EP0636145A1 - Synthetic polypeptides derived from the hiv envelope glycoprotein - Google Patents

Synthetic polypeptides derived from the hiv envelope glycoprotein

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Publication number
EP0636145A1
EP0636145A1 EP93909057A EP93909057A EP0636145A1 EP 0636145 A1 EP0636145 A1 EP 0636145A1 EP 93909057 A EP93909057 A EP 93909057A EP 93909057 A EP93909057 A EP 93909057A EP 0636145 A1 EP0636145 A1 EP 0636145A1
Authority
EP
European Patent Office
Prior art keywords
leu
gln
hiv
gly
thr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP93909057A
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German (de)
English (en)
French (fr)
Inventor
Robert Vincent Fishleigh
Barry Robson
Roger Aston
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Protherics Medicines Development Ltd
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Proteus Molecular Design Ltd
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Application filed by Proteus Molecular Design Ltd filed Critical Proteus Molecular Design Ltd
Publication of EP0636145A1 publication Critical patent/EP0636145A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • 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
    • C12N2740/16011—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

Definitions

  • the present invention relates to synthetic polypeptides. It particularly relates to synthetic polypeptides which emulate the three-dimensional structures and/or electrostatic surfaces and/or other physical, chemical and structural properties of specific regions of viral envelope proteins. It is of particular interest to the design of vaccines, immunologically active therapeutic agents, diagnostics and other medical or scientific agents in relation to the Human Immunodeficiency Virus (HIV) known to be the causative agent of Acquired Immune Deficiency Syndrome (AIDS) .
  • HIV Human Immunodeficiency Virus
  • AIDS Acquired Immune Deficiency Syndrome
  • An object of the present invention is the development of synthetic polypeptides which can elicit the production of antibodies to the HIV virus, and most preferably neutralising antibodies, that is, antibodies which prevent infection by and/or limit the spread of the HIV virus by passive or active immunisation. Passive immunisation with such antibodies may constitute an effective means of treatment of AIDS patients thus controlling the spread of the virus within and between individuals and hence slow or halt the progress of the disease.
  • Our invention provides a synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV) r said polypeptide consisting substantially of an amino acid sequence of formula (I) :-
  • R 1 is selected from Gln-Gln-R 4 —R 5 , Gln-R 4 -
  • R 4 -R gr j or R. J is absent;
  • R 2 i iss aann aammiinnoo aacciidd rreessii ⁇ due selected from Gin, Lys, Glu or Arg;
  • R- i iss aann aammiinnoo aacciidd residue selected from lie, Thr or Ala;
  • R 4 is His or Glu; 5 is Leu or Met; and
  • X and Y may each independently be absent or independently be one or more amino acid residues. Normally, when R 1 is Gln-Gln-R 4 -R 5 and X and Y are present, X and Y are not homologous with the natural envelope protein sequence. More particularly, when X and/or Y are present and R, is as defined above X and Y do not provide or form part of an antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus.
  • Peptides according to formula I above without X and Y being present will of course be useful, for example, in the production of antibodies to the HIV. Such peptides will be especially effective when conjugated to a carrier molecule.
  • X or Y may be any length but preferably less than 20 amino acids, more preferably less than 10, eg. 3 to 6.
  • the sequence according to formula I may constitute a protein with X and Y being major portions of the protein with the antigenic sequence being, for example, part of an exposed loop on a globular protein.
  • R 5 is either Leu or Met, more preferably Leu
  • R 4 if present is His or Glu, more preferably His.
  • R 2 is preferably Gin, Lys or Glu, more preferably Gin and R- is preferably lie.
  • R 1 is Gln-Gln-R-R 5 or is absent and when present, R 4 is preferably His and R 5 is Leu. Also it is preferable that R 2 is Leu and R 3 is Gin.
  • polypeptide of formula (I) consist of the sequences:-'
  • X and Y are as defined above although it is preferred that if X or Y are present they are relatively short sequences.
  • X is absent and Y is 2 or 3 residues long, e.g. Gly-Cys or Gly-Cys-Ala.
  • sequences la and lb it is preferred that X is absent and Y is Gly-Cys in sequence la and is Gly-Cys or Gly-Cys-Ala in sequence lb; such C-terminal extensions provide alternate sites for coupling to a carrier.
  • Polypeptides according to formula I resemble certain epitopic portions of HIV 1 envelope protein.
  • polypeptide according to the invention consists substantially of an amino acid sequence of formula (II) :-
  • R_ is selected from Gln-Gln-Glu-R 5 , Gln-
  • Glu-R 5 , Glu-R j , R- or R 1 is absent; R 3 is Thr or Ala; R 5 is Met or Leu; and X and Y are as defined above.
  • R 5 if present, is Met and R 3 is preferably Thr.
  • R t is Gln- Gln-Glu-Met or absent.
  • X is absent and Y is 2 or 3 residues long, e.g. Gly-Cys or Gly-Cys-Ala.
  • Polypeptides according to formula II are similar to certain epitopes of HIV 2 envelope proteins.
  • Preferred polypeptide sequences according to the invention were chosen on the basis of their topographical similarity to one or more antigenic determinants of the HIV envelope proteins.
  • an antigenic determinant to which a given polypeptide was originally designed to be an analogue may also show topographical similarity to one or more other regions of the HIV envelope proteins possibly due to duplication of ancestral genes, or because the polypeptide is an analogue of a discontinuous determinant, or because the polypeptides have been designed to be polyvalent.
  • a discontinuous epitope may be viewed,as being composed of closely opposed sequential epitopes which may be of antigenic significance in their own right and a polyvalent polypeptide may contain two or more (continuous or discontinuous) determinant analogues in a single polypeptide chain, thus providing a means to simultaneously elicit the production of a range of antibodies which will recognise two or more determinants on the HIV envelope proteins.
  • Peptides according to the invention may be synthesised for example using either standard 9- fluorenyl-methoxycarbonyl (F-Moc) chemistry (see, for example, Atherton, E. and Sheppard, R. C. (1985) J. Chem. Soc. Chem. Comm. 165) or standard butyloxycarbonate (T-Boc) chemistry.
  • F-Moc 9- fluorenyl-methoxycarbonyl
  • T-Boc standard butyloxycarbonate
  • chromatographic analyses including high performance liquid chromatography, and spectrographic analyses, may for example be employed for this purpose. All the sequences herein are stated using the standard I.U.P.A.C.
  • amino acid residues defined as follows: Gly-Glycine, Ala-Alanine, Val-Valine, Leu-Leucine, Ile-Isoleucine, Ser-Serine, Thr-Threonine, Asp-Aspartic acid, Glu- Glutamic acid, Asn-Asparagine, Gln-Glutamine, Lys- Lysine, His-Histidine, Arg-Arginine, Phe-Phenylalanine, Tyr-Tyrosine, Trp-Tryptophan, Cys-Cysteine, Met- Methionine and Pro-Proline.
  • polypeptides according to the invention or antibodies thereto may be administered on their own or with other agents such as 3*-azido-3 •-deoxythymidine (AZT) (zidovudine) , which acts at a different level by interfering with the replication of the genetic material of the virus, and/or HIV protease inhibitors, which block the action of an enzyme essential to the development of the virus.
  • ZT 3*-azido-3 •-deoxythymidine
  • HIV protease inhibitors which block the action of an enzyme essential to the development of the virus.
  • Polypeptides according to the invention may be used to raise antibodies which will cross-react with envelope proteins produced by a wide range of HIV 1 and/or HIV 2 strains. Our analyses have shown that, since the conformational/topographic/electrostatic properties of polypeptides according to the invention are such that they are highly likely to elicit the production of antibodies which will cross-react with HIV envelope proteins from several or many strains, further advantages may arise from combining several variant polypeptides in a larger polypeptide.
  • Such a polypeptide may have the general formula (III) :
  • F and G may each independently be a polypeptide according to any one of Formulae I to lib
  • L is a linking sequence
  • a, b and c are each independently 0 or 1
  • m and n are each positive numbers e.g. between 1 and 10 inclusive.
  • L is preferably a short, conformationally flexible section of polypeptide chain such as, for example and without limit (Seq. I.D. No: 5) Gly-Gly-Gly-Gly-Gly, (Seq. I.D. No: 6) Gly-Pro-Gly-Pro- Gly-Pro or (Seq. I.D. No: 7) Gly-Ser-Ala-Gly-Ser-Gly- Ala. It should be clear that each repeat may optionally have a different variant of a polypeptide according to the invention.
  • Polyvalent determinant analogues as defined by Formula III are referred to as pseudohomopolyvalent, wherein variants of essentially the same determinant analogue are repeated in a single polypeptide chain.
  • simple homopolyvalent polypeptide immunogens which contain multiple copies of the same variant of one of the determinant analogues according to any one of formulae I to lib, would also be expected to be effective, and are also included within the scope of the present invention.
  • Pseudohomopolyvalent immunogenic polypeptides are expected to be particularly valuable as vaccines, where they should elicit the production of a range of (neutralising) antibodies with a similar but non-identical underlying specificity, which between them would cross-react with envelope protein from a wider range of HIV strains, and would thus be more effective at conferring protective immunity.
  • heteropolyvalent polypeptides which contain one or more copies, in any order, of one of the polypeptides according to the present invention and one or more other polypeptide analogues of determinant analogues.
  • Such polypeptides which are provided for in the present invention, have the general formula (IV) :
  • F is a polypeptide according to any one of Formulae I to lib
  • G is a polypeptide according to any one of Formulae I to lib or other sequence
  • m and n are each positive numbers e.g. between 1 and 10 inclusive
  • d and e are each independently 0 or 1.
  • "L" is preferably a short, conformationally flexible section of polypeptide chain such as, for example and without limit (Seq. I.D. No: 5) Gly-Gly-Gly-Gly-Gly, (Seq. I.D. No: 6) Gly-Pro-Gly-Pro-Gly-Pro or (Seq. I.D. No: 7) Gly- Ser-Ala-Gly-Ser-Gly-Ala.
  • any antigenically significant subfragments and/or antigenically significant variants of the above-identified polypeptide sequences which retain the general form and function of the parent polypeptide are included within the scope of this invention.
  • substitution of any of the specific residues by residues having comparable conformational and/or physical properties, including substitution by rare amino acids (e.g. D-stereoisomers) or synthetic amino acid analogues is included.
  • substitution of a residue by another in the same Set is included within the ambit of the invention;
  • D-stereoisomers of all amino acid types may be substituted, for example, D-Phe, D-Tyr and D-Trp.
  • X and Y if present may independently include one or more segments of protein sequence with the ability to act as a T-cell epitope.
  • Lys, Arg, His, Glu, Asp, Asn, Gin, Ser, Thr, Pro appear to act as T-cell epitopes in at least some instances (Rothbard, J.B. & Taylor, W.R. (1988) . A sequence pattern in common to T-cell epitopes. The EMBO Journal 7(1): 93-100). Similarly segments can be of the sequence 1'-2*-3 '-4*-5* , wherein l 1 is equivalent to 1 as defined earlier, 2 1 to 2, 3* and 4 1 to 3, and 5" to 4 (ibid).
  • each epitope may be of the type defined above or may be of other structure and may be separated by spacer segments of any length or composition (preferably less than five amino acid residues in length) and comprise for example residues selected from Gly, Ala, Pro, Asn, Thr, Ser or polyfunctional linkers such as non- ⁇ amino acids. It is possible for a C- or N-terminal linker to represent a complete protein, thus obviating the possible need for conjugation to a carrier protein.
  • analogue according to the invention and containing a retro-inverso amino acid may have the formula:
  • R is any functional group, e.g. a glycine side chain
  • Al and A2 are preferably each a copy of one of the analogues defined herein (but not necessarily the same) attached by its N- or C-terminal end.
  • T-cell epitopes may optionally be included as discussed earlier.
  • Retro-inverso modification of peptides involves the reversal of one or more peptide bonds to create analogues more resistant than the original molecule to enzymatic degradation and offer one convenient route to the generation of branched immunogens which contain a high concentration of epitope for a medium to large immunogen.
  • the use of these compounds in large-scale solution synthesis of retro-inverso analogues of short- chain biologically active peptides has great potential. It should be noted that analogues incorporating retro-inverso amino acid derivatives cannot be made directly using a recombinant DNA system.
  • the basic analogues can, and they can then be purified and chemically linked to the retro-inverso amino-acids using standard peptide/organic chemistry.
  • a practical and convenient novel procedure for the solid-phase synthesis on polyamide-type resin of retro-inverso peptides has been described recently [Gazerro, H., Pinori, M. & Verdini, A.S. (1990) .
  • polypeptides are optionally linked to a carrier molecule, either through chemical groups within the polypeptides themselves or through additional amino acids added at either the C- or N-terminus, and which may be separated from the polypeptides themselves or surrounded by one or more additional ami ⁇ o acids, in order to render them optimal for their immunological function.
  • linkages are suitable and include for example use of the side chains of Tyr, Cys and Lys residues.
  • Suitable carriers include, for example, purified protein derivative of tuberculin (PPD) , tetanus toxoid, cholera toxin and its B subunit, ovalbumin, bovine serum albumin, soybean trypsin inhibitor, muramyl dipeptide and analogues thereof, and Braun's lipoprotein although other suitable carriers will be readily apparent to the skilled person.
  • PPD tuberculin
  • tetanus toxoid cholera toxin and its B subunit
  • ovalbumin bovine serum albumin
  • soybean trypsin inhibitor muramyl dipeptide and analogues thereof
  • Braun's lipoprotein although other suitable carriers will be readily apparent to the skilled person.
  • multiple antigen peptides may be used such as those comprising a pol lysyl core, e.g. heptalysyl, bearing reactive amino termini.
  • Polypeptide antigens according to the invention may be reacted with, or synthesised
  • PPD is expected to be a preferred carrier for use in such countries.
  • the mode of coupling the polypeptide to the carrier will depend on the nature of the materials to be coupled.
  • a lysine residue in the carrier may be coupled to a C-terminal or other cysteine residue in a polypeptide by treatment with N- ⁇
  • polypeptides may be administered by any route (eg parenteral, nasal, oral, rectal, intra-vaginal) , with or without the use of conventional adjuvants (such as aluminium hydroxide or, when not for administration to humans, Freund's complete or incomplete adjuvants) and/or other immunopotentiating agents.
  • adjuvants such as aluminium hydroxide or, when not for administration to humans, Freund's complete or incomplete adjuvants
  • the invention also includes formulation of polypeptides according to the invention in slow-release forms, such as a sub- dermal implant or depot comprising, for example, liposomes (Allison, A.C. & Gregoriadis, G.
  • polypeptides according to the invention may be synthesised by any conventional method, either directly using manual or automated peptide synthesis techniques as mentioned above, or indirectly by RNA or DNA synthesis and conventional techniques of molecular biology and genetic engineering. Such techniques may be used to produce hybrid proteins containing one or more of the polypeptides inserted into another polypeptide sequence.
  • Another aspect of the present invention therefore provides a DNA molecule coding for at least one synthetic polypeptide according to the invention, preferably incorporated into a suitable expression vector replicable in microorganisms or in mammalian, insect, plant, fungal or other cells.
  • the DNA may also be part of the DNA sequence for a longer product e.g. the polypeptides may be expressed as parts of other proteins into which they have been inserted by genetic engineering.
  • One practical guide to such techniques is "Molecular cloning: a laboratory manual" by Sambrook, J., Fritsch, E.F. and Maniatis, T. (2nd Edition, 1989).
  • Polypeptides according to the invention may be used either alone or linked to an appropriate carrier, as: (a) Peptide vaccines, for use to prevent infection by one or more strain of HIV; (b) As ligands in assays of, for example, sera from HIV positive patients;
  • the invention further provides for genetically engineered forms or sub-components, especially V H regions, of antibodies raised against the polypeptides, and of humanised forms of antibodies initially raised against the polypeptides in other animals, using techniques described in the literature; and
  • a further aspect of the invention provides a kit for detecting HIV or antibodies against HIV which comprises at least one synthetic polypeptide according to the invention.
  • the preparation of polyclonal or monoclonal antibodies, humanised forms of such antibodies see, for example, Thompson K. M. et al (1986) Immunology .58., 157- 160) , single domain antibodies (see, for example, Ward, E. S., Gussow, D. , Griffiths, A. D. , Jones, P. and
  • Antibodies according to the invention are, inter alia, of use in a method of diagnosing mammalian HIV infection which comprises incubating a sample of tissue or body fluid of mammal with an effective amount of antibody as described herein and determining whether, and if desired the extent to which and/or rate at which, cross-reaction between said sample and said antibody occurs. Diagnostic kits which contain at least one of said antibodies also form part of this invention.
  • a further aspect of the invention provides synthetic polypeptides for use in therapy or prophylaxis of mammalian HIV infection and/or stimulating the mammalian immune system and/or blocking the cellular receptors for the HIV virus and for the preparation of medicaments suitable for such uses.
  • pharmaceutical compositions containing, as active ingredient, at least one polypeptide or polypeptide-carrier conjugate as described herein in association with one or more pharmaceutically acceptable adjuvant carrier and/or excipient.
  • the compositions may be formulated for oral, rectal, nasal or especially parenteral administration (including intra-CNS administration) .
  • the invention further provides a method of therapy or prophylaxis of mammalian HIV infection and/or of stimulating the mammalian immune system and/or of blocking the cellular receptors for the HIV virus, which comprises administering an effective amount of a polypeptide as hereinbefore defined, either in isolation or in combination with other agents for the treatment of AIDS such as AZT and/or inhibitors of the HIV protease.
  • a polypeptide as hereinbefore defined, either in isolation or in combination with other agents for the treatment of AIDS such as AZT and/or inhibitors of the HIV protease.
  • the C-terminal Gly-Cys-Ala was introduced to provide an alternative coupling site to the carrier.
  • the peptide was cleaved from the resin in the presence of trifluoroacetic acid and subsequent purification of peptide was achieved by gel filtration, ion exchange chromatography and reverse-phase high performance liquid chromatography. The purity of the resultant peptide was in excess of 85%.
  • the C-terminal alanine was included to assist the conjugation.
  • the peptide was dissolved in phosphate-buffered saline (PBS; 5mg/ml) and mixed with an equal* volume of ovalbumin (5mg/ml) prior to the addition of glutaraldehyde to a final concentration of 0.1%(w/v).
  • PBS phosphate-buffered saline
  • ovalbumin 5mg/ml
  • the conjugate mixture was allowed to stand for 30 minutes prior to emulsi ication with Freund's adjuvant.
  • Each sheep (5 animals in each group) was immunised with the equivalent of 250 ⁇ g of peptide in Freund's Complete Adjuvant (FCA) .
  • FCA Freund's Complete Adjuvant
  • Each animal was given a booster injection at 2 weeks and then again at 5-6 weeks with a " similar amount of peptide in Freund's Incomplete Adjuvant (FIA) .
  • Blood samples were taken 7-10 days after the final booster injection and assayed for binding to HIV gpl60
  • HIV-1 strains Two HIV-1 strains were used in the study. One of these was a fully-characterised, well-recognised and widely-used HIV-1 strain named GB8 available from Dr G. Farrarr, Centre for Applied Microbiology and Research (CAMR) , Porton Down, Salisbury, UK and discussed in AIDS 1: 147-150 (1987). The other was the RF strain of HIV-1 used in the syncytiu inhibition assay. RF strain of HIV-1 is known from Science 224: 497-500 (1984) . Sequences from two or more HIV-1 isolates from a single individual have been obtained and the capabilities of the test materials to inhibit growth and replication of these virus strains were assessed comparatively. The isolates were obtained from Dr. G.
  • Farrarr under designations GB8A, GB8B and GB8D.
  • Cell lines H9, C8166 and . JM are known to support replication or show syncytium formation when infected with HIV-1 'laboratory strains' of GB8 or RF.
  • This assay measures the capability of the anti-peptide antisera to prevent attachment and infection of susceptible cells by live, cell-free virus. The measurement of this is determined by syncytium counts.
  • Test antisera were inactivated by heat at 56°C for 30 minutes, diluted appropriately in RPMI 1640 and incubated with an equal volume of HIV-1 containing fluid of known titre for 30 minutes at room temperature (RT) .
  • a CD4+ cell line (JM) at a known concentration and highly susceptible to infection with HIV-1 GB8 strain was exposed to the HIV-1 antiserum mixture, left for 2 hours at 37°C, washed, resuspended in cell culture medium, incubated and assessed by syncytium counts at specified times. Neutralising activity of the antisera was assessed by reduction in syncytium counts compared to controls.
  • Controls included with each batch of tests were known negative and positive sera replacing the test antiserum in an otherwise identical procedure.
  • This assay determines the ability of the anti-peptide antisera to inhibit replication of HIV-1 subsequent to cellular infection and measures levels of extracellular and intracellular HIV-1 p24 antigen. In addition, syncytium detection and enumeration are performed.
  • a highly susceptible CD4+ cell line was exposed to HIV-1 at a known multiplicity of infection and incubated for 2 hours at 37°C. The cells were then washed 3 times with RPMI 1640 and an equal volume of appropriately diluted, heat-inactivated test antiserum was added. The infected cell serum suspension was incubated for 1 hour at 37°C, examined microscopically and the culture medium sampled at specified time-points subsequently (at days 3 and 5) for measurement of p24 antigen. Intracellular p24 antigen levels were determined at similar time points following lysis of the cells using appropriate lysis buffer. Samples to be tested .for HIV-1 p24 antigen by ELISA were stored until the assays were performed. Syncytium counts were measured on day 5.
  • Controls for each batch of tests included known negative and positive sera replacing the test antiserum in an otherwise identical procedure.
  • This assay determines the capability of anti-peptide antisera to prevent spread of live HIV-1 from infected to uninfected cells and to prevent fusion between cells mediated through reactivity between the virus glycoprotein (gpl60) and the CD4 molecule.
  • the measurement is carried out by syncytium detection and enumeration.
  • Controls for each batch of tests included known positive and negative sera replacing the test serum in an otherwise identical procedure.
  • Sera 17 and 37 show comparable activity in terms of reduction in syncytium counts at day 5 in the replication inhibition assay as demonstrated in Table 2. Further, replication is prevented as indicated by p24 antigen determination. It should be noted that the sera used in this assay were not diluted. Table 2: Assay for replication inhibition of HIV-1 (strain GB8) by anti-peptide sheep sera in vitro
  • Table 3 gives data for serum 17 in the syncytium inhibition assay. A dilution of 1:100 reduced the inhibiting activity of the serum to a little less than 50%. This test suggests how even diluting the serum may prevent cell fusion and hence spread of live HIV.
  • W091/00903 is a known to be an immunodominant peptide related to the HIV envelope protein and is known to recognise substantially all HIV-1 infected individuals. This peptide was used to investigate cross-reactivity and confirm the suitability of the protocol.
  • the sera to be tested were diluted 1/100 or 1/200 using dilution buffer. 0.1 ml was added to each well, and the plates incubated for 1 hour at 37°C. This was done in triplicate for each sample. 5. The plates were washed three times for 2 minutes with washing buffer.
  • the human sera samples tested were as follows:
  • control contained no serum, but the total volume was maintained by addition of 0.1 ml of dilution buffer.
  • HIV-1+ serum samples had detectable antibodies to the HIV-1 (10A) peptide.
  • Table 4 Reactivity of HIV positive sera with peptides 2A, 2B and 10A.
  • HIV-2+ sera from a number of patients contain antibodies which react specifically with the HIV-2 peptides according to the formulae Ila and lib of the invention.
  • mice Immunisation of mice with a peptide according to formula la conjugated to a carrier protein
  • Example 1 was synthesised and purified as described in Example 1.
  • the peptide was conjugated to ovalbumin using either glutaraldehyde or MBS (M-maleimidobenzoyl-N-hydroxy- succinimide ester) as the coupling agent.
  • Glutaraldehyde The peptide was dissolved in PBS (5 mg/ml) and ovalbumin added to give a molar ratio of 50 amino acids of carrier per 1 mole of peptide. The total volume was adjusted to 2 ml with PBS. 2 ml of 0.2% solution of glutaraldehyde in PBS was slowly added to the stirring mixture and the solution left stirring at room temperature for 1 hour. The mixture was dialysed against large volumes of PBS overnight and stored at -20°C for not longer than 3 weeks.
  • MBS 0.1 ml of a solution of MBS (25 mg/ml in PBS) was added to a stirring solution of ovalbumin (10 mg/ml in PBS) and the reaction mixture stirred at room temperature for 30 mins.
  • the activated ovalbumin was then separated from free MBS by gel filtration on a Sephadex G-25 column equilibrated with PBS.
  • Peptide was dissolved in PBS and added to the activated ovalbumin so that a final concentration of 1 mole of peptide for each 50 amino acids of carrier resulted.
  • the pH was adjusted to 7-7.5 and the reaction stirred for three hours at room temperature.
  • the mixture was dialysed against large volumes of PBS overnight and stored at -20°C for not longer than 3 weeks.
  • a procedure described by Habeeb can be used (Habeeb, A.F.S.A. , Anal. Biochem. , 14, 328 (1966)).
  • the free amino acids are determined by the trinitrobenzene sulphonic acid (TNBS) method using borate buffer, pH 9.0.
  • the absorbence of the solution is read at 335 nm and the percentage of modified free amino acid groups is calculated.
  • the free thiol assay as described by Ellman can also be used to assess the efficiency of conjugation (Anderson, W.L. and Wetlaufer, D.B., Anal. Biochem., 67. 493 (1975))).
  • mice C57 Black mice were pre-bled 2-3 days before the primary inoculation, the sera separated and stored at -20°C. This was the negative control mouse sera.
  • Each mouse was immunised with 40 ⁇ g of peptide in FCA and subsequently with 20 ⁇ g of peptide in FIA 2 to 3 weeks later.
  • the inoculation volume was 0.1 ml in each case and all injections were given subcutaneously. Animals were bled immediately prior to the booster injection, and then two weeks and four weeks later.
  • the ELISA protocol was as described in Example 2, except that sera were diluted 1/50 and 1/100 in dilution buffer, and the antibody used was alkaline phosphatase conjugated goat anti-mouse IgG.
  • mice had high levels of antibodies to the peptide after only one immunisation with the peptide according to formula la conjugated to ovalbumin by the specific linkage using MBS. This is a strong and specific response.

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EP93909057A 1992-04-16 1993-04-16 Synthetic polypeptides derived from the hiv envelope glycoprotein Withdrawn EP0636145A1 (en)

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GB9208428 1992-04-16
GB929208428A GB9208428D0 (en) 1992-04-16 1992-04-16 Synthetic polypeptides
PCT/GB1993/000808 WO1993021218A1 (en) 1992-04-16 1993-04-16 Synthetic polypeptides derived from the hiv envelope glycoprotein

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JP (1) JPH07505878A (ko)
KR (1) KR950700933A (ko)
CN (1) CN1082053A (ko)
AU (1) AU3960493A (ko)
CA (1) CA2118033A1 (ko)
GB (1) GB9208428D0 (ko)
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US6210873B1 (en) 1987-08-28 2001-04-03 Board Of Regents, The University Of Texas System Methods and compositions for the priming of specific cytotoxic T-lymphocyte response
US5128319A (en) 1987-08-28 1992-07-07 Board Of Regents, The University Of Texas System Prophylaxis and therapy of acquired immunodeficiency syndrome
KR950702839A (ko) 1992-08-27 1995-08-23 스티븐 딕 레트로, 인버소-, 및 레트로- 인버소 합성 펩티드 유사체(retro-, inverso-, and retro-inverso synthetic peptide analogues)
US5603933A (en) * 1993-08-31 1997-02-18 Board Of Regents, The University Of Texas CD4 peptides for binding to viral envelope proteins
AUPM411994A0 (en) * 1994-02-25 1994-03-24 Deakin Research Limited Epitopes
FR2717081B1 (fr) * 1994-03-14 1996-06-21 Centre Nat Rech Scient Rétropeptides, anticorps dirigés contre ces derniers, et leurs utilisations pour la vaccination et le diagnostic in vitro.
US5705522A (en) * 1995-09-15 1998-01-06 Compagnie De Developpement Aguettant S.A. Compounds having anti-inflammatory and anti-viral activity, compositions of these, alone and in combination with reverse transcriptase inhibitors
CN100366633C (zh) * 2006-04-18 2008-02-06 河北师范大学 棕点湍蛙抗病毒多肽及其在制药中的应用
JP2008173077A (ja) * 2007-01-22 2008-07-31 National Institute Of Advanced Industrial & Technology 膜タンパク質の発現を解析するモニタータンパク質

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GB8714802D0 (en) * 1987-06-24 1987-07-29 Proteus Biotech Ltd Synthetic polypeptides
EP0330359A3 (en) * 1988-02-25 1991-06-05 Bio-Rad Laboratories, Inc. Composition useful in the diagnosis and treating of hiv-1 infection
WO1989010416A1 (en) * 1988-04-20 1989-11-02 Trustees Of The University Of Pennsylvania PROTECTIVE PEPTIDES DERIVED FROM HUMAN IMMUNODEFICIENCY VIRUS-1 gp160
CA2003383A1 (en) * 1988-11-23 1990-05-23 Sushil G. Devare Synthetic dna derived recombinant hiv antigens
GB9005829D0 (en) * 1990-03-15 1990-05-09 Proteus Biotech Ltd Synthetic polypeptides

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JPH07505878A (ja) 1995-06-29
ZA932648B (en) 1994-10-15
CN1082053A (zh) 1994-02-16
KR950700933A (ko) 1995-02-20
AU3960493A (en) 1993-11-18
WO1993021218A1 (en) 1993-10-28
CA2118033A1 (en) 1993-10-28

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