WO2008059018A2 - Vaccin pour vacciner des espèces félines et canines contre le virus de la grippe - Google Patents

Vaccin pour vacciner des espèces félines et canines contre le virus de la grippe Download PDF

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WO2008059018A2
WO2008059018A2 PCT/EP2007/062392 EP2007062392W WO2008059018A2 WO 2008059018 A2 WO2008059018 A2 WO 2008059018A2 EP 2007062392 W EP2007062392 W EP 2007062392W WO 2008059018 A2 WO2008059018 A2 WO 2008059018A2
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virus
vaccine
influenza
cats
influenza virus
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PCT/EP2007/062392
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WO2008059018A3 (fr
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Fengsheng Lin
Ian Tarpey
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Intervet International B.V.
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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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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

Definitions

  • Vaccine for vaccinating felines and canines against influenza virus Vaccine for vaccinating felines and canines against influenza virus.
  • the present invention is concerned with a vaccine for vaccinating felines and canines against influenza virus.
  • Influenza virus is a RNA virus of the family Orthomyxoviridae (the influenza viruses) capable of infecting birds and mammals. Influenza viruses have a segmented genome of eight negative sense, single strands (segments) of RNA, abbreviated as PB2, PB1 , PA, HA, NP, NA, M and NS.
  • the HA segment encodes the hemagluttinin protein, which is an antigenic protein found in the protein coat (viral envelope) of the viral particle. The protein is involved in cellular entry of the virus.
  • the NA segment encodes the neuraminidase, which is an antigenic glycosylated enzyme also found on the surface of the influenza viral particle. It facilitates the release of progeny virus from the cell.
  • influenza viruses There are three types of influenza viruses: A, B, and C.
  • influenza types A, B, and C viruses Humans can be infected with influenza types A, B, and C viruses.
  • Type C may not be a true influenza virus and usually causes only mild or asymptomatic disease.
  • Influenza B virus usually causes a minor illness, but it does have the potential to cause more severe disease in older persons.
  • Influenza type A viruses can infect people, birds, pigs, horses, and other animals, but wild birds are the natural hosts for these viruses. Typically, wild birds do not become sick when they are infected with avian influenza A viruses. However, domestic poultry, such as turkeys and chickens, can become very sick and die from avian influenza, and some avian influenza A viruses also can cause serious disease and death in wild birds.
  • influenza A viruses are further classified by subtype on the basis of the two main surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). No different subtypes of H and N have been identified for influenza B and C. There are 16 known HA subtypes and 9 known NA subtypes for Influenza A viruses. For example, an ⁇ 5N1 " virus has an HA 5 protein and an NA 1 protein.
  • Antigenic drift occurs in both influenza A and B viruses. It is part of the continuing occurrence of new influenza strains that differ form their ancestors by mutations (point mutations) in the HA and NA genes. The amount of change can be subtle or dramatic. This process allows influenza viruses to change and re-infect people repeatedly through their lifetime, and is the reason why flu vaccines need seasonal updates. The emergence of new strains may lead to the occurrence of strains that are substantially different from the previous strain and thus may result in a large flu epidemic. The most recent example of such large-scale epidemic in Hong Kong was the attack of influenza A H3N2 Sydney in the first few months of 1998.
  • the second type of antigenic variation "antigenic shift”.
  • a genetic shift can occur when two different influenza viruses, co-infecting the same host, exchange a whole genomic segment, thereby giving rise to a new 'reassortanf of a whole new subtype.
  • Such a genetic shift may result in a yet unknown viral subtype -for example, from H1 N1 to H2N2 in 1957 and from H2N2 to H3N2 in 1968, that will be new for the host's immune system.
  • the occurrence of a genetic shift may give rise to an influenza pandemic. The most recent pandemic threat occurred in Hong Kong SAR.
  • H5N1 avian subtype of influenza A was found to be associated with serious disease in humans, while the H5 subtype influenza virus was previously confined to avian species and was for the first time found to infect humans.
  • Subtypes of influenza A that are currently circulating among people worldwide include H1 N1 , H1 N2, and H3N2 viruses.
  • H5N1 avian influenza
  • the virus is highly contagious and already over 200 million domestic birds have either been culled or died following infection. So far it has claimed approximately 100 human lives.
  • Avian influenza virus has also been known to infect cats and other felidae. Indeed in 2003/2004 more than 147 tigers and two leopards in zoos in Thailand, reportedly fed on infected chicken carcasses, died or had to be euthanased for animal welfare reasons. More recently between October 2005 and February 2006 FAO field veterinarians have reported unusual high cat mortality in Iraq and Indonesia in the vicinity of H5N1 outbreaks in poultry.
  • H5N1 infection was confirmed by Friedrich Loeffler Institute (the first organisation isolated feline influenza virus H5N1 ) in 3 dead cats on the Baltic Sea island of Ruegen (Germany) where over 100 wild birds had been found dead on the island during previous weeks.
  • the potential for infection of and spread between domestic cats has been shown in laboratory trials.
  • Rotterdam demonstrated systemic spread of human influenza virus (H5N1 ) in domestic cats infected by respiratory, digestive, and cat-to-cat contact (Rimmelzwaan, et al.,Am. J. Pathol. ,16891 ), 176-183, 2006). All cats were infected with H5N1 virus and showed clinical signs of disease such as fever, lethargy, laboured breathing, and virus was detected in throat nasal, rectal swabs and many internal organs, regardless of the original site of infection.
  • H5N1 influenza is also capable of infecting dogs. (Songserm T. et al., Emerging Infectious Diseases, VoI 12, No. 1 1 , November
  • An efficacious vaccine for vaccinating cats or dogs against influenza should prevent the vaccinated animals from getting physically ill, or even die, due to a subsequent infection with the influenza virus.
  • a vaccine for vaccinating cats and dogs against influenza infection should not only prevent the animals from getting ill or die due to an influenza infection, but should preferably also significantly reduce the amount of virus a cats may excrete into their environment in amounts that would allow infection. When the cats excrete hardly any or no virus the changes that it will infect other animals or even humans are minimized.
  • the present invention provides a vaccine for vaccinating felines and canines against influenza virus, comprising an inactivated influenza virus of the A (H5) type.
  • A influenza virus
  • feline is meant a member of the felidae family, including domestic cats, and other felines such as lions, tigers, leopard, jaguar, puma, and cheetah, and other wild cats such as the lynx, caracal, and bobcat.
  • feline The most familiar feline is the domestic cat or house cat (subspecies Felis silvestris catus). With “canine” is meant a member of the family canidae including dogs, wolves, foxes and coyotes. Of course the most familiar canine is the domestic dog.
  • a vaccine according to the invention is very suitable for use in felines, such as domestic cats, and canines (e.g. dogs) that come into regular contact with other animals, but also with birds which may be infected with an avian influenza virus (such as the very pathogenic H5N1 type), and also with humans.
  • a vaccine according to the invention aims at protecting felines and canines against disease caused by infection with an (avian) influenza virus, especially of the influenza A (H5) type. Furthermore the vaccine aims at preventing the animals from excreting substantial amounts of such a virus into the environment in case they become infected.
  • Influenza vaccines for vaccinating poultry are known in the art.
  • avian influenza vaccines for vaccinating poultry are based on influenza strains such as H5N2 or H5N6 strains.
  • An example of such a poultry vaccine is Nobilis® Influenza H5, an inactivated avian influenza type A H5N2 virus (A/Chicken/mexico/232-CPA/94) water-in-oil emulsion vaccine manufactured by Intervet International, Boxmeer, the Netherlands.
  • This vaccine comprises the inactivated virus and an adjuvant ("GNE" adjuvant, which is a proprietary product Intervet International, the Netherlands).
  • a vaccine according to the invention may be based on any suitable strain of the A (H5) type influenza virus, e.g. H5N2, H5N3 or H5N6.
  • a suitable vaccine may even be based on a H5N1 strain (optionally modified to reduce its virulence), it is preferred to base the vaccine on a subtype that is different in its Neuraminidase type from the one actually causing the infection (wild type virus).
  • This difference in the neuraminidase type between wild type strain and vaccine strain allows for the differentiation between vaccinated and infected animals, by using differential diagnostic techniques based on the detection of the neuraminidase subtype of the virus.
  • a vaccine that allows vaccinated animals to be discriminated from infected animals is commonly referred to as a "marker vaccine".
  • the strains useful in a vaccine according to the invention may be isolated wild type strains or may be strains that are created with the aid of recombinant DNA techniques such as "reverse genetics", or may be strains which are reassortants wherein segments of different existing influenza type A strains are combined (in creating reassortants, usually a segment of a wild type strain is combined with the backbone of a safe vaccine strain referred to as the "donor strain").
  • donor strain for each subtype different strains may exist which can be used in a vaccine according to the invention, or segments of different strains may be combined or modified. Good results were obtained with a vaccine according to the invention, based on an influenza A strain of the subtype H5N6 (strain A/duck/postdam/2243/84).
  • a vaccine according to the invention preferably contains a suitable adjuvant.
  • an adjuvant is meant a substance that increases the ability of an antigen to stimulate the immune system.
  • Different types of adjuvants are known in the art, for example Freunds complete adjuvant (FCA), which is prepared as a water in oil emulsion and comprises a mixture of a non-metabolizable oil (mineral oil), a surfactant (Arlacel A), and mycobacteria (M. tuberculosis or M. butyricum), or Freunds incomplete adjuvant which is the same oil/surfactant mixture as FCA but does not contain any mycobacteria.
  • FCA non-metabolizable oil
  • Glalacel A surfactant
  • mycobacteria M. tuberculosis or M. butyricum
  • Freunds incomplete adjuvant which is the same oil/surfactant mixture as FCA but does not contain any mycobacteria.
  • Alternative adjuvants include other oil/surfactant based adjuvants in which different surfactants are combined with either a non-metabolizable mineral oil, a metabolizable oil, or a mixture of the two. They may be prepared for use as an emulsion with aqueous Ag solution. Examples of such adjuvants are Montanide ISA Adjuvants [Seppic, Paris, France]. Other adjuvants known in the art include those marketed by MVP laboratories Inc., such as Emulsigen, Carbigen or Polygen. Further adjuvants known in the art are adjuvants based on an dl- alpha tocopherol (vitamin E), a substance known for its antioxidant effects on the cell membranes.
  • vitamin E dl- alpha tocopherol
  • Adjuvants of this type are disclosed in EP382271.
  • An example of such an adjuvant is Diluvac Forte (Intervet International BV).
  • Other adjuvants known in the art include aluminium salt adjuvants.
  • adjuvants are used that are effective, but also well tolerated by the animal and do not give rise to severe or painful local reactions at the injection site.
  • Adjuvated inactivated viral vaccines for cats and dogs are known in the art.
  • Nobivac Rabies Intervet International BV which contains inactivated rabies virus and is adjuvated with aluminumphosphate.
  • a vaccine according to the invention can be used to vaccinate cats and dogs to prevent them from getting physically ill, or even die, from an infection with and influenza virus, more particular, a type A (H5) influenza virus such as the very pathogenic H5N1 strains.
  • vaccinating felines and/or canines with a vaccine according to the invention will prevent the vaccinated animal from excreting the virus into its environment.
  • viruses can mutate rapidly and they have a tendency to acquire genes from flu viruses that infect other animal species. If avian and human influenza viruses were to simultaneously infect a person or animal, the two viruses might swap genes. The result could be a new virus that is readily transmissible between humans and against which humans would have no natural immunity. Such an event could trigger a worldwide influenza pandemic.
  • the vaccine will limit the spread of the virus and will thereby limit the risk that other animals or humans that may be in contact with an infected cat will get infected.
  • the virus that forms the basis for a vaccine according to the invention may be produced by methods known to the person skilled in the art.
  • influenza vaccines are at present mostly produced in fertilized chicken eggs (egg-based influenza vaccine production).
  • the influenza virus is injected into the eggs and accumulates in the fluid surrounding the embryo. The embryo becomes infected so that the virus can multiply. After several days of incubation, the eggs are opened to harvest the virus. The harvested virus is purified, chemically inactivated and used to produce a vaccine.
  • An alternative way of producing flu vaccine is based on cell or tissue cultures (cell- culture based influenza vaccine production). Mammalian kidney cells are preferably used for these cell cultures. Useful cells include those known in the art such as canine kidney cells, for example MDCK cells, or monkey kidney cells such as Vero cells.
  • Both egg-derived- and cell-culture derived influenza virus can be used in the manufacture of a vaccine according to the invention. Based on immunization results obtained with vaccines according to the invention of both types (egg-based versus cell-culture based), cell culture derived antigen is preferred. Good results were obtained when antigen was used that was derived from a culture of MDCK cells.
  • a vaccine according to the invention is therefore preferably based on MDCK derived influenza virus if the H5N6 subtype (strain A/duck/postdam/2243/84), and adjuvated with a vitamin E based adjuvant such as Diluvac Forte.
  • Example 1 Vaccine virus strain: subtype H5N6, strain A/duck/postdam/2243/84.
  • roller bottles were seeded with appropriate cell density of MDCK cells. After cells become confluent, the cells were infected with the virus at MOI of . The infected cells were incubated at 36°C - 38°C until CPE complete. Supernatant and cells are harvested. Normally live virus titres are between 10 60 TCID 50 /ml and 10 85 TCID 50 /ml.
  • the bulk antigen was stored at 2°C to 8 0 C, and was inactivated by addition of formaldehyde to a final concentration of 0.1 % and by incubation at 37°C for 48 hrs.
  • Sodium metabisulfite will be added to inactivated bulk antigen to neutralise formaldehyde residual for 30 min at room temperature.
  • the pH was adjusted to pH7.2 by the addition of sodium bicarbonate.
  • Example 2 testing of different formulations in vaccination of cats.
  • the antigens derived from eggs and MDCK cells were formulated into 6 test vaccines with following adjuvants.
  • Table 1 test vaccines used in animal trial.
  • the finished products were aliquoted into one dose vials (1.0ml/vial) which were stored at at 2°C to 8 0 C.
  • test vaccines 1 to 6 above Seven groups of 5 SPF cats aged between 8 and 9 weeks at the beginning of the study were used. For the first 6 groups each group were immunised subcutaneously with one dose of one of the test vaccines (test vaccines 1 to 6 above) twice on the back of the neck with a four week interval. Group 7 was kept as un-vaccinated controls.
  • test vaccine No. 4 MDCK cells derived antigen formulated in Diluvac Forte
  • group 7 The 5 un-vaccinated controls (group 7) were transferred to isolation units on day 19 post the secondary vaccination. They were challenged oro-nasally with 10 60 EID 50 /dose of feline influenza H5N1 virus R606/06 hp strain on day 27 post the secondary vaccination. Oro-pharyngeal and rectal swabs were taken from each challenged cats regularly for the detection of virus.
  • the cats in groups 1 to 3, 5 and 6 were clinically monitored between day -1 and 56 post primary immunisation, and the cats in groups 4 and 7 were clinically monitored between day -1 and 46 post primary immunisation. This was because that cats in groups 4 and 7 were transferred to isolation units for challenge on day 47 post primary vaccination.
  • the daily score for each cat ⁇ 3 is not considered to be significant to practising veterinary surgeons and pet owners.
  • the daily score for each cat ⁇ 5, especially long lasting one (more than 14 days), is considered to be unacceptable.
  • Individual data is listed in Tables 1 to 6.
  • Two cats did not have any local reactions. Two cats had daily score of 1 for 1 and 2 days, respectively. The remaining one had a score of 1 for 17 days.
  • One cat in group 3 showed increased rectal temperature of 39.7°C on the day of but prior to the primary vaccination.
  • One cat in group 4 had rectal temperature of 40 0 C on day 46 post primary vaccination.
  • HAI titre equal to and greater than 4log 2 is considered to be protective. All cats had antibodies against avian influenza H5N6 virus higher than 4.0log 2 after the first vaccination with the exception of one cat in group 1 , which was sero-negative. After the secondary vaccination all cats in all groups had HAI titre equal to and greater than 5.3log 2 For each adjuvant, it seems that MDCK cells derived antigen is slightly more immunogenic than egg derived antigen. Antibody titres were lower in group 1 than that in other 5 groups.
  • CT-value ⁇ 30 is considered to be viral genome positive; CT-value ⁇ 40 is considered to be negative; and CT-value between 31 and 39 is generally negative by virus isolation in 9-1 1 day old SPF chicken embryonated eggs, but occasionally positive with a very low titre.
  • a few swab suspensions were also inoculated into allantoic cavity in 9-1 1 day old SPF chicken embryonated eggs for further confirmation by virus isolation and the presence or absence of challenge virus in samples was confirmed by HA.
  • the aim of the study was to determine whether the formulations of H5N6 antigen in various amount of adjuvant is safe and can induce a protective level of immunity in dogs.
  • Virus used to prepare vaccine in this study was subtype H5N6, strain A/duck/potsdam/2243/84, which was propagated on MDCK cells as described in Example 1 , and inactivated as described in Example 1.
  • H5N6 antigen The content of H5N6 antigen was determined by a standard method of haemagglutination (HA) assay.
  • Polygen (co-polymer base) was purchased from MVP Laboratories, Inc. US and used for the formulation of the test vaccines. Polygen was added to the inactivated antigen prepared above to give a final concentration of either 10% (v/v) (test vaccine 1 ) or 15% (test vaccine 2) and mixed thoroughly under aseptic conditions. The antigen content of vaccine was between 136 and 162 haemagglutinating units per ml (dose). The formulated test vaccines were stored at 2°C to 8°C.
  • the six dogs were randomly divided into two groups of three.
  • the dogs in groups 1 and 2 were immunised subcutaneously with 1.0 ml of test vaccine 1 (containing 10% Polygen) or test vaccine 2 (containing 15% Polygen) twice at a 4 week interval (into separate sites),. All dogs were clinically monitored daily including local reactions at injection sites until day 10 post second immunisation. In addition, the rectal temperatures were measured at 4 hours post each immunisation.
  • H5N6 antigen The measurement of antibodies against H5N6 antigen was carried out by a standard method of haemagglutination-inhibition (HAI) assay.
  • the antigen used was beta- propiolactone inactivated A/duck/potsdam/2243/84 H5N6 virus.
  • antibody titre is expressed as log 2 . ⁇ 3.3 log 2 is considered as negative and ⁇ 3.3 log 2 is considered as positive.
  • Example 2 The study in cats reflected in Example 2 indicates that cats, which were immunised with the identical antigen formulated in another adjuvant (Diluvac Forte), produced a similar level of HI antibody. The vaccinated cats were fully protected from highly pathogenic H5N1 virus challenge. Therefore, data generated from the study in cats suggests that both formulations used in this study could induce a protective level of immunity in dogs against highly pathogenic influenza H5H1 virus challenge, and that the immunity could last at least 6 months following two vaccinations.

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Abstract

La présente invention concerne un vaccin pour vacciner des espèces félines et canines contre le virus de la grippe, comprenant un virus de la grippe inactivé du type A (H5). Un vaccin selon l'invention vise à protéger les animaux contre la maladie provoquée par une infection par un virus de la grippe (aviaire), notamment de la grippe du type A (H5). De plus, le vaccin vise à empêcher les chats ou les chiens d'excréter des quantités substantielles d'un tel virus dans l'environnement, au cas où ils deviendraient infectés. Un vaccin selon l'invention peut être basé sur n'importe quelle souche appropriée du virus de la grippe du type A (H5), par exemple H5N2, H5N3 or H5N6.
PCT/EP2007/062392 2006-11-15 2007-11-15 Vaccin pour vacciner des espèces félines et canines contre le virus de la grippe WO2008059018A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7674469B2 (en) 2006-10-25 2010-03-09 Internet International B.V. Feline influenza vaccine and method of use

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