OA16305A - Vaccines against pregnancy-associated malaria. - Google Patents

Abstract

The present invention relates to specific regions of the N-terminal portion of the VAR2CSA protein and to the use of such specific regions in the prevention of pregnancy-associated malaria. The invention also provides immunogenic compositions and vaccines that are useful for preventing malaria in pregnant women.

Description

The présent invention relates to the use of spécifie régions of the N-tcrminal portion of the VAR2CSA protein in the prévention or treatment of pregnancyassociated malaria.

Background of the Invention

Malaria is the most frequent parasitic infectious disease in the woild. It is caused by a eukaryotic microorganism of the Plasmodium genns which is transinitted through bit ing b y a female mosquito (Anupheles). Several species of Plasmodium can infect human beings, but Plasmodium fait iparum is the most frequent and most 15 pathogenic species and the species that is rcsponsible for dcadly cases. Once introduced în the blood, the parasite infects hepatic cells, in which it proliférâtes, before circulating again in the blood and invading red blood cells (érythrocytes). Malaria affects about a hundred co un trie s in the world, in particular poor tropical régions of Africa, Asia and South America, Africa being by far the most affected 20 continent. The World Health Organization estimâtes that malaria is responsible for 225 millions of cases of fever and approximately one million dcaths annually (World Malaria Report, WHO, 2010). Currently available means of fîghting against malaria infection include anti-malaria drugs (in particular chloroquine and quinine) and action against mosquitoes, vectors of the parasite. However. the situation is ail the more 25 worrisome that for several years, the parasites bave developed increased résistance to drues and mosquitoes hâve developed résistance to insecticide. Today, there are no vaccines available against malaria.

Malaria affects mainly children of less than 5 years of âge and prégnant women. in particular primigravidae (i.e., women who are prégnant for the first time). Prégnant 30 women are particularly vulnérable because the placenta constitutes a target where (X parasites can accumulate. In prégnant women, malaria infection can cause a large variety of damaging effects: spontaneous abortion, early delivery, low weight at birth. congénital transmission, and néonatal death. In the African régions where malaria is endetnic, 3 to 5% of newborn deaths can be imputed to pregnancy-associated malaria.

Furthermorc, it is also a real danger for the mother who can suffer from sometinies severe. or even deadly, anaemia,

Today prévention of malaria in prégnant women is achieved by préventive administration of sulfadoxine/pyrimethamine (Cot el al., Br. Med. Bull., 2003, 67: 137-148). However, this intermittent treatment cannot provide a prévention against 10 malaria during the entire pregnancy; firstly because administration of the drugs only takes place from the 20^lh week of pregnancy (the tératogénie risks during embryogenesis being too high); secondly because the treatment involves two curative doses of sulfadoxine/pyrimethamine administered at one month interval, which only provides partial médicinal protection; and third ly because the efficacy of I5 sulfadoxine/pyrimethamine is very strongly decreasing in ail malaria endemic zones due to a rise in parasite résistance (Cot el al., Am. J. Trop. Med. Hyg., 1998, 59: 813822; WHO/HTM/MALZ2005J 103. Geneva: World Health Organization; ter Kuile e! al., JAMA, 2007, 297: 2603-2616; Mockenhaupt ei al., J. Infect. Ois., 2008, 198: 1545-1549; Briand el al., J. Infect. Dis., 2009, 991-1001; Hanington el al., Proc. Natl.

Acad. Sci. USA. 2009, 106: 9027-9032). Drugs are currently tested in this context, and numei'ous efforts are focuscd toward the development of a vaccine against placenta! malaria. The possibility of vaccinating prégnant women or prepubertal girls would offer several obvtous advantages over the sulfadoxine/pyrimethamine treatment, since the préventive protection would be temporally extended, and 25 probably of higher quality.

One of the contemplated vaccinal strategies to flght against pregnancyassociated malaria is to re-create the natural protective immunity. Indeed, the clinical severity of malaria caused by Plasmodium faleipanim is, at least partly. linked to alterations undergone by infected érythrocytes. These alterations aie induced by 30 proteins of the parasite that are exported to the surface of érythrocytes during the phase of development in blood. Some of these surface proteins of the PfEMPI (Plasmodium falcipartim Erythrocyte Membrane Protein 1) family, confcr novcl cytoadherence properties to infected érythrocytes. The infected érythrocytes bind to the internai walls of blood vessels. thereby becoming unavailable foi' transport towards purging organs of the immune System, whose rôle is to destroy cells recognized as abnormal. In pregnancy-associated malaria, infected érythrocytes adhéré to chondroitin sulfate A (CSA:. a sulfated glycosaminoglycan présent in the placenta. After several pregnancies, women acquire protective antibodies that block this adhérence, One vaccinal strategy is to re-create this protective immunity by blocking the attachment of infected érythrocytes to the placenta.

The VAR2CSA protein. one ofthe members ofthe PfEMPl family, is ciirrently the object of numerous research projects with the goal of developing a vaccine specifically adapted to prégnant women (Tuikue Ndam et al., J. Infect. Dis,, 2005, 192: 331-335; Chia et al., J. Infect. Dis., 2005, 192: 1284-1293; Tuikue Ndam el al., J. Infect. Dis., 2006, 193: 713-720; Dabiback el al., PLoS Pathogens, 2006, 2: 10691082; Badaut et ai., Mol. Biochem. Parasitol.. 2007, 15:89-99; Khattab et al.. Paras i toi. Res„ 2007. 101: 767-774; Guitard et al., Malaria J„ 2008, 11:7-10; Gui tard et ai.. Malaria J.. 2010, 9: 165; Gangnard et al.. Mol. Biochem. Parasitol,, 2010, 173 : 1 15-122; Gnidehou et al., Mol. Biochem. Parasitol., 2010, 5( 10) : e 13105). Although these studies are rendered diffîcult by VAR2CSA polymorphism, Phase I trials aie nevertheless contemplated. Furthermore. the full-length extracellular domain of this protein has recent!y been expressed in a heterologous System (Srivastava et al., Proc. Natl. Acad. Sci. USA, 2010. 107: 4884-4889; Khunrae et a!., J. Mol. Biol., 2010,397: 826-834), and antibodies induced against this construct showed a very high antiadhesion IgG titer. However, technological constraints in the optimal production of such a large antigen question the use of full length VAR2CSA in vaccine development. Furthermore, the development of new vaccinal approaches will have to take into account the numerous immunodoniinani epitopes that do no induce ’hntiadherent” antibodies.

Therefore, it appears to be crucial to continue exploring and developing new strategies to fight and prevent pregnancy-associated malaria,

Summary of the Invention

In their study of the modulation of the immune response to Plasmodium falcipantm during pregiiancy, the présent invenlors have generated overlapping fragments of the sequence of the \'ar2csa gene from the FCR3 parasite strain. ρζ introduced these fragments into plasmids, and intramuscularly injected each of the plasmids obtained in mice and in rabbits. They hâve identified the N-term inal portion of VAR2CSA, and more specifically the sub-region consisting in the DBLIx domain, the Idl inter-domain and the DBL2x domain (i.e., NTS-DBLlx-ld I-DBL2x), as being 5 the région of VAR2CSA that contains epitopes capable of inducing in vivo production of antibodîes that block the binding ot' Plasmodium falriparum-ïnfected érythrocytes to CSA. They then produced a recombinant protein corresponding to that particular portion of VAR2CSA and confitmed. via protein vaccination, the observations made with genetic vaccination, f urther results hâve allowed the Idl-DBL2x région of the 10 VAR2CSA protein to be identified as the minimal antigenic région of VAR2CSA that is involved in the acquisition of protcctive immunity against placenta! séquestration taking place during pregnancy-associated malaria.

Consequently, in a first aspect, the présent invention relates to the use of polypeptides corresponding to and polynucleotîdes encoding spécifie régions of the 15 N-terminal portion of the VAR2CSA protein in the fight against placental malaria.

More specifically, the présent invention provides an isolated or purified polypeptide consisting of the NTS-DBLI x-ldl -DBL2x région of the VAR2CSA protein, or a biologically active fragment of the NTS-DBLI x-Id l-DBL2x région, for the treatment or prévention of pregnancy-associated malaria, wherein the biologically 20 active fragment comprises at least the Idl-DBL2x région ofthe VAR2CSA protein.

In certain embodiments, the NTS-DBL lx-ldl-DBL2x région has the sequence set forth in SEQ ID NO: l, or a homologous sequence thereof.

In certain embodiments, the Idl-DBL2x has the sequence set forth in SEQ ID NO: 2, or a homologous sequence thereof.

In certain embodiments. the isolated or purified polypeptide consists of the ld l DBL2x région of the VAR2CSA protein.

The invention also provides a fusion protein for use in the treatment or prévention of pregnancy-associated malaria. A fusion protein according to the invention consists of a polypeptide as described herein fused to a fusion partner 30 selected from the group consisting of maltose binding protein, signal sequence of the maltose binding protein, poly-histidine tag, S-Tag. glutathione-S-transferase, thioredoxin, β-galactosidase, streptavidin. dihydrol'olate reductase, pelB signal sequence, ompA signal sequence, signai sequence of alkaline phosphatase, green fluorescent protein (GF^;P). toxins. human growth hormone, interleukin-2 (!L-2). granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony 5 stimulating factor (G-CSF), c a Ici ton in. iritei feron-beta, interferon-alpha, glucagon like peptide I (GLP-l), glucagon like peptide 2 (GLP-2), PA toxin, parathyroid hormone (PTH(l-34) and ΡΊΉ(I-84)). butyrylcholinesterase. glucocerebrosida.se (GBA), and excndin-4,

The présent invention also provides an isolated or purified polynucleotide foi' l 0 use in the treatment or prévention of pregrtancy-associated malaria, the polynucleotide consisting of a sequence encoding a polypeptide or a fusion protein according to the invention and éléments necessary to the in vitro or in vivo expression of said polypeptide or fusion protein.

In another aspect, the présent invention provides a cloning or expression vector 15 comprising at least one polynucleotide described above, Cloning or expression vectors according to the invention allow the expression, in host cells of a polypeptide consisting of the NTS-DBLl x-ld I-DBL2x région of the VAR2CSA protein, or a biologically active fragment thereof comprising at least the ldi-DBL2x région of VAR2CSA. These vectors may be pliages, plasmids, cosmids, or viruses. The NTS20 DBLlx-ldl-DBL2x région, or biologically active fragment thereof, may be f'used to a fusion partner has described herein.

The invention also provides host cells (transformed or transfected) comprising at least on polynucleotide or at least one vector as described above. The host cells may be bacteria, yeast, insect cells or mammal cells.

ln another aspect, the invention provides an immunogenic composition comprising at least one pharmaceutically acceptable excipient or carrier and at least one member of the group consisting of polypeptides according to the invention, fusion proteins according to the invention, polynucléotides according to the invention, and vectors according to the invention. Preferably, such an immunogenic composition can induce antibodies that prevent adhérence of Plasmodium Jaliipanirn-infecteà érythrocytes to the placenta receptor CSA. A.

In a related aspect, the invention relates to vaccines against pregnancyassociated malaria. More specifically. the invention provides a DNA vaccine comprising a DNA, naked or formulated. comprising and expressing in vivo, a nucléotide sequence encoding a polypeptide consisting of the NTS-DBLlx-IdlDBL2x région of the VAR2CSA protein, or a biologically active fragment thereof. fused or not to one or more fusion partners, as described herein. The invention also provides a protein vaccine comprising a polypeptide or a fusion protein of the invention. Preferably, the vaccines according to the présent invention induce antibodies that prevent Plasmodium faltiparum-ïntected érythrocytes from attaching to the placenta receptor CSA. Vaccines according to the invention may further comprise an adjuvant.

In a related aspect, the invention relates to methods of treatment or prévention of pregnancy-associated malaria. More specifically, the invention provides a method for inducing a protective immune response against Plasmodium falcipartim in a female human being, the method comprising a stop of administering an effective amount of an immunogenic composition or vaccine according to the invention. The invention also provides a method of vaccinating a female human being against Plasmodium falciparum. the method comprising a step of administering an effective amount of a vaccine of the invention, in particular a DNA vaccine or a protein vaccine described herein. The methods of treatment or prévention of pregnancy-associated malaria are mainly intended to women in âge of bearing children (in particular postpubertal girls and priinieravidac women) and to prepubertal girls. In certain preferred embodiments, a method of treatment or prévention of pregnancy-associated malaria induces, in the female human being treated, the production of antibodies that prevent Plasmodium faleiparum-infecled érythrocytes from attaching to the placenta receptor CSA. In the methods of treatment or prévention pregnancy-associated malaria according to the invention, the immunogenic composition or vaccine may be administered by any suitable route.

These and other objects, advantages and features of the présent invention will become apparent to those of ordinary ski 11 in the art having read the following detailed description of the preferred embodiments. A

Brief Description of (he Drawing

Figure l shows the surface reactivity and anti-adhesion capacity of mice antisera to various VAR2CSA constructs. (.A) is a schematic représentation ofthe 13 overlapping VAR2CSA constructs made in a pVAXl dérivative vector. (B) Blood samples before rmmunization and from fui I bleeds at D75 were pooled for each group of 5 mice to constitute the pre-bleed pool (PbP) and immune pool (IP), respectively. The flow reactivity shown (black fi lied histograms) for each pool is defined as the Médian Fluorescence Intensity (ΜΓΙ) ratio (MH test / MPI négative control). The négative controls were stained only with secondary FiTC-conjugated anti-mouse antibody. Binding inhibition of infected érythrocytes to decorin (bovine CSPG) was measured using sérum in a l:5 dilution (white filled histograms). 'Phe degree of inhibition was defined as [1 - (bound infected érythrocytes with test sérum / bound infected érythrocytes without sérum)].

Figure 2 shows that NTS-DBL1x-ldl-DBL2x induces adhesion-inhibitory lgGs in different animal species. (Λ) Two rabbits intmunized with plasmids encoding the NTS-DBLlx-ldl-DBL2x of the FCR3 var2csa variant acquired anti-adhesion antibodies to both CFR3-Be\Vo and BB3-BeWo infected érythrocytes from the second immunization. (B) purifîed IgGs induced against the NTS-DBLlx-ldlDBL2x région and purifîed IgGs induced against DBL6e recognize native VAR2CSA on the suri ace of FCR3-BeWo infected érythrocytes. However, they did not recognized unselected FCR3 infected érythrocytes (data not shown). IgGs purifîed from animais before vaccination did not label the surface of FCR3-BeWo infected érythrocytes. (C) Purifîed anti-NTS-DBLlx-Idi-DBL2x IgGs specifically inhibit binding of FCR3-BeWo infected érythrocytes to CDPG.

Figure 3 is an SDS-PAGE1 of NTS l)BLlx-ld!-l)BL2x of VAR2CSA purifîed on a I mi. HisSelect (Ni²⁺), i mL Capto S HP (IFX), and I ml. of Heparin HP column (Hep), respectively. Samples of2.5 and 5 pg ofthe purifîed recombinant protein were loaded for yield comparison.

Figure 4 shows that expert mental ly induced or naturally acquired antibodies against NTS-DBLlx-Idi-DBL2x target common epitopes. (A) Anti-adhesion capacity in a dilution sériés of hyperimmune mice antisera on the binding of FCR3BeWo infected érythrocytes to CSPG. The proportion of infected érythrocytes binding to CSPG in the presence of the indicated dilutions of the D75 antiserum is shown compared to control binding without compétition. Antisera induced by DNA immunization with the full length (empty histograms) or NTS-DBL2x (stripped histograms) or by the baculovirus-expressed recombinant NTS-DBL2x (dotted histograms) were used. BSA îndicates the binding of infected érythrocytes to bovine sérum albumin. (B) Compétitive récognition of recombinant NTS-DBLJ x-ld IDBL2x between spécifie antibodies induced b y genetic immunization versus protein immunization. Sera from protein-vaccinated mice and the corresponding pre-bleed are the corn pet ing antibodies. D75 sérum front DNA vaccinated rabbit is used as non competing antibodies. (C) Compétitive récognition of recombinant NTS-DBLlx-ld I DBL2x between antibodies produced by genetic immunization in rabbits and naturally acquired in the plasma of malaria-exposed prégnant women from Bénin. The competing sera are: D?5 sérum from DNA vaccinated rabbit, and the corresponding rabbit pre-immune sérum. The non-competing antibodies are represented by a pool of Beninese multigravidae plasma.

Figure 5 shows pregnancy-specificity and parity-dependency of plasma IgG reactivity to VAR2CSA NTS-DBLlx-Idl-DBL2x recombinant protein. ELISA was carried out on plates coated with 0.5 mg/mL of PfAMAl (A) and recombinant NTSDBLlx-ld l-DBL2x domain of VAR2CSA (B). The IgG plasma levels are expressed as optical densities (OD) and are shown for unexposed prégnant French women (LJPFW. n=20), malaria-exposed Beninese men (EMB, n=20), malaria-exposed Sencgalesc children (ECS, n=20), malaria-exposed Sencgalese men (EMS, n=20), and two malaria-exposed prégnant women areas; Bénin [primigravidae, (EPGB, n=20) and multigravidae (EMGB, n=20)] and Sénégal [primigravidae, (EPG, n=20) and multigravidae (EMG. n=20)J.

Figure 6 shows that naturally acquired IgGs against VAR2CS/X NTS-DBLlxIdl-DBL2x target strain-transcendent anti-adhesion epitopes. Spécifie human IgGs to NTS-DBLlx-Idl-DBL2x were affinity-purified from a pool of plasma fiom I0 multigravid women that previously showed anti-adhesion capacities. The FCR3BeWo and HB3-BeWo infected érythrocytes were incubated with different concentrations (Ί2,5. 25 or 50 pg/mL) of the purified human anti-NTS-DBLlx-IdlDBL2x IgG and the actîvity was compared lo binding without competitor or soluble competing CSA. None of the infected érythrocytes bound to BSA. Y

Figure 7 shows the functional capacity of the antibodies targeting different portions of the N-terminal région of VAR2CSA. The graph shows the ability of immune sérum to spécifie constructs to inhibit binding to CSA by VAR2CSA expressing FCR3 and HB3 infected érythrocytes.

Figure 8 is a scheme showing the different portions of the NTS-DBI.lx-IdlDBL2x that hâve been tested to further icfine the important proteclive epitope région.

Detailed Description of Certain Preferred Embodiments

The présent invention generally relates to the use of spécifie régions of the extracellular domain of the VAR2CSA protein of the FCR.3 parasite line for I0 prévention and/or treatment of pregnancy-associated malaria.

I - NTS-DBLlx-Idl-DBL2x and Biologicalty Active Fragments thereof

NTS-l)FLIx-ldl-l)BL2x~derived Polypeptides and Polynucleotides

The présent invention relates to polynucleotides and polypeptides derived from the extracellular domain of VAR2CSA and involved in pregnancy-associated malaria.

and to their use in the treatment and/or prévention of pregnancy-associated malaria.

The var2csa gene has been isolated and sequeneed for several parasite strains, including FCR3 (CienBank Accession Number: AY372123'). The sequence of the corresponding VAR2CSA has been deduced (GenBank Accession Number: AAQ73926.1).

More specifically, the présent invention provides isolated polypeptides that consist of the NTS-DBLlx-Idl-DBL2x région of the VAR2CSA protein, or a biologically fragment thereof that comprises at least the Idl-DBL2x région of the VAR2CSA protein.

The terrn “isolated”, as used herein in reference to a polypeptide or polynucleotide, means a polypeptide or polynucleotide, which by virtue of its origin or manipulation is separated from at least some of the components with which it is naturally associated or with which it is associated when initially obtained. By “isolated”, it is alternatively or addîtionalIÿ meant that the polypeptide or polynucleotide of interest is produced, synthesized and/or purified by the hand of man.

U

The terms “protein”, “polypeptide” and polypeptide sequence” are used herein interchangeably. They refer to a sequence of amino acids (either in their neutral (uncharged) lorms or as salts, and either unmodified or modified by glycosylation, side chain oxidation. or phosphorylation) that are linked through peptide bonds. In 5 certain embodiments, the amino acid sequence is a full-length native protein. In other embodiments, the amino acid sequence is a smaller portion of the full-length protein. In still other embodiments, the amino acid sequence is modified by additional substituents attached to the amino acid side chains, such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical 10 conversions of the chains such as oxidation of sulfydryl groups. Thus, the term “protein” (or its équivalent terms) is intended to include the amino acid sequence of the full-length native protein, or a portion thereof, subject to those modifications that do not signifïcantly change its spécifie properties. In particular, the term “protein” encompasses protein isoforms, i.e., variants that are encoded by the same gene, but 15 that differ in their pl or MW, or both. Such isoforms can differ in their amino acid sequence (e.g., as a resuit of allelic variation, alternative splicing or lîmited proteolysis), or in the alternative, may avise from differential post-translational modification (e.g., glycosylation, acylation, phosphorylation).

The terms “fragment”, “portion” and “région” are used herein interchangeably.

When used herein in reference to a protein, they refer to a polypeptide having an amino acid sequence of at least 5 consecutive amino acid residues (preferably, at least about: 10, 15, 20, 25. 30. 35, 40. 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250 or more consecutive amino acid residues) of the amino acid sequence of the protein. The fragment of a protein may or may not possess a functional activity of the protein.

The term “biologically active”, as used herein to characterize a protein variant, analogue or fragment, refers to a molécule thaï shares sufficient amino acid sequence identity or homology with the protein to exhibit similar or identical properties to the protein. For example, a biologically active fragment of NTS-DBLlx-ldl-DBL2x is a fragment that retains the ability of the NTS-DBLlx-ldl-DBL2x région of VAR2CSA 30 to induce the production of antibodies that prevent adhérence of Plasmodium falciparum-infected érythrocytes to the placenta receptor CSA.

The terms “NTS-DBLIx-idl-DBL2x”. NTS-DBLI x-DBL2x and “NTSDBL2x” are used herein interchangeably. They refer to a N-temninal sequence (NTS) of VAR2CSA consisting of the following subdomains: Duffy-binding-like domain Ix (DBLlx), interdomain l (Id l ) and Duffy-binding-like domain 2x (DBL2x ) of 5 VAR2CSA. In certain embodiments, NTS-DBLI x-Idl-DBL.2x has the sequence corresponding amino acids 8 to 866 of VAR2CSA, i.e., has the sequence set forth in SEQ ID NO: l, or a homologous sequence thereof.

The term “Idl-DBL2x refers to a polypeptide consisting of the following subdomains: the interdomain i (Idh and Duffy-binding-like domain 2x (DBL2x) of 10 VAR2CSA. In certain embodiments, ldl-DBL2x has the sequence corresponding amino acids 392 to 866 of VAR2CSA, i.e., has the sequence set forth in SEQ ID NO: 2, or a homologous sequence thereof.

The term “homologous” (or “homology”), as used herein, is synonymous with the term “identity” and refers to the sequence simîlarîty between two polypeptide I5 molécules. When a position in both compared sequences is occupied by the same amino acid residue, the respective molécules are then homologous at that position. The percentage of homology between two sequences corresponds to the number of matching or homologous positions shared by the two sequences divided by the number of positions compared and multiplicd by 100. Generally, a comparison is 20 made when two sequences are aligned to give maximum homology. The optimal alignment of sequences may be performed manually or using softwares (such as GAP, BESTFIT, BLASTP, BLASTN, FASTA, and TFASTA, which are available on the NCBl site or in Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI). Homologous amino acid sequences share identical or similar amino 25 acid sequences. Similar residues are conservative substitutions for, or “allowed point mutations” of. corresponding amino acid residues in a reference sequence. “Conservative substitutions of a residue in a reference sequence are substitutions that are physical I y or functionally si mil ai to the corresponding reference residue, e.g. that hâve a similar size, shape, electric charge, chemical properties. including the ability to 30 form covalent or hydrogen bonds, or the like. Particularly preferred conservative substitutions are those fulfilling the criteria defined for an “accepted point mutation as described by Dayhoff ei cil. (Atlas of Protein Sequence and Structure”, 1978. Nat. Biomed. Res. Foundation. Washington, DC, Suppl. 3, 22: 354-352), l l

The présent invention also provides fusion proteins consisting of at least one polypeptide described herein fused to at least fusion partner.

The terms “fusion partner” and “fusion partner sequence” are used herein interchangeably, and refer to an amino acid sequence that confers to the fusion protein one or more désirable properties. Thus. a fusion partner may be a protein thaï improves the expression of the NTS-DBLl x-Id l-DBL2x région, or biologically active fragment thereof, in host cells during préparation of the fusion protein, and/or a protein that facilitâtes purification of the fusion protein, and/or a protein that increases the stabilitv (e.g., plasma stabiiity) of the fusion protein (compared to the stabiiity of the non-fused protein), and/or a protein that improves or facilitâtes administration of the fusion protein to the subject being treated, and/or a protein that increases the desired therapeutic effect (for example by increasing the immune and vaccinal response). and/or a protein exhibiting a désirable biological or therapeutic activity.

Fusion partners that can be used in the context of the invention include, but are not limited to, maltose binding protein, signal sequence of the maltose binding protein, poly-histidine segments capable of binding metallic ions, S-Tag, glutathioneS-transfcrase, thioredoxin, β-galactosidasc, streptavidin, dihydrofolate rcductase, pelB signal sequence, ompA signal sequence, signal sequence of alkaline phosphatase, green fluorescent protein (GFP), toxins such as, for example, E. Coli enterotoxin L.T or B-subunit thereof, a domain of tetanus toxin fragment C, choiera toxin or B-subunit thereof, CTAI-DD. Other fusion partners may be human growth hormone, an immunostimulating cytokine such as: interleuk.in-2 (lL-2), a growth factor such as granulocyte macrophage colony slimulating factor (GM-CSF). granulocyte colony stimulating factor (G-CSF), peptides or hormones such as: calcitonin, interferon-beta. interferon-alpha, glucagon like peptide l (GLP-l), glucagon like peptide 2 (GLP-2). PA toxin, parathyroid hormone (PTH( i-34) and PTH(l-84)), butyrylcholinesterase. ghicocerebrosidase (GBA), and exendin-4.

The présent invention also provides isolated polynucleotides for use in the treatment or prévention of pregnancy-associated malaria, the polynucleotides consisting of a sequence encoding a polypeptide or a fusion protein according to the invention and éléments necessary to the in vitro or in vivo expression of said polypeptide or fusion protein. Preferably, the éléments necessary to the in vitro or in A vivo expression of the polypeptide or fusion protein are operably Itnked to the polynucieotide sequence to be transcri bed.

The terms “nucleic acid sequence”. “nucleic acid”, nucleic acid molécule”, “polynucieotide” and “oligonucleotide” are used herein intejchangeably. They refer to a given sequence of nucléotides, modified or not, which defines a région of a nucleic acid molécule and which may be either under the l'orm a single strain or double strain DNAs or under the form of transcription products thereof.

The terms “éléments necessary to the in vitro or in vivo expression of the polypeptide” and “éléments necessary to the ni vitro or in vivo transcription of the polynucieotide” are used herein intejchangeably. They refer to sequences known in the ail that allow the expression, and optionally lhe régulation, of a polypeptide (or the transcription of the polynucieotide sequence encoding the polypeptide) in a host cell or in vivo. Such éléments include at least a transcription initiation sequence (also called promoter) and a transciiption tennination sequence that are fu net ion in a host cell or in vivo. The term “operably linked” refers to a functional link between the regulatory sequences and the nucleic acid sequence that they control.

Préparation of NTS-l)BLlx-Idl-l)l1l.2x-derived Polypeptides and Polynucleotides

The polynucleotides and polypeptides of the pj'esent invention may be prepared using any suitable method known in the art.

Techniques to isolate or clone a gene or a nucléotide sequence encoding a spécifie domain of a protein are known in the art and include, for example, isolation from genomic DNA, préparation from cDNA. or combination of these methods. Cloning a gene, or an acid nucleic sequence encoding a spécifie domain of a protein, from genomic DNA may be performed for ex ample using a polymerase chain reaction (PCR) or b y screening expression Ii brade s to detect cloned DNA fragments with identical structural characteristics (Innis et al.. “PCR: Λ Guide to Method and Application”, 1990, Academie Press: New York). Other amplification methods of nucleic acid molécules known in the art may be used, such as for example, ligase chain reaction (LCR), ligation activated transcription (l.AT) and Nucleic Acid Sequence Based Amplification (NASBA). It is also po>rible to use a chemical method of synthesis to préparé a polynucieotide sequence Chemical methods of synthesis of DNA or RNA strains are known to those skilled in the an, and involve the use of commercially available automatic synthesizers.

Methods to prépare polypeptides sequences 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.), I997, Academie Press: San Diego, CA) and recombinant methods (Sambrook et al., Molecular Cloning; Λ Laboraiory Manual, 2^ Ed., I989, Cold Spring I larbor Press: C.old Spring, NY) using host cells.

A recombinant method for the production of NTS-DBI.Ix-Idl-DBL2x is described in the Examples provided below.

Cloning or Expression Vectors

The présent invention also relates to cloning or expression vectors that allow expression of NTS-DBI.lx-Idl-DBI.2x, or biologically active fragments thereof, in host cells. More specifically, the présent invention provides cloning or expression vectors comprising at least one polynucleotide or one fusion protein described herein. '5 The cloning or expression vectors may be phages, plasmids, cosmids orviruses.

Host cells transformed or transfected with a polynucleotide or cloning or expression vector described herein are also encompassed în the présent invention. Such host cells may be bacteria, yeast, insect cells or mammal cells,

II - Inimunogenic Compositions and Vaccines

Polypeptides and polynucleotides of the invention are particularly suitable for use as drugs in the management of malaria in prégnant women. indeed as demonstrated in the Examples section, the polypeptides of the invention are antieenic régions of the VAR2CSA protein involved in the acquisition of protective immunity against the placental séquestration thaï takes place during pregnancy-associated malaria: and the polynucleotides of the invention encode these antieenic régions. They may be used as such, or under a modified form, as an inimunogenic composition or a vaccine.

A suitable modification of polypeptides according to the invention is conjugation. Conjugates according to the invention comprise at least one polypeptide 30 of the invention liked to a carrier. Conjugates may be obtained by coupling the polypeptide peptide to a physiological! y acceptable, non-toxic, natural or synthetic carrier via a covalent bound. The carrier may be selected to increase the immunogenic properties of the polypeptide.

Methods for the préparation of such conjugates are known in the art. For example, international application number WO 2006/124712 describes methods of préparation of conjugates comprising a plurality of ami génie peptides of Plasmodium falciparum linked to a protein carrier that improves the antigens immunogenicity.

Preferred carriers according to the invention include, but are not limited to, viral particles, lipids such as for example C16-CI8 lipids. polylysines, poly(DL-alanine)poly(Lysine)s, nitrocellulose, polystyrène microparticles, latex beads, biodégradable polymers, polyphosphoglycane microparticles, protein carriers such as OPMC (outer membrane protein coinplex of Neissena meningitidîs) or improved OPMC, BSA (bovine sérum albumin), TT (tetanus toxoid), ovalbumin, KLH (heyhole limpet hemocyanin), THY (bovine thyroglobulin), HbSAg and HBcAg of hepatitis B virus, rotavirus capside protein, protein LI of human papilloma virus, VLP (virus like particle) of types 6, I l and 16, tuberculin PPD (purified protein dérivative).

The polypeptides, fusion proteins, conjugales, polynucleotides and vectors of the invention may advantageous!y be used as therapeutic agents, in particular formulated as immunogenic compositions or vaccines.

imm mnogenie Compositions

An immunogenic composition according to the invention generally comprises at least one pharmaceutically acceptable carrier or excipient and at least one member of the group consisting of polypeptides described herein, fusion proteins described herein, conjugates described herein, polynucleotides described herein, cloning or expression vectors described herein. and any combination thereof. The term “pharmaceutically acceptable carrier or excipient” refers to a carrier medium which does not interfère with the effectiveness of the biological activity of the active ingredient(s) and which is not excessively loxic to the individual at the concentration at which it is administered. The term includes solvents, dispersion, media, coatings. antibacterial and antifungal agents, isotonie agents, and adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the ail (see for example Pi'miriglon's Pharmaceutical Sciences”,

E.W. Martin, I8^lh Ed.. 1990, Mack Publishing Co.: Easton. PA, which is incorporated herein by reference in its entirely).

The formulation of an immunogenic composition according to the présent invention may vary depending on the dosage and administration route selected. After 5 formulation with at least one pharmaceutically acceptable carrier or excipient, an immunogenic composition according to the invention may be administered under any form suitable for human administration, for example solid or liquid form. One ski lied in the art knows how to select carriers and/or excipients suitable to a given formulation.

Injectable préparations, for example stérile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents, and suspending agents. The stérile injectable préparation may also be a stérile injectable solution, suspension or émulsion in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 2,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P. and isotonie sodium chioride solution. In addition, stérile, fixed oils are conventionally employed as a solution or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid may also be used in the préparation of injectable formulations.

Stérile liquid carriers are useful in stérile liquid form compositions for parentéral administration.

Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of stérile solid compositions which can be dissolved or dispersed in stérile water or other stérile 25 injectable medium prior to use. Liquid pharmaceutical compositions that are stérile solutions or suspensions can be administered for example, by întravenous, intramuscular, intraperitoneal or subcutaneous injection. Injection may be via single push or by graduai infusion. Where necessary or desired. the composition may include a local anesthetic to case pain at the site of injection.

In order to pro Ion g the effect of an active ingrédient, it is often désirable to slow the absorption of the ingrédient from subcutaneous or intramuscular injection. Delaying absorption of a parenterally administered active ingrédient may be accomplished by dissolving or suspendine the ingrédient in an oil vehicle. Injectable depot forms are made by formine m iciO-encap.su lated matrices of the active ingrédient in biodégradable polymers such as polylactide-polyglycolide. Dépending upon the ratio of active ingrédient to polymer and the nature of the particular polymer 5 employed, the rate of ingrédient release can be controlled. Examples of other biodégradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the active ingrédient in liposomes or mîcroemulsions which are compatible with body tissues.

Liquid dosage forms for oral administration include, but are not limited to, 10 pharmaceutically acceptable émulsions, microemidsions. solutions, suspensions, syrups, élixirs, and pressurized compositions. In addition to the active principles, the liquid dosage form may contain inert diluents commonly used in the art such as, for example, water or other solvent, solubilising agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyi alcohol, benzyi 15 benzoate, propylene glycol, l ,3-butyienc glycol, di methyl form amide, oils (in particular. cotton seed, ground mit. corn, germ. olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol. polyethylene glycols, and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, suspending agents, preservatives, 20 sweetening, flavourîng, and perfuming agents, thickening agents, colors, viscosity rcgulators, stabilizers or osmo-regulators. Examples of suitable liquid carriers for oral administration include water (potcntially contaîning additives as above, e.g., cellulose dérivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols such as glycols) and their dérivatives. 25 and oils (e.g., fractionated coconut oil and arachis oil). For pressurized compositions, the liquid carrier can be halogenated hydrocaibon or other pharmaceutically acceptable propellant.

Solid dosage forms for oral administration include, for example, capsules, tablets, pills, powders, and granules. In such solid dosage forms. active ingrédients 30 may be mixed with at least one inert. physiologicalIy acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and one or more of: (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannital, and silicic acid:

(b) binders such as, for example, carboxymethylcellulose, alginates, gélatine, polyvinylpyrrolidone. sucrose. and acacia; (c) humectants such as gîycerol;

(d) disintegrating agents such as agar-agar. calcium carbonate, polato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarding agents such as paraffin; absorption accelerators such as quatemary ammonium compounds; (g) wetting agents such as, for example, cetyl alcohol and gîycerol monostearate; (h) absorbents such as kaolin and bentonite clay; and (i) lubricants such as talc, calcium stéarate, magnésium stéarate, solid polyethylene glycols, sodium lauryl sulphate, and mixtures thereof. Other excipients suitable for solid formulations include surface modifying agents such as non-ionic and anionic surface modifying agents. Représentative examples of surface modifying agents include, but are not limited to, poloxamcr 188, benzalkonium chloride, calcium stéarate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloïdal silicon dioxide, phosphates, sodium dodecylsulfate, magnésium aluminum silicate, and triethanolamine. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

In addition, in certain instances, it is expected that the inventive compositions may be disposed within transdermal devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the active ingrédient by either passive or active release mechamsms. Transdermal administrations include ail administrations across the surface of the body and the inner linings of bodily passage including épithélial and mucosal tissues. Such administrations may be carried out using the présent compositions in lotions, creams, foams, patches, suspensions, and solutions.

Transdermal administration may be accomplished through the use of a transdermal patch containing active ingrédients and a carrier that is non-toxic to the skin, and allows the delivery of the ingrédient for systemic absorption into the bloodstream via the skin. The carrier may take any number of forms such as créants and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid émulsions of either the oil-in-water or waler-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingrédient may be suitable. A variety of occlusive devices may be used to release the active ingrédient into the bloodstream such as a semi-permeable membrane covering a réservoir containing the active ingrédient with or wîthout a carrier, or a matrix containing the active ingrédient.

In certain preferred embodiments. the immunogenic compositions and vaccines of the invention may comprise orte or more adjuvants used in combination. Examples of suitable classical adjuvants include Montanide et/ou TAlum. Other suitable adjuvants include, but are not limited to, incomplète Freund’s adjuvant, QS2I, SBQS2, MF59, naL'l’, PHL. GpG DNA, calcium phosphate, dehydrated calcium sulfate, PLG, CT, LT B, CT/LT, AS02A, aluminium phosphate, aluminium hydroxide, monophosphoryl lipid A (MPL), a saponin. vitamin A, and varions water-in-oil émulsions prepared from biodégradable oils such as squalene and/or tocopherol, Qui! A, Ribi Detox, CRL-1005, L-121 and combinations thereof.

Immunogenic compositions and vaccines according to the invention may further comprise at least one antigen spécifie of preerythrocytic stages (CSP, TR AP, LSA-l, LSA-3, SALSA, STARP, EXP-l), asexual erythrocytic stages (MSP-l, MSP-3. AMA-I, EBA-175, GLURP, MSP-2. MSP-4, MSP-5, RAP-2, RESA, PtEMP-l, synthetic GPl toxin) or sexual erythrocytic sages (PfS25).

Vaccines, Protein Vaccines, DNA Vaccines

A vaccine against pregnancy-associated malaria according to the présent invention generally comprises at least polypeptide described herein, at least one polynucleotide described herein, or at least one cloning or expression vector described herein, and is used to induce, in treated subjects, antibodies capable of inhibiting the binding of infected érythrocytes to CSA. In particular, the invention provides a DNA vaccine (also called plasmid vaccine or polynucleotide vaccine) against placenta! malaria, The invention also provides a protein vaccine (also called polypeptide vaccine) against placenta! malaria.

Protein Vaccines

More specifically, the invention provides a protein vaccine comprising a polypeptide consisting of the NTS-DBLlx-ldl-DBL2x région of the VAR2CSA protein, or a biologically active fragment thereof comprising at least the Idl-DBL2x région of the VAR2CSA protein. In certain preferred embodiments, the NTS-DBLl xldl-DBL2x région has the sequence set forth in SEQ ID NO: l, or a homologous ΰζ sequence thereof. In certain preferred embodiments, the !dl-DBL2x région has the sequence set forth in SEQ ID NO: 2, or a homologous sequence thereof. In certain embodiments, the polypeptide is fused to al Ieast one fusion partner, as described herein.

The administration of a protein vaccine according to the présent invention may be peiformed using any suitable route, such as for example, intravenously, subcutaneously, intradermically, orally, topically or systemically.

DNA Vaccines

The présent invention also relates to a DNA vaccine against pregnancyassociated malaria. Genetic vaccination or DNA vaccination is aimed at inducing an immune response and consists in the direct introduction, in certain cells, of a gene or a nucléotide sequence encoding a vaccinal antigen or of a purified DNA plasmid comprising a sequence encoding a vaccinal antigen. In the Examples presented herein, DNA vaccination was performed on muscle cells. However, DNA vaccination may be performed on other types of cells, such as for example, cells of the skin. Examples of methods of administration of a DNA vaccine include, but are not limited to, intra-muscular injection, particle “bombardment” to the skin, and nasal administration. The DNA pénétrâtes in the targeted muscle cells, skin cells or other types of cells; and these cells then synthesize the antigen. The synthesized antigen is presented to the immune System, and initiâtes a response (the production of antibodies that have the ability, in case of infection, to specifically recognize that particular antigen on the parasite). The vaccine is thus produced locally by the organism of the immunized individual. This method of vaccination is simple and inexpensive, and présents important advantages in ternis of efficiency. Indeed, the antigen thus produced is generally under the form of the native peptide sequence, fused or not to one or more peptidic sequences (fusion partners). Furthermore, it is produced in a temporally extended fashion by cells of the organism, and this lengthy production and présentation of the antigen should prevent the need of booster vaccines. In addition. DNA vaccines are chemically defined and thermally stable, which reduces the need to maintain an unbroken cold chain.

Therefore, the présent invention provides a DNA vaccine comprising a naked DNA, in particular a circulai' vaccinal plasmid (either super-coi lcd or not) or a linear γ

DNA molécule, comprising and expressing in vivo a nucléotide sequence encodirtg a polypeptide consisting of the NTS-DBElx-Idl-DBL2x région of the VAR2CSA protein, or a biologically active fragment thereof comprising at least the Idl-DBL2x région of VAR2CSA. The tenu “naked DNA, as used herein, has its art understood ineaning and refers to a DNA transcription unit under the form of a polynucleotide sequence comprising at least one nucléotide sequence encoding a vaccine antigen and éléments necessary to the expression of the nucléotide sequence in vivo. Polynucleotides according to the invention may advantageously be inserted into a plasmid such as DNA-CSP, Nyvac pf7, VR1020, VR1012, etc.

The éléments necessary to the expression of a nucléotide sequence in vivo include, but are not limited to, a promoter or transcription initiation région, and a transcription termination région that are functîonal in a human cell. In addition, sequences that increase the genetic expression, such as introns, “enhancer” séquences and “leader” sequences are often necessary for the expression of a sequence encoding an immunogenic protein. As known in the art, thèse éléments are preferably operably lirked to the nucléotide sequence that is to be transcribed.

Examples of promoters useful in DNA vaccines, in particular, in DNA vaccines intended to be used in human vaccination, include, but are not limited to, SV40 virus promoter, mouse mammary tumor virus-like virus (MMTV) promoter, HIV virus promoter, Moioney virus promoter, cytomégalovirus (C.MV) promoter, Epstein-Ban· virus (EBV) promoter, Rous sarcoma virus (RSV), as well as promoters of human genes such as actin gene promoter, myosin gene promoter, hemoglobulin gene promoter, muscle creatin gene promoter, and metallothionein gene promoter.

One skilled in the art knows how to construct a DNA vaccine.

The naked DNA may also be incorporated into a drug carrier. Examples of suitable drug carriers include, but are not limited to, biodégradable microcap.sules. immunostimulating complexes, liposomes, catiotiic lipîds, and livc, attenuated vaccine vectors such as viruses and bacteria.

A DNA vaccine according to the invention may also be administered in combination with an agent that improves or favors the pénétration of a vaccine genetic material into cells. Thus, a DNA vaccine may be formulated to contain such an agent or be administered at substantially the same time as such an agent. Examples of agents that improve the pénétration of a vaccine genetic material into cells include. but are not limited to, esters of benzoic acid, anilides, a midi nés, urethanes, and hydrochloride salts thereof (U.S. Pat. No. 6.248.565). The administration of DNA to cells may be improved using chemical vectors (such as. for example, cationic 5 polymers or cationic lipids), physical technical such as eiectroporation. sonoporation. magnetofection, etc, or using viral vectors such as adenoviruses. etc.

III Uses of Immunogenic Compositions and Vaccines

The immunogenic compositions and vaccines according to the présent invention may be used to immunize female human beings (and more specifically prepubertal 10 girls and women in âge of bearing children, in particular postpubertal girls or primigravtdae) with the goal of preventing pregnancy-associated malaria.

Consequently, the invention relates to methods of treatment or prévention of pregnancy-associated malaria. More specifically. the invention provide a method for inducing a protective immune response against Plasmodium Julciparum in a female 15 human being, the method comprising a step of administering, to the female human being, an effective amount of an immunogenic composition or vaccine described herein. The invention also provides a method of vaccination of a female human being against pregnancy-relatcd malaria, the method comprising a step of administering. to the female human being, an effective amount of a vaccine of the invention, in 20 particular a DNA vaccine or a protein vaccine described herein.

As used herein, the term “effective amount” refers to any amount of an immunogenic composition or vaccine that is suffïcient to fulfil its intended purpose(s). For example, in certain embodiments of the présent invention, the purpose(s) may be: to prevent pregnancy-associated malaria, and/or to induce the production of antibodies 25 that inhibit binding of P. Jalciparum-infected érythrocytes to placenta! CSA. and/or to treat pregnancy-associated malaria.

In these methods, administration of an immunogenic composition or a vaccine may be performed using any suitable joute (e.g., orally, parentally. mucosally). In certain embodiments, a DNA vaccine is administered intramuscularly, intradermically 30 oi' mucosally. In other embodiments, a protein vaccine is administered intraveinously. sub-cutaneously, intradermaily, orally, topically or systemically.

An immunogenic composition or a vaccine according to the invention may be administered in a single dose or in several doses. The attending physician will know, or will know how to détermine, the efficient dose and appropriate administration regimen to be used in a given protocol of immunization or vaccination.

IV - Kits

The présent invention also provides pharmaceutical packs or kits for the prévention of pregnancy-associated malaria. More specifically, a pharmaceutical pack or kit comprises materials that are necessary to perform a vaccination according to the invention. Generally, a kit comprises an immunogenic composition or vaccine I0 according to the invention, and instructions to perform the vaccination. Optionally, the kit can further comprise means to perform a vaccination.

The kit will comprise one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles) containing one or more ingrédients of an inventive immunogenic composition or vaccine, allowing administration according to the invention. Different I5 ingrédients of a pharmaceutical pack or kit may bc supplied in a solid (e.g., lyophilized) or liquid form. Each ingrédient will generally be suîtable as aliquoted in its respective container or provided in a concentrated form. Pharmaceutical packs or kits may include media for the reconstitution of lyophilized ingrédients. Individual containers of the kits will preferably be maintained in close 20 confinement for commercial sale.

Optionally associated with the containei(s) can be a notice or package insert in the form prescribed by a governmental agency regulaling the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The notice of package insert 25 may contain instructions for use of an immunogenic composition or vaccine according to methods of vaccination or treatment disclosed herein.

An identifier, e.g., a bar code, radio frequency, ID tags, etc., may be présent in or on the kit. The identifier can be used, for example, to uniquely identîfy the kit for purposes of quality control, inventory control, tracking movement between

*16305

Unless specified otherwise. ali the technical and scientific terms used herein hâve the same meaning as that generally understood by a régulai' expert in the fteld of this invention. Similariy, any publications, patent applications, patents and any other référencés mentioned herein are included by reference.

The following examples and the figures are described to illustrate some embodiments of the procedures described above and should in no way be considered to be a limitation of the scope of the invention.

Examples

The following examples describe some of the preferred modes of making and 10 practicing the présent invention. However. it should be understood that the examples are for illustrative purposes only and are not meant to linut the scope of the invention. F’urthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the spécification, is not intended to suggest that experiments were aciually performed or data were actually obtained. Some of the results presented 15 below hâve been described in a scientific paper (Bigey et al., “The NTS-DBL2x région of VAR2CSA induces cross-reactive antibodies that inhibit adhesion of Plasmodium isolâtes to Chondroitin-Sulfate A”, J. Infect. Dis., 2011, in press), which is incorporated herein by reference in its entirety.

The studies presented below were approved by the Comité Consultatif de 20 Déontologie et d’Ethique of the Research Institute for Development (France), the cthîcal committee of the Ministry of Health (Sénégal), and the ethics committee of Health Science Faculty (University of Abomey-Calavi, Bénin). All procedures, including the animal immunization procedures, complied with European and National régulations.

Example 1: Identification, Production and Analysisof NTS-DBLlx-ldl-DBL2x ln the study presented in this section, the possibilité* of identifying functionally important VAR2CSA régions that can induce IgGs with high adhesion inhibitory capacity has been investigating. Using intramuscular plasmid DNA electrotransfer. antibodies induced against a spécifie région of VAR2CA, the NTS-DBLlx-Idl30 DBL2x, were shown to efficient! y block parasite binding to CSA at a similar level as antibodies induced against the t’ull-length extracelluiar domain of VAR2CA. The présent work highlights an important achievement towards development of a protective vaccine against placenta! malaria.

Materials and Methods

Parasites and Human Plasma. ht rmz>-propagated P. faitiparum parasites FCR3, 5 and HB3 grown in 0+ érythrocytes without human sérum, as previously described (Cranmer et al., Trans. R. Soc. Trop. Med. Hyg., 1997, 91: 363-365), were used in this study. Antibody reactivity with infected érythrocytes was tested on unselected cultures and cultures selected for infected érythrocytes adhesion to CSA. Cultures were seiected following several panning on choriocarcinoma cell line BeWo, as 10 described (Haase étal., Infect. Immun,, 2006. 74: 3035-3038).

Primary fîeld P. falciparum isolâtes and plasma samples were collected from a cohort of prégnant women enrolled in the ongoing STOPPAM project in the district of Corné, located 70 km West from the economical capital of Bénin. Cotonou (I luynth et al.. Malar. J., 20ll, 3l;l0(l): 72). The isolâtes were obtained either from the I5 peripheral blood of childrcn below the age of 5 years (N=5) and from prégnant women (N=24), or from placental blood at delivery (N-6). Peripheral blood isolâtes were maintained tti vitro for no more than 48 hours before testing.

Plasma samples from a previous study conductcd in Sénégal were also used (Tuikue Ndam et al., J, Infect. Dis., 2004, 190: 2001-2009).

Animal Immnnization and Antibody Screening. The var2cxa gene from the FCR3 parasite genome and a corresponding synthetic gene with a codon-optimized sequence (GenBank accession no. G U 2495 98) as described in Khunrae et al. (Khunrae et al.. J. Mol, Biol., 2010, 397: 826-8340 were used as cioning templates. Using construits comprise of single and multiple domains of VAR2CA proteins. sequences were cloned into a pVaxl vector backbone (Invilrogen) in which the original cytomégalovirus (CMV) promoter was replaced with the CMV promoter of the pCMVb plasmid (Clontech), and fused to the mEPO signal sequence. as already described (Trollet et al.. Infect. Immun.. 2009, 77: 2221-2229).

In vitro immunizations were carried out on 6-week old Swiss female mice (Janvier, France) and on 2-month old New-Zealand rabbits (Grimaud, France).

Electrotransfer experiments were carried out on mice, as previously described (Avril es al., PLoS One, 2011. 7:6(2): e!6622). Briefly, mice were anesthetized by intraperitoneal injection of 0.3 mL of a mix of ketamine (100 mg/mL) and xylazine ( 10 mg/mL) in 150 mM NaCl. Hind legs were shaved. Plasmid DNA (40 pg) in saline was injected into the tibial cranial muscle. After injection, transcutaneous electric puises (8 puises of 200 V/cm and 20 ms duration at a frequency of 2 Hz) were applied by two stainless steel external plate électrodes placed abut 5 mm apart, at each side of the leg (Trollet ei al., infect. Immun., 2009, 77: 2221-2229).

For rabbit immunization, animais were anesthetized by intramuscular injection of a mix of ketamine (35 mg/kg) and xylazine (5 mg/kg). The backs of the rabbits were shaved, and 300 pg of plasmid DNA in plasmid were injected in 5 different sites of each longissiiHus dorsi muscle with a 3-needle electrode device. After injection, electrical puises (8 puises of 120 V/cm and 20 ms duration at a frequency of 2 Hz) were applied at each injection site by means of a 3-needle electrode device.

AH animais (mice and rabbits) were immunized three times: at days 0, 30 and 60, and antisera were collected 15 days after the second and the last immunization (i.e.. at days 45 and 75).

For protein immunization, mouse antisera were also produced by the intraperitoneal injection of 10 pg of the recombinant protein in 50 mL, mixed with an equal volume of Alugel. Micc were immunized three times: at days 0, 30 and 60. Antisera were collected 15 days after the final boosting injection (i.e., at day 75).

IgG Préparation. Total IgG was manually purified from final bleed mice/rabbit sera on a Hi-Trap Protein G HP column according to the manufacturées recommendations (GE Healthcare). Construct-specific IgGs were affinity purified from plasma pools of women exposed to pregnancy-associated malaria and from exposed male using HiTrap NHS-activated HP columns (GE Healthcare) on which the corresponding recombinant protein was coupled following the manu facturer's recommendations.

Antibody Reactivity with falciparum Laboratory Lines and Field Isolâtes, In rtew-propagated P. falciparum parasites FC R 3 and HB3 were repeated! y panned on the human choriocarcinoma cell line BeWo, as previously described (Haase et al.. Infect. Immun., 2006, 74: 3035-3038). The derived CSA-adhering infected érythrocytes (FCR3-BeWo. HB3-Bcwo) and 35 primary field P. Jdlriparum isolâtes collected at Corné, southwestern Bénin (YadouJeton et al., Malaria J„ 2010. 9: 204) were analyzed to détermine the reactivity ofthe antibodies generated.

Flow cytometry (FACS Calibur. Beckman Coulter) was used to test the reactivity of sera of vaccinated animais to the surface of infected érythrocytes, as previously described (Barfod et al.. J. ïmmunol., 2010, 185: 7553-7561). In brief. CSA-selected parasite cultures or fielcï parasite isolâtes were enriched to contain late trophozoite and shizont stage parasites by exposure to a strong magnetic field (VarioMACS and CS columns, Miltenyï). Aliquots (2 x IÛ⁵ infected érythrocytes) were labelled using cthidium bromide and sequentially exposed to mouse/human sérum and anti-mouse/human IgG-FITC (Invitrogen). AU samples relating to a particular parasite isolate were processed and analyzed in a single assay.

Protein Expression, Purification and Evaluation. The NTS-DBLlx-ldl-DBL2x région of the var2csa gene from FC R 3 parasite line (synthetic gene) was cloned into the baculovirus vector pAcGP67-A (BD Biosciences) upstream of a histidine tag in the C-terminal end of the construct. This construct was made to allow translation from amino acid N9 to amino acid A864. Linearized Bakph6 Baculovirus DNA (BD Biosciences) was cotransfected with pAcGP67-A into Sf9 insect cells for production of recombinant virus particles. Hi5 insect cells grown in 600 mL serum-free media (Gibco, 10486) were infected with 18 mL of 2ⁿⁱⁱ amplification of the recombinant virus particles. After 2 days of induction, the cells were centrifuged (8.000g, 4°C, 10 minutes) and the supematant was filtered using two 10 kDa NMWC PES membranes (0.45 pm) (GE Healthcaie). The supematant was then concentrated to 30 mL and diafiltered six times on an ÀKTA crossflow (GE Healthcare) with buffer A (10 mM sodium phosphate, pH 7.4, 500 mM NaCl). The retentale was recovered from the System and filtered (0.2 pm), Before loading on the HisSelcct column, imidazole (Sigma-Aldrich) (150 pl.. 1 M, pH 7.4) was added to the sample. giving a final imidazole concentration of 15 pM. The bound protein was eluted with buffer A + 200 mM imidazole (HisSelect). The eluted protein was subjected to gel filtration.

Spécifie récognition of the purified protein was evaluated in ELISA using plasma samples from prégnant womeii of Bénin and Sénégal, unexposed prégnant French women, and malaria-exposed chiidren (from Sénégal) and men (from Bénin and from Sénégal).

X

Inhibition of Infected Erythrocytes to CSPG by Spécifie IgG. The static assays employed to evaluate the capacity of the antibodies to interfère with CSA-specific adhesion of infected érythrocytes was described in detail elsewhere (Fried el al.. Methods Mol. Med., 2002, 72: 555-560). In this assay, plates were coated overnight 5 at 4^1>C with 20 pL of ligand: l% BSA, 5 pg/mL decorin: CSPG (Chondroitin Sulfate IProtcoglycan, Sigma) or 50 pg/mL bovine CSA (Sigma) diluted in PBS. Each spot was subsequently blocked with 3% BSA in PBS for 30 minutes at room température, i Late-stage-infected érythrocytes were also blocked in BSA/RPMI for 30 minutes at i

room température. Parasite suspensions adjusted to 20% parasite density were 10 incubated with sérum (I:5 final dilution) or purified IgG (0.01 mg/mL to l mg/mL final concentration) or 500 pg/mL soluble CSA for 30 minutes at room température I before they were allowed to bind to ligand for 15 minutes at room température. Nonadhering cells were removed by an automated washing System. Spots were fixed with l.5% glutaraldehyde in PBS and adhering infected érythrocytes was quantified by 15 microscopy.

Compétition ELISA. Prior to compétition ELISA, the anti-NTS- DBLlx-ldlDBL2x IgG titer was determined in plasma pools composed of samples from exposed multigravid women from Bénin. DNA-vaccinated rabbits and protein-immunized mice (plasma pools from D75). Microtiter plates (Nunc) were coated with 20 recombinant NTS-DBLlx-ldl-DBL2x (0.5 pg/mL in PBS). After the plates were saturated with blocking buffer (PBS, 0.5 M NaCI, i% Triton Χ-Ι00, l% BSA) for I | hour at room température, they were incubated for l hour at room température with increasing dilutions of the competing plasma (plasma pools from D75 DNAvaccinated rabbits or protein-vaccinated mice against NTS-DBLlx-ldl-DBL2x). Pre25 immune sera pools of rabbit or mouse or plasma pooi fjom unexposcd Erench prégnant women were used as négative control. Plates were washed four times with washing buffer (PBS, 0.5 M NaCI, l% Triton X-100. pH 7.4) and incubated with a fixed dilution of one plasma/serum (Plasma pools from D75 DNA-vaccinated rabbits or sera from malaiia-exposed Beninese multîgravidae) for l hourat room température.

The spécifie secondary antibody directed against the non-competing antibodies (goat anti-human IgG HRP, Sigma-Aldrich. goat anti-mouse IgG MRP or goat anti-rabbit IgG HRP. Sigma) diluted 1:4000 in blocking biif’fe: was added, and incubated for 1 hour at room température. After a 4-times washing, antibody reactivity of nocompeting plasma/serum was visualized at 450 nm following the addition of TMB (tetramethylbenzidine). The percent réduction in antibody reactivity in the presence of a competitor was calculated as follows: I00 x [OD competitor antibody ! OD 5 without competitor antibody]

Results

Plasmid DNA immunization induced high titer surface réactivé antibodies.

A total of 13 plasmids representîng single and overlapping multiple domains of VAR2CSA from the FCR3 parasite line were constiucted and used for immunization I0 (Figure IA). The single and overlapping domains of VAR2CSA that were tested are;

NTS-DBLÎx-Idl-DBL2x (corresponding to amino acids 8 to 866); DBL2x-ld2 (amino acids 446-1208); Id2-DBL3x (amino acids 870-1575); DBL3x-4e (amino acids 1168-1987); DBL4e(a) (amino acids 1576-1987); DBL4c(b) (amino acids [5831989); DBL4E-5E (amino acids 1576-2313); DBL5e (amino acids 1982-2313);

DBL5e-6e (amino acids 1982-2673); DBL6e (amino acids 2314-2673); NTS-DBL3x (amino acids 9-1572); DBL3x-DBL6c (amino acids 1168-2673), and NTS-DBL6e (amino acids 1-2673).

Ail immunîzations with single to triple-domains construis of VAR2CSA induced the formation of polyclonal antibodies with a high ELISA titer (> 1 x 10⁵) 20 following intramuscular plasmid electrotranfer. However. for plasmids containing more than 3000 bp of coding sequence. effective humoral immune response in ali vaccinated animais, both mice and rabbits, required the use of a codon-optimized sequence (GenBank Accession Number GIJ249598). Although ail single and muitidomains of VAR2CSA could induce antibodies reacting with native VAR2CSA on 25 the surface of the CSA ad heri ne-erythrocytes infected with the homologous FCR3, constnjcts containing DBLlx, DBL2x, DBL5E and DBL6e were the most efficient in inducing surface reactive antibodies (Figure JB). Noue of the polyclonal antiVAR2CSA antisera recognized the érythrocytes infected with the non-CSA adhèrent FCR3 parasite line.

Antibodies induced against VAR2CSA inhibit binding of infected érythrocytes to chondroitin sulfate proteoglycan (CSPG). A Pétri dish-based static binding assay was used to screen sera for their ability to inhibit parasite binding to CSA. Of ail the FCR3 VAR2CSA régions tested. only sequences located between the N-terminal sequence (NTS) and the DBL3x appeared to irtduce inhibitory antibodies (Figure l B). Highly inhibitory antibodies were obtained with the fulllength extracellular VAR2CSA construct, which total ly inhibited binding. interestingly. similar inhibition was seen with scia from animais (both mice and rabbits) vaccinated with the NTS-DBLlx-Idl-DBL2x construct. lu addition, the inhibitory activity of sera from NTS-DBLix-IdI-DBL2x vaccinated animais was investigated on a heterologous parasite line; the CSA adhèrent HB3 line. The same pattern of inhibition was observed (Figure 2A).

To confirm that the inhibition observed with NTS-DBLI x-Id l -DBL2x antiserum was mcdîated by IgG, IgGs were purified and tested for binding inhibition activity. The purified IgGs recognized the surface of BeWo-selected FC R 3 infected érythrocytes (Figure 2B). The purified IgGs inhibited 100% of the binding of infected érythrocytes to CSA at a concentration of 0.5 mg/mL (Figure 2D).

Antibodies induced against NTS-DBI Jx-ldl-I)BL2x specifically recognized isolâtes from prégnant women. f low cytometry analysis cleai ly demonstrated that murine anti-NTS-DBl,Ix-ld)-DBL2x antibodies specifically recognize the surface of placent al malaria parasites among the field isolâtes. Thirty five (35) isolâtes were analyzed by flow cytometry in this study, including 24 periphcral blood isolated from prégnant women. Six placental isolâtes and 2! of the 24 peripheral blood isolâtes from prégnant women were recognized by polyclonal murine antibodies while noue of the 5 children isolâtes tested were labelled.

Of the 21 isolâtes from prégnant women that reacted with anti-NTS-DBLlx-ld l DBL2x antibodies by flow' cytometry, 16 showed spécifie adhesion to CSPG. while 5 isolâtes did not bind. Among the 3 peripheral blood isolâtes that w'ere not labelled in flow cytometry, 2 bound to CSPG but their interaction could not be abrogated b y soluble CSA, and l isolate did not bind.

Fîfteen samples containing sufficient amount of parasite were further processed in binding inhibition assay. These comprised 14 isolâtes li'oin peripheral blood samples and one placental isolate. The binding to CSA of I2 of the 15 prégnant women isolâtes tested as highly inhibited by spécifie anti-NTS-DBLlx-Idl-DBL2x sera (see Table l below).

Table 1. Adhesion inhibitory capacity of spécifie antibodies induced against NTSDBL2x on P. Jalcipartim-infecled érythrocytes from naturally infected prégnant women in Bénin,

isolâtes	Sound IEs /mm2 on BSA	Bound IEs /mm2 on CSPG	MFI (ratio to négative control)	O/ /o prebleed inhibition	% anti-NTSD8L2x inhibition	% CSA inhibition
CM0425	1	1035	3.2	0.00	41.75	92.21
WP0182	2	339	3.8	23.92	74.75	94.16
CM0375	5	63	1.6	0.00	51.32	94.23
WP0140	2.5	244	1.8	16.36	61.65	98.98
WP0161	1	1301	21 0	32.26	87.34	90.75
WP0168	2	473	6.4	0.00	42.35	94.16
CM0437	1	736	4.1	29 89	94.56	93.34
WP0200	4.5	185	1.4	0.00	31.79	68.29
CM445	1.5	218	2.2	28.69	76.27	87.62
AK366	0.5	357	4.3	15.91	63.35	95.99
AK357	2.5	337	1.5	20.66	58.04	91.12
WP203	1	161	4.2	31.26	82.68	85.87
1MH016	2	178	2.0	16.48	53.92	85.04
1MMCH	2.5	394	3.1	1.89	72.87	86.48
CM307	1	623	5.9	17.83	89.45	96.30

Animais immunized with recombinant NTS-DBLlx-ldl -DBL2x or DNA electrotransfer produced antibodies of similar specificity. Murine polyclonal antibodies induced either by recombinant protein or plasmid DNA of NTS-DBLlxldl-DBL2x showed similar reactivity. The reactivity to érythrocytes surface and inhibitory activity on binding to CSA were similar on BeWo-selected FCR3 infected érythrocytes. The inhibitory activity was compared in dilution sériés of sera from mice immunized with either the full-length construct or NTS-DBLlx-ld l-DBL2x (both DNA and protein immunization). Down to the dilution 1:100, sera from mice vaccinated with full-length DNA construct or recombinant NTS-DBLlx-ldl-DBL2x totally inhibited binding of infected érythrocytes (Figure 4A). The inhibitory capacity of the sérum samples following plasmid DNA immunization with the full-length construct or by protein vaccination with NTS-DBLlx-ld 1 -DBL2x was seen at subséquent dilutions, these sera were diluted 1:5000 before inhibition vanished (Figure 4A). This observation clearly strcnglhens the importance of the NTS-DBLlxldl-DBL2x région of VAR2CSA in eliciting adhesion-inhibitory antibodies by vaccination.

Antibodies induced in animais by vaccination with NTS-DBLlx-ldl-DBL2x target the same epitopes as naturally acquired antibodies. The recombinant NTSDBLlx-Idl-DBL2x produced in insect cells w?s recognized by plasma from malaria exposed prégnant women from Bénin and Sénégal in a parity-dépendent manner (Figure 5). This NTS-DBLl x-ld I-DBL2x was used in compétition ELIS A to analyze target epitopes among antibodies induced in animais by plasmid DNA immunization and protein immunization, as well as the naturally acquired antibodies against the NTS-DBLlx-IdLDBL2x région of VAR2CSA in prégnant women. A mutual inhibition pattern was observed in the ability of ail three antisera to recognize the recombinant NTS-DBLlx-Id!-DBL2x protein. The inhibition pattern between sera from DNA immunization s and protein i m muni zat ions was concentrai ion -dépendent (Figure 4B). A similar inhibition was observed when antibodies in a human plasma pool from exposed multigravidae competed with spécifie anti-sera from rabbits (Figure 4C).

The naturally acquired human IgG against VA R CSA NTS-DBLlx-ldlDBL2x inhibit adhesion of infected érythrocytes to CSA. Plasma samples from women included in the STOPPAM project are routinely analyzed foi’ anti-adhesion capacity on the FCR3-BeWo parasite lines. The recombinant NTS-DBLlx-ld lDBL2x protein was used to affinity-purity IgG from plasma of malaria-exposed Beninese prégnant women (selected for having a high anti-adhesion activity on CSAbinding parasite lines). Interestingly, naturally-acquired antibodies targeting the NTSDBLl x-ldl-DBL2x of VAR2CSA demonstrated anti-adhesion activity. This activity was shown both on FCR3-BeWo and HB3-HeWo parasite lines, with a clear concentration-dependent effect of purified IgG (Figure 6). This is the first time that naturally acquired antibodies to a spécifie VAR2CSA région have been shown to inhibit P. falciparum infected érythrocytes binding toCSA.

Discussion

Molecular details of the interaction of the P. fahipantm ligand VAR2CSA with the placenta] receptor CSA are currentiy not well delineated, but recent studies suggest that the binding site dépends on a higher-order architecture in which DBL domains and the interdomain régions of VAR2CSA fold together to form a ligandbinding pocket (Khunrae et al., J. Mol. Biol,, 20i(). 397: 826-834; Dahlback et al., Trends Parasitol., 2010, 26: 230-235). However. polyclonal antibodies induced by immunization with the recombinant extracellular part of VAR2CSA highly inhibit binding of infec’ed érythrocytes to CSA in vitro (Khimrae et al., J. Mol. Biol., 2010, 397: 826-834). This suggests that protective inimunity to placental malaria acquired A, over a few pregnancies in areas of intense P. fait ipantrn transmission that corrélâtes with levels of anti-adhesion antibodies (Duffy et al.. Infect. Immun.. 2003. 71: 66206623) is mostly mediated by anti-VAR2CSA IgCis. Nevertheless, a recent work reports that immunization with full-length VAR2CSA did not induce potent cross5 inhibitory antibodies (Avril et al.. PLoS One, 201 i. 7:6(2): C16622).

Although antibody response may directly inhibit infected érythrocytes adhesion placenta, it also might be implicated in opsonization (Keen et al., PLoS Med, 2007. 4(5):e 181 ; Feng ei al., J. Infect. Dis., 2009, 15:200(2):299-306).

VAR2CSA thus appears as an important candidate for vaccine development.

However sequence analyses among parasites hâve shown that il is a polymorphie protein compo.sed of alternating areas of substantial intercional polymorphism (Bockhorst et al., Mol, Biochem. Parasitol., 2007, 155: 103-112; Fernandez et al.. Malaria J,, 2008. 7: 170). The rationale for deveioping an effective VAR2SA-based vaccine against placental malaria thus requires définition of VAR2CSA areas containing functionally important epitopes that transcend this interclonal diversity. In the présent sluciy, full-length and truncated VAR2CSA constructs were tested for their ability to induce adhesion inhibitory antibodies.

The DNA vaccine technology that has proven efficient on various pathogens and tumor antigens (Kutzler et al., Nat. Rev. Genet., 2008, 9; 776-788), was successfully used here with the P. falciparum var2csa gene. The res urgence in interesl for such concept observed in the last few years is due to several technical improvements such as codon optîmization strategies, novel formulations and more effective delivery approaches. The delivery of eleclrical puises after intramuscuïar plasmîd DNA infection particularly enhanced DNA uptake and resulted in a stronger and more spécifie humoral response when the antigen was fused to a leader sequence (Trollet et al.. Infect. Immun., 2009, 77: 2221-2229). Several clinical trial based on this approach are currently ongoing in the fields of cancer and infectious diseases. One of these trials that started in July 2010 targets Plasmodium falciparum malaria.

In the présent study, a strong immune response was obtained both în mice and in rabbits vaccinated with VAR2CSA genetic fragments that were fused to mEPO leader sequence. Interestingly. ail antibodies induced were able to recognize the native protein expressed on the surface of érythrocytes infected with the homologous FCR3 parasite line. In line with data previous reported by Khunrae et al. (Khunrae et al., J. Mol. Biol., 2010. 397: 826-834), the plasmid encoding the full-length extracellular pan of the protein induced a robust humoral response that compietely blocked infected érythrocytes binding to CS PG. However, the major finding of this study is 5 that a shorter construct of the N-terminal moiety of VAR2CSA corresponding to

NTS-DBLlx-Idi-DBL2x was able to induce high potency antibodies with similar inhibitory capacity as those elicited against the fuil-lenglh VAR2CSA. Moreover, compétition ELISA analysis revealed thaï antibodies raised by experimental immunization (plasmid DNA or purified recombinant protein) or those naturally I0 acquired by prégnant women to this particular région of VAR2CSA predominantly target similar épitopes, This resuit is in line with others that reported that prégnant women do acquire cross-reactive antibodies (Elliott et al., Infect. Immun.. 2005, 73(5): 5903-5907; Beeson et al., J. Infect. Dis.. 2006, 193(5): 721-730). This suggests that vaccination may repioduce, at least partially, natural acquired immunity against 15 placental malaria.

Recombinant NTS-DBLIx-Id!-DBL2x expressed in insect cells was specifically recognized by sera from malaria-exposed women in a parity-dependent manner supporting the fact that this recombinant protein exhibîts important targets of the immune response against VAR2CSA. Murine polyclonal antibodies raised against 20 this construct from the FCR3 parasite strain stained the surface of most isolâtes from prégnant women of Bénin. Remarkably, antibodies raised against a single variant of NTS-DBLlx-Id l-DBL2x showed consistent inhibitory activity against several isolâtes originating from prégnant women. Actually, the binding of infected érythrocytes to CSPG/CSA of 12 out of the 15 prégnant women isolâtes tested was inhibited by more 25 than 50%. This highlights the existence of functionally important epitopes within this région of VAR2CSA that are shared b y most placenta-sequestering P. fait ipar um isolâtes. However, ail isolâtes were not inhibited as a probable conséquence of antigenic poiymorphism. Possible mcchanisms of action include that anti-NTSDBIJx-Idl-DBL2x antibodies inhibit infected érythrocytes adhesion to CSPG/CSA 30 by blocking a single unique CSA binding-site exhibited in the quaternary structure of VAR2CSA, or by modifying the assembly of such high ordered structure médiat in g the binding of native VAR2CSA to CSA (Nielsen et al., Infect, immun., 2009. 77: 2482-2487). A

The results présent ed here clearly indicate that antibody récognition of just a few VAR2CSA variants containing key epîtopes might be sufficient to markedly affect the binding of VAR2CSA-expressing infected érythrocytes lo CSA.

Of particular interest, maternai antibodies purified with the recombinant NTSDBL lx-Id!-DBL2x reacted with both BeWo-selected FCR3 and HB3 stiaîns, and showed high inhibitory activity on these two distinct parasite lines. This indicates that the inhibitory properties of anti-VAR2CSA antibodies observed in the current study are of biological sig ni finance in the acquired immune protection to piacental malaria, h was recently shown that some VAR2C’SA-specific human monoclonal IgG s from P. ftilciparum-euposed women can exhibit some moderate degree of adhesion inhibition that increases with their combination (Barfod et ciL, J. Immunol., 2010, 185: 75537561 ). To the bcst of the inventor’s knowledge, the présent study clearly shows functional evidence on a spécifie area of VAR2CSA that is a target of significant naturally acquired anti-adhesion antibodies.

In conclusion, genetic immunization by intramuscular plasmid electrotransfer represents a general technology for fast and efficient screening of immunogenic domains within large proteins of which optimal production as recombinant proteins are technically demanding. This work showed that a truncated N-terminal région of VAR2CSA was a major target of anti-adhesion immune response in piacental malaria, and therefore an attractive vaccine target. Further studies are required to ascertain the impact of sequence variation within this particular VAR2CSA région to its potential for cross-reactivity.

Example 2: Identification, Production and Analysis of Id l-DBL2x

In the study presented in this section, the inventons hâve investigated the possibility of identifying functionally important VAR2CSA régions, in particular functional ly important régions ofthe NTS-DBLIx-id-DBL2x portion of VAR2CSA, which can induce IgG with high adhesion inhibitory capacity,

Materials and Methods

To further refine the important protective epitope région, five additional constructs were buîlt based upon the NTS-DBI.l x-DBL2x sequence, encoding NTSDBLlx, NTS-DBLlx-Idl, Idl, ldi-DBL2x. and DBL2x, as shown in Figure 8. DNA sequences encoding the subfragments of NTS-DBLlx-DBL2x were clones into a pVAXI vector backbone (Invitrogen) as already described (Trollet et al., Infect. Immun.. 2009, 77: 2221-2229). Mice were immunized with these constructs as described above in Example l.

Results

The results obtained are presented on Figure 7. Ail constructs but the Idl successfully raised an immune response. The NTS-DBLlx, NTS-DBLlx-ldl and ldl-DBL2x fragments raised high titer immune response, comparable to that obtained with the full extracellular part NTS-DBLI χ-6ε. Among these constructs. binding inhibitory capacity of infected érythrocytes to CSA was not found with NTS-DBLlx antiserums, highlighting that the VAR2CSA minimal construct inducing anti-adhesion antibodies is beyond the DBLIx domain. However. the construct made of Idl alone did not induce significant immune response. This refined experiment allowed the identification of ldl-DBL2x (corresponding to the sequence from amino acid 392 to amino acid 866 of VAR2CSA - i.e., SEQ ID NO: 2) as the minimal région concentrating the main anti-adhesion epitopes.

Other Embodiments

Other embodiments of the invention will be apparent to those skilled in the art from a considération of the spécification or practice of the invention disciosed herein. It îs intended that the spécification and examples be considered as exemplary only. with the true scope of the invention being indicated by the following claims.

Claims (13)

1. Isolated polypeptide consisting of the NTS- DBL l x-ld l -DBL2x région of the VAR2CSA protein, or a biologically active fragment thereof, for use in the treatment or prévention of pregnancy-associated malaria, wherein the biologically active fragment comprises the Idl-DBL2x région of the VAR2CSA protein.

2. Isolated polypeptide according to claim I, wherein the isolated polypeptide consists of the Idl-DBL2x région of the VAR2CSA protein.

3. isolated polypeptide according to claim I or claim 2, wherein the ldl-DBL2x région of the VAR2CSA protein has the sequence set forth in SEQ ID NO: 2.

4. Isolated polypeptide according to any one of daims l to 3. wherein the NTSDBLlx-Idl-DBL2x région of the VAR2CSA protein has the sequence set forth in SEQ ID NC): l.

5. Fusion protein consisting of at least one polypeptide according to any one of daims l to 4 fused to at ieast one fusion partner for use in the treatment or prévention of pregnancy-associated malaria, wherein the fusion partner is selected from the group consisting of maltose binding protein, signal sequence of the maltose binding protein, poly-histidine tag, S-Tag, glutathione-Stransferase, thioredoxin, β-galactosidase, streptavidin, dihydrofolate reductase, pelB signal sequence, ompA signal sequence, signal sequence of alkaline phosphatase, green fluorescent protein (GFP), toxins, human growth hormone, interleukin-2 (IL-2), granulocyte macrophage col on y stimulât! ng factor (GMCSF), granulocyte col on y stimulât ing factor (G-CS F), calcitonin. interferonbeta, interferon-alpha, glucagon like peptide I (GLP-1). glucagon like peptide 2 (GLP-2), PA toxin, parathyroid hormone (PTH11-34) and PTH(l-84)), b utyryl cholinestérase, glucocerebrosidase (G B A), and exendin-4.

6. Isolated polynucleotide consisting of a sequence encoding a polypeptide according to any one of daims 1 to 4 or a sequence encoding a fusion protein according to daim 5 and éléments necessary to the in viiro or in vivo expression of said polypeptide or fusion protein, for use in the treatment or prévention of pregnancy-associated malaria.

7. Cloning or expression vector comprising at least one polynucleotide according to claim 6.

S. Host cell comprising at least one polynucleotide according to claim 6 or at least one vector according to claim 7.

9. lmmunogenic composition comprising at least one pharmaceutically acceptable carrier or excipient and at least one member of the group consisting of polypeptides according to any one of claims l to 4, fusion proteins according to claim 5, polynucleotides according to claim 6, and cloning or expression vectors according to claim 7.

10. DNA vaccine against pregnancy-associated malaria comprising a naked DNA comprising a nucléotide sequence encoding a polypeptide according to any one of claims l to 4 or a fusion protein according to claim 5 and éléments necessary to the in vivo expression of said polypeptide or fusion protein.

11. Protein vaccine against pregnancy-associated malaria comprising a polypeptide according to any one of claims l to 4 or a fusion protein according to claim 5.

12. Vaccine according to claim 10 or claim 11 further comprising at least one adjuvant.

13. Method of preventing or treating pregnancy-associated malaria in a female subject comprising a stop of administering a therapeutically effective amount of an immiinogenic composition of claim 9 or a vaccine according to any one of claims 10 to 12.

14. Method according to claim 13, wherein the female subject is a prepubertal giri, a postpubertal girl or a primigravidae woman. Z