CN117224667A - Avian influenza and newcastle disease virus vaccine composition and application thereof - Google Patents
Avian influenza and newcastle disease virus vaccine composition and application thereof Download PDFInfo
- Publication number
- CN117224667A CN117224667A CN202311104479.9A CN202311104479A CN117224667A CN 117224667 A CN117224667 A CN 117224667A CN 202311104479 A CN202311104479 A CN 202311104479A CN 117224667 A CN117224667 A CN 117224667A
- Authority
- CN
- China
- Prior art keywords
- strain
- virus
- vaccine
- newcastle disease
- avian influenza
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 94
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 54
- 241000711404 Avian avulavirus 1 Species 0.000 title claims abstract description 45
- 206010064097 avian influenza Diseases 0.000 title claims abstract description 43
- 208000002979 Influenza in Birds Diseases 0.000 title claims abstract description 34
- 239000000203 mixture Substances 0.000 title claims abstract description 25
- 238000004321 preservation Methods 0.000 claims abstract description 22
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 238000004519 manufacturing process Methods 0.000 claims abstract description 6
- 230000002265 prevention Effects 0.000 claims abstract description 3
- 241000700605 Viruses Species 0.000 claims description 120
- 239000007788 liquid Substances 0.000 claims description 71
- 210000003837 chick embryo Anatomy 0.000 claims description 50
- 239000000427 antigen Substances 0.000 claims description 18
- 102000036639 antigens Human genes 0.000 claims description 18
- 108091007433 antigens Proteins 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 16
- 238000003756 stirring Methods 0.000 claims description 16
- 241000342557 H7N9 subtype Species 0.000 claims description 14
- 239000012071 phase Substances 0.000 claims description 10
- 238000001816 cooling Methods 0.000 claims description 9
- 239000000839 emulsion Substances 0.000 claims description 9
- 229940031551 inactivated vaccine Drugs 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 230000000415 inactivating effect Effects 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000004945 emulsification Methods 0.000 claims description 6
- 238000003306 harvesting Methods 0.000 claims description 6
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 6
- 229920000053 polysorbate 80 Polymers 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 4
- 239000008346 aqueous phase Substances 0.000 claims description 3
- 230000001804 emulsifying effect Effects 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 230000003612 virological effect Effects 0.000 claims description 3
- 239000002671 adjuvant Substances 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims description 2
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 244000144977 poultry Species 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 5
- 229940031348 multivalent vaccine Drugs 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract 1
- 241000287828 Gallus gallus Species 0.000 description 69
- 235000013330 chicken meat Nutrition 0.000 description 69
- 101710154606 Hemagglutinin Proteins 0.000 description 62
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 62
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 62
- 101710176177 Protein A56 Proteins 0.000 description 62
- 239000000185 hemagglutinin Substances 0.000 description 61
- 208000010359 Newcastle Disease Diseases 0.000 description 33
- 108090000623 proteins and genes Proteins 0.000 description 31
- 230000003053 immunization Effects 0.000 description 26
- 238000002649 immunization Methods 0.000 description 26
- 210000001161 mammalian embryo Anatomy 0.000 description 24
- 101150039660 HA gene Proteins 0.000 description 20
- 231100000419 toxicity Toxicity 0.000 description 20
- 230000001988 toxicity Effects 0.000 description 20
- 238000003776 cleavage reaction Methods 0.000 description 15
- 235000013594 poultry meat Nutrition 0.000 description 15
- 230000007017 scission Effects 0.000 description 15
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 14
- 238000011081 inoculation Methods 0.000 description 13
- 210000002966 serum Anatomy 0.000 description 13
- 101150008820 HN gene Proteins 0.000 description 12
- 210000003555 cloaca Anatomy 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 235000013601 eggs Nutrition 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 230000001717 pathogenic effect Effects 0.000 description 10
- 230000007918 pathogenicity Effects 0.000 description 9
- 238000002255 vaccination Methods 0.000 description 9
- 230000005540 biological transmission Effects 0.000 description 8
- 239000008098 formaldehyde solution Substances 0.000 description 8
- 230000002779 inactivation Effects 0.000 description 8
- 125000003275 alpha amino acid group Chemical group 0.000 description 7
- 102000005348 Neuraminidase Human genes 0.000 description 6
- 108010006232 Neuraminidase Proteins 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 210000002257 embryonic structure Anatomy 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 238000010802 RNA extraction kit Methods 0.000 description 5
- 238000009395 breeding Methods 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000002441 reversible effect Effects 0.000 description 5
- 208000035473 Communicable disease Diseases 0.000 description 4
- 108020000999 Viral RNA Proteins 0.000 description 4
- 210000001691 amnion Anatomy 0.000 description 4
- 229940031567 attenuated vaccine Drugs 0.000 description 4
- 210000003711 chorioallantoic membrane Anatomy 0.000 description 4
- 230000008030 elimination Effects 0.000 description 4
- 238000003379 elimination reaction Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 238000010008 shearing Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 241000271566 Aves Species 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- 229940124873 Influenza virus vaccine Drugs 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000006978 adaptation Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 229940012466 egg shell membrane Drugs 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 244000144992 flock Species 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 229960003971 influenza vaccine Drugs 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 101710194807 Protective antigen Proteins 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000013190 sterility testing Methods 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000048433 H5N6 subtype Species 0.000 description 1
- 241000617520 H5N8 subtype Species 0.000 description 1
- 101710175243 Major antigen Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical group CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229940031416 bivalent vaccine Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000003278 egg shell Anatomy 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 231100000206 health hazard Toxicity 0.000 description 1
- 238000010211 hemagglutination inhibition (HI) assay Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 210000000867 larynx Anatomy 0.000 description 1
- 229940023832 live vector-vaccine Drugs 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000009374 poultry farming Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an avian influenza and newcastle disease virus vaccine composition and application thereof, wherein the vaccine composition comprises a compound having a preservation number of CCTCC NO: artificially recombined H5N6 influenza virus H5-Re13 strain of V202159 with a preservation number of CCTCCNO: artificially recombined H5N8 influenza virus H5-Re14 strain of V202160 with a preservation number of CCTCC NO: the artificially recombined H7N9 influenza virus H7-Re4 strain of V202161 and the preservation number is CCTCC NO: an artificially recombinant strain of newcastle disease virus rLa-VII of V202334. The vaccine provided by the invention is a multivalent vaccine, achieves the immune protection effect of 'one-needle multi-disease prevention', reduces the poultry immune burden and the production cost, and has a wide market prospect and important application value.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to an avian influenza and newcastle disease virus vaccine composition and application thereof.
Background
China is a large poultry raising country, and the annual stock quantity is more than 160 hundred million. The poultry farming industry is an important component of agriculture in China, and the development of the poultry industry is significant for farmers to get rich. The highly pathogenic avian influenza and newcastle disease are the most important 2 infectious diseases which harm the poultry industry, especially the chicken industry, and have great effects on effectively preventing and controlling the infectious diseases, ensuring the healthy development of the poultry industry, supplying poultry meat products and promoting the income increase of peasants.
Highly pathogenic avian influenza is an acute and virulent avian infectious disease caused by H5 and H7 subtype avian influenza virus (avian influenza virus, AIV), while H5 and H7 subtype AIV can also infect humans, causing significant public health hazards. Subtype H5 AIV advances into different HA branches (0-9), wherein the 2.3.4.4b branch and the 2.3.4.4H branch H5 virus are main branches of AIV which are harmful to poultry industry in China recently, and the 2.3.4.4b branch virus HAs accumulated in the world to cause death or destruction of more than 2 hundred million poultry, thereby attracting global high attention. The subtype H7 AIV appears in domestic poultry in 2013 and continuously causes 5 times of human influenza epidemic situation, the virus is mutated from low pathogenicity to high pathogenicity in early 2017, and epidemic situation is caused in a plurality of provincial chicken flocks; recently, the virus still exists in the chicken flock in the Bohai sea area in the northern ring of China, and the virus constitutes an important hazard to the health of the chicken flock. Vaccine immunization is an important means for controlling H5 and H7 subtype avian influenza, but there is no cross protection between H5 and H7 subtype AIVs, and cross protection between different branched H5 subtype AIVs is low.
Newcastle Disease (ND) is an acute, febrile, septicemic and highly contagious disease of birds caused by newcastle disease virus (Newcastledisease virus, NDV), has high morbidity and mortality, and is another major infectious disease that endangers the poultry industry. Newcastle disease viruses can be classified into genotype II, III, IV, V, VI, VIb subtypes. In recent years, the gene VII type newcastle disease virus is the virus which is most widely distributed in China and is most seriously damaged. After the conventional newcastle disease vaccine is immunized, the gene VII type newcastle disease virus is difficult to effectively control, atypical newcastle disease frequently occurs, and the method has important significance for effective prevention and control.
Vaccine immunization is an important measure for preventing highly pathogenic H5/H7 subtype avian influenza and newcastle disease. Currently, no vaccine for 2.3.4.4H branch H5 subtype avian influenza, 2.3.4.4b branch H5 subtype avian influenza, H7 subtype avian influenza and newcastle disease is available in China, and poultry can be effectively prevented by respectively inoculating an avian influenza (H5 + H7 subtype) vaccine and a newcastle disease vaccine. Because the H5/H7 avian influenza and newcastle disease vaccines are required to be immunized for multiple times in the production of laying hens and breeding birds, the immunization times are too high due to the simultaneous inoculation of the 2-class vaccines, the poultry is excessively burdened with immunization, and the breeding cost is greatly increased. The bird flu (H5 + H7) and newcastle disease bigeminal vaccine capable of preventing multiple diseases by one needle is developed and applied, so that the poultry immunity burden can be obviously reduced, the cost is saved, the breeding income is increased, and the vaccine is a clinical urgent-needed product, and has important clinical application value and broad market prospect.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: provides an avian influenza (H5+H27) and newcastle disease virus vaccine composition, a preparation method and application thereof; the bird flu (H5+H27) and newcastle disease bigeminal vaccine capable of preventing multiple diseases by one needle is developed, after the vaccine is developed successfully and pushed to be applied, the poultry immunity burden is greatly reduced, the breeding cost is saved, the vibration of the rural breeding industry is effectively promoted, and the vaccine is a vaccine product which is required in clinic in situ, and has wide market prospect and application value.
The technical scheme of the invention is as follows: an avian influenza and newcastle disease virus vaccine composition, wherein the vaccine composition comprises a vaccine composition with a preservation number of CCTCC NO: artificially recombined H5N6 avian influenza virus H5-Re13 strain of V202159 with the preservation number of CCTCC NO: artificially recombined H5N8 avian influenza virus H5-Re14 strain of V202160 with the preservation number of CCTCC NO: artificially recombined H7N9 avian influenza virus H7-Re4 strain of V202161 and CCTCC NO: four viral antigens of the artificially recombined newcastle disease virus rLa-VII strain of V202334.
Further, the ratio of the H5-Re13 strain, the H5-Re14 strain, the H7-Re4 strain and the rLa-VII strain virus antigen is 1:1:1:1.
The vaccine composition is applied to the preparation of medicaments for preventing diseases caused by H5 subtype avian influenza 2.3.4.4H branch viruses, H5 subtype avian influenza 2.3.4.4b branch viruses, H7N9 subtype avian influenza viruses and newcastle disease viruses.
Artificially recombined newcastle disease virus rLa-VII strain is preserved in China center for type culture collection with a preservation number of CCTCC NO: v202334.
The rLa-VII strain is used for preparing medicines for preventing diseases caused by newcastle disease viruses.
The invention also discloses a preparation method of the vaccine composition, which comprises the following steps:
inoculating the H5-Re13 strain, the H5-Re14 strain, the H7-Re4 strain and the rLa-VII strain into chick embryos respectively, incubating, harvesting virus liquid, concentrating for 4 times respectively, inactivating by formaldehyde, mixing according to the ratio of 1:1:1:1 of antigen content, and adding a white oil adjuvant to prepare the oil emulsion inactivated vaccine.
Further, the method for preparing the inactivated vaccine of the oil emulsion comprises the following steps:
(1) Preparing an oil phase: taking 95 parts of white oil for injection and 80 parts of span, uniformly mixing, sterilizing at 121 ℃ under high pressure, and cooling for later use;
(2) Preparing an aqueous phase: taking 7 parts of Tween 80 after sterilization and cooling, adding 23.25 parts of virus concentrates of H5-Re13 strain, H5-Re14 strain, H7-Re4 strain and rLa-VII strain, and fully stirring until the Tween 80 is completely dissolved;
(3) Emulsification: pouring 3 parts of the oil phase obtained in the step (1) into an emulsifying tank for stirring, slowly adding 1 part of the water phase obtained in the step (2), and fully stirring for emulsification; and (5) quantitatively packaging.
The H5-Re13 strain (with the preservation number of CCTCC NO: V202159 and the patent number of ZL 202111216162.5) of the artificially recombined H5N6 influenza virus HAs the surface antigen Hemagglutinin (HA) of the H5 subtype avian influenza virus A/dock/Fujian/S1424/2020 (H5N 6), but the HA cleavage site is typical low pathogenicity avian influenza virus molecular characteristics (-RETR-), and the HA gene branch is 2.3.4.4H branch, which belongs to the main flow HA gene branch of the H5 subtype avian influenza virus in China. The strain uses PB2, PB1, PA, NP, M and NS of widely used chicken embryo high titer adaptation strain A/PR/8/34 (H1N 1) as internal gene donors of recombinant viruses, takes 2.3.4.4H branched H5N6 virus A/dock/Fujian/S1424/2020 (H5N 6) surface antigen Hemagglutinin (HA) and Neuraminidase (NA) genes as surface gene donors, and modifies an HA cleavage site into typical low pathogenicity avian influenza virus molecular characteristics (-RETR-), and constructs the recombinant virus H5-Re13 strain through reverse genetic manipulation technology. The recombinant virus reacts positively with H5-Re13 strain serum, reacts negatively with H7, H9 subtype bird flu positive serum and newcastle disease and egg drop syndrome positive serum, the amino acid sequence of HA gene is completely consistent with that of A/dock/Fujian/S1424/2020 (H5N 6) surface antigen Hemagglutinin (HA), but the cleavage site is of low pathogenicity molecular characteristics.
The H5-Re14 strain (with the preservation number of CCTCC NO: V202160 and the patent number of ZL 202111217660.1) of the artificially recombined H5N8 influenza virus HAs the surface antigen Hemagglutinin (HA) of the H5 subtype avian influenza virus A/swan/Shanxi/4-1/2020 (H5N 8), but the HA cleavage site is typical low pathogenicity avian influenza virus molecular characteristics (-RETR-), and the HA gene branch is a 2.3.4.4b branch, and belongs to the main flow HA gene branch of the H5 subtype avian influenza virus in China. The strain uses PB2, PB1, PA, NP, M and NS of widely used chicken embryo high titer adaptation strain A/PR/8/34 (H1N 1) as internal gene donors of recombinant viruses, takes 2.3.4.4b branched H5N8 virus A/swan/Shanxi/4-1/2020 (H5N 8) surface antigen Hemagglutinin (HA) and Neuraminidase (NA) genes as surface gene donors, and modifies the HA cleavage site into typical low pathogenic avian influenza virus molecular characteristics (-RETR-), and constructs the recombinant virus H5-Re14 strain by reverse genetic manipulation technology. The recombinant virus reacts positively with H5-Re14 strain serum, reacts negatively with H7, H9 subtype bird flu positive serum and newcastle disease and egg drop syndrome positive serum, the amino acid sequence of the HA gene is completely consistent with that of A/swan/Shanxi/4-1/2020 (H5N 8) surface antigen Hemagglutinin (HA), but the cleavage site is of low pathogenicity molecular characteristics.
The H7-Re4 strain (with the preservation number of CCTCC NO: V202161 and the patent number of ZL 202111217661.6) of the artificially recombined H7N9 influenza virus HAs the surface antigen Hemagglutinin (HA) of the H7 subtype avian influenza virus A/chicken/Yunnan/SD024/2021 (H7N 9), but the amino acid sequence of an HA cleavage site is-PKGR-, and the HA gene branch belongs to the main stream HA gene branch of the H7 subtype avian influenza virus in China. The strain uses PB2, PB1, PA, NP, M and NS of widely used chicken embryo high titer adaptation strain A/PR/8/34 (H1N 1) as internal gene donors of recombinant viruses, uses H7N9 virus A/chicken/Yunnan/SD024/2021 (H7N 9) surface antigen Hemagglutinin (HA) and Neuraminidase (NA) genes as surface gene donors, and modifies an HA cleavage site into typical low pathogenic avian influenza virus molecular characteristics (-PKGR-), and constructs the recombinant virus H7-Re4 strain through reverse genetic manipulation technology. The recombinant virus HAs positive reaction of H7N9 virus and H7-Re4 strain serum, negative reaction of H5 and H9 subtype bird flu positive serum and newcastle disease and egg drop syndrome positive serum, and the amino acid sequence of HA gene is completely identical with that of A/chicken/Yunnan/SD024/2021 (H7N 9) surface antigen Hemagglutinin (HA), but the cleavage site is low pathogenic molecule characteristic.
The artificially recombined newcastle disease virus rLa-VII strain (with the preservation number of CCTCC NO: V202334) has a gene VII type newcastle disease virus chicken/Hebei/38/2006 surface antigen Hemagglutinin (HN), and the gene VII type belongs to the mainstream genotype of the newcastle disease virus in China. The strain uses a Newcastle disease virus LaSota attenuated vaccine strain widely used worldwide as a carrier, and replaces an HN gene of an NDV LaSota vaccine strain by a HN gene (SEQ ID No. 1) of a gene VII type Newcastle disease virus chicken/Hebei/38/2006 strain through a reverse genetic operation technology, so as to construct a recombinant virus rLa-VII strain. The recombinant virus has the protective antigen (HN gene) of the gene VII Newcastle disease virulent, retains the low pathogenicity and high titer chick embryo growth characteristics of LaSota attenuated vaccine strain, has low vaccine production cost, and is convenient for large-scale production.
Compared with the prior art, the invention has the following beneficial effects:
the vaccine composition provided by the invention comprises H5-Re14 strain H5N8 subtype avian influenza virus vaccine, H5-Re13 strain H5N6 subtype avian influenza virus vaccine, H7-Re4 strain H7N9 subtype avian influenza virus vaccine and rLa-VII strain newcastle disease virus vaccine, is multivalent vaccine, achieves the immune protection effect of 'one needle for preventing multiple diseases', reduces the poultry immune burden and the production cost, and has wide market prospect and important application value.
Preservation information:
newcastle disease virus (Newcastledisease virus) NDV harbin/ND rLa-VII/2022, namely Newcastle disease virus rLa-VII, is preserved in China center for type culture collection (CCTCC NO) of Wuhan in China at 5-month 8 of 2023: v202334.
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.
Abbreviation and key term definitions
Avian Influenza Virus (AIV)
Newcastle Disease Virus (NDV)
Hemagglutinin protein (HA)
Hemagglutination inhibition assay (HI)
Half-lethal dose of 100 times chickens (100 CLD) 50 )
Half-number infection of chick Embryo (EID) 50 )
Experimental materials
1. Virus strain
Vaccine compositions use vaccine strains: an artificially recombinant H5N6 influenza virus H5-Re13 strain carrying H5 subtype 2.3.4.4H branched virus HA gene (preservation number is CCTCC NO: V202159, patent number ZL 202111216162.5), an artificially recombinant H5N8 influenza virus H5-Re14 strain carrying H5 subtype 2.3.4.4b branched virus HA gene (preservation number is CCTCC NO: V202160, patent number ZL 202111217660.1), an artificially recombinant H7N9 influenza virus H7-Re4 strain carrying H7N9 subtype avian influenza virus HA gene (preservation number is CCTCC NO: V202161, patent number ZL 202111217661.6), an artificially recombinant newcastle disease virus rLa-VII strain carrying gene VII type newcastle disease virus HN gene (La sota strain with a newcastle disease virus of type VII inserted into the HN gene of the newcastle/Hebei/38/2006, la-VII strain with a preservation number of CCTCC NO: V202334), and the like were constructed by the national institutes of Harbin and the national institutes of Harmonious of agriculture.
Vaccine composition evaluation use strains: 2.3.4.4H branched H5N6 Virus A/dock/Fujian/S1424/2020 (H5N 6) (abbreviated DK/FJ/S1424/20) was isolated from a Duck sample in the year 2020; 2.3.4.4b Branch H5N8 Virus A/swan/Shanxi/4-1/2020 (H5N 8) (abbreviated SW/SX/4-1/20) was isolated from a Swan sample of the year 2020; subtype H7 avian influenza virus A/chicken/Yunnan/SD024/2021 (H7N 9) (CK/YN/SD 024/21 for short) was isolated from 2021 Yunnan chicken sample; the gene VII type newcastle disease virus chicken/Hebei/38/2006 (CK/HeB/38/2006 for short) is separated from chicken samples in 2006, and the viruses are highly pathogenic to chicken and are separated and identified by Harbin veterinary institute of China academy of agricultural sciences and are stored in a biosafety third-level laboratory of the institute. The applicant ensures that the biomaterial can be provided open within twenty years from the date of application.
2. Chick embryo and SPF chick
SPF chick embryo and SPF chick are purchased from Harbin veterinary research institute of China academy of agricultural sciences/national poultry experimental animal resource library, and susceptible chick embryo is purchased from Harbin local farm and then hatched.
3. Main reagent
The viral RNA extraction kit was purchased from beijing tiangen biochemical technology limited; reverse transcription kit (ReverTra Ace RT Kit) was purchased from Toyobo corporation of japan; ex-taq enzyme was purchased from Takara company; PCR product purification KIT (CYCLE-PURE-KIT) was purchased from Omega company; the gel recovery kit (Wizard SV Gel and PCR Clean-Up System) was purchased from Promega corporation; sequencing kit (ABI BigDye Terminator v 3.1) was purchased from Thermo Fisher, inc. of America.
4. Primer(s)
Analysis and comparison, specific primers for synthesizing H5-Re13 strain, H5-Re14 strain, H7-Re4 strain, virus main antigen HA gene and rLa-VII strain virus antigen HN gene are designed, and the synthetic primer sequences are shown in Table 1.
Table 1 primer sequences for identifying major antigen genes of various vaccines
Example 1: preparation and identification of H5 subtype 2.3.4.4H branched avian influenza vaccine components
Preparation of 1H5 subtype 2.3.4.4H branched virus liquid
1.1 inoculation H5 subtype 2.3.4.4H branched recombinant avian influenza H5-Re13 strain is diluted to 10 with sterilized normal saline -4 The allantoic cavity is inoculated with 10-11 days old healthy susceptible chick embryo, each embryo is 0.1ml, the pinhole is sealed after inoculation, and the chick embryo is incubated at 36 ℃ without turning eggs.
1.2 incubating and observing the chick embryo, after inoculation, the chick embryo is irradiated every day, incubated for 72 hours, taken out completely no matter dead or not, the air chamber is erected upwards, and the chick embryo is placed at 2-8 ℃ for cooling for 12 hours.
1.3 harvesting the cooled chick embryo, taking out, sterilizing the air chamber part, then aseptically stripping the eggshell membrane of the air chamber part, shearing the chorioallantoic membrane and the amniotic membrane, sucking the embryo liquid, placing the embryo liquid in the same sterilized container, and sampling and measuring the HA titer of each group. The results show that the HA prices of the virus liquid harvested after inoculation are 1:256.
1.4 concentration of virus liquid the H5-Re13 strain virus liquid is clarified by proper method, the large granular substances in the virus liquid are removed, and after the virus liquid becomes transparent or semitransparent solution, the H5-Re13 strain virus liquid is concentrated to 1/4 of original volume, and the HA valence is monitored. The results show that the HA titer of the H5-Re13 strain virus liquid is 1:1024 after concentration.
1.5 inactivation the concentrated H5-Re13 strain virus solution is mixed in a large container, and a few milliliters of sample is sucked out for measuring the virus value. Adding formaldehyde solution into the rest embryo liquid, stirring while adding to thoroughly mix. The final concentration of formaldehyde solution was 0.2%. Inactivating for 24 hours at 37 ℃ with continuous stirring. 10 SPF chick embryos of 10 days old are inoculated with inactivated H5-Re13 strain virus liquid by allantoic cavity, and each egg is 0.1ml. The chicken embryo solution is harvested after incubation for 72 hours at 36 ℃ and the HA titer is determined. The result shows that the HA price is negative after the inactivated allantoic fluid is inoculated into chick embryos; blind transmission for 1 generation, still negative, indicates complete inactivation.
Identification of 2H5 subtype 2.3.4.4H branched virus liquid
2.1HA Gene identification virus liquid 200 μl was taken, viral RNA was extracted with RNA extraction kit, gene fragments containing HA cleavage sites were amplified by RT-PCR method with upstream primer ATGCATACAAAATCATCAAGACAGG and downstream primer AGTCCAGACATCTAGGAATCCGTCT, and the amplified PCR products were sequenced with a full automatic sequencer. The results show that the amino acid sequence of the HA gene cleavage site of the amplified virus liquid should be typical of low pathogenic molecules of-RETR-and completely coincide with the 800 th to 1311 th nucleotide sequence of the HA gene coding region of the disclosed H5 subtype 2.3.4.4H branched DK/FJ/S1424/20 (H5N 6) virus.
2.2 sterile taking the inactivated H5-Re13 strain virus liquid, and performing sterile inspection. The results showed that the virus liquid grew aseptically.
Example 2: preparation and identification of H5 subtype 2.3.4.4b branched avian influenza vaccine components
Preparation of 1H5 subtype 2.3.4.4b branched virus liquid
1.1 inoculation H5 subtype 2.3.4.4b branched recombinant avian influenza H5-Re14 strain is diluted to 10 with sterilized normal saline -4 The allantoic cavity is inoculated with 10-11 days old healthy susceptible chick embryo, each embryo is 0.1ml, the pinhole is sealed after inoculation, and the chick embryo is incubated at 36 ℃ without turning eggs.
1.2 incubating and observing the chick embryo, after inoculation, the chick embryo is irradiated every day, incubated for 72 hours, taken out completely no matter dead or not, the air chamber is erected upwards, and the chick embryo is placed at 2-8 ℃ for cooling for 12 hours.
1.3 harvesting the cooled chick embryo, taking out, sterilizing the air chamber part, then aseptically stripping the eggshell membrane of the air chamber part, shearing the chorioallantoic membrane and the amniotic membrane, sucking the embryo liquid, placing the embryo liquid in the same sterilized container, and sampling and measuring the HA titer of each group. The results show that the HA valence of the virus liquid is 1:256.
1.4 concentration of virus liquid the H5-Re14 strain virus liquid is clarified by a proper method, the large particle substances in the virus liquid are removed, and after the virus liquid becomes transparent or semitransparent solution, the H5-Re14 strain virus liquid is concentrated to 1/4 of the original volume, and the HA valence is detected. The results show that the HA titer of the H5-Re14 strain virus liquid is 1:1024.
1.5 inactivation the concentrated H5-Re14 strain virus solution is mixed in a large container, and a few milliliters of sample is sucked out for measuring the virus value. Adding formaldehyde solution into the rest embryo liquid, stirring while adding to thoroughly mix. The final concentration of formaldehyde solution was 0.2%. Inactivating for 24 hours at 37 ℃ with continuous stirring. 10 SPF chick embryos of 10 days old are inoculated with inactivated H5-Re14 strain virus liquid by allantoic cavity, and each egg is 0.1ml. The chicken embryo solution is harvested after incubation for 72 hours at 36 ℃ and the HA titer is determined. The result shows that the HA price is negative after the inactivated allantoic fluid is inoculated into chick embryos; blind transmission for 1 generation, still negative, indicates complete inactivation.
Identification of 2H5-Re14 strain virus liquid
2.1HA Gene identification 200. Mu.l of the harvested virus liquid was taken, viral RNA was extracted with RNA extraction kit, gene fragments containing HA cleavage sites were amplified by RT-PCR method using the upstream primer AAGAAAGGGGACTCAACAATTATGA and the downstream primer CCTTTCTAAGTTATTAAACTCCCTT, and the amplified PCR products were sequenced by full automatic sequencer. The results show that the amplified virus liquid HA gene cleavage site amino acid sequence should be typical of the low pathogenic molecular profile of-RETR-and be identical to the nucleotide sequence of the region 820 to 1260 of the HA gene coding region of the disclosed H5 subtype 2.3.4.4b branched SW/SX/4-1/20 (H5N 8) (2.3.4.4b branched) virus.
2.2 sterile test of Virus liquid inactivated H5-Re14 Strain Virus liquid is taken for test. The results showed that the virus liquid grew aseptically.
Example 3: preparation and identification of H7 subtype avian influenza vaccine components
Preparation of 1H7 subtype avian influenza virus liquid
1.1 inoculating H7 subtype recombinant avian influenza H7-Re4 strain virus seed is diluted to 10 with sterilized normal saline -4 The allantoic cavity is inoculated with 10-11 days old healthy susceptible chick embryo, each embryo is 0.1ml, the pinhole is sealed after inoculation, and the chick embryo is incubated at 36 ℃ without turning eggs.
1.2 incubating and observing the chick embryo, after inoculation, the chick embryo is irradiated every day, incubated for 72 hours, taken out completely no matter dead or not, the air chamber is erected upwards, and the chick embryo is placed at 2-8 ℃ for cooling for 12 hours.
1.3 harvesting the cooled chick embryo, taking out, sterilizing the air chamber part, then aseptically stripping the eggshell membrane of the air chamber part, shearing the chorioallantoic membrane and the amniotic membrane, sucking the embryo liquid, placing the embryo liquid in the same sterilized container, and sampling and measuring the HA titer of each group. The results show that the HA number of the harvested viruses is 1:256.
1.4 concentration of virus liquid the H7-Re4 strain virus liquid is clarified by proper method, and the large granular substances in the virus liquid are removed, so that after the virus liquid becomes transparent or semitransparent solution, the H7-Re4 strain virus liquid is concentrated to 1/4 of original volume, and when the HA titer of H7-Re4 strain virus liquid is 1:1024, the virus liquid can be used for preparing seedlings.
1.5 inactivation the concentrated H7-Re4 strain virus solution is mixed in a large container, and a few milliliters of sample is sucked out for measuring the virus value. Adding formaldehyde solution into the rest embryo liquid, stirring while adding to thoroughly mix. The final concentration of formaldehyde solution was 0.2%. Inactivating for 24 hours at 37 ℃ with continuous stirring. 10 SPF chick embryos of 10 days old are inoculated with inactivated H7-Re4 strain virus liquid by allantoic cavity, and each egg is 0.1ml. The chicken embryo solution is harvested after incubation for 72 hours at 36 ℃ and the HA titer is determined. The result shows that the HA price is negative after the inactivated allantoic fluid is inoculated into chick embryos; blind transmission for 1 generation, still negative, indicates complete inactivation.
Identification of 2H7-Re4 strain virus liquid
2.1HA Gene identification virus seed 200. Mu.l, RNA extraction kit to extract virus RNA, upstream primer ATAGCTCCAGATCGTGCAAGCTTCC and downstream primer TTATAGAATCTCTGGTCCAATTTAT to amplify gene fragment containing receptor binding site and HA cleavage site by RT-PCR method, and full automatic sequencing to analyze the amplified PCR product. The result shows that the amino acid sequence of the HA gene cleavage site of the H7-Re4 strain is-PKGR-, accords with the low-pathogenicity molecular characteristics of the avian influenza H7 subtype virus, and is completely consistent with the nucleotide sequence of the region from 781 st to 1294 th of the HA gene coding region of the disclosed H7N9 subtype CK/YN/SD024/21 (H7N 9) virus.
2.2 testing the inactivated H7-Re4 strain virus liquid, and testing. The results showed that the virus liquid grew aseptically.
Example 4: preparation and identification of vaccine components of Newcastle disease ND rLa-VII strain
Preparation of 1ND rLa-VII strain virus liquid
Construction of ND rLa-VII strain virus was performed with reference to construction of avian influenza (subtype H5) recombinant newcastle disease live vector vaccine (rLa-H5 strain) (Ge, JVI, 2007): a recombinant virus rLa-VII strain is constructed by taking a Newcastle disease virus LaSota attenuated vaccine strain widely used worldwide as a carrier, and replacing the HN gene of the NDV LaSota vaccine strain by the HN gene (SEQ ID No. 1) of a gene VII type Newcastle disease virus chicken/Hebei/38/2006 strain through a reverse genetic operation technology. The recombinant virus has the protective antigen (HN gene) of the gene VII Newcastle disease virulent, retains the low pathogenicity and high titer chick embryo growth characteristics of LaSota attenuated vaccine strain, has low vaccine production cost, and is convenient for large-scale production.
1.1 inoculation the seed is diluted to 10 with sterile physiological saline -4 The allantoic cavity is inoculated with 10-11 days old healthy susceptible chick embryo, each embryo is 0.1ml, the pinhole is sealed after inoculation, and the chick embryo is incubated at 37 ℃ without turning eggs.
1.2 incubation and observation after chick embryo inoculation, eggs were photographed daily and the dead chick embryos were discarded within 24 hours. Taking out the dead chick embryo at any time after 24 hours until 96 hours, taking out all the chick embryos no matter whether the chick embryo is dead or not, standing the chick embryo upwards, and cooling the chick embryo at the temperature of 2-8 ℃ for 12 hours.
1.3 harvesting cooled chick embryos, taking out, sterilizing the air chamber part by using iodine tincture, then stripping egg shell membranes of the air chamber part by using aseptic operation, shearing chorioallantoic membranes and amniotic membranes (preventing egg yolk from cracking), sucking embryo liquid, placing the embryo liquid in a same sterilized container, and measuring the HA titer by sampling each group to be not lower than 1:256. Preserving the harvested embryo liquid at 2-8 ℃ before inactivating for no more than 3 days.
1.4 concentration of virus liquid the ND rLa-VII strain virus liquid is clarified by a proper method, the large particle matters in the virus liquid are removed, the virus liquid becomes transparent or semitransparent solution, the rLa-VII strain virus liquid is concentrated to 1/4 of the original volume, and the HA valence of the virus liquid is detected. The result shows that the HA titer of the concentrated rLa-VII strain virus liquid is 1:1024.
1.5 inactivation the concentrated ND rLa-VII strain virus solution was mixed in a large vessel and several ml samples were aspirated for the assay. Adding formaldehyde solution into the rest embryo liquid, stirring while adding to thoroughly mix. The final concentration of formaldehyde solution was 0.2%. Inactivating for 24 hours at 37 ℃ with continuous stirring. 10 SPF chick embryos of 10 days old are inoculated with inactivated rLa-VII strain virus liquid by allantoic cavity, and each strain is 0.1ml. The chicken embryo solution is harvested after incubation for 72 hours at 36 ℃ and the HA titer is determined. The result shows that the HA price is negative after the inactivated allantoic fluid is inoculated into chick embryos; blind transmission for 1 generation, still negative, indicates complete inactivation.
Identification of 2ND rLa-VII strain Virus liquid
2.1HN Gene identification 200. Mu.l of virus seed was taken, viral RNA was extracted with RNA extraction kit, HN gene fragment was amplified by RT-PCR method using the upstream primer CTGTTATTGGCAGTGTAGC and the downstream primer ATAAAGTGCCTGGATGGT, and the amplified PCR product was subjected to sequence analysis by full automatic sequencer. The results show that the amplified fragment of the virus liquid HA gene is completely consistent with the HN gene full-length nucleotide sequence of the gene VII type CK/HeB/38/2006 (ND). The HN gene sequence is shown as SEQ ID No. 1.
2.2 sterile test of inactivated Virus liquid the inactivated ND rLa-VII strain Virus liquid is taken for test. The results showed that the inactivated virus liquid grew aseptically.
Example 5: avian influenza (H5 + H7), newcastle disease bivalent vaccine preparation and immune effect evaluation
1 preparation of vaccine
1.1 preparation of oil phase 95 parts of white oil for injection, span-80 parts, mixing uniformly, sterilizing at 121 ℃ for 20 minutes under high pressure, and cooling for later use.
1.2 preparing water phase, taking 7 parts of sterilized and cooled Tween-80, adding 23.25 parts of qualified H5-Re13 strain, H5-Re14 strain, H7-Re4 strain and ND rLa-VII strain virus liquid, and fully stirring until the Tween-80 is completely dissolved.
1.3, 3 parts of oil phase is taken by emulsification, poured into an emulsifying tank and stirred, 1 part of water phase is slowly added, and full stirring and emulsification are carried out; quantitatively packaging, sealing, and sticking labels.
3 batches of vaccine were prepared consecutively as described above, lots 2022001, 2022002, 2022003, respectively.
1.4 physical Properties of vaccine and sterility testing
1.4.1 trait testing was performed as follows.
1.4.1.1 the appearance of the vaccine is observed to observe the color and state of the vaccine, and the vaccine is milky uniform emulsion.
1.4.1.2 the preparation is prepared by taking a clean straw, sucking a little vaccine drop on the surface of cold water, and observing the state. The results show that the vaccine does not spread on the cold water surface for 7 seconds.
1.4.1.3 stability 10ml of the aspirated vaccine was added to a centrifuge tube, centrifuged at 3000r/min for 15 minutes, and the amount of water precipitated at the bottom of the tube was measured. The results showed that no aqueous phase separated out after centrifugation of the vaccine.
1.4.1.4 viscosity the viscosity distribution of 3 batches of vaccine was 58.6cP, 60.5cP, 59.2cP as measured in accordance with the current annex of the chinese beast pharmacopoeia.
1.4.2 sterility testing and formaldehyde content determination were performed according to the annex of the current "Chinese veterinary pharmacopoeia". The results show that 3 batches of vaccine have no bacterial pollution and the enhanced content is in the range of 0.03-0.04%.
Table 23 test results for vaccine characteristics, sterility and Formaldehyde content
2. Safety assessment of vaccine
10 SPF chickens with 4 weeks of age are used for each vaccine batch, each neck is injected with 2.0ml of vaccine subcutaneously or intramuscularly in the chest, 10 vaccine batches are observed continuously for 14 days, all vaccine is healthy and no local or systemic adverse reaction caused by the vaccine occurs. The result shows that the vaccine has good safety to chickens.
Table 33 vaccine safety test results
3. Evaluation of vaccine immunopotency
3.1 vaccine immunization against H5 subtype 2.3.4.4H branched avian influenza Virus
3.1.1 method 3 batches of the prepared oil emulsion inactivated vaccine were immunized with 0.3 ml/dose of 30 SPF chickens of 3 weeks old in different vaccination modes, and 10 SPF chickens were additionally provided as a blank group without vaccination. At week 3 after immunization, serum was collected and isolated, and HI antibody assay was performed; referring to the existing vaccine immune toxicity attack method, the H5 subtype 2.3.4.4H branch DK/FJ/S1424/2020 (H5N 6) strain (nasal cavity infection mode, 100CLD50 dose each) is used for carrying out artificial toxicity attack efficacy assessment, after toxicity attack, the death and condition of the tested chicken are observed, throat and cloaca swab samples are collected 3 days and 5 days after toxicity attack, 9-11 days old chicken embryos are inoculated, the toxicity elimination condition is detected (HA is negative after the throat or cloaca swab samples are inoculated to the chicken embryos, the chicken embryos are inoculated again for blind transmission generation, the HA price is still negative, the sample is judged to be negative, the throat and cloaca samples are both negative, and the toxicity elimination of the chicken is indicated), so that the vaccine is evaluated for the immune protection efficacy of the H5 subtype 2.3.4.4H branch viruses.
3.1.2 antibody measurement results when SPF chickens were immunized with 3 vaccine batches, respectively, for 3 weeks, blood was collected and the antibodies were measured. The results showed that the average value of H5 subtype H5-Re13 strain HI antibodies after 3 weeks of vaccine immunization was 7.8log2, 7.6log2 and 7.8log2, respectively, and the results are shown in Table 4 for the control group antibodies which were all < 1log 2.
TABLE 4 determination of HI antibody to H5 subtype 2.3.4.4H branched avian influenza Virus strain after vaccine immunization
3.1.2 challenge results 3 batches of vaccine were challenge with H5 subtype 2.3.4.4H branch strains after immunization of SPF chickens for 21 days, all vaccine immunized groups of chickens were healthy and alive, and the number of virus isolation negative numbers were 10/10; the control group was all ill, dead, and 10/10 virus isolated positive. The results are detailed in Table 5.
TABLE 5 results of vaccine immunization of chickens against H5 subtype 2.3.4.4H branched avian influenza virus
* Indicating that the control chickens all died within 5 days after the challenge.
3.2 vaccine immunization against H5 subtype 2.3.4.4b branched avian influenza Virus
3.2.1 method 3 batches of oil emulsion inactivated vaccine prepared were immunized with 0.3 ml/dose of 30 SPF chickens of 3 weeks old in different vaccination modes, and 10 SPF chickens were additionally provided as blank groups without vaccination. At week 3 after immunization, serum was collected and isolated, and HI antibody assay was performed; and using H5 subtype 2.3.4.4b branch SW/SX/4-1/2020 (H5N 8) strain (nasal infection mode, 100CLD50 dose each) to evaluate the artificial toxicity attack efficacy, observing the morbidity, mortality and condition of the tested chicken after toxicity attack, collecting 3-day and 5-day throat and cloaca swab samples after toxicity attack, inoculating 9-11 day old chick embryos, detecting the toxicity elimination condition (HA is a negative sample after the throat or cloaca swab samples are inoculated to the chick embryos, inoculating the chick embryos again for blind transmission generation, judging that the sample is negative if the HA valence is still negative, judging that the throat and cloaca samples are negative, indicating that the chicken is negative in toxicity attack), and evaluating the immunoprophylaxis efficacy of the vaccine to the H5 subtype avian influenza 2.3.4.4b branch viruses.
3.2.2 antibody measurement results when SPF chickens were immunized with 3 vaccine batches, respectively, for 3 weeks, blood was collected and the antibodies were measured. The results showed that the average value of H5 subtype H5-Re14 strain HI antibodies after 3 weeks of vaccine immunization was 7.7log2, 8.0log2 and 7.8log2, respectively, and the results are shown in Table 6 for the control group antibodies which were all < 1log 2.
TABLE 6 determination of HI antibody to H5 subtype 2.3.4.4b branched avian influenza Virus strain after vaccine immunization
3.2.3 results of challenge after 3 batches of vaccine were immunized against SPF chickens for 21 days, the virus was separately challenged with H5 subtype 2.3.4.4b branched strains, all immunized groups of chickens were healthy and live, and the number of virus isolation negatives was 10/10; the control group was all ill, dead, and 10/10 virus isolated positive. The results are detailed in Table 7.
TABLE 7 results of immunization of chickens with vaccine against H5 subtype 2.3.4.4b branched avian influenza virus strain
* Indicating that the control chickens all died within 5 days after the challenge.
3.3 vaccine immunization against H7N9 subtype avian influenza Virus
3.3.1 method 3 batches of oil emulsion inactivated vaccine prepared were immunized with 0.3 ml/dose of 30 SPF chickens of 3 weeks old in different vaccination modes, and 10 SPF chickens were additionally provided as blank groups without vaccination. At week 3 after immunization, serum was collected and isolated, and HI antibody assay was performed; and the H7N9 subtype CK/YN/SD024/21 (H7N 9) strain (nasal infection mode, 100CLD50 doses each) is used for evaluating the artificial toxicity attacking efficacy, the disease death and the condition of the tested chickens are observed after toxicity attack, throat and cloaca swab samples are collected for 3 days and 5 days after toxicity attack, 9-11 days old chick embryos are inoculated, the toxicity expelling condition is detected (HA is a negative sample after the throat or cloaca swab samples are inoculated to the chick embryos, the chick embryos are inoculated again for blind transmission generation, the HA price is still negative, the sample is judged to be negative, and the throat and cloaca samples are negative, so that the chicken is proved to be negative in toxicity expelling) so as to evaluate the immunoprophylaxis of the vaccine to the H7N9 subtype avian influenza virus.
3.3.2 antibody measurement results when SPF chickens were immunized with 3 vaccine batches, respectively, for 3 weeks, blood was collected and the antibodies were measured. The results showed that the average value of H7 subtype H7-Re4 strain HI antibodies after 3 weeks of immunization of SPF chickens with the vaccine was 7.9log2, 7.7log2 and 8.0log2, respectively, and the results are shown in Table 8 in detail for the antibodies of the control group < 1log 2.
TABLE 8 determination of H7 subtype avian influenza HI antibody after immunization
3.3.3 challenge results when 3 batches of vaccine were immunized against SPF chickens for 3 weeks, respectively, H7N9 subtype avian influenza virulent strain was challenged. Within the 14-day observation period, all SPF chickens in each immune group survive, do not attack or expel toxin, and are protected by 10/10; the control group was all ill and dead, and 10/10 virus was positive for isolation. The results are detailed in Table 9.
TABLE 9 results of immunization of chickens with vaccine against H7N9 subtype avian influenza virus
* Indicating that the control chickens all died within 5 days after the challenge.
3.4 prophylactic Effect of vaccine immunization on Newcastle disease Virus
3.4.1 method according to the efficacy test method of the current newcastle disease related inactivated vaccine (vaccination test using conventional dose of 0.5 ml/1/25), the prepared 3 batches of oil emulsion inactivated vaccine were immunized with 20 μl/1 dose of 30 SPF chickens at 3 weeks of age by different vaccination routes, and 10 SPF chickens were additionally provided as blank control group without vaccination. At week 3 after immunization, serum was collected and isolated, and HI antibody assay was performed; and using the gene VII strain of Newcastle disease virus CK/HeB/38/2006 (intramuscular injection mode, 10 each 5 EID 50 Dose), evaluating the artificial toxicity attack efficacy, observing the morbidity, mortality and conditions of the test chicken after toxicity attack, collecting throat and cloaca swab samples of 3 days and 5 days after toxicity attack, inoculating 9-11 days old chicken embryos, detecting the toxicity elimination condition (HA is a negative sample after the throat or cloaca swab samples are inoculated to the chicken embryos, inoculating the chicken embryos again for blind transmission, and judging that the sample is negative if the HA price is still negative; the samples of the larynx and cloaca are negative, which indicates that the chicken is negative in toxin expelling) so as to evaluate the immunoprophylaxis effect of the vaccine on the gene VII type newcastle disease virus.
3.4.2 antibody measurement results when SPF chickens were immunized with 3 vaccine batches for 3 weeks, the antibodies were collected and measured. The results showed that the average value of newcastle disease HI antibodies after vaccine immunization of SPF chickens was 5.9log2, 5.9log2 and 6.1log2, respectively, and the antibodies of the control group were all < 1log2, and the results are detailed in Table 10.
TABLE 10 results of Newcastle disease HI antibody assay after immunization
3.4.3 challenge results when 3 batches of vaccine were immunized against SPF chickens for 3 weeks, respectively, the gene VII type newcastle disease virus strain was challenged. Within the 14-day observation period, all SPF chickens in each immune group survive, do not attack or expel toxin, and are protected by 10/10; the control group was all ill and dead, and 10/10 virus was positive for isolation. The results are detailed in Table 11.
Table 11 results of vaccine immunization against Gene VII Newcastle disease Virus
* Indicating that the control chickens all died within 5 days after the challenge.
The results show that the avian influenza (H5+H27) and newcastle disease vaccine composition can induce ideal HI antibody level by intramuscular injection and neck subcutaneous immunization, can completely resist the attack of 2.3.4.4H and 2.3.4.4b branch H5 subtype avian influenza virus, H7N9 subtype avian influenza virus and ND virus, has the immune function of simultaneously preventing chicken highly pathogenic avian influenza (H5+H2) and newcastle disease, and achieves the effect of preventing multiple diseases by one needle.
Claims (7)
1. The avian influenza and newcastle disease virus vaccine composition is characterized in that the vaccine composition contains a vaccine composition with a preservation number of CCTCC NO: artificially recombined H5N6 influenza virus H5-Re13 strain of V202159 with a preservation number of CCTCC NO: artificially recombined H5N8 influenza virus H5-Re14 strain of V202160 with a preservation number of CCTCC NO: the artificially recombined H7N9 influenza virus H7-Re4 strain of V202161 and the preservation number is CCTCC NO: four viral antigens of the artificially recombined newcastle disease virus rLa-VII strain of V202334.
2. The vaccine composition of claim 1, wherein the ratio of H5-Re13 strain, H5-Re14 strain, H7-Re4 strain and rLa-vii strain viral antigens is 1:1:1:1.
3. Use of the vaccine composition of claim 1 or 2 in the preparation of a medicament for preventing diseases caused by H5 subtype avian influenza 2.3.4.4H branch virus, H5 subtype avian influenza 2.3.4.4b branch virus, H7N9 subtype avian influenza virus and newcastle disease virus.
4. Artificially recombined newcastle disease virus rLa-VII strain is preserved in China center for type culture collection with a preservation number of CCTCC NO: v202334.
5. Use of a rLa-vii strain according to claim 4 in the manufacture of a medicament for the prevention of a disease caused by newcastle disease virus.
6. A method of preparing a vaccine composition as claimed in claim 1 or 2, characterised by the steps of:
inoculating the H5-Re13 strain, the H5-Re14 strain, the H7-Re4 strain and the rLa-VII strain into chick embryos respectively, incubating, harvesting virus liquid, concentrating for 4 times respectively, inactivating by formaldehyde, mixing according to the ratio of 1:1:1:1 of antigen content, and adding a white oil adjuvant to prepare the oil emulsion inactivated vaccine.
7. The preparation method of the oil emulsion inactivated vaccine according to claim 6, wherein the preparation method comprises the following steps:
(1) Preparing an oil phase: taking 95 parts of white oil for injection and 80 parts of span, uniformly mixing, sterilizing at 121 ℃ under high pressure, and cooling for later use;
(2) Preparing an aqueous phase: taking 7 parts of Tween 80 after sterilization and cooling, adding 23.25 parts of virus concentrates of H5-Re13 strain, H5-Re14 strain, H7-Re4 strain and rLa-VII strain, and fully stirring until the Tween 80 is completely dissolved;
(3) Emulsification: pouring 3 parts of the oil phase obtained in the step (1) into an emulsifying tank for stirring, slowly adding 1 part of the water phase obtained in the step (2), and fully stirring for emulsification; and (5) quantitatively packaging.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311104479.9A CN117224667A (en) | 2023-08-30 | 2023-08-30 | Avian influenza and newcastle disease virus vaccine composition and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311104479.9A CN117224667A (en) | 2023-08-30 | 2023-08-30 | Avian influenza and newcastle disease virus vaccine composition and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117224667A true CN117224667A (en) | 2023-12-15 |
Family
ID=89085332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311104479.9A Pending CN117224667A (en) | 2023-08-30 | 2023-08-30 | Avian influenza and newcastle disease virus vaccine composition and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117224667A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101491672A (en) * | 2009-02-24 | 2009-07-29 | 乾元浩生物股份有限公司 | Chicken new castle disease-bird influenza dual oil-emulsion inactivated vaccine and preparation method thereof |
| CN102302775A (en) * | 2011-07-06 | 2012-01-04 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine of Newcastle disease and H9 subtype avian influenza and preparation method thereof |
| CN107281479A (en) * | 2016-03-31 | 2017-10-24 | 普莱柯生物工程股份有限公司 | A kind of genotype Ⅶ NDV low virulent strain, vaccine combination and its application |
| CN113913394A (en) * | 2021-10-19 | 2022-01-11 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Artificial recombinant H5N6 influenza virus and preparation method and application thereof |
| CN113913396A (en) * | 2021-10-19 | 2022-01-11 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Artificial recombinant H7N9 influenza virus and preparation method and application thereof |
| CN113913395A (en) * | 2021-10-19 | 2022-01-11 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Artificial recombinant H5N8 influenza virus and preparation method and application thereof |
| CN114524862A (en) * | 2021-12-16 | 2022-05-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Construction and application of avian influenza (H5+ H7) trivalent DNA vaccine |
-
2023
- 2023-08-30 CN CN202311104479.9A patent/CN117224667A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101491672A (en) * | 2009-02-24 | 2009-07-29 | 乾元浩生物股份有限公司 | Chicken new castle disease-bird influenza dual oil-emulsion inactivated vaccine and preparation method thereof |
| CN102302775A (en) * | 2011-07-06 | 2012-01-04 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine of Newcastle disease and H9 subtype avian influenza and preparation method thereof |
| CN107281479A (en) * | 2016-03-31 | 2017-10-24 | 普莱柯生物工程股份有限公司 | A kind of genotype Ⅶ NDV low virulent strain, vaccine combination and its application |
| CN113913394A (en) * | 2021-10-19 | 2022-01-11 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Artificial recombinant H5N6 influenza virus and preparation method and application thereof |
| CN113913396A (en) * | 2021-10-19 | 2022-01-11 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Artificial recombinant H7N9 influenza virus and preparation method and application thereof |
| CN113913395A (en) * | 2021-10-19 | 2022-01-11 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Artificial recombinant H5N8 influenza virus and preparation method and application thereof |
| CN114524862A (en) * | 2021-12-16 | 2022-05-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Construction and application of avian influenza (H5+ H7) trivalent DNA vaccine |
Non-Patent Citations (2)
| Title |
|---|
| YA-WEN BU等: "Recombinant Newcastle disease virus (NDV) La Sota expressing the haemagglutinin–neuraminidase protein of genotype VII NDV shows improved protection efficacy against NDV challenge", AVIAN PATHOLOGY, vol. 48, no. 2, 10 December 2018 (2018-12-10), pages 91 - 97 * |
| 农业农村部: "中华人民共和国农业农村部公告第513号", pages 1, Retrieved from the Internet <URL:http://www.moa.gov.cn/nybgb/2022/202202/202203/t20220324_6393768.htm> * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101508977B (en) | Separation identification and purification process for chicken source H9N2 avian influenza virus strain and uses thereof | |
| CN111000993B (en) | Bivalent inactivated vaccine for duck viral hepatitis and duck reovirus disease and preparation method thereof | |
| CN101508978A (en) | Separation identification and purification process for chicken source H9N2 avian influenza virus strain and uses thereof | |
| CN110680914B (en) | Triple inactivated vaccine and preparation method thereof | |
| CN109097340B (en) | Avian adenovirus, quadruple vaccine and preparation method thereof | |
| CN108465107B (en) | Duck type 2 adenovirus and Muscovy duck parvovirus disease combined inactivated vaccine | |
| CN101843900B (en) | Bird flu inactivated vaccine and preparation method thereof | |
| CN104922665A (en) | Triple inactivated vaccine for newcastle disease, infectious bronchitis and H9 subtype avian influenza | |
| CN109055320B (en) | Infectious bronchitis virus isolate and application thereof in vaccine preparation | |
| CN105802918B (en) | Chicken's infectious bronchitis nephritis strain and its vaccine composition, preparation method and application | |
| CN106065030A (en) | Yolk antibody of chicken inclusion body hepatitis and preparation method thereof | |
| CN116286670A (en) | Novel duck reovirus and application thereof in preparation of inactivated vaccine and egg yolk antibody | |
| CN117224667A (en) | Avian influenza and newcastle disease virus vaccine composition and application thereof | |
| CN117224666A (en) | Avian influenza and newcastle disease virus bivalent pentavalent vaccine composition and application thereof | |
| CN111150840B (en) | Tetrad vaccine for poultry and preparation method and application thereof | |
| CN113151189B (en) | H9N2 avian influenza virus, inactivated vaccine and preparation method thereof | |
| CN109679926B (en) | Avian influenza virus H9 subtype JXD strain and application thereof | |
| CN116875564B (en) | H9 subtype avian influenza virus strain and culture method thereof | |
| CN109091670B (en) | Avian influenza virus H9 subtype bivalent inactivated vaccine and preparation method thereof | |
| CN114015658B (en) | Bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma | |
| CN113249339B (en) | H3 subtype canine influenza virus strain, inactivated vaccine and preparation method thereof | |
| CN115261335B (en) | Oral immune avian influenza inactivated vaccine | |
| CN108714211B (en) | Goose parvovirus and duck type 2 adenovirus bivalent inactivated vaccine | |
| CN102747045A (en) | Swine influenza virus H1N1 SIVTJ inactivated vaccine and preparation method thereof | |
| RU2323740C1 (en) | "NOVOSIBIRSKY" STRAIN OF BIRD FLUE Influenzae virus avicum TO CONTROL IMMUNOGENIC AND ANTIGENIC ACTIVITY OF VACCINES AND TO MANUFACTURE BIOMEDICINES FOR DIAGNOSTICS AND SPECIFIC PREVENTION OF BIRD FLUE |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |