WO2013021163A1 - Marqueurs peptidiques et leurs utilisations - Google Patents

Marqueurs peptidiques et leurs utilisations Download PDF

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Publication number
WO2013021163A1
WO2013021163A1 PCT/GB2012/050790 GB2012050790W WO2013021163A1 WO 2013021163 A1 WO2013021163 A1 WO 2013021163A1 GB 2012050790 W GB2012050790 W GB 2012050790W WO 2013021163 A1 WO2013021163 A1 WO 2013021163A1
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sequence
peptide
peptides
dengue
dengue virus
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PCT/GB2012/050790
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English (en)
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Gathsaurie Neelika MALAVIGE
Graham Ogg
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Isis Innovation Limited
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Publication of WO2013021163A1 publication Critical patent/WO2013021163A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—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
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to markers for detecting dengue virus infection, and to the use of the markers for diagnostic and monitoring purposes.
  • the invention also relates to the use of the markers for the induction of immune responses for preventative and therapeutic purposes and for detecting and monitoring the immune responses following intervention.
  • all references as cited herein are incorporated by reference in their entireties.
  • Dengue viral infections have become one of the most important mosquito borne viral infections in the world and are one of the major emerging infectious diseases. It is estimated that 2.1 million cases of dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS) occur every year resulting in 21 ,000 deaths. There are four dengue virus serotypes (DEN1 -4) which are closely related (Weaver and Vasilakis Infect Genet Evol 2009;9(4):523-40). Initial infection with a particular serotype is known as a primary infection, which is usually asymptomatic or results in mild disease manifestations. However, subsequent infection with other serotypes (secondary dengue infections) may lead to severe disease which manifests in the form of DHF/DSS. The pathophysiology of dengue viral infections and factors that result in severe clinical disease is poorly understood. However, studies have suggested that immunopathological, host genetic and viral factors all contribute to the occurrence of severe disease.
  • CD4+ and CD8+ T cell epitopes have been identified from patients with acute dengue infection, which have been shown to be highly cross reactive. Cross reactive T cells are thought to contribute to immunopathology, which occurs in severe dengue infections, by altering the cytokine profiles during secondary infection. So far all dengue virus (DV) specific T cell epitopes have been identified from patients with acute dengue infection and from healthy volunteers who received live attenuated monovalent dengue vaccines. T cells specific for these epitopes have been shown to produce a large amount of proinflammatory cytokines and are thus thought to contribute to disease pathogenesis.
  • DV dengue virus
  • serotype specific T cell epitopes Although some serotype specific T cell epitopes have also been identified in patients with acute infection, all these serotype specific T cell epitopes identified show >55% homology between the four DV serotypes (Appanna et al Clin Vaccine Immunol 2007; 14(8):969-77; Vaughan et al Viral Immunol 2010 Jun;23(3):259-84).
  • the present invention provides one or more peptides selected from the list comprising a peptide of Sequence ID No: 1 to 103, 1 A, I B, 1 1 A, 1 1 B, 15A, 15B, 18A, 18B, 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41 B, 44A, 44B, 61 A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A, 72B, 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A and 94B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95%. 98%o or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the one or more isolated peptides correspond to epitopes that are unique to different dengue virus serotypes or show >70%>, 75%, 80%, 85%, 90%, 95% or more specificity to individual serotypes.
  • peptides with the sequence of Sequence ID Nos: 1 to 21 , 103, 1A, I B, 1 1 A, 1 1 B, 15A, 15B, 18A and 18B are all epitopes specific to Dengue virus serotype 1 ;
  • peptides with the sequence of Sequence ID Nos: 22 to 48, 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41 B, 44A and 44B are all epitopes specific to Dengue virus serotype 2;
  • peptides with the sequence of Sequence ID Nos: 49 to 74, 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A and 72B are all epitopes specific to Den
  • peptides with the sequence of Sequence ID Nos: 1A, I B, 1 1A, 1 1B, 15A, 15B, 18A and 18B are all epitopes specific to Dengue virus serotype 1 ; peptides with the sequence of Sequence ID Nos: 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41B, 44A and 44B are all epitopes specific to Dengue virus serotype 2; peptides with the sequence of Sequence ID Nos: 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A and 72B are all epitopes specific to Dengue virus serotype 3 ; and peptides with the sequence of Sequence ID Nos: 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A and 94B are all epitopes specific to Dengue virus serotype 4.
  • peptides with the sequence of Sequence ID Nos: I B, 1 1 B, 15B, and 18B are all epitopes specific to Dengue virus serotype 1 ; peptides with the sequence of Sequence ID Nos: 30B, 36B, 37B, 41 B and 44B are all epitopes specific to Dengue virus serotype 2; peptides with the sequence of Sequence ID Nos: 61 B, 63B, 67B, 70B and 72B are all epitopes specific to Dengue virus serotype 3 ; and peptides with the sequence of Sequence ID Nos: 79B, 84B, 86B, 93B and 94B are all epitopes specific to Dengue virus serotype 4.
  • peptides with the sequence of Sequence ID Nos: 1A, 1 1 A, 15 A, and 18A are all epitopes specific to Dengue virus serotype 1 ; peptides with the sequence of Sequence ID Nos: 30A, 36A, 37A, 41A and 44A are all epitopes specific to Dengue virus serotype 2; peptides with the sequence of Sequence ID Nos: 61A, 63A, 67A, 70A and 72A are all epitopes specific to Dengue virus serotype 3 ; and peptides with the sequence of Sequence ID Nos: 79A, 84A, 86A, 93A and 94A are all epitopes specific to Dengue virus serotype 4.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No: 1A, I B, 1 1A, 1 1 B, 15A, 15B, 18A, 18B, 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41 B, 44A, 44B, 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A, 72B, 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A and 94B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No: 1A, 1 1 A, 15A, 18A, 30A, 36A, 37A, 41 A, 44A, 61A, 63A, 67A, 70A, 72A, 79A, 84A, 86A, 93A and 94A, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No: I B, 1 1 B, 15B, 18B, 30B, 36B, 37B, 41 B, 44B, 61 B, 63B, 67B, 70B, 72B, 79B, 84B, 86B, 93B and 94B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No: 1A, I B, 1 1A, 1 1 B, 15A, 15B, 18A and 18B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No: 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41 B, 44A and 44B, or a peptide with at least about 70%, 75%), 80%), 85%o, 90% or 95%> or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No: 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A and 72B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A and 94B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : I B, 1 1 B, 15B and 1 8B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : 30B, 36B, 37B, 41 B and 44B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : 61 B, 63B, 67B, 70B and 72B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : 79B, 84B, 86B, 93B and 94B, or a peptide with at least about 70%, 75%, 80%, 85%o, 90%) or 95%) or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : 1 A, 1 1 A, 15A and 1 8A, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : 30A, 36A, 37A, 41 A and 44A, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : 61 A, 63A, 67A, 70A and 72A, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : 79A, 84A, 86A, 93A and 94A, or a peptide with at least about 70%, 75%), 80%), 85%o, 90%) or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the aforementioned sequences.
  • the present invention provides one or more peptides selected from the list comprising a peptide of Sequence ID No : 1 A, I B, 1 1 A, 1 1 B, 15A, 15B, 1 8A, 1 8B, 30A, 30B, 36A, 36B, 37A, 37B, 41 A, 41 B, 44A, 44B, 61 A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A, 72B, 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A and 94B.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : I B, 1 1 B, 15B, 1 8B, 30B, 36B, 37B, 41 B, 44B, 61 B, 63B, 67B, 70B, 72B, 79B, 84B, 86B, 93B and 94B.
  • the present invention may provide one or more peptides selected from the list comprising a peptide of Sequence ID No : 1 A, 1 1 A, 15A, 1 8A, 30A, 36A, 37A, 41 A, 44A, 61 A, 63A, 67A, 70A, 72A, 79A, 84A, 86A, 93A and 94A.
  • the peptides correspond to serotype specific, highly conserved, non-cross reactive T-cell and/or antibody epitopes in the dengue virus.
  • the peptides correspond to T-cell epitopes.
  • the peptides referred to above are unique immunogenic regions of a particular dengue virus serotype, these regions are not shared by other dengue virus serotypes and thus display no cross-reactivity between serotypes.
  • Sequence ID No : 1 is unique to dengue virus serotype 1 and will not cross react with immune responses from a subj ect exposed to any other dengue virus serotype.
  • Sequence ID No : 93 is unique to dengue virus serotype 4 and will not cross react with immune responses from a subj ect exposed to any other dengue virus serotype.
  • a peptide analog or derivative of a peptide of the invention may have one or more deletion, insertion, or modification of any amino acid residue, including the N or C- terminal residue.
  • the peptide may be acetylated, acetylated, alkylated, citrullinated, glycosylated, and the like.
  • the peptide may also comprise additional amino acids either at the C or N terminal end, or at both ends.
  • the peptide may include up to 10 additional amino acids at either, or both, the C and N terminal end.
  • the peptide may include up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids at either, or both, the C and N terminal end.
  • the peptide may consist of up to 40 amino acid residues.
  • the peptide may consist of up to 35 amino acid residues.
  • the peptide may consist of up to 30 amino acid residues.
  • the peptide, analog or derivative may be part of a fusion protein.
  • the present invention also relates to a fusion protein comprising a peptide of the invention.
  • the fusion protein may comprise multiple peptides as described herein or comprise at least one peptide as described herein and an unrelated sequence.
  • a fusion partner may, for example, assist in presenting the T cell and/or antibody epitope (an immunological fusion partner), or may assist in expressing the peptide (an expression enhancer) at higher yields than the native recombinant peptide.
  • Certain preferred fusion partners are both immunological and expression enhancing fusion partners.
  • Other fusion partners may be selected so as to increase the solubility of the peptide.
  • Still further fusion partners include affinity tags, which facilitate purification of the peptide. Fusion proteins may generally be prepared using standard techniques, including chemical conjugation.
  • a fusion protein is expressed as a recombinant protein allowing the production of increased levels relative to a non- fused peptide in an expression system.
  • the invention also encompasses sequences based on those above which include modifications and changes which do not affect the function of the peptide.
  • the amino acid substitutions may be considered to be conservative in that they are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Percentage sequence identity is defined as the percentage of amino acids in a sequence that are identical with the amino acids in a provided sequence after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent sequence identity can be achieved in many ways that are well known to the man skilled in the art, and include, for example, using BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool).
  • Variations in percent identity may be due, for example, to amino acid substitutions, insertions or deletions.
  • the isolated peptides of the present invention can be prepared in a number of suitable ways known in the art.
  • the peptide, analog or derivative may be isolated from a natural system, or it may be synthetically or recombinantly produced. Synthesised peptides may be produced by standard chemical methods, including synthesis by automated procedure. Recombinant peptides may be used in a purified form. Alternatively, the supernatant from cells expressing the recombinant peptide may be used.
  • a further aspect of the invention provides a nucleic acid (e.g. polynucleotide) encoding a peptide of the invention.
  • the nucleic acid according to the present invention may be DNA, cDNA, PNA, CNA, RNA or combinations thereof and it may or may not contain introns as long as it codes for the peptide.
  • a still further aspect of the invention provides an expression vector capable of expressing a polypeptide according to the invention.
  • the specific peptides referred to above it is possible to determine which dengue virus serotype or serotypes an individual subject has been exposed to or to detect or monitor the dengue-specific immune response following prophylactic vaccination or therapeutic intervention.
  • the specific peptides reflect epitopes unique to a particular dengue serotype, such that on infection of a subject with a specific serotype the subject may raise an immune response to one or more of the unique epitopes, depending on the dengue serotype which caused the infection.
  • the nature of a current or previous dengue infection can be determined. For example, if a subject has been exposed to infection by a dengue virus serotype 1 , then if a blood or tissue sample or culture of cells derived from a sample is obtained from this subject and treated with one or more peptides unique to dengue virus serotype 1 , for example Sequence ID No: 1 , an immune response may be detected.
  • An immune response may be detected by stimulation of cytokine production, for example IFN-gamma, upon exposure of T cells and/or antibodies in a sample to one or more peptides, for example, a peptide of Sequence ID No: 1.
  • the dengue-specific T cells may be detected by other means, including proliferation of T cells in response to one or more of the peptides or by proteomic analysis or RNA expression analysis including expression microarray, nucleotide sequencing or polymerase chain reaction.
  • the dengue-specific T cells may be detected in the samples or in cultures derived from the samples, using multimeric complexes of HLA molecules folded with dengue virus specific peptides.
  • Dengue specific antibody responses may be detected by ELISA or by functional responses such as viral neutralisation or inhibition of replication.
  • the invention provides a method of determining the dengue infection status of a subject, comprising the steps of:
  • step (b) determining the T-cell and/or antibody response in the sample or a culture derived from the sample to one or more of the peptides selected from the list comprising a peptide of Sequence ID No: 1 to 103, 1A, I B, 1 1A, 1 1 B, 15A, 15B, 18A, 18B, 30A, 36A, 36B, 30B, 37A, 37B, 41A, 41 B, 44A, 44B, 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A, 72B, 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A and 94B or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of an aforementioned sequence.
  • the T-cell and/or antibody response to one or more peptides selected from the list comprising a peptide of Sequence ID No: I B, 1 1 B, 15B, 1 8B, 30B, 36B, 37B, 41 B, 44B, 61 B, 63B, 67B, 70B, 72B, 79B, 84B, 86B, 93B and 94B is determined.
  • the T-cell and/or antibody response to one or more peptides selected from the list comprising a peptide of Sequence ID No: 1A, 1 1 A, 15A, 1 8A, 30A, 36A, 37A, 41A, 44A, 61A, 63A, 67A, 70A, 72A, 79A, 84A, 86A, 93A and 94A is determined.
  • step (b) by determining in step (b) the response to one or more peptides with the sequence of Sequence ID No: 1 to 21 , 103, 1A, IB, 1 1A, 1 1 B, 15A, 15B, 18A or 18B, preferably with one or more peptides of Sequence ID no: 1A, I B, 1 1A, 1 1 B, 15A, 15B, 18A and 18B; more preferably with one or more peptides of Sequence ID no: I B, 1 1 B, 15B or 18B, or of Sequence ID no: 1A, 1 1A, 15A or 18A, the method of the invention can be used to determine whether a subject has been infected with dengue virus serotype 1.
  • step (b) by determining in step (b) the response to one or more peptides with the sequence of Sequence ID No: 22 to 48, 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41 B, 44A or 44B, preferably with one or more peptides of Sequence ID no: 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41 B, 44A or 44B, more preferably with one or more peptides of Sequence ID no: 30B, 36B, 37B, 41 B or 44B, or of Sequence ID no : 30A, 36A, 37A, 41 A or 44A, the method of the invention can be used to determine whether a subject has been infected with dengue virus serotype 2.
  • step (b) by determining in step (b) the response to one or more peptides with the sequence of Sequence ID No: 49 to 74, 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A or 72B, preferably with one or more peptides of Sequence ID no: 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A or 72B, more preferably with one or more peptides of Sequence ID no: 61 B, 63B, 67B, 70B or 72B, or of Sequence ID no : 61A, 63A, 67A, 70A or 72A, the method of the invention can be used to determine whether a subject has been infected with dengue virus serotype 3.
  • step (b) by determining in step (b) the response to one or more peptides with the sequence of Sequence ID No: 75 to 102, 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A or 94B, preferably with one or more peptides of Sequence ID no: 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A or 94B, more preferably with one or more peptides of Sequence ID no: 79B, 84B, 86B, 93B or 94B, or of Sequence ID no : 79A, 84A, 86A, 93 A or 94A, the method of the invention can be used to determine whether a subject has been infected with dengue virus serotype 4.
  • dengue infection status includes any present or past infection with one or more dengue virus serotypes.
  • dengue infection status also includes the dengue-specific immune response following prophylactic vaccination or therapeutic intervention.
  • dengue infection status includes, without limitation, determining whether a subject has currently or previously been infected with dengue virus, and preferably where a subject has had a current or previous infection with which serotypes of dengue virus the subject has been infected.
  • the sample may be a sample of blood or other tissues or body fluids.
  • the sample is preferably blood or blood derived.
  • the method of the invention does not comprise the step of obtaining a sample from a human subject.
  • the presence of a T-cell response may be determined by screening for an increased level of cytokines, such as IFN-gamma, in a sample in the presence of one or more peptides according to the invention.
  • cytokine levels may be determined by any appropriate assay, for example an immunoassay, such as an ELISpot assay or ELISA assay or multiplex bead system or proteomic analysis or RNA expression analysis including expression microarray, nucleotide sequencing or polymerase chain reaction.
  • the presence of an antibody response may be detected by ELISA or assay such as viral neutralisation or inhibition of viral replication.
  • a sample displays a T-cell and/or an antibody response to one or more peptides of the invention this may be used to determine that the subject from whom the sample was obtained was currently infected with a dengue virus, and/or had previously been infected with a dengue virus.
  • the serotype of the dengue virus or viruses which caused the infection can be determined.
  • the present invention provides an improved diagnostic or monitoring assay for the diagnosis of dengue virus infection, and more particularly to detect whether a subject has previously been silently (asymptomatically) infected with dengue. More specifically, the method of the invention allows infection by specific serotypes of dengue virus to be detected and distinguished.
  • the present invention would provide a tool to monitor silent infection and disease transmission in the community. This would also provide information regarding past infection with multiple dengue virus serotypes.
  • the only available method to detect past infection is by detection of dengue virus specific IgG antibodies. This assay would not reveal if a person has had one past dengue infection or several and if so the past infecting dengue virus serotype.
  • Neutralization assays can be used to detect past infecting dengue virus serotype 2-3 months after an acute dengue illness.
  • this assay can be unreliable. Therefore, this invention would provide valuable insight to immune responses to the dengue virus in individuals who experience asymptomatic infection.
  • the present invention also relates, in a preferred embodiment, to a diagnostic or monitoring marker or a set of diagnostic or monitoring markers comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, peptides of the group consisting of Sequence ID Nos.
  • the diagnostic or monitoring markers for dengue 1 may comprise one or more, or two or more, or three or more, or four or more etc, of a peptide of Sequence ID Nos: 1A, I B, 1 1A, 1 1 B, 15A, 15B, 18A and 18B, or of a peptide of Sequence ID Nos: I B, 1 1 B, 15B, and 18B, or of a peptide of Sequence ID Nos: 1A, 1 1 A, 15A and 18A, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of an aforementioned sequence.
  • the diagnostic or monitoring markers for dengue 2 may comprise one or more, or two or more, or three or more, or four or more etc, of a peptide of Sequence ID Nos: 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41 B, 44A and 44B, or a peptide of Sequence ID Nos: 30B, 36B, 37B, 41 B, and 44B, or a peptide of Sequence ID Nos: 30A, 36A, 37A, 41A, and 44A, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of an aforementioned sequence.
  • the diagnostic or monitoring markers for dengue 3 may comprise one or more, or two or more, or three or more, or four or more etc of a peptide of Sequence ID Nos: 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A and 72B, or a peptide of Sequence ID Nos: 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A and 72B, or a peptide of Sequence ID Nos: 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A and 72B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of an aforementioned sequence.
  • the diagnostic or monitoring markers for dengue 4 may comprise one or more, or two or more, or three or more, or four or more etc, of a peptide of Sequence ID Nos : 79B, 84B, 86B, 93B, and 94B, or a peptide of Sequence ID Nos: 79A, 84A, 86A, 93A and 94A, or a peptide of Sequence ID Nos: 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A and 94B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of an aforementioned sequence.
  • the diagnostic or monitoring markers for dengue 1 may comprise one or more, or two or more, or three or more, or four or more etc, of a peptide of Sequence ID Nos : I B, 1 1 B, 15B and 1 8B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of an aforementioned sequence.
  • the diagnostic or monitoring markers for dengue 2 may comprise one or more, or two or more, or three or more, or four or more etc, of a peptide of Sequence ID Nos : 30B, 36B, 37B, 41 B and 44B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of an aforementioned sequence.
  • the diagnostic or monitoring markers for dengue 3 may comprise one or more, or two or more, or three or more, or four or more etc, of a peptide of Sequence ID Nos : 61 B, 63B, 67B, 70B and 72B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95%) or more identity with one of the aforementioned sequences, or a peptide analog or derivative of an aforementioned sequence.
  • the diagnostic or monitoring markers for dengue 4 may comprise one or more, or two or more, or three or more, or four or more etc, of a peptide of Sequence ID Nos : 79B, 84B, 86B, 93B and 94B, or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95%) or more identity with one of the aforementioned sequences, or a peptide analog or derivative of an aforementioned sequence.
  • a preferred diagnostic or monitoring marker in accordance with the present invention relates to a diagnostic marker or a set of diagnostic markers comprising at least two polypeptides of the group consisting of Sequence ID Nos: 1A, I B, 1 1A, 1 1 B, 15A, 15B, 18A, 18B, 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41 B, 44A, 44B, 61A, 61 B, 63A, 63B, 67A, 67B, 70A, 70B, 72A, 72B, 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A and 94B or a peptide with at least about 70%, 75%, 80%, 85%, 90% or 95% or more identity with one of the aforementioned sequences, or a peptide analog or derivative of one of the sequences.
  • the present invention provides an immunogenic composition comprising at least one peptide according to the invention, wherein the composition is capable of eliciting an immune response when administered to a human or non-human animal.
  • An immunogenic composition is a composition that is capable of eliciting an immune response to at least one peptide when the composition is administered to a subject.
  • the subject is a human or non-human animal, more preferably a human or non-human mammal.
  • an immunogenic composition of the invention elicits a serotype specific immune response, the specificity being determined by the one or more peptides in the composition.
  • the immune response may be humoral or cellular.
  • the immunogenic composition of the invention is capable of eliciting serotype specific protective or therapeutic immunity.
  • the immune response elicited by the composition of the invention affects the ability of dengue virus to infect an immunised human (prophylactic) or may modify the severity of disease associated with previous infection (therapeutic).
  • the ability of a dengue virus to infect a human immunised with the composition of the invention is impeded or prevented. This may be achieved in a number of ways.
  • the immune response elicited may recognise and destroy the dengue virus. Alternatively, or additionally, the immune response elicited may impede or prevent replication of the dengue virus. Alternatively, or additionally, the immune response elicited may impede or prevent the dengue virus causing disease in the human or non-human animal.
  • An immunogenic composition may comprise one or more peptides derived from the same or different dengue virus serotypes.
  • an immunogenic composition may comprise one or more peptides derived from epitopes for dengue virus serotype 1 , or dengue virus serotype 2, or dengue virus serotype 3 or dengue virus serotype 4.
  • an immunogenic composition according to the invention may comprise two or more peptides wherein at least two of the peptides are derived from different dengue virus serotypes.
  • at least one dengue virus serotype 1 peptide and at least one dengue virus serotype 2 peptide are included in the composition.
  • the composition may also comprise all other possible variations, including at least one peptide for each dengue virus serotype.
  • the immunogenic composition may be given as part of routine vaccination or tailored to a particular individual. For example, if an individual has been shown to have been exposed to dengue virus serotype 1 , then an immunogenic composition may b e administered which is specific to one or more of dengue virus serotype 2, dengue virus serotype 3 or dengue virus serotype 4; or perhaps just to what is predicted or known to be the most prevalent dengue virus serotype in a particular area.
  • the immunogenic composition may modify the disease associated with subsequent infection with a different serotype.
  • the peptides may be arranged to be administered simultaneously in a single composition, or sequentially in a series of compositions each containing one or more peptides according to the invention.
  • composition of the invention may also comprise a further one or more antigens, in addition to the one or more peptides of the invention.
  • the composition may be used to elicit/produce a protective immune response when administered to a subject.
  • the protective immune response may cause the dengue virus to be killed upon infecting the subject, or it may prevent or inhibit the dengue virus from replicating and/or from causing disease.
  • the composition may be used as a prophylactic or a therapeutic vaccine directed to the dengue virus.
  • the invention provides a pharmaceutical composition comprising at least one peptide according to the invention and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition is capable of producing a protective immune response to the dengue virus.
  • the phrase "producing a protective immune response" as used herein means that the composition is capable of generating a protective or therapeutic response in a host organism, such as a human or a non-human mammal, to whom it is administered.
  • a protective or therapeutic immune response protects against current or subsequent infection by a dengue virus.
  • the immune response may eliminate or reduce the level of infection by reducing replication of the dengue virus or by affecting the mode of action of the dengue virus to reduce disease.
  • Suitable acceptable excipients and carriers will be well known to those skilled in the art. These may include solid or liquid carriers. Suitable liquid carriers include water and saline.
  • the proteins of the composition may be formulated into an emulsion or they may be formulated into biodegradable microspheres or liposomes.
  • composition of the invention may further comprise an adjuvant.
  • adjuvants will be well known to those skilled in the art, and may include Freund's Incomplete Adjuvant (for use in animals), and metal salts, such as aluminium or calcium salts.
  • the composition may also comprise polymers or other agents to control the consistency of the composition, and/or to control the release of the antigen/secreted protein from the composition.
  • composition may also comprise other agents such as diluents, which may include water, saline, glycerol or other suitable alcohols etc; wetting or emulsifying agents; buffering agents; thickening agents for example cellulose or cellulose derivatives; preservatives; detergents, antimicrobial agents; and the like.
  • diluents which may include water, saline, glycerol or other suitable alcohols etc; wetting or emulsifying agents; buffering agents; thickening agents for example cellulose or cellulose derivatives; preservatives; detergents, antimicrobial agents; and the like.
  • the active ingredients in the composition are greater than 50% pure, usually greater than 80%> pure, often greater than 90%> pure and more preferably greater than 95%, 98%o or 99% pure. With active ingredients approaching 100%) pure, for example about 99.5%) pure or about 99.9% pure, being used most often.
  • the composition of the present invention may be used as a vaccine against infections caused by the dengue virus.
  • the vaccine may be administered prophylactically to those at risk of exposure to the dengue virus, and/or therapeutically to persons who have already been exposed to the dengue virus to prevent further infection with the other dengue virus serotypes.
  • the composition comprises an immunologically effective amount of a peptide according to the invention.
  • An "immunologically effective amount" of a peptide is an amount that when administered to an individual, either in a single dose or in a series of doses, is effective for treatment or prevention of infection by the dengue virus. This amount will vary depending upon the health and physical condition of the individual to be treated and on the antigen.
  • a composition according to the invention may be for oral, systemic, parenteral, topical, mucosal, intramuscular, intravenous, intraperitoneal, intradermal, subcutaneous, intranasal, intravaginal, intrarectal, transdermal, sublingual, inhalation or aerosol administration.
  • the composition may be arranged to be administered as a single dose or as part of a multiple dose schedule. Multiple doses may be administered as a primary immunisation followed by one or more booster immunisations. Suitable timings between priming and boosting immunisations can be routinely determined.
  • a composition according to the invention may be used in isolation, or it may be combined with one or more other immunogenic or vaccine compositions, and/or with one or more other therapeutic regimes.
  • the present invention provides the use of one or more peptides of the invention in the preparation of a medicament for eliciting an immune response.
  • the medicament may be used for the prophylactic or therapeutic vaccination of subjects against the dengue virus.
  • the medicament may be a prophylactic or a therapeutic vaccine.
  • the invention provides a composition comprising one or more peptides of the invention for use in generating an immune response to the dengue virus.
  • the immune response may be prophylactic or therapeutic.
  • the composition may be for use as a vaccine.
  • the present invention provides a method of protecting a human or non-human animal from the effects of infection by the dengue virus comprising administering to the human or non-human animal a composition according to any other aspect of the invention.
  • the composition may be a vaccine.
  • the invention provides a method for raising an immune response in a human or non-human animal comprising administering a pharmaceutical composition according to the invention to the human or non-human animal.
  • the immune response is preferably protective.
  • the method may raise a booster respons e in a patient that has already been primed.
  • the immune response may be prophylactic or therapeutic.
  • One way to check the efficacy of a therapeutic treatment comprising administration of a composition according to the invention involves monitoring for the dengue virus infection after administration of the composition.
  • One way to check the efficacy of a prophylactic treatment comprising administration of a composition according to the invention involves monitoring immune responses to the dengue virus after administration of the composition.
  • the invention provides the use of one or more peptides according to the invention in the preparation of a medicament for use in the immunisation of human or non-human mammals against infection by the dengue virus.
  • the invention provides a kit for use in inducing an immune response in an organism, comprising an immunogenic or vaccine composition according to the invention and instructions relating to administration.
  • compositions according to the invention may be useful as diagnostic and monitoring reagents and as a measure of the immune competence of a vaccinee.
  • Figure 1 - illustrates cultured IFNy ELISpot responses to the peptides from
  • DEN- 1 dengue virus serotype 1
  • DEN-2 dengue virus serotype 2
  • DEN-3 dengue virus serotype 3
  • DEN-4 dengue virus serotype 4
  • Figure 2A - illustrates IFNy ELISpot responses to DEN-3 peptide 3 specific line, incubated with DEN-3 peptide 3 directly added to T cells and in the presence or absence of anti-DR, anti-DP or anti-DQ antibodies.
  • FIG. 2B - illustrates IFNy ELISpot responses using DEN-3 peptide 3 specific line, incubated with DEN-3 peptide 3 directly added to T cells and DRB 1 * 1501 expressing L cells pulsed with the peptide and L cells unpulsed used as antigen presenting cells to the T cell line.
  • Figure 3 - illustrates ex vivo IFNy ELISpot responses to the identified serotype specific, non-cross reactive and highly conserved peptides of DEN- 1 , DEN2, DEN-3 and DEN-4 serotypes, in a cohort of healthy immune donors following natural exposure.
  • the horizontal bar shows the mean+3 SD of the irrelevant peptide control.
  • Figure 4 - is a table illustrating the sequences, regions of the dengue virus an other details of serotype specific, non cross reactive and highly conserve peptides of the four dengue virus serotypes.
  • Figure 5 - depicts the degree of conservation of the identified peptides, in the published dengue virus sequences. Degree of conservation was assessed by the use of the virus variation resource on dengue virus sequence data base available at :
  • Figure 6 - is a table illustrating the response to two dengue 1 peptides in 5 patients with acute dengue infection due to dengue 1 (DEN- 1 ).
  • A indicates the first sample from the patient taken at day 4-5
  • B indicates the second sample from the same patient taken on day 6-7.
  • the patients with acute DEN- 1 infection only reacted to the peptides from the DEN- 1 serotype, no responses to peptides from of other serotypes (data not shown) were observed.
  • Responses are expressed as number of IFNy producing cells per million PBMC
  • Sequence ID No: 64 ATLLVWHTWQKQTQRS
  • Sequence ID No: 65 KNFQTTPGTFQTTTGE
  • Sequence ID No: 68 TSIGLICVIASSGMLWMAEVPLQ
  • Sequence ID No: 78 SNHGVTATITPRSPSVEVK
  • Sequence ID No: 84 AMTTTLSIPHDLMELIDGIS
  • Sequence ID No: 85 AMTTVLSIPHDLMELIDGIS
  • Sequence ID No: 1A L( /R)TEVTNPA(V/I) (Seq ID no: 104 in Sequence listing)
  • Sequence ID No: 79A RDVNKEKVVGR(V/I)ISSTP(L/F)AE (Seq ID no: 105 in Sequence listing)
  • Sequence ID No: 30A IMTG(D/E)I(K/R)G(I/T)MQ(A/V) (Seq ID no: 106 in Sequence listing)
  • Sequence ID No: 1 1 A SLVASVELPN(S/C)L(E/D)ELGDGLAMGIMI (Seq ID no: 107 in Sequence listing)
  • Sequence ID No: 15A GSLGCKPLTMFLIAENKIWG (Seq ID no: 108 in Sequence listing)
  • Sequence ID No: 36A SSQQK(T/A)DWIPL(A/V)L(T/A)(I/V)KGLNP (Seq ID no: 109 in Sequence listing)
  • Sequence ID No: 61A RENLLLGVGLAMA(T/A)TLQLPE (Seq ID no: 1 10 in Sequence listing)
  • Sequence ID No: 84A AMTT(T/V)(L/F)SIPHDLMELIDGIS (Seq ID no: 1 1 1 in Sequence listing)
  • Sequence ID No: 37A (D/E)QAEISGSSPILSITISEDG (Seq ID no: 1 13 in Sequence listing)
  • Sequence ID No: 63A TM(R/K)IK(D/N)DETENILTVLLKTA (Seq ID no: 1 14 in Sequence listing)
  • Sequence ID No: 18A TSGTYVSAIAQA(K/R)ASQE (Seq ID no: 1 15 in Sequence listing)
  • Sequence ID No: 41A L(T/A)L(N/S)LITEMGRLPTFMTQK(A/T) (Seq ID no: 1 16 in Sequence listing)
  • Sequence ID No: 67A IALDLVTEIGRVP(S/T)HLA(H/Y)RT (Seq ID no: 1 17 in Sequence listing)
  • Sequence ID No: 94A IYVIL(T/A)ILTII(G/A/S)L(I/N) (Seq ID no: 1 19 in Sequence listing)
  • Sequence ID No: 44A (S/N)RLNALG(K/R)SEFQI (Seq ID no: 120 in Sequence listing)
  • Sequence ID No: 72A HVNAEPETPNMDVIGERI(K/R)R (Seq ID no: 121 in Sequence listing)
  • Sequence ID No: 70A K(K/R/E)LNQL(S/P)RK(E/D)FDL (Seq ID no: 122 in Sequence listing)
  • Sequence ID No: 30B IMTGDIKGIMQA (Seq ID no: 125 in Sequence listing) Sequence ID No: 1 1 B: SLVASVELPNSLEELGDGLAMGIMI (Seq ID no: 126 in Sequence listing)
  • Sequence ID No: 15B GSLGCKPLTMFLIAENKIWG (Seq ID no: 127 in Sequence listing)
  • Sequence ID No: 36B SSQQKTDWIPLALTIKGLNP (Seq ID no: 128 in Sequence listing)
  • Sequence ID No: 84B AMTTTLSIPHDLMELIDGIS (Seq ID no: 130 in Sequence listing)
  • Sequence ID No: 86B IVTQFDNTQVGTLA (Seq ID no: 131 in Sequence listing)
  • Sequence ID No: 37B DQAEISGSSPILSITISEDG (Seq ID no: 132 in Sequence listing)
  • Sequence ID No: 63B TMRIKDDETENILTVLLKTA (Seq ID no: 133 in Sequence listing)
  • Sequence ID No: 18B TSGTYVSAIAQAKASQE (Seq ID no: 134 in Sequence listing)
  • Sequence ID No: 41 B LTLNLITEMGRLPTFMTQKA (Seq ID no: 135 in Sequence listing)
  • Sequence ID No: 67B IALDLVTEIGRVPSHLAHRT (Seq ID no: 136 in Sequence listing)
  • Sequence ID No: 93B ILTEIASLPTYLSSRAKL (Seq ID no: 137 in Sequence listing)
  • Sequence ID No: 94B IYVILTILTIIGLI (Seq ID no: 138 in Sequence listing)
  • Sequence ID No: 44B SRLNALGKSEFQI (Seq ID no: 139 in Sequence listing)
  • Sequence ID No: 72B HVNAEPETPNMDVIGERIKR (Seq ID no: 140 in Sequence listing)
  • PBMC Peripheral blood mononuclear cells
  • FCS fetal calf serum
  • PBMC from each donor were incubated with the peptides of each dengue virus serotype peptide pool consisting of all overlapping peptides. Briefly, 5.0 > ⁇ 10 6 PBMCs were incubated for 10 days with 200 ⁇ 1 of 40 ⁇ peptide pool in a 24 well plate. IL-2 was added on day 3 and 7 at a concentration of l OOunits/ml. All cell lines were routinely maintained in RPMI 1640 supplemented with 2mM L-glutamine, l OOIU/ml penicillin and 100 ⁇ g/ml plus 10% human serum at 37°C, in 5% CO 2 .
  • T cell lines were tested after 10 days culture using a peptide matrix in order to identify individual peptide responses in a 96 well ELISpot plate.
  • Peptides of each dengue virus serotype were arranged in to 9 peptide pools, with each pool consisting of 5 to 8 peptides and each peptide present in two different pools. Therefore, each peptide would drive a response in two different pools.
  • each peptide response was confirmed at least twice. All peptides that induced an IFN- ⁇ response of more than mean+ 3 standard deviations of the irrelevant peptide were considered positive.
  • ELISpot assays were performed as previously described. Briefly, ELISpot plates (Millipore Corp., Bedford, Massachusetts, USA) were coated with anti-human IFNy antibody overnight (Mabtech AB, Nacka, Sweden) . The plates were washed six times with RPMI 1640 and incubated for 1 hour with RPMI- 1640 and 10% FCS . 0. 1 x l O 6 PBMC were added to a final volume of 200 ⁇ . Peptide was added at a final concentration of 10 ⁇ . All peptides were tested in duplicate. PHA was always included as a positive control and an irrelevant peptide (SARS peptide) was included as a negative control.
  • SARS peptide irrelevant peptide
  • the plates were incubated overnight at 37°C and 5% CO 2 .
  • the cells were removed and the plates developed with a second biotinylated Ab to human IFNy and washed a further six times.
  • the plates were developed with streptavidin- alkaline phosphatase (Mabtech AB) and colorimetric substrate, and the spots enumerated using an automated ELISpot reader. Background (cells plus media) was subtracted and data expressed as number of spot-forming units (SFU) per 106 PBMC.
  • SFU spot-forming units
  • Ex vivo ELISpot assays were performed as previously described in 24 dengue immune donors and 5 dengue seronegative donors. For ex vivo ELISpot assays, 0.1 * 10 6 PBMC were added to a final volume of 200 ⁇ . Peptide was added at a final concentration of 10 ⁇ . All peptides were tested in duplicate. PHA was always included as a positive control and an irrelevant peptide (SARS peptide) was included as a negative control. Ex vivo responses were only assessed for the immunogenic peptides identified by the cultured ELISpot assays. Background (cells plus media) was subtracted and data expressed as number of spot-forming units (SFU) per 106 PBMC. All peptides that induced an IFN- ⁇ response of more than mean+3 standard deviations of the irrelevant peptide were considered positive.
  • SFU spot-forming units
  • ex vivo- PBMC or T-cell lines were stimulated at l xl O 6 to 2xl 0 6 /ml in RPMI 1640 plus 10% FCS with the relevant peptides (20 ⁇ 1 of ⁇ peptide) for 16 hours according to manufacturers instructions in the presence of Brefeldin A (BD GolgiStopTM). Cells were washed and stained with anti CD3 (FITC), anti CD4 (PerCP) (BD Biosciences) and anti CD8 (PE).
  • FITC anti CD3
  • PerCP PerCP
  • PE anti CD8
  • Murine fibroblast cell lines transfected with HLA-DRB 1 * 15 were maintained in Dulbecco modified Eagle medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 U/mL penicillin, and 50 ⁇ g/mL streptomycin at 37°C with 5% C0 2 .
  • DMEM Dulbecco modified Eagle medium
  • FCS fetal calf serum
  • 2 mM L-glutamine 50 U/mL penicillin
  • streptomycin 50 ⁇ g/mL streptomycin at 37°C with 5% C0 2 .
  • HLA-DR L243
  • HLA-DQ SPV-L3
  • HLA-DP Leinco Technologies; H127
  • Murine fibroblast cell lines were initially pulsed with ⁇ ⁇ of 40 ⁇ peptide for 1 hour at 37°C, in 5% CO 2 . They were then washed three times in RPMI plus 10% fetal calf serum and used as antigen presenting cells to washed T cells harvested from cell cultures.
  • the peptides that were recognized by individuals who were naturally exposed to the dengue virus are shown in Figure 4.
  • the donors recognized 4 peptides of the 23 20mer peptides in DEN- 1 ; 5 peptides of the 35 20mer peptides of DEN-2, 5 peptides of the 35 peptides of the DEN-3 and 5 peptides of the 28 20mer peptides of DEN-4 ( Figure 4).
  • All dengue immune donors responded to the peptides of at least 2 dengue virus serotypes.
  • peptides had ⁇ 15% homology between each peptide except for 30%) homology for 4 peptides (DEN- 1 peptide with DEN- 1 pep 1 1 , DEN-2 pep 33 , DEN-4 pep 12, DEN-2 pep l l , DEN-3 pep 1 1 and DEN-2 peptide 17 with DEN-3 pep 21 and DEN-3 pep l l with DEN-4 pep 19).
  • the peptides of a particular serotype had ⁇ 45 % homology with serotype of the other viruses except for the following peptides.
  • DEN3 pep21 (ID70A, 70B) shared 53% homology with the NS5 regions of DEN- 1 and DEN-4 serotypes.
  • DEN- 1 pep l (ID 1A, I B), which derived from the envelop protein of the DEN- 1 serotype shared 50% homology with the envelop protein of the DEN-2 virus.
  • DEN- 1 pep20 (ID 18A, 18B) which is derived from the NS3 region of DEN- 1 , shares 58% homology with the DEN- 4 NS3 region, 64% homology with the DEN2 NS3 region and 47% homology with the DEN3 NS3 region.
  • DEN-3 pep3 shares 55% homology with the NS2B region of the DEN- 1 serotype.
  • peptides identified which were from the NS2A region 1 peptide each was from DEN-2 and DEN-3, 2 peptides from DEN-4 and 3 of the peptides were from DEN- 1. 3/7 of these peptides were from the region represented by amino acids (aa) 99 to 133, and 2/7 peptides were from the region represented by aa 184 to 216.
  • One peptide from DEN-4 was from the aa 135 to 148 and one peptide from the DEN- 1 was from aa 157 to 174.
  • peptide number 16 (Sequence ID No: 15B) from the DEN- 1 (GSLGCKPLTMFLIAENKIWG), which is from aal 97 to 216 of DEN- 1 was found to 100%) conserved within all published dengue virus serotype 1 sequences.
  • DEN-2 peptide 33 (Sequence ID No: 36B) (SSQQKTDWIPLALTIKGLNP), which was also from the same region of NS2A (184 to 203) was again found to be conserved except for a change in one amino acid. In some variants the amino acid alanine in position 6 was replaced by threonine. The sequence in the current study had this substitution.
  • Peptide 10 (Sequence ID No: 84B) of DEN-4 (AMTTTLSIPHDLMELIDGIS), had the amino acid leucine in position 6 replaced by isoleucine in some sequences.
  • HLA-DR, DQ and DP blocking antibodies were initially used to determine which of these molecules were involved in presenting these peptides. All 3 of these MHC class II molecules were found to be involved in presenting these peptides. Interestingly, the most frequently recognized peptides (peptide 21 and peptide 28 of DEN-3, peptide 19 of DEN-4, peptide 1 and peptide 33 of DEN-2) were found to be restricted through HLA- DP.
  • peptide 18 of DEN-2 was found to include an epitope with restriction through HLA-DQ*06, as complete blocking of the responses to this peptide was achieved by HLA-DQ antibodies in two HLA-DQ*06 homozygous individuals.
  • PMBCs were obtained from 5 patients with acute dengue fever. Elispot assays were performed using peptides according to the invention representing all 4 serotypes. A positive response was only observed with peptides 1 1 and 16 ( Figure 8), both peptides are dengue 1 specific. The results depicted clearly demonstrate the serotype specificity of the peptides.
  • Assays were performed using IP lined ninety-six well multi-screen plates (Millipore (UK) Ltd). Wells were coated with 50 ⁇ 1 of IFN- ⁇ catcher antibody (mAb 1.D 1 K, Mabtech) diluted to 5 ⁇ in sterile PBS pH7.4 and incubated 16h at 4°C. Plates were washed 6 times and blocked with 200 ⁇ 1 RI O per well for 1 hour at 37°C. PBMC were diluted to 1 x l OVml in hR10 and ⁇ ⁇ was added to each well. Peptides were added to a final concentration of ⁇ ⁇ and the plates were incubated overnight ( 16h) at 37°C.
  • IFN- ⁇ catcher antibody diluted to 5 ⁇ in sterile PBS pH7.4
  • PHA was used as the positive control at 2 ⁇ g/ml per well and RPMI alone as a negative control.
  • An irrelevant peptide was used to assess non-specific IFN- ⁇ responses.
  • the plates were developed by addition of ⁇ g/ml of biotin-linked anti- IFN- ⁇ mAb (mAb 7-B6- l -Biotin, Mabtech) as a detection antibody, which is subsequently conjugated to streptavidin alkaline phosphatase (Mabtech), and visualised using an AP conjugate substrate kit (Biorad, 170-643, as instructions). Spots were enumerated using an automated AID ELISpot reader. Background (cells plus irrelevant peptide) was subtracted and data expressed as number of spot-forming units (s.f.u.) per 10 6 PBMC.
  • DEN-4 has found to be associated with milder disease. Although the DEN-4 serotype was not prevalent among patients with DHF/DSS in Sri Lanka, it is possible that it caused the majority of the silent dengue infections as it resulted in milder clinical disease.

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Abstract

L'invention concerne un ou plusieurs peptides qui sont sélectionnés dans la liste comprenant un peptide correspondant au SEQ ID No : 1 à 103, 1A, 1B, 11A, 11B, 15A, 15B, 18A, 18B, 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41B, 44A, 44B, 61A, 61B, 63A, 63B, 67A, 67B, 70A, 70B, 72A, 72B, 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A et 94B, ou un peptide identique à au moins environ 70%, 75%, 80%, 85%, 90% ou 95%, 98% ou plus à une des séquences mentionnées ci-dessus, ou un peptide analogue ou dérivé d'une des séquences mentionnées ci-dessus. L'invention concerne en outre l'utilisation d'un ou de plusieurs de ces peptides. L'invention concerne en particulier une méthode pour déterminer l'état d'un sujet infecté par la dengue, comprenant les étapes consistant à : (a) utiliser un échantillon d'un matériau contenant des lymphocytes T et/ou des anticorps prélevés sur le sujet ; (b) déterminer la réponse du lymphocyte T et/ou de l'anticorps dans l'échantillon, ou une culture dérivée de l'échantillon, à un ou plusieurs peptides qui sont sélectionnés parmi un ou plusieurs des peptides suivants : i) un peptide correspondant au SEQ ID No : 1A, 1B, 11A, 11B, 15A, 15B, 18A et 18B; ii) un peptide correspondant au SEQ ID No : 30A, 30B, 36A, 36B, 37A, 37B, 41A, 41B, 44A et 44B; iii) un peptide correspondant au SEQ ID No :61A, 61B, 63A, 63B, 67A, 67B, 70A, 70B, 72A et 72B; et iv) un peptide correspondant au SEQ ID No: 79A, 79B, 84A, 84B, 86A, 86B, 93A, 93B, 94A et 94B; ou un peptide identique à au moins environ 70% ou plus à une des séquences mentionnées ci-dessus, ou un peptide analogue ou dérivé d'une des séquences mentionnées ci-dessus.
PCT/GB2012/050790 2011-04-11 2012-04-11 Marqueurs peptidiques et leurs utilisations WO2013021163A1 (fr)

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GBGB1106069.6A GB201106069D0 (en) 2011-04-11 2011-04-11 Peptide markers and uses thereof
GB1106069.6 2011-04-11

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US10556929B2 (en) * 2011-06-29 2020-02-11 Emergex Vaccines Holding Ltd. Cytotoxic T lymphocyte inducing immunogens for prevention treatment and diagnosis of dengue virus infection

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WO2005014627A1 (fr) * 2003-08-07 2005-02-17 International Centre For Genetic Engineering And Biotechnology Proteines a epitopes multiples de dengue recombinantes en tant qu'intermediaires de diagnostic

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10556929B2 (en) * 2011-06-29 2020-02-11 Emergex Vaccines Holding Ltd. Cytotoxic T lymphocyte inducing immunogens for prevention treatment and diagnosis of dengue virus infection

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