U.S. patent application number 11/376124 was filed with the patent office on 2006-10-26 for proteins involved in cytoadhesion of plasmodium falciparum ring-stage-infected erythrocytes, antibodies which bind to the proteins, and methods for detecting infection, stage of infection and vaccines for protecting against infection.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Pierre Buffet, Juerg Gysin, Bruno Pouvelle, Artur Scherf.
Application Number | 20060240491 11/376124 |
Document ID | / |
Family ID | 22772644 |
Filed Date | 2006-10-26 |
United States Patent
Application |
20060240491 |
Kind Code |
A1 |
Gysin; Juerg ; et
al. |
October 26, 2006 |
Proteins involved in cytoadhesion of plasmodium falciparum
ring-stage-infected erythrocytes, antibodies which bind to the
proteins, and methods for detecting infection, stage of infection
and vaccines for protecting against infection
Abstract
The present invention provides hybridomas and antibodies which
specifically react with native P. falciparum at a surface of ring
stage infected erythrocytes and uses thereof.
Inventors: |
Gysin; Juerg;
(Saint-Zacharie, FR) ; Pouvelle; Bruno;
(Saint-Maximin, FR) ; Scherf; Artur; (Paris,
FR) ; Buffet; Pierre; (Paris, FR) |
Correspondence
Address: |
C. IRVIN MCCLELLAND;OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
INSTITUT PASTEUR
Paris Cedex 15
FR
Centre National De La Recherche Scient.
Paris Cedex 14
FR
|
Family ID: |
22772644 |
Appl. No.: |
11/376124 |
Filed: |
March 16, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10305956 |
Nov 29, 2002 |
|
|
|
11376124 |
Mar 16, 2006 |
|
|
|
PCT/EP01/06874 |
May 30, 2001 |
|
|
|
10305956 |
Nov 29, 2002 |
|
|
|
60207952 |
May 31, 2000 |
|
|
|
Current U.S.
Class: |
435/7.22 ;
435/258.2; 530/350; 530/388.6 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61K 39/00 20130101; A61K 2039/505 20130101; C07K 14/445 20130101;
A61P 33/06 20180101; Y02A 50/412 20180101 |
Class at
Publication: |
435/007.22 ;
530/350; 530/388.6; 435/258.2 |
International
Class: |
G01N 33/569 20060101
G01N033/569; C12N 1/10 20060101 C12N001/10; C07K 14/445 20060101
C07K014/445; C07K 16/20 20060101 C07K016/20 |
Claims
1-22. (canceled)
23. A hybridoma cell that produces an antibody which specifically
reacts with native Plasmodium falciparum protein at a surface of
ring-sage infected erythrocytes and does not specifically react
with mature trophozoite and schizonte infected erythrocytes.
24. The hybridoma cell of claim 23, which is hybridoma Pf26G1/B4
deposited at the CNCM on Feb. 23, 2001 under accession number
I-2635.
25. An antibody produced by the hybridoma cell of claim 23.
26. A composition comprising the antibody of claim 25 and at least
one pharmaceutically acceptable carrier.
27. A method of inhibiting cytoadhesion of ring-stage infected
erythrocytes to endothelial cells of brain and placenta in an
individual, the method comprising administering the antibody of
claim 25 to the individual in an amount sufficient to inhibit
cytoadhesion or ring-stage infected erythrocytes to endothelial
cells of brain and placenta.
28. A method of inhibiting merozoite invasion of erythrocytes in an
individual, the method comprising administering the antibody of
claim 25 to the individual in an amount sufficient to inhibit
merozoite invasion of erythrocytes.
29. A method of treating malaria in an individual, the method
comprising administering the antibody of claim 23 to the individual
to treat malaria in the individual.
30. An antibody which binds to an isolated ring surface protein-1
(RSP-1).
31. The antibody of claim 30, which is a monoclonal antibody.
32. The antibody of claim 30, which is a polyclonal antibody.
33. A composition comprising the antibody of claim 30 and at least
one pharmaceutically acceptable carrier.
34. A composition comprising the antibody of claim 31 and at least
one pharmaceutically acceptable carrier.
35. A composition comprising the antibody of claim 32 and at least
one pharmaceutically acceptable carrier.
36. A method of inhibiting cytoadhesion of ring-stage infected
erythrocytes to endothelial cells of brain and placenta in an
individual, the method comprising administering the antibody of
claim 30 to the individual in an amount sufficient to inhibit
cytoadhesion or ring-stage infected erythrocytes to endothelial
cells of brain and placenta.
37. A method of inhibiting merozoite invasion of erythrocytes in an
individual, the method comprising administering the antibody of
claim 30 to the individual in an amount sufficient to inhibit
merozoite invasion of erythrocytes.
38. A method of treating malaria in an individual, the method
comprising administering the antibody of claim 30 to the individual
to treat malaria in the individual.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention provides the RSP-1 and RSP-2 proteins
which are involved in the cytoadhesion of P. falciparum during
ring-stage infection of erythrocytes, antibodies which bind to the
proteins, methods of screening for a P. falciparum infection,
methods of determining the infective stage of P. falciparum and
vaccines for protecting individuals from Plasmodium sp.
infections.
[0003] 2. Description of the Background
[0004] A common pathological characteristic in P. falciparum
infection is the cytoadbesion of mature-stage infected erythrocytes
([E) to host endothelium and syncytiotrophoblasts. Massive
accumulation of IE in the brain microvascular or placenta is
strongly correlated with severe form of malaria.sup.1. Extensive
binding of IE to placental CSA is associated with physiopathology
during pregnancy.sup.2,3. The adhesive phenotype of IE correlates
with the appearance of POEMP1 at the erythrocyte surface (approx.
16 hours after merozoite invasion) and therefore only early
blood-stage (ring-stage) IE are seen in the peripheral blood Here
we describe results that challenge the existing view of blood-stage
IE biology. We demonstrate the specific adhesion of IE, during the
early ring-stage, to endothelial cell lines from brain and lung and
to placental syncytiotrophoblasts. Later in the blood-stage
development of these IE, trophozolites switch to an exclusively
chondroitin-sulphate A (CSA) cytoadhesion phenotype. Therefore,
adhesion to an individual endothelial cell or syncytiotrophoblast
may occur throughout the blood stage cycle, suggesting that there
are non-circulating (cryptic) parasite subpopulations in malaria
patients. We detected two novel parasite proteins on the surface of
ring-stage IE. These proteins disappear shortly after the start of
PfEMP1-mediated adhesion. These data have important implications
for epidemiological studies, parasite tissue tropism and malarial
disease outcome.
SUMMARY OF INVENTION
[0005] Thus, an object of the present invention is an isolated
RSP-1 protein which mediates Plasmodium falciparum ring-stage
adhesion to endothelial cells and is approximately 200 kilodaltons
in size as determined by SDS-polyacrylamide gel
electrophoresis.
[0006] Another object of the present invention is an isolated RSP-2
protein which mediates Plasmodium falciparum ring-stage adhesion to
endothelial cells and is approximately 40 kilodaltons in size as
determined by SDS-polyacrylamide gel electrophoresis.
[0007] Another object of the present invention is an antibody which
binds to RSP-1 or RSP-2.
[0008] Another object of the present invention is a method of
detecting the presence of a Plasmodium species in a sample
comprising contacting said sample with the RSP-1 or RSP-2 antibody
andidentifying an interaction between the antibody and the
Plasmodium species in said sample, wherein said interaction
indicates the presence of the Plasmodium species, preferably where
the Plasmodium species is P. falciparum.
[0009] Another object of the present invention is a method of
detecting the presence of a Plasmodium antibody in a sample
comprising contacting said sample with the isolated RSP-1 or RSP-2
protein; and identifying an interaction between the protein and the
Plasmodium antibody in said sample, wherein said interaction
indicates the presence of the Plasmodium, preferably where the
Plasmodium species is P. falciparum.
[0010] Another object of the present invention is a method of
diagnosing a Plasmodium falciparum blood-stage cycle in an
individual suspected of being infected with Plasmodium falciparum
comprising obtaining a biological sample from said individual;
contacting said sample with an RSP-1 and/or and RSP-2 antibody; and
identifying an interaction between the antibody and an antigen in
said sample, wherein said interaction indicates a ring-stage
infection.
[0011] Another object of the present invention is a method of
diagnosing the Plasmodium falciparum blood-stage cycle in an
individual suspected of being infected with Plasmodium falciparum
comprising obtaining a biological sample from said individual;
contacting said sample with the RSP-1 or RSP-2 protein; and
identifying an interaction between the protein and an antibody in
said sample, wherein said interaction indicates a ring-stage
infection.
[0012] Another object of the present invention is an immunogenic
composition comprising the isolated RSP-1 and/or RSP-2 protein and
a pharmaceutical acceptable carrier and further wherein the
immunogenic composition is a vaccine.
[0013] Another object of the present invention is a method of
protecting an individual against a Plasmodium falciparum infection
comprising administering RSP-1 and/or RSP-2 to said individual in
an amount sufficient to induce an immune response in said
individual.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1: Ring-stage IE.sup.CSA cytoadhesion to various cells
and tissues. IE.sup.CSA from highly synchronized cultures
cytoadhered to (A) cryosections of liquid nitrogen snap-frozen
uninfected human placenta biopsy samples (R, ring- and
T,.trophozoite stage) or (B) SBEC ID monolayer (Nikon E800,
.times.1000). (C) Cytoadhesion of synchronized PA.sup.CSA IE, at
the 8th hour after invasion, to monolayers of SBEC ID, HUVEC, HLEC
primo explants, HMEC, C32 and CHO cells and to cryosections of
liquid nitrogen snap-frozen uninfected human placenta biopsy
samples. After extensive washing, the number of cytoadherent IE per
high-power fields of placental cryosections or per mm.sup.2 of cell
monolayer counted on 4 random fields (0.25 mm.sup.2 area at
.times.300 magnification, Olympus CK2) over the entire surface of
each sample was determined. The values for each experiment were
standarized to 5% parasitaemia, and the results expresed as a mean
cytoadhesion value.+-.SD. (D) Highly synchronized populations of
PA.sup.CSA and PA.sup.CD36 IE were mixed at the 8th hours of the
cycle to give an initial population, the phenotype distribution of
which was determined 24 hours later by cytoadhesion inhibition
microassays. The rest of the mixture was immediately subjected to
selection by cytoadhesion of SBEC ID. The cytoadherent IE were
cultured and the phenotype distribution of the result population
determined. The percentage cytoadhesion obtained for each
inhibitor. CSA (.box-solid.) and anti-CD36 FA6-152 monoclonal
antibodies ( ) gives the inverse proportion of each phenotype in
the IE populations.
[0015] FIG. 2: Cytoadhesion throughout the blood-stage cycle is
mediated by a switch in adhesion phenotype. (A) Determination of
the inhibitory activity of CSA (.box-solid.) and chondroitinase ABC
(.quadrature.) on the cytoadhesion of SBEC 1D of highly
synchronized PA.sup.CSA IE, every 4 hours throughout the cycle. (B)
Cytoadhesion of ring-stage PA.sup.CSA IE to SBEC ID 8 hours
post-invasion in the presence of dermatan-sulphate (CSB),
chondroitin-6-sulphate (CSC), keratan sulphate (Ker), hyaluronic
acid (HA), heparin (Hep), or after treatment of the target cells
with chondroitinase ABC (Case ABC) and B (Case B), hyaluronaste
lyase (H Lyase) and heparinase II (Hepase).
[0016] FIG. 3: Adhesive ring-stage IE express novel ring
stae-specific surface molecules that are targets o the immune
response. (A) Identification of a high-molecular weight
.sup.125I-labeled surface antigen on a ring-stage IE. The
separation of SDS extracts of the iodinated surface FCR3.sup.CSA at
various time points after merozoite re-invasion (7, 14, 21 and 32
hours) is shown. A single labeled band of approximately 200 kDa
(RSP-1) is detected from the early ring (7 hour) to early
trophozoite stages (21 hour). A second labeled band of
approximately 400 kDa appears at 14 hours and is detected until he
end of the cycle. .sup.35S-labeled ring-stage IE extracts show a
200 kDa band that co-migrates with RSP-1. The 200 and 400 kDa bands
are not seen in control erythrocytes (data not shown). (B) A pool
of immune sera from pregnant women (Cameroon) immunoprecipitated
two major protease-sensitive proteins of approximately 200 and 40
kDa Later in the life cycle, the var.sup.CSA molecule (400 kDa) is
immunopurified. Lane 1: trophozoite stage 32 hours post-infection)
followed by .sup.125I-labeled young- ring-stage (lane 2) and
trypsin (tryp) treatment before immunoprecipitation: 10 .mu.g/ml
trypsin (lane 3), 100 .mu.g/ml trypsin (lane 4) and 100 .mu.g/ml
.alpha.-chymotrypsin (chymo; lane 5). .sup.35S-methionine labeled
ring-stage IE SDS extract immunoprecipated with the serum pool
(lane 6). (C) Sensitivity of cytoadhesion to the treatment
ring-stage IE with different concentrations of trypsin or
.alpha.-chmotrypsi. (D) Immunolabeling of ring-stage (R) and
trophozoite-stage (T) PA.sup.CSA IE with a pool of 5 sera from
Senegalese and Cameroonian patients living in areas of endemic
malaria. Antibodies at the surface of the IE were detected with an
FITC-conjugated anti-human IgG and were observed by EPR microscopy
(CELLscan). (E) Cytoadhesion inhibition by sera obtained from a
primigravida (1), and multigravida (25 and 46) women, a child (61)
and a male adult (1613). The percentage cytoadhesion was obtained
by comparing the binding obtained in the presence of each serum
with a control carried out with a pool of sera from volunteers who
had never contracted malaria.
DETAILED DESCRIPTION OF THE INVENTION
[0017] All patents and publications mentioned herein are
incorporated herein by reference to the extent allowed by law for
the purpose of describing and disclosing the proteins, enzymes,
vectors, host cells, and methodologies reported therein that might
be used with the present invention. However, nothing herein is to
be construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention.
[0018] The RSP-1 and RSP-2 proteins of this invention may be
purified to substantial purity by standard techniques well known in
the art, including selective precipitation with such substances as
ammonium sulfate, column chromatography, immunopurification
methods, and others. See, for instance, R. Scopes, Protein
Purification: Principles and Practice, Springer-Verlag: New York
(1982).
[0019] In addition, provided with the purified proteins of the
present invention one of skill in the art will be able to obtain a
amino acid sequence from which the polynucleotide sequence which
encodes the RSP-1 and RSP-2 proteins can be obtained. Methods for
protein sequencing and isolation of a polynucleotide sequence are
known in the art and include polynucleotide amplification using
primers derived from the amino acid sequence of the purified
proteins. These and other methods are disclosed in Current
Protocols in Molecular Biology, F. M. Ausebel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc. (2000) and Maniatis et al.
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,
1988.
[0020] Having obtained the polynucleotide sequences encoding the
RSP-1 and RSP-2 proteins, the polynucleotide sequences can be
constructed in recombinant expression vectors for expression of the
genes in transfected cells. Molecular cloning techniques to achieve
these ends are known in the art. A wide variety of cloning and in
vitro amplification methods suitable for the construction of
recombinant nucleic acids are well-known to persons of skill.
Examples of these techniques and instructions sufficient to direct
persons of skill through many cloning exercises are found in Berger
and Kimmel, Guide to Molecular Cloning Techniques, Methods in
Enzymology volume 152 Academic Press, Inc., San Diego, Calif.
(Berger); and Current Protocols in Molecular Biology, F. M. Ausubel
et al., eds., Current Protocols, a joint venture between Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(2000).
[0021] Cell cultures that may be used in the present invention,
include cell lines and cultured cells from tissue or blood samples
is well known in the art Freshney (Culture of Animal Cells, a
Manual of Basic Technique, third edition Wiley-Liss, New York
(1994)) and the references cited therein provides a general guide
to the culture of cells.
[0022] Proteins produced by recombinant DNA technology may be
purified by standard techniques well known to those of skill in the
art. These proteins can be directly expressed or expressed as a
fusion protein. The protein can then be purified by a combination
of cell lysis (e.g., sonication) and affinity chromatography. For
fusion products, subsequent digestion of the fusion protein with an
appropriate proteolytic enzyme releases the RSP-1 or RSP-2 protein
sequences.
[0023] The proteins of the invention can be used to raise
monoclonal antibodies specific for RSP-1 or RSP-2. The antibodies
can be used for diagnosis of malarial infection or as therapeutic
agents to inhibit binding of merozoites to erythrocytes. The
production of monoclonal antibodies against a desired antigen is
well known to those of skill in the art The multitude of techniques
available to those skilled in the art for production and
manipulation of various immunoglobulin molecules can thus be
readily applied to inhibit binding. As used herein, the terms
"immunoglobulin" and "antibody" refer to a protein consisting of
one or more polypeptides substantially encoded by immunoglobulin
genes. Immunoglobulins may exist in a variety of forms besides
antibodies, including for example, Fv, Fab, and F(ab).sub.2, as
well as in single chains.
[0024] Antibodies which bind the proteins of the invention may be
produced by a variety of means. The production of non-human
monoclonal antibodies, e.g., murine, lagomorpha, equine, etc., is
well known and may be accomplished by, for example, immunizing the
animal with a preparation containing the polypeptide.
Antibody-producing cells obtained from the immunized animals are
immortalized and screened. Methods of producing polyclonal and
monoclonal antibodies are known to those of skill in the art. See,
e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene,
N.Y.; and Harlow and Lane (1989) Antibodies: A Laboratory Manual
Cold Spring Harbor Press, N.Y. Specific monoclonal and polyclonal
antibodies will usually bind with a Kd of at least about 0.1 mM,
more usually at least about 1 .mu.M, and most preferably at least
about 0.1 .mu.M or better.
[0025] The invention relates also to hybridoma and especially to
the hybridoma named Pf26G1/B4 deposited at the CNCM (Paris, France)
on Feb. 23, 2001 under accession number I-2635.
[0026] This hybridoma is specific to chondroitin-sulphate A (CSA)
cytoadhesion phenotype. It secretes monoclonal antibodies B4 which
react with the native P. falciparum proteins at the surface of
ring-infected erythrocytes but not with the mature trophozoite and
schizonte-infected erythrocytes. B4 inhibits the adhesion of
ring-infected erythrocytes and also the re-invasion of the
erythrocytes by the merozoites.
[0027] The proteins and polynucleotides of the invention can be
used in diagnostic applications for the detection of Plasmodium
parasites or nucleic acids in a biological sample. The presence of
parasites can be detected using several well recognized specific
binding assays based on immunological results. For example, labeled
antibodies to polypeptides of the invention can be used to detect
Plasmodium in a biological sample. Alternatively, labelled
polypeptides of the invention can be used to detect the presence of
antibodies to RSP-1 or RSP-2 in a biological sample. For a review
of the general procedures in diagnostic immunoassays, see Basic and
Clinical Immunology 7th Edition (D. Stites and A. Terr ed.)
1991.
[0028] In addition, modified polypeptides, antibodies or other
compounds capable of inhibiting the interaction between RSP-1 and
RSP-2 and erythrocytes can be assayed for biological activity. For
instance, polypeptides can be recombinantly expressed on the
surface of cells and the ability of the cells to bind erythrocytes
can be measured as described below. Alternatively, peptides or
antibodies can tested for the ability to inhibit binding between
erythrocytes and Plasmodium and/or RSP-1 and/or RSP-2.
[0029] Cell-free assays can also be used to measure binding of
RSP-1 or RSP-2 polypeptides, for example, the sample can be
immobilized on a solid surface and binding of labeled RSP-1 or
RSP-2 can be determined. Many assay formats employ labeled assay
components. The labeling systems can be in a variety of forms. The
label (detectable moiety) may be coupled directly or indirectly to
the desired component of the assay according to methods well known
in the art. A wide variety of labels may be used. The component may
be labeled by any one of several methods. The most common method of
detection is the use of autoradiography with .sup.3H, .sup.125I,
.sup.35S, .sup.14C, or .sup.32P labeled compounds or the like.
Non-radioactive labels include ligands which bind to labeled
antibodies, fluorophores, chemiluminescent agents, enzymes, and
antibodies which can serve as specific binding pair members for a
labeled ligand. The choice of label depends on sensitivity
required, ease of conjugation with the compound, stability
requirements, and available instrumentation.
[0030] In the case of the use nucleic acids for diagnostic
purposes, standard nucleic hybridization techniques can be used to
detect the presence of the genes identified here, RSP-1 and/or
RSP-2. If desired, nucleic acids in the sample may first be
amplified using standard procedures such as PCR. Diagnostic kits
comprising the appropriate primers and probes can also be
prepared.
[0031] RSP-1 and RSP-2 are useful in therapeutic and prophylactic
applications for the treatment of malaria. Pharmaceutical
compositions of the invention are suitable for use in a variety of
drug delivery systems. Suitable formulations for use in the present
invention are found in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief
review of methods for drug delivery, see, Langer, Science
249:1527-1533 (1990). The compositions are suitable for single
administrations or a series of administrations. When given as a
series, inoculations subsequent to the initial administration are
given to boost the immune response and are typically referred to as
booster inoculations.
[0032] The pharmaceutical compositions of the invention are
intended for parenteral, topical, oral or local administration.
Preferably, the pharmaceutical compositions are administered
parenterally, e.g., intravenously, subcutaneously, intradermally,
or intramuscularly. Thus, the invention provides compositions for
parenteral administration that comprise a solution of the agents
described above dissolved or suspended in an acceptable carrier,
preferably an aqueous carrier. A variety of aqueous carriers may be
used, e.g., water, buffered water, 0.4% saline, 0.3% glycine,
hyaluronic acid and the like. These compositions may be sterilized
by conventional, well known ster iization techniques, or may be
sterile filtered. The resulting aqueous solutions may be packaged
for use as is, or lyophilized, the lyophilized preparation being
combined with a sterile solution prior to administration. The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents, wetting agents and the like, for example, sodium acetate,
sodium lactate, sodium chloride, potassium chloride, calcium
chloride; sorbitan monolaurate, triethanolamine oleate, etc.
[0033] For solid compositions, conventional nontoxic solid carriers
may be used which include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10-95% of active ingredient and more preferably at a
concentration of 25%-75%.
[0034] For aerosol administation, the polypeptides are preferably
supplied in finely divided form along with a surfactant and
propellant. The surfactant must, of course, be nontoxic, and
preferably soluble in the propellant. Representative of such agents
are the esters or partial esters of fatty acids containing from 6
to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,
stearic, linoleic, linolenic, olesteric and oleic acids with an
aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters,
such as mixed or natural glycerides may be employed. A carrier can
also be included, as desired, as with, e.g., lecithin for
intranasal delivery.
[0035] The amount administered to the patient will vary depending
upon what is being administered, the state of the patient and the
manner of administration. In therapeutic applications, compositions
are administered to a patient already suffering from malaria in an
amount sufficient to inhibit spread of the parasite through
erythrocytes and thus cure or at least partially arrest the
symptoms of the disease and its complications. An amount adequate
to accomplish this is defined as "therapeutically effective dose."
Amounts effective for this use will depend on the severity of the
disease, the particular composition, and the weight and general
state of the patient.
[0036] Alternatively, the polypeptides of the invention can be used
prophylactically as vaccines. The vaccines of the invention contain
as an active ingredient an immunogenically effective amount of the
binding domain polypeptide or of a recombinant virus as described
herein. The immune response may include the generation of
antibodies; activation of cytotoxic T lymphocytes (CTL) against
cells presenting peptides derived from RSP-1 and/or RSP-2, or other
mechanism well )mown in the art See e.g. Paul Fundamental
Immunology Second Edition published by Raven press New York
(incorporated herein by reference) for a description of immune
response. Useful carriers are well known in the art, and include,
for example, thyroglobulin, albumins such as human serum albumin,
tetanus toxoid, polyamino acids such as poly(D-lysine:D-glutamic
acid), influenza, hepatitis B virus core protein, hepatitis B virus
recombinant vaccine. The vaccines can also contain a
physiologically tolerable (acceptable) diluent such as water,
phosphate buffered saline, or saline, and further typically include
an adjuvant. Adjuvants such as incomplete Freund's adjuvant,
aluminum phosphate, aluminum hydroxide, or alum are materials well
known in the art. The DNA or RNA encoding RSP-1 or RSP-2 may be
introduced into patients to obtain an immune response to the
polypeptides which the polynucleotide encodes.
[0037] Vaccine compositions containing the proteins, nucleic acids
or viruses of the invention are administered to a patient to elicit
a protective immune response against the polypeptide. A "protective
immune response" is one which prevents or inhibits the spread of
the parasite through erythrocytes and thus at least partially
prevent the symptoms of the disease and its complications. An
amount sufficient to accomplish this is defined as an
"immunogenically effective dose." Amounts effective for this use
will depend on the composition, the manner of administration, the
weight and general state of health of the patient.
[0038] After immunization the efficacy of the vaccine can be
assessed by production of antibodies or immune cells that recognize
the antigen, as assessed by specific lytic activity or specific
cytokine production or by tumor regression. One skilled in the art
would know the conventional methods to assess the aforementioned
parameters.
[0039] Having generally described this- invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only, and are not intended to be limiting unless otherwise
specified.
EXAMPLES
Parasites
[0040] The following parasite isolates were used in this study:
Palo-Alto (FUP)1.sup.13, IPL/BRE1.sup.14, FCR3 subpopulations
panned on CSA, CD36 and ICAM-1.sup.15, two isolates desequestered
using soluble CSA from infected human placentas (42.sup.CSA and
939.sup.CSA3) and a CSA-selected population of a field isolate
collected from the peripheral bloodstream (A53.sup.CSA). The IE
were cultured in RPMI 1640 containing bicarbonate, glutamine, 0.2%
glucose, 50 .mu.M hypoxanthine, 10 g/ml gentamicin and 10% human
AB.sup.+ serum, containing O.sup.+ eythrocytes, at 37.degree. C. in
a humidified atmosphere containing 5% O.sub.2, 5% CO.sub.2 and 90%
N.sub.2. IE cultures were synchronized by selecting ring-stage
parasites using multiple 5% sorbitol treatments until the parasites
reinvaded erythrocytes within 4 h.
Selection by Panning
[0041] Subpopulations of the Palo-Alto (FUP)1, (PA), IPL/BRE1
(BRE1), FCR3 strains and of the A53 isolate were selected by three
successive pannings of mature stage IE on cellular CSA as
previously described.sup.15, using SBEC 17.sup.16. In addition,
subpopulations of PA and FCR3 were selected by three successive
pannings of mature stage IE on cellular CD36 or ICAM-1, using
chondroitinated SBEC C2 and 3A.sup.15. Highly synchronsized
ring-stage IE cultures were panned on SBEC 1D expressing CSA, CD36
and ICAM-1, as previously described. The cells were washed
extensively to remove non-cytoadherent IE and were then incubated
in culture medium for 24 hours to allow ring-stage IE to mature.
RBC were added and the cultures grown as previously described.
Cytoadhesion and Cytoadhesion Inhibition Assays
[0042] Gelatin-enriched preparations of mature-stage IE were
resuspended at a concentration of 5.times.10.sup.6 IE/ml in
cytoadhesion medium at pH 6.8. Cytoadhesion microassays were then
performed on 12-well IFA slides (Institut Pasteur, Paris) as
previously described.sup.15.
[0043] Ring-stage cytoadhesion assays were performed with
endothelial cells as described above and with placental
cryosections as previously described.sup.11, with 1 to 10%
parasitaemia (a 1.times.10.sup.7 IE/ml suspension).
[0044] For cytoadhesion inhibition assays, the IE were incubated
with SBEC in the presence of 2.5 .mu.g/ml thrombospondin, 0.1 mg/ml
soluble CSA, dermatan sulphate (CSB), chondroitin-6-sulphate (CSC),
keratin sulphate (Ker), hyaluronic acid (HA), heparin (Hep) (Fluka,
France), or with SBEC previously incubated for 1 hour at 37.degree.
C. with 1 U/ml Case ABC (Fluka, France), 1 to 10 U/ml heparinase
III (Hepase III) (Sigma, France) or 5 .mu.g/ml anti-CD36 FA6-152
Mab (gift from Dr. Edelman). Inhibition assays were also carried
out in the presence of sera obtained from Senegalese and
Cameroonian patients living in areas of endemic malaria. The sera
were obtained from primigravida and multigravida women, a male
adult and a child, absorbed onto O.sup.+ human blood and SBEC ID,
and tested at a dilution of 1/20. The results were compared to
those for cytoadhesion in the presence of a 1/20 dilution of a pool
of control sera from volunteers who had never contacted
malaria.
[0045] The protease sensitivity of ring-stage IE cytoadhesion was
analyzed using 5 .mu.l of packed PA.sup.CSA IE (8 hours
post-invasion). IE were incubated with 10 or 100 .mu.g/ml of
trypsin TPCK (Signa) or 100 g/ml of -chymotrypsin TLCK (Sigma) for
30 minutes at 37.degree. C. The digestion was topped by adding
culture medium containing 10% human plasma. The cells were then
washed in cytoadhesion medium and allowed to cytoadhere to SBEC ID,
as previously described, using untreated PA.sup.CSA as a
control.
Surface Immunolabeling of IE
[0046] 100 .mu.l of a pool of 5 sera from Senegalese and
Cameroonian patients living in an area of endemic malaria was
adsorbed onto 30 1 of O.sup.+ human blood, once at 37.degree. C.
and once at room temperature. 5 .mu.m of highly synchronized ring-
or trophozoite-stage PA.sup.CSA IE were incubated on ice for 45
minutes with 100 .mu.l of the pool of sera diluted 1/10 in
cytoadhesion medium. The IE were washed three times in cytoadhesion
medium and incubated for 45 minutes on ice with FITC-conjugated
anti-human IgG (Sigma, F-6380). After a final wash, the IE were
observed by EPR microscopy (CELLscan, Scanalytics, Billerica,
MA.sup.17).
Surface Iodination and Metabolic Labeling of IE
[0047] Synchronized mature-stage IE previously selected on CSA and
CD36 by the receptor panning procedure.sup.18 were enriched to
>75% by the gelatin technique and hen diluted with fresh
crythrocyes to obtain approximately 20% ring-stage forms at the
next cycle. Surface iodination was performed using the
lactoperoxidase method.sup.7, 7, 14, 21 and 32 hours after
re-invasion. Metabolic labeling was performed by adding 2 mCi
.sup.35S-methionine to a 5 ml culture flask at the late
schizont-stage. The culture was stopped 14 hours after re-invasion.
Sequential extraction with 1% Triton X-100 then 2% SDS was carried
out, followed by protease treatment (TPCK-treated trypsin and
-achmotrypsin TLCK (Sigma, Lt. Louis) as previously described).
Samples iodinated or metabolically labeled were separated on a
5%-17.5% gradient acrylamide gel, which was then dried and placed
against Kodak Bio Max MS1 film. Prestained protein markers were
purchased from Life Technologies, Gaithersburg, Md. and New England
BioLabs Inc., Beverly, Mass.
Statistical Analysis
[0048] The results of IE adhesion, cytoadhesion and cytoadhesion
inhibition assays are expressed as means.+-.SE. The Mann-Whitney
test was used to evaluate the statistical significance of data from
cytoadhesion inhibition assays and to compare cytoadhesion levels.
TABLE-US-00001 TABLE 1 Cytoadhesion of IE.sub.S.sup.CSA to Saimiri
brain endothelial cells ID IE.sup.CSA cytoadhesion during the first
2 hours of the cycle 1E 4 H 8 H 12 H 16 20 H Laboratory strains
PA.sup.CSA 64 .+-. 42 73 .+-. 59 122 .+-. 177 Bre.sup.CSA 35 .+-.
15 38 .+-. 19 128 .+-. 59 FCR.sup.CSA 278 .+-. 210 237 .+-. 124 86
.+-. 21 Peripheral Blood ioslate A53.sup.CSA 46 .+-. 29 32 .+-. 21
53 .+-. 33 Placential Isolates 42.sup.CSA 29 .+-. 26 28 .+-. 12 51
.+-. 37 939.sup.CSA 47 .+-. 38 57 .+-. 50 71 .+-. 37
Data are the mean number (.+-.SD) of cytoadherent IE/mm.sup.2 of
SBEc ID monolayer (mean of quadruplicate spots). Nd: not done. The
shadowed values correspond to PfEMP1-mediated cytoadhesion.
Results
[0049] The peripheral blood of pregnant women infection with P.
falciparum may be devoid of circulating ring-stage IE.sup.4,5 or
contain IE with little or no CSA binding phenotype.sup.2,3 despite
the massive binding of IE to placenta CSA. This led us to
investigate whether ring-stage IE from pregnant women are able to
avoid circulating in the peripheral blood. We tested ring-stage
adhesion to placenta and various cell types. Highly synchronized
young ring-stage IE of P. falciparum isolate Palo-Alto (FUP).sup.1,
prepared in vitro and selected for binding to CSA (PA.sup.CSA),
adhered to syncytiotrophoblasts in placenta cryosections and
monolayers of cultured Saimiri brain endothelial cells (SBEC)
(FIGS. 1A, B and C). Binding was observed immediately after
merozoite reinvasion and continued throughout the ring-stage cycle
(Table 1). There was extensive specific binding of ring-stage IE to
SBEC ID and melanoma C32 cells (approx. 70 IE/mm.sup.2). The level
of binding to human brain endothelial cells (HBEC), human lung
endothelial cells (HLEC) and CHO cells was low but significant
(.gtoreq.5 IE/mm.sup.2 of cell monolayer) (FIG. 1C). Finally, no
ring-stage IE cytoadhesion to human umbilical vein endothelial
cells (HUVEC, primo explants) and human dermal endothelial cells
(HMEC-1) was detected. We investigated additional genetically
different CSA-binding isolates: one from the peripheral blood of a
child (A53.sup.CSA), two placental isolates, 939.sup.CSA and
42.sup.CSA, and two isolates cultured in vitro (FCR3.sup.CSA and
Bre1.sup.CSA). The results obtained confirmed that ring-stage IE
bound to endothelial cells (Table 1).
[0050] Ring-stage adhesion was not detected in PA and FCR3
parasites selected for binding at the trophozoite stage to CD36 or
ICAM-1 (<1 bound IE/mm.sup.2 of SBEC 1D). These data suggest
that the CSA-binding phenotype is connected to ring-stage adhesion
by an unknown mechanism. We investigaged this by mixing equal
numbers of highly synchronized ring-stage IE (8 hours after
re-invasion) selected for bind to CSA and CD36 and allowing them to
bind to a monolayer of SBEC 1D. Bound ring-stage IE were cultured
and their phenotypes were assessed at the trophozoite stage by
measuring the sensitivity to phenotype-specific inhibitors (soluble
CSA mAb directed against CD 36) of adhesion. Almost all trophozoite
binding was CSA-dependent whereas the non-selected IE combinations
were inhibited similarly by both inhibitors (FIG. 1D). Similar
selection was observed for the binding for mature IE to CSA if a
mixture of IE subpopulations binding to CSA and ICAM-1 was tested
(data not shown). Therefore, for the three phenotypes tested, the
ability to cytoadhere before the trophozoite stage was strictly
linked to the CSA-binding phenotype of mature IE.
[0051] To identify the host receptor involved in ring-stage
adhesion, we tested the inhibitory activity of CSA throughout the
cycle. We observed that ring-stage IE.sup.CSA cytoadhesion was
insensitive to 0.1 mg.ml of CSA and to the prior treating of the
target cells with 1 U/ml of chondroitinase ABC (Case ABC) until the
16th hour after invasion (FIG. 2A). At this time, with the
beginning of knob formation and surface expression of the
var.sup.CSA gene, the inhibitory effects on binding of CSA and Case
ABC were first seen. Inhibition was maximal at hour 24. Thus, all
IE.sup.CSA cytoadhere throughout the blood-stage cycle, switching
from CSA-independent receptor interaction to a CSA-dependent
phenotype 16 hours after invasion. We tested the possible
involvement of various mature-stage IE adhesion receptors.
Thrombospondin had no effect on IE binding to SBEC 1D (data not
shown). Transfected CHO 745 (CSA.sup.-) expressing CD36, ICAM-1,
VCAM or E-selectin at their surface showed non-specific
cytoadhesion of ring-stage IE similar to that of the CHO-745
control cells (.ltoreq.bound IEs/mm.sup.2). We also investigated
the possible inhibitory effects of various glycosaminoglycans and
their corresponding enzymes (FIG. 2B). The inhibition obtained with
dermatan-sulphate (CSB) was not specific as Case ABC and B had no
significant inhibitory activity. Hyaluronic acid had no activity
whereas hyaluronate lyase had a low level of inhibitory activity,
probably due to its secondary capacity to digest heparin and
heparan sulphates. Heparin (100 m/ml) and heparinase gave about 50%
inhibition, SBEC ID do not express heparin at their surfare.
Instead, they express heparan sulphate proteoglycans.sup.6, and it
is probably by completion with or digestion of SBEC 1D heparan
sulphate that heparin and heparinase II partially inhibit
ring-stage cytoadhesion. We are currently purifying SBEC 1D heparan
sulphates to test their inhibition of ring-stage IE.sup.CSA
cytoadhesion.
[0052] As ring-stage IE.sup.CSA adhesion is not mediated by CSA, we
thought it likely that a novel parasite surface molecule mediated
binding to endothelial cells and syncytiotrophoblasts in early
blood stage parasites. Surface iodination of FCR3.sup.CSA
ring-stage IE identified a molecule of approximately 200 kDa that
was absent from control erythrocytes and is referred to here as
"ring surface protein-1" (RSP-1) (FIG. 3). We immunoprecipitated
surface-iodinated ring-stage IE extracts using a pool of serum from
ultigravida women from Cameroon. The sera used recognized the 200
kDa molecule and a second molecule of approximately 40 kDa, termed
RSP-2. RSP-2 was not detectable in total parasite extracts because
it co-migrates with a band strongly labeled in uninfected
erythrocytes. Parasite proteins identical in size to RSP-1 and
RSP-2 were found in S-methionine labeled protein extracts from
ring-stage IE. RSP-1 and RSP-2 were efficiently extracted in 2% SDS
and were degraded by trypsin (100 .mu.m/ml) or -chymotirpsin (100
g/ml) treatment (FIGS. 3A and B, and data not shown). Ring-stage IE
adhesion was substantially inhibited at a protease concentration of
100 .mu.g/ml (FIG. 3C), consistent with the involvement of RSP-1
and RSP-2 in the adhesion process. Both molecules were detected at
the surface of young ring-stage IE but neither was present in
mature trophozoites. In trophozoite IE, a large molecule,
approximately 400 kDa in size, was detected at the IE surface
(between 14 and 21 h post-invasion, FIGS. 3A and B) at a time
coinciding with the switch in adhesive phenotype. The 400 kDa
molecule of FCR3.sup.CSA IE was identified in a previous study as
the var gene product, which mediates the adhesion of mature forms
to CSA. The RPS-1, RPS-2 and var.sup.CSA molecule are naturally
immunogenic and were efficiently immunoprecipitated by 8 sera from
pregnant women (FIG. 3B and data not shown). These sera react with
the surface of ring-stage and trophozoite-stage IE (FIG. 3E). Sera
from malaria patients from. Cameroon/Senegal (pregnant women, male
adults and children) blocked the cytoadhesion of ring-stage IE to
endothelial cells (FIG. 3F).
[0053] Our work challenges current views concerning the blood-stage
biology of P. falciparum. It is generally accepted that ring-stage
IE circulate in the blood and that adhesive properties become
evident with the expression of the PfEMP1 molecule at the IE
surface approximately 14 to 16 hours after invasion. Here we
describe for the first time the specific adhesion of young ring IE
to endothelial cells from critical target organs such as the brain
and lung and to syncytiotrophoblasts. The differences between
placental and peripheral blood parasitaemia and phenotype
distribution observed in infected pregnant women can be accounted
for by our findings. We suggest that the adhesion of ring-stage IE
to placenta syncytiotrphoblasts precedes the CSA-binding of
mature-stage IE, leading to a cryptic, or at least partially
cryptic, life cycle of parasites with this adhesive phenotype.
Evidence that ring-stage IE may cytoadhere in patients other than
pregnant women comes from a recent study on sequestration of P.
falciparum in the human brain.sup.8. All developmental stages were
observed in brain vessels of patients dying from cerebral malaria.
Some vessels clearly contained large numbers of ring-stage IE but
the nature of the interaction is unknown. As CSA is present in the
brain microvasulature.sup.9-11, IE subpopulations may adhere to the
same host cell throughout the blood stage cycle. Clearly, the
absence or under-representation of specific virulent adhesive
phenotypes in the bloodstream has a major impact on clinical
studies based on peripheral blood-stage parasites. The role of
ring-stage adhesion in tissue tropism should also be investigated.
It is tempting to speculate that the massive accumulation of
CSA-binding parasites observed in the placenta, for example, is due
to the initial binding of rings.
[0054] The level of ring-stage adhesion to endothelial cells is
markedly lower than that of trophozoite binding. This may be due to
differences in the strength of the interaction between ligand and
receptor pairs or to there being fewer ring-stage adhesion
receptors than CSA molecules. Preliminary data obtained in
flow-based assays, indicate an order to magnitude difference in
strength of interaction between the ring and mature stages.
Ring-stage adhesion is presumably maximal in the placenta, where
blood flow is much lower than in other vascular beds.
[0055] The switch between two different adhesive phenotypes during
the 48-hour blood-stage cycle is an entirely new phenomenon in the
biology of P. falciparum. The expression pattern of IE surface
molecules throughout the blood-stage cycle coincides with the
observed change in adhesive phenotype, thus suggesting a role for
RSP-1 and/or RSP-2 in ring-stage E adhesion. In parasite that do
not present ring-stage adhesion (CD36 phenotype), surface molecules
with molecular masses similar to those of RSP-1 and RSP-2 were
detected (data not shown). It is unclear whether RSP-1 and RSP-2
are members of a ene family or if phenotype-specific
post-translational modifications of IE surface molecules.sup.12 are
responsible for the differences in adhesive features of rings.
[0056] Finally, the novel ring-stage IE surface molecules RSP-1 and
RSP-2 are nature targets of the antibody-mediated immune response
capable of blocking ring-stage adhesion. These antigens were
therefore potential vaccine candidates that could reduce the
severity of this major disease.
[0057] Obviously, numerous modifications and variations on the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
REFERENCES
[0058] 1. Miller, L. H., Good, M. F. & Milton, G. Malaria
pathogenesis. Science 264, 1878-1883 (1994).
[0059] 2. Fried, M. & Duffy, P. E. Adherence of Plasmodium
falciparum to chondroitin sulfate A in the human placenta. Science
272, 1502-1504 (1996).
[0060] 3. Gysin, J., Pouvelle, B. Fievet, N., Schert, A &
Lepolard, C. Ex vivo desequestration of Plasmodium
falciparum-infected erythrocytes from human placenta by chondroitin
sulfate A. Infect Immun. 67, 6596-6602 (1999).
[0061] 4. Watkinson, M. & Rushton, D. J. Plasmodial
pigmentation of placenta and outcome of pregnancy in West African
moths. Brit. Med. J. 287, 251-254 (1983).
[0062] 5. Matteelli, A. et al, Malaria and anaemia in pregnant
women in urban Zanzibar, Tanzania Ann. Trop. Med. Parasitol.
88,475-483 (1994).
[0063] 6. Fusai, T. et al, Characterisation of the chondroitin
sulphate of Saimiri brain microvascular endothelial cells involved
in P. falciparum cytoadhesion. Mol. Biochem. Parasitol., in press
(2000).
[0064] 7. Buffet, P. A. et al. Plasmodium falciparum domain
mediating adhesion to chondroitin sulfate A: A receptor for human
placental infection. Proc. Natl. Acad. Sci. U.S.A. 96, 12743-12748
(1999).
[0065] 8. Silamut, K et al. A quantitative analysis of the
microvascular sequestration of malaria parasites in the human
brain. Am. J. Pathol. 155, 395-410 (1999).
[0066] 9. Boffa, M. C., Jackman, R. W., Peyri, N. & George, B.
Thrombomodulin in the central nervous system. Nouv. Rev. Fr.
Hematol. 33, 423-429 (1991).
[0067] 10. Wong, V. L., Hofman, F. M. Ishii, H. & Fisher, M.
Regional distribution of thrombomodulin in human brain. Brain Res.
556, 105 (1991).
[0068] 11. Gysin, J., Pouvelle, B., Le Tonqueze, M., Edelman, L.
& Boffa, M. C., Chondroitin sulfate of thrombomodulin is an
adhesion receptor for Plasmodium falciparum-infected erythrocyics.
Mol. Biochem. Parasitol. 88, 267-271 (1997).
[0069] 12. Fernandez, V. Hommel, M. chen, Q., Hagblom, P. &
Wahlgren, M. Small, clonally variant antigens expressed on the
surface of the Plasmodium falciparum-infected erythrocyte are
encoded by the rif gene family and are the target of human immune
responses. J. Exp. Med. 190, 1393-1404 (1999).
[0070] 13. Gysin, J. & Fandeur, T. Saimiri sciureus (Karyotype
14-7) an alternative experimental model of Plasmodium falciparum
infection Am. J. Trop Med. Hyg. 32, 461-467 (1983).
[0071] 14. Robert, C. et al Chondroitin-4-sulphate (proteoglycan),
a receptor for Plasmodium falciparum-infected erythrocyte adherence
on brain microvascular endothelial cells. Res. Immunol. 146,
383-393 (1995).
[0072] 15. Pouvelle, B. Fusai, T. Lepolard, C. & Gysin, J.
biological and biochemical characteristics of cytoadhesion of
Plasmodium falciparum-infected erythrocytes to
chondroitin-4-sulfate. Infect. Immun. 66, 4950-4956 (1998).
[0073] 16. Gay, F. et al, Isolation and characterization of brain
microvascular endothelial cells from Saimiri monkeys. An in vitro
model for sequestration of Plasmodium falciparum-infected
erythrocytes. J. Immunol. Meth. 184, 15-28 (1995).
[0074] 17. Carter, K. C. et al. A three-dimensional view of
precursor messenger RNA metabolism within the mammalian nucleus.
Science 259, 1330-1335 (1993).
[0075] 18. Scherf, A. et al. Antigenic variation in malaria in situ
switching, relaxed and mutally exclusive transcription of var genes
during intra-erythrocytic development in Plasmodium falciparum.
Embo J. 17, 5418-5426 (1998).
* * * * *