EP0516721A1 - Antigenic proteins of plasmodium - Google Patents

Antigenic proteins of plasmodium

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Publication number
EP0516721A1
EP0516721A1 EP91905182A EP91905182A EP0516721A1 EP 0516721 A1 EP0516721 A1 EP 0516721A1 EP 91905182 A EP91905182 A EP 91905182A EP 91905182 A EP91905182 A EP 91905182A EP 0516721 A1 EP0516721 A1 EP 0516721A1
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EP
European Patent Office
Prior art keywords
protein
dna fragment
merozoite
plasmodium
encodes
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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.)
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Application number
EP91905182A
Other languages
German (de)
French (fr)
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EP0516721A4 (en
Inventor
Paul Andrew Waters
Thomas F. Mccutchan
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US Department of Health and Human Services
US Department of Commerce
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US Department of Health and Human Services
US Department of Commerce
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Publication of EP0516721A1 publication Critical patent/EP0516721A1/en
Publication of EP0516721A4 publication Critical patent/EP0516721A4/en
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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
    • 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, in general, to merozoite antigen proteins of simian or simian-like species of Plasmodium suitable for use as vaccines against malaria infection.
  • the invention further relates to DNA sequences encoding such proteins, to recombinant DNA molecules that include such sequences and to cells trans- formed therewith.
  • the disease contributes substantially to infant mortality in endemic areas and remains a severe and debilitating illness for those who remain afflicted with it as adults .
  • the causative agent of malaria is a protozoan of the genus Plasmodium. Individual species within the genus appear to have a restricted host range for the animals they infect. Despite species differences in host range, the life cycles, mode of infection, biochemistry and genetics of the various Plasmodium species are markedly similar.
  • Plasmodium The life cycle of Plasmodium is complex, with the organism undergoing several distinct morphological chang ⁇ es, involving the participation of a mammalian host and a mosquito vector.
  • the parasite in the sporozoite form, is introduced to the mammalian host through the bite of the mosquito vector.
  • the sporozoite rapidly disappear from the blood stream and are next found as intracellular parasites of liver parenchymal cells.
  • a blood infection ensues, characterized by the well-known clinical symptoms of malaria after a complex series of morphological and biochemical transitions.
  • the parasite is then found in the red blood cells, where it continues its development. Substantial amounts of the parasite may be obtained from the red blood cells of infected patients.
  • Vaccine development to provide protective immunity against malaria infection has been thwarted by the fact -that the parasite's life cycle in the mammalian host is primarily intracellular. Except for brief periods of time, the parasite is protected from contact with the immune system. Two stages in the parasite's life cycle- during which it becomes briefly exposed to the immune system are: (1) the interval following initial infection before sporozoite have successfully invaded the cells of the liver, and (2) the interval during which merozoites leave infected red blood cells and enter uninfected red blood cells.
  • the present invention discloses antigenic proteins useful as vaccines to provide immunity against merozoite forms of the parasite.
  • the present invention to provides merozoite antigen proteins of simian or simian-like species of Plasmodium useful use as vaccines to provide protection against malaria in humans and animals.
  • the present invention relates to a substantially pure form of a merozoite antigen protein isolatable from P. mknowle ⁇ i having a molecular weight of 64.5 kDa.
  • the present inven ⁇ tion relates to a substantially pure form of a merozoite antigen protein isolatable from P. vivax homologous to the P. mknowlesi protein described above.
  • the present invention relates to a DNA fragment encoding the above- described merozoite antigen protein of P. *knowle ⁇ i .
  • the present invention relates to a DNA fragment encoding the above- described merozoite antigen protein of P. vivax.
  • the present invention relates to a recombinant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite antigen protein of P. knowlesi .
  • the present invention relates to a reco binant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite antigen protein of P. vivax.
  • the present invention relates to a host cell transformed with the above-described recombinant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite antigen protein of P. knowlesi .
  • the present invention relates to a host cell transformed with the above-described recombinant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite antigen protein of P. vivax.
  • the present invention relates to a process of producing the above- described merozoite protein of P. knowlesi .
  • the method comprises culturing the host cell transformed with the above-described recombinant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite antigen protein of P. knowlesi , under conditions such that the DNA fragment is expressed and the merozoite antigen protein thereby produced.
  • the present invention relates to a process of producing the above- described merozoite protein of P. vivax.
  • the method comprises culturing the host cell transformed with the above-described recombinant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite protein of P. vivax, under conditions such that the DNA fragment is expressed and the merozoite protein thereby produced.
  • Figure 1A-1I shows the gene sequence of the 66 JcDa merozoite surface antigen of Plasmodium knowlesi (PK66) , a partial sequence of its analogue from Plasmodium vivax
  • the sequence of the gene (K) encoding the PK66 is shown on the upper line. Above this sequence is given the partial gene (V) sequence of PV66. PV66 is only given where there is a difference with PK66. Beneath the PK66 gene is the translation of its major open reading frame (K) . Both the gene and its translation are aligned with the translation product of the gene designated AMA-1 (Peterson et al, Mol. Cell. Biol. 9, 3151-3155, 1989), designated herein as PF83 (F), as well as the translation of the partial gene PV66 (V) . An asterisk indicates identity at the amino acid level and a period indicates conservative substitution. The region of PF83 which is not co-linear with PK66 is indicated.
  • Figure 2B shows the distribution of PK66 on a mature schizont of P. knowlesi and during erythrocyte invasion.
  • PK66 maintain an association after cleavage.
  • Figure 3A shows the immmune precipitation of prepa ⁇ ration A with rabbit sera (Lanes 1-7) and of preparation B (Lanes 8-11) .
  • the antibody preparations are as given in the appropriate lanes.
  • External peptide is serum raised against a syn ⁇ thetic peptide to a hydrophilic region of the predicted sequence of PK66 and does not appear to recognize the protein.
  • Rabbit anti-66 is a rabbit polyclonal antiserum raised against the affinity purified PK66 and has been described (Gamier et al., J. Mol. Biol.
  • Mab R3/1C2 has also been described previously (Deans et al., Clin. Exp. Immunol. 49, 297-309, 1982).
  • the arrows indicate the migration of PK66 in lanes 1-7 and, in lanes 8-11, from the top PK66, and its processed fragments of 44 and 20 kDa.
  • Figure 3B shows the western blot analysis of prepa- ration C.
  • FIG. 1 shows the position of migration of PK66 (top) , and the processed fragments of 44 and 20 kDa respectively.
  • Figure 4 shows the gene hybridization of PK66 cDNA to genomic DNA of different species of Plasmodium.
  • Hybridization of PK66 cDNA to the DNA of other species of Plasmodium reveals that homologues exist in the simian branch of the Plasmodium genus.
  • the present invention relates to merozoite antigen- ic proteins of simian or simian-like species of Plasmodium (for example, P. knowlesi , P. vivax, P. ovale, P. fragile and P. cynomologi , as distinguished from species such as P. falciparum) suitable for use as vaccines protective against malaria in humans and animals.
  • the proteins can also be used in the design of anti-malarial drugs which bind to the protein in a manner such that erythrocyte invasion is prevented.
  • the invention further relates to DNA sequences (fragments) encoding all, or unique portions (i.e., at least 5 amino acids), of such proteins.
  • the invention also relates to recombinant molecules ' containing such DNA sequences, and to cells transformed therewith.
  • the present invention relates to DNA sequences (including cDNA sequences) that encode the entire amino acid sequence for P. knowlesi or P. vivax given in Figure 1 (the specific DNA sequence given in Figure 1 being only an example) , or any unique portion thereof.
  • the present inven ⁇ tion relates to a recombinant DNA molecule that includes a vector and a DNA sequence encoding the merozoite antigen protein of P. knowlesi (advantageously, a DNA sequence encoding the protein shown in Figure 1 or a protein having the immunogenic properties of that protein) .
  • the present invention relates to a recombinant DNA molecule that includes a vector and a DNA sequence encoding the merozoite antigen protein of P. vivax (advantageously, a DNA sequence encoding the protein shown in Figure 1 or a protein having the immunogenic properties of that protein) .
  • the vector can take the form of a virus or a plasmid (for example, pUC19 or vaccinia virus) .
  • the DNA sequence can be present in the vector operably linked to regulatory elements, includ ⁇ ing, for example, a promoter.
  • the recombinant molecule can be suitable for transforming procaryotic or eucaryotic cells, advantageously vertebrate cells, and especially mammalian cells.
  • the present invention relates to host cells transformed with the above-described recombinant molecules.
  • the host can be procaryotic (for example, bacterial), lower eucaryotic (i.e., fungal, including yeast) or higher eucaryotic (i.e., mammalian, including human). Transformation can be effected using methods known in the art.
  • the present inven ⁇ tion relates to a vaccine protective against malaria.
  • the vaccine comprises the merozoite antigenic protein de ⁇ scribed above (and a pharmaceutically acceptable carrier) in an amount sufficient to protect against malarial infection.
  • the vaccine can be given parenterally.
  • the protein can be present in a purified form or in a virus, (advantageously, vaccinia virus; that is, the DNA sequence encoding the protein can be incorporated into a vaccinia virus in a manner such that the DNA sequence is expressed) .
  • purified proteins it may be advantageous to include adjuvants known in the art (for example, alum or Freund's adjuvant).
  • adjuvants known in the art
  • an enhancer is albumin.
  • compositions of matter containing a Plasmodium protein having 55% amino acids sequence identity with PK66 or fragment thereof may be used as immunogens to raise antibodies protective against malaria.
  • the protective antibodies are usually best elicited by a series of 2-3 dosings about 2-3 weeks apart. The series can be repeated when circulating antibody concen ⁇ tration drops.
  • the antigens of the invention can also be used as diagnostics to determine the presence of circulating antibodies against malaria.
  • the antigens can be presented attached to a solid support such a microtiter plate.
  • Antigens may be used in standard antigen-antibody tests to detect the presence of antibodies against the protein in the blood. Examples of such assays include immunofluores- cence tests or ELISA tests .
  • Antibodies raised against the protein of the invention can be used to detect antigen level in patient tissues, including blood.
  • PK66 The 66 kDa merozoite surface antigen (PK66) of Plasmodium knowlesi , a simian malaria, possesses immuno- - genie properties that are thought to originate from a role in parasite invasion of erythrocytes .
  • F(ab) fragments of inhibitory, PK66 specific monoclonal antibodies (MABS) uniquely retain the ability to inhibit erythrocyte inva ⁇ sion by the merozoite. (Thomas et al., Mol. Biocheirt. Parasitol. 13, 187-199, 1984). This implies that PK66, and/or its processed products of 44 and 42 kDA, act as erythrocyte-specific receptors .
  • Rhesus monkeys have been successfully vaccinated with a combination of PK66 and the clinically promising adjuvant, saponin (Deans et al., Parasite Immunol. 10, 535-552, 1988), that, followed by infection, generated a strong immunity (Deans et al., Parasite Immunol. 10, 535-552, 1988).
  • PK66 Plasmodium vivax
  • PV66 Plasmodium vivax
  • PK66 changes after schizont rupture in a co-ordinate manner associated with merozoite invasion.
  • the protein is concentrated at the apical end prior to rupture, following which it can distribute itself entirely across the surface of the free merozoite.
  • immunofluorescence studies indicate that PK66 is excluded from the erythrocyte at, and behind, the invasion interface.
  • the products of PK66 process ⁇ ing which occurs around the time of schizont rupture (Deans et al. , Mol. Biochem. Parasitol. 11, 189-204, 1984), retain a stable association with one another.
  • PK66 appears to be integrally related to invasion of the red cell (Thomas et al., Mol. Biochem. Parasitol. 13, 187-199, 1984; and Deans et al., Clin. Exp. Immunol. 49, 297-309, 1982).
  • cDNA clones expressing PK66 have been identified using a monospecific polyclonal rabbit serum (Deans et al., Mol. Biochem. Parasitol., 26, 155-166, 1987).
  • a full length cDNA sequence ( Figure 1) contains a 564 amino acid open reading frame of 64.5 kDa molecular weight. Its identity was confirmed by sera raised to a synthetic peptide from the C-terminu ⁇ (CT serum) of the translated sequence (see below).
  • the remainder of the two proteins are essentially collinear (residues 54-564 in P. knowlesi , and residues 98-622 in P. falciparum) . 55% of the residues in PK66 are identical in PF83. Excluding conservative substitutions, only 12.5% of the residues are completely nonhomologous.
  • the partial PV66 protein shows considerably more homology to PK66 (86.5% identity) and resembles PK66 in its relative similarity to PF83 (58.6% identity and lack of a 4 aa. region at position 393 in PK66).
  • the structurally significant amino acids, cysteine and proline, are positionally conserved between all three proteins (hatched and boxed in Figure 1).
  • the Robson predictive algorithm determines that the collinear portion of the three proteins have almost identical secondary struc ⁇ tures.
  • the charge and hydropathic profiles are also very similar.
  • Identity between the three proteins is regionalized; the C-terminus is an outstanding example where 29 of 30 residues are identical. This region has no assigned function but the preservation of identity argues its importance.
  • the two conserved tyrosine residues at the exact C-terminus are potential phosphorylation sites which may be significant in the biological role of this protein.
  • a search of the NBRF database revealed no entries with significant homologies to the intact proteins or to the more highly conserved regions. P. knowlesi and P.
  • falciparum are evolutionarily well separated within the genus, and their genomes have very different base composition (McCutchan et al. , Sci ⁇ ence, 225, 808-811, 1984). This is reflected in the fact that the genes encoding the highly conserved proteins, PK66 and PF83, are sufficiently different to prevent cross hybridization, even at low stringency. This indicates that the protein is vital to the merozoites of both species and remarkably little variation is tolerable. Consistent with this, an analogue can be detected in phylogentically diverse species of Plasmodium spp. For example, by immunofluorescence using CT serum, analogues can be shown in P. falciparum and P. berghei despite gene hybridization being negative ( Figure 2A) . Southern analy ⁇ sis with PK66 cDNA shows the gene to be distributed throughout the simian branch of Plasmodium.
  • PK66 was localized by immunofluorescence with inhibitory MAB (Deans et al., Clin. Exp. Immunol. 49, 297- 309, 1982) and CT serum. Its surface distribution was modulated during the period between schizont rupture and successful invasion of the erythrocyte (Deans et al., Clin. Exp. Immunol. 49, 297-309, 1982). While the previ ⁇ ously described general surface distribution was observed (Deans et al. , Clin. Exp. Immunol. 49, 297-309, 1982) ( Figure 2A) , discrete merozoites within mature schizonts demonstrated predominantly apical staining. This was also seen with the merozoites of P.
  • PK66 Full length PK66, and its analogues, have features expected of integral membrane proteins (Deans et al., Mol. Biochem. Parasitol. 11, 189- 204, 1984; and Deans et al., Clin. Exp. Immunol. 49, 297- 309, 1982).
  • the observed distribution indicates that PK66 originates apically, either as a localized surface compo ⁇ nent or as a constituent of organelles, such as the rhoptry, dense granules or the micronemes.
  • Other apically concentrated merozoite vaccine candidates, such as RESA (Brown et al., J. Exp. Med. 162, 774-779, 1986) are thought to originate from internal organelles, but this is not found on the merozoite surface.
  • PK66 is clearly associated with the surface of the merozoite after schizont rupture.
  • PK66 is not carried into the erythrocyte during invasion (Peterson et al., Mol. Cell. Biol. 9, 3151-3155, 1989) ( Figure 2B), but remains associated with the inva ⁇ sion interface and the areas of the merozoite that have not yet entered the erythrocyte. Dependant upon the attitude of the invaded cell relative to the observer, the staining pattern may also progress down the side of the parasite during invasion ( Figure 2B).
  • a lambda gtll expression library constructed with cDNA from mature schizonts of P. knowlesi was screened with rabbit polyclonal serum monospecific for the PK66 (Gamier et al., J. Mol. Biol. 120, 97-120, 1978). Reactive colonies were plaque purified and the inserts subcloned into the pGEM series of vectors (Promega). PV66 was isolated by PCR amplification of P. vivax genomic DNA using degenerate primers with Bam HI ends.
  • PK66 CT serum was prepared as described (Dame et al., Science 225, 593-599, 1984). Parasites were prepared for IFAT analysis using cold methanol as fixative. • Fluorescent secondary antibodies were used at concentra ⁇ tions recommended by the manufacturer (Kirkegaard & Perry) . Microscopy and photography were carried out using an Axiophot microscope (Zeiss) and Tri X-pan film (Kodak) according to manufacturers' instructions. The distribution of PK66 analogues within the genus Plasmodium is shown in Figure 2A.
  • CT serum staining was revealed using a fluores- cein conjugated anti-rabbit secondary antibody.
  • 13C11 and R31C2 staining were revealed using rhodamine conjugated anti-mouse and anti-rat secondary antibody preparations, respectively, and photographed as above.
  • Example 3 Parasites extracts are referred to by letter (A, B or C). Metabolic labelling with [ 35 S]-methionine was carried out essentially as described in Deans et al. Glin. Exp. Immunol. 49, 297-309, 1982. Preparation A was labelled for 2h in the presence of protease inhibitors chymostatin and leupeptin and no schizont rupture was observed. Preparation B was labelled for llh and appre ⁇ ciable schizont bursting and merozoite release had oc- curred. Preparation C was an unlabelled parasite extract of a mature culture with ruptured schizonts prepared for Western analysis.

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Abstract

L'invention se rapporte à des séquences d'ADN qui codent pour des protéines antigéniques mérozoïtes de plasmodium du singe ou d'espèces simiennes et qui sont utilisables comme vaccins contre les infections paludéennes chez l'homme et chez les animaux. L'invention se rapporte également à des molécules d'ADN recombinantes qui contiennent ces séquences et à des cellules transformées à l'aide de celles-ci.The invention relates to DNA sequences which code for merozoite antigenic proteins of plasmodium from monkeys or ape species and which can be used as vaccines against malaria infections in humans and animals. The invention also relates to recombinant DNA molecules which contain these sequences and to cells transformed therewith.

Description

ANTIGENIC PROTEINS OF PLASMODIUM
BACKGROUND OF THE INVENTION Technical Field The present invention relates, in general, to merozoite antigen proteins of simian or simian-like species of Plasmodium suitable for use as vaccines against malaria infection. The invention further relates to DNA sequences encoding such proteins, to recombinant DNA molecules that include such sequences and to cells trans- formed therewith.
State of the Art Malaria constitutes a worldwide public health hazard of enormous economic and medical significance. The disease contributes substantially to infant mortality in endemic areas and remains a severe and debilitating illness for those who remain afflicted with it as adults . Despite advances in the techniques of mosquito abatement and improved public health measures, the disease is still endemic in large portions of the world. The causative agent of malaria is a protozoan of the genus Plasmodium. Individual species within the genus appear to have a restricted host range for the animals they infect. Despite species differences in host range, the life cycles, mode of infection, biochemistry and genetics of the various Plasmodium species are markedly similar.
The life cycle of Plasmodium is complex, with the organism undergoing several distinct morphological chang¬ es, involving the participation of a mammalian host and a mosquito vector. The parasite, in the sporozoite form, is introduced to the mammalian host through the bite of the mosquito vector. The sporozoite rapidly disappear from the blood stream and are next found as intracellular parasites of liver parenchymal cells. A blood infection ensues, characterized by the well-known clinical symptoms of malaria after a complex series of morphological and biochemical transitions. The parasite is then found in the red blood cells, where it continues its development. Substantial amounts of the parasite may be obtained from the red blood cells of infected patients.
Vaccine development, to provide protective immunity against malaria infection has been thwarted by the fact -that the parasite's life cycle in the mammalian host is primarily intracellular. Except for brief periods of time, the parasite is protected from contact with the immune system. Two stages in the parasite's life cycle- during which it becomes briefly exposed to the immune system are: (1) the interval following initial infection before sporozoite have successfully invaded the cells of the liver, and (2) the interval during which merozoites leave infected red blood cells and enter uninfected red blood cells. The present invention discloses antigenic proteins useful as vaccines to provide immunity against merozoite forms of the parasite.
SUMMARY OF THE INVENTION * The present invention to provides merozoite antigen proteins of simian or simian-like species of Plasmodium useful use as vaccines to provide protection against malaria in humans and animals.
In one specific embodiment, the present invention relates to a substantially pure form of a merozoite antigen protein isolatable from P. mknowleεi having a molecular weight of 64.5 kDa.
In another specific embodiment, the present inven¬ tion relates to a substantially pure form of a merozoite antigen protein isolatable from P. vivax homologous to the P. mknowlesi protein described above. In still another specific embodiment, the present invention relates to a DNA fragment encoding the above- described merozoite antigen protein of P. *knowleεi .
In yet another specific embodiment, the present invention relates to a DNA fragment encoding the above- described merozoite antigen protein of P. vivax.
In a further specific embodiment, the present invention relates to a recombinant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite antigen protein of P. knowlesi .
In a further specific embodiment, the present invention relates to a reco binant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite antigen protein of P. vivax.
In yet another specific embodiment, the present invention relates to a host cell transformed with the above-described recombinant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite antigen protein of P. knowlesi .
In yet another specific embodiment, the present invention relates to a host cell transformed with the above-described recombinant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite antigen protein of P. vivax.
In still another specific embodiment, the present invention relates to a process of producing the above- described merozoite protein of P. knowlesi . The method comprises culturing the host cell transformed with the above-described recombinant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite antigen protein of P. knowlesi , under conditions such that the DNA fragment is expressed and the merozoite antigen protein thereby produced.
In yet another specific embodiment, the present invention relates to a process of producing the above- described merozoite protein of P. vivax. The method comprises culturing the host cell transformed with the above-described recombinant DNA molecule comprising a vector, and the above-described DNA fragment encoding the merozoite protein of P. vivax, under conditions such that the DNA fragment is expressed and the merozoite protein thereby produced. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A-1I shows the gene sequence of the 66 JcDa merozoite surface antigen of Plasmodium knowlesi (PK66) , a partial sequence of its analogue from Plasmodium vivax
SUBSTITUTESHEET (PV66) and a comparison of the encoded protein with PF83, the analogue from Plasmodium falciparum.
The sequence of the gene (K) encoding the PK66 is shown on the upper line. Above this sequence is given the partial gene (V) sequence of PV66. PV66 is only given where there is a difference with PK66. Beneath the PK66 gene is the translation of its major open reading frame (K) . Both the gene and its translation are aligned with the translation product of the gene designated AMA-1 (Peterson et al, Mol. Cell. Biol. 9, 3151-3155, 1989), designated herein as PF83 (F), as well as the translation of the partial gene PV66 (V) . An asterisk indicates identity at the amino acid level and a period indicates conservative substitution. The region of PF83 which is not co-linear with PK66 is indicated. Both the predicted signal region and transmembrane anchor regions are desig¬ nated. The conserved cysteine (hatched boxes) and proline (empty boxes) residues are indicated. Finally, the C- ter inal peptide used to generate the immune rabbit serum used in the Western blot analysis and in the immunofluo- rescence studies is also shown. The gene and protein sequences of PK66 are numbered as well as the protein sequence of PF83. The PV66 partial gene and protein sequences are not numbered. Figure 2A shows distribution of PK66 Analogues within the Genus Plasmodium.
Immunofluorescence reactivity of rabbit serum immunized with the C-terminal peptide of PK66. The reactivity is illustrated with a bursting schizont of P. knowlesi , intact schizonts of P. falciparum, a bursting schizont of P. falciparum and free merozoites of P. berghei .
Figure 2B shows the distribution of PK66 on a mature schizont of P. knowlesi and during erythrocyte invasion.
Immune fluorescence visualization of the distribu¬ tion of PK66: (I) prior to schizont rupture, (II) after attachment of the merozoite to the erythrocyte, (III) - (VI) invasion of the erythrocyte. The three columns depict, from the left, phase pictures of CT serum and 13C11 (which recognizes gp230) stained parasite material except in line (VI) where R31C2 replaced 13C11. Figures 3A and 3B show the processed fragments of
PK66 maintain an association after cleavage.
Figure 3A shows the immmune precipitation of prepa¬ ration A with rabbit sera (Lanes 1-7) and of preparation B (Lanes 8-11) . The antibody preparations are as given in the appropriate lanes. The sera used in the two sets of CT serum absorptions are from different animals. Key: pre- = pre-immunization bleed; post- = post-immunization bleed. External peptide is serum raised against a syn¬ thetic peptide to a hydrophilic region of the predicted sequence of PK66 and does not appear to recognize the protein. Rabbit anti-66 is a rabbit polyclonal antiserum raised against the affinity purified PK66 and has been described (Gamier et al., J. Mol. Biol. 120, 97-120, 1978) . Mab R3/1C2 has also been described previously (Deans et al., Clin. Exp. Immunol. 49, 297-309, 1982). The arrows indicate the migration of PK66 in lanes 1-7 and, in lanes 8-11, from the top PK66, and its processed fragments of 44 and 20 kDa.
Figure 3B shows the western blot analysis of prepa- ration C.
Lane 1, Rabbit prebleed; Lane 2, Rabbit anti-PK66 serum; Lane 3, CT serum. The arrows show the position of migration of PK66 (top) , and the processed fragments of 44 and 20 kDa respectively. Figure 4 shows the gene hybridization of PK66 cDNA to genomic DNA of different species of Plasmodium.
Hybridization of PK66 cDNA to the DNA of other species of Plasmodium reveals that homologues exist in the simian branch of the Plasmodium genus. Southern blot of Hind III digested genomic DNA probed with clone 821 cDNA. Lanes contain DNA from: 1) P. fragile , 2 ) P. knowlesi , 3) P. inui , 4) P. vivax (Gombak) , and 5) P. vivax (London). Size markers are Hind III digested lambda DNA and migrated as indicated, the upper mark corresponds to 23.6 kbp, the lower 2.0 kbp.
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to merozoite antigen- ic proteins of simian or simian-like species of Plasmodium (for example, P. knowlesi , P. vivax, P. ovale, P. fragile and P. cynomologi , as distinguished from species such as P. falciparum) suitable for use as vaccines protective against malaria in humans and animals. The proteins can also be used in the design of anti-malarial drugs which bind to the protein in a manner such that erythrocyte invasion is prevented. The invention further relates to DNA sequences (fragments) encoding all, or unique portions (i.e., at least 5 amino acids), of such proteins. The invention also relates to recombinant molecules' containing such DNA sequences, and to cells transformed therewith.
In a specific embodiment, the present invention relates to DNA sequences (including cDNA sequences) that encode the entire amino acid sequence for P. knowlesi or P. vivax given in Figure 1 (the specific DNA sequence given in Figure 1 being only an example) , or any unique portion thereof.
In another specific embodiment, the present inven¬ tion relates to a recombinant DNA molecule that includes a vector and a DNA sequence encoding the merozoite antigen protein of P. knowlesi (advantageously, a DNA sequence encoding the protein shown in Figure 1 or a protein having the immunogenic properties of that protein) . In still another specific embodiment, the present invention relates to a recombinant DNA molecule that includes a vector and a DNA sequence encoding the merozoite antigen protein of P. vivax (advantageously, a DNA sequence encoding the protein shown in Figure 1 or a protein having the immunogenic properties of that protein) . In each case, the vector can take the form of a virus or a plasmid (for example, pUC19 or vaccinia virus) . The DNA sequence can be present in the vector operably linked to regulatory elements, includ¬ ing, for example, a promoter. The recombinant molecule can be suitable for transforming procaryotic or eucaryotic cells, advantageously vertebrate cells, and especially mammalian cells.
In a further specific embodiment, the present invention relates to host cells transformed with the above-described recombinant molecules. The host can be procaryotic (for example, bacterial), lower eucaryotic (i.e., fungal, including yeast) or higher eucaryotic (i.e., mammalian, including human). Transformation can be effected using methods known in the art.
In another specific embodiment, the present inven¬ tion relates to a vaccine protective against malaria. The vaccine comprises the merozoite antigenic protein de¬ scribed above (and a pharmaceutically acceptable carrier) in an amount sufficient to protect against malarial infection. The vaccine can be given parenterally. The protein can be present in a purified form or in a virus, (advantageously, vaccinia virus; that is, the DNA sequence encoding the protein can be incorporated into a vaccinia virus in a manner such that the DNA sequence is expressed) . Where purified proteins are used, it may be advantageous to include adjuvants known in the art (for example, alum or Freund's adjuvant). Other components used to enhance antigenicity can also be added. An example of such, an enhancer is albumin.
Compositions of matter containing a Plasmodium protein having 55% amino acids sequence identity with PK66 or fragment thereof may be used as immunogens to raise antibodies protective against malaria. The protective antibodies are usually best elicited by a series of 2-3 dosings about 2-3 weeks apart. The series can be repeated when circulating antibody concen¬ tration drops.
The antigens of the invention can also be used as diagnostics to determine the presence of circulating antibodies against malaria. The antigens can be presented attached to a solid support such a microtiter plate.
Antigens may be used in standard antigen-antibody tests to detect the presence of antibodies against the protein in the blood. Examples of such assays include immunofluores- cence tests or ELISA tests .
Antibodies raised against the protein of the invention can be used to detect antigen level in patient tissues, including blood.
The 66 kDa merozoite surface antigen (PK66) of Plasmodium knowlesi , a simian malaria, possesses immuno- - genie properties that are thought to originate from a role in parasite invasion of erythrocytes . F(ab) fragments of inhibitory, PK66 specific monoclonal antibodies (MABS) uniquely retain the ability to inhibit erythrocyte inva¬ sion by the merozoite. (Thomas et al., Mol. Biocheirt. Parasitol. 13, 187-199, 1984). This implies that PK66, and/or its processed products of 44 and 42 kDA, act as erythrocyte-specific receptors . Rhesus monkeys have been successfully vaccinated with a combination of PK66 and the clinically promising adjuvant, saponin (Deans et al., Parasite Immunol. 10, 535-552, 1988), that, followed by infection, generated a strong immunity (Deans et al., Parasite Immunol. 10, 535-552, 1988).
Disclosed herein is the complete sequence of PK66 and a partial sequence of its analogue from Plasmodium vivax (PV66). It is further demonstrated that highly conserved analogues exist throughout Plasmodium including a recently reported gene from Plasodium falciparum (Peter¬ son et al., Mol. Cell. Biol. 9, 3151-3155, 1989)." These analogues are highly promising vaccination candidates.
The distribution of PK66 changes after schizont rupture in a co-ordinate manner associated with merozoite invasion. The protein is concentrated at the apical end prior to rupture, following which it can distribute itself entirely across the surface of the free merozoite. During invasion, immunofluorescence studies indicate that PK66 is excluded from the erythrocyte at, and behind, the invasion interface. Significantly, the products of PK66 process¬ ing, which occurs around the time of schizont rupture (Deans et al. , Mol. Biochem. Parasitol. 11, 189-204, 1984), retain a stable association with one another.
PK66 appears to be integrally related to invasion of the red cell (Thomas et al., Mol. Biochem. Parasitol. 13, 187-199, 1984; and Deans et al., Clin. Exp. Immunol. 49, 297-309, 1982). Here, cDNA clones expressing PK66 have been identified using a monospecific polyclonal rabbit serum (Deans et al., Mol. Biochem. Parasitol., 26, 155-166, 1987). A full length cDNA sequence (Figure 1) contains a 564 amino acid open reading frame of 64.5 kDa molecular weight. Its identity was confirmed by sera raised to a synthetic peptide from the C-terminuε (CT serum) of the translated sequence (see below).
Study of animal models of malaria indicates that biologically relevant molecules may have homologues in the human pathogens. A highly similar protein has recently been reported in P. falciparum (Peterson et al., Mol. Cell. Biol. 9, 3151-3155, 1989) which is referred to herein as PF83. The two proteins are compared in Figure 1, together with a partial sequence for the homologue isolated from P. vivax (PV66). Comparison of the predict¬ ed amino acid sequences of PK66 and PF83 reveal striking conservation of the major features. Although the proteins are of different size, the additional portion of PF83 resides entirely within a single region at the N-terminus of the mature protein and may reflect adaptation to the particular host environment. The remainder of the two proteins are essentially collinear (residues 54-564 in P. knowlesi , and residues 98-622 in P. falciparum) . 55% of the residues in PK66 are identical in PF83. Excluding conservative substitutions, only 12.5% of the residues are completely nonhomologous. The partial PV66 protein shows considerably more homology to PK66 (86.5% identity) and resembles PK66 in its relative similarity to PF83 (58.6% identity and lack of a 4 aa. region at position 393 in PK66). The structurally significant amino acids, cysteine and proline, are positionally conserved between all three proteins (hatched and boxed in Figure 1). The Robson predictive algorithm (Gamier et al., J. Mol. Biol. 120, 97-120, 1978), determines that the collinear portion of the three proteins have almost identical secondary struc¬ tures. The charge and hydropathic profiles are also very similar. Identity between the three proteins is regionalized; the C-terminus is an outstanding example where 29 of 30 residues are identical. This region has no assigned function but the preservation of identity argues its importance. The two conserved tyrosine residues at the exact C-terminus are potential phosphorylation sites which may be significant in the biological role of this protein. A search of the NBRF database revealed no entries with significant homologies to the intact proteins or to the more highly conserved regions. P. knowlesi and P. falciparum are evolutionarily well separated within the genus, and their genomes have very different base composition (McCutchan et al. , Sci¬ ence, 225, 808-811, 1984). This is reflected in the fact that the genes encoding the highly conserved proteins, PK66 and PF83, are sufficiently different to prevent cross hybridization, even at low stringency. This indicates that the protein is vital to the merozoites of both species and remarkably little variation is tolerable. Consistent with this, an analogue can be detected in phylogentically diverse species of Plasmodium spp. For example, by immunofluorescence using CT serum, analogues can be shown in P. falciparum and P. berghei despite gene hybridization being negative (Figure 2A) . Southern analy¬ sis with PK66 cDNA shows the gene to be distributed throughout the simian branch of Plasmodium.
METHODS PK66 was localized by immunofluorescence with inhibitory MAB (Deans et al., Clin. Exp. Immunol. 49, 297- 309, 1982) and CT serum. Its surface distribution was modulated during the period between schizont rupture and successful invasion of the erythrocyte (Deans et al., Clin. Exp. Immunol. 49, 297-309, 1982). While the previ¬ ously described general surface distribution was observed (Deans et al. , Clin. Exp. Immunol. 49, 297-309, 1982) (Figure 2A) , discrete merozoites within mature schizonts demonstrated predominantly apical staining. This was also seen with the merozoites of P. berghei and P. falciparum using the CT serum (Figure 2A) . Full length PK66, and its analogues, have features expected of integral membrane proteins (Deans et al., Mol. Biochem. Parasitol. 11, 189- 204, 1984; and Deans et al., Clin. Exp. Immunol. 49, 297- 309, 1982). The observed distribution indicates that PK66 originates apically, either as a localized surface compo¬ nent or as a constituent of organelles, such as the rhoptry, dense granules or the micronemes. Other apically concentrated merozoite vaccine candidates, such as RESA (Brown et al., J. Exp. Med. 162, 774-779, 1986) are thought to originate from internal organelles, but this is not found on the merozoite surface. In contrast, PK66 is clearly associated with the surface of the merozoite after schizont rupture.
PK66 is not carried into the erythrocyte during invasion (Peterson et al., Mol. Cell. Biol. 9, 3151-3155, 1989) (Figure 2B), but remains associated with the inva¬ sion interface and the areas of the merozoite that have not yet entered the erythrocyte. Dependant upon the attitude of the invaded cell relative to the observer, the staining pattern may also progress down the side of the parasite during invasion (Figure 2B). These observations indicate that PK66 associates with, or is excluded at, the boundary of parasite penetration.
A model in conformance with the above observations indicates a role for PK66 in reorientation of the parasite on the surface of erythrocytes, although its precise function and the basis of MAB inhibition remains unclear. A potential vaccine candidate would be expected to exhibit minimal strain variation. Evidence thus-far obtained indicates that this is the case for both PK66 and PF83. Full length sequence data for PK66 from two strains of P. knowlesi indicates only two amino acid changes, neither of al., Parasite Immunol. 10, 535-552, 1988). Comparison of full length PF83 sequence from 3 strains reveals approxi¬ mately 95% identity and most of the substitutions are conservative. Example 1
A lambda gtll expression library constructed with cDNA from mature schizonts of P. knowlesi (Hudson et al. , J. Mol. Biol. 203, 707-714, 1988) was screened with rabbit polyclonal serum monospecific for the PK66 (Gamier et al., J. Mol. Biol. 120, 97-120, 1978). Reactive colonies were plaque purified and the inserts subcloned into the pGEM series of vectors (Promega). PV66 was isolated by PCR amplification of P. vivax genomic DNA using degenerate primers with Bam HI ends. Amplified fragments were cloned into PUC19, and 20 different clones from two amplification reactions were pooled for sequencing. Sequencing was performed on denatured double stranded templates (Hattori et al., Anal. Biochem. 52, 232-238, 1986) using Sequenase according to the manufacturer's instructions (US Biochemicals) . Sequence data was compiled either manually or with the composition programs associated with Staden- Plus™ software (Amersham) . Alignments were produced with the Clusta 14 program (D. Higgins et al. , Gene 73, 237- 244, 1988; and D. Higgins et al., CABIOS 5, 151-153, 1989) kindly supplied by Dr. D. Higgins and manually optimized. The gene sequence of PK66 and PV66 is shown in Figure 1, along with a comparison of the encoded PK66 protein with PF83, the analogue from P. falciparum.
Example 2 A) PK66 CT serum was prepared as described (Dame et al., Science 225, 593-599, 1984). Parasites were prepared for IFAT analysis using cold methanol as fixative. • Fluorescent secondary antibodies were used at concentra¬ tions recommended by the manufacturer (Kirkegaard & Perry) . Microscopy and photography were carried out using an Axiophot microscope (Zeiss) and Tri X-pan film (Kodak) according to manufacturers' instructions. The distribution of PK66 analogues within the genus Plasmodium is shown in Figure 2A.
B) CT serum staining was revealed using a fluores- cein conjugated anti-rabbit secondary antibody. 13C11 and R31C2 staining were revealed using rhodamine conjugated anti-mouse and anti-rat secondary antibody preparations, respectively, and photographed as above.
The distribution of PK66 on a mature schizont of P. knowlesi and during erythrocyte invasion is shown in Figure 2B.
Example 3 Parasites extracts are referred to by letter (A, B or C). Metabolic labelling with [35S]-methionine was carried out essentially as described in Deans et al. Glin. Exp. Immunol. 49, 297-309, 1982. Preparation A was labelled for 2h in the presence of protease inhibitors chymostatin and leupeptin and no schizont rupture was observed. Preparation B was labelled for llh and appre¬ ciable schizont bursting and merozoite release had oc- curred. Preparation C was an unlabelled parasite extract of a mature culture with ruptured schizonts prepared for Western analysis. These and unlabelled parasite extracts were prepared as described (Deans et al, Clin. Exp. Immunol. 49, 297-3099, 1982). Immune-precipitation was carried out and the precipitates or unlabelled parasite extract were fraction¬ ated on 10% or 7.5% SDS-PAGE systems as described (Deans et al, Clin. Exp. Immunol. 49, 297-309, 1982). The results are shown in Figure 3A. Western blotting was also carried out as previously published (Gamier et al., J. Mol. Biol. 120, 97-120, 1978). The blotted extracts were probed with appropriate rabbit antisera at a 1 in 200 dilution and visualized using alkaline phosphatase-conjugated secondary antibodies according to the manufacturer's instructions (Promega). The results are shown in Figure 3B. The entire contents of all references cited herein- above are hereby incorporated by reference and relied upon.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, an artisan will appreciate that it is likely that proteins similar to those described herein will exist in other human and animal parasites of medical and agricultural significance (for example, Babesia and Toxoplas a). Therefore, the present invention includes within its scope such proteins.

Claims

WHAT IS CLAIMED IS:
1. A substantially pure form of a merozoite antigenic protein isolatable from simian and simian-like species of Plasmodium or a recombinant protein analogous thereto wherein binding of said protein present in said merozoite to antibody specific therefor inhibits erythro¬ cyte invasion by said merozoite.
2. A DNA fragment that encodes said protein according to claim 1.
3. A recombinant DNA molecule comprising:
(i) a vector, and
(ii) said DNA fragment according to claim 2.
4. The recombinant DNA molecule according to claim 3, wherein said vector is a viral vector.
5. The recombinant DNA molecule according to claim
3, wherein said DNA fragment encodes the amino acid sequence set forth in Figure 1, or a unique portion of that sequence.
6. A host cell transformed with the recombinant DNA molecule according to claim 3.
7. The host cell according to claim 6, wherein said cell is a eucaryotic cell.
8. A process of producing a substantially pure form of a merozoite antigenic protein isolatable from simian and simian-like species of Plasmodium, which comprises culturing the cell according to claim 6, under conditions such that said DNA fragment is expressed and said protein thereby produced, and isolating said protein.
9. The protein according to claim 1, wherein said species of Plasmodium is P. knowlesi .
10. The protein according to claim 9, wherein said protein has an amino acid sequence corresponding to that shown in Figure 1, or a unique portion thereof.
11. A DNA fragment that encodes the protein accord- ing to claim 9.
12. The DNA fragment according to claim 11, wherein said DNA fragment encodes the amino acid sequence set forth in Figure 1, or a unique portion thereof.
13. The protein according to claim 1, wherein said species of Plasmodium is P. vivax.
14. The protein according to claim 13, wherein said protein has an amino acid sequence corresponding to that shown in Figure 1, or a unique portion thereof.
15. A DNA fragment that encodes the protein accord¬ ing to claim 13.
16. The DNA fragment according to claim 15, wherein said DNA fragment encodes the amino acid sequence set forth in Figure 1, or a unique portion thereof.
17. A composition of matter comprising antigenic protein of claim 1 or a unique fragment thereof in a pharmaceutical carrier.
18. The composition of claim 17 further comprising an adjuvant.
19. The composition of claim 18 wherein the adju¬ vant is alum.
20. A composition of matter comprising an antigenic protein of claim 1, or unique fragment thereof, on a solid support.
21. The composition of claim 20 wherein the solid support is a microtiter plate.
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Title
CHEMICAL ABSTRACTS, vol. 101, no. 11, 10 September 1984, Columbus, Ohio, US; abstract no. 87145b, J.A. DEAN ET AL. 'Biosynthesis of a putative protective Plasmodium knowlesi merozoite antigen' page 335 ;column R ; *
MOL. BIOCHEM. PARASITOL. vol. 13, no. 2, 1984, ELSEVIER SCIENCE PUB., NEW YORK, US; pages 187 - 199 A.W. THOMAS ET AL. 'The Fab fragments of monoclonal IgG to a merozoite surface antigen inhibit Plasmodium knowlesi invasion of erythrocytes' *
PARASITE IMMUNOLOGY vol. 10, no. 5, September 1988, BLACKWELL SCIENTIFIC PUB.,OXFORD,UK; pages 535 - 552 J.A. DEANS ET AL. 'Vaccination trials in rhesus monkeys with a minor, invariant, Plasmodium knowlesi 66kD merozoite antigen' *
See also references of WO9113161A1 *

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