WO1991008756A1 - Novel malarial sporozoite peptide antigens - Google Patents

Novel malarial sporozoite peptide antigens Download PDF

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Publication number
WO1991008756A1
WO1991008756A1 PCT/US1990/007194 US9007194W WO9108756A1 WO 1991008756 A1 WO1991008756 A1 WO 1991008756A1 US 9007194 W US9007194 W US 9007194W WO 9108756 A1 WO9108756 A1 WO 9108756A1
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protein
sporozoites
antigenic
berghei
falciparum
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PCT/US1990/007194
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French (fr)
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Michael R. Hollingdale
Barbara Sina
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Biomedical Research Institute
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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 the isolation, identification and purification of a specific poly- peptide antigen, which when used as an inoculant elicits an immunogenicity and protection against malarial sporo ⁇ zoites.
  • the invention further relates to the use of this antigen as a vaccine for humans and animals.
  • Malaria is a multi-stage disease which is charac ⁇ teristically initiated by the bite of an infected anopheles mosquito. Through its bite, the mosquito injects malarial sporozoites into a host. The sporo ⁇ zoites rapidly invade the host's liver, and the parasite then multiplies asexually in the liver parenchymal cells.
  • the second phase of the disease involves the invasion of host red blood cells by the merozoites.
  • the erythrocytic phase of malaria is cyclic, as new merozoites are periodically released from ruptured red blood cells. New merozoites are also produced in the invaded host red blood cells. The red blood cells also rupture and release merozoites, which then invade more red blood cells.
  • the erythro ⁇ cytic phase of malaria is the symptomatic or clinical phase in which paroxysms of chills, fever and sweating are observed. Other characteristic symptoms include anemia, splenomegaly and a chronic relapsing course of symptoms.
  • the circumsporozoite protein of the major human species contains a central immunodominant repeat region containing the sequences (NANP) 37 (NVDP) 4 and flanking regions. Within the flanking regions, several short sequences have been described that function as T-cell epitomes, and one of these, Nl (Lys-Leu-Lys-Gln-Pro) , was identified by the inventors as the ligand by which sporozoites recognize hepatocyte receptors leading to sporozoite invasion. An Nl derived peptide, (Lys-Leu-Gln-Pro) is undergoing vaccine trials in mice. Circumsporozoite proteins are detected through- out exoerythrocytic development and may be a target of cytotoxic T-cell activity that may also serve to protect against sporozoite infection.
  • Identifying antigens specific to the sporozoite would be a significant step in developing an effective vaccine against malaria.
  • Such a vaccine which creates host immunity to the sporozoites would be especially useful, because it would protect the host at the earliest stage of the disease.
  • the present invention relates to novel antigenic proteins of the malarial sporozoite.
  • the proteins are found on the surface of either Plasmodium falciparum or Plasmodium berghei sporozoites.
  • the proteins have molecular weights of 42 kilodaltons (KD) and 54 KD, as measured by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) .
  • the 42 KD protein desig ⁇ nated Pf/Pb 42
  • has similar properties to the 54 KD protein designated Pf/Pb 54
  • Pf 42 isolated from P_j_.
  • falciparum is either identical to or substantially homologous to Pb 42 (isolated from £__. berghei)
  • Pf 54 is either identical to or substantially homologous to Pb 54 (isolated from P___ berghei)
  • the invention further relates to fragments or portions of these proteins which are also antigenic, including epitopes.
  • the gene(s) coding for the protein(s) have been cloned and sequenced.
  • the proteins from either species also immunoreact with antibodies from P___ falciparum or P___. berghei immunized mice and rabbits, and naturally protected humans. Furthermore, _j_ falci ⁇ parum antisera cross reacts with the P__. berghei anti ⁇ genic proteins and P___ berghei antisera cross-reacts with the P JJ . falciparum antigenic proteins. In purified form, the antigenic proteins may be used as vaccines for animals or humans. BRIEF DESCRIPTION OF THE FIGURES
  • Figure 1 shows an immunoelectron micrograph of P. falciparum sporozoites reacted with rabbit antibodies to P. berghei sporozoites;
  • Figure 2 shows an immunoelectron micrograph of P ⁇ . falciparum sporozoites reacted with mouse antibodies to P. berghia sporozoites;
  • Figure 3 shows control ⁇ berghei sporozoites reacted with mouse anti-circumsporozoite protein anti ⁇ bodies; and
  • Figure 4 is a representation of western blots in which P___ berghei and JE s _ falciparum antigens are reacted and cross-reacted with £___ berghei and £ ⁇ falciparum antisera.
  • the present invention relates to malarial sporo ⁇ zoite protein antigens or fragments thereof which are antigenic.
  • the proteins are related peptides or poly- peptides which can only be isolated from either P. falciparum sporozoites or P___. berghei sporozoites. Based on their isolation from P___ falciparum (Pf) sporozoites and E__. berghei (Pb) sporozoites, and their respective molecular weights of 42 KD and 54 KD, the related proteins are designated Pf/Pb 42 and Pf/Pb 54. The proteins are also described together herein due to their related properties, and when referred to together, are designated Pf/Pb 42/54.
  • Pf/Pb 42 has similar properties to Pf/Pb 54. Also Pf 42 (isolated from P___. falpiparum) is either identical to or substantially homologous to Pb 42 (isolated from P. berghei) . Similarly, Pf 54 is either identical to or substantially homologous to Pb 54.
  • the proteins are local ⁇ ized on the surface of P ⁇ . falciparum sporozoites.
  • _P__. falciparum sporozoites were reacted with rabbit antibodies to £__. berghei sporozoites
  • the £___ falciparum sporozoites were reacted with mouse antibodies to P_ s _ berghei sporozoites.
  • These cross-reactive antibodies label the surface of the sporozoites as shown by the black spots of gold particles.
  • Circumsporozoite protein is a known antigen on the surface of sporozoites.
  • the banded proteins from the SDS- PAGE molecular weight determination are transferred to a nitrocellulose filter and quenched with blotting buffer containing 5% bovine serum albumin. After wash ⁇ ing, the filter is added to blotting buffer containing a 1/100 dilution of mouse or rabbit antiserum. The filter is then inoculated in blotting buffer containing 125 I-labelled second antibody (i.e. either anti-mouse or anti-rabbit depending on the primary antibody) . Exposure of the filter to X-ray film reveals the antibody/antigen reaction of the 42 KD protein and the 54 KD protein.
  • I-labelled second antibody i.e. either anti-mouse or anti-rabbit depending on the primary antibody
  • falciparum sporozoites reacting with _j_ falciparum antisera fail to differentiate the 42 KD and 54 KD bands. This occurs because the known circumsporozoite protein of molecular weight about 44 KD reacts so strongly with the antisera that its band over ⁇ whelms the neighboring bands at 42 KD and 54 KD.
  • the circumsporozoite protein does not cross-react as do the 42 KD protein and the 54 KD pro- tein. Therefore, when £___ berghei sporozoites are reacted with E__. falciparum antisera, the bands at 42 KD and 54 KD show up on the Western blot. Similarly, when P. falciparum sporozoites are reacted with P berghei antisera, the 42 KD and 54 KD bands also show up on the Western blot.
  • the £__. falciparum sporozoites and P___. berghei sporo ⁇ zoites from which the Pf/Pb 42/54 proteins are isolated are produced using similar procedures. Particularly, female Anopheles Stephensi mosquitoes which are five days old are: 1) infected with £__. berghei sporozoites by feeding on £__. berghei-infected mice; or 2) infected with P. falciparum sporozoites by feeding on cultured P. falciparum-infected human blood contained in small glass chambers which are covered with a membrane. Both the ]______. falciparum sporozoites and the p___.
  • berg ⁇ hei sporozoites are recovered from the mosquitoes in the same manner. However, the P ⁇ berghei sporozoites are isolated from the mosquitoes 18 days following infection, whereas the P_j_ falciparum sporozoites are recovered 14 days following infection.
  • the sporozoite-infected salivary glands of the mosquitoes are dissected manually in a minimal volume of MEM medium containing 10% fetal bovine serum. The glands are then transferred to a Wheaton ® glass homogenizer and disrupted by several strokes. The sporozoites are released from the ruptured glands and counted in a hemo- cytometer. Finally, the sporozoites are pelleted in a microfuge for five minutes, and resuspended at a concentration of 5000 sporozoites per microliter.
  • the antigenic proteins (Pb 42, Pf 42, Pb 54 and Pf 54) are isolated and purified using conventional techniques. Suitable techniques include column chroma- tography, HPLC and immunoaffinity chromatography.
  • the antigenic proteins can also be produced by genetic engineering techniques. Briefly, the 42 KD and 54 KD bands on the SDS-PAGE gels are cut out, crushed and allowed to sit in buffer to elute the antigen/antibody complex. Alternatively, the Western blots are cut out and dissolved in DMSO. Trichloroacetic acid is then used, to precipitate the antigen/antibody complexes, and a solution of high salt concentration dissociates the complexes so that the antigens may be recovered.
  • CSP-2 molecular weight 42kd/54kd
  • CSP-2 encod ⁇ ing clones are identified by screening a mung bean nuclease-treated P. falciparum genomic DNA/lambda gtll library (McCutchan et al., Science 225 f 625 (1984)) with rabbit anti-P. berghei sporozoite antisera.
  • Lambda gtll clones produce fusion proteins containing / 8-galacto- sidase and the encoded malaria sequence which react with the corresponding antibody.
  • the DNA sequences of clones 3, 4 and 6 are set forth as SEQ. ID NO. 1, SEQ. ID NO. 2, and SEQ. ID NO. 3, respectively, in the sequnce listing.
  • the corresponding amino acids sequences are set forth as SEQ. ID NO. 4, SEQ. ID NO. 5 and SEQ. ID NO. 6, respec ⁇ tively, in the sequence listing.
  • Full-length DNA sequences are identified using the three clones as probes of genomic DNA, before or after amplification as described below.
  • a genomic DNA library is prepared by conventional techniques.
  • the three clones are confirmed to contain DNA sequences for CSP-2 by: 1.
  • 9-galactosidase fusion protein produced from the cloned sequence reacted specifically with the rabbit anti-P. berghei sporozoite antisera on a western blot.
  • Other E. coli proteins, 3-galactosidase alone and another 3-galactosidase-malaria antigen fusion protein did not react.
  • the malaria insert sequence from the lambda gtll clones is transferred to a maltose binding protein encoding plas id vector (New England Biolabs) .
  • Recombinant fusion protein is purified by amylose affinity chromatography and injected with complete Freund's adjuvent into outbred mice. Mice are boosted with the recombinant protein in incomplete Freund's adjuvent every two weeks. Mice immunized with the recombinant protein produced antibody which reacted with P. falci ⁇ parum and P. berghei CSP-2 on western blots of sporozoites.
  • the anti-recombinant protein mouse sera was also shown to block the invasion of HepG2 hepatoma cells in vitro, as did the anti-CSP-2 monoclonal antibody Mab 63.
  • N- and C- terminal amino acid sequences are determined using conventional techniques. Then oligonucleotide primers are produced for use in a poly erase catalyzed reaction ("PCR") (United States Patent 4,683,202) of genomic P. falciparum or P. berg ⁇ hei DNA. Fragments or portions of the proteins are also antigenic, and the a ino acid sequences of the fragments can also be determined.
  • the epitopes can be determined using conventional epitope mapping techniques. Such techniques have been described in Gullich, W. , et al., Biochem. 20. 2173 (1981); Grimm, R. , et al., Z. Natur- forsch 41c. 993 (1986); MacKenzie, D.
  • PCR can also be used to produce by genetic engineering the larger antigenic protein portions or fragments.
  • the smaller epitope specific fragments can be produced by genetic engineering or conventional peptide synthesis techniques.
  • the DNA coding for the Pf/Pb 42/54 proteins or fragments thereof is then cloned into appropriate replication or expression vectors in accordance with techniques well known in the art.
  • Expression vectors are produced using appropriate replicons and control sequences determined by the host utilized to produce the recombinant proteins.
  • the antisera used in reactions with the Pf/Pb 42/54 proteins are produced by a variety of techniques. These techniques include the biting of animals by infected mosquitoes, injection of attenuated sporozoites into animals, or injection of sporozoites to animals on chloroquine prophylaxis.
  • Vaccines are made from the purified antigenic proteins or by attaching a carrier to natural, synthe- sized or cloned Pf/Pb 42/54 proteins as described above.
  • Useful carriers include tetanus toxoid linked to the protein through tyrosine, Lipid A ® , hepatitis B antigen, keyhole limpet hemocyanin or any microorganism to which the protein may be linked.
  • tetanus toxoid linked to tyrosine is a preferred carrier.
  • the vaccines may further contain additional malarial antigens to produce a multi-valent vaccine.
  • a "live" vaccine is made by incorporating a DNA sequence coding for the Pf/Pb 42/54 proteins or antigenic fragments thereof into a suitable virus vector, such as vaccinia virus.
  • a DNA sequence is incorporated into vaccinia virus according to United States Patent 4,769,330 and Piccini, A. et al., Meth.Enzy ol. 153. 545 (1987), both incorporated herein by reference.
  • the modified vacinia virus may further contain DNA sequences coding for additional malarial antigenic peptides to produce a single, multivalent malarial vaccine.
  • a multivalent malarial vaccine may also be produced using several different modified vaccinia viruses, each containing a different malarial antigenic peptide.
  • Monoclonal antibodies can be produced according to established procedures (Kohler & Milstein, Nature 256, 495 (1975)). Briefly, female BALB/C mice are immunized intraperitoneally repeatedly with lysates of I ⁇ _ falci- paru - or P___ berghei-infected MOLT 3 cells emulsified in complete Freund's adjuvant (50%). Sensitized spleen cells are fused with P3X63-Ag8.653 myeloma cells using PEG 1500. Heterokaryons are selected in HAT medium, cloned and screened for reactivity to P___ falciparum and P. berghei antigens in a capture ELISA.
  • Anopheles Stephensi mosquitoes (female, 5 days old) were infected with P___ berghei by feeding on P___ berghei- infected mice.
  • Other A___ Stephensi mosquitoes (female, 5 days old) were infected with _P__, falciparum sporozoites by feeding on small glass chambers covered by a membrane and containing cultured £__. falciparum-infected human blood.
  • the sporozoite-infected salivary glands (either species) were then dissected manually in a minimal volume of MEM medium containing 10% fetal bovine serum. The glands were transferred to a Wheaton ® glass homo- genizer and disrupted by several strokes.
  • the sporo- zoites were then released from the ruptured glands, and counted in a hemocytometer. Finally, the sporozoites were pelleted in a microfuge for 5 minutes and resus- pended at a concentration of 5,000 sporozoites per microliter. The _______ berghei sporozoites were recovered 18 days after infection of the mosquitoes, and the p___ falciparum sporozoites were recovered 14 days after infection of the mosquitoes.
  • mice and rabbits were immunized against ______ berghei sporozoites and £_>_ falciparum sporozoites by mosquito bite or by injection.
  • Either P___ falciparum or p___ berghei-infected mosqui ⁇ toes were used to bite either outbred mice or rabbis. Each mouse was bitten by 10 mosquitoes, and each rabbit was bitten by 50 mosquitoes. This was done once a week for 4 weeks. Antisera was then collected from the mice and rabbits using conven ⁇ tional techniques.
  • Freshly isolated sporozoites from the salivary glands of infected Anopheles Stephensi mosquitoes were suspended at a concentration of 5,000/ microliter ( ⁇ l) in SDS sample buffer (10 ml final volume containing 0.5 mg 10% SDS, 62.5 ⁇ l 1M Tris-HCl (pH 6.8), 0.1 mg glycerol, 20 ⁇ l 0.1% Bromophenol blue). Ten ⁇ l of sample was then loaded per lane of gel (equivalent to 50,000 sporozoites).
  • the 10% SDS-polyacrylamide gel which was used was approximately 3 mm thick, 10 cm long and 14 cm wide, with 2.5 cm stacking gel and a 1 cm agarose plug to seal the bottom of the plates.
  • the 10% polyacrylamide gel solution for 1 gel con ⁇ tained: a) 10 ml 30% acrylamide solution; b) 7.6 ml lower gel buffer; c) 11.2 ml distilled water; d) 14 ⁇ l TEMED; and e) 200 ⁇ l 10% ammonium sulphate; and the 30% acrylamide solution contained: a) 29.2 g acrylamide; and b) 0.8 g bis-acrylamide.
  • the final volume of solution was 100 ml.
  • the samples were loaded under the running buffer in the top chamber. BRL ® prestained protein molecular weight standards were run in adjacent lanes. The gel was then electrophoresed at room temperature at 150 volts for about 6 hours to reveal the bands at 42 KD and 54 KD.
  • the running buffer contained: a) 6.05 g Tris base; b) 28.8 g glycine; and c) 3.0 g sodium dodecylsulphate; for a final volume of 2 liters.
  • the lower gel buffer contained: a) 1.5 M Tris-HCl (pH 8.8); and b) 0.6% sodium dodecylsulphate.
  • the stacking gel solution for 1 gel contained: a) 2.8 ml stacking gel buffer; b) 1.4 ml 30% acrylamide solution; c) 6.8 ml distilled water; d) 100 ⁇ l 10% ammonium persulphate; and e) 10.5 ⁇ l TEMED; and the stacking gel buffer contained: a) 0.5 M Tris-HCl (pH 6.8); and b) 0.4% sodium dodecylsulphate.
  • Example 3 The gel from Example 3 was removed from the plates. The stacking gel and agarose plug were removed with a scalpel. A gel-sized nitrocellulose filter soaked in trans ⁇ fer buffer was placed over the gel and sandwiched between saturated 3MM paper sheets in an electroblotting frame. The frame was placed in a transfer tank contain ⁇ ing 5 liters of transfer buffer and electroblotted over- night at 25 volts or for 6 hours at 50 volts, at 4°C.
  • the transfer buffer contained: a) 500ml 10 X stock; b) 1000 ml methanol; and c) 3500 ml distilled water; and the 10 X stock contained: a) 60 g Tris base; and b) 302.8 g glycine to make a final volume of 2 liters.
  • the filter was immedi- ately quenched with blotting buffer containing 5% bovine serum albumin (BSA) for 30 minutes. After washing twice with blotting buffer for 10 to 15 minutes, the filter was added to a minimal volume of blotting buffer con ⁇ taining a 1/100 dilution of mouse or rabbit antiserum of Example 2. After incubating at room temperature for 1-2 hours with agitation, the incubation was continued at 4°C overnight.
  • BSA bovine serum albumin
  • the filter was washed three times with blotting buffer and incubated with a minimal volume of blotting buffer contai •ni•ng 125I-labelled second antibody (i.e., either anti-mouse or anti-rabbit depending on the primary antibody) at about 5xl0 5 counts per minute per ml for 2-4 hours at room temperature with agitation.
  • the blot was washed extensively with blotting buffer and exposed to X-ray film to reveal the antibody/antigen reactions.
  • the blotting buffer contained: a) 10 mM Tris-HCl (pH 7.2); b) 1 mM EDTA; c) 150 mM NaCl; d) 0.1% Tween 20; and e) 0.1% BSA.
  • mice inoculated Three groups of Balb/c mice are inoculated. Each group receives a different preparation. The mice in Groups 1 and 2 are immunized with a vaccine, while the mice in Group 3 are controls.
  • Each group of immunized mice receives a different vaccine.
  • Group 1 receives the purified Pf/Pb 42 protein
  • Group 2 receives the purified Pf/Pb 54 protein.
  • the initial dose of each vaccine comprises 50 ⁇ g of vaccine in 0.1 ml of Freund's complete adjuvant.
  • Each group also receives two boosts of the vaccine comprising 50 ⁇ g in 0.1 ml of Freund's incomplete adjuvant. The boosts are given at intervals of two weeks.
  • the mice in control Group 3 did not receive anything.
  • Sera from the mice of each group is collected two weeks after the last boost of vaccine. The sera is then tested by immunofluorescence antibody assay and inhibi ⁇ tion of sporozoite invasion assay after challenge with P___ berghei sporozoites and challenge with P___ falciparum sporozoites.
  • mice exhibit protection against P. berghei sporozoite challenge and P___ falciparum sporo ⁇ zoite challenge.
  • mice in Control Group 3 do not exhibit any anti-sporozoite activity.
  • CSP-2 molecular weight 42kd/54kd
  • CSP-2 encoding clones were identified by screening a ung bean nuclease treated P. falciparum genomic DNAS/lambda gtll library (McCutchan et al.. Science 225. 625 (1984)) with rabbit anti-P. berghei sporozoite antisera.
  • Lambda gtll clones produce fusion proteins containing ⁇ -galactosi- dase and the encoded malaria sequence which react with the corresponding antibody.
  • Positive phage plaques were rescreened and purified. None of the positive clones reacted with the P. falciparum CS protein repeat sequence specific monoclonal antibody ZA10 (Nartin et al., J.EXP.Med. 156. 20-30 (1982)).
  • Clones 3, 4 and 6 were identified and found to contain malaria DNA inserts of approximately 120 bp, 144 bp and 120 bp, respectively. Each insert was subcloned into the EcoRI site of the plasmid pUCl ⁇ (Promega) for sequencing using the dideoxynucleotide method of Sanger et al., Proc.Nat.Acad.Sci.USA 74. 5463-67 (1977).
  • the DNA sequences and corresponding amino acid sequences of clones 3, 4 and 6 are set forth in the sequence listing as SEQ. ID NO. 1, SEQ. ID NO. 2 and SEQ. ID NO. 3, res- pectively (for DNA sequences) and SEQ. ID NO. 4, SEQ. ID NO.
  • Clones 3, 4 and 6 were confirmed to encode CSP-2 peptides by the following experiments. 1. /3-galactosidase fusion proteins produced from the cloned sequences reacted specifically with the rabbit anti-P. berghei sporozoite antisera on a western blot. Other E. coli proteins, such as ⁇ -galactosidase alone and another 3-galactosidase-malaria antigen fusion protein, did not react.
  • the malaria insert sequences from the lambda gtll clones were transferred to maltose binding protein encoding plasmid vectors (New England Biolabs) .
  • Recombinant fusion proteins were purified by amylose affinity chromatography and injected with complete Freund's adjuvent into outbred mice. Mice were boosted with the recombinant proteins in incomplete Freund's adjuvent every two weeks. Mice immunized with the recombinant protein produced antibody which reacted with P. falci ⁇ parum and P. berghei CSP-2 on western blots of sporo- zoites.
  • the anti-recombinant protein mouse sera was also shown to block the invasion of HepG2 hepatoma cells in vitro f as did the anti-CSP-2 monoclonal antibody Mab 63.
  • ADDRESS Venable, Baetjer, Howard & Civiletti

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Abstract

The present invention relates to novel malarial sporozoite peptide antigens and DNA sequences coding therefor. The antigenic proteins are located on the surface of either Plasmodium falciparum sporozoites or Plasmodium berghei sporozoites, and have molecular weights of 42 kilodaltons and 54 kilodaltons. P. falciparum antisera cross-reacts with the antigenic proteins or P. berghei sporozoites and P. berghei antisera cross-reacts with the antigenic proteins or P. falciparum sporozoites.

Description

TITLE OF THE INVENTION
NOVEL MALARIAL SPOROZOITE PEPTIDE ANTIGENS
BACKGROUND OF THE INVENTION
The present invention relates to the isolation, identification and purification of a specific poly- peptide antigen, which when used as an inoculant elicits an immunogenicity and protection against malarial sporo¬ zoites. The invention further relates to the use of this antigen as a vaccine for humans and animals. Malaria is a multi-stage disease which is charac¬ teristically initiated by the bite of an infected anopheles mosquito. Through its bite, the mosquito injects malarial sporozoites into a host. The sporo¬ zoites rapidly invade the host's liver, and the parasite then multiplies asexually in the liver parenchymal cells. Following the multiplication, there is a period of maturation which typically lasts from five days to four weeks, after which the liver cells rupture and release merozoites into the host's blood. During the above-described stages of the disease the host is asymptomatic, and the disease is referred to as being in its exoerythrocytic phase.
The second phase of the disease, referred to as the erythroσytic phase, involves the invasion of host red blood cells by the merozoites. The erythrocytic phase of malaria is cyclic, as new merozoites are periodically released from ruptured red blood cells. New merozoites are also produced in the invaded host red blood cells. The red blood cells also rupture and release merozoites, which then invade more red blood cells. The erythro¬ cytic phase of malaria is the symptomatic or clinical phase in which paroxysms of chills, fever and sweating are observed. Other characteristic symptoms include anemia, splenomegaly and a chronic relapsing course of symptoms. It has been observed that humans in malaria endemic regions develop anti-sporozoite antibodies which are thought to be protective, and which recognize the circumsporozoite protein. The circumsporozoite proteins of many malaria (Plasmodium) species have been cloned and sequenced, and candidate subunit vaccines have been tested for immunogenicity and protection in rodents, and in human clinical trials.
The circumsporozoite protein of the major human species, Plasmodium falciparum. contains a central immunodominant repeat region containing the sequences (NANP)37(NVDP)4 and flanking regions. Within the flanking regions, several short sequences have been described that function as T-cell epitomes, and one of these, Nl (Lys-Leu-Lys-Gln-Pro) , was identified by the inventors as the ligand by which sporozoites recognize hepatocyte receptors leading to sporozoite invasion. An Nl derived peptide, (Lys-Leu-Gln-Pro) is undergoing vaccine trials in mice. Circumsporozoite proteins are detected through- out exoerythrocytic development and may be a target of cytotoxic T-cell activity that may also serve to protect against sporozoite infection.
As recognized by Nussenzweig et al. (United States Patent 4,466,917), the major problem in attempting to develop antibodies capable of inactivating sporozoites is that sporozoites may be obtained only from infected mosquitoes, and therefore are available in extremely limited quantity. However, it has been found that the sporozoites are highly antigenic (see Mulligan et al., J. Malar. Inst. India 4., 25 (1941) ; Nussenzweig et al. , Nature 216. 160 (1967) ; Vanderberg et al., J. Paraistol. 54. 1175 (1968); and Nussenzweig et al., Mil. Med. 134. 1176 (1979)).
Identifying antigens specific to the sporozoite would be a significant step in developing an effective vaccine against malaria. Such a vaccine which creates host immunity to the sporozoites would be especially useful, because it would protect the host at the earliest stage of the disease.
SUMMARY OF THE INVENTION
The present invention relates to novel antigenic proteins of the malarial sporozoite. The proteins are found on the surface of either Plasmodium falciparum or Plasmodium berghei sporozoites. The proteins have molecular weights of 42 kilodaltons (KD) and 54 KD, as measured by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) . The 42 KD protein (desig¬ nated Pf/Pb 42) has similar properties to the 54 KD protein (designated Pf/Pb 54) and are sometimes referred to herein as related polypeptides or related proteins. Furthermore, Pf 42 (isolated from P_j_. falciparum) is either identical to or substantially homologous to Pb 42 (isolated from £__. berghei) , and Pf 54 (isolated from P. falciparum) is either identical to or substantially homologous to Pb 54 (isolated from P___ berghei) . The invention further relates to fragments or portions of these proteins which are also antigenic, including epitopes. The gene(s) coding for the protein(s) have been cloned and sequenced.
The proteins from either species (P^. falciparum or P. berghei) also immunoreact with antibodies from P___ falciparum or P___. berghei immunized mice and rabbits, and naturally protected humans. Furthermore, _j_ falci¬ parum antisera cross reacts with the P__. berghei anti¬ genic proteins and P___ berghei antisera cross-reacts with the PJJ. falciparum antigenic proteins. In purified form, the antigenic proteins may be used as vaccines for animals or humans. BRIEF DESCRIPTION OF THE FIGURES
The various objects, advantages and novel features of the invention will be more readily appreciated from the following detailed description when read in co junc- tion with the appended figures, in which:
Figure 1 shows an immunoelectron micrograph of P. falciparum sporozoites reacted with rabbit antibodies to P. berghei sporozoites;
Figure 2 shows an immunoelectron micrograph of P^. falciparum sporozoites reacted with mouse antibodies to P. berghia sporozoites;
Figure 3 shows control ^ berghei sporozoites reacted with mouse anti-circumsporozoite protein anti¬ bodies; and Figure 4 is a representation of western blots in which P___ berghei and JEs_ falciparum antigens are reacted and cross-reacted with £___ berghei and £^ falciparum antisera.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to malarial sporo¬ zoite protein antigens or fragments thereof which are antigenic. The proteins are related peptides or poly- peptides which can only be isolated from either P. falciparum sporozoites or P___. berghei sporozoites. Based on their isolation from P___ falciparum (Pf) sporozoites and E__. berghei (Pb) sporozoites, and their respective molecular weights of 42 KD and 54 KD, the related proteins are designated Pf/Pb 42 and Pf/Pb 54. The proteins are also described together herein due to their related properties, and when referred to together, are designated Pf/Pb 42/54.
Pf/Pb 42 has similar properties to Pf/Pb 54. Also Pf 42 (isolated from P___. falpiparum) is either identical to or substantially homologous to Pb 42 (isolated from P. berghei) . Similarly, Pf 54 is either identical to or substantially homologous to Pb 54.
As seen in Figures 1 and 2, the proteins are local¬ ized on the surface of P^. falciparum sporozoites. In Figure 1, _P__. falciparum sporozoites were reacted with rabbit antibodies to £__. berghei sporozoites, and in Figure 2, the £___ falciparum sporozoites were reacted with mouse antibodies to P_s_ berghei sporozoites. These cross-reactive antibodies label the surface of the sporozoites as shown by the black spots of gold particles.
Particularly, fixed parasites were sectioned and then reacted with the primary antibody, followed by rabbit anti-mouse antibodies if required. Finally, the parasites were reacted with protein A conjugated to colloidal gold.
The control Pi berghei sporozoites shown in Figure 3 were reacted with mouse anti-circumsporozoite protein antibodies. Circumsporozoite protein is a known antigen on the surface of sporozoites.
Although not shown in the figures, similar reac¬ tivity to that seen in Figure 1 was observed when rabbit antibodies to p___ falciparum sporozoites were reacted with ;_____. berghei sporozoites. The molecular weights of these related proteins were found to be 42 KD and 54 KD as measured by SDS- PAGE. Particularly, fresh sporozoites were isolated from the salivary glands of infected Anopheles Stephensi mosquitoes, and suspended in SDS sample buffer at a con- centration of 5,000 sporozoites/microliter (μl) . Ten μl of sample (i.e. 50,000 sporozoites) was then loaded per lane of 10% SDS-polyacrylamide gel, and run next to BRL® prestained protein molecular weight standard in adjacent lanes. The molecular weights of the proteins (42 KD and 54 KD) coincide with the range of molecular weights of well characterized circumsporozoite proteins of both the P. falciparum species and p___ berghei species. There¬ fore, Pf/Pb 42/54 was initially identified as a cross- reactive antigen (i.e. sera from mice or rabbits immunized with the protein of one species detect the protein in the other species) .
However, Western blotting techniques further clarified the properties and distinctiveness of Pf/Pb 42/54. As shown in the Western blots of Figure 4, P. berghei sporozoites reacted with sera from £_»_ fal- ciparum-immunized mice and rabbits. Furthermore, sera from naturally protected humans also recognizes the 42 KD and 54 KD proteins.
Particularly, the banded proteins from the SDS- PAGE molecular weight determination are transferred to a nitrocellulose filter and quenched with blotting buffer containing 5% bovine serum albumin. After wash¬ ing, the filter is added to blotting buffer containing a 1/100 dilution of mouse or rabbit antiserum. The filter is then inoculated in blotting buffer containing 125I-labelled second antibody (i.e. either anti-mouse or anti-rabbit depending on the primary antibody) . Exposure of the filter to X-ray film reveals the antibody/antigen reaction of the 42 KD protein and the 54 KD protein.
It has also been found that the Pj_. falciparum sporozoites react with sera from P___ berghei-inoculated rabbits or mice that have been kept on chloroquine prophylaxis. Furthermore, the P___ falciparum sporozoites react with sera from mice which were inoculated with gamma-irradiated attenuated P___ berghei sporozoites. The antigenic proteins (Pf 42, Pf 54, Pb 42 and Pb 54) were identified due to their cross reactivity. Western blots of ^ berghei sporozoites reacting with P. berghei antisera, and £__. falciparum sporozoites reacting with _j_ falciparum antisera fail to differentiate the 42 KD and 54 KD bands. This occurs because the known circumsporozoite protein of molecular weight about 44 KD reacts so strongly with the antisera that its band over¬ whelms the neighboring bands at 42 KD and 54 KD.
However, the circumsporozoite protein does not cross-react as do the 42 KD protein and the 54 KD pro- tein. Therefore, when £___ berghei sporozoites are reacted with E__. falciparum antisera, the bands at 42 KD and 54 KD show up on the Western blot. Similarly, when P. falciparum sporozoites are reacted with P berghei antisera, the 42 KD and 54 KD bands also show up on the Western blot.
The £__. falciparum sporozoites and P___. berghei sporo¬ zoites from which the Pf/Pb 42/54 proteins are isolated are produced using similar procedures. Particularly, female Anopheles Stephensi mosquitoes which are five days old are: 1) infected with £__. berghei sporozoites by feeding on £__. berghei-infected mice; or 2) infected with P. falciparum sporozoites by feeding on cultured P. falciparum-infected human blood contained in small glass chambers which are covered with a membrane. Both the ]_____. falciparum sporozoites and the p___. berg¬ hei sporozoites are recovered from the mosquitoes in the same manner. However, the P^ berghei sporozoites are isolated from the mosquitoes 18 days following infection, whereas the P_j_ falciparum sporozoites are recovered 14 days following infection.
The sporozoite-infected salivary glands of the mosquitoes are dissected manually in a minimal volume of MEM medium containing 10% fetal bovine serum. The glands are then transferred to a Wheaton® glass homogenizer and disrupted by several strokes. The sporozoites are released from the ruptured glands and counted in a hemo- cytometer. Finally, the sporozoites are pelleted in a microfuge for five minutes, and resuspended at a concentration of 5000 sporozoites per microliter. The antigenic proteins (Pb 42, Pf 42, Pb 54 and Pf 54) are isolated and purified using conventional techniques. Suitable techniques include column chroma- tography, HPLC and immunoaffinity chromatography. The antigenic proteins can also be produced by genetic engineering techniques. Briefly, the 42 KD and 54 KD bands on the SDS-PAGE gels are cut out, crushed and allowed to sit in buffer to elute the antigen/antibody complex. Alternatively, the Western blots are cut out and dissolved in DMSO. Trichloroacetic acid is then used, to precipitate the antigen/antibody complexes, and a solution of high salt concentration dissociates the complexes so that the antigens may be recovered.
A portion of the gene encoding the P. falciparum sporozoite antigenic protein CSP-2 (molecular weight 42kd/54kd) has been cloned and sequenced. CSP-2 encod¬ ing clones are identified by screening a mung bean nuclease-treated P. falciparum genomic DNA/lambda gtll library (McCutchan et al., Science 225f 625 (1984)) with rabbit anti-P. berghei sporozoite antisera. Lambda gtll clones produce fusion proteins containing /8-galacto- sidase and the encoded malaria sequence which react with the corresponding antibody. Positive phage plaques were rescreened and purified. None of the positive clones reacted with the £. falciparum CS protein repeat sequence specific monoclonal antibody 2A10 (Nardin et al., J.Exp.Med. 156. 20-30 (1982)). Three clones are identified as clones 3, 4 and 6, and are found to contain malaria DNA inserts of approxi¬ mately 120 bp, 144 bp and 120 bp, respectivelyj. Each insert is subcloned into the EcoRI site of the plasmid pϋC18 (Promega) for sequencing using the dideoxynucleo- tide method of Sanger et al. (Proc.Nat.Acad.Sci.USA 74. 5143-5467 (1977)). The DNA sequences of clones 3, 4 and 6 are set forth as SEQ. ID NO. 1, SEQ. ID NO. 2, and SEQ. ID NO. 3, respectively, in the sequnce listing. The corresponding amino acids sequences are set forth as SEQ. ID NO. 4, SEQ. ID NO. 5 and SEQ. ID NO. 6, respec¬ tively, in the sequence listing. Full-length DNA sequences are identified using the three clones as probes of genomic DNA, before or after amplification as described below. A genomic DNA library is prepared by conventional techniques. The three clones are confirmed to contain DNA sequences for CSP-2 by: 1. 9-galactosidase fusion protein produced from the cloned sequence reacted specifically with the rabbit anti-P. berghei sporozoite antisera on a western blot. Other E. coli proteins, 3-galactosidase alone and another 3-galactosidase-malaria antigen fusion protein, did not react.
2. /9-galactosidase fusion proteins produced from the cloned sequence are used to affinity-purify CSP-2 specific antibody. Antibody bound by the fusion protein after incubation with rabbit anti-P. berghei sporozoite antisera is eluted and subsequently is shown to react only with CSP-2 on western blots of P. falciparum sporozoites.
3. For high levels of expression and rapid puri¬ fication of large quantities of recombinant protein, the malaria insert sequence from the lambda gtll clones is transferred to a maltose binding protein encoding plas id vector (New England Biolabs) . Recombinant fusion protein is purified by amylose affinity chromatography and injected with complete Freund's adjuvent into outbred mice. Mice are boosted with the recombinant protein in incomplete Freund's adjuvent every two weeks. Mice immunized with the recombinant protein produced antibody which reacted with P. falci¬ parum and P. berghei CSP-2 on western blots of sporozoites. The anti-recombinant protein mouse sera was also shown to block the invasion of HepG2 hepatoma cells in vitro, as did the anti-CSP-2 monoclonal antibody Mab 63.
Alternatively, the N- and C- terminal amino acid sequences are determined using conventional techniques. Then oligonucleotide primers are produced for use in a poly erase catalyzed reaction ("PCR") (United States Patent 4,683,202) of genomic P. falciparum or P. berg¬ hei DNA. Fragments or portions of the proteins are also antigenic, and the a ino acid sequences of the fragments can also be determined. The epitopes can be determined using conventional epitope mapping techniques. Such techniques have been described in Gullich, W. , et al., Biochem. 20. 2173 (1981); Grimm, R. , et al., Z. Natur- forsch 41c. 993 (1986); MacKenzie, D. et al., Biochem. 23. 6544 (1984); Tzartos, S. et al., Proc. Natl. Acad. Sci. USA 85. 2899 (1988); Mehra, V. et al. , Proc. Natl. Acad. Sci. USA 83. 7013 (1986) ; and Nunberg, J., et al., Proc. Natl. Acad. Sci. USA 81. 3675 (1984) . PCR can also be used to produce by genetic engineering the larger antigenic protein portions or fragments. The smaller epitope specific fragments can be produced by genetic engineering or conventional peptide synthesis techniques.
The DNA coding for the Pf/Pb 42/54 proteins or fragments thereof is then cloned into appropriate replication or expression vectors in accordance with techniques well known in the art. Expression vectors are produced using appropriate replicons and control sequences determined by the host utilized to produce the recombinant proteins. The antisera used in reactions with the Pf/Pb 42/54 proteins are produced by a variety of techniques. These techniques include the biting of animals by infected mosquitoes, injection of attenuated sporozoites into animals, or injection of sporozoites to animals on chloroquine prophylaxis.
When rabbits or mice are subjected to the biting of infected mosquitoes to produce antisera, outbred rabbits are bitten by 50 infected mosquitoes (P_j_ falciparum or P. berghei) once a week for four weeks, and outbred mice are bitten by 10 infected mosquitoes once a week for four weeks. However, when attenuated sporozoites are used to produce antisera, P___ falciparum- or _______ berghei- infected mosquitoes are subjected to gamma-irradiation with a 60Co source. The mosquito salivary glands are then dissected, and the sporozoites recovered and counted. Then, 50,000-100,000 attenuated sporozoites are intravenously inoculated into each animal once a week for four weeks.
Vaccines are made from the purified antigenic proteins or by attaching a carrier to natural, synthe- sized or cloned Pf/Pb 42/54 proteins as described above. Useful carriers include tetanus toxoid linked to the protein through tyrosine, Lipid A®, hepatitis B antigen, keyhole limpet hemocyanin or any microorganism to which the protein may be linked. However tetanus toxoid linked to tyrosine is a preferred carrier. The vaccines may further contain additional malarial antigens to produce a multi-valent vaccine. Alternatively, a "live" vaccine is made by incorporating a DNA sequence coding for the Pf/Pb 42/54 proteins or antigenic fragments thereof into a suitable virus vector, such as vaccinia virus. A DNA sequence is incorporated into vaccinia virus according to United States Patent 4,769,330 and Piccini, A. et al., Meth.Enzy ol. 153. 545 (1987), both incorporated herein by reference. The modified vacinia virus may further contain DNA sequences coding for additional malarial antigenic peptides to produce a single, multivalent malarial vaccine. A multivalent malarial vaccine may also be produced using several different modified vaccinia viruses, each containing a different malarial antigenic peptide.
Monoclonal antibodies can be produced according to established procedures (Kohler & Milstein, Nature 256, 495 (1975)). Briefly, female BALB/C mice are immunized intraperitoneally repeatedly with lysates of I\_ falci- paru - or P___ berghei-infected MOLT 3 cells emulsified in complete Freund's adjuvant (50%). Sensitized spleen cells are fused with P3X63-Ag8.653 myeloma cells using PEG 1500. Heterokaryons are selected in HAT medium, cloned and screened for reactivity to P___ falciparum and P. berghei antigens in a capture ELISA. The IgG frac¬ tion of polyclonal human anti-P. falciparum and poly- clonal mouse anti-P. berghei are coated onto wells of microtiter dishes. £__. falciparum and £__. berghei (pro¬ duced in MOLT 3 cells) and culture supernatants are added simultaneously to the wells. Bound urine anti¬ bodies are detected with an enzyme-labelled anti-mouse Ig antibody.
The present invention is further illustrated by reference to the following examples. These examples are provided for illustrative purposes, and are in no way intended to limit the scope of the invention.
EXAMPLE 1
Isolation of £__, falciparum Sporozoites and P. berghei Sporozoites
Anopheles Stephensi mosquitoes (female, 5 days old) were infected with P___ berghei by feeding on P___ berghei- infected mice. Other A___ Stephensi mosquitoes (female, 5 days old) were infected with _P__, falciparum sporozoites by feeding on small glass chambers covered by a membrane and containing cultured £__. falciparum-infected human blood. The sporozoite-infected salivary glands (either species) were then dissected manually in a minimal volume of MEM medium containing 10% fetal bovine serum. The glands were transferred to a Wheaton® glass homo- genizer and disrupted by several strokes. The sporo- zoites were then released from the ruptured glands, and counted in a hemocytometer. Finally, the sporozoites were pelleted in a microfuge for 5 minutes and resus- pended at a concentration of 5,000 sporozoites per microliter. The _______ berghei sporozoites were recovered 18 days after infection of the mosquitoes, and the p___ falciparum sporozoites were recovered 14 days after infection of the mosquitoes.
EXAMPLE 2
Immunization of Animals to £__, berghei Sporozoites and P. falciparum Sporozoites
Mice and rabbits were immunized against ______ berghei sporozoites and £_>_ falciparum sporozoites by mosquito bite or by injection.
A. By Bite
Either P___ falciparum or p___ berghei-infected mosqui¬ toes (from Example 1 above) were used to bite either outbred mice or rabbis. Each mouse was bitten by 10 mosquitoes, and each rabbit was bitten by 50 mosquitoes. This was done once a week for 4 weeks. Antisera was then collected from the mice and rabbits using conven¬ tional techniques.
B. By Injection P. berghei-infected mosquitoes (from Example 1) were gamma-irradiated with a ^Co source. The mosquitoes were then dissected as above in Example 1, sporozoites were counted, and 50,000-100,000 sporozoites were inoculated intravenously into each mouse. This was repeated weekly for four weeks. Antisera was then collected from the mice using conventional techniques.
EXAMPLE 3
Molecular Weight Determination of Pf/Pb 42/54
Freshly isolated sporozoites from the salivary glands of infected Anopheles Stephensi mosquitoes (Exa ple 1) were suspended at a concentration of 5,000/ microliter (μl) in SDS sample buffer (10 ml final volume containing 0.5 mg 10% SDS, 62.5 μl 1M Tris-HCl (pH 6.8), 0.1 mg glycerol, 20 μl 0.1% Bromophenol blue). Ten μl of sample was then loaded per lane of gel (equivalent to 50,000 sporozoites).
The 10% SDS-polyacrylamide gel which was used was approximately 3 mm thick, 10 cm long and 14 cm wide, with 2.5 cm stacking gel and a 1 cm agarose plug to seal the bottom of the plates.
The 10% polyacrylamide gel solution for 1 gel con¬ tained: a) 10 ml 30% acrylamide solution; b) 7.6 ml lower gel buffer; c) 11.2 ml distilled water; d) 14 μl TEMED; and e) 200 μl 10% ammonium sulphate; and the 30% acrylamide solution contained: a) 29.2 g acrylamide; and b) 0.8 g bis-acrylamide.
The final volume of solution was 100 ml.
The samples were loaded under the running buffer in the top chamber. BRL® prestained protein molecular weight standards were run in adjacent lanes. The gel was then electrophoresed at room temperature at 150 volts for about 6 hours to reveal the bands at 42 KD and 54 KD.
The running buffer contained: a) 6.05 g Tris base; b) 28.8 g glycine; and c) 3.0 g sodium dodecylsulphate; for a final volume of 2 liters.
The lower gel buffer contained: a) 1.5 M Tris-HCl (pH 8.8); and b) 0.6% sodium dodecylsulphate. The stacking gel solution for 1 gel contained: a) 2.8 ml stacking gel buffer; b) 1.4 ml 30% acrylamide solution; c) 6.8 ml distilled water; d) 100 μl 10% ammonium persulphate; and e) 10.5 μl TEMED; and the stacking gel buffer contained: a) 0.5 M Tris-HCl (pH 6.8); and b) 0.4% sodium dodecylsulphate.
EXAMPLE 4
Western Blots of Pf/Pb 42/54
The gel from Example 3 was removed from the plates. The stacking gel and agarose plug were removed with a scalpel. A gel-sized nitrocellulose filter soaked in trans¬ fer buffer was placed over the gel and sandwiched between saturated 3MM paper sheets in an electroblotting frame. The frame was placed in a transfer tank contain¬ ing 5 liters of transfer buffer and electroblotted over- night at 25 volts or for 6 hours at 50 volts, at 4°C. The transfer buffer contained: a) 500ml 10 X stock; b) 1000 ml methanol; and c) 3500 ml distilled water; and the 10 X stock contained: a) 60 g Tris base; and b) 302.8 g glycine to make a final volume of 2 liters.
After the electroblotting, the filter was immedi- ately quenched with blotting buffer containing 5% bovine serum albumin (BSA) for 30 minutes. After washing twice with blotting buffer for 10 to 15 minutes, the filter was added to a minimal volume of blotting buffer con¬ taining a 1/100 dilution of mouse or rabbit antiserum of Example 2. After incubating at room temperature for 1-2 hours with agitation, the incubation was continued at 4°C overnight. The filter was washed three times with blotting buffer and incubated with a minimal volume of blotting buffer contai •ni•ng 125I-labelled second antibody (i.e., either anti-mouse or anti-rabbit depending on the primary antibody) at about 5xl05 counts per minute per ml for 2-4 hours at room temperature with agitation. The blot was washed extensively with blotting buffer and exposed to X-ray film to reveal the antibody/antigen reactions.
The blotting buffer contained: a) 10 mM Tris-HCl (pH 7.2); b) 1 mM EDTA; c) 150 mM NaCl; d) 0.1% Tween 20; and e) 0.1% BSA.
EXAMPLE 5
Anti-Sporozoite Immunogenicity Elicited By Vaccines
Comprising the Pf/Pb 42/54 Antigen
Three groups of Balb/c mice are inoculated. Each group receives a different preparation. The mice in Groups 1 and 2 are immunized with a vaccine, while the mice in Group 3 are controls.
Each group of immunized mice receives a different vaccine. Group 1 receives the purified Pf/Pb 42 protein, and Group 2 receives the purified Pf/Pb 54 protein. The initial dose of each vaccine comprises 50 μg of vaccine in 0.1 ml of Freund's complete adjuvant. Each group also receives two boosts of the vaccine comprising 50μg in 0.1 ml of Freund's incomplete adjuvant. The boosts are given at intervals of two weeks. The mice in control Group 3 did not receive anything. Sera from the mice of each group is collected two weeks after the last boost of vaccine. The sera is then tested by immunofluorescence antibody assay and inhibi¬ tion of sporozoite invasion assay after challenge with P___ berghei sporozoites and challenge with P___ falciparum sporozoites.
The Groups 1 and 2 mice exhibit protection against P. berghei sporozoite challenge and P___ falciparum sporo¬ zoite challenge. The mice in Control Group 3 do not exhibit any anti-sporozoite activity.
EXAMPLE 6 Cloning the CSP-2 Gene
A portion of the gene encoding the P. falciparum sporozoite antigenic protein CSP-2 (molecular weight 42kd/54kd) was cloned and sequenced as follows. CSP-2 encoding clones were identified by screening a ung bean nuclease treated P. falciparum genomic DNAS/lambda gtll library (McCutchan et al.. Science 225. 625 (1984)) with rabbit anti-P. berghei sporozoite antisera. Lambda gtll clones produce fusion proteins containing ø-galactosi- dase and the encoded malaria sequence which react with the corresponding antibody. Positive phage plaques were rescreened and purified. None of the positive clones reacted with the P. falciparum CS protein repeat sequence specific monoclonal antibody ZA10 (Nartin et al., J.EXP.Med. 156. 20-30 (1982)).
Clones 3, 4 and 6 were identified and found to contain malaria DNA inserts of approximately 120 bp, 144 bp and 120 bp, respectively. Each insert was subcloned into the EcoRI site of the plasmid pUClδ (Promega) for sequencing using the dideoxynucleotide method of Sanger et al., Proc.Nat.Acad.Sci.USA 74. 5463-67 (1977). The DNA sequences and corresponding amino acid sequences of clones 3, 4 and 6 are set forth in the sequence listing as SEQ. ID NO. 1, SEQ. ID NO. 2 and SEQ. ID NO. 3, res- pectively (for DNA sequences) and SEQ. ID NO. 4, SEQ. ID NO. 5 and SEQ. ID NO. 6, respectively (for the amino acid sequence) . Clones 3, 4 and 6 were confirmed to encode CSP-2 peptides by the following experiments. 1. /3-galactosidase fusion proteins produced from the cloned sequences reacted specifically with the rabbit anti-P. berghei sporozoite antisera on a western blot. Other E. coli proteins, such as β-galactosidase alone and another 3-galactosidase-malaria antigen fusion protein, did not react.
2. ,3-galactosidase fusion proteins produced from the cloned sequences were used to affinity-purify CSP-2 specific antibody. Antibody bound by the fusion protein after incubation with rabbit anti-P. berghei sporozoite antisera was eluted and subsequently was shown to react only with CSP-2 on western blots of P. falciparum sporo¬ zoites.
3. For high levels of expression and rapid puri¬ fication of large quantities of recombinant protein, the malaria insert sequences from the lambda gtll clones were transferred to maltose binding protein encoding plasmid vectors (New England Biolabs) . Recombinant fusion proteins were purified by amylose affinity chromatography and injected with complete Freund's adjuvent into outbred mice. Mice were boosted with the recombinant proteins in incomplete Freund's adjuvent every two weeks. Mice immunized with the recombinant protein produced antibody which reacted with P. falci¬ parum and P. berghei CSP-2 on western blots of sporo- zoites. The anti-recombinant protein mouse sera was also shown to block the invasion of HepG2 hepatoma cells in vitrof as did the anti-CSP-2 monoclonal antibody Mab 63.
While the invention has been described in connec- tion with specific embodiments thereof, it will be understood that it is capable of further modifications. This disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims.
SEOUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANTS: Michael R. Hollingdale
Barbara Sina
(ii) TITLE OF THE INVENTION: Novel Malarial
Sporozoite Peptide Antigens
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS
(A) ADDRESS: Venable, Baetjer, Howard & Civiletti
(B) STREET: 1201 New York Avenue, N.W. ,
Suite 1000
(C) CITY: Washington
(D) STATE: District of Columbia (E) COUNTRY: United States of America
(F) ZIP: 20005
(V) COMPUTER READABLE FORM
(A) MEDIUM TYPE:
(B) COMPUTER: (C) OPERATING SYSTEM:
(D) SOFTWARE:
(vi) CURRENT APPLICATION DATA
(A) APPLICATION NO. :
(B) FILING DATE: (C) CLASSIFICATION:
(vii) ATTORNEY/AGENT INFORMATION
(A) NAME: Jeffrey L. Ihnen
(B) REG. NO.: 28,957
(C) REFERENCE/DOCKET NO. : 18602-91240 (viϋ) TELECOMMUNICATION INFORMATION
(A) TELEPHONE: (202) 962-4810
(B) TELECOPIER: (202) 962-8300
(2) INFORMATION FOR SEQ. ID NO. 1:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 117 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(iii) SEQUENCE DESCRIPTION: SEQ. ID NO. 1
GC AGC AAC AAC AAC AAC AAC AGC AGC AAC AAC AGC AAC 39 AGC AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC 78 AGC AAC AAC AAC AAC AAC AAC AAC AGC AAC AAC AAC AGC 117
(2) INFORMATION FOR SEQ. ID NO. 2:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 144 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(iii) SEQUENCE DESCRIPTION: SEQ. ID NO. 2
AGC AAC AAC AAC AAC AAC AGC AGC AAC AAC AGC AAC AGC 39
AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC AGC 78 AAC AAC AAC AAC AAC AAC AAC AAC AGC AAC AAC AAC AGC 117
AAC AAC AAC AAC AAC AAC AAC AGC AAC 144 (2) INFORMATION FOR SEQ. ID NO. 3:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 120 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(iii) SEQUENCE DESCRIPTION: SEQ. ID NO. 3
AGC AGC AAC AAC AAC AAC AAC AGC AGC AAC AAC AGC AAC 39 AGC AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC AGC 78
AGC AAC AAC AAC AAC AAC AAC AAC AAC AGC AAC AAC AAC 117
AGC 120
(2) INFORMATION FOR SEQ. ID NO. 4:
(i) SEQUENCE CHARACTERISTICS (A) LENGTH: 39 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) SEQUENCE DESCRIPTION: SEQ. ID NO. 4
Ser Ser Asn Asn Asn Asn Asn Ser Ser Asn Asn Ser Asn 13 Ser Asn Asn Asn. Asn Asn Asn Asn Asn Asn Asn Asn Asn 26 Ser Asn Asn Asn Asn Asn Asn Asn Ser Asn Asn Asn Ser 39
(2) INFORMATION FOR SEQ. ID NO. 5:
(i) SEQUENCE CHARACTERISTICS (A) LENGTH: 48 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) SEQUENCE DESCRIPTION: SEQ. ID NO. 5
Ser Asn Asn Asn Asn Asn Ser Ser Asn Asn Ser Asn Ser 13 Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Ser 26
Asn Asn Asn Asn Asn Asn Asn Asn Ser Asn Asn Asn Ser 39
Asn Asn Asn Asn Asn Asn Asn Ser Asn 48
(2) INFORMATION FOR SEQ. ID NO. 6:
(i) SEQUENCE CHARACTERISTICS (A) LENGTH: 40 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) SEQUENCE DESCRIPTION: SEQ. ID NO. 6
Ser Ser Asn Asn Asn Asn Asn Ser Ser Asn Asn Ser Asn 13
Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn 26
Ser Asn Asn Asn Asn Asn Asn Asn Asn Ser Asn Asn Asn 39
Ser 40

Claims

WHAT IS CLAIMED IS:
1. An antigenic protein comprising a sporozoite polypeptide selected from the group consisting of Pf 42 derived from Plasmodium falciparum- Pb 42 derived from Plasmodium berghei. Pf 54 derived from Plasmodium falciparum. Pb 54 derived from Plasmodium berghei. antigenic fragments thereof or epitopes thereof.
2. The antigenic protein of claim 1 comprising Pf 42 in purified form.
3. The antigenic protein of claim 1 comprising Pb 42 in purified form.
4. The antigenic protein of claim l comprising Pf 54 in purified form.
5. The antigenic protein of claim 1 comprising Pb 54 in purified form.
6. An essentially pure protein, said protein being: a) antigenic; b) present on the surface of Plasmodium falciparum sporozoites; c) of a molecular weight of about 42 kilo¬ daltons by sodium dodecylsulphate-polyacrylamide gel electrophoresis; and d) cross-reactive with Plasmodium berghei antisera.
7. An essentially pure protein, said protein being: a) antigenic; b) present on the surface of Plasmodium falciparum sporozoites; c) of a molecular weight of about 54 kilo¬ daltons by sodium dodecylsulphate-polyacrylamide gel electrophoresis; and d) cross reactive with Plasmodium berghei antisera.
8. An essentially pure protein, said protein being: a) antigenic; b) present on the surface of Plasmodium berghei sporozoites; c) of a molecular weight of about 42 kilo¬ daltons by sodium dodecylsulphate-polyacrylamide gel electrophoresis; d) cross-reactive with Plasmodium falciparum antisera.
9. An essentially pure protein, said protein being: a) antigenic; b) present on the surface of Plasmodium berghei sporozoites; c) of a molecular weight of about 54 kilo- daltons by sodium dodecylsulphate-polyacrylamide gel electrophoresis; d) cross-reactive with Plasmodium falciparum antisera.
10. The protein of any of the precceding claims further comprising a carrier linked to said polypeptide.
11. The protein of claim 10 wherein the carrier is tetanus toxoid linked to the polypeptide through tyrosine.
12. A malarial sporozoite vaccine comprising the antigenic protein of any of the preceeding claims.
13. The vaccine of claim 12 further comprising one or more different malarial antigenic peptides.
14. A DNA sequence coding for the protein of any of claims 1-9.
15. A live vaccine comprising an immunologically acceptable virus modified to contain the DNA sequence of claim 14.
16. The live vaccine of claim 15 further modified to contain one or more DNA sequences coding for different malarial antigenic peptides.
PCT/US1990/007194 1989-12-12 1990-12-12 Novel malarial sporozoite peptide antigens WO1991008756A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2005219839B2 (en) * 2004-03-01 2011-11-24 Immune Disease Institute, Inc Natural IgM antibodies and inhibitors thereof
US9243059B2 (en) 2013-03-12 2016-01-26 Decimmune Therapeutics, Inc. Humanized anti-N2 antibodies and methods of treating ischemia-reperfusion injury

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2037151A1 (en) * 1990-03-23 1991-09-24 F. Hoffmann-La Roche Ag Plasmodium sporozoite antigen

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4707357A (en) * 1984-06-26 1987-11-17 The United States Of America As Represented By The Secretary Of The Army Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor
EP0289110A2 (en) * 1987-02-02 1988-11-02 Swiss Serum and Vaccine Institute Berne Conjugate malaria vaccine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4707357A (en) * 1984-06-26 1987-11-17 The United States Of America As Represented By The Secretary Of The Army Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor
EP0289110A2 (en) * 1987-02-02 1988-11-02 Swiss Serum and Vaccine Institute Berne Conjugate malaria vaccine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
IMMUNOLOGY LETTERS, Volume 25, issued 1990, HOLLINGDALE et al., "Non-Cs Pre-Erythrocytic Protective Antigens", pages 71-76. *
TECHNOLOGICAL ADVANCES IN VACCINE DEVELOPMENT, Published 1988, by ALAN R. LISS. INC. see page 295-309. *

Cited By (6)

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AU2005219839B9 (en) * 2004-03-01 2011-12-22 Immune Disease Institute, Inc Natural IgM antibodies and inhibitors thereof
US9657060B2 (en) 2004-03-01 2017-05-23 Children's Medical Center Corporation Natural IgM antibodies and inhibitors thereof
US9914751B2 (en) 2004-03-01 2018-03-13 Children's Medical Center Corporation Natural IGM antibodies and inhibitors thereof
US9243059B2 (en) 2013-03-12 2016-01-26 Decimmune Therapeutics, Inc. Humanized anti-N2 antibodies and methods of treating ischemia-reperfusion injury
US9409977B2 (en) 2013-03-12 2016-08-09 Decimmune Therapeutics, Inc. Humanized, anti-N2 antibodies

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