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EP2945648A1 - Impfstoffe gegen schwangerschaftsassoziierte malaria - Google Patents

Impfstoffe gegen schwangerschaftsassoziierte malaria

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Publication number
EP2945648A1
EP2945648A1 EP14701072.2A EP14701072A EP2945648A1 EP 2945648 A1 EP2945648 A1 EP 2945648A1 EP 14701072 A EP14701072 A EP 14701072A EP 2945648 A1 EP2945648 A1 EP 2945648A1
Authority
EP
European Patent Office
Prior art keywords
idl
sequence
protein
seq
var2csa
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14701072.2A
Other languages
English (en)
French (fr)
Inventor
Nicaise Tuikue Ndam
Philippe Deloron
Justin DORITCHAMOU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut de Recherche pour le Developpement IRD
Original Assignee
Institut de Recherche pour le Developpement IRD
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Publication of EP2945648A1 publication Critical patent/EP2945648A1/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of combinations of specific regions of the N-terminal portion of the VAR2CSA protein from different families or parasitic lines of Plasmodium jalciparum in the prevention and / or treatment of gestational malaria.
  • Malaria (or malaria) is the most common parasitic infection observed in the world. It is due to a parasite of the genus Plasmodium, which is transmitted during the bite by a female mosquito (Anopheles). Several species of Plasmodium can infect humans, but Plasmodium jalciparum is the most common and pathogenic species and the one responsible for fatal cases. Once introduced into the bloodstream, the parasite infects liver cells, in which it proliferates, before circulating again in the blood and invading red blood cells (erythrocytes or red blood cells). Malaria affects some 100 countries worldwide, particularly in the less-favored tropical areas of Africa, Asia and Latin America; Africa being by far the most affected continent.
  • malaria infection can cause a range of adverse effects: spontaneous abortion, premature delivery, low birth weight, congenital transmission, and neonatal death. In areas of Africa where malaria is endemic, 3 to 5% of infant deaths at birth can be attributed to gestational malaria. In addition, it also presents a real danger for the mother who can suffer from anemia sometimes severe or even fatal.
  • sulfadoxine-pyrimethamine Cot et al, Br Med Med, 2003, 67: 137-148.
  • the erythrocytes are then able to attach to the inner wall of the blood vessels, thereby preventing the transport of infected erythrocytes to the purifying organs of the immune system, one of whose roles is to destroy the cells recognized as abnormal.
  • parasitized erythrocytes attach to a sugar, chondroitin sulfate A (CSA), present in the placenta.
  • CSA chondroitin sulfate A
  • women acquire protective antibodies that block this adhesion.
  • One of the vaccine strategies is to recreate this protective immunity by blocking the adhesion of infected erythrocytes to the placenta.
  • the protein VAR2CSA one of the proteins of the PfEMP1 family, is currently the subject of much research with a view to obtaining a specific vaccine for pregnant women (Tuikue Ndam et al., J. Infect Dis., 2005, 192: 331-335, Chia et al., J. Infect Dis., 2005, 192: 1284-1293, Tuikue Ndam et al, J.
  • the present inventors have previously identified the N-terminal region of VAR2CSA, and in particular the NTS-DBLlx-Idl-DBL2x region consisting of the DBLlx domain, Interdomain Idl, and the DBL2x domain, as being that which contains epitopes capable of inducing antibodies blocking the adhesion of Plasmodium falciparum-infected red cells to the CSA, and the Idl-DBL2x subregion as the minimal antigenic region of VAR2CSA involved in the acquisition of protective immunity to placental sequestration that occurs in gestational malaria (WO 2012/014073).
  • the inventors By analyzing the sequences of parasitic isolates from pregnant to benign women, the inventors also demonstrated a segregation of parasitic variants in interdomain ID1 of VAR2CSA. This new area of dichotomy has never been described so far.
  • the sequence alignments led to the establishment of two consensus sequences representative of the Idl interdomain of VAR2CSA: the first Idl A (or SEQ ID NO: 11) corresponds to a newly identified cluster, and the second IdlB (or SEQ ID NO: 12) corresponds to the other group of sequences (which include the FCR3 line and the 3D7 line).
  • a first aspect of the present invention relates to the use of combinations of polypeptide sequences or polynucleotides corresponding to the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein from different Plasmodium parasitic families. falciparum, in the management of malaria of the pregnant woman.
  • the present invention relates to the use of isolated or purified polypeptide or polynucleotide sequences corresponding to the NTS-region.
  • the present invention relates to a combination of at least two polypeptides isolated or purified for use in the treatment or prevention of gestational malaria, the first isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of a first parasitic family of Plasmodium jalciparum, and the second isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of a second Parasitic family of Plasmodium jalciparum, for use in the treatment or prevention of gestational malaria.
  • the first family is the Plasmodium jalciparum parasitic family, the VAR2CSA protein of which is characterized by an interdomain Id1 which has the sequence sequence SEQ ID NO: 11 or which is encoded by the consensus nucleic sequence SEQ ID NO: 13, and the second family is the parasitic family of Plasmodium jalciparum whose VAR2CSA protein is characterized by an interdomain Id1 which has for sequence the consensus sequence SEQ ID NO: 12 or which is encoded by the consensus nucleic sequence SEQ ID NO: 14.
  • the second parasitic family of Plasmodium jalciparum comprises the parasitic line FCR3 and the parasitic line 3D7.
  • a combination according to the invention is characterized in that it consists of three isolated or purified polypeptides, the first isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the Plasmodium falciparum parasite family, the VAR2CSA protein of which is characterized by an Idl interdomain which is sequenced by the SEQ consensus sequence ID NO: 11 or which is encoded by the consensus nucleic acid sequence SEQ ID NO 13, the second isolated or purified polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line, and the isolated or purified third polypeptide consisting of the NTS-DBL1x-Id1-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line.
  • the NTS-DBL1x-Id1-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 1 or a homologous sequence of SEQ ID NO: 1, and the NTS-DBLlx-Idl region.
  • -DBL2x of the VAR2CSA protein of line 3D7 has the sequence SEQ ID NO: 5 or a homologous sequence of SEQ ID NO: 5.
  • VAR2CSA of the FCR3 line has the sequence SEQ ID NO: 3 or a homologous sequence of SEQ ID NO: 3
  • the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 7 or a homologous sequence
  • the present invention also relates to a combination of at least two isolated or purified fusion proteins for use in the treatment or prevention of gestational malaria, wherein the combination corresponds to a combination of at least two isolated or purified polypeptides as described herein wherein each of the polypeptides is fused to a fusion partner sequence.
  • each of the fusion partner sequences is independently selected from the group consisting of the maltose binding protein, the signal sequence of the maltose binding protein, an S-tag, glutathione-S-transferase, thioredoxin , ⁇ -galactosidase, streptavidin, dihydrofolate reductase, pelB signal sequence, ompA signal sequence, alkaline phosphatase signal sequence, green fluorescent protein, toxins, human growth hormone, interleukin -2 (IL-2), the granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), calcitonin, interferon-beta, interferon-alpha, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), PA toxin, parathyroid hormones (PTH (1-34) and PTH (1-84)), butyrylcholinesterase,
  • the invention also relates to a combination of at least two polynucleotides isolated or purified for use in the treatment or prevention of gestational malaria, wherein each of the isolated or purified polynucleotides encodes a polypeptide as defined above or for a fusion protein such as than previously defined. More specifically, the invention relates to a combination of at least two isolated or purified polynucleotides, the first isolated or purified polynucleotide encoding a first polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the protein.
  • the first family is the Plasmodium jalciparum parasitic family, the VAR2CSA protein of which is characterized by an interdomain Id1 which has the sequence sequence SEQ ID NO: 11 or which is encoded by the consensus sequence SEQ ID NO: 13 and the second family is the parasitic family of Plasmodium jalciparum whose VAR2CSA protein is characterized by an interdomain Id1 which has for sequence the consensus sequence SEQ ID NO: 12 or which is encoded by the consensus sequence SEQ ID NO: 14.
  • the second parasitic family of Plasmodium jalciparum comprises the parasitic strain FCR3 and the parasitic line
  • the combination according to the invention consists of three isolated or purified polynucleotides, the first isolated or purified polynucleotide encoding a first polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of the Plasmodium falciparum parasitic family, the VAR2CSA protein of which is characterized by an interdomain Id1 which has for sequence the consensus sequence SEQ ID NO: 11 or which is encoded by the consensus sequence SEQ ID NO: 13 or for a first protein of fusion comprising the first polypeptide, and containing the elements necessary for the expression of said first polypeptide or of said first fusion protein in vitro and / or in vivo; the second isolated or purified polynucleotide encoding a second polypeptide consist
  • the NTS-DBL1x-Id1-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 1 or a homologous sequence of SEQ ID NO: 1, and the NTS-DBLlx-Idl region.
  • -DBL2x of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 5 or a homologous sequence of SEQ ID NO: 5.
  • the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 3 or a homologous sequence of SEQ ID NO: 3
  • the Idl-DBL2x region of the VAR2CSA protein of the Line 3D7 has the sequence SEQ ID NO: 7 or a homologous sequence of SEQ ID NO: 7.
  • the isolated or purified second polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 2, a sequence homologous to SEQ ID NO: 2, SEQ ID NO: 4, a homologous sequence of SEQ ID NO: 4, SEQ ID NO: 6, a homologous sequence of SEQ ID NO: 6, SEQ ID NO: 8, and a homologous sequence of SEQ ID NO: 8.
  • a second and third isolated or purified polynucleotide is present in the second isolated or purified polynucleotide preferably comprises a sequence selected from the group consisting of SEQ ID NO: 2, a homologous sequence of SEQ ID NO: 2, SEQ ID NO: 4, a homologous sequence of SEQ ID NO: 4, and the third isolated or purified polynucleotide preferably comprises a sequence selected from the group consisting of SEQ ID NO: 6, a sequence homologous to SEQ ID NO: 6, SEQ ID NO: 8, and a homologous sequence of SEQ ID NO: 8.
  • the invention relates to an immunogenic composition
  • an immunogenic composition comprising at least one pharmaceutically acceptable carrier or excipient and at least one combination of polypeptides or fusion proteins or polynucleotides according to the invention.
  • an immunogenic composition is characterized in that it is capable of inducing antibodies that totally inhibit the adhesion of Plasmodium falciparum-infected erythrocytes to the placental CSA (chondroitin sulfate A) receptor.
  • the invention also relates to vaccines against gestational malaria.
  • the invention provides a vaccine comprising at least one combination of polypeptides or fusion proteins according to the invention, or at least one combination of polynucleotides according to the invention.
  • the polynucleotides of the combination present in a vaccine are inserted into at least one plasmid.
  • the vaccines according to the invention are characterized in that they are capable of inducing antibodies which totally inhibit the adhesion of Plasmodium falciparum infected erythrocytes to the placental CSA receptor.
  • the vaccines described herein may further include an adjuvant.
  • the invention in another variant of this aspect, relates to methods of treating or preventing gestational malaria.
  • the invention provides a method for inducing a protective immune response against Plasmodium falciparum in a human female, the method comprising a step of administering an effective amount of a composition immunogen or vaccine described herein.
  • the invention also provides a method of vaccinating a female human against Plasmodium falciparum, the method comprising a step of administering an effective amount of a vaccine, particularly a DNA vaccine described herein or a protein vaccine described here.
  • Methods of treatment and prevention of gestational malaria are primarily aimed at pre-pubertal girls and women of childbearing age.
  • a method of treating or preventing gestational malaria is characterized in that it induces, in female humans, antibodies that prevent the adhesion of Plasmodium falciparum-infected erythrocytes to CSA placental receiver.
  • administration of the immunogenic composition or vaccine can be by any suitable route.
  • the invention in another aspect, relates to a gestational malaria vaccination kit comprising at least one combination, or at least one immunogenic composition or at least one vaccine according to the invention and instructions for performing a vaccination against gestational malaria.
  • the at least two polypeptides or at least two fusion proteins or at least two polynucleotides of the combination, immunogenic composition or vaccine are separately provided in the kit.
  • the present invention relates to the use of combinations of specific regions of the N-terminal portion of the VAR2CSA protein from different parasitic families of Plasmodium falciparum, in the prevention and / or treatment of gestational malaria. .
  • the present invention relates to combinations of at least two polynucleotides or at least two polypeptides derived from the N-terminal portion of the extracellular domain of VAR2CSA of NTS-DBLlx-Idl-DBL2x and Idl-DBL2x. different parasitic families of Plasmodium falciparum, as well as their applications in the management of gestational malaria.
  • the invention relates to combinations of at least two polynucleotides or at least two polypeptides derived from the N-terminal region of the extracellular domain of VAR2CSA from two parasitic families of Plasmodium falciparum demonstrated by the inventors, and of which the segregation takes place in interdomain Idl of VAR2CSA.
  • the first family which is a newly identified cluster is characterized by a VAR2CSA protein having an Idl interdomain having for sequence the consensus sequence SEQ ID NO: 11 or which is encoded by the consensus nucleic sequence SEQ ID NO: 13.
  • gaTTAtaTAAAGgaTgATCCTTATTCC gaTTAtaTAAAGgaTgATCCTTATTCC.
  • GTTCTTTTGGAACA TCgTtTTCTTATgAAAaTAGTgTAA ...
  • the second family which is a cluster including the FCR3 line and the line
  • 3F7 is characterized by a VAR2CSA protein having an Idl interdomain having for sequence the consensus sequence SEQ ID NO: 12 or which is encoded by the consensus nucleotide sequence SEQ ID NO: 14.
  • SEQ ID NO: 12 is characterized by a VAR2CSA protein having an Idl interdomain having for sequence the consensus sequence SEQ ID NO: 12 or which is encoded by the consensus nucleotide sequence SEQ ID NO: 14.
  • the second parasitic family of Plasmodium falciparum demonstrated by the inventors comprises the parasitic strain FCR3 and the parasitic line 3D7.
  • VAR2CSA has been isolated from several parasitic strains including FCR3 (GenBank Accession Number: AY372123) and 3D7 (GenBank Accession Number: AE014188.3) and sequenced.
  • the corresponding VAR2CSA protein has been deduced (GenBank Accession Number: AAQ73926.1 for the FCR3 line and GenBank Accession Number: AAN36095.1 for the 3D7 line).
  • isolated or purified refers to a polypeptide or polynucleotide which, by its origin or manipulation, is separated from at least some components with which it is naturally associated. Alternatively or additionally, by “isolated or purified” is meant a polypeptide or polynucleotide that is produced or synthesized by humans.
  • NPS-DBLlx-DBL2x and “NTS-DBLlx-Idl-DBL2x” are here used interchangeably.
  • VAR2CSA constituted by the domains: Duffy-binding-like domain lx (DBLlx), interdomain 1 (Idl) and Duffy-binding-like domain 2x (DBL2x).
  • DBLlx Duffy-binding-like domain lx
  • Idl interdomain 1
  • DBL2x Duffy-binding-like domain 2x
  • Idl-DBL2x refers to the VAR2CSA region consisting of interdomain 1 (Idl) and the Duffy-binding-like domain 2x (DBL2x).
  • the polypeptide of a combination according to the invention consisting of the NTS-DBLlx-Idl-DBL2x region of the VAR2CSA protein of the parasitic line FCR3 has the sequence SEQ ID NO: 1, a homologous sequence of the sequence SEQ ID NO: 1 or a modified sequence of the sequence SEQ ID NO: 1; and the polypeptide of a combination according to the invention consisting of the NTS-DBLlx-Idl-DBL2x region of the VAR2CSA protein of the 3D7 parasite line has the sequence SEQ ID NO: 5, a sequence homologous to the sequence SEQ ID NO: Or a modified sequence of the sequence SEQ ID NO: 5.
  • peptide protein
  • peptide sequence polypeptide sequence
  • polypeptide polypeptide
  • peptide sequence homologous to the sequence SEQ ID NO: X is meant any peptide sequence which differs from the sequence SEQ ID NO: X by substitution, deletion, and / or insertion of an amino acid or of a reduced number amino acid positions at positions such that these homologous peptide sequences have substantially the same biological properties as the NTS-DBL1x-Idl-DBL2x region or the Idl-DBL2x region of VAR2CSA of the FCR3 line or the lineage 3D7.
  • a homologous peptide sequence has a percentage of identity such that it is at least 75% identical to the sequence SEQ ID NO: X, preferably at least 85%, more preferably at least 95%.
  • percentage of identity or “homology” between two nucleotide sequences or two peptide sequences, it is meant to designate a percentage of nucleotides or identical amino acid residues between the two sequences to be compared, obtained after optimal alignment. This percentage is purely statistical and the differences between the two sequences are randomly distributed over the entire length of the sequence.
  • optimal alignment and “best alignment”, which are used interchangeably herein, refer to the alignment for which the percent identity determined as described below is the highest.
  • the optimal alignment of the sequences necessary for comparison can be done manually or by means of computer software (GAP, BESTFIT, BLASTP, BLASTN, FASTA, and TFASTA, which are available either on the NCBI site or in Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI).
  • the percentage of identity between two nucleotide sequences or two peptide sequences is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the number of identical positions. total of positions compared and multiplying the result by 100.
  • modified peptide sequence of the sequence SEQ ID NO: X is meant any polypeptide sequence which differs from SEQ ID NO: X by one or more modifications, such as, for example, post-translational cellular modifications (eg “editing” ", glycosylation, sulfation, etc.).
  • the present invention also relates to combinations of at least two fusion proteins for the treatment or prevention of gestational malaria, wherein each of the fusion proteins consists of an isolated or purified polypeptide as defined above fused to a given fusion partner sequence. .
  • fusion partner sequence is meant herein a peptide sequence which confers on the fusion protein one or more desirable properties.
  • a fusion partner sequence may consist of a protein that promotes the expression of the NTS-DBLlx-Idl-DBL2x region or of the Idl-DBL2x region in the cell.
  • fusion protein eg comparison with a sequence corresponding to non-fused NTS-DBLlx-Idl-DBL2x or Idl-DBL2x, and / or to a protein that promotes the administration of the fusion protein to the vaccinated subject, and / or to a protein that increases the desired therapeutic effect (for example by increasing the immune and vaccine response) and / or a protein exhibiting biological or therapeutic activity.
  • Fusion partners that may be used in the context of the present invention include, without limitation, the maltose binding protein, the signal sequence of the maltose binding protein, poly-histidine segments capable of binding metal ions, an -Tag, glutathione-S-transferase, thioredoxin, ⁇ -galactosidase, streptavidin, dihydrofolate reductase, the pelB signal sequence, the ompA signal sequence, the alkaline phosphatase signal sequence, green fluorescent protein, a toxin such as for example enterotoxin LT of E.
  • fusion partners may be human growth hormone, an immunostimulatory cytokine such as: interleukin-2 (IL-2), a growth factor such as the granulocyte macrophage colony stimulating factor (GM-CSF), the granulocyte colony stimulating factor (G-CSF), peptides or hormones such as calcitonin, interferon-beta, interferon-alpha, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), PA toxin, parathyroid hormone (PTH (1-34) and PTH (1-84)), butyrylcholinesterase, glucocerebrosidase (GBA), and exendin-4.
  • IL-2 interleukin-2
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • peptides or hormones such as calcitonin, interferon-beta, interferon-alpha,
  • the new cluster demonstrated by the present inventors is characterized by a VAR2CSA protein whose interdomain Id1 has as sequence the consensus sequence SEQ ID NO: 11 or is coded by the consensus sequence SEQ ID NO: 13.
  • the other cluster highlighted by the present inventors is characterized by a VAR2CSA protein whose interdomain Idl has as sequence the consensus sequence SEQ ID NO: 12 or is encoded by the consensus nucleotide sequence SEQ ID NO: 14.
  • Consensus sequence is meant an idealized sequence of a given region of a protein in which each position represents the amino acid met most frequently.
  • the consensus groups were established by comparison of real sequences.
  • the present invention also relates to combinations of at least two polynucleotides isolated or purified for use in the treatment or prevention of gestational malaria, wherein each of the isolated or purified polynucleotides encodes a polypeptide as defined above or for a fusion protein such as as defined above, and contains the elements necessary for the expression, in vitro and / or in vivo, of said polypeptide or of said fusion protein.
  • the invention relates to a combination of at least two polynucleotides, wherein each of the polynucleotides encodes a polypeptide consisting of the NTS-DBLlx-Idl-DBL2x region or the Idl-DBL2x region of the VAR2CSA protein of a lineage or parasitic family of Plasmodium falciparum given or for a fusion protein comprising said polypeptide, and wherein each of the isolated or purified polynucleotides contains the elements necessary for the expression of said polypeptide or said fusion protein in vitro or in vivo.
  • nucleotide sequence used interchangeably herein. By these terms is meant to designate a precise sequence of nucleotides, modified or not, for defining a region of a nucleic acid, and which may correspond to double-stranded DNA or single-stranded DNA as well as to transcription products. of these DNAs.
  • the elements necessary for the expression of a nucleotide sequence in vivo include, for example, a promoter, a transcription initiation region, and a transcription termination region that are functional in a mammalian cell, preferably a cell. human.
  • sequences that increase gene expression such as introns, enhancer sequences, and "leader" sequences, are often necessary for the expression of a coding sequence for an immunogenic protein.
  • these elements are preferably operably linked to the nucleotide sequence to be expressed.
  • the terms "operably linked” and “operably linked” are used interchangeably and refer to a functional link between the regulatory sequences and the nucleic acid sequence that they control.
  • promoters useful in the context of the present invention include, but are not limited to, promoters of SV40 virus, mouse mammary tumor virus (MMTV), HIV virus, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), as well as promoters of human genes such as the human actin, myosin, hemoglobin, muscle creatine and metallothionein.
  • nucleotide sequence consisting of a given nucleotide sequence and the elements necessary for the expression of this nucleotide sequence are within the skill of those skilled in the art.
  • the nucleotide sequence of the polynucleotide which encodes the NTS-DBL1x-Id1-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 2 or a homologous sequence of SEQ ID NO: 2
  • the nucleotide sequence of the polynucleotide which encodes the NTS-DBL1-Idl-DBL2x region of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 6 or a homologous sequence of SEQ ID NO: 6.
  • the nucleotide sequence of the polynucleotide which codes for the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line has the sequence SEQ ID NO: 4 or a homologous sequence of SEQ ID NO: 4
  • the nucleotide sequence of the isolated or purified polynucleotide which encodes the Idl-DBL2x region of the VAR2CSA protein of the 3D7 line has the sequence SEQ ID NO: 8 or a homologous sequence of SEQ ID NO: 8.
  • nucleotide sequence homologous to the sequence SEQ ID NO: X is meant any nucleotide sequence which differs from the sequence SEQ ID NO: X by substitution, deletion, and / or insertion of a nucleotide, or of a reduced number nucleotides, at positions such that these sequences encode the same polynucleotide or substantially the same polynucleotide as SEQ ID NO: X.
  • a “nucleotide sequence homologous to the sequence SEQ ID NO: X” is preferably a homologous sequence of SEQ ID NO: X which results from the degeneracy of the genetic code.
  • the polynucleotides, fusion proteins, and polypeptides of the present invention may be prepared by any suitable method.
  • Techniques for isolating or cloning a gene or nucleotide sequence encoding a specific domain of a protein are known in the art and include isolation from genomic DNA, preparation from complementary DNA, or combination of these methods.
  • the cloning of a gene, or a nucleotide sequence coding for a specific domain of a protein, from a genomic DNA can be carried out for example by using a polymerase chain reaction (PCR) or by screening libraries. expression to detect cloned DNA fragments with identical structural characteristics (Innis et al., "PCR: A Guide to Method and Application", 1990, Academy Press: New York).
  • nucleic acid amplification methods known to those skilled in the art can be used, such as, for example, ligase chain reaction (LCR), ligation-activated transcription (LAT) and NASBA (Nucleic) Acid Sequence Based Amplification). It is also possible to use a chemical synthesis method to prepare a nucleotide sequence. The methods of total chemical synthesis of DNA or RNA strands are known to those skilled in the art, and use commercial automated synthesizers.
  • LCR ligase chain reaction
  • LAT ligation-activated transcription
  • NASBA Nucleic Acid Sequence Based Amplification
  • Methods for preparing a peptide sequence include chemical methods (RB Merrifield, J. Am Chem Soc 1963, 85: 2149-2154, “Solid Phase Peptide Synthesis", Methods in Enzymology, GB Fields (Ed), 1997). Academic Press: San Diego, CA), and recombinant methods (Sambrook et al, Molecular Cloning. A Laboratory Manual, 2nd Ed, 1989, Cold Spring Harbor Press. Cold Spring, NY) using host cells (particularly in the case of fusion proteins).
  • the polynucleotides or fusion proteins or polypeptides may be present in any proportions.
  • the two components may in particular be present in equal amounts.
  • the three components may in particular be present in equal amounts.
  • the combinations according to the invention are particularly suitable for use as medicaments in the management of gestational malaria. Indeed, as demonstrated by the inventors, these combinations make it possible to induce anti-adherence antibodies with a broad spectrum of activity. As such, they can be used, as such or in a modified form, as an immunogenic composition or vaccine.
  • conjugates comprise at least one of the polypeptides of a combination according to the invention, linked to a support.
  • Conjugates can be obtained by coupling via a convalent bond between a polypeptide and a physiologically acceptable, non-toxic, natural or synthetic carrier, capable, for example, of increasing the immunogenic nature of the polypeptide.
  • conjugates mention may be made by way of example, the application WO 2006/124712, which describes methods for preparing conjugates comprising a plurality of antigenic peptides of Plasmodium falciparum bound to a carrier protein improving the immunogenicity of said antigens.
  • the preferred supports according to the invention are chosen from viral particles, lipids, for example lipids of C16-C18 type, polylysines, poly (DL-alanine) -poly (Lysine) s, nitrocellulose, microparticles of polystyrene, microparticles of latex beads, biodegradable polymers, polyphosphoglycan microparticles, carrier proteins such as OPMC (outer membrane protein complex of Neisseria meningitidis) or improved OPMC, BSA (bovine serum albumin), TT (tetanus toxoid), ovalbumin, KLH (heyhole limpet hemocyanin), THY (bovine thyroglobulin), HbSAg and HBcAg of the hepatitis B virus, rotavirus capsid proteins, human papillomavirus LI protein, virus type particle (VLP) type 6, 11 and 16, PPD (purified protein derivative) tuberculin.
  • OPMC
  • An immunogenic composition according to the invention comprises, in addition to a combination described herein, a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable carrier or excipient refers to any vehicle or medium which does not interfere with the effectiveness of the biological activity of the active ingredient of the composition, and which is not toxic to the individual to be treated. the concentration at which it is administered.
  • the use of such carriers or excipients for the formulation of active substances is well known in the art ("Remington's Pharmaceutical Sciences", EW Martin, 18 th Ed., 1990, Mack Publishing Co.: Easton, PA).
  • an immunogenic composition of the present invention may vary depending on the route of administration and the dosage. After formulation with at least one pharmaceutically acceptable carrier or excipient, an immunogenic composition of the invention may be administered in any form suitable for administration to a human, for example in solid or liquid form. Those skilled in the art know how to select the most suitable vehicles and excipients for the preparation of a certain type of formulation. Thus, for example, excipients such as water, 2-3-butanediol, isotonic sodium chloride solution, synthetic mono or diglycerides, and oleic acid are often used for the formulation of injectable preparations.
  • excipients such as water, 2-3-butanediol, isotonic sodium chloride solution, synthetic mono or diglycerides, and oleic acid are often used for the formulation of injectable preparations.
  • Liquid compositions including emulsions, microemulsions, solutions, suspensions, syrups, etc., can be formulated in the presence of solvents, solubilizers, emulsifiers, oils, fatty acids and other additives. as suspending agents, preservatives, viscosizing agents, etc.
  • the solid compositions for oral administration may be formulated in the presence of an inert excipient such as sodium citrate, and optionally additives such as binding agents, humectants, disintegrating agents, absorption accelerators, lubricating agents, etc.
  • the immunogenic compositions and vaccines of the invention may comprise one or more adjuvants used in combination.
  • adjuvants such as Montanide and / or Alum may be used.
  • adjuvants such as QS21, SBQS2, MF59, mLT, PHL, CpG DNA, calcium phosphate, dehydrated calcium sulfate, PLG, CT, LTB, CT / LT, AS02A are also suitable.
  • the immunogenic compositions and vaccines according to the invention may further comprise at least one specific antigen of the pre-erythrocyte stages (CSP, TRAP, LSA-1, LSA-3, SALSA, STARP, EXP-1), asexual erythrocytes (MSP-1, MSP-3, AMA-1, EBA-175, GLURP, MSP-2, MSP-4, MSP-5, RAP-2, RESA, PfEMP-1, synthesized GPI toxin) or sexed (PfS25).
  • CSP pre-erythrocyte stages
  • TRAP pre-erythrocyte stages
  • LSA-1 LSA-1
  • LSA-3 SALSA
  • SALSA STARP
  • EXP-1 asexual erythrocytes
  • MSP-1, MSP-3, AMA-1, EBA-175, GLURP, MSP-2, MSP-4, MSP-5, RAP-2, RESA, PfEMP-1, synthesized GPI toxin or s
  • a vaccine against gestational malaria comprises at least one combination described herein and is used to induce in antibodies vaccinated antibodies inhibiting cytoadherence to CSA.
  • the invention relates to a DNA vaccine (also called plasmid vaccine or polunucleotide vaccine) against gestational malaria.
  • the invention also relates to a protein vaccine (also called polypeptide vaccine) against gestational malaria. Protein vaccines
  • the present invention therefore relates to a protein vaccine comprising a combination of at least two polypeptides as described above or a combination of at least two fusion proteins as described above.
  • a protein vaccine may be by any appropriate route, such as, for example, intravenously, subcutaneously, intradermally, orally, topically or systemically.
  • the present invention also relates to a DNA vaccine against gestational malaria.
  • the aim of genetic vaccination or DNA vaccination is to induce an immune response and consists in introducing directly into certain cells of the body a polynucleotide gene or sequence coding for a vaccinal antigen or a purified plasmid of DNA containing a coding sequence. for the vaccine antigen.
  • the cells in question are, in the example of the invention, muscle cells, but any other type of cell may be suitable, for example cells of the skin.
  • Administration is non-exclusive, by intramuscular injection or by "bombardment" of particles on the skin or nasally. DNA enters the muscle cells, skin cells or others; and these cells then themselves synthesize the antigen.
  • the antigen is presented to the immune system and triggers a response (the production of antibodies capable, during an infection, of specifically recognizing this antigen on the parasite).
  • the vaccine is therefore produced, locally, by the body of the individual to be immunized.
  • This method of vaccination, simple and inexpensive, has significant advantages in terms of effectiveness: the antigen thus produced is generally in its native peptide sequence, fused or not to one or more peptide sequences (fusion partners). Above all, it is produced in a prolonged way by the cells of the body, and this durable presentation of the antigen to the immune system should make it possible to avoid the recourse to the recalls.
  • DNA vaccines are chemically defined and thermally stable, which reduces the need to maintain the cold chain.
  • the present invention therefore relates to a DNA vaccine comprising a combination of at least two polynucleotides as described above.
  • a polynucleotide of a combination of the invention may be a naked DNA, in particular a circular vaccinia plasmid, supercoiled or not, or a linear DNA molecule incorporating and expressing in vivo a nucleotide sequence coding for the NTS-region. DBLlx-Idl-DBL2x or for the Idl-DBL2x region of the VAR2CSA protein of the FCR3 line or the 3D7 line or the new cluster.
  • naked DNA is meant, as is now commonly accepted, a DNA transcription unit in the form of a polynucleotide sequence comprising at least one nucleotide sequence coding for a vaccine antigen and the elements necessary for its in vivo expression.
  • the polynucleotides of a combination according to the invention may advantageously be inserted into a plasmid of the DNA-CSP type, Nyvac pf7, VR1020, VR1012, etc.
  • naked DNA is incorporated into a drug carrier.
  • suitable drug vectors include, but are not limited to, biodegradable microcapsules, immunostimulant complexes, liposomes, cationic lipids, and genetically attenuated live vectors such as viruses and bacteria.
  • a DNA vaccine of the invention may also be administered in conjunction with an agent that enhances the penetration of the vaccine genetic material into treated cells.
  • the DNA vaccine can be formulated to contain such an agent or be administered at the same time as such an agent.
  • agents that enhance the penetration of vaccine genetic material into treated cells include, but are not limited to, benzoic acid esters, anilides, amidines, urethanes, and their hydrochloride salts (US Patent 6,248,565).
  • Administration of DNA to cells may be promoted by chemical vectors (such as, for example, cationic polymers or cationic lipids), physical techniques such as electroporation, sonoporation, magnetofection, etc., or still viral vectors such as viruses associated with adenovirus, etc.
  • the immunogenic compositions and vaccines can be advantageously used to immunize female human beings (prepubertal girls and women of childbearing age) in the context of preventive gestational malaria therapy.
  • the invention also relates to methods of treating or preventing gestational malaria.
  • the invention provides a method for inducing a protective immune response against Plasmodium falciparum in a human female, the method comprising a step of administering an effective amount of an immunogenic composition or vaccine described right here.
  • the invention also provides a method of vaccinating a female human against Plasmodium falciparum, the method comprising a step of administering an effective amount of a vaccine, particularly a DNA vaccine or a protein vaccine described herein.
  • a method of treating or preventing gestational malaria is characterized in that it induces, in female humans, antibodies that prevent the adhesion of Plasmodium falciparum-infected erythrocytes to CSA placental receiver.
  • the administration of the immunogenic composition or the vaccine can be done by any appropriate route (oral, parenteral, mucosal).
  • administration of a DNA vaccine is intramuscular, intradermal, or mucosal.
  • administration of a protein vaccine is, for example, intravenous, subcutaneous, intradermal, oral, topical or systemic.
  • An immunogenic composition or a vaccine according to the invention may be administered in a single dose or in several doses. Those skilled in the art will be able to determine the effective dose of immunizing protein or DNA for use in each immunization or vaccination protocol.
  • kits for the prevention of gestational malaria More specifically, the kit comprises material useful for carrying out a vaccination according to the method of the invention.
  • a kit comprises a combination, an immunogenic composition or a vaccine according to the invention, and instructions for performing a vaccination against gestational malaria.
  • the kit may further include means for performing the vaccination.
  • kits according to the invention are configured such that the components of a combination according to the invention are provided separately (for example in different containers). Such a configuration allows both the simultaneous administration and the sequential administration of the components of the combination.
  • simultaneous administration is meant here a administration of the components, together or separately, at approximately the same time (for example, at an interval of 5, 10, 15 or 30 minutes each). other).
  • sequential administration is meant here an administration of the components separately and at different times (for example at different times of the same day, or at one or more days of interval).
  • the kit may comprise reagents or solutions for the preparation of the composition to be administered.
  • the various components of the kit may be provided in solid form (for example in freeze-dried form) or in liquid form.
  • a kit may optionally include a container each containing reagents or solutions, and / or containers (test tubes, flasks, etc.) to effect the preparation of the composition to be administered.
  • a notice in the form prescribed by a government agency regulating the sale and use of pharmaceutical products may be included in the kit.
  • Isolates of Plasmodium falciparum Field samples of infected erythrocytes were obtained from pregnant women at the Suru Lowski maternity hospital in the eastern region of Cotonou. The study site is characterized by hyper-endemic malaria in the lagoon zone and a high rate of malaria transmission with two peaks corresponding to the two rainy seasons (Akogbeto et al., Parasitologia, 1992, 34: 147-154). Peripheral venous blood was collected in vacutainers containing anticoagulant citrate phosphate dextrose adenine (CPDA), during prenatal visits and at the time of delivery. The erythrocytes were separated from the plasma and washed with RPMI 1640 (Lonza).
  • CPDA citrate phosphate dextrose adenine
  • erythrocyte pellets 200 ⁇ l were homogenized in 10 volumes of TRIzol reagent (Invitrogen) and frozen at -80 ° C. until extraction of the total RNA or frozen at -20 ° C. moment of extraction of the total DNA.
  • the pellets of erythrocytes infected with ring stage parasites were immediately cultured in vitro to obtain aged trophozoites as previously described (Trager et al., Science, 1976, 193: 673-675).
  • the isolates were grown in vessels containing RPMI 1640 supplemented with HEPES and L-glutamine (Lonza Biowhittaker), 0.3 g / L 1-glutamine, 0.05 g / L gentamicin, 5 g / L The albumax.
  • the culture vessels were placed under a 92.5% N2, 2% O2 and 5.5% CO2 atmosphere and incubated at 37 ° C for no more than 48 hours prior to testing.
  • the parasitic laboratory strains FCR3, HB3 and NF54 were cultured in 0+ erythrocytes and were selected after several panning steps on the BeWo cell line as previously described (Haase et al., Infect Immun., 2006, 74: 3035). -3038).
  • DNA Extraction and Genotyping msp The DNA was extracted from 100 pellet with the GeneJet Genomic Purification Kit (Fermentas) according to the manufacturer's recommendations. The mspl and msp2 genes were amplified by nested PCR using specific primers (Snounou et al., Trans R Soc Trop Med Hyg, 1999, 93: 369-374). The multiplicity of infection (MOI) was determined for each sample.
  • MOI multiplicity of infection
  • RNA extraction, cDNA synthesis and Genotyping of DBL2x The RNAs were extracted from frozen samples stored in the TRIzol reagent (In vitro gen) according to the manufacturer's recommendations. Total RNAs were treated with DNAse I (Invitrogen) for 15 minutes at room temperature according to the manufacturer's recommendations to eliminate any potential contamination with genomic DNA (gDNA). The absence of gDNA in the RNA samples was confirmed by a lack of amplification after 40 cycles of a real-time PCR with primers targeting housekeeping seryl-tRNA synthetase gene (Salanti et al, Mol Microbiol, 2003 49: 179-191).
  • RNA without DNA was performed with Thermoscript (Invitrogen) and random hexamer primers for 1 hour at 50 ° C in a total volume of 20 ⁇ L as recommended by the manufacturer.
  • DSM dimorphic sequence motif
  • DBL2x from VAR2CSA of Plasmodium falciparum isolates (Sander et al., PLoS One, 2009, 4: e6667), this domain was selected to amplify the cDNA of each isolate with a primer pair (5'-TTAYCCCCAAGAACACA-3 'and 5'-TTTT A ATTTTTTCC ATG A A-3 ').
  • Reactions were performed with high fidelity Taq Fusion Polymerase (to obtain higher yields and lower mutation rates) under the following conditions: 94 ° C for 1 minute followed by 35 cycles at 94 ° C for 30 minutes. seconds, 50 ° C for 30 seconds and 68 ° C for 50 seconds, and final extension at 68 ° C for 10 minutes.
  • the PCR products were digested with the restriction enzymes BstCI (which cuts the FCR3 type DSMs) and Hppyl88I (which cuts the 3D7 DSMs) for 1 hour at 50 ° C and 37 ° C, respectively.
  • the digested products were separated by electrophoresis on 1.5% agarose gel.
  • the NTS-DBL1x-Idl-DBL2x fragment and the entire sequence of the optimized varlcsa gene of FCR3 and 3D7 parasite lines were used to produce specific anti-VAR2CSA IgGs, by DNA vaccination as previously described (Bigey et al., J. Infect Dis ., 2011, 204: 1125-1133). Briefly, the DNA sequences were cloned into a vector derived from pVax1 and fused to the mEPO signal sequence as previously described (Trollet et al., Infect Immun., 2009, 77: 2221-2229).
  • Immunizations were performed on Swiss female mice aged 6 weeks and rabbits from New Zealand (January-France). The mice were anesthetized by intraperitoneal injection with a ketamine-xylazine mixture and in the case of rabbits the injection was made at 5 sites along each longissimus dorsi muscle. DNA transfer was performed by transcutaneous electrical pulses applied by two stainless steel electrodes placed at each injection site. The animals were immunized on days 0, 30 and 60, and the antisera were collected on days 0 and 15 after each of the immunizations. Total IgG was purified in the final mouse or rabbit serum sample on a Hi-Trap column to which the corresponding recombinant protein had been coupled in accordance with the manufacturer's recommendations (GE Healthcare).
  • Suspensions enriched with erythrocytes infected with aged trophozoites were obtained by filtration on a magnetic column (VarioMACS, Miltenyi). The suspension, whose parasite density was adjusted to 20% in 1 ⁇ 10 5 cells, was then blocked for 30 minutes at room temperature (RT) in a solution of 3% BSA diluted in RPMI. The erythrocyte preparation was incubated with purified IgG (0.25 mg / mL) or 500 ⁇ g / mL soluble SCA (Sigma), and the cells were then adhered for 15 minutes at room temperature on Petri dishes. pre-coated with different ligands. The non-adherent cells were removed by an automatic washing system. The spots were fixed with 1.5% glutaraldehyde in PBS, stained with Giemsa solution and the infected infected erythrocytes were counted under a microscope.
  • Figure 1 is a graph showing the distribution of the dimorphic sequence signatures of the DBL2x region of VAR2CSA among parasites infecting pregnant women.
  • the dimorphic sequence motif (DSM) in DBL2x that distinguishes VAR2CSA alleles into two subgroups (types 3D7 and FCR3) were genotyped in parasites collected from pregnant women in Benin.
  • the proportions of type 3D7 (white histograms), type FCR3 (striped histograms) and mixing of the two genotypes (black histograms) are presented.
  • the multiplicity of infection (MOI) is indicated for each category.
  • Figure 2 is a graph showing the recognition of VAR2CSA expressed in IgG field isolates directed against the different constructs of Var2CSA.
  • VAR2CSA expressed by the IgGs induced against the VAR2CSA constructs (NTS-DBL2X / 3D7 and NTS-DBL2X / FCR3) and against the entire extracellular domain of VAR2CSA (NTS-DBL1-68 / FCR3) of the strain FCR3 has been studied on field isolates by flow cytometry.
  • the parasites were separated according to their category DSM (DSM 3D7, DSM FCR3 or DSM Blend FCR3 / 3D7).
  • the bars indicate the average MFI ratio (MFI corresponding to the reactivity of the IgG of the hyperimmune animal / MFI corresponding to the reactivity of the IgG of the same pre-immune animal.
  • Figure 3 includes two graphs showing the IgG adhesion inhibitory activity induced against each of the two 'serotypes' of NTS-DBL1x-Idl-DBL2x on field isolates. Inhibitory adhesion activity was assessed on 18 Plasmodium falciparum isolates freshly obtained from pregnant women. The data is presented as percent inhibition for each antibody normalized by the CSA inhibition value (used as a reference for maximum adhesion inhibition). Histograms are presented for each of the DSM types detected in the isolate: ⁇ for DSMs of type 3D7, m for DSMs of type FCR3, and ⁇ for DSMs of mixture of the two genotypes.
  • Figure 4 is a series of three graphs showing the inhibition profile of antibodies on strains selected and adapted and binding to the CSA.
  • the FCR3, HB3 and NF54 strains selected on Bewo cells were used to evaluate the ability of the antibodies induced to inhibit the adhesion of the 3 laboratory strains to CSPG.
  • the in vitro functionality of the antibody mixtures was also evaluated. The values presented are normalized with the inhibition value of the CSA.
  • Figure 5 is a representation of the phylogenetic relationships between sequences spanning the ID1-DBL2X region of VAR2CSA generated from the CDNA of 123 parasite isolates of pregnant women.
  • the phylogenetic tree illustrates the phylogenetic relationship between the different sequences, with the groupings of dichotomous variants defined in DBL2X that discriminates between the 3D7 and FCR3 clusters and the clusters of a new cluster whose identity is defined in the Idl region of VAR2CSA. Bootstrap values are shown at each main branch.
  • DSM type 3D7 transcripts were found in 54 isolates (44%) and FCR3 type DSM transcripts were found in 47 isolates (39%) ( Figure 1). In 21 isolates (17%), both types of transcripts were detected, suggesting a mixture of genotypes. No differences were observed between the MOIs of the isolates of the groups determined after the typing of varlcsa (the determined MOIs are 3.05 in isolates expressing type 3D7, 2.98 in isolates expressing type CFR3, and 3.6 in the isolates with a mixture of the two DSM signatures).
  • the anti-NTS-DBLlx-Idl-DBL2x IgG specific to varlcsa variants of 3D7 and FCR3, and the IgGs induced against the entire extracellular domain of VAR2CSA were used in this experiment.
  • IgG induced against the entire extracellular domain of VAR2CSA was used to estimate the level of absolute recognition of the native VAR2CSA protein expressed on the surface of infected erythrocytes.
  • a high level of reactivity was observed with antibodies induced against the entire extracellular domain of VAR2CSA compared to antibodies directed against the NTS-DBLlx-Idl-DBL2x domain of VAR2CSA regardless of the origin of the latter (P ⁇ 0.05 ).
  • Soluble CSA was used as a reference for maximum inhibition of adhesion.
  • the degree of parasite inhibition by anti-NTS-DBL1x-Idl-DBL2x antibodies of FCR3 and 3D7 variants was normalized to CSA inhibition.
  • the average antibody inhibitory activity on all isolates was 80% [interquartile range: 50.8 - 100] for the anti-NTS-DBL1x-Idl-DBL2x antibody of the FCR3 variant and 97% [interquartile range: 55.3 - 100] for the anti-NTS-DBL1x-Idl-DBL2x antibody of the variant 3D7.
  • Different modes of inhibition were observed with the antibodies as a function of the types of varlcsa isolates.
  • isolates OPT144 and OPT161 are not inhibited by the antibodies induced against serotype FCR3, and interestingly, these isolates bearing DSM of type 3D7 have been strongly inhibited by IgG specific to the NTS-DBL1x-Idl-domain. DBL2x of the variant 3D7.
  • a similar profile was obtained with the OPT105 isolate which is more strongly inhibited with the DSM serotype-specific IgG homologous to the FCR3 variant and less well inhibited with the 3D7 variant-specific anti-NTS-DBLlx-Idl-DBL2x IgG.
  • the mixture of the anti-NTS-DBL1-Idl-DBL2x IgGs of the FCR3 line and the 3D7 line retains the property of completely inhibiting the adhesion of all the CSPG-infected erythrocytes.
  • Identification of a third cluster Sequence variations of the NTS-DBLlx-Idl-DBL2x domain of the VAR2CSA protein of the transcript of 123 parasite isolates of pregnant women from Benin were analyzed. Total RNA was extracted from freshly collected parasites, and the cDNA was synthesized. The var2csa gene was amplified from the cDNA using a high fidelity enzyme (Fusion), and universal primers were used. Ten (10) clones were sequenced for each isolate and the sequences were generated by multiple alignment of the protein and nucleic sequences.
  • VAR2CSA contains conserved anti-adherence epitopes and indicates that the development of an effective vaccine based on VAR2CSA will require the combination of a limited number of VAR2CSA variants.
  • the combination of the three major variants of VAR2CSA used in this work will be essential to develop an effective vaccine against placental malaria.

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