FIELD OF INVENTION

The invention relates to a chimaeric peptide for use as a vaccine or treatment for HIV. hi particular, the invention relates to a chimaeric peptide comprising a CCR5 epitope and a T cell epitope.

BACKGROUND

The chemokine receptor CCR5 is required by many strains of HIV for cellular entry. Accordingly, it provides a potential target for molecules such as antibodies designed to block HIV transmission. It has been suggested that CCR5 is a potential vaccine target, using a vaccine that generates antibody production.

Most vaccines in current use stimulate strong neutralising antibody responses, which play an important part in mediating protection. However, classical vaccination strategies applied to HIV infection, in which an antibody response to the virus itself is generated, have proved to be ineffective. A significant proportion of antibody against the virus is non-neutralising, and indeed some of this antibody may enhance viral infectivity [1;2] The majority of neutralising antibody is directed against the gpl20 glycoprotein on the viral surface. This protein shows extensive variability, and most antibodies against it are in fact strain specific, limiting the application of this immunogen in protecting at risk populations. Some cross-strain neutralising antibodies have been reported [3;4] and efforts are in progress to focus the immune response on the epitopes recognised by these antibodies; however, alternative strategies to inhibit HIV are of interest.

An alternative target for HIV blocking antibodies are the receptors used by virus to gain entry into the cell. The CD4 molecules play a key role in T cell function, and antibodies against it are likely to be pathogenic. However, the CCR5 chemokine receptor offers a more attractive target. This receptor is absent in approximately 1% of the Caucasian population [5], and these individuals show no gross symptoms of immunodeficiency. CCR5 deficiency is associated with almost complete protection against HIV infection [7;8] and even CCR5 heterozygous individuals, which show haplotype insufficiency, show a slower progression to AIDS [9]. Genetically determined overexpression of CCR5 ligand, CL3L1, is also associated with partial protection [10]. Finally, small molecular weight CCR5 antagonists can block HIV entry, and one such inhibitor is in clinical use [H]. Thus, CCR5 levels are quantitatively, as well as qualitatively, a key parameter in determining the course of HIV infection, and the subsequent development of AIDS. Several groups have recently investigated the possibility of raising antibodies against CCR5 [12-16], using recombinant proteins, recombinant viruses or synthetic cyclic peptides. The results have been encouraging, suggesting that it is possible to break tolerance to CCR5 in vivo, that autoantibody production is not associated with any overt pathology, and that the antibodies can inhibit viral replication in vitro and in vivo. The safety of autoantigen driven vaccine strategies remains of concern, however. For example, a trial of therapeutic vaccination in Alzheimer patients using the amyloid fragment A/3, was discontinued because of adverse side effects attributed to the autoimmune response [17]. However, there was no direct correlation between the pathology and antibody titre in these patients, suggesting the possibility that the damage was due to autoimmune cellular responses. Cellular autoimmune responses against the CCR5 receptor are also likely to be pathogenic, since they may lead to elimination of dendritic cells, macrophages, T cells and any other cell types which express this receptor. The inventor has, therefore, explored the possibility of raising an immune response to the CCR5 receptor, using a very short N terminal fragment of the receptor, coupled to a well characterised epitope of tetanus toxoid [18;19]. Since the immunogen contained only a small number of amino acids of CCR5 sequence, the possibility of including a CD4 or CD8 T cell auto-epitope is removed. The inventor has shown that this chimaeric peptide can indeed stimulate antibody which recognises intact receptor, and that this antibody can induce inhibition of HIV infectivity.

SUMMARY OF THE INVENTION

The invention provides a chimaeric peptide comprising a CCR5 epitope comprising an amino acid sequence comprising between 4 and 9 contiguous amino acid residues found contiguously in the extracellular portions of a CCR5 receptor, the chimaeric peptide further comprising a T helper cell epitope.

The peptide of the invention comprises two epitopes, that is two peptides that are recognised by, and stimulate a response from, the immune system. The first epitope is a CCR5 epitope, used to stimulate the production of antibodies to the CCR5 receptor. The CCR5 epitope comprises between 4 and 9, such as at least 5, or at least 6, and such as no more than 8, particularly 7 contiguous amino acid residues found contiguously in the extracellular regions of CCR5. It is particularly preferred that the region of CCR5 is the N terminal region. This means that any part of the CCR5 epitope may contain more than 9, or more than 8, or more than 7 amino acid residues but that no more than 9, preferably no more than 8, even more preferably no more than 7 contiguous amino acid residues may be found contiguously in one of the extracellular regions of CCR5, especially the N terminal. This is to avoid using an accidental CD4 or CD8 autoepitope. The CCR5 epitope comprises at least 4, more preferably at least 5, even more preferably at least 6 contiguous amino acid residues found contiguously in one of the extracellular regions of CCR5. This is to allow a specific response to CCR5 to be generated.

The CCR5 epitope is preferably selected from those shown in table 1. In particular the CCR5 epitope is preferably selected from those in column 1 of table 1. Those epitopes are taken from the N-terminal sequence of CCR5. This region is preferred since it may adopt a looser configuration than the other extracellular loops, and hence increase the possibility that the peptides will stimulate antibody recognising the receptor.

The T helper cell epitope is an epitope that stimulates T helper cells of the immune system. It is known in the art that antibody responses produced by B cells to a specific region of a protein or peptide require simultaneous recognition of another part of that protein or peptide by T helper cells. This is known as B/T cell collaboration. This may be mimicked in the chimaeric peptide containing the CCR5 epitope (as the B cell epitope) by linking the CCR5 epitope to a T helper cell epitope. Strong T helper cell epitopes which are recognised by the immune T cells of a large proportion of individuals are known in the art and are referred to as promiscuous epitopes. One example of such an epitope is from the tetanus toxoid, but any other promiscuous epitope may be used. Other promiscuous epitopes are described in Mustafa AS, Shaban FA, ProPred analysis and experimental evaluation of promiscuous T-cell epitopes of three major secreted antigens of Mycobacterium tuberculosis. Tuberculosis (Edinb). 2006 Mar;86(2): 115-24; Agadjanyan MG, Ghochikyan A, Petrushina I, Vasilevko V, Movsesyan N, Mkrtichyan M, Saing T, Cribbs DH. Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide. J Immunol. 2005 Feb 1 ; 174(3): 1580-6; Al-Attiyah R, Shaban FA, Wiker HG, Oftung F, Mustafa AS. Synthetic peptides identify promiscuous human ThI cell epitopes of the secreted mycobacterial antigen MPB70. Infect Immun. 2003 Apr;71(4): 1953-60; Panina-Bordignon P, Tan A, Termijtelen A, Demotz S, Corradin G, Lanzavecchia A. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol. 1989 Dec;19(12):2237-42.; and Muller CP, Ammerlaan W, Fleckenstein B, Krauss S, Kalbacher H, Schneider F, Jung G, Wiesmϋller KH. Activation of T cells by the ragged tail of MHC class II- presented peptides of the measles virus fusion protein, hit Immunol. 1996 Apr;8(4):445-56. The T helper cell epitope is preferably taken from the tetanus toxoid and preferably comprises or has the following sequence: VDDALINSTKIYSYFPSV.

The two epitopes may be linked by a spacer to provide conformational separation between the two epitopes. For example the spacer may comprise a number, such as 2, 3 or 4 amino acids. Multiple spacers may be used. Such spacers are well known in the art. A preferred spacer has the sequence GPSL or GPSLC or CGPSL. When one of GPSLC and CGPSL is used as the spacer, the CCR5 epitope may comprise a further C residue at its opposite end to the end attached to the spacer.

The epitopes may be linked, possibly via the spacer, with either the N terminal of the CCR5 epitope linked to the C terminal of the T helper cell epitope or with the C terminal of the CCR5 epitope linked to the T helper cell epitope N terminal. The peptides of the invention may comprise further amino acids in addition to the epitopes and the spacer, when used. For example, the peptide may include more than one T helper cell epitope, especially two or three T helper cell epitopes, and/or may comprise more than one CCR5 epitope, especially two or three CCR5 epitopes. In particular, the peptide may comprise a plurality, especially two or three of each type of epitope. The epitopes may be arranged in any order. When more than one of each type of epitope is included, the CCR5 epitopes may be the same or different and the T helper cell epitopes may be the same or different. When a plurality of one or both types of epitope is included, the epitopes are preferably separated by spacers.

Also, the epitopes may be flanked by other amino acids. For example, amino acids may be included that encourage the peptide to fold into a conformation that mimics the conformation of the region from which the CCR5 epitope is taken. This might be achieved by including cysteine residues that will encourage the peptide to fold by forming disulphide bonds. Alternatively this might include lysine/arginine and glutamic/aspartic acid residues that can constrain the peptide by forming intrachain peptide bonds. The peptide may include additional immunostimulatory regions such as immunostimulatory epitopes from invasin proteins especially from Yersinia species. Such epitopes are well known in the art. It may also be useful to make lipid modifications to the T helper cell eptiope. Such modifications are described in US 5,843,446.

Further provided by the invention is an antibody that binds specifically to one of the peptides of the invention. In particular, there is provided an antibody that binds to the CCR5 epitope in the peptide of the invention. Preferably, the antibody binds to CCR5 and more preferably binds to the CCR5 and blocks that receptor.

Also provided by the invention is a pharmaceutical composition comprising a peptide or an antibody according to the invention.

The composition is suitable for administration to patients. In addition to the peptide or antibody, it may comprise one or more appropriate pharmaceutical excipient(s) such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. The preparation of pharmaceutical compositions and the use of excipients is well known in the art. Other active compounds may also be included. The pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. It may be possible to create compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes. For example, the administration may be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.

Solutions or suspensions used for intradermal or subcutaneous application typically include at least one of the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and tonicity agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases. Such preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials.

Solutions or suspensions used for intravenous administration include a carrier such as physiological saline, bacteriostatic water, ethanol, or polyol. In all cases, the composition must be sterile and fluid for easy syringability. Proper fluidity can often be obtained using lecithin or surfactants. The composition must also be stable under the conditions of manufacture and storage. Prevention of microorganisms can be achieved with antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents (sugar), polyalcohols (mannitol and sorbitol), or sodium chloride may, be included in the composition. Prolonged absorption of the composition can be accomplished by adding an agent which delays absorption, e.g., aluminium monostearate and gelatin. Oral compositions include an inert diluent or edible carrier. The composition can be enclosed in gelatin or compressed into tablets. For the purpose of oral administration, the antibodies can be incorporated with excipients and prepared as tablets or capsules, for example. The oral composition may also contain, for example, a binder, an excipient, a lubricant and flavourings.

Compositions may also be administered by a transmucosal or transdermal route. For example, antibodies that comprise a Fc portion may be capable of crossing mucous membranes in the intestine, mouth, or lungs (via Fc receptors). Transmucosal administration can be accomplished through the use of lozenges, nasal sprays, inhalers, or suppositories. Transdermal administration can also be accomplished through the use of composition containing ointments, salves, gels, or creams known in the art. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used.

For administration by inhalation, antibodies are delivered in an aerosol spray from a pressured container or dispenser, which contains a propellant (e. g., liquid or gas) or a nebulizer.

hi certain embodiments, peptides and antibodies of this invention are prepared with carriers to protect the antibodies against rapid elimination from the body.

Biodegradable polymers (e. g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid) are often used.

Methods for the preparation of such pharmaceutical compositions are known by those skilled in the art.

Peptides, antibodies or compositions according to the invention may be administered in therapeutically effective amounts, as determined, based on, for example, the patient's weight, gender, age and medical condition. The peptides, antibodies or compositions may be administered in a single dose, as a bolus or as continuous therapy. The pharmaceutical composition may also comprise an adjuvant, especially an adjuvant that enhances the immunogenicity of the peptides. Such adjuvants may or may not be immunogenic themselves, but may for example attract immune cells to the peptide or may retain the peptide near the site of interest or site of administration.

Immuno stimulatory adjuvants include aluminium salts ("alum"), lipopolysaccharides and immunomodulators. These and many other immunostimulatory adjuvants are well known in the art and are described in EP 1237930. Adjuvants that produce the maximum response for the lowest dose and that result in the fewest and weakest side effects are preferred.

The pharmaceutical composition may, in particular, be an immunising composition that includes an effective amount of one or more peptides according to the invention to induce the production of antibodies that bind to the CCR5. Such formulations would be readily known to those skilled in the art and include formulations for immediate release and for sustained release. The appropriate dosage regime would be known to one skilled in the art, the amount of compositions per dose unit usually being between 0.5 μg and 1.0 mg per kilo of body weight. Such a composition may be administered in single or multiple doses and may be for administration before or after a subject's exposure to HIV.

Also provided by the invention is a peptide or antibody according to the invention for use in therapy. In particular, the peptide or antibody is for the use in the prevention or treatment of an infection with HIV. Prevention or treatment of an infection with HIV means that the peptides or antibodies may be used to prevent the entry of HIV into an individual's cell and, accordingly, prevent viral replication.

The peptides or antibodies of the invention may be administered alone or a number of different peptides according to the invention may be administered together. Also the peptides or antibodies may be administered with other biologically active molecules, especially molecules that bind to CCR5, such as appropriate small molecules, RANTES, MEP Ia and MEPI/3. Such molecules may also be included in a pharmaceutical composition with the peptides and antibodies.

Further provided is a method of preventing or treating a HIV infection comprising administering a peptide, antibody or composition according to the invention to a subject in need thereof.

The peptides, antibodies and pharmaceutical compositions of the invention may also be used in any other therapies in which the modulation especially the blocking of CCR5 would be useful. Such therapies include the prevention, treatment or alleviation of rheumatoid arthritis; treatment of experimental autoimmune myocarditis; treatment of chronic inflammation, particularly of the respiratory tract; treatment of graft rejection; treatment of T helper 1 -mediated auto-immune diseases, and infectious diseases involving CCR5; and treatment of diabetes.

The peptides according to the invention may be made by any standard means, such as synthetic chemical methods well known to those skilled in the art. For example the peptides may be made by solid phase synthesis. Alternatively the peptides may be made using recombinant DNA technology. The invention therefore provides an isolated nucleic acid molecule encoding a peptide according to the invention. Further provided is a vector comprising that nucleic acid molecule and a host cell comprising the vector. Any appropriate vector and host cell may be used and would be known to the skilled person.

The peptides, antibodies and nucleic acids of the invention are preferably isolated. The term "isolated" refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it was derived. The term also refers to preparations where the isolated protein is sufficiently pure for pharmaceutical compositions; or at least 70-80% (w/w) pure; or at least 80-90% (w/w) pure; or at least 90-95% pure; or at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure. The term 'peptide' as used herein means an amino acid polymer and includes polypeptides. There is no limit on the size of the peptides, but the peptides preferably comprise between 15 and 200 amino acids, more preferably between 15 and 100 amino acids, most preferably between 15 and 50 amino acids. The amino acids may be naturally occurring amino acids or may be artificial or modified amino acids. Particular modifications of interest include glycosylations, or the use of sulphoamino acids. Sulphoamino acids are found naturally on CCR5, especially at the N-terminal. (Bannert, N., S. Craig, M. Farzan, D. Sogah, N. V. Santo, H. Choe, and J. Sodroski. 2001. Sialylated O-glycans and sulfated tyrosines in the NH2-terminal domain of CC chemokine receptor 5 contribute to high affinity binding of chemokines. J.Exp.Med. 194:1661.)

The term CCR5 is used to refer to a human chemokine receptor and to the homologues thereto found in other species such as primates and rodents. The term preferably means the human receptor. The term is well known in the art, and is primarily expressed by T cells, dendritic cells, macrophages and microglia. The extracellular regions of CCR5 are those regions that are on the extracellular surface of a cell membrane in which CCR5 is found and are shown in figure 7..

A T helper cell epitope is an epitope that is recognised by T helper cells. Specifically, the term refers to promiscuous epitopes that are capable of eliciting T helper cell responses in a large number of individuals having diverse MHC haplotypes. Such epitopes are able to elicit potent antibody responses in a wide and genetically diverse population.

When the T helper cell epitope is derived from a natural source, it is derived from a natural source that is different to the source of the CCR5 epitope. In other words, the T helper cell epitope is not recognized as part of a self molecule in the mammal subject immunized according to the method of the present invention.

The T helper cell epitope is also preferably selected not only for a capacity to cause immune responses in most members of a given population, but also for a capacity to cause memory/recall responses. When the mammal is human, the vast majority of human subjects/patients receiving immunotherapy with the chimaeric peptide of the present invention will already have been immunized with the paediatric vaccines (i.e., measles+mumps+rubella and diphtheria+pertussis+tetanus vaccines) and, possibly, the hepatitis B virus vaccine. These patients have therefore been previously exposed to at least one of the T helper cell epitopes present in chimaeric paediatric vaccines. Prior exposure to a T helper cell epitope through immunization with the standard vaccines should establish T helper cell clones which can immediately proliferate upon administration of the chimaeric peptide (i.e., a recall response), thereby stimulating rapid B cell responses to the chimaeric peptide. In addition, the use of T helper cell peptide epitopes avoid recognition of the immunising antigen by preexisting antibody which is stimulated by previous vaccination, and which can suppress the subsequent response to immunization, a problem encountered when intact toxin molecules are used to elicit T helper cell responses.

The T helper cell epitopes in the chimaeric peptide of the invention are promiscuous but are unlikely to be universal. This characteristic means that the T helper cell epitopes are reactive in a large segment of an outbred population expressing different MHC antigens (reactive in 50 to 90% of the population), but not in all members of that population. To provide a comprehensive, approaching universal, immune reactivity for an internal peptide cleavage product, a combination of chimaeric peptides with different T helper cell epitopes can be prepared. For example, a combination of four chimaeric peptides with promiscuous T helper cell epitopes from tetanus and pertussis toxins, measles virus F protein and HBsAg may be more effective. Alternatively, one chimaeric peptide may contain a plurality of T helper cell epitopes.

Promiscuous T helper cell epitopes often share common structural features. For example, promiscuous T helper cell epitopes range in size from about 12 to about 20 residues. Amphipathic helices are a common feature of the T helper cell epitopes. An amphipathic helix is defined by an α-helical structure with hydrophobic amino acid residues dominating the surrounding faces. T helper cell epitopes frequently contain additional primary amino acid patterns such as a GIy or a charged reside followed by two to three hydrophobic residues followed in turn by a charged or polar residue. T helper cell epitopes often obey the 1, 4, 5, 8 rule, where a positively charged residue is followed by hydrophobic residues at the fourth, fifth and eighth positions after the charged residue. Since all of these structures are composed of common hydrophobic, charged and polar amino acids, each structure can exist simultaneously within a single T helper cell epitope.

The T helper cell epitope is a sequence of amino acids (natural or non-natural) that contains a T helper cell epitope. A T helper cell epitope can be a continuous or discontinous epitope. Hence, not every amino acid of T helper cell is necessarily part of the epitope. The term T helper cell epitope encompasses analogs and segments of T helper cell epitopes, providing those analogs and segments are capable of enhancing or stimulating an immune response to the CCR5 epitope. Immunodominant T helper cell epitopes are broadly reactive in animal and human populations with widely divergent MHC types (Celis et al., 1988 ; Demotz et al., 1989 ; and Chong et al.,1992). The T helper cell domain of the chimeric peptides of the present invention has from about 10 to about 50 amino acids residues and preferably from about 10 to about 30 amino acids residues. When multiple T helper cell epitopes are present, then each T helper cell epitope is independently the same or different.

T helper cell epitope analogs may include substitutions, deletions and insertions of one to about five amino acid residues in the T helper cell epitope. T helper cell segments are contiguous portions of a T helper cell epitope that are sufficient to enhance or stimulate an immune response to the internal peptide cleavage product. An example of T helper cell segments is a series of overlapping peptides that are derived from a single longer peptide.

The T helper cell epitopes of the present invention include, for example hepatitis B surface antigen T helper cell epitopes, pertussis toxin T helper cell epitopes, tetanus toxin T helper cell epitopes, measles virus F protein T helper cell epitope, Chlamydia trachomitis major outer membrane protein T helper cell epitopes), diphtheria toxin T helper cell epitopes, Plasmodium falciparum circumsporozoite T helper cell epitopes, Schistosoma mansoni triose phosphate isomerase T helper cell epitopes, Escherichia coli T helper cell epitopes. A spacer is one or more residues separating the CCR5 and T helper cell epitopes. Immunogenicity can be improved through the addition of spacer residues. Often the spacer residue is one or more Glycine residues. In addition to physically separating the T helper cell epitope from the CCR5 epitope, the spacer residues can disrupt any artificial secondary structures created by the joining of the T helper cell epitope with the CCR5 epitope, and thereby eliminate interference between the T and/or B cell responses. The conformational separation between the helper epitope and the antibody eliciting domain thus permits more efficient interactions between the presented immunogen and the appropriate T helper cell and B cells. The spacer amino acid residues can be naturally-occurring amino acids or non-naturally-occurring amino acids, which include, but are not limited to,-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, gamma-amino butyric acid, homoserine, citrulline and the like.

The term "antibody" is well known in the art. Herein it means an immunoglobulin or any functional fragment thereof. It encompasses any polypeptide that has an antigen- binding site. It includes but is not limited to monoclonal, polyclonal, monospecific, polyspecific, non-specific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. The term "antibody" encompasses antibody fragments such as Fab, F (ab ¹ ) 2, Fv, scFv, Fd, dAb, and any other antibody fragments that retain antigen-binding function. Typically, such fragments would comprise an antigen-binding domain. When preceded by the word "intact" the term "antibody" means a whole antibody molecule, namely two heavy chains, each with one variable region and three constant regions, and two light chains, each with one variable region and one constant region.

Intact antibodies are also known as immunoglobulins (Ig). As indicated above, intact antibodies comprise light chains and heavy chains. Light chains are classified into two isotypes, and heavy chains are classified into five isotypes (A, D, E, G, and M). Some heavy chain isotypes are further divided into isotype subclasses, e. g., IgGl, IgG2, IgG3, and IgG4. It is particularly preferred that the antibodies of the invention are IgG antibodies, hi particular, IgG2b and IgG2a antibodies are preferred. The domain and three dimensional structures of different antibodies are known in the art. The light chain is composed of a constant domain (C) and an N-terminal variable domain (V). The heavy chain is composed of three or four constant domains (C _H ), a hinge region, and a N-terminal variable domain (V _H ). The C _H adjacent to the V _H domain is designated C _{HI .} The V _H and V _L domains contain four regions of conserved sequence called framework (FR) regions (FRl, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequence called complementarity determining regions (CDR). The CDRs (CDRl, CDR2, and CDR3) contain most of the antibody amino acids that specifically binds antigen. Heavy chain CDRs are denoted Hl, H2, and H3, while light chain CDRs are denoted Ll, L2, and L3. The term CDR is well known in the art. One skilled in the art would be able to recognise CDRs in an antibody or fragment by using Kabat numbering and the amino acids found either side of the CDRs.

The Fab fragment (Fragment antigen-binding) consists of V _H , C _H I, V _L and C _L domains covalently linked by a disulfide bond between the constant regions. The Fv fragment is smaller and consists of V _H and V _L domains non-covalently linked. To overcome the tendency of non-covalently linked domains to dissociate, a single chain Fv fragment (scFv) can be constructed. The scFv contains a flexible polypeptide that links the C- terminus of V _H to the N-terminus of V _L , or the C-terminus of V _L to the N-terminus of V _H . A 15-mer (GIy ₄ Ser) ₃ peptide may be used as a linker, but other linkers are well known.

The antibodies of the invention are preferably able to bind to CCR5. Further they are preferably able to block that receptor, that is to prevent entry of HIV to a cell via the CCR5. It is possible to screen for these functions using techniques well known in the art. A functional fragment is an antibody fragment that is still able to bind CCR5. Further, a functional fragment is preferably able to block CCR5.

The terms "antigen-binding site", "antigen-binding domain" and "antigen-binding fragment" mean the part of an antibody that specifically binds antigen. The part of the antigen that is recognised and bound by the antibody is referred to as the "epitope". An antigen-binding domain usually comprises variable regions from both the light chain (V _L ) and the heavy chain (V _H ), but it does not have to comprise both. Antigen- binding fragments include Fab fragments (monovalent fragments consisting of the VL, V _H , C _L and C _HI domains); F(ab') ₂ fragments (bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region); Fd fragments (the two V _H and C _HI domains); Fv fragments (V _L or V _R domains, dAb fragments (Ward et al., (1989) Nature 341: 544-546), one or more complementarity determining regions (CDR); and single chain Fvs. The various antibody fragments can be obtained using conventional techniques known to those with skill in the art. It is possible to screen for the functionality of the fragments, e.g. binding and agonising a receptor using techniques known in the art.

As is known in the art, it is possible to use murine antibodies from mice and rats for therapy in humans. However, rodent antibodies tend to provoke strong Human anti- Murine Antibody (HAMA) immune responses which restricts their usefulness for repeated application in the same patient. Hence, the antibodies according to the invention are preferably chimeric, humanised (CDR grafted or reshaped).

The term "chimeric" refers to antibodies in which the whole of the variable regions of a mouse or rat antibody are expressed along with human constant regions. This provides the antibody with human effector functions and also reduces immunogenicity (HAMA) caused by the murine Fc region.

"Humanised" antibodies (also called CDR grafted or reshaped antibodies)" are an alternative to chimeric antibodies in which only the complimentarity determining regions from the rodent antibody V-regions are combined with framework regions from human V-regions. The idea is that these antibodies should be more human-like than chimeric and thus perhaps less immunogenic than chimeric antibodies.

It is also possible to obtain fully human antibodies from transgenic mice or other transgenic animals. Transgenic mice have been created which have a repertoire of human immunoglobulin germline gene segments. These mice when immunised thus make human like antibodies. B cells from such immunised mice may be used in the production of monoclonal antibodies.

All of these types of antibodies are encompassed by the invention.

The invention will now be discussed in detail, by way of example only, with reference to the figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The sequences of all peptides used in the study, showing the sequences derived from CCR5, the linker sequence and the Tetanus toxoid T cell epitope sequence.

Figure 2. Antipeptide antibody responses in rabbits immunized with peptides CCR5.1 and CCR5.2 a) Antisera from the final bleed from rabbits immunized with CCR5.1 (left panel) or CCR5.2 (right panel) were diluted 1:100 in blocking buffer, and then diluted over a range of threefold dilutions as shown. Anti body reactivity was tested by ELISA against peptides CCR5.3 (left panel) and CCR5.4 (right panel) as described in materials and methods. Each point shows the mean of duplicate wells. Each rabbit serum was tested at least three times, b) Antibody titre from prebleed (PB) or test bleeds collected at different times post-immunization as shown. Each point shows the mean of duplicate wells. Titres of 100 are shown as zero, c) Cross reactivity of antisera to heterologous peptides. Rabbits 1-3 were immunized with peptide CCR5.1 (with N-terminal methionine) while rabbits 4-6 were immunized withCCR5.2 (without N-terminal methionine) . Each serum was then tested against the homologous and heterologous peptide as shown, and titre determined by ELISA. Asterix indicates titre <3000. Figure 3. AntiCCR5 antibody responses in rabbits immunized with peptides CCR5.1 and CCR5.2. Final bleed antisera from rabbits 1-6 (see fig 2) were tested for binding to CCR5+/CD4+ U87 cells (diamonds) or control CD4+ U87 cells (squares) by flow cytometry, a) Mean fluorescent intensity at various dilutions for each rabbit, b) A representative histogram for prebleed (filled histogram) or final bleed (solid line) for rabbit 2 serum, tested against CCR5+/CD4+ U87 cells (top panel) or control CD4+ U87 cells. Each serum was tested by flow cytometry at least four times, c) Control staining of a monoclonal antiCCR5 (solid line) or isotype control (filled histogram) from the Centralized AIDS Repository. Figure 4. Antibody responses in rabbits immunized with peptide CCR5.5. a) Antipeptide titre in final bleeds from rabbits 7-12 , measured by ELISA using CCR5.3 peptide as target. Rabbits 7-9 were immunized with CCR5.5/KLH conjugate with CFA, rabbits 10-12 were immunized with CCR5.5 peptide coupled to magnetic beads with Alum. Ctrl shows the results using rabbit serum from an immunized rabbit. Asterisk shows titre < 100. The figure shows one representative experiment, from at least three with each serum, b) Final bleed antisera from rabbits 7-12 were tested for binding to CCR5/CD4+ U87 cells by flow cytometry. Each panel shows mean fluorescent intensity at dilutions of 1 :10, 1:30, 1:90 and 1 :270 for each rabbit. Ctrl shows the results using sera from an unimmunized rabbit. The figure shows one representative experiment, from at least three with each serum.

Figure 5. Purification of antibody from serum, a) Antipeptide binding activity of unpurified serum (A), flow through from affinity column of peptide CCR5.3 (B), and pooled eluate from affinity column (C) from rabbit 2 and rabbit 10. The titre of each sample was measured by ELISA using CCR5.3 as target. The purified antibody preparations were first adjusted to a concentration of 400 μg/ml. Asterisk indicate titre less than 1000. b) The purified antibody preparations, or an equal concentration of purified rabbit Ig were tested for binding to CCR5/CD4 expressing U87 cells or control CD4 expressing U87 cells by flow cytometry. Top two panels shows representative histograms of rabbit 2 purified antibody (80 μg/ml solid line) or control Ig (80 μg/ml , shaded) staining of CCR5+CD4+ U87 cells (left) or control CD4+ U87 cells (right). Bottom two panels show Mean Fluorescent Intensity at different concentrations of purified antibody from sera of rabbit 2 (diamonds), rabbit 10 (squares) or control rabbit Ig, tested either on CD4+CCR5+ expressing cells (left) or CD4+ expressing cells (right), c) 100 μl purified antibody (40 μg protein) from rabbit 2 serum was adsorbed on 2xlO ⁷ CCR5+CD4+ U87 cells (squares) or 2xlO ⁷ CD4+ U87 cells (triangles) for 1 hour at 4 ⁰ C. The cells were removed by centrifugation and the supernatant tested for binding by flow cytometry at different concentrations of protein. The figure shows results for one of two experiments, d) The same two samples of adsorbed antibody as in c) were tested for binding to peptide CCR5.3 by ELISA.

Figure 6. Inhibitory activity of antiCCR5 antibodies. Human macrophage cultures were infected with HIV BaL at various MOIs in the presence of anti-CCR5 purified antibody (80 μg/ml) or control rabbit Ig. A) Representative p24 staining after seven days of culture. B) Average % of p24 positive cells/well of triplicate cultures (one experiment of three).

Figure 7 shows the CCR5 sequence showing intra and extracellular domains.

Figure 8 shows the immunisation schedule used in experiment 3. Figure 9 shows the CCR5 receptor sequence showing the immunogen sequences.

Figure 10 shows the Vaccines and test antigens used in experiment 3.

Figures 11 and 12 show the antibody responses after immunisation in experiment 3.

Figures 13 to 15 show antibody response to cells expressing CCR5.

Figure 16 shows the expression of CCR5 on spleen cells from immunised mice.

EXAMPLES

Methods

Peptides

The sequence of the peptides used in this study are shown in Fig 1. All peptides were synthesised by the Protein and Peptide Chemistry Department at Cancer Research UK.

Purity was greater than 80% as measured by HPLC. Aliquots of lyophilised peptide were dissolved in phosphate buffered saline to give stock solutions of 1 mg/ml, and stored at -2O ⁰ C. Peptide CCR5.5 was initially dissolved in a small volume of DMSO, and then diluted to 1 mg/ml in PBS. Peptide CCR5 was also supplied coupled to Keyhole Limpet Haemocyanin (KLH) via an additional terminal cysteine, using the protocol described below for bovine serum albumin (BSA).

Cell lines U87 cells ( a human glioma derived line) expressing CD4 and CCR5 (U87- CD4-CCR5) were obtained from Drs. Littman and Deng, via the Centralised Facility for AIDS Reagents (Repository Reference ARP069 and ARP072) (http://www.nibsc.ac.uk/spotlight/aidsreagent/) supported by EU Programme AVA/MRC (contract QLKZ-CT- 1999-00609) and the UK Medical Research Council. The cells were maintained in selection medium (15% foetal calf serum, DMEM) containing G418 (300μg/ml) and puromycin (1 μg/ml, only for CCR5 expressing cells) as described in detail in the data sheet for these cells lines. The human glioma cell line NP2, expressing CD4 and CCR5 or CXCR4 [20], were maintained at 37 ⁰ C and 5% CO ₂ in Dulbecco's modified Eagle medium (Invitrogen, United Kingdom) supplemented with 10% foetal calf serum and 500 U/ml penicillin and streptomycin.

Primary human monocyte derived macrophages. Human blood samples were obtained from healthy volunteers. The study was approved by the joint University College London/ University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants. Peripheral blood mononuclear cells prepared by standard density gradient protocols were seeded into 48-well plates, 2 xlO ⁶ cells /well. After one hour at 37°C nonadherent cells (lymphocytes) were removed and adherent monocytes were incubated in RPMI 1640 with 10% autologous heat-inactivated human serum supplemented with 20 ng/mL macrophage colony stimulating factor (M CSF) (R&D systems) for three days. The media was then refreshed (without additional M CSF), removing any remaining non-adherent cells. This protocol yields 1 xlO ⁵ macrophages, at >95% purity by morphology and CD 14 staining.

Immunisations.

Two sets of immunisations were carried out at the Biological Services Unit, UCL.

Experiment 1

Three rabbits were immunised with each peptide CCR5.1 and CCR5.2. Primary immunisations were carried out with peptide (lmg/ml) emulsified in complete Freund's adjuvant (CFA, Sigma), at four subcutaneous sites. The rabbits were boosted at day 15, 22, 29 and 42 with peptide emulsified in incomplete Freund's adjuvant (IFA). All rabbits were sacrificed and exsanguinated by cardiac puncture on day 63. Serum (preimmune, test and final) were collected using standard methods, and stored at -20 ⁰ C until needed.

Experiment 2 Three rabbits were immunised with peptide CCR5.5 conjugated to KLH. Primary immunisations were carried out with conjugate (0.4 mg/ml) emulsified with CFA, at four subcutaneous sites. Rabbits were boosted on days 14, 21, 28 and 44 with KLH/peptide conjugate (0.2 mg/ml) emulsified with EFA. A further three rabbits were immunised with peptide CCR5.5 bound to magnetic strep tavidin beads (M-PVA SAVl, Chemagen). Beads (25 mg) were washed in PBS, and mixed with peptide CCR5.5 (100 μg). After 15 minutes, the suspension of beads was mixed with adjuvant (Imject® ALUM, Pierce), and shaken vigorously to produce an emulsion. Immunisation schedule was as for CCR5.5/KLH, except that the same bead/pep tide suspension was used for all immunisations. All rabbits were sacrificed and exsanguinated by cardiac puncture on day 63. Serum (preimmune, test and final) were collected using standard methods, and stored at -20° C until needed. Conjugation of peptides to BSA CCR5.3 and CCR5.4 were conjugated to BSA using sulphosuccinimimidyl 4-(N-maleimidomethyl) cyclohexane (Sulpho-SMCC), Sigma Aldrich, Poole, Dorset). Briefly, 0.6 μmoles BSA were reacted with 17 μmoles Sulpho-SMCC in phosphate buffered saline (PBS) for one hour. The BSA/SMCC conjugates were exchanged into sodium phosphate buffer (0.1 M, pH6) using PD-10 desalting column (Amersham Biosciences). Meanwhile, 10 μmoles of each peptide were dissolved in 1 ml borate buffer (0.1 M, pH 8) and reduced by the addition of sodium borohydride (130 micromoles); excess borohydride was destroyed by brief acidification. Each reduced peptide solution was mixed with half the BSA/SMCC, and incubated overnight at 4 ⁰ C. The final conjugate was dialysed against PBS and stored frozen in aliquots. Conjugation of BSA/peptide to Sepharose. 15-20 mg of each peptide/BSA conjugate was coupled to CNBr-activated Sepharose 4B (Sigma SAldrich) using the manufacturer's instructions. The coupled matrix was extensively washed in high (pH8) and low (pH4) pH buffers, and stored in PBS/azide until used for affinity purification.

Antibody Purification CCR5.3/BSA/Sepharose (approximately 1 ml) was washed with 20 ml PBS. 5-10 ml serum from immunised rabbits was mixed with the Sepharose and incubated for 20 minutes to allow binding to the immunoadsorbent. The flow through was then collected, and the column was washed with a further 20 ml PBS. Bound antibody was eluted with 5 ml glycine solution (0.1 M, pH 2.5), and 400 μl fractions collected. 6μl Tris solution (IM) was added to each collecting tube to bring the pH back to neutral as rapidly as possible. Each fraction was tested for CCR5.3 binding activity by ELISA, and fractions containing the highest titres (typically fractions 4-8) were pooled, and filter sterilised. For functional assays, the purified antibody was exchanged into complete culture medium using PD-10 columns, following manufacturer's instructions. Antibody radiolabelling. 40μg purifed antibody was mixed with 0.2 mCi NaI125 (Amersham) and 10 μg chloramine T (Sigma) and incubated for 60 seconds. The reaction was terminated by adding sodium metabisulfite (lOmg), and excess 1125 quenched by addition of potassium iodide (10 mg). Labelled protein was purified from reactants by column chromatography on a PDlO column (Amersham Biosciences) according to manufacturers' instructions, and stored in PBS at 4 ⁰ C until used. Specific activity was about 1.2 x 10 ⁶ cpm per μg protein.

ELISA

96 well ELISA plates (Nunc, ??) were coated with BSA/peptide conjugates incoating buffer (0.1 M sodium bicarbonate, pH8.5), overnight at 4 ⁰ C . A concentration of 0.5-1 μg protein per well was chosen based on initial titration experiments, and was used for all experiments shown. All wells were washed (washing buffer PBS 0.1% Tween 20) and blocked by addition of 2% low fat milk (Tesco) in PBS for at least two hours at room temperature. Primary sera were diluted in washing buffer and added at the concentrations shown, for 1.5-3 hours at room temperature. All wells were washed three times, and alkaline phosphatase coupled sheep anti-rabbit (Sigma) antibody (1 :2000 dilution) was added for one hour at room temperature. The wells were again washed twice, and then developed using substrate (PNPP FAST, Sigma) using manufactures instructions. Results are shown either as optical density (405 nm) or as log titre. The titre is calculated as the largest dilution factor which gives a reading above twice the mean background optical density (no first layer).

Flow Cytometry U87 transfected cells (5xlO ⁴ ) were incubated in 50 μl PBS containing 10% goat serum (Sigma) and 0.1% sodium azide (blocking buffer) for 30 minutes at 4 ⁰ C. Test or control sera at various dilutions (in blocking buffer) were added, and incubated for 2-3 hours at 4 ⁰ C. Cells were washed three times by centrifugation, and incubated in goat anti-rabbit FITC (R&D Systems) for a further hour. Cells were washed twice more, fixed in 3.8% formaldehyde, and analysed by flow cytometry. As a positive control some cells were also stained with a monoclonal rat anti-human CCR5 obtained from Drs. J. McKeating and C. Shotton, via the Centralised Facility for AIDS Reagents (Repository Reference ARP3214.1) (http://www.nibsc.ac.uk/spotlight/aidsreagent/) supported by EU Programme AVA/MRC (contract QLKZ-CT- 1999-00609) and the UK Medical Research Council.

Functional Assays of Blocking Activity

Inhibition of HIV on transfected NP2 cells. 5x10 ³ NP2 cells stably expressing CD4 and either CCR5 or CXCR4 were seeded into a 96 well plate 24h prior to treatment with media containing lOμM TAK779 (NIBSC, UK) or purified CCR5 antibodies. The cells were incubated at 37 ⁰ C for Ih after which the existing media was replaced with fresh supplemented media and 100 focus-forming units of virus. The HIV-I strains SF 162, BaL and the primary isolate SL2 were used in these experiments. After 2h at 37°C the cells were washed twice and supplemented media was placed back onto the cells for a further 48h. The cells were then fixed in ice cold acetone-methanol (1:1) and stained in situ for p24 expression as described previously [21].

Inhibition of HIV on primary human macrophages Non-adherent peripheral blood lymphocytes (PBLs) from these cell preparations were used to propagate the CCR5 tropic HIV 1 strain, BaL. 1 xlO ⁶ /ml PBLs were activated for three days in RPMI 1640 with 20% FCS and 0.5 μg/ml PHA (Sigma). They were then inoculated with HIV virus using a MOI of 1, and cultured in RPMI 1640 with 20%FCS and 20 U/ml IL2. At 3-4 day intervals, the cell culture supernatant was collected and additional PHA- stimulated PBLs were added to maintain the cell density at 1 xlO ⁶ /ml. Cell culture supernatants containing PBL derived HIV were filtered through 0.45μm filters and used to inoculate 6-day old macrophage cultures with an MOI of 1. Culture supernatants from infected MDM, containing MDM- derived HIV 1 BaL, was collected at weekly intervals,centrifuged at 40Og for 5 mins and filtered through 0.45μm filter to remove cellular debris. MDM-derived virus suspensions were then ultracentrifuged through a 20% sucrose buffer and resuspended in appropriate media for subsequent infection experiments. All virus preparations were titrated on the NP2 astrocytoma cell line stably transfected with CD4 and either CCR5 or CXCR4, as previously described [21]. For virus neutralization assay macrophage cultures were pre-incubated with affinity purified rabbit anti CCR5 immunoglobulin or pre-immune rabbit IgG for 1 hour at room temperature and then inoculated with HTV-I BaL using doubling dilutions of virus prepared in media with anti CCR5 or control antibody. The inoculum was removed after overnight incubation and the intracellular p24 staining assessed 3-4 days later as described [21].

Results

The sequences of the first two peptides used for immunisation are shown in fig 1. CCR5.1 incorporates the first seven amino acids of the full length sequence of human CCR5, followed by a linker/spacer and an MHC class II promiscuous T cell epitope from tetanus toxoid (REF). Remarkably, there was no information available to us from the literature about whether the methionine at position 1 is still present or is cleaved before CCR5 is exported to the cell membrane. A second peptide, in which the methionine was absent was therefore tested in parallel. Sera were first tested by ELISA, using as target antigen peptides CCR5.3 and CCR5.4, which contain the first twelve amino acids of the CCR5 receptor (fig 1), with (CCR5.3) or without (CCR5.4) the initial methionine. Each peptide was conjugate via an additional C-terminal cysteine to BSA. A representative ELISA for all six immunised sera is shown in fig 2a, and showed that all rabbits gave strong antibody responses to the N-terminal CCR5 sequences to which they had been immunised, with titres of greater than 20,000 (9, Log3). Titres rose rapidly after 1 or 2 boosts (fig 2b), and appear to be reaching a plateau by the time the rabbits are sacrificed. The specificity of the response was highly restricted by the N-terminal amino acid of the immunising peptide. Thus the sera from the three rabbits immunised with ethionine containing peptide preferentially recognised the target peptide with an N-terminal methionine (fig 2c, left), while the sera from rabbits immunised with peptide without methionine preferentially recognised peptides without methionine (fig 2c, right). The sera were then tested for binding to CCR5 by flow cytometry, using CCR5 transfectants as targets. A summary showing mean fluorescence intensities at a variety of concentration for all six rabbits is shown in fig 3 a, and a representative fluorescence histogram for one rabbit (rabbit 2) is shown in fig 3b. Two of the three sera recognising the methionine containing N-terminal peptide bound CCR5 transfected cells, but not to controls. In contrast, none of the sera from the three rabbits immunised with the truncated peptide bound to CCR5. All further experiments were therefore focused on the methionine containing sequence. The immunogenicity of the peptides may have been limited by the presence of only a single T helper cell epitope in the immunising peptide, and/or by the fact that peptide immunogens are intrinsically univalent. Two additional sets of immunisation were therefore carried out with peptide CCR5.5 (fig 1), which is identical to CCR5.1, except for the addition of a C-terminal biotinylated cysteine. The C-terminal cysteine was used to couple to a strong "carrier protein", KLH, and the conjugate emulsified in Freund's adjuvant was used to immunise rabbits 7-9. In parallel, the biotin moiety was used to couple to inert streptavidin coated magnetic beads, and the beads were resuspended in Alum adjuvant and used to immunise rabbits 10-12. The titres of all sera at the time the rabbits were sacrificed tested against CCR5.3 is shown in fig 4. The titres were generally somewhat lower than those obtained with the first set of immunisations, with the lowest titres obtained following bead/ Alum immunisation. However, in all cases the titres were at least one hundred fold higher than control or preimmune sera, which gave titres of less than 4(log3, 100). The six sera were further tested for binding to CCR5 receptor as above (fig 4b). None of the three sera from rabbits immunised with KLH conjugate showed significant binding to the transfectants. In contrast two of three mice immunised with CCR5.1 conjugated magnetic beads showed significant binding to CCR5 transfected cells (fig 4b), but not controls (not shown). Interestingly, there was little correlation between the titre of antibody reactivity measured by ELISA, and the level of binding to the cell surface. The results shown in figs 2-4 demonstrate a very large difference between the titre of anti-peptide antibody as measured by ELISA (typically, binding is seen at concentrations of 1:1000 to 1:10000), and the binding to intact receptor, as measured by flow cytometry. To investigate this further, the antibody with anti-peptide activity was purified from serum of rabbit 2 (first set of immunisations) and rabbit 10 (second set of immunisations) by affinity chromatography, using peptide CCR5.3/BSA conjugate bound to Sepaharose 4B. The titre of antibody remained high after purification (fig 5a), although when adjusted for volume of purified antibody, recovery of active antibody was about 50% for both sera.

In order to test whether the anti-peptide antibody fraction contained the antibody which bound to the CCR5 receptor, the two purified antibody preparations were further tested for binding to CCR5 expressing transfectants (fig 5b). Both purified samples bound specifically to CCR5 transfectants, but not to control cells. In order to estimate the proportion of total peptide binding antibody which bound to CCR5, a purified antibody sample was absorbed on excess target CCR5 cells (2x10 ⁷ ) or on control cells not expressing CCR5, and then tested for binding to CCR5 (fig 5c) and to CCR5.3 peptide (Fig 5d). Adsorption on receptor expressing cells removed the majority of receptor binding activity as expected (fig 5c), but did not significantly alter the peptide binding ability as measured by ELISA (fig 5d). Thus receptor binding activity can be attributed to a small proportion of anti-peptide binding antibody. In order to try and obtain more quantitative figures for antibody concentration and antibody affinity, purified antibody was labelled to high specificity (approximately 1.5 x 10 ⁶ cpm/μg protein) by chloramine iodination. Approximately 50% of this activity bound to CCR5.3/BSA/Sepharose beads, the remaining 50% representing either nonspecific protein which copurified from the affinity column, or antibody damaged/destroyed by the iodination procedure. Specific binding to CCR5 transfectants was measured, either by comparing binding to control cells without CCR5, or by the addition of a large excess of unlabelled antibody. In both cases, only 0.1-0.2% of the activity bound to transfected cells, even under optimal conditions of cell numbers/antibody concentration. This low % binding is consistent with the binding data shown in fig 5c/d. The level of binding was too low to allow any accurate measurements of affinity/antibody concentration. Although binding of antibody to CCR5 required large concentrations of antibody, we tested both unpurified sera and purified antibody in HIV neutralisation assays. Initial experiments were carried out using several strains and NP2 CCR5 transfectants. Antisera/purified antibodies (concentrations of 1 :5 or 1 :10, or around 40μg/ml purified antibody) consistently showed some inhibition in infectivity compared to control pre-bleed sera, or purified rabbit Ig, but the degree of inhibition was small (typically 10- 20%, not shown), hi comparison the CCR5 inhibitor TAK779 gave inhibition of around 50% in this model, variable ranging between 20 and 70%. hi contrast, inhibition experiments using primary macrophage cultures as targets, showed strong inhibition of inhibition (fig 6).

Discussion

The primary objective of this study was to test the hypothesis that a short N-terminal fragment of the CCR5 would stimulate antibodies which recognize the intact receptor on the cell surface, and to characterize these antibodies. The results suggest that the basic hypothesis is correct, but suggest that the three dimensional conformation of this domain may have structural constraints not previous appreciated. The basic strategy uses a chimaeric immunogen containing a short N-terminal sequence of CCR5 to act as the B cell immunogen, and a C-terminal tetanus toxoid T helper cell epitope, separated by a four amino acid linker region. Immunization with these chimaeric peptides successfully generated a strong antibody response to a peptide coding the N terminal sequence of CCR5. The antibodies generated were highly focused on the N terminal itself, since inclusion or removal of an additional methionine generated antisera with minimal cross reactivity. Although numbers of animals tested were small, no antisera to the methionine-less peptide recognized the intact receptor, suggesting that this amino acid is retained at the cell surface. Although CCR5 protein has not been sequenced, these results are consistent with the known preference of methionine aminopeptidase, which cleaves methionine inefficiently where the adjacent amino acid has a large side chain (e.g. aspartic acid in this example)[22]. Similarly, a methionine at position 1 is retained in the three dimensional structure of rhodopsin, a G protein coupled receptor homologue of CCR5 [23]. This observation raises some questions about recent attempts to determine or model structures for the CCR5 N-terminal peptide in complex with HTV gpl20, since in these studies the N- terminal methionine is generally absent [24;25].

Three different immunogens were used in this study, the chimaeric peptide alone (with CFA), the chimaeric peptide coupled to KLH (with CFA), and the chimaeric peptide coupled to lμm streptavidin magnetic beads (with Alum). All three stimulated good titres of anti-peptide antigen, and there was no apparent advantage to incorporating additional T cell epitopes by coupling to KLH. Lower titres of anti- peptide antibody were obtained using Alum as adjuvant, consistent with the known weaker adjuvant activity of Alum compared to CFA. However, other factors, such as the concentration of peptide, and the influence of magnetic beads may also have played a role, and these different variables need to be analysed further. Although all three antigens stimulated anti-peptide response, the KLH-peptide conjugate failed to stimulate significant antibody which recognized CCR5 receptor by flow cytometry. The reasons for this remain speculative, but the much lower concentration of peptide injected using the conjugate (at least 10-100 fold less) may be an important consideration. In both sets of rabbits whose sera did show antireceptor activity one rabbit failed to respond, perhaps reflecting the genetic variability in these outbred strains.

Although four rabbits did detect cell surface CCR5 by flow cytometry, the concentration of antibody required was much higher than that required to detect peptide by ELISA (microgram versus nanogram/ml). Although other factors may play a role, the data supports the hypothesis that the antibody is made up of two populations with different specificities, one major species which recognizes the peptide, but not the intact protein, and a minor species which recognizes both. In support of this hypothesis, absorption with CCR5 expressing cells decreases the titre of cell binding antibody significantly, but does not detectably change the titre of anti- peptide activity. Furthermore, absorption of radio labelled antibody with CCR5 expressing cells only removes about 0.1-0.2% of the total labelled population, although at least 50% can still bind peptide-coupled sepharose beads. If correct, the hypothesis suggests that the N-terminal peptide of CCR5 may have a well-ordered structure, and that alternative peptidomimetics may provide more successful immunogens. Although the binding studies suggested it would be difficult to saturate CCR5 on target cells using concentrations of serum or purified antibody which were achievable in cell culture, we tested the ability of the antibodies to block HIV entry and replication, in both transfected cell lines, and primary human macrophage cultures. Unsurprisingly, the antibodies were poor inhibitors of HIV infectivity as measured on NP2 CCR5 transfectants, which had very high levels of receptor expression (not shown) and were difficult or impossible to saturate. The antibodies showed a reasonably good level of inhibition when tested using primary macrophage targets. The reasons for this difference were not explored, but a plausible hypothesis is the extremely low level of CCR5 receptor expression on the primary macrophages compared to the transfected cell lines. In conclusion, this study demonstrates that very short linear peptide epitopes, which cannot code for intact T cell auto epitopes, may be used to generate an antibody response to the CCR5 protein. Further studies will be needed to test whether this strategy will successfully break tolerance in an auto antigen setting, and if so whether development of autoantibody is associated with any pathology, and whether it can provide any protection against virus transmission.

Experiment 3

Mice were immunised according to the schedule shown in figure 8, using the vaccines shown in figure 10. Some time after vaccination, the mice were bled and the blood tested for antibody response to the immunising peptide. Ninety percent of the mice showed a response, which was maintained over a long period. Unimmunised mice not responsive. The blood was also tested for a response to CCR5 -expressing cells. Most mice were non-responsive, showing the majority of the antibodies raised do not remain in the blood but rather bind to the CCR5 on cells, as required to prevent the entry of HIV into those cells.

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Table 1 Sequences of examples of CCR5 epitopes that could be used as part of chimaeric peptides (see fig 7 for numbering)