Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DIFFERENTIALLY EXPRESSED LEISHMANIA GENES AND PROTEINS
The present invention is related to molecular cloning of
Leishmania genes and, in particular, to the cloning of
amastigote differentially expressed genes from Leishmania
donovani.
Leishmania potozoans are the causative agents of human
leishmaniasis, which includes a spectrum of diseases ranging
from self-healing skin ulcers to fatal visceral infections.
Human leishmaniasis is caused by at least thirteen different
species and subspecies of parasites of the genus Leishmania.
Leishmaniasis has been reported from about eighty countries and
probably some 400,000 new cases occur each year. Recently, the
World Health Organization has reported 12 million people to be
infected (ref. 1 - a listing of the references appears at the
end of the disclosure).
L. donovani causes visceral leishmaniasis, also known as
kala-azar. L. brasiliensis causes mucotaneous leishmaniasis
and L. major causes cutaneous leishmaniasis. Untreated
visceral leishmaniasis is usually fatal and mucocutaneous
leishmaniasis produces mutilation by destruction of the naso-
oropharyngeal cavity and, in some cases, death.
In addition, a major health problem has been created in
areas of high infection when blood is collected for transfusion
purposes. Since blood is a carrier of the parasites, blood
from an infected individual may be unknowingly transferred to a
healthy individual.
The Leishmania protozoans exist as extracellular
flagellated promastigotes in the alimentary tract of the
sandfly in the free-living state, and are transmitted to the
mammalian host through the bite of the insect vector. Once
introduced, the promastigotes are taken up by
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2~Q~3~
macrophages, rapidly differentiate into non-flagellated
amastigotes and start to multiply within the
phagolysosomal compartment. As the infected cells
rupture, amastigotes subsequently infect other
macrophages giving rise to the various symptoms
associated with leishmaniasis (refs. 1 and 2). In this
manner, it is the amastigote form of the parasite which
is responsible for the pathology in humans.
While in the midgut of the insect, newly transformed
promastigotes, functionally avirulent, progressively
acquire capacity for infection and migrate to the
mouthparts (ref. 3). This process, termed the
metacyclogenesis, which occurs only in promastigotes, is
concurrent with the differential expression of major
surface glycoconjugates which mediate the migration of
promastigotes in the alimentary tract and prepare the
organism for the serum environment (refs. 4 and S). In
comparison, the promastigote to amastigote
cytodifferentiation is a profound morphological and
physiological transformation. During the promastigote to
amastigote differentiation, the parasite looses its
flagellum, rounds-up, changes its glycoconjugate coat
(refs. 6, 7 and 8) and expresses a set of metabolic
enzymes optimally active at low pH. The survival of the
parasite inside the macrophage phagolysosome is
associated with its ability to down-regulate many
effector and accessory functions of its host cell,
including oxygen metabolite-mediated killing and the
capacity of the macrophage to act as an efficient antigen
presenting cell (reviewed in, for example, ref. 9).
Leishmaniasis is, therefore, a serious disease and
various types of vaccines against the disease have been
developed, including live parasites; frozen promastigotes
from culture; sonicated promastigotes; gamma-irradiated
live promastigotes; and formalin-killed promastigotes
treated with glucan (reviewed in, for example, ref. 10).
5 ~ ~ ~
However, none of these approaches have provided satisfactory
results.
The promastigote-amastigote differentiation is important
to the establishment of infection. It would be desirable to
identify genes and gene products that are differentially
expressed when the amastigotes are present in macrophages.
Joshi, et al. describe L. donovani genes that are
expressed at about two-fold higher in in vitro generated and
maintained "amastigotes" compared to promastigotes (ref. 11).
The present invention is directed towards the provision of
a Leishmania protein that is differentially expressed in the
amastigote stage when the Leishmania organism is present within
macrophages and genes encoding the differentially expressed
protein. The amastigote differentially expressed gene and
protein are useful for the preparation of vaccines against
disease caused by Leishmania, the diagnosis of infection by
Leishmania and as tools for the generation of immunological
reagents and the generation of attenuated variants of
Leishmania.
In accordance with one aspect of the present invention,
there is provided a purified and isolated DNA molecule, the
molecule comprising at least a portion coding for a
differentially expressed gene of a Leishmania organism, the
differentially expressed gene being expressed at an increased
level when the amastigote form of the Leishmania organism is
present within a macrophage. The increased level of expression
maybe at least about a ten-fold increase in expression. In one
embodiment of the present invention, the differentially
expressed gene may be a virulence gene of the Leishmania
organism and may be required for maintenance of infection by
the amastigote form of the Leishmania organism.
In a further aspect of the invention, the differentially
expressed virulence gene is functionally disabled by, for
example, deletion or mutagenesis, such as insertional
mutagenesis, to produce an attenuated Leishmania organism for
~.~
CA 0210~38 1998-11-16
use as, for example, a live vaccine. Conveniently, strains of
Leishmania from which differentially expressed genes may be isolated
include Leishmania donovani.
Further aspects of the invention include the protein encoded by
the differentially expressed gene, and the use of the protein in
vaccination and diagnosis. Additional aspects of the invention
include an attenuated strain of Leishmania in which the virulence gene
is disabled and a vaccine comprising the same.
In the following description, reference is made to the
accompanying drawings, in which:
Figure 1 shows a schematic outline of the amastigote cDNA
library construction and differential screening with amastigote and
promastigote-specific cDNA probes. An example of an amastigote-
specific cDNA clone is indicated by an arrow on the colony
hybridization autoradiogram;
Figure 2 shows a restriction enzyme and size analysis of
Leishmania donovani amastigote-specific cDNA clones;
Figure 3 shows a Southern blot analysis of Leishmania donovani
amastigote-specific cDNA clones;
Figure 4 shows a Northern blot analysis to ~em~qtrate that A2-
specific transcripts are present in amastigote-infected macrophages
but not promastigotes;
Figure 5 shows a Southern blot analysis to ~mo~qtrate that A2
transcripts are encoded by a multigene family;
Figure 6 shows a restriction map of plasmid pGECO 90 that
contains the L. donovani A2 gene;
Figure 7 shows a restriction map of a genomic clone of the A2
gene and its relationship to A2-related cDNAs;
Figure 8 shows the nucleotide sequence (SEQ ID NO: 2) and
deduced amino acid sequence (SEQ ID NO: 3) of the open reading frame
II (ORF II) of the Leishm~n;a donovani A2 gene as well as the
nucleotide sequence of the full-length XhoI to XbaI fragment (SEQ ID
no.: l);
Figure 9 shows the homology between the Leishm~n-a donovani A2
protein (SEQ ID NO: 3) and the Plasmodium
5 ~ 5 ~ ~
falciparum S antigen (SEQ ID NO: 4) within the repeated
subunits of these proteins;
Figure 10 shows the construction of a plasmid pET 16b/ORF
II+ for expression of the A2 protein;
Figure 11 shows the presence of antibodies against A2
fusion protein in kala-azar immune serum by
immunoprecipitation; and
Figure 12 shows the specific recognition of A2 fusion
protein by kala-azar sera by Western blot analysis.
Referring to Figure 1, there is illustrated a method used
for isolating a Leismania gene differentially expressed during
the amastigote stage in the life cycle thereof. The method
comprises the steps of (a) constructing a cDNA library from the
Leishmania organism in the amastigote stage in the life cycle
thereof; (b) constructing a first mixture of cDNA probes
specific for the amastigote stage in the life cycle; (c)
constructing a second mixture of cDNA probes specific for the
promastigote stage in the life cycle; (d) separately probing
the cDNA library with the amastigote and promastigote-specific
cDNA probes in order to identify cDNA clones that are
recognized by the amastigote mixture of cDNA probes but not the
promastigote mixture of cDNA probes; and (e) isolating the cDNA
clones identified in step (d).
The amastigote-specific cDNA clones identified by the
above procedure can be further characterized by restriction
enzyme analysis and their relatedness determined by Southern
hybridization studies. To determine if cDNA clones identified
by the above procedure represent amastigote-specific clones
that are expressed at a higher level (more than about ten-fold
higher) when the amastigote form of the Leishmania organism is
present within macrophages, macrophages were infected with
amastigotes and differentially-expressed gene transcripts were
detected by Northern blot analysis. In an embodiment of the
present invention, the differentially expressed Leishmania gene
is L. donovani gene that is expressed at an increased level
when the amastigote form of the Leishmania organism is present
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5 3 8 ~t
within a macrophage. The intracellular environment of the
macrophage has an acidic pH of, for example, about 4.5. The
differentially expressed genes include those having sequences,
such as the DNA sequence set out in Figure 8 (SEQ ID Nos: 1
and 2) or its complementary strand; and DNA sequences which
hybridize under stringent conditions to such DNA sequences.
Such differentially expressed gene sequences include the A2
gene of L. donovani having the DNA sequence set out in Figure 8
and the lnvention includes a cDNA clone encoding the A2 gene
depicted in Figure 8, which clone may be in the form of a
plasmid, particularly that designated pGEC0 90 (Figure 6),
which has ATCC accession number ATCC 75510.
The differentially expressed genes may encode proteins,
such as the 22 kD A2 protein (SEQ ID No: 2) being encoded by
the longest open reading frame (ORF II) of the A2 gene. Most
of the predicted A2 protein is composed of a repetitive
sequence consisting of a stretch of ten amino acids repeated
nineteen times (Figure 8). Since each unit of this repeat
contains two serines, two valines, two leucines and two
prolines separated from each other by five residues, the
repeated region also may be considered as a stretch of five
amino acids repeated thirty-eight times. The amino acid
sequence of the A2 protein has homology with an S-antigen of
Plasmodium falciparum (SEQ ID NO: 4), as shown in Figure 9. As
with the L. donovani A2 protein, the carboxy-terminal portion
of the S-antigen of P. falciparum Vietnamese isolate VI is
composed of a stretch of eleven amino acids repeated nineteen
times; the repeated units of both proteins are 50% identical
and 80~ homologous.
Life cycle stage specific genes from Leishmania may be
isolated in the present invention. Some of these genes are
required for transition between the life cycle stages and
include virulence genes of the Leishmania parasite, such as
virulence genes that are required for maintenance of infection
by the amastigote form of the Leishmania organism. These
3 ~ -~
virulence genes may be functionally disabled by, for example,
deletion or mutation, including insertional mutagenesis and,
furthermore, the wild-type Leishmania gene may be replaced by
the functionally disabled gene. The virulence genes may be
functionally disabled by, for example, replacing the A2 gene by
a selectable antibiotic resistance gene by homologous
recombination following transformation of the Leishmania
organism with a fragment of DNA containing the antibiotic
resistance gene flanked by 5'- and 3'- non-coding DNA
sequences. This process can be used to generate attenuated
variants of Leishmania and the residual pathogenicity of the
attenuated variants can be assessed in mice and hamsters pigs.
It is likely that deletion of genes that are selectively
expressed in the human host environment (that being when the
Leishmania organism is inside the macrophage cell) result in an
attenuated strain which cannot survive in humans but generates
a protective immune response. Attenuated strains of Leishmania
would be useful as live vaccines against the diseases caused by
Leishmania and such attenuated strains form an aspect of the
present invention.
2 ~
Differentially expressed genes and proteins of
Leishmania typified by the embodiments described herein
are advantageous as:
- antigens for vaccination again~t the diseases
caused by Leishmania.
- diagnostic reagents including hybridization
probes, antigens and the means for producing
specific antisera for (for example) detecting
infection by Leishmania.
- target genes for functional
disablement for the generation of
attenuated Leishmania variants.
Vaccines comprising an effective amount of the
protein encoded by the differentially expressed genes or
of an attenuated strain of Leishmania and a
physiologically-acceptable carrier therefor may utilize
an adjuvant as the carrier and the protein may be
presented to the immune system of the host in combination
with an ISCOM or liposome. The vaccine may be formulated
to be administered to a host in an injectable form,
intranasally or orally, to immunize the host against
disease.
BIOLOGICAL DEPOSITS
A plasmid pGECO 90 described and referred to herein
was deposited with the American Type Culture Collection
(ATCC) located at Rockville, Maryland, USA, pursuant to
the Budapest Treaty on July 28, 1993 and prior to the
filing of this application and assigned the ATCC
accession number 75510. A diagram of this plasmid is
shown in Figure 6. The plasmid contains the A2 gene of
L. donovani described herein. The plasmid will become
available to the public upon grant of a patent based upon
this United States patent application. The invention
described and claimed herein is not to be limited in
scope by the material deposited, since the deposited
embodiment is intended only as an illustration of the
2 1~J~ 3~
invention. Any equivalent materials are within the scope
of the invention.
EXAMPLES
The above disclosure generally describes the present
invention. A more complete understanding can be obtained
by reference to the following specific Examples. These
Examples are described solely for purposes of
illustration and are not intended to limit the scope of
the invention. Changes in form and substitution of
equivalents are contemplated as circumstances may suggest
or render expedient. Although specific terms have been
employed herein, such terms are intended in a descriptive
sense and not for purposes of limitations.
Methods of molecular genetics and protein
biochemistry used but not explicitly described in this
disclosure and these Examples, are amply reported in the
scientific literature and are well within the ability of
those skilled in the art.
Example 1
This Example describes culturing and isolation of
Leishmania organisms.
Amastigotes of the L. donovani Ethiopian LV9 strain
were harvested from spleens of infected female gold
Symian hamsters and purified as described previously
(ref. 12). Briefly, parasites were released from tissue
using an homogenizer, the mixture was centrifuged three
times at 100xg to remove cellular debris, and amastigotes
were pelleted at 1500xg. The pellet was resuspended in
0.17 M sodium acetate to lyse contaminating red blood
cells and amastigotes were recovered by centrifugation at
1500xg. Organisms were incubated at 37 C in complete
RPMI medium (RPMI 1640 supplemented with 10~ endotoxin
free heat-inactivated FBS, 10 ml of lM HEPES pH 7.3, 100
U of penicillin and 100 U of streptomycin per ml) for 18
hours prior to RNA extraction. After this period of
incubation and multiple washes, the amastigote
lo ~ 5 ~
preparation was still physiologically active and relatively
free of host cell contamination. To obtain promastigotes, LV9
strain amastigotes were allowed to differentiate in complete
RPMI medium at 26 C, and cultured for at least seven days in the
same medium before use (ref. 12).
Promastigotes of the L. donovani Sudanese strain lS2D were
cultivated and passaged in complete RPMI medium at 26 C.
Amastigote-like organisms of the lS2D strain were cultivated as
described by Doyle et al. (ref. 13). The Sudanese strains lS2D
and lS2D (wt) were obtained from Dr. S. Turco, the University
of Kentucky, USA. The lS2D (wt) promastigotes were adapted to
grow in axenic conditions and had lost the ability to transform
into infective promastigotes.
Example 2
This Example describes the preparation of and screening of
a Leishmania cDNA library.
A method for isolating a Leishmania gene differentially
expressed during the amastigote stage in the life cycle of the
organism is illustrated in Figure 1.
Total RNA of amastigotes and promastigotes was prepared by
the guanidinium isothiocyanate method using RNAzolTM
(Cinna/biotecx Laboratories International Inc., Friendswood,
TX); poly A+ RNA was selected by oligo dT cellulose
chromatography (grade 7:Pharmacia) as described by Sambrook et
al. (ref. 14). A total of 10 mg of amastigote mRNA was used to
construct an E RI/ Xho I unidirectional cDNA library of 106
clones in the l ZAP II vector (Stratagene)i hemi-methylated
cDNA was produced using the manufacturers reagents and
protocols. About 40,000 amastigote and promastigote-specific
clones of the primary library were screened differentially with
amastigote and promastigote stage-specific gene probes. The
cDNA probes were prepared using oligo dT12_18 primer (Pharmacia)
and M-MLV reverse transciptase (BRL) following protocols
previously described (ref. 15). Duplicate filters were
hybridized with each probe for 18 h at 42 C in 50~ formamide, 6X
~ 1 ~ 5 5 ~
11
SSC, 5X Denhardt's solution, 5% dextran sulfate. Membranes
then were washed twice at room temperature in lX SSC for 20
min, twice at 55 C in lX SSC, 0.1% SDS and then autoradiographed
on Kodak X-OMATTM films with an intensifying screen for 18 to 72
hours. Areas on the plates containing putative clones of
interest were picked and the phage pools were submitted to a
second round of screening. An example of an amastigote-
specific cDNA clone is indicated by the arrow on the plaque
hybridization autoradiogram of Figure 1.
Although cDNA clones representing promastigote-specific
transcripts were more abundant than clones representing
amastigote-specific transcripts, seven independent cDNA clones
which only hybridized with amastigote-specific probes were
isolated and termed 2, 3, 5, 6, 8, 9, 11. For each cDNA clone
isolated, a Bluescript plasmid derivative was excised from the
lZAP II recombinant phages in vivo using the helper phage R-
408.Example 3
This Example describes the characterization of amastigote-
specific cDNA clones.
The insert size of each of the Bluescript plasmids
corresponding to the amastigote-specific cDNA clones was
determined by restriction enzyme digestion and agarose gel
electrophoresis (Figure 2). Recombinant plasmids (A2, A3, A5,
A6, A8, A9 and A11) were digested with Eco RI and Xho I to
excise the cDNA inserts. Fragments were separated on a 1%
agarose gel and stained with ethidium bromide. The cDNA
inserts varied from 0.5 kb (A5) to 1.8 kb and A8 contained an
internal E RI site. To determine if the amastigote-specific
cDNA clones contain
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21~5~33
common sequences, Southern blot hybridization analysis of
the Bluescript plasmids corresponding to the amastigote-
specific cDNA clones was performed using clone A2 and
clone A6 specific probes (Figure 3).
For Southern blot analysis, 10 ~g of total DNA was
cut to completion with the restriction enzymes Eco RI and
Xho I and separated on a 1~ agarose gel. The restriction
fragments were transferred to nylon membranes using
standard procedures (ref. 16) and duplicates hybridized
with ~_32p dCTP nick-translated probes representing the
inserts of the cDNA clones A2 (0.9kb) or A6 (0.6kb). The
A2 probe recognized five cDNAs (A2, A3, A8, A9 and A11)
and the A6 cDNA only hybridized to itself. Thus, this
Southern blot analysis indicated that cDNA clones A2, A3,
A8, A9 and A11 contained homologous sequences but A5 and
A6 were clones of unrelated amastigote-specific
transcripts.
To confirm that the A2 series of clones represented
Leishmania genes that were differentially expressed when
the Leishmania organism is present in macrophages
compared to expression in the free-living promastigotes,
Northern blot analysis was performed. Total RNA was
extracted from bone marrow-derived macrophages (BMM), L.
donovani LV9-infected BMM (IBMM) and L. donovani LV9
promastigotes (PRO). Murine bone marrow-derived
macrophage cultures and L. donovani amastigote in vitro
infections were carried out as previously described (ref.
12). The RNA species (15 ~g) were separated on an
agarose gel and stained with ethidium bromide prior to
transfer (Figure 4, right panel). The RNA was denatured
by glyoxal treatment and transferred to a nylon membrane.
The Northern blot was hybridized with labelled cDNA A2
(0.9 kb) fragment, as previously described (ref. 12)
(Figure 4, left panel). This probe recognized
predominantly a 3.5 kb transcript present in amastigote-
infected macrophages but not in promastigotes or in non-
2 l ~533
infected macrophages. This analysis showed that the A2gene was differentially expressed at an increased level
in amastigotes when they were present in macrophages
compared to a free-living existence and that the
increased expression was at least a ten fold increase.
Exam~le 4
This Example describes the genomic arrangement and
sequencing of the Leishmania donovani amastigote-specific
A2 gene.
Regulation of transcription is one of the unusual
features of the genetics of trypanosomatids. Copies of
a gene or related genes are often clustered in tandem
arrays on the same chromosome and a unique promoter
region regulates expression of the cluster.
Transcription leads to the synthesis of a polycistronic
RNA molecule which is cleaved into monomeric units by
trans-splicing prior to translation. The genomic
arrangement of A2 related gene(s) was investigated by
Southern blot analysis to determine whether it represents
a multigene family. Total DNA was digested to completion
with several restriction enzymes (E: Eco RI, S: SalI, X:
Xba I, C: Cla I, P: Pvu II). For double digests, the DNA
was first cut to completion with Cla I or Pvu II, the DNA
precipitated and resuspended in the appropriate buffer
for the second digestion. Restriction fragments were
separated on a 0.7~ agarose gel, transferred to a nylon
membrane and hybridized with a 0.5 kb Pst I/Xho I
fragment of the A2 cDNA insert nick-translated with ~_32p
dCTP. For each digest, the hybridization pattern
displayed a series of bands of different intensities,
clearly showing that many copies of the gene were present
in the genome (Figure 5). Moreover, common bands at
about 6 to 8 kb for the Eco RI, Xba I and Sal I digests
suggested an arrangement in tandem arrays. However, the
presence of at least two other bands in each lane
suggested that more than one cluster existed, each
14 ~ 3~ ~
cluster belng flanked by restriction fragments of different
sizes. Alternatively, clusters also may carry copies of
unrelated genes or intergenic regions of variable sizes.
To identify the protein coding region of A2, genomic
clones carrying the A2 gene sequence were isolated. A partial
genomic library containing 6 to 10 kb Eco RI fragments was
constructed in the lambda ZAP II vector (Stratagene). More
than 2,000 clones were screened on duplicate filters with
probes prepared with the A2 cDNA using techniques and
hybridization conditions described in Example 2. Eight clones
were isolated and purified. Bluescript plasmid derivatives
were excised from recombinant 1 phages as for cDNA clones.
The 1.9 kb Xho I/ Eco RI insert fragment of the A2
Bluescript clone was subcloned into the Bluescript phagemids KS+
and KS- for sequencing. Nested deletions were carried out on
both plasmids using Exo III exonuclease and S1 nuclease.
Sequencing reactions were performed on single-strand DNA
templates using the M13K07 helper phage according to published
procedures (ref. 17) with the DeazaTM G/A sequencing mixes
(Pharmacia) and d35ATP or d35CTP radio-isotopes. Reactions were
analysed on 6% denaturing gels. The inserts of the genomic
clones were mapped with several restriction enzymes and
displayed similar patterns, except some inserts were longer
than others. One of these clones, pGECO 90 (as shown in Figure
6), was selected for further characterization. Figure 7 shows
the restriction map of the insert of pGEC0 90 and how it
corresponds to the A2 related cDNAs. The restriction enzymes
shown in Figure 7 are S: Sal I, P: Pst I, O: Xho I, X: Xba I,
E: Eco RI, M: Sma I. Plasmid pGECO 90 contained unique sites
for Sal I and Xba I, but no Cla I site, and this was consistent
with the Southern blot analysis shown in Figure 5. The DNA
sequence flanking the Eco RI site on this genomic clone was
determined and shown to correspond exactly to the related
portion of the A8 cDNA, confirming that this fragment
represented one unit of the tandem array.
~3 ~
7~
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The DNA sequence of the 1.9 kb Xho I/ Eco RI fragment of
the pGECO 90 genomic clone corresponding to the 3.5 kb A2
transcript was determined (Figure 8) and compared to the cDNA's
sequences. The longest open reading frame (ORF II) found was
contained in the X I/ Xba I 1.1 kb fragment and potentially
encoded a 22 kD protein product (A2 protein). Stop codons were
observed in two other frames and upstream from the initiating
ATG. Most of this predicted A2 protein was composed of a
repetitive sequence consisting of a stretch of ten amino acids
repeated nineteen times. Since each unit of this repeat
contains two serines, two valines, two leucines and two
prolines separated from each other by five residues, the
repeated region could also be considered as a stretch of five
amino acids repeated thirty-eight times. The only hydrophobic
domain was located at the amino terminal portion and may
correspond to a signal peptide. The predicted amino acid
sequence was compared with proteins reported in the Swiss-Prot
database version using a Fasta algorithm (Canada Institute for
Scientific and Technical Information: Scientific Numeric
Database Service). The most striking identity was observed
with an S-antigen of Plasmodium falciparum Vietnamese isolate
VI. The alignment of the A2 protein sequence (A2) with the
carboxyl-terminal portion of the S-antigen of P. falciparum
isolate VI is shown in Figure 9. Identical residues are
indicated by dashes and homologous amino acids by dots. As
with the L. donovani A2 protein, the carboxy-terminal portion
of this antigen of P. falciparum Vietnamese isolate IV is
composed of a stretch of eleven amino acids repeated nineteen
times. The repeated units of both proteins are 50% identical
and 80% homologous. The S-antigen, as the CS-antigens of
Plasmodium, are
.~..
~'
2lass33
16
proteins which are stage-specific, being expressed in the
m~mmAlian host but not in the insect host. Therefore,
the A2 and S-antigen genes from unrelated human
infectious protozoa are expressed specifically in the
m~mm~lian host and encode similar proteins. Thus, the A2
and S-antigen proteins may perform similar functions and
may be required to enable these protozoa to survive in
humans and functional disablement of the A2 sequences in
L. donovani may be expected to result in a non-infective
promastigote useful as a live attenuated vaccine for
leishmaniasis.
ExamPle 5
This Example describes the functional disablement of
differentially expressed genes in Leishmania.
One approach for the development of attenuated
strains of Leishmania is to functionally disable
amastigote-specific genes (such as the A2 gene) from the
Leishmania genome (by for example deletion) using
homologous recombination. Deletion of genes from
protozoa (such as Leishmania) has been described (ref.
18). This procedure involves cloning DNA fragments 5'-
and 3'- to the A2 gene and constructing a plasmid vector
that contains these flanking DNA sequences sandwiching a
neomycin resistance gene. This 5'- neo 3'- fragment of
DNA then is used to transform L. donovani promastigotes
to G418 resistance. L. donovani is diploid and deletion
one allele of the A2 gene in such G418 resistant strains
can be determined by Southern blot hybridization using A2
specific probes. The second A2 allele then can be
deleted by constructing a second deleting vector
containing the 5~- and 3~- A2 flanking sequences
sandwiching a hygromycin resistance gene. Following
transformation colonies are selected on medium containing
G418 and hygromycin. Deletion of both copies of the A2
gene can be confirmed by Southern blot hybridization.
2 1~ 3 ~
17
Example 6
This Example describes the expression of the L.
donovani amastigote-specific A2 gene and the recognition
of the A2 gene product by kala-azar immune sera.
To produce the A2 protein in a heterologous system,
the coding region from the initiating ATG to the Xba I
restriction site (see Figure 8) was subcloned in the pET
16B expression vector in frame with the HIS-TAG (Figure
10). The A2 fusion protein of 27 kD was produced in an
in vitro transcription-translation assay (TNT system,
Promega) using the pET16b/ORF II plasmid and a negative
control pBluescript/p53 plasmid, encoding the human p53
protein. The in vitro translated HIS-TAG/A2 35S-labelled
protein was immunoprecipitated with kala-azar immune
serum and analyzed by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (Figure 11). Kala-
azar is a term used to describe the disease caused by L.
donovani. The kala-azar immune serum was obtained form
a patient suffering from visceral leishmaniasis and
reacted strongly against L. donovani antigens on ELISA.
In Figure 11, Lanes 1 and 2 contained the labelled
proteins A2 and p53, respectively, prior to
immunoprecipitation analysis. Lanes 3 and 4 contained
proteins A2 and p53, respectively, immunoprecipitated
with the kala-azar immune serum (L1) and Lanes 5 and 6
contained proteins A2 and p53, respectively,
immunoprecipitated with a control human serum (TXC). The
kala-azar serum did not react against the negative
control protein human p53 but did immunoprecipitate the
A2 gene-product. Neither of the proteins were
immunoprecipitated by the control human serum. This
analysis showed that the product of the L. donovani A2
gene was specifically recognized by kala-azar immune
serum.
To confirm the specificity of the immune reaction,
the pET 16b/ORF II plasmid coding for the recombinant A2
18 ~ 3~ ~
fusion protein and a negative control plasmid pET 16b with no
insert, were introduced into E. coli. Expression was induced
with IPTG, and total lysates of the recombinant E. coli cells
separated by SDS-PAGE and analyzed by Western blot analysis
using the kala-azar immune serum described above (see Figure
12). In Figure 12, Lane 1 contained E. coli/pET 16b cells and
Lane 2 contained E. coli/pET 16b/ORF II cells. The kala-azar
serum reacted specifically with protein products of 27.5 and 25
kD in the lysates of cells containing the pET 16b/ORF II
plasmid (Lane 2). The 25 kD protein probably corresponded to
the A2 protein without the HIS-TAG since the A2 sequence did
contain its own initiating ATG. The serum did not react
specifically with protein from E. coli lysates containing the
control pET 16b plasmid (Lane 1). These data confirmed that
the ORF II of the A2 gene encoded a L. donovani protein (A2)
that was antigenic in patients with visceral leishmaniasis.
In summary of this disclosure, the present invention
provides differentially expressed genes and proteins of
Leishmania, including the A2 gene expressed at significantly
higher levels in the amastigote stage of the life cycle when
the Leishmania organism is present in macrophages than in the
promastigote stage. Modifications are possible within the
scope of this invention.
. =
21~ 3~
REFERENCES
1. WHO, Tropical Disease Report, 1989. pp 85-92.
2. Turco, S.J., and Descoteaux, A. 1992. The
Lipophosphoglycan of Leishmania parasites. Annu.
Rev. Microbiol. 46:65-94.
3. Sacks, D.L. 1989. Metacyclogenesis in Leishmania
promastigotes. Exp. Parasitology. 69:100-103.
4. Sacks D.L., and da Silva, R.P. 1987. The
generation of infective stage L. major
promastigotes is associated with the cell-surface
expression and release of a developmentally
regulated glycolipid. J. Immunol. 139:3099-3106.
5. Sacks, D.L., Brodin T.N., Turco, S.J. 1990.
Developmental modification of the
lipophosphoglycan from L. Major promastigotes
during metacyclogenesis. Mol. Biochemical
Parasitol. 42:225-234.
6. Medina-Acosta, E., Karess, R.E., Schwartz H., and
Russell, D.G. 1989. The promastigote surface
protease (gp63) of Leishmania is expressed but
differentially processed and localized in the
amastigote stage. Mol. Biochemical Parasitol.
37:263-274.
7. Turco, S.J. and Sacks, D.L. 1991. Expression of
stage-specific lipophosphoglycan in Leishmania
maior amastigotes. Mol. Biochemical Parasitol.
45:91-100.
8. McConville, M.J., and Blackwell J.M. 1991.
Developmental changes in the glycosylated
phosphatidylinositols of L. donovani J. Biol.
Chem. 260:15170-15179.
9. Bogdan, C., Rollinghoff M., and Solbach, W. 1990.
Evasion strategies of Leishmania parasites.
Parasitol. Today. 6:183-187.
2 ~ 3 8
10. Modabber, F. 1989. Experiences with vaccines
against cutaneous leishmaniasis: of men and mice.
Parasitol. 98:S49-S60.
11. Joshi, M., Dwyer, D.M., and Nakhasi, H.L. 1993.
Cloning and characterization of differentially
expressed genes from in vitro-grown "amastigotes"
of Leishmania donovani. Mol. Biochemical
Parasitol. 58:345-354.
12. Descoteaux, A., and Matlashewski, G. 1989. c-fos
and tumor necrosis factor gene expression in
Leishmania donovani-infected macrophages.
Mol.Cell. Biol. 9:5223-5227.
13. Doyle, P.S. Engel, J.C., Pimenta, P.F.P. da
Silva, P. and Dwyer. 1991. Leishmania donovani:
Long-term culture of axenic amastigotes at 37 C.
Exp. Parasitol. 73:326-334.
14. Sambrook, J., Fritsch, E.F., and Maniatis. 1989.
Molecular cloning. A laboratory guide. Cold
Spring Harbor Laboratories Press, New York. pp
7.26-7.29.
15. Sambrook, J., Fritsch, E.F., and Maniatis. 1989.
Molecular cloning. A laboratory guide. Cold
Spring Harbor Laboratories Press, New York. pp
10.44-10.45.
16. Sambrook, J., Fritsch, E.F., and Maniatis. 1989.
Molecular cloning. A laboratory guide. Cold
Spring Harbor Laboratories Press, New York. pp
9.38-9.40.
17. Sambrook, J., Fritsch, E.F., and Maniatis. 1989.
Molecular cloning. A laboratory guide. Cold
Spring Harbor Laboratories Press, New York. pp
4.48.
18. Cruz,A., and Beverley, S.M. 1990. Gene-
replacement in parasitic protozoa. Nature
348:171-173.
SEQUENCE LISTING ~ 3 8
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: McGill University
(B) STREET: 845 Sherbrooke Street West
(C) CITY: Montreal
(D) STATE: Quebec
(E) C~UN 1 K-Y: Canada
(F) POSTAL CODE (ZIP): H3A 2T5
(A) NAME: Gregory Matlashewski
(B) STREET: 2571 Chestnut Circle
(C) CITY: St-Lazare
(D) STATE: Quebec
(E) C~UN LK~: Canada
(F) POSTAL CODE (ZIP): J0P lV0
(A) NAME: Hugues Charest
(B) STREET: 1930 Sommet-Trinite
(C) CITY: St-Bruno
(D) STATE: Quebec
(E) C~ UN 1 ~ Y: Canada
(F) POSTAL CODE (ZIP): H3V 4P6
(ii) TITLE OF lNv~NLION: DIFFERENTIALLY EXPRESSED LEISHMANIA GENES AND
PROTEINS
(iii) NUMBER OF S~UU~N~S: 4
(iv) 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 #1.25 (EPO)
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1091 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xi) S~:YU~N~: DESCRIPTION: SEQ ID NO:1:
GAGCTCCCCC AGCGACCCTC TCGGCAACGC GAGCGCCCCA GTCCCCCCAC GCACAACTTT 60
GACCGAGCAC AATGAAGATC CGCAGCGTGC GTCCGCTTGT GGTGTTGCTG GTGTGCGTCG 120
CGGCGGTGCT CGCACTCAGC GCCTCCGCTG AGCCGCACAA GGCGGCCGTT GACGTCGGCC 180
CG~lClCC~L TGGCCCGCAG lCC~lCGGCC CG~l~l~l~l TGGCCCGCAG GCTGTTGGCC 240
CG~lClCC~L TGGCCCGCAG TCCGTCGGCC CG~ L TGGCCCGCAG GCTGTTGGCC 300
CG~l~l~l~l TGGCCCGCAG lCC~llGGCC CG~ CC~l TGGCCCGCTC TCCGTTGGCC 360
CGCAGTCTGT TGGCCCGCTC LCC~LlGGCT CGCAGTCCGT CGGCCCGCTC L-l~LlGGTC 420
CGCAGTCCGT CGGCCCGCTC lCC~LlGGCC CGCAGGCTGT TGGCCCGCTC TCCGTTGGCC 480
CGCAGTCCGT CGGCCCGCTC L~llGGCC CGCAGGCTGT TGGCCCGCTC L~l~LLGGCC 540
~.
22 ~ 3 ~
CGCAGTCCGT TGGCCCGCTC lCCGllGGCC CGCAGTCTGT TGGCCCGCTC TCCGTTGGCT 600
CGCAGTCCGT CGGCCCGCTC L-l~llGGTC CGCAGTCCGT CGGCCCGCTC TCCGTTGGCC 660
CGCAGTCTGT CGGCCCGCTC LCC~LlGGCC CGCAGTCCGT CGGCCCGCTC lCC~llGGTC 720
CGCAGTCCGT TGGCCCGCTC lCC~llGGCC CGCAGTCCGT TGACGTTTCT CCG~l~l~ll 780
AAGGCTCGGC GTCCGCTTTC CGGTGTGCGT AAAGTATATG CCATGAGGCA TGGTGACGAG 840
GCAAACCTTG TCAGCAATGT GGCATTATCG TACCCGTGCA AGAGCAACAG CAGAGCTGAG 900
TGTTCAGGTG GCCACAGCAC CACGCTCCTG TGACACTCCG TGGGGTGTGT GTGACCTTGG 960
CTGCTGTTGC CAGGCGGATG AACTGCGAGG GCCACAGCAG CGCAAGTGCC GCTTCCAACC 1020
TTGCGACTTT CACGCCACAG ACGCATAGCA GCGCCCTGCC TGTCGCGGCG CATGCGGGCA 1080
AGCCATCTAG A 1091
(2) INFORMATION FOR SEQ ID NO:2:
(i) S~yu~N~ CHARACTERISTICS:
(A) LENGTH: 711 base pairs
(B) TYPE: nucleic acid
(C) STRANn~nN~.~S: single
(D) TOPOLOGY: linear
(xi) S~yu~ DESCRIPTION: SEQ ID NO:2:
ATGAAGATCC GCAGCGTGCG TCCGCTTGTG GTGTTGCTGG TGTGCGTCGC GGCGGTGCTC 60
GCACTCAGCG CCTCCGCTGA GCCGCACAAG GCGGCCGTTG ACGTCGGCCC G~l~lCCGll 120
GGCCCGCAGT CCGTCGGCCC G~l~lCl~ll GGCCCGCAGG CTGTTGGCCC GCTCTCCGTT 180
GGCCCGCAGT CCGTCGGCCC G~1~L~1~11 GGCCCGCAGG CTGTTGGCCC G~L~1~1~11 240
GGCCCGCAGT CCGTTGGCCC GCTCTCCGTT GGCCCGCTCT CCGTTGGCCC GCA~L~l~ll 300
GGCCCGCTCT CCGTTGGCTC GCA~lCC~lC GGCCCGCTCT CTGTTGGTCC GCAGTCCGTC 360
GGCCCGCTCT CCGTTGGCCC GCAGGCTGTT GGCCCGCTCT CCGTTGGCCC GCAGTCCGTC 420
GGCCCGCTCT CTGTTGGCCC GCAGGCTGTT GGCCCGCTCT CTGTTGGCCC GCA~LCC~ll 480
GGCCCGCTCT CCGTTGGCCC GCA~L~l~ll GGCCCGCTCT CCGTTGGCTC GCA~LCC~lC 540
GGCCCGCTCT CTGTTGGTCC GCA~lCC~lC GGCCCGCTCT CCGTTGGCCC GCA~l~l~lC 600
GGCCCGCTCT CCGTTGGCCC GCAGTCCGTC GGCCCGCTCT CCGTTGGTCC GCA~lCC~Ll 660
GGCCCGCTCT CCGTTGGCCC GCAGTCCGTT GAC~lll~lC CG~l~l~lLA A 711
(2) INFORMATION FOR SEQ ID NO:3:
(i) ~uu~ CHARACTERISTICS:
(A) LENGTH: 236 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~.
5 3 ~
(Xi) S~:yU~N~ DESCRIPTION: SEQ ID NO:3:
Met Lys Ile Arg Ser Val Arg Pro Leu Val Val Leu Leu Val Cys Val
1 5 10 15
Ala Ala Val Leu Ala Leu Ser Ala Ser Ala Glu Pro His Lys Ala Ala
Val Asp Val Gly Pro Leu Ser Val Gly Pro Gln Ser Val Gly Pro Leu
Ser Val Gly Pro Gln Ala Val Gly Pro Leu Ser Val Gly Pro Gln Ser
Val Gly Pro Leu Ser Val Gly Pro Gln Ala Val Gly Pro Leu Ser Val
Gly Pro Gln Ser Val Gly Pro Leu Ser Val Gly Pro Leu Ser Val Gly
Pro Gln Ser Val Gly Pro Leu Ser Val Gly Ser Gln Ser Val Gly Pro
100 105 110
Leu Ser Val Gly Pro Gln Ser Val Gly Pro Leu Ser Val Gly Pro Gln
115 120 125
Ala Val Gly Pro Leu Ser Val Gly Pro Gln Ser Val Gly Pro Leu Ser
130 135 140
Val Gly Pro Gln Ala Val Gly Pro Leu Ser Val Gly Pro Gln Ser Val
145 150 155 160
Gly Pro Leu Ser Val Gly Pro Gln Ser Val Gly Pro Leu Ser Val Gly
165 170 175
Ser Gln Ser Val Gly Pro Leu Ser Val Gly Pro Gln Ser Val Gly Pro
180 185 190
Leu Ser Val Gly Pro Gln Ser Val Gly Pro Leu Ser Val Gly Pro Gln
195 200 205
Ser Val Gly Pro Leu Ser Val Gly Pro Gln Ser Val Gly Pro Leu Ser
210 215 220
Val Gly Pro Gln Ser Val Asp Val Ser Pro Val Ser
225 230 235
(2) INFORMATION FOR SEQ ID NO:4:
(i) S~yu~N~ CHARACTERISTICS:
(A) LENGTH: 269 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xi ) S~U~N~ DESCRIPTION: SEQ ID NO:4:
Pro Gly Ser Glu Gly Pro Lys Gly Thr Gly Gly Pro Gly Ser Glu Gly
1 5 10 15
Pro Lys Gly Thr Gly Gly Pro Gly Ser Glu Gly Pro Lys Gly Thr Gly
23a
Gly Pro Gly Ser Glu Gly Pro Lys Gly Thr Gly Gly Pro Gly Ser Glu
Gly Pro Lys Gly Thr Gly Gly Pro Gly Ser Glu Gly Pro Lys Gly Thr
Gly Gly Pro Gly Ser Glu Gly Pro Lys Gly Thr Gly Gly Pro Gly Ser
Glu Gly Pro Lys Gly Thr Gly Gly Pro Gly Ser Glu Gly Pro Lys Gly
Thr Gly Gly Pro Gly Ser Glu Gly Pro Lys Gly Thr Gly Gly Pro Gly
100 105 110
Ser Glu Gly Pro Lys Gly Thr Gly Gly Pro Gly Ser Glu Gly Pro Lys
115 120 125
Gly Thr Gly Gly Pro Gly Ser Glu Gly Pro Lys Gly Thr Gly Gly Pro
130 135 140
Gly Ser Glu Gly Pro Lys Gly Thr Gly Gly Pro Gly Ser Glu Gly Pro
145 150 155 160
Lys Gly Thr Gly Gly Pro Gly Ser Glu Gly Pro Lys Gly Thr Gly Gly
165 170 175
Pro Gly Ser Glu Ser Pro Lys Gly Thr Gly Gly Pro Gly Ser Glu Gly
180 185 190
Pro Lys Gly Thr Gly Gly Pro Gly Ser Glu Gly Pro Lys Gly Thr Gly
195 200 205
Pro Lys Gly Thr Gly Gly Pro Gly Ser Glu Ala Gly Thr Glu Gly Pro
210 215 220
Lys Gly Thr Gly Gly Pro Gly Ser Glu Ala Gly Thr Glu Gly Pro Lys
225 230 235 240
Gly Thr Gly Gly Pro Gly Ser Gly Gly Glu His Ser His Asn Lys Lys
245 250 255
Lys Ser Lys Lys Ser Ile Met Asn Met Leu Ile Gly Val
260 265
= .
.~
L~
~ i ~ ~
