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WO1999028445A1 - Vaccin contre la grippe attenue vivant - Google Patents

Vaccin contre la grippe attenue vivant Download PDF

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Publication number
WO1999028445A1
WO1999028445A1 PCT/KR1998/000384 KR9800384W WO9928445A1 WO 1999028445 A1 WO1999028445 A1 WO 1999028445A1 KR 9800384 W KR9800384 W KR 9800384W WO 9928445 A1 WO9928445 A1 WO 9928445A1
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Prior art keywords
virus
influenza virus
htca
influenza
cold
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Inventor
Balk Lin Seong
Kwang Hee Lee
Jin Won Youn
Suk Joon Kim
Kyung Ho Cheoun
Jeong-Hyun Kim
Hong Gi Kim
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CJ Corp
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CJ Corp
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • C12N7/08—Inactivation or attenuation by serial passage of virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a novel cold-adapted influenza virus which is useful in the preparation of an safe and effective influenza vaccine.
  • the present invention relates to a process for producing a cold-adapted influenza virus by conducting passage culture of influenza virus capable of actively growing in fertilized egg selected as parent virus, in the fertilized egg at low temperatures, cold-adapted influenza viruses prepared by said process, a recombinant cold-adapted influenza virus prepared by co-infecting the host cell with said cold adapted influenza virus and a wild type influenza virus, and an influenza vaccine comprising said cold adapted influenza virus and/or said recombinant cold adapted influenza virus.
  • influenza virus is virulent and infectious, and occasionally causes severe disease to many animals. It infects humans, live stock, and other animals including birds, horses, seals, whales and mink, via their respiratory systems. The influenza virus primarily infects the upper respiratory system but may penetrate into the lung to cause the fatal diseases.
  • influenza virus is able to modify its surface antigens, the haemagglutinin (hereinafter referred to as "HA”) and the neuraminidase (hereinafter referred to as "N A”), by the antigenic shift/antigenic drift. Therefore, it is difficult to prevent the influenza virus by vaccine. To prevent the spread of the influenza virus, it is necessary to predict the character of the next influenza virus so that an immunologically effective vaccine can be made.
  • HA haemagglutinin
  • N A neuraminidase
  • influenza viruses which can be actively grown in the fertilized egg are usually used in the preparation of the vaccine for the targeted virus.
  • the six genes of the A/PR/8/34 or A/X-31 except for HA and NA are typically used in preparing the vaccine against the influenza virus A ⁇ Influenza, Plenum Medical Book Company, 291, 1987).
  • the currently available influenza vaccines are a killed vaccine.
  • the examples of the killed vaccine include whole virion vaccine, disrupted virus vaccine, subunit vaccine and the like.
  • the whole virion vaccine is prepared by inactivating the whole virion, first developed in early 1930's, with formalin or ⁇ -propiolactone and purifying it.
  • the disrupted virus vaccine is prepared by treating virion with an non-ionic detergent, for example, Tween-ether mixture, sodium desoxycholate, Triton N101, ethylmethyl- ammoniumbromide and the like, and purifying it.
  • the subunit vaccine is made by isolating the surface antigens HA and NA of virus and purifying them.
  • the killed vaccine has been primarily used in prevention of the flu and its preventive effect was reported to be from about 70 to 80%.
  • the killed vaccine has some problems in its preparation and application to humans.
  • its immunity lasts for a short period, in particular in adults.
  • Live vaccines have been investigated. They should be non-toxic or slightly toxic, and also be genetically stable so that toxicity does not revive after administration. Attenuated live vaccines and methods for the preparation of the vaccines have been suggested. The examples include a temperature-sensitive mutant prepared by mutagenesis ⁇ J. Infect. Dis. 128, 479, 1973), excellent replicable mutant at low temperatures screened following successive passages at low temperatures ⁇ Vaccine 3, 335, 1985), and host-range mutant that can not replicate in humans while rapidly replicating in the other animals ⁇ Science 218, 1330, 1982).
  • the most available virus as live vaccine virus is the influenza virus strain produced by cold adaptation method. Two cold-adapted attenuated strains have been used in Russia for years. One is A/Leningrad/134/17/57(H2N2) strain obtained by 17 passages of the virus at 25 "C. It was reported that the virus has PA, NP and M protein mutations which involve temperature sensitivity ⁇ J.Gen. Virol. 53, 215, 1981). Another is A/Leningrad/134/47/57(H2N2) strain obtained by 47 passages of the virus at 25 ° C. This virus is known to include PB2, PA, NP, M, and NS protein mutations which involve temperature sensitivity ⁇ Infect. Immun 44, 730, 1984). The A/Leningrad/134/
  • A/ Ann Arbor/6/60(H2N2) cold-adapted virus ⁇ Nature 213, 612, 1967.
  • the cold-adapted A AnnArbor/6/60 strain was obtained by passage of the virus in primary chick kidney cells and fertilized eggs at temperatures of 33°C, 30°C and 25°C. This strain is temperature sensitive in that it does not replicate at 38 ° C to 39°C, at which wild type virus is replicable, but well replicate at 25°C. It also elicits toxicity-reduced phenotype in that it little replicate at the upper and lower respiratory tract ⁇ Rev. Infect. Dis. 2, 421, 1980; Arch. Virol. 64, 171, 1980). The analysis of the virus genomes revealed that mutations are found in all of eight genomic genes ⁇ Virology 167, 554-567, 1988).
  • a recombinant virus was prepared using the parent A/ Ann Arbor/6/60 and the influenza virus HlNl or H3N2 and was applied to humans. It was characterized by attenuation, immunogenicity, especially induction of IgA, genetic stability, and little virulence.
  • the live vaccine containing cold-adapted viruses have several advantages over the killed vaccine.
  • the purpose of the invention is to provide a new cold-adapted influenza virus useful in preparation of toxicity-reduced, genetically stable and effective live influenza vaccine.
  • the invention was accomplished by conducting passage culture of A/X-31 virus in fertilized egg at low temperature of 24 ° C to obtain cold-adapted virus, co-infecting the resulting cold-adapted virus together with wild type influenza virus to obtain recombinant influenza virus, and confirming their excellent growth and greatly reduced toxicity.
  • the present invention is to provide a novel cold-adapted influenza virus which exhibits temperature sensitivity, is actively grown in fertilized egg, and exposes greatly reduced toxicity in animals including humans. It is therefore useful in preparation live influenza vaccine.
  • the present invention is to provide a cDNA gene of said cold- adapted influenza virus.
  • the present invention is also to provide a process for preparing said cold adapted influenza virus which comprises conducting passage culture of an influenza virus, which is actively grown in fertilized egg, in a manner of injecting the influenza virus into the fertilized egg, culturing the egg at low temperatures for several days to amplify the virus, recovering the virus from the allantoic fluid of the egg, and again injecting the recovered virus in another fertilized egg and repeatedly carrying out the next steps.
  • the present invention is also to provide a recombinant influenza virus prepared by co-infecting fertilized egg with the above cold-adapted virus and wild type influenza virus. Additionally, the present invention is to provide a use of the above cold-adapted influenza virus or the recombinant influenza virus in preparation of live influenza vaccine.
  • Figure 1 shows the comparison of toxicities induced by infecting rats with 50 ⁇ l (10 7 pfu/ml) of influenza virus A X-31 and 50 ⁇ l of cold adapted influenza virus HTCA- A101 via their nasal cavities (D: A/X-31 virus infection; O: HTCA-AlOl virus infection; ⁇ : PBS control)
  • Figure 2 shows the changes of average weight of live mice at the indicated times after they were infected with various concentrations of wild type A/PR 8/34 virus via their nasal cavities ( ⁇ : PBS control; D: 10 4 pfu virus infection; O: 10 5 pfu virus infection; ⁇ : 10 6 pfu virus infection; ⁇ : 10 7 pfu virus infection; •: 10 8 pfu virus infection).
  • Figure 3 shows the number of dead mice at the indicated times after they were infected with various concentrations of wild-type A/PR/8/34 virus via their nasal cavities ( ⁇ : PBS control; D: 10 4 pfu virus infection; 0: 10 5 pfu virus infection; ⁇ : 10 7 pfu virus infection; •: 10 8 pfu virus infection).
  • the cold-adapted influenza virus is prepared by passage culture of influenza virus which is actively grown in fertilized egg, at low temperatures.
  • a X-31 virus and B/Yamagata/16/88 were used as parent influenza virus type A and type B, respectively.
  • passage culture at low temperatures reduces growth rate of viruses.
  • virus with excellent growth capability in fertilized egg was selected as parent.
  • the passage culture was carried out by inj ecting the selected influenza virus into fertilized egg, culturing the egg at low temperatures of from 30°C to 24°C for several days to amplify the virus, recovering the virus from the allantoic fluid of the egg, and again injecting the recovered virus in another fertilized egg, culturing the egg at the same low temperature for the same days to amplify the virus, recovering the virus from the allantoic fluid of the egg, and repeating the cycle several times. If excessive amount of the recovered virus from the allantoic fluid is injected into the fertilized egg, then defective interfering particles form and reduce the growth of the virus. Thus, it is preferable to using the recovered virus from the allantoic fluid as the least amount.
  • the resulting cold-adapted virus was more actively grown at low temperatures of 30°C or less, compared to the parent virus, but was not grown at body temperature (about 37 ° C). It can elicit immunity with greatly reduced toxicity and thus is believed to be useful for attenuated vaccine.
  • the A X-31 virus was injected into fertilized egg, and then 19 passages at 30°C, 40 passages at 27 ° C, and 33 passages at 24°C were carried out in turn to obtain cold-adapted influenza virus A.
  • the resulting virus A was referred to as HTC A- A101.
  • the B/Yamagata/ 16/88 virus was injected into fertilized egg, and then 8 passages at 30°C, 11 passages at 27 ° C, and 21 passages at 24 ° C were carried out in turn to obtain cold-adapted influenza virus B.
  • the resulting virus B was referred to as HTCA-B102.
  • Another cold-adapted influenza virus B was obtained by injecting the B/Lee/40 virus into fertilized egg, and then performing 7 passages at 30 ° C, 36 passages at 27°C, and 32 passages at 24 ° C. It was referred to as HTCA-B 104.
  • RNAs were isolated from the cultured viruses and the RT-PCR (reverse-transcription polymerase chain reaction) was conducted using the isolated RNAs as template to construct cDNAs. Specifically, primers for the RT-PCR were designed from PB2, PB 1 ,
  • RNA. PB2, PB1 and PA protein cDNAs coding for polymerase are represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • NP protein cDNA coding for nucleoprotein is represented by SEQ ID NO: 4.
  • M and NS protein cDNAs are represented by SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the HTCA-AlOl virus genes were compared with those of A/X-31 virus (parent strain of the HTCA-AlOl), A/PR/8/34 virus (parent strain of the A/X-31 ), and other influenza viruses registered in
  • influenza virus of the invention In order to confirm whether the cold-adapted influenza virus of the invention is an attenuated virus, its plaque-forming capability was tested by culturing the virus in MDCK cell at varied temperatures. The result was that the influenza virus of the invention was characterized by cold adaptive and temperature sensitive phenotypes.
  • the virus of the invention is industrially useful.
  • the present invention provides a recombinant influenza virus prepared by co- infecting the fertilized egg with the cold adapted influenza virus and the wild type influenza virus. After mammalian cells are co-infected with the cold adapted influenza virus according to the invention and the wild-type pathogenic influenza virus, they are cultured for a sufficient time. Genes are exchanged from both viruses during the culture to generate several recombinant viruses. The cold adapted recombinant virus is screened from them using an antibody against a cold adaptive strain. The plaque inhibition assay is then carried out to confirm whether the screened virus is a recombinant virus.
  • the fertilized egg is co-infected with the cold-adapted virus HTCA- A101 according to the invention and the highly toxic virus HlNl or H3N2, and is cultured. Then, the recombinant influenza virus is screened by the plaque inhibition assay.
  • the screened recombinant influenza virus was cultured in MDCK cells or fertilized eggs at varied temperatures.
  • the recombinant influenza virus proved to be cold adaptive and temperature sensitive and was also confirmed as exhibiting greatly reduced toxicity.
  • the nasal cavity of a mouse or a rabbit was infected with the virus to determine how the condition of the experimental animal changed as time passed. The toxicity of the recombinant influenza virus was thereby found to be low enough to be used for live influenza vaccine.
  • the fertilized eggs purchased from SPAFAS, USA were cultured in a cultivator at 37°C for 10 days.
  • the cultured eggs were examined so that only eggs showing good embryo growth were selected.
  • the selected eggs were used for virus infection.
  • the injection area was washed with 70% alcohol solution and a hole was punched in the eggshell.
  • the amount of the injected virus was between 0.01 and 0.001 HAU.
  • the hole of the eggshell was sealed with candle wax and the egg was cultivated in a cultivator at 30 ° C, 27°C and 24 ° C for 3 or 4 days. Thereafter, the eggs were placed at 4°C for at least 5 hours to inactivate the embryo. The air sac area of the inactivated egg was sheared with scissors and allantoic fluid was collected. The amount of the virus in the fluid was measured by haemagglutination assay. The fluid was filtered with 0.2 ⁇ m syringe filter and was diluted as necessary with PBS. The dilution was injected into a new egg. In this manner, the passage culture was conducted up to 19 passages at 30"C, 40 passages at 27 ° C, and 33 passages at 24 ° C, to obtain the cold adapted influenza virus
  • the resulting cold adapted influenza virus A was referred to as HTC A-A 101 and was deposited under accession number of KCTC 0400BP at the Korea Research Institute of Bioscience and Biotechnology Korean Collection for Type Cultures, Taejon, Republic of Korea on November 11, 1997. This was done in accordance with the Budapest Treaty for the international regulation of the deposit of microorganisms for the purpose of patenting.
  • the cold adapted influenza virus B was obtained in the same manner as in Example 1 above, except that B/Yamagata/16/88 was used as influenza virus B, and the passage culture was conducted up to 8 passages at 30°C, 11 passages at 27°C, and 21 passages at 24°C.
  • the resulting virus was referred to as HTCA-B102 and was deposited under accession number of KCTC 0401 BP at the Korea Research Institute of Bioscience and Biotechnology Korean Collection for Type Cultures, Taejon, Republic of Korea on November 11, 1997. This was done in accordance with the Budapest Treaty for the international regulation of the deposit of microorganisms for the purpose of patenting.
  • the cold adapted influenza virus B was obtained in the same manner as in Example 1 above, except that B/Lee/40 was used as influenza virus B, and the passage culture was conducted up to 7 passages at 30°C, 36 passages at 27 ° C, and 32 passages at 24 ° C.
  • the resulting virus was referred to as HTCA-B104 and was deposited under accession number of KCTC 040 IBP at the Korea Research Institute of Bioscience and Biotechnology Korean Collection for Type Cultures, Taejon, Republic of Korea on November 11, 1997. This was done in accordance with the Budapest Treaty for the international regulation of the deposit of microorganisms for the purpose of patenting.
  • the genes were sequenced and their base sequences were compared to identify at the molecular dimension the mutations accumulated in the HTC A- A 101 virus during the cold adaptation.
  • the gene cloning of the A/X-31 and the HTCA-A101 was carried out as follows: The monolayer of MDCK cells originating from canine kidney was infected with each virus at 5 moi. The A/X-31 was cultured at 37°C for 6 hours, whereas the HTCA-AlOl was cultured at 30 ° C for 6 hours. Thereafter, cell portions were taken and the RNAs were isolated using QIAGEN's RNeasy Total RNA Kit. The primers for the polymerase chain reaction (PCR) were designed from PB2, PBl and PA protein genes. The PCR reaction yielded the products containing the intact genes of each influenza virus.
  • PCR polymerase chain reaction
  • RNA concentration was measured at optical absorbency of 260 nm.
  • the RT-PCR was carried out using the Superscript Preamplification System. 2 ⁇ g of RNAs were used as a template for PB2, PBl and PA protein genes, whereas 2 ⁇ g of RNAs were used as a template for NP, M and NS protein genes.
  • the base sequences of the primers used in the RT-PCR are shown in Table 1 below. Table 1
  • the PCR product amplified by the above procedure was purified using the Qiaquick PCR Purification Kit.
  • the purified product was phosphorylated by T4 polynucleotide kinase, which was purchased from NEB, and an electrophoresis was conducted on 1% agarose gel. The portion corresponding to the desired size was separated from the agarose gel, and was purified using GENOMED's JetSorb Gel Extraction Kit.
  • the genes obtained by this method were used as a template for determining the nucleotide sequence.
  • pUC18 plasmid was digested with restriction enzyme Hindi, which is commercially available from NEB, and the excised vector was then isolated by an electrophoresis on 1% agarose gel, and was then purified using the JetSorb Gel Extraction Kit. The purified fragment was combined with the above obtained genes and the resulting plasmid was transformed into E. coli. This constructed recombinant plasmid containing the virus gene will be used as a cloning vector.
  • the Applied Biosystem's automatic sequencer was used in determining the base sequence.
  • the Applied Biosystem's DNA/RNA synthesizer was used to prepare the primers.
  • sequences of the primers were designed at intervals of 300 nucleotides from the reported nucleotide sequence of the A PR/8/34 gene. Table 2 below shows the sequences of the primers.
  • the A/X-31 is a recombinant virus in which 6 internal genes were inherited from the A PR 8/34, and the surface genes HA and NA were inherited from the A/HK/68. Because the A/X-31 was adapted in the fertilized egg for a long time, it contains many mutations and therefore does not appears to be virulent to humans.
  • the genes of the A/PR/8/34 were compared with the genes of the A/X-31 to detect the base and amino acid mutations which occurred during its adaptation in the fertilized egg (Tables 3 a, 4a,
  • Tables 3c, 4c, 5c, 6c and 7c of other influenza viruses (Tables 3b, 4b, 5b, 6b, 7b and 8b) registered in the Genbank Database, using the clustal V program (Gene 73, 237-244, 1988).
  • the nucleotide sequences of the A/PR/8/34 genes were compared with the nucleotide sequences of the A/X-31 genes.
  • the comparison revealed that 71 mutations occuned in the A/X-31 gene. Only 24 base mutations resulted in amino acid substitutions.
  • the substituted amino acids were compared with the corresponding amino acids of other influenza virus proteins registered in the Genbank Database.
  • the result was that 9 distinct mutations were observed (2 mutations in PB2, 2 mutations in PBl, 1 mutation in PA, 2 mutations in Ml and 2 mutations in M2). These mutations were different from those reported to involve host-specificity or attenuation. However, because few mutations involving host-specificity or attenuation have been reported up to now, it is possible that mutations in the A/X-31 involve attenuation of the virus and substantial growth of the virus in the fertilized egg.
  • the mutations were detected in the HTC A- A 101 which occurred during the cold adaptation of the A/X-31 virus.
  • the base sequences of the A/X-31 genes were compared with the base sequences of the HTC A- A 101 genes. 30 base mutations in the HTC A- A 101 genes were observed, and 16 amino acid substitutions were found to have occuned in the HTCA-A 101 protein.
  • the mutated HTCA-A 101 proteins were compared with the proteins of Genbank's viruses. Some distinct amino acid mutations were observed (2 mutations in PB2, 2 mutations in PBl , 2 mutations in NP, 2 mutations in Ml and 1 mutation in M2), including host specificity-involving mutation (1 mutation in Ml), and attenuation-involving mutation (1 mutation in M2). Therefore, it is believed that the HTCA-A 101 virus was attenuated by a specific gene mutation which is distinct from any known mutations of cold adapted viruses.
  • the PB2 protein gene of the A/X-31 was compared with that of the A/PR/8/34 virus and other influenza strains registered in the Genebank Database. Table 3a below shows the results. Table 3b below indicates the Genbank's influenza viruses used to compare the amino acid sequence of the PB2 protein. In addition, the PB2 protein gene of the HTCA-AlOl was compared with the PB2 protein gene of the A/X-31 and other Genbank's strains. Table 3 c below shows the results.
  • the PB2 protein is a partial component of the polymerase complex. It specifically recognizes and binds 5'-cap of the host mRNAs ⁇ PANS 78, 7355-7359, 1981; NAR 10,
  • the PB2 protein of the HTCA-A 101 comprises glutamine at amino acid 110, whereas the PB2 proteins of other viruses registered in the Genbank Database all comprises histidine at amino acid 110.
  • the PB2 protein of the HTCA-AlOl comprises serine at amino acid 588.
  • the amino acid 588 in the PB2 protein of the known human-infecting viruses is isoleucine.
  • the swine- or bird- infecting viruses comprise alanine and the horse-infecting viruses comprises threonine at amino acid 588. Even though there are no available reports, the data in Table 3c above suggests the possibility that the amino acid 588 in the PB2 protein involves the host- specificity.
  • the PBl protein gene of the A X-31 was compared with that of the A PR 8/34 and other influenza strains registered in the Genbank Database. Table 4a below shows the results. Table 4b below indicates the Genbank's influenza viruses used to compare the amino acid sequence of the PBl protein. The PBl protein of the HTCA-AlOl was compared with that of the A/X-31 virus and other Genbank's strains. Table 4c below shows the results.
  • the PB 1 protein is a partial component of the polymerase complex and is known to initiate the synthesis of RNA strands by transcription ⁇ Cell 34, 609, 1983 ; J. Virol. 70. 6390-6394, 1996).
  • Mutations V91A and G684E of the four amino acid substitutions indicate that amino acids 91 and 684 in the PBl protein of the A/X-31 were reverted to those originally present in the A/PR/8/34. Thus, the V91 A and G684 mutations do not appear to contribute to cold adaptation and temperature sensitivity. However, because glutamic acid and threonine were substituted for lysine and alanine at amino acids 121 and 240 in other influenza strains, it is believed that the amino acid 121 and 240 mutations significantly affect attenuation.
  • the PA protein gene of the A/X-31 was compared with that of the A/PR/8/34 and other Genbank's influenza strains. Table 5a below shows the results. Table 5b indicates the Genbank's influenza strains used to compare the amino acid of the PA protein. In addition, the PA protein gene of the HTCA-A 101 was compared with that of the A/X-31. Table 5c below shows the results. Table 5a
  • PA protein gene two of six base mutations resulted in amino acid variations. These varied amino acids are observed in most influenza viruses and thus are not likely to contribute to attenuation. However, because the interaction between one gene and another gene may result in an unexpected phenotype, it should not be concluded that the two amino acid variations have not contributed to attenuation.
  • the NP protein gene of the A/X-31 was compared with that of the A PR/8/34 and Genbank's registered influenza viruses. Table 6a below shows the results. Table 6b below indicates the Gnebank's influenza viruses used to compare the amino acid sequence of the NP protein. In addition, the NP protein gene of the HTCA-AlOl was compared to that of the A/X-31 and the Genbank's other influenza viruses. Table 6c below shows the results.
  • the NP protein is a multi-functional protein and is a major component of the RNP complex.
  • the NP protein is known to bind template RNAs and terminate the transcription.
  • Most influenza viruses conserve glutamic acid and threonine at amino acids
  • NP protein 18 and 130 of the NP protein, respectively. These amino acids were replaced with glycine and methionine, respectively, in the HTCA-AlOl. It is likely that the two amino acid variations in the NP protein contribute to the attenuation of the virus. In particular, because the NP protein were reported to involve host-specificity ( Virology 147, 287-294, 1985), it is believed that the two amino acid mutations contribute to host-specificity.
  • the M protein gene of the A/X-31 was compared with that of the A/PR 8/34 and Genbank's registered influenza viruses. Table 7a below shows the results. Table 7b below indicates the Gnebank's influenza viruses used to compare the amino acid sequence of the M protein. In addition, the M protein gene of the HTCA-AlOl was compared to that of the A/X-31 and the Genbank's other influenza viruses. Table 6c below shows the results.
  • the spliced M protein gene express Ml and M2 proteins from its transcript mRNA.
  • the Ml protein is a peripheral lipid protein which combines the envelop with the RNP complex.
  • the Ml protein consists of a lipophilic region at its amino terminus and a RNP complex interacting domain at its carboxy terminus.
  • the M2 protein regulates the concentration of hydrogen ion in the virus as an ion channel.
  • the M protein of the HTCA-AlOl includes five amino acid variations and its variation percentage is highest.
  • the Ml protein of Genbank's influenza viruses conserves proline and asparagine at amino acids 54 and 94, respectively. These amino acids were replaced with serine and glutamic acid, respectively in the Ml protein of the HTCA-AlOl.
  • amino acid 137 of the Ml protein and the amino acid 86 of the M2 protein involve host-specificity (J. Virol. 57. 697-700, 1986).
  • the amino acid 137of the Ml protein in the known human-infecting viruses is alanine
  • the amino acid 137 of the Ml protein in the known swine or bird- infecting influenza viruses is threonine. Therefore, it is likely that the amino acid mutation A137T in the Ml protein of the HTCA-AlOl changes human-specificity to nonhuman-specificity.
  • the M2 protein of the known human-infecting influenza virus conserves alanine at amino acid 86, whereas the M2 protein of swine or bird-infecting influenza virus conserves valine at amino acid 86.
  • the mutation A86T in the M2 protein of the HTCA-AlOl is not observed in known influenza virus strains.
  • Arbor/6/60/ca virus and the A/Leningrad/57/ca virus which are cunently used as influenza live vaccine virus, conserve serine and threonine at amino acid 86 of the M2 protein, respectively, similar to the HTCA-AlOl. Therefore, the above amino acid mutations do not induce certain host-specificity but can contribute to the attenuation of the virus as in the A/ Ann Arbor/6/60/ca virus and the A Leningrad/57/ca virus.
  • the NS protein gene of the A/X-31 was compared with that of the A/PR/8/34 and Genbank's registered influenza viruses. Table 8a below shows the results. Table 8b below indicates the Genbank's influenza viruses used to compare the amino acid sequence of the NS protein. In addition, the NS protein gene of the HTCA-AlOl was compared to that of the A/X-31 and the Genbank's influenza viruses. Table 8c below shows the results. Table 8a
  • the spliced NS protein gene expresses the NS1 and NS2 proteins as in the M protein gene.
  • the NS1 protein prevents poly A RNA from moving out of nucleus
  • the NS1 protein inhibits the splicing of the pre-mR A ⁇ EMBO. J. 13,
  • the NS1 protein functions as an enhancer for the translation of the virus mRNA.
  • the function of the NS2 protein is little known. There is a report that it assists the normal replication of the short length RNA (J. Gen. Virol. 75, 43-53, 1994).
  • the NS protein of the HTCA-AlOl includes only one mutation at amino acid 98. In contrast, known influenza viruses conserve methionine or isoleucine at amino acid 98 of the NS protein.
  • Leucine is unusually found at amino acid 98 of the NS protein in some known influenza viruses, and can also be induced through serial cold adaptation of the virus.
  • A/X-31 genomes (except HA and NA) were analyzed. Even more mutations were observed in the A/X-31, compared to the parent A/PR/8/34. It appears that prolonged adaptation of the influenza virus in the fertilized egg resulted in many mutations, contributing to attenuation and substantial growth of the virus.
  • comparison of the A X-31 and the HTCA-A 101 revealed that mutations occuned in all of six genomes during the cold adaptation.
  • One mutation in the M protein gene was common in the HTCA-AlOl and in known cold-adapted viruses. However, the other mutations in the HTCA-A101 were different from those in known cold- adapted viruses.
  • the HTCA-AlOl is distinguishable from known influenza vaccine viruses. Therefore, the HTCA-AlOl is a novel cold adapted mutant virus.
  • the plaque assay was conducted on MDCK cells at varied temperatures to observe the attenuation degree of the HTCA-AlOl.
  • MDCK cells were enriched up to 90% density on a 6-well plate. After removing media, the MDCK cell layer was infected with 200 ⁇ l of virus solution in which 2 HAU of virus were diluted to 10000 times by
  • the cell layer was overlaid with DMEM medium containing 0.5% Seakem Agarose, 0.2% bovine serum albumin and 15 ⁇ g/ ⁇ l of trypsin and was cultured for 3 days at 25°C, 30"C, 37°C or 39°C. Thereafter, the plaque size was measured, and the results are indicated in Table 9 below. Where the culture was conducted at 25 ° C, the plaque was observed 8 day after the culture.
  • the A/X-31 virus formed normal plaques of 3 mm and 2 mm when it was cultured at 37 ° C and 39°C, respectively.
  • the plaques of 0.5 mm or less were formed at 30°C and no plaques were observed at 25°C. That is, the virus was not adapted at the temperatures of from 25°C to 30°C.
  • the HTCA-AlOl virus was cultured at 30°C, the plaque of 4 mm was formed. Even when the HTCA-AlOl virus was cultured at 25°C, the plaque of 1 mm was formed 8 days after the culture. Therefore, the HTCA-AlOl exhibits cold adaptation phenotype.
  • the HTCA- AlOl virus formed no plaques at 39 ° C. This demonstrates that the HTCA-AlOl virus exhibits temperature sensitivity phenotype.
  • the HTCA-AlOl virus was similar to A/X- 31 virus in that both formed plaques of 2 mm at 37°C. However, the microscopic observation of the culture of HTCA-AlOl virus at 37°C revealed that necrosis was formed at the center and was shaped as an asterisk. This demonstrates that the HTCA- A101 is temperature-sensitive at 37°C in contrast with wild type influenza virus.
  • the HTCA-AlOl virus which was derived from cold adaptation of A X-31 virus, possesses a character of a toxicity-reduced strain which is inhibited at a living body temperature while being actively grown at low temperatures.
  • the HTCA-AlOl virus is useful in preparation of live influenza vaccine.
  • the attenuation degree of the HTCA-A 101 virus was examined by comparing the growth of the HTCA-AlOl virus and the A/X-31 virus in the fertilized egg.
  • the examination was conducted in a similar manner to the above Experimental Example 1 except that an initial infection amount was as much as 10 HAU and the cultivation was canied out at 25 ° C, 30 ° C or 37°C.
  • the results are indicated in Table 10 below.
  • A/X-31 virus is more actively grown with the titer of 3x 10 4 HAU/ml at 37°C, compared to other influenza viruses.
  • the A/X-31 virus maintains the same growth rate at 30"C.
  • the growth rate of the HTCA-AlOl virus at 30 ° C is the same as that of the parent virus A/X-31.
  • HTCA-AlOl virus at 37 ° C was reduced by 1/10 of the parent A/X-31, and thus the HTCA-AlOl is clearly temperature-sensitive.
  • the HTCA-AlOl virus was grown at 25 °C with lxl 0 3 HAU/ml. This high growth rate indicates that the HTCA-AlOl exhibits cold adaptation phenotype.
  • the influenza vaccine can be prepared at reduced costs by using the cold adapted virus.
  • mice Groups of five 6-week-old BALB/c mice, purchased from Charles River, Japan, were used in this toxicity test. The mice were anesthetized by inhalation of isoflurane and then were infected through nasal inhalation with HTCA-AlOl virus at 10 7 pfu/ml, 10 6 pfu ml, 10 5 pfu/ml, 10 4 pfu ml or 10 3 pfu/ml or with 50 ⁇ l of A/X-31 virus solution. The lethality and the change in body weight were observed as time was elapsed ( Figure 1).
  • the HTCA-AlOl virus of the invention will expose greatly reduced toxicity by undergoing cold adaptation.
  • influenza virus A Two types of influenza virus A, H IN 1 and H3N2, and one type of influenza virus
  • the inventors developed a recombinant virus using the cold adapted virus HTCA-AlOl and recently prevailing extremely toxic influenza virus and prepared a trivalent vaccine using the recombinant virus.
  • A/Singapore/6/86 virus was used in preparing the desired recombinant HlNl. After egg was co-infected with the HTCA-AlOl virus and the A/Singapore/6/86 virus, it was cultured in a cultivator at 30 ° C for 2 days. The allantoic fluid was collected and the plaque assay was conducted on MDCK cell at 27°C. Then, 0.5 ⁇ l/ml of polyclonal antibody was added to medium. The amount of the antibody was as much as the minimum concentration which inhibits formation of the plaque. After 3 days were elapsed, the formed plaques were recovered. After another egg was again infected with the recovered plaque, they were cultured at 27 ° C for 3 days. The allantoic fluid was collected to confirm whether the virus was amplified. The plaque inhibition assay was applied to the amplified viruses to detect the resulting recombinant viruses. In this assay,
  • the plaque inhibition assay shows that the resulting recombinant virus forms the large plaque at 1 ⁇ l/ml of the antibody, whereas the HTCA-AlOl virus forms the small plaque at 3 ⁇ l/ml of the antibody. (6-2) Preparation of recombinant influenza virus type H3N2
  • A/Shangdong/9/93 virus was used in preparing the desired recombinant H3N2. After egg was co-infected with A/Shangdong/9/93 virus and HTCA-AlOl virus, it was cultured at 30 ° C. The allantoic fluid was collected and were again injected into another egg. Then, 10 ⁇ l, 2 ⁇ l or 0.5 ⁇ l/ml of antibodies were injected into the egg. After culturing egg at 30°C for 3 days, the allantoic fluid was recovered to confirm the amplification of the viruses. The egg containing 2 ⁇ l/ml or 1 ⁇ l/ml of antibody were infected with the amplified viruses and then was cultured at 27°C for 3 days. It was confirmed that the viruses were amplified in the egg containing 2 ⁇ l/ml of the antibody.
  • the growth of the amplified viruses in the egg containing 10 ⁇ l/ml of the antibody was confirmed.
  • the same plaque inhibition assay as above was carried out on the viruses grown in the egg containing 10 ⁇ l/ml of the antibody.
  • the result was that the viruses formed the plaques on MDCK cells containing 0.7 ⁇ l/ml of antibody.
  • the resulting recombinant viruses were cultured in eggs without addition of antibodies, their growth was almost similar with that of the HTCA-AlOl virus. Therefore, the character of the recombinant virus did not chang.
  • influenza vaccine was administered via nasal cavity of ICR mouse at 8.3 x 10 4 TCID 50 (50% tissue culture infectious dose)/g/B.W., ' 4.15 x 10 4 TCID 50 /g/B.W., 2.08 x 10 4 TCID 50 /g/B.W., 1.04 x 10 4 TCID 50 /g/B.W. or 0.52 x 10 4
  • Acute toxicity was tested when one drop of the influenza vaccine was administered to New Zealand White rabbits.
  • the amounts of the vaccine to be given were 8.3 x 10 4 TCID 50 /g/B.W., 4.15 x 10 4 TCID 50 /g/B.W., 2.075 x 10 4 TCID 50 /g/B.W., 1.0375 x 10 4 TCID 50 /g/B. W. or 0.51875 x 10 4 TCID 50 /g/B. W.
  • the results were same as did in the mice. That is, neither deaths nor change in clinical symptoms occuned at any dosages, and no changes due to the administration of the vaccine were observed at the autopsy.
  • 8.3 x 10 1 TCID 50 /g/days, 8.3 x 10 2 TCID 50 /g/days or 8.3 x 10 3 TCID 50 /g/days were administered, once per day and 7 times per week, for 4 weeks.
  • the result was the neither deaths nor changes in clinical symptoms, such as loss or gain in weight and food intake, occuned.
  • the blood and serum tests showed that the administration of the influenza vaccine did cause no changes.
  • a pathological histological examination revealed that there were no changes which may be caused by dose-dependent vaccine administration. Consequently, the influenza vaccine of the invention were nontoxic for the 4-week successive drop administration of 8.3 x 10 3 TCID 50 /g/days to mice.

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Abstract

La présente invention porte sur un procédé de production d'un virus de la grippe adapté au froid en effectuant des passages dans un milieu de culture du virus de la grippe capable de se développer activement dans un oeuf fécondé sélectionné comme virus parent, à des températures basses comprises entre 30 °C et 24 °C. L'invention porte également sur les virus de la grippe adaptés au froid HTCA-A101, HTCA-B102, et HTCA-B104 préparés selon le procédé de l'invention, sur un virus de la grippe adapté au froid recombiné obtenu en co-infectant la cellule hôte avec le virus de la grippe adapté au froid et un virus de la grippe de type sauvage, et sur un vaccin contre la grippe comprenant le virus de la grippe adapté au froid et/ou le virus de la grippe adapté au froid recombiné.
PCT/KR1998/000384 1997-11-29 1998-11-30 Vaccin contre la grippe attenue vivant Ceased WO1999028445A1 (fr)

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US7270990B2 (en) 2003-06-20 2007-09-18 Microbix Biosystems, Inc. Virus production
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