EP3497206A1 - Variantes du virus de la bronchite infectieuse (bi) et compositions, utilisations et procédés les concernant - Google Patents

Variantes du virus de la bronchite infectieuse (bi) et compositions, utilisations et procédés les concernant

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Publication number
EP3497206A1
EP3497206A1 EP17755136.3A EP17755136A EP3497206A1 EP 3497206 A1 EP3497206 A1 EP 3497206A1 EP 17755136 A EP17755136 A EP 17755136A EP 3497206 A1 EP3497206 A1 EP 3497206A1
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European Patent Office
Prior art keywords
virus
nucleic acid
seq
amino acid
nos
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP17755136.3A
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German (de)
English (en)
Inventor
Martin LIMAN
Theresa OLDOPP
Jennifer HANEKE
Wiebke BIELENBERG
Natalie VOGEL
Swaantje RÖNCHEN
Diana PETZOLDT
Klaus-Peter BEHR
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Anicon Labor GmbH
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Anicon Labor GmbH
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Publication of EP3497206A1 publication Critical patent/EP3497206A1/fr
Withdrawn legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C12N2770/20011—Coronaviridae
    • C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • IB Infectious Bronchitis
  • the invention is directed to agents, compositions, uses and methods of a novel variant of the infectious bronchitis virus (IB virus).
  • IB virus infectious bronchitis virus
  • the new variant is also referred to here as "IB80”.
  • the invention provides the isolated infectious bronchitis virus variants, referred to as I B viruses, referred to in particular in the National Collection of Pathogenic Viruses (NCPV), Culture Collections, Public Health England, ( Porton Down, Salisbury, SP4 OJG, UK) on 16 June 2016 under accession number 16061601 and on 7 July 2016 under accession number 16070701 under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
  • NCPV National Collection of Pathogenic Viruses
  • IB Infectious bronchitis
  • IBV Infectious Bronchitis Virus
  • Viruses are infectious particles that spread as virions outside of cells (extracellularly) through transmission, but as viruses can only multiply within a suitable host cell (intracellularly). All viruses contain (propagate and propagate) genetic information, but lack the ability to replicate independently and have their own metabolism and therefore rely on the metabolism of a host cell, so virologists largely agree that viruses are not reckless with living things So far, about 3,000 types of viruses have been identified and viruses infect cells from eukaryotes (plants, fungi, animals, humans) and prokaryotes (bacteria and archaea).
  • the coronavirus is a member of the family Coronaviridae, which is a family within the order Nidovirales.
  • the Coronaviridae family is subdivided into two subfamilies based on phylogenetic characteristics and host range, and the genera alpha, beta and gamma (formerly groups 1, 2 and 3 of the ancient genus coronavirus) and deltacoronavirus, as well as torovirus and bafinovirus.
  • the coronavirus is the subfamily Coronavirinae, belonging to the genus gamma coronavirus assigned.
  • the species name according to ICTV is: Avian Coronavirus.
  • infectious bronchitis virus ie the causative agent of infectious bronchitis
  • this includes, for example, the former species turkey / turkey coronavirus (TCoV), pheasant coronavirus (PhCoV), duck coronavirus, geese coronavirus and Pigeon coronavirus.
  • ToV turkey / turkey coronavirus
  • PrCoV pheasant coronavirus
  • duck coronavirus geese coronavirus
  • geese coronavirus geese coronavirus
  • Pigeon coronavirus avirus with genome lengths.
  • RNA polymerase of other RNA viruses In contrast to the usually high error rate of the RNA polymerase of other RNA viruses, which leads to a restriction of the genome length to about 10,000 nucleotides, in coronaviruses a relatively high genetic stability, inter alia, by a 3'-5'-exoribonuclease function of the protein Nsp-14 reached.
  • the single-stranded RNA genome of the coronaviruses is about 27,600 to 31,000 nucleotides (nt) long, which coronaviruses have the longest genome of all known RNA viruses.
  • the 120 to 160 nm virions have a virus envelope in which 3 or 4 different membrane proteins are embedded:
  • the large, glycosylated S protein (180 to 220 kDa) forms as a trimer with its club-shaped, about 20 nm outwardly projecting spikes (Peplomere) the characteristic, coronal appearance of the coronaviruses (lat. Corona: wreath, crown).
  • a second membrane protein E (9 to 12 kDa) is present.
  • the human coronavirus OC43 and the coronaviruses of group 2 additionally contain the HE protein (hemagglutin esterase protein, 65 kDa).
  • the M-protein (23 to 35 kDa), which is also anchored in the sheath, faces inwards and covers the inside of the virus envelope (matrix protein). Inside is a helical nucleoprotein complex. This consists of the nucleoprotein N (50 to 60 kDa), which is complexed with a strand of single-stranded RNA of positive polarity. Certain outward-protruding amino acid residues of the N protein interact with the matrix protein M such that the capsid is associated with the membrane interior.
  • coronaviridae virus family cause very different diseases in various vertebrates such as mammals, rodents, fish and birds.
  • Coronaviruses are genetically highly variable and individual virus species can also infect several host species by overrunning the species barrier.
  • the entry portals of the virus are mainly the lid conjunctiva and mucous membranes of the upper digestive tract and the respiratory system.
  • the transmission is mainly as a droplet infection, with dust particles and droplets loaded with viruses can travel long distances.
  • the virus colonizes the ciliary epithelium of the respiratory tract, but also the reproductive tract and the kidneys can be affected.
  • IB Infectious bronchitis
  • False-tube infections later lead to laying disorders such as pale, thin-skinned or deformed eggs, diaper eggs, markedly reduced or completely missing laying activity ("false larks”) and reduced hatching rate.
  • the duration of the disease and the clinical symptoms can vary.Furthermore, the disease is complicated by secondary bacterial infections Some virus strains may be followed by a kidney infection, which can lead to high mortality from kidney failure or toxemia (toxinemia) .
  • toxemia toxinemia
  • the clinical manifestations allow a presumptive diagnosis and the diagnosis can be made by pathological examination of dead birds as well as by molecular biological, serological and virological detection methods In terms of genetic diagnosis, all respiratory diseases or pathogens which impair the genitourinary tract may be considered.
  • Viral infections such as infectious laryngotracheitis, Newcastle disease and egg drop syndrome, as well as bacterial infections such as mycoplasmosis or infectious chicken cold, as well as non-infectious diseases (eg feeding errors, management errors) should be differentiated.
  • Treatment of infectious bronchitis is possibly symptomatic possible. Therefore, the control is based primarily on the prophylactic vaccine, which can take place immediately after hatching of the broiler or during the first few weeks of life of the animals.
  • the vaccination in endangered areas should be repeated in the vaccination program at regular intervals in accordance with the instructions of the vaccine manufacturer, possibly also with the use of different vaccine variants.
  • Infectious bronchitis is an acute and highly contagious disease of the respiratory tract of chickens, as explained earlier.
  • the disease is caused by the infectious bronchitis virus (IBV), a coronavirus, and is characterized by respiratory and genitourinary symptoms including wheezing, sneezing, tracheal rattling and nasal discharge.
  • IBV infectious bronchitis virus
  • respiratory and genitourinary symptoms including wheezing, sneezing, tracheal rattling and nasal discharge.
  • severe respiratory distress and nephritis can occur.
  • In laying hens can. Shortness of breath, (significant) decline in egg production and loss of egg quality in the interior (watery egg white) and externalities (pale eggs (brownlegs), thin-shelled eggs, soft, deformed, rough shells, missing shells (wind eggs)).
  • IBV infectious bronchitis virus
  • Coronaviruses contain the largest known viral RNA genome by number of nucleotides, at approximately 30,000 bases. The RNA forms a single strand and a single segment.
  • IBV diversity is based on an error of transcription that is of high relevance when it occurs in genomic sequences that code for proteins involved in targeting or inducing immune responses. Due to transcriptional errors, variants may occur which have an evolutionary advantage in disease-susceptible chickens for this variant. Very large genomic changes can take place, e.g. with complete gene replacement, by reassortment in replication, generating multiple subgenomic mRNAs and allowing reassortment in coinfections, thus summarizing the current IBV problem as follows:
  • IBV Infectious Bronchitis Virus
  • IBV Infectious Bronchitis Virus
  • the virus has a broad spectrum of antigenically and genetically different types of virus, which means prevention and control of this disease. makes gers extremely complex.
  • the I B virus is mainly found in chicken.
  • the I B virus or IBV-like and other avian coronaviruses can be found in pets and wildlife, including domestic poultry. Partridges. Geese, pigeons, guinea fowls. Teals.
  • the IBV is a single-stranded, positive-oriented RNA virus of the family Coronaviridae, genus Gammacoronavirus (Cavanagh and Naqi, 2003, International Committee on Taxonomy of Viruses, http://www.ictvonline.org/virustaxonomy.asp).
  • the viral genome contains two untranslated regions (UTRs) at the 5 'and 3' ends (Boursnell et al., 1987; Ziebuhr et al., 2000). two overlapping open reading frames (ORFs).
  • the S protein (-3462 nt) located in the surface of the viral membrane is the most important trigger for neutralizing antibodies (Cavanagh and Naqi, 1997, Winter et al., 2008) and is useful for viral binding and invasion into the virus Host cells (Cavanagh et al, 1986a, Koch et al, 1990, Niesters et al, 1987). It is cleaved post-translationally at a cleavage site with multiple basic amino acids (Cavanagh et al., 1986b) into the amino-terminal S1 (-535 amino acids) and the carboxyl-terminal S2 subunit (-627 amino acids).
  • IB serotypes can differ by 20% to 25% at the genomic level and in up to 50% of the amino acids of the S1 protein (Cavanagh et al., 2005) has received considerable attention (Cavanagh and Gelb 2008). guided. Such variability can lead to important biological differences between strains, and as a result of a limited number of amino acid changes in the spike protein, novel serotype variants can arise. Nucleotide heterogeneity is widespread in the S1 part of the S gene and is largely within three different hypervariable regions (HVRS) of the S1 gene (aa 38-67, 91-141 and 274-387) (Cavanagh et al., 1988; most commonly Moore et al., 1997).
  • HVRS hypervariable regions
  • genotype, serotype or protector type-based approaches do not always classify IB viruses in the same way.
  • analyzes of S1 sequence data are the most commonly used methods for assigning IBV strains to groups that appear to have been arbitrarily subdivided into genetic types, genotypes, cladding, or clusters.
  • the infectious bronchitis virus is the cause of a highly contagious disease in the global poultry industry, causing serious economic losses.
  • the virus exists in a variety of genetically different virus types. Both phylogenetic analysis and pairwise similarity measurements between nucleotide or amino acid sequences were used to classify IBV strains. Until recently, however, there was no consensus about the method by which to compare IBV sequences. Heterogeneous designations of genetic groups that are incompatible with phylogenetic history have been adopted, leading to confusing coexistence of several systems of genotyping. However, the acceptance of an internationally recognized viral nomenclature is crucial to conducting well-founded studies and to gaining and evaluating new insights into the epidemiology and evolution of IBV.
  • Valastro et al. a published in 2016 (Valastro V., Holmes EC, Britton P., Fusaro A., Jackwood MW, Cattoli G., Monne I., S1 gene-based phylogeny of infectious bronchitis virus: An attampt to harmonize virus classification , Infection, Genetics and Evolution, 39 (2016), 349-364), a new classification scheme based on phylogenetic relationships (based on the S1 gene) that can be updated and revised as new S1 sequences become available. Valastro et al.
  • UV Unique Variant
  • the object of the invention is to provide Bases for the detection, control or prophylactic strategy provide, in particular, the here disclosed new IBV variant (IB80) should be affected as a pathogen.
  • the present invention thus aims at a significant advance in solving the particular problems posed by the IB80 virus in poultry keeping, with the aim of providing faster, more reliable and effective means of controlling, particularly the diagnosis, prevention and / or treatment of infectious agents To provide bronchitis in birds.
  • the object on which the invention is based is achieved by providing the agent specified in the claims, such as the IB virus according to the invention (IB80), the nucleic acid according to the invention, the protein according to the invention, the novel protein Vector, antibody, vaccine, the use specified in the claims of the invention IB virus and the pharmaceutical mixture according to the invention.
  • the agent specified in the claims such as the IB virus according to the invention (IB80), the nucleic acid according to the invention, the protein according to the invention, the novel protein Vector, antibody, vaccine, the use specified in the claims of the invention IB virus and the pharmaceutical mixture according to the invention.
  • novel agents, uses, mixtures and methods of the invention described herein are directed to a novel variant IB80 and its closest relative, the agents, uses, mixtures and methods of the invention being useful for the diagnosis and prevention of IB80 virus infections ; including the associated development of in vitro diagnostics, vaccines and the prevention of infectious bronchitis, and associated symptoms or clinical pictures, as further explained below.
  • the present invention is based on the isolation and identification of a novel IB virus (IB80) in birds, especially chickens.
  • IB80 This IB virus (including variants of the invention) is also referred to herein as "IB80.”
  • IB80 was isolated from the material obtained from a monitoring study of a clinically normal chicken flock at that time, and a pool of cecal tonsils was used for culture Organ material was homogenized in 1xPBS including gentamycin and amphotericin B at room temperature, then centrifuged and the supernatant filtered with a 0.45 ⁇ filter, followed by incubation of the filtrate at room temperature, appropriate methods and conditions to be used to isolate virus material are known to the person skilled in the art.
  • the isolated virus is a member of the family Coronaviridae, a family of RNA viruses with the largest known genomes. Accordingly, the invention relates to the isolated IB virus which belongs morphologically and phylogenetically to known members of the subfamily Coronavirinae and, therein, of the genus Gamma coronavirus, the species Avian coronavirus.
  • infectious bronchitis IB is a viral disease of birds, which mainly affects domestic chicken and pheasant, and the causative agent is Infectious Bronchitis Virus (IBV), a coronavirus.
  • the invention relates to a birds and, in particular, chickens-related, isolated IB virus as described and defined herein.
  • the invention herein relates to an isolated IB virus, corresponding to accession number 16061601 (June 16, 2016) or accession number 16070701 (July 7, 2016), which deposits, as noted above under "Filing Declaration", in the National Collection of Pathogenic Viruses (NCPV), Culture Collections, Public Health England, (Porton Down, Salisbury, SP4 0JG, UK)
  • NCPV National Collection of Pathogenic Viruses
  • the invention relates to an isolated IB virus comprising a) one or more nucleic acid segments selected from the group consisting from nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9,
  • one or more proteins comprising or consisting of an amino acid chain selected from the group of the amino acid sequences SEQ ID NOs: 1 1, 12, 13, 14, 15, 16, 17, 18, 19 and 20,
  • nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10 and / or
  • one or more proteins comprising or consisting of an amino acid chain with an identity of> 85% to the amino acid sequence SEQ ID NO: 13 or to a
  • Amino acid sequence selected from the group of amino acid sequences with a Identity of> 98% to one of the amino acid sequences SEQ ID NOs: 1 1, 12, 14, 15, 16, 17, 18, 19 or 20.
  • the invention also relates to an isolated IB virus comprising under d) preferably a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 90%, more preferably> 95%, even more preferably> 98% and all particularly preferably> 99%, to SEQ ID NO: 3; and / or preferably a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 99% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9 or 10.
  • the Invention also an isolated IB virus comprising e) preferably one or more proteins, comprising or consisting of an amino acid chain with an identity of> 90%, more preferably of> 95%, even more preferably of> 98% and most preferably of > 99%, to the amino acid sequence SEQ ID NO: 13; and / or preferably to an amino acid sequence selected from the group of amino acid sequences with an identity of> 99% to one of the amino acid sequences SEQ ID NOs: 1 1, 12, 14, 15, 16, 17, 18, 19 or 20.
  • An isolated IB virus (IB80) according to the invention as defined above may in one embodiment of the invention be dead or attenuated (attenuated) or vital.
  • the invention relates to the complete genomic sequence of the isolated IB virus according to the invention, birds and in particular chickens.
  • the invention also relates to nucleic acid molecules isolated from the IB virus according to the invention, as defined above, or fragments thereof.
  • the invention also relates, as indicated above, to proteins or polypeptides isolated from the IB virus according to the invention, as defined above and / or encoded by a nucleic acid as defined above, including viral proteins isolated from cells which are immunogenic are infected with the IB virus but are not present in comparable uninfected cells; or fragments thereof.
  • the invention relates to a vector comprising a nucleic acid segment as defined above. Furthermore, in one aspect, the invention relates to an isolated antibody or isolated antigen-binding fragment thereof which immunospecifically binds to a virus, protein or nucleic acid as defined above.
  • the invention further relates in one aspect to a vaccine comprising a therapeutically and / or prophylactically effective amount of an IB virus, as defined above, or a therapeutically and / or prophylactically effective amount of one or more nucleic acids, as defined above and / or one or more proteins, as defined above, and a pharmaceutically acceptable carrier.
  • a vaccine, also called vaccination, is the administration of a vaccine to protect against a (communicable) disease is used to activate the immune system against specific substances, for example vaccines have been developed as a preventive measure against infectious diseases
  • a preventive vaccine against an infectious disease is based on a specific, active immunization against the pathogen and is therefore sometimes called an active vaccine
  • the aim of active vaccination is to enable the body's own immune system to respond to infection with the pathogen so rapidly and effectively that it results in no or only a weakened infectious disease.
  • Attenuated vaccines live vaccines, attenuated vaccines
  • inactivated vaccines dead vaccines
  • toxoid vaccines toxoid vaccines
  • passive vaccination also known as immunization
  • the vaccine has been given orally (mouth, "oral”) or nasal (nasal), also transdermally with dermal patches, and most of the active vaccines are administered intramuscularly
  • mouth, "oral” or nasal
  • nasal also transdermally with dermal patches
  • active vaccines are administered intramuscularly
  • eye drops eye drops
  • the active vaccine corresponds to the vaccine in the medical sense and is based on an active immunization. In doing so, the immune system is stimulated to produce pathogen-specific immunocompetence without having to go through the infectious disease itself.
  • the live vaccine contains attenuated, still reproducible pathogens that do not trigger the disease in immunocompetent vaccine. In a dead vaccine, however, these pathogens were killed; or there are only fragments of the pathogen.
  • Upon ingress of the vaccine into the body its proteins and / or sugar molecules are recognized as foreign body antigens by circulating and / or viable immune-competent white blood cells.
  • the primary immune response through pathogen-specific imprinting of immunocompetent lymphocytes in the form of long-lived memory cells.
  • Critical to the protection of a later infection is that for the body, the antigens of the vaccine are largely similar to those of the infectious disease agent. If the infection occurs, the memory cells on the invaded pathogen recognize the antigens of the former vaccine and cause lymphocytes to differentiate into short-lived plasma cells that produce antibodies, on the other hand to T lymphocytes and NK cells, which represent the cellular defense.
  • the vaccine should thus cause the persistence of immunity against the pathogen, so that it does not come to infection after infection due to specific and rapidly induced immune response to infectious disease.
  • Toxoid vaccines containing only the biologically inactive component (toxoid) of the toxin of a pathogen are also among the dead vaccines. They do not diminish the multiplication of the pathogens in the body. In case of an infection, they do not interrupt these, but prevent the outbreak of the clinical disease in the vaccine in so far that here the toxins of the pathogens are not effective.
  • Different live vaccines can be administered either simultaneously or at intervals of a few weeks. For dead vaccines, also in combination with live vaccines, the same applies. The applications should always follow the manufacturer's instructions.
  • a passive vaccination is indicated if there is a risk of suffering a serious infectious disease through contact with the pathogen, but the affected person's immune system is not affected by a prior vaccination or by an earlier (possibly also symptomless, "silent fever") infection
  • the affected person is injected with an immune serum that contains high concentrations of antibodies against the pathogen, since the immune system in this case does not actively produce its own antibodies, but rather it does so to the body of
  • This is preferably a passive immunization of the vaccine using preferably monoclonal antibodies produced by genetic engineering on cell cultures or, if these are not available, extracts from the blood of subjects who have already undergone the infectious disease in question , or out the blood of animals that were specifically infected with the pathogen (convalescent serum).
  • the advantage of immune serums is the faster onset of protection: the antibodies do not have to be formed within one to two weeks, but are available immediately after the injection of the immune serum for infection control.
  • the disadvantage is that the protection only lasts for a few weeks. Thereafter, the administered antibodies are degraded by the recipient and his organism is threatened by repeated infection with the same pathogen again. This is due to the fact that the immune system is not stimulated by the administration of immune serum, via memory cells to form their own immune memory with respect to the pathogens. If the immune serum is derived from animal or human, comes as a further disadvantage that it may contain traces of foreign protein or polysaccharides of the donor apart from the desired antibodies.
  • the recipient's immune system then initiates a cascade of immunological reactions against these alien components.
  • the antibodies enriched in the vaccine serum are excreted more quickly and thus remain effective shorter than desired.
  • repeated administration of external serum of the same species may result in an unwanted allergic reaction of the recipient in the form of a serum sickness or an allergic shock. Therefore, if possible, such immune sera are replaced by monoclonal antibodies.
  • the invention relates to a use of an IB virus (IB80) according to the invention, as defined above, or one or more nucleic acids, as defined above, and / or one or more proteins, such as those above are defined for the preparation of a vaccine for the prevention of a virus, as defined above, symptoms or diseases.
  • IB virus IB80
  • Symptoms or clinical pictures of the disease resulting from IBV infection affect the portal of entry or replication sites of the virus, in particular the connective tissue of the upper digestive and respiratory tract, as well as the ciliary epithelium of the respiratory tract, the reproductive tract and the kidneys.
  • respiratory distress, nasal discharge, wheezing respiratory sounds, sneezing and conjunctivitis should be mentioned.
  • the symptoms of the disease include general disorders with no appetite, decrease in water intake, oviduct infections, laying disorders such as pale, thin-shelled, deformed eggs, wind eggs, significantly reduced or completely missing laying activity (“false Leger”) and reduced hatching rate Kidney failure or resulting toxemia (toxemia) lead to the death of the affected animals, whereas a superficial serious respiratory problem, a tracheal occlusion may be due to the accumulation of mucus and other inflammatory products deaths.
  • the invention also relates to a use of an IB virus, as defined above, or one or more nucleic acids, as defined above, and / or one or more proteins, as defined above, for the production monoclonal or polyclonal antibody.
  • the invention also relates to a pharmaceutical mixture comprising a therapeutically and / or prophylactically effective amount of an antibody or an isolated antigen-binding fragment thereof for the therapeutic or prophylactic treatment of symptoms or diseases caused by a virus, as defined herein. Further aspects relating to the invention will be explained below in order to further illustrate the scope and meaning of the present invention.
  • the invention relates to the use of the isolated IB80 for diagnostic and therapeutic procedures.
  • the invention provides a method of detecting an antibody in a biological sample that is immunospecific for the genus gamma coronavirus, and the species Avian coronavirus, in particular for infectious bronchitis virus (IBV), and more particularly variant IB80, is, of the invention described herein at least one isolated IB virus according to the invention or one of the proteins or polypeptides according to the invention as described herein are used.
  • the invention provides a method for screening for an antibody that immunospecifically binds and neutralizes the infectious bronchitis virus (IB80) variant.
  • an antibody is useful for passive immunization or immunotherapy of an IB80 infected subject.
  • the invention provides isolated antibodies or antigen-binding fragments thereof, which immunospecifically bind to the above-described Infectious Bronchitis Virus (IB80) of the invention.
  • IB80 Infectious Bronchitis Virus
  • the invention relates to methods of detecting the presence, activity or expression of IB80, or fragments thereof, in a biological material such as cells, blood, saliva, urine, feces, etc.
  • the invention relates to a method of detecting the infectious bronchitis virus (IB80) of the present invention to detect its presence in a biological sample, the method comprising: (a) contacting the sample with an agent that selectively targets an infectious agent Bronchitis virus (IB80) binds; and (b) detecting if the agent binds to the infectious bronchitis virus (IB80) in the sample.
  • the invention relates to a method for determining the presence of the polypeptide of the invention in a biological sample, the method comprising: (a) contacting the biological sample with an agent that selectively binds to the polypeptide; and (b) detecting if the agent binds to the polypeptide in the sample.
  • the invention relates to a method for detecting the presence of a nucleic acid molecule derived from the IB80 of the invention in a biological sample, the method comprising: (a) contacting the biological sample with an agent that selectively binds to the nucleic acid binds; and (b) detecting if the agent binds to a nucleic acid in the sample.
  • the invention relates to a method for propagating the infectious bronchitis virus (IB80) according to the invention in host cells, cell lines and / or egg systems, for example hatching eggs, comprising infecting the host cells, cell lines and / or egg systems, for example hatching eggs, with the infectious agent isolated according to the invention lethal bronchitis virus (IB80), culturing the host cells, cell lines and / or egg systems, such as hatching eggs, to allow the virus to replicate and harvesting the resulting virions.
  • the present invention therefore also relates to the infectious bronchitis virus (IB80) infected host cells, cell lines and / or egg systems, for example hatching eggs.
  • infectious bronchitis virus (IB80) according to the invention is the deposit in NCPV in which infectious virus according to the invention (IB80) was deposited.
  • Host cells or suitable cell lines are generally available via cell banks, egg systems such as hatching eggs via the respective producers.
  • the invention relates to a method for detecting the presence of an antibody that immunologically binds IB80 in a biological sample, the method comprising: (a) contacting the biological sample with the host cell of the invention; and (b) detecting the antibody bound to the host cell.
  • the invention relates to vaccine preparations comprising the infectious bronchitis virus (IB80) of the invention, including recombinant and chimeric forms of the virus, the nucleic acid molecules comprised by the virus, or protein subunits of the virus.
  • the invention also relates to a vaccine formulation comprising a therapeutically or prophylactically effective amount of the Infectious Bronchitis Virus (IB80) of the invention, and a pharmaceutically acceptable carrier.
  • the invention relates to a vaccine formulation comprising a therapeutically or prophylactically effective amount of a protein extract of the Infectious Bronchitis Virus (IBV) of the invention or a subunit thereof and a pharmaceutically acceptable carrier.
  • the invention features a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; or a complement thereof, and a pharmaceutically acceptable carrier.
  • a nucleic acid molecule comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a
  • the invention features a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising any of the nucleic acid sequences of the invention or a complement thereof, and a pharmaceutically acceptable carrier.
  • the invention relates to an immunogenic formulation comprising an immunogenically effective amount of the Infectious Bronchitis Virus (IB80) of the invention and a pharmaceutically acceptable carrier.
  • the invention features an immunogenic formulation comprising an immunogenically effective amount of a protein extract of the Infectious Bronchitis Virus of the invention (IB80), or a subunit thereof, and a pharmaceutically acceptable carrier.
  • the invention features an immunogenic formulation comprising an immunogenically effective amount of a nucleic acid molecule comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; or a complement thereof, and a pharmaceutically acceptable carrier.
  • the invention features an immunogenic formulation comprising an immunogenically effective amount of a nucleic acid molecule containing one of the other nucleic acid sequences of the invention or a complement thereof, and a pharmaceutically acceptable carrier.
  • the invention features an immunogenic formulation comprising an immunogenically effective amount of one of the polypeptides of the invention.
  • the present invention relates to pharmaceutical compositions comprising antiviral agents of the present invention and a pharmaceutically acceptable carrier.
  • the antiviral agent of the invention is an antibody that immunospecifically binds Infectious Bronchitis Virus (IB80).
  • the antiviral agent is a polypeptide or protein of the present invention or a nucleic acid molecule of the present invention.
  • the invention relates to a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of an anti-infective bronchitis virus (IB80) agent and a pharmaceutically acceptable carrier.
  • kits containing compositions and formulations of the present invention relate to kits containing compositions and formulations of the present invention.
  • the invention relates to a kit comprising a container containing the immunogenic formulation of the invention.
  • the invention relates to a kit comprising a container containing the vaccine formulation of the invention.
  • the invention relates to a kit comprising a container containing the pharmaceutical composition of the invention.
  • the invention relates to a kit comprising a container containing the vaccine formulation of the invention.
  • the invention in another aspect, relates to a method of identifying a subject infected with the Infectious Bronchitis Virus (IB80) of the invention comprising: (a) recovering the total RNA from a biological sample obtained from the subject; (b) reverse transcription of total RNA to obtain cDNA; and (c) amplifying the cDNA using a set of primers derived from a nucleic acid sequence of the Infectious Bronchitis Virus (IB80) of the invention.
  • the invention furthermore relates to the use of the sequence information of the isolated infectious bronchitis virus (IB80) according to the invention for diagnostic and therapeutic methods.
  • IB80 isolated infectious bronchitis virus
  • the present invention relates to methods for the screening of antiviral agents that inhibit the infectious ability or replication of infectious bronchitis virus (IB80) or other variants thereof.
  • the invention further relates to methods for the production of recombinant or chimeric forms of infectious bronchitis virus (IB80).
  • the present invention is not limited to particular embodiments described, as it will be understood that they may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Due to the sequence divergence of IB80 relative to all previously detected avian viruses, the present invention finds utility in the design of diagnostic assays to monitor IB80 disease in animal subjects, particularly birds, and to develop potent antivirals and vaccines , The IB virus of the present invention (IB80) is genetically> 20% different in the S1 gene from all other known IB viruses.
  • an antibody or an (antigen binding) fragment of an antibody that immunospecifically binds to a polypeptide of the invention refers to an antibody or fragment thereof that is immunospecifically attached to a virus of the invention, a protein of the invention, or to the (IBV) nucleotide sequence comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; coded polypeptide binds.
  • proteins according to the invention herein associated with the term "antibody” or "(antigen-binding) fragment of an antibody” comprise one or more proteins, comprising or consisting of an amino acid chain selected from the group of the amino acid sequences SEQ ID NOs: 11, 12 , 13, 14, 15, 16, 17, 18, 19 and 20, or one or more proteins comprising or consisting of an amino acid chain having an identity of> 85% to the amino acid sequence SEQ ID NO: 13 or to an amino acid sequence selected from Group of amino acid sequences with an identity of> 98% to one of the amino acid sequences SEQ ID NOs: 1 1, 12, 14, 15, 16, 17, 18, 19 or 20.
  • an antibody or fragment thereof immunospecifically binding to the polypeptide of the invention may cross-react with other antigens ilik or a fragment thereof, the immunospecifically binds to a polypeptide of the invention, not with other antigens.
  • An antibody or fragment thereof immunospecifically binding to the polypeptide of the invention can be identified, for example, by immunoassays or other methods known to those skilled in the art or otherwise as described herein.
  • immunospecific or the term “immunospecificity” is a term familiar to a person skilled in the art and frequently used in connection with antibodies or antigens.
  • immunospecific or “immunospecificity” generally describes the ability of a substance or compound, including nucleic acids, peptides, polypeptides, fragments thereof, to specifically react with an antibody molecule directed against it for antibody-antigen binding. This binding can be detected by immunological methods.
  • the terms mentioned are thus associated with the specific immune response which takes place after contact with, for example, an antigen-related immunological reaction of an affected subject;
  • the immune response optionally also includes, for example, the formation of specific T lymphocytes with said substance or compound, including nucleic acids, peptides, polypeptides, fragments thereof, or T lymphocytes specifically reactive with the antigen in an animal subject.
  • the presence of an immunospecific binding is tested in an experiment as described in Example 5.
  • an “isolated” or “purified” peptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived or substantially free of chemical precursors or other chemicals when chemically synthesized
  • the term “substantially free of cellular material” includes preparations of a polypeptide / protein in which the polypeptide / protein is separate from cellular components of the cells from which it is isolated or recombinantly produced.
  • a polypeptide / protein that is substantially free of cellular material includes preparations of the polypeptide / protein that are contaminating with less than about 30%, 20%, 10%, 5%, 2.5%, and 1% (dry weight), respectively Protein.
  • the polypeptide / protein when it is recombinantly produced, it is also substantially free of culture medium, ie, the culture medium constitutes less than about 20%, 10% or 5% of the volume of the protein preparation.
  • the culture medium constitutes less than about 20%, 10% or 5% of the volume of the protein preparation.
  • a polypeptide / protein when produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, ie it is separated from chemical precursors or other chemicals involved in the synthesis of the protein. Accordingly, such preparations less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or other compounds than the polypeptide / protein fragment of interest.
  • the polypeptides / proteins are isolated or purified.
  • isolated refers to a particular biological or synthetic material separated from its natural or synthetic environment, in particular a virus, a viral component, a nucleic acid, RNA, DNA, cDNA, fragments thereof, nucleic acid segments, a protein, a Amino acid sequence, fragments thereof, an antibody or an antigen-binding fragment thereof, and the like materials, said particular material preferably being substantially free of other concomitant materials of any nature, for example, accompanying nucleic acids, proteins, lipids, carbohydrates, or other materials which the particular isolated material may be generally associated under natural or synthetic conditions, for example by association with another cellular material, culture medium or in a synthesis medium.
  • an "isolated" nucleic acid molecule or fragment thereof or a nucleic acid segment is thus, for example, each one or one that is separated and purified from other nucleic acid molecules present in the natural source of the nucleic acid molecule.
  • an "isolated" nucleic acid molecule such as an RNA molecule or cDNA molecule, may be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or others Chemicals when chemically synthesized.
  • the nucleic acid molecules encoding polypeptides / proteins of the invention are isolated or purified.
  • isolated nucleic acid molecule does not include nucleic acid and the like that is a member of a library but has not been separated and purified from the other nucleic acid molecules contained in the library.
  • fragment as used herein includes the indicated fragment lengths and all integers therebetween, including the endpoints specified in a specified range, and including any length to full length of a protein, polypeptide or nucleic acid .
  • having a biological activity of the protein or “having biological activities of the polypeptides of the invention” refers to the properties of the polypeptides or proteins which have a common biological activity, similar or identical structural domain, and / or sufficient amino acid identity to which A nucleotide sequence comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid
  • proteins of the invention herein associated with the term "with a biological activity of the protein” or "with biological activities of the polypeptides of the invention” comprise one or more proteins comprising or consisting of an amino acid chain selected from the group of the amino acid sequences SEQ ID NOs: 1 1, 12, 13, 14, 15, 16, 17, 18, 19 and 20; or one or more proteins, comprising or consisting of an amino acid chain with an identity of> 85% to the amino acid sequence SEQ ID NO: 13 or to an amino acid sequence selected from the group of amino acid sequences with an identity of> 98% to one of the amino acid sequences SEQ ID NOs : 1 1, 12, 14, 15, 16, 17, 18, 19 or 20.
  • the above-mentioned preferred% -dentities apply here as well to the above SEQ ID NOs.
  • Such common biological activities of the polypeptides of the invention include antigenicity and immunogenicity.
  • under stringent conditions refers to hybridization and washing conditions in which nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with each other remain hybridized to each other .
  • hybridization conditions are described, for example but not limited to, in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6; Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., New York (1986), pp. 75-78 and 84-87. and Molecular Cloning, Cold Spring Harbor Laboratory, NY (1982), pp. 387-389, and are well known to those skilled in the art.
  • a preferred, non-limiting example of stringent hybridization conditions is hybridization in 6x sodium chloride / sodium citrate (SSC), 0.5% SDS at about 68 ° C, followed by one or more washes in 2x SSC, 0.5% SDS room temperature.
  • SSC sodium chloride / sodium citrate
  • Another preferred, non-limiting example of stringent hybridization studies The hybridization in 6 x SSC at about 45 ° C is followed by one or more washes in 0.2 SSC, 0.1% SDS at about 50-65 ° C.
  • variant refers to either a naturally occurring genetic mutant of IB80 or a recombinantly produced variation of this IBV variant, each of which has one or more mutations in its genome compared to IBV, which is a or a plurality of nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10.
  • variant may also refer to either a naturally occurring variant of a given peptide or a recombinantly produced variation of a given peptide or protein in which one or more amino acid residues have been modified by amino acid substitution, addition or deletion.
  • homology refers to sequence similarity or, alternatively, sequence identity between two or more polynucleotide sequences, or two or more polypeptide sequences.
  • percent identity and % -ldentity as applied to polynucleotide sequences refer to the percentage of identical nucleotide matches between at least two polynucleotide sequences aligned using a standard algorithm can, in a standardized and reproducible manner, insert gaps into the compared sequences to optimize the alignment between two sequences and thus achieve a more meaningful comparison of the two sequences.
  • the percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, the percent identity may be determined using the default parameters (default parameter) of the CLUSTAL V algorithm as incorporated in version 3.12e of the MEGALIGN Sequence Alignment program LASERGENE software packages, a set of molecular biological analysis programs (DNASTAR, Madison, Wis.). CLUSTAL V is described in Higgins, DG and PM Sharp (1989; CABIOS 5: 151-153) and in Higgins, DG et al. (1992, CABIOS 8: 189-191).
  • the Clustal W algorithm is a widely used alignment program that is suitable for alignments of more than two sequences (multi-sequence alignment).
  • the Clustal W program uses a progressive alignment using the neighbor-joining algorithm to create a multiple sequence alignment.
  • BLAST Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST 2 Sequences can also be interactively accessed and used on the Internet via the NCBI World Wide Web site.
  • the "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below).
  • BLAST programs are commonly used with gap and other parameters set to the default settings. For example, to compare two nucleotide sequences, one can use blastn with the "BLAST 2 Sequences” version 2.0.12 tool (April 21, 2000), which is set to default parameters.
  • Percent identity can be measured over the length of an entire defined sequence, such as defined by a particular SEQ ID number, or measured for a shorter length, for example, over the length of a fragment taken from a larger defined sequence , For example, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100 or at least 200 consecutive nucleotides. Such lengths are exemplary only, and it is understood that any fragment length, supported by the sequences shown herein, in tables, figures, or sequence listings, can be used to describe a length over which the percent identity can be measured.
  • percent identity and % -ldentity refer to the percentage of identical residue matches between at least two aligned polypeptide sequences using a standardized algorithm. Methods of polypeptide sequence alignment are well known, and some alignment methods take conservative amino acid substitutions into account.Such conservative substitutions ("substitutions"), discussed above, generally conserve charge and hydrophobicity at the site of substitution, thereby reducing the structure (and therefore function) of the polypeptide.
  • percent similarity and percent similarity as applied to polypeptide sequences refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two aligned polypeptide sequences using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
  • the PAM250 matrix is selected as the default residual weight table.
  • NCBI BLAST software series can be used.
  • the "BLAST 2 Use Sequences' version 2.0.12 tool (April 21, 2000) with blastp set to default parameter.
  • standard parameters can be for example: matrix: BLOSUM62; Open gap: 1 1 and expansion gap: 1 sanction; Gap x drop-off: 50; Expect: 10; Word size: 3; Filter: on.
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example as defined by a particular SEQ ID number, or may be measured for a shorter length, for example, along the length of a fragment removed from a larger, defined polypeptide sequence, for example For example, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residuals.
  • a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residuals are exemplary only and it is understood that any fragment length, supported by the sequences shown herein, in the tables, figures or sequence listings, can be used to describe a length over which the percent identity can be measured.
  • the term "agent” includes any chemical, biochemical or biological molecule; such as small molecules, proteins, polypeptides, antibodies, nucleic acid molecules, including DNA or RNA, and the like.
  • the present invention is based on the isolation and identification of a new variant of infectious bronchitis virus, IB80, and its sequencing.
  • IB80 was isolated from chickens with infectious bronchitis.
  • the isolated virus has a single-stranded RNA and is a member of the family Coronaviridae, a family of positive-oriented RNA viruses of the genus Gamma coronavirus, the species Avian coronavirus.
  • the invention relates to the isolated IB virus that is morphologically and phylogenetically related to known members of the coronaviridae, and more particularly the gammacoronaviruses.
  • the invention relates to an isolated IB virus containing a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent conditions with one of the sequences defined according to the invention, the sequences according to the invention preferably being selected from the group: One or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9;
  • a nucleic acid of SEQ ID NO: 10 or
  • a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9 or 10; or wherein the isolated IB virus-containing nucleic acid molecule has a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID has NO.
  • the IB virus is killed.
  • the IB virus is attenuated.
  • the infectivity of the attenuated IB virus is reduced.
  • the infectivity is reduced at least 5x, 10x, 25x, 50x, 100x, 250x, 500x or 10,000x.
  • the replication ability of the attenuated IB virus is reduced.
  • the replication ability of the attenuated IB virus is reduced at least 5x, 10x, 25x, 50x, 100x, 250x, 500x, 1,000x, or 10,000x.
  • the ability of the attenuated IB virus to reduce protein synthesis is reduced.
  • the ability of the attenuated IB protein to protein synthesis is reduced at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1,000-fold or 10,000-fold ,
  • the ability of the attenuated IB virus to reduce assembly is reduced.
  • the ability of the attenuated IB virus to assemble is at least 5x, 10x, 25x, 50x, 100x, 250x, 500x, 1,000x, or 10,000x reduced.
  • the cytopathic effect of the attenuated IB virus is reduced.
  • the cytopathic effect of the attenuated IB virus is reduced at least 5 times, 10 times, 25 times, 50 times, 100 times, 250 times, 500 times, 1,000 times or 10,000 times
  • the invention provides the complete genomic sequence of the IB virus.
  • the IB virus comprises a nucleotide sequence of SEQ ID NO: 10 corresponding to the complete virus.
  • the invention relates to nucleic acid molecules isolated from IBV or fragments thereof.
  • the isolated nucleic acid molecule comprises one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9 or 10; or a complement thereof.
  • the above-mentioned preferred% -dentities also apply to the above SEQ ID NOs.
  • the nucleic acid molecule comprises a nucleotide sequence of at least 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 , 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 4600, 4700, 4800 or 4900 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 10 or a complement thereof; or at least 5000, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500 or 6600 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 10, or a complement thereof.
  • the isolated nucleic acid molecule comprises a nucleotide sequence which, for the IBV amino acid sequence, comprises one or more proteins, comprising or consisting of an amino acid chain selected from the group of the amino acid sequences SEQ ID NOs: 1 1, 12, 13, 14, 15, 16 , 17, 18, 19 and 20; or for one or more proteins, comprising or consisting of an amino acid chain with an identity of> 85% to the amino acid sequence SEQ ID NO: 13 or to an amino acid sequence selected from the group of amino acid sequences with an identity of> 98% to one of the amino acid sequences SEQ ID NOs: 1 1, 12, 14, 15, 16, 17, 18, 19 or 20; or a complement of the nucleotide sequence encoding the IBV amino acid sequences of the aforementioned SEQ ID NOs.
  • the isolated nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs: comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5 , 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; or a complement thereof, wherein the hybridizing nucleic acid molecule encodes an amino acid sequence having a biological activity exhibited by a polypeptide encoded by the nucleotide sequence of the aforementioned SEQ ID NOs.
  • the above SEQ ID NOs also apply here above preferred% -ldentticianen.
  • the invention relates to proteins or polypeptides, including viral proteins, which are isolated from cells infected with the IB virus, such as host cells, cell lines and / or egg systems, for example hatching eggs, but which are not in comparable uninfected ones Cells, such as host cells, cell lines and / or egg systems, such as hatching eggs, are present.
  • the amino acid sequences of one or more proteins comprising or consisting of an amino acid chain are selected from the group of the amino acid sequences SEQ ID NOs: 1 1, 12, 13, 14, 15, 16, 17, 18, 19 and 20 ; or one or more proteins, comprising or consisting of an amino acid chain with an identity of> 85% to the amino acid sequence SEQ ID NO: 13 or to an amino acid sequence selected from the group of amino acid sequences with an identity of> 98% to one of the amino acid sequences SEQ ID NOs : 1 1, 12, 14, 15, 16, 17, 18, 19 or 20; set out, or a fragment thereof.
  • the polypeptides or proteins of the present invention have a biological activity of the protein (including antigenicity and / or immunogenicity) which is represented by the sequence comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; is encoded.
  • the sequence comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an
  • polypeptides or proteins of the present invention have a biological activity of at least one protein having the amino acid sequence (including antigenicity and / or immunogenicity), such as this amino acid sequence, or a fragment thereof, set forth hereinabove.
  • amino acid sequence including antigenicity and / or immunogenicity
  • this amino acid sequence or a fragment thereof, set forth hereinabove.
  • preferred% -dentities also apply to the above SEQ ID NOs.
  • the invention relates to an isolated polypeptide encoded by the above-described nucleic acid molecule of the invention.
  • the isolated polypeptide comprises the amino acid sequence selected from the group consisting of: a) one or more proteins, comprising or consisting of an amino acid chain selected from the group of the amino acid sequences SEQ ID NOs: 1 1, 12, 13, 14 , 15, 16, 17, 18, 19 and 20; or one or more proteins, comprising or consisting of one Amino acid chain with an identity of> 85% to the amino acid sequence SEQ ID NO: 13 or to an amino acid sequence selected from the group of amino acid sequences with an identity of> 98% to one of the amino acid sequences SEQ ID NOs: 1 1, 12, 14, 15, 16 , 17, 18, 19 or 20; and b) an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence according to a).
  • the isolated polypeptide comprises the amino acid sequence of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 210, 220, 230 , 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 450, 500, 550, 600, 610, 620, 630, 640 , 650, 660, 670, 700, 710, 720, 730, 750, 800, 850, 900, 950, 1000, 1050, 1100, 150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 , 1600, 1650, 1700, 1750, 1800 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2160, 2170, 2180, 2190 or 2200 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs set forth
  • the invention relates to the use of an isolated IB virus for diagnostic and therapeutic procedures.
  • the invention provides a method for detecting an antibody that is immunospecific in a biological sample for the IB virus using the isolated IB virus of the invention described herein, or any of the proteins or polypeptides of the invention described herein.
  • the invention relates to a method of screening for an antibody that binds immunospecifically and neutralizes IBV or a combination of IBVs. Such an antibody is useful for passive immunization or immunotherapy of an IBV-infected subject.
  • the invention relates to an isolated antibody or antigen-binding fragment thereof, which immunospecifically binds to an IB virus genus of the invention described above.
  • the isolated antibody or an antigen-binding fragment thereof neutralizes a genus of the IB virus.
  • the isolated antibody or an antigen-binding fragment thereof immunospecifically binds to the above-described polypeptide of the invention.
  • the invention further relates to antibodies which specifically bind a polypeptide of the invention comprising a nucleotide sequence comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 , 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or with an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9 or 10; a fragment thereof, or encoded by a nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to one of the aforementioned nucleotide sequences, and / or any IB80 epitope having one or more biological activities of a polypeptide of the invention.
  • polypeptides comprise the proteins according to the invention, ie one or more proteins, comprising or consisting of an amino acid chain selected from the group of the amino acid sequences SEQ ID NOs: 1 1, 12, 13, 14, 15, 16, 17, 18, 19 and 20; or one or more proteins, comprising or consisting of an amino acid chain with an identity of> 85% to the amino acid sequence SEQ ID NO: 13 or to an amino acid sequence selected from the group of amino acid sequences with an identity of> 98% to one of the amino acid sequences SEQ ID NOs : 1 1, 12, 14, 15, 16, 17, 18, 19 or 20.
  • the above-mentioned preferred% identities apply here as well to the above SEQ ID NOs.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, bispecific, multispecific, human, humanized, chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') 2 fragments, disulfide-linked Fvs, intrabodies, and fragments containing either a VL or VH domain or even a complementarity determining region (CDR) that specifically binds to a polypeptide of the invention.
  • CDR complementarity determining region
  • the invention relates to methods for detecting the presence, activity or expression of the IB virus of the invention in a biological material such as cells, blood, saliva, urine and the like.
  • the increased or decreased activity or expression of the IB virus in a sample relative to a control sample can be determined by contacting the biological material with an agent that directly or indirectly recognizes the presence, activity or expression of the IB virus can.
  • the detection means are the antibodies or nucleic acid molecules of the present invention.
  • the invention relates to a method for detecting the presence of the above-described IB virus of the invention in a biological sample, the method comprising: (a) contacting the sample with an agent that selectively binds to the IB virus; and (b) detecting if the agent binds to the IB virus in the sample.
  • the biological sample is selected from the group consisting of cells; Blood; Serum; Plasma; Kot; tracheal, choanal or cloacal and conjunctival smears.
  • that is Agent that binds to the IB virus an antibody.
  • the agent that binds to the IB virus is a nucleic acid molecule comprising the nucleotide sequence comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; or a complement thereof.
  • the above-mentioned preferred% -dentities also apply to the above SEQ ID NOs.
  • the agent that binds to the IB virus is a nucleic acid molecule comprising a nucleotide sequence of at least 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 , 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500 , 4000, 4500, 4600, 4700, 4800, 4900, 5000, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500 or 6600 contiguous nucleotides of the nucleotide sequence of one of the aforementioned SEQ ID NOs.
  • the invention relates to a method for determining the presence of the above-described polypeptide of the invention in a biological sample, the method comprising: (a) contacting the biological sample with an agent that selectively binds to the polypeptide; and (b) detecting if the agent binds to the polypeptide in the sample.
  • the biological sample is selected from the group consisting of cells; Blood; Serum; Plasma; Kot; tracheal, choanal or cloacal and conjunctival smears.
  • the agent that binds to the polypeptide is an antibody or antigen-binding fragment thereof.
  • the invention relates to a method for detecting the presence of a first nucleic acid molecule derived from the above-described IB virus of the invention in a biological sample, the method comprising: (a) contacting the biological sample with an agent which selectively the nucleic acid binds; and (b) detecting if the agent binds to the nucleotide in the sample.
  • the agent that binds to the first nucleic acid molecule is a second nucleic acid molecule comprising the nucleotide sequence comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4 , 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprehensive a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8 , 9 or 10; or a complement thereof.
  • the second nucleic acid molecule comprises at least 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 4600, 4700, 4800, 4900, 5000, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500 or 6600 contiguous nucleotides of the nucleotide sequence of any one of the aforementioned SEQ ID NOs, or a complement thereof.
  • the invention relates to a method for propagating the IB80 in host cells, comprising infecting the host cells, cell lines and / or egg systems, for example hatching eggs, with an isolated Infectious Bronchitis Virus of the invention described above, culturing the host cells, cell lines and / or egg systems, such as hatching eggs, to allow the virus to replicate and harvesting resulting virions.
  • the present invention also relates to host cells, cell lines and / or egg systems, for example hatching eggs, which are infected with the virus according to the invention described above.
  • the host cell is a bird cell; According to the invention, all bird species are suitable, in particular chickens.
  • the egg system particularly relates to hatching eggs.
  • the egg system relates to embryonated eggs; For example, so-called SPF eggs, “Serumeier”, “Clean Eggs” are to be mentioned here in particular.
  • SPF eggs so-called SPF eggs, "Serumeier", "Clean Eggs” are to be mentioned here in particular.
  • the invention relates to a method of detecting the presence of an antibody in a biological sample immunologically binding IB virus, the method comprising: (a) contacting the biological sample with the above-described host cell of the invention; and (b) detecting the antibody bound to the cell.
  • the invention relates to vaccine preparations, including the IB virus of the invention, including recombinant and chimeric forms of the virus, the nucleic acid molecules or protein subunits of the virus comprised by the virus.
  • the vaccine preparations comprise the present the invention, the live but attenuated IB virus, with or without pharmaceutically acceptable carriers, including adjuvants.
  • the vaccine preparations of the invention comprise an inactivated or killed IB virus, variants of the IB virus or a combination thereof, with or without pharmaceutically acceptable carriers, including adjuvants.
  • Such attenuated or inactivated viruses can be produced by a series of passages of the virus in a host system, eg, a cell line, by inactivation methods, or by producing recombinant or chimeric forms of the virus. Accordingly, the present invention further relates to methods of producing the recombinant or chimeric forms of the IB viruses of the invention described herein.
  • the invention relates to a vaccine formulation comprising a therapeutically or prophylactically effective amount of the IB virus of the invention described above and a pharmaceutically acceptable carrier.
  • the invention relates to a vaccine formulation comprising a therapeutically or prophylactically effective amount of a protein extract of the IB virus of the invention described above, or a subunit thereof, and a pharmaceutically acceptable carrier.
  • the invention relates to a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; or a complement thereof, and a pharmaceutically acceptable carrier.
  • a nucleic acid molecule comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of
  • the invention relates to a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising any of the above-described nucleotide sequences of the invention, or a complement thereof, and a pharmaceutically acceptable carrier.
  • the invention features a vaccine formulation comprising a therapeutically or prophylactically effective amount of a protein extract of the IB virus of the invention described above, or a subunit thereof, and a pharmaceutically acceptable carrier.
  • the invention relates to a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; or a complement thereof, and a pharmaceutically acceptable carrier.
  • a nucleic acid molecule comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of
  • the invention relates to a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising any of the nucleotide sequences of the invention described above, or a complement thereof, and a pharmaceutically acceptable carrier.
  • the vaccine preparations of the present invention comprise a nucleic acid or a fragment of IB80, for example virus number 16061601 or accession number 16070701, or nucleic acid molecules comprising one or more nucleic acid segments selected from the group consisting of nucleic acids Sequences SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; or a fragment thereof.
  • the vaccine preparations comprise a protein / polypeptide of the invention encoded by any of the nucleotide sequences set forth above, or a fragment thereof.
  • the vaccine preparations contain proteins / polypeptides of the invention comprising or consisting of an amino acid chain selected from the group of the amino acid sequences SEQ ID NOs: 1 1, 12, 13, 14, 15, 16, 17, 18, 19 and 20; or one or more proteins, comprising or consisting of an amino acid chain with an identity of> 85% to the amino acid sequence SEQ ID NO: 13 or to an amino acid sequence selected from the group of amino acid sequences with an identity of> 98% to one of the amino acid sequences SEQ ID NOs: 1 1, 12, 14, 15, 16, 17, 18, 19 or 20; or as encoded by any of the above nucleotide sequences of the invention, or a fragment thereof.
  • the present invention relates to methods for treating, alleviating, managing or preventing infectious bronchitis (this generally includes other symptoms caused by IB80) by the vaccine preparations or antibodies of the present invention alone or in combination with adjuvants, or other pharmaceutical acceptable excipients.
  • the present invention relates to methods for treating, alleviating, managing or preventing infectious bronchitis by administering the compositions and formulations of the invention, including the vaccine preparations or antibodies of the present invention, alone or in combination with antiviral drugs [eg amantadine, rimantadine, gancyclovir, Acyclovir, ribavirin, penciclovir, oseltamivir, foscarnet zidovudine (AZT), didanosine (DDL), lamivudine (3TC), zalcitabine (ddC), stavudine (d4T), nevirapine, delavirdine, indinavir, ritonavir, vidarabine, nelfinavir, saquinavir, Relenza, Tamiflu, pleconaril, interferons, etc.], with steroids and corticosteroids such as prednisone, cortisone, fluticasone and glucocortic drugs
  • the invention features an immunogenic formulation comprising an immunogenically effective amount of the IB virus of the invention described above and a pharmaceutically acceptable carrier.
  • the invention features an immunogenic formulation comprising an immunogenically effective amount of a protein extract of the IB80 virus of the present invention, or a subunit thereof, and a pharmaceutically acceptable carrier.
  • the invention features an immunogenic formulation comprising an immunogenically effective amount of a nucleic acid molecule comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; a combination thereof or a complement thereof, and a pharmaceutically acceptable carrier.
  • the above-mentioned preferred% -dentities also apply to the above SEQ ID NOs.
  • the invention features an immunogenic formulation comprising an immunogenically effective amount of a nucleic acid molecule containing one of the nucleotide sequences of the invention described above, or a complement thereof, and a pharmaceutically acceptable carrier.
  • the invention relates to an immunogenic formulation comprising an immunogenically effective amount of any of the polypeptides of the invention described above.
  • the present invention relates to pharmaceutical compositions comprising antiviral agents of the present invention and a pharmaceutically acceptable carrier.
  • the antiviral agent of the invention is an antibody that immunospecifically binds IB80 or an IB80 epitope.
  • the antiviral agent is a polypeptide or protein of the present invention or nucleic acid molecule of the invention.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of an anti-I B virus agent and a pharmaceutically acceptable carrier.
  • the anti-IB80 agent is an antibody or antigen-binding fragment thereof, which immunospecifically binds to the virus of accession number 16061601 or 16070701, or to polypeptides or proteins derived therefrom.
  • the anti-IB80 agent is a nucleic acid molecule comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; a combination thereof or a fragment thereof.
  • the anti-IB80 agent is a polypeptide encoded by a nucleic acid molecule comprising any of the nucleotide sequences set forth above, a combination thereof, or a fragment thereof having a biological activity of the polypeptide.
  • the above-mentioned preferred% -dentities also apply to the above SEQ ID NOs.
  • kits comprising compositions and formulations of the present invention.
  • the invention relates to a kit, comprising a container containing the immunogenic formulation of the invention described above.
  • the invention relates to a kit including a container containing the above-described pharmaceutical composition of the invention.
  • the invention relates to a kit, including a container containing the vaccine formulation of the invention described above.
  • the invention relates to a method of identifying a subject infected with the IB80 of the invention described above, including: (a) recovering the total RNA from a biological sample obtained from the subject; (b) reverse transcription of the total RNA to obtain cDNA; and (c) amplifying the cDNA using a set of primers derived from a nucleotide sequence of the IB virus of the invention described above.
  • the set of primers is derived from the nucleotide sequence of the genome of the IB virus of Accession No. 16061601 or 16070701. In another, the set of primers is derived from one of the nucleotide sequences of the invention described above, or a complement thereof.
  • the invention further relates to the use of the sequence information of the isolated IB80 virus for diagnostic and therapeutic procedures.
  • the invention relates to nucleic acid molecules suitable for use as primers, consisting of or including any of the nucleotide sequences of the invention set forth above, or a complement thereof, or at least a portion of the nucleotide sequence thereof.
  • the invention relates to nucleic acid molecules suitable for hybridization to the IB80 nucleic acid of the invention; including, but not limited to, PCR primers, reverse transcriptase primers, probes for Southern analysis, Northern blots, probes for real-time PCR, or other nucleic acid hybridization assays for detection of IB80 nucleic acids, for example consisting of or including one of the nucleotide sequences of the invention set forth above, a combination thereof, a complement thereof, or a portion thereof.
  • the invention further encompasses chimeric or recombinant IB80 viruses that are fully or partially encoded by the nucleotide sequences.
  • the present invention relates to methods for screening antiviral agents that inhibit the infectivity or replication of IB80, including variants thereof.
  • the invention further relates to methods for producing recombinant or chimeric forms of IB80.
  • the invention relates to vaccine preparations, including the IB80 virus, including recombinant and chimeric forms of the IB80 virus or subunits of the IB80 virus.
  • the present invention encompasses recombinant or chimeric viruses encoded by viral vectors derived from the genome of the IB80 virus of the invention described herein or natural variants thereof.
  • the recombinant virus is a virus derived from the IB80 virus of accession number 16061601 or 16070701.
  • natural variants of the IB80 viruses of the invention described herein include one or more mutations, including, but not limited to, point mutations, rearrangements, insertions, deletions, etc., in the genomic sequence. It is recognized that the mutations may or may not lead to a phenotypic change.
  • a chimeric virus of the invention is a recombinant IB80 virus further comprising a heterologous nucleotide sequence.
  • a chimeric virus may be encoded by a nucleotide sequence in which heterologous nucleotide sequences have been added to the genome or in which endogenous or native nucleotide sequences have been replaced by heterologous nucleotide sequences.
  • the chimeric viruses are encoded by the viral vectors of the invention which further comprise a heterologous nucleotide sequence.
  • a chimeric virus is encoded by a viral vector that may or may not contain nucleic acids that are non-native to the viral genome.
  • a chimeric virus is preferably encoded by a viral vector in which heterologous nucleotide sequences have been added, inserted, or substituted for native or non-native sequences.
  • the chimeric virus may be encoded by nucleotide sequences derived from different species or variants of the IB virus.
  • the chimeric virus is going through Nucleotide sequences encoding antigenic, derived from different species or variants of the IB virus polypeptides.
  • a chimeric virus may be of particular use for the production of recombinant vaccines for protection against two or more viruses (Tao et al., J. Viral., 72, 295- 2961, Durbin et al., 2000, J. Viral , 6821-6831, Skiadopoulos et al., 1998, J. Viral., 72, 1762-1768 (1998), Teng et al., 2000, J. Viral., 74, 9317-9321).
  • a virus vector derived from the IB80 virus that expresses one or more proteins of variants of the IB80 virus will protect a subject vaccinated with such a vector against both native IB80 virus and variant infections
  • Attenuated and replication-deficient viruses may be useful for vaccination with live vaccines, as has been suggested for other viruses.
  • the heterologous sequence to be incorporated into the viral vectors, which encode the recombinant or chimeric viruses of the invention includes sequences derived from or derived from different species or variants of IB80.
  • the chimeras or recombinant viruses of the invention are encoded by viral vectors derived from viral genomes wherein one or more sequences, intergenic regions, termini sequences or portions or entire ORF have been replaced by a heterologous or non-native sequence are.
  • the chimeric viruses of the invention are encoded by viral vectors derived from viral genomes wherein one or more heterologous sequences have been inserted or added into the vector.
  • the selection of the viral vector may depend on the species of the subject to be treated or protected for viral infection.
  • An attenuated IB80 virus can be used to provide the antigenic sequences.
  • the viral vectors can be constructed to provide antigenic sequences conferring protection against infection thereof by the IB80 of the invention and natural variants thereof.
  • the viral vectors can be engineered to provide one, two, three or more antigenic sequences.
  • the antigenic sequences may be derived from the same virus, from different species or variants of the same virus type or from different viruses.
  • the expression products and / or recombinant or chimeric virions obtained in accordance with the invention can be advantageously used in vaccine formulations.
  • the expression products and chimeric virions of the present invention can be engineered to produce vaccines against a broad spectrum of pathogens, including viral and bacterial antigens, tumor antigens, allergen antigens, and autoantigens involved in autoimmune diseases.
  • pathogens including viral and bacterial antigens, tumor antigens, allergen antigens, and autoantigens involved in autoimmune diseases.
  • One way to achieve this goal is to modify existing IB80 genes to contain foreign sequences in their respective external domains.
  • these chimeric viruses can be used to induce a protective immune response against the pathogen from which these determinants are derived.
  • the chimeric virions of the present invention can be engineered to confer vaccines for the protection of a subject from IB80 virus infections and variants thereof.
  • the present invention further relates to the use of viral vectors and recombinant or chimeric viruses of the invention to formulate vaccines against a broad spectrum of viruses and / or antigens.
  • the present invention also encompasses recombinant viruses of the invention, including a viral vector derived from the IB80 or variants thereof, which contains sequences resulting in a virus having a phenotype more suitable for use in vaccine formulations, e.g. an attenuated phenotype or with improved antigenicity.
  • the mutations and modifications may be in coding regions, in intergenic regions and in the leader and trailer sequences of the virus.
  • the invention relates to a host cell with a nucleic acid or a vector according to the invention.
  • Plasmid or viral vectors containing the polymerase components of the IB80 virus are generated in prokaryotic cells for expression of the components in the appropriate cell types (bacteria, insect cells, eukaryotic cells).
  • Full-length or partial copies of the IB80 genome containing plasmid or viral vectors are generated in prokaryotic cells for the expression of viral nucleic acids in vitro or in vivo.
  • the latter vectors contain if appropriate, other viral sequences for the production of chimeric viruses or chimeric virus proteins, they may be missing parts of the viral genome for the production of a replication-defective virus, and they may contain mutations, deletions or insertions to produce attenuated viruses.
  • the present invention relates to a host cell, cell line and / or egg system, for example Brutei, which is infected with an IB80 virus of accession number 16061601 or 16070701. Host cells or cell lines are generally available via cell banks, egg systems such as hatching eggs via the respective producers.
  • IBV Infectious copies of the IBV (as wild-type, attenuated, replication defective, or chimeric) are optionally prepared upon co-expression of the polymerase components according to the prior art technologies described above.
  • eukaryotic cells that transiently or stably express one or more full-length or partially expressed IB80 proteins are optionally used.
  • such cells are produced by transfection (proteins or nucleic acid vectors), infection (viral vectors) or transduction (viral vectors), and are useful for complementation of said wild-type, attenuated, replication-defective, or chimeric viruses.
  • the viral vectors and chimeric viruses of the present invention may modulate a subject's immune system by stimulating a humoral immune response, a cellular immune response, or by stimulating tolerance to an antigen.
  • a subject means preferably humans, primates, horses, cows, sheep, pigs, goats, dogs, cats, birds and rodents.
  • a subject means: bird species, especially chickens.
  • the invention relates to a proteinaceous molecule or IB80 virus-specific viral protein or a functional fragment thereof encoded by a nucleic acid according to the invention.
  • useful proteinaceous molecules are derived from any of the genes or genomic fragments derivable from the virus of the invention.
  • Such molecules, or their antigenic fragments as provided herein, are useful, for example, in diagnostic methods or kits and in pharmaceutical compositions such as subunit vaccines.
  • polypeptides which are replaced by a A nucleotide sequence comprising one or more nucleic acid segments selected from the group consisting of nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; or antigenic fragments thereof, for inclusion as an antigen or immunogenic subunit, but the inactivated whole virus can also be used.
  • the above-mentioned preferred% -dentities also apply to the above SEQ ID NOs.
  • those proteinaceous substances that are encoded by recombinant nucleic acid fragments of the IB80 genome, preferably those that are within the preferred limits and dimensions of ORFs, particularly to elicit specific IBV antibody or T cell responses, whether in vivo (eg for protective or therapeutic purposes or for the provision of diagnostic antibodies) or in vitro (eg by phage display technique or another technique useful for the production of synthetic antibodies).
  • IB80 polypeptides are within the scope of the present invention, including substitutions, alterations, modifications of amino acid or other alterations of amino acid that increase the function or immunogenic propensity of the immunogen or vaccine of the present invention or not change.
  • post translational modifications are equally within the scope of the present invention, illustrative including incorporation of naturally occurring amino acid (s), phosphorylation, glycosylation, sulfation, and addition of side groups such as biotinylation, fluorophores, lumiphors, radioactive groups, antigens, or other molecules ,
  • peptides and proteins are recombinantly expressed in eukaryotic cells.
  • eukaryotic cells include yeast, HeLa cells, 293 cells, COS cells, Chinese hamster ovary (CHO) cells, and many other cell types known in the art.
  • yeast yeast
  • HeLa cells yeast
  • 293 cells yeast
  • COS cells Chinese hamster ovary
  • Both eukaryotic and prokaryotic expression systems and cells are available, for example from Invitrogen Corp., Carlsbad, CA. It is understood that cell-free expression systems can be operated similarly.
  • an immunogenic polypeptide is a full-length IB80 protein.
  • an immunogen is full-length IB80 protein comprising one or more proteins comprising or consisting of an amino acid chain selected from the group of amino acid sequences SEQ ID NOs: 1 1, 12, 13, 14, 15, 16, 17, 18, 19 and 20; or one or more proteins, comprising or consisting of an amino acid chain with an identity of> 85% to the amino acid sequence SEQ ID NO: 13 or to an amino acid sequence selected from the group of amino acid sequences with an identity of> 98% to one of the amino acid sequences SEQ ID NOs : 1 1, 12, 14, 15, 16, 17, 18, 19 or 20. or a fragment thereof as described herein.
  • the above-mentioned preferred% -dentities also apply to the above SEQ ID NOs.
  • an immunogen has a minimum of 5 amino acids.
  • an immunogen is preferably a polypeptide.
  • the terms immunogen, polypeptide and antigen are used interchangeably. Modifications and changes may be made in the structure of the immunogens of the invention which are the subject of the application and still provide a molecule having similar or improved properties as the wild-type sequence (eg, a conservative amino acid substitution). For example, certain amino acids are optionally substituted by other amino acids in a sequence without appreciable loss of immunogenic activity.
  • polypeptide sequence Because it is the interactive capacity and nature of a polypeptide that defines the biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and still obtain a polypeptide having the same or improved properties.
  • a polypeptide is used that has less or more immunogenic activity compared to the wild-type sequence.
  • the hydropathic index of amino acids is preferably considered.
  • the importance of the hydropathic amino acid index in conferring the interactive biological function on a polypeptide is well understood in the art. It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or value and still yield a polypeptide having similar biological activity. Each amino acid has been assigned a hydropathicity index based on its hydrophobicity and charge characteristics.
  • indices are for example after Kyte and Doolittle: isoleucine (+4,5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1, 9); Alanine (+1, 8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1, 3); Proline (-1, 6); Histidine (-3,2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (- 3,5); Lysine (-3.9); and arginine (-4.5).
  • the relative hydropathic character of the amino acid determines the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that one amino acid can be replaced by another amino acid with a similar hydropathy index and still give a functionally equivalent immunogen. In such changes, preference is given to the substitution of amino acids whose hydropathic indices are within ⁇ 2, more preferably within ⁇ 1, and those within ⁇ 0.5 are most preferred.
  • amino acid substitutions generally rely on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take into account various of the above properties and that are well known to those skilled in the art include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin , His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lle: Leu, Val), (Leu : lle, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: ll , Leu).
  • the embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide and immunogen as set forth above.
  • the embodiments of the polypeptides and immunogens optionally include variants having about 50%, 60%, 70%, 80%, 90% and 95% sequence identity to the polypeptide of interest.
  • the invention relates to vaccine formulations for the prevention and treatment of infections with IB80 virus.
  • the vaccine of the invention comprises recombinant and chimeric viruses of the IB80 virus.
  • the virus is attenuated.
  • inactivated vaccine formulations are prepared using conventional techniques to "kill" the viruses.
  • Inactivated vaccines are "dead” in the sense that their infectivity has been destroyed and no residual replicable virus is more detectable. Ideally, the infectivity of the virus is destroyed without affecting its immunogenicity.
  • the virus may be in cell culture or in egg System, for example in Brutei, especially in the allantois of a bird embryo (such as in particular in the allantoic chicken embryo) are grown, for example, purified by zonal ultra-centrifugation, for example by formaldehyde, ß-propiolactone, and / or other methods are inactivated and pooled.
  • the resulting vaccine can be inoculated intramuscularly, subcutaneously or intranasally.
  • Inactivated viruses are optionally formulated with a suitable adjuvant to enhance the immunological response.
  • suitable adjuvants illustratively include, but are not limited to, mineral gels, for example, aluminum hydroxide; surfactants such as lysolecithin, pluronic polyols, polyanions; peptides; Oil emulsions; and potentially useful animal or human adjuvants such as BCG and Corynebacterium parvum.
  • the present invention also relates to formulations of DNA vaccines comprising a nucleic acid or a fragment of the inventive IB virus, for example the virus of accession number 16061601 or 16070701 or nucleic acid molecules comprising one or more nucleic acid segments selected from the group consisting of Nucleic acids of the sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9; or comprising a nucleic acid of SEQ ID NO: 10; or comprising a nucleic acid comprising or consisting of a nucleic acid segment having an identity of> 85% to SEQ ID NO: 3 or having an identity of> 98% to one of SEQ ID NOs: 1, 2, 4, 5, 6, 7 , 8, 9 or 10; or a fragment thereof.
  • a nucleic acid or a fragment of the inventive IB virus for example the virus of accession number 16061601 or 16070701 or nucleic acid molecules comprising one or more nucleic acid segments selected from the group consisting of Nucleic acids of the sequences SEQ
  • the DNA vaccine formulations of the present invention comprise a nucleic acid or fragment thereof encoding antibodies that immunologically bind IB viruses.
  • a vaccine DNA comprises a viral vector as derived from the IB virus, a bacterial plasmid or other expression vector carrying an insert with a nucleic acid molecule of the present invention operably linked to one or more Control elements to thereby enable expression of the proteins encoded by the nucleic acid molecule in a vaccinated subject.
  • Such vectors can be produced by recombinant DNA technology as recombinant or chimeric viral vectors carrying a nucleic acid molecule of the present invention.
  • a nucleic acid as used herein refers to single-stranded or double-stranded molecules, optionally DNA, including nucleotide bases A, T, C and G, or RNA, including bases A, U (replaces T), C and G.
  • the nucleic acid may be a coding strand or its complement.
  • Nucleic acids are optionally sequence identical to the sequence that occurs naturally or include alternative codons that encode the same amino acid as found in the naturally occurring sequence.
  • nucleic acids optionally include codons that are conservative substitutions of amino acids, as are well known in the art.
  • isolated nucleic acid means a nucleic acid separate from or substantially free of at least some of the other components of the naturally occurring organism, for example, usually the cell structural components found associated with nucleic acids in a cellular environment and / or other nucleic acids.
  • the isolation of nucleic acids is illustratively achieved by techniques such as lysis followed by phenol and chloroform extraction followed by ethanol precipitation of the nucleic acids.
  • the nucleic acids of this invention are illustratively isolated from cells according to methods well known in the art for the isolation of nucleic acids.
  • the nucleic acid encoding the peptide or polypeptide of the present invention is part of a recombinant nucleic acid construct comprising any combination of restriction sites and / or functional elements well known in the art to facilitate molecular cloning and other recombinant DNA manipulations ,
  • the present invention further relates to a recombinant nucleic acid construct containing a nucleic acid encoding a polypeptide of this invention.
  • a cDNA version of the (recombinant) polynucleotide it may be advantageous to use a cDNA version of the (recombinant) polynucleotide. It is believed that the use of a cDNA version offers advantages in that the size of the gene can generally be substantially smaller and can be more easily used to transfect the target cell than will a genomic gene, typically up to an order of magnitude larger will be as the cDNA gene. However, the invention does not exclude the possibility that, if desired, a genomic version of a particular gene may be used.
  • engineered cells are synonymous with “host cells” and are intended to refer to a cell or cell line into which an exogenous DNA segment or gene such as a cDNA or gene, has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells that do not contain a recombinantly-introduced exogenous DNA segment or gene.
  • a host cell, or cell line is optionally a naturally occurring cell or cell line transformed or transfected with an exogenous DNA segment or gene, or a cell or cell line that is unmodified.
  • a host cell preferably does not have a naturally occurring coding spike protein gene.
  • Constructed cells or cell lines are thus cells which have a gene or genes introduced by the hand of humans.
  • Recombinant cells illustratively include those having introduced cDNA or genomic DNA or RNA, and also include genes positioned adjacent to a promoter that is not naturally associated with the particular gene introduced.
  • an expression vector containing a polynucleotide under the control of one or more promoters is prepared.
  • a coding sequence "under the control" of a promoter one positions the 5 'end of the translational initiation site of the reading frame generally between about 1 and 50 nucleotides "downstream" of (i.e., 3' of) the chosen promoter.
  • the upstream promoter stimulates transcription of the inserted DNA and promotes expression of the encoded recombinant protein. This is the meaning of "recombinant expression” as used in context herein.
  • expression vectors containing the appropriate nucleic acids and transcriptional / translational control sequences to achieve protein or peptide expression in a variety of host expression systems.
  • Available cell types for expression include, but are not limited to, bacteria such as E. coli and B. subtilis transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors.
  • Eukaryotic expression systems may also be used, such as yeasts, baculovirus and insect cells, or HEK cells. The depletion of IB80 virus
  • the IB80 virus of the present invention or variants thereof are optionally genetically engineered to have an attenuated phenotype.
  • the viruses of the invention exhibit an attenuated phenotype in a subject to which the virus is administered as a vaccine. Attenuation can be achieved by any method known to a person skilled in the art.
  • the attenuated phenotype of the viruses of the invention is effected, for example, by using a virus which, of course, does not replicate well in an intended host species, e.g., by diminished replication of the viral genome, by a reduced ability of the virus To infect the host cell or by a reduced ability of the viral proteins to mount an infectious viral particle relative to the wild-type species of the virus.
  • Attenuation Under attenuation (weaknesses, diminishing) or also virulence reduction, attenuation, in microbiology one understands the deliberate reduction of the pathogenic properties of a pathogen (virulence), while at the same time maintaining or only minimizing its ability to multiply. Attenuation also aims at maintaining the surface properties of the pathogen (epitopes), which are essential for the immune defense, and thus its immunogenicity. Therefore, attenuation is one way to make live vaccines for active immunization. The methods which can be used for this purpose are well known to the person skilled in the art.
  • the natural property of the pathogen is exploited in the beginning, although in a host that is unfavorable to it, it can initially grow small, but often does not cause any disease. This is explained, for example, in the case of viruses in that those receptors of the virus surface which allow uptake into a specific target cell are not adapted to the cells of the new host. Furthermore, in cell cultures, the genes for immune evasion and some virulence factors are unnecessary and are therefore often deleted.
  • those mutants of the pathogen are selected, which can still multiply and be replicated in a transmission to the original host (such as humans) in lesser numbers or with fewer disease symptoms. Attenuation is / can also the cultivation of the pathogen at less favorable, low Temperatures (about 25 ° C) can be used, in which the pathogens can also lose their virulence.
  • a monoclonal antibody is directed against exactly one specific epitope of an antigen.
  • animals must be immunized and then their plasma cells (from spleen or lymph nodes) recovered. Since the plasma cells have lost the capacity for cell division, a fusion with tumor cells must first take place.
  • the resulting cell hybrids are given by the plasma cells the property of producing and secreting a specific antibody and of the tumor cell's ability to divide theoretically infinitely in culture, and thus to theoretically live for an infinitely long time.
  • the present invention encompasses pharmaceutical mixtures (compositions), including antiviral agents of the present invention.
  • the antiviral agent is preferably an antibody that immunospecifically binds and neutralizes the IB80 virus or variants thereof or any proteins derived therefrom.
  • the antiviral agent is a polypeptide or nucleic acid molecule of the invention.
  • the pharmaceutical compositions are useful as antiviral prophylactic agents are illustratively administered to a subject when the subject has been exposed or is expected to be exposed to a virus.
  • Various delivery systems are known and understood by one of ordinary skill in the art to prepare them to administer the pharmaceutical composition of the invention.
  • compositions and pharmaceutical mixtures (compositions) of the invention may be administered by any suitable route, for example by infusion or bolus injection, through absorption through epithelial or mucosal linings (e.g., oral mucosa, kloakal and intestinal mucosa, etc.), optionally together with other biologically active agents , administered.
  • the administration is systemic or local. In a preferred embodiment, it is desirable to introduce the agents and pharmaceutical mixtures (compositions) of the invention into the lung by any suitable route. Pulmonary administration may also be accomplished, for example, by use of an inhaler or nebuliser, and formulation with an aerosolizing agent.
  • the administration of the drinking water or the administration by means of spray are preferred, and are particularly important for IB viruses, e.g. the viruses of the invention (IB80), is of importance.
  • IB80 the viruses of the invention
  • eye-drop application of the vaccine in which each animal is individually supplemented with a drop of the agent and pharmaceutical mixtures (compositions) of the invention , The solution is dropped into the eye.
  • the present invention also relates to a method for detecting an antibody which binds immunospecifically to the IB80 virus of the invention, for example, in a biological sample, including, for example, blood, serum, plasma, saliva, urate, stool, etc., from a subject present under one IBV infection suffers.
  • a sample for example, with the IB80 virus or a genomic nucleic acid sequence according to the invention is immobilized in contact directly on a substrate and the virus-bound antibody detected directly or indirectly by suitable methods.
  • Detection methods are well known to those skilled in the art, and include, for example Hybridization methods, labeling methods, detection probes,
  • Immunofluorescence assay It may be in vitro or in v / Vo methods.
  • the invention also relates to a method for the identification of agents or substances which inhibit the ability of the IBV virus, in particular the IB80, to infect a host or a host cell.
  • the invention relates to methods of identifying agents or substances that inhibit the ability of the IBV virus, particularly IB80, to replicate in a host or host cell. Any technique known to those skilled in the art can be used for this purpose.
  • the invention relates to methods for identifying agents or substances that replicate the ability of the IBV virus, particularly the IB80, to replicate in a species of bird, or more specifically, in poultry, eg, chickens. More particularly, the invention relates to methods of identifying agents or substances that inhibit the ability of the IBV virus, particularly the IB80, to infect a species of bird or, in particular, poultry, such as chickens. In certain embodiments, the invention relates to methods of identifying agents or substances that inhibit the ability of the IBV virus, particularly the IB80, to replicate in host cells, cell lines and / or egg systems, for example in hatching eggs.
  • the material used for virus isolation comes from the monitoring of a clinically normal chicken flock at that time.
  • a pool of caecaline salmon was used for cultural cultivation.
  • This organ material was mixed in 15 ml tubes with 1x PBS, pH 7-7.4 with 50 ⁇ g / ml gentamycin and 2.5 ⁇ g / ml amphotericin B, in the ratio 1:10.
  • the sample was homogenized in FastPrep-24 (MP Biomedicals) for 30 sec at room temperature. To clarify the organ material this was then centrifuged at 2,000 x g and 4 ° C for 20 minutes and the supernatant filtered with a 0.45 ⁇ filter. This was followed by incubation of the filtrate at room temperature for 30 minutes.
  • the virus replication was carried out by direct passage of the filtrate of the reconditioned cecal tonsils in SPF chicken eggs (VALO BioMedia). For this purpose, 200 ⁇ of this material were injected into the allantoic cavity (AH) of eggs (gross 10) with a cannula (0.5 ⁇ 16 mm) in the sterile workbench (clean room class A). Then the opening in the egg was closed with Uhu Alleskleber. Incubation was for 7 days at 37-38 ° C and a relative humidity of about 60%.
  • the inoculated eggs were regularly sheared on Absterber. After the 7 days, the eggs were chilled for 4 hours at 4 ° C. Subsequently, the eggs were opened with sterile scissors in the area of the air bubble and the harvest of the allantoic fluid by means of a transfer pipette. The harvest of the inoculated eggs was pooled.
  • the harvest was stored at -20 ° C.
  • the first passage crop was sterilized under sterile conditions with 1: 100 1xPBS, pH 7-7.4, and re-seeded at 200 ⁇ per egg into the allantoic cavity of SPF hatching eggs. These were sealed with Uhu Alleskleber and incubated for 4 days at 37 ° C and a relative humidity of about 50% for 4 days and shaved daily. A mortal within the first 24 hours after inoculation was rejected as nonspecific. The remaining eggs were chilled on day 4 for about 4 hours at 4 ° C and then their allantoic fluid was harvested as a pool under sterile conditions.
  • the allantoic fluid was again diluted 1: 100 with 1xPBS as inoculated into the allantoic cavity of SPF hatching eggs and incubated for 4 days. A mortal within the first 24 hours after inoculation was again discarded as nonspecific. The remaining eggs were chilled on day 4 for about 4 hours at 4 ° C and then their allantoic fluid was harvested as a pool under sterile conditions and frozen in 1-2 ml aliquots at -80 ° C.
  • RNA purification from the harvested allantoic fluid of a sample was carried out with the QIAamp Viral RNA Mini Kit® (Qiagen) according to the manufacturer's instructions. Subsequently, the IBV status of the samples was checked by means of a real-time PCR using the Kylt® IB Virus Kit (Kylt) according to the manufacturer's instructions. The IBV score was positive at a CT of 13.86.
  • the sequencing of the genome of the IB virus isolate was carried out according to the dideoxymethod of Sanger (Sanger and Coulsen, 1977) and was carried out by means of a 2-step RT-PCR protocol:
  • Reverse transcription was performed using the GoScript TM Reverse Transcription System (Promega) as recommended by the manufacturer. In this case, the reverse transcription on the one hand with the random primers contained in the kit (cDNA A) and on the other with 14 IBV-specific primers (cDNA B, Franzo et al., 2015, modified, see Table 1) performed (see Table 1). For the reverse transcription, 20 pmol of the primer mix (1.43 pmol per primer) were used for a reaction batch of 20 .mu. ⁇ .
  • the final protocol for the 20 ⁇ assay is composed as follows: 4 ⁇ of RNA purification of the sample, 1 ⁇ primer mix (random primer or IBV-specific primer mix), 4 ⁇ GoScript TM 5X Reaction Buffer, 2 , 4 ⁇ M 25 mM MgCl 2 , 1 ⁇ M PCR Nucleotide Mix, 0.5 ⁇ M Recombinant RNasin® Ribonuclease Inhibitor, 1 ⁇ M GoScript TM Reverse Transcriptase, 6, 1 ⁇ M nuclease-free water.
  • the temperature profile corresponded to the specifications of the manufacturer.
  • the protocol for the 20 ⁇ approach is composed as follows: 2 ⁇ cDNA, 2 ⁇ 10X Buffer for KOD Hot Start DNA Polymerase®, 1, 2 ⁇ M 25 mM MgS0 4 , 2 ⁇ _ dNTPs (2 mM each), 0.8 iL Forward Primer (10 ⁇ ), 0.8 ⁇ Reverse Primer (10 ⁇ ), 0.4 ⁇ KOD Hot Start DNA Polymerase® (1U / ⁇ ), 10.8 ⁇ nuclease-free water. After initial denaturation for 2 min at 95 ° C, 40 cycles were performed at 95 ° C for 20 sec, 60 ° C for 10 sec, and 70 ° C for 40 sec each (deviations for certain primer pairs [1-14]) are tabulated 3).
  • the purification of the PCR products was carried out using the QIAquick PCR Purification Kit® (Qiagen) according to the manufacturer's instructions.
  • the DNA concentration of the purified PCR products was determined using the NanoDrop® measurement principle and a NanoDrop® 2000c spectrophotometer.
  • the sequencing reaction was performed by MWG Eurofins (Ebersbach, Germany) using the Mix2Seq Kit according to the manufacturer's instructions.
  • Each PCR product was sequenced with the PCR primers (forward and reverse) and usually with an additional primer located further in the middle of the product. For PCR product 10, no additional intermediate primer was needed for sequencing. For PCR products 2 and 11, two internal primers were used for sequencing (see Table 5).
  • the evaluation of the sequencing was carried out with the software DNASTAR Lasergene. First, the individual chromatograms were checked and tailored. The individual fragments were then aligned to a reference sequence to assemble the entire genome sequence.
  • antigens eg allantoic fluid, egg skin, embryo homogenate, cell culture material can each be loaded with IB80 (or preferably IB80) - Particles)
  • Pipette 100 [iL into each well of the plate; cover with adhesive foil (also with all following steps!); Keep plates at 30 ° C for 2 hours.
  • Block Add 200 [iL blocking solution to each well; Keep plates at 30 ° C for 2 hours.
  • Evaluation The evaluation is carried out in relation to a test series which has been treated in exactly the same way as the antigen-loaded test series in which, however, an antigen is not contained (control). If a higher antibody binding is detected in the antigen-loaded samples, immunospecificity of the antibody tested is present. Used solutions:

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Abstract

L'invention concerne des agents, des compositions, des utilisations et des procédés ciblant une nouvelle variante du virus de la bronchite infectieuse (virus de la BI).
EP17755136.3A 2016-08-12 2017-08-14 Variantes du virus de la bronchite infectieuse (bi) et compositions, utilisations et procédés les concernant Withdrawn EP3497206A1 (fr)

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