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WO1990006130A1 - Nouvel antigene peptidique du sporozoite et du parasite exoerythrocytere de la malaria - Google Patents

Nouvel antigene peptidique du sporozoite et du parasite exoerythrocytere de la malaria Download PDF

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Publication number
WO1990006130A1
WO1990006130A1 PCT/US1989/005335 US8905335W WO9006130A1 WO 1990006130 A1 WO1990006130 A1 WO 1990006130A1 US 8905335 W US8905335 W US 8905335W WO 9006130 A1 WO9006130 A1 WO 9006130A1
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Prior art keywords
antigenic protein
gln
glu
amino acid
leu
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PCT/US1989/005335
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English (en)
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Michael R. Hollingdale
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Biomedical Research Institute
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Publication of WO1990006130A1 publication Critical patent/WO1990006130A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • 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 and identification of a specific polypeptide antigen, which when used as an inoculant elicits an immunogenicity and protection against malarial sporozoites and exoerythrocytic parasites.
  • the invention furthermore, relates to the isolation and identification of a specific polypeptide antigen, which when used as an inoculant elicits an immunogenicity and protection against malarial sporozoites and exoerythrocytic parasites.
  • Malaria is a multi-stage disease which is characteristically initiated by the bite of an infected anopheles mosquito. Through its bite, the mosquito
  • the erythrocytic phase involves the invasion of host red blood cells by the merozoites.
  • the erythrocytic ' phase of malaria is cyclic, as new merozoites are periodically
  • the erythrocytic 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, sequenced, and candidate subunit vaccines tested for immunogenicity and protection in rodents, and in human clinical trials.
  • 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-Lys-Gln-Pro) is undergoing vaccine trials in mice. Circumsporozoite proteins are detected throughout exoerythrocytic development and may be a target of cytotoxic T-cell activity that may also serve to protect against sporozoite infection.
  • SUBSTITUTESHEET specific antigen (LSA) .
  • LSA SUBSTITUTESHEET specific antigen
  • a fragment of the liver-stage- specific antigen was cloned and sequenced and consists of 3 ⁇ repeats of a 17 amino acid sequence, which contains a major sequence Glu-Gln-Gln-Ser-Asp-Leu-Glu- Gln-Glu-Arg-Leu-Ala-Lys-Glu-Lys-Leu-Gln and a minor sequence Gly-Gln-Gln-Ser-Asp-Leu-Glu-Gln-Glu-Arg-Leu- Ala-Lys-Glu-Lys-Leu-Gln Human antibodies affinity purified by Guerin-Marchand et al.
  • a peptide corresponding to one repeat Glu-Gln-Gln- Ser-Asp-Leu-Glu-Gln-Glu-Arg-Leu-Ala-Lys-Glu-Lys-Leu-Gln was synthesized and reacted in ELISA assays with the affinity purified human antibodies as well as 10 human sera from Africa.
  • Identifying antigens specific to the exoerythrocytic stage parasite and/or particularly the sporozoite would be significant in developing an effective vaccine against malaria.
  • Such a vaccine which creates host immunity to the ⁇ sporozoites and/or exoerythrocytic parasites would be very useful, because it would protect the host at the earliest stage of the disease.
  • the present invention relates to a novel antigenic protein of the malaria sporozoite and exoerythrocytic stage parasite.
  • the protein is a polypeptide which has the amino acid se-guence X-Gln-Gln-Ser-A.sp or the amino acid sequence Asp-Ser-Gln-Gln-X on at least one terminal end thereof where X may be either Glu or Gly.
  • X may be either Glu or Gly.
  • a portion of the N-terminus of the protein has also been sequenced and cloned.
  • the invention further relates to a vaccine comprising the novel antigenic protein with a carrier linked to one of the terminal amino acids.
  • a carrier linked to one of the terminal amino acids.
  • One effective carrier is tetanus toxoid linked to a terminal amino acid through tyrosine.
  • the most effective vaccine isthepeptide, Leu-Glu-Gln-Glu-Arg-Leu-Ala-Lys-Glu-Lys- Leu-Gln-Glu-Gln-Gln-Ser-Asp, with the tetanus toxoid/- tyrosine carrier linked to Asp.
  • Figure 1 shows the amino acid sequence of the Guerin-Marchand peptide compared to the T-cell epitope, Nl?
  • Figure 2 shows a comparison of the amino acid sequences of the Guerin-Marchand peptide, the novel peptide of the present invention and the T-cell epitope, Nl;
  • Figure 3 shows the amino acid sequences of two effective vaccines against malaria sporozoites
  • Figure 4 shows the amino acid sequences of two experimental vaccines which were ineffective in protecting a host against malaria sporozoites;
  • Figure 5 shows the nucleotide sequence of the N- terminal portion of the Plasmodium falciparum protein gene
  • Figure 6 shows a schematic diagram of . the Plasmodium falciparum protein gene displaying the locations of its major sequence, minor sequence and a degenerative sequence.
  • the present invention relates to a malarial sporozoite and exoerythrocytic stage antigen.
  • the antigen is a peptide or polypeptide which has the amino acid sequence X-Gln-Glu-Ser-Asp or the amino acid sequence Asp-Ser-Gln-Gln-X on at least one terminal end thereof where X is either- ⁇ Glu or Gly.
  • the Guerin-Marchand peptide contains a sequence closely similar to the Nl peptide, which the inventors have found is the ligand by which sporozoites recognize hepatocyte receptors.
  • Figure 1 shows a comparison of the similar amino acid sequences of the Guerin-Marchand peptide and the Nl peptide.
  • This novel peptide starts at the sixth amino acid of the Guerin-Marchand peptide and comprises 17 amino acids of the Guerin-Marchand repeat.
  • This peptide has a molecular weight of about 3000 to about 4000.
  • a comparison of similar amino acid sequences of the Guerin-Marchand peptide, the novel peptide and the Nl peptide is shown in Figure 2.
  • the novel peptide was synthesized such that the region of ho ology with the Nl peptide is in the center and not at a terminal end as with the Guerin-Marchand peptide. Usin ⁇ £ ELISA, rabbit
  • a new vaccine construct was synthesized. This new vaccine construct was used in mice to test for protection to P_ a _ ber ⁇ hei sporozoite challenge.
  • the new vaccine construct comprised the Guerin-Marchand peptide linked at its glutamic acid terminus through cysteine to the carrier, keyhole limpet hemocyanin.
  • mice Groups of ten mice were immunized with the . new vaccine and a control vaccine comprising keyhole limpet hemocyanin linked to cysteine. The sera from the mice was then tested in ELISA with Guerin-Marchand peptide or keyhole limpet hemocyanin as the antigen.
  • mice responded well to both vaccines, with a boosting response indicating that the keyhole limpet hemocyanin serves as a T-cell epitope (it is unknown if Guerin-Marchand peptide contains B-cell epitomes or T-
  • mice immunized with the new vaccine developed specific antibodies to the Guerin- Marchand peptide. However, only one of these mice reacted in the immunofluorescence antibody assay with .P ⁇ . ber ⁇ hei sporozoites. In contrast, four mice immunized with the control vaccine also developed antibodies which recognized P ⁇ , ber ⁇ hei sporozoites by immunofluorescence antibody assay. All of the mice were challenged with Pj. ber ⁇ hei sporozoites, but about 50% of the new vaccine mice were protected and about 50% of the control vaccine mice were protected.
  • Useful carriers include tetanus/toxoid linked to the peptide through tyrosine, Lipid A ® , hepatitis B antigen and any microorganism to which the peptide may be linked.
  • the tetanus toxoid/tyrosine carrier was selected and four vaccine constructs were made by linking the carrier to the N-terminal or C-terminal amino acid of the Guerin- Marchand peptide ( Figure 1) and the novel peptide ( Figure 2) .
  • TT-Y-EQQ carrier linked to the glutamic acid terminus of the Guerin-Marchand peptide (EQQ peptide shown in Figure 2)
  • EQQ-Y-TT carrier linked to the glutamine terminus of the Guerin-Marchand peptide (EQQ peptide shown in Figure 2)
  • TT-Y-LEQ carrier linked to the leucine terminus of the novel peptide (LEQ peptide shown in Figure 2)
  • LEQ-Y-TT carrier linked to the aspartic acid terminus of the novel peptide (LEQ peptide shown in Figure 2) .
  • mice Groups of 10 Balb/c mice were immunized with each vaccine given initially in Freund's complete adjuvant followed by two boosts in Freund's incomplete adjuvant. Control groups of mice received tetanus toxoid alone, Freund's adjuvant only or nothing. Sera from each mouse taken after the first and second boost were tested in ELISA for reactivity against each vaccine candidate. Sera from each group are being tested by IFA and ISI with P J . ber ⁇ hei sporozoites.
  • EQQ-Y-TT vaccine was more immunogenic than TT-Y-EQQ
  • TT-Y-LEQ was more immunogenic than LEQ-Y-TT
  • TT-Y-LEQ was the most immunogenic of the four vaccines. This suggested: a) that the relationship (i.e., end) that the tetanus toxoid was linked to the peptide affects immunogenicity, and differed for EQQ and LEQ; and b) that merely hooking tetanus toxoid to a -peptide does not predictively increase immunogenicity.
  • mice immunized with either EQQ vaccines recognized only EQQ peptides
  • mice immunized with either LEQ vaccines recognized only LEQ peptides.
  • the antibody response elicited by EQQ was specific for EQQ
  • the response elicited by LEQ was specific for LEQ.
  • Sera from each group were also tested by IFA and ISI with I ⁇ . ber ⁇ hei or P_ s _ falciparum sporozoites.
  • mice SUBSTITUTE SHEET All of -the mice were then challenged with P. ber ⁇ hei sporozoites. The only mice which exhibited protection against the P ber ⁇ hei sporozoites were some of those which had been inoculated with TT-Y-EQQ and some of those which had been inoculated with LEQ-Y-TT. A higher percentage of mice in the group inoculated with LEQ-Y-TT exhibited immunogenicity to the P ⁇ ber ⁇ hei sporozoites than those inoculated with TT-Y-EQQ. No mice in the other two groups (inoculated with EQQ-Y-TT and TT-Y-LEQ, respectively) exhibited immunogenicity to challenge with P ber ⁇ hei sporozoites.
  • the LEQ-Y-TT vaccine confers some protection to challenge with . ber ⁇ hei sporozoites, and minor protection is exhibited with the TT-Y-EQQ vaccine.
  • Preliminary experiments testing a variety of overlapping peptides by ELISA as described by Geysen et al. in Proc. Natl. Acad. Sci. 81, 3998 (1984) and Proc. Natl. Acad. Sci. 82, 178 (1985) were performed to map the actual amino acid sequence recognized by the mouse anti-TT-Y- LEQ serum.
  • the results of these experiments suggested that the active epitope is the 5 amino acids adjacent to the carrier, TT-Y- (i.e., Leu-Glu-Gln-Glu-Arg) .
  • TT-Y- i.e., Leu-Glu-Gln-Glu-Arg
  • the antibodies to the effective vaccine LEQ-Y-TT do not recognize TT-Y-EQQ, and the antibodies to the effective vaccine TT-Y-EQQ do not recognize LEQ-Y-TT. Therefore, . the antibody response cannot be primarily directed to the conserved central sequence common to both vaccines (Leu-Glu-Gln-Glu-Arg-Leu-Ala-Lys-Gly-Lys-
  • stearic property is crucial to anti-peptide recognition, and might be imposed by the carrier, TT-Y-.
  • the stearic property was noted because (as discussed above) , the antibody to TT-Y-EQQ (which has the pertinent 5 amino acid sequence Glu-Gln-Gln-Ser-Asp) does not recognize LEQ-Y-TT (which has the reverse pertinent 5 amino acid sequence Asp-Ser-Gln-Gln-Glu) .
  • LEQ-Y-TT which has the reverse pertinent 5 amino acid sequence Asp-Ser-Gln-Gln-Glu
  • LEQ-Y-TT Glu-Gln-Gln-Ser-Asp-T-TT, reversed
  • TT-Y-Asp-Ser-Gln-Gln-Glu TT-Y-Asp-Ser-Gln-Gln-Glu
  • SUBSTITUTESHEET Additional peptides with the crucial protective site five amino acid sequences (X-Gln-Gln-Ser-Asp or Asp-Ser-Gln-Gln-X) where X is Glu or Gly can be synthesized using conventional peptide synthesis . techniques or by cloning using DNA coding for the desired peptide.
  • the peptides are in the form of one or more consecutive repeats of the sequences.
  • One to five repeats of the pertinent amino acid sequences are preferred for the synthesized proteins, i.e. (X-Gln-Gln- Ser-Asp) n or (Asp-Ser-Gln-Gln-X) n where n is 1 to 5.
  • the peptide comprises three repeats of the sequence, i.e., (X-Gln-Gln-Ser-Asp) 3 or (Asp-Ser-Gln-Gln-X) 3 .
  • a portion of the gene for the novel Plasmodium falciparum sporozoite antigenic protein has been sequenced and cloned.
  • the portion of the gene which has been sequenced codes for the N-terminus of the protein.
  • the portion of the gene which has been sequenced is shown in Figure 5.
  • an oligonucleotide probe was constructed with the sequence
  • the sequence of the probe was based on the known 17 amino acid sequence of the novel protein (Glu-Gln-Gln-. Ser-Asp-Leu-Glu-Gln-Glu-Arg-Leu-Ala-Lys-Glu-Lys-Leu- Gln) , and its corresponding nucleotide sequence.
  • a j_ falciparum mung bean/gt 11 library (McCutchan et al.. Science 225. 625 (1984)) is prepared, and then screened with the oligonucleotide probe. The clones are checked and res ⁇ reened, and two positive clones are recovered. Using southern blot analysis, the two clones are determined to have insert sizes of 0.7kb and 1.6kb, and are labeled #13 and #1813.
  • the protein has been isolated from the pGEM13 plasmid and is characterized by at least 23 repeats containing either a major sequence, a minor repeat or a single degenerative repeat.
  • the major sequence is:
  • the minor repeat is:
  • the degenerative repeat is:
  • Proteins synthesized from 1 to 1000 copies of all of the above sequences i.e. the major sequence, the minor repeat, the degenerative sequence and the Asp-Asp-Asp- Asp-Lys-Lys-Lys sequence
  • the protein shown in Figure 5 (which is synthesized from the isolated gene portion) is also suitable for eliciting a sporozoite antibody response in test animals.
  • Vaccines may be made by attaching a carrier to the synthesized or cloned peptides as described above at the appropriate terminal amino acid.
  • a carrier for (X-Gln-Gln-Ser- Asp) n the carrier is attached to terminal amino acid, Asp.
  • Asp-Ser-Gln-Gln-X the carrier is attached to terminal amino acid, Glu or Gly.
  • SUBSTITUTESHEET include tetanus toxoid linked to the appropriate terminal amino acid through tyrosine, Lipid A*, hepatitis B antigen and or microorganism to which the peptide may be linked.
  • the preferred carrier is tetanus toxoid linked to the appropriate terminal amino atid through tyrosine.
  • mice Seven groups of Balb/c mice were inoculated. Each group received a different preparation. The mice in Groups 1-4 were immunized with a vaccine, while the mice in Groups 5-7 were controls.
  • Each group of immunized mice received a different vaccine.
  • Group 1 received the TT-Y-EQQ vaccine (shown in Figure 3) .
  • Group 2 received the EQQ-Y-TT vaccine (shown in Figure 4) .
  • Group 3 received the TT-Y-LEQ vaccine (shown in Figure 4) , and
  • Group 4 received the LEQ-Y-TT vaccine (shown in Figure 3).
  • the initial dose of each vaccine comprised 50 ⁇ g of vaccine in 0.1ml of Freund's complete adjuvant.
  • Each group also received two boosts of the vaccine 50tg in 0.1ml of Freund's incomplete adjuvant. The boosts were given at intervals of two weeks.
  • mice in the control groups received the following: Group 5 received tetanus toxoid alone; Group 6 received Freund's adjuvant alone; and Group 7 did not receive anything.
  • mice in Groups 1 and 4 exhibited protection against P_s_ ber ⁇ hei sporozoite challenge. Twelve percent of the mice in Group 1 showed P_ s . ber ⁇ hei sporozoite protection, while twenty-two percent of the mice in Group 4 showed similar protection.
  • the other two vaccine groups (Groups 2 and 3) and the control groups (Groups 5-7) did not exhibit any anti-sporozoite activity. The results of the tests are shown below in Tables 1 and 2.
  • mice #2 and #4 developed ELISA antibodies to LEQ-Y-TT a j TT-Y-LEQ, and both were protected. Also LEQ-Y-TT vaccine was more immunogenic than TT-Y-EQQ.
  • the Guerin-Marchand peptide ( Figure 1) was synthesized using conventional peptide synthesis techniques. The peptide was then coupled to the carrier poly-L-lysine and
  • the in vitro ISI assay was used with cultured human hepatoma (HepG2-A16) cells.
  • the rabbit sera was diluted to a number of concentrations in complete medium and added to the HepG2-Al6 . cultures. Control cultures contained no rabbit sera.
  • P. falciparum sporozoites and JP ⁇ ber ⁇ hei sporozoites were then added to separate control and test cultures in amounts of about 12,000 to about 40,000 sporozoites per culture.
  • the cultures were incubated at 37 ⁇ C in 5% C0 2 in air for three hours, then rinsed two times with Dulbecco's phosphate-buffered saline (PBS) , fixed with methanol, and rinsed two times again in PBS.
  • Sporozoites that had entered cells were visualized in fixed cultures by immunoperoxidase antibody assay (IPA) (Hollingdale et al., Trans. R. Soc. Trop. Med. Hy ⁇ . 76. 624 (1982)).
  • IPA immunoperoxidase antibody assay
  • Th ' e IPA was carried out by treating the fixed cultures with a monoclonal antibody to P ⁇ . falciparum or to P ⁇ . ber ⁇ hei. followed by incubation with anti-mouse immunoglobulins conjugatedwithhorseradishperoxidase, and stained with 3,3-diaminobenzidine. The number of sporozoites that had invaded cultured cells was determined by counting the parasites present in the entire preparation on a Nikon microscope at 20Ox magnification with a dark blue filter.
  • a 32 P-labeled oligonucleotide probe based on the novel peptide was prepared using conventional techniques.
  • the titre and insert rate of a mung bean/gt 11 phage library were determined using conventional techniques.
  • a 5ml preculture of Y1090 in LB + Amp (50 ⁇ g/ l) + 0.2% maltose was grown at 37 ⁇ C overnight.
  • a series dilution of phages in SM medium (Maniatis et al. , Molecular Clonin ⁇ , A Laboratory Manual. Cold Spring Harbor Laboratory (1982)) was prepared and stored at 4°C.
  • a top agar medium of LB + 0.7% agar was melted in a microwave oven and then cooled to 50 ⁇ C, and stock solutions of 2% X-gal, 0.1M IPTG and Amp (5mg/ml) were warmed.
  • the Y1090 culture (0.3 ml) was infected by mixing it with 10-20/il of series dilution of phage library. The infected culture was incubated at room temperature for 30 minutes, and then 10ml of 50 ⁇ C top agar, lOO ⁇ l of Ampicillin stock, 20 ⁇ l of IPTG stock and 20 ⁇ l of X-gal stock were added and mixed. The mixture was poured onto an LB-agar plate containing ampicillin, and the plate was allowed to stand for 30 minutes at room temperature, and then was turned upside down and incubated at 37°C overnight.
  • the white colonies were then used to plate out 10,000 plaques per 188mm plate with LB and ampicillin. These plates were incubated at 37°C for six hours until the edges of the plaques reached each other, and then stored at 4 ⁇ C for 1.5 hours. Numbered nitrocellulose filters were then placed on each plate and needled three times. After 60 -20-
  • the filters were removed and immersed in denaturing solution (1.5M NaCl, 0.5M NaOH) for 60 seconds.
  • denaturing solution 1.5M NaCl, 0.5M NaOH
  • the filter was then transferred into a neutralizing solution (1.5M NaCl, 0.5M Tris-Cl) for 5 minutes, rinsed * 5 with SSPE and dried. Additional filters were prepared in the same manner, and then all the filters were baked at 80'C in a vacuum oven.
  • the filters were then prehybridized and hybridized against the 32 P-labeled oligonucleotide probe at 45*C 0 overnight, and washed in 6x SSC/0.1% SDS at room temperature, and once at 45 ⁇ C for 15 minutes. After autoradiographing, the positive plaques were picked up and suspended together with agar in 200 ⁇ l of SM medium and stored at 4 ⁇ C overnight. 5 Verification of the positive phage mixture was then performed by Slot-blot hybridization. A nitrocellulose filter was wetted in water and 6x SSC for 5 minutes, and mounted in an S&S minifold blotter. The minifold blotter was used to slot-blot 20 ⁇ l of the phage mixture with a 0 gentle vacuum.
  • the purified inserts were ligated into EcoRI-cleaved plasmid pUC13 (Pharmacia) and pGEM72f (Promega) , and the ligation was transformed into DH5a cells (BRL) .
  • the recombinant plasmids containing the 0.7kb and 1.6kb inserts were then analyzed with various commercially available enzymes including BamHI and EcoRI. Large scale preparation of the recombinant plasmids, pUCJ16 and pGEM13 (1.6kb), and pGEM1813 (0.7kb) was also carried out using the techniques of Maniatis et al., supra.
  • a sequencing gel was prepared by mixing 42 grams urea (ultrapure, BRL #5505UA) , 20ml instaPAGE (sequencing grade, 19:1 acrylamide:bisacrylamide, IBI #70014), 10ml lOx TBE and sufficient deionized water to make 100ml. The ingredients were mixed on a magnetic stirrer with a little heat to solubilize the urea. The resulting gel was 8% acrylamide, 7M urea and lx TBE. -22-
  • TEMED was then added to the above acryla ide solution and mixed. The mixture was then poured between two glass plates with 0.4mm spacers at both edges. The gel polymerized in 30 minutes and was usable in 6 hours.
  • the glass plates Prior to pouring of the mixture, the glass plates were thoroughly cleaned, and the larger plate was siliconized as described by Maniatis et al, supra.
  • the smaller plate was coated with a solution made from 4ml of a mixture of 50 ⁇ l MOTS (methacryloxy-propyltrimethoxysilane) in 10ml pure ethanol, and 120 ⁇ l of acetic acid. After the coating had dried, the plate was rinsed with ethanol and recoated.
  • MOTS methacryloxy-propyltrimethoxysilane
  • the plasmid DNA from pUC 16 and pGEM1813 was then denatured by alkali.
  • a plasmid DNA solution was made in water to a concentration of 0.5 ⁇ g/ ⁇ l.
  • lO ⁇ l of each plasmid DNA solution (pUCJ16 and pGEM1813) was mixed with 2 ⁇ l DEP-H 2 0 and 8 ⁇ l 0.5N NaOH to a final concentration of 0.2N NaOH.
  • the mixture was incubated at room temperature for 5 minutes, and then neutralized with 8 ⁇ l of 5M NH_,Ac (pH 7.5), mixed and spun.
  • the DNA was precipitated by adding 80 ⁇ l of reagent pure ethanol and placing the mixture on dry ice for 5 minutes.
  • the mixture was then centrifuged in a cold room for 10 minutes, washed in 80% ethanol once, and recentrifuged for 10 minutes. The supernatant was aspirated and the ethanol dried in a speed vacuum to leave a DNA pellet which was dissolved in lO ⁇ l of water.
  • ddTTP 2.5 ⁇ l of ddTTP was added to the T tube.
  • 4 ⁇ l of dGTP Labeling Mix was diluted with 16 ⁇ l distilled water, and the sequenase enzyme was diluted 1:8 in ice-cold TE buffer.
  • a mixture of lO ⁇ l annealed plasmid DNA- primer, l ⁇ l 0.1M DTT, 2 ⁇ l diluted labeling mix, 0.5 ⁇ l (a- 35S) dATP (lOOOCi/mmol, lO ⁇ Ci/ ⁇ l) and 2 ⁇ l diluted sequenase was incubated at room temperature for 10 minutes (The age of the s-isotope was less than half life) .
  • the gel was run for 4 hours and a second group of samples were loaded. The gel was run for another 5 hours. The electrophoresis was then stopped and the bigger plate was removed. The gel on the small plate was then soaked in a tray with 2000ml 10% acetic acid and 12% methanol for 30 minutes.. The gel was dried in a hood at room temperature overnight.
  • mice inoculated Seven groups of Babl/c mice are inoculated. Each group receives a different preparation. The mice in Groups 1-4 are" immunized with a vaccine, while the mice in Groups 5-7 are controls.
  • mice in Group 1 receive a vaccine comprising the degenerative repeat polypeptide (Glu-Asn-Arg-Gln-Glu-Asp- Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Lys-Leu-Gln) .
  • Mice in Group 2 receive a vaccine comprising the polypeptide Asp- Asp-Asp-Asp-Lys-Lys-Lys.
  • Mice in Group 3 receive a vaccine comprising the polypeptide shown in Figure 5
  • mice in Group 4 receive a vaccine comprising the polypeptide cloned from the protein gene inserted in pGEM72f to make plasmid PGEM13.
  • each vaccine comprises 50 ⁇ l of vaccine in 0.1ml of Freund's complete adjuvant.
  • the mice in each group also receive two boosts of 50 ⁇ l of vaccine in 0.1ml of Freund's incomplete adjuvant. The boosts are given at two week intervals.
  • mice in the control groups receive the same compositions as the controls in Example 1; i.e. Group 5 receives tetanus toxoid alone, Group 6 receives Freund's adjuvant alone and Group 7 does not receive anything.
  • mice in Groups 1-4 All show protection against I ⁇ . ber ⁇ hei and P falciparum sporozoite challenge.
  • the control groups did not show any anti-sporozoite activity.

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Abstract

La présente invention se rapporte à une nouvelle protéine antigénique du sporozoïte de la malaria et du parasite de cette maladie en phase exoérythrocytère. La protéine antigénique est un polypeptide qui comporte sur son extrémité terminale C, la séquence d'acides aminés X-Gln-Gln-Ser-Asp ou la séquence d'acides aminés Asp-Ser-Gln-Gln-X, où X représente Glu ou Gly. Une partie de la terminaison N de la protéine a été mise en séquence et clonée. La présente invention se rapporte en outre à un vaccin contenant la protéine antigénique épurée liée à un excipient, tel qu'un toxoïde du tétanos lié à l'acide aminé à terminaison C par la tyrosine.
PCT/US1989/005335 1988-11-30 1989-11-30 Nouvel antigene peptidique du sporozoite et du parasite exoerythrocytere de la malaria WO1990006130A1 (fr)

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US27823488A 1988-11-30 1988-11-30
US278,234 1988-11-30
US33720489A 1989-04-12 1989-04-12
US337,204 1989-04-12

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2672290A1 (fr) * 1991-02-05 1992-08-07 Pasteur Institut Sequences peptidiques specifiques des stades hepatiques de p. falciparum porteuses d'epitopes capables de stimuler les lymphocytes t.
US5589343A (en) * 1987-02-09 1996-12-31 Institut Pasteur Antibodies which bind to molecules containing at least one peptide sequence carrying one or several epitopes characteristic of a liver stage antigen produced by P. falciparum in hepatocytes
US6270771B1 (en) 1988-10-06 2001-08-07 Institut Pasteur Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes
US9266930B1 (en) * 1993-03-05 2016-02-23 Epimmune Inc. Inducing cellular immune responses to Plasmodium falciparum using peptide and nucleic acid compositions

Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0254862A1 (fr) * 1986-06-26 1988-02-03 BEHRINGWERKE Aktiengesellschaft Vaccins contre les parasites protozoiques
EP0289110A2 (fr) * 1987-02-02 1988-11-02 Swiss Serum and Vaccine Institute Berne Vaccin conjugué contre la malaria

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0254862A1 (fr) * 1986-06-26 1988-02-03 BEHRINGWERKE Aktiengesellschaft Vaccins contre les parasites protozoiques
EP0289110A2 (fr) * 1987-02-02 1988-11-02 Swiss Serum and Vaccine Institute Berne Vaccin conjugué contre la malaria

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NATURE, Vol. 329, September 1987 Claudine Guerin-Marchand et al: "A liver-stage-specific antigen of Plasmodium falciparum characterized by gene cloning ", see page 164 and the whole article. *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5589343A (en) * 1987-02-09 1996-12-31 Institut Pasteur Antibodies which bind to molecules containing at least one peptide sequence carrying one or several epitopes characteristic of a liver stage antigen produced by P. falciparum in hepatocytes
US5599542A (en) * 1987-02-09 1997-02-04 Institut Pasteur Molecules containing at least one peptide sequence carrying one or several epitopes characteristic of a liver stage antigen produced by P. falciparum in hepatocytes and compositions containing them
US5602031A (en) * 1987-02-09 1997-02-11 Institut Pasteur DNA encoding molecules containing at least one peptide sequence carrying one or several epitopes characteristic of a liver stage antigen produced by p. falciparum in hepatocytes and compositions containing them
US7087231B2 (en) 1987-02-09 2006-08-08 Institut Pasteur Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes
US6270771B1 (en) 1988-10-06 2001-08-07 Institut Pasteur Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes
FR2672290A1 (fr) * 1991-02-05 1992-08-07 Pasteur Institut Sequences peptidiques specifiques des stades hepatiques de p. falciparum porteuses d'epitopes capables de stimuler les lymphocytes t.
WO1992013884A1 (fr) * 1991-02-05 1992-08-20 Institut Pasteur Sequences peptidiques specifiques des stades hepatiques de p. falciparum porteuses d'epitopes capables de stimuler les lymphocytes t.
US6319502B1 (en) 1991-02-05 2001-11-20 Institut Pasteur Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes
US7332595B2 (en) 1991-02-05 2008-02-19 Institut Pasteur DNA sequences encoding peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes
US7438917B2 (en) 1991-02-05 2008-10-21 Institut Pasteur Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes
US9266930B1 (en) * 1993-03-05 2016-02-23 Epimmune Inc. Inducing cellular immune responses to Plasmodium falciparum using peptide and nucleic acid compositions

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