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.

Claims

1. An expression vector comprising a polynucleotide encoding PfRipr polypeptide and a suitable signal sequence, said polynucleotide operably linked to an expression control sequence which induces the PfRipr polypeptide expression in a Drosophila derived cell wherein the polynucleotide comprises SEQ ID NO:14, wherein the PfRipr polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.

2. A Drosophila cell comprising the expression vector of claim 1.

3. An immunogenic composition comprising the vector of claim 1 and a physiologically or pharmaceutically acceptable carrier, diluent or excipient.

4. The immunogenic composition of claim 3 further comprising an adjuvant.

5. A method of producing Plasmodium falciparum (Pf) Ripr polypeptide comprising transfecting a Drosophila cell with a vector of claim 1 and isolating PfRipr from said cells.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) 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.

(2) 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.

(3) FIG. 3A to 3K 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

(4) SEQ ID NO: 1: PfRipr Full length (FL) nucleotide sequence;

(5) SEQ ID NO: 2: PfCyrPa nucleotide sequence;

(6) SEQ ID NO: 3: PfRh5 version 1 nucleotide sequence;

(7) SEQ ID NO: 4: PfRh5 version 2 nucleotide sequence;

(8) SEQ ID NO: 5: PfEBA175 nucleotide sequence;

(9) SEQ ID NO: 6: PfRiprFL amino acid sequence;

(10) SEQ ID NO: 7: PfCyrPa amino acid sequence;

(11) SEQ ID NO: 8: PfRh5 Full length (FL) amino acid sequence;

(12) SEQ ID NO: 9: PfRh5 version 1 amino acid sequence;

(13) SEQ ID NO: 10: PfEBA175 amino acid sequence;

(14) SEQ ID NO: 11: pExpreS2-1 vector nucleotide sequence;

(15) SEQ ID NO: 12: pExpreS2-2 vector nucleotide sequence;

(16) SEQ ID NO: 13: pExpreS2-PAC vector nucleotide sequence;

(17) SEQ ID NO: 14: pExpreS2-1 vector comprising PfRiprFL;

(18) SEQ ID NO: 15: pExpreS2-2 vector comprising PfRh5 version 1;

(19) SEQ ID NO: 16: pExpreS2-PAC vector comprising PfCyrPa;

(20) SEQ ID NO: 17: pExpreS2-1 vector comprising PfEBA175;

(21) SEQ ID NO: 18: PfRh5 version 2 amino acid sequence;

(22) SEQ ID NO: 19: His-tag nucleotide sequence;

(23) SEQ ID NO: 20: StrepII purification tag nucleotide sequence;

(24) SEQ ID NO: 21: N-terminal secretion signal nucleotide sequence;

(25) SEQ ID NO: 22: Flag-tag nucleotide sequence;

(26) SEQ ID NO: 23: His-tag amino acid sequence;

(27) SEQ ID NO: 24: StrepII purification tag amino acid sequence;

(28) SEQ ID NO: 25: N-terminal secretion signal amino acid sequence;

(29) SEQ ID NO: 26: Flag-tag amino acid sequence;

(30) SEQ ID NO: 27: PfEBA175 amino acid sequence (760-1271 W2MEF).

LIST OF TABLES

(31) Table 1: Exemplary substitutions.

(32) Table 2: Constructs present in each cell line.

(33) Table 3: Lane order of gel from FIG. 2A.

DETAILED DISCUSSION OF EMBODIMENTS

(34) 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.

(35) 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.

(36) 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).

(37) 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.

(38) 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.

(39) 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.

(40) 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.

(41) 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).

(42) 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.

(43) 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.

(44) The biologically active fragment may have the same or similar activity as, or an enhanced activity/immunogenicity relative to a reference polypeptide.

(45) 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.

(46) 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.

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

(48) 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.

(49) TABLE-US-00001 TABLE 1 Original Residue Exemplary 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

(50) 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.

(51) 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.

(52) 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) lo, 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),=34 (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.

(53) 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.

(54) 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.

(55) 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.

(56) 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.

(57) 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.

(58) 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.

(59) Polynucleotides

(60) 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.

(61) 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.

(62) 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.

(63) 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).

(64) 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.

(65) Expression Systems

(66) 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.

(67) 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.

(68) 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).

(69) 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.

(70) In one embodiment the Arthropoda cell is a Drosophila cell. Methods of culturing Drosphila 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.

(71) 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.

(72) 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.

(73) 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.

(74) 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).

(75) 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.

(76) 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.

(77) 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.

(78) 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.

(79) 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).

(80) 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.

(81) 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.

(82) 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.

(83) 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.

(84) 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.

(85) In some embodiment, the mixture or complex or composition further comprises EBA175 as described herein.

(86) 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).

(87) 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).

(88) 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.

(89) 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 polyetheylene 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 manitol, 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.

(90) 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.

(91) 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.

(92) 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.

(93) 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 saponin, ISCOMATRIX 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) polyptide), 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.

(94) 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.

(95) 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.

(96) 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.

(97) 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.

(98) 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.

(99) 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.

(100) 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.

(101) 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.

(102) 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.

(103) Immunoassay

(104) 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.

(105) 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.

(106) 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.

(107) 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.

(108) 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.

(109) 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.

(110) 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.

(111) 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.

(112) 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.

(113) 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.

(114) 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.

(115) 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), -D-galactosidease. In an embodiment, the enzyme is HRP.

(116) 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.

(117) 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.

(118) Kits

(119) 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.

(120) 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.

(121) The following methods are used in practising the present description.

(122) Vectors.

(123) 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.

(124) Cell Transfection.

(125) 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 810.sup.6 cells/ml and incubated at 25 C. at 115 rpm. On the day of transfection the cells were split by centrifugation and resuspended in Excell420 medium at a concentration of 210.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 l ExpreS.sup.2 Insect-TR 5 (ExpreS2ion Biotechnologies, Denmark) was added to the cell suspension followed by 12.5 g DNA of the above vectors comprising PfRiprFL, EBA175, PfRh5 or PfCyrPa and mixed. The cells were then incubated for 3 hours at 25 C. before 1 mL of FBS was added. The day after transfection selection was added at the following concentrations: Zeocin 2000 g/mL; G418 4000 g/mL; and Puromycin 100 g/mL.

(126) For the next two weeks the T-flask was counted every 3-4 day and the cells were diluted to 110.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 810.sup.6 cells/ml in a 250 ml shake flask (Signma-Aldrich; CLS431144). The cells were subsequently frozen in CryoStor CS10 (Sigma; C2874-100ML) for later use.

(127) 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.

(128) Production of PfRipr in WAVE Bioreactor.

(129) 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 810.sup.6 cells/mL with a volume of 5000 mL Excell420 at 25 C. The system parameters were set to 20 rpm, 8 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 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.

(130) Immunization Protocol for Raising Antibodies Against the PfRIPR/PfRh5/PfCyrPa complex.

(131) 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 g. Since the molecular weight of Ripr is roughly twice that of PfRh5 and PfCyrPa to ensure 100 g of assembled complex 55 g of each of PfRh5 and PfCyrPa was added to 125 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.

(132) Analysis of Antibodies Raised Against the PfRIPR/PfRh5/PfCyrPa Complex.

(133) Serum was obtained from blood taken two weeks after the third immunization in the immunization schedule and IgG prepared by protein A sepharose chromatography.

(134) The following non-limiting examples are provided.

Example 1: Comparison of PfRipr Expression; PfRipr Synergistic Effect

(135) 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 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

(136) 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.

(137) The samples from each cell line were prepared by mixing with buffer and reducing agent and incubation at 95 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.

(138) 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-13 pExpreS2-1 vector comprising PfRiprFL, pExpreS2-2 vector comprising PfRh5 and pExpreS2-PAC vector comprising PfCyrPa

(139) TABLE-US-00003 TABLE 3 Lane Sample Comment Cell line Construct Tag 1 TJ1438 WHTP-12 PfCyrPa 2 TJ1463 WHTZGP-13 PfRiprFL, PfRh5, PfCyrPa 3 TJ1463 Un-reduced WHTZGP-13 PfRiprFL, PfRh5, 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-13 PfRiprFL, PfRh5, PfCyrPa 9 TJ1463 Un-reduced WHTZGP-13 PfRiprFL, PfRh5, PfCyrPa 10 Seeblue Plus2 11 TJ1343 WHTZ-04 PfRiprFL 12 TJ1463 WHTZGP-13 PfRiprFL, PfRh5, PfCyrPa 13 TJ1463 Un-reduced WHTZGP-13 PfRiprFL, PfRh5, Strep PfCyrPa 14 TJ1483 Control WHTZ-04 PfRiprFL 15 TJ1484 Additive 1 WHTZ-04 PfRiprFL 16 TJ1485 Additive 2 WHTZ-04 PfRiprFL 17 Empty

(140) 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.

(141) 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.

(142) 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.

(143) 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).

(144) 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.

(145) 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

(146) 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.

(147) 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).

(148) The growth inhibition single cycle assay was performed according to methods described in Malkin et al. 2005.

REFERENCES

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