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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
  • C07K14/445—Plasmodium
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This invention is directed to compositions and methods for inhibiting hepatocyte invasion by malarial sporozoites. More specifically, the invention is directed to (a) ligands and mimetics thereof for the hepatocyte plasma membrane receptor for the circumsporozoite protein and peptides (and polypeptides) based on a portion of the circumsporozoite protein that constitutes an essential part of the specific ligand for this receptor; and (b) methods using such peptides to inhibit malaria sporozoite invasion of liver cells.
• Malaria is transmitted by the bite of the Anopheles mosquito. Minutes after infection, sporozoites (the mosquito- hosted stage of the malarial parasite) enter hepatocytes of the susceptible mammal where they multiply by schizogony and develop into exoerythrocytic forms ("EEF"). Except in highly endemic areas, the number of parasites inoculated by a single mosquito is small, probably below 100, but malarial infection has high efficiency. This coupled with the uniqueness of the target (the victim's liver cells) suggests that hepatocyte invasion is receptor-mediated. However, neither the structure of the receptors nor that of the ligands had been elucidated.
• the circumsporozoite protein a malarial stage- and species-specific protein that uniformly covers the surface membrane of sporozoites isolated from mosquito salivary glands, constitutes one of the main proteins expressed by mature infective sporozoites, and would be a candidate ligand for a hepatic cell receptor if such a receptor existed.
• CS protein The circumsporozoite (CS) protein has been extensively investigated (reviewed in Nussenzweig and Nussenzweig, Adv. Immunol. 45_:283-334, 1989) .
• the sequences of CS proteins from several malarial species have been elucidated and their main structural and antigenic properties which show substantial interspecies similarities have been described in Doolan et al. , Infect. Immunol. 0:675-682, 1992; Lockyer et al., Mol. Biochem. Parasitol. 37:275, 1989; De La Cruz et al. , J. Biol. Chem. 2_6_2:11925-11940, 1987; McCutchan et al.
• All CS proteins contain (i) a species-specific immunodominant repeat domain encompassing about one-half of their molecule; (ii) two pairs of cysteines in the C-terminal region, and (iii) two relatively short stretches of conserved amino acid sequences flanking the repeat domain.
• the N-terminal proximal conserved sequence (Region I) is the smaller of the two conserved regions and has been described in Dame, J.B. et al. , Science 225:593-599, 1984.
• One group of investigators reported that peptides corresponding to Region I bind to hepatocytes and that antibodies against this region inhibit invasion (Aley, S.B. et al. , . Exp. Med. 164,:1915-1921, 1986) but to the knowledge of the present inventors, there has been no follow up or independent confirma ⁇ tion of these studies.
• Region II The conserved sequence proximal to the C-terminal (Region II) surrounds the first pair of cysteines on the C- terminal side of the repeat domain.
• Region II was initially described by Dame et al. , supra. but has now been redefined by the present inventors and as redefined will hereafter be referred to as Region 11+.
• Region 11+ is highly homologous to a cell-adhesion domain of thrombospondin (Prater et al. , J. Cell. Biol. 112:1031-1040. 1991; Tuszynski, G.P. et al. , Exp. Cell. Res. 182:473-481.
• Region II as defined by Dame et al. did not prove to be immunogenic and was discarded as a candidate for a malaria vaccine. No function was attributed to it. CS regions adjacent to Region II, however, were shown to be immunogenic (See, e.g., U.S.P. 4,915,942). Region 11+ was redefined based on extensive homology, considering not only P. knowlesi and P. falciparum (as Dame et al. had done) but also considering many other malarial species. See. Table 1 below.
• CS proteins have been extensively investigated and a large amount of information has been accumu ⁇ lated on their structure, immunological properties and evolution, their function remains unknown.
• the participation of the CS protein in hepatocyte invasion has been suggested by the observation that Fab fragments of monoclonal antibodies against the repeats inhibit sporozoite infectivity in vitro and in vivo.
• the ligand (if any) recognized by hepatocyte receptors did not seem to be in the repeats, in view of the fact that sporozoites of different species (the CS proteins of which have different repeat units) infect the liver of the same host.
• Objects of the invention include the discovery of novel agents, including but not limited to ligands, receptor derived agents, and mimetics, and methods that are useful in inhibiting circumsporozoite protein binding to and sporozoite invasion of hepatocytes and in designing drugs and agents useful for the same purposes.
• a further object of the invention includes the isolation and/or identification of the hepatocyte CS receptor, cleavage products thereof, and the corresponding ligands.
• a peptide inhibitor for the binding of a circumsporozoite polypeptide to receptors of hepatocytes from malaria-susceptible mammals.
• the inhibitor has an amino acid sequence selected from the group consisting of:
• Region 11+ of a circumsporozoite protein the Region 11+ containing the subsequence CSVTCG;
• fragments of the Region 11+ containing at least a portion of the adhesion ligand for the receptors, the portion comprising at least one cysteine of the Region 11+;
• peptide constructs comprising (a) (i) or (ii) and (b) at least one other fragment of the amino acid sequence of the circumsporozoite protein, the constructs having no substantial ability to elicit the formation of antibodies recognizing the immunodominant epitope of the circumsporozoite protein.
• Peptide inhibitors also include dimers, multimers, and/or aggregates of any of the above, and particularly homo- dimers, -multimers, or -aggregates of any of the above, as well as structural, such as those having a similar three-dimensional configuration, or chemically functional mimetics thereof.
• a peptide inhibitor for the binding of a circumsporozoite polypeptide to basolateral plasma membrane of hepatocytes from malaria-susceptible mammals is provided.
• the inhibitor has an amino acid sequence selected from the group consisting of:
• Region 11+ of a circumsporozoite protein the Region 11+ containing the subsequence CSVTCG;
• fragments of the Region 11+ containing at least a portion of the adhesion ligand for the receptors, the portion comprising at least one cysteine of Region 11+;
• peptide constructs comprising (a) (i) or (ii) and (b) at least one other fragment of the amino acid sequence of the circumsporozoite protein, the constructs having no substantial ability to elicit the formation of antibodies recognizing the immunodominant epitope of the circumsporozoite protein.
• Peptide inhibitors also include dimers, multimers, and/or aggregates of any of the above, and particularly homo- dimers, -multimers, and/or -aggregates of any of the above. Additional inhibitors for the binding of a CS polypeptide or polypeptides as described above to a hepatocyte receptor in a malarial susceptible mammal are provided. These inhibitors comprise a cleavage product of a heparan sulfate proteoglycan from the surface of the hepatocyte. Structural and chemically functional mimetics thereof are provided as well.
• Also contemplated is a method of inhibiting the binding of a circumsporozoite polypeptide to hepatocytes susceptible to sporozoite invasion comprising: supplying to the environment of the hepatocytes the peptide inhibitor(s) or mimetic(s) above in an amount effective to inhibit the binding, no later than exposure of the hepatocytes to the circumsporozoite protein.
• Peptides consisting essentially of Region 11+ of the circumsporozoite protein and dimers, multimers and/or aggregates thereof, and mimetics thereof are also provided.
• a method of delivering or targeting a substance to hepatocytes is provided.
• the substance is combined with the inhibitors above, and the resultant complex is administered to the individual.
• Figure 2 Shows photomicrographs illustrating the binding of CS polypeptides to sinusoids in liver sections or to human hepatocyte cell line HepG2. Binding is revealed with anti-repeat MAb 2A10 followed by a conjugate of rat anti-mouse
• Panel 2A Binding of 12.5 ⁇ g/ml CS27IVC (control) ;
• Panel 2B Binding of 12.5 ⁇ g/ml falciparum-1;
• Panel 2C Inhibition of binding of CS27IVC by 250 ⁇ g/ml PfRII+;
• Panel 2E Binding of CSFZ to HepG2.
• Figure 3 Panel A shows photomicrographs of the binding of CS polypeptides to human hepatocyte microvilli within the Space of Disse (arrows or lateral membranes of adjacent hepatocytes (arrowheads) ) , but no binding in bile canaliculi (BC) or endothelia cells (EC) .
• FIG. 3 Panel B shows a higher magnification of the Space of Disse of 3A showing aggregates of CS polypeptide (CS27IVC) binding to hepatocyte microvilli. Binding is revealed as in Fig. 2 but with gold instead of fluorescein.
• Panel B binding of CS27IVC to rat liver -- aggregated gold particles are shown under lysosomes of K but not on K surface;
• Panel C binding of CS27IVC to .rat liver cell membrane fractions
• Panel D non-binding of CS27IVC to rat liver mitochondrium and rough endoplasmic reticulum.
• Figure 5 Top panel shows the FPLC elution profile of CS27IVC following passage through a Superose column. Ordinate: ODxlO "2 ; Abscissa: fraction number.
• Bottom panel shows aggregates of CS in fraction 9 and monomers of CS in fractions 12-14. Molecular weight markers shown on top.
• Figure 6 A graph showing binding of CS to HepG2 cells. Fluorescence indicates amounts of bound protein.
• Figure 7 A graph of percent inhibition by RII+ peptides of the binding of CSFZ (Cys) to HepG2 cells as a function of RII+ concentration.
• Figure 8a An electron micrograph of labeled human liver sections treated with chondroitinase.
• D Space of Disse; Arrows: lateral domain of the hepatocyte plasma membrane;
• Figure 9 A graphic illustration of the inhibition of CS binding to HepG2 cells by glycosa inoglycans expressed as percent inhibition v. concentration of glycosaminoglycan.
• Figure 10 A graphic illustration of the inhibition of CS binding to HepG2 cells by heparitinase digestion expressed as percent inhibition v. concentration of heparitinase.
• Figure 11a An electron micrograph of labeled rat kidney sections.
• BM basement membrane
• PT proximal tube
• Figure lib An electron micrograph of labeled rat binding sections.
• BC Bowman capsule Arrows: Laminae rarae CL: capillary lumen P: podocyte BS: Bowman Space
• Figure 13 A photograph of an SDS-PAGE.
• Figure 14 A graphic illustration of trypsin-release and CS-precipitation of HepG2 cell HSPG expressed as percent of total incorporated cpm released v. trypsin concentration.
• Figure 15 A graphic illustration of the ion exchange chromatography of trypsin-released HepG2 cell HSPG.
• Figure 16 A photograph of an SDS-PAGE.
• Figure 17 A photograph of an SDS-PAGE.
• Figure 18 A graphic illustration of molecular sieving chromatography of purified HSPG released by trypsin from HepG2 cells.
• Figure 19 A schematic representation of CS proteins.
• Figure 20 A graphic illustration of Vivax-1 and Vivax-2 binding to sulfatides and cholesterol-3-sulfate expressed as cpm v. concentration of peptide.
• Figure 21 A graphic illustration of the binding of Falciparum-2 to sulfatides but not to sulfatide analogues expressed as cpm v. concentration of peptide.
• Figure 22 A graphic illustration of the failure of Vivax-2 to bind the analogous of cholesterol-3-sulfate expressed as cpm v. concentration of steroid.
• Figure 23 A graphic illustration of the binding of
• Figure 24 A Western Blot of CS proteins bound to sulfatides.
• Figure 25 A graphic illustration of the inhibition of CS protein binding to sulfatide and cholesterol-sulfatide by reduction and alkylation expressed as cpm v. concentration of sporozoite equivalent.
• Figure 26 A graphic illustration of the inhibition of CS protein binding to sulfatide and cholesterol-sulfatide by reduction and alkylation expressed as cpm v. concentration of peptide.
• Figures 27A and B Photographs of the anti-peptide serum recognition of glutaraldehyde fixed sporozoites.
• Figure 28A A graphic illustration of the binding of CS27IVC to heparin sepharose.
• Figure 28B A photograph of an SDS-PAGE.
• Figure 29 A graphic illustration of the CS27IVC functions obtained by incubation with fixed HepG2 cells.
• Figures 30 A, B, and C Graphic illustrations of ligand clearance.
• Figures 31A, B and C Photomicrographs of mouse livers and kidneys stained with FITC labelled rtiAb 2A10.
• Figures 32A and B Photomicrographs of mouse liver after intravenous injection of CS27IVC.
• peptide as used herein includes polypeptides and the term polypeptides as used herein includes peptides.
• the structure of the CS receptor is of importance in drug design, one of the advantages of the present invention is that knowledge of the receptor structure is not required.
• the hepatocyte CS receptor and the corresponding ligand can serve as a basis for rational drug design and DNA or drug delivery. For example, the incorporation of the Region 11+ amino acid sequence into the envelope protein of a recombinant virus may enhance its capture by hepatocytes .
• the peptides or constructs (which include the dimers, multimers, or aggregates described above) of the invention should have no substantial ability to elicit the formation of antibodies recognizing the immunodominant epitope of CS that would diminish the effectiveness of the peptides or constructs.
• these peptides or constructs may be recognized by antibodies raised by conjugating the peptides or constructs to a separate immunogenic component.
• Recombinant P. ivax and P. falciparum proteins and polypeptide fragments thereof can be made, e.g. in accordance with now well-known recombinant techniques, see, e.g., Barr, P.J. et al., J. Exp. Med. 165_:1160-1171, 1987 and U.S.P. Nos . 4,997,647 and 4,880,734 and European Patent Publication No. 460716. Their sequences as well as other recombinant methods for making them have been published in Dame, J.B. et al.,
• Vivax-2 Includes the entire N-terminal moiety of P.vivax
• CS protein the repeats Region 11+ (as redefined by the present inventors) and 11 amino acid residues downstream from the end of the repeats through a leucine residue.
• Vivax-2 has no free sulfhydryl groups as determined by Ellman reac ⁇ tion.
• Falciparum-2 The corresponding fragment of P.falciparum CS protein,also terminating at the leucine residue
• Falciparum-2 also has no free sulfhydryl groups as determined by Ellman reaction.
• Vivax-1 Same as Vivax-2 minus most of Region 11+, termi- nating with the proline residue immediately preceding the first cysteine of Region 11+.
• Falciparum-1 Same as Falciparum-2 minus most of Region 11+, terminating with the proline residue preceding the first cysteine of Region 11+ (P.falciparum residues 43-348) .
• Region II in P.falciparum has been redefined by the present inventors as: E-W-S-P-C-S-V-T-C-G-N-G-I-Q-V-R-I-K
• the conserved motif EWXXCXVTCGXGXXXRXK/R encompassing a hepatocyte ligand for malaria sporozoites, is contained in the C-terminus of the CS protein.
• the recognition by the liver receptor requires the presence of the cluster of positively charged amino acids RXK/R at the N-terminus, and these peptides (RXK/R) specifically inhibit host cell invasion by sporozoites and binding of CS to its liver receptor.
• CSFZ(Cys) Consists of residues 27-123 plus NANP (a single instance) plus residues 300-411 of P.falciparum CS protein.
• P.falciparum derived peptides were synthesized as described in Houghten, R.A., Proc. Nat'l. Acad. Sci. USA 82:5131-5135, 1985.
• Pfl contains most of P.falciparum Region 11+.
• PflA Identical to Pfl except alanine residues are substituted for the two cysteines.
• PflB Identical to Pfl except the sulfhydryl groups on both cysteines have been blocked with acetamide groups.
• Pf2 Contains most of Region 11+.
• Pf2A Identical to Pf2 except the first cysteine has been replaced by an alanine residue.
• Pf25C A scrambled version of Pf2.
• Pf3 Contains only a C-proximal moiety of Region 11+.
• PbRII+ Contains Region 11+ from P.berghei.
• Pf70 Contains most of P.falciparum Region I.
• Pf4 Contains only a C-proximal moiety of Region 11+ except for a single amino acid deletion.
• Monoclonal antibody 2A10 (prepared according to Nardin, E.H. et al. , J. Ex . Med. 156:20-30, 1982) is directed against an epitope contained in the repeat - (NANP) n - region of P.falciparum CS protein and recognizes the amino acid sequence PDPNANPN found 5' of Region II in the repeat-less recombinant polypeptide CSFZ (Cys) (Burkot, T.R. et al., Parasite Immunol. 13:161-170. 1991).
• Monoclonal antibody 2E6 reacts with the liver stage of P.berghei. Such antibodies can be made by methods known to those skilled in the art.
• Polyclonal antisera against P.berghei CS Region II were raised by immunizing rabbits with peptide PbRII coupled to key hole limpet hemocyanin with glutaraldehyde.
• a rabbit was immunized once with 500 ⁇ g of the peptide-adjuvant conjugate in complete Freund's adjuvant and then boosted monthly (four times) with the same amount of conjugate in incomplete Freund's adjuvant.
• the antisera recognize the peptide PbRII+ dried onto plastic wells, and this binding is inhibited by soluble PbRII ⁇ . The difficulty of making such antisera testifies to the non- immunogenic character of Region 11+.
• the sections were first incubated with the peptide at various concentrations in BSA/TPBS for 1 hour at 37°C, washed six times with BSA/TPBS, and then stained with 2.5 ⁇ g/ml CS recombinant protein as described above. In a few experiments, the tissues were fixed for 10 min. with methanol, acetone, or 4% paraformaldehyde containing 0.5% glutaraldehyde.
• Rat or mouse liver tissue or hepatocyte subcellular fractions from Example 2 were fixed in PBS containing 1% glutaraldehyde (grade 1, Sigma, St. Louis, Missouri) and 4% paraformaldehyde (Kodak, Rochester, New York) , dehydrated in ethanol, and embedded in LR White (Polysciences, Warrington, Washington) . (Frevert et al. , Infect, and Immun. 60:2349-2360, June 1992.) Normal human liver was embedded in Lowicryl K4M (Ted Pella, Redding, CA) . Ultrathin sections were labelled by incubating them sequentially with 10-50 ⁇ g/ml CS27IVC, CSFZ
• HepG2 cells (ATCC number HB8065, Rockville, Maryland; Knowles, B.P., et al. , Science 2_09_:497-499, 1980) were grown on slides (Cel-Line Associates, Inc., Newfield, New Jersey) overnight in minimum essential medium with 10% fetal calf serum (FCS-MEM; GIBCO, Grand Island, New York) , 1 mM L-glutamine (GIBCO) , 3 mg/ml glucose (Sigma) , 1 x nonessential amino acids (GIBCO) , 50 ⁇ g/ml penicillin, and 100 ⁇ g/ml streptomycin (GIBCO) .
• MAb 2A10 at a concentration of 10 ⁇ g/ml for 30 min. and goat anti-mouse immunoglobulin conjugated to alkaline phosphatase (Boehringer Mannheim) for 30 min. Bound enzyme was revealed by the addition of the fluorescent substrate, 1 mM 4- methylumbelliferyl phosphate in 100 mM Tris-HCl, 100 mM NaCl, and 5 mM MgCl 2 (pH 9.5) . After 15 min., fluorescence was read in a Fluoroskan II plate reader (Flow Lab Inc., McLean, Virginia) with excitation filter 350 nm and emission filter 460 nm. In the peptide inhibition experiments, wells were first incubated for 1 hr.
• Superose 12 sizing column Pulacia
• the protein was eluted in the same buffer using a flow rate of 0.2 ml/min.
• a high molecular weight fraction (69kDa) co-eluted with thyroglobulin.
• sporozoites were preincubated in FCS-MEM alone, or FCS-MEM containing anti-Region II IgG, or with preimmune sera IgG for 30 min. at 4°C.
• Sporozoites (5 x 10 4 ) , in a volume of 100 ⁇ l, were added to each well. The medium was replenished after 2 hr. and changed after 18 and 28 hr. Each point was performed in quadruplicate.
• sporozoites were first incubated with the HepG2 cells for 2 hr in FCS-MEM.
• FCS-MEM containing the peptide at a concentration of 250 ⁇ g/ml (or the IgGs at a concentration of 700 ⁇ g/ml) was then added to the cultures, and incubation proceeded for two additional hours.
• FCS-MEM was changed after 18 and 28 hr. All cultures were fixed with cold methanol containing 0.3% H 2 0 2 after 48 hr. Wells were blocked with BSA/TPBS, incubated for 45 min. with 10 ⁇ g/ml MAb 2E6 directed against the exoerythrocytic forms of the parasite, washed three times with BSA/PBS, incubated for 45 min.
• CS27IVC Frozen sections of various rat organs were incubated with recombinant CS27IVC polypeptide. Tissue-bound CS was revealed with MAb 2A10 and fluorescence microscopy. Strong staining was observed in liver sections with concentrations of CS27IVC within the range 50-5 ⁇ g/ml. The results are shown in Fig. 2 Panel 2A. The staining closely followed the sinusoidal spaces of the hepatic lobules, indicating CS binding to the hepatocyte membrane. Other areas of the liver sections and sections of other organs (spleen, lung, heart or brain) were not stained. Control sections (incubated with CS27IVC in the absence of antibody or vice versa) were not stained either.
• CSFZ Cys
• Falciparum-2 which contains Region I and II as well as the repeats bound to the liver sections at 25 ⁇ g/ml;
• Peptides, PbRII+, Pfl and Pf2 all containing substan ⁇ tially Region 11+ inhibited the binding of CS27IVC with the inhibition being complete at 250 ⁇ g/ml of peptide PbRII+.
• Recombinant polypeptide Falciparum-1 which contains Region I and the repeats but not Region 11+ did not bind to liver sections even at concentrations as high as 250 ⁇ g/ml.
• the same assay system was used to evaluate the ability of the various synthetic peptides to inhibit the binding of CSFZ (Cys) to the HepG2 cells.
• Paraformaldehyde- fixed immobilized HepG2 cells were pre-incubated with peptides at concentrations between 0 and 250 ⁇ g/ml, washed, and incubated with CSFZ (Cys) at 2.5 ⁇ g/ml. After washing, the cells were incubated with MAb 2A10 followed by anti-mouse IgG conjugated to alkaline phosphatase. Bound enzyme was revealed by a fluorescent substrate, methylumbelliferyl phosphate. Each peptide concentration was assayed in triplicate wells and the means of the fluorescent reading were calculated.
• CS protein labels human lysosomes and also binds to the lateral hepatocyte cell membrane (arrowheads).
• M stands for mitochondrium.
• lysosomes (L) of Kupffer cells (K) are labelled but not the Kupffer cell surface nor the endothelial cell membrane (arrowheads) whereas the hepatocyte microvilli in the space of Disse are heavily labelled.
• a rat liver cell membrane shows labelling of those membrane fragments that contain microvilli whereas other membranes (possibly bile canaliculi shown by arrowheads) are not labelled and contaminating cell organ cells (*) are not labelled.
• rat cell liver fractions containing mostly mitochondria (M) and rough endoplasmic reticulum (arrowheads) are not labelled.
• Example 11 Aggregated, Region 11+-Containing CS
• HepG2 cells were incubated with P.berghei sporozoites in the presence of varying amounts of P.berghei peptide PbRII ⁇ or control peptides. The number of exoerythrocytic forms
• EEF EEF of the parasite that developed in the HepG2 cells were counted two days later. Because the viability and infectivity of sporozoites vary greatly, multiple experiments were performed so the results are statistically significant.
• HepG2 cells were plated at a density of 0.5 x 10 6 /ml, incubated for two hours with 50,000 P.berghei sporozoites per well in the presence of PbRII ⁇ or other control peptides, or simply media, as indicated in Table 2 below. Cultures were grown for two days, fixed and stained with MAb 2E6 followed by goat anti- mouse IgG conjugated to horseradish peroxidase.
• b P values were calculated using one-way analysis of variance (ANOVA) , corrected by the
• PbRII+ was effective in inhibiting EEF if it had been present during the initial phases of invasion. Two hours after addition of the sporozoites PbRII+ was no longer effective.
• Several rabbits were hyperimmunized with PbRII+ conjugated to keyhole limpet hemocyanin. The an iserum titer of the animals was high ( ⁇ 20,000 by ELISA) . Nevertheless, only one antiserum reacted weakly with sporozoites (1:1000 by indirect immunofluorescence) .
• the IgG fraction of this antiserum at 700 ⁇ g/ml significantly inhibited sporozoite invasion of HepG2 cells while preimmune IgG had no effect.
• b P values were calculated using one-way analysis of variance (ANOVA) , corrected by the
• CS in fact aggregated CS, recognizes specifically the basolateral domain of hepatocyte cell membrane. This specificity predicts the existence of a receptor on the hepatocyte cell surface.
• the ligand for this receptor (which is characterized below) resides within Region 11+ of the CS protein, as defined by the present inventors.
• the peptide Pfl an example of a peptide consisting essentially of Region 11+, competes very effectively with recombinant CS and (at micromolar concentrations) with the P.berghei sporozoites.
• the peptide Pf2 which lacks the amino acids PCSVT competes much less effectively indicating that the missing sequence is part of the adhesion ligand (nevertheless, peptide Pf2 also binds to the same site as the CS protein) .
• the cysteines in Region 11+ are also important because analogs of Pfl lacking only these cysteines were totally inactive.
• peptides containing essential parts, or the entirety, of Region 11+ can be easily identified using one or more of the above-described assays and the overlapping peptide method, which is a peptide screening technique well-known in the art and no more than routine experimentation.
• peptides formed by omitting progressively one-by-one C-terminal amino acids from Regions 11+ of different malarial species can be tested for CS- binding inhibitory activity. It has already been determined that the N-terminal and the positively charged amino acids of
• Region 11+ are important.
• peptides and peptide-containing constructs within the present invention include the following: (A) Peptides that inhibit the binding of CS protein
• CS protein includes recombinant CS polypeptides and entire sporozoites
• Such peptides should possess substantial CS-binding inhibitory activity (e.g., not substan ⁇ tially less than that of Pf2) and, if intended for use in vivo, should not elicit a substantial immune response from the host.
• Such peptides may be as small as the minimum CS- binding inhibitory amino acid sequence from Region 11+ or as large as CSFZ (Cys), i.e., consisting essentially of the CS- protein minus the immunodominant region.
• Such peptides should be soluble in aqueous media. Region II of Dame is excluded from the present invention.
• peptides within the invention that compete for binding with CS should preferably be dimeric, multimeric, or aggregated. This is consistent with the finding that CS aggre ⁇ gates bind to hepatocytes and CS monomers do not.
• Region 11+ is not itself immunogenic, immunogenic and non-immunogenic peptides incorporating it are nevertheless useful in inhibiting sporozoite invasion of hepatocytes.
• the absence of immunogenicity of Region 11+ can be used to advantage in vivo, because the host to whom such peptides are being administered will not mount an immune response against them.
• Region 11+-containing peptides or peptidomimetics can be administered to malaria susceptible subjects, for example intravenously, at sufficiently high concentrations to compete effectively with a subsequent challenge with sporozoites or to attenuate the severity of subsequent infection.
• concentrations can be determined by means known to one of ordinary skill in the art. For example, optimum concentrations can be established using serially diluted preparations of the peptide in connection with a suitable testing procedure in rodents injected with P. berghei or P. yoelii. Preferred concentrations range from about 1 to about lO g in a mouse and from about 10 to about 100 mg in a human.
• Suitable vehicles for administration include, but are not limited to, isotonic saline.
• the peptides of the present invention can also be encapsulated in liposomes, the encapsulation having been described by Brenner, D., J.M.C.I. 28 . :1436, 1989; Anderson, P. et al. , Cancer Research 50:1853, 1990; and Anderson, P. et al . , J. Immunotherapy 12 . :19, 1992.
• Peptides consisting of Region 11+ or of the ligand adhesion portion thereof, are not expected to be toxic to the malaria-susceptible mammalian hosts because they were not toxic when administered to mice.
• Region 11+ peptides Such administration of Region 11+ peptides would have to be preventive because they will have no effect on the blood stages or on the development of the liver stages of the parasite. Simultaneous infection and administration is about the limit of the CS-binding inhibitory effectiveness of Region 11+ peptides.
• Region 11+ peptides could be used to select monoclonal antibodies in vitro using the phage display technology ⁇ See, Barbas et al., Proc. Nat'l. Acad. Sci. U.S.A. 8J3:7978 (1991) . These can be human or humanized monoclonal antibodies. Human origin or humanization would cause the immune system of the host to be "blind" to the antibodies. If administered to travelers, these antibodies to Region 11+ would bind to the ligand adhesion site of the CS protein (or sporozoites) and thus prevent liver invasion through the hepatocyte receptor. Human chimeric and humanized antibodies of various predetermined specificities are engineered currently, See, e.g.
• Region 11+ The nonantigenic nature of Region 11+ precludes the use of peptides containing Region 11+ (and also containing an antigenic determinant) in vaccines. Nevertheless, peptides consisting essentially of the immunogenic amino acid sequences immediately following or preceding Region 11+ of the CS protein can be used to immunize susceptible hosts. (See, Good et al . , Annu. Rev. Immunol. . 6:663-688, 1988) . It seems likely that these antibodies would sterically hinder Region 11+, and prevent infection.
• such an antigenic C-proximal amino acid sequence is the sequence N K P K D Q L D Y Q N D I Q.
• Other examples of such antigen sequences include, but are not limited to:
• mimetics are typically non-peptide compositions that maintain the activity of the corresponding peptides because of structural and/or chemical functionality similarities.
• advantages of mimetics are their relative lack of antigenicity and their ability to withstand degradation to which peptides are susceptible.
• Region 11+ peptides would be useful in drug design, i.e. in the construction of peptidomimetic molecules (for use in chemoprophylaxis) that bind to the CS hepatocyte receptor with sufficient affinity to inhibit the subsequent binding of sporozoites.
• An in vitro assay system for this purpose has been described above in Examples 4 and 8- 12. It could employ for example HepG2 cells as targets and would test the ability of recombinant CS proteins to bind to their receptors in the presence or absence of a designed putative drug based on Region 11+. The drugs which inhibit this binding would then be tested for their effectiveness in inhibiting sporozoite invasion of HepG2 cells, and in rodent malaria models (P. berghei and P.
• Peptides consisting essentially of Region 11+, or its ligand adhesion subregions (see Table 1) , can also be used for such drug screening and are more convenient for this purpose than recombinant CS-constructs.
• Region 11+ derived peptides could be labelled and used exactly as the recombinant CS protein. The effectiveness of a drug would be accessed by its ability to inhibit the accumulation of the labelled Region 11+ peptide in the liver of mice or other animals.
• the peptides or mimetics above can be combined with a substance to be delivered to a hepatocyte.
• substances include, but are not limited to, DNA, such as genes, therapeutic agents such as drugs or other pharmaceutically active agents, or the like.
• the peptide or mimetic can be combined with the substance to form a complex through means known to those skilled in the art such as substitution, insertion, or conjugation with the peptide or mimetic. See, Mulligan, Science 260:926-932 (May 14, 1993); A.D. Miller, Hum. Gene Ther. 2:5 (1990) : N. Jones and T. Shenk, Cell 16:683 (1979); K.L. Berkner, BioTechniques 6:616 (1988); F.L.
• the peptide or mimetic can be incorporated into the envelope protein of a recombinant virus to enhance or achieve the capture of the recombinant virus by the hepatocyte.
• complexes will deliver or target the substance to the hepatocyte when administered to a mammal.
• the substance will be delivered in a single or a cumulative therapeutically effective amount which can be determined by means known to those skilled in the art such as by developing a matrix and assigning a dosage to each point in the matrix.
• Administration of these complexes can be by any manner known to those skilled in the art, including but not limited to, oral or parenteral administration.
• the Hepatocyte Membrane Receptor for CS Protein The existence of a receptor for the CS protein in the basolateral domain of hepatocyte plasma membranes has been shown in the experiments by the localization and specificity of the binding and the resemblance of the foregoing experimental results to other receptor-ligand interactions involving different proteins and their receptors.
• CS receptors or the basolateral domain of the plasma membrane of hepatocytes have been purified and identified as heparan sulfate proteoglycans (HSPG) of 400-700,000 Mr, which are tightly associated with the cell membrane. This characterization is based on observations made at the tissue, cellular, and molecular levels. Additionally, it has been determined that CS protein binds to the receptor via the particular motif CSVTCGXXXXXRXR. Previous work had suggested that the receptor was a sulfated glycoconjugate because the same motif, CSVTCGXXXXXRXR, determined the binding of the CS to sulfatide and cholesterol- 3-sulfate.
• HSPG heparan sulfate proteoglycans
• liver, kidney tissue, or HepG2 cells (ATCC HB8065 - Rockville, Maryland) metabolically labeled with carrier-free Na 2 35 S0 4 (Amersham - Arlington, Illinois) or with 35 S-methionine and cysteine (Tran 35 S-label; ICN - Costa Mesa, California) were used.
• carrier-free Na 2 35 S0 4 Amersham - Arlington, Illinois
• 35 S-methionine and cysteine Tran 35 S-label; ICN - Costa Mesa, California
• ligands for the putative receptors four recombinant CS proteins (Cerami et al. , Cell 70:1021-1033 , 1992) were used. Two of these were E.
• coli-derived Hoffmann- LaRoche: CS27IVC, which contains Region I, Region 11+ and a full representation of the repeat domain as is described above; CSFZ (Cys) , which is identical to CS27IVC, except that it contained only a single copy of the repetitive sequence NANP and is described above.
• the HepG2 binding assays were performed in Removawell tissue culture plates (Dynatech Laboratories - Chantilly, Virginia) . For the immunoelectron microscopy studies, glutaraldehyde (grade I - Sigma - St. Louis, Missouri) and paraformaldehyde (Kodak -
• Ultrathin sections were sequentially labeled with 10-50 ⁇ g/ml CS27IVC, 15 ⁇ g/ml mAb 2A10 and a 1:30 dilution of PAG15. Prior to immunolabeling, part of the sections were incubated for 120 min at 37°C with heparitinase and chondroitinase ABC (both ICN) using concentrations of 20 U/ml and 2 U/ml, respectively. Control specimens were incubated only with the gold markers or with mAb 2A10 and PAG15.
• lysis buffer A 1% Triton X-100, 100 mM NaCl, 50 mM Tris-HCl, pH 7.2, 1% bovine
• Adherent HepG2 cells labeled with Na 2 35 S0 4 as described above were washed 3x with cold PBS, and then incubated for 5 minutes at 4°C with PBS containing 0, 2.5, 5, 10, 20 or 40 ⁇ g/ml trypsin. The supernatants were removed, and 200 ⁇ g/ml soybean trypsin inhibitor (Sigma) was added. The trypsin- treated cells were then scraped off in PBS/BSA, and extracts were prepared in lysis buffer A as above.
• the Staph A cells were washed 3 times with the lysis buffer A, 2 times with lysis buffer B (10 mM Tris-HCl, pH 7.2, containing 100 mM NaCl and 0.5% NP40) , 1 time with 50 mM Tris-HCl, ph 7.2, and were processed in this buffer for electrophoresis under reducing and non-reducing conditions.
• 35 S0 4 -labeled immunoprecipitates were examined on either 5% or 3 to 20% gradient polyacrylamide gels (Laemmli, Nature 128:2009-2012. 1970). The gels were fixed with 10% glacial acetic acid and 30% methanol, impregnated for 30 min in 1 M salicylic acid, dried and exposed to Kodak X-Omat AR film at -70°C.
• the combined positive fractions from 3 column runs were pooled and were concentrated to a final volume of 1 ml. Part of this preparation was subjected to hydrolysis and amino acid and amino sugar analysis.
• amino acid analysis the hydrolysis was with 6N HC1 at 110°C for 22 hours, and for amino sugars with 4N HC1 at 100°C for 7 hours. Analysis was performed using Waters Maxima software, 510 pump, and 490 detector. A Waters Novapak C8, 15 centimeter column was used.
• HepG2 cells were deposited in 96-well Removawell tissue culture plates and allowed to grow overnight in MEM (Gibco) containing 10% FCS, 1 mM L-glutamine (Gibco) , 3 mg/ml glucose (Sigma) and lx non-essential amino acids (Gibco) .
• the HepG2 cells were fixed with 4% paraformaldehyde in TBS (50 mM Tris-HCl, pH 7.5, 137 mM NaCl, 2 mM KC1) , were washed 3 times with TBS, and were stored at 4°C in BSA/TBS until use.
• CSFZ (Cys) at 5 ⁇ g/ml was incubated with increasing amounts of the presumed inhibitors at 37°C for 15 min. Fifty ⁇ l of these mixtures were added to the cells, incubated for 1 hr at 37°C, washed 3x with TBS/0.05% Tween and then sequentially incubated with 50 ⁇ l of mAb 2A10 at a concentration of 10 ⁇ g/ml in TBS/BSA buffer for 30 min at 37°C, and 1:5000 dilution of goat anti-mouse IgG conjugated to alkaline phosphatase (Boehringer) for 30 minutes.
• Bound enzyme was revealed by the addition of the fluorescent substrate, 4-methylumbelliferyl-phosphate (Sigma) in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl and 5 mM MgCl 2 . After 15 minutes, the fluorescence was measured in a Fluoroskan II plate reader (ICN) .
• ICN Fluoroskan II plate reader
• HepG2 cells were treated with either heparitinase (Sigma) or chondroitinase ABC (ICN) before incubation with the CS.
• Heparitinase treatment was performed in 0.05 M acetate buffer, pH 6.0, containing 1 mg/ml BSA and 1 mM PMSF, 5 ⁇ g/ml leupeptin and pepstatin.
• Cells were treated for 3 hours at 37°C and then were washed 3x with TBS before protein was added.
• Chondroitinase ABC treatment was performed overnight at 37°C in 0.1 M Tris/HCl, pH 8.0, 0.03 M sodium acetate and 0.1% BSA, and washed 3x with TBS.
• Enzyme-treated cells were then incubated with CS as outlined above.
• FIGS. 8a and 8b are electron micrographs of Lowicryl K4M-embedded sections.
• the section of Figure 8a was treated with 2 U/ml chondroitinase ABC (ICN) for 2 hour at 37° before the immunolabeling.
• the typical CS label on the space of Disse (D) and on the lateral domain of the hepatocyte plasma membrane (arrows) and on lysosomes (L) was unaltered.
• Example 14 Inhibition of CS Binding to HepG2 Cells Proteoglycans were preincubated with 5 ⁇ g/ml of
• CSFZ(Cys) were added to paraformaldehyde-fixed HepG2 cells. Binding of CS to the HepG2 cells was revealed by mAb 2A10, followed by anti-mouse alkaline phosphatase-conjugated IgG. Bound enzyme was revealed by a fluorescent substrate, 4-methylumbelliferyl-phosphate. Each point represents the mean of triplicates. Percent inhibition was calculated by comparison to CSFZ(Cys) preincubated in medium alone.
• Figure 9 illustrates that the CS binding to HepG2 cells was inhibited by heparin, heparan-sulfate, dextran- sulfate and fucoidan, but not by chondroitin sulfate and dextran.
• HepG2 cells were preincubated with varying amounts of enzyme, washed and then incubated with 5 ⁇ g/ml of CSFZ(Cys) .
• CS binding was revealed as described in Example 14.
• Heparitinase and chondroitinase ABC were used at initial concentrations of 5 U/ml and 1 U/ml, respectively. As shown in Figure 10, heparitinase treatment prevented CS binding, while chondroitinase ABC treatment had no effect. Each point represents the mean of triplicates, and percent inhibition was calculated by comparison to cells that were not treated with enzyme.
• Electron micrographs of LR White-embedded rat kidney sections labeled with CS27IVC, mAb 2A10, and PAG15 were studied. A highly selective CS staining in the rat kidney in a pattern consistent with proteoglycan involvement was detected.
• Figure 11a illustrates that in the glomerulus, the CS binding was restricted to the basement membrane of the Bowman capsule (BC) and predominantly to the laminae rarae (arrowheads) , as well as to the proximal tubules, Henle loop, distal tubules, and collecting tubules.
• the glomerular basement membrane below the fenestrated capillary endothelium (arrows) was unstained;
• Figure lib illustrates that the epithelia of the distal tubule (DT) show CS label only on the laminae rarae of the basement membrane (BM) .
• the epithelia of the proximal tubule (PT) are also stained on their basolateral domains and lysosomes (L) .
• the apical microvilli and all the other kidney epithelia were negative.
• the proximal epithelia showed intracellular CS staining of the lysosomes.
• kidney sections were treated either with heparitinase or with chondroitinase ABC prior to CS staining as in Example 15. Again, only heparitinase abolished the staining.
• Example 17 CS Binding to Heparan-Sulfate Proteoglycans Extracts of cells metabolically labeled with 35 S0 4 were incubated for 30 minutes at 4°C with 32 ⁇ g/ml of recombinant CSFZ(Cys) or 64 ⁇ g/ml Falc-1, and were immunoprecipitated with mAb 2A10 and Staph A cells. From the total liver lysate, CSFZ(Cys) co-immunoprecipitated sulfated molecules which migrated as a smear on top of the SDS-PAGE. Immunoprecipitation with Falc-1 was negative.
• Sub-confluent HepG2 cells were incubated with various concentrations of trypsin for 5 minutes at 4°C. Sulfate label was released in the supernatant in a dose-dependent manner, and maximum release (50% of the total incorporated cpm) was reached at 20 ⁇ g/ml trypsin as illustrated in Figure 14. Independently of the trypsin concentration, about 80% of the released HSPG was co-immunoprecipitated by 32 ⁇ g/ml CSFZ(Cys) and 10.8 ⁇ g/ml mAb 2A10. Under the same conditions, Falc-1 was negative.
• the cell- associated, and the trypsin-released molecules ran similarly as high molecular weight smears on top of the gel, indicating that mild trypsinization of the HepG2 cells does not lead to extensive degradation of the proteoglycan core proteins (compare patterns in Figure 13 and in insert of Figure 17) .
• the cells were treated with 0-100 U/ml phosphatidylinositol- specific phospholipase C (PI-PLC) for 60 minutes at 37°C or with 0-500 ⁇ g/ml heparin for 10 minutes at 25°C, respectively. Neither treatment lead to the specific re-lease of sulfate- labeled molecules.
• PI-PLC phosphatidylinositol- specific phospholipase C
• HepG2 cells were metabolically labeled with 35 S-methionine and 35 S-cysteine for 3 hours. As revealed by SDS-PAGE, the HepG2 cell extracts contained a very large number of radiolabeled proteins. However, following mild trypsinization as above (20 ⁇ g/ml, 5 minutes 4°C) , no counts were released in the supernatant above background.
• 35 S0 4 -labeled trypsin-released proteoglycan (total supernatant) was precipitated by CSFZ(Cys) (32 ⁇ g/ml), Falc-2 (64 ⁇ g/ml) , CS27IVC (64 ⁇ g/ml) and Falc-1 (64 ⁇ g/ml) .
• the labeled molecules were incubated with CSFZ(Cys), CS27IVC, and Falc-2 at equivalent concentrations of Region 11+, and co- immunoprecipitated with the mAb 2A10.
• Subconfluent sulfate-labeled HepG2 cells were incubated with 20 ⁇ g/ml trypsin for 5 minutes at 4°C, and the supernatants were loaded onto an anion exchange chromatography
• Pharmacia mono-Q column These were eluted with a 0 to 2 M NaCl gradient in the presence of 7 M urea.
• the radiolabelled proteoglycan was eluted from the column as a sharp peak at a NaCl concentration of 0.6 to 0.8 M as illustrated in Figure 15, and about 80% of the counts in this peak were co- immunoprecipitated by CSFZ(Cys). Protein was not detectable in the labeled peaks.
• One ml fractions were collected and counted.
• Figure 17 illustrates that heparin, but not chondroitin sulfate, inhibited co-immunoprecipitation.
• Heparitinase and pronase, but not chondroitinase ABC degraded the CS receptors.
• CSFZ(Cys) 32 ⁇ g/ml precipitated the purified proteoglycans as a high molecular weight smear (lane 1) .
• Heparin (lane 2) but not chondroitin sulfate (lane 3) inhibited the precipitation.
• Pronase (lane 4) and heparitinase (lane 5) but not chondroitinase ABC (lane 6) , degraded the high molecular weight band.
• Thyroglobulin (669 kDa) , apoferritin (443 kDa) , jS-amylase (200 kDa) and alcohol dehydrogenase (150 kDa) were used as molecular weight markers. Fractions (500 ⁇ l) were collected and counted. Fractions 13-22 (19,000 cpm, derived from 7xl0 7 HepG2 cells), and subjected to amino acid with hexosamine analysis. Results are shown in Table 4. On the basis of the amino acid analyses, the sample contained 2.8 ⁇ g of the proteoglycan.
• Examples 13-20 indicate that the binding of CS constructs to tissue sections and to HepG2 cells is specifically inhibited by heparitinase treatment of the cells, and by the presence of heparin in the incubation medium.
• the CS receptors are sulfated molecules of 400 - 700,000 Mr, which migrate as a smear on SDS polyacrylamide gels and are digested by heparitinase. The size of the individual GAG chains, and of the core protein, remain to be determined. After pronase digestion, the sulfated molecules are significantly reduced in size to about 200,000 Mr, perhaps representing remnants of the core protein linked to multiple GAG chains.
• Biosynthetically labeled, sulfated molecules are rapidly removed from the surface membrane of HepG2 cells by low concentrations of trypsin, and more than 80% of the labeled released molecules are precipitated with the CS .
• the 35 S0 4 - labeled molecules from the membrane of HepG2 cells were purified by two chromatographic steps. By molecular sieving chromatography in the presence of 7 M urea, their apparent molecular weight is between 400 and 700 kDa, and on a molar basis they contain 40% glucosamine, the amino sugar found in heparan sulfate GAG chains.
• the smaller amounts of galactosamine may be derived from 0-linked oligosaccharides, or from chondroitin sulfate GAG chains, known to be present in syndecans.
• the amino acid analysis reflects only the composition of the trypsin-resistant portion of the core protein and reveals the presence of a high content of serine and glycine.
• GAGs are known to be assembled on serine residues of the core protein, frequently within the amino acid sequence acidic-X-Ser-Gly-acidic (Bourdon et al. , P.N.A.S. USA, 84:3194- 3198, 1987; Zimmerman and Ruoslahti, EMBO J. 1:2975-2981, 1989.
• HSPGs are ubiquitous constituents of mammalian cell surfaces, and they may behave as integral membrane proteins (Brandan and Hirschberg, J. Biol. Chem. 26_4:10520-10526, 1989; Kjellen et al., J. Biol. Chem. 2_55:10407-10413, 1980; Kjellen et al., P.N.A.S. USA. 21:5371-5375, 1981; Stow et al. , J. Cell. Biol. 100:975-980, 1985) , or peripheral membrane proteins which can be released from the cell surface by treatment with heparin or high ionic strength (Brandan and Hirschberg, J. Biol. Chem.
• CS receptors share some properties with the syndecan family of HSPGs.
• Circumsporozoite proteins The yeast-derived recombinant CS proteins Vivax-1 and -2, Falciparum-1, and -2 were obtained from the Chiron Corporation, Emeryville CA.
• Figure 19 illustrates the recombinant proteins Vivax- 2, Falciparum-2, Vivax-1 and Falciparum-1 and their relation to the entire CS protein.
• the bottom half of the panel shows the sequence of Region II from P. vivax. P. falciparum and P. berghei. Amino acids shared by all three proteins are enclosed in boxes.
• P. berghei sporozoites Partially purified salivary gland P. berghei sporozoites were extracted for 10 minutes at room temperature with 0.5% NP-40, 150 mM NaCl, 25 mM Tris pH 8.8, and protease inhibitors: ImM PMSF, 0.25 ⁇ g/ml Pepstatin, 5 ⁇ g/ml Leupeptin, 5 ⁇ g/ml Aprotinin, 5 ⁇ g/ml Antipain (Boehringer Mannheim - Indianapolis, IN) . After centrifugation, the supernatants were used in the solid phase binding assay described below. In some experiments, extraction was performed in the presence of 50 mM iodoacetamide (IA) to alkylate any free sulfhydryl groups in the CS protein.
• IA iodoacetamide
• Vivax-2 and P. berghei CS proteins were respectively reduced with 10 mM or 75 mM dithiothreitol (DTT) for 30 minutes at room temperature and alkylated with excess IA for 30 minutes at room temperature.
• DTT dithiothreitol
• the proteins were then passed through a Sephadex G-25 column to remove DTT and IA, and then assayed for binding to the various lipids and steroids.
• Antibodies The following monoclonal antibodies were used: 2F2, 2A10, and 3D11 (Yoshida et al. Science 207:71-73. 1980; Nardin, et al. J. Exp. Med. 156:20-30, 1982), which recognize the repeat-containing domains of P. vivax. P. falciparum. and P. berghei CS proteins respectively, and 2E6, which recognizes the liver stage of P. berghei.
• the first six amino acids of the peptide consist of a motif shared by thrombospondin and Region II of the CS proteins.
• Steroids, glycolipids and anionic polymers were obtained from Sigma (St. Louis, MO), except for 5-cholenic-3-sulfate.
• Solid phase binding assays Thirty ⁇ l of lipids or steroids dissolved in methanol (20-250 ⁇ g/ml ) were dried into wells of microassay plates (Cat.# 3911 - Falcon - Oxnard, CA) . The plates were then blocked with 1% BSA/PBS pH 7.4 (BSA/PBS) for 30 minutes.
• BSA/PBS BSA/PBS pH 7.4
• Thin layer chromatography The purity of the various lipids and steroids was analyzed by thin layer chromatography (plates 7011-04 - J.T. Baker - Phillipsburg, NJ) using the solvent system CHCl 3 /MeOH/conc.NH 4 OH, 80/20/0.4.
• SDS-PAGE and Western blotting SDS-PAGE was performed in 10% slab gels.
• the gel contents were electrophoretically transferred to Immobilon (Millipore Corp. - Bedford, MA (Towbin, et al. P.N.A.S. USA 26:4350-4354, 1979.
• TlTe membrane was then blocked for 1 hour with BSA/PBS, incubated for 1 hour with specific antibodies (mAbs or rabbit antisera), washed 3 times with PBS/0.5% Tween-20 and then incubated with gcat anti-mpuse or anti-rabbit Ig coupled to alkaline-phosphatase (Sigma) for 1 hour.
• the bound enzyme was developed with bromochlorophenol blue and nitrotetrazolium blue in 0.1 M Tris, 0.1 M NaCl, 5 mM MgCl 2 .
• Infection assay Semiconfluent monolayers pf HepG2 cells were grown in 10% FCS/MEM on 8 chamber slides (#4808 Lab-tek - Naperville, IL) for 24 hours prior to each experiment.
• Sporozoites were pre-incubated in media alone or with the various polymers for 15 minutes at 4°C. 5xl0 4 sporozoites, in a volume of 10 ⁇ l, were added per chamber. After 4 hours of incubation at 37°C, the medium containing the sporozoites was gently aspirated, and 0.5 ml MEM/10% FCS was added. Fresh medium was added at 18 hours and 28 hours, and then cells were fixed with methanol containing 0.3% H 2 0 2 at 48 hours. After fixation, the wells were blocked with 10% FCS/PBS and were incubated with 10 ⁇ g/ml of mAb 2E6.
• Binding of the mAb was detected with a pclyclonal goat anti-mouse Ig conjugated to horseradish peroxidase.
• the bound enzyme was revealed with 1 mg/ml 3,3' -diaminobenzidine in 50 mM Tris pH 7.6, 0.01% H 2 0 2 .
• the parasites in each well are counted under a 40X light microscope objective.
• P. berghei and P. falciparum sporozoites were dissected from mosquito salivary glands, fixed with 0.1% glutaraldehyde, and then dried into the wells of IFA slides. The sporozoites were incubated with polyclonal rabbit anti-CSVTCGSGIRVRKRKGSNKKAEDL sera (diluted 1:20 in PBS) for 1 hour at 37°C, washed with PBS, incubated with FITC-labeled goat anti-mouse in PBS for 1 hour at 37°C. Circumsporozoite reaction: The P.
• berghei sporozoites were incubated for 30 minutes at room temperature with 5 ⁇ g/ml of mAb 3D11 in 10%FCS/PBS, in the presence or absence of 50 ⁇ g/ml dextran sulfate, and scored for the typical precipitate formation under the light microscope.
• Iodination The labelling of mAbs with 125 I was performed with Iodpgen (Pierce, Rockford, IL) according to the manufacturer's instructions.
• Vivax-2 bound to sulfatide and to cholesterol-3-sulfate, but not to the asialoganglioside or the galactocerebroside I and II.
• Falciparum-2 like Vivax-2, specifically bound sulfatide and not trisialoganglioside, monosialogangliosides- GM1 or GM2, galactocerebrosides type I or II, disialogangliosides-GDla or GDlb, or cholesterol. Falciparum-1
• Vivax-2 bound to cholesterol-3-sulfate, and the binding increased proportionally to the amount of plastic- immobilized ligand. No binding was observed with the following analogues: cholesterol, 5-cholenic acid-3/3-ol-sulfate, 5- cholenic acid-3/3-ol, lithocholic acid-3-sulfate, lithocholic acid, androsterone sulfate (5-Androsten-3/3-ol-17-one sulfate) and dehydroepiandrosterone (5-Androsten-3/?-ol-17-one) .
• Example 25 Western Blot of CS Proteins to Sulfatides
• the P. berghei CS protein usually appears as 2 bands.
• the band of lower Mr is the membrane form, and the band of higher Mr may represent an intracellular precursor.
• Yoshida et al. J. Exp. Med. 154:1225-1236. 1981.
• NP-40 extracts of P. berghei sporozoites and Falciparum-2 were prepared in the presence of protease inhibitors and alkylating agents, and were incubated with sulfatide coated wells.
• the bound material was eluted with SDS sample-buffer and subjected to Western Blotting in the presence (lanes 1-3) and in the absence (lanes 4 and 5) of 2-ME. This material was also run on 10% SDS PAGE, transblotted, and revealed with rtiAbs 3D11 or 2A10, followed by goat anti-mouse linked to alkaline phosphatase.
• Vivax-2 (.12 ⁇ g/well) and P. berghei sporozoite extract (35,000 sporozoite equivalents/well) were mixed with inhibitor as illustrated in Table 5, and were incubated in wells coated with 1.4 nmol/well of sulfatide or cholesterol-sulfate. Binding was revealed with either mAb 2F2 or mAb 3D11, followed by iodinated protein A. Percent inhibition is based on the results of sextuplicate wells and calculated by comparison with wells in which no inhibitor was added.
• VIVAX-2 CHOLESTERO - DEXTRAN-SULFATE l ⁇ g/ml 12.2
• CS protein 10 ⁇ g/ml 63 lOO ⁇ g/ml 79 The binding of Vivax-2 and of the native P. berghei protein to sulfatide was inhibited by dextran-sulfate. Fifty percent inhibition of binding was achieved at dextran-sulfate concentrations between 5 and 10 ⁇ g/ml. The inhibition was specific, and not simply a consequence of electrostatic interactions.
• chondroitin-sulfate A chondroitin-sulfate A
• chondroitin-sulfate C dermatan sulfate
• heparan- sulfate keratan-sulfate
• hyaluronic acid had no effect at concentrations of 1 mg/ml
• chondroitin-sulfate B was only slightly inhibitory at 1 mg/ml.
• Example 28 Region 11+ of the CS Protein Is Exposed on the Surface of Sporozoites
• HepG2 cells were plated at a density of 0.5xl0 6 , infected with P. berghei sporozoites (50,000/well) in the presence of dextran, dextran-sulfate (molecular weight 5,000), chondroitin-sulfate A, heparan-sulfate or media alone and then incubated for 48 hours.
• the cells were fixed and stained with mAb 2E6, followed by goat-anti-mouse linked to horseradish peroxidase. Results are illustrated in Table 6. Numbers represent schizonts counted/20 fields under 40 X magnification in a light microscope.
• the CSP reaction was performed on the treated parasites. This reaction is only observed in infective sporozoites, and can be used as a crude measurement of viability. Sporozoites which had been incubated for 1 hour at 37°C in 10% FCS/PBS containing 50 ⁇ g/ml of dextran sulfate, developed CSP reactions in the same proportion and with similar intensity as sporozoites incubated in medium alone.
• proteins with seemingly unrelated functions such as the adhesive molecules thrombospondin and von Willebrand factor; the complement protein properdin; PyPSSP2, another sporozoite protein; terminal complement components F-spondin and UNC-5; the leech anticoagulant antistasin, the coat proteins of sporozoites (the CS protein) and of Herpes simplex I virus; and two non-characterized proteins, TRAP from the blood stage of malaria parasites and EtHL6 from Eimeria.
• proteins with seemingly unrelated functions such as the adhesive molecules thrombospondin and von Willebrand factor; the complement protein properdin; PyPSSP2, another sporozoite protein; terminal complement components F-spondin and UNC-5; the leech anticoagulant antistasin, the coat proteins of sporozoites (the CS protein) and of Herpes simplex I virus; and two non-characterized proteins, TRAP from the blood stage of malaria parasites and EtHL6 from Eimeria.
• Examples 21-29 indicate that the motif itself binds both to sulfatide and to cholesterol-3-sulfate. As shown in Table 5, dextran sulfate inhibited sulfatide binding to CS at much lower concentrations than those required to inhibit cholesterol-3-sulfate binding, perhaps reflecting the lower avidity of the latter compound for CS.
• glycosammoglycan chains in different organs may depend upon both their structural features and their availability to the relevant ligands.
• Cleavage products of the heparan sulfate proteoglycans are typically prepared by trypsin or protease cleavage or degradation. These cleavage products are not the same as the entire proteoglycan but retain the ability to bind the ligands or mimetics described above.
• the monoclonal antibody mAb 2A10 (Nardin et al., J. Exp. Med. 156:20, 1982) is directed against an epitope contained in the (NNP) n repeat domain of the P. falciparum CS.
• mice Balb/C males, from Taconic Farms, weighing between 15 and 20 grams were used.
• Fluorescein Isothiocyanate (FITC) Labeling of mAb 2A10 The antibody labeling was performed as in Harlow et al. , Antibodies, A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Laboratories (1988) .
• red blood cells Mouse blood (200 ⁇ l) was collected from the retroorbital sinus, and washed with phosphate buffered saline (PBS) , pH 7.4 containing 1% BSA (BSA/PBS) . The RBC pellet was incubated with 10 ⁇ Ci of Na 51 Cr0 4 for 30 minutes, and then washed with BSA/PBS.
• PBS phosphate buffered saline
• BSA/PBS 1% BSA
• the free iodide was removed by filtration in Sephadex G-25 (Isolab Inc., Akron, Ohio) , and dialysis against 50 mM Tris, 75 mM NaCl, pH 7.4 (buffer A) .
• CS27IVC Multimers Radiolabeled CS27IVC was applied to a 1 ml Heparin-Sepharose column (Sigma Chemical C, St. Louis, MO) which had been preequilibrated with 50 mM Tris, 75 mM NaCl, 1% BSA, 0.05% Tween-20 (Biorad, Hercules, Ca) pH
• HepG2 cells 10 5 HepG2 cells (ATCC number HB8065, Rockville, Maryland) were deposited in 96 microtiter wells of Removawell plates (Dynatech Laboratories, Chantilly, VA) and grown overnight in minimum essential medium with 10% fetal calf serum (Gibo, Grand Island, NY) , ImM L- glutamine (Gibco) , 3 mg/ml glucose (Sigma) , 1 x nonessential amino acids (Gibo) , 50 ⁇ g/ml penicillin, and 100 ⁇ g/ml streptomycin (Gibco) .
• the cells were fixed with 4% paraformaldehyde, washed three times with TBS (25mM Tris-Cl, pH 7.4, 138mM NaCl) , and stored at 4°C in buffer B.
• TBS 25mM Tris-Cl, pH 7.4, 138mM NaCl
• the wells were incubated with serial dilutions of the iodinated proteins for 1 hour, washed three times with buffer B, and counted in an LKB gamma counter. Cerami et al. , Cell 2 ⁇ :1021 (1992)
• mice were anesthetized with ether, and injected with 10 5 cpm of 125 I-labeled CS27IVC or Falc-1 via the periorbital sinus. At two, five and fifteen minutes after injection, the mice were exsanguinated, the organs removed, rinsed in TBS, blotted dry on filter paper, and counted for radioactivity. To estimate the amounts of blood contaminating the various organs, we repeated the same procedure in mice injected with 100 ⁇ l (1.5xl0 5 cpm) of 51 Cr-labeled RBC. The mean volumes of blood in the liver, spleen and kidney of the exsanguinated animals were 75, 24, and 35 ⁇ l respectively. In all other organs, the amount of blood was negligible, i.e., less than 15 ⁇ l. The percent of injected dose of CS which was retained in the various organs was calculated as described as follows:
• % retained cpm corrected cpm per organ x 100 total injected dose
• mice were injected i.v. with 100 ⁇ g of unlabeled CS27IVC or Falc-1 via the retroorbital plexus.
• the mice were exsanguinated five minutes after injection, the organs removed, rinsed in TBS, blotted dry, and then snap frozen in liquid nitrogen for light microscopy.
• electron microscopy the organs were cut into small pieces and fixed in a mixture of 0.1% glutaraldehyde and 4% paraformaldehyde.
• the frozen tissue was embedded in Tissue Tek O.C.T. (Miles Inc., Naperville, Illinois) and cut into 5 ⁇ m sections. Sections were dried for 30 minutes, fixed for 10 minutes in 4% paraformaldehyde, and either used immediately or stored at 4°C in PBS containing 1% BSA, 0.5% Tween-20. After blocking with the same buffer, the sections were incubated with mAb 2A10 conjugated to FITC, and examined in a fluorescence microscope.
• Electron Microscopy The fixed liver specimens were dehydrated in ethanol and embedded in Lowicryl K4M at -20°C (Frevert et al. , Infect, and Immun. 0:2349 (1992)). After UV polymerization, sections were cut with a RMC MT-7 ultramicrotome and stained by sequential incubation with 15 ⁇ g/ml mAb 2A10 and protein A gold 10 nm (PAG10) (1:30, Amersham, Arlington, Illinois). Cerami et al. , Cell 70:1021 (1992) . Control sections were strained only with the gold conjugate. Photographs were taken with a Zeiss EM 910 electron microscope.
• CS multimers bind to the GAG chains of HSPG and were isolated by affinity chromatography on heparin-sepharose.
• 125 I-labeled CS27IVC was applied to a heparin- sepharose column pre-equilibrated in buffer A. The column was washed with 5 ml of the same buffer, and the bound CS27IVC was eluted with 50 mM Tris, 1.5 M NaCl, pH 7.4. The ordinates represent the cpm in 10 ⁇ l of each fraction ( Figure 28A) . Ten ⁇ l of selected fractions were run on 10% SDS-PAGE under non ⁇ reducing conditions, the gel was dried and subjected to radioautography (Figure 28B) .
• CS27IVC As shown in Fig. 28A, about 30% of the radiolabeled CS27IVC bound to the heparan-sepharose column, and the bound molecules were eluted with a buffer containing a high salt concentration. SDS-PAGE analysis under non-reducing conditions showed that the break-through peak contained only CS monomers, while the heparin-binding material contained various CS multimers or aggregates as well as monomers (Fig. 28B) . Under reducing conditions, a single band corresponding to a CS monomer was detected in proteins under both peaks, indicating that the CS multimers consisted of mixed aggregates of disulfide linked dimers, trimers, etc. and non-covalently bound monomeric CS.
• Fig. 29 shows that the CS27IVC fractions obtained by filtration in sizing columns (Cerami et al. , Cell 70:1021 (1992)), and by affinity chromatography on heparin-sepharose have similar properties, i.e., only the CS multimers or aggregates bind to HepG2 cells.
• Example 31 Clearance of CS27IVC and Falc-1
• Example 32 The CS27IVC Aggregates Accumulate on the Microvilli of Hepatocytes
• mice were injected with 100 ⁇ g CS27IVC or Falc-1, and 5 minutes later they were killed. The livers and kidneys were removed, washed in TBS, blotted dry and snap frozen in liquid nitrogen. Frozen tissues were stained with mAb 2A10 conjugated to FITC.
• cleavage products of the heparan sulfate proteoglycan of an hepatocyte or a mimetic thereof can be utilized to target or deliver a substance to an hepatocyte as described above.

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