Classifications

• • C—CHEMISTRY; METALLURGY
  • 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
• • 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
• • 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
• • 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
• • 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
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16151—Methods of production or purification of viral material

Definitions

• This invention relates to a process for viral replication in mammalian cells and particularly viral replication of human influenza virus in Vero cell culture.
• HA hemagglutinin
• the major influenza virus glycoprotein hemagglutinin (HA)
• HA hemagglutinin
• Post- translational cleavage of the HA forms two subunits, HA1 and HA2, joined by a disulfide bond. Cleavage is essential to the production of infectious virus; virions containing uncleaved HA are non-infectious. The process can occur intracellularly or extracellularly.
• the HAS of human, swine, and most avian influenza virus strains cannot be cleaved by ubiquitous intracellular proteases.
• influenza viruses grown in mammalian cells possess structurally homogenous hemagglutinin molecules (Has) that are identical to the predominant Has of the original clinical isolate [Katz et al, Virology, Vol. 165 (1988), pp. 446-456; Robertson et al, Virology, Vol. 179 (1990), pp. 35-40].
• influenza viruses grown in mammalian cells elicit neutralizing and hemagglutinin inhibition (HI) antibodies in human sera more readily and at higher titers than do their egg-grown counterparts.
• MDCK Madin-Darbin canine kidney
• Influenza viruses can be propagated in several types of primary cell cultures including chick embryo kidney, chick embryo lungs, monkey kidney, canine kidney, bovine kidney, chick kidney, guinea pig kidney, and chick embryo fibroblasts.
• primary tissue cultures are unlikely to be useful as a substrate for vaccine production for several reasons, including contamination by various endogenous agents, the variable quality of the cells, different sensitivities to variants of the same virus, and, of course, high cost and difficulties in obtaining and preparing the tissue cultures.
• Diploid tissue cultures such as WI-38, have been used to produce vaccines against poliomyelitis, adenovirus types 4 and 7, rubella, measles, and rabies viruses.
• human diploid (MRC-5) cells can support the growth of influenza viruses, such systems have stringent growth media requirements and are expensive to maintain, making them suboptimal for large-scale production of vaccines. Disclosure of the Invention
• This invention of a process for ensuring replication of human influenza virus at a low multiplicity of infection in a mammalian cell line involves maintaining a consistent minimum concentration of trypsin (about 0.05 ⁇ g/ml) in the culture medium.
• Vero cells are sensitive to a spectrum of viruses, including: enteroviruses, measles and parainfluenza viruses, herpes viruses, andenoviruses, rhabdoviruses and some arboviruses. Low-passage- number Vero cells lack tumorigenicity, do not contain adventitious viruses and can support efficient proliferation of many types of viruses. This cell line has been used successfully for the production of vaccines against poliomyelitis and rabies. The Vero cell line is suitable for cultivation of infectious influenza A viruses and for primary isolation of currently circulating influenza A (H3N2) strains.
• H3N2 currently circulating influenza A
• H1N1 influenza A/England/1/53 [HG] strain to Vero cell culture, its growth characteristics and antigenic stability, and the likelihood of obtaining high yields of viral proteins with Vero is compared to MDCK cells.
• Vero (WHO) cells When infected with influenza A virus at a multiplicity of at least 0.005 TCID 50 per cell, Vero (WHO) cells produced yields of virus comparable to those produced in Madin-Darby canine kidney (MDCK) cells. However, at lower multiplicities of infection, multicycle growth was blocked early in the course of infection, the progress of the cytopathic effect was stopped, and the final virus yields were low.
• MDCK Madin-Darby canine kidney
• trypsin concentrations of at least 0.05 ⁇ q/ml in the cell culture were essential for securing high virus yields and that a concentration of about 0.1 ⁇ g/ml was optimal when the multiplicity of infection ranged from about 1 ⁇ 10 -5 and 1 ⁇ 10 -6 TCID 50 per cell; satisfactory results were obtained at about 5 ⁇ 10 -7 TCID 50 Per cell.
• trypsin had to be maintained from about 0.05 to 0.5 ⁇ g/ml to secure adequate yields of virus.
• a higher volume of maintenance medium per area of cell monolayer also required slight improvement of multicycle virus growth at low input doses, most likely because of a lower concentration of trypsin-inactivating factor in the medium.
• the trypsin- inactivating factor in cell cultures belongs to the class of proteins that inhibit proteinases
• our findings of a putative inhibitor of trypsin activity may be of value in the studies of serine proteinase inhibitors.
• the inhibitors of this family although numerous and extensively studied, are mostly derived from such substances as plants, bovine pancreas, human and animal plasma, tissues of invertebrate species, etc. For this reason, their structure in enzymological properties are far better known than their biosynthesis, intracellular transport and secretion mechanism. Thus, inhibitors produced by cultured cells may prove to be a valuable asset.
• the best mode for carrying out the invention there are described several preferred embodiments to illustrate the invention. However, it is to be understood that the invention is not intended to be limited to the specific embodiments contained therein. Best Mode for Carrying Out the Invention
• the Vero (WHO) cell line deposit no. 1297, was obtained from the American Type Tissue Collection at the level of the 134th passage.
• the cells were cultivated as monolayers in Falcon Labware 250 cm 3 flasks at 37°C and 5% Co 2 in a growth medium of Eagles minimal essential medium (MEM) supplemented with 10% unheated fetal calf serum.
• MEM Eagles minimal essential medium
• MDCK Madin-Darby canine kidney
• LLC-MK 2 rhesus monkey kidney
• the medium used was MEM with 5% fetal calf serum heated 30 minutes at 56°C.
• RPMI 1640 medium was used with 5% heated fetal calf serum.
• the cells were grown either in 50 cm 3 flasks or in 6-well, 24-well, and 96-well plates (Falcon Labware). Cell monolayers were washed three times with PBS and overlaid with maintenance medium. The latter had the same composition as the growth medium for each cell line, the serum being omitted and 0.3% bovine serum albumin (BSA) added. Unless otherwise stated, the maintenance medium contained TPCK-trypsin (Worthington) at 1.0 ⁇ g/ml. Plaque assays were performed with TPCK-treated trypsin (2.5 ⁇ g/ml).
• Vero (WHO)-adapted influenza A/England/1/53 (H1N1) [HG], A/FW/1/50 (H1N1), and A/Aichi/2/68-PR/8/34 (H3N2) [X-31] viruses were used. The viruses were passaged 5 times in Vero (WHO) cell cultures, and the final stock virus preparations contained about 10 7.3 to 10 8.25 TCID 50 /0.2 ml and about 32 to about 128 HAU. In the preliminary experiments, the Vero (WHO)-adapted A/Rome/49 (H1N1) strain was used (10 6.7 TCID 50 /0.2 ml, about 15 to about 32 HAU).
• HA and infectivity titration were performed essentially as described in "Advanced Laboratory Techniques for Influenza Diagnosis" [Immunol. Ser. 6, pp. 51-57 (1975)]. HA titrations were done in mirotiter plates. Infectivity was measured by an end point titration technique in MDCK cells grown in 96-well plates with CPE evaluation at 72h postinfection.
• BAAMC Na-benzoyl-L- arginine-7-amido-4-methylcoumarin-hydro-chloride
• the substrate was dissolved to a final concentration of about 0.2 Mm in a buffer containing 50 Mm of Tris-Hcl, Ph about 8.0, 10 Mm CaCl 2 and 1% DMSO.
• a 0.1 ml sample of trypsin-containing cell culture fluid was added to about 0.9 ml of BAAMC solution and incubated at 37°C for 1 hour. The samples were placed on ice and assayed in a Perkin-Elmer MPF-44B fluorescence spectrophotometer at activation and emission wavelengths of about 380 and 460 nm, respectively.
• Vero cells were infected with the A/England/1/53 (H1N1) [High Growth, HG] strain of influenza virus, a reassortant containing the gene segments coding for the two surface glycoproteins (HA and NA) from A/England/1/53 (H1N1) and the remaining six genes from A/PR/8/34 (H1N1).
• H1N1 A/England/1/53
• HA and NA two surface glycoproteins
• H1N1N1 the virus was left to adsorb for 1 hour at 37°C, after which the monolayer was washed twice with warm phosphate buffered saline (PBS) solution to remove the unadsorbed viruses.
• PBS phosphate buffered saline
• Serum-free MEM with 0.3% bovine serum albumin (BSA) was then added; the maintenance medium contained
• TPCK-treated trypsin at about 1.0 ⁇ g/ml.
• the input dose of virus was 10 -2 -10 -3 PFU/cell.
• the material for further passage was collected 72 hours postinfection (p.i.), with trypsin (final concentration, about 1.0 ⁇ g/ml) added at 48 hours p.i..
• Cells were infected with serial 10-fold dilutions of virus, which were added to the washed cell monolayer without previous adsorption.
• Virus accumulation was estimated by visual determination of the cytopathic effect (CPE) and HA titration of culture fluid at different times p.i. (24, 48 and 72 hours). Infectivity titrations were performed in 96-well plates.
• Tissue culture infectious doses (TCID 50 /ml and egg infectious doses (EID) 50 /ml values were calculated by the formula of Karber [Arch, Exp. Path. Pharmak., Vol. 162, pp. 480-483 (1931).
• Virus-containing culture fluids were concentrated in an Amicon system and purified by differential sedimentation through 25-70% sucrose gradients. Whole virus protein estimates were made by the method of Bradford (1976). To determine the yield of HA protein in virus grown in Vero and MDCK cells, the virus proteins were separated by gradient (4-20%) SDS-PAGE and intensity of Coomassie blue-stained protein bands was quantified by densitometry.
• Influenza A viruses were isolated from the throat washings of patients with clinical signs of influenza and collected in PBS to which 0.7% BSA was added.
• Cell culture both Vero and MDCK
• embryonated chicken eggs were infected directly with freshly collected (not frozen) throat washings.
• Chicken eggs were inoculated amniotically and allantoically.
• Clinical samples used for isolation were inoculated undiluted and at 10 -1 and 10 -2 dilutions and incubated for 72-96 hours. Trypsin was added at 0 and 48 hours p.i. (about 1.0 ⁇ g/ml) and tested for virus replication with chicken and guinea pig erythrocytes. Each sample was given at least two passages in chicken eggs or cell cultures before being considered negative.
• Monolayer antibodies to the A/Baylor/5700/82 (H1N1) and A/Baylor 11515/82 (H1N1) strains were prepared by the method of Köhler and Milstein (1976).
• Polyclonal antisera to influenza A/England/ 1/53 virus (20 passages in Vero cells) were prepared in chickens by intravenous injection of virus-containing culture fluid.
• HA and HI reactions were performed in microtiter plates with about 0.5% (v/v) chicken erythrocytes.
• Guinea pig erythrocytes (about 0.4% v/v) were used to analyze primary influenza A isolates from the 1993-1994 winter epidemic season.
• RNA was isolated by treating virus-containing allantoic or culture fluids with proteinase K and sodium dodecyl sulfate and then extracting the product with phenol-chloroform (1:1) and ethanol precipitation as previously described (Bean et al, 1980). Viral RNA was converted to cDNA with the use of U12 (5'AGCGAAAGCAGG3') and AMV reverse transcriptase.
• U12 5'AGCGAAAGCAGG3'
• AMV reverse transcriptase The sequences of the oligonucleotide primers used in this study for molecular characterization of internal genes (PB2, PB1, PA, NS and M) are available on request.
• Amplification proceeded through a total of 35 cycles of denaturation at 95°C (1 min), annealing at 50°C (1 min), and primer extension at 74°C (3 min).
• Amplified DNAs were analyzed by electrophoresis, visualized with ethidium bromide and then purified with either the MagicTM PCR Preps DNA purification system (Promega, Madison, WI) or the Geneclean ® kit (BIO 101, La Jolla, CA) according to the manufacturers' instructions.
• Nucleotide sequencing was performed dideoxynucleotide chain termination method with the fmolTM DNA sequencing system (Promega). The reaction products were separated on 6% polyacrylamide-7M urea gels, 0.4 mm thick.
• Vero and MDCk cell monolayers were infected with the Vero-adapted influenza virus strain A/England/1/53 [HG] at 10 -3 PFU/cell multiplicity of infection, trypsin (about 1.0 ⁇ g/ml) was included in the medium. Infected and control cell monolayers were fixed at 48 hours postinoculation in cacodylate-buffered 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in graded series of alcohols and embedded in Spurr low-viscosity embedding medium (Ladd Research Industries, Burlington, VT).
• trypsin about 1.0 ⁇ g/ml
• Influenza viruses can replicate to high titers in a limited number of mammalian cells, provided that trypsin is present for cleavage of the HA molecule.
• a virus repository was screened and a master strain was selected that would replicate sufficiently in the mammalian epithelial-like cell line.
• MDCK cells which are widely used to isolate and culture viruses, were included in the study as a reference.
• the influenza A virus strains that we examined had been isolated from a wide range of human and avian hosts, and represent 12 of the 14 HA (not H5 and H7) and 9 NA subtypes.
• Viruses were passaged three times in Vero and MDCk cells, and the virus yield was estimated from HA and infectivity titers. Of the 72 strains investigated, 65 (90.3%) replicated to the level that can be detected by HA titration in Vero cells after the first passage and 37 (51.4%) after the second. By comparison, all strains could replicate in MDCK cells during the first and second passages.
• Six humans and four avian influenza A viruses were selected as strains with the highest growth potential (Table 4), among which A/England/1/53 (H1N1) [HG] virus was chosen for further adaptation to Vero cells.
• [HG] strain selected for adaptation to growth in Vero cells is a reassortant between the original A/England/1/53 strain and
• A/PR/8/34 Six genes of the reassortant encoded internal proteins of A/PR/8/34 and two surface glycoproteins of A/England/1/53.
• Viral protein yield is an important feature of any system used to produce influenza virus vaccines.
• To establish the amount of virus-specific proteins that can be obtained from Vero cells we compared the protein yields of A/England/1/53 [HG] (20-passage) virus after replication in Vero and MDCK cells (Table 7). Determination of the HA protein yield was done using SDS-PAGE separated virus proteins and was quantitated by densitometry. Tests of culture fluids indicated that approximately 6 ⁇ 10 8 of infected cells could produce 4.38 mg of virus protein in Vero and 4.13 mg in MDCk cells. It was also possible to obtain viral proteins from disrupted virus-infected cells of either type; the protein yields were lower than in the supernatant but there was no significant difference between the cell types in the amount of virus protein.
• MDCK cells provide the most sensitive host cell system for the primary isolation of influenza viruses.
• Vero cells have been successfully used to isolate parainfluenza and mumps viruses, but they were judged unsuitable for the isolation of influenza viruses.
• We tested nine clinical specimens collected during the 1993-1994 epidemic season in three culture systems (Vero and MDCK cells and embryonated chicken eggs).
• Six influenza A (H3N2) strains were isolated in Vero cells, seven in MDCK cells and only two in embryonated chicken eggs (Table 9). Two samples failed to yield virus in any host system.
• CPE observed 48-72 hours after inoculation was the only evidence of virus reproduction.
• HA activity was detectable on the second passage, and, by the third passage, the positive samples produced both CPEs and HA titers that ranged from 2-32.
• guinea pig erythrocytes it was necessary to use guinea pig erythrocytes to determine HA titers, chicken erythrocytes failed to be agglutinated as was first described by Burnet and Bull.
• influenza A H2N2
• virions were released from the apical surface of Vero cells, a feature typical of epithelial cells infected with influenza virus.
• the budding virions in MDCK and Vero cells appeared filamentous.
• a fraction of infected cells in both systems showed cytopathological changes indicative of apoptosis.
• the nuclear changes included fragmentation and condensation of chromatin, margination of chromatin to the nuclear envelope, and blebbing of the nuclear envelope.
• the cytoplasmic changes consisted of condensation, extensive vacuolation, and blebbing and vesiculation of the plasma membrane to form "apoptotic bodies.”
• the histochemical assay consisted of addition of digoxigenin-labeled nucleotides to the 3'-OH ends of broken DNA with use of terminal deoxynucleotidyl transferase and detection of the added nucleotides by reactivity with fluorescein-labeled antidigoxigenin antibodies.
• the infected Vero and MDCK cells showed 20% and 30% positive cells, respectively, by this assay, whereas the uninfected cells were negative.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Virology (AREA)
• General Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Genetics & Genomics (AREA)
• Mycology (AREA)
• Zoology (AREA)
• Epidemiology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biochemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Wood Science & Technology (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• General Engineering & Computer Science (AREA)
• Pulmonology (AREA)
• Gastroenterology & Hepatology (AREA)
• Biophysics (AREA)
• Biotechnology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Catalysts (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Seasonings (AREA)

Priority Applications (6)

Applications Claiming Priority (3)

Publications (1)

Family

ID=25679347

Family Applications (1)

Country Status (6)

Cited By (10)

Families Citing this family (1)

Citations (1)

Family Cites Families (1)

• 1995
  • 1995-11-13 JP JP8516287A patent/JPH11509081A/ja not_active Ceased
  • 1995-11-13 AU AU41589/96A patent/AU694592B2/en not_active Expired
  • 1995-11-13 NZ NZ296861A patent/NZ296861A/en not_active IP Right Cessation
  • 1995-11-13 WO PCT/US1995/014814 patent/WO1996015232A1/en not_active Application Discontinuation
  • 1995-11-13 EP EP95939952A patent/EP0808361A4/en not_active Withdrawn
• 1997
  • 1997-05-15 NO NO972239A patent/NO972239L/no not_active Application Discontinuation

Patent Citations (1)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events