WO2015055772A1 - Combination of plasmodium merozoite surface proteins msp4 and 1 and uses thereof - Google Patents

Combination of plasmodium merozoite surface proteins msp4 and 1 and uses thereof Download PDF

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Publication number
WO2015055772A1
WO2015055772A1 PCT/EP2014/072236 EP2014072236W WO2015055772A1 WO 2015055772 A1 WO2015055772 A1 WO 2015055772A1 EP 2014072236 W EP2014072236 W EP 2014072236W WO 2015055772 A1 WO2015055772 A1 WO 2015055772A1
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seq
another embodiment
fragments
variants
pfmsplpl9
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PCT/EP2014/072236
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French (fr)
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Shirley Longacre
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Shirley Longacre
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    • 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
    • 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 treatment and prevention of malaria. More specifically, the present invention relates to vaccines against malaria, comprising a combination of two Plasmodium merozoite surface proteins, namely MSP4 and MSPl, variants or fragments thereof.
  • Plasmodium malaria parasites take an unacceptable toll on human health in tropical and sub-tropical developing countries. Plasmodium falciparum, which causes the most severe, life-threatening disease, is becoming multi-drug resistant, exacerbating the need for an effective vaccine.
  • Blood stage extracellular merozoites invade erythrocytes, and merozoite surface proteins (MSPs) are promising vaccine candidates because they are accessible to host immune effectors such as antibodies, complement, and neutrophil phagocytes, and they mediate a critical process for parasite survival. Indeed, high antibody titers to some MSPs in the sera of endemic residents have been associated with protection against clinical episodes of malaria, and many current blood stage vaccine candidates are recombinant MSP analogues.
  • WO97/30159 and EP 1 808 443 both describe the use of a recombinant protein containing a C-terminal fragment of Plasmodium MSP-1 as a vaccine candidate.
  • both MSPlp42 and/or MSPlpl9 showed substantial, but adjuvant dependent efficacy (Perera et al, 1998, Infect Immun 66: 1500- 1506).
  • high antibody titers to baculovirus PfMSPlpl9 in exposed individuals were correlated with protection against clinical episodes in an age adjusted, multivariate analysis (Perraut et al, 2005, J. Infect. Dis. 191: 264-271). P.
  • MSP4 and MSP5 have more recently joined the panoply of potential MSP vaccine candidates.
  • WO2007/060550 describes recombinant MSP4 and MSP5 proteins, and use thereof in diagnostic methods, kits, vaccines or anti-parasite therapy. Both proteins have a single C-terminal epidermal-growth-factor (EGF) like domain and are anchored to the merozoite surface by a glycosyl-phosphatidyl-inositol (GPI) moiety, similar to MSP1.
  • GEF epidermal-growth-factor
  • GPI glycosyl-phosphatidyl-inositol
  • PfMSP4 and PfMSP5 Baculovirus soluble recombinant analogues of PfMSP4 and PfMSP5 have been produced as potential vaccine candidates, including a C-terminal protease resistant form of PfMSP4, lacking most of the polymorphic region, PfMSP4p20.
  • the Applicant herein provides data suggesting much enhanced potency for a vaccine comprising a combination of MSP4 and MSP1 proteins, variants or fragments thereof.
  • Anti-merozoite antibodies in the sera of malaria endemic populations have been shown to induce antibody dependent respiratory burst (ADRB) activity by blood neutrophils, which is highly correlated with protection from clinical episodes of malaria (Joos et al, 2010, PLoS ONE 5(3):e9871).
  • the Applicant demonstrates that by depleting antibodies to both MSPlpl9 and MSP4p20 from the sera of immune individuals, the protective ADRB activity is decreased in a synergistic manner (as shown in the Examples), supporting the use of a combination of both antigens to enhance the efficacy of a malaria vaccine.
  • the present invention thus relates to a composition comprising MSP4 and MSP1, variants or fragments thereof.
  • Another object of the invention is a pharmaceutical composition comprising MSP4 and MSPl, variants or fragments thereof in combination with at least one pharmaceutically acceptable carrier.
  • Another object of the invention is a medicament comprising MSP4 and MSPl, variants or fragments thereof.
  • Another object of the invention is a vaccine against malaria comprising MSP4 and MSPl, variants or fragments thereof.
  • the vaccine of the invention further comprises at least one pharmaceutically acceptable adjuvant.
  • said adjuvant is Montanide ISA51.
  • said fragment of MSP4 is MSP4p20 and/or said fragment of MSPl is MSPlpl9.
  • said fragments of MSP4 are PfMSP4p20 and PvMSP4p40 and/or said fragment of MSPl is PfMSPlpl9 and PvMSPlpl9.
  • the present invention also relates to the composition, pharmaceutical composition, medicament or vaccine as hereinabove described, for treating malaria.
  • Polypeptide is essentially free of association with other proteins or polypeptides, for example as a purification product of recombinant host cell culture or as a purified product from a non-recombinant source.
  • Treating refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • a subject or mammal is successfully "treated” for an infection if, after receiving a therapeutic amount of the pharmaceutical composition or medicament according to the invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; reduction of the parasite replication, inactivation of the parasite and/or relief to some extent from one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality, and improvement in quality of life issues.
  • the above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. - "About": preceding a figure means plus or minus 10% of the value of said figure.
  • the present invention relates to a composition comprising MSPl and MSP4, fragments or variants thereof.
  • MSPl and MSP4 are merozoite surface proteins.
  • MSPl is synthesized in hepatic and erythrocytic schizonts as a membrane-bound precursor of about 200 kDa, which is processed in two steps leading first to 42 kDa (MSPlp42) and then to 19 kDa (MSPlpl9) GPI anchored C-terminal molecules around the time of merozoite release.
  • MSPlpl9 is composed almost entirely of two cysteine- rich epidermal growth factor (EGF)-like domains that form a tightly packed, disc-like structure.
  • EGF epidermal growth factor
  • MSP4 gene encodes a 272 amino acid protein containing a single C-terminal EGF- like domain and GPI membrane attachment motif.
  • MSP4 is MSP 4 from Plasmodium falciparum (accession number AF295318).
  • MSPl and/or MSP4 are derived from a species of Plasmodium infectious for humans, preferably a Plasmodium selected from the group comprising or consisting of Plasmodium vivax and Plasmodium falciparum.
  • MSPl and MSP4 are derived from Plasmodium falciparum, and are named, respectively, PfMSPl and PfMSP4.
  • MSPl and MSP4 are derived from Plasmodium vivax, and are named, respectively, PvMSPl and PvMSP4.
  • the composition comprises PfMSPl and PfMSP4, fragments or variants thereof.
  • the composition comprises PvMSPl and PvMSP4, fragments or variants thereof.
  • the composition comprises PfMSPl and PvMSP4, fragments or variants thereof. In another embodiment of the invention, the composition comprises PvMSPl and PfMSP4, fragments or variants thereof.
  • the composition comprises PfMSPl, PvMSPl and PfMSP4, fragments or variants thereof.
  • the composition comprises PvMSPl, PfMSPl and PvMSP4, fragments or variants thereof.
  • the composition comprises PfMSPl, PvMSP4 and PfMSP4, fragments or variants thereof.
  • the composition comprises PvMSPl, PfMSP4 and PvMSP4, fragments or variants thereof. In another embodiment of the invention, the composition comprises PfMSPl, PvMSPl, PvMSP4 and PfMSP4, fragments or variants thereof.
  • a variant of MSPl or MSP4 has an amino acid sequence comprising at least 100, 150 or at least 200 contiguous amino acids of said MSPl or MSP4 sequences.
  • a variant of MSPl or MSP4 has the amino acid sequence of MSPl or MSP4 and additional N-terminal or C-terminal amino acids, wherein the number of additional amino acids ranges from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 to 500, preferably from 10 to 200, more preferably from 10 to 150 nucleotides.
  • a variant of MSPl or MSP4 has an amino acid sequence that typically differs from the amino acid sequence of MSP1 or MSP4 in one or more substitutions, deletions, additions and/or insertions.
  • said substitutions, deletions, additions and/or insertions may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • a variant of MSP1 or MSP4 has an amino acid sequence of at least 25, preferably of at least 50, 100, 150, 200, amino acids having at least 75%, 80%, 90%, 95%, or at least 96%, 97%, 98%, 99% identity with the amino acid sequence of MSP1 or MSP4.
  • identity refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms”). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. ⁇ 2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)).
  • the BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra).
  • NCBI National Center for Biotechnology Information
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • a fragment of MSPl or MSP4 comprises at least 25 contiguous amino acids, preferably at least 50, 100, 150, 200 or at least 250 amino acids, preferably contiguous amino acids, of MSPl or MSP4 respectively.
  • the fragment of MSPl is MSPlpl9.
  • the MSPlpl9 fragment corresponds to a membrane-associated 19-kDa COOH-terminal fragment of the protein MSPl.
  • the fragment of MSPl is PvMSPlpl9 or PfMSPl, preferably PfMSPlpl9.
  • PfMSPlpl9 has a sequence of at least 93, 100, 150, 200 or 250 contiguous amino acids comprising the following sequence:
  • PfMSPlpl9 comprises or consists in SEQ ID NO: 1.
  • PfMSPlpl9 has a sequence of at least 93, 100, 150, 200 or 250 contiguous amino acids comprising the following sequence:
  • PfMSPlpl9 comprises or consists in SEQ ID NO: 2.
  • PfMSPlpl9 has a sequence of at least 155, 200 or 250 contiguous amino acids comprising the following sequence:
  • PfMSPlpl9 has a sequence of at least 155, 200 or 250 contiguous amino acids comprising the following sequence:
  • PfMSPlpl9 comprises or consists in SEQ ID NO: 3 or SEQ ID NO: 187.
  • PfMSPlpl9 comprises or consists in SEQ ID NO: 3 or SEQ ID NO: 187 wherein the E and/or F amino acids residues at position 25 and 26 respectively are deleted or replaced by another amino acid residue. In one embodiment, PfMSPlpl9 comprises or consists in SEQ ID NO: 3 or SEQ ID NO: 187 wherein both the E and F amino acids residues at position 25 and 26 respectively are deleted.
  • PfMSPlpl9 has a sequence of at least 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 200 or 250 contiguous amino acids comprising the sequences SEQ ID NO: 4-44.
  • PfMSPlpl9 comprises or consists in a sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 44.
  • QDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTC NENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS SEQ ID NO: 7).
  • GKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENP NPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 10).
  • PfMSPlpl9 comprises or consists in a sequence selected from the group consisting of SEQ ID NO: 20 to SEQ ID NO: 44 wherein the E and/or F amino acids residues corresponding to positions 25 and 26 respectively in SEQ ID NO: 3 or SEQ ID NO: 187 are deleted or replaced by another amino acid residue.
  • PfMSPlpl9 comprises or consists in SEQ ID NO: 20 to SEQ ID NO: 44 wherein both the E and F amino acids residues corresponding to positions 25 and 26 respectively in SEQ ID NO: 3 or SEQ ID NO: 187 are deleted.
  • PfMSPlpl9 has a sequence of at least 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising the sequences SEQ ID NO: 45-85.
  • PfMSPlpl9 comprises or consists in a sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 85.
  • EAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDER EECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCEC TKPDS YPLFDGIFCS (SEQ ID NO: 73).
  • PfMSPlpl9 comprises or consists in a sequence selected from the group consisting of SEQ ID NO: 61 to SEQ ID NO: 85 wherein the E and/or F amino acids residues corresponding to positions 25 and 26 respectively in SEQ ID NO: 3 or SEQ ID NO: 187 are deleted or replaced by another amino acid residue.
  • PfMSPlpl9 comprises or consists in SEQ ID NO: 61 to SEQ ID NO: 85 wherein both the E and F amino acids residues corresponding to positions 25 and 26 respectively in SEQ ID NO: 3 or SEQ ID NO: 187 are deleted.
  • PfMSPlpl9 has a sequence comprising or consisting of SEQ ID NO: 1-44 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in N-terminal and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in C-terminal.
  • PfMSPlpl9 has a sequence comprising or consisting of SEQ ID NO: 45-85 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in N-terminal and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in C-terminal.
  • PfMSPlpl9 has a sequence as set forth herein, wherein at least one of the five following dimorphic positions is present: 14E/Q, 61K/T, 70N/S, 71G/R, 86L/F (wherein said positions 14, 61, 70, 71 and 86 corresponds to the positions within the sequences SEQ ID NO: 1 or SEQ ID NO: 2).
  • PfMSPlpl9 has a sequence as set forth herein but different from SEQ ID NO: 1 or SEQ ID NO: 2
  • the skilled artisan would know which dimorphic positions is equivalent to the dimorphic positions 14E/Q, 61K/T, 70N/S, 71G/R, 86L/F within these sequences.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85 wherein the dimorphic position 14E/Q or the equivalent thereof is present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98,
  • PfMSPlpl9 has a sequence of at least 94, 95, 95, 97, 98, 99,
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic position or the equivalent thereof 71G/R is present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic position 86L/F or the equivalent thereof is present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q and 61K/T or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q and 70N/S or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q and 71G/R or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 61K/T and 70N/S or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising SEQ ID NO: 1- 85, wherein the dimorphic positions 61K/T and 71G/R or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 61K/T and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 70N/S and 71G/R or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising SEQ ID NO: 1- 85, wherein the dimorphic positions 70N/S and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 71G/R and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T and 70N/S or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T and 71G/R or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 70N/S and 71G/R or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 70N/S and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 71G/R and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-64, wherein the dimorphic positions 61K/T, 70N/S and 71G/R or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 61K/T, 71G/R and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 61K/T, 70N/S and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 70N/S, 71G/R and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T, 70N/S and 71G/R or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T, 70N/S and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T, 71G/R and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 61K/T, 70N/S, 71G/R and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 70N/S, 71G/R and 86L/F or the equivalents thereof are present.
  • PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T, 70N/S, 71G/R and 86L/F or the equivalents
  • PvMSPlpl9 has a sequence of at least 89, 90, 100, 150, 200 or 250 contiguous amino acids comprising the following sequence:
  • PvMSPlpl9 comprises or consists in SEQ ID NO: 86.
  • PvMSPlpl9 has a sequence of at least 118, 119, 120, 125, 130, 135, 140, 145, 150, 155, 200 or 250 contiguous amino acids comprising the following sequence:
  • PvMSPlpl9 comprises or consists in SEQ ID NO: 185.
  • PvMSPlpl9 comprises or consists in SEQ ID NO: 185 wherein the E and/or F amino acids residues corresponding to positions 14 and 15 respectively are deleted or replaced by another amino acid residue. In one embodiment, PvMSPlpl9 comprises or consists in SEQ ID NO: 185 wherein both the E and F amino acids residues corresponding to positions 14 and 15 respectively are deleted.
  • PvMSPlpl9 has a sequence comprising or consisting in SEQ ID NO: 86 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in N-terminal and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in C-terminal.
  • PvMSPlpl9 has a sequence comprising or consisting in SEQ ID NO: 185 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in N-terminal and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in C-terminal.
  • MSPlpl9 has a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 187, preferably, MSPlpl9 has a sequence SEQ ID NO: 3 or SEQ ID NO: 187.
  • PvMSPlpl9 has a sequence of at least 89, 90, 100, 150, 200 or 250 contiguous amino acids comprising SEQ ID NO: 86 wherein the dimorphic position 69K/E is present. In another embodiment, PvMSPlpl9 has a sequence of at least 118, 119, 120, 125, 130, 135, 140, 145, 150, 155, 200 or 250 contiguous amino acids comprising SEQ ID NO: 185 wherein the dimorphic position 98K/E is present.
  • fragments of MSP4 include, without limitation, MSP4p20 (such as, for example, PfMSP4p20 or PvMSP4p20), MSP4p30, MSP4p40, which are described in the patent application WO2007/060550, the content of which is incorporated herein by reference.
  • the MSP4 fragment is a MSP4 exo-antigen (minus C-terminal hydrophobic amino acids of the GPI attachment site, thus allowing protein secretion) with a deletion of 30 amino acids from a polymorphic region near the N-terminus.
  • MSP4p30 This fragment is referred to as MSP4p30.
  • the MSP4 fragment is a MSP4 exo-antigen (minus C-terminal hydrophobic amino acids of the GPI attachment site, thus allowing protein secretion) without a deletion of 30 amino acids from a polymorphic region near the N-terminus.
  • MSP4p40 is the full-length gene product.
  • the MSP4 fragment is a 20 kDa polypeptide corresponding approximately to the C-terminal half of MSP4, starting around the sequence KSPKE motif and including the EGF domain. Upstream supplementary amino acids could be included, in particular amino acids of the MSP4p40 sequence localized upstream of the KSPKE motif. This fragment is referred to as MSP4p20.
  • PfMSP4p20 has a sequence of at least 114, 125, 150, 200 or 250 contiguous amino acids comprising the following sequence:
  • PfMSP4p20 comprises or consists in SEQ ID NO: 87. In another embodiment, PfMSP4p20 has a sequence of at least 119, 125, 150, 200 or 250 contiguous amino acids comprising the following sequence:
  • PfMSP4p20 comprises or consists in SEQ ID NO: 88. This sequence may also be referred to as MSP4p21ss2.
  • PfMSP4p20 has a sequence of at least 120, 125, 133, 140, 150, 200 or 250 contiguous amino acids comprising the following sequence: MRILGEEKPNVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHV GEEEDHNEGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDD KLCEYVGNRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 89).
  • PfMSP4p20 comprises or consists in SEQ ID NO: 89. This sequence may also be referred to as MSP4p21ssl. In another embodiment, PfMSP4p20 has a sequence of at least 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 140, 150, 200 or 250 contiguous amino acids comprising SEQ ID NO: 90 to SEQ ID NO: 107. In one embodiment, PfMSP4p20 comprises or consists in a sequence selected in the group comprising SEQ ID NO: 90 to SEQ ID NO: 107.
  • NVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEG EGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGN RRVKCKCKEGYKLEGIECVE (SEQ ID NO: 99).
  • PfMSP4p40 has a sequence comprising or consisting in the following sequence:
  • PfMSP4p30 has a sequence comprising or consisting in the following sequence: MWrVKFLIVVHFFIICTINFDKLYISYSYNIVPENGRMLNMRILAAEKKDEKEAS EQGEESHKKENSQESANGKDDVKEEKKTNEKKDDGKTDKVQEKVLEKSPKES QMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEHEEEEEHEEDDDD EDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVKCKCKEGYKLEGI ECVE (SEQ ID NO: 186).
  • PvMSP4p40 has a sequence comprising or consisting in the following sequence:
  • the fragment of PvMSP4p40 has a sequence comprising or consisting in the following sequence:
  • the fragment of PvMSP4p40 has a sequence comprising at least 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 200 or 250 contiguous amino acids comprising SEQ ID NO: l l l to SEQ
  • MSP4 or a fragment thereof has a sequence SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, preferably has a sequence SEQ ID NO: 87.
  • the composition comprises PfMSPlpl9 and PvMSP4p20, fragments or variants thereof. In another embodiment of the invention, the composition comprises PfMSPlpl9 and PvMSP4p40, fragments or variants thereof.
  • composition comprises PvMSPlpl9 and PvMSP4p20, fragments or variants thereof.
  • composition comprises PvMSPlpl9 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9 and PfMSP4p20, fragments or variants thereof.
  • composition comprises PvMSPlpl9 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20 and PvMSP4p20, fragments or variants thereof.
  • composition comprises PfMSPlpl9, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPl l9, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PvMSPlpl9 and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p40, PvMSPlpl9 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9 and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9 and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20 and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p40, PvMSP4p40 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
  • composition comprises PfMSPlpl9 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9 and PfMSP4p30, fragments or variants thereof.
  • composition comprises PvMSPlpl9 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSPlpl9 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSPlpl9 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p30 and PfMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p30 and PfMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSPlpl9, PvMSP4p20 and PfMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSPlpl9, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSPlpl9, PvMSP4p30 and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSPlpl9, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSPlpl9, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p40 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p40 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p40 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p40 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p40 and PvMSP4p30, fragments or variants thereof.
  • composition comprises PfMSPlpl9, PvMSPlpl9,
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
  • composition comprises PfMSPlpl9, PvMSPlpl9,
  • the composition comprises PfMSPlpl9, PfMSP4p20, and
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20, PvMSP4p40 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20, PvMSP4p40 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p30 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p30 and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p30 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p30 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p30 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p30 and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p30 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p30 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p30 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p30 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p30 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p30 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p20, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
  • the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
  • the MSPl and/or MSP4 protein, fragment or variant thereof carries a C-terminal glycosyl-phosphatidyl-inositol group (GPI).
  • GPI glycosyl-phosphatidyl-inositol group
  • the anchor peptide that signals for GPI addition has the following amino acids sequence: SSNFLGISFLLILMLILYSFI (SEQ ID NO: 184).
  • the MSPl and/or MSP4 protein, fragment or variant thereof has glycosylation groups, such as, for example, O-glycosylation groups or N-glycosylation groups.
  • the MSPl and/or MSP4 protein, fragment or variant thereof is expressed with a His-tag, such as, for example, a 6His-tag, a 7His-tag, a 8His-tag, a 9His-tag or a lOHis-tag.
  • the His-tag is located near the C- terminus end of the MSPl and/or MSP4 protein, fragment or variant thereof.
  • the MSPl and/or MSP4 protein, fragment or variant thereof is expressed with another affinity tag well known from the skilled artisan.
  • affinity tags include, but are not limited to, Hemaglutinin Tag, Poly Arginine Tag, Myc Tag, Strep Tag, S-Tag, HAT Tag, 3x Flag Tag, Calmodulin- binding peptide Tag, SBP Tag, Chitin binding domain Tag, GST Tag, Maltose-Binding protein Tag, Fluorescent Protein Tag, T7 Tag, V5 Tag and Xpress Tag.
  • MSPl and/or MSP4, variants or fragments thereof are recombinant polypeptides.
  • MSPl and/or MSP4 variants or fragments thereof can be produced by any production system such as E. coli, yeast or baculovirus-insect cell expression system known in the art.
  • MSPl and/or MSP4, variants or fragments thereof is/are produced in the baculovirus-insect cell expression system or other non-bacterial, non-fungal system.
  • MSPlpl9 and MSPlpl9-GPI molecules can be obtained from the baculoviruses deposited at the CNCM on February 1, 1996 as described in WO97/30158 or on November 10, 2005 as described in EP1808443:
  • MSP4p40, MSP4p30 and MSP4p21 molecules can be obtained from the baculoviruses deposited at the CNCM on November 10, 2005 or on November 21, 2006 as described in WO2007/060550: Identification reference Registration numbers
  • MSP1 and/or MSP4, variants or fragments are expressed as soluble, secreted polypeptides.
  • MSP1 and/or MSP4, variants or fragments are isolated, purified or substantially purified molecules.
  • the present invention also relates to a pharmaceutical composition comprising MSP4 and MSP1, variants or fragments thereof, and at least one pharmaceutically acceptable excipient.
  • a pharmaceutically acceptable excipient refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biologies standards.
  • the pharmaceutical composition comprises a composition as hereinabove described.
  • the present invention also relates to a medicament comprising MSP4 and MSP1, variants or fragments thereof.
  • the medicament of the invention comprises a composition as hereinabove described.
  • the present invention also relates to an immunogenic composition comprising MSP4 and MSPl, variants or fragments thereof.
  • the immunogenic composition of the invention comprises a composition as hereinabove described.
  • the present invention also relates to a vaccine comprising MSP4 and MSPl, variants or fragments thereof, as hereinabove described.
  • vaccine is used to describe a preparation intended for active immunological prophylaxis, i.e. capable of eliciting an immune response which protects against infection by Plasmodium and/or against clinical symptoms typically associated with either mild or more severe life-threatening forms of malaria.
  • Examples of clinical symptoms of malaria include, but are not limited to, fever, fatigue, vomiting, headaches, shivering, joint pain, hemolytic anemia, jaundice, hemoglobin in the urine, retinal damage, convulsions, neurological symptoms associated with cerebral malaria (including for example abnormal posturing, nystagmus, conjugate gaze palsy, opisthotonus, seizures, or coma), respiratory distress, metabolic acidosis, noncardiogenic pulmonary oedema, concomitant pneumonia, severe anaemia, splenomegaly, hepatomegaly, hypoglycemia, and hemoglobinuria with renal failure.
  • said vaccine may or may not further comprise an adjuvant.
  • adjuvants include, but are not limited to, Montanide ISA51, Montanide ISA 720, Freund's adjuvant, alum, liposomes.
  • the vaccine of the invention comprises MSPl and MSP4, variants or fragments thereof, in association with the adjuvant Montanide ISA51.
  • the vaccine of the invention comprises MSPlpl9 and MSP4p20, or variants and fragments thereof, in association with the adjuvant Montanide ISA51.
  • the vaccine of the invention comprises MSPlpl9 having the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 187, preferably SEQ ID NO: 3 or SEQ ID NO: 187 and MSP4p20 having the sequence SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, preferably SEQ ID NO: 88, or variants and fragments thereof, in association with the adjuvant Montanide ISA51.
  • said vaccine may further comprise polymer additives.
  • said vaccine may further comprise one or more further antigens of Plasmodium such as MSP5, MSP3 or GLURP or LSA antigens (LSA1, LSA3, LSA5 or combination thereof).
  • the vaccine of the invention comprises MSP1, MSP4 and MSP5, variants or fragments thereof, preferably in association with an adjuvant, more preferably with Montanide IS A51.
  • the vaccine of the invention comprises MSPlpl9, MSP4p20 and MSP5 or variants and fragments thereof, in association with the adjuvant Montanide ISA51.
  • One object of the invention is the use of the composition, pharmaceutical composition, medicament or vaccine as described herein above against malaria.
  • One object of the invention is a composition, pharmaceutical composition, medicament or vaccine as described hereinabove for treating or preventing malaria.
  • One object of the invention is a method for treating malaria in a subject in need thereof, comprising administering an effective amount of the composition, pharmaceutical composition, medicament or vaccine as described herein above.
  • an "effective amount” means level or amount of agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of malaria; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of malaria; (3) bringing about ameliorations of the symptoms of malaria; (4) reducing the severity or incidence of malaria; or (5) curing malaria.
  • An effective amount may be administered prior to the onset of malaria, for a prophylactic or preventive action. Alternatively or additionally, the effective amount may be administered after initiation of malaria, for a therapeutic action.
  • said method is for treating malaria, in particular Plasmodium falciparum malaria and/or Plasmodium vivax malaria.
  • the effective amount ranges from about 1 to 250 ⁇ g per antigen, preferably from about 1 to 100 ⁇ g per antigen, preferably from about 1 to 50 ⁇ g per antigen.
  • the composition, pharmaceutical composition, medicament or vaccine of the invention comprises from about 1 ⁇ g/ml to about 5 mg/ml of each antigen, preferably from about 1 ⁇ g/ml to about 2 mg/ml, more preferably from about 1 ⁇ g/ml to about 1 mg/ml.
  • the subject is a mammal, preferably a human.
  • the major purpose of the immune response in a malaria-infected mammal is to inactivate the malaria parasites and to facilitate malaria parasite killing and clearance of parasite infected red blood cells.
  • the B-cell arm of the immune response has the major responsibility for inactivating blood-stage malaria parasites.
  • the principal manner in which this is achieved is by neutralization of infectivity (inhibition of erythrocyte invasion) and antibody dependent cellular cytotoxicity (ADCC) likely mediated by blood neutrophils in the case of malaria.
  • ADCC antibody dependent cellular cytotoxicity
  • the target antigen must be conserved to be effective against re-infection with other parasite strains, and capable of inducing T helper cell activity (CD4+) to generate a long-lived memory response.
  • T cell mediated mechanism for destruction of parasite infected cells is provided by cytotoxic (CD8+) T lymphocytes (CTL) that could recognize MSP1 and/or MSP4 antigens expressed in combination with Class I histocompatibility antigens at the surface of hepatic cells in the preerythrocytic phase of infection.
  • CTL cytotoxic T lymphocytes
  • the vaccine of the invention aims at raising neutralizing antibodies that either inhibit erythrocyte invasion by merozoites or opsonize merozoites to facilitate both their phagocytosis by neutrophils and the induction of neutrophil respiratory bursts known to be toxic for parasitized erythrocytes in order to inactivate blood forms of the parasite, reduce the viability of the parasite in vivo, or inhibit or prevent parasite replication in vivo.
  • the methods for administering the composition, pharmaceutical composition, medicament or vaccine of the present invention may vary and include intramuscular, subcutaneous, intradermal, transdermal, intravenous, buccal, oral, sublingual, or nasal administration.
  • the composition, pharmaceutical composition, medicament or vaccine of the invention is in a form adapted for injection, preferably selected from the group comprising solutions, such as, for example, sterile aqueous solutions, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms and the like.
  • solutions such as, for example, sterile aqueous solutions, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms and the like.
  • the composition, pharmaceutical composition, medicament or vaccine of the invention may be lyophilized.
  • Vaccine compositions may thus be presented in a freeze-dried (lyophilized) form.
  • the MSP1 and MSP4 proteins according to the invention are combined with one or more lyophilisation auxiliary substances.
  • lyophilisation auxiliary substances are well known by the one skilled in the art. Lyophilization of auxiliary substances encompasses sugars like lactose and mannitol.
  • the composition, pharmaceutical composition, medicament or vaccine of the invention is in a form adapted for transdermal administration, preferably is a form selected from ointment, paste, cream, foam, lotion, emulsion (a macroemulsion, a microemulsion, or a nanoemulsion), patch, such as, for example, transdermal patch, gel, liposomal forms and the like.
  • the present invention thus also relates to a patch comprising the composition, pharmaceutical composition, medicament or vaccine of the invention.
  • the patch of the invention is made of a polymeric material, such as, for example, polyvinylalcohol, gelatin, cellulose or derivatives thereof, Poly-N- vinylpyrrolidone and mixtures thereof.
  • the composition, pharmaceutical composition, medicament or vaccine may be mixed with stabilizers, e.g. to protect degradation-prone proteins from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze-drying efficiency.
  • stabilizers include, but are not limited to, SPGA (Sucrose-Phosphate- Glutamate-Albumin), carbohydrates (such as, for example, sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (such as, for example, albumin or casein or degradation products thereof), mixtures of amino acids such as lysine or glycine, and buffers, such as alkali metal phosphates.
  • SPGA Sucrose-Phosphate- Glutamate-Albumin
  • carbohydrates such as, for example, sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose
  • proteins such as, for example, albumin or casein or degradation products thereof
  • the immunization schedule may depend upon several factors, such as, for example, the susceptibility of the subject to infection and the age of the subject.
  • a single dose of the vaccine of the invention may be administered to the subject or a primary course of immunization may be followed in which several doses at intervals of time are administered. Subsequent doses used as boosters can be administered as needed following the primary course.
  • the immunization schedule comprises one administration of the vaccine of the invention. In another embodiment, the immunization schedule comprises two or three administrations of the vaccine of the invention.
  • the composition, pharmaceutical composition, medicament or vaccine of the invention is administered to the subject in an amount sufficient to induce immune responses that prevent or inhibit parasite infection and replication in vivo so as to reduce the parasite burden in the subject and diminish clinical symptoms.
  • an immunogenic response may be obtained by administering the MSP1 and MSP4 antigens, fragments or variants thereof, in an amount ranging from about 1 to 250 ⁇ g per antigen per dose, preferably from about 1 to 100 ⁇ g per antigen per dose, preferably from about 1 to 50 ⁇ g per antigen per dose.
  • Figure 1 is a histogram showing the impact of PfMSPlpl9 specific antibodies on ADRB activity.
  • Figure 2 is a graph showing the specificity of the antibody depletion protocol.
  • the pool of 30 hyper-immune sera from Dielmo (HIS) were depleted of antibodies specific for MSP4p20, and titered before (triangles) and after (circles) depletion, either against (A) MSP4p20 or (B) MSPlpl9.
  • Figure 3 is a histogram showing the effect of anti-MSPlpl9 and/or anti-MSP4p20 depletion on ADRB activity.
  • ADRB activity measured before depletion corresponds to 100% ADRB activity.
  • Figure 4 is a graph showing IgG responses to MSPl and MSP4 in OD ratio, as a function of clinical accesses (0 vs 1 or 2 vs >2 accesses).
  • Figure 5 is a combination of graphs showing antibody responses in symptomatic malaria in two villages of Cote d'lsian.
  • A Titers of antibodies to MSPl or MSP4 (MFI: mean fluorescence intensity).
  • B Parasite density in blood, expressed in trophozoite per microliter.
  • C Antibody response to somatic antigens of Plasmodium, expressed in OD ratio.
  • Soluble secreted recombinant proteins corresponding to PfMSPlpl9 and PfMSP4p20 were produced in the baculovirus / insect cell expression system by replacing the C- terminal GPI-modification signal sequence in each case with a hexa-histidine tag (Bonnet et al, 2006, Vaccine 24:5997-6008). Briefly, the MSP4p20 antigen corresponded to the C-terminal half of MSP4, lacking most polymorphic sites near the N-terminus.
  • Recombinant proteins were produced in High Five (Invitrogen) insect cells infected with recombinant baculovirus, and were purified by metalloaffinity chromatography using a C-terminal hexa-histidine tag as previously described (Bonnet et al, 2006, Vaccine 24:5997-6008).
  • Antigens were coated on Immulon-4 plates (Dynatech) at 0.5 ⁇ g.mL "1 .
  • the ELISA protocol has been previously described (Perraut et al, 2003, J Infect Dis 188 : 1940- 1950).
  • the HIS pool or sera from individuals with high ELISA responses for the different MSP test antigens were selected. Each serum (100 was diluted 1:3 in PBS and incubated in solution with 50 ⁇ g of MSP recombinant hexa-histidine tagged protein for 30 min at room temperature (RT) to allow efficient antigen-antibody binding. Packed TALON Metal Affinity Resin (Ozyme) pre-equilibrated with PBS (200 ⁇ ), was added and incubated with gentle mixing for 3 hours at RT, to allow efficient antigen-antibody complex binding via the C-terminal hexa-histidine tag.
  • TALON Metal Affinity Resin Ozyme
  • P. falciparum parasites (FUP/CB, an FCR3-like background) were maintained in continuous culture on 0 + erythrocytes in RPMI supplemented with 0.5% Albumax and 1 ⁇ g.mL "1 gentamycin, in candle jars (Diouf et al, 2002, Dakar Med 47:5-11). After centrifugation of cultures with greater than 5% parasitemia for 5 min at 400xg, pelleted RBCs were replaced in culture, and merozoites were collected from culture supernatants following centrifugation for 20 min at 1500xg. The concentrated merozoite extracts were stored at -20°C until use.
  • Chemiluminescence was measured at 37°C using opaque 96- well plates (Berthold), and a MicroLumat Plus 96 luminometer (Berthold), controlled with Window software. Chemiluminescence was recorded as relative light units (rlu). A 4 mg/ml stock solution of isoluminol (4-aminophthalhydrazide; Sigma) prepared in DMSO, was stored in aliquots at -20°C in the dark, and used at a final concentration of 0.04 mg/ml in sterile PBS. To facilitate rapid handling, only 40-50 wells per plate were used.
  • ADRB (rlu maximum sample / rlu maximum HIS) x 1000 where rlu maximum HIS is an average of the first and last wells on the plate.
  • the ADRB assay measures primarily MSP specific IgG functionality
  • MSPlpl9 or MSP4p20 the prevalence and levels of IgG specific for two MSP antigens, MSPlpl9 or MSP4p20, were first determined in the sera from Dielmo (holoendemic, 116 individuals) and Ndiop (mesoendemic, 114 individuals). Those with ELISA OD ratios above background at a 1:200 dilution were considered to be responders for prevalence evaluations. In Dielmo, 73% and 88% of sera analyzed recognized MSPlpl9 and MSP4p20, respectively. Corresponding determinations of prevalence for Ndiop were 86% and 78%.
  • ADRB readout was compared using either P. falciparum D10 (D10-PfM3') or transgenic D10 merozoites, in which PfMSPlpl9 has been replaced by the non-cross reactive P. chabaudi homologue, PcMSPlpl9 (PcMEGF) (O'Donnell et al, 2001, J Exp Med 193: 1403-1412).
  • Figure 2 shows an example of anti- MSP4p20 depletion from the HIS pool.
  • the HIS was titered for either MSP4p20 ( Figure 2A) or MSPlpl9 ( Figure 2B) specific antibodies before and after depletion.
  • the results show that the depletion protocol is specific and highly effective.
  • Median titer units decreased from 511 to 1 after anti-MSPlpl9 depletion, and from 816 to 8 after anti-MSP4p20 depletion, giving mean efficiencies of >99%.
  • ELISA analysis on a batch of PfMSPlpl9 and PfMSP4p20 has been carried out on a set of villagers from Ndiop withdrawn before rainy season in year 2000 (cross sectional prospective study). ELISA was done using the two Ags combined in the same plate (1 ⁇ g/ml for coating) according to standard procedure. Multiplex Analysis was done using custom, Elisa-derived Luminex Fluorescent magnetic bead based assay (Magpix System, Millipore). Beads from different regions were covalently linked to MSPlpl9 and MSP4p20 and analysed in the same samples using adapted technique derived from those described with Luminex 100 device.
  • a Poisson regression model was used to analyze the relationship between antibody response(s), prior to the transmission season, and incidence of malaria attacks during the follow-up period.
  • Optimal age stratification was based on the age distribution of the parasitologic and clinical data available for this setting ie age cut-offs of 15 and 30 years old.
  • the follow-up time was calculated as the number of days actually spent in the village during the five months of follow-up, excluding individuals absent from the village for more than 30 days during the follow-up period.
  • the follow- up time was adjusted for individuals who experienced malaria attacks, by excluding from the days at risk a period of 10 days after the diagnosed malaria attack.
  • the analysis included 144 individuals, who experienced a total of 129 clinical episodes during the follow-up period. There was a significant relationship with protection against clinical malaria in an age independent un-stratified model for both Ags on plate: P ⁇ 0.001, IRR 0.874 [0.81-0.93].
  • the model for analysis included dichotomized stratified age of individuals with high versus low IgG responses. High IgG responses were defined as OD ratio > mean for PfMSP4/PfMSPl association. In this model, high Ab responses were significantly associated with protection to both Ags on plate: P ⁇ 0.001, IRR 0.469 [0.32-0.68].
  • Antibody responses against MSP1 and MSP4 were measured in consultants recruited in sentinel health centers of Cote d'lrium from 2 different geographical settings as detailed in Table 4.

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Abstract

The present invention relates to a composition and vaccine against malaria.

Description

COMBINATION OF PLASMODIUM MEROZOITE SURFACE PROTEINS MSP4 AND 1 AND USES THEREOF
FIELD OF INVENTION The present invention relates to the treatment and prevention of malaria. More specifically, the present invention relates to vaccines against malaria, comprising a combination of two Plasmodium merozoite surface proteins, namely MSP4 and MSPl, variants or fragments thereof.
BACKGROUND OF INVENTION
Plasmodium malaria parasites take an unacceptable toll on human health in tropical and sub-tropical developing countries. Plasmodium falciparum, which causes the most severe, life-threatening disease, is becoming multi-drug resistant, exacerbating the need for an effective vaccine. Blood stage extracellular merozoites invade erythrocytes, and merozoite surface proteins (MSPs) are promising vaccine candidates because they are accessible to host immune effectors such as antibodies, complement, and neutrophil phagocytes, and they mediate a critical process for parasite survival. Indeed, high antibody titers to some MSPs in the sera of endemic residents have been associated with protection against clinical episodes of malaria, and many current blood stage vaccine candidates are recombinant MSP analogues.
WO97/30159 and EP 1 808 443 both describe the use of a recombinant protein containing a C-terminal fragment of Plasmodium MSP-1 as a vaccine candidate. In primate vaccination-challenge trials, both MSPlp42 and/or MSPlpl9 showed substantial, but adjuvant dependent efficacy (Perera et al, 1998, Infect Immun 66: 1500- 1506). In addition, high antibody titers to baculovirus PfMSPlpl9 in exposed individuals, were correlated with protection against clinical episodes in an age adjusted, multivariate analysis (Perraut et al, 2005, J. Infect. Dis. 191: 264-271). P. falciparum MSP4 and MSP5 have more recently joined the panoply of potential MSP vaccine candidates. For example, WO2007/060550 describes recombinant MSP4 and MSP5 proteins, and use thereof in diagnostic methods, kits, vaccines or anti-parasite therapy. Both proteins have a single C-terminal epidermal-growth-factor (EGF) like domain and are anchored to the merozoite surface by a glycosyl-phosphatidyl-inositol (GPI) moiety, similar to MSP1. PfMSP4, but not PfMSP5, is essential for parasite survival, although the latter may be required for optimal parasite growth in vivo. Baculovirus soluble recombinant analogues of PfMSP4 and PfMSP5 have been produced as potential vaccine candidates, including a C-terminal protease resistant form of PfMSP4, lacking most of the polymorphic region, PfMSP4p20.
However, to the Applicant's knowledge, no malaria vaccine composition containing either MSP1 or MSP4 and showing good efficacy in humans has been approved and marketed yet.
The Applicant herein provides data suggesting much enhanced potency for a vaccine comprising a combination of MSP4 and MSP1 proteins, variants or fragments thereof. Anti-merozoite antibodies in the sera of malaria endemic populations have been shown to induce antibody dependent respiratory burst (ADRB) activity by blood neutrophils, which is highly correlated with protection from clinical episodes of malaria (Joos et al, 2010, PLoS ONE 5(3):e9871). Here, the Applicant demonstrates that by depleting antibodies to both MSPlpl9 and MSP4p20 from the sera of immune individuals, the protective ADRB activity is decreased in a synergistic manner (as shown in the Examples), supporting the use of a combination of both antigens to enhance the efficacy of a malaria vaccine.
SUMMARY
The present invention thus relates to a composition comprising MSP4 and MSP1, variants or fragments thereof. Another object of the invention is a pharmaceutical composition comprising MSP4 and MSPl, variants or fragments thereof in combination with at least one pharmaceutically acceptable carrier.
Another object of the invention is a medicament comprising MSP4 and MSPl, variants or fragments thereof.
Another object of the invention is a vaccine against malaria comprising MSP4 and MSPl, variants or fragments thereof. In one embodiment, the vaccine of the invention further comprises at least one pharmaceutically acceptable adjuvant. In one embodiment, said adjuvant is Montanide ISA51. In one embodiment, said fragment of MSP4 is MSP4p20 and/or said fragment of MSPl is MSPlpl9. In another embodiment of the invention, said fragments of MSP4 are PfMSP4p20 and PvMSP4p40 and/or said fragment of MSPl is PfMSPlpl9 and PvMSPlpl9.
The present invention also relates to the composition, pharmaceutical composition, medicament or vaccine as hereinabove described, for treating malaria.
DEFINITIONS
In the present invention, the following terms have the following meanings:
"Purified": this term means that the polypeptide is essentially free of association with other proteins or polypeptides, for example as a purification product of recombinant host cell culture or as a purified product from a non-recombinant source.
"Treating" or "treatment": this term refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for an infection if, after receiving a therapeutic amount of the pharmaceutical composition or medicament according to the invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; reduction of the parasite replication, inactivation of the parasite and/or relief to some extent from one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. - "About": preceding a figure means plus or minus 10% of the value of said figure.
DETAILED DESCRIPTION
The present invention relates to a composition comprising MSPl and MSP4, fragments or variants thereof. MSPl and MSP4 are merozoite surface proteins.
MSPl is synthesized in hepatic and erythrocytic schizonts as a membrane-bound precursor of about 200 kDa, which is processed in two steps leading first to 42 kDa (MSPlp42) and then to 19 kDa (MSPlpl9) GPI anchored C-terminal molecules around the time of merozoite release. MSPlpl9 is composed almost entirely of two cysteine- rich epidermal growth factor (EGF)-like domains that form a tightly packed, disc-like structure.
The MSP4 gene encodes a 272 amino acid protein containing a single C-terminal EGF- like domain and GPI membrane attachment motif. An example of MSP4 is MSP 4 from Plasmodium falciparum (accession number AF295318). In one embodiment of the invention, MSPl and/or MSP4 are derived from a species of Plasmodium infectious for humans, preferably a Plasmodium selected from the group comprising or consisting of Plasmodium vivax and Plasmodium falciparum. In a first embodiment, MSPl and MSP4 are derived from Plasmodium falciparum, and are named, respectively, PfMSPl and PfMSP4. In a second embodiment, MSPl and MSP4 are derived from Plasmodium vivax, and are named, respectively, PvMSPl and PvMSP4.
In one embodiment of the invention, the composition comprises PfMSPl and PfMSP4, fragments or variants thereof.
In another embodiment of the invention, the composition comprises PvMSPl and PvMSP4, fragments or variants thereof.
In another embodiment of the invention, the composition comprises PfMSPl and PvMSP4, fragments or variants thereof. In another embodiment of the invention, the composition comprises PvMSPl and PfMSP4, fragments or variants thereof.
In another embodiment of the invention, the composition comprises PfMSPl, PvMSPl and PfMSP4, fragments or variants thereof.
In another embodiment of the invention, the composition comprises PvMSPl, PfMSPl and PvMSP4, fragments or variants thereof.
In another embodiment of the invention, the composition comprises PfMSPl, PvMSP4 and PfMSP4, fragments or variants thereof.
In another embodiment of the invention, the composition comprises PvMSPl, PfMSP4 and PvMSP4, fragments or variants thereof. In another embodiment of the invention, the composition comprises PfMSPl, PvMSPl, PvMSP4 and PfMSP4, fragments or variants thereof.
In one embodiment, a variant of MSPl or MSP4 has an amino acid sequence comprising at least 100, 150 or at least 200 contiguous amino acids of said MSPl or MSP4 sequences. In another embodiment, a variant of MSPl or MSP4 has the amino acid sequence of MSPl or MSP4 and additional N-terminal or C-terminal amino acids, wherein the number of additional amino acids ranges from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 to 500, preferably from 10 to 200, more preferably from 10 to 150 nucleotides. In another embodiment, a variant of MSPl or MSP4 has an amino acid sequence that typically differs from the amino acid sequence of MSP1 or MSP4 in one or more substitutions, deletions, additions and/or insertions. In one embodiment, said substitutions, deletions, additions and/or insertions may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In another embodiment, a variant of MSP1 or MSP4 has an amino acid sequence of at least 25, preferably of at least 50, 100, 150, 200, amino acids having at least 75%, 80%, 90%, 95%, or at least 96%, 97%, 98%, 99% identity with the amino acid sequence of MSP1 or MSP4.
The term "identity" or "identical", when used in a relationship between the sequences of two or more polypeptides, refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. \2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity.
In one embodiment of the invention, a fragment of MSPl or MSP4 comprises at least 25 contiguous amino acids, preferably at least 50, 100, 150, 200 or at least 250 amino acids, preferably contiguous amino acids, of MSPl or MSP4 respectively.
In one embodiment, the fragment of MSPl is MSPlpl9. The MSPlpl9 fragment corresponds to a membrane-associated 19-kDa COOH-terminal fragment of the protein MSPl.
In one embodiment, the fragment of MSPl is PvMSPlpl9 or PfMSPl, preferably PfMSPlpl9.
In one embodiment, PfMSPlpl9 has a sequence of at least 93, 100, 150, 200 or 250 contiguous amino acids comprising the following sequence:
NISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENN GGCDADAKCTEEDSGSNGKKITCECTKPDS YPLFDGIFCS (SEQ ID NO: 1). In one embodiment, PfMSPlpl9 comprises or consists in SEQ ID NO: 1.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 100, 150, 200 or 250 contiguous amino acids comprising the following sequence:
NIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENN GGCDADAKCTEEDSGSNGKKITCECTKPDS YPLFDGIFCS (SEQ ID NO: 2). In one embodiment, PfMSPlpl9 comprises or consists in SEQ ID NO: 2.
In another embodiment, PfMSPlpl9 has a sequence of at least 155, 200 or 250 contiguous amino acids comprising the following sequence:
VTHESYQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQC PENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEE D SGSNGKKITCECTKPDS YPLFDGIFCS (SEQ ID NO: 3). In another embodiment, PfMSPlpl9 has a sequence of at least 155, 200 or 250 contiguous amino acids comprising the following sequence:
VTHESYQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQC PENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEE D SGSNGKKITCECTKPDS YPLFDGIFCS (SEQ ID NO: 187).
In one embodiment, PfMSPlpl9 comprises or consists in SEQ ID NO: 3 or SEQ ID NO: 187.
In one embodiment, PfMSPlpl9 comprises or consists in SEQ ID NO: 3 or SEQ ID NO: 187 wherein the E and/or F amino acids residues at position 25 and 26 respectively are deleted or replaced by another amino acid residue. In one embodiment, PfMSPlpl9 comprises or consists in SEQ ID NO: 3 or SEQ ID NO: 187 wherein both the E and F amino acids residues at position 25 and 26 respectively are deleted.
In another embodiment, PfMSPlpl9 has a sequence of at least 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 200 or 250 contiguous amino acids comprising the sequences SEQ ID NO: 4-44. In one embodiment, PfMSPlpl9 comprises or consists in a sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 44. LNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNEN NGGCD AD AKCTEEDSGSNGKKITCECTKPDS YPLFDGIFCS (SEQ ID NO: 4).
MLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNE NNGGCD AD AKCTEEDSGSNGKKITCECTKPDS YPLFDGIFCS (SEQ ID NO: 5).
DMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCN ENNGGCD AD AKCTEEDSGSNGKKITCECTKPDS YPLFDGIFCS (SEQ ID NO: 6). QDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTC NENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 7).
FQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPT CNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 8).
KFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNP TCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 9).
GKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENP NPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 10).
EGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENP NPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 11).
IEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVEN PNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 12).
LIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVE NPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 13).
KLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCV ENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 14).
SKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKC VENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS
(SEQ ID NO: 15). ISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKC VENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS
(SEQ ID NO: 16).
IISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDK CVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS
(SEQ ID NO: 17).
TIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGD KCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS
(SEQ ID NO: 18).
NTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEG DKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFC
S (SEQ ID NO: 19).
FNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQE GDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIF CS (SEQ ID NO: 20).
EFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQE GDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIF CS (SEQ ID NO: 21).
YEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYK QEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFD GIFCS (SEQ ID NO: 22).
GYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNY KQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLF DGIFCS (SEQ ID NO: 23).
TGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNY KQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLF DGIFCS (SEQ ID NO: 24). LTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLN YKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPL FDGIFCS (SEQ ID NO: 25).
VLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLL NYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYP LFDGIFCS (SEQ ID NO: 26).
AVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCL LNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSY PLFDGIFCS (SEQ ID NO: 27).
DAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECK CLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPD SYPLFDGIFCS (SEQ ID NO: 28).
EDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECK CLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPD SYPLFDGIFCS (SEQ ID NO: 29).
FEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREEC KCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKP DSYPLFDGIFCS (SEQ ID NO: 30).
AFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREE CKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECT KPDSYPLFDGIFCS (SEQ ID NO: 31).
EAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDERE ECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECT KPDSYPLFDGIFCS (SEQ ID NO: 32).
LEAFED AVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDER EECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCEC TKPDSYPLFDGIFCS (SEQ ID NO: 33). KLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDE REECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCE CTKPDSYPLFDGIFCS (SEQ ID NO: 34).
KKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHL DEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKIT CECTKPDSYPLFDGIFCS (SEQ ID NO: 35).
VKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRH LDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKI TCECTKPDSYPLFDGIFCS (SEQ ID NO: 36).
LVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFR HLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGK KITCECTKPDSYPLFDGIFCS (SEQ ID NO: 37).
ELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFR HLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGK KITCECTKPDSYPLFDGIFCS (SEQ ID NO: 38).
QELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCF RHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNG KKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 39).
YQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENSG CFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGS NGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 40).
SYQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENS GCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSG SNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 41).
ESYQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPENS GCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSG SNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 42). HESYQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPE NSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEED SGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 43).
THESYQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNISQHQCVKKQCPE NSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEED SGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 44).
In one embodiment, PfMSPlpl9 comprises or consists in a sequence selected from the group consisting of SEQ ID NO: 20 to SEQ ID NO: 44 wherein the E and/or F amino acids residues corresponding to positions 25 and 26 respectively in SEQ ID NO: 3 or SEQ ID NO: 187 are deleted or replaced by another amino acid residue. In one embodiment, PfMSPlpl9 comprises or consists in SEQ ID NO: 20 to SEQ ID NO: 44 wherein both the E and F amino acids residues corresponding to positions 25 and 26 respectively in SEQ ID NO: 3 or SEQ ID NO: 187 are deleted.
In another embodiment, PfMSPlpl9 has a sequence of at least 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising the sequences SEQ ID NO: 45-85. In one embodiment, PfMSPlpl9 comprises or consists in a sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 85.
LNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNEN NGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 45).
MLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNE NNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 46). DMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTC NENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 47). QDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPT CNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 48).
FQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPT CNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 49).
KFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPN PTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 50).
GKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENP NPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 51).
EGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVEN PNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 52).
IEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVEN PNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 53).
LIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVE NPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 54).
KLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCV ENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 55).
SKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKC VENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS
(SEQ ID NO: 56). ISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDK CVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS
(SEQ ID NO: 57).
IISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDK CVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS
(SEQ ID NO: 58).
TIIS KLIEGKFQDMLNI AQHQC VKKQCPENS GCFRHLDEREECKCLLN YKQEGD KCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS
(SEQ ID NO: 59).
NTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEG DKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFC
S (SEQ ID NO: 60).
FNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQE GDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIF CS (SEQ ID NO: 61).
EFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQ EGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGI FCS (SEQ ID NO: 62).
YEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNYK QEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFD GIFCS (SEQ ID NO: 63).
GYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLNY KQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLF DGIFCS (SEQ ID NO: 64).
TGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLN YKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPL FDGIFCS (SEQ ID NO: 65). LTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLLN YKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPL FDGIFCS (SEQ ID NO: 66).
VLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKCLL NYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYP LFDGIFCS (SEQ ID NO: 67).
AVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECKC LLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDS YPLFDGIFCS (SEQ ID NO: 68).
DAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREECK CLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPD S YPLFDGIFCS (SEQ ID NO: 69).
EDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREEC KCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKP DSYPLFDGIFCS (SEQ ID NO: 70).
FEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDEREEC KCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKP DSYPLFDGIFCS (SEQ ID NO: 71).
AFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDERE ECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECT KPDSYPLFDGIFCS (SEQ ID NO: 72).
EAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDER EECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCEC TKPDS YPLFDGIFCS (SEQ ID NO: 73).
LEAFED AVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLDE REECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCE CTKPDSYPLFDGIFCS (SEQ ID NO: 74). KLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHLD EREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITC ECTKPDSYPLFDGIFCS (SEQ ID NO: 75).
KKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRHL DEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKIT CECTKPDSYPLFDGIFCS (SEQ ID NO: 76).
VKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFRH LDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKI TCECTKPDSYPLFDGIFCS (SEQ ID NO: 77).
LVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCFR HLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGK KITCECTKPDSYPLFDGIFCS (SEQ ID NO: 78).
ELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGCF RHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNG KKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 79).
QELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSGC FRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSN GKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 80).
YQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENSG CFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGS NGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 81).
SYQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPENS GCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSG SNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 82).
ESYQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPEN SGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDS GSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 83). HESYQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCPE NSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEED SGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 84).
THESYQELVKKLEAFEDAVLTGYEFNTIISKLIEGKFQDMLNIAQHQCVKKQCP ENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEE DSGSNGKKITCECTKPDSYPLFDGIFCS (SEQ ID NO: 85).
In one embodiment, PfMSPlpl9 comprises or consists in a sequence selected from the group consisting of SEQ ID NO: 61 to SEQ ID NO: 85 wherein the E and/or F amino acids residues corresponding to positions 25 and 26 respectively in SEQ ID NO: 3 or SEQ ID NO: 187 are deleted or replaced by another amino acid residue. In one embodiment, PfMSPlpl9 comprises or consists in SEQ ID NO: 61 to SEQ ID NO: 85 wherein both the E and F amino acids residues corresponding to positions 25 and 26 respectively in SEQ ID NO: 3 or SEQ ID NO: 187 are deleted.
In another embodiment, PfMSPlpl9 has a sequence comprising or consisting of SEQ ID NO: 1-44 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in N-terminal and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in C-terminal.
In another embodiment, PfMSPlpl9 has a sequence comprising or consisting of SEQ ID NO: 45-85 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in N-terminal and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in C-terminal.
In another embodiment, PfMSPlpl9 has a sequence as set forth herein, wherein at least one of the five following dimorphic positions is present: 14E/Q, 61K/T, 70N/S, 71G/R, 86L/F (wherein said positions 14, 61, 70, 71 and 86 corresponds to the positions within the sequences SEQ ID NO: 1 or SEQ ID NO: 2). Should PfMSPlpl9 has a sequence as set forth herein but different from SEQ ID NO: 1 or SEQ ID NO: 2, the skilled artisan would know which dimorphic positions is equivalent to the dimorphic positions 14E/Q, 61K/T, 70N/S, 71G/R, 86L/F within these sequences. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85 wherein the dimorphic position 14E/Q or the equivalent thereof is present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98,
99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,
134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic position 61K/T or the equivalent thereof is present. In another embodiment, PfMSPlpl9 has a sequence of at least 94, 95, 95, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic position 70N/S or the equivalent thereof is present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic position or the equivalent thereof 71G/R is present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic position 86L/F or the equivalent thereof is present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q and 61K/T or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q and 70N/S or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q and 71G/R or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q and 86L/F or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 61K/T and 70N/S or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising SEQ ID NO: 1- 85, wherein the dimorphic positions 61K/T and 71G/R or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 61K/T and 86L/F or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 70N/S and 71G/R or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising SEQ ID NO: 1- 85, wherein the dimorphic positions 70N/S and 86L/F or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 71G/R and 86L/F or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T and 70N/S or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T and 71G/R or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T and 86L/F or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 70N/S and 71G/R or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 70N/S and 86L/F or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 71G/R and 86L/F or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-64, wherein the dimorphic positions 61K/T, 70N/S and 71G/R or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 61K/T, 71G/R and 86L/F or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 61K/T, 70N/S and 86L/F or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 70N/S, 71G/R and 86L/F or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T, 70N/S and 71G/R or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T, 70N/S and 86L/F or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T, 71G/R and 86L/F or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 61K/T, 70N/S, 71G/R and 86L/F or the equivalents thereof are present.
In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 70N/S, 71G/R and 86L/F or the equivalents thereof are present. In another embodiment, PfMSPlpl9 has a sequence of at least 93, 94, 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 148, 150, 151, 152, 153, 154, 155, 200 or 250 contiguous amino acids comprising or consisting of SEQ ID NO: 1-85, wherein the dimorphic positions 14E/Q, 61K/T, 70N/S, 71G/R and 86L/F or the equivalents thereof are present.
In one embodiment, PvMSPlpl9 has a sequence of at least 89, 90, 100, 150, 200 or 250 contiguous amino acids comprising the following sequence:
MSSEHTCIDTNVPDNAACYRYLDGTEEWRCLLTFKEEGGKCVPASNVTCKDN NGGCAPEAECKMTDSNKIVCKCTKEGSEPLFEGVFCS (SEQ ID NO: 86).
In one embodiment, PvMSPlpl9 comprises or consists in SEQ ID NO: 86. In another embodiment, PvMSPlpl9 has a sequence of at least 118, 119, 120, 125, 130, 135, 140, 145, 150, 155, 200 or 250 contiguous amino acids comprising the following sequence:
ETESYKQLVANVDEFILSQLLNVQTQLLTMSSEHTCIDTNVPDNAACYRYLDGT EEWRCLLTFKEEGGKCVPASNVTCKDNNGGCAPEAECKMTDSNKIVCKCTKE GSEPLFEGVFCS (SEQ ID NO: 185).
In one embodiment, PvMSPlpl9 comprises or consists in SEQ ID NO: 185.
In one embodiment, PvMSPlpl9 comprises or consists in SEQ ID NO: 185 wherein the E and/or F amino acids residues corresponding to positions 14 and 15 respectively are deleted or replaced by another amino acid residue. In one embodiment, PvMSPlpl9 comprises or consists in SEQ ID NO: 185 wherein both the E and F amino acids residues corresponding to positions 14 and 15 respectively are deleted.
In another embodiment, PvMSPlpl9 has a sequence comprising or consisting in SEQ ID NO: 86 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in N-terminal and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in C-terminal.
In another embodiment, PvMSPlpl9 has a sequence comprising or consisting in SEQ ID NO: 185 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in N-terminal and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in C-terminal.
According to an embodiment, MSPlpl9 has a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 187, preferably, MSPlpl9 has a sequence SEQ ID NO: 3 or SEQ ID NO: 187.
In another embodiment, PvMSPlpl9 has a sequence of at least 89, 90, 100, 150, 200 or 250 contiguous amino acids comprising SEQ ID NO: 86 wherein the dimorphic position 69K/E is present. In another embodiment, PvMSPlpl9 has a sequence of at least 118, 119, 120, 125, 130, 135, 140, 145, 150, 155, 200 or 250 contiguous amino acids comprising SEQ ID NO: 185 wherein the dimorphic position 98K/E is present.
Examples of fragments of MSP4 include, without limitation, MSP4p20 (such as, for example, PfMSP4p20 or PvMSP4p20), MSP4p30, MSP4p40, which are described in the patent application WO2007/060550, the content of which is incorporated herein by reference.
In one embodiment, the MSP4 fragment is a MSP4 exo-antigen (minus C-terminal hydrophobic amino acids of the GPI attachment site, thus allowing protein secretion) with a deletion of 30 amino acids from a polymorphic region near the N-terminus. This fragment is referred to as MSP4p30.
In another embodiment, the MSP4 fragment is a MSP4 exo-antigen (minus C-terminal hydrophobic amino acids of the GPI attachment site, thus allowing protein secretion) without a deletion of 30 amino acids from a polymorphic region near the N-terminus. This fragment is referred to as MSP4p40 and is the full-length gene product.
In another embodiment, the MSP4 fragment is a 20 kDa polypeptide corresponding approximately to the C-terminal half of MSP4, starting around the sequence KSPKE motif and including the EGF domain. Upstream supplementary amino acids could be included, in particular amino acids of the MSP4p40 sequence localized upstream of the KSPKE motif. This fragment is referred to as MSP4p20.
In one embodiment, PfMSP4p20 has a sequence of at least 114, 125, 150, 200 or 250 contiguous amino acids comprising the following sequence:
KSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEHEEEEEH EEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVKCKCKEG YKLEGIECVE (SEQ ID NO: 87).
In one embodiment, PfMSP4p20 comprises or consists in SEQ ID NO: 87. In another embodiment, PfMSP4p20 has a sequence of at least 119, 125, 150, 200 or 250 contiguous amino acids comprising the following sequence:
MRILEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEHE EEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVKC KCKEGYKLEGIECVE (SEQ ID NO: 88).
In one embodiment, PfMSP4p20 comprises or consists in SEQ ID NO: 88. This sequence may also be referred to as MSP4p21ss2.
In another embodiment, PfMSP4p20 has a sequence of at least 120, 125, 133, 140, 150, 200 or 250 contiguous amino acids comprising the following sequence: MRILGEEKPNVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHV GEEEDHNEGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDD KLCEYVGNRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 89).
In one embodiment, PfMSP4p20 comprises or consists in SEQ ID NO: 89. This sequence may also be referred to as MSP4p21ssl. In another embodiment, PfMSP4p20 has a sequence of at least 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 140, 150, 200 or 250 contiguous amino acids comprising SEQ ID NO: 90 to SEQ ID NO: 107. In one embodiment, PfMSP4p20 comprises or consists in a sequence selected in the group comprising SEQ ID NO: 90 to SEQ ID NO: 107. EKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEHEEEEE HEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVKCKCKE GYKLEGIECVE (SEQ ID NO: 90).
LEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEHEEEE EHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVKCKCK EGYKLEGIECVE (SEQ ID NO: 91 ) . SLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEHEEE EEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVKCKC KEGYKLEGIECVE (SEQ ID NO: 92).
TSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEHEE EEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVKCK C KEGYKLEGIECVE (SEQ ID NO: 93).
STSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEHEE EEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVKCK C KEGYKLEGIECVE (SEQ ID NO: 94). VSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEH EEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVK CKCKEGYKLEGIECVE (SEQ ID NO: 95).
GVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGE HEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRV KCKCKEGYKLEGIECVE (SEQ ID NO: 96).
DGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEG EHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRR VKCKCKEGYKLEGIECVE (SEQ ID NO: 97).
VDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGE GEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNR RVKCKCKEGYKLEGIECVE (SEQ ID NO: 98).
NVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEG EGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGN RRVKCKCKEGYKLEGIECVE (SEQ ID NO: 99). PNVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNE GEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVG NRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 100). KPNVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHN EGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVG NRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 101).
EKPNVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDH NEGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYV GNRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 102).
EEKPNVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEED HNEGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEY VGNRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 103). GEEKPNVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEE DHNEGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCE YVGNRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 104).
LGEEKPNVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEE EDHNEGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLC EYVGNRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 105).
ILGEEKPNVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEE EDHNEGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLC EYVGNRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 106).
RILGEEKPNVDGVSTSLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGE EEDHNEGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKL CEYVGNRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 107).
In one embodiment, PfMSP4p40 has a sequence comprising or consisting in the following sequence:
MWrVKFLIVVHFFIICTINFDKLYISYSYNIVPENGRMLNMRILGEEKPNVDGVST SNTPGGNEASSASPNLADAAEKKDEKEASEQGEESHKKENSQESANGKDDVKE EKKTNEKKDDGKTDKVQEKVLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNS GGHVGEEEDHNEGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGG CGDDKLCEYVGNRRVKCKCKEGYKLEGIECVE (SEQ ID NO: 108).
In one embodiment, PfMSP4p30 has a sequence comprising or consisting in the following sequence: MWrVKFLIVVHFFIICTINFDKLYISYSYNIVPENGRMLNMRILAAEKKDEKEAS EQGEESHKKENSQESANGKDDVKEEKKTNEKKDDGKTDKVQEKVLEKSPKES QMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEHEEEEEHEEDDDD EDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVKCKCKEGYKLEGI ECVE (SEQ ID NO: 186). In one embodiment, PvMSP4p40 has a sequence comprising or consisting in the following sequence:
GRILGEGGEQSGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANS ATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQD GEDYDDAEDDDLYELSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKP GYKLVGTECVE (SEQ ID NO: 109).
In one embodiment, the fragment of PvMSP4p40 has a sequence comprising or consisting in the following sequence:
AEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEV DENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 110).
In one embodiment, the fragment of PvMSP4p40 has a sequence comprising at least 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 200 or 250 contiguous amino acids comprising SEQ ID NO: l l l to SEQ ID NO: 183. In one embodiment, the fragment of PvMSP4p40 comprises or consists in a sequence selected from the group comprising SEQ ID NO: 111 to SEQ ID NO: 183.
SAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSE VDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 111).
HSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKS EVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 112).
DHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEK SEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 113).
GDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYE KSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 114). TGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKY EKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE
(SEQ ID NO: 115).
QTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDK YEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE
(SEQ ID NO: 116).
GQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDD KYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE
(SEQ ID NO: 117).
GGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDD DKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECV
E (SEQ ID NO: 118). SGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAED DDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTEC VE (SEQ ID NO: 119).
GSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAE DDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTE CVE (SEQ ID NO: 120).
PGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDA EDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGT ECVE (SEQ ID NO: 121). TPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDD AEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVG TECVE (SEQ ID NO: 122).
STPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDD AEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVG TECVE (SEQ ID NO: 123).
NSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYD DAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLV GTECVE (SEQ ID NO: 124).
QNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDY DDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKL VGTECVE (SEQ ID NO: 125).
PQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGED YDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYK LVGTECVE (SEQ ID NO: 126). GPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGE DYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGY KL VGTECVE (SEQ ID NO: 127). TGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDG EDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPG YKLVGTECVE (SEQ ID NO: 128).
ATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQD GEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKP GYKLVGTECVE (SEQ ID NO: 129).
SATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQ DGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCK PGYKLVGTECVE (SEQ ID NO: 130).
NSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDE QDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLC KPGYKLVGTECVE (SEQ ID NO: 131).
ANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGD EQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCL C KPGYKLVGTECVE (SEQ ID NO: 132).
PANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHG DEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKC LCKPGYKLVGTECVE (SEQ ID NO: 133).
DPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDH GDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVK CLCKPGYKLVGTECVE (SEQ ID NO: 134).
SDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDD HGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIV KCLCKPGYKLVGTECVE (SEQ ID NO: 135).
GSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGD DHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGI VKCLCKPGYKLVGTECVE (SEQ ID NO: 136). GGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHG DDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGK GIVKCLCKPGYKLVGTECVE (SEQ ID NO: 137).
SGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHG DDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGK GIVKCLCKPGYKLVGTECVE (SEQ ID NO: 138).
GSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDH GDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLG KGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 139).
SGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDD HGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENL GKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 140).
SSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGD DHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICEN LGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 141).
GSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHG DDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICE NLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 142).
AGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDH GDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKIC ENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 143).
PAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDD HGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKI CENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 144).
PPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGD DHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDD KICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 145). SPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGD DHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDD KICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 146).
PSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHG DDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCGD D KICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 147).
GPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDH GDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGCG DDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 148).
GGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGDD HGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGGC GDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 149).
SGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQGD DHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNGG CGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 150).
SSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQG DDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNNG GCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 151).
GSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNEQ GDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDNN GGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 152).
GGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNE QGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDN NGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 153).
SGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYNE QGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLDN NGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 154). LSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDYN EQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCLD NNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 155).
GLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGDY NEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLCL DNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 156).
GGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENGD YNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANLC LDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 157).
SGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAENG DYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENANL CLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 158).
SSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAEN GDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENAN LCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 159).
DSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEAE NGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDENA NLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 160).
GDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAEA ENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDEN ANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 161).
SGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSAE AENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVDE NANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 162). SSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHSA EAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEVD ENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 163).
GSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDHS AEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSEV DENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 164).
GGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGDH SAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKSE VDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 165).
SGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTGD HSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEKS EVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 166).
SSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQTG DHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYEK SEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 167).
GSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQT GDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKYE KSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE (SEQ ID NO: 168).
GGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQ TGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKY EKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE
(SEQ ID NO: 169). SGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGGQ TGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDKY EKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE
(SEQ ID NO: 170).
ASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSGG QTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDDK YEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE
(SEQ ID NO: 171).
GASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGSG GQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDDD KYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECVE
(SEQ ID NO: 172).
GGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPGS GGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAEDD DKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTECV
E (SEQ ID NO: 173).
SGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTPG SGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAED DDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTEC VE (SEQ ID NO: 174).
QSGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNSTP GSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDAE DDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVGTE CVE (SEQ ID NO: 175).
EQSGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQNS TPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDD AEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVG TECVE (SEQ ID NO: 176). GEQSGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQN STPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDD AEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLVG TECVE (SEQ ID NO: 177).
GGEQSGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGPQ NSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYD DAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLV GTECVE (SEQ ID NO: 178).
EGGEQSGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATGP QNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDY DDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKL VGTECVE (SEQ ID NO: 179).
GEGGEQSGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSATG PQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGED YDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYK LVGTECVE (SEQ ID NO: 180).
LGEGGEQSGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSAT GPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGE DYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGY KL VGTECVE (SEQ ID NO: 181).
ILGEGGEQSGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSAT GPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGE DYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGY KL VGTECVE (SEQ ID NO: 182).
RILGEGGEQSGGASGGSSGGSSGDSSGGLSGGSSGGPSPPAGSSGSGGSDPANSA TGPQNSTPGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDG EDYDDAEDDDKYEKSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPG YKL VGTECVE (SEQ ID NO: 183). According to an embodiment, MSP4 or a fragment thereof has a sequence SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, preferably has a sequence SEQ ID NO: 87.
In one embodiment of the invention, the composition comprises PfMSPlpl9 and PvMSP4p20, fragments or variants thereof. In another embodiment of the invention, the composition comprises PfMSPlpl9 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9 and PvMSP4p40, fragments or variants thereof.
In another embodiment of the invention, the composition comprises PfMSPlpl9 and PfMSP4p20, fragments or variants thereof.
In another embodiment of the invention, the composition comprises PfMSPlpl9 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPl l9, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PvMSPlpl9 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p40, PvMSPlpl9 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9 and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20 and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p40, PvMSP4p40 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSPlpl9 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSPlpl9 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p30 and PfMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p30 and PfMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSPlpl9, PvMSP4p20 and PfMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSPlpl9, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSPlpl9, PvMSP4p30 and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSPlpl9, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSPlpl9, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p40 and PfMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p40 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p20 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40, PvMSP4p30, PfMSP4p40 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p40 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p40 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9,
PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9,
PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p40 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20,
PfMSP4p40, PfMSP4p30, PvMSP4p20, PvMSP4p40 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20, PvMSP4p40 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40, PfMSP4p30, PvMSP4p20, PvMSP4p40 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p30 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p30 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p30 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p30 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p30 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p30 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p30 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p30 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p30 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p30 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p30 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p30 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p40 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p40, PvMSP4p30 and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30 and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30 and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PvMSP4p20, PvMSP4p30, PvMSP4p40, and PfMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p20, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p20, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PfMSP4p40, and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p40, and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p20, PfMSP4p40, PvMSP4p40, and PvMSP4p30, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p30, fragments or variants thereof.
In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p20, and PvMSP4p40, fragments or variants thereof. In another embodiment, the composition comprises PfMSPlpl9, PvMSPlpl9, PfMSP4p40, PfMSP4p30, PvMSP4p40, and PvMSP4p30, fragments or variants thereof.
In one embodiment of the invention, the MSPl and/or MSP4 protein, fragment or variant thereof carries a C-terminal glycosyl-phosphatidyl-inositol group (GPI).
Such GPI group has been described in EP 1808443 which is incorporated herein by reference. In one embodiment, the anchor peptide that signals for GPI addition has the following amino acids sequence: SSNFLGISFLLILMLILYSFI (SEQ ID NO: 184).
In one embodiment of the invention, the MSPl and/or MSP4 protein, fragment or variant thereof has glycosylation groups, such as, for example, O-glycosylation groups or N-glycosylation groups. In one embodiment of the invention, the MSPl and/or MSP4 protein, fragment or variant thereof is expressed with a His-tag, such as, for example, a 6His-tag, a 7His-tag, a 8His-tag, a 9His-tag or a lOHis-tag. Preferably, the His-tag is located near the C- terminus end of the MSPl and/or MSP4 protein, fragment or variant thereof.
In one embodiment of the invention, the MSPl and/or MSP4 protein, fragment or variant thereof is expressed with another affinity tag well known from the skilled artisan. Examples of affinity tags include, but are not limited to, Hemaglutinin Tag, Poly Arginine Tag, Myc Tag, Strep Tag, S-Tag, HAT Tag, 3x Flag Tag, Calmodulin- binding peptide Tag, SBP Tag, Chitin binding domain Tag, GST Tag, Maltose-Binding protein Tag, Fluorescent Protein Tag, T7 Tag, V5 Tag and Xpress Tag. In one embodiment, MSPl and/or MSP4, variants or fragments thereof are recombinant polypeptides. MSPl and/or MSP4, variants or fragments thereof can be produced by any production system such as E. coli, yeast or baculovirus-insect cell expression system known in the art. Preferably, MSPl and/or MSP4, variants or fragments thereof is/are produced in the baculovirus-insect cell expression system or other non-bacterial, non-fungal system.
In one embodiment of the invention, MSPlpl9 and MSPlpl9-GPI molecules can be obtained from the baculoviruses deposited at the CNCM on February 1, 1996 as described in WO97/30158 or on November 10, 2005 as described in EP1808443:
Figure imgf000058_0001
In the Table above, "A" stands for anchored and the corresponding proteins comprise a GPI sequence, while "S" stands for soluble (and the corresponding proteins do not comprise GPI sequence). Moreover, "Pc" stands for Plasmodium cynomolgi.
In one embodiment of the invention, MSP4p40, MSP4p30 and MSP4p21 molecules can be obtained from the baculoviruses deposited at the CNCM on November 10, 2005 or on November 21, 2006 as described in WO2007/060550: Identification reference Registration numbers
PfMSP4p30 1-3513 pfMSP4p40 1-3514
PfMSP4p21ssl 1-3695
PfMSP4p21ss2 1-3696
PvMSP4 1-3694
In one embodiment, MSP1 and/or MSP4, variants or fragments are expressed as soluble, secreted polypeptides.
In one embodiment, MSP1 and/or MSP4, variants or fragments are isolated, purified or substantially purified molecules. The present invention also relates to a pharmaceutical composition comprising MSP4 and MSP1, variants or fragments thereof, and at least one pharmaceutically acceptable excipient. As used herein, a "pharmaceutically acceptable excipient" refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biologies standards.
In one embodiment of the invention, the pharmaceutical composition comprises a composition as hereinabove described.
The present invention also relates to a medicament comprising MSP4 and MSP1, variants or fragments thereof. In one embodiment, the medicament of the invention comprises a composition as hereinabove described. The present invention also relates to an immunogenic composition comprising MSP4 and MSPl, variants or fragments thereof. In one embodiment, the immunogenic composition of the invention comprises a composition as hereinabove described.
The present invention also relates to a vaccine comprising MSP4 and MSPl, variants or fragments thereof, as hereinabove described.
The term vaccine is used to describe a preparation intended for active immunological prophylaxis, i.e. capable of eliciting an immune response which protects against infection by Plasmodium and/or against clinical symptoms typically associated with either mild or more severe life-threatening forms of malaria. Examples of clinical symptoms of malaria include, but are not limited to, fever, fatigue, vomiting, headaches, shivering, joint pain, hemolytic anemia, jaundice, hemoglobin in the urine, retinal damage, convulsions, neurological symptoms associated with cerebral malaria (including for example abnormal posturing, nystagmus, conjugate gaze palsy, opisthotonus, seizures, or coma), respiratory distress, metabolic acidosis, noncardiogenic pulmonary oedema, concomitant pneumonia, severe anaemia, splenomegaly, hepatomegaly, hypoglycemia, and hemoglobinuria with renal failure.
In one embodiment, said vaccine may or may not further comprise an adjuvant.
Examples of adjuvants include, but are not limited to, Montanide ISA51, Montanide ISA 720, Freund's adjuvant, alum, liposomes. In one embodiment, the vaccine of the invention comprises MSPl and MSP4, variants or fragments thereof, in association with the adjuvant Montanide ISA51.
Preferably, the vaccine of the invention comprises MSPlpl9 and MSP4p20, or variants and fragments thereof, in association with the adjuvant Montanide ISA51.
Preferably, the vaccine of the invention comprises MSPlpl9 having the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 187, preferably SEQ ID NO: 3 or SEQ ID NO: 187 and MSP4p20 having the sequence SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, preferably SEQ ID NO: 88, or variants and fragments thereof, in association with the adjuvant Montanide ISA51. In another embodiment, said vaccine may further comprise polymer additives.
In another embodiment, said vaccine may further comprise one or more further antigens of Plasmodium such as MSP5, MSP3 or GLURP or LSA antigens (LSA1, LSA3, LSA5 or combination thereof). In one embodiment, the vaccine of the invention comprises MSP1, MSP4 and MSP5, variants or fragments thereof, preferably in association with an adjuvant, more preferably with Montanide IS A51.
Preferably, the vaccine of the invention comprises MSPlpl9, MSP4p20 and MSP5 or variants and fragments thereof, in association with the adjuvant Montanide ISA51. One object of the invention is the use of the composition, pharmaceutical composition, medicament or vaccine as described herein above against malaria.
One object of the invention is a composition, pharmaceutical composition, medicament or vaccine as described hereinabove for treating or preventing malaria.
One object of the invention is a method for treating malaria in a subject in need thereof, comprising administering an effective amount of the composition, pharmaceutical composition, medicament or vaccine as described herein above.
As used herein, an "effective amount" means level or amount of agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of malaria; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of malaria; (3) bringing about ameliorations of the symptoms of malaria; (4) reducing the severity or incidence of malaria; or (5) curing malaria. An effective amount may be administered prior to the onset of malaria, for a prophylactic or preventive action. Alternatively or additionally, the effective amount may be administered after initiation of malaria, for a therapeutic action. In one embodiment, said method is for treating malaria, in particular Plasmodium falciparum malaria and/or Plasmodium vivax malaria.
In one embodiment, the effective amount ranges from about 1 to 250 μg per antigen, preferably from about 1 to 100 μg per antigen, preferably from about 1 to 50 μg per antigen.
In one embodiment, the composition, pharmaceutical composition, medicament or vaccine of the invention comprises from about 1 μg/ml to about 5 mg/ml of each antigen, preferably from about 1 μg/ml to about 2 mg/ml, more preferably from about 1 μg/ml to about 1 mg/ml. In one embodiment, the subject is a mammal, preferably a human.
The major purpose of the immune response in a malaria-infected mammal is to inactivate the malaria parasites and to facilitate malaria parasite killing and clearance of parasite infected red blood cells. The B-cell arm of the immune response has the major responsibility for inactivating blood-stage malaria parasites. The principal manner in which this is achieved is by neutralization of infectivity (inhibition of erythrocyte invasion) and antibody dependent cellular cytotoxicity (ADCC) likely mediated by blood neutrophils in the case of malaria. The target antigen must be conserved to be effective against re-infection with other parasite strains, and capable of inducing T helper cell activity (CD4+) to generate a long-lived memory response. The T cell mediated mechanism for destruction of parasite infected cells is provided by cytotoxic (CD8+) T lymphocytes (CTL) that could recognize MSP1 and/or MSP4 antigens expressed in combination with Class I histocompatibility antigens at the surface of hepatic cells in the preerythrocytic phase of infection.
In one embodiment, the vaccine of the invention aims at raising neutralizing antibodies that either inhibit erythrocyte invasion by merozoites or opsonize merozoites to facilitate both their phagocytosis by neutrophils and the induction of neutrophil respiratory bursts known to be toxic for parasitized erythrocytes in order to inactivate blood forms of the parasite, reduce the viability of the parasite in vivo, or inhibit or prevent parasite replication in vivo. The methods for administering the composition, pharmaceutical composition, medicament or vaccine of the present invention may vary and include intramuscular, subcutaneous, intradermal, transdermal, intravenous, buccal, oral, sublingual, or nasal administration. In one embodiment, the composition, pharmaceutical composition, medicament or vaccine of the invention is in a form adapted for injection, preferably selected from the group comprising solutions, such as, for example, sterile aqueous solutions, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms and the like.
According to an embodiment and for storage purposes, the composition, pharmaceutical composition, medicament or vaccine of the invention may be lyophilized. Vaccine compositions may thus be presented in a freeze-dried (lyophilized) form. In said embodiment, the MSP1 and MSP4 proteins according to the invention are combined with one or more lyophilisation auxiliary substances. Various lyophilisation auxiliary substances are well known by the one skilled in the art. Lyophilization of auxiliary substances encompasses sugars like lactose and mannitol.
In one embodiment, the composition, pharmaceutical composition, medicament or vaccine of the invention is in a form adapted for transdermal administration, preferably is a form selected from ointment, paste, cream, foam, lotion, emulsion (a macroemulsion, a microemulsion, or a nanoemulsion), patch, such as, for example, transdermal patch, gel, liposomal forms and the like.
The present invention thus also relates to a patch comprising the composition, pharmaceutical composition, medicament or vaccine of the invention. In one embodiment, the patch of the invention is made of a polymeric material, such as, for example, polyvinylalcohol, gelatin, cellulose or derivatives thereof, Poly-N- vinylpyrrolidone and mixtures thereof.
In some embodiments, the composition, pharmaceutical composition, medicament or vaccine may be mixed with stabilizers, e.g. to protect degradation-prone proteins from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze-drying efficiency. Useful stabilisers include, but are not limited to, SPGA (Sucrose-Phosphate- Glutamate-Albumin), carbohydrates (such as, for example, sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (such as, for example, albumin or casein or degradation products thereof), mixtures of amino acids such as lysine or glycine, and buffers, such as alkali metal phosphates.
The skilled artisan will easily understand that the immunization schedule may depend upon several factors, such as, for example, the susceptibility of the subject to infection and the age of the subject. A single dose of the vaccine of the invention may be administered to the subject or a primary course of immunization may be followed in which several doses at intervals of time are administered. Subsequent doses used as boosters can be administered as needed following the primary course. In one embodiment, the immunization schedule comprises one administration of the vaccine of the invention. In another embodiment, the immunization schedule comprises two or three administrations of the vaccine of the invention.
In one embodiment, the composition, pharmaceutical composition, medicament or vaccine of the invention is administered to the subject in an amount sufficient to induce immune responses that prevent or inhibit parasite infection and replication in vivo so as to reduce the parasite burden in the subject and diminish clinical symptoms. In one embodiment, an immunogenic response may be obtained by administering the MSP1 and MSP4 antigens, fragments or variants thereof, in an amount ranging from about 1 to 250 μg per antigen per dose, preferably from about 1 to 100 μg per antigen per dose, preferably from about 1 to 50 μg per antigen per dose.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a histogram showing the impact of PfMSPlpl9 specific antibodies on ADRB activity. ADRB chemiluminescence readouts (rlu) for 21 sera having high anti- MSPlpl9 OD-ratios, were compared using either P. falciparum D10 merozoites (D10- PfM3', grey bars) or transgenic D10 merozoites with P. falciparum MSPlpl9 replaced by the P. chabaudi homologue (DIO-PcMEGF, black bars).
Figure 2 is a graph showing the specificity of the antibody depletion protocol. The pool of 30 hyper-immune sera from Dielmo (HIS) were depleted of antibodies specific for MSP4p20, and titered before (triangles) and after (circles) depletion, either against (A) MSP4p20 or (B) MSPlpl9.
Figure 3 is a histogram showing the effect of anti-MSPlpl9 and/or anti-MSP4p20 depletion on ADRB activity. ADRB activity measured before depletion corresponds to 100% ADRB activity. Figure 4 is a graph showing IgG responses to MSPl and MSP4 in OD ratio, as a function of clinical accesses (0 vs 1 or 2 vs >2 accesses).
Figure 5 is a combination of graphs showing antibody responses in symptomatic malaria in two villages of Cote d'lvoire. (A) Titers of antibodies to MSPl or MSP4 (MFI: mean fluorescence intensity). (B) Parasite density in blood, expressed in trophozoite per microliter. (C) Antibody response to somatic antigens of Plasmodium, expressed in OD ratio.
EXAMPLES
The present invention is further illustrated by the following examples. EXAMPLE 1 Methods Study site
Two villages in Senegal with different malaria epidemiology were studied. In Dielmo malaria transmission is perennial because a nearby river maintains continuous mosquito breeding sites. In Ndiop, transmission is strictly seasonal and depends on the rainy season that normally occurs from July to September. This differing transmission is reflected in relatively constant P. falciparum prevalence rates of 80% in Dielmo compared to seasonal rates in Ndiop that change from 20% in the dry season to 70% in the rainy season. Consequently, the peak incidence of clinical malaria in Dielmo occurs in very young children of 2-5 years and declines rapidly with age, whereas in Ndiop peak incidence is in children of 5-10 years with only a gradual decrease in age. A field research station with a dispensary was built for the project in each village to identify and treat all episodes of morbidity. After venous puncture, RBCs were removed by centrifugation and plasma was stored at -20°C.
Antigens
Soluble secreted recombinant proteins corresponding to PfMSPlpl9 and PfMSP4p20 were produced in the baculovirus / insect cell expression system by replacing the C- terminal GPI-modification signal sequence in each case with a hexa-histidine tag (Bonnet et al, 2006, Vaccine 24:5997-6008). Briefly, the MSP4p20 antigen corresponded to the C-terminal half of MSP4, lacking most polymorphic sites near the N-terminus. Recombinant proteins were produced in High Five (Invitrogen) insect cells infected with recombinant baculovirus, and were purified by metalloaffinity chromatography using a C-terminal hexa-histidine tag as previously described (Bonnet et al, 2006, Vaccine 24:5997-6008).
ELISA analysis
Antigens were coated on Immulon-4 plates (Dynatech) at 0.5 μg.mL"1. The ELISA protocol has been previously described (Perraut et al, 2003, J Infect Dis 188 : 1940- 1950). For reliable inter-assay comparisons, results were expressed as OD-ratio = OD- sample / OD-naive, and used to measure the prevalence and levels of specific antibodies to each antigen at a 1:200 dilution. To monitor specific antibody depletions quantitatively, samples were analyzed at 1:200, 1:400 and 1:800 dilutions, and an arbitrary titer was extrapolated using a four-parameter logistic fit from a standardized positive control regression curve on each plate, determined using a pool of 30 hyperimmune sera (HIS) mostly from adult residents of Dielmo (mean age: 36 yr, range: 9-73 yr) and 2-fold dilutions starting at 1:200. A non-immune, negative control serum (NIS) was obtained commercially (Valbiotech, France). Serum depletion
The HIS pool or sera from individuals with high ELISA responses for the different MSP test antigens were selected. Each serum (100 was diluted 1:3 in PBS and incubated in solution with 50 μg of MSP recombinant hexa-histidine tagged protein for 30 min at room temperature (RT) to allow efficient antigen-antibody binding. Packed TALON Metal Affinity Resin (Ozyme) pre-equilibrated with PBS (200 μί), was added and incubated with gentle mixing for 3 hours at RT, to allow efficient antigen-antibody complex binding via the C-terminal hexa-histidine tag. Depleted sera were recovered in the supernatant after centrifugation without further dilution, so that initial and depleted sera were directly comparable. Individual sera were depleted of antibodies specific for PfMSPlpl9 or PfMSP4p20 alone, or for both PfMSPlpl9 and PfMSP4p20 (consecutive depletions).
Parasite culture and merozoite extraction
P. falciparum parasites (FUP/CB, an FCR3-like background) were maintained in continuous culture on 0+ erythrocytes in RPMI supplemented with 0.5% Albumax and 1 μg.mL"1 gentamycin, in candle jars (Diouf et al, 2002, Dakar Med 47:5-11). After centrifugation of cultures with greater than 5% parasitemia for 5 min at 400xg, pelleted RBCs were replaced in culture, and merozoites were collected from culture supernatants following centrifugation for 20 min at 1500xg. The concentrated merozoite extracts were stored at -20°C until use.
/' V/ V preparation
Blood samples from healthy adults presenting for routine blood tests at the Pasteur Institute, Dakar were obtained with informed written consent approved by the Ethical Committee of the Institut Pasteur de Dakar. Samples from 6-7 donors (4 mL each), were collected into EDTA-K3 tubes, layered onto Ficoll-Hystopaque (density 1.077, Sigma) and centrifuged at RT for 30 min at 400xg. PMN were harvested at the ficoll- RBC interface. Residual RBCs were lysed by incubation in 8.32 g.L21 NH4C1, 0.8 g.L21 sodium bicarbonate, and 0.043 g.L21 EDTA 8 min at 4uC and centrifuged at 400xg for 5 min. Purified effector cells were washed twice with Hank's balanced salt solution (HBSS), enumerated using trypan blue, and resuspended in PBS at 1-5.107 cells mL21, depending on the quantity of PMN collected on a given day and the number of wells tested. Within this concentration range there was no effect on readout under the conditions of the assay. The major contaminants were cellular debris and RBC. Viability (verified before every experiment) was .99% and PMNs constituted 90-99% of leucocytes. PMN were used immediately after harvest and purification, usually within 2-3 hr following sample collection.
Chemiluminescence monitoring of PMN ROS generation
Chemiluminescence was measured at 37°C using opaque 96- well plates (Berthold), and a MicroLumat Plus 96 luminometer (Berthold), controlled with Window software. Chemiluminescence was recorded as relative light units (rlu). A 4 mg/ml stock solution of isoluminol (4-aminophthalhydrazide; Sigma) prepared in DMSO, was stored in aliquots at -20°C in the dark, and used at a final concentration of 0.04 mg/ml in sterile PBS. To facilitate rapid handling, only 40-50 wells per plate were used. Merozoite pellets (40 μΐ), or alternatively normal or parasitized red cell pellets, were incubated with 10 μΐ of test or control sera or IgG for at least 30 min at 37°C. PMN (100 μΐ at 1- 5.107 cells/ml) and isoluminol (100 μΐ) were loaded rapidly using an Eppendorf Multipipette 4780. Plate reading started immediately, and each well was monitored automatically by the luminometer for 1 sec every minute for 1 hr. The parameter used for this work was maximal rlu, which was generally observed within minutes of initiating the reactions under the conditions used. Data are represented as a standardized activity index of Antibody Dependent Respiratory Burst (ADRB) calculated as:
ADRB = (rlu maximum sample / rlu maximum HIS) x 1000 where rlu maximum HIS is an average of the first and last wells on the plate.
Statistical analysis of ADRB assay data
ELISA and/or ADRB data were analyzed for statistical significance using the Wilcoxon signed rank test and the Spearman rank correlation test for non-normally distributed data, and only P values <0.05 were considered significant. Results
Relationships between levels of IgG specific for MSPlpl9 and MSP4p20 in holoendemic and mesoendemic sera
Since the ADRB assay measures primarily MSP specific IgG functionality, the prevalence and levels of IgG specific for two MSP antigens, MSPlpl9 or MSP4p20, were first determined in the sera from Dielmo (holoendemic, 116 individuals) and Ndiop (mesoendemic, 114 individuals). Those with ELISA OD ratios above background at a 1:200 dilution were considered to be responders for prevalence evaluations. In Dielmo, 73% and 88% of sera analyzed recognized MSPlpl9 and MSP4p20, respectively. Corresponding determinations of prevalence for Ndiop were 86% and 78%. Correlations between IgG levels to both antigens in individual sera from both villages, was tested using the Spearman rank test. As shown in Table 1, there were highly significant positive correlations of IgG responses to PfMSPlpl9 and PfMSP4p20 (rho from 0.37 to 0.58, P<0.0001), indicating that individuals tended to respond with similar relative intensities to both antigens.
Table 1
Figure imgf000069_0001
All correlations (rho) are highly significant (P < 0.0001) for both areas (Spearman rank test).
Relationships between levels of IgG for MSPlpl9 or MSP4p20 and ADRB activity in individual sera
To determine if IgGs specific for the two MSP antigens might have a role in mediating ADRB activity, a statistical analysis was carried out using ADRB index and ELISA OD-ratio values determined for each serum (230 samples). Tables 2 and 3 shows that highly significant correlations were found between the ADRB index and IgG responses to MSPlpl9 and MSP4p20 in both villages (P < 0.0003). Nevertheless, when stratified by age group, some correlations were no longer significant using the Bonferroni correction, and this was different for the two villages (Tables 2 and 3).
Table 2. NDIOP - mesoendemic area
Figure imgf000070_0001
Significant correlations (rho) are noted with their P values (Spearman rank test).
Non significant correlations are stipulated NS.
Impact of MS Pip 19 antibodies on ADRB activity using isogenic merozoites
A role for MSPlpl9 specific IgG in the ADRB assay was investigated using 21 individual sera with high anti-MSPlpl9 ELISA OD-ratios, and the HIS pool of 30 sera from immune residents of Dielmo. ADRB readout (rlu) was compared using either P. falciparum D10 (D10-PfM3') or transgenic D10 merozoites, in which PfMSPlpl9 has been replaced by the non-cross reactive P. chabaudi homologue, PcMSPlpl9 (PcMEGF) (O'Donnell et al, 2001, J Exp Med 193: 1403-1412). Thus, differences in the observed chemiluminescence signal using these two parasite strains, can be attributed to antibodies specific for PfMSPlpl9. Figure 1 shows that the transgenic merozoites elicited highly significant (P<0.0001) reduced chemiluminescence for all individual sera, varying from 7-54% inhibition, with a mean of 32%. Importantly, the same level of reduced chemiluminescence was also observed using the HIS pool of 30 antisera, none of which were previously analyzed or selected for anti-MSPlpl9 responders. These results indicate that antibodies induced by natural P. falciparum infection to this single small conserved antigen, are major activators of extra-cellular reactive oxygen species (ROS) release by neutrophils.
Efficacy of antigen specific IgG depletion
Since the use of isogenic merozoites was relevant only for evaluating MSPlpl9-specific antibody contribution to ADRB readout, another technique was developed, involving depletion of serum antibodies specific for other antigens, and ELISA monitoring for efficiency of depletion as described in Methods. Figure 2 shows an example of anti- MSP4p20 depletion from the HIS pool. The HIS was titered for either MSP4p20 (Figure 2A) or MSPlpl9 (Figure 2B) specific antibodies before and after depletion. The results show that the depletion protocol is specific and highly effective. Median titer units decreased from 511 to 1 after anti-MSPlpl9 depletion, and from 816 to 8 after anti-MSP4p20 depletion, giving mean efficiencies of >99%.
Impact of MSPlpl9 and MSP4p20 specific IgG depletion on ADRB activity
The effects of specific IgG depletion on ADRB activity are shown in Figure 3. Anti- MSPlpl9 depletion in individual immune sera resulted in a highly significant 29,2% average reduction of the ADRB index, confirming the results obtained using isogenic merozoites (Figure 3). Moreover, anti-MSP4p20 depletion in individual sera with high anti-MSP4p20 ELISA OD-ratios, reduced the average ADRB index by about 36% (Figure 3). Consecutive depletion of IgGs specific for both MSPlpl9 and MSP4p20 in individual sera, diminished the ADRB signal by an average of 72.9% (Figure 3).
These results thus demonstrate a surprising and unexpected synergy, which is a potentiating effect, between the depletion of MSP-1 and MSP-4. This data strongly support the use of a vaccine combining MSP-1 and MSP-4 antigens for treating or preventing malaria in subjects in need thereof. EXAMPLE 2: Analysis of antibody responses to MSP1 and MSP4 regarding clinical outcome
Method
ELISA analysis on a batch of PfMSPlpl9 and PfMSP4p20 has been carried out on a set of villagers from Ndiop withdrawn before rainy season in year 2000 (cross sectional prospective study). ELISA was done using the two Ags combined in the same plate (1 μg/ml for coating) according to standard procedure. Multiplex Analysis was done using custom, Elisa-derived Luminex Fluorescent magnetic bead based assay (Magpix System, Millipore). Beads from different regions were covalently linked to MSPlpl9 and MSP4p20 and analysed in the same samples using adapted technique derived from those described with Luminex 100 device.
A Poisson regression model was used to analyze the relationship between antibody response(s), prior to the transmission season, and incidence of malaria attacks during the follow-up period. Optimal age stratification was based on the age distribution of the parasitologic and clinical data available for this setting ie age cut-offs of 15 and 30 years old. For each villager, the follow-up time was calculated as the number of days actually spent in the village during the five months of follow-up, excluding individuals absent from the village for more than 30 days during the follow-up period. The follow- up time was adjusted for individuals who experienced malaria attacks, by excluding from the days at risk a period of 10 days after the diagnosed malaria attack.
The analysis included 144 individuals, who experienced a total of 129 clinical episodes during the follow-up period. There was a significant relationship with protection against clinical malaria in an age independent un-stratified model for both Ags on plate: P <0.001, IRR = 0.874 [0.81-0.93]. The model for analysis included dichotomized stratified age of individuals with high versus low IgG responses. High IgG responses were defined as OD ratio > mean for PfMSP4/PfMSPl association. In this model, high Ab responses were significantly associated with protection to both Ags on plate: P <0.001, IRR = 0.469 [0.32-0.68]. When stratifying IgG responses as function of No of clinical accesses (Fig. 1; 0 vs 1 or 2 vs >2 clinical accesses) we observe a significant decrease of levels of IgG to MSP1 and MSP4 with increasing morbidity as recorded after the rainy season (Figure 4).
Taken together, these data are in favor of a positive association of high levels of IgG to MSP1 and MSP4 for protection (2.13 less risk of malaria attack).
EXAMPLE 3: Analysis of antibody responses in symptomatic malaria in Cote d'lvoire
Antibody responses against MSP1 and MSP4 were measured in consultants recruited in sentinel health centers of Cote d'lvoire from 2 different geographical settings as detailed in Table 4.
Table 4
Figure imgf000073_0001
Long lasting impregnated nets: individual inquiry for use of bednets before consultation * The mean national level of prevalence of clinical malaria in Cote d'lvoire is 100 %o
In these settings, present levels of malaria transmission are high. In Cote d'lvoire individuals shows a less systematic use of bednets than in Senegal, thus resulting in higher risk for malaria, particularly in the fast growing population of townships such Abobo. IgG responses measured with custom ELISA multiplex assay show (Fig 5) highly significant level of antibody responses to MSPl and MSP4 during infection (Figure 5 A) that relates inversely to the level of parasitaemia accurately measured on the day of consultation (Figure 5B). Such difference in Ab responses between settings is not strictly reflected by use of somatic antigens (Figure 5C).
These results can be interpreted by the fact that in the village of Korogho, where the background of immunity remains high (strong transmission, lower LLIN's protection), the boosting of Ab responses by infection is responsible for significantly lower symptomatic densities of circulating parasite correlating with very high levels of IgG against PfMSPl and PfMSP4. Of note, the use of multiplex beads assay discriminate higher levels of Ab responses to PfMSP4 vs PfMSPl, contrary to ELISA using somatic Ag.
Therefore, these results demonstrate that lower prevalence of clinical malaria and lower parasitemia are associated with a higher amount of antibodies directed to MSPl and MPS4 (Figure 5A), but not with a general increase of antibodies directed to total schizont antigens (Figure 5C). Consequently, these results strongly support the use of a combination of MSPl and MSP4 antigens for preventing malaria.

Claims

A composition comprising MSP4 and MSP1, variants or fragments thereof.
A pharmaceutical composition comprising MSP4 and MSP1, variants or fragments thereof in combination with at least one pharmaceutically acceptable carrier.
A medicament comprising MSP4 and MSP1, variants or fragments thereof.
A vaccine against malaria comprising MSP4 and MSP1, variants or fragments thereof.
The vaccine according to claim 4, further comprising at least one pharmaceutically acceptable adjuvant.
The vaccine according to claim 5, wherein said adjuvant is Montanide ISA51.
The composition according to claim 1, the pharmaceutical composition according to claim 2, the medicament according to claim 3 or the vaccine according to claims 4 to 6, wherein said fragment of MSP4 is MSP4p20 and/or said fragment of MSPl is MSPlpl9.
The composition according to claim 1, the pharmaceutical composition according to claim 2, the medicament according to claim 3 or the vaccine according to claims 4 to 6, wherein said fragments of MSP4 are PfMSP4p20 and PvMSP4p40 and/or said fragment of MSP1 is PfMSPlpl9 and PvMSPlpl9.
The composition, pharmaceutical composition, medicament or vaccine according to anyone of claims 1-8, for treating malaria.
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