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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/002—Protozoa antigens
  • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
• • 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

• the present invention relates to conjugates of an antigen and a carrier protein useful for providing protective immunity against malaria. More particularly, the present invention relates to conjugates of a peptide and a carrier protein useful for providing protective immunity against the sporozoite stage of malaria.
• the target antigens of these anti-sporozoite antisera have been identified by monoclonal antibodies. They belong to a family of polypeptides (circumsporozoite surface- or CS-proteins) that normally cover the entire surface membrane of the sporozoite but are shed upon reaction (cross-linkirg) with antibodies.
• CS proteins contain strongly immuno- dominant repeated epitopes. Monoclonal antibodies to these epitopes neutralize sporozoite infectivity both j Ln vitro and in vivo.
• the immunodominant epitope comprises the sequence H-(Asn-Ala- Asn-Pro) 3 -OH — also designated as (NANP)3. This epi- tope is found in Plasmodium falciparum strains from all endemic areas of the world, and is represented many times in the CS molecule. (Enea, et al. 1984. Science: 225, 628; Dame, et al. 1984, Science: 225, 593; Zavala, F. et al.. Fed. Proc. _43_: 1808, 1984 and J. Immunol, in press).
• Such a vaccine should not only be effective in conferring (or boosting) immunity, but should be easy and inexpensive to produce in a mass scale, in view of the 5 great number of persons in need of immunization.'
• a malaria vaccine using synthetic short length peptides as an immunization agent would be advantageous over another vaccine that used whole CS proteins as the immunization agents because of ease of manufacture, lower 0 cost and large supply of immunogen.
• conjugates of a peptide comprising the immunodominant epitope of P.falci ⁇ parum CS protein and a carrier are effective in raising 5 high titers of antibodies jLn vivo. These antibodies recognize sporozoites and neutralize sporozoite infectivity in vitro by a vigorous CSP reaction.
• the preferred peptide is H-(Asn-Ala-Asn-Pro) 3 -OH — also designated (NANP)-. — i.e., a dodecapeptide consisting of the amino acid sequence 0 NANP tandemly repeated three times, and the preferred carrier is tetanus toxoid.
• Figs-. 1-3 are plots of radioactivity counts per minute observed in immunoradiometric assays against the reciprocal serum dilution from rabbits immunized with conjugates according to the present invention.
• Fig. 4 is a plot of the results of an immuno ⁇ radiometric assay of rabbit antisera raised against the conjugate of the present invention in the presence of in ⁇ creasing concentrations of (NANP), in the fluid phase.
• Fig. 5 depicts the results of Western blotting of
• Plasmodium falciparum extracts revealed by rabbit antisera raised against (a) Plasmodium falciparum sporozoite ex ⁇ tracts; (b) the conjugate of the present invention with complete Freund's adjuvant; (c) normal rabbit serum; and (d) the conjugate of the present invention in incomplete Freund's adjuvant.
• Fig. 6 is a plot of the results of an immuno ⁇ radiometric assay of rabbit antisera raised against a conjugate according to the present invention performed in the presence of increasing concentrations of P. falciparum and P. berghei sporozoite extracts.
• Fig. 7 depicts the proportion of positive serum reactions with (NANP).. in humans from endemic areas according to the age of the human subjects.
• Fig. 8 is a histogram of the result of an immuno ⁇ radiometric assay of the same human sera, as those used to generate Figure 7; the assay was conducted in the presence or absence of competing (NANP) 3 or another peptide in the fluid phase.
• the present conjugates have been found to gener ⁇ ate high titers of anti-P. falciparum sporozoite antibodies by immunization of rabbits, mice and aotus monkeys, whether these conjugates were emulsified in complete or incomplete Freund's adjuvant.
• the conjugates elicited anti-sporozoite antibodies in rabbits even in the absence of adjuvant.
• the antibody titers increase with the amount of antigen injected.
• the present conjugate is a good candidate for developing a malaria vaccine, especially one that could be used to immunize humans in different geo ⁇ graphical areas.
• the present conjugates have been prepared by conjugation of (NANP with tetanus-toxoid.
• tetanus-toxoid is an immuniza ⁇ tion agent in its own right.
• examples are: diphtheria toxoid (available from many commercial sources: Lederle Laboratories, Pearl River, N.Y.; Merrell Dow Pharmaceuti- cals, Cincinnati, Ohio; Eli Lilly & Co., Indianapolis, Indiana et al) other proteins and polysaccharides well- known for that purpose as well as synthetic peptides and polymers comprising lysine and arginine groups.
• conjugates have been prepared using glutaraldehyde as a coupling reagent.
• glutaraldehyde as a coupling reagent.
• other coupling procedures are readily available, such as one using water soluble carbodiimides (J ⁇ Biol. Chem. 242 2447-2453, 1967) or bis-diazobenzidine [following addition of an extra tyrosine residue at the N-terminal of
• NANP 3 Proc. Nat'l Acad. Sci., 2Z ⁇ 5 197-5200, 1980] or malimidobenzoyl-N-hydroxy succinimide ester [following addition of an extra cysteine residue or other sulphydrils to the N-terminal of (NANP)_ see Proc. Nat'l Acad. Sci. ,
• a particularly preferred embodiment of the present invention lies in the addition of a cysteine residue to the N-terminal of the peptide arid the use of malimido benzoyle-N-hydroxy succinimide ester as a coupling reagent.
• Freund's complete (as well as incomplete) adjuvant was used as an adjuvant.
• the function of an adjuvant is to enhance the immune response. Any adjuvant suitable for use in vaccine preparations can be used.
• an adjuvant advantageously increases the immunogenicity of a conjugate and is therefore preferably included.
• suitable adjuvants are aluminum phosphate, aluminum hydroxide, muramyl dipeptide or derivatives et al.
• the present invention is described further below by reference to particularly preferred embodiments. How ⁇ ever, as will be readily recognized by persons of ordinary skill in the art, a number of modifications, additions, and substitutions may be made without departing from the scope or spirit of the present invention as disclosed in this specification, the accompanying claims and the appended drawings. The purpose of the following examples is to illustrate the present invention but not to limit its scope.
• Example 1 Synthesis and Purification of (NANP)-
• NANP dodecapeptide
• Boc-Pro-OCH 2 -Pam-resin (2 g, 0.4 mmol substitution per gram of resin) was placed into the reaction vessel of a modified Beckman 990 synthesizer (Beckman Instruments, Palo Alto, California). Synthesis was performed using a computer, which optimized the coupling steps.
• the protected dodecameric peptide-resin was deprotected batchwise (0.5 gram) by HF-anisole (9:1, v/v, 10 ml) for 60 minutes at 0°C.
• the cleavage yield was 91% based on back hydro ⁇ lysis of the resin by 6N HCl.
• the purity of the crude peptide was determined to be greater than 85% by high pressure liquid chromatography on a reverse-phase C-18 column (4.6 x 250 mm manufactured by Vydac, Hesperia, Calif.) using an aqueous CF 3 C0 H and CH3CN gradient system as follows: eluant A contained 100 ml H 2 0 and 0.05 ml CH 3 ⁇ 0 H, and eluant B contained 60 ml H 2 0, 40 ml CH 3 CN and 0.05 ml CF 3 C0 2 H. The system was eluted at
• the eluting system consisted of 750 ml of eluent A (712.5 ml H 2 0, 37.5 ml CH3CN and 0.375 ml of CF 3 C0 2 H and 800 ml of eluent B (480 ml H 2 0, 320 ml CH 3 CN and 0.4 ml CF 3 C0 2 H) .
• the system was eluted at 1.5ml/min. Fractions were collected at 5 ml/min in a linear eluent B gradient (0-100%B) in 16 hr by an LDC pump. Detection was at 215 nm and a major symmetrical peak was detected between fractions 43 and 57.
• Boc-Pro-OH (20.66 g, 96 mmol ) and DCC (9.89 g, 48 mmol) are reacted in DMF (400 ml) for 1 hr, filtered and the resultant preformed symmetric anhydride is added to hydroxy- methyl-resin (20 g; 0.8 meq/g; 16 mmol) in the presence of 4-dimethylaminopyridine (0.586 g; 4.8 mmol).
• the slurry is shaken for 24 hr at room temperature.
• An aliquot (approx. 50 mg) is hydrolyzed with 1 ml of 1:1 propionic acid/HCl in a sealed tube at 150° for 1 hr.
• Boc-Asn-Pro-Hydroxymethyl-resin 3 5 Boc-Asn-OH (7.02 g, 30.24 mmol, 4.0 equiv.) is added to Prohydroxymethyl-resin (2_, 7.56 mmol) in 300 ml of CH 2 C1 2 and agitated for 5 min.
• Dicyclohexylcarbodiimide (6.23 g, 30.24 mmol, 4.0 equiv.) is added and agitation proceeds for 60 min.
• Diisopropylethylamine (3 ml) is added o (1% by volume) and agitation continued for an additional 15 min.
• the reaction mixture is filtered and washed 3 times with 300 ml CH 2 Cl 2 .
• An aliquot of resin (approx. 1.5 mg) is removed and monitored by the Ninhydrin Reaction as follows: The peptide-resin is placed in a small test tube 5 and treated with 3 drops each of solutions A, B and C
• Boc-Asn-Pro-hydroxy ethyl-resin, 3_ (7.56 mmol) is charged into a 1000 mL reaction vessel, attached to a Kraft Shaker and subjected to the washing, deprotection and neutralization procedures specified in Step 2. Coupl ⁇ ing reactions and washings are then carried out with 30.24 mmol (4 equiv.) of Boc-amino acids by the DDC procedure specified in Step 3 (or via HOBt-ester) in the sequence shown:
• the reaction mixture is filtered (to remove dicyclohexylurea) and added to the peptide resin [which was washed with 300 ml of DMF prior to, and subsequent to, the addition of the HOBt-ester] and agitated for 1 hr.
• the washing cycles are otherwise identical to the protocol for the DCC-coupling procedure.
• the N a ⁇ amino group of Cys is acetylated with a solution of acetic anhydride (150 ml) :Pyridine (150 ml) for 1 hr.
• peptide-resin _4_ (10.5 g) is placed in an HF-reaction vessel and 10.5 ml of dithioethane is added. Liquid HF (94.5 ml) is condensed into the reaction vessel and stirring proceeds at 0 ⁇ for 1 hr. Following evaporation to dryness _in vacuo the residue is treated with 500 mL of EtOAc and filtered. The precipitate is extracted 4 times with 80 ml each of TFA and evaporated. The oily residue is triturated 4 times with 300 mL of anhydrous ether and dried _in vacuo to give 1.98 g of crude J3_.
• the 1.98 g of crude 5_ is dissolved in 30 ml of H 2 0 (containing 0.1% TFA), filtered through an 0.8 micron Type AA Millipore filter and refiltered through a 0.45 mi- cron Type HA Millipore filter.
• the filtrate (total volume, 45 ml) is charged onto a Nucleosil C.g reversed-phase column (2.54 x 25 cm) [previously equilibrated with 5% acetonitrile (containing 0.1% TFA)-H 2 0 (containing 0.1% TFA)].
• the column is eluted (flow rate, 5 ml/min) with a solvent system consisting of acetonitrile (containing 0.1% TFA) - water (containing 0.1% TFA) in a linear gradient mode from 5% acetonitrile to 25% acetonitrile in 120 min using an LDC Constametric IIG with a Gradient Master and Spectromonitor III Detector and LKB Fraction Collector.
• a solvent system consisting of acetonitrile (containing 0.1% TFA) - water (containing 0.1% TFA) in a linear gradient mode from 5% acetonitrile to 25% acetonitrile in 120 min using an LDC Constametric IIG with a Gradient Master and Spectromonitor III Detector and LKB Fraction Collector.
• the crude product was subjected to purification by HPLC as in 5_.
• the monitoring of the purifi- cation was exactly as in 5_.
• tetanus toxoid suitable for use in humans for vaccination is also commercially available from Burroughts Wellcome Research Triangle Park, N.C., or from Wyeth Laboratories, Dir. of Am. Home Products Corp., Philadelphia, Penn.
• Equal volumes of TT (1 mg/ml) and (NANP), (1 mg/ml) were mixed; a solution 0.37% glutaraldehyde in water was added to a final concedntration of 0.02%. After incubation for six hours at room temperature, the mixture was extensively dialyzed against distilled water for 48 hours and lyophilized.
• the polymerized toxoid and peptide recovery ranged between 68 and 80% by weight.
• the preparation contained less than 1% of free peptide.
• the resulting material was resuspended in 2 ml of phosphate buffered saline (pH 7.4) and kept in the re ⁇ frigerator.
• adjuvant was not used, a booster of the same dose of vaccine was given subcutaneoulsy two weeks after the first injection.
• the adjuvant mixture was prepared by emulsifying equal volumes of the vaccine described in Example 2 (at a concentration of 2 mg/ ml) in the adjuvant.
• antibody titers (define-i as the serum dilution giving 10 3cpm in the IRMA) between 1,000 and
• FIG. 2 shows the antibody titers obtained when
• 0.1 mg of antigen [(NANP)--TT] was injected in three rabbits with incomplete Freund's adjuvant.
• the serum titers were 320-80). Pre-immune sera were negative.
• Fig. 3 shows the antibody titers obtained when 0.1 mg of antigen was injected in the absence of any
• Freund's adjuvant The titers ranged between 80 and 10. cpm. Again, pre-immune sera were negative. No reactivity was observed when the plates were coated with peptide alone. The serum titers remained practically unchanged for at least 10 weeks after immunization.
• Example 4 The same rabbit sera used in the immunoradio- metric assay of Example 4 were also assayed for reactivity with the surface membrane of glutaraldehyde-fixed sporozo ⁇ ites of P. falciparum.
• the immunofluorescence assay was disclosed in Nardin, E.H., et al (1979) Science 206:597. Fixed parasite preparations were obtained by incubation with 0.1% gluta- raldehyde for 10 min at room temperature. The sporozoites were washed and resuspended to a concentration of 3-5 x
• the sporozoites were distributed in multiple-well slides, air dried and stored at -70°C.
• Example 4 The assay of Example 4 was repeated except that increasing concentrations of (NANP) 3 peptide were added to the antisera in the incubation mixture.
• Another immunoradiometric assay was performed to determine the proportion of the anti-co.njugate antibodies that reacted with active CS protein.
• the assay used a constant dilution of a rabbit antiserum to the conjugate (1/100) in the presence of increasing concentrations of P. falciparum sporozoite extract. As shown in Fig. 6, the reactivity of the anti ⁇ body with the bound (NANP) 3 diminished to about 30% of control (no sporozoite extract.) levels. This means that 70% of the reactivity of the anti-conjugate antibodies was absorbed by the CS protein of P. falciparum. The in ⁇ hibitory effect was specific, since it was not observed with extracts of sporozoites of Plasmodium berghei.
• Example 7 Western Blotting Western blotting was used to measure the ability of the anti-conjugate antisera to react with the CS protein and its precursors. Western blotting was performed as follows:
• Sporozoite extracts (10 /ml) were subjected to electrophoresis in a 10% sodium dodecyl sulfate poly- acrylamide gel.
• the separated proteins were electropho- retically transferred to nitrocellulose sheets (as dis ⁇ closed by Towbin, H. et al., Electrophoretic Transfer of -Proteins From Polyacrylamide Gels to Nitrocellulose Sheets, Proc. Nat'l. Adad. Sci. (USA) 76:4350-4354 (1979)).
• the nitrocellulose paper was saturated with PBS containing 5% BSA and 10% normal goat serum for 2 hours at 37 ⁇ C.
• the various lanes were cut and each lane was incubated as fol ⁇ lows: (1) with rabbit antiserum against whole P. falcipa ⁇ rum extract; (2) with anti-[ (NANP ⁇ -TT] (from immuniza ⁇ tion with complete Freund's adjuvant); (3) with normal (preimmune) rabbit serum; and (4) with anti-[ (NANP),-TT] from immunization with incomplete Freund's adjuvant.
• the antiserum against whole P. falciparu sporozoites was used as a control.
• the rabbit was immunized with contaminated crude material obtained from the salivary glands of mosquitoes infected with P. falciparum sporozoites. Immunoglobulin from the serum of one rabbit was purified by chromatography on diethylamino-ethyl cellulose (DEAE-Cellulose) and used in sporozoite _in. vitro neutrali ⁇ zation experiments in accordanced with the Hollingdale procedure: J. Immunol. 132:909(1984). Parasites were obtained from salivary glands of laboratory-bred mosquitoes infected by membrane feeding with cultures of P. falciparum blood stages. Salivary glands were pooled in heat-inacti ⁇ vated human serum, disrupted by trituration and counted. All studies were carried out with human hepatoma (Hep
• Intracellular (exoerythrocytic) forms Example 9: Recognition of (NANP), by Human Antibodies to P.falciparum
• Example 4 Sera from 58 individuals from the Gambia, West Africa (an endemic region) and from 29 healthy blood donors in New York City (not an endemic region) were analyzed by IRMA for the presence of antibodies that would recognize (NANP),.
• the assay of Example 4 was employed, except that the second antibody was 1 ⁇ [I] - labeled, affinity purified rabbit anti-human IgG (Sp.act. about 5 x 10 cpm/ microgram) was used.
• the second antibody was used: either 25 [l]-labeled, affinity- purified goat anti-human IgG (gamma) or anti-human IgM(mu) , both from Kirkegaard & Perry Laboratories, Gaithersburg, MD.
• Non-specific binding of antibody to the wells was determined for each individual serum sample by omitting ( NANP) 3 from the wells.
• the number of cpm in the control wells was 300-800 (for 1/10 serum dilution).
• the non ⁇ specific cpm was subtracted from the experimental results.
• the difference (specific binding) is referred to as / ⁇ cpm.
• the average __ cpm of a tenfold dilution of the normal sera was 259 + 155. This value, plus or minus three standard deviations (724 cpm) was defined as the normal rar- d e.
• the antibody type was IgG.
• the specificity of the antisera-(NANP), reac ⁇ tion was tested by the inhibition assay of Example 4, i.e. by preincubation of the antisera with a solution of (NANP) (50 microg ams/ml) . The results are shown in Figure 8.
• an IFA was performed in accordance with the method of Example 5. The purpose was to find the proportion of human antibodies that did not recognize (NANP) .

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  • 1986-03-26 ES ES553425A patent/ES8800273A1/es not_active Expired
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