H5N6 Recombinant Influenza Virus, A Composition For Preparing The Same, And A Vaccine Composition Containing The Same

Abstract

The present invention relates to an mammalian non-pathogenic, highly-productive-in-embryonated-egg, heat-resistant, and attenuated clade 2.3.4.4c H5N6 recombinant influenza A virus, and a vaccine composition including the recombinant influenza virus and artificial H5 gene as active ingredients.

Claims

1. An H5N6 recombinant influenza virus, comprising one or more proteins selected from the group consisting of: a hemagglutinin (HA) protein of influenza virus H5N6 strain, wherein the hemagglutinin protein is a hemagglutinin protein in which the 103.sup.rd amino acid from the N-terminus is mutated in the amino acid sequence of SEQ ID NO: 1; neuraminidase (NA) of H5N6 strain; polymerase subunit B2 (PB2) of low pathogenic influenza virus; polymerase subunit B1 (PB1) of influenza virus H1N1 strain; polymerase subunit A (PA) of influenza virus H1N1 strain; nucleocapsid (nucleoprotein: NP) of influenza virus H1N1 strain or influenza virus H5N1 strain; matrix protein (matrix: M) of influenza virus H1N1 strain or low pathogenic influenza virus; and nonstructural protein (NS) of influenza virus H1N1 strain or influenza virus H9N2.

2. The H5N6 recombinant influenza virus of claim 1, wherein in the hemagglutinin protein, histidine (H), which is the 103.sup.rd amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1, is mutated to tyrosine (Y).

3. The H5N6 recombinant influenza virus of claim 1, wherein the low pathogenic influenza virus is low pathogenic influenza virus 01310 strain.

4. The H5N6 recombinant influenza virus of claim 1, wherein the influenza virus H1N1 strain is A/Puerto Rico/8/34(PR8).

5. The H5N6 recombinant influenza virus of claim 1, wherein the influenza virus H5N1 strain is A/wild duck/Korea/SNU50-5/2009.

6. The H5N6 recombinant influenza virus of claim 1, wherein the influenza virus H9N2 strain is A/chicken/Korea/KBNP-0028/00.

7. The H5N6 recombinant influenza virus of claim 1, wherein the H5N6 recombinant influenza virus is a virus deposited as Accession No. KCTC14261BP.

8. The H5N6 recombinant influenza virus of claim 1, wherein the H5N6 recombinant influenza virus is a virus deposited as Accession No. KCTC14391BP.

9. A composition for preparing an H5N6 recombinant influenza virus, comprising: a polynucleotide encoding a hemagglutinin protein (HA) of influenza virus H5N6 strain, wherein hemagglutinin protein is a hemagglutinin protein in which the 103.sup.rd amino acid from the N-terminus is mutated in the amino acid sequence of SEQ ID NO: 1; a polynucleotide encoding neuraminidase (NA) of H5N6 strain; a polynucleotide encoding polymerase subunit B2 (PB2) of low pathogenic influenza virus; a polynucleotide encoding polymerase subunit B1 (PB 1) of influenza virus H1N1 strain; a polynucleotide encoding polymerase subunit A (PA) of influenza virus H1N1 strain; a polynucleotide encoding nucleocapsid (nucleoprotein: NP) of influenza virus H1N1 strain or influenza virus H5N1 strain; a polynucleotide encoding matrix protein (matrix: M) of influenza virus H1N1 strain or low pathogenic influenza virus; and a polynucleotide encoding nonstructural protein (NS) of influenza virus H1N1 strain or influenza virus H9N2.

10. The composition of claim 9, wherein the polynucleotide is comprised in a vector.

11. A cell transformed with the composition for preparing an H5N6 recombinant influenza virus according to claim 9.

12. The cell of claim 11, wherein the cell is selected from the group consisting of A549, 293T, MDCK, Vero, DF1, PK15, and ST1 cells.

13. A vaccine composition, comprising the H5N6 recombinant influenza virus of claim 1.

Description

DESCRIPTION OF DRAWINGS

[0065] FIG. 1 is a schematic diagram of the genome combination of an mammalian non-pathogenic, highly-productive-in-embryonated-egg, heat-resistant, and attenuated clade 2.3.4.4c H5N6 recombinant influenza A virus.

[0066] FIG. 2 is a result of confirming the proliferation of rH5N6-310PB2 and rH5N6-H103Y-310PB2 viruses in MDCK cells or A549 cells, which are mammalian cells.

[0067] FIG. 3 is a result of confirming the heat resistance of rH5N6-310PB2 and rH5N6-H103Y-310PB2 viruses.

[0068] FIG. 4 is a result of confirming the acid resistance of rH5N6-310PB2 and rH5N6-H103Y-310PB2 viruses.

[0069] FIG. 5 is a result of confirming the proteins of rH5N6-H103Y-310PB2 and rH5N6-310PB2 in embryonated eggs through SDS-PAGE to measure the antigen amount.

MODES OF THE INVENTION

[0070] Hereinafter, the present invention will be described in detail by exemplary embodiments. However, the following exemplary embodiments are only illustrative of the present invention, and the present invention is not limited by the following exemplary embodiments.

EXAMPLE 1

Construction and Characteristic Analysis of Recombinant Virus Having HA and NA Genomes of Highly Pathogenic H5N6 Avian Influenza Virus and PB2 Genome Segment of 01310

[0071] 1-1. Construction of HA Genomic Plasmid with Mutated HA Protein of Influenza Virus

[0072] By collecting the sequences of the HA and NA proteins of the highly pathogenic avian influenza virus H5N6 which was isolated in Korea, the HA and NA genome sequences with the highest coincidence were selected, and the cleavage site was changed to ASGR to synthesize the HA gene and the NA gene, from which pathogenicity was removed, respectively, and these were cloned into a Hoffman vector (Patent No. 0862758). In the case of the synthesized HA protein of H5N6, in order to substitute the 103.sup.rd amino acid from histidine to tyrosine, the nucleotide constituting the codon of the corresponding amino acid in the HA genomic gene was substituted from CAC to TAC, and a set of about 30 bp complementary primers having the same sequence in both directions was prepared around the same. By using primers and the Muta-Direct Site Directed. Mutagenesis Kit (iNtRon Co., South Korea), the HA genome cloning vector plasmid of the synthesized H5N6 virus, in which the genome of the 103.sup.rd amino acid portion of the HA protein was substituted from CAC to TAC, was constructed.

[0073] 1-2. Production of Recombinant Virus

[0074] For the construction of recombinant influenza virus, Dr. Hoffman's reverse genetics vector system (Patent No. 0862758) was used.

[0075] Specifically, the Hoffman vector into which the HA and NA genome fragments of the synthesized highly pathogenic H5N6 avian influenza virus were cloned (Patent No. 0862758), the Hoffman vector into which the 01310 PB2 genome fragment was cloned (Patent No. 0862758), and the Hoffman vector into which PB1, PA, NP, M, and NS genome fragments of PR8 were cloned (Patent No. 0862758), the Hoffman vector into which the 01310 M genomic fragment was cloned, the Hoffman vector into which the SNU50-5 NP genomic fragment was cloned, and the Hoffman vector plasmid into which the 0028 NS fragment was cloned were prepared.

[0076] 293T cells (Life Resources Center, KCTC) were suspended in DMEM (GIBCO BRL) medium containing 5% (v/v) FBS in a 6-well cell culture vessel, added to each well, and attached for 24 hours. After removing the medium, 0.8 mL of Opti-MEM medium (Invitrogen Co., USA) was added.

[0077] All of the 8 prepared plasmids were placed in an amount of 300 ng each in one 1.5 mL tube, and the Opti-MEM medium was added to final 2.5 μL. In another 1.5 mL tube, 6 μL of the plus reagent (Invitrogen Co., USA) and 69 μL of the Opti-MEM medium were added and mixed, and after mixing by adding to a 1.5 mL tube containing the plasmids, it was reacted at room temperature for 15 minutes.

[0078] After 15 minutes of the reaction, 4 μL of lipofectamine (Invitrogen Co.) and 96 μL of Opti-MEM were mixed, and by taking 100 μL, it was added to the tube with the plasmids, followed by further reaction for 15 minutes. 200 μL, of the obtained reaction product was added to each well containing the 293T cells. After incubating the 6-well culture vessel at 5% CO2 and 37° C. for 20 hours, 10 μg of trypsin (2.5 μg/μL) per well and 1 mL of the opti-MEM medium were added, and the supernatant was harvested after 24 hours to inoculate 200 μL of the harvested stock solution into 10 to 11-day-old SPF embryonated eggs (Sunrise Co., NY) by the allantoic route. After incubating the inoculated embryonated eggs at 37° C. for 3 days, the allantoic fluid was harvested to determine whether hemagglutination occurred, and as a result, all showed positive hemagglutination. The hemagglutination titer of this recombinant virus was measured and diluted 100 times, and the virus (E2) proliferated in embryonated eggs by the same method was stored at −70° C. and used in the experiment.

TABLE-US-00001 TABLE 1 Recombinant virus HA NA PB2 PB1 PA NP M NS rH5N6 H5 N6 PR8 PR8 PR8 PR8 PR8 PR8 rH5N6-H103Y H5-H103Y N6 PR8 PR8 PR8 PR8 PR8 PR8 rH5N6-310PB2 H5 N6 01310 PR8 PR8 PR8 PR8 PR8 rH5N6-IG H5 N6 01310 PR8 PR8 50-5 01310 0028 rH5N6-H103Y-310PB2 H5-H103Y N6 01310 PR8 PR8 PR8 PR8 PR8 rH5N6-H103Y-IG H5-H103Y N6 01310 PR8 PR8 50-5 01310 0028

[0079] 1-3. Measurement of Viral Titer

[0080] In order to measure the proliferation titer (50% embryo infection dose, EID.sub.50/mL) of the recombinant viruses (E2) in chicken embryos, each of the recombinant viruses was diluted in decimal by 10.sup.−1 to 10.sup.−9 with a phosphate buffer solution and inoculated by 100 μL into five 10 to 11-day-old SPF embryonated eggs for each dilution factor by the allantoic route. Then, after 3 days of incubation, the allantoic fluid was harvested, and the viral titer (EID.sub.50/mL) was measured according to the calculation formula of the Spearman-Karber method by determining whether hemagglutination occurred with red blood cells of the chicken.

[0081] 1-4. Comparison of Proliferative Properties in 10-Day-Old Embryonated Eggs

[0082] Based on the viral titer (EID.sub.50/mL (log 10)) obtained in Example 1-3 above, the virus at 100 EID.sub.50 was inoculated by 100 μL into each of five 10-day-old embryonated eggs by the allantoic route. Then, after 3 days of incubation, the allantoic fluid was harvested, and EID.sub.50/mL was measured in the same manner as above, and the result of comparing the proliferative properties in embryonated eggs is shown in Table 1 below. Looking at the viral titer (EID.sub.50/mL (log 10)), it was confirmed that the proliferation titer in embryonated eggs showed high proliferation of more than 10.sup.9.0 EID.sub.50/mL in all four viruses, rH5N6, rH5N6-H103Y, rH5N6-310PB2, and rH5N6-H103Y-310PB2. The rH5N6-IG and rH5N6-H103Y-IG viruses with modified NP, M, and NS genes also showed proliferative properties of about 10.sup.9.0 EID.sub.50/mL.

TABLE-US-00002 TABLE 2 Recombinant virus EID.sub.50/mL (log 10) rH5N6 9.08 ± 0.14 rH5N6-H103Y 9.03 ± 0.31 rH5N6-310PB2 9.33 ± 0.29 rH5N6-IG 9.25 ± 0.25 rH5N6-H103Y-310PB2 9.58 ± 0.14 rH5N6-H103Y-IG 8.92 ± 0.38

[0083] 1-5. Confirmation of Proliferation of Recombinant Virus in Mammalian Cells

[0084] In order to compare whether each recombinant virus proliferates in mammalian cells, the growth curves in the Madin-Darby Canine Kidney (MDCK) cell line and the A549 cell line were determined. In the case of the MDCK cell line, 10% fetal bovine serum (FBS) was added to Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies Co., CA, USA) and maintained, and in the case of the A549 cell line, 10% FBS was added to DMEM/F12 (Life Technologies Co., CA, USA) medium and maintained. Two types of the cell lines were each formed into a mono-layer on a 12-well cell culture plate, and then, 5×10.sup.5/0.5 mL of each recombinant virus was inoculated, and 100 μL of the supernatant was obtained every 0, 24, 48, and 72 hours. The obtained supernatant was diluted in decimal by 10.sup.−1 to 10.sup.−9 and inoculated into MDCK cells in which a mono-layer was formed in a 96-well cell culture plate, and the Tissue Culture infectious Dose (TCID.sub.50/0.1 mL) was measured, and the result is shown in FIG. 2. In both MDCK cells and. A549 cells, it was confirmed that rH5N6 proliferated at the level of rPR8, whereas rH5N6-H103Y proliferated similarly in MDCK cells, but the proliferation decreased in A549 cells, and rH5N6-310PB2 and rH5N6-H103Y-310PB2 could not proliferate in both of the two cells.

[0085] 1-6. Comparison of Heat Resistance

[0086] In order to confirm whether H103Y actually increases heat resistance and increases proliferation in embryonated eggs, mammalian cell lines and mice, the rH5N6-310PB2 and rH5N6-H103Y-310PB2 viruses were treated at 55° C. for 0, 15, 30, 45, 60, and 90 minutes, respectively, and the result of comparing the HA titers through the HA, test is shown in FIG. 2. Unlike rH5N6-310PB2, in the case of rH5N6-H103Y-310PB2, it was confirmed that the HA protein was stable up to 90 minutes to increase heat resistance (FIG. 3).

[0087] 1-7. Comparison of Acid Resistance

[0088] In order to observe the effect of H103Y on acid resistance, after rH5N6 and rH5N6-H103Y viruses were reacted for 1 hour in various environments from pH 5.0 to pH 6.0, respectively, these were inoculated into mammalian cell lines and eggs, and as a result, it was confirmed that rH5N6-H103Y lost its maturity at low pH and lost proliferation at pH 5.2 or less in MDCK cells, and at pH 5.0, it lost proliferative properties such that it was inactivated more easily in an acidic environment. It is known that influenza virus isolated from humans undergoes fusion of the HA protein at a lower pH, and avian influenza exhibits activity at a relatively high pH, and it was found that the rH5N6-H103Y-310PB2 virus had a lower risk of transmission to the human body because the activity was removed at low pH (FIG. 4).

[0089] 1-8. Comparison of Antigen Amount

[0090] In order to determine whether the antigen amount increases when H103Y is applied, the same amounts of the rH5N6-310PB2 and rH5N6-H103Y-3 10PB2 viruses were separated by ultracentrifugation of the allantoic fluid, and the total amount of virus protein was measured through the Bicinchonic Acid (BCA) assay, and the amount of each virus constituent protein was compared through SDS-PAGE. Whereas rH5N6-H103Y-310PB2 had a slightly lower EID.sub.50/1 mL than that of rH5N6-310PB2, which is the proliferation titer of in embryonated eggs, it was confirmed that the antigen amount compared to the proliferation titer was more excellent because the amounts of the HA titer and the total virus protein and the amount of each protein in SDS-PAGE were more excellent. (FIG. 5).

TABLE-US-00003 TABLE 3 Total protein EID.sub.50/1 mL amount of virus (log 10) HA titer (μg/mL) rH5N6-310PB2 9.92 ± 0.38  64.00 ± 0.00 1325.42 rH5N6-H103Y- 9.42 ± 0.14 107.63 ± 0.89 2008.75 310PB2

[0091] 1-9. Comparison of Stimulation Ability of Humoral Immunity

[0092] The rH5N6-H103Y-310PB2 had a more excellent antigen content at similar proliferative titers compared to rH5N6-310PB2, and when it was actually inactivated and inoculated into chickens and ducks with an oil emulsion vaccine, it was determined whether the antibody formation was further increased. When inoculated to 3-week-old chickens, the rH5N6-H103Y-310PB2-inoculated group at the 3.sup.rd week of inoculation showed an average of 172.3, about twice as much antibody titer compared to the rH5N6-310PB2-inoculated group, and while the degree was low in ducks, rH5N6-H103Y-310PB2 showed a better ability to stimulate humoral immunity as an inactivated oil emulsion vaccine.

TABLE-US-00004 TABLE 4 Antibody titer (geometric mean, 95% confidence Vaccina- Inac- interval) tion age, tivated Vaccination Vaccination Vaccination species vaccine week 0 week 3 week 4 3 weeks rH5N6- <2 98.70 90.51 old, 310PB2 (64.13-151.9) (42.38-193.3) chicken rH5N6- <2 172.3 152.2 H103Y- (88.28-356.4) (68.06-340.4) 310PB2 Negative <2 <2 <2 control 2 weeks rH5N6- <2 14.86 12.00 old, 310PB2 (6.95-22.77) (7.22-16.78) duck rH5N6- <2 20.16 18.66 H103Y- (9.49-42.83) (9.84-35.41) 310PB2 Negative <2 <2 <2 control

[0093] 1-10. Comparison of Stimulation Ability of Cellular Immunity

[0094] When rH5N6-IG was inoculated into embryonated eggs with a similar proliferative titer compared to rH5N6-310PB2, the obtained antigen amount was lower, and it was actually inactivated, resulting in lower antibody formation when inoculated into chickens as an oil emulsion vaccine. When inoculated to 3-week-old chickens, the rH5N6-310P132-inoculated group showed an average of 118.5 at the 3.sup.rd week of inoculation, and in the case of the rH5N6-IG-inoculated group, it was 64, showing an antibody titer about twice as low. On Days 1, 3, 5, and 7 after attacking and inoculating with the outdoor strain H5N6 virus, the excretion of the virus into the throat cavity and total excretion cavity was terminated earlier, and it was confirmed that the antibody formation ability was low when the internal genes were substituted with the genome fragments derived from avian influenza virus, but viral clearance was performed more effectively.

TABLE-US-00005 TABLE 5 Antibody titer (geometric mean, 95% confidence Vaccina- Inac- interval) tion age, tivated Vaccination Vaccination Vaccination species vaccine week 0 week 3 week 4 3 weeks rH5N6- <2 118.5 118.5 old, 310PB2 (78.14-179.7) (72.28-194.3) chicken rH5N6-IG <2 64.00 80.63 (43.91-93.28) (55.32-117.5) Negative <2 <2 <2 control

TABLE-US-00006 TABLE 6 Virus release rate Vaccination Inactivated Throat cavity Total excretion cavity age, species vaccine Day 1 Day 3 Day 5 Day 7 Day 1 Day 3 Day 5 Day 7 3 weeks old, rH5N6-310PB2 2/9 7/9 4/9 3/9 2/9 6/9 4/9 2/9 chicken rH5N6-IG 4/9 4/9 4/9 0/9 1/9 5/9 4/9 0/9 Negative control 9/9 9/9 Dead 7/9 9/9 Dead

[0095] [Depositary Institution]

[0096] Name of depositary institution: Korea Research Institute of Bioscience and Biotechnology

[0097] Accession number: KCTC14391BP

[0098] Date of deposit: Nov. 27, 2020

[0099] Name of depositary institution: Korea Research Institute of Bioscience and. Biotechnology

[0100] Accession number: KCTC14261BP

[0101] Date of deposit: Aug. 4, 2020