Influenza virus live vaccine and method of preparation and use

Abstract

The present application relates to the technical field of biology, and particularly provides an influenza virus live vaccine, a preparation method and application, aiming at solving the problems of single immunization invalidity, cell immunity loss, insufficient broad-spectrum protection and high safety risk of the influenza vaccine in the prior art. Therefore, the present application provides an influenza virus live vaccine, comprising an influenza virus without attenuation treatment. The influenza virus live vaccine of the present application breaks through the safety limit of the traditional attenuated live vaccine, and for the first time proves that the unattenuated influenza epidemic strain (such as H1N1 PR8 strain) can realize safe and efficient immune response through subcutaneous inoculation, thereby providing a brand-new technical path for the development of the influenza vaccine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically providing a live influenza virus vaccine, its preparation method, and its application. Background Technology

[0002] Influenza (flu) is a respiratory and other organ disease caused by the influenza virus. It occurs to varying degrees every year in the spring and winter, and even in other seasons, among healthy children and adults. It is usually an acute and contagious disease.

[0003] Influenza virus is the pathogen that causes influenza. It belongs to the negative-sense single-stranded RNA virus family, and its genome consists of eight independent RNA segments (named segments 1-8), with a total nucleic acid length of approximately 13.6 kb. These eight segments encode ten proteins, eight of which are structural proteins, including PB1, PB2, PA, HA, NA, NP, M1, and M2, while NS1 and NS2 are non-structural proteins. Influenza viruses are divided into human influenza viruses and animal influenza viruses. Human influenza viruses are further classified into three types: A, B, and C.

[0004] Viral replication primarily relies on viral ribonucleoproteins (vRNPs). The ribonucleoprotein of influenza A virus, composed of viral RNA, an RNA polymerase (RdRp) complex, and nucleoproteins (NPs), is the smallest unit of viral replication; viral proteins can only be expressed on this structural basis. The RdRp in the vRNP structure consists of three subunits (PA, PB2, and PB1). PB1 is located at the core of the trimer, and its N-terminus and C-terminus form stable protein complexes with the C-terminus of the PA subunit and the N-terminus of the PB2 subunit, respectively, through non-covalent bonds (such as hydrophobic interactions, hydrogen bonds, and van der Waals forces).

[0005] Although various antiviral drugs can be used to treat influenza, due to the rapid mutation of the influenza virus, there are sporadic outbreaks and epidemics of influenza around the world every year. Correct influenza vaccination can effectively reduce the incidence of related diseases caused by influenza virus infection.

[0006] Currently, existing influenza vaccines have the following significant limitations:

[0007] ① Inactivated vaccines have weak immunogenicity, require adjuvants to elicit an effective response, and the induced antibodies have a short duration of action and cannot activate an effective cellular immune response, thus their immune protection effect is limited.

[0008] ② Although live attenuated vaccines can mimic natural infection, there is a risk of virulence reversion, which may cause respiratory symptoms, and currently they are limited to mucosal administration.

[0009] ③ Recombinant protein vaccines, split vaccines, and VLP vaccines all require more than one booster dose to achieve basic protection, and are highly dependent on antigen matching, with weak cross-protection against heterologous strains.

[0010] In summary, existing influenza vaccines generally face problems such as poor efficacy of a single immunization, insufficient cellular immunity, and poor broad-spectrum protection.

[0011] Accordingly, a new technical solution is needed in this field to solve the above-mentioned technical problems. Summary of the Invention

[0012] The present invention aims to solve the above-mentioned technical problems, namely, to solve the problems that existing influenza vaccines generally suffer from poor single-dose immunization effect, insufficient cellular immunity, and poor broad-spectrum protection.

[0013] In a first aspect, the present invention provides a live influenza virus vaccine, wherein the vaccine comprises an unattenuated influenza virus.

[0014] In the preferred embodiment of the above-mentioned live influenza virus vaccine, the live influenza virus vaccine does not contain adjuvant.

[0015] In the preferred embodiment of the above-mentioned live influenza virus vaccine, the formulation of the live influenza virus vaccine is suitable for subcutaneous injection.

[0016] In the preferred embodiment of the above-mentioned live influenza virus vaccine, the viral titer of the unattenuated influenza virus is 10. 1 ~10 8 TCID 50 / mL, and the residual amount of host cell DNA is ≤25ng / mL, and the residual amount of host cell protein is ≤100μg / mL.

[0017] In the preferred technical embodiment of the above-mentioned live influenza virus vaccine, the unattenuated influenza virus is influenza A H1N1, influenza A H3N2, influenza B Victoria lineage and / or influenza B Yamagata lineage.

[0018] In a second aspect, the present invention provides a method for preparing the aforementioned live influenza virus vaccine, comprising the following steps:

[0019] S1. Culture: Influenza virus is inoculated into virus cultures of MDCK cells, Vero cells or chicken embryos and cultured to obtain virus harvest fluid;

[0020] S2. Purification: The virus harvested solution is subjected to deep filtration, ultrafiltration concentration, nuclease treatment and chromatography purification in sequence to obtain the original solution;

[0021] S3. Formulation: The stock solution is sterilized and filtered, and a diluent is added to prepare a liquid formulation, or a preservative is added and then freeze-dried to prepare a freeze-dried formulation.

[0022] In the preferred embodiment of the above preparation method, when the influenza virus is inoculated into MDCK cells, the culture in step S1 is performed as follows:

[0023] S11. Wash the culture container with MDCK cells that have grown at 80-100% confluence at least twice with PBS;

[0024] S12. Mix the influenza virus with DMEM-F12 medium without bovine serum to obtain the inoculum solution;

[0025] S13. Add the inoculum to the cleaned culture container and incubate;

[0026] S14. Add DMEM-F12 medium without bovine serum and add TPCK trypsin or a substitute.

[0027] S15. Continue culturing to obtain virus harvest fluid.

[0028] In the preferred embodiment of the above preparation method, the purification in step S2 includes:

[0029] S21. The virus harvested fluid is subjected to deep filtration to obtain filtrate;

[0030] S22. The filtrate is concentrated by ultrafiltration to obtain a concentrated solution;

[0031] S23. Add nuclease and MgCl2 to the concentrated solution, incubate overnight or perform enzyme digestion to obtain enzyme digestion solution;

[0032] S24. The enzyme digestion solution is filtered and purified by chromatography. The collected chromatographic solutions are combined to obtain the original solution.

[0033] In the preferred embodiment of the above preparation method, in step S24, the chromatographic purification includes equilibration, and the equilibration buffer used is a solution containing 20~60mM PBS, 0.5~1.5mM MgCl2, 50~150mM NaCl and pH 6.5~8.0.

[0034] In a third aspect, the present invention provides the use of the described live influenza virus vaccine in the preparation of a medicament for the prevention of influenza virus via subcutaneous injection.

[0035] In the preferred embodiment of the above application, the drug is administered via a single-dose or multi-dose immunization procedure.

[0036] The live influenza virus vaccine of the present invention has the following technical effects:

[0037] 1. The live influenza virus vaccine of this invention overcomes the safety limitations of traditional attenuated live vaccines, demonstrating for the first time that unattenuated circulating influenza strains (such as H1N1 PR8 strain) can achieve a safe and efficient immune response through subcutaneous inoculation, providing a novel technical pathway for influenza vaccine development. Viral replication is strictly confined to draining lymph nodes and the spleen, and is completely cleared within 7-15 days after inoculation. No viral load or pathological damage was detected in vital organs such as the heart, brain, and kidneys. A single immunization can induce high levels of neutralizing antibodies, and mouse challenge experiments show 100% protective efficacy, indicating that this immunization strategy can induce significant immune protection.

[0038] 2. The formulation of the live influenza virus vaccine of the present invention is suitable for subcutaneous injection, indicating that by changing the route of administration of live virus to subcutaneous injection, the spread of the virus to the whole body can be effectively prevented, and the potential risk of systemic infection can be eliminated.

[0039] 3. The live influenza virus vaccine of this invention utilizes the natural structural advantage of intact viral antigens to successfully activate cross-immune responses against conserved epitopes such as HA. Compared to subunit vaccines, mRNA vaccines, split vaccines, VLP vaccines, and viral vector vaccines, the live virus vaccine completely preserves viral antigenic epitopes, exhibiting stronger broad-spectrum protective potential and providing a new solution to address the rapid mutation of influenza viruses. Attached Figure Description

[0040] The preferred embodiments of the present invention are described below with reference to the accompanying drawings, in which:

[0041] Figure 1 This is a process flow diagram of the method for preparing the live influenza virus vaccine of the present invention;

[0042] Figure 2 This is a process flow diagram of the culture process in the preparation method of the live influenza virus vaccine of the present invention;

[0043] Figure 3 This is a process flow diagram of purification in the method for preparing live influenza virus vaccine of the present invention;

[0044] Figure 4 A schematic diagram of subcutaneous immunization challenge with high, medium, and low doses of live influenza PR8 virus vaccine in BALB / c mice and the results of weight changes after immunization;

[0045] Figure 5 The results of the challenge protection experiment after immunization with high, medium and low doses of influenza PR8 strain;

[0046] Figure 6 The results are for the detection of high-dose influenza PR8 strain live vaccine subcutaneously immunized BALB / c mice;

[0047] Figure 7 Results of immune challenge experiments with live and inactivated influenza vaccines;

[0048] Figure 8 Results of cross-protection and heterologous challenge experiments with live influenza vaccines;

[0049] Figure 9 Immunization challenge experiments for live virus vaccines, recombinant protein vaccines, and VLP vaccines;

[0050] Figure 10 The results of qRT-PCR detection of viral load in various tissues and organs of mice challenged after a single immunization with live viral vaccine, recombinant protein vaccine, and VLP vaccine were obtained.

[0051] Figure 11 The results show the viral load in organs of challenged mice after a single immunization with live viral vaccine, recombinant protein, and VLP booster immunization. Detailed Implementation

[0052] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0053] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0054] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0055] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0056] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0057] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0058] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0060] As noted in the background section, existing influenza vaccines generally suffer from problems such as ineffective single-dose immunization, lack of cellular immunity, insufficient broad-spectrum protection, and high safety risks. This invention provides a live influenza virus vaccine, in which the virus is not attenuated during preparation and is administered via subcutaneous injection, inducing highly effective protection with a single immunization.

[0061] Specifically, in a first aspect, the present invention provides a live influenza virus vaccine, comprising an unattenuated influenza virus.

[0062] The live influenza virus vaccine provided by this invention contains unattenuated influenza virus. It is administered via subcutaneous injection and a single immunization can induce good immune protection.

[0063] Preferably, the live influenza virus vaccine does not contain adjuvant.

[0064] More preferably, the formulation of the influenza virus live vaccine is suitable for subcutaneous injection.

[0065] Attenuated live influenza vaccines, such as AstraZeneca's FluMist and Changchun BCHT's live attenuated vaccines, are nasal spray formulations. However, this invention achieves a subcutaneous injection formulation, overcoming the misconception that traditional live viral vaccines cannot be injected. Furthermore, subcutaneous injection can induce a better immune response or protective effect.

[0066] More preferably, the viral titer of the unattenuated influenza virus is 10. 1 ~10 8 TCID 50 / mL, and the residual amount of host cell DNA is ≤25ng / mL, and the residual amount of host cell protein is ≤100μg / mL.

[0067] Current technology generally holds that live viral vaccines must undergo attenuation to ensure safety. However, this invention directly uses unattenuated wild-type strains, administered subcutaneously, which not only elicits a strong immune response but also does not cause clinical infection symptoms. This discovery breaks through the technical prejudice that "wild-type strains inevitably lead to infection" and overturns the traditional understanding that "live viral vaccines must be attenuated." Experiments have shown that viral replication is strictly limited to draining lymph nodes and the spleen, and is completely cleared within 7 days after inoculation. Furthermore, no viral load or pathological damage was detected in vital organs such as the heart, brain, and kidneys. A single immunization can induce high levels of neutralizing antibodies, and challenge experiments showed a 100% survival rate in mice, indicating a significant immunoprotective effect.

[0068] More preferably, the unattenuated influenza virus is influenza A H1N1, influenza A H3N2, influenza B Victoria lineage and / or influenza B Yamagata lineage.

[0069] Furthermore, in a second aspect, the present invention provides a method for preparing the aforementioned live influenza virus vaccine, such as... Figure 1 As shown, the preparation method is carried out according to the following steps:

[0070] S1. Culture: Influenza virus is inoculated into virus cultures of MDCK cells, Vero cells or chicken embryos and cultured to obtain virus harvest fluid;

[0071] S2. Purification: The virus harvested solution is subjected to deep filtration, ultrafiltration concentration, nuclease treatment and chromatography purification in sequence to obtain the original solution;

[0072] S3. Formulation: The stock solution is sterilized and filtered, and a diluent is added to prepare a liquid formulation, or a preservative is added and then freeze-dried to prepare a freeze-dried formulation.

[0073] Traditional inactivated vaccines (such as Fluzone) require the addition of adjuvants and multiple administrations. This invention, through a combination of serum-free MDCK cell culture and a specific purification process, allows the unattenuated virus to retain its complete antigenicity, inducing more durable humoral and cellular immunity with a single subcutaneous injection. This differs from the conventional understanding that "vaccines require adjuvants to enhance their effect," thus overcoming cognitive biases regarding existing vaccine technologies and achieving adjuvant-free vaccines that induce long-lasting immunity with a single immunization.

[0074] Specifically, when choosing chicken embryo culture, select healthy 9-11 day old chicken embryos, inoculate them with influenza virus, incubate at 33-35℃ for 48-72 hours, collect allantoic fluid, and centrifuge to remove impurities.

[0075] In some preferred embodiments, such as Figure 2 As shown, when influenza virus is inoculated into MDCK cells, the culture in step S1 is performed as follows:

[0076] S11. Wash the culture container with MDCK cells that have grown at 80-100% confluence at least twice with PBS;

[0077] S12. Mix the influenza virus with DMEM-F12 medium without bovine serum to obtain the inoculum solution;

[0078] S13. Add the inoculum to the cleaned culture container and incubate;

[0079] S14, add DMEM-F12 medium without bovine serum and add TPCK trypsin;

[0080] S15. Continue culturing to obtain virus harvest fluid.

[0081] In some specific embodiments, in step S12, the volume ratio of influenza virus to DMEM-F12 culture medium without bovine serum is 1:10 to 1:10000, preferably 1:100.

[0082] In some specific embodiments, in step S13, the incubation is carried out at 30~37°C and 0~5% CO2 for 0.5~3 hours; preferably, it is carried out at 34°C and 5% CO2 for 2 hours.

[0083] In some specific embodiments, in step S14, the addition of TPCK trypsin is to a final concentration of 0~10 μg / mL, preferably 1 μg / mL.

[0084] In some specific embodiments, in step S15, the continued culture time is 24 to 72 hours, preferably 48 hours.

[0085] In some preferred embodiments, such as Figure 3 As shown, the purification in step S2 includes:

[0086] S21. The virus harvested fluid is subjected to deep filtration to obtain filtrate;

[0087] S22. The filtrate is concentrated by ultrafiltration to obtain a concentrated solution;

[0088] S23. Add nuclease and MgCl2 to the concentrated solution, incubate overnight or perform enzyme digestion to obtain enzyme digestion solution;

[0089] S24. After filtering the enzyme digestion solution, perform chromatographic purification, combine the collected chromatographic solutions, and obtain the original solution.

[0090] In some specific embodiments, in step S21, the deep filtration uses a 0.8+0.6μm deep filter.

[0091] In some specific embodiments, in step S22, the ultrafiltration concentration is performed using a hollow fiber column of 100KD~500KD, preferably 300KD.

[0092] In some specific embodiments, in step S23, the addition of nuclease and MgCl2 involves adding nuclease to a final concentration of 100-1000 U / mL, preferably 250 U / mL, and adding MgCl2 to a final concentration of 1-10 mM, preferably 2 mM; the incubation is carried out at 37°C.

[0093] In some specific embodiments, in step S24, the filtration is performed using a 0.45μm filter; the chromatographic purification is performed using Core 700 packing material.

[0094] In some preferred embodiments, step S24, the chromatographic purification includes equilibration, wherein the equilibration buffer is a solution containing 20-60 mM PBS, 0.5-1.5 mM MgCl2, 50-150 mM NaCl, and pH 6.5-8.0.

[0095] Existing technologies suggest that wild-type influenza viruses are prone to mutation during in vitro culture. This invention utilizes MDCK cell culture technology to amplify influenza viruses, maintaining the genetic stability of unattenuated viruses (H1N1 / H3N2) during continuous passage. This overcomes the technical obstacle of "difficulty in industrializing wild-type viruses" and solves the technical problem of "stability of wild-type viruses in cell culture."

[0096] In some specific embodiments, in step S3, the filtration is performed through a 0.22 μm filter.

[0097] It should be noted that in step S3, the diluent or protectant is a commonly used diluent or protectant in the vaccine preparation process in this field. The present invention does not make specific limitations here. For example, the diluent can be PBS, physiological saline or other buffer solutions, and the protectant can be human serum albumin, bovine serum albumin, sucrose, glycine, etc.

[0098] In this invention, the preparation method does not include a toxicity reduction step.

[0099] It should be noted that, in this invention, the attenuation treatment refers to the operation of reducing the virulence of influenza virus through artificial means (such as gene recombination, continuous passage or chemical modification).

[0100] In a third aspect, the present invention provides the use of the described live influenza virus vaccine in the preparation of a medicament for the prevention of influenza virus via subcutaneous injection.

[0101] Specifically, the drug is administered via a single-dose or multi-dose immunization schedule.

[0102] The following detailed embodiments illustrate the influenza virus live vaccine, its preparation method, and its application.

[0103] Example 1

[0104] This embodiment relates to the cultivation, purification, and formulation of a live viral vaccine.

[0105] S1, Cultivation

[0106] S11. Wash the MDCK cells (T175 culture flask) that have grown to 80-100% confluence twice with 10mL PBS;

[0107] S12. Take 100 μl of H1N1 pdm09 and mix it with 10 mL of 0% bovine serum / DMEM-F12 medium to obtain the inoculum solution;

[0108] S13. Add the inoculum to the cleaned T175 bottle and incubate at 34°C and 5% CO2 for 2 hours.

[0109] S14. Add 20 mL of 0% bovine serum / DMEM-F12 and 30 μl of 1 mg / mL TPCK trypsin (final concentration 1 μg / mL).

[0110] In S15 and T175 culture flasks, culture at 34℃ and 5% CO2 for 48 hours until cytopathic effect reaches 90%. Transfer the supernatant to a 50mL centrifuge tube, centrifuge at 3000rpm and 4℃ for 10min, and aliquot into T75 cell culture flasks (approximately 200mL). Store at -80℃.

[0111] S2, Purification

[0112] S21, Deep Filtration:

[0113] Deep filtration was performed using a Sartorius GF 0.80+0.6μm filter. The solution was pre-washed with 200mL, top-washed with 40mL, and approximately 250mL of clear liquid was harvested.

[0114] S22, Ultrafiltration Concentration:

[0115] The clarified liquid was concentrated using ultrafiltration with a 300KD hollow fiber column.

[0116] S23, Nuclease treatment:

[0117] Add 1 KU of nuclease to a final concentration of 250 U / ml; then add 47 μL of 2 M MgCl2 to a final concentration of 2 mM (1:1000 addition), and digest overnight at 2-8°C.

[0118] S24. Chromatographic purification:

[0119] A 0.45μm filter is preferred for filtration;

[0120] Core700 chromatography purification:

[0121] Regeneration: Buffer C (1M NaOH + 30% isopropanol);

[0122] Equilibration: buffer A (40mM PBS, 1mM MgCl2, 100mM NaCl, pH 7.5);

[0123] Sample loading: 5 mL / min, volume approximately 45.08 mL;

[0124] Collection: Approximately 90 mL of eluent

[0125] S3, Preparation

[0126] S31. Combine the collected chromatographic solutions to obtain the original solution;

[0127] S33. Preparation: Filter the stock solution using a 0.22μm syringe filter; add diluent (PBS) according to the concentration and dispense into 200μL×20 vials and 500μL×16 vials, freeze at -80℃ to obtain the H1N1 pdm09 strain live vaccine.

[0128] Example 2

[0129] Unlike Example 1, the virus used in step S12 is the H1N1 PR8 strain.

[0130] Example 3

[0131] Unlike Example 1, the virus used in step S12 is H3N2 influenza A.

[0132] Example 4

[0133] Unlike Example 1, the virus used in step S12 is of the Victoria lineage.

[0134] Example 5

[0135] Unlike Example 1, the virus used in step S12 is of the Yamagata lineage beta.

[0136] Example 6

[0137] Unlike Example 1, in step S24, the equilibration buffer A is a solution containing 20 mM PBS, 0.5 mM MgCl2, and 50 mM NaCl with a pH of 6.5.

[0138] Example 7

[0139] Unlike Example 1, in step S24, the equilibration buffer A is a solution containing 60 mM PBS, 1.5 mM MgCl2, 150 mM NaCl, and pH 8.0.

[0140] Example 8

[0141] The difference from Example 1 is:

[0142] In step S13, the volume ratio of influenza virus to DMEM-F12 medium without bovine serum is 1:1000.

[0143] In step S14, the incubation is carried out at 34°C and 5% CO2 for 1.5 hours.

[0144] In step S15, the TPCK trypsin is added to a final concentration of 5 μg / mL;

[0145] In step S16, the continued culturing time is 72 hours;

[0146] In step S22, the ultrafiltration concentration is performed using a 300KD hollow fiber column;

[0147] In step S23, the addition of nuclease and MgCl2 involves adding nuclease to a final concentration of 500 U / mL and adding MgCl2 to a final concentration of 5 mM; the incubation is carried out at 37°C.

[0148] Example 9

[0149] The difference from Example 1 is:

[0150] In step S13, the volume ratio of influenza virus to DMEM-F12 medium without bovine serum is 1:10000.

[0151] In step S14, the incubation is carried out at 34°C and 5% CO2 for 3 hours;

[0152] In step S15, the addition of TPCK trypsin is to be done to a final concentration of 10 μg / mL;

[0153] In step S16, the continued culturing time is 72 hours;

[0154] In step S22, the ultrafiltration concentration is performed using a 300KD hollow fiber column;

[0155] In step S23, the addition of nuclease and MgCl2 involves adding nuclease to a final concentration of 1000 U / mL and adding MgCl2 to a final concentration of 10 mM; the incubation is carried out at 37°C.

[0156] Example 10

[0157] The difference from Example 1 is:

[0158] In step S13, the volume ratio of influenza virus to DMEM-F12 medium without bovine serum is 1:10.

[0159] In step S14, the incubation is carried out at 37°C for 0.5 hours;

[0160] In step S15, TPCK trypsin was not added;

[0161] In step S16, the continued culturing time is 72 hours;

[0162] In step S22, the ultrafiltration concentration is performed using a 300KD hollow fiber column;

[0163] In step S23, the addition of nuclease and MgCl2 involves adding nuclease to a final concentration of 100 U / mL and adding MgCl2 to a final concentration of 1 mM; the incubation is carried out at 37°C.

[0164] Experimental Example 1

[0165] The quality of the live influenza virus vaccine stock solution prepared in each embodiment was tested.

[0166] Detection methods: The viral titer, residual host cell DNA, and residual host cell protein of the live influenza vaccine stock solution in each embodiment were detected according to the methods in the Chinese Pharmacopoeia.

[0167] The results are shown in Table 1:

[0168] Table 1

[0169]

[0170] Experimental Example 2

[0171] This trial involved a complete protection trial using a single immunization with a high- or medium-dose live vaccine.

[0172] (1) Experimental steps:

[0173] Twenty-five 6-8 week old female BALB / c mice were divided into 5 groups of 5 each. Each group was subcutaneously injected with PR8 strain live vaccine (derived from Example 2) (high-dose group: 5 × 10⁻⁶). 4 TCID 50 / ani, medium-dose group 5×10 3 TCID 50 / only and low-dose group 5×TCID 50 / ani), PBS (nasal drops) control group and PBS (subcutaneous injection) control group;

[0174] 21 days post-immunization, the low, medium, and high dose groups and the PBS (subcutaneous injection) control group received 100×LD50. 50 The process of PR8 strain virus challenge via nasal drops is as follows: Figure 4 As shown in Figure A;

[0175] Monitor weight and survival rate;

[0176] Protection against PR8 strain challenge was performed 28 days post-immunization; viral load in lung / brain / liver / enteroside was detected by qRT-PCR on days 4, 6, 8, and 14 post-challenge.

[0177] (2) Experimental results:

[0178] The changes in body weight of BALB / c mice immunized subcutaneously with high, medium, and low doses of live influenza PR8 strain virus vaccine, compared with those in the PBS (subcutaneous injection) and PBS (nasal drop) control groups, are shown below. Figure 4 As shown in Figure B.

[0179] from Figure 4 The results in Figure B show that: after subcutaneous injection of high, medium, and low doses of influenza PR8 strain live vaccine into BALB / c mice, and subsequent challenge protection experiments were conducted on mice in the PBS (subcutaneous injection) control group and the PBS (nasal drop) control group, the body weight changes in the high, medium, and low dose groups were <5%. Figure 4 (Figure B) No respiratory distress or abnormal activity was observed.

[0180] The results of the challenge protection experiment for the high, medium, and low dose groups, the PBS (subcutaneous injection) control group, and the PBS (nasal drops) control group are as follows: Figure 5 As shown. Among them, Figure 5 Figure A shows the results of weight changes after immune challenge; Figure 5 Figure B shows the survival rate results after immune challenge; Figure 5 Figures C through F show the results of viral load detection in various tissues and organs of mice after challenge by qRT-PCR. Figure C shows the viral load detection results in lung tissue, Figure D shows the viral load detection results in brain tissue, Figure E shows the viral load detection results in liver tissue, and Figure F shows the viral load detection results in intestinal tissue.

[0181] from Figure 5 It can be seen that the protection rate of low, medium and high dose immunization groups in the challenge protection experiment was 100%. Figure 5 (Figure B).

[0182] like Figure 5 As shown, high and medium doses of the live viral vaccine immunization group can effectively protect BALB / c mice from high lethal doses (100×LD50). 50 The virus was infected with the PR8 strain. The PBS (subcutaneous injection) control group died 5 days after challenge, and a large number of viral gene copies were detected in the lungs (small amounts of virus were also found in brain and liver tissues); the medium-dose group (5×10⁻⁶)... 2 TCID 50 The immunized group had normal body weight and 100% survival rate after challenge, and the viral load in lung tissue decreased to 10. 2 ~10 3 copies / μL (PBS (subcutaneous injection) control group was 10 6 ~10 7 copies / μL) Figure 5 Figure C). High-dose group (5×10) 4 TCID 50 After a single immunization (per animal), the protection rate against challenge was 100%, and the viral load in organs such as lung tissue, brain tissue, liver, and intestines was below the detection limit (<10 copies / μL). Figure 5 (Figures C, D, E, and F).

[0183] Conclusion: A single high-dose subcutaneous immunization can achieve complete protection in mice, and the virus can be rapidly cleared, indicating that it can induce good immunogenicity and protective effect.

[0184] Experimental Example 3

[0185] This trial involved a biodistribution study of a live viral vaccine (viral replication limitation and histopathological study).

[0186] (1) Experimental steps:

[0187] Female BALB / c mice aged 6-8 weeks were subcutaneously injected with PR8 strain live vaccine (derived from Example 2) (5×10 4 TCID 50 / ani (subcutaneous immunization group) and intranasal immunization with PR8 strain live virus vaccine (50×TCID) 50 Five animals were included in each of the following groups: (nose-in-nasal immunization group) and PBS control group (NC).

[0188] On days 3 and 7 post-immunization, mice subcutaneously immunized with the PR8 strain live vaccine were dissected, and lung, liver, heart, injection site skin, intestinal tissue, spleen, and lymph nodes were collected. The viral load in each tissue and organ was detected by qRT-PCR. On day 3 post-immunization, tissues and organs from the subcutaneous immunization group, the intranasal immunization group, and the control group were collected to prepare pathological sections and perform HE staining for observation. On days 7, 14, and 21 post-immunization, tissues and organs from the subcutaneously immunized group were collected to prepare pathological sections and perform HE staining for observation.

[0189] (2) Experimental results:

[0190] Experimental results are as follows Figure 6 As shown, Figures A-1 and A-2 are the results of qRT-PCR detection of viral load in various tissues and organs of mice on days 3 and 7 after subcutaneous immunization with PR8 strain live vaccine, respectively; Figure B is the HE staining result of pathological sections of lung tissue from immunized mice; Figure C is the HE staining result of pathological sections of heart tissue from immunized mice; Figure D is the HE staining result of pathological sections of kidney tissue from immunized mice; Figure E is the HE staining result of pathological sections of brain tissue from immunized mice; and Figure F is the HE staining result of pathological sections of intestinal tissue from immunized mice.

[0191] from Figure 6 The results show that: PR8 strain live vaccine (5×10 4 TCID 50 (each animal) was immunized for 3 and 7 days, and the viral load in various tissues and organs was measured. The test results are as follows: Figure 6 Figures A-1 and A-2 show that 3 days post-immunization, low levels of nucleic acid copy numbers (10T) were detected by qRT-PCR in lymph nodes, spleen, and skin at the injection site. 2 ~10 3 copies / μL); completely cleared 7 days after immunization (<10 copies / μL); 1 (copies / μL).

[0192] In addition, the PR8 strain live vaccine was administered via nasal drops (100 LD). 50 ) Infection group and PR8 strain live vaccine subcutaneously (5×10) 4 TCID50 The pathological changes in the lungs of immunized mice (number of mice) were examined, and the results are as follows: Figure 6 Figure B shows that after subcutaneous immunization with the PR8 strain live vaccine, no significant changes were observed in the pathological changes of mouse lung tissue; however, after intranasal infection of mice with the PR8 strain live vaccine... Figure 6 The mice in Figure B showed lung congestion, significant hemorrhage, alveolar thickening, and inflammatory cell infiltration, confirming that the live virus vaccine was confined to the injection site and posed no risk of systemic spread. Furthermore, no significant abnormalities were observed in the pathological examination of the heart, kidneys, brain tissue, and intestines of mice in all groups (e.g., [Figure B]). Figure 6 (Figures C-F) showed no obvious inflammatory cell infiltration.

[0193] Conclusion: After vaccination with PR8 strain live virus, the virus is strictly confined to the injection site and immune organs, cleared within 7 days, and there is no risk of systemic spread.

[0194] Test Example 4

[0195] This experiment involved cross-protection of different strains (H1N1 pdm09 subcutaneous immunization with challenge of H1N1-PR8 strain).

[0196] (1) Experimental steps:

[0197] Twenty-five female Balb / c mice aged 6-8 weeks were subcutaneously injected with a live H1N1 pdm09 virus vaccine (derived from Example 1) (high-dose group 5 × 10⁻⁶). 5 TCID 50 / ani, medium-dose group 5×10 3 TCID 50 / only and low-dose group 50×TCID 50 The study included three groups of animals: a control group (PBS(SC) group) receiving subcutaneous injection of PBS solution and a negative control group (PBS group) receiving nasal drops of PBS solution (without immunization or challenge). Each group consisted of 5 animals.

[0198] On day 21 post-immunization, the low, medium, and high dose groups and the PBS (SC) group were administered PR8 strain virus (100×LD). 50 Nasal droplet infection challenge, respectively recorded as pdm09(50 TCID) 50 SC) / PR8 group, pdm09 (5×10 3 TCID 50 SC) / PR8 group, 5×10 5 TCID 50 The SC / PR8 group and the PBS(SC) / PR8 group were used for challenge with PBS solution and were designated as the PBS / PBS group.

[0199] The body weight and survival rate of all groups were monitored, and the PR8 strain virus (100 LD) was detected by qRT-PCR. 50 The viral load in the lungs of each group after nasal droplet infection challenge.

[0200] (2) Experimental results:

[0201] Experimental results are as follows Figure 7 As shown, where, Figure 7 Figure A shows the changes in body weight of mice challenged after immunization with the H1N1-pdm09 live vaccine; Figure 7 Figure B shows the survival rate of mice challenged after immunization with the H1N1-pdm09 virus live vaccine; Figure 7 Figure C shows the viral load in mouse lung tissue after challenge, detected by qRT-PCR. Figure 7 The D-plot shows the viral load in the heart of mice after challenge, as detected by qRT-PCR.

[0202] The results showed that ( Figure 7 Balb / c mice subcutaneously immunized with the high-dose H1N1 (pdm09) group showed no abnormal weight changes; the medium-dose group experienced a decrease followed by a recovery in weight; and the low-dose group and the PBS (SC) / PR8 group showed a continuous decrease in weight within one week. Figure 7 (Figure A), H1N1pdm09 virus live vaccine (5×10) 5 TCID 50 The survival rate was 100% for each animal, with an 80% survival rate in the medium-dose group, while all animals in the low-dose group and the PBS(SC) / PR8 group died. Figure 7 Figure B); H1N1 pdm09 virus live vaccine (5×10 5 TCID 50 / animal) lung virus load was 10 3 copies / μL (PBS(SC) / PR8 control group 10) 7 copies / μL, reduced by 10,000 times) Figure 7 (Figure C); the viral load in the lungs of the medium-dose group was 10 5 The number of copies / μL was approximately reduced by about 100-fold, and there was no difference between the low-dose group and the PBS(SC) / PR8 control group. Figure 7 (Figure C).

[0203] Conclusion: Live influenza vaccines provide potent cross-protection through the advantage of whole viral components.

[0204] Experimental Example 5

[0205] This trial case involves a protective defect test of inactivated vaccines.

[0206] (1) Experimental steps:

[0207] Twenty-five female Balb / c mice aged 6-8 weeks were divided into groups of five and subcutaneously injected with β-propiolactone-inactivated PR8 strain whole virus vaccine (with a viral titer of 5 × 10⁻⁶ before inactivation). 5 TCID 50 (Ina-PR8(5×10)) 5 TCID 50 (SC group), subcutaneous injection of PR8 strain live vaccine (derived from Example 2) 5×10 5 TCID 50 / group (PR8(5×10)) 5 TCID 50 Group SC) and subcutaneous injection of PR8 strain live vaccine (from Example 2) 5×10 6 TCID 50 / group (PR8(5×10)) 6 TCID 50 The study included two groups: a positive control group (PBS(SC) group) and a negative control group (PBS group) receiving subcutaneous PBS injection and nasal drops without immunization or challenge.

[0208] On day 21 post-immunization, Ina-PR8 (5×10) 5 TCID 50 SC group, PR8 (5×10) 5 TCID 50 SC group, PR8 (5×10) 6 TCID 50 The SC group and the PBS (SC) group used PR8 strain virus (100×LD) 50 Nasal drops for drug administration, recorded as Ina-PR8 (5×10) 5 TCID 50 SC) / PR8 group, PR8 (5×10 5 TCID 50 SC) / PR8 group, PR8 (5×10 6 TCID 50 The SC / PR8 group and the PBS(SC) / PR8 group; the PBS group was challenged with PBS solution and was denoted as the PBS / PBS group.

[0209] The body weight and survival rate of all groups were monitored, and the PR8 strain virus (100×LD) was detected by qRT-PCR. 50 The viral load in the lungs of each group after nasal droplet infection challenge.

[0210] (2) Experimental results:

[0211] Experimental results are as follows Figure 8 As shown, where, Figure 8 Figure A shows the changes in body weight of challenged mice after immunization with PR8 strain whole virus inactivated vaccine and different dose groups of live virus vaccine; Figure 8 Figure B shows the survival rate of mice challenged after immunization with PR8 strain whole virus inactivated vaccine and different dose groups of live virus vaccine; Figure 8 Figure C shows the results of qRT-PCR detection of viral load in mouse lung tissue after challenge.

[0212] Experimental results show that ( Figure 8 ): No abnormalities were observed in the body weight of Balb / c mice subcutaneously immunized with the PR8 strain live vaccine at any dose group, and all mice survived after challenge. Figure 8 Figure A); After a single immunization with the PR8 strain whole virus inactivated vaccine, the patient's weight initially decreased and then recovered, with a survival rate of 60% after challenge. Figure 8 (Figure B), and the viral load in the lung tissue after challenge is >10. 7 copies / μL ( Figure 8 Figure C); All members of the positive control (PBS(SC) / PR8) group died. Figure 8 (Figure B), viral load in lung tissue >10 8 Approximately copies / μL ( Figure 8 (Figure C).

[0213] Conclusion: The PR8 strain live vaccine provides better protection than the whole virus inactivated vaccine.

[0214] Experimental Example 6

[0215] This trial case involves a recombinant protein vaccine that requires booster immunization testing.

[0216] (1) Experimental steps:

[0217] Fifty female Balb / c mice aged 6-8 weeks were used. Five G1 mice were immunized once, subcutaneously injected with PR8-HA trimer (4.5 μg) + adjuvant (CpG + aluminum hydroxide). They were challenged 21 days later (100 LD50). 50 PR8 strain virus challenged via nasal drops); G2: 5 mice were boosted with subcutaneous injections of PR8-HA trimer (4.5 μg) + adjuvant (CpG + aluminum hydroxide) twice (days 0 and 21), and challenged with virus 100×LD for 42 days later. 50 PR8 strain virus challenge via intranasal droplet); G3: 5 mice were immunized once with H1N1 VLP (non-PR8 strain) vaccine (3μg) + adjuvant (CpG + aluminum hydroxide), and challenged 21 days later (100 LD50). 50PR8 strain virus challenge via intranasal instillation); G4: 5 mice were booster immunized twice with H1N1 VLP (non-PR8 strain) vaccine (3μg) + adjuvant (CpG + aluminum hydroxide) (days 0 and 21); 42 days later, 100 LD50 was administered. 50 PR8 strain virus challenge via nasal drops; additional subcutaneous injection of 5×10 5 PR8 virus live vaccine (derived from Example 2) group, PBS solution (subcutaneous injection) positive control group (PBS(SC) group) (100 LD) 50 Two groups (n=5 each) were used to challenge PR8 strain virus via nasal drops, a negative control group (PBS group) with PBS solution (nasal drops), and a challenge group (PBS group) without immunization or challenge. The challenge was performed on days 21 and 42, respectively.

[0218] Monitor body weight and survival rate; detect viral load in lung tissue, heart, brain tissue, and kidneys using qRT-PCR.

[0219] 42 days later, the booster immunization group was given PR8 strain virus (100×LD). 50 Nasal drops for detoxification;

[0220] Monitor body weight and survival rate; detect viral load in lung tissue, heart, brain tissue, and kidneys using qRT-PCR.

[0221] (2) Experimental results:

[0222] Immune challenge and experimental results as follows Figure 9 , Figure 10 and Figure 11 As shown, where, Figure 9 Figure A is a schematic diagram of immune challenge; Figure 9 Figures B-1 and B-2 show the changes in body weight and survival rate of challenged mice after a single immunization (21 days). Figure 9 Figures C-1 and C-2 show the changes in body weight and survival rate of mice after challenge following booster immunization (42 days). Figure 9 The D-plot shows the serum antibody levels 14 days after immunization using a micro-neutralization assay. Figure 9 Figure E shows the antibody levels in serum 21 days after immunization, detected using the micro-cell method. Figure 9 The F-plot shows the serum antibody levels detected by the cell micro-neutralization method 28 days after immunization; Figure 9 The G-plot shows the serum antibody levels detected by the micro-neutralization method 35 days after immunization.

[0223] Experimental results show that ( Figure 9 No anti-HA antibodies were detected after a single immunization with PR8-HA, the survival rate after challenge was 0%, and the neutralizing antibody against PR8 strain was below the detection limit (<1:20). Figure 9(Figure D); After challenge with the virus, the survival rate of mice in both the single-immunized and booster-immunized VLP groups was 0%, and the viral load of influenza virus HA in lung, heart, brain, and kidney tissues was >10. 6 copies / μL ( Figure 10 For the results of a single immunization and Figure 11 (As a result of enhanced immunity). Among them, Figure 10 In the figures, A shows the viral load in the lung tissue of challenged mice after a single immunization; B shows the viral load in the heart of challenged mice after a single immunization; C shows the viral load in the brain tissue of challenged mice after a single immunization; and D shows the viral load in the kidneys of challenged mice after a single immunization. Figure 11 In the figures, A shows the viral load in the lung tissue of mice challenged after booster immunization; B shows the viral load in the heart of mice challenged after booster immunization; C shows the viral load in the brain tissue of mice challenged after booster immunization; and D shows the viral load in the kidneys of mice challenged after booster immunization.

[0224] Following two immunizations with the recombinant protein (Day 35), the anti-HA antibody titer was 1:200, while in the live viral vaccine group, the anti-HA antibody titer was 1:800 on day 35. Viral load in various tissues was measured 21 days post-immunization; the residual viral load in the lungs of the live viral vaccine immunization group was <10. 3 copies / μL, while there was no significant difference between the PR8-HA protein single immunization group, the VLP single immunization group and the positive control (non-immunized group challenged only) (PBS(SC) / PR8) group (10 6 (copies / μL); viral load was measured in tissue samples 42 days post-immunization. The viral load in lung tissue of the live virus vaccine immunization group and the PR8-HA protein booster immunization group was 10 copies / μL). 2 ~10 3 copies / μL, while there was no significant difference between the VLP booster immunization group and the positive control (non-immunized group challenged only) (PBS(SC) / PR8) group (10 copies / μL). 6 (copies / μL).

[0225] Conclusion: Recombinant protein vaccines rely on booster immunization and adjuvants, and their immunoprotective effect is significantly lower than that of live viral vaccines.

[0226] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A live influenza virus vaccine, characterized in that, The live influenza virus vaccine is prepared by adding a diluent or protectant to an unattenuated influenza virus; the diluent is physiological saline or a buffer solution, and the protectant is human serum albumin, bovine serum albumin, sucrose, or glycine; the live influenza virus vaccine is prepared by the following steps: S1. Culture: Influenza virus H1N1 PR8 strain was inoculated into MDCK cells and cultured to obtain virus harvest fluid; S2. Purification: The virus harvested solution is subjected to deep filtration, ultrafiltration concentration, nuclease treatment and chromatography purification in sequence to obtain the original solution; S3. Formulation: The stock solution is sterilized and filtered, and a diluent is added to prepare a liquid formulation, or a preservative is added and then freeze-dried to prepare a freeze-dried formulation. In step S1, the cultivation is performed according to the following steps: S11. Wash the culture container with MDCK cells that have grown at 80-100% confluence with PBS; S12. Mix the influenza virus H1N1 PR8 strain with DMEM-F12 medium without bovine serum to obtain the inoculation solution; S13. Add the inoculum to the cleaned culture container and incubate at 34°C and 5% CO2 for 2 hours. S14, add DMEM-F12 medium without bovine serum and add TPCK trypsin; S15. Continue culturing at 34℃ and 5% CO2 until MDCK cell cytopathic effect reaches 90%. Harvest the virus culture supernatant, centrifuge, and collect the supernatant to obtain the virus harvest solution. In step S2, the purification includes: S21. The virus harvested liquid is subjected to deep filtration to obtain a clear liquid; S22. The clarified liquid is concentrated by ultrafiltration to obtain a concentrated liquid; S23. Add nuclease and MgCl2 to the concentrated solution, and digest overnight at 2-8°C to obtain the digestion solution; S24. The enzyme digestion solution is filtered and purified by chromatography. The collected chromatographic solutions are combined to obtain the original solution. In step S24, the chromatographic purification includes the following steps: First, filter using a 0.45μm filter; Then it is regenerated with buffer C, which consists of 1M NaOH and 30% isopropanol; Then equilibrate with buffer A, which consists of 40 mM PBS, 1 mM MgCl2 and 100 mM NaCl, and the pH of buffer A is 7.5; Then load the sample at a rate of 5 mL / min; Finally, collect the eluent; The formulation of the live influenza virus vaccine is suitable for subcutaneous injection.

2. The live influenza virus vaccine according to claim 1, characterized in that, The live influenza vaccine described does not contain adjuvants.

3. The live influenza virus vaccine according to claim 1, characterized in that, The viral titer of the unattenuated influenza virus was 10. 1 ~10 8 TCID 50 / mL, and the residual amount of host cell DNA is ≤25ng / mL, and the residual amount of host cell protein is ≤100μg / mL.

4. A method for preparing a live influenza virus vaccine according to any one of claims 1 to 3, characterized in that, The live influenza virus vaccine is prepared by adding an unattenuated influenza virus to a diluent or a protective agent; the diluent is physiological saline or a buffer solution, and the protective agent is human serum albumin, bovine serum albumin, sucrose, or glycine; the preparation method is carried out according to the following steps: S1. Culture: Influenza virus H1N1 PR8 strain was inoculated into MDCK cells and cultured to obtain virus harvest fluid; S2. Purification: The virus harvested solution is subjected to deep filtration, ultrafiltration concentration, nuclease treatment and chromatography purification in sequence to obtain the original solution; S3. Formulation: The stock solution is sterilized and filtered, and a diluent is added to prepare a liquid formulation, or a preservative is added and then freeze-dried to prepare a freeze-dried formulation. The cultivation process described in step S1 is performed as follows: S11. Wash the culture container with MDCK cells that have grown at 80-100% confluence with PBS; S12. Mix the influenza virus H1N1 PR8 strain with DMEM-F12 medium without bovine serum to obtain the inoculation solution; S13. Add the inoculum to the cleaned culture container and incubate at 34°C and 5% CO2 for 2 hours. S14, add DMEM-F12 medium without bovine serum and add TPCK trypsin; S15. Continue culturing at 34℃ and 5% CO2 until MDCK cell cytopathic effect reaches 90%. Harvest the virus culture supernatant, centrifuge, and collect the supernatant to obtain the virus harvest solution. The purification described in step S2 includes: S21. The virus harvested liquid is subjected to deep filtration to obtain a clear liquid; S22. The clarified liquid is concentrated by ultrafiltration to obtain a concentrated liquid; S23. Add nuclease and MgCl2 to the concentrated solution, and digest overnight at 2-8°C to obtain the digestion solution; S24. The enzyme digestion solution is filtered and purified by chromatography. The collected chromatographic solutions are combined to obtain the original solution. In step S24, the chromatographic purification includes the following steps: First, filter using a 0.45μm filter; Then it is regenerated with buffer C, which consists of 1M NaOH and 30% isopropanol; Then equilibrate with buffer A, which consists of 40 mM PBS, 1 mM MgCl2 and 100 mM NaCl, and the pH of buffer A is 7.5; Then load the sample at a rate of 5 mL / min; Finally, collect the eluent; The formulation of the live influenza virus vaccine is suitable for subcutaneous injection.

5. The use of the live influenza virus vaccine according to any one of claims 1 to 3 in the preparation of a medicament for the prevention of influenza virus by subcutaneous injection.

6. The application according to claim 5, characterized in that, The drug is administered via a single-dose or multiple-dose immunization program.

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