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  • 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
  • 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
  • A61K39/245—Herpetoviridae, e.g. herpes simplex 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
  • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
  • A61K39/27—Equine rhinopneumonitis virus
• • 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
  • A61P31/20—Antivirals for DNA viruses
  • A61P31/22—Antivirals for DNA viruses for herpes 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
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
• • 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
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells 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
  • A61K2039/51—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
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/543—Mucosal route intranasal
• • 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
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
  • C12N2710/16734—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
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
  • C12N2710/16761—Methods of inactivation or attenuation
  • C12N2710/16763—Methods of inactivation or attenuation by chemical treatment

Definitions

• the present invention relates to an equine abortion virus mutant, a process for the preparation of said mutant, use of said mutant and live vaccines derived from said mutant
• Equine abortion virus (EHV-1 ), a herpes virus, is a major equine pathogen responsible for viral-induced abortion, neurological disease such as paresis, infections of the upper respiratory tract, and neonatal foal disease (NFD) NFD results from close to term transplacental infection of fetuses, which are born weak with severe respiratory disease and some with jaundice due to liver infection by EHV-1
• EHV-1 Equine rhinopneumonitis virus
• rhinopneumomtis acute respiratory tract disease
• Rhinopneumonitis is characterized by fever, anorexia, and profuse serous nasal discharge that later becomes mucopuruient
• EHV4 infection causes abortion in pregnant mares
• EHV1 and EHV4 establish persistent, lifelong latent infections Upon reactivation the viruses cause recurrent disease, accompanied by virus shedding and transmission to other animals
• the present invention provides for such vaccines
• the present invention provides for an EHV-1 Ts mutant as deposited at the European Collection of Animal Cell Culture (ECACC), Salisbury, Wiltshire SP4 OJG, UK on 10 June 1999 under accession number V99061001 , and progeny thereof
• EHV-1 Ts mutants according to the invention are furthermore phenotypically characterized in that
• the EHV-1 Ts mutants according to the invention have the advantage that replication is restricted to the upper respiratory tract of conventional equidae with no or limited ensuing viraemia
• the Ts mutants are safe for pregnant mares while giving rise to significant immune stimulation following growth in the upper respiratory tract.
• the Ts mutants are not readily back-passaged form animal to animal thus limited in their potential for transmission and reversion
• progeny ' is defined to include also all strains obtained by further serial passage of the deposited EHV-1 Ts mutant.
• a temperature sensitive mutant is defined as a mutant virus which has an impaired growth at or above a certain temperature at which the wild type has a normal growth
• the EHV-1 Ts mutants according to the present invention are characterized in that they are temperature sensitive at the body temperature of the host animal
• the EHV-1 Ts mutants of the present invention do not replicate above a temperature of 38.5 to 39 0°C
• the EHV-1 Ts mutants according to the invention do not replicate at a temperature of 38 5°C
• small plaques are defined as plaques that are at least half to one third the size of the plaques formed by the wild-type parent strain in equine cells
• the "limited abiity to cause viraemia” is defined as the ability to cause no or low grade (that is, just detectable) vireamia for 1 to 3 or 4 days in some animals with respect to the ability of the parent strain to cause viraemia
• Temperature sensitive EHV-1 mutants according to the invention can be obtained by treatment of infected bovine, equine or other permissive ceil cultures at 34°C with non-toxic concentrations of a mutagens such as 5-bromo-2-deoxy undine, azacytidine and the like during viral replication in vitro, followed by biological cloning of progeny virus from said treated cultures in bovine or equine or other permissive cell lines
• a composition in particular a vaccine composition, comprising an EHV-1 Ts-mutant according to the invention, and a pharmaceutically acceptable carrier or vehicle
• a (vaccine) composition according to the invention comprises the EHV1 Ts-mutant deposited at the ECACC, Salisbury, UK having accession number V99061001 and/or progeny thereof
• Pharmaceutical acceptable carriers or vehicles that are suitable for use in a
• the vaccine compositions according to the invention are safe and can be used to protect the equidae clinically and virologically against infections with EHV-1 and to protect against virus-induced abortions and paresis
• the vaccine according to the invention was found to stop trans-placental infection, thus protecting the newborn foal from the effects of neonatal foal disease
• the vaccine composition according to the present invention can be administered not only to horses but also to other animals that are susceptible to EHV-1 infection such as donkeys, zebra's and the like Cattle which have been reported to be susceptible to EHV-1 and EHV-4 infection can also be treated with the vaccine according to the invention
• vaccines comprising an EHV-1 Ts- mutant according to the invention not only protect against EHV-1 infections but also against the disease and the associated virus shedding following EHV-4 infection
• a vaccine can be useful to obtain cross-protection in the vaccinated equidae
• Said vaccines give rise to improved protection thus effectively blocking infection with wild-type viruses
• Vaccine compositions according to the invention can be prepared following standard procedures
• a vaccine according to the invention preferably is a live vaccine
• the seed virus of the EHV-Ts mutant can be grown on a cell culture, such as primary or secondary bovine kidney or equine cells
• the viruses thus grown can be harvested by collecting the tissue cell culture fluids and/or cells
• the yield of the viruses can be promoted by techniques that improve the liberation of the infective particles from the growth substrate, e g sonication
• the live vaccine may be prepared in the form of a suspension or may be lyophilized
• compositions suitable for use in a vaccine according to the invention are sterile water, saline, aqueous buffers such as PBS and the like
• a vaccine according to the invention may comprise other additives such as adjuvants, stabilizers, anti-oxidants and others
• Suitable stabilizers are for example carbohydrates including sorbitol, mannitol, starch, sucrose, dextran and glucose, proteins and degradation products thereof including but not limited to albumin and casein, protein-containing agents such as bovine serum or skimmed milk, and buffers including but not limited to alkali metal phosphates In lyophilized vaccine compositions it is preferable to add one or more stabilizers
• Suitable adjuvants include but are not limited to aluminum hydroxide, phosphate or oxide, amphigen, tocopherols, monophosphenyl lipid A, muramyl dipeptide, oil emulsions, glucans, carbomers, block-copolymers, cytokines and saponins such as Quil A
• the amount of adjuvant added depends on the nature of the adjuvant itself
• EHV-1 Ts mutants are preferably administered to conventional, seronegative animals varying in ages from a few days to several years, including those in-foal
• the vaccine can be administered to the animals via non- parenterally administration routes, including but not limited to intradermal, oral, spraying, aerosol, mtra-ocular, and intranasal administration
• the vaccine can be administered via parenteral administration routes
• the vaccine is administered intradermally or intranasally
• the EHV-1 Ts mutant virus is administered in an amount that is effective to induce protection against EHV-1 infection
• the dose generally will depend on the route of administration, the time of administration, as well as age, health and diet of the animal to be vaccinated
• the virus can be administered in an amount between 10 2 and 10 9 pfu/dose per animal, preferably between 10 3 and 10 5 pfu/dose and more preferably at 10 4 pfu/dose per animal
• the vaccines according to the invention also may be given simultaneously or concomitantly with other live or inactivated vaccines
• These additional vaccines can be administered non-parenterally or parenterally
• the additional vaccines are recommended for parenteral administration EXAMPLES
• tissue culture medium 25 ml
• tissue culture medium 25 ml
• 40 ⁇ g/ml of 5-bromo-2-deoxy undine was incubated at 34°C
• the culture was harvested (frozen at -40°C and then thawed at 37°C), dialyzed overnight at 4°C against PBS, titrated for EHV-1 mfectivity in ED cells at 37°C and subsequently cloned at 34°C in ED cells grown in 96-well microtitration plates.
• EHV-1 strain TS C147 has EHV-4 like characteristics
• a parameter for the differentiation between EHV-1 and EHV-4 is their ability to replicate in rabbit kidney (RK13) cells EHV-1 strains replicate well in RK13 cells but the cells are refractory to EHV-4 strains EHV-1 strain TS C147 at MSV+1 ° level, its wild type parent EHV-1 strain, EHV-1 strain deficient in immediate early gene (EHV-1 IE), pathogenic EHV-1 strain CHLi and a field EHV-4 isolate were titrated in parallel at 37°C in RK13 cells and Equine dermal (ED) cells Results given in Table 2 show that the 4 EHV-1 strains, including strain TS C147 and EHV-4 strain replicated in ED cells but EHV-1 strain TS C147 and EHV-4 strain did not grow in RK13 cells.
• Titers given as ⁇ 1 1 log 10 TCID 50 /ml represent no EHV-1 CPE detected in 4 x 200 ⁇ l of the lowest dilution (10 ' ) in the titratio ⁇
• Vaccine virus grew to low titers (nasal mucus peak titers 1 5 to 3 0 log 10

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• 2000
  • 2000-09-11 RU RU2002109231A patent/RU2252253C2/ru not_active IP Right Cessation
  • 2000-09-11 WO PCT/EP2000/008944 patent/WO2001017553A1/en not_active Application Discontinuation
  • 2000-09-11 HU HU0202738A patent/HUP0202738A3/hu unknown
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  • 2000-09-11 EP EP20000974378 patent/EP1216054A1/en not_active Withdrawn
  • 2000-09-11 JP JP2001521341A patent/JP2004532601A/ja not_active Withdrawn
  • 2000-09-11 AU AU12711/01A patent/AU778157B2/en not_active Ceased

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