BINDING AND SIGNALING

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Application No. 62/110,018, filed January 30, 2015, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is SBRI155178_ST25.txt. The text file is 5 KB; was created on January 27, 2016; and is being submitted via EFS-Web with the filing of the specification.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under IROIGMIOI 183-01 Al awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

Eph receptors and their corresponding natural ephrin ligands are membrane-bound proteins that require direct cell-cell interactions for Eph receptor activation. Eph/ephrin signaling has been implicated in the regulation of a host of processes critical to embryonic development including axon guidance, formation of tissue boundaries, cell migration, and segmentation. Additionally, Eph/ephrin signaling has recently been identified to play a critical role in the maintenance of several processes during adulthood including long-term potentiation, angiogenesis, and stem cell differentiation and cancer.

Despite advances in the art, there remains a need for compositions and methods to modulate ephrin cell signaling to address a variety of Eph receptor-related conditions. The present disclosure addresses this and related needs. SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the present disclosure is directed to an isolated polypeptide corresponding to or derived from a 6-Cys protein of Plasmodium. In some embodiments, the polypeptide comprises a first amino acid sequence corresponding to a 6-Cys domain from Plasmodium. In some embodiments, the polypeptide further comprises a second amino acid sequence corresponding to a 6-Cys domain from Plasmodium, wherein the second amino acid sequence is the same or different as the first amino acid sequence.

In another aspect the present disclosure is directed to a pharmaceutical composition comprising the described polypeptide.

In another aspect the present disclosure is directed to a nucleic acid encoding the described polypeptide.

In another aspect the present disclosure is directed to a vector comprising the described nucleic acid.

In another aspect the present disclosure is directed to a cell comprising the described vector.

In another aspect, the present disclosure is directed to a method of modulating binding of a natural ligand to an Eph receptor, comprising administering to a cell expressing the Eph receptor a sufficient amount of the polypeptide described herein.

In another aspect, the present disclosure is directed to a method of modulating signaling of an Eph receptor in a cell, comprising administering to a cell expressing the Eph receptor a sufficient amount of the polypeptide described herein.

In another aspect, the present disclosure is directed to a method of inhibiting the proliferation of a cancer cell, comprising contacting the cell with a sufficient amount of the polypeptide described herein.

In another aspect, the present disclosure is directed to a method of treating a disease characterized by elevated Eph receptor signaling, elevated Eph receptor expression, and/or elevated Eph receptor ligand expression, comprising administering to a subject in need an effective amount of the polypeptide described herein. In one embodiment, the disease is a cancer or cataract. In another embodiment, the disease is caused by hepatocyte infection by a Plasmodium parasite.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURES 1A-1K illustrate that Plasmodium sporozoites invade hepatocytes with high EphA2 expression. (1A) Hepal-6 cells were infected with P. yoelii sporozoites and visualized by immunofluorescence 24 hours after infection. The upper panel shows EphA2 distribution in an exemplary Hepal-6 cell. The middle panel shows UIS4 expression in the exemplary Hepal-6 cell. The bottom panel is an overlay of the two individual images that combines the signals for EphA2 and UIS4 in the cell. The scale bar is 5 mm. In FIGURES IB to ID, Hepal-6 cells were infected with 10 ⁵ P. yoelii sporozoites: (IB) shows the distribution of EphA2 1.5 hours after infection (mEphA2, mouse EphA2); in (1C), EphA2 levels are compared between parasite-infected and uninfected cells; and (ID) shows parasite-infection rates in EphA2 ^hi § ^h and EphA2 ^low cells (Py, P. yoelii). The numbers in the bars are the percentages of infected cells within each subset. In FIGURES IE to 1G, BALB/c mice were infected with 10 ⁶ P. yoelii sporozoites by intravenous injection. Hepatocytes were analyzed as in (IB) to (ID). In FIGURES 1H to 1J, HC-04 cells were infected with 10 ⁵ P. falciparum sporozoites (hEphA2, human EphA2). Analyses were performed as in (IB) to (ID). (IK) Hepal-6 cells were incubated with EphA2 -blocking antibody (D4A2) or immunoglobulinG (IgG) as a control 30 min before infection with 10 ⁵ P. yoelii sporozoites. The infection rate was normalized to the infection rate in the presence of IgG. Each figure represents at least three independent experiments. The bar graphs show means with standard deviations.

FIGURES 2A-2G illustrate that EphA2 affects PVM formation. (2A) Time sequence showing the maintenance of P. yoelii infection in Hepal-6 cells. Maintenance of infection was defined as the infection rate at a given point divided by the infection rate at 1.5 hours after infection. Error bars indicate the standard deviation of biological replicates. (2B) EphA2( ^~/~ ) or age-matched wildtype (WT) mice were infected with 10 ⁵ P. yoelii sporozoites. Infection was assessed by quantitative polymerase chain reaction 42 hours after infection (Py 18s, P. yoelii 18S ribosomal RNA; mGAPDH, mouse glyceraldehyde-3 -phosphate dehydrogenase). Error bars show SEM. (2C) EphA2( ^~/~ ) or strain-matched WT mice were infected with 10 ² P. yoelii sporozoites. Patency was monitored daily by thin smear. The horizontal bar indicates the median. (2D) A yUIS4- Myc parasite in a Hepal-6 cell 24 hours after infection. The scale bar is 5 mm. (2E and 2F) yUIS4-Myc parasites were used to infect Hepal-6 cells, and the cells were analyzed for the presence of the PVM (UIS4P ^0S ) after 24 hours (MFI, median florescence intensity). (2G) PyUIS4-Myc infected Hepal-6 cells were assessed for permeability after 48 hours. Each figure represents at least three independent experiments. (2E) to (2G) show means with standard deviations.

FIGURES 3A-3E illustrate that P36 interacts with EphA2. (3A) 2 ^χ 10 ⁵ WT or p52 ^~ /p36- P. yoelii parasites were used to infect Hepal-6 cells. Levels of EphA2 were monitored in infected and uninfected cells. (3B) Plasmodium yoelii sporozoites were used to infect Hepal-6 cells 30 min after treatment with IgG or EphA2-blocking antibody (aEphA2). Infection levels were normalized to the infection rate in the presence of IgG. (3C and 3D) Hepal-6 cells were incubated with recombinant P52 or P36, alone or in combination with 10 min of EphrinAl treatment. Immunoblots show levels of pEphA2 (pY772). (3E) 2 x 10 ⁵ WT or p52 ^' /p36 ^{^} /sapl ^{^} P. falciparum sporozoites were used to infect 6 x 10 ⁵ HC-04 cells. EphA2 levels were measured in infected and uninfected cells. Each figure represents at least three independent experiments. The bar graphs show means with standard deviations.

FIGURES 4A-4C illustrate that EphA2 levels are increased in more susceptible hepatocytes. (4A) Hepal-6 cells were stained for EphA2 and DNA and monitored by flow cytometry. EphA2 levels are higher in cells with higher DNA content. (4B) Hepatocytes were isolated by collagenase-mediated perfusion and percoll gradient from BALB/cJ or BALB/cByJ mice and then stained with EphA2 and assessed by flow cytometry. BALB/cByJ mice have higher levels of EphA2 than BALB/cJ mice. (4C) Plated human hepatocytes infected with P. falciparum show a higher percentage of cells with 8n DNA content, and lower percentage of 2n cells, than uninfected cells from the same culture. Results are from single hepatocyte donor; results are representative of multiple donors.

FIGURES 5A and 5B illustrate that EphA2 levels vary more within a single culture than between cultures. (5 A) Image showing variability of EphA2 levels between hepatoma cells in a Hepal-6 culture. EphA2 staining is depicted in red (indicated with arrows), DNA is visualized by DAPI in blue. Scale bar is 10 μιη. (5B) Hepal-6 cells were passaged for one month and lysed every 1-3 passages. Levels of EphA2 were assessed by western blot and pActin was used as a loading control.

FIGURES 6A-6E. (6A) Gating strategy for identifying CSP-positive cells, EphA2high and EphA2 ^low cells. (6B) Plasmodium sporozoites preferentially invade hepatocytes with high EphA2 expression in vitro and in vivo. Histograms depict the distribution of surface levels of EphA2 in uninfected (light green) and infected (dark green) Hepal-6 cells. (6C) Surface EphA2 levels in infected Hepal-6 cells are higher than uninfected Hepal-6 cells within a single culture. EphA2 levels are monitored by EphA2-PE and expressed as median fluorescence intensity (MFI). (6D) Infection rates within EphA2 ^hi§h and EphA2 ^low populations are illustrated. The percentages within each bar graph represent the proportion of infected cells within each subset of EphA2 expression. (6E) Hepal-6 cells were infected with P. yoelii parasites at a MOI of 0.3. The highest 10, 20, 30, 40 or 50% of EphA2-expressing hepatoma cells were gated, then the infection rate within that subset is shown. The infection rate in the total culture is depicted with a red line.

FIGURES 7A-7C illustrate that Dasatinib treatment does not impact initial parasite infection. (7 A) Schematic describing EphA2 structure. (7B) Hepal-6 cells were stimulated with 1 μg/mL EphrinAl-Fc for 10 min with or without 2 h pretreatment with Dasatinib. Cells were lysed and levels of activated EphA2 (pY772) was assessed by western blot. (7C) Untreated or Dasatinib treated Hepa 1-6 cells were infected with P. yoelii sporozoites. Infection rate was assessed by staining with an antibody against CSP and flow cytometry. These data demonstrate that Dasatinib treatment does not impact infection. FIGURES 8 A and 8B. (8 A) Cell division is increased in EphA2high cells. Hepal-6 cells were stained for EphA2 and the cell cycle progression marker Ki-67 and assessed by flow cytometry. EphA2 ^hi sh cells have a greater proportion of cycling cells than EphA2 ^low cells. (8B) Hepatocytes of EphA2( ^~ / ^~ ) mice have similar cell division rates when compared to hepatocytes of WT mice. Livers from three WT BALB/cByJ mice and three EphA2( ^_/~ ) mice were dissociated by collagenase-mediated perfusion. Hepatocytes were stained for the cell cycle marker Ki-67.

FIGURE 9 illustrates that p52Vp36- parasites are rapidly cleared from liver in vivo. BALB/cJ mice were infected with 10 ⁵ p52 ^~ , p36 ^~ or WT P. yoelii sporozoites. Mice were sacrificed 3 h after infection and assessed for liver stage burden by qRT-PCR.

FIGURE 10 illustrates the verification and characterization of recombinant protein produced in HEK293 cells. Purified PyP52 (~160-kDa peak; indicated) and PyP36 (~100-kDa peak; indicated) were separated by size-exclusion chromatography on a calibrated Superdex 200 column (molecular weight standards are overlayed in black). Migration patterns of PyP52 and PyP36 under denaturing conditions (SDS-PAGE; inset) suggest that the recombinant proteins are glycosylated and dimerize under native conditions.

DETAILED DESCRIPTION

The present disclosure addresses a need for modulating Ephrin receptor ("Eph receptor") binding and signaling in cells.

In humans, there are about 14 different known Eph receptors, which are membrane-bound receptors that bind to various ephrin ligands. There are currently about 10 different endogenous ephrin ligands for the 14 Eph receptors, which provide substantial cross-talk among the various Eph receptors. See, e.g., Tognolini, M., et al., "Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor," ChemMedChem. 7(6): 1071-1083 (2012), incorporated herein by reference in its entirety. Both Eph receptors and their corresponding ephrin ligands are membrane-bound proteins that require direct cell-cell interactions for Eph receptor activation. Eph/ephrin signaling has been implicated in the regulation of a host of processes critical to embryonic development including axon guidance, formation of tissue boundaries, cell migration, and segmentation. Additionally, Eph/ephrin signaling has recently been identified to play a critical role in the maintenance of several processes during adulthood including long-term potentiation, angiogenesis, and stem cell differentiation and cancer.

As described in more detail below, the present inventors have produced new insight into the molecular recognition events that govern successful Plasmodium sporozoite infection of host hepatocytes. Namely, the inventors demonstrated that Plasmodium 6-Cys proteins (e.g., P36 and P52) bind the EphA2 receptor on hepatocytes leading to the formation of the intracellular parasitophorous vacuole membrane (PVM) essential for successful infection. Further, inventors have shown that a subfragment of the parasitic protein P36 alone blocks binding of the EphA2 receptor by its natural ligand, EphrinAl . Significantly, in this interaction P36 does not activate the EphA2 receptor and, thus, provides means to prevent any human disease which is driven by EphA2 activation, including malaria disease. The findings also have significance beyond possible application to the treatment of malaria. High EphA2 receptor expression in cancer cells is correlated to a poor prognosis associated with recurrence due to enhanced metastasis (Tandon, M, et al., "Emerging strategies for EphA2 receptor targeting for cancer therapeutics," Expert Opinion Therapeutic Targets 75(1):31-51 (2011), incorporated herein by reference in its entirety). Thus, the ability of P36 protein to block EphA2 receptor activation will be useful and clinically relevant, particularly, for the inhibition of formation of metastases or, generally for the treatment of cancer. Moreover, considering the substantial cross-talk between natural ephrin ligands and Eph receptors, the parasitic ligands described herein have potential applicability to modulation of other Eph ligands to influence a host of other signaling pathways and resulting diseases.

In accordance with the foregoing, in one aspect the present disclosure provides a polypeptide comprising a first amino acid sequence corresponding to a 6-Cys protein from Plasmodium, or derivative thereof. In some embodiments, the polypeptide is isolated or purified from its natural environment in the Plasmodium. In some embodiments, the polypeptide is produced recombinantly from a non-Plasmodium cell according to standard protocols in the art. In some embodiments, the polypeptide comprises a first amino acid sequence with at least 10 contiguous amino acids from SEQ ID NO:2. SEQ ID NO: 2 is an amino acid sequence of a truncated form of Plasmodium yoelii P36, which specifically corresponds to amino acid positions 74-356 of the full-length wild type P36 protein. The indicated sequence lacks the wild-type transmembrane domains but retains two 6-Cys domains, as described in more detail below. An exemplary nucleic acid encoding SEQ ID NO:2 is set forth in SEQ ID NO: l, which is modified from the wild-type encoding sequence for codon bias for optimal expression in human cells. In some embodiments, the polypeptide can comprise a first amino acid sequence with at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or more, or any range derivable therein, of contiguous amino acids of SEQ ID NO:2.

In some embodiments, the first amino acid sequence comprises a domain that corresponds to a 6-Cys domain in a 6-Cys protein from Plasmodium. In some embodiments, the 6-Cys protein is P36. In some embodiments, the Plasmodium is Plasmodium yoelii .

The 6-Cys domains are believed to be unique to Plasmodium parasites and are involved in a variety functions throughout the parasite's life cycle. The domains and Plasmodium proteins that contain them have been described elsewhere. See, e.g., Annoura, T., et al., "Two Plasmodium 6-Cys family-related proteins have distinct and critical roles in liver-stage development," The FASEB Journal 25:2158-2170 (2014)), incorporated herein by reference in its entirety. In one stage, various 6-Cys proteins are expressed on the surface of the sporozoite stage of the Plasmodium life cycle stage. As described in more detail below, the inventors have discovered that the certain 6-Cys proteins on the surface on the sporozoite stage are critical for the selective and successful invasion of host hepatocyte cells due to their ability to bind to Eph receptors present on the hepatocyte surface.

The binding of the 6-Cys proteins (e.g., P52 and P36) to the Eph receptor is believed to be specifically due to the 6-Cys domains in the 6-Cys proteins, which are situated in the extracellular space of the sporozoite. This is supported by the data described below that a truncated form of the P36 protein, as represented by the sequence set forth in SEQ ID NO:2, which contains two 6-Cys domains, retains the capacity to bind Eph receptor. Furthermore, while there is no sequence similarity between 6-Cys domains and endogenous ephrin ligands, similarities in the secondary structures have been described. See, e.g., Arredondo, S.A., et al., "Structure of the Plasmodium 6- cysteine s48/45 domain," Proc Natl Acad Sci U S A 109: 6692 (2012), incorporated herein by reference in its entirety. Accordingly, in one embodiment, the polypeptide comprises a first amino acid sequence corresponding to a 6-Cys domain from Plasmodium. The 6-Cys domain can be any 6-Cys domain known from the Plasmodium genus. See, e.g., Arredondo, S.A., et al., (2012). In another embodiment, the polypeptide further comprises a second amino acid sequence that also corresponds to a 6-Cys domain from Plasmodium. The first and second polypeptides can have the same sequence or can have different or variant sequences. Either 6-Cys domain can be any 6-Cys domain known from the Plasmodium genus. See, e.g., Arredondo, S.A., et al., (2012). The first and second domains can be contiguous or can be separated by linker amino acids of variable length. The linker amino acid sequence can be naturally occurring or synthetic (man-made).

In some embodiments, the polypeptide is not naturally occurring, but is the result of human intervention. Accordingly, the polypeptide can be a purified or isolated form of a naturally occurring polypeptide. The terms "isolated" and "purified" refer the removal of the naturally occurring polypeptide from some, most or all of other molecular structures in its natural environment in the extracellular space of the Plasmodium sporozoite. In this regard, the purified or isolated form of any polypeptide described herein possesses markedly different characteristics from the naturally occurring form of the polypeptide. For example, without being bound to any particular theory, it is believed that P52 and P36 might naturally form a complex in the Plasmodium sporozoite, which enables P36 to contact the EphA2 receptor on a host hepatocyte. The contact is believed to lead to the formation of a parasitophorous vacuole membrane (PVM) that contributes to a successful and productive infection in the cell. However, it has been theorized that without the participation of P52 in the complex, the P36 is not positioned correctly and fails to correctly interact with the host Eph2A receptor in a way to initiate the formation of the PVM. The isolated or purified version of the polypeptide may possess the wild- type sequence, or be a truncated or otherwise altered version of a wild-type sequence. Being isolated or purified, the polypeptide encompassed by this description is not in a complex that would occur within the naturally occurring Plasmodium and, thus, does not trigger the formation of a PVM within a hepatocyte.

The polypeptide can be produced using any techniques, such as recombinant protein expression, that are familiar and well-known in the art. An exemplary approach is illustrated in the description below.

In some embodiments, the sequence of the polypeptide is not naturally occurring. In this regard, the sequence of the polypeptide may differ from a naturally occurring 6-Cys protein by virtue of lacking one or more amino acids that appear in the naturally occurring version. Thus, the polypeptide may have a sequence that corresponds to a subsequence of a naturally occurring 6-Cys protein. In other embodiments, the polypeptide will not correspond precisely with a subsequence of a naturally occurring 6-Cys protein, but rather will contain at least one or more variations (e.g., one or more deletions, additions, and/or mutations) in the naturally occurring 6-Cys protein subsequence.

In one specific embodiment, the first amino acid sequence is one of A) the amino acids sequence corresponding to residue positions 18 to 114 of SEQ ID NO:2, or an amino acid sequence with at least 80% identity thereto, and B) the amino acids sequence corresponding to residue positions 141 to 267 of SEQ ID NO:2, or an amino acid sequence with at least 80% identity thereto. As indicated herein, SEQ ID NO:2 sets forth the sequence of a P36 fragment that lacks the wild-type transmembrane domains. These amino acid subsequences of SEQ ID NO:2 indicated above correspond to the two separate 6-Cys domains within the P36 fragment. Accordingly, the polypeptide of the present disclosure can have a sequence corresponding to either one of these indicated domains, or have a sequence with at least 80% identity thereto.

In some embodiments, the polypeptide has a first and second 6-Cys domain. Thus, in some embodiments, the first and second 6-Cys domains correspond to residue positions 18 to 114 of SEQ ID NO:2 or an amino acid sequence with at least 80% identity thereto. Alternatively, in some embodiments, the first and second 6-Cys domains correspond to residue positions 141 to 267 of SEQ ID NO:2, or an amino acid sequence with at least 80% identity thereto. In yet additional embodiments, the polypeptide contains both of the amino acid sequences corresponding to residue positions 18 to 114 of SEQ ID NO:2, or an amino acid sequence with at least 80% identity thereto, as the first 6-Cys domain, and residue positions 141 to 267 of SEQ ID NO:2, or an amino acid sequence with at least 80% identity thereto, as the second 6-Cys domain. As described above, the first and second 6-Cys domains can be contiguous or can be separated by linker amino acids of variable length. In some embodiments, the first and second 6-Cys domains are linked by a linker amino acid sequence that is naturally occurring or synthetic (man-made).

In some embodiments, the polypeptide comprises the amino acid sequence corresponding to residue positions 18 to 267 of SEQ ID NO:2, or a sequence with at least 70%) identity thereto. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, or a sequence with at least 60%> identity the amino acid sequence set forth in SEQ ID NO:2 (such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%), 98%), 99%), or any range therein, sequence identity thereto).

As used herein, the term "polypeptide" generally refers to a macromolecule of multiple amino acids linked by peptide (amide) bonds. As used herein, an "amino acid" refers to any of the naturally occurring amino acids found in proteins, D-stereoisomers of the naturally occurring amino acids (e.g., D-threonine), unnatural amino acids, and chemically modified amino acids. Each of these types of amino acids is not mutually exclusive. a-Amino acids comprise a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom, and a distinctive group referred to as a "side chain." The side chains of naturally occurring amino acids are well-known in the art and include, for example, hydrogen (e.g., as in glycine), alkyl (e.g., as in alanine, valine, leucine, isoleucine, proline), substituted alkyl (e.g., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine), arylalkyl (e.g., as in phenylalanine and tryptophan), substituted arylalkyl (e.g., as in tyrosine), and heteroarylalkyl (e.g., as in histidine).

The following abbreviations are used for the 20 naturally occurring canonical amino acids: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

Unnatural amino acids (that is, those that are not naturally found in proteins) are also known in the art, as set forth in, for example, Mol. Cell. Biol., 9:251 (1989); J. Amer. Chem. Soc, 772:4011-4030 (1990); J. Amer. Chem. Soc, 56: 1280-1283 (1991); J. Amer. Chem. Soc, 773:9276-9286 (1991); and all references cited herein in their entireties, β- and γ-amino acids are known in the art and are also contemplated herein as unnatural amino acids.

The polypeptide can also have chemically modified amino acids, which refers to an amino acid whose side chain has been chemically modified. For example, a side chain may be modified to comprise a signaling moiety, such as a fluorophore or a radiolabel. A side chain may be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group. Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.

Finally, persons of ordinary skill in the art will readily appreciate that the polypeptide can encompass a polypeptide derivative, such as a type of peptidomimetic where a canonical chemical aspect of the polypeptide is modified. As used herein, the term "peptidomimetic" refers to compounds whose essential elements (pharmacophore) mimic a natural peptide or polypeptide in 3D space, and which retain the ability to interact with the biological target (e.g., a receptor) and produce the same biological effect as an unmodified, canonical polypeptide structure. However, peptidomimetics are designed to circumvent some of the problems associated with a natural peptide: e.g., stability against proteolysis (duration of activity) and poor bioavailability. Certain other properties, such as receptor selectivity or potency, often can be substantially improved. The structural modifications that result in peptidomimetics are well-known and have been described elsewhere. See, e.g., Vagner, J., et al., "Peptidomimetics, a synthetic tool of drug discovery," Curr Opin Chem Biol. 72(3):292-296 (2008), incorporated herein by reference in its entirety.

As used herein, the terms "percent identity" or "percent identical" refer to the percentage of amino acid residues in a polypeptide sequence (or nucleotides in a nucleic acid sequence) that are identical with the amino acid sequence (or nucleic acid sequence) of a specified molecule, after aligning the sequences to achieve the maximum percent identity. Alignments can include the introduction of gaps in the sequences to be aligned to maximize the percent identity. In any embodiment described herein, described sequences can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical, or have any range of identity derivable therein.

In another aspect, the present disclosure provides a pharmaceutical composition comprising any embodiment of the polypeptide described above. Formulation strategies involving active polypeptides for treatments of various conditions are well-known in the art.

In another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding any embodiment of the polypeptide described above.

It should be noted that, as used herein, the term "nucleic acid" refers to any polymer molecule that comprises multiple nucleotide subunits (i.e., a polynucleotide).

Nucleic acids encompassed by the present disclosure can include deoxyribonucleotide polymer (DNA), ribonucleotide polymer (RNA), cDNA or a synthetic nucleic acid known in the art.

Nucleotide subunits of the nucleic acid polymers can be naturally occurring or artificial or modified. A nucleotide typically contains a nucleobase, a sugar, and at least one phosphate group. The nucleobase is typically heterocyclic. Canonical nucleobases include purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T) (or typically in RNA, uracil (U) instead of thymine (T)), and cytosine (C)). The sugar is typically a pentose sugar. Suitable sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate, or triphosphate. These are generally referred to herein as nucleotides or nucleotide residues to indicate the subunit. Without specific identification, the general terms nucleotides, nucleotide residues, and the like, are not intended to imply any specific structure or identity. The nucleotides can also be synthetic or modified.

Nucleotide subunits of the nucleic acid polymers can be naturally occurring or artificial or modified. A nucleotide typically contains a nucleobase, a sugar, and at least one phosphate group. The nucleobase is typically heterocyclic. Canonical nucleobases include purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T) (or typically in RNA, uracil (U) instead of thymine (T)), and cytosine (C). The sugar is typically a pentose sugar. Suitable sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate, or triphosphate. These are generally referred to herein as nucleotides or nucleotide residues to indicate the subunit. Without specific identification, the general terms nucleotides, nucleotide residues, and the like, are not intended to imply any specific structure or identity. The nucleotides can also be synthetic or modified.

In another aspect, the disclosure provides vectors comprising the nucleic acid sequences described herein, such as a vector comprising a nucleic acid sequence encoding the polypeptide described above.

Any vector described herein can further comprise a promoter sequence. Any vector described herein can further comprise a constitutive promoter or inducible promoter appropriate for the expression system to be used, as known in the art. A promoter may comprise an inducible promoter. An inducible promoter may comprise an acetamide-inducible promoter.

Also provided are cultured cells transfected with any vector described herein, or progeny thereof wherein the cell is capable of expressing a polypeptide comprising a 6-Cys domain, as described above. The cell can be prokaryotic or eukaryotic, such as insect or mammalian.

In another aspect, the present disclosure provides a method of modulating binding of a natural ligand to an Eph receptor. In one embodiment, the method comprises administering to a cell expressing the Eph receptor a sufficient amount of the polypeptide described herein above.

As described above, humans have approximately 12 distinct Eph receptors and about 10 ephrin ligands that bind to the Eph receptors. Typically, there is substantial "cross-talk" meaning that a single ephrin ligand can bind to, and cause signaling through, multiple Eph receptors. Accordingly, the Eph receptor can be any known Eph receptor, such as EphAl, EphA2, Eph A3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphAlO, EphBl, EphB2, EphB3, EphB4, EphB5, and EphB6. In some embodiments, the Eph receptor is an Eph2A receptor. In some embodiments, the Eph receptor is a human or rodent Eph receptor.

In some embodiments, the natural ligand is any ephrin ligand. In some embodiments, the ligand is EphrinAl ligand. In some embodiments, the natural ligand is a 6-Cys protein expressed on the surface of a, Plasmodium sporozoite.

As used herein, the term "modulating" refers to causing change in the binding of the natural ligand to the receptor. In some embodiments, the modulation is a prevention or inhibition. For example, the polypeptide may compete with or block binding of the natural ligand to the Eph receptor.

Because the modulation of binding can lead to a subsequent modulation of signaling, the disclosure also provides in another aspect a method of modulating Eph receptor signaling in a cell, comprising contacting the cell with a polypeptide described herein. As above, the Eph receptor can be any Eph receptor known in the art, for example human EphA2 receptor. In this context, the modulation of Eph receptor signaling refers to changing the cell-signaling that normally results from binding of the Eph receptor to its cognate or natural ligand, as described above. Thus, without being bound to any particular theory, the application of a described polypeptide can cause a change in the signaling by modulating the availability of the Eph receptor for binding by the natural ligand. The modulation of signaling can be observed using any known technique, such as described in more detail below. In some embodiments, the modulation of signaling is a reduction in Eph receptor signaling that would be observed by a natural ligand without the presence of the polypeptide described herein.

The cell can be a eukaryotic cell, including a mammalian cell, such as rodent or human. The cell can be in vivo, ex vivo, or in vitro.

Considering the well-known connection of Eph receptor signaling to various diseases, including cancer, cataracts, and malaria, in another aspect the present disclosure also provides a method of treating a disease characterized by elevated Eph receptor signaling, elevated Eph receptor expression, and/or elevated Eph receptor ligand expression, comprising administering to a subject in need an effective amount of the polypeptide described herein. In some embodiments, the method comprises administering to the subject an effective amount of a therapeutic formulation as described herein. As used herein, the term "treating a disease" indicates inducing any beneficial therapeutic effect on presence or progression of the disease. There is not necessarily any implication that treatment is required to result in a full cure. Thus, the effects can include slowing or inhibiting the progression of any aspect of the disease, inhibiting or reducing the chance of the establishment of the disease, reducing or inhibiting the physical effects of the disease, and the like.

In some embodiments, the disease is a cancer, such as any solid tumor cancer that is correlated with elevated Eph receptor signaling, Eph receptor expression, and/or Eph receptor ligand expression. See, e.g., Tandon, M, et al., "Emerging strategies for EphA2 receptor targeting for cancer therapeutics," Expert Opinion Therapeutic Targets 75(1):31- 51 (2011), incorporated herein by reference in its entirety. In other embodiments, the disease is caused by hepatocyte infection by Plasmodium.

In another aspect, the present disclosure provides a method for inhibiting tumor growth in a mammalian subject. The method comprises administering a sufficient (or therapeutic) amount of the polypeptide described herein to the subject. In one embodiment, the administration of the polypeptide (or a formulation containing the polypeptide) results in an inhibition or reduction in the angiogenesis in the immediate environment of the tumor. In another embodiment, the administration of the polypeptide results in an inhibition or reduction in the cell proliferation or cell survival of transformed cells in the tumor. In yet another embodiment, the administration of the polypeptide results in an inhibition or reduction in the tumor cell migration, which can also result in a reduction or inhibition in the potential for metastasis of the tumor.

It is noted that, as used herein, the use of the term "or" in the claims means "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Following long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the claims or specification, denotes one or more, unless specifically noted.

The practice of the present disclosure employs, unless otherwise indicated, conventional immunological and molecular biological techniques and pharmacology within the skill of the art. Such techniques are well-known to the skilled worker, and are explained fully in the literature. See, e.g., Coligan, Dunn, Ploegh, Speicher and Wingfield "Current Protocols in Protein Science" (1999), Volume I and II (John Wiley & Sons Inc.); Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989), 2 ^nd Edition (Cold Spring Harbor Laboratory Press); and Prescott, Harley and Klein "Microbiology" (1999), 4 ^th Edition (WBC McGraw-Hill). Additionally, such considerations as routes of administration, antigen dose, number, frequency of administrations, and appropriate formulations are all matters of optimization within the scope of the ordinary skill in the art.

All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. However, publications mentioned herein are cited for the purpose of describing and disclosing the protocols, reagents, and the like, which are reported in the publications and which might be used in connection with the invention.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to." Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above," and "below," and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. It is understood that, when combinations, subsets, interactions, groups, etc., of these materials are disclosed, each of various individual and collective combinations is specifically contemplated, even though specific reference to each and every single combination and permutation of these compounds may not be explicitly disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in the described methods. Thus, specific elements of any foregoing embodiments can be combined or substituted for elements in other embodiments. For example, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. Additionally, it is understood that the embodiments described herein can be implemented using any suitable material such as those described elsewhere herein or as known in the art.

The following describes the discovery that a, Plasmodium 6-Cys protein binds to a human Eph receptor to mediate successful hepatocyte infection by the sporozoite. This study demonstrated that Furthermore, the study demonstrates the surprising discovery that a truncated form of the 6-Cys protein comprising two 6-Cys domains can engage the human Eph receptor to block binding and signaling by the cognate human ligand, without causing signaling itself.

Title of Study:

Malaria parasites target the hepatocyte receptor EphA2 for successful host infection

Abstract:

The invasion of a suitable host hepatocyte by mosquito-transmitted Plasmodium sporozoites is an essential early step in successful malaria parasite infection. Yet precisely how sporozoites target their host cell and facilitate productive infection remains largely unknown. We found that the hepatocyte EphA2 receptor was critical for establishing a permissive intracellular replication compartment, the parasitophorous vacuole. Sporozoites productively infected hepatocytes with high EphA2 expression, and the deletion of EphA2 protected mice from liver infection. Lack of host EphA2 phenocopied the lack of the sporozoite proteins P52 and P36. Our data suggest that P36 engages EphA2, which is likely to be a key step in establishing the permissive replication compartment.

Introduction, Results, and Discussion:

Malaria infections place a tremendous burden on global health (1). Their causative agents, Plasmodium parasites, are transmitted to mammals as sporozoites by the bite of Anopheles mosquitoes. After entry into a capillary, sporozoites are carried to the liver, where they pass through multiple cells before recognizing and invading hepatocytes. During invasion, the sporozoite forms a protective parasitophorous vacuole made of hepatocyte plasma membrane, which ensconces the parasite, establishes the intrahepatocytic replication niche, and supports successful infection. Highly sulfated proteoglycans are known to provide a signal to sporozoites to invade the liver parenchyma (2, 3), and hepatocyte CD81 and scavenger receptor B l are important for hepatocyte infection (4-6). Beyond this, the molecular mechanisms underlying infection remain poorly understood.

Hepatocytes exhibit differential susceptibility to infection. Sporozoites preferentially enter polyploidy hepatocytes (7). Also, BALB/cByJ mice are more susceptible than BALB/cJ mice to Plasmodium yoelii sporozoite infection (8). To identify potential host receptors that might contribute to differential susceptibility, we used an antibody array to assess the levels of 28 activated receptors in the livers of BALB/cJ and BALB/cByJ mice. Nine receptors, including EphA2, were present at significantly (P < 0.01) and substantially elevated levels in highly susceptible BALB/cByJ mice (Table 1, bold entries). Polyploid hepatocytes also expressed higher levels of EphA2 (FIGURES 4A and 4B).

Table 1 : Assessment of the activity of 28 receptors in BALB/cJ and BALB/cByJ mouse livers.

Receptor* BALB/cBvJ signal BALB/cJ signal** p-value

InsR 1.269 1.000 0.013

EphAl 1.187 1.000 0.014

TrkB-NTRK2 1.137 1.000 0.015

FGFR4 1.159 1.000 0.015

Ron-MSTIR 1.280 1.000 0.018

FGFR1 1.157 1.000 0.020

VEGFR2-KDR 1.211 1.000 0.022

EphB4 1.260 1.000 0.028

EphA3 1.242 1.000 0.038

Tyro-3-Dtk 1.156 1.000 0.047

HER2-ErbB2 1.207 1.000 0.048

Axl 1.164 1.000 0.057

TrkA-NTRKl 1.193 1.000 0.074

ALK 1.323 1.000 0.088

FGFR3 1.168 1.000 0.090

FLT3-Flk2 1.223 1.000 0.103

Ret 0.953 1.000 0.599

**Receptors are depicted in bold if they have >20% increase in signal in BALB/cByJ mice with a p-value of <0.01

** All receptor levels were normalized to the average level in BALB/cJ mice

In metazoans, Eph receptors and their cognate Ephrin ligands mediate cell-cell contact (9), making EphA2 a candidate to mediate the hepatocyte-sporozoite interaction. Furthermore, an Ephrin-like fold is present in the parasite's 6-Cys protein family (10). Although Hepal-6 cells (a murine hepatocyte line) expressed EphA2 consistently across passages, variation within a culture was substantial (FIGURES 5A and 5B). We therefore postulated that if EphA2 mediates sporozoite invasion, susceptibilities might vary within a culture of Hepal-6 cells. We infected Hepal-6 cells with P. yoelii sporozoites; after 24 hours, we assessed parasites in hepatocytes that expressed high levels of EphA2 (FIGURE lA).We also observed this by flow cytometry 1.5 hours after infection (FIGURES IB and 6A), and parasite-infected cells exhibited significantly increased levels of both total (FIGURE 1C) and surface (FIGURES 6B to 6D) EphA2. Similarly, the frequency of infection in cells in the top 50% of EphA2 expression (EphA2 ^hi gh) was elevated compared with infection frequency in cells in the bottom 50% (EphA2 ^low ) (FIGURE ID). When we included only the top 40, 30, 20, or 10% of EphA2-expressing cells in the EphA2 ^hi S ^h gate, the preference was even more pronounced (FIGURE 6E).

We next challenged BALB/c mice with 10 ⁶ P. yoelii sporozoites and isolated hepatocytes after 3 hours. We again observed a strong parasite preference for EphA2 ^hi gh hepatocytes (FIGURE IE to 1G). Finally, we tested whether the preference for infection of EphA2 ^hi Sh hepatocytes is also present in the human parasites by infecting HC-04 hepatocytes with P. falciparum. We observed elevated levels of EphA2 in infected cells and a higher proportion of sporozoite-containing cells in the EphA2 ^hi § ^h population (FIGURE lH to 1J).

EphA2 has an extracellular ligand-binding region and an intracellular kinase domain, which mediates downstream signaling. To assess whether interaction with the extracellular portion of EphA2 is critical for Plasmodium infection, we infected hepatocytes in the presence of an antibody that binds extracellular EphA2. The presence of the antibody reduced sporozoite infection in a dose dependent manner (FIGURE IK). In contrast, inhibiting the kinase domain of EphA2 did not inhibit infection (FIGURES 7A to 7C). Thus, the extracellular portion of EphA2 facilitates Plasmodium invasion of hepatocytes.

To test whether EphA2 levels are important for liver-stage parasite survival and development, we measured infection rates in EphA2 ^hi § ^h and EphA2 ^low cells over the course of 48 hours, normalizing each infection rate to the rate at 1.5 hours after infection. Whereas the number of EphA2 ^hi sh infected cells was maintained throughout the course of infection, the number of EphA2 ^low infected cells decreased over time (FIGURE 2A). This difference could not be accounted for by division rates, because we observed lower levels of host cell division among EphA2 ^low cells. Thus, our results may in fact underestimate the impact of EphA2 on infected cell survival (FIGURES 8A and 8B). When we infected EphA2( ^~/~ ) and wild-type mice with 10 ⁵ P. yoelii sporozoites, we observed a large decrease in liver-stage burden after 42 hours in EphA2( ^~/~ ) mice (FIGURE 2B). EphA2( ^~/~ ) mice also exhibited a delay in the onset of blood-stage infection by 1 to 3 days (FIGURE 2C). Thus, without EphA2, the host is far less susceptible to productive parasite liver infection.

The parasitophorous vacuole membrane (PVM) is critical for liver-stage development. One liver stage PVM-resident protein, UIS4, is highly expressed after invasion when it is exported to the PVM (11), making it a useful marker. We constructed a P. yoelii parasite line, iyUIS4-Myc, which expressed a UIS4-Myc fusion protein driven by the endogenous UIS4 promoter (FIGURE 2D). This allowed us to monitor PVM prevalence (UIS4P ^0S ) in infected cells by flow cytometry. Most of the UIS4P ^0S infected host cells were in the EphA2 ^hi S ^h category (FIGURE 2E). Similarly, the level of EphA2 expression was higher in UIS4P ^0S infected cells than in UIS4 ^ne § infected cells (FIGURE 2F). Thus, sporozoites not only preferentially entered EphA2 ^hi§h cells, but invasion accompanied by PVM formation was far more effective in these cells. UIS4 ^ne§ infected hepatocytes suffered a higher frequency of cell death (FIGURE 2G).

Two members of the 6-Cys family of parasite proteins (12, 13), P52 and P36, are expressed in sporozoites, are important for the invasion of hepatocytes (14-16), and are critical for PVM formation (14). In mouse livers, parasites without P52 or P36 were almost entirely eliminated within 3 hours after infection (FIGURE 9).We tested whether the lack of P52 and P36 phenocopies the lack of host EphA2 and found that p52 ^~ /p36 ^~ P. yoelii sporozoites exhibited a reduced preference for EphA2 ^hi sh cells (FIGURE 3 A). The related 6-Cys protein P12 shows structural similarity to the mammalian ligand for EphA2, EphrinAl (10).

We showed that an interaction in the extracellular region of EphA2 was required for sporozoite entry using an EphA2-blocking antibody (FIGURE IK). Therefore, we next asked whether the presence of P36 and P52 was required for the antibody to block sporozoite entry. The EphA2 antibody blocked infection for wild-type P. yoelii sporozoites, but p52 ^~ /p36- sporozoite entry was not affected (FIGURE 3B). These data suggest that P36 or P52 engages EphA2 at the point of host cell invasion. We next tested whether P52 or P36 could directly impede the interaction between EphrinAl and EphA2 on the hepatocyte surface, which results in EphA2 activation. When we added EphrinAl in the presence of P36 to Hepal-6 cells, P36 blocked the activation of EphA2 (FIGURES 3C and 3D). P52, however, did not block EphrinAl -mediated activation of EphA2 (FIGURES 3C and 3D). To determine whether the interaction between EphA2 and P36 also occurs in human parasites, we assessed levels of EphA2 in P. falciparum wild-type or p52-/p36 ^~ /sapl- parasite-infected HC-04 cells. The P52-P36-deficient P. falciparum sporozoites exhibited partially reduced selectivity for EphA2 ^hi§h HC-04 cells compared with P. falciparum wild-type sporozoites (FIGURE 3E). Thus, P36 engages EphA2 but does not trigger its activation in rodent and human parasites.

We have shown that both host EphA2 and parasite 6-Cys proteins have a role in sporozoite invasion of hepatocytes and the establishment of the growth-permissive intracellular niche. Without either component, the parasite can still enter hepatocytes, but it does so without a PVM, which can result in death of the infected hepatocyte. The convergence of infection-permissive phenotypes is best explained by an interaction between parasite P36 and hepatocyte EphA2 when the PVM is formed. This role for EphA2 in hepatocyte infection does not preclude the possibility that additional hepatocyte receptors may be critical for infection. Interventional strategies aimed at either EphA2 or sporozoite 6-Cys proteins might block parasite infection before the onset of clinical malaria.

Methods and Materials

Mosquito Rearing and Sporozoite Production: For P. yoelii sporozoite production, female 6-8-week-old Swiss Webster (SW) mice (Harlan, Indianapolis, IN) were injected with blood stage P. yoelii (17XNL) parasites to begin the growth cycle. Animal handling was conducted according to Institutional Animal Care and Use Committee-approved protocols. Female A. stephensi mosquitoes were allowed to feed on infected mice after gametocyte exflagellation was observed. Salivary gland sporozoites were isolated according to standard procedures at days 14 or 15 post blood meal. For each experiment, salivary glands were isolated in parallel in order to ensure sporozoites were extracted from salivary glands under the same conditions.

Cell Lines and Culture: Hepal-6 Cells were purchased from ATCC. HC04 cells were a kind gift from Jetsumon Sattabongkot Prachumsri. Cells were maintained in DMEM complete media (Dulbecco's Modified Eagle Medium (Cellgro, Manassas, VA), supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 100 IU/mL penicillin (Cellgro), 100 μg/mL streptomycin (Cellgro), 2.5 μg/mL fungizone (HyClone/ Thermo Fisher, Waltham, MA) and split 1-2 times weekly.

Primary human hepatocyte experiments: Cryopreserved Human Hepatocytes from single donor (Triangle Research, Research Triangle Park, NC) were plated on collagen-coated 12-well plates at a density of 800k live cells per well. Cells were maintained in InvitroGro HI Hepatocyte Media supplemented with Torpedo antibiotic mix (BioreclamationlVT, Baltimore, MD) for 24 hours at 37°C with 5% C0 ₂ . 100k P. falciparum sporozoites per well were added and incubated as above for 90 minutes. Cells were then detached from plate using trypsin, fixed in Cytoperm/Cytofix (BD Biosciences, Franklin Lake, NJ) on ice for 15 minutes, and stained for parasite CSP as described above [antibody 2A10, conjugated to Alexa-488]. After staining, cells were incubated with DNA staining buffer [1 μΜ Sytox Blue (Thermo Fisher Scientific, Waltham, MA), 0.4 mg/mL RNAse A (Therm oFisher), and 5 mM EDTA in PBS] at room temperature for 30 minutes before flow cytometry on an LSRII (BD Biosciences), with analysis on FlowJo software (Tree Star, Ashland, OR).

Antibody Array: Livers were collected from 7 BALB/cJ and 7 BALB/cByJ mice and flash frozen in liquid nitrogen. Frozen livers were then ground using RetschlOO Planetary Ball Mill. Cyroground liver powder was resuspended at 30 μg/mL in IX Cell Lysis buffer supplemented with ImM PMSF. Lysates were centrifuged for 10 minutes at 4°C and supernatant was used in the assay. PathScan Antibody arrays (Cell Signaling Technology) were used to assess levels of active RTKs according to manufacturer instructions. Signal was captured using GenePix 2000 microarray scanner. Spots were aligned and signal analyzed using Mapix software (Innopsys, Chicago, IL). Immunofluorescence Assay: 1.5xl0 ⁵ Hepal-6 cells were seeded in DMEM complete medium in each well of an eight-well Permanox slide. Cells were infected with 5xl0 ⁴ P. yoelii sporozoites. Slides were centrifuged for 3 min at 515 x g in a hanging- bucket centrifuge to aid in sporozoite invasion. After 90 min, we removed media that contained sporozoites that had not infected the cells and added fresh media only. We allowed LSs to develop for 24 hours or 48 hours, at which time cells were fixed with 4% paraformaldehyde, blocked, and permeabilized for 1 hour in PBS with the addition of 0.1% Triton X-100 and 2% BSA. Staining steps were performed in PBS supplemented with 2% BSA. We stained cells using an anti-EphA2 antibody (Cell Signaling Technology, clone D4A2) at a 1 : 175 dilution, and a monoclonal antibody to UIS4 (made against recombinant UIS4 by Promab, clone 8E11B3), used at a final concentration of 16.7 μg/ml. Cells were incubated with primary antibodies at 4°C overnight and then washed several times, and antibodies were visualized with the use of AlexaFluor-488 goat anti-mouse and AlexaFluor-594 goat anti-rabbit secondary antibody (Life Technologies, Grand Island, NY, USA). We used DAPI (1 μg/mL) stain to visualize both hepatocyte and parasite nuclei.

Cloning, protein expression and purification of PyP52 and PyP36: Sequences for PyP52 (UniProt:Q7K5V2) and PyP36 (UniProt:Q7RPW4) were optimized for human codon bias and synthesized commercially (Integrated DNA Technologies, San Jose, CA, USA). For PyP52, the endogenous leader sequence (residues 1-24) was replaced by the tissue plasminogen activator (tPA -UniProt:P00750) signal peptide (residues 1-23) to promote protein secretion. The transmembrane domain was removed (residues 458-480) and replaced by a GS linker and poly-Histidine tag (8X HIS). The final expression construct, containing PyP52 amino acid residues 25-457, was placed under CMV promotion for expression in mammalian cell culture. For the PyP36 expression construct, the two amino-terminus transmembrane domains (residues 25-44 and 51-73) were replaced by the tPA signal, and a GS linker 8XHIS tag was added to the carboxyl- terminus. The final PyP36 expression containing amino acids 74-356 was placed under the CMV promoter for expression in mammalian cells.

The expression constructs were used to transfect suspension HEK293 cells maintained in antibiotic free, serum free FreeStyle 293 Expression medium (Life Technologies) using 293 -Free transfection reagent (EMD Millipore, Billerica, MA, USA), according to manufacturer's instructions. Five to six days post-transfection, culture supernatants were harvested and clarified by centrifugation/filtration. The supernatants were then supplemented with 350 mM NaCl (final concentration) and 0.2% sodium azide (final concentration) prior to binding to Ni-NTA agarose. The protein- bound resin was treated with the wash buffer (25 mM Tris pH 8, 300 mM NaCl, 20 mM imidazole), and the protein was eluted with the elution buffer (25 mM Tris pH 7.4, 300 mM NaCl, 200 mM imidazole). Purified protein was further separated by size-exclusion chromatography using a HiLoad 16/600 Superdex-200 pg column (GE Healthcare) in HBS-E (10 mM Hepes pH 7, 150 mM NaCl, 2 mM EDTA) to remove contaminants and protein aggregates. Final purity was assessed by analytical size exclusion chromatography. Quality control analysis by Coomassie gel and size-exclusion trace is shown in FIGURE 10.

Cell Treatments, Lysis and Western blots: Hepal-6 cells were plated at 6 x 10 ⁵ cells per well of a 12-well plate in DMEM complete media. Where indicated, cells were treated with complete media only, 10 μg/mL recombinant P52 (described above), 10 μg/mL recombinant P36 (described above), or a combination of both for 30 min. EphrinAl-Fc (R&D Systems, Minneapolis, MN, USA) was supplemented at 1 μg/mL 10 min before cell lysis, alone or in combination with recombinant P52 or P36. Dasatinib (Cell Signaling Technology, Danvers, MA) was used at 200 nM. Cells were lysed in SDS lysis buffer (2% SDS, 50mM Tris-HCl, 5% glycerol, 5 mM EDTA, 1 mM NaF, lOmM β- glycerophosphate, 1 mM PMSF, 1 mM activated Na ₃ V0 ₄ , 1 mM DTT, 1% phosphatase inhibitor cocktail 2; Sigma-Aldrich, St. Louis, MO, USA), 1% PhosSTOP Phosphatase Inhibitor Cocktail Tablet (Roche, Indianapolis, IN, USA), filtered for 30 min at 4000 rpm through AcroPrep Advance Filter Plates (Pall Corporation, Port Washington, NY, USA) and stored at -80°C. Western blots were performed according to manufacturer instruction with the iBlot Dry Transfer System (Life Technologies, Carlsbad, CA, USA). Membranes were probed with antibodies against p-EphA2( clone D4A2) (pY772) (Cell Signaling Technology, Danvers, MA) at a dilution of 1 : 1000 and pActin (clone 8H10D10) (Cell Signaling Technology, Danvers, MA) at a dilution of 1 :2000 in a solution of 5% BSA and 0.1% TritonXlOO. Signals from immunoblots were detected using either an Alexa 680- conjugated anti-rabbit antibody or an Alexa 800-conjugated anti-mouse antibody used at 1 :7500 in the same solution as above (LI-COR Biosciences). Membranes were visualized using an Odyssey infrared imaging system (LI-COR Biosciences).

In vivo experiments in EphA2 ^("/_) mice: EphA2 ^("/_) mice were purchased from Jackson labs. WT controls were crossed with EphA2 ^("/_) mice, then F2 animals were generated by crossing heterozygotes and littermates were used as controls. Mice were infected i.v. with 10 ⁵ P. yoelii sporozoites via tail-vein injection. Animals were sacrificed at 42 h post-infection and liver tissue was harvested in TRIzol (Life Technologies, Carlsbad, CA, USA). Animal handling was conducted according to Institutional Animal Care and Use Committee-approved protocols.

Quantification of liver burden by real-time RT-PCR: Total RNA was extracted using TRIzol reagent. cDNA synthesis was performed using the QuantiTect Reverse Transcription Kit according to the manufacturer's instructions (Qiagen, Germantown, MD, USA). All PCR amplification cycles were performed at 95°C for 30 s for DNA denaturation, and 60°C for 4 min for primer annealing and DNA strands extension. Parasite 18S was amplified using primers with sequences: 5'- GGGGATTGGTTTTGACGTTTTTGCG-3 ' (SEQ ID NO:3) and 5'- AAGCATTAAATAAAGCGAATACATCCTTAT-3' (SEQ ID NO:4). Mouse GAPDH was amplified using sequences 5'-CCTCAACTACATGGTTTACAT-3' (SEQ ID NO: 5) and 5 ' -GCTCCTGGAAGATGGTGATG-3 ' (SEQ ID NO:6). For quantitative PCR (qPCR), a standard curve was generated using 1 :4 dilutions of a reference cDNA sample for PCR amplification of all target PCR products. Experimental samples were compared to this standard curve to give a relative abundance of transcript.

Quantification of liver stages, EphA2 level and PVM formation by FACS: For Cells were cultured as described above. Plasmodium yoelii infections, Hepal-6 cells were seeded at lxlO ⁵ cells/cm ² , and infected with P. yoelii sporozoites at an MOI of 0.3, 16-24 hrs following plating. For Plasmodium falciparum infections, HC04 cells were seeded lxlO ⁵ cells/cm2, and infected with P. falciparum sporozoites at an MOI of 0.3, 16-24 hrs following plating. At the desired time point, cells were detached using a Cell Dissociation Buffer, enzyme-free (Life Technologies). Unless specifically indicated, total, not surface EphA2 was monitored. Surface EphA2 was measured using an antibody against EphA2 (R&D Systems, Minneapolis, MN, Clone #233720) conjugated to PE in 2% BSA prior to permeabilization, after fixation with 4% PFA. Total mouse EphA2 was measured using an antibody against EphA2 conjugated to APC (R&D Systems, Minneapolis, MN, Clone #233720) following permeabilization with Perm/Fix solution (BD Biosciences) with 2% BSA in PBS. Permeabilized cells were blocked in Perm/Wash buffer (BD Biosciences Franklin Lakes, NT, USA) supplemented with 2% BSA. Additional staining steps were performed in the same buffer. Cells were stained using the monoclonal antibody (clone 2F6, for P. yoelii and 2A10 for P. falciparum) to circumsporozoite protein (CSP) conjugated to Alexa Fluor® 647 at a final concentration of 2 μg/mL. Human EphA2 was only monitored after permiabilization according to manufacturers' specifications. For PVM identification, P. yoelii parasites which express a UIS4-Myc fusion were detected using a human c-Myc antibody (clone 9E10) (R&D Systems, Minneapolis, MN, USA) according to manufacturer's specifications. Permeability dye from Invitrogen (LIVE/DEAD® Fixable Yellow Dead Cell Stain Kit, for 405 nm excitation) was used to manufacturer's specifications. All populations were identified by FACS. Analysis was performed on BD LSRII. Flow cytometric analysis was performed using FlowJo software (TreeStar). All experimental conditions were tested in biological triplicate. All data is representative of three independent experiments.

Statistical Methods: Unless otherwise indicated, p-values were determined using a two-tailed end t-test for samples with unequal variance.

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While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.