CN106939355A - A kind of screening of influenza virus attenuated live vaccines strain and authentication method - Google Patents

Abstract

The invention relates to a method for screening and identifying influenza virus attenuated live vaccine strains, and belongs to the technical field of biopharmaceuticals. The present invention researches and develops a method for obtaining attenuated live vaccine candidate strains by constructing and screening a high-density random insertion mutant library mediated by Mu phage transposons, and develops a set of methods for the obtained A technical system for systematic evaluation of attenuated live vaccine strains. These methods can not only be directly applied to the screening and evaluation of influenza virus attenuated live vaccine strains, but also have extensive reference significance for the research and development of other virus attenuated live vaccines.

Description

A screening and identification method for attenuated live vaccine strain of influenza virus

technical field

The invention relates to a method for screening and identifying influenza virus attenuated live vaccine strains, belonging to the technical field of biopharmaceuticals.

Background technique

Influenza is a respiratory infectious disease that seriously threatens human health and public health safety. The main method of preventing and treating influenza at present is that patients apply antiviral drugs to treat and normal population is immunized. However, due to the general drug resistance of influenza viruses that have been circulating in recent years, the curative effect of currently used anti-influenza virus drugs is very limited, and the treatment of influenza faces the situation that there is no drug available. Therefore, vaccination has become the most basic and effective means in the current influenza prevention and control system. There are two kinds of influenza vaccines currently in use, one is an inactivated influenza virus vaccine, and the other is an attenuated live influenza virus vaccine. China uses an inactivated flu vaccine.

Traditional inactivated vaccines use chicken embryos as production substrates, which poses many problems in their application. First of all, there will be some components of chicken embryos left in the influenza vaccine produced from chicken embryos, which often cause more serious side effects in vaccinators, which makes it impossible for some people to receive influenza vaccines at all; in order to reduce the side effects of vaccination , now undergoes a complex purification process to remove egg protein, which in turn increases the time and economic cost of vaccine production. Secondly, the inactivated vaccines currently used are not very effective for infants and the elderly, and the main reason may be that the heterologous antigens in the chicken embryo vaccine disperse the limited immune response ability of these populations. Related to differences in antigenic micromodification in avian and human cells. In addition, when a new type of influenza virus emerges, it generally takes nearly 6 months to produce a new vaccine using the chicken embryo production system of the current vaccine, which often prevents us from producing it in time when facing an influenza epidemic or pandemic and stockpile sufficient vaccines. And some avian influenza viruses may not be able to produce vaccines for this type of virus through chicken embryos at all due to their high lethality to chicken embryos. Due to its advantages in mediating cellular immunity and mucosal immunity, the live attenuated influenza vaccine can provide a very good immune protection effect to the human body. These cold-adapted attenuated vaccines continuously pass the wild-type influenza virus under non-viral physiological conditions, so that the virus acquires a certain mutation during the cold-adapted process, and then selects from the mutated virus that can be used as an attenuated live vaccine. strain. This method is very time-consuming, and the amount of mutant virus that can be obtained by cold adaptation is very limited, resulting in very little selectivity in the candidate screening process. in addition. The mutant virus obtained by cold adaptation often has one or several point mutations in the virus genome. This mutation is very prone to reverse mutations in the subsequent vaccine production and immunization process, which may cause the reversion of the vaccine and cause disease. Nevertheless, the existing cold-adapted attenuated vaccines are all developed by relevant foreign units, and my country does not have its own independent intellectual property rights of attenuated vaccine strains, which hinders the development of live attenuated influenza virus vaccines in China to a certain extent. production and application. It can be seen that the development of a new screening and evaluation system for live attenuated influenza vaccines, the development of attenuated vaccine strains with independent intellectual property rights, and the overcoming of the shortcomings of the traditional attenuated vaccine development system are urgent needs in response to the current severe influenza prevention and control situation. High economic value.

Contents of the invention

The present invention researches and develops a method for obtaining attenuated live vaccine candidate strains by constructing and screening a high-density random insertion mutant library mediated by Mu phage transposons, and develops a set of methods for the obtained A technical system for systematic evaluation of attenuated live vaccine strains. These methods can not only be directly applied to the screening and evaluation of influenza virus attenuated live vaccine strains, but also have extensive reference significance for the research and development of other virus attenuated live vaccines.

To achieve the above object, the present invention adopts the following technical solutions:

A method for screening and identifying influenza virus attenuated live vaccine strains, characterized in that it comprises the following steps:

(1) Using Mu phage transposon-mediated random insertion technology to establish a high-density mutation library of the M gene:

Using the Mu phage transposon-mediated random insertion mutagenesis kit from Finnzymes company to insert a 15nt oligonucleotide sequence of 5'-NNNNNTGCGGCCGCA-3' between each base of the influenza virus A/WSN/1933M gene to obtain High-density mutation library of influenza virus M gene;

(2) Obtain virus mutant library through influenza virus reverse genetics technology:

The plasmid carrying the M gene mutant was transformed into Escherichia coli DH10B competent cells by electroporation, the M gene mutant library plasmid was extracted from the recombinant strain, and then the A/WSN/33H1N1 influenza virus 8 plasmid virus was reverse genetically Scientific operating system to obtain virus mutant library;

(3) Analyze the components of the virus mutant library by second-generation sequencing technology:

Get the virus mutant library virus that step (2) obtains and passage on MDCK cell, utilize TRIzol reagent to extract virus RNA then, and carry out reverse transcription to produce corresponding cDNA according to reverse transcription kit iScriptTM cDNA Synthesis kit to each RNA, Using the cDNA as a template, the specific forward primers 5'-AGCAAAAGCAGGTAGATATT-3', 5'-GGGGCCAAAGAAATAGCACT-3', 5'-TCCTAGCTCCAGTGCTGGTC-3' of the three M genes were used to specifically reverse the inserted sequence of the Vic marker. Perform PCR amplification on the primers, set a repeat of the fluorescently labeled PCR product obtained by PCR and use a 96-capillary 3730xl DNA analyzer for sequencing with the Liz-500 molecular weight standard, and use ABI software to analyze the generated data according to the following standards, removing PCR Non-specific data generated by processes, primers, and sequencing instruments;

(4) Screening candidate strains of influenza virus attenuated live vaccine in mice:

First, the mutant library virus was concentrated by ultracentrifugation, and its titer was determined for subsequent mouse infection experiments. The virus was infected by intranasal instillation of 6-8 week-old C57/B6 mice, respectively. On the second day, the fourth day, the sixth day and the eighth day, the lung tissue of the mouse was collected and homogenized, and the total RNA was extracted from the lung tissue homogenate with TRIzol reagent, and the samples were analyzed according to the method of the above-mentioned step (3). The M gene of the virus is sequenced and analyzed qualitatively and quantitatively, and the candidate strains of attenuated live vaccines are determined according to the presence of different M gene mutant viruses in each sample;

(5) Genetic stability and safety evaluation of attenuated live vaccine candidate strains:

(a) Isolation and phenotypic identification of attenuated live vaccines: firstly, the above-mentioned candidate strains of attenuated live vaccines were monocloned, and W7-757, W7-791 and W7-797 which could replicate effectively in MDCK cells Three viruses were amplified, and the W7-791 virus with better replication ability and lower cytotoxicity was screened out;

(b) Detection of the genetic stability of the attenuated live vaccine: Passage the W7-791 virus in MDCK cells and mice, and determine the sequence of the M gene in the gene sequence of the virus obtained from the cells or mouse lung homogenate, Confirm that the mutation of the M gene of W7-791 virus can be stably inherited;

(c) Safety evaluation of the attenuated vaccine: immunization of 6-8 week-old mice with different titers of W7-791 virus, no weight loss and influenza symptoms were found in the mice; 15-day-old newborn BALB/c mice Different titers of W7-791 or wild-type WSN virus of 10 ⁴ TCID50 were inoculated intranasally, and the body weight and lung lesions of the mice were detected. W7-791-inoculated mice did not observe the same symptoms as wild-type WSN virus-infected mice. Weight loss and lung lesions confirmed that the W7-791 virus was the attenuated live vaccine strain of influenza virus.

Further, the conditions of electroconversion in step (2) are 2.0kV, 200Ω, 25μF.

Further, the specific method for obtaining the virus mutant library in step (2) is: transfer the cultured HEK293T cells to a 6-well culture plate, and when the confluence of the cells reaches 80-90%, according to the operation instructions of the transfection reagent, the Mix the plasmid with the inserted mutant M gene and the plasmid containing the other 7 gene segments of influenza virus in equal amounts, mix with the transfection reagent in proportion, incubate at room temperature for 15 minutes, add dropwise to the HEK293T cell culture medium, and incubate at 37°C with 5% CO2 After culturing in the box for 48 hours, the supernatant of transfected cells was collected, and the virus was inoculated on MDCK cells for amplification. After 48 hours of infection, the virus was collected, and part of the virus was frozen for future use.

Further, PCR enzyme KOD Hot-Start polymerase of Novagen is used in step (3), and the reaction conditions of PCR are pre-denaturation 95°C, 10min; denaturation 95°C, 45s; annealing 52°C, 30s; extension 72°C, 90s; Run 30 cycles; final extension at 72°C for 10 min.

Further, the method for removing non-specific data in step (3) is: (a) all data meet the standard default detection level; (b) because the initial 70bp of the sequence has a strong non-specific background, it is removed; ( c) All sequences are aligned with the DNA sequence corresponding to the M gene of influenza virus; (d) The sequencing data are compared with wild-type A/WSN/1933 virus-infected cells, uninfected virus cells, and different gene libraries. normalization processing.

Further, the criteria for determining the attenuated vaccine candidate strains in step (4) are: the virus can replicate effectively after infection and be cleared by the body in 6-8 days.

Further, the titer in step (5) is 10 ⁶ -10 ⁸ TCID50.

The attenuated live vaccine strain of influenza virus screened by the method of the present invention has been preserved in the China Microbiological Culture Preservation Management Committee Common Microbial Strain Preservation Center, and the address of the preservation unit is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing Institute of Microbiology, deposit number is CGMCC No.13784, classification and name: influenza A virus, deposit date: 2017-02-21. Its M gene has the sequence shown in SEQ ID No.1.

Advantages and beneficial effects of the present invention

Mu phage transposon-mediated random insertion technology can quickly and high-throughput obtain high-density mutant libraries of any gene, combined with the influenza virus reverse genetics operating system, can obtain a large-capacity influenza virus mutant library, the The virus mutant library provides a material basis for screening attenuated vaccine strains of influenza virus. This is better than the traditional method of constructing attenuated vaccine strains through cold adaptation mutation. In addition, the invention comprehensively uses the emerging second-generation high-throughput sequencing technology and the vaccine screening technology for screening virus mutant libraries in mice to obtain attenuated live vaccine candidate strains. This technology also has high-throughput and fast speed. And the characteristics of reliable results can greatly shorten the development cycle of attenuated vaccines. Finally, the present invention has developed a systematic and comprehensive vaccine immune effect evaluation system, which can not only scientifically evaluate the immune protection that vaccines can provide for the body, but also clarify the specific mechanism of immune protection provided by vaccines to a certain extent. Provide assistance for further improvement and optimization.

Description of drawings

Figure 1 Schematic diagram of the establishment of the transposon-mediated M gene fragment mutant influenza virus library;

Figure 2 Gene analysis diagram of the M gene mutant library;

Figure 3. Genotyping results at different times after intranasally infecting mice with the M gene mutant library;

Fig. 4. The distribution overview (A) of the different mutant clones isolated by the primary mutation on the M gene; the above-mentioned M gene mutant plasmid is transfected into a schematic diagram of the change in virus titer in 293T cells (B);

Figure 5. Schematic diagram of the impact of influenza virus on the viability of MDCK cells;

Figure 6. Schematic diagram of the change in virus titer infecting MDCK cells;

Fig. 7. The schematic diagram of the influence of influenza virus strain (W7-791) of the present invention and wild influenza virus strain (WT-WSN) on mice;

Fig. 8. After the mouse is inoculated with the influenza virus strain (W7-791) of the present invention, the schematic diagram of the mouse's resistance to the same subtype influenza virus;

Figure 9. Schematic diagram of the immune response of mice to the influenza virus strain (W7-791) of the present invention.

Among them, (A) in Fig. 1 uses transposon-mediated mutation to randomly insert 15 bases on the influenza virus (A/WSN/1933) M gene fragment to obtain a high-density mutated M gene plasmid library. The obtained M gene mutant plasmid library and other seven wild-type influenza virus gene reverse genetics manipulation plasmids were co-transfected to establish a mutant virus library. Cell supernatants containing mutant virus pools were used to infect MDCK cells or directly infect mice. The lungs and lymphoid organs of infected mice were collected at different time points to isolate the virus. Mutations that lead to virus growth slowdown were identified by genotyping and the in vivo and in vitro infection effects were further evaluated; (B) PCR amplification was performed with gene-specific primers and primers that recognized 15nt inserts, and the obtained PCR products were sequenced. The positions of all mutation points in Curry can be obtained. (C) Influenza mutant libraries can be screened in vivo or in vitro. Essential and non-essential regions of the genome can be identified by comparing genotyping results of unscreened and screened libraries.

In Figure 2, a summary of the results of the genetic analysis of the overall mutation pool of the influenza virus A WSN(H1N1) M1 and M2 proteins (upper part). Columns indicate insertion sites in genes. All mutant pools were transfected into 293T cells and amplified for several rounds in MDCK cells, labeled for passages 1-4. The fluorescence intensity represented by the peak reflects the amount of viral RNA (lower panel).

In Fig. 3, 8 6-8 week old C57/B6 mice were infected with intranasal drops of M gene fragment mutant virus library. The lungs were collected and genotyped at different time points. Each peak represents the amount of viral RNA at the insertion site. PBS-treated and wild-type WSN-infected lung homogenates served as positive controls.

In Fig. 4, the single virus clone (A) screened out from the M gene mutation pool is the distribution overview of 67 different mutant clones isolated on the M gene by primary mutation. (B) The above-mentioned M gene mutant plasmid was transfected into 293T cells, and the cell supernatant was collected and the virus titer was determined.

In Figure 6, MDCK cells were infected with 0.25 MOI of wild-type WSN virus and W7-791 virus to detect virus titers at different time points.

In Fig. 7, (A, C) body weight of mice inoculated with 10 ⁶ , 10 ⁷ or 10 ⁸ TCID50 of W7-791 or wild-type WSN virus; (B, D) virus titers on the fourth and sixth days after inoculation Determination; (E) Mouse body weight monitoring after inoculation of W7-791, WSN or PBS into newborn BALB/c mice.

In Figure 8, (A) Schematic diagram of mouse immunization and virus infection; (BC) 5 mice in each group were immunized with 10 ⁵ PFU of W7-791 or the same volume of PBS by intranasal drip, and then inoculated with 4 times MLD50 of WSN after one month of immunization Viruses, the body weight and survival status of the mice were regularly detected after virus infection; (DE) 5 mice in each group were immunized with 10 ⁵ PFU of W7-791 or PBS by intranasal drip, and one month after the immunization, they were inoculated with 4 times the PR8 virus of MLD50, and the virus The body weight and survival status of the mice were detected regularly after infection. *** represents P-value <0.001.

In Figure 9, (A) detection of virus titer in mouse lung homogenate; (B) detection of HAI activity in immunized mouse serum; (C) detection of anti-influenza virus antibody in immunized mouse serum; (D) microneutralization assay Neutralizing antibody titers in sera of W7-791 immunized mice; (E-F) The sera of W7-791 immunized mice were adopted into non-immunized mice, and lethal doses of WSN and HK68/H3 viruses were inoculated 24 hours later, observed and Record the survival rate of mice at each time point; (G-H) T cells from W7-791 immunized mice were adopted into non-immunized mice, and lethal doses of WSN and HK68/H3 viruses were inoculated 24 hours later, and the time points were observed and recorded. Rat survival rate.

detailed description

Below in conjunction with accompanying drawing, the present invention is specifically described and explained by embodiment:

The specific technical scheme of the novel influenza virus attenuated live vaccine screening and evaluation method established by the present invention is shown in Figure 1, and is specifically described as follows:

1. Using Mu phage transposon-mediated random insertion technology to establish a high-density mutation library of M gene

First, insert 5'-NNNNNTGCGGCCGCA-3 between each base of the influenza virus A/WSN/1933M gene according to the operating steps of the Mu phage transposon-mediated random insertion mutagenesis kit (MGS kit, Finnzymes) from Finnzymes Company 'This 15nt long oligonucleotide sequence, thereby obtaining a high-density mutation library of the influenza virus M gene (as shown in Figure 1A; Figure 2).

2. Obtain virus mutant library through influenza virus reverse genetics technology

The plasmid carrying the M gene mutant was transformed into Escherichia coli (E.coli) DH10B competent cells by electroporation, and the electroporation conditions were 2.0kV, 200Ω, 25μF (ElectroMax™ DH10B, Invitrogen). The M gene mutant library plasmid was extracted from the recombinant strain, and then the A/WSN/33 (H1N1) influenza virus 8 plasmid virus reverse genetics operating system established by Hoffmann et al. (PNAS, 2000) was used to obtain the virus mutant library. The specific method is: the cultured HEK293T cells are transferred to a 6-well culture plate, and when the confluence of the cells reaches 80-90%, according to the operation instructions of the transfection reagent, the plasmid containing the insertion mutant M gene and the other 7 genes of the influenza virus are mixed. The plasmids of the fragments were mixed in equal amounts, mixed with the transfection reagent in proportion, incubated at room temperature for 15 min, added dropwise to the HEK293T cell culture medium, and cultured in a 5% CO2 incubator at 37°C for 48 h, and the transfected cell supernatant was collected, and Viruses were inoculated on MDCK cells for amplification. Virus was collected after 48 hours of infection, and part of the virus was frozen for future use (as shown in Figure 1A).

3. Analysis of virus mutant library components by second-generation sequencing technology

Get the above-mentioned obtained virus mutant library virus and passage on MDCK cells, then utilize TRIzol reagent (Invitrogen) to extract viral RNA, and each RNA is according to the requirement of reverse transcription kit iScriptTM cDNA Synthesiskit (Bio-Rad) operating instructions Reverse transcription was performed to generate the corresponding cDNA. Using the cDNA as a template, use the specific forward primers (5'-AGCAAAAGCAGGTAGATATT-3', 5'-GGGGCCAAAGAAATAGCACT-3', 5'-TCCTAGCTCCAGTGCTGGTC-3') of the three M genes to be specific to the inserted sequence of the Vic marker The positive reverse primer was used for PCR, and the PCR enzyme KOD Hot-Start polymerase of Novagen was used for PCR. The reaction conditions of PCR were pre-denaturation at 95°C for 10 min (1 cycle); denaturation at 95°C for 45 s; annealing at 52°C for 30 s; extension at 72°C for 90 s; running for 30 cycles; and final extension at 72°C for 10 min (1 cycle). Fluorescence-labeled PCR products obtained by PCR (one repetition set) and Liz-500 molecular weight standards (AppliedBiosystems) were sequenced using a 96-capillary 3730xl DNA analyzer (3730xl DNA Analyzer, AppliedBiosystems) (as shown in Figure 1B). The generated data was analyzed using ABI software according to the following criteria, (1) all data met the standard default detection level; (2) the initial 70bp of the sequence had a strong non-specific background, so it was removed; (3) all All the sequences were aligned with the DNA sequence corresponding to the influenza virus M gene; (4) The sequencing data were normalized relative to wild-type A/WSN/1933 virus-infected cells, uninfected virus cells, and different gene library controls This process removes non-specific data generated by the PCR process, primers, and sequencing instruments.

4. Screening of Candidate Strains of Influenza Virus Attenuated Live Vaccine in Mice

In the present invention, we use the mouse model and the above-mentioned second generation sequencing technology to identify the method for the components of the virus mutant library to screen the influenza virus attenuated live vaccine candidate strain (as shown in Figure 1A) from the M gene mutant virus library. First, the mutant library virus was concentrated by ultracentrifugation, and its virus titer was determined for subsequent mouse infection experiments. The virus infected 6-8 week-old C57/B6 mice by intranasal instillation, with 8 mice in each group. The lung tissues of the mice were harvested on the second day, fourth day, sixth day and eighth day after infection, respectively. Homogenate treatment was performed, total RNA was extracted from the lung tissue homogenate with TRIzol reagent, and the virus M gene in the sample was sequenced and quantitatively analyzed according to the method in 3 above. In the experiment, the total RNA extracted from the lung tissue of mice treated with PBS and wild-type WSN virus was used as a control. Utilize the above-mentioned method 3 to carry out qualitative and quantitative analysis to the sequence of M gene in the extracted RNA, to determine the existence situation of different M gene mutant viruses in each sample. We observed three viruses with different replication kinetics (Figure 3). As shown in Figure 3, cluster A viruses, which have effective replication capabilities, may cause disease just like wild-type viruses; cluster B mutant viruses may affect the structure and function of viral genes and proteins due to mutations or cause The virus escapes the loss of the host's immune response function and is severely weakened and grows slowly. Since these viruses can hardly survive in the body, they cannot effectively stimulate the body to generate an immune response, so they are not ideal vaccine candidate strains. In contrast, cluster C viruses, although efficiently replicating in the first six days post-infection, are cleared by the body by day 6-8, at which point the presence of these viruses is barely detectable. Such a virus can stimulate the body to produce a strong immune response, but because it cannot continue to replicate and cannot cause disease, it can be used as a candidate strain for attenuated live vaccines.

5. Genetic stability and safety evaluation of candidate attenuated live vaccine strains

A good attenuated live vaccine needs to be absolutely safe, and its phenotype and genotype need to be stably inherited between generations. Therefore, it is a very important part of this technical system to conduct a systematic and comprehensive safety and genetic stability evaluation of the attenuated vaccine candidate strains obtained through the above screening. To this end, we conducted the following experiments: (1) Isolation and phenotypic identification of attenuated viruses: We first monocloned the above-mentioned cluster C viruses, and obtained 67 virus clones in total. And three strains of viruses W7-757, W7-791 and W7-797 which can effectively replicate in MDCK cells were amplified ( FIG. 4 ). Among them, W7-791 showed better replication ability and lower cytotoxicity (Fig. 5, Fig. 6). Therefore, we preliminarily believe that W7-791 may be an ideal attenuated vaccine candidate strain; (2) Genetic stability test of the attenuated vaccine: In order to ensure that the vaccine will not undergo back mutation and the attenuated vaccine will return to the ancestors, we will W7-791 The virus was passaged a series of times in MDCK cells and mice, and the gene sequence of the virus obtained from the cells or mouse lung homogenate, especially the sequence of the M gene, was determined. We found a mutation in the M gene of the W7-791 virus It can be stably inherited, and no insertion mutation, deletion or back mutation will occur. And with the increase of passage times, the titer of W7-791 virus also gradually decreased. This shows that the mutation and phenotype of w7-791 virus can be inherited stably. (3) Vaccine safety assessment: 6-8 week-old mice were immunized with different titers of W7-791 virus, even when the virus inoculation amount of each mouse was as high as 10 ⁷ TCID50, we did not find that the mice developed body weight Drops and flu symptoms. In contrast, mice infected with 10 ³ TCID50 of wild-type WSN virus developed obvious flu symptoms and lost body weight. The viral load of mice infected with w7-791 at 6 days was 100 times lower than that of mice infected with wild-type WSN virus and H3 subtype virus (Fig. 7A, B, C, D). If we observed the lungs of the mice 4 days after infection, we found that the lungs of the PBS group and W7-791-infected mice had no obvious lesions, while the mice infected with wild-type WSN virus showed severe lung tissue damage. In order to further confirm the safety of W7-791, we intranasally inoculated 15-day-old neonatal BALB/c mice with different amounts (10 ⁶ , 10 ⁷ or 10 ⁸ TCID50) of W7-791 or 10 ⁴ TCID50 of wild-type WSN The detection results of virus, mouse body weight and lung lesions showed that the weight loss and lung lesions of W7-791-inoculated mice were not observed as in wild-type WSN virus-infected mice (Fig. 7E). These results all indicate that the influenza virus mutant strain W7-791 obtained by our screening is a new attenuated strain with limited replication in vitro and in vivo, and has high safety for both adult and newborn mice.

6. Systematic evaluation of the protective effect of candidate vaccine strains

After the candidate vaccine strain is determined, it is necessary to evaluate the immune protection it can provide to the body. Wherein it mainly involves the following contents: (1) Immunological protection detection: immunize mice with W7-791, and in one month after immunization, use 4 times of MLD50 wild-type WSN virus or PR8 virus to infect mice. We found that the mice in the non-immunized group lost weight severely and died during the experiment, while the mice immunized with W7-791 maintained a normal weight and did not show any symptoms of influenza (as shown in Figure 8A-E). (2) Detection of humoral immunity level: Influenza-specific antibodies or virus neutralizing antibodies in serum of immunized mice can be determined by influenza virus hemagglutination inhibition test or virus neutralization test. The results of antibody detection in immunized mice showed that the mice immunized with W7-791 only produced WSN virus-specific antibodies, but no antibodies against PR8 virus, HK68(H3N1), and Wis(H3N2) viruses (as shown in Figure 9A-C) . However, when the serum of W7-791 immunized mice was adoptively transferred to non-immunized mice, when these mice were infected with various viruses, blood clearance of immunized mice could provide partial protection against WSN itself, but could not protect other Virus infection of mice (Fig. 9D-F). This suggests that humoral immunity is not the only source of immunity conferred by the W7-791 strain. (3) Detection of cellular immune response level: adoptively transfer T lymphocytes from W7-791 immunized mice to unimmunized mice, and then infect mice with different wild-type influenza viruses, and observe that the adopted T lymphocytes may be mice The immunity that can be provided to determine the role of T cell immunity in vaccine protection. We found that when T cells from W7-791 immunized mice were adoptively transferred to unimmunized mice, the mice could obtain partial broad-spectrum protection, thereby reducing the mice's ability to be infected by various influenza viruses to a certain extent. The degree of onset and disease symptoms (Fig. 9D-F). This shows that W7-791 can effectively induce the body to produce a protective T cell immune response, which is also in line with the immune characteristics of the attenuated live influenza virus vaccine.

SEQUENCE LISTING

<110> Suzhou Institute of Systems Medicine

<120> A method for screening and identifying influenza virus attenuated live vaccine strains

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Claims (5)

1. a screening and identification method of influenza virus attenuated live vaccine strain, it is characterized in that comprising the following steps: (1) Using Mu phage transposon-mediated random insertion technology to establish a high-density mutation library of the M gene: Using the Mu phage transposon-mediated random insertion mutagenesis kit from Finnzymes company, a 15nt long oligonucleotide sequence of 5'-NNNNNTGCGGCCGCA-3' was inserted between the bases of the M gene of influenza virus A/WSN/1933, Thereby obtaining a high-density mutation library of influenza virus M gene; (2) Obtain virus mutant library through influenza virus reverse genetics technology: The plasmid carrying the M gene mutant was transformed into Escherichia coli DH10B competent cells by electroporation, the M gene mutant library plasmid was extracted from the recombinant strain, and then reversed using the A/WSN/33 H1N1 influenza virus 8 plasmid virus The genetics operating system acquires a library of virus mutants; (3) Analysis of virus mutant library components by second-generation sequencing technology: Get the virus mutant library virus that step (2) obtains and passage on MDCK cell, utilize TRIzol reagent to extract virus RNA then, and carry out reverse transcription to produce corresponding cDNA according to reverse transcription kit iScriptTM cDNA Synthesis kit to each RNA, Using the cDNA as a template, use three specific forward primers of the M gene, the sequences of which are SEQ ID No.2, SEQ ID No.3, and SEQ ID No.4, respectively, and the insertion sequence of the Vic marker is specifically reversed The primers are used for PCR amplification, and the fluorescence-labeled PCR products obtained by PCR are set to repeat once and the Liz-500 molecular weight standard is used for sequencing with a 96-capillary 3730xl DNA analyzer. The generated data is analyzed using ABI software to remove the PCR process, primers and Non-specific data generated by sequencing instruments; (4) Screening candidate strains of influenza virus attenuated live vaccine in mice: First, the mutant library virus was concentrated by ultracentrifugation, and its titer was determined for subsequent mouse infection experiments. The virus was infected by intranasal instillation of 6-8 week-old C57/B6 mice, respectively. The lung tissue of the mice was harvested on the second day, the fourth day, the sixth day and the eighth day and homogenized, and the total RNA was extracted from the homogenate of the lung tissue with TRIzol reagent, and the samples were analyzed according to the method in the above step (3). The M gene of the virus is sequenced and analyzed qualitatively and quantitatively, and the candidate strains of attenuated live vaccines are determined according to the presence of different M gene mutant viruses in each sample; (5) Genetic stability and safety evaluation of attenuated live vaccine candidate strains: (a) Isolation and phenotypic identification of attenuated live vaccines: firstly, the above-mentioned attenuated live vaccine candidate strains were monocloned, and among them, W7-757, W7-791 and W7-797 that could replicate effectively in MDCK cells Three viruses were amplified, and the W7-791 virus with better replication ability and lower cytotoxicity was initially screened; (b) Detection of the genetic stability of the attenuated live vaccine: Passage the W7-791 virus in MDCK cells and mice, and determine the sequence of the M gene in the gene sequence of the virus obtained from the cells or mouse lung homogenate, Confirm that the mutation of the M gene of W7-791 virus can be stably inherited; (c) Safety evaluation of the attenuated vaccine: immunized 6-8 week-old mice with different titers of W7-791 virus, no weight loss and influenza symptoms were found in the mice; 15-day-old newborn BALB/c mice were given Different titers of W7-791 or 10 ⁴ TCID50 of wild-type WSN virus were inoculated intranasally, and the body weight and lung lesions of the mice were detected. W7-791 inoculated mice did not observe the same symptoms as wild-type WSN virus-infected mice. Weight loss and lung lesions confirmed that the W7-791 virus was the attenuated live vaccine strain of influenza virus. 2. The method for screening and identifying influenza virus attenuated live vaccine strains according to claim 1, characterized in that: the electroporation conditions in step (2) are 2.0kV, 200Ω, 25μF. 3. The method for screening and identifying influenza virus attenuated live vaccine strains according to claim 1, characterized in that: the specific method for obtaining the virus mutant library in step (2) is: the cultured HEK293T cells are transferred to 6-well culture In the plate, when the confluence of the cells reaches 80-90%, according to the operation instructions of the transfection reagent, mix the plasmid containing the insertion mutant M gene and the plasmid containing the other 7 gene fragments of influenza virus in equal amounts, and mix them with the transfection reagent in proportion Mix well, incubate at room temperature for 15 min, add dropwise to HEK293T cell culture medium, incubate in 37°C, 5% CO2 incubator for 48 h, collect transfected cell supernatant, inoculate the virus on MDCK cells for amplification, and infect After 48 hours, the virus was collected, and part of the virus was frozen for future use. 4. The method for screening and identification of attenuated live vaccine strains of influenza virus according to claim 1, characterized in that: PCR enzyme KOD Hot-Start polymerase from Novagen is used for PCR in step (3), and the reaction condition of PCR is pre-denaturation 95°C, 10 min; denaturation at 95°C, 45s; annealing at 52°C, 30s; extension at 72°C, 90s; 30 cycles of operation; final extension at 72°C for 10 min. 5. The method for screening and identification of attenuated live vaccine strains of influenza virus according to claim 1, characterized in that: the criteria for determining candidate strains of attenuated live vaccines in step (4) are: the virus can be released in mice after infection The lungs replicate efficiently but are cleared by the body by 6-8 days post-infection.