Note: Descriptions are shown in the official language in which they were submitted.
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
2183977
~Wo 95/23166 PCT/AU95/00090
-- 3
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
_
2183977
Wo 95/23166 PCr/AU95/OOo90
-- 4
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--
-- 6
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 --
-- 8
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 --
- 10 -
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
-- 11 -
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
2183
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
- 13 -
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
-- 14 --
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
- 15 --
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
-- 16 -
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
-- 17 -
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.
- Atherton. E., Cameron, L.R. & Sheppard, R.C. (1988)
Peptide synthe3is. Part 10. U3e of pentafluorophenyl
e3ters of f luorenylmethoxycarbonylamino acid3 in 301id
phase peptide synthesi3. Tetrahedron, 44, 843-857.
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.
Peptide Protein Res., 24, 553-556.
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.
(1986) Peptide synthesis. Part 9. Solid-pha3e
3ynthe3i3 of melanin cnncPntrating hormone u3ing a
~ nnt;nllnu3-flow polyamide mechod. J. Chem. Soc. Perkin
Trans I, 3 61- 3 67 .
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 ~--
-- 18 --
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.
M. Jolivet, L. I~ise, ~. Gras-Masse, A. Tartar, P.
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.
F. Sinigaglia, M. Guttinger, ~. Kilguæ, D.M. Doran, H.
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
- 19 -
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.
Miller & J.A. Berzofsky (1988) Parasite polymorphism
present within minimal T cell epitopes of Pla6modium
falciparum circumsporozoite protein . J. Immunol ., 140 ,
1645 -1650 .
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 .
K.J. Kim, S. McKinness & C.R. Manclark (1990)
Determination of T cell epitopes on the S1 subunit of
pertussis toxin. J. Immunol., 144, 3529-3534.
A. Sette, A. Lamont, S. Buus, S.M. Colon, C. Miles & H.M.
Grey (1989) Effect of conformational propensity of
peptide antigens in their interaction with HMC class II
molecules. Failure to document the importance of regular
secondary structure. J. Immunol., 143, 1268-1273.
M.J. Francis, C.M. Fry, D.J. Rowlands, F. Brown, J.L.
Bittle, R. Houghten h R.A. Lerner (1985) Immunological
priming with synthetic peptides of foot and mouth disease
virus. J. Gen. Virol. 66, 2347.
C.J. Hackett, B. Dietzschold, W. Herhard, B. Ghrist, R.
Knorr, D. Gillessen & F. Melchers (1983) Tnflll~n7~ virus
site recognized by a murine helper T cell specific for H1
strains. J. Exp. Med. 158, 294.
J.L. Hurwitz, E. Heber-Katz, C.J. Hackett & W.J. Gerhard
(1984) Characterization of the murine TH response to
influenza virus hemaglutinin: evidence for three major
specificities. Immunol 133, 3371.
J.R. Lamb, D.D. Eckels, P. Lake, J.N. Woody & N. Green
(1982) Human T cell clones recognize chemically
8ynth~;7~1 peptides o_ inflll~n7~ hemagglutinin. Nature
300, 66.
WO 95123166 ~ pcrlA~95looo9o --
- 20 -
R.I. Macfarlan, B. Dietzschold, T.J. Wiktor, M. Kiel, R.
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.
D.R. Milich, G.B. Thornton, A. ~T.A~hlAn, M.K. McNamara &
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.
62, 4465-4473.
20 C.D. Partidos, C.M. Stanley & M.W. Steward (1991) Immune
responses in mice following immunization with chimeric
synthetic peptides repr~ nt; n~ B and T cell epitopes of
measles virus proteins. J. gen. Vir. 72, 1293-1299.
A.S. Rr~s~nth~l (1978) Determinant selectio~ and
25 macrophage function in genetic control of the immune
response . Inui1unol . Rev. 4 0, 14 6 .
J.W. Thomas, W. Danho, E. Bullesbach, J. Fohles & A.S.
Rosenthal (1981) Immune response gene control of
determinant selection. III.: Polypeptide f,dyl -tc of
30 insulin are differentially recognized by T but not by B
cells in insulin immune guinea pigs. J. Ilrununol. 126-,
1095.
~WO 951~166 2 1 8 3~9 7~7 r PCT/AU95100090
- 21 --
~ ~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
-- 22 --
(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
WO 95/23166 PCTIAU95/00090
-- 23 --
(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
- 25 --
(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
- 26 --
(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
2183977
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
1 0
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 --
- 28 -
(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
15 20
(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
20 1 5 10
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
- 29 --
(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
5 10
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
20 1 5 10
( 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
-- 31 -
(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
1 5 10
(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
-- 34 --
(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
20 1 5 10
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
15 20
(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
2183977
t ~ f~ n
WO 95/23166 PCT1AU9510009
-- 37 -
(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
