CA2183977A1

CA2183977A1 - Synthetic inverso or retro-inverso t-cell epitopes

Info

Publication number: CA2183977A1
Application number: CA002183977A
Authority: CA
Inventors: Alfio Comis; Margaret Isabel Tyler; Peter Fischer
Original assignee: Individual
Current assignee: Deakin Research Pty Ltd
Priority date: 1994-02-25
Filing date: 1995-02-24
Publication date: 1995-08-31
Also published as: EP0751960A4; ZA951591B; JPH09509182A; WO1995023166A1; AUPM411994A0; EP0751960A1

Abstract

Synthetic T cell epitope analogues of native T cell epitopes which are partially or completely inverso or retro-inverso modified with respect to the native T cell epitope are shown to be effective as T cell epitopes. 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.

Description

2i83~`77 Wo 9S/23166 PCT1A1395/00090 SYNTHETIC INVERSO OR RETRO-INVERSO T-CELL EPITOPES
TECHNI~'~T FTT~T r) The present inventio~ relates to synthetic T cell epitope analogues of native T cell epitopes with partial 5 or complete inver30 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 lO vaccines comprising the T cell epitope analogues, to methods of preparing vaccines comprising these T cell epitope analogues, and to ant i ho~ R generated using these T cell epitope analogues.
B~CRGROUND ART
The ster~ol-h-m; qtry of polypeptides can be described in terms of the topo~ h ~i c~ 1 aL, a~y ~ - t of the side chains of the amino acid residues about the polypeptide h~khnn~ which is defined by the peptide bonds between the amino acid residues and the ~-carbon atoms of the bonded residues. In addition, polypeptide h~nkhnn~q have distinct termini and thus direction.
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 ~n~nt j l rs of L-amino acids and form peptides which are herein referred to as inverso peptides, that is, peptides corr~Rpnn~1in~ to native peptides but made up of D-amino acids rather than L-amino acids .
3 0 Retro - inverso modif ication 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 3 ~ peptide sequence . A retro - inverso analogue thus has reversed termini and reversed direction of peptide bonds Wo 95/23166 218 3 ~ ~7 PCr/AUs5/ooogo 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 se~uence is 5reversed and replaced with enantiomeric amino acid residues. Since the retro-inverted portion of such an r analogue has reversed amino and carboxyl termini, the amino acid residues fl;~nk;n~ the retro-inverted portion are replaced by side-chain-analogous ~Y-substituted 10 geminal ~ m; n~ - thanes and malonates, respectively .
Processes for synthesis of retro-inverso peptide analogues (sonelli et al ., 1984 ; Verdini and Viscomi , 1985) and some processes for the solid-phase synthesis of partial retro-inverso peptide analogues have been 15 described (pesBi et al., 1987).
It has been observed that due to the stereospecif icity of enzymes with respect to their substrates, r~rl ~ -nt of L-amino acid residues with D-amino acid residues in peptide substrates generally 20 abolishes proteolytic enzyme recognition and/or activity, although exceptions are known.
Peptide hormones have been of particular interest as targets or retro-inversion, presumably because their analogues would have potential use as therapeutic agents.
25 Partial, and in a few cases complete, retro-inverso analogues of a number of peptide h~ AA have been prepared and tested (see, for example, Goodman and Chorev, 19 81 ) .
Complete or P~r~Pn~r1 partial retro-inverso analogues 30 have generally been found to be devoid of biological activity. The lack of biological activity has been attributed to possible complex structural changes caused by ~Yt~nf~d modification, the presence of reversed chain termini or the presence of proline residues in the 35 seguences. Some partial retro-inverso analogues, that is peptides in which only selected residues were modified, on the other hand, have been shown to retain or enhance biological activity. Retro-inversion has also found ~Wo 95/23166 PCT/AU95/00090 application in the area of rational design of enzyme inhibitors .
The fact that retro-inversion of biologically active peptides has met with Dnly limited success in retaining 5 or Pnh: Innl n~ the activity of the native peptide is probably due to several reasons. Although structurally very similar, it was realised early that peptides and their retro-enantiomers are topologically not identical and crystal structure and solution conformation studies 10 have borne this out. Biological activity of a peptide hormone or neurotransmitter depends primarily on its dynamic int~r~ctlnn with a receptor, as well as on transduction processes of the peptide-receptor complex.
It is now clear that such interactions are complex 15 processes involving multiple conformational and topological properties. Consequently it is not surpri8ing that a retro - inverso analogue may not be able to mimic all of these propertie5.
In order to activate the ,~ ]1~ component of the 20 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 maj or 25 histocompatibility complex (MHC). 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 30 transported to the surface of APCs. The peptides capa~le of binding MEIC molecules are 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 3~ (Jolivet et al., 1990) therefore need to be designed with regard to these ~acts. 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 _ Wo 95/23166 PCr/AU95/OOo90 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 nt; f i cation of appropriate T-cell epitope ~ cocktails ' potentially useful in synthetic vaccines is therefore an active field of research (Schwartz, 1936).
DIS(:~DOSrrr~r' QF T~IE LNV~;Nll~.)N
Def;nitions Throughout the specification and claims ~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.
Throughout the spf~;f;~tion and claims "inver80 modified" refers to a peptide which is made up of D-amino acids in which the amino acid residues are a8sembled in the same direction as the native peptide with respect to which it is inverso modif ied .
LllLuuy~luuL the specification and claims ~'retro-inverOEo modified~ refers to a peptide which i8 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 i9 retro-inverso ~fiecl.
Throughout t~e specification and claims the term '~native~ refers to any sequence of L amino acids used as a starting sequence for the preparation of partial or complete retro, inverso or retro-inverso analogues.
The term "peptide" as used throughout the specif ication and claims is to be understood to include peptides of any length.
~l~Luu~l~uut the specification and claims the term ~antigenic fragment" refers to a peptide which is a portion of an antigen which itself is immunogenic or capable of binding antibodies.
The term ~antigen" as used throughout the ~p~ if; c :~tion and claims is to be understood to include immunogens as the context requires.

2183g7~ ~ ~
Wo 95/23166 ~ PCT/AU9S/00090 Throughout the ~3pecif ication and claims the term "antigen analogue" refers to a peptide moIecule capable of mimicking the immunological activity of the native peptide antigen with respect to which it is partially or 5 completely retro, inverso or retro-inverso modified.
Retro peptides are made up of ~-amino acids and are peptides in which the amino acid residues are assembled in opposite direction to the, native peptide sequence.
Throughout the specification and claims the term "T-lO cell epitope analogue~ refers to a peptide moleculecapable of mimicking the immunological activity of the native T-cell epitope with respect to which it is partially or completely inverso or retro-inverso modif ied .
Partial modification includes analogues in which as few a8 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 20 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 25 administration. Further, incorporation of D-amino acids has potential f or 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-30 cell epitope counterparts it follows that, generally,these analogues can be expected to be successful since T-cell epitope - M~tC molecule interactions are not flln~' ~Al ly different from case to case.
In a first aspect the invention provides a synthetic 35 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 n~tive T cell epitope.

wog5l23l66 7}~;~ PCr/AU9SI00090--The T cell epitope analogues o~ the present invention sti~ltlAte immune responsiveness when used in place oi their native T cell epitope counterparts in vaccines .
The eff~icacy of T cell epitope analogues of the invention is illustrated with respect to the malaria T
cell epitopes of Example 2.
In a second aspect 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 vet~r;n~r~lly acceptable carrier, diluent, excipient and/or adjuvant. Typically, 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. Preferably the T cell epitope analogue is conjugated to the s 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 c~ntintl~us peptide.

2 0 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, ~IV, cholera, malaria, influenza, rabies or ~;rhth~ria causing agents, or toxins such as robustoxin, heat labile toxin of pathogenic ~scherichia coli strains and Shiga toxin from Shigella dysenteriae. Other B cell épitopes of interest include epitopes of Amyloid ~ protein (Alzheimer' s disease) and human chorionic gonadotropin and gonadotropin releasing hormone (contraceptive vaccines).
The B cell epitope i8 preferably a retro, retro-inverso or inverso antigen analogue.
Preferred T ell epitope :analogues of the invention WO gsn3l66 218 3 ~177 PCr/Aussloooso are analogues of:
Diphtheria toxin:
E~-Gln-Val -Val -His-Asn-Ser-Tyr-Asn-Arg-Pro-Ala-Tyr-Ser-Pro - Gly- OH l SEQ ID NO: 1 ) Pertussis toxin:
H-His -Arg-Met-Gln-Glu-Ala-Val -Glu-Ala-Glu -Arg-Ala-Gly-Arg-OH (SEQ ID NO: 2) Malaria CSA protein:
H-Pro-Ser-Asp-Lys-His-Ile-Glu-Gln-Tyr-Leu-Lys-Lys-Ile-Lys-Asn-Ser-Ile-Ser-OH (SEQ ID NO: 3) Malaria CSB protein:
H-His-Ile-Glu-Gln-Tyr-Leu-Lys-Lys-Ile-Lys-Asn-Ser-Ile-Ser-OH (SEQ ID NO: 4) Malaria CST3 protein:
H-Gly-Asp-Ile-Glu-Lys-Lys-Ile-Ala-Lys-Met-Glu-Lys-Ala-Ser-Ser-Val-Phe-Asn-Val-Val-Asn-Ser-OH (SEQ ID NO: 5) Hen egg lysozy~ne:
H-Cys-Ser-Ala-Leu-Leu-Ser-Ser-Asp- Ile-Thr-Ala-Ser-Val -Asn-Cys-Ala-OH (SEQ ID NO:6) Ovalbumin:
H- Ile -Ser-Gln-Ala -Val -His -Ala -Ala-His -Ala-Glu- Ile-Asn-Glu-OH (SEQ ID NO: 7) and H-Tyr-Thr-Tyr-Thr-Val -His-Ala-Ala-His -Ala-Tyr-Thr-Tyr-Thr-OH (SEQ ID NO: 8) Other pref erred T cell epitope analogues are analogues of:
Measles Virus F and H glycoproteins: (Partidos C.D. et al, 1991) MVF:258-277 H-Gly-Ile-Leu-Glu-Ser-Arg-Gly-Ile-Lys-Ala-Arg-Ile-Thr-His-Val-Asp-Thr-Glu-Ser-Tyr-OH
(SEQ ID NO: 9) MVF:288-302 H-Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-His-Arg-Leu-Glu-Gly-Val-OH (SEQ ID NO: 10) Respiratory syncytial virus lA protein: ~Nicholas J.A. et al, 1988) RSlA:45-60 H-Cys-Glu-Tyr-Asn-Val-Phe-His-Asn-Lys-Thr-Phe-Glu-Leu-Pro-Arg-Ala-OH (SEQ ID NO: 11) Influenza halnagglutinin A/PR/8/34 Mt.S.:
-~ l8~ t~
Wo 95l23~66 ~ - PCr/AU95lO0090 --109-ll9 (Hackett C J. et al 1983) (SEQ ID NO: 12) 130-140 (Hurwitz J.J. et al 1984) (SEQ ID NO: 13) 302-313 (Lamb J.R. et al 1982; Hurwitz J.I.. et al 1984) (SEQ ID NO: 14) 5Pork Insulin:
(A) 4-14 (Rosenthal A.S. 197B) (SEQ ID NO: 15) (B) 5-16 (Thomas J.W. et al 1981) (SBQ ID NO: 16) Hepatitis B virus pre S:
120-132 (Milich D.R. et al 1986) (SEQ ID NO: 17) 10 E~epatitis B virus major surface antigen:
38-52 (Milich D.R. et al 1985) (SEQ ID NO: 18) 95-109 " (SEQ ID NO: lg) 140-154 ~' (SEQ ID NO: 20) Foot and mouth virus VPl:
15 141-160 (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:
22) In a third aspect 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.
In a f ourth aspect the invention provides antibodies produced by ' ~ s~tion of a host with a vaccine of the second aspect.
In a fifth 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.
In a sixth aspect the invention provides a method of preparing a vaccine of the second a6pect comprising conjugating a T cell epitope analogue of the first aspect to a B cell epitope or ~ n~ a T cell epitope analogue of the first aspect with a B cell epitope and ~ n~ an effective amount of the resulting mixture or conjugate with a pharmaceutically or veterinarally acceptable carrier, diluent, excipient and/or adjuvant.
~ac i~es ' che i ,en-~on ca~ be fc~ _ late~ us g 21g~7~ ~ ~
Wo 95123166 PCr/AU95100090 g standard methods in the art of vaccine formulation.
Selection of appropriate diluents, carriers, excipients and/or adjuvants can be made in accordance with standard techniques in the art.
Vaccines of the invention may be administered to hosts in need of such treatment by injection. Vaccine~
- incorporating D-amino acid ct~n~:~;n~n~ analogues may also be administered orally.
ABBREVIATIONS
BOP (benzotriazolyloxy) tris (dimethylamino) phosphonium hexafluorophosphate (Castro' s reagent ) DMF dimethyl formamide E~ISA enzyme-linked immunosorbent assay Fmoc 9-fluorenylmethoxycarbonyl HP~C high-perf ormance liquid chromatography Ig immunoglobulin in inverso i.p. intraperitoneal no normal (native) PBS phosphate buffered saline (lO mM phosphate, 150mM NaCl, pH 7.4) Pfp pentafluorophenyl PVC polyvinylchloride ri retro-inverso TFA trifluoroacetic acid Amino Acids:
~ -amino acids are lndicated by an upper case fo~lowed by lower case lettering e.g. Ala indicates

3 o I. - alanine .
D-amino acids are indicated by all lower case abbreviations, e.g. ala indicates D-alanine.
BRIEF DESCRIPTION OF THE FIGIJRES
Figure 1 shows the results of a cell proliferation 35 experiment conducted using the T-cell epitope peptides wo9~/23166 ?,~83 ~ f ' PCT/AU95/OoO90 --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-Pro1 3-OI~
(SEQ ID NO: 23) alone or together with either.no or 5 riMalCST3.
Figure 3 shows antibody production measured in mice immunized with the B-cell epitope ~- (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 X- (Asn-Ala-Asn-Pro) 3-OH
(SEQ ID NO: 23) alone or together with either ~o (SEQ ID
NO: 4 ) or riMalCSB protein.
Figure 5 3hows antibody production measured in mice 15 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: l ) or riDiphT .
Figure 6 shows antibody production measured in mice immunized with the B-cell epitope H- (Asn-Ala-Asn-Pro) 3-OH
20 (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
25 NO: 7) or riOvalT.
BEST MODE OF CARRYING OUT THE INVENTION
T cell epitope analogues of the inYention are prepared by standard techniques for the preparation of and D amino acid ~nntiqln;n~ peptides, particularly as 3 0 outlined in Example l .
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 iniectable vaccines.
35 Effective amounts of Tcell-epitope analogues to be incorporated in the ~accines can be determined in accordance with scandard methods. Conjugation techniques ~ 21839`.~77.-.
Wo 9~/23166 PCTIAU9~l00090 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 i ; ~ation of the host is 5 desired or where the host is being used to produce antibodies for exogenous use.
The invention is further described in the following examples which are illustrative of the invention but in no way limiting on its scope.
EXAMPJ.E 1 Pe~tide Svnthesis 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 &
15 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 20 ~auantitatively with the appropriate preformed C-terminal Fmoc-amino acid symmetrical anhydrides, in the presence of 0.2 molar equivalent3 of N,N-dimethylaminopyridine and N-methylmorpholine. The Polyhipe resin, derivatised with Fmoc-Rink linker (Rink, 1987) did not require 25 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-h~d,~"~yl,~,izotriazole coupling (Hudson, 1988). The progress of each syntheEis was monitored 30 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, cr~nt;~;n;n~ a 35 suitable mixture of scavenger chemicals (Tam, 1988).
After filtration and vacuum evaporation, the peptides were triturated with diethyl ether, collected by WO 95/23166 - 12 - PCTIAUgS/00090 --centrifugation and lyophilised from aqueous ammonium bicarbonate solution.
All peptides then underwent an initial desalting and purif ication step by column chromatography on suitable 5 gel filtration media in aqueQus ~olvents. Afterwards they were purif ied to homogeneity by reversed-phase HPLC
using water-acetonitrile ~cnn~;n1ng o . 05-0.1~ TFA) gradient elution. The purity of the synthetic peptides was further aRsessed by gas-phase acid hydrolysis/amino 10 acid analysis (B; ~ y~::L et al ., 1987) and, if deemed necessary, by automated gas-phase sequencing (Hunkapiller & Hood, 1983) .
EXAMP~F 2 Malaria T-cell e~i~o~e ~et:tides It has been shown that nonresponsiveness to the malaria immuno-lnm1n~nt B-cell epitope (Asn-Ala-Asn-Pro)x (SEQ ID NO: 23) of the p7iqRmnr~ m 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 reglon of the _ circumsporozoite protein from which the peptide stems is apparently conserved in different parasite isolates.
The following peptides were prepared according to the usual protocols:
noMal CST3 H- Gly-Asp - I le - Glu - Lys - Lys - I le -Ala - Lys - Met -Glu-Lys-Ala-Ser-Ser-Val-Phe-Asn-Val-Val-Asn-Ser-OH (SEQ ID NO: 5) inMalCST3 H-Gly-asp-ile-glu-lys-lys-ile-ala-lys-met-glu - lys - ala- ser- ser -val -phe - Asn -val -val - asn -ser -OH5 riMalCST3 H-ser-asn-val-val-asn-phe-val-ser-~er-ala- - -lys -glu-met -lys-ala-aile- lys -lys-glu-aile-asp-Gly-OH

~18`3977 Wo 9S/23166 ~ PCr/AU95/0009 sALB/c mice were immunised subcutaneously at the base of the tail with the above T-cell epitope peptides emul6ified in an e~aual volume of complete Freund' 5 adjuvant. Ten days later, the animals were killed by cervical dislocation and the inguinal and popliteal lymph nodes removed. A cell suspension from the lymph nodes was prepared and the cells cultured in the presence of various concentrations of the test antigen, as well as a non-related control antigen. ~ Cell proliferation was lo quantitated by measuring the incorporation into the cell~
of radiolabelled thymidine. Results from the experiment are shown in Fig. 1.
When animals were primed with any form of the peptide and the animals ~ cultured T cells challenged with the same peptide, proliferation was observed in every case. Upon priming with one form of a peptide and challenging with either of the other two forms, some activation was observed in each case.
In order to remove any potential effects due to non-3pecific cell proliferation, the T cell assay method was improved as follows:
A cell suspension from the lymph nodes was centrifuged on Ficoll-Isopaque to separate 1- nn1l~lear 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 f rom 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 ~ n~nmi n~nt B-cell epitope H- (Asn-Ala-Asn-Pro)3-OH (SEQ ID NO: 23) of the circumsporozoite protein were measured following intraperitoneal injection Wo 95/23166 ~ 7; ¦ ~ PCTIAU95/OOO9o of Balb/c mice. ~ One hundred mi~:L.y, q of B-cell epitope were administered in an e~ual volume of Freund ' s complete adjuvant either alone or in a mixture (l:l) with either noMalCST3 (SEQ ID NO: 5) or riMalCST3. As a 5 negative control, a further group of mice were immunised with either noMalCST3 ~SEQ ID NO: 5) or riMalCST3 in the ab~ence of the B-cell epitope. Three weeks after priming, mice were boosted by the same route and with the same dose of peptide in incomplete Freund's adjuvant. A
lO second booster i~jection was given two weeks after the f irst 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 15 immunosorbent assay (ELISA) . Titres of ilnt; ho~li es against the B-cell epitope were det~rrni n~rl in microtitre plates coated overnight at 4C with O . 5 mi- ~ ~,y ~ ~ of synthetic peptide cross-linked to ovalbumin.
Low titre of antibodies were measured in mice 20 immunised with the B-cell epitope alone, however, much higher titre of antibodies was observed in each case in mice co-immunised with the same peptide and either form of the T-cell epitope (Fig.2) . All together, these f indings demonstrate the potential usefulness of 25 riMalCST3 and inMalCST3 as vaccine, ~ntq; the cellular immune respon~qe they elicit is responsive to the normal antigen.
Antibody response to the same B-cell epitope was also measured using five more T-cell epitopes selected 30 from the literature and synthesized in the following f orms:
M;~l ~ria circums~orozoite Protein:
noMa l CSA ( Good e t al, l 9 8 7 ):
H-Pro-Ser-Asp-Lys -His - Ile-Glu-Gln-Tyr-Leu-Lys -Lys - Ile-35 Lys-Asn-Ser-Ile-Ser-NH2 (SEQ ID NO: 3) riMalCSA:
H-ser-ile-ser-asn-lys -ile-lys-lys-leu-tyr-gln-glu-ile-his - lys -asp- ser-pro -NH2 ra ~
2183977.
Wo 9S/23166 ~ PCr/AU95l00090 noMalCSB (Good et al, 1988):
H-Hls-Ile-Glu-Gln-Tyr-Leu-Lys-Lys-Ile-~ys-Asn-Ser-Ile-Ser-N~2 (SEQ ID NO~
riMa l CSB:
5 H-ser-ile-ser-asn-lys-ile-lys-lys-leu-tyr-gln-glu-ile-his -NH2 DiPhtheria toxin:
noDipT (Bixler et al, 1989) H-Gln-Val -Val -His-Asn-Ser-Tyr-Asn-Arg-Pro-Ala-Tyr-Ser-10 Pro-Gly-NH2 (SEQ ID NO:1) riDipT:
H-Gly-pro-ser-tyr-ala-pro-arg-asn-tyr-eer-asn-his-val-val -gln-NH2 Pertu3sis t~r; n noPertT (Kim et al, 1990) (SEQ ID NO: 2):
H-His -Arg-Met-Gln-Glu-Ala-Val -Glu-Ala-Glu-Arg-Ala-Gly-Arg-NH2 riPertT:
H-arg-Gly-ala-arg-glu-ala-glu-val -ala-glu-gln-met-arg-20 his-NH2 Oval hllmi n noOvalT (Sette et al, 1989) (SEQ ID NO: 7):
H- Ile-Ser-Gln-Ala-Val -His-Ala-Ala-His-Ala-Glu- Ile-Asn-Glu -NH2 2 5 riOvalT:
H-glu-asn- ile-glu-ala-his-ala-ala-his-val -ala-gln-ser-ile-NH2 The synthesis of the above peptides was performed on Polyhipe Rink resin. The side chain protecting groups 3 0 used were: t -butyl f or serine, threonine, aspartic acid, glutamic acid and tyrosine; trityl for histidine, glutamine and asparagine; t-butoxycarbonyl for lysine and 2,2,5,7,8-p~nt: -t~lyl chroman-6-sulphonyl for arginine.
For diphtheria and pertussis peptides, cleavage and side-35 chain deprotection were accomplished by reaction of thepeptidyl resins for 90 min at 0C with lM
trimethylsilylbromide-thioanisole in TFA containing 0.25M
1, 2-ethanedithiol (5% v/v) and water (59~ v/v) in TFA at 218391~7,~
Wo 95/23166 PCT/A~95100090 room temperature for 90 min.
In each case the mice developed very low titres against the B-cell epitope when immunised with the B-cell epitope alone, but produced much higher antibody titre 5 when a mixture of the B-cell epitope and any o~ the T-cell epitopes in either no- or ri- form were used in the immunogen formulation (Fig. 3-7).
INDT~sTR~AT~ AppIlI~'ATI~)N
T cell epitope analogues in accordance with the lO invention have a range of potential applications in eliciting ~ ic responses in a host. These analogues can be used in the treatment and/or prophylaxis of diseases, and therapy of disease states. In particular, these analogues can be used in vaccines in 15 animals, including humans for protection against pathogens and the like.

2~83g77 ~ ~ ~
wo ssr23l66 ~ PcrlAuss/oooso R~ N~;S
Arshady, R., Atherton, E., Clive, D.L.J. & Sheppard, R.C.
(1981) Peptide synthesis. Part 1. Preparation and use of polar supports based on poly (dimethylacrylamide~ . J.
Chem. Soc. Perkin Trans. I, 529-537.
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Bidlingmeyer, B.A., Tarvin, T.L. & Cohen, S.A. (1987) Amino acid analy3is of 3ubmicrogram hydrolyzate 3amples.
In "Method3 in Protein Sequence Analy3is", Walsh, K.A.
(Ed. ), pp . 229-245, The Humana Press .
Bonelli, F., Pessi, A. & Verdini, A.S. (1984) Solid phase 1~ 3ynthesi3 of retro-inver30 peptide analogue3. Int. J.
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Carpino, L.A. & Han, G.Y. (1972) The 9-fluorenylmethoxy-carbonyl amino-protecting group. J. Org. Chem., 37, 3404 -3409 .
Eberle, A.N., Atherton, E., Dryland, A. & Sheppard, R.C.
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Goodman, M. & Chorev, M. (1981) The synthe3i3 and confirmational analysis of retro-inverso analogues of biologically active molecules . In ' Per3pective3 in Peptide Chemi3try'; Karger, Basel; pp. 283-294.
Hancock, W.S. & Batter3by, J.E. (1976) A new micro-test 3 0 for the detection of incomplete coupling reactions in solid-phase peptide synthesis using 2, 4, 6-trinitrobenzene-sulphonic acid. Anal. Biochem., 71, 260-264.
Hudson, D. (1988) Methodological implications of 3imultaneous solid-pha3e peptide 3ynthesis. 1.
Compari30n of different coupling procedures. J. Org.
Chem., 53 , 617-624 .

WO 95/23166 2 ~ ~ 3 B i ~ PCr/AU95/00090 ~--Hunkapillar, M.W. & Hood, L.E. (1983) Protein sequence analysiæ: automated microsequencing. Science, 219, 650-659 .
Pesæi, A., Pinori, M., Verdini, A. S . & Viscomi, G. C.
~1987) Totally solid phase synthesis of peptide (8) c-.,n~A;nlnS retro-inverted peptide bond, uæing croæælinked sarcoæinyl copolymer as support. European Patent 97994-B, 30 Sep. 1987 (8739) .
Tam, J.P. (1988) Acid deprotéction reactions in peptide synthesis. In 'Macromolecular Sequencing and Synthesis, Selected Methods and Application ', pp . 153 -184; Alan R .
Liss, Inc.
Verdini, A.S. & Viscomi, G.C. ~ (1985) Synthesis, resolution, and assignment of configuration of potent hypotensive retro-inverso bradykinin potentiating peptide 5a(BPP5a) analogues. J. Chem. Soc. Perkin Trans. I, 697-701 .
H. Rink (1987) Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methylester resin.
Tetrahedron Lett., 28,3787-37go B.J. Spalding ~1992) In hot pursuit of an XIV vaccine.
Bio/Technology, 10, 24-29 R.A. Wirtz, J.F. Duncan, E.K. Njelesoni, I. Schneider, A.E. Brown, C.N. Oster, JBO Were and H.K. Webster (1989) Bull WHO, 67, 535-542 . ELISA method for detecting Plasmodium falciparum circumsporozoite antibody.
Steward, M.W. h Howard, C.R. (1987) Synthetic peptides: a next generation of vaccines? Immunol. Today, 8, 51-58.
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Audibert & L. Chedid (1990) Polyvalent synthetic vaccines: relationship between T epitopes and immunogenicity. Vaccine, 8, 35-40 .
R.H. Schwartz (1986) The value of synthetic peptideæ as vaccines for eliciting T-cell immunity. Current Topics ~icro}~iol. Imnlunol., 130, 79-84.
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Matile, ~I. Etlinger, A. Trzeciak, D. Gillesæen & J.R.L.
Pi~k (1988) A malaria T-cell epitope recognized in Wo 95/23166 2 1 8 3 ~ ~ 7 i PCT/AUsS/OOogn association with most human MHC class II molecules.
Nature, 3~6, 778-780.
M.F. Good, W.L. Maloy, M.N. Lunde, H. Margalit, J.L.
Cornette, G.L. Smith, B. Moss, L.H. Miller & J.A.
- 5 Berzofsky (1987) Construction of synthetic immunogen: use of new T-helper epitope on malaria circumsporozoite protein. Science, 235, 1058-1062.
M.F. Good, D. Pombo, D.L. Maloy, V.F. De La Cruz, L.H.
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G. Bixler, S. Pillai & R. Insel (1989) T-cell epitope as carriers molecule f or conj ugate vaccines . WO 8 9 / 0 6 9 74 .
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Houghten, R.A. Lerner, J.G. Sutcliffe & H. Koprowski (1984) T cell responses to cleaved rabies glycoprotein and to synthetic peptides. J. Inununol. 133, 2748.
5 D.R. Milich, D.L. Peterson, G.G. Leroux-Roels, R.A.
Lerner & F.V. Chisari (1985) Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg) .
VI. Fine specificity. J. Ilrcnunol. 134, 4203.
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10 F.V. Chisari (1986) T and B cell recognition of native and synthetic pre-S region determinants on HBsAg. In Nodern Approache~ to Vaccines. R. Chanock, R.A. Lerner and F Brown, eds. Cold Spring Harbor Laboratories, New York .
15 J.A. Nicholas, M.A. Mitchell, M.E. Levely, K.L. Rubino, J.H. Kinner, N.K. Harn & C.W. Smith (1988) Mapping an antibody binding site and a T cell stimulating site on the lA protein of respiratory syncytial virus. J. Virol.
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cells in insulin immune guinea pigs. J. Ilrununol. 126-, 1095.

~WO 951~166 2 1 8 3~9 7~7 r PCT/AU95100090 ~ ~u NS~; LISTING
( 1 ) GENERAl. INFORMATION:
(i) APPLICA~T: Deakin Research Limited, N/A N/A
Comis, Alf io Fischer, Peter Tyler, Margaret I
(ii) TITLE OF INVENTION: EPITOPES
(iii) NUMBER OF ~ U~;N~S: 23 (iv) CORRESPONDENCE ADDRESS:
(A) AnnRRS.C:~.: Gri~ith EIack & Co (B) STREET: Level 8, 168 Walker Street (C) CITY: North sydney (D) STATE: New South Wales (E) COUNTRY: Australia (F) ZIP: 2060 (V) ~ Ul~;~ R~n~R~.~ FORM:
(A) MEDI~ TYPE: Floppy disk (B) ~:U..~U'l'~;~: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1. 25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUM~3ER: AU PM 4119 (B) FILING DATB: 25-FEB-1994 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Kurts, Ann D
(B) REGISTRATION NUMBER: N/A
30 (C) ~ ;N~;/DOCKET NUMBER: P21192 Wo 9S/23166 21~ 3 9 i~ PCT/AU95/00090 ~a (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 61 2 957 5944 (B) TELEFAX: 61 2 957 628~3 (C) TELEX: AA26547 5 ( 2 ) INFORMATION FOR SEQ ID NO :1:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) sTR~Nn~n~ single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ( i i i ) ~Y ~r~ CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SO~ROE:
(A) ORGANISM: Corynebacterium diphtheriae (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Gln Val Val His A~n Ser Tyr Asn Arg Pro Ala Tyr Ser 2 0 Pro Gly (2) INFORMATION FOR SEQ ID NO:2:
U~;N~ RAt'T~RT.C~TICS:
(A) LENGTE~: 14 amino acids (B) TYPE: amino acid (C) sTR~Nn~nN~ single ~D) TOPOLOGY: linear 218~977 (ii) MO:I.ECULE TYPE: peptide ( i i i ) HYPOTHETI CAL: NO
- (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: interrLal (vi ) ORIGINAL SOURCE:
(A) ORGANISM: Bordetella pertu~3is (xi) ~ U~;N~: DESCRIPTION: SEQ ID NO:2:
His Arg Met Gln Glu Ala Val Glu Ala Glu Arg Ala Gly l C Arg (2) INFORMATION FOR SEQ ID NO:3:
(i) ~;5,1U~:N~L. CHARACTERISTICS:
(A) ~ENGTH: 18 amino acids (B) TYPE: amino acid (C) STR~Nll~n~ : single ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide (iii) ~Y~ ~L1CAL: NO
( iv) ANTI - SENSE: NO
(v) FRAGMENT TYPE: internal (vi ) ORIGINAL SOURCE:
(A) ~RGANISM: Pla~modium falc:iparum 21839Z~
Wo 95/23166 - 24 - PCr/~U9slooo9o (xi ) ~ ;UU~;N(_'~ DESCRIPTION: SEQ ID NO: 3:
Pro= Ser Asp Lys His Ile Glu Gln Tyr Leu Lys Lys Ile Lys Asn Ser Ile Ser (2) INFORMATION FOR SEQ ID NO:4:
U~ TARA(~T~T.CTICS:
(A) LENGTH: 14 amlno acids (B) TYPE: amino acid (C) sT~ANn~nNRc~ single ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: peptide ( i i i ) HYPOTHET I CAL: NQ
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: ; nt~rn~l (Xi) ~;~;UU~N~; DESCRIPTION: SEQ ID NO:4:
His Ile Glu Gln Tyr Leu Lys Lys Ile Lys Asn Ser Ile Ser (2) INFORMATION FOR SEQ ID: NO-5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) sTRANn~nN~c single . (D) TOPOLOGY: linear .

21839~7 ~ `
WO 9512316G PCT/A~195/00090 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAI: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Plasmodium falciparum (xi) ~;s,u~ ; DESCRIPTION: SEQ ID NO:5:
Gly Asp Ile Glu ~ys Lys Ile Ala Lys Met Glu Lys Ala Ser Ser Val Phe Asn Val Val Asn Ser (2) INFORMATION FOR SEQ ID NO:6:
( i ) SEQUENCE CHI~RACTERISTICS:
(A) l.ENGTH: 16 amino acids (B) TYPE: amino acid (C) STRzNnRnNR.~.~ single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTE~ETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGME~IT TYPE: internal .

2183977 ~
WO 9~/23166 PCT/AU9~/00090 (Xi) ~ ,2U~;NC`J~ DESCRIPTION: SEQ ID NO:6:
CYB Ser Ala ~eu ~eu Ser Ser A~p Ile ~hr Ala Ser Val Asn Cys Ala ~ ~

~2) INFORMATION FOR SEQ ID NO:7:
u~ ; CHARACTERISTICS:
(A) I.ENGTH: 14 amino acids (B) TYPE: amino acid 10 (C) S'rRANn~n~ S: single ( D ) TOPO~OGY: 1 inear (ii) MOLECU~E TYPE: peptide ( i i i ) li Y ~ CAJ~: NO
(iv) ANTI-SENSE: NO
15 (v) FRAGMENT TYPE: internal (Xi) Y~;(..!U~;N~; DESCRIPTION: SEQ ID NO:7:
Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu ( 2 ) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENG~H: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Wo 95/23166 - 27 - PCT/AU95/00090 ( i i ) MOLECULE TYPE: peptide ( i i i ) HYPOTHET I CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Tyr Thr Tyr Thr Val His Ala Ala His Ala Tyr Thr Tyr Thr ( 2 ) INFORMATION FOR SEQ ID NO: 9:
~ QU~;N(:I~; CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) sTRAl~n~nN~ single (D) TOPOLOGY: linear 1~ (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Measles V~ S

WO9~/23166 2183977 PCr/AU9~/00090 --(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr His Val Asp Thr Glu Ser Tyr (2) INFORMATION FOR SEQ ID NO:10:
(i) ~;~U~;N(:~; r~TARZ~rT~RT.~TICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STR~Nn~n~ s single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO .
( iv ) ANT I - SENSE: NO
1 5 ( v ) FRAGMENT TYPE: ; n t .o rn :~1 (vi ) ORIGINAL SOURCE:
(A) ORGANISM: Measles virus (Xi) ~ U~;N~ DESCRIPTION: SEQ ID NO:10:
Leu Ser Glu Ile Lys Gly Val Ile Val His l~rg Leu Glu Gly Val (2) INFORMATION FOR SEQ ID NO:11:
;S.?U~N( :~; CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid ~ -Wo 95/23166 2 ~ 8 3 9 7 7 ` PCr/AU95/00090 (C) STRANn~llN~cs: single ( D ) TOPOl,OGY: l inear (ii) MOLECULE TYPE: peptide (iii) ~Y~L~ll~L: NO
5 ~iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Respiratory syncytial virus (Xi) ~ ,2U~;N(~:~; DESCRIPTION: SEQ ID NO:11:
Cys Glu Tyr Asn Val Phe His Asn Lys Thr Phe Glu Leu Pro Arg Ala ( 2 ) INFORMATION FOR SEQ ID NO :12:
(i) ~ !U~;N~l:; CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANnFi~nN~.~.C: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ( i i i ) 11 Y ~0'1'11~;'1' L CAL: ~0 (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal WO gs/23l66 218 3 9 7 7 3 o - PCT/AU95/00090 (vi ) ORIGINAL SOURCE:
(A) ORGANISM: Inf luenza virus (Xi) ~ U~N(:~ DESCRIPTION: SEQ ID NO:12:
Ser Ser Phe Glu Arg Phe Glu I1P Phe Pro Lys l 5 10 ( 2 ) INFORMATION FOR SEQ ID NO :13:
;Uu~;N~; CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANn~nN~ : single ( D ) TOPOLOGY: 1 i near (ii) MOLECULE TYPE: peptide ( iii ) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ~
(A) ORGANISM: Influenza viruæ
(Xi) ~ ;UU~;NC~; DESCRIPTION: SEQ ID NO:13:
Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys ( 2 ) INFORMATION FOR SEQ ID NO :14:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 12 amlno acids (B) TYPE: amino acid Wo 95/23166 218 3 9 7 7 ~ ~ ~CT/AI~95/00090 (C) STRANDEDNESS: single (D) TOPOLOGY: linear:
(ii) MOLECULE TYPE: peptide ( i i i ) ~ Y ~U ~ CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: interna (vi ) ORIGINAL SOURCE:
(A) ORGANISM: In~luenza virus (Xi) ~ 5,?U~;N(:~; DESCRIPTION: SEQ ID NO:14:
Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met ( 2 ) INFORMATION FOR SEQ ID NO :15:
U~;N~ R~TRR~.CTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) sTR~NnFm~ single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ( i i i ) HYPOTHETI CAL: NO
(iv) ANTI-SENSE: NO
( v) FRAGMENT TYPE: 1 n t P rn ~1 (vi ) ORIGINAL SOURCE:
(A) ORGANISM: pig W095/~166 218 3 ~ i i PCTIAU95/00090 ~
_ 32 _ (xi) SEQUENCE DESCRIPTION: S
EQ ID NO:15:

Glu Gln Cys Cys Thr Ser Ile Cy9 Ser Leu Tyr (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE ~ARA~
T~RT~TICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: 8 i ngle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) ~Y~o~ CAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: i nt~rn~1 (vi) ORIGINAL SOURCE:

(A) ORGANISM- pig (xi) SEQUENCE DESCRIPTION: S
EQ ID NO:16:

His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr l 5 10 (2) INFORMATION FOR SEQ ID NO.17:

(i) ~U~N~ CHARACTERISTICS:

(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANn~TlNE~: single (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: peptide WO 9S/23166 _ ~3' ~ ` PCT/AU95/00090 (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE:; ntf~rn;31 (vi) ORIGINAL SOURCE:
(A) ORGANISM: Hepatitis B virus (Xi) ~ U~:N(:~; DESCRIPTION: SEQ ID NO:17:
Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr ~eu Gln (2) INFORMATION FOR SEQ ID NO:18:
10 (i) ~ ,lU~N~ R~t~T~RT~sTIcs:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: peptide ( i i i ) HYPOTHETI CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOUROE:
2 0 (A) ORGANISM: Hepatitis B virus .

2 1 8 3 ~7 ~
Wo 95/23166 ~ ~ - PCT/AU95100090 (Xi) ~:UUI~N~:~; DESCRIPTION: SEQ ID NO:18:
Ser l.eu Asn Phe ~eu Gly Gly Thr Thr Val Cys ~.eu Gly 5 ~ 10 Gln Asn ~2) INFORMATION FOR SEQ ID NO:19:
( i ) SEQUENCE CHARACTERISTICS:
(A) 3.ENGTH: 15 amino acids (B) TYPE: amino acid (C) sTR~Nn~nN~c5 gingle (D) TOPOLOGY: linear ( ii ) MO~ECULE TYPE: peptide ( iii ) HYPOTHETI CAL: NO
(iv) ANTI-SENSE: NO
15 (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOTJRCE: =
(A) ORGANISM: Hepatitis B virus (Xi) ~ !U~;N(:~ DESCRIPTION: SEQ ID NO:19:
~eu Val Leu Leu Asp Tyr Gln Gly Met ~eu Pro Val Cys Pro I.eu (2) INFORMATION FOR SEQ ID NO:20:
2ul~N~ R1~'TT:RT~5TIcs:
25 (A) ~ENGTH: 15 amino acids (B) TYPE: amino acid 2 1 8 3 9 i 7 L . ~ pCT/AlJ95100090 WO 9~/23166 _ 35 (C) s~RANn~nN~.cs single ( D ) TOPOLOGY: 1 inear ( ii ) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (vi ) ORIGINAL SOURCE:
(~) ORGANISM: Hepatitis B virus (Xi) ~;UU~;N~-'~ DESCRIPTION: SEQ ID NO:20:
Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser (2) INFORMATION FOR SEQ ID NO:21:
(i) ~ UU~;N(~:~; CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid ( C ) ST~ ANn~nNE .~ S: s ingle ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: peptide ( i i i ) HYPOTHBTI CAL: NO
(iv) ANTI-SBNSE: NO
(v) FRAGMENT TYPE: internal Wo 9S/23166 ~1~3 3 ~ ~ 7 ; PCT/AU9S/OOO90 (vi) ORIGINAL SOURCE:
(A) ORGANISM: Foot and mouth diseaee virus (Xi) ~i~.2U~;N~:~; DESCRIPTION: SEQ ID ~0:21:
Val Pro Asn Leu Arg Gly Asp Leu Gln Val Leu Ala Gln l 5 10 Lys Val Ala Arg Thr Leu Pro (2) INFORMATION FOR SEQ ID NO:22:
(i) Y~;~U~;N~ Rz~(~T~RT.c~TICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid ( C ) STR ~n~nNE~ : 8 ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) XYPOTXETICAL: NO
(iv) ANTI-SENSE: NO
( v ) FRAGMENT TYPE: ; n ~ P rn ~1 (vi) ORIGINAL SOUROE:
(A) ORGANISM: Ra~ies virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Asp Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met t ~ f~ n (2) INFORMATION FOR SEQ ID NO:23:
;UU~;N:.:~ CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANn~nN~.q.q: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: ~O
(iv) ANTI-SENSE: NO
l0 (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Plasmodium ~alciparum (Xi) ~i~UU~;N~:~; DESCRIPTION: SEQ ID NO:23:
Asn Ala Asn Pro

Claims

- 38 -

1. A synthetic peptide T cell epitope analogue of a native T cell epitope which analogue is partially or completely inverso modified with respect to the native T
cell epitope.

2. A synthetic peptide T cell epitope analogue of a native T cell epitope which analogue is partially or completely retro-inverso modified with respect to the native T cell epitope.

3. A synthetic peptide T cell epitope analogue acoording to claim 1 or claim 2 wherein the native T cell epitope is selected from the group consisting of:
H-Gln-Val-Val-His-Asn-Ser-Tyr-Asn-Arg-Pro-Ala-Tyr-Ser-Pro-Gly-OH, from diphtheria toxin (SEQ ID NO: 1);
H-His-Arg-Met-Gln-Glu-Ala-Val-Glu-Ala-Glu-Arg-Ala-Gly-Arg-OH, from pertussis toxin (SEQ ID NO: 2);
H-Pro-Ser-Asp-Lys-His-Ile-Glu-Gln-Tyr-Leu-Lys-Lys-Ile-Lys-Asn-Ser-Ile-Ser-OH, from malaria CSA protein (SEQ ID
NO: 3);
H-His-Ile-Glu-Gln-Tyr-Leu-Lys-Lys-Ile-Lys-Asn-Ser-Ile-Ser-OH, from malaria CSB protein (SEQ ID NO: 4);
H-Gly-Asp-Ile-Glu-Lys-Lys-Ile-Ala-Lys-Met-Glu-Lys-Ala-Ser-Ser-Val-Phe-Asn-Val-Val-Asn-Ser-OH, from malaria CST3 protein (SEQ ID NO: 5);
H-Cys-Ser-Ala-Leu-Leu-Ser-Ser-Asp-Ile-Thr-Ala-Ser-Val-Asn-Cys-Ala-OH, from hen egg lysozyme (SEQ ID NO: 6) ;
H-Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-OH (SEQ ID nO: 7) and H-Tyr-Thr-Tyr-Thr-Val-His-Ala-Ala-His-Ala-Tyr-Thr-Tyr-Thr-OH (SEQ ID NO: 8), from ovalbumin;
MVF:258-277 H-Gly-Ile-Leu-Glu-Ser-Arg-Gly-Ile-Lys-Ala-Arg-Ile-Thr-His-Val-Asp-Thr-Glu-Ser-Tyr-OH
(SEQ ID NO: 9) and MVF:288-302 H-Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-His-Arg-Leu-Glu-Gly-Val-OH (SEQ ID NO: 10), from measles virus F and H glycoproteins ;

RS1A:45-60 H-Cys-Glu-Tyr-Asn-Val-Phe-His-Asn-Lys-Thr-Phe-Glu-Leu-Pro-Arg-Ala-OH (SEQ ID NO: 11), from respiratory syncytial virus 1A protein;
Influenza hamagglutinin A/PR/8/34 Mt.S.: residues 109-119 (SEQ ID NO: 12), 130-140 (SEQ ID NO: 13), and 302-313 (SEQ ID NO: 14);
residues (A)4-14 (SEQ ID NO: 15) and (B)5-16 (SEQ ID NO:
16) from pork insulin;
Hepatitis B virus pre S residues 120-132 (SEQ ID NO: 17);
Hepatitis B virus major surface antigen: residues 38-52 (SEQ ID NO: 18), 95-109 (SEQ ID NO: 19), and 140-154 (SEQ
ID NO: 20);
Foot and mouth virus VP1: residues 141-160 (SEQ ID NO:
21); and Rabies virus-spike glycoprotein precursor: residues 32-44 (SEQ ID NO: 22).

4. A T cell epitope analogue according to claim 2 or claim 3 wherein the amino acid residues flanking the retro-inverted sequence are substituted by side-chain-analogous .alpha.-substituted geminal-diaminomethanes and malonates.

5. A vaccine comprising a T cell epitope analogue according to any one of claims 1 to 4 together with a B cell epitope and a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

6. A vaccine according to claim 5 wherein the T
cell epitope analogue is conjugated to the B cell epitope.

7. A vaccine according to claim 5 which is a cocktail of T cell epitope analogues and B cell epitopes tailored to the condition against which vaccination is required.

8. A vaccine according to claim 5 wherein the B
cell epitope is a peptide or polypeptide of any length whose amino acid sequences stem from:
polypeptides of a pathogen including poliomyelitis, hepatitis B, foot and mouth disease of livestock, tetanus, pertussis, HIV, cholera, malaria, influenza, rabies or diphtheria causing agents;
a toxin including robustoxin, heat labile toxin of pathogenic Escherichia coli strains and Shiga toxin from Shigella dysenteriae;
Amyloid .beta. protein;
human chorionic gonadotropin;
or gonadotropin releasing hormone.

9. A vaccine according to claim 5 wherein the B cell epitope is a retro, retro-inverso or inverso antigen analogue.

10. A method of vaccinating a host in need of such treatment which method comprises administering an effective amount of a vaccine according to claim 5 to the host.

11. A method of preparing a T cell epitope analogue according to claim 1 or 2, the method comprising synthesising a partially or completely inverso or retro-inverso analogue of the native T cell epitope.

12. Antibodies produced by immunisation of a host with a vaccine according to claim 5.

13. A method of preparing a vaccine according to claim 5 which method comprises: conjugating a T cell epitope analogue according to claim 1 or claim 2 to a B
cell epitope, or admixing a T cell epitope analogue according to claim 1 or claim 2 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.