EP0344257A1 - RHOPTRY ANTIGEN OF $i(PLASMODIUM FALCIPARUM) - Google Patents
RHOPTRY ANTIGEN OF $i(PLASMODIUM FALCIPARUM)Info
- Publication number
- EP0344257A1 EP0344257A1 EP89900001A EP89900001A EP0344257A1 EP 0344257 A1 EP0344257 A1 EP 0344257A1 EP 89900001 A EP89900001 A EP 89900001A EP 89900001 A EP89900001 A EP 89900001A EP 0344257 A1 EP0344257 A1 EP 0344257A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- antigen
- antigenic fragment
- detergent
- triton
- rhoptry
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 239000000427 antigen Substances 0.000 title claims abstract description 58
- 102000036639 antigens Human genes 0.000 title claims abstract description 58
- 108091007433 antigens Proteins 0.000 title claims abstract description 58
- 241000223960 Plasmodium falciparum Species 0.000 title claims abstract description 24
- 239000003599 detergent Substances 0.000 claims abstract description 15
- 210000004369 blood Anatomy 0.000 claims abstract description 8
- 239000008280 blood Substances 0.000 claims abstract description 8
- 210000003936 merozoite Anatomy 0.000 claims abstract description 7
- 230000004807 localization Effects 0.000 claims abstract description 5
- TWJNQYPJQDRXPH-UHFFFAOYSA-N 2-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C#N TWJNQYPJQDRXPH-UHFFFAOYSA-N 0.000 claims abstract description 3
- 102000003886 Glycoproteins Human genes 0.000 claims abstract description 3
- 108090000288 Glycoproteins Proteins 0.000 claims abstract description 3
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 claims abstract description 3
- 235000021360 Myristic acid Nutrition 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 13
- 244000045947 parasite Species 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 11
- 230000000890 antigenic effect Effects 0.000 claims description 10
- 229920001184 polypeptide Polymers 0.000 claims description 9
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 9
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- 102000053602 DNA Human genes 0.000 claims 3
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- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000003071 parasitic effect Effects 0.000 abstract 1
- IDOQDZANRZQBTP-UHFFFAOYSA-N 2-[2-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1OCCO IDOQDZANRZQBTP-UHFFFAOYSA-N 0.000 description 32
- 229920004929 Triton X-114 Polymers 0.000 description 32
- 206010035500 Plasmodium falciparum infection Diseases 0.000 description 15
- 239000000020 Nitrocellulose Substances 0.000 description 11
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- 108090000623 proteins and genes Proteins 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 102000018697 Membrane Proteins Human genes 0.000 description 7
- 108010052285 Membrane Proteins Proteins 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
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- 241000588724 Escherichia coli Species 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 5
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- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 239000002198 insoluble material Substances 0.000 description 2
- 230000001320 lysogenic effect Effects 0.000 description 2
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- 238000012546 transfer Methods 0.000 description 2
- 210000003812 trophozoite Anatomy 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
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- VDQQXEISLMTGAB-UHFFFAOYSA-N chloramine T Chemical compound [Na+].CC1=CC=C(S(=O)(=O)[N-]Cl)C=C1 VDQQXEISLMTGAB-UHFFFAOYSA-N 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 238000005534 hematocrit Methods 0.000 description 1
- 230000008076 immune mechanism Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
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- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002195 soluble material Substances 0.000 description 1
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- 239000000600 sorbitol Substances 0.000 description 1
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- 230000001360 synchronised effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/002—Protozoa antigens
- A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
- C07K14/445—Plasmodium
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- This invention relates to the identification of an antigen of the asexual blood stages of Plasmodium falciparum. which is potentially capable of generating an immune response and antibodies which are able to inhibit the growth of the parasite, and to the use of this antigen and antibodies to it in immunization, diagnostic and treatment methods.
- Rhoptry antigens are of particular interest in the study of malarial immunology and the mechanism of host protection to parasites due to their accessibility to host immune mechanisms and because of their likely involvement in the erythrocytic invasion process.
- a number of rhoptry antigens have been shown to be capable of inducing immune responses that in vivo or in vitro inhibit the growth of the asexual blood stages of P. falciparum. (1,2).
- a rhoptry antigen of the asexual blood stages of Plasmodium falciparum which is characterised by: (i) having an apparent molecular weight of approximately 55 kDa; (ii) being a glycoprotein.
- the antigen is a polypeptide having a primary structure which includes the amino acid sequence set out in Figure 2, or an antigenic fragment thereof.
- the invention also provides a method for actively immunising a host against Plasmodium falciparum which method comprises administering to the host an antigen according to the present invention, or an antigenic fragment thereof.
- the invention provides a vaccine comprising ' an antigen of the present invention, or an antigenic fragment thereof, a pharmaceutically acceptable carrier or diluent, and optionally an adjuvant. Further features of the present invention will become apparent from the detailed description in the following Example. EXAMPLE
- FC27 P.falciparum isolate FCQ27/PNG (FC27) used in these studies has been described elsewhere (2).
- HTPBS human tonicity phosphate buffered saline
- Triton X-114 solubilization and phase separation.
- Triton X-114 solubilization and separation of hydrophobic, hydrophilic and insoluble fractions was performed essentially as described by Bordier (4) with the following modifications.
- Triton X-114 (purchased from Fluka Ag., Switzerland) was precondensed in human tonicity phosphate buffered saline (HTPBS) .
- HTPBS human tonicity phosphate buffered saline
- a 1 ml aliquot of prepacked parasitized cells was solubilized in 15ml of 0.5% Triton X-114 for 90min on ice, with mild vortexing at lOmin intervals.
- a 1 ml sample of the total material was removed and snap frozen. The remaining 15ml were then centrifuged at 10,000xg for 15min at 4°C to remove insoluble material.
- the supernatent was removed and this step repeated.
- the sedimented material (insoluble pellet) was then washed a further three times in 0.5% Triton X-114 before being snap frozen in 1ml of HTPBS.
- the detergent-soluble material was then carefully layered over a 10ml sucrose cushion of cold 6% sucrose, 0.06% Triton X-114, placed for 5 min in a 37°C waterbath and then centrifuged at 500xg for 5min at 37°C. After centrifugation the 15ml detergent-depleted upper layer was collected and chilled on ice. The 10ml sucrose cushion was discarded and the detergent- enriched pellet (l-2ml) was resuspended on ice with 10ml of cold HTPBS.
- the resuspended detergent- enriched phase was again layered over a sucrose cushion, brought to 37° for 5min and re-pelleted by centrifugation. After this second precipitation, the detergent-enriched pellet was resuspended to 5ml in HTPBS and snap frozen.
- the detergent-depleted upper layer from the sucrose cushion separation was further depleted of hydrophobic proteins by adding 1ml of 11.4% Triton X-114 on ice, vortexing into solution, warming to 37°C for 5min, then centrifuging and discarding the Triton X-114 pellet. This cycle was repeated three times. The remaining detergent-depleted aqueous solution (aqueous phase) was then snap frozen. All samples were stored at -70°C until analysis. Electrophoresis and immunoblottin ⁇ .
- Samples for sodium dodecyl sulphate polyacrylamide gel electrophoresis were processed under reducing conditions and electrophoresed on 10% slab gels. Gels to be analysed for protein were stained with Coomassie brilliant blue. Samples to be analysed by immunoblo ting were fractionated on 10% slab gels and electrophoretically transferred to nitrocellulose sheets. After transfer the nitrocellulose was blocked with 5% (W/V) skim milk powder in HTPBS (Blotto) and probed with sera or affinity purified human antibodies diluted appropriately in Blotto.
- Protein A (Pharmacia Fine Chemicals, Uppsala, Sweden) was iodinated by the chloramine T method to a specific activity of 40 ⁇ Ci/ ⁇ g ⁇ .
- Antibodies were affinity purified from human serum or plasma on Triton X-114 soluble antigens electrophoretically transferred to nitrocellulose as described previously (5) and modified in this instance as follows. Reduced samples of Triton X-114 extracted membrane antigens were electrophoretically separated and transferred to nitrocellulose. Radioactive 14C high molecular weight markers
- the affinity-purified antibodies were diluted 1:2 in Blotto to probe immunoblots and ⁇ Amp3 cDNA library filters, or concentrated using a Centricon microconcentrator (Amicon) for immunofluorescence assays.
- Antibodies were also eluted directly from immunopositive ⁇ Amp3 clones grown as lawns on nitrocellulose. Filters with lysed lawns were pre-eluted with borate and glycine buffers, incubated with serea, and the monospecific antibodies eluted as above. This second method of affinity purification required the removal of anti E.coli antibodies which" was achieved with sonicates and lawns of control ⁇ Amp3 clones. Identification of cDNA clones expressing relevant poly ep i antigens.
- FCQ27/PNG isolate cDNA library Details of the FCQ27/PNG isolate cDNA library, its amplification in ⁇ Amp3 and lysogenic expression in E.coli have been described (6). Detection of antigen-expressing clones by in situ colony immunoassay with human antibodies has also been described (6). Positive clones were grown and induced in liquid culture and extracted with sample buffer for electrophoresis as described (7) . Samples were electrophoresed on 10% SDS-PAGE slab gels and either stained for protein or im ⁇ tunoblotted and analysed with affinity purified antibodies, to detect the presence or absence of stable fusion polypeptides with ⁇ -galactosidase. Antibody depletion.
- ⁇ Amp3 phage were isolated from immunopositive clones and the cDNA insert extracted by EcoRI digestion as described. The purified insert was subcloned into M13 vectors for single stranded sequence determined by the dideoxy method of Sanger (8,9) and into pBTA224 (a modification of PUR-290) kindly provided by Gary Coburn (Biotechnology Australia Pty.Ltd.).
- the plasmid vector pBTA224 is an expressing vector in E.coli cells, and clones were rescreened for immunoreactivity by colony immunoassay. Indirect immunofluorescence.
- Triton X-114 extracts of £___. falciparum infected erythrocytes were fractionated on 10% SDS-PAGE, electrophoretically transferred to nitrocellulose, and probed with either (a), pooled adult PNG sera, (b) , affinity purified polyclonal human antibodies, or (c), affinity purified monospecific human antibodies.
- lanes are (1), uninfected erythrocyte control (2) , total unfractionated material, (3), aqueous phase, (4), Triton phase, (5), insoluble material.
- Fig 3 Rhoptry localization of Ag512 with affinity purified monospecific antibodies used in indirect immunofluorescence assays on asynchronous blood films. Pictured is a mature schizont with a punctate pattern of fluorescence characteristic of a rhoptry localized antigen. A subset of P.falciparum antigens partition into Triton X-114.
- Antibodies affinity purified on lawns of selected clones were used in colony immunoassays on the array of ten clones and to probe immunoblots of parasite antigens. Seven of the clones were found to be siblings encoding the Mr 55,000 antigen from the Triton X-114 phase. The most immunoreactive was chosen as the type clone and designated Ag512.
- Stage-specific parasite preparations were Triton X-114 extracted, fractionated by SDS-PAGE and immunoblots probed with PNG sera.
- the antigen corresponding to Ag512 was present in all stages of the parasite life cycle, but was least abundant in the asexual ring and trophozoite stages and most abundant in late schizont preparations. In the late schizont and merozoite stages, the antigen was totally soluble in the detergent Triton X-114, but in ring and trophozoite stages it partitioned into both the Triton X-114 phase and the insoluble pellet.
- Triton X-114 extracts were prepared from five strains of P.falciparum giving asychronously in culture. The antigen was present in all strains and there were no apparent strain-related differences in the size or immunoreactivity of the antigen. Nucleotide and amino acid sequence of Ao512.
- the nucleotide sequence of AG512 together with the predicted translation is given in Fig.2.
- the cDNA clone does not encode the complete coding sequence. It contains one open reading frame which is in frame with ⁇ -galactosidase, and the clone produces a fusion polypeptide that is unstable in E. coli.
- the sequence is 839 base pairs long and contains no lengthy repetitive element.
- the sequence does not contain a hydrophobic stretch of amino acids.
- the encoded polypeptide has a predicted molecular weight of 33,534 daltons.
- Ag512 corresponds to a putative rhoptrv antigen.
- Affinity purified human antibodies to Ag512 were prepared as described, concentrated, and used in indirect immunofluorescence assays on asynchronous blood films. The fluorescence pattern observed was consistent with localisation of the antigen in the rhoptry organelles of merozoites.
- This Example shows the temperature dependent Triton X-114 detergent separation of integral membrane protein antigens of P.falciparum.
- One of the antigens that partitioned to the Triton X-114 phase was identified as CRA, a well characterised antigen of known primary structure which is typical of an integral membrane protein.
- the other dominant antigens that partition into the Triton X-114 clustered into the Mr 40-55,000 range and apparently did not correspond to antigens that had been previously cloned.
- These proteins, after transfer to. nitrocellulose were used to affinity purify polyclonal monospecific human antibodies which were used to isolate a clone designated Ag512, corresponding to a merozoite rhoptry protein, from a ⁇ Amp3 cDNA expression library.
Abstract
Un antigène toxonémique des étapes sanguines asexuées du plasmodium falciparum se caractérise: (i) par un poids moléculaire apparent d'environ 55kDa; (ii) par sa constitution en glycoprotéine contenant de l'acide myristique; (iii) par sa présence dans une zone de localisation restreinte avec des mérozoïtes compatibles avec une position toxonémique; et (iv) par sa capacité à être extrait dans la phase détergente lorsque des antigènes parasites sont séparés par division en phase avec un détergent.A toxonemic antigen of the asexual blood stages of plasmodium falciparum is characterized: (i) by an apparent molecular weight of approximately 55kDa; (ii) by its constitution as a glycoprotein containing myristic acid; (iii) by its presence in a restricted localization zone with merozoites compatible with a toxonemic position; and (iv) by its ability to be extracted in the detergent phase when parasitic antigens are separated by division in phase with a detergent.
Description
RHOPTRY ANTIGEN OF PLASMODIUM FALCIPARUM
This invention relates to the identification of an antigen of the asexual blood stages of Plasmodium falciparum. which is potentially capable of generating an immune response and antibodies which are able to inhibit the growth of the parasite, and to the use of this antigen and antibodies to it in immunization, diagnostic and treatment methods.
Rhoptry antigens are of particular interest in the study of malarial immunology and the mechanism of host protection to parasites due to their accessibility to host immune mechanisms and because of their likely involvement in the erythrocytic invasion process. A number of rhoptry antigens have been shown to be capable of inducing immune responses that in vivo or in vitro inhibit the growth of the asexual blood stages of P. falciparum. (1,2).
According to one aspect of the present invention, there is provided a rhoptry antigen of the asexual blood stages of Plasmodium falciparum , which is characterised by: (i) having an apparent molecular weight of approximately 55 kDa; (ii) being a glycoprotein. incorporating myristic acid; (iii) being present in a restricted localisation with merozoites consistent with a rhoptry location; and (iv) being extracted into the detergent phase when parasite antigens are separated by phase partitioning with detergent, for example with the detergent Triton X-114; or an antigenic fragment thereof.
Preferably, the antigen is a polypeptide having a primary structure which includes the amino acid sequence set out in Figure 2, or an antigenic fragment thereof.
The invention also provides a method for actively immunising a host against Plasmodium falciparum which method comprises administering to the host an antigen according to the present invention, or an antigenic fragment thereof.
The invention provides a vaccine comprising ' an antigen of the present invention, or an antigenic fragment thereof, a pharmaceutically acceptable carrier or diluent, and optionally an adjuvant. Further features of the present invention will become apparent from the detailed description in the following Example.
EXAMPLE
In this example, selective partitioning of integral membrane proteins into the detergent phase upon temperature-dependent phase separation of aqueous solutions of the detergent Triton X-114, has been used to enrich for putative integral membrane proteins of P.falciparum. Human antibodies obtained from patients from Papua New Guinea were then affinity-purified on these antigens after they had been blotted onto nitrocellulose. The purified human antibodies were used to identify recombinant clones of Escherichia coli expressing polypeptide fragments corresponding to these antigens. Materials and Methods Parasites.
The origin of P.falciparum isolate FCQ27/PNG (FC27) used in these studies has been described elsewhere (2). Parasites synchronized by sorbitol treatment were cultured at 0.25% haematocrit with parasitaemias ranging from 2-7%. All samples were washed free of medium by centrifugation and substitution with human tonicity phosphate buffered saline (HTPBS) . Cells were then pelleted, and 1ml packed cell aliquots were snap frozen and stored at -70°C until further processing.
Triton X-114 solubilization and phase separation.
Triton X-114 solubilization and separation of hydrophobic, hydrophilic and insoluble fractions was performed essentially as described by Bordier (4) with the following modifications. Triton X-114 (purchased from Fluka Ag., Switzerland) was precondensed in human tonicity phosphate buffered saline (HTPBS) . A 1 ml aliquot of prepacked parasitized cells was solubilized in 15ml of 0.5%
Triton X-114 for 90min on ice, with mild vortexing at lOmin intervals. A 1 ml sample of the total material was removed and snap frozen. The remaining 15ml were then centrifuged at 10,000xg for 15min at 4°C to remove insoluble material. The supernatent was removed and this step repeated. The sedimented material (insoluble pellet) was then washed a further three times in 0.5% Triton X-114 before being snap frozen in 1ml of HTPBS. The detergent-soluble material was then carefully layered over a 10ml sucrose cushion of cold 6% sucrose, 0.06% Triton X-114, placed for 5 min in a 37°C waterbath and then centrifuged at 500xg for 5min at 37°C. After centrifugation the 15ml detergent-depleted upper layer was collected and chilled on ice. The 10ml sucrose cushion was discarded and the detergent- enriched pellet (l-2ml) was resuspended on ice with 10ml of cold HTPBS. The resuspended detergent- enriched phase was again layered over a sucrose cushion, brought to 37° for 5min and re-pelleted by centrifugation. After this second precipitation, the detergent-enriched pellet was resuspended to 5ml in HTPBS and snap frozen. The detergent-depleted upper layer from the sucrose cushion separation was further depleted of hydrophobic proteins by adding 1ml of 11.4% Triton X-114 on ice, vortexing into solution, warming to 37°C for 5min, then centrifuging and discarding the Triton X-114 pellet. This cycle was repeated three times. The remaining detergent-depleted aqueous solution (aqueous phase) was then snap frozen. All samples were stored at -70°C until analysis.
Electrophoresis and immunoblottinσ.
Samples for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were processed under reducing conditions and electrophoresed on 10% slab gels. Gels to be analysed for protein were stained with Coomassie brilliant blue. Samples to be analysed by immunoblo ting were fractionated on 10% slab gels and electrophoretically transferred to nitrocellulose sheets. After transfer the nitrocellulose was blocked with 5% (W/V) skim milk powder in HTPBS (Blotto) and probed with sera or affinity purified human antibodies diluted appropriately in Blotto.
Bound antibody was detected by probing with
125I-protein A followed by autoradiography.
Protein A (Pharmacia Fine Chemicals, Uppsala, Sweden) was iodinated by the chloramine T method to a specific activity of 40μCi/μg~ .
Affinity purification of polyclonal monospecific human antibodies.
Antibodies were affinity purified from human serum or plasma on Triton X-114 soluble antigens electrophoretically transferred to nitrocellulose as described previously (5) and modified in this instance as follows. Reduced samples of Triton X-114 extracted membrane antigens were electrophoretically separated and transferred to nitrocellulose. Radioactive 14C high molecular weight markers
(Amersham) were used to determine the region of interest. Strips of nitrocellulose corresponding to regions of interest were incubated for 8hrs at 4°C with sera or plasma from individuals exposed to malaria. Sera was removed and the strips washed vigorously over 2 hours with 6 changes of Blotto,
three changes of HTPBS, followed by a 15 minute wash, in borate buffer (0.1 M glycine, 0.15 M sodium chloride, pH 2.6) for 10 minutes. Eluted antibodies were immediately neutralized with 2 M Tris-HCl pH 8.0 and stored at 4°C with 0.05% sodium azide. The affinity-purified antibodies were diluted 1:2 in Blotto to probe immunoblots and λAmp3 cDNA library filters, or concentrated using a Centricon microconcentrator (Amicon) for immunofluorescence assays. Antibodies were also eluted directly from immunopositive λAmp3 clones grown as lawns on nitrocellulose. Filters with lysed lawns were pre-eluted with borate and glycine buffers, incubated with serea, and the monospecific antibodies eluted as above. This second method of affinity purification required the removal of anti E.coli antibodies which" was achieved with sonicates and lawns of control λAmp3 clones. Identification of cDNA clones expressing relevant poly ep i antigens.
Details of the FCQ27/PNG isolate cDNA library, its amplification in λAmp3 and lysogenic expression in E.coli have been described (6). Detection of antigen-expressing clones by in situ colony immunoassay with human antibodies has also been described (6). Positive clones were grown and induced in liquid culture and extracted with sample buffer for electrophoresis as described (7) . Samples were electrophoresed on 10% SDS-PAGE slab gels and either stained for protein or imπtunoblotted and analysed with affinity purified antibodies, to detect the presence or absence of stable fusion polypeptides with β-galactosidase.
Antibody depletion.
One ml cultures of immunopositive clones were induced, pelleted by centrifugation, and the liquid medium removed. Pellets were frozen and thawed three times and the lysed cells resuspended in lml HTPBS. A 20μl aliquot of PNG sera was added and incubated with the lysate for 3hrs at 4°C. The cellular debris was then spun down at 10,000xg and the supernatant used to probe Triton X-114 extracted and immunoblotted parasite membrane protein antigens in order to determine the degree of antibody depletion attributable to the induced fusion polypeptide. Subcloninσ of immunopositive λAmp3 clones. λAmp3 phage were isolated from immunopositive clones and the cDNA insert extracted by EcoRI digestion as described. The purified insert was subcloned into M13 vectors for single stranded sequence determined by the dideoxy method of Sanger (8,9) and into pBTA224 (a modification of PUR-290) kindly provided by Gary Coburn (Biotechnology Australia Pty.Ltd.). The plasmid vector pBTA224 is an expressing vector in E.coli cells, and clones were rescreened for immunoreactivity by colony immunoassay. Indirect immunofluorescence.
Thin blood films of parasitized erythrocytes from asynchronous cultures of P.falciparum were air dried and fixed in 100% acetone at -20°C for 20 minutes. Slides were incubated with concentrated affinity-purified monospecific human antibodies, with fluorescein-conjugated sheep antihuman Ig antiserum as second antibody. Parasite nuclei were counterstained with ethidium bromide.
LEGENDS TO FIGURES Fiα 1:
Triton X-114 extracts of £___. falciparum infected erythrocytes were fractionated on 10% SDS-PAGE, electrophoretically transferred to nitrocellulose, and probed with either (a), pooled adult PNG sera, (b) , affinity purified polyclonal human antibodies, or (c), affinity purified monospecific human antibodies. In (a) and (b), lanes are (1), uninfected erythrocyte control (2) , total unfractionated material, (3), aqueous phase, (4), Triton phase, (5), insoluble material.
In (c), human monospecific antibodies were affinity purified from pooled adult PNG sera on a filter lawn of Ag512.(l), control strip of Triton X-114 phase antigens probed with the pooled sera, ( 2 ) , affinity purified anti Ag512 probed to a duplicate filter strip. Fiqt 2:
Partial nucleotide sequence of Ag512 with the predicted amino acid sequence of the single open reading frame. Fig 3: Rhoptry localization of Ag512 with affinity purified monospecific antibodies used in indirect immunofluorescence assays on asynchronous blood films. Pictured is a mature schizont with a punctate pattern of fluorescence characteristic of a rhoptry localized antigen.
A subset of P.falciparum antigens partition into Triton X-114.
When sera from individuals repeatedly infected with P.falciparum are used to probe immunoblots of P.falciparum antigens fractionated by phase separation in Triton X-114 several putative integral membrane protein antigens are identified. The most dominant of these had relative molecular masses (Mr) of 21,000, 35,000, 42,000, 50,000 and 55,000 as determined SDS-PAGE analysis. When the same antigen extracts were probed with antisera raised against or affinity purified on a number of cloned P.falciparum antigens it was shown that the Mr 21,000 Triton X-114 soluble antigen was the circumsporozoite protein related antigen (CRA) . This molecule is known to have a 26 amino acid hydrophobic sequence typical of integral membrane proteins (10), confirming that the procedures selects for such molecules. None of the other Triton X-114 soluble antigens appeared to correspond to any of the many P.falciparum antigens that have been previously characterised.
In order to obtain purified antibodies specific for the set of proteins of Mr 35-55,000 Triton X-114 extracts of P.falciparum were electrophoretically fractionated and transferred to nitrocellulose. The appropriate region of the nitrocellulose filter was then excised and used as an adsorbent for affinity purification of the corresponding antibodies from human serum (Materials and Method) . The purified antibodies were tested for specificity by reacting them with fractionated P.falciparum proteins. It is clearly evident in Fig.lb that these purified antibodies react almost
exclusively with those Mr 35-55,000 antigens that - selectively partition into the Triton X-114 phase.
In order to isolate clones expressing these antigens, a library of λAmp3 clones expressing P.falciparum cDNA sequences was screened with the affinity-purified antibodies. Ten positive lysogenic clones that were detected by this procedure were replated for single colonies. Selected colonies correspond to Triton X-114 soluble antigens.
Antibodies affinity purified on lawns of selected clones were used in colony immunoassays on the array of ten clones and to probe immunoblots of parasite antigens. Seven of the clones were found to be siblings encoding the Mr 55,000 antigen from the Triton X-114 phase. The most immunoreactive was chosen as the type clone and designated Ag512.
Analysis of clones by SDS-PAGE followed by protein staining or immunoblotting showed that all seven sibling clones of Ag512 produced unstable fusion proteins with β-galactosidase. Immuno sera were absorbed with sonicates of the most immunoreactive cDNA clone, Ag512, and then used to probe immunoblots of Triton X-114 extracts of P.falciparum. Sonicates of this cDNA clone totally removed all detectable antibody reactivity to the corresponding parasite antigens. Thus this cDNA clone apparently encodes the dominant naturally immunogenic epitopes in this antigen. Stage and strain specificity.
Stage-specific parasite preparations were Triton X-114 extracted, fractionated by SDS-PAGE and immunoblots probed with PNG sera. The antigen corresponding to Ag512 was present in all stages of
the parasite life cycle, but was least abundant in the asexual ring and trophozoite stages and most abundant in late schizont preparations. In the late schizont and merozoite stages, the antigen was totally soluble in the detergent Triton X-114, but in ring and trophozoite stages it partitioned into both the Triton X-114 phase and the insoluble pellet. Triton X-114 extracts were prepared from five strains of P.falciparum giving asychronously in culture. The antigen was present in all strains and there were no apparent strain-related differences in the size or immunoreactivity of the antigen. Nucleotide and amino acid sequence of Ao512.
The nucleotide sequence of AG512 together with the predicted translation is given in Fig.2. The cDNA clone does not encode the complete coding sequence. It contains one open reading frame which is in frame with β-galactosidase, and the clone produces a fusion polypeptide that is unstable in E. coli. The sequence is 839 base pairs long and contains no lengthy repetitive element. The sequence does not contain a hydrophobic stretch of amino acids. The encoded polypeptide has a predicted molecular weight of 33,534 daltons. Ag512 corresponds to a putative rhoptrv antigen.
Affinity purified human antibodies to Ag512 were prepared as described, concentrated, and used in indirect immunofluorescence assays on asynchronous blood films. The fluorescence pattern observed was consistent with localisation of the antigen in the rhoptry organelles of merozoites.
DISCUSSION
This Example shows the temperature dependent Triton X-114 detergent separation of integral membrane protein antigens of P.falciparum. One of the antigens that partitioned to the Triton X-114 phase was identified as CRA, a well characterised antigen of known primary structure which is typical of an integral membrane protein. The other dominant antigens that partition into the Triton X-114 clustered into the Mr 40-55,000 range and apparently did not correspond to antigens that had been previously cloned. These proteins, after transfer to. nitrocellulose were used to affinity purify polyclonal monospecific human antibodies which were used to isolate a clone designated Ag512, corresponding to a merozoite rhoptry protein, from a λAmp3 cDNA expression library. Antibodies to Ag512 reacted with a Triton X-114 soluble protein of Mr 55,000 which was present in all strains of P.falciparum examined and present in all of the asexual life cycle stages. Indirect immunofluorescent microscopy with these antibodies gave strong staining on mature stages with a "double dot" grape-like pattern on merozoites a pattern characteristic of antibodies to rhoptry antigens. Part of the primary structure of the Ag512 related antigen has been deduced from the nucleotide sequence of the cDNA insert in clone Ag512.
REFERENCES
1. Holder, A.A., Freeman, A.A. (1981) Nature. 294 : 361-364.
2. Perrin, L.H., Merkli, B., Gabra, M.S., Stocker, J.W., Chizzolini, C, Richie, R. (1985). J.Clin.Invest. 7_5_ : 1718-1721.
3. Chen, P., Lamont, G., Elliot, T., Kidson, C, Brown, G., Mitchell, G., Stace, J. and Alpers, M. (1980). South East Asian J.Trop. Med.Pub.Hlth. 11 : 435-440.
4. Bordier, C. (1981). J.Biol.Chem. 256, 1604-1607.
5. Beal, J.A., Mitchell, G.F. (1986), J.Immunol. Methods. 86, 217-223.
6. Kemp, D.J., Coppel, R.L., Cowman,A.F., Saint, R.B., Brown, G.V., Anders, R.F, (1983), Proc.Natl.Acad.Sci. USA 60 : 3787-3791.
7. Anders, R.F., Coppel, R.L., Brown, G.V., Saint, R.B., Cowman, A.F., Lingelbach, K.R., Mitchell, G.F. and Kemp, D.J. (1984). Molee. Biol.Med. 2, 177-192.
8. Messing, J. and Vieira, J. (1982). Gene 19. 269-276.
9. Sanger, F., Nicklen, S., Coulson, A.A. (1977) Proc.Natl.Acad.Sci. USA 7. : 5463-5467.
10. Hope, I.A., Mackay, M. , Hyde, J.E. (1985) Nucl.Acids Res. 12, 269-279.
Claims
1. A rhoptry antigen of the asexual blood stages of Plasmodium falciparum, which is characterised by: (i) having an apparent molecular weight of approximately 55kDa; (ii) being a glycoprotein incorporating myristic acid; (iii) being present in a restricted localisation with merozoites consistent with a rhoptry location; and (iv) being extracted into the detergent phase when parasite antigens are separated by phase partitioning with detergent; or an antigenic fragment thereof.
2. An antigen according to claim 1, which is antigen Ag512 described herein, or an antigenic fragment thereof.
3. An antigen according to claim 1, having a primary structure which includes the amino acid sequence set out in Figure 2, or an antigenic fragment thereof.
4. A vaccine composition comprising an antigen according to any one of claims 1 to 3, or an antigenic fragment thereof, and a pharmaceutically acceptable carrier or diluent.
5. A composition according to claim 4, further comprising an adjuvant.
6. A method for actively immunising a host against Plasmodium falciparum. which method comprises administering to the host an antigen according to anyone of claims 1 to 3, or an antigenic fragment thereof, or a vaccine composition according to claim 4 or claim 5.
7. A recombinant DNA molecule comprising all or a portion of a nucleotide sequence which is capable of being expressed as a polypeptide having the antigenicity of an antigen according to any one of claims 1 to 3, or an antigenic fragment thereof, or a recombinant cloning vehicle or vector or a host cell comprising a said recombinant DNA molecule.
8. A recombinant DNA molecule, recombinant cloning vehicle or vector or host cell according to claim 7, wherein said nucleotide sequence is a sequence as set out in Figure 2.
9. A synthetic polypeptide prepared by expression of all or a portion of a nucleotide sequence according to claim 7 or claim 8.
10. A vaccine composition comprising a synthetic polypeptide according to claim 9.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU56/86 | 1987-12-01 | ||
| AUPI568687 | 1987-12-01 |
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| Publication Number | Publication Date |
|---|---|
| EP0344257A1 true EP0344257A1 (en) | 1989-12-06 |
| EP0344257A4 EP0344257A4 (en) | 1990-04-09 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP19890900001 Withdrawn EP0344257A4 (en) | 1987-12-01 | 1988-12-01 | RHOPTRY ANTIGEN OF -i(PLASMODIUM FALCIPARUM). |
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| Country | Link |
|---|---|
| EP (1) | EP0344257A4 (en) |
| JP (1) | JPH02502337A (en) |
| KR (1) | KR900700599A (en) |
| AU (1) | AU608096B2 (en) |
| GB (1) | GB2220943A (en) |
| IL (1) | IL88558A0 (en) |
| WO (1) | WO1989005348A1 (en) |
| ZA (1) | ZA888983B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU5851390A (en) * | 1989-06-19 | 1991-01-08 | Statens Serum Institut | A malaria vaccine |
| CA2088478C (en) * | 1990-08-02 | 2002-06-11 | Allan J. Saul | Cloning and expression of a rhoptry associated protein of p. falciparum |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1984002917A1 (en) * | 1983-01-28 | 1984-08-02 | Inst Medical W & E Hall | EXPRESSION OF PLASMODIUM FALCIPARUM POLYPEPTIDES FROM CLONED cDNA |
| WO1985003725A1 (en) * | 1984-02-20 | 1985-08-29 | Biogen N.V. | Dna sequences, recombinant dna and processes for producing antigens of plasmodium falciparum |
-
1988
- 1988-11-30 ZA ZA888983A patent/ZA888983B/en unknown
- 1988-12-01 EP EP19890900001 patent/EP0344257A4/en not_active Withdrawn
- 1988-12-01 IL IL88558A patent/IL88558A0/en unknown
- 1988-12-01 WO PCT/AU1988/000463 patent/WO1989005348A1/en not_active Application Discontinuation
- 1988-12-01 AU AU26095/88A patent/AU608096B2/en not_active Ceased
- 1988-12-01 GB GB8916546A patent/GB2220943A/en not_active Withdrawn
- 1988-12-01 JP JP1500010A patent/JPH02502337A/en active Pending
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- 1989-07-31 KR KR1019890701431A patent/KR900700599A/en not_active Application Discontinuation
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1984002917A1 (en) * | 1983-01-28 | 1984-08-02 | Inst Medical W & E Hall | EXPRESSION OF PLASMODIUM FALCIPARUM POLYPEPTIDES FROM CLONED cDNA |
| WO1985003725A1 (en) * | 1984-02-20 | 1985-08-29 | Biogen N.V. | Dna sequences, recombinant dna and processes for producing antigens of plasmodium falciparum |
Non-Patent Citations (4)
| Title |
|---|
| BIOTECHNOLOGY, vol. 3, no. 8, August 1985, pages 729-740, New York US; J.V. RAVETCH et al.:"Molecular genetic strategies for the development of anti-malarial vaccines" * |
| MOLECULAR AND BIOCHEMICAL PARASITOLOGY, vol. 25, 1987, pages 73-81, Elsevier Science Publishers, Amsterdam, NL; R.L. COPPEL et al.: "A cDNA clone expressing a rhoptry protein of Plasmodium falciparum" * |
| PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, vol. 85, July 1988, pages 5195-5199, Washington, D.C., US; J.A. SMYTHE et al.: "Identification of two integral membrane proteins of Plasmodium falciparum" * |
| See also references of WO8905348A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0344257A4 (en) | 1990-04-09 |
| WO1989005348A1 (en) | 1989-06-15 |
| GB2220943A (en) | 1990-01-24 |
| KR900700599A (en) | 1990-08-16 |
| ZA888983B (en) | 1989-08-30 |
| GB8916546D0 (en) | 1989-11-01 |
| JPH02502337A (en) | 1990-08-02 |
| AU2609588A (en) | 1989-07-05 |
| IL88558A0 (en) | 1989-07-31 |
| AU608096B2 (en) | 1991-03-21 |
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