T helper cell epitopes

FIELD OF THE INVENTION

The present invention relates to T helper cell epitopes derived from Canine Distemper Virus (CDV). The present invention relates to compositions including at least one T helper cell epitope and optionally B cell epitopes and/or CTL epitopes.

BACKGROUND OF THE INVENTION

For any peptide to be able to induce an effective antibody response it must contain particular sequences of amino acids known as epitopes that are recognised by the immune system. In particular, for antibody responses, epitopes need to be recognised by specific immunoglobulin (Ig) receptors present on the surface of B lymphocytes. It is these cells which ultimately differentiate into plasma cells capable of producing antibody specific for that epitope. In addition to these B cell epitopes, the immunogen must also contain epitopes that are presented by antigen presenting cells (APC) to specific receptors present on helper T lymphocytes, the cells which are necessary to provide the signals required for the B cells to differentiate into antibody producing cells.

In the case of viral infections and in many cases of cancer, antibody is of limited benefit in recovery and the immune system responds with cytotoxic T cells (CTL) which are able to kill the virus-infected or cancer cell. Like helper T cells, CTL are first activated by interaction with APC bearing their specific peptide epitope presented on the surface, this time in association with MHC class I rather than class II molecules. Once activated the CTL can engage a target cell bearing the sane peptide/class I complex and cause its lysis. It is also becoming apparent that helper T cells play a role in this process; before the APC is capable of activating the CTL it must first receive signals from the helper T cell to upregulate the expression of the necessary costimulatory molecules.

Helper T cell epitopes are bound by molecules present on the surface of APCs that are coded by class II genes of the major histocompatibility complex (MHC). The complex of the class II molecule and peptide epitope is then recognised by specific T-cell receptors (TCR) on the surface of T helper lymphocytes. In this way the T cell, presented with an antigenic epitope in the context of an MHC molecule, can be activated and provide the necessary signals for the B lymphocyte to differentiate. Traditionally the source of helper T cell epitopes for a peptide immunogen is a carrier protein to which peptides are covalently coupled but this coupling procedure can introduce other problems such as modification of the antigenic determinant during the coupling process and the induction of antibodies against the carrier at the expense of antibodies which are directed toward the peptide (Schutze, M. P., Leclerc, C. Jolivet, M. Audibert, F. Chedid, L. Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. J Immunol. 1985, 135, 2319-2322; DiJohn, D., Torrese, J. R. Murillo, J. Herrington, D. A. et al. Effect of priming with carrier on response to conjugate vaccine. The Lancet. 1989, 2, 1415-1416). Furthermore, the use of irrelevant proteins in the preparation introduces issues of quality control. The choice of appropriate carrier proteins is very important in designing peptide vaccines and their selection is limited by factors such as toxicity and feasibility of their large scale production. There are other limitations to this approach including the size of the peptide load that can be coupled and the dose of carrier that can be safely administered (Audibert, F. a. C., L. 1984. Modern approaches to vaccines. Molecular and chemical basis of virus virulence and immunogenicity., Cold Spring Harbor Laboratory, New York.). Although carrier molecules allow the induction of a strong immune response they are also associated with undesirable effects such as suppression of the anti-peptide antibody response (Herzenberg, L. A. and Tokuhisa, T. 1980. Carrier-priming leads to hapten-specific suppression. Nature 285:664; Schutze, M. P., Leclerc, C., Jolivet, M., Audibert, F., and Chedid, L. 1985. Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. J Immunol 135:2319; Etlinger, H. M., Felix, A. M., Gillessen, D., Heimer, E. P., Just, M., Pink, J. R., Sinigaglia, F., Sturchler, D., Takacs, B., Trzeciak, A., and et, a. 1988. Assessment in humans of a synthetic peptide-based vaccine against the sporozoite stage of the human malaria parasite,

Plasmodium falciparum

. J Immunol 140:626).

In general then, an immunogen must contain epitopes capable of being recognised by helper T cells in addition to the epitopes that will be recognised by surface Ig or by the receptors present on cytotoxic T cells. It should be realised that these types of epitopes may be very different. For B cell epitopes, conformation is important as the B cell receptor binds directly to the native immunogen. In contrast, epitopes recognised by T cells are not dependent on conformational integrity of the epitope and consist of short sequences of approximately nine amino acids for CTL and slightly longer sequences, with less restriction on length, for helper T cells. The only requirements for these epitopes are that they can be accommodated in the binding cleft of the class I or class II molecule respectively and that the complex is then able to engage the T-cell receptor. The class II molecule's binding site is open at both ends allowing a much greater variation in the length of the peptides bound (Brown, J. H., T. S. Jardetzky, J. C. Gorga, L. J. Stern, R. G. Urban, J. L. Strominger and D. C. Wiley. 1993. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1, Nature 364:33) with epitopes as short as 8 amino acid residues being reported (Fahrer, A. M., Geysen, H. M., White, D. O., Jackon, D. C. and Brown, L. E. Analysis of the requirements for class II-restricted T-cell recognition of a single determinant reveals considerable diversity in the T-cell response and degeneracy of peptide binding to I-Ed J. Immunol. 1995. 155: 2849-2857).

Canine distemper virus (CDV) belongs to the subgroup of morbillivirus of paramyxovirus family of negative-stranded RNA viruses. Other viruses which are members of this group are measles virus and rinderpest virus. Development of peptide based vaccines has aroused considerable interest in identification of B and T cell epitopes from sequences of proteins. The rationale for using T cell epitopes from proteins such as the F protein of CDV is that young dogs are inoculated against CDV in early life and will therefore possess helper T cells specific for helper T cell epitopes present on this protein. Subsequent exposure to a vaccine which contains one or more of the epitopes will therefore result in recruitment of existing helper T cells and consequently an enhanced immune response. Such helper T cell epitopes could, however, be administered to unprimed animals and still induce an immune response. The present inventors aimed to identify canine T cell epitopes from the sequence of CDV fusion protein so that these epitopes can then be used in the design of peptide based vaccines, in particular, for the canine and related species.

LHRH (Luteinisiing hormone releasing hormone) is a ten amino acids long peptide hormone whose sequence is conserved in mammals. It is secreted by the hypothalamus and controls the reproductive physiology of both males and females. The principle of development of LHRH-based immunocontraceptive vaccines is based on observations that antibodies to LHRH block the action of the hormone on pituitary secretion of luteinising hormone and follicle stimulating hormone, leading to gonadal atrophy and sterility in mammals.

Most LHRH vaccines that have been developed consist of LHRH chemically conjugated to protein carriers to provide T cell help for the generation of anti-LHRH antibodies. It has been shown that upon repeated inoculation of LHRH-protein carrier conjugates the anti-LHRH titre decreases due to the phenomenon known as “carrier induced epitope suppression”. One aim of the present inventors is to replace protein carriers in the vaccines with defined T helper epitopes (TH-epitopes) so as to eliminate “carrier induced epitope suppression”.

SUMMARY OF THE INVENTION

The present inventors have identified a number of 17 residue peptides each of which includes a T helper cell epitope. As will be readily appreciated the majority of these peptides are not minimal T helper cell epitopes. Typically class II molecules have been shown to be associated with peptides as short as 8 amino acids (Fahrer et al., 1995 ibid) but usually of 12-19 amino acids (Chicz, R. M., Urban, R. G., Gorga, J. C., Vignali, D. A. A., Lane, W. S. and Strominger, J. L. Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles, J Exp Med 1993, 178, 27-47; Chicz, R. M., Urban, R. G., Lane, W. S., Gorga, J. C., Stern, L. J., Vignali, D. A. A. and Strominger, J. L. Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size. Nature 1992, 358, 754-8), although, peptides up to 25 amino acids in length have been reported to bind to class II (reviewed in Rammensee, H.-G. Chemistry of peptide associated with class I and class II molecules. Curr Opin Immunol 1995, 7, 85-95.).

Thus peptide epitopes that range in length between 8 and 25 amino acid residues can bind to class II molecules. The shorter peptides are “core” epitopes that may have less activity than longer sequences but it is a trivial exercise to truncate longer sequences at the N- or the C-terminus to yield shorter sequences that have the same or better activity than the parent sequence .

Accordingly in a first aspect the present invention consists in a T helper cell epitope, the epitope being contained within a peptide sequence selected from the group consisting of SSKTQTHTQQDRPPQPS (SEQ ID NO:1); QPSTELEETRTSRARHS (SEQ ID NO:2); RHSTTSAQRSTHYDPRT (SEQ ID NO:3); PRTSDRPVSYTMNRTRS (SEQ ID NO:4); TRSRKQTSHRLKNIPVH (SEQ ID NO:5); SHQYLVIKLIPNASLIE (SEQ ID NO:6); IGTDNVHYKIMTRPSHQ (SEQ ID NO:7); YKIMTRPSHQYLVIKLI (SEQ ID NO:8); KLIPNASLIENCTKAEL (SEQ ID NO:9); AELGEYEKLLNSVLEPI (SEQ ID NO:10); KLLNSVLEPINQALTLM (SEQ ID NO:11); EPINQALTLMTKNVKPL (SEQ ID NO:12); FAGVVLAGVALGVATAA (SEQ ID NO:13); GVALGVATAAQITAGIA (SEQ ID NO:14); TAAQITAGIALHQSNLN (SEQ ID NO:15); GIALHQSNLNAQAIQSL (SEQ ID NO:16); NLNAQAIQSLRTSLEQS (SEQ ID NO:17); QSLRTSLEQSNKAIEEI (SEQ ID NO:18); EQSNKAIEEIREATQET (SEQ ID NO:19); TELLSIFGPSLRDPISA (SEQ ID NO:20); PRYIATNGYLISNFDES (SEQ ID NO:21); CIRGDTSSCARTLVSGT (SEQ ID NO:22); DESSCVFVSESAICSQN (SEQ ID NO:23); TSTIINQSPDKLLTFIA (SEQ ID NO:24); SPDKLLTFIASDTCPLV (SEQ ID NO:25) and SGRRQRRFAGVVLAGVA (SEQ ID NO:26).

In a second aspect the present invention consists in a composition for use in raising an immune response in an animal, the composition comprising at least one T helper cell epitope, the at least one T helper cell epitope being contained within a peptide sequence selected from the group consisting of SSKTQTHTQQDRPPQPS (SEQ ID NO:1); QPSTELEETRTSRARHS (SEQ ID NO:2); RHSTTSAQRSTHYDPRT (SEQ ID NO:3); PRTSDRPVSYTMNRTRS (SEQ ID NO:4); TRSRKQTSHRLKNIPVH (SEQ ID NO:5); SHQYLVIKLIPNASLIE (SEQ ID NO:6); IGTDNVHYKIMTRPSHQ (SEQ ID NO:7); YKIMTRPSHQYLVIKLI (SEQ ID NO:8); KLIPNASLIENCTKAEL (SEQ ID NO:9); AELGEYEKLLNSVLEPI (SEQ ID NO:10); KLLNSVLEPINQALTLM (SEQ ID NO:11); EPINQALTLMTKNVKPL (SEQ ID NO:12); FAGVVLAGVALGVATAA (SEQ ID NO:13); GVALGVATAAQITAGIA (SEQ ID NO:14); TAAQITAGIALHQSNLN (SEQ ID NO:15); GIALHQSNLNAQAIQSL (SEQ ID NO:16); NLNAQAIQSLRTSLEQS (SEQ ID NO:17); QSLRTSLEQSNKAIEEI (SEQ ID NO:18); EQSNKAIEEIREATQET (SEQ ID NO:19); TELLSIFGPSLRDPISA (SEQ ID NO:20); PRYIATNGYLISNFDES (SEQ ID NO:21); CIRGDTSSCARTLVSGT (SEQ ID NO:22); DESSCVFVSESAICSQN (SEQ ID NO:23); TSTIINQSPDKLLTFIA (SEQ ID NO:24); SPDKLLTFIASDTCPLV (SEQ ID NO:25) and SGRRQRRFAGVVLAGVA (SEQ ID NO:26).

In a preferred embodiment of the present invention the composition comprises at least one peptide selected from the group consisting of SSKTQTHTQQDRPPQPS (SEQ ID NO:1); QPSTELEETRTSRARHS (SEQ ID NO:2); RHSTTSAQRSTHYDPRT (SEQ ID NO:3); PRTSDRPVSYTMNRTRS (SEQ ID NO:4); TRSRKQTSHRLKNIPVH (SEQ ID NO:5); SHQYLVIKLIPNASLIE (SEQ ID NO:6); IGTDNVHYKIMTRPSHQ (SEQ ID NO:7); YKIMTRPSHQYLVIKLI (SEQ ID NO:8); KLIPNASLIENCTKAEL (SEQ ID NO:9); AELGEYEKLLNSVLEPI (SEQ ID NO:10); KLLNSVLEPINQALTLM (SEQ ID NO:11); EPINQALTLMTKNVKPL (SEQ ID NO:12); FAGVVLAGVALGVATAA (SEQ ID NO:13); GVALGVATAAQITAGIA (SEQ ID NO:14); TAAQITAGIALHQSNLN (SEQ ID NO:15); GIALHQSNLNAQAIQSL (SEQ ID NO:16); NLNAQAIQSLRTSLEQS (SEQ ID NO:17); QSLRTSLEQSNKAIEEI (SEQ ID NO:18); EQSNKAIEEIREATQET (SEQ ID NO:19); TELLSIFGPSLRDPISA (SEQ ID NO:20); PRYIATNGYLISNFDES (SEQ ID NO:21); CIRGDTSSCARTLVSGT (SEQ ID NO:22); DESSCVFVSESAICSQN (SEQ ID NO:23); TSTIINQSPDKLLTFIA (SEQ ID NO:24); SPDKLLTFIASDTCPLV (SEQ ID NO:25) and SGRRQRRFAGVVLAGVA (SEQ ID NO:26).

It is further preferred that the composition further comprises at least one B cell epitope and/or at least one CTL epitope.

In yet another preferred embodiment the at least one B cell epitope and/or the at least one CTL epitope are linked to at least one of the T helper cell epitopes. It is also preferred that the composition comprises a plurality of epitope constructs in which each comprises at least one T helper cell epitope and at least one B cell epitope. Alternatively the composition may comprises a plurality of epitope constructs in which each comprises at least one T helper cell epitope and at least one CTL epitope.

It will be understood that the B cell epitope or CTL epitope may be any epitope. A currently preferred B cell epitope is an LHRH B cell epitope.

The composition of the present invention may comprises a plurality of T helper cell epitopes. These epitopes may be singular or be linked together to form a single polypeptide. It will be understood that where the epitopes are linked to together in a single polypeptide the epitopes may be contiguous or spaced apart by additional amino acids which are not themselves part of the T helper cell epitopes.

As discussed above in one embodiment the T helper cell epitopes and at least one B cell epitope and/or at least one CTL epitope in which the epitopes are linked. This may be done by simple covalent linkage of the peptides. In another embodiment the epitopes are polymerised, most preferably such as described in PCT/AU98/00076, the disclosure of which is incorporated herein by reference.

In yet another preferred embodiment the composition further comprises a pharmaceutically acceptable excipient, preferably an adjuvant.

In a further aspect the present invention consists in a method of inducing an immune response in an animal, the method comprising administering to the animal the composition of the second aspect of the present invention.

Pharmaceutically acceptable carriers or diluents include those used in compositions suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. They are non-toxic to recipients at the dosages and concentrations employed. Representative examples of pharmaceutically acceptable carriers or diluents include, but are not limited to water, isotonic solutions which are preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline) and can also contain one or more of, mannitol, lactose, trehalose, dextrose, glycerol, ethanol or polypeptides (such as human serum albumin). The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

As mentioned it is preferred that the composition includes an adjuvant. As will be understood an “adjuvant” means a composition comprised of one or more substances that enhances the immunogenicity and efficacy of a vaccine composition. Non-limiting examples of suitable adjuvants include squalane and squalene (or other oils of animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-derived adjuvants such as

Corynebacterium parvum

; Propionibocterium-derived adjuvants such as

Propionibocterium acne; Mycobactenum bovis

(Bacille Calmette and Guerin or BCG); interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1; tumour necrosis factor; interferons such as gamma interferon; combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOM adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A derivatives; dextran sulfate; DEAE-Dextran or with aluminium phosphate; carboxypolymethylene such as Carbopol′ EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g. U.S. Pat. No. 5,047,238); vaccinia or animal poxvirus proteins, sub-viral particle adjuvants such as cholera toxin, or mixtures thereof.

As will be recognised by those skilled in the art modifications may be made to the peptides of the present invention without complete abrogation of biological activity. These modifications include additions, deletions and substitutions, in particular conservative substitutions. It is intended that peptides including such modifications which do not result in complete loss of activity as T helper cell epitopes are within the scope of the present invention.

Whilst the concept of substitution is well known in the field the types of substitutions envisaged are set out below.

Original

Exemplary

Preferred

Residue

Substitutions

Substitutions

Ala (A)

val; leu; ile

val

Arg (R)

lys; gln; asn

lys

Asn (N)

gln; his; lys; arg

gln

Asp (D)

glu

glu

Cys (C)

ser

ser

Gln (Q)

asn

asn

Glu (E)

asp

asp

Gly (G)

pro

pro

His (H)

asn; gln; lvs; arg

arg

Ile (I)

leu; val; met; ala; phe

leu

norleucine

Leu (L)

norleucine, ile; val; met;

ile

ala; phe

Lys (K)

arg; gln; asn

arg

Met (M)

leu; phe; ile;

leu

Phe (F)

leu; val; ile; ala

leu

Pro (P)

gly

gly

Ser (S)

thr

thr

Thr (T

ser

ser

Trp (W)

tyr

tyr

Tyr (Y)

trp; phe; thr; ser

phe

Val (V)

ile; leu; met; phe ala;

leu

norleucine

Another type of modification of the peptides envisaged include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides.

Examples of side chain modifications contemplated by the present invention include, but are not limited to, modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH

4

: amidation with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5′-phosphate followed by reduction with NaBH

4

.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine. sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid; 2-thienyl alanine and/or D-isomers of amino acids.

The peptides of the present invention may be derived from CDV. Alternatively, the peptide or combination of peptide epitopes may be produced by recombinant DNA technology. It is, however, preferred that the peptides are produced synthetically using methods well known in the field. For example, the peptides may be synthesised using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled “Peptide Synthesis” by Atherton and Sheppard which is included in a publication entitled “Synthetic Vaccines” edited by Nicholson and published by Blackwell Scientific Publications. Preferably a solid phase support is utilised which may be polystyrene gel beads wherein the polystyrene may be cross-linked with a small proportion of divinylbenzene (e.g. 1%) which is further swollen by lipophilic solvents such as dicliloromethane or more polar solvents such as dimethylformamide (DMF). The polystyrene may be functionalised with chloromethyl or aminomethyl groups. Alternatively, cross-linked and functionalised polydimethyl-acrylamide gel is used which may be highly solvated and swollen by DMF and other dipolar aprotic solvents. Other supports can be utilised based on polyethylene glycol which is usually grafted or otherwise attached to the surface of inert polystyrene beads. In a preferred form, use may be made of commercial solid supports or resins which are selected from PAL-PEG-PS, PAC-PEG-PS, KA, KR or TGR.

In solid state synthesis, use is made of reversible blocking groups which have the dual function of masking unwanted reactivity in the α-amino, carboxy or side chain functional groups and of destroying the dipolar character of amino acids and peptides which render them inactive. Such functional groups can be selected from t-butyl esters of the structure RCO—OCMe

3

—CO. Use may also be made of the corresponding benzyl esters having the structure RCO—OCH

2

—C

6

H

5

and urethanes having the structure C

6

H

5

CH

2

OCO—NHR which are known as the benzyloxycarbonyl or Z-derivatives and any Me

3

—COCO—NHR, which are known as t-butoxyl carbonyl, or Boc derivatives. Use may also be made of derivatives of fluorenyl methanol and especially the fluorenyl-methoxy carbonyl or Fmoc group. Each of these types of protecting group is capable of independent cleavage in the presence of one other so that frequent use is made, for example, of BOC-benzyl and Fmoc-tertiary butyl protection strategies.

Reference also should be made to a condensing agent to link the amino and carboxy groups of protected amino acids or peptides. This may be done by activating the carboxy group so that it reacts spontaneously with a free primary or secondary amine. Activated esters such as those derived from p-nitrophenol and pentafluorophenol may be used for this purpose. Their reactivity may be increased by addition of catalysts such as 1-hydroxybenzotriazole. Esters of triazine DHBT (as discussed on page 215-216 of the abovementioned Nicholson reference) also may be used. Other acylating species are formed in situ by treatment of the carboxylic acid (i.e. the N-alpha-protected amino acid or peptide) with a condensing reagent and are reacted immediately with the amino component (the carboxy or C-protected amino acid or peptide).

Dicyclohexylcarbodiimide, the BOP reagent (referred to on page 216 of the Nicholson reference), O′Benzotriazole-N,N,N′N′-tetra methyl-uronium hexafluorophosphate (HBTU) and its analogous tetrafluoroborate are frequently used condensing agents.

The attachment of the first amino acid to the solid phase support may be carried out using BOC-amino acids in any suitable manner. In one method BOC amino acids are attached to chloromethyl resin by warming the triethyl ammonium salts with the resin. Fmoc-amino acids may be coupled to the p-alkoxybenzyl alcohol resin in similar manner. Alternatively, use may be made of various linkage agents or “handles” to join the first amino acid to the resin. In this regard, p-hydroxymethyl phenylacetic acid linked to aminomethyl polystyrene may be used for this purpose.

Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

DETAILED DESCRIPTION OF THE INVENTION

In order that the nature of the present invention may be more readily understood preferred forms there of will now be described with reference to the following non-limiting examples.

FIGURE LEGENDS

FIG.

1

. Amino acid sequence of the fusion protein of CDV

FIG.

2

. Stimulation indices to Th-epitope P25 and its truncated versions from dogs immunised with P25-LHRH. (X-axis concentration of peptides nmoles/well)

FIG.

3

. Stimulation indices to Th-epitope P27 and its truncated 15-mer from dogs immunised with P27-LHRH. (X-axis concentration of peptides nmoles/well)

FIG.

4

. Stimulation indices to Th-epitope P35 and its truncated versions from dogs immunised with P35-LHRH. (X-axis concentration of peptides nmoles/well)

EXAMPLE 1

Identification of T Helper Cell Epitopes

Methods and Results

Towards identification of canine T cell epitopes 94, 17 residue overlapping peptides were designed encompassing the entire sequence of fusion protein of canine distemper virus (CDV). The 17 mer peptides were numbered sequentially for identification starting from the N-terminus The sequence of the fusion protein of CDV as determined by Barrett et al 1987 (Virus Res. 8, 373-386) is shown in FIG.

1

. The peptides were used in T-cell proliferation assays using peripheral blood lymphocytes (PBMC) from dogs immunised with Canvac™ 3 in 1 vaccine (CSL Limited) which contains live CDV.

Initially, four dogs were used and they were boosted with the Canvac™ 3 in 1 vaccine twice with four to six weeks between each vaccination. The dogs were bled after each booster vaccination and the PBMCs were tested against the peptides. No significant proliferation to peptides was observed.

Since CDV has been reported to be lymphotropic and the vaccine consists of live CDV, there was the possibility that it may be sequestered in lymphoid organs preventing significant numbers of precursor T cells entering the peripheral system. To increase the frequency of peripheral blood anti-CDV T cells dogs were boosted with heat killed CDV (obtained as a pellet from virus culture medium, CSL Limited). Two weeks later, the dogs were bled and the PBMCs tested for proliferation against the peptides. Again there was no proliferation to the peptide antigens.

An alternate strategy was used to increase the precursor frequency of specific T cells recognising the CDV peptides. Fresh PBMCs obtained from these hyperimmunised dogs were subjected to stimulation in vitro with pools of all 94 peptides for 30 minutes at 37° C. The cells were then washed to remove any excess peptides and cultured for 7 days. This population of T cells was then tested with autologous APCs with every single peptide as the antigen.

Table 1 shows the peptides to which significant (stimulation index >2) levels of proliferation were observed.

To confirm this observation, the same four dogs were bled again, five weeks after receiving the dose of killed virus. The PBMCs were stimulated in vitro with pools of either all 94 peptides or peptides 21-40 (because most of the activity was in this region) and after 7 days of culture the stimulated T cells were tested against individual peptides. Significant stimulatory indices were obtained with all peptides, confirming the above results. Four more dogs which received only one dose of 3 in 1 vaccine were tested using the in vitro stimulation method and all four dogs responded to the majority of peptides shown in Table 2.

The above peptides were also tested on cells from additional dogs, with results shown in Table 3. Peptides P64, P74 and P75 were also shown to react strongly with peripheral blood mononuclear cells from dogs of various breeds immunized with CDV (Table 4), and are therefore identified as strong T-helper epitopes.

TABLE 1

Identification of canine T cell epitopes from the sequence of fusion

protein of CDV.

Beagle

Beagle

Beagle

Beagle

Foxhound

Foxhound

Foxhound

Foxhound

Peptides

(Dog #18)

(Dog #19)

(Dog #20)

(Dog #21)

p2

2*

<2

8

3.9

p4

4.9

<2

3.3

4.6

p6

2.5

<2

4

5.1

p10

2.3

<2

3.2

9.1

p24

5.8

9.9

2.8

29

p25

3.2

11.9

4.5

17

p27

3.3

34

6.7

14.8

p29

3.5

42

4.4

<2

p35

3.1

57

3.3

22

p36

6.7

3.7

3.3

16

p37

6.9

10.9

8.2

26

p38

2.8

6.7

3.6

4.2

p47

3.3

85.7

2.9

1.9

p62

<2

51

5.6

4.2

p68

6.6

<2

<2

11.7

*Stimulatory index

TABLE 2

Identification of canine T cell epitopes from the sequence of fusion

protein of CDV.

Beagle

Beagle

Beagle

Beagle

Foxhound

Foxhound

Foxhound

Foxhound

Peptides

(Dog #70)

(Dog #71)

(Dog #72)

(Dog #73)

p8

2.2

p22

2.6

p24

3.2

2.2

p25

1.5

2.9

2

12

p27

2.7

3.5

4.8

p28

2

p29

2

6

p33

1.6

p35

1.7

6.8

p37

1.7

p62

3

TABLE 3

Identification of canine T cell epitopes from the sequence of fusion

protein of CDV.

Kelpie

Kelpie

Foxhound

Foxhound

Peptides

(Dog #125)

(Dog #126)

p23

3.2

p27

4.5

8.5

p28

1.9

p29

3.6

p33

6

p34

2.1

p35

3.8

10

p36

3

p37

2.5

p38

2.2

p39

2.9

p47

2.7

p62

2.4

p68

2.9

TABLE 4

Identification of canine T cell epitopes from the sequence of fusion

protein of CDV.

Beagle

Beagle

Beagle

Beagle

Poodle

Foxhound

Foxhound

Foxhound

Foxhound

Peptides

Shitzu

#18

#19

#20

#21

p64

50.0

2.5

2.5

p74

4.0

1.7

6.0

p75

10

2.5

7.2

Once again the same peptides and one additional peptide P32 were tested on cells from additional dogs. These peptides were also shown to react strongly with peripheral blood mononuclear cells from dogs of various breeds immunised with CDV (Table 5), and are therefore identified as strong T-helper epitopes.

In conclusion, 26 peptides were identified as canine T helper cell epitopes in the fusion protein of CDV. The sequences of each of these peptides are set out in Table 6.

These T helper cell epitopes will have usefulness as components of animal, in particular, canine vaccines, either simply as synthetic peptide based vaccines and as additions to vaccines containing more complex antigens.

TABLE 5

Identification of canine T cell epitopes from the sequence of fusion

protein of CDV.

Poodle

Grey

Fox

Terrier

Kelpie

Border

Peptides

Shitzu

hound

Terrier

Cross

Pointer

Collie

P2

140

<2

<2

<2

2.6

2

P4

44

2

<2

2

3.5

2

P6

38

<2

<2

<2

<2

2

P8

100

2

<2

<2

2.8

2

P10

50

2

2.2

2.1

2.4

3

P25

<2

<2

2.6

<2

2.6

<2

P29

2

<2

<2

2

<2

<2

P32

<2

2

<2

<2

<2

<2

P33

<2

<2

<2

<2

2

2

P35

<2

<2

2.2

<2

2

2

P37

<2

<2

<2

2

2

<2

P62

24

<2

<2

<2

<2

<2

P64

50

<2

<2

<2

<2

<2

P68

5

<2

<2

<2

<2

<2

P74

4

<2

<2

<2

<2

<2

P75

10

<2

<2

<2

<2

<2

P2

SSKTQTHTQQDRPPQPS (SEQ ID NO:1)

P4

QPSTELEETRTSRARHS (SEQ ID NO:2)

P6

RHSTTSAQRSTHYDPRT (SEQ ID NO:3)

P8

PRTSDRPVSYTMNRTRS (SEQ ID NO:4)

P10

TRSRKQTSHRLKNIPVH (SEQ ID NO:5)

P24

SHQYLVIKLIPNASLIE (SEQ ID NO:6)

P22

IGTDNVHYKIMTRPSHQ (SEQ ID NO:7)

P23

YKIMTRPSHQYLVIKLI (SEQ ID NO:8)

P25

KLIPNASLIENCTKAEL (SEQ ID NO:9)

P27

AELGEYEKLLNSVLEPI (SEQ ID NO:10)

P28

KLLNSVLEPINQALTLM (SEQ ID NO:11)

P29

EPINQALTLMTKNVKPL (SEQ ID NO:12)

P32

SGRRQRRFAGVVLAGVA (SEQ ID NO:26)

P33

FAGVVLAGVALGVATAA (SEQ ID NO:13)

P34

GVALGVATAAQITAGIA (SEQ ID NO:14)

P35

TAAQITAGIALHQSNLN (SEQ ID NO:15)

GIALHQSNLNAQAIQSL (SEQ ID NO:16)

P37

NLNAQAIQSLRTSLEQS (SEQ ID NO:17)

P38

QSLRTSLEQSNKAIEEI (SEQ ID NO:18)

P39

EQSNKAIEEIREATQET (SEQ ID NO:19)

P47

TELLSIFGPSLRDPISA (SEQ ID NO:20)

P62

PRYIATNGYLISNFDES (SEQ ID NO:21)

P68

CIRGDTSSCARTLVSGT (SEQ ID NQ:22)

P64

DESSCVFVSESAICSQN (SEQ ID NO:23)

P74

TSTIINQSPDKLLTFIA (SEQ ID NO:24)

P75

SPDKLLTFIASDTCPLV (SEQ ID NO:25)

Selected sequences of the identified T-cell epitopes were tested for their ability to induce an antibody response to a linked B-cell epitope. Trials were conducted in dogs for assessment of antibody responses. The T-cell epitopes were linked to the B cell epitope LHRH (leuteinising hormone releasing hormone), with the T-cell epitope at the N-terminus and LHRH positioned at the carboxy terminus.

Peptides were synthesised using standard chemistry with Fmoc protection. All peptides were purified to at least 50% purity and the product checked by mass spectroscopy.

The peptides were produced as contiguous T-cell-B cell determinants. The LHRC sequence of Pyro Glu-His-Trp-Ser-Tyr-Gly-Leu Arg Pro-Gly, or variations of it, was linked to the carboxyl terminus of each respective CDV T helper epitope.

In-vivo evaluation of some of the T-helper epitopes was conducted in two trials, by vaccination of dogs with T-helper-LHRH sequences.

EXAMPLE 2(TRIAL K9-5)

A total of 14 dogs of mixed sex were used in this trial. All had been previously vaccinated with a live CDV vaccine and had also been vaccinated against LHRH.

Vaccine Formulation

Test peptides P25, P27, P35 from CDV were synthesised with LHRH at the C terminus of each T-helper epitope. The LHRH sequence used was the full 10 amino acids of the native LHRH. Each of the vaccine constructs, together with a control peptide comprising a mouse influenza T-cell epitope linked to a repeat malarial B-cell epitope (sequence shown in table below) were purified to −50-90% purity. All peptides were dissolved in 4M urea before dilution with sterile saline to an appropriate volume to give 40nmoles per 1 mL dose. Iscomatrix™ was added to a final concentration of 150 μg /1 mL dose as adjuvant together with thiomersal preservative (0.01%).

ISCOM™ or Immunostimulating Complex (Barr, Sjolander and Cox, 1998, Advanced Drug Delivery Systems 32: 247-271) are a well characterised class of adjuvant comprised of a complex of phospholipid, cholesterol and saponin, usually with a protein incorporated into the complex. Where the complex is formed in the absence of protein antigen, then this complex is termed Iscomatrix™. The saponin used in the preparation of this adjuvant was Quil A.

Vaccination, Blood Samples and Assays

All dogs were vaccinated with a 1 mL dose, delivered in the scruff of the neck. Vaccinations were given at 0 and 4 weeks and venous blood samples were obtained at intervals during the trial.

Effective T-cell help was determined by measuring the antibody response to LHRH by ELISA. Biological effectiveness of the peptide based vaccine was determined by measuring the levels of progesterone in female dogs and testosterone in male dogs.

TABLE 7

Trial Groups

Peptide

Dog Nos.

Control (SEQ ID NO:30) -ALNNRFQIKGVELKS-(NANP)3

104, 998

P26 -LHRH 1-10

70, 73,

127, 993

P27 -LHRH 1-10

20, 94,

101, 105

P35 -LHRH 1-10

19, 96,

100, 102

Results

Pre-existing low antibody levels to LHRH were present in all dogs due to immunisation previously with a different vaccine. The control group of dogs exhibited a slow decrease in antibody levels.

Dogs immunised with P25-LHRH, P27-LHRH and P35-LHRH all showed strong antibody responses to the B-cell epitope (LHRH). This response persisted to 6 weeks post boost vaccination (see Table 8).

The biological potency of the vaccine was demonstrated by a significant reduction in progesterone or testosterone levels (see Tables 9 and 10).

TABLE 8

Anti LHRH Titres

Anti LHRH Titres

2 wks post

6wks post

Peptide

Dog No

Prebleeds

boost

boost

Control

104

1258

1975

1936

998

2559

1982

1947

Average

1794

1978

1941

Range

1258-2559

1975-1982

1936-1947

P25-LHRH

70

856

24245

16697

(1-10)

73

42665

16922

127

1361

21485

19662

993

577

24879

15119

Average

886

23120

17242

Range

0-1361

21485-42665

15119-19662

p27-LHRH

20

747

29653

8423

(1-10)

94

41247

22759

101

4256

52724

17353

105

944

12600

8366

Average

2004

25774

12049

Range

747-4256

12600-52724

8366-22759

p35-LHRH

19

665

18033

6228

(1-10)

96

1621

26583

5744

100

580

17255

4829

102

180

11740

2963

Average

323

14233

3783

Range

180-1621

11740-26583

2963-6228

TABLE 9

Progesterone results (nmol/L)

Dog

4wks post

2wks post

6wks post

Peptide

No.

primary

boost

boost

Control

998

5.17

4.28

<0

p25-LHRH

127

3.04

4.83

<0

(1-10)

993

1.7

0.87

<0

p27-LHRH

101

0.42

0.14

<0

(1-10)

p35-LHRH

96

31.76

2.15

<0

(1-10)

100

<0

<0

<0

TABLE 10

Testosterone results (nmol/L)

Dog

4wks post

2wks post

6wks post

Peptide

No.

primary

boost

boost

Control

104

9.69

2.51

3.31

p25-LHRH

70

<0

<0

<0

(1-10)

73

5.38

<0

<0

p27-LHRH

20

1.04

<0

<0

(1-10)

94

3.33

<0

<0

105

>47.7

<0

<0

p35-LHRH

19

4.3

2.77

4.55

(1-10)

102

6.72

<0

<0

The effectiveness of selected T-cell epitopes from the F-protein of CDV in providing T-cell help to elicit antibody responses in dogs proves that the identified sequences are functional. These results also validate the scientific approach and usefulness of the in vitro screening method for identifying T-helper epitope sequences within vivo activity.

EXAMPLE 3 (TRIAL K9-8)

A total of 35 dogs mixed sex were used in this trial. All had been previously vaccinated with a live CDV vaccine but had not been vaccinated against LHRH.

Vaccine Formulation

The T-Helper epitopes were linked to a truncated form of LHRH, containing amino acids 2 to 10 of the native 10 amino acid sequence, as shown below:

2-10 (SEQ ID NO:29)His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.

All vaccines were formulated as for Example 2, ie each 1 mL dose of vaccine contained 40 nmoles of peptide, 150 μg Iscomatrix™, and thiomersal as preservative.

Where dogs were vaccinated with a pool of peptides, the concentration of each peptide was adjusted to give equal concentrations and a total amount of 40 nmoles of LHRH epitope per 1 mL dose.

Vaccination, Blood Samples and Assays

All dogs were vaccinated with a 1 mL dose, delivered in the scruff of the neck. Vaccinations were given at 0 and 4 weeks and venous blood samples were obtained at intervals during the trial. Effective T-cell help was determined by measuring the antibody response to LHRH by ELISA. Biological effectiveness of the peptide based vaccine was determined by measuring the levels of progesterone in female dogs and testosterone in male dogs.

TABLE 11

Trial Groups

Peptide Group

Dog Nos.

P25-LHRH 2-10

211, 195, 197, 181

P27-LHRH 2-10

203, 191, 186, 201

P35-LHRH 2-10

217, 198, 187, 196

Pool: P25-LHRH 2-10,

212, 193, 178, 216, Y3

P27-LHRH 2-10, P35-LHRH 2-10

P2-LHRH 2-10

194, 199, 179, 220

P8-LHRH 2-10

Y4, Y6, 160, 200

P62-LHRH 2-10

219, 185, 221, 177

P75-LHRH 2-10

189, 222, 202, 176

Unvaccinated controls

190, 159

Results

Strong antibody responses to LHRH were demonstrated in dogs immunised with the T-cell-LHRH constructs with the T-cell epitopes P25, P27, P35, P62, P75, and the pool of T-cell-LHRH peptides comprising a combination of T-cell epitopes P25, P27 and P35 (see Table 1Z).

Low to undetectable antibody responses were seen in dogs immunised with P2 and P8-LHRH peptides (see Table 12). This was concluded to indicate that these T-cell peptides were not well recognised by Beagle-Foxhound dogs, which is consistent with their identification using PBMCs' from other dog breeds. The initial screening in Beagle foxhound dogs indicated that this breed of dog does not respond to these 2 T-cell epitopes.

As is well understood by those skilled in the art of peptide vaccines the response to individual peptides is genetically determined. The class II Major Histocompatability Complex (MHC II) is polymorphic. Class II molecules at the cell surface function to bind peptides for presentation to T-cells, which is required as part of the activation process for T-cells, including helper T-cells. The allelic forms of MHC class II bind discrete sets of peptide antigens, and thus the response to those antigens is genetically determined. Thus the results are interpreted to indicate that the Beagle—Foxhound breed of dog does not possess the appropriate MHC-II alleles to respond to P2 and P8, but that other breeds of dog do, eg. the Poodle Shitzu breed that were used to identify these peptides.

Control dogs showed no change in antibody levels to LHRH during the trial period and hormone levels were within normal ranges for the age and sex of the dogs (see Table 12).

TABLE 12

Anti-LHRH Titres

Anti LHRH Titres

4 wks after

2 wks post

4 wks post

Peptide

Group

Dog No

primary

boost

boost

Control

1

159

0

0

0

1

190

0

0

0

GMT

Pool

2

Y3

1860

55659

95038

2

178

17900

416036

486793

2

193

8770

211369

189143

2

212

3766

121411

135293

2

216

8378

294769

642293

GMT

6207

177292

237798

P25-LHRH

3

181

1893

152264

131643

3

195

31197

205906

455193

3

197

14423

337698

240543

3

211

20607

142798

131643

GMT

11510

193037

214229

P27-LHRH

4

186

0

11206

17263

4

191

0

59154

125493

4

201

0

17041

34103

4

203

0

1000

857

GMT

0

18523

26698

P35-LHRH

5

187

2009

141775

55797

5

196

4868

237208

158040

5

198

1539

154375

68307

5

217

0

121050

40822

GMT

2469

103085

58002

P2-LHRH

6

179

0

0

0

6

194

0

0

0

6

199

0

0

0

6

220

0

0

0

GMT

P8-LHRH

7

Y4

0

0

0

7

Y6

0

0

0

7

160

0

1200

ND

7

200

0

8000

2227

GMT

P62-LHRH

8

177

1242

3821

2985

8

185

0

146581

67461

8

219

0

29353

28282

8

221

2697

231473

156549

GMT

1830

44167

30728

P75-LHRH

9

176

0

12177

5559

9

189

0

15795

17155

9

202

0

2121

2216

9

222

0

9787

7879

GMT

11201

8746

EXAMPLE 4

In Vitro T Cell Proliferation Assays to Demonstrate Recognition of Th-epitope Incorporated in the Peptide Vaccines

To demonstrate recognition of the Th-epitope within the peptide immunogen PBMCs obtained from dogs immunised with peptide vaccines (dogs from Example 2) were tested against the respective Th-epitopes. The assay was carried out without the enrichment of PBMCs. PBMCs obtained from Ficoll gradient purification were directly tested against the respective Th-epitope and its truncated versions. The study demonstrated that all the dogs immunised with peptide vaccines responded to the Th-epitope incorporated confirming that T-cell activity resides in the respective sequences (FIGS.

2

-

4

). Truncated versions of the respective Th-epitopes were also tested to more closely define the T-cell activity within the sequences. It was observed that for P25 the full sequence of 17 residues was better than the shorter peptides of 15 and 12 residues, each truncated from the N-terminus of the sequence (FIG.

2

). This implies that the T-cell activity is towards the N-terminus or middle of the 17-residue peptide.

A similar observation was made with P27, the 17 residue long peptide was a better simulator than the 15-mer truncated from the N-terminus (FIG.

3

). This observation again suggested that the T-cell activity may reside towards the middle or the N-terminus of the full length peptide.

In the case of P35 and its shorter versions, except for one dog (#102), the other three dogs responded as well to the 12 residue peptide as to the full length 17 residue one (FIG.

4

). In dog #102 the 15 residue peptide was more stimulatory than the full length peptide. From this it can be deduced that that the first two residues in the sequence of P35 may not be essential and that the activity is towards the middle or C-terminus of the peptide.

EXAMPLE 5

Trial in BALB/c Mice

The canine vaccines with CDV-F derived Th-epitopes and LHRH used in Example 3 were also used to immunise BALB/c mice to investigate if the Th-epitopes would be functional in a different animal species.

Vaccine Formulation

All vaccines were formulated as for Example 3 except that they were diluted further so that 100 μl doses contained 2.7 nmoles of peptide and 10 μg of Iscomatrix™ and thiomersol as preservative.

Vaccination, Blood Samples and Assays

Mice were vaccinated with 100 μl of the vaccine at the base of tail. Vaccinations were given at 0 and 4 weeks and animals bled at intervals after each vaccination from the retro-orbital plexus. Effective T-cell help was determined by measuring the antibody response to OHM by ELISA.

Results

Mice immunised with P25-LHRH and pool of peptides comprising of P25-LHRH P27-LHRH and P35-LHRH generated high antibody titres to LHRH Peptides P35 and P75 generated low antibody titres whereas mice immunised P2, P8 and P62 had undetectable levels of anti-LHRH antibodies (Table 13).

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

TABLE 13

Anti-LHRH Antibody titers in mice immunised with CDV-F derived T cell epitope-LHRH vaccines

4 weeks post first vaccination

2 weeks post second vaccination

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Groups

1

2

3

4

5

1

2

3

4

5

Group

<100

<100

<100

<100

1 (control)

Group 2

100

126

200

126

200

16,000

16,000

16,000

16,000

16,000

(pool)

Group 3

126

400

282

100

282

16,000

16,000

16,000

16,000

16,000

(p25-LHRH)

Group 5

<100

<100

<100

<100

<100

1,412

800

<100

<100

<100

(p35-LHRH)

Group 6

<100

<100

<100

<100

(p2-LHRH)

Group 7

<100

<100

<100

<100

<100

<100

<100

<100

(p6-LHRH

Group 8

<100

<100

<100

126

126

<100

(p62-LHRH

Group 9

<100

<100

<100

<100

<100

<100

<100

316

3,162

<100

(p75-LHRH

30

1

17

PRT

canine distemper virus

1
Ser Ser Lys Thr Gln Thr His Thr Gln Gln Asp Arg Pro Pro Gln Pro
1 5 10 15
Ser

2

17

PRT

canine distemper virus

2
Gln Pro Ser Thr Glu Leu Glu Glu Thr Arg Thr Ser Arg Ala Arg His
1 5 10 15
Ser

3

17

PRT

canine distemper virus

3
Arg His Ser Thr Thr Ser Ala Gln Arg Ser Thr His Tyr Asp Pro Arg
1 5 10 15
Thr

4

17

PRT

canine distemper virus

4
Pro Arg Thr Ser Asp Arg Pro Val Ser Tyr Thr Met Asn Arg Thr Arg
1 5 10 15
Ser

5

17

PRT

canine distemper virus

5
Thr Arg Ser Arg Lys Gln Thr Ser His Arg Leu Lys Asn Ile Pro Val
1 5 10 15
His

6

17

PRT

canine distemper virus

6
Ser His Gln Tyr Leu Val Ile Lys Leu Ile Pro Asn Ala Ser Leu Ile
1 5 10 15
Glu

7

17

PRT

canine distemper virus

7
Ile Gly Thr Asp Asn Val His Tyr Lys Ile Met Thr Arg Pro Ser His
1 5 10 15
Gln

8

17

PRT

canine distemper virus

8
Tyr Lys Ile Met Thr Arg Pro Ser His Gln Tyr Leu Val Ile Lys Leu
1 5 10 15
Ile

9

17

PRT

canine distemper virus

9
Lys Leu Ile Pro Asn Ala Ser Leu Ile Glu Asn Cys Thr Lys Ala Glu
1 5 10 15
Leu

10

17

PRT

canine distemper virus

10
Ala Glu Leu Gly Glu Tyr Glu Lys Leu Leu Asn Ser Val Leu Glu Pro
1 5 10 15
Ile

11

17

PRT

canine distemper virus

11
Lys Leu Leu Asn Ser Val Leu Glu Pro Ile Asn Gln Ala Leu Thr Leu
1 5 10 15
Met

12

17

PRT

canine distemper virus

12
Glu Pro Ile Asn Gln Ala Leu Thr Leu Met Thr Lys Asn Val Lys Pro
1 5 10 15
Leu

13

17

PRT

canine distemper virus

13
Phe Ala Gly Val Val Leu Ala Gly Val Ala Leu Gly Val Ala Thr Ala
1 5 10 15
Ala

14

17

PRT

canine distemper virus

14
Gly Val Ala Leu Gly Val Ala Thr Ala Ala Gln Ile Thr Ala Gly Ile
1 5 10 15
Ala

15

17

PRT

canine distemper virus

15
Thr Ala Ala Gln Ile Thr Ala Gly Ile Ala Leu His Gln Ser Asn Leu
1 5 10 15
Asn

16

17

PRT

canine distemper virus

16
Gly Ile Ala Leu His Gln Ser Asn Leu Asn Ala Gln Ala Ile Gln Ser
1 5 10 15
Leu

17

17

PRT

canine distemper virus

17
Asn Leu Asn Ala Gln Ala Ile Gln Ser Leu Arg Thr Ser Leu Glu Gln
1 5 10 15
Ser

18

17

PRT

canine distemper virus

18
Gln Ser Leu Arg Thr Ser Leu Glu Gln Ser Asn Lys Ala Ile Glu Glu
1 5 10 15
Ile

19

17

PRT

canine distemper virus

19
Glu Gln Ser Asn Lys Ala Ile Glu Glu Ile Arg Glu Ala Thr Gln Glu
1 5 10 15
Thr

20

17

PRT

canine distemper virus

20
Thr Glu Leu Leu Ser Ile Phe Gly Pro Ser Leu Arg Asp Pro Ile Ser
1 5 10 15
Ala

21

17

PRT

canine distemper virus

21
Pro Arg Tyr Ile Ala Thr Asn Gly Tyr Leu Ile Ser Asn Phe Asp Glu
1 5 10 15
Ser

22

17

PRT

canine distemper virus

22
Cys Ile Arg Gly Asp Thr Ser Ser Cys Ala Arg Thr Leu Val Ser Gly
1 5 10 15
Thr

23

17

PRT

canine distemper virus

23
Asp Glu Ser Ser Cys Val Phe Val Ser Glu Ser Ala Ile Cys Ser Gln
1 5 10 15
Asn

24

17

PRT

canine distemper virus

24
Thr Ser Thr Ile Ile Asn Gln Ser Pro Asp Lys Leu Leu Thr Phe Ile
1 5 10 15
Ala

25

17

PRT

canine distemper virus

25
Ser Pro Asp Lys Leu Leu Thr Phe Ile Ala Ser Asp Thr Cys Pro Leu
1 5 10 15
Val

26

17

PRT

canine distemper virus

26
Ser Gly Arg Arg Gln Arg Arg Phe Ala Gly Val Val Leu Ala Gly Val
1 5 10 15
Ala

27

662

PRT

canine distemper virus

27
Met His Arg Gly Ile Pro Lys Ser Ser Lys Thr Gln Thr His Thr Gln
1 5 10 15
Gln Asp Arg Pro Pro Gln Pro Ser Thr Glu Leu Glu Glu Thr Arg Thr
20 25 30
Ser Arg Ala Arg His Ser Thr Thr Ser Ala Gln Arg Ser Thr His Tyr
35 40 45
Asp Pro Arg Thr Ser Asp Arg Pro Val Ser Tyr Thr Met Asn Arg Thr
50 55 60
Arg Ser Arg Lys Gln Thr Ser His Arg Leu Lys Asn Ile Pro Val His
65 70 75 80
Gly Asn His Glu Ala Thr Ile Gln His Ile Pro Glu Ser Val Ser Lys
85 90 95
Gly Ala Arg Ser Gln Ile Glu Arg Arg Gln Pro Asn Ala Ile Asn Ser
100 105 110
Gly Ser His Cys Thr Trp Leu Val Leu Trp Cys Leu Gly Met Ala Ser
115 120 125
Leu Phe Leu Cys Ser Lys Ala Gln Ile His Trp Asp Asn Leu Ser Thr
130 135 140
Ile Gly Ile Ile Gly Thr Asp Asn Val His Tyr Lys Ile Met Thr Arg
145 150 155 160
Pro Ser His Gln Tyr Leu Val Ile Lys Leu Ile Pro Asn Ala Ser Leu
165 170 175
Ile Glu Asn Cys Thr Lys Ala Glu Leu Gly Glu Tyr Glu Lys Leu Leu
180 185 190
Asn Ser Val Leu Glu Pro Ile Asn Gln Ala Leu Thr Leu Met Thr Lys
195 200 205
Asn Val Lys Pro Leu Gln Ser Leu Gly Ser Gly Arg Arg Gln Arg Arg
210 215 220
Phe Ala Gly Val Val Leu Ala Gly Val Ala Leu Gly Val Ala Thr Ala
225 230 235 240
Ala Gln Ile Thr Ala Gly Ile Ala Leu His Gln Ser Asn Leu Asn Ala
245 250 255
Gln Ala Ile Gln Ser Leu Arg Thr Ser Leu Glu Gln Ser Asn Lys Ala
260 265 270
Ile Glu Glu Ile Arg Glu Ala Thr Gln Glu Thr Val Ile Ala Val Gln
275 280 285
Gly Val Gln Asp Tyr Val Asn Asn Glu Leu Val Pro Ala Met Gln His
290 295 300
Met Ser Cys Glu Leu Val Gly Gln Arg Leu Gly Leu Arg Leu Leu Arg
305 310 315 320
Tyr Tyr Thr Glu Leu Leu Ser Ile Phe Gly Pro Ser Leu Arg Asp Pro
325 330 335
Ile Ser Ala Glu Ile Ser Ile Gln Ala Leu Ile Tyr Ala Leu Gly Gly
340 345 350
Glu Ile His Lys Ile Leu Glu Lys Leu Gly Tyr Ser Gly Ser Asp Met
355 360 365
Ile Ala Ile Leu Glu Ser Arg Gly Ile Lys Thr Lys Ile Thr His Val
370 375 380
Asp Leu Pro Gly Lys Phe Ile Ile Leu Ser Ile Ser Tyr Pro Thr Leu
385 390 395 400
Ser Glu Val Lys Gly Val Ile Val His Arg Leu Glu Ala Val Ser Tyr
405 410 415
Asn Ile Gly Ser Gln Glu Trp Tyr Thr Thr Val Pro Arg Tyr Ile Ala
420 425 430
Thr Asn Gly Tyr Leu Ile Ser Asn Phe Asp Glu Ser Ser Cys Val Phe
435 440 445
Val Ser Glu Ser Ala Ile Cys Ser Gln Asn Ser Leu Tyr Pro Met Ser
450 455 460
Pro Leu Leu Gln Gln Cys Ile Arg Gly Asp Thr Ser Ser Cys Ala Arg
465 470 475 480
Thr Leu Val Ser Gly Thr Met Gly Asn Lys Phe Ile Leu Ser Lys Gly
485 490 495
Asn Ile Val Ala Asn Cys Ala Ser Ile Leu Cys Lys Cys Tyr Ser Thr
500 505 510
Ser Thr Ile Ile Asn Gln Ser Pro Asp Lys Leu Leu Thr Phe Ile Ala
515 520 525
Ser Asp Thr Cys Pro Leu Val Glu Ile Asp Gly Ala Thr Ile Gln Val
530 535 540
Gly Gly Arg Gln Tyr Pro Asp Met Val Tyr Glu Gly Lys Val Ala Leu
545 550 555 560
Gly Pro Ala Ile Ser Leu Asp Arg Leu Asp Val Gly Thr Asn Leu Gly
565 570 575
Asn Ala Leu Lys Lys Leu Asp Asp Ala Lys Val Leu Ile Asp Ser Ser
580 585 590
Asn Gln Ile Leu Glu Thr Val Arg Arg Ser Ser Phe Asn Phe Gly Ser
595 600 605
Leu Leu Ser Val Pro Ile Leu Ser Cys Thr Ala Leu Ala Leu Leu Leu
610 615 620
Leu Ile Tyr Cys Cys Lys Arg Arg Tyr Gln Gln Thr Leu Lys Gln His
625 630 635 640
Thr Lys Val Asp Pro Ala Phe Lys Pro Asp Leu Thr Gly Thr Ser Lys
645 650 655
Ser Tyr Val Arg Ser Leu
660

28

10

PRT

Canis sp.

MOD_RES

(1)

pyroglutamic acid

28
Glu His Trp Ser Tyr Gly Leu Arg Pro Gly
1 5 10

29

9

PRT

Canis sp.

29
His Trp Ser Tyr Gly Leu Arg Pro Gly
1 5

30

27

PRT

Artificial Sequence

Description of Artificial Sequence Control
peptide

30
Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Asn
1 5 10 15
Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro
20 25

Number	Date	Country	Kind
PP8533	Feb 1999	AU
PQ2013	Aug 1999	AU

T helper cell epitopes

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Priority Claims (2)

PCT Information

Foreign Referenced Citations (1)

Non-Patent Literature Citations (7)

Entry
Partidos et al , Journal of General Virology, 1990, vol. 71, pp. 2099-2105.*
Visser et al, “Fusion protein nucleotide sequence similarities . . . ,” Journal of General Virology, vol. 74, pp. 1989-1994 (1993).
Curran et al, “The fusion protein gene of phocine distemper virus: nucleotide . . . ,” Arch Virol, vol. 126, pp. 159-169 (1992).
Kovamees et al, “The nucleotide sequence and deduced amino acid composition of the . . . ,” Journal of General Virology, vol. 72, pp. 2959-2966 (1991).
Barrett et al, “The nucleotide sequence of the gene encoding . . . ,” Virus Research, vol. 8, pp. 373-386 (1987).
Liermann et al, “Genetic Analysis of the Central Untranslated . . . ,” Virus Genes, vol. 17, No. 3, pp. 259-270 (1998).
Bolt et al, “Nucleotide and deducted amino acid sequences . . . ,” Virus Research, vol. 34, pp. 291-304 (1994).