AU4578893A

AU4578893A - Peptides that mimic gp120 hiv epitope

Info

Publication number: AU4578893A
Application number: AU45788/93A
Authority: AU
Inventors: Peter Jonathan Gasking Butler; Graeme Norman Varey Hacking
Original assignee: Medical Research Council
Current assignee: Medical Research Council
Priority date: 1992-07-16
Filing date: 1993-07-16
Publication date: 1994-02-14
Anticipated expiration: 2013-07-16
Also published as: EP0654080A1; ZA935165B; GB9215129D0; WO1994002614A1; JPH07509237A; AU681591B2; CA2140151A1

Description

Title PEPTIDES THAT MIMIC GP120 HIV EPITOPE.

Field of the Invention

This invention concerns developments relating to human immunodeficiency viruses (HIV).

Background to the Invention

The envelope glycoprotein (env) of human immunodeficiency virus (HIV) is highly variable between independent

isolates (reference 1) and this variation is even more marked between HIV-1 and HIV-2(2). This variation is seen not only between independent isolates, but also for sequential isolates of virus from an individual patient (3, 4). Not surprisingly, similar sequence variation has been observed between the human and simian

immunodeficiency viruses (SIV) and also within SIV even when isolated from the same species of simian (5-7).

Not only is the variation greater in the envelope

glycoprotein than in other gene products of HIV, but even within this one protein the variation is concentrated into specific variable regions (mostly in the surface portion (gp120) generated by the proteolytic maturation of the initial gene product), with other regions being less variable (8, 9). Unfortunately the most variable regions are often also the most immunogenic (10), so that the virus partially evades the host's immune response and establishes a persistent infection.

This variability presents problems for diagnostic

techniques based upon specific interactions, with separate or mixed reagents usually being employed to test samples for both HIV-1 and HIV-2. This variability also presents problems for any possible vaccine or immune therapy, since any suitable agent will have to give a response towards the many variant virus strains as well as towards HIV-1 and HIV-2.

The present invention is based on the discovery by the inventors of a hitherto unrecognised highly conserved undecapeptide in gp 120 of env, starting from position 122 (on our alignment of HIV-1 env sequences), which is present in HIV-1, HIV-2 and also SIV. This sequence is totally conserved as Lys-Pro-Cys-Val-Lys-Leu-Thr-Pro-Leu-Cys-Val (Seq. Id. No. 1, also referred to as sequence A) in all listed isolates of both types of HIV, including the "highly divergent" HIV-2 isolates D194 and D205 ( 11), and only undergoes conservative changes at the threonine and second valine in all isolates of SIV except in the highly divergent African mandrill isolate (12) wherein threonine and both leucines are mutated.

In this specification amino acids are either identified by their full names, by conventional 3 letter abbreviations or by conventional 1 letter symbols, as listed in Table 1.

Table 2 gives amino acid sequence data for a number of different isolates of HIV-1, HIV-2 and SIV compared with the highly conserved consensus sequence. The consensus sequence is based upon an absolute majority; for individual isolates a dash indicates agreement with the consensus, otherwise the amino acid is shown.

Comparison of the nucleotide sequences in the gene coding for this peptide also shows a high degree of conservation, although the redundancy of the genetic code allows

slightly wider variation.

Sequence information was taken from the Los Alamos Data

Base.

Earlier reports have shown that the imraunodominant regions of gp120 are largely in the variable loops, particularly V3 (e.g. 10). Limited studies with peptides have included the conserved region we describe (e.g. 13). These

peptides showed very little immunogenicity. This is now thought to be because no precautions were taken to protect the amino acid side chains from modification during conjugation to the carrier protein.

The conserved sequence identified above is referred to in EP 0298633 of Proteus Biotechnology Limited as the

possible basis of a vaccine to promote immunity against at least one strain of HIV. The sequence is stated to have been chosen on the basis of its topographical similarity to at least one other antigenic determinant of the HIV envelope proteins. However, there is no indication in EP 0298633 of an appreciation of the high degree of

conservation of the sequence between different strains of HIV-1, HIV-2 and SIV.

EP 0298633 also discloses linking the C terminal of the conserved sequence identified above to a further sequence chosen in similar manner, and it is stated in this document that the inessential C terminal dipeptide Cys-Val may possibly be omitted, and that the first Cys residue of the sequence may be cross linked to another Cys residue in the molecule via an intramolecular disulphide bridge.

The present invention is further based on the appreciation that correct presentation of the conserved seqence is very important for eliciting an immune response. It is now known that in its native form the conserved sequence identified above is cross-linked via intramolecular disulphide bridges to a further sequence of gp120

different from that proposed in EP 0298633. This further sequence (sequence B) is also highly conserved within strains of both HIV-1 and HIV-2, with only limited

mutations between the two types. SIV isolates show strong similarities to either HIV-1 (SIV_CPZ) or HIV-2 (all other strains of SIV) (Table 3). It is further now appreciated that both cysteine residues of the conserved sequence identified above are involved in respective disulphide bridges in the native confirmation (14), and it is

believed that both cysteine residues are required to elicit a proper immune response, contrary to the statement in EP 0298633 that one of the cysteine residues is

inessential. The strong conservation of this second peptide is fully compatible with it forming an essential structural conformation together with the conserved sequence.

In order to demonstrate this, the present inventors have carried out work using the conserved sequence identified above with both cysteine residues derivatised to

approximate to one half of a disulphide bond, thus

mimicking native presentation. Initial immunogenic studies on this peptide show that the peptide is immunogenic when presented coupled to an appropriate carrier protein and that the resulting antisera react with native gp 120 in permeabilised cells. Work has been carried out using the conserved sequence identified above linked to a further sequence via disulphide bridges from both cysteine residues of each sequence in appropriate orientation.

Summary of the Invention

In one aspect the present invention provides a molecule comprising a peptide having the amino acid sequence lysine-proline-cysteine-valine-lysine-leucine-threonineproline-leucine-cysteine-valine (Seq. Id. No. 1), wherein each cysteine residue is disulphide bridged to a further cysteine residue or is derivatised to simulate part of a disulphide bond, or functionally equivalent variants of such a peptide which mimic the immunogenic behaviour of an epitope of gp120 of HIV env.

The strict conservation of a peptide sequence within the external envelope protein of HIV, coupled with the near conservation in the related SIV, suggests that the

sequence that is the basis of the invention is essential for the function of the env-protein and hence unlikely to change further in other, as yet uncharacterised, isolates. Searches have shown that the sequence is not present in other proteins in the EMBL Data Base and so it appears to be specific to immunodeficiency viruses. The invention therefore offers a possible fixed sequence that has many possible implications for the design of diagnostic, immunogenic agents, and therapeutic agents, as will be explained below. As explained above, it is important that each cysteine residue contributes to a respective disulphide bridge (actual or simulated by suitable derivatisation) for the molecule to mimic accurately behaviour of the

corresponding sequence of gp120. This can be achieved by suitable derivatisation of the cysteine residues, e.g. as the S-acetamidomethyl derivative. This can alternatively be achieved by cross-linking the cysteine residues to respective cysteine residues in a further peptide

sequence, which can either be a continuation of the peptide of the invention, spaced with a suitable linker of peptide or other nature, or a separate peptide. One preferred cross-linking arrangement, which is based on the actual cross-linking now known to occur in gp120 is as follows :

Sequence A is the uniquely conserved sequence on which the present invention is based, and Sequence B is based on the sequence of amino acids 219 to 230 of HIV-1 and 210 to 221 of HIV-2, with mutations indicated below in order of frequency. Sequence B is thus fairly conservative

although different sequences may be optimal for HIV-1 and HIV-2 (Table 3).

Desired peptide sequences can be readily synthesised in conventional manner, e.g. using Fmoc techniques.

Alternatively, peptide sequences can be produced by recombinant DNA techniques in known manner.

A further aspect of the invention thus provides a DNA molecule coding for a peptide molecule in accordance with the invention, preferably incorporated into a suitable expression vector.

It will be apparent that the molecule of the invention, based on the highly conserved sequence, can be modified in a variety of different ways without significantly

affecting the functionally important immunogenic behaviour of the molecule. Possible modifications to the or each peptide sequence include the following:

1) One or more individual amino acids can be substituted by amino acids having comparable properties e.g. as follows:

V substituted by I

T substituted by S

K substituted by R

L substituted by I, V or M

2) One or more of the normal peptide linkages can be substituted by isosteric replacements. Possible

replacement linkages are well known to those skilled in the art of peptide synthesis and are reviewed, for

example, in reference 15. Examples would include: the use of the azapeptide linkage, where the alpha-carbon of the amino acid is replaced by a nitrogen atom (i.e. -NH-NR-CO-); a reduced amide bond, giving a methyl amine (i.e. -NH-CHR-CH₂-); thiomethyl groups, with the CO replaced by CH and the NH by S (i.e. -S-CHR-CH₂-); replacement of both the NH and CO groups with unsaturated carbon atoms (i.e. =CH-CHR-CH=); or replacement of the NH group with a methylene (i.e. -CH₂-CHR-CO-).

3) One, or both, pairs of disulphide-bridged cysteine residues (i.e. cystine residues) can be substituted by a cystine analogue such as:

4) One or more of the amino acids can be replaced by a "retro-inverso" amino acid, i.e. a bifunctional amine having a functional group corresponding to an amino acid, as discussed in WO91/13909.

5) One or more non-essential amino acids can be

deleted.

6) One or more additional amino acids not significantly affecting function can be included.

7) Structural analogs mimicking the 3-dimensional structure of the peptide can be used in place of the peptide. It has been shown that a molecule in accordance with the invention can elicit an immune response. One possible use of the molecule is therefore as the basis of a potential vaccine against AIDS and AIDS related conditions.

In a further aspect the invention thus provides a vaccine against AIDS and AIDS related conditions, comprising a molecule in accordance with the invention.

For this purpose, the molecule of the invention may optionally be linked to a carrier molecule, possibly via chemical groups of amino acids of the conserved sequence or via additional amino acids added at the C- or N-terminus. Many suitable linkages are known, e.g. using the side chains of Tyr residues. Suitable carriers include, e.g., keyhole limpet hemocyanin (KLH), serum albumin, purified protein derivative of tuberculin (PPD), ovalbumin, non-protein carriers and many others. It is thought the lysine residues in the conserved sequence may be of importance in the molecule of the invention, and for this reason the epsilon-amino groups of lysine residues are preferably protected in known manner during a

conjugation reaction to link a carrier, to prevent

unwanted reaction, with the groups being subsequently regenerated before use of the conjugate.

The molecule of the invention may alternatively be

presented as a possible live vaccine, e.g. as part of the coat of a genetically modified virus such as polio or vaccinia.

The vaccine of the invention may be administered in conventional manner, e.g. by injection, orally etc., with or without use of conventional adjuvants such as Freund's complete or incomplete adjuvant or aluminium hydroxide, and with or without other immunopotentiating agents.

Diluents suitable for use in formulating the vaccine for administration are also well known, including distilled water, phosphate-buffered saline, and buffer solutions such as citrate buffer.

Molecules in accordance with the invention may further find use in the treatment (prophylactic or curative) of AIDS and related conditions, by acting either to displace the binding of the HIV virus to human or animal cells or by disturbing the 3-dimensional organisation of the virus.

A further aspect of the invention thus provides a method for the prophylaxis or treatment of AIDS or related conditions, comprising administering an effective amount of a molecule in accordance with the invention.

In a further aspect, the invention provides a

pharmaceutical composition containing, as an active ingredient, a molecule in accordance with the invention, possibly in association with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients.

The invention also provides use of a molecule in

accordance with the invention for the preparation of a medicament for the therapy or prophylactic treatment of AIDS or related conditions.

Molecules which bind to the conserved sequence on which the invention is based, particularly antibodies, antibody-related molecules and structural analogs thereof, are also of possible use as agents in the treatment and diagnosis of AIDS and related conditions.

In a further aspect the invention thus provides a molecule that binds to the conserved sequence that is the basis of the invention, particularly an antibody, an antigen binding site of an antibody or a structural analog

thereof.

Techniques for making antibodies (monoclonal and

polyclonal) are well known to those skilled in the art.

Variants of antibodies (including an antigen binding site), such as chimeric antibodies, humanised antibodies, veneered antibodies, and engineered antibodies generally are included within the scope of the invention.

Techniques for the production of such variants are also well known to those skilled in the art.

With a knowledge of the 3-dimensional structure of the conserved sequence in gp 120, it is possible to design and construct synthetic molecules that will bind to the sequence. Suitable techniques for this purpose are disclosed e.g. in references 16 and 17, which involve the use of computer-modelling to design potential inhibitors to renin.

Antibodies and other molecules which bind to the conserved sequence on which the invention is based can be used for therapeutic (prophylactic and curative) and diagnostic purposes in a number of different ways, including the following:-

1) For passive immunisation by suitable administration of antibodies, preferably humanised antibodies to patients. 2) To activate complement or mediate antibody dependent cellular cytotoxicity (ADCC) by use of antibodies of suitable subclass or isotype (possibly obtained by

appropriate antibody engineering) to be capable of

performing the desired function.

3) For targetted delivery of toxins or other agents, e.g. by use of immunotoxins comprising conjugates of antibody and a cytotoxic moiety, for binding directly or indirectly to the target conserved sequence of gp 120.

4) For targetted delivery of highly immunogenic materials to the surface of HIV-infected cells, leading to possible ablation of such cells by either the humoral or cellular immune system of the host.

5) For detection of HIV, e.g. using a variety of

immunoassay techniques.

Techniques for performing all of the above are well known to those skilled in the art.

In another aspect the invention thus covers use of a molecule which binds to the conserved sequence of the invention for therapeutic or diagnostic purposes.

The invention also includes within its scope methods and kits for detecting HIV, antibodies against HIV or

infection with HIV using molecules in accordance with the invention.

The invention will be further described, by way of

illustration, in the following examples. The first example concerns immunological studies on a peptide incorporating the highly conserved seqence, which show that the peptide is immunogenic, as presented, and that the antisera react with native gp 120 in infected cells. The example refers to Figures 1 to 4 of the accompanying drawings, in which:

Figure 1 is a series of photographs showing

immunofluorescence of cells, some expressing gp 120 from

HIV-1, treated with sera obtained from a rabbit before and after treatment with the peptide, with the serum treated with peptide before application to the cells in some cases;

Figure 2 is a series of photographs showing

immunofluorescence of cells, either uninfected or infected with different isolates of HIV-1, treated with sera obtained from a rabbit before and after treatment with the peptide;

Figure 3 is a series of photographs showing

immunofluorescence of cells, uninfected or infected, treated with sera obtained from two different rabbits before and after treatment with the peptide; and

Figure 4 is a series of photographs showing

immunofluorescence of cells, either uninfected or infected with HIV-2, treated with sera obtained from a rabbit before and after treatment with the peptide.

The second example concerns immunological studies on a peptide incorporating the highly conserved sequence A disulphide-bridged in an antiparallel fashion to the moderately conserved sequence B, which show that the peptide is immunogenic, as presented, and that the

antiserum reacts with HIV-1 to neutralise its infectivity. The example refers to Figure 5 of the accompanying

drawings in which:

Figure 5 is a graph of virus present in the supernatant fluid above tissue culture cells, measured by the activity of viral reverse transcriptase in incorporating ³H-dTTP, at various times after attempted infection with virus (50

TC1D₅₀) treated for 60 minutes, at 37°C, with the

indicated serum (or phosphate buffered saline, PBS) diluted 1:20 with RPMI/10%FCS.

Examples

Analysis of HIV envelope glycoprotein sequences for conservation

HIV and SIV sequences used for the analysis were from the Los Alamos Data Base. Sequence comparisons were performed on a VAX 8600 computer, using HOMED, Version 3.30, from P A Stockwell, Otago University, Dunedin, New Zealand.

Alignment of the sequences of the envelope glycoprotein sequences of HIV-1 and HIV-2 isolates showed a number of regions with varying degrees of conservation. However most of these were unconserved when comparison was also made to SIV sequences and only a single region of

significant size was still conserved. This occurs in gp 120, immediately before the first hypervariable (V1) loop (9), and the conservation is shown in Table 2. The undecapeptide is absolutely conserved in all isolates of HIV and the changes in SIV are highly conservative, with only changes of T to S and V to I in most isolates (and L to I, T to N and L to Y in the, otherwise, highly

divergent African mandrill isolate GB1).

The sequence of 11 amino acids appears to be unique to HIV and SIV, with no matches of more than 5 contiguous

residues being found on searching the EMBO and SwissProt sequence data bases (or 6 residues, allowing gaps). The restricted sequence variation of this region of the otherwise highly variable gp 120 suggests that it might be essential for HIV viability and hence not be able to vary significantly and yet maintain infectious progeny

virions.

Alignment of the sequences also showed a high degree of conservation of a second region, immediately after the second hypervariable (V2) loop (9) and disulphide-bridged to both cysteine residues in the first, uniquely conserved region (14), and the conservation is shown in Table 3. The conservation is greatest between the cysteine residues forming the disulphide bridges and each HIV Type is mutated only very conservatively, with the SIV isolates resembling one or other HIV Type. This further suggests that the antiparallel cross-linked peptides form a

structure in gp 120 essential for viability of the virus.

Example 1

Based on this analysis, experiments were carried out using a synthetic peptide based on the highly conserved sequence of the invention.

All chemicals were of analytical grade and were used without further purification, unless otherwise mentioned. Peptide Synthesis

A peptide having the sequence KPCVKLTPLCVTLY (Seq. Id. No.4) was synthesised by Fmoc continuous flow solid phase synthesis using an automatic synthesiser (LKB Biolynx). Fmoc amino acid pentafluorophenyl esters were used as acylating species throughout, except for Thr where the ester of 2,3-dihydro-3-hydroxy-4-oxo-benzotriazine was preferred. Kieselguhr supported polydimethyl-acrylamide functionalised with the hydroxymethylphenoxy acetic acid linkage and norleucine as internal reference amino acid (18,19) was used as solid support.

The peptide was cleaved from the resin using

trifluoroacetic acid/phenol/triethylsilane (23ml/1g/1ml for 500mg of peptide-resin assembly) which effected simultaneous cleavage of the tert-butyl based side chain protecting groups except for the acid stable Acm

(acetamidomethyl) on the side chain of Cys. The resin was removed by filtration and washed with a little neat trifluoroacetic acid (TFA). The solvent was removed from the combined TFA filtrates by rotary evaporation under reduced pressure. The oily residue was dissolved in 0.1% aq. TFA (20ml) and extracted with diethyl ether (5 × 20ml) and the solvent removed from the combined aqueous phases by freeze drying to yield a white powder (20.9 umols, 76% yield).

The peptide was analysed by HPLC after synthesis and found to be at least 98% pure and was therefore used, without further purification, for coupling to KLH for immunisation of rabbits.

Antiserum production The peptide was coupled, in about 30-fold molar excess, to KLH, essentially by the protocol of Bassiri and Utiger (20) as modified by Kiberstis et al. (21). Since we were particularly concerned to present the peptide in a

condition as near to that in the native protein as

possible, the Cys residues were left as the S-acetamidomethyl derivative, which was chosen to

approximate to one half of a disulphide bond, and the epsilon-amino groups of the Lys residues were reversibly protected during the coupling reaction, which would otherwise have converted them to hydroxyl groups, by modification with 2,3-dimethylmaleic anhydride (10-fold molar excess) at pH 9.0, prior to coupling, with

subsequent unblocking at pH 6.5, 20ºC, for 8h (22).

Three New Zealand white rabbits (known as Ping, Pong and Pang) (conventionally bred from Rosemeal Rabbits Ltd., UK) were immunised with the equivalent of 0.6mg peptide in Freunds complete adjuvant, by multiple subcutaneous injections and subsequently given 2 boosts subcutaneously with the equivalent of 0.3mg peptide in incomplete Freunds adjuvant. Preimmune and post-immunisation blood samples were taken, together with the final immune blood, and sera prepared by allowing the samples to clot at room

temperature.

Immune responses were monitored by ELISA against the peptide bound to wells of microtitre plates, which were subseqently blocked with bovine serum albumin. Horse radish peroxidase-conjugated goat anti-rabbit antibody ( DAKO Ltd.) was used as the second antibody and 1,2-phenylenediamine, dihydrochloride (DAKO Ltd.) as the substrate. ELISA of the rabit preimmune sera and also sera after immunisation showed that all 3 rabbits developed a

response to the peptide as an immunogen, when presented coupled to keyhole limpet hemocyanin (Table 4). The final bleeds give enhancements (greater than or equal to 4-fold compared to preimmune serum) at dilutions of 1:6400 or 1:3200, suggesting that the peptide was immunogenic when presented as the conjugate on KLH.

Immunofluorescence study of binding of antisera to HIV-infected cells

Binding of antibodies in the rabbit sera to native gp 120 was assayed using confocal immunofluorescent microscopy (23) with the Bio-Rad MRC 600 confocal microscope, with fluorescein isothiocyanate (FΙTC)-labelled goat anti-rabbit IgG (Sigma Chemical Company), as the second

antibody to show the rabbit antibody. Positive control rabbit antiserum against intact gρ120 was a kind gift of Dr Rod Daniels of the National Institute for Medical

Research, London. The test cells were the human T-lymphoblastoid cell line, C8166, which is non-productively infected with human T-cell lymphotropic virus Type-1 (24). These were grown in RPMI/10% fetal calf serum and infected with HIV-1 BRU (25), HIV-1 SF2 (26), or else uninfected as a control. Cells (10 in 15ul) were transferred to the wells of poly-L-lysine treated microscope slides and allowed to bind for 30 min at room temperature, before being fixed with 4% glutaraldehyde then treated with 0.1% Triton X100 to permeabilise the plasma membranes.

Antisera at appropriate dilutions in PBS were added and allowed to bind for 60min at room temperature before the cells were washed. Fluorescein-labelled second antibody was then added (at the dilution recommended by the

supplier) and allowed to bind for 60min before washing in PBS. The slides were then examined in the confocal microscope, to detect antibody binding, and appropriate specimens photographed.

Binding of immune sera to cells expressing gp 120 of HIV- 1_IIIB was determined, using both preimmune sera and also uninfected cells as controls. Typical fields for one rabbit (Pang) are shown in Figure 1. In the upper panel of this Figure, cells were either not expressing gp 120 (a,b and f) or expressing (all other photographs) and were treated with either preimmune serum (a and e) or immune serum (all other photographs). In the lower panel of this Figure, all cells were expressing gp 120 and were treated with immune serum from one rabbit (Pang) either without peptide (i and m) or after incubation with peptide A (j and n), or from another rabbit (Ping) again without peptide (k and o) or after incubation with peptide A (l and p).

Figure 1 shows major binding only with the immune sera and gp 120 expressing cells, while low binding is seen for serum onto non-expressing cells or for preimmune serum onto infected cells. The distribution of binding of immune serum to the gp 120 expressing cells is very similar to that seen for control serum, from a rabbit immunised with gp 120, binding to expresssing, but not non-expressing, cells. Moreover, the binding of immune serum to infected cells is blocked by the peptide, indicating that it is to a specific site on the gp 120.

Binding of the immune sera to HIV-derived material in infected cells was determined, using both preimmune sera and also uninfected cells as controls. Typical fields for one rabbit (Ping), with HIV-BRU, are shown in Figure 2. In this Figure, cells were either uninfected (a,b,e and f) or infected with HIV-1 BRU (c and d) or SF2 (g and h), while the serum was either preimmune (a, c, e and g) or the immune (b, d, f and h). Bar represents 25 um.

Figure 2 shows major binding only with the immune serum and infected cells, while low binding is seen for either serum onto uninfected cells or for preimmune serum on infected cells. The distribution of the binding of immune serum to the infected cells is similar to that seen for control serum, from a rabbit immunised with gp 120, binding to infected cells, but not uninfected cells.

Repetition of these experiments with cells infected with HIV-1 SF 2 showed very similar results (Figure 1 g and h), confirming that the reaction is independent of the HIV strain employed.

Results of similar tests with sera from the 2 further rabbits (Pong and Pang) and HIV-1 SF2 are shown in Figure 3. Figure 3a shows results using either preimmune serum (upper left) or immune serum (remaining pictures) from Pong with infected cells in each case, and Figure 3b shows results using immune serum from Pang, with either

uninfected cells (upper left) or infected cells (remaining pictures). Bar represents 25 um.

Similar binding was seen with the sera from the rabbit Pong to cells infected with HIV-1 BRU or SF2 (Figure 3a). The third rabbit (Pang) again showed binding of its hyperimmune serum, but in this case the preimmune serum also bound to HIV-1 BRU-infected cells and so no conclusion can be drawn about the response. However, cells infected with HIV-1 SF 2 showed results very similar to those with the other rabbits, with both the preimmune and immune sera (Figure 3b) and, with this strain of virus, the preimmune serum did not bind and so binding could be shown to result from immunisation.

Figure 4 shows binding of sera from one rabbit (Pong) to cells infected with HIV-2. The cells were either

uninfected (a and b) or infected with HIV-2 _CAM2, and were treated with either preimmune (a and c) or immune serum (b and d). Again binding is seen only with the immune serum onto infected cells.

These results demonstrate that the highly conserved sequence of gp 120 in the form presented, i.e. coupled to keyhole limpet hemocyanin and with the cysteine residues in a configuration mimicking disulphide bridges and the epsilon-amino groups of the lysine residues maintained, gave a good immune response in the 3 rabbits tested. The resulting antisera bind to native gp 120 from a number of strains of HIV-1, and to HIV-2.

This extent of conservation, coupled with the surface accessibility of the epitope, suggests that this peptide may have some functional role in the native protein. One possible role could be in binding to a receptor additional to the well characterised CD4.

Example 2

Further experiments have been carried out using a peptide including the conserved sequence of the invention (A chain peptide) cross linked by disulphide bridges to a further peptide (B chain peptide) based on the cross linked sequences naturally occurring in gp 120.

The sequences of A chain peptide and B chain peptide (with cross-linking indicated) are as follows:

Further experiments were carried out using a disulphide-bridged peptide based upon the sequences A and B of the invention.

Peptide Synthesis

Peptides having the sequences DQSLKPCVKLTPLCVTLY (A chain) (Seq. Id.No.6) and LINCNRSAIKESCPKVSF (B chain)

(Seq. Id.No.5) were synthesised as described in Example 1, but with the cysteine residues protected by Acm on

cysteine 7 and Trityl on cysteine 14 of the A chain, and by Trityl on cysteine 4 and Acm on cysteine 13 of the B chain. Trityl groups were removed from each peptide by acidolysis and the resulting cysteine in the A chain then reacted with 2-dipyridyl disulphide and gel filtered to remove excess reagent. The activated peptide was then reacted with 1.5-fold excess of B chain and the singly disulphide-bridged peptide purified on Whatman CM52 cation exchange resin.

The second disulphide-bridge was then formed specifically by reaction with 10 equivalents of I₂ in methanol. The peptide was finally purified by gel filtration.

Antiserum Production

The disulphide-bridged peptide was coupled to KLH exactly as described in Example 1, with the same protection for the lysine residues. Two rabbits (Tristan and König

Marke) were immunised by subcutaneous injection of the KLH conjugate in Freunds complete adjuvant, followed by a single boost with the same conjugate in Freunds incomplete adjuvant. Subsequent boosts were with peptide alone in Freunds incomplete adjuvant.

Immune responses were analysed by ELISA against

biotinylated peptide bound to streptavidin coated wells of microtitre plates. The development reagents were the same as those in Example 1.

ELISA of the rabbit preimmune sera and also sera after immunisation showed that both rabbits developed a response to the peptide as immunogen, when presented as described (Table 5). The final bleeds gave enhancements (greater than 10-fold compared to the preimmune sera) at ddilutions of 1:16000.

Study of Virus Neutralisation by Antisera

Samples (50 tissue culture infective doses (TCID₅₀)) of HIV-1_BRU were incubated with 1:20 dilutions of either preimmune or immune sera from one rabbit (Tristan), or as controls with PBS or serum from a rabbit immunised with gp 120 from HIV-1_BRU, for 60 minutes at 37°C. The treated virus was then used to infect C8166 tissue culture cells and the course of infection followed by standard methods, measuring the virus present in the supernatant over the cells by the reverse transcriptase activity released by Triton X100, following the incorporation of ³H-dTTP with poly-A as template and oligo-dT as primer.

Figure 5 shows that the virus in the supernatant rose about 1000 fold between days 7 and 12 after infection with the virus incubated with the control of PBS, while

incubation of the virus with antiserum against gp 120 prevented any sign of infection. The preimmune serum did not affect the infection, but the immune serum was as effective in preventing infection as the anti-gp 120 control serum.

Immunisation with the disulphide-bridged peptide of the invention, by the method described above, therefore led to the presence of antibodies in the serum which would neutralise the virus infectivity. Moreover the sequence of the B chain in the immunogen is not identical to that of the equivalent peptide in HIV-1 _RU and yet

neutralisation is found, suggesting that the overall peptide will lead to antibodies reacting with variants of HIV-1 or HIV-2, although better rection with the specific HIV Types might be obtained with one peptide species for each as discussed above.

Table 1

Abbreviations for amino acids

Amino acid Three-letter One-letter abbreviation symbol

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D

Cysteine Cys C

Glutamine Gln Q

Glutamic acid Glu E

Glycine Gly G

Histidine His H

Isoleucine Ile I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V Table 2

10 20

Consensus DQSLKPCVKLTPLCVTLNC-D (Seq. Id. No. 7)

1 HIV-1 : Z 6 - - - - - - - - - - - - - - - - - - - -T-

2 HIV-1: HXB 2 - - - - - - - - - - - - - - - S-K-T-

3 HIV-1: ELI - - - - - - - - - - - - - - - - - - - -S-

4 HIV-1: HAN - - - - - - - - - - - - - - - - - - K -T-

5 HIV-1: CDC 451 - - - - - - - - - - - - - - - - - - - -T-

6 HIV-1: BRVA - - - - - - - - - - - - - - - - - - - -H-

7 HIV-1: JFL - - - - - - - - - - - - - - - - - - - -IN

8 HIV-1: BRU - - - - - - - - - - - - - - - -S-K-T-

9 HIV-1: JH 3 - - - - - - - - - - - - - - - - - - - -I-

10 HIV-1: JR-CSF - - - - - - - - - - - - - - - - - - --K-

11 HIV-1: JR-FL - - - - - - - - - - - - - - - - - - - -K-

12 HIV-1: ALA 1 - - - - - - - - - - - - - - - - - -H-I-

13 HIV-1: JY 1 - -N- - - - - - - - - - - - - - - - -TN

14 HIV-1: MAL - - - - - - - - - - - - - - - - - - - -TN

15 HIV-1: MFA - - - - - - - - - - - - - - - -N-K-T-

16 HIV-1: MN - - - - - - - - - - - - - - - - - - - -T-

17 HIV-1: NDK - - - - - - - - - - - - - - - - - - - -T-

18 HIV-1: NY 5 - - - - - - - - - - - - - - - - -T-

19 HIV-1: OYI - - - - - - - - - - - - - - - - - -D-T-

20 HIV-1: RF - - - - - - - - - - - - - - - - - - - -T-

21 HIV-1: SC - - - - - - - - - - - - - - - - - -TN

22 KIV-1: SF 162 - - - - - - - - - - - - - - - -H-TN

23 HIV-1: SF 2 - - - - - - - - - - - - - - - - - - -T-

24 HIV-1: SF 33 - - - - - - - - - - - - - - - - - - -T-

25 HIV-1: WMJ 2 - - - - - - - - - - - - - - - - - - -I-

26 HIV-1: Z2 Z6 - - - - - - - - - - - - - - - - - I-

27 HIV-1: Z 321 - - - - - - - - - - - - - - - - -S-HN

28 HIV-2: ST ET-I - - - - - - - - - - -AMR-NS

29 HIV-2: GH 1 ET-I - - - - - - - - - - -AMS-NS

30 HIV-2: ROD ET-I - - - - - - - - - - -AMK-SS

31 HIV-2: BEN ET-I - - - - - - - - - - -AM--SR

32 HIV-2: ISY ET-I - - - - - - - - - - - M--NA

33 HIV-2: NIH Z ET-I - - - - - - - - - - -AM- -TR

34 HIV-2: D 194 ET-I - - - - - - - - - - -AM- -NI

35 HIV-2: D 205 ET-I - - - - - - - - - - -AM--SK

36 SIV: CPZ Q-SK

37 SIV: MNE ET-I - - - - - - - - - -I-MK-NK

38 SIV: SMM-PBJ ET- I - - - - - - - - - -I-MR-NK

39 SIV: SMM-H4 ET-I - - - - - - - - - -I -AMR-NK (IAMR is Seq. Id. No. 8)

40 SIV: MM 239 ET-I - - - - - -S - - -I-MR-NK

41 SIV: MM 251 ET-I - - - - - -S - - -I-MR-NK

42 SIV: MM 142 ET-I - - - - - -S- - -I-MR-NK

43 SIV: AGM 3 E-T- - - - - - -S- - -IKMS-VE (IKMS is Seq. Id. No. 9)

44 SIV: AGM 155 E-T S- --IKM- -VE

45 SIV: TYO-1 E-TM S- - -IKM- -VE

46 SIV: MN-DGB1 -TII - - - - -IN-Y--KMQ-QE Table 3

10 20

Consensus LI-CNTSVITQACPKVSF (Sea. Id. No. 10),

1 HIV-1 : Z 6 - -N - - - - A - - - - - - - - - -

2 HIV-1 : HXB 2 -TS - - - - - - - - - - - - - - -

3 HIV-1: ELI - - N - - - - A - - - - - - - - - -

4 HIV-1: HAN - - H- - R - - - - - - - - - - - -

5 HIV-1: CDC 451 - - N - - - - - - - - - - - - - - -

6 HIV- 1 : BRVA - - S - - - - - - - - - - - - - T -

7 HIV-1: JFL - -N - - - -T - - - - - - - - - -

8 HIV-1: BRU -TS - - - - - - - - - - - - - - -

9 HIV-1: JH 3 - - S - - - - - - - - - - - - I - -

10 HIV-1: JR-CSF - - S - - - - - - - - - - - - - - -

11 HIV-1: JR-FL - - S - D - - - - - - - - - - I - -

12 HIV-1: ALA 1 - - S - - - - - - - - - - - - - - -

13 HIV-1: JY 1 - -N - - - -A - - - - - - - -T-

14 HIV-1: MAL - -N - - - - - - - - - - - - -T-

15 HIV-1: MFA - - S - - - - - - - - - - - - - - -

16 HIV-1: MN - - S - - - - - - - - - - - -I - -

17 HIV-1: NDK - -N-D - -T - - - - - - - I - -

18 HIV-1: NY 5 - - N - D - - - - - - - - - - - - -

19 HIV-1: OYI - - H - - - -T - - - - - - -I- -

20 HIV-1: RF - -H- -S - - - - - - - - - - - -

21 HIV-1: SC - - N - - - - - - - - - - - - - - -

22 HIV-1: SF 162 - - N - - - - - - - - - - - - - - -

23 HIV-1: SF 2 - -H- -R - - - - - - - - - - - -

24 HIV-1: SF 33 - -H- -S - - - - -T - - - - - -

25 HIV-1: WMJ 2 - -N - - - - - - - - - - - - - - -

26 HIV-1: Z2 Z6 - -N - - - -A - - - - - - - - - -

27 HIV-1: Z 321 - -N - - - -A - - - - - - - - - -

28 HIV-2: ST MNH - - - - - - -ES-D-HYW

29 HIV-2: GH 1 MNH - - - - - - -ES-D-HYW

30 HIV-2: ROD MNH - - - - - - -ES-D-HYW

31 HIV-2: BEN MRH - - - - I -KES-D-HYW

32 HIV-2: ISY MNH - - - - - - -ES-D-HYW

33 HIV-2: NIH Z MNH - - - - - - -ES-D-HYW

34 HIV-2: D 194 MRH - - - - - -KES-D-HYW

35 SIV : CPZ I -N - - -TAH - - - - - - T - -

36 SIV: MNE MNH - - - - - -QES-D-HYW

37 SIV: SMM-PBJ MNH - - - - - -QES-D-HYW

38 SIV: SMM-H4 MHH - - - - - -QES-D-HYW

39 SIV: MM 239 MNH - - - - - -QES-D-HYW

40 SIV MM 251 MNH - - - - - -QES-D-HYW

41 SIV: MM 142 MNH - - - - - -QEC-D-DYW

42 SIV AGM 3 M-H- -D- - -KE- - D -TYW

43 SIV AGM 155 M-H- -D- - -KE- -D-TYW

44 SIV TY0-1 M-H- -D- - -KE- -D-TYW

45 SIV MN-DGB1 MNH- -E- -N-ED-Q-GLL Tab le 4. ELISA reactivities of sera from rabbits used for immunisation studies.

Serum No peptide added Ping Pang Pong

dilution to well Preimmune Immune serum Preimmune Immune serum Preimmune Immune serum

1 :3200 0.045 0.029 0.354 0.104 0.337 0.130 0.566

1 :6400 0.051 0.034 0.183 0.052 0.210 0.089 0.363

Table 5. ELISA reactivities of sera from rabbits immunised with HB-AIDS2.

Serum No peptide added Tristan König Marke

dilution to well Preimmune Immune serum Preimmu Immune serum

1:2000 0.083 0.056 4.000 0.074 3.889

1:4000 0.076 0.059 4.000 0.067 2.556

1:8000 0.070 0.060 4.000 0.063 1.467

1:16000 0.083 0.081 2.817 0.077 0.880

References

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8. Starcich, B R, Hahn, B H, Shaw, G M, McNeely, P D, Modrow, S, Wolf, H, Parks, E S, Parks, W P, Josephs, S F, Gallo, R C & Wong-Staal, F (1986) Cell 45, 637-648. 9. Modrow, S, Hahn, B H, Shaw, G M, Gallo, R C, Wong-Staal, F & Wolf, H (1987) J Virol. 61, 570-578.

10. Rusche, J R, Javaherian, K, McDanal, C, Petro, J, Lynn, D L, Grimaila, R, Langlois, A, Gallo, R C, Arthur, L O, Fischinger, P J, Bolognesi, D P, Putney, S D &

Matthews, T J (1988) Proc. Nat. Acad. Sci. USA 85, 3198-3202.

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Gojobori, T & Hayami, M (1989) Nature (Lond.) 341, 539-541.

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Franchini, G, Kalyanaraman, V S & Gallo, R C (1983)

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Alizon, M (1985) Cell 40, 9-17.

26. Sanchez-Pescadoe, R, Power, M D, Barr, P J, Steimer, K S, Stempien, M M, Brown-Shimer, S L, Gee, W W, Renard, A, Randolph, A, Levy, J A, Dina, D & Luciw, P A (1985) Science (Wash.) 227, 484-492.

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(A ) NAME : Medical Research Council

(B) STREET: 20 Park Crescent

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(F) POSTAL CODE (ZIP) : WIN 4AL

(G) TELEPHONE : (071) 636 5422

(H) TELEFAX: (071) 323 1331

(A) NAME: Butler, Peter J. G.

(B) STREET: 2 Heron's Close

(C) CITY: Cambridge

(E) COUNTRY: United Kingdom

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(A) NAME: Hacking, Graeme N. V.

(B) STREET: Selwyn College

(C) CITY: Cambridge

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(ii) TITLE OF INVENTION: Developments relating to human immunodeficiency viruses

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Claims

1. A molecule comprising a peptide having the amino acid sequence lysine-proline-cysteine-valine-lysine-leucinethreonine-proline-leucine-cysteine-valine (Seq. Id.No.1), wherein each cysteine residue is disulphide bridged to a further cysteine residue or is derivatised to simulate part of a disulphide bond, or functionally equivalent variants of such a peptide which mimic the immunogenic behaviour of an epitope of gρ120 of HIV env.

2. A molecule according to claim 1, wherein each cysteine residue is derivatised as a S-acetamidomethyl derivative.

3. A molecule according to claim 1, cross-linked to a further peptide sequence.

4. A molecule acording to claim 3, cross-linked as follows:

5. A molecule according to any one of claims 1 to 4, wherein the or each peptide sequence is modified by one or more individual amino acids having been substituted by amino acids having comparable properties.

6. A molecule according to any one of claims 1 to 5, wherein the or each peptide sequence is modified by one or more of the normal peptide linkages having been

substituted by isosteric replacements.

7. A molecule according to any one of claims 1 to 6, wherein the or each peptide sequence is modified by one or both pairs of disulphide-bridged cysteine residues having been substituted by a cystine analogue.

8. A molecule according to any one of claims 1 to 7, wherein the or each peptide sequence is modified by one or more of the amino acids having been replaced by a "retro-inverso" amino acid.

9. A molecule according to any one of claims 1 to 8, wherein the or each peptide sequence is modified by one or more non-essential amino acids having been deleted.

10. A molecule accordig to any one of claims 1 to 9, wherein the or each peptide sequence is modified by inclusion of one or more additional amino acids not significantly affecting function.

11. A DNA molecule coding for a peptide molecule in accordance with any one of the preceding claims.

12. A molecule according to claim 11, incorporated into a suitable expression vector.

13. A vaccine against AIDS and AIDS related conditions, comprising a molecule in accordance with any one of claims 1 to 10.

14. A vaccine according to claim 13, wherein the molecule of any one of claims 1 to 10 is linked to a carrier molecule.

15. A vaccine according to claim 13, wherein the molecule of any one of claims 1 to 10 is presented as a live vaccine.

16. A method for the prophylaxis or treatment of AIDS or related conditions, comprising administering an effective amount of a molecule in accordance with any one of claims 1 to 10.

17. A pharmaceutical composition containing, as an active ingredient, a molecule in accordance with any one of claims 1 to 10, possibly in association with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients.

18. Use of a molecule in accordance with any one of claims 1 to 10, for the preparation of a medicament for the therapy or prophylactic treatment of AIDS or related conditions.

19. A molecule that binds to the molecule of any one of claims 1 to 10, particularly an antibody, an antigen binding site of an antibody or a structural analog

thereof.

20. Use of a molecule which binds to the molecule of any one of claims 1 to 10 for therapeutic or diagnostic purposes. AMENDED CLAIMS

[received by the International Bureau on 20 December 1993 (20.12.93)

original claims 1-20 replaced by amended claims 1-21 (4 pages) ]

1 . A molecule comprising a peptide having the amino acid sequence lysine-proline-cysteine-valine-lysine-leucinethreonine-proline-leucine-cysteine-valine ( Seq . Id . No . 1 ) , wherein each cysteine residue is disulphide bridged to a further cysteine residue or is derivatised to simulate part of a disulphide bond , or functionally equivalent variants of such a peptide which mimic the immunogenic behaviour of an epitope of gp120 of HIV env .

2. A molecule according to claim 1 , wherein each cysteine residue is derivatised as a S-acetamidomethyl derivative .

3. A molecule according to claim 1 , cross-linked to a f urther peptide sequence .

4. A molecule acording to claim 3 , cross-linked as follows :

5. A molecule according to any one of claims 1 to 4 , wherein the or each peptide sequence is modified by one or more individual amino acids having been substituted by amino acids having comparable properties .

6. A molcule according to claim 5 , comprising the

f ollowing sequences , cross-linked as indicated :

7. A molecule according to any one of claims 1 to 6, wherein the or each peptide sequence is modified by one or more of the normal peptide linkages having been

substituted by isosteric replacements.

8. A molecule according to any one of claims 1 to 7 wherein the or each peptide sequence is modified by one or both pairs of disulphide-bridged cysteine residues having been substituted by a cystine analogue.

9. A molecule according to any one of claims 1 to 8, wherein the or each peptide sequence is modified by one or more of the amino acids having been replaced by a "retro-inverso" amino acid.

10. A molecule according to any one of claims 1 to 9, wherein the or each peptide sequence is modified by one or more non-essential amino acids having been deleted.

11. A molecule according to any one of claims 1 to 10, wherein the or each peptide sequence is modified by inclusion of one or more additional amino acids not significantly affecting function.

12. A DNA molecule coding for a peptide molecule in accordance with any one of the preceding claims.

13. A molecule according to claim 12, incorporated into a suitable expression vector.

14. A vaccine against AIDS and AIDS related conditions, comprising a molecule in accordance with any one of claims 1 to 11.

15. A vaccine according to claim 14, wherein the molecule of any one of claims 1 to 11 is linked to a carrier molecule.

16. A vaccine according to claim 14, wherein the molecule of any one of claims 1 to 11 is presented as a live vaccine.

17. A method for the prophylaxis or treatment of AIDS or related conditions, comprising administering an effective amount of a molecule in accordance with any one of claims 1 to 11.

18. A pharmaceutical composition containing, as an active ingredient, a molecule in accordance with any one of claims 1 to 11, possibly in association with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients.

19. Use of a molecule in accordance with any one of claims 1 to 11, for the preparation of a medicament for the therapy or prophylactic treatment of AIDS or related conditions.

20. A molecule that binds to the molecule of any one of claims 1 to 11, particularly an antibody, an antigen binding site of an antibody or a structural analog thereof.

21. Use of a molecule which binds to the molecule of any one of claims 1 to 11 for therapeutic or diagnostic purposes.