EP0477301A1 - Peptides et anticoprs derives de diagnostic et de therapie d'une infection provoquee par le htlv-1, et de vaccination contre celui-ci - Google Patents

Peptides et anticoprs derives de diagnostic et de therapie d'une infection provoquee par le htlv-1, et de vaccination contre celui-ci

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Publication number
EP0477301A1
EP0477301A1 EP90917762A EP90917762A EP0477301A1 EP 0477301 A1 EP0477301 A1 EP 0477301A1 EP 90917762 A EP90917762 A EP 90917762A EP 90917762 A EP90917762 A EP 90917762A EP 0477301 A1 EP0477301 A1 EP 0477301A1
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EP
European Patent Office
Prior art keywords
leu
ser
pro
lys
asn
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP90917762A
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German (de)
English (en)
Inventor
Anders Vahlne
Bo Svennerholm
Lars Rymo
Peter Horal
Stig Jeannsson
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Maxim Pharmaceuticals Inc
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Syntello AB
Syntello Inc
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Publication of EP0477301A1 publication Critical patent/EP0477301A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to peptides, the sequences of which correspond to antigenic regions of an immunologically important protein of HTLV-1, These peptides are useful as diagnostic reagents for detecting the presence of antibodies to HTLV-1 and may also be useful as immunogens in compositions and methods to elicit antibodies against HTLV-1 in animals and man.
  • the etiologic agent of adult T-cell leukemia/lymphoma (ATL) has been identified as HTLV-1 (human T-cell lymphotropic virus type 1.) See e.g., Sarngadharan et.al., "Human T-cell Leukemia Viruses", in: Virology, B.N.
  • HTLV-1 and Human T cell Lymphotropic Virus Type II are antigenically related members of a family of oncogenic retroviruses sharing a tropism for T lymphocytes and a correlation with lymphoproliferative diseases. Due to the degree of homology between HTLV-2 and HTLV1, serological studies have been unable to differentiate between infection by HTLV-1 and HTLV-2. Unequivocal differentiation of HTLV-1 and HTLV-2 requires virus isolation and/or molecular identification. Although HTLV-2 has not been definitely associated with human disease, intravenous drug abusers (IVDAs) previously thought to be infected with HTLV-1 have now been found to be infected with HTLV-2.
  • IVDAs intravenous drug abusers
  • HTLV-2 infection may be quite common among IVDAs. Tedder et.al., "Low Prevalance in UK of HTLV-1 and HTLV-II Infection in Subjects With AIDS, With Extended Lymphodenopathy, and at Risk of Aids", Lancet, 2:125-128; Robert-Gurott et.al., "Prevalence of Antibodies to HTLV-1, -II and -III in Intravenous Drug Abusers from an AIDS Endemic Region", J.A.M.A., 255:3133-3137 (1986). In view of the association of HTLV-2 with AIDS, virus isolation is particulary difficult and dangerous.
  • An automated blood screening test format capable of readily detecting HTLV-1 and distinguishing HTLV-1 from HTLV-2 infections is also critical to a supply of uninfected blood.
  • Methods for detection of HTLV-1 infection in general, measure exposure to the virus by detecting and quantifying antibodies to HTLV-1 antigens in blood, sera, and bloodderived products. Such assays can be used to aid diagnosis of ATL and TSP and to screen blood and blood products for previous exposure to HTLV-1 thereby preventing further infections.
  • ELISA enzyme-linked immunosorbent assay
  • Other methods may utilize Western blotting techniques to detect HTLV-1 specific antibodies in test samples.
  • immunoassay including but not limited to radioimmunoassays, agglutination tests or indirect immunoflourescence, in addition to ELISA and Western blots, can be adapted, by use of specific reagents, for the detection of HTLV-1 a and antibodies thereto.
  • the source of antigens for these assays may include but are not limited to antigeni ⁇ proteins obtained from HTLV-1 infected T cell lines and antigens produced by recombinant DNA techniques.
  • the use of antigens obtained from these sources has significant drawbacks.
  • HTLV-1 per se in continuous cell lines must be performed in high risk (P3 containment) laboratories due the danger to investigators who may become adversely exposed to the virus. It is also likely that the use of T cell derived HTLV-1 antigens can produce false negative and false positive results in ELISA tests. For example, by analogy, in measuring exposure to the AIDS virus, there have been false negative and false positive results reported with ELISA tests using whole virus HIV-1 antigens obtained from cell lines. Gurtler et.al., "Sensitivity and Specificity of Commercial ELISA kits For Screening Anti-LAV/HTLV-III". J. Virol. Met., 15:11-23 (1987).
  • Exhaustive purification of viral antigens from cell lines can also conceivably destroy immunogenicity of immunologically important proteins or otherwise inactivate antigens, thereby producing reagents that result in false negative reactions.
  • false negative reactions using livevirus-derived antigens may occur because of steric hindrance whereby antibodies cannot react with their specific antigens because the reaction is blocked by the presence of other antigens and antibodies in the reaction mixture.
  • proteins isolated from live virus can be unsuitable for vaccination due to the risk of contamination by whole virus or viral genetic material.
  • ELISA tests to detect HTLV-1 infection may also employ immunologically important viral proteins produced by cloning portions of the HTLV-1 genome in bacteria.
  • HTLV-1 The complete nucleotide sequence of HTLV-1 has been reported by Seiki et.al., "Human Adult T-cell Leukemia Virus: Complete Nucleotide Sequence of the Provirus Genome Integrated in Leukemia Cell DNA", Proc. Nat. Acad. Sci. USA, 80:3618-3622 (1983).
  • the viral envelope glycoproteins and core proteins, respectively encoded by the env and gag genes of HTLV-1 are apparently the antigens recognized by antibodies in the sera of patients with HTLV-1 infection.
  • Immunologically important HTLV-1 antigens which are present in the viral envelope and core, may be prepared by cloning portions of the HTLV-1 genome in various expression systems such as bacteria, yeast or vaccinia. Such recombinant antigens may be used in diagnosis and as potential vaccine compositions as has been done for HIV-1 proteins. See, e.g.
  • HTLV-1 antigens produced by recombinant DNA methods must still be exhaustively purified to avoid false positive reactions in the ELISA due to any antibody reactivity to antigens of the expression system which may contaminate the HTLV-1 antigen preparation. Also, denaturation of HTLV-1 antigens during purification may destroy important antigen activity.
  • recombinant proteins purified from bacteria or yeast are often contaminated with bacterial or yeast proteins. Even minute amounts of these contaminants are capable of causing adverse reactions. While HTLV-1 antigens produced by recombinant techniques may be an improvement over antigens obtained from virus-infected cell cultures, the recombinant proteins still may not provide reagents that give as accurate a diagnosis as possible. Because of the nature of the disease and the need for accurate results, other reagents must be developed to approach 100 % accuracy and specificity in diagnosis of HTLV-1.
  • Proteins contain a number of epitopes or antigenic determinants which are the regions of the proteins which comprise the binding sites for specific antibodies. In general, proteins contain between 5 to 10 epitopes, each of which comprises a sequence of 6 to 8 amino acids. Epitopes can be either continuous, in which case the 6 to 8 amino acids are present in linear sequence, or discontinuous, in which the amino acids that form the epitope are brought together by the three dimensional folding of the protein. Even though an epitope constitutes only a relatively few amino acids, its reactivity with an antibody is influenced by the amino acids in the protein which surround the epitope.
  • Recombinant peptides usually about 100 amino acids long, or more, will fold to produce a tertiary structure mimicking that of the intact protein and allowing discontinuous epitopes to form.
  • linear internal epitopes might be hidden within the molecule and unaccessible to their specific antibodies.
  • synthetic peptides if encompassing major antigenic determinants of a protein, have potential as immunogenic compositions, including vaccines and diagnostic reagents.
  • Peptides have several advantages in specific antibody production and reactivity. The exact sequence of the peptide can be selected from the amino acid sequence as actually determined by amino acid sequencing of a protein or predicted from the DNA sequence coding for the protein.
  • the use of specific synthetic peptides eliminates the need for using the full-length protein in the production of or assay for specific antibodies.
  • the solid phase peptide synthetic techniques of Merrifield and coworkers allow for essentially unlimited quantities of the synthesized peptide of interest to be chemically produced.
  • peptides corresponding to such regions may not always be useful in large-scale screening and diagnosis for example, antigenicity may be lost because the peptide is not in a proper spacial orientation which is recognized by antibodies which react with the protein.
  • HIV-1 and HIV-2 there is significant genetic variability within each of these two virus groups leading to many serotypes of the viruses. This has put a significant constraint on choosing a region of a protein from which to derive a peptide for use in screening and diagnosis and in formulating vaccines.
  • certain immunodominant portions of HIV-1 and HIV-2 proteins have been found to be relatively invariant.
  • the amino acid sequence of SP-71 is: Pro-TyrVal-Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu. Copeland et.al., "Envelope Proteins of Human T-cell Leukemia Virus Type I: Characterization by Antisera to Synthetic Peptides and Identification of Natural Epitope", J. Immunol., 137:2945-2951 (1986), synthesized three additional HTLV-1 peptides which correspond to regions of the protein product encoded by the env gene of HTLV-1.
  • SP-70 which is located near the C terminus of the major surface glycoprotein gp46, had antigenic activity but reacted with only 4/12 sera from HTLV-1 positive patients.
  • Peptide SP-70 is encoded by the nucleotide sequence of the HTLV-1 genome encompassing base pairs 6066-6098 and has the amino acid sequence: Pro-Pro-Phe-Ser-Leu-Ser-Pro-Val-Pro-Thr-Leu-NH 2 .
  • Figure 2 depicts the results of epitope mapping of the HTLV-1 gag gene encoded proteins (gp15, gp19 and gp24).
  • the numbers on the X-axis represent the amino acid number of the first ( amino-terminal) amino acid of the respective hexapeptide.
  • the Y-axis shows the corresponding optical density reading obtained for each peptide with two pooled HTLV-1 positive sera.
  • novel synthetic peptides corresponding to epitopes of HTLV-1 gag and HTLV-1 env proteins are provided. These peptides are useful, alone or in combination, uncoupled or coupled to other molecules, in selective diagnostic methods for detecting HTLV-1 infections, in immunization against HTLV-1 infection and in production of polyclonal and monoclonal antibodies.
  • novel peptides are useful in tests to diagnose HTLV-1 infection or prior exposure to the virus and as immunogens in compositions to elicit the production in animals including man of antibodies against HTLV-1.
  • the peptides encompassed by the invention comprise oligopeptides having amino acid sequences containing therein sequences which comprise continuous (linear) epitopes reactive with HTLV-1 specific antibodies.
  • gag-1 112-130 DSDPQIPPPYVEPTAPQVL 74/81 gag-2 131-150 PVMHPHGAPPNHRPWQMKDL 0/6 gag-3 41-61 YDFHQLKKFLKIAQFDPTAKD 0/6 gag-4 206-226 DSLISEAETRGITGYNPLAGP 0/6 gag-5 265-281 SILQGLEQPYHAFVERL 1/5
  • V 239-261 VLYSPNVSVPSSSSTPLLYPSLA 81/91
  • V1 239-261 VLYSPNVSVPSPSSTPLLYPSLA 4/6 F 55-77 LALSADQALQPPCPNLVSYSSYH 2/17
  • Isoleucine I Valine V The thirty five different peptides were bound to the wells of microtiter plates and screened for reactivity with six sera obtained from HTLV-1 positive patients using an enzyme linked immunoabsorbent assay (EIA). The eleven peptides that gave a positive reaction with one or more of the serum samples were then further tested with an additional 65 HTLV- 1 positive serum samples.
  • the amino acid sequences of the peptides as well as the fraction of HTLV-1 positive sera that reacted with each peptide are shown in Table 1.
  • One peptide from the amino-terminal half of gp46 (O-HTLV-1 amino acids 89 to 110) gave a positive reaction with the HTLV-1 positive sera.
  • the peptides of the present invention derived from the sequence of gp21 yielded four peptides that had a positive reaction with HTLV-1 positive antisera.
  • the present invention has revealed two regions in gp21 containing epitopes immunogenic in man ( Figure 1).
  • One region of the aminoterminal half of gp21 corresponding to amino acids 345-404 was defined by three peptides, AA-HTLV-1 (amino acids 345-367), BB-HTLV-1 (amino acids 363 to 385) and A-HTLV-1 (amino acids 381 to 404).
  • the other region containing an epitope was the carboy-terminus of gp21 as defined by peptides GG-HTLV-1 (amino acids 466 to 488).
  • GG-HTLV-1 amino acids 466 to 488
  • the peptides of Figure 1 was synthetized with an Applied Biosystems 430A peptide synthetizer. The peptides were cleaved from the solid phase and the protective side chains were removed with hydrogen fluoride. The peptides were coupled to bovine serum albumin (BSA) with SPDP according to Pharmacia's method (Pharmacia, Uppsala, Sweden). To the original peptide sequence cystein has been added at the C-terminus in order to facilitate the coupling reaction with SPDP. The peptide BSA complex was then loated onto microtiter plates (NUNC, Denmark). The sera were tested for reactivity against the peptides in 1/50 dilution in indirect ELISA.
  • BSA bovine serum albumin
  • SPDP Pharmacia's method
  • Another series of peptides were synthesized in order to determine the epitopes of HTLV-1 gag gene products. A different screening strategy was used compared to the one described above. Hexamer peptides overlapping one another by five amino acid residues and covering the entire gag gene product were synthesized on polyethylene rods as described by Geysen et.al. (1984) "Use of Peptide Synthesis to Probe Viral Antigens for Epitopes to a Resolution of a Single Amino Acid", Proc. Natl. Acad. Sci. USA, 81:3998-4002, the frame for the peptide synthesis was moved one amino acid C-terminally for each new peptide (a copy was made for each individual peptide 1).
  • the numbers on the x_axis corresponds to the amino acid coordinats for the first (N-terminal) amino acid of the respective hexa peptide.
  • the Y-axis shows the absorbany value for each peptide against the pool of HTLV-1 positive sera.
  • the invention thus encompasses the immunplogically reactive peptides and functionally equivalent variants thereof, which do not significantly affect the antigenic properties of the peptides, corresponding to regions of the envelope glycoprotein encoded by the env gene of HTLV-1.
  • the peptides were synthesized by known solid phase peptide synthesis techniques. See e.g., Merrifield and Barany, The Peptides: Analysis, Synthesis, Biology (1980), vol. 1, Gross and Gonhofer, eds., Academic Press, newYork, Chap. 1.
  • the synthesis also allows for one or two amino acids not corresponding to the original protein sequence to be added to the amino or carboxyl terminus of the peptides. Such extra amino acids are useful for coupling the peptides to each other, to another peptide, to a large carrier protein or to a solid support.
  • Amino acids that are useful for these purposes include but are not limited to tyrosine, lysine, glutamine acid, aspartic acid, cysteine and derivatives thereof. Additional protein modification techniques may be used, e.g., NH 2 -acetylation or COOH-terminal amidation, to provide additional means for coupling the peptides to another protein or peptide molecule or to a support.
  • Analogues are peptides which are functionally equivalent to the present peptides but which contain nonnaturally occuring amino acids.
  • Homologs are peptides which have conservatively substituted amino acids or correspond to peptides encoded by the genome of any other isolate of HTLV-1.
  • Conservative amino acid substitutions include but are not limited to: glycine, alanine; valine, isoleucine, leucine; asparagine, glutamine; aspartic acid, glutamine acid; serine,threonine; lysine, arginine; and phenylalanine, tyrosine.
  • the peptides encompassed by the invention comprise amino acid sequences each containing at least one continuous (linear) epitope reactive with HTLV-1 antibodies.
  • derivatives of the peptides containing less thatn the complete amino acid sequences shown below are embodied by the present invention provided they contain at least one linear epitope recognized by antibodies specific to HTLV-1.
  • the expression, "the peptides" thus includes the amino acid sequences give, analogues, homologs and epitopes derived therefrom.
  • the novel peptides corresponding to the HTLV-1 proteins are set forth below.
  • the numbering of the amino acid residues has been previously described by Seiki et.al., Proc. Natl. Acad. Sci. USA, 80:3618-3622 (1983).
  • the amino acid abbreviations are: Ala, alanine; Arg, arginine; Asn. asparagine; Asp.
  • Cys cysteine; Gin, glutamine; Glu, glutamic acid; Gly, glycine; His, histidine; lie, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Trp, tryptophan; Tyr, Tyrosine; Val, valine.
  • X is either a hydrogen molecule of the amino terminal NH 2 group of the peptide or an additional amino acid bonded to the amino terminal NH 2 group of the peptide, the additional amino acid being selected to facilitate coupling of the peptide to peptides, proteins or other support;
  • Y is absent or a cysteine residue; and
  • Z is a hydroxyl group or amino group.
  • Peptide I-HTLV-1 is derived from the env protein of HTLV-1.
  • Peptide I-HTLV-1 in which X is hydrogen, Y is a cysteine residue and Z is a hydroxyl group is particularly preferred.
  • Peptide AA-HTLV-1 is derived from the env protein of HTLV-1.
  • Peptide AA-HTLV-1 in which X is hydrogen, Y is a cysteine residue and Z is a hydroxyl group is particularly preferred.
  • Peptide BB-HTLV-1 is derived from the env protein of HTLV- 1.
  • Peptide BB-HTLV-1 in which X is hydrogen, Y is a cysteine residue and Z is a hydroxyl group is particularly preferred.
  • Peptide GG-HTLV-1 is derived from the env protein of HTLV-1.
  • Peptide GG-HTLV-1 in which X is hydrogen, Y is a cysteine residue and Z is a hydroxyl group is particularly preferred.
  • Peptide O-HTLV-1 is derived from the env protein of HTLV-1.
  • Peptide O-HTLV-1 in which X is hydrogen, Y is a cysteine residue and Z is a hydroxyl group is particularly preferred.
  • Peptide T-HTLV-1 is derived from the env protein of HTLV-1.
  • Peptide T-HTLV-1 in which X is hydrogen, Y is a cysteine residue and Z is a hydroxyl group is particularly preferred.
  • Peptide V-HTLv-1 is derived from the env protein of HTLV-1.
  • Peptide V-HTLV-1 in which X is hydrogen, Y is a cysteine residue and Z is a hydroxyl group is particularly preferred.
  • Peptide X-HTLV-1 is derived from the env protein of HTLV-1.
  • Peptide X-HTLV-1 in which X is hydrogen, Y is a cysteine residue and Z is a hydroxyl group is particularly preferred.
  • Peptide GAG-l-HTLV-1 is derived from the gag protein of HTLV-1.
  • Peptide GAG-l-HTLV-1 in which X is hydrogen, Y is a cysteine residue and Z is a hydroxyl group is particularly preferred.
  • the peptides can be used alone or in combination in methods for detection of antibodies to HTLV-1 or HTLV-1 associated antigens.
  • Antibodies can be found in biological samples including but not limited to sera, other body fluids, tissue samples and other samples which may contain antibodies to HTLV-1. Particularly H-HTLV-1, 0-HTLV-1 and T-HTLV-1 can be used to distinguish HTLV-1 infection from HTLV-2 infection.
  • the invention is useful for screening blood and blood-derived products with a high degree if reliability and specificity.
  • the peptides are also useful as vaccines to protect against future infection by HTLV-1
  • the invention also provides monoclonal and polyclonal antibodies which specifically recognize the peptides.
  • the methods which use the peptides to detect the presence of HTLV-1 specific antibodies in the sample involve contacting the sample with at least one of the peptides under condi tions which allow the formation of an immunological complex between the peptide and any antibodies to HTLV-1 that may be present in the sample. The formation of an immunological complex, if any, indicating the presence of antibodies to HTLV-1 in the sample, is then detected and measured by suitable means.
  • Such detection methods include but are not limited to homogeneous and heterogeneous binding immunoassays, such as radioimmunoassays (RIA), ELISA and Western blot analyses. Further, the assay protocols using the novel peptides allow for competitive and non-competitive binding studies to be performed. The screening methods are rapid, efficient and allow for simultaneous screening of numerous samples.
  • homogeneous and heterogeneous binding immunoassays such as radioimmunoassays (RIA), ELISA and Western blot analyses.
  • the peptides may be labeled (signal-generating) or unlabeled depending on the type of assay used.
  • Labels which may be coupled to the peptides are those known is the art and include but are not limited to enzymes, radionuclides, fluorogenic and chromogenic substrates, cofactors, biotin/avidin, colloidal gold, and magnetic particles.
  • the peptides can be coupled by any means known in the art to other peptides, solid supports and carrier proteins.
  • Such solid supports include but are not limited to polystyrene or polyvinyl microtiter plates, glass tubes or glass beads and chromatographic supports, such as paper, cellulose and cellulose derivates, and silica.
  • Carrier proteins include but are not limited to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH).
  • Preferred assay techniques especially for largescale clinical screening of patient sera and blood and blood-derived products are ELISA, agglutination and Western blot techniques, ELISA tests being particularly preferred for speed, the ability to test numerous samples simultaneously and ease of automation.
  • the ELISA tests employing the peptides desc ribed above are based on those currently in use for detecting other viruses.
  • the peptides of the invention are conveniently bonded to the inside surface of microtiter wells.
  • the peptides may be directly bonded by hydrophobic interactions to the microtiter well, or attached covalently by means known in the art to a carrier protein, such as BSA, with the resulting conjugate being used to coat the wells.
  • the peptides were used in a concentration of approximately between 1 to 100 ⁇ /ml although this range is not limiting, for instance as much as 500 ⁇ /ml of a peptide may be required for the assay to be successful. Generally the peptides are used in concentration of between 10 to 100 ⁇ /ml for coating.
  • Samples including but not limited to body fluids and tissue samples, are then added to the peptide coated wells, where an immunological complex forms if antibodies to HTLV-1 are present in the sample.
  • a signal generating means may be added to aid detection of complex formation.
  • a detectable signal is produced if HTLV-1 specific antibodies are present in the sample.
  • Agglutination assays are commonly used in Japan. Either latex or erythrocytes can be used in the technique. The methods used in agglutination assays are well known in the art of blood screens.
  • the peptides of the invention may also be formulated into compositions for use as immunogens. These immunogenes can be used as or to elicit production of antibodies in animals and man against HTLV-1.
  • the antibodies produced can be either polyclonal or monoclonal.
  • an immunogenically effective amount of at least one of the peptides is admixed with a physiologically acceptable carrier suitable for administration to animals including man.
  • the peptides may be covalently attached to each other, to other peptides, to a protein carrier or to other carriers, incorporated into liposomes or other such vesicles, or complexed with an adjuvant or adsorbent as is known in the vaccine art.
  • the peptides are not complexed with the above and merely admixed with a physiologically acceptable carrier such as normal saline or a buffering compound suitable for administration to animals including man.
  • a physiologically acceptable carrier such as normal saline or a buffering compound suitable for administration to animals including man.
  • the immunogenically effective amounts of the peptides of the invention must be determined empirically. Factors to be considered include the immunogenicity of the native peptide, wether or not the peptide will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier and route of administration for the composition, i.e. intravenous, intramuscular, subcutaneous, etc., and number of immunizing does to be administered. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.
  • the invention also encompasses antibodies made in response to the peptide and which recognize the peptides.
  • Such antibodies can be either polyclonal or monoclonal. Methods for making antibodies are well known in the art.
  • the protecting groups were removed from the synthesized peptide and the peptide was cleaved from the solid support resin by treatment at 0°C with anhydrous hydroflouric acid (HF) combining 10 % anisole and 10 % dimethylsulfide as scavenging agents. After cleavage, the HF in the sample was purged under a stream of N 2 , with removal of any residual HF accomplished by subjecting the sample to vacuum at 0°C. The peptides were extracted from the resin by treatment with trifluouroacetic acid (TFA) which was then removed by evaporation at room temperature. Following TFA removal, the peptides were precipitated and washed with anhydrous ether.
  • TFA trifluouroacetic acid
  • the peptides Prior to use in specific assays, the peptides can be further purified, if desired, by reverse phase high performance liquid chromatography (HPLC).
  • HPLC reverse phase high performance liquid chromatography
  • a particularly suited column for such purification is the reverse-phase Vydak C-18 column using a water (TFA) - acetonitrile (TFA) gradient to elute the peptides.
  • Table 2 A particularly suited column for such purification is the reverse-phase Vydak C-18 column using a water (TFA) - acetonitrile (TFA) gradient to elute the peptides.
  • N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, Pharmacia) is then dissolved in ethanol to a final concentration of 5-40mM.
  • concentration of SPDP is determined by measuring the reactive ester according to the Pharmacia Fine Chemicals SPDP brochure.
  • 2-pyridyldisulfide residues are introduced into the BSA prepared as above by adding ten SPDP equivalents to each BSA equivalent.
  • the SPDP solution is introduced to the BSA solution with stirring. The mixture is then incubated for 15-30 minutes at room temperature.
  • the pyridyldisulfide-BSA mixture is aliquoted into 6 equal volumes each of which is applied to a PD-10 column.
  • the columns are equilibrated and the product is eluted with 10 % acetic acid in water.
  • the degree of substitution is measured according to the Pharmacia Fine Chemicals SPDP brochure.
  • the recovery of BSA is generally 90-120 mg and the degree of substitution is approximately 7 with a range of 6-8.
  • the peptide solution is made by mixing 25 mg peptide and an amount of the pyridyldisulfide-BSA solution to make seven peptide equivalents to one BSA equivalent. The mixture is incubated for 18-48 hours at room temperature. The released 2-thiopyridone is removed by running the reaction mixture over a column (2.0cm 2 , 80-100 ml) packed with Sephadex G-25 (Pharmacia-LKB), equilibrated with 10 % acetic acid in water. The product is eluted with 10 % acetic acid in water. The fractions with A 280 greater than 0.5 are then collected and pooled. The pooled volume is approximately 30-40 ml. The A 280 of the pooled fractions is measured and the BSA concentration is calculated. The pooled fractions are stored at 4° C and are stable for months.
  • the peptide-BSA conjugate is then diluted in coating buffer (50mM NaCo 3 , 0.15M Nacl, pH 9.5) to 60 mg/ml.
  • coating buffer 50mM NaCo 3 , 0.15M Nacl, pH 9.5
  • the pH of the solution is checked and adjusted with 1-5M NaOH, to 9.5.
  • the liquid is aspirated from the wells.
  • the microtiter plates are placed in a safety cabinet and air dried for about three hours.
  • the plates can be stored for a long time at +4° C or -20° C in any closed container, for instance on sealed aluminium bags.
  • a drying agent such as silica gel aids in preservation of the plates.
  • the peptides were used in an ELISA test to measure their immunologic reactivity. All peptides were run in parallel ELISA tests against serum samples positive for antibodies to HTLV-2, serum samples positive for antibodies to HTLV-1 and 10 blood donor sera negative for HTLV-1/HTLV-2. The sera were also tested against HTLV-1 peptides which have been previously described in PCT patent publication, W089-08664.
  • microtiter plates are prepared as in Example 2. If the plates have been stored they may first be brought to room temperature and then they may be pre-soaked for ten minutes in wash buffer (0.05 % Tween 20 in PBS). The presoak solution is then aspirated from the wells prior to use.
  • wash buffer 0.05 % Tween 20 in PBS
  • the serum samples are each diluted 1:50 in serum dilution solution (1 % BSA in wash buffer). An aliquot of 100 ⁇ l diluted serum is placed in each well and the plates are incubated for 90 minutes at 37° C in a humidifier. After the incubation, the plates are washed three times with wash buffer.
  • Anti-human immunoglobulin G (IgG) conjugate (Jackson, from Labassco, art. nr. 10.4999999, 109-056-003, Alkaline Phosphatase) is dissolved in 0.5 ml H 2 O, aliquoted and frozen.
  • Frozen aliquots are thawed and diluted 1 : 5000 in serum dilution buffer. An aliquot of 100 ⁇ l is added to each well.
  • the plates are incubated for 90 minutes at 37° C in a humidified chamber. After the incubation, the plates are washed three times with wash buffer. Alkaline phosphatase substrate (Sigma, tablets) is dissolved in substrate dilution buffer (50mM Na 2 CO 3 , ImM MgCl 2 ) to a final concentration of 1 mg/ml. An aliquot of 200 ⁇ l is added to each well. The plates are incubated for approximately 35 minutes at room temperature. If desired the reaction can be stopped by the addition of 100 ⁇ l 3M NaOH per well.
  • substrate dilution buffer 50mM Na 2 CO 3 , ImM MgCl 2
  • the plates are read at 405 nm. The higher the absorbance, the greater the amount of bound antibody.
  • HTLV-2 Peptides derived from HTLV-2, homologous to A-HTLV-1; AA-HTLV-1, B-HTLV-1, C-HTLV-1, HH-HTLV-1, V-HTLV-1, and X-HTLV- 1 were made by the method described in Example 1.
  • the HTLV- 2 peptides are described in U.S. Patent Application Serial No. 07/434,239, filed Nov. 13, 1989.
  • the HTLV-2 amino acid sequences were derived from the nucleotide sequence described by Shimitohno (1985) "Complete Nucleotide Sequence of an Infectious Clone of Human T cell Leukemia Virus Type II: An Open Reading Frame for the Protease", Proc. Nat'l. Acad. Sci. U.S.A.
  • the serum samples used in this screening test were confirmed HTLV-2 positive by PCR analysis.
  • the sera designated HT-201- HT-220 were obtained from Serologicals Inc., Pensacola, Fla. Previous researchers were unable to distinguish between antibodies specific for either HTLV-1 or HTLV-2 in these sera by any of Western blot analyses, ELISA tests and immu nofluorescense assays.
  • Table 3 shows the results obtained by the ELISA test.
  • the negative controls are sera negative for both HTLV-1 and HTLV-2 and are designated NC-1 and NC-2, the HTLV-1 positive serum is designated HTLV-1.
  • the patient sera HT-201-HT-220 are designated 201-220.
  • the results presented are absorbance readings at 405nm.
  • the results presented in Table 3 clearly show that H-HTLV-2, O-HTLV-2, T-HTLV-2 and Gag-1-HTLV-2 react strongly with antibodies present in HTLV-2 infected patient sera. All of the peptides react poorly with the sera designated HT-218 and HT-219. These sera were found to be only weakly positive with previous ELISA tests indicating that the level of HTLV-2 specific antibodies in these sera was low.
  • the HTLV-2 peptides react well with HTLV-2 infected patient sera and poorly with antibodies present in HTLV-1 infected patient sera.
  • the Gag-1-HTLV-2 peptide is not as specific as the other three peptides.
  • HTLV-1 peptides were compared to H-HTLV-2, O-HTLV-2, and T-HTLV-2, by testing against both HTLV-1 and HTLV-2 positive sera.
  • the patient sera were obtained from Dr. William Hall, Cornell University, N.Y. Table 4 shows the results obtained.
  • the sera designated 2a, 2b, 2c, 2d, 2e and 2f were obtained from five different patients and were HTLV-2 positive as determined by polymerase chain reaction (PCR) analysis and were also human immunodeficiency virus (HIV) positive.
  • PCR polymerase chain reaction
  • the sera designated 1a, 1b, 1c, 1d, 1e and 1f were obtained from five different patients and were HTLV-1 positive as determined by PCR analysis. Patients 1a, 1b and 1c have adult T cell leukemia and patients 1d, 1e and 1f are HIV positive. Sera designated HIV-1 were obtained from patients who are neither HTLV-1 nor HTLV-2 positive but are HIV positive. Sera designated NL-1 and NL-2 are negative controls obtained from patients not infected with either HTLV-1 or HTLV-2. The numbers shown in Table 4 are the average of two experiments and are the absorbance readings at 405nm.
  • novel synthetic peptides which correspond to regions of proteins encoded by the env and gag genes of HTLV-1, clearly provide unique reagents for a sensitive assays for the presence of antibodies to HTLV-1.
  • peptides H-HTLV-2 clearly discriminate between antibodies which recognize HTLV-1 and antibodies which recognize HTLV-2. It may in some cases be appropriate to use at least two peptides in assays, since two peptides provide a higher frequency of sero-positivity or sensitivity as compared to one peptide. This also menas that the frequency of false negative reactions decreases.

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Abstract

L'invention concerne des peptides correspondant aux régions de protéines codées par le génome du HTLV-1 (virus lymphotropique de type 1 des cellules T humaines). Ces peptides réagissent immunologiquement avec des anticorps spécifiques du HTLV-1. Certains de ces peptides sont capables de faire une distinction entre une infection provoquée par le HTLV-1 et une infection provoquée par le HTLV-2. Les peptides sont utiles dans des analyses de détection d'une infection provoquée par le HTLV-1 ou d'une exposition à ce dernier, ainsi que dans des compositions destinées à mettre en lumière la production d'anticorps contre le HTLV-1 chez l'animal et chez l'homme. L'invention concerne également les anticorps mis en lumière en réaction aux anticorps.
EP90917762A 1989-06-13 1990-06-12 Peptides et anticoprs derives de diagnostic et de therapie d'une infection provoquee par le htlv-1, et de vaccination contre celui-ci Withdrawn EP0477301A1 (fr)

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SE8902148 1989-06-13
SE8902148A SE467542B (sv) 1989-06-13 1989-06-13 Syntetiska peptidantigener, immuniserande komposition och immunanalys foer htlv-1 antikroppar

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US5643714A (en) * 1986-12-31 1997-07-01 Genelabs Technologies, Inc. Method and assay for HTLV
JP2807287B2 (ja) * 1989-10-13 1998-10-08 株式会社医学生物学研究所 ペプチドおよびその用途
FI910245L (fi) * 1990-01-24 1991-07-25 United Biomedical Inc Syntetiska peptidkompositioner med immunoreaktivitet mot htlv-antikroppar.
AU641554B2 (en) * 1990-08-29 1993-09-23 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Novel peptide antigens and immunoassays, test kits and vaccines using the same
US5849475A (en) * 1990-10-12 1998-12-15 Benjamin Rovinski et al Immunoassay diagnostic kit containing antigens derived from self-assembled, non-infectious, non-replicating, immunogenic retrovirus-like particles comprising modified HIV genomes and chimeric envelope glycoproteins
US5773211A (en) * 1991-07-10 1998-06-30 Abbott Laboratories Differentiation of HTLV-I and HTLV-II using synthetic peptides
JP3421336B2 (ja) * 1991-07-10 2003-06-30 アボツト・ラボラトリーズ 合成ペプチドを用いてのｈｔｌｖ−▲ｉ▼とｈｔｌｖ−▲ｉｉ▼との区別
CA2117378C (fr) * 1992-02-24 2003-04-15 Steven K. H. Foung Dosage et methode de detection de htlv-i/htlv-ii
US6406841B1 (en) 1993-07-01 2002-06-18 Abbott Laboratories Methods for the detection of HTLV-II antibodies employing novel HTLV-II NRA envelope peptides
JPH09510009A (ja) * 1993-12-20 1997-10-07 アボツト・ラボラトリーズ 合成ペプチドを用いるｈｔｌｖ−ｉおよびｈｔｌｖ−ｉｉの識別
FR2844514B1 (fr) * 2002-09-16 2007-10-19 Neovacs Produit immunogene stable comprenant des heterocomplexes antigeniques, compositions les contenant et procede de preparation
JP2015215244A (ja) * 2014-05-12 2015-12-03 富士レビオ株式会社 免疫測定における抗ｈｔｌｖ抗体の検出感度を向上させる方法

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US4572800A (en) * 1982-09-30 1986-02-25 Otsuka Pharmaceutical Co., Ltd. Human leukemia virus-related peptides, a process for production thereof, antibodies of the peptides and a process for production of the antibodies
US4525300A (en) * 1983-01-07 1985-06-25 Japanese Foundation For Cancer Research Human leukemia virus-related peptides, antibodies of the peptides and a process for production of the antibodies
JPS6028993A (ja) * 1983-07-22 1985-02-14 Otsuka Pharmaceut Co Ltd ヒト白血病ウイルス関連ペプチド
US4689398A (en) * 1984-06-20 1987-08-25 Ortho Diagnostic Systems Inc. HTLV test using synthetic peptides
US4833071A (en) * 1987-01-09 1989-05-23 United Biomedical, Inc. Peptide composition as anitgen for detection of antibodies to HTLV-I, as a vaccine for ATL and methods therefor
WO1989006543A1 (fr) * 1988-01-12 1989-07-27 Genelabs Incorporated Antigene peptidique de l'htlv-i et procede d'analyse utilisant un tel antigene
JPH02209889A (ja) * 1988-02-08 1990-08-21 Univ Duke 合成親水性ペプチド
US5017687A (en) * 1988-03-10 1991-05-21 Virovahl, S.A. Peptides for the detection of HTLV-1 infection
JPH0628993A (ja) * 1992-07-10 1994-02-04 Fujitsu Ltd 電子ビーム装置

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AU638727B2 (en) 1993-07-08
SE467542B (sv) 1992-08-03
AU5945390A (en) 1991-01-08
HUT61323A (en) 1992-12-28
JPH04506077A (ja) 1992-10-22
SE8902148D0 (sv) 1989-06-13
WO1990015820A1 (fr) 1990-12-27
HU906639D0 (en) 1992-02-28
KR920702688A (ko) 1992-10-06
AU4890693A (en) 1993-12-16
AU654240B2 (en) 1994-10-27
SE8902148L (sv) 1990-12-14
JP2650217B2 (ja) 1997-09-03
FI915702A7 (fi) 1991-12-03
KR0151860B1 (ko) 1998-10-01
NZ234049A (en) 1993-02-25
ZA904565B (en) 1991-03-27
FI915702A0 (fi) 1991-12-03
CA2062713A1 (fr) 1990-12-14
EP0814090A1 (fr) 1997-12-29

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