SYNTHETIC POLYPEPTIDES

Synthetic Polypeptides 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 I and HIV II 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 40, 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), said polypeptide consisting substantially of an amino acid sequence of formula (I): X-R1 -R2-R3-R4- -R6-R7-Trp-Gly- Cys-R8--R10-R11-R12-Cys-Y (I) wherein R1 is Asp or Glu; R2 is an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, Asn or Gln; R3 is an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, Asp, Glu, Asn, Lys, His, Gln or Arg; R4, R5 and R11 are each independently any amino acid residue; R6 and R8 are each independently an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, or Asn; R7 is an amino acid residue selected from Gly, Ala, Val, Leu, Ile, Ser, Thr, Asn, Gln, Phe, Tyr, Trp, Cys, Met or Pro;

; 4 and R12 are each independently an amino acid residue selected from Gly, Ala, Leu, Ile, Val, Met, Cys, Phe, Tyr or Trp; R10 is an amino acid residue selected from Lys, His or Arg; and X and Y may each independently be absent or independently be one or more, e.g. 3, additional amino acid residues.

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. 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, R2 is selected from Gln or Thr, R3 is selected from Ser, Asn, Gln, Arg or Ala, R4 is selected from Leu, Ile, Gln or Arg, R5 is either Leu or Lys, R6 is either Gly or Asn, R7 is selected from Gly, Ala, Leu, Ile, Val, Met, Cys, Phe, Tyr, Trp or Ser, R8 is either Ser or Ala, R9 is either Gly or Phe, R10 is either Arg or Lys, R11 is selected from Leu, His, Ile, Gln and R12 is selected from Ala, Ile or Val. The Cys residues at positions 10 and 16 may optionally be linked by an intra-molecular disulphide bridge.

One preferred form of polypeptide according to the invention consists substantially of an amino acid sequence of formula (II): X-Asp-Gln-R3 - Leu-R5 -Gly-R7-Trp-Gly- Cys-Ser-Gly-Lys-R11-R12-Cys-Y (Il) wherein R3R5, R11, R12, X and Y are as defined above; and R7 is an amino acid residue selected from Gly, Ala, Leu, Ile, Val, Met, Cys, Phe, Tyr or Trp.

Preferably in a sequence according to formula (II), R3 is selected from Ser, Asn, Gln and Arg, R11 is selected from Leu, His or Ile and R12 is Ile or Ala.

Advantageously, R7 is selected from Ile, Phe, Met, Val or Leu. More preferably R3 is either Ser or Asn and R5 is Lys.

A preferred form of polypeptide of formula (II) according to the invention consists of the sequence: X-Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys-Y wherein X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an intra-molecular disulphide bridge.

Another preferred form of polypeptide of Formula (II) comprises R3 selected from Gln or Arg, R5 is Leu, R7 is selected from Ile, Phe and Met, R11 is selected from Leu, His or Ile, and R12 is Ala. A preferred sequence has the formula: X-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys-Y wherein X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an intra-molecular disulphide bridge.

Polypeptides according to formula II resemble certain epitopic portions of HIV I envelope protein.

Another preferred form of polypeptide according to the invention consists substantially of an amino acid sequence of formula (III) :- X-R1-R2-R3-R4-R5-Asn-Ser-Trp-Gly-Cys-Ala- Phe-Arg-Gln-Val-Cys-Y (III) wherein R1 is either Glu or Asp; R2 is Thr or Gln; R3 is an amino acid selected from Ser, Asn Arg, Gln or Ala; R4 is an amino acid selected from Leu, Ile, Arg or Gln; R5 is Lys or Leu; and wherein X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an intra-molecular disulphide bridge.

A preferred form of a polypeptide according to Formula III consists substantially of an amino acid sequence of formula (IIIa): X-Glu-Thr-R3-R4-Lys-Asn-Ser-Trp-Gly Cys-Ala-Phe-Arg-Gln-Val-Cys-Y (IIIa) wherein R3, is an amino acid residue selected from Ser, Asn, Arg, Gln or Ala, R4 is an amino acid residue selected from Leu, Ile, Arg or Gln, X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an intra-molecular disulphide bridge. It is preferred that R3 is Ser when R4 is Ile and when R3 is Ala R4 is either Arg or Gln but preferably Arg.

Polypeptides according to formula III are similar to certain epitopes of HIV II envelope proteins.

Preferred polypeptide sequences according to the invention were chosen on the basis of their topographical similarity to more than one antigenic determinant 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 intheir 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 9fluorenyl-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 excess of 85%, should be carefully checked, and particular attention be given to the correctness of internal disulphide bridging arrangements when present.

Various chromatographic analyses, including high performance liquid chromatography, and spectrographic analyses, including Raman spectroscopy, 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.

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 I and/or HIV II 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 (IV): [La?F]m?[Lb?G]n?LC (IV) wherein F and G may each independently be a polypeptide according to any one of Formulae I to IIIa, 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 Gly-Gly-Gly-Gly Gly, Gly-Pro-Gly-Pro-Gly-Pro or 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 IV 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 IIIa, 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 (V): Ld?[G?L]m?F?[L?G]n?Le (V) wherein F is a polypeptide according to any one of Formulae I to IIIa, G is a polypeptide according to any one of Formulae I to IIIa 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 Gly-Gly-Gly-Gly-Gly, Gly-Pro-Gly-Pro-Gly-Pro or Gly-Ser-Ala-Gly-Ser-Gly-Ala.

In preferred forms of peptide V, G may comprise a polypeptide according to any one of Formula I to IIIa or the sequence: X-Gln-Gln-Glu-Lys-Asn-Gly-Gly-Glu-Leu-Y wherein each Gly may independently be replaced with any other amino acid and/or X and Y may each independently be absent or one or more e.g. three amino acid residues or G may comprise some other polypeptide sequence related to antigenic proteins from HIV.

It is to be understood that any antigenically significant sub fragments 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 (but naturally occurring, 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, Ile, Phe, Tyr, Trp and Met; Set 2 - Ser, Thr, Asn and Gln; Set 3 - Asp and Glu; Set 4 - Lys, His and Arg; Set 5 - Asn and Asp; Set 6 - Glu and Gln; 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. Ile, 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, Gln, Gly), and 4 is a polar amino acid (e.g. Lys, Arg, His, Glu, Asp, Asn, Gln, 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 1' is equivalent to 1 as defined earlier, 2' to 2, 3' and 4' 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) which may be of the type defined above or may be of other structure and which may be separated by spacer segments of any length or composition, preferably less than five amino acid residues in length and comprising for example residues selected from Gly, Ala, Pro, Asn, Thr, Ser or polyfunctional linkers such as non-a 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: EMI10.1

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 shortchain 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. & BR< 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 amino 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.

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-a -maleimidobutyryloxy-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 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 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 strains of HIV; (b) As ligands in assays of, for example, serum from HIV positive patients; (c) As quality control agents in testing, for example, binding levels of antibodies raised against the polypeptides; (d) As antigenic agents for the generation of monoclonal or polyclonal antibodies by immunisation of an appropriate animal, such antibodies being of use for (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 immunoassay techniques known in the art, inter alia, sandwich assay, competitive and noncompetitive 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, 157160), 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 adjuvants, carriers and/or excipients. 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 basic peptide (a) with sequence Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys was synthesised using standard solid-phase F-moc methodologies and an intra-molecular disulphide bridge was formed between the two Cys residues. 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; Smg/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 emulsification with Freund's adjuvant. Each sheep (5 animals/group) was immunised with 250 ssg of peptide in Freund's complete adjuvant (FCA) and subsequently (14 days) challenged with a further similar quantity in Freund's incomplete adjuvant (FIA). Further challenges were performed after a period of 3-4 weeks in FIA. Blood samples were taken 7-10 days post-challenge and assayed for binding to HIV envelope protein.

The determination of anti-synthetic peptide antibodies which recognise HIV envelope protein was undertaken by employing a recombinant vaccinia virus (see, for example, Macket, M. and Smith, G. L. (1986) J.

gen. Virol. 67, 2067, for general methodology) which has been constructed to express the HIV envelope proteins on the surface of infected cells. This method of assay is preferred as binding to the antigen is measured on the surface of virus infected cells and hence may be more representative than binding to solution phase antigen (e.g. because certain potential epitopes on the envelope proteins may be masked in vivo through interaction with membrane phospholipids). CVl monkey kidney cells, grown in monolayer cultures in 96-well microtitre plates, were infected with the recombinant vaccinia virus. The virus construct contained the gene encoding the HIV envelope glycoprotein and this is known to be processed and expressed on the surface of the infected cells.

Antisera from sheep were assayed in duplicate. A quantity of 50 ssl of antiserum at various dilutions was added to the wells and incubated for 4 hours prior to washing (twice) and adding a second peroxidase-labelled anti-sheep antiserum. Positive wells were determined by reading the optical density in a microplate reader.

The anti-peptide antisera were found to cross-react with high affinity with HIV envelope protein expressed on the surface of infected cells (see Tables 1 and 1A).

These results confirm that these anti-peptide antibodies would be valuable as research tools and as diagnostic agents.

Table 1 Cross-reaction between serum from sheep immunised with conjugate of peptide (a) and recombinant HIV envelope protein expressed on the surface of monkey kidney cells Cross reaction EMI17.1

Control monkey Monkey kidney kidney cell line cell line expressing HIV envelope protein Serum from immunised sheep Control serum Table 1A Typical titres of positive antisera from sheep immunised with a conjugate of peptide (a) when reacted with recombinant HIV envelope protein expressed on the surface of monkey kidney cells Mean optical density value EMI17.2

(serum dilution) 1ample 1 1 1 1 Serum sample 50 250

1250 31250 156250 (titre) Control sheep 0.344 0.288 0.197 0.012 0.016 serum Peptide (a) 0.736 0.636 0.432 0.140 0.052 treated sheep serum diluted 1/5000 The above table shows that even with significant dilution of the serum from test sheep (relative to the control) there is a greatly increased titre.

Example 2 One role of neutralising antibodies is to inhibit the transmission of virus from infected to non-infected cells. Preventing or reducing the rate of transmission of virus from T-cells to macrophages is crucial since this may extend the life of AIDS patients considerably Anti-peptide antisera were assayed for their ability to inhibit syncitia formation in vitro. Syncitia are multinucleated giant cells which result from "bridge"-formation between HIV-infected cells.

The C-terminally extended form of basic peptide (a) with sequence Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys and containing an intra-molecular disulphide bridge between the two Cys residues was synthesised, purified, conjugated and used to immunise groups of sheep as described in Example 1. The anti-peptide antisera were used in an in vitro HIV neutralisation assay. The anti-peptide antibodies were introduced into an in vitro culture of human T-lymphocytes infected with a highly virulent strain of HIV, and uninfected human macrophages. The anti-peptide antisera were found to inhibit syncitia formation amongst this mixed population of T-cells and macrophages exposed to a virulent strain of HIV I, i.e. they inhibit spread of HIV infection from T-cell to macrophage.

The antibodies were found to prevent infection of the macrophages with HIV, and are therefore neutralising in this in vitro assay, whereas the macrophages became infected in a control culture, as shown in Table 2.

Table 2 Neutralisation of virulent strain of HIV in in vitro culture of HIV-infected human T-lymphocytes and uninfected human macrophages. "+++" indicates a high level of infection and "-" indicates effective protection against infection.

Cell culture Level of infection of macrophages of macrophages HIV infected T-lympho cytes and uninfected macrophages only HIV infected T-lympho cytes, uninfected macrophages and anti peptide(a) antibodies peptide (a) antibodies Also, positive antisera to peptide (a) were evaluated in a syncitium assay. This assay determines the capability of antisera to prevent spread of live HIV from infected to uninfected cells and to prevent fusion between cells mediated through reactivity between the virus glycoprotein (gp160) and the CD4 molecule.

Measurement is carried out by syncitium detection and enumeration.

The syncitium assay is performed as follows: a known concentration of an HIV producer (CD+4) cell line supporting active virus replication is washed three times and mixed with the antiserum under test and used at specified dilutions. After incubation for 30 minutes at 37"C these cells are mixed, at specified proportions with an indicator CD4+ cell line, highly susceptible to HIV infection and syncitium induction. The cells are observed daily for syncitium formation.

Antibodies to peptide(a) were found to inhibit syncitium formation as shown in Table 3.

Table 3 Sync it ium inhibition by antisera between HIV infected producer cells and an indicator cell line Serum Sample Syncitium inhibition Anti-peptide (a) antiserum + (3/5 animals) Normal sheep serum Example 3 A panel of sera from HIV-1 infected individuals in various stages of progression to acquired immunodeficiency syndrome disease (AIDS), ranging from asymptomatic to full-blow symptoms were obtained in order to produce a representative picture of the reactivity of sera from such patients with the synthetic peptides in ELISA.

The ELISA procedure determines the degree of reactivity of the synthetic peptides (analogous to regions of either gp120 or the gp41 transmembrane protein of HIV-1) with antibody to the surface glycoprotein of HIV-1 in sera of HIV-1 positive individuals.

One hundred antisera from HIV positive individuals were reacted with synthetic peptide (a). Two of these sera (from asymptomatic individuals) contained HIV neutralising antibodies and cross-reacted with peptide (a) as shown in Table 4.

Table 4 Cross reactivity of peptide (a) with two HIV positive antisera from asymptomatic individuals EMI21.1

samle OD (etide (a)) Buffer 0.056 Normal serum 1/100 0.476 I/iooo 0.140 HIV positive (1) 1/100 > 2.0 1/1000 > 2.0 HIV positive (2) 1/100 > 2.0 > 2.0 ) replicates 1.37 )