WO1994006464A1 - Compositions et procedes d'inhibition de l'invasion des hepatocytes par les sporozoïtes malariques - Google Patents

Compositions et procedes d'inhibition de l'invasion des hepatocytes par les sporozoïtes malariques Download PDF

Info

Publication number
WO1994006464A1
WO1994006464A1 PCT/US1993/008800 US9308800W WO9406464A1 WO 1994006464 A1 WO1994006464 A1 WO 1994006464A1 US 9308800 W US9308800 W US 9308800W WO 9406464 A1 WO9406464 A1 WO 9406464A1
Authority
WO
WIPO (PCT)
Prior art keywords
region
peptide
circumsporozoite
protein
binding
Prior art date
Application number
PCT/US1993/008800
Other languages
English (en)
Inventor
Carla Cerami
Ute Frevert
Photini Sinnis
Victor Nussenzweig
Original Assignee
New York University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by New York University filed Critical New York University
Priority to AU51307/93A priority Critical patent/AU5130793A/en
Publication of WO1994006464A1 publication Critical patent/WO1994006464A1/fr

Links

Classifications

    • 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
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • 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 (a) to ligands for the hepatocyte plasma membrane receptor for the circumsporozoite protein and peptides based on a portion of the circumsporozoite protein that constitutes an essential part of the specific ligand for this receptor; and (b) to methods using such peptides to inhibit circumsporozoite invasion of liver cells. BACKGROUND OF THE INVENTION Malaria is transmitted by the bite of the Anopheles mosquito.
  • 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 ⁇ v- 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 and constitutes one of the main proteins expressed by mature infective sporozoites would be a candidate ligand for a hepatic cell receptor if such a receptor existed.
  • the circumsporozoite (CS) protein has been extensively investigated (reviewed in Nussenzweig and Nussenzweig, Adv.
  • 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.
  • 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 22_5:593-599, 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., J. Exp. Med.
  • Region II The C-terminal proximal conserved sequence (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.
  • Objects of the invention include the discovery of novel agents 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, especially for chemo-prophylaxis as well as immuno-prophylaxis against malaria.
  • 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+;
  • 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:
  • 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.
  • a method of inhibiting the binding of a circumsporozoite polypeptide to hepatocytes susceptible to sporozoite invasion comprising: supplying to the environment of the hepatocytes an amount effective to inhibit the binding of the peptide inhibi- cor(s) above, no later than exposure of the hepatocytes to the circumsporozoite protein.
  • a peptide consisting essentially of Region 11+ of the circumsporozoite protein is also provided.
  • Figure 1 is a schematic representation of various CS recombinant polypeptides.
  • 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 IgG conjugated to fluorescein isothiocyanate.
  • Panel 2A Binding of 12.5 ⁇ g/ml CS271VC (control) ; Panel 2B Binding of 12.5 ⁇ g/ml falciparu -1; Panel 2C Inhibition of binding of CS271VC by 250 ⁇ g/ml PfRII+; Panel 2D No inhibition of binding of CS271VC by 50 " 0 ⁇ g/ml Pf3; 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 D non-binding of CS27IVC to rat liver mitochondrium and rough endoplasmic reticulum.
  • FIG. 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.
  • P.vivax 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., Science 225:593-599. 1984; and McCutchan, T.F. et al. , Science 230:1381- 1383, 1985.
  • 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 Ell an reac ⁇ tion.
  • Falciparum-2 The corresponding fragment of P.falciparum CS protein,also terminating at the leucine residue (P.falciparum CS residues 43-391) . 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 of preceding the first cysteine of Region 11+ (P.falciparum residues 43-348) .
  • Region 11+ 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
  • Regions 11+ for other malarial species and the extensive homology among them are shown in Table 1 below.
  • CS27IVC Consists from N- to C-terminal of residues 27-123 plus (NANPNVDP) 3 plus (NANP) 21 plus residues 300- 411 of the P.falciparum CS protein. See. Takacs et al., J. Immunol . Meth. 143:231-240. 1991; Hochuli et al . , J. Chromat. 411:177-184, 1987.
  • 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 constructed 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 substitut- ed 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.
  • Pf Contains only a C-proximal moiety of Region 11+ except for a single amino acid deletion.
  • Antibodies Monoclonal antibody 2A10 (prepared according to Nardin,
  • 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+.
  • Rats were euthanized with C0 2 . Small pieces of the liver, spleen, heart, brain and lung were removed, snap frozen in liquid nitrogen, embedded in Tissue Tek O.C.T. (Miles, Inc., Naperville, Illinois), and cut into 5 ⁇ m sections. Sections were dried for 30 mm, fixed for 10 min.
  • 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 I mun. 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 CS271V, CSFZ (Cys) , Falc-2, or Falc-1 for 30 min.; 15 ⁇ g/ml MAb 2A10 for 30 min.; protein A-gold 15 nm (PAG15, 1:30; Amersham, Arlington, Illinois) or goat anti-mouse IgG gold 10 nm (GAM10, 1:30; Amersham) for 30 min. Control specimens were incubated in the absence of CS and only with the gold conjugates. Photographs were taken with a Philips EM 301 electron microscope.
  • HepG2 cells (ATCC number HB8065, Rockville, Maryland; Knowles, B.P., et al. , Science 209:497-499, 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 penicil ⁇ lin, 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, l mM 4- methylumbelliferyl phosphate in 100 mM Tris-HCl, 100 mMNaCl, 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. at 37°C with peptides diluted in gelatin/TPBS, washed three times with gelatin/TPBS, and then incubated with recombinant CSFZ
  • Example 6 Western Blotting of Recombinant CS Polypeptides Western blotting was conducted as described by Towbin, H.T. et al., Proc. Natl. Acad. Sci. USA 7£:4350-4354, 1979. Aliquots of the fractions obtained from the FPLC analysis were run on a 7.5% SDS-polyacrylamide gel under nonreducing conditions and electrophoretically transferred to Immobilon membrane
  • Falciparum-1 at a concentration of 1 mg/ml was incubated with 250 mM Traut's reagent (Pierce Chemical Co., Rockford, Illinois) in 50 mM triethylamine-hydrochloric acid (TEA-HC1) (pH 8.0), l mMMg(Ac) 2 , and 50 mM KC1 for 20 min. on ice.
  • Traut's reagent 0.5 M stock was prepared immediately before use in a solution containing equal volumes of 1 M TEA-HC1 and 1 M TEA-free base.
  • 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
  • FCS-MEM 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 MAb2A10 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. 2A. The staining closely followed the sinusoidal spaces of the hepatic lobules, indicating CS binding to the hepatic 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. The same pattern was observed using air-dried (instead of frozen) sections or in sections fixed with various fixtures (4% p-formaldehyde alone or in combination with 0.5% glutaralde ⁇ hyde, acetone or methanol) .
  • Region I and the repeats but not Region 11+ did not bind to liver sections even at concentrations as high as 250 ⁇ g/ml.
  • Pfl open circles; Pf2: dark circles; Pf3: open upright triangles; PflA dark square; PflB open inverted triangles; Pf2A dark triangle; Pf70 open diamond; Pf25C dark diamond. Standard deviations were no greater than +.5% of the plotted mean.
  • Rat liver tissue was embedded in LR white and sections were labelled with CS27IVC, MAb 2A10 and goat-antimouse IgG - gold conjugate.
  • Gold particles were found in areas of hepatocyte membrane exposed to the bloodstream, namely the microvilli protruding in the Space of Disse and to lateral membranes of adjacent hepatocyte up to the tight junctions that seal the bile canaliculi. Other regions of the hepatocyte plasma were not stained, and neither were cell membranes of Kupffer cells or endothelia. Liver sections incubated with falciparum-1 protein which lacks Region 11+ or sections incubated only with monoclonal antibody 2A10 and/or with the gold conjugate were negative.
  • Intracellular hepatocyte labelling with CS27IVC was seen on the lysosomes in the vicinity of bile canaliculi possibly reflecting receptor internalization and degradation.
  • the bile ducts themselves were negative. Lysosomal staining was also seen in Kupffer cells and, occasionally, in cells containing lipid droplets. On the whole, labelling was extremely localized.
  • 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 mitochondrium (M) and rough endoplasmic reticulum (arrowheads) are not labelled.
  • Example 11 Aggregated, Region 11+-Containing CS
  • the gold conjugates employed in the foregoing liver section experiments contained no aggregates. However, the staining pattern was always patchy which suggested that the receptors were clustered, or the CS was aggregated, or both. Recombinant CS27IVC was subjected to molecular sieving as detailed above to isolate the monomeric form of the protein
  • Falciparum-1 aggregates were formed by introduction of sulfhydryl groups followed by air oxidation. The presence of aggregates or monomers in the active and inactive fractions, respectively, was confirmed by SDS-PAGE under reducing and nonreducing conditions followed by Western blotting using MAb 2A10. Reducing conditions resulted in the observation of a single band in all samples. Nonreducing conditions preserved several bands of increasing molecular weight in the FPLC fraction that had showed activity. (See. Example 5 above) . The results are depicted in Figure 5 which is a plot of OD v. FPLC fraction number. It shows that the CS protein eluted in two peaks, with the first one (fraction 9) corresponding to the aggregated form and the second one (Fractions 12-14) corresponding mainly to monomeric forms.
  • 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") 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°/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.
  • HepG2 cells were plated at a density of 0.5 x 10 6 /ml, incubated for 2 hr. with P.berghei sporozoites (50, per well) in the presence of the PbRII+, negative control peptides (Pf4 and CD59) , or media alone. Cultu were grown for 2 days, fixed, and stained with MAb 2E6, followed by goat anti-mouse immunoglobu conjugated to horseradish peroxidase. Numbers in the fourth column represent the average schizonts coun per 20 fields under 20 x magnification of a light microscope in quadruplicate wells ( ⁇ the stand deviation) .
  • EEF exoerythrocytic forms.
  • P values were calculated using one-way analysis of variance (ANOVA) , corrected by the Bonferr method.
  • NS not significant.
  • c In this experiment, peptides were added to the culture 2 hr. after the addition of the parasites, af completion of invasion.
  • 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 antiserum 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.
  • 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 overlap ⁇ ping peptide method, which is a peptide screening technique well- known in the art and no more than routine experimentation.
  • the overlap ⁇ ping peptide method is a peptide screening technique well- known in the art and no more than routine experimentation.
  • Table 1 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 of Region 11+ is important. The only remaining questions fall into the category of whether, e.g., E W T P C S V T C G V G V derived from P.
  • vivax Region 11+ would have inhibitory activity and whether the higher order structures (e.g., the small peptide loop formed by sulfide bonds beteen the two cysteines) are essential for inhibitory activity.
  • peptides and peptide-containing constructs within the present invention include the following:
  • CS protein includes recombinant CS polypeptides and entire sporozoites
  • Such peptides should possess substantial CS-binding inihibitory 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 against the immunodominant (repetitive) epitope of the CS protein.
  • Such peptides may be as small as the minimum CS-binding inhibitory amino acid sequence from Region 11+ or as large as CSFZ, 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.
  • Region 11+ is not itself particularly immunogenic, 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 can be administered to malaria susceptible subjects, for example intravenously, at sufficiently high concentrations to compete effectively with a subsequent challenge with sporozoites.
  • 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. Preferred concentrations range from about 1 to about lOmg 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 encapulated in liposomes, the encapsulation having been described by Brenner, D. , J.M.C.I. 78:1436. 1989; Anderson, P. et al., Cancer Research .50:1853, 1990; and Anderson, P. et al. , J. I munotherapy 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 sporozoites are targeted to the liver of the infected mammal so efficiently and quickly that therapeutic administration would not be effective, as shown by Example 12 above. 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 raise antibodies in vitro.
  • These can be human or humanized monoclonal antibodies. Human origin or hu anization would cause the immune system of the host to be "blind" to the antibodies, and raising them against Region 11+ would cause them to 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. Presta, L.G. Curr. Op. Struct. Biol. 2:593-596, 1992; and Burton, D.R., Hospital Practice. August 15, 1992, 67-74 and references cited in each.
  • the amount of monoclonal antibody administered should be sufficient to achieve a blood level ranging from about 1 to about 10 mg/ml.
  • In vitro elicitation of antibodies can be performed according to methods known to those skilled in the art.
  • Region 11+ does not preclude use of peptides containing Region 11+ (and also containing an antigenic determinant) in vaccines.
  • 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.
  • antigen sequences include, but are not limited to: 1. P S D K H I E Q Y L K K I K N S I (TH2R) ,
  • TH2R and TH3R include only a few interspecies amino acid substitutions, and therefore, polymorphism would not be an impediment to incorporat ⁇ ing them in a malaria vaccine preparation.
  • Region 11+ peptides would be useful in drug design, i.e. in the construction of 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 largely described above. 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.
  • Peptides consisting essentially of Region 11+, or its ligand adhesion subregions can also be used for such drug screening and are more covenient for this purpose than recombi- nant CS-constructs.
  • Sucn Region 11+ derived peptides could be labelled and used exactly as the recombinant CS protein, or their binding to hepatocytes could be assessed previously, and the unbound peptide could be measured.
  • this receptor is not known, but there are indications that it may be a sulfated macromolecule and most likely a heparan sulfate proteoglycan.
  • other proteins bearing the Region 11+ motif bind specifically to sulfated compounds.
  • Recombinant CS proteins from P. vivax (vivax-1) and P. falciparum (falciparum-1) containing Region 11+ (but not proteins lacking Region 11+ such as vivax-2 and falciparum-2) also specifically bind to sulfatides as well as to cholesterol-3-sulfate. This binding is abrogated by reduction or alkylation of the recombinant CS constructs.
  • the two cysteines in Region 11+ form a disulfide bond forming a small peptide loop which appears important in receptor recogni ⁇ tion.
  • the binding of the recombinant constructs as well as live sporozoites is inhibited by dextran sulfate, which makes it likely that the receptor is a sulfated glycoconjugate.
  • the dextran sulfate should be present before contact of the CS polypeptide (or of the sporozoites) with the hepatocytes (in much the same manner as described in Example 12 above for the inhibitory ability of Region 11+ peptides present in the extra- hepatocytic medium prior to challenge with live sporozoites) .
  • CS polypeptides do not bind to gangliosides or steroids that are structurally similar to sulfatides and choles ⁇ terol sulfate but are not sulfated; and they do not bind to polyanionic compounds (including sulfated polyanionic compounds) such as heparan-sulfate, chondroitin-sulfate, and hyaluronic acid.
  • polyanionic compounds including sulfated polyanionic compounds
  • dextran does not appreciably inhibit sporozoite invasion of hepatocytes, which is consistent with the conclusion that a sulfated receptor ' structure is involved.
  • CS receptor Although the structure of the CS receptor is of importance in drug design, one of the advantages of the present invention is that it does not regain knowledge of the receptor structure.
  • the peptides or constructs should have no substantial ability to elicit the formation of antibodies recognizing the immunodominant epitope of CS that would diminish the effective ⁇ ness of the peptides or constructs.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des compositions et des procédés d'inhibition de l'invasion des hépatocytes par des sporozoïtes malariques. L'invention porte plus particulièrement sur: a) des ligands du récepteur de la membrane plasmique hépatocytaire de la protéine ainsi que des peptides de circumsporozoïte basés sur une partie de la protéine circumsporozoïte qui constitue une partie essentielle du ligand de ce récepteur; et b) des procédés utilisant ces peptides en vue d'inhiber l'invasion des cellules hépatiques par les circumsporozoïtes.
PCT/US1993/008800 1992-09-17 1993-09-17 Compositions et procedes d'inhibition de l'invasion des hepatocytes par les sporozoïtes malariques WO1994006464A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU51307/93A AU5130793A (en) 1992-09-17 1993-09-17 Compositions and methods for inhibiting hepatocyte invasion by malarial sporozoites

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US94703392A 1992-09-17 1992-09-17
US07/947,033 1992-09-17

Publications (1)

Publication Number Publication Date
WO1994006464A1 true WO1994006464A1 (fr) 1994-03-31

Family

ID=25485402

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/008800 WO1994006464A1 (fr) 1992-09-17 1993-09-17 Compositions et procedes d'inhibition de l'invasion des hepatocytes par les sporozoïtes malariques

Country Status (2)

Country Link
AU (1) AU5130793A (fr)
WO (1) WO1994006464A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0730466A1 (fr) * 1993-09-10 1996-09-11 New York University Compositions et procedes destines a inhiber l'invasion des hepatocytes par les sporozo tes du paludisme
FR2766190A1 (fr) * 1997-07-16 1999-01-22 Univ Clermont Auvergne Nouveaux peptides et polypeptides utiles dans la regeneration du systeme nerveux
WO2000049146A1 (fr) * 1999-02-18 2000-08-24 Rmf Dictagene S.A. Vaccin contre la malaria
EP1404877A2 (fr) * 2001-07-02 2004-04-07 Bioincept, LLC. Nouveaux dosages pour le facteur preimplantatoire et peptides du facteur preimplantatoire
WO2005044995A2 (fr) * 2003-11-05 2005-05-19 Sloan-Kettering Institute For Cancer Research Utilisations de peptide deformylase humaine
US8222211B2 (en) 2001-07-02 2012-07-17 Bioincept, Llc Methods of administering PIF agonist peptides and uses thereof
US8454967B2 (en) 2004-10-22 2013-06-04 Bioincept Llc Compositions and methods for modulating the immune system
US9266930B1 (en) 1993-03-05 2016-02-23 Epimmune Inc. Inducing cellular immune responses to Plasmodium falciparum using peptide and nucleic acid compositions
US9737585B2 (en) 2011-03-02 2017-08-22 Bioincept, Llc Compositions and methods for treatment of intracellular damage and bacterial infection
US11090355B2 (en) 2015-08-28 2021-08-17 Bioincept, Llc Compositions and methods for the treatment of neurodamage
US11096987B2 (en) 2015-08-28 2021-08-24 Bioincept, Llc Mutant peptides and methods of treating subjects using the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. EXP. MED., Vol. 164, issued December 1986, ALEY et al., "Synthetic Peptides from Circumsporozoite Proteins of Plasmodium Falciparum and Plasmodium Knowlesi Recognize the Human Hepatoma Cell Line HepG2-A16 In Vitro", see pages 1915-1922. *
SYNTHETIC PEPTIDES IN BIOLOGY AND MEDICINE, issued 1985, E. RUOSLAHTI et al., "Synthetic Peptides in Analysis of Cell Adhesion", see pages 191-197. *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9266930B1 (en) 1993-03-05 2016-02-23 Epimmune Inc. Inducing cellular immune responses to Plasmodium falciparum using peptide and nucleic acid compositions
EP0730466A4 (fr) * 1993-09-10 1999-04-14 Univ New York Compositions et procedes destines a inhiber l'invasion des hepatocytes par les sporozo tes du paludisme
EP0730466A1 (fr) * 1993-09-10 1996-09-11 New York University Compositions et procedes destines a inhiber l'invasion des hepatocytes par les sporozo tes du paludisme
US6995140B1 (en) 1997-07-16 2006-02-07 Nucleica Peptides and polypeptides useful for regenerating the nervous system
FR2766190A1 (fr) * 1997-07-16 1999-01-22 Univ Clermont Auvergne Nouveaux peptides et polypeptides utiles dans la regeneration du systeme nerveux
WO1999003890A1 (fr) * 1997-07-16 1999-01-28 Nucleica Nouveaux peptides et polypeptides utiles dans la regeneration du systeme nerveux
WO2000049146A1 (fr) * 1999-02-18 2000-08-24 Rmf Dictagene S.A. Vaccin contre la malaria
US6579524B1 (en) 1999-02-18 2003-06-17 Rmf Dictagene S.A. Malaria vaccine
US7670850B2 (en) 2001-07-02 2010-03-02 Bioincept, Llc Assays for preimplantation factor and preimplantation factor peptides
US8222211B2 (en) 2001-07-02 2012-07-17 Bioincept, Llc Methods of administering PIF agonist peptides and uses thereof
EP1404877A2 (fr) * 2001-07-02 2004-04-07 Bioincept, LLC. Nouveaux dosages pour le facteur preimplantatoire et peptides du facteur preimplantatoire
US7670852B2 (en) 2001-07-02 2010-03-02 Bioincept, Llc Assays for preimplantation factor and preimplantation factor peptides
CN102174079B (zh) * 2001-07-02 2013-09-11 倍奥英赛普特有限责任公司 用于植入前因子的新型检测方法以及植入前因子肽
US7670851B2 (en) 2001-07-02 2010-03-02 Bioincept, Llc Assays for preimplantation factor and preimplantation factor peptides
US7678582B2 (en) 2001-07-02 2010-03-16 Bioincept, Llc Assays for preimplantation factor and preimplantation factor peptides
US7695977B2 (en) 2001-07-02 2010-04-13 Bioincept, Llc Assays for preimplantation factor and preimplantation factor peptides
US8012700B2 (en) 2001-07-02 2011-09-06 Bioincept, Llc Assays for preimplantation factor and preimplantation factor peptides
EP1404877A4 (fr) * 2001-07-02 2007-07-11 Bioincept Llc Nouveaux dosages pour le facteur preimplantatoire et peptides du facteur preimplantatoire
WO2005044995A2 (fr) * 2003-11-05 2005-05-19 Sloan-Kettering Institute For Cancer Research Utilisations de peptide deformylase humaine
WO2005044995A3 (fr) * 2003-11-05 2007-11-22 Sloan Kettering Inst Cancer Utilisations de peptide deformylase humaine
US8454967B2 (en) 2004-10-22 2013-06-04 Bioincept Llc Compositions and methods for modulating the immune system
US10071131B2 (en) 2006-02-03 2018-09-11 Bioincept, Llc Compositions and methods for modulating the immune system
US10806771B2 (en) 2006-02-03 2020-10-20 Bioincept, Llc Compositions and methods for modulating the immune system
US9737585B2 (en) 2011-03-02 2017-08-22 Bioincept, Llc Compositions and methods for treatment of intracellular damage and bacterial infection
US10953071B2 (en) 2011-03-02 2021-03-23 Bioincept, Llc Compositions and methods for treatment of intracellular damage and bacterial infection
US11090355B2 (en) 2015-08-28 2021-08-17 Bioincept, Llc Compositions and methods for the treatment of neurodamage
US11096987B2 (en) 2015-08-28 2021-08-24 Bioincept, Llc Mutant peptides and methods of treating subjects using the same

Also Published As

Publication number Publication date
AU5130793A (en) 1994-04-12

Similar Documents

Publication Publication Date Title
Howard et al. Secretion of a malarial histidine-rich protein (Pf HRP II) from Plasmodium falciparum-infected erythrocytes.
Cerami et al. The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites
US7438917B2 (en) Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes
Theisen et al. The glutamate-rich protein (GLURP) of Plasmodium falciparum is a target for antibody-dependent monocyte-mediated inhibition of parasite growth in vitro
US7211256B2 (en) Plasmodium falciparum antigens inducing protective antibodies
Cerami et al. Binding of malarial circumsporozoite protein to sulfatides [Gal (3-SO4) β1-Cer] and cholesterol-3-sulfate and its dependence on disulfide bond formation between cysteines in region II
WO1994006464A1 (fr) Compositions et procedes d'inhibition de l'invasion des hepatocytes par les sporozoïtes malariques
Charoenvit et al. Development of two monoclonal antibodies against Plasmodium falciparum sporozoite surface protein 2 and mapping of B-cell epitopes
US6669945B1 (en) Universal T-cell epitopes for anti-malarial vaccines
Chatterjee et al. Characterization of domains of the phosphoriboprotein P0 of Plasmodium falciparum
Bharadwaj et al. Induction of protective immune responses by immunization with linear multiepitope peptides based on conserved sequences from Plasmodium falciparum antigens
AU656914B2 (en) New peptides and their use
EP0216901A1 (fr) Conjugue proteinique immunogenique porteur d'antigenes, destine a etre utilise dans un vaccin contre la malaria
Flueck et al. Evaluation of two long synthetic merozoite surface protein 2 peptides as malaria vaccine candidates
AHLBORG et al. Epitope specificity and capacity to inhibit parasite growth in vitro of human antibodies to repeat sequences of the Plasmodium falciparum antigen Ag332
US20060240491A1 (en) Proteins involved in cytoadhesion of plasmodium falciparum ring-stage-infected erythrocytes, antibodies which bind to the proteins, and methods for detecting infection, stage of infection and vaccines for protecting against infection
Gabriel et al. Identification of a Plasmodium chaubaudi antigen present in the membrane of ring stage infected erythrocytes
EP1526178B1 (fr) Famille de gènes du type MSP-3
US20030119733A1 (en) Compositions and methods for inhibiting hepatocyte invasion by malarial sporozoites
Chappel et al. IgG 4 Pf NPNA-1 a human anti-Plasmodium falciparum sporozoite monoclonal antibody cloned from a protected individual inhibits parasite invasion of hepatocytes
US20050169928A1 (en) Antibodies which bind to proteins involved in cytoadhesion of plasmodlum falciparum ring-stage-infected erythrocytes
EP0730466A1 (fr) Compositions et procedes destines a inhiber l'invasion des hepatocytes par les sporozo tes du paludisme
Siddique et al. Antibodies to nonrepeat sequences of antigen Pf155/RESA of Plasmodium falciparum inhibit parasite growth in vitro
US20020164652A1 (en) Conformational antigens and antibodies recognizing said antigens, a process for the efficient generation of monoclonal antibodies to native or conformational antigens expressed or carried by eukaryotic cells, a process for the selection of conformational antigens, use of monoclonal antibodies for therapeutical, diagnostic or vaccine applications
Sulzer et al. Antibodies to the ring-infected erythrocyte surface antigen of Plasmodium falciparum elicited by infection with Plasmodium malariae

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA