WO2015094001A1 - Viral peptides - Google Patents

Viral peptides Download PDF

Info

Publication number
WO2015094001A1
WO2015094001A1 PCT/PT2014/000077 PT2014000077W WO2015094001A1 WO 2015094001 A1 WO2015094001 A1 WO 2015094001A1 PT 2014000077 W PT2014000077 W PT 2014000077W WO 2015094001 A1 WO2015094001 A1 WO 2015094001A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
eiav
nev
animal
peptides
Prior art date
Application number
PCT/PT2014/000077
Other languages
French (fr)
Inventor
Isabel Maria Fidalgo dos Santos Silva CARVALHO
Alexandre Simão Vieira PIRES
Original Assignee
Equigerminal Sa
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Equigerminal Sa filed Critical Equigerminal Sa
Priority to GB1515545.0A priority Critical patent/GB2526222B/en
Priority to US15/104,249 priority patent/US20160367659A1/en
Priority to EP14824566.5A priority patent/EP3083659B1/en
Publication of WO2015094001A1 publication Critical patent/WO2015094001A1/en

Links

Classifications

    • 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
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. human immunodeficiency virus [HIV], visna-maedi virus or equine infectious anaemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • Various embodiments of the present invention generally relate to biotechnology. Further, various embodiments relate to veterinary medicine. In particular, the invention relates to antigenic peptides of equine viruses and uses thereof.
  • Equine infectious anemia or swamp fever is an ancient lentiviral disease of Equids.
  • the Swamp fever or equine infectious anemia is the only retroviral disease known to affect Equidae.
  • the EIAV Equine infectious anemia virus
  • the EIAV Equine infectious anemia virus retrovirus is known, historically, to be the first viral agent responsible for an animal disease, the Swamp fever in horses.
  • EIAV infections have been reported in the United Kingdom (UK), France, Belgium, Romania, Slovenia, Austria and Germany, but very little is know about the EIAV sequences responsible for these infections.
  • the full length EIAV genome of European strains is restricted to Ireland strains isolated from a 2006 outbreak (Quinlivan M et al., 2013). Nevertheless, even in the continents where the disease is prevalent, reports of EIAV in naturally infected horses or viral sequence variations among primary isolates are limited.
  • Recently some EIAV isolates from Pennsylvania, US and Japan were recovered by 'in vivo' viral transfer experiments between EIAV seropositive and naive horses. The retrieved viral sequences showed more diverse and divergent EIAV strains.
  • the identified EIAV strains were detected in horses determined as seropositive by the Coggins test (also known as the AGID or Agar Gel Diffusion test). The AGID test detects antibodies against the major core protein of EIAV (p26).
  • the 01 E official test to diagnose the presence of EIA has been the presence of antibodies specific for the disease in the serum of affected animals using the Coggins or agar gel diffusion test (AGID) (described in U.S. Patent No. 3.929.982 and U.S. Patent No.3.932.601).
  • Fidalgo-Carvalho (2008; PhD thesis, University of Porto, Portugal) identified 33 horses that tested positive for EIAV in the immunoblot test, but that tested negative in the Coggins test and commercial ELISAs directed to the EIAV core protein.
  • the immunoblot test uses gradient purified EIAV viral particles obtained from cell culture supernatants of equine cell lines infected with EIAV PV . a biological EIAV isolate. In the immunoblot test linear epitopes of all EIAV viral proteins, gag derived (p26, p15 and p1 1), envelope proteins (gp90 and gp45), and accessory proteins can be detected by specific sera antibodies.
  • the present invention is based on the use of peptides for therapeutic and/or diagnostics purposes of an EIAV and/or NEV infection of horses.
  • the inventors used viral peptides (identified by mass spectrometry) from EIAV seropositive horses.
  • the viral peptides were obtained from viral supernatants which originated from macrophage cell cultures isolated from EIAV seropositive horses.
  • EIAV seropositive horses were identified by immunoblot techniques, previously these horses were considered to be EIAV negative in commercially available ELISAs and in the Coggins test.
  • the present invention provides peptide-based immunodiagnostics techniques with the similar sensitivity and specificity of immunoblot techniques.
  • the invention provides tools for the detection of divergent EIAV and/or NEV strains or subtypes.
  • the diagnostic methods of the present invention are capable of identifying EIAV and/or NEV infection in animals which current diagnostic methods do not detect an infection.
  • the present invention provides a composition comprising one or more peptides selected from the group consisting of:
  • SRLLESRGPTTSEK (SEQ ID NO: 1);
  • NAPLLSKVSP (SEQ ID NO: 6);
  • MAQCIVNR SEQ ID NO: 7
  • KPNVEGRYGLSRSETNK SEQ ID NO: 8
  • QQGPEAPLPSLQVAEVPK SEQ ID NO: 9
  • PHAGSTQTEWPK SEQ ID NO: 10
  • AKLEESFPGK (SEQ ID NO 26);
  • NGSLAGESIIIR (SEQ ID NO 27);
  • NITFNSSAGGDLEIT SEQ ID NO 28
  • FGNKTTIIFTK SEQ ID NO 33
  • VNVSVTNNNTTTNV SEQ ID NO 35
  • TLQYLALTALVTPK SEQ ID NO 39
  • PTTPVTPAPGVGEISKELAQGK SEQ ID NO 40
  • WSSALQYLIPR SEQ ID NO 41
  • PVWAEPVK SEQ ID NO 42
  • FPSIVGRPR (SEQ ID NO 48);
  • VAPEEHPVLLTEAPLNPK (SEQ ID NO 52);
  • AVFPSIVGRPR (SEQ ID NO 53);
  • AVFPSIVGR (SEQ ID NO 55);
  • RGILTLK (SEQ ID NO 58);
  • WGIAHTTGIPGNSQGQAMVER SEQ ID NO 59
  • HLVDQLIRDLK SEQ ID NO 60
  • TTISFSK (SEQ ID NO 66);
  • DIVLLENGK (SEQ ID NO 71);
  • NRLEILK (SEQ ID NO 74); ADDVAVLQ DALG R (SEQ ID NO 75);
  • variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76; and/or comprising one or more polynucleotide sequences encoding one or more of the peptides;
  • AGCAGGCTGCTGGAGAGCAGGGGCCCCACCACCAGCGAGAAG SEQ ID NO 77;
  • CAGCCCAGCCTGCCCACCGGCAGCGAGGAGCTGAAG SEQ ID NO 93
  • GAGAAGATCGAGCAGCTGAGG (SEQ ID NO 96);
  • AACGGCAGCCTGGCCGGCGAGAGCATCATCATCAGG (SEQ ID NO 102);
  • GCCGTGATCCTGCTGCTGGACAGGCTGAGG SEQ ID NO 104
  • CTGTTCGTGAGCGTGCTGCAGAGG SEQ ID NO 120
  • GCCGCCGAGCAGAAGGCCAGCCCCCCCAGCCTGACCCCCAAG SEQ ID NO 121
  • ATGACCCAGATCATGTTCGAGACCTTC (SEQ ID NO 125); GAGGAGGAGGTGGCCGCCCTGGTGATCGACAACGGCAGCGGCATGTGCAAG (SEQ ID NO 126);
  • CAGACCATCATCCCCACCAACAAGGACGTGGACGAGAAG SEQ ID NO 139
  • AAGAACTACGGCAAGCTGGACAAG SEQ ID NO 140
  • CTGCCCCCCCTGAGCATCCTGAAG (SEQ ID NO 147);
  • ATCTTCCTGATCAACCTGGCCTTCCTGATCAAG (SEQ ID NO 148);
  • AACAGGCTGGAGATCCTGAAG (SEQ ID NO 149);
  • the present invention provides a vector capable of encoding one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
  • the present invention provides a vector comprising one or more polynucleotide sequences encoding one or more of the peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76;
  • polynucleotide sequences selected from the group consisting of SEQ ID NOs 77 to 152 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
  • the present invention provides an antibody capable of binding to one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
  • the present invention provides in another aspect, a pharmaceutical composition
  • a pharmaceutical composition comprising: one or more peptides according to the present invention.
  • the present invention provides a composition according to the present invention, and/or a vector according to the present invention, and/or antibody according to the present invention, and/or a pharmaceutical composition according to the present invention for use in the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV and/or NEV in an animal.
  • the present invention provides, in a further aspect, a kit comprising:
  • composition according to the present invention and/or a vector according to the present invention; and/or
  • the present invention provides, in another aspect, a diagnostic method comprising obtaining a sample from an animal and determining the presence or absence in said sample of antibodies which are capable of binding to one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76
  • the present invention provides a kit comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76 and optionally instructions for determining the presence or absence in an animal sample of antibodies capable of binding to one or more said peptides.
  • the present invention provides, in a further aspect, a kit comprising one or more antibodies according to the present invention and optionally instructions for determining the presence or absence in an animal sample of peptides capable of binding to said antibodies.
  • an oligonucleotide sequence (e.g. a primer or a probe) comprising or consisting of a polynucleotide sequence capable of encoding one or more of the peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76
  • polynucleotide sequences selected from the group consisting of SEQ ID NOs: 77 to 152 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
  • the present invention provides a kit comprising one or more oligonucleotide sequences according to the present invention and optionally instructions for determining the presence or absence in an animal sample of said polynucleotide sequence.
  • the present invention provides a method for controlling EIAV and/or NEV disease in a group of animals comprising the identification of EIAV and/or NEV infection in an animal using the diagnostic method according to the present invention.
  • the present invention provides a method for controlling EIAV and/or NEV disease in a group of animals comprising the identification of EIAV and/or NEV infection in an animal using the diagnostic method according to the present invention and the isolation of an EIAV and/or NEV infected animal from other animals.
  • the present invention provides, in another aspect, the use of a composition according to the present invention, and/or a vector according to the present invention, and/or an antibody according to the present invention, and/or a pharmaceutical composition according to the present invention, for the manufacture of a medicament for the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV and/or NEV in an animal.
  • the present invention provides a method for the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV and/or NEV in an animal, wherein said method comprising administering to an animal one or more compositions according to the present invention, and/or one or more vectors according to the present invention, and/or one or more antibodies according to the present invention, and/or one or more pharmaceutical compositions according to the present invention.
  • FIG. 1 Immunoblot determining equine infectious anemia (EIAV) seropositiveness.
  • Figure 2 NEV cell lines established from AGID negative horses showed positive reverse transcriptase activity, cytopathic effects and syncytia cell formation.
  • Fig.2.A1 and 2.A2 - NEV.MaA Macrophage-like cells 2,3 weeks after culturing show Syncytia Cell Formation. Syncytia are indicated by arrows.
  • Fig. 2.B NEV.MaA Macrophage-like cell after passaging (cell line).
  • Fig. 2.C NEV.MaA. VpC: Macrophage-like cell line 5 days after infection with NEV viral particles C.
  • Fig.2.D1 Reverse transcriptase (RT) activity in viral supernatants NEV.MaA cells infected with NEV viral particles 5 days after infection. RT activity was measured by EnZCheck RT assay. Fig.2.D2 shows standard curve obtained with EnZCheck Assay for lambda DNA.
  • Figure 3A NEV purified viral particles: viral RNA.
  • Figure 3B NEV purified viral particles: cDNA and viral proteins.
  • FIG. 4A Cytopathic effects (CPE) and viability of infected cells.
  • MaC and ED mock cells Fig. 4A
  • MaC or ED cells infected with NEV virus Fig. 4B
  • ED cells infected with EIAV-Wyoming Fig. 4C
  • Presto blue cell viability assays of infected ED cells at day 11 post infection Fig. 4D).
  • FIG. 1 Dose response curves for mab#4G6E4.
  • B Dose responses curve and inhibition rates of NEV seronegative and seropositive samples.
  • C Representative inhibition rates (%l) of NEV seropositive and one seronegative. Inhibition rates were obtained for a SEQ ID NO: 159 peptide concentration of 96 ng/mL of peptide and mab#4G6E4 IgG concentration of 70ng/mL
  • EIAV equine infectious anaemia virus
  • EIAV causes equine infectious anaemia (EIA) in equines.
  • Equine infectious anaemia is also known as swamp fever in horses.
  • EIAV can infect donkeys (Equus asinus) (Cook et al., 2001) and mules (Spyrou et al., 2003).
  • EIAV infected horses can present three different disease states during infection: acute/sub-acute, chronic and inapparent.
  • EIAV is transmitted by bloodsucking insects.
  • the virus (EIAV) is endemic in the Americas, parts of Europe, the Middle and Far East, Russia, and South Africa.
  • the virus is a lentivirus.
  • EIAV can be transmitted through blood, saliva, milk, and body secretions. Transmission is primarily through biting flies, such as the horse-fly and deer-fly. The virus can survive up to 4 hours in the carrier. Contaminated surgical equipment and recycled needles and syringes, and bits can transmit EIAV. Further, mares can transmit EIAV to their foals via the placenta. The risk of transmitting the disease is greatest when an infected horse is ill, as the blood levels of the virus are then high.
  • the EIA incubation period lasts usually one to three weeks, but may be as long as three months.
  • EIA acute form of EIA is a sudden onset of the disease at full-force.
  • Clinical signs include high fever, anemia (due to the breakdown of red blood cells), thrombocytopenia, weakness, swelling of the lower abdomen and legs, weak pulse, irregular heartbeat, tachypneia, petechiae on the mucous membrane, diarrhoea and blood stained feces.
  • Thrombocytopenia is a consistent hematological finding and one of the earliest hematological abnormalities detected in acutely infected horses (Clabough et al., 1991 ; Crawford et al., 1996).
  • Neurological signs are also reported in EIA infected horses (Oaks et al., 2004). Occasionally, death occurs during the acute infection, and the equine may die suddenly. After the initial bout, the majority of the horses may become asymptomatic.
  • the subacute form of EIA is a slower, less severe progression of the disease. Symptoms include recurrent fever, weight loss, an enlarged spleen (felt during a rectal examination), anemia, and swelling of the lower chest, abdominal wall, penile sheath, scrotum, and legs.
  • EIA chronic recurring EIA signs that vary from mild illness and failure to thrive to fever, depression, petechial hemorrhages on the mucous membranes, weight loss, edema, and sometimes death.
  • the chronic form of EIA is where an equine tires easily and is unsuitable for work.
  • the equine may have a recurrent fever and anemia; the equine may relapse to the subacute or acute form even several years after the original attack.
  • the majority of infected horses become life long inapparent carriers with no overt clinical abnormalities as a result of infection (Coggins, 1984; Leroux et al., 2004; McGuire et al., 990), yet still tests positive for EIA antibodies. Such an equine can still pass on the virus.
  • EIA may cause abortion in pregnant mares. This may occur at any time during the pregnancy if there is a relapse when the virus enters the blood. Most infected mares will abort, however some give birth to healthy foals. The foals are not necessarily infected.
  • the present inventors have discovered a new and previously uncharacterised virus, which was obtained from horses with discordant results for EIAV testing i.e. the horses were positive for EIAV in an immunoblot but negative for EIAV in both the AGID test and an ELISA.
  • NEV Newcastle disease virus New Equine Virus
  • ECAAC European Collection of Cell Cultures
  • Other workers in the field may refer to NEV as EIAV or EIAV-like.
  • the present inventors have identified a number of NEV peptides which find use in the present invention.
  • the NEV virus as described herein may comprise or contain any of SEQ ID NOS: 1- 76.
  • the NEV virus as described herein may comprise or contain any of SEQ ID NOS: 77-
  • NEV peptides of the present invention examples include.
  • SRLLESRGPTTSEK (SEQ ID NO: 1);
  • STFPPTPV SEQ ID NO: 5
  • NAPLLSKVSP (SEQ ID NO: 6);
  • MAQCIVNR SEQ ID NO: 7
  • EALDKIEEIQNKNKQK (SEQ ID NO 18); PPIPVGGIYK (SEQ ID NO 19);
  • AKLEESFPGK (SEQ ID NO 26);
  • NGSLAGESIIIR (SEQ ID NO 27);
  • NITFNSSAGGDLEIT SEQ ID NO 28
  • FGNKTTIIFTK SEQ ID NO 33
  • VNVSVTNNNTTTNV SEQ ID NO 35
  • PTTPVTPAPGVGEISKELAQGK SEQ ID NO 40
  • WSSALQYLIPR SEQ ID NO 41
  • PVWAEPVK SEQ ID NO 42
  • FPSIVGRPR (SEQ ID NO 48);
  • VAPEEHPVLLTEAPLNPK (SEQ ID NO 52);
  • AVFPSIVGRPR (SEQ ID NO 53); YPIEHGIVTNWDDMEK (SEQ ID NO 54);
  • AVFPSIVGR (SEQ ID NO 55);
  • RGILTLK (SEQ ID NO 58);
  • HLVDQLIRDLK (SEQ ID NO 60);
  • TTISFSK (SEQ ID NO 66);
  • DIVLLENGK (SEQ ID NO 71);
  • NRLEILK (SEQ ID NO 74);
  • ADDVAVLQDALGR (SEQ ID NO 75);
  • variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
  • NEV peptides of the present invention are selected from the group consisting of SEQ ID NOs 1 to 14.
  • nucleotide sequences encoding NEV peptides of the present invention include polynucleotide sequences encoding:
  • SRLLESRGPTTSEK (SEQ ID NO: 1);
  • NAPLLSKVSP (SEQ ID NO: 6);
  • MAQCIVNR SEQ ID NO: 7
  • KPNVEGRYGLSRSETNK SEQ ID NO: 8
  • QQGPEAPLPSLQVAEVPK SEQ ID NO: 9
  • PHAGSTQTEWPK SEQ ID NO: 10
  • AKLEESFPGK (SEQ ID NO 26);
  • NGSLAGESIIIR (SEQ ID NO 27);
  • NITFNSSAGGDLEIT SEQ ID NO 28
  • FGNKTTIIFTK SEQ ID NO 33
  • VNVSVTNNNTTTNV SEQ ID NO 35
  • TLQYLALTALVTPK SEQ ID NO 39
  • PTTPVTPAPGVGEISKELAQGK SEQ ID NO 40
  • WSSALQYLIPR SEQ ID NO 41
  • PVWAEPVK SEQ ID NO 42
  • FPSIVGRPR (SEQ ID NO 48);
  • VAPEEHPVLLTEAPLNPK (SEQ ID NO 52);
  • AVFPSIVGRPR (SEQ ID NO 53);
  • AVFPSIVGR (SEQ ID NO 55);
  • RGILTLK (SEQ ID NO 58);
  • WGIAHTTGIPGNSQGQAMVER SEQ ID NO 59
  • HLVDQLIRDLK SEQ ID NO 60
  • TTISFSK (SEQ ID NO 66);
  • DIVLLENGK (SEQ ID NO 71);
  • NRLEILK (SEQ ID NO 74); ADDVAVLQDALGR (SEQ ID NO 75);
  • variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
  • nucleotide sequences encoding NEV peptides of the present invention are selected from the group consisting of polynucleotide sequences encoding any one of SEQ ID NOs 1 to 14.
  • nucleotide sequences encoding NEV peptides of the present invention include:
  • AGCAGGCTGCTGGAGAGCAGGGGCCCCACCACCAGCGAGAAG SEQ ID NO 77;
  • CAGCCCAGCCTGCCCACCGGCAGCGAGGAGCTGAAG SEQ ID NO 93
  • GAGAAGATCGAGCAGCTGAGG (SEQ ID NO 96);
  • ATCGTGGAGCTGCTGGGCAGG (SEQ ID NO 98);
  • AACGGCAGCCTGGCCGGCGAGAGCATCATCATCAGG (SEQ ID NO 102);
  • GCCGTGATCCTGCTGCTGGACAGGCTGAGG SEQ ID NO 104
  • GAGAGCTTCCCCAACAAG (SEQ ID NO 107);
  • CTGTTCGTGAGCGTGCTGCAGAGG SEQ ID NO 120
  • GCCGCCGAGCAGAAGGCCAGCCCCCCCAGCCTGACCCCCAAG SEQ ID NO 121
  • CCCCTG AG CCTG CCCCTG AAG ATC (SEQ ID NO 122); TTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID NO 123);
  • ATGACCCAGATCATGTTCGAGACCTTC (SEQ ID NO 125);
  • G ACAGCTACGTG GG CG ACG AG G CCCAG AG CAAG AG G (SEQ ID NO 132);
  • CAGACCATCATCCCCACCAACAAGGACGTGGACGAGAAG SEQ ID NO 139
  • AAGAACTACGGCAAGCTGGACAAG SEQ ID NO 140
  • CTGCCCCCCCTGAGCATCCTGAAG (SEQ ID NO 147);
  • ATCTTCCTGATCAACCTGGCCTTCCTGATCAAG (SEQ ID NO 148);
  • AACAGGCTGGAGATCCTGAAG (SEQ ID NO 149);
  • GCCGACGACGTGGCCGTGCTGCAGGACGCCCTGGGCAGG (SEQ ID NO 150); CTGAACAAGAGCCTGGAGCAGCTGAGG (SEQ ID NO 151); and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
  • nucleotide sequences encoding NEV peptides of the present invention are selected from the group consisting of SEQ ID NOs 77 to 89.
  • NEV a novel virus
  • the peptides of the invention and/or the novel viral strain or variant as described herein may be denoted as "EIAV-like".
  • EIAV-like it is meant that the virus and/or peptides were derived from horses with discordant results obtained from EIAV testing i.e. the horses were positive using an EIAV immunoblot but negative using the AGID test and an ELISA. None of the identified peptides belonged to the known EIAV proteome. Some of the peptides were found to have sequence identity with retroviral peptides. Some of the peptides were found to have sequence identity to part of the HIV-1 proteome.
  • Table 2 shows the viral proteins which have the highest sequence identity (similarity) to SEQ ID NOs 1-76 of the present invention.
  • Both EIAV and NEV can infect animals such as equines.
  • the animal is an equid.
  • Examples of equids include horses, donkeys, mules, hinnys and zebras.
  • the animal is a horse.
  • a peptide according to the present invention, and/or a nucleotide sequence according to the present invention, and/or a vector according to the present invention, and/or an antibody according to the present invention, and/or a pharmaceutical composition according to the present invention is capable of inducing an immune response against EIAV and/or NEV in an animal.
  • a peptide according to the present invention, and/or a nucleotide sequence according to the present invention, and/or a vector according to the present invention, and/or an antibody according to the present invention, and/or a pharmaceutical composition according to the present invention is capable of inducing a protective immune response against EIAV and/or NEV in an animal.
  • treatment of equine infectious anaemia refers to a reduction in EIA symptoms in the treated animal.
  • the animal may no longer be classified as having an acute form of EIA but now has the subacute form, or the chronic form, or the inapparent form and no longer shows any EIA symptoms - the virus may, however, still be detected in the animal but at levels below, for example, 10 5 copies of EIAV RNA/mL plasma.
  • the EIAV e.g. the EIAV of the present description
  • the viral titre in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the EIAV viral titre of the animal before treatment.
  • thrombocytopenia in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the thrombocytopenia of the animal before treatment.
  • the peaks of the fever in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the temperature of the animal before treatment.
  • the treatment of equine infectious anaemia occurs by a reduction in the level of an EAIV virus in an animal.
  • treatment of NEV refers to a reduction in NEV symptoms in the treated animal.
  • the animal may no longer be classified as having an acute form of NEV but now has the subacute form, or the chronic form, or the inapparent form and no longer shows any NEV symptoms - the virus may, however, still be detected in the animal but at levels below, for example, 10 s copies of NEV RNA/mL plasma.
  • the NEV e.g. the NEV of the present description
  • the NEV viral titre in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the NEV viral titre of the animal before treatment.
  • thrombocytopenia in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the thrombocytopenia of the animal before treatment.
  • the peaks of the fever in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the temperature of the animal before treatment.
  • the treatment of NEV occurs by a reduction in the level of an NEV virus in an animal.
  • the viral titre may be determined in a sample from an animal such as a blood sample, a blood serum sample, a plasma sample, a saliva sample, a sputum sample, a urine sample, a semen sample, a fecal sample, a milk sample, a biopsy sample, a lymph node biopsy sample, and/or a sweat sample.
  • a sample from an animal such as a blood sample, a blood serum sample, a plasma sample, a saliva sample, a sputum sample, a urine sample, a semen sample, a fecal sample, a milk sample, a biopsy sample, a lymph node biopsy sample, and/or a sweat sample.
  • prevention of equine infectious anaemia refers to a treated animal not developing EIA symptoms if the treated animal is exposed to the equine infectious anaemia virus, for example by exposure to a bloodsucking insect carrying the virus.
  • the treated animal did not have any EIA symptoms before treatment and/or did not have any detectable EIAV in a sample such as a blood sample before treatment.
  • commercially available tests such as the Coggins test and/or the diagnostic test of the present invention may be used to determine (i.e. detect) if there is an EIAV titre in an animal.
  • prevention of NEV refers to a treated animal not developing NEV symptoms if the treated animal is exposed to the NEV virus, for example by exposure to a bloodsucking insect carrying the virus. The treated animal did not have any NEV symptoms before treatment and/or did not have any detectable NEV in a sample such as a blood sample before treatment.
  • treatment of an infection by an EIAV refers to a reduction in EIAV (e.g. the EIAV of the present description) viral titre in a sample from the treated animal when compared to the EIAV viral titre of the animal before treatment. For instance, after treatment, the EIAV (e.g.
  • viral titre in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the viral titre of the animal before treatment.
  • a reduction in the level of an EIAV virus in an animal may prevent the development of EIA symptoms.
  • treatment of an infection by a NEV refers to a reduction in NEV (e.g. the NEV of the present description) viral titre in a sample from the treated animal when compared to the NEV viral titre of the animal before treatment.
  • NEV e.g. the NEV of the present description
  • the NEV (e.g. the NEV of the present description) viral titre in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the viral titre of the animal before treatment.
  • a reduction in the level of an NEV virus in an animal may prevent the development of NEV symptoms.
  • prevention of an infection by an EIAV refers to a treated animal not developing a detectable EIAV titre if the treated animal is exposed to the equine infectious anaemia virus, for example by exposure to a bloodsucking insect carrying the virus.
  • the treated animal did not have any detectable EIAV in a sample such as a blood sample before treatment.
  • commercially available tests such as the Coggins test and/or the diagnostic test of the present invention may be used to determine (i.e. detect) if there is a EIAV titre in an animal.
  • prevention of an infection by a NEV refers to a treated animal not developing a detectable NEV titre if the treated animal is exposed to the NEV virus, for example by exposure to a bloodsucking insect carrying the virus. The treated animal did not have any detectable NEV in a sample such as a blood sample before treatment.
  • animals which are infected with EIAV and/or NEV can be identified.
  • Animals with an EIAV and/or NEV infection may be isolated from other animals (e.g. animals which do not have the EIAV and/or NEV virus).
  • animals e.g. animals which do not have the EIAV and/or NEV virus.
  • this helps to prevent the spread of EIAV and/or NEV infection from infected animals to those which are not infected thereby controlling EIAV/NEV infection and/or EIAV/NEV within a group of animals.
  • Animals with an EIAV and/or NEV infection may be monitored (by using a kit according to the present invention or by using a diagnostic method of the present invention) to determine the progression of the EIAV/NEV infection and/or determine the progression of EIAV/NEV.
  • Animals with an EIAV and/or NEV infection may be isolated from other animals (e.g. animals which do not have the EIAV and/or NEV virus) once the level of infection and/or the progression of EIAV and/or NEV has reached a critical point.
  • animals should be isolated during febrile episodes when rectal temperatures are above 39°C, platelets levels are below 105000/ ⁇ of blood and viremia is at least of 10 5 copies of EIAV and/or NEV RNA mL plasma.
  • care can be taken to ensure that medical equipment used on an EIAV and/or NEV infected animal is not used on an animal which does not have a EIAV and/or NEV infection.
  • this helps to prevent the spread of EIAV and/or NEV infection from infected animals to those which are not infected thereby controlling EIAV and/or NEV disease with a group of animals.
  • an animal identified as having EIAV and/or NEV is euthanized.
  • animals which are euthanized are those with frequent febrile episodes and animals which are lethargic or in lateral recumbence.
  • An animal having EIAV and/or NEV may be euthanized when the viremia peaks are frequent.
  • the present invention encompasses the use of NEV peptides in the discrimination of NEV- infected animals, and in the diagnosis, treatment and prevention of NEV infection in NEV- infected animals.
  • the present invention encompasses the use of NEV peptides in the discrimination of EIAV-infected animals, and in the diagnosis, treatment and prevention of EIAV infection in EIAV-infected animals.
  • the present inventors have identified EIAV reference sera (from horses) that are positive for an NEV fusion peptide of the invention.
  • NEV virus described herein has been deposited by Equigerminal SA, Parque tecnologico de Cantanhede, nucleo 4 lote 4, Cantanhede, 3060-197 Portugal under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure at European Collection of Cell Cultures (ECAAC), Culture Collections, Public Health England, Porton Down, Salisbury, Wiltshire UK SP4 0JG on 2 December 2014 under accession number 14120201 , Other aspects of the present invention relate to the above vira! deposit made at the ECAAC depository under accession number 14120201.
  • the present invention also relates to peptide and nucleotide sequences obtainable from this deposit.
  • the present invention also relates to nucleotide sequences capable of hybridising to nucleotide sequences obtainable from the deposit.
  • the NEV virus of the deposit may also be referred to as EIAV-like in the deposit.
  • a vector is a tool that allows or facilitates the transfer of an entity from one environment to another.
  • some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a host and/or a target cell for the purpose of replicating the vectors comprising the nucleotide sequences of the present invention and/or expressing the proteins of the invention encoded by the nucleotide sequences of the present invention.
  • vectors used in recombinant DNA techniques include but are not limited to plasmids, chromosomes, artificial chromosomes or viruses.
  • vector includes expression vectors and/or transformation vectors.
  • expression vector means a construct capable of in vivo or in vitro or ex wo expression.
  • the term "expression vector” refers to a DNA construct containing a DNA coding sequence (e.g., gene sequence) that is operably linked to one or more suitable control sequence(s) capable of affecting expression of the coding sequence in a host.
  • control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
  • the plasmid is the most commonly used form of expression vector. However, the description is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art.
  • operably linked refers to juxtaposition wherein the elements are in an arrangement allowing them to be functionally related.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the coding sequence.
  • the nucleotide sequence of the present invention is operably linked to a transcription unit.
  • each transcription unit as described herein are regions of nucleic acid containing coding sequences and the signals for achieving expression of those coding sequences independently of any other coding sequences.
  • each transcription unit generally comprises at least a promoter, an optional enhancer and a polyadenylation signal.
  • transformation vector means a construct capable of being transferred from one species to another.
  • the vectors comprising nucleotide sequences encoding polypeptides of the present invention may be administered directly as "a naked nucleic acid construct"; in one embodiment the vector comprises flanking sequences homologous to the host cell genome.
  • naked DNA refers to a plasmid comprising a nucleotide sequence encoding a polypeptide of the present invention together with a short promoter region to control its production. It is called “naked” DNA because the plasmids are not carried in any delivery vehicle.
  • a DNA plasmid enters a host cell, such as a eukaryotic cell, the proteins it encodes (such as NEV peptides of the present invention) are transcribed and translated within the cell.
  • the vectors comprising nucleotide sequences of the present invention may be introduced into suitable host cells using a variety of non-viral techniques known in the art, such as transfection, transformation, electroporation and biolistic transformation.
  • transfection refers to a process using a non-viral vector to deliver a gene to a target mammalian cell.
  • Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), multivalent cations such as spermine, cationic lipids or polylysine, 1 , 2, -bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.
  • CFAs cationic facial amphiphiles
  • nucleic acid constructs Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents.
  • transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectamTM and transfectamTM).
  • cationic agents for example calcium phosphate and DEAE-dextran
  • lipofectants for example lipofectamTM and transfectamTM.
  • nucleic acid constructs are mixed with the transfection agent to produce a composition.
  • the vectors comprising nucleotide sequences of the present invention may be introduced into suitable host cells using a variety of viral techniques which are known in the art, such as for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses.
  • the vector is a recombinant viral vectors.
  • Suitable recombinant viral vectors include but are not limited to adenovirus vectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, a retroviral vector, lentiviral vectors, baculoviral vectors, pox viral vectors or parvovirus vectors (see Kestler et a/1999 Human Gene Ther 10(10):1619- 32).
  • AAV adeno-associated viral
  • herpes-virus vectors a retroviral vector
  • lentiviral vectors lentiviral vectors
  • baculoviral vectors pox viral vectors or parvovirus vectors (see Kestler et a/1999 Human Gene Ther 10(10):1619- 32).
  • gene delivery is mediated by viral infection of a target cell.
  • NEV as described herein may be used as a vector to deliver a gene of interest (GOI).
  • GOI gene of interest
  • NEV as described herein, or an element thereof may be used as a vector to deliver a gene of interest to a cell, part of a cell, a tissue or an organism.
  • the NEV viral vector may be an attenuated virus, or may have one or more relevant and/or functional parts deleted. Retroviral vectors
  • retroviruses include but are not limited to: murine leukemia virus (MLV), human immunodeficiency virus (HIV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
  • MMV murine leukemia virus
  • HMV human immunodeficiency virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • Fujinami sarcoma virus FuSV
  • Moloney murine leukemia virus Mo-MLV
  • FBR MSV FBR murine osteosarcoma virus
  • vectors for use in accordance with the present invention are recombinant viral vectors, in particular recombinant retroviral vectors (RRV) such as lentiviral vectors.
  • RRV retroviral vectors
  • RRV recombinant retroviral vector
  • the term "recombinant retroviral vector” refers to a vector with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell includes reverse transcription and integration into the target cell genome.
  • the RRV carries non-viral coding sequences which are to be delivered by the vector to the target cell.
  • An RRV is incapable of independent replication to produce infectious retroviral particles within the final target cell.
  • the RRV lacks a functional gag-pol and/or env gene and/or other genes essential for replication.
  • the vector of the present invention may be configured as a split-intron vector. A split intron vector is described in PCT patent application WO 99/15683.
  • Lentiviruses can be divided into primate and non-primate groups.
  • primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV).
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • lentivirus family and other types of retroviruses are that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et a/1992 EMBO. J 11 : 3053-3058; Lewis and Emerman 1994 J. Virol. 68: 510-516).
  • retroviruses - such as MLV - are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
  • Adenoviruses in one embodiment of the present invention, the features of adenoviruses may be combined with the genetic stability of retroviruses/lentiviruses which can be used to transduce target cells to become transient retroviral producer cells capable of stably infect neighbouring cells.
  • retroviral producer cells which are engineered to express a NEV peptide of the present invention can be implanted in organisms such as animals for use in the treatment or prevention of diseases such as EIA, NEV and/or for the treatment or prevention of EIAV and/or NEV infection.
  • vectors for use in accordance with the present invention are recombinant pox viral vectors such as fowl pox virus (FPV), entomopox virus, vaccinia virus such as NYVAC, canarypox virus, MVA or other non-replicating viral vector systems such as those described for example in WO 95/30018.
  • FV fowl pox virus
  • entomopox virus vaccinia virus
  • vaccinia virus such as NYVAC
  • canarypox virus canarypox virus
  • MVA non-replicating viral vector systems
  • the nucleotide sequences encoding the NEV peptides of the present invention may be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleotide sequence in a compatible host cell.
  • a method of making the polypeptide of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell.
  • a nucleotide sequence of present invention which is inserted into a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence, such as the coding sequence of the NEV peptide of the present invention by the host cell, i.e. the vector is an expression vector.
  • the polypeptide produced by a host recombinant cell may be secreted or may be contained intracellular ⁇ depending on the sequence and/or the vector used.
  • expression vectors containing the polypeptide coding sequences can be designed with signal sequences which direct secretion of the polypeptide coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • the vectors of the present invention may be transformed or transfected into a suitable host cell and/or a target cell as described below to provide for expression of a polypeptide of the present invention.
  • This process may comprise culturing a host cell and/or target cell transformed with an expression vector under conditions to provide for expression by the vector of a coding sequence encoding the NEV peptide of the present invention and optionally recovering the expressed polypeptide.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • the expression of the polypeptide of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, polypeptide production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.
  • Host and/or target cells comprising nucleotide sequences of the present invention may be used to express the polypeptides of the present invention under in vitro, in vivo and ex vivo conditions.
  • host cell and/or target cell includes any cell derivable from a suitable organism which a vector is capable of transfecting or transducing.
  • host and/or target cells can include but are not limited to cells capable of expressing the polypeptides of the present invention under in vitro, in vivo and ex vivo conditions. Examples of such cells include but are not limited to white blood cells such as macrophages.
  • Further examples include stem cells, progenitor cells, endothelial cells respiratory airway epithelial cells, hepatocytes, muscle cells, cardiac myocytes, synoviocytes, primary mammary epithelial cess and post-mitotically terminally differentiated non-replicating cells such as monocytes, macrophages and/or neurons.
  • the cell is an animal cell.
  • the cell is an equine cell.
  • the term "organism" includes any suitable organism. In one embodiment, the organism is an animal. In one embodiment, the organism is an equine.
  • polypeptides of the invention may be produced using prokaryotic cells as host cells
  • eukaryotic cells are used, for example yeast, plant, insect or mammalian cells, in particular equine cells.
  • Suitable host cells include bacteria such as E. coli, yeast, plant, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
  • the present description also provides a method comprising transforming a host and/or target cell with a or the nucleotide sequence(s) of the present invention.
  • transformed cell means a host cell and/or a target cell having a modified genetic structure. With the present description, a cell has a modified genetic structure when a vector according to the present invention has been introduced into the cell.
  • Host cells and/or a target cells may be cultured under suitable conditions which allow expression of the polypeptide of the invention.
  • nucleotide sequence(s) comprising culturing a transformed host cell - which cell has been transformed with a or the nucleotide sequence(s) according to the present invention under conditions suitable for the expression of the polypeptide encoded by said nucleotide sequence(s).
  • the present description also provides a method comprising culturing a transformed host cell - which cell has been transformed with a or the nucleotide sequence(s) according to the present invention or a homologue, or fragment thereof - under conditions suitable for the expression of the polypeptide encoded by said nucleotide sequence(s); and then recovering said polypeptide from the transformed host cell culture.
  • the polypeptide of the present invention can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. The polypeptide may be purified and isolated in a manner known perse.
  • the present description also encompasses gene therapy whereby the NEV-encoding nucleotide sequence(s) of the present invention is regulated in vivo or in vitro or ex vivo.
  • expression regulation may be accomplished by administering compounds that bind to the NEV-encoding nucleotide sequence(s) of the present invention, or control regions associated with the NEV-encoding nucleotide sequence of the present invention, or its corresponding RNA transcript to modify the rate of transcription or translation.
  • Control sequences operably linked to sequences encoding the NEV peptide of the present invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell and/or target cell in which the expression vector is designed to be used. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
  • promoter is well-known in the art and is used in the normal sense of the art, e.g. as an RNA polymerase binding site.
  • the term encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
  • the promoter is typically selected from promoters which are functional in mammalian, cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used.
  • the promoter is typically derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, ⁇ -actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase).
  • the promoters referred to herein may be tissue-specific promoters. That is, they are capable of driving transcription of an NEV-encoding nucleotide sequence(s) in one tissue while remaining largely “silent" in other tissue types.
  • the promoters of the present invention may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors.
  • Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.
  • MMLV LTR Moloney murine leukaemia virus long terminal repeat
  • RSV rous sarcoma virus
  • CMV human cytomegalovirus
  • the promoters may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
  • any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences.
  • Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • the term “enhancer” includes a DNA sequence which binds to other protein components of the transcription initiation complex and thus facilitates the initiation of transcription directed by its associated promoter.
  • the pharmaceutical composition may be any pharmaceutical composition.
  • the pharmaceutical composition is to be administered orally, enterally, rectally or parenterally.
  • the composition may be an edible composition. "Edible” means a material that is approved for human or animal consumption.
  • compositions are administered orally in the form of an oral paste or a powder to be dissolved into the drinking water.
  • pharmaceutical compositions may also be administered by injectable routes such as subcutaneous, intramuscular or intravenous injection.
  • compositions may be for animal usage in veterinary medicine.
  • suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the "Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and PJ Weller.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.
  • suitable diluents include ethanol, glycerol and water.
  • compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
  • Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • compositions, pharmaceutical compositions, or vectors of the present invention may be adapted for oral, rectal, vaginal, intrauterine, parenteral, intramuscular, intraperitoneal, intraarterial, intra-articular, intra-hoof, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
  • compositions, pharmaceutical compositions, or vectors of the present invention are adapted for oral, rectal, vaginal, parenteral, nasal, buccal or sublingual routes of administration.
  • compositions, pharmaceutical compositions, or vectors of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, pastes, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
  • transdermal administration is by use of a skin patch.
  • the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin.
  • the active ingredient can also be incorporated into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
  • compositions, pharmaceutical compositions, or vectors may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
  • a person of ordinary skill in the art can easily determine an appropriate dose of the polypeptide or a polypeptide sequence encoding said polypeptide or polynucleotide sequence to administer to a subject without undue experimentation.
  • a veterinarian will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the polypeptide, the stability and length of action of the polypeptide, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
  • the dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • the composition or the pharmaceutical composition comprises a combination of different peptides selected from the group consisting of SEQ ID NOs 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
  • SEQ ID No 20 An example of a fragment of SEQ ID No 20 is QEQNPPPSVSLRSLFG (SEQ ID No 153).
  • SEQ ID No 154 encodes QEQNPPPSVSLRSLFG (SEQ ID No 153).
  • SEQ ID No 59 An example of a fragment of SEQ ID No 59 is WGIAHTTGIPGNSQGQAM (SEQ ID No 155).
  • composition or the pharmaceutical composition comprises a combination of different peptides selected from the group consisting of SEQ ID NOs 1 to 14 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 14.
  • the composition or the pharmaceutical composition comprises at least two, three, four, five or six peptides selected from the group consisting of SEQ ID NOs 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
  • compositions or the pharmaceutical composition further comprise at least one known EIAV peptide.
  • EIAV peptides include the composition or the pharmaceutical composition comprising (i) QEQNPPPSVSLRSLFG (from SEQ ID NO 20), (ii) EALDKIEEIQNKNKQK (SEQ ID NO 18) and (iii) LLNGSLAGESIIIR (SEQ ID NO 34).
  • composition or pharmaceutical composition comprises (i) QEQNPPPSVSLRSLFG (from SEQ ID NO 20), (ii) EALDKIEEIQNKNKQK (SEQ ID NO 18), (iii) LLNGSLAGESIIIR (SEQ ID NO 34), and (iv) QPSLPTGSEELK (SEQ ID NO 17) and/or (v) WGIAHTTGIPGNSQGQAM (from SEQ ID NO 59).
  • the composition or the pharmaceutical composition comprises at least two, three, four, five or six polynucleotide sequences encoding peptides selected from the group consisting of SEQ ID NOs 1 to 76 and variants, homoiogues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
  • the composition or the pharmaceutical composition comprises at least two, three, four, or five peptides polynucleotide sequences encoding selected from the group consisting of SEQ ID NOs 18, 34, 17, 153 and 155.
  • the composition or the pharmaceutical composition comprises at least two, three, four, five or six polynucleotide sequences selected from the group consisting of SEQ ID NOs 77 to 152 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
  • the composition or the pharmaceutical composition comprises at least two, three, four or five polynucleotide sequences selected from the group consisting of SEQ ID NOs 152, 109, 93, 154, and 156.
  • compositions or the pharmaceutical composition comprise a combination of at least one peptide of the present invention and at least one nucleotide sequence of the present invention.
  • compositions or the pharmaceutical composition further comprise at least one known EAIV peptide and/or at least one nucleotide sequence encoding a known EIAV peptide.
  • the peptides may be combined in the form of a fusion protein - that is- the peptides of the invention may be covalently linked together.
  • one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 or variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any of SEQ ID NOs 1 to 76 may be linked together as a fusion protein.
  • said two peptides may be SEQ ID NO: 18 and SEQ ID NO: 153, or variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 18 and SEQ ID NO: 153.
  • one or more peptides of the invention are linked together as a fusion protein by a linker peptide.
  • Peptide of the invention - linker peptide - peptide of the invention where "-" represent a covalent bond.
  • linker peptide is a continuous, unbranched chain of amino acids.
  • linker peptides may be any length, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length.
  • said linker peptide is 3 amino acids in length.
  • the linker peptide comprises a repetitive amino acid sequence.
  • the linker peptide consists of alanine residues.
  • the linker peptide is AAA.
  • the fusion protein of the invention may be represented by one or more of the following structures:
  • EALDKIEEIQNKNKQKAAAQEQNPPPSVSLRSLFGNDPL (SEQ ID NO: 157).
  • the fusion protein consists of or comprises the following sequence:
  • the peptides of the invention which form part of the fusion protein may be truncated by any number of amino acids at either the N-terminus or the C-terminus.
  • the peptides may be truncated by 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • EALDKIEEIQNKNKQKAAAQEQNPPPSVSLPvSLFG (SEQ ID NO: 159).
  • the present invention provides a polynucleotide sequence encoding a fusion protein as described herein.
  • the present invention also includes diagnostic methods and kits for the detection and measurement of one or more NEV peptides of the present invention in a sample, the measurement of one or more antibodies of the present invention, the measurement of one or more polynucleotide sequences of the present invention.
  • the peptides of the present invention may be used in a diagnostic method and kit to permit detection of circulating equine infectious anaemia virus (EIAV) and/or new equine virus (NEV) which, in certain situations, may indicate the progression of a disease state.
  • EIAV circulating equine infectious anaemia virus
  • NEV new equine virus
  • An antibody of the present invention that, for example, possesses high binding specificity can be used to establish easy to use kits for rapid, reliable, sensitive, and specific measurement of NEV peptides of the present invention in samples such as plasma, urine, and in cell culture media.
  • the antibody of the present invention may also be used in a diagnostic method and kit to permit detection of circulating EIAV and/or NEV which, in certain situations, may indicate the progression of a disease state.
  • polynucleotide sequences of the present invention may be used in a diagnostic method and kit to permit detection of circulating EIAV and/or NEV which, in certain situations, may indicate the progression of a disease state.
  • the diagnostic method according to the present invention determines the viral titre of an equine infectious anaemia virus (EIAV) and/or NEV in an animal. This may enable the progression of EIAV/NEV infection and/or EIAV/NEV disease progression to be monitored.
  • EIAV equine infectious anaemia virus
  • the viral titre may be determined in a sample from an animal such as a blood sample, a blood serum sample, a plasma sample, a saliva sample, a sputum sample, a urine sample, a semen sample, a biopsy sample, a fecal sample, a milk sample, a lymph node biopsy sample, and/or a sweat sample.
  • a sample from an animal such as a blood sample, a blood serum sample, a plasma sample, a saliva sample, a sputum sample, a urine sample, a semen sample, a biopsy sample, a fecal sample, a milk sample, a lymph node biopsy sample, and/or a sweat sample.
  • kits and diagnostic methods of the present invention may include but are not limited to the following techniques; competitive and non-competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, infrared assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips, or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry.
  • competitive and non-competitive assays radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, infrared assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips, or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry.
  • competitive and non-competitive assays radioimmunoa
  • the presence or absence in a sample of one or more polynucleotide sequence encoding one or more peptides according to the present invention can be determined by using, for example, PCR (such as quantitative PCR, digital PCR), nucleic acid isothermal amplification, (such as Loop mediated isothermal amplification), and/or hybridisation techniques (such as Southern blotting).
  • a primer for use, for example, in a PCR assay may be based on a polynucleotide sequence encoding a peptide of the present invention and/or a polynucleotide sequence of the present invention.
  • primers are between 15 to 40 nucleotides in length. In some embodiments, the primers are between 18 to 26 nucleotides in length.
  • a probe for use, for example, in a hybridisation assay may be based on a polynucleotide sequence encoding a peptide of the present invention and/or a polynucleotide sequence of the present invention.
  • probes are at least 15 nucleotides in length.
  • the primers are at least 20, 30 or 40 nucleotides in length. In some embodiments, the primers are between 15 and 200 nucleotides in length.
  • a combination of primers and/or probes may be used to determine the presence or absence in a sample of one or more polynucleotide sequence encoding one or more peptides according to the present invention.
  • the sample may be blood, blood serum, plasma, saliva, sputum, urine, fecal biopsy, lymph node biopsy, milk, semen, and/or sweat.
  • NEV peptides of the present invention By determining the presence of NEV peptides of the present invention and/or the presence of NEV antibodies of the present invention and/or the presence of polynucleotide sequences encoding peptides of the present invention in a sample from an animal it can be determined that an animal has been infected with an equine infectious anaemia virus (EIAV) and/or NEV.
  • EIAV equine infectious anaemia virus
  • the diagnostic methods of the present invention are typically carried out ex vivo or in vitro.
  • compositions according to the invention and/or a vector according to the invention, and/or a pharmaceutical composition according to the invention, and/or an antibody according to the invention may also be used in a kit or assay for the diagnosis or prevention of EIAV and/or NEV, and/or the diagnosis or prevention of an infection by an EIAV and/or NEV in an animal.
  • the scope of the present invention encompasses polynucleotide sequences encoding NEV peptides.
  • amino acid sequence encompassed by scope the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism. In one embodiment, however, the amino acid sequence encompassed by scope of the present invention may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
  • the nucleotide sequence encompassed by the scope of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA).
  • the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232).
  • a nucleotide sequence encoding either a peptide of the present invention may be identified and/or isolated and/or purified from any cell or organism producing said peptide.
  • Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences.
  • DNA amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or purified.
  • the nucleotide sequence encoding the peptide may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al., (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al., (1984) EMBO J. 3, p 801-805.
  • the phosphoroamidite method oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.
  • the nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).
  • the scope of the present invention also encompasses NEV peptides as defined herein.
  • the amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
  • the peptide encompassed in the present invention may be used in conjunction with other peptide (e.g. known EIAV peptides).
  • the present invention also covers a combination of peptides wherein the combination comprises the peptide of the present invention and another peptide, which may be another peptide according to the present invention.
  • amino acid sequence when relating to and when encompassed by the per se scope of the present invention is not a native peptide.
  • native peptide means an entire peptide that is in its native environment and when it has been expressed by its native nucleotide sequence.
  • Antibodies may be produced by standard techniques, such as by immunisation with the peptide of the invention or by using a phage display library.
  • the term "antibody”, unless specified to the contrary, includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library, as well as mimetics thereof.
  • Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
  • the antibodies and fragments thereof may be humanised antibodies. Neutralising antibodies, i.e., those which inhibit biological activity of the substance polypeptides, are especially preferred for diagnostics and therapeutics.
  • polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, llama, etc.) and/or avian is immunised with the sequence of the present invention (or a sequence comprising an immunological epitope thereof). Depending on the host species, various adjuvants may be used to increase immunological response.
  • a selected mammal e.g., mouse, rabbit, goat, horse, llama, etc.
  • avian is immunised with the sequence of the present invention (or a sequence comprising an immunological epitope thereof).
  • various adjuvants may be used to increase immunological response.
  • Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the peptide of the present invention (or a sequence comprising an immunological epitope thereof) contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
  • Monoclonal antibodies directed against the sequence of the present invention can also be readily produced by one skilled in the art and include, but are not limited to, the hybridoma technique Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et a/., (1983) Immunol Today 4:72; Cote et al., (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan Rickman Liss Inc, pp 77-96).
  • polynucleotide sequence and “nucleotide sequence” are used interchangeably herein.
  • the present invention also encompasses the use of sequences having a degree of sequence identity or sequence homology with amino acid sequence(s) of a polypeptide described herein (e.g. variants, homologues and derivatives) or of any nucleotide sequence encoding such a polypeptide (hereinafter referred to as a "homologous sequence(s)").
  • homologue means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences.
  • the term “homology” can be equated with "identity”.
  • a homologous sequence is taken to include an amino acid or a nucleotide sequence which may be at least 50, 60, 70, 75, 80, 85 or 90% identical, in some embodiments at least 95, 96, 97, 98 or 99% identical to the subject sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • a homologous sequence is taken to include an amino acid sequence or nucleotide sequence which has one or several additions, deletions and/or substitutions compared with the subject sequence.
  • the present invention relates to the use of a protein whose amino acid sequence is represented herein or a protein derived from this (parent) protein by substitution, deletion or addition of one or several amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more amino acids, such as 10 or more than 10 amino acids in the amino acid sequence of the parent protein and having the activity of the parent protein.
  • the present invention relates to the use of a nucleic acid sequence (or gene) encoding a protein whose amino acid sequence is represented herein or encoding a protein derived from this (parent) protein by substitution, deletion or addition of one or several amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more amino acids, such as 10 or more than 10 amino acids in the amino acid sequence of the parent protein and having the activity of the parent protein.
  • the homologous amino acid sequence and/or nucleotide sequence may provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the polypeptide.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • % homology can be measured in terms of identity
  • the alignment process itself is typically not based on an all-or-nothing pair comparison.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSU 62 matrix - the default matrix for the BLAST suite of programs.
  • Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI package.
  • percentage homologies may be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).
  • % homology for example % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result. Should Gap Penalties be used when determining sequence identity, then the following parameters can be used for pairwise alignment for example:
  • CLUSTAL may be used with the gap penalty and gap extension set as defined above.
  • the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, for example over at least 30 contiguous nucleotides, for example over at least 40 contiguous nucleotides, for example over at least 50 contiguous nucleotides, for example over at least 60 contiguous nucleotides, for example over at least 100 contiguous nucleotides, for example over at least 200 contiguous nucleotides, for example over at least 300 contiguous nucleotides.
  • the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.
  • sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • Non-homologous substitution may also occur i.e.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine thienylalanine
  • naphthylalanine phenylglycine
  • Replacements may also be made by unnatural amino acids include; alpha* and alpha- disubstituted* amino acids, N-alkyl amino acids * , lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine * , p-CI-phenylalanine*, p-Br-phenylalanine * , p-l- phenylalanine*, L-allyl-glycine * , ⁇ -alanine*, L-a-amino butyric acid*, L-y-amino butyric acid * , L-a-amino isobutyric acid*, L-s-amino caproic acid , 7-amino heptanoic acid*, L-methionine sulfone* * , L-norleucine*, L-norvaline * , p-nitro-L-phenylalanine*, L-hydroxyproline , L- thioproline
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ -alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or ⁇ -alanine residues.
  • a further form of variation involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the ⁇ -carbon substituent group is on the residue's nitrogen atom rather than the a- carbon.
  • the nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides.
  • a number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention.
  • the present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative or fragment thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
  • Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways.
  • Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations.
  • other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
  • Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
  • Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • hybridisation shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
  • nucleotide sequences can hybridise to a probe of the present invention under stringent conditions (e.g. 50°C and 0.2xSSC).
  • nucleotide sequences can hybridise to a probe of the present invention under high stringent conditions (e.g. 65°C and O.lxSSC).
  • a composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76; and/or comprising one or more polynucleotide sequences encoding one or more of the peptides;
  • polynucleotide sequences selected from the group consisting of SEQ ID NOs: 77 to 152, and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
  • a pharmaceutical composition comprising:
  • a diagnostic method comprising obtaining a sample from an animal and determining the presence or absence in said sample of antibodies which are capable of binding to one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76
  • a kit comprising one or more peptides selected from the group consisting of SEQ ID Nos 1 to 76 and optionally instructions for determining the presence or absence in an animal sample of antibodies capable of binding to one or more said peptides.
  • a kit comprising one or more antibodies according to paragraph 4 and optionally instructions for determining the presence or absence in an animal sample of peptides capable of binding to said antibodies.
  • An oligonucleotide sequence (e.g. a primer or a probe) comprising or consisting of a polynucleotide sequence capable of encoding one or more of the peptides selected from the group consisting of SEQ ID NOs: 1 to 76
  • kits comprising one or more oligonucleotide sequences according to paragraph 15 and optionally instructions for determining the presence or absence in an animal sample of said polynucleotide sequence.
  • a method for controlling EIAV disease in a group of animals comprising the identification of EIAV infection in an animal using the diagnostic method according to any one of paragraphs 9 to 12 and, optionally, the isolation of an EIAV infected animal from other animals.
  • compositions according to paragraph 1 , and/or a vector according to paragraph 2, and/or a pharmaceutical composition according to paragraph 3, and/or an antibody according to paragraph 4, for the manufacture of a medicament for the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV in an animal.
  • a method for the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV in an animal comprising administering to an animal one or more compositions according to paragraph 1 , and/or one or more vectors according to paragraph 2, and/or one or more antibodies according to paragraph 3, and/or one or more pharmaceutical compositions according to paragraph 4.
  • compositions and/or a vector and/or a pharmaceutical composition substantially as described herein with reference to any one of the Examples and/or drawings for use in the treatment or prevention of equine infectious anaemia and/or treatment or prevention of an infection by an EIAV in an animal.
  • the inventors have developed a peptide based immunodiagnostic assay that has been validated by comparing results on reference and field samples with the immunoblot test.
  • the peptide ELISA has similar sensitivity and specificity of the immunoblot techniques for the detection of EIAV infected horses.
  • the ELISA is based in peptides identified in purified viral particles obtained from EIAV AG ID negative field samples. The purified viral particles were obtained from primary macrophages cell lines established from seropositive horses.
  • the EIAV AGID negative strains were also considered negative in ELISAs directed to p26 core protein as also to the dual antigen gp45 ELISAs (Eradikit ELISA, In3 Diagnostics, Italy).
  • the retrieved peptides were short peptides with generally less than 30 amino acids. These peptides had a good sensitivity and specificity. More than 62 peptides were identified from EIAV AGID-negative viral purified strains by mass spectrometry; these peptide were screened against public viral databases (UNIPROT KB or NCBI) and twenty three peptides of interest were selected which were found that were present in a EIAV AGID negative gene bank that belongs to Equigerminal - these peptides are shown in Table 1. None of the identified peptides belonged to the EIAV proteome. 45 of the 62 peptides were found to have sequence identity with retroviral peptides.
  • Table 1 - Table 1 details peptide sequences and the polynucleotide sequence which encodes the peptide.
  • the polynucleotide sequence shown as SEQ ID NO 77 encodes the peptide shown as SEQ ID NO 1.
  • AKLEESFPGK (SEQ ID NO GCCAAGCTGGAGGAGAGCTTCCCCGGCAAG (SEQ ID NO 26) 101)
  • NGSLAGESIIIR (SEQ ID NO AACG GCAGCCTGGCCGGCGAGAG C ATC ATCATC AGG (SEQ 27) ID NO 102)
  • NITFNSSAGGDLEIT SEQ ID AACATCACCTTCAACAGCAGCGCCGGCGGCGACCTGGAGAT NO 28
  • CACC SEQ ID NO 10
  • FGNKTTIIFTK SEQ ID NO TTCGGCAACAAGACCACCATCATCTTCACCAAG (SEQ ID NO 33) 1078
  • IGCQHSRIGITLPR (SEQ ID ATCGGCTGCCAGCACAGCAGGATCGGCATCACCCTGCCCA NO 38) GG (SEQ ID NO 113)
  • TLQYLALTALVTPK SEQ ID ACCCTGCAGTACCTGGCCCTGACCGCCCTGGTGACCCCCAA NO 39
  • WSSALQYLIPR SEQ ID NO GTG GTG AG C AGCG CCCTG CAGTACCTG ATCCCCAG G (SEQ 41) ID NO 116)
  • PVWAEPVK (SEQ ID NO 42) CCCGTGTGGGCCGAGCCCGTGAAG (SEQ ID NO 117)
  • VKGNDLLK (SEQ ID NO 43) GTGAAGGGCAACGACCTGCTGAAG (SEQ ID NO 118)
  • EDLNLQDWK (SEQ ID NO 44) GAGGACCTGAACCTGCAGGACTGGAAG (SEQ ID NO 119)
  • AAEQKASPPSLTPK SEQ ID GCCGCCGAGCAGAAGGCCAGCCCCCCCAGCCTGACCCCCA NO 46
  • FPSIVGRPR (SEQ ID NO 48) TTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID NO 123)
  • IWHHTFYNELR SEQ ID NO ATCTG G CACC AC ACCTTCTACAACG AGCTG AG G (SEQ ID NO 49) 124)
  • MTQIMFETF (SEQ ID NO 50) ATGACCCAGATCATGTTCGAGACCTTC (SEQ ID NO 125)
  • VAPEEHPVLLTEAPLNPK GTGGCCCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCC
  • AVFPSIVGRPR (SEQ ID NO GCCGTGTTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID 53) NO 128)
  • AVFPSIVGR (SEQ ID NO 55) GCCGTGTTCCCCAGCATCGTGGGCAGG(SEQ ID NO 130) IGRKDAERQLLSPGNAR ATCGGCAGGAAGGACGCCGAGAGGCAGCTGCTGAGCCCCG (SEQ ID NO 56) GCAACGCCAGG (SEQ ID NO 131 )
  • RGILTLK (SEQ ID NO 58) AGGGGCATCCTGACCCTGAAG (SEQ ID NO 133)
  • HLVDQLIRDLK (SEQ ID NO CACCTGGTGGACCAGCTGATCAGGGACCTGAAG (SEQ ID 60) NO 135)
  • INRELLK (SEQ ID NO 63) ATCAACAG GG AG CTGCTG AAG (SEQ ID NO 38)
  • QTIIPTNKDVDEK (SEQ ID NO CAGACCATCATCCCCACCAACAAGGACGTGGACGAGAAG 64) (SEQ ID NO 139)
  • KNYGKLDK (SEQ ID NO 65) AAG AACTACGG CAAGCTGG ACAAG (SEQ ID NO 140)
  • TTISFSK (SEQ ID NO 66) ACCACCATCAGCTTCAGCAAG (SEQ ID NO 141)
  • QDRTTISFSK (SEQ ID NO 67) CAGGACAGGACCACCATCAGCTTCAGCAAG (SEQ ID NO 67)
  • DIVLLENGK (SEQ ID NO 71) GACATCGTGCTGCTGGAGAACGGCAAG (SEQ ID NO 146)
  • IFLINLAFLIK (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGATCAAG (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGATCAAG (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGATCAAG (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGATCAAG (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGATCAAG (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGATCAAG (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGATCAAG (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGATCAAG (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGATCAAG (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGA
  • NRLEILK (SEQ ID NO 74) AACAGGCTGGAGATCCTGAAG (SEQ ID NO 149)
  • ADDVAVLQDALGR (SEQ ID GCCGACGACGTGGCCGTGCTGCAGGACGCCCTGGGCAGG NO 75) (SEQ ID NO 150)
  • LNKSLEQLR (SEQ ID NO 76) CTG AACAAG AG CCTGG AG CAG CTG AG G (SEQ ID NO 151)
  • the inventors carried out ELISA assays using the above identified short synthetic peptides in Table 1 , which are easy to produce, and control and which can be used as target antigens for the detection of anti-NEV antibodies.
  • Equine infectious anemia (EIAV) seropositiveness was first determined by immunoblot ( Figure 1).
  • EIAV Wyoming EIAVWYO viral strain (ATCC VR-778) Malmquist et al., 1973) was cultured in a permissive macrophage-like cell line EML-3C cell line (ATCC CRL-2996) previously established for EIAV replication studies (Fidalgo-Carvalho et al., 2009).
  • the cells were seeded in T75cm 2 flasks prior to infection. After two days cell cultures were infected with EIAVWYO- Tissue culture supernatants were screened for Reverse Transcriptase (RT) activity by using the Enzcheck reverse transcriptase kit (Molecular Probes, Invitrogen) according to manufacturer instructions.
  • RT Reverse Transcriptase
  • RT positive cell culture supernatants were submitted to sequential centrifugations steps. To remove cell debris supernatants were centrifuged at 500 rcf for 10 minutes, supernatants moved to a clean tube and centrifuged again at 3200 rcf for 20 minutes. The RT positive supernatants were then filtered by a syringe minisart 0.45 micron filter (Sartorius) and viral particles concentrated to 0.5-1 mL in a Vivaspin 6 > 100,000 MWCO PES (Sartorius) accordingly manufacturers instructions.
  • Sartorius syringe minisart 0.45 micron filter
  • EIA viral particles were then lysed and submitted to SDS-PAGE gel system by using 10% Bis-Tris gels Novex NuPAGE in a 2D well lane and run in a MES buffer. After separation the gels were transferred to nitrocellulose membranes in 7 minutes by using the iBIot dry blotting system (Invitrogen) accordingly manufacturer instructions.
  • Membrane Strips were cut and incubated with horse sera at 1:50 or 1 :100 dilution in blocking buffer for 1 hour at 37°C. Strips were washed three times with TBS 0.005% Tween20 and incubated, for 30 minutes at 37°C, with goat secondary anti-horse IgG (H+L) antibody labelled with Horseradish peroxidase in a 1:4000 dilution.
  • a non-animal blocking buffer Nap-Blocker, G-Biosciences
  • the secondary antibody was washed out three times, strips passed in a PBS solution and revealed with Ampliflu Red (Sigma-aldrich) fluorescent substrate and visualized with a 570 nm filter at in a Gel doc EZ (Biorad). Results were analysed by using the Image lab software (Biorad).
  • EIAV positive reference sera were used in the immunoblot: the World organization for animal health (OIE) EIAV positive reference sera kindly provided by the European Reference Center for Equine diseases (ANSES, France), and the EIA strong, medium and weak reference sera purchased to VMRD diagnostic (Pullman, Washington DC).
  • EIAV PV blotted membranes As a positive control seropositive EIAV AGID negative were also tested in EIAV PV blotted membranes kindly provided by Doctor Charles Issel (University of Kentucky). The results obtained with the 'in house' EIAVWYO blotted membranes were similar to the results obtained with UK EIAVpv membranes.
  • the EIAV PV viral strain is a strain derived from EIAVWYO (Lichtenstein et a/., 2006).
  • the inventors compared the pattern of the OIE reference sera and the EIAV AGID negative samples.
  • the immunoblot pattern of EIAV AGID positive samples such as the OIE reference sera, can be visualised. This pattern it is usually composed of a three-band pattern in the region of the p26, the gp45 and gp120 proteins that correspond to the binding of anti-p26, anti- gp45, and to the anti-gp120 specific antibodies.
  • the VMRD reference sera a more broad reactivity against different EIAV proteins (not shown) can be visualized.
  • the pattern of EIAV AGID positive and negative reactors can be compared.
  • the EIAV AGID negative like the EIAV AGID positive reactors, possesses the three-band pattern characteristic of EIAV seropositiveness. However, these reactors are considered negative in the AGID assay and/or in the ELISA (Eradikit, In3 Diagnostic, Italy).
  • the EIAV immunoblot (IB) positive sera were submitted to AGID testing by using an ID. Vet EIAV AGID kit (ID. Vet, France). The AGID was performed accordingly the manufacturer instructions. Moreover, 1 :2 and 1 :3 dilutions of the EIAV antigen (p26 recombinant protein) and positive control were performed in order to visualize weak reactivity against the p26 antigen in EIAV IB positive samples. As results none of the seropositives detected in the immunoblot were positive in AGID, neither with the non-diluted nor diluted reagents.
  • EIAV AGID negative sera were analysed by EIAV commercially available ELISAs using antibodies raised against the p26 antigen, or against the dual antigen p26 and gp45, the Eradikit (IN3.Diagnostic, Italy). The sera were serial diluted from 1 :6 to 1 :20 dilution. The results showed that EIAV AGID negative sera were also considered as negative in all the dilutions tested in this ELISA. Establishment of NEV macrophage-like cell lines from EIAV AGID negative horses.
  • EIAV AGID negative strains For isolation and viral propagation of EIAV AGID negative strains, three different macrophage cell lines were established from EIAV AGID negative infected horses. Peripheral blood was collect from 3 different EIAV seropositive horses and processed according to Fidalgo-Carvalho et al., (2009).
  • the macrophage-like cells were able to divide, being passaged and remained in culture for 3-4 months.
  • the macrophage-like cells obtained were similar to the EML-3C reported in Fidalgo-Carvalho et al., (2009), but during the first weeks of culture showed syncytia cell formation and cell death ( Figure 2A1 and Figure 2A2). After 3 weeks the cells were able to recover ( Figure 2B) and showed low levels of reverse transcriptase activity measured in cell supernatants by the Enzcheck reverse transcriptase assay (Invitrogen) ( Figure 2.D1 and 2.D2).
  • Viral particles were isolated from RT positive cell culture supernatants from NEV macrophage-like cell lines A (NEV_MaA), and/or NEV_MaB and/or NEV_MaC. No evident signs of cell stress or death were visualized. The viral particles were isolated as described in the above section. Viral transfer
  • the inventors performed viral transfer among the NEV cells lines generated. Viral particles of MaA cells were used to infect NEV-MaB cells, or NEV-MaC cells and vice-versa. Surprisingly, the cell cultures of NEV-MaC infected with vpB, and NEV-MaA infected with vpC showed an increased reverse transcription activity (in cell culture supernatants) and marked cythopathic events after 7-10 days of infections (Figure 2C) and all cells died 15 days post-infection. The viral particles obtained from NEV-MaC-VpB and/or NEV-MaA-vpC infection were isolated, centrifuged, filtered in a 0.2 ⁇ and concentrated in Vivaspin6 100 kda as described above.
  • viral particles were used for further analysis of the proteome by mass spectrometry.
  • the purified viral particles were also used for viral genome analysis.
  • Viral RNA was extracted from isolated and purified viral particles by using the Trizol LS method, or by using the Pure-link viral RNA/DNA mini-kit (Invitrogen) according to the manufacturer instructions (Figure 3A).
  • the purified viral particles were run in a 10% SDS-PAGE and different bands were digested and injected for mass spectrometry analysis. The results showed a wide diverse spectrum and several retroviral peptides were identified in the EIAV AGID negative viral particles. None of the peptides retrieved belonged to the known EIAV AGID positive strains ( Figure 3B).
  • the inventors performed in vitro serial passages of NEV virus into MaC cell line as described above (viral transfer).
  • MaC monolayers cells in T75cm 2 or T175cm 2 flasks were infected with 1 ml of NEV virus and incubated at 37°C, 5%C02 in a humid chamber for 7 days in DMEM high glucose medium without phenol red (Gibco, Life Technologies), supplemented with 10% of fetal bovine serum, 2% glutamax (Gibco, Life Technologies) and 1% of Penicilin-Streptomicin (Gibco, Life Technologies).
  • the supernatant medium of named passage 1 was serially passaged in MaC cells as described above for 10 times.
  • FIG. 4 the inventors compared cell micrograph of day 7 non-infected cells (Fig 4A) versus NEV infected cells. The infected cells showed an increased number of induced syncytia cell formation and cell death. (Fig. 4B - MaC cells). These cytopathic effects begin to appear at day 4 post-infection (p.i), cell monolayers were partially destroyed at day 5-7 p.i. and completely destroyed at 10- 12 days p.i.
  • NEV is a highly cytopathic virus in Equine Dermal cells.
  • Equine Dermal Cells (ED) (NB-6, ATCC CCL-57) and its cytopathogenicity compared to EIAV_Wyoming.
  • EIAV-Wyoming (ATCC-VR-778) is reported as an in vivo pathogenic virus that can replicate in Equine Dermal cells with slight cytopathic effects (CPE) (Malmquist WA et al., 1973, Orego A, et al, 1982).
  • ED cells were seeded 24 to 72 hours prior to infection in T75cm 2 flasks. Highly confluent (80-90%) cells were infected with 1ml of 0.2 pm filtered NEV viral supernatants or 1 MOI of EIAV-Wyoming, and incubated at 37°C 5% C02 for 5 or more days.
  • the amplified 494 bp of the EIAV pol gene was previously cloned into Topo TA pCR4 vector to obtain the plasmid construct pCR4.EIAVpol.
  • This plasmid was further used to construct a standard curve by performing 10-fold serial dilutions in duplicates.
  • the retrieved standard curve had a R 2 of 0.998 and was able to detect 4 viral particles.
  • viral supernatant obtained from day 6 cell cultures of ED cells infected with EIAV-Wyoming contained 9.18x10 " " viral particles/ml.
  • the results showed that EIAV Wyoming virus replicates at high levels in ED cells without significant CPE.
  • the RT-qPCR assay failed to amplify the NEV genome.
  • Presto Blue cell viability assays (Molecular Probes, Life Technologies) were performed in cells infected with NEV or EIAV Wyoming (ATCC-VR-778). Presto blue cellular viability assays were performed in quintuplicates following the manufacturer's instructions. ED cells were seeded into 96-well plates 24-48 hours before infection. Highly confluent cell monolayers were infected with 20 ⁇ of NEV viral supernatant of day 7, or 1 MOI of EIAV Wyoming strain and allowed to incubate for 11 days. Presto blue reagent was added to the cultures, incubated for 16 hours and absorbance read at 570 nm in a Multiskan Go ELISA reader (Thermo Scientific).
  • the viability of NEV infected cells showed a statistically significant difference (Mann Whitney test) when compared to non-infected or EIAV-Wyoming infected cells (Fig.4D).
  • cell viability of EIAV Wyoming infected cells and mock cells showed similar absorbance (Fig.4D) confirming the microscopic data (Fig. 4C) and indicating that contrary to NEV, the EIAV-Wyoming doesn't induce evident CPE on ED cells at day 11.
  • NEV virus possesses Aspartic protease activity
  • cell infections were performed in serum free media conditions. Day 5-7 p.i. viral particles were concentrated and extensively washed with HBSS in Vivaspin 100kDa (Sartorius) columns. Concentrated viral particles were lysed in 0.1% SDS and submitted to analytical size-exclusion chromatography on a Superdex 200 5/150 GL (GE Healthcare Life Sciences) column connected to a Prominence HPLC system (Shimadzu Corporation, Tokyo, Japan). The column was equilibrated in 20 mM phosphate buffer pH 7.5 containing 150 mM NaCI.
  • protease activity was determined by fluorescence assays in 96-well plates in a Gemini EM Fluorescence Microplate Reader, using the HIV Protease Substrate 1 Arg-Glu(EDANS)- Ser-Gln-Asn-Tyr-Pro-lle-Val-Gln-Lys(DABCYL)-Arg (Sigma Aldrich) or the Phe-Phe substrate.
  • HIV Protease Substrate 1 Arg-Glu(EDANS)- Ser-Gln-Asn-Tyr-Pro-lle-Val-Gln-Lys(DABCYL)-Arg Sigma Aldrich
  • viral particles were assayed for activity at 37°C in buffers ranging between pH 3 and 6 (50 mM sodium acetate pH 3.0, 4.0, 5.0, and 6.0; 50 mM Tris-HCI pH) containing 100 mM NaCI.
  • Indinavir (Sigma Aldrich), a specific inhibitors of HIV protease, or pepstatin were used.
  • the protease was pre-incubated in the presence of the inhibitor.
  • the Pesptatin or Indinavir (10 ⁇ ) was again incubated with viral particles for 10 minutes at room temperature in 50 mM sodium acetate pH 5 or pH 6.0 containing 100 mM NaC.
  • the rate of substrate hydrolysis was monitored for 3 hours by the increase of intensity with excitation/emission wavelengths of 328/393 nm.
  • the detection of antibodies directed against theNEV viruses is based, in the example below, on the principle of the immunoenzyme technique of the "double-antigen sandwich" type.
  • the test is based on the use, on the one hand, of a microplate (solid phase) sensitized with purified antigens, and, on the other hand, of a conjugate constituted by a peptide according to the invention labelled with peroxidase, which take an NEV antibody to be detected into a sandwich.
  • the assay protocol used is as follows: 100 ⁇ of each serum sample tested, diluted to [3/4], are distributed in a well of the sensitized microplate and the mixture is homogenized. After incubation of the mixture under adhesive film for 60 minutes at 37°C, followed by washing of the microplate by means of a Tris NaCI buffer, 100 ⁇ of labelled conjugate (peroxidase- labelled NEV peptide according to the invention) are added to each well. The mixture is incubated under adhesive film for 30 minutes at 18-30°C.
  • a sample is considered to be positive (carrier of anti-NEV antibodies) if the OD obtained is greater than that of the cut-off, and negative if its OD is lower than that of the cut-off.
  • the peptides were dissolved in 50% (v/v) acetic acid, water, or DMSO, at a concentration of 5 mg/ml.
  • the stock solutions were diluted in 0.10 M sodium bicarbonate (pH 9.6) such that the concentrations of the peptides are 1 pg/ml.
  • 100 ⁇ of the dilute solution were added to each of the wells of type B microtitration plates from Nunc, Denmark. The filled test plates were incubated at 20°C for 18 hours.
  • Monoclonal antibodies against h-lgG were prepared in accordance with the method of Koehler and Milstein, Nature 256: 495, 1975, with different monoclonal antibodies having the same antigen specificity being identified by the method described by Stahli et al., J. of Immunological Methods 32: 297-304 (1980).
  • the monoclonal antibody fraction (4 mg of protein/ml) was reacted with N-gammamaleimidobutyloxysuccinimide (GMBS) in accordance with Tanamori et al., J. Immunol. Meth. 62: 123-131 (1983).
  • GMBS N-gammamaleimidobutyloxysuccinimide
  • 2- iminothiolane hydrochloride from Sigma, Cat. No. 1 6256
  • horseradish peroxidase from Boehringer Mannheim, Cat. No. 413470
  • An antibody/POD conjugate was prepared from the GMBS/antibody conjugate and the iminothiolane/POD conjugate as described by Tanamori et al., supra.
  • the resulting solution of the IgG/POD conjugate had a protein content of 360 ⁇ /ml.
  • the ratio of POD to IgG was 2.8.
  • the solution was subsequently diluted to 500 ng/ml IgG/POD using a solution of 50 ml/l fetal calf serum. (FCS, from Biochrom KG, Berlin) and 5 g/l polyoxyethylene (20) sorbitan monolaurate (Tween 20) in PBS, and was given the designation anti-lgG/POD conjugate.
  • FCS polyoxyethylene (20) sorbitan monolaurate
  • the anti-lgG/POD conjugate was diluted 1 :100 to 1 :20,000 with Tris buffer (pH 7.4, containing 0.5% Tween 20), and then a series of 1 :26 final dilutions in conjugate buffer (0.1 M1-amino-2-(hydroxymethyl)-1 ,3- propanediol (Tris), 0.1 M sodium chloride (NaCI) and 0.1 % Tween 20, pH 8.4) is prepared.
  • TMB tetramethylbenzidine
  • Stock solution 1 TMB dihydrochloride was dissolved with stirring in double-distilled water at a concentration of 5 g/l (16 mmol/1), and this solution was adjusted to pH 1.5 using 5 N hydrochloric acid. Penicillin G was added to this solution with stirring, up to a final concentration of 200 mg/l (0.56 mmol/l).
  • Stock solution 2 1.4 ml of glacial acetic acid, 1.5 ml of 1 N NaOH and 250 mg (3 mmol) of H 2 0 2 , as a urea/hydrogen peroxide adduct, were added to 900 ml of double-distilled water. After these substances had dissolved completely, the solution was made up to 1 litre using double-distilled water.
  • TMB substrate preparation One part by volume of stock solution 1 and 10 parts by volume of stock solution 2 were mixed together.
  • test solutions were aspirated and the wells were in each case washed five times with washing buffer containing 1 g/l Tween 20 in PBS. After that, 100 ⁇ of conjugate were added to each of the wells, a preliminary dilution of 1 :3000 in Tris buffer (pH 7.4, 0.5% Tween 20) and a final dilution of 1 :26 in conjugate buffer preferably being selected.
  • the magnetic particle reagents are to be prepared according to the manufacturers recommended protocol.
  • Dynal AS is the manufacturer of the Dynabeads, which are employed.
  • the magnetic particles coated with ligand are called Reagent 1.
  • a peptide according to the invention is covalently coupled to the pre-activated surface of the magnetic particles. It is also possible to physically absorb the peptide to the surface of the magnetic particles.
  • the concentration of particles in Reagent 1 is within the range from 1 mg/ml to 15 mg/ml.
  • the particle size varies between 0.2 ⁇ to 15 pm.
  • the concentration of peptides is within the range from 1 ng/mg particle to 1 mg/mg particle.
  • the concentration of peptides may be within the range from 1 ng/mg particle to 0.01 mg/mg particle, or preferably within the range from 0.01 mg/mg particle to 1 mg/mg particle.
  • the anti horse Ig Alkaline Phosphatase (AP) conjugated antibody reagent is prepared according to the recommended protocol of Dako AS. This protocol is a standard procedure in this field. This reagent is called Reagent 2.
  • the substrate solution phenolphtaleine-monophosphate is to be prepared according to the recommended protocol of Fluka AG. This protocol is a standard procedure in this field.
  • the substrate solution is called Reagent 3.
  • the washing and incubation buffer which is used is standard 0.05M tris-base buffer with the following additional compounds; Tween 20 (0.01% to 0.1%), glycerol (0.1% to 10%) and sodium chloride (0.2% to 0.1%).
  • the assay procedure comprises an incubation step wherein 1 drop of Reagent 1 is mixed with 2 drops of washing buffer in each well. After mixing, 30 ⁇ of sample is added and the solution is incubated for 5 minutes. The magnetic particles can be trapped by a magnet and the liquid removed, before the magnet is separated. Then the wells are washed twice in 4 drops of washing solution, before incubation with Reagent 2. 1 drop of Reagent 2 is added with 2 drops of washing buffer and the solution is incubated for 5 minutes.
  • the immunoassay kit could be used in detection of antibodies, induced either by NEV virus or NEV-specific peptides or proteins, for instance the peptides of the present invention.
  • Immunomodulator e.g. therapeutic or prophylactic vaccine.
  • At least one of the peptides of the invention selected from the group of sequences SEQ ID NO: 1 to SEQ ID NO: 76 can form antigens and constitute the active principle of an immunomodulator e.g. a prophylactic or therapeutic vaccine intended to provide protection against the equine retrovirus (EIAV) or NEV.
  • the immunomodulator may include compounds having beneficial effects in protecting or stimulating the hosts immune system for instance interleukins, interferons, granulocyte macrophage growth factors, haematopoietic growth factors or similar.
  • the immunomodulator further contains an adjuvant or vehicle; for example, the adjuvant or vehicle is Monophosphoryl Lipid A (MPL(R)) possibly with alum, Freund's adjuvant (complete or incomplete) or aluminium hydroxyd.
  • MPL(R) Monophosphoryl Lipid A
  • the optimal amount of adjuvant/vehicle will depend on the type(s) which is chosen.
  • the peptides of the present invention may be modified by C-terminal addition of a single fatty acid such as a single palmitoyi chain to form a lipopeptide vaccine. Further the lipopeptides can be introduced into liposome membranes by the freeze-thaw method resulting in liposomes bearing the peptide ligands on their surface.
  • the peptides of the present invention or compositions of the present invention or pharmaceutical compositions of the present invention or immunomodulator formulation can be freeze-dried prior to storage.
  • the freeze-dried peptides can be dissolved in sterile water to a final concentration of 0.1-100 mg/ml.
  • the peptides of the present invention or compositions of the present invention or pharmaceutical compositions of the present invention or the immunomodulator may be stored, for example, at low temperature, in ampoules containing one or more dosage units, ready for use.
  • a typical dosage unit of a peptide according to the invention is within the concentration range: 0.05 g-1 mg per kg bodyweight, preferably within 0, 15 ⁇ g-0.15 mg per kg body weight.
  • a suitable dose will depend on the body weight of the animal, the type of disease, severity of condition, administration route and several other factors.
  • the peptides of the present invention or compositions of the present invention or pharmaceutical compositions of the present invention may be used as a therapeutic vaccine.
  • the vaccine When used as a therapeutic vaccine, the vaccine might be administered up to 12 times, through injections. Further boosters might follow and can in some cases take place throughout the animal life.
  • the peptides In preparation of an injection solution the peptides are typically dissolved in sterile water at a final concentration of 1 mg/ml per peptide. Typically an injection volume is 100 ⁇ to 200 ⁇ (2x100 ⁇ ).
  • the peptide is preferably co-administered with a suitable adjuvant and/or a granulocyte-macrophage growth factor for instance Leucomax(R) (Schering Plough) made within a concentration range of from 0.1 mg/ml to 1 mg/ml, or according to the manufacturers recommendations for use.
  • a suitable adjuvant and/or a granulocyte-macrophage growth factor for instance Leucomax(R) (Schering Plough) made within a concentration range of from 0.1 mg/ml to 1 mg/ml, or according to the manufacturers recommendations for use.
  • peptides may be administered simultaneously or sequentially.
  • Suitable administration may be intracutaneous, subcutaneous, intravenous, peroral, intramuscular, intranasal, mucosal or any other suitable route.
  • Booster administrations may be required in order to maintain protection.
  • the immunomodulators according to the invention may be useful not only in prevention of infection, but also in treatment of infection.
  • An immunomodulator comprising one or more peptides selected from the group consisting of SEQ ID NO: 1 to and 76 are prepared.
  • the freeze-dried peptides are dissolved in sterile water at a final concentration of 4 mg/ml.
  • a preparation of a granulocyte-macrophage-colony stimulating factor (GM-CSF) is also prepared, according to the manufacturers directions for use, to a final concentration of 0.3 mg/ml.
  • the solutions are administered intracutaneously or intramuscularly or intradermal.
  • a typical injection dose is 100 ⁇ .
  • An antigen solution or suspension is mixed with equal parts of Freund's adjuvant of Behring, complete or incomplete, and is then finely emulsified by being drawn up into, and vigorously pressed out of, an injection syringe, or with a homogenator.
  • the emulsion should remain stable for at least 30 minutes.
  • the antigen-adjuvant emulsions is best injected subcutaneously, or intramuscularly or intradermal as a depot.
  • c-ELISA competitive enzyme-linked immunosorbent
  • Mouse monoclonal antibodies were produced against SEQ ID NO: 18 or SEQ ID NO: 20.
  • a monoclonal antibody mab#4G6E4 was directed against SEQ ID NO: 20.. Furthermore this mab was tested against SEQ ID NO: 20. or the fusion peptide SEQ ID NO: 159. by ELISA procedures.
  • mab#4G6E4 was diluted in the antibody dilution buffer, 3% BSA blocker (Thermoscientific).
  • a rapid (2 hours) c-ELISA procedure was performed as follows: 70 ng/mL of mab#4G6E4 alone (50 microliters) diluted in dilution buffer was added to peptide-coated wells and incubated for 1hour at 37°C. After 1 hour of incubation plates were extensively washed with 300 microliters of PBS-Tween20 (0.05%) for three times.
  • the plate was after that incubated with a secondary goat anti-mouse IgG- HRP (Sigma Aldrich) at 1 :2500 dilution and incubated for 30 minutes at 37°C. Plates were washed again four times with 300 microliters of PBS-Tween20 (0.05%) and signal developed by adding 50 microliters of fresh TMB reagent (Sigma Aldrich) for 15 minutes at room temperature. After colour development the reaction was stopped by adding 50 microliters of 1 N H 2 S0 (Sigma Aldrich). Determination of absorbance was obtained by reading plates at 450 nm in a Multiskan Go ELISA reader (Thermoscientific).
  • the mab#4G6E4 was incubated in the presence of a NEV negative control sera or positive control sera and incubated as previously described in wells coated with serial dilutions of the fusion peptide set forth as SEQ ID NO: 159. For that, 25 microliters of mab#4G6E4 and 25 microliters of diluted equine sera were added in coated wells and incubated together for 1hour at 37°C. Serum or mab#4G6E4 were diluted in 0.025% Casein blocking buffer in PBS (Thermoscientific, Pierce).
  • Results showed EC 50 , Hill slope, bottom and top values of mixtures of mab#4G6E4 and seronegative sera were statistically different (p ⁇ 0.0001) from the EC 50> Hill slope, bottom and top values of mixtures of mab#4G6E4 and seropositive sera.
  • the R 2 retrieved for each dose response curve were always higher than 0.980.
  • FIG. 6C we show representative inhibition rates (%l) of NEV seropositive and one seronegative. Inhibition rates were obtained for a SEQ ID NO: 159 peptide concentration of 96 ng/mL of peptide and mab#4G6E4 IgG concentration of 70ng/mL.
  • SRLLESRGPTTSEK (SEQ ID NO: 1);
  • STFPPTPV SEQ ID NO: 5
  • NAPLLSKVSP (SEQ ID NO: 6);
  • MAQCIVNR SEQ ID NO: 7
  • AKLEESFPGK (SEQ ID NO 26);
  • NGSLAGESIIIR (SEQ ID NO 27);
  • NITFNSSAGGDLEIT SEQ ID NO 28
  • ESFPNK SEQ ID NO 32
  • FGNKTTIIFTK SEQ ID NO 33
  • VNVSVTNNNTTTNV SEQ ID NO 35
  • PTTPVTPAPGVGEISKELAQGK SEQ ID NO 40
  • WSSALQYLIPR SEQ ID NO 41
  • PVWAEPVK SEQ ID NO 42
  • FPSIVGRPR (SEQ ID NO 48);
  • VAPEEHPVLLTEAPLNPK (SEQ ID NO 52);
  • AVFPSIVGRPR (SEQ ID NO 53);
  • AVFPSIVGR (SEQ ID NO 55);
  • RGILTLK (SEQ ID NO 58);
  • WGIAHTTGIPGNSQGQAMVER SEQ ID NO 59
  • HLVDQLIRDLK SEQ ID NO 60
  • TTISFSK (SEQ ID NO 66);
  • QDRTTISFSK (SEQ ID NO 67); AQGRAIAHK (SEQ ID NO 68);
  • DIVLLENGK (SEQ ID NO 71);
  • NRLEILK (SEQ ID NO 74);
  • ADDVAVLQDALGR (SEQ ID NO 75);
  • AGCAGGCTGCTGGAGAGCAGGGGCCCCACCACCAGCGAGAAG SEQ ID NO 77;
  • CAGCCCAGCCTGCCCACCGGCAGCGAGGAGCTGAAG SEQ ID NO 93
  • GAGAAGATCGAGCAGCTGAGG (SEQ ID NO 96);
  • ATCGTGGAGCTGCTGGGCAGG (SEQ ID NO 98); ATCTGGGGCAACGTGACCTGGATGGAGTGGGAGAGG (SEQ ID NO 99);
  • AACGGCAGCCTGGCCGGCGAGAGCATCATCATCAGG (SEQ ID NO 102);
  • GCCGTGATCCTGCTGCTGGACAGGCTGAGG SEQ ID NO 104
  • CTGTTCGTGAGCGTGCTGCAGAGG SEQ I D NO 120
  • GCCGCCGAGCAGAAGGCCAGCCCCCCCAGCCTGACCCCCAAG SEQ ID NO 121

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Communicable Diseases (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

A composition comprising one or more peptides selected from the group consisting of SEQ ID NOs 1 to 76; and/or comprising one or more polynucleotide sequences encoding one or more of the peptides selected from the group consisting of SEQ ID NOs 1 to 76;and/or comprising one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs 77 to 152.

Description

VIRAL PEPTIDES
FIELD OF THE INVENTION
Various embodiments of the present invention generally relate to biotechnology. Further, various embodiments relate to veterinary medicine. In particular, the invention relates to antigenic peptides of equine viruses and uses thereof.
BACKGROUND OF THE INVENTION
Equine infectious anemia (EIA) or swamp fever is an ancient lentiviral disease of Equids. The Swamp fever or equine infectious anemia is the only retroviral disease known to affect Equidae.
The EIAV (Equine infectious anemia virus) retrovirus is known, historically, to be the first viral agent responsible for an animal disease, the Swamp fever in horses.
The disease was first reported in Europe, in France, in 1843 (Lignee, 1843). At the present it is considered to be distributed worldwide. In the last years, several clinical cases of swamp fever were reported by the World Organization for Animal Health (OIE) in the European horse population. However, knowledge about the EIAV European primary isolates sequences is scarce. EIAV sequences are limited to long terminal repeats or gag sequences reported in Greece (Spyrou et al., 2003), Ireland (Mooney J et al., 2006, Quinlivan M et al., 2007), Italy, Romania and Slovenia (Cappeli et al., 2011 , Cappomacio et al., 2012, Kuhar et al., 2014). EIAV infections have been reported in the United Kingdom (UK), France, Belgium, Romania, Slovenia, Austria and Germany, but very little is know about the EIAV sequences responsible for these infections. The full length EIAV genome of European strains is restricted to Ireland strains isolated from a 2006 outbreak (Quinlivan M et al., 2013). Nevertheless, even in the continents where the disease is prevalent, reports of EIAV in naturally infected horses or viral sequence variations among primary isolates are limited. Recently some EIAV isolates from Pennsylvania, US and Japan were recovered by 'in vivo' viral transfer experiments between EIAV seropositive and naive horses. The retrieved viral sequences showed more diverse and divergent EIAV strains. The identified EIAV strains were detected in horses determined as seropositive by the Coggins test (also known as the AGID or Agar Gel Diffusion test). The AGID test detects antibodies against the major core protein of EIAV (p26).
The ancient origin of this virus, geographical dispersion, persistence over time in countries with monitoring plans, and the ability of EIAV to be highly variable suggest that much remains to be learned about this virus. Means and methods for diagnosis and treatment are thus needed.
Currently, there is no known cure for the disease. Diagnosis, quarantine/isolation or elimination of seropositive animals is the only way to control the disease. The current immunodiagnostic tests for the detection of anti-EIAV antibodies in the serum of horses infected with EIAV utilize the whole virus as an antigen, or viral recombinant proteins.
The 01 E official test to diagnose the presence of EIA has been the presence of antibodies specific for the disease in the serum of affected animals using the Coggins or agar gel diffusion test (AGID) (described in U.S. Patent No. 3.929.982 and U.S. Patent No.3.932.601).
In the Coggins test, prepared antigens are used to test serum for the presence of anti-EIAV antibodies. In these assays the used antigens are whole EIAV viral proteins produced in cell lines or p26 recombinant proteins. Although they brought great progress, these immunoassays still do not permit detection of all the serums of subjects infected with EIAV divergent viral strains.
Fidalgo-Carvalho (2008; PhD thesis, University of Porto, Portugal) identified 33 horses that tested positive for EIAV in the immunoblot test, but that tested negative in the Coggins test and commercial ELISAs directed to the EIAV core protein.
Recently Issel and colleagues (2013) have shown the fragilities of the Coggins test, and documented the immunoblot as the more reliable test for determining EIAV seropositiveness. When first deployed, the immunoblot test was recommended as a useful adjunct for diagnosis because samples from EIAV- infected equids with low levels of anti-p26 antibodies (that may be falsely reported as negative on the AGID test) generally had higher levels of reactivity against the envelope proteins. The immunoblot test uses gradient purified EIAV viral particles obtained from cell culture supernatants of equine cell lines infected with EIAVPV. a biological EIAV isolate. In the immunoblot test linear epitopes of all EIAV viral proteins, gag derived (p26, p15 and p1 1), envelope proteins (gp90 and gp45), and accessory proteins can be detected by specific sera antibodies.
The available ELISA tests have been validated by comparing results on reference and field samples with those in AGID tests, with cut-off points adjusted so that results are in agreement to a high degree of significant statistical accuracy. The majority of commercially available test kits in AGID and ELISA formats use the p26 antigen only. Two ELISA kits also incorporate a synthetic antigen determinant of the transmembrane protein gp45.
STATEMENT OF INVENTION
The present invention is based on the use of peptides for therapeutic and/or diagnostics purposes of an EIAV and/or NEV infection of horses. The inventors used viral peptides (identified by mass spectrometry) from EIAV seropositive horses. The viral peptides were obtained from viral supernatants which originated from macrophage cell cultures isolated from EIAV seropositive horses. EIAV seropositive horses were identified by immunoblot techniques, previously these horses were considered to be EIAV negative in commercially available ELISAs and in the Coggins test. The present invention provides peptide-based immunodiagnostics techniques with the similar sensitivity and specificity of immunoblot techniques. Further, the invention provides tools for the detection of divergent EIAV and/or NEV strains or subtypes. Advantageously, the diagnostic methods of the present invention are capable of identifying EIAV and/or NEV infection in animals which current diagnostic methods do not detect an infection. Advantageously, this means that more animals (e.g. horses) with EIAV and/or NEV infection (i.e. seropositive animals) can be identified. The present invention provides a composition comprising one or more peptides selected from the group consisting of:
SRLLESRGPTTSEK (SEQ ID NO: 1);
TLKE VLGG EAAVR (SEQ ID NO: 2);
PCRSKNILPV (SEQ ID NO: 3);
DPCVHSVDVLLR (SEQ ID NO: 4); STFPPTPV (SEQ ID NO: 5);
NAPLLSKVSP (SEQ ID NO: 6);
MAQCIVNR (SEQ ID NO: 7);
KPNVEGRYGLSRSETNK (SEQ ID NO: 8); QQGPEAPLPSLQVAEVPK (SEQ ID NO: 9); PHAGSTQTEWPK (SEQ ID NO: 10);
PIQINACK (SEQ ID NO: 11);
GGMRESWDGGQV (SEQ ID NO: 12);
ESLVSKGFR (SEQ ID NO: 13);
CSLVLGE QIKRIR (SEQ ID NO: 14);
LDKWEKIR (SEQ ID NO 15);
QMMIAASEK (SEQ ID NO 16);
QPSLPTGSEELK (SEQ ID NO 17);
EALDKIEEIQNKNKQK (SEQ ID NO 18);
PPIPVGGIYK (SEQ ID NO 19);
QEQNPPPSVSLRSLFGNDPL(SEQ ID NO 20); EKIEQLR (SEQ ID NO 21);
DLLLIVAR (SEQ ID NO 22);
IVELLGR (SEQ ID NO 23);
IWGNVTWMEWER (SEQ ID NO 24);
LKDLFPNK(SEQ ID NO 25);
AKLEESFPGK (SEQ ID NO 26);
NGSLAGESIIIR (SEQ ID NO 27);
NITFNSSAGGDLEIT (SEQ ID NO 28);
AVILLLDRLR(SEQ ID NO 29);
GPGIHIGKR ( SEQ ID NO 30);
VIICSASK (SEQ ID NO 31);
ESFPNK (SEQ ID NO 32);
FGNKTTIIFTK (SEQ ID NO 33);
LLNGSLAGESIIIR (SEQ ID NO 34);
VNVSVTNNNTTTNV (SEQ ID NO 35);
AILHILRR (SEQ ID NO 36);
DLLALDK (SEQ ID NO 37);
IGCQHSRIGITLPR (SEQ ID NO 38);
TLQYLALTALVTPK (SEQ ID NO 39); PTTPVTPAPGVGEISKELAQGK (SEQ ID NO 40); WSSALQYLIPR (SEQ ID NO 41);
PVWAEPVK (SEQ ID NO 42);
VKGNDLLK (SEQ ID NO 43);
EDLNLQDWK (SEQ ID NO 44);
LFVSVLQR (SEQ ID NO 45);
AAEQKASPPSLTPK (SEQ ID NO 46);
PLSLPLKI (SEQ ID NO 47);
FPSIVGRPR (SEQ ID NO 48);
IWHHTFYNELR (SEQ ID NO 49);
MTQIMFETF (SEQ ID NO 50);
EEEVAALVIDNGSGMCK (SEQ ID NO 51);
VAPEEHPVLLTEAPLNPK (SEQ ID NO 52);
AVFPSIVGRPR (SEQ ID NO 53);
YPIEHGIVTNWDDMEK (SEQ ID NO 54);
AVFPSIVGR (SEQ ID NO 55);
IGRKDAERQLLSPGNAR (SEQ ID NO 56);
DSYVG DEAQSKR (SEQ ID NO 57);
RGILTLK (SEQ ID NO 58);
WGIAHTTGIPGNSQGQAMVER (SEQ ID NO 59); HLVDQLIRDLK (SEQ ID NO 60);
YEQLQLQAR (SEQ ID NO 61);
TITLEVEPSDTIENVK (SEQ ID NO 62);
INRELLK (SEQ ID NO 63);
QTIIPTNKDVDEK (SEQ ID NO 64);
KNYGKLDK (SEQ ID NO 65);
TTISFSK (SEQ ID NO 66);
QDRTTISFSK (SEQ ID NO 67);
AQGRAIAHK (SEQ ID NO 68);
MAQTQLVPVK (SEQ ID NO 69);
QELIPPCK (SEQ ID NO 70);
DIVLLENGK (SEQ ID NO 71);
LPPLSILK (SEQ ID NO 72);
IFLINLAFLIK (SEQ ID NO 73);
NRLEILK (SEQ ID NO 74); ADDVAVLQ DALG R (SEQ ID NO 75);
LNKSLEQLR (SEQ ID NO 76); and
variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76; and/or comprising one or more polynucleotide sequences encoding one or more of the peptides;
and/or comprising one or more polynucleotide sequences selected from the group consisting of:
AGCAGGCTGCTGGAGAGCAGGGGCCCCACCACCAGCGAGAAG (SEQ ID NO 77);
ACCCTGAAAGAGGTTCTTGGTGGTGAAGCAGCCGTGCGC (SEQ ID NO 78); CCCTGCCGGAGCAAGAACATCCTGCCCGTG (SEQ ID NO 79);
GACCCCTGCGTGCACAGCGTGGACGTGCTGCTGAGG (SEQ ID NO 80);
TCCACATTTCCTCCCACTCCCGTC (SEQ ID NO 81);
AATGCCCCATTACTCTCAAAAGTGTCCCCA (SEQ ID NO 82);
ATG G CAC AGTGTATCGTTAACCG C (SEQ ID NO 83);
AAGCCCAACGTGGAGGGCAGGTACGGCCTGAGCAGGAGCGAGACCAACAAG(S EQ ID NO 84);
CAGCAAGGCCCAGAGGCGCCACTGCCCAGCCTGCAGGTTGCAGAAGTGCCTA AA (SEQ ID NO 85);
CCCCATGCGGGCTCCACTCAGACAGAGTGGCCCAAA (SEQ ID NO 86);
CCAATACAGATCAATGCTTGCAAA (SEQ ID NO 87);
GGTGGAATGCGTGAGTCATGGGATGGAGGACAAGTT (SEQ ID NO 88);
GAATCTCTGGTGTCAAAAGGGTTTCGG (SEQ ID NO 89);
TG CTCATTAGTCCTTGG G GAG ATG CAAATCAA A (SEQ ID NO 90);
CTG G ACAAGTG G G AG AAG ATCAG G (SEQ ID NO 91);
CAGATGATGATCGCCGCCAGCGAGAAG(SEQ ID NO 92);
CAGCCCAGCCTGCCCACCGGCAGCGAGGAGCTGAAG (SEQ ID NO 93);
GAGGCCCTGGACAAGATCGAGGAGATCCAGAACAAGAACAAGCAGAAG (SEQ ID NO 152);
CCCCCCATCCCCGTGGGCGGCATCTACAAG (SEQ ID NO 94);
CAGGAGCAGAACCCCCCCCCCAGCGTGAGCCTGAGGAGCCTGTTCGGCAACG ACCCCCTG(SEQ ID NO 95);
GAGAAGATCGAGCAGCTGAGG (SEQ ID NO 96);
GACCTGCTGCTGATCGTGGCCAGG (SEQ ID NO 97); ATCGTGGAGCTGCTGGGCAGG (SEQ ID NO 98);
ATCTGGGGCAACGTGACCTGGATGGAGTGGGAGAGG (SEQ ID NO 99);
CTGAAGGACCTGTTCCCCAACAAG (SEQ ID NO 100);
GCCAAGCTGGAGGAGAGCTTCCCCGGCAAG (SEQ ID NO 101);
AACGGCAGCCTGGCCGGCGAGAGCATCATCATCAGG (SEQ ID NO 102);
AACATCACCTTCAACAGCAGCGCCGGCGGCGACCTGGAGATCACC (SEQ ID NO 103);
GCCGTGATCCTGCTGCTGGACAGGCTGAGG (SEQ ID NO 104);
GGCCCCGGCATCCACATCGGCAAGAGG (SEQ ID NO 105);
GTG ATC ATCTG C AG CG CC AG C AAG (SEQ ID NO 106);
GAG AG CTTCCCCAAC AAG (SEQ ID NO 107);
TTCGGCAACAAGACCACCATCATCTTCACCAAG (SEQ ID NO 108);
CTGCTGAACGGCAGCCTGGCCGGCGAGAGCATCATCATCAGG (SEQ ID NO
109) ;
GTGAACGTGAGCGTGACCAACAACAACACCACCACCAACGTG (SEQ ID NO
110) ;
GCCATCCTGCACATCCTGAGGAGG (SEQ ID NO 111);
GACCTGCTGGCCCTGGACAAG (SEQ ID NO 112);
ATCGGCTGCCAGCACAGCAGGATCGGCATCACCCTGCCCAGG (SEQ ID NO
113) ;
ACCCTGCAGTACCTGGCCCTGACCGCCCTGGTGACCCCCAAG (SEQ ID NO
114) ;
CCCACCACCCCCGTGACCCCCGCCCCCGGCGTGGGCGAGATCAGCAAGGAGC TGGCCCAGGGCAAG (SEQ ID NO 115);
GTGGTGAGCAGCGCCCTGCAGTACCTGATCCCCAGG (SEQ ID NO 116);
CCCGTGTGGGCCGAGCCCGTGAAG (SEQ ID NO 1 17);
GTGAAGGGCAACGACCTGCTGAAG (SEQ ID NO 118);
GAGGACCTGAACCTGCAGGACTGGAAG (SEQ ID NO 119);
CTGTTCGTGAGCGTGCTGCAGAGG (SEQ ID NO 120);
GCCGCCGAGCAGAAGGCCAGCCCCCCCAGCCTGACCCCCAAG (SEQ ID NO 121);
CCCCTGAGCCTGCCCCTGAAGATC (SEQ ID NO 122);
TTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID NO 123);
ATCTGG CACCAC ACCTTCTAC AACG AG CTG AG G (SEQ ID NO 124);
ATGACCCAGATCATGTTCGAGACCTTC (SEQ ID NO 125); GAGGAGGAGGTGGCCGCCCTGGTGATCGACAACGGCAGCGGCATGTGCAAG (SEQ ID NO 126);
GTGGCCCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCA AG (SEQ ID NO 127);
GCCGTGTTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID NO 128);
TACCCCATCGAGCACGGCATCGTGACCAACTGGGACGACATGGAGAAG (SEQ ID NO 129);
GCCGTGTTCCCCAGCATCGTGGGCAGG(SEQ ID NO 130);
ATCGGCAGGAAGGACGCCGAGAGGCAGCTGCTGAGCCCCGGCAACGCCAGG
(SEQ ID NO 131);
GACAGCTACGTGGGCGACGAGGCCCAGAGCAAGAGG (SEQ ID NO 132);
AGGGGCATCCTGACCCTGAAG (SEQ ID NO 133);
TGGGGCATCGCCCACACCACCGGCATCCCCGGCAACAGCCAGGGCCAGGCCA TGGTGGAGAGG (SEQ ID NO 134);
CACCTGGTGGACCAGCTGATCAGGGACCTGAAG (SEQ ID NO 135);
TACGAGCAGCTGCAGCTGCAGGCCAGG (SEQ ID NO 136);
ACCATCACCCTGGAGGTGGAGCCCAGCGACACCATCGAGAACGTGAAG (SEQ ID NO 137);
ATC AACAG G G AG CTGCTG AAG (SEQ ID NO 138);
CAGACCATCATCCCCACCAACAAGGACGTGGACGAGAAG (SEQ ID NO 139); AAGAACTACGGCAAGCTGGACAAG (SEQ ID NO 140);
ACC ACCATCAG CTTC AG C AAG (SEQ ID NO 141);
CAGGACAGGACCACCATCAGCTTCAGCAAG (SEQ ID NO 142);
GCCCAGGGCAGGGCCATCGCCCACAAG (SEQ ID NO 143);
ATGGCCCAGACCCAGCTGGTGCCCGTGAAG (SEQ ID NO 144);
CAGGAGCTGATCCCCCCCTGCAAG (SEQ ID NO 145);
GACATCGTGCTGCTGGAGAACGGCAAG (SEQ ID NO 146);
CTGCCCCCCCTGAGCATCCTGAAG (SEQ ID NO 147);
ATCTTCCTGATCAACCTGGCCTTCCTGATCAAG (SEQ ID NO 148);
AACAGGCTGGAGATCCTGAAG (SEQ ID NO 149);
GCCGACGACGTGGCCGTGCTGCAGGACGCCCTGGGCAGG (SEQ ID NO 150); CTGAACAAGAGCCTGGAGCAGCTGAGG (SEQ ID NO 151); and
variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152. In another aspect, the present invention provides a vector capable of encoding one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
In a further aspect, the present invention provides a vector comprising one or more polynucleotide sequences encoding one or more of the peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76;
and/or comprising one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs 77 to 152 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
In a further aspect, the present invention provides an antibody capable of binding to one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
The present invention provides in another aspect, a pharmaceutical composition comprising: one or more peptides according to the present invention; and/or
one or more polynucleotide sequences according to the present invention; and/or one or more vectors according to the present invention; and/or
one or more antibodies according to the present invention;
and a pharmaceutically acceptable carrier, vehicle, diluent or excipient.
In a further aspect, the present invention provides a composition according to the present invention, and/or a vector according to the present invention, and/or antibody according to the present invention, and/or a pharmaceutical composition according to the present invention for use in the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV and/or NEV in an animal.
The present invention provides, in a further aspect, a kit comprising:
a composition according to the present invention; and/or a vector according to the present invention; and/or
one or more antibodies according to the present invention; and/or
a pharmaceutical composition according to the present invention;
and optionally instructions for administration to an animal.
The present invention provides, in another aspect, a diagnostic method comprising obtaining a sample from an animal and determining the presence or absence in said sample of antibodies which are capable of binding to one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76
and/or determining the presence or absence in said sample of one or more of peptides of the present invention;
and/or determining the presence or absence in said sample of one or more polynucleotide sequence of the present invention.
In another aspect, the present invention provides a kit comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76 and optionally instructions for determining the presence or absence in an animal sample of antibodies capable of binding to one or more said peptides.
The present invention provides, in a further aspect, a kit comprising one or more antibodies according to the present invention and optionally instructions for determining the presence or absence in an animal sample of peptides capable of binding to said antibodies.
The present invention provides, in another aspect, an oligonucleotide sequence (e.g. a primer or a probe) comprising or consisting of a polynucleotide sequence capable of encoding one or more of the peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76
and/or comprising or consisting of one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs: 77 to 152 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
In a further aspect, the present invention provides a kit comprising one or more oligonucleotide sequences according to the present invention and optionally instructions for determining the presence or absence in an animal sample of said polynucleotide sequence.
In another aspect, the present invention provides a method for controlling EIAV and/or NEV disease in a group of animals comprising the identification of EIAV and/or NEV infection in an animal using the diagnostic method according to the present invention.
In another aspect, the present invention provides a method for controlling EIAV and/or NEV disease in a group of animals comprising the identification of EIAV and/or NEV infection in an animal using the diagnostic method according to the present invention and the isolation of an EIAV and/or NEV infected animal from other animals.
The present invention provides, in another aspect, the use of a composition according to the present invention, and/or a vector according to the present invention, and/or an antibody according to the present invention, and/or a pharmaceutical composition according to the present invention, for the manufacture of a medicament for the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV and/or NEV in an animal.
In a further aspect, the present invention provides a method for the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV and/or NEV in an animal, wherein said method comprising administering to an animal one or more compositions according to the present invention, and/or one or more vectors according to the present invention, and/or one or more antibodies according to the present invention, and/or one or more pharmaceutical compositions according to the present invention.
BRIEF DESCRIPTION OF FIGURES
Figure 1. Immunoblot determining equine infectious anemia (EIAV) seropositiveness.
Figure 2. NEV cell lines established from AGID negative horses showed positive reverse transcriptase activity, cytopathic effects and syncytia cell formation. Fig.2.A1 and 2.A2 - NEV.MaA Macrophage-like cells 2,3 weeks after culturing show Syncytia Cell Formation. Syncytia are indicated by arrows. Fig. 2.B - NEV.MaA Macrophage-like cell after passaging (cell line). Fig. 2.C - NEV.MaA. VpC: Macrophage-like cell line 5 days after infection with NEV viral particles C. Fig.2.D1 Reverse transcriptase (RT) activity in viral supernatants NEV.MaA cells infected with NEV viral particles 5 days after infection. RT activity was measured by EnZCheck RT assay. Fig.2.D2 shows standard curve obtained with EnZCheck Assay for lambda DNA.
Figure 3A. NEV purified viral particles: viral RNA.
Figure 3B. NEV purified viral particles: cDNA and viral proteins.
Figure 4. Cytopathic effects (CPE) and viability of infected cells. MaC and ED mock cells (Fig. 4A), MaC or ED cells infected with NEV virus (Fig. 4B) and ED cells infected with EIAV-Wyoming (Fig. 4C) after 7 days of infection. Presto blue cell viability assays of infected ED cells at day 11 post infection (Fig. 4D).
Figure 5. In vitro cleavage activity of viral protein fractions analyzed by size-exclusion chromatography. Cleavage of Phe-Phe substrate at pH=3 (Fig. 5A), pH=4 (Fig. 5B) and its inhibition by Indinavir or pepstatin (10 μΜ). Cleavage of the specific HIV Protease Substrate 1 Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-lle-Val-Gln-Lys(DABCYL)-Arg and its inhibition by Indinavir (10 μΜ) (Fig. 5C).
Figure 6. (A) Dose response curves for mab#4G6E4. (B) Dose responses curve and inhibition rates of NEV seronegative and seropositive samples. (C) Representative inhibition rates (%l) of NEV seropositive and one seronegative. Inhibition rates were obtained for a SEQ ID NO: 159 peptide concentration of 96 ng/mL of peptide and mab#4G6E4 IgG concentration of 70ng/mL
DETAILED DESCRIPTION
EIAV
EIAV (equine infectious anaemia virus) is a retrovirus.
EIAV causes equine infectious anaemia (EIA) in equines. Equine infectious anaemia is also known as swamp fever in horses. In addition to horses (Equus caballus) EIAV can infect donkeys (Equus asinus) (Cook et al., 2001) and mules (Spyrou et al., 2003). However, a wide range of host susceptibility to disease expression is exhibited among these species (Cook et al., 2001; Hammond et al., 2000; Spyrou et al., 2003). EIAV infected horses can present three different disease states during infection: acute/sub-acute, chronic and inapparent.
EIAV is transmitted by bloodsucking insects. The virus (EIAV) is endemic in the Americas, parts of Europe, the Middle and Far East, Russia, and South Africa. The virus is a lentivirus. EIAV can be transmitted through blood, saliva, milk, and body secretions. Transmission is primarily through biting flies, such as the horse-fly and deer-fly. The virus can survive up to 4 hours in the carrier. Contaminated surgical equipment and recycled needles and syringes, and bits can transmit EIAV. Further, mares can transmit EIAV to their foals via the placenta. The risk of transmitting the disease is greatest when an infected horse is ill, as the blood levels of the virus are then high.
The EIA incubation period lasts usually one to three weeks, but may be as long as three months.
The most notable of the signs of disease are the concurrent development of febrile episodes (defined as rectal temperatures above 39°C), thrombocytopenia (defined as platelets levels bellow 105000/μΙ of blood), that are typically accompanied by viremia at least of 105 copies of EIAV RNA/mL plasma.
The acute form of EIA is a sudden onset of the disease at full-force. Clinical signs include high fever, anemia (due to the breakdown of red blood cells), thrombocytopenia, weakness, swelling of the lower abdomen and legs, weak pulse, irregular heartbeat, tachypneia, petechiae on the mucous membrane, diarrhoea and blood stained feces. Thrombocytopenia is a consistent hematological finding and one of the earliest hematological abnormalities detected in acutely infected horses (Clabough et al., 1991 ; Crawford et al., 1996). Neurological signs are also reported in EIA infected horses (Oaks et al., 2004). Occasionally, death occurs during the acute infection, and the equine may die suddenly. After the initial bout, the majority of the horses may become asymptomatic.
The subacute form of EIA is a slower, less severe progression of the disease. Symptoms include recurrent fever, weight loss, an enlarged spleen (felt during a rectal examination), anemia, and swelling of the lower chest, abdominal wall, penile sheath, scrotum, and legs.
Some develop chronic recurring EIA signs that vary from mild illness and failure to thrive to fever, depression, petechial hemorrhages on the mucous membranes, weight loss, edema, and sometimes death. The chronic form of EIA is where an equine tires easily and is unsuitable for work. The equine may have a recurrent fever and anemia; the equine may relapse to the subacute or acute form even several years after the original attack. The majority of infected horses become life long inapparent carriers with no overt clinical abnormalities as a result of infection (Coggins, 1984; Leroux et al., 2004; McGuire et al., 990), yet still tests positive for EIA antibodies. Such an equine can still pass on the virus.
In contrast to the pathogenesis observed in infected horses, no evident clinical signs result from EIAV in in vivo experimental infections of donkeys and mules. Indeed these Equids behave as inapparent carriers from the onset of infection (Cook et al., 2001 ; Spyrou et al., 2003).
EIA may cause abortion in pregnant mares. This may occur at any time during the pregnancy if there is a relapse when the virus enters the blood. Most infected mares will abort, however some give birth to healthy foals. The foals are not necessarily infected.
The present inventors have discovered a new and previously uncharacterised virus, which was obtained from horses with discordant results for EIAV testing i.e. the horses were positive for EIAV in an immunoblot but negative for EIAV in both the AGID test and an ELISA.
The present inventors have named this new uncharacterised virus New Equine Virus (NEV). NEV may be obtainable from or may be similar to a virus obtainable from the deposit made at the European Collection of Cell Cultures (ECAAC), Culture Collections, under accession number 14120201. Other workers in the field may refer to NEV as EIAV or EIAV-like.
The present inventors have identified a number of NEV peptides which find use in the present invention. Thus, the NEV virus as described herein may comprise or contain any of SEQ ID NOS: 1- 76.
Thus, the NEV virus as described herein may comprise or contain any of SEQ ID NOS: 77-
152.
Examples of NEV peptides of the present invention include.
SRLLESRGPTTSEK (SEQ ID NO: 1);
TLKEVLGGEAAVR (SEQ ID NO: 2);
PCRSKNILPV (SEQ ID NO: 3);
DPCVHSVDVLLR (SEQ ID NO: 4);
STFPPTPV (SEQ ID NO: 5);
NAPLLSKVSP (SEQ ID NO: 6);
MAQCIVNR (SEQ ID NO: 7);
KPNVEGRYGLSRSETNK (SEQ ID NO: 8);
QQGPEAPLPSLQVAEVPK (SEQ ID NO: 9);
PH AG STQTE WPK (SEQ ID NO: 10);
PIQINACK (SEQ ID NO: 11);
GGMRESWDGGQV (SEQ ID NO: 12);
ESLVSKGFR (SEQ ID NO: 13);
CSLVLGEMQIKRIR (SEQ ID NO: 14);
LDKWEKIR (SEQ ID NO 15);
QMMIAASEK (SEQ ID NO 16);
QPSLPTGSEELK (SEQ ID NO 17);
EALDKIEEIQNKNKQK (SEQ ID NO 18); PPIPVGGIYK (SEQ ID NO 19);
QEQNPPPSVSLRSLFGNDPL (SEQ ID NO 20); EKIEQLR (SEQ ID NO 21);
DLLLIVAR (SEQ ID NO 22);
I ELLGR (SEQ ID NO 23);
IWGNVTWMEWER (SEQ ID NO 24);
LKDLFPNK (SEQ ID NO 25);
AKLEESFPGK (SEQ ID NO 26);
NGSLAGESIIIR (SEQ ID NO 27);
NITFNSSAGGDLEIT (SEQ ID NO 28);
AVILLLDRLR (SEQ ID NO 29);
GPGIHIGKR (SEQ ID NO 30);
VIICSASK (SEQ ID NO 31);
ESFPNK (SEQ ID NO 32);
FGNKTTIIFTK (SEQ ID NO 33);
LLNGSLAGESIIIR (SEQ ID NO 34);
VNVSVTNNNTTTNV (SEQ ID NO 35);
AILHILRR (SEQ ID NO 36);
DLLALDK (SEQ ID NO 37);
IGCQHSRIGITLPR (SEQ ID NO 38);
TLQYLALTALVTPK (SEQ ID NO 39);
PTTPVTPAPGVGEISKELAQGK (SEQ ID NO 40); WSSALQYLIPR (SEQ ID NO 41);
PVWAEPVK (SEQ ID NO 42);
VKGNDLLK (SEQ ID NO 43);
EDLNLQDWK (SEQ ID NO 44);
LFVSVLQR (SEQ ID NO 45);
AAEQKASPPSLTPK (SEQ ID NO 46);
PLSLPLKI (SEQ ID NO 47);
FPSIVGRPR (SEQ ID NO 48);
IWHHTFYNELR (SEQ ID NO 49);
MTQIMFETF (SEQ ID NO 50);
EEEVAALVIDNGSGMCK (SEQ ID NO 51);
VAPEEHPVLLTEAPLNPK (SEQ ID NO 52);
AVFPSIVGRPR (SEQ ID NO 53); YPIEHGIVTNWDDMEK (SEQ ID NO 54);
AVFPSIVGR (SEQ ID NO 55);
IGRKDAERQLLSPGNAR (SEQ ID NO 56);
DSYVGDEAQSKR (SEQ ID NO 57);
RGILTLK (SEQ ID NO 58);
WGIAHTTGIPGNSQGQAMVER (SEQ ID NO 59);
HLVDQLIRDLK (SEQ ID NO 60);
YEQLQLQAR (SEQ ID NO 61);
TITLEVEPSDTIENVK (SEQ ID NO 62);
INRELLK (SEQ ID NO 63);
QTIIPTNKDVDEK (SEQ ID NO 64);
KNYGKLDK (SEQ ID NO 65);
TTISFSK (SEQ ID NO 66);
QDRTTISFSK (SEQ ID NO 67);
AQGRAIAHK (SEQ ID NO 68);
MAQTQLVPVK (SEQ ID NO 69);
QELIPPCK (SEQ ID NO 70);
DIVLLENGK (SEQ ID NO 71);
LPPLSILK (SEQ ID NO 72);
IFLINLAFLIK (SEQ ID NO 73);
NRLEILK (SEQ ID NO 74);
ADDVAVLQDALGR (SEQ ID NO 75);
LNKSLEQLR (SEQ ID NO 76); and
variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
In one embodiment, NEV peptides of the present invention are selected from the group consisting of SEQ ID NOs 1 to 14.
Examples of nucleotide sequences encoding NEV peptides of the present invention include polynucleotide sequences encoding:
SRLLESRGPTTSEK (SEQ ID NO: 1);
TLKEVLGG EAAVR (SEQ ID NO: 2);
PCRSKNILPV (SEQ ID NO: 3);
DPCVHSVDVLLR (SEQ ID NO: 4); STFPPTPV (SEQ ID NO: 5);
NAPLLSKVSP (SEQ ID NO: 6);
MAQCIVNR (SEQ ID NO: 7);
KPNVEGRYGLSRSETNK (SEQ ID NO: 8); QQGPEAPLPSLQVAEVPK (SEQ ID NO: 9); PHAGSTQTEWPK (SEQ ID NO: 10);
PIQINACK (SEQ ID NO: 11);
GGMRESWDGGQV (SEQ ID NO: 12);
ESLVSKGFR (SEQ ID NO: 13);
CSLVLGEMQIKRIR (SEQ ID NO: 14);
LDKWEKIR (SEQ ID NO 15);
QMMIAASEK (SEQ ID NO 16);
QPSLPTGSEELK (SEQ ID NO 17);
EALDKIEEIQNKNKQK (SEQ ID NO 18);
PPIPVGGIYK (SEQ ID NO 19);
QEQNPPPSVSLRSLFGNDPL(SEQ ID NO 20); EKIEQLR (SEQ ID NO 21);
DLLLIVAR (SEQ ID NO 22);
IVELLGR (SEQ ID NO 23);
IWGNVTWMEWER (SEQ ID NO 24);
LKDLFPNK(SEQ ID NO 25);
AKLEESFPGK (SEQ ID NO 26);
NGSLAGESIIIR (SEQ ID NO 27);
NITFNSSAGGDLEIT (SEQ ID NO 28);
AVILLLDRLR(SEQ ID NO 29);
GPGIHIGKR ( SEQ ID NO 30);
VIICSASK (SEQ ID N0 31);
ESFPNK (SEQ ID NO 32);
FGNKTTIIFTK (SEQ ID NO 33);
LLNGSLAGESIIIR (SEQ ID NO 34);
VNVSVTNNNTTTNV (SEQ ID NO 35);
AILHILRR (SEQ ID NO 36);
DLLALDK (SEQ ID NO 37);
IGCQHSRIGITLPR (SEQ ID NO 38);
TLQYLALTALVTPK (SEQ ID NO 39); PTTPVTPAPGVGEISKELAQGK (SEQ ID NO 40); WSSALQYLIPR (SEQ ID NO 41);
PVWAEPVK (SEQ ID NO 42);
VKGNDLLK (SEQ ID NO 43);
EDLNLQDWK (SEQ ID NO 44);
LFVSVLQR (SEQ ID NO 45);
AAEQKASPPSLTPK (SEQ ID NO 46);
PLSLPLKI (SEQ ID NO 47);
FPSIVGRPR (SEQ ID NO 48);
IWHHTFYNELR (SEQ ID NO 49);
MTQIMFETF (SEQ ID NO 50);
EEEVAALVIDNGSGMCK (SEQ ID NO 51);
VAPEEHPVLLTEAPLNPK (SEQ ID NO 52);
AVFPSIVGRPR (SEQ ID NO 53);
YPIEHGIVTNWDDMEK (SEQ ID NO 54);
AVFPSIVGR (SEQ ID NO 55);
IGRKDAERQLLSPGNAR (SEQ ID NO 56);
DSYVGDEAQSKR (SEQ ID NO 57);
RGILTLK (SEQ ID NO 58);
WGIAHTTGIPGNSQGQAMVER (SEQ ID NO 59); HLVDQLIRDLK (SEQ ID NO 60);
YEQLQLQAR (SEQ ID NO 61);
TITLEVEPSDTI ENVK (SEQ ID NO 62);
INRELLK (SEQ ID NO 63);
QTIIPTNKDVDEK (SEQ ID NO 64);
KNYGKLDK (SEQ ID NO 65);
TTISFSK (SEQ ID NO 66);
QDRTTISFSK (SEQ ID NO 67);
AQGRAIAHK (SEQ ID NO 68);
MAQTQLVPVK (SEQ ID NO 69);
QELIPPCK (SEQ ID NO 70);
DIVLLENGK (SEQ ID NO 71);
LPPLSILK (SEQ ID NO 72);
IFLINLAFLIK (SEQ ID NO 73);
NRLEILK (SEQ ID NO 74); ADDVAVLQDALGR (SEQ ID NO 75);
LNKSLEQLR (SEQ ID NO 76); and
variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
In one embodiment, nucleotide sequences encoding NEV peptides of the present invention are selected from the group consisting of polynucleotide sequences encoding any one of SEQ ID NOs 1 to 14.
Further examples of nucleotide sequences encoding NEV peptides of the present invention include:
AGCAGGCTGCTGGAGAGCAGGGGCCCCACCACCAGCGAGAAG (SEQ ID NO 77);
ACCCTGAAAGAGGTTCTTGGTGGTGAAGCAGCCGTGCGC (SEQ ID NO 78); CCCTGCCGGAGCAAGAACATCCTGCCCGTG (SEQ ID NO 79);
GACCCCTGCGTGCACAGCGTGGACGTGCTGCTGAGG (SEQ ID NO 80);
TCCACATTTCCTCCCACTCCCGTC (SEQ ID NO 81);
AATGCCCCATTACTCTCAAAAGTGTCCCCA (SEQ ID NO 82);
ATG G CACAGTGTATCGTTAACCG C (SEQ ID NO 83);
AAGCCCAACGTGGAGGGCAGGTACGGCCTGAGCAGGAGCGAGACCAACAAG(S EQ ID NO 84);
CAGCAAGGCCCAGAGGCGCCACTGCCCAGCCTGCAGGTTGCAGAAGTGCCTA AA (SEQ ID NO 85);
CCCCATGCGGGCTCCACTCAGACAGAGTGGCCCAAA (SEQ ID NO 86);
CCAATACAGATCAATGCTTGCAAA (SEQ ID NO 87);
GGTGGAATGCGTGAGTCATGGGATGGAGGACAAGTT (SEQ ID NO 88);
GAATCTCTGGTGTCAAAAGGGTTTCGG (SEQ ID NO 89);
TGCTCATTAGTCCTTGGGGAGATGCAAATCAAA (SEQ ID NO 90);
CTGGACAAGTGGGAGAAGATCAGG (SEQ ID NO 91);
CAGATGATGATCGCCGCCAGCGAGAAG(SEQ ID NO 92);
CAGCCCAGCCTGCCCACCGGCAGCGAGGAGCTGAAG (SEQ ID NO 93);
GAGGCCCTGGACAAGATCGAGGAGATCCAGAACAAGAACAAGCAGAAG (SEQ ID NO 152);
CCCCCCATCCCCGTGGGCGGCATCTACAAG (SEQ ID NO 94);
CAGGAGCAGAACCCCCCCCCCAGCGTGAGCCTGAGGAGCCTGTTCGGCAACG ACCCCCTG(SEQ ID NO 95);
GAGAAGATCGAGCAGCTGAGG (SEQ ID NO 96);
GACCTGCTGCTGATCGTGGCCAGG (SEQ ID NO 97);
ATCGTGGAGCTGCTGGGCAGG (SEQ ID NO 98);
ATCTGGGGCAACGTGACCTGGATGGAGTGGGAGAGG (SEQ ID NO 99);
CTGAAGGACCTGTTCCCCAACAAG (SEQ ID NO 100);
GCCAAGCTGGAGGAGAGCTTCCCCGGCAAG (SEQ ID NO 101);
AACGGCAGCCTGGCCGGCGAGAGCATCATCATCAGG (SEQ ID NO 102);
AACATCACCTTCAACAGCAGCGCCGGCGGCGACCTGGAGATCACC (SEQ ID NO 103);
GCCGTGATCCTGCTGCTGGACAGGCTGAGG (SEQ ID NO 104);
GGCCCCGGCATCCACATCGGCAAGAGG (SEQ ID NO 105);
GTG ATC ATCTG C AG CG CC AG CAAG (SEQ ID NO 106);
GAGAGCTTCCCCAACAAG (SEQ ID NO 107);
TTCGGCAACAAGACCACCATCATCTTCACCAAG (SEQ ID NO 108);
CTG CTG AACG GCAGCCTGGCCGGCGAGAG CATC ATC ATCAG G (SEQ ID NO
109) ;
GTGAACGTGAGCGTGACCAACAACAACACCACCACCAACGTG (SEQ ID NO
110) ;
GCCATCCTGCACATCCTGAGGAGG (SEQ ID NO 111);
GACCTGCTGGCCCTGGACAAG (SEQ ID NO 112);
ATCGGCTGCCAGCACAGCAGGATCGGCATCACCCTGCCCAGG (SEQ ID NO
113) ;
ACCCTGCAGTACCTGGCCCTGACCGCCCTGGTGACCCCCAAG (SEQ ID NO
114) ;
CCCACCACCCCCGTGACCCCCGCCCCCGGCGTGGGCGAGATCAGCAAGGAGC TGGCCCAGGGCAAG (SEQ ID NO 115);
GTGGTGAGCAGCGCCCTGCAGTACCTGATCCCCAGG (SEQ ID NO 116);
CCCGTGTGGGCCGAGCCCGTGAAG (SEQ ID NO 117);
GTGAAGGGCAACGACCTGCTGAAG (SEQ ID NO 118);
GAGGACCTGAACCTGCAGGACTGGAAG (SEQ ID NO 119);
CTGTTCGTGAGCGTGCTGCAGAGG (SEQ ID NO 120);
GCCGCCGAGCAGAAGGCCAGCCCCCCCAGCCTGACCCCCAAG (SEQ ID NO 121);
CCCCTG AG CCTG CCCCTG AAG ATC (SEQ ID NO 122); TTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID NO 123);
ATCTGGCACCACACCTTCTACAACGAGCTGAGG (SEQ ID NO 124);
ATGACCCAGATCATGTTCGAGACCTTC (SEQ ID NO 125);
GAGGAGGAGGTGGCCGCCCTGGTGATCGACAACGGCAGCGGCATGTGCAAG
(SEQ ID NO 26);
GTGGCCCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCA AG (SEQ ID NO 127);
GCCGTGTTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID NO 128);
TACCCCATCGAGCACGGCATCGTGACCAACTGGGACGACATGGAGAAG (SEQ ID NO 129);
GCCGTGTTCCCCAGCATCGTGGGCAGG(SEQ ID NO 130);
ATCGGCAGGAAGGACGCCGAGAGGCAGCTGCTGAGCCCCGGCAACGCCAGG
(SEQ ID NO 131);
G ACAGCTACGTG GG CG ACG AG G CCCAG AG CAAG AG G (SEQ ID NO 132);
AGGGGCATCCTGACCCTGAAG (SEQ ID NO 133);
TGGGGCATCGCCCACACCACCGGCATCCCCGGCAACAGCCAGGGCCAGGCCA TGGTGGAGAGG (SEQ ID NO 134);
CACCTGGTGGACCAGCTGATCAGGGACCTGAAG (SEQ ID NO 135);
TACGAGCAGCTGCAGCTGCAGGCCAGG (SEQ ID NO 136);
ACCATCACCCTGGAGGTGGAGCCCAGCGACACCATCGAGAACGTGAAG (SEQ ID NO 137);
ATCAACAGGGAGCTGCTGAAG (SEQ ID NO 138);
CAGACCATCATCCCCACCAACAAGGACGTGGACGAGAAG (SEQ ID NO 139); AAGAACTACGGCAAGCTGGACAAG (SEQ ID NO 140);
ACCACCATCAG CTTCAG CAAG (SEQ ID NO 141);
CAGGACAGGACCACCATCAGCTTCAGCAAG (SEQ ID NO 142);
GCCCAGGGCAGGGCCATCGCCCACAAG (SEQ ID NO 143);
ATGGCCCAGACCCAGCTGGTGCCCGTGAAG (SEQ ID NO 144);
CAGGAGCTGATCCCCCCCTGCAAG (SEQ ID NO 145);
G ACATCGTG CTG CTGG AG AACGG CAAG (SEQ ID NO 146);
CTGCCCCCCCTGAGCATCCTGAAG (SEQ ID NO 147);
ATCTTCCTGATCAACCTGGCCTTCCTGATCAAG (SEQ ID NO 148);
AACAGGCTGGAGATCCTGAAG (SEQ ID NO 149);
GCCGACGACGTGGCCGTGCTGCAGGACGCCCTGGGCAGG (SEQ ID NO 150); CTGAACAAGAGCCTGGAGCAGCTGAGG (SEQ ID NO 151); and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
In one embodiment, nucleotide sequences encoding NEV peptides of the present invention are selected from the group consisting of SEQ ID NOs 77 to 89.
The peptides of the present invention and the nucleotide sequences of the present invention are derived from a novel virus, NEV. Without wishing to be bound by theory, NEV identified by the present inventors represents a completely new and uncharacterised virus.
In some embodiments, the peptides of the invention and/or the novel viral strain or variant as described herein may be denoted as "EIAV-like". By "EIAV-like" it is meant that the virus and/or peptides were derived from horses with discordant results obtained from EIAV testing i.e. the horses were positive using an EIAV immunoblot but negative using the AGID test and an ELISA. None of the identified peptides belonged to the known EIAV proteome. Some of the peptides were found to have sequence identity with retroviral peptides. Some of the peptides were found to have sequence identity to part of the HIV-1 proteome.
Table 2 shows the viral proteins which have the highest sequence identity (similarity) to SEQ ID NOs 1-76 of the present invention.
Table 2. Similarity of peptides of the invention to known viral peptides (if any)
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Both EIAV and NEV can infect animals such as equines. In one embodiment the animal is an equid.
Examples of equids include horses, donkeys, mules, hinnys and zebras.
In some embodiments, the terms "equid" and "equine" are used interchangeably.
In another embodiment, the animal is a horse.
In one embodiment a peptide according to the present invention, and/or a nucleotide sequence according to the present invention, and/or a vector according to the present invention, and/or an antibody according to the present invention, and/or a pharmaceutical composition according to the present invention is capable of inducing an immune response against EIAV and/or NEV in an animal. In another embodiment, a peptide according to the present invention, and/or a nucleotide sequence according to the present invention, and/or a vector according to the present invention, and/or an antibody according to the present invention, and/or a pharmaceutical composition according to the present invention is capable of inducing a protective immune response against EIAV and/or NEV in an animal.
As used herein the term "treatment of equine infectious anaemia" refers to a reduction in EIA symptoms in the treated animal. For example, the animal may no longer be classified as having an acute form of EIA but now has the subacute form, or the chronic form, or the inapparent form and no longer shows any EIA symptoms - the virus may, however, still be detected in the animal but at levels below, for example, 105 copies of EIAV RNA/mL plasma. For instance, after treatment, the EIAV (e.g. the EIAV of the present description) viral titre in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the EIAV viral titre of the animal before treatment. In another example, thrombocytopenia in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the thrombocytopenia of the animal before treatment. In a further example, the peaks of the fever in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the temperature of the animal before treatment. Without wishing to be bound by theory, the treatment of equine infectious anaemia occurs by a reduction in the level of an EAIV virus in an animal. As used herein the term "treatment of NEV" refers to a reduction in NEV symptoms in the treated animal. For example, the animal may no longer be classified as having an acute form of NEV but now has the subacute form, or the chronic form, or the inapparent form and no longer shows any NEV symptoms - the virus may, however, still be detected in the animal but at levels below, for example, 10s copies of NEV RNA/mL plasma. For instance, after treatment, the NEV (e.g. the NEV of the present description) viral titre in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the NEV viral titre of the animal before treatment. In another example, thrombocytopenia in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the thrombocytopenia of the animal before treatment. In a further example, the peaks of the fever in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the temperature of the animal before treatment. Without wishing to be bound by theory, the treatment of NEV occurs by a reduction in the level of an NEV virus in an animal.
The viral titre may be determined in a sample from an animal such as a blood sample, a blood serum sample, a plasma sample, a saliva sample, a sputum sample, a urine sample, a semen sample, a fecal sample, a milk sample, a biopsy sample, a lymph node biopsy sample, and/or a sweat sample.
As used herein the term "prevention of equine infectious anaemia" refers to a treated animal not developing EIA symptoms if the treated animal is exposed to the equine infectious anaemia virus, for example by exposure to a bloodsucking insect carrying the virus. The treated animal did not have any EIA symptoms before treatment and/or did not have any detectable EIAV in a sample such as a blood sample before treatment. For example, commercially available tests such as the Coggins test and/or the diagnostic test of the present invention may be used to determine (i.e. detect) if there is an EIAV titre in an animal. As used herein the term "prevention of NEV" refers to a treated animal not developing NEV symptoms if the treated animal is exposed to the NEV virus, for example by exposure to a bloodsucking insect carrying the virus. The treated animal did not have any NEV symptoms before treatment and/or did not have any detectable NEV in a sample such as a blood sample before treatment. As used herein the term "treatment of an infection by an EIAV" refers to a reduction in EIAV (e.g. the EIAV of the present description) viral titre in a sample from the treated animal when compared to the EIAV viral titre of the animal before treatment. For instance, after treatment, the EIAV (e.g. the EIAV of the present description) viral titre in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the viral titre of the animal before treatment. Without wishing to be bound by theory, a reduction in the level of an EIAV virus in an animal may prevent the development of EIA symptoms.
As used herein the term "treatment of an infection by a NEV" refers to a reduction in NEV (e.g. the NEV of the present description) viral titre in a sample from the treated animal when compared to the NEV viral titre of the animal before treatment. For instance, after treatment, the NEV (e.g. the NEV of the present description) viral titre in the treated animal may be reduced by at least 10%, 20%, 30%, 40% or 50% when compared to the viral titre of the animal before treatment. Without wishing to be bound by theory, a reduction in the level of an NEV virus in an animal may prevent the development of NEV symptoms.
As used herein the term "prevention of an infection by an EIAV" refers to a treated animal not developing a detectable EIAV titre if the treated animal is exposed to the equine infectious anaemia virus, for example by exposure to a bloodsucking insect carrying the virus. The treated animal did not have any detectable EIAV in a sample such as a blood sample before treatment. For example, commercially available tests such as the Coggins test and/or the diagnostic test of the present invention may be used to determine (i.e. detect) if there is a EIAV titre in an animal.
As used herein the term "prevention of an infection by a NEV" refers to a treated animal not developing a detectable NEV titre if the treated animal is exposed to the NEV virus, for example by exposure to a bloodsucking insect carrying the virus. The treated animal did not have any detectable NEV in a sample such as a blood sample before treatment.
By using a kit according to the present invention or by using a diagnostic method of the present invention, animals which are infected with EIAV and/or NEV can be identified.
Animals with an EIAV and/or NEV infection may be isolated from other animals (e.g. animals which do not have the EIAV and/or NEV virus). Advantageously, this helps to prevent the spread of EIAV and/or NEV infection from infected animals to those which are not infected thereby controlling EIAV/NEV infection and/or EIAV/NEV within a group of animals.
Animals with an EIAV and/or NEV infection may be monitored (by using a kit according to the present invention or by using a diagnostic method of the present invention) to determine the progression of the EIAV/NEV infection and/or determine the progression of EIAV/NEV. Animals with an EIAV and/or NEV infection may be isolated from other animals (e.g. animals which do not have the EIAV and/or NEV virus) once the level of infection and/or the progression of EIAV and/or NEV has reached a critical point. Typically animals should be isolated during febrile episodes when rectal temperatures are above 39°C, platelets levels are below 105000/μΙ of blood and viremia is at least of 105 copies of EIAV and/or NEV RNA mL plasma.
In addition or alternatively, by identifying animals with an EIAV and/or NEV infection (by using a kit according to the present invention or by using a diagnostic method of the present invention) care can be taken to ensure that medical equipment used on an EIAV and/or NEV infected animal is not used on an animal which does not have a EIAV and/or NEV infection. Advantageously, this helps to prevent the spread of EIAV and/or NEV infection from infected animals to those which are not infected thereby controlling EIAV and/or NEV disease with a group of animals.
In some embodiments, an animal identified as having EIAV and/or NEV is euthanized. Typically animals which are euthanized are those with frequent febrile episodes and animals which are lethargic or in lateral recumbence. An animal having EIAV and/or NEV may be euthanized when the viremia peaks are frequent.
The present invention encompasses the use of NEV peptides in the discrimination of NEV- infected animals, and in the diagnosis, treatment and prevention of NEV infection in NEV- infected animals. Similarly, the present invention encompasses the use of NEV peptides in the discrimination of EIAV-infected animals, and in the diagnosis, treatment and prevention of EIAV infection in EIAV-infected animals. For example, the present inventors have identified EIAV reference sera (from horses) that are positive for an NEV fusion peptide of the invention. Deposit
The NEV virus described herein has been deposited by Equigerminal SA, Parque tecnologico de Cantanhede, nucleo 4 lote 4, Cantanhede, 3060-197 Portugal under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure at European Collection of Cell Cultures (ECAAC), Culture Collections, Public Health England, Porton Down, Salisbury, Wiltshire UK SP4 0JG on 2 December 2014 under accession number 14120201 , Other aspects of the present invention relate to the above vira! deposit made at the ECAAC depository under accession number 14120201.
The present invention also relates to peptide and nucleotide sequences obtainable from this deposit.
The present invention also relates to nucleotide sequences capable of hybridising to nucleotide sequences obtainable from the deposit.
The NEV virus of the deposit may also be referred to as EIAV-like in the deposit.
Vectors
As it is well known in the art, a vector is a tool that allows or facilitates the transfer of an entity from one environment to another, In accordance with the present invention, and by way of example, some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a host and/or a target cell for the purpose of replicating the vectors comprising the nucleotide sequences of the present invention and/or expressing the proteins of the invention encoded by the nucleotide sequences of the present invention. Examples of vectors used in recombinant DNA techniques include but are not limited to plasmids, chromosomes, artificial chromosomes or viruses.
The term "vector" includes expression vectors and/or transformation vectors. The term "expression vector" means a construct capable of in vivo or in vitro or ex wo expression.
As used herein, the term "expression vector" refers to a DNA construct containing a DNA coding sequence (e.g., gene sequence) that is operably linked to one or more suitable control sequence(s) capable of affecting expression of the coding sequence in a host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. The plasmid is the most commonly used form of expression vector. However, the description is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art.
The term "operably linked" refers to juxtaposition wherein the elements are in an arrangement allowing them to be functionally related. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the coding sequence. In one embodiment, the nucleotide sequence of the present invention is operably linked to a transcription unit.
The term "transcription unit(s)" as described herein are regions of nucleic acid containing coding sequences and the signals for achieving expression of those coding sequences independently of any other coding sequences. Thus, each transcription unit generally comprises at least a promoter, an optional enhancer and a polyadenylation signal.
The term "transformation vector" means a construct capable of being transferred from one species to another.
"Naked DNA"
The vectors comprising nucleotide sequences encoding polypeptides of the present invention may be administered directly as "a naked nucleic acid construct"; in one embodiment the vector comprises flanking sequences homologous to the host cell genome. As used herein, the term "naked DNA" refers to a plasmid comprising a nucleotide sequence encoding a polypeptide of the present invention together with a short promoter region to control its production. It is called "naked" DNA because the plasmids are not carried in any delivery vehicle. When such a DNA plasmid enters a host cell, such as a eukaryotic cell, the proteins it encodes (such as NEV peptides of the present invention) are transcribed and translated within the cell.
Non-viral delivery
Alternatively, the vectors comprising nucleotide sequences of the present invention may be introduced into suitable host cells using a variety of non-viral techniques known in the art, such as transfection, transformation, electroporation and biolistic transformation.
As used herein, the term "transfection" refers to a process using a non-viral vector to deliver a gene to a target mammalian cell.
Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), multivalent cations such as spermine, cationic lipids or polylysine, 1 , 2, -bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.
Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents. Examples of these agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam™ and transfectam™). Typically, nucleic acid constructs are mixed with the transfection agent to produce a composition.
Viral vectors
Alternatively, the vectors comprising nucleotide sequences of the present invention may be introduced into suitable host cells using a variety of viral techniques which are known in the art, such as for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses.
In one embodiment the vector is a recombinant viral vectors. Suitable recombinant viral vectors include but are not limited to adenovirus vectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, a retroviral vector, lentiviral vectors, baculoviral vectors, pox viral vectors or parvovirus vectors (see Kestler et a/1999 Human Gene Ther 10(10):1619- 32). In the case of viral vectors, gene delivery is mediated by viral infection of a target cell.
NEV as described herein may be used as a vector to deliver a gene of interest (GOI). For example NEV as described herein, or an element thereof, may be used as a vector to deliver a gene of interest to a cell, part of a cell, a tissue or an organism. In such embodiments, the NEV viral vector may be an attenuated virus, or may have one or more relevant and/or functional parts deleted. Retroviral vectors
Examples of retroviruses include but are not limited to: murine leukemia virus (MLV), human immunodeficiency virus (HIV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
In one embodiment vectors for use in accordance with the present invention are recombinant viral vectors, in particular recombinant retroviral vectors (RRV) such as lentiviral vectors.
The term "recombinant retroviral vector" (RRV) refers to a vector with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell includes reverse transcription and integration into the target cell genome. The RRV carries non-viral coding sequences which are to be delivered by the vector to the target cell. An RRV is incapable of independent replication to produce infectious retroviral particles within the final target cell. Usually the RRV lacks a functional gag-pol and/or env gene and/or other genes essential for replication. The vector of the present invention may be configured as a split-intron vector. A split intron vector is described in PCT patent application WO 99/15683.
A detailed list of retroviruses may be found in Coffin et al ("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).
Lentiviral vectors
Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the prototype "slow virus" visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
A distinction between the lentivirus family and other types of retroviruses is that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et a/1992 EMBO. J 11 : 3053-3058; Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, other retroviruses - such as MLV - are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
Adenoviruses In one embodiment of the present invention, the features of adenoviruses may be combined with the genetic stability of retroviruses/lentiviruses which can be used to transduce target cells to become transient retroviral producer cells capable of stably infect neighbouring cells. Such retroviral producer cells which are engineered to express a NEV peptide of the present invention can be implanted in organisms such as animals for use in the treatment or prevention of diseases such as EIA, NEV and/or for the treatment or prevention of EIAV and/or NEV infection.
Pox viruses In one embodiment, vectors for use in accordance with the present invention are recombinant pox viral vectors such as fowl pox virus (FPV), entomopox virus, vaccinia virus such as NYVAC, canarypox virus, MVA or other non-replicating viral vector systems such as those described for example in WO 95/30018. Replication vectors
The nucleotide sequences encoding the NEV peptides of the present invention may be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleotide sequence in a compatible host cell. Thus in one embodiment there is provided a method of making the polypeptide of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Expression vector
In one embodiment, a nucleotide sequence of present invention which is inserted into a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence, such as the coding sequence of the NEV peptide of the present invention by the host cell, i.e. the vector is an expression vector. The polypeptide produced by a host recombinant cell may be secreted or may be contained intracellular^ depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing the polypeptide coding sequences can be designed with signal sequences which direct secretion of the polypeptide coding sequences through a particular prokaryotic or eukaryotic cell membrane.
Expression in vitro
The vectors of the present invention may be transformed or transfected into a suitable host cell and/or a target cell as described below to provide for expression of a polypeptide of the present invention. This process may comprise culturing a host cell and/or target cell transformed with an expression vector under conditions to provide for expression by the vector of a coding sequence encoding the NEV peptide of the present invention and optionally recovering the expressed polypeptide. The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. The expression of the polypeptide of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, polypeptide production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG. Host/target cells
Host and/or target cells comprising nucleotide sequences of the present invention may be used to express the polypeptides of the present invention under in vitro, in vivo and ex vivo conditions.
The term "host cell and/or target cell" includes any cell derivable from a suitable organism which a vector is capable of transfecting or transducing. Examples of host and/or target cells can include but are not limited to cells capable of expressing the polypeptides of the present invention under in vitro, in vivo and ex vivo conditions. Examples of such cells include but are not limited to white blood cells such as macrophages. Further examples include stem cells, progenitor cells, endothelial cells respiratory airway epithelial cells, hepatocytes, muscle cells, cardiac myocytes, synoviocytes, primary mammary epithelial cess and post-mitotically terminally differentiated non-replicating cells such as monocytes, macrophages and/or neurons.
In one embodiment, the cell is an animal cell.
In one embodiment, the cell is an equine cell. The term "organism" includes any suitable organism. In one embodiment, the organism is an animal. In one embodiment, the organism is an equine.
Although the polypeptides of the invention may be produced using prokaryotic cells as host cells, in one embodiment eukaryotic cells are used, for example yeast, plant, insect or mammalian cells, in particular equine cells. Suitable host cells include bacteria such as E. coli, yeast, plant, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
The present description also provides a method comprising transforming a host and/or target cell with a or the nucleotide sequence(s) of the present invention.
The term "transformed cell" means a host cell and/or a target cell having a modified genetic structure. With the present description, a cell has a modified genetic structure when a vector according to the present invention has been introduced into the cell.
Host cells and/or a target cells may be cultured under suitable conditions which allow expression of the polypeptide of the invention.
There is also provided a method comprising culturing a transformed host cell - which cell has been transformed with a or the nucleotide sequence(s) according to the present invention under conditions suitable for the expression of the polypeptide encoded by said nucleotide sequence(s).
The present description also provides a method comprising culturing a transformed host cell - which cell has been transformed with a or the nucleotide sequence(s) according to the present invention or a homologue, or fragment thereof - under conditions suitable for the expression of the polypeptide encoded by said nucleotide sequence(s); and then recovering said polypeptide from the transformed host cell culture. The polypeptide of the present invention can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. The polypeptide may be purified and isolated in a manner known perse.
Regulation of expression in vivo or in vitro or ex vivo
The present description also encompasses gene therapy whereby the NEV-encoding nucleotide sequence(s) of the present invention is regulated in vivo or in vitro or ex vivo. For example, expression regulation may be accomplished by administering compounds that bind to the NEV-encoding nucleotide sequence(s) of the present invention, or control regions associated with the NEV-encoding nucleotide sequence of the present invention, or its corresponding RNA transcript to modify the rate of transcription or translation. Control sequences
Control sequences operably linked to sequences encoding the NEV peptide of the present invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell and/or target cell in which the expression vector is designed to be used. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
Promoters
The term promoter is well-known in the art and is used in the normal sense of the art, e.g. as an RNA polymerase binding site. The term encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
The promoter is typically selected from promoters which are functional in mammalian, cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used. The promoter is typically derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, β-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase).
Tissue-specific promoters
The promoters referred to herein may be tissue-specific promoters. That is, they are capable of driving transcription of an NEV-encoding nucleotide sequence(s) in one tissue while remaining largely "silent" in other tissue types.
Inducible promoters The promoters of the present invention may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.
It may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
Enhancer
In addition, any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
The term "enhancer" includes a DNA sequence which binds to other protein components of the transcription initiation complex and thus facilitates the initiation of transcription directed by its associated promoter.
Pharmaceutical composition
The pharmaceutical composition may be any pharmaceutical composition. In one aspect, the pharmaceutical composition is to be administered orally, enterally, rectally or parenterally. For example, the composition may be an edible composition. "Edible" means a material that is approved for human or animal consumption.
Typically in equine medicine, pharmaceutical compositions are administered orally in the form of an oral paste or a powder to be dissolved into the drinking water. Further in equine medicine, pharmaceutical compositions may also be administered by injectable routes such as subcutaneous, intramuscular or intravenous injection.
The pharmaceutical compositions may be for animal usage in veterinary medicine. Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the "Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and PJ Weller.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
Administration
The compositions, pharmaceutical compositions, or vectors of the present invention may be adapted for oral, rectal, vaginal, intrauterine, parenteral, intramuscular, intraperitoneal, intraarterial, intra-articular, intra-hoof, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
In one aspect, the compositions, pharmaceutical compositions, or vectors of the present invention are adapted for oral, rectal, vaginal, parenteral, nasal, buccal or sublingual routes of administration.
In a further aspect, the compositions, pharmaceutical compositions, or vectors of the present invention are adapted for oral administration.
For oral administration, particular use is made of compressed tablets, pills, tablets, gels, drops, pastes, and capsules.
Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intraarticular, intrauterine, intrahoof, intrathecal^, subcutaneously, intradermally, intraperitoneally, intracutane, subcutane, peroral, muscosal or intramuscularly, and which are prepared from sterile or sterilisable solutions. The compositions, pharmaceutical compositions, or vectors of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, pastes, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. In another example, the active ingredient can also be incorporated into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
Compositions, pharmaceutical compositions, or vectors may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
Dosage
A person of ordinary skill in the art can easily determine an appropriate dose of the polypeptide or a polypeptide sequence encoding said polypeptide or polynucleotide sequence to administer to a subject without undue experimentation. Typically, a veterinarian will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the polypeptide, the stability and length of action of the polypeptide, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Combinations
In one embodiment, the composition or the pharmaceutical composition comprises a combination of different peptides selected from the group consisting of SEQ ID NOs 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
An example of a fragment of SEQ ID No 20 is QEQNPPPSVSLRSLFG (SEQ ID No 153).
An example of a fragment of SEQ ID No 95 is
CAGGAGCAGAACCCCCCCCCCAGCGTGAGCCTGAGGAGCCTGTTCGGC (SEQ ID No 154). SEQ ID No 154 encodes QEQNPPPSVSLRSLFG (SEQ ID No 153).
An example of a fragment of SEQ ID No 59 is WGIAHTTGIPGNSQGQAM (SEQ ID No 155).
An example of a fragment of SEQ ID No 134 is
TGGGGCATCGCCCACACCACCGGCATCCCCGGCAACAGCCAGGGCCAGGCCATG
(SEQ ID No 156). SEQ ID No 156 encodes WGIAHTTGIPGNSQGQAM (SEQ ID NO 155).
For example, the composition or the pharmaceutical composition comprises a combination of different peptides selected from the group consisting of SEQ ID NOs 1 to 14 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 14.
In one embodiment, the composition or the pharmaceutical composition comprises at least two, three, four, five or six peptides selected from the group consisting of SEQ ID NOs 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
For Example, the composition or the pharmaceutical composition comprises at least two, three, four, or five peptides selected from the group consisting of SEQ ID NOs 18, 34, 17, 20 and 59 and homoiogues thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 18, 34, 17, 153 and 154.
In one embodiment, the composition or the pharmaceutical composition comprises at least two, three, four, or five peptides selected from the group consisting of SEQ ID NOs 18, 34, 17, 153 and 155.
In some embodiments, the compositions or the pharmaceutical composition further comprise at least one known EIAV peptide. Examples of combinations of peptides include the composition or the pharmaceutical composition comprising (i) QEQNPPPSVSLRSLFG (from SEQ ID NO 20), (ii) EALDKIEEIQNKNKQK (SEQ ID NO 18) and (iii) LLNGSLAGESIIIR (SEQ ID NO 34). In another example, the composition or pharmaceutical composition comprises (i) QEQNPPPSVSLRSLFG (from SEQ ID NO 20), (ii) EALDKIEEIQNKNKQK (SEQ ID NO 18), (iii) LLNGSLAGESIIIR (SEQ ID NO 34), and (iv) QPSLPTGSEELK (SEQ ID NO 17) and/or (v) WGIAHTTGIPGNSQGQAM (from SEQ ID NO 59).
In one embodiment, the composition or the pharmaceutical composition comprises a combination of different polynucleotide sequences encoding different peptides selected from the group consisting of SEQ ID NOs 1 to 76 and variants, homoiogues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76.
In one embodiment, the composition or the pharmaceutical composition comprises at least two, three, four, five or six polynucleotide sequences encoding peptides selected from the group consisting of SEQ ID NOs 1 to 76 and variants, homoiogues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76. For example, the composition or the pharmaceutical composition comprises at least two, three, four, or five polynucleotide sequences encoding peptides selected from the group consisting of SEQ ID NOs 18, 34, 17, 20 and 59 and homologues thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 18, 34, 17, 20 and 59.
In one example, the composition or the pharmaceutical composition comprises at least two, three, four, or five peptides polynucleotide sequences encoding selected from the group consisting of SEQ ID NOs 18, 34, 17, 153 and 155.
In one embodiment, the composition or the pharmaceutical composition comprises a combination of different polynucleotide sequences selected from the group consisting of SEQ ID NOs 77 to 152 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
In one embodiment, the composition or the pharmaceutical composition comprises at least two, three, four, five or six polynucleotide sequences selected from the group consisting of SEQ ID NOs 77 to 152 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
For example, the composition or the pharmaceutical composition comprises at least two, three, four or five polynucleotide sequences selected from the group consisting of SEQ ID NOs 152, 109, 93, 95 and 134 and homologues thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 152, 109, 93, 95 and 134.
In one embodiment, the composition or the pharmaceutical composition comprises at least two, three, four or five polynucleotide sequences selected from the group consisting of SEQ ID NOs 152, 109, 93, 154, and 156.
In some embodiments, the compositions or the pharmaceutical composition further comprise at least one nucleotide sequence encoding a known EIAV peptide.
In some embodiments, the compositions or the pharmaceutical composition comprise a combination of at least one peptide of the present invention and at least one nucleotide sequence of the present invention. In further embodiments the compositions or the pharmaceutical composition further comprise at least one known EAIV peptide and/or at least one nucleotide sequence encoding a known EIAV peptide.
Fusion proteins
In any of the above combinations of peptides, the peptides may be combined in the form of a fusion protein - that is- the peptides of the invention may be covalently linked together.
In the fusion protein, the peptides of the invention may be directly fused together, or may be separated by a linker peptide.
Thus, one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 or variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any of SEQ ID NOs 1 to 76 may be linked together as a fusion protein.
In a preferred embodiment, any two peptides selected from the group consisting of SEQ ID NOs: 1 to 76 are linked together as a fusion protein.
For example, said two peptides may be SEQ ID NO: 18 and SEQ ID NO: 20, or variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 18 and SEQ ID NO: 20.
For example, said two peptides may be SEQ ID NO: 18 and SEQ ID NO: 153, or variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 18 and SEQ ID NO: 153.
In one embodiment, one or more peptides of the invention are linked together as a fusion protein by a linker peptide.
By "linker peptide" it is meant any collection or chain of amino acids that are covalently linked together. The linker peptide is also covalently linked to one or more peptides of the invention. By way of example, a generic representation of a fusion protein of the invention comprising two peptides of the invention is as follows:
Peptide of the invention - linker peptide - peptide of the invention where "-" represent a covalent bond.
In a preferred embodiment, the linker peptide is a continuous, unbranched chain of amino acids. Thus, linker peptides may be any length, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length.
In a preferred embodiment, the linker peptide is between 2 and 10 amino acid residues in length.
In a particularly preferred embodiment, said linker peptide is 3 amino acids in length.
The linker peptide may consist of or comprise any type of amino acid. For example, the linker peptide may consist of or comprise non-polar amino acids. In another embodiment, the linker peptide may consist of or comprise amino acid residues having aliphatic side chains.
In yet another embodiment, the linker peptide comprises a repetitive amino acid sequence. In a preferred embodiment, the linker peptide consists of alanine residues. In a particularly preferred embodiment, the linker peptide is AAA. In a preferred embodiment, the fusion protein of the invention may be represented by one or more of the following structures:
SEQ ID NO: 20 - linker peptide - SEQ ID NO: 18;
SEQ ID NO: 18 - linker peptide - SEQ ID NO: 20;
SEQ ID NO: 18 - linker peptide - SEQ ID NO: 153;
SEQ ID NO: 153 - linker peptide - SEQ ID NO: 18;
SEQ ID NO: 20 - AAA - SEQ ID NO: 18;
SEQ ID NO: 18 - AAA - SEQ ID NO: 20;
SEQ ID NO: 18 - AAA - SEQ ID NO: 153;
SEQ ID NO: 153 - AAA - SEQ ID NO: 18; In one embodiment, the fusion protein consists of or comprises the following sequence:
EALDKIEEIQNKNKQKAAAQEQNPPPSVSLRSLFGNDPL (SEQ ID NO: 157).
In one embodiment, the fusion protein consists of or comprises the following sequence:
QEQNPPPSVSLRSLFGNDPLAAAEALDKIEEIQNKNKQK (SEQ ID NO: 158). In other embodiments, the peptides of the invention which form part of the fusion protein may be truncated by any number of amino acids at either the N-terminus or the C-terminus. For example, the peptides may be truncated by 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
In a highly preferred embodiment, the fusion protein consists of or comprises the following sequence:
EALDKIEEIQNKNKQKAAAQEQNPPPSVSLPvSLFG (SEQ ID NO: 159).
In one aspect, the composition of the invention may comprise a fusion protein consisting of or comprising the sequence set forth in SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159.
In a further aspect, the present invention provides a polynucleotide sequence encoding a fusion protein as described herein.
Diagnostic kits
The present invention also includes diagnostic methods and kits for the detection and measurement of one or more NEV peptides of the present invention in a sample, the measurement of one or more antibodies of the present invention, the measurement of one or more polynucleotide sequences of the present invention.
The peptides of the present invention may be used in a diagnostic method and kit to permit detection of circulating equine infectious anaemia virus (EIAV) and/or new equine virus (NEV) which, in certain situations, may indicate the progression of a disease state. An antibody of the present invention that, for example, possesses high binding specificity can be used to establish easy to use kits for rapid, reliable, sensitive, and specific measurement of NEV peptides of the present invention in samples such as plasma, urine, and in cell culture media. The antibody of the present invention may also be used in a diagnostic method and kit to permit detection of circulating EIAV and/or NEV which, in certain situations, may indicate the progression of a disease state.
The polynucleotide sequences of the present invention may be used in a diagnostic method and kit to permit detection of circulating EIAV and/or NEV which, in certain situations, may indicate the progression of a disease state.
In one embodiment, the diagnostic method according to the present invention determines the presence or absence of an EIAV and/or NEV in an animal.
In one embodiment, the diagnostic method according to the present invention determines the viral titre of an equine infectious anaemia virus (EIAV) and/or NEV in an animal. This may enable the progression of EIAV/NEV infection and/or EIAV/NEV disease progression to be monitored.
The viral titre (e.g. NEV viral titre) may be determined in a sample from an animal such as a blood sample, a blood serum sample, a plasma sample, a saliva sample, a sputum sample, a urine sample, a semen sample, a biopsy sample, a fecal sample, a milk sample, a lymph node biopsy sample, and/or a sweat sample.
The kits and diagnostic methods of the present invention may include but are not limited to the following techniques; competitive and non-competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, infrared assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips, or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry. For each kit the range, sensitivity, precision, reliability, specificity and reproducibility of the assay are established. Intraassay and interassay variation is established at 20%, 50% and 80% points on the standard curves of displacement or activity. The presence or absence in a sample from an animal of antibodies which are capable of binding to one or more NEV peptides of the present invention can be determined using, for example, enzyme-linked immunosorbent assays (ELISA). The presence or absence in a sample from an animal of one or more NEV peptides according to the present invention can be determined using, for example, enzyme-linked immunosorbent assays (ELISA).
The ELISA assay may be an indirect ELISA in which peptides of the present invention are immobilized onto, for example, a microtitre plate.
The ELISA assay may be a sandwich ELISA in which antibodies of the present invention are immobilized onto, for example, a microtitre plate. The antibodies may be obtained from the antisera of equines (e.g. horses) which have been infected with NEV peptides of the present invention and/or NEV viruses as mentioned herein. Alternatively, the antibodies may be obtained from animals, such as mice, which have been infected with NEV peptides of the present invention and/or NEV viruses as mentioned herein.
The presence or absence in a sample of one or more polynucleotide sequence encoding one or more peptides according to the present invention can be determined by using, for example, PCR (such as quantitative PCR, digital PCR), nucleic acid isothermal amplification, (such as Loop mediated isothermal amplification), and/or hybridisation techniques (such as Southern blotting). A primer for use, for example, in a PCR assay may be based on a polynucleotide sequence encoding a peptide of the present invention and/or a polynucleotide sequence of the present invention.
Typically primers are between 15 to 40 nucleotides in length. In some embodiments, the primers are between 18 to 26 nucleotides in length.
A probe for use, for example, in a hybridisation assay (such as Southern hybridisation) may be based on a polynucleotide sequence encoding a peptide of the present invention and/or a polynucleotide sequence of the present invention. Typically probes are at least 15 nucleotides in length. In some embodiments, the primers are at least 20, 30 or 40 nucleotides in length. In some embodiments, the primers are between 15 and 200 nucleotides in length.
A combination of primers and/or probes may be used to determine the presence or absence in a sample of one or more polynucleotide sequence encoding one or more peptides according to the present invention.
The sample as referred to herein is obtained/obtainable from an animal. In one embodiment, the sample is obtained/obtainable from an equine such as a horse, a donkey, a mule, a hinny, or a zebra.
The sample may be blood, blood serum, plasma, saliva, sputum, urine, fecal biopsy, lymph node biopsy, milk, semen, and/or sweat.
By determining the presence of NEV peptides of the present invention and/or the presence of NEV antibodies of the present invention and/or the presence of polynucleotide sequences encoding peptides of the present invention in a sample from an animal it can be determined that an animal has been infected with an equine infectious anaemia virus (EIAV) and/or NEV.
The diagnostic methods of the present invention are typically carried out ex vivo or in vitro.
Compositions according to the invention and/or a vector according to the invention, and/or a pharmaceutical composition according to the invention, and/or an antibody according to the invention may also be used in a kit or assay for the diagnosis or prevention of EIAV and/or NEV, and/or the diagnosis or prevention of an infection by an EIAV and/or NEV in an animal.
Polynucleotide sequence
The scope of the present invention encompasses polynucleotide sequences encoding NEV peptides.
The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti-sense strand. The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. In one embodiment it means cDNA sequence coding for the present invention.
In a preferred embodiment, the nucleotide sequence when relating to and when encompassed by the per se scope of the present invention does not include the native nucleotide sequence when in its natural environment and when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. For ease of reference, herein this embodiment is called the "non-native nucleotide sequence". In this regard, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. However, the amino acid sequence encompassed by scope the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism. In one embodiment, however, the amino acid sequence encompassed by scope of the present invention may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
Typically, the nucleotide sequence encompassed by the scope of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232). Preparation of the nucleotide sequence
A nucleotide sequence encoding either a peptide of the present invention may be identified and/or isolated and/or purified from any cell or organism producing said peptide. Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences. By way of example, DNA amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or purified.
By way of further example, a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the peptide. If the amino acid sequence is known, labelled oligonucleotide probes may be synthesised and used to identify clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a similar known gene could be used to identify clones. In the latter case, hybridisation and washing conditions of lower stringency are used.
Alternatively, clones comprising the peptides of the present invention could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar plates containing a substrate for the peptide thereby allowing clones expressing the peptide to be identified.
In a yet further alternative, the nucleotide sequence encoding the peptide may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al., (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al., (1984) EMBO J. 3, p 801-805. In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.
The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).
Amino acid sequences
The scope of the present invention also encompasses NEV peptides as defined herein. The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
The peptide encompassed in the present invention may be used in conjunction with other peptide (e.g. known EIAV peptides). Thus the present invention also covers a combination of peptides wherein the combination comprises the peptide of the present invention and another peptide, which may be another peptide according to the present invention.
Preferably the amino acid sequence when relating to and when encompassed by the per se scope of the present invention is not a native peptide. In this regard, the term "native peptide" means an entire peptide that is in its native environment and when it has been expressed by its native nucleotide sequence.
Antibodies
One aspect of the present invention relates to amino acids that are immunologically reactive with the NEV peptides of the present invention.
Antibodies may be produced by standard techniques, such as by immunisation with the peptide of the invention or by using a phage display library.
For the purposes of this invention, the term "antibody", unless specified to the contrary, includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library, as well as mimetics thereof. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. Furthermore, the antibodies and fragments thereof may be humanised antibodies. Neutralising antibodies, i.e., those which inhibit biological activity of the substance polypeptides, are especially preferred for diagnostics and therapeutics.
If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, llama, etc.) and/or avian is immunised with the sequence of the present invention (or a sequence comprising an immunological epitope thereof). Depending on the host species, various adjuvants may be used to increase immunological response.
Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the peptide of the present invention (or a sequence comprising an immunological epitope thereof) contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
Monoclonal antibodies directed against the sequence of the present invention (or a sequence comprising an immunological epitope thereof) can also be readily produced by one skilled in the art and include, but are not limited to, the hybridoma technique Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et a/., (1983) Immunol Today 4:72; Cote et al., (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan Rickman Liss Inc, pp 77-96). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity may be used (Morrison et al., (1984) Proc Natl Acad Sci 81 :6851-6855; Neuberger er al., (1984) Nature 312:604-608; Takeda er al., (1985) Nature 314:452-454).
Alternatively, techniques described for the production of single chain antibodies (US Patent No. 4,946,779) can be adapted to produce the substance specific single chain antibodies.
Antibody fragments which contain specific binding sites for the substance may also be generated. For example, such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al., (1989) Science 256:1275-128 1). Sequence Identity or Sequence Homology
The terms "peptide", "polypeptide", "polypeptide sequence", "protein" and "amino acid sequence" are used interchangeably herein.
The terms "polynucleotide sequence" and "nucleotide sequence" are used interchangeably herein. The present invention also encompasses the use of sequences having a degree of sequence identity or sequence homology with amino acid sequence(s) of a polypeptide described herein (e.g. variants, homologues and derivatives) or of any nucleotide sequence encoding such a polypeptide (hereinafter referred to as a "homologous sequence(s)"). Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity".
In the present context, a homologous sequence is taken to include an amino acid or a nucleotide sequence which may be at least 50, 60, 70, 75, 80, 85 or 90% identical, in some embodiments at least 95, 96, 97, 98 or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. In some embodiments, a homologous sequence is taken to include an amino acid sequence or nucleotide sequence which has one or several additions, deletions and/or substitutions compared with the subject sequence.
In some embodiments, the present invention relates to the use of a protein whose amino acid sequence is represented herein or a protein derived from this (parent) protein by substitution, deletion or addition of one or several amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more amino acids, such as 10 or more than 10 amino acids in the amino acid sequence of the parent protein and having the activity of the parent protein. In some embodiments, the present invention relates to the use of a nucleic acid sequence (or gene) encoding a protein whose amino acid sequence is represented herein or encoding a protein derived from this (parent) protein by substitution, deletion or addition of one or several amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more amino acids, such as 10 or more than 10 amino acids in the amino acid sequence of the parent protein and having the activity of the parent protein.
In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 50, 60, 70, 75, 85 or 90% identical, in some embodiments at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding a polypeptide described herein (the subject sequence). Typically, the homologues will comprise the same or equivalent sequences that code for the domain(s) etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
The homologous amino acid sequence and/or nucleotide sequence may provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the polypeptide.
In some aspects, an amino acid sequence as described herein has at least 50, 60, 70, 75, 80, 85 or 90% identity, in some embodiments at least 95, 96, 97, 98 or 99% identity to the subject sequence.
In some aspects, a nucleotide sequence as described herein has at least 50, 60, 70, 75, 80, 85 or 90% identity, in some embodiments at least 95, 96, 97, 98 or 99% identity to the subject sequence.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalizing unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. Typically the default values are used when using such software for sequence comparisons.
Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4th Ed - Chapter 18), BLAST 2 (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.qov). FASTA (Altschul et al 1990 J. Mol. Biol. 403-410) and AlignX for example. At least BLAST, BLAST 2 and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60).
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSU 62 matrix - the default matrix for the BLAST suite of programs. Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI package.
Alternatively, percentage homologies may be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).
Once the software has produced an optimal alignment, it is possible to calculate % homology, for example % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. Should Gap Penalties be used when determining sequence identity, then the following parameters can be used for pairwise alignment for example:
Figure imgf000061_0001
Figure imgf000061_0002
In one embodiment, CLUSTAL may be used with the gap penalty and gap extension set as defined above.
In one embodiment, the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, for example over at least 30 contiguous nucleotides, for example over at least 40 contiguous nucleotides, for example over at least 50 contiguous nucleotides, for example over at least 60 contiguous nucleotides, for example over at least 100 contiguous nucleotides, for example over at least 200 contiguous nucleotides, for example over at least 300 contiguous nucleotides. In one embodiment, the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.
The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Figure imgf000062_0001
The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.
Replacements may also be made by unnatural amino acids include; alpha* and alpha- disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-CI-phenylalanine*, p-Br-phenylalanine*, p-l- phenylalanine*, L-allyl-glycine*, β-alanine*, L-a-amino butyric acid*, L-y-amino butyric acid*, L-a-amino isobutyric acid*, L-s-amino caproic acid , 7-amino heptanoic acid*, L-methionine sulfone**, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline , L- thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl- Phe*, L-Phe (4-amino) , L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1 ,2,3,4- tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid * and L-Phe (4-benzyl)*. The notation * has been utilised for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.
Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the a- carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et a/., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.
The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention.
The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative or fragment thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
Hybridisation
'
The term "hybridisation" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies. In one embodiment, nucleotide sequences can hybridise to a probe of the present invention under stringent conditions (e.g. 50°C and 0.2xSSC).
In another embodiment, nucleotide sequences can hybridise to a probe of the present invention under high stringent conditions (e.g. 65°C and O.lxSSC).
The invention is further described by way of the following numbered paragraphs:
1. A composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76; and/or comprising one or more polynucleotide sequences encoding one or more of the peptides;
and/or comprising one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs: 77 to 152, and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
2. A vector capable of encoding one or more peptides as defined in paragraph 1 or a vector comprising one or more nucleotide sequences as defined in paragraph 1.
3. An antibody capable of binding to one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76.
4. A pharmaceutical composition comprising:
one or more peptides as defined in paragraph 1 ; and/or
one or more polynucleotide sequences as defined in paragraph 1 ; and/or
one or more vectors according to paragraph 2; and/or
one or more antibodies according to paragraph 3;
and a pharmaceutically acceptable carrier, vehicle, diluent or excipient.
5. A composition according to paragraph 1 , and/or a vector according to paragraph 2, and/or an antibody according to paragraph 3, and/or a pharmaceutical composition according to paragraph 4 for use in the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV in an animal.
6. The composition and/or vector and/or antibody and/or pharmaceutical composition according to paragraph 5 for the use according to paragraph 5 wherein said animal is an equine. 7. The composition and/or vector and/or pharmaceutical composition and/or antibody according to paragraph 5 for the use according to paragraph 5 wherein said animal is a horse.
8. A kit comprising:
a composition according to paragraph 1 ; and/or
a vector according to paragraph 2; and/or
an antibody according to paragraph 3; and/or
a pharmaceutical composition according to paragraph 4;
and optionally instructions for administration to an animal.
9. A diagnostic method comprising obtaining a sample from an animal and determining the presence or absence in said sample of antibodies which are capable of binding to one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76
and/or determining the presence or absence in said sample of one or more of said peptides; and/or determining the presence or absence in said sample of one or more polynucleotide sequence as defined in paragraph 1. 10. The diagnostic method according to paragraph 9 wherein said method determines the presence or absence of an equine infectious anaemia virus (EIAV) in an animal and/or the diagnostic method according to paragraph 9 determines the viral titre of an equine infectious anaemia virus (EIAV) in an animal. 11. The diagnostic method according to paragraph 9 or 10 wherein said animal is an equine.
12. The diagnostic method according to one of paragraphs 9 to 11 wherein said animal is a horse.
13. A kit comprising one or more peptides selected from the group consisting of SEQ ID Nos 1 to 76 and optionally instructions for determining the presence or absence in an animal sample of antibodies capable of binding to one or more said peptides.
A kit comprising one or more antibodies according to paragraph 4 and optionally instructions for determining the presence or absence in an animal sample of peptides capable of binding to said antibodies.
15. An oligonucleotide sequence (e.g. a primer or a probe) comprising or consisting of a polynucleotide sequence capable of encoding one or more of the peptides selected from the group consisting of SEQ ID NOs: 1 to 76
and/or comprising or consisting of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 77 to 152. 16. A kit comprising one or more oligonucleotide sequences according to paragraph 15 and optionally instructions for determining the presence or absence in an animal sample of said polynucleotide sequence.
17. A method for controlling EIAV disease in a group of animals comprising the identification of EIAV infection in an animal using the diagnostic method according to any one of paragraphs 9 to 12 and, optionally, the isolation of an EIAV infected animal from other animals.
18. Use of a composition according to paragraph 1 , and/or a vector according to paragraph 2, and/or a pharmaceutical composition according to paragraph 3, and/or an antibody according to paragraph 4, for the manufacture of a medicament for the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV in an animal. 19. A method for the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV in an animal, wherein said method comprising administering to an animal one or more compositions according to paragraph 1 , and/or one or more vectors according to paragraph 2, and/or one or more antibodies according to paragraph 3, and/or one or more pharmaceutical compositions according to paragraph 4.
20. A peptide substantially as described herein with reference to any one of the Examples and/or drawings. 21. A polynucleotide substantially as described herein with reference to any one of the Examples and/or drawings.
22. An antibody substantially as described herein with reference to any one of the Examples and/or drawings.
23. An oligonucleotide substantially as described herein with reference to any one of the Examples and/or drawings.
24. A vector substantially as described herein with reference to any one of the Examples and/or drawings.
25. A pharmaceutical composition substantially as described herein with reference to any one of the Examples and/or drawings.
26. A kit substantially as described herein with reference to any one of the Examples and/or drawings.
27. A composition and/or a vector and/or a pharmaceutical composition substantially as described herein with reference to any one of the Examples and/or drawings for use in the treatment or prevention of equine infectious anaemia and/or treatment or prevention of an infection by an EIAV in an animal.
28. A diagnostic method substantially as described herein with reference to any one of the Examples and/or drawings.
29. A use substantially as described herein with reference to any one of the Examples and/or drawings.
30. A method substantially as described herein with reference to any one of the Examples and/or drawings for the treatment or prevention of equine infectious anaemia and/or treatment or prevention of an infection by an EIAV in an animal.
31. A composition according to paragraph 1, and/or a vector according to paragraph 2 and/or an antibody according to paragraph 3, and/or a pharmaceutical composition according to paragraph 4 for use as an immunomodulator. 32. A composition according to paragraph 1 , and/or a vector according to paragraph 2 and/or an antibody according to paragraph 3, and/or a pharmaceutical composition according to paragraph 4 for use as a vaccine.
The present invention is further described by way of the following non-limiting examples:
EXAMPLES
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Col d Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; and E. M. Shevach and W. Strober, 1992 and periodic supplements, Current Protocols in Immunology, John Wiley & Sons, New York, NY. Each of these general texts is herein incorporated by reference.
The inventors have developed a peptide based immunodiagnostic assay that has been validated by comparing results on reference and field samples with the immunoblot test. The peptide ELISA has similar sensitivity and specificity of the immunoblot techniques for the detection of EIAV infected horses. The ELISA is based in peptides identified in purified viral particles obtained from EIAV AG ID negative field samples. The purified viral particles were obtained from primary macrophages cell lines established from seropositive horses. The EIAV AGID negative strains were also considered negative in ELISAs directed to p26 core protein as also to the dual antigen gp45 ELISAs (Eradikit ELISA, In3 Diagnostics, Italy).
The retrieved peptides were short peptides with generally less than 30 amino acids. These peptides had a good sensitivity and specificity. More than 62 peptides were identified from EIAV AGID-negative viral purified strains by mass spectrometry; these peptide were screened against public viral databases (UNIPROT KB or NCBI) and twenty three peptides of interest were selected which were found that were present in a EIAV AGID negative gene bank that belongs to Equigerminal - these peptides are shown in Table 1. None of the identified peptides belonged to the EIAV proteome. 45 of the 62 peptides were found to have sequence identity with retroviral peptides. 26 of the 62 peptides were found to have sequence identity to part of the HIV-1 proteome, Table 1 - Table 1 details peptide sequences and the polynucleotide sequence which encodes the peptide. For example, the polynucleotide sequence shown as SEQ ID NO 77 encodes the peptide shown as SEQ ID NO 1.
Figure imgf000072_0001
AKLEESFPGK (SEQ ID NO GCCAAGCTGGAGGAGAGCTTCCCCGGCAAG (SEQ ID NO 26) 101)
NGSLAGESIIIR (SEQ ID NO AACG GCAGCCTGGCCGGCGAGAG C ATC ATCATC AGG (SEQ 27) ID NO 102)
NITFNSSAGGDLEIT (SEQ ID AACATCACCTTCAACAGCAGCGCCGGCGGCGACCTGGAGAT NO 28) CACC (SEQ ID NO 103)
AVILLLDRLR (SEQ ID NO 29) GCCGTGATCCTGCTGCTGGACAGGCTGAGG (SEQ ID NO
104)
GPGIHIGKR ( SEQ ID NO 30) GGCCCCGGCATCCACATCGGCAAGAGG (SEQ ID NO 105)
VIICSASK (SEQ ID NO 31) GTGATCATCTGCAGCGCCAGCAAG (SEQ ID NO 106)
ESFPNK (SEQ ID NO 32) GAGAGCTTCCCCAACAAG (SEQ ID NO 107)
FGNKTTIIFTK (SEQ ID NO TTCGGCAACAAGACCACCATCATCTTCACCAAG (SEQ ID NO 33) 108)
LLNGSLAGESIIIR (SEQ ID CTGCTGAACGG CAG CCTGG CCGG CGAGAGCATCATCATCA NO 34) GG (SEQ ID NO 109)
VNVSVTNNNTTTNV (SEQ ID GTGAACGTGAGCGTGACCAACAACAACACCACCACCAACGT NO 35) G (SEQ ID NO 1 10)
AILHILRR (SEQ ID NO 36) GCCATCCTGCACATCCTGAGGAGG (SEQ ID NO 11 1)
DLLALDK (SEQ ID NO 37) GACCTGCTGGCCCTGGACAAG (SEQ ID NO 112)
IGCQHSRIGITLPR (SEQ ID ATCGGCTGCCAGCACAGCAGGATCGGCATCACCCTGCCCA NO 38) GG (SEQ ID NO 113)
TLQYLALTALVTPK (SEQ ID ACCCTGCAGTACCTGGCCCTGACCGCCCTGGTGACCCCCAA NO 39) G (SEQ ID NO 114)
PTTPVTPAPGVGEISKELAQG CCCACCACCCCCGTGACCCCCGCCCCCGGCGTGGGCGAGA
(SEQ ID NO 40) TCAGCAAGGAGCTGGCCCAG
GGCAAG (SEQ ID NO 115)
WSSALQYLIPR (SEQ ID NO GTG GTG AG C AGCG CCCTG CAGTACCTG ATCCCCAG G (SEQ 41) ID NO 116)
PVWAEPVK (SEQ ID NO 42) CCCGTGTGGGCCGAGCCCGTGAAG (SEQ ID NO 117)
VKGNDLLK (SEQ ID NO 43) GTGAAGGGCAACGACCTGCTGAAG (SEQ ID NO 118)
EDLNLQDWK (SEQ ID NO 44) GAGGACCTGAACCTGCAGGACTGGAAG (SEQ ID NO 119)
LFVSVLQR (SEQ ID NO 45) CTGTTCGTGAGCGTGCTGCAGAGG (SEQ ID NO 120)
AAEQKASPPSLTPK (SEQ ID GCCGCCGAGCAGAAGGCCAGCCCCCCCAGCCTGACCCCCA NO 46) AG (SEQ ID NO 121)
PLSLPLKI (SEQ ID NO 47) CCCCTGAGCCTGCCCCTGAAGATC (SEQ ID NO 122)
FPSIVGRPR (SEQ ID NO 48) TTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID NO 123)
IWHHTFYNELR (SEQ ID NO ATCTG G CACC AC ACCTTCTACAACG AGCTG AG G (SEQ ID NO 49) 124)
MTQIMFETF (SEQ ID NO 50) ATGACCCAGATCATGTTCGAGACCTTC (SEQ ID NO 125)
EEEVAALVIDNGSGMCK GAGGAGGAGGTGGCCGCCCTGGTGATCGACAACGGCAGCG
(SEQ ID NO 51) GCATGTGCAAG (SEQ ID NO 126)
VAPEEHPVLLTEAPLNPK GTGGCCCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCC
(SEQ ID NO 52) CCCTGAACCCCAAG (SEQ ID NO 127)
AVFPSIVGRPR (SEQ ID NO GCCGTGTTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID 53) NO 128)
YPIEHGIVTNWDDMEK (SEQ TACCCCATCGAGCACGGCATCGTGACCAACTGGGACGACAT ID NO 54) GGAGAAG (SEQ ID NO 129)
AVFPSIVGR (SEQ ID NO 55) GCCGTGTTCCCCAGCATCGTGGGCAGG(SEQ ID NO 130) IGRKDAERQLLSPGNAR ATCGGCAGGAAGGACGCCGAGAGGCAGCTGCTGAGCCCCG (SEQ ID NO 56) GCAACGCCAGG (SEQ ID NO 131 )
DS YVG DEAQSKR (SEQ ID GACAGCTACGTGGGCGACGAGGCCCAGAGCAAGAGG (SEQ NO 57) ID NO 132)
RGILTLK (SEQ ID NO 58) AGGGGCATCCTGACCCTGAAG (SEQ ID NO 133)
WGIAHTTGIPGNSQGQAMVE TGGGGCATCGCCCACACCACCGGCATCCCCGGCAACAGCC
R (SEQ ID NO 59) AGGGCCAGG CCATG GTG GAG
AGG (SEQ ID NO 134)
HLVDQLIRDLK (SEQ ID NO CACCTGGTGGACCAGCTGATCAGGGACCTGAAG (SEQ ID 60) NO 135)
YEQLQLQAR (SEQ ID NO 61) T ACG AG CAG CTG C AGCTG CAGG C C AG G (SEQ ID NO 136)
TITLEVEPSDTIENVK (SEQ ID ACCATCACCCTGGAGGTGGAGCCCAGCGACACCATCGAGA NO 62) ACGTGAAG (SEQ ID NO 137)
INRELLK (SEQ ID NO 63) ATCAACAG GG AG CTGCTG AAG (SEQ ID NO 38)
QTIIPTNKDVDEK (SEQ ID NO CAGACCATCATCCCCACCAACAAGGACGTGGACGAGAAG 64) (SEQ ID NO 139)
KNYGKLDK (SEQ ID NO 65) AAG AACTACGG CAAGCTGG ACAAG (SEQ ID NO 140)
TTISFSK (SEQ ID NO 66) ACCACCATCAGCTTCAGCAAG (SEQ ID NO 141)
QDRTTISFSK (SEQ ID NO 67) CAGGACAGGACCACCATCAGCTTCAGCAAG (SEQ ID NO
142)
AQGRAIAHK (SEQ ID NO 68) GCCCAGGGCAGGGCCATCGCCCACAAG (SEQ ID NO 143)
MAQTQLVPVK (SEQ ID NO ATGGCCCAGACCCAGCTGGTGCCCGTGAAG (SEQ ID NO 69) 144)
QELIPPCK (SEQ ID NO 70) CAGGAGCTGATCCCCCCCTGCAAG (SEQ ID NO 145)
DIVLLENGK (SEQ ID NO 71) GACATCGTGCTGCTGGAGAACGGCAAG (SEQ ID NO 146)
LPPLSILK (SEQ ID NO 72) CTGCCCCCCCTGAGCATCCTGAAG (SEQ ID NO 147)
IFLINLAFLIK (SEQ ID NO 73) ATCTTCCTGATCAACCTGGCGTTCCTGATCAAG (SEQ ID NO
148)
NRLEILK (SEQ ID NO 74) AACAGGCTGGAGATCCTGAAG (SEQ ID NO 149)
ADDVAVLQDALGR (SEQ ID GCCGACGACGTGGCCGTGCTGCAGGACGCCCTGGGCAGG NO 75) (SEQ ID NO 150)
LNKSLEQLR (SEQ ID NO 76) CTG AACAAG AG CCTGG AG CAG CTG AG G (SEQ ID NO 151)
The inventors carried out ELISA assays using the above identified short synthetic peptides in Table 1 , which are easy to produce, and control and which can be used as target antigens for the detection of anti-NEV antibodies.
This trend towards smaller antigens however is accompanied by a risk that the synthesized epitope is not able to assume a rigid conformation that is recognized by the antibody.
Although the number of serum samples tested in each of these cases is very limited, specificity was found to be very high (95%-100%) with small synthetic peptides but the overall sensitivity varied between 80 and 100%. In the only example where 100% sensitivity was attained only a few samples had been tested. Also, the use of a synthetic peptide based ELISA can also overcome the use of the impurities present in the antigens preparations resultant from protein production in bacterial or mammalian cells that are also responsible for unacceptably high levels of false positive results which can cause healthy horse to be eliminated by common policies of EIAV control.
Material and Methods
EIAV seropositive and AGID negative sera.
Equine infectious anemia (EIAV) seropositiveness was first determined by immunoblot (Figure 1).
EIAV Wyoming (EIAVWYO viral strain (ATCC VR-778) Malmquist et al., 1973) was cultured in a permissive macrophage-like cell line EML-3C cell line (ATCC CRL-2996) previously established for EIAV replication studies (Fidalgo-Carvalho et al., 2009). The cells were seeded in T75cm2 flasks prior to infection. After two days cell cultures were infected with EIAVWYO- Tissue culture supernatants were screened for Reverse Transcriptase (RT) activity by using the Enzcheck reverse transcriptase kit (Molecular Probes, Invitrogen) according to manufacturer instructions. RT positive cell culture supernatants were submitted to sequential centrifugations steps. To remove cell debris supernatants were centrifuged at 500 rcf for 10 minutes, supernatants moved to a clean tube and centrifuged again at 3200 rcf for 20 minutes. The RT positive supernatants were then filtered by a syringe minisart 0.45 micron filter (Sartorius) and viral particles concentrated to 0.5-1 mL in a Vivaspin 6 > 100,000 MWCO PES (Sartorius) accordingly manufacturers instructions.
EIA viral particles were then lysed and submitted to SDS-PAGE gel system by using 10% Bis-Tris gels Novex NuPAGE in a 2D well lane and run in a MES buffer. After separation the gels were transferred to nitrocellulose membranes in 7 minutes by using the iBIot dry blotting system (Invitrogen) accordingly manufacturer instructions.
After blotting the membranes were blocked with a non-animal blocking buffer (Nap-Blocker, G-Biosciences) for two hours at room temperature. Membrane Strips were cut and incubated with horse sera at 1:50 or 1 :100 dilution in blocking buffer for 1 hour at 37°C. Strips were washed three times with TBS 0.005% Tween20 and incubated, for 30 minutes at 37°C, with goat secondary anti-horse IgG (H+L) antibody labelled with Horseradish peroxidase in a 1:4000 dilution.
The secondary antibody was washed out three times, strips passed in a PBS solution and revealed with Ampliflu Red (Sigma-aldrich) fluorescent substrate and visualized with a 570 nm filter at in a Gel doc EZ (Biorad). Results were analysed by using the Image lab software (Biorad). Four different EIAV positive reference sera were used in the immunoblot: the World organization for animal health (OIE) EIAV positive reference sera kindly provided by the European Reference Center for Equine diseases (ANSES, France), and the EIA strong, medium and weak reference sera purchased to VMRD diagnostic (Pullman, Washington DC).
As a positive control seropositive EIAV AGID negative were also tested in EIAVPV blotted membranes kindly provided by Doctor Charles Issel (University of Kentucky). The results obtained with the 'in house' EIAVWYO blotted membranes were similar to the results obtained with UK EIAVpv membranes. The EIAVPV viral strain is a strain derived from EIAVWYO (Lichtenstein et a/., 2006).
In Figure 1 the inventors compared the pattern of the OIE reference sera and the EIAV AGID negative samples. In this Figure the immunoblot pattern of EIAV AGID positive samples, such as the OIE reference sera, can be visualised. This pattern it is usually composed of a three-band pattern in the region of the p26, the gp45 and gp120 proteins that correspond to the binding of anti-p26, anti- gp45, and to the anti-gp120 specific antibodies. However, with the VMRD reference sera, a more broad reactivity against different EIAV proteins (not shown) can be visualized. In Figure 1 the pattern of EIAV AGID positive and negative reactors can be compared. The EIAV AGID negative, like the EIAV AGID positive reactors, possesses the three-band pattern characteristic of EIAV seropositiveness. However, these reactors are considered negative in the AGID assay and/or in the ELISA (Eradikit, In3 Diagnostic, Italy).
EIAV AGID and ELISA negative sera
The EIAV immunoblot (IB) positive sera were submitted to AGID testing by using an ID. Vet EIAV AGID kit (ID. Vet, France). The AGID was performed accordingly the manufacturer instructions. Moreover, 1 :2 and 1 :3 dilutions of the EIAV antigen (p26 recombinant protein) and positive control were performed in order to visualize weak reactivity against the p26 antigen in EIAV IB positive samples. As results none of the seropositives detected in the immunoblot were positive in AGID, neither with the non-diluted nor diluted reagents.
Moreover, EIAV AGID negative sera were analysed by EIAV commercially available ELISAs using antibodies raised against the p26 antigen, or against the dual antigen p26 and gp45, the Eradikit (IN3.Diagnostic, Italy). The sera were serial diluted from 1 :6 to 1 :20 dilution. The results showed that EIAV AGID negative sera were also considered as negative in all the dilutions tested in this ELISA. Establishment of NEV macrophage-like cell lines from EIAV AGID negative horses.
For isolation and viral propagation of EIAV AGID negative strains, three different macrophage cell lines were established from EIAV AGID negative infected horses. Peripheral blood was collect from 3 different EIAV seropositive horses and processed according to Fidalgo-Carvalho et al., (2009).
The macrophage-like cells were able to divide, being passaged and remained in culture for 3-4 months. The macrophage-like cells obtained were similar to the EML-3C reported in Fidalgo-Carvalho et al., (2009), but during the first weeks of culture showed syncytia cell formation and cell death (Figure 2A1 and Figure 2A2). After 3 weeks the cells were able to recover (Figure 2B) and showed low levels of reverse transcriptase activity measured in cell supernatants by the Enzcheck reverse transcriptase assay (Invitrogen) (Figure 2.D1 and 2.D2). Viral particles (vp) were isolated from RT positive cell culture supernatants from NEV macrophage-like cell lines A (NEV_MaA), and/or NEV_MaB and/or NEV_MaC. No evident signs of cell stress or death were visualized. The viral particles were isolated as described in the above section. Viral transfer
Further the inventors performed viral transfer among the NEV cells lines generated. Viral particles of MaA cells were used to infect NEV-MaB cells, or NEV-MaC cells and vice-versa. Surprisingly, the cell cultures of NEV-MaC infected with vpB, and NEV-MaA infected with vpC showed an increased reverse transcription activity (in cell culture supernatants) and marked cythopathic events after 7-10 days of infections (Figure 2C) and all cells died 15 days post-infection. The viral particles obtained from NEV-MaC-VpB and/or NEV-MaA-vpC infection were isolated, centrifuged, filtered in a 0.2μηι and concentrated in Vivaspin6 100 kda as described above.
Moreover these viral particles were used for further analysis of the proteome by mass spectrometry. The purified viral particles were also used for viral genome analysis.
Viral R A extraction
Viral RNA was extracted from isolated and purified viral particles by using the Trizol LS method, or by using the Pure-link viral RNA/DNA mini-kit (Invitrogen) according to the manufacturer instructions (Figure 3A).
EIAV AGID negative proteome
The purified viral particles were run in a 10% SDS-PAGE and different bands were digested and injected for mass spectrometry analysis. The results showed a wide diverse spectrum and several retroviral peptides were identified in the EIAV AGID negative viral particles. None of the peptides retrieved belonged to the known EIAV AGID positive strains (Figure 3B).
Serial passages of NEV virus and gain of virulence
To propagate the undescribed virus, the inventors performed in vitro serial passages of NEV virus into MaC cell line as described above (viral transfer). MaC monolayers cells in T75cm2 or T175cm2 flasks were infected with 1 ml of NEV virus and incubated at 37°C, 5%C02 in a humid chamber for 7 days in DMEM high glucose medium without phenol red (Gibco, Life Technologies), supplemented with 10% of fetal bovine serum, 2% glutamax (Gibco, Life Technologies) and 1% of Penicilin-Streptomicin (Gibco, Life Technologies). The supernatant medium of named passage 1 was serially passaged in MaC cells as described above for 10 times. After serial passage #10, virulence become more marked. In Figure 4 the inventors compared cell micrograph of day 7 non-infected cells (Fig 4A) versus NEV infected cells. The infected cells showed an increased number of induced syncytia cell formation and cell death. (Fig. 4B - MaC cells). These cytopathic effects begin to appear at day 4 post-infection (p.i), cell monolayers were partially destroyed at day 5-7 p.i. and completely destroyed at 10- 12 days p.i.
NEV is a highly cytopathic virus in Equine Dermal cells.
Viral propagation of the NEV virus was further tested in Equine Dermal Cells (ED) (NB-6, ATCC CCL-57) and its cytopathogenicity compared to EIAV_Wyoming.
EIAV-Wyoming (ATCC-VR-778) is reported as an in vivo pathogenic virus that can replicate in Equine Dermal cells with slight cytopathic effects (CPE) (Malmquist WA et al., 1973, Orego A, et al, 1982).
ED cells were seeded 24 to 72 hours prior to infection in T75cm2 flasks. Highly confluent (80-90%) cells were infected with 1ml of 0.2 pm filtered NEV viral supernatants or 1 MOI of EIAV-Wyoming, and incubated at 37°C 5% C02 for 5 or more days.
At day 5-7, cell death was evident with visible cell monolayer destruction. In ED cells the NEV induced CPE (Fig. 4B - ED cells) were very similar to those described above for MaC cells (Fig. 4B - MaC cells). However, the ED cells infected with EIAV- Wyoming failed to induce marked CPE and cell death at day 7 or day 12 (Fig 4C - ED cells).
Moreover, to quantify EIAV viral replication activity we developed a reverse transcriptase and qPCR assay by using the gene specific primers El A (3595-3615) forward primer 5'- GGGGTGGTATTATTCGTGGCT-3' and EIA (4060-4088) reverse primer 5'- TTTTTCTAATTTGTGTGCCTTGATATGCT 3' directed to the pol region. The assay performed with Superscript III Reverse transcriptase (Life Technologies) and SsoFast™ EvaGreen® supermix (Bio-Rad) according to the manufacturer's instructions was run in a CFX96 Bio-Rad apparatus. The amplified 494 bp of the EIAV pol gene was previously cloned into Topo TA pCR4 vector to obtain the plasmid construct pCR4.EIAVpol. This plasmid was further used to construct a standard curve by performing 10-fold serial dilutions in duplicates. The retrieved standard curve had a R2 of 0.998 and was able to detect 4 viral particles. According to the obtained standard curve, viral supernatant obtained from day 6 cell cultures of ED cells infected with EIAV-Wyoming (Fig.4C) contained 9.18x10"" viral particles/ml. The results showed that EIAV Wyoming virus replicates at high levels in ED cells without significant CPE. Moreover, the RT-qPCR assay failed to amplify the NEV genome.
To better evaluate the pathogenicity of NEV in ED cells, Presto Blue cell viability assays (Molecular Probes, Life Technologies) were performed in cells infected with NEV or EIAV Wyoming (ATCC-VR-778). Presto blue cellular viability assays were performed in quintuplicates following the manufacturer's instructions. ED cells were seeded into 96-well plates 24-48 hours before infection. Highly confluent cell monolayers were infected with 20μΙ of NEV viral supernatant of day 7, or 1 MOI of EIAV Wyoming strain and allowed to incubate for 11 days. Presto blue reagent was added to the cultures, incubated for 16 hours and absorbance read at 570 nm in a Multiskan Go ELISA reader (Thermo Scientific).
The results showed that the viability of NEV infected cells was decreased to less than one third of the absorbance values of mock cells. The viability of NEV infected cells showed a statistically significant difference (Mann Whitney test) when compared to non-infected or EIAV-Wyoming infected cells (Fig.4D). Moreover, cell viability of EIAV Wyoming infected cells and mock cells showed similar absorbance (Fig.4D) confirming the microscopic data (Fig. 4C) and indicating that contrary to NEV, the EIAV-Wyoming doesn't induce evident CPE on ED cells at day 11.
NEV virus possesses Aspartic protease activity
To assess for in vitro proteolytic cleavage activity, cell infections were performed in serum free media conditions. Day 5-7 p.i. viral particles were concentrated and extensively washed with HBSS in Vivaspin 100kDa (Sartorius) columns. Concentrated viral particles were lysed in 0.1% SDS and submitted to analytical size-exclusion chromatography on a Superdex 200 5/150 GL (GE Healthcare Life Sciences) column connected to a Prominence HPLC system (Shimadzu Corporation, Tokyo, Japan). The column was equilibrated in 20 mM phosphate buffer pH 7.5 containing 150 mM NaCI.
The protease activity was determined by fluorescence assays in 96-well plates in a Gemini EM Fluorescence Microplate Reader, using the HIV Protease Substrate 1 Arg-Glu(EDANS)- Ser-Gln-Asn-Tyr-Pro-lle-Val-Gln-Lys(DABCYL)-Arg (Sigma Aldrich) or the Phe-Phe substrate. For determination of the pH profile, viral particles were assayed for activity at 37°C in buffers ranging between pH 3 and 6 (50 mM sodium acetate pH 3.0, 4.0, 5.0, and 6.0; 50 mM Tris-HCI pH) containing 100 mM NaCI. To test the specificity of enzymatic cleavage, Indinavir (Sigma Aldrich), a specific inhibitors of HIV protease, or pepstatin were used. The protease was pre-incubated in the presence of the inhibitor. The Pesptatin or Indinavir (10 μΜ) was again incubated with viral particles for 10 minutes at room temperature in 50 mM sodium acetate pH 5 or pH 6.0 containing 100 mM NaC. The rate of substrate hydrolysis was monitored for 3 hours by the increase of intensity with excitation/emission wavelengths of 328/393 nm.
Our results demonstrated that fraction 12, 15 and 16 of NEV viral particles were able to cleave HIV substrate 1 (Fig 5C) at pH=5 and Phe-Phe substrate at pH=3 and 4 (Fig. 5A and B). Moreover, pepstatin or Indinavir were able to inhibit the protease activity of NEV fractions (Fig. 5A, B and C).
Example 1
Immunoenzyme Assay Using the Peroxidase-NEV Peptide Conjugates The detection of antibodies directed against theNEV viruses is based, in the example below, on the principle of the immunoenzyme technique of the "double-antigen sandwich" type. The test is based on the use, on the one hand, of a microplate (solid phase) sensitized with purified antigens, and, on the other hand, of a conjugate constituted by a peptide according to the invention labelled with peroxidase, which take an NEV antibody to be detected into a sandwich.
The assay protocol used is as follows: 100 μΙ of each serum sample tested, diluted to [3/4], are distributed in a well of the sensitized microplate and the mixture is homogenized. After incubation of the mixture under adhesive film for 60 minutes at 37°C, followed by washing of the microplate by means of a Tris NaCI buffer, 100 μΙ of labelled conjugate (peroxidase- labelled NEV peptide according to the invention) are added to each well. The mixture is incubated under adhesive film for 30 minutes at 18-30°C.
After removal (by washing with the above-mentioned Tris NaCI buffer) of the conjugate fraction that has remained free, the presence of the immobilized enzyme on the complexes is revealed. To that end, 80 μΙ of a revelation solution (containing H202 substrate in a solution of citric acid and sodium acetate, and a chromogenic compound, tetramethylbenzidine or TMB) are added to each well. After incubation of the mixture again in darkness for 30 minutes at 18-30°C, the revelation is stopped by addition of 100 μΙ of 1 N sulfuric acid. The optical density is read on a spectrophotometer at 450/620 nm.
The presence or absence of anti-NEV antibodies is determined by comparing, for each sample tested, the recorded optical density ("OD") with that of the calculated cut-off (cut- off=average of the OD of negative samples+0.100 OD unit).
A sample is considered to be positive (carrier of anti-NEV antibodies) if the OD obtained is greater than that of the cut-off, and negative if its OD is lower than that of the cut-off.
Example 2
Indirect Immunoassay for the NEV Detection of Specific Antibodies Synthesis of the Peptides According to the Invention was carried out in accordance with Barani, G. and Merrifield, R. B. in The Peptides, Analysis, Synthesis and Biology, Vol. 2, Academic Press, Ed. Erhard Gross, Johannes Meyerhofer. The crude peptide was purified by HPLC.
Example 2a
Preparation of Peptide Solutions and Coating of Micro-Titration Plates with These Peptides
The peptides were dissolved in 50% (v/v) acetic acid, water, or DMSO, at a concentration of 5 mg/ml. The stock solutions were diluted in 0.10 M sodium bicarbonate (pH 9.6) such that the concentrations of the peptides are 1 pg/ml. 100 μΙ of the dilute solution were added to each of the wells of type B microtitration plates from Nunc, Denmark. The filled test plates were incubated at 20°C for 18 hours. The solutions were then sucked off and the wells were rinsed 3-4 times with 300 μΙ of a 10 g/l solution of bovine serum albumin in phosphate- buffered physiological sodium chloride solution (PBS, pH 7.4), and the test plates were then dried over silica gel at 20°C.
Example 2b
Preparation of a Peroxidase-Labelled Antibody Against Horse Immunoglobulin of the IgG Class (h-lgG), and also TMB Substrate for Detection
Monoclonal antibodies against h-lgG were prepared in accordance with the method of Koehler and Milstein, Nature 256: 495, 1975, with different monoclonal antibodies having the same antigen specificity being identified by the method described by Stahli et al., J. of Immunological Methods 32: 297-304 (1980). Following purification by gel chromatography and dialysis against PBS buffer, pH 7.4, the monoclonal antibody fraction (4 mg of protein/ml) was reacted with N-gammamaleimidobutyloxysuccinimide (GMBS) in accordance with Tanamori et al., J. Immunol. Meth. 62: 123-131 (1983). In parallel with this, 2- iminothiolane hydrochloride (from Sigma, Cat. No. 1 6256) was reacted with horseradish peroxidase (POD, from Boehringer Mannheim, Cat. No. 413470) in accordance with King et al. Biochem. 17: 1499-1506 (1978). An antibody/POD conjugate was prepared from the GMBS/antibody conjugate and the iminothiolane/POD conjugate as described by Tanamori et al., supra. The resulting solution of the IgG/POD conjugate had a protein content of 360 μΙ/ml. The ratio of POD to IgG was 2.8. The solution was subsequently diluted to 500 ng/ml IgG/POD using a solution of 50 ml/l fetal calf serum. (FCS, from Biochrom KG, Berlin) and 5 g/l polyoxyethylene (20) sorbitan monolaurate (Tween 20) in PBS, and was given the designation anti-lgG/POD conjugate. For use in the ELISA, the anti-lgG/POD conjugate was diluted 1 :100 to 1 :20,000 with Tris buffer (pH 7.4, containing 0.5% Tween 20), and then a series of 1 :26 final dilutions in conjugate buffer (0.1 M1-amino-2-(hydroxymethyl)-1 ,3- propanediol (Tris), 0.1 M sodium chloride (NaCI) and 0.1 % Tween 20, pH 8.4) is prepared. For detecting anti-human IgG/POD, the present inventors used a substrate system, or a substrate preparation, composed of hydrogen peroxide and tetramethylbenzidine (TMB), which was prepared from two stock solutions as follows: Stock solution 1: TMB dihydrochloride was dissolved with stirring in double-distilled water at a concentration of 5 g/l (16 mmol/1), and this solution was adjusted to pH 1.5 using 5 N hydrochloric acid. Penicillin G was added to this solution with stirring, up to a final concentration of 200 mg/l (0.56 mmol/l). Stock solution 2:1.4 ml of glacial acetic acid, 1.5 ml of 1 N NaOH and 250 mg (3 mmol) of H202, as a urea/hydrogen peroxide adduct, were added to 900 ml of double-distilled water. After these substances had dissolved completely, the solution was made up to 1 litre using double-distilled water. TMB substrate preparation: One part by volume of stock solution 1 and 10 parts by volume of stock solution 2 were mixed together.
Example 2c
Determination of Horse Antibodies of the Immunoglobulin G Class Against peptides in an ELISA Using the Peptide According to the Invention
50 μΙ of serum or plasma were added to 50 μΙ of sample buffer, containing 0.3 M Tris, 0.3 M NaCI, 20% bovine serum and 0.1% Tween 20, in wells of coated microtiter plates which were prepared in accordance with the Example 2b. After the plates had been incubated at 37°C. for 30 minutes, the test solutions were aspirated and the wells were in each case washed five times with washing buffer containing 1 g/l Tween 20 in PBS. After that, 100 μΙ of conjugate were added to each of the wells, a preliminary dilution of 1 :3000 in Tris buffer (pH 7.4, 0.5% Tween 20) and a final dilution of 1 :26 in conjugate buffer preferably being selected. After the plates had been incubated at 37°C. for 30 minutes, the contents of the wells were aspirated and the wells were once again in each case washed five times. Subsequently, 100 μΙ of TMB substrate preparation were added to each well and the plates were incubated at 20-22°C for 30 minutes; the reaction was then stopped by adding 100 μΙ of 1 normal sulfuric acid. The extinction of the colored solution was measured at a wavelength of 450 nm (E450) against a blank value of PBS.
The reactivities of Western-blot anti-NEV negative and Western-blot anti-NEV positive samples are compared on microtitration plates which are coated, on the one hand, with the synthetic peptide. Example 3
Immunoassay for Detection of Antibodies Induced by NEV.
The magnetic particle reagents are to be prepared according to the manufacturers recommended protocol. Dynal AS, is the manufacturer of the Dynabeads, which are employed. The magnetic particles coated with ligand are called Reagent 1. A peptide according to the invention is covalently coupled to the pre-activated surface of the magnetic particles. It is also possible to physically absorb the peptide to the surface of the magnetic particles. The concentration of particles in Reagent 1 is within the range from 1 mg/ml to 15 mg/ml. The particle size varies between 0.2 μηη to 15 pm. The concentration of peptides is within the range from 1 ng/mg particle to 1 mg/mg particle. For example the concentration of peptides may be within the range from 1 ng/mg particle to 0.01 mg/mg particle, or preferably within the range from 0.01 mg/mg particle to 1 mg/mg particle. The anti horse Ig Alkaline Phosphatase (AP) conjugated antibody reagent is prepared according to the recommended protocol of Dako AS. This protocol is a standard procedure in this field. This reagent is called Reagent 2.
The substrate solution phenolphtaleine-monophosphate is to be prepared according to the recommended protocol of Fluka AG. This protocol is a standard procedure in this field. The substrate solution is called Reagent 3.
The washing and incubation buffer which is used is standard 0.05M tris-base buffer with the following additional compounds; Tween 20 (0.01% to 0.1%), glycerol (0.1% to 10%) and sodium chloride (0.2% to 0.1%). The assay procedure comprises an incubation step wherein 1 drop of Reagent 1 is mixed with 2 drops of washing buffer in each well. After mixing, 30 μΙ of sample is added and the solution is incubated for 5 minutes. The magnetic particles can be trapped by a magnet and the liquid removed, before the magnet is separated. Then the wells are washed twice in 4 drops of washing solution, before incubation with Reagent 2. 1 drop of Reagent 2 is added with 2 drops of washing buffer and the solution is incubated for 5 minutes. The magnetic particles can be trapped by a magnet and the liquid removed, before the magnet is separated. Then the washing step is repeated before incubation with Reagent 3. Two drops of Reagent 3 are added to each well and the solution is incubated for 3 minutes. The results can be read against a white background. Positive results are red (3+=strong red) whereas negative results are clearly light yellow/brown solutions as obtained in the negative control. The immunoassay kit could be used in detection of antibodies, induced either by NEV virus or NEV-specific peptides or proteins, for instance the peptides of the present invention.
Example 4
Immunomodulator (e.g. therapeutic or prophylactic vaccine).
At least one of the peptides of the invention, selected from the group of sequences SEQ ID NO: 1 to SEQ ID NO: 76 can form antigens and constitute the active principle of an immunomodulator e.g. a prophylactic or therapeutic vaccine intended to provide protection against the equine retrovirus (EIAV) or NEV. The immunomodulator may include compounds having beneficial effects in protecting or stimulating the hosts immune system for instance interleukins, interferons, granulocyte macrophage growth factors, haematopoietic growth factors or similar. In one embodiment the immunomodulator further contains an adjuvant or vehicle; for example, the adjuvant or vehicle is Monophosphoryl Lipid A (MPL(R)) possibly with alum, Freund's adjuvant (complete or incomplete) or aluminium hydroxyd. The optimal amount of adjuvant/vehicle will depend on the type(s) which is chosen.
The peptides of the present invention may be modified by C-terminal addition of a single fatty acid such as a single palmitoyi chain to form a lipopeptide vaccine. Further the lipopeptides can be introduced into liposome membranes by the freeze-thaw method resulting in liposomes bearing the peptide ligands on their surface.
The peptides of the present invention or compositions of the present invention or pharmaceutical compositions of the present invention or immunomodulator formulation can be freeze-dried prior to storage. The freeze-dried peptides can be dissolved in sterile water to a final concentration of 0.1-100 mg/ml. The peptides of the present invention or compositions of the present invention or pharmaceutical compositions of the present invention or the immunomodulator may be stored, for example, at low temperature, in ampoules containing one or more dosage units, ready for use.
A typical dosage unit of a peptide according to the invention is within the concentration range: 0.05 g-1 mg per kg bodyweight, preferably within 0, 15 μg-0.15 mg per kg body weight. Persons skilled in the art will appreciate that a suitable dose will depend on the body weight of the animal, the type of disease, severity of condition, administration route and several other factors.
The peptides of the present invention or compositions of the present invention or pharmaceutical compositions of the present invention may be used as a therapeutic vaccine. When used as a therapeutic vaccine, the vaccine might be administered up to 12 times, through injections. Further boosters might follow and can in some cases take place throughout the animal life. In preparation of an injection solution the peptides are typically dissolved in sterile water at a final concentration of 1 mg/ml per peptide. Typically an injection volume is 100 μΙ to 200 μΙ (2x100 μΙ). The peptide is preferably co-administered with a suitable adjuvant and/or a granulocyte-macrophage growth factor for instance Leucomax(R) (Schering Plough) made within a concentration range of from 0.1 mg/ml to 1 mg/ml, or according to the manufacturers recommendations for use.
These peptides may be administered simultaneously or sequentially. Suitable administration may be intracutaneous, subcutaneous, intravenous, peroral, intramuscular, intranasal, mucosal or any other suitable route. Booster administrations may be required in order to maintain protection. For persons skilled in the art it will be understood that the immunomodulators according to the invention may be useful not only in prevention of infection, but also in treatment of infection.
Example 5
An immunomodulator comprising one or more peptides selected from the group consisting of SEQ ID NO: 1 to and 76 are prepared. The freeze-dried peptides are dissolved in sterile water at a final concentration of 4 mg/ml. A preparation of a granulocyte-macrophage-colony stimulating factor (GM-CSF) is also prepared, according to the manufacturers directions for use, to a final concentration of 0.3 mg/ml. The solutions are administered intracutaneously or intramuscularly or intradermal. A typical injection dose is 100 μΙ.
Example 6
An antigen solution or suspension is mixed with equal parts of Freund's adjuvant of Behring, complete or incomplete, and is then finely emulsified by being drawn up into, and vigorously pressed out of, an injection syringe, or with a homogenator. The emulsion should remain stable for at least 30 minutes. The antigen-adjuvant emulsions is best injected subcutaneously, or intramuscularly or intradermal as a depot.
Example 7
Development of a fast and sensitive peptide based c-ELISA to identify NEV infected animals
A highly sensitive peptide based competitive enzyme-linked immunosorbent (c-ELISA) procedure was developed to detect sera antibodies against NEV.
Mouse monoclonal antibodies (mab) were produced against SEQ ID NO: 18 or SEQ ID NO: 20.
A monoclonal antibody mab#4G6E4 was directed against SEQ ID NO: 20.. Furthermore this mab was tested against SEQ ID NO: 20. or the fusion peptide SEQ ID NO: 159. by ELISA procedures.
For that, serial dilutions of peptides in duplicates (1 :1.3 or 1 :1.4 in 0.05M carbonate buffer pH=9.3) ranging between 5 to 500 ng/mL were covalently linked to 96 microwell Poylsorp amino plates (Nunc, Thermofisher) for two hours at room temperature. To remove unbound peptide, plates were washed three times with PBS-Tween20 (0.005%) in an automated Wellwasher apparatus (Thermoscientific). After washing, plates were blocked by adding 300 microliters/well of Superblock T20 in PBS (Thermoscientific, Pierce). Superblock T20 was immediately removed and plates inverted till drying out. After blocked and dried plates were ready for c-ELISA or to be stored at 4-8°C for further testing.
To compare the affinity of mab#4G6E4 for the peptide SEQ ID NO: 20 or for the fusion peptide SEQ ID NO: 159 mab#4G6E4 was diluted in the antibody dilution buffer, 3% BSA blocker (Thermoscientific). A rapid (2 hours) c-ELISA procedure was performed as follows: 70 ng/mL of mab#4G6E4 alone (50 microliters) diluted in dilution buffer was added to peptide-coated wells and incubated for 1hour at 37°C. After 1 hour of incubation plates were extensively washed with 300 microliters of PBS-Tween20 (0.05%) for three times. The plate was after that incubated with a secondary goat anti-mouse IgG- HRP (Sigma Aldrich) at 1 :2500 dilution and incubated for 30 minutes at 37°C. Plates were washed again four times with 300 microliters of PBS-Tween20 (0.05%) and signal developed by adding 50 microliters of fresh TMB reagent (Sigma Aldrich) for 15 minutes at room temperature. After colour development the reaction was stopped by adding 50 microliters of 1 N H2S0 (Sigma Aldrich). Determination of absorbance was obtained by reading plates at 450 nm in a Multiskan Go ELISA reader (Thermoscientific).
Dose responses curves and inhibition rate
Dose responses curves were analysed by GraphPad Prism 6.0 software by using the four parameter function log(agonist) vs response with a variable slope. In these conditions the EC50 of peptide SEQ ID NO: 159 was 163.5 ng/mL and 193.7 ng/mL for the SEQ ID NO: 20 peptide. The Hill slope was very high for both peptides 7.2 for SEQ ID NO: 159 and 6.6 for SEQ ID NO: 20. The results are indicating high affinity between the mab and the peptide sequences (Figure 6A1).
Furthermore dose response curves were optimized in order to decrease the Hill Slope and turn the assay more sensitive. For that the dilution buffer was replaced by 0.025% of casein blocking (instead of 3%BSA). The results showed (Figure 6A2) that in these conditions the mab#4G6E4 (70ng/mL) is highly specific and able to bind less than 25 ng/mL of the fusion peptide. In these conditions the EC50 was decreased to 59 ng/mL and Hill Slope to 2.52, the R2 was 0.9997. In Figure 6A1 we show dose response curves for SEQ ID NO: 20 and SEQ ID NO: 159 and mab#4G6E4 in 3% BSA. In Figure 6A2 we show dose responses curves for mab#4G6E4 diluted in 0.025% of casein.
Competitive ELISA
To develop a competitive ELISA the mab#4G6E4 was incubated in the presence of a NEV negative control sera or positive control sera and incubated as previously described in wells coated with serial dilutions of the fusion peptide set forth as SEQ ID NO: 159. For that, 25 microliters of mab#4G6E4 and 25 microliters of diluted equine sera were added in coated wells and incubated together for 1hour at 37°C. Serum or mab#4G6E4 were diluted in 0.025% Casein blocking buffer in PBS (Thermoscientific, Pierce).
Data were analysed by using GraphPad PRISM6.0 version software. Different dose response curves were generated by the nonlinear regression, log(agonist)vs response with a variable slope - four parameter function.
The inhibition of mAb#4G6E4 binding was calculated per each peptide dilution as a percent inhibition of the mAb with reference negative serum, using the following formula: % inhibition (%l) = 100 - [(test serum ODx100)/(mean negative control OD)]. The test serum was considered to be antibody-positive if the %l was≥ 30%.
Moreover in the presence of seropositive equine sera (diluted 1 :10 or 1 :100) mab#4G6E4 binding to SEQ ID NO: 159 was evident with decreased signal due to inhibition/competition per the peptide. The same effect was not shown in the presence of seronegative sera. The EC50, Hill slope, top and bottom values of the dose curve response were calculated for different dose curve responses in the presence of seropositive or seronegative sera. Different dose responses curves were analysed and compared with each other using the extra sum of squares F test. Results showed EC50, Hill slope, bottom and top values of mixtures of mab#4G6E4 and seronegative sera were statistically different (p<0.0001) from the EC50> Hill slope, bottom and top values of mixtures of mab#4G6E4 and seropositive sera. The R2 retrieved for each dose response curve were always higher than 0.980.
In Figure 6B we present dose responses curve and inhibition rates of NEV seronegative and seropositives samples.
In Figure 6C we show representative inhibition rates (%l) of NEV seropositive and one seronegative. Inhibition rates were obtained for a SEQ ID NO: 159 peptide concentration of 96 ng/mL of peptide and mab#4G6E4 IgG concentration of 70ng/mL.
SEQUENCES
Below are the polypeptide and polynucleotide sequences referred to in the present application:
SRLLESRGPTTSEK (SEQ ID NO: 1);
TLKEVLGGEAAVR (SEQ ID NO: 2);
PCRSKNILPV (SEQ ID NO: 3);
DPCVHSVDVLLR (SEQ ID NO: 4);
STFPPTPV (SEQ ID NO: 5);
NAPLLSKVSP (SEQ ID NO: 6);
MAQCIVNR (SEQ ID NO: 7);
KPNVEGRYGLSRSETNK (SEQ ID NO: 8);
QQGPEAPLPSLQVAEVPK (SEQ ID NO: 9);
PHAGSTQTEWPK (SEQ ID NO: 10);
PIQINACK (SEQ ID NO: 11);
GGMRESWDGGQV (SEQ ID NO: 12);
ESLVSKGFR (SEQ ID NO: 13);
CSLVLGEMQIKRIR (SEQ ID NO: 14);
LDKWEKIR (SEQ ID NO 15);
QMMIAASEK (SEQ ID NO 16);
QPSLPTGSEELK (SEQ ID NO 17);
EALDKIEEIQNKNKQK (SEQ ID NO 18);
PPIPVGGIYK (SEQ ID NO 19);
QEQNPPPSVSLRSLFGNDPL (SEQ ID NO 20);
EKIEQLR (SEQ ID NO 21);
DLLLIVAR (SEQ ID NO 22);
IVELLGR (SEQ ID NO 23);
IWGNVTWMEWER (SEQ ID NO 24);
LKDLFPNK (SEQ ID NO 25);
AKLEESFPGK (SEQ ID NO 26);
NGSLAGESIIIR (SEQ ID NO 27);
NITFNSSAGGDLEIT (SEQ ID NO 28);
AVILLLDRLR (SEQ ID NO 29);
GPGIHIGKR (SEQ ID NO 30);
VIICSASK (SEQ ID NO 31);
ESFPNK (SEQ ID NO 32); FGNKTTIIFTK (SEQ ID NO 33);
LLNGSLAGESIIIR (SEQ ID NO 34);
VNVSVTNNNTTTNV (SEQ ID NO 35);
AILHILRR (SEQ ID NO 36);
DLLALDK (SEQ ID NO 37);
IGCQHSRIGITLPR (SEQ ID NO 38);
TLQYLALTALVTPK (SEQ ID NO 39);
PTTPVTPAPGVGEISKELAQGK (SEQ ID NO 40); WSSALQYLIPR (SEQ ID NO 41);
PVWAEPVK (SEQ ID NO 42);
VKGNDLLK (SEQ ID NO 43);
EDLNLQDWK (SEQ ID NO 44);
LFVSVLQR (SEQ ID NO 45);
AAEQKASPPSLTPK (SEQ ID NO 46);
PLSLPLKI (SEQ ID NO 47);
FPSIVGRPR (SEQ ID NO 48);
IWHHTFYNELR (SEQ ID NO 49);
MTQIMFETF (SEQ ID NO 50);
EEEVAALVIDNGSGMCK (SEQ ID NO 51);
VAPEEHPVLLTEAPLNPK (SEQ ID NO 52);
AVFPSIVGRPR (SEQ ID NO 53);
YPIEHGIVTNWDDMEK (SEQ ID NO 54);
AVFPSIVGR (SEQ ID NO 55);
IGRKDAERQLLSPGNAR (SEQ ID NO 56);
DSYVGDEAQSKR (SEQ ID NO 57);
RGILTLK (SEQ ID NO 58);
WGIAHTTGIPGNSQGQAMVER (SEQ ID NO 59); HLVDQLIRDLK (SEQ ID NO 60);
YEQLQLQAR (SEQ ID NO 61);
TITLEVEPSDTIENVK (SEQ ID NO 62);
INRELLK (SEQ ID NO 63);
QTIIPTNKDVDEK (SEQ ID NO 64);
KNYGKLDK (SEQ ID NO 65);
TTISFSK (SEQ ID NO 66);
QDRTTISFSK (SEQ ID NO 67); AQGRAIAHK (SEQ ID NO 68);
MAQTQLVPVK (SEQ ID NO 69);
QELIPPCK (SEQ ID NO 70);
DIVLLENGK (SEQ ID NO 71);
LPPLSILK (SEQ ID NO 72);
IFLINLAFLIK (SEQ ID NO 73);
NRLEILK (SEQ ID NO 74);
ADDVAVLQDALGR (SEQ ID NO 75);
LNKSLEQLR (SEQ ID NO 76);
AGCAGGCTGCTGGAGAGCAGGGGCCCCACCACCAGCGAGAAG (SEQ ID NO 77);
ACCCTGAAAGAGGTTCTTGGTGGTGAAGCAGCCGTGCGC (SEQ ID NO 78); CCCTGCCGGAGCAAGAACATCCTGCCCGTG (SEQ ID NO 79);
GACCCCTGCGTGCACAGCGTGGACGTGCTGCTGAGG (SEQ ID NO 80);
TCCACATTTCCTCCCACTCCCGTC (SEQ ID NO 81);
AATGCCCCATTACTCTCAAAAGTGTCCCCA (SEQ ID NO 82);
ATGGCACAGTGTATCGTTAACCGC (SEQ ID NO 83);
AAGCCCAACGTGGAGGGCAGGTACGGCCTGAGCAGGAGCGAGACCAACAAG
(SEQ ID NO 84);
CAGCAAGGCCCAGAGGCGCCACTGCCCAGCCTGCAGGTTGCAGAAGTGCCTA AA (SEQ ID NO 85);
CCCCATGCGGGCTCCACTCAGACAGAGTGGCCCAAA (SEQ ID NO 86);
CCAATACAGATCAATGCTTGCAAA (SEQ ID NO 87);
GGTGGAATGCGTGAGTCATGGGATGGAGGACAAGTT (SEQ ID NO 88);
GAATCTCTGGTGTCAAAAGGGTTTCGG (SEQ ID NO 89);
TGCTCATTAGTCCTTGGGGAGATGCAAATCAAA (SEQ ID NO 90);
CTGGACAAGTGGGAGAAGATCAGG (SEQ ID NO 91);
CAGATGATGATCGCCGCCAGCGAGAAG (SEQ ID NO 92);
CAGCCCAGCCTGCCCACCGGCAGCGAGGAGCTGAAG (SEQ ID NO 93);
CCCCCCATCCCCGTGGGCGGCATCTACAAG (SEQ ID NO 94);
CAGGAGCAGAACCCCCCCCCCAGCGTGAGCCTGAGGAGCCTGTTCGGCAACG ACCCCCTG (SEQ ID NO 95);
GAGAAGATCGAGCAGCTGAGG (SEQ ID NO 96);
GACCTGCTGCTGATCGTGGCCAGG (SEQ ID NO 97);
ATCGTGGAGCTGCTGGGCAGG (SEQ ID NO 98); ATCTGGGGCAACGTGACCTGGATGGAGTGGGAGAGG (SEQ ID NO 99);
CTGAAGGACCTGTTCCCCAACAAG (SEQ ID NO 100);
GCCAAGCTGGAGGAGAGCTTCCCCGGCAAG (SEQ ID NO 101);
AACGGCAGCCTGGCCGGCGAGAGCATCATCATCAGG (SEQ ID NO 102);
AACATCACCTTCAACAGCAGCGCCGGCGGCGACCTGGAGATCACC (SEQ ID NO 103);
GCCGTGATCCTGCTGCTGGACAGGCTGAGG (SEQ ID NO 104);
GGCCCCGGCATCCACATCGGCAAGAGG (SEQ ID NO 105);
GTGATCATCTGCAGCGCCAGCAAG (SEQ ID NO 106);
GAG AG CTTCCCCAAC AAG (SEQ ID NO 107);
TTCGGCAACAAGACCACCATCATCTTCACCAAG (SEQ ID NO 108);
CTGCTGAACGGCAGCCTGGCCGGCGAGAGCATCATCATCAGG (SEQ ID NO
109) ;
GTGAACGTGAGCGTGACCAACAACAACACCACCACCAACGTG (SEQ ID NO
1 10) ;
G CCATCCTG C AC ATCCTG AGG AG G (SEQ ID NO 111);
GACCTGCTGGCCCTGGACAAG (SEQ ID NO 112);
ATCGGCTGCCAGCACAGCAGGATCGGCATCACCCTGCCCAGG (SEQ ID NO
1 13) ;
ACCCTGCAGTACCTGGCCCTGACCGCCCTGGTGACCCCCAAG (SEQ ID NO
1 14) ;
CCCACCACCCCCGTGACCCCCGCCCCCGGCGTGGGCGAGATCAGCAAGGAGC TGGCCCAGGGCAAG (SEQ ID NO 115);
GTGGTGAGCAGCGCCCTGCAGTACCTGATCCCCAGG (SEQ ID NO 116);
CCCGTGTGGGCCGAGCCCGTGAAG (SEQ ID NO 117);
GTGAAGGGCAACGACCTGCTGAAG (SEQ ID NO 1 18);
GAGGACCTGAACCTGCAGGACTGGAAG (SEQ ID NO 119);
CTGTTCGTGAGCGTGCTGCAGAGG (SEQ I D NO 120) ;
GCCGCCGAGCAGAAGGCCAGCCCCCCCAGCCTGACCCCCAAG (SEQ ID NO 121);
CCCCTG AG CCTG CCCCTG AAG ATC (SEQ ID NO 122);
TTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID NO 123);
ATCTGG C ACCACACCTTCTAC AACG AG CTG AG G (SEQ ID NO 124);
ATGACCCAGATCATGTTCGAGACCTTC (SEQ ID NO 125);
GAGGAGGAGGTGGCCGCCCTGGTGATCGACAACGGCAGCGGCATGTGCAAG (SEQ ID NO 126);
GTGGCCCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCA AG (SEQ ID NO 127);
GCCGTGTTCCCCAGCATCGTGGGCAGGCCCAGG (SEQ ID NO 128);
TACCCC ATCG AG CACG G CATCGTG ACC AACTG GG ACG AC ATG G AG AAG (SEQ
ID NO 129);
GCCGTGTTCCCCAGCATCGTGGGCAGG (SEQ ID NO 130);
ATCGGCAGGAAGGACGCCGAGAGGCAGCTGCTGAGCCCCGGCAACGCCAGG
(SEQ ID NO 131);
GACAGCTACGTGGGCGACGAGGCCCAGAGCAAGAGG (SEQ ID NO 132);
AGGGGCATCCTGACCCTGAAG (SEQ ID NO 133);
TGGGGCATCGCCCACACCACCGGCATCCCCGGCAACAGCCAGGGCCAGGCCA TGGTGGAGAGG (SEQ ID NO 134);
CACCTGGTGGACCAGCTGATCAGGGACCTGAAG (SEQ ID NO 135);
TACGAGCAGCTGCAGCTGCAGGCCAGG (SEQ ID NO 136);
ACCATCACCCTGGAGGTGGAGCCCAGCGACACCATCGAGAACGTGAAG (SEQ ID NO 137);
ATCAACAGGGAGCTGCTGAAG (SEQ ID NO 138);
CAGACCATCATCCCCACCAACAAGGACGTGGACGAGAAG (SEQ ID NO 139); AAG AACTACG G CAAG CTGG ACAAG (SEQ ID NO 140);
ACC ACCATC AG CTTCAG C AAG (SEQ ID NO 141);
CAGGACAGGACCACCATCAGCTTCAGCAAG (SEQ ID NO 142);
G CCCAG GG CAGG G CC ATCG CCCAC AAG (SEQ ID NO 143);
ATGGCCCAGACCCAGCTGGTGCCCGTGAAG (SEQ ID NO 144);
CAGGAGCTGATCCCCCCCTGCAAG (SEQ ID NO 145);
GACATCGTGCTGCTGGAGAACGGCAAG (SEQ ID NO 146);
CTGCCCCCCCTGAGCATCCTGAAG (SEQ ID NO 147);
ATCTTCCTGATCAACCTGGCCTTCCTGATCAAG (SEQ ID NO 148);
AACAGGCTGGAGATCCTGAAG (SEQ ID NO 149);
GCCGACGACGTGGCCGTGCTGCAGGACGCCCTGGGCAGG (SEQ ID NO 150); CTGAACAAGAGCCTGGAGCAGCTGAGG (SEQ ID NO 151);
GAGGCCCTGGACAAGATCGAGGAGATCCAGAACAAGAACAAGCAGAAG (SEQ ID NO 152);
QEQNPPPSVSLRSLFG (SEQ ID No 153);
CAGGAGCAGAACCCCCCCCCCAGCGTGAGCCTGAGGAGCCTGTTCGGC (SEQ ID No 154);
WGIAHTTGIPGNSQGQAM (SEQ ID No 155);
TGGGGCATCGCCCACACCACCGGCATCCCCGGCAAGAGCCAGGGCCAGGCCA TG (SEQ ID No 156);
EALDKIEEIQNKNKQKAAAQEQNPPPSVSLRSLFGNDPL (SEQ ID NO: 157); QEQNPPPSVSLRSLFGNDPLAAAEALDKIEEIQNKNKQK (SEQ ID NO: 158); EALDKIEEIQNKNKQKAAAQEQNPPPSVSLRSLFG (SEQ ID NO: 159).
REFERENCES
Lignee, M., 1843, Memoire et observation sur une maladie du sang, connue sous le nom d'anhemie hydrohemie cachexie aqueuse du cheval. Rec. Med. Vet. 20, 30.
Spyrou V, Papanastassopoulou M, Koumbati M, Nikolakaki SV, Koptopoulos G. Molecular analysis of the proviral DNA of equine infectious anemia virus in mules in Greece. Virus Res. 2005 Jan;107(1):63-72. PubMed PMID: 15567035.
Mooney J, Flynn O, Sammin D. Equine infectious anaemia in Ireland: characterisation of the virus. Vet Rec. 2006 Oct 21 ;159(17):570. PubMed PMID: 17056658.
Quinlivan M, Cook F, Kenna R, Callinan JJ, Cullinane A. Genetic characterization by composite sequence analysis of a new pathogenic field strain of equine infectious anemia virus from the 2006 outbreak in Ireland. J Gen Virol. 2013 Mar;94(Pt 3):612-22. doi: 10.1099/vir.0.047191-0. Epub 2012 Nov 21. PubMed PMID: 23175240.
Cappelli K, Capomaccio S, Cook FR, Felicetti M, Marenzoni ML, Coppola G, Verini-Supplizi A, Coletti M, Passamonti F. Molecular detection, epidemiology, and genetic characterization of novel European field isolates of equine infectious anemia virus. J Clin Microbiol. 2011 Jan;49(1):27-33. doi: 10.1128/JCM.01311-10. Epub 2010 Nov 17. PubMed PMID: 21084503; PubMed Central PMCID: PMC3020406.
Quinlivan M, Cook RF, Cullinane A. Real-time quantitative RT-PCR and PCR assays for a novel European field isolate of equine infectious anaemia virus based on sequence determination of the gag gene. Vet Rec. 2007 May 5;160(18):611-8. PubMed PMID: 17483378.
Cappelli K, Capomaccio S, Cook FR, Felicetti M, Marenzoni ML, Coppola G, Verini-Supplizi A, Coletti M, Passamonti F. Molecular detection, epidemiology, and genetic characterization of novel European field isolates of equine infectious anemia virus. J Clin Microbiol. 201 1 Jan;49(1):27-33. doi: 10.1128/JCM.01311-10. Epub 2010 Nov 17. PubMed PMID: 21084503; PubMed Central PMCID: PMC3020406.
Capomaccio S, Cappelli K, Cook RF, Nardi F, Gifford R, Marenzoni ML, Passamonti F. Geographic structuring of global EIAV isolates: a single origin for New World strains? Virus Res. 2012 Feb;163(2):656-9. doi:10.1016/j.virusres.2011.11.011. Epub 2011 Nov 22. PubMed PMID: 22119901.
Kuhar U, Zavrsnik J, Toplak I, Malovrh T. Detection and molecular characterisation of equine infectious anaemia virus from field outbreaks in Slovenia. Equine Vet J. 2014 May;46(3):386-91. doi: 10.1111/evj.12138. Epub 2013 Sep 9. PubMed PMID: 23834226.
Scicluna MT, Issel CJ, Cook FR, Manna G, Cersini A, Rosone F, Frontoso R, Caprioli A, Antonetti V, Autorino GL. Is a diagnostic system based exclusively on agar gel immunodiffusion adequate for controlling the spread of equine infectious anaemia? Vet Microbiol. 2013 Jul 26;165(1-2):123-34. doi: 10.1016/j.vetmic.2013.02.027. Epub 2013 Mar 28. PubMed PMID: 23618837.
Quinlivan M, Cook F, Kenna R, Callinan JJ, Cullinane A. Genetic characterization by composite sequence analysis of a new pathogenic field strain of equine infectious anemia virus from the 2006 outbreak in Ireland. J Gen Virol. 2013 Mar;94(Pt 3):612-22. doi: 10.1099/vir.0.047191-0. Epub 2012 Nov 21. PubMed PMID: 23175240.
Issel CJ, Scicluna MT, Cook SJ, Cook RF, Caprioli A, Ricci I, Rosone F, Craigo JK, Montelaro RC, Autorino GL. Challenges and proposed solutions for more accurate serological diagnosis of equine infectious anaemia. Vet Rec. 2013 Feb 23;172(8):210. doi: 10.1136/vr-2012-100735. Epub 2012 Nov 16. PubMed PMID: 23161812; PubMed Central PMCID: PMC3593188.
Capomaccio S, Willand ZA, Cook SJ, Issel CJ, Santos EM, Reis JK, Cook RF. Detection, molecular characterization and phylogenetic analysis of full-length equine infectious anemia (EIAV) gag genes isolated from Shackleford Banks wild horses. Vet Microbiol. 2012 Jun 15;157(3-4):320-32. doi: 10.1016/j.vetmic.2012.01.015. Epub 2012 Jan 18. PubMed PMID: 22310073.
Murakami K, Konishi M, Kameyama K, Shibahara T. Detection of equine infectious anaemia virus in native Japanese ponies. Vet Rec. 2012 Jul 21 ;171 (3):72. doi: 10.1 136/vr.100459. Epub 2012 Jul 10. PubMed PMID: 22781341.
Craigo JK, Barnes S, Zhang B, Cook SJ, Howe L, Issel CJ, Montelaro RC. An EIAV field isolate reveals much higher levels of subtype variability than currently reported for the equine lentivirus family. Retrovirology. 2009 Oct 20;6:95. doi: 10.1186/1742-4690-6-95. PubMed PMID: 19843328; PubMed Central PMCID: PMC2770520.
Boldbaatar B, Bazartseren T, Koba R, Murakami H, Oguma K, Murakami K, Sentsui H. Amplification of complete gag gene sequences from geographically distinct equine infectious anemia virus isolates. J Virol Methods. 2013 Apr; 189(1 ):41-6. doi: 10.1016/j.jviromet.2012.12.010. Epub 2013 Jan 1 1. PubMed PMID: 23318370.
Dong JB, Zhu W, Cook FR, Goto Y, Horii Y, Haga T. Identification of a novel equine infectious anemia virus field strain isolated from feral horses in southern Japan. J Gen Virol. 2013 Feb;94(Pt 2):360-5. doi: 10.1099/vir.0.047498-0. Epub 2012 Oct 24. PubMed PMID: 23100364.
Murakami K, Konishi M, Kameyama K, Shibahara T. Detection of equine infectious anaemia virus in native Japanese ponies. Vet Rec. 2012 Jul 21 ;171 (3):72. doi: 10.1136/vr.100459. Epub 2012 Jul 10. PubMed PMID: 22781341.
Pagamjav O, Kobayashi K, Murakami H, Tabata Y, Miura Y, Boldbaatar B, Sentsui H. Serological survey of equine viral diseases in Mongolia. Microbiol Immunol. 201 1 Apr;55(4):289-92. doi: 10.111 1/j.1348-0421.2011.00312.x. PubMed PMID: 21447048.
Sentsui H, Inoshima Y, Murakami K, Akashi H, Purevtseren B, Pagmajav O, Sugiura T. Cross reaction of recombinant equine infectious anemia virus antigen to heterologous strains and application for serological survey among horses in the field. Microbiol Immunol. 2001 ,45(1 ):45-50. PubMed PMID: 11270606.
Clabough DL, Gebhard D, Flaherty MT, Whetter LE, Perry ST, Coggins L, Fuller FJ. Immune-mediated thrombocytopenia in horses infected with equine infectious anemia virus. J Virol. 1991 Nov;65(11):6242-51. PubMed PMID: 1717720; PubMed Central PMCID: PMC250322.
Crawford TB, Wardrop KJ, Tornquist SJ, Reilich E, Meyers KM, McGuire TC. A primary production deficit in the thrombocytopenia of equine infectious anemia. J Virol. 1996 Nov;70(11):7842-50. PubMed PMID: 8892906; PubMed Central PMCID: PMC190855.
Oaks JL, Long MT, Baszler TV. Leukoencephalitis associated with selective viral replication in the brain of a pony with experimental chronic equine infectious anemia virus infection. Vet Pathol. 2004 Sep;41(5):527-32. PubMed PMID: 15347829.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. A composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 1 to 76; and/or comprising one or more polynucleotide sequences encoding one or more of the peptides;
and/or comprising one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs: 77 to 152, and variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 77 to 152.
2. The composition according to claim 1 , wherein said one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof are linked together as a fusion protein.
3. The composition according to claim 1 or claim 2, wherein any two peptides selected from the group consisting of SEQ ID NOs: 1 to 76 and variants, homologues, fragments or derivatives thereof are linked together as a fusion protein.
4. The composition according to claim 3, wherein said two peptides are SEQ ID NO: 18 and SEQ ID NO: 20, or variants, homologues, fragments or derivatives thereof having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 18 and SEQ ID NO: 20.
5. The composition according to any one of claims 2 to 4, further wherein said peptides are linked together as a fusion protein by a linker peptide.
6. The composition of claim 5, wherein said linker peptide is between 2 and 10 amino acid residues in length.
7. The composition of claim 6, wherein said linker peptide is 3 amino acids in length.
8. The composition of any one of claims 5 to 7, wherein said linker peptide consists of non- polar amino acids.
9. The composition of any one of claims 5 to 8, wherein said linker peptide consists of amino acid residues having aliphatic side chains.
10. The composition of any one of claims 5 to 9, wherein said linker peptide comprises a repetitive amino acid sequence.
1 1. The composition of any one of claims 5 to 10, wherein said linker peptide consists of alanine residues.
12. The composition of any one of claims 5 to 11 , wherein said linker peptide is AAA.
13. The composition according to any one of claims 2 to 12, wherein said fusion protein comprises the sequence QEQNPPPSVSLRSLFGNDPLAAAEALDKIEEIQNKNKQK (SEQ ID NO: 158) or EALDKIEEIQNKNKQKAAAQEQNPPPSVSLRSLFG (SEQ ID NO: 159).
14. A polynucleotide sequence encoding the fusion protein of any one of claims 2 to 13.
15. A vector capable of encoding one or more peptides as defined in any one of claims 1 to 13, or a vector comprising one or more polynucleotide sequences as defined in claim 1 or claim 14.
16. An antibody capable of binding to one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76.
17. A pharmaceutical composition comprising:
one or more peptides as defined in any one of claims 1 to 13; and/or
one or more polynucleotide sequences as defined in claim 1 or claim 14; and/or one or more vectors according to claim 15; and/or
one or more antibodies according to claim 16;
and a pharmaceutically acceptable carrier, vehicle, diluent or excipient.
18. A composition according to any one of claims 1 to 13, and/or a vector according to claim 15, and/or an antibody according to claim 16, and/or a pharmaceutical composition according to claim 17 for use in the treatment or prevention of equine infectious anaemia, and/or the treatment or prevention of an infection by an equine infectious anaemia virus (EIAV) and/or new equine virus ( EV) in an animal.
19. The composition and/or vector and/or antibody and/or pharmaceutical composition according to claim 18 for the use according to claim 18 wherein said animal is an equine.
20. The composition and/or vector and/or pharmaceutical composition and/or antibody according to claim 18 for the use according to claim 18 wherein said animal is a horse.
21. A kit comprising:
a composition according to any one of claims 1 to 13; and/or
a vector according to claim 15; and/or
an antibody according to claim 16; and/or
a pharmaceutical composition according to claim 17;
and optionally instructions for administration to an animal.
22. A diagnostic method comprising obtaining a sample from an animal and determining the presence or absence in said sample of antibodies which are capable of binding to one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 76
and/or determining the presence or absence in said sample of one or more of said peptides; and/or determining the presence or absence in said sample of one or more polynucleotide sequences as defined in claim 1 or claim 14.
23. The diagnostic method according to claim 22 wherein said method determines the presence or absence of an EIAV and/or NEV in an animal, and/or the diagnostic method according to claim 22 determines the viral titre of an EIAV and/or NEV in an animal.
24. The diagnostic method according to claim 22 or 23 wherein said animal is an equine.
25. The diagnostic method according to one of claims 22 to 24 wherein said animal is a horse.
26. A kit comprising one or more peptides selected from the group consisting of SEQ ID NO:s 1 to 76 and optionally instructions for determining the presence or absence in an animal sample of antibodies capable of binding to one or more said peptides.
27. A kit comprising one or more antibodies according to claim 16 and optionally instructions for determining the presence or absence in an animal sample of peptides capable of binding to said antibodies.
28. An oligonucleotide sequence (e.g. a primer or a probe) comprising or consisting of a polynucleotide sequence capable of encoding one or more of the peptides selected from the group consisting of SEQ ID NOs: 1 to 76
and/or comprising or consisting of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 77 to 152.
29. A kit comprising one or more oligonucleotide sequences according to claim 28 and optionally instructions for determining the presence or absence in an animal sample of said polynucleotide sequence.
30. A method for controlling EIAV and/or NEV disease in a group of animals comprising the identification of EIAV and/or NEV infection in an animal using the diagnostic method according to any one of claims 22 to 25 and, optionally, the isolation of an EIAV and/or NEV infected animal from other animals.
31. Use of a composition according to any one of claims 1 to 13, and/or a vector according to claim 15, and/or a pharmaceutical composition according to claim 17, and/or an antibody according to claim 16, for the manufacture of a medicament for the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV and/or NEV in an animal.
32. Use of a composition according to any one of claims 1 to 13, and/or a vector according to claim 15, and/or a pharmaceutical composition according to claim 17, and/or an antibody according to claim 16 in a kit or assay for the diagnosis or prevention of equine infectious anaemia and/or the diagnosis or prevention of an infection by an EIAV and/or NEV in an animal.
33. A method for the treatment or prevention of equine infectious anaemia and/or the treatment or prevention of an infection by an EIAV and/or NEV in an animal, wherein said method comprises administering to an animal one or more compositions according to any one of claims 1 to 13, and/or one or more vectors according to claim 15, and/or one or more antibodies according to claim 16, and/or one or more pharmaceutical compositions according to claim 17.
34. A peptide substantially as described herein with reference to any one of the Examples and/or drawings.
35. A polynucleotide substantially as described herein with reference to any one of the Examples and/or drawings.
36. An antibody substantially as described herein with reference to any one of the Examples and/or drawings.
37. An oligonucleotide substantially as described herein with reference to any one of the Examples and/or drawings.
38. A vector substantially as described herein with reference to any one of the Examples and/or drawings.
39. A pharmaceutical composition substantially as described herein with reference to any one of the Examples and/or drawings.
40. A kit substantially as described herein with reference to any one of the Examples and/or drawings.
41. A composition and/or a vector and/or a pharmaceutical composition substantially as described herein with reference to any one of the Examples and/or drawings for use in the treatment or prevention of equine infectious anaemia and/or treatment or prevention of an infection by an EIAV and/or NEV in an animal.
42. A diagnostic method substantially as described herein with reference to any one of the Examples and/or drawings.
43. A use substantially as described herein with reference to any one of the Examples and/or drawings.
44. A method substantially as described herein with reference to any one of the Examples and/or drawings for the treatment or prevention of equine infectious anaemia and/or treatment or prevention of an infection by an EIAV and/or NEV in an animal.
PCT/PT2014/000077 2013-12-19 2014-12-19 Viral peptides WO2015094001A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
GB1515545.0A GB2526222B (en) 2013-12-19 2014-12-19 Viral peptides
US15/104,249 US20160367659A1 (en) 2013-12-19 2014-12-19 Viral peptides
EP14824566.5A EP3083659B1 (en) 2013-12-19 2014-12-19 Viral peptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1322574.3A GB201322574D0 (en) 2013-12-19 2013-12-19 Retroviral peptides
GB1322574.3 2013-12-19

Publications (1)

Publication Number Publication Date
WO2015094001A1 true WO2015094001A1 (en) 2015-06-25

Family

ID=50071139

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/PT2014/000077 WO2015094001A1 (en) 2013-12-19 2014-12-19 Viral peptides

Country Status (5)

Country Link
US (1) US20160367659A1 (en)
EP (1) EP3083659B1 (en)
GB (2) GB201322574D0 (en)
PT (1) PT3083659T (en)
WO (1) WO2015094001A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108715600A (en) * 2018-04-12 2018-10-30 中国科学院南海海洋研究所 A kind of oligopeptides and its preparation method and application promoting Intestinal epithelial cells proliferation and migration

Citations (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3929982A (en) 1971-11-05 1975-12-30 Cornell Res Foundation Inc Method and composition for the diagnosis of equine infectous anemia virus infection by using agar-gel-immunodiffusion reaction
US3932601A (en) 1971-04-07 1976-01-13 Cornell Research Foundation, Inc. Method and composition for the diagnosis of equine infectious anemia virus infection by using agar-gel-immunodiffusion reaction
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4946779A (en) 1981-04-13 1990-08-07 Takeda Chemical Industries, Ltd. Pseudo-aminosugars, their production and use
WO1995030018A2 (en) 1994-04-29 1995-11-09 Immuno Aktiengesellschaft Recombinant poxviruses with foreign polynucleotides in essential regions
EP0743364A2 (en) * 1995-05-18 1996-11-20 Commissariat A L'energie Atomique Nucleic acid fragments derived form the HIV-1 genome, corresponding fragments and their application as reactives for risk evaluation of HIV-1 mother-foetal transmission
US5693325A (en) * 1994-03-15 1997-12-02 Molecumetics, Ltd. Peptide vaccines and methods relating thereto
WO1998032771A1 (en) * 1997-01-29 1998-07-30 Toray Industries, Inc. Chimeric proteins, heterodimer complexes thereof and substitute for platelet
WO1999015683A1 (en) 1997-09-25 1999-04-01 Oxford Biomedica (Uk) Limited Retroviral vectors comprising a functional splice donor site and a functional splice acceptor site
WO2001024810A1 (en) * 1999-10-05 2001-04-12 Epimmune Inc. Inducing cellular immune responses to human immunodeficiency virus-1 using peptide and nucleic acid compositions
WO2002048345A2 (en) * 2000-12-13 2002-06-20 Deltagen, Inc. Transgenic mice containing glutamate receptor (grik5) gene disruptions
WO2002054081A2 (en) * 2000-12-29 2002-07-11 Oxford Glycosciences (Uk) Limited Proteins, genes and their use for diagnosis and treatment of kidney response
WO2002069691A2 (en) * 2001-03-01 2002-09-12 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention, Technology Transfer Office Immunogenic hiv peptides for use as reagents and vaccines
WO2004081186A2 (en) * 2003-03-10 2004-09-23 Applera Corporation Single nucleotide polymorphisms associated with stenosis, methods of detection and uses thereof
WO2004083418A1 (en) * 2003-03-17 2004-09-30 Merck & Co., Inc. Adenovirus serotype 24 vectors, nucleic acids and virus produced thereby
JP2004361227A (en) * 2003-06-04 2004-12-24 Tohoku Univ Method for identifying amyloid beta bond protein
WO2005015206A2 (en) * 2003-08-08 2005-02-17 Genova Ltd. Secreted polypeptide species associated with cardiovascular disorders
WO2005032495A2 (en) * 2003-10-03 2005-04-14 Bayer Pharmaceuticals Corporation Gene expression profiles and methods of use
WO2005100399A2 (en) * 2004-04-06 2005-10-27 Genentech, Inc. Dr5 antibodies and uses thereof
US20050271676A1 (en) * 1993-03-05 2005-12-08 Epimmune Inc. Inducing cellular immune responses to human immunodeficiency virus-1 using peptide and nucleic acid compositions
US20050282744A1 (en) * 2003-07-11 2005-12-22 Tony Hollingsworth Compositions and methods for preventing or treating cancer
WO2006034005A2 (en) * 2004-09-17 2006-03-30 University Of Utah Research Foundation Imaging reporters of transgene expression
WO2006086111A2 (en) * 2005-02-10 2006-08-17 Cell Signaling Technology, Inc. Reagents for the detection of protein phosphorylation in leukemia signaling pathways
WO2008054514A2 (en) * 2006-04-21 2008-05-08 Wyeth Differential expression profiling analysis of cell culture phenotypes and the uses thereof
US20080263730A1 (en) * 1999-05-07 2008-10-23 Andersen Scott E Nucleic acid molecules and other molecules associated with plants
WO2009074350A2 (en) * 2007-12-13 2009-06-18 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. Serum proteomic for finding diagnostic markers and for monitoring therapeutical intervention in treatment of hepatocellular carcinoma
WO2009097692A1 (en) * 2008-02-07 2009-08-13 Siu K W Michael Biomarkers for head-and-neck cancers and precancers
US20110065756A1 (en) * 2009-09-17 2011-03-17 De Taeye Bart M Methods and compositions for treatment of obesity-related diseases
WO2012088513A2 (en) * 2010-12-23 2012-06-28 Fox Chase Cancer Center Inhibiting the integration of retrovirus dna into host cell dna and screening compounds capable of the same
WO2012102963A1 (en) * 2011-01-26 2012-08-02 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Urine biomarkers for prediction of recovery after acute kidney injury : proteomics
WO2013151669A1 (en) * 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins

Patent Citations (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3932601A (en) 1971-04-07 1976-01-13 Cornell Research Foundation, Inc. Method and composition for the diagnosis of equine infectious anemia virus infection by using agar-gel-immunodiffusion reaction
US3929982A (en) 1971-11-05 1975-12-30 Cornell Res Foundation Inc Method and composition for the diagnosis of equine infectous anemia virus infection by using agar-gel-immunodiffusion reaction
US4946779A (en) 1981-04-13 1990-08-07 Takeda Chemical Industries, Ltd. Pseudo-aminosugars, their production and use
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (en) 1985-03-28 1990-11-27 Cetus Corp
US20050271676A1 (en) * 1993-03-05 2005-12-08 Epimmune Inc. Inducing cellular immune responses to human immunodeficiency virus-1 using peptide and nucleic acid compositions
US5693325A (en) * 1994-03-15 1997-12-02 Molecumetics, Ltd. Peptide vaccines and methods relating thereto
WO1995030018A2 (en) 1994-04-29 1995-11-09 Immuno Aktiengesellschaft Recombinant poxviruses with foreign polynucleotides in essential regions
EP0743364A2 (en) * 1995-05-18 1996-11-20 Commissariat A L'energie Atomique Nucleic acid fragments derived form the HIV-1 genome, corresponding fragments and their application as reactives for risk evaluation of HIV-1 mother-foetal transmission
WO1998032771A1 (en) * 1997-01-29 1998-07-30 Toray Industries, Inc. Chimeric proteins, heterodimer complexes thereof and substitute for platelet
WO1999015683A1 (en) 1997-09-25 1999-04-01 Oxford Biomedica (Uk) Limited Retroviral vectors comprising a functional splice donor site and a functional splice acceptor site
US20080263730A1 (en) * 1999-05-07 2008-10-23 Andersen Scott E Nucleic acid molecules and other molecules associated with plants
WO2001024810A1 (en) * 1999-10-05 2001-04-12 Epimmune Inc. Inducing cellular immune responses to human immunodeficiency virus-1 using peptide and nucleic acid compositions
WO2002048345A2 (en) * 2000-12-13 2002-06-20 Deltagen, Inc. Transgenic mice containing glutamate receptor (grik5) gene disruptions
WO2002054081A2 (en) * 2000-12-29 2002-07-11 Oxford Glycosciences (Uk) Limited Proteins, genes and their use for diagnosis and treatment of kidney response
WO2002069691A2 (en) * 2001-03-01 2002-09-12 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention, Technology Transfer Office Immunogenic hiv peptides for use as reagents and vaccines
WO2004081186A2 (en) * 2003-03-10 2004-09-23 Applera Corporation Single nucleotide polymorphisms associated with stenosis, methods of detection and uses thereof
WO2004083418A1 (en) * 2003-03-17 2004-09-30 Merck & Co., Inc. Adenovirus serotype 24 vectors, nucleic acids and virus produced thereby
JP2004361227A (en) * 2003-06-04 2004-12-24 Tohoku Univ Method for identifying amyloid beta bond protein
US20050282744A1 (en) * 2003-07-11 2005-12-22 Tony Hollingsworth Compositions and methods for preventing or treating cancer
WO2005015206A2 (en) * 2003-08-08 2005-02-17 Genova Ltd. Secreted polypeptide species associated with cardiovascular disorders
WO2005032495A2 (en) * 2003-10-03 2005-04-14 Bayer Pharmaceuticals Corporation Gene expression profiles and methods of use
WO2005100399A2 (en) * 2004-04-06 2005-10-27 Genentech, Inc. Dr5 antibodies and uses thereof
WO2006034005A2 (en) * 2004-09-17 2006-03-30 University Of Utah Research Foundation Imaging reporters of transgene expression
WO2006086111A2 (en) * 2005-02-10 2006-08-17 Cell Signaling Technology, Inc. Reagents for the detection of protein phosphorylation in leukemia signaling pathways
WO2008054514A2 (en) * 2006-04-21 2008-05-08 Wyeth Differential expression profiling analysis of cell culture phenotypes and the uses thereof
WO2009074350A2 (en) * 2007-12-13 2009-06-18 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. Serum proteomic for finding diagnostic markers and for monitoring therapeutical intervention in treatment of hepatocellular carcinoma
WO2009097692A1 (en) * 2008-02-07 2009-08-13 Siu K W Michael Biomarkers for head-and-neck cancers and precancers
US20110065756A1 (en) * 2009-09-17 2011-03-17 De Taeye Bart M Methods and compositions for treatment of obesity-related diseases
WO2012088513A2 (en) * 2010-12-23 2012-06-28 Fox Chase Cancer Center Inhibiting the integration of retrovirus dna into host cell dna and screening compounds capable of the same
WO2012102963A1 (en) * 2011-01-26 2012-08-02 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Urine biomarkers for prediction of recovery after acute kidney injury : proteomics
WO2013151669A1 (en) * 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
WO2013151671A1 (en) * 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of cosmetic proteins and peptides

Non-Patent Citations (72)

* Cited by examiner, † Cited by third party
Title
"Handbook of Pharmaceutical Excipients", 1994
"Oligonucleotide Synthesis: A Practical Approach", 1984, IRL PRESS
"Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING CO.
ALTSCHUL ET AL., J. MOL. BIOL., 1990, pages 403 - 410
AUSUBEL, F. . ET AL.: "Current Protocols in Molecular Biology", 1995, JOHN WILEY & SONS
AUSUBEL: "Short Protocols in Molecular Biology", 1999
B. ROE; J. CRABTREE; A. KAHN: "DNA Isolation and Sequencing: Essential Techniques", 1996, JOHN WILEY & SONS
BARANI, G.; MERRIFIELD, R. B.: "The Peptides, Analysis, Synthesis and Biology", vol. 2, ACADEMIC PRESS
BEUCAGE S.L. ET AL., TETRAHEDRON LETTERS, vol. 22, 1981, pages 859 - 18
BOLDBAATAR B; BAZARTSEREN T; KOBA R; MURAKAMI H; OGUMA K; MURAKAMI K; SENTSUI H.: "Amplification of complete gag gene sequences from geographically distinct equine infectious anemia virus isolates.", J VIROL METHODS., vol. 189, no. 1, 11 January 2013 (2013-01-11), pages 41 - 6
CAPOMACCIO S; CAPPELLI K; COOK RF; NARDI F; GIFFORD R; MARENZONI ML; PASSAMONTI F.: "Geographic structuring of global EIAV isolates: a single origin for New World strains?", VIRUS RES., vol. 163, no. 2, 22 November 2011 (2011-11-22), pages 656 - 9
CAPOMACCIO S; WILLAND ZA; COOK SJ; ISSEL CJ; SANTOS EM; REIS JK; COOK RF.: "Detection, molecular characterization and phylogenetic analysis of full-length equine infectious anemia (EIAV) gag genes isolated from Shackleford Banks wild horses.", VET MICROBIOL., vol. 157, no. 3-4, 8 January 2012 (2012-01-08), pages 320 - 32
CAPPELLI K; CAPOMACCIO S; COOK F; FELICETTI M; MARENZONI L; COPPOLA G; VERINI-SUPPLIZI A; COLETTI M; PASSAMONTI F.: "Molecular detection, epidemiology, and genetic characterization of novel European field isolates of equine infectious anemia virus.", J CLIN MICROBIOL., vol. 49, no. 1, 17 November 2010 (2010-11-17), pages 27 - 33
CAPPELLI K; CAPOMACCIO S; COOK FR; FELICETTI M; MARENZONI ML; COPPOLA G; VERINI-SUPPLIZI A; COLETTI M; PASSAMONTI F.: "Molecular detection, epidemiology, and genetic characterization of novel European field isolates of equine infectious anemia virus.", J CLIN MICROBIOL., vol. 49, no. 1, 17 November 2010 (2010-11-17), pages 27 - 33
CARUTHERS H ET AL., NUC ACIDS RES SYMP SER, 1980, pages 215 - 23
CLABOUGH DL; GEBHARD D; FLAHERTY MT; WHETTER LE; PERRY ST; COGGINS L; FULLER FJ.: "Immune-mediated thrombocytopenia in horses infected with equine infectious anemia virus", J VIROL., vol. 65, no. 11, November 1991 (1991-11-01), pages 6242 - 51
COFFIN: "Retroviruses", 1997, COLD SPRING HARBOUR LABORATORY PRESS, pages: 758 - 763
COLE ET AL.: "Monoclonal Antibodies and Cancer Therapy", 1985, ALAN RICKMAN LISS INC, pages: 77 - 96
COTE ET AL., PROC NATL ACAD SCI, vol. 80, 1983, pages 2026 - 2030
CRAIGO JK; BARNES S; ZHANG B; COOK SJ; HOWE L; LSSEL CJ; MONTELARO RC.: "An EIAV field isolate reveals much higher levels of subtype variability than currently reported for the equine lentivirus family.", RETROVIROLOGY., vol. 6, 20 October 2009 (2009-10-20), pages 95
CRAWFORD TB; WARDROP KJ; TORNQUIST SJ; REILICH E; MEYERS KM; MCGUIRE TC.: "A primary production deficit in the thrombocytopenia of equine infectious anemia.", J VIROL, vol. 70, no. 11, November 1996 (1996-11-01), pages 7842 - 50
D. M. J. LILLEY; J. E. DAHLBERG: "Methods in Enzymology", 1992, ACADEMIC PRESS, article "Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA"
DATABASE EMBL [online] 20 October 2006 (2006-10-20), XP002736721, retrieved from EBI Database accession no. CU197709 *
DATABASE EMBL [online] 23 July 2010 (2010-07-23), XP002736728, retrieved from EBI Database accession no. FN566618 *
DATABASE EMBL [online] 23 October 2003 (2003-10-23), XP002736726, retrieved from EBI Database accession no. CG714943 *
DATABASE EMBL [online] 31 January 2013 (2013-01-31), XP002736724, retrieved from EBI Database accession no. JR948040 *
DATABASE EMBL [online] 31 January 2013 (2013-01-31), XP002736725, retrieved from EBI Database accession no. JR949410 *
DATABASE EMBL [online] 31 January 2013 (2013-01-31), XP002736727, retrieved from EBI Database accession no. JR948186 *
DATABASE EMBL [online] 31 October 2009 (2009-10-31), XP002736722, retrieved from EBI Database accession no. CP001220 *
DATABASE EMBL [online] 6 September 2002 (2002-09-06), XP002736723, retrieved from EBI Database accession no. AC129777 *
DATABASE EMBL [online] 9 August 2007 (2007-08-09), XP002736682, retrieved from EBI Database accession no. AC207823 *
DATABASE UniProt [online] 1 November 1999 (1999-11-01), XP002736729, Database accession no. Q9W7Z6 *
DONG JB; ZHU W; COOK FR; GOTO Y; HORII Y; HAGA T. I: "dentification of a novel equine infectious anemia virus field strain isolated from feral horses in southern Japan.", J GEN VIROL., vol. 94, 24 October 2012 (2012-10-24), pages 360 - 5
E. M. SHEVACH; W. STROBER: "Current Protocols in Immunology,", 1992, JOHN WILEY & SONS
FEMS MICROBIOL LETT, vol. 174, no. 2, 1999, pages 247 - 50
FEMS MICROBIOL LETT, vol. 177, no. 1, 1999, pages 187 - 8
HIGGINS DG; SHARP PM, GENE, vol. 73, no. 1, 1988, pages 237 - 244
HORN T ET AL., NUC ACIDS RES SYMP SER, 1980, pages 225 - 232
HORWELL DC, TRENDS BIOTECHNOL., vol. 13, no. 4, 1995, pages 132 - 134
HUSE WD ET AL., SCIENCE, vol. 256, 1989, pages 1 75 - 128 1
ISSEL CJ; SCICLUNA MT; COOK SJ; COOK RF; CAPRIOLI A; RICCI , ROSONE F; CRAIGO JK; MONTELARO RC; AUTORINO GL.: "Challenges and proposed solutions for more accurate serological diagnosis of equine infectious anaemia.", VET REC., vol. 172, no. 8, 16 November 2012 (2012-11-16), pages 210
J. M. POLAK; JAMES O'D. MCGEE: "In Situ Hybridization: Principles and Practice", 1990, OXFORD UNIVERSITY PRESS
J. SAMBROOK; E. F. FRITSCH; T. MANIATIS: "Molecular Cloning: A Laboratory Manual", 1989, COL D SPRING HARBOR LABORATORY PRESS, pages: 1 - 3
KESTLER, HUMAN GENE THER, vol. 10, no. 10, 1999, pages 1619 - 32
KING ET AL., BIOCHEM., vol. 17, 1978, pages 1499 - 1506
KOEHLER; MILSTEIN, NATURE, vol. 256, 1975, pages 495
KOEHLER; MILSTEIN, NATURE, vol. 256, 1975, pages 495 - 497
KOSBOR ET AL., IMMUNOL TODAY, vol. 4, 1983, pages 72
KUHAR U; ZAVRSNIK J; TOPLAK , MALOVRH T.: "Detection and molecular characterisation of equine infectious anaemia virus from field outbreaks in Slovenia.", EQUINE VET J., vol. 46, no. 3, 9 September 2013 (2013-09-09), pages 386 - 91
LEWIS, EMBO. J, vol. 11, 1992, pages 3053 - 3058
LEWIS; EMERMAN, VIROL., vol. 68, 1994, pages 510 - 516
LIGNEE, M.: "Memoire et observation sur une maladie du sang, connuesous le nom d'anhemie hydrohemie cachexie aqueuse du cheval.", REC. MED. VET., vol. 20, pages 30
MATTHES ET AL., EMBO J., vol. 3, 1984, pages 801 - 805
MOONEY J; FLYNN O; SAMMIN D.: "Equine infectious anaemia in Ireland: characterisation of the virus", VET REC., vol. 159, no. 17, 21 October 2006 (2006-10-21), pages 570
MORRISON ET AL., PROC NATL ACAD SCI, vol. 81, 1984, pages 6851 - 6855
MURAKAMI K; KONISHI M; KAMEYAMA K; SHIBAHARA T.: "Detection of equine infectious anaemia virus in native Japanese ponies", VET REC., vol. 171, no. 3, 10 July 2012 (2012-07-10), pages 72
MURAKAMI K; KONISHI M; KAMEYAMA K; SHIBAHARA T.: "Detection of equine infectious anaemia virus in native Japanese ponies.", VET REC., vol. 171, no. 3, 10 July 2012 (2012-07-10), pages 72
NEUBERGER ET AL., NATURE, vol. 312, 1984, pages 604 - 608
OAKS JL; LONG MT; BASZLER TV.: "Leukoencephalitis associated with selective viral replication in the brain of a pony with experimental chronic equine infectious anemia virus infection.", VET PATHOL., vol. 41, no. 5, September 2004 (2004-09-01), pages 527 - 32
PAGAMJAV O; KOBAYASHI K; MURAKAMI H; TA ATA Y; MIURA Y; BOLDBAATAR B; SENTSUI H.: "Serological survey of equine viral diseases in Mongolia.", MICROBIOL IMMUNOL., vol. 55, no. 4, April 2011 (2011-04-01), pages 289 - 92
QUINLIVAN M; COOK F; KENNA R; CALLINAN JJ; CULLINANE A.: "Genetic characterization by composite sequence analysis of a new pathogenic field strain of equine infectious anemia virus from the 2006 outbreak in Ireland.", GEN VIROL., vol. 94, March 2013 (2013-03-01), pages 612 - 22
QUINLIVAN M; COOK F; KENNA R; CALLINAN JJ; CULLINANE A.: "Genetic characterization by composite sequence analysis of a new pathogenic field strain of equine infectious anemia virus from the 2006 outbreak in Ireland.", J GEN VIROL., vol. 94, 21 November 2012 (2012-11-21), pages 612 - 22
QUINLIVAN M; COOK RF; CULLINANE A.: "Real-time quantitative RT-PCR and PCR assays for a novel European field isolate of equine infectious anaemia virus based on sequence determination of the gag gene", VET REC., vol. 160, no. 18, 5 May 2007 (2007-05-05), pages 611 - 8
SAIKI R K ET AL., SCIENCE, vol. 239, 1988, pages 487 - 491
SCICLUNA MT; ISSEL CJ; COOK FR; MANNA G; CERSINI A; ROSONE F; FRONTOSO R; CAPRIOLI A; ANTONETTI V; AUTORINO GL.: "Is a diagnostic system based exclusively on agar gel immunodiffusion adequate for controlling the spread of equine infectious anaemia?", VET MICROBIOL., vol. 165, no. 1-2, 28 March 2013 (2013-03-28), pages 123 - 34
SENTSUI H; INOSHIMA Y; MURAKAMI K; AKASHI H; PUREVTSEREN B; PAGMAJAV 0; SUGIURA .: "Cross reaction of recombinant equine infectious anemia virus antigen to heterologous strains and application for serological survey among horses in the field.", MICROBIOL IMMUNOL., vol. 45, no. 1, 2001, pages 45 - 50
SIMON RJ ET AL., PN S, vol. 89, no. 0, 1992, pages 9367 - 9371
SPYROU V; PAPANASTASSOPOULOU M; KOUMBATI M; NIKOLAKAKI SV; KOPTOPOULOS G.: "Molecular analysis of the proviral DNA of equine infectious anemia virus in mules in Greece.", VIRUS RES., vol. 107, no. 1, January 2005 (2005-01-01), pages 63 - 72
STAHLI ET AL., J. OF IMMUNOLOGICAL METHODS, vol. 32, 1980, pages 297 - 304
TAKEDA ET AL., NATURE, vol. 314, 1985, pages 452 - 454
TANAMORI ET AL., J. IMMUNOL. METH, vol. 62, 1983, pages 123 - 131
WOLFF; TRUBETSKOY, NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 421

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108715600A (en) * 2018-04-12 2018-10-30 中国科学院南海海洋研究所 A kind of oligopeptides and its preparation method and application promoting Intestinal epithelial cells proliferation and migration

Also Published As

Publication number Publication date
PT3083659T (en) 2021-11-26
EP3083659B1 (en) 2021-07-28
EP3083659A1 (en) 2016-10-26
GB2526222B (en) 2017-03-22
US20160367659A1 (en) 2016-12-22
GB201322574D0 (en) 2014-02-05
GB201515545D0 (en) 2015-10-14
GB2526222A (en) 2015-11-18

Similar Documents

Publication Publication Date Title
ES2397627T3 (en) Endogenous retroviruses regulated by an increase in prostate cancer
CA1265648A (en) Leukemia-associated virus immunogen, vaccine and assay
US8183349B2 (en) Markers of XMRV infection and uses thereof
JP2008043337A (en) Composition and method for the treatment and diagnosis of breast cancer
JP4270523B2 (en) Compositions and methods for the treatment and diagnosis of breast cancer
KR100204258B1 (en) Recombinant feline coronavirus s proteins
ES2227525T3 (en) MULTIPLE SCLEROSIS VIRUSES.
Forti et al. Identification of a novel overlapping sequential E epitope (E′) on the bovine leukaemia virus SU glycoprotein and analysis of immunological data
Battles et al. Immunological characterization of the gag gene products of bovine immunodeficiency virus
Jin et al. Infection of CD4+ T lymphocytes by the human T cell leukemia virus type 1 is mediated by the glucose transporter GLUT-1: evidence using antibodies specific to the receptor's large extracellular domain
Fujii et al. Expression of human immunodeficiency virus type 1 Nef antigen on the surface of acutely and persistently infected human T cells
EP3083659B1 (en) Viral peptides
CA2454879A1 (en) Retrovirus isolated from mantle histiocytes in mantle cell lymphoma
US8088910B2 (en) Isolated multiple sclerosis (MS)-associated retrovirus (MSRV) nucleic acids corresponding to the gag region
NO331637B1 (en) Isolated DNA molecules, purified polypeptides and their use, antibodies, vectors, diagnostic sets and methods for screening and prognostic evaluation of human retrovirus in breast cancer.
JP2007159489A (en) Species-specific antigen of trichinella and method for testing infection of trichinella utilizing the antigen
KR101411272B1 (en) Hiv vif mutnats
Sherman et al. Sequence analysis of an immunogenic and neutralizing domain of the human T-cell lymphoma/leukemia virus type I gp46 surface membrane protein among various primate T-cell lymphoma/leukemia virus isolates including those from a patient with both HTLV-I-associated myelopathy and adult T-cell leukemia
JPH06500928A (en) Methods for diagnosing and treating chronic fatigue immunodeficiency syndrome and compositions therefor
KR20220047442A (en) A Novel Composition for Preventing or Treating Coronavirus Infectious Diseases
Betemps et al. Overexpression and purification of an immunologically reactive His–BIV capsid fusion protein
WO1995029240A1 (en) Isolation and characterization of a novel primate t-cell lymphotropic virus and the use of this virus or components thereof in diagnostics assays and vaccines
Kashiwase et al. Characterization of one monoclonal antibody against feline immunodeficiency virus p24 and its application to antigen capture ELISA
CA2576902C (en) Method and device for detecting feline immunodeficiency virus
Magdalena et al. Occurrence of Equine Foamy Virus Infection in Horses from Poland

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14824566

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 1515545

Country of ref document: GB

Kind code of ref document: A

Free format text: PCT FILING DATE = 20141219

WWE Wipo information: entry into national phase

Ref document number: 1515545.0

Country of ref document: GB

WWE Wipo information: entry into national phase

Ref document number: 15104249

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2014824566

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2014824566

Country of ref document: EP