PLASMODIUM FALCIPARUM VIRULENCE FACTOR VAR O

FACTEUR DE VIRULENCE VAR O DU PLASMODIUM FALCIPARUM

English Abstract

The present invention relates to the discovery of the var O gene specific to
the Palo Alto
FUP/SP-O strain of Plasmodium falciparum and the corresponding protein, an
adhesin
that mediates rosetting and auto-agglutination of red blood cells in malaria
pathogenesis.
Novel biological tools and methods of use of the foregoing are disclosed
including nucleic
acid molecules, polypeptides, antibodies, recombinant vectors, recombinant
host cells,
and corresponding diagnostic and therapeutic applications of the foregoing.

Claims

36
CLAIMS
1. An isolated or purified polynucleotide having a nucleic acid sequence being
at
least 65% identical to any one of SEQ ID NO 1, 13 to 21 and fragments thereof.
2. An isolated or purified polynucleotide having a nucleic acid sequence being
at
least 80% identical to any one of SEQ ID NO 1, 13 to 21 and fragments thereof.
3. An isolated or purified polynucleotide having a nucleic acid sequence being
at
least 95% identical to any one of SEQ ID NO 1, 13 to 21 and fragments thereof.
4. An isolated or purified oligonucleotide which can be used as a primer for
hybridization with a polynucleotide as defined in any one of claims 1 to 3.
5. The isolated or purified oligonucleotide of claim 4 comprising a nucleotide
sequence selected from the group consisting of SEQ ID No 22 à 39.
6. An isolated or purified polypeptide comprising an amino acid sequence
encoded
by a polynucleotide sequence of any one of claims 1 to 3 and fragments
thereof.
7. An isolated or purified polypeptide having an amino acid sequence being at
least 80% identical to SEQ ID NO 2 and fragments thereof.
8. An isolated or purified polypeptide having an amino acid sequence being at
least 95% identical to SEQ ID NO 2 and fragments thereof.
9. An isolated or purified polypeptide having an amino acid sequence
substantially
the same or having 100% identity to SEQ ID NO 2 and fragments thereof.
10. An isolated and purified polypeptide comprising an amino acid sequence
encoded by a nucleic acid hybridizing under stringent conditions to the
complement of the polynucleotide of any one of claims 1 to 3 and having the
ability




37

to induce cytoadherence in cells infected Plasmodium related species.

11. The isolated and purified polypeptide of any one of claims 6 to 10,
comprising
at least one amino acid sequence selected from the group consisting of amino
acid sequence having SEQ ID NO. 3, SEQ ID NO.4, SEQ ID NO. 5, SEQ ID NO.6,
SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ
ID NO.12, and biologically active fragments thereof.

12. A cloning or expression vector comprising a polynucleotide sequence as
defined in any one of claims 1 to 3.

13. The cloning or expression vector of claim 12, wherein said vector is
capable of
directing expression of the polypeptide encoded by said polynucleotide
sequence
in a vector-containing cell.

14. The cloning or expression vector of claim 13, is selected from the group
consisting of pET22b- Clone G, pET22b-Clone A, pET22b-Clone B, pET22b-
Clone C, pET22b-Clone D, pET22b-Clone G, pET22b-Clone L, pET22b-Clone M,
and pET22b-Clone O.

15. A transformed or transfected cell containing the polynucleotide sequence
as
defined in any one of claims 1 to 3.

16. A transformed or transfected cell containing a cloning or expression
vector of
any one of claims 12 to 14.

17. The cell of claim 15 or 16, wherein said cell consists of a Escherichia
coli
bacterium.

18. The cell of claim 17, wherein the Escherichia coli bacterium is selected
from
the group consisting of the cells deposited under accession numbers CNCM I-
2929 and CNCM I-2930.




38

19. An antibody that specifically binds to the isolated or purified
polypeptide as
defined in any one of claims 6 to 11 and/or fragments thereof.

20. The antibody of claim 19, wherein said antibody consists of a monoclonal
or
polyclonal antibody.

21. A composition comprising the isolated or purified polynucleotide of any
one of
claims 1 to 3; the isolated or purified polypeptide of any one of claims 6 to
11;
and/or the antibody of claim 19 or 20, and an acceptable carrier.

22. The composition according to claim 21, for preventing and/or treating a
Plasmodium species related disease.

23. The composition according to claim 22, wherein the disease is malaria.

24. A vaccine comprising the isolated or purified polynucleotide of any one of
claims 6 to 11; the isolated or purified polypeptide of any one of claims 6 to
11;
and/or the antibody of claim 19 or 20, and an acceptable carrier.

25. The vaccine according to claim 24, for preventing and/or treating malaria.

26. A method for treating and/or preventing malaria in a mammal, comprising
the
step of administering to the mammal an effective amount of:

-the isolated or purified polynucleotide as defined in any one
of claims 1 to 3;

-the isolated or purified polypeptide as defined in any one of
claims 6 to 11;

-the composition as defined in any one of claims 21 to 22;

-the antibody of claim 19 or 20;
and/or

-the vaccine as defined in any one of claims 24 to 25.




39

27. An in vitro diagnostic method for the detection of the presence or absence
of
antibodies indicative of Plasmodium species, which bind with the polypeptide
according to any one of claims 6 to 11 to form an immune complex, comprising
the
steps of

a) contacting the polypeptide according to any one of claims 6 to 9 with a
biological sample for a time and under conditions sufficient to form an immune
complex; and

b) detecting the presence or absence of the immune complex formed in a).

28. The method of claim 27, wherein the Plasmodium species consist of P.
falciparum.

29. A diagnostic kit for the detection of the presence or absence of
antibodies
indicative of Plasmodium species, comprising:

- a polypeptide according to any one of claims 6 to 9;

- a reagent to detect polypeptide-antibody immune complex;

- a biological reference sample lacking antibodies that immunologically bind
with said peptide; and

- a comparison sample comprising antibodies which can specifically bind to
said peptide;

wherein said polypeptide, reagent, biological reference sample, and comparison
sample are present in an amount sufficient to perform said detection.

30. The diagnostic kit according to claim 29, wherein the P. species consists
of P.
falciparum.

31. An in vitro diagnostic method for the detection of the presence or absence
of
polypeptides indicative of Plasmodium species, which bind with the antibody of
claim 18 or 19 to form an immune complex, comprising the steps of:

a) contacting the antibody of the invention with a biological sample for a
time and under conditions sufficient to form an immune complex; and




40

b) detecting the presence or absence of the immune complex formed in
a).

32. An in vitro diagnostic method for the detection of the presence or absence
of a
polynucleotide indicative of Plasmodium species, comprising the steps of:

a) contacting at least one oligonucleotide according to claim 4 or 5 with a
biological sample for a time and under conditions sufficient for said
oligonucleotide to hybridize to said polynucleotide; and

b) detecting the presence or absence of an hybridization between said
oligonucleotide and polynucleotide.

33. The method of claim 31 and 32, wherein the Plasmodium species consist of
P.
falciparum.

34. A diagnostic kit for the detection of the presence or absence of
polypeptides
indicative of Plasmodium species, comprising:

- an antibody according to any one of claims 19 and 20;

- a reagent to detect polypeptide-antibody immune complex;

- a biological reference sample lacking polypeptides that immunologically
bind with said antibody; and

- a comparison sample comprising polypeptides which can specifically bind
to said antibody;

wherein said antibody, reagent, biological reference sample, and comparison
sample are present in an amount sufficient to perform said detection.

35. A diagnostic kit for the detection of the presence or absence of
polynucleotide
indicative of Plasmodium species, comprising:

- at least one oligonucleotide according to claim 4 or 5;

- a reagent to detect polynucleotide-oligonucleotide hybridization complex;

- a biological reference sample lacking polynucleotides that hybridise with
said oligonucleotide; and

- a comparison sample comprising polynucleotides which can specifically




41

hybridise to said oligonucleotide;

wherein said oligonucleotide, reagent, biological reference sample, and
comparison sample are present in an amount sufficient to perform said
detection.

Description

CA 02409897 2002-10-25
1
PLASMODIUM FALCIPARUM VIRULENCE FACTOR VAR O
FIELD OF THE INVENTION
The present invention relates to Plasmodium parasite virulence factors
involved in the
pathogenesis of malarial infections. More particularly, this invention
concerns novel
peptide, polypeptide and vaccine compositions for the diagnosis, treatment and
prevention of malaria.
BACKGROUND OF THE INVENTION
Malariotherapy infections in neurosyphilitic patients conducted in the first
half of the
20~h century have shown that Plasmodium species, in particular, Plasmodium
falciparum
strains differ in the clinical manifestations they provoke (James S.P. et al.
(1932) Proc. R.
Soc. Med. 25, 1153-1181). This observation was confirmed in experimental
infections of
Saimiri sciureus monkeys (Fandeur T. et al. (1996). Exp. Parasitol. 84, 1-15)
and
suggests the existence of parasite virulence factors. Identification of the
parasite factors
which critically influence infection outcome is pivotal in devising
intervention strategies
aiming at preventing or curing malaria morbidity.
To date, little is known about the parasite factors involved at the various
steps of the
pathogenesis cascade. The current view on P. falciparum pathogenesis is that
severe
malaria results from an inappropriate immune response to high parasite
density, with
excessive production of pro-inflammatory cytokines and massive colonisation of
specific
organs by parasites expressing specific adhesin(s). The relationship between
mild and
severe malaria is uncertain. The fact that spreading of drug resistance is
associated with
an increased malaria-attributable mortality (Trape J.F. (2001) Am. J. Trop.
Med. Hyg. 64
S 12-17) suggests a direct continuum, with progression to severe complications
if mild
3CI malaria is not rapidly treated.
It is clear that parasite density is a critical component of clinical malaria.
In children
living in endemic areas acute malaria is defined as febrile episodes with
possibly
additional symptoms associated with a parasite density above a certain
threshold (Rogier
C. et al. (1996). Am. J. Trop. Med. Hyg. 54, 613-619). In severe cases,
parasite density is

CA 02409897 2002-10-25
higher than in matched mild cases (Robert F. et al. (1996). Trans. R. Soc.
Trop. Med.
Hyg. 90, 704-711; Pain A. et al. (2001) Proc. Natl Acad. Sci. U.S.A. 98 (4),
1805-1810;
Heddini A. et al. (2001 ) Infect. Immun. 68, 5849-5856) and correlates with
prognosis
(Molyneux M.E. et al. (1989). Quat. J. Med. 71, 446-474). This strongly
suggests that the
parasite factors that favour an increased multiplication rate/development are
contributing
to pathogenesis. Indeed, parasites from severe malaria Thai patients have a 3-
fold higher
in vitro multiplication potential and a lower selectivity for red blood cells
than parasites
from mild malaria patients (Chotivanich K. et al. (2000) J. Inf. Dis. 181,
1206-1209).
The concept that expression of parasite adhesins promotes massive organ-
specific
sequestration has only been validated for the placenta (Fried M. & Duffy P.E.
(1996)
Science 272, 1502-1504; Beeson J.G. et al. 2001. Trends Parasitol. 17(7), 331-
337). So
far, the capacity of infected red blood cells with mature parasite stages to
bind uninfected
red blood cells (called "rosetting") {Carlson J. et al. 1990. The Lancet 336,
1457-1460;
Rowe A. et al. (1995). Infect. Immun. 63, 2323-2336) or to form large auto-
agglutinates of
infected red blood cells (Pain A. et al. (2001 ) Proc. Natl Acad. Sci. U.S.A.
98 (4), 1805-
1810; Roberts D.J. ef al. (2000). The Lancet 355, 1427-1428) associated with
the
absence of antibodies disrupting rosettes (Carlson J. et al. 1990. The Lancet
336, 1457-
1460) are the only factors associated with severity in African children. The
large cellular
aggregates formed by rosettes and/or auto-agglutinates provoke microvascular
obstruction. Such a sequestration is not restricted to specific organs, since
it involves the
physical size of the cellular agglutinate rather than local binding of the
infected red blood
cells (IRBC) to the endothelial lining through specific ligand / receptor
interactions. Thus,
targetting (preventing or reverting) rosettinglauto-agglutination should have
broader
consequences than preventing homing to certain territories by targetting the
specific
ligand.
So far, identification of parasite factors contributing to pathology in humans
has
relied on association studies, looking for specific parasite characteristics
associated with
more or less severe clinical forms (Robert F. et al. {1996). Trans. R. Soc.
Trop. Med. Hyg.
90, 704-711; Pain A. et al. (2001) Proc. Natl Acad. Sci. U.S.A. 98 (4), 1805-
1810; Heddini
A. et al. (2001 ) Infect. Immun. 69, 5849-5856; Carlson J. et al. 1990. The
Lancet 336,
1457-1460; Roberts D.J. et al. (2000). The Lancet 355, 1427-1428; Newbold C.I.
et al.
(1997). Am. J. Trop. Med. Hyg. 57, 389-398; Ariey F. et al. (2001 ). J.
Infect. Dis., 184,
237-241; Kun J.F. et a!. (1998). Trans. R. Soc. Trop. Med. Hyg. 92(1), 110-
114).

CA 02409897 2002-10-25
3
Interpretation of these studies is difficult because of the very large field
Plasmodium
falciparum diversity, with numerous strains circulating in any given place.
Investigation of
variant phenotypic adhesion specificity is further complicated by the rapid
var switching
rate, resulting in clonal phenotypic heterogeneity with numerous adhesive
phenotypes
expressed at the time of blood collection (Newbold C.I. et al. (1997). Am. J.
Trop. Med.
Hyg. 57, 389-398). The number of possible receptors, the list of which is most
probably
not closed, is so large that an exhaustive analysis of binding properties of
patient isolates
cannot be conducted.
Isolates with rosette-forming parasites [67% -100%] (Carlson J. et al. 1990.
The
Lancet 336, 1457-1460; Rowe A. et al. (1995). Infect. Immun. 63, 2323-2336;
Rogerson
S.J. et aL (2000) Infect. Immun., 68 391-393) or autoagglutinating parasites
[47-86] (Pain
A. et al. (2001 ) Proc. Natl Acad. Sci. U.S.A. 98 (4), 1805-1810; Roberts D.J.
et al. (2000).
The Lancet 355, 1427-1428) are frequent in African children and in adults (Ho
M. et aL
(1991 ) Infect. Immun. 59, 2135-2139; Rogerson S.J. ef al. (2000) Infect.
Immun., 68 391-
393), but the percentage of all infected erythrocytes forming cellular
aggregates within an
isolate is low. Rosette-forming infected erythrocytes account for 2.8-9% of
the all infected
red blood cells (Rogerson S.J. ef al. (2000) Infect. Immun., 68 391-393), 1%
in mild and
5% in severe malaria (Rowe A, et al. (1995). Infect. Immun. 63, 2323-2336) and
autoagglutinating erythrocytes for 6.6% of ail IRBC present in severe cases
and 2.1 % mild
cases (Roberts D.J. et al. (2000). The Lancet 355, 1427-1428). Thus, the
association of
the capacity of rosetting and autoagglutination with severity was
statistically significant in
several studies, but figures rather unsatisfactory.
Several factors limit such studies in humans and their interpretation. The
inclusion
criteria applied differ from one study to the other e.g. high parasite density
is >4%
(Heddini A. et al. (2001) Infect. Immun. 69, 5849-5856), 0.2% (Roberts D.J. et
al. (2000).
The Lancet 355, 1427-1428) or 0.3% (Roberts D.J. et al. (2000). The Lancet
355, 1427-
1428), associated with removal of monocytes and granulocytes (Heddini A. et
al. (2001 )
Infect. Immun. 69, 5849-5856), presence or not of platelets in the in vitro
assays (Pain A.
et al. (2001) Proc. Natl Acad. Sci. U.S.A. 98 (4), 1805-1810) etc.... Adhesive
phenotypes
can only be evidenced and hence studied after in vitro maturation until
pigmented stage.
Not all parasites mature to that stage in vitro (Heddini A. et al. (2001 )
Infect. Immun. 69,
5849-5856; Carlson J. et al. 1990. The Lancet 336, 1457-1460; Roberts D.J. et
al. (2000).
The Lancet 355, 1427-1428. The association of certain adhesive phenotypes with
severity

CA 02409897 2002-10-25
4
is therefore based upon only a fraction of the circulating parasite pool.
Furthermore, these
studies all make the assumption that circulating parasites are fairly
representative of the
sequestered pool, which is actually causing disease. However, recent data
indicate that
such is not the case and that in 86% of the cases examined some sequestered
genotypes
(strains) are not present in the peripheral blood (Schleiermacher D., et al.
2002 Inf. Genet.
Evol. 46, 1-9).
The molecular mechanisms underlying cytoadherence in malaria parasites have
been
recently clarified. The cytoadherence phenotype acquired by mature Plasmodium
falciparum-infected erythrocytes is mediated by variant PfEMP1 adhesins
exposed onto
the surface of infected RBC from thetrophozoite stage on. PfEMP1 adhesins are
encoded
by a repertoire of approximately 50 var genes (Smith et al., (2000) Mol.
Biochem.
Parasitol., 110, 293-310; Smith et al., (2001 ) Trends Parasitol., 17 (11 )
538-545).
The structural organisation established in 1995 by two independent groups
has been clarified by numerous subsequent studies. In brief, var genes have
two
exons, exons 1 and 2. Exon 2 codes for a relatively well conserved domain
implicated in interaction with the erythrocyte cytosqueleton. Exon 1 codes for
the
variable extra-cellular region of the molecule and has a modular organisation
with
Duffy Binding Like domains (known as "DBL"), Cysteine Rich Interdomain Regions
(known as "CIDR") and C2 domains. Based on sequence homology, the DBL
domains are grouped into five distinct classes (alpha to epsilon) and the CIDR
into
three classes (alpha to gamma). Within each class, there exist unique
consensus
motifs that can be used to characterise and classify PfEMPI molecules(Smith et
al., (2000) Mol. Biochem. Parasitol., 110, 293-310; Smith et al., (2001 )
Trends
Parasitol. 17 (11 ) 538-545). The arrangement and sequence of DBL and CIDR
differ between different PfEMP1 proteins.
The prototypical PfEMP1 extracellular region consists in a NTS (a globular
N terminal segment) followed by a duplicated arrangement of the DBL-CIDR
tandem. The first tandem is almost invariably DBLlalpha-CIDR1alpha and the
second is generally DBL2delta-CIDR2beta. The number of DBL domains varies
from 2-7 and the number of CIDR varies from 1-2. DBLbeta is invariably

CA 02409897 2002-10-25
associated with C2. Mapping of the PfEMPI adhesive domains has indicated that
rosetting is associated with DBL1 alpha , binding to ICAM-1 with the tandem
DBLbeta -C2, binding to CD31 with the DBLdelta , binding to CSA is associated
with DBLgamma and binding to CD36 with CIDR1 alpha(Smith et al., (2001 )
5 Trends Parasitol. 17 (11 ) 538-545).
Three var genes associated with rosetting have been described to date.
Firstly, the
var gene 2182041 (Y13402 in Genbank) has been associated with the rosetting
phenotype of the R29 clone in the strain It (which de facto is FCR3) (Rowe A.
et al.
(1997). Nature. 388, 292-295). The gene has 4 DBL domains and 1 CIDR (see
i:lgure 2).
The first DBL1-CIDR1 association is atypical it consists in DBL1alpha-
CIDRlgamma .The
rosetting receptor on the erythrocyte surface has been identified. It is
Complement
Receptor 1 (CR1 ). Common CR1 African polymorphism reduced binding of IRBC.
The
domain responsible for rosetting is DBL1aIpha. Expression of the different
DBL1 - 4 and
CIDR1 domains onto the surface of COS cells identified DBL1 alpha as the
single domain
binding erythrocytes.
Secondly, there is the var gene 2961468 also called FCR3S1.2 from the FCR3
strain (Chen Q. et al. (1998). J. Exp. Med.187, 15-23). This gene has been
identified as
mediating rosetting by single-cell PCR of micro-manipulated rosette forming
cells. It is a
very short gene, which contains two DBL domains (1alpha & 2delta ) and two
CIDR (alpha
and beta) (see figure 2). This gene codes for a multi-adhesive protein. The
DBL1alpha
GST binds heparin-Sepharose, glycosaminoglycans and binds erythrocytes.
Lastly, there is the var gene 15991381 Flick et al. (2001 ) Science 293, 2098-
2100
(AF366567 in Genbank) derived from clone TM284S2 which forms giant rosettes,
mixed
rosettes and autoagglutinates. The var gene 15991381 codes for a PfEMP1
adhesin
which binds IgG and through this IgG bridge, binds to the placenta IgG
receptor. It has
four DBL1 domains and two CIDRs (see figure 2). The six extra-cellular domains
were
expressed as GST-fusion proteins. This showed that DBL2beta is the IgG binding
domain.
The demonstration that specific parasite factors contribute to pathology
requires an
experimental model of infection, where the size of the inoculum, the time of
injection, the
parasite strains and possible surface phenotype be well defined and as
homogeneous as

CA 02409897 2002-10-25
s
possible. Comparison of the course of infection induced by 14 different
strains in the
splenectomized Saimiri sciureus (a highly susceptible model of infection) has
shown
substantial differences in the course of infection, some strains inducing
afulminant (lethal
if untreated) infection, whereas others induce self-curing infections reaching
moderate,
low or very low peak parasite densities (Fandeur T. et al. (1996). Exp.
Parasitol. 84, 1-15).
Phenotyping and genotyping these strains for more than 20 characters showed
that the
lethal strains Palo Alto FUP/SP, and 2 other ones collected from fatal human
cases,
formed rosettes and large autoagglutinates (Fandeur T. et al. (1996). Exp.
Parasitol. 84,
1-15).
To study the possible contribution of rosetting in pathogenesis, two antigenic
variants
of the Palo Alto FUP/SP line, variants O and R were studied. Variant O forms
rosettes and
auto-agglutinates while variant R, which is derived from variant O under
immune pressure,
no longer forms rosettes or autoagglutinates (Fandeur T et al., (1995) J Exp
Med 181,
I5 283-295). While both variants have the same genetic make-up, they
nevertheless present
distinct adhesive phenotypes. To minimize sequestration and gain access to
circulating
pigmented parasites, removal of the spleen is necessary (David P. et al.
(1983) Proc. Natl
Acad. Sci. U.S.A. 80, 5075-5079). While there are few experimental models of
infection
available to identify virulence factors, there exists a model of infection
with the O and R
Palo Alto variants in the splenectomized Saimiri sciureus monkey. The large
cellular
aggregates which are normally filtered by the spleen in spleen-intact
individuals, circulate
in splenectomized monkeys (Contamin H. ef al. (2000) Microbes & Infection 2.
945-954).
Unlike what is observed in human infections, a very large proportion of
parasites (80%
and over) are rosette-forming or autoagglutinating within the var O
population.
Thus, there is a need to identify virulence factors of Plasmodium parasite
since it is
clear from the above discussion that identification of the parasite factors
which critically
influence infection outcome is pivotal in devising intervention strategies
aiming at
preventing or curing malaria morbidity.
The present invention fulfills that need and also other needs, which will be
apparent to those skilled in the art upon reading the following specification.

CA 02409897 2002-10-25
7
SUMMARY OF THE INVENTION
The present invention concerns the characterization of a var gene, more
particularly, the var O gene.
The present invention also concerns at least one polypeptide encoded by the
var
O gene and expressed in Plasmodium species.
More precisely, an object of the present invention is to provide an isolated
or
purified polynucleotide having a nucleic acid sequence being at least 65%
identical to any
one of SEQ ID NO 1, SEQ ID N0.13 to SEQ ID N0.21 and fragments thereof.
Another object of the present invention is to provide an isolated or purified
polypeptide comprising an amino acid sequence encoded by the polynucleotide
sequence
as defined above and/or biologically active fragments thereof.
A further object of the present invention is to provide an isolated or
purified
polypeptide having at least 80% sequence identity with amino acid sequence of
SEQ ID
NO 2.
Another object of the invention is to provide an isolated or purified
oligonucleotide
which can be used as a primer for hybridization with a polynucleotide of the
invention.
Yet another object of the present invention is to provide an isolated and
purified
polypeptide comprising an amino acid sequence encoded by a nucleic acid
hybridizing
under stringent conditions to the complement of the polynucleotide of the
present
invention and having the ability to induce cytoadherence in cells infected
Plasmodium
related species.
Still another object of the present invention is to provide a cloning or
expression
vector comprising a polynucleotide sequence having SEQ !D NO. 1 and 13 to 21.
!n a
particular embodiment, the present invention is directed to a plasmid
comprising at least
one var O gene fragment selected from the group consisting of the plasmids
deposited
under number CNCM I-2929 and CNCM I-2930.

CA 02409897 2002-10-25
Also another object of the present invention is to provide a transformed or
transfected cell containing the polynucleotide sequence of the present
invention.
Still a further object of the present invention is to provide a host cell
comprising the
cloning or expression vector of the instant invention.
Another object of the present invention is to provide a recombinantEscherichia
coli
cell selected from the group consisting of the cells deposited under number
CNCM I-2929
and CNCM I-2930.
Yet another object of the present invention is to provide an antibody that
specifically binds to the isolated or purified polypeptide of the instant
invention.
Still another object of the present invention is to provide a composition
comprising
the isolated or purified polynucleotide and/or the isolated or purified
polypeptide of the
instant invention; andlor the antibody specific to the polypeptide encoded by
the
polynucleotide of the invention or biologically active fragments thereof, and
an acceptable
carrier.
zo
Also another object of the present invention is to provide a vaccine
comprising the
isolated or purified polynucleotide and/or the isolated or purified
polypeptide and/or the
antibody of the instant invention, and an acceptable carrier.
Yet another object of the present invention is to provide a method for
treating
and/or preventing a Plasmodium species related disease, for example malaria,
in a
mammal, comprising the step of administering to the mammal an effective amount
of
-the isolated or purified polynucleotide, polypeptide, antibody,
composition and/or vaccine of the instant invention.
Still another object of the present invention is to provide an in vitro
diagnostic
method for the detection of the presence or absence of antibodies indicative
of
Plasmodium species, which bind with the polypeptide of the present invention
to form an
immune complex, comprising the steps of
a) contacting the polypeptide of the invention with a biological sample for a
time and under

CA 02409897 2002-10-25
conditions sufficient to form an immune complex; and
b) detecting the presence or absence of the immune complex formed in a).
The present invention also provides a diagnostic kit for the detection of the
presence or absence of antibodies indicative of Plasmodium species,
comprising:
- a polypeptide of the invention;
- a reagent to detect polypeptide-antibody immune complex;
- a biological reference sample lacking antibodies that immunologically bind
with
said polypeptide; and
- a comparison sample comprising antibodies which can specifically bind to
said
polypeptide;
wherein said polypeptide, reagent, biological reference sample, and comparison
sample
are present in an amount sufficient to perform said detection.
Still another object of the present invention is to provide an in vitro
diagnostic
method for the detection of the presence or absence of polypeptides indicative
of
Plasmodium species, which bind with the antibody of the present invention to
form an
immune complex, comprising the steps of:
a) contacting the antibody of the invention with a biological sample for a
time and under
conditions sufficient to form an immune complex; and
b) detecting the presence or absence of the immune complex formed in a).
Still another object of the present invention is to provide an in vitro
diagnostic
method for the detection of the presence or absence of a polynucleotide
indicative of
Plasmodium species, comprising the steps of:
a) contacting at least one oligonucieotide of the invention with a biological
sample
for a time and under conditions sufficient for said oligonucleotide to
hybridize to
said polynucleotide; and
b) detecting the presence or absence of an hybridization between said
oligonucleotide and polynucleotide.
Still another object of the present invention is to provide a diagnostic kit
for
the detection of the presence or absence of polypeptide antibodies indicative
of
Plasmodium species, comprising:

CA 02409897 2002-10-25
- an antibody of the present invention;
- a reagent to detect polypeptide-antibody immune complex;
- a biological reference sample lacking polypeptides that immunologically
bind with said antibody; and
5 - a comparison sample comprising polypeptides which can specifically bind
to said antibody;
wherein said antibody, reagent, biological reference sample, and comparison
sample are present in an amount sufficient to perform said detection.
10 Yet another object of the present invention is to provide a diagnostic kit
for
the detection of the presence or absence of polynucleotide indicative of
Plasmodium species, comprising:
- an oligonucleotide of the present invention;
- a reagent to detect polynucleotide-oligonucleotide hybridization complex;
- a biological reference sample lacking polynucleotides that hybridise with
said oligonucleotide; and
- a comparison sample comprising polynucleotides which can specifically
hybridise to said oligonucleotide;
wherein said oligonucleotide, reagent, biological reference sample, and
comparison sample are present in an amount sufficient to perform said
detection.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation of the structure of exon 1 of the var O
gene and the
sub-cloned domains expressed as recombinant proteins.
Figure 2 is a schematic representation of the Paio Alto var O deduced protein
domains
and scores of homology with other var protein domains.
Figure 3 is the nucleic acid sequence and the deduced amino acid sequence of
the var O
gene and identified as SEQ ID NO. 1.
Figure 4 is the amino acid sequence of the polypeptide encoded by the var O
gene and

CA 02409897 2002-10-25
11
identified as SEQ ID NO. 2.
Figure 5 is the amino acid sequence of the DBL 1 alpha domain encoded by the
var O
gene and identified as SEQ ID NO. 3.
Figure 6 is the amino acid sequence of the DBL 2 beta domain encoded by the
var O
gene and identified as SEQ ID NO. 4.
Figure 7 is the amino acid sequence of the DBL 3 gamma domain encoded by the
var O
gene and identified as SEQ ID NO. 5.
Figure 8 is the amino acid sequence of the DBL 4 epsilon domain encoded by the
var O
gene and identified as SEQ 1D NO. 6.
Figure 9 is the amino acid sequence of the DBL 5 epsilon domain encoded by the
var O
gene and identified as SEQ ID N0.7.
Figure 10 is the amino acid sequence of the CIRD gamma domain encoded by
thevar O
gene and identified as SEQ ID NO. 8.
Figure 11 is the amino acid sequence of the C2 domain encoded by the var O
gene and
identified as SEQ ID NO. 9.
Figure 12 is the amino acid sequence of the ID 3 domain encoded by thevar0
gene and
identified as SEQ ID N0.10.
Figure 13 is the amino acid sequence of the ID 4 domain encoded by thevar O
gene and
identified as SEQ ID NO. 11.
Figure 14 is the amino acid sequence of the transmembrane segment encoded by
the var
3U O gene and identified as SEQ ID NO. 12.
Figure 15 is the nucleic acid sequence of the DBL 1 alpha domain of the var O
gene
identified as SEQ ID NO. 13.
Figure 16 is the nucleic acid sequence of the DBL 2 beta domain of the var O
gene

CA 02409897 2002-10-25
12
identified as SEQ ID NO. 14.
Figure 17 is the nucleic acid sequence of the C2 domain of the var O gene
identified as
SEQ ID NO. 19.
Figure 18 is the nucleic acid sequence of the DBL 3 gamma domain of the var O
gene
identified as SEQ ID NO. 15.
Figure 19 is the nucleic acid sequence of the ID 3 inter-domain of the var O
gene
identified as SEQ ID NO. 20.
Figure 20 is the nucleic acid sequence of the DBL 4 epsilon domain of the var
O gene
identified as SEQ ID NO. 16.
Figure 21 is the nucleic acid sequence of the ID 4 inter-domain of the var O
gene
identified as SEQ ID NO. 21.
Figure 22 is the nucleic acid sequence of the DBL 5 epsilon domain of the var
O gene
identified as SEQ ID N0.17.
Figure 23 is the nucleic acid sequence of the DBL1 alpha CIRD gamma tandem
domain of
the var0 gene identified as SEQ ID NO. 18.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a polynucleotide encoding a Plasmodium
virulence
factor and its use in the preparation of compositions and vaccines. More
specifically, the
present invention is concerned with compositions, vaccines and methods for
providing an
immune response and/or a protective immunity to mammals against a Plasmodium
species as well as oligonucleotides and methods for the diagnosis of
Plasmodium
infection.
A non-exhaustive list of P. species against which the methods, compositions
and
vaccines of the invention may be useful, includes those which affect humans
and
preferably those that cause malaria, such as P. vivax, P. ovate, P. malariae
and P.
falciparum. In a preferred embodiment, the compositions, vaccines and methods
of the

CA 02409897 2002-10-25
13
present invention will be useful against disorders caused by P. falciparum.
As used herein, the term "immune response" refers to the T cell response or
the
increased serum levels of antibodies to an antigen, or presence of
neutralizing antibodies
~~ to an antigen, such as a Plasmodium falciparum virulence factor, for
instance, a var
peptide. The term "immune response" is to be understood as including a humoral
response, a cellular response and an inflammatory response.
The term "protection" or "protective immunity" refers herein to the ability of
the
1C1 serum antibodies and cellular response induced during immunization to
protect (partially
or totally) against malaria caused by an infectious agent, such as aP.
falciparum. Thus, a
mammal immunized by the compositions or vaccines of the invention will
experience
limited growth and spread of an infectious P. falciparum.
15 As used herein, the term "protection" also means cure of an ongoing
infection for
instance by administration of a component reducing parasite density by
disrupting cellular
interaction of the parasite with host cells or autoagglutination.
As used herein, the term "mammal" refers to any mammal that is susceptible to
be
20 infected by a Plasmodium species causing malaria. Among the mammals which
are
known to be potentially infected by a P. species, there are humans, apes,
birds, and
bovines.
1. Polynucleotides and polypeptides
2~~
In a first embodiment, the present invention concerns an isolated or purified
polynucleotide encoding a P, falciparum virulence factor, namely the var O
protein.
Therefore, the polynucleotide of the invention has a nucleic acid sequence
which is at
least 65% identical, more particularly 80 % identical and even more
particularly 95%
30 identical to any one of SEQ ID NO 1, 13 to 21 as shown in figures 3, 12 to
23.
As used herein, the terms "Isolated or Purified" means altered "by the hand of
man"
from its natural state, i.e., if it occurs in nature, it has been changed or
removed from its
original environment, or both. For example, a polynucleotide or a
protein/peptide naturally
3~~ present in a living organism is neither "isolated" nor purified, the same
polynucleotide

CA 02409897 2002-10-25
14
separated from the coexisting materials of its natural state, obtained by
cloning,
amplification and/or chemical synthesis is "isolated" as the term is employed
herein.
Moreover, a polynucleotide or a protein/peptide that is introduced into an
organism by
transformation, genetic manipulation or by any other recombinant method is
"isolated"
even if it is still present in said organism.
Amino acid or nucleotide sequence "identity" and "similarity" are determined
from an
optimal global alignment between the two sequences being compared. An optimal
global
alignment is achieved using, for example, the Needleman-Wunsch algorithm
(Needleman
and Wunsch, 1970, J. Mol. Biol. 48:443-453). "Identity" means that an amino
acid or
nucleotide at a particular position in a first polypeptide or polynucleotide
is identical to a
corresponding amino acid or nucleotide in a second polypeptide or
polynucleotide that is
in an optimal global alignment with the first palypeptide or polynucleotide .
In contrast to
identity, "similarity" encompasses amino acids that are conservative
substitutions. A
"conservative" substitution is any substitution that has a positive score in
the blosum62
substitution matrix (Hentikoff and Hentikoff, 1992, Proc. Natl. Acad. Sci. USA
89: 10915-
10919). By the statement "sequence A is n% similar to sequence B" is meant
that n% of
the positions of an optimal global alignment between sequences A and B
consists of
identical residues or nucleotides and conservative substitutions. By the
statement
"sequence A is n% identical to sequence B" is meant that n% of the positions
of an
optimal global alignment between sequences A and B consists of identical
residues or
nucleotides.
As used herein, the term "polynucleotide(s)" generally refers to any
polyribonucleotide or poly-deoxyribonucleotide, which may be unmodified RNA or
DNA or
modified RNA or DNA. This definition includes, without limitation, single- and
double-
stranded DNA, DNA that is a mixture of single- and double-stranded regions or
single-,
double- and triple-stranded regions, single- and double-stranded RNA, and RNA
that is
mixture of single- and double-stranded regions, hybrid molecules comprising
DNA and
RNA that may be single-stranded or, more typically, double-stranded, or triple-
stranded
regions, or a mixture of single- and double-stranded regions. In addition,
"polynucleotide"
as used herein refers to triple-stranded regions comprising RNA or DNA or both
RNA and
DNA. The strands in such regions may be from the same molecule or from
different
molecules. The regions may include all of one or more of the molecules, but
more typically
involve only a region of some of the molecules. One of the molecules of a
triple-helical

CA 02409897 2002-10-25
region often is an oligonucleotide. As used herein, the term
"polynucleotide(s)" also
includes DNAs or RNAs as described above that contain one or more modified
bases.
Thus, DNAs or RNAs with backbones modified for stability or for other reasons
are
"polynucleotide(s)" as that term is intended herein. Moreover, DNAs or RNAs
comprising
5 unusual bases, such as inosine, or modified bases, such as tritylated bases,
to name just
two examples, are polynucleotides as the term is used herein. It will be
appreciated that a
great variety of modifications have been made to DNA and RNA that serve many
useful
purposes known to those of skill in the art. "Polynucleotide(s)" embraces
short
polynucleotides or fragments often referred to as oligonucleotide(s). The term
10 "polynucleotide(s)" as it is employed herein thus embraces such chemically,
enzymatically
or metabolically modified forms of polynucleotides, as well as the chemical
forms of DNA
and RNA characteristic of viruses and cells, including, for example, simple
and complex
cells which exhibits the same biological function as the polypeptide encoded
by SEQ ID
N0.1. The term "polynucleotide(s)" also embraces short nucleotides or
fragments, often
15 referred to as "oligonucleotides", that due to mutagenesis are not 100%
identical but
nevertheless code for the same amino acid sequence.
In a second embodiment, the present invention concerns an isolated or purified
polypeptide comprising an amino acid sequence encoded by a polynucleotide as
defined
previously. The polypeptide of the present invention preferably has an amino
sequence
having at least 80% homology, or even preferably 85% homology to part or all
of SEQ ID
N0:2 as shown in figure 4.
Yet, more preferably, the polypeptide comprises an amino acid sequence
substantially the same or having 100% identity with SEQ ID N0:2.
According to a preferred embodiment, the polypeptide of the present invention
comprises at least one amino acid sequence selected from the group consisting
of amino
acid sequence having SEQ ID NO. 3 (figure 5), SEQ ID N0.4 (figure 6), SEQ ID
NO. 5
(figure 7), SEQ ID N0.6 (figure 8), SEQ ID N0.7 (figure 9), SEQ ID N0.8
(figure 10), SEQ
ID N0.9 (figure 11 ), SEQ ID N0.10 (figure 12), SEQ ID N0.11 (figure 13), SEQ
ID N0.12
(figure 14), and biologically active fragments thereof.
As used herein, the expression "biological active" refers to a polypeptide or
fragments)
thereof that substantially retain the capacity of forming var O-receptor
complex.

CA 02409897 2002-10-25
16
According to another preferred embodiment, the isolated and purified
polypeptide of
the present invention comprises an amino acid sequence encoded by a nucleic
acid which
hybridizes under stringent conditions to the complement of SEQ ID NO 1 or
fragments
thereof. Such a polypeptide has the ability to induce cytoadherence in cells
infected with
Plasmodium related species. As used herein, to hybridize under conditions of a
specified
stringency describes the stability of hybrids formed between two single-
stranded DNA
fragments and refers to the conditions of ionic strength and temperature at
which such
hybrids are washed, following annealing under conditions of stringency less
than or equal
to that of the washing step. Typically high, medium and low stringency
encompass the
following conditions or equivalent conditions thereto
1 ) high stringency : 0. 1 x SSPE or SSC, 0. 1 % SDS, 65° C
2) medium stringency : 0. 2 x SSPE or SSC, 0. 1 % SDS, 50° C
3) low stringency : 1. 0 x SSPE or SSC, 0. 1 % SDS, 50° C.
As used herein, the term "polypeptide(s)" refers to any peptide or protein
comprising
two or more amino acids joined to each other by peptide bonds or modified
peptide bonds.
"Polypeptide(s)" refers to both short chains, commonly referred to as
peptides,
oligopeptides and oligomers and to longer chains generally referred to as
proteins. A
peptide according to the invention preferably comprises from 2 to 20 amino
acids, more
preferably from 2 to 10 amino acids, and most preferably from 2 to 5 amino
acids.
Polypeptides may contain amino acids other than the 20 gene-encoded amino
acids.
"Polypeptide(s)" include those modified either by natural processes, such as
processing
and other post-translational modifications, but also by chemical modification
techniques.
Such modifications are well described in basic texts and in more detailed
monographs, as
well as in a voluminous research literature, and they are well known to those
of skill in the
art. It will be appreciated that the same type of modification may be present
in the same or
varying degree at several sites in a given polypeptide. Also, a given
polypeptide may
contain many types of modifications. Modifications can occur anywhere in a
polypeptide,
including the peptide backbone, the amino acid side-chains, and the amino or
carboxyl
termini. Modifications include, for example, acetylation, acylation, ADP-
ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a heme
moiety, covalent
attachment of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid
derivative, covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide
bond formation, demethylation, formation of cysteine, formation of
pyroglutamate,

CA 02409897 2002-10-25
17
formylation, gamma-carboxylation, GP6 anchor formation, hydroxylation,
iodination,
methylation, myristoylation, oxidation, proteolytic processing,
phosphorylation, prenylation,
racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation
of glutamic
acid residues, hydroxylation, selenoylation, sulfation and transfer-RNA
mediated addition
of amino acids to proteins, such as arginylation, and ubiquitination. See, for
instance:
PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,
W.H. Freeman and Company, New York (1993); Wold, F., Posttranslational Protein
Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL
COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New
York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); and Rattan et
al., Protein
Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci.
663: 48-
62(1992). Polypeptides may be branched or cyclic, with or without branching.
Cyclic,
branched and branched circular polypeptides may result from post-translational
natural
processes and may be made by entirely synthetic methods, as well.
The present invention concerns also the fragments of said polypeptide
containing
between 2 to 20 amino acids. The fragment may further be a molecule (natural
or
synthetic) that inhibits the interaction ofvar O protein with its receptor.
Thus, the fragment
may be an analog, an antibody or a molecule specifically designed to bind the
active site
of var O protein (site of interaction of var O protein with its receptor).
2. Vectors and Cells
In a third embodiment, the invention is also directed to a host, such as a
genetically modified cell, comprising any of the polynucleotide sequence
according to the
invention and more preferably, a host capable of expressing the polypeptide
encoded by
this polynucleotide.
The host cell may be any type of cell (a transiently-transfected mammalian
cell
line, an isolated primary cell, or insect cell, yeast (Saccharomyces
cerevisiae,
Ktuyveromyces lactis, Pichia pastoris), plant cell, microorganism, or a
bacterium (such as
E. colt'. More preferably the host is Escherichia coli bacterium. The
following biological
deposits named IMP 537 and IMP 538 relating to Escherichia coli comprising an
expression vector encoding for DBL1-CIDR domains and DBL1-DBLS domains were
registered at the Collection Nationale des Cultures de Microorganismes (CNCM)
under

CA 02409897 2002-10-25
18
accession numbers I-2929 and I-2930 on August 30, 2002, respectively.
In a fourth embodiment, the invention is further directed to cloning or
expression
vector comprising a polynucleotide sequence as defined above, and more
particularly
directed to a cloning or expression vector which is capable of directing
expression of the
polypeptide encoded by the polynucleotide sequence in a vector-containing
cell.
As used herein, the term "vector" refers to a polynucleotide construct
designed for
transduction/transfection of one or more cell types. Vectors may be, for
example, "cloning
vectors" which are designed for isolation, propagation and replication of
inserted
nucleotides, "expression vectors" which are designed for expression of a
nucleotide
sequence in a host cell, or a "viral vector" which is designed to result in
the production of a
recombinant virus or virus-like particle, or "shuttle vectors", which comprise
the attributes
of more than one type of vector.
A number of vectors suitable for stable transfection of cells and bacteria are
available to the public (e.g. plasmids, adenoviruses, baculoviruses, yeast
baculoviruses,
plant viruses, adeno-associated viruses, retroviruses, Herpes Simplex Viruses,
Alphaviruses, Lentiviruses), as are methods for constructing such cell lines.
It will be
understood that the present invention encompasses any type of vector
comprising any of
the polynucleotide molecule of the invention.
3. Antibodies
In a fifth embodiment, the invention features purified antibodies that
specifically
bind to the isolated or purified polypeptide as defined above or fragments
thereof, and
more particularly to a protein encoded by the P, falciparum var O gene. The
antibodies of
the invention may be prepared by a variety of methods using the var O protein
or
polypeptides described above. For example, the var O polypeptide, or antigenic
fragments
thereof, may be administered to an animal in order to induce the production
ofpolyclonal
antibodies. Alternatively, antibodies used as described herein may be
monoclonal
antibodies, which are prepared using hybridoma technology (see, e.g.,
Hammerling et al.,
In Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, 1981 ).
As mentioned above, the present invention is preferably directed to antibodies
that

CA 02409897 2002-10-25
19
specifically bind P. falciparum var O polypeptides, or fragments thereof. In
particular, the
invention features "neutralizing" antibodies. By "neutralizing" antibodies is
meant
antibodies that interfere with any of the biological activities of any of
theP. falciparum var
O polypeptides, particularly the ability of P, falciparum to induce the
rosettinglautoagglutination cytoadherence phenotype of infected and non-
infected red
blood cells. Any standard assay known to one skilled in the art may be used to
assess
potentially neutralizing antibodies. Once produced, monoclonal and polyclonal
antibodies
are preferably tested for specific var O proteins recognition by Western blot,
immunoprecipitation analysis or any other suitable method.
Antibodies that recognize var O expressing cells and anitbodies that
specifically
recognize var0 proteins (or fragments var0), such as those described herein,
are
considered useful to the invention. Such an antibody may be used in any
standard
immunodetection method for the detection, quantification, and purification of
var0
proteins. The antibody may be a monoclonal or a polyclonal antibody and may be
modified for diagnostic purposes. The antibodies of the invention may, for
example, be
used in an immunoassay to monitor var0 expression levels, to determine the
amount of
var0 or fragment thereof in a biological sample and evaluate the presence or
not of a
var0 strain of Plasmodium. In addition, the antibodies may be coupled to
compounds for
diagnostic and/or therapeutic uses such as gold particles, alkaline
phosphatase,
peroxidase for imaging and therapy. The antibodies may also be labeled (e.g.
immunofluorescence) for easier detection.
With respect to antibodies of the invention, the term "specifically binds to"
refers to
antibodies that bind with a relatively high affinity to one or more epitopes
of a protein of
interest, but which do not substantially recognize and bind molecules other
than the
ones) of interest. As used herein, the term "relatively high affinity" means a
binding
affinity between the antibody and the protein of interest of at least 106 M-',
and preferably
of at least about 10' M-' and even more preferably 10a M~' to 10'° M-'.
Determination of
such affinity is preferably conducted under standard competitive binding
immunoassay
conditions which is common knowledge to one skilled in the art. As used
herein,
"antibody" and "antibodies" include all of the possibilities mentioned
hereinafter:
antibodies or fragments thereof obtained by purification, proteolytic
treatment or by
genetic engineering, artificial constructs comprising antibodies or fragments
thereof and
artificial constructs designed to mimic the binding of antibodies or fragments
thereof. Such

CA 02409897 2002-10-25
antibodies are discussed in Colcher et al. (Q J Nucl Med 1998; 42: 225-241 ).
They include
complete antibodies, F(ab')2 fragments, Fab fragments, Fv fragments, scFv
fragments,
other fragments, CDR peptides and mimetics. These can easily be obtained and
prepared
by those skilled in the art. For example, enzyme digestion can be used to
obtain F(ab')2
5 and Fab fragments by subjecting an IgG molecule to pepsin or papain cleavage
respectively. Recombinant antibodies are also covered by the present
invention.
Alternatively, the antibody of the invention may be an antibody derivative.
Such an
antibody may comprise an antigen-binding region linked or not to a non-
immunoglobulin
10 region. The antigen binding region is an antibody light chain variable
domain or heavy
chain variable domain. Typically, the antibody comprises both light and heavy
chain
variable domains, that can be inserted in constructs such as single chain Fv
(scFv)
fragments, disulfide-stabilized Fv (dsFv) fragments, multimeric scFv
fragments, diabodies,
minibodies or other related forms (Colcher et al. Q JNucl Med 1998; 42: 225-
241 ). Such a
i5 derivatized antibody may sometimes be preferable since it is devoid of the
Fc portion of
the natural antibody that can bind to several effectors of the immune system
and elicit an
immune response when administered to a human or an animal. Indeed, derivatized
antibody normally do not lead to immuno-complex disease and complement
activation
(type III hypersensitivity reaction).
Alternatively, a non-immunoglobulin region is fused to the antigen-binding
region of the antibody of the invention. The non-immunoglobulin region is
typically a non-immunoglobulin moiety and may be an enzyme, a region derived
from a protein having known binding specificity, a region derived from a
protein
toxin or indeed from any protein expressed by a gene, or a chemical entity
showing inhibitory or blocking activity(ies) against the Plasmodium virulence-
associated polypeptide. The two regions of that modified antibody may be
connected via a cleavable or a permanent linker sequence.
Preferably, the antibody of the invention is a human or animal immunoglobulin
such as IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE or IgD carrying rat or mouse
variable
regions (chimeric) or CDRs (humanized or "animalized"). Furthermore, the
antibody of the
invention may also be conjugated to any suitable carrier known to one skilled
in the art in
order to provide, for instance, a specific delivery and prolonged retention of
the

CA 02409897 2002-10-25
21
antibody, either in a targeted local area or for a systemic application.
The term "humanized antibody" refers to an antibody derived from a non-human
antibody, typically murine, that retains or substantially retains the antigen-
binding
properties of the parent antibody but which is less immunogenic in humans.
This may be
achieved by various methods including (a) grafting only the non-human CDRs
onto human
framework and constant regions with or without retention of critical framework
residues, or
(b) transplanting the entire non-human variable domains, but "cloaking" them
with a
human-like section by replacement of surface residues. Such methods are well
known to
one skilled in the art.
As mentioned above, the antibody of the invention is immunologically specific
to
the polypeptide of the present invention and immunological derivatives
thereof. As used
herein, the term "immunological derivative" refers to a polypeptide that
possesses an
immunological activity that is substantially similar to the immunological
activity of the
whole polypeptide, and such immunological activity refers to the capacity of
stimulating
the production of antibodies immunologically specific to the Plasmodium
virulence-
associated protein or derivative thereof. The term "immunological derivative"
therefore
encompass "fragments", "segments", "variants", or "analogs" of a polypeptide.
4. Compositions and vaccines
The polypeptides of the present invention, the polynucleotides coding the
same,
and polyclonal or monoclonal antibodies produced according to the invention,
may be
used in many ways for the diagnosis, the treatment or the prevention of
Plasmodium
related diseases and in particular malaria.
In a sixth embodiment, the present invention relates to a composition for
eliciting
an immune response or a protective immunity against aP. species. According to
a related
aspect, the present invention relates to a vaccine for preventing and/or
treating a
Plasmodium associated malarial disease. As used herein, the term "treating"
refers to a
process by which the symptoms of malaria are alleviated or completely
eliminated. As
used herein, the term "preventing" refers to a process by which a Plasmodium
associated
malarial disease is obstructed or delayed. The composition or the vaccine of
the invention
comprises a polynucleotide, a polypeptide and/or an antibody as defined above
and an

CA 02409897 2002-10-25
22
acceptable carrier.
As used herein, the expression "an acceptable carrier" means a vehicle for
containing the polynucleotide, a polypeptide and/or an antibody that can be
injected into a
mammalian host without adverse effects. Suitable carriers known in the art
include, but
are not limited to, gold particles, sterile water, saline, glucose, dextrose,
or buffered
solutions. Carriers may include auxiliary agents including, but not limited
to, diluents,
stabilizers (i. e., sugars and amino acids), preservatives, wetting agents,
emulsifying
agents, pH buffering agents, viscosity enhancing additives, colors and the
like.
Further agents can be added to the composition and vaccine of the invention.
For
instance, the composition of the invention may also comprise agents such as
drugs,
irnmunostimulants (such as a-interferon, p-interferon, y-interferon,
granulocyte
macrophage colony stimulator factor (GM-CSF), macrophage colony stimulator
factor (M-
CSF), interleukin 2 (/L2), interleukin 12 (/L_12), and CpG oligonucleotides),
antioxidants,
surfactants, flavoring agents, volatile oils, buffering agents, dispersants,
propellants, and
preservatives. For preparing such compositions, methods well known in the art
may be
used.
The amount of polynucleotide, a polypeptide and/or an antibody present in
the compositions or in the vaccines of the present invention is preferably a
therapeutically effective amount. A therapeutically effective amount of
polynucleotide, a polypeptide and/or an antibody is that amount necessary to
allow
the same to pertorm their immunological role without causing, overly negative
effects in the host to which the composition is administered. The exact amount
of
polynucleotide, a polypeptide and/or an antibody to be used and the
compositionlvaccine to be administered will vary according to factors such as
the
type of condition being treated, the mode of administration, as well as the
other
ingredients in the composition.
5. Methods of use
In a seventh embodiment, the present invention relates to methods for treating
and/or

CA 02409897 2002-10-25
23
preventing Plasmodium related diseases, such as malaria in a mammal are
provided.
The method comprises the step of administering to the mammal an effective
amount of
the isolated or purified polynucleotide, the isolated or purified polypeptide,
the composition
as defined above and/or the vaccine as defined above.
The vaccine, antibody and composition of the invention may be given to a
mammal
through various routes of administration. For instance, the composition may be
administered in the form of sterile injectable preparations, such as sterile
injectable
aqueous or oleaginous suspensions, These suspensions may be formulated
according to
techniques known in the art using suitable dispersing or wetting agents and
suspending
agents. The sterile injectable preparations may also be sterile injectable
solutions or
suspensions in non-toxic parenterally-acceptable diluents or solvents. They
may be given
parenterally, for example intravenously, intramuscularly or sub-cutaneously by
injection,
by infusion or per os. The vaccine and the composition of the invention may
also be
formulated as creams, ointments, lotions, gels, drops, suppositories, sprays,
liquids or
powders for topical administration. They may also be administered into the
airways of a
subject by way of a pressurized aerosol dispenser, a nasal sprayer, a
nebulizer, a
metered dose inhaler, a dry powder inhaler, or a capsule. Suitable dosages
will vary,
depending upon factors such as the amount of each of the components in the
composition, the desired effect (short or long term), the route of
administration, the age
and the weight of the mammal to be treated. Any other methods well known in
the art
may be used for administering the vaccine, antibody and the composition of the
invention.
The present invention is also directed to an in vitro diagnostic method for
the detection
of the presence or absence of antibodies indicative of Plasmodium species (for
instance
P. falciparum), which bind with the polypeptide as defined above to form an
immune
complex. Such method comprises the steps of
a) contacting the polypeptide of the present invention with a biological
sample for a
time and under conditions sufficient to form an immune complex; and
b) detecting the presence or absence of the immune complex formed in a).
In a further embodiment, a diagnostic kit for the detection of the presence or
absence
of antibodies indicative of Plasmodium species is provided. Accordingly, the
kit comprises:
- a polypeptide as defined above;

CA 02409897 2002-10-25
24
- a reagent to detect polypeptide-antibody immune complex;
- a biological reference sample lacking antibodies that immunologically bind
with
the polypeptide; and
- a comparison sample comprising antibodies which can specifically bind to the
polypeptide;
wherein the polypeptide, reagent, biological reference sample, and comparison
sample are present in an amount sufficient to perform the detection.
The present invention also proposes an in vitro diagnostic method for the
detection
of the presence or absence of polypeptides indicative of Plasmodium species,
which bind
with the antibody of the present invention to form an immune complex,
comprising the
steps of:
a) contacting the antibody of the invention with a biological sample for a
time and
under conditions sufficient to form an immune complex; and
b) detecting the presence or absence of the immune complex formed in a).
In a further embodiment, a diagnostic kit for the detection of the presence or
absence of polypeptides indicative of Plasmodium species is provided.
Accordingly, the kit
comprises:
- an antibody as defined above;
- a reagent to detect polypeptide-antibody immune complex;
- a biological reference sample lacking polypeptides that irnmunologically
bind with
the antibody; and
- a comparison sample comprising polypeptides which can specifically bind to
the
antibody;
wherein said antibody, reagent, biological reference sample, and comparison
sample are
present in an amount sufficient to perform the detection.
Yet, in another embodiment, an in vitro diagnostic method for the detection of
the
presence or absence of a polynucleotide indicative of Plasmodium species is
provided,
accordingly, the method comprises the steps of:
a) contacting at least one oligonucleotide as defined above with a
biological sample for a time and under conditions sufficient for said
oligonucleotide to hybridize to said polynucleotide; and
b) detecting the presence or absence of an hybridization between the

CA 02409897 2002-10-25
oligonucleotide and the polynucleotide.
Yet, according to a further embodiment, a diagnostic kit for the detection of
the
presence or absence of polynucleotide indicative of Plasmodium species is
provided.
5 accordingly, the kit comprises:
- an oligonucleotide as defined above;
- a reagent to detect polynucleotide-oligonucleotide hybridization complex;
- a biological reference sample lacking polynucleotides that hybridise with
the
oligonucleotide; and
10 - a comparison sample comprising polynucleotides which can specifically
hybridise
to the oligonucleotide;
wherein said oligonucleotide, reagent, biological reference sample, and
comparison
sample are present in an amount sufficient to perform the detection.
15 In a preferred embodiment, the oligonucleotide referred to in the
diagnostic methods and
kits hybridises to the polypeptide having SEQ ID NO 18 or fragments thereof.
The present invention will be more readily understood by referring to the
following
example. This example is illustrative of the wide range of applicability of
the present
20 invention and is not intended to limit its scope. Modifications and
variations can be made
therein without departing from the spirit and scope of the invention. Although
any methods
and materials similar or equivalent to those described herein can be used in
the practice
for testing of the present invention, the preferred methods and materials are
described.

CA 02409897 2002-10-25
26
FY~MPI F
Cloning and characterization of the P. falciparum var O gene.
Characterization of the var O gene as one virulence factor was done using the
experimental model of infection with the O and R Palo Alto variants in
thesplenectomized
Saimiri sciureus monkey.
The large cellular aggregates which are normally filtered by the spleen in
spleen-
intact individuals, circulate in splenectomized monkeys (Contamin H. et al.
(2000)
Microbes & Infection 2. 945-954}. Unlike what is observed in human infections,
a very
large proportion of parasites (80% and over) are rosette-forming or
autoagglutinating
within the var O population. This provides the opportunity to study the
biological properties
of that variant with a high degree of confidence.
The inventors of the present invention compared the growth rate of variant
O and variant R in vivo. This growth rate comparison has variant O to have a
higher multiplication rate in vivo than its sibling var R. The calculated
multiplication
rate (increase in parasite density) is of 2.7 per day for the var O parasites
as
opposed to 1.7 per day for the var R parasites. Since the O variant is the
largely
dominant clone within the population, the inventors could conclude with a fair
degree of confidence to the contribution of the rosetting phenotype of variant
O to
increased multiplication rate. This indicates that rosetting is indeed a
virulence
factor. The rapid multiplication rate of the O variant is further indicated by
the
observation of re-emerging rosetting/autoagglutinating parasites from two
independent variant lines, namely var R and "pic2", that were propagated in
the
Saimiri monkey most probably due to outgrowth of parasites that had switched
back to express the var O type.
Consecutive infections with the same variant have shown that the absence of
antibodies reacting with the var O surface, as determined by Fluorescence
Activated Cell
Sorter or by agglutination on the day of infection, is usually associated with
sensitivity to
subsequent infection by variant O. Inversely, the inventors have observed that
the

CA 02409897 2002-10-25
27
presence of antibodies reacting with the surface is associated with failure of
the var O
parasites to develop.
Both lines of arguments suggest that the rosetting/autoagglutination
cytoadherence phenotype associated with var O parasites represents a virulence
factor, and that specific immunity against that phenotype protects against
further
infection by this variant. This is consistent with published associations
obtained in
human studies. The results show a direct correlation of a highly dominant
cytoadherence phenotype (rosettinglautoagglutination) with specific infection
outcome.
The specific var gene [var O], specifically expressed by rosetting FUP/SP
parasites, which has been identified by RT-PCR as being the dominant var gene
expressed by var O parasites was characterized by gene walking and RT-PCR.
In order to identify the DBL1 domain of the var gene specifically expressed by
the O parasites, the inventors proceeded by RT-PCR using degenerate primers
targeted to the DBL1 domain. O and R parasite RNAs were extracted from highly
parasited blood samples (over 20% infected red blood cells) containing a 70 to
90%
proportion of mature parasites.
Several sets of primers described in the literature were tested: UNIEBP (Smith
J.
D. et al., (1995), Cell 82:101-110), VarA5.1/VarE3.2 (Hernandez-Rivas et al.,
(1997)
Mol. Cell. Biol. 17:604-611 ), and DBL1.1/DBL1.2 (Chen et al., (1998) J. Exp.
Med.
187:15-23), alpha AF/alpha BR (Taylor et al., (2000) MBP 105:13-23).
Following RT-PCR amplification using universal primers (alpha AF alpha BR), 69
DBL1 sequences (49 clones from the O cDNAs and 20 clones from the R cDNAs)
were
analyzed. In all, 18 different DBL1 sequences were obtained from these
samples. This
relatively important diversity indicates that the universal primers permit the
identification
of a large number of sequences. In fact, the parasites propagated in the
splenectomized monkey develap asynchronously and the enrichment method,
consisting of eliminating trophozoites and schizonts by sorbitol treatment and
allowing
rings to mature in culture over 24 hours, offered variable rates among
samples. The
purified RNA from these samples therefore contained transcripts from non
mature

CA 02409897 2002-10-25
28
forms of parasites, abortive transcripts that do not correspond to the
majority sequence
translated by mature forms.
In order to identify the relevant sequences, it was therefore necessary to
analyze
a great number of clones. Alignment of these 69 sequences evidenced specific
majority
DBL1 clones for parasites O and R. These specific sequences respectively
represent
60% and 25% of clones O and R analyzed.
As illustrated below in Table 1, the analogous sequences for the O parasites
break down into the following: a group of 28 identical sequences (specifically
identified
from O cDNAs), a group of 12 identical sequences, a group of 2 identical
sequences
and 7 unique sequences from 49 analyzed clones. For the R parasites, the 20
sequences analyzed break down as follows: 2 groups of 5 identical sequences
(one of
which is also expressed by the O parasites), 2 groups of 2 identical sequences
and 6
unique sequences.
parasites


DBL1 Sequences var O -varR -


1


2 1.2........................._...._._....__...._2;
_._...._._._.___...__......._....._....__.__..
..~


3
~___.........._......_............._.___..._._.._...__.___~._....~


4 1..._...__.._........_......__.......__..._._._.~
._......._..
~..._..._..._...._._......_.. ....._.__.._.__._....._....__..___.


1..........,....._...._..._......._.._._..
___._.._.._.._..._..___...__~
L...~......_ . _ __...._...... ... _ _____._._._._..___.._.___.~


6 1


7 1


8 1


9 1


10 1


11


12 2


13 1


14 1


15 1


16 1


17 1


18 1


Total 49 20



CA 02409897 2002-10-25
29
Table 1. Distribution of the different sequences of DBL1 clones obtained after
RT-
PCR using universal primers (alpha AF/alpha BR) for the extracted RNAs of O
and
R parasites propagated in the splenectomized monkey.
Using a set of UNIEBP degenerated primers, of less restrained specificity,
different amplification profiles were again observed with O and R parasite
cDNAs. A
450bp band was mainly detected in the O samples and inversely, a 500 by band
was
mainly detected in samples R confirming that the difference between O and R
parasites
is at the phenotypic (gene expression) level. As predicted, amplification of
genomic
sequences result in identical profiles. Alignment with the var genes described
in the
literature indicates two different DBL3 domains amplified using the UNIEBP
primers.
This result was reproduced with 8 different parasite RNA preparations (4 Os'
and 4 Rs'). The two bands corresponding to specific amplification products
were
sequenced.
As a result of these analyses, two specific domains of the var O gene : the
DBL1
domain and the DBL3 domain were identified. Comparison of these sequences with
those described in the literature showed that the var O DBL1alpha domain
identified
presents a strong homology with that encoded by the var gene implicated in
rosetting
decribed by Rowe et a1.((1997) Nature 388:292-295) but little homology with
the other
DBL1alpha domain implicated in rosetting described by Chen et a1.((1998) J.
Exp. Med.
187:15-23) (Figure 2).
From these specific sequences, the inventors were able to define new primers
and proceed with the sequencing of the entire var O gene. Two strategies were
developed: (1 ) chromosome walking which permitted to obtain several fragments
on
either side of the identified domains, and (2) RT-PCR, which by combining
specific
primers and conserved primers generated two large fragments, a DBL1-DBL3
fragments and a DB3-exonll.
Analysis of these different fragments allowed the inventors to define an open
reading frame of 7378 by disclosed in figure 3.
Exon 1 which codes for the extracellular part of the molecule, features five
DBL

CA 02409897 2002-10-25
domains, one CIDR domain adjacent to the DBL1 domain and threeinterdomains
(C2,
ID3 and ID4). Alignment of the sequences with the DBL and CIDR domains
described
in Smith et al.'s phylogenetic study (2000) MBP 110:293-310) allowed the
inventors to
identify and class the var O domains according to the new nomenclature. As
illustrated
5 in figure 1, the DBL 1 domain corresponds to the DBLalpha group, the DBL2
belongs to
the DBLbeta group, the DBL3 to the DBLgamma group, the DBL4 and DBL5 to the
DBLepsilon group, and finally, the CIDR corresponds to CIDRgamma.
The 2459 amino acid deduced var O protein sequence delimits the different
10 protein domains (with conserved amino acids underlined) which can be seen
in figures
5 to 14.
The 2459 amino acid deduced var O protein sequence shows five DBL and one
CIDR domains, with a particular organization (figure 1 ). The var O protein is
a
15 composite of domains, each of which shows a best identity match with a
different
PfEMP1 protein for a specific domain. Var O differs from the recently
described and
well conserved var sub-families which share homology all over their sequences
(Salanti
A. et al. (2002) Mol. Biochem. Parasitol. 122, 111-115; Rowe J.A. et al.
(2002) J. Inf.
Dis. 185, 1207-1211 ), the prototype being the var CSA gene (Buffet P. et al.
(1999)
20 Proc. Natl Acad. Sci. U.S.A. 96, 12743-12748). Var O differs substantially
from the
previously chracterized var CSA gene(Buffet P. et al. (1999) Proc. Natl Acad.
Sci.
U.S.A. 96, 12743-12748). Maximal identity observed was 64%, suggesting unique
features to this protein (Figure 2).
25 The deduced var O protein sequence has several important features. The
DBL1alpha-CIDR1gamma association is atypical and has been so far described in
only
one case, namely the PfEMP1 protein coding for the rosette-forming variant R29
of the
It line. The R29 DBL1 alpha domain has been implicated in binding to the
erythrocyte
receptor CR1. Homology with the R29 PfEMP1 protein is restricted to this
region.
30 Additional domains share substantial identity with domains from other
PfEMP1
molecules that have been implicated in other binding specificities.
The var O PfEMP1 protein presents the so far unique and unexpected feature of
having no CIDRalpha domain. CIDRalpha is responsible for binding to CD36. The
absence of a canonical CD36 binding domain in var O PfEMP1 is consistent with
the

CA 02409897 2002-10-25
31
incapacity to demonstrate binding to human CD36. This is particularly
interesting
because CD36 binding is a comman phenotype in Plasmodium falciparum isolates,
with quite important consequences in terms of stimulation of the immune
system.
CIDRalpha has been shown to stimulate naive CD4 T cells to produce interferon
(IFN)
gamma and interleukin (IL) 10 (Allsop C.E.M. et al. (2002) J. Inf. Dis. 185,
812-819).
Intact infected red blood cells cytoadhere to dendritic cells via a CIDRalpha
/ CD36
interaction. Upon binding on dendritic cells, they inhibit dendritic cell
maturation and
function (Urban B. et al. (1999) Nature 400, 73-77). In this regard, the
absence of a
CIDRalpha from the var O PfEMP1 protein is particularly interesting as it
should
present the unexpected advantage no such impairment of T cell function and
hence a
better immunogenicity of the head domain.
The various DBL domains, the CIDR domain, the variable inter-domains and the
DBL1 alpha-CIDR1 gamma head structure have been sub-cloned for expression in a
bacterial expression system, the pET system, for expression into the
periplasmic space
(Novagene). The pET system is a proven expression system for a large number of
recombinant molecules, particularly those possessing numerous disulfide bonds.
The pET22b vector was chosen in order to increase the chances of obtaining
soluble and active proteins. This vector carries the pelB signal sequence that
promotes
periplasmic orientation of target proteins. This cellular compartment provides
a
favorable environment for disulfide bond formation and correct protein
folding. The
pET22b vector also carries a C terminal poly-histidine tail allowing
recombinant
molecule detection and affinity purification.
Cloning step was effected in two stages. Recombinant plasmids were first
isolated from the Novablue strain. Two bacterial strains namely, BL21 (DE3)
and
BL21 (DE3)PIysS, possessing the T7 polymerase gene under the control of the
IPTG-
inducible IacUVS promoter, were then transformed with these plasmids and used
for
overexpression.
Use of the pET system required the preparation of new inserts providing
adequate cloning sites for unidirectional insertion into the pET22b vector. To
date, only
the DBL1 domain of the var genes has been implicated in red blood cell
adhesion,
however, certain associations of tandem domains have been observed (e.g.
DBLalpha

CA 02409897 2002-10-25
32
CIDRalpha, DBLbeta C2). These associations might have a functional
explanation. For
these reasons, the DBLalpha-CIDR segment (Figure 1 ) was also expressed. The
different constructs were isolated from the Escherichia coli K12 Novablue
strain
Novagene) endA1 hsdRl7 (rk,2 -- mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac
jF' pro
A+B+ !ac n ZOM15 : : Tn 10 (TcF~)J, then transformed into the expression
strains
BL21 (DE3) and BL21 (DE3)PIysS. These bacterial clones were registered at the
Authorized International depositary known as the Collection Nationale de
Cultures de
Microorganismes {CNCM) on August 30, 2002 under accession numbers I-2929
(clone
IMP 537- clone V) and I-2930 (clone IMP 538).
Clone N°IMP 529 Escherichia coli K12 Novablue strain ( Novagene)
endA1
hsdRl7 (rk,2 ' mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B+ lac
i° ZAM15
Tn10 (TcR)J carries plasmid pET22b-Clone G containing the DBL1 domain coding
sequence e.g. nucleotides 283 to 1206 of the var O Palo Alto nucleotide
sequence as
shown in figure 15. The following set of amplification primers were used:
SEQ ID NO. 22: sens SDBLIB : 5'TAC AAC GAG GAT CCA AAG CCT TGT TAT GGA AGG
(+2aa);
SEQ ID NO. 23: anti-sens 3DBLIX : 5'AGT TTT TTT CCA CTC GAG AAT TTC ATA GAA
TAT TTA
AGG TTT TG.
Clone N°IMP 530 Escherichia coli K12 Novablue strain ( Novagene)
endA1
hsdR17 (rk,2 - mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+8+ lac
~° ZOM15
TnlO (TcR)J carries plasmid pET22b-Clone A containing the DBL2 domain coding
sequence, e.g. nucleotides 2497 to 3312 of the var O Palo Alto nucleotide
sequence as
shown in figure 16. The following set of amplification primers were used
SEQ ID NO. 24: sens 5DBL2B : 5'CGT TCT GGT TAG GAT CCT AAA GGA CCA TGT ACA G;
SEQ ID NO. 25: anti-sees 3DBL2X : 5'TGC AAT TTT TGC TTT CTC GAG TAA ATC CTT
GTA
TTT TTG TTC.
Clone N°IMP 531 Escherichia coli K12 Novablue strain ( Novagene)
endA1
hsdRl7 (rk,2 - mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+8+ lac
i° Z~M15
TnlO (TcR)J carries plasmid pET22b-Clone B containing the C2 domain coding
sequence,
e.g. nucleotides 3301 to 3735 of the var O Palo Alto nucleotide sequence as
shown in
figure 17. The following set of amplification primers were used:
SEQ ID NO 26: Sens 51D2B : 5'TGG AAA CAA ATG GAT CCA AAA TAC AAG GAT TTA TAC;

CA 02409897 2002-10-25
33
SEQ ID NO 27: Anti-sens 31D2X : 5'CCA ATT TAC CTC GAG TTT TAT ATT GCA CCC ATA
TAT
TGA.
Clone N°IMP 532 Escherichia coli K12 Novablue strain ( Novagene)
endA1
hsdRl7 (rk,z - mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B' lac
i° ZOM15
Tn10 (TcR)J carries plasmid pET22b-Clone C containing the DBL3 domain coding
sequence, e.g. nucleotides 3713 to 4461 of the var O Palo Alto nucleotide
sequence as
shown in figure 18. The following set of amplification primers were used
SEQ ID NO 28: Sens 5DBL3B : 5'AAG GGC GAA ACG GAT CCA ATA TAT GGG TG;
SEQ ID NO 29: Anti-sens 3DBL3X : 5'TTT TCC TTT CTC GAG TGT AAA TTT TTG GCT TCG
TTT
TTC.
Clone N°IMP 533 Escherichia coli K12 Novablue strain ( Novagene)
endA1
hsdRl7 (rk,z - mk,z +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B+ lac ~Q
ZOM15
Tn10 (TcR)J carries plasmid pET22b-Clone D containing the ID3 domain coding
sequence,
e.g. nucleotides 4444 to 4896 of the var O Palo Alto nucleotide sequence as
shown in
figure 19. The following set of amplification primers were used
SEQ ID NO 30: Sens 51D3B : AAA ACT CAA TAG GAT CCA CGA AGC CAA AAA TTT ACA
AGA;
SEQ ID NO 31: Anti-sens 31D3X : AGA AAC TTC CTC GAG TTT ACA TGT TTC CCC ATT
TTG
ATT.
Clone N°IMP 534 Escherichia coli K12 Novablue strain ( Novagene)
endA1
hsdR17 (rk,z - mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B+ !ac
i° Z~M15
TnlO (TcR)J carries plasmid pET22b-Clone L containing the DBL4 domain coding
sequence, e.g. nucleotides 4891 to 5799 of the var O Palo Alto nucleotide
sequence as
shown in figure 20. The following set of amplification primers were used
SEQ ID NO 32: Sens 5DBL4B : AAT CAA AAT GGG GAT CCA TGT AAA TTT AAA GAA GTT;
SEQ ID NO 33: Anti-sens 3DBL4X : AGC ATT TTT CTC GAG TTC TTT ATA TGC TTT GTT
TTG
TGT TTC.
Clone N°IMP 535 Escherichia coli K12 Novablue strain ( Novagene)
endA1
hsdRl7 (rk,z - mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B+ lac
~° Z~M15
TnlO (TcR)J carries plasmid pET22b-Clone O containing the ID4 domain coding
sequence,
e.g. nucleotides 5779 to 6111 of the var O de Palo Alto nucleotide sequence as
shown in
figure 21. The following set of amplification primers were used

CA 02409897 2002-10-25
34
SEQ ID NO 34: Sens 51D4B : GCC CAA TTG GAT CCA CAA AAC AAA GCA TAT AAA G;
SEQ ID NO 35: Anti-sans 31D4X : GAA ATT TTT CTC GAG ACA ACT ACC TAT ACC ATA
CT'f
CTT.
Clone N°IMP 536 Escherichia coli K12 Novablue strain ( Novagene)
endA1
hsdRl7 (rk~2 - mk~2 +) supE44 thi-1 recA 1 gyrA96 relA1 lac [F' pro A+B+ lac
~° ZOM15
TnlO (TcR)j carries plasmid pET22b-Clone M coding for the DBLS domain, e.g.
nucleotides 6103 to 6813 of the var O Palo Alto nucleotide sequence as shown
in figure
22. The following set of amplification primers were used
SEQ ID NO 36: Sans 5DBL5B : TGT AAG AAG TAG GAT CCA GGT AGT TGT CCA GAA;
SEQ ID NO 37: Anti-sans 3DBL5X : TAG TGT CTT CTC GAG TTC TTT ATC ATA TTT ATC
CTT
TTG AAT TTC.
Clone N°IMP 537 Novablue strain ( Novagene) endA1 hsdR17 (rk,2 -
mk,2 +)
supE44 thi-1 recA1 gyrA96 relA1 lac [F' pro A''8+ !ac i° 7~M15 : : TnlO
(TcR)J
carries plasmid pET22b-Clone V containing the DBL1-CIDR tandem domain, e.g.
coding
sequence, e.g. nucleotides 283 to 2520 of the var O Palo Alto nucleotide
sequence as
shown in figure 23. The following set of amplification primers were used
SEQ ID NO 38: Sans 5DBLIB : 5'TAC AAC GAG GAT CCA AAG CCT TGT TAT GGA AGG;
SEQ ID NO 39: Anti-sans 3CIDRX : 5' TCC TAT TTT AAA CCT CTC GAG ATT TTT ACC
TGT
ACA TGG.
Expression assays of the different domains were also conducted. The bacterial
protein extracts, induced and non-induced, were analyzed by SDS-PAGE
(Coomassie
blue staining), and western blot (detection with a monoclonal anti-his
antibody).
Different expression rates are observed depending on the construct. The C2
domain is
recognized by E1 variant var O infected monkey antibodies.
This collection of clones represents a unique tool to investigate by the
methods
known to the person skilled in the art the binding specificity of the various
domains,
identify the naturally immunogenic regions, and to devise a rational
intervention and
diagnosis based on that molecule.
As an example of the usefulness of such a compehensive array of sub-clones is
the observation that interdomain 2 called C2 induces antibodies in monkeys
infected
with var O parasites. A collection of polyclonal and monoclonal antibodies
that

CA 02409897 2002-10-25
recognize the different epitopes encoded by the var O gene and its different
domains
may be produced by the methods known by the person skilled in the art.
Interventions based on this molecule will reduce the incidence of clinical
5 malaria by interfering with in vivo propagation, capillary obstruction and
possibly
reduce in this way systemic inflammation. A particularly novel aspect of the
invention is the inclusion of the DBt-1 alpha-CI DR1 gamma expression product
for further analysis, including of possible immunomodulatory activities. The
invention concerns also the collection of individual domains, including the
non
10 adhesive domains sub-cloned into an Escherichia coli expression vector.
This
forms a comprehensive tool for analysis of naturally acquired immune response
and a versatile tool for a final vaccine composition including one or multiple
individual domains.