EP0546031A1 - Enterovirus peptides - Google Patents

Enterovirus peptides

Info

Publication number
EP0546031A1
EP0546031A1 EP91915656A EP91915656A EP0546031A1 EP 0546031 A1 EP0546031 A1 EP 0546031A1 EP 91915656 A EP91915656 A EP 91915656A EP 91915656 A EP91915656 A EP 91915656A EP 0546031 A1 EP0546031 A1 EP 0546031A1
Authority
EP
European Patent Office
Prior art keywords
thr
val
ala
gly
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91915656A
Other languages
German (de)
French (fr)
Inventor
Jonas Blomberg
Rüdiger Pipkorn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Replico Medical AB
Original Assignee
Replico Medical AB
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 Replico Medical AB filed Critical Replico Medical AB
Publication of EP0546031A1 publication Critical patent/EP0546031A1/en
Withdrawn legal-status Critical Current

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
    • 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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32311—Enterovirus
    • C12N2770/32322—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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32622—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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32711—Rhinovirus
    • C12N2770/32722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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 new peptides, to a diagnostic antigen, to the use of said diagnostic antigen in immunoassays and to a vaccine containing said diagnostic antigen.
  • Picornaviruses are a family of small RNA viruses which are responsible for a variety of diseases in humans and animals.
  • the diseases may be severe, like poliomyelitis (caused by polioviruses types 1, 2, and 3), hepatitis (caused by hepatitis A virus) and generalized neonatal enterovirus infection (often caused by Coxsackie and ECHO viruses), as well as mild, like the common cold (caused by rhinoviruses).
  • poliomyelitis caused by polioviruses types 1, 2, and 3
  • hepatitis caused by hepatitis A virus
  • generalized neonatal enterovirus infection often caused by Coxsackie and ECHO viruses
  • a clinical virological laboratory receives many samples every year for diagnosis of picornavirus infections.
  • the picornavirus family is divided into the enterovirus, rhinovirus and hepatitis A subfamilies.
  • the enteroviruses and hepatitis A viruses are the ones where a diagnosis is most often sought.
  • the enteroviruses are further divided into polio-, Coxsackie A, Coxsackie B and ECHO viruses on the basis of their pathogenicity in mice.
  • the enteroviral disease where a diagnosis is most often required is aseptic meningitis, but myocarditis, encephalitis, generalized neonatal infection and hand-, foot and mouth disease also constitute rather frequent clinical problems.
  • Flavivirus is another virus family which e.g. causes yellow fever and encephalitis. These diseases are spread by mosquitos and ticks and constitute a major worldwide problem.
  • the Dengueviruses and TBE-virus are types of flaviviruses. There is currently no therapy for these infections, so the diagnosis of picornavirus and flavivirus infections serves mostly to exclude other diseases or to give
  • the diagnosis of a picornavirus infection can be made either by virus isolation or by serology.
  • a serum sample taken early in the course of the infection an acute phase serum
  • a serum taken 1-3 weeks later a convalescent phase serum
  • an antibody assay most commonly a complement fixation assay (see e.g. Sever JL. Application of a microtechnique to viral serological investigations.
  • the antigen then is a purified or semipurified preparation from picornavirus-infected cells. If non-heat-treated virus antigens are used, a relatively type-specific antibody assay is obtained. If a heattreated viral antigen is used, a more group-specific assay is obtained. Presumably, the virus undergoes a conformational change upon heating, exposing group-specific antigenie determinants. The conformational change mostly seems to involve the N-terminus of VP1 [Fricks CE, Hogle JM. Cell-induced conformational change in poliovirus: Externalization of the amino terminus of VP1 is responsible for liposome binding. J. Virol. 64:193-1945 (1990)1.
  • One object of the present invention is to provide new peptides which can be used as a diagnostic antigen in immunoassays.
  • Another object of the present invention is to use a diagnostic antigen for more specific diagnosing of diseases or infections caused by picornavirus and/or flavivirus and/or discriminating between false and true diagnosed HIV-1 positive sera.
  • Still another object of the present invention is to provide a vaccine based on said peptides against diseases or infections caused by picornavirus and/or flavivirus.
  • the present invention relates to peptides of the formula:
  • the present invention relates to a diagnostic antigen having the ability to detect antibodies against at least one type of picornavirus and flavivirus in sera, and/or having the ability of binding to antibodies with a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or cluster of epitopes of HIV-1 p17, wherein said antigen mainly comprises an antigen selected from the peptides according to claim 1 and antigenic parts thereof.
  • the present invention relates to the use of at least one diagnostic antigen for diagnosis of infections caused by picornaviruses and/or flaviviruses, wherein the diagnostic antigen or antigens having the ability to detect antibodies against at least one type of picornavirus and flavivirus are added to a serum from a subject suspected to have a recent or previous picornavirus and/or flavivirus infection, and possible antigen/ antibody complexes formed are detected using an immunoassay, the diagnosis of said recent or previous infection(s) caused by picornaviruses and/or flaviviruses being established based either upon demonstration of antibodies in a single serum or upon a comparison of the amounts of antibodies detected in both acute and convalescent phase sera, and if said amount in the convalescent phase serum is significantly larger than in the acute phase serum, the subject donating the serum suffers or has suffered from a picorna- and/or flavivirus infection, wherein the antigen containing the peptides
  • Y 1 is Ala or Thr and Y 2 is Val or Ile, is primarily used for diagnosis of diseases caused by yellow fever virus and Japanese encephalitis virus, e) H-X-R 1 -Thr-Asp-Y 1 -Gly-His-Asp-Thr-Val-Y 2 -R 2 -X-Z,
  • Y 1 is Ser or Thr and Y 2 is Ile or Val, is primarily used for diagnosis of diseases caused by
  • Y 1 is His or Tyr and Y 2 is Ile or Thr, is primarily used for diagnosis of diseases caused by
  • R 1 and R 2 represent optional amino acid residues, wherein R 1 and R 2 together represent at most 25 amino acid residues, and Z represents -NH- or -OH.
  • the present invention relates to the use of a diagnostic antigen for discriminating between a false and true diagnosed HIV-1 positive serum sample, which has first been diagnosed as positive in a standard HIV-1 antibody screening EIA test and then found to exhibit a p17 pattern of serological activity in an electrophoretic immunoblot assay, wherein at least one diagnostic antigen having the ability of binding to antibodies which have a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or cluster of epitopes of HIV-1 p17, optionally coupled to a carrier, is added to said serum sample, and possible antigen/antibody complexes formed are detected using an immunoassay, the discrimination being established such that said serum sample is false HIV-1 positive if said complexes are detected, and true HIV-1 positive if said complexes are not detected.
  • the present invention relates to a vaccine composition which comprises as an immunizing component at least one picornavirus and/or flavivirus antigen selected from at least one of the peptides above.
  • one X represents a chemical bond and the other X represents a chemical bond or a coupling-facilitating sequence of at least 4, and preferably 8, particular amino acid residues, which are all chosen from the group consisting of -Thr- and -Ser-.
  • X is an amino acid sequence, it can be located either in the C- or N-terminus of the peptides but not in both ends at the same time. If it is for example located in the C-terminus, X in the N-terminus corresponds to a chemical bond and vice versa. However, X can be a bond at both ends of the peptide according to the present invention.
  • the amino acid sequence acts as a coupling-facilitating spacer, which permits proper binding to the carrier to which the peptides according to the present invention will be bound during the discrimination method.
  • the sequence X should not be an amino acid
  • amino acids threonine and serine also fulfill these requirements particularily well, and any one of the amino acids in said sequence X can be threonine or serine.
  • the number of amino acids in this spacer sequence should be at least 4, but in a preferred embodiment according to the present invention 8 amino acids are used.
  • R 1 and R 2 are optional amino acid residues and together represent at most 25 amino acid residues, preferably 20. Further, this means that one or both of R 1 and R 2 can be a chemical bond, i.e. contain no amino acids. If R 1 is a sequence containing e.g. 25 amino acid residues, R 2 must be a chemical bond and vice versa.
  • antigenic parts thereof as used herein means antigenic parts in the essential amino acid sequence between R 1 and R 2 in the peptides according to the present invention.
  • polypeptides according to the present invention can be bound via the amino acid sequence X to a carrier by physical/chemical interaction, as for example covalent binding, ionic binding, hydrogen binding or hydrophobic binding.
  • covalent binding are ester, ether and disulfide binding.
  • carrier as used herein should be interpreted broadly, and it may be any material which is compatible with and not adversely affects the method according to the present invention, for example resins, microplates, plastic surfaces, gels, matrixes etc.
  • epitope as used herein means antigenic or immunogenic determinant and relates to a specific part of a structure of an antigen inducing an immuno response, and the antibodies produced are directed against this part.
  • the immunoassay method according to the present invention can be an enzyme immunoassay (EIA), radioimmunoassay (RIA), immunoassay involving metal labelling, fluorescence immunoassay (FIA) or an immunoassay in which the peptide is soluble and inhibits another reaction.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • FIA fluorescence immunoassay
  • the vaccine composition according to the present invention comprises at least one picornavirus and/or flavivirus antigen selected from the peptides according to the present invention together with a non-toxic pharmaceutically acceptable carrier and/or a diluent in an amount effective to protect a subject from diseases caused by picornavirus and/or flavivirus.
  • the vaccine composition comprises an antigen adjuvant in an amount which together with an amount of said picornavirus and/or flavivirus antigen(s) is effec tive to protect a subject from diseases caused by picornaviruses and/or flaviviruses.
  • the vaccine composition additionally comprises one or more buffers and/or preservatives.
  • the peptides according to the invention are derived from sequences which are exposed upon heat inactivation of picornavirus or are situated on the other envelope proteins of flavivirus particles, and they are able to detect broadly cross-reactive picornavirus and flavivirus antibodies. This makes it possible to diagnose picornaviral and/or flaviviral infections or diseases by the use of only one or a few peptides from the highly conserved antigenically active picornaviral and flaviviral sequence.
  • the peptide sequences according to the present invention can be said to derive from a common basic structure which can be expressed as the consensus sequence
  • picornavirus this sequence is situated at one site in VP1 in the amino acids 20-40. In flavivirus, it is situated about 80 amino acids from the transmembrane region of the E-glucoprotein. In both cases, the sequence participates in interactions with the outer membrane of the host cell. In both cases, the sequence is also concealed to antibodies during part of the replication cycle of the virus, but becomes accessible to them, for instance when the virus has come into contact with the outer membrane of the cell.
  • the invention can be considered to belong to a previously unknown class of sequences having a common structure and function.
  • the peptides can be used for diagnosing diseases caused by both picornavirus and flavivirus, the peptides can, because of their common origin, structure and function, however with a certain, appropriate
  • amino acids be comprised by the same inventive concept.
  • Many or all of the peptides, preferably 3-4, according to the present invention can be included in one and the same assay for diagnosing different
  • lamely peptide (f) directed against Denguevirus was found, for instance, to strongly bind to a peptide
  • the ability of producing neutralising antibodies is one of the characteristics required of a vaccine, it must be considered highly likely that the peptides according to the present invention are usable as antigens in vaccine compositions against the above-mentioned picornavirus- and/or flavivirus-induced diseases.
  • Table 1 refers to the alignment of some different viral amino acid sequences from the region of similarity with HIV-1 p17.
  • the peptides according to the present invention can be used as a diagnostic antigen to detect antibodies in both acute and convalescent phase sera. If the amount of antibody detected in the convalescent serum is significantly larger than in the acute serum, the person donating the blood samples is believed to suffer from a picornavirus infection.
  • the expression "significantly larger” as used herein is a standard concept in this area (see J.
  • antibodies belonging to the IgM class and reactive with at least one of the picornaviral and/or flaviviral peptides described in the present invention are detected in one or more sera from a patient, then the patient is likely to suffer from a disease caused by a picornavirus and/or a flavivirus.
  • the peptides according to the present invention can further be used as a supplementary test during the
  • anti-p17 reaction can be abrogated by preabsorption or competition with one of the peptides according to the present invention, it is unlikely that the anti-p17 reaction is caused by antibodies to a picornavirus and/or flavivirus.
  • the as yet unpublished Swedish patent application 8903985-3 filed on November 27, 1989, also by Replico AB, describes new peptides, diagnostic antigens containing said peptides and having the ability of binding to anti ⁇ bodies which have a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or a cluster of epitopes of HIV-1 p17, and a method of discriminating between a false and true diagnosed HIV-1 positive serum sample using an immunoassay.
  • the technical background, proper definitions, and some of the methods used in connection with the use of the peptides according to the present invention in the p17 area are described in said Swedish patent application.
  • HIV-1 EIB reactivities may give rise to HIV-1 electrophoretic immunoblot patterns which are hard to interpret.
  • HIV-1 EIB reactivities is characterized by a reactivity with HIV-1 p17, but with no other HIV-1 protein. The reactivity with p17 peptides in sera from healthy Swedes which had the p17 pattern of serological reactivity has been studied.
  • Figure 1 shows absorption of five p17 positive sera with resin without peptide, resin with HIV-1 gag 118-140, resin with HTLV-I gag 111-130 (the C-terminus of HTLV-I), and resin with Coxsackie B1 VP125-53.
  • the intensity of the p17 band in EIB (y axis) was measured with a
  • Figure 2 shows the serological reactivity of a peptide derived from Coxsackie B1 VP1 25-53 with
  • p17-derived peptides were compared with that of a Coxsackie B1-derived peptide. 10 p17 positive sera, 19 HIV seronegative blood donors, 6 sera from confirmed HIV-1 seropositive persons (1 adult and 5 children, aged 1-7), and acute and convalescent sera from six patients with aseptic meningitis with significant (at least fourfold) titre rises in complement fixation antibody tests against Coxsackie B1-B6 antigen were analyzed.
  • Coxsackie antibodies were measured, by titration in standard microtitre complement fixation antibody tests with Coxsackie B1, B2, B3, B4, B5, and B6 antigens.
  • the diagnostic antigens according to the present invention can be utilized in methods for the same purpose as the claimed use of said diagnostic antigens.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

On décrit des peptides et un antigène diagnostique contenant l'un desdits peptides et possédant la capacité de détecter les anticorps agissant contre au moins un type de picornavirus et de flavivirus. L'utilisation dudit antigène diasgnostique pour diagnostiquer des infections causées par le picornavirus et/ou le flavivirus et pour discriminer entre des sérums diagnostiqués comme étant faux séropositifs et vrais séropositifs pour le VIH-1 p17 est aussi décrite, ainsi que des vaccins contre les maladies causées par des picornavirus et/ou des flavivirus.Peptides and a diagnostic antigen containing one of said peptides and having the capacity to detect antibodies acting against at least one type of picornavirus and flavivirus are described. The use of said diasgnostic antigen for diagnosing infections caused by picornavirus and / or flavivirus and for discriminating between sera diagnosed as being false positive and true HIV positive for p-1 is also described, as well as vaccines against the diseases caused by picornaviruses and / or flaviviruses.

Description

EMTEROVTRUS PEPTIDES
The present invention relates to new peptides, to a diagnostic antigen, to the use of said diagnostic antigen in immunoassays and to a vaccine containing said diagnostic antigen.
Background art
Picornaviruses are a family of small RNA viruses which are responsible for a variety of diseases in humans and animals. The diseases may be severe, like poliomyelitis (caused by polioviruses types 1, 2, and 3), hepatitis (caused by hepatitis A virus) and generalized neonatal enterovirus infection (often caused by Coxsackie and ECHO viruses), as well as mild, like the common cold (caused by rhinoviruses). Picornaviruses then are
significant pathogens, and the diagnosis of infections caused by them is an important medical task. A clinical virological laboratory receives many samples every year for diagnosis of picornavirus infections. The picornavirus family is divided into the enterovirus, rhinovirus and hepatitis A subfamilies. Among these, the enteroviruses and hepatitis A viruses are the ones where a diagnosis is most often sought. The enteroviruses are further divided into polio-, Coxsackie A, Coxsackie B and ECHO viruses on the basis of their pathogenicity in mice. The enteroviral disease where a diagnosis is most often required is aseptic meningitis, but myocarditis, encephalitis, generalized neonatal infection and hand-, foot and mouth disease also constitute rather frequent clinical problems.
Flavivirus is another virus family which e.g. causes yellow fever and encephalitis. These diseases are spread by mosquitos and ticks and constitute a major worldwide problem. The Dengueviruses and TBE-virus are types of flaviviruses. There is currently no therapy for these infections, so the diagnosis of picornavirus and flavivirus infections serves mostly to exclude other diseases or to give
epidemiological information.
The diagnosis of a picornavirus infection can be made either by virus isolation or by serology. In the latter case, for enteroviruses, a serum sample taken early in the course of the infection (an acute phase serum) and a serum taken 1-3 weeks later (a convalescent phase serum) are analyzed with an antibody assay, most commonly a complement fixation assay (see e.g. Sever JL. Application of a microtechnique to viral serological investigations.
J. Immunol, 88:320-329). The antigen then is a purified or semipurified preparation from picornavirus-infected cells. If non-heat-treated virus antigens are used, a relatively type-specific antibody assay is obtained. If a heattreated viral antigen is used, a more group-specific assay is obtained. Presumably, the virus undergoes a conformational change upon heating, exposing group-specific antigenie determinants. The conformational change mostly seems to involve the N-terminus of VP1 [Fricks CE, Hogle JM. Cell-induced conformational change in poliovirus: Externalization of the amino terminus of VP1 is responsible for liposome binding. J. Virol. 64:193-1945 (1990)1. There are some indications that antibodies to this portion of VP1 can influence the course of infection [Chow M, Yabrov R, Bittle J, Hogle JM, Baltimore D. Synthetic peptides from four separate regions of the poliovirus capsid protein VP1 induce neutralizing antibodies. Proc Natl Acad Sci USA 82:910-914 (1985)1, this making it interesting also as a candidate for vaccination purposes. For flaviviruses, a variety of serological techniques exist, all of which however have great limitations due to lack of sensitivity or specificity.
Thus, there is a need for a means for a more exact diagnosis of diseases caused by picornavirus and flavi virus, which could further specify and facilitate the treatment of the disease concerned.
Further, there is a need for vaccines for different diseases caused by picornavirus and flavivirus.
One object of the present invention is to provide new peptides which can be used as a diagnostic antigen in immunoassays.
Another object of the present invention is to use a diagnostic antigen for more specific diagnosing of diseases or infections caused by picornavirus and/or flavivirus and/or discriminating between false and true diagnosed HIV-1 positive sera.
Still another object of the present invention is to provide a vaccine based on said peptides against diseases or infections caused by picornavirus and/or flavivirus.
All these objects are achieved by the present invention.
Description of the invention
In one aspect, the present invention relates to peptides of the formula:
a) H-X-R1-Ala-Y1-Glu-Thr-Gly-His-Thr-Ser-Gln-Val- R2-X-Z, wherein Y1 is Ala or Val,
b) H-X-R1-Ala-Y1-Glu-Thr-Gly-Ala-Thr-Asn-Pro-Leu- R2-X-Z, wherein Y1 is Ala or Val,
c) H-X-R1-Ala-Ala-Glu-Thr-Gly-His-Thr-Ser-Y1-Val- R 2-X-Z, wherein Y1 is Ser or Asn,
d) H-X-R1-Y1-Asp-Thr-Gly-His-Gly-Thr-Val-Y2-R2-X-Z, wherein Y 1 is Ala or Thr and Y2 is Val or Ile, e) H-X-R1-Thr-Asp-Y1-Gly-His-Asp-Thr-Val-Y2-R2-X-Z, wherein Y1 is Ser or Thr and Y2 is Ile or Val, and
f) H-X-R1-Ala-Glu-Thr-Gln-Y1-Gly-Thr-Y2-Val-R2-X-Z, wherein Y 1 is His or Tyr and Y2 is Ile or Thr, in which one X represents a chemical bond and the other X represents a chemical bond or a coupling-facilitating amino acid sequence, R 1 and R2 represent optional amino acid residues, wherein R 1 and R2 together represent at most 25 amino acid residues, and Z represents -NH2 or -OH. In another aspect, the present invention relates to a diagnostic antigen having the ability to detect antibodies against at least one type of picornavirus and flavivirus in sera, and/or having the ability of binding to antibodies with a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or cluster of epitopes of HIV-1 p17, wherein said antigen mainly comprises an antigen selected from the peptides according to claim 1 and antigenic parts thereof.
In still another aspect, the present invention relates to the use of at least one diagnostic antigen for diagnosis of infections caused by picornaviruses and/or flaviviruses, wherein the diagnostic antigen or antigens having the ability to detect antibodies against at least one type of picornavirus and flavivirus are added to a serum from a subject suspected to have a recent or previous picornavirus and/or flavivirus infection, and possible antigen/ antibody complexes formed are detected using an immunoassay, the diagnosis of said recent or previous infection(s) caused by picornaviruses and/or flaviviruses being established based either upon demonstration of antibodies in a single serum or upon a comparison of the amounts of antibodies detected in both acute and convalescent phase sera, and if said amount in the convalescent phase serum is significantly larger than in the acute phase serum, the subject donating the serum suffers or has suffered from a picorna- and/or flavivirus infection, wherein the antigen containing the peptides
a) H-X-R1-Ala-Y1-Glu-Thr-Gly-His-Thr-Ser-Gln-Val-R2-X-Z, wherein Y1 is Ala or Val, is primarily used for diagnosis of diseases caused by Coxsackievirus, b) H-X-R1-Ala-Y1-Glu-Thr-Gly-Ala-Thr-Asn-Pro-Leu-R2-X-Z, wherein Y1 is Ala or Val, is primarily used for diagnosis of diseases caused by poliovirus,
c) H-X-R1-Ala-Ala-Glu-Thr-Gly-His-Thr-Ser-Y1-Val-R2-X-Z, wherein Y1 is Ser or Asn, is primarily used for diagnosis of deseases caused by rhinovirus,
d) H-X-R1-Y1-Asp-Thr-Gly-His-Gly-Thr-Val-Y2-R2-X-Z,
wherein Y 1 is Ala or Thr and Y2 is Val or Ile, is primarily used for diagnosis of diseases caused by yellow fever virus and Japanese encephalitis virus, e) H-X-R1-Thr-Asp-Y1-Gly-His-Asp-Thr-Val-Y2-R2-X-Z,
wherein Y 1 is Ser or Thr and Y2 is Ile or Val, is primarily used for diagnosis of diseases caused by
TBE-virus,
and
f) H-X-R1-Ala-Glu-Thr-Gln-Y1-Gly-Thr-Y2-Val-R2-X-Z,
wherein Y 1 is His or Tyr and Y2 is Ile or Thr, is primarily used for diagnosis of diseases caused by
Dengue- and rhinovirus,
in which one X represents a chemical bond and the other X represents a chemical bond or a coupling-facilitating amino acid sequence, R 1 and R2 represent optional amino acid residues, wherein R 1 and R2 together represent at most 25 amino acid residues, and Z represents -NH- or -OH.
In still another aspect, the present invention relates to the use of a diagnostic antigen for discriminating between a false and true diagnosed HIV-1 positive serum sample, which has first been diagnosed as positive in a standard HIV-1 antibody screening EIA test and then found to exhibit a p17 pattern of serological activity in an electrophoretic immunoblot assay, wherein at least one diagnostic antigen having the ability of binding to antibodies which have a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or cluster of epitopes of HIV-1 p17, optionally coupled to a carrier, is added to said serum sample, and possible antigen/antibody complexes formed are detected using an immunoassay, the discrimination being established such that said serum sample is false HIV-1 positive if said complexes are detected, and true HIV-1 positive if said complexes are not detected. In still another aspect, the present invention relates to a vaccine composition which comprises as an immunizing component at least one picornavirus and/or flavivirus antigen selected from at least one of the peptides above.
Other characteristics and features of the present invention appear from the attached claims.
In the peptides according to the present invention, one X represents a chemical bond and the other X represents a chemical bond or a coupling-facilitating sequence of at least 4, and preferably 8, particular amino acid residues, which are all chosen from the group consisting of -Thr- and -Ser-. When X is an amino acid sequence, it can be located either in the C- or N-terminus of the peptides but not in both ends at the same time. If it is for example located in the C-terminus, X in the N-terminus corresponds to a chemical bond and vice versa. However, X can be a bond at both ends of the peptide according to the present invention. The amino acid sequence acts as a coupling-facilitating spacer, which permits proper binding to the carrier to which the peptides according to the present invention will be bound during the discrimination method. The sequence X should not be an amino acid
sequence which adversely affects the result of the diagnosis method. Accordingly, it should not have too high a charge or be too hydrophobiσ and it should not disturb the conformation of the peptides. The amino acids threonine and serine also fulfill these requirements particularily well, and any one of the amino acids in said sequence X can be threonine or serine. The number of amino acids in this spacer sequence should be at least 4, but in a preferred embodiment according to the present invention 8 amino acids are used.
R 1 and R2 are optional amino acid residues and together represent at most 25 amino acid residues, preferably 20. Further, this means that one or both of R 1 and R2 can be a chemical bond, i.e. contain no amino acids. If R1 is a sequence containing e.g. 25 amino acid residues, R2 must be a chemical bond and vice versa.
The expression "and antigenic parts thereof as used herein means antigenic parts in the essential amino acid sequence between R 1 and R2 in the peptides according to the present invention.
The polypeptides according to the present invention can be bound via the amino acid sequence X to a carrier by physical/chemical interaction, as for example covalent binding, ionic binding, hydrogen binding or hydrophobic binding. Examples of covalent binding are ester, ether and disulfide binding.
The expression "carrier" as used herein should be interpreted broadly, and it may be any material which is compatible with and not adversely affects the method according to the present invention, for example resins, microplates, plastic surfaces, gels, matrixes etc.
The expression "epitope" as used herein means antigenic or immunogenic determinant and relates to a specific part of a structure of an antigen inducing an immuno response, and the antibodies produced are directed against this part.
The immunoassay method according to the present invention can be an enzyme immunoassay (EIA), radioimmunoassay (RIA), immunoassay involving metal labelling, fluorescence immunoassay (FIA) or an immunoassay in which the peptide is soluble and inhibits another reaction.
The vaccine composition according to the present invention comprises at least one picornavirus and/or flavivirus antigen selected from the peptides according to the present invention together with a non-toxic pharmaceutically acceptable carrier and/or a diluent in an amount effective to protect a subject from diseases caused by picornavirus and/or flavivirus.
Further, the vaccine composition comprises an antigen adjuvant in an amount which together with an amount of said picornavirus and/or flavivirus antigen(s) is effec tive to protect a subject from diseases caused by picornaviruses and/or flaviviruses.
Further, the vaccine composition additionally comprises one or more buffers and/or preservatives.
The peptides according to the invention are derived from sequences which are exposed upon heat inactivation of picornavirus or are situated on the other envelope proteins of flavivirus particles, and they are able to detect broadly cross-reactive picornavirus and flavivirus antibodies. This makes it possible to diagnose picornaviral and/or flaviviral infections or diseases by the use of only one or a few peptides from the highly conserved antigenically active picornaviral and flaviviral sequence.
The location of this sequence on the envelope protein, its pronounced evolutionary conservation in flaviviruses and the proven antigeniσity of analogous sequences in picornaviruses make this sequence especially accessible for antibodies suitable for diagnostic and immunisation purposes.
The peptide sequences according to the present invention can be said to derive from a common basic structure which can be expressed as the consensus sequence
aaDTGHxLxV where the capital letters relate to amino acids of strong or complete conservation, the small letters relate to amino acids present in more than half of the cases, and x relates to any amino acid. This common basic structure has been discovered on one and the same occasion and has been described in an article by Blomberg &
Medstrand in New Biologist 2:1044-1046 (1990). In
picornavirus, this sequence is situated at one site in VP1 in the amino acids 20-40. In flavivirus, it is situated about 80 amino acids from the transmembrane region of the E-glucoprotein. In both cases, the sequence participates in interactions with the outer membrane of the host cell. In both cases, the sequence is also concealed to antibodies during part of the replication cycle of the virus, but becomes accessible to them, for instance when the virus has come into contact with the outer membrane of the cell. Thus, the peptides according to the present
invention can be considered to belong to a previously unknown class of sequences having a common structure and function. Although the peptides can be used for diagnosing diseases caused by both picornavirus and flavivirus, the peptides can, because of their common origin, structure and function, however with a certain, appropriate
variation of the amino acids, be comprised by the same inventive concept. Many or all of the peptides, preferably 3-4, according to the present invention can be included in one and the same assay for diagnosing different
picornavirus- and flavivirus-induced diseases.
As to the presence of the peptides according to the present invention in vaccine compositions, it may initially be stated that the above-mentioned natural basic sequence is included in a region where it has earIler been demonstrated that peptides give rise to neutralising anti¬bodies in rabbit immunisation. In these experiments, however, the peptides according to the present invention were not used, but yet closely related peptide sequences. The evolutionary conservation of said basic sequence confers e.g. the advantage of producing a vaccine inhibiting infection involving many different cold viruses.
As to peptides deriving from flavivirus, the inventor, together with Dr Vincent Deubel at the Arbovirus laboratory at the Pasteur Institute in Paris, has tested the ability of previously known monoclonal antibodies having a virus-neutralising effect, of binding to the
peptides according to the present invention. One of them, lamely peptide (f) directed against Denguevirus, was found, for instance, to strongly bind to a peptide
containing the Dengue variant of the common basic
structure. Since the ability of producing neutralising antibodies is one of the characteristics required of a vaccine, it must be considered highly likely that the peptides according to the present invention are usable as antigens in vaccine compositions against the above-mentioned picornavirus- and/or flavivirus-induced diseases.
Table 1 refers to the alignment of some different viral amino acid sequences from the region of similarity with HIV-1 p17.
The peptides according to the present invention can be used as a diagnostic antigen to detect antibodies in both acute and convalescent phase sera. If the amount of antibody detected in the convalescent serum is significantly larger than in the acute serum, the person donating the blood samples is believed to suffer from a picornavirus infection. The expression "significantly larger" as used herein is a standard concept in this area (see J.
Blomberg, I. Nilsson and M. Andersson. 1983. Viral antibody screening system that uses a standardized single dilution immunoglobulin 6 enzyme immunoassay with multiple antigens. J. Clin. Microbiol. 17:1081-1091.)
If antibodies belonging to the IgM class and reactive with at least one of the picornaviral and/or flaviviral peptides described in the present invention are detected in one or more sera from a patient, then the patient is likely to suffer from a disease caused by a picornavirus and/or a flavivirus.
The peptides according to the present invention can further be used as a supplementary test during the
investigation of an antibody reaction with HIV-1 p17. If the anti-p17 reaction can be abrogated by preabsorption or competition with one of the peptides according to the present invention, it is unlikely that the anti-p17 reaction is caused by antibodies to a picornavirus and/or flavivirus.
The as yet unpublished Swedish patent application 8903985-3, filed on November 27, 1989, also by Replico AB, describes new peptides, diagnostic antigens containing said peptides and having the ability of binding to anti¬bodies which have a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or a cluster of epitopes of HIV-1 p17, and a method of discriminating between a false and true diagnosed HIV-1 positive serum sample using an immunoassay. The technical background, proper definitions, and some of the methods used in connection with the use of the peptides according to the present invention in the p17 area are described in said Swedish patent application.
After the introduction of large-scale HIV-1 antibody testing, it has been observed that a small portion of sera from humans unlikely to be HIV-1 infected contains antibodies to one or some HIV-1 proteins, mostly derived from the gag part of the molecule. Such reactivities may give rise to HIV-1 electrophoretic immunoblot patterns which are hard to interpret. One of the most common types of such HIV-1 EIB reactivities is characterized by a reactivity with HIV-1 p17, but with no other HIV-1 protein. The reactivity with p17 peptides in sera from healthy Swedes which had the p17 pattern of serological reactivity has been studied. By direct binding and absorption experiments it has been shown that, in contrast to EIB confirmed HIV-1 antibody positive sera, the p17 positive sera reacted with peptides from the C-terminus of p17. It has furthermore been shown that they contain a stretch highly similar to a conserved sequence from the N-terminus of VP1 of some enteroviruses, and to a lesser extent, flaviviruses.
Brief description of the drawings
Figure 1 shows absorption of five p17 positive sera with resin without peptide, resin with HIV-1 gag 118-140, resin with HTLV-I gag 111-130 (the C-terminus of HTLV-I), and resin with Coxsackie B1 VP125-53. The intensity of the p17 band in EIB (y axis) was measured with a
densitometer.
Figure 2 shows the serological reactivity of a peptide derived from Coxsackie B1 VP1 25-53 with
a/ acute and convalescent phase sera from five cases of aseptic meningitis with significant Coxsackie CF titre rises,
b/ 10 p17 reactive sera,
c/ 19 HIV seronegative blood donors and
d/ 6 confirmed HIV-1 antibody positive sera. Experiment with a Coxsackie B1 VPl derived peptide
A peptide from amino acids 25-53 of the Coxsackie B1 VP1 sequence (Iizuka, N. , S. Kuge and A. Nomoto. 1987.
Complete nucleotide sequence of the genome of Coxsackievirus B1. Virology 156:64-73.) was synthesized, and its ability to absorb p17 antibodies was compared with that of HIV-1 hxb2 gag 118-140 (Fig. 1). In five out of five sera tested, this peptide in resin-bound form absorbed out all p17 EIB reactivity. HIV-1 hxb2 gag 118-140 also absorbed these antibodies, but less completely. Absorption with resin alone, or with HTLV-I gag 111-130 did not affect the p17 EIB reactivity. The p17 reactive antibodies in these sera thus must bind to epitopes simulated by the resinbound Coxsackie-derived peptide. The peptide was present in vast molar excess compared to antibodies, thus favouring absorption also of low-affinity antibodies.
The reactivity of p17-derived peptides was compared with that of a Coxsackie B1-derived peptide. 10 p17 positive sera, 19 HIV seronegative blood donors, 6 sera from confirmed HIV-1 seropositive persons (1 adult and 5 children, aged 1-7), and acute and convalescent sera from six patients with aseptic meningitis with significant (at least fourfold) titre rises in complement fixation antibody tests against Coxsackie B1-B6 antigen were analyzed.
As to antibody activity against the Coxsackie peptide among the p17 reactive sera diluted 1/200, it was found that they did not react significantly more than blood donor sera (Fig 2). This may indicate that the Coxsackie peptide, when absorbed to plastic, does not assume a conformation suitable for binding of p17 reactive antibodies, or that it can bind many other antibodies, which obscures a possible correlation. Six HIV-1 confirmed positive sera with clear p17 bands reacted weakly. Five of the sera were selected from children, to reduce the likelihood of previous exposure to a Coxsackie-like virus. Thus, there was no cross-reactivity of p17 antibodies arising during HIV-1 infection to the Coxsackie peptide. Finally, six serum pairs were tested from patients with aseptic meningitis who had significant titre rises in the complement fixation test with Coxsackie B1-B6 antigen. It was found that five exhibited clear rises in absorbance difference and one reacted strongly on both sera (Fig. 2). A CF antibody titre rise in this system indicates an enterovirus infection, not necessarily with Coxsackie Bl. When the same sera were tested at a dilution of 1/50, almost all reacted strongly (not shown), attesting to the high antigenicity and frequent recognition of this peptide sequence. The Coxsackie peptide may then be a group-reactive reagent suitable for serological diagnosis of
enterovirus infections. The cross-reaction between the Coxsackie B1 VP1 peptide and HIV-1 hxb2 p17 thus was unidirectional, from the enterovirus to HIV-1, but not vice versa. Antibody reactions with the Coxsackie peptide were very common in Swedish sera diluted 1/50. Only a few of them can bind also to HIV-1 p17 in EIB.
The immunoassays used are described in the Swedish patent application above. Coxsackie antibodies were measured, by titration in standard microtitre complement fixation antibody tests with Coxsackie B1, B2, B3, B4, B5, and B6 antigens.
The diagnostic antigens according to the present invention can be utilized in methods for the same purpose as the claimed use of said diagnostic antigens.

Claims

1. Peptides of the formula
a) H-X-R1-Ala-Y1-Glu-Thr-Gly-His-Thr-Ser-Gln- Val-R2-X-Z, wherein Y1 is Ala or Val,
b) H-X-R1-Ala-Y1-Glu-Thr-Gly-Ala-Thr-Asn-Pro- Leu-R2-X-Z, wherein Y1 is Ala or Val,
c) H-X-R1-Ala-Ala-Glu-Thr-Gly-His-Thr-Ser-Y1- Val-R2-X-Z, wherein Y1 is Ser or Asn,
d) H-X-R1-Y1-Asp-Thr-Gly-His-Gly-Thr-Val-Y2-R2- X-Z, wherein Y1 is Ala or Thr and Y2 is Val or Ile,
e) H-X-R1-Thr-Asp-Y1-Gly-His-Asp-Thr-Val-Y2-R2- X-Z, wherein Y1 is Ser or Thr and Y2 is Ile or Val,
and
f) H-X-R1-Ala-Glu-Thr-Gln-Y1-Gly-Thr-Y2-Val-R2- X-Z, wherein Y1 is His or Tyr and Y2 is Ile or Thr,
in which one X represents a chemical bond and the other X represents a chemical bond or a coupling-facilitating amino acid sequence, R 1 and R2 represent optional amino acid residues, wherein R 1 and R2 together represent at most 25 amino acid residues, and Z represents -NH2 or -OH.
2. Peptides according to claim 1, wherein the
coupling-facilitating amino acid sequence represents at least 4, preferably 8, amino acid residues, which are each selected from the group consisting of -Thr- and -Ser-, and R 1 and R2 together preferably represent at most 20 amino acid residues.
3. A diagnostic antigen having the ability to detect antibodies against at least one type of picornavirus and flavivirus in sera, and/or having the ability of binding to antibodies with a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or cluster of epitopes of HIV-1 p17, c h a r a c t e r i s e d in that it mainly comprises an antigen selected from the peptides according to claim 1 and antigenic parts thereof.
4. Use of a diagnostic antigen according to claim 3 for the diagnosis of Infections caused by picornaviruses and/or flaviviruses, wherein a diagnostic antigen having the ability to detect antibodies against at least one type of picornavirus and flavivirus is added to a serum from a subject suspected to have a recent or previous picorna-virus and/or flavivirus infection, and possible antigen/antibody complexes formed are detected using an immunoassay, the diagnosis of said recent or previous infection or infections caused by picornavirus and/or flavivirus being established based either upon demonstration of antibodies in a single serum or upon a comparison of the amounts of antibodies detected in both acute and convalescent sera, and if said amount in the convalescent serum is significantly larger than that in the acute serum, the subject donating the serum is likely to suffer from a picorna- and/or flavivirus infection, wherein the antigen containing the peptide
a) H-X-R1-Ala-Y1-Glu-Thr-Gly-His-Thr-Ser-Gln- Val-R2-X-Z, wherein Y1 is Ala or Val, is primarily used for diagnosis of diseases caused by Coxsackievirus,
b) H-X-R1-Ala-Y1-Glu-Thr-Gly-Ala-Thr-Asn-Pro- Leu-R2-X-Z, wherein Y1 is Ala or Val, is primarily used for diagnosis of diseases caused by poliovirus,
c) H-X-R1-Ala-Ala-Glu-Thr-Gly-His-Thr-Ser-Y1- Val-R 2-X-Z, wherein Y1 is Ser or Asn, is primarily used for diagnosis of diseases caused by rhinovirus,
d) H-X-R1-Y1-Asp-Thr-Gly-His-Gly-Thr-Val-Y2-R2- X-Z, wherein Y1 is Ala or Thr and Y2 is Val or Ile, is primarily used for diagnosis of diseases caused by yellow fever virus and Japanese encephalitis virus,
e) H-X-R1-Thr-Asp-Y1-Gly-His-Asp-Thr-Val-Y2-R2- X-Z, wherein Y1 is Ser or Thr and Y2 is Ile or Val, is primarily used for diagnosis of diseases caused by TBE-virus,
and
f) H-X-R1-Ala-Glu-Thr-Gln-Y1-Gly-Thr-Y2-Val-R2- X-Z, wherein Y1 is His or Tyr and Y2 is Ile or Thr, is primarily used for diagnosis of diseases caused by Dengue- and rhinovirus, in which one X represents a chemical bond and the other X represents a chemical bond or a coupling-facilitating amino acid sequence, R 1 and R2 represent optional amino acid residues, wherein R 1 and R2 together represent at most 25 amino acid residues, and Z represents -NH2 or -OH.
5. Use of a diagnostic antigen according to claim 3 for discriminating between a false and true diagnosed HIV-1 positive serum sample, which has first been diagnosed as positive in a standard HIV-1 antibody screening EIA test and then found to exhibit a p17 pattern of serological activity in an electrophoretic immunoblot assay, wherein at least one diagnostic antigen having the ability of binding to antibodies which have a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or cluster of epitopes of HIV-1 p17,
optionally coupled to a carrier, is added to said serum sample, and possible antigen/antibody complexes formed are detected using an immunoassay, the discrimination being established such that said serum sample is false HIV-1 positive if said complexes are detected, and true HIV-I positive if said complexes are not detected.
6. Use according to claims 4 and 5, c h a r a c t e r i s e d in that the immunoassay for diagnosis of diseases caused by picornavirus and/or flavivirus and for detection of said antibody/antigen complex, respectively, is an enzyme immunoassay (EIA), radioimmuno assay (RIA), immunoassay involving metal labelling, fluorescence immunoassay (FIA) or an immunoassay in which the peptide is soluble and inhibits another reaction.
7. Use according to claims 4 and 5, c h a r a c t e r i s e d in that the carrier is a resin, microplate, plastic surface, gel or matrix.
8. Use according to claims 4-7, c h a r a c t e r i s e d in that in the diagnosis and discrimination use is simultaneously made of many or all, preferably 3-4, of the diagnostic antigens according to claim 3.
9. A vaccine composition, c h a r a c t e r i s e d in that it comprises as an immunizing component, at least one antigen selected from at least one peptide from the group consisting of
a) H-X-R1-Ala-Y1-Glu-Thr-Gly-His-Thr-Ser-Gln- Val-R2-X-Z, wherein Y1 is Ala or Val,
b) H-X-R1-Ala-Y1-Glu-Thr-Gly-Ala-Thr-Asn-Pro- Leu-R2-X-Z, wherein Y1 is Ala or Val,
c) H-X-R1-Ala-Ala-Glu-Thr-Gly-His-Thr-Ser-Y1- Val-R2-X-Z, wherein Y1 is Ser or Asn,
d) H-X-R1-Y1-Asp-Thr-Gly-His-Gly-Thr-Val-Y2-R2- X-Z, wherein Y1 is Ala or Thr and Y2 is Val or Ile,
e) H-X-R1-Thr-Asp-Y1-Gly-His-Asp-Thr-Val-Y2-R2- X-Z, wherein Y1 is Ser or Thr and Y2 is Ile or Val, and
f) H-X-R1-Ala-Glu-Thr-Gln-Y1-Gly-Thr-Y2-Val-R2- X-Z, wherein Y1 is His or Tyr and Y2 is Ile or Thr,
in which one X represents a chemical bond and the other X represents a chemical bond or a coupling-facilitating amino acid sequence, R 1 and R2 represent optional amino acid residues, wherein R 1 and R2 together represent at most 25 amino acid residues, and Z represents -NH- or -OH, and antigenic parts thereof, together with a non-toxic pharmaceutically acceptable carrier and/or diluent.
10. A vaccine composition according to claim 9, c h a r a c t e r i s e d in that it comprises said picorna- and/or flavivirus antigen or antigens in an amount effective to protect a subject from diseases caused by picornaviruses and/or flaviviruses.
11. A vaccine composition according to claim 9, c h a r a c t e r i s ed in that it further comprises an antigen adjuvant in an amount which together with an amount of said picornavirus and/or flavivirus antigen or is effective to protect a subject from diseases caused by picornaviruses and/or flaviviruses.
EP91915656A 1990-08-16 1991-08-16 Enterovirus peptides Withdrawn EP0546031A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9002671A SE470074B (en) 1990-08-16 1990-08-16 Method for diagnosis of picorene and / or flavivirus infection, peptides, diagnostic antigens and vaccine composition with these peptides
SE9002671 1990-08-16

Publications (1)

Publication Number Publication Date
EP0546031A1 true EP0546031A1 (en) 1993-06-16

Family

ID=20380170

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91915656A Withdrawn EP0546031A1 (en) 1990-08-16 1991-08-16 Enterovirus peptides

Country Status (4)

Country Link
EP (1) EP0546031A1 (en)
AU (1) AU649473B2 (en)
SE (1) SE470074B (en)
WO (1) WO1992003475A1 (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4776075B2 (en) 1998-12-31 2011-09-21 ノバルティス バクシンズ アンド ダイアグノスティックス，インコーポレーテッド Modified HIVENV polypeptide
CA2452015C (en) 2001-07-05 2012-07-03 Chiron Corporation Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof
US20030170614A1 (en) 2001-08-31 2003-09-11 Megede Jan Zur Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
BE1022174B1 (en) * 2013-03-15 2016-02-24 Glaxosmithkline Biologicals S.A. VACCINE

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT384363B (en) * 1982-10-05 1987-11-10 Immuno Ag METHOD FOR USE OF ANTIGENS REACTIVE PEPTIDES FOR THE PRODUCTION OF VACCINES ACTIVE AGAINST TBE VIRUS INFECTIONS
EP0220273B2 (en) * 1985-04-29 2007-01-17 Bio-Rad Laboratories, Inc. Synthetic antigens for the detection of aids-related disease
JPH0768267B2 (en) * 1986-06-05 1995-07-26 財団法人阪大微生物病研究会 Flavivirus antigen
WO1988003032A1 (en) * 1986-10-27 1988-05-05 Fournier Maurielle J Diagnosis of and vaccine for japanese encephalitis virus and related viruses
DE3853693D1 (en) * 1987-03-20 1995-06-08 Immuno Ag TBE virus western subtype DNA and RNA molecules, polypeptides encoded by these molecules, and their use.
JPH084508B2 (en) * 1987-09-16 1996-01-24 国立予防衛生研究所長 Recombinant vaccinia virus
GB8821077D0 (en) * 1988-09-08 1988-10-05 Wellcome Found Peptides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9203475A1 *

Also Published As

Publication number Publication date
SE470074B (en) 1993-11-01
SE9002671L (en) 1992-02-17
SE9002671D0 (en) 1990-08-16
AU8400291A (en) 1992-03-17
WO1992003475A1 (en) 1992-03-05
AU649473B2 (en) 1994-05-26

Similar Documents

Publication Publication Date Title
JP2763215B2 (en) Synthetic peptides specific for detecting antibodies to HCV, diagnosing HCV infection and its prevention as a vaccine
KR940000755B1 (en) Synthetic peptides specific for the detection of antibodies to hcv
KR870000702B1 (en) The synthetic antigen peptide manufacturing method
JP3156200B2 (en) New HCV isolate
JP3514680B2 (en) Hepatitis C virus (HCV) polypeptide
Zhao et al. A comprehensive study of neutralizing antigenic sites on the hepatitis E virus (HEV) capsid by constructing, clustering, and characterizing a tool box
JPH04253998A (en) C-type hepatitis assay
Jiang et al. Sapporo-like human caliciviruses are genetically and antigenically diverse
Cello et al. Identification of group-common linear epitopes in structural and nonstructural proteins of enteroviruses by using synthetic peptides
JPH03503284A (en) Synthetic peptide antigen for detecting HTLV-1 infection
JPH07501442A (en) Hepatitis C virus test
Wang et al. Immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognized by human and woodchuck antibodies
CN113607952B (en) African swine fever virus blocking ELISA antibody detection kit and preparation method and application thereof
Lennette et al. Picornaviridae: the enteroviruses (polioviruses, coxsackieviruses, echoviruses)
CN105418738A (en) A-type antigen polypeptide, fusion antigen polypeptide and vaccine of foot and mouth disease virus
US6153378A (en) Diagnosis of, and vaccination against, a positive stranded RNA virus using an isolated, unprocessed polypeptide encoded by a substantially complete genome of such virus
JPH06510861A (en) Hepatitis C assay
Yeung et al. The cell attachment proteins of type 1 and type 3 reovirus are differentially susceptible to trypsin and chymotrypsin
AU649473B2 (en) Enterovirus peptides
US5843450A (en) Hepatitis GB Virus synthetic peptides and uses thereof
Mitchell et al. Identification of rubella virus T-cell epitopes recognized in anamnestic response to RA27/3 vaccine: associations with boost in neutralizing antibody titer
Kurstak et al. Enzyme immunoassays and related procedures in diagnostic medical virology
RU2811996C1 (en) Test system for determining titer of antibodies against 146s particles of foot-and-mouth disease virus genotype sat-2/iv using elisa liquid-phase
RU2821044C1 (en) Test system for detecting antibodies to structural proteins of foot-and-mouth disease virus strain "sat-1/kenya/2017" using liquid-phase blocking indirect "sandwich" version of elisa
JPH06510289A (en) Hepatitis C assay using recombinant antigen derived from NS5 region

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19930316

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

17Q First examination report despatched

Effective date: 19950530

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19980105