Note: Descriptions are shown in the official language in which they were submitted.
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CHIMERIC GENE FORMED OF THE DNA SEQUENCES THAT ENCODE THE
ANTIGENIC DETERMINANTS OF FOUR PROTEINS OF L. INFANTUM
AND PROTEIN ENCODED BY SAID GENE, AND PHARMACUETICAL
COMPOSITION USEFUL FOR PREVENTING AND/OR TREATING
LEISHMANIASIS, IN ANIMALS OR HUMANS.
DESCRIPTION
OBJECT OF THE INVENTION
The present specification relates to an application
for an Invention Patent, regarding a chimeric gene formed
of the DNA sequences that encode the antigenic
determinants of four proteins of L. infantum, and to
proteins encoded by said chimeric gene, useful for the
prevention or treatment of Leishmaniasis, in particular
canine Leishmaniasis. The obvious purpose of this lies
in using the gene sequence or the protein obtained from
the chimeric gene for providing pharmaceutical
compositions for preventing or treating Leishmaniasis, in
particular canine Leishmaniasis, that can be present in
the body of a patient, for instance as a vaccine or a
monoclonal antibody preparation. This patient does not
have to be a dog but can also be a human being who
suffers from diseases that involve immuno-depression. T o
achieve this, a chimeric gene will be produced that
encodes a protein called MSPQ consisting of a chimeric
product originating from an "in vitro" synthesis of a
chimeric gene constructed "ad hoc", which contains five
of the antigenic determinants of four different proteins.
The product is configured as highly sensitive and
specific for -for instance - generating a protective
immune responds against canine Leishmaniasis, or for
preparing antibodies against canine Leishmaniasis
FIELD OF THE INVENTION
This invention is of utility within the industry
dedicated to the manufacture of pharmaceutical products
in general.
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BACKGROUND OF THE INVENTION
The parasitic protozoa of the Leishmania genus are
the aetiological agents that cause Leishmaniasis, a range
of diseases that have a world-wide distribution and that
are characterised in that they give rise to a wide
variety of clinical symptoms.
The main forms of Leishmaniasis are zoonotic in
nature and humans are considered as secondary hosts.
The species denoted L. Infantum, widely distributed
throughout many Mediterranean areas is the cause of
visceral Leishmaniasis (LV) in humans and dogs.
In fact, dogs infected with L. infantum are the
main animal reserve of this parasite, particularly during
the long incubation period before the clinical symptoms
can be observed.
The epidemiological data indicate that there is a
direct correlation between the prevalence of canine
Leishmaniasis and the transmission of the parasite to
humans. Fo/ this reason, it is crucial to detect the
disease or infection early on in campaigns undertaken to
control the spread of the disease.
The parasite is transmitted to the host vertebrate
as a flagellate promastigote, by means of a bite of a fly
of the family "Phlebotominae", and the parasite enter the
cells of the mononuclear phages where they differentiate
and reproduce as amastigotes, within the phago-lisosomal
structure.
The infected cells gather in certain tissues,
mainly spleen, liver and lymph nodes. It is estimated
that around 15 million people are infected with
Leishmaniasis, and every year in the world 500,000 new
clinical cases appear in the world, mainly in the
underdeveloped and developing world.
In the south-western countries of Europe, Visceral
Leishmaniasis (VL), is a zoonotic disease caused by the
L. Infantum species, as was mentioned earlier. Recent
data derived from epidemiological studies indicate that
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there is an alarming incidence of this infection.
In Italy the reported data for incidence of VL
ranges from 14.4% to 37% according to the region.
In Portugal, more particularly in the area around
Lisbon, seropositive rates of 8.4% have been found and in
the region of the French Maritime Alps different centres
of prevalence have been found that vary between 3.2% and
17.1%.
In Spain, the prevalence of Leishmaniasis depends
on the zone being studied. In Catalonia an average
incidence rate of 9.3% has been observed although in some
hot-spots a prevalence of infected dogs of up to 18% has
been found.
On the Island of Mallorca, the incidence rate is
14%, and other rates that have been found are: 2.4% in
Murcia, 8.8% in Granada, from 10 to 15% in Salamanca,
5.25% in the province of Madrid, and 14% in Caceres.
Although the number of cases of VL in humans caused
by L. infantum can be considered relatively low, the high
percentage of patients with immuno-depression that become
infected by Leishmania could be related to the high level
of this illness in dogs.
In fact, in the South of Europe, 50% of adults that
are infected by Leishmaniasis are also patients infected
by the HIV virus. On the other hand, according to these
data of Leishmania-HIV co-infection, it has been
estimated that the level of infection (by parasites) can
be one or two orders of magnitude higher than this figure
due to the existence of a large number of undetected
infections.
A common characteristic of the different types of
Leishmania infection is that it induces a strong humoral
response in the host. Therefore, diagnostic methods based
on serological techniques are currently the most widely
used.
It has been described that these antibodies are
detected even during the asymptomatic phase of the
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disease in natural and experimental infections.
The sensitivity and specificity of these methods
depends on the type, source and purity of the antigen
used. In
immunological processes that are currently
commercialised, complete promastigotes and preparations
more or less prepared from these are used as a source of
antigen. This method normally leads to cross-reactions
with serum from patients suffering from leprosy,
tuberculosis, African tripanosomiasis, Chagas disease,
malaria and other parasitosis.
The sensitivity and specificity of the serologic
methods depend on the type, source and purity of the
employed antigen. During the last years a great number of
Leishmania antigens have been characterised, some of them
can be considered as proteins specific to the parasite.
Among these proteins specific to the parasite, the
surface protease GP63, the surface glycoprotein gp46 and
the lipophosphoglicane associated KMP-11 protein deserve
a mention.
An additional group of Leishmania antigens are
formed of evolutionarily conserved proteins, such as
kinesine, thermal shock proteins, actin and tubulin.
As part of a strategy to develop a specific
serological diagnostic system for Leishmaniasis canine, a
laboratory based project has been undertaken to identify
the antigens of L. infantum, by means of a immuno-
detection search of an expression library for genes of L.
infantum using dog serum with active visceral
Leishmaniasis.
It has been observed that most of the antigens
isolated by this method belong to the family of proteins
conserved during the course of evolution. The
identification of the B epitopes of these antigens
indicate, however, that in all cases the antigenic
determinants were localised in regions that were not well
conserved.
In particular, the acidic ribosomal proteins LiP2a
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and LiP2b are recognised by more than 80% of the VL
serums.
It has been confirmed that these proteins contain
disease specific antigenic determinants, and that the
5 recombinant proteins LiP2a and LiP2b, from which a
fragment had been removed, could be used as a specific
instrument able to distinguish between VL and Chages
disease.
It has also been shown that the PO ribosomal
protein of L. infantum, very highly conserved on the
evolutionary scale, is recognised by a high percentage of
VL dog serums. Furthermore, the antigenic determinants
are found exclusively on the C-terminus of the protein,
that is to say, in the region that has been poorly
conserved during the course of evolution.
It has been observed that in 78% of the VL dog
serums, antigens against H2A protein are also present,
and it has been confirmed that despite the sequence
identity in all the H2A proteins among eukaryotic
organisms, the humoral response to this protein in VL
serums is particularly provoked by determinants specific
to the Leishmania protein H2A.
The antigenic determinants recognised by the VL dog
serums are found at both termini of the H2A protein.
The obvious solution to the problem currently
encountered in this art would be to have an invention
that would allow the assembly of a synthetic chimeric
gene that contained the DNA regions encoding the
antigenic determinants specific to the proteins LiP2a,
LiP2b, LiP0, and H2A, with a view to constructing a
protein rich in antigenic determinants.
However, as far as the applicant is aware, there is
currently no invention that contains the characteristics
described as ideal, with a view to reaching the desired
aim. This aim is the construction of a protein rich in
antigenic determinants, arising from the assembly of a
chimeric synthetic gene, that contains the DNA regions
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encoding the antigenic determinants specific to the
aforementioned proteins.
DESCRIPTION OF THE INVENTION
In a first aspect, the invention relates to a
chimeric gene formed by the DNA sequences that encode
antigenic determinants of four proteins of L.infantum,
useful for preventing or treating canine Leishmaniasis
In a further aspect, the invention relates to a
protein encoded by said chimeric gene, containing one or
more of the antigenic determinants of four proteins of
L.infantum encoded by the chimeric gene.
The invention further relates to method for
preventing and/or treating canine Leishmaniasis in a
human being or an animal. In this therapeutic method, the
chimeric gene of the invention or the protein encoded by
it can be used. Also, antibodies against the protein
encoded by the chimeric gene of the invention, or a
antigenic part thereof such as an epitope, can be used.
In further aspects, the invention relates to
pharmaceutical compositions for the prevention and/oi
treatment, in humans and/or animals, of Leishmaniasis
comprising an active substance derived from or directed
against the chimeric gene of the invention and/or the
protein encoded by it, or parts thereof. The active
substance is preferably such that it can be used in a
pharmaceutical composition for the treatment and/or
prevention of Leishmaniasis.
In particular, the pharmaceutical composition will
be in a form of a vaccin, containing the protein encoded
by the chimeric gene of the invention, or one or more
parts thereof, containing one or more of the antigenic
determinants of the protein encoded by the chimeric gene
of the invention.
In a further embodiment, the pharmaceutical
composition of the invention comprises antibodies
directed to the protein encoded by the chimeric gene of
the invention, or parts thereof.
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The pharmaceutical preparations of the invention
may further contain all known adjuvants, solvents,
buffers etc. known per se for pharmaceutical compositions
and/or vaccines.
In a further aspect, the invention relates to a
method for the treatment or prevention of Leishmaniasis
using a pharmaceutical composition or a vaccin according
to the invention, or a preparation comprising antibodies
directed against the protein encoded by the chimeric gene
of the invention.
This method will generally comprise administering
an active substance directed against the chimeric gene or
the protein to a human being or animal, such as a dog, in
a pharmaceutically active amount.
For the prevention of Leishmaniasis, a vaccin
comprising the protein encoded by the chimeric gene, or
encoding one or more parts of said protein comprising one
or more of the antigenic determinants, will be
adminstered to a human being to elicit a protective
immune response.
Administration of the preparations, antibodies
and/or vaccins of the invention may be carried out in a
manner known per se, such as orally, intramuscularly,
intravenously, subcutaneously, by (drip)infusion etc..
Preferably, the preparation or a vaccin is injected,
whereas with an antibody preparation, an infusion can be
used.
It should be noted that when herein, reference is
made to the chimeric gene of the invention, this term
also encompasses nucleic acid sequences that can
hybridize with the sequence mentioned below under
moderate or stringent hybridizing conditions.
In this context, heterologous hybridisation
conditions can be as follows: hybridisation in 6 x SSC
(20xSSC per 1000 ml : 175.3 g NaC1, 107.1 g sodium
citrate . 5H20, pH 7.0), 0.1% SDS, 0.05%
sodium
pyrophosphate, 5* Denhardt's solution (100 x Denhardt's
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solution per 500 ml : 10 g Ficoll"-400, 10 g polyvinyl-
pyrrolidone, 10 g Bovine Serum Albumin (Pentax Fraction
V)) and 20 1g/m1 denatured herring sperm DNA at 56 C for
18-24 hrs followed by two 30 min. washes in 5 x SSC, 0.1
% SDS at 56 C and two 30 min. washes in 2 x SSC, 0.1% SDS
at 56 C.
For instance, sequences that can hybridize with the
sequence mentioned below include mutant DNA sequences
which encode proteins with the same biological function
as the protein encoded by the sequence mentioned
hereinbelow. Such mutant sequences can comprise one or
more nucleotide deletions, substitutions and/or additions
to the sequence mentioned below. Preferably, the mutant
sequences still have at least 50%, more preferably at
least 70%, even more preferably more than 90 % nucleotide
homology with the sequence given hereinbelow.
The term chimeric gene as used herein also
encompasses nucleic acid sequences that comprise one or
more parts of the sequence mentioned hereinbelow.
Preferably, such sequences comprise at least 10%, more
preferably at least 30%, more preferably at least 50% of
the nucleotide sequence given hereinbelow. Such sequences
may comprise a contiguous fragment of the sequence
mentioned hereinbelow, or two or more fragments of the
sequence given below that have been combined in and/or
incorporated into a single DNA sequence.
It should be noted that when herein, reference is
made to a protein encoded by the chimeric gene ofthe
invention, this term also includes mutant proteins that
still essentially have the same biological function.
Such mutant proteins can comprise one or more amimo acid
deletions, substitutions and/or additions compared to the
protein encoded by the sequence mentioned below.
Preferably, the mutant proteins still have at least 50%,
more preferably at least 70%, even more preferably more
than 90 % amino acid homology with the sequence given
hereinbelow.
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The term protein also encompasses fragments of the
protein encoded by the chimeric gene of the invention.
Such fragments preferably still show the biological
activity of the full protein. Preferably, such proteins
comprise at least 30%, more preferably at least 50% of
the amino acid sequence of the full protein. Also, two or
more fragments of the full protein encoded by the
chimeric gene of the invention may be combined to form a
single protein.
More specifically, the invention relates to a
chimeric gene formed by the DNA sequences that encode
antigenic determinants of four proteins of L. infantum,
encoding a protein useful for pharmacological purposes,
in particular for the prevention and/or treatment of
Leishmaniasis, in particular canine Leishmaniasis, and
obtaining the final product or the construction of the
chimeric gene that encodes a polypeptide that contains
all the selected antigenic determinants, characterised in
that it uses a cloning strategy in which the clone that
expresses the protein rLiPO-Ct-Q is used as an initial
vector, and to this vector, by means of the use of
suitable restriction sites, fragments Of DNA are
sequentially added that encode the proteins LiP2a-Q,
LiP2b-Q, LiH2A-Ct-Q, LiH2A-Nt-Q, and after each step of
cloning the correct orientation of each one of the
inserts is deduced and the size of the expression
products, the complete sequence of nucleotides of the
final clone pPQV finally being determined and the deduced
sequence of amino acids is
MBP IEGRPLTPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGYARRIGA 50
SGAPRISEFSVKAAAQSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVV
140
PNISKAMAKKKGGKKGKATPSAPEFGDSSRPMSTKYLAAYALASLSKASPSQAD
157
, 35 VEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGV
211
TASAAGDAAPAAAAAKKDEPEEEADDDMGPSVRDPMQYLAAYALVALSGKTPSK
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265
STAGAGAGAVAEAKKEEPEEEEADDDMGPVDLOPAAAAPAAPSAAAKAAPEESD
374
EDDFGMGGLF (SEQ ID NO: 3)
5 Said
chimeric gene preferably encodes a polypeptide
generated with a molecular weight of 38 kD and an
isoelectric point of 7.37.
The invention als relates to a pharmaceutical
composition for the prevention and treatment, in humans
10 or animals, of Leishmaniasis formed
a- by the protein Chimera Q (SEQ ID NO:1), or a variant of this
protein which contains modifications or substitutions of
conserved amino acids, administered to a subject (human
or animal), either
b- in isolated form or together with any physiological
adjuvant via the intraperitoneal, subcutaneous or
intramuscular routes.
Also, the invention relates to a vaccine capable of
stimulating the production of antibodies which recognise
the Leishmania parasite, formed
a- by the protein Chimera Q or a variant of this
protein which differs from protein Q in conserved amino
acids administered to a subject (human or animal), either
b- in isolated form or together with any physiological
adjuvant via the intraperitoneal, subcutaneous or
intramuscular routes.
Another aspect of the invention comprises a
pharmaceutical composition for the prevention and
treatment, in humans or animals, of Leishmaniasis formed
=
a- by protein Q, or a variant of this protein which
contains, modifications or substitutions of conserved
amino acids, bound to protein LiHsp70, complete or
fragmented, administered to a subject (human or animal),
either
b- in isolated form or together with any physiological
adjuvant via the intraperitoneal, subvutaneous or
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intramuscular routes.
A further pharmaceutical composition of tpe
invention for the prevention and treatment, in humans ,or
animals, of Leishmaniasis can be formed:
a- by any DNA
vector carrying the sequence which
encodes the protein Chimera Q (SEQ ID NO:2), or a variant of this
sequence which contains modifications or substitutions of
nucleotides which code for conserved amino acids,'
administered to a subject (human or animal), either
b- by the intramuscular or subcutaneous routes.
In yet another aspect, the invention relates to a
pharmaceutical composition for the prevention and
treatment, in humans or animals, of Leishmaniasis formed
a- by any DNA vector carrying 1- the sequence which
encodes the protein Chimera Q, or a variant of this
sequence which contains modifications or substitutions of
nucleotides which code for conserved amino acids, and 2 -
the sequence which encodes the protein LiHsp70, or
variants of the same which differ in conserved amino
acids, administered to a subject (human or animal),
either
b- by the intramuscular, subcutaneous or intramuscular
route.
The invention also relates to a protein useful for
pharmacological purposes, in particular for the
prevention and/or treatment of Leishmaniasis, in
particular canine Leishmaniasis, having the amino acid
sequence:
MBP IEGRPLTPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGYARRIGA 50
SGAPRISEFSVKAAAQSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVV
140
PNISKAMAKKKGGKKGKATPSAPEFGDSSRPMSTKYLAAYALASLSKASPSQAD
157
VEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGV
211
TASAAGDAAPAAAAAKKDEPEEEADDDMGPSVRDPMQYLAAYALVALSGKTPSK
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265
STAGAGAGAVAEAKKEEPEEEEADDDMGPVDLOPAAAAPAAPSAAAKAAPEESD
374
EDDFGMGGLF
or a mutant or fragment thereof that can be used for
generating a protective immune response in a human or
animal against Leishmaniasis, and to a pharmaceutical
composition for the prevention and treatment, in humans
or animals, of Leishmaniasis, comprising this protein or
a mutant or fragment thereof that can be used for
generating a protective immune response in a human or
animal against Leishmaniasis.
Also, the invention relates to a vaccine capable of
stimulating the production of antibodies which recognise
the Leishmania parasite, comprising the protein mentioned
above or a mutant or fragment thereof that can be used
for generating a protective immune response in a human or
animal against Leismaniasis.
A further aspect of the invention encompasses a
pharmaceutical composition for the prevention and
treatment, in humans or animals, of Leishmaniasis,
comprising antibodies directed against the protein
mentioned above or a mutant or fragment thereof,
preferably containing one or more antigenic determinants
such as an epitope.
The invention further relates to a method for the
prevention or treatment of Leishmaniasis in a human or
animal, comprising administering to the human or animal a
pharmaceutical composition as described above, or to a
method for preventing Leishmaniasis in a human or animal,
comprising administering to the human or animal a vaccine
as described above.
The chimeric gene formed of the DNA sequences that
encode the antigenic determinants of four proteins of L.
infantum and protein obtained, useful for preventing
and/or treating Leishmaniasis, that the invention
proposes, in its own right constitutes an obvious novelty
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within its field of application, as according to the
invention, a synthetic chimeric gene is produced that as
it is obtained by assembly, containing the DNA region
encoding the antigenic determinants specific to the
proteins LiP2a, LiP2b, LiP0 and H2A, thus constructing a
protein rich in antigenic determinants. The chimeric gene
obtained is expressed in Escherichia coli and the product
has been analysed with respect to its antigenic
properties. The results confirm that this chimeric
protein maintains all the antigenic determinants of the
parent proteins and that it constitutes a relevant
pharmaceutically useful element for canine VL, with a
sensibility that oscillates between 80% to 93%, and a
specificity of between 96% to 100%.
More particularly, the chimeric gene formed by the
DNA sequences that encode the antigenic determinants of
four proteins of L. infantum and the protein encoded by
at, useful for the prevention and/or treatment of canine
Leishmaniasis and protein obtained object of the
invention, is produced by means of the following stages,
namely:
Construction of the chimeric gene. Methodology.
Cloning strategy.
Cloning of DNA sequences that encode antigenic
determinants of the histone protein H2A.
Cloning of the sequences that encode rLiP2a-Q and
rLiP2b-Q.
Cloning of the sequence rLiP0-Q.
Cloning of the chimeric gene.
- Construction of the chimeric gene from the
construction of intermediate products.
Cloning of epitopes specific to the L. infantum
antigens.
- Construction of the final product
Construction of the chimeric gene that encodes a
polypeptide that contains all the selected antigenic
determinants.
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- optionally expression of the sequence thus obtained.
- Evaluation of the final product.
Serums.
Purification of proteins
Electrophoresis of proteins and immuno-analysis.
Measurements by Fast-ELISA
- Evaluation of the final product.
Antigenic properties.
Sensitivity and specificity of the chimeric protein
CP in the serum diagnosis of canine VL.
The strategy followed by the cloning of DNA
sequences that encode each one of the selected antigenic
determinants is the same in all cases, and in a first
step, the sequence of interest is amplified by means of a
PCR and the use of specific oligonucleotides that contain
targets for restriction enzymes at the extremes.
For the cloning step, the amplified product is
directed by means of the appropriate restriction enzyme
and it is inserted in the corresponding restriction site
of the plasmid pUC18.
After sequencing the DNA, the insert is recovered
and sub-cloned to the corresponding restriction site of
the modified plasmid denominated pMAL-c2. The
modification is made by inserting a termination codon
downstream of the target HindIII in the polylinker of
pMal-c2, denominating the resulting plasmid pMAL-c2'.
Regarding the cloning of the DNA sequence that
encodes the antigenic determinants of the histone protein
H2A, it should be pointed out that the cDNA of the clone
cL71, that encodes the histone H2A of L. infantum, is
used as a template for the PCR reactions, and for the DNA
amplification, that encodes the N-terminal region of the
histone H2A, more exactly rLiH2A-Nt-Q, the following
oligonucleotides are used: sense 5'
-
= CCTTIAGCTACTCCTCGCAGCGCCAAG-3' (SEQIIDNK):4) (position 84-104 of the
sequence cL71); antisense 5'CCTGGGGGCGCCAGAGGCACCGATGCG-
3' (SEQ ID NO:5) (inverse and complimentary to position 204-224 of the
CA 02256124 2000-07-24
sequence cL71) .
The sequences that are included in the
oligonucleotides for the cloning and that are not present
in the parent sequence cL-71 are marked in boldface type.
5 The amplified DNA fragment is cloned directly from
the restriction site XmnI of pMAI-c2*.
The fragment is sequenced by means of the initiator
#1234 malE and the antigenic C-terminal region of histone
H2A, in particular rLiH2A-Ct-Q, is amplified with the
10 following oligonucleotides. These are:
Sense, 5'-GAATTCTCCGTAAGGCGGCCGCGCAG-3' (SEQ ID NO: 6) (position
276-296 of the sequence cL71).
Antisense, 5'-GAATTCGGGCGCGCTCGGTGTCGCCTTGCC (SEQ ID NO:?)
(inverse and complimentary to the positions 456-476 of
15 the plasmid cL71).
A triplet that encodes proline (indicated as GGG
after the underlined letters) is included in the anti-
sense oligonucleotide, the restriction site EccRI that is
included in both oligonucleotides for cloning is
indicated by underlining.
Regarding the cloning of the sequences that encode
rLiP2a-Q, it should be pointed out that the regions of
interest are amplified by PCR from cDNAs encoding LiP2a
and LiP2b.
The oligonucleotides that are used for constructing
the expression clone LiP2a-Q, are the following.
Sense, 5'-GTCGACCCCATGCAGTACCTCGCCGCGTAC-3' (SEQ ID NO:8).
Anti-sense, GTCGACGGGGCCCATGTCATCATCGGCCTC-3' (SEQ ID NO: 9).
it should be pointed out that the Sail restriction
sites added to the 5' extremes of the oligonucleotides
have been underlined.
When constructing the expression clone LiP2b-Q, the
oligonucleotides used were:
Sense, 5'-TCTAGACCCGCCATGTCGTCGTCTTCCTCGCC-3' (SEQ ID NO: 10).
Anti-sense, TCTAGAGGGGCCATGTCGTCGTCGGCCTC-3' (SEQ ID NO: 11).
At the 5' extremes of the oligonucleotides the
restrictions sites are included for the enzyme XbaI
_
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16
(underlined), and due to the cloning needs, an additional
triplet, encoding a proline residue, is included
downstream of the restriction site.
Regarding the cloning of the sequence rLiP0-Q, it
should be pointed out that the cloning of the DNA
sequence of the C-terminal region of the protein PO of L.
infantum is carried out by amplifying a clone of cDNA
called L27 and the following oligonucleotides:
Sense, 5'-IODGCAGCCCGCCGCTGCCGCGCCGGCCGCC-3' (SEQIIIDNO:11)
(positions 1-24 of the L27 cDNA) and the initiator of the
pUC18 sequence (#1211), the amplified DNA is directed by
the enzymes PstI+HindIII, with later insertion into the
plasmid pMAL-c2.
The resulting clone is denominated pPQI and it
should be noted that the restriction site PstI is
included in the nucleotide with sense (underlined
sequence) and that the restriction target HindIII is
present in the cDNA L27.
Regarding the cloning of the chimeric gene, it
should be pointed out that the DNA sequences that encode
the five antigenic determinants are assembled into a
chimeric gene, and this assembly is carried out on the
clone pPQI, to which the codifying regions for the
antigenic regions LiP2a-Q are added sequentially in the
3' direction (naming the results of cloning pPQ2), LiP2b-
Q (clone pPQ3), LiH2a-Ct-Q (clone pPQ4) and LiH2A-Nt-Q
(clone pPQ5).
Finally, the insert obtained after the SacI+HindIII
digestion of the final clone pPQ5 is inserted into the
pQE31 expression plasmid, naming the resulting clone pPQ.
DESCRIPTION OF THE DRAWINGS
To complete the description that is being made and
with the aim of aiding the understanding of the
characteristics of the invention, the present disclosure
is accompanied, as an integral part thereof, by a set of
plans of illustrative nature that are not limiting. The
following is represented:
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Figure number 1.
Corresponds to a graphic
representation of each one of the recombinant proteins
fused to the maltose binding protein. These will be
purified by affinity chromatography on an amylose column
as is represented in Figure number 4.
Figure number 2. Sample of the different vectors
considered to obtain the chimeric gene object of the
invention, from which the pertinent protein destined to
carry out an accurate diagnostic on animals or human
beings that show symptoms of Leishmaniasis will be
extracted.
Figure number 3. Corresponds to the identification
of the protein obtained from the chimeric gene, the
preparation of which is represented in Figure number 2.
Figure number 4. Corresponds to a chromatographic
representation of affinity chromatography on an amylose
column, and after the purification process' the
recombinant proteins submitted to electrophoresis.
Figure number 5. Shows finally a synthesised
graphical representation of the reactivity of a wide
variety of canine serums, divided into three groups. The
first group contain animals with real infection by L.
infantum. The second group includes serum obtained from
dogs with various clinical symptoms but that are not
infected with Leishmania, and a third group is made up of
fifteen control serums from healthy dogs. This figure
demonstrates the value of the invention for carrying out
serological diagnosis of VL.
Figure numberd 6a and 6b show the immunological
response in 4 hamsters injected intraperitoneally with 5
micrograms of protein Q and 5 micrograms of protein
LiHsp70. The graph shows the humoral response (weeks vs
optical density). These figures show that all the animals
respond immunologically to protein Q, and that starting
from the second week after immunisation the response
against protein Q was very high and this response
increased after the second immunisation (week 4), whereas
CA 02256124 2012-10-17
18
the response against protein LiHsp70 was lower than that
observed against Q.
PREFERRED EMBODIMENT OF THE INVENTION
The chimeric gene formed from the DNA sequences
that encode the antigenic determinants of four proteins
of L. infantum, useful for the serological diagnosis of
canine Leishmaniasis and the protein obtained that is
being proposed are constituted from the construction of
intermediate products. In a first instance, cloning of
epitopes specific to the antigens of L. infantum is
carried out, which is configured on the basis of earlier
studies on the antigenic properties of four protein
antigens of L. infantum (LiP2a, PiP0, LiP2b, LiH2a),
which allow the existence of B epitopes to be defined for
these proteins, and which are specifically recognised by
the canine serums of VL.
With a view to improving the antigenic specificity
of these antigens with respect to the proteins of L.
infantum, the specific antigenic determinants are cloned
from these proteins. After deleting certain regions of
these proteins these can be recognised by serums from
animals that are carriers of VL and other different
diseases.
By using the specific oligonucleotides and
amplification by PCR of regions specific to the genes
LiP2a, LiP2b, PO and H2A, several clones are constructed
that express the recombinant proteins rLiPO-Ct-Q, rLiP2a-
Q, rLiP2b-Q, rL1H2A-Ct-Q and rLiH2A-Nt-Q, just has been
detailed in the description of the invention relating to
the methodology, where the cloning details are described.
The recombinant proteins used are the following:
- rLiPO-Ct-Q, which corresponds to the 30 C-
terminal residues of the ribosomal protein LIP .
- rLiP2a-Q and rLiP2b-Q, that are derived from the
ribosomal proteins LiP2a and LiP2b respectively.
- Two sub-regions of the histone H2A, that
correspond to the 46 N-terminus residues (xLiH2A-Nt-Q),
CA 02256124 1999-10-22
19
and to the 67 C-terminus residues (residues (xLiH2A-Ct-
Q).
Each one of the recombinant proteins fused to the
maltose binding protein (MBP) is expressed in E. Coll, as
represented in Figure number 1, and they were purified by
affinity chromatography on a amylose column. After the
process of purification the electrophoresis was carried
out on the recombinant proteins (lanes 1 to 5) in Figure
number 3.
With the aim of analysing whether the recombinant
proteins were recognised by VL canine serums, a Western
blot was incubated, containing the recombinant proteins
in a mixture of three VL canine serums. Given that all
these proteins are recognised by the serums, it is
concluded that the antigenic determinants present in the
parent proteins are maintained in the recombinant
proteins.
The antigenic properties of the recombinant
proteins are compared with the antigenic determinants of
the parent antigens by means of a FAST ELISA, testing
against a collection of 26 VL canine serums, just as is
shown in the section of Figure number 1, and the fact
that the serums showed a similar reactivity value, both
against the selected antigenic regions and the
corresponding complete proteins, demonstrates that no
alteration to the antigenic epitope has occurred during
the cloning procedure.
In regard to the construction of the final product,
more exactly of the chimeric gene that encodes a
polypeptide that contains all the selected antigenic
determinants, it should be pointed out that the cloning
strategy is indicated following Figure number 2 section
A. The
intermediate products generated during the
process are shown.
A clone that expresses the proteins rLiPO-Ct-Q
(pPQI) is used as the initial vector, and the fragments
of DNA that encode the proteins rLiPO-Ct-Q, rLiP2a-Q,
CA 02256124 1999-10-22
rLiP2b-Q, rLiH2A-Ct-Q and rLiH2A-Nt-Q are added
sequentially using appropriate restriction sites.
After each cloning step, the correct orientation of
each one of the inserts is deduced from the size of the
5 expression products, and finally the complete nucleotide
sequence of the final clone pPQV is determined and the
amino acid sequence deduced from the sequence represented
in Figure number 3.
The polypeptide generated has a molecular mass of
10 38 kD, with an isoelectric point of 7.37, including
spacer sequences encoding proline, underlined in Figure
number 3. The aim of doing this is to efficiently
separate the antigenic domains and avoid possible
tertiary conformations that could interfere with the
15 stability and antigenicity of the final product.
The expression and recovery of each of the
intermediate products is shown in figure number 4, boxes
A and B. As was expected, after each addition, the size
of the expression product in the vector pMAL gradually
20 increases until reaching a molecular weight of 80 kDa.
Included in this are the sizes of the proteins rLiH2A-Ct
and rLiH2A-Ct, observing a certain degree of rupture
during purification.
The chimeric gene was also cloned in the plasmid
pQE, a vector that allows the expression of proteins with
a fragment of 6 histidines at the extreme N-terminus.
The resulting clone and the recombinant proteins
are denominated pPQ and PQ respectively.
The level of expression of the protein in bacteria
transformed with the pPQ plasmid and the purified
proteins are shown in Figure number 4, referred to in
particular with a D, with the protein PQ, purified by
affinity chromatography in denaturising conditions is
more stable that the recombinant protein pPQV represented
in Figure number 4, in box E.
In order to evaluate the final product a series of
materials were used, and obviously some techniques, as is
CA 02256124 1999-10-22
21
described below.
Serums of VL obtained from dogs of different
origins are used. The animals are evaluated clinically
and analytically in the pertinent laboratory, generally
in a Department of Parasitology, and all the positive
serums are assayed for indirect immuno-fluoresence (IIF).
The presence of amastigotes of the parasites of
these animals is confirmed by direct observation of the
popliteal and pleescapular lymph nodes, and a second
group of 33 serums of VL originating from other regions,
were given a positive diagnosis in the ELISA against
total protein extracts of the parasite and/or by IIF.
The serums of dogs affected by different diseases
that were not VL are obtained from different origins.
Within this group serums from the following infections
are found:
Mesocestoides spp.
Dyphylidium caninum
Uncinaria stenocephala
Toxocara canis
Dipetalonema dranunculoides
Demodex canis
Babesia canis
Ehrlichia cannis
Ricketsia ricketsiae.
The rest of the serums were obtained from dogs that
exhibited various clinical symptoms that were not related
to any infective process, and the serum controls were
obtained from fifteen carefully controlled healthy
animals.
Purification of the recombinant proteins expressed
by the clones pMA1-c2 is carried out by affinity
chromatography on amylose columns, and the purification
of the recombinant protein expressed by the clone pPQ was
performed on Ni-NTA resin columns in denaturising
conditions (Qiagen).
For analysing the proteins electrophoresis on 10%
CA 02256124 2008-02-06
22
polyacrimide gels in the presence of SDS was carried out
under standard conditions. Immunological analysis of the
proteins separated by electrophoresis was carried out on
nitrocellulose membranes to which the proteins had been
transferred. The transferred proteins were blocked with
dried 5% skimmed milk in a PBS buffer with 0.5% Tween"20.
The filters were sequentially brought into contact
with primary and secondary anti-serum in blocking
solutions and an immuno-conjugate labelled with
peroxidase was used as second antibody, visualising the
specific binding by means of an ECL system.
The Fast-ELISA was used instead of the classic
ELISA, and the sensitisation of the antigen was carried
out for 12 hours at room temperature.
The plates were sensitised with 100 pl of antigen
whose concentration in all cases was 2 Ag/ml.
After sensitising the wells the plates were
incubated for 1 hour with blocking solution (0.5%
powdered skimmed milk dissolved in PBS - 0.5% Tween"20
and the serums were diluted three hundred fold in
blocking solution).
The wells were incubated with serum for 2 hours at
room temperature, and after exposure to the antibody the
wells were washed with PBS-Tween"20.
Antibodies labelled with peroxidase were used as
second antibodies at a dilution of 1:2000 and the colour
of the reaction was developed using the substrate ortho-
phenylenediamine, measuring the absorption at 450 rim.
In regard to evaluation of the final product, it
should be pointed out that the antigenic properties were
determined by means of the pertinent study of the
reactivity of the VL canine serums against the chimeric
protein and against each one of the intermediate products
in a "Western blot" assay. All the intermediate products
maintained their antigenicity as well as did the final
pPQV product, throughout the whole of the cloning
CA 02256124 1999-10-22
23
process.
It should also be pointed out that the recombinant
protein expressed by the pPQ plasmid was recognised by
the VL serums. With a view to analysing with greater
precision the antigenic properties of the chimeric
protein and the intermediate products, an analysis of the
reactivity of a wide variety of VL canine serums was
performed by means of a fast-ELISA against the
recombinant proteins, as is shown in the section F of
Figure number 4. It can be highlighted that the
absorption values and the sensitivity of the different
intermediate products of cloning increases after each
addition stop. It should also be pointed out that the
protein pQI is recognised by most of the VL serums and
the protein PQII equally by most of the serums. This
proportion is greater for the protein PQIII, and the
proteins PQIV, PQV and PQ are recognised by practically
all the serums.
According to what has been discussed above, the
percentage of recognition shown by the serums was similar
both in the case of assaying the chimeric proteins PQV
and PQ, and of assaying a mixture of recombinant proteins
rLiPO-Ct-Q, rLiP2a, rLiP2b and rLiH2A. It was seen that
the antigenic properties of each one of the 5 selected
antigenic regions are present in the PQ expression
product, and therefore this product can be used for
diagnosis instead of a mixture of the antigens expressed
individually.
With a view to determining whether the chimeric
protein can be used for canine VL serum diagnosis, and
according to the pertinent analysis of a wide variety of
canine serums against this protein, bearing in mind that
according to the clinical characteristics of the animals,
the canine serums have been classified into three groups.
A first group consisted of serum of dogs with a real L.
infantum infection. A second group was composed of serums
of dogs that had various clinical symptoms without being
CA 02256124 2012-10-17
24
infected with Leishmania, including dogs infected with
parasites different to Leishmania incorporated into this
group. The rest of the serums originated from dogs that
exhibit clinical symptoms that could be confused with
those observed during Leishmaniasis,
The third group was made up of control serums,
originated from serums of healthy dogs.
In figure number 5 the average values of reactivity
are shown for each group of serums, the reactivity of the
VL serums reaching an average reactivity value of 0.8
(S.D. = 0.4).
Within this group the reactivity of 12 serums is
less than 0.35, while the reactivity of 10 serums reaches
values of between 0.35 and 0.5. It is observed that the
reactivity over 23 serums varies between 0.5 and 1.0,
with 14 serums showing a reactivity greater than 1Ø
The average absorption value of the serums of the
second group, that is to say, the group in which animals
infected with Leishmania parasites and parasites
different to Leishmania parasites, is 0.2 (S.D. = 0.05)
and the reactivity of the control serums, that is to say,
the third group, is 0.1 (S.D. = 0.003).
The data presented above indicate that the chimeric
protein PQ in the FAST ELISA has a sensitivity of 80% for
the VL diagnosis, if the cut-off value is defined as the
average reactivity value of the serums of group 2 plus
three S.D.'s (that is to say 0.35).
The sensitivity of the assayed group reaches 93%,
if the cut-off value is defined by the reactivity values
of the control group, and the data indicate that the
protein CP has a specificity of 96% for the VL diagnosis,
when the cut-off value is defined by the aforementioned
serums of group 2, and only two serums from group 2
showed reactivity between 0.35 and 0.40.
100%
specificity in the assay was reached when the reactivity
values of healthy dogs were considered.
The process to be used is the following:
CA 02256124 2008-02-06
1.- The microtitre plates are covered with
.antibodies by incubated 100 Al of a solution that
contains 1 Ag/m1 of antigen dissolved in a buffer PBS -
0.5% Tween 20 - 5% skimmed milk (Buffer A).
5 The incubation is performed for 12 hours at room
temperature, and then the plates are washed three times
with the same buffer containing no antigen. The dry
antigenated plates could be maintained at room
temperature.
10 2.- A first incubation of the wells was carried out
with the serum of animal at a dilution of 1/200 in buffer
A. The incubation lasts for 1 hour.
3.- The wells are washed with buffer A, as
described in point 1, three times with a wash flask.
15 4.- They are incubated with a second antibody (IgG
labelled with peroxide) diluted 1:2000 in buffer A,
carrying out the incubation for 1 hour.
5.- The wells are washed once again with buffer A
three times, as was indicated in the third section, that
20 is to say with a wash flask.
6.- The reactivity is revealed using the substrate
= ortho-phenylenediamine and the absorption measured at 450
nm.
The protein used for the diagnosis extracted from
25 the chimeric gene is identified as follows:
MBP IEGRPLTPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGYARRIGA 50
SGAPRISEFSVKAAAQSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVV
140
PNISKAMAKKKGGKKGKATPSAPEFGDSSRPMSTKYLAAYALASLSKASPSQAD
157
VEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGV
211
TASAAGDAAPAAAAAKKDEPEEEADDDMGPSVRDPMQYLAAYALVALSGKTPSK
265 t-
STAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKAAPEESD
374
EDDFGMGGLF
CA 02256124 2008-02-06
26
It is not considered necessary to extend this
description in order that someone skilled in the art can
understand the scope of the invention and the advantages
that it confers.
The materials, form, size and disposition of the
elements are susceptible to change, provided it does not
suppose a change in the essence of the invention.
=
The terms in which this disclosure has been written
should always be considered as broad in nature and not
limiting.
Experimental
The protozoan parasites of the genus Leishmania are
responsible for causing leishmaniasis, a symptomatically
complex disease which essentially affects men and animals
in tropical and subtropical regions. It is estimated that
the number of new cases of human visceral leishmaniasis
can reach the number of 500,000, there being a minimum of
several tens of millions of persons affected.
Additionally the number of cutaneous and mucocutaneous
leishmaniasis can be of the order of 2.000.000 per year
(Modabber F., Development of vaccines against leishmaniasis.
Scand J Infect Dis Suppl. 1990; 76:72-8).
Although the
persons at risk of contracting the different types of
leishmaniasis can be estimated in about 350 million, the
number of persons with real infection can be much higher,
due to the fact that there are no clear estimations of the
real cases of asymptomatic infections and because of the
existence of cryptic infections (Alvar et al., 1996).
In
fact, leishmaniasis can be considered within the global
context as an infection/ disease of endemic nature, situated
between the 4th and 5th place in the ranking of parasitic
diseases with world-wide repercussion.
Three main forms of leishmaniasis can be
distinguished: cutaneous, mucocutaneous and visceral, the
characteristics of which mostly depend upon the localisation
of the parasite, the species to which it belongs and the
clinical manifestations it produces.
CA 02256124 1999-10-22
27
(Coutinho et al., 1987; Cuba et al., 1988). The species
distributed along Asia and certain regions in the
Mediterranean area bring about the presence of the
cutaneous form, with localised ulceration which, in many
cases, heal spontaneously. These manifestations are
caused by L. major and L. tropica. L. aethiopica
(Mediterranean, Asia, Africa) also induces the cutaneous
form of leishmaniasis, although its manifestation is more
diffuse. In America, the species L. mexicana produces the
cutaneous form with a generalised localisation that does
not usually heal spontaneously. The mucocutaneous form of
the disease in humans is caused by L. brasiliensis and is
characterised by the presence of cutaneous lesions in
oronasal and pharyngeal regions, bringing about the
destruction of the mucosae. In America, Europe, Africa
and Asia, the most frequent form of leishmaniasis is the
visceral form, caused by L. chagasi, L. donovani and L.
infantum. This form of leishmaniasis is characterised by
clinical symptoms associated to fever, anaemia and an
intense hepato/splenomegalia, which is lethal if it is
not treated suitably at the right time. In the advanced
form of the disease, the host is incapable of developing
an effective immune response. All of these forms of
leishmaniasis are also detected in canids and some
rodents which, in fact, constitute the main reservoirs of
the parasite (Liew & O'Donnell., 1993). The health
problem generated by L. infantum in the Mediterranean
basin is serious because there is a high incidence of the
infection / disease in dogs, and the vector insect is
very extended (Abranches et al., 1991). It is calculated
that between 7% and 20% of all canids are infected by
Leishmania, reaching 30%, in some areas of Spain where it
is endemic (Garcia Alonso., 1994). This fact constitutes
a serious veterinary problem which additionally increases
the risk of contagion, fundamentally by immunodepressed
persons (Acedo et al., 1996). In Europe, there are some
11 million dogs at risk of infection by Leishmania.
CA 02256124 1999-10-22
28
L. infantum, like the rest of the species in the
genus has a dimorphic biological cycle. The intermediate
hosts are insects of the Psychodidae family, genus
Phlebotomus. In the Mediterranean area of Europe, it has
been demonstrated that the species P. arias and P.
perniciosus (Sanchia Marin et al., 1991); Alves at al.,
1991; Alves and Riveiro, 1991; Maroli et al., 1994) are
the main vectors, although the vectors P. papatosi, P.
longicuspis and P. sergenti are also present (Sanchis
Mann et al., 1986; Martinez Ortega, 1986; Morillas
Marquez et al., 1991; Rosado et al., 1995a, 1995b). When
the parasite is ingested by the vector together with the
blood of a vertebrate host, it places itself in the gut
in an extracellular form, it transforms into a
promastigote and it divides. The infective forms migrate
towards the pharynx and the proboscis, from where they
will be inoculated into a new vertebrate host (Chang et
al., 1985). The promastigotes are characterised in that
they have a flagellum and an elongated shape of some 15
to 20 Am in length, with a rounded posterior end and a
sharp anterior end. The nucleus is situated in central
position and the kinetoplast at the anterior end
(Zuckerman and Lainson, 1977). In culture media, the
parasites exhibit a certain degree of morphological
variability (Chang and Hendricks, 1985). After the
inoculation of the promastigotes in the skin of the
vertebrate host, the establishment, or not, of the
infection depends essentially upon two factors: the
existence of a suitable cell population -macrophages- and
other cells of the phagocytic mononuclear system, and the
ability of the parasite to survive and multiply itself in
the interior of these cells.
The first step in the penetration of Leishmania
into macrophages is the approximation and adherence to
the plasma membrane of the target cell. In vitro studies
seem to indicate that there is no direct chemotactic
CA 02256124 1999-10-22
29
attraction of the promastigotes over the macrophages
(Bray, 1983). Within the environment of tissues, free
promastigotes activate complement by the alternative
pathway, bringing about the formation of a concentration
gradient of fraction C5a, which attracts macrophages and
other inflammatory cells towards the site of inoculation
(Bray and Alexander, 1987). Once the promastigote is
within a parasitophorous vacuole, the lysosomes fuse to
it forming a phagolysosome. In infections caused by other
micro-organisms, the phagolysosome is the organelle
responsible for the lysis and elimination by means of
several mechanisms such as the production of toxic oxygen
radicals, by an oxidative metabolic process (Keblanoff,
1980), the action of hydrolytic lysosomal enzymes,
cationic proteins and low pH (Bray and Alexander, 1987).
The survival of the parasite in the phagolysosome is a
function of its ability to resist and avoid said
mechanisms.
The existence of an immune response against
parasitisation by Leishmania which is both humoral and
cellular was discovered from the first moments in which
the disease was studied, and has been revised in numerous
occasions (Maael and Behin, 1981; Pearson et al., 1983;
Behin and Louis, 1984; Howard, 1985; Liew and O'Donnell,
1993). The type of humoral response depends upon the form
of leishmaniasis. In the cutaneous form, the humoral
response is very weak, whereas in the visceral type a
high antibody response is observed (Liew and O'Donnell,
1993). In the cutaneous affections there is a remarkable
cell-mediated response, detectable both in vivo by means
of delayed type hypersensitivity tests (DTH), as well as
in vitro by means of lymphoblastic transformation tests
and macrophage migration inhibition tests. In these cases
the titre of serum antibodies is normally low and
directly related to the seriousness of the process
(Howard, 1985; Liew and O'Donnell, 1993). Once the
amastigotes are within the macrophages, the resolution of
CA 02256124 1999-10-22
the infection depends essentially upon the cell-mediated
immune mechanisms (Pearson et al., 1983; Coutinho et al.,
1987; Kubbs et al., 1988). The cellular response is
determined by the joint action of macrophages, B cells,
5 several sub-populations of T-cells, and the different
lymphokines secreted by all of them. The
parasitised
macrophage processes the Leishmania antigens and
expresses them on its surface by a process mediated by
the class II Major Histocompatibility Complex (MHC-II).
10 Additionally, the macrophage secretes IL-1, which acts as
a second activating signal for the T-lymphocyte (Antezac
and Gorman, 1989). In humans, visceral leishmaniasis or
kala-azar is characterised by a weakened or absent
cellular response, detectable both by the absence of
15 delayed hypersensitivity (DTH) (Turk and Bryceson, 1971)
and by cell proliferation methods (Haldar et al., 1983;
Ghose at al., 1979; Howard, 1985). Absence of
proliferation of T-cells is detected even in the presence
of mitogens such as concanavelin A or phytohaemagglutinin
20 (Reiner and Finke, 1983; NTriez-Moreno, 1991) and
inhibition in the production of IL-2 by stimulated T-
cells (Reiner and Finke, 1983; Carvalho et al, 1985).
In mice, the population of T-lymphocytes (CD4
phenotype) is heterogeneous and can be divided into at
25 least two sub-populations according to the lymphokines
they produce (Mosmann and Coffman, 1987). These cells are
essential in the development of protective immunity
against cutaneous leishmaniasis (sub-population Th-1) and
are at the same time involved in the suppression of the
30 protective immune response (sub-population Th-2) (Liew
and O'Donnell, 1993), T cells induced in resistant mice
C57BL/6 or cured BALB/c mice are predominantly of the Th-
1 type, whereas the cells in uncured BALB/c mice are of
the Th-2 type (Lockaley et al., 1987; Sadick et al.,
1987; Heinzel et al, 1989, 1991). In general, the
lymphokines secreted by these cells favour the
development of the cell line which produces them and has
CA 02256124 1999-10-22
31
an antagonic effect on the development of the other sub-
population (Reiner and Lockaley, 1993). Thus, IL-4 and
IL-10 produced by Th-2 cells contribute to the
progression of the infection, favouring the development
of this line. Additionally, they can act directly on the
macrophage, not permitting its activation. However, in
mice susceptible of infection by Leishmania, deficient
in the gene which encodes IL-4, contradictory results
have been obtained. In some cases it has been observed
that the absence of this cytokine redirects the response
and increases the resistance to the infection (Satoskat
et al., 1995) whereas in others no differences are
observed in the level of infection of the mice (Noben et
al, 1996). In genetically resistant mice it has been
observed that the absence of expression of the genes of
IFN-y or its receptor (Swihart et al., 1995), CD40 or the
ligand of 0D40, increases the susceptibility to the
infection (Campbell et al., 1996; Kamanaka et al, 1996;
Soong et al., 1996). The interaction of CD40 and its
ligand is necessary for the production of the cytokine
IFN-y necessary to direct the response towards Th-1. Mice
with a resistant genetic base, which develop a Th-1 type
response, become susceptible if they are deficient in the
expression of IL-12., developing a Th-2 type response
(Mattner et al., 1996). Recent data suggests that the
production of IL-12 is important to direct the response
towards Th-1, and that the absence of IL-4 can avoid the
Th-2 response (Guier at al., 1996).
There are a large number of Leishmania proteins
which have an antigenic character (Jaffe et al., 1990;
Rolland-Burger et al., 1991; Mary et al., 1992)
essentially characterised by "Western Blot" methods. In
the serum of patients and dogs infected by Leishmania it
has been possible to detect the presence of antibodies
against membrane proteins such as gp63 (Shreffler et al.,
1993; Morales et al., 1997) gp 46 (Burns et al., 1991),
PSA (Kahl and McMahon-Pratt, 1987; Jimenez-Ruiz et al.,
CA 02256124 1999-10-22
32
1998) and KPM-11 (Berberich et al., 1997). Additionally
several antigens of cytoplasmatic intracellular
localisation have been characterised such as: Hsp70
(Macfarlane et al., 1990; Wallace et al., 1992; Quijada
et al., 1996a; Quijada et al., 1996b); Hsp83 (Angel et
al., 1996); LIP2a, LIP2b, LILIPO, H2A, H3 (Soto et al.,
1993; Soto, 1994; Soto et al., 1995a; Soto et al., 1995b;
Soto et al., 1995c; Soto et al., 1996) and a protein
related to kinesin, K39 of L. chagasi (Burns et al.,
1993). The reactivity of the antibodies, which recognise
conserved proteins present in the serum of dogs infected
by L. infantum is directed towards the least conserved
areas of these proteins (Wallace et al., 1992; Soto et
al., 1995a). Some of the membrane proteins are very
antigenic in natural infections (Berberich et al., 1997;
Jimenez-Ruiz et al., 1998). In all the cases of natural
infection there is a great restriction in the humoral
response against the proteins because the antibodies
developed during the infective process recognise very
restricted areas of the same ( Soto, 1994; Soto et al.,
1993; Soto et al., 1995a; Soto et al., 1995b; Quijada et
al., 1996a; Quijada et al., 1996b ; Soto et al., 1996).
The levels of IgG normally correspond to the intensity of
the infection (Moray et al., 1987; Mimori et al., 1987)
and according to Howard (1985), reflect the degree and
the duration of antigenic stimulation determined by the
parasitic load. A Leishmania antigen homologous to the
type C kinase receptors (LACK) has recently been
described which produces an early response of the Th-2
type (Mougneau et al., 1995; Julia et al., 1996). In mice
transgenic for the LACK antigen and with a genetic
background susceptible to the infection, the induction of
tolerance to this antigen protects against the infection
by Leishmania major. The anti-Leishmania antibodies can
destroy the promastigotes in vitro in the presence of
complement (Pearson and Steibigel., 1980; Mosser and
Edelson., 1984), promote phagocytosis (Herman., 1980) and
CA 02256124 1999-10-22
33
induce the adherence of several particles to the surface
of promastigotes and amastigotes (Dwyer, 1976).
The pathologic reaction seems to go in parallel
with the density of parasitised macrophages. In visceral
leishmaniasis the macrophages generally distribute
themselves in a diffuse manner throughout the different
organic tissues (Andrade and Andrade, 1966; Oliveira et
al., 1985). The type of inflammation is is constituted by
an important cellular infiltrate with a predominance of
lymphocytes and plamatic cells together with hyperplasia
of phagocytic cells (Veress et al., 1977; Carvalho et
al., 1985; Hassan et al., 1986). The inflammation brings
about alterations in the physiology of the affected
organs producing serious systemic alterations (Ridley,
1987). In some occasions local granulomatous inflammation
occurs, with appearance of granulomae and microgranulomae
in the different organs. These granulomae are constituted
by macrophages and histiocytes (affected by parasites or
not) surrounded by plasmatic cells and lymphocytes and,
in some cases, by fibroblasts (McElrath et al., 1988).
This inflammatory process is accompanied by an organic
reaction with the appearance of characteristic lesions in
the affected organs. Some authors have found deposits of
amyloid substance in virtually the totality of the organs
(Martinez-Gomez et al., 1980). Some authors have
formulated the hypothesis that the damages produced by
the disease are not directly attributable to the
aethiologic agent but to the organic reaction triggered.
Vaccine against Leishmania
The intense immunity which follows the recovery
from cutaneous leishmaniasis has given a great impulse to
the development of prophylactic vaccines against this
disease. This immunity is derived from the induction of a
T response which has associated to it the production of
inflammatory cytokines which activate macrophages and
destroy the parasites. The immunological memory in the
CA 02256124 1999-10-22
34
cases of infection is probably maintained by the
persistent presence of the parasite in the host in a
process known as concomitant immunity (Aebischer et al.,
1993).
The first studies regarding vaccination against
Leishmania in the decade of the 40's used live parasites
as immunogens. These studies led to the production of
vaccines which produced significant protection against
subsequent re-infection. However, the knowledge of the
possibility that live organisms could produce real
infections led to such vaccination programmes not to take
place for very long and, on the contrary, interest
focused upon vaccines based upon dead parasites. These
studies provided the first evidence on the possibility of
producing effective vaccines by inoculation of parasites.
The clinical trials which used immunisation with
dead Leishmania promastigotes also began in the decade of
the 40's. These vaccines yielded remarkable successes as
a certain degree of protection was observed, which could
oscillate between 0 and 82% (Grimaldi, 1995) depending on
the population. These vaccines had a smaller effect than
live parasite vaccines. The isolation of avirulent clones
of L. major which protect mice against infection has also
demonstrated that an attenuated vaccine is possible
(Handmann and McConville., 1990). However, the ignorance
of the mutations which lead to the loss of virulence and
the risk of production of virulent revertants make this
type of vaccination currently unacceptable.
Recently there has been an important progress
towards the identification of molecularly defined
candidate vaccines, such as gp63 ( ), gp46/M2 ( ), the
surface antigen related to the latter antigen known as
PSA-2 (Xu et al., 1995; Handmann et al., 1995) and the
proteins dp72 and gp70-2 (Rachamin and Jaffe, 1993), the
LACK protein ( ) and Kmpll ( ).
Specifically, the T
epitopes present in protein gp63 have been identified,
_
CA 02256124 1999-10-22
resulting in that only some of them are capable of
inducing a T response, both of Th-1 as of Th-2 (Russo et
al., 1993b). These antigens induce significant protection
in model animals when they are administered with
5 adjuvants. ?rotein PSA-2 of Leishmania is capable of
protecting against infection by L. major by inducing a
Th-1 response. To evaluate the mechanism of protection of
this protein as a vaccine against Leishmania in humans,
its ability to induce T-cell proliferation was studied,
10 in patients which had suffered leishmaniasis and had
recovered from it. It was observed that the protein is
capable of bringing about a strong proliferation of the
T-cells of these individuals, but not of controls with no
prior history of infection. The response was of the Th-1
15 type, as was demonstrated by the cytokine induction
pattern (Kemp et al., 1998).
Sub-unit vaccines have focused strongly on protein
antigens because they are easy to identify, isolate and
clone. However, it is necessary to take into account that
20 not all potential vaccine molecules have to be proteins.
In fact, lipophosphoglycan (LPG) plays an essential role
in the establishment of infection. Vaccination with LPG
can protect against infection with L. major. In spite of
the existence of a dogma which states that T-cells do not
25 recognise non-proteinaceous antigens, the LPG molecule
seems to be presented to T-cells by Langerhans cells of
the skin. (Moll, H. 1989; Moll et al., 1995).
Additionally, there is evidence which has demonstrated
that microbial glycolipids and other non-proteinaceous
30 molecules can recognise T-cells when they are presented
via the CD-1 route (Procelli, 1996). Although there is no
clear evidence that protein KMP11 associated to LPG
induces a protective response, it has been proved that
the proteinaceous fraction associated to LPG is capable
35 of inducing a T response and IFN-T, whereas the LPG
fraction without protein is not (Jardim et al., 1991).
It is normally accepted that sub-unit vaccines
CA 02256124 1999-10-22
36
although protective, only induce a short term immunity.
This problem may not be important in endemic areas, where
the individuals can be periodically boosted by cause of
natural infections. A major problem in the use of sub-
units may arise from the fact that there may not be a
response to a single antigen in a genetically diverse
population. A cocktail of antigens containing B and T
inducers may overcome this drawback. It has recently been
published that an extract of membrane proteins of
Leishmania infantum, when injected intraperitoneally, is
capable of conferring protection against the virulent
promastigote forms of this parasite, and that this
protection is greater when the proteins are encapsulated
in positively charged liposomes (Afrin and Ali., 1997)
Adjuvanticity and protective immunity elicited by
Leishmania donovani antigens encapsulated in positively
charged liposomes. Infection and Immunity p 2371-2377.
Vaccination with nucleic acids carrying genes which
encode Leishmania proteins involves the administration of
genetic material of the parasite to the host. This DNA is
taken up by the cells and is introduced into the nucleus
where it is transcribed and subsequently translated in
the cytoplasm. The advantage of this type of vaccination
is that it is possible to direct the immune response by
means of the MHC-I or the MHC-II route (Wahren., 1996).
The antigens produced intracellularly are processed in
the cell and the peptides generated are presented on the
cell surface in association with MHC-I molecules. The
consequence would be that these molecules would give rise
to the induction of cytotoxic T-cells. The antigens
produced in an extracellular environment would be
specially taken up by specialised antigen-presenting
cells, processed and presented on their surface bound to
MHC-II molecules, resulting in the induction and
activation of CD4+ cells which secrete cytokines which
regulate the effector mechanisms of other cells of the
immune system.
CA 02256124 1999-10-22
37
The first DNA vector to be administered as a
vaccine contained the gp63 gene (Xu and Liew., 1995).
Also, the PSA-2 gene has been introduced into a plasmid
and it has been observed that it generates a Th-1
response and induction of protection. Vaccination with
DNA plasmids which contain Ag-2 induce a Th-1 response
and protect against infection with L. major, while Ag-2
in stimulatory immune complexes elicits a combined Th-1
and Th-2 response and does not protect despite the fact
that IFN-1, is induced (Sjolander et al., 1998). Equally,
the gene encoding the LACK protein has been administered
subcutaneously to BALB/c mice, in an expression vector
which expresses the protein under the control of the
cytomegalovirus promoter, and protection against
infection with L. major has been observed (Gurunathan et
al., 1997). In almost all cases in which DNA has been
administered, the route has been intramuscular, although
intradermal injection of particulate DNA must also be
explored, as it requires a smaller amount of DNA . Other
immunisation systems use vectors such as Salmonella, BCG
or Vaccinia virus. It is interesting to remark that the
inclusion of the gp63 gene in BCG is capable of inducing
protection against L. major (Connell et al., 1993).
The gene which encodes protein gp63 has also been
introduced into gene delta araC under the control of the
rac promotor in an attenuated Salmonella typhimurium.
Oral administration of lx109 colonies of the transformed
bacterium induces a T response within the scope of both
Th-1 and of cytotoxic cells against mastocytoma cells
which express gp63 (Gonzalez et al., 1998). Protein gp63
in the form of gp63-ISCOM5 complex induces protection in
mice, evidenced by the reduction in inflammation and
suppression of lesions. In serum, there are antibodies of
the IgG2a type and, additionally, it is possible to
observe a Tb-1 response by induction of IL-2, IFN-1, and
IL-10. No DTH response was observed (Papadopoulou, G. et
al., 1998). Salmonella typhi Nramp 1 transformed with
CA 02256124 1999-10-22
38
gp63 elicits a Th-1 response with induction of IL-2 and
IFN-y, and a strong resolution of the lesions is detected
(Soo et al., 1998). A protein of L. pifanoi known as P-4
induces significant protection against infection by
Leishmania. Recent studies in humans with cutaneous
leishmaniasis indicate that this protein or the peptides
derived from it are capable of making T cells
proliferate. There is no induction of IL-4, whereas IFN-y
is induced (Haberer et al., 1996). Aro-A and aro-D
mutants of Salmonella typhi transformed with IL-2, IFN-y
and TNF-ci administered orally may serve as therapeutic
systems against infection by L. major. It is interesting
to observe that in these patients there is a greater
induction of iNOS (Xu, D. et al., 1998). The gp&3 gene
has also been cloned in Aro-A and Aro-D mutants, and it
has been observed that after oral administration, the
protein encoded is capable of inducing significant
protection against infection with L. major (Xu, D. et
al., 1995). This same protein is capable of inducing
protection when it is administered fused to several
promoters in specific varieties (GID105 and GID106) of
Salmonella (McSorley et al., 1997).
An artificial protein denominated Q has recently
been described by our group, which is composed of several
antigenic fragments from 4 proteins of Leishmania
infantum (more specifically, Lip2a, Lip2b, PO and H2A),
which, after being used as antigen, has proven to have an
important value for the diagnosis of canine
leishmaniasis, with a 93% sensitivity and a 100%
specificity when compared with sera of control animals
which are not infected (Soto et al., 1998). Equally, our
group has demonstrated that protein hsp70 of Leishmania
infantum is an important target of the immune response in
infections caused by infection with this parasite
(Quijada et al., 1996a; Quijada et al., 1996b).
With the object of exploring the possibility that
protein Q may be used to design protection systems
CA 02256124 1999-10-22
39
against infection by Leishmania infantum, both on its own
as in combination with Hsp70, three series of experiments
were designed using the hamster as a model. An experiment
was designed to check whether immunisation with Protein Q
protected the animals against infection on the short
term, another to check whether immunisation protected
them on the long term and the third was to check this
protective effect after immunisation with the two
proteins together. It was thereafter observed both from
the short term analysis as well as from the long term
analysis, that protein Q was capable of eliciting an
immune response which reduces the parasitic load both in
the Liver and in the Spleen after infection by Leishmania
infantum in most of the immunised animals, and that
immunisation with the proteins Q+Hsp70 also induced a
significant response against both proteins and lead to a
significant reduction of the parasitic load in most of
the immunised animals.
Immunisation with Protein Q
Example 1
4 animals were immunised with 5 micrograms of
protein Q dissolved in 40 microlitres of Freund's
adjuvant, and another 4 animals were immunised with the
same amount of adjuvant emulsified with 40 microlitres of
PBS saline solution without the protein. In the first
immunisation, Freund's complete adjuvant was employed
combined with the protein, while in the two subsequent
immunisations, incomplete Freund's adjuvant was used
mixed with the protein in the same proportion of
protein/adjuvant. Three intraperitoneal immunisations
were carried out at 15 day intervals. Starting from the
second week after each immunisation and throughout the
whole period of immunisation, blood samples were
extracted to measure the humoral response against protein
Q in ELISA assays. It was observed that already in the
second week after the first immunisation there is a
positive IgG response against protein Q, and that this
CA 02256124 1999-10-22
response was high after the second week after infection,
and increased with time of immunisation until reaching a
titre of 1/100.000. Equally, it was observed that immune
response against protein Q was not modified significantly
5 after the infection with the parasite Fig. 1.
Fifteen days after the third immunisation, the
animals were infected with a dose of 105 promastigote
parasites, differentiated from infective amastigotes
10 originating from an infected hamster. It had been
previously checked that the inoculum was capable of
inducing a strong parasitemia together with the disease
four months after having administered the parasites, in
100% of the animals infected. Table 1 indicates the level
15 of parasitemia per mg of tissue both in liver and in the
spleen of the control and the vaccinated animals. It is
possible to observe that in all of the vaccinated animals
the parasitic load in the liver decreases with respect to
the controls, and that this happens in a very significant
20 manner in 75% of them. When the parasitic load in the
spleen is examined, it possible to observe that also in
75% of the animals this load was significantly lower than
that of the controls, the RPL reaching 83%-86%. The
animal in which the RPL was 20% in the liver, had a 48%
25 one in the spleen.
Table 1. Parasitic load in the liver and spleen of
hamsters vaccinated with protein Q via the
intraperitoneal route. After four weeks of
30 infection, the parasitic load was measured by the
method of limit dilutions. The parasitic load is
expressed as parasites per milligram of tissue. RPL
= reduction in parasitic load in %.
35 MouseLiver(RPL)Spleen(RPL)
1 4 + 1(71%)49 + 3(83%)
2 3 + 2(78%)39 + 2(86%)
CA 02256124 1999-10-22
41
3 5 + 1(64%)50 + 4(83%)
4 11 + 3(20%)153 + 10(48%)
Controls
4 animals. Mean.14 + 5295 + 30
Example 2
In order to verify the effect of vaccination with
protein Q on the reduction of the parasitic load on the
long term, using another route of inoculation, 4 animals
were injected subcutaneously with 5 micrograms of protein
Q dissolved in 40 microlitres of PBS and mixed with 40
microlitres of Freund's adjuvant. In the first
immunisation, Freund's complete adjuvant was employed,
while in the two subsequent ones, incomplete Freund's
adjuvant was used as indicated above. Vaccination was
administered in three doses spaced at 15 day intervals.
Fifteen days after the third immunisation they were
administered an inoculum of 10 infective parasites.
During all of the immunisation period and throughout the
whole of the infection (five months) blood was extracted
to determine the kind of humoral response against protein
Q and against the total proteins of the parasite. Figure
2 shows that already after the second week of
immunisation, the response against protein Q was
positive, as in the previous case, in three of the mice,
and that the response against the protein was very high
after two weeks of the first immunisation. The response
kept on being very high after the remaining
immunisations, reaching a titre of 1/75.000 on the week
following the third immunisation. In one of the animals
the response against protein Q was slower in relation to
time, although the response reached the level of that in
the other animals by the end of the experiment.
Consequently, from the data derived both from example 1
and from example 2, it is possible to conclude that the
CA 02256124 1999-10-22
42
degree of the immune response against the protein, by
both routes, intraperitoneal and subcutaneous, is very
rapid, although the response via the intraperitoneal
route attained higher titres in the same times (1/75.000
versus 1/30.000). Figure 3 indicates the response against
protein Q in the control animals. It is possible to
observe that reactivity against this protein is detected
on the 14th week after infection, which is when the first
symptoms attributable to a potential leishmaniasis begin
to be detected in the animals infected. Table 2 shows the
levels of parasitemia in the liver and spleen of the
control and vaccinated animals. It can be seen that 50%
of the animals were protected at the level of the liver,
in the sense that the reduction in the level of
parasitemia was very high (87-89%). One of the animals
was not protected, whereas in another of the animals the
reduction of the parasitic load was of 22%. On the
contrary, the reduction of the parasitic load was of 98-
99% in 100% of the animals at the level of the spleen. It
was observed that there is no relation between the degree
of protection and the reduction of the parasitic load.
Table 2. Parasitic load in the liver and spleen of
hamsters vaccinated with protein Q by the
subcutaneous route. After 20 weeks of infection,
the parasitic load was measured by the method of
limit dilutions. The parasitic load is expressed as
parasites per milligram of tissue. RPL = reduction
in parasitic load in %.
MouseLiver(RPL)Spleen(RPL)
5 1.4 x 106(22%)1.0 x 107(98%)
6 2.0 x 105(89%)4.9 x 105(99.9%)
7 2.4 x 106(87%)3.3 x 105(99.9%)
8 1.9 x 106(0%)6.3 x 106(99.9%)
_ .
CA 02256124 1999-10-22
43
Controls
4 animals. Mean. 1.8x108 + 1.2x105 5.2 x1 08
2.6x107
With the object of testing if protein Q could be
used to design protection systems in formulations which
contained protein LiHsp70 of Leishmania infantum, an
experiment was carried out in Balb/c mice. It was
observed that after immunisation, the parasitic load both
in the liver as in the spleen was reduced significantly,
being in some of the animals, four orders of magnitude
lower.
CA 02256124 1999-10-22
44
Immunisations with protein Q + protein Hsp70 in Freund's
Adjuvant.
Example 3
Each one of 4 hamsters were injected
intraperitoneally with 5 micrograms of protein Q and 5
micrograms cf protein LiHsp70 dissolved in 40 microlitres
of PBS and emulsified in 40 microlitres of Freund's
adjuvant. In the first immunisation, Freund's complete
adjuvant was employed, while in the two subsequent ones,
incomplete Freund's adjuvant was used as indicated above.
Vaccination was administered in three doses spaced at 15
day intervals.
Fifteen days after the third immunisation they were
administered an inoculum of 10 infective parasites.
Every two weeks, for all of the immunisation period and
throughout the whole of the infection, blood was
extracted from them to determine the degree of humoral
response against protein Q and against protein LiHsp70.
45 days after the third dose they were administered 10'
parasites via the intracardiac route and were sacrificed
at week 22. Figure 6a shows that all the animals respond
immunologically to protein Q, and that starting from the
second week after immunisation the response against
protein Q was very high and this response increased after
the second immunisation (week 4). The response against
protein LiHsp70 throughout the whole time of the
experiment was also positive, although lower than that
observed against Q, vide Fig 6b. The difference between
the parasitLc load in the spleen of control and immunised
animals was of 3 orders of magnitude, measured in
parasites per milligram of tissue.
CA 02256124 2008-02-06
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: C.B.F. Leti S.A.
(ii) TITLE OF INVENTION: Chimeric Gene Formed of the DNA Sequence
that Encode the Antigenic Determinants of Four Proteins of
L. Infantum, and Protein Encoded by Said Gene, and
Pharamaceutical Composition Useful for ...
(iii) NUMBER OF SEQUENCES: 12
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BERESKIN & PARR
(B) STREET: 40 King Street West
(C) CITY: Toronto
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) ZIP: M5H 3Y2
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,256,124
(B) FILING DATE: 23-DEC-1998
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gravelle, Micheline
(B) REGISTRATION NUMBER: 4189
(C) REFERENCE/DOCKET NUMBER: 444-153
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 364-7311
(B) TELEFAX: (416) 361-1398
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 412 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Met Arg Gly Ser His His His His His His Thr Asp Pro His Ala Ser
1 5 10 15
Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile Glu Gly
20 25 30
Arg Pro Leu Ala Thr Pro Arg Ser Ala Lys Lys Ala Val Arg Lys Ser
35 40 45
,
CA 02256124 2008-02-06
. ,
46
Gly Ser Lys Ser Ala Lys Cys Gly Leu Ile Phe Pro Val Gly Arg Val
50 55 60
Gly Gly Met Met Arg Arg Gly Gln Tyr Ala Arg Arg Ile Gly Ala Ser
65 70 75 80
Gly Ala Pro Arg Ile Ser Glu Phe Ser Val Lys Ala Ala Ala Gln Ser
85 90 95
Gly Lys Lys Arg Cys Arg Leu Asn Pro Arg Thr Val Met Leu Ala Ala
100 105 110
Arg His Asp Asp Asp Ile Gly Thr Leu Leu Lys Asn Val Thr Leu Ser
115 120 125
His Ser Gly Val Val Pro Asn Ile Ser Lys Ala Met Ala Lys Lys Lys
130 135 140
Gly Gly Lys Lys Gly Lys Ala Thr Pro Ser Ala Pro Glu Phe Gly Ser
145 150 155 160
Ser Arg Pro Met Ser Thr Lys Tyr Leu Ala Ala Tyr Ala Leu Ala Ser
165 170 175
Leu Ser Lys Ala Ser Pro Ser Gln Ala Asp Val Glu Ala Ile Cys Lys
180 185 190
Ala Val His Ile Asp Val Asp Gln Ala Thr Leu Ala Phe Val Met Glu
195 200 205
Ser Val Thr Gly Arg Asp Val Ala Thr Leu Ile Ala Glu Gly Ala Ala
210 215 220
Lys Met Ser Ala Met Pro Ala Ala Ser Ser Gly Ala Ala Ala Gly Val
225 230 235 240
Thr Ala Ser Ala Ala Gly Asp Ala Ala Pro Ala Ala Ala Ala Ala Lys
245 250 255
Lys Asp Glu Pro Glu Glu Glu Ala Asp Asp Asp Met Gly Pro Ser Arg
260 265 270
Val Asp Pro Met Gln Tyr Leu Ala Ala Tyr Ala Leu Val Ala Leu Ser
275 280 285
Gly Lys Thr Pro Ser Lys Ala Asp Val Gln Ala Val Leu Lys Ala Ala
290 295 300
Gly Val Ala Val Asp Ala Ser Arg Val Asp Ala Val Phe Gln Glu Val
305 310 315 320
Glu Gly Lys Ser Phe Asp Ala Leu Val Ala Glu Gly Arg Thr Lys Leu
325 330 335
Val Gly Ser Gly Ser Ala Ala Pro Ala Gly Ala Val Ser Thr Ala Gly
340 345 350
Ala Gly Ala Gly Ala Val Ala Glu Ala Lys Lys Glu Glu Pro Glu Glu
355 360 365
' CA 02256124 2008-02-06
, .
,
. .
47
Glu Glu Ala Asp Asp Asp Met Gly Pro Val Asp Leu Gin Pro Ala Ala
370 375 380
Ala Ala Pro Ala Ala Pro Ser Ala Ala Ala Lys Glu Glu Pro Glu Glu
385 390 395 400
Ser Asp Glu Asp Asp Phe Gly Met Gly Gly Leu Phe
405 410
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1436 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
atgagaggat ctcaccacca ccaccaccac acggatccgc atgcgagctc gaacaacaac 60
aacaataaca ataacaacaa cctcgggatc gagggaaggc ctttagctac tcctcgcagc 120
gccaagaagg ccgtccgcaa gagcggctcc aagtccgcga aatgtggtct gatcttcccg 180
gtgggccgcg tcggcgggat gatgcgccgc ggccagtacg ctcgccgcat cggtgcctct 240
ggcgccccca ggatttcaga attctccgtg aaggcggccg cgcagagcgg gaagaagcgg 300
tgccgcctga acccgcgcac cgtgatgctg gccgcgcgcc acgacgacga catcggcacg 360
cttctgaaga acgtgacctt gtctcacagc ggcgttgtgc cgaacatcag caaggcgatg 420
gcaaagaaga agggcggcaa gaagggcaag gcgacaccga gcgcgcccga attcggatcc 480
tctagaccca tgtccaccaa gtacctcgcc gcgtacgctc tggcctccct gagcaaggcg 540
tccccgtctc aggcggacgt ggaggctatc tgcaaggccg tccacatcga cgtcgaccag 600
gccaccctcg cctttgtgat ggagagcgtt acgggacgcg acgtggccac cctgatcgcg 660
gagggcgccg cgaagatgag cgcgatgccg gcggccagct ctggtgccgc tgctggcgtc 720
actgcttccg ctgcgggtga tgcggctccg gctgccgccg ccgcgaagaa ggacgagccc 780
gaggaggagg ccgacgacga catgggcccc tctagagtcg accccatgca gtacctcgcc 840
gcgtacgccc tcgtggcgct gtctggcaag acgccgtcga aggcggacgt tcaggctgtc 900
ctgaaggccg ccggcgttgc cgtggatgcc tcccgcgtgg atgccgtctt ccaggaggtg 960
gagggcaaga gcttcgatgc gctggtggcc gagggccgca cgaagctggt gggctctggc 1020
tctgccgctc ctgctggcgc tgtctccact gctggtgccg gcgctggcgc ggtggccgag 1080
gcgaagaagg aggagcccga ggaggaggag gccgatgatg acatgggccc cgtcgacctg 1140
cagcccgccg ctgccgcgcc ggccgcccct agcgccgctg ccaaggagga gccggaggag 1200
CA 02256124 2008-02-06
48
agcgacgagg acgacttcgg catgggcggt ctcttctaag cgactcgcca tctcttagcc 1260
tccttgtggt gcgcttgagg tgctctcgct ctgcttctcc ttgcagtgtt ggctgactct 1320
ggcgggtatg tgccgtcgca ttacacccac ctctcccacc cctttgccct acgcgctcgc 1380
atgcgcaatc cgtgaatcat cgagggaagt ctctctgggt ggcagtgggt aagctt 1436
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 328 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ile Glu Gly Arg Pro Leu Thr Pro Arg Ser Ala Lys Lys Ala Val Arg
1 5 10 15
Lys Ser Gly Ser Lys Ser Ala Lys Cys Gly Leu Ile Phe Pro Val Gly
20 25 30
Arg Val Gly Gly Met Met Arg Arg Gly Tyr Ala Arg Arg Ile Gly Ala
35 40 45
Ser Gly Ala Pro Arg Ile Ser Glu Phe Ser Val Lys Ala Ala Ala Gln
50 55 60
Ser Gly Lys Lys Arg Cys Arg Leu Asn Pro Arg Thr Val Met Leu Ala
65 70 75 80
Ala Arg His Asp Asp Asp Ile Gly Thr Leu Leu Lys Asn Val Thr Leu
85 90 95
Ser His Ser Gly Val Val Pro Asn Ile Ser Lys Ala Met Ala Lys Lys
100 105 110
Lys Gly Gly Lys Lys Gly Lys Ala Thr Pro Ser Ala Pro Glu Phe Gly
115 120 125
Asp Ser Ser Arg Pro Met Ser Thr Lys Tyr Leu Ala Ala Tyr Ala Leu
130 135 140
Ala Ser Leu Ser Lys Ala Ser Pro Ser Gln Ala Asp Val Glu Ala Ile
145 150 155 160
Cys Lys Ala Val His Ile Asp Val Asp Gln Ala Thr Leu Ala Phe Val
165 170 175
Met Glu Ser Val Thr Gly Arg Asp Val Ala Thr Leu Ile Ala Glu Gly
180 185 190
Ala Ala Lys Met Ser Ala Met Pro Ala Ala Ser Ser Gly Ala Ala Ala
195 200 205
Gly Val Thr Ala Ser Ala Ala Gly Asp Ala Ala Pro Ala Ala Ala Ala
CA 02256124 2008-02-06
49
210 215 220
Ala Lys Lys Asp Glu Pro Glu Glu Glu Ala Asp Asp Asp Met Gly Pro
225 230 235 240
Ser Val Arg Asp Pro Met Gin Tyr Leu Ala Ala Tyr Ala Leu Val Ala
245 250 255
Leu Ser Gly Lys Thr Pro Ser Lys Ser Thr Ala Gly Ala Gly Ala Gly
260 265 270
Ala Val Ala Glu Ala Lys Lys Glu Glu Pro Glu Glu Glu Glu Ala Asp
275 280 285
Asp Asp Met Gly Pro Val Asp Leu Gln Pro Ala Ala Ala Ala Pro Ala
290 295 300
Ala Pro Ser Ala Ala Ala Lys Ala Ala Pro Glu Glu Ser Asp Glu Asp
305 310 315 320
Asp Phe Gly Met Gly Gly Leu Phe
325
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCTTTAGCTA CTCCTCGCAG CGCCAAG 27
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CCTGGGGGCG CCAGAGGCAC CGATGCG 27
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02256124 2008-02-06
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GAATTCTCCG TAAGGCGGCC GCGCAG 26
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GAATTCGGGC GCGCTCGGTG TCGCCTTGCC 30
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GTCGACCCCA TGCAGTACCT CGCCGCGTAC 30
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GTCGACGGGG CCCATGTCAT CATCGGCCTC 30
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
TCTAGACCCG CCATGTCGTC GTCTTCCTCG CC 32
CA 02256124 2008-02-06
51
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
TCTAGAGGGG CCATGTCGTC GTCGGCCTC 29
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CTGCAGCCCG CCGCTGCCGC GCCGGCCGCC 30
