WO2014112945A1 - Souches d'entérovirus 71 sensibles à la température, adaptées au froid, et procédés de développement de souches virales sensibles à la température, adaptées au froid - Google Patents

Souches d'entérovirus 71 sensibles à la température, adaptées au froid, et procédés de développement de souches virales sensibles à la température, adaptées au froid Download PDF

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Publication number
WO2014112945A1
WO2014112945A1 PCT/SG2013/000027 SG2013000027W WO2014112945A1 WO 2014112945 A1 WO2014112945 A1 WO 2014112945A1 SG 2013000027 W SG2013000027 W SG 2013000027W WO 2014112945 A1 WO2014112945 A1 WO 2014112945A1
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Prior art keywords
virus
enterovirus
cold
temperature sensitive
strain
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PCT/SG2013/000027
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English (en)
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Kaw Bing CHUA
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Temasek Life Sciences Laboratory Limited
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Priority to SG11201505468VA priority Critical patent/SG11201505468VA/en
Priority to PCT/SG2013/000027 priority patent/WO2014112945A1/fr
Priority to CN201380070773.0A priority patent/CN104918636B/zh
Priority to JP2015553680A priority patent/JP6117379B2/ja
Priority to MYPI2015001671A priority patent/MY185184A/en
Priority to TW103101883A priority patent/TWI705140B/zh
Publication of WO2014112945A1 publication Critical patent/WO2014112945A1/fr
Priority to HK16101297.0A priority patent/HK1213474A1/zh

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32311—Enterovirus
    • C12N2770/32321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32311—Enterovirus
    • C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to cold-adapted temperature sensitive Enterovirus 71 strains, particularly to the cold-adapted temperature sensitive Enterovirus 71 strains EV71 :TLLPP20 and EV7T.TLLaP20.
  • the present invention also relates to processes of developing cold-adapted temperature sensitive virus strains, particularly RNA virus strains. .
  • HFMD Hand foot and mouth disease
  • enterovirus 71 EV71
  • CA16 coxsackievirus A16
  • EV71 has been known to cause severe neurological diseases with deaths especially in young children.
  • Human enterovirus 71 is a small non-enveloped virus of approximately 30 nm in size with a single-stranded positive RNA genome of about 7,450 nucleotides. The virus is classified as Human enterovirus. A species under the genus Enterovirus within the family Picornaviridae (Alexander et al., 1994; Melnick, 1996).
  • EV71 is divided into three major genogroups (denoted A, B, and C), and genogroups B and C are further subdivided into subgenogroups Bl to B5 and CI to C5, based on phylogenetic analysis of its major capsid protein (VP1) gene (Bible et al., 2008).
  • VP1 major capsid protein
  • the virus was first isolated from a child with aseptic meningitis in California, USA and subsequently characterized as a new serotype of the genus Enterovirus (Schmidt et al., 1974). In the years following its initial isolation, outbreaks of HFMD with complications due to the virus were reported in various parts of the world (Blomberg et al., 1974; Kennett et al., 1974; Deibel et al., 1975; Hagiwara et al., 1978).
  • Live attenuated vaccines represent one of the first successful methods of vaccination, dated back to the 18 century when the British doctor Edward Jenner began using cowpox virus to vaccinate children against the devastating disease of smallpox.
  • Live attenuated vaccines use live viruses or microorganisms that have been weakened so that they are unable to cause disease, yet induce protective immune response.
  • Traditional, classical and genetic methods have been successful to some extent in attenuating viruses and microorganism for use as live attenuated vaccine. 44"48
  • Traditional method uses naturally occurring related organisms that are avirulent in humans, such as, use of cowpox or vaccinia viruses.
  • Classical method involves rounds of growth of virulent viruses or microorganism under conditions that attenuated them, such as, in tissue culture or harsh liquid media. Genetic method utilizes modern molecular biotechnology to manipulate the genomes to reduce their virulence (Huygelen, 1997; Robinson, 2008; Coleman et al., 2008; Lauring et al., 2010; Kenney et al., 2011).
  • the wild-type virus is plaque- selected via plaque assay technique by culturing the virus in suitable cells incubated at lower temperature. The selected virus strain is subsequently passaged repeatedly at the targeted lower incubation temperature (Hagiwara et al., 1982; Hashimoto and Hagiwara, 1983; Richman and Murphy, 1997).
  • virus strains which can be used to treat viral diseases which retain phenotypic and genetic stability in specific in vitro cell culture conditions and do not exhibit neuro-virulence in monkeys following intravenous inoculation. It is also desired to develop cold-adapted temperature sensitive strains of viruses, including RNA viruses, that are derived following serial passages in cell-culture.
  • the present invention relates to cold-adapted temperature sensitive Enterovirus 71 strains, particularly to the cold-adapted temperature sensitive Enterovirus 71 strains EV71:TLLPP20 and EV71 :TLLaP20.
  • the present invention also relates to processes of developing the cold-adapted temperature sensitive virus strains, particularly RNA virus strains. .
  • the present invention provides the cold-adapted temperature sensitive Enterovirus 71 strains.
  • the cold-adapted temperature sensitive Enterovirus 71 strain is EV71:TLLpP20 as described herein.
  • the cold- adapted temperature sensitive Enterovirus 71 strain is EV71 :TLLaP20 as described herein.
  • the Enterovirus 71 strains of the present invention are prepared by a method to attenuate Enterovirus 71 using temperature sensitivity as a phenotypic marker.
  • the method is an in vitro laboratory process to change the biologic growth characteristic of the virus to adapt for optimal replication at incubation temperature of below 30° C.
  • the adaption process which is described in detail below, is carried out in a systematic stepwise manner of incremental lower incubation temperature for culturing the virus until the targeted temperature chosen for optimal replication of the virus is achieved.
  • the present invention provides a composition comprising ihe cold- adapted temperature sensitive Enterovirus 71 strains described herein.
  • the composition comprises an effective amount of the virus strains described herein.
  • the composition comprises one or more physiologically or pharmaceutically acceptable carriers.
  • the composition is a vaccine.
  • Vaccines containing a cold-adapted temperature sensitive Enterovirus 71 strain described herein are prepared using techniques well known to the skilled artisan. Such vaccines are useful for providing immunity against the parent virus strain by administering the vaccine to a subject, such as a human subject, using techniques well known to the skilled artisan.
  • the present invention provides a method of eliciting a protective immune response in a subject, such as a human subject, which comprising administering to a subject a prophylactically or therapeutically or immunologically effective amount of a cold- adapted temperature sensitive Enterovirus 71 strain described herein.
  • the protective immune response protects the subject against a disease caused by Enterovirus 71.
  • the disease is hand, foot and mouth disease.
  • the disease is aseptic meningitis.
  • the disease is encephalitis.
  • the disease is poliomyelitis-like paralysis.
  • a cold- adapted temperature sensitive Enterovirus 71 strain described herein is administered as a vaccine.
  • the subject has been exposed to wild-type Enterovirus 71.
  • the administration of a cold-adapted temperature sensitive Enterovirus 71 strain described herein prevents a subject, such as a human subject, from becoming afflicted with an Enterovirus 71 -associated disease.
  • the subject has been exposed to wild-type Enterovirus 71.
  • the administration of a cold-adapted temperature sensitive Enterovirus 71 strain described herein delays the onset of or slows the rate of progression of an Enterovirus 71 -associated disease in a virus-infected subject, such as a human subject.
  • the present invention provides a method to attenuate a virus using temperature sensitivity as a phenotypic marker.
  • the method develops cold-adapted temperature sensitive virus strains.
  • the method of the present invention is an in vitro laboratory process to change the biologic growth characteristic of the virus to adapt for optimal replication at incubation temperature of below 30° C.
  • the adaption process is carried out in a systematic stepwise manner of incremental lower incubation temperature for culturing the virus until the targeted temperature chosen for optimal replication of the virus is achieved.
  • the method comprises the following steps: (i) preparing a reference stock of parental wild-type virus, (ii) incubating a culture of cells infected with the reference stock of parental wild-type virus at higher multiplicity of infection (MOI) and an incubation temperature of about 34° C to about 36° C, preferably about 34° C, for five or more passages until a full cytopathic effect (CPE) is obtained at each passage using an inoculum of a lower MOI and a shorter period of incubation for each passage to obtain full CPE, (iii) incubating a culture of cells infected with the resultant virus of the previous step at higher MOI and an incubation temperature of about 1° C to about 3° C lower than in the previous step for five or more passages until a full cytopathic effect (CPE) is obtained at each passage using an inoculum of a lower MOI and a shorter period of incubation for each passage to obtain full CPE, and (
  • the reference stock of parental wild-type virus is prepared by incubating a culture of cells infected with a wild-type virus at a temperature of about 36° C to about 38° C, preferably about 37° C for one or two passages until a full cytopathic effect (CPE) is obtained. Aliquots of culture supernatant containing the produced virus are placed in vials or other suitable storage devices. This culture supernatant serves as a reference stock of parental wild-type virus. The reference parental wild-type virus is used for subsequent attenuation process. In another embodiment, aliquots of the reference stock of parental wild- type virus are stored at a suitable temperature, such as at -80° C.
  • CPE cytopathic effect
  • the virus is any virus.
  • the virus is an PvNA virus.
  • the R A virus is a plus strand RNA virus.
  • the virus is a member of the Picornaviridae family.
  • the virus is a member of the Enterovirus genus.
  • the virus is Enterovirus 71 (EV71).
  • the virus is cocksackievirus A16 (CA16). The method of the present invention is useful in producing cold-adapted temperature sensitive virus strains of any of these viruses, including but not limited to cold-adapted temperature sensitive strains of EV71 and CA16.
  • the cell to be infected by the virus is any cell which is permissive for the growth of the virus.
  • the cells are Vero cells (ATCC CCL- 81).
  • the cells are maintained by regular passage in a medium suitable for growth of the cells.
  • the Vero cells are cultured in Dulbecco's modified Eagles's medium (DMEM) supplemented with 10% fetal calf serum (FCS).
  • DMEM Dulbecco's modified Eagles's medium
  • FCS fetal calf serum
  • Vero cells maintained in DMEM supplemented with 1 % FCS is used for production of the parental wild-type virus, virus culture, attenuation, titration and assessment of temperature sensitive phenotype.
  • the DMEM is supplemented with 1% FCS for adapting the virus strains for replication in successively lowered temperature of incubation.
  • the present invention also relates to the cold-adapted temperature strains of virus produced by the method described herein.
  • the cold-adapated temperature viruses produced by the method described herein are useful in the production of vaccines using techniques well known to the skilled artisan. Such vaccines are useful for providing immunity against the parent virus strain by administering the vaccine to a subject using techniques well known to the skilled artisan.
  • Figure la shows a peripheral blood mononuclear cell (arrow), derived from the blood of a monkey on day 4 after given an intravenous dose of enterovirus 71 (EV71 :TLLpP20), staining positive by indirect immunofluorescence assay using a commercial monoclonal antibody specific for the virus.
  • enterovirus 71 EV71 :TLLpP20
  • Figure lb shows photograph of GelRed-stained electrophoresed agarose gels showing One-step RT-PCR amplified products of tissues derived from Day 4 post-immunized monkeys (2202F, 289 IF) using an oligonucleotide primer pair specific for detection of enterovirus 71. Expected size of RT-PCR amplified product is 427 bp.
  • the lanes in the two gels are as follows.
  • Lane 1 100 bp DNA ladder; Lane 2: 2202F-Heart; Lane 3: 2202F-Spleen; Lane 4: 2202F-Lymph Node; Lane 5: 2202F-Kidney; Lane 6: 2202F-Liver; Lane 7: 2891F-Heart; Lane 8: 2891F-Spleen; Lane 9: 2891F-Lymph Node; Lane 10: 2891F-Kidney; Lane 11 : 2891F-Liver; Lane 12: 2202F-Brain stem (Pons); Lane 13: 2202F-Brain stem (medulla oblongata); Lane 14: 2202F-Cortex (Gyrus); Lane 15: 2202F-Spinal Cord (Cervical); Lane 16: 2202F-Spinal Cord (Lumbar); Lane 17: 2202F-Spinal Cord (Thoracic); Lane 18: 2891F- Brain stem (medulla oblongata); Lane 19: 2891F- Brain stem
  • the present invention relates to cold-adapted temperature sensitive Enterovirus 71 strains, particularly to the cold-adapted temperature sensitive Enterovirus 71 strains EV71 :TLLpP20 and EV71 :TLLaP20.
  • the present invention also relates to processes of developing the cold-adapted temperature sensitive virus strains, particularly RNA virus strains.
  • the present invention provides the cold-adapted temperature sensitive.
  • the cold-adapted temperature sensitive Enterovirus 71 strain is EV71 :TLLpP20 as described herein.
  • the cold-adapted temperature sensitive Enterovirus 71 strain is EV71 :TLLaP20 as described herein.
  • EV71 :TLLPP20 was deposited on 25 October 2012 under terms of the Budapest Treaty with the American Type Culture Collection located at 10801 University Boulevard, Manassas, Virginia 20110, USA and assigned Accession Number PTA-13285.
  • EV71 :TLLaP20 was deposited on 25 October 2012 under terms of the Budapest Treaty with American Type Culture Collection, and assigned Accession Number PTA- 13284.
  • the Enterovirus 71 strains of the present invention are prepared by a method to attenuate Enterovirus 71 using temperature sensitivity as a phenotypic marker.
  • the method is an in vitro laboratory process to change the biologic growth characteristic of the virus to adapt for optimal replication at incubation temperature of below 30° C.
  • the adaption process is carried out in a systematic stepwise manner of incremental lower incubation temperature for culturing the virus until the targeted temperature chosen for optimal replication of the virus is achieved.
  • preparing a reference stock of parental wild-type virus (i) preparing a reference stock of parental wild-type virus, (ii) incubating a culture of cells infected with the reference stock of parental wild-type virus at higher multiplicity of infection (MOI) and an incubation temperature of about 34° C for five or more passages until a full cytopathic effect (CPE) is obtained at each passage using an inoculum of a lower MOI and a shorter period of incubation for each passage to obtain full CPE, (iii) incubating a culture of cells infected with the resultant virus of the previous step at higher MOI and an incubation temperature of about 1° C to about 3° C lower than in the previous step for five or more passages until a full cytopathic effect (CPE) is obtained at each passage using an inoculum of a lower MOI and a shorter period of incubation for each passage to obtain full CPE, and (iv) repeating step (iii) in a systematic stepwise
  • the reference stock of parental wild-type virus is prepared by incubating a culture of cells infected with a wild- type virus at a temperature of about 36° C to about 38° C, preferably about 37° C for one or two passages until a full cytopathic effect (CPE) is obtained. Aliquots of culture supernatant containing the produced virus are placed in vials or other suitable storage devices. This culture supernatant serves as a reference stock of parental wild-type virus. The reference parental wild-type virus is used for subsequent attenuation process. In another embodiment, aliquots of the reference stock of parental wild-type virus are stored at a suitable temperature, such as at -80° C.
  • CPE cytopathic effect
  • the cell to be infected by the virus is any cell which is permissive for the growth of the virus.
  • the cells are Vero cells (ATCC CCL- 81).
  • the cells are maintained by regular passage in a medium suitable for growth of the cells.
  • the Vero cells are cultured in Dulbecco's modified Eagles's medium (DMEM) supplemented with 10% fetal calf serum (FCS).
  • DMEM Dulbecco's modified Eagles's medium
  • FCS fetal calf serum
  • Vero cells maintained in DMEM supplemented with 1% FCS is used for production of the parental wild-type virus, virus culture, attenuation, titration and assessment of temperature sensitive phenotype.
  • the DMEM is supplemented with 1 % FCS for adapting the virus strains for replication in successively lowered temperature of incubation.
  • the virus is passaged in each step of the process by obtaining a clarified culture supernatant containing the virus and passing this supernatant into each successive fresh new culture flask of cells, e.g., Vero cells, as soon as the virus obtains a full cytopathic effect (CPE).
  • the culture supernatant containing the virus is passed at a higher multiplicity of infection (MOI) of 20 at the beginning of each successive change to a lower temperature of incubation.
  • MOI multiplicity of infection
  • the new culture flask containing freshly confluent monolayer Vero cells is inoculated with the same MOI of virus for at least three more passages before subsequently reduced to a lower MOI of 5 to 10.
  • the attenuation process is then moved on to the next phase of successive lower incubation temperature after passaging for at least three more times at a MOI of 5 to 10.
  • the number of days required for each passage depends on how fast the virus is adapted at each phase of incrementally lower incubation temperature. The skilled artisan will readily know when a full CPE is reached. Further details of the method for preparing the cold-adapted temperature sensitive Enterovirus 71 strains of the present invention are described below.
  • the present invention provides a composition comprising the cold- adapted temperature sensitive Enterovirus 71 strains described herein.
  • the composition comprises an effective amount of the virus strains described herein.
  • the composition comprises one or more physiologically or pharmaceutically acceptable carriers.
  • the composition is a vaccine.
  • Vaccines containing a cold-adapted temperature sensitive Enterovirus 71 strain described herein are prepared using techniques well known to the skilled artisan. Such vaccines are useful for providing immunity against the parent virus strain by administering the vaccine to a subject, such as a human subject, using techniques well known to the skilled artisan.
  • a cold-adapted temperature sensitive Enterovirus 71 strain described herein where used to elicit a protective immune response in a subject or to prevent a subject from becoming afflicted with a virus-associated disease or to delay the onset of or slow the rate of progression of a virus-associated disease, is administered to the subject in the form of a composition additionally comprising one or more a physiologically or pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known to the skilled artisan and include, but are not limited to, one or more of 0.01 M - 0.1 M and preferably 0.05 M phosphate buffer, phosphate-buffered saline (PBS), or 0.9% saline.
  • Such carriers also include aqueous or non-aqueous solutions, suspensions, and emulsions.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • Solid compositions may comprise nontoxic solid carriers such as, for example, glucose, sucrose, mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate and magnesium carbonate.
  • an agent or composition is preferably formulated with a nontoxic surfactant, for example,, esters or partial esters of C6 to C22 fatty acids or natural glycerides, and a propellant. Additional carriers such as lecithin may be included to facilitate intranasal delivery.
  • Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives and other additives, such as, for example, antimicrobials, antioxidants and chelating agents, which enhance the shelf life and/or effectiveness of the active ingredients.
  • auxiliary substances such as wetting or emulsifying agents, preservatives and other additives, such as, for example, antimicrobials, antioxidants and chelating agents, which enhance the shelf life and/or effectiveness of the active ingredients.
  • auxiliary substances such as wetting or emulsifying agents, preservatives and other additives, such as, for example, antimicrobials, antioxidants and chelating agents, which enhance the shelf life and/or effectiveness of the active ingredients.
  • the instant compositions can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to a subject.
  • the present invention provides a method of eliciting a protective immune response in a subject, such as a human subject, which comprising administering to a subject a prophylactically or therapeutically or immunologically effective amount of a cold- adapted temperature sensitive Enterovirus 71 strain described herein. Accordingly, the present invention also provides a cold-adapted temperature sensitive Enterovirus 71 strain or a composition comprising the cold-adapted temperature sensitive Enterovirus 71 strain for use in eliciting a protective immune response in a subject.
  • the present invention also provides the use of cold-adapted temperature sensitive Enterovirus 71 strain or a composition comprising the cold-adapted temperature sensitive Enterovirus 71 strain for the manufacture of a medicament for eliciting a protective immune response in a subject.
  • the protective immune response protects the subject against a disease caused by Enterovirus 71.
  • the disease is hand, foot and mouth disease.
  • the disease is aseptic meningitis.
  • the disease is encephalitis.
  • the disease is poliomyelitis-like paralysis.
  • a cold-adapted temperature sensitive Enterovirus 71 strain described herein is administered as a vaccine.
  • the subject has been exposed to wild-type Enterovirus 71.
  • Exposed to an Enterovirus 71 means contact with the Enterovirus 71 such that an infection could result.
  • administration of a cold-adapted temperature sensitive Enterovirus 71 strain described herein prevents a subject, such as a human subject, from becoming afflicted with an Enterovirus 71 -associated disease.
  • the present invention also provides a cold-adapted temperature sensitive Enterovirus 71 strain or a composition comprising the cold- adapted temperature sensitive Enterovirus 71 strain for use in preventing a subject, such as a human subject, from becoming afflicted with an Enterovirus 71 -associated disease.
  • the present invention also provides the use of cold-adapted temperature sensitive Enterovirus 71 strain or a composition comprising the coldradapted temperature sensitive Enterovirus 71 strain for the manufacture of a medicament for preventing a subject, such as a human subject, from becoming afflicted with an Enterovirus 71-associated disease.
  • a subject such as a human subject
  • the subject has been exposed to wild- type Enterovirus 71.
  • the administration of a cold-adapted temperature sensitive Enterovirus 71 strain described herein delays the onset of or slows the rate of progression of an Enterovirus 71-associated disease in a virus-infected subject, such as a human subject.
  • the present invention also provides a cold-adapted temperature sensitive Enterovirus 71 strain or a composition comprising the cold-adapted temperature sensitive Enterovirus 71 strain for use in delaying the onset of or slows the rate of progression of an Enterovirus 71 -associated disease in a virus-infected subject, such as a human subject.
  • the present invention also provides the use of cold-adapted temperature sensitive Enterovirus 71 strain or a composition comprising the cold-adapted temperature sensitive Enterovirus 71 strain for the manufacture of a medicament for delaying the onset of or slows the rate of progression of an Enterovirus 71 -associated disease in a virus-infected subject, such as a human subject.
  • administering means delivering using any of the various methods and delivery systems known to those skilled in the art.
  • Administering can be performed, for example, intraperitoneally, intracerebrally, intravenously, orally, transmucosally, subcutaneously, transdermally, intradermally, intramuscularly, topically, parenterally, via implant, intrathecally, intralymphatically, intralesionally, pericardially, or epidurally.
  • An agent or composition may also be administered in an aerosol, such as for pulmonary and/or intranasal delivery.
  • Administering may be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • Eliciting a protective immune response in a subject can be accomplished, for example, by administering a primary dose of a vaccine to a subject, followed after a suitable period of time by one or more subsequent administrations of the vaccine.
  • a suitable period of time between administrations of the vaccine may readily be determined by one skilled in the art, and is usually on the order of several weeks to months.
  • the present invention is not limited, however, to any particular method, route or frequency of administration.
  • a “prophylactically effective dose” or “a immunologically effective dose” is any amount of a vaccine that, when administered to a subject prone to viral infection or prone to affliction with a virus-associated disorder, induces in the subject an immune response that protects the subject from becoming infected by the virus or afflicted with the disorder.
  • Protecting the subject means either reducing the likelihood of the subject's becoming infected with the virus, or lessening the likelihood of the disorder's onset in the subject, by at least twofold, preferably at least ten-fold.
  • a "prophylactically effective dose” induces in the subject an immune response that completely prevents the subject from becoming infected by the virus or prevents the onset of the disorder in the subject entirely.
  • inventions of any of the instant immunization and therapeutic methods may further comprise administering to the subject at least one adjuvant.
  • adjuvant shall mean any agent suitable for enhancing the immunogenicity of an antigen and boosting an immune response in a subject.
  • Numerous adjuvants, including particulate adjuvants, suitable for use with both protein- and nucleic acid-based vaccines, and methods of combining adjuvants with antigens, are well known to the skilled artisan.
  • Adjuvants suitable for use with protein immunization include, but are not limited to, alum, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FLA), alum adjuvants, saponin-based adjuvants, such as Quil A, and QS- 21, and the like.
  • the present invention also provides a method to attenuate a virus using temperature sensitivity as a phenotypic marker.
  • the method develops cold- adapted temperature sensitive virus strains.
  • the method of the present invention is an in vitro laboratory process to change the biologic growth characteristic of the virus to adapt for optimal replication at incubation temperature of below 30° C.
  • the adaption process is carried out in a systematic stepwise manner of incremental lower incubation temperature for culturing the virus until the targeted temperature chosen for optimal replication of the virus is achieved.
  • the method comprises the following steps: (i) preparing a reference stock of parental wild-type virus, (ii) incubating a culture of cells infected with the reference stock of parental wild-type virus at higher multiplicity of infection (MOI) and an incubation temperature of about 34° C to about 36° C, preferably about 34° C, for five or more passages until a full cytopathic effect (CPE) is obtained at each passage using an inoculum of a lower MOI and a shorter period of incubation for each passage to obtain full CPE, (iii) incubating a culture of cells infected with the resultant virus of the previous step at higher MOI and an incubation temperature of about 1° C to about 3° C lower than in the previous step for five or more passages until a full cytopathic effect (CPE) is obtained at each passage using an inoculum of a lower MOI and a shorter period of incubation for each passage to obtain full CPE
  • the targeted temperature is about 26° C to about 29° C, preferably about 28° C.
  • the incrementally lower incubation temperature is a temperature lowered about 1° C to about 2° C.
  • the reference stock of parental wild-type virus is prepared by incubating a culture of cells infected with a wild-type virus at a temperature of about 36° C to about 38° C, preferably about 37° C for one or two passages until a full cytopathic effect (CPE) is obtained. Aliquots of culture supernatant containing the produced virus are placed in vials or other suitable storage devices. This culture supernatant serves as a reference stock of parental wild-type virus. The reference parental wild-type virus is used for subsequent attenuation process. In another embodiment, aliquots of the reference stock of parental wild-type virus are stored at a suitable temperature, such as at -80° C.
  • the virus is passaged in each step of the process by obtaining a clarified culture supernatant containing the virus and passing this supernatant into each successive fresh new culture flask of cells, e.g., Vero cells, as soon as the virus obtains a full cytopathic effect (CPE).
  • the culture supernatant containing the virus is passed at a higher multiplicity of infection (MOI) of 20 at the beginning of each successive change to a lower temperature of incubation.
  • MOI multiplicity of infection
  • the new culture flask containing freshly confluent monolayer Vero cells is inoculated with the same MOI of virus for at least three more passages before subsequently reduced to a lower MOI of 5 to 10.
  • the attenuation process is then moved on to the next phase of successive lower incubation temperature after passaging for at least three more times at a MOI of 5 to 10.
  • the number of days required for each passage depends on how fast the virus is adapted at each phase of incrementally lower incubation temperature. The skilled artisan will readily know when a full CPE is reached.
  • the virus is any virus.
  • the virus is an RNA virus.
  • the R A virus is a plus strand RNA virus.
  • the virus is a member of the Picornaviridae family.
  • the virus is a member of the Enterovirus genus.
  • the virus is Enterovirus 71 (EV71).
  • the virus is cocksackievirus A16 (CA16). The method of the present invention is useful in producing cold-adapted temperature sensitive virus strains of any of these viruses, including but not limited to cold-adapted temperature sensitive strains of EV71 and CA16.
  • the cell to be infected by the virus is any cell which is permissive for the growth of the virus.
  • the cells are Vero cells' (ATCC CCL- 81).
  • the cells are maintained by regular passage in a medium suitable for growth of the cells.
  • the Vero cells are cultured in Dulbecco's modified Eagles's medium (DMEM) supplemented with 10% fetal calf serum (FCS).
  • DMEM Dulbecco's modified Eagles's medium
  • FCS fetal calf serum
  • Vero cells maintained in DMEM supplemented with 1% FCS is used for production of the parental wild-type virus, virus culture, attenuation, titration and assessment of temperature sensitive phenotype.
  • the DMEM is supplemented with 1% FCS for adapting the virus strains for replication in successively lowered temperature of incubation.
  • the present invention also relates to the cold-adapted temperature strains of virus produced by the method described herein.
  • the cold-adapted temperature viruses produced by the method described herein are useful in the production of vaccines using techniques well known to the skilled artisan. Such vaccines are useful for providing immunity against the parent virus strain by administering the vaccine to a subject using techniques well known to the skilled artisan.
  • the present method is applicable to the production of cold-adapted temperature sensitive viruses of the Picornaviridae family and of the Enterovirus genus.
  • the applicability has been demonstrated herein by the production of a cold-adapted temperature sensitive strains of EV71 (TLLa) and EV71 ( ⁇ ) which are derived following serial passages in cell-culture at incremental lower temperature of incubation.
  • the EV71 ( ⁇ ) strain retains phenotypic and genetic stability in specific in vitro cell culture conditions and does not exhibit neuro- virulence in monkeys following intravenous inoculation.
  • the applicability has also been demonstrated herein by the production of a cold-adapted temperature sensitive CA16 which is derived following serial passages in cell-culture at incremental lower temperature of incubation.
  • the invention also provides a kit for immunization of a subject with of a cold-adapted temperature sensitive Enterovirus 71 strain described herein.
  • the kit comprises a cold-adapted temperature sensitive Enterovirus 71 strain described herein, a pharmaceutically acceptable carrier, an applicator, and an instructional material for the use thereof.
  • the invention includes other embodiments of kits that are known to the skilled artisan.
  • the instructions can provide any information that is useful for directing the administration of the of a cold-adapted temperature sensitive Enterovirus 71 strain described herein.
  • RNA Interference RNA Interference
  • RNAi The Nuts & Bolts ofsiRNA Technology, DNA Press, 2003; Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Human Press, Totowa, NJ, 2004; Sohail, Gene Silencing by RNA Interference: Technology and Application, CRC, 2004.
  • Vero cells maintained by regular passage in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) were used for virus culture, attenuation, titration and assessment of temperature sensitive phenotype.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • the new culture flask containing freshly confluent monolayer Vero cells was inoculated with the same MOI of virus for three more passages before subsequently reduced to a lower MOI of 5 to 10.
  • the attenuation process was then moved on to the next successive lower incubation temperature after passaging for at least three more times.
  • the succeeding incremental lower temperatures of incubations for developing cold- adapted temperature sensitive strains of enterovirus. 71 in this example were 34° C, 32° C, 30° C, 29° C, and 28° C.
  • Virus Titration was determined by microtitration assay in Vero cells in accordance with the method described in Polio Laboratory Manual 2004 of World Health Organization with minor modification and virus titer was calculated as 50% cell culture infectious dose (CCID5 0 ) per milliliter following the method of Reed and Muench (1938). Briefly, following treatment with equal volume of chloroform to disperse virus aggregates, a 10- fold serial dilution of the clarified virus supernatant was made in DMEM containing 1% FCS.
  • Vero cell monolayers (10 4 cells per well) in 96-well flat-bottom tissue culture plate were inoculated with 100 ⁇ the serially diluted of each virus stock and incubated in an ambient of 5% C0 2 at each respective incubation temperature for 5 days prior to observation for the presence of CPE.
  • Temperature-sensitivity assay Two approaches were used to assess the growth characteristic of the virus strains in Vero cells at incubation temperature of 28° C, 37° C and 39.5° C. The first approach assessed the number of days taken for the virus strain to cause full CPE in infected cells (replication kinetic) and second approach assessed the titer of the virus strain in cells incubated at each specific tested temperature. Briefly, in the first approach, the growth medium of three T-25 tissue culture flasks containing confluent monolayer Vero cells of similar age were replaced with maintenance medium (DMEM with 1% FCS).
  • DMEM maintenance medium
  • the medium in each flask was then allowed to equilibrate to the specified temperature to be tested by placing in respective incubators for 1 hour and subsequently inoculated with the virus strain at a dose of 10 multiplicity of infection (MOI). If no CPE was noted at the end of 10-day culture, the supernatant was passed into a new of flask of monolayer Vero cells and similarly incubated for another 10 days. It was taken as no virus replication if no CPE was noted after second passage. In the Second approach, Vero cell suspension of density 10 4 cells per 100 ⁇ was seeded into each well of three 96-well cell-culture plates and incubated at 37° C in an ambient of 5% C0 2 .
  • MOI multiplicity of infection
  • each cell-culture plate was then allowed to equilibrate to the specific temperature to be tested by placing in respective incubators for 1 hour.
  • the cells in each well were subsequently inoculated with 100 ⁇ of 10-fold serial dilutions of the virus strain before being transferred to incubators of respective temperature and incubated for 5 days prior to observation for the presence of CPE.
  • RNA Extraction, RT-PCR and Sequencing Viral genomic RNA was extracted from the culture fluid of infected cells at full CPE using a commercially available Viral RNA Extraction Kit (Qiagen, Germany). First-strand synthesis was performed with EV71 -specific primers using Superscript II RNA polymerase (InvitrOgen, USA), and subsequent PCR with 18 degenerate primer pairs was done using GoTaq Green PCR mix (Promega, USA). Fragments generated were sequenced using BigDye Terminator sequencing kit (Applied Biosystems, USA).
  • 5 'RACE was performed to determine the 5'-UTR viral sequence by ligating the 5'- cordycepin-blocked adaptor DT88 (5'-GAA GAG AAG GTG GAA ATG GCG TTT TTG G- cordycepin-3'; SEQ ED NO:l) to the 5'-end of the EV71 cDNA using standard T4 DNA Ligase (Fermentas, USA), and standard PCR was performed afterwards using a DT88-complementary primer (5'-CCA AAA CGC CAT TTC CAC CTT CTC TTC 3'; SEQ ID NO:2) and an EV71- specific primer (5 '-ATT CAG GGG CCG GAG GAC TAC-3'; SEQ ID NO:3).
  • 3'-RACE was also performed to determine the 3'-UTR viral sequence using an oligo-dT primer (Li et al., 2005).
  • the remaining two monkeys were given respective equivalent booster dose of EV71 :TLLpP20 at day 14 PI after blood samples were collected for assessment of anti-EV71 antibodies. They were put to sleep 16 days after receiving the booster dose and CNS tissues were collected for histopathological study at autopsy.
  • CNS tissue specimens cervical, cerebellum, basal ganglia, brain-stem and spinal cord
  • non-neural tissues lymph node, spleen, liver, kidney, lung and heart
  • PBS phosphate buffered saline
  • the spinal cord was sectioned horizontally 10 times at cervical, 8 times at thoracic and 10 times at lumbar levels respectively.
  • Paraffin sections 6 ⁇ in thickness, were stained with haematoxylin and eosin (H&E) and with Luxol-fast blue/cresyl violet (Kluver-Barrera method) after paraffin removal and rehydration process.
  • H&E haematoxylin and eosin
  • Luxol-fast blue/cresyl violet Kluver-Barrera method
  • PBMC Peripheral blood mononuclear cells
  • Ficoll-PaqueTM PLUS Ficoll-PaqueTM PLUS
  • aliquots of PBMC suspension were seeded onto wells of Teflon-coated slide for detection of EV71 antigen by indirect immunofluorescence assay using commercial detecting monoclonal antibodies (Cat. No. 3360, Light Diagnostics, USA).
  • Virus isolation was carried out by inoculating PBMC suspension into wells of 24-well cell culture plate containing monolayer Vero cells.
  • Virus isolation on each serum specimen was performed by inoculating 50 ⁇ and 100 ⁇ of serum into respective wells of 24-well culture plate containing monolayer Vero cells. Monkey tissues were lightly washed with two exchanges of sterile PBS and homogenized by grinding using mortal and pastel inside a class II biosafety cabinet. Tissue homogenates (10%, w/v) prepared in DMEM was clarified by centrifugation at 1000 g for 10 minutes. The clarified supernatant was filtered through a 0.22 micron syringe filter and virus isolation was performed by inoculating 100 ⁇ and 200 ⁇ of the filtrate. A 10% stool suspension was made in PBS and clarified by centrifugation at 1000 g for 10 minutes.
  • virus isolation was performed by inoculating 100 ⁇ and 200 ⁇ of stool filtrate. All virus isolation work on monkeys' samples was performed in duplicate with one set of the inoculated cell culture incubated at 28° C and the other at 37° C.
  • RNA extraction and purification kit (Qiagen, Germany) was used to extract viral genomic RNA from serum, PBMCs and clarified tissue homogenates.
  • a commercial one-step RT-PCR kit (Qiagen, Germany) and a consensus oligonucleotide primer-pair (Sense: 5'-CACCCTTGTGATAGCAT GGATCAG-3 ' (SEQ ID NO:4); Anti-sense: 5 '-GTGAATTAAGAACRCAYCGTGTYT-3 ' (SEQ ID NO:5)) that amplified the proximal third of VP1 gene of all EV71 genotypes were used for molecular amplification and detection of EV71 -specific viral RNA after extraction from tissues.
  • the neutralizing antibodies titer of monkeys was determined by micro-neutralizing assay using Vero cells in accordance with the method described in Polio Laboratory Manual 2004 of World Health Organization with minor modification.
  • the concentration of each genotype of EV71 used for neutralization was 100 CCDD50 per 100 ⁇ .
  • the virus neutralization assay was performed using 96-well flat bottom culture plate. A serial 2-fold dilution of each serum sample was prepared in duplicate at a volume of 100 ⁇ DMEM (1% FCS) starting at 1 :10 dilution. An equal volume (100 ⁇ ) of virus working stock in DMEM (1% FCS) containing 100 CCID50 EV71 was added into each well of the diluted sera and incubated for two hours at 37° C.
  • the original three parental EV71 viruses used to derive the cold-adapted strains caused full CPE in Vero cells within 3 days after inoculation at a virus inoculum of 10 MOI and incubation temperature of 37.5° C.
  • the original parental EV71 viruses caused full CPE in Vero cells within 5 days at incubation temperature of 39.5° C.
  • all the three original parental EV71 viruses derived from first two passages in Vero cells cultured at 37.5° C did not cause CPE in Vero cells at a virus inoculum of 10 MOI and incubation temperature of 28° C. No CPE was also noted after a blind passage at the end of 10 days culture at 28° C.
  • the absence of virus replication in the inoculated Vero cells was further supported by negative staining of suspended cells presence in the culture supernatant fluid at the end of 10 days culture using commercial detecting monoclonal antibodies against EV71 by indirect immunofluorescence assay.
  • Incubation temperature of 28° C and 37° C was used to assay the virus replication titer of three cold-adapted strains and the assay was repeated at least 4 times.
  • the titer of EV71 :TLL was 1 XI 0 8 CCH3 50 /ml and 2 to 3 XI O 7 CCID 5 o/ml when the titrated cultures were incubated at 28° C and 37° C respectively.
  • EV71 :TLLa gave a virus titer of 1 X10 8 CdD 50 /ml at an incubation temperature of 28° C and a titer of 1 to 2 XI 0 5 5-6 CCJD 50 /m ⁇ at 37° C.
  • EV71 :TLLp gave a titer of 2 to 5 XI 0 8 CCTD 50 /m ⁇ at an incubation temperature of 28° C and a titer of 1 X10 7 CCIDso/ml at 37° C.
  • the stability of cold-adaptation of EV71 :TLLa and EV71 :TLLP was assessed by passaging both virus strains for another 20 passages in Vero cells at an incubation temperature of 28° C and a virus inoculum of 10 MOI After an additional 20 passages, EV71 :TLLa caused full CPE in cells incubated at 28° C within 2 days PI but failed to cause CPE in cells incubated at 37° C even after 2 blind passages.
  • EV71 :TLLp maintained the same cold- adapted phenotype in term of growth kinetic and virus titer at both incubation temperature of 28° C and 37° C after an additional 20 passages.
  • the stability of cold-adaptation of EV71 :TLLP was assessed further by passaging another 20 more passages (40 additional passages from the 100 th passage) under the same culture conditions and it was found to remain similar cold- adapted temperature sensitive phenotype.
  • the virus remained inability to produce viable infectious particles (lack of positive immunofluorescent staining cells) in Vero cells at incubation temperature of 39.5° C after 3 successive passages in cells incubated at 37° C and inability to cause full CPE in cell culture at incubation temperature of 39.5° C at 6 th repeated passage.
  • CPE Cytopathic Effect
  • the growth characteristic and titer of the virus at each passage was cultured or titrated in Vero cells at incubation temperature of 28° C, 37° C and 39.5° C.
  • the number of nucleotide changes between original parental wild-type, EV71 :TLLp, EV71 :TLL P20 and EV71 :TLLpP40 is shown in Table 3.
  • the complete genomes of virus strains derived from temperature sensitive reversion study by 6 successive passaging at incubation temperature of 37° C were also sequenced and number of nucleotide changes with respect to that of EV71 :TLLpP20 is shown in Table 4.
  • NT nucleotide
  • AA amino acids
  • NT nucleotides
  • AA corresponding amino acids
  • NT nucleotides
  • AA amino acids
  • X10 8 CCE 5o ml developed a spike of low grade fever (39.3° C) on day 3 post-inoculation. None of the monkeys had weight loss on reweighing at the time they were sacrificed under deep anesthesia.
  • Virological Investigations Virus isolation was performed on monkeys' sera, PBMC, stool samples and all autopsied tissues using Vero cells at incubation temperature of 28° C and 37° C. No virus was isolated from any of the monkeys' samples despite a blind passage after 10 days of.culture., One additional blind passaging in Vero cells were carried out for sera, PBMC samples and those tissue homogenates that were tested positive for EV71 by RT-PCR.
  • EV71 antigen was detected in a few PBMC derived from heparinised blood of two monkeys, 2891F (received 1 X10 7 CCID 50 of EV71:TLLj3P20) and 2890M (received 1 XI 0 8 CCID 50 of EV71 :TLLpP20), collected on Day 4 PI by indirect immunofluorescence assay ( Figure la) using commercial monoclonal antibodies. No virus antigen was detected in PBMC harvested from heparinized blood of monkeys "collected on Day 8 PI.
  • EV71 specific genomic sequence was detected in serum samples of all monkeys collected on both Day 4 and day 8 PI by RT-PCR. Of the non-neuronal tissues, only EV71 genomic sequence was detected in spleen homogenate of 2 monkeys (2202F and 289 IF) ( Figure lb). No EV71 genomic sequence was detected in any of the neuronal tissues homogenates.
  • the genome of EV71 :TLLPP20 present in the serum samples of two monkeys (2889M and 2890M) collected on both Day 4 and Day 8 PI were extracted and completely sequenced. The number of nucleotides (NT) and corresponding amino acids (AA) mutations or reversion that occurred in each of the genomic segments of virus strains present in the sera in comparison with the genome of EV71 : ⁇ 20 is shown in Table 5.
  • NT nucleotide
  • AA corresponding amino acids
  • Monkey Humoral Immune Response Assay for the presence of binding antibodies (IgM and IgG) in the sera of monkeys given intravenous EV71 :TLLpP20 was performed by indirect immunofluorescence assay using in-house prepared infected Vero cells as antigen. The titers of anti-EV71 IgM and IgG present in the blood of the two remaining monkeys (2889M and 2890M) collected on Day 14 PI, prior given an equivalent intravenous booster dose, and Day 30 PI (16 days post-booster) are shown in Table 6.
  • the titer of neutralizing antibodies against a number of genotypes of EV71 (A, B3, B4, B5, CI and C5) presence in sera of two monkeys after given intravenous EV71 :TLLPP20 as determined by micro-neutralization assay
  • Vero cells maintained by regular passages in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum
  • FCS coxsackievirus A16
  • CA16 parental wild-type coxsackievirus A16
  • the virus was plaque purified once in accordance with the technique described previously. After plaque purification, the virus stock designated as the respective parental wild-type strain was prepared following two passages in Vero cells at 37° C. All virus stock and respective passaged strains were stored in minus 80° C freezer.
  • Virus Titration was determined by microtitration assay in Vero cells in accordance with the method described in Polio Laboratory Manual 2004 of World Health Organization with minor modification and virus titer was calculated as 50% cell culture infectious dose (CCIDso) per milliliter following the method of Reed & Munch (1938). Briefly, following treatment with equal volume of chloroform to disperse virus aggregates, a 10-fold serial dilution of the clarified virus supernatant was made in DMEM containing 1% FCS.
  • Vero cell monolayers (10 4 cells per well) in 96-well flat-bottom tissue culture plate were inoculated with 100 ⁇ the serially diluted of each virus stock and incubated in an ambient of 5% C0 2 at each respective incubation temperature for 5 days prior to observation for the presence of CPE.
  • Temperature-Sensitive Assay Two approaches were used to assess the growth characteristic of the cold-adapted virus strain in Vero cells at incubation temperature of 28° C, 37° C and 39.5° C. The first approach assessed the number of days taken for the virus strain to cause full CPE in infected cells (replication kinetic) and second approach assessed the titre of the virus strain in cells incubated at each specific tested temperature.
  • the growth medium of three T-25 tissue culture flasks containing confluent monolayer Vero cells of similar age were replaced with maintenance medium (DMEM with 1% FCS).
  • the medium in each flask was then allowed to equilibrate to the specified temperature to be tested by placing in respective incubators for 1 hour and subsequently inoculated with the virus strain at a dose of 10 multiplicity of infection (MOI). If no CPE was noted at the end of 10-day culture, the supernatant was passed into a new of flask of monolayer Vero cells and similarly incubated for another 10 days. It was taken as no virus replication if no CPE was noted after second passage.
  • MOI multiplicity of infection
  • Vero cell suspension of density 10 4 cells per 100 ⁇ was seeded into each well of three 96-well cell-culture plates and incubated at 37° C in an ambient of 5% C0 2. After 10 hours of incubation, each cell-culture plate was then allowed to equilibrate to the specific temperature to be tested by placing in respective incubators for 1 hour. The cells in each well were subsequently inoculated with 100 ⁇ of 10-fold serial dilutions of the virus strain before being transferred to incubators of respective temperature and incubated for 5 days prior to observation for the presence of CPE.
  • Incubation temperature of 28°' C and 37° C was used to assay the virus replication titer of cold-adapted temperature sensitive strain of CA16.
  • the titre of cold-adapted temperature sensitive strain of CA16 was 1 XI 0 8 CdD 50 /ml and 1 XI 0 7 CCID 5 o/ml when the titrated culture plates were incubated at 28° C and 37° C respectively.
  • the use of the terms "a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
  • Huygelen C The concept of virus attenuation in the eighteenth and early nineteenth centuries. Biologicals 1997; 25: 339-45.

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Abstract

La présente invention concerne des souches d'entérovirus 71 sensibles à la température, adaptées au froid, en particulier les souches d'entérovirus 71 sensibles à la température, adaptées au froid, EV71 : TLLpP20 et EV71 : TLLaP20. La présente invention concerne également des procédés de développement des souches virales sensibles à la température, adaptées au froid.
PCT/SG2013/000027 2013-01-18 2013-01-18 Souches d'entérovirus 71 sensibles à la température, adaptées au froid, et procédés de développement de souches virales sensibles à la température, adaptées au froid WO2014112945A1 (fr)

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SG11201505468VA SG11201505468VA (en) 2013-01-18 2013-01-18 Cold-adapted temperature sensitive strains of enterovirus 71 and processes of developing cold-adapted temperature sensitive virus strains
PCT/SG2013/000027 WO2014112945A1 (fr) 2013-01-18 2013-01-18 Souches d'entérovirus 71 sensibles à la température, adaptées au froid, et procédés de développement de souches virales sensibles à la température, adaptées au froid
CN201380070773.0A CN104918636B (zh) 2013-01-18 2013-01-18 肠病毒71的冷适应型温度敏感株及开发冷适应型温度敏感性病毒株的方法
JP2015553680A JP6117379B2 (ja) 2013-01-18 2013-01-18 エンテロウイルス７１の低温適応温度感受性株および低温適応温度感受性ウイルス株を発展させるプロセス
MYPI2015001671A MY185184A (en) 2013-01-18 2013-01-18 Cold-adapted temperature sensitive strains of enterovirus 71 and processes of developing cold-adapted temperature sensitive virus strains
TW103101883A TWI705140B (zh) 2013-01-18 2014-01-17 適於冷之腸病毒７１溫度敏感株及發展適於冷之溫度敏感病毒株之方法
HK16101297.0A HK1213474A1 (zh) 2013-01-18 2016-02-04 腸病毒 的冷適應型溫度敏感株及開發冷適應型溫度敏感性病毒株的方法

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WO2015190999A1 (fr) * 2014-06-12 2015-12-17 Temasek Life Sciences Laboratory Limited Développement d'entérovirus chimériques sensibles à la température adaptés à un froid stable
TWI685566B (zh) * 2014-06-12 2020-02-21 新加坡商淡馬錫生命科學研究院公司 穩定之適於冷之溫度敏感型嵌合腸病毒之發展
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US20180195048A1 (en) * 2015-02-13 2018-07-12 Takeda Vaccines, Inc. Methods for producing virus for vaccine production
CN114107221A (zh) * 2015-02-13 2022-03-01 武田疫苗股份有限公司 用于产生制备疫苗的病毒的方法

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