Heat-resistant H1N1 subtype influenza virus mutant vaccine strain and application thereof

Abstract

The invention relates to a heat-resistant H1N1 subtype influenza virus mutant vaccine strain and application thereof. The invention belongs to the technical field of virus gene engineering, and particularly discloses a heat-resistant H1N1 subtype influenza virus mutant vaccine strain and application thereof. According to the H1N1 subtype influenza virus mutant vaccine strain and the preparation method thereof, the HA protein head region, which is a key region influencing the virus thermal stability, is mutated, the virus thermal stability can be remarkably improved only by mutating three amino acid sites, meanwhile, the virus still has good thermal stability after being inactivated, the H1N1 subtype influenza virus mutant vaccine strain prepared by HA protein point mutation has the preservation number of CCTCC NO: V202471, and the H1N1 subtype influenza virus mutant vaccine strain can be used for preparing the H1N1 subtype influenza virus mutant vaccine strain. And immune animals can be induced to generate high-level HI antibodies.

Description

Technical Field

[0001] The present invention belongs to the technical field of viral genetic engineering, and in particular, relates to a heat-resistant H1N1 subtype influenza virus mutant vaccine strain and application thereof. Background Art

[0002] Influenza is an infectious disease caused by influenza virus, which is characterized by invading the host's respiratory system. Every year, about 1 billion people are infected with influenza, and 300,000 to 500,000 people die from influenza. After a latent period of 1-2 days, humans often develop clinical symptoms characterized by high fever, dry cough, sore throat, headache, general fatigue, muscle and joint pain. Influenza virus is widely prevalent around the world, seriously endangering human health and public health security.

[0003] Influenza virus belongs to orthomyxovirus, which is a single-stranded negative-strand RNA virus. Influenza virus particles are mostly filamentous or spherical, with a diameter of about 80 to 120 nm. Influenza virus RNA mainly includes 8 gene segments, namely neuraminidase (NA), hemagglutinin protein (HA), nucleoprotein (NP), matrix protein (M1), polymerase protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and non-structural protein (NS1). According to the serotype, influenza virus can be divided into four types: A (A), B (B), C (C) and D (D). According to the structure and sequence characteristics of HA and NA, influenza A virus can be divided into 18 HA subtypes and 11 NA subtypes. Since there is no proofreading mechanism in the replication cycle of influenza virus, avian influenza virus replication is prone to mutation, resulting in antigenic drift. At the same time, since the influenza genome is segmented, different subtypes of influenza virus will undergo gene segment recombination when infecting the same host, so influenza virus is very easy to mutate, which increases the difficulty of influenza virus prevention and control.

[0004] Vaccination is considered the best measure to prevent and control influenza infection. Currently, there are three types of approved influenza vaccines, namely inactivated influenza virus vaccine, live attenuated influenza virus vaccine and recombinant influenza virus vaccine. These vaccines exert immune protection by inducing the body to produce neutralizing antibodies against the variable region of HA protein antigen.

[0005] Most vaccines have poor thermal stability and have high temperature requirements during transportation and storage. Cold chain is a necessary condition for vaccines during transportation and storage, but the cost of cold chain is high and the temperature is prone to unexpected conditions during use, which will cause the titer of live vaccine viruses to decrease or inactivate, ultimately resulting in incomplete immunity or immune failure, leading to severe infection after immunization. According to incomplete statistics, nearly 50% of some types of vaccines are eventually discarded due to poor thermal stability. Therefore, improving the thermal stability of vaccine strains and developing heat-resistant vaccines are one of the important directions for the development of new vaccines.

[0006] In order to obtain H1N1 subtype influenza virus with better heat resistance, the patent document "A heat-resistant H1N1 subtype influenza virus mutant and its preparation method and application" with the authorization announcement number CN112111466B obtains a mutant with certain heat resistance by mutating the amino acids at positions 2, 91, 158, 252 and 516 of the HA protein. The present invention can obtain H1N1 subtype influenza virus with better heat resistance by precisely mutating the amino acids at positions 60, 62 and 91 of the HA protein head region. At the same time, after inactivation, the heat-resistant strain is more stable than the parent strain and can be stored at a higher temperature. Most reports on improving the thermal stability of influenza vaccines use the method of adding heat-resistant protective agents. For example, the patent document "A kind of influenza virus subunit vaccine protective agent and its application" with application number CN201510583080.2 discloses a vaccine protective agent with sucrose, arginine, sodium glutamate and genetically recombinant human serum albumin as main ingredients. The mutant strains obtained by the reverse genetics method of the present invention are independent of the vaccine protective agent and have good thermal stability after inactivation. Summary of the invention

[0007] The object of the present invention is to provide a mutant HA mutant protein of H1N1 subtype influenza virus, wherein the protein is shown in SEQ ID NO.2.

[0008] Another object of the present invention is to provide the use of HA mutant protein in preparing heat-resistant H1N1 subtype influenza virus mutant vaccine strain.

[0009] Another object of the present invention is to provide a heat-resistant H1N1 subtype influenza virus mutant vaccine strain, the deposit number of the vaccine strain is: CCTCC NO: V202471.

[0010] The last object of the present invention is to provide the use of the above-mentioned heat-resistant H1N1 vaccine strain in the preparation of a drug for treating or preventing H1N1 subtype influenza virus infection.

[0011] In order to achieve the above object, the present invention adopts the following technical measures:

[0012] The applicant artificially performed site-directed mutagenesis on the HA protein of the H1N1 subtype influenza virus, fusion-expressed the mutant, and rescued the mutant virus. It was found that the H1N1 subtype influenza virus mutant strain rescued using the HA-head-N3 protein mutant had the strongest heat resistance. The HA-head-N3 protein is shown in SEQ ID NO.2.

[0013] The HA-head-N3 protein described above, preferably, the polynucleotide encoding the same is shown in SEQ ID NO.1.

[0014] The expression vector for expressing HA-head-N3 protein is also within the protection scope of the present invention.

[0015] The protection scope of the present invention also includes:

[0016] Use of the above HA-head-N3 protein in preparing a recombinant H1N1 subtype influenza virus strain;

[0017] In the above application, preferably, the virus strain is a heat-resistant virus strain;

[0018] In the above application, preferably, the heat-resistant temperature of the heat-resistant virus strain is ≤56°C; the heat-resistant temperature of the inactivated virus is ≤42°C;

[0019] The above-mentioned application is preferably to co-transfect the plasmid expressing HA-head-N3 protein with other plasmids expressing H1N1 subtype influenza virus PB2, PB1, PA, NA, NP, M and NS into infected cells to complete the assembly and proliferation of the virus.

[0020] A heat-resistant H1N1 subtype influenza virus mutant vaccine strain prepared using HA-head-N3 protein, the deposit number of the vaccine strain is: CCTCC NO: V202471.

[0021] Application of the heat-resistant H1N1 subtype influenza virus mutant vaccine strain in the preparation of a drug for treating or preventing H1N1 subtype influenza virus infection.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] To improve the thermal stability of influenza vaccines, most of them adopt the method of adding heat-resistant protective agents. The traditional method is to avoid vaccine inactivation by filling with substances such as sucrose, arginine, sodium glutamate and genetically recombinant human albumin. The mutant strains obtained by the present invention through reverse genetics methods do not rely on vaccine protective agents, and the inactivated virus has good thermal stability. In recent years, with the development of viral genetic engineering technology, it has also been found that mutations in some sites of the HA protein of the H1N1 subtype influenza virus can enhance the thermal stability of the virus. These sites can be used to enhance the thermal stability of the virus through genetic modification methods, but these sites are random. The present invention mutates the HA protein head region, a key area that affects the thermal stability of the virus. It only needs to mutate 3 amino acid sites to significantly improve the thermal stability of the virus. At the same time, the virus still has good thermal stability after inactivation, and is an ideal strain for the creation of inactivated heat-resistant vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the genetic structure of influenza virus mutants.

[0025] Figure 2 These are the results of the HA thermal stability test of influenza virus mutant strains.

[0026] Figure 3 These are the results of infectivity testing of influenza virus mutant strains after heat treatment.

[0027] Figure 4 This is the cell proliferation curve of the influenza virus mutant strain.

[0028] Figure 5 Inactivated influenza virus stability test.

[0029] Figure 6 Determination of antibody levels to inactivated influenza virus vaccine.

[0030] Figure 7 Evaluation of the protective efficacy of inactivated influenza virus vaccines. DETAILED DESCRIPTION

[0031] The present invention is further explained below in conjunction with the examples and drawings, but the content of the present invention is not limited to the content described in the following examples. The technical solutions described in the present invention, unless otherwise specified, are all conventional technologies in the art, and the reagents or materials, unless otherwise specified, are all derived from commercial channels. The present invention takes the H1N1 subtype influenza virus strain PR8 as an example, and uses the HA-head-N3 provided by the present invention to prepare a heat-resistant vaccine strain. Other commonly used H1N1 subtype influenza virus strains in the art can also be used as vaccine strain carriers to prepare heat-resistant H1N1 subtype influenza virus strains.

[0032] Embodiment 1;

[0033] Construction of HA transcription plasmid for H1N1 subtype influenza virus mutant strain and virus rescue

[0034] 1. HA gene mutation sequence

[0035] Three mutant HA gene sequences (HA-head-N3, HA-stalk-N3, HA-head-P3) were obtained by gene synthesis ( Figure 1 ). The mutation scheme of HA-head-N3 is that the R at position 60 of the head region of the spatial structure of the HA protein is mutated to S, the K at position 62 is mutated to N, and the R at position 91 is mutated to N (the protein sequence of HA-head-N3 is shown in SEQ ID NO.2, and the amino acid sequence encoding it is shown in SEQ ID NO.1). The mutation scheme of HA-stalk-N3 is that the N at position 390 is mutated to D, the N at position 490 is mutated to D, and the N at position 498 is mutated to D. The mutation scheme of HA-head-P3 is that the S at position 53 is mutated to K, the Q at position 68 is mutated to H, and the N at position 71 is mutated to K. The synthesized genes were connected to the cloning plasmid (ph w2000) respectively.

[0036] The cloned plasmid containing the synthetic gene was used as a template, and the mutant HA gene was amplified using the high-fidelity DNA polymerase Invitrogen PlatinumSuper Fi DNA, with the primers: 5′-ATGAAGGCAAACCTA-3′, 5′-AGTAGAAACAAGGGTGTT-3′. The PCR product was detected by agarose gel electrophoresis, and the specific target band was recovered using a DNA gel recovery kit to obtain the mutant HA gene fragment.

[0037] 2. Connection and identification of mutant HA gene transcription plasmid

[0038] A pair of PCR primers (5′-GACGGTACCGTCTCCAATAAC-3′, 5′-CCAACTTCGGAGGTCGAC-3′) were designed and synthesized to amplify all sequences except the HA gene in the transcription plasmid PR8-HA (doi.org / 10.1038 / s41598-019-44813-z).

[0039] Through primer extension, partial HA gene sequences were introduced at both ends of the PCR product, so that the PCR product and the mutant HA gene fragment had a consistent sequence that could be used for homologous recombination. The target band was detected by agarose gel electrophoresis, and the PCR amplified band was purified and recovered by a DNA purification kit to obtain the PR8 strain HA gene transcription plasmid (excluding the HA gene) fragment.

[0040] The mutant HA gene fragment and the PR8 strain HA gene transcription plasmid (excluding the HA gene) fragment were In-fusion connected using the In-Fusion HD Cloning Kit, and transformed into DH5α competent cells. The resistant LB plates were coated and inverted for 16 hours before picking up single colonies for PCR and sequencing identification. The positive colonies were expanded and the plasmids were extracted to obtain the mutant HA gene transcription plasmids HA-head-N3 (the present invention), HA-stalk-N3 (control group), and HA-head-P3 (control group) of the PR8 strain.

[0041] 3. Rescue of mutant viruses

[0042] When MDCK cells were cultured to 80% density, the mutant HA gene transcription plasmid and the remaining 7 gene transcription plasmids of the PR8 strain (pPR8-PB2, pPR8-PB1, pPR8-PA, pPR8-NA, pPR8-NP, pPR8-M and pPR8-NS) (doi.org / 10.1038 / s41598-019-44813-z) were co-transfected into MDCK cells using Lipofectamine 2000. After 6 hours of transfection, the supernatant was discarded and the maintenance medium (serum-free DMEM medium containing 1 μg / ml TPCK-treated pancreatic enzyme) was replaced. After 96 hours, the culture supernatant was collected and filtered with a 0.22 μm filter; then inoculated with 9-10 day old SPF chicken embryos, cultured for 48-72 hours, and the virus chicken embryo allantoic fluid was collected. The point mutation of the virus in the allantoic fluid was verified by PCR and sequencing analysis, and the results showed that the mutation site of the mutant strain was consistent with expectations. Recombinant H1N1 influenza virus strains with HA gene point mutations, rPR8-HA / head-N3 (the present invention), rPR8-HA / stalk-N3 (control group), and rPR8-HA / head-P3 (control group), were obtained.

[0043] The recombinant H1N1 influenza virus strain rPR8-HA / head-N3 of the present invention was sent to the China Center for Type Culture Collection for preservation on August 30, 2024, and was classified and named: H1N1 subtype influenza virus rPR8-HA / head-N3, with a preservation number of: CCTCC NO: V202471, and the address is: Wuhan University, Wuhan, China.

[0044] Embodiment 2:

[0045] Thermal stability test of recombinant H1N1 influenza virus strain with HA gene point mutation

[0046] 1. Determination of HA thermal stability

[0047] 9-day-old SPF chicken embryos were infected with influenza viruses of rPR8-HA / head-N3 (prepared in Example 1 of the present invention), rPR8-HA / stalk-N3 (control group), rPR8-HA / head-P3 (control group) and parent strain PR8 (control group), and allantoic fluid was collected. 100 μL / tube was heat-treated in a 56°C water bath. Three replicates were set up, and the virus allantoic fluid was taken out at 0, 10, 30, 60, 120, and 180 min, respectively, and quickly placed on ice to detect the hemagglutination (HA) titer of the virus. The titer change was statistically analyzed to obtain Figure 2 .

[0048] like Figure 2 As shown, after 10 minutes of treatment, the HA titer of rPR8-HA / head-P3 had dropped to 0, after 30 minutes of heat treatment of rPR8-HA / stalk-N3 and PR8, the HA titer had dropped to 0, and after 180 minutes of heat treatment of rPR8-HA / head-N3, the HA titer dropped by 2.8log2.

[0049] Therefore, compared with the parent PR8 strain, the three mutant viruses showed completely different thermal stability. The thermal stability of the rPR8-HA / head-N3 strain was significantly improved, the thermal stability of the rPR8-HA / head-P3 strain was significantly reduced, and the thermal stability of the rPR8-HA / stalk-N3 strain did not change significantly. Therefore, among the three mutant strains, only the rPR8-HA / head-N3 strain had a significantly improved thermal stability.

[0050] 2. Determination of infectivity of influenza virus mutants after heat treatment

[0051] The allantoic fluid of influenza viruses of rPR8-HA / head-N3 (the present invention), rPR8-HA / stalk-N3 (control group), rPR8-HA / head-P3 (control group) and parent strain PR8 (control group) were treated at 56°C for 5, 15 and 30 min, and the treated viruses were placed in an ice bath. After the ice bath, the samples were inoculated into MDCK cells for TCID 50 Detection.

[0052] TCID 50 Changes, get Figure 3 .like Figure 3 As shown, after 5 min of rPR8-HA / head-P3 treatment, TCID 50 After heat treatment for 15 min, the TCID 50 The TCID 50Decreased by 4log2.

[0053] Therefore, compared with the parent PR8 strain, the three mutant viruses showed different thermal stability, the thermal stability of the rPR8-HA / head-N3 strain was significantly improved, the thermal stability of the rPR8-HA / head-P3 strain was significantly reduced, and the thermal stability of the rPR8-HA / stalk-N3 strain did not change significantly. Therefore, among the three mutant strains, only the thermal stability of the rPR8-HA / head-N3 strain was significantly improved.

[0054] Embodiment 3:

[0055] Determination of proliferation characteristics of recombinant influenza virus rPR8-HA / head-N3 strain

[0056] To analyze whether the point mutation affects the proliferation titer of rPR8-HA / head-N3, the proliferation of rPR8-HA / head-N3 strain and parent PR8 in chicken embryos and cells was compared. The virus was propagated by chicken embryo inoculation, and the chicken embryo 50% infectious dose of rPR8-HA / head-N3 strain and PR8 strain were 10 8.82 and 10 8.64 EID 50 / ml.

[0057] The recombinant virus with an infection dose of 0.001 MOI was used to infect MDCK cells grown to about 70-80% in 12-well plates. After incubation for 2 hours, the cells were washed three times with PBS, and maintenance solution (serum-free DMEM medium containing 1 μg / ml TPCK-treated pancreatic enzyme) was added and cultured at 37°C and 5% CO2. The supernatant of the cells was collected at 0, 12, 24, 36, 48, 60 and 72 hours and stored in a -80°C refrigerator. The virus titer in the cell supernatant at each time point was measured, and the virus growth curve was drawn ( Figure 4 ). The rPR8-HA / head-N3 strain and the PR8 strain had similar growth curves, indicating that the point mutation did not affect the proliferation characteristics of rPR8-HA / head-N3.

[0058] Embodiment 4:

[0059] Virus β-propiolactone inactivation test

[0060] After the rPR8-HA / head-N3 virus allantoic fluid was inactivated by β-propiolactone at a final concentration of 0.1% or 0.2% at 37°C and 4°C for 1 hour, 100 μL was taken to inoculate 5 9-day-old SPF chicken embryos, and the chicken embryo allantoic fluid was harvested and the HA titer was tested. If the HA was negative, it was considered to be completely inactivated. The inactivation test showed that the virus allantoic fluid was completely inactivated by β-propiolactone at a final concentration of 0.2% at 37°C for 1 hour. When the inactivation was complete, the HA hemagglutination titer of the virus allantoic fluid remained unchanged (Table 1).

[0061] Table 1

[0062]

[0063] Embodiment 5:

[0064] Inactivated influenza virus stability test

[0065] The poison price is 10 8 EID 50 The rPR8-HA / head-N3, rPR8-HA / stalk-N3, rPR8-HA / head-P3 and the parental strain PR8 were inactivated and placed in a 42°C incubator. Samples were taken every 10 days to test the HA titer, and three replicates were set for each sample. Figure 5 The results showed that the prepared rPR8-HA / head-N3 still maintained good thermal stability at 42°C after inactivation, and the hemagglutination titer of the virus decreased by less than 2 after 30 days. 1 The hemagglutination titers of the control groups rPR8-HA / stalk-N3, rPR8-HA / he ad-P3 and the parental strain PR8 have increased from the initial 2 8 Down to 2 3 The results are as follows. This indicates that the inactivated rPR8-HA / head-N3 has good thermal stability.

[0066] The oil phase was prepared by using white oil, aluminum stearate and Siben-80 in a volume ratio of 92:2:6. The toxicity of the above preparation was 10 8 EID 50 The rPR8-HA / head-N3, rPR8-HA / stalk-N3, rPR8-HA / head-P3 and the parental strain PR8 were inactivated and heat treated at 42°C for 30 days, and then emulsified at a volume ratio of 3:1 between the virus liquid and the oil phase to obtain an inactivated vaccine, which was used in the following examples.

[0067] Embodiment 6:

[0068] Determination of antibody levels in inactivated influenza virus vaccine

[0069] The rPR8-HA / head-N3, rPR8-HA / stalk-N3, rPR8-HA / head-P3 and parental strain PR8 inactivated vaccines prepared in Example 5 were inoculated into 8-week-old female BALB / c mice (0.2 mL / mouse), 10 mice in each group, and blood was collected every week after immunization to determine the HI antibody level. The results showed that after two weeks of immunization, the average HI antibody level of rPR8-HA / stalk-N3, rPR8-HA / head-P3 and parental strain PR8 inactivated vaccines was lower than 10 3 The average antibody of rPR8-HA / head-N3 inactivated vaccine is 2 7.33 Therefore, the rPR8-HA / head-N3 strain still has good thermal stability after inactivation, and can still induce immune animals to produce high levels of HI antibodies after being stored at high temperature for 30 days ( Figure 6 ).

[0070] Embodiment 7:

[0071] The protection rate of rPR8-HA / head-N3 inactivated vaccine against virus infection:

[0072] The rPR8-HA / head-N3, rPR8-HA / stalk-N3, rPR8-HA / head-P3 and parental strain PR8 inactivated vaccine immunization group prepared in Example 5, 8 weeks, 10 female BALB / c mice each, were immunized according to the method of Example 6. After 14 days of immunization, 10 4 EID 50 / 0.1mL / mouse H1N1 virus was used for the challenge test by nose drops and eye drops. The mice were observed for 7 consecutive days, and the status of the mice was observed and the death rate was recorded every day. Figure 7 ), the results showed that the rPR8-HA / head-N3 inactivated vaccine could still achieve a 90% protection rate after immunization after being treated at 42°C for 30 days, but the protection rates of rPR8-HA / stalk-N3, rPR8-HA / head-P3 and PR8 inactivated vaccines were all lower than 30%. Therefore, the rPR8-HA / head-N3 strain still had good thermal stability after inactivation, and the immunized mice still had a high degree of protection.

Claims

1. An artificially synthesized HA mutant protein, wherein the HA mutant protein is shown in SEQ ID NO.

2.

2. A polynucleotide encoding the mutant protein according to claim 1.

3. The polynucleotide according to claim 2, wherein the sequence of the polynucleotide is shown in SEQ ID NO.

1.

4. An expression vector for expressing the mutant protein according to claim 1.

5. Use of the mutant protein according to claim 1, the polynucleotide according to claim 2 or the expression vector according to claim 4 in preparing a recombinant H1N1 subtype influenza virus strain.

6. The use according to claim 5, wherein the virus strain is a heat-resistant virus strain.

7. The use according to claim 6, wherein the heat-resistant temperature of the heat-resistant virus strain is ≤56°C; and the heat-resistant temperature of the inactivated virus is ≤42°C.

8. The use according to claim 5, wherein the use process comprises co-transfecting a plasmid expressing HA-head-N3 protein with other plasmids expressing H1N1 subtype influenza virus PB2, PB1, PA, NA, NP, M and NS into infected cells to complete the assembly and proliferation of the virus.

9. A heat-resistant H1N1 subtype influenza virus mutant vaccine strain prepared using the protein of claim 1, wherein the deposit number of the vaccine strain is: CCTCC NO: V202471.

10. Use of the heat-resistant H1N1 subtype influenza virus mutant vaccine strain according to claim 9 in the preparation of a vaccine for treating or preventing H1N1 subtype influenza virus infection.

Images