EP2945648A1

EP2945648A1 - Vaccines against pregnancy-associated malaria

Info

Publication number: EP2945648A1
Application number: EP14701072.2A
Authority: EP
Inventors: Nicaise Tuikue Ndam; Philippe Deloron; Justin DORITCHAMOU
Original assignee: Institut de Recherche pour le Developpement IRD
Current assignee: Institut de Recherche pour le Developpement IRD
Priority date: 2013-01-21
Filing date: 2014-01-21
Publication date: 2015-11-25
Also published as: FR3001152B1; FR3001152A1; US9855321B2; WO2014111597A1; US20160136253A1; AP2015008615A0

Abstract

The present invention relates to combinations of polypeptides or of polynucleotides corresponding to a specific region of the N-terminal portion of the VAR2CSA protein of different parasitic families or lines of Plasmodium falciparum, and to their use in the prevention of pregnancy-associated malaria. The invention also relates to immunogenic compositions and to vaccines useful for preventing malaria in pregnant women.

Description

VACCINES AGAINST GESTATIONAL MALARIA

Parent Application

The present international application claims the priority of the French application number FR 13 50508 filed on January 21, 2013, the content of which is hereby incorporated by reference in its entirety.

Field of the Invention

The present invention relates to the use of combinations of specific regions of the N-terminal portion of the VAR2CSA protein from different families or parasitic lines of Plasmodium jalciparum in the prevention and / or treatment of gestational malaria.

Context of the Invention

Malaria (or malaria) is the most common parasitic infection observed in the world. It is due to a parasite of the genus Plasmodium, which is transmitted during the bite by a female mosquito (Anopheles). Several species of Plasmodium can infect humans, but Plasmodium jalciparum is the most common and pathogenic species and the one responsible for fatal cases. Once introduced into the bloodstream, the parasite infects liver cells, in which it proliferates, before circulating again in the blood and invading red blood cells (erythrocytes or red blood cells). Malaria affects some 100 countries worldwide, particularly in the less-favored tropical areas of Africa, Asia and Latin America; Africa being by far the most affected continent. According to estimates by the World Health Organization, malaria is responsible for 225 million cases of fevers and about 1 million deaths annually (World Malaria Report, WHO, 2010). The existing control measures are antimalarial drugs (the best known are chloroquinine and quinine) and the fight against mosquitoes vectors of the parasite. But the situation is all the more worrying because for several years, parasites are developing more and more resistance to drugs and mosquitoes develop resistance to insecticides. No vaccine is available today. Malaria mainly affects children under 5 and pregnant women, especially primigest (that is, pregnant for the first time). first time). Pregnant women are particularly vulnerable because the placenta is a target where parasites can accumulate. In pregnant women, malaria infection can cause a range of adverse effects: spontaneous abortion, premature delivery, low birth weight, congenital transmission, and neonatal death. In areas of Africa where malaria is endemic, 3 to 5% of infant deaths at birth can be attributed to gestational malaria. In addition, it also presents a real danger for the mother who can suffer from anemia sometimes severe or even fatal. Currently, the prevention of malaria in pregnant women relies on preventive treatment with sulfadoxine-pyrimethamine (Cot et al, Br Med Med, 2003, 67: 137-148). But this intermittent treatment is not able to provide malaria prevention during the entire pregnancy: first, because the drug is administered only from the 20 ^th week of pregnancy (the teratogenic risk during embryogenesis being considered too important); secondly, because it is based on two sulfadoxine-pyrimethamine curative doses spaced at least one month apart, providing only partial drug coverage; and third, because the efficacy of sulfadoxine-pyrimethamine is decreasing very widely in all malaria-endemic areas due to the expansion of parasite resistance (Cot et al, Am. J. Trop Hyg., 1998, 59: 813-822, WHO / HTM / MAL / 2005.1103, Geneva: World Health Organization, Kuile et al, JAMA, 2007, 297: 2603-2616, Mockenhaupt et al, J. Infect. Dis., 2008, 198: 1545-1549, Briand et al., J. Infect Dis., 2009, 991-1001, Harrington et al, Proc Natl Acad Sci USA, 2009, 106: 9027-9032. ). Drugs are currently being studied in this context, and many efforts are also focused on developing a vaccine for gestational malaria. The possibility of vaccinating the pregnant woman or the pre-pubertal girl would be an obvious advantage over drug treatment, as the preventive coverage of the pregnancy would be extended, and potentially of better quality.

One of the vaccine strategies envisaged to fight against gestational malaria is to recreate the natural protective immunity. Indeed, the clinical severity of malaria due to Plasmodium falciparum is partly linked to alterations infected erythrocytes. These alterations are induced by parasite proteins that are exported to the surface of erythrocytes during the development phase in the blood. Some of these parasite-encoded PfEMPl family proteins (Plasmodium falciparum Erythrocyte Membrane Protein 1) confer new cytoadherence properties on erythrocytes. The erythrocytes are then able to attach to the inner wall of the blood vessels, thereby preventing the transport of infected erythrocytes to the purifying organs of the immune system, one of whose roles is to destroy the cells recognized as abnormal. In the case of gestational malaria, parasitized erythrocytes attach to a sugar, chondroitin sulfate A (CSA), present in the placenta. After several pregnancies, women acquire protective antibodies that block this adhesion. One of the vaccine strategies is to recreate this protective immunity by blocking the adhesion of infected erythrocytes to the placenta. The protein VAR2CSA, one of the proteins of the PfEMP1 family, is currently the subject of much research with a view to obtaining a specific vaccine for pregnant women (Tuikue Ndam et al., J. Infect Dis., 2005, 192: 331-335, Chia et al., J. Infect Dis., 2005, 192: 1284-1293, Tuikue Ndam et al, J. Infect Dis., 2006, 193: 713-720, Dahlback et al, PLoS Pathogens, 2006, 2: 1069-1082, Badaut et al., Mol Biochem Parasitai, 2007, 15: 89-99, Khattab et al, Parasitai Res, 2007, 101: 767-774, Guitard et al, Malaria J., 2008, 11: 7-10, Guitard et al, Malaria J., 2010, 9: 165, Gangnard et al, Mol Biochem Parasitai, 2010, 173: 115-122, Gnidehou et al, Mol. Biochem Parasitai, 2010, 5 (10): el3105, WO 2012/014073). This work is hampered by VAR2CSA polymorphism, but phase 1 trials are nevertheless being considered. The entire extracellular domain of this protein has been expressed in a heterologous system (Srivastava et al, Proc Natl Acad Sci, USA, 2010, 107: 4884-4889, Khunrae et al., J. Mol Biol, 2010, 397). 826-834), and the antibodies induced against this extracellular domain show a very high level of anti-adhesion. However, the development of new vaccine approaches will have to take into account the many immunodominant epitopes that do not induce anti-adherence antibodies.

It is therefore crucial to continue to explore and develop new strategies for the control and prevention of gestational malaria. Summary of the Invention

The present inventors have previously identified the N-terminal region of VAR2CSA, and in particular the NTS-DBLlx-Idl-DBL2x region consisting of the DBLlx domain, Interdomain Idl, and the DBL2x domain, as being that which contains epitopes capable of inducing antibodies blocking the adhesion of Plasmodium falciparum-infected red cells to the CSA, and the Idl-DBL2x subregion as the minimal antigenic region of VAR2CSA involved in the acquisition of protective immunity to placental sequestration that occurs in gestational malaria (WO 2012/014073). They have now discovered that the combination of antibodies induced against the VAR2CSA NTS-DBLlx-Idl-DBL2x region of the two Plasmodium falciparum FCR3 and 3D7 parasite lines completely inhibit the binding of parasitized erythrocytes to chondroitin sulfate A (CSA) as well. placental isolates of pregnant women only on well-characterized laboratory strains. In comparison, the antibodies induced against the VAR2CSA NTS-DBL1x-Id1-DBL2x region of each of the two parasitic FCR3 and 3D7 parasite lines of Plasmodium falciparum used separately only have a percent inhibition of about 70%. By analyzing the sequences of parasitic isolates from pregnant to benign women, the inventors also demonstrated a segregation of parasitic variants in interdomain ID1 of VAR2CSA. This new area of dichotomy has never been described so far. The sequence alignments led to the establishment of two consensus sequences representative of the Idl interdomain of VAR2CSA: the first Idl A (or SEQ ID NO: 11) corresponds to a newly identified cluster, and the second IdlB (or SEQ ID NO: 12) corresponds to the other group of sequences (which include the FCR3 line and the 3D7 line).

Accordingly, a first aspect of the present invention relates to the use of combinations of polypeptide sequences or polynucleotides corresponding to the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein from different Plasmodium parasitic families. falciparum, in the management of malaria of the pregnant woman.

More specifically, the present invention relates to the use of isolated or purified polypeptide or polynucleotide sequences corresponding to the NTS-region. DBLlx-Idl-DBL2x or to the Idl-DBL2x region of the VAR2CSA protein from Plasmodium jalciparum parasites whose VAR2CS A protein is characterized by an interdomain Id1 which has as sequence the consensus sequence SEQ ID NO: 11 (or which is coded by the consensus nucleic sequence SEQ ID NO: 13) and optionally polypeptide sequences or polynucleotides corresponding to the NTS-DBLlx-Idl-DBL2x region or to the Idl-DBL2x region of the VAR2CSA protein from Plasmodium jalciparum parasites whose VAR2CSA protein is characterized by an interdomain Id1 which has as sequence the consensus sequence SEQ ID NO: 12 (or which is encoded by the consensus nucleic sequence SEQ ID NO: 14).

In particular, the present invention relates to a combination of at least two polypeptides isolated or purified for use in the treatment or prevention of gestational malaria, the first isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of a first parasitic family of Plasmodium jalciparum, and the second isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of a second Parasitic family of Plasmodium jalciparum, for use in the treatment or prevention of gestational malaria. In certain preferred embodiments, the first family is the Plasmodium jalciparum parasitic family, the VAR2CSA protein of which is characterized by an interdomain Id1 which has the sequence sequence SEQ ID NO: 11 or which is encoded by the consensus nucleic sequence SEQ ID NO: 13, and the second family is the parasitic family of Plasmodium jalciparum whose VAR2CSA protein is characterized by an interdomain Id1 which has for sequence the consensus sequence SEQ ID NO: 12 or which is encoded by the consensus nucleic sequence SEQ ID NO: 14.

In some embodiments, the second parasitic family of Plasmodium jalciparum comprises the parasitic line FCR3 and the parasitic line 3D7.

In certain embodiments, a combination according to the invention is characterized in that it consists of three isolated or purified polypeptides, the first isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the Plasmodium falciparum parasite family, the VAR2CSA protein of which is characterized by an Idl interdomain which is sequenced by the SEQ consensus sequence ID NO: 11 or which is encoded by the consensus nucleic acid sequence SEQ ID NO 13, the second isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line, and the isolated or purified third polypeptide consisting of the NTS-DBL1x-Id1-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line. In some embodiments, the NTS-DBL1x-Id1-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 1 or a homologous sequence of SEQ ID NO: 1, and the NTS-DBLlx-Idl region. -DBL2x of the VAR2CSA protein of line 3D7 has the sequence SEQ ID NO: 5 or a homologous sequence of SEQ ID NO: 5. In some embodiments, the Idl-DBL2x region of the protein

VAR2CSA of the FCR3 line has the sequence SEQ ID NO: 3 or a homologous sequence of SEQ ID NO: 3, and the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 7 or a homologous sequence The present invention also relates to a combination of at least two isolated or purified fusion proteins for use in the treatment or prevention of gestational malaria, wherein the combination corresponds to a combination of at least two isolated or purified polypeptides as described herein wherein each of the polypeptides is fused to a fusion partner sequence.

In some embodiments, each of the fusion partner sequences is independently selected from the group consisting of the maltose binding protein, the signal sequence of the maltose binding protein, an S-tag, glutathione-S-transferase, thioredoxin , β-galactosidase, streptavidin, dihydrofolate reductase, pelB signal sequence, ompA signal sequence, alkaline phosphatase signal sequence, green fluorescent protein, toxins, human growth hormone, interleukin -2 (IL-2), the granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), calcitonin, interferon-beta, interferon-alpha, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), PA toxin, parathyroid hormones (PTH (1-34) and PTH (1-84)), butyrylcholinesterase, glucocerebrosidase (GBA), and exendin-4.

The invention also relates to a combination of at least two polynucleotides isolated or purified for use in the treatment or prevention of gestational malaria, wherein each of the isolated or purified polynucleotides encodes a polypeptide as defined above or for a fusion protein such as than previously defined. More specifically, the invention relates to a combination of at least two isolated or purified polynucleotides, the first isolated or purified polynucleotide encoding a first polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the protein. VAR2CSA of a first parasitic family of Plasmodium jalciparum or for a first fusion protein comprising the first polypeptide, and containing the elements necessary for the expression of said first polypeptide or of said first fusion protein in vitro and / or in vivo; and the second isolated or purified polynucleotide encoding a second polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of a second parasitic family of Plasmodium jalciparum or a second fusion protein comprising the second polypeptide, and containing the elements necessary for the expression of said second polypeptide or said second fusion protein in vitro and / or in vivo, for use in the treatment or prevention of gestational malaria.

In some embodiments, the first family is the Plasmodium jalciparum parasitic family, the VAR2CSA protein of which is characterized by an interdomain Id1 which has the sequence sequence SEQ ID NO: 11 or which is encoded by the consensus sequence SEQ ID NO: 13 and the second family is the parasitic family of Plasmodium jalciparum whose VAR2CSA protein is characterized by an interdomain Id1 which has for sequence the consensus sequence SEQ ID NO: 12 or which is encoded by the consensus sequence SEQ ID NO: 14.

In some embodiments, the second parasitic family of Plasmodium jalciparum comprises the parasitic strain FCR3 and the parasitic line In some embodiments, the combination according to the invention consists of three isolated or purified polynucleotides, the first isolated or purified polynucleotide encoding a first polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the Plasmodium falciparum parasitic family, the VAR2CSA protein of which is characterized by an interdomain Id1 which has for sequence the consensus sequence SEQ ID NO: 11 or which is encoded by the consensus sequence SEQ ID NO: 13 or for a first protein of fusion comprising the first polypeptide, and containing the elements necessary for the expression of said first polypeptide or of said first fusion protein in vitro and / or in vivo; the second isolated or purified polynucleotide encoding a second polypeptide consisting of the NTS-DBL1x-Id1-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line or for a second fusion protein comprising the second polypeptide, and containing the elements necessary for the expression of said second polypeptide or of said second fusion protein in vitro and / or in vivo; and the third isolated or purified polynucleotide encoding a third polypeptide consisting of the NTS-DBL1x-Id1-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line or for a third fusion protein comprising the third polypeptide, and containing the elements necessary for the expression of said third polypeptide or said third fusion protein in vitro and / or in vivo.

In some embodiments, the NTS-DBL1x-Id1-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 1 or a homologous sequence of SEQ ID NO: 1, and the NTS-DBLlx-Idl region. -DBL2x of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 5 or a homologous sequence of SEQ ID NO: 5.

In certain embodiments, the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 3 or a homologous sequence of SEQ ID NO: 3, and the Idl-DBL2x region of the VAR2CSA protein of the Line 3D7 has the sequence SEQ ID NO: 7 or a homologous sequence of SEQ ID NO: 7.

In some embodiments, the isolated or purified second polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 2, a sequence homologous to SEQ ID NO: 2, SEQ ID NO: 4, a homologous sequence of SEQ ID NO: 4, SEQ ID NO: 6, a homologous sequence of SEQ ID NO: 6, SEQ ID NO: 8, and a homologous sequence of SEQ ID NO: 8.

In embodiments where a second and third isolated or purified polynucleotide is present in the second isolated or purified polynucleotide preferably comprises a sequence selected from the group consisting of SEQ ID NO: 2, a homologous sequence of SEQ ID NO: 2, SEQ ID NO: 4, a homologous sequence of SEQ ID NO: 4, and the third isolated or purified polynucleotide preferably comprises a sequence selected from the group consisting of SEQ ID NO: 6, a sequence homologous to SEQ ID NO: 6, SEQ ID NO: 8, and a homologous sequence of SEQ ID NO: 8.

In another aspect, the invention relates to an immunogenic composition comprising at least one pharmaceutically acceptable carrier or excipient and at least one combination of polypeptides or fusion proteins or polynucleotides according to the invention. Preferably, such an immunogenic composition is characterized in that it is capable of inducing antibodies that totally inhibit the adhesion of Plasmodium falciparum-infected erythrocytes to the placental CSA (chondroitin sulfate A) receptor.

In a variant of this aspect, the invention also relates to vaccines against gestational malaria. In particular, the invention provides a vaccine comprising at least one combination of polypeptides or fusion proteins according to the invention, or at least one combination of polynucleotides according to the invention. In some embodiments, the polynucleotides of the combination present in a vaccine are inserted into at least one plasmid. Preferably, the vaccines according to the invention are characterized in that they are capable of inducing antibodies which totally inhibit the adhesion of Plasmodium falciparum infected erythrocytes to the placental CSA receptor. The vaccines described herein may further include an adjuvant.

In another variant of this aspect, the invention relates to methods of treating or preventing gestational malaria. In particular, the invention provides a method for inducing a protective immune response against Plasmodium falciparum in a human female, the method comprising a step of administering an effective amount of a composition immunogen or vaccine described herein. The invention also provides a method of vaccinating a female human against Plasmodium falciparum, the method comprising a step of administering an effective amount of a vaccine, particularly a DNA vaccine described herein or a protein vaccine described here. Methods of treatment and prevention of gestational malaria are primarily aimed at pre-pubertal girls and women of childbearing age. In certain preferred embodiments, a method of treating or preventing gestational malaria is characterized in that it induces, in female humans, antibodies that prevent the adhesion of Plasmodium falciparum-infected erythrocytes to CSA placental receiver. In methods of treating or preventing gestational malaria according to the invention, administration of the immunogenic composition or vaccine can be by any suitable route.

In another aspect, the invention relates to a gestational malaria vaccination kit comprising at least one combination, or at least one immunogenic composition or at least one vaccine according to the invention and instructions for performing a vaccination against gestational malaria. In some embodiments, the at least two polypeptides or at least two fusion proteins or at least two polynucleotides of the combination, immunogenic composition or vaccine are separately provided in the kit.

A more detailed description of some preferred embodiments of the invention is given below.

Detailed Description of the Invention

In general, the present invention relates to the use of combinations of specific regions of the N-terminal portion of the VAR2CSA protein from different parasitic families of Plasmodium falciparum, in the prevention and / or treatment of gestational malaria. .

I - NTS-DBLlx-Idl-DBL2x and Idl-DBL2x

The present invention relates to combinations of at least two polynucleotides or at least two polypeptides derived from the N-terminal portion of the extracellular domain of VAR2CSA of NTS-DBLlx-Idl-DBL2x and Idl-DBL2x. different parasitic families of Plasmodium falciparum, as well as their applications in the management of gestational malaria. In particular, the invention relates to combinations of at least two polynucleotides or at least two polypeptides derived from the N-terminal region of the extracellular domain of VAR2CSA from two parasitic families of Plasmodium falciparum demonstrated by the inventors, and of which the segregation takes place in interdomain Idl of VAR2CSA.

The first family which is a newly identified cluster is characterized by a VAR2CSA protein having an Idl interdomain having for sequence the consensus sequence SEQ ID NO: 11 or which is encoded by the consensus nucleic sequence SEQ ID NO: 13.

SEQ ID NO: 11

dY! K. #PYS. EyGKLLkFDNTNAFkESiT. nkNVCsCSgnEKliis # GSssS. SFGT SfSY # nS! . TSnKRkECkQIKFSGNKNNMNInICSTQD. # nLLVkl ## LLKgFC # tc dt. iG. VEVVsE # NCEEQYKKLLPcLEKCT! LnCNECNKTrcKpLKK. #EkWIWgKp kq .. aGLQkE

or "!" is one of I and V, "#" is one of N, D, Q, E, B, and Z, and "." is either a gap, or a position where no consensus has been reached.

SEQ ID NO: 13

gaTTAtaTAAAGgaTgATCCTTATTCC. cAGAAtATGGAAAACTATTAAaaTTTGAT AACACTAATGCATTTAaaGAAtcTatAACAT. TaAcAAaAATGTATGTTcTTGtAGt ggtaaTGAAAAATtGatcAtAtCaGAaGGATCATcAaGTTCA. GTTCTTTTGGAACA TCgTtTTCTTATgAAAaTAGTgTAA ... CATCaAAtAAgAGAAAaGAATGTaaACAA ATAAAATTTAGTGGTAATAAAAATAATATGAATATTAAtATATGTT. CCACGCAGGA T .. aAcAAtTTgTTGGTAaAATTagAGGAgTTATTGAAAgGTTTTTGCgATAcATgT GacacTgaTAtTGGAG ... TTGAGGTAGTTaGTGAGaAtAATTGCGAAGAGCAATAT AAAAAACTGCTCCCCTgTCTTGAGAAATGcACTgTTTTGAaTTGTAATGAATGCAAT AAAACTcgATgTAAAccaTTAAAAAAGgaacAAGAAAaATGGATtTGGggtAAAcca aaacaagaagctGcaGGgTTgCAAaAAGAa

or "." is either a gap, or a position where no consensus has been reached. The second family which is a cluster including the FCR3 line and the line

3F7 is characterized by a VAR2CSA protein having an Idl interdomain having for sequence the consensus sequence SEQ ID NO: 12 or which is encoded by the consensus nucleotide sequence SEQ ID NO: 14. SEQ ID NO: 12

. Y! KdDPYsaEY. TKLSFI lNsSDa # tsSeki. knnDEvCNcNESel ssVgqaqtS. PSS # KtCiTHSsIkaNKKKvCKdVKLG! r # nDKdLk! CVIEdtsLsGV # NCCcqDlL giLQEncsD.NksgSSSNGSC # nkn ##. C # knL # kvlASLtNgYKc # KCKSeqSkkn n .. WiWkK. SENSE. GLQkE

SEQ ID NO: 13

aaTTAtaTAAAgGaTGATCCTTATTcCgcAGAATATgcAACtAAATTATCATTTATT ttAAATtCATCAGATgCtaAtAcTtCGTCTGaAaaAata. aAAAaaATaATGATGAA GtATGTAACtgTAATGAATCAGaAATTtCATctGTTGgACaGGcAcaAAcATCgGgT ccgTcGTCgaAtAAAaCATGTAtCACACATAGcTccATAaaAgCTAATAAGAAAAAA GtATGTAAAgATGTAAAGTTGGGTgTTcgTgAaAAtGATAAaGaTTTGAaAaTATGC GTAATTGAGgAcactTCCTTaaGTGGTGTTGAaAATTGTTGTTgCcAAGATTTaTTG gGAATtCTTCAAGAAaaTtGtAgTGATAAtAA. vs . at . GTGgATCTAGTTCTAATGGT AGTTGTgATAAcAAaAaTcAGGAaG. ATGTgAAAaaAAtTTAGAaaAAGtacTTGCA TCTTTAactAATgGTTATAAAtgCgAcAAATGTAAATCTGgAacATCAA. Aa .... T AAcAAaAaaTGGAtATGGAaAAAAT. CtcTGGTaatgaagaaGGATTACAAaAaGAA where "." is either a gap, or a position where no consensus has been reached. The second parasitic family of Plasmodium falciparum demonstrated by the inventors comprises the parasitic strain FCR3 and the parasitic line 3D7. The gene encoding VAR2CSA has been isolated from several parasitic strains including FCR3 (GenBank Accession Number: AY372123) and 3D7 (GenBank Accession Number: AE014188.3) and sequenced. The corresponding VAR2CSA protein has been deduced (GenBank Accession Number: AAQ73926.1 for the FCR3 line and GenBank Accession Number: AAN36095.1 for the 3D7 line).

The term "isolated or purified", as used herein to describe a polypeptide or polynucleotide, refers to a polypeptide or polynucleotide which, by its origin or manipulation, is separated from at least some components with which it is naturally associated. Alternatively or additionally, by "isolated or purified" is meant a polypeptide or polynucleotide that is produced or synthesized by humans. The terms "NTS-DBLlx-DBL2x" and "NTS-DBLlx-Idl-DBL2x" are here used interchangeably. They denote the region of VAR2CSA constituted by the domains: Duffy-binding-like domain lx (DBLlx), interdomain 1 (Idl) and Duffy-binding-like domain 2x (DBL2x). The term "Idl-DBL2x" refers to the VAR2CSA region consisting of interdomain 1 (Idl) and the Duffy-binding-like domain 2x (DBL2x).

In certain preferred embodiments, the polypeptide of a combination according to the invention consisting of the NTS-DBLlx-Idl-DBL2x region of the VAR2CSA protein of the parasitic line FCR3 has the sequence SEQ ID NO: 1, a homologous sequence of the sequence SEQ ID NO: 1 or a modified sequence of the sequence SEQ ID NO: 1; and the polypeptide of a combination according to the invention consisting of the NTS-DBLlx-Idl-DBL2x region of the VAR2CSA protein of the 3D7 parasite line has the sequence SEQ ID NO: 5, a sequence homologous to the sequence SEQ ID NO: Or a modified sequence of the sequence SEQ ID NO: 5. In other preferred embodiments, the polypeptide of a combination according to the invention consisting of the Idl-DBL2x region of the VAR2CSA protein of the parasitic line FCR3 a the sequence SEQ ID NO: 3, a sequence homologous to the sequence SEQ ID NO: 3 or a modified sequence of the sequence SEQ ID NO: 3, and the polypeptide of a combination according to the invention consisting of the region Idl-DBL2x of the VAR2CSA protein of the parasitic line 3D7 has the sequence SEQ ID NO: 7, a sequence homologous to the sequence SEQ ID NO: 7 or a modified sequence of the sequence SEQ ID NO: 7.

The terms "peptide", "protein", "peptide sequence", "polypeptide sequence", and "polypeptide" are used interchangeably herein. By these terms is meant to designate a precise sequence of amino acids, modified or not, linked together by peptide bonds.

By "peptide sequence homologous to the sequence SEQ ID NO: X" is meant any peptide sequence which differs from the sequence SEQ ID NO: X by substitution, deletion, and / or insertion of an amino acid or of a reduced number amino acid positions at positions such that these homologous peptide sequences have substantially the same biological properties as the NTS-DBL1x-Idl-DBL2x region or the Idl-DBL2x region of VAR2CSA of the FCR3 line or the lineage 3D7. Preferably, such a homologous peptide sequence has a percentage of identity such that it is at least 75% identical to the sequence SEQ ID NO: X, preferably at least 85%, more preferably at least 95%.

By "percentage of identity" or "homology" between two nucleotide sequences or two peptide sequences, it is meant to designate a percentage of nucleotides or identical amino acid residues between the two sequences to be compared, obtained after optimal alignment. This percentage is purely statistical and the differences between the two sequences are randomly distributed over the entire length of the sequence. The terms "optimal alignment" and "best alignment", which are used interchangeably herein, refer to the alignment for which the percent identity determined as described below is the highest. The optimal alignment of the sequences necessary for comparison can be done manually or by means of computer software (GAP, BESTFIT, BLASTP, BLASTN, FASTA, and TFASTA, which are available either on the NCBI site or in Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI). The percentage of identity between two nucleotide sequences or two peptide sequences is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the number of identical positions. total of positions compared and multiplying the result by 100.

By "modified peptide sequence of the sequence SEQ ID NO: X" is meant any polypeptide sequence which differs from SEQ ID NO: X by one or more modifications, such as, for example, post-translational cellular modifications (eg "editing" ", glycosylation, sulfation, etc.). The present invention also relates to combinations of at least two fusion proteins for the treatment or prevention of gestational malaria, wherein each of the fusion proteins consists of an isolated or purified polypeptide as defined above fused to a given fusion partner sequence. .

By "fusion partner sequence" is meant herein a peptide sequence which confers on the fusion protein one or more desirable properties. Thus, a fusion partner sequence may consist of a protein that promotes the expression of the NTS-DBLlx-Idl-DBL2x region or of the Idl-DBL2x region in the cell. host during the preparation of the fusion protein, and / or a protein which facilitates the purification of the fusion protein, and / or a protein which increases the stability (eg plasma stability) of the fusion protein (eg comparison with a sequence corresponding to non-fused NTS-DBLlx-Idl-DBL2x or Idl-DBL2x), and / or to a protein that promotes the administration of the fusion protein to the vaccinated subject, and / or to a protein that increases the desired therapeutic effect (for example by increasing the immune and vaccine response) and / or a protein exhibiting biological or therapeutic activity.

Fusion partners that may be used in the context of the present invention include, without limitation, the maltose binding protein, the signal sequence of the maltose binding protein, poly-histidine segments capable of binding metal ions, an -Tag, glutathione-S-transferase, thioredoxin, β-galactosidase, streptavidin, dihydrofolate reductase, the pelB signal sequence, the ompA signal sequence, the alkaline phosphatase signal sequence, green fluorescent protein, a toxin such as for example enterotoxin LT of E. coli or its B subunit, a domain of the tetanus toxin fragment C, cholera toxin or its subunit B, CTA1-DD. Other fusion partners may be human growth hormone, an immunostimulatory cytokine such as: interleukin-2 (IL-2), a growth factor such as the granulocyte macrophage colony stimulating factor (GM-CSF), the granulocyte colony stimulating factor (G-CSF), peptides or hormones such as calcitonin, interferon-beta, interferon-alpha, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), PA toxin, parathyroid hormone (PTH (1-34) and PTH (1-84)), butyrylcholinesterase, glucocerebrosidase (GBA), and exendin-4. The new cluster demonstrated by the present inventors is characterized by a VAR2CSA protein whose interdomain Id1 has as sequence the consensus sequence SEQ ID NO: 11 or is coded by the consensus sequence SEQ ID NO: 13. The other cluster highlighted by the present inventors is characterized by a VAR2CSA protein whose interdomain Idl has as sequence the consensus sequence SEQ ID NO: 12 or is encoded by the consensus nucleotide sequence SEQ ID NO: 14. By "consensus sequence" is meant an idealized sequence of a given region of a protein in which each position represents the amino acid met most frequently. The consensus groups were established by comparison of real sequences.

The present invention also relates to combinations of at least two polynucleotides isolated or purified for use in the treatment or prevention of gestational malaria, wherein each of the isolated or purified polynucleotides encodes a polypeptide as defined above or for a fusion protein such as as defined above, and contains the elements necessary for the expression, in vitro and / or in vivo, of said polypeptide or of said fusion protein. More specifically, the invention relates to a combination of at least two polynucleotides, wherein each of the polynucleotides encodes a polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of a lineage or parasitic family of Plasmodium falciparum given or for a fusion protein comprising said polypeptide, and wherein each of the isolated or purified polynucleotides contains the elements necessary for the expression of said polypeptide or said fusion protein in vitro or in vivo.

The terms "nucleotide sequence", "nucleic acid", "nucleic sequence", "polynucleotide" and "oligonucleotide" are used interchangeably herein. By these terms is meant to designate a precise sequence of nucleotides, modified or not, for defining a region of a nucleic acid, and which may correspond to double-stranded DNA or single-stranded DNA as well as to transcription products. of these DNAs.

The elements necessary for the expression of a nucleotide sequence in vivo include, for example, a promoter, a transcription initiation region, and a transcription termination region that are functional in a mammalian cell, preferably a cell. human. In addition, sequences that increase gene expression, such as introns, enhancer sequences, and "leader" sequences, are often necessary for the expression of a coding sequence for an immunogenic protein. As is known in the art, these elements are preferably operably linked to the nucleotide sequence to be expressed. The terms "operably linked" and "operably linked" are used interchangeably and refer to a functional link between the regulatory sequences and the nucleic acid sequence that they control. Examples of promoters useful in the context of the present invention include, but are not limited to, promoters of SV40 virus, mouse mammary tumor virus (MMTV), HIV virus, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), as well as promoters of human genes such as the human actin, myosin, hemoglobin, muscle creatine and metallothionein.

The construction of a polynucleotide consisting of a given nucleotide sequence and the elements necessary for the expression of this nucleotide sequence are within the skill of those skilled in the art.

In certain preferred embodiments, the nucleotide sequence of the polynucleotide which encodes the NTS-DBL1x-Id1-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 2 or a homologous sequence of SEQ ID NO: 2, and the nucleotide sequence of the polynucleotide which encodes the NTS-DBL1-Idl-DBL2x region of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 6 or a homologous sequence of SEQ ID NO: 6.

In other preferred embodiments, the nucleotide sequence of the polynucleotide which codes for the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 4 or a homologous sequence of SEQ ID NO: 4, and the nucleotide sequence of the isolated or purified polynucleotide which encodes the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 8 or a homologous sequence of SEQ ID NO: 8.

By "nucleotide sequence homologous to the sequence SEQ ID NO: X" is meant any nucleotide sequence which differs from the sequence SEQ ID NO: X by substitution, deletion, and / or insertion of a nucleotide, or of a reduced number nucleotides, at positions such that these sequences encode the same polynucleotide or substantially the same polynucleotide as SEQ ID NO: X. A "nucleotide sequence homologous to the sequence SEQ ID NO: X" is preferably a homologous sequence of SEQ ID NO: X which results from the degeneracy of the genetic code. Preparations for Polynucleotides and Polypeptides Relating to NTS-DBLlx-Idl-DBL2x and Idl-DBL2x

The polynucleotides, fusion proteins, and polypeptides of the present invention may be prepared by any suitable method. Techniques for isolating or cloning a gene or nucleotide sequence encoding a specific domain of a protein are known in the art and include isolation from genomic DNA, preparation from complementary DNA, or combination of these methods. The cloning of a gene, or a nucleotide sequence coding for a specific domain of a protein, from a genomic DNA can be carried out for example by using a polymerase chain reaction (PCR) or by screening libraries. expression to detect cloned DNA fragments with identical structural characteristics (Innis et al., "PCR: A Guide to Method and Application", 1990, Academy Press: New York). Other methods of nucleic acid amplification known to those skilled in the art can be used, such as, for example, ligase chain reaction (LCR), ligation-activated transcription (LAT) and NASBA (Nucleic) Acid Sequence Based Amplification). It is also possible to use a chemical synthesis method to prepare a nucleotide sequence. The methods of total chemical synthesis of DNA or RNA strands are known to those skilled in the art, and use commercial automated synthesizers.

Methods for preparing a peptide sequence include chemical methods (RB Merrifield, J. Am Chem Soc 1963, 85: 2149-2154, "Solid Phase Peptide Synthesis", Methods in Enzymology, GB Fields (Ed), 1997). Academic Press: San Diego, CA), and recombinant methods (Sambrook et al, Molecular Cloning. A Laboratory Manual, ^2nd Ed, 1989, Cold Spring Harbor Press. Cold Spring, NY) using host cells ( particularly in the case of fusion proteins).

Composition of a combination

In a combination according to the invention, the polynucleotides or fusion proteins or polypeptides may be present in any proportions. In embodiments where a combination of the invention consists of only two components (two polypeptides, two fusion proteins or two polynucleotides), the two components may in particular be present in equal amounts. In embodiments where a combination of the invention consists of three components (three polypeptides, three fusion proteins or three polynucleotides), the three components may in particular be present in equal amounts.

The respective amounts of the components of a combination may be determined and / or optimized by those skilled in the art depending on the use of the combination and / or the nature of its components.

II - Immunogenic Compositions and Vaccines

The combinations according to the invention are particularly suitable for use as medicaments in the management of gestational malaria. Indeed, as demonstrated by the inventors, these combinations make it possible to induce anti-adherence antibodies with a broad spectrum of activity. As such, they can be used, as such or in a modified form, as an immunogenic composition or vaccine.

An appropriate modification of the polypeptides contained in a combination according to the invention is the production of conjugates. These comprise at least one of the polypeptides of a combination according to the invention, linked to a support. Conjugates can be obtained by coupling via a convalent bond between a polypeptide and a physiologically acceptable, non-toxic, natural or synthetic carrier, capable, for example, of increasing the immunogenic nature of the polypeptide.

As regards the conjugates, mention may be made by way of example, the application WO 2006/124712, which describes methods for preparing conjugates comprising a plurality of antigenic peptides of Plasmodium falciparum bound to a carrier protein improving the immunogenicity of said antigens.

The preferred supports according to the invention are chosen from viral particles, lipids, for example lipids of C16-C18 type, polylysines, poly (DL-alanine) -poly (Lysine) s, nitrocellulose, microparticles of polystyrene, microparticles of latex beads, biodegradable polymers, polyphosphoglycan microparticles, carrier proteins such as OPMC (outer membrane protein complex of Neisseria meningitidis) or improved OPMC, BSA (bovine serum albumin), TT (tetanus toxoid), ovalbumin, KLH (heyhole limpet hemocyanin), THY (bovine thyroglobulin), HbSAg and HBcAg of the hepatitis B virus, rotavirus capsid proteins, human papillomavirus LI protein, virus type particle (VLP) type 6, 11 and 16, PPD (purified protein derivative) tuberculin.

Immunogenic compositions

An immunogenic composition according to the invention comprises, in addition to a combination described herein, a pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable carrier or excipient" refers to any vehicle or medium which does not interfere with the effectiveness of the biological activity of the active ingredient of the composition, and which is not toxic to the individual to be treated. the concentration at which it is administered. The use of such carriers or excipients for the formulation of active substances is well known in the art ("Remington's Pharmaceutical Sciences", EW Martin, 18 ^th Ed., 1990, Mack Publishing Co.: Easton, PA).

The formulation of an immunogenic composition of the present invention may vary depending on the route of administration and the dosage. After formulation with at least one pharmaceutically acceptable carrier or excipient, an immunogenic composition of the invention may be administered in any form suitable for administration to a human, for example in solid or liquid form. Those skilled in the art know how to select the most suitable vehicles and excipients for the preparation of a certain type of formulation. Thus, for example, excipients such as water, 2-3-butanediol, isotonic sodium chloride solution, synthetic mono or diglycerides, and oleic acid are often used for the formulation of injectable preparations. Liquid compositions, including emulsions, microemulsions, solutions, suspensions, syrups, etc., can be formulated in the presence of solvents, solubilizers, emulsifiers, oils, fatty acids and other additives. as suspending agents, preservatives, viscosizing agents, etc. The solid compositions for oral administration may be formulated in the presence of an inert excipient such as sodium citrate, and optionally additives such as binding agents, humectants, disintegrating agents, absorption accelerators, lubricating agents, etc. According to a preferred embodiment, the immunogenic compositions and vaccines of the invention may comprise one or more adjuvants used in combination. Conventional adjuvants such as Montanide and / or Alum may be used. However other adjuvants such as QS21, SBQS2, MF59, mLT, PHL, CpG DNA, calcium phosphate, dehydrated calcium sulfate, PLG, CT, LTB, CT / LT, AS02A are also suitable.

The immunogenic compositions and vaccines according to the invention may further comprise at least one specific antigen of the pre-erythrocyte stages (CSP, TRAP, LSA-1, LSA-3, SALSA, STARP, EXP-1), asexual erythrocytes (MSP-1, MSP-3, AMA-1, EBA-175, GLURP, MSP-2, MSP-4, MSP-5, RAP-2, RESA, PfEMP-1, synthesized GPI toxin) or sexed (PfS25).

Vaccines, Polypeptide Vaccines, DNA Vaccines

In general, a vaccine against gestational malaria according to the invention comprises at least one combination described herein and is used to induce in antibodies vaccinated antibodies inhibiting cytoadherence to CSA. In particular, the invention relates to a DNA vaccine (also called plasmid vaccine or polunucleotide vaccine) against gestational malaria. The invention also relates to a protein vaccine (also called polypeptide vaccine) against gestational malaria. Protein vaccines

The present invention therefore relates to a protein vaccine comprising a combination of at least two polypeptides as described above or a combination of at least two fusion proteins as described above.

The preparation of a protein vaccine, which can be done chemically or biochemically (recombinant protein) is within the skill of the skilled person.

The administration of a protein vaccine may be by any appropriate route, such as, for example, intravenously, subcutaneously, intradermally, orally, topically or systemically. DNA vaccines

The present invention also relates to a DNA vaccine against gestational malaria. The aim of genetic vaccination or DNA vaccination is to induce an immune response and consists in introducing directly into certain cells of the body a polynucleotide gene or sequence coding for a vaccinal antigen or a purified plasmid of DNA containing a coding sequence. for the vaccine antigen. The cells in question are, in the example of the invention, muscle cells, but any other type of cell may be suitable, for example cells of the skin. Administration is non-exclusive, by intramuscular injection or by "bombardment" of particles on the skin or nasally. DNA enters the muscle cells, skin cells or others; and these cells then themselves synthesize the antigen. The antigen is presented to the immune system and triggers a response (the production of antibodies capable, during an infection, of specifically recognizing this antigen on the parasite). The vaccine is therefore produced, locally, by the body of the individual to be immunized. This method of vaccination, simple and inexpensive, has significant advantages in terms of effectiveness: the antigen thus produced is generally in its native peptide sequence, fused or not to one or more peptide sequences (fusion partners). Above all, it is produced in a prolonged way by the cells of the body, and this durable presentation of the antigen to the immune system should make it possible to avoid the recourse to the recalls. In addition, DNA vaccines are chemically defined and thermally stable, which reduces the need to maintain the cold chain.

The present invention therefore relates to a DNA vaccine comprising a combination of at least two polynucleotides as described above. A polynucleotide of a combination of the invention may be a naked DNA, in particular a circular vaccinia plasmid, supercoiled or not, or a linear DNA molecule incorporating and expressing in vivo a nucleotide sequence coding for the NTS-region. DBLlx-Idl-DBL2x or for the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line or the 3D7 line or the new cluster. By "naked DNA" is meant, as is now commonly accepted, a DNA transcription unit in the form of a polynucleotide sequence comprising at least one nucleotide sequence coding for a vaccine antigen and the elements necessary for its in vivo expression. The polynucleotides of a combination according to the invention may advantageously be inserted into a plasmid of the DNA-CSP type, Nyvac pf7, VR1020, VR1012, etc.

It is also contemplated that naked DNA is incorporated into a drug carrier. Examples of suitable drug vectors include, but are not limited to, biodegradable microcapsules, immunostimulant complexes, liposomes, cationic lipids, and genetically attenuated live vectors such as viruses and bacteria.

A DNA vaccine of the invention may also be administered in conjunction with an agent that enhances the penetration of the vaccine genetic material into treated cells. Thus the DNA vaccine can be formulated to contain such an agent or be administered at the same time as such an agent. Examples of agents that enhance the penetration of vaccine genetic material into treated cells include, but are not limited to, benzoic acid esters, anilides, amidines, urethanes, and their hydrochloride salts (US Patent 6,248,565). . Administration of DNA to cells may be promoted by chemical vectors (such as, for example, cationic polymers or cationic lipids), physical techniques such as electroporation, sonoporation, magnetofection, etc., or still viral vectors such as viruses associated with adenovirus, etc.

III - Uses of Immunogenic Compositions and Vaccines

The immunogenic compositions and vaccines can be advantageously used to immunize female human beings (prepubertal girls and women of childbearing age) in the context of preventive gestational malaria therapy.

Accordingly, the invention also relates to methods of treating or preventing gestational malaria. In particular, the invention provides a method for inducing a protective immune response against Plasmodium falciparum in a human female, the method comprising a step of administering an effective amount of an immunogenic composition or vaccine described right here. The invention also provides a method of vaccinating a female human against Plasmodium falciparum, the method comprising a step of administering an effective amount of a vaccine, particularly a DNA vaccine or a protein vaccine described herein.

In certain preferred embodiments, a method of treating or preventing gestational malaria is characterized in that it induces, in female humans, antibodies that prevent the adhesion of Plasmodium falciparum-infected erythrocytes to CSA placental receiver.

In the methods of treatment or prevention of gestational malaria according to the invention, the administration of the immunogenic composition or the vaccine can be done by any appropriate route (oral, parenteral, mucosal). In some embodiments, administration of a DNA vaccine is intramuscular, intradermal, or mucosal. In other embodiments, the administration of a protein vaccine is, for example, intravenous, subcutaneous, intradermal, oral, topical or systemic.

An immunogenic composition or a vaccine according to the invention may be administered in a single dose or in several doses. Those skilled in the art will be able to determine the effective dose of immunizing protein or DNA for use in each immunization or vaccination protocol.

IV - Kits

The present invention also provides a kit for the prevention of gestational malaria. More specifically, the kit comprises material useful for carrying out a vaccination according to the method of the invention. In general, a kit comprises a combination, an immunogenic composition or a vaccine according to the invention, and instructions for performing a vaccination against gestational malaria. Optionally, the kit may further include means for performing the vaccination.

In some embodiments, a kit according to the invention is configured such that the components of a combination according to the invention are provided separately (for example in different containers). Such a configuration allows both the simultaneous administration and the sequential administration of the components of the combination. By "simultaneous administration" is meant here a administration of the components, together or separately, at approximately the same time (for example, at an interval of 5, 10, 15 or 30 minutes each). other). By "sequential administration" is meant here an administration of the components separately and at different times (for example at different times of the same day, or at one or more days of interval).

The kit may comprise reagents or solutions for the preparation of the composition to be administered. The various components of the kit may be provided in solid form (for example in freeze-dried form) or in liquid form. A kit may optionally include a container each containing reagents or solutions, and / or containers (test tubes, flasks, etc.) to effect the preparation of the composition to be administered. Finally, a notice in the form prescribed by a government agency regulating the sale and use of pharmaceutical products may be included in the kit.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an ordinary specialist in the field to which this invention belongs. Likewise, all publications, patent applications, patents and all other references mentioned herein are incorporated by reference.

The following examples and figures are presented to illustrate certain embodiments of the procedures described above and should in no way be considered as a limit to the scope of the invention. Examples

The following examples describe some embodiments of the present invention. However, it is understood that the examples are presented for illustrative purposes only and in no way limit the scope of the invention.

It has been shown that antibodies against constructs corresponding to a region of the N-terminal portion of VAR2CSA of a single parasite variant could inhibit only 70% of CSA adhesion of pregnant isolates (Bigey et al. J Infect Dis., 2011, 204: 1125-1133, Bordbar et al, Vaccine, 2012, 30: 1343-1348). The inventors have previously identified a dimorphic region in the DBL2x domain of VAR2CSA (Sander et al., PLoS One, 2009, 4: e6667), leading to two distinct phylogenetic categories of type FCR3 and type 3D7. In the study presented below, the inventors studied the prevalence of variant of dimorphic DBL2x among Plasmodium falciparum parasites isolated from samples obtained from pregnant women in Benin and the inhibitory adhesion capacity of specific antibodies induced against the NTS-DBLlx-Idl-DBL2x region of each serotype on isolated infected erythrocytes pregnant women. Methods and Materials Used

Isolates of Plasmodium falciparum. Pregnant women were included in this study only after their consent was obtained in writing. The study was approved by the IRD Advisory Committee on Ethics and Ethics (Research Institute for Development), and the ethics committee of the Faculty of Health Sciences (University of Abomey-Calavi, Benign). All procedures used in this study comply with European and National regulations. The animal immunization procedure is in accordance with the guidelines of FELASA (Federation of Laboratory Animal Science Associations) and has been approved by the Committee of Ethics in Animal Experimentation affiliated to the University Paris Descartes.

Isolates of Plasmodium falciparum. Field samples of infected erythrocytes were obtained from pregnant women at the Suru Léré maternity hospital in the eastern region of Cotonou. The study site is characterized by hyper-endemic malaria in the lagoon zone and a high rate of malaria transmission with two peaks corresponding to the two rainy seasons (Akogbeto et al., Parasitologia, 1992, 34: 147-154). Peripheral venous blood was collected in vacutainers containing anticoagulant citrate phosphate dextrose adenine (CPDA), during prenatal visits and at the time of delivery. The erythrocytes were separated from the plasma and washed with RPMI 1640 (Lonza). 200 μl of erythrocyte pellets were homogenized in 10 volumes of TRIzol reagent (Invitrogen) and frozen at -80 ° C. until extraction of the total RNA or frozen at -20 ° C. moment of extraction of the total DNA. The pellets of erythrocytes infected with ring stage parasites (ring or young trophozoite) were immediately cultured in vitro to obtain aged trophozoites as previously described (Trager et al., Science, 1976, 193: 673-675). Briefly, the isolates were grown in vessels containing RPMI 1640 supplemented with HEPES and L-glutamine (Lonza Biowhittaker), 0.3 g / L 1-glutamine, 0.05 g / L gentamicin, 5 g / L The albumax. The culture vessels were placed under a 92.5% N2, 2% O2 and 5.5% CO2 atmosphere and incubated at 37 ° C for no more than 48 hours prior to testing. The parasitic laboratory strains FCR3, HB3 and NF54 were cultured in 0+ erythrocytes and were selected after several panning steps on the BeWo cell line as previously described (Haase et al., Infect Immun., 2006, 74: 3035). -3038).

DNA Extraction and Genotyping msp. The DNA was extracted from 100 pellet with the GeneJet Genomic Purification Kit (Fermentas) according to the manufacturer's recommendations. The mspl and msp2 genes were amplified by nested PCR using specific primers (Snounou et al., Trans R Soc Trop Med Hyg, 1999, 93: 369-374). The multiplicity of infection (MOI) was determined for each sample.

RNA extraction, cDNA synthesis and Genotyping of DBL2x. The RNAs were extracted from frozen samples stored in the TRIzol reagent (In vitro gen) according to the manufacturer's recommendations. Total RNAs were treated with DNAse I (Invitrogen) for 15 minutes at room temperature according to the manufacturer's recommendations to eliminate any potential contamination with genomic DNA (gDNA). The absence of gDNA in the RNA samples was confirmed by a lack of amplification after 40 cycles of a real-time PCR with primers targeting housekeeping seryl-tRNA synthetase gene (Salanti et al, Mol Microbiol, 2003 49: 179-191). Reverse transcription of RNA (without DNA) was performed with Thermoscript (Invitrogen) and random hexamer primers for 1 hour at 50 ° C in a total volume of 20 μL as recommended by the manufacturer. For genotyping of the dimorphic sequence motif (DSM) in the domain

DBL2x from VAR2CSA of Plasmodium falciparum isolates (Sander et al., PLoS One, 2009, 4: e6667), this domain was selected to amplify the cDNA of each isolate with a primer pair (5'-TTAYCCCCAAGAACACA-3 'and 5'-TTTT A ATTTTTTCC ATG A A-3 '). Reactions were performed with high fidelity Taq Fusion Polymerase (to obtain higher yields and lower mutation rates) under the following conditions: 94 ° C for 1 minute followed by 35 cycles at 94 ° C for 30 minutes. seconds, 50 ° C for 30 seconds and 68 ° C for 50 seconds, and final extension at 68 ° C for 10 minutes. The PCR products were digested with the restriction enzymes BstCI (which cuts the FCR3 type DSMs) and Hppyl88I (which cuts the 3D7 DSMs) for 1 hour at 50 ° C and 37 ° C, respectively. The digested products were separated by electrophoresis on 1.5% agarose gel.

Antibody Production and IgG Preparation. The NTS-DBL1x-Idl-DBL2x fragment and the entire sequence of the optimized varlcsa gene of FCR3 and 3D7 parasite lines were used to produce specific anti-VAR2CSA IgGs, by DNA vaccination as previously described (Bigey et al., J. Infect Dis ., 2011, 204: 1125-1133). Briefly, the DNA sequences were cloned into a vector derived from pVax1 and fused to the mEPO signal sequence as previously described (Trollet et al., Infect Immun., 2009, 77: 2221-2229). Immunizations were performed on Swiss female mice aged 6 weeks and rabbits from New Zealand (January-France). The mice were anesthetized by intraperitoneal injection with a ketamine-xylazine mixture and in the case of rabbits the injection was made at 5 sites along each longissimus dorsi muscle. DNA transfer was performed by transcutaneous electrical pulses applied by two stainless steel electrodes placed at each injection site. The animals were immunized on days 0, 30 and 60, and the antisera were collected on days 0 and 15 after each of the immunizations. Total IgG was purified in the final mouse or rabbit serum sample on a Hi-Trap column to which the corresponding recombinant protein had been coupled in accordance with the manufacturer's recommendations (GE Healthcare).

Flow cytometry. The reactivity of purified IgG against the surface of Plasmodium falciparum infected erythrocytes was analyzed by flow cytometry (FACS Calibur) as previously described (Magistrado et al., Vaccine, 2011, 29: 437-443). In brief, infected CSA adherent erythrocytes (FCR3-Bewo and HB3-Bewo) and parasitic field isolates were enriched by exposure to a high intensity magnetic field (VarioMACS and CS columns, Miltenyi). For each test, 2 × 10 ⁵ infected erythrocytes were labeled with ethidium bromide, and sequentially exposed to rabbit IgG, and then to a FITC-conjugated anti-rabbit antibody (Invitrogen). Data was acquired and analyzed, and the median fluorescence intensity (MFI) was determined. The marking of the VAR2CSA surface is considered positive if the ratio of MFI is greater than 1.2 (MFI with IgG post-immunization at day 75 / MFI with pre-immunization IgG at day 0) as described by Magistrado et al. (Vaccine, 2011, 29: 437-443).

Adhesion Inhibition Tests. The ability of antibodies to inhibit adhesion of infected red cells to chondroitin sulfate (CSPG) was determined by a Petri dish assay previously described (Bigey et al., J. Infect Dis., 2011, 204: 1125-1133). . Briefly, petri dishes (Falcon 351029) were covered with spots of 100 x 15 mm 20 μL of 5 mg / mL CSPG-Decorin (Sigma) or 10 μg / mL bovine serum albumin (BSA). ) diluted in PBS. The spots were incubated overnight at 4 ° C in a humid chamber. Each spot was blocked with 3% BSA in PBS for 30 minutes at 37 ° C. Suspensions enriched with erythrocytes infected with aged trophozoites were obtained by filtration on a magnetic column (VarioMACS, Miltenyi). The suspension, whose parasite density was adjusted to 20% in 1 × 10 ⁵ cells, was then blocked for 30 minutes at room temperature (RT) in a solution of 3% BSA diluted in RPMI. The erythrocyte preparation was incubated with purified IgG (0.25 mg / mL) or 500 μg / mL soluble SCA (Sigma), and the cells were then adhered for 15 minutes at room temperature on Petri dishes. pre-coated with different ligands. The non-adherent cells were removed by an automatic washing system. The spots were fixed with 1.5% glutaraldehyde in PBS, stained with Giemsa solution and the infected infected erythrocytes were counted under a microscope.

Legends of Figures

Figure 1 is a graph showing the distribution of the dimorphic sequence signatures of the DBL2x region of VAR2CSA among parasites infecting pregnant women. The dimorphic sequence motif (DSM) in DBL2x that distinguishes VAR2CSA alleles into two subgroups (types 3D7 and FCR3) were genotyped in parasites collected from pregnant women in Benin. The proportions of type 3D7 (white histograms), type FCR3 (striped histograms) and mixing of the two genotypes (black histograms) are presented. The multiplicity of infection (MOI) is indicated for each category. Figure 2 is a graph showing the recognition of VAR2CSA expressed in IgG field isolates directed against the different constructs of Var2CSA. The recognition capacity of VAR2CSA expressed by the IgGs induced against the VAR2CSA constructs (NTS-DBL2X / 3D7 and NTS-DBL2X / FCR3) and against the entire extracellular domain of VAR2CSA (NTS-DBL1-68 / FCR3) of the strain FCR3 has been studied on field isolates by flow cytometry. The parasites were separated according to their category DSM (DSM 3D7, DSM FCR3 or DSM Blend FCR3 / 3D7). The bars indicate the average MFI ratio (MFI corresponding to the reactivity of the IgG of the hyperimmune animal / MFI corresponding to the reactivity of the IgG of the same pre-immune animal.

Figure 3 includes two graphs showing the IgG adhesion inhibitory activity induced against each of the two 'serotypes' of NTS-DBL1x-Idl-DBL2x on field isolates. Inhibitory adhesion activity was assessed on 18 Plasmodium falciparum isolates freshly obtained from pregnant women. The data is presented as percent inhibition for each antibody normalized by the CSA inhibition value (used as a reference for maximum adhesion inhibition). Histograms are presented for each of the DSM types detected in the isolate: □ for DSMs of type 3D7, m for DSMs of type FCR3, and ■ for DSMs of mixture of the two genotypes. Figure 4 is a series of three graphs showing the inhibition profile of antibodies on strains selected and adapted and binding to the CSA. The FCR3, HB3 and NF54 strains selected on Bewo cells (FCR3-Bewo, HB36-Bewo and NF54-Bewo) were used to evaluate the ability of the antibodies induced to inhibit the adhesion of the 3 laboratory strains to CSPG. The in vitro functionality of the antibody mixtures was also evaluated. The values presented are normalized with the inhibition value of the CSA.

Figure 5 is a representation of the phylogenetic relationships between sequences spanning the ID1-DBL2X region of VAR2CSA generated from the CDNA of 123 parasite isolates of pregnant women. The phylogenetic tree illustrates the phylogenetic relationship between the different sequences, with the groupings of dichotomous variants defined in DBL2X that discriminates between the 3D7 and FCR3 clusters and the clusters of a new cluster whose identity is defined in the Idl region of VAR2CSA. Bootstrap values are shown at each main branch.

Results

Determination of the Multiplicity of Infection and Molecular Typing of Var2csa Among Isolated Parasites in Pregnant Women. A total of 122 parasites were collected, and it was determined by genotyping msp-2 that the multiplicity of infections (MOI) is 3.10 distinct genotypes per isolate with a range of 1 to 7. A PCR method -RFLP was developed and implemented on all isolates to study the distribution of the two major varlcsa variants among the Benin field isolates. Analysis of the dimorphic region of varlcsa in isolates of pregnant women revealed the presence of at least one of the two dimorphic variants described by Sander et al. (PLoS One, 2009, 4: e6667) in all parasites. DSM type 3D7 transcripts were found in 54 isolates (44%) and FCR3 type DSM transcripts were found in 47 isolates (39%) (Figure 1). In 21 isolates (17%), both types of transcripts were detected, suggesting a mixture of genotypes. No differences were observed between the MOIs of the isolates of the groups determined after the typing of varlcsa (the determined MOIs are 3.05 in isolates expressing type 3D7, 2.98 in isolates expressing type CFR3, and 3.6 in the isolates with a mixture of the two DSM signatures).

Specifically Induced IgG against the NTS-DBLlx-Idl-DBL2x Region of the FCR3 and 3D7 Lines recognize all Isolais of Pregnant Women. The level of recognition of VAR2SCA expressed on the surface of erythrocytes was estimated immunologically on 47 Plasmodium jalciparum-infected erythrocytes isolated from pregnant women. Among these isolates, it was found that 16 of them have DSM type 3D7, 21 have DSM type FCR3 and a mixture of genotypes has been detected in 10 isolates. Infected erythrocytes were labeled and evaluated by flow cytometry using purified IgG from sera from vaccinated animals. The anti-NTS-DBLlx-Idl-DBL2x IgG specific to varlcsa variants of 3D7 and FCR3, and the IgGs induced against the entire extracellular domain of VAR2CSA were used in this experiment. IgG induced against the entire extracellular domain of VAR2CSA was used to estimate the level of absolute recognition of the native VAR2CSA protein expressed on the surface of infected erythrocytes. A high level of reactivity was observed with antibodies induced against the entire extracellular domain of VAR2CSA compared to antibodies directed against the NTS-DBLlx-Idl-DBL2x domain of VAR2CSA regardless of the origin of the latter (P <0.05 ). However, no significant difference was observed between the levels of recognition of the native VAR2CSA protein by anti-NTS-DBL1x-Idl-DBL2x IgGs induced against the FCR3 variant and against the 3D7 variant (P = 0.59). The inventors then investigated whether the surface recognition of the antibodies was influenced by the genotype of varlcsa. The isolates were divided according to the type of DSM of their varlcsa. Data from infected erythrocytes labeled with each antibody were separated according to the DSM of the isolates. No significant difference was observed in the recognition of VAR2CSA by each antibody tested regardless of the type of DSM (Figure 2).

Properties of Inhibition of the Adhesion of anti-NTS-DBLlx-Idl-DBL2x IgG Specific for Variants FCR3 and 3D7. The analysis of the inhibitory activity of the antibodies was also carried out on 18 samples of infected erythrocytes binding to CSA obtained from pregnant women from Benin. The typing of the DSMs of these isolates revealed that 6 of them (33%) express the 3D7 type DSM of varlcsa; 8 (44%) express the FCR3 type DSM of varlcsa; and 4 (22%) are mixtures of both types (Figure 3).

Soluble CSA was used as a reference for maximum inhibition of adhesion. The degree of parasite inhibition by anti-NTS-DBL1x-Idl-DBL2x antibodies of FCR3 and 3D7 variants was normalized to CSA inhibition. The average antibody inhibitory activity on all isolates was 80% [interquartile range: 50.8 - 100] for the anti-NTS-DBL1x-Idl-DBL2x antibody of the FCR3 variant and 97% [interquartile range: 55.3 - 100] for the anti-NTS-DBL1x-Idl-DBL2x antibody of the variant 3D7. Different modes of inhibition were observed with the antibodies as a function of the types of varlcsa isolates. 16 of the 18 isolates tested (89%) were effectively inhibited by the antibodies induced against the NTS-DBL1x-Idl-DBL2x domain of the FCR3 variant (43-100%) whereas a strong inhibitory activity of the antibodies induced against the NTS domain -DBLlx-Idl-DBL2x variant 3D7 (50-100%) was observed in 15 of the 18 isolates (84% tested). However, it appears that the activities of the two types of antibodies on the different isolates tested differ each other. The inhibitory effect of one of the two types of antibody may be weak in one isolate while the antibodies induced against the other serotype may exhibit a high inhibitory effect on the same parasite. For example, isolates OPT144 and OPT161 are not inhibited by the antibodies induced against serotype FCR3, and interestingly, these isolates bearing DSM of type 3D7 have been strongly inhibited by IgG specific to the NTS-DBL1x-Idl-domain. DBL2x of the variant 3D7. A similar profile was obtained with the OPT105 isolate which is more strongly inhibited with the DSM serotype-specific IgG homologous to the FCR3 variant and less well inhibited with the 3D7 variant-specific anti-NTS-DBLlx-Idl-DBL2x IgG. In contrast, in two isolates with 3D7 serotypes (OPT127 and OPT141), better inhibition was observed with antibodies induced against the opposite FCR3 serotype. In general, complementarity of inhibition was observed on field isolates, with a substantial increase in the extent of adhesion inhibition of field isolates to CSPG now reaching 100% of currently inhibited isolates. In addition, 8 out of 10 isolates (80%) with a 3D7 DSM of varlcsa were inhibited with anti-NTS-DBLlx-Idl-DBL2x antibodies induced against the homologous DSM variant while all isolates with DSM-type FCR3 of varlcsa were efficiently inhibited by anti-NTS-DBLlx-Idl-DBL2x IgG specific FCR3 serotypes. The ability of antibodies to inhibit CSA adhesion of infected erythrocytes expressing VAR2CSA and the behavior of the mixture of the two antibodies induced against the NTS-DBLlx-Idl-DBL2x domain of VAR2CSA were studied on laboratory strains and selected strains. by Bewo: FCR3 and HB3 (which have a DSM type FCR3) and NF54 (which a background of type 3D7). High inhibitory activity was achieved with antibodies to infected erythrocytes belonging to the homologous DSM. The anti-NTS-DBLlx-Idl-DBL2x antibodies induced against the FCR3 variant completely (100%) inhibit the CSA adhesion of the FCR23 and HB3 lines (FIG. 4). However, only partial inhibition of these lines (20% to 40%) was observed with the anti-NTS-DBL1x-Idl-DBL2x IgG induced against the 3D7 variant, which also strongly inhibit the adhesion to CSA of the NF54 parasite. . In addition, the mixture of the anti-NTS-DBL1-Idl-DBL2x IgGs of the FCR3 line and the 3D7 line retains the property of completely inhibiting the adhesion of all the CSPG-infected erythrocytes. Identification of a third cluster. Sequence variations of the NTS-DBLlx-Idl-DBL2x domain of the VAR2CSA protein of the transcript of 123 parasite isolates of pregnant women from Benin were analyzed. Total RNA was extracted from freshly collected parasites, and the cDNA was synthesized. The var2csa gene was amplified from the cDNA using a high fidelity enzyme (Fusion), and universal primers were used. Ten (10) clones were sequenced for each isolate and the sequences were generated by multiple alignment of the protein and nucleic sequences.

The results of the analyzes clearly showed a segregation of the parasitic variants in the Idl region of VAR2CSA. This new dichotomy domain, which has not been described so far, is located in a critical region of the VAR2CSA protein that is essential to its CSA binding properties. This very clear segregation of sequences will have implications in combining a limited number of variants required for an optimal vaccine based on VAR2CSA.

The sequence alignments led to the establishment of two representative consensus Interdomain Id1 sequences of VAR2CSA. The first, ID1A or SEQ ID NO: 11 corresponds to the newly identified cluster, and the second ID1B or SEQ ID NO: 12 corresponds to the other group of sequences. conclusions

This work demonstrates that the NTS-DBL2X region of VAR2CSA contains conserved anti-adherence epitopes and indicates that the development of an effective vaccine based on VAR2CSA will require the combination of a limited number of VAR2CSA variants. The combination of the three major variants of VAR2CSA used in this work will be essential to develop an effective vaccine against placental malaria.

Claims

claims

A combination of at least two isolated or purified polypeptides, the first isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of a first parasitic family of Plasmodium jalciparum, and the second isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of a second parasitic family of Plasmodium jalciparum, for use in the treatment or prevention of gestational malaria.

Combination for use according to Claim 1, characterized in that the first family is the parasitic family of Plasmodium jalciparum, the VAR2CSA protein of which is characterized by an interdomain Id1 which has the sequence sequence SEQ ID NO: 11 or which is coded by the consensus sequence SEQ ID NO: 13, and the second family is the parasitic family of Plasmodium jalciparum, the VAR2CSA protein of which is characterized by an interdomain Id1 which is sequenced by the consensus sequence SEQ ID NO: 12 or which is encoded by the sequence consensus SEQ ID NO: 14.

Combination for use according to Claim 2, characterized in that the second parasitic family of Plasmodium jalciparum comprises the parasitic line FCR3 and the parasitic line 3D7.

Combination for use according to claim 3, characterized in that it consists of three isolated or purified polypeptides, the first isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the protein VAR2CSA of the Plasmodium jalciparum parasitic family, the VAR2CSA protein of which is characterized by an Idl interdomain having for sequence the consensus sequence SEQ ID NO: 11, the second isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the region Idl-DBL2x of the VAR2CSA protein of the FCR3 line, and the third isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line. Combination for use according to claim 3 or claim 4, characterized in that the NTS-DBLlx-Idl-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 1 or a homologous sequence of SEQ ID NO: 1, and the NTS-DBLlx-Idl-DBL2x region of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 5 or a homologous sequence of SEQ ID NO: 5.

Combination for use according to claim 3 or claim 4, characterized in that the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 3 or a homologous sequence of SEQ ID NO: 3, and the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 7 or a homologous sequence of SEQ ID NO: 7.

Combination for use according to one of claims 1 to 6, characterized in that each of the at least two isolated or purified polypeptides is fused to a fusion partner sequence, thereby forming a fusion protein.

Combination for use according to claim 7, characterized in that each of the fusion partner sequences is independently selected from the group consisting of the maltose binding protein, the signal sequence of the maltose binding protein, an S-tag, the glutathione-S-transferase, thioredoxin, β-galactosidase, streptavidin, dihydrofolate reductase, pelB signal sequence, ompA signal sequence, alkaline phosphatase signal sequence, green fluorescent protein, toxins, human growth hormone, interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), calcitonin, interferon-beta, interferon-alpha, glucagon like peptide 1 (GLP-1), glucagon like peptide 2 (GLP-2), PA toxin, parathyroid hormones (PTH (1-34) and PTH (1-84)), butyrylcholinesterase, glucocerebrosidase (GBA), and exendin-4.

Combination of at least two isolated or purified polynucleotides, the first isolated or purified polynucleotide encoding a first polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the protein VAR2CSA of a first parasitic family of Plasmodium jalciparum or for a first fusion protein comprising the first polypeptide, and containing the elements necessary for the expression of said first polypeptide or of said first fusion protein in vitro and / or in vivo; and the second isolated or purified polynucleotide encoding a second polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of a second Plasmodium falciparum parasitic family or a second fusion protein comprising the second polypeptide, and containing the elements necessary for the expression of said second polypeptide or said second fusion protein in vitro and / or in vivo, for use in the treatment or prevention of gestational malaria.

Combination for use according to claim 9, characterized in that the first family is the parasitic family of Plasmodium falciparum whose VAR2CSA protein is characterized by an interdomain Id1 having for sequence the consensus sequence SEQ ID NO: 11 and the second family is the parasitic family of Plasmodium falciparum, the VAR2CSA protein of which is characterized by an Idl interdomain having for sequence the consensus sequence SEQ ID NO: 12.

Combination for use according to claim 9, characterized in that the first family is the parasitic family Plasmodium falciparum whose VAR2CSA protein is characterized by an interdomain Idl encoded by the consensus sequence SEQ ID NO: 13 and the second family is the parasitic family Plasmodium falciparum whose VAR2CSA protein is characterized by an interdomain Idl encoded by the consensus sequence SEQ ID NO: 14.

Combination for use according to claim 10 or claim 11, characterized in that the second parasitic family of Plasmodium falciparum comprises the parasitic line FCR3 and the parasitic line 3D7.

Combination for use according to claim 12, characterized in that it consists of three isolated or purified polynucleotides, the first isolated or purified polynucleotide encoding a first polypeptide consisting of in the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the Plasmodium jalciparum parasitic family, the VAR2CSA protein of which is characterized by an Idl interdomain having for sequence the consensus sequence SEQ ID NO: 11 or being encoded by the consensus sequence SEQ ID NO: 13 or for a first fusion protein comprising the first polypeptide, and containing the elements necessary for the expression of said first polypeptide or said first fusion protein in vitro and / or in vivo; the second isolated or purified polynucleotide encoding a second polypeptide consisting of the NTS-DBL1x-Id1-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line or for a second fusion protein comprising the second polypeptide, and containing the elements necessary for the expression of said second polypeptide or of said second fusion protein in vitro and / or in vivo; and the third isolated or purified polynucleotide encoding a third polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line or for a third fusion protein comprising the third polypeptide, and containing the elements necessary for the expression of said third polypeptide or said third fusion protein in vitro and / or in vivo.

Combination for use according to Claim 12 or Claim 13, characterized in that the NTS-DBLlx-Idl-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 1 or a homologous sequence of SEQ ID NO: 1, and the NTS-DBLlx-Idl-DBL2x region of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 5 or a homologous sequence of SEQ ID NO: 5.

Combination for use according to Claim 12 or Claim 13, characterized in that the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 3 or a homologous sequence of SEQ ID NO: 3, and the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 7 or a homologous sequence of SEQ ID NO: 7. Combination for use according to claim 12 or claim 13, characterized in that the second isolated or purified polynucleotide, and optionally the third isolated or purified polynucleotide, comprises a sequence selected from the group consisting of SEQ ID NO: 2, a homologous sequence of SEQ ID NO: 2, SEQ ID NO: 4, a homologous sequence of SEQ ID NO: 4, SEQ ID NO: 6, a homologous sequence of SEQ ID NO: 6, SEQ ID NO: 8, and a sequence homologue of SEQ ID NO: 8.

An immunogenic composition comprising at least one pharmaceutically acceptable carrier or excipient and at least one combination as claimed in any one of claims 1 to 16.

Use of an immunogenic composition according to claim 17 in the prevention or treatment of gestational malaria.

Gestational malaria vaccine comprising at least one combination according to any one of claims 1 to 16.

DNA vaccine against gestational malaria comprising at least one combination according to any one of claims 9 to 16, characterized in that the polynucleotides of the combination are inserted into at least one plasmid.

The vaccine of claim 19 or claim 20 further comprising at least one adjuvant.

Gestational malaria vaccination kit comprising at least one combination according to any one of claims 1 to 16, or an immunogenic composition according to claim 17 or a vaccine according to any one of claims 19 to 21, and instructions for carrying out vaccination against gestational malaria.

Kit according to claim 22, characterized in that the at least two polypeptides or at least two fusion proteins or at least two polynucleotides of the combination are provided separately in the kit.