The invention provides isolated placental P. falciparum polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof. The invention also provides isolated nucleic acid molecules encoding the placental P. falciparum polypeptides of the invention, compositions comprising one or more placental P. falciparum polypeptides of the invention, methods for inducing an immune response against the placental P. falciparum polypeptides, and methods for treating and diagnosing placental malaria.

Description of the Invention

SUMMARY OF THE INVENTION

One aspect of the invention provides isolated placental P. falciparum polypeptides. In some embodiments, the isolated placental P. falciparum polypeptides comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24. In some embodiments, the placental P. falciparum proteins are preferentially recognized by sera from multigravidas than by sera from primigravidas and/or sera from males. The isolated placental P. falciparum polypeptides of the invention may be recombinant or synthetic polypeptides. In some embodiments, the polypeptides of the invention are immunogenic derivatives of polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24.

Another aspect of the invention provides isolated nucleic acid molecules encoding the placental P. falciparum polypeptides of the invention. Thus, some embodiments provide an isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof.

A further aspect of the invention provides compositions comprising one or more placental P. falciparum polypeptides of the invention and a pharmaceutically acceptable carrier. Thus, some embodiments provide an immunogenic composition comprising an isolated polypeptide and a pharmaceutically acceptable carrier, wherein the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof. In some embodiments, the compositions of the invention are immunogenic compositions for inducing immune responses, such as vaccine compositions.

In another aspect, the invention provides methods for inducing an immune response against placental P. falciparum parasites, comprising administering an immunogenic composition comprising an effective amount of one or more placental P. falciparum polypeptides of the invention. Thus, in some embodiments the invention provides a method for inducing an immune response in a mammalian subject against Plasmodium falciparum, comprising administering to the host a composition comprising an effective amount of at least one isolated polypeptide selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof.

Yet another aspect of the invention provides methods for treating a subject in need thereof, comprising administering to a subject in need thereof an immunogenic composition comprising an effective amount of one or more placental P. falciparum polypeptides of the invention. Thus, in some embodiments the invention provides a method for treating a human subject in need thereof, comprising administering to a human subject an immunogenic composition comprising at least one isolated polypeptide selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof.

The invention also provides expression vectors encoding the placental P. falciparum polypeptides of the invention, host cells comprising such expression vectors; antibodies that bind specifically to the placental P. falciparum polypeptides of the invention, or immunogenic derivatives thereof; and diagnostic assays for detecting the presence of the placental P. falciparum polypeptides of the invention, or nucleic acid molecules encoding them.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In one aspect, the invention provides novel proteins expressed by placental P. falciparum parasites. In some embodiments, these proteins are expressed on the surface of red blood cells infected by Plasmodium falciparum parasite, as shown in EXAMPLE 1. In some embodiments, the genes encoding these proteins are upregulated in placental P. falciparum parasites, as shown in EXAMPLE 4. Placental P. falciparum proteins are preferentially recognized by sera from multigravidas than by sera from primigravidas and/or sera from males, as shown in EXAMPLES 2 and 5.

Thus, one aspect of the invention provides isolated placental P. falciparum polypeptides. In some embodiments, the isolated placental P. falciparum polypeptides comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24. The sequences of these proteins, the nucleotide sequences encoding them, and annotation information may be obtained from the Plasmodium Genome Database (http://plasmodb.org/; Kissinger et. al (2002) Nature 419: 490-492) under the protein/gene ID numbers provided in Tables 1, 2 and 4 (see Original Patent), and are herein incorporated by reference. The isolated placental P. falciparum polypeptides of the invention may be recombinant or synthetic full-length polypeptides, or immunogenic derivatives thereof, as further described below. Accordingly, some embodiments of the invention provide an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof.

As used herein, the term "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. Included within the definition are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, PNA, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

The placental P. falciparum polypeptides of the invention may be full-length polypeptides, immunogenic derivatives or domains of full-length polypeptides, or immunogenic variants thereof. As used herein, the term "immunogenic" refers to the ability of a polypeptide to elicit a humoral and/or cellular immune response, whether alone or when linked to a carrier, in the presence or absence of an adjuvant. Thus, an immunogenic portion of a full-length placental P. falciparum polypeptide of the invention refers to a portion of the full-length polypeptide that is capable of eliciting an immune response against the corresponding full-length polypeptide. The term "immunogenic derivative or domain" encompasses any polypeptide that includes at least 5 to 8 amino acids (such as, for example, 10 to 50 amino acids or 30 to 200 amino acids) and that is capable of inducing an immune response to the full-length polypeptide, such as a truncated form, epitope, or other derivative. The term "epitope" refers to a linear array of 3 to 10 amino acids aligned along the surface of a protein. In a linear epitope, the amino acids are joined sequentially and follow the primary structure of the protein. In a conformational epitope, residues are not joined sequentially, but lie linearly along the surface due to the conformation (folding) of the protein. With respect to conformational epitopes, the length of the epitope-defining sequence can be subject to wide variations. The portions of the primer structure of the antigen between the residues defining the epitope may not be critical to the structure of the conformational epitope. For example, deletion or substitution of these intervening sequences may not affect the conformational epitope provided sequences critical to epitope conformation are maintained (e.g., cysteines involved in disulfide bonding, glycosylation sites, etc.). A conformational epitope may also be formed by 2 or more essential regions of subunits of a homo-oligomer or hetero-oligomer. Other immunogenic derivatives may be prepared by the addition, deletion, substitution, or rearrangement of amino acids or by chemical modifications thereof.

Methods of predicting immunogenic regions in a polypeptide are well-known in the art. For example, a polypeptide sequence may be analyzed using the DNASTAR program by several algorithms, including prediction of hydrophilicity according to the Kyte-Doolittle method, surface probability according to the Emini method, and antigenicity according to the Jameson-Wolf method. Other epitope prediction approaches are known in the art (see, e.g., Moise & De Groot (2006) Nat. Biotechnol. 24(7):791-2).

In some embodiments, the immunogenic derivatives of the placental P. falciparum proteins of the invention include 5 to 10, 10 to 50, 20 to 200, or 40 to 300 contiguous amino acids of a full-length polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24. Exemplary immunogenic derivatives of the polypeptides of the invention include, but are not limited to, polypeptides comprising amino acids 572 to 877 of SEQ ID NO:11, amino acids 1 to 500 of SEQ ID NO:3, amino acids 50 to 750 of SEQ ID NO:4, amino acids 751-1471 of SEQ ID NO:4, amino acids 370 to 670 of SEQ ID NO:8, amino acids 2000 to 2500 of SEQ ID NO:8, or amino acids 34 to 347 of SEQ ID NO:13.

Immunogenic derivatives of the polypeptides of the invention, which may be useful in the preparation of vaccines, may be prepared by expression of the appropriate gene fragments or by peptide synthesis, using methods well known in the art, as further described below. Exemplary methods for recombinant expression of immunogenic derivatives of the invention are provided in EXAMPLES 3 and 5.

An immunogenic derivative may be a fusion polypeptide containing additional sequences encoding one or more epitopes for other Plasmodium immunogens, or other non-Plasmodium immunogens. Alternatively, the immunogenic derivative of the invention can be fused to a carrier polypeptide (such as Hepatitis B surface or core antigen) or to another carrier that has immunostimulating properties, as in the case of an adjuvant, or that otherwise enhances the immune response to the protein or derivative thereof, or that is useful in expressing, purifying or formulating the protein or derivative thereof. The proteins or immunogenic derivatives thereof which are useful in the invention may be chemically conjugated to a macromolecule using a conventional linking agent such as glutaraldehyde (Geerlings et al. (1988) J. Immunol. Methods 106: 239-244).

In some embodiments, the placental P. falciparum polypeptides of the invention include immunogenic derivatives with more than 80% amino acid sequence identity (such as more than 90% sequence identity, more than 95% amino acid sequence identity, or more than 99% sequence identity) to the sequences defined in SEQ ID NOs:1-4 and 6-23. The terms "identical" or percent "identity", in the context of two or more amino acid sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

It is recognized that amino acid positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. The scoring of conservative substitutions can be calculated according to, for example, the algorithm of Meyers & Millers (1988) Computer Applic. Biol. Sci. 4:11-17.

A "comparison window" includes reference to a segment of contiguous positions, such as between about 25 and about 600 positions, or between about 50 to 200 positions, or between about 100 and 150 positions, over which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by a local homology algorithm (Smith & Waterman (1981) Adv. Appl. Math. 2:482), by a global alignment algorithm (Needleman & Wunsch (1970) J. Mol. Biol. 48:443), by search for similarity methods (Pearson & Lipman (1988) Proc. Natl. Acad. Sci. USA. 85:2444; Altschul et al. (1997) Nucl. Acids Res. 25(17):3389-402), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and BLAST in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), typically using the default settings, or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (1994) Ausubel et al., eds.). For example, BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences that are more than 80% identical to the amino acid sequence of SEQ ID NOs:1-4 and 6-24.

One example of a useful algorithm implementation is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a dendrogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle (1987) J. Mol. Evol. 35:351-60. The method used is similar to the method described by Higgins & Sharp (1989) CABIOS 5:151-3. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster can then be aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. A series of such pairwise alignments that includes increasingly dissimilar sequences and clusters of sequences at each iteration produces the final alignment.

In some embodiments, the placental P. falciparum polypeptides of the invention include variants of the wild-type polypeptides. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants may be naturally occurring allelic or interspecies variants (e.g., variants from different P. falciparum strains), or they may be prepared by site-specific mutagenesis of nucleotides in the DNA encoding protein. Site-specific mutagenesis may be performed using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Variant target protein fragments having up to about 100-150 amino acid residues may be prepared by in vitro synthesis using established techniques. Conservative substitution tables providing functionally similar amino acids are well known in the art (Henikoff & Henikoff (1992) Proc. Natl. Acad. Sci. USA. 89:10915-9)

Amino acid substitutions are typically of single residues. Insertions usually will be on the order of from about 1 to about 20 amino acids, although considerably longer insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases, deletions may be much longer. Substitutions, deletions, and insertions or any combinations thereof may be used to arrive at a final derivative.

In some embodiments, the placental P. falciparum polypeptides of the invention are recombinant polypeptides. The term "recombinant polypeptide" refers to a protein produced by recombinant expression methods, such as, for example, in prokaryotic or eukaryotic host cells, or in cell-free in vitro expression systems, as described in detail below.

The placental P. falciparum polypeptides of the invention are typically expressed using an expression vector and purified. Expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, expression vectors include transcriptional and translational regulatory nucleic acid sequences operably linked to the nucleic acid encoding the target protein. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Operably linked DNA sequences may be contiguous or non-contiguous. Methods for linking DNA sequences are well-known in the art and include use of the polymerase chain reaction and ligation. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the target protein; for example, transcriptional and translational regulatory nucleic acid sequences from E. coli are preferably used to express the target protein in E. coli.

Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells. Methods for expressing polypeptides are well known in the art (e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory; Berger and Kimmel (1987) Guide to Molecular Cloning Techniques, Methods in Enzymology, vol. 152, Academic Press, Inc., San Diego, Calif.; Ausubel et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY)

In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art.

An expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to a sequence in the host cell genome, and preferably two homologous sequences that flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.

In addition, an expression vector may include a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary depending on the host cell used.

The placental P. falciparum polypeptides of the invention may be produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a placental P. falciparum polypeptide, under the appropriate conditions to induce or cause expression of the placental P. falciparum polypeptide. The conditions appropriate for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art using routine experimentation. For example, the growth and proliferation of the host cell may be optimized for the use of constitutive promoters in the expression vector, and appropriate growth conditions for induction are provided for use of an inducible promoter. In addition, in some embodiments, the timing of the harvest is important, for example, when using baculoviral systems. One of skill in the art will recognize that the coding sequences may be optimized for expression in the selected host cells.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Host cells include, but are not limited to, Drosophila melanogaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, Hep G2 cells, THP1 cell line (a macrophage cell line), and human cells and cell lines.

In some embodiments, the placental P. falciparum polypeptides are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral systems. Promoters from viral genes are frequently used in mammalian expression systems, because the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter. Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. Examples of transcription terminator and polyadenylation signals include those derived from SV40.

The placental P. falciparum polypeptides of the invention may be cloned using DNA amplification methods, such as the polymerase chain method (PCR) (see e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbour, N.Y.; Berger & Kimmel (1987) Methods in Enzymology. Vol. 152: Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego, Calif.; Co et al. (1992) J. Immunol. 148:1149). Thus, for example, a nucleic acid molecule encoding a placental P. falciparum polypeptide may be PCR amplified using a sense primer containing one restriction site and an antisense primer containing another restriction site. This will produce a nucleic acid encoding the desired sequence or subsequence having terminal restriction sites. This nucleic acid can then easily be ligated into a vector having appropriate corresponding restriction sites. Suitable PCR primers are easily chosen by one of skill in the art based on the sequence to be expressed. Appropriate restriction sites can also be added by site-directed mutagenesis (see Gillman & Smith (1979) Gene 8: 81-97; Roberts et al. (1987) Nature 328: 731-4).

The methods of introducing exogenous nucleic acid into host cells are well known in the art, and will vary with the host cell used. Suitable techniques include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

In some embodiments, the placental P. falciparum polypeptides of the invention are expressed in bacterial systems. Bacterial expression systems are well known in the art. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. The expression vector may also include a signal peptide sequence that provides for secretion of the target protein in bacteria. The placental P. falciparum polypeptide is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). The expression vector may also include an epitope tag providing for affinity purification of the target protein. The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes that render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways. These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others. The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others. An exemplary method for expressing placental P. falciparum polypeptides of the invention using a bacterial expression system is described in EXAMPLES 2 and 3.

The placental P. falciparum polypeptides of the invention may also be produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art. The placental P. falciparum polypeptides may also be produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.

The placental P. falciparum polypeptides of the invention may be produced in a cell-free expression system in vitro using an expression vector containing nucleic acid encoding a placental P. falciparum polypeptide, under the appropriate conditions to induce or cause expression of the placental P. falciparum polypeptide in vitro. Cell-free in vitro expression systems are well known in the art. An exemplary method for expressing placental P. falciparum polypeptides of the invention using a cell-free in vitro expression system is described in EXAMPLE 3.

The placental P. falciparum polypeptides of the invention may also be made as a fusion proteins, using techniques that are well known in the art. For example, a placental P. falciparum polypeptides may be made as a fusion protein to increase expression or to link it with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. Exemplary tags include the myc epitope and 6-histidine. The epitope tag is generally placed at the amino- or carboxyl-terminus of the target protein. The presence of such epitope-tagged forms of a target protein can be detected using an antibody against the tag polypeptide. Thus, the epitope tag enables the target proteins to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 (Field et al. (1988) Mol. Cell. Biol. 8:2159-65); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al. (1985) Mol. Cell. Biol. 5:3610-6); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsly et al. (1990) Prot. Eng. 3(6):547-53). Other tag polypeptides include the Flag-peptide (Hopp et al. (1988) BioTechnol. 6:1204-10); the KT3 epitope peptide (Martin et al. (1992) Science 255:192-4); tubulin epitope peptide (Skinner et al. (1991) J. Biol. Chem. 266:15163-6); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA. 87:6393-7).

Covalent modifications of placental P. falciparum polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a target protein with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a target protein. Derivatization with bifunctional agents is useful, for instance, for crosslinking a target protein to a water-insoluble support matrix or surface for use in screening assays. Commonly used crosslinking agents include, but are not limited to, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

The placental P. falciparum polypeptides of the invention may be purified or isolated after expression. The terms "isolated" "purified" or "biologically pure" refer to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. The term "purified" denotes that a protein gives rise to essentially one band in an electrophoretic gel. For example, it means that the protein is at least 85% pure, such as at least 95% pure, such as at least 99% pure. The term "isolated polypeptides" also includes polypeptides in situ within recombinant host cells, since at least one component of the polypeptide natural environment will not be present. Generally, however, isolated polypeptide will be prepared by at least one purification step.

The placental P. falciparum polypeptides of the invention may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the target protein may be purified using an antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. Suitable purification techniques are standard in the art (see generally R. Scopes (1982) Protein Purification, Springer-Verlag, N.Y.; Deutcher (1990) Methods in Enzymology vol. 182: Guide to Protein Purification, Academic Press, Inc. N.Y.). The degree of purification necessary will vary depending on the use of the polypeptide. In some instances no purification may be necessary.

Some embodiments of the invention provide synthetic placental P. falciparum polypeptides. Polypeptides having up to about 100-150 amino acid residues may be prepared by in vitro synthesis using established techniques. Synthetic polypeptides may be prepared by chemical synthesis (such as solid phase peptide synthesis) using methods known in the art, such as those described in Merrifield et al. (1964) J. Am. Chem. Soc. 85:2149, Houghten et al. (1985) Proc. Natl. Acad. Sci. USA, 82:51:32, and Stewart & Young (1984) Solid phase peptide synthesis, Pierce Chem Co., Rockford, Ill. Such polypeptides can be synthesized with or without a methionine on the amino terminus. Chemically synthesized placental P. falciparum proteins of the invention and immunogenic derivatives thereof may be oxidized using methods set forth in these references to form disulfide bridges. Further, peptidomimetics that structurally and/or functionally resemble a polypeptide embodiment may be made. Several approaches to make peptidomimetics that resemble polypeptides have been described (see, e.g., U.S. Pat. Nos. 5,288,707; 5,552,534; 5,811,515; 5,817,626; 5,817,879; 5,821,231; and 5,874,529).

Another aspect of the invention provides isolated nucleic acid molecules encoding the placental P. falciparum polypeptides of the invention. Thus, some embodiments provide an isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof. The term "isolated nucleic acid molecule(s)" refers to a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) encoding placental P. falciparum polypeptides or immunogenic derivatives thereof. The sequence of these nucleic acid molecules may be different to the any naturally-occurring sequences encoding the placental P. falciparum polypeptides of the invention but that due to the degeneracy of the genetic code, still encode a placental P. falciparum polypeptide. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.

Another aspect of the invention provides expression vectors encoding the placental P. falciparum polypeptides of the invention. Another aspect of the invention provides host cells comprising expression vectors encoding the placental P. falciparum polypeptides of the invention.

Another aspect of the invention provides antibodies that bind specifically to the placental P. falciparum polypeptides of the invention, or immunogenic derivatives thereof. The term "antibody" refers to an intact immunoglobulin, or to an antigen-binding portion of an immunoglobulin that competes with the intact antibody for specific binding to a protein or fragment of a protein of the present invention. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies. Antigen-binding portions of an immunoglobulin of the present invention can be produced by various techniques including, but not limited to recombinant DNA techniques and enzymatic or chemical cleavage of intact antibodies.

An "isolated antibody" as used herein is an antibody that (1) is not associated with naturally-associated components, including other naturally-associated antibodies, that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. The terms "bind specifically" and "specific binding" refer to the ability of an antibody of the present invention to bind to a first molecular species in preference to binding to other molecular species with which the antibody and first molecular species are admixed. An antibody is said specifically to "recognize" a first molecular species when it can bind specifically to that first molecular species. In the present invention the first molecular species is a placental P. falciparum polypeptide of the invention, or immunogenic derivative thereof.

Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a placental P. falciparum polypeptide of the invention, or an immunogenic derivative thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler & Milstein (1975) Nature 256:495. In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. Suitable immortalized cell lines for the production of monoclonal antibodies are well-known in the art (see, e.g., Goding (1986) Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103; Kozbor (1984) J. Immunol. 133:3001; Brodeur et al. (1987) Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63).

The binding specificity of monoclonal antibodies produced by the hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody may, for example, be determined by the Scatchard analysis of Munson & Pollard (1980) Anal. Biochem. 107:220.

The monoclonal antibodies may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567, herein incorporated by reference. Monoclonal antibodies may be isolated using phage display libraries (Hoogenboom & Winter (1991) J. Mol. Biol. 227:381; Marks et al. (1991) J. Mol. Biol. 222:581).

The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

The antibodies may also be human or humanized antibodies, bispecific antibodies, or heteroconjugate antibodies. Methods for preparing human or humanized antibodies, bispecific antibodies, or heteroconjugate antibodies are well known in the art and described, for example, in Desnoyers et al., U.S. Pat. No. 7,084,258, herein incorporated by reference.

The antibodies that specifically bind to the placental P. falciparum polypeptides of the invention may be used in diagnostic assays, for example, to detect the presence of placental malaria parasites, or as therapeutic or prophylactic agents for treating or preventing infection by P. falciparum. The term "therapeutic agent" refers to an agent capable of treating a malaria infection. The term "prophylactic agent" refers to an agent capable of preventing an infection by P. falciparum. In some embodiments, the antibodies may be used to treat subjects at risk of developing or suffering from pregnancy malaria by passive immunization.

In general, this will comprise administering a therapeutically or prophylactically effective amount of one or more antibodies of the present invention to a subject susceptible to malaria or a subject exhibiting a malaria infection. Any active form of the antibody can be administered, including Fab and F(ab').sub.2 fragments. Treatment of individuals having malaria infection may comprise the administration of a therapeutically effective amount of antibodies of the present invention. The dosage of administered antibodies will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, as well as other factors know to those of skill in the art. An appropriate effective amount can be readily determined using only routine experimentation. Effective amounts and routes of administration for therapeutic and prophylactic applications are further described below.

Another aspect of the invention provides compositions comprising one or more placental P. falciparum polypeptides of the invention and a pharmaceutically acceptable carrier. Thus, some embodiments provide an immunogenic composition comprising an isolated polypeptide and a pharmaceutically acceptable carrier, wherein the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof. In some embodiments, the compositions of the invention are immunogenic compositions for inducing immune responses, such as vaccine compositions. A "vaccine" is an immunogenic composition capable of eliciting protection against infection by Plasmodium parasites and/or malarial disease, whether partial or complete. A vaccine that is used for treatment of an infected individual may be referred to as a therapeutic vaccine. The immunogenic compositions of the invention may also be used to elicit antibodies in a species that is not infectable by P. falciparum, for example to raise antibodies in rabbits or mice.

The invention further provides methods for preparing an immunogenic composition, by suspending and packaging one or more placental P. falciparum polypeptides of the invention in a suitable pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carrier include sterile water or sterile physiological salt solution, particularly phosphate buffered saline (PBS), as is well known in the art.

The immunogenic compositions of the invention generally also include an adjuvant. Adjuvants are well known in the art (see, for example, Vaccine Design--The Subunit and Adjuvant Approach (1995) Pharmaceutical Biotechnology, Volume 6 (eds. Powell, M. F., & Newman, M. J.) Plenum Press, New York and London, ISBN 0-306-44867-X). Exemplary adjuvants include complete Freund's adjuvant (CFA) that is not used in humans, incomplete Freund's adjuvant (IFA), squalene, squalane and alum (e.g., Alhydrogel.TM., Superfos, Denmark), which are materials well known in the art, and are available commercially from several sources. Other exemplary adjuvants include the adjuvants described in Lanar et al., U.S. Pat. No. 7,029,685, and U.S. Patent Publication No. 2006/0073171, herein incorporated by reference.

In some embodiments, the immunogenic composition is a vaccine composition. Vaccine preparation is generally described in New Trends and Developments in Vaccine (eds. Voller et al.), University Park Press, Baltimore, Md., U.S.A., 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Pat. No. 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No. 4,474,757.

The amount of immunogen(s) present in each vaccine dose is selected as an amount that induces an immune response (such as an immunoprotective response) without significant, adverse side effects in typical vaccines. The term "immune response" refers to an acquired and enhanced degree of protective immunity against Plasmodium infection or malarial disease, for example, complete or partial protection against infection or disease following subsequent exposure to malaria parasites. The amount of immunogen present in each dose will vary depending upon which specific immunogens are employed, and other factors. Generally, it is expected that each dose will comprise a total of 1-1000 micrograms of protein, such as 1-200 micrograms or 10-100 micrograms or 5-50 micrograms. Following an initial vaccination, subjects will generally receive one or more boosts. An optimal amount for a particular vaccine, as well as the number and frequency of boosts, can be determined empirically by standard studies involving observation of immune responses in subjects.

The vaccine compositions of the invention may be administered by any suitable method of administration known in the art, including, but not limited to, intradermally, subcutaneously, intramuscularly, intraperitoneally, orally, ocularly (e.g., as an eye spray), and intravenously. Vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations that are suitable for other modes of administration include suppositories and, in some cases, oral formulation or by nasal spray. For suppositories, traditional binders and carriers can include, for example, polyalkalene glycols or triglycerides. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.

In some embodiments, the vaccine compositions of the invention are DNA vaccines comprising a nucleic acid molecule encoding one or more placental P. falciparum polypeptides of the invention. Thus, some embodiments provide an immunogenic composition comprising a nucleic acid molecule encoding a polypeptide and a pharmaceutically acceptable carrier, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof. Methods for preparing and administering a DNA vaccine expressing Plasmodium proteins are known in the art and have been previously described (see, e.g., Doolan & Hoffman (2001) Int. J. Parasitol. 31:753-62; Narum et al., U.S. Pat. No. 7,078,507, herein incorporated by reference. In some embodiments, the vaccine compositions of the invention are viral vaccines comprising a viral vector encoding one or more placental P. falciparum polypeptides of the invention. Exemplary viral vectors for use in the vaccine compositions of the invention include, but are not limited to, vaccinia viral vectors (such as vectors based on modified vaccinia virus or avian pox viruses), adenoviral vectors, and yellow fever viral vectors (see, e.g., Imoukhuede et al. (2006) Vaccine, in press; Miao et al. (2006) Vaccine, in press; Tao et al. (2005) J. Exp. Med. 201:201-9). Methods for preparing and administering viral vaccine expressing Plasmodium proteins are known in the art and have been previously described (see, e.g., Imoukhuede et al. (2006) Vaccine, in press; Miao et al. (2006) Vaccine, in press; Tao et al. (2005)J. Exp. Med. 201:201-9).

Another aspect of the invention provides methods for inducing an immune response against placental P. falciparum parasites, comprising administering an immunogenic composition comprising an effective amount of one or more placental P. falciparum polypeptides of the invention. Thus, in some embodiments the invention provides a method for inducing an immune response in a mammalian subject against Plasmodium falciparum, comprising administering to the host a composition comprising an effect amount of at least one isolated polypeptide selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof. Exemplary mammalian subjects for the methods of inducing an immune response include, but are not limited to, humans, goats, rabbits, and mice. In some embodiments the mammalian subject is a human subject.

Another aspect of the invention provides methods for treating a subject in need thereof, comprising administering to a subject in need thereof an immunogenic composition comprising an effective amount of one or more placental P. falciparum polypeptides of the invention. Thus, in some embodiments the invention provides a method for treating a human subject in need thereof, comprising administering to a human subject an immunogenic composition comprising at least one isolated polypeptide selected from the group consisting of SEQ ID NOs:1-4 and 6-24, and immunogenic derivatives thereof.

The term "treating" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. In some embodiments, the subjects to be treated are human subjects suffering from malaria, such as, for example, placental malaria. In some embodiments, the subjects to be treated are human subjects at risk for contracting malaria, including, but not limited to women at risk for contracting placental malaria. The subjects to be treated may or may not have previously been infected by P. falciparum parasites.

The term "effective amount" for a therapeutic or prophylactic treatment refers to an amount or dosage of a composition sufficient to induce a desired response (e.g., an immunogenic response) in the individual to which it is administered. Preferably, the effective amount is sufficient to effect treatment, as defined above. The effective amount and method of administration of a particular therapeutic or prophylactic treatment may vary based on the individual patient and the stage of the disease, as well as other factors known to those of skill in the art. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosages for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be taken into account include the prevalence of P. falciparum in the geographical vicinity of the patient, the severity of the disease state of the patient, age, and weight of the patient, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. An appropriate effective amount can be readily determined using only routine experimentation. Several doses may be needed per individual in order to achieve a sufficient response to effect treatment. Suitable regimes for initial administration and follow-up administration (e.g., booster shots) are also variable, but are typified by an initial administration followed in intervals (weeks or months) by a subsequent administration.

The production of antibodies elicited by a treatment is readily ascertained by obtaining a plasma or serum sample from the subject to which an immunogenic composition is administered and assaying the antibodies therein for their ability to bind to the polypeptide(s) used to elicit the immune response to P. falciparum parasites, such as placental parasites. Exemplary methods include, but are not limited to, ELISA assays or by other immunoassays such as a Western blots, as is well known in the art. Another method for measuring the production of antibodies is by using an indirect immunofluorescence assay (IFA).

Antibodies to one or more of the placental P. falciparum parasites of the invention may be isolated from the blood of the host mammal using well known techniques, and then reconstituted into a second vaccine for passive immunization, as is also well known. Similar techniques are used for gamma-globulin immunizations of humans. For example, antiserum from one or a number of immunized hosts can be precipitated in aqueous ammonium sulfate (typically at 40-50 percent of saturation), and the precipitated antibodies purified chromatographically (e.g., affinity chromatography).

In another aspect, the invention provides diagnostic and screening agents and assays, which may be protein-based or nucleic acid-based. These agents and assays may be used to detect the presence of the placental P. falciparum polypeptides of the invention, or nucleic acid molecules encoding them, in order to determine whether a subject is suffering from or is likely to suffer from malaria, particularly pregnancy malaria. Many techniques may be used, including, but not limited to, ELISA, sandwich assays, immunoprecipitation, immunoblots, hybridization techniques, and PCR.

In some embodiments, the placental P. falciparum polypeptides of the invention are used for the detection of antibodies in a subject. In some embodiments, antibodies to the placental P. falciparum polypeptides of the invention are used to detect the presence of these polypeptides. Diagnostic immunoassay procedures are standard in the art (see, e.g., Basic and Clinical Immunology (1991) 7th ed., Stites, D., & Terr, A.) Exemplary methods may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide. Such labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays that amplify the signals from the immune complex are also known, such as assays that utilize biotin and avidin or streptavidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

Some embodiments provide methods for the in vitro diagnosis of malaria in a subject likely to be infected by P. falciparum, comprising (a) contacting a biological sample comprising antibodies from a subject with one or more placental P. falciparum polypeptides of the invention under conditions enabling the formation of antigen/antibody complexes between the polypeptides and the antibodies, and (b) detecting the formation of antigen/antibody complexes. Examples of biological samples that may be used to perform this method are red blood cells, white blood cells, serum or urine. Conditions enabling the formation of antigen/antibody complexes are well known in the art.

The invention also provides methods for monitoring the immune status of a subject vaccinated against infection or disease caused by P. falciparum, comprising (a) contacting a biological sample comprising antibodies from a subject with one or more placental P. falciparum polypeptides of the invention under conditions enabling the formation of antigen/antibody complexes between the polypeptides and the antibodies, and (b) detecting the formation of antigen/antibody complexes.

In the diagnostic and monitoring methods described above, the biological sample may be further contacted with one or several antigenic peptides originating from other Plasmodium antigens.

In some embodiments, the diagnostic and screening agents and assays are nucleic acid-based. Exemplary diagnostic and screening agents for use in nucleic acid-based assays include nucleic acid probes complementary to nucleic acid molecules encoding P. falciparum polypeptides of the invention. Nucleic-acid based diagnostic and screening assays are well known in the art. Exemplary diagnostic and screening assays to be used in this aspect of the invention are described in Scherf et al., U.S. Pat. No. 6,855,323, herein incorporated by reference.

The invention also provides kits which are useful for carrying out the present invention. The kits may include a first container means containing the vaccine or antibodies of the invention. The kit may also include other container means containing solutions necessary or convenient for carrying out the invention. The container means can be made of glass, plastic or foil and can be a vial, bottle, pouch, tube, bag, etc. The kit may also contain written information, such as procedures for carrying out the present invention or analytical information, such as the amount of reagent contained in the first container means. The container means may be in another container means, e.g. a box or a bag, along with the written information.
 

Claim 1 of 4 Claims

1. An immunogenic composition comprising an amount of an isolated polypeptide effective for inducing an immune response against placental Plasmodium falciparum parasites in a mammalian subject and a pharmaceutically acceptable carrier, wherein the isolated polypeptide has at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO:20 and said immunogenic composition further comprises an adjuvant.

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