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  • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
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Definitions

• the present invention relates to synthetic T cell epitope analogues of native T cell epitopes with partial or complete inverso or retro-inverso modifications. These T cell epitope analogues stimulate immune responsiveness when used in place of their native T cell epitope counterparts in vaccines.
• the invention further relates to the use of these T cell epitope analogues, to vaccines comprising the T cell epitope analogues, to methods of preparing vaccines comprising these T cell epitope analogues, and to antibodies generated using these T cell epitope analogues.
• polypeptide backbone which is defined by the peptide bonds between the amino acid residues and the ⁇ -carbon atoms of the bonded residues.
• polypeptide backbones have distinct termini and thus direction.
• L-amino acids The majority of naturally occurring amino acids are L-amino acids. Naturally occurring polypeptides are largely comprised of L-amino acids. D-amino acids are the enantiomers of L-amino acids and form peptides which are herein referred to as inverso peptides, that is, peptides corresponding to native peptides but made up of D-amino acids rather than L-amino acids.
• Retro-inverso modification of naturally occurring polypeptides involves the synthetic assemblage of amino acids with ⁇ -carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e. D- or D-allo-amino acids, in reverse order with respect to the native peptide sequence.
• a retro-inverso analogue thus has reversed termini and reversed direction of peptide bonds while approximately maintaining the topology of the side chains as in the native peptide sequence.
• Partial retro-inverso peptide analogues are polypeptides in which only part of the sequence is reversed and replaced with enantiomeric amino acid residues. Since the retro-inverted portion of such an analogue has reversed amino and carboxyl termini, the amino acid residues flanking the retro-inverted portion are replaced by side-chain-analogous ⁇ -substituted geminal-diaminomethanes and malonates, respectively.
• Processes for synthesis of retro-inverso peptide analogues (Bonelli et al . , 1984; Verdini and Viscomi, 1985) and some processes for the solid-phase synthesis of partial retro-inverso peptide analogues have been described (Pessi et al . , 1987).
• Peptide hormones have been of particular interest as targets for retro-inversion, presumably because their analogues would have potential use as therapeutic agents. Partial, and in a few cases complete, retro-inverso analogues of a number of peptide hormones have been prepared and tested (see, for example, Goodman and Chorev, 1981) .
• T cells In order to activate the cellular component of the immune system a vaccine must present T-cell epitopes, as well as pathogen-specific B-cell epitopes. T cells fail to recognise soluble antigen. They require its presentation on the surface of antigen presenting cells (APC) in association with molecules encoded by the major histocompatibility complex (MHO . In the case of large proteins which constitute conventional vaccines, the protein undergoes enzymatic digestion intracellularly. Some of the resulting peptide fragments can bind to MHC molecules and the peptide-MHC complexes are then transported to the surface of APCs. The peptides capable of binding MHC molecules are T-cell epitopes.
• APC antigen presenting cells
• T-cell epitopes Because of the genetic restriction of the MHC, the sequences which can act as T-cell epitopes may vary amongst individuals in an outbred population. Totally synthetic vaccines (Jolivet et al . , 1990) therefore need to be designed with regard to these facts. While it is possible to provide T-cell epitopes in a peptide vaccine by conjugation of the relevant B-cell epitope peptides to a carrier protein such as tetanus toxoid, this is not desirable because it negates the inherent advantages of a peptide vaccine, e. g. chemical stability and ease of production. The identification of appropriate T-cell epitope 'cocktails' potentially useful in synthetic vaccines is therefore an active field of research (Schwartz, 1986) .
• retro modified refers to a peptide which is made up of L-amino acids in which the amino acid residues are assembled in opposite direction to the native peptide with respect to which it is retro modified.
• verso modified refers to a peptide which is made up of D-amino acids in which the amino acid residues are assembled in the same direction as the native peptide with respect to which it is inverso modified.
• retro- inverso modified refers to a peptide which is made up of D-amino acids in which the amino acid residues are assembled in the opposite direction to the native peptide with respect to which it is retro-inverso modified.
• peptide as used throughout the specification and claims is to be understood to include peptides of any length.
• antigenic fragment refers to a peptide which is a portion of an antigen which itself is immunogenic or capable of binding antibodies.
• antigen as used throughout the specification and claims is to be understood to include immunogens as the context requires.
• antiigen analogue refers to a peptide molecule capable of mimicking the immunological activity of the native peptide antigen with respect to which it is partially or completely retro, inverso or retro-inverso modified. Retro peptides are made up of L-amino acids and are peptides in which the amino acid residues are assembled in opposite direction to the native peptide sequence.
• T- cell epitope analogue refers to a peptide molecule capable of mimicking the immunological activity of the native T-cell epitope with respect to which it is partially or completely inverso or retro-inverso modified. Partial modification includes analogues in which as few as two consecutive residues are modified. Typically at least 5 or 6 consecutive residues are modified.
• the present invention relates to partially or completely inverso or retro-inverso modified T-cell epitope analogues of native T cell epitopes which stimulate immune responsiveness when used in place of their native T cell epitope counterparts in vaccines. Incorporation of D-amino acids into T-cell epitope analogues increases their stability to degradation after administration.
• incorporation of D-amino acids has potential for oral administration of analogues. Having shown that particular retro-inverso or inverso T-cell epitope analogues can stimulate immune responsiveness when used in the place of their native T- cell epitope counterparts it follows that, generally, these analogues can be expected to be successful since T- cell epitope - MHC molecule interactions are not fundamentally different from case to case.
• the invention provides a synthetic peptide T cell epitope analogue of a native T cell epitope, which analogue is partially or completely inverso or retro-inverso modified with respect to the native T cell epitope.
• the T cell epitope analogues of the present invention stimulate immune responsiveness when used in place of their native T cell epitope counterparts in vaccines.
• the efficacy of T cell epitope analogues of the invention is illustrated with respect to the malaria T cell epitopes of Example 2.
• the invention provides a vaccine comprising a T cell epitope analogue of the first aspect together with a B cell epitope and a pharmaceutically or veterinarally acceptable carrier, diluent, excipient and/or adjuvant.
• the vaccines of the invention are cocktails of T cell epitope analogues and B cell epitopes tailored to the condition against which vaccination is required.
• the T cell epitope analogue is conjugated to the B cell epitope.
• the B cell epitope is conjugated to the T cell epitope by standard chemical conjugation techniques or the conjugate is synthesized as a continuous peptide.
• the B cell epitope can be provided as any epitope, or any intact molecule providing the epitope, against which an antibody response is required.
• the B cell epitopes to be incorporated into vaccines in accordance with the invention include peptides or polypeptides of any length whose amino acid sequences stem from polypeptides of pathogens such as poliomyelitis, hepatitis B, foot and mouth disease of livestock, tetanus, pertussis, HIV, cholera, malaria, influenza, rabies or diphtheria causing agents, or toxins such as robustoxin, heat labile toxin of pathogenic
• Escherichia coli strains and Shiga toxin from Shigella dysenteriae Other B cell epitopes of interest include epitopes of Amyloid ⁇ protein (Alzheimer's disease) and human chorionic gonadotropin and gonadotropin releasing hormone (contraceptive vaccines) .
• the B cell epitope is preferably a retro, retro- inverso or inverso antigen analogue.
• Preferred T cell epitope analogues of the invention are analogues of :
• Malaria CST3 protein H-Gly-Asp-Ile-Glu-Lys-Lys-Ile-Ala-Lys-Met-Glu-Lys-Ala-
• T cell epitope analogues are analogues of:
• Foot and mouth virus VP1 141-160 (Francis M.J. et al 1985) (SEQ ID NO: 21) Rabies virus-spike glycoprotein precursor: 32-44 (Macfarlan R.I. et al 1984) (SEQ ID NO:
• the invention provides a method of vaccinating a host in need of such treatment which method comprises administering an effective amount of a vaccine according to the second aspect to the host.
• the invention provides antibodies produced by immunisation of a host with a vaccine of the second aspect.
• the invention provides a method of preparing a T cell epitope analogue of the invention comprising synthesising a partially or completely inverso or retro-inverso peptide comprising the analogue.
• the invention provides a method of preparing a vaccine of the second aspect comprising conjugating a T cell epitope analogue of the first aspect to a B cell epitope or admixing a T cell epitope analogue of the first aspect with a B cell epitope and admixing an effective amount of the resulting mixture or conjugate with a pharmaceutically or veterinarally acceptable carrier, diluent, excipient and/or adjuvant.
• Vaccines of the invention can be formulated using standard methods in the art of vaccine formulation.
• Vaccines of the invention may be administered to hosts in need of such treatment by injection.
• Vaccines incorporating D-amino acid containing analogues may also be administered orally.
• PBS phosphate buffered saline (10 mM phosphate, 150mM NaCl, pH 7.4)
• L-amino acids are indicated by an upper case followed by lower case lettering e.g. Ala indicates L-alanine.
• D-amino acids are indicated by all lower case abbreviations, e.g. ala indicates D-alanine.
• FIGURES Figure 1 shows the results of a cell proliferation experiment conducted using the T-cell epitope peptides noMalCST3 (SEQ ID NO: 5) , inMalCST3 and riMalCST3.
• Figure 2 shows antibody production measured in mice immunized with the B-cell epitope H- (Asn-Ala-Asn-Pro) 3 -OH (SEQ ID NO: 23) alone or together with either no or riMalCST3.
• Figure 3 shows antibody production measured in mice immunized with the B-cell epitope H- (Asn-Ala-Asn-Pro) 3 -OH (SEQ ID NO: 23) alone or together with either no (SEQ ID NO: 3) or riMalCSA protein.
• Figure 4 shows antibody production measured in mice immunized with the B-cell epitope H- (Asn-Ala-Asn-Pro) 3 -OH (SEQ ID NO: 23) alone or together with either no (SEQ ID NO: 4) or riMalCSB protein.
• Figure 5 shows antibody production measured in mice immunized with the B-cell epitope H- (Asn-Ala-Asn-Pro) 3 -OH (SEQ ID NO: 23) alone or together with either no (SEQ ID NO: 1) or riDiphT.
• Figure 6 shows antibody production measured in mice immunized with the B-cell epitope H- (Asn-Ala-Asn-Pro) 3 -OH (SEQ ID NO: 23) alone or together with either no (SEQ ID NO: 2) or riPertT.
• Figure 7 shows antibody production measured in mice immunized with the B-cell epitope H- (Asn-Ala-Asn-Pro) 3 -OH (SEQ ID NO: 23) alone or together with either no (SEQ ID NO: 7) or riOvalT.
• T cell epitope analogues of the invention are prepared by standard techniques for the preparation of L and D amino acid containing peptides, particularly as outlined in Example 1.
• Vaccines of the invention are formulated by standard techniques for vaccine formulation using standard carriers, diluents excipients and/or adjuvants suitable for the formulation of oral or injectable vaccines. Effective amounts of Tcell-epitope analogues to be incorporated in the vaccines can be determined in accordance with standard methods. Conjugation techniques where used are standard chemical conjugation techniques.
• the vaccination regimes used are standard regimes for the vaccination of animal or human hosts. These regimes can be used where immunisation of the host is desired or where the host is being used to produce antibodies for exogenous use.
• Peptides were synthesised by a solid-phase method on polyamide (Arshady et al . , 1981) or Polyhipe supports using side-chain protected Fmoc amino acids (Carpino & Han, 1972) , essentially as described by Eberle et al . (1986) . Only pure amino acid derivatives, obtained commercially or by synthesis, were used.
• the polyamide synthesis resins, derivatised with p-alkoxybenzyl alcohol-based linkage agents, were esterified quantitatively with the appropriate preformed C-terminal Fmoc-amino acid symmetrical anhydrides, in the presence of 0.2 molar equivalents of N,N-dimethylaminopyridine and N-methylmorpholine.
• the Polyhipe resin, derivatised with Fmoc-Rink linker did not require esterification of the first amino acid linked to it.
• Chain elongation was carried out using Fmoc-amino acid pentafluorophenyl esters (Atherton et al . , 1988) or Castro's reagent/l-hydroxybenzotriazole coupling (Hudson, 1988) .
• the progress of each synthesis was monitored using a specific colour test (Hancock & Battersby, 1976) and/or amino acid analysis of acid-hydrolysed peptidyl resin samples .
• the peptides were cleaved from the resins and side-chain deprotected with the aid of TFA, containing a suitable mixture of scavenger chemicals (Tarn, 1988) . After filtration and vacuum evaporation, the peptides were triturated with diethyl ether, collected by centrifugation and lyophilised from aqueous ammonium bicarbonate solution.
• All peptides then underwent an initial desalting and purification step by column chromatography on suitable gel filtration media in aqueous solvents. Afterwards they were purified to homogeneity by reversed-phase HPLC using water-acetonitrile (containing 0.05-0.1% TFA) gradient elution. The purity of the synthetic peptides was further assessed by gas-phase acid hydrolysis/amino acid analysis (Bidlingmeyer et al . , 1987) and, if deemed necessary, by automated gas-phase sequencing (Hunkapiller & Hood, 1983) .
• EXAMPLE 2 Malaria T-cell epitope peptides It has been shown that nonresponsiveness to the malaria immunodominant B-cell epitope (Asn-Ala-Asn-Pro) x (SEQ ID NO: 23) of the Plasmodium falciparum circumsporozoite protein can be overcome in the presence of a particular T-cell epitope peptide from the same protein (Sinigaglia et al , 1988) . The peptide in question, unlike most T-cell epitopes, is recognised in association with most human MHC class II molecules and has been suggested as a suitable component of a synthetic peptide vaccine against malaria. The region of the circumsporozoite protein from which the peptide stems is apparently conserved in different parasite isolates.
• the T cell assay method was improved as follows:
• a cell suspension from the lymph nodes was centrifuged on Ficoll-Isopaque to separate mononuclear cells from erythrocytes.
• the resulting cell preparation was washed extensively in PBS and incubated with Dynabeads coated with anti-mouse IgG to remove B- lymphocytes.
• the cells from this preparation were then cultured in the presence of various concentrations of the test antigen, as well as a non-related control antigen.
• Cell proliferation was quantitated by measuring the incorporation into the cells of radiolabelled thymidine and or by the use of Promega Cell Titer 96 AQ kit. Again efficacy of the T cell epitope analogues was demonstrated.
• Antibody responses to synthetic peptides representing the immunodominant B-cell epitope H- (Asn- Ala-Asn-Pro) 3 -OH (SEQ ID NO: 23) of the circumsporozoite protein were measured following intraperitoneal injection of Balb/c mice.
• One hundred microgrammes of B-cell epitope were administered in an equal volume of Freund' s complete adjuvant either alone or in a mixture (1:1) with either noMalCST3 (SEQ ID NO: 5) or riMalCST3.
• As a negative control a further group of mice were immunised with either noMalCST3 (SEQ ID NO: 5) or riMalCST3 in the absence of the B-cell epitope.
• mice were boosted by the same route and with the same dose of peptide in incomplete Freund' s adjuvant.
• a second booster injection was given two weeks after the first with the same dose of antigen in incomplete Freund's adjuvant.
• Blood samples were taken five days later by retro-ocular bleeding and, after centrifugation, the sera was immediately used in an enzyme-linked immunosorbent assay (ELISA) .
• ELISA enzyme-linked immunosorbent assay
• Antibody response to the same B-cell epitope was also measured using five more T-cell epitopes selected from the literature and synthesized in the following forms:
• riMalCSB H-ser-ile-ser-asn-lys-ile-lys-lys-leu-tyr-gln-glu-ile- his-NH 2
• Diphtheria toxin noDipT (Bixler et al , 1989)
• Pertussis toxin noPertT (Kim et al , 1990) (SEQ ID NO: 2) :
• Ovalbumin noOvalT (Sette et al , 1989) (SEQ ID NO: 7) :
• the synthesis of the above peptides was performed on Polyhipe Rink resin.
• the side chain protecting groups used were: t-butyl for serine, threonine, aspartic acid, glutamic acid and tyrosine; trityl for histidine, glutamine and asparagine; t-butoxycarbonyl for lysine and
• cleavage and side- chain deprotection were accomplished by reaction of the peptidyl resins for 90 min at 0°C with 1M trimethylsilylbromide-thioanisole in TFA containing 0.25M
• mice developed very low titres against the B-cell epitope when immunised with the B-cell epitope alone, but produced much higher antibody titre when a mixture of the B-cell epitope and any of the T- cell epitopes in either no- or ri- form were used in the immunogen formulation (Fig. 3-7) .
• T cell epitope analogues in accordance with the invention have a range of potential applications in eliciting immunogenic responses in a host.
• These analogues can be used in the treatment and/or prophylaxis of diseases, and therapy of disease states.
• these analogues can be used in vaccines in animals, including humans for protection against pathogens and the like.
• ORGANISM Corynebacterium diphtheriae
• ORGANISM Bordetella pertussis
• ORGANISM Plasmodium falciparum (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :
• ORGANISM Respiratory syncytial virus

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