WO1986005790A1

WO1986005790A1 - Immunogenic antigen-carrier protein conjugate for use in a vaccine against malaria

Info

Publication number: WO1986005790A1
Application number: PCT/US1986/000627
Authority: WO
Inventors: Victor N. Nussenzweig; Fidel P. Zavala
Original assignee: New York University
Priority date: 1985-03-28
Filing date: 1986-03-27
Publication date: 1986-10-09
Also published as: EP0216901A4; IL78292A0; ZA862376B; AU5695686A; ES8800273A1; PT82282A; ES553425A0; PT82282B; EP0216901A1; IE860846L; JPS62502340A

Abstract

A conjugate comprising a synthetic immunogenic peptide, which hasan amino acid sequence corresponding to that of an immunodominant epitope of the circumsporozoite protein of the malaria parasite P. falciparum and a carrier protein selected from the group consisting of carrier proteins used in vaccine preparations.

Description

-1-

IMMUNOGENIC AN IGEN-CARRIER PROTEIN CONJUGATE FOR USE IN A VACCINE AGAINST MALARIA

The United States government has rights in this invention by virtue of Grants No. DPE-0453-C-00-2002-00 from the Department of State, Agency for International Development and 5R01-AI-17429-03 from the Department of Health and Human Services.

The present application incorporates by refer¬ ence the entire disclosures of:

(a) U.S. Patent No. 4,466,917 of Nussenzweig, R. , et al, issued on August 21, 1984 and entitled Malaria Vaccine;

(b) Assignee's co-pending U.S. Patent Applica¬ tion Serial No. 574,553 of Ellis, J. et al, filed on January 27, 1984 and entitled "Protective Peptide Antigen";

(c) Assignee's co-pending U.S. Patent Applica¬ tion Serial No. 633,147 of Ellis, J. et al, filed on July 23, 1984 and entitled "Protective Peptide Antigen Corresponding to Plasmodium falciparum Circumsporozoite Protein."

(d) Assignee's co-pending- U.S. Patent Applica¬ tion Serial No. 649,903 of Vergara, U. et al, filed on October 26, 1984 and entitled "Cross-Reactive and Protec¬ tive Epitopes of Circumsporozoite Proteins"; and

(e) Assignee's co-pending U.S. Patent Applica¬ tion Serial No. 695,257 of Nussenzweig, V. et al, filed on January 28, 1985 and entitled "Immunodominant Epitope of the Circumsporozoite Surface Protein."

-2- Background of the Invention

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.

It is known that inoculation of relatively small amounts of X-irradiated sporozoites into rodents, primates and humans results in protective immunity. The immunity is stage-specific (i.e. it protects against the sporozoite stage of malaria but not against the blood stages) and in most instances species-specific (i.e. inoculation with sporozoites of a single species usually confers immunity only against that species) but not strain-specific (innocu- lation with sporozoites of one species originating from one particular endemic area confers immunity against sporozo¬ ites of the same species originating from other endemic areas) . It is also known that incubation of every species of sporozoites with antisera from any host immunized with X-irradiated sporozoites of the same species results in the formation of tail-like precipitate on the sporozoite surface. (This reaction is known as "circumsporozoite pre- σipitation reacton" or "CSP reaction.") It also results in complete neutralization (loss) of sporozoite infectivity.

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.

All known CS proteins contain strongly immuno- dominant repeated epitopes. Monoclonal antibodies to these epitopes neutralize sporozoite infectivity both _jLn vitro and in vivo. ^■ The gene corresponding to the CS protein of P. falciparum, a human malaria species, has been cloned. 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).

•0 The ultimate goal of all this research is to develop a preventive vaccine against malaria..

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. Summary of the Invention

It has now been discovered that 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.

It has also been discovered that most or all antibodies in human sera from endemic areas recognize a synthetic peptide, (NANP)₃. This further supports the 5 notion that (NANP)₃ indeed represents faithfully the -4- repetitive epitope of P. falciparum CS protein. Therefore, the conjugates of the present invention are -useful in the development of a protective vaccine against malaria.

The present invention is further described in detail below.

Brief Description Of The Drawing

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. Detailed Description Of The Invention

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.

Most of the antibodies raised against the conju¬ gate of the present invention recognize the P.falciparum CS protein and neutralize sporozoite infectivity j vitro at low concentrations (below about 0.2 micrograms/ml) .

The antibody titers increase with the amount of antigen injected.

Most antibodies to sporozoites in human sera from endemic areas react with (NANP)-, and confirm that the epitope of the P. falciparum CS protein is not strain- specific. Accordingly, this epitope would not give rise to strain-specific antibodies.

Therefore, 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. In addition to being a carrier protein, tetanus-toxoid is an immuniza¬ tion agent in its own right. However, there are many other such carriers that could by used. 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. Use of these other carriers is fully expected to give rise to- effective conjugates. -6- In some of the examples below, the conjugates have been prepared using glutaraldehyde as a coupling reagent. However, 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^ϊ5197-5200, 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. ,

7^:3403-3407, 1981]. 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.

In the present invention, 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.

Although the present results indicate that the presence of an adjuvant in a vaccine preparation is not essential, an adjuvant advantageously increases the immunogenicity of a conjugate and is therefore preferably included. Other 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)-

The dodecapeptide (NANP), was synthesized by the solid-phase method of Merrifield, R.B. (1962) Fed. Proc. Fed. Am. Soc. Ex. Biol., 21:412; and (1963) J. Chem. Soc, 85:2149.

The attachment of the C-terminal amino acid resi¬ due, Boc-Pro, was onto hydroxymethyl-Pam-[copoly(styrene-1% divinylbenzene) ]-resin support which was synthesized from underivated polystyrene resin from Bio-Rad, Richmond, California, (as disclosed by Mitchell, A.R. et al, 1976 J. Am. Chem. Soc. 98:7357) to prevent loss of peptide chains during synthesis.

The thus prepared Boc-Pro-OCH₂-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 CF₃C0 H and CH3CN gradient system as follows: eluant A contained 100 ml H₂0 and 0.05 ml CH₃ ^~0 H, and eluant B contained 60 ml H₂0, 40 ml CH₃CN and 0.05 ml CF₃C0₂H. The system was eluted at

1 ml/min in a linear gradient of 10%B to 85 %B in 30 min in a Waters Associates, (Milford, MA.) HPLC system. De¬ tection was at 215 nm and a major symmetrical peak was de¬ tected at 11.4 minutes. This peak accounted for more than 85% of the crude peptide content and contained the correct amino acid ratios for (NANP),. Preparative purification (60 mg) was carried out in a low-pressure liquid chromatography system on a 2.5 x 30 cm Michel-Miller column.

The eluting system consisted of 750 ml of eluent A (712.5 ml H₂0, 37.5 ml CH3CN and 0.375 ml of CF₃C0₂H and 800 ml of eluent B (480 ml H₂0, 320 ml CH₃CN and 0.4 ml CF₃C0₂H) . 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. The fractions were collected, the CF₃C0₂H was neutralized by concentrate NH4OH and CH₃CN was removed by vacuum. The aqueous portion was lyophilized. The purified peptide gave a single symmetrical peak upon reverse phase analytical high pressure liquid chromatography. On amino acid analysis, the peptide gave Asp:Ala:Pro, 2.02:1:0.99 (theoretical value 2:1:1). The results of preparative scale were used to optimize the coupling steps in the synthesizer and to program the com¬ puter accordingly.

Example la: Synthesis of Ac-Cys(NANP)₃*-OH and Cys(NANP)₃-OH Boc-Pro-hydroxymethyl-resin, 1

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. Amino acid analysis reveals a substitution level of 0.36 mmol/g. The resin is stirred in CH₂cl₂:pyridine (400 ml: 12.94 ml) and benzoyl chloride, (18.75 ml, 160 mmol) added and stirring continued at-0^β for 30 min and at room temperature for 1 hr. The -9- reaction mixture is filtered and washed with CH₂C1₂ (3 x 350 ml), DMF (2 x 350 ml), CH₂C1₂ (2 x^tJO-ml), MeOH (2 x 350 ml) and dried _in vacuo to give 21.6 g of 3_. Pro-Hydroxymethyl-resin, 2 Boc-Pro-Hydroxymethyl-resin, j_ (21 g; 0.36 mol/g;

7.56 mmol) is washed with 600 ml of CH-C1₂, deprotected with 300 ml of 50% TFA-CH₂C1₂ for 1 in, washed with 300 mL of CH₂C1₂ and deprotected again with 300 ml of 50% F -CH₂Cl₂ for 20 min. The reaction mixture is washed 4 0 times with 300 ml of CH₂cl₂ and neutralized by washing 2 times with 300 ml of 8% DIEA-CH₂Cl₂ (5 min each), 2 times with 300 ml of CH₂Cl₂, 2 times with 300 ml of 2-propanol and 6 times with 300 ml of CH₂cl₂. 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 CH₂C1₂ 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 CH₂Cl₂. 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

[Solution A: 500 mg ninhydrin in 10 ml of EtOH; Solution B: 80 g phenol in 20 ml EtOH; Solution C: 2 ml 0.001M KCN in 100 ml pyridine]. The tube is heated at 95-100^* for 5 min and the beads and solution are examined visually. The ₀ peptide coupling reaction is determined to be incomplete if the Ninhydrin Reaction is positive and gives either a blue solution or blue beads. If the Ninhydrin Reaction is positive the entire coupling cycle is repeated. Ac-Cys(Dmb)-Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala- ₅ Asn-Pro-hydroxymethyl-resin, 4

The 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:

Coupling

No. Residue Acid Amounts Coupling Procedure

1 11 Ala 11.5 g DCC

2 10 Asn 35.0 g HOBt-Ester

3 9 Pro 22.0 g DCC

4 8 Asn 21.0 g HOBt-Ester

5 7 Ala 11.5 g DCC

6 6 Asn 28.0 g HOBt-Ester

7 5 Pro 19.5 g DCC

8 4 Asn 35.0 g HOBt-Ester

9 3_ Ala 17.2 g DCC

10 2 Asn 35.0 g HOBt-Ester

11 1 . Cys(Dmb) 31.0 g DCC A modified protocol is used for the 1-hydroxyben- zotriazole (HOBt)-dicyclohexylcarbodiimide (DCC) coupling procedure. In these cases the Boc-amino acids (4 equiv.) dissolved in 300 ml of DMF, in a separate flask, and reacted with 1-hydroxybenzotriazole (5.09 g, 33.26 mmol, 4.4 equiv.) for 45 min. 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. After the completion of the 11 coupling reactions and final deprotection with 50% TFA-CH₂C1₂, the N^a~ amino group of Cys is acetylated with a solution of acetic anhydride (150 ml) :Pyridine (150 ml) for 1 hr. The peptide-resin _4_, is finally washed 4 times with 300 ml of CH₂C1₂ and dried _in vacuo to give -20.5 g of 4. Ac-Cys-Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-OH,

Ac-Cys(NANP)₃-QH, 5

A portion of 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^β 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 H₂0 (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)-H₂0 (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. [Settings: wavelength: 215 nm; recorder speed: 1 mm/min; sensitivity: 2.0 AUFS; fractions: 1 min (5 ml)/fraction.

Aliquots are analyzed by HPLC using an LDC Con¬ stametric IIG equipped with a Gradient Master Spectromoni¬ tor III Detector and Miσromeritics 725 Autoinjector. [Settings: wavelength: 206 nm; column: Lichrosorb RP-8 (5 micron); eluant: acetonitrile - 0.1M HC10₄ (pH 2.5); gradient: linear, 8% acetonitrile to 20% acetonitrile in 20 min; sensitivity: 0.2 AUFS]. The product emerges in frac¬ tions 55 to 65 which are combined, evaporated and lyophi- lized to give 577 mg of pure product. Side-bands (frac¬ tions 50-54 and 66-70, 421- mg) are also obtained. Yield: 914 mg (19.6%). Amino Acid Anal. <6M HCl; 150°, 1 h) : Asp- 5.85; Pro, 3.02; Ala, 3.11; Cys, 1.13.

63.9] ° (£ 0.86, 0.2N AcOH). (+) FAB Mass Spectroscopy (6 KV) : Calcd. for C₅₃Hg₁N_{l 9}0_2QS, 1352.56; Found, 1352. ¹H-NMR Spectroscopy (DMSO-dg): 1.18 (3H,d, J=7Hz, CH₃ of Ala), 1.22 (6H,d,J=7Hz, 2 x CH₃ of Ala), 1.89 (3H,s,NAc). Analytical HPLC: Column, Lichrosorb RP-8 (5 micron); Eluant, (A) 0.1M HC10₄ (pH 2.5) - (B) Acetoni¬ trile in a linear gradient mode from 5%{B) to 15%(B) in 30 min; Detection, 206 nm; Purity estimated to be greater than 95% (retention time: 23 min). H-Cys-Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-OH,

Cys(NANP)₃-OH, 6

A 1 g portion of peptide-resin (from _4) which was deprotected with 50% TFA-CH₂C1₂ but not N^a-acetylated, was subjected to an HF cleavage and work-up as in 5_. Yield: 192 mg.

The crude product was subjected to purification by HPLC as in 5_. The mixture was loaded onto a Nucleosil ^c _ιo reversed phase column (2.54 x 25 cm) and the column eluted (flow rate = 5 ml/min) with a solvent system consist¬ ing of acetonitrile (containing 0.1% TFA)-water (containing 0.1% TFA) in a linear gradient mode from 7% acetonitrile to 22% acetonitrile in 180 min. The monitoring of the purifi- cation was exactly as in 5_. The product emerged in frac¬ tions 86-93 which were pooled, evaporated and lyophilized to give 61.2 mg (0.047 mmol, 12.7%) of Cys(NANP)₃-OH. Sidebands (fractions 84-85 and 94-97, 19.2 mg) were also obtained. Total yield: 15.5%. The product was shown to be homogeneous in the described analytical HPLC system (retention time = 18 min) and gave the expected amino acid composition (6N HCl-TGA; 110°; 24 h): Asp, 5.96; Pro, 3.13; Ala, 3.07. Example 2: Antigen-Carrier Conjugation Fluid tetanus toxoid (TT) supplied by the Pasteur

Institute, Paris, France, was dialyzed against distilled water for 48 hours and lyophilized. Partially purified 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. By HPLC analysis, 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.

Two lots of antigen were thus prepared and used to immunize groups of rabbits with 1.0 or 0.1 mg protein in the presence or absence of Freund's adjuvant — complete or incomplete. Example 3: Immunization

Rabbits (2-2.5 kg) were injected in one hind foot pad and opposite thigh intramuscularly with a total of 2 ml of the vaccine preparation of Example 2, either emulsified in Freund's adjuvant (both complete and incomplete) or diluted in PBS. The total amount of injected protein was 1 mg or 0.1 mg per rabbit. When 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.

The rabbits were bled four weeks after immuniza- tion. - Example 4: Solid-Phase Immunoradiometric (IRMA) Assays

Flexible polyvinylchloride microtiter plates (Dynatech Laboratories, Inc., Alexandria, Virginia) were incubated with 150 microliters of 50 micrograms/ml bovine serum albumin ( adio-immunoassay grade, Sigma Chemical Co., Inc., St. Louis, Mo.) for four hours at 37°C. After wash¬ ing several times with phosphate-buffered saline (pH 7.4), 20 microliters of a solution containing 100 micrograms per ml of (NANP)₃ and 0.25% glutaraldehyde by volume were placed in each well and incubated at room temperature for two hours. The wells were washed three times with PBS and incubated overnight with 150 microliters of PBS containing 1% bovine serum albumin (BSA) and 0.5M ethanolamine at pH 7.5 (PBS-BSA-Eth buffer). Rabbit serum samples (obtained four weeks after injection) were diluted in buffer PBS-BSA-Eth containing 0.5% Tween-20, and 30 microliters were placed in each well. After incubation for one hour at room temperature, each well was washed three times with PBS-BSA-Eth containing 0.5% Tween-20 (ICI Americas, Inc., Wilmington, Delaware) to eliminate unbound material.

Thirty microliters (7 x 10⁴ counts per minute) of 125[I]-labeled, affinity-purified, goat anti-rabbit immunoglobulin (Miles-Yeda, Elkart, Ind.) were placed in each well to label the bound antisera. The wells were washed, cut and counted.

The results of the immunoradiometric assays are shown in Figures 1-3.

In Fig. 1, antibody titers (define-i as the serum dilution giving 10 3cpm in the IRMA) between 1,000 and

10,000 were found in six rabbits immunized with 1 mg of antigen [ (NANP) -TT] in incomplete Freund's adjuvant

(single dose). Both lots of conjugate vaccine appear to be equally effective. Antisera from lot 1 rabbits are represented by white circles for the first rabbit, black circles for the second rabbit and white squares for the third rabbit. Antisera from lot 2 rabbits are represented by black triangles, white up-right triangles and white inverted triangles, respectively.

The results of immunoradiometric assays conducted with antisera obtained by immunization with (NANP) -TT in complete Freund's adjuvant were identical (not shown).

A control immunoradiometric assay using pre- im une rabbit serum gave negative results (30-50 cpm above background) . 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.

The above results indicate that effective immuni¬ zation can occur with (NANP) conjugated to tetanus toxoid, preferably emulsified in an adjuvant. The quantity of antibodies produced increases with the amount of antigen injected. Antisporozoite antibodies that recognize the dodecapeptide (NANP)-.. were elicited even in the absence of adjuvants. Example 5: Indirect Immunofluorescence Assay

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

10 /ml. The sporozoites were distributed in multiple-well slides, air dried and stored at -70°C.

The results are summarized in the inserts of Figures 1-3. The correlation between antibody titers (ob¬ tained by immunofluorescence) with those obtained in IRMA was highly significant (p < 0.001) by the Spearman rank correlation coefficient. Example 6: Inhibitory Effect of (NANP)-. On Immunoradiometric Assay.

The assay of Example 4 was repeated except that increasing concentrations of (NANP)₃ peptide were added to the antisera in the incubation mixture.

A constant (1/1,000) dilution of rabbit antisera to the present conjugate was incubated with serial dilu¬ tions of (NANP)₃. (NANP)₃ effectively inhibits the immunochemical reaction between rabbit anti-conjugate antisera and (NANP)₃ bound to the wells.

The results, shown in Fig. 4, demonstrate the specificity of the peptide-antiserum reaction.

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^βC. 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.

After extensive washing in PBS containing 1% BSA, the strips were incubated for two hours at room temperature with affinity-purified 195[l]-labeled goat anti-rabbit IgG. The strips were washed, dried and exposed to auto- radiography. The results are shown in Figure 5. The two top bands correspond to the intracellular precursor (67,000 Mr) and membrane (58,000 Mr) forms of the CS protein. Some additional unidentified antigens of lower Mr (probably of mosquito origir ) have been revealed by the antiserum against whole P. falciparum sporozoites (lane 1). Anti- P. falciparum activity was absent in lane 3, as expeσted) . Example 8: Sporozoite Neutralization by Anti-Conjugate Antiser The procedure employed was disclosed in Holling- dale, M.R., et al (1984) J. Immunol., 132:9£r9.

λ 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

G2-A1G from American Type Culture Collection, Rockville, Md.) cells cultured in Eagle's minimum essential medium (GIBCO, Grand Island, N.Y. ) supplemented with 1% human serum. The results, summarized in Table I, below, show that immune rabbit IgG inhibited parasite development in a dose-dependent manner. Very strong neutralization (between 40-80%) took place even at concentrations of total IgG as low as 2 micrograms/ml (of which not more than 10% is likely to be (NANP) -specified antibody). When the anti- conjugate antibodies were removed from the IgG by immunoaf- finity chromatography using (NANP) as the adsorbent, no inhibition in parasite development was observed (Experiment #4). The removal of antipeptide antibodies from the IgG fraction was acertained by immunoradiomet ic assay in accordance with the method of Example 4.

Table I

Inhibition of P . falciparum sporozoite " in vitro" infectivity by antibodies to ( NANP ) ,

Experi ent Identif i- Number added Origin Concentration EEF* Percent Number cation per culture ________ microgram/ml inhibition

1 7G8 25x10³ pre-immune 200 180 anti(NANP)₃ 200 26 76

2 134 26

2 NF54 17x10³ pre-immune 100 203 - anti(NANP)₃ 100 172 15

20 41 80

10 21 90

_. 45 78

1 131 36

0.1 198 3

3 7G8 27x10³ pre-immune 10,0 237 -_ anti(NANP)₃ 100 66 72

20 11 95

2 106 55

1 78 12

0.1 216 9

4 NF54 26x10³ pre-immune 20 240 - anti(NANP) 20 35 86

5 76 68

2 150 38

0.2 200 17 anti(NANP)₃ 20 228 7 adsorbed with 5 248 0

(NANP),

Intracellular (exoerythrocytic) forms Example 9: Recognition of (NANP), by Human Antibodies to P.falciparum

To determine whether human antibodies also recog¬ nize the repeated epitope of P.falciparum CS protein, the reactivity of such antibodies with (NANP), was tested.

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.

To determine the immunoglobulin class 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-_de.

The assay results are plotted in Figure 7.

The percentage of positive sera (^__ cpm > 724) in endemic areas increased with age, ranging from 25% in children (age 1 to 14 years) to 84% in adults over 34 years old. Most positive sera had titers higher than 1/200. 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.

The antibody-peptide binding was completely in¬ hibited by presence of (NANP) in the liquid phase. By contrast, presence of an unrelated synthetic dodecapeptide (co esponding to the repeated epitope of P.knowlesi) failed to inhibit the antisera-(NANP) binding.

Example 10: Indirect Immunofluorescence Assay (IFA)

To detect human antibodies directed to the sur¬ face membrande of P.falciparum sporozoites 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) .

Randomly selected IRMA-negative and IRMA-positive sera from individuals older than 20 years and living in a malaria endemic area were tested for antibody specificity to sporozoites either in the presence or in the absence of competing (NANP)₃ (50 micrograms/ml) . The results are summarized in Table II, below.

TABLE II

IRA with glutaraldehyde

Identification IRMA with (NANP)₃ as fixed sporozoites as of se um antigen ( Δ cpm)* antigen Serum Titer* Serum Titer in the presence of (NANP) **

G.Z. 9201 4096 320

IDA 4851 1280 <20

8017 3539 640 <20

7930 3501 640 <20

7979 3311 640 <20

7973 2735 320 <20

P-2 2473 320 <20

P-5 2024 320 <20

8012 1765 640 <20

Normal 163 <10 N.D.

7981 168 <10 N.D.

8074 133 20 N.D.

7878 96 <10 N.D.

P-12 75 <10 N.D.

8312 72 <10 N.D.

P-11 -13 <10 N.D.

8286 -91 20 N.D.

7907 -103 <10 N.D.

When the results of IFA and IRMA were compared by a non-parametric method (Spearman Rank Correlation), the r was 0.87 (p<0.001). The results of the IRMA in the dilution samples of the positive sera are shown in Figure 2.

** Serum samples were incubated with 50 ug/ml (NANP) for 2 hours at room temperature before performing the IFA. N.D. = not done. The results of the immunoradiometric assay corre¬ lated highly (Spearman rank correlation test) with those of the IFA (p < 0.001). The presence of (NANP) substan¬ tially reduced the titers of IRMA-positive sera. The results of Examples 9 and 10 demonstrate that most human antibodies detected by immunofluorescence (and therefore recognizing only the surface antigen of P.falci¬ parum) were in fact directed against the immunodominant epitope of the P.falciparum CS protein and recognized (NANP),. These results also highlight the strong immuno- dominance of the repetitive epitope of the CS protein in man and demonstrate the existence in humans of B-cells that recognize the repeated epitope of the P.falciparum CS protein. It is expected that these B-cells can be made to respond to a synthetic peptide vaccine either to confer primary immunity or to boost the natural immunity of individuals living in endemic areas.

Claims

We claim :

1. A conjugate of an immunogenic peptide said peptide having an amino acid sequence corresponding to that of an immunodominant epitope of P.falciparum circumsporozoite protein and a carrier protein selected from the group consist¬ ing of carrier proteins used in vaccine preparations.

2. The conjugate of claim 1 wherein said epitope consists essentially of a tandem repeat of the amino-acid sequence Asn-Ala-Asn-Pro.

3. The conjugate of claim 2 wherein said peptide has been chemically synthesized.

4. The conjugate of claim 3, wherein said peptide has the amino-acid sequence

Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro.

5. The conjugate of claim 1, wherein said carrier protein is selected from the group consisting of diphtheria toxoid, tetanus toxoid, and synthetic random copolymers of amino acids containing lysine or arginine or combinations thereof.

6. The conjugate of claim 4, wherein said protein is tetanus toxoid.

7. A component of a vaccine against the P.falci¬ parum malaria parasite consisting essentially of the conjugate of claim 1.

8. A component of a vaccine against the P.falcipa¬ rum malaria parasite consisting essentially of the conjugate of claim 2.

9. A component of a vaccine against the P.falci¬ parum malaria parasite consisting essentially of the con¬ jugate of claim 4.

10. A component of a vaccine against malaria consisting essentially of the conjugate of claim 6.

11. The conjugate of claim 4 wherein said epitope consists essentially of a tandem repeat of the amino acid sequence Asn-Ala-Asn-Pro from the C- to the N-terminal.

0

12. The conjugate of claim 11 wherein said peptide has been chemically synthesized.

13. The conjugate of claim 4 wherein said peptide has the amino acid sequence Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro- Asn-Ala-Asn-Pro from the N- to the C-terminal.

14. The conjugate of claim 13 wherein said protein is tetanus toxoid.

15. A component of a vaccine against the P.falci¬ parum malaria parasite consisting essentially of the conjugate of claim 11.

16. A component of a vaccine against the P.falci¬ parum malaria parasite consisting essentially of the conjugate of claim 13.

17. A component of the vaccine against malaria con¬ sisting essentially of the conjugate of claim 14.

18. The conjugate of claim 11 wherein said peptide has the sequence Cys-Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala- Asn-Pro from the N-terminal to the C-terminal.

19. The conjugate of claim 18 wherein said peptide has been chemically synthesized.

20. The conjugate of claim 11 wherein said peptide has the amino acid sequence Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro- Asn-Ala-Asn-Pro-Cys from the C-terminal to the N-terminal.

21. The conjugate of claim 20 wherein said peptide is chemically synthesized.

22. The conjugate of claim 18 wherein said peptide is tetanus toxoid.

23. The conjugate of claim 20 wherein said peptide is tetanus toxoid.

24. A component of the vaccine against the P.falci- parum malaria parasite consisting essentially of the conjugate of clatim 18.

25. A component of the vaccine against the P.falci- parum malaria parasite consisting essentially of the conjugate of claim 20.

26. A vaccine against the sporozoite stage of the P.falciparum malaria parasite comprising a conjugate of a peptide having the sequence Cys-Asn-Ala-Asn-Pro-Asn-Ala-Asn- Pro-Asn-Ala-Asn-Pro,

a carrier protein and a vaccine adjuvant, said vaccine suitable for immunizing mammals.