WO2009156448A1 - Attenuated pestivirus - Google Patents
Attenuated pestivirus Download PDFInfo
- Publication number
- WO2009156448A1 WO2009156448A1 PCT/EP2009/057911 EP2009057911W WO2009156448A1 WO 2009156448 A1 WO2009156448 A1 WO 2009156448A1 EP 2009057911 W EP2009057911 W EP 2009057911W WO 2009156448 A1 WO2009156448 A1 WO 2009156448A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- attenuated
- pro
- pestivirus
- bvdv
- residue
- Prior art date
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Classifications
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- 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
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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
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
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- 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
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- 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
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- 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
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24361—Methods of inactivation or attenuation
- C12N2770/24362—Methods of inactivation or attenuation by genetic engineering
Definitions
- the present invention relates to the field of animal health and in particular to attenuated pestiviruses such as classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV) or border disease virus (BDV).
- CSFV classical swine fever virus
- BVDV bovine viral diarrhea virus
- BDV border disease virus
- Pestiviruses are causative agents of economically important diseases of animals in many countries worldwide.
- Presently known virus isolates have been grouped into four different species which together form one genus within the family Flaviviridae.
- CSFV Classical swine fever virus
- hog cholera virus is responsible for classical swine fever (CSF) or hog cholera (HC) (Moennig and Plagemann, 1992; Thiel et al., 1996).
- Border disease virus (BDV) is typically found in sheep and causes border disease
- Pestiviruses are small enveloped viruses with a single stranded RNA genome of positive polarity lacking both 5' cap and 3' poly(A) sequences.
- the viral genome codes for a polyprotein of about 4000 amino acids giving rise to final cleavage products by co- and posttranslational processing involving cellular and viral proteases.
- the viral proteins are arranged in the polyprotein in the order NH 2 -N pro -C-E rns -El-E2-p7-NS2-NS3-NS4A-NS4B- NS5A-NS5B-COOH (Lindenbach and Rice, 2001).
- E ms lacks a typical membrane anchor and is secreted in considerable amounts from the infected cells; this protein has been reported to exhibit RNase activity (Hulst et al., 1994; Schneider et al., 1993; Windisch et al., 1996). The function of this enzymatic activity for the viral life cycle is presently unknown. The enzymatic activity depends on the presence of two stretches of amino acids conserved between the pestivirus E ras and different known RNases of plant and fungal origin. Both of these conserved sequences contain a histidine residue (Schneider et al., 1993). Inactivation of the RNase activity residing within the E ms results in an attenuated apathogenic pestivirus which is capable to be used as a modified live vaccine (WO 99/64604).
- the pestivirus glycoprotein E ms is expressed on the surface of virions and in infected cells as a disulfide-linked homodimer.
- the most C-terminal cysteine residue forms the intermolecular disulfide bond between two E ms monomers, resulting in the E ms homodimer (Schneider et al., 1993).
- N pr0 represents the first protein encoded by the long open reading frame in the pestivirus RNA.
- N pr0 represents a nonstructural protein that has protease activity and cleaves itself of the nascent polyprotein (Stark et al., 1993; Wiskerchen et al., 1991) presumably already during translation.
- N pro is a cysteine protease (R ⁇ menapf et al., 1998) that is not essential for virus replication (Tratschin et al., 1998).
- N pr0 somehow interferes with the cellular antiviral defense so that it can be hypothesized to modulate the immune system within an infected host (R ⁇ ggli et al., 2003). Mayer and coworkers presented indications for an attenuation of CSFV in consequence of a deletion of the N pr0 gene (Mayer et al., 2004).
- BVDV vaccines for the prevention and treatment of BVDV infections still have drawbacks (Oirschot et al. 1999). Vaccines against the classical BVDV-I provide only partial protection from BVDV-2 infection, and vaccinated dams may produce calves that are persistently infected with virulent BVD V-2 (Bolin et al., 1991, Ridpath et al., 1994). This problem is probably due to the great antigenic diversity between type 1 and type 2 strains which is most pronounced in the glycoprotein E2, the major antigen for virus neutralization (Tijssen et al., 1996). Most monoclonal antibodies against type 1 strains fail to bind to type 2 viruses (Ridpath et al., 1994).
- Vaccines comprising attenuated or killed viruses or viral proteins expressed in heterologous expression systems have been generated for CSFV and BVDV and are presently used.
- Conventional BVDV life vaccines are typically generated by cell culture passages resulting in viruses with attenuated virulence in the target species.
- the structural basis of the attenuation of BVDV used as life vaccines is not known. Therefore it is not possible to assess the molecular stability of the attenuation process.
- These vaccines although attenuated, are most often associated with safety problems regarding use in breeding animals.
- the vaccine viruses may cross the placenta of pregnant animals, e.g. cows and lead to clinical manifestations in the fetus and/or the induction of persistently infected calves.
- the international patent application WO2005/111201 provides a new generation of a modified live pestivirus vaccine, which comprises a multiple modified pestivirus, having at least one mutation in the coding sequence for glycoprotein E ms and at least another mutation in the coding sequence for N pro , wherein said mutation in the coding sequence for glycoprotein E ms leads to inactivation of RNase activity residing in E ms and/or said mutation in the coding sequence for N pro leads to inactivation of said N pro .
- Attenuated pestiviruses such as BVDV
- pathogenic pestiviruses such as BVDV
- compositions and vaccines comprising said attenuated pesitiviruses, such as BVDV.
- the technical problem underlying the present invention is to provide new attenuated pestiviruses, referably an attenuated BVDV for use as live attenuated vaccines.
- Such improved attenuated pestivirus should especially (i) not cross the placenta themselves and (ii) induce an immunity that prevents viral transmission across the placenta and thereby prevents pregnancy problems like abortion of the fetus or birth of persistently calves from infected host animals in the case of BVDV infection.
- Fig. 1 Growth curves of viruses EP #82(4), EP #98(1) and TF #283(1).
- Fig. 2 average clinical score for group 1 / 2 / 3
- Fig. 3 average body temperature of group 1 / 2 / 3
- Fig. 4 body temperature of group 1 infected with TF #283 (1)
- Fig. 5 body temperature of group 2 infected with EP #82 (4)
- Fig. 6 body temperature of group 3 infected with TF #1347 / TF #230 / 3
- Fig. 8 WBC counts of group 2
- Fig. 9 WBC counts of group 3
- SEQ ID NO: 1 (CSF V_wt) amino acid sequence of CSFV wildtype SEQ ID NO:2 (CSFV d) amino acid sequence of CSFV comprising a deletion of cystein at amino acid position 438 as compared to SEQ ID NO:1 SEQ ID NO:3 (CSF V_S) amino acid sequence of CSFV comprising a cystein/serine substitution at amino acid position 438 as compared to SEQ ID NO:1 SEQ ID NO:4 (BVDV_Ke9_wt) amino acid sequence of BVDV type 1 wildtype SEQ ID NO:5 (BVDV_Ke9_d) amino acid sequence of BVDV type 1 comprising a deletion of cystein at amino acid position 441 as compared to SEQ ID NO:4 SEQ ID NO:6 (BVDV_Ke9_S) amino acid sequence of BVDV type 1 comprising a cystein/serine substitution at amino acid position 441 as compared to SEQ ID NO:4 SEQ ID NO:7 (B VD V
- SEQ ID NO:8 (BVDV NY d) amino acid sequence of BVDV type 2 comprising a deletion of cystein at amino acid position 441 as compared to SEQ ID NO:7
- SEQ ID NO:9 (BVDVJNY S) amino acid sequence of BVDV type 2 comprising a cystein/serine substitution at amino acid position 441 as compared to SEQ ID NO:8
- SEQ ID NO: 10 (BDV_x818_wt) amino acid sequence of BDV wildtype SEQ ID NO: 1 1 (BDV_x818_d) amino acid sequence of BDV comprising a deletion of cystein at amino acid position 439 as compared to SEQ ID NO: 10
- SEQ ID NO: 12 (BDV_x818_S) amino acid sequence of BDV comprising a cystein/serine substitution at amino acid position 439 as compared to SEQ ID NO: 10
- one aspect of the present patent application relates to a recombinant attenuated pestivirus, wherein said recombinant attenuated pestivirus does not produce a dimeric E ms glycoprotein.
- that pestivirus is selected from the group consisting of CSFV, BVDV and BDV, including any subtype of any of these pestiviruses.
- the present invention also relates to attenuated pestiviruses, wherein said attenuated pestiviruses do not produce a dimeric E ms glycoprotein and having at least a mutation in the N pr0 protein, wherein said mutation in the N pro protein leads to inactivation of said N pro protein.
- that pestivirus is selected from the group consisting of CSFV, BVDV and BDV, including any subtype of any of these pestiviruses.
- the present invention relates to an immunogenic composition
- an attenuated pestivirus wherein said attenuated pestivirus does not produce a dimeric E ms glycoprotein.
- that pestivirus has at least a further mutation in the N pr0 protein, wherein said mutation in the N pro protein leads to inactivation of said N pro protein.
- Preferred pestiviruses are selected from the group consisting of CSFV, BVDV and BDV, including any subtype of any of these pestiviruses.
- the present invention also relates to a method for attenuating a pestivirus, comprising modifications in the E ms glycoprotein of said pestivirus in such that said attenuated pestivirus does not produce a dimeric E ms glycoprotein.
- that pestivirus is selected from the group consisting of CSFV, BVDV and BDV, including any subtype of any of these pestiviruses.
- Pestivirus refers to all members of the genus Pestivirus, including BVDV, CSFV and BDV, within the family Flaviviridae.
- CSFV refers to all viruses belonging to species of classical swine fever virus (CSFV) in the genus Pestivirus within the family Flaviviridae.
- BVDV refers to all viruses belonging to species bovine viral diarrhea virus (BVDV) type 1 (BVDV-I) and BVDV type 2 (BVDV-2) in the genus Pestivirus within the family Flaviviridae (Heinz et al., 2000).
- BVDV bovine viral diarrhea virus
- BVDV-I bovine viral diarrhea virus
- BVDV-2 BVDV type 2
- the more classical BVDV type 1 strains and the more recently recognized BVDV type 2 strains display some limited but distinctive differences in nucleotide and amino acid sequences.
- N pro as understood herein relates to the first protein encoded by the viral open reading frame and cleaves itself from the rest of the synthesized polyprotein (Stark, et al., J. Virol. 67:7088-7093 (1993); Wiskerchen, et al., Virol. 65:4508-4514 (1991)). Said term, depending on the context, may also relate to the remaining “N pro " amino acids after mutation of the encoding nucleotide sequence or to the coding nucleotide sequence for said protein itself. "Protease activity residing in N pro " relates to the polypeptide cleavage activity of said "N p ⁇ 0 "
- E ms as used herein relates to the glycoprotein E ms which represents a structural component of the pestivirus virion (Thiel et al., 1991). E rns lacks a typical membrane anchor and is secreted in considerable amounts from the infected cells; this protein has been reported to exhibit RNase activity (Hulst et al., 1994; Schneider et al., 1993; Windisch et al., 1996). It should be noted that the term glycoprotein EO is often used synonymously to glycoprotein E ms in publications. Said term, depending on the context, may also relate to the mutated “E ms " protein after mutation of the encoding nucleotide sequence or to the coding nucleotide sequence for said protein itself.
- RNase activity residing in glycoprotein E ms relates to the RNA cleavage activity of said glycoprotein, i.e. the ability of the glycoprotein E ms to hydrolyze RNA.
- activation of the RNase activity residing in said glycoprotein refers to the inability or reduced capability of a modified glycoprotein E ms to hydrolyze RNA as compared to the unmodified wild type of said glycoprotein E ms .
- Attenuation means that there is a statistically significant difference between the virulence of attenuated pestivirus or BVDV particles of the present invention, wherein said attenuated viral particles being attenuated by a method described herein, and wild-type pestivirus or BVDV isolates from which said attenuated pestivirus or BVDV particles have been derived, for the predominant clinical parameters, in case of BVDV for diarrhea, pyrexia and lethality in animals infected with the same dose, preferably 6x10 6 TCIDs O .
- said attenuated BVDV particles do not cause diarrhea, pyrexia and lethality and thus may be used in a vaccine.
- Inactivation of E ms as used herein means RNase activity not significantly above the level measured for noninfected control cells in an RNase assay as described in Meyers et al, 1999. "Not significantly above the level measured for noninfected control cells in an RNase assay as described in Meyers et al., 1999, means for example, that the RNase activity is less than 150% compared to the noninfected control cells.
- Inactivation of N pro means the prevention or considerable reduction of the probable immunemodulating activity of N pro by mutation.
- this mutation prevents or considerably reduces the interference of N pro with the induction of an interferon response by the infected cells as described by R ⁇ ggli et al., (2003).
- the inactivation of N pro would allow the cell to mount a normal interferon response.
- the "dimeric E ms glycoproteins” means a homodimer of two momomers of E ras glycoproteins. It should be noted that the two monomers of the E ms glycoprotein which build the homodimer may comprise a certain level of sequence diversity within their amino acid sequence. In this context, "a certain level of sequence diversity” shall mean that the monomers forming the homodimer show at least 80%, preferably 90%, more preferably 95%, even more preferably 98% sequence homology in respect to the amino acid sequence of the E ms gene region.
- non-dimeric E ms glycoprotein shall mean, but is not limted to an E ras glycoprotein that is not capable to form a detectable amount of homodimer with a second E ms glycoprotein.
- non-dimeric E ms glycoprotein shall mean, but is not limted to an E ms glycoprotein that is not capable to any homodimer with a second E ms glycoprotein
- Processing signal as used herein relates to a substance that ensures the generation of a functional N-terminal of the C protein of the pestivirus, preferably of BVDV, in particular a substance selected from the group of ubiquitin, LC3, SUMO-I, NEDD8, GATE-16 and GABA(A)RAP. Also proteases selected from the group of Intein, picornavirus 3C, caridovirus 2A, and pi 5 of rabbit hemorrhagic disease virus are understood as “processing signals” as used herein. Similarly, 2A proteins of aphtoviruses or related sequences that promote the expression of two separate proteins by translational discontinuity are included int eh term "Processing signal”. Any other similar processing signal known to the skilled person that ensures the generation of a functional N-terminal of the C protein shall also be comprised in the term "processing signal”.
- Protein C or “C protein” or “C-protein” as used herein relates to a structural component of the pestivirus virion (Thiel et al., 1991).
- Protein C is the capsid or core protein of pestiviruses. Said term, depending on the context, may also relate to the "Protein C” with one or several amino acids exchanges resulting from mutation of the encoding nucleotide sequence.
- a ,,fragment" according to the invention is any subunit of a polynucleotide molecule according to the invention, i.e. any subset.
- said fragment is characterized in that it is shorter than the DNA covering the full length viral genome.
- a functional variant of the nucleotide molecule according to the invention is a nucleotide molecule which possesses a biological activity (either functional or structural) that is substantially similar to the nucleotide molecule according to the invention.
- the term functional variant also includes ,,a fragment", ,,a functional variant", ,,variant based on the degenerative nucleic acid code" or ,,chemical derivative".
- Such a functional variant e.g. may carry one or several nucleotide exchanges, deletions or insertions.
- Said functional variant at least partially retains its biological activity, e.g. function as an infectious clone or a vaccine strain, or even exhibits improved biological activity.
- “Possess a biological activity that is substantially similar” means with respect to the pestiviruses provided herewith, for example, that said pestivirus is attenuated in a manner described herein and result in a non-pathogenic virus suitable for the production of live attenuated virus, which loss ability to pass the placenta but mediates an immune response after vaccination.
- a ,variant based on the degenerative nature of the genetic code is a variant resulting from the fact that a certain amino acid may be encoded by several different nucleotide triplets. Said variant at least partially retains its biological activity, or even exhibits improved biological activity.
- a molecule is substantially similar" to another molecule if both molecules have substantially similar nucleotide sequences or biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that te ⁇ n is used herein if the nucleotide sequence is not identical, and two molecules which have a similar nucleotide sequence are considered variants as that term is used herein even if their biological activity is not identical.
- a mutation as used herein relates to modifications in the nucleic acid molecules encoding the proteins / amino acids according to the invention.
- Said mutations relate to, but are not limited to, substitutions (replacement of one or several nucleotides/base pairs), deletions (removal of one or several nucleotides/base pairs), and/or insertions (addition of one or several nucleotides/base pairs).
- mutation may be a single mutation or several mutations, therefore, often the term "mutation(s)" is used and relates to both a single mutation and several mutations.
- Said mutations include, but are not limited to point mutations (single nucleotide mutations) or larger mutations wherein e.g.
- vaccine refers to a pharmaceutical composition comprising at least one immunologically active component that induces an immunological response in an animal and possibly but not necessarily one or more additional components that enhance the immunological activity of said active component.
- a vaccine may additionally comprise further components typical to pharmaceutical compostions.
- the immunologically active component of a vaccine may comprise complete virus particles in either their original form or as attenuated particles in a so called modified live vaccine (MLV) or particles inactivated by appropriate methods in a so called killed vaccine (KV).
- MMV modified live vaccine
- KV killed vaccine
- the immunologically active component of a vaccine may comprise appropriate elements of said organisms (subunit vaccines) whereby these elements are generated either by destroying the whole particle or the growth cultures containing such particles and optionally subsequent purification steps yielding the desired structure(s), or by synthetic processes including an appropriate manipulation by use of a suitable system based on, for example, bacteria, insects, mammalian or other species plus optionally subsequent isolation and purification procedures, or by induction of said synthetic processes in the animal needing a vaccine by direct incorporation of genetic material using suitable pharmaceutical compositions (polynucleotide vaccination).
- a vaccine may comprise one or simultaneously more than one of the elements described above.
- the term "vaccine” as understood herein is a vaccine for veterinary use comprising antigenic substances and is administered for the purpose of inducing a specific and active immunity against a disease provoked by a pestivirus infection, preferably by a BVDV infection.
- the attenuated pestivirus in particular the attenuated BVDV as described herein, confer active immunity that may be transferred passively via maternal antibodies against the immunogens it contains and sometimes also against antigenically related organisms.
- a vaccine of the invention refers to a vaccine as defined above, wherein one immunologically active component is a BVDV or of pestiviral origin or derived from a nucleotide sequence that is more than 70% homologous to any known pestivirus sequence (sense or antisense).
- live vaccine refers to a vaccine comprising a living, in particular, a living viral active component.
- adjuvants like e.g. aluminiumhydroxide, mineral or other oils or ancillary molecules added to the vaccine or generated by the body after the respective induction by such additional components, like but not restricted to interferons, interleukins or growth factors.
- a "pharmaceutical composition” essentially consists of one or more ingredients capable of modifying physiological e.g. immunological functions of the organism it is administered to, or of organisms living in or on the organism.
- the term includes, but is not restricted to, antibiotics or antiparasitics, as well as other constituents commonly used to achieve certain other objectives like, but not limited to, processing traits, sterility, stability, feasibility to administer the composition via enteral or parenteral routes such as oral, intranasal, intravenous, intramuscular, subcutaneous, intradermal or other suitable route, tolerance after administration, controlled release properties.
- Such a pharmaceutical composition could be prepared as follows: Cell culture supernatant of an infected cell culture is mixed with a stabilizer (e.g. spermidine and/or BSA (bovine serum albumin)) and the mixture is subsequently lyophilized or dehydrated by other methods. Prior to vaccination, said mixture is then rehydrated in aqueous (e.g. saline, PBS (phosphate buffered saline)) or non-aqueous solutions (e.g. oil emulsion, aluminum-based adjuvant).
- a stabilizer e.g. spermidine and/or BSA (bovine serum albumin)
- BSA bovine serum albumin
- pestiviruses lacking the ability to form homodimers of the E ms glycoprotein are well attenuated and suitable candidates for a modified live vaccine for the prophylaxis and/or treatment of animals against a pestivirus infection (see example section for more details).
- one aspect of the present patent application relates to an attenuated pestivirus, wherein said attenuated pestivirus does not produce a dimeric E ras glycoprotein.
- that pestivirus is selected from the group consisting of CSFV, BVDV and BDV, including any subtype of any of these pestiviruses.
- the pestivirus lacking the ability to form homodimers of the E ms glycoprotein can be established by recombinant bioengineering techniques.
- a deletion or substitution at least of the cystein at amino acid position 438 of the CSFV pestivirus results in such a recombinant attenuated CSFV pestivirus lacking the ability to form E ms homodimers, because such E ms glycoprotein is not longer able to form intermolecular disulfide bonds between to E ms monomers.
- BVDV pestivirus type 1 and/or 2
- a deletion or substitution of at least the cystein at amino acid position 441 of the BVDV pestivirus results in such a recombinant attenuated BVDV pestivirus lacking the ability to form E ms homodimers.
- a deletion or substitution of at least the cystein at amino acid position 439 of the BDV pestivirus results in such a recombinant attenuated BDV pestivirus lacking the ability to form E ms homodimers.
- Preferred substitutions are cystein/serine substitutions.
- any other substitutions of the last C-terminal cystein within the E ms glycoprotein e.g. Cys438 of CSFV, Cys441 of BVDV type 1 or 2, Cys439 of BDV
- substitutions of the last C-terminal cystein within the E ms glycoprotein are also within the meaning of the present invention.
- the key element of the present invention is to provide a pestivirus that is not longer able to form homodimers of the E ms glycoprotein. Homodimer formation can be prevented by a deletion or substitution of at least the last C-terminal cystein within the E ms glycoprotein as decribed above, because any such pestiviruses are not longer capable to form intermolecular disulfide bonds between two E ms glycoprotein monomers.
- homodimerisation between two monomers of the E ms glycoprotein can also be inhibited by a modification of the amino acid environment of the last C-terminal cystein of the E ras glycoprotein, if this modification leads, for instance, to a change of the charge and/or conformation of the direct environment of the last C-terminal cystein in a manner that the intermolecular disulfide bonds can not be formed any more.
- an insertion, deletion or substitution of prolin close to that last C- te ⁇ ninal cystein may change the conformation of the E ms glycoprotein in such that the last C- terminal cystein of the E ms glycoprotein is not longer exposed in a manner that it can form intermolecular disulfide bonds with a second E ms glycoprotein.
- modifications of the cystein environment with result in a strong positive or negative polarity of such environment e.g by insertion of or substitution by positive charged amino acids such as argnin, lysine and/or histidine; by insertion of or substitution by negative charged amino acids such as aspratate or glutamate
- positive charged amino acids such as argnin, lysine and/or histidine
- negative charged amino acids such as aspratate or glutamate
- the present invention does not only relate to recombinant attenuated pestiviruses, wherein the last C-terminal cystein of such E ms glycoprotein is deleted or substituted by a non-cystein residue, it also relates to any modified attenuated pestivirus, wherein the formation of homodimers of the E ms glycoprotein of said pestivirus is (in general) inhibited by a deletion, insertion or modification.
- such modification is introduced closely to the last C-terminal cystein of the E ms glycoprotein, preferably such modification affects the amino acids between the amino acid positions 410 to 470, preferably 420 to 460.
- one aspect of the present invention relates to a recombinant attenuated pestivirus, wherein said attenuated pestivirus does not produce a dimeric E ms glycoprotein.
- the carboxy-terminus of the E ms glycoprotein of said attenuated pestivirus is modified by a deletion, insertion or substitution.
- At least the last C-terminal Cystein-residue of the E ms glyoprotein of said attenuated pestivirus is deleted or substituted by non-Cys amino acid residue.
- At least the most last Cystein-residue in the E ms glycoprotein of said attenuated pestivirus is deleted.
- said attenuated pestivirus selected from the group consisting of CSFV, BVDV type 1 and/or 2, and BDV.
- said attenuated pestivirus is a CSF pestivirus.
- At least the Cystein-residue at amino acid position 438 according to SEQ ID NO: 1 of the E ms glyoprotein of said attenuated CSFV is deleted or substituted by a non Cystein-residue.
- said attenunated pestivirus is a BVD type 1 and/or 2 pestivirus.
- At least the Cystein-residue at amino acid position 441 according to SEQ ID NO: 4 (BVDV type 1) or SEQ ID NO:7 (BVDV type 2) of the E ms glyoprotein of said attenuated BVDV is deleted or substituted by a non Cystein-residue.
- said attenunated pestivirus is a BDV pestivirus.
- at least the Cystein-residue at amino acid position 439 according to SEQ ID NO: 10 of the E ms glyoprotein of said attenuated BDV is deleted or substituted by a non Cystein-residue.
- WO 99/64604 it is described that the inactivation of the RNAse activity residing within the E ms also results in an attenuated apathogenic pestivirus, which is capable to be used as a modified live vaccine. According to a further aspect of the present invention, both modifications can be combined which would result in an attenuated pestivirus, wherein the RNAse activity residing within the E ms is inactivated by a deletion, insertion or substitution and which is not capable to form any E ms dimers.
- Suitable modifications of the glycoprotein E ms which result in RNase negative E ms glycoproteins are for example, the single substitutions/deletions: S298G, H300K, H300L, H300R, H300del, W3O3G, P304del, E305A, C308G, R343G, E345del, W346G, K348A, H349K, H349L, H349del, H349Q, H349SV (mutation H349S and insertion of V), K348R, W351P, W351G, W351L, W351K, W351H; the double substitutions/deletions: H300L/H349L, K348del/H349del, H349del/G350del, E345del/H349del, W303G/E305A, H300K/H349K, H300K/H349L and the triple deletions: L2
- the putative active site of the RNase is represented by the conserved E ms sequences SLHGIWPEKICTG and/or LQRHEWNKHGWCNWFHIEPW (sequence of the BVDV-2 New York' 93 protein given here in an exemplary manner; minor changes can possibly be found in other pestivirus sequences but the identity of the motif will always be obvious for an expert in the field.
- the corresponding amino acid sequences of BVDV-I CP7 would be SLHGIWPEKICTG and/or LQRHEWNKHGWCNWYNIEPW and that of CSFV Alfort/T ⁇ bingen SLHGIWPEKICKG and/or LQRHEWNKHGWCNWYNIDPW).
- the invention further relates to a BVDV according to the invention, wherein said RNase negative mutation(s) in the coding sequence for glycoprotein E rns are located in the nucleotide sequence coding for the conserved E ras sequence SLHGIWPEKICTG and/or LQRHEWNKHGWCNWFHIEPW.
- RNase negative mutation(s) in the coding sequence for glycoprotein E rns are located in the nucleotide sequence coding for the conserved E ras sequence SLHGIWPEKICTG and/or LQRHEWNKHGWCNWFHIEPW.
- These sequences are representing the putative active site of the RNase.
- the sequences SLHGI WPEKIC and RHEWNKHGWCNW of the putative E ms active site are even more conserved across pestiviruses.
- the present invention also relates to attenuated pestiviruses, wherein said attenuated pestiviruses do not produce a dimeric E RNS glycoprotein and having at least a mutation in the N pro protein, wherein said mutation in the N pro protein leads to inactivation of said N pro protein.
- that pestivirus is selected from the group consisting of CSFV, BVDV and BDV, including any subtype of any of these pestiviruses.
- Inactivation of the N pro is achieved in pestiviruses, in particular BVDV of the specified formula described more in detail below, wherein between 0 and all amino acids of N pro are present; ubiquitin or LC3 or another sequence serving as processing signal (e.g. SUMO-I, NEDD8, GATE- 16,GABA(A)RAP, or proteases like e.g. Intein, picornavirus 3C, caridovirus 2A, or pi 5 of rabbit hemorrhagic disease virus, or sequences like aphtovirus 2A that lead to discontinuous translation) is present or absent.
- ubiquitin or LC3 or another sequence serving as processing signal e.g. SUMO-I, NEDD8, GATE- 16,GABA(A)RAP, or proteases like e.g. Intein, picornavirus 3C, caridovirus 2A, or pi 5 of rabbit hemorrhagic disease virus, or sequences like aphtovirus 2A that lead
- the invention relates to a pestivirus, in particular to BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula: [N pro ] ⁇ -[PS] y -[C-term] and wherein:
- [N pr0 ] relates to the N pro portion of said polyprotein, wherein "x" represents the number of amino acids of the N pro present in the polyprotein;
- [PS] relates to a processing signal selected from: ubiquitin, LC3, SUMO-I, NEDD8,
- GATE-16 or GABA(A)RAP or proteases like e.g. Intein, picornavirus 3C, caridovirus 2A, or pi 5 of rabbit hemorrhagic disease virus or any processing signal known to the skilled person that ensures the generation of a functional N-terminal of the C-protein.
- [C-term] relates to the complete pestivirus, in particular the complete BVDV polyprotein except for N 1 " "0 , but including the capsid (C)-protein and any other protein present in the pestivirus polyprotein, in particular in the BVDV polyprotein including the carboxyterminal NS5B.
- the glycoprotein E ms in said [C-term] is mutated, in such that the RNase activity residing in the glycoprotein E ms is inactivated.
- any other protein present in the pestivirus polyprotein /BVDV polyprotein relates to E ras , El, E2, p7, NS2, NS3, NS4A, NS4B and NS5A, wherin glycoprotein E ms is mutated, preferably as disclosed herein (see above), in such that the RNase activity residing in the glycoprotein E ms is inactivated.
- the pestivirus, in particular the BVDV according to the invention has a C-protein which is not mutated except for the amino acid at position 2 which is changed from D to N.
- the invention relates to a pestivirus, in particular to BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula: [ N pro ]i-[PS]o-[C-te ⁇ n] and wherein the definitions are as defined above.
- the invention relates to a pestivirus, in particular BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula:
- the invention relates to a pestivirus, in particular to BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula: [ N pro ] 3 -[PS] 0 -[C-term] and wherein the definitions are as defined above.
- BVDV A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by the N pro sequence EL and the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B.
- the invention relates to a BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula:
- the invention relates to a pestivirus, in particular to BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula:
- BVDV [ N pro ] 4 -[PS]o-[C-term] and wherein the definitions are as defined above.
- N-terminal methionine is followed by the N pro sequence ELF and the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B.
- the invention relates to a BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pr0 lead to an encoded polyprotein as characterized by the following formula:
- the invention relates to pestivirus, in particular to BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula:
- BVDV A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by the N pro sequence ELFSN and the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B.
- the invention relates to a BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula:
- the invention relates to a pestivirus, in particular to BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula:
- the invention relates to a BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula:
- the invention relates to a pestivirus, in particular BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula:
- PS is any of the PS disclosed above, preferably selected from the group of ubiquitin or LC3.
- BVDV A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by any 21 or 28 N pr0 amino acids, ubiquitin or LC3 and the C-protein.
- the invention relates to a BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula:
- Ubiquitin is a well known highly conserved cellular protein of 76 amino acids. Among other functions, ubiquitin is a key player in protein catabolism since conjugation with ubiquitin can mark a protein for degradation via the proteasome. Ubiquitin conjugated with or fused to other proteins via the carboxyterminal glycin can be cleaved off by cellular ubiquitin-specific proteases. Thus, fusion of a protein to the carboxyterminus of ubiquitin will usually result in defined proteolytic cleavage of the fusion protein into its components when expressed within a cell.
- LC3 (light chain 3 of microtubule associated proteins) represents a cellular protein of 125 amino acids that serves a variety of functions (length given for bovine LC3). Recently, a fundamental role of the protein in autophagy has been defined. During this process, LC3 is activated by carboxyterminal cleavage. Thereby, a new carboxyterminus is generated that consists of glycine. LC3 is then conjugated via the carboxyterminal glycine to phosphatidylethanolamine present in the membranes of autophagic vesicles. Because of this process, a protein fused to the carboxyterminus of LC3 will be cleaved off by a cellular protease at a defined position.
- the invention relates to a pestivirus, preferably to BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula selected from the group of :
- y is 0 (no PS present).
- the invention relates to a pestivirus, preferably to BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pr0 lead to an encoded polyprotein as characterized by the following formula selected from the group of : M-[PS]o-[C-term]; MEL-[PS]o-[C-term]; MELF-[PS] 0 -[C-term]; MELFS-[PS]o-[C-term]; MELFSN-[PS]o-[C-term];
- the invention relates to a pestivirus, preferably to BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pr0 lead to an encoded polyprotein as characterized by the following formula selected from the group of :
- the invention relates to a pestivirus, preferably to BVDV according to the invention, wherein said mutation(s) in the coding sequence for N pro lead to an encoded polyprotein as characterized by the following formula selected from the group of :
- compositions comprising a pestivirus, in particular a BVDV according to the invention, and a solution.
- additional components which may be comprised in said composition (see also Remington's Pharmaceutical Sciences. (1990). 18th ed. Mack Publ, Easton).
- the expert may use known injectable, physiologically acceptable sterile solutions.
- aqueous isotonic solutions such as e.g. saline or corresponding plasma protein solutions are readily available.
- the pharmaceutical compositions may be present as lyophylisates or dry preparations, which can be reconstituted with a known injectable solution directly before use under sterile conditions, e.g. as a kit of parts.
- the final preparation of the immunogenic compositions of the present invention are prepared for e.g. injection by mixing said pestivirus, preferably BVDV according to the invention with a sterile physiologically acceptable solution, that may be supplemented with known carrier substances or/and additives (e.g. serum albumin, dextrose, sodium bisulfite, EDTA).
- Said solution may be based on a physiologically acceptable solvent, e.g. an aqueous solution between pH 7 and 8.
- the pH may be stabilised by a pharmaceutically acceptable buffer.
- the solution may also contain further stabilising agents like a detergent like Tween 20, serum albumin such as BSA (bovine serum albumin), ascorbic acid, and/or spermidine.
- composition may also comprise adjuvants, e.g. aluminiumhydroxide, mineral or other oils or ancillary molecules added to the vaccine or generated by the body after the respective induction by such additional components, like but not restricted to interferons, interleukins or growth factors.
- adjuvants e.g. aluminiumhydroxide, mineral or other oils or ancillary molecules added to the vaccine or generated by the body after the respective induction by such additional components, like but not restricted to interferons, interleukins or growth factors.
- the pestivirus in particular BVDV may be solved in: Pestivims (preferably BVDV) IQr - I ⁇ 8 TCID50
- the immunogenic composition is first lyophilized or dehydrated by other methods, then, prior to vaccination, said composition is rehydrated in aqueous (e.g. saline, PBS (phosphate buffered saline)) or non-aqueous solutions (e.g. oil emulsion (mineral oil, or vegetable/metabolizable oil based/single or double emulsion based), aluminum-based, carbomer based adjuvant).
- aqueous e.g. saline, PBS (phosphate buffered saline)
- non-aqueous solutions e.g. oil emulsion (mineral oil, or vegetable/metabolizable oil based/single or double emulsion based), aluminum-based, carbomer based adjuvant.
- the immunogenic composition according to the invention is capable to induce an immunological response in an animal. More preferred, the immunogenic composition according to the invention is a vaccine.
- a vaccine as understood herein comprises a pestivirus, in particular BVDV according to the invention and is defined above (section "definitions")
- the immunogenic composition according to the invention further comprises a pharmaceutically acceptable carrier or excipient.
- a pharmaceutically acceptable carrier or excipient Several carriers or excipients are disclosed above.
- the composition may comprise, if aimed at injections or infusion, substances for preparing isotonic solutions, preservatives such as p-hydroxybenzoates, stabilizers, such as alkalisalts of ethylendiamintetracetic acid, possibly also containing emulsifying and/ or dispersing.
- the immunogenic composition according to the invention may be applied intradermally, intratracheally, or intravaginally.
- the composition preferably may be applied intramuscularly or intranasally.
- the immunogenic compositions according to the invention can be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.
- the invention also relates to the use of a pestivirus, in particular BVDV according to the invention in the manufacture of a vaccine for the prophylaxis and treatment of pestiviral infections, in particular of BVDV infections.
- polynucleotide molecule comprsing the nucleic acid coding for a pestivirus, in particular for a BVDV according to the invention, or a fragment, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.
- said polynucleotide molecule is DNA.
- said polynucleotide molecule is RNA.
- said polynucleotide molecule also comprises the nucleotide sequence of a functional 5'- and/or 3 '-non-translated region of a pestivirus, in particular of BVDV.
- nucleotide sequences known in the art which represents the basis for the production of a polynucleotide molecule coding for a pestivirus attenuated according to the present invention.
- nuclecic acid sequences of wild-type sequences of several members of pesti viruses are listed below: Border disease virus
- Another important aspect of the invention is a method for attenuating a pestivirus which results in a pestivirus according to the invention, comprising modifying the E ms glycoprotein of said pestivirus in such that said attenuated pestivirus does not produce a dimeric E ms glycoprotein.
- the carboxy-terminus of the E ms glycoprotein of said pestivirus is modified by a deletion, insertion or substitution.
- at least the last C-terminal Cystein-residue of the E ms glyoprotein of said pestivirus is deleted or substituted by a non-Cys amino acid residue.
- At least the last C-ternimal Cystein-residue in the E ms glycoprotein of said pestivirus is deleted.
- said pestivirus selected from the group consisting of CSFV, BVDV type 1 and/or 2, and BDV.
- said attenunated pestivirus is a CSF pestivirus.
- at least the Cystein-residue at amino acid position 438 according to SEQ ID NO: 1 of the E ms glyoprotein of said CSFV pestivirus is deleted or is substituted by a non Cystein-residue.
- said pestivirus is a BVD type 1 and/or 2 pestivirus.
- At least the Cystein-residue at amino acid position 441 according to SEQ ID NO: 4 (BVDV type 1) or SEQ ID NO:7 (BVDV type 2) of the E ms glyoprotein of said BVDV is deleted or substituted by a non Cystein-residue.
- said pestivirus is a BD pestivirus.
- at least the Cystein-residue at amino acid position 439 according to SEQ ID NO: 10 of the E ms glyoprotein of said BDV is deleted or is substituted by a non Cystein-residue.
- the present invention provides a method for attenuating a pestivirus, characterized in that the glycoprotein E ms is modified in such that the RNAse activity residing within the E ms is inactivated by a deletion, insertion or substitution, and in such that said pestivirus is not capable to form any E ms dimer.
- said pestivirus is selected from the group consisting of CSFV, BVDV, and BDV, including any subtypes thereof.
- the present invention provides a method for attenuating a pestivirus, characterized in that glycoprotein E rns is modified by a deletion, insertion or substitution in such that said pestivirus is not capable to form any E ms dimer and by modifying the N pro protein by a deletion, insertion or substitution in such that the N pr0 protein is inactivated.
- said method comprises the steps: a) reverse transcription of a wild-type pestivirus nucleotide sequence into a cDNA; b) cloning said cDNA; c) introducing mutations selected from the group of deletions, insertion mutations and/or substitution mutations into said cDNA, wherein said mutations are located in the coding sequence encoding glycoprotein E ms , or the coding sequence encoding glycoprotein E ms and the protease N pro , d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of pestivirus cDNA into RNA in vitro or upon infection of suitable cells.
- Yet another important embodiment of the invention is a method of treatment of disease caused by a pestivirus, wherein a pestivirus according to the invention or a composition according to the invention is administered to an animal in need thereof at a suitable dosis as known to the skilled person and the reduction of symptoms of said pestivirus infection.
- Yet another important embodiment of the invention is a method of treatment of disease caused by BVDV, wherein a BVDV according to the invention or a composition according to the invention is administered to an animal in need thereof at a suitable dosis as known to the skilled person and the reduction of symptoms of BVDV infection such as viremia and leukopenia and/or pyrexia and/or diarrhea is monitored.
- the objective of the study TV#26 was to define the outcome of clinical signs after infection of pigs with a CSFV Alfort/T ⁇ bingen mutant exhibiting a deletion of the cysteine codon at position 438 in the polypeptide (171 in the E ms sequence). This mutation prevents E ms dimerization (EP #82(4)).
- an RNase negative variant of CSFV Alfort/T ⁇ bingen with mutation H297K and wt virus were used.
- the mutants show only a slightly reduced growth rate between 24 to 70 h p.i.in cell culture compared to the parental strain. It will have to be tested in further experiments whether this difference is significant (Fig. 1).
- the CSFV mutants (first group: TF #283(1) / second group: EP #82 (4)) and CSFV wild type virus (EP #98(1)) were applied intramuscularly and intranasally on day 0 (0 dpi). Each animal received 10 KID50 virus in 1.5 ml DMEM. Two-thirds of the suspension were applied intranasally (0.5 ml per nostril). For better i.m. application the last 0.5 ml of virus was filled up to 2 ml with DMEM and were injected in the muscle brachiocephalicus.
- virus suspension was collected after different dilution and transport steps: a) original virus (stock solution) b) first dilution step with DMEM as diluent to obtain a virus concentration of 10 5 ' 824 KID 50 / ml (10 6 KID 50 /1.5 ml) directly to -70 0 C c) This sample was taken along "on ice" with the inoculum and handled the same way. After returning from the stable this sample was frozen at -70 °C immediately. d) second dilution step for i.m. application (see above)
- Blood was taken from the jugular vein for BC isolation, leukocyte counting, plasma isolation, FACS analysis and serum preparation according to table 3. After infection, animals were monitored for 22 days and rectal temperatures were recorded daily or, in the late stage of the experiment, every second day.
- Pigs were observed daily for general health status.
- the animals of group #3 challenged with the wild type virus had to be killed prematurely on day 9 p.i. because of considerable signs of CSF.
- groups infected with the RNase negative mutant and the dimerization incompetent mutant all animals showed signs of disease typical for CSF.
- the severity of symptoms in group 1 and 2 were significantly less distinctive.
- Rectal temperatures were recorded on -3 and -2 dpi and daily or every second day from 0 dpi up to 20 dpi.
- group 1 and 2 every animal reached the critical temperature of 40 0 C on 4 / 5 or 6 days post infection. The temperature recovered in all animals of these groups to normal values till 10 dpi or 1 1 dpi.
- body temperature increased from day 4 post infection and no descent was detectable until euthanasia.
- WBC counts were determined in a haemocytometer, "Neubauer chamber", by standard laboratory procedure. For all animals a reduction of WBC was detectable during the study. The surviving animals showed an increase to almost normal values until study termination.
- Serum neutralisation assays will be performed using SP50 as test virus on day -1 before infection, 9 days post infection for group 3 and 22 dpi for group 1 and 2.
- the dimerization has been found to be crucial for the biological functions but not enzymatic activity of other RNases, it can be hypothesized that the attenuation of CSFV in consequence of the prevention of E ms dimer formation and the abrogation of the RNase activity rely on the same principles of the virus host interaction, namely the blockage of the E ms RNase effect.
- the prevention of dimer formation is most likely equivalent to blocking the RNase activity, even though the virus is able to express an active RNase.
- Bovine viral diarrhea virus a review. J. Am. Vet. Med.Assoc. 190: 1449- 1458.
- Glycoprotein E2 of classical swine fever virus expression in insect cells and identification as a ribonuclease. Virology 200: 558-565.
- Kit, M. and S. Kit. 1991 Sensitive glycoprotein gill blocking ELISA to distinguish between pseudorabies (Aujeszky's disease) -infected and vaccinated pigs.
- N-terminal protease of pestiviruses identification of putative catalytic residues by site directed mutagenesis. J. Virol. 72: 2544-2547.
- Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J. Virology 64, 3563-3569.
- Pestivirus gene expression the first protein product of the bovine viral diarrhea virus large open reading frame, p20, possesses proteolytic activity J. Virol. 65:4508-4514.
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Priority Applications (9)
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EP09769297.4A EP2303320B1 (en) | 2008-06-25 | 2009-06-24 | Attenuated pestivirus |
JP2011515376A JP5642672B2 (en) | 2008-06-25 | 2009-06-24 | Attenuated pestivirus |
BRPI0914560A BRPI0914560A2 (en) | 2008-06-25 | 2009-06-24 | attenuated pestivirus |
NZ589723A NZ589723A (en) | 2008-06-25 | 2009-06-24 | Attenuated pestivirus wherein the attenuated pestivirus does not produce a dimeric erns glycoprotein |
AU2009264206A AU2009264206B2 (en) | 2008-06-25 | 2009-06-24 | Attenuated pestivirus |
CN2009801243281A CN102076356A (en) | 2008-06-25 | 2009-06-24 | Attenuated pestivirus |
CA2729105A CA2729105A1 (en) | 2008-06-25 | 2009-06-24 | Attenuated pestivirus |
MX2010013437A MX2010013437A (en) | 2008-06-25 | 2009-06-24 | Attenuated pestivirus. |
US13/000,962 US8895026B2 (en) | 2008-06-25 | 2009-06-24 | Attenuated pestivirus |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8778355B2 (en) | 2001-09-06 | 2014-07-15 | Boehringer Ingelheim Vetmedica Gmbh | Infectious bovine viral diarrhea virus |
US8846054B2 (en) | 2009-01-09 | 2014-09-30 | Boehringer Ingelheim Vetmedica, Inc. | Method of treating pregnant cows and/or heifers |
US8895286B2 (en) | 1998-06-05 | 2014-11-25 | Boehringer Ingelheim Vetmedica Gmbh | Attenuated pestiviruses |
US8895026B2 (en) | 2008-06-25 | 2014-11-25 | Boehringer Ingelheim Vetmedica Gmbh | Attenuated pestivirus |
JP2015528455A (en) * | 2012-08-31 | 2015-09-28 | ノバルティス アーゲー | Stabilized protein for immunization against STAPHYLOCOCUSAUREUS |
JP2017510268A (en) * | 2014-03-31 | 2017-04-13 | ベーリンガー インゲルハイム フェトメディカ ゲーエムベーハーBoehringer Ingelheim Vetmedica GmbH | Improved modular antigen transport molecules and uses thereof |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7572455B2 (en) * | 2004-05-19 | 2009-08-11 | Boehringer Ingelheim Vetmedica Gmbh | Vaccine comprising an attenuated pestivirus |
UY29915A1 (en) * | 2005-11-15 | 2007-06-29 | Boehringer Ingelheim Vetmed | COMBINED VACCINE THAT INCLUDES A DAMAGED VIRUS OF THE BOVINE VIRAL DIARRHEA |
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CN110177799A (en) | 2016-12-22 | 2019-08-27 | 维也纳兽医大学 | Cause the separation of the novel pestivirus of A type congenital tremors |
KR20220009960A (en) * | 2019-04-18 | 2022-01-25 | 베링거 잉겔하임 베트메디카 (차이나) 코포레이션 리미티드 | Recombinant Classical Swine Fever Virus |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0982402A1 (en) * | 1998-08-14 | 2000-03-01 | Stichting Instituut voor Dierhouderij en Diergezondheid (ID-DLO) | Pestivirus vaccination |
WO2005111201A1 (en) * | 2004-05-19 | 2005-11-24 | Boehringer Ingelheim Vetmedica Gmbh | Vaccine comprising an attenuated pestivirus |
Family Cites Families (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4719177A (en) * | 1981-04-20 | 1988-01-12 | Massachusetts Institute Of Technology | Production of complementary DNA representing RNA viral sequences by recombinant DNA methods and uses therefor |
US5206163A (en) * | 1985-07-08 | 1993-04-27 | Chiron Corporation | DNA encoding bovine diarrhea virus protein |
ATE164885T1 (en) | 1986-01-27 | 1998-04-15 | Syntro Corp | WEAKENED HERPES VIRUSES, HERPES VIRUSES CONTAINING FOREIGN DNA CODING AMINO ACID SEQUENCES AND VACCINES CONTAINING SAME |
US6001613A (en) * | 1996-05-24 | 1999-12-14 | Board Of Regents Of University Of Nebraska | Plasmid bearing a cDNA copy of the genome of bovine viral diarrhea virus, chimeric derivatives thereof, and method of producing an infectious bovine viral diarrhea virus using said plasmid |
EP0965639A1 (en) | 1998-06-05 | 1999-12-22 | Boehringer Ingelheim Vetmedica Gmbh | Attenuated pestiviruses |
US7179473B2 (en) * | 1998-06-05 | 2007-02-20 | Boehringer Ingelheim Vetmedica Gmbh | Attenuated pestiviruses |
US6168942B1 (en) * | 1998-11-10 | 2001-01-02 | Pfizer Inc. | Attenuated forms of bovine viral diarrhea virus |
EP1104676A1 (en) | 1999-11-30 | 2001-06-06 | Boehringer Ingelheim Vetmedica Gmbh | Safe attenuated bovine viral diarrhea viruses for use in pregnant cows |
CA2363493C (en) * | 2000-11-22 | 2006-06-06 | Xuemei Cao | Attenuated forms of bovine viral diarrhea virus |
US20030147914A1 (en) * | 2001-07-02 | 2003-08-07 | Pfizer Inc. | Mycoplasma bovis vaccine and methods of reducing pneumonia in animals |
US7135561B2 (en) * | 2001-09-06 | 2006-11-14 | Boehringer Ingelheim Vetmedica Gmbh | Infectious bovine viral diarrhea virus clone |
DE10143813A1 (en) | 2001-09-06 | 2003-04-10 | Boehringer Ingelheim Vetmed | Infectious bovine viral diarrhea virus clone |
EA011578B1 (en) * | 2002-08-26 | 2009-04-28 | Пфайзер Продактс Инк. | Immunogenic composition for preventing cattle from infections of respiratory and reproductive systems and use thereof |
US20040185056A1 (en) * | 2003-01-03 | 2004-09-23 | Board Of Regents, University Of Nebraska-Lincoln | Vaccines containing bovine herpesvirus 1 attenuated by mutation in latency-related gene |
US7563449B2 (en) * | 2003-04-21 | 2009-07-21 | Pfizer Inc, | Methods for reducing cattle reproductive diseases |
US7361357B2 (en) * | 2003-07-29 | 2008-04-22 | Pfizer Inc. | Safe mutant viral vaccines |
US7572455B2 (en) * | 2004-05-19 | 2009-08-11 | Boehringer Ingelheim Vetmedica Gmbh | Vaccine comprising an attenuated pestivirus |
UY29915A1 (en) | 2005-11-15 | 2007-06-29 | Boehringer Ingelheim Vetmed | COMBINED VACCINE THAT INCLUDES A DAMAGED VIRUS OF THE BOVINE VIRAL DIARRHEA |
UY31930A (en) * | 2008-06-25 | 2010-01-29 | Boheringer Ingelheim Pharma Kg | RECOMBINANT DAMAGED PESTIVIRUS, IN PARTICULAR TO CSFV, BVDV OR RECOMBINANT DAMPED BDV |
US8846054B2 (en) * | 2009-01-09 | 2014-09-30 | Boehringer Ingelheim Vetmedica, Inc. | Method of treating pregnant cows and/or heifers |
-
2009
- 2009-06-23 UY UY0001031930A patent/UY31930A/en not_active Application Discontinuation
- 2009-06-24 CN CN201410035977.7A patent/CN103721250A/en active Pending
- 2009-06-24 AU AU2009264206A patent/AU2009264206B2/en not_active Expired - Fee Related
- 2009-06-24 MX MX2010013437A patent/MX2010013437A/en active IP Right Grant
- 2009-06-24 KR KR1020107028302A patent/KR20110036538A/en not_active Application Discontinuation
- 2009-06-24 CA CA2729105A patent/CA2729105A1/en not_active Abandoned
- 2009-06-24 AR ARP090102333A patent/AR072302A1/en active Pending
- 2009-06-24 CN CN2009801243281A patent/CN102076356A/en active Pending
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- 2009-06-24 EP EP09769297.4A patent/EP2303320B1/en active Active
- 2009-06-24 JP JP2011515376A patent/JP5642672B2/en not_active Expired - Fee Related
- 2009-06-24 US US13/000,962 patent/US8895026B2/en active Active
- 2009-06-24 SG SG2014010581A patent/SG2014010581A/en unknown
- 2009-06-24 WO PCT/EP2009/057911 patent/WO2009156448A1/en active Application Filing
- 2009-06-24 BR BRPI0914560A patent/BRPI0914560A2/en not_active IP Right Cessation
-
2010
- 2010-12-22 CO CO10160843A patent/CO6280496A2/en not_active Application Discontinuation
- 2010-12-24 CL CL2010001569A patent/CL2010001569A1/en unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0982402A1 (en) * | 1998-08-14 | 2000-03-01 | Stichting Instituut voor Dierhouderij en Diergezondheid (ID-DLO) | Pestivirus vaccination |
WO2005111201A1 (en) * | 2004-05-19 | 2005-11-24 | Boehringer Ingelheim Vetmedica Gmbh | Vaccine comprising an attenuated pestivirus |
Non-Patent Citations (1)
Title |
---|
VAN GENNIP H G P ET AL: "Dimerisation of glycoprotein Erns of classical swine fever virus is not essential for viral replication and infection", ARCHIVES OF VIROLOGY ; OFFICIAL JOURNAL OF THE VIROLOGY DIVISIONOF THE INTERNATIONAL UNION OF MICROBIOLOGICAL SOCIETIES, SPRINGER-VERLAG, VI, vol. 150, no. 11, 1 November 2005 (2005-11-01), pages 2271 - 2286, XP019378420, ISSN: 1432-8798 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8895286B2 (en) | 1998-06-05 | 2014-11-25 | Boehringer Ingelheim Vetmedica Gmbh | Attenuated pestiviruses |
US8778355B2 (en) | 2001-09-06 | 2014-07-15 | Boehringer Ingelheim Vetmedica Gmbh | Infectious bovine viral diarrhea virus |
US8895026B2 (en) | 2008-06-25 | 2014-11-25 | Boehringer Ingelheim Vetmedica Gmbh | Attenuated pestivirus |
US8846054B2 (en) | 2009-01-09 | 2014-09-30 | Boehringer Ingelheim Vetmedica, Inc. | Method of treating pregnant cows and/or heifers |
JP2015528455A (en) * | 2012-08-31 | 2015-09-28 | ノバルティス アーゲー | Stabilized protein for immunization against STAPHYLOCOCUSAUREUS |
JP2017510268A (en) * | 2014-03-31 | 2017-04-13 | ベーリンガー インゲルハイム フェトメディカ ゲーエムベーハーBoehringer Ingelheim Vetmedica GmbH | Improved modular antigen transport molecules and uses thereof |
US10919945B2 (en) | 2014-03-31 | 2021-02-16 | Boehringer Ingelheim Vetmedica Gmbh | Modular antigen transportation molecules and uses therof |
Also Published As
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KR20110036538A (en) | 2011-04-07 |
JP5642672B2 (en) | 2014-12-17 |
MX2010013437A (en) | 2011-01-14 |
BRPI0914560A2 (en) | 2015-12-15 |
AU2009264206B2 (en) | 2014-12-18 |
CA2729105A1 (en) | 2009-12-30 |
CL2010001569A1 (en) | 2011-09-16 |
CN102076356A (en) | 2011-05-25 |
EP2303320A1 (en) | 2011-04-06 |
AU2009264206A1 (en) | 2009-12-30 |
AR072302A1 (en) | 2010-08-18 |
NZ589723A (en) | 2013-01-25 |
US8895026B2 (en) | 2014-11-25 |
CN103721250A (en) | 2014-04-16 |
US20110117126A1 (en) | 2011-05-19 |
EP2303320B1 (en) | 2014-09-10 |
CO6280496A2 (en) | 2011-05-20 |
JP2012530681A (en) | 2012-12-06 |
SG2014010581A (en) | 2014-05-29 |
UY31930A (en) | 2010-01-29 |
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