[0202] The experimental procedure is the same as in Example 2.

[0203] 5. Experimental Results:

[0204] Figures 12-14 show the results of the humoral immune response. Figure 12 shows the humoral immune response against H1N1; Figure 13 shows the humoral immune response against H3N2; and Figure 14 shows the humoral immune response against BV.

[0205] Figures 15 and 16 show the results of the cellular immune response. Figure 15 shows the IFN-γ ELISPOT results for D35; Figure 16 shows the IL-2 ELISPOT results for D35.

[0206] The results above show that different doses of CpG ODN combined with aluminum adjuvant can produce strong humoral and cellular immune responses, which are much stronger than those of commercially available vaccines. In particular, the dual-adjuvant influenza vaccine with 20-80 μg CpG ODN combined with 400 μg aluminum hydroxide adjuvant showed superior humoral and cellular immune responses, and the dual-adjuvant influenza vaccine with 80 μg CpG ODN combined with 400 μg aluminum hydroxide adjuvant showed the best immunogenicity.

[0207] Example 5: Evaluation of the immunogenicity and protective effect against challenge of CpG ODN adjuvanted influenza vaccine in ferrets.

[0208] In this embodiment, ferrets were used as an animal model. They were immunized with a CpG ODN-adjuvanted trivalent influenza vaccine or a commercially available vaccine and then challenged with the virus. Serum IgG, hemagglutination inhibition titer, viral load in nasal wash fluid, and viral load in the lungs were measured after immunization, and lung histopathological analysis was performed using hematoxylin and eosin (HE) staining. The immunogenicity of the CpG ODN-adjuvanted trivalent influenza vaccine in ferrets and its protective effect after challenge with the viral strain were evaluated.

[0209] 1. Experimental materials:

[0210] Female ferrets aged 5-6 months were provided by Wuxi Coral Reef Biotechnology Co., Ltd.

[0211] The components of the influenza virus split vaccine (CpG adjuvant) (trivalent vaccine - low dose adjuvant) are shown in Group B of Table 1 in Example 2;

[0212] The influenza virus split vaccine (CpG adjuvant) (trivalent vaccine - high dose adjuvant) has the following components as shown in Group C of Table 1 in Example 2;

[0213] Commercially available trivalent influenza virus split vaccine;

[0214] H1N1 A / Victoria / 4897 / 2022 (Brand Code: NIBSC(22 / 316));

[0215] H3N2 IVR-228 (Brand Code: NIBSC(21 / 246))

[0216] H3N2 A / Hong Kong / 4801 / 2014 (Brand: ATCC, Item No.: VR1990);

[0217] Influenza B / Austria / 1359417 / 2021 (Brand code: NIBSC(22 / 204));

[0218] 20% Turkey Red Blood Cells (Kewei Biotechnology, KW-0120R-20);

[0219] 1% guinea pig erythrocytes (Sempervir, SBJ-RBC-GP001);

[0220] Serum-free cell cryopreservation solution (NewSemi Biotechnology Co., Ltd., C40100);

[0221] 1×PBS buffer (Biosharp, BL302A);

[0222] Anti-Ferret Ig G H&L (HRP) (abcam, ab112770);

[0223] ELISA coating buffer (10× coating solution) (absin, abs9290);

[0224] ELISA Supplemental Solution Set (SEKCR02).

[0225] ELISA coating antigen Influenza A [Victoria / 4897 / 2022] Hemagglutinin (HA) Protein, His Tag (MALS verified), Viral subtype A / Victoria / 4897 / 2022 (H1N1), (Acro, HA1-V52H8);

[0226] ELISA coating antigen Influenza A [A / Darwin / 9 / 2021(H3N2)] Hemagglutinin (HA) Protein, His Tag (MALS verified), Viral subtype Influenza Virus Infection IVR-228(H3N2), (Acro, HA2-V52H6);

[0227] ELISA coating antigen Influenza B [Austria / 1359417 / 2021(B / Victoria lineage)] Hemagglutinin (HA) Protein, His Tag, Viral subtype B / Austria / 1359417 / 2021(B / Victoria lineage), (Acro, HAE-V52H3).

[0228] 2. Experimental methods:

[0229] Female ferrets aged 5-6 months were selected and divided into 13 groups, of which G1, G5, and G9 were model groups, and G13 was the normal group. The specific grouping is shown in the table below:

[0230] Ferrets were immunized on Day 0 and Day 21. Peripheral blood was collected on Day 21 (before the second immunization) and Day 42 (before challenge). Serum was separated and serum IgG (3 antigens) was detected by indirect ELISA. The steps included: coating with antigen, washing, blocking, washing, adding 3-fold serially diluted serum and incubating, discarding serum, washing, adding Ferret HRP-IgG secondary antibody diluted 1:1500, discarding secondary antibody, washing, adding chromogenic solution and incubating, adding stop solution, and reading the absorbance values ​​at OD 450nm and OD 630nm. For the detection of hemagglutination inhibition titers (3 viral subtypes), please refer to Example 2. When detecting the hemagglutination inhibition titers in serum against the H1N1 A / Victoria / 4897 / 2022 and Influenza B / Austria / 1359417 / 2021 strains, 1% turkey red blood cells were used; when detecting the hemagglutination inhibition titers in serum against the H3N2 IVR-228 strain, 1% guinea pig red blood cells were used.

[0231] Day 43: Animals were challenged via nasal drops (1 mL / animal). Before challenge, body temperature and weight were measured and recorded, and clinical symptoms were observed once daily until the end of the experiment. After challenge, nasal wash fluid was collected on days 1, 3, and 5, and the viral load in the nasal wash fluid was detected using the TCID50 assay. On day 6 post-challenge, the experimental animals were euthanized. The complete lungs were harvested, weighed, and the right lung was weighed. The tissue was homogenized, and the viral load in the lungs was detected using the TCID50 and qPCR methods.

[0232] The left lung was fixed with 10% formalin, and after sectioning, HE staining was performed for pathological evaluation. The evaluation focused on the extent of lung lesions, bronchioles, pulmonary arterioles, alveolar inflammation, and alveolar wall thickness. The scoring criteria are shown in the table below.

[0233] Evaluation criteria for HE staining of lung tissue

[0234] Data were processed using Office Excel 2017 and GraphPad Prism 9.0, and are expressed as Mean ± SEM and GMT ± SD. One-way and two-way ANOVA were used for analysis, and Tukey's test was employed to assess the significance of differences between groups. For comparisons between two groups, a two-tailed t-test was used. A p-value < 0.05 was considered statistically significant between the two groups.

[0235] 3. Experimental Results:

[0236] The results of specific binding antibody IgG against H1N1, H3N2, and BV are shown in Figure 17-19 (Two-way ANOVA: ***p<0.001 vs. model group, *p<0.05 vs. model group). As shown in Figure 17-19, no significant specific IgG antibody titers against H1N1, H3N2, and BV were detected in the serum of animals in the normal group, model group, and commercially available vaccine group. Higher titers of specific IgG antibodies against H1N1, H3N2, and BV were detected in the serum of animals in the tested trivalent vaccine group, with Day 42 showing higher titers than Day 21, and a clear dose-dependent relationship was observed.

[0237] The results of hemagglutination inhibition titer assays are shown in Figures 20-22 (Two-way ANOVA: ***p<0.001 vs. model group, **p<0.01 vs. model group, *p<0.05 vs. model group; ###p<0.001 vs. low-dose trivalent vaccine group; ##p<0.01 vs. low-dose trivalent vaccine group). As shown in the figures, compared to the model group, the serum of animals in the commercially available vaccine group showed lower hemagglutination inhibition titers against H1N1, H3N2, and BV. The serum of both the high- and low-dose trivalent vaccine groups contained high titers of hemagglutination inhibition against H1N1, H3N2, and BV (p<0.05), and this showed a clear dose-dependent effect.

[0238] The viral load results in the nasal wash fluid are shown in Figures 23-25. Figure 23 (Two-way ANOVA: ###p<0.001 vs. normal group; ***p<0.001 vs. model group, **p<0.01 vs. model group) shows that, against H1N1 A / Victoria / 4897 / 2022 challenge, compared with the normal group, the nasal wash fluid of the model group animals had higher viral load titers on Day 1, Day 3, and Day 5 (p<0.001), and showed a gradually decreasing trend. Compared with the model group, the viral load in the nasal wash fluid of the commercially available vaccine group did not differ significantly at different time points. The viral load in the nasal wash fluid of the tested trivalent vaccine group was significantly reduced on Day 3 and Day 5 (p<0.01), and a clear dose-response relationship was observed. Figure 24 (Two-way ANOVA: ###p<0.001 vs. normal group, #p<0.05 vs. normal group; **p<0.01 vs. model group, *p<0.05 vs. model group; T-test: &&p<0.01 vs. model group, &p<0.05 vs. model group) shows that, against H3N2 A / Hong Kong / 4801 / 2014 challenge, compared with the normal group, the nasal wash fluid of the model group animals had higher viral load titers on Day 1, Day 3, and Day 5 (p<0.05), and showed a gradually decreasing trend; compared with the model group, the viral load of nasal wash fluid in the commercially available vaccine group did not differ significantly at different time points; the viral load of nasal wash fluid in the low-dose trivalent vaccine group showed a decreasing trend on Day 1, Day 3, and Day 5, with Day... There was a significant difference at 1 day (p<0.05); the viral load in the nasal wash of the high-dose trivalent vaccine group was significantly reduced at Day 1, Day 3 and Day 5 (p<0.05).Figure 25 (Two-way ANOVA: ###p<0.001 vs. normal group; ***p<0.001 vs. model group, *p<0.05 vs. model group; T-test: &p<0.05 vs. model group) shows that, against Influenza B / Austria / 1359417 / 2021 challenge, compared with the normal group, the nasal wash fluid of the model group animals had higher viral load titers on Day 1, Day 3, and Day 5 (p<0.001), and showed a gradually decreasing trend; compared with the model group, the viral load of nasal wash fluid in the commercially available vaccine group did not differ significantly on Day 1 and Day 3, but decreased significantly on Day 5 (p<0.05); the viral load of nasal wash fluid in the high- and low-dose trivalent vaccine groups on Day 1, Day 3, and Day 5 showed a significant decrease on Day 5. The viral load decreased significantly at 5 days (p<0.05), with larger decreases at Day 3 and Day 5 (p<0.001). At Day 5, the viral load in the high- and low-dose trivalent vaccine groups was below the detection limit.

[0239] The results of the TCID50 assay for viral load in the lungs are shown in Figure 26 (One-way ANOVA: *p<0.05 vs. model group). As shown in the figure, no significant viral load was detected in the lungs of animals in groups G1-G4 after challenge with H1N1 A / Victoria / 4897 / 2022; similarly, no significant viral load was detected in the lungs of animals in groups G5-G8 after challenge with H3N2 A / Hong Kong / 4801 / 2014; and after challenge with Influenza B / Austria / 1359417 / 2021, significant viral load was detected in the lungs of 3 animals in the G9 model group (6 animals), while no significant viral load was detected in the remaining animals. Compared with the model group, the viral load in the lungs of all three vaccine-immunized groups was significantly reduced (p<0.05).

[0240] The results of pulmonary viral load detection by qPCR are shown in Figures 27-29 (One-way ANOVA: ###p<0.001 vs. normal group; ***p<0.001 vs. model group, *p<0.05 vs. model group; T-test: &p<0.05 vs. model group). As shown in Figure 27, when challenged with H1N1 A / Victoria / 4897 / 2022, the pulmonary viral load in the model group was significantly higher than that in the normal group (p<0.001). Compared with the model group, there was no significant difference in the commercially available vaccine group. The high and low doses of the trivalent vaccine significantly reduced the pulmonary viral load (p<0.001), with a reduction greater than 2Log10. As shown in Figures 28 and 29, when challenged with H3N2 A / Hong Kong / 4801 / 2014 and Influenza B / Austria / 1359417 / 2021, the viral load in the lungs of the model group showed an increasing trend compared with the normal group, but the overall level was low and there was no statistically significant difference (p>0.05). Compared with the model group, there was no significant difference in the commercially available vaccines. The viral load in the lungs of the high- and low-dose trivalent vaccines was significantly reduced (p<0.05), close to the detection threshold.

[0241] The results of HE staining analysis of the lungs are shown in Figures 30-38. As shown in Figures 30-32 (T-test: ##p<0.01 vs. normal group; *p<0.05 vs. model group), compared with the normal group, the model group showed a significant increase in the extent of inflammatory lesions in the lungs, alveolar inflammatory cell infiltration, and peribronchial and arteriolar inflammatory cell infiltration scores after H1N1 A / Victoria / 4897 / 2022 challenge (p<0.01). Compared with the model group, there were no significant changes in the extent of inflammatory lesions in the lungs, alveolar inflammatory cell infiltration, and peribronchial and arteriolar inflammatory cell infiltration scores in the commercially available vaccine group. The extent of inflammatory lesions in the lungs was significantly reduced in the trivalent vaccine-low-dose group (p<0.05), but there was no significant improvement in alveolar inflammatory cell infiltration and peribronchial and arteriolar inflammatory cell infiltration scores. The extent of inflammatory lesions in the lungs, as well as alveolar inflammatory cell infiltration and peribronchial and arteriolar inflammatory cell infiltration scores, were significantly reduced in the trivalent vaccine-high-dose group (p<0.05).

[0242] As shown in Figures 33-35 (T-test: #p<0.05 vs. normal group), after challenge with H3N2 A / Hong Kong / 4801 / 2014 virus, compared with the normal group, the model group showed a significant increase in the extent of inflammatory lesions in the lungs (p<0.05), and an upward trend in the scores of alveolar inflammatory cell infiltration and peribronchial and arteriolar inflammatory cell infiltration. Compared with the model group, the commercially available vaccine group showed no significant change in lung pathology; the high- and low-dose trivalent vaccine groups showed an improvement trend in the extent of inflammatory lesions in the lungs, as well as the scores of alveolar inflammatory cell infiltration and peribronchial and arteriolar inflammatory cell infiltration.

[0243] As shown in Figures 36-38 (T-test: ###p<0.001 vs. normal group; #p<0.05 vs. normal group; **p<0.01 vs. model group; *p<0.05 vs. model group), compared with the normal group, the model group showed a significant increase in the extent of inflammatory lesions in the lungs, as well as the scores for alveolar inflammatory cell infiltration and peribronchial and arteriolar inflammatory cell infiltration (p<0.05) after challenge with Influenza B / Austria / 1359417 / 2021 virus (p<0.05). Compared with the model group, the lung pathology in the commercially available vaccine group and the low-dose trivalent vaccine group showed a trend of improvement, but there was no statistical difference (p>0.05); the high-dose trivalent vaccine group showed a significant decrease in the extent of inflammatory lesions in the lungs, as well as the scores for alveolar inflammatory cell infiltration and peribronchial and arteriolar inflammatory cell infiltration (p<0.05).

[0244] in conclusion:

[0245] (1) After being challenged with H1N1 A / Victoria / 4897 / 2022, H3N2 A / Hong Kong / 4801 / 2014, and Influenza B / Austria / 1359417 / 2021, the animals showed a decrease in body weight and an increase in body temperature. The main clinical symptoms were sneezing and runny nose. The nasal washing fluid of the upper respiratory tract contained a high viral load, and the lungs of the lower respiratory tract showed obvious pathological changes mainly characterized by inflammation. This indicates that the challenge models of the three subtypes of influenza were successfully established.

[0246] (2) After two immunizations with commercially available vaccines, the specific IgG and HAI values ​​of H1N1 A / Victoria / 4897 / 2022, H3N2IVR-228, and Influenza B / Austria / 1359417 / 2021 subtypes in serum were low; no protective effect was observed against H1N1 A / Victoria / 4897 / 2022, H3N2 A / Hong Kong / 4801 / 2014, and Influenza B / Austria / 1359417 / 2021 subtypes.

[0247] (3) After two immunizations with the tested trivalent vaccine, the serum had high specific IgG and HAI values ​​for H1N1A / Victoria / 4897 / 2022, H3N2 IVR-228, and Influenza B / Austria / 1359417 / 2021 subtypes, indicating good immunogenicity in vivo and a clear dose-response relationship. Against challenge with H1N1 A / Victoria / 4897 / 2022, H3N2 A / Hong Kong / 4801 / 2014, and Influenza B / Austria / 1359417 / 2021, the trivalent vaccine showed protective effects in multiple aspects, including clinical symptoms, viral shedding in nasal wash fluid of the upper respiratory tract, viral load in the lungs of the lower respiratory tract, and lung pathology, and also showed a clear dose-dependent effect.

[0248] (4) Although the tested trivalent vaccine components do not contain the H3N2 A / Hong Kong / 4801 / 2014 HA antigen, they show protection against H3N2 A / Hong Kong / 4801 / 2014 challenge, indicating that the tested trivalent vaccine has certain cross-protection against different subtypes of influenza.

[0249] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0250] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An adjuvanted influenza vaccine, characterized in that, The vaccine includes a CpG adjuvant selected from at least one of CpG HP007, CpG PV001, CpG 7909, and CpG 1018, wherein CpG HP007 has the nucleotide sequence shown in SEQ ID NO: 1; CpG PV001 has the sequence shown in SEQ ID NO: 2; CpG 7909 has the sequence shown in SEQ ID NO: 3; and CpG 1018 has the sequence shown in SEQ ID NO:

4.

2. The adjuvanted influenza vaccine of claim 1, characterized in that, The vaccine is a single-adjuvant vaccine, and the content of the CpG adjuvant is 200-3000 μg / ml, preferably 200-1000 μg / ml, and more preferably 300-400 μg / ml.

3. The adjuvanted influenza vaccine according to claim 1, characterized in that, The vaccine is a dual-adjuvant vaccine containing both CpG adjuvant and aluminum adjuvant. In this case, the content of CpG adjuvant is 20-1000 μg / ml, preferably 20-200 μg / ml, and more preferably 160 μg / ml.

4. The adjuvanted influenza vaccine according to claim 3, characterized in that, The aluminum adjuvant is selected from one or more of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, and amorphous aluminum hydroxyphosphate adjuvant.

5. The adjuvanted influenza vaccine according to claim 3, characterized in that, The aluminum adjuvant content is 0.6-1.0 mg / ml, preferably 0.8 mg / ml.

6. The adjuvanted influenza vaccine according to claim 3, characterized in that, The mass ratio of the CpG adjuvant to the aluminum adjuvant is (10-500):(300-500).

7. The adjuvanted influenza vaccine according to any one of claims 1-6, characterized in that, The influenza vaccine includes immunogenic substances, wherein the immunogenic substances are immunogenic substances of influenza A virus and / or influenza B virus.

8. The adjuvanted influenza vaccine according to claim 7, characterized in that, The immunogenic substances of the influenza A virus and / or influenza B virus include influenza virus split vaccine stock solutions, including at least one of a monovalent stock solution containing influenza A virus hemagglutinin and a monovalent stock solution containing influenza B virus hemagglutinin; in particular, the monovalent stock solution containing influenza A virus hemagglutinin includes at least one of a monovalent stock solution containing H1N1 influenza A virus hemagglutinin and a monovalent stock solution containing H3N2 influenza A virus hemagglutinin; the monovalent stock solution containing influenza B virus hemagglutinin includes at least one of a monovalent stock solution containing BV influenza B virus hemagglutinin and a monovalent stock solution containing BY influenza B virus hemagglutinin.

9. The adjuvanted influenza vaccine according to claim 7, characterized in that, Each immunogenic substance is present at a concentration of 7.5-30 μg / ml; preferably, at 15-30 μg / ml.

10. The adjuvanted influenza vaccine according to claim 2, characterized in that, The mass ratio of the CpG adjuvant to each immunogenic substance is (100-1500):(3.75-15).

11. The adjuvanted influenza vaccine according to claim 1, characterized in that, The adjuvanted influenza vaccine is a multivalent influenza vaccine.

12. The adjuvanted influenza vaccine according to claim 11, characterized in that, The multivalent influenza vaccine includes an immunogenic substance selected from at least one of the following: H1N1 influenza A virus immunogenic substance, H3N2 influenza A virus immunogenic substance, BV influenza B virus immunogenic substance, and BY influenza B virus immunogenic substance.

13. The adjuvanted influenza vaccine according to claim 1, characterized in that, The subjects of the adjuvanted influenza vaccine were humans.

14. The adjuvanted influenza vaccine according to claim 13, characterized in that, The vaccine is a single-adjuvant vaccine, and the content of CpG adjuvant in each dose of the vaccine is 100-1500 μg, preferably 100-500 μg, and more preferably 160 μg.

15. The adjuvanted influenza vaccine according to claim 13, characterized in that, The vaccine is a dual-adjuvant vaccine containing both CpG adjuvant and aluminum adjuvant, wherein each dose of vaccine contains 10-500 μg of CpG adjuvant, preferably 10-100 μg, more preferably 80 μg; and 0.3-0.5 mg of aluminum adjuvant, preferably 0.4 mg.

16. The adjuvanted influenza vaccine according to claim 13, characterized in that, In each dose of the vaccine, each immunogenic substance is present in a quantity of 3.75-15 μg; preferably, in a quantity of 7.5-15 μg.

17. The adjuvanted influenza vaccine of claim 13, characterized in that, Each dose of vaccine contains 0.5 ml.

18. The adjuvanted influenza vaccine according to any one of claims 13-17, characterized in that, Each person receives 1 to 3 doses of the adjuvanted vaccine.

19. The use of the adjuvanted influenza vaccine according to any one of claims 1-17 in any of the following: 1) Use in the preparation of products for the treatment and / or prevention of diseases caused by influenza pathogens; 2) Application in the preparation of products that can enhance cellular immune activity against influenza pathogens; 3) Application in the preparation of products that can enhance humoral immune activity against influenza pathogens.

20. The use according to claim 19, characterized in that, The influenza pathogen is either influenza A virus or influenza B virus.

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