AU3960493A - Synthetic polypeptides derived from the HIV envelope glycoprotein - Google Patents
Synthetic polypeptides derived from the HIV envelope glycoproteinInfo
- Publication number
- AU3960493A AU3960493A AU39604/93A AU3960493A AU3960493A AU 3960493 A AU3960493 A AU 3960493A AU 39604/93 A AU39604/93 A AU 39604/93A AU 3960493 A AU3960493 A AU 3960493A AU 3960493 A AU3960493 A AU 3960493A
- Authority
- AU
- Australia
- Prior art keywords
- leu
- hiv
- gln
- polypeptide
- gly
- 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.)
- Abandoned
Links
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- BDHFUVZGWQCTTF-UHFFFAOYSA-N sulfonic acid Chemical compound OS(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-N 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 239000003656 tris buffered saline Substances 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 229960001005 tuberculin Drugs 0.000 description 1
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Description
SYNTHETIC POLYPEPTIDES DERIVED FROM THE HIV ENVELOPE GLYCOPROTEIN.
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) . Within the last decade AIDS has emerged as an important medical problem throughout the world and there is currently an urgent need for agents for the study, diagnosis, treatment and/or prevention of infection by the HIV, the causative agent of the disease. With the availability of the amino acid sequences of proteins produced by the HIV 1 and HIV 2 viruses (see, for example, Ratner, L. et al., Nature 313. 277 (1985); Meusing, M.A. et al., Nature 313, 450 (1985); Wain- Hobson, S. et al., Cell 4_0 , 9 (1985)), it has been possible to devise synthetic polypeptides which emulate the antigenic properties of the viral envelope proteins.
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) :-
X-R1-Leu-R2-Leu-Thr-Val-Trp-Gly-R*5-Lys-Y
(I) wherein R1 is selected from Gln-Gln-R4—R5, Gln-R4-
H~, R4-Rgr j or R.J is absent; R2 iiss aann aammiinnoo aacciidd rreessii<due selected from Gin, Lys, Glu or Arg;
R- iiss aann aammiinnoo aacciidd residue selected from lie, Thr or Ala;
R4 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 R1 is Gln-Gln-R4-R5 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. However, when X or Y are present they may be any length but preferably less than 20 amino acids, more preferably less than 10, eg. 3 to 6. It will of course be appreciated that 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.
Preferably, if R1 is present, R5 is either Leu or Met, more preferably Leu, and R4 if present, is His or Glu, more preferably His. R2 is preferably Gin, Lys or Glu, more preferably Gin and R- is preferably lie. Preferably in a sequence according to formula (I) , R1 is Gln-Gln-R-R5 or is absent and when present, R4 is preferably His and R5 is Leu. Also it is preferable that R2 is Leu and R3 is Gin.
Preferred forms of polypeptide of formula (I) according to the invention consist of the sequences:-'
Seq. I.D. No: 1
X-Gln-Gln-His-Leu-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Y
(la) ; and
Seq. I.D. No: 2
X-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Y (lb)
wherein X and Y are as defined above although it is preferred that if X or Y are present they are relatively short sequences. Preferably, X is absent and Y is 2 or 3 residues long, e.g. Gly-Cys or Gly-Cys-Ala. With regard to sequences la and lb above, 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.
Another preferred form of polypeptide according to the invention consists substantially of an amino acid sequence of formula (II) :-
X-R1-Leu-Arg-Leu-Thr-Val-Trp-Gly-R3-Lys-Y (II)
wherein R_, is selected from Gln-Gln-Glu-R5, Gln-
Glu-R5, Glu-Rj, R- or R1 is absent; R3 is Thr or Ala; R5 is Met or Leu; and X and Y are as defined above.
Preferably, R5, if present, is Met and R3 is preferably Thr. Also, it is preferable that Rt is Gln- Gln-Glu-Met or absent. Preferred forms of polypeptide according to formula II consist of the sequences:
Seq. I.D. No: 3
X-Gln-Gln-Glu-Met-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Y (Ha) ; and
Seq. I.D. No: 4
X-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Y (lib) '.
Preferably, 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. For example, 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. The correctness of the structure and the level of purity, which will normally be in excess of 85%, should be carefully checked. Various 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. three-letter-code abbreviations for 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. For therapeutic applications, 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.
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) :
[ a-F]m-[Lb-G]n-Lc (III)
wherein 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 and 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. In addition, 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. There would also be advantages in constructing 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) :
Ld-[G-L]m-F-[L-G]n-Le (IV)
wherein 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, and 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.
It is to be understood that 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. In particular, the 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. For example, substitution of a residue by another in the same Set, as defined below, is included within the ambit of the invention; Set 1 - Ala, Val, Leu, lie, Phe, Tyr, Trp and Met; Set 2 - Ser, Thr, Asn and Gin; Set 3 - Asp and Glu; Set 4 - Lys, His and Arg; Set 5 - Asn and Asp; Set 6 - Glu and Gin; Set 7 - Gly, Ala, Pro, Ser and Thr. D-stereoisomers of all amino acid types, may be substituted, for example, D-Phe, D-Tyr and D-Trp.
In preferred embodiments of the invention, 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. For example, segments of amino acid sequence of the general formula 1-2-3-4, where 1 is Gly or a charged amino acid (e.g. Lys, His, Arg, Asp or
Glu) , 2 is a hydrophobic amino acid (e.g. lie, Leu, Val, Met, Tyr, Phe, Trp, Ala) , 3 is either a hydrophobic amino acid (as defined above) or an uncharged polar amino acid (e.g. Asn, Ser, Thr, Pro, Gin, Gly) , and 4 is a polar amino acid (e.g. 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 l1 is equivalent to 1 as defined earlier, 21 to 2, 3* and 41 to 3, and 5" to 4 (ibid). Both forms are included within the scope of the present invention and ■ one or more T-cell epitopes (preferably less than five) may be incorporated into a polypeptide according to any one of formulae I to lib. Therefore 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-α 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.
Also included within the scope of this invention are derivatives of the polypeptide according to formula I in which X or Y are or include a "retro-inverso" amino acid, i.e. a bifunctional amine having a functional group corresponding to an amino acid. For example an analogue according to the invention and containing a retro-inverso amino acid may have the formula:
R I Al - N - C - N - A2
I I I H H H
where R is any functional group, e.g. a glycine side chain, and 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. However, 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) . A new general procedure for the solid-phase synthesis of retro-inverso peptides. In "Innovation and Perspectives in Solid Phase Synthesis" Ed. Roger Epton. SPCC (UK) Ltd, Birmingham, UK] .
The 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 amiήo acids, in order to render them optimal for their immunological function. Many 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. For example, 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 on, the amino termini and different polypeptide antigens may be reacted with the same core or carrier.
When using PPD as a carrier for polypeptides according to the invention, a higher titre of antibodies is achieved if the recipient of the polypeptide-PPD conjugate is already tuberculin sensitive, e.g. by virtue of earlier BCG vaccination. In the case of a human vaccine it is worth noting that in the UK and many other countries the population is routinely offered BCG vaccination and is therefore largely PPD-sensitive.
Hence 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. For example, a lysine residue in the carrier may be coupled to a C-terminal or other cysteine residue in a polypeptide by treatment with N-γ
-malei idobutyryloxy-succinimide (Kitagawa, T. & Ackawa, T. (1976) J. Biochem. 79., 233) . Other coupling reactions and reagents have been described in the literature.
The polypeptides, either alone or linked to a carrier molecule, 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. 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. (1974) Nature (London) 252, 252) or biodegradable microcapsules manufactured from co-polymers of lactic acid and glycolic acids (Gresser, J. D. and Sanderson, J. E. (1984) in "Biopolymer Controlled Release Systems" pp 127-138, Ed. D. L. Wise). It is to be understood that the 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;
(c) As quality control agents in testing, for example, binding levels of antibodies raised against the
polypeptides;
(d) As immunogenic agents for the generation of monoclonal or polyclonal antibodies by immunisation of an appropriate animal, such antibodies being of use or (i) the scientific study of the HIV virus, (ii) as diagnostic agents, e.g. as part of histochemical reagents, (iii) for the passive immunisation of HIV patients, either as a treatment for AIDS in itself, or in combination with other agents such as, for example AZT and/or HIV protease inhibitors, and (iv) as a means of targeting other agents (e.g. AZT or HIV protease inhibitors) to HIV infected cells expressing HIV envelope proteins on their surfaces, such agents either . being linked covalently or otherwise associated, e.g. as in liposomes containing such agents and incorporating antibodies raised against any of the antigenic polypeptides. The invention further provides for genetically engineered forms or sub-components, especially VH 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
(e) The treatment of HIV infections, either by displacing the binding of HIV virus to human or animal cells or by disturbing the three-dimensional ' organisation of the virus in vivo; as well as aiding the scientific study of HIV viruses in vitro.
In respect of detection and diagnosis, of HIV or antibodies against HIV, the skilled person will be aware of a variety of im unoassay techniques known in the art, inter alia, sandwich assay, competitive and non- competitive assays and the use of direct and indirect labelling. 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
Winter, G. (1989) Nature 341. 544-546) , and antibodies which might cross the blood-brain barrier, which bind specifically to a synthetic polypeptide according to the present invention, may be carried out by conventional means and such antibodies are considered to form part of this invention. 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. Also included are 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. The following examples are intended to illustrate the invention and are not limiting in any way.
Example 1
A C-terminally extended form of peptide lb of sequence (Seq. I.D. No: 8)
Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Gly-Cys-Ala
was synthesised using standard solid-phase F-moc methodologies (Atherton, E. and Sheppard, R.C. , 1985;
J.Chem. Soc. Comm. 165-166) . 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). 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 μg of peptide in Freund's Complete Adjuvant (FCA) . 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 envelope protein.
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.
(a) Assays of inhibition of live HIV-I anti-peptide antisera
Three standard assay procedures have been used to assess the inhibition of live HIV-1 by anti-peptide antisera.
(i) Neutralisation assay
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.
(ii) Inhibition Assay
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.
(iii) Syncytium assay
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.
A known concentration of an HIV-1 producer (CD4+) cell line supporting active virus replication (H9 cells chronically infected with HIV-1 RF strain) was washed 3 times and mixed with the antiserum under test used at specified dilutions. After incubation for 30 minutes at 37°C these cells were mixed in specified proportions with an indicator CD4+ cell line (C8166) , highly susceptible to HIV-1 infection and syncytium induction. These cells were observed daily for syncytium formation.
Controls for each batch of tests included known positive and negative sera replacing the test serum in an otherwise identical procedure.
RESULTS
Neutralisation assay
A number of sera raised to peptide lb exhibited in vitro neutralising activity against HIV-1 GB8 strains. Table 1 indicates greater activity of serum 17 against all GB8 sequential isolates. The assay demonstrates that the sera neutralise cell-free virus.
Table 1 : Anti-peptide sheep sera showing in vitro neutralising activity against HIV-1 GB8 strains
* = mean no. of synctia in test/mean no. of syncytia in control (% reduction) (-) = reduction < 50%
Replication inhibition assay
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
P9 = positive human serum NSS = normal sheep serum
Syncytium inhibition assay
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.
Table 3: Syncytium inhibiting activing of anti-peptide against HIV-1 (strain H9-RF) in vitro
* = Mean number of syncytia in test/Mean number of syncytia in control (% reduction)
Example 2
ELISA reactivity of HIV-2 positive human sera with peptides according to formulae Ila and lib
A C-terminally extended peptide of formula Ha having the sequence (Seq. I.D. No: 9)
Gln-Gln-Glu-Met-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr- Lys-Gly-Cys (2A)
and a C-terminally extended peptide of formula lib having the sequence (Seq. I.D. No: 10)
Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Gly-Cys (2B)
were synthesised and purified as described in Example 1. The peptides were then used in an ELISA with HIV positive human sera. Peptide "10A" (disclosed in Example 1 of our copending application published as
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.
ELISA protocol
1. 100 μl of coating buffer, containing 20 μg/ml of peptide, was added to each well of the ELISA plates
(Dynetech) and the plates incubated overnight at 4°C.
2. Plates were washed once in Tris buffered saline and dried thoroughly.
3. 200 μl of blocking buffer was added to each well, the plates shaken for 10 seconds and incubated for 1 hour at 37°C.
4. 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.
6. 100 μl of alkaline phosphatase conjugated goat anti-human IgG (1/2000 in dilution buffer) was added to each well and plates incubated for 1 hour at 37°C.
7. The wells were washed as in (5) .
8. 50 μl of alkaline phosphatase buffer was added to each well followed by 50 μl of substrate (10 mg/ml in water) and the plates incubated for 15 mins at 37°C. 9. The absorbence was read immediately at 414 nm.
The human sera samples tested were as follows:
20 known HIV-2 positive sera that had previously been shown to exhibit no cross-reactivity with HIV-1 sera. All twenty patients came from the Ivory Coast.
5 known HIV-2 positive sera that had previously been shown to exhibit no cross-reactivity with HIV-1 sera. All five patients came from Africa but not the Ivory Coast. 7 known HIV-1 positive sera that had been shown to exhibit no cross-reactivity with HIV-2 sera.
10 known HIV negative sera samples from age and sex matched subjects.
The control contained no serum, but the total volume was maintained by addition of 0.1 ml of dilution buffer.
It is clear from the results in Table 4 below that :
1) No cross reactivity between HIV-1 and HIV-2 peptides was observed, that is- HIV-2 sera did not recognise the HIV-1 peptide and HIV-1 sera did not recognise the HIV-2 peptides;
2) No peptide specific activity was found in the HIV negative samples;
3) There is a distinction in reactivities between sera of patients who came from the Ivory Coast, and patients who came from other parts of the African continent; and
4) The 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.
s expec e , no posi ive resu s occurre wi e con ro
These results show that 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.
Example 3
Immunisation of mice with a peptide according to formula la conjugated to a carrier protein
A C-terminally extended according to formula la having the sequence (Seq. I.D. No: 11)
Gln-Gln-His-Leu-Leu-Gln-Leu-Thr-Val-Trp- Gly-Ile-Lys-Gly-Cys (1A)
was synthesised and purified as described in Example 1.
Conjugation
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.
To determine the peptide/carrier protein ratio a procedure described by Habeeb can be used (Habeeb, A.F.S.A. , Anal. Biochem. , 14, 328 (1966)). In this, 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))).
Immunisation
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 μg of peptide in FCA and
subsequently with 20 μg 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.
ELISA
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.
Results
1. 2/24 (8%) animals responded to the peptide according to formula la conjugated using glutaraldehyde when compared to controls after the third bleeding. Thus it is possible to raise antibodies against this peptide when it has been non-specifically conjugated to a carrier.
2. 14/24 (58%) of the 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.
The results show that the peptide according to formula la, when conjugated to ovalbumin using MBS, is a powerful immunogen inducing very high levels of antibody after a single immunisation and that antibodies can be raised even if the peptide is non-specifically bound to ovalbumin.
SEQUENCE LISTING Number of Sequences 11
(1) Information for Seq. I.D. No: 1
(i) Characteristation of sequence:
(A) Length: 13 Amino acids
(B) Type: Amino acid (D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 1
Gin Gin His Leu Leu Gin Leu Thr Val Trp Gly lie Lys 1 5 10
(2) Information for Seq. I.D. No: 2
(i) Characteristation of sequence:
(A) Length: 9 Amino acids
(B) Type: Amino acid (D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 2
Leu Gin Leu Thr Val Trp Gly lie Lys 1 5
(3) Information for Seq. I.D. No: 3
(i) Characteristation of sequence:
(A) Length: 13 Amino acids
(B) Type: Amino acid (D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 3
Gin Gin Glu Met Leu Arg Leu Thr Val Trp Gly Thr Lys 1 5 10
(4) Information for Seq. I.D. No: 4
(i) Characteristation of sequence:
(A) Length: 9 Amino acids
(B) Type: Amino acid (D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 4
Leu Arg Leu Thr Val Trp Gly Thr Lys 1 5
(5) Information for Seq. I.D. No: 5
(i) Characteristation of sequence:
(A) Length: 5 Amino acids
(B) Type: Amino acid (D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 5
Gly Gly Gly Gly Gly 1 5
(6) Information for Seq. I.D. No: 6
(i) Characteristation of sequence:
(A) Length: 6 Amino acids
(B) Type: Amino acid (D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 6
Gly Pro Gly Pro Gly Pro 1 5
(7) Information for Seq. I.D. No: 7
(i) Characteristation of sequence:
(A) Length: 7 Amino acids
(B) Type: Amino acid
(D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 7
Gly Ser Ala Gly Ser Gly Ala 1 5
(8) Information for Seq. I.D. No: 8
(i) Characteristation of sequence:
(A) Length: 12 Amino acids
(B) Type: Amino acid (D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 8
Leu Gin Leu Thr Val Trp Gly lie Lys Gly Cys Ala 1 5 10
(9) Information for Seq. I.D. No: 9
(i) Characteristation of sequence:
(A) Length: 15 Amino acids
(B) Type: Amino acid (D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 9
Gin Gin Glu Met Leu Arg Leu Thr Val Trp Gly Thr Lys Gly Cys 1 5 10 15
(10) Information for Seq. I.D. No: 10
(i) Characteristation of sequence:
(A) Length: 11 Amino acids
(B) Type: Amino acid (D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 10
Leu Arg Leu Thr Val Trp Gly Thr Lys Gly Cys 1 5 10
(11) Information for Seq. I.D. No: 11
(i) Characteristation of sequence:
(A) Length: 15 Amino acids
(B) Type: Amino acid (D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. No: 11
Gin Gin His Leu Leu Gin Leu Thr Val Trp Gly lie Lys Gly Cys 1 5 10 15
Claims (29)
1. A synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV) , said polypeptide consisting substantially of an amino acid sequence of formula (I):-
X-R.,-Leu-R-,-Leu-Thr-Val-Trp-Gly-R3-Lys-Y
(I) wherein R1 is selected from Gln-Gln-R4-R5, Gln-R4-Rg,
R4-R5, R- or R. is absent; R2 is an amino acid residue selected from
Gin, Lys, Glu or Arg; R3 is an amino acid residue selected from lie, Thr or Ala; R4 is His or Glu; R5 is Leu or Met; and X and Y may each independently be absent or independently be one or more amino acid residues.
2. A synthetic polypeptide as claimed in claim 1 wherein R- is selected from Gin, Lys and Glu, and R3 is lie.
3. A synthetic polypeptide as claimed in claim 1 or claim 2 consisting of the sequence (Seq. I.D. No: 1) X-Gln-Gln-His-Leu-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Y
(la); or (Se;. I.D. No: 2)
X-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Y (lb)
wherein X and Y are as defined in claim 1.
4. A synthetic polypeptide as claimed in claim 1 consisting substantially of an amino acid sequence of formula (II):-
X-R^Leu-Arg-Leu-Thr-Val-Trp-Gly-Rj-Lys-Y (II) wherein R1 is selected from Gln-Gln-Glu-R5, Gln-Glu- R5, Glu-R5, R5 or R1 is absent; R3 is Thr or Ala; R5 is Met or Leu; and X and Y are as defined in claim 1.
5. A synthetic polypeptide as claimed in claim 4 consisting of the sequence (Seq. I.D. No: 3)
X-Gln-Gln-Glu-Met-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Y
(Ila) ; or (Seq. I.D. No: 4) X-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Y (lib) ;
6. A synthetic polypeptide as claimed in any one of the preceding claims wherein X is absent and is Y Gly-Cys or Gly-Cys-Ala.
7. A synthetic polypeptide of general formula (III)
[-- -F] -[Lh-G] -L. (HI)
wherein 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 and m and n are each positive numbers e.g. between 1 and 10 inclusive.
8. A synthetic polypeptide of general formula (IV):
L-tG-L*] m-F-fL-G3π-Le (IV)
wherein 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, and d and e are each independently 0 or 1.
9. A synthetic polypeptide which comprises an antigenically sigificant subfragment and/or antigenically significant variant of any one of the polypeptide sequences as claimed in claims 1 to 8.
10. A synthetic polypeptide as claimed in any one of claims 1 to 5 or claims 7 to 9 additionally comprising a T- cell epitope.
11. A synthetic polypeptide as claimed in any one of claims 1 to 5 or claims 7 to 10 including a retro-inverso amino acid.
12. A synthetic polypeptide-as claimed in any one of the preceding claims linked to a carrier.
13. A DNA molecule coding for at least one synthetic polypeptide as claimed in any one of claims 1 to 10.
14. A vaccine comprising at least one synthetic polypeptide as claimed in any one of claims 1 to 12 effective to promote prophylaxis against HIV infection.
15. A kit for detecting HIV or antibodies against HIV which comprises at least one synthetic polypeptide as claimed in any one of claims 1 to 12.
16. A pharmaceutical composition containing, as active ingredient, at least one synthetic polypeptide as claimed in any one of claims 1 to 12 in association with one or more pharmaceutically acceptable adjuvant, carrier and/or excepient.
17. A pharmaceutical composition as claimed in claim 16, further comprising AZT and/or a HIV protease inhibitor.
18. A method of detecting HIV or antibodies against HIV or antigen binding fragments thereof, which comprises incubating a sample with at least one polypeptide as claimed in any one of claims 1 to 12.
19. An antibody or antigen binding fragment thereof which specifically binds to a synthetic polypeptide as claimed in any one of claims 1 to 12.
20. A diagnostic kit for detecting HIV or antibodies against HIV which contains at least one antibody or binding fragment thereof as claimed in claim 19.
21. A method of detecting HIV or antibodies against HIV or antigen binding fragments thereof, which comprises incubating a sample with at least one one antibody or binding fragment thereof as claimed in claim 19.
22. A pharmaceutical composition containing, as active ingredient an antibody or antigen binding fragment thereof as claimed in claim 19, in association with one or more pharmaceutically acceptable adjuvant, carrier and/or excepient.
23. A pharmaceutical composition as claimed in claim 22, further comprising AZT and/or a HIV protease inhibitor.
24. A method of diagnosing HIV infection which comprises incubating a sample of tissue or body fluid of a mammal with an effective amount of an antibody or binding fragment thereof as claimed in claim 19 and determining whether, and if desired the extent to which and/or the rate at which cross-reaction between said sample and said antibody occurs.
25. An anti-idiotypic antibody raised against an antibody or antigen binding fragment, as claimed in claim 19.
26. Use of a synthetic polypeptide as claimed in any one of claims 1 to 12 in the preparation of a medicament for the therapeutic or prophylatic treatment of mammalian HIV infection and/or for stimulating the mammalion immune system and/or blocking the cellular receptors for the HIV virus.
27. A method of therapy or prophylaxis of mammalian HIV infection and/or for stimulating the mammalion immune system and/or locking the cellular receptors for the HIV virus, which comprises administering an effective amount of a polypeptide as claimed in any one of claims 1 to 12.
28. A process for the manufacture of a synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV) , said polypeptide consisting substantially of an amino acid sequence of formula (I) :-
X-R.,-Leu-R2-Leu-Thr-Val-Trp-Gly-R3-Lys-Y
(I) wherein R. is selected from Gln-Gln-R4-R5, Gln-R4—Rg, R4-R5, R- or R1 is absent; R2 is an amino acid residue selected from
Gin, Lys, Glu or Arg;
R3 iiss aann aammiinnoo aacciidd residue selected from lie, Thr or Ala;
R4 is His or Glu;
R5 is Leu or Met; and
X and Y may each independently be absent or independently be one or more amino acid residues, the process comprising the steps of coupling the residues using chemical, biological or recombinant techniques known per se and isolating the polypeptide.
29. A process for the manufacture of an antibody which specifically binds to a synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV) , said polypeptide consisting substantially of an amino acid sequence of formula (I):-
X-R^Leu-R^Leu-Thr-Val-Trp-Gly-R-j-Lys-Y
(I) wherein R1 is selected from Gln-Gln-R-R5, Gln-R4-R5, R4-R5, R5 or R1 is absent; R2 is an amino acid residue selected from
Gin, Lys, Glu or Arg;
R3 iiss aann aammiinnoo aacciidd residue selected from lie, Thr or Ala;
R4 is His or Glu;
R5 is Leu or Met; and
X and Y may each independently be absent or independently be one or more amino acid residues, the process comprising immunising a non-human mammal with said polypeptide and isolating the antibody.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9208428 | 1992-04-16 | ||
GB929208428A GB9208428D0 (en) | 1992-04-16 | 1992-04-16 | Synthetic polypeptides |
Publications (1)
Publication Number | Publication Date |
---|---|
AU3960493A true AU3960493A (en) | 1993-11-18 |
Family
ID=10714175
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU39604/93A Abandoned AU3960493A (en) | 1992-04-16 | 1993-04-16 | Synthetic polypeptides derived from the HIV envelope glycoprotein |
Country Status (9)
Country | Link |
---|---|
EP (1) | EP0636145A1 (en) |
JP (1) | JPH07505878A (en) |
KR (1) | KR950700933A (en) |
CN (1) | CN1082053A (en) |
AU (1) | AU3960493A (en) |
CA (1) | CA2118033A1 (en) |
GB (1) | GB9208428D0 (en) |
WO (1) | WO1993021218A1 (en) |
ZA (1) | ZA932648B (en) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5128319A (en) | 1987-08-28 | 1992-07-07 | Board Of Regents, The University Of Texas System | Prophylaxis and therapy of acquired immunodeficiency syndrome |
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 |
US6261569B1 (en) | 1992-08-27 | 2001-07-17 | Deakin Research Limited | 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 (en) * | 1994-03-14 | 1996-06-21 | Centre Nat Rech Scient | Retropeptides, antibodies to the latter, and their uses for vaccination and in vitro diagnosis. |
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 (en) * | 2006-04-18 | 2008-02-06 | 河北师范大学 | Antiviral polypeptide from Zongdian Tuanwa frog and its application in pharmacy |
JP2008173077A (en) * | 2007-01-22 | 2008-07-31 | National Institute Of Advanced Industrial & Technology | Monitor protein for analyzing expression of membrane protein |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
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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 |
AU3557889A (en) * | 1988-04-20 | 1989-11-24 | Trustees Of The University Of Pennsylvania, The | 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 |
-
1992
- 1992-04-16 GB GB929208428A patent/GB9208428D0/en active Pending
-
1993
- 1993-04-15 ZA ZA932648A patent/ZA932648B/en unknown
- 1993-04-16 CN CN93105907A patent/CN1082053A/en active Pending
- 1993-04-16 EP EP93909057A patent/EP0636145A1/en not_active Withdrawn
- 1993-04-16 JP JP5518145A patent/JPH07505878A/en active Pending
- 1993-04-16 CA CA002118033A patent/CA2118033A1/en not_active Abandoned
- 1993-04-16 WO PCT/GB1993/000808 patent/WO1993021218A1/en not_active Application Discontinuation
- 1993-04-16 AU AU39604/93A patent/AU3960493A/en not_active Abandoned
-
1994
- 1994-10-14 KR KR1019940703649A patent/KR950700933A/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
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CA2118033A1 (en) | 1993-10-28 |
KR950700933A (en) | 1995-02-20 |
EP0636145A1 (en) | 1995-02-01 |
ZA932648B (en) | 1994-10-15 |
GB9208428D0 (en) | 1992-06-03 |
JPH07505878A (en) | 1995-06-29 |
CN1082053A (en) | 1994-02-16 |
WO1993021218A1 (en) | 1993-10-28 |
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