MALARIA VACCINE AND METHODS FOR PRODUCING SAME

Abstract

The present description relates to malaria vaccines comprising Plasmodium falciparum (Pf) polypeptide complexes and methods of producing the same. The Pf polypeptides in complexes or in a partially complexed arrangement may comprise two or more of the following polypeptides: PfRipr, PfCyrPa and PfRh5. Drosophila cells and expression vectors are also described.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present invention is a Divisional of co-pending application Ser. No. 16/488,999, filed Aug. 27, 2019, which is a national stage application which claims priority from PCT Application No. PCT/AU2018/050155 filed Feb. 23, 2018, and AU Application No. AU2017900648 filed Feb. 27, 2017. Applicants claim the benefits of 35 U.S.C. .sctn. 120 as to the said co-pending application and PCT application, and priority under 35 U.S.C. .sctn. 119 as to the said AU application, and the entire disclosures of all applications are incorporated herein by reference in their entireties.

FIELD

[0002] The present description relates to malaria vaccines comprising Plasmodium falciparum (Pf) polypeptides and methods of producing the same.

BACKGROUND

[0003] Bibliographic details of references in the subject specification are also listed at the end of the specification.

[0004] Reference to any prior art in this specification is not, and should not be taken as, acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0005] Malaria in man is a tropical disease resulting from infection by one celled protozoan parasites of the genus Plasmodium which are deposited in to the blood stream when an infected mosquito takes a blood meal. Plasmodium falciparum and Plasmodium vivax are responsible for the most common and severe forms of malaria and despite recent significant reductions in the frequency of infection and deaths, last year it is estimated that over 200 million people were affected with approximately 400,000 deaths. Children in sub-Saharan Africa are particularly vulnerable to P. falciparum and account for most deaths from malaria. The parasite undergo a complex life cycle in man with rounds of infection and replication in erythrocytes or reticulocytes causing many of the early clinical symptoms of malaria, i.e., fever, shaking chills, headache, malaise, myalgia and fatigue. Current guidelines suggest that all new subjects with P. falciparum malaria should be admitted to hospital initially because of the speed with which patients can worse.

[0006] Subunit malaria vaccines are still in development 20 years after they were widely heralded as the most promising way forward for protecting vulnerable populations from Malaria. Much research has been undertaken during this period and knowledge concerning the molecular immunobiology of the host parasite relationship has accumulated. There are currently no available vaccines for malaria. The most advanced vaccine currently undergoing development is RTS,S which targets the liver stages of the parasite life cycle.

[0007] Another proposed vaccine approach is to target blood stage (merozoite) parasites. A large number of proteins are produced by the merozoite stage of the parasite that congregate at least in part at the apical end or complex of the merozoite prior to or at the time of erythrocyte engagement. Plasmodium merozoite proteins also form erythrocyte cell surface proteins that act as receptors for the invading merozoite. Illustrative merozoite proteins include MSP1, MSP2, MSP4, MSP5, MSP10, Pf12, Pf38, Pf92, Pf113, ASP, RAMA, EBA140, EBA175, EBA181, EBL1, AMA1, MTRAP, MSP3, MSP6, H101, H103, MSP7, Pf41, RAP1, RAP2, RAP3, RHopH1, RhopH3, Rh1, Rh4, Rh2b, Rh5, SPATR, PTRAMP, TLP, Pf34, PF14_0344, PF10_0323, PFF0335c, AARP, MSP3.4, MSP3.8, MSRP1, MSRP2, MSRP3, RON1, RON2, 4, 5 and 8) RON3, RON6, Pf12p, MSP9, GAMA, and PF11_0373. The merozoite surface proteins (MSPs) are thought to function early in merozoite invasion (a process which takes place in less than two minutes) and are highly polymorphic. The erythrocyte binding antigens (PfEBAs) and reticulocyte binding protein (PfRHs) are related to P. virax duffy binding proteins/reticulocyte binding proteins respectively and they orchestrate a variety of interations that result in host cell invasion by poorly defined pathways that display strain specific permutations. RH5 is more conserved PfRH merozoite secreted protein that binds the Ok blood group antigen, basagin. Importantly antibodies raised against RH5 are able to prevent parasite growth in vitro and the blocking effect is not strain specific. In this case, the ability to produce a immunogenic RH5 able to engender inhibitory antibodies has been a problem for vaccine developers. Others have looked at the ability of antibodies against other merozoite proteins to enhance invasion blocking and found, for example that antibodies against PfRH4 and PfRH5 work synergistically to enhance invasion blocking effects.

[0008] These molecules are proposed as vaccine candidates based on their surface location as extracellular proteins and/or proposed role in host cell invasion and development within the host cell. One of the problems with this approach generally is the antigenic variability or allele polymorphism of the blood stage antigens which limits the ability of one allele of the protein to protect against different alleles present in the population of Plasmodium alleles. Another problem is redundancy in the host invasion pathway which means that blocking one pathway may not affect merozoite invasion levels.

[0009] Another set of problems faced by malaria vaccine developers in the field is the production of adequate amount of protein in a suitable form. For example, Plasmodium polynucleotides have a very high A:T content which leads to low codon usage compatibility in heterologous expression systems. The large size of some of these proteins and the presence of long stretches of repetitive amino acid sequences also hampers their expression in heterologous expression systems. Endogenous Plasmodium proteins are not modified by N-linked glycans thus expression in most available eukaryotic expression systems may lead to inappropriate or non-native glycosylations or disulfide bonding which foreshadow unpredictable effects on the immunogenicity of vaccine candidates. One approach with membrane proteins has been to express hydrophilic ectodomains without the more hydrophobic domain, or to express only about 200 bp fragments of the ectodomain recombinantly.

[0010] Accordingly, it remains challenging to develop and produce Plasmodium derived antigens, including modified or truncated forms that are able to engender effective immunity against blood stage forms of the parasite.

SUMMARY

[0011] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0012] As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a single composition, as well as two or more compositions; reference to "an agent" includes one agent, as well as two or more agents; reference to "the disclosure" includes single and multiple aspects of the disclosure and so forth.

[0013] Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of sequence identifiers is provided after the brief description of the drawings. A sequence listing is provided after the claims.

[0014] Plasmodium falciparum (Pf) Ripr polypeptide is a peripheral membrane protein of over 1000 amino acids comprising 10 EGF domains mostly at the C-terminal portion of the polypeptide. EGF domains comprise multiple conserved cysteine residues which can lead to complex aggregate or multimer formation upon expression in vitro. Such polypeptides are typically difficult to express in prokaryotic expression systems and when expressed in E. coli, the present inventors identified that antibodies produced against the polypeptide were not inhibitory.

[0015] In one embodiment, the specification provides a modified arthropoda cell, modified to express a Plasmodium falciparum (Pf) Ripr polypeptide from a heterologous polynucleotide encoding a PfRipr polypeptide. In one embodiment, the cell is an insect cell. In one embodiment, the heterologous polypeptide comprises at least initially (prior to cleavage) a suitable signal sequence. In one embodiment, the polynucleotide is operably linked to an expression control sequence which induces PfRipr polypeptide expression in the cell. As determined herein, PfRipr polypeptide expressed in the insect cell induces parasite invasion inhibitory antibodies when administered to a subject.

[0016] In one embodiment, the specification provides a modified Drosophila derived cell modified to over express a Plasmodium falciparum (Pf) Ripr polypeptide (antigen) from a heterologous polynucleotide encoding a PfRipr polypeptide. In one embodiment, the heterologous polypeptide comprises at least initially (prior to cleavage) a suitable signal sequence. In one embodiment, the signal sequences is an insect signal sequence to direct the polypeptide through the host cell secretory pathway. In one embodiment, the polynucleotide is operably linked to an expression control sequence which induces PfRipr polypeptide expression in the cell. In one embodiment, insect promoter sequences are routinely employed. As determined herein, PfRipr polypeptide expressed in the Drosophila cell induces parasite and specifically merozoite invasion inhibitory antibodies when administered to a subject.

[0017] An illustrative Drosophila derived cell is a Drosophila melanogaster derived cell, such as a Scheider 2 cell line (S2), S3 cells or a derivative of the Kc cell line used for recombinant protein production. A wide variety of vectors in general allow for stable expression of recombinant proteins in insect cells. However, the production of such a large full length PfRipr polypeptide in Drosophila derived S2 cells was not a routine matter.

[0018] Other insect host cell expression systems are known in the art and include Spodoptera frugiperda derived cells, such as Sf9, Sf21 cell lines. Many types of viruses can infect insect cells and Baculovirus expression systems are widely employed to express recombinant proteins transiently or stably in insect cells.

[0019] Illustrative signal sequences include the insect BiP leader sequence, the honeybee melittin, signal peptide. As known to those of skill in the art, co-expression of BiP/Grp78 with a protein disulfide isomerase can enhance secreted expression in insect cells. Putative optimal signal sequences are tested to determine the optimal performance.

[0020] In one embodiment, the Pf polypeptides are expressed as soluble non-aggregated forms.

[0021] Expression may be transient or stable. Based upon the present disclosure, a wide range of selection procedures are known in the art to identify stable cell lines expressing high levels of antigenic Pf polypeptide, including a PfRipr polypeptide, and a PfRH5 polypeptide and a PfCyrPa polypeptide.

[0022] Expression control sequences promote optimal transcription, processing, RNA export and translation of the subject Pf polypeptide/s. Transcription regulatory sequences, i.e., promoters/enhancers are selected for high levels of transcription. Putative optimal expression control sequences are tested to determine the optimal performance. Illustrative promoters include the actin 5 promoter, and PpIE promoters, OpIE (OpIE1/OpIE2 for example) promoters, and KanR promoters.

[0023] In one embodiment, the insect cell line grows in suspension, is tolerant to osmolarity changes, and does not require CO.sub.2 for cultivation. Protein is expressed into the culture media without requirement for cell lysis and thus purification steps are reduced.

[0024] Reference herein to a Plasmodium "polypeptide", includes reference to an "antigen" or "one or more epitopes" and refers to proteins or parts or portions thereof that comprise naturally occurring amino acid sequences, alleles, recombinantly produced modified forms, processed products, or modified or truncated (biologically active fragments) forms thereof which induce antibodies in a subject that are effective in inhibiting the invasion of host cells by merozoites. In one embodiment the Pf polypeptide is a polypeptide that is secreted from the merozoite. Illustrative truncated forms include the ectodomain together with all or part of a transmembrane domain/GPI anchor. Illustrative modified forms include polypeptides with one or more amino acid deleted, substituted or modified. In one embodiment, modified or truncated forms essentially retain or improve on the antigenicity and/or stability of the unmodified or naturally occurring polypeptide sequence. Modified forms may be modified to ensure certain host cell post-translational modifications are avoided or reduced in the Pf polypeptide while others are enhanced to allow optimal folding and antigenicity. In one illustrative embodiment, modified forms may have amino acid substitutions, for example to Ala or Gly, to reduce host cell glycosylation events or to enhance stability for example in the case of unpaired cysteines.

[0025] In one embodiment, modified and truncated forms of Plasmodium polypeptides display at least one characteristic selected from the group comprising (i) reduced binding to non-invasion inhibitory antibodies, (ii) at least substantially the same binding to invasion inhibitory antibodies, (iii) elicits the production of lower titers of non-neutralizing antibodies than invasion inhibitory antibodies, (iv) elicits the production of invasion inhibitory antibodies, (v) elicits the production of broadly invasion inhibitory antibodies against more than one allele of the antigen/s, (vi) optionally elicits the production of higher titers of invasion inhibitory antibodies; and (vii) optionally elicits the production of high titers of broadly invasion inhibitory antibodies. Specifically, in one embodiment a modified or truncated polypeptide displays an enhanced ability relative to the unmodified form to elicit antibodies that facilitate invasion inhibition. Inhibition by an antibody includes complete inhibition of invasion and a significant reduction in invasion such as 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% or less than 30% invasion. By co-administering Plasmodium polypeptides, PfRipr, PfRh5, PfCyrPa individually or as a mixture or or as a complex, combinations of antibodies are generated against PfRipr, PfRh5, PfCyrPa that effectively inhibit growth of Plasmodium blood stages within the host. In another embodiment, modified and truncated forms may also be modified to comprise expression, detection, purification or anchorage tags to facilitate their isolation, purification, detection or administration etc. Tags may individually perform several such as enhancing folding and stability and facilitating purification. Several tags are known in the art and all such forms may be tested and employed. Modified and truncated forms may also comprise linker sequences to aid, for example, in their presentation to the immune system.

[0026] Illustrative purification tags include N-terminal hexa-His tag, biotin, Fc and Strepll (SAWSHPQFEK).

[0027] Reference to a Pf polypeptide/protein means one or more of PfRipr, PfRh5, PfCyrPa and PfEBA175 polypeptides or antigens, and includes any allele of the protein or modified or truncated forms thereof which induce inhibitory antibodies in a subject. In one embodiment, a Pf polypeptide may be expressed as a monomer. In one embodiment, a Pf polypeptide may be expressed as a homodimer. In one embodiment, a Pf polypeptide may be expressed as a homotrimer.

[0028] Reference to a Pf polypeptide/protein means one or more of PfRipr, PfRh5, PfCyrPa and PfEBA175 polypeptides or antigens, and includes any allele of the protein or modified or truncated forms thereof which induce inhibitory antibodies in a subject. In one embodiment, a Pf polypeptide may be expressed as a monomer. In one embodiment, a Pf polypeptide may be expressed as a homodimer. In one embodiment, a Pf polypeptide may be expressed as a homotrimer.

[0029] Reference to a Pf protein complex, or Pf protein mixture means substantially a complex or substantially a mixture comprising two or more of PfRipr, PfRh5, PfCyrPa and PfEBA175 polypeptides or antigens, and includes any allele of the protein or modified or truncated forms thereof which induce inhibitory antibodies in a subject.

[0030] In one embodiment, the Pf protein may be glycosylated.

[0031] In one embodiment, the PfRipr is essentially full length, lacking its signal sequence. The PfRipr signal sequence (MKLCILLAVVAFVGLSLG) is cleaved and removed during secretion. In an embodiment, PfRipr is expressed as a soluble monomer, a soluble homodimer or a combination thereof.

[0032] Illustrative strains or genotypes from which the polypeptides or their sequences may be derived include 7G8, 3D7 (reference strain), DD2, HB3, W2MEF, D10, GHANA1, T994, CSL-2, E8B, MCAMP, PREICH, RO-33, V1_S, SANTALUCIA, SENEGAL3404, K1, FCR-3, FCC-2, and D6.

[0033] "Derived from" includes directly or indirectly derived from.

[0034] In an embodiment, PfRipr comprises the amino acid sequence set forth in SEQ ID NO: 6 or a sequence or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identical thereto, or a biologically or immunologically active fragment thereof.

[0035] In an embodiment, PfRh5 comprises the amino acid sequence set forth in SEQ ID NO: 8 or 9; or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identical to SEQ ID NO: 8 or 9; or a biologically or immunological active fragment thereof.

[0036] In an embodiment, PfCyrPa comprises the amino acid sequence set forth in SEQ ID NO: 7 or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identical thereto, or a biologically or immunologically active fragment thereof.

[0037] In an embodiment, PfEBA175 comprises the amino acid sequence set forth in SEQ ID NO: 10 or 27 or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identical thereto, or a biologically or immunologically active fragment thereof.

[0038] In one embodiment, the heterologous polynucleotide encoding a PfRipr polypeptide and a suitable signal sequence operably linked to a promoter which induces PfRipr polypeptide expression in the cell comprises the sequence set forth in SEQ ID NO: 14.

[0039] In another embodiment, the cell further comprises a heterologous polynucleotide encoding P/12115 polypeptide and a suitable signal sequence operably linked to a promoter which induces polypeptide expression the cell. In an embodiment, the polynucleotide comprises the sequence set forth in SEQ ID NO: 15. In one embodiment, co-expression of PfRipr and PfRh5 polypeptides in a Drosophila culture cell induces increased yields of both polypeptides compared to mono-expression of either polypeptide in the same cells.

[0040] In one embodiment, the cell further comprises a heterologous polynucleotide encoding a PfCyrPa polypeptide and a suitable signal sequence operably linked to a promoter which induces high level of PfCyrPa polypeptide expression in the cell. In an embodiment, the polynucleotide comprises the sequence set forth in SEQ ID NO: 16.

[0041] In one embodiment PfEBA175 is expressed together with PfRipr, PfRh5, PfCyrPa in the expression host cell.

[0042] Thus in one embodiment, the cell of the present invention may express a PfRipr, a PfRh5, or a PfCyrPa polypeptide, individually, a PfRipr and a PfRh5 polypeptide together, a PfRh5 and a PfCyrPa polypeptide together, a PfRipr and a PfCyrPa polypeptide together, or a PfRipr and a PfRh5 and a PfCyrPa polypeptide together. Reference to "together" includes substantially as a complex and/or substantially as an uncomplexed mixture of protein.

[0043] In another embodiment, the expression host cell expresses a PfRipr, a PfRh5, a PfCyrPa or a PfEBA175 polypeptide individually, a PfRipr, a PfRh5 and a PfEBA175 polypeptide together, or a PfRh5, a PfCyrPa and a PfEBA175 polypeptide together, or a PfRipr, a PfCyrPa and a PfEBA175, or a PfRipr and a PfRh5 and a PfCyrPa polypeptide and a PfEBA175 polypeptide together.

[0044] In one embodiment, the heterologous polynucleotide or vector expressing the subject polypeptides encodes one or two or three or four heterologous polypeptides.

[0045] In one embodiment, A Drosophila derived cell modified to express a Plasmodium falciparum (Pf) Ripr polypeptide, a PfRh5 polypeptide and a PfCyrPa polypeptide from a heterologous polynucleotide encoding a PfRipr polypeptide and a suitable signal sequence operably linked to a promoter which induces high level of PfRipr polypeptide expression, a PfRh5 polypeptide and a suitable signal sequence operably linked to a promoter which induces high level of PfRh5 polypeptide expression and a PfCyrPa polypeptide and a suitable signal sequence operably linked to a promoter which induces high level of PfCyrPa polypeptide expression in a Drosophila derived cell wherein the PfRipr polypeptide, PfRh5 polypeptide and PfCyrPa polypeptide from said cell induces parasite invasion inhibitory antibodies when administered to a subject.

[0046] In one embodiment the cell further comprises a heterologous polynucleotide encoding a PfEBA175 polypeptide and a suitable signal sequence operably linked to a promoter which induces EBA175 polypeptide expression in the cell. In one embodiment amino acids 760-1298 of EBA175 allele derived from 3D7 strain are expressed (SEQ ID NO: 17). In one embodiment, amino acids 761-1271 of EBA175 allele derived from W2MEF are expressed (SEQ ID NO: 27).

[0047] Thus the cell of the present invention may express a PfRipr polypeptide and one or more of a PfRh5 polypeptide, a PfCyrPa polypeptide, and a PfEBA175 polypeptide. In one embodiment, the cell may express a PfRipr polypeptide and a PfRh5 polypeptide, or a PfRipr polypeptide and a PfCyrPa polypeptide, or a PfRipr polypeptide and a PfEBA175 polypeptide. In one embodiment, the cell may express a PfRipr polypeptide, a PfRh5 polypeptide and a PfCyrPa polypeptide.

[0048] In one embodiment PfRipr is expressed in a Drosophila cell. In one embodiment PfRh5 and/or PfCyraPa and/or PfEBA175 are expressed in an expression system other than a Drosophila cell. A wide range of expression systems are known in the art and some are described herein. Protein expression systems suitable for clinical biomanufacture include baculovirus expression systems and mammalian expression systems and prokaryotic expression systems.

[0049] In another aspect the description provides an expression vector comprising, a polynucleotide encoding a PfRipr polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces PfRipr polypeptide expression in a Drosophila continuous culture cell, wherein the PfRipr polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.

[0050] In another aspect the description provides an expression vector comprising, a polynucleotide encoding a PfRh5 polypeptide including a truncated form and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces PfRh5 polypeptide expression in an expression host cell, wherein the PfRh5 polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.

[0051] In on embodiment, the PfRh5 polypeptide is a truncated from of PfRh5 polypeptide comprising amino acids D126 through to Q526 of full length PfRh5. In one embodiment the truncated form of PfRh5 is formed in S2 cell culture.

[0052] In another aspect the description provides an expression vector comprising, a polynucleotide encoding PfCyrPa polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces high level of PfCyrPa polypeptide expression in an expression host cell, wherein the PfCyrPa polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.

[0053] In one embodiment, the description provides an expression vector comprising, a polynucleotide encoding a PfEBA175 polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces PfEBA175 polypeptide expression in an expression host cell, wherein the PfEBA175 polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.

[0054] In one embodiment, the expression host cell and expression vector are compliant with current good manufacturing practices.

[0055] In another aspect the description provides an expression vector comprising, a polynucleotide encoding a PfRh5 polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces high level of PfRh5 polypeptide expression in a Drosophila continuous culture cell, wherein the PfRh5 polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject, and wherein the polypeptide is a truncated from of PfRh5 polypeptide comprising amino acids D126 through to Q526 of full length PfRh5.

[0056] In another aspect the description provides an expression vector comprising, a polynucleotide encoding PfCyrPa polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces high level of PfCyrPa polypeptide expression in a Drosophila continuous culture cell, wherein the PfCyrPa polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.

[0057] In one embodiment, the description provides an expression vector comprising, a polynucleotide encoding a PfEBA175 polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces PfEBA175 polypeptide expression in a Drosophila continuous culture cell, wherein the PfEBA175 polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.

[0058] In one embodiment the polynucleotide encoding a PfEBA175 polypeptide and a suitable signal sequence operably linked to a promoter induces EBA175 polypeptide expression in a host cell. In an embodiment, the polynucleotide comprises the sequence set forth in SEQ ID NO: 17.

[0059] In one embodiment, amino acids 760-1298 of EBA175 allele derived from the 3D7 strain are encoded.

[0060] In one embodiment, amino acids 761-1271 of EBA175 allele derived from W2MEF are encoded.

[0061] In another aspect the description provides a Drosophila cell comprising one, two or three vectors selected from a vector as described herein. In an embodiment, the one, two or three vectors are stably transfected into the Drosophila cell. In an embodiment, the one, two or three vectors are transiently transfected into the Drosophila cell.

[0062] In another aspect the description provides a Pf polypeptide or Pf protein complex or Pf protein mixture purified from the cell as described herein.

[0063] In another aspect the description provides a purified Pf protein complex or mixture wherein the complex or mixture comprises PfRipr polypeptide, PfRh5 polypeptide comprising only amino acids D126 through to Q526 of full length PfRh5 (lacking amino acids 1 to 125), and PfCyrPa polypeptide, and wherein the purified Pf protein complex or mixture induces parasite invasion inhibitory antibodies when administered to a subject.

[0064] In another aspect the description provides an immunogenic composition comprising the purified Pf complex or mixture as described herein. Thus, in one embodiment, the immunogenic composition comprises a PfRipr polypeptide, a PfRh5 polypeptide, and a PfCyrPa polypeptide. In one embodiment, the PfRh5 polypeptide comprises only amino acids D126 through to Q526 of full length PfRh5 (lacking amino acids 1 to 125) or a modified form thereof. In one embodiment, the immunogenic composition further comprises a PfEBA175 polypeptide. In one embodiment, the EBA175 polypeptide comprises amino acids 760-1298 of EBA175 allele derived from the 3D7 strain. In one embodiment, the EBA175 polypeptide comprises amino acids 761-1271 of EBA175 allele derived from W2MEF.

[0065] In another aspect the description provides a Virus like particle (VLP) or virosome comprising the purified Pf protein or complex or mixture as described herein.

[0066] Thus in one embodiment, the description provides VLP or virosome comprising one or more of PfRipr polypeptide, a PfRh5 polypeptide, a PfCyrPa polypeptide, and a PfEBA175 polypeptide. In one embodiment, the VLP or virosome is assembled using a conjugation system to covalently attach the Pf protein to the surface of the VLP or virosome to form a medium or high density array of Pf polypeptide. VLP or virosome particles may be prepared using one, two, three or four Pf polypeptides, or complexes of two or three Pf polypeptides as described herein. Single Pf polypeptide VLPs or virosomes may then be combined to form substantially mixtures of Pf polypeptides as described herein.

[0067] Thus, in one embodiment, the VLP or virosome or mixture thereof comprises a PfRipr polypeptide, a PfRh5 polypeptide, and a PfCyrPa polypeptide. In one embodiment, the PfRh5 polypeptide comprises only amino acids D126 through to Q526 of full length PfRh5 (lacking amino acids 1 to 125) or a modified form thereof. In one embodiment, the VLP or virosome or substantially mixture thereof further comprises a PfEBA175 polypeptide. In one embodiment, the EBA175 polypeptide comprises amino acids 760-1298 of EBA175 allele derived from the 3D7 strain. In one embodiment, the EBA175 polypeptide comprises amino acids 761-1271 of EBA175 allele derived from W2MEF.

[0068] In one embodiment the description provides an immunogenic composition comprising the purified Pf protein or complex or mixture as described herein or the VLP or virosome as described herein, and a pharmaceutically acceptable carrier such as a diluent or excipient, or an adjuvant.

[0069] The term "carrier" applied to pharmaceutical compositions refers to a diluent, excipient, or vehicle with which the Pf protein mixture or complex is administered. Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

[0070] In one embodiment the description provides a method of producing a Plasmodium falciparum (Pf) Ripr polypeptide comprising transfecting a Drosophila derived cell with a vector of as described herein and isolating PfRipr from said cells. In one embodiment, the Pf polypeptides comprise one or more tags to facilitate isolation and purification.

[0071] In one embodiment the description provides a method of producing a Plasmodium falciparum (Pf) polypeptide mixture or complex comprising transfecting a population of Drosophila derived cells with one, two, three or four vectors of as described herein. In one embodiment, the method comprises culturing the cells to identify a stable cell line expressing one or more Pf polypeptides. In one embodiment, the method comprises culturing a Drosophila derived cell line comprising one or more vectors as described herein and isolating the Pf polypeptides or complex comprising same from cell culture media. In one embodiment, the Pf polypeptide/s comprise/s one or more tags to facilitate isolation and purification.

[0072] In one embodiment the description provides a method of producing an immune response in a subject, the method comprising a administering to the subject an effective amount of the immunogenic composition as described herein.

[0073] In one embodiment the description provides a method of treating and/or preventing malaria caused by P. falciparum in a subject, the method comprising administering to the subject an effective amount of the immunogenic composition as described herein.

[0074] In one embodiment the description provides an immunogenic composition comprising the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein for use in the treatment and/or prophylaxis of P. falciparum malaria.

[0075] In one embodiment the description provides an immunogenic composition comprising the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein for use in the manufacture of a preparation for the treatment or prophylaxis of P. falciparum malaria.

[0076] In another embodiment, the immunogenic composition further comprises a pharmaceutically or physiologically acceptable carrier or diluent.

[0077] In a further embodiment, the immunogenic composition further comprises an adjuvant. In one illustrative embodiment, the adjuvant is a saponin based adjuvant.

[0078] In one illustrative embodiment, the adjuvant is a Virus-like particle (VLP).

[0079] In a further embodiment the description provides a kit for detecting if a subject (a) has or had a P. falciparum malaria infection or (b) has been vaccinated against P. falciparum malaria, the kit comprising:

[0080] (i) a Pf polypeptide or Pf protein complex or Pf protein mixture as described herein capable of binding an anti-P. falciparum malaria antibody and forming and antigen-antibody complex, and

[0081] (ii) one or more antigen-antibody complex detection reagents.

[0082] In a further embodiment the present description provides a method for producing a purified antibody against one or more of the herein described proteins produced in a Drosophila derived cell, comprising administering to a subject an immunologically effective amount of the protein, mixture or complex, and isolating and purifying the antibody produced.

[0083] In an embodiment the description provides an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein.

[0084] In one embodiment, antibodies generated include those that at least partially neutralize an important part of the malaria life cycle such as host cell invasion or schizogony. In some embodiments, antibodies generated against the present Pf polypeptide mixture or complex, inhibit the formation of a complex between PfRipr, PfRh5, a PfCyrPa and thereby

[0085] The present description also provides for use of the herein described proteins, mixtures or complex in, or in the manufacture of a diagnostic agent (such as an antibody or protein mixture) for, the diagnosis or monitoring of infection by Plasmodium falciparum or monitoring of an anti-malaria treatment protocol.

[0086] In other aspects, screening methods are provided employing the instant proteins, mixtures and complexes to identify binding molecules such as antibodies, antigen-binding fragments or molecules, ligands, peptides, organic or inorganic molecules.

[0087] In vitro expression of proteins PfRipr, PfRh5, a PfCyrPa which form a complex in vivo facilitates screening assays to identify and test binding agents or other molecules able to prevent the complex formation or subsequent erythrocyte cell invasion.

[0088] In another aspect, the present description provides a kit or a solid or semi-solid substrate comprising one or more of the herein described proteins, mixtures or complex. The kits or substrates of the present invention are contemplated for use in diagnostic, prognostic, therapeutic or prophylactic applications as well as for use in designing and/or screening Plasmodium binding molecules or Plasmodium receptor molecules. The kits and substrates are also useful in monitoring the efficacy of a treatment protocol against Plasmodium falciparum. In an embodiment the present description provides a kit for detecting if a subject has a P. falciparum infection, the kit comprising:

[0089] (i) an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein capable of binding a P. falciparum malaria antigen, and

[0090] (ii) one or more antigen-antibody complex detection reagents.

[0091] In an embodiment the present description provides a kit for monitoring treatment of a subject having a P. falciparum infection, the kit comprising:

[0092] (i) an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein capable of binding a P. falciparum malaria antigen, and

[0093] (ii) one or more antigen-antibody complex detection reagents.

[0094] In an embodiment the present description provides a kit as described herein, wherein the kit is for use in an immunoassay. In an embodiment the kit is an Enzyme Linked Immunosorbent Assay (ELISA)-type kit. In an embodiment the kit is an indirect ELISA type kit.

[0095] ELISA-type assays and kits are widely used in the art. In one illustrative embodiment kit comprise one or more detection reagents include/s a binding reagent which binds the antigen-antibody complex. In an embodiment the binding reagent is selected from: (i) protein G or a recombinant version thereof; (ii) protein A or a recombinant version thereof; (iii) isolated and/or recombinant protein A/G; (iv) protein L or a recombinant version thereof; or (v) an antibody or fragment thereof. In an embodiment the binding reagent is detectably labeled. In an embodiment the binding reagent is linked to an enzyme. In an embodiment the one or more detection reagents includes an enzyme substrate.

[0096] In an embodiment the present description provides a kit as described herein, wherein the Pf polypeptide, Pf protein complex, Pf protein mixture or isolated antibody raised against the Pf polypeptide, Pf protein complex or Pf protein mixture is conjugated to a solid support. In an embodiment the solid support is a microtitre well or part of a point of care device such as a microfluidic or immunographic device.

[0097] In an embodiment, the steps, features, integers, compositions and/or compounds disclosed herein or indicated in the specification of this application individually or collectively, and any and all combinations of two or more of said steps or features.

[0098] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0099] The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments of the present disclosure.

[0100] Many modifications will be apparent to those skilled in the art without departing from the scope of the present disclosure.

[0101] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

BRIEF DESCRIPTION OF THE FIGURES

[0102] FIG. 1 show the analysis of gene expression from different vectors. (A) shows the comparison of PfRipr expression from a cell line comprising vectors expressing PfRiprFL only (lane 2 sample 1491, cell line WHTZ-04; lane 3 sample 1483, cell line WHTZ-04), PfRiprFL and PfRh5 (lane 4, sample 1320, cell line WHTZG-10), or PfRiprFL and PfRh5 and PfCypra (lane 5, sample 1482, cell line WHTZGP-13) compared to the See Blue Plus2 ladder (lane 1). (B) shows the comparison of expression of PfRiprFL and PfRh5 and PfCypra in cell lines expressing 1, 2 or 3 vectors. The samples run in each lane are listed in Table 3.

[0103] FIG. 2 shows that antibodies against full-length PfRipr inhibit the growth of Plasmodium falciparum parasites. P. falciparum 3D7 parasites were incubated with serially diluted purified IgG from either non-immune rabbit serum (NS) or anti-Ripr antiserum (R1682). The parasitaemia in triplicate wells was counted after 96 hours (2 invasion cycles) and error bars represent the standard deviation of the mean values in triplicate wells.

[0104] FIG. 3A to 3L shows the PfCyrPa, PfRipr PfRh5, PfEBA175 nucleotide and protein sequences and sequences of the pExpreS2-1, pExpreS2-2, pExpreS2-PAC expression vectors.

KEY TO SEQUENCE LISTING

[0105] SEQ ID NO: 1: PfRipr Full length (FL) nucleotide sequence; SEQ ID NO: 2: PfCyrPa nucleotide sequence; SEQ ID NO: 3: PfRh5 version 1 nucleotide sequence; SEQ ID NO: 4: PfRh5 version 2 nucleotide sequence; SEQ ID NO: 5: PfEBA175 nucleotide sequence; SEQ ID NO: 6: PfRiprFL amino acid sequence; SEQ ID NO: 7: PfCyrPa amino acid sequence; SEQ ID NO: 8: PfRh5 Full length (FL) amino acid sequence; SEQ ID NO: 9: PfRh5 version 1 amino acid sequence; SEQ ID NO: 10: PfEBA175 amino acid sequence; SEQ ID NO: 11: pExpreS2-1 vector nucleotide sequence; SEQ ID NO: 12: pExpreS2-2 vector nucleotide sequence; SEQ ID NO: 13: pExpreS2-PAC vector nucleotide sequence; SEQ ID NO: 14: pExpreS2-1 vector comprising PfRiprFL; SEQ ID NO: 15: pExpreS2-2 vector comprising PfRh5 version 1; SEQ ID NO: 16: pExpreS2-PAC vector comprising PfCyrPa; SEQ ID NO: 17: pExpreS2-1 vector comprising PfEBA175; SEQ ID NO: 18: PfRh5 version 2 amino acid sequence; SEQ ID NO: 19: His-tag nucleotide sequence; SEQ ID NO: 20: StrepII purification tag nucleotide sequence; SEQ ID NO: 21: N-terminal secretion signal nucleotide sequence; SEQ ID NO: 22: Flag-tag nucleotide sequence; SEQ ID NO: 23: His-tag amino acid sequence; SEQ ID NO: 24: StrepII purification tag amino acid sequence; SEQ ID NO: 25: N-terminal secretion signal amino acid sequence; SEQ ID NO: 26: Flag-tag amino acid sequence; SEQ ID NO: 27: PfEBA175 amino acid sequence (760-1271 W2MEF).

LIST OF TABLES

[0106] Table 1: Exemplary substitutions. Table 2: Constructs present in each cell line. Table 3: Lane order of gel from FIG. 2A.

DETAILED DISCUSSION OF EMBODIMENTS

[0107] The subject disclosure is not limited to particular screening procedures for agents, specific formulations of agents and various medical methodologies, as such may vary. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any materials and methods similar or equivalent to those described herein can be used to practice or test the present disclosure. Practitioners are particularly directed to and Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; Colowick and Kaplan, eds., Methods In Enzymology, Academic Press, Inc.; Weir and Blackwell, eds., Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, 1986; Remington's Pharmaceutical Sciences (18th ed., Mack Easton, Pa. (1990)), for definitions and terms of the art and other methods known to the person skilled in the art.

[0108] The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

[0109] As used herein "subjects" contemplated in the present description are humans or animals including laboratory or art-accepted test or vehicle animals. In an embodiment, the subject is a mammal. Preferably, the subject is a human, however the present description extends to treatment and/or prophylaxis of other mammalian patients including primates and laboratory test animals (e.g. mice, rabbits, rats, guinea pigs).

[0110] The terms "protein" and "polypeptide" are generally used interchangeably herein. A polypeptide may be defined by the extent of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by having a greater % identity to one reference amino acid sequence than to another. The % identity of a polypeptide to a reference amino acid sequence is typically determined by GAP analysis (Needleman and Wunsch, 1970; GCG program) with parameters of a gap creation penalty=5, and a gap extension penalty=0.3. In an embodiment, the query sequence is at least 50 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. In an embodiment, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. In another embodiment, the query sequence is at least 150 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 150 amino acids. Preferably, the GAP analysis aligns two sequences over their entire length. Preferably, the polypeptide has an antigenic activity of at least 10% of the activity of the reference polypeptide.

[0111] With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided herein will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide/protein comprises an amino acid sequence which is at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

[0112] As used herein, the term "Pf polypeptide" or "Pf protein" refers to a protein from Plasmodium falciparum, a protozoan parasite of the genus Plasmodium that causes malaria in humans.

[0113] As used herein, the term "Ripr" refers to the Rh5 interacting protein and "PfRipr" refers to the Rh5 interacting protein from Plasmodium falciparum. In an embodiment, the PfRipr protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotide sequence that is at least 50%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 1 or a biologically active fragment thereof. In an embodiment, PfRipr has an amino acid sequence that is at least 45%, or at least 50%, or at least 54%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6 or a biological active fragment thereof.

[0114] As used herein, the term "a Rh5 polypeptide" refers to reticulocyte-binding protein homolog 5 and "a PfRh5 polypeptide" refers to reticulocyte-binding protein homolog from Plasmodium falciparum. In an embodiment, the PfRh5 polypeptide is encoded by nucleotide sequence set forth in SEQ ID NO: 3 or 4 or a nucleotide sequence that is at least 50%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 3 or 4 or a biologically active fragment thereof. In an embodiment, a PfRh5 polypeptide has an amino acid sequence that is at least 45%, or at least 50%, or at least 54%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8, 9 or 18 or a biological active fragment thereof. In an embodiment, the PfRh5 polypeptide comprising amino acids D126 through to Q526 of full length PfRh5 (SEQ ID NO: 18).

[0115] As used herein, the term "a CyrPa polypeptide" refers to the Plasmodium cysteine-rich protective antigen and "a PfCyrPa polypeptide" refers to the Plasmodium cysteine-rich protective antigen from Plasmodium falciparum. In an embodiment, the PfCyrPa polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO: 2 or a nucleotide sequence that is at least 50%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 2 or a biologically active fragment thereof. In an embodiment, PfCyrPa has an amino acid sequence that is at least 45%, or at least 50%, or at least 54%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identical to the sequence set forth in SEQ ID NO: 7 or a biological active fragment thereof.

[0116] As used herein, the term "a EBA175 polypeptide" refers to the Erythrocyte binding antigen-175 and PfEBA175'' refers to the Plasmodium Erythrocyte binding antigen-175 from Plasmodium falciparum. In an embodiment, the PfEB175 protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 5 or a nucleotide sequence that is at least 50%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 5 or a biologically active fragment thereof. In an embodiment, PfEB175 has an amino acid sequence that is at least 45%, or at least 50%, or at least 54%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identical the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO:11 or a biological active fragment thereof.

[0117] The biologically active fragment may have the same or similar activity as, or an enhanced activity/immunogenicity relative to a reference polypeptide.

[0118] The Pf proteins as described herein may comprise one or more mutations e.g. an amino acid substitution, deletion or insertion or may comprise a chemical analog not present in the naturally occurring protein. Chemical analogs contemplated include modification of side chains, incorporation of unnatural amino acids and/or their derivatives during synthesis and the use of linkers or cross-linkers or other methods to inter alia impose conformational constraints.

[0119] Amino acid sequence mutants of the polypeptides defined herein can be prepared by introducing appropriate nucleotide changes into a polynucleotide defined herein, or by in vitro synthesis of the desired polypeptide. Such mutants include for example, deletions, insertions, or substitutions of residues within the amino acid sequence. A combination of deletions, insertions and substitutions can be made to arrive at the final protein, provided that the final polypeptide product possesses the desired characteristics.

[0120] Mutant (altered) polypeptides can be prepared using any technique known in the art, for example, using directed evolution or rational design strategies.

[0121] In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series for example, by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site. Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues. Substitution mutants have at least one amino acid residue in the polypeptide removed and a different residue inserted in its place. Such conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".

TABLE-US-00001 TABLE 1 Original Exemplary Residue Substitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; his Asp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, ala His (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; phe Lys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S) thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe, ala

[0122] In one embodiment a mutant/variant polypeptide has only, or not more than, one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 1. Mutants with desired activity may be engineered using standard procedures in the art such as by performing random mutagenesis, targeted mutagenesis, or saturation mutagenesis on known genes of interest, or by subjecting different genes to DNA shuffling.

[0123] Modified Pf polypeptides are tested for their ability to bind to, engender or compete for binding with invasion inhibitory or non-inhibitory antibodies (binding agents) that bind to Pf merozoites in vitro or in vivo. Such assays are commonly employed in the art.

[0124] In an embodiment the mutation may comprise the insertion of a "linker". The term "linker" is used herein to refer to a short, flexible, polypeptide sequence of one or more amino acid residues in length. In an embodiment, the mutation comprises removal of one or more amino acid residues in the polypeptide and replacement with one or more residues with a linker. In an embodiment, the linker permits disulfide bond linkages between cysteine residues leading to retention of the native or "wild-type" disulfide linkages, and in particular retention of the ability to bind to conformation dependent antibodies. Suitable linker sequences are discussed in review articles by George and Heringa, 2002, and Argos, 1990, and may consist of up to 20 amino acid residues such as Gly and Ser, and include, and comprise amino acids selected from the sequence group consisting of Gly, Ser, Ala, Thr and Arg, more particularly Gly- and Ser-Ser-Gly (GSSG). Suitable linkers include, by way of example, the sequences (Gly) 2-Ala-(Gly)2, (Gly)5 or (Gly)s (see Sabourin et al., 2007), (Gly)6, (Gly)7 or (Gly)1o, Gly-Ser-Gly-Ser-Gly (see Dipti et al., 2006), -21(Gly) 4 (see Gly-Ala-Gly, (Gly)2-Arg-(Gly) 2-Ser (see Bellamy-McIntyre et al., 2007), (Gly-Gly-Gly-Gly-Ser), =3.about.4 (see Arai et al., 2006), and Ser-(Gly) 2-Ser-Gly (see Bahrami et al., 2007). One linker may have the sequence Gly-(Ser)2-Gly disclosed herein. It will be understood that selection of suitable linker is a matter of routine experimentation for a person skilled in this field, and the modified polypeptide contemplated herein is not limited to the particular linker sequences disclosed herein.

[0125] As used herein, the term "Pf protein complex" refers to a complex comprising a PfRipr polypeptide, a PfCyrPa polypeptide and a PfRh5 polypeptide. The polypeptides co-localise microscopically and form a functional interacting complex during invasion in vivo and may be co-purified and migrate together in non-reducing SDS-PAGE.

[0126] In one embodiment, the complex or proteins are produced using the cells, expression vectors and methods as described herein. The Pf protein complex may comprise PfRipr and PfCyrPa. The Pf protein complex may comprise PfRipr and PfRh5. The Pf protein complex may comprise PfRipr, PfCyrPa and PfRh5. The Pf protein complex or mixture may comprise PfRipr, PfCyrPa and PfRh5. A mixture may include the three polypeptides in an uncomplexed arrangement or in a partially complexed arrangement.

[0127] Reference to "Co-expression" includes for example expression in a cell of two or more vectors per cell, and includes one vector expressing two or more Plasmodium polypeptides.

[0128] As used herein, the term "Pf protein mixture" refers to mixture comprising two or more Pf proteins wherein the two or more proteins are not present as a complex.

[0129] In one embodiment, the proteins are produced using the cells, expression vectors and methods as described herein. In one embodiment the Pf protein mixture or complex contains or consists essentially of PfRipr and PfCyrPa. In one embodiment, the Pf protein mixture or complex contains or consists essentially of PfRipr and PfRh5. In one embodiment the Pf protein mixture or complex contains or consists essentially of PfRipr, PfCyrPa and PfRh5.

[0130] The term "isolated" and "purified" isolated means material that is substantially or essentially free from other components that normally accompany it in its native state. For example, an "isolated nucleic acid molecule" refers to a nucleic acid or polynucleotide, isolated from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. In particular, an isolated Pf polypeptide, Pf protein complex or Pf protein mixture includes in vitro isolation and/or purification of a protein from its natural cellular environment, and from association with other components of the cell. Without limitation, an isolated nucleic acid, polynucleotide, peptide, or polypeptide can refer to a native sequence that is isolated by purification or to a sequence that is produced by recombinant or synthetic means. In an embodiment, the Pf polypeptides as described herein may be isolated or purified as a Pf protein complex. In an embodiment, the Pf polypeptides as described herein may be isolated individually or as a Pf protein mixture and subsequently folded into a Pf protein complex.

Polynucleotides

[0131] As used herein, the term "polynucleotide" or "nucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A "heterologous polynucleotide" of the invention may be of genomic, cDNA, semisynthetic, or synthetic origin, double-stranded or single-stranded and by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature. The following are non-limiting examples of polynucleotides: messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, chimeric DNA of any sequence.

[0132] As used herein, an "isolated polynucleotide" refers to a polynucleotide which has been separated from the polynucleotide sequences with which it is associated or linked in its native state, or a non-naturally occurring polynucleotide. As used herein, an "exogenous polynucleotide" or "heterologous polynucleotide" refers to a polynucleotide produced or originating outside a particular host organism or cell.

[0133] The heterologous polynucleotide described herein encoding Pf polypeptides comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different to that found in nature. Such sequences may include a promoter, nucleotides encoding a signal sequences or sequences comprising or encoding a tag to aid expression and/or purification. In one embodiment, the heterologous polynucleotide comprises a promoter which allows for a high level of polypeptide expression when in a cell. The heterologous polynucleotide may comprise an N-terminal secretion signal sequence. In an embodiment the N-terminal secretion signal comprises the amino acid sequence "MKLCILLAVVAFVGLSLG" (SEQ ID NO: 25). The heterologous polynucleotide may comprise or encode a tag to aid purification of the expressed polypeptide. In one embodiment the tag is a His-tag comprising the amino acid sequence "HHHHHH" (SEQ ID NO: 23). In an embodiment, the tag is a StrepII purification tag comprising the amino acid sequence "SAWSHPQFEK". In one embodiment the tag is a Flag-tag comprising the amino acid sequence "DYKDDDDK". In another embodiment, the heterologous polynucleotide may comprise a sequence which aids the formation of virus like particles. In an embodiment, the tag facilitates controlled assembly of one or more Pf proteins as described herein in desirable (such as immune-protective) proportions in a VLP.

[0134] Polynucleotides of the invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Polynucleotides which have mutations relative to a reference sequence can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis or DNA shuffling on the nucleic acid as described above).

[0135] In an embodiment, the polynucleotide may be codon optimised to increase expression of the isolated and/or recombinant protein or antigenic fragment thereof in a particular host cell. For example, the polynucleotide may by codon optimised for expression in Drosophila. The term "codon optimised" refers to modification of the codon encoding a particular amino acid to increase the expression of a protein in a given host cell. In one embodiment, endogenous glycosylation sites are retained.

Expression Systems

[0136] The selection of a suitable host organism for expression purposes is determined by various factors which are well known in the art. Factors to be considered include, for example, compatibility with the selected vector, toxicity of the expression product, expression characteristics, immunogenicity of the expressed product, necessary biological safety precautions and costs.

[0137] In some instances it may be desirable to insert the heterologous polynucleotide into an expression vector. In an embodiment, the expression vector may be transferred into a cell and the cell used to produce a Pf protein, Pf protein complex, or Pf protein mixture as described herein.

[0138] As used herein, a "vector" or "expression vector" is a DNA or RNA vector that is capable of transforming a host cell and effecting expression of one or more polynucleotides. In one embodiment, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic. Expression vectors include any vectors that function (i.e., direct gene expression) in host expression cells of the present invention. The vector may be, e.g., a plasmid, virus, artificial chromosome, or a bacteriophage. Such vectors include chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophages, and viruses, vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids and phagemids. In an embodiment, the vector is suitable for expression of Pf protein, Pf protein complex, or Pf protein mixture in an Arthropoda host cell such as a Drosophila cell. In an embodiment, the vector is a DNA polynucleotide comprising a nucleotide sequence having promoter activity in a Drosophila derived cell. In an embodiment, the vector is a viral vector such as a baculovirus (such as for example, AcMNPV that infects Lepidopteran derived cells such as Sf- or Hi-5 cells) retrovirus, a lentivirus, an adenovirus, a herpes virus, a poxvirus or an adeno-associated viral vector. Viral infected cells have a finite life span due to lysis of infected cells. Non-lytic expression systems are also available where the expression vector is stably integrated into the insect genome. Here, expression and secretion does not require cell lysis, making purification straightforward. Expression may be induced or constitutive depending upon the expression control sequence employed. In an embodiment, the vector is pExpreS2-1, pExpreS2-2 pExpreS2-PAC (Expres2ion Biotecnologies, Denmark).

[0139] A person skilled in the art will appreciate that a Pf polypeptide, Pf protein complex or Pf protein mixture as described herein can be produced in cell culture by expression of a heterologous polynucleotide as described herein in a host cell. In an embodiment, the host cell is an Arthropoda cell. A person skilled in the art would appreciate that the Arthropoda cell can be any Arthropoda cell which can be cultured in vitro and in which Pf polypeptide, Pf protein complex or Pf protein mixture can be expressed. In one embodiment, the cells are from a "continuous cell line" or can be cultured in "continuous cell culture". As used herein, the term "continuous cell culture" refers to a culture comprising of a single cell type that can be serially propagated in cell culture for a limited number of cell divisions or indefinitely. The cells as described herein can be cultured in any cell culture medium that allows the expansion of the cells in vitro.

[0140] In one embodiment the Arthropoda cell is a Drosophila cell. Methods of culturing Drosophila cells, including large scale production using a bioreactor, are known in the art and are described for example in Swiech et al (2008). In one example, the cells are cultured in at least a 500 mL, a 1 L, a 1.5 L, a 2 L, a 2.5 L or a 3 L volume.

[0141] "Cells," "insect cells", "Drosophila derived cell" "host expression cells," "transformed host cells," and the like are terms that not only refer to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0142] A person skilled in the art will appreciate that the Pf protein, Pf protein complex, or Pf protein mixture as described herein (which can include purified protein or a subunit thereof or a viral particle or subunit thereof comprising the isolated and/or recombinant protein or an antigenic fragment thereof) can be purified by any method known to a person skilled in the art, including, for example one or more of the following steps: centrifugation, microfiltration, antibody purification, depth filtration ultrafiltration, diafiltration, precipitation, bead chromatography (for example size exclusion chromatography, ion exchange chromatography or affinity chromatography), membrane adsorber (for example ion exchange chromatography or affinity chromatography). The Pf proteins as described herein may comprise a tag, such as a His-tag or Flag-tag, which aids purification of the isolated and/or recombinant protein or antigenic fragment thereof.

[0143] The present description enables a composition comprising a Pf polypeptide complex or mixture of polypeptides wherein the Pf polypeptide complex or mixture is substantially free of Pf molecules other than a soluble PfRipr polypeptide, a soluble PfCyrPa polypeptide and a soluble PfRh5 polypeptide.

[0144] As used herein the phrases "substantially free" refers to a composition comprising less than 30%, 29%, 28%, 27%, 26%, `25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less than 1% aggregated Pf polypeptide (by weight).

[0145] In one embodiment, the proportion of Pf non-monomeric forms is less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% by weight.

[0146] In a particular embodiment, the compositions are substantially free of aggregated Pf polypeptide having less than 1% or less than 0.1% aggregated Pf molecules.

[0147] In another embodiment, the present description enables a composition comprising a Pf polypeptide complex comprising or consisting of a PfRipr polypeptide, a PfCyrPa polypeptide and a PfRh5 polypeptide wherein the Pf polypeptide complex is substantially free or depleted of uncomplexed Pf molecules.

[0148] In another embodiment, the present description enables a composition comprising a Pf polypeptide complex comprising a PfRipr polypeptide and a PfCyrPa polypeptide, or a PfRipr polypeptide and a PfRh5 polypeptide, or a PfCyrPa and a PfRipr polypeptide, a wherein the composition further comprises a PfRipr polypeptide, a PfCyrPa polypeptide or a PfRh5 polypeptide in uncomplexed form.

[0149] As used herein the phrases "substantially free or depleted" refers to a composition comprising less than 30%, 29%, 28%, 27%, 26%, `25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less than 1% uncomplexed Pf polypeptide (by weight).

[0150] In one embodiment, the proportion of uncomplexed Pf polypeptide is less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% by weight.

[0151] In a particular embodiment, the immunogenic compositions are substantially free of uncomplexed Pf polypeptide having less than 1% or less than 0.1% uncomplexed Pf molecules.

[0152] In one embodiment reference to a "mixture of Pf polypeptides" includes a proportion of complex polypeptide such as a composition comprising from 0% to 80% complexed Pf polypeptide and more than 20% uncomplexed Pf polypeptide. In one embodiment reference to a "mixture of Pf polypeptides" includes a composition having more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (by weight) of uncomplexed Pf polypeptide.

[0153] In a related aspect, the immunogenic composition or VLP is enriched for or comprises a Pf polypeptide complex comprising two or three of a PfRipr polypeptide, a PfCyrPa polypeptide and a PfRh5 polypeptide and includes preparations of the composition having more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (by weight) of the Pf complex. In an embodiment, the composition or VLP further comprises up to 30% uncomplexed Pf polypeptide selected from one or more of a PfRipr polypeptide, a PfCyrPa polypeptide, PfRh5 polypeptide and an EBA175 polypeptide.

[0154] In another embodiment, the present description enables a composition comprising a Pf polypeptide complex comprising a PfRipr polypeptide, a PfCyrPa polypeptide and a PfRh5 polypeptide, wherein the Pf polypeptide complex is conjugated at high density to a virus like particle or virosome. In another embodiment individual polypeptides of the complex are displayed separately on the VLP. Illustrative VLPs are known in the art and includes those using conjugation systems such as the SpyTag/SpyCatcher system and variations thereof. In one illustrative embodiment one or more of the Pf polypeptides is genetically modified to comprise a c-terminal Spycatcher tag which is used to display the complex via covalent interaction with a Spytag attached to Acinetobacter phage AP205 VLPs modified to display at least one tag per VLP subunit.

[0155] In some embodiment, the mixture or complex or composition further comprises EBA175 as described herein.

[0156] The term "immunogenic composition", "vaccine" or "vaccine composition as used herein refers to a pharmaceutical composition comprising at least one immunologically active component that induces an immunological response in a subject and possibly but not necessarily one or more additional components that enhance the immunological activity of said active component (for example an adjuvant).

[0157] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solids or solvents (such as phosphate buffered saline buffers, water, saline) dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. The use of such media and agents for pharmaceutically active substances is well known in the art. Immunogenic compositions are described in a number of sources that are well known and readily available to those skilled in the art, for example, Remington's Pharmaceutical Sciences (Martin E. W., Easton Pa., Mack Publishing Company, 19.sup.th ed., 1995).

[0158] An immunogenic composition is formulated to be compatible with its intended route of administration, e.g., local or systemic. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, nasal, topical, transdermal, transmucosal, and rectal administration. Oral and nasal administration include administration via inhalation. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0159] Immunogenic compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, non-aqueous solutions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminium monostearate or gelatine.

[0160] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatine capsules. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatine; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.

[0161] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays, drops, or suppositories. For transdermal administration, the active compound (e.g., polynucleotides of the description) are formulated into ointments, salves, gels, or creams, as generally known in the art.

[0162] The immunogenic compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0163] In one embodiment, the immunogenic composition further comprises an adjuvant. Subject responses to immunogens can be enhanced if administered as a mixture with one or more adjuvants. Immune adjuvants typically function in one or more of the following ways: (1) immunomodulation (2) enhanced presentation (3) CTL production (4) targeting; and/or (5) depot generation. Illustrative adjuvants include: particulate or non-particulate adjuvants, complete Freund's adjuvant (CFA), aluminum salts, emulsions, ISCOMS, LPS derivatives such as MPL and derivatives thereof such as 3D, mycobacterial derived proteins such as muramyl di- or tri-peptides, particular saponins from Quillaja saponaria, such as QS21 and ISCOPREP.TM. saponin, ISCOMATRIX.TM. adjuvant, and peptides, such as thymosin alpha 1 or a VLP. In some embodiments, a VLP includes proteins from one or more of the following: an influenza virus (e.g., a hemaglutinin (HA) or neuraminidase (NA) polypeptide), Hepatitis B virus (e.g., a core or capsid polypeptide), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human papilloma virus, HIV, RNA-phages, Q-phage (e.g., a coat protein), GA-phage, fr-phage, AP205 phage, a Ty (e.g., retrotransposon Ty protein p1). See, e.g., WO03/024480, WO03/024481, WO08/061243, and WO07/098186. An extensive description of adjuvants can be found in Cox and Coulter, "Advances in Adjuvant Technology and Application", in Animal Parasite Control Utilizing Biotechnology, Chapter 4, Ed. Young, W. K., CRC Press 1992, and in Cox and Coulter, Vaccine 15(3): 248-256, 1997.

[0164] The description includes a method for prophylactic or therapeutic treatment of Plasmodium infection in a patient, which comprises administration to the patient of an effective amount of a composition as described herein. Reference herein to "treatment" is to be understood in its broadest context. Accordingly, the term "prophylactic treatment" includes treatment to protect the patient against infection or to reduce the likelihood of infection. Similarly, the term "therapeutic treatment" of infection does not necessarily imply that the patient is treated until total recovery from infection, and includes amelioration of the symptoms of infection as well as reducing the severity of, or eliminating, the infection.

[0165] The immunogenic composition as described herein is administered in an effective amount. An "effective amount" means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of the infection. The amount varies depending upon the health and physical condition of the individual to be treated, the racial background of the individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. If necessary, the administration of an effective amount may be repeated one or several times. The actual amount administered will be determined both by the nature of the infection which is being treated and by the rate at which the active immunogen or composition as described herein is being administered.

[0166] With regard to "prophylactic treatment" which can include vaccination a subject may be administered with one or more doses to achieve an effective immune response.

[0167] In accordance with the present description, the composition as described herein is administered to a patient by a parenteral or non-parenteral routes of administration. Parenteral administration includes any route of administration that is not through the alimentary canal (that is, not enteral), including administration by injection, infusion and the like. Administration by injection includes, by way of example, into a vein (intravenous), an artery (intraarterial), a muscle (intramuscular) and under the skin (subcutaneous). The composition as described herein may also be administered in a depot or slow release formulation, for example, subcutaneously, intradermally or intramuscularly, in a dosage which is sufficient to obtain the desired pharmacological effect.

[0168] In accordance with the methods and uses as described herein, a subject may receive a therapeutically effective amount of the composition as described herein in one or more doses. A person skilled in the art will understand that the composition as described herein, in particular a vaccine composition may be administered to a subject more than once and can be administered on any appropriate schedule, e.g., from one or more times per day to one or more times per week; including once every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3 months, 6 months, or more, or any variation thereon. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat, prevent or ameliorate a condition in a subject, including but not limited to previous treatments, the general health and/or age of the subject, and other diseases present. The skilled person will further understand that the compositions as described herein may be administered with one or more other immunogens suitable for the methods and uses described herein.

[0169] Sustained-release preparations that may be prepared are particularly convenient for inducing immune responses. Examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. Liposomes may be used which are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30% cholesterol, the selected proportion being adjusted for the optimal therapy.

[0170] Stabilization of proteins may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. The in vivo half-life of proteins may be extended using techniques known in the art, including, for example, by the attachment of other elements such as polyethyleneglycol (PEG) groups.

[0171] Prime-boost immunization strategies as disclosed in the art are contemplated. See for example International Publication No. WO/2003/047617. Thus, compositions may be in the form of a vaccine, priming or boosting agent.

[0172] The present description enables antibodies or antigen binding portions raised against the herein described Pf polypeptide or Pf protein complex or Pf protein mixture, and their derivatives, by routine protocols.

Immunoassay

[0173] A person skilled in the art will appreciate that a Pf protein, Pf protein complex, or Pf protein mixture as described herein or an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein are suitable for use in an immunoassay. Exemplary immunoassay formats include immunoblot, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and enzyme immunoassay and assays suitable for point of care such as microfluidic assays and immunochromatographic device formats.

[0174] In an embodiment an immunoassay as described herein may comprise immobilizing an antigen, for example a Pf protein, Pf protein complex, or Pf protein mixture as described herein, to a solid support e.g. a membrane or microtitre well. A sample from a subject is then brought into physical relation with the Pf protein, Pf protein complex, or Pf protein mixture, and anti-P. falciparum malaria antibody/antibodies in the sample are bound or `captured` by Pf protein, Pf protein complex, or Pf protein mixture. Binding of an anti-P. falciparum malaria antibody/antibodies to the Pf protein, Pf protein complex, or Pf protein mixture results in an antigen-antibody complex. The antigen-antibody complex is then detected by one or more detection reagents.

[0175] In an alternate embodiment an immunoassay as described herein may comprise immobilizing an antibody, for example an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein to a solid support. A sample from a subject is then brought into physical relation with the isolated antibodies and P. falciparum malaria antigens in the sample are bound or `captured` by the isolated antibody forming an antigen-antibody complex. The antigen-antibody complex is then detected by one or more detection reagents.

[0176] In an embodiment, the immunoassay is an ELISA. A person skilled in the art will appreciate that the ELISA may be a colorimetric, chemiluminescent or fluorescent. Furthermore, a person skilled in the art will appreciate that the ELISA can be a direct or an indirect ELISA.

[0177] In a direct ELISA the antigen-antibody complex is bound (or detected) by a binding reagent that is detectably labelled. In an indirect ELISA the binding reagent may be conjugated to an enzyme which acts upon an enzyme substrate to produce a detectable signal. Alternatively, in an indirect ELISA the antigen-antibody complex is bound by a first binding reagent (e.g. a primary antibody), which is bound by a second binding reagent (e.g. a secondary antibody), wherein the second binding reagent is detectably labelled or is conjugated to an enzyme which acts upon an enzyme substrate to produce a detectable signal.

[0178] It will be appreciated by a person skilled in the art that detection reagents may include, but is not limited to, one or more of the following: a binding reagent, a binding reagent conjugated to a detectable label, a binding reagent conjugated to an enzyme, an enzyme substrate, a wash buffer, a blocking buffer, and a solution for stopping an enzymatic reaction.

[0179] As used herein the term "binding reagent" refers to any reagent that binds to the antigen-antibody complex as described herein. In an embodiment, the binding reagent is a native or isolated and/or recombinant protein of microbial origin that binds to mammalian immunoglobulin molecules. In an embodiment the binding reagent, may bind to an Fc region in the antigen-antibody complex. In an embodiment, the binding reagent may bind to a VL-kappa present in the antigen-antibody complex. In an embodiment, the binding reagent binds to IgG, IgM IgA, IgE and/or IgD immunoglobulins. In an embodiment, the binding reagent binds to IgG antibodies. In an embodiment, the binding reagent is protein G or a recombinant version thereof, protein A or a recombinant version thereof, isolated and/or recombinant protein A/G, protein L or a recombinant version thereof.

[0180] In an embodiment, the binding reagent is an antibody or an antigen binding fragment thereof. These include immunoglobulins, immunoglobulin fragments or non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. The term "antibody" as used herein includes monoclonal antibodies, bispecific antibodies, fusion diabodies, triabodies, heteroconjugate antibodies, chimeric antibodies including intact molecules as well as fragments thereof, and other antibody-like molecules. Antibodies include modifications in a variety of forms including, for example, but not limited to, domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light (VL) and heavy chain (VH) variable regions which may be joined directly or through a linker, or Fd fragments containing the heavy chain variable region and the CH1 domain.

[0181] A scFv consisting of the variable regions of the heavy and light chains linked together to form a single-chain antibody (Bird et al., 1988; Huston et al., 1988) and oligomers of scFvs such as diabodies and triabodies are also encompassed by the term "antibody". Also encompassed are fragments of antibodies such as Fab, (Fab')2 and FabFc2 fragments which contain the variable regions and parts of the constant regions. Complementarity determining region (CDR)-grafted antibody fragments and oligomers of antibody fragments are also encompassed. The heavy and light chain components of an Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region. The antibody may be of animal (for example mouse, rabbit or rat) or may be chimeric (Morrison et al., 1984). The antibody may be produced by any method known in the art.

[0182] The antibodies may be Fv regions comprising a variable light (VL) and a variable heavy (VH) chain in which the light and heavy chains may be joined directly or through a linker. As used herein a linker refers to a molecule that is covalently linked to the light and heavy chain and provides enough spacing and flexibility between the two chains such that they are able to achieve a conformation in which they are capable of specifically binding the epitope to which they are directed. Protein linkers are particularly preferred as they may be expressed as an intrinsic component of the Ig portion of the fusion polypeptide.

[0183] In an embodiment, the binding reagent comprises a detectable label. In an embodiment, the binding reagent is conjugated to an enzyme. In an embodiment, the binding reagent is conjugated to a protein such as biotin or streptavidin so that it may interact with a detectable label or enzyme that is streptavidin or biotin conjugated.

[0184] In an embodiment, the detection reagent comprises an enzyme. In an embodiment, the enzyme, may be, but is not limited to being alkaline phosphatase (AP), a peroxidisae such as horseradish peroxidase enzyme (HRP), .beta.-D-galactosidease. In an embodiment, the enzyme is HRP.

[0185] A person skilled in the art will appreciate that the enzyme substrate can be any enzyme substrate suitable for use with one of the aforementioned enzymes wherein when acted upon by an enzyme, a detectable signal is produced. A person skilled in the art will appreciate that the enzyme substrate may be, for example, a colorimetric substrate, chemiluminescent substrate or fluorescent substrate.

[0186] In an embodiment, the solid support may be, but is not limited to being a membrane or microtitre well. In an embodiment, the solid support may be polyethylene, polypropylene or glass. In an embodiment, the solid support is a microtitre well. In an embodiment, the microtitre well is the well of a 96-well or 384-well plate.

Kits

[0187] A person skilled in the art would appreciate that the a Pf protein, Pf protein complex, or Pf protein mixture as described herein may be packaged in a kit suitable for detecting the presence of a P. falciparum malaria infection or the presence or detecting vaccination against a P. falciparum malaria infection. A person skilled in the art would further appreciate that an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein may be packaged in a kit suitable for detecting if a subject has a P. falciparum infection. Such kits may contain one or more immunoassays suitable for detecting a positive or negative marker.

[0188] In an embodiment, the kit is for use in an immunoassay. In an embodiment, the kit is an Enzyme Linked Immunosorbent Assay (ELISA) kit. In an embodiment, the kit is an indirect ELISA kit. In an embodiment, the one or more detection reagents of the kit at least includes a binding reagent which binds the antigen-antibody complex. In an embodiment, the kit as described herein is suitable for high-throughput screening. The term "high-throughput screening" refers to screening methods that can be used to test or assess more than one sample at a time and that can reduce the time for testing multiple samples.

[0189] The following methods are used in practising the present description.

[0190] Vectors. The PfRiprFL (SEQ ID NO: 1) and PfEBA175 (SEQ ID NO: 5) nucleotide sequences were inserted into the pExpreS2-1 expression vector (ExpreS.sup.2ion Biotechnologies, Denmark; SEQ ID NO: 11) which has a marker allowing for Zeocin selection. The PfRh5 sequence (SEQ ID NO: 4) was inserted in to the pExpreS2-2 expression vector (ExpreS.sup.2ion Biotechnologies, Denmark; SEQ ID NO: 12) which has a marker allowing for G418 selection and the PfCyrPa sequence (SEQ ID NO: 2) was inserted into the pExpreS2-PAC expression vector (ExpreS.sup.2ion Biotechnologies, Denmark; SEQ ID NO: 13) which has a marker allowing for Puromycin selection.

[0191] Cell transfection. The above vectors comprising PfRiprFL, PfEBA175, PfRh5 or PfCyrPa were transfected into Drosophila melanogaster ExpreS.sup.2 S2 cells (ExpreS.sup.2ion Biotechnologies, Denmark). On the day before transfection ExpreS.sup.2 S2 cells were split by centrifugation and resuspended in Excell420 Serum-Free Medium for Insect Cells (Sigma Aldrich; 14420C) at a density of 8.times.10.sup.6 cells/m1 and incubated at 25.degree. C. at 115 rpm. On the day of transfection the cells were split by centrifugation and resuspended in Excell420 medium at a concentration of 2.times.10.sup.6 cells/mL. A volume of 5 mL of the cell suspension was transferred to a T25 T-flask (CELLSTAR; GR-690160) and 50 .mu.l ExpreS.sup.2 Insect-TR 5.times. (ExpreS2ion Biotechnologies, Denmark) was added to the cell suspension followed by 12.5 .mu.g DNA of the above vectors comprising PfRiprFL, EBA175, PfRh5 or PfCyrPa and mixed. The cells were then incubated for 3 hours at 25.degree. C. before 1 mL of FBS was added. The day after transfection selection was added at the following concentrations: Zeocin 2000 .mu.g/mL; G418 4000 .mu.g/mL; and Puromycin 100 .mu.g/mL.

[0192] For the next two weeks the T-flask was counted every 3-4 day and the cells were diluted to 1.times.10.sup.6 cells/ml by adding the appropriate amount of fresh Excell420 medium comprising 10% FBS and selection to a final volume of 6 mL. After two weeks the cell suspension was transferred to a T75 flask (CELLSTAR; GR-658170) and 4 mL of Excell420 comprising 10% FBS and selection added. After 4 days, an additional 5 mL of media with selection was added. The cells were then transferred to a 125 mL shake flask (Sigma-Aldrich; CLS431143) after the cells had recovered and 10 ml of Excell420 added. After 4 days, the cells were split by centrifugation and resuspended in 50 ml Excell420 at a density of 8.times.10.sup.6 cells/ml in a 250 ml shake flask (Signma-Aldrich; CLS431144). The cells were subsequently frozen in CryoStor.RTM. CS10 (Sigma; C2874-100ML) for later use.

[0193] The cell lines for co-expression was created by retransfecting a cell line already comprising a vector expressing one of the proteins PfRiprFL, PfEBA175, PfRh5 or PfCyrPa as described above with a second vector. The retransfection was the same as the initial transfection protocol. In this manor Rh5 was transfected into the cell line expressing PfRiprFL. A triple expressing cell line was created by transfecting PfCyrPa in the PfRiprFL, and PfRh5 expressing cell line.

[0194] Production of PfRipr in WAVE Bioreactor. The production of PfRipr was performed by expanding the S2 cell line in shake flasks to a final volume of 1200 mL in Excell420 media (Sigma Aldrich; 14420C) in a 5 l shake flask (Thomson Instrument Company; 931116). The disposable WAVE Bioreactor system (GE Healthcare; WAVE Base 20/50) was used for the final production. The bag (GE Healthcare; CB0050L10-01) was inoculated to a density of 8.times.10.sup.6 cells/mL with a volume of 5000 mL Excell420 at 25.degree. C. The system parameters were set to 20 rpm, 8.degree. angle, and 0.85 l/min of atmospheric air. After three days, an additional 20 mL of Excell420 was added to the bag and the parameters adjusted to 26 rpm, 9.degree. angle, and 1.50 l/min of atmospheric air for 3 days. The contents of the bag were harvested by centrifugation and subsequently sterile filtered using 0.22 um filters.

[0195] Immunization Protocol for raising antibodies against the PfRIPR/PfRh5/PfCyrPa complex. Antigens were prepared for immunization by mixing the three individual proteins (PfRIPR/P/Rh5/PfCyrPa) expressed separately in equal molar ratio (1.1.1) based on quantitation of the individual proteins by UV spectroscopy. For each immunization the total antigen quantity of the combined proteins was 100 .mu.g. Since the molecular weight of Ripr is roughly twice that of PfRh5 and PfCyrPa to ensure 100 .mu.g of assembled complex 55 .mu.g of each of PfRh5 and PfCyrPa was added to 125 .mu.g of PfRipr. All proteins are in PBS pH 7.4. Previous analyses showed that complex formation occurs immediately and with high affinity (data not shown). Alternatively, a complex or mixture of proteins isolated from a cell expressing PfRIPR, PfRh5 and PfCyrPa may be used for immunizations. The protein mixture was then mixed with Freund's adjuvant (FA) before injection into a rabbit. A three dose schedule was followed with one immunization in complete FA followed at 28-day intervals with two immunization in incomplete FA.

[0196] Analysis of antibodies raised against the PfRIPR/PfRh5/PfCyrPa complex. Serum was obtained from blood taken two weeks after the third immunization in the immunization schedule and IgG prepared by protein A sepharose chromatography.

[0197] The following non-limiting examples are provided.

Example 1: Comparison of PfRipr Expression; PfRipr Synergistic Effect

[0198] Samples were taken from the three cell lines at day three of culture and analyzed by western blot to compare PfRipr expression as shown in FIG. 1A. The lane order is as follows: lane 1, SeeBlue Plus2 ladder (Life Technologies); lane 2, sample 1491, cell line WHTZ-04 expressing PfRiprFL only; lane 3, sample 1483, cell line WHTZ-04 expressing PfRiprFL only; lane 4, sample 1320, cell line WHTZG-10 expressing PfRiprFL and PfRh5; lane 5, sample 1482, cell line WHTZGP-13 expressing PfRiprFL and PfRh5 and PfCyrPa. The samples from each cell line were prepared by mixing with buffer and reducing agent and incubation at 95.degree. C. for five minutes. The gel was loaded and run for 35 minutes at 165V and blotted with an iBlott (Life Technologies). The membrane was blotted with anti-Strep (QIAGEN; 34850) and anti-mouse-HRP (DAKO; P0447) as secondary antibody for detection of PfRipr. The blot was then detected by enhanced chemiluminescence. The two samples of WHTZ-04 (from two different productions) show similar expression level. Both WHTZG-10 and WHTZGP-13 show similar expression level and an increased level compared to WHTZ-04. Co-expression of PfRh5 appears to increase the expression of PfRipr. The addition of PfCyrPa does not seem to further increase the expression of PfRipr.

Example 2: Vector Expression Analysis

[0199] Vectors comprising PfRiprFL, PfRh5, PfCyrPa as outlined above in the Vectors section were transfected separately and in combination with each other in ExpreS2 S2 cells. Each established cell line with their respective expression constructs are listed in the Table 2. The theoretical size of the PfRiprFL, PfRh5, PfCyrPa proteins is 126.67 kDa, 53.80 kDa, and 40.18 kDa, respectively.

[0200] The samples from each cell line were prepared by mixing with buffer and reducing agent and incubation at 95.degree. C. for five minutes. Reducing agent was omitted in the lanes noted as un-reduced. The samples were loaded in the order listed below in Table 3. The gel was run for 35 minutes at 165V and blotted with an iBlotter. The membrane was blotted with anti-His or anti-Strep and anti-mouse-HRP as secondary antibody. The blot was then detected by enhanced chemiluminescence and imaged. Results are shown in FIG. 1B.

TABLE-US-00002 TABLE 2 Cell line Constructs WHTZ-04 pExpreS2-1 vector comprising PfRiprFL WHTG-09 pExpreS2-2 vector comprising PfRh5 WHTP-12 pExpreS2-PAC vector comprising PfCyrPa WHTZG-10 pExpreS2-1 vector comprising PfRiprFL and pExpreS2-2 vector comprising PfRh5 WHTZGP- pExpreS2-1 vector comprising PfRiprFL, 13 pExpreS2-2 vector comprising PfRh5 and pExpreS2-PAC vector comprising PfCyrPa

TABLE-US-00003 TABLE 3 Lane Sample Comment Cell line Construct Tag 1 TJ1438 WHTP-12 PfCyrPa 2 TJ1463 WHTZGP- PfRiprFL, PfRh5, 13 PfCyrPa 3 TJ1463 Un-reduced WHTZGP- PfRiprFL, PfRh5, 13 PfCyrPa 4 Seeblue Plus2 5 TJ1322 WHTG-09 PfRh5 His 6 TJ1320 WHTZG-10 PfRiprFL, PfRh5 7 TJ1320 Un-reduced WHTZG-10 PfRiprFL, PfRh5 8 TJ1463 WHTZGP- PfRiprFL, PfRh5, 13 PfCyrPa 9 TJ1463 Un-reduced WHTZGP- PfRiprFL, PfRh5, 13 PfCyrPa 10 Seeblue Plus2 11 TJ1343 WHTZ-04 PfRiprFL 12 TJ1463 WHTZGP- PfRiprFL, PfRh5, 13 PfCyrPa 13 TJ1463 Un-reduced WHTZGP- PfRiprFL, PfRh5, 13 PfCyrPa Strep 14 TJ1483 Control WHTZ-04 PfRiprFL 15 TJ1484 Additive 1 WHTZ-04 PfRiprFL 16 TJ1485 Additive 2 WHTZ-04 PfRiprFL 17 Empty

[0201] Lane 1, 5, and 11 of FIG. 1B shows PfCyrPa, PfRh5 and PfRiprFL expressed individually from vectors. The observed size of the proteins corresponds to the theoretical size outlined above. The co-expression of PfRipr and PfRh5 (Lane 6 and 7) also shows expression. The reduced samples in lane 6 shows a clear band of WRh5. For the un-reduced sample (Lane 7), the band of PfRh5 is still visible though not as bright as the reduced sample. The high molecular weight bands is a normal background we see for our cells in His-tag western blots.

[0202] The triple-expression of PfRipr, PfRh5, and PfCyrPa (Lane 2, 3, 8, 9, 12, and 13) is also positive for all three constructs. The His-tag shows a positive band for both Rh5 and PfCyrPa in the reduced samples (Lane 2 and 8) while only, what is presumably PfCyrPa, one band is visible in the un-reduced samples (Lane 3 and 9). The band appears as a triplet and is slightly smaller compared to the reduced sample.

[0203] The Strep-tag shows a clear band in both the reduced and un-reduced samples. The un-reduced sample appears to be slightly smaller compared to the reduced sample.

[0204] Two additives were evaluated to determine if the expression level of PfRipr could be improved. No obvious difference in intensity is observed for the three samples (Lane 14 through 16).

[0205] All three constructs are expressed individually and confirmed by western blot and the observed size of each construct correspond to the theoretical size. The co-transfection of PfRipr and PfRh5 was successfully performed and stable cell line expressing both constructs was created.

[0206] The triple-expression of PfRipr, PfRh5, and PfCyrPa was successful and the cell line expresses all three constructs. The two additives tested did not have any obvious effect on the yield of PfRipr.

Example 3: Antibodies Against Full-Length Ripr Inhibit the Growth of Plasmodium falciparum Parasites

[0207] Anti-Ripr antibodies were raised by immunizing a rabbit three times with 100 ug purified Ripr_FL protein in Freund's complete/incomplete adjuvant at day 0, 28 and 56. Serum was obtained from blood taken two weeks after the third immunization and IgG prepared by protein A sepharose chromatography.

[0208] To assess if antibodies raised against full-length Ripr inhibit the growth of P. falciparum, P. falciparum 3D7 parasites were incubated with serially diluted purified IgG from either non-immune rabbit serum (NS) or anti-Ripr antiserum (R1682). The parasitaemia in triplicate wells was counted after 96 hours (2 invasion cycles). In the presence of anti-Ripr IgG, merozoite invasion of erythrocytes was inhibited by 55% compared with non-immune rabbit IgG (FIG. 2).

[0209] The growth inhibition single cycle assay was performed according to methods described in Malkin et al. 2005.

REFERENCES

[0210] Argos J, 1990, Mol Biol 20; 211(4):943-58.

[0211] Bahrami et al., 2007, Virol 5; 363(2):303-9.

[0212] Bird et al., 1988, Science 242:423-426.

[0213] George and Heringa, 2002, Protein Eng 15(11):871-9.

[0214] Huston et al., 1988, Proc Natl Acad Sci USA 85:5879-5883.

[0215] Malkin et al., 2005, Infect Immun 73: 3677-3685.

[0216] Morrison et al., 1984, Proc Natl Acad Sci USA 81:6851-6855.

[0217] Sabourin et al., 2007, Yeast 24(1):39-45).

[0218] Swiech et al., 2008, Cytotechnology 57:61-66.

Sequence CWU 1

1

2713207DNAPlasmodium falciparum 1atcgatctga tcgagggcat cttctacgag aagaacgaga tcgacaagct gaccttcagc 60ctggatcacc gcgtgcgcga taacctgaag accgacctga tcctgaacaa caacggcgag 120aacgattacg cctacctgaa caaatacgtg tacaccatcc tgaaccgcga cagcaccgag 180aagatcaaga ccttcttcag ccacaacaag gacatgaagt cctgcgacta cttcatcagc 240aaggagtaca acagcagcga caagaccaac cagatctgct acaagaagac gttctgcggc 300gtcgtgatcc ccaacagcga ggagattaag acgaacaaga tcaccaacga taagctgtac 360tgcgcccact tcaacagcac ccacatcatc atctactaca tcagccagcc cctgctgctg 420gagccccacg tggtgtacga ggagaccttt ttcgagaagg gcaagaacga ccagatcaac 480tgccagggca tgtacatctc cctgcgctcc gtgcatgtgc acacccacaa cgccatcctg 540cagcaggaga ccctgaccta catcaagaac ctgtgcgacg gcaagaacaa ctgcaagttc 600gacttcgaca gcattaagta cgagaacaag agcctgaccc actacctgtt cttcatcaac 660atccagtacc agtgcatcag ccccctgaac ctgcaggaga atgagatgtg cgacgtgtac 720aacgacgata cgcacaaggc cacgtgcaaa tacggcttca acaagatcga gctgctgaag 780aatgtgtgcg aggagaacta ccgctgcacc caggatatct gcagcgtgaa ccagttctgc 840gacggcgaga atgagacctg cacgtgcaag accagcctgc tgcccagcgc caagaacaat 900tgcgagtaca acgatctgtg caccgtgctg aactgccccg agaactcgac ctgcgagcag 960atcggcaatg gcaagaaggc cgagtgcaag tgcgagaacg gcaagtacta ccacaacaac 1020aagtgctaca ccaagaacga tctggagctg gccatcaaga ttgagcccca caagaaggag 1080aagttctata agaacaacct gtaccagggc aaggccctga agcccgagta catcttcatg 1140cagtgcgaga atggcttcag catcgaagtg atcaacgcct acgtgtcctg ctaccgcgtg 1200tccttcaatc tgaacaagct gaaatacgtg accgagagcc tgaagaagat gtgcgacgga 1260aagaccaagt gcgcctacgg caacaccatc gatcccatcg atgatctgaa ccaccacaac 1320atctgcaaca acttcaacac gatctttaag tatgactacc tgtgcgtgtt caacaaccag 1380aacatcacct ccgacaagaa cagccatctg cacagcaaca tccccagcct gtacaactcc 1440agcatcctgc ccgatatcaa caagagcaag ttccacctga tcagccgcaa cagccgcacc 1500aaccagtacc cccacaacaa tatcagtatg ctggagatcc agaatgagat cagcagccac 1560aactccaacc agttctccac cgatccccac accaactcga acaacatcaa caacatgaat 1620atcaagaagg tggagatctt ccgcagccgc ttcagctcca agctgcagtg ccagggcggc 1680aagatcaaca tcgacaaggc cattctgaag ggcggcgagg gctgcaatga tctgctgctg 1740accaacagcc tgaagtccta ctgcaacgac ctgagcgagt gcgatatcgg cctgatctac 1800cacttcgata cctactgcat caatgaccag tacctgttcg tgtcctacag ctgcagcaac 1860ctgtgcaaca agtgccacaa caactccacg tgctacggca accgcttcaa ctacgattgc 1920ttctgcgata acccctacat ctcgaagtac ggaaacaagc tgtgcgagcg ccccaacgat 1980tgcgagagcg tgctgtgctc ccagaaccaa gtgtgccaga tcctgccgaa tgataagctg 2040atctgccagt gcgaggaggg ctacaagaac gtgaagggaa aatgcgtgcc ggataacaag 2100tgcgatctga gctgccccag caacaaagtg tgcgtgatcg agaatggaaa gcagacctgc 2160aagtgctccg agcgcttcgt gctggagaac ggcgtgtgca tctgcgccaa cgattacaag 2220atggaggatg gcatcaactg cattgccaag aacaagtgca agcgcaagga gtacgagaat 2280atctgcacca accccaacga gatgtgcgcc tacaatgagg agaccgatat cgtgaagtgc 2340gagtgcaagg agcactacta ccgcagcagc cgcggagagt gcattctgaa cgactactgc 2400aaggacatca attgcaagga gaacgaggag tgcagcatcg tgaacttcaa gccagagtgc 2460gtgtgcaagg agaacctgaa gaagaacaac aagggcgagt gcatctacga gaacagctgc 2520ctgatcaacg agggcaactg ccccaaggat agcaagtgca tctatcgcga gtacaagccc 2580cacgagtgcg tgtgcaacaa gcagggacac gtggccgtga atggcaaatg cgtgctggag 2640gataagtgcg tgcacaacaa gaagtgcagc gagaacagca tctgcgtgaa cgtgatgaac 2700aaggagccaa tctgcgtgtg cacctacaac tactacaaga aggacggcgt gtgcctgatc 2760cagaacccct gcctgaagga taacggcggc tgctcccgca actccgagtg caccttcaag 2820tacagcaaga tcaactgcac gtgcaaggag aactacaaga acaaggatga tagctgcgtg 2880cccaacacga acgagtacga tgagagcttc accttccagt ataacgacga cgccagcatc 2940atcctgggcg cctgcggcat gatcgagttc agctacatct acaaccagat tatctggaag 3000attaacaact cgaaggagtc ctacgtgttc tactacgatt accccaccgc cggcaacatc 3060gaggtgcaga ttaagaatga gattttccac acgatcatct atctgaagaa gaagatcggc 3120aacagcgtga tctacgacga tttccaggtg gaccaccaga cctgcatcta tgagaatgtg 3180ttttactaca gcaaccagaa tagcgcc 32072999DNAPlasmodium falciparum 2agccgccacg tgttcatccg caccgagctg agcttcatca agaacaacgt gccctgcatc 60cgcgacatgt tcttcatcta caagcgcgag ctgtacaaca tctgcctgga tgatctgaag 120ggcgaggagg atgagaccca catctacgtg cagaagaaag tgaaggacag ctggatcacc 180ctgaacgacc tgttcaagga gaccgatctg accggacgcc cccacatctt cgcctacgtg 240gacgtggagg agatcatcat tctgctgtgc gaggatgagg agttcagcaa ccgcaagaag 300gatatgacct gccaccgctt ctacagcaac gatggcaagg agtacaacaa cagcgagatc 360accatcagcg actacatcct gaaggataag ctgctgtcca gctacgtgtc cctgcccctg 420aagatcgaga accgcgagta cttcctgatc tgcggcgtgt ccccctacaa gttcaaggat 480gataacaaga aggacgacat cctgtgcatg gccagccacg ataagggcga gacctggggc 540accaagatcg tgattaagta cgacaactac aagctgggcg tgcagtactt cttcctgcgc 600ccctacatca gcaagaacga tctgagcttc cacttctacg tgggcgacaa catcaacaac 660gtgaagaacg tgaacttcat cgagtgcacc cacgagaagg atctggagtt cgtgtgctcc 720aaccgcgatt ttctgaagga caacaaggtg ctgcaggatg tgtccaccct gaatgatgag 780tacatcgtgt cctacggcaa cgacaacaac ttcgccgagt gctacatctt cttcaacaac 840gagaacagca tcctgatcaa gcccgagaag tacggcaaca ccaccgccgg atgctacggc 900ggcaccttcg tgaagattga tgagaaccgc accctgttca tctactccag cagccagggc 960atctacaaca tccacaccat ctactacgcc aactacgag 99931200DNAPlasmodium falciparum 3gacgtgaaga acaacgagga ctacaagaac gtggactata agaatgtgaa cttcctgcag 60taccacttca aggagctgag caactacaat atcgccaaca gcatcgacat tctgcaggag 120aaggagggcc acctggattt cgtgatcatc ccccactaca cctttctgga ctactacaag 180cacctgagct acaactccat ctaccacaag agcagcacct acggcaagtg cattgccgtg 240gatgccttca tcaagaagat caacgagacc tacgacaaag tgaagtccaa gtgcaacgac 300atcaagaacg acctgatcgc cacgatcaag aagctggagc acccctacga tatcaacaac 360aagaacgatg acagctaccg ctacgacatc agcgaggaga tcgacgataa gtccgaggag 420acggacgacg agaccgagga ggtggaggat agcatccagg ataccgatag caaccacacc 480cccagcaaca agaagaagaa tgatctgatg aaccgcacct ttaagaagat gatggacgag 540tacaatacga aaaagaagaa gctgatcaag tgcatcaaga atcacgagaa cgacttcaac 600aagatctgca tggacatgaa gaactacggc accaacctgt tcgagcagct gtcctgctac 660aacaacaact tctgcaacac caacggcatc cgctaccact acgatgagta catccacaag 720ctgatcctga gcgtgaagtc gaagaacctg aacaaggatc tgagcgacat gaccaacatc 780ctgcagcaga gcgagctgct gctgaccaat ctgaacaaga agatgggcag ctacatctac 840atcgacacca tcaagttcat tcacaaggag atgaagcaca tcttcaaccg catcgagtac 900cacaccaaga tcatcaacga caagacgaag atcattcagg acaagatcaa gctgaacatc 960tggcgcacct tccagaagga tgagctgctg aagcgcatcc tggatatgag caacgagtac 1020agcctgttca tcaccagcga tcatctgcgc cagatgctgt acaacacctt ctacagcaag 1080gagaagcacc tgaacaacat cttccaccac ctgatctacg tgctgcagat gaagttcaac 1140gacgtgccca tcaagatgga gtacttccag acctataaga agaacaagcc cctgacccag 120041200DNAPlasmodium falciparum 4gacgtgaaga acaacgagga ctacaagaac gtggactata agaatgtgaa cttcctgcag 60taccacttca aggagctgag caactacaat atcgccaaca gcatcgacat tctgcaggag 120aaggagggcc acctggattt cgtgatcatc ccccactaca cctttctgga ctactacaag 180cacctgagct acaactccat ctaccacaag agcagcacct acggcaagtg cattgccgtg 240gatgccttca tcaagaagat caacgagacc tacgacaaag tgaagtccaa gtgcaacgac 300atcaagaacg acctgatcgc cacgatcaag aagctggagc acccctacga tatcaacaac 360aagaacgatg acagctaccg ctacgacatc agcgaggaga tcgacgataa gtccgaggag 420acggacgacg agaccgagga ggtggaggat agcatccagg ataccgatag caaccacacc 480cccagcaaca agaagaagaa tgatctgatg aaccgcacct ttaagaagat gatggacgag 540tacaatacga aaaagaagaa gctgatcaag tgcatcaaga atcacgagaa cgacttcaac 600aagatctgca tggacatgaa gaactacggc accaacctgt tcgagcagct gtcctgctac 660aacaacaact tctgcaacac caacggcatc cgctaccact acgatgagta catccacaag 720ctgatcctga gcgtgaagtc gaagaacctg aacaaggatc tgagcgacat gaccaacatc 780ctgcagcaga gcgagctgct gctgaccaat ctgaacaaga agatgggcag ctacatctac 840atcgacacca tcaagttcat tcacaaggag atgaagcaca tcttcaaccg catcgagtac 900cacaccaaga tcatcaacga caagacgaag atcattcagg acaagatcaa gctgaacatc 960tggcgcacct tccagaagga tgagctgctg aagcgcatcc tggatatgag caacgagtac 1020agcctgttca tcaccagcga tcatctgcgc cagatgctgt acaacacctt ctacagcaag 1080gagaagcacc tgaacaacat cttccaccac ctgatctacg tgctgcagat gaagttcaac 1140gacgtgccca tcaagatgga gtacttccag acctataaga agaacaagcc cctgacccag 120051620DNAPlasmodium falciparum 5caggaggccg tgccagagga gagcaccgag attgcccacc gcaccgagac ccgcaccgat 60gagcgcaaga atcaggagcc cgccaacaag gatctgaaga acccccagca gagcgtgggc 120gagaacggca cgaaggatct gctgcaggag gatctgggag gcagccgcag cgaggatgaa 180gtgacccagg agttcggcgt gaaccacggc atccccaagg gcgaggatca gaccctggga 240aagtccgatg ccatccccaa catcggcgag cccgagaccg gaatcagtac caccgaggag 300tcccgccacg aggagggcca taacaagcag gccctgagca cctccgtgga tgagcccgag 360ctgagcgata ccctgcagct gcacgaggat accaaggaga acgataagct gcccctggag 420agcagcacca tcaccagccc aaccgagagc ggcagcagcg ataccgagga gaccccatcc 480attagcgagg gcccgaaggg caacgagcag aagaagcgcg acgacgatag cctgagcaag 540atcagcgtgt cccccgagaa cagccgccca gagaccgatg ccaaggatac cagcaacctg 600ctgaagctga agggcgacgt ggacatcagc atgcccaagg ccgtgatcgg cagctccccc 660aacgataaca tcaacgtgac cgagcagggc gacaacatct cgggcgtgaa cagcaagccc 720ctgtccgatg atgtgcgccc cgataagaac catgaggaag tgaaggagca caccagcaac 780agcgataacg tgcagcagtc cggcggcatc gtgaacatga acgtggagaa ggagctgaag 840gacaccctgg agaaccccag ctccagcctg gatgagggaa aggcccatga ggagctgtcc 900gagcccaacc tgtccagcga tcaggatatg agcaacaccc caggccccct ggataacacc 960tcggaggaga cgaccgagcg catcagcaac aacgagtaca aagtgaacga gcgcgagggc 1020gagcgcaccc tgaccaagga gtatgaggat atcgtgctga agtcccacat gaaccgcgag 1080agcgacgatg gcgagctgta cgatgagaac agcgatctga gcaccgtgaa cgatgagtcc 1140gaggatgccg aggccaagat gaagggcaat gataccagcg agatgagcca caacagcagc 1200cagcacatcg agagcgatca gcagaagaac gatatgaaga ccgtgggcga cctgggcacc 1260acccacgtgc agaatgagat ctccgtgccc gtgaccggcg agatcgatga gaagctgcgc 1320gagagcaagg agtccaagat ccacaaggcc gaggaggagc gcctgagcca caccgatatc 1380cacaagatca accccgagga tcgcaactcc aacaccctgc acctgaagga tatccgcaac 1440gaggagaatg agcgccacct gacgaaccag aacatcaaca tcagccagga gcgcgacctg 1500cagaagcacg gcttccacac catgaacaac ctgcacggcg acggcgtgtc cgagcgcagc 1560cagatcaatc actcgcacca cggcaaccgc caggatcgcg gaggaaatag tggaagcgcc 162061067PRTPlasmodium falciparum 6Ile Asp Leu Ile Glu Gly Ile Phe Tyr Glu Lys Asn Glu Ile Asp Lys1 5 10 15Leu Thr Phe Ser Leu Asp His Arg Val Arg Asp Asn Leu Lys Thr Asp 20 25 30Leu Ile Leu Asn Asn Asn Gly Glu Asn Asp Tyr Ala Tyr Leu Asn Lys 35 40 45Tyr Val Tyr Thr Ile Leu Asn Arg Asp Ser Thr Glu Lys Ile Lys Thr 50 55 60Phe Phe Ser His Asn Lys Asp Met Lys Ser Cys Asp Tyr Phe Ile Ser65 70 75 80Lys Glu Tyr Asn Ser Ser Asp Lys Thr Asn Gln Ile Cys Tyr Lys Lys 85 90 95Thr Phe Cys Gly Val Val Ile Pro Asn Ser Glu Glu Ile Lys Thr Asn 100 105 110Lys Ile Thr Asn Asp Lys Leu Tyr Cys Ala His Phe Asn Ser Thr His 115 120 125Ile Ile Ile Tyr Tyr Ile Ser Gln Pro Leu Leu Leu Glu Pro His Val 130 135 140Val Tyr Glu Glu Thr Phe Phe Glu Lys Gly Lys Asn Asp Gln Ile Asn145 150 155 160Cys Gln Gly Met Tyr Ile Ser Leu Arg Ser Val His Val His Thr His 165 170 175Asn Ala Ile Leu Gln Gln Glu Thr Leu Thr Tyr Ile Lys Asn Leu Cys 180 185 190Asp Gly Lys Asn Asn Cys Lys Phe Asp Phe Asp Ser Ile Lys Tyr Glu 195 200 205Asn Lys Ser Leu Thr His Tyr Leu Phe Phe Ile Asn Ile Gln Tyr Gln 210 215 220Cys Ile Ser Pro Leu Asn Leu Gln Glu Asn Glu Met Cys Asp Val Tyr225 230 235 240Asn Asp Asp Thr His Lys Ala Thr Cys Lys Tyr Gly Phe Asn Lys Ile 245 250 255Glu Leu Leu Lys Asn Val Cys Glu Glu Asn Tyr Arg Cys Thr Gln Asp 260 265 270Ile Cys Ser Val Asn Gln Phe Cys Asp Gly Glu Asn Glu Thr Cys Thr 275 280 285Cys Lys Thr Ser Leu Leu Pro Ser Ala Lys Asn Asn Cys Glu Tyr Asn 290 295 300Asp Leu Cys Thr Val Leu Asn Cys Pro Glu Asn Ser Thr Cys Glu Gln305 310 315 320Ile Gly Asn Gly Lys Lys Ala Glu Cys Lys Cys Glu Asn Gly Lys Tyr 325 330 335Tyr His Asn Asn Lys Cys Tyr Thr Lys Asn Asp Leu Glu Leu Ala Ile 340 345 350Lys Ile Glu Pro His Lys Lys Glu Lys Phe Tyr Lys Asn Asn Leu Tyr 355 360 365Gln Gly Lys Ala Leu Lys Pro Glu Tyr Ile Phe Met Gln Cys Glu Asn 370 375 380Gly Phe Ser Ile Glu Val Ile Asn Ala Tyr Val Ser Cys Tyr Arg Val385 390 395 400Ser Phe Asn Leu Asn Lys Leu Lys Tyr Val Thr Glu Ser Leu Lys Lys 405 410 415Met Cys Asp Gly Lys Thr Lys Cys Ala Tyr Gly Asn Thr Ile Asp Pro 420 425 430Ile Asp Asp Leu Asn His His Asn Ile Cys Asn Asn Phe Asn Thr Ile 435 440 445Phe Lys Tyr Asp Tyr Leu Cys Val Phe Asn Asn Gln Asn Ile Thr Ser 450 455 460Asp Lys Asn Ser His Leu His Ser Asn Ile Pro Ser Leu Tyr Asn Ser465 470 475 480Ser Ile Leu Pro Asp Ile Asn Lys Ser Lys Phe His Leu Ile Ser Arg 485 490 495Asn Ser Arg Thr Asn Gln Tyr Pro His Asn Asn Ile Ser Met Leu Glu 500 505 510Ile Gln Asn Glu Ile Ser Ser His Asn Ser Asn Gln Phe Ser Thr Asp 515 520 525Pro His Thr Asn Ser Asn Asn Ile Asn Asn Met Asn Ile Lys Lys Val 530 535 540Glu Ile Phe Arg Ser Arg Phe Ser Ser Lys Leu Gln Cys Gln Gly Gly545 550 555 560Lys Ile Asn Ile Asp Lys Ala Ile Leu Lys Gly Gly Glu Gly Cys Asn 565 570 575Asp Leu Leu Leu Thr Asn Ser Leu Lys Ser Tyr Cys Asn Asp Leu Ser 580 585 590Glu Cys Asp Ile Gly Leu Ile Tyr His Phe Asp Thr Tyr Cys Ile Asn 595 600 605Asp Gln Tyr Leu Phe Val Ser Tyr Ser Cys Ser Asn Leu Cys Asn Lys 610 615 620Cys His Asn Asn Ser Thr Cys Tyr Gly Asn Arg Phe Asn Tyr Asp Cys625 630 635 640Phe Cys Asp Asn Pro Tyr Ile Ser Lys Tyr Gly Asn Lys Leu Cys Glu 645 650 655Arg Pro Asn Asp Cys Glu Ser Val Leu Cys Ser Gln Asn Gln Val Cys 660 665 670Gln Ile Leu Pro Asn Asp Lys Leu Ile Cys Gln Cys Glu Glu Gly Tyr 675 680 685Lys Asn Val Lys Gly Lys Cys Val Pro Asp Asn Lys Cys Asp Leu Ser 690 695 700Cys Pro Ser Asn Lys Val Cys Val Ile Glu Asn Gly Lys Gln Thr Cys705 710 715 720Lys Cys Ser Glu Arg Phe Val Leu Glu Asn Gly Val Cys Ile Cys Ala 725 730 735Asn Asp Tyr Lys Met Glu Asp Gly Ile Asn Cys Ile Ala Lys Asn Lys 740 745 750Cys Lys Arg Lys Glu Tyr Glu Asn Ile Cys Thr Asn Pro Asn Glu Met 755 760 765Cys Ala Tyr Asn Glu Glu Thr Asp Ile Val Lys Cys Glu Cys Lys Glu 770 775 780His Tyr Tyr Arg Ser Ser Arg Gly Glu Cys Ile Leu Asn Asp Tyr Cys785 790 795 800Lys Asp Ile Asn Cys Lys Glu Asn Glu Glu Cys Ser Ile Val Asn Phe 805 810 815Lys Pro Glu Cys Val Cys Lys Glu Asn Leu Lys Lys Asn Asn Lys Gly 820 825 830Glu Cys Ile Tyr Glu Asn Ser Cys Leu Ile Asn Glu Gly Asn Cys Pro 835 840 845Lys Asp Ser Lys Cys Ile Tyr Arg Glu Tyr Lys Pro His Glu Cys Val 850 855 860Cys Asn Lys Gln Gly His Val Ala Val Asn Gly Lys Cys Val Leu Glu865 870 875 880Asp Lys Cys Val His Asn Lys Lys Cys Ser Glu Asn Ser Ile Cys Val 885 890 895Asn Val Met Asn Lys Glu Pro Ile Cys Val Cys Thr Tyr Asn Tyr Tyr 900 905 910Lys Lys Asp Gly Val Cys Leu Ile Gln Asn Pro Cys Leu Lys Asp Asn 915 920 925Gly Gly Cys Ser Arg Asn Ser Glu Cys Thr Phe Lys Tyr Ser Lys Ile 930 935 940Asn Cys Thr Cys Lys Glu Asn Tyr Lys Asn Lys Asp Asp Ser Cys Val945 950 955 960Pro Asn Thr Asn Glu Tyr Asp Glu Ser Phe Thr Phe Gln Tyr Asn Asp 965 970 975Asp Ala Ser Ile Ile Leu Gly Ala Cys Gly Met Ile Glu Phe Ser Tyr 980 985 990Ile Tyr Asn Gln Ile Ile Trp Lys Ile Asn Asn Ser Lys Glu Ser Tyr 995 1000 1005Val Phe Tyr Tyr Asp Tyr Pro Thr Ala Gly Asn Ile Glu Val Gln 1010 1015 1020Ile Lys Asn Glu Ile Phe His Thr Ile Ile Tyr Leu Lys Lys Lys 1025 1030 1035Ile Gly Asn Ser Val Ile Tyr Asp Asp Phe Gln Val Asp His Gln 1040 1045 1050Thr Cys Ile Tyr Glu Asn Val Phe Tyr Tyr Ser Asn Gln Asn 1055 1060 10657333PRTPlasmodium falciparum 7Ser Arg His Val Phe Ile Arg Thr Glu Leu Ser Phe Ile Lys Asn Asn1 5

10 15Val Pro Cys Ile Arg Asp Met Phe Phe Ile Tyr Lys Arg Glu Leu Tyr 20 25 30Asn Ile Cys Leu Asp Asp Leu Lys Gly Glu Glu Asp Glu Thr His Ile 35 40 45Tyr Val Gln Lys Lys Val Lys Asp Ser Trp Ile Thr Leu Asn Asp Leu 50 55 60Phe Lys Glu Thr Asp Leu Thr Gly Arg Pro His Ile Phe Ala Tyr Val65 70 75 80Asp Val Glu Glu Ile Ile Ile Leu Leu Cys Glu Asp Glu Glu Phe Ser 85 90 95Asn Arg Lys Lys Asp Met Thr Cys His Arg Phe Tyr Ser Asn Asp Gly 100 105 110Lys Glu Tyr Asn Asn Ser Glu Ile Thr Ile Ser Asp Tyr Ile Leu Lys 115 120 125Asp Lys Leu Leu Ser Ser Tyr Val Ser Leu Pro Leu Lys Ile Glu Asn 130 135 140Arg Glu Tyr Phe Leu Ile Cys Gly Val Ser Pro Tyr Lys Phe Lys Asp145 150 155 160Asp Asn Lys Lys Asp Asp Ile Leu Cys Met Ala Ser His Asp Lys Gly 165 170 175Glu Thr Trp Gly Thr Lys Ile Val Ile Lys Tyr Asp Asn Tyr Lys Leu 180 185 190Gly Val Gln Tyr Phe Phe Leu Arg Pro Tyr Ile Ser Lys Asn Asp Leu 195 200 205Ser Phe His Phe Tyr Val Gly Asp Asn Ile Asn Asn Val Lys Asn Val 210 215 220Asn Phe Ile Glu Cys Thr His Glu Lys Asp Leu Glu Phe Val Cys Ser225 230 235 240Asn Arg Asp Phe Leu Lys Asp Asn Lys Val Leu Gln Asp Val Ser Thr 245 250 255Leu Asn Asp Glu Tyr Ile Val Ser Tyr Gly Asn Asp Asn Asn Phe Ala 260 265 270Glu Cys Tyr Ile Phe Phe Asn Asn Glu Asn Ser Ile Leu Ile Lys Pro 275 280 285Glu Lys Tyr Gly Asn Thr Thr Ala Gly Cys Tyr Gly Gly Thr Phe Val 290 295 300Lys Ile Asp Glu Asn Arg Thr Leu Phe Ile Tyr Ser Ser Ser Gln Gly305 310 315 320Ile Tyr Asn Ile His Thr Ile Tyr Tyr Ala Asn Tyr Glu 325 3308526PRTPlasmodium falciparum 8Met Ile Arg Ile Lys Lys Lys Leu Ile Leu Thr Ile Ile Tyr Ile His1 5 10 15Leu Phe Ile Leu Asn Arg Leu Ser Phe Glu Asn Ala Ile Lys Lys Thr 20 25 30Lys Asn Gln Glu Asn Asn Leu Thr Leu Leu Pro Ile Lys Ser Thr Glu 35 40 45Glu Glu Lys Asp Asp Ile Lys Asn Gly Lys Asp Ile Lys Lys Glu Ile 50 55 60Asp Asn Asp Lys Glu Asn Ile Lys Thr Asn Asn Ala Lys Asp His Ser65 70 75 80Thr Tyr Ile Lys Ser Tyr Leu Asn Thr Asn Val Asn Asp Gly Leu Lys 85 90 95Tyr Leu Phe Ile Pro Ser His Asn Ser Phe Ile Lys Lys Tyr Ser Val 100 105 110Phe Asn Gln Ile Asn Asp Gly Met Leu Leu Asn Glu Lys Asn Asp Val 115 120 125Lys Asn Asn Glu Asp Tyr Lys Asn Val Asp Tyr Lys Asn Val Asn Phe 130 135 140Leu Gln Tyr His Phe Lys Glu Leu Ser Asn Tyr Asn Ile Ala Asn Ser145 150 155 160Ile Asp Ile Leu Gln Glu Lys Glu Gly His Leu Asp Phe Val Ile Ile 165 170 175Pro His Tyr Thr Phe Leu Asp Tyr Tyr Lys His Leu Ser Tyr Asn Ser 180 185 190Ile Tyr His Lys Ser Ser Thr Tyr Gly Lys Cys Ile Ala Val Asp Ala 195 200 205Phe Ile Lys Lys Ile Asn Glu Thr Tyr Asp Lys Val Lys Ser Lys Cys 210 215 220Asn Asp Ile Lys Asn Asp Leu Ile Ala Thr Ile Lys Lys Leu Glu His225 230 235 240Pro Tyr Asp Ile Asn Asn Lys Asn Asp Asp Ser Tyr Arg Tyr Asp Ile 245 250 255Ser Glu Glu Ile Asp Asp Lys Ser Glu Glu Thr Asp Asp Glu Thr Glu 260 265 270Glu Val Glu Asp Ser Ile Gln Asp Thr Asp Ser Asn His Thr Pro Ser 275 280 285Asn Lys Lys Lys Asn Asp Leu Met Asn Arg Thr Phe Lys Lys Met Met 290 295 300Asp Glu Tyr Asn Thr Lys Lys Lys Lys Leu Ile Lys Cys Ile Lys Asn305 310 315 320His Glu Asn Asp Phe Asn Lys Ile Cys Met Asp Met Lys Asn Tyr Gly 325 330 335Thr Asn Leu Phe Glu Gln Leu Ser Cys Tyr Asn Asn Asn Phe Cys Asn 340 345 350Thr Asn Gly Ile Arg Tyr His Tyr Asp Glu Tyr Ile His Lys Leu Ile 355 360 365Leu Ser Val Lys Ser Lys Asn Leu Asn Lys Asp Leu Ser Asp Met Thr 370 375 380Asn Ile Leu Gln Gln Ser Glu Leu Leu Leu Thr Asn Leu Asn Lys Lys385 390 395 400Met Gly Ser Tyr Ile Tyr Ile Asp Thr Ile Lys Phe Ile His Lys Glu 405 410 415Met Lys His Ile Phe Asn Arg Ile Glu Tyr His Thr Lys Ile Ile Asn 420 425 430Asp Lys Thr Lys Ile Ile Gln Asp Lys Ile Lys Leu Asn Ile Trp Arg 435 440 445Thr Phe Gln Lys Asp Glu Leu Leu Lys Arg Ile Leu Asp Met Ser Asn 450 455 460Glu Tyr Ser Leu Phe Ile Thr Ser Asp His Leu Arg Gln Met Leu Tyr465 470 475 480Asn Thr Phe Tyr Ser Lys Glu Lys His Leu Asn Asn Ile Phe His His 485 490 495Leu Ile Tyr Val Leu Gln Met Lys Phe Asn Asp Val Pro Ile Lys Met 500 505 510Glu Tyr Phe Gln Thr Tyr Lys Lys Asn Lys Pro Leu Thr Gln 515 520 5259400PRTPlasmodium falciparum 9Asp Val Lys Asn Asn Glu Asp Tyr Lys Asn Val Asp Tyr Lys Asn Val1 5 10 15Asn Phe Leu Gln Tyr His Phe Lys Glu Leu Ser Asn Tyr Asn Ile Ala 20 25 30Asn Ser Ile Asp Ile Leu Gln Glu Lys Glu Gly His Leu Asp Phe Val 35 40 45Ile Ile Pro His Tyr Thr Phe Leu Asp Tyr Tyr Lys His Leu Ser Tyr 50 55 60Asn Ser Ile Tyr His Lys Ser Ser Thr Tyr Gly Lys Cys Ile Ala Val65 70 75 80Asp Ala Phe Ile Lys Lys Ile Asn Glu Thr Tyr Asp Lys Val Lys Ser 85 90 95Lys Cys Asn Asp Ile Lys Asn Asp Leu Ile Ala Thr Ile Lys Lys Leu 100 105 110Glu His Pro Tyr Asp Ile Asn Asn Lys Asn Asp Asp Ser Tyr Arg Tyr 115 120 125Asp Ile Ser Glu Glu Ile Asp Asp Lys Ser Glu Glu Thr Asp Asp Glu 130 135 140Thr Glu Glu Val Glu Asp Ser Ile Gln Asp Thr Asp Ser Asn His Thr145 150 155 160Pro Ser Asn Lys Lys Lys Asn Asp Leu Met Asn Arg Thr Phe Lys Lys 165 170 175Met Met Asp Glu Tyr Asn Thr Lys Lys Lys Lys Leu Ile Lys Cys Ile 180 185 190Lys Asn His Glu Asn Asp Phe Asn Lys Ile Cys Met Asp Met Lys Asn 195 200 205Tyr Gly Thr Asn Leu Phe Glu Gln Leu Ser Cys Tyr Asn Asn Asn Phe 210 215 220Cys Asn Thr Asn Gly Ile Arg Tyr His Tyr Asp Glu Tyr Ile His Lys225 230 235 240Leu Ile Leu Ser Val Lys Ser Lys Asn Leu Asn Lys Asp Leu Ser Asp 245 250 255Met Thr Asn Ile Leu Gln Gln Ser Glu Leu Leu Leu Thr Asn Leu Asn 260 265 270Lys Lys Met Gly Ser Tyr Ile Tyr Ile Asp Thr Ile Lys Phe Ile His 275 280 285Lys Glu Met Lys His Ile Phe Asn Arg Ile Glu Tyr His Thr Lys Ile 290 295 300Ile Asn Asp Lys Thr Lys Ile Ile Gln Asp Lys Ile Lys Leu Asn Ile305 310 315 320Trp Arg Thr Phe Gln Lys Asp Glu Leu Leu Lys Arg Ile Leu Asp Met 325 330 335Ser Asn Glu Tyr Ser Leu Phe Ile Thr Ser Asp His Leu Arg Gln Met 340 345 350Leu Tyr Asn Thr Phe Tyr Ser Lys Glu Lys His Leu Asn Asn Ile Phe 355 360 365His His Leu Ile Tyr Val Leu Gln Met Lys Phe Asn Asp Val Pro Ile 370 375 380Lys Met Glu Tyr Phe Gln Thr Tyr Lys Lys Asn Lys Pro Leu Thr Gln385 390 395 40010538PRTPlasmodium falciparum 10Gln Glu Ala Val Pro Glu Glu Ser Thr Glu Ile Ala His Arg Thr Glu1 5 10 15Thr Arg Thr Asp Glu Arg Lys Asn Gln Glu Pro Ala Asn Lys Asp Leu 20 25 30Lys Asn Pro Gln Gln Ser Val Gly Glu Asn Gly Thr Lys Asp Leu Leu 35 40 45Gln Glu Asp Leu Gly Gly Ser Arg Ser Glu Asp Glu Val Thr Gln Glu 50 55 60Phe Gly Val Asn His Gly Ile Pro Lys Gly Glu Asp Gln Thr Leu Gly65 70 75 80Lys Ser Asp Ala Ile Pro Asn Ile Gly Glu Pro Glu Thr Gly Ile Ser 85 90 95Thr Thr Glu Glu Ser Arg His Glu Glu Gly His Asn Lys Gln Ala Leu 100 105 110Ser Thr Ser Val Asp Glu Pro Glu Leu Ser Asp Thr Leu Gln Leu His 115 120 125Glu Asp Thr Lys Glu Asn Asp Lys Leu Pro Leu Glu Ser Ser Thr Ile 130 135 140Thr Ser Pro Thr Glu Ser Gly Ser Ser Asp Thr Glu Glu Thr Pro Ser145 150 155 160Ile Ser Glu Gly Pro Lys Gly Asn Glu Gln Lys Lys Arg Asp Asp Asp 165 170 175Ser Leu Ser Lys Ile Ser Val Ser Pro Glu Asn Ser Arg Pro Glu Thr 180 185 190Asp Ala Lys Asp Thr Ser Asn Leu Leu Lys Leu Lys Gly Asp Val Asp 195 200 205Ile Ser Met Pro Lys Ala Val Ile Gly Ser Ser Pro Asn Asp Asn Ile 210 215 220Asn Val Thr Glu Gln Gly Asp Asn Ile Ser Gly Val Asn Ser Lys Pro225 230 235 240Leu Ser Asp Asp Val Arg Pro Asp Lys Asn His Glu Glu Val Lys Glu 245 250 255His Thr Ser Asn Ser Asp Asn Val Gln Gln Ser Gly Gly Ile Val Asn 260 265 270Met Asn Val Glu Lys Glu Leu Lys Asp Thr Leu Glu Asn Pro Ser Ser 275 280 285Ser Leu Asp Glu Gly Lys Ala His Glu Glu Leu Ser Glu Pro Asn Leu 290 295 300Ser Ser Asp Gln Asp Met Ser Asn Thr Pro Gly Pro Leu Asp Asn Thr305 310 315 320Ser Glu Glu Thr Thr Glu Arg Ile Ser Asn Asn Glu Tyr Lys Val Asn 325 330 335Glu Arg Glu Gly Glu Arg Thr Leu Thr Lys Glu Tyr Glu Asp Ile Val 340 345 350Leu Lys Ser His Met Asn Arg Glu Ser Asp Asp Gly Glu Leu Tyr Asp 355 360 365Glu Asn Ser Asp Leu Ser Thr Val Asn Asp Glu Ser Glu Asp Ala Glu 370 375 380Ala Lys Met Lys Gly Asn Asp Thr Ser Glu Met Ser His Asn Ser Ser385 390 395 400Gln His Ile Glu Ser Asp Gln Gln Lys Asn Asp Met Lys Thr Val Gly 405 410 415Asp Leu Gly Thr Thr His Val Gln Asn Glu Ile Ser Val Pro Val Thr 420 425 430Gly Glu Ile Asp Glu Lys Leu Arg Glu Ser Lys Glu Ser Lys Ile His 435 440 445Lys Ala Glu Glu Glu Arg Leu Ser His Thr Asp Ile His Lys Ile Asn 450 455 460Pro Glu Asp Arg Asn Ser Asn Thr Leu His Leu Lys Asp Ile Arg Asn465 470 475 480Glu Glu Asn Glu Arg His Leu Thr Asn Gln Asn Ile Asn Ile Ser Gln 485 490 495Glu Arg Asp Leu Gln Lys His Gly Phe His Thr Met Asn Asn Leu His 500 505 510Gly Asp Gly Val Ser Glu Arg Ser Gln Ile Asn His Ser His His Gly 515 520 525Asn Arg Gln Asp Arg Gly Gly Asn Ser Gly 530 535112531DNAArtificial SequenceVector 11cgtaagtatc aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag 60acagagaaga ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc 120ctttctctcc acaggaattc tggatcctct agaccggtca tatgcggccg cggatcgatc 180gatatctgac taaatcttag tttgtattgt catgttttaa tacaatatgt tatgtttaaa 240tatgttttta ataaatttta taaaataatt tcaactttta ttgtaacaac attgtccatt 300tacacactcc tttcaagcgc gtgggactcg atgctcggcg ccactcaaag gcggtaatac 360ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 420aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 480acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 540gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 600ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac 660gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 720cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 780taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 840atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 900cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 960cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 1020ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 1080ctcagtggaa cgaaaactca cgttaaggga ttttgcatgc gctaagcggg ctttataaaa 1140cgggctgcgg gaccagtttt catatcacta ccgtttgagt tcttgtgctg tgtggatact 1200cctcccgaca ccgaattaat tcggatctct gcaagggatt ttggtcatga acaataaaac 1260tgtctgctta cataaacagt aatacaaggg gtgttcatag tataatacga ctcactatag 1320gagggccacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc 1380cggagcggtc gagttctgga ccgaccggct cgggttcagc cgggacttcg tggaggacga 1440cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt 1500ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga 1560gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat 1620cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca 1680cttcgtggcc gaggagcagg actgaccgac gccgaccaac accgccggtc cgacggcggc 1740ccacgggtcc caggggggtc gacctcgaaa cttgtttatt gcagcttata atggttacaa 1800ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 1860tggtttgtcc aaactcatca atgtatctta tcatgtcttc acgtaataag tgtgcggcta 1920gcagtcaact actagtgaat gccctactag aagatgtgtg ttgcacaaaa tgtccctgga 1980ataaccaatt tgaagtgcag atagcagtaa acgtaagcta atatgaatat tatttaactg 2040taatgtttta atatcgctgg acattactaa taaacccact ataaacacat gtacatatgt 2100atgttttggc atacaatgag tagttgggga aaaaatgtgt aaaagcaccg tgaccatcac 2160agcataaaga taaccagctg aagtatcgaa tatgagtaac ccccaaattg aatcacatgc 2220cgcaactgat aggacccatg gaagtacact cttcatggcg atatacaaga cacacacaag 2280cacgaacacc cagttgcgga ggaaattctc cgtaaatgaa aacccaatcg gcgaacaatt 2340catacccata tatggtaaaa gttttgaacg cgacttaaga gcgccggagt ataaatagag 2400gcgcttcgtc tacggagcga caattcaatt caaacaagca aagtgaacac gtcgctaagc 2460gaaagctaag caaataaaca agcgcagctg aacaagctaa acaatctgca gaagcttggt 2520accctcgagc t 2531122799DNAArtificial SequenceVector 12aattcggatc tctgcaaggg attttggtca tgaacaataa aactgtctgc ttacataaac 60agtaatacaa ggggtgttca tagtataata cgactcacta taggagggcc accatgagcc 120acatccagcg cgaaaccagc tgcagccgtc cgcgcctgaa cagcaacatg gatgccgatc 180tgtacggcta caaatgggcc cgcgataacg tgggccagag cggcgctacc atctaccgcc 240tgtacggcaa accggatgcc ccggaactgt tcctgaaaca cggcaaaggc agcgtggcca 300acgatgtgac cgatgaaatg gtgcgcctga actggctgac cgagttcatg ccgctgccga 360ccatcaaaca cttcatccgc accccggatg atgcctggct gctgaccacc gccattccgg 420gcaaaaccgc cttccaggtg ctggaagaat acccggatag cggcgaaaac atcgtggatg 480ccctggccgt gttcctgcgc cgcctgcaca gcatcccggt gtgcaactgc ccgttcaaca 540gcgatcgcgt gttccgcctg gctcaggccc agagccgcat gaacaacggc ctggtggatg 600ccagcgattt cgatgatgaa cgcaacggct ggccggtgga acaggtgtgg aaagagatgc 660acaaactgct gccgttcagc ccggattccg tggtgaccca cggcgatttc agcctggata 720acctgatctt cgatgagggc aaactgatcg gctgcatcga tgtgggccgc gtgggcattg 780ccgatcgcta ccaggatctg gccatcctgt ggaactgcct gggcgagttc agcccgagcc 840tgcagaaacg cctgttccag aagtacggca tcgataaccc ggatatgaac aaactgcagt 900tccacctgat gctggatgag ttcttctaat aagtcgacct cgaaacttgt ttattgcagc 960ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 1020actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tcttcacgta 1080ataagtgtgc ggctagcagt caactactag tgaatgccct actagaagat gtgtgttgca 1140caaaatgtcc ctggaataac caatttgaag tgcagatagc agtaaacgta agctaatatg 1200aatattattt aactgtaatg ttttaatatc gctggacatt actaataaac ccactataaa 1260cacatgtaca tatgtatgtt ttggcataca atgagtagtt ggggaaaaaa tgtgtaaaag 1320caccgtgacc atcacagcat aaagataacc agctgaagta tcgaatatga gtaaccccca 1380aattgaatca catgccgcaa ctgataggac ccatggaagt acactcttca tggcgatata 1440caagacacac acaagcacga acacccagtt gcggaggaaa ttctccgtaa atgaaaaccc

1500aatcggcgaa caattcatac ccatatatgg taaaagtttt gaacgcgagc gaacttaaga 1560gcgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt caaacaagca 1620aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg aacaagctaa 1680acaatctgca gaagcttggt accctcgagc tcagctgaat tctggatcct ctagaccggt 1740catatgcggc cgcggatcga tcgatatctg actaaatctt agtttgtatt gtcatgtttt 1800aatacaatat gttatgttta aatatgtttt taataaattt tataaaataa tttcaacttt 1860tattgtaaca acattgtcca tttacacact cctttcaagc gcgtgggact cgatgctcgg 1920cgccactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 1980atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 2040ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 2100cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 2160tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 2220gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 2280aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 2340tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 2400aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 2460aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 2520ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 2580ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 2640atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttgcat 2700gcgctaagcg ggctttataa aacgggctgc gggaccagtt ttcatatcac taccgtttga 2760gttcttgtgc tgtgtggata ctcctcccga caccgaatt 2799132709DNAArtificial SequenceVector 13taattcggat ctctgcaagg gattttggtc atgaacaata aaactgtctg cttacataaa 60cagtaataca aggggtgttc atagtataat acgactcact ataggagggc caccatgacc 120gagtacaagc ccaccgtgcg cctggccacc cgcgacgatg tgccacgcgc cgtgcgcacc 180ctggccgccg cctttgccga ttatccagcc acccgccata ccgtggaccc cgatcgccat 240attgagcgcg tgaccgagct gcaggagctg ttcctgaccc gcgtgggcct ggatattggc 300aaagtgtggg tggccgatga cggagccgcc gtggccgtgt ggaccacccc agagagtgtg 360gaggccggag ccgtgttcgc cgagattgga ccacgcatgg ccgagctgag tggaagtcgc 420ctggccgccc agcagcagat ggagggactg ctggccccac accgcccaaa ggagccagcc 480tggtttctgg ccaccgtggg agtgtcccca gatcaccagg gaaagggact gggaagtgcc 540gtggtgctgc caggcgtgga ggccgccgag cgcgccggcg tgccagcctt tctggagacc 600agtgccccac gcaacctgcc cttttacgag cgcctgggct ttaccgtgac cgccgatgtg 660gaggtgccag agggcccacg cacctggtgc atgacccgca agccaggcgc ctaagtcgac 720ctcgaaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 780tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 840tatcttatca tgtcttcacg taataagtgt gcggctagca gtcaactact agtgaatgcc 900ctactagaag atgtgtgttg cacaaaatgt ccctggaata accaatttga agtgcagata 960gcagtaaacg taagctaata tgaatattat ttaactgtaa tgttttaata tcgctggaca 1020ttactaataa acccactata aacacatgta catatgtatg ttttggcata caatgagtag 1080ttggggaaaa aatgtgtaaa agcaccgtga ccatcacagc ataaagataa ccagctgaag 1140tatcgaatat gagtaacccc caaattgaat cacatgccgc aactgatagg acccatggaa 1200gtacactctt catggcgata tacaagacac acacaagcac gaacacccag ttgcggagga 1260aattctccgt aaatgaaaac ccaatcggcg aacaattcat acccatatat ggtaaaagtt 1320ttgaacgcga gcgaacttaa gagcgccgga gtataaatag aggcgcttcg tctacggagc 1380gacaattcaa ttcaaacaag caaagtgaac acgtcgctaa gcgaaagcta agcaaataaa 1440caagcgcagc tgaacaagct aaacaatctg cagaagcttg gtaccctcga gctcgtaagt 1500atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc gagacagaga 1560agactcttgc gtttctgata ggcacctatt ggtcttactg acatccactt tgcctttctc 1620tccacaggaa ttctggatcc tctagaccgg tcatatgcgg ccgcggatcg atcgatatct 1680gactaaatct tagtttgtat tgtcatgttt taatacaata tgttatgttt aaatatgttt 1740ttaataaatt ttataaaata atttcaactt ttattgtaac aacattgtcc atttacacac 1800tcctttcaag cgcgtgggac tcgatgctcg gcgccactca aaggcggtaa tacggttatc 1860cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 1920gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 1980tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 2040ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 2100atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 2160gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 2220tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 2280cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 2340cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 2400tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 2460cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 2520cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 2580gaacgaaaac tcacgttaag ggattttgca tgcgctaagc gggctttata aaacgggctg 2640cgggaccagt tttcatatca ctaccgtttg agttcttgtg ctgtgtggat actcctcccg 2700acaccgaat 2709145807DNAArtificial SequenceVector 14cgtaagtatc aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag 60acagagaaga ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc 120ctttctctcc acaggaattc gccaccatga agctgtgcat cctgctggcc gtggtggcct 180tcgtgggact gagcctgggc atcgatctga tcgagggcat cttctacgag aagaacgaga 240tcgacaagct gaccttcagc ctggatcacc gcgtgcgcga taacctgaag accgacctga 300tcctgaacaa caacggcgag aacgattacg cctacctgaa caaatacgtg tacaccatcc 360tgaaccgcga cagcaccgag aagatcaaga ccttcttcag ccacaacaag gacatgaagt 420cctgcgacta cttcatcagc aaggagtaca acagcagcga caagaccaac cagatctgct 480acaagaagac gttctgcggc gtcgtgatcc ccaacagcga ggagattaag acgaacaaga 540tcaccaacga taagctgtac tgcgcccact tcaacagcac ccacatcatc atctactaca 600tcagccagcc cctgctgctg gagccccacg tggtgtacga ggagaccttt ttcgagaagg 660gcaagaacga ccagatcaac tgccagggca tgtacatctc cctgcgctcc gtgcatgtgc 720acacccacaa cgccatcctg cagcaggaga ccctgaccta catcaagaac ctgtgcgacg 780gcaagaacaa ctgcaagttc gacttcgaca gcattaagta cgagaacaag agcctgaccc 840actacctgtt cttcatcaac atccagtacc agtgcatcag ccccctgaac ctgcaggaga 900atgagatgtg cgacgtgtac aacgacgata cgcacaaggc cacgtgcaaa tacggcttca 960acaagatcga gctgctgaag aatgtgtgcg aggagaacta ccgctgcacc caggatatct 1020gcagcgtgaa ccagttctgc gacggcgaga atgagacctg cacgtgcaag accagcctgc 1080tgcccagcgc caagaacaat tgcgagtaca acgatctgtg caccgtgctg aactgccccg 1140agaactcgac ctgcgagcag atcggcaatg gcaagaaggc cgagtgcaag tgcgagaacg 1200gcaagtacta ccacaacaac aagtgctaca ccaagaacga tctggagctg gccatcaaga 1260ttgagcccca caagaaggag aagttctata agaacaacct gtaccagggc aaggccctga 1320agcccgagta catcttcatg cagtgcgaga atggcttcag catcgaagtg atcaacgcct 1380acgtgtcctg ctaccgcgtg tccttcaatc tgaacaagct gaaatacgtg accgagagcc 1440tgaagaagat gtgcgacgga aagaccaagt gcgcctacgg caacaccatc gatcccatcg 1500atgatctgaa ccaccacaac atctgcaaca acttcaacac gatctttaag tatgactacc 1560tgtgcgtgtt caacaaccag aacatcacct ccgacaagaa cagccatctg cacagcaaca 1620tccccagcct gtacaactcc agcatcctgc ccgatatcaa caagagcaag ttccacctga 1680tcagccgcaa cagccgcacc aaccagtacc cccacaacaa tatcagtatg ctggagatcc 1740agaatgagat cagcagccac aactccaacc agttctccac cgatccccac accaactcga 1800acaacatcaa caacatgaat atcaagaagg tggagatctt ccgcagccgc ttcagctcca 1860agctgcagtg ccagggcggc aagatcaaca tcgacaaggc cattctgaag ggcggcgagg 1920gctgcaatga tctgctgctg accaacagcc tgaagtccta ctgcaacgac ctgagcgagt 1980gcgatatcgg cctgatctac cacttcgata cctactgcat caatgaccag tacctgttcg 2040tgtcctacag ctgcagcaac ctgtgcaaca agtgccacaa caactccacg tgctacggca 2100accgcttcaa ctacgattgc ttctgcgata acccctacat ctcgaagtac ggaaacaagc 2160tgtgcgagcg ccccaacgat tgcgagagcg tgctgtgctc ccagaaccaa gtgtgccaga 2220tcctgccgaa tgataagctg atctgccagt gcgaggaggg ctacaagaac gtgaagggaa 2280aatgcgtgcc ggataacaag tgcgatctga gctgccccag caacaaagtg tgcgtgatcg 2340agaatggaaa gcagacctgc aagtgctccg agcgcttcgt gctggagaac ggcgtgtgca 2400tctgcgccaa cgattacaag atggaggatg gcatcaactg cattgccaag aacaagtgca 2460agcgcaagga gtacgagaat atctgcacca accccaacga gatgtgcgcc tacaatgagg 2520agaccgatat cgtgaagtgc gagtgcaagg agcactacta ccgcagcagc cgcggagagt 2580gcattctgaa cgactactgc aaggacatca attgcaagga gaacgaggag tgcagcatcg 2640tgaacttcaa gccagagtgc gtgtgcaagg agaacctgaa gaagaacaac aagggcgagt 2700gcatctacga gaacagctgc ctgatcaacg agggcaactg ccccaaggat agcaagtgca 2760tctatcgcga gtacaagccc cacgagtgcg tgtgcaacaa gcagggacac gtggccgtga 2820atggcaaatg cgtgctggag gataagtgcg tgcacaacaa gaagtgcagc gagaacagca 2880tctgcgtgaa cgtgatgaac aaggagccaa tctgcgtgtg cacctacaac tactacaaga 2940aggacggcgt gtgcctgatc cagaacccct gcctgaagga taacggcggc tgctcccgca 3000actccgagtg caccttcaag tacagcaaga tcaactgcac gtgcaaggag aactacaaga 3060acaaggatga tagctgcgtg cccaacacga acgagtacga tgagagcttc accttccagt 3120ataacgacga cgccagcatc atcctgggcg cctgcggcat gatcgagttc agctacatct 3180acaaccagat tatctggaag attaacaact cgaaggagtc ctacgtgttc tactacgatt 3240accccaccgc cggcaacatc gaggtgcaga ttaagaatga gattttccac acgatcatct 3300atctgaagaa gaagatcggc aacagcgtga tctacgacga tttccaggtg gaccaccaga 3360cctgcatcta tgagaatgtg ttttactaca gcaaccagaa tagcgcctgg tcccaccccc 3420agttcgagaa atgagcggcc gcggatcgat cgatatctga ctaaatctta gtttgtattg 3480tcatgtttta atacaatatg ttatgtttaa atatgttttt aataaatttt ataaaataat 3540ttcaactttt attgtaacaa cattgtccat ttacacactc ctttcaagcg cgtgggactc 3600gatgctcggc gccactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 3660ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 3720ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 3780cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 3840ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 3900tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 3960gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 4020tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 4080gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 4140tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 4200ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 4260agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 4320gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 4380attttgcatg cgctaagcgg gctttataaa acgggctgcg ggaccagttt tcatatcact 4440accgtttgag ttcttgtgct gtgtggatac tcctcccgac accgaattaa ttcggatctc 4500tgcaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg 4560ggtgttcata gtataatacg actcactata ggagggccac catggccaag ttgaccagtg 4620ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc 4680tcgggttcag ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga 4740ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt 4800gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc 4860gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg 4920ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgaccga 4980cgccgaccaa caccgccggt ccgacggcgg cccacgggtc ccaggggggt cgacctcgaa 5040acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 5100ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 5160atcatgtctt cacgtaataa gtgtgcggct agcagtcaac tactagtgaa tgccctacta 5220gaagatgtgt gttgcacaaa atgtccctgg aataaccaat ttgaagtgca gatagcagta 5280aacgtaagct aatatgaata ttatttaact gtaatgtttt aatatcgctg gacattacta 5340ataaacccac tataaacaca tgtacatatg tatgttttgg catacaatga gtagttgggg 5400aaaaaatgtg taaaagcacc gtgaccatca cagcataaag ataaccagct gaagtatcga 5460atatgagtaa cccccaaatt gaatcacatg ccgcaactga taggacccat ggaagtacac 5520tcttcatggc gatatacaag acacacacaa gcacgaacac ccagttgcgg aggaaattct 5580ccgtaaatga aaacccaatc ggcgaacaat tcatacccat atatggtaaa agttttgaac 5640gcgagcgaac ttaagagcgc cggagtataa atagaggcgc ttcgtctacg gagcgacaat 5700tcaattcaaa caagcaaagt gaacacgtcg ctaagcgaaa gctaagcaaa taaacaagcg 5760cagctgaaca agctaaacaa tctgcagaag cttggtaccc tcgagct 5807154135DNAArtificial SequenceVector 15aattcggatc tctgcaaggg attttggtca tgaacaataa aactgtctgc ttacataaac 60agtaatacaa ggggtgttca tagtataata cgactcacta taggagggcc accatgagcc 120acatccagcg cgaaaccagc tgcagccgtc cgcgcctgaa cagcaacatg gatgccgatc 180tgtacggcta caaatgggcc cgcgataacg tgggccagag cggcgctacc atctaccgcc 240tgtacggcaa accggatgcc ccggaactgt tcctgaaaca cggcaaaggc agcgtggcca 300acgatgtgac cgatgaaatg gtgcgcctga actggctgac cgagttcatg ccgctgccga 360ccatcaaaca cttcatccgc accccggatg atgcctggct gctgaccacc gccattccgg 420gcaaaaccgc cttccaggtg ctggaagaat acccggatag cggcgaaaac atcgtggatg 480ccctggccgt gttcctgcgc cgcctgcaca gcatcccggt gtgcaactgc ccgttcaaca 540gcgatcgcgt gttccgcctg gctcaggccc agagccgcat gaacaacggc ctggtggatg 600ccagcgattt cgatgatgaa cgcaacggct ggccggtgga acaggtgtgg aaagagatgc 660acaaactgct gccgttcagc ccggattccg tggtgaccca cggcgatttc agcctggata 720acctgatctt cgatgagggc aaactgatcg gctgcatcga tgtgggccgc gtgggcattg 780ccgatcgcta ccaggatctg gccatcctgt ggaactgcct gggcgagttc agcccgagcc 840tgcagaaacg cctgttccag aagtacggca tcgataaccc ggatatgaac aaactgcagt 900tccacctgat gctggatgag ttcttctaat aagtcgacct cgaaacttgt ttattgcagc 960ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 1020actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tcttcacgta 1080ataagtgtgc ggctagcagt caactactag tgaatgccct actagaagat gtgtgttgca 1140caaaatgtcc ctggaataac caatttgaag tgcagatagc agtaaacgta agctaatatg 1200aatattattt aactgtaatg ttttaatatc gctggacatt actaataaac ccactataaa 1260cacatgtaca tatgtatgtt ttggcataca atgagtagtt ggggaaaaaa tgtgtaaaag 1320caccgtgacc atcacagcat aaagataacc agctgaagta tcgaatatga gtaaccccca 1380aattgaatca catgccgcaa ctgataggac ccatggaagt acactcttca tggcgatata 1440caagacacac acaagcacga acacccagtt gcggaggaaa ttctccgtaa atgaaaaccc 1500aatcggcgaa caattcatac ccatatatgg taaaagtttt gaacgcgagc gaacttaaga 1560gcgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt caaacaagca 1620aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg aacaagctaa 1680acaatctgca gaagcttggt accctcgagc tcagctgaat tcgccaccat gaagctgtgc 1740atcctgctgg ccgtggtggc cttcgtggga ctgagtctgg gcgacgtgaa gaacaacgag 1800gactacaaga acgtggacta taagaatgtg aacttcctgc agtaccactt caaggagctg 1860agcaactaca atatcgccaa cagcatcgac attctgcagg agaaggaggg ccacctggat 1920ttcgtgatca tcccccacta cacctttctg gactactaca agcacctgag ctacaactcc 1980atctaccaca agagcagcac ctacggcaag tgcattgccg tggatgcctt catcaagaag 2040atcaacgaga cctacgacaa agtgaagtcc aagtgcaacg acatcaagaa cgacctgatc 2100gccacgatca agaagctgga gcacccctac gatatcaaca acaagaacga tgacagctac 2160cgctacgaca tcagcgagga gatcgacgat aagtccgagg agacggacga cgagaccgag 2220gaggtggagg atagcatcca ggataccgat agcaaccaca cccccagcaa caagaagaag 2280aatgatctga tgaaccgcac ctttaagaag atgatggacg agtacaatac gaaaaagaag 2340aagctgatca agtgcatcaa gaatcacgag aacgacttca acaagatctg catggacatg 2400aagaactacg gcaccaacct gttcgagcag ctgtcctgct acaacaacaa cttctgcaac 2460accaacggca tccgctacca ctacgatgag tacatccaca agctgatcct gagcgtgaag 2520tcgaagaacc tgaacaagga tctgagcgac atgaccaaca tcctgcagca gagcgagctg 2580ctgctgacca atctgaacaa gaagatgggc agctacatct acatcgacac catcaagttc 2640attcacaagg agatgaagca catcttcaac cgcatcgagt accacaccaa gatcatcaac 2700gacaagacga agatcattca ggacaagatc aagctgaaca tctggcgcac cttccagaag 2760gatgagctgc tgaagcgcat cctggatatg agcaacgagt acagcctgtt catcaccagc 2820gatcatctgc gccagatgct gtacaacacc ttctacagca aggagaagca cctgaacaac 2880atcttccacc acctgatcta cgtgctgcag atgaagttca acgacgtgcc catcaagatg 2940gagtacttcc agacctataa gaagaacaag cccctgaccc agcaccacca tcaccaccac 3000aagcgccgct ggaagaagaa ctttatcgcc gtgtccgccg ccaaccgctt caagaagatt 3060agcagcagcg gagccctgta ggcggccgcg gatcgatcga tatctgacta aatcttagtt 3120tgtattgtca tgttttaata caatatgtta tgtttaaata tgtttttaat aaattttata 3180aaataatttc aacttttatt gtaacaacat tgtccattta cacactcctt tcaagcgcgt 3240gggactcgat gctcggcgcc actcaaaggc ggtaatacgg ttatccacag aatcagggga 3300taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 3360cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 3420ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 3480aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 3540tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt 3600gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 3660cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 3720ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 3780cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 3840gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 3900cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 3960tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 4020ttaagggatt ttgcatgcgc taagcgggct ttataaaacg ggctgcggga ccagttttca 4080tatcactacc gtttgagttc ttgtgctgtg tggatactcc tcccgacacc gaatt 4135163766DNAArtificial SequenceVector 16taattcggat ctctgcaagg gattttggtc atgaacaata aaactgtctg cttacataaa 60cagtaataca aggggtgttc atagtataat acgactcact ataggagggc caccatgacc 120gagtacaagc ccaccgtgcg cctggccacc cgcgacgatg tgccacgcgc cgtgcgcacc 180ctggccgccg cctttgccga ttatccagcc acccgccata ccgtggaccc cgatcgccat 240attgagcgcg tgaccgagct gcaggagctg ttcctgaccc gcgtgggcct ggatattggc 300aaagtgtggg tggccgatga cggagccgcc gtggccgtgt ggaccacccc agagagtgtg 360gaggccggag ccgtgttcgc cgagattgga ccacgcatgg ccgagctgag tggaagtcgc 420ctggccgccc agcagcagat ggagggactg ctggccccac accgcccaaa ggagccagcc 480tggtttctgg ccaccgtggg agtgtcccca gatcaccagg gaaagggact gggaagtgcc 540gtggtgctgc caggcgtgga ggccgccgag cgcgccggcg tgccagcctt tctggagacc 600agtgccccac gcaacctgcc cttttacgag cgcctgggct ttaccgtgac cgccgatgtg 660gaggtgccag agggcccacg cacctggtgc atgacccgca agccaggcgc ctaagtcgac 720ctcgaaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 780tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 840tatcttatca tgtcttcacg taataagtgt gcggctagca gtcaactact agtgaatgcc

900ctactagaag atgtgtgttg cacaaaatgt ccctggaata accaatttga agtgcagata 960gcagtaaacg taagctaata tgaatattat ttaactgtaa tgttttaata tcgctggaca 1020ttactaataa acccactata aacacatgta catatgtatg ttttggcata caatgagtag 1080ttggggaaaa aatgtgtaaa agcaccgtga ccatcacagc ataaagataa ccagctgaag 1140tatcgaatat gagtaacccc caaattgaat cacatgccgc aactgatagg acccatggaa 1200gtacactctt catggcgata tacaagacac acacaagcac gaacacccag ttgcggagga 1260aattctccgt aaatgaaaac ccaatcggcg aacaattcat acccatatat ggtaaaagtt 1320ttgaacgcga gcgaacttaa gagcgccgga gtataaatag aggcgcttcg tctacggagc 1380gacaattcaa ttcaaacaag caaagtgaac acgtcgctaa gcgaaagcta agcaaataaa 1440caagcgcagc tgaacaagct aaacaatctg cagaagcttg gtaccctcga gctcgtaagt 1500atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc gagacagaga 1560agactcttgc gtttctgata ggcacctatt ggtcttactg acatccactt tgcctttctc 1620tccacaggaa ttcgccacca tgaagctgtg catcctgctg gccgtggtgg ccttcgtggg 1680actgagtctg ggaagccgcc acgtgttcat ccgcaccgag ctgagcttca tcaagaacaa 1740cgtgccctgc atccgcgaca tgttcttcat ctacaagcgc gagctgtaca acatctgcct 1800ggatgatctg aagggcgagg aggatgagac ccacatctac gtgcagaaga aagtgaagga 1860cagctggatc accctgaacg acctgttcaa ggagaccgat ctgaccggac gcccccacat 1920cttcgcctac gtggacgtgg aggagatcat cattctgctg tgcgaggatg aggagttcag 1980caaccgcaag aaggatatga cctgccaccg cttctacagc aacgatggca aggagtacaa 2040caacagcgag atcaccatca gcgactacat cctgaaggat aagctgctgt ccagctacgt 2100gtccctgccc ctgaagatcg agaaccgcga gtacttcctg atctgcggcg tgtcccccta 2160caagttcaag gatgataaca agaaggacga catcctgtgc atggccagcc acgataaggg 2220cgagacctgg ggcaccaaga tcgtgattaa gtacgacaac tacaagctgg gcgtgcagta 2280cttcttcctg cgcccctaca tcagcaagaa cgatctgagc ttccacttct acgtgggcga 2340caacatcaac aacgtgaaga acgtgaactt catcgagtgc acccacgaga aggatctgga 2400gttcgtgtgc tccaaccgcg attttctgaa ggacaacaag gtgctgcagg atgtgtccac 2460cctgaatgat gagtacatcg tgtcctacgg caacgacaac aacttcgccg agtgctacat 2520cttcttcaac aacgagaaca gcatcctgat caagcccgag aagtacggca acaccaccgc 2580cggatgctac ggcggcacct tcgtgaagat tgatgagaac cgcaccctgt tcatctactc 2640cagcagccag ggcatctaca acatccacac catctactac gccaactacg agcaccacca 2700ccatcaccac taagcggccg cggatcgatc gatatctgac taaatcttag tttgtattgt 2760catgttttaa tacaatatgt tatgtttaaa tatgttttta ataaatttta taaaataatt 2820tcaactttta ttgtaacaac attgtccatt tacacactcc tttcaagcgc gtgggactcg 2880atgctcggcg ccactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 2940gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 3000tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 3060agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 3120tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 3180cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg 3240ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 3300ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 3360ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 3420ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 3480cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 3540gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 3600atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 3660ttttgcatgc gctaagcggg ctttataaaa cgggctgcgg gaccagtttt catatcacta 3720ccgtttgagt tcttgtgctg tgtggatact cctcccgaca ccgaat 3766174220DNAArtificial SequenceVector 17cgtaagtatc aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag 60acagagaaga ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc 120ctttctctcc acaggaattc gccaccatga agctgtgcat cctgctggcc gtggtggcct 180tcgtgggact gagtctggga caggaggccg tgccagagga gagcaccgag attgcccacc 240gcaccgagac ccgcaccgat gagcgcaaga atcaggagcc cgccaacaag gatctgaaga 300acccccagca gagcgtgggc gagaacggca cgaaggatct gctgcaggag gatctgggag 360gcagccgcag cgaggatgaa gtgacccagg agttcggcgt gaaccacggc atccccaagg 420gcgaggatca gaccctggga aagtccgatg ccatccccaa catcggcgag cccgagaccg 480gaatcagtac caccgaggag tcccgccacg aggagggcca taacaagcag gccctgagca 540cctccgtgga tgagcccgag ctgagcgata ccctgcagct gcacgaggat accaaggaga 600acgataagct gcccctggag agcagcacca tcaccagccc aaccgagagc ggcagcagcg 660ataccgagga gaccccatcc attagcgagg gcccgaaggg caacgagcag aagaagcgcg 720acgacgatag cctgagcaag atcagcgtgt cccccgagaa cagccgccca gagaccgatg 780ccaaggatac cagcaacctg ctgaagctga agggcgacgt ggacatcagc atgcccaagg 840ccgtgatcgg cagctccccc aacgataaca tcaacgtgac cgagcagggc gacaacatct 900cgggcgtgaa cagcaagccc ctgtccgatg atgtgcgccc cgataagaac catgaggaag 960tgaaggagca caccagcaac agcgataacg tgcagcagtc cggcggcatc gtgaacatga 1020acgtggagaa ggagctgaag gacaccctgg agaaccccag ctccagcctg gatgagggaa 1080aggcccatga ggagctgtcc gagcccaacc tgtccagcga tcaggatatg agcaacaccc 1140caggccccct ggataacacc tcggaggaga cgaccgagcg catcagcaac aacgagtaca 1200aagtgaacga gcgcgagggc gagcgcaccc tgaccaagga gtatgaggat atcgtgctga 1260agtcccacat gaaccgcgag agcgacgatg gcgagctgta cgatgagaac agcgatctga 1320gcaccgtgaa cgatgagtcc gaggatgccg aggccaagat gaagggcaat gataccagcg 1380agatgagcca caacagcagc cagcacatcg agagcgatca gcagaagaac gatatgaaga 1440ccgtgggcga cctgggcacc acccacgtgc agaatgagat ctccgtgccc gtgaccggcg 1500agatcgatga gaagctgcgc gagagcaagg agtccaagat ccacaaggcc gaggaggagc 1560gcctgagcca caccgatatc cacaagatca accccgagga tcgcaactcc aacaccctgc 1620acctgaagga tatccgcaac gaggagaatg agcgccacct gacgaaccag aacatcaaca 1680tcagccagga gcgcgacctg cagaagcacg gcttccacac catgaacaac ctgcacggcg 1740acggcgtgtc cgagcgcagc cagatcaatc actcgcacca cggcaaccgc caggatcgcg 1800gaggaaatag tggaagcgcc tggtcccacc cccagttcga gaaatgagcg gccgcggatc 1860gatcgatatc tgactaaatc ttagtttgta ttgtcatgtt ttaatacaat atgttatgtt 1920taaatatgtt tttaataaat tttataaaat aatttcaact tttattgtaa caacattgtc 1980catttacaca ctcctttcaa gcgcgtggga ctcgatgctc ggcgccactc aaaggcggta 2040atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 2100caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 2160cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 2220taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 2280ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc 2340tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 2400gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 2460ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 2520aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 2580aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 2640agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 2700cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 2760gacgctcagt ggaacgaaaa ctcacgttaa gggattttgc atgcgctaag cgggctttat 2820aaaacgggct gcgggaccag ttttcatatc actaccgttt gagttcttgt gctgtgtgga 2880tactcctccc gacaccgaat taattcggat ctctgcaagg gattttggtc atgaacaata 2940aaactgtctg cttacataaa cagtaataca aggggtgttc atagtataat acgactcact 3000ataggagggc caccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg 3060tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt cagccgggac ttcgtggagg 3120acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc 3180aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg 3240ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg 3300agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg 3360tgcacttcgt ggccgaggag caggactgac cgacgccgac caacaccgcc ggtccgacgg 3420cggcccacgg gtcccagggg ggtcgacctc gaaacttgtt tattgcagct tataatggtt 3480acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 3540gttgtggttt gtccaaactc atcaatgtat cttatcatgt cttcacgtaa taagtgtgcg 3600gctagcagtc aactactagt gaatgcccta ctagaagatg tgtgttgcac aaaatgtccc 3660tggaataacc aatttgaagt gcagatagca gtaaacgtaa gctaatatga atattattta 3720actgtaatgt tttaatatcg ctggacatta ctaataaacc cactataaac acatgtacat 3780atgtatgttt tggcatacaa tgagtagttg gggaaaaaat gtgtaaaagc accgtgacca 3840tcacagcata aagataacca gctgaagtat cgaatatgag taacccccaa attgaatcac 3900atgccgcaac tgataggacc catggaagta cactcttcat ggcgatatac aagacacaca 3960caagcacgaa cacccagttg cggaggaaat tctccgtaaa tgaaaaccca atcggcgaac 4020aattcatacc catatatggt aaaagttttg aacgcgagcg aacttaagag cgccggagta 4080taaatagagg cgcttcgtct acggagcgac aattcaattc aaacaagcaa agtgaacacg 4140tcgctaagcg aaagctaagc aaataaacaa gcgcagctga acaagctaaa caatctgcag 4200aagcttggta ccctcgagct 422018400PRTPlasmodium falciparum 18Asp Val Lys Asn Asn Glu Asp Tyr Lys Asn Val Asp Tyr Lys Asn Val1 5 10 15Asn Phe Leu Gln Tyr His Phe Lys Glu Leu Ser Asn Tyr Asn Ile Ala 20 25 30Asn Ser Ile Asp Ile Leu Gln Glu Lys Glu Gly His Leu Asp Phe Val 35 40 45Ile Ile Pro His Tyr Thr Phe Leu Asp Tyr Tyr Lys His Leu Ser Tyr 50 55 60Asn Ser Ile Tyr His Lys Ser Ser Thr Tyr Gly Lys Cys Ile Ala Val65 70 75 80Asp Ala Phe Ile Lys Lys Ile Asn Glu Thr Tyr Asp Lys Val Lys Ser 85 90 95Lys Cys Asn Asp Ile Lys Asn Asp Leu Ile Ala Thr Ile Lys Lys Leu 100 105 110Glu His Pro Tyr Asp Ile Asn Asn Lys Asn Asp Asp Ser Tyr Arg Tyr 115 120 125Asp Ile Ser Glu Glu Ile Asp Asp Lys Ser Glu Glu Thr Asp Asp Glu 130 135 140Thr Glu Glu Val Glu Asp Ser Ile Gln Asp Thr Asp Ser Asn His Thr145 150 155 160Pro Ser Asn Lys Lys Lys Asn Asp Leu Met Asn Arg Thr Phe Lys Lys 165 170 175Met Met Asp Glu Tyr Asn Thr Lys Lys Lys Lys Leu Ile Lys Cys Ile 180 185 190Lys Asn His Glu Asn Asp Phe Asn Lys Ile Cys Met Asp Met Lys Asn 195 200 205Tyr Gly Thr Asn Leu Phe Glu Gln Leu Ser Cys Tyr Asn Asn Asn Phe 210 215 220Cys Asn Thr Asn Gly Ile Arg Tyr His Tyr Asp Glu Tyr Ile His Lys225 230 235 240Leu Ile Leu Ser Val Lys Ser Lys Asn Leu Asn Lys Asp Leu Ser Asp 245 250 255Met Thr Asn Ile Leu Gln Gln Ser Glu Leu Leu Leu Thr Asn Leu Asn 260 265 270Lys Lys Met Gly Ser Tyr Ile Tyr Ile Asp Thr Ile Lys Phe Ile His 275 280 285Lys Glu Met Lys His Ile Phe Asn Arg Ile Glu Tyr His Thr Lys Ile 290 295 300Ile Asn Asp Lys Thr Lys Ile Ile Gln Asp Lys Ile Lys Leu Asn Ile305 310 315 320Trp Arg Thr Phe Gln Lys Asp Glu Leu Leu Lys Arg Ile Leu Asp Met 325 330 335Ser Asn Glu Tyr Ser Leu Phe Ile Thr Ser Asp His Leu Arg Gln Met 340 345 350Leu Tyr Asn Thr Phe Tyr Ser Lys Glu Lys His Leu Asn Asn Ile Phe 355 360 365His His Leu Ile Tyr Val Leu Gln Met Lys Phe Asn Asp Val Pro Ile 370 375 380Lys Met Glu Tyr Phe Gln Thr Tyr Lys Lys Asn Lys Pro Leu Thr Gln385 390 395 4001918DNAArtificial SequenceExpression tag 19caccaccatc accaccac 182024DNAArtificial SequenceExpression tag 20tggtcccacc cccagttcga gaaa 242154DNAArtificial SequenceExpression tag 21atgaagctgt gcatcctgct ggccgtggtg gccttcgtgg gactgagtct ggga 542224DNAArtificial SequenceExpression tag 22gactacaagg atgacgacga caag 24236PRTArtificial SequenceExpression tag 23His His His His His His1 5248PRTArtificial SequenceExpression tag 24Trp Ser His Pro Gln Phe Glu Lys1 52518PRTArtificial SequenceExpression tag 25Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser1 5 10 15Leu Gly268PRTArtificial SequenceExpression tag 26Asp Tyr Lys Asp Asp Asp Asp Lys1 527511PRTPlasmodium falciparum 27Gln Glu Ala Val Pro Glu Glu Ser Thr Glu Ile Ala His Arg Thr Glu1 5 10 15Thr Arg Thr Asp Glu Arg Lys Asn Gln Glu Pro Ala Asn Lys Asp Leu 20 25 30Lys Asn Pro Gln Gln Ser Val Gly Glu Asn Gly Thr Lys Asp Leu Leu 35 40 45Gln Glu Asp Leu Gly Gly Ser Arg Ser Glu Asp Glu Val Thr Gln Glu 50 55 60Phe Gly Val Asn His Gly Ile Pro Lys Gly Glu Asp Gln Thr Leu Gly65 70 75 80Lys Ser Asp Ala Ile Pro Asn Ile Gly Glu Pro Glu Thr Gly Ile Ser 85 90 95Thr Thr Glu Glu Ser Arg His Glu Glu Gly His Asn Lys Gln Ala Leu 100 105 110Ser Thr Ser Val Asp Glu Pro Glu Leu Ser Asp Thr Leu Gln Leu His 115 120 125Glu Asp Thr Lys Glu Asn Asp Lys Leu Pro Leu Glu Ser Ser Thr Ile 130 135 140Thr Ser Pro Thr Glu Ser Gly Ser Ser Asp Thr Glu Glu Thr Pro Ser145 150 155 160Ile Ser Glu Gly Pro Lys Gly Asn Glu Gln Lys Lys Arg Asp Asp Asp 165 170 175Ser Leu Ser Lys Ile Ser Val Ser Pro Glu Asn Ser Arg Pro Glu Thr 180 185 190Asp Ala Lys Asp Thr Ser Asn Leu Leu Lys Leu Lys Gly Asp Val Asp 195 200 205Ile Ser Met Pro Lys Ala Val Ile Gly Ser Ser Pro Asn Asp Asn Ile 210 215 220Asn Val Thr Glu Gln Gly Asp Asn Ile Ser Gly Val Asn Ser Lys Pro225 230 235 240Leu Ser Asp Asp Val Arg Pro Asp Lys Asn His Glu Glu Val Lys Glu 245 250 255His Thr Ser Asn Ser Asp Asn Val Gln Gln Ser Gly Gly Ile Val Asn 260 265 270Met Asn Val Glu Lys Glu Leu Lys Asp Thr Leu Glu Asn Pro Ser Ser 275 280 285Ser Leu Asp Glu Gly Lys Ala His Glu Glu Leu Ser Glu Pro Asn Leu 290 295 300Ser Ser Asp Gln Asp Met Ser Asn Thr Pro Gly Pro Leu Asp Asn Thr305 310 315 320Ser Glu Glu Thr Thr Glu Arg Ile Ser Asn Asn Glu Tyr Lys Val Asn 325 330 335Glu Arg Glu Gly Glu Arg Thr Leu Thr Lys Glu Tyr Glu Asp Ile Val 340 345 350Leu Lys Ser His Met Asn Arg Glu Ser Asp Asp Gly Glu Leu Tyr Asp 355 360 365Glu Asn Ser Asp Leu Ser Thr Val Asn Asp Glu Ser Glu Asp Ala Glu 370 375 380Ala Lys Met Lys Gly Asn Asp Thr Ser Glu Met Ser His Asn Ser Ser385 390 395 400Gln His Ile Glu Ser Asp Gln Gln Lys Asn Asp Met Lys Thr Val Gly 405 410 415Asp Leu Gly Thr Thr His Val Gln Asn Glu Ile Ser Val Pro Val Thr 420 425 430Gly Glu Ile Asp Glu Lys Leu Arg Glu Ser Lys Glu Ser Lys Ile His 435 440 445Lys Ala Glu Glu Glu Arg Leu Ser His Thr Asp Ile His Lys Ile Asn 450 455 460Pro Glu Asp Arg Asn Ser Asn Thr Leu His Leu Lys Asp Ile Arg Asn465 470 475 480Glu Glu Asn Glu Arg His Leu Thr Asn Gln Asn Ile Asn Ile Ser Gln 485 490 495Glu Arg Asp Leu Gln Lys His Gly Phe His Thr Met Asn Asn Leu 500 505 510

Claims

1.-24. (canceled)

25. A Drosophila derived cell modified to express a Plasmodium falciparum (Pf) Ripr polypeptide from a heterologous polynucleotide encoding a PfRipr polypeptide and a suitable signal sequence, further comprising a heterologous polynucleotide encoding PfRh5 polypeptide and a suitable signal sequence, wherein each polynucleotide is operably linked to an expression control sequence which induces the Pf polypeptide expression in the cell and wherein the PfRipr polypeptide and the PfRh5 polypeptide from said cell induce merozoite invasion inhibitory antibodies when administered to a subject.

26. The cell of claim 25, wherein co-expression of PfRipr and PfRh5 polypeptides in the cell induces increased yields of either or both polypeptides compared to mono-expression of either polypeptide.

27. The cell of claim 25, further comprising a heterologous polynucleotide encoding a PfCyrPa polypeptide and a suitable signal sequence, operably linked to an expression control sequence which induces PfCyrPa polypeptide expression in the cell, wherein the PfRipr polypeptide, PfRh5 polypeptide and PfCyrPa polypeptide from said cell induce parasite invasion inhibitory antibodies when administered to a subject.

28. The cell of claim 25, further comprising a heterologous polynucleotide encoding a PfEBA175 polypeptide and a suitable signal sequence, operably linked to a promoter which induces EBA175 polypeptide expression in the cell.

29. The cell of claim 27, further comprising a heterologous polynucleotide encoding a PfEBA175 polypeptide and a suitable signal sequence, operably linked to a promoter which induces EBA175 polypeptide expression in the cell.

30. A Drosophila cell comprising an expression vector comprising a polynucleotide encoding PfRipr polypeptide and a suitable signal sequence and an expression vector comprising a polynucleotide encoding PfRh5 polypeptide and a suitable signal sequence, wherein each polynucleotide is operably linked to an expression control sequence which induces Pf Ripr and PfRh5 polypeptide expression in a Drosophila derived cell, wherein the PfRipr polypeptide and the PfRh5 polypeptide from the cell induces parasite growth inhibitory antibodies when administered to a subject.

31. A Drosophila cell of claim 30 further comprising an expression vector comprising a polynucleotide encoding a PfCyrPa polypeptide and a suitable signal sequence, said polynucleotide operably linked to an expression control sequence which induces PfCyrPa polypeptide expression in a Drosophila derived cell, wherein the PfCyrPa polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.

32. An expression vector comprising a polynucleotide encoding PfRipr polypeptide and a suitable signal sequence and a polynucleotide encoding PfRh5 polypeptide and a suitable signal sequence, wherein each polynucleotide is operably linked to an expression control sequence which induces PfRipr and PfRh5 polypeptide expression in a Drosophila derived cell, wherein the PfRipr polypeptide and the PfRh5 polypeptide from the cell induces parasite growth inhibitory antibodies when administered to a subject.

33. A Drosophila cell comprising the expression vector of claim 32.

34. A Drosophila cell comprising the expression vector of claim 32 and further comprising an expression vector comprising a polynucleotide encoding a PfCyrPa polypeptide and a suitable signal sequence, said polynucleotide operably linked to an expression control sequence which induces PfCyrPa polypeptide expression in a Drosophila derived cell, wherein the PfCyrPa polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.

35. An immunogenic composition comprising an expression vector comprising a polynucleotide encoding PfRipr polypeptide and a suitable signal sequence and an expression vector comprising a polynucleotide encoding PfRh5 polypeptide and a suitable signal sequence, wherein each polynucleotide is operably linked to an expression control sequence which induces PfRipr and PfRh5 polypeptide expression, and a physiologically or pharmaceutically acceptable carrier, diluent or excipient.

36. The immunogenic composition of claim 35, further comprising an expression vector comprising a polynucleotide encoding a PfCyrPa polypeptide and a suitable signal sequence, said polynucleotide operably linked to an expression control sequence which induces PfCyrPa polypeptide expression, and a physiologically or pharmaceutically acceptable carrier, diluent or excipient.

37. The immunogenic composition of claim 35 further comprising an adjuvant.

38. The immunogenic composition of claim 36 further comprising an adjuvant.

39. An immunogenic composition comprising the vector of claim 32 and a physiologically or pharmaceutically acceptable carrier, diluent or excipient.

40. The immunogenic composition of claim 39 further comprising an adjuvant.

41. A method of producing PfRipr and PfRh5 polypeptide comprising transfecting a Drosophila cell with a vector of claim 32 and isolating PfRipr and PfRh5 polypeptide from said cells.

42. A Pf protein complex or Pf protein mixture purified from the cell of claim 25.