WO2007025420A1 - Souche de faible virulence du vaccin recombinant lasota de la maladie de newcastle exprimant la protéine ha du virus h5 de la grippe aviaire - Google Patents

Souche de faible virulence du vaccin recombinant lasota de la maladie de newcastle exprimant la protéine ha du virus h5 de la grippe aviaire Download PDF

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WO2007025420A1
WO2007025420A1 PCT/CN2006/000056 CN2006000056W WO2007025420A1 WO 2007025420 A1 WO2007025420 A1 WO 2007025420A1 CN 2006000056 W CN2006000056 W CN 2006000056W WO 2007025420 A1 WO2007025420 A1 WO 2007025420A1
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newcastle disease
vaccine strain
attenuated vaccine
virus
rlasota
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PCT/CN2006/000056
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Chinese (zh)
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Zhigao Bu
Hualan Chen
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Zhigao Bu
Hualan Chen
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    • C12N2760/18011—Paramyxoviridae
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    • C12N2760/18143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the field of recombinant viral vaccines, and more particularly to a recombinant Newcastle disease LaSota attenuated vaccine strain expressing a gene encoding a wild type or mutant avian influenza virus H5 subtype hemagglutinin (HA) protein, more specifically
  • the recombinant Newcastle disease LaSota attenuated vaccine strains were rLasota-H5wtHA and rLasota-H5mutHA.
  • the invention also discloses a method for preparing the recombinant Newcastle disease LaSota attenuated vaccine strain and the application of the recombinant Newcastle disease LaSota attenuated vaccine strain in preparing a vaccine for preventing avian influenza. Background technique
  • Newcastle disease virus is a non-segmented single-stranded negative-strand RNA virus, which is an important member of the Paramyxoviridae family and a model virus.
  • Recombinant NDV has extraordinary advantages as a live virus vaccine vector: NDV attenuated vaccine including LaSota strain has been used for poultry epidemic prevention for a long time, and its safety and efficacy have been fully proved; NDV inheritance is relatively stable, only one serotype, poison The possibility of recombination and virulence reversion between plants is extremely small; the replication process is completed in the cell paddle, from RNA to RNA, there is no possibility of DNA phase and cell genome integration; NDV attenuated vaccine can induce systemic humoral immunity at the same time, local The formation of mucosal immunity and cellular immunity forms a more comprehensive and accurate immune protection; it can be used in a variety of ways by drinking water, spraying, nasal drops, eye drops or injection; NDV has high titer of chicken embryo growth characteristics, Production costs are extremely low (1
  • the reverse genetic operation of negative-strand RNA viruses is a process of producing new viruses by manipulating viral genomic cDNA.
  • the basic process is: 1 assembling a complete viral genome (or recombinant genome) cDNA clone, precisely at the 5' end. After being affixed to the T7 promoter, 3, the terminal is precisely fused to the self-cleaving nuclease sequence and the T7 transcription termination signal, constitutes a genomic cDNA transcription template; 2 is a genomic cDNA transcription template and a transcription-related functional structural protein necessary for initiating viral replication.
  • NP nuclear protein
  • P phosphoprotein
  • polymerase proteins polymerase proteins
  • RNA virus I'2, 3'4'5'6
  • the NDV genome is 15186 nucleotides in length and, like other paramyxoviruses, includes nuclear protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), and lectin neuraminidase protein (HN). ), and the six large transcriptional coding units of the large polymerase protein (L) (Fig. 1A).
  • NDV nuclear protein
  • P phosphoprotein
  • M matrix protein
  • F fusion protein
  • HN lectin neuraminidase protein
  • L large polymerase protein
  • Avian influenza is an important disease that jeopardizes the development of the world poultry industry. Highly pathogenic avian influenza can cause 100% of deaths in infected poultry and is classified as a Class A severe infectious disease by the OIE.
  • the H5 subtype has historically caused an outbreak of avian flu. Since the end of 2003 to the beginning of 2004, Asian countries such as South Korea, Japan, Vietnam, Thailand, Indonesia, Cambodia, Laos, and mainland China have successively outbreaked H5 subtype highly pathogenic avian influenza, and existing avian influenza inactivated vaccines and poultry pox live carriers.
  • the vaccine has the advantages of safe and immune protection, but it still has the disadvantages of high manufacturing cost and relatively inconvenient use. It is of great practical significance to develop a new generation of vaccines that are efficient, safe, low-cost and easy to use.
  • Avian Influenza is an avian infection and/or disease syndrome caused by Avian Influenza Virus (AIV).
  • AIV is taxonomically classified as: Viral (Vira) - Orthomyxoviridae ( Orthomyxoviridae) - Influenza Virus A and B - Avian Influenza Virus o
  • the avian influenza virus belongs to the influenza A virus of the genus Orthomyxoviridae, and the genome consists of 8 single-strand negative chains. Composition of RNA fragments.
  • the surface structural proteins hemagglutinin (HA) and neuraminidase (NA) are different in antigenicity and are classified into different subtypes.
  • Hemagglutinin is the main immunogenic protein of avian influenza virus, which induces the production of antibody-specific specific humoral and cellular immune responses. Anti-HA antibodies can interfere with the binding of viruses to sialic acid receptors. The process of fusion of the viral envelope with the endocytic membrane neutralizes the infection of the virus.
  • the virulence of AIV is closely related to the amino acid sequence of its surface structural protein HA cleavage site.
  • the low virulence AIV HA cleavage site has only one basic amino acid arginine (R).
  • R basic amino acid arginine
  • High pathogenicity H5 and H7 subtypes The AIV HA cleavage site contains a contiguous number of basic amino acid residues, RKKR, which can be recognized and cleaved by proteases widely present in various cells in the body, thus having a wide range of tissue tropism. Once infected, it can cause systemic spread and lead to rapid death. versus The highly pathogenic H5 and H7 subtypes AIV are potentially more harmful to humans than the subtypes of influenza viruses such as H1, H2, H3 and H9, which can spread systemically and may spread throughout the body. Quickly die.
  • NDV B1 strain expressing H7 subtype HA gene is NDV and The survival protection of H7 subtype highly pathogenic avian influenza lethal challenge is only 60% and 40%, respectively, and does not prevent the replication and release of the virus in vivo (12) .
  • the deficiency and defects of the live virus vector itself, and the cost of use, etc. it has not been widely applied in production practice. Summary of the invention
  • the present inventors further improved the immunogenicity of the avian influenza virus-expressing antigen, and constructed a recombinant NDV live vector two-week attenuated vaccine rLasota-H5wtHA and rLasota-H5mutHA expressing wild-type and mutant avian influenza virus HA immunogen proteins.
  • Animals are immunized by various methods such as nose drops, eye drops, intramuscular injection and even drinking water, spray inhalation, etc. to induce a protective immune response against avian influenza, and are used for the prevention of immunity against avian Newcastle disease and avian influenza.
  • a recombinant Newcastle disease LaSota attenuated vaccine strain which expresses a gene encoding a wild type or mutant avian influenza virus H5 subtype hemagglutinin (HA) protein.
  • the gene encoding the wild type HA protein has the nucleotide sequence set forth in SEQ ID No. 1.
  • the gene encoding the mutant HA protein has the nucleotide sequence shown in SEQ ID No. 2.
  • the Newcastle disease LaSota attenuated vaccine strain is AV1615, and more preferably the recombinant Newcastle disease LaSota attenuated vaccine strain is rLasota-H5wtHA and rLasota-H5mutHA.
  • Still another object of the present invention is to provide a method for producing the above-described recombinant Newcastle disease LaSota attenuated vaccine strain, the method comprising:
  • a transcription plasmid comprising a wild type or mutant avian influenza virus inserted therein Genomic cDNA sequence of the Newcastle disease LaSota attenuated vaccine strain of the H5 subtype HA protein gene (wild type or mutant HA gene);
  • transcriptional helper plasmids comprising a cDNA sequence encoding the nuclear protein (NP) of the Newcastle disease LaSota attenuated vaccine strain, and a phosphoprotein (P) encoding the Newcastle disease LaSota attenuated vaccine strain a cDNA sequence, and a cDNA sequence encoding the large polymerase protein (L) of the Newcastle disease LaSota attenuated vaccine strain;
  • the gene of the HA protein was inserted into the artificially introduced Pmel site between the genomes P and M of the Newcastle disease LaSota attenuated vaccine strain.
  • the LaSota attenuated vaccine strain is AV1615.
  • the genomic cDNA sequence included in the transcription plasmid is located after the T7 promoter, and the genomic cDNA transcription is constituted before the sequence encoding the self-cleaving nuclease and the T7 transcription terminator. template.
  • the self-shearing nuclease is a hepatitis D virus ribozyme (Rib).
  • the cDNA sequence encoding the nuclear protein (NP) of the Newcastle disease LaSota attenuated vaccine strain in the transcriptional helper plasmid, and the phosphoric acid encoding the Newcastle disease LaSota attenuated vaccine strain are included.
  • the cDNA sequence of the protein (P) and the cDNA sequence of the large polymerase protein (L) encoding the Newcastle disease LaSota attenuated vaccine strain are located after the T7 promoter.
  • the transcription plasmid is pBRN-FL-H5wtHA (also known as pBR-FL-H5HA t ) or pBR -FL-H5 mutHA (also known as P BRN-FL-H5HAmut), and the transcriptional helper plasmid is plasmid pBSNP, pBSP And pBSL.
  • the host cell is BHK-21.
  • the present invention also provides the use of the above-mentioned recombinant Newcastle disease LaSota attenuated vaccine strain (especially rLasota-H5wtHA and rLasota-H5mutHA) for preparing a vaccine for preventing avian influenza.
  • LaSota attenuated vaccine strain especially rLasota-H5wtHA and rLasota-H5mutHA
  • the present invention amplifies 10 cDNA fragments of the DV vaccine strain LaSota by RT-PCR, and splices the overlapping portions of the fragments to assemble a full-length cDNA clone.
  • the sequence determination results have been registered in GenBank with accession number AY845400.
  • the HA gene was recombined into P and M of the NDV vaccine strain LaSota, respectively.
  • RNA is synthesized by co-transfection into cells infected with poxvirus expressing T7 polymerase. This RNA is transcribed and replicated under the action of P, P and L proteins. The transfected supernatant was inoculated with SPF embryos to obtain an infectious rescue virus from cDNA. It was confirmed by RT-PCR and genomic cDNA sequence analysis that Lasota derived strains r Lasota-H5wtHA and rLasota-H5mutHA with artificial genetic markers and wild-type or mutant HA genes were rescued.
  • NP nuclear protein
  • P phosphoprotein
  • L large polymerase protein
  • the proliferative characteristics of the rescued virus on chicken embryos were similar to those of the wild-type LaSota vaccine strain, and the blood coagulation price was as high as 2 12 .
  • the above recombinant NDV, rLasota-H5wtHA and rLasota-H5mutHA can be used as a bivalent attenuated vaccine against the attenuated vaccine against avian influenza A and Newcastle disease and avian influenza, which does not interfere with the current general application.
  • Avian influenza epidemiology serological surveillance can be used as a bivalent attenuated vaccine against the attenuated vaccine against avian influenza A and Newcastle disease and avian influenza, which does not interfere with the current general application.
  • HA as an RNA viral envelope protein disease, may be involved in the surface of the viral envelope of recombinant NDV, and may play a role in cell invasion and cell invasion.
  • the present invention manually deletes four basic amino acids (-RKKR-) at the cleavage site by PCR, and mutates another amino acid to form a mutant low-pathogenic form H5 subtype HA gene (mutHA gene).
  • SEQ ID No 2 used to construct a recombinant Newcastle disease LaSota bivalent vaccine strain expressing the H5 subtype avian influenza virus HA antigen.
  • FIG. 1 Assembly of full-length NDV cDNA from subgenomic overlapping cDNA fragments generated from high fidelity RT-PCR.
  • the cDNA fragments were ligated at a consensus restriction site and assembled in the transcription plasmid pBR322, and the RBZ and T7 terminator sequences were pre-cloned in the transcription plasmid PBR322 at Eco?/ and See the instructions for details).
  • (A) shows the first and last nucleotides of the entire full-length genome of the parental NDV.
  • B A cDNA clone of NDV containing the GFP gene is displayed at the top, and the horizontal line below the genetic map shows the position of a single cDNA.
  • FIG. 1 Nucleotide changes introduced into the modified enzyme site by RT-PCR and sequenced by using the PRISM kit (Perkin-Elmer) and Applied Biosystems ABB 10 automated sequencer. Boxed is a nucleotide substitution introduced in pBR l-10 by PCR mutagenesis (mutation from A to G;).
  • FIG. 3A and D NDV-H5wtHA infects BHK-21 cells with an MOI of 1
  • 3B and E NDV-H5mutHA infected BHK-21 cells with MOI of 1
  • Fig. 3C and F NDV LaSota strain control infected BHK-21 cells with MOI of 1, and infected BHK cells were fixed in methanol 20 hours after infection.
  • Chicken anti-H5 subtype avian influenza virus high serum Fig.
  • FIG. 4 Recombinant Newcastle disease virus rLasota-H5wtHA and rLasota-H5mutHA expression H5HA protein blot analysis.
  • Lane 1 Protein labeling
  • Lane 2 Chicken embryo primary cells (CEF) infected with rLasota-H5wtHA
  • Lane 3 CEF cells infected with rLasota-H5mutHA
  • Lane 4 CEF cells infected with rLaSota
  • Lane 5 normal CEF cells
  • Lane 6 H5 subtype highly pathogenic avian influenza virus inoculation Chicken embryo allantoic fluid
  • Lane 7 Newcastle disease virus LaSota attenuated vaccine strain inoculated 9 to 10 days old SPF chicken embryo allantoic fluid
  • Lane 8 SPF chicken embryo allantoic fluid.
  • Figure 5 Comparison of kinetics of chicken embryo growth in recombinant Newcastle disease virus live vector vaccine.
  • FIG. 7 DNA sequence of the pBTRT plasmid.
  • the first italic part T7 promoter; underlined part: ribozyme sequence; second underlined italic part: T7 terminator.
  • FIG 8. P BRN-FL-H5wtHA Plasmid map.
  • Figure 9 DNA sequence of the pBRN-FL-H5wtHA plasmid. Underlined italic part: wtHA gene sequence.
  • Figure 10. Plasmid map of pBRN-FL-H5mutHA.
  • Example 1 Recombinant Newcastle disease expressing wild-type or mutant avian influenza virus H5 subtype hemagglutinin (HA) protein
  • LaSota attenuated vaccine strains BHK-21 cells (milk hamster kidney cell ATCC CCL-10), medium is DMEM containing 10% fetal bovine serum (Hyclone) and lg/ml G418 (Dulbecco's modified Eagle's medium); DV Lasota vaccine strain AV1615 (purchased From the China Veterinary Microorganisms Collection and Management Center (CVCC). Inoculated 9-10 years old SPF chicken embryos were harvested at -70 °C after storage. The chicken anti-NDV high serum was prepared by our laboratory (Chu, HP, G. Snell, DJ Alexander, and GC Schild. 1982.
  • H5 subtype highly pathogenic avian influenza virus HPAIV
  • HPAIV A/Goose/Guangdong/96/l/H5Nl isolate [GD/1/96(H5N1) ⁇ (The earliest isolated H5 subtype highly pathogenic bird in China Influenza virus, Tang Xiuying et al. Identification of Chinese avian influenza epidemic strains.
  • the genomic RNA transcription vector pBTRT was constructed with the low copy cloning vector pBR322 (Invitrogen) and inserted into the T7 promoter (T7 promotor), the hepatitis D virus ribozyme (Rib) and the T7 transcription termination signal (T7 terminal) at the EcoR a I site. Built by the lab itself.
  • the DNA fragment cloned between the T7 promoter and the ribozyme can be transcribed under the action of T7 RNA polymerase, and due to the autocatalytic function of Rib, the 3' end of the transcript can be ensured to be exactly identical to the cloned DNA fragment.
  • H5mutHA gene gene full-length DNA sequence shown in Figure 11 underlined italic part, sequence listing SEQ ID No 2
  • the T7 RNA polymerase promoter was prefixed to the 5' end of the full-length cDNA fragment, and a self-catalyzing hepatitis delta ribozyme (GenBank X04451) and a T7 transcription termination signal were ligated to the cDNA fragment.
  • the constructed plasmids were named pBRN-FL-H5wtHA and pBRN-FL-H5mutHA, respectively.
  • Genomic RNA was extracted by conventional method (animal virology, second edition); the whole gene component was 10 fragments (F1-F10) with overlapping ends and RT-PCR amplification.
  • the cDNA fragment was cloned into the pBluescript (Clontech) Smal site and confirmed by sequence analysis to be identical to the viral genomic RNA sequence; the sequence assay results were registered in GenBank under accession number AY845400.
  • a methylated J3 ⁇ 4d site was selected at 6172 bp of the Lasota vaccine strain genomic cDNA, and the sequence was TCTAGATCA, which was mutated to TCTAGACCA by PCR, so that it was no longer recognized by methylase.
  • the restriction endonuclease the restriction enzyme cleavage site existing in the overlapping portion of the adjacent fragment is ligated to assemble the complete NDV genomic cDNA (Fig. 1A), and the wild type H5 subtype avian influenza virus is separately And mutant HA genes H5wtHA and H5mutHA genes (wtHA: with Trizol
  • Upstream primer 5 (Invitrogen) proposed the IBDV genome, and after reverse transcription, the gene was amplified by PCR. The following primers were added to the system. Upstream primer 5 '
  • the transcription plasmids pBRN-FL-H5wtHA and pBRN-FL-H5nmtHA (Fig. IB); the open reading frame (ORF) cDNAs expressing the nuclear protein (NP), phosphoprotein (P) and large polymerase protein (L) genes are followed by The clones were cloned downstream of the pBluescript II (+/-) plasmid T7 promoter and constituted the transcriptional helper plasmids pBSNP, pBSP and pBSL, respectively.
  • BHK-21 cells were first co-transfected with pBRN-FL-H5wtHA and P BRN-FL-H5mutHA and a helper plasmid expressing NDV NP, P, L protein, respectively.
  • the fusion protein F0 of NDV must be lysed into F1 and F2 to be infectious.
  • BHK-21 cells can not secrete the protease required for cleavage of F0 protein.
  • TPCK toluenesulfonylalanyl chloride. Sigma
  • Viral positive allantoic fluid was harvested as the F1 generation of the rescued viruses rLasota-H5wtHA and rLasota-H5mutHA. Further RT-PCR and sequence analysis showed that the 6178-point base of the F1 generation rescued the viral genomic cDNA was C, but not the C of the original LaSota parental strain, which was completely consistent with the expectation (Fig. 2). The results showed that the infectious progeny viruses rLasota-H5wtHA and rLasota-H5mutHA were successfully rescued by NDV LaSota vaccine strain genomic cDNA clone by reverse genetic manipulation technique. More specifically, the experimental steps are as follows:
  • the transcription plasmid and helper plasmids pBRN-FL-H5wtHA or pBRN-FL-H5mutHA, pBSNP, pBSP and pBSL were 5 g, 2.5, respectively.
  • co-transfected BHK-21 cells, using the CaP04 transfection kit (Invitrogene) the operation was carried out according to the kit instructions.
  • the transfection mixture was discarded, cells were shocked with PBS containing 10% DMSO for 2.5 minutes, added to complete DMEM overnight, replaced with serum-free medium the next day, and added with TPC (1 ⁇ ⁇ /ml) After 2-3 days of incubation, the culture supernatant was harvested, filtered through a 0.22 um pore filter, and inoculated with SP-11 embryoid sac cavity for 9-11 days; the SPF embryo after inoculation was further cultured, 3-5 days, and the chicken embryo was taken.
  • the NDV LaSota vaccine strain transiently infects mammalian cells cultured in vitro.
  • rLasota-H5wtHA and rLasota-H5mutHA viruses in BHK-21 cells and expression of viral antigens, both of which have an infection with MOI of 1 to infect approximately 70-80% of monolayer BHK-21 cells (Fig. 3A) And B)
  • the NDV wild-type LaSota vaccine strain infected cells as control Fig. 3C
  • the cells showed early CPE (cytopathic) phenomenon 20 hours after infection, and immediately indirectly immunized with NDV high-free SPF chicken positive serum as detection antibody. Fluorescence staining, as a result, strong positive reactions were observed under fluorescence microscopy of the three virus-infected cells (Fig. 3A, B and C). More specifically, the experimental steps were as follows:
  • FITC Fluorescein-labeled rabbit anti-chicken IgG secondary antibody
  • CEF chicken embryo fibroblast
  • Antigen high serum was primary antibody, horseradish peroxidase (HRP) labeled rabbit anti-mouse goat anti-mouse IgG (Sigma) secondary antibody, 1:2500 times diluted with PBST, DAB (diaminobiphenyl gum, Sigma) After 3 to 5 minutes of color, the reaction was quenched with deionized water. The results are shown in Figure 4, demonstrating that recombinant NDV expresses wild-type and mutant AVI HA proteins, respectively.
  • HRP horseradish peroxidase
  • rLasota-H5wtHA and rLasota-H5mutHA were positive for HA antigen infection in CEF, and Newcastle disease Lasota vaccine strain virus infection and HA antigen detection without CEF were negative; H5N1 subtype highly pathogenic avian influenza
  • the virus GD/1/96 strain was positive for HA antigen inoculated with SPF chicken embryo allantoic fluid, while the Newcastle disease Lasota vaccine strain virus inoculation and non-inoculated SPF chicken embryo allantoic fluid HA antigen were negative.
  • H5 subtype avian influenza virus HA antigen was correctly expressed in the recombinant Newcastle disease virus rLasota-H5mutHA and rLasota-H5wtHA.
  • Example 4 Growth characteristics and pathogenic characteristics of rNDV in chicken embryos To determine the growth characteristics of chick embryos of rLasota-H5wtHA and rLasota-H5mutHA rescued by reverse genetic manipulation and their pathogenicity to chicken embryos, virus embryos will be rescued. The F1 generation was inoculated with lxl0 4 EID 5Q for 9 to 10 days of ⁇ SPF chicken embryo allantoic cavity.
  • the wild-type Newcastle disease virus LaSota vaccine strain (rLaSota) rescued by reverse genetic manipulation did not kill SPF chicken embryos within 120 hours. Allantoic fluid was harvested at 24 hours, 48 hours, 72 hours and 96 hours after inoculation. EID 5Q the cyst fluid were 10- 8 '5, 10' 8 ⁇ 6, 10 '1 ⁇ and 10' 9.4.
  • rLasota-H5wtHA and rLasota-H5mutHA were inoculated for 9 to 10 days in the same dose route as SPF chicken embryo chorioallantoic cavity, and SPF chicken embryos were also not killed within 120 hours; rLasota-H5wtHA harvested at 24 hours, 48 hours, 72 hours and 96 hours after inoculation allantoic fluid per milliliter of allantoic fluid were then EID 5G 10- 8 ⁇ 2, 10 ⁇ 8 ⁇ 6, 1 (T 9 'Q and 1 ( ⁇ 8 ⁇ 5.
  • rLasota-H5wtHA and rLasota-H5mutHA were compared with the reverse genetic manipulation to rescue the wild-type Lasota vaccine strain (rLaSota), rLaSota, for pathogenicity analysis.
  • Intracerebral pathogenicity index (ICPI), intravenous pathogenicity index (IVPI) and average embryonic lethal time (MDT) were determined according to the recommendations of the International Organization for Animal Health (O.I.E.).
  • the LaSota live vaccine strain for newborn pheasant should have an ICPI of around 0.4 or below 0.4.
  • the rLasota-H5mutHA virus not only maintained the low pathogenicity of the NDV Lasota strain, AV1615, but was also weakened.
  • the above results indicate that the recombinant NDV maintains the high titer growth characteristics and low pathogenicity of the parental LaSota vaccine strain in the SPF dock.
  • Virus strains Chicken embryos mean brain pathogenicity index Intravenous pathogenicity index F protein cleavage site Dead time (hours) (ICPI) ** (IVPI) ** Sequence analysis *** ( MDT ) **
  • rLasota >120 0.35 0 GGRQGR L rLasota-H5mutHA >120 0 0 GGRQGR L rLasota-H5wtHA >120 0 0 GGRQGR L
  • rLasota-H5wtHA was used as an example.
  • Chicken embryos were amplified by F1 generation of allantoic acid 2xlO ID 5Q .
  • Artificial immunization of 12 7-day-old white henhen SPF chicks (provided by SPF Experimental Animal Center of Harbin Veterinary Research Institute), and another set of non-immune control group 8; immunization group and non-immune control group were respectively isolated in air negative pressure filtration In the device.
  • the blood samples from the wing veins were routinely tested for specific hemagglutination inhibitory antibodies against Newcastle disease and H5 subtype avian influenza.
  • the rLasota-H5wtHA and rLasota-H5mutHA viruses were each compared with the NDV Lasota vaccine strain AV1615 for induction of protective antibody immunoreactivity.
  • the results are shown in Tables 2 and 3, indicating that both the recombinant viruses rLasota-H5wtHA and rLasota-H5mutHA are capable of simultaneously inducing a protective antibody immunoreactivity against NDV and AIV.
  • Table 2 H5 subtype avian influenza Newcastle disease virus live vector bivalent vaccine (rLasota-H5wtHA) immunized 1 week old SPF chicks induced protective antibody immune response
  • the vaccine is immunized on the 7th day by intranasal and intraocular injection, with a total volume of 100 ⁇ per feather;
  • rLasota-H5wtHA and rLasota-H5mutHA viruses were used to immunize 1 week old SPF chicks with two H5 subtype avian influenza Newcastle disease virus live vector bivalent vaccines, and to evaluate the lethality of H5 subtype ⁇ pathogenic avian influenza. Immune protection.
  • the results are shown in Tables 6 and 7.
  • the results showed that the rLasota-H5wtHA and rLasota-H5mutHA virus recombinant vaccines immunized chickens with 100% complete immune protection against Newcastle disease virulent strains and H5 subtype highly pathogenic avian influenza virus. No disease, no death; H5 subtype high pathogenic avian influenza virus completely prevented respiratory and digestive tract virus emissions after attack.
  • the non-coding region between the P gene and the M gene in the genome was a foreign gene insertion site, and a HA immunogen gene expressing the H5 subtype ⁇ pathogenic avian influenza virus wild type and an artificial deletion cleavage site with a plurality of bases were constructed.
  • Recombinant NDV strain of mutant HA immunogenic gene, rLasota-H5wtHA and rLasota-H5mutHA as a bivalent attenuated vaccine candidate against Newcastle disease and H5 subtype highly pathogenic avian influenza, and biosafety Evaluation. Studies have shown that the NDV genome inserts exogenous reporter genes or immunogenic genes at different sites, and the biological characteristics, low pathogenicity and genetic stability of the genome are maintained by successive high passages of cells or chicken embryos.
  • the immunoassay results of the recombinant viruses rLasota-H5wtHA and rLasota-H5mutHA showed that the rLasota-H5mutHA and rLasota-H5mutHA recombinant vaccines can produce 100% of the lethal attack of H5 subtype highly pathogenic avian influenza and Newcastle disease virulent immunity.
  • Fully immunoprotective, the ability to induce protective antibodies is comparable to existing inactivated vaccines, and has an advantage in inducing important mucosal and cellular immunity; maintaining a safe, effective, high titer of the parental LaSota vaccine strain on newborn chicks
  • the advantages of chicken embryo growth characteristics, ease of use, etc.; environmental and social benefits are significant, compared with the traditional avian influenza vaccine, the same dose of vaccine production is only one percent of the chicken embryo, the product volume is only one thousandth, and the production
  • the use of mineral oil is not required, and the effect of traditional oil emulsion inactivated vaccine injection on immunization against commercial chickens is completely avoided.
  • Newcastle disease virus as a live virus vector has great advantages in constructing a double-valent vaccine against avian influenza and Newcastle disease.
  • Avian influenza and Newcastle disease have been classified as Class A severe infectious diseases by the OIE. They are an important disease that jeopardizes the development of the world poultry industry. Avian influenza also has extremely important public health significance.
  • the attenuated vaccine used in Newcastle disease prevention in China is at least 10 billion feathers per year.
  • the application of NDV attenuated vaccine, especially LaSota vaccine strain is an indispensable immunization program for all newborn chicks in China.
  • Newcastle disease virus (NDV) is a non-segmented single-stranded negative-strand RNA virus with clear genomic structure and functional background. Only one serotype is genetically stable.
  • the foreign gene inserted in recombinant NDV is high in cells or chicken embryos. Stable expression can still be maintained after sub-passage, and is very suitable as an expression or vaccine vector.
  • the safety and efficacy of the NDV attenuated LaSota vaccine strain has been fully proved; the live vaccination vaccine can simultaneously induce systemic humoral immunity, local mucosal immunity and cellular immunity to form a more comprehensive and accurate immune protection; , spray, nose, eye or injection a variety of ways to the seedlings, the use of extremely convenient; DV has a sputum titer of chicken embryo growth characteristics, production costs are extremely low.
  • rLasota-H5mutHA recombinant vaccine will make the H5 subtype highly pathogenic avian influenza vaccine control almost no additional manufacturing and use costs, and the country can save at least tens of millions of dollars of epidemic prevention funds and a large number of society every year. Labor costs, and reduce the adverse stress response of immunized subjects.
  • the available data show that this vaccine has great social, economic and environmental benefits compared with the existing Newcastle disease and avian influenza vaccines, and the domestic and international markets have broad prospects.
  • Newcastle disease virus as a vaccine carrier is an internationally advanced new technology. If the progress is further accelerated, H5 Asia The avian influenza Newcastle disease virus live vector vaccine is expected to become the first live vaccine for the negative-strand RNA virus in the world.

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Abstract

L’invention concerne la souche de faible virulence du vaccin recombinant Lasota de la maladie de Newcastle exprimant la protéine hémagglutinine sauvage ou mutante (HA) du virus H5 de la grippe aviaire. En particulier, les souches de faible virulence du vaccin recombinant Lasota de la maladie de Newcastle sont la rLasota-H5wtHA et la rLasota-H5mutHA. La présente invention a aussi pour objet une méthode pour préparer des souches de faible virulence et l'utilisation de ces souches pour préparer un vaccin afin de prévenir la grippe aviaire.
PCT/CN2006/000056 2005-09-02 2006-01-13 Souche de faible virulence du vaccin recombinant lasota de la maladie de newcastle exprimant la protéine ha du virus h5 de la grippe aviaire WO2007025420A1 (fr)

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CN107188934A (zh) 2012-02-13 2017-09-22 高等教育联邦系统-匹兹堡大学 以计算方式优化的人和禽h5n1流感的广泛反应性抗原
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WO2010058236A1 (fr) * 2008-11-19 2010-05-27 Laboratorio Avi-Mex, S.A. De C.V. Vaccin recombinant à vecteur viral inactivé
CN117224666A (zh) * 2023-08-30 2023-12-15 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） 一种禽流感、新城疫病毒二联五价疫苗组合物及其应用
CN117224667A (zh) * 2023-08-30 2023-12-15 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） 一种禽流感、新城疫病毒疫苗组合物及其应用
CN117224667B (zh) * 2023-08-30 2024-05-28 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） 一种禽流感、新城疫病毒疫苗组合物及其应用
CN117224666B (zh) * 2023-08-30 2024-05-28 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） 一种禽流感、新城疫病毒二联五价疫苗组合物及其应用

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