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WO1999062945A2 - Peptides universels - Google Patents

Peptides universels Download PDF

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Publication number
WO1999062945A2
WO1999062945A2 PCT/US1999/012446 US9912446W WO9962945A2 WO 1999062945 A2 WO1999062945 A2 WO 1999062945A2 US 9912446 W US9912446 W US 9912446W WO 9962945 A2 WO9962945 A2 WO 9962945A2
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WO
WIPO (PCT)
Prior art keywords
seq
peptide
group
amino acid
sequence
Prior art date
Application number
PCT/US1999/012446
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English (en)
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WO1999062945A9 (fr
WO1999062945A3 (fr
Inventor
Mohammed Afzal Chowdhury
David Bernstein
Marvin A. Motsenbocker
Original Assignee
Peptide Solutions, Inc.
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.)
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Publication date
Priority claimed from PCT/US1999/001726 external-priority patent/WO1999038887A1/fr
Application filed by Peptide Solutions, Inc. filed Critical Peptide Solutions, Inc.
Priority to AU45463/99A priority Critical patent/AU4546399A/en
Publication of WO1999062945A2 publication Critical patent/WO1999062945A2/fr
Publication of WO1999062945A9 publication Critical patent/WO1999062945A9/fr
Publication of WO1999062945A3 publication Critical patent/WO1999062945A3/fr

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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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates in part, to diagnostic tests and antigens used in such tests.
  • Embodiments of the invention relate to peptide sequences and sequence modifications of such peptides that cross react immunologically with a wide range of antigens such as from HIV-1 Group O virus strains.
  • HIV Human Immunodeficiency Virus
  • HTLV-ffl Human Immunodeficiency Virus
  • AIDS Acquired Immunodeficiency Syndrome
  • Infection by this virus results in the appearance of antibodies in the blood that react against various molecular parts of the virus, particularly the envelope proteins gp41 and gpl20.
  • the antibody-based binding reaction between antibodies from an infected patient and a viral antigen(s) is used in various methods for detection of HIV infection, such as latex agglutination and ELISA.
  • retro viruses HIV has received the most attention recently because of the widespread damage caused by this virus.
  • the first tests developed to detect HIV infection contained whole viral lysates for reaction with antibodies from a blood sample. Occasionally, however, these tests yield false results due to nonspecific binding reactions with one or more antibody binding sites, i.e. , "epitopes" of the various proteins found in a lysate. Consequently, all positive results from these tests must be confirmed by further testing with another method such as Western Blot assay.
  • gp41 immunodominant region is known to be important due to its key role in presenting epitopes during HIV infection (Gnann et al , J. Infect. Dis., 156:261-267 1987 and /. Virol, 61:2639-2641 1987). Peptides that share sequence similarity with this region should be cross-reactive with HIV.
  • the immunodominant region of gp41 comprises a major heptapeptide loop epitope that has been well studied (Wang et al, Proc. Natl Acad. Sci.
  • RNA virion such as an HIV virion
  • RNA replication has a very short generation time and forms many copies of genetic material within a cell by RNA replication.
  • RNA polymerases replicases
  • the virus yield of a single cell generally is a population of genomes, each with one or more (usually subtle or silent) changes from an average sequence.
  • the polypeptide sequence of a virus was isolated twice from an individual over a 3 month interval and was shown to have had radically changed its amino acid sequence in an important (normally) conserved region of envelope protein that is known to be highly immunogenic. Eberle et al. , J. Vir. Meth. 67: 85,88 (1997). This kind of rapid and drastic change in the immunogenic portion of the envelope protein is a major detection and therapy problem. The HIV antigen diversity problem is seen at different levels of viral classification.
  • HIV-1 has been subdivided into several types (from A to I) with a more distinct Group O.
  • Group O exhibits 55-70% homology with the other HIV-1 Groups, and is regarded by some researchers as a new group. Accordingly, and to reflect the close relation of types A-I, which were first found, these types are grouped together as Group M (for major).
  • the 25 amino acid long portion of variant CM.4974-95 differs from variant MVP5180 by a single conservative substitution of arginine for lysine.
  • peptide antigens that react with HIV-1, HIV-1 Group O and HIV-2, when present as a mixture, can react with most sera of patients infected with HIV.
  • Drawbacks of this strategy include, among other things, a higher degree of non-specific binding reactions due to a greater variety of proteins used and a limitation in the amount of total antigen that can be added to a test that typically has a small solid phase surface or reaction volume. If a single peptide having broad reactivity could be used in place of two or more peptides, significant improvements to assay quality thus can be achieved. Reasons for this include, inter alia, a greater amount of peptide can be dissolved and used in an assay, and less unnecessary peptide sequence (which causes non-specific binding) is exposed to the sample.
  • One embodiment of the invention is a peptide useful for detecting HIV-1 Group O infection, having a length of between 16 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 1, wherein XI is selected from the group consisting of N, Q, G, S, T, and A; X2 is R or K; X3 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M; and X4 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M, and wherein at least one of the two X4 residues is selected from the group consisting of N, Q, G, S, T, H, A, P and M.
  • Another embodiment of the invention is a peptide useful for detecting HIV-1 Group O infection, having a length between 26 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 2, wherein X is a helix of at least 5 amino acids, XI is selected from the group consisting of N, Q, G, S, T, D, N, H and A, X2 is R, K, P or E, X3 is selected from the group consisting of I, L, and V, X4 is selected from the group consisting of T, S and A, and X5 is at least one amino acid long.
  • Another embodiment of the invention is a peptide useful for detecting HIV-1 Group O infection, having a length between 36 and 100 amino acid residues, and comprising a core sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 and derivatives thereof that contain one or more conservative amino acid substitutions
  • Another embodiment of the invention is a peptide between 26 and 100 amino acids long that substantially reacts with Group O HIV-1 test specimens and that comprises a 17 amino acid long immunodominant region, the region having a sequence shown by SEQ ID NO: 8.
  • Another embodiment of the invention is a peptide useful for detecting HIV-1 Group
  • O infection having a length of between 16 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 1, wherein XI is selected from the group consisting of N, Q, G, S, T, and A; X2 is selected from the group consisting of R, K, T and N; X3 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M; and X4 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M, and wherein at least one of the two X4 residues is selected from the group consisting of N, Q, G, S, T, H, A, P and M.
  • Another embodiment of the invention is a peptide useful for detecting HIV-1 Group O infection, having a length of between 17 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 8, wherein X2 is selected from the group consisting of R, K, T and N; X5 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M; and wherein at least one of the two X5 residues is selected from the group consisting of N, Q, G, S, T, H, A, P and M.
  • Yet another embodiment of the invention is a peptide useful for detecting HIV-1 Group O infection, having a length of between 17 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 17, wherein X4 is selected from the group consisting of R, K, N, E and A; X5 is selected from the group consisting of G, N, E and D; X6 is R or K; X7 is selected from the group consisting of N, Q, L, I, V and P; X8 is selected from the group consisting of N, Q, L, I and V; X12 is selected from the group consisting of S, T and A; X13 is selected from the group consisting of I, L, V and A; X14 is selected from the group consisting of K, R and E; X15 is W or T; X16 is N or H; and X17 is R or K.
  • X4 is selected from the group consisting of R, K, N, E and A
  • X5 is selected from the group consist
  • Yet another embodiment of the invention is a peptide useful for detecting HIV-1 infection, comprising a sequence of at least 35 amino acids from a gp41 immunodominant region, wherein the peptide sequence comprises at least 5 amino acids in an alpha helix structure on the amino terminal side of the immunodominant region, and wherein the alpha helix structure is determined by Chou-Fasman Conformational parameters from a peptide analysis computer program.
  • Yet a further embodiment of the invention is a reagent for immunological detection of anti-HIV antibody in a blood sample, comprising a dried antigen that, upon rewetting with water or a clinical sample, substantially reacts with antibodies from patients exposed to HIV- 1 group M virus and with antibodies from patients exposed to HIV-1 Group O virus, wherein the antigen is between 16-50 amino acids long and possesses a sequence described herein.
  • Yet another embodiment of the invention is a method of detecting HIV-1 Group O infection, comprising incubating a blood sample or blood derivative with a peptide described herein, followed by determination of binding between antibody in the blood sample or blood derivative and the peptide.
  • kits for determining infection with HIV-1 Group O comprising, an instruction booklet and a device for detecting the presence of anti- HIV- 1 Group O antibody in a blood sample or blood derivative, wherein the device comprises a peptide described herein.
  • Another embodiment of the invention is a method of improving reactivity of an antigenic peptide for a diagnostic test of an infectious agent, comprising replacing a hydrophobic amino acid in a known immunoactive portion of the peptide with a hydrophilic amino acid.
  • Yet another embodiment of the invention is an improved diagnostic test peptide antigen for the detection of an infectious agent, wherein the peptide has a sequence that corresponds to a naturally occurring sequence and wherein at least one hydrophobic amino acid residue from the naturally occurring sequence is replaced by a hydrophilic amino acid residue
  • Yet another embodiment of the invention is an improved diagnostic test peptide antigen for the detection of an infectious agent, wherein the peptide has a sequence that corresponds to a naturally occurring sequence and wherein two hydrophobic amino acid residues from the naturally occurring sequence are replaced by hydrophilic amino acid residues.
  • Yet another embodiment of the invention is an improved diagnostic test peptide antigen for the detection of an infectious agent, wherein the peptide has a sequence that corresponds to a naturally occurring sequence and wherein three hydrophobic amino acid residues from the naturally occurring sequence are replaced by hydrophilic amino acid residues.
  • Yet another embodiment of the invention is an improved diagnostic test peptide antigen for the detection of an infectious agent, wherein the peptide has a sequence that corresponds to a naturally occurring sequence and wherein four hydrophobic amino acid residues from the naturally occurring sequence are replaced by hydrophilic amino acid residues.
  • a further embodiment of the invention is a peptide between 26 and 100 amino acids long having a central portion of at least 16 amino acids that corresponds in sequence identity to an immunodominant region of an antigen protein, and at least one amino acid at each end of the central portion, wherein the peptide is chemically synthesized and at least one hydrophobic amino acid of the immunodominant region has been replaced with a hydrophilic amino acid.
  • Another embodiment of the invention is an improved immunogenic therapeutic peptide for the treatment or prevention of infection by an infectious agent, the improvement comprising replacing at least a L, I, or V of a naturally occurring sequence of the peptide with an amino acid selected from the group consisting of A, S, T, G and N.
  • Another embodiment of the invention is a peptide antigen that comprises an amino acid sequence selected from the sequences listed in Figure 2.
  • Figure 1 shows allowable amino acid substitutions in an antigenic sequence of gp41 in accordance with one embodiment of the claimed invention.
  • Figure 2 shows representative advantageous sequences for HIV-1 peptide antigens, (SEQ ID NOs: 1-26) and for hepatitis C peptide antigens (SEQ ID NOs: 28-30, 32-229) in accordance with the claimed invention.
  • SEQ ID Nos. 7 and 27 are naturally occurring sequences of proteins that correspond to HIV-1 and hepatitis C respectively.
  • Figure 3 shows data obtained from testing HIV-1 infected blood samples with known antigen sequences and with sequences according to embodiments of the claimed invention, as described in Example 1.
  • Figure 4 shows data obtained from testing HIV negative, HIV-1 positive, HIV-1 O group positive, and HIV-2 positive samples with an antigen having a native sequence from HIV-1 (MVP5180) and with an antigen (SEQ ID NO: 5) that differs from this native sequence by three amino acid substitutions.
  • Figure 5 is a representative set of peptide Chou Fasman conformational parameters for a peptide obtained by software in accordance with one embodiment of the claimed invention.
  • a fourth strategy is to constrain the structure of an epitopic sequence by forming a covalent crosslink (such as a cystine bridge) within the peptide.
  • Embodiments of the claimed invention provide peptide antigens for diagnostic testing and therapy of disease having sequences that differ from naturally occurring peptide sequences. These peptides immunologically cross-react with a wide variety of mutated forms of antigens and are particularly useful for testing and treatment of retroviral disease such as HIV or HCV infection, where rapid mutation of the disease vector is a concern.
  • the peptides are from about 16 (e.g. 16) to about 100 (e.g. 100) amino acid residues long and preferably from 25 to 50 amino acids long.
  • the antigens also may possess one or more other advantageous characteristics such as improved water solubility or improved immunological reactivity compared to antigens having epitopic sequences from naturally occurring strains such as (in the case of HIV-1) ANT70 and MVP5180.
  • Preferred peptides that have been modified according to principles of the claimed invention differ greatly from the naturally occurring forms and are not identified as belonging to any particular viral Group or strain.
  • many new transgenic therapies transgenically express a protein that binds specifically to (one or more) binding partners such as other proteins or peptide hormones.
  • a protein having suitable binding characteristics By expressing a protein having suitable binding characteristics, the desired binding reactions are favored.
  • an expressed protein such as a therapeutic transgenic protein
  • the methods of the invention are particularly useful for these applications because they alleviate problems that occur when a shorter intermediate sized peptide is prepared from a larger protein. That is, methods of the invention can stabilize a peptide structure, allowing the peptide to remain more soluble and even to bind more advantageously to its intended target.
  • intermediate peptides obtained from a larger binding protein are improved by alterations as described below.
  • the inventive methods can enhance the biological and/or immunological reactivity of a peptide by improving its solubility and/or by increasing the association (binding constant) between the peptide and an intended binding partner.
  • the inventors have obtained data that verifies the use of certain embodiments for improving binding between certain peptides and antibodies made against various strains of HIV. However, binding between peptides and other molecules can be improved as well by practice of the methods.
  • peptides include, for example, portions (or complete sequences) that correspond to the following peptides.
  • Lymphokines and Interferons IL-1, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFN- alpha , IFN- beta , IFN- gamma.
  • Hormones and Growth Factors nerve growth factor, somatotropin, somatomedins, parathormone, FSH, LH, EGF, TSH, THS-releasing factor, HGH, GRHR, PDGF, IGF-I, IGF-II, TGF- beta , GM-CSF, M-CSF, G-CSF, erythropoetin.
  • Tumor Markers and Tumor Suppressors beta -HCG, 4-N- acetylgalactosaminyltransferase, GM2, GD2, GD3, MAGE-1, MAGE-2, MAGE-3, MUC- 1, MUC-2, MUC-3, MUC-4, MUC-18, ICAM-1, C-CAM, V-CAM, ELAM, NM23, EGFR, E-cadherin, N-CAM, CEA, DCC, PSA, Her2-neu, UTAA, melanoma antigen p75, K19, HKer 8, pMel 17, tyrosinase related proteins 1 and 2, p97, ⁇ 53, RB, APC, DCC, NF-1, NF-2, WT-1 , MEN-I, MEN-II, BRCA1 , VHL, FCC and MCC.
  • Complement Cascade Proteins and Receptors Clq, Clr, Cls, C4, C2, Factor D, Factor B, properdin, C3, C5, C6, C7, C8, C9, Cllnh, Factor H, C4b-binding protein, DAF, membrane cofactor protein, anaphylatoxin inactivator S protein, HRF, MIRL, CR1, CR2, CR3, CR4, C3a/C4a receptor, C5a receptor.
  • HIV gag, pol, gpl20, vif, tat, rev, nef, vpr, vpu, vpx
  • HSV ribonucleotide reductase, alpha -TIF, ICP4, ICP8, ICP35, LAT-related proteins, gB, gC, gD, gE, gH, gl, gJ
  • influenza hemagluttinin, neuraminidase, PB1, PB2, PA, NP, Ml, M2, NS1, NS2), papillomaviruses (El, E2, E3, E4, E5a, E5b, E6, E7, E8, LI, L2) adenovirus (E1A, E1B, E2, E3, E4, E5, LI, L2, L3, L4, L5), Epstein-Barr Virus (EBNA), Hepatitis B Virus (gp27, gp
  • a desired epitope (or binding site) of the protein or peptide is selected and then altered and/or combined with another sequence(s) to produce a new peptide according to four strategies outlined below.
  • hydrophobic to hydrophilic amino acid residue shift can be that the new peptide may have greater solubility in water.
  • An increase in water solubility can lead directly to improved diagnostic assay or vaccine performance by allowing a greater amount of peptide to be used.
  • This attribute also facilitates the use of more than one peptide together in the same solution without causing a precipitate at higher concentrations of one or more of the peptides.
  • a peptide antigen according to the invention is greater than 16 amino acid residues long but smaller than 100 amino acid residues long. This size range is termed "intermediate size.”
  • the upper size limit reflects the fact that an intermediate size peptide according to the invention is shorter than most proteins, which have tertiary structure due to folding of the peptide sequence. In a protein, the polypeptide chain folds upon itself (forms tertiary structure) to, among other things, allow mutual association of hydrophobic residues in order to maximize entropy of a water solution that contains the polypeptide.
  • Intermediate sized peptides in accordance with the invention on the other hand, generally are smaller, generally fold less and have less tertiary structure than an intact protein but have secondary structure.
  • Their minimum size limit of 16 amino acids reflects the fact that peptides smaller than 16 residues long generally have little structure outside the primary structure of amino acid sequence and are less improved by making an alteration according to the claimed embodiment.
  • intermediate sized peptides were synthesized having additional substitutions of hydrophilic amino acid residues for hydrophobic residues. These peptides have sequences that correspond to (i.e., at least half of the amino acids correspond in identity with) naturally-occurring sequences.
  • the synthesized peptides showed greater specificity for HIV-1 O Group specimens compared to peptides that have sequences that are identical to sequences from naturally occurring proteins. That is, the peptide sequences of the claimed invention exhibit different immunological characteristics than the corresponding sequences of naturally occurring proteins. The different characteristics can include a loss of one or more immunological properties, exemplified by the loss of HIV-2 reactivity for peptides obtained from a naturally-occurring HIV-1 envelope protein sequence.
  • the inventors theorize that altering a hydrophobic amino acid such as leucine, valine and isoleucine etc. to a hydrophilic amino acid such as glutamine, asparagine, serine, threonine etc., particularly in an immunodominant (or biologically active) region of a protein, helps prevent structural instability when present in an intermediate sized (16-100 residue-long) peptide that lacks complex protein (i.e. tertiary structure).
  • the inventors theorize that hydrophobic amino acid residues in a large protein come together to form an interior oily pocket that excludes water and stabilize the structure of the complete large protein.
  • a peptide antigen less than about 100 amino acids e.g. less than 100 amino acids
  • particularly less than about 75 amino acids e.g. less than 75 amino acids
  • more particularly less than about 50 amino acids e.g. less than 50 amino acids
  • individual hydrophobic residues no longer can avoid water by optimally coming together and instead randomly are exposed to water and increase disorder of the peptide in water.
  • the disorder contributes to less stable and unrecognizable epitopic structures which react less well or react less specifically with antibodies directed against the native undenatured protein, which is more ordered.
  • the increased disorder is alleviated by decreasing the hydrophobic character of the hydrophobic residue, preferably by substituting the amino acid with a more hydrophilic residue.
  • it is particularly advantageous to alter leucine or isoleucine to glutamine because of the similarity in sizes of these amino acids, although other related changes are desirable and contemplated as described elsewhere.
  • Some embodiments of the invention pertain specifically to epitopes useful for diagnosis and therapy of infectious disease agents.
  • many biological activities arise from binding reactions between a protein or peptide and another agent, such as a cell surface receptor, or specific binding protein inside a cell.
  • the invention is useful for making intermediate sized peptides having improved binding activity for these biological effects because the improved stability of the intermediate sized peptides provides greater opportunity for binding between the peptide and the in-vivo binding partner, such as a cell surface receptor or intracellular receptor.
  • One embodiment in this vein is to improve the binding reactivity of a peptide having a sequence obtained from a larger protein, wherein the larger protein acts as a binding partner in vivo.
  • the binding partner may be for example, a membrane protein that has a portion that binds to a blood factor.
  • This embodiment of the invention is particularly useful to modify leucine zipper regions of proteins when preparing a peptide portion (less than the whole protein sequence) that lacks at least some of the secondary or tertiary (folding) structure of the protein.
  • a "hydrophobic zipper" binding mechanism with leucines playing a major role is believed to be important in protein folding dynamics, as described in Proc. Nat'l Acad. Sci. USA 90: 1953, (1993) and Science 263:536, 1994).
  • This embodiment of the invention is particularly applicable for obtaining intermediate sized peptides from proteins of this protein class described in these two publications.
  • the embodiment of replacing one or more hydrophobic amino acids with one or more hydrophilic amino acids particularly relates to intermediate sized peptides from 16 amino acids to 100 amino acids in length, and more particularly to peptides between 25 to 50 amino acids, 36 to 50 amino acids and 41 to 50 amino acids.
  • the improved effect is seen particularly with intermediate sized peptides because, at very small sizes of less than about 16 (e.g., 16), and particularly less than 10 amino acids, the epitope recognized by an antibody more closely resembles the primary structure of the short segment, namely, the individual amino acid residues themselves. That is, antibody reactivity (if any) to such a short peptide arises primarily from chemical characteristics of the amino acid residues themselves.
  • peptides between about 25 to 100 amino acid residues long, and particularly 25-50 amino acids long advantageously are used. These intermediate sized antigens are larger than short pieces studied by Horal, Aleanzi and others, and have more advantageous secondary structure in water solution. In this case, altering a hydrophobic amino acid to a hydrophilic amino acid provides an advantage to the peptide.
  • peptide antigens of most interest for diagnostics and therapy generally have more advantageous secondary and tertiary structures which are more sensitive to disruption by a hydrophobic residue, yet the hydrophobic residue(s) present in these peptides need a large protein for proper orientation.
  • the claimed invention is exemplified by, for example, altering a leucine to a glutamine but works well with shifts of other hydrophobic amino acids such as I, V, M, F and W to hydrophilic amino acids, and even to hydrophilic charged amino acids.
  • Most advantageous in this aspect is to replace a leucine, which has a three carbon long residue with a methyl group attached, with a glutamine, which also has a three carbon long residue with an additional amine group attached.
  • a peptide between 25 and 50 amino acids long is used for diagnostic tests that has only one hydrophobic residue within an 8 residue long portion. Altering this hydrophobic residue to a hydrophilic residue improves reactivity (sensitivity and/or selectivity). In yet another embodiment, 2 hydrophobic residues within an 8 amino acid long portion exist and at least one of these is altered to a hydrophilic amino acid to provide the benefit. Altering 2 or more residues within a short region can provide great improvement to solubility and the ability to incorporate the peptide, alone or with other peptide(s) in a diagnostic test reagent or therapeutic agent.
  • an isoleucine, leucine, valine, or methionine is replaced with glutamine.
  • any of these hydrophobic amino acids is replaced with asparagine.
  • any of these hydrophobic amino acids is replaced with threonine, serine, alanine or glycine.
  • any of these hydrophobic amino acids is replaced with histidine or proline.
  • any of these hydrophobic amino acids is replaced with aspartic acid, glutamic acid, arginine or lysine.
  • a phenyl alanine can be converted to a glutamine.
  • a phenyl alanine can be converted to any of the other hydrophilic amino acids.
  • methods are contemplated in which:
  • intermediate peptide sequence is reviewed to determine the presence of a leucine, isoleucine, valine, methionine, or other hydrophobic amino acid; (2) at least one such hydrophobic amino acid in the sequence is changed to a less hydrophobic, or preferably, hydrophilic amino acid as described herein; and (3) an intermediate peptide is synthesized having the new sequence.
  • at least one leucine or isoleucine is changed to an arginine and/or arginine.
  • a computer modeling software program such as "Peptide Companion” advantageously is used and a specific alteration is chosen, using the program, to maintain the predicted pre-existing secondary or tertiary structure of the protein.
  • Secondary structure in this context refers to polypeptide helix or pleated sheet that forms primarily by multiple hydrogen bonding between peptide bond hydrogen and oxygen. Most advantageous is alpha helix structure that forms within a stretch of the peptide.
  • the alpha helix on the amino terminal side of this region is important to stabilize the antigen structure. The degree of stabilization has a great influence on performance of a peptide used in diagnosis or therapy.
  • the extra 5 amino acids, "RARLQ” provide a more stable peptide by virtue of extending the alpha helix at the amino terminal side of the immunodominant region.
  • QMWRANDEGHLKFPS Peptides that comprise one or more of these combinations are contemplated in the invention.
  • Advantage amino acids may be, for example, determined by predictions from a peptide analysis software program, "Peptide Companion Version 1.24 for Windows" from Peptides International, Inc. Louisville, Kentucky 40299 U.S.A.
  • the Chou-Fasman Conformational parameters are used in determining which amino acids can be changed within the helix in a manner to preserve the helix, with corresponding advantageous antigenicity of the peptide.
  • an antigen works better if it includes at least about 5 amino acids (e.g. five) to the amino terminal side of the immunodominant region. In alternative embodiments, this portion may be 6, 7, 8, 9, 10, 11, 12 or more amino acids long. In advantageous embodiments this added portion, (or at least a part that is adjacent to the immunodominant region) is in the form of a helix as described above.
  • methods are contemplated for preparing improved intermediate sized peptides for diagnostics and therapy of disease, comprising: (1) obtaining the sequence of an intermediate-sized peptide, or portion of a larger protein; (2) computing secondary structure information for at least a portion of the sequence from step (1); (3) determining one or more specific amino acid substitutions to the portion studied, that allow greater predicted secondary structure compared to the sequence from step (1); and (4) synthesizing an intermediate sized peptide having the sequence determined from step (3).
  • alpha helix information is obtained from step (2) and step (3) is carried out by substituting various amino acids until at least one is found that gives a sequence with greater predicted alpha helix structure.
  • a peptide antigen could be improved to provide greater specificity if a basic amino acid such as arginine or lysine in the antigen is replaced with another non-basic hydrophilic amino acid. Such substitution most advantageously is made for an arginine or lysine that is in an alpha helix but not in an immunodominant region.
  • the new amino acid should be chosen to maintain the helix structure.
  • One embodiment of the invention accordingly, is a method to improve an intermediate sized peptide reagent used in a diagnostic assay, comprising the step of replacing an arginine or lysine of the peptide with another amino acid that lacks a positive charge in the buffer or aqueous solution used.
  • the arginine or lysine is at the amino terminus of the peptide and the peptide has two positive charges at the amino terminus, by virtue of the amino terminus amino group and the side group of the lysine or arginine.
  • one of the positive charges is removed when the basic lysine or arginine is replaced with the non-basic amino acid.
  • the entire peptide sequence is reviewed to determine whether two or more positively charged amino acids are present within a 8 amino acid long portion. If found, at least one of the two positive charges is removed by replacing basic amino acid(s) with a non-basic amino acid such as glutamine.
  • a basic amino acid within an intermediate sized peptide but outside of a known epitopic sequence of the peptide is altered to a non-basic amino acid.
  • arginine is altered to a glutamine.
  • the inventors have used the method to improve a peptide for HIV-1 diagnosis by converting an arginine of the peptide into a non-basic acid.
  • lysine and even histidine which forms a positive charge in many physiological pH solutions profitably may be altered by the method.
  • a basic amino acid residue (having a positive charge at physiological pH) forms undesirable ionic bonds with negative charges of other molecules or with negative surfaces. These bonds can result in non-specific binding, thus causing a false positive assay result, or improper non-specific binding in the case of a therapeutic application.
  • the inventors further point out that a positive charge (from a basic amino acid) within an intact protein, when positioned near a negative charge, would not readily form a strong ionic bond with another molecule or with a negatively charged surface.
  • the basic amino acid is more free to react outside the molecule that contains it, leading to high background and/or taise positives when the peptide is used as an immobilized binding partner in an assay.
  • removing the positive charge will improve assay performance.
  • the method works best for a basic amino acid(s) outside the desired known epitopic sequence because in most cases background ionic binding is to be alleviated while maintaining epitopic reactivity of the peptide.
  • the inventors further note that the method works best for removing positive charge(s) in particular. Removing a negative charge from the peptide is less helpful, probably because most surfaces of interest for diagnostics tests are negatively charged.
  • false positive assay results were alleviated by replacing an arginine residue at the position 12 residues to the amino terminal side of the gp41 immunodominant loop region, with the non-basic amino acid glutamine. Data was obtained showing that replacement of this arginine with glutamine provides enhanced antigen selectivity and somewhat less antigen sensitivity, associated with removal of false positive results.
  • a constrained "portion" in this context means a segment of at least 5 amino acids, preferably at least 6, more preferably at least 8 and in some cases 13 amino acids long or more.
  • constrained means that the conformational movement of the portion (and thus the structure of the epitope) is restrained by cross-linking between two terminal amino acids, one at each end of the portion that comprises the epitope.
  • the peptide structure that is recognized by the immune system after administration of the construct in a vaccine may be larger than the portion that is bound by cross-linked terminal amino acids.
  • the constrained portion may form a larger epitope site with another section of the peptide construct as a tertiary structure (complex between different regions of the peptide construct) although in preferred embodiments the constrained portion, which optionally includes the terminal amino acids, itself forms the epitope.
  • the epitope may be smaller than the portion between the terminal amino acids and, in some cases a helix is formed within the constrained portion. In most embodiments a complete helix does not form, and in some cases no helix structure would form. In every case, however, the epitope primarily (i.e. more than half of the amino acids that create the epitope) is formed by amino acids within the bounded portion, or a tertiary structure is formed wherein the bounded portion forms a stable complex (non-covalently formed) with a peptide region outside the constrained portion and a sequence from the constrained portion by a spacer region.
  • the spacer region if used, preferably is between 3 and 10 amino acids and more preferably between 5 and 6 amino acids long.
  • the terminal amino acids of an epitopic region are cross-linked by forming at least one covalent bond between them.
  • cross-linking occurs by the formation of a sulphur-sulphur bond via formation of a cystine from oxidation of two cysteines.
  • This type of crosslinking is preferred in cases where the cystine bridge itself forms part of a desired epitope.
  • Formation of a cystine cross-link from two cysteines is readily carried out by known procedures that cause two thiol groups on the same peptide to oxidize and form a dicysteine (cystine) in the presence of oxygen.
  • a cystine bridge is particularly preferred for use with some V3 loop epitopes, as illustrated in the examples.
  • a side chain amide bond-forming group may be placed at the N-terminus of an epitope sequence and another amino acid with a side chain amide bond-forming group is placed at the C-terminus of the peptide.
  • the side chain amide bond-forming groups of the N-terminal and C- terminal residues are joined to form a cyclized structure which constrains the epitopic sequence.
  • the sequence is 6 amino acids long and forms an ⁇ -helix within the loop as described in U.S. No. 98/20036.
  • a larger peptide (less than 75 amino acids, particularly less than 50 amino acids) lock any sequence of, for example, six amino acid residues within a larger peptide into, for example, a helix by importing two residues with side chain amide bond-forming groups into the N-terminal amino acid position and the C-terminal position amino acid position flanking the sequence of six amino acid residues.
  • the side chain amide bond-forming groups of the N-terminal and C-terminal flanking residues are made to form a cyclic structure which mimics the conformation of the ⁇ -helix. Regions 5 amino acids long and regions greater than 6 amino acids long, of course, also can be used as exemplified in this specification and often will form particular helix structures.
  • constrained peptides of this embodiment there are at least two general methods for constructing constrained peptides of this embodiment: (1) synthesis of a linear peptide comprising a pair of residues that flank an amino acid sequence that is five to thirteen residues in length, wherein the two flanking residues are independently selected from amino acid residues having side chain amide bond-forming groups, followed by bridging the side chain amide bond-forming groups of the flanking residues with a linker or peptide coupling reagent (i.e.
  • flanking amino acid residues include amino acids with side chains carrying a free carboxy group, such as aminopropanedioic acid, aspartate, glutamate, 2-aminohexanedioic acid, and 2- aminoheptanedioic acid, and amino acids with side chains carrying a free amino group, such as 2,3-diaminopropanoicacid (2,3-diaminopropionicacid), 2,4-diaminobutanoicacid (2,4-diaminobutyricacid), 2,5-diaminopentanoic acid, lysine and ornithine.
  • the functional groups on either side may be used such as thiol (SH) or hydroxyl (OH) groups.
  • a native peptide antigen sequence can be varied outside of an immunodominant epitopic region in order to react immunologically with a more diverse range of antibodies. This is particularly important where the disease causing organism is an RNA virus and rapidly mutates new antigen structures.
  • sequence variation is added to a peptide containing a gp41 immunodominant region according to a formula shown by Figure 1, to form a peptide that reacts with a wider variety of HIV-1 infected blood specimens.
  • Figure 1 shows allowable changes to the core immunoreactive part of a peptide (positions 594-609) according to this embodiment.
  • an HIV peptide antigen according to this embodiment of the claimed invention is from about 25 (e.g., 25) to about 100 (e.g., 100) amino acids long. More advantageously, the HIV peptide antigen is from about 36 (e.g., 36) to about 50 (e.g., 50) amino acids long. Even more preferably, this antigen includes at least about 5 amino acids, from about 21 amino acids to about 29 amino acids (e.g., 21 to 29 amino acids) from the cysteine at the N-terminal side of the heptapeptide loop. This segment on the N-terminal side of the loop preferably forms an alpha helix.
  • the Hopp acrophilicity scale peptide profile should be about at least in 75% agreement (e.g., 75% or more) with the profile of the classical HIV-1 group M strain B sequence, as determined by peptide analysis with "Peptide Companion Version 1.24 for Windows” software from Peptides International, Inc. Louisville, Kentucky 40299 U.S.A.
  • strain B also means the same as “Group B” or "clade B.
  • the sequence denoted as strain B of group M has been published by the Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (HUMAN RETRO VIRUSES AND AIDS 1996).
  • a universal peptide sequence according to this embodiment preferably has a Janin accessibility scale peptide profile that is in about at least 80% agreement (eg. , 80% or more) with the sequence profile of the classical HIV-1 M strain B Group, as determined by this software. Also preferred is a sequence having a Hopp and Woods hydrophilicity scale peptide profile that is at least in 75% agreement with the profile of the classical HIV-1 M strain B Group.
  • the Kyte and Doolittle hydropathy scale profile of the peptide should be in at least 80% agreement with the profile of the classical HIV-1 M strain B Group (all determined by the Peptide Companion software.)
  • Table 1 the algorithm shown in Table 1 , and in accordance with the method detailed herein, it is advantageous to use these computer derived profiles to help determine which alterations of which amino acid(s) will work best in the sequence.
  • Antigens that cross-react with the immunodominant region of the gp41 envelope protein of HIV are contemplated as embodiments of the claimed invention as exemplified above. Combinations of substitutions are particularly advantageous. Specific examples of these antigens are peptides that comprise (i.e. contain in whole or in part) peptide sequences shown as SEQ ID Nos. 1 through 20 in Figure 2. The inventors realized that they could mix one or more peptide antigens according to the invention with recombinant antigen at a higher concentration if the peptide is made more hydrophilic by amino acid substitution as described herein.
  • antigens that have at least one substitution of a hydrophilic amino acid (such as glutamine or arginine) for an aliphatic amino acid (such as leucine, isoleucine or valine) from a naturally occurring sequence are particularly advantageous.
  • a hydrophilic amino acid such as glutamine or arginine
  • an aliphatic amino acid such as leucine, isoleucine or valine
  • Figure 4 shows that two alterations of leucine to glutamine and alteration of arginine to glutamine in a 36 mer portion of a natural HIV-1 sequence (MVP5180) removed reactivity of this sequence to an entire group of non HIV-1 samples, in this case, HIV-2 infected samples.
  • substitution of a valine for an isoleucine within the cysteine loop region of the HIV-1 gp 41 protein, and substitution of a basic amino acid such as arginine or lysine at the eighth position to the carboxy terminal side of the cystine loop is particularly advantageous and can lead to a peptide having altered immunological characteristics. Sequences of representative altered peptides having these and related changes are shown in Figure 2, and described more generally by the language of the claims. Of course, other alterations are contemplated in accordance with the discoveries detailed herein.
  • the helix turn profile and Chou Fasman conformational parameters shown in Figure 5 are useful for predicting an advantageous peptide that contains a cystine loop sequence found in many naturally existing HIV strains.
  • the following sequences are particularly useful to form the cystine loop region of HIV-1 having the advantageous peptide turn profile: SEQ ID NO: 21: CAGKQVC; SEQ ID NO: 22: CAGRLVC; SEQ ID NO: 23: CADRQVC; SEQ ID NO: 24: CANRQVC; SEQ ID NO: 25: CAGRQVC; and SEQ ID NO: 26: CAGKLVC.
  • a peptide that comprises a sequence chosen from this list advantageously further comprises one or more amino acids at both ends of the cystine loop region.
  • HCV Hepatitis C Virus
  • an epitope sequence of HCV that is within an intermediate sized peptide is cross-linked as described in section 4 above.
  • the cross- linking stabilizes the epitopic structure, increasing its reactivity with antibodies and with other components of the immune system. It is further preferred to modify one or more hydrophobic residues within the epitope by substituting a hydrophilic form of an amino acid for a hydrophobic form, and to add (or increase) secondary structure outside the chosen epitopic region.
  • SEQ ID NOs: 43 through 229 depict representative examples of sequences according to the invention that specify intermediate length peptides useful as antigens for detecting HCV infection.
  • SEQ ID NOs: 35 through 42 show sequences from the HCV core region residues 5 through 21, and particularly residues 5 through 15, wherein the desired epitopic region is constrained by placement within a cystine loop. These sequences also show a helix region outside of the loop, which is preferred to further improve reactivity of the selected epitopic sequence.
  • SEQ ID NOs: 108-115 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.
  • the peptide having two or more basic amino acids within an 8 amino acid long segment, and particularly having 3 or 4 basic amino acids within a 6 amino acid long segment, as shown in this group be used together with a glycosaminoglycan such as heparin or chondroitin sulfate. That is, to prevent non-specific binding of the antigen to a negatively charged surface used in the assay, it is preferred to add a glycosaminoglycan to the assay kit, solid phase, wash solution or the like.
  • SEQ ID NOs: 43 through 50 show sequences from the HCV core region residues 44 through 55, and particularly residues 44 through 53, wherein the desired epitopic region is constrained by placement within a cystine loop. These sequences also show a helix region outside of the loop, which is preferred to further improve reactivity of the selected epitopic sequence.
  • SEQ ID NOs: 116 through 123 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.
  • SEQ ID NOs: 51 and 52 show sequences from the HCV core region residues 61 through 74 wherein a terminal arginine has been replaced with a non-basic amino acid in accordance with an embodiment of the invention.
  • SEQ ID NOs: 124 and 125 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.
  • SEQ ID NOs: 53 through 60 show sequences from the HCV core region residues 62 through 71, wherein the desired epitopic region is constrained by placement within a cystine loop. These sequences also show a helix region outside of the loop, which is preferred to further improve reactivity of the selected epitopic sequence.
  • SEQ ID NOs: 126 through 133 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.
  • SEQ ID NOs: 61 through 70 show sequences from the HCV non-structural 4 region (NS4) residues 1933 through 1947, particularly residues 1937 through 1945, wherein, as shown in SEQ ID NOs: 63 through 70, the desired epitopic region preferably is constrained by placement within a cystine loop. These sequences also show a helix region outside of the loop, which is preferred to further improve reactivity of the selected epitopic sequence.
  • SEQ ID NOs: 134 through 143 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.
  • SEQ ID NOs: 71 through 99 show sequences from the HCV El region residues 208 through 226, and particularly residues 211 through 222, wherein the desired epitopic region is constrained by placement within a cystine loop.
  • 18 amino acid long regions are terminated by cysteines.
  • the cysteines are oxidized to form intra-chain cystines in order to constrain and stabilize the epitope structure.
  • SEQ ID NOs: 144 through 172 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.
  • SEQ ID NOs: 100 through 107 show sequences from the HCV El region residues 208 through 226, and particularly residues 211 through 222, wherein the desired epitopic region preferably is constrained by terminal cystines.
  • the cysteines are oxidized to form intra-chain cystines in order to constrain and stabilize the epitope structure.
  • These sequences also show a helix region outside of the loop, which is preferred to further improve reactivity of the selected epitopic sequence.
  • SEQ ID NOs: 173 through 180 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.
  • SEQ ID NOs: 181 through 184 show representative sequences useful for detecting antibodies against the HCV core region.
  • SEQ ID NOs: 185 through 194 show related sequences for the NS3 protein.
  • SEQ ID NOs: 195 through 209 show related sequences for the NS4 protein.
  • SEQ ID NOs: 210 through 229 show related sequences for the NS5 protein.
  • sequences can be determined according to methods of the invention and are contemplated.
  • combinations of two or more, particularly three or more, more particularly 4 or more peptides are used together.
  • the embodiment of altering a peptide to make it more hydrophilic as described above is particularly helpful to obtain multiple peptides of the same epitopic site but of alternative viral strains. It is preferred that peptides used in combination be made more hydrophilic so that they could be used at higher concentration.
  • Peptide Antigens that Correspond to Other Disease Organisms differ from naturally occurring proteins and peptides, and provide improved diagnostic assay and therapeutic results compared to the use of sequences obtained from naturally occurring molecules. Further embodiments according to the invention provide improved tests and vaccines for intermediate size antigens that correspond to other disease agents such as HTLV-I and HTLV-II virus as well.
  • Disease agents such as those exemplified herein as well as others contain antigens with hydrophobic amino acid residues of leucine, isoleucine, valine, methionine and phenyl alanine. These residues can contribute to structural instability when a peptide antigen mimic from about 25 to about 100 amino acids long, advantageously from about 36 to about 50 amino acids and more advantageously between about 41 to about 50 amino acids long is prepared (de novo or by removal) from a larger protein sequence.
  • hydrophobic amino acid can be replaced with a charged amino acid such as arginine for leucine.
  • the hydrophobic amino acid most advantageously is replaced with a hydrophilic uncharged amino acid having a similar size to the original hydrophobic amino acid.
  • the other strategies for improving intermediate sized antigens by for example, removing a positive charge and increasing the amount of alpha helix are useful to prepare antigens corresponding to other disease-causing organisms and are contemplated.
  • These antigens generally are more stable than the corresponding natural sequence antigens and can be used advantageously in improved immunoassays and immunotherapies.
  • Embodiments of the claimed invention advantageously allow an increase in the amount of antigen used in an immunodiagnostic assay (or therapy) by making the antigen more hydrophilic.
  • This increase in antigen used for specific binding reaction(s) can lead directly to more advantageous sensitivity as well as more advantageous reactivity with a broader range of HIV-1 Group O specimens when applied to HIV infection testing.
  • Analogous improvements in the use of other peptide antigens for other disease organisms such as HTLV-I and HTLV-II virus are possible.
  • Peptides of the invention can be used in diagnostic tests that employ antigen-antibody binding for detection of a disease agent. It is preferred to use a very easy, rapid (three minutes) dot-blot assay method as described in co-pending application U.S. App. Ser. No. 09/069,935 "Multiple Readout Immunoassay with Improved Resistance to Interferences" (Attorney Docket No. 073294/0173 filed April 30, 1998, incorporated herein in its entirety by reference.) However, the inventive antigens also can be used in diagnostic methods that require these very long incubation time periods and multiple steps.
  • the test device has a housing comprised of a water impermeable material in which other test components such as an absorbent pad with a reagent layer, filter and a reagent used to obtain a test result are held.
  • the housing has an opening to admit a fluid sample. The housing comes apart during use so that the user can remove the filter to expose the reagent layer for application of a reagent and/or wash fluid.
  • a sleeve that holds the filter is removably attached to the housing such that contact of the filter is favored over contact of the sleeve with the surface of the reagent layer.
  • the sleeve is attached to the housing by a bayonet mount.
  • the sleeve is removed and further optional reagent solution and a wash solution are added directly to the reagent layer.
  • a sample is added to the device and further processing is carried out at a separate location or after storage of the device for a few hours.
  • the sleeve remains attached to the housing to prevent or delay the release of moisture from the device until the later processing steps are carried out.
  • the housing also may contain a cover to protect the opening and further guard against the release of moisture.
  • Multiple housings can be incorporated into a multi-test unit to allow high volume testing.
  • the latter embodiment is acceptable for infectious disease testing of blood samples at blood banks.
  • a 32 well multiple-test device having overall dimensions of 3.5 inches by 6.75 inches
  • a 48 well multiple-test device having overall dimensions of 5.125 inches by 6.75 inches
  • a ninety six well multiple-test device having overall dimensions of 6.75 inches by 9.875 inches.
  • Each of these multiple-test devices has a well-size (for admission of a sample) of 0.75 inches.
  • the 32 well device is particularly advantageous and is desirably configured as a single array of 4 eight member rows.
  • 4 (or 8) test devices that correspond in size to a column (or row) of a microtiter plate are used in applications where intermediate numbers of samples are processed.
  • the housing and other parts of the test device are constructed from well-known materials in accordance with well-known methods of the prior art.
  • Material suitable for the invention should not interfere with the production of a detectable signal and should have a reasonable inherent strength, or strength can be provided by means of a supplemental support, such as, for example, by forming a nitrocellulose layer onto an absorbent pad, by means of a suspension of nitrocellulose.
  • the test device positions parts with a positioning "sleeve" to allow even fluid flow between the parts without interference by the sleeve itself, and the parts are arranged to minimize transverse flow.
  • the device uses friction-held parts and water swellable parts to allow fluid to more evenly flow through junctions between the parts and a dispersing layer downstream of the filter to help disperse fluid more evenly to the reagent layer, where the reagent layer is integrated with absorbent material to form a single unit.
  • the physical assembly of components from known materials within the housing generally will be understood to a skilled artisan but for clarity, further details are provided in the above- referenced applications in the form of definitions of some terms used in the claims.
  • An antigen for an HIV test is immobilized onto the reagent layer portion of the absorbent pad by absorption, via spotting a water solution of the antigen.
  • the optimum amount of antigen to use is determined by methods accepted in the art. The inventors used approximately 100 ng of antigen per test for the HIV-1 embodiments.
  • Acceptable antigens for use as a device for hepatitis C testing include, for example, peptides of modified "HCV regions" known as core, NS3, NS4 and NS5, as discussed by Feucht et al. in J. Clin. Microbiol. 33:620-624 (1995), having one or more amino acid substitutions as described for HIV test antigens in the present specification.
  • Representative examples taken from an immunoreactive region of the NS4 protein are shown in Figure 2 as SEQ ID NO: 27-30.
  • the three intermediate size peptides of SEQ ID NO: 28-30 are derived from the natural sequence shown in SEQ ID NO: 27.
  • Representative examples taken from the core protein of hepatitis C virus are shown as SEQ ID NO: 31-34.
  • the three intermediate size peptides of SEQ ID NO: 32-34 are derived from the natural sequence shown in SEQ ID NO: 31.
  • the claimed invention of replacing one or more hydrophobic residue(s) with hydrophilic residue(s), as exemplified by SEQ ID NO: 28-30 and SEQ ID NO: 32-34 is particularly advantageous for hepatitis C testing because a mixture of several antigens typically are used together in order to detect a suitably wide range of hepatitis C infections.
  • the claimed invention allows a larger amount and/or variety of peptide to be employed as binding agent for testing hepatitis C.
  • Antigens useful for testing of exposure to other pathogens such as those responsible for lyme disease, toxoplasmosis, and other microorganisms such as rubella, mycoplasma, cytomegalo virus, herpes, HTLVI, HTLVII, Hepatitis B, and chlamydia are known and also can be modified according to the principles enumerated herein. Chemically synthesized peptides and recombinant proteins can be immobilized within devices as claimed by routine methods, such as spotting a water solution of the antigen onto a nitrocellulose membrane or membrane layer. Antigens in accordance with the invention can be used for therapy (prevention and/or treatment) of infection according to well known methods in the art, such as those described in the above-cited co-pending applications.
  • the peptides of the invention can be prepared using any suitable means. Because of their relatively short size (generally, less than 100 amino acids, advantageously less than 75, more advantageously less than 50 and conveniently less than 40), the peptides can be synthesized in solution or on a solid support in accordance with conventional peptide synthesis techniques. Various automatic synthesizers are commercially available (for example, from Applied Biosystems) and can be used in accordance with known protocols. See, for example, Stewart and Young, SOLID PHASE PEPTIDE SYNTHESIS (2d. ed. , Pierce Chemical Co., 1984); Tarn et. al , J. Am. Chem.
  • DNA-derived proteins or peptides which comprise one or more peptide sequences of the invention, can be used to prepare the HIV cytotoxic T cell epitopes identified herein or identified using the methods disclosed herein.
  • a recombinant peptide of the claimed invention is prepared in which the amino acid sequence is altered so as to present more effectively epitopes of peptide regions described herein to stimulate a cytotoxic T lymphocyte response.
  • a polypeptide is used that incorporates several T cell epitopes into a single polypeptide.
  • coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et. al. , J. Am. Chem. Soc , 103, 3185 (1981), modification can be made simply by substituting the appropriate base(s) for those encoding the native peptide sequence.
  • the coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available.
  • the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in a suitable cellular host.
  • promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence.
  • the resulting expression vectors are transformed into suitable bacterial hosts.
  • Yeast or mammalian cell hosts may also be used, employing suitable vectors and control sequences.
  • At least one additional amino acid is added to at least one terminus of a peptide of the claimed invention.
  • Such added amino acid(s) facilitates linking the peptide to another peptide, coupling to a carrier, or coupling to a support.
  • the added amino acid(s) also can be chosen to alter the physical, chemical or biological properties of the peptide, such as, for example adding another epitope for T-cell stimulation.
  • Suitable amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, and the like, can be introduced at the C- or N-terminus of the peptide.
  • a peptide of the invention can differ from the natural sequence by being modified by terminal-NH sub 2 acylation, e.g. , acetylation, or thioglycolic acid amidation, terminal-carboxyl amidation, e.g. , ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule, thereby providing a linker function.
  • terminal-NH sub 2 acylation e.g. , acetylation, or thioglycolic acid amidation, terminal-carboxyl amidation, e.g. , ammonia, methylamine, etc.
  • these modifications may provide sites for linking to a support or other molecule, thereby providing a linker function.
  • the peptides of the claimed invention or analogs or homologs thereof may be further modified beyond the sequence considerations given above, as necessary to provide certain other desired attributes, e.g. , improved pharmacological characteristics, while increasing or at least retaining substantially the biological activity of the unmodified peptide.
  • the peptides can be modified by extending, decreasing or substituting amino acids in the peptide sequence by, for example, the addition or deletion of suitable amino acids on either the amino terminal or carboxyl terminal end, or both, of peptides derived from the sequences disclosed herein.
  • substitutions for HIV-1 testing are described by, for example, SEQ ID Nos. 1-6, further conservative substitutions are possible and sometimes desirable for HIV-1 testing.
  • conservative substitutions is meant replacing an amino acid residue with another that is biologically and/or chemically similar, e.g. , one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • Other amino acid substitutions are provided as groups within individual claims.
  • the portion of the peptide sequence that is intended to mimic an antigen of HIV will not differ by more than about 30% from any of the sequences provided herein, except where additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, for example, ease of linking or coupling, and the like.
  • additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, for example, ease of linking or coupling, and the like.
  • regions of the peptide sequences are highly variable, it may be desirable to vary one or more particular amino acids to mimic more effectively differing epitopes of different HIV strains.
  • the contributions made by the side chains of the residues can be probed via a systematic replacement of individual residues with a suitable amino acid, such as Gly or Ala.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art as cited in U.S. No. 5,703,057 (citing Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant peptide which in turn defines the interaction of the peptide with other molecules, for example, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a peptide with similar biological activity, i.e., still obtain a biological functionally equivalent peptide.
  • substitution of amino acids whose hydropathic indices are within +- 2 is preferred, those which are within +- 1 are particularly preferred, and those within +- 0.5 are even more particularly preferred.
  • hydrophihcity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +- 1) glutamate (+3.0 +- 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0) threonine (-0.4); proline (-0.5 +- 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0) methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3) phenylalanine (-2.5); tryptophan (-3.4).
  • amino acid can be substituted for another having a similar hydrophihcity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent peptide.
  • substitution of amino acids whose hydrophihcity values are within +- 2 is preferred, those which are within +- 1 are particularly preferred, and those within +- 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophihcity, charge, size, and the like.
  • substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Peptides that tolerate multiple amino acid substitutions generally incorporate small, relatively neutral molecules, e.g., Ala, Gly, Pro, or similar residues.
  • the number and types of residues that can be substituted, added or subtracted will depend on the spacing necessary between the essential epitopic points and certain conformational and functional attributes that are sought.
  • types of residues it is intended, e.g., to distinguish between hydrophobic and hydrophilic residues, among other attributes. If desired, increased binding affinity of peptide analogs to can also be achieved by such alterations.
  • any spacer substitutions, additions or deletions between epitopic and/or conformationally important residues will employ amino acids or moieties chosen to avoid stearic and charge interference that might disrupt intramolecular binding of the peptides and intermolecular binding of peptides to other molecules.
  • Peptides that tolerate multiple substitutions while retaining the desired immunological activity also may be synthesized as D-amino acid-containing peptides.
  • Such peptides may be synthesized as "inverso" or “retro-inverso” forms, that is, by replacing L-amino acids of a sequence with D-amino acids, or by reversing the sequence of the amino acids and replacing one or more L-amino acids with D-amino acids.
  • the D-peptides are substantially more resistant to peptidases, and therefore are more stable in serum and tissues compared to their L-peptide counterparts, the stability of D-peptides under physiological conditions may more than compensate for a difference in affinity compared to the corresponding L-peptide.
  • L-amino acid-containing peptides with or without substitutions can be capped with a D-amino acid to inhibit exopeptidase destruction of the antigenic peptide.
  • an advantageous embodiment is to prepare the peptide by chemical synthesis.
  • the peptide is made recombinantly.
  • modifications, including conservative modifications, are best carried out by changing a DNA sequence that codes for the peptide.
  • the following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table:
  • Biologically functional universal peptides can be prepared through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • site-specific mutagenesis is well known in the art, as exemplified by various publications.
  • the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • Site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • peptides were prepared from sequences shown in Figure 2 and were tested as antigens for HIV-1 testing of blood samples.
  • the test method is described in co- pending U.S. patent application "Multiple Readout Immunoassay for Improved Resistance to Interferences. " Five HIV Group O blood specimens obtained from Africa, two HIV Group O blood specimens obtained from the United States and one non-Group O blood specimen were tested. Each blood specimen was tested at no dilution, 10 times dilution, 100 times dilution and 1000 times dilution with peptides according to embodiments of the claimed invention and with two previously known peptides, as shown in Figure 3.
  • the peptide is incorporated into a diagnostic test according to the procedure of Example 1. Ten HIV-1 group O infected samples are tested according to the procedure of Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1 Group O virus antigen.
  • a peptide is prepared having a sequence shown by SEQ ID NO: 10.
  • the peptide is incorporated into a diagnostic test according to the procedure of Example 1.
  • Example 4 Ten HIV-1 group O infected samples are tested according to the procedure of Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1 Group O virus antigen.
  • Example 4
  • a peptide is prepared having a sequence shown by SEQ ID NO: 11. The peptide is incorporated into a diagnostic test according to the procedure used in Example
  • Example 1 and all test results are positive, indicating the presence of antibodies to HIV-1
  • a peptide is prepared having a sequence shown by SEQ ID NO: 12.
  • the peptide is incorporated into a diagnostic test according to the procedure used in Example
  • a peptide is prepared having a sequence shown by SEQ ID NO: 13.
  • the peptide is incorporated into a diagnostic test according to the procedure used in Example 1. Ten HIV-1 group O infected samples are tested according to the procedure used in Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1 Group O virus antigen.
  • a peptide is prepared having a sequence shown by SEQ ID NO: 14.
  • the peptide is incorporated into a diagnostic test according to the procedure used in Example 1. Ten HIV-1 group O infected samples are tested according to the procedure used in Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1 Group O virus antigen.
  • a peptide is prepared having a sequence shown by SEQ ID NO: 15.
  • the peptide is incorporated into a diagnostic test according to the procedure used in Example
  • a peptide is prepared having a sequence shown by SEQ ID NO: 16.
  • the peptide is incorporated into a diagnostic test according to the procedure used in Example 1. Ten HIV-1 group O infected samples are tested according to the procedure used in Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1 Group O virus antigen.
  • a peptide is prepared having a sequence shown by SEQ ID NO: 18.
  • the peptide is incorporated into a diagnostic test according to the procedure used in Example 1. Ten HIV-1 group O infected samples are tested according to the procedure used in Example 1.
  • Example 1 and all test results are positive, indicating the presence of antibodies to HIV-1
  • a peptide is prepared having a sequence shown by SEQ ID NO: 19.
  • the peptide is incorporated into a diagnostic test according to the procedure used in Example 1. Ten HIV-1 group O infected samples are tested according to the procedure used in Example 1.
  • Example 1 and all test results are positive, indicating the presence of antibodies to HIV-1
  • a peptide is prepared having a sequence shown by SEQ ID NO: 20.
  • the peptide is incorporated into a diagnostic test according to the procedure used in Example
  • Example 1 and all test results are positive, indicating the presence of antibodies to HIV-1 Group O virus antigen.
  • Peptides having a substitution for an isoleucine within the cystine loop and for a threonine at eight positions to the carboxyl terminal side of the cystine loop are useful for
  • a peptide is prepared having a sequence shown by SEQ ID NO: 30.
  • the peptide is incorporated into a diagnostic test according to the procedure used in Example 1.
  • a hepatitis C infected sample is tested according to the procedure used in Example 1 , and the test result is positive, indicating the presence of antibodies to hepatitis C virus antigen.

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Abstract

L'invention concerne des antigènes peptidiques améliorés, permettant d'effectuer des essais diagnostiques et de traiter des maladies, différents des séquences peptidiques naturelles. Ces peptides se croisent immunologiquement avec une grande variété de formes d'antigènes mutés, et sont particulièrement utiles pour effectuer des essais diagnostiques et traiter une maladie rétrovirale telle qu'une infection HIV, dans laquelle la mutation rapide du vecteur de la maladie pose un problème. Les peptides présentent une longueur d'environ 25 à 100 résidus d'acide aminé, une réactivité avantageuse, et peuvent également posséder une ou plusieurs autres caractéristiques avantageuses, telles qu'une solubilité dans l'eau ou une réactivité immunologique améliorées par rapport aux souches naturelles, telles que ANT70 et MVP5180. Des altérations avantageuses comprennent la substitution d'un ou plusieurs résidus hydrophobes par des résidus hydrophiles, l'élimination d'une charge positive d'acide aminé basique pour réduire une liaison non spécifique, et accroître une structure secondaire peptidique par addition d'un ou plusieurs acides aminés, de façon à former ou étendre une hélice alpha dans le peptide. Un des exemples de l'invention dont la protection est demandée consiste en la substitution d'une leucine de HIV. Les peptides qui ont été modifiés selon les principes de l'invention sont très différents des formes naturelles, et sont identifiés comme appartenant à n'importe quelle souche ou groupe viral particuliers.
PCT/US1999/012446 1998-06-05 1999-06-04 Peptides universels WO1999062945A2 (fr)

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US8822998P 1998-06-05 1998-06-05
US60/088,229 1998-06-05
US9870598P 1998-09-01 1998-09-01
US60/098,705 1998-09-01
US10042298P 1998-09-15 1998-09-15
US60/100,422 1998-09-15
PCT/US1999/001726 WO1999038887A1 (fr) 1998-01-28 1999-01-28 Peptides de vih-1 divergents
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003018045A1 (fr) * 2001-08-29 2003-03-06 Tianjin Fusogen Biotech Co., Ltd Medicament a utiliser pour le traitement des infections a vih, composition et utilisations correspondantes

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AU2683792A (en) * 1991-09-16 1993-04-27 Genelabs Technologies, Inc. Peptide based hepatitis c virus immunoassays
DK0591914T4 (da) * 1992-10-06 2010-05-25 Siemens Healthcare Diagnostics Retrovirus fra HIV-gruppen og dets anvendelse
DE4405810A1 (de) * 1994-02-23 1995-08-24 Behringwerke Ag Von einem Retrovirus aus der HIV-Gruppe abgeleitete Peptide und deren Verwendung
AU715731B2 (en) * 1994-10-20 2000-02-10 Institut Pasteur Nucleotide sequences of HIV-1 group (or subtype) O retrovirus antigens
FR2731013B1 (fr) * 1995-02-27 1997-05-16 Inst Nat Sante Rech Med Vih-1 de groupe o, fragments desdits virus, ainsi que leurs applications
US5977299A (en) * 1997-04-07 1999-11-02 Dade Behring Marburg Gmbh Activated peptides and conjugates
WO1999004011A2 (fr) * 1997-07-18 1999-01-28 Innogenetics N.V. Antigenes du groupe o du vih-1 et leurs utilisations
WO1999045395A1 (fr) * 1998-03-04 1999-09-10 Universal Healthwatch, Inc. Test de confirmation rapide d'infections microbiennes

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003018045A1 (fr) * 2001-08-29 2003-03-06 Tianjin Fusogen Biotech Co., Ltd Medicament a utiliser pour le traitement des infections a vih, composition et utilisations correspondantes

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