OA17444A - Vaccines against gestational malaria. - Google Patents

Abstract

The present invention relates to combinations of polypeptides or of polynucleotides corresponding to a specific region of the part N-terminal portion of the VAR2CSA protein of different parasitic families or lines of Plasmodium falciparum and 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

INSTITUT DE RECHERCHE POUR LE DEVELOPPEMENT (IRD)

Immeuble le Sextant,44 Bd Dunkerque, CS90009, F-13002 Marseille 2 (FRANCE)

INVENTEURS:

1) TUIKUE NDAM, Nicaise; 54, boulevard Garibaldî, F-75015 Paris (FR).

2) DELORON, Philippe; 26, rue de Rochechouart, F-75009 Paris (FR).

3) DORITCHAMOU, Justin; IRD-UMR216,4 avenue de l'Observatoire, F-75006 Pari (FR).

VACCINES AGAINST PREGNANC Y- ASSOCIATED MALARIA Parent Application

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

Field of the invention

The présent invention relates to the use of combinations of spécifie régions of the N-termînal portion of the VAR2CSA protein derived from various parasitic families or lines of Plasmodium falciparum in the prévention and/or treatment of pregnancy-associated malaria.

Context of the invention

Malaria (paludism) is the commonest of the parasitic infections observed in the world. It is due to a parasite of the genus Plasmodium, which is transmitted by the bite of a female mosquito (Anopheles). Several Plasmodium species can infect humans, but Plasmodium falciparum is the commonest and most pathogenîc species and the one that is responsible for fatal cases. Once introduced into the blood, the parasite infects the liver cells, in which it proliférâtes, before circulating in the blood again and invading the red blood cells (érythrocytes). Malaria affects about a hundred countries in the world, particularly in the disadvantaged tropical zones of Africa, Asia and Latin America; Africa being the continent most affected by far, According to the estimâtes of the World Health Organization, malaria is responsible annually for 225 million cases of fever and about l million deaths (World Malaria Report, WHO, 2010). Existing means for combating it are antimalarials (the best known being chloroquine and quinine) and tackling the mosquitoes that are the vectors of the parasite. However, the situation is more worrying than some years ago; the parasites are becoming more and more résistant to the médications and the mosquitoes are developing résistance to insecticides. No vaccine is available at présent.

Malaria mainly affects children under 5 years of âge and prégnant women, in particular primigravidae (i.e. women prégnant for the first time). Prégnant women are particularly vulnérable as the placenta constitutes a target where the parasites may accumulatc. In prégnant women. malaria! infection may cause a wholc range ot dctrimental effccts: spontaneous aborlion. prématuré birth. inadéquate birth weight. congénital transmission, and néonatal death. In zones of Africa where malaria is endémie, 3 to 5% of the dcaths of children al birth may be ascribcd to prcgnancyassociated malaria. Morcover, it also présents a real danger for the niother. who may suffer anémia, which is sometimes severe, or even fatal.

Currently, prévention of malaria in prégnant women is based on prophylactic treatment with sulfadoxinc-pyrinicthaniine (Cot et al., Br. Med. Bull.. 2003. 67: 137148). However, this intermittent treatment is not able to provide prévention of malaria throughout prcgnancy: firslly, because the drug is only administered start ing from the 20th week of prcgnancy (as the tératogénie risk during embryogenesis is considered too great); secondly, hecause it is based on two administrations of suJfadoxine-pyrimcthamine at therapeutic dose at least one month apart, which only provides partial médicinal protection; and thirdly, because the efficacy of sulfadoxine-pyrimethamine is now decreasing considerably in ail malaria-endemic régions owing to expansion of parasitic résistance (Cot et al., Am. J. Trop. Med. Hyg., 1998, 59: 813-822; WHO/HTM/MAL/2005.1103. Gcneva: World Health Organization; ter 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). Médicinal products are currently under investigation in this context, and much effort is also focused on development of a vaccine against pregnancy-associated malaria. The possibility of vaccinating prégnant women or prepubescent girls would constitute an obvious advantage over drug treatment, in that préventive cover of pregnancy would be extended, and would potentially be of better quality.

One of the vaccine strategies envisaged for combating pregnancy-associated malaria is to recreate naturel protective irnmunity. In fact, the clinical severity of malaria due to Plasmodium falciparum is partly connected with changes of the infected érythrocytes. These changes are induced by the proteins of the parasite, which are exported to the surface of the érythrocytes during the development stage in the blood. Certain of these surface proteins of the PfEMPl family (Plasmodium falciparum Erythrocyte Membrane Protein 1 ), encoded by the parasites, confer new cytoadhérence properties on the érythrocytes. The érythrocytes are then capable of

ΙΟ adhcring lo the inside wall of blood vessels. thereby preventing transport of the infected érythrocytes lo the purifying organs ofthe immune system. one ofthe rôles of which is to destrov cells recognized as abnormal. In the case of pregnancyassoeiatcd malaria, the parasitizcd érythrocytes adhère to a sugar. uhondroitin sulfate A (CSA), présent in the placenta. After several pregnancies. women acquire protective antibodies that block this adhesion. One vaccine strategy is to reercate this protective immunity. by blocking adhesion of the infected érythrocytes to the placenta.

The VAR2CSA protein, one ofthe proteins ofthe PfEMPI family, is currently the subject of many investigations with a view to obtaining a spécifie vaccine for prégnant women (Tuikuc 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; Dahlbâck et al., PLoS Pathogens, 2006, 2: 1069-1082; Badaut et al., Mol. Biochem. Parasitol., 2007, 15: 89-99; Khattab et al., Parasitol. Res., 2007, 101: 767774; Guitard et al., Malaria J., 2008, 11:7-10; Guitard et al., Malaria J., 2010, 9: 165; Gai gnard et al., Mol. Biochem. Parasitol., 2010, 173: 115-122; Gnidehou et al., Mol. Biochem. Parasitol., 2010, 5(10): el3105; WO 2012/014073). These works corne up against polymorphism of VAR2CSA, but phase 1 studies are nevertheless envisaged. The whole of the extracellular domain of this protein has been expressed in a heterologous system (Srivastava et al., Proc. Natl. Acad. Soi. USA, 2010, 107: 48844889; Khunrae et al., J. Mol. Biol., 2010, 397: 826-834), and the antibodies induced against this extracellular domain display a very high level of antiadherence. However, development of new vaccine approaches will hâve to take into account the numerous immunodominant epitopes that do not induce antiadherence” antibodies.

It therefore appears crucial to continue to explore and develop new strategies for combating and preventing pregnancy-associated malaria.

Sunimary ofthe invention

The présent inventais hâve previously identified the N-temiinal région of VAR2CSA, and notably the NTS-DBLlx-Idl-DBL2x région consisting of the DBLlx domain, the ldi interdomain and the DBL2x domain, as being the région that contains epitopes capable of inducing, in vivo, antibodies that block adhérence of érythrocytes infected by Plasmodium falciparum to CSA, and the Idl-DBL2x subrcgion as being the minimal aiiligenic région of VAR2CSA involvcd in the acquisition of protective inununity with respect to the placenta! séquestration that occurs in prcgnancy-associated malaria (WO 2012/014073). Thev bave now discovered that the combination of antibodies induced against the NTS-DBLlx-ldlDBL2x région of VAR2CSA of the two parasite lines FCR3 and 3D7 of Plasmodium falciparum completely inhibit fixation of the parasitized érythrocytes to chondroitin sulfate A (CSA) both on placental field isolâtes from prégnant women and on wellcharacterized laboratory strains. For comparison. the antibodies induced against the NTS-DBLlx-idl-DBL2x région of VAR2CSA of each of the two parasite lines FCR3 and 3D7 of Plasmodium falciparum used separately only display a percentage inhibition of about 70%. By analyzing the sequences of parasitic isolâtes from prégnant women in Bénin, the inventons also demonstrated ségrégation of the parasitic variants in the Idl interdomain of VAR2CSA. This new dichotomy domain has never been described previously. Sequence alignments led to the establishment of two consensus sequences représentative of the ldi interdomain of VAR2CSA; the first ldlA (or SEQ ID NO: 11) corresponds to a recently identified cluster, and the second ldi B (or SEQ ID NO: 12) corresponds to the other group of sequences (of which the line FCR3 and the line 3D7 form part).

Accordingly, a first aspect of the présent invention relates to the use of combinations of polypeptide or polynucleotide sequences corresponding to the NTSDBLlx-ldl-DBL2x région or to the Idl-DBL2x région of the VAR2CSA protein derived from different parasitic families of Plasmodium falciparum, in the management of malaria in prégnant women.

More specifically, the présent invention relates to the use of isolated or purified polypeptide or polynucleotide sequences corresponding to the NTS-DBLlx-IdlDBL2x région or to the ldl-DBL2x région of the VAR2CSA protein derived from Plasmodium falciparum parasites whose VAR2CSA protein is characterized by an Idl interdomain having as sequence the consensus sequence SEQ ID NO: 11 (or that is encoded by the consensus nucleic acid sequence SEQ ID NO: 13) and optionally from polypeptide or polynucleotide sequences corresponding to the NTS-DBLIxldl-DBL2x région or to the Idl-DBL2x région of the VAR2CSA protein derived from Plasmodium falciparum parasites whose VAR2CSA protein is characterized by ^λ an kl l interdomain that has as sequence the consensus sequence SEQ ID NO: 12 (or thaï is encoded by ihe consensus nucleic acid sequence SEQ ID NO: 14).

In particular. the présent invention relates lo a combination of al least two isolated or purified polypeptides for lise in the treatment or prévention of prcgnancyassociated malaria, the first isolated or purified polypeptide consisting of the NTSDBLlx-kll-DBL2x région or the ldl-DBL2x région of the VAR2CSA protein of a first parasitic family of Plasmodium faiciparum, and the second isolated or purified polypeptide consisting of the NTS-DBLlx-ldl-DBL2x région or the kll-DBL2x région of the VAR2CSA protein of a second parasitic family of Plasmodium faiciparum, for use in the treatment or prévention of pregnancy-associated malaria.

In certain preferred embodiments. the first family is the parasitic family of Plasmodium faiciparum whose VAR2CSA protein is characterized by an kl l interdomain that has as sequence the consensus sequence SEQ ID NO: l J or that is encoded by the consensus nucleic acid sequence SEQ ID NO: 13, and the second family is the parasitic family of Plasmodium faiciparum whose VAR2CSA protein is characterized by an Idl interdomain that has as sequence the consensus sequence SEQ ID NO: 12 or that is encoded by the consensus nucleic acid sequence SEQ ID NO: 14.

In certain embodiments, the second parasitic family of Plasmodium faiciparum 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 région or the Idl-DBL2x région of the VAR2CSA protein of the parasitic family of Plasmodium faiciparum whose VAR2CSA protein is characterized by an Idl interdomain that has as sequence the consensus sequence SEQ ID NO: Il or that is encoded by the consensus nucleic acid sequence SEQ ID NO 13, the second isolated or purified polypeptide consisting of the NTS-DBLlx-ldl-DBL2x région or the ldlDBL2x région of the VAR2CSA protein of the FCR3 line, and the third isolated or purified polypeptide consisting of the NTS-DBLIx-Idl-DBL2x région or the IdlDBL2x région of the VAR2CSA protein ofthe 3D7 line.

lu certain embodiments. lhe NTS-DBLI x-ldl-DBL2x région of lhe VAR2CSA protein ofthe FCR3 line has the sequence SEQ ID NO: ] or a homologous sequence of SEQ ID NO: I, and the NTS-DBL l x-kll-DBL2x région ofthe VAR2CSA protein ofthe 3D7 line has the sequence SEQ ID NO: 5 or a homologous sequence of SEQ ID NO: 5.

In certain embodiments. lhe ldl-DBL2x région oftlie VAR2CSA protein ofthe FCR3 line has the sequence SEQ ID NO: 3 or a homologous sequence of SEQ ID NO: 3. and the ldl-DBL2x région ofthe VAR2CSA protein of tlie 3D7 line has the sequence SEQ ID NO: 7 or a homologous sequence of SEQ ID NO: 7.

The présent invention also relates to a combination of at least two isolated or purified fusion proteins for use in the treatment or prévention of pregnancyassociated malaria, where the combination corresponds to a combination of at least two isolated or purified polypeptides as described here in which each of the polypeptides is fused to a fusion partner sequence.

In certain embodiments, each of the fusion partner sequences is selected independently 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, the signal sequence pelB, the signal sequence ompA, the signal sequence of alkaline phosphatase, green fluorescent protein, toxins, human growth hormone, inteiieukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), calcitonîn, interferon beta, interferon alfa, glucagon-like peptide 1 (GLP-I), glucagon-like peptide 2 (GLP-2), toxin PA, parathyroid hormones (PTHQ-34) and PTH(l-84)), butyrylcholinesterase, glucocerebrosidase (GBA), and exendin-4.

The invention also relates to a combination of at least two isolated or purified polynucleotides for use in the treatment or prévention of pregnancy-associated malaria, where each of the isolated or purified polynucleotides encodes a polypeptide as defined above or for a fusion protein as defined above. More precisely, 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 région or the ldl-DBL2x région of the VAR2CSA protein of a first parasilie family of Plasmodium falciparum or l’or a Inst fusion protein comprising the llrst polypeptide, and containing tlie cléments necessary for 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 5 polypeptide consisting of the NTS-DBL 1 x-ld l-DBL2x région or the ldl-DBL2x région of the VAR2CSA protein of a second parasilie family of Plasmodium falciparum or for a second fusion protein comprising the second polypeptide, and containing the éléments necessary for expression of said second polypeptide or of said second fusion protein in vitro and/or in vivo, for use in the treatment or 10 prévention of pregnancy-associated malaria.

In certain embodiments, the first family is the parasitic family of Plasmodium falciparum whose VAR2CSA protein is characterized by an ldi interdomain that has as sequence the consensus sequence SEQ ID NO; 11 or that is encoded by the consensus sequence SEQ ID NO: 13 and the second family is the parasitic family of 15 Plasmodium falciparum whose VAR2CSA protein is characterized by an ldi interdomain that has as sequence the consensus sequence SEQ ID NO: 12 or that is encoded by the consensus sequence SEQ ID NO: 14,

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

ln certain 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-DBL 1 x-ld l-DBL2x région or the ldl-DBL2x région of the VAR2CSA protein of the parasitic family of Plasmodium falciparum whose VAR2CSA protein is characterized by an Idl interdomain that has as sequence the consensus sequence SEQ ID NO: 11 or that is encoded by the consensus sequence SEQ ID NO: 13 or for a first fusion protein comprising the first polypeptide, and containing the éléments necessary for 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 NTSDBL] x-ldl-DBL2x région or the ldl-DBL2x région of the VAR2CSA protein of the FCR3 line or for a second fusion protein comprising the second polypeptide, and containing the éléments necessary for 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 ofthe NTS-DBLl x-ld IDBL2.X région or the Id l -DBL2x région ofthe VAR2CS.A protein of the 3D7 line or for a thîrd fusion protein comprising the third polypeptide, and containing the éléments necessary for expression of said third polypeptide or of said third fusion protein in vitro and/or in vivo.

In certain embodiments, the NTS-DBL l x-ld l -DBL2x région of the VAR2CSA protein ofthe FC R 3 line has the sequence SEQ ID NO: l or a Homo logo us sequence ofSEQ ID NO; l. and the NTS-DBLl.x-ld l-DBL2x région ofthe VAR2CSA protein ofthe 3D7 line has the sequence SEQ ID NO: 5 or a homologous sequence of SEQ IDNO: 5.

In certain embodiments. the ldl-DBL2x région ofthe 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 région ofthe VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO; 7 or a homologous sequence of SEQ ID NO: 7.

In certain embodiments, the second îsolated 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 homologous sequence of SEQ ID NO: 8.

In the embodiments where a second and a third îsolated or purified polynucleotide are présent, the second îsolated 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 îsolated or purified polynucleotide preferably comprises a sequence selected from the group consisting of 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 another aspect, the invention relates to an immunogenic composition comprising at least one pharmaceutically acceptable vehicle or excipient and at least one combination of polypeptides or of fusion proteins or of polynucleotides according to the invention. Preferably, an immunogenic composition of this kind is characterized in that it is capable of inducing antibodies that completely inhibit the adhesion of érythrocytes infected by Plasmodium falcipurum to the placental receptor CSA (chondroitin sulfate A).

In a variant of this aspect, the invention also relaies to vaccines against pregnancy-associated malaria. In particular, the invention supplies a vaccine comprising at least one combination of polypeptides or of fusion proteins according to the invention, or at least one combination of polynucleotidcs according to the invention. In certain embodiments. the polynucleotides of the combination présent in a vaccine are inserted in at least one plasmid. Preferably. the vaccines according to the invention are characterized in that they are capable of inducing antibodies that completely inhibit the adhesion of érythrocytes infected by Plasmodium falciparum to the placental receptor CSA. The vaccines described herc may further comprise an adjuvant.

In another variant of this aspect, the invention relates to methods of treating or preventing prcgnancy-associated malaria. In particular, the invention supplies a method for inducing a protective immune response against Plasmodium falciparum in a human being of the female sex, said method comprising a step of administering an effective amount of an immunogcnic composition or of a vaccine described here. The invention also supplies a method of vaccinaiing a human being of the female sex against Plasmodium falciparum, said method comprising a step of administering an effective amount of a vaccine, in particular of a DNA vaccine described here or of a protein vaccine described here. The methods of treating and preventing pregnancyassociated malaria mainly apply to prepubescent girls and to women of reproductive âge. In certain preferred embodiments, a method of treating or preventing pregnancyassocîated malaria is characterized in that it induces, in human beings of the female sex, antibodies that prevent the adhesion of érythrocytes infected by Plasmodium falciparum to the placental receptor CSA. In the methods of treating or preventing pregnancy-associated malaria according to the invention, the immunogcnic composition or vaccine may be administered by any suitable route.

In another aspect, the invention relates to a kit for vaccinating against pregnancy-associated malaria comprising at least one combination, or at least one immunogenic composition or at least one vaccine according to the invention and instructions for carrying oui vaccination against pregnancy-associated malaria. In certain embodiments, the at least two polypeptides or at least two fusion proteins or at least i\\ o polynucleotides ofthe combination, ofthe immunogcnic composition or ofthe vaccine arc supplied separately in the kit,

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

Detailed description ofthe invention

In general, the présent invention relates to the use of combinations of spécifie régions of the N-terminal portion of the VAR2CSA protein dcrix'ed from different parasitic families of Plasmodium Jalciparum in the prévention and/or treatment of pregnancy-associated malaria.

I - NTS-DBLlx-Id1-DBL2x and Idl-DBL2x

Polynucleotides and polypeptides relating to NTS-DBLlx-ldl-DBLlx and IdlDBL2x

The présent invention relates to combinations of at least two polynucleotides or of at least two polypeptides derived from the N-tenninal portion of the cxtraccllular domain of VAR2CSA of different parasitic families of Plasmodium Jalciparum, as well as to their applications in the management of pregnancy-associated malaria. In particuiar, the invention relates to combinations of at least two polynucleotides or of at least two polypeptides derived from the N-terminal région of the extracellular domain of VAR2CSA of two parasitic families of Plasmodium Jalciparum demonstrated by the inventors, and whose ségrégation takes place in the ldi interdomain of VAR2CSA.

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

SEQ 1DNO: 11 dY!K.#PYS.EyGKLLkFDNTNAFkESiT.nkNVCsCSgnEKliis#GSssS .SFGT SfSY#nS!.TSnKRkECkQIKFSGNKNNMNInICSTQD.ttnLLVkl;LLKgFC#tcd t.iG.VEVVsEftNCEEQYKKLLPcLEKCT!LnCNECNKTrcKpLKK.#EkWIWgKpk g..aGLQkE where ! is one of l ami V. is one of N. D, Q. E. B. and Z. and is either a gap.

or a position where no consensus has been attained.

SEQ ID NO: 13 gaTTAtaTAAAGgaTgATCCTTATTCC.cAGAAtATGGAAAACTATTAAaaTTTGAT

AACACTAATGCATTTAaaGAAtcTatAACAT.TaAcAAaAATGTATGTTcTTGtAGt ggtaaTGAAAAATtGatcAtAtCaGAaGGATCATcAaGTTCA.GTTCTTTTGGAACA TCgTtTTCTTATgAAAaTAGTgTAA...CATCaAAtAAgAGAAAaGAATGTaaACAA

ATAAAATTTAGTGGTAATAAAAATAATATGAATATTAAtATATGTT.CCACGCAGGA

T..aAcAAtTTgTTGGTAaAATTagAGGAgTTATTGAAAgGTTTTTGCgATAcATgT GacacTgaTAtTGGAG...TTGAGGTAGTTaGTGAGaAtAATTGCGAAGAGCAATAT AAAAAACTGCTCCCCTgTCTTGAGAAATGcACTgTTTTGAaTTGTAATGAATGCAAT

AAAACTcgATgTAAAccaTTAAAAAAGgaacAAGAAAaATGGATtTGGggtAAAcca aaacaagaagctGcaGGgTTgCAAaAAGAa where is either a gap, or a position where no consensus has been attained.

The second family, w'hich is a cluster comprising the F CR. 3 line and the 3F7 line, is characterizcd by a VAR2CSA protein having an ldi interdomain having as sequence the consensus sequence SEQ ID NO: !2 or that is encodcd by the consensus nucleic acid sequence SEQ ID NO: 14.

SEQ ID NO: 12 .Y !KdDPYsaEY.TKLSFIlNsSDa#tsSeki.knnDEvCNcNESelssVgqaqtS. psS#KtCiTHSsIkaNKKKvCKdVKLG!rttnDKdLk!CVIEdtsLsGV#NCCcqDlL giLQEncsD.NksgSSSNGSC#nkn;.C#knL#kvlASLtNgYKc#KCKSeqSkknn ..WiWkK.sGne.GLQkE where ! 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 attained.

SEQ ID NO: 13 aaTTAtaTAAAgGaTGATCCTTATTcCgcAGAATATgcAACtAAATTATCATTTATT ttAAATtCATCAGATgCtaAtAcTtCGTCTGaAaaAata.aAAAaaATaATGATGAA GtATGTAACtgTAATGAATCAGaAATTtCATctGTTGgACaGGcAcaAAcATCgGgT ccgTcGTCgaAtAAAaCATGTAtCACACATAGcTccATAaaAgCTAATAAGAAAAAA GtATGTAAAgATGTAAAGTTGGGTgTTcgTgAaAAtGATAAaGaTTTGAaAaTATGC GTAATTGAGgAcactTCCTTaaGTGGTGTTGAaAATTGTTGTTgCcAAGATTTaTTG gGAATtCTTCAAGAAaaTtGtAgTGATAAtAA. c. a .GTGgATCTAGTTCTAATGGT AGTTGTgATAAcAAaAaTcAGGAaG.ATGTgAAAaaAAtTTAGAaaAAGtacTTGCA TCTTTAactAATgGTTATAAAtgCgAcAAATGTAAATCTGgAacATCAA.Aa., . ,T AAcAAaAaaTGGAtATGGAaAAAAT.CtcTGGTaatgaagaaGGATTACAAaAaGAA where îs either a gap, or a position where no consensus has been attained,

The second parasitic family of Plasmodium falciparum demonstrated by the inventors comprises the parasitic line FCR3 and the parasitic line 3D7.

The gene encoding VAR2CSA was isolated in several parasitic strains including FCR3 (GenBank Accession Number: AY372I23) and 3D7 (GenBank Accession Number: AE014188.3) and sequcnced. The corresponding VAR2CSA protein was dedueed (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 here for qualîfyiiig a polypeptide or polynucleotide, dénotés a polypeptide or polynucleotide which, owing to its origin or its manipulation, is separated from at least certain components with which it is naturally associated. Altematively or additionally, isolated or purified means a polypeptide or polynucleotide that is produced or synthesized by humans.

The terms NTS-DBLlx-DBL2x and NTS-DBLlx-ldl-DBL2x are used here indiscriminately. They dénoté the région of VAR2CSA consîsting of the domains: Duffy-binding-like domain lx (DBLlx), interdomain l (Idl) and Duffybinding-like domain 2x (DBL2x). The terni ldl-DBL2x dénotés the région of VAR2CSA consîsting of the interdomain I (ldi) and the Duffy-binding-like domain 2x (DBL2x).

In certain preferred embodîments, the polypeptide of a combination according to the invention consîsting of the NTS-DBLlx-Idl-DBL2x région of the VAR2CSA protein of the parasitic FCR3 line has the sequence SEQ ID NO: l, a homologous sequence of the sequence SEQ ID NO: l or a modified sequence of the sequence SEQ ID NO; l; and the polypeptide of a combination according to the invention consîsting of the NTS-DBLlx-Idl-DBL2x région of the VAR2CSA protein of the parasitic line 3D7 has the sequence SEQ ID NO: 5, a homologous sequence of the sequence SEQ ID NO: 5 or a modified sequence of the sequence SEQ ID NO; 5.

ln other preferred embodiments. lhe polypeptide of a combination according to the int ention consisting of lhe ldl-DBL2x région of the VAR2CSA protein of the parasilic FC R 3 line has the sequence SEQ ID NO: 3. a homologous sequence of the sequence SEQ ID NO: 3 or a modified sequence of the sequence SEQ ID NO: 3. and 5 îhe polypeptide of a combination according to the invention consisting of the Jd I DBL2x région of the VAR2CSA protein of the parasitic line 3D7 has the sequence SEQ ID NO: 7. a homologous sequence of 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, I0 and polypeptide are used here indiscriminatcly. These tenus are intended to dénoté a précisé chain of amino acids, modified or not, bound to one another by peptide bonds.

Homologous peptide sequence of the sequence SEQ ID NO: X means any peptide sequence that differs from the sequence SEQ ID NO: X b y substitution, 15 délétion, and/or insertion of an amino acid or of a reduced number of amino acids, at positions such that these homologous peptide sequences hâve substantially the same biological properties as the NTS-DBLlx-ldl-DBL2x région or the Idl-DBL2x région of VAR2CSA of the FCR3 line or 3D7 line, Preferably, a homologous peptide sequence of this kind has a percentage identity such that it is identical to at least 75% 20 of the sequence SEQ ID NO: X, preferably at least 85%, even more preferably at least 95%.

Percentage identity or homology between two nucléotide sequences or two peptide sequences dénotés a percentage of nucléotides or of amino acid residues that are identical between the two sequences to be compared, obtained after optimal 25 alignment. This percentage is purely statistical and the différences between the two sequences are distributed at random and over the entire length of the sequence. The ternis optimal alignment and best alignment, which are used interchangeably here, dénoté the alignment for which the percentage identity detemiined as described below is highest. Optimal alignment of the sequences, necessary for comparison, 30 may be carried out manually or by means of computer software (GAP, BESTFIT, BLASTP, BLASTN, FASTA, and TFASTA, which are available either on the NCBI website, or in the Wisconsin Genetics Software Package, Genetics Computer Group,

Madison, WI). The percentage identity between two nucléotide sequences or two

peptide sequences is calculated by determining the number of identical positions for which the nucléotide or the amino acid residue is idenlical between tlie two sequences. by dividing this number of identical positions by lhe total number of positions compared and by multiplying the resuit obtained by l 00.

Modified peptide sequence of the sequence SEQ ID NO: X means any polypeptide sequence that differs from SEQ ID NO: X by one or more modifications, for c.xample post-translatîonal cellular modifications (e.g. editing. glycosylation, sulfation. etc.).

The présent invention also relates to combinations of at least two fusion proteins for treating or preventing pregnancy-associatcd malaria, where each of the fusion proteins consists of an isolated or purified polypeptide as defined above fused to a given fusion sequence partner.

Herc, fusion sequence partner means a peptide sequence that endows the fusion protein with one or more désirable properties. Thus, a fusion sequence partner may consist of a protein that promûtes expression of the NTS-DBLlx-Idl-DBL2x région or of the Idl-DBL2x région in the host cell during préparation of the fusion protein, and/or of a protein that facilitâtes purification of the fusion protein, and/or of a protein that increases the stability (e.g. plasma stability) of the fusion protein (by comparison with a sequence correspondîng to unfused NTS-DBLlx-kll-DBL2x or ldi -DBL2x), and/or of a protein that promûtes administration of the fusion protein to the subject being vaccinated, and/or of a protein that increases the required therapeutic effect (for example by increasing the immune and vaccine response) and/or of a protein displaying biological or therapeutic activity.

The fusion partners that may be used in the context of the présent invention include, without limitation, maltose-binding protein, the signal sequence of maltosebinding protein, polyhistidine segments capable of binding métal ions, an S-Tag, glutalhione-S-transferase, thioredoxin, β-galactosîdase, streptavidin, dihydrofolate reductase, the signal sequence pelB, lhe signal sequence ompA, the signal sequence of alkaline phosphatase, green fluorescent protein, a toxin such as, for example, the enterotoxin LT of E. coli or its subunit B, a domain of the C fragment of tetanus toxin, the choiera toxin or its subunit B, CTA l-DD. Other fusion partners may be human growth hormone, an imniunostimulating cytokine such as: interleukin-2 (IL14

ΙΟ

2), a grow th taclor such as granuloc>le macrophage colonx siinnilating factor (GMCSF), granulocyte colony slimulating factor (G-CSF). peptides or hormones such as calcitonin. interferon beta. interferon alfa, gkicagon-like peptide I (GLP-l). glucagon-like peptide 2 (GLP-2). PA toxin. parathyroid hormone (PTPl( I-34) and PTH( l -84)), bulyrylcholineslcrase. gkicoccrebiosidasc (GBA). and exendin-4.

The novel cluster demonstrated by the présent inventors is characterized by a VAR2CSA protein whose kl I interdomain lias as sequence die consensus sequence SEQ ID NO: ll or is encoded by the consensus sequence SEQ ID NO: 13. The other cluster demonstrated by the présent inventors is characterized by a VAR2CSA protein whose ldi interdomain has as sequence the consensus sequence SEQ ID NO: 12 or is encoded by the consensus nucleic acid sequence SEQ ID NO: I4. Consensus sequence means an idealized sequence of a given région of a protein in which each position represents the amino acid encountered most frequently. The consensus sequences were established by comparing real sequences.

The présent invention also relates to combinations of at least two isolated or purified polynucleotides for use in the treatment or prévention of pregnancyassociated malaria, where each of the isolated or purified polynucleotides encodes a polypeptide as defined above or for a fusion protein as defined above, and contains the éléments necessary for expression, in vitro and/or in vivo, of said polypeptide or of said fusion protein. More precisely, the invention relates to a combination of at least two polynucleotides, where each of the polynucleotides encodes a polypeptide consisting of the NTS-DBLlx-Idl-DBL2x région or the Idl-DBL2x région of the VAR2CSA protein of a given parasitic line or family of Plasmodium falciparum or for a fusion protein comprising said polypeptide, and where each of the isolated or purified polynucleotides contains the éléments necessary for expression of said polypeptide or of said fusion protein in vitro or in vivo.

The terms nucléotide sequence, nucleic acid, nucleic acid sequence, polynucleotide and oligonucleotide are used here indiscriminately. These tenus dénoté a précisé chain of nucléotides, modified or not, allowing a région of a nucleic acid to be defined, and which may correspond both to a double-stranded DNA or a single-stranded DNA and to transcription products of these DNAs.

Tlie éléments necessar\ for expression of a nucléotide sequence in vivo include. for example. a promoter. a transcription start région, and a transcription lermination région, which are fùnctional in a mammalian cell, preferably a human cell. Morcovcr, sequences thaï increase gene expression, such as introns. enhancer sequences and leader sequences. are often ncccssary for expression of a sequence encoding an înimunogenic protein. As is known in the prior art. these éléments are preferably bound operationallv to the nucléotide sequence that must be expressed. The tenus linked operationally and linked in an opeiatîonal manner are used indiscriminately and refer to a fùnctional link between the regulatory sequences and the nucleic acid sequence that they control.

Examples of promoters useful in the context of the présent invention include non-cxhaustively the promoters of the SV40 virus, of the mouse mammary tumor virus (MMTV), of the HIV virus, of the Moloney virus, of the cytomégalovirus (CMV), of the Epstein-Barr virus (EBV), of the Rous sarcoma virus (RSV), as well as the promoters of human genes such as the human gene of actin, of myosin, of hemoglobin, of mu seul ar creati ne and of metallothionein.

The construction of a polynucleotide consisting of a given nucléotide sequence and of the éléments necessary for expression of this nucléotide sequence is within the compétence of a person skilled in the art.

In certain preferred embodiments, the nucléotide sequence of the polynucleotide that encodes the NTS-DBLlx-IdI-DBL2x région 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 nucléotide sequence of the polynucleotide that encodes the NTS-DBL!x-Idl-DBL2x région 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 nucléotide sequence of the polynucleotide that encodes the Idl-DBL2x région 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 nucléotide sequence of the isolated or purified polynucleotide that encodes the IdlDBL2x région of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 8 or a homologous sequence of SEQ ID NO: 8.

Honiologous nucléotide sequence of tlie sequence SEQ ID NO: X means any nucléotide sequence thaï differs from the sequence SEQ ID NO: X by substitution, délétion, and'or insertion of a nucléotide, or of a reduced number of nucléotides, at positions such that these séquences encode the same polynucleotide or suhslanlially tlie same polynucleotide as SEQ ID NO: X. A honiologous nucléotide sequence of the sequence SEQ ID NO: X” is preferably a honiologous sequence of SEQ ID NO: X that results from degencration of the genctic code.

Préparations of polynucleotides and polypeptides relatiiig to .XTS-DBLlx-IdlDBL2x and Idl-DBL2x

The polynucleotides. fusion proteins and polypeptides of the présent invention may be prepared by any suitable method.

The techniques for isolating or cloning a gene or a nucléotide sequence encoding a spécifie domain of a protein are known in the prior art and include isolation from genomic DNA, préparation from compJemcntary DNA, or a combination of these methods. The cloning of a gene, or of a nucléotide sequence encoding a spécifie domain of a protein, starting from a genomic DNA may be carried out for example using a polymerase chain réaction (PCR) or by screening expression libraries to detect cloned DNA fragments with identîcal structural characteristics (Innis et al., PCR: A Guide to Method and Application, 1990, Academie Press: New York). Other methods of amplification of nucleic acids known by a person skilled in the art may be used, for ex amp le a ligase chain reaction (LCR), a ligation activated transcription (LAT) and the NASBA (nucleic acid sequence based amplification) technique. It is also possible to use a method of chemical synthesis to préparé a nucléotide sequence. The methods of complété chemical synthesis of strands of DNA or of RNA are known by a person skilled in the art, and use commercial automated synthesizers.

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

Composition ofa combination

In a combinai ion according lo lhe invention, lhe polynuclcoiidcs or fusion proteins or polypeptides may be présent in any proportions. In the embodiments where a combination of lhe invention only consists of two components (two polypeptides, two fusion proteins or two polynucleotidcs), the two components may în particular be présent in equal amounts. In the embodiments where a combination of the invention consists of three components (three polypeptides, three fusion proteins or three polynucleotidcs). the three components may in particular be présent in equal amounts.

The respective amounts of lhe components of a combination may bc determined and/or optimized by a person skilled in the art as a function ofthe use of the combination and-'or ofthe nature ofits components.

II- 1 nimunogcnic Compositions and Vaccines

The combinations according to the invention are particularly suitable for use as médicinal products in the management of pregnancy-associated malaria. In fa et. as the inventors hâve demonstrated, these combinations make it possible to induce antiadherence antibodies with a broad spectrum of activity. Accordingly, they may be used. as they are or in a modified form, as an immunogenic composition or vaccine.

A suitable modification of the polypeptides contained in a combination according to the invention is the production of conjugales. The latter comprise at least one of the polypeptides of a combination according to the invention, bound to a support. The conjugates may be obtained by coupling via a covalent bond between a polypeptide and a physiologically acceptable nontoxic, natural or synthetic support that is able, for example, to increase the immunogenic character of the polypeptide.

Regarding the conjugates, we may mention as an example application WO 2006/124712, which describes methods for preparing conjugates comprising a plurality of antigénie peptides of Plasmodium falciparum bound to a supporting protein improving the immunogenicity of said antigens.

The preferred supports according to the invention are selected from the viral particles, lipids, for example lipids of the C16-C18 type, polylysines, poly(DLalanîne)-poly(lysine)s, nîtrocellulosc, polystyrène microparticlcs, microparticles of latex beads, biodégradable polymers, microparticles of polyphosphoglycans,

ΙΟ supporting proleins such as OMPC (outer membrane protein complex of Neisscria meningitidis) or improved OMPC. BSA (bovine sérum album in). TT (tcîanus toxoid). ovalbumin. KLH (keyhole limpet hcmocyanin). THY (bovine thyroglobulin). HbSAg and HBcAg of hcpatitis B virus, rolavirus capsid proteins. protein Ll of human papillonna virus. VLP (virus like particle) of type 6, l l and 16. tuberculin PPD (purified protein dérivative).

Immunogenic compositions

An immunogenic composition according to the invention comprises, in addition to a combination described here. a pharmaceutically acceptable vehicle or excipient. The term pharmaceutically acceptable vehicle or excipient dénotés any vehicle or medium that does not interfère with the effectiveness of the biological activity ofthe active principle of the composition, and that is not toxic for an individual at the concentration at which it is administered. The use of such vehicles or excipients for formulating active substances is well known in the ail (Remingion's Pharmaceitiical Sciences, E.W. Martin, I8'^h Ed., 1990, Mack PublishingCo.: Easton, PA).

The fonnulation of an immunogenic composition of the présent invention may vary as a function of the route of administration and the dosage. After formulation with at least one pharmaceutically acceptable vehicle or excipient, an immunogenic. composition of the invention may be administered in any form suitable for administration to a human being, for ex ample in solid or liquid form. A person skilled in the art knows how to select the most suitable vehicles and excipients for preparing a certain type of formulation. Thus, for example, excipients such as water, 2-3-butanediol, isotonie solution of sodium chloride, synthetic mono- or diglycerides, and oieîc acid are often used for formulating injectable préparations. Liquid compositions, including émulsions, microemulsions, solutions, suspensions, syrups, etc., may be formulated in the presence of solvents, solubilizers, emulsifiers, oils, fatty acids and other additives such as suspendîng agents, preservatives, viscosity modifiers, etc. The solid compositions for administration by the oral route may be formulated in the presence of an inert excipient such as sodium citrate, and optionally additives such as binders, humectants, disintegrants, absorption accelerators, lubricants, etc.

According to n preferred embodiment. the immunogcnic compositions and the vaccines of the invention may comprise one or more adjuvants used in combination. Conventional adjuvants such as Montanidc and/or alum may be used. However, other adjuvants such as QS2I. SBQS2. MF59. ml.T. PHI.. CpG DNA. calcium phosphate, dchyd rated calcium sulfate. P LG. CT. LT B. CT· LT. AS02A arc also suitable.

The immunogenic compositions and vaccines according to the invention may further comprise at least one spécifie antigen of the proérythrocyte stages (CSP. TR.AP. LSA-l. LSA-3, SALSA. STARP. EXP-l). ascxual érythrocyte stages (MSPI, MSP-3, AMA-l. EBA-I75, GLURP, MSP-2. MSP-4. MSP-5. RAP-2, RESA. PfEMP-l. synthetic GPI toxin) or sexual stages (PtS25).

Vaccines, Polypeptide Vaccines, DNA Vaccines

In general, a vaccine against pregnancy-associated malaria according to the invention comprises at least one combination described here and is used for inducing, in the vaccinated subjccts, antibodies that inhibit cytoadhcrcnce to CSA. In particular, the invention relates to a DNA vaccine (also called plasmid vaccine or polynueleotide vaccine) against pregnancy-associated malaria. The invention also relates to a protein vaccine (also called polypeptide vaccine) against pregnancyassociated malaria.

Protein vaccines

The présent 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.

Préparation of a protein vaccine, which may be doue by a chemical or biochemical route (recombinant protein), is within the compétence of a person skilled in the art.

A protein vaccine may be administered by any suitable route, for example by the intravenous, subcutaneous, intradermal, oral, topical or systemic route.

DNA vaccines

The présent invention also relates to a DNA vaccine against pregnancyassociated malaria. The purpose of gcnetic vaccination or DNA vaccination is to induce an immune response and consists of introducing a gene or a polynueleotide sequence encoding a vaccinal antigen or a purified DNA plasmid containing a sequence encoding the vaccinal antigen directly into certain cells of the body, The cells in question are. in the examplc of the invention, muscle cells, but any other type of cell may be suitable, for examplc cells of lhe skin. Administration is performed. noit-exclusivcly, b y intram uscu kir injection or b y bombardmenl of particles on lhe skin or else by the nasal route. The DNA pénétrâtes into the muscle cells. cells of the skin or others; and these cells then synthesize the antigen themselves. The antigen is presented to the immune system and triggers a response (production of antibodies which. during infection, arc capable of specifically recognizing this antigen on the parasite). The vaccine is therefore produced locally by the body of lhe îndividual to be imntunized. This method of vaccination, which is simple and inexpensive, offers important advantages in ternis of efficacy: the antigen thus produced is generally in the form of its native peptide sequence, fused or not to one or more peptide sequences (fusion partners). In particular, it is produced over an extended period by the body's cells, and this long-lasting présentation of the antigen to the immune system should make it possible to avoid the need for booster doses. Moreover. DNA vaccines are chemically defined and themially stable, which reduces the need to maintain the cold chain.

The présent invention therefore relates to a DNA vaccine comprising a combination of at least two polynuclcotides as described above. A polynucleotide of a combination of the invention may be bare DNA, in particular a circulai- vaccine plasmid, supercoiled or not, or a linear DNA molécule, incorporatîng and expressing in vivo a nucléotide sequence encoding the NTS-DBLlx-ldl-DBL2x région or for the Idl-DBL2x région of the VAR2CSA protein of the FCR3 line or 3D7 line or of the new cluster. Bare DNA means, as is commonly accepted at présent, a DNA transcription unit in the form of a polynucleotide sequence comprising at least one nucléotide sequence encoding a vaccinal antigen and the cléments necessary for expression thereof in vivo. The polynucleotides of a combination according to the invention may advantageously be inserted in a plasmid of type DNA-CSP, Nyvac pf7, VR1020, VR1012, etc.

It îs also envisaged that the bare DNA is incorporated in a médicinal product vector. Examples of suitable médicinal product vectors include, non-exhaustively, biodégradable micro caps nies, immunostimulating complexes, liposomes, cationic lipids and genctically altenualcd live vectors such as viruses and bacleria.

A DNA vaccine of the invention may also be administered in conjonction with an agent that improves the pénétration of (he gcnctic material oflhe vaccine into the cells that are treated. Thus. the DNA vaccine may be formulated to contaîn such an agent or may be administered at tlie same time as such an agent. Examples of agents that improve the pénétration of tlie genetic material of the vaccine into the treated cells include. non-exhaustively. benzoic acid esters, anilidcs. amidincs. urethanes. and hydrochloride salts thereof (US patent 6,248.565). Administration of the DNA to the cells may be pronioted by chemical vectors (for example, cationic polynters or cationic lipids), physical techniques such as electroporation, sonoporation, magnetofeclion, etc., or viral vectors such as the adenovirus-associated viruses. etc.

III - Uses of the Immunogenic Compositions and Vaccines

The immunogenic compositions and the vaccines may be used advantageously for immunizing human beings of the female sex (prepubescent girls and women of reproductive âge) in the context of prophylactic therapy of prcgnancy-associated malaria.

Conséquent!y, the invention also relates to methods of treating or preventing pregnancy-associated malaria. In particular, the invention supplies a method for inducing a protective immune response against Plasmodium falciparum in a human being of the female sex, said method comprising a step of administering an effective amount of an immunogenic composition or of a vaccine described here. The invention also supplies a method of vaccinating a human being of the female sex against Plasmodium falciparum, said method comprising a step of administering an effective amount of a vaccine, in particular a DNA vaccine or a protein vaccine described here.

In certain preferred embodiments, a method of treating or preventing pregnancy-associated malaria is characterized in that it induces, in human beings of the female sex, antibodies that prevent the adhesion of érythrocytes infected by Plasmodium falciparum to the placenta! receptor CSA.

In the methods of treating or preventing pregnancy-associated malaria according to the invention, the immunogenic composition or vaccine may be

2?^ administered b y any suitable method (oral, parentéral, mucosal route). In certain embodiments. a DNA vaccine is administered by the ml camuse u kir. intradennal. or mucosal route. In other embodiments. a protein vaccine is administered for example by the intravenous. subeutaneous. inlradermal, oral, topical or systemic route.

An immunogcnic composition or a vaccine according to the invention may be administered in a single dose or in several doses. A person skilled in the art will bc able to déterminé the effective dose of immunizing protein or of DNA lo be used in each immunization or vaccination protocol.

IV - Kits

The présent invention also relates to a kit for prophylaxis against pregnancyassociatcd malaria. More specifically, the kit comprises materials useful for carrying out vaccination by the method of the invention. In general, a kit comprises a combination, an immunogcnic composition or a vaccine according to the invention, and instructions for carrying out vaccination against pregnancy-associatcd malaria. Optionally, the kit may further comprise means for perfomiing vaccination.

In certain embodiments, a kit according to the invention is configured in such a way that the components of a combination according to the invention are supplied separately (for example in different containers). Such a configuration allows both simultaneous administration and sequential administration of the components of the combination. Here, simultaneous administration means administration of the components, together or separately, at approximately the same time (for example, at an interval of 5, 10, 15 or 30 minutes from onc other). Here, sequential administration means administration of the components separately and at different times (for example at different times on the same day, or with an interval of one or more days).

The kit may comprise reagents or solutions for preparing the composition to be administered. The various components of the kit may be supplied in solid form (for example in lyophilized form) or in liquid form. A kit may optionally include a container containing each of the reagents or solutions, and/or containers (test tubes, bottles, etc.) for carrying out préparation of the composition lo be administered.

Finally, a notice in the form prescribed by a government agcncy regulating the sale and use of pharmaceutical products may be included in the kit.

Unless they arc defined otherw ise. ail lhe technical and scicnlific ternis used herc hâve the saine meaning as that commonly undersiuod by an ordinary specialist in the field to which this invention bclougs. Moreover. ail the publications, patent applications, ail the patents and ail other référencés mcntioned heie are incorporated by reference.

The following examples and the figures arc presented for illusirating certain embodiments of the procedures described above and must in no case be regarded as a limit to the scope of the invention.

Examples

The following examples describe certain embodiments of the présent invention. However, it is understood that the examples are only presented for purposes of illustration and they do not limit in any instance the scope of the invention.

It has been demonstrated that antibodies to constructs corresponding to a région of the N-tenninal portion of VAR2CSA of a single parasitic variant can only inhibit 70% of the adhérence of isolâtes from prégnant women to CSA (Bigey et al., J Infect Dis., 2011, 204: 1125-1133; Bordbar et al.. Vaccine, 2012, 30: 1343-1348). The inventors had previously identified a dimorphic région in the DBL2x domain of VAR2CSA (Sander et al., PLoS One, 2009, 4: e6667), leading to two distinct phylogenetic categories of type FCR3 and of type 3D7. In the study presented below, the inventors studied the prevalence of the types of dimorphic variant of DBL2x among the Plasmodium faiciparum parasites isolated from samples obtained from prégnant women in Bénin and the capacity for inhibiting the adhesion of spécifie antibodies induced against the NTS-DBLlx-Idl-DBL2x région of each serotype on the infected érythrocytes isolated from prégnant women.

Methods and Materials Used

Plasmodium faiciparum isolâtes. Prégnant women were only included in the présent study after their written consent had been obtained. The study was approved by the Consultative Committee of Deontology and Ethics of the IRD (Institut de Recherche pour le Développement: Research Institutc for Development), and the ethics committee of the health sciences faculty (Univcrsity of Abomey-Calavi, Bénin). Ail the procedures used in the présent study comply with European and

National régulations, l he procedure for anima! immunization compiles with the directive of FEl.ASA (Fédération of Laboratory Animal Science Associations) and was approved by the Et hies Committee with respect to animal experiments affilia lcd to the Descartes Universitv. Paris.

P/asniodiuin falciparnm isolâtes. The field samples of infectcd érythrocytes were obtained from prégnant women at the Suru Lérc maiernity hospital in the Eastern région of Cotonou. The study site is characterized by hyperendemie malaria in the lagoon région and a high rate of malaria transmission with two peaks corresponding to the two rainy seasons (Akogbeto et al.. Parasilologia, 1992. 34: 147-154). Periphera] venous blood was collected in Vacutainers containing the anticoagulant citrate-phosphate-dextrose-adenine (CPDA). during prénatal visits and at the moment of childbirth. The érythrocytes were separated from the plasma and washed with RPMI 1640 (Lonza). 200 pL of red blood cell residues were homogenized in 10 volumes of TRlzol reagent (Invitrogen) and frozen at -80“C until the moment of extraction of the total RNA or frozen at -20°C until lhe moment of extraction ofthe total DNA. The residues ofthe érythrocytes infccted by parasites at the ring (or young trophozoite) stage were immediately eultured in vitro to obtain aged trophozoites as described previously (Trager et aL, Science, 1976, 193: 673675). Briefly, the isolâtes were eultured in containers containing RPMI 1640 with addition of HEPES and L-glutamine (Lonza Biowhittaker), 0.3 g/L l-glutamine, 0.05 g/L gentamicin, 5 g/L Albumax. The culture vessels were placed under an atmosphère of a mixture of 92.5% Ni, 2% O₂ and 5.5% CO;> and were incubated at 37°C for not more than 48 hours before the assays. The parasitic laboratory strains FCR3, HB3 and NF54 were eultured in O+ érythrocytes and were selected after several panning steps for the BeWo cell line as described previously (Haase et al., Infect. Immune., 2006, 74: 3035-3038).

Extraction of DNA and msp Genotyping. The DNA was extracted from 100 pL of blood sédiment with the Gene J et Genomic Purification kit (Fermentas) according to the manufacturées recommendations. The genes mspl and nisp2 were amplified by nested PCR using spécifie primers (Snounou et aL, Trans R Soc Trop

Med Hyg, 1999, 93: 369-374). The multiplicity of infection (MOI) was determined for each sample.

Extraction of RNA, Synthesis o f cDNA and Genotyping ofthe l)BL2x. The RNAs were extracted from froz.cn samples prescrved in TRlznl reagcnl (Invitrogen) according to the manufacturcr's recommendations. The total RNAs were treated with DNAse l (Invitrogen) for 15 minutes at room température according to the ma nu facturer's recommendations to remove any potential contamination by genomie DNA (gDNA). Absence ofgDNA in the samples of RNA was continued by absence of amplification after 40 cycles of real-time PCR with primers targeting the housekeeping gene seryi-tRNA synlheutse (Salanti et al,. Mol Microbiol. 2003. 49: I79-191). Reverse transcription of the RNA (without DNA) was performed with Thermoscript (Invitrogen) and random hexameric primers for I hour at 50^l,C in a total volume of 20 pL according to the manufacturées recommendations.

For genotyping of the dimorphic sequence motif (DSM) in the DBL2x domain of VAR2CSA of the Plasmodium Jalciparum isolâtes (Sandcr et al., PLoS One, 2009, 4: e6667), this domain was selected for amplifying the cDNA of each îsolate with a primer pair (5'-TTAYCCCCAAGAACACA-3' and 5'TTTTAAATTTTTTCCATGAA-3'). The reactions were carried out with highfidelity Fusion Taq Polymerase (to obtain higher yields and lower mutation rates) in the following conditions: 94°C for l minute, followed by 35 cycles at 94°C for 30 seconds, 50°C for 30 seconds and 68°C for 50 seconds, and a final extension at 68°C for 10 minutes, The PCR products were digested with the restriction enzymes BstCl (whîch eut the DSMs of type FCR3) and HppyI881 (which eut the DSMs of type 3D7) for l hour at 50^cC and 37°C, respectively. The digested products were separated by electrophoresis on l .5% agarose gel.

Production ofthe antibodies and préparation of the IgGs. The fragment NTSDBLlx-Idl-DBL2x and the entire sequence of the optimized var2csa gene of the parasite lines FCR3 and 3D7 were used for producing spécifie antiVAR2CSA IgGs, by DNA vaccination as described previously (Bîgey et al., J Infect Dis., 20II, 204: Il25-Il33). Briefly, the DNA sequences were cloned into a vector derived from pVaxl and fused to the signal sequence mEPO as described previously (Trollet et al., Infect Immune., 2009, 77: 2221-2229). Immunizations were performed on female Swiss mice aged 6 weeks and on New Zcaland rabbits (Janvier-France). The mice were anesthetized by intraperitoneal injection with a ketamine-xylazine mixture and in the case of the rabbits, injection was carried out at 5 sites along each longissimns dorsi muscle. DNA transfcr was performed by transcutonemis electric puises applied b y iwo sluinlcss steel électrodes placed at each injection site. The animais were immunized on days 0, 30 and 60. and the antisera were collected on days 0 and 15 after each immunization. The total IgGs were purified in the final mouse or rabbit sérum sample on a Hi-Trap column on which the corresponding rccombinant protein had been coupled according to the manufaeturer’s recommendations (GE Healthcare).

Flow cylometry. The react î vi tv of the IgG s purified toward the surface of the érythrocytes infected by Plasmodium falciparum was analyzed by llow cytometry (FACS Calibur) as described previously (Magistrado et al.. Vaccine, 2011. 29: 437443). B ri et! y, the infected érythrocytes adhering to CSA (FCR3-Bcwo and HB3Bewo) and the parasitic field isolâtes were enriched by exposure to a high-intensity magnetic field (VarioMACS and CS columns, Miltenyi). For each assay, 2x10’^ infected érythrocytes were labeled with ethidium bromide, and exposed sequentially to a rabbit IgG, and then to an anti-rabbit antibody conjugated to F1TC (lnvitrogen). The data were acquired and analyzed, and the médian fluorescence intensity (MF!) was detennined. The labeling of the surface ofVAR2CSA is regarded as positive if the MFI ratio is greater than 1.2 (MFI with IgG post-immunization on day 75 / MFI with IgG pre-immunization on day 0) as described by Magistrado et al. (Vaccine, 2011,29:437-443).

Adhesion inhibition assays. The capacity of the antibodies for inhibiting adhesion of the infected érythrocytes to chondroitin sulfate (CSPG) was detennined by an assay in Pétri dishes described previously (Bigey et al., J Infect Dis., 2011, 204: 1125-1133). Briefly, Pétri dishes (Falcon 351029) were covered with 100 x 15 mm spots of 20 pL of 5 mg/mL CSPG-Decorin (Sigma) or of 10 pg/mL of bovine sérum albumin (BSA) diluted in PBS. The spots were incubated ovemight at 4C in a humid chamber. Each spot was blocked with 3% BSA in PBS for 30 minutes at 37C. Suspensions enriched with érythrocytes infected with aged trophozoites were obtained by filtration on a magnetic column (VarioMACS, Miltenyi). The suspension, the parasite density of which was adjusted to 20% in lxlO⁵ cells, was then blocked for 30 minutes at room température (RT) in 3% BSA solution diluted in RPM1. The érythrocyte préparation was incubated with purified IgGs (0.25 mg/mL) or 500 pg/mL of soluble CSA (Sigma), and the cells were then left to adhéré for 15 minutes at room tempéraiurc on Pétri dishes prccoated with different ligands. The nonadhering cells were removed by an automatic washing system. The spots were fixed with l.5% of glutaraldchyde in PBS. stained with Giemsa solution and lhe adhering infected érythrocytes were countcd using a microscope.

Legcnds of the figures

Fig. I is a graph showing lhe distribution of the dimorphic sequence signatures of the DBL2x région of VAR2CSA among the parasites infecting prégnant uomen. The dimorphic sequence motif (DSM) in DBL2x which dislinguishes the allèles of VAR2CSA in two subgroups (types 3D7 and FCR3) were genotyped in parasites 10 collected from prégnant wotnen in Bénin. The proportions of type 3D7 (white histograms), type FCR3 (shaded histograms) and of mixture of the two génotypes (black histograms) arc shown. The multipliciîy of infection (MOI) is stated for each category.

Fig- 2 îs a graph showing the récognition of VAR2CSA expressed in field 15 isolâtes by the IgGs directed against the various constructs of Var2CSA. The capacity for récognition of VAR2CSA expressed by the IgGs induced against the constructs of VAR2CSA (NTS-DBL2X/3D7 and NTS-DBL2X/FCR3) and against the entire extracellular domain of VAR2CSA (NTS-DBL l -6e/FCR3) of the FCR3 strain was studied on field isolâtes by flow cytomctry. The parasites were separated 20 as a function of their DSM category (DSM 3D7, DSM FCR3 or DSM Mixture FCR3/3D7). The bars indicate mean value of the MFI ratio (MFI corresponding to the reactivity of the IgG of the hyper-immune animal / MFI correspondîng to the reactivîty of the IgG of the same animal pre-immunization.

Fig. 3 comprises two graphs showing the adhesion inhibiting activity of IgG induced against each of the two 'sérotypes' of NTS-DBL lx-Idl-DBL2x on field isolâtes. The adhesion inhibiting activity was evaluated on 18 Plasmodium falciparum isolâtes freshly obtained from prégnant women. The data are presented in the form of percentage inhibition for each antibody normalized by the value of inhibition of CSA (used as a reference of maximum inhibition of adhesion). The histograms are presented for each of the types of DSM detected in the isolatc: □ for the DSMs of type 3D7, ® for the DSMs of type FCR3, and b for the DSMs of a mixture of the two génotypes.

Fig. 4 is a stries of three graphs show ing the inhibition profile of the antibodies on selected and adapted strains thaï bind to CSA. The strains FCR3. HB3 and NF54 selected on Bewo cells (FC'R3-Bewo. IIB36-Bc\vo and NF54-Bcwo) were used for evalualing the capacity of the induced antibodies for inhibiling the adhérence of the 3 laboratory strains to CSPG. The in vitro functionality of the mixtures of antibodies was also evaluated. The values presented are normalizcd with the value for inhibition ofCSA.

Fig. 5 is a représentation of the phylogenclic relations between the sequences covering the région 1D1-DBL2X of VAR2CSA generated from the cDXA of I23 parasitic isolâtes from prégnant women. The phylogenetic tree illustrâtes the phylogenelic relation between the different sequences. with the groupings of the dichotomie variants defined in the DBL2X that discriminâtes the clusters 3D7 and FCR3 and the groupings of a new cluster whose identity is defined in the ldi région of VAR2CSA. The bootstrap values are indicated at each principal bifurcation.

Results

Détermination of the Multiplicity of Infection and Molecular Typing of var2csa among the Parasites Isolated from Prégnant Women. In total, 122 parasites were collected, and it was determined by genotyping of msp-2 that the multiplicity of infection (MOI) is 3.10 distinct génotypes per isolate with a range from l to 7. A PCR-RFLP method was developed and implemented on ail the isolâtes to study the distribution of the two major variants of var2csa among the field isolâtes from Bénin. Analysis of the dimorphic région of var2csa in the isolâtes from prégnant women revealed the presence of at least one of the two dimorphic variants described by Sander et al. (PLoS One, 2009, 4: e6667) in ail the parasites. The transcripts of DSM of type 3D7 were found in 54 isolâtes (44%) and the transcripts of DSM of type FCR3 were found in 47 isolâtes (39%) (Fig. I). In 21 isolâtes (17%), both types of transcripts were detected, suggesting a mixture of génotypes. No différence was observed between the MOI values of the isolâtes of the groups determined after typing of var2csa (the MOI values determined are 3.05 in the isolâtes expressing type 3D7, 2.98 in the isolâtes expressing type CFR3 and 3.6 in the isolâtes with a mixture of both DSM signatures).

IgG Specifically Induced against the NTS-DBLlx-ldl-DBL2x Région of the

FCR3 and 3D~ Lines recognize ail lhe Isolâtes front Prégnant Jï’o/nen. The récognition level of VAR2SCA expressed on lhe surface of lhe érythrocytes was estimated immunologicallv on 47 érythrocytes infected by Plasmodium fidciparnnt 5 isolated from prégnant wonien. Among these isolâtes, il was found thaï 16 ot'lhem bave the DSM of type 3D7. 21 bave lhe DSM of type FCR3 and a mixture of génotypes was detectcd in 10 isolâtes, The infected érythrocytes were labeled and cvaluated by flow cytomctry using purified IgGs from sera of vaccinated animais, The anti-NTS-DBL l x-ld I-DBL2x IgGs spécifie to the var2csu variants of 3D7 and JO FCR3. and the IgGs induced against the entire extraccllular domain of VAR2CSA were used in this experiment. The IgGs induced against the entire extraccllular domain of VAR2CSA were used for estimating the absolute récognition level of the native VAR2CSA protein expressed on the surface of the infected érythrocytes. A high JeveJ of reactivity was observed with antibodies induced against the entire 15 extracellular domain of VAR2CSA compared to the antibodies directed against the

NTS-DBLI x-kll-DBL2x domain of VAR2CSA whatever the origin of the latter (P < 0,05). However, no significant différence was observed between the récognition levels of the VAR2CSA native protein by the anti-NTS-DBLIx-Idl-DBL2x IgGs induced against the FC R 3 variant and against the 3D7 variant (P = 0.59). The 20 inventors then investigated whether the surface récognition of the antibodies was influenced b y the génotype of var2csa. The isolâtes were divided up as a function of the type of DSM of their var2esa. The data of the infected érythrocytes labeled by each antibody were separated as a function of the DSM of the isolâtes. No signîficant différence was observed at the level of récognition of VAR2CSA by each antibody 25 tested whatever the type of DSM (Fig. 2).

Adhesion Inhibition Properties of the anti-NTS-DBL Ix-Idl-DBL2x IgGs Spécifie of the FCR3 and 3D7 Variants. Analysis of the inhibitory activity of the antibodies was also performed on 18 samples of infected érythrocytes binding to CSA obtained from prégnant woinen in Bénin, Typing of the DSMs of these isolâtes 30 revealed that 6 of them (33%) express the DSM of type 3D7 of var2csa; 8 (44%) express the DSM of type FCR3 of var2csa', and 4 (22%) are mixtures of the two types (Fig. 3).

Soluble CSA was used ns reference of maximum inhibition of adhesion. Tlie degree of inhibition ofthe parasites by the anti-NTS-DBLlx-ldl-DBl.2x antibodies ofthe FCR3 and 3D7 variants was nonnalized to the inhibition of CSA. The mean value of lhe inhibitory activity of the antibodies on ail the isolâtes îs 80% [interquartilc range: 50.8 - 100] for the anti-NTS-DBL l x-ld l-DBL2x antibody of the FCR3 variant and 97% [interquartilc range: 55.3 - 100] for the anti-NTS-DBL lxldl-DBL2x antibody of the 3D7 variant. Different modes of inhibition were observed with the antibodies depending on the types of var/csci ofthe isolâtes. 16 of the 18 isolâtes tested (89%) were eiïeclively inhibited by the antibodies induccd against the NTS-DBLI x-ld l-DBL2x domain of the FCR3 variant (43-100%) whereas a strong inhibitory activity of the antibodies induced against the NTSDBLlx-ldl-DBL2x domain of the 3D7 variant (50-100%) was observed on 15 of the 18 isolâtes (84% tested). However, it appears that the activitics of the two types of antibodies on the various isolâtes tested differ from one another. The inhibitory effect of one of the two types of antibodies may be wcak in one îsolate whereas the antibodies induccd against the other serotype may hâve a strong inhibitory effect on the same parasite. For example, the isolâtes OPT144 and OPT161 arc not inhibited by the antibodies induced against the FCR3 serotype, and interestingly, these isolâtes bearing the DSM of type 3D7 were strongly inhibited by the spécifie IgGs of the NTS-DBLlx-Idl-DBL2x domain of the 3D7 variant. A similar profile was obtained with the isolate OPT105, which is more strongly inhibited with the spécifie IgGs of the homologous DSM serotype of the FCR3 variant and less well inhibited with the anti-NTS-DBL !x-Idl-DBL2x IgGs spécifie to the 3D7 variant. Converse!y, in two isolâtes with 3D7 serotypes (OPT127 and OPT141), better inhibition was observed with antibodies induced against the opposite FCR3 serotype. Generally, complementarity of inhibition was observed on the field isolâtes, with a substantial increase in the extent of inhibition of adhérence of the field isolâtes to CSPG, now reaching 100% of the isolâtes currently inhibited. Moreover, 8 of the 10 isolâtes (80%) with a DSM of type 3D7 of var2csa were inhibited with anti-NTS-DBLlxldl-DBL2x antibodies induced against the homologous DSM variant whereas ail the isolâtes with the DSM of type FCR3 of var2csa were effectively inhibited by the anti-NTS-DBLlx-Idl-DBL2x IgGs spécifie to the FCR3 serotype.

The capacity of the anlibodics for inhibiting adhérence ol infected crytlwtcs expressing VAR2CSA to CSA and the behavior ol'tlie mixture ofthe two aniiàtlics induced against the NTS-DBl.!x-kll-DBL2x domain of VAR2CSA were studiion lhe laboratory strains and strains selected by Bewo: FCR3 and HB3 (which tec a DSM of type FCR3) and NF54 (which has a background of type 3D7).i|igh inhibitory activity was obtained with antibodies to infected érythrocytes bclon^ to the liomologous DSM. The anti-NTS-DBLlx-ldl-DBL2x antibodies induced aginst the FC'R3 variant completcly (100%) inhibit the adhérence of the FCR23 an<lB3 lines to CSA (Fig. 4). However. only partial inhibition of these lines (20% was observed with the anti-NTS-DBLlx-ldl-DBL2x IgGs induced against thcîD7 variant, which moreover strongly inhibit the adhérence ofthe NF54 parasite lofSA. Moreover, the mixture of the anti-NTS-DBLlx-ldl-DBL2x IgGs ofthe FCRSlinc and ofthe 3D7 line conserve the praperty of completcly inhibiting the adhere&c of ail the infected érythrocytes to CS PG.

Identification of a third cluster. The sequence variations of the NTS-DJLlxldl-DBL2x domain of the VAR2CSA protein of the transcript of 123 paasitic isolâtes from prégnant women in Bénin were analyzed. The total RNA was exlnctcd from the freshly collected parasites, and the cDNA was synthesized. The vjr2csa gene was amplified from the cDNA using a high-fidelity enzyme (Fusion), and universal primera were used. Ten (10) clones were sequenced for each isolate and the sequences were generated by multiple alignment of the protein and nucleic acid sequences.

The results of the analyses clearly demonstrated ségrégation of the parasitic variants in the ldi région of VAR2CSA. This new dichotomy domain, which has not been described hitherto, is located in a critical région of the VAR2CSA protein that is essentîal for its CSA binding properties. This very clean ségrégation of the sequences will hâve implications in the combination of a limited number of variants required for an optimal vaccine based on VAR2CSA.

The sequence alignments led to the establishment of two consensus sequences représentative of the ldi interdomain of VAR2CSA. The iîrst, 1D1A or SEQ ID NO: 11 corresponds to the recently identified cluster, and the second, ID1B or SEQ ID NO: 12 corresponds to the other group of sequences.

Conclusions

This work démon strates thaï the NTS-DBL2X région ot'VAR2CSA contains conserved an ti-adhérence épitopes and indicates that the development of an effective vaccine based on VAR2CSA will require combination of a restricted number of 5 VAR2CSA variants. Tbc combination of the three major variants of VAR2CSA used in this work will be essential for devcloping an effective vaccine against placental malaria.

Claims

Claims (4)

1. I. A combination of at least two isolated or purified polypeptides, tlie first isolated or purified polypeptide consisting of the NTS-DBLI x-kl I-DBL
2. 2x région or the ldl-DBL2x région of the VAR2CSA protein of a first parasitic 5 family of Plasmodium falciparum, and the second isolated or purified polypeptide consisting of the NTS-DBLlx-ldl-DBL2x région or the kilDBL2x région of the VAR2CSA protein of a second parasitic family of Plasmodium falciparum. for use in the treatment or prévention of pregnancyassociated malaria.

   10 2. The combination for the use as claimed in claim l, characterized în that the first family is the parasitic family of Plasmodium falciparum whose VAR2CSA protein is characterized by an ldi interdomain that has as sequence the consensus sequence SEQ ID NO: 11 or that is encoded by the consensus sequence SEQ ID NO: 13, and the second family is the parasitic 15 family of Plasmodium falciparum whose VAR2CSA protein is characterized by an ld l interdomain that has as sequence the consensus sequence SEQ ID NO: 12 or that is encoded by the consensus sequence SEQ ID NO: 14.
3. 3. The combination for the use as claimed in claim 2, characterized in that the second parasitic family of Plasmodium falciparum comprises the parasitic

   20 line FCR3 and the parasitic line 3D7.
4. 4. The combination for the use as claimed in 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 région or the IdlDBL2x région of the VAR2CSA protein of the parasitic family of

   25 Plasmodium falciparum whose VAR2CSA protein is characterized by an Idl interdomain having as sequence the consensus sequence SEQ ID NO: 11, the second isolated or purified polypeptide consisting of the NTS-DBLlx-IdlDBL2x région or the ldl-DBL2x région of the VAR2CSA protein of the FCR3 line, and the third isolated or purified polypeptide consisting of the 30 NTS-DBLlx-Idl-DBL2x région or the Idl-DBL2x région of the VAR2CSA