CN-121337972-B - Influenza virus live vaccine, preparation method and application

CN121337972BCN 121337972 BCN121337972 BCN 121337972BCN-121337972-B

Abstract

The invention relates to the technical field of biology, and particularly provides an influenza virus live vaccine, a preparation method and application thereof. Aims at solving the problems of invalid single immunization, cell immune deficiency, insufficient broad-spectrum protection and high safety risk of the influenza vaccine in the prior art. To this end, the invention provides a live influenza virus vaccine comprising influenza virus that has not been attenuated. The influenza virus live vaccine breaks through the safety limit of the traditional attenuated live vaccine, and the invention proves that the influenza epidemic strain (such as H1N1 PR8 strain) which is not attenuated can realize safe and efficient immune response through subcutaneous inoculation for the first time, thereby providing a brand-new technical path for developing the influenza vaccine.

Inventors

ZHOU RONG
SU XIAOBO
GAO WENJUAN
ZHOU CHENGXING
LI LEI
MA SHUZHI
TIAN XINGUI
LIU TIANTIAN
WANG YU
SHE WEIBIN
LI XIAO

Assignees

呼研所生物安全科技(广州)股份有限公司
厦门联合呼吸健康研究院

Dates

Publication Date: 20260508
Application Date: 20251216

Claims (6)

1. The influenza virus live vaccine is characterized by being prepared by adding diluent or protective agent into influenza virus which is not subjected to attenuation treatment, wherein the diluent is physiological saline or buffer solution, the protective agent is human blood albumin, bovine blood albumin, sucrose or glycine, and the influenza virus live vaccine is prepared by the following steps: S1, culturing, namely inoculating an influenza virus H1N1 PR8 strain into MDCK cells, and culturing to obtain a virus harvest liquid; s2, purifying, namely sequentially carrying out deep filtration, ultrafiltration concentration, nuclease treatment and chromatographic purification on the virus harvest liquid to obtain a stock solution; s3, sterilizing and filtering the stock solution, adding a diluent to prepare a liquid preparation or adding a protective agent to prepare a freeze-dried preparation after freeze-drying; Wherein, in step S1, the culture is operated as follows: s11, cleaning a culture container growing with MDCK cells with 80-100% confluence by using PBS; S12, mixing an influenza virus H1N1 PR8 strain with a DMEM-F12 culture medium without bovine serum to obtain an inoculation liquid; s13, adding the inoculation liquid into a cleaned culture container, and incubating for 2 hours at 34 ℃ and 5% CO 2 ; s14, supplementing a DMEM-F12 culture medium without bovine serum, and adding TPCK pancreatin; S15, continuously culturing under the conditions of 34 ℃ and 5% CO 2 until the MDCK cell lesion reaches 90%, harvesting virus culture supernatant, centrifuging, and taking the supernatant to obtain a virus harvest liquid; in step S2, the purification includes: s21, carrying out deep filtration on the virus harvest liquid to obtain a clarified liquid; s22, performing ultrafiltration concentration on the clarified liquid to obtain concentrated solution; S23, adding nuclease and MgCl 2 into the concentrated solution, and performing enzyme digestion at 2-8 ℃ overnight to obtain an enzyme digestion solution; S24, filtering the enzyme cutting liquid, performing chromatographic purification, and combining chromatographic collection liquid to obtain a stock solution; in step S24, the chromatographic purification includes the steps of: First, filtration was performed using a 0.45 μm filter; Then regenerating with buffer C, wherein the buffer C consists of 1M NaOH and 30% isopropanol; Then balancing by using buffer A, wherein the buffer A consists of 40mM PBS, 1mM MgCl 2 and 100mM NaCl, and the pH of the buffer A is 7.5; Then loading at a rate of 5 mL/min; Finally collecting the eluent; The preparation form of the influenza virus live vaccine is suitable for subcutaneous injection.
2. The live influenza virus vaccine of claim 1, wherein the live influenza virus vaccine is devoid of an adjuvant.
3. The live influenza virus vaccine according to claim 1, wherein the non-attenuated influenza virus has a viral titer of 10 1 ～10 8 TCID 50 /mL and a host cell DNA residue of 25ng/mL or less and a host cell protein residue of 100 μg/mL or less.
4. The preparation method of the influenza virus live vaccine according to any one of claims 1-3, which is characterized in that the influenza virus live vaccine is prepared by adding diluent or protective agent into influenza virus which is not subjected to attenuation treatment, wherein the diluent is normal saline or buffer solution, the protective agent is human blood albumin, bovine blood albumin, sucrose or glycine, and the preparation method is operated according to the following steps: S1, culturing, namely inoculating an influenza virus H1N1 PR8 strain into MDCK cells, and culturing to obtain a virus harvest liquid; s2, purifying, namely sequentially carrying out deep filtration, ultrafiltration concentration, nuclease treatment and chromatographic purification on the virus harvest liquid to obtain a stock solution; s3, sterilizing and filtering the stock solution, adding a diluent to prepare a liquid preparation or adding a protective agent to prepare a freeze-dried preparation after freeze-drying; wherein, the culture in the step S1 is operated according to the following steps: s11, cleaning a culture container growing with MDCK cells with 80-100% confluence by using PBS; S12, mixing an influenza virus H1N1 PR8 strain with a DMEM-F12 culture medium without bovine serum to obtain an inoculation liquid; s13, adding the inoculation liquid into a cleaned culture container, and incubating for 2 hours at 34 ℃ and 5% CO 2 ; s14, supplementing a DMEM-F12 culture medium without bovine serum, and adding TPCK pancreatin; S15, continuously culturing under the conditions of 34 ℃ and 5% CO 2 until the MDCK cell lesion reaches 90%, harvesting virus culture supernatant, centrifuging, and taking the supernatant to obtain a virus harvest liquid; The purification of step S2 comprises: s21, carrying out deep filtration on the virus harvest liquid to obtain a clarified liquid; s22, performing ultrafiltration concentration on the clarified liquid to obtain concentrated solution; S23, adding nuclease and MgCl 2 into the concentrated solution, and performing enzyme digestion at 2-8 ℃ overnight to obtain an enzyme digestion solution; S24, filtering the enzyme cutting liquid, performing chromatographic purification, and combining chromatographic collection liquid to obtain a stock solution; in step S24, the chromatographic purification includes the steps of: First, filtration was performed using a 0.45 μm filter; Then regenerating with buffer C, wherein the buffer C consists of 1M NaOH and 30% isopropanol; Then balancing by using buffer A, wherein the buffer A consists of 40mM PBS, 1mM MgCl 2 and 100mM NaCl, and the pH of the buffer A is 7.5; Then loading at a rate of 5 mL/min; Finally collecting the eluent; The preparation form of the influenza virus live vaccine is suitable for subcutaneous injection.
5. The use of an influenza virus live vaccine according to any one of claims 1 to 3 in the manufacture of a medicament for the prophylaxis of influenza virus by subcutaneous injection.
6. The use according to claim 5, wherein the medicament is administered in a single dose or in a multiple dose immunization schedule.

Description

Influenza virus live vaccine, preparation method and application Technical Field The invention relates to the technical field of biology, and particularly provides an influenza virus live vaccine, a preparation method and application thereof. Background Influenza (Influenza) is a disease of the respiratory tract and other organs caused by the Influenza virus, and is a disease that is usually acute and infectious in healthy children and adults, with different degrees of epidemic each year in spring and winter, and even in other seasons. Influenza viruses are causative agents of influenza, and belong to the minus-strand single-stranded RNA viruses whose genome consists of a total of 8 independent RNA fragments (designated as fragments 1-8, respectively), with a total nucleic acid length of about 13.6kb. These 8 fragments together encode 10 proteins, 8 of which are structural proteins, including PB1, PB2, PA, HA, NA, NP, M1, M2, while NS1, NS2 are non-structural proteins. Influenza viruses are classified into human influenza viruses and animal influenza viruses, and human influenza viruses are classified into three types of a (a), B (B), and C (C). Viral replication relies primarily on viral ribonucleoprotein (vRNPs). The ribonucleoprotein of influenza a virus consists of viral RNA, RNA polymerase (RdRp) complex and Nucleoprotein (NP), which is the smallest replicative unit of the virus on which the viral proteins can be expressed. The RdRp in vRNPs structure consists of 3 subunits (PA, PB2, PB 1), PB1 is located in the core of the trimer, and its N-and C-termini form stable protein complexes with the C-terminus of the PA subunit and the N-terminus of the PB2 subunit, respectively, via non-covalent bonds (e.g.hydrophobic interactions, hydrogen bonding, van der Waals forces, etc.). Although various antiviral drugs can be used for treating influenza viruses, the influenza viruses are rapidly changed, so that the influenza is sporadic and epidemic and outbreak occur every year worldwide, and the related diseases caused by influenza virus infection can be effectively reduced by correctly inoculating influenza vaccines. Currently, existing influenza vaccines have the following significant limitations: ① The inactivated vaccine has weak immunogenicity, can excite effective response only by relying on an adjuvant, has short duration of induced antibodies, can not activate effective cellular immune response, and has limited immune protection effect; ② Attenuated live vaccines can simulate natural infection, but have the risk of virulence reversion, possibly cause respiratory symptoms, and are currently limited to mucosal inoculation routes; ③ The recombinant protein vaccine, the split vaccine and the VLP vaccine all need more than 1 dose of booster immunity to achieve basic protection, and have high dependence on antigen matching property and weak cross protection capability on heterologous strains. In conclusion, the influenza vaccine in the prior art generally has the problems of poor single immunization effect, insufficient cellular immunity, poor broad-spectrum protection effect and the like. Accordingly, there is a need in the art for a new solution to the above-mentioned technical problems. Disclosure of Invention The invention aims to solve the technical problems that the influenza vaccine in the prior art generally faces the problems of poor single immunization effect, insufficient cellular immunity, poor broad-spectrum protection effect and the like. In a first aspect, the invention provides a live influenza virus vaccine comprising an influenza virus that has not been attenuated. In the preferred technical scheme of the influenza virus live vaccine, the influenza virus live vaccine does not contain an adjuvant. In the preferred technical scheme of the influenza virus live vaccine, the preparation form of the influenza virus live vaccine is suitable for subcutaneous injection. In the preferred technical scheme of the influenza virus live vaccine, the virus titer of the influenza virus which is not subjected to the attenuation treatment is10 1~108TCID50/mL, the DNA residue of the host cell is less than or equal to 25ng/mL, and the protein residue of the host cell is less than or equal to 100 mug/mL. In the preferred technical scheme of the influenza virus live vaccine, the influenza virus which is not subjected to attenuation treatment is H1N1, H3N2, victoria pedigree B and/or Yamagata pedigree B. In a second aspect, the invention provides a method for preparing the influenza virus live vaccine, which comprises the following steps: S1, culturing, namely inoculating influenza virus into virus cultures of MDCK cells, vero cells or chick embryos, and culturing to obtain virus harvest liquid; s2, purifying, namely sequentially carrying out deep filtration, ultrafiltration concentration, nuclease treatment and chromatographic purification on the virus harvest liquid to obtain a stock solution; and S3, sterilizing a