VACCINES AGAINST PREGNANCY-ASSOCIATED MALARIA

The present invention relates to the use of specific regions of the N-terminal portion of the VAR2CSA protein in the prevention or treatment of pregnancy-associated malaria in a female subject.

Malaria is the most frequent parasitic infectious disease in the world. It is caused by a eukaryotic microorganism of the Plasmodium genus which is transmitted through biting by a female mosquito (Anopheles). Several species of Plasmodium can infect human beings, but Plasmodium falciparum is the most frequent and most pathogenic species and the species that is responsible for deadly cases. Once introduced in the blood, the parasite infects hepatic cells, in which it proliferates, before circulating again in the blood and invading red blood cells (erythrocytes). Malaria affects about a hundred countries in the world, in particular poor tropical regions of Africa, Asia and South America, Africa being by far the most affected continent. The World Health Organization estimates that malaria is responsible for 225 millions of cases of fever and approximately one million deaths annually (World Malaria Report, WHO, 2010). Currently available means of fighting against malaria infection include anti-malaria drugs (in particular chloroquine and quinine) and action against mosquitoes, vectors of the parasite. However, the situation is all the more worrisome that for several years, the parasites have developed increased resistance to drugs, and mosquitoes have developed resistance to insecticide. Today, there are no vaccines available against malaria.

Malaria affects mainly children of less than 5 years of age and pregnant women, in particular primigravidae (i.e., women who are pregnant for the first time). Pregnant women are particularly vulnerable because the placenta constitutes a target where parasites can accumulate. In pregnant women, malaria infection can cause a large variety of damaging effects: spontaneous abortion, early delivery, low weight at birth, congenital transmission, and neonatal death. In the African regions where malaria is endemic, 3 to 5% of newborn deaths can be imputed to pregnancy-associated malaria. Furthermore, it is also a real danger for the mother who can suffer from sometimes severe, or even deadly, anaemia.

Today, prevention of malaria in pregnant women is achieved by preventive administration of sulfadoxine/pyrimethamine (Cot et al., Br. Med. Bull., 2003, 67: 137-148). However, this intermittent treatment cannot provide a prevention against malaria during the entire pregnancy; firstly because administration of the drugs only takes place from the 20^th week of pregnancy (the teratogenic 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 medicinal protection; and thirdly because the efficacy of sulfadoxine/pyrimethamine is very strongly decreasing in all malaria endemic zones due to a rise in parasite resistance (Cot et al., Am. J. Trop. Med. Hyg., 1998, 59: 813-822; WHO/HTM/MAL/2005.1103. Geneva: 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). Drugs are currently tested in this context, and numerous efforts are focused toward the development of a vaccine against placental malaria. The possibility of vaccinating pregnant women or prepubertal girls would offer several obvious advantages over the sulfadoxine/pyrimethamine treatment, since the preventive protection would be temporally extended, and probably of higher quality.

One of the contemplated vaccinal strategies to fight against pregnancy-associated malaria is to re-create the natural protective immunity. Indeed, the clinical severity of malaria caused by Plasmodium falciparum is, at least partly, linked to alterations undergone by infected erythrocytes. These alterations are induced by proteins of the parasite that are exported to the surface of erythrocytes during the phase of development in blood. Some of these surface proteins of the PfEMP1 (Plasmodium falciparum Erythrocyte Membrane Protein 1) family, confer novel cytoadherence properties to infected erythrocytes. The infected erythrocytes bind to the internal walls of blood vessels, thereby becoming unavailable for transport towards purging organs of the immune system, whose role is to destroy cells recognized as abnormal. In pregnancy-associated malaria, infected erythrocytes adhere to chondroitin sulfate A (CSA), a sulfated glycosaminoglycan present in the placenta. After several pregnancies, women acquire protective antibodies that block this adherence. One vaccinal strategy is to re-create this protective immunity by blocking the attachment of infected erythrocytes to the placenta.

The VAR2CSA protein, one of the members of the PfEMP1 family, is currently the object of numerous research projects with the goal of developing a vaccine specifically adapted to pregnant women (Tuikue Ndam et al., J. Infect. Dis., 2005, 192: 331-335; Chia et al., J. Infect. Dis., 2005, 192: 1284-1293; Tuikue Ndam et al., J. Infect. Dis., 2006, 193: 713-720; Dahlbäck 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: 767-774; Guitard et al., Malaria J., 2008, 11: 7-10; Guitard et al., Malaria J., 2010, 9: 165; Gangnard et al., Mol. Biochem. Parasitol., 2010, 173: 115-122; Gnidehou et al., Mol. Biochem. Parasitol., 2010, 5(10): e13105). Although these studies are rendered difficult by VAR2CSA polymorphism, Phase I trials are nevertheless contemplated. Furthermore, the full-length extracellular domain of this protein has recently been expressed in a heterologous system (Srivastava et al., Proc. Natl. Acad. Sci. USA, 2010, 107: 4884-4889; Khunrae et al., J. Mol. Biol., 2010, 397: 826-834), and antibodies induced against this construct showed a very high anti-adhesion 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 immunodominant epitopes that do no induce "antiadherent" antibodies.

Nielsen et al. (Infection and Immunity, 2009, 77: 2482-2487) have described several polypeptides corresponding to different regions of VAR2CSA, and have demonstrated that among the polypeptides tested, the antibodies against DBL4 gave the best results with respect to the binding inhibition of infected erythrocytes to CSA. After the filing date of the priority document (FR 10 56294 of July 30, 2010) of the present patent, Srivastava Anand et al. (PLOS ONE, May 2011, 6: e20270) showed that the smallest region within VAR2CSA with similar binding properties to those of the full-length VAR2CSA was DBL1x-DBL3x; and Dahlbäck et al. (J. Biol. Chem, May 2011, 286: 15908-15912) reported that the core of the CSA-binding was situated in the DBL1x-Id1-DBL2x-DBL3x region.

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

In their study of the modulation of the immune response to Plasmodium falciparum during pregnancy, the present inventors have generated overlapping fragments of the sequence of the var2csa gene from the FCR3 parasite strain, introduced these fragments into plasmids, and intramuscularly injected each of the plasmids obtained in mice and in rabbits. They have identified the N-terminal portion of VAR2CSA, and more specifically the sub-region consisting in the DBL1x domain, the Id1 inter-domain and the DBL2x domain (i.e., NTS-DBL1x-Id1-DBL2x), as being the region of VAR2CSA that contains epitopes capable of inducing in vivo production of antibodies that block the binding of Plasmodium falciparum-infected erythrocytes to CSA. They then produced a recombinant protein corresponding to that particular portion of VAR2CSA and confirmed, via protein vaccination, the observations made with genetic vaccination. Further results have allowed the Id1-DBL2x region of the VAR2CSA protein to be identified as the minimal antigenic region of VAR2CSA that is involved in the acquisition of protective immunity against placental sequestration taking place during pregnancy-associated malaria.

Consequently, in a first aspect, the present invention relates to the use of polypeptides corresponding to and polynucleotides encoding specific regions of the N-terminal portion of the VAR2CSA protein in the fight against placental malaria.

More specifically, the present invention provides an isolated polypeptide consisting of the NTS-DBL1x-Id1-DBL2x region of the VAR2CSA protein for use in the treatment or prevention of pregnancy-associated malaria, wherein the NTS-DBL1x-Id1-DBL2x region of the VAR2CSA protein has the sequence set forth in SEQ ID NO: 1.

The present invention also provides an isolated polypeptide consisting of the Id1-DBL2x region of the VAR2CSA protein for use in the treatment or prevention of pregnancy-associated malaria, wherein the Id1-DBL2x region of the VAR2CSA protein has the sequence set forth in SEQ ID NO: 2.

The invention also provides a fusion protein consisting of at least one polypeptide as defined above fused to at least one fusion partner, for use in the treatment or prevention of pregnancy-associated malaria, wherein the fusion partner is human growth hormone, interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), calcitonin, interferon-beta, interferon-alpha, glucagon like peptide 1 (GLP-1), glucagon like peptide 2 (GLP-2), PA toxin, parathyroid hormone (PTH(1-34) and PTH(1-84)), butyrylcholinesterase, glucocerebrosidase (GBA), and exendin-4.

The present invention also provides an isolated polynucleotide for use in the treatment or prevention of pregnancy-associated malaria, the polynucleotide consisting of a sequence encoding a polypeptide or a fusion protein as defined above and elements necessary to the in vivo expression of said polypeptide or fusion protein.

In another aspect, the invention provides an immunogenic composition comprising at least one pharmaceutically acceptable excipient or carrier and a cloning or expression vector for use in the treatment or prevention of pregnancy-associated malaria in a female subject, wherein said cloning or expression vector comprises at least one polynucleotide as defined above. The present invention also provides an immunogenic composition for use in the treatment or prevention of pregnancy-associated malaria in a female subject, wherein said immunogenic composition comprises at least one pharmaceutically acceptable carrier or excipient and at least one member of the group consisting of polypeptides as defined above, fusion proteins as defined above, and polynucleotides as defined above, vectors as defined above. Preferably, such an immunogenic composition can induce antibodies that prevent adherence of Plasmodium falciparum-infected erythrocytes to the placenta receptor CSA.

In a related aspect, the invention relates to vaccines against pregnancy-associated malaria. More specifically, the invention provides a DNA vaccine for use in the treatment or prevention of pregnancy-associated malaria in a female subject, wherein said DNA vaccine comprises a naked DNA comprising a nucleotide sequence encoding a polypeptide as defined above or a fusion protein as defined above, and elements necessary to the in vivo expression of said polypeptide or fusion protein. In certain embodiments, the naked DNA is inserted into a plasmid. The invention also provides a protein vaccine for use in the treatment or prevention of pregnancy-associated malaria in a female subject, wherein said protein vaccine comprises a polypeptide as defined above or a fusion protein as defined above. Preferably, the vaccines according to the present invention induce antibodies that prevent Plasmodium falciparum-infected erythrocytes from attaching to the placenta receptor CSA. Vaccines according to the invention may further comprise an adjuvant.

In certain embodiments, the immunogenic composition according to the present invention is intended for the use in the treatment or prevention of pregnancy-associated malaria in a prepubertal girl or a woman in age of bearing children.

The present document also describes methods of treatment or prevention of pregnancy-associated malaria. More specifically, is described herein a method for inducing a protective immune response against Plasmodium falciparum in a female human being, the method comprising a step of administering an effective amount of an immunogenic composition or vaccine defined above. Also described is 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 prevention of pregnancy-associated malaria are mainly intended to women in age of bearing children (in particular postpubertal girls and primigravidae women) and to prepubertal girls. A method of treatment or prevention of pregnancy-associated malaria described herein, induces, in the female human being treated, the production of antibodies that prevent Plasmodium falciparum-infected erythrocytes from attaching to the placenta receptor CSA. In the methods of treatment or prevention pregnancy-associated malaria described herein, the immunogenic composition or vaccine may be administered by any suitable route.

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

• Figure 1 shows the surface reactivity and anti-adhesion capacity of mice antisera to various VAR2CSA constructs. (A) is a schematic representation of the 13 overlapping VAR2CSA constructs made in a pVAX1 derivative vector. (B) Blood samples before immunization and from full 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 filled histograms) for each pool is defined as the Median Fluorescence Intensity (MFI) ratio (MFI test / MFI negative control). The negative controls were stained only with secondary FITC-conjugated anti-mouse antibody. Binding inhibition of infected erythrocytes to decorin (bovine CSPG) was measured using serum in a 1:5 dilution (white filled histograms). The degree of inhibition was defined as [1 - (bound infected erythrocytes with test serum / bound infected erythrocytes without serum)].
• Figure 2 shows that NTS-DBL1x-Id1-DBL2x induces adhesion-inhibitory IgGs in different animal species. (A) Two rabbits immunized with plasmids encoding the NTS-DBL1x-Id1-DBL2x of the FCR3 var2csa variant acquired anti-adhesion antibodies to both CFR3-BeWo and HB3-BeWo infected erythrocytes from the second immunization. (B) purified IgGs induced against the NTS-DBL1x-Id1-DBL2x region and purified IgGs induced against DBL6e recognize native VAR2CSA on the surface of FCR3-BeWo infected erythrocytes. However, they did not recognize unselected FCR3 infected erythrocytes (data not shown). IgGs purified from animals before vaccination did not label the surface of FCR3-BeWo infected erythrocytes. (C) Purified anti-NTS-DBL1x-Id1-DBL2x IgGs specifically inhibit binding of FCR3-BeWo infected erythrocytes to CDPG.
• Figure 3 is an SDS-PAGE of NTS-DBL1x-Id1-DBL2x of VAR2CSA purified on a 1 mL HisSelect (Ni²⁺), 1 mL Capto S HP (IEX), and 1 mL of Heparin HP column (Hep), respectively. Samples of 2.5 and 5 µg of the purified recombinant protein were loaded for yield comparison.
• Figure 4 shows that experimentally induced or naturally acquired antibodies against NTS-DBL1x-Id1-DBL2x target common epitopes. (A) Anti-adhesion capacity in a dilution series of hyperimmune mice antisera on the binding of FCR3-BeWo infected erythrocytes to CSPG. The proportion of infected erythrocytes binding to CSPG in the presence of the indicated dilutions of the D75 antiserum is shown compared to control binding without competition. 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 indicates the binding of infected erythrocytes to bovine serum albumin. (B) Competitive recognition of recombinant NTS-DBL1x-Id1-DBL2x between specific antibodies induced by genetic immunization versus protein immunization. Sera from protein-vaccinated mice and the corresponding pre-bleed are the competing antibodies. D75 serum from DNA vaccinated rabbit is used as non-competing antibodies. (C) Competitive recognition of recombinant NTS-DBL1x-Id1-DBL2x between antibodies produced by genetic immunization in rabbits and naturally acquired in the plasma of malaria-exposed pregnant women from Benin. The competing sera are: D75 serum from DNA vaccinated rabbit, and the corresponding rabbit pre-immune serum. 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-DBL1x-Id1-DBL2x recombinant protein. ELISA was carried out on plates coated with 0.5 mg/mL of PfAMA1 (A) and recombinant NTS-DBL1x-Id1-DBL2x domain of VAR2CSA (B). The IgG plasma levels are expressed as optical densities (OD) and are shown for unexposed pregnant French women (UPFW, n=20), malaria-exposed Beninese men (EMB, n=20), malaria-exposed Senegalese children (ECS, n=20), malaria-exposed Senegalese men (EMS, n=20), and two malaria-exposed pregnant women areas: Benin [primigravidae, (EPGB, n=20) and multigravidae (EMGB, n=20)] and Senegal [primigravidae, (EPG, n=20) and multigravidae (EMG, n=20)].
• Figure 6 shows that naturally acquired IgGs against VAR2CSA NTS-DBL1x-Id1-DBL2x target strain-transcendent anti-adhesion epitopes. Specific human IgGs to NTS-DBL1x-Id1-DBL2x were affinity-purified from a pool of plasma from 10 multigravid women that previously showed anti-adhesion capacities. The FCR3-BeWo and HB3-BeWo infected erythrocytes were incubated with different concentrations (12.5, 25 or 50 µg/mL) of the purified human anti-NTS-DBL1x-Id1-DBL2x IgG and the activity was compared to binding without competitor or soluble competing CSA. None of the infected erythrocytes bound to BSA.
• Figure 7 shows the functional capacity of the antibodies targeting different portions of the N-terminal region of VAR2CSA. The graph shows the ability of immune serum to specific constructs to inhibit binding to CSA by VAR2CSA expressing FCR3 and HB3 infected erythrocytes.
• Figure 8 is a scheme showing the different portions of the NTS-DBL1x-Id1-DBL2x that have been tested to further refine the important protective epitope region.

The present invention generally relates to the use of specific regions of the extracellular domain of the VAR2CSA protein of the FCR3 parasite line for prevention and/or treatment of pregnancy-associated malaria.

Are described herein, specific polynucleotides and polypeptides derived from the extracellular domain of VAR2CSA and involved in pregnancy-associated malaria, and their use in the treatment and/or prevention of pregnancy-associated malaria. The var2csa gene has been isolated and sequenced for several parasite strains, including FCR3 (GenBank Accession Number: AY372123). The sequence of the corresponding VAR2CSA has been deduced (GenBank Accession Number: AAQ73926.1).

More specifically, isolated polypeptides are described that consist of the NTS-DBL1x-Idl-DBL2x region of the VAR2CSA protein, or a biologically fragment thereof that comprises at least the Id1-DBL2x region of the VAR2CSA protein.

The term "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 additionally meant that the polypeptide or polynucleotide of interest is produced, synthesized and/or purified by the hand of man.

The terms "protein", "polypeptide" and "polypeptide sequence" are used herein interchangeably. They refer to a sequence of amino acids (either in their neutral (uncharged) forms or as salts, and either unmodified or modified by glycosylation, side chain oxidation, or phosphorylation) that are linked through peptide bonds. The amino acid sequence may be a full-length native protein. Alternatively, the amino acid sequence may be a smaller portion of the full-length protein. The amino acid sequence may be 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 conversions of the chains such as oxidation of sulfydryl groups. Thus, the term "protein" (or its equivalent 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 significantly change its specific properties. In particular, the term "protein" encompasses protein isoforms, i.e., variants that are encoded by the same gene, but that differ in their pI or MW, or both. Such isoforms can differ in their amino acid sequence (e.g., as a result of allelic variation, alternative splicing or limited proteolysis), or in the alternative, may arise from differential post-translational modification (e.g., glycosylation, acylation, phosphorylation).

The terms "fragment", "portion" and "region" 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 molecule that 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-DBL1x-Id1-DBL2x is a fragment that retains the ability of the NTS-DBL1x-Id1-DBL2x region of VAR2CSA to induce the production of antibodies that prevent adherence of Plasmodium falciparum-infected erythrocytes to the placenta receptor CSA.

The terms "NTS-DBL1x-Id1-DBL2x", "NTS-DBL1x-DBL2x" and "NTS-DBL2x" are used herein interchangeably. They refer to a N-terminal sequence (NTS) of VAR2CSA consisting of the following subdomains: Duffy-binding-like domain 1x (DBL1x), interdomain 1 (Id1) and Duffy-binding-like domain 2x (DBL2x) of VAR2CSA. Preferably, NTS-DBL1x-Id1-DBL2x has the sequence corresponding amino acids 8 to 866 of VAR2CSA, i.e., has the sequence set forth in SEQ ID NO: 1, or a homologous sequence thereof.

The term "Id1-DBL2x" refers to a polypeptide consisting of the following subdomains: the interdomain 1 (Id1) and Duffy-binding-like domain 2x (DBL2x) of VAR2CSA. Preferably, Id1-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 similarity between two polypeptide molecules. When a position in both compared sequences is occupied by the same amino acid residue, the respective molecules 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 multiplied by 100. Generally, a comparison is 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 NCBI site or in Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI). Homologous amino acid sequences share identical or similar amino 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 physically or functionally similar to the corresponding reference residue, e.g. that have a similar size, shape, electric charge, chemical properties, including the ability to 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 et al. ("Atlas of Protein Sequence and Structure", 1978, Nat. Biomed. Res. Foundation, Washington, DC, Suppl. 3, 22: 354-352).

Also disclosed herein are 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 desirable properties. Thus, a fusion partner may be a protein that improves the expression of the NTS-DBL1x-Id1-DBL2x region, or biologically active fragment thereof, in host cells during preparation of the fusion protein, and/or a protein that facilitates purification of the fusion protein, and/or a protein that increases the stability (e.g., plasma stability) of the fusion protein (compared to the stability of the non-fused protein), and/or a protein that improves or facilitates 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 desirable biological or therapeutic activity.

Fusion partners that can be used 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, glutathione-S-transferase, thioredoxin, β-galactosidase, streptavidin, dihydrofolate reductase, pelB signal sequence, ompA signal sequence, signal sequence of alkaline phosphatase, green fluorescent protein (GFP), toxins such as, for example, E. Coli enterotoxin LT or B-subunit thereof, a domain of tetanus toxin fragment C, cholera toxin or B-subunit thereof, CTA1-DD. Other preferred fusion partners may be human growth hormone, an immunostimulating cytokine such as: interleukin-2 (IL-2), a growth factor such as granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), peptides or hormones such as: calcitonin, interferon-beta, interferon-alpha, glucagon like peptide 1 (GLP-1), glucagon like peptide 2 (GLP-2), PA toxin, parathyroid hormone (PTH(1-34) and PTH(1-84)), butyrylcholinesterase, glucocerebrosidase (GBA), and exendin-4.

Also described are isolated polynucleotides for use in the treatment or prevention of pregnancy-associated malaria, the polynucleotides consisting of a sequence encoding a polypeptide or a fusion protein as described herein and elements necessary to the in vitro or in vivo expression of said polypeptide or fusion protein. Preferably, the elements necessary to the in vitro or in vivo expression of the polypeptide or fusion protein are operably linked to the polynucleotide sequence to be transcribed.

The terms "nucleic acid sequence", "nucleic acid", nucleic acid molecule", "polynucleotide" and "oligonucleotide" are used herein interchangeably. They refer to a given sequence of nucleotides, modified or not, which defines a region of a nucleic acid molecule and which may be either under the form a single strain or double strain DNAs or under the form of transcription products thereof.

The terms "elements necessary to the in vitro or in vivo expression of the polypeptide" and "elements necessary to the in vitro or in vivo transcription of the polynucleotide" are used herein interchangeably. They refer to sequences known in the art that allow the expression, and optionally the regulation, of a polypeptide (or the transcription of the polynucleotide sequence encoding the polypeptide) in a host cell or in vivo. Such elements include at least a transcription initiation sequence (also called promoter) and a transcription termination sequence that are function 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.

The polynucleotides and polypeptides described herein may be prepared using any suitable method known in the art.

Techniques to isolate or clone a gene or a nucleotide sequence encoding a specific domain of a protein are known in the art and include, for example, isolation from genomic DNA, preparation from cDNA, or combination of these methods. Cloning a gene, or an acid nucleic sequence encoding a specific domain of a protein, from genomic DNA may be performed for example using a polymerase chain reaction (PCR) or by screening expression libraries to detect cloned DNA fragments with identical structural characteristics (Innis et al., "PCR: A Guide to Method and Application", 1990, Academic Press: New York). Other amplification methods of nucleic acid molecules known in the art may be used, such as for example, ligase chain reaction (LCR), ligation activated transcription (LAT) and Nucleic Acid Sequence Based Amplification (NASBA). It is also possible to use a chemical method of synthesis to prepare a polynucleotide sequence. Chemical methods of synthesis of DNA or RNA strains are known to those skilled in the art, and involve the use of commercially available automatic synthesizers.

Methods to prepare 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.), 1997, Academic Press: San Diego, CA) and recombinant methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Press: Cold Spring, NY) using host cells.

A recombinant method for the production of NTS-DBL1x-Id1-DBL2x is described in the Examples provided below.

Also disclosed herein are cloning or expression vectors that allow expression of NTS-DBL1x-Id1-DBL2x, or biologically active fragments thereof, in host cells. More specifically, are disclosed herein cloning or expression vectors comprising at least one polynucleotide or one fusion protein described herein. The cloning or expression vectors may be phages, plasmids, cosmids or viruses.

Also described are host cells transformed or transfected with a polynucleotide or cloning or expression vector described herein. Such host cells may be bacteria, yeast, insect cells or mammal cells.

Polypeptides and polynucleotides described herein are particularly suitable for use as drugs in the management of malaria in pregnant women. Indeed as demonstrated in the Examples section, the polypeptides described herein are antigenic regions of the VAR2CSA protein involved in the acquisition of protective immunity against the placental sequestration that takes place during pregnancy-associated malaria; and the polynucleotides described herein encode these antigenic regions. They may be used as such, or under a modified form, as an immunogenic composition or a vaccine.

A suitable modification of polypeptides described herein is conjugation. Conjugates comprise at least one polypeptide described herein linked to a carrier. Conjugates may be obtained by coupling the polypeptide to a physiologically 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 preparation of such conjugates are known in the art. For example, international application number WO 2006/124712 describes methods of preparation of conjugates comprising a plurality of antigenic peptides of Plasmodium falciparum linked to a protein carrier that improves the antigens immunogenicity.

Preferred carriers include, but are not limited to, viral particles, lipids such as for example C16-C18 lipids, polylysines, poly(DL-alanine)-poly(Lysine)s, nitrocellulose, polystyrene microparticles, latex beads, biodegradable polymers, polyphosphoglycane microparticles, protein carriers such as OPMC (outer membrane protein complex of Neisseria meningitidis) or improved OPMC, BSA (bovine serum albumin), TT (tetanus toxoid), ovalbumin, KLH (heyhole limpet hemocyanin), THY (bovine thyroglobulin), HbSAg and HBcAg of hepatitis B virus, rotavirus capside protein, protein L1 of human papilloma virus, VLP (virus like particle) of types 6, 11 and 16, tuberculin PPD (purified protein derivative).

The polypeptides, fusion proteins, conjugates, polynucleotides and vectors described herein may advantageously be used as therapeutic agents, in particular formulated as immunogenic compositions or vaccines.

An immunogenic composition described herein 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 interfere with the effectiveness of the biological activity of the active ingredient(s) and which is not excessively toxic to the individual at the concentration at which it is administered. The term includes solvents, dispersion, media, coatings, antibacterial and antifungal agents, isotonic agents, and adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art (see for example "Remington's Pharmaceutical Sciences", E.W. Martin, 18th Ed., 1990, Mack Publishing Co.: Easton, PA).

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

Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents, and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion 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 isotonic sodium chloride solution. In addition, sterile, 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 preparation of injectable formulations. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.

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

In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the ingredient from subcutaneous or intramuscular injection. Delaying absorption of a parenterally administered active ingredient may be accomplished by dissolving or suspending the ingredient in an oil vehicle. Injectable depot forms are made by forming micro-encapsulated matrices of the active ingredient in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active ingredient to polymer and the nature of the particular polymer employed, the rate of ingredient release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the active ingredient in liposomes or microemulsions which are compatible with body tissues.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, 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, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cotton seed, ground nut, 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, sweetening, flavouring, and perfuming agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Examples of suitable liquid carriers for oral administration include water (potentially containing additives as above, e.g., cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols such as glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For pressurized compositions, the liquid carrier can be halogenated hydrocarbon 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 ingredients may be mixed with at least one inert, physiologically 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, gelatine, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (h) absorbents such as kaolin and bentonite clay; and (i) lubricants such as talc, calcium stearate, magnesium stearate, 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. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium 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 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 ingredient by either passive or active release mechanisms. Transdermal administrations include all administrations across the surface of the body and the inner linings of bodily passage including epithelial and mucosal tissues. Such administrations may be carried out using the present compositions in lotions, creams, foams, patches, suspensions, and solutions.

Transdermal administration may be accomplished through the use of a transdermal patch containing active ingredients and a carrier that is non-toxic to the skin, and allows the delivery of the ingredient for systemic absorption into the bloodstream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may be suitable. A variety of occlusive devices may be used to release the active ingredient into the bloodstream such as a semi-permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.

Preferably, the immunogenic compositions and vaccines described herein may comprise one or more adjuvants used in combination. Examples of suitable classical adjuvants include Montanide et/ou l'Alum. Other suitable adjuvants include, but are not limited to, incomplete Freund's adjuvant, QS21, SBQS2, MF59, mLT, PHL, CpG DNA, calcium phosphate, dehydrated calcium sulfate, PLG, CT, LTB, CT/LT, AS02A, aluminium phosphate, aluminium hydroxide, monophosphoryl lipid A (MPL), a saponin, vitamin A, and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol, Quil A, Ribi Detox, CRL-1005, L-121 and combinations thereof.

Immunogenic compositions and vaccines described herein may further comprise at least one antigen specific of preerythrocytic stages (CSP, TRAP, LSA-1, LSA-3, SALSA, STARP, EXP-1), asexual erythrocytic stages (MSP-1, MSP-3, AMA-1, EBA-175, GLURP, MSP-2, MSP-4, MSP-5, RAP-2, RESA, PfEMP-1, synthetic GPI toxin) or sexual erythrocytic sages (PfS25).

A vaccine against pregnancy-associated malaria described herein 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 erythrocytes to CSA. In particular, is described herein a DNA vaccine (also called plasmid vaccine or polynucleotide vaccine) against placental malaria. Is also described herein a protein vaccine (also called polypeptide vaccine) against placental malaria.

More specifically, is described herein a protein vaccine comprising a polypeptide consisting of the NTS-DBL1x-Id1-DBL2x region of the VAR2CSA protein, or a biologically active fragment thereof comprising at least the Id1-DBL2x region of the VAR2CSA protein. The NTS-DBL1x-Id1-DBL2x region may have the sequence set forth in SEQ ID NO: 1, or a homologous sequence thereof. The Id1-DBL2x region may have the sequence set forth in SEQ ID NO: 2, or a homologous sequence thereof. The polypeptide may be fused to at least one fusion partner, as described herein.

The administration of a protein vaccine described herein may be performed using any suitable route, such as for example, intravenously, sub-cutaneously, intradermically, orally, topically or systemically.

Also described herein is a DNA vaccine against pregnancy-associated 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 nucleotide 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, intramuscular injection, particle "bombardment" to the skin, and nasal administration. The DNA penetrates 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 initiates 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 presents important advantages in terms 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 presentation 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, is described herein a DNA vaccine comprising a naked DNA, in particular a circular vaccinal plasmid (either super-coiled or not) or a linear DNA molecule, comprising and expressing in vivo a nucleotide sequence encoding a polypeptide consisting of the NTS-DBL1x-Id1-DBL2x region of the VAR2CSA protein, or a biologically active fragment thereof comprising at least the Id1-DBL2x region of VAR2CSA. The term "naked DNA", as used herein, has its art understood meaning and refers to a DNA transcription unit under the form of a polynucleotide sequence comprising at least one nucleotide sequence encoding a vaccine antigen and elements necessary to the expression of the nucleotide sequence in vivo. Polynucleotides described herein may advantageously be inserted into a plasmid such as DNA-CSP, Nyvac pf7, VR1020, VR1012, etc.

The elements necessary to the expression of a nucleotide sequence in vivo include, but are not limited to, a promoter or transcription initiation region, and a transcription termination region that are functional in a human cell. In addition, sequences that increase the genetic expression, such as introns, "enhancer" sequences and "leader" sequences are often necessary for the expression of a sequence encoding an immunogenic protein. As known in the art, these elements are preferably operably linked to the nucleotide 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, Moloney virus promoter, cytomegalovirus (CMV) promoter, Epstein-Barr 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, biodegradable microcapsules, immunostimulating complexes, liposomes, cationic lipids, and live, attenuated vaccine vectors such as viruses and bacteria.

A DNA vaccine described herein may also be administered in combination with an agent that improves or favors the penetration 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 penetration of a vaccine genetic material into cells include, but are not limited to, esters of benzoic acid, anilides, amidines, 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 polymers or cationic lipids), physical technics such as electroporation, sonoporation, magnetofection, etc, or using viral vectors such as adenoviruses, etc.

The immunogenic compositions and vaccines described herein may be used to immunize female human beings (and more specifically prepubertal girls and women in age of bearing children, in particular postpubertal girls or primigravidae) with the goal of preventing pregnancy-associated malaria.

Consequently, are described herein methods of treatment or prevention of pregnancy-associated malaria. More specifically, is disclosed herein a method for inducing a protective immune response against Plasmodium falciparum in a female 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. Also disclosed is a method of vaccination of a female human being against pregnancy-related malaria, the method comprising a step of administering, to the female human being, an effective amount of a vaccine described herein, in 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 sufficient to fulfil its intended purpose(s). For example, the purpose(s) may be: to prevent pregnancy-associated malaria, and/or to induce the production of antibodies that inhibit binding of P. falciparum-infected erythrocytes to placental 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 route (e.g., orally, parentally, mucosally). A DNA vaccine may be administered intramuscularly, intradermically or mucosally. A protein vaccine may be administered intraveinously, sub-cutaneously, intradermally, orally, topically or systemically.

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

Also described herein are pharmaceutical packs or kits for the prevention of pregnancy-associated malaria. More specifically, a pharmaceutical pack or kit comprises materials that are necessary to perform a vaccination as described herein. Generally, a kit comprises an immunogenic composition or vaccine described herein, 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 ingredients of the immunogenic composition or vaccine, allowing administration according to a method described herein. Different ingredients of a pharmaceutical pack or kit may be supplied in a solid (e.g., lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Pharmaceutical packs or kits may include media for the reconstitution of lyophilized ingredients. Individual containers of the kits will preferably be maintained in close confinement for commercial sale.

Optionally associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating 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 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 present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for purposes of quality control, inventory control, tracking movement between workstations, etc.

Unless specified otherwise, all the technical and scientific terms used herein have the same meaning as that generally understood by a regular expert in the field of this invention. Similarly, any publications, patent applications, patents and any other references 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.

The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or data were actually obtained. Some of the results presented below have been described in a scientific paper (Bigey et al., "The NTS-DBL2x region of VAR2CSA induces cross-reactive antibodies that inhibit adhesion of Plasmodium isolates to Chondroitin-Sulfate A", J. Infect. Dis., 2011, 204(7): 1125-1133).

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

In the study presented in this section, the possibility of identifying functionally important VAR2CSA regions that can induce IgGs with high adhesion inhibitory capacity has been investigated. Using intramuscular plasmid DNA electrotransfer, antibodies induced against a specific region of VAR2CA, the NTS-DBL1x-Id1-DBL2x, were shown to efficiently block parasite binding to CSA at a similar level as antibodies induced against the full-length extracellular domain of VAR2CA. The present work highlights an important achievement towards development of a protective vaccine against placental malaria.

Parasites and Human Plasma.In vitro-propagated P. falciparum parasites FCR3, and HB3 grown in O+ erythrocytes without human serum, 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 erythrocytes was tested on unselected cultures and cultures selected for infected erythrocytes adhesion to CSA. Cultures were selected following several panning on choriocarcinoma cell line BeWo, as described (Haase et al., Infect. Immun., 2006, 74: 3035-3038).

Primary field P. falciparum isolates and plasma samples were collected from a cohort of pregnant women enrolled in the ongoing STOPPAM project in the district of Comé, located 70 km West from the economical capital of Benin, Cotonou (Huynth et al., Malar. J., 2011, 31;10(1): 72). The isolates were obtained either from the peripheral blood of children below the age of 5 years (N=5) and from pregnant women (N=24), or from placental blood at delivery (N=6). Peripheral blood isolates were maintained in vitro for no more than 48 hours before testing.

Plasma samples from a previous study conducted in Senegal were also used (Tuikue Ndam et al., J. Infect. Dis., 2004, 190: 2001-2009).Animal Immunization and Antibody Screening. The var2csa gene from the FCR3 parasite genome and a corresponding synthetic gene with a codon-optimized sequence (GenBank accession no. GU249598) as described in Khunrae et al. (Khunrae et al., J. Mol. Biol., 2010, 397: 826-8340) were used as cloning templates. Using constructs comprised of single and multiple domains of VAR2CA proteins, sequences were cloned into a pVax1 vector backbone (Invitrogen) in which the original cytomegalovirus (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 et al., PLoS One, 2011, 7:6(2): e16622). 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 µg) in saline was injected into the tibial cranial muscle. After injection, transcutaneous electric pulses (8 pulses of 200 V/cm and 20 ms duration at a frequency of 2 Hz) were applied by two stainless steel external plate electrodes placed about 5 mm apart, at each side of the leg (Trollet et al., Infect. Immun., 2009, 77: 2221-2229).

For rabbit immunization, animals 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 µg of plasmid DNA in plasmid were injected in 5 different sites of each longissimus dorsi muscle with a 3-needle electrode device. After injection, electrical pulses (8 pulses 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.

All animals (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 intraperitoneal injection of 10 µg of the recombinant protein in 50 mL, mixed with an equal volume of Alugel. Mice 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 Preparation. Total IgG was manually purified from final bleed mice/rabbit sera on a Hi-Trap Protein G HP column according to the manufacturer's 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 manufacturer's recommendations.Antibody Reactivity with P. falciparum Laboratory Lines and Field Isolates.In vitro-propagated P. falciparum parasites FCR3 and HB3 were repeatedly 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 erythrocytes (FCR3-BeWo, HB3-Bewo) and 35 primary field P. falciparum isolates collected at Comé, southwestern Benin (Yadouleton et al., Malaria J., 2010, 9: 204) were analyzed to determine the reactivity of the antibodies generated.

Flow cytometry (FACS Calibur, Beckman Coulter) was used to test the reactivity of sera of vaccinated animals to the surface of infected erythrocytes, as previously described (Barfod et al., J. Immunol., 2010, 185: 7553-7561). In brief, CSA-selected parasite cultures or field parasite isolates were enriched to contain late trophozoite and shizont stage parasites by exposure to a strong magnetic field (VarioMACS and CS columns, Miltenyi). Aliquots (2 x 10⁵ infected erythrocytes) were labelled using ethidium bromide and sequentially exposed to mouse/human serum and anti-mouse/human IgG-FITC (Invitrogen). All samples relating to a particular parasite isolate were processed and analyzed in a single assay.Protein Expression, Purification and Evaluation. The NTS-DBL1x-Id1-DBL2x region of the var2csa gene from FCR3 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 Bakpb6 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 2^nd amplification of the recombinant virus particles. After 2 days of induction, the cells were centrifuged (8.000g, 4°C, 10 minutes) and the supernatant was filtered using two 10 kDa NMWC PES membranes (0.45 µm) (GE Healthcare). The supernatant was then concentrated to 30 mL and diafiltered six times on an ÄKTA crossflow (GE Healthcare) with buffer A (10 mM sodium phosphate, pH 7.4, 500 mM NaCl). The retentate was recovered from the system and filtered (0.2 µm). Before loading on the HisSelect column, imidazole (Sigma-Aldrich) (150 µL, 1 M, pH 7.4) was added to the sample, giving a final imidazole concentration of 15 µM. The bound protein was eluted with buffer A + 200 mM imidazole (HisSelect). The eluted protein was subjected to gel filtration.

Specific recognition of the purified protein was evaluated in ELISA using plasma samples from pregnant women of Benin and Senegal, unexposed pregnant French women, and malaria-exposed children (from Senegal) and men (from Benin and from Senegal).Inhibition of Infected Erythrocytes to CSPG by Specific IgG. The static assays employed to evaluate the capacity of the antibodies to interfere with CSA-specific adhesion of infected erythrocytes was described in detail elsewhere (Fried et al., Methods Mol. Med., 2002, 72: 555-560). In this assay, plates were coated overnight at 4°C with 20 µL of ligand: 1% BSA, 5 µg/mL decorin: CSPG (Chondroitin Sulfate Proteoglycan, Sigma) or 50 µg/mL bovine CSA (Sigma) diluted in PBS. Each spot was subsequently blocked with 3% BSA in PBS for 30 minutes at room temperature. Late-stage-infected erythrocytes were also blocked in BSA/RPMI for 30 minutes at room temperature. Parasite suspensions adjusted to 20% parasite density were incubated with serum (1:5 final dilution) or purified IgG (0.01 mg/mL to 1 mg/mL final concentration) or 500 µg/mL soluble CSA for 30 minutes at room temperature before they were allowed to bind to ligand for 15 minutes at room temperature. Non-adhering cells were removed by an automated washing system. Spots were fixed with 1.5% glutaraldehyde in PBS and adhering infected erythrocytes was quantified by microscopy.Competition ELISA. Prior to competition ELISA, the anti-NTS-DBL1x-Id1-DBL2x IgG titer was determined in plasma pools composed of samples from exposed multigravid women from Benin, DNA-vaccinated rabbits and protein-immunized mice (plasma pools from D75). Microtiter plates (Nunc) were coated with recombinant NTS-DBL1x-Id1-DBL2x (0.5 µg/mL in PBS). After the plates were saturated with blocking buffer (PBS, 0.5 M NaCl, 1% Triton X-100, 1% BSA) for 1 hour at room temperature, they were incubated for 1 hour at room temperature with increasing dilutions of the competing plasma (plasma pools from D75 DNA-vaccinated rabbits or protein-vaccinated mice against NTS-DBL1x-Id1-DBL2x). Pre-immune sera pools of rabbit or mouse or plasma pool from unexposed French pregnant women were used as negative control. Plates were washed four times with washing buffer (PBS, 0.5 M NaCl, 1% 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 malaria-exposed Beninese multigravidae) for 1 hour at room temperature. The specific secondary antibody directed against the non-competing antibodies (goat anti-human IgG HRP, Sigma-Aldrich, goat anti-mouse IgG HRP or goat anti-rabbit IgG HRP, Sigma) diluted 1:4000 in blocking buffer was added, and incubated for 1 hour at room temperature. After a 4-times washing, antibody reactivity of no-competing plasma/serum was visualized at 450 nm following the addition of TMB (tetramethylbenzidine). The percent reduction in antibody reactivity in the presence of a competitor was calculated as follows: 100 x [OD competitor antibody / OD without competitor antibody].

Plasmid DNA immunization induced high titer surface reactive antibodies. A total of 13 plasmids representing single and overlapping multiple domains of VAR2CSA from the FCR3 parasite line were constructed and used for immunization (Figure 1A). The single and overlapping domains of VAR2CSA that were tested are: NTS-DBL1x-Id1-DBL2x (corresponding to amino acids 8 to 866); DBL2x-Id2 (amino acids 446-1208); Id2-DBL3x (amino acids 870-1575); DBL3x-4ε (amino acids 1168-1987); DBL4ε(a) (amino acids 1576-1987); DBL4ε(b) (amino acids 1583-1989); DBL4ε-5ε (amino acids 1576-2313); DBL5ε (amino acids 1982-2313); DBL5ε-6ε (amino acids 1982-2673); DBL6ε (amino acids 2314-2673); NTS-DBL3x (amino acids 9-1572); DBL3x-DBL6ε (amino acids 1168-2673); and NTS-DBL6ε (amino acids 1-2673).

All immunizations with single to triple-domains constructs of VAR2CSA induced the formation of polyclonal antibodies with a high ELISA titer (> 1x10⁵) following intramuscular plasmid electrotranfer. However, for plasmids containing more than 3000 bp of coding sequence, effective humoral immune response in all vaccinated animals, both mice and rabbits, required the use of a codon-optimized sequence (GenBank Accession Number GU249598). Although all single and multi-domains of VAR2CSA could induce antibodies reacting with native VAR2CSA on the surface of the CSA adhering-erythrocytes infected with the homologous FCR3, constructs containing DBL1x, DBL2x, DBL5ε and DBL6ε were the most efficient in inducing surface reactive antibodies (Figure 1B). None of the polyclonal anti-VAR2CSA antisera recognized the erythrocytes infected with the non-CSA adherent FCR3 parasite line.

Antibodies induced against VAR2CSA inhibit binding of infected erythrocytes to chondroitin sulfate proteoglycan (CSPG). A Petri dish-based static binding assay was used to screen sera for their ability to inhibit parasite binding to CSA. Of all the FCR3 VAR2CSA regions tested, only sequences located between the N-terminal sequence (NTS) and the DBL3x appeared to induce inhibitory antibodies (Figure 1B). Highly inhibitory antibodies were obtained with the full-length extracellular VAR2CSA construct, which totally inhibited binding. Interestingly, similar inhibition was seen with sera from animals (both mice and rabbits) vaccinated with the NTS-DBL1x-Id1-DBL2x construct. In addition, the inhibitory activity of sera from NTS-DBL1x-Id1-DBL2x vaccinated animals was investigated on a heterologous parasite line; the CSA adherent HB3 line. The same pattern of inhibition was observed (Figure 2A).

To confirm that the inhibition observed with NTS-DBL1x-Id1-DBL2x antiserum was mediated by IgG, IgGs were purified and tested for binding inhibition activity. The purified IgGs recognized the surface of BeWo-selected FCR3 infected erythrocytes (Figure 2B). The purified IgGs inhibited 100% of the binding of infected erythrocytes to CSA at a concentration of 0.5 mg/mL (Figure 2D).

Antibodies induced against NTS-DBL1x-Id1-DBL2x specifically recognized isolates from pregnant women. Flow cytometry analysis clearly demonstrated that murine anti-NTS-DBL1x-Id1-DBL2x antibodies specifically recognize the surface of placental malaria parasites among the field isolates. Thirty five (35) isolates were analyzed by flow cytometry in this study, including 24 peripheral blood isolated from pregnant women. Six placental isolates and 21 of the 24 peripheral blood isolates from pregnant women were recognized by polyclonal murine antibodies while none of the 5 children isolates tested were labelled.

Of the 21 isolates from pregnant women that reacted with anti-NTS-DBL1x-Id1-DBL2x antibodies by flow cytometry, 16 showed specific adhesion to CSPG, while 5 isolates did not bind. Among the 3 peripheral blood isolates that were not labelled in flow cytometry, 2 bound to CSPG but their interaction could not be abrogated by soluble CSA, and 1 isolate did not bind.

Fifteen samples containing sufficient amount of parasite were further processed in binding inhibition assay. These comprised 14 isolates from peripheral blood samples and one placental isolate. The binding to CSA of 12 of the 15 pregnant women isolates tested as highly inhibited by specific anti-NTS-DBL1x-Id1-DBL2x sera (see Table 1 below).

Animals immunized with recombinant NTS-DBL1x-Id1-DBL2x or DNA electrotransfer produced antibodies of similar specificity. Murine polyclonal antibodies induced either by recombinant protein or plasmid DNA of NTS-DBL1x-Id1-DBL2x showed similar reactivity. The reactivity to erythrocytes surface and inhibitory activity on binding to CSA were similar on BeWo-selected FCR3 infected erythrocytes. The inhibitory activity was compared in dilution series of sera from mice immunized with either the full-length construct or NTS-DBL1x-Id1-DBL2x (both DNA and protein immunization). Down to the dilution 1:100, sera from mice vaccinated with full-length DNA construct or recombinant NTS-DBL1x-Id1-DBL2x totally inhibited binding of infected erythrocytes (Figure 4A). The inhibitory capacity of the serum samples following plasmid DNA immunization with the full-length construct or by protein vaccination with NTS-DBL1x-Id1-DBL2x was seen at subsequent dilutions, these sera were diluted 1:5000 before inhibition vanished (Figure 4A). This observation clearly strengthens the importance of the NTS-DBL1x-Id1-DBL2x region of VAR2CSA in eliciting adhesion-inhibitory antibodies by vaccination.

Antibodies induced in animals by vaccination with NTS-DBL1x-Id1-DBL2x target the same epitopes as naturally acquired antibodies. The recombinant NTS-DBL1x-Id1-DBL2x produced in insect cells was recognized by plasma from malaria exposed pregnant women from Benin and Senegal in a parity-dependent manner (Figure 5). This NTS-DBL1x-Id1-DBL2x was used in competition ELISA to analyze target epitopes among antibodies induced in animals by plasmid DNA immunization and protein immunization, as well as the naturally acquired antibodies against the NTS-DBL1x-Id1-DBL2x region of VAR2CSA in pregnant women. A mutual inhibition pattern was observed in the ability of all three antisera to recognize the recombinant NTS-DBL1x-Id1-DBL2x protein. The inhibition pattern between sera from DNA immunizations and protein immunizations was concentration-dependent (Figure 4B). A similar inhibition was observed when antibodies in a human plasma pool from exposed multigravidae competed with specific anti-sera from rabbits (Figure 4C).

The naturally acquired human IgG against VARCSA NTS-DBL1x-Id1-DBL2x inhibit adhesion of infected erythrocytes to CSA. Plasma samples from women included in the STOPPAM project are routinely analyzed for anti-adhesion capacity on the FCR3-BeWo parasite lines. The recombinant NTS-DBL1x-Id1-DBL2x protein was used to affinity-purity IgG from plasma of malaria-exposed Beninese pregnant women (selected for having a high anti-adhesion activity on CSA-binding parasite lines). Interestingly, naturally-acquired antibodies targeting the NTS-DBL1x-Id1-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 specific VAR2CSA region have been shown to inhibit P. falciparum infected erythrocytes binding to CSA.

Molecular details of the interaction of the P. falciparum ligand VAR2CSA with the placental receptor CSA are currently not well delineated, but recent studies suggest that the binding site depends on a higher-order architecture in which DBL domains and the interdomain regions of VAR2CSA fold together to form a ligand-binding pocket (Khunrae et al., J. Mol. Biol., 2010, 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 infected erythrocytes to CSA in vitro (Khunrae et al., J. Mol. Biol., 2010, 397: 826-834). This suggests that protective immunity to placental malaria acquired over a few pregnancies in areas of intense P. falciparum transmission that correlates with levels of anti-adhesion antibodies (Duffy et al., Infect. Immun., 2003, 71: 6620-6623) is mostly mediated by anti-VAR2CSA IgGs. Nevertheless, a recent work reports that immunization with full-length VAR2CSA did not induce potent cross-inhibitory antibodies (Avril et al., PLoS One, 2011, 7:6(2): e16622).

Although antibody response may directly inhibit infected erythrocytes adhesion placenta, it also might be implicated in opsonization (Keen et al., PLoS Med, 2007, 4(5):e181; Feng et 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 have shown that it is a polymorphic protein composed of alternating areas of substantial interclonal polymorphism (Bockhorst et al., Mol. Biochem. Parasitol., 2007, 155: 103-112; Fernandez et al., Malaria J., 2008, 7: 170). The rationale for developing an effective VAR2SA-based vaccine against placental malaria thus requires definition of VAR2CSA areas containing functionally important epitopes that transcend this interclonal diversity. In the present study, 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 resurgence in interest for such concept observed in the last few years is due to several technical improvements such as codon optimization strategies, novel formulations and more effective delivery approaches. The delivery of electrical pulses after intramuscular plasmid DNA infection particularly enhanced DNA uptake and resulted in a stronger and more specific humoral response when the antigen was fused to a leader sequence (Trollet et al., Infect. Immun., 2009, 77: 2221-2229). Several clinical trials 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 present study, a strong immune response was obtained both in mice and in rabbits vaccinated with VAR2CSA genetic fragments that were fused to mEPO leader sequence. Interestingly, all antibodies induced were able to recognize the native protein expressed on the surface of erythrocytes 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 part of the protein induced a robust humoral response that completely blocked infected erythrocytes binding to CSPG. However, the major finding of this study is that a shorter construct of the N-terminal moiety of VAR2CSA corresponding to NTS-DBL1x-Id1-DBL2x was able to induce high potency antibodies with similar inhibitory capacity as those elicited against the full-length VAR2CSA. Moreover, competition ELISA analysis revealed that antibodies raised by experimental immunization (plasmid DNA or purified recombinant protein) or those naturally acquired by pregnant women to this particular region of VAR2CSA predominantly target similar epitopes. This result is in line with others that reported that pregnant 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 reproduce, at least partially, natural acquired immunity against placental malaria.

Recombinant NTS-DBL1x-Id1-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 exhibits important targets of the immune response against VAR2CSA. Murine polyclonal antibodies raised against this construct from the FCR3 parasite strain stained the surface of most isolates from pregnant women of Benin. Remarkably, antibodies raised against a single variant of NTS-DBL1x-Id1-DBL2x showed consistent inhibitory activity against several isolates originating from pregnant women. Actually, the binding of infected erythrocytes to CSPG/CSA of 12 out of the 15 pregnant women isolates tested was inhibited by more than 50%. This highlights the existence of functionally important epitopes within this region of VAR2CSA that are shared by most placenta-sequestering P. falciparum isolates. However, all isolates were not inhibited as a probable consequence of antigenic polymorphism. Possible mechanisms of action include that anti-NTS-DBL1x-Id1-DBL2x antibodies inhibit infected erythrocytes adhesion to CSPG/CSA 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 mediating the binding of native VAR2CSA to CSA (Nielsen et al., Infect. Immun., 2009, 77: 2482-2487).

The results presented here clearly indicate that antibody recognition of just a few VAR2CSA variants containing key epitopes might be sufficient to markedly affect the binding of VAR2CSA-expressing infected erythrocytes to CSA.

Of particular interest, maternal antibodies purified with the recombinant NTS-DBL1x-Id1-DBL2x reacted with both BeWo-selected FCR3 and HB3 strains, 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 significance in the acquired immune protection to placental malaria. It was recently shown that some VAR2CSA-specific human monoclonal IgGs from P. falciparum-exposed women can exhibit some moderate degree of adhesion inhibition that increases with their combination (Barfod et al., J. Immunol., 2010, 185: 7553-7561). To the best of the inventor's knowledge, the present study clearly shows functional evidence on a specific 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 region of VAR2CSA was a major target of anti-adhesion immune response in placental malaria, and therefore an attractive vaccine target. Further studies are required to ascertain the impact of sequence variation within this particular VAR2CSA region to its potential for cross-reactivity.

In the study presented in this section, the inventors have investigated the possibility of identifying functionally important VAR2CSA regions, in particular functionally important regions of the NTS-DBL1x-Id-DBL2x portion of VAR2CSA, which can induce IgG with high adhesion inhibitory capacity.

To further refine the important protective epitope region, five additional constructs were built based upon the NTS-DBL1x-DBL2x sequence, encoding NTS-DBL1x, NTS-DBL1x-Id1, Id1, Id1-DBL2x, and DBL2x, as shown in Figure 8. DNA sequences encoding the subfragments of NTS-DBL1x-DBL2x were cloned into a pVAX1 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 1.

The results obtained are presented on Figure 7. All constructs but the Id1 successfully raised an immune response. The NTS-DBL1x, NTS-DBL1x-Id1 and Id1-DBL2x fragments raised high titer immune response, comparable to that obtained with the full extracellular part NTS-DBL1x-6ε. Among these constructs, binding inhibitory capacity of infected erythrocytes to CSA was not found with NTS-DBL1x antiserums, highlighting that the VAR2CSA minimal construct inducing anti-adhesion antibodies is beyond the DBL1x domain. However, the construct made of Id1 alone did not induce significant immune response. This refined experiment allowed the identification of Id1-DBL2x (corresponding to the sequence from amino acid 392 to amino acid 866 of VAR2CSA - i.e., SEQ ID NO: 2) as the minimal region concentrating the main anti-adhesion epitopes.

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

• <110> IRD Deloron, Philippe
• <120> Vaccines against pregnancy-associated malaria
• <130> BCT 110329 QT
• <150> FR 10 56294 <151> 2010-07-30
• <160> 2
• <170> PatentIn version 3.5
• <210> 1 <211> 858 <212> PRT <213> Plasmodium falciparum
• <400> 1
• <210> 2 <211> 475 <212> PRT <213> Plasmodium falciparum
• <400> 2