EP0215059A1

EP0215059A1 - cDNA CODING FOR PLASMODIUM FALCIPARUM GLYCOPHORIN BINDING PROTEINS WEIGHING 130,000 DALTONS AND 155,000 DALTONS

Info

Publication number: EP0215059A1
Application number: EP86901657A
Authority: EP
Inventors: Jeffrey V. Ravetch; Margaret E. Perkins
Original assignee: Rockefeller University; Memorial Sloan Kettering Cancer Center
Current assignee: Rockefeller University; Memorial Sloan Kettering Cancer Center
Priority date: 1985-02-20
Filing date: 1986-02-19
Publication date: 1987-03-25
Also published as: AU5540886A; EP0215059A4; WO1986004922A1; JPS62502933A

Abstract

De l'ADNc du gène de codage des protéines qui se lient à la glycophorine du P. falciparum présente une séquence répétitive en tandem de 150 nucléotides qui code pour une unité répétitive en tandem d'acides aminés dans les protéines. L'invention concerne également des vecteurs contenant l'ADNc, des microorganismes transformés et des anticorps produits par immunisation aux protéines. On a découvert que la protéine de liaison avec la glycophorine existe sous deux formes. La première, qui pèse 155.000 daltons, semble être le précurseur de la deuxième, qui pèse environ 130.000 daltons.Protein coding gene cDNA that binds to P. falciparum glycophorin has a tandem repeat sequence of 150 nucleotides that encodes a tandem repeat unit of amino acids in proteins. The invention also relates to vectors containing cDNA, transformed microorganisms and antibodies produced by protein immunization. It has been discovered that the glycophorin binding protein exists in two forms. The first, which weighs 155,000 daltons, seems to be the precursor of the second, which weighs around 130,000 daltons.

Description

cDNA CODING FOR PLASMODIUM FALCIPARUM

GLYCOPHORIN BINDING PROTEINS WEIGHING

130,000 DALTONS AND 155,000 DALTONS

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

This invention relates to a gene for a glycophorin binding protein of the Plasmodiuπt falciparum merozoite. More specifically, the invention relates to cDNA clones for the gene which codes for this protein, vectors containing the cDNA, microorganisms transformed by introduction of this cDNA, antibodies to the protein, the protein itself, and methods of producing these.

Plasmodia are protozoan parasites which cause malarial infections in mammals. Different species are responsible for the disease in different mammals, with

Plasmodium falciparum the major cause of the disease in humans.

The life cycle of Plasmodia is complex, and is similar from species to species. Usually there are three stages in the life cycle. The first, known as the sporozoite stage, is the form in which the protozoa is introduced to blood of a host, generally by a mosquito bite. The second stage, the asexual blood stage includes the extraerythrocytic merozoite and the intraerythrocytic schizont, follows rapidly after introduction to the host. This stage is responsible for the clinical manifestations of mararia. The cycle is completed when the parasite enters into the sexual cycle, the gamotocytic form, and is reingested by a mosquito.

Malaria has remained a serious health problem. An immunogenic response is not always protective in the mammalian host, because the protozoa are rapidly sequested in liver cells or erythrocytes where immunogenic response is not effective.

While it may be thought that a vaccine could be prepared to allow an immunogenic response to be mounted, such a vaccine has not been available, for several reasons. Identification of immunodominant antigens for each stage of the life cycle is required, with the ability to obtain sufficient quantities of immunogen in order to mount an immune response. An immunogenic response occurs when an antibody is raised to an antigen, usually a foreign protein or proteinaceous molecule. A vaccine operates by introducing to a subject a sample of inactivated antigens. Antibodies are raised, whether the antigen is active or not, and usually the antibodies remain effective when an active form of the antigen is actually introduced.

Preparation of malaria vaccines have been hampered because of the different stages in the Plasmodia life cycle. Antibodies are very specific, often responding only to one antigen. Malarial antigens are known to be surface proteins of the protozoa, and these vary from stage to stage. A vaccine to malaria must, therefore, consist of several different antigens, each of which will raise an antibody to a stage, specific antigens. Kolata, Science, 226, 679-682 (1984) .

As the merozoite stage is the precursor to the intraerythrocytic stage of Plasmodia, it is logical to attempt to stem the infection when the protozoa are in this stage. A vaccine should, therefore, be directed against surface proteins of this stage, more than any other. The surface of merozoite stage protozoa has proven to be very complex, however, containing many different molecules. _. The role of each of these in the infection process is not clear. If a vaccine is to be prepared, it should, ideally, be directed against a surface protein which is characterized completely or to some degree, is essential to parasite survival, and is causative for infection.

Two P^ falciparum merozoite surface proteins have been found which satisfy the above criteria. The proteins have molecular weights of 155,000 and 130,000 respectively, recognize host erythrocytes, and interact with high affinity, and specificity with the erythrocyte receptor, glycophorin. Antibodies directed against these proteins have been shown to inhibit merozoite invasion of erythrocytes. Perkins, J. Exp. Med. 160_,, 788 (1984) . Production of antibodies to these proteins requires, samples of the proteins themselves. Small quantities of protein have been obtained for these proteins, with large scale isolation proving extremely difficult. Additionally, inherent problems with purifying the proteins, once obtained from the merozoites, are factors which deter one from obtaining the proteins in this way.

Recent advances in DNA technology provide an alternative path for obtaining a particular protein. If the gene or genes coding for a particular protein are identified, it is possible to obtain complementary DNA, or "cDNA" for that gene. "Complementary DNA" is a sequence of nucleotides which is identical, or nearly identical to the DNA coding for a protein. One obtains cDNA by first obtaining RNA complementary to the gene in question ("mRNA") , and then synthesizing a complementary DNA molecule, i.e., cDNA, to that mRNA molecule. The cDNA is then inserted into appropriate vectors, and/or microorganisms, which then produce the protein for which the cDNA codes. In this way, a protein which may otherwise be difficult or impossible to obtain, becomes readily available.

Hence it is an object of this invention to obtain cDNA coding for proteins of P^ falciparum merozoites.

It is a further object of this invention to produce antibodies to P^ falciparum. merozoite proteins which may then be used to combat P^ falciparum injection.

How these as well as other objects of the invention are accomplished will be seen from the disclosure which now follows.

DESCRIPTION OF RELATED ART

Recent studies have shown that different species of

Plasmodia show tandemly repeating sequences of amino acids.

See, e.g., Coppel et al, Nature 306, 751-756 (1983) ; Koenen et al, Nature 311, 387 (1984) (P. falciparum erythrocyte stage antigens) ; Godson et al, Nature 305, 29-33 (1983) ;

(circumsporozoite antigen of P^ knowlesi) ; Dame et al,

Science 225, 593 (1984) ; Enea et al, Science 225, 628 (1984)

(circumsporozoite antigen of P^ falciparum) ; Ravetch et al, Nature (1984) (312, 616) , (histidine rich protein of P. lophuae) .

SUMMARY OF THE INVENTION

cDNA to __;_ falciparum merozoite surface proteins weighing approximately 130,000 daltons and 155,000 daltons, and which specifically bind to erythrocyte surface glycophorin is disclosed. The cDNA exhibits a repeating sequence of 150 nucleotides, and codes for a protein with a 50 amino acid tandem repeat sequence. The subject proteins, identified hereafter as GBP 130 - ("glycophorin binding proteins of 130,000 daltons"), or GBP 155,000 ("glycophorin binding proteins of 155,000 daltons) are coded for by a gene which is conserved in 4 strains of P^ falciparum, (including Gambia and Honduras), and is expressed as a 6.6 kb mRNA, which accumulates in late schizonts.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts Western blot analysis of fusion proteins expressed in E^ coli between P_^ falciparum GBP 130 and -galactosidase. Figure 2 shows the restriction map for cDNA for P. falciparum GBP 130 and GBP "155.

Figure 3 shows the nucleotide sequence of cDNA for P. falciparum GBP 130 and GBP 155.

Figure 4 shows the 150 nucleotide tandem repeat sequence of cDNA for P^ falciparum GBP 130 and GBP 155.

Figure 5 depicts comparative immunofluorescence patterns using goat anti-rabbit antisera and rabbit anti-mouse antisera.

Figure 6 shows comparative Western blot analysis of merozoite protein lysates.

Figure 7 compares restriction enzyme digested DNA of two strains of P_^ falciparum.

Figure 8 shows observation of RNA species at various stages in the __j_ falciparum life cycle. Figure 9 shows the tandem repeat sequence of 50 amino acids coded for by the cDNA. DETAILED DESCRIPTION OF THE INVENTION

I. ISOLATION AND EXPRESSION

OF cDNA CLONES FOR GBP 155/130

The method of Villa-Komaroff et al, P.N.A.S. 7J5 3727 (1978), and Okayama et al, Mol. Cell. Biol 2 , 161 (1982) , was used to construct a cDNA library to late stage schizonts of P_^ falciparum. "Late stage" refers to the period of time following infection, which in this case, is 42 hours. The cDNA library referred to supra is obtained by ligating the late stage schizont DNA into the expression vector pUC-9, and then proceeding in the matter described supra. This produces a library of 50,000 recombinants, which are then screened for expression of antigenic determinants recognized by rabbit antisera directed against GBP 130, and GBP 155 (glycophorin binding proteins of 155,000 daltons) . In situ colony immunoassay is used for screening.

Ten clones are obtained which express the desired antigenic determinant, as shown in Table 1.

The ten selected clones are further characterized. The plasmid DNA is transformed into JM103, a strain of E. coli which can be induced by IPTG, so as to identify fusion protein between the p -galactosidase and pUC-9, and the cDNA. Extracts from induced, - and uninduced cultures, are fractionated on 12.5% SDS polyacrylamide gels, transferred to nitrocellulose filters, and are then probed with rabbit anti-GBPl55/130, or pre-immune normal rabbit sera.

Four isolates demonstrate inducible protein which react with immune sera, while other isolates demonstrate constitutive expression. This may be seen in attached Figure 1. Figure 1 shows Western blot analysis of fusion proteins expressed in E__;_ coli between P^ falciparum GBP 155/130, and

P-galactosidase. Identical extracts, equivalent to 200 ul of

7 E. coli, or 8x10 bacteria, of induced (I) , or uninduced (U)

JM103 cultures containing cDNA clones expressing GBP determinants are treated as described supra, and then are treated with 125I protein A. Note induction for clones 6,

11, 12, and 14, with constitutive expression for clones 1, 5 and 10. No protein band is observed with pUC-9 controls. II. NUCLEOTIDE SEQUENCE OF cDNA FOR CLONES

CODING FOR GBP 155/130, AND PRIMARY AMINO ACID SEQUENCE FOR GBP 155/130

Clone 6, which showed inducible expression, and contained the largest cDNA insert, was analyzed further. Restriction and sequencing resulted in the restriction map shown in Figure 2, and the nucleotide sequence of Figure 3.

It is found that there is a continuous open reading frame, starting at position 1, and ending at position 672 of the clone. Additionally, a repeat DNA sequence of 150 nucleotides is found, which codes for 50 amino acids. This sequence is shown at Figure 9. The repeat sequence may be found at Figure 4.

The clone analyzed shows four repeat units, which terminate at positions 673-675, with the nucleotide sequence

TAA. An A-T rich sequence follows, which has been shown to be characteristic of non-coding in Plasmodia. Ozaki et al,

Cell, 3_4_, 815-822 (1983) ; Dame et al, Science 225, 593-599

(1984) ; Ravetch et al, Nature, (1984) (312, 616) . The clone begins with a repeat, which is a consequence of the cDNA cloning strategy.

Analysis of two other cross-hybridizing clones (clones 8 and 155-1) show the tandemly repeat sequency in 4-6 copies. All of the clones cross-hybridize, and react with rabbit anti-GBP antiserum. One may conclude from this, that an epitope, recognized by the sera, is encoded within the 50 amino acid repeat.

III. CHARACTERIZATION OF THE REPEATING SEQUENCE

In order to more fully characterize the cDNA repeat sequence, cDNA clones expressing the sequence were induced in the JM103 strains of E^ coli with IPTG. Extracts are then prepared, and used to immunize mice. As controls, mice were immunized from E__;_ coli extracts transformed by vector sequences alone. Antisera raised are used in immuno- fluorescence studies, and in Western blot analysis. More specifically, smears of P_^ falciparum culture are fixed in acetone for 10 minutes, at 4°C. The smears are dried, and then incubated at room temperature for 30 minutes with either mouse Λ -pGBP155/130 from clone 6, as expressed in E^ coli, or rabbit anti-GBPl55/130 antiserum. The smears are washed extensively in PBS, and are incubated either with FITC goat anti-rabbit IgG (Cappel) , or rabbit anti-mouse IgG. These results are shown in Figure 5.

The right side of Figure 5 presents the immuno- fluorescence pattern from mouse antisera to the repeat domain of GBP155/130. The pattern shows schizont and merozoite immunofluorescence. This is consistent with staining of a surface protein. The left panel, using rabbit anti-GBPl55/130 antiserum, shows a similar pattern.

Further characterization of merozoite proteins recognized by the antisera is undertaken. Lysates of merozoite proteins are fractionated on 10% SDS-polyacryl- amide gels, transferred to nitrocellulose, and are then stained with the mouse antisera raised to the repeat sequence expressed in E^ coli. . Similar treatment is undertaken with rabbit anti

GBP155/130 antisera. As controls, normal mouse sera, or mouse sera to vector alone are used. Western blots are prepared, and these are shown in Figure 6. Lanes 1 and 6 are the controls, i.e., normal mouse sera (Lane 1), and mouse sera to vector alone (Lane. 6) . Lane 5 is the result of the mouse antisera treatment, lane 4, that of rabbit.

When culture supernatant, and culture supernatant boiled for 5 minutes are immunoblotted, protein bands of 130,000, 120,000, and 110,000, identical to those in lanes 4 and 5 are obtained. This indicates that the proteins recognized by the mouse and rabbit antisera are released into the supernatant, and are heat stable.

Previous studies by Perkins, J. Exp. Med. , 160, 788 (1984) , had found two immuno-precipitable protein products which bind to glycophorin-acrylamide, at weight of 155,000 and 130,000. The 130,000 dalton protein band, detected on the Western blots of Figure 6, co-migrates with this immuno- precipitable protein (as shown in lanes 3, 4 and 5). This shows correspondence between the repeat sequence coded for by the cDNA clones, and the glycophorin binding proteins. In addition, the E^ coli fusion proteins expressing GBP determinants appear to compete with the binding of the schizont synthesized 155 and 130 kd glycophorin binding proteins to glycophorin acrlamide. This suggests that the protein sequence encoded by the pGBP-6 cDNA clone show 3 encodes the glycophorin binding site of both GPB155 and 130.

IV. GENOMIC STRUCTURE AND

EXPRESSION OF GBP155/130 cDNA

Radiolabelled DNA of clone 6 is used to identify DNA fragments of restriction enzyme digested P_^ falciparum DNA. Two strains were used: Honduras, and Gambia, and six restriction enzymes were used: Ahalll, XmnI, Xbal, AccI, Hindlll, and EcoRl. The digested DNA was fractionated on 0.75% agarose, transferred to nitrocellulose, and probed with labeled cDNA, (50% formamide, 10% dextran sulfate 40°, washed in 0.1 x SSC, at 52°C) . Figure 7 shows that there is no difference in the strains. Hence, the gene is conserved from species to species.

Stage specificity of the gene's expression determined by isolating RNA from ring, trophozoite, and schizont infected erythrocytes. The RNA obtained in this way is size fractionated under denaturing conditions, and is probed with radiolabelled cDNA. Figure 8 shows that an RNA species of 6.6 kilobases is observed, which reaches highest steady state levels during late schizogeny.

By cloning the cDNA, one may obtain quantities of GBP155/130 which may then be used to produce antibodies to the antigenic protein. Vaccination with GBP155/130 may well serve as a way of reducing or alleviating malarial infection in humans, caused by P_^ falciparum.

Expression of the gene for GBP155/130 in prokaryotes, as has been described herein, offers an opportunity to test effectiveness of different proteins domains in the infecting process. A 50 amino acid tandem repeat sequence has been found in GBP155/130, coded for by a 150 nucleotide tandem repeat sequence. The sequences are conserved in two strains of P^ falciparum, and are likely candidates for use in the TABLE 1

GBP130 Expressed GBP130

Clone Insert 1 :b_P) Determinant (kd) Inducability

1 400 18

5 550 20

6 1200 22 ++

8+ 850 40, 30 +/^■

9+ 700 30, 25 +/-

10 400 22

11 550 21 ++

12 550 21 ++

14 500 19 +

155-1 850 30

Characterization of 10 independent cDNA-clones expressing antigenic determinants of P. falciparum glycophorin-binding protein. cDNA clones were digested with PstI to identify the insert size. E. coli extracts were prepared from IPTG induced or uninduced cultures of JM103 transformed with the recombinant expression plastnids, separated by 12.5% SDS-PAGE, Western blotted and probed with rabit anti GBP150, 130

125 antisera followed by I-protein A.

+ Larger protein species may represent fusions between the 10 amino terminal residues of J -galactosidase, the insert, and the carboxy 145 amino acids of the bacterial enzyme. These clones demonstrate a lac-t- phenotype on JM103, a lac z- strain, suggesting functional complementation.

Claims

What is claimed is:

1. cDNA expressing a P_^ falciparum merozoite protein which binds to glycophorin.

2. cDNA of claim 1, wherein said cDNA expresses a P _ falciparum merozoite protein of about 130,000 daltons.

3. cDNA of claim 1, wherein said cDNA expresses a P. falciparum merozoite protein of about 155,000 daltons.

4. cDNA of claim 1, where said cDNA has the nucleotide sequence of Figure 3.

5. cDNA of claim 1 having the restriction map of

Figure 2.

6. cDNA of claim 1, where said cDNA comprises an essentially repeating nucleotide sequence.

7. cDNA of claim 1, where said cDNA comprises the nucleotide sequence:

TTA ACT AGT GCC GAT CCA GAA GGT CAA ATA ATG AGA GAA TAT GCT

GCT GAT CCA GAA TAT CGT AAA GAC TTA GAA ATA TTT CAT AAA ATA

TTG ACT AAT ACC GAT CCA AAT GAT GAA GTA GAA AGA CAA AAT GCT GAT AAT AAC GAA GCA

8. Vectors containing cDNA for a P^ falciparum merozoite protein which binds to glycophorin.

9. Vectors of claim 8 containing cDNA for a 130,000 dalton P^ falciparum merozoite protein.

10. Vectors of claim 8 containing cDNA for a 155,000 dalton P__;_ falciparum merozoite protein.

11. Vectors as in claim 8, where said vector is pUC-9.

12. Microorganisms transformed by the cDNA for a P. falciparum merozoite protein GBP155/130 which binds to glycophorin.

13. Microorganisms of claim 12, where said micro¬ organisms are E__j_ coli.

14. Microorganisms of claim 13, where the microorganisms are the strain JM103-.

15. Microorganisms of claim 12, where the microorganisms are IPTG induced.

16. P__;_ falciparum merozoite glycophorin binding proteins prepared by transcription of cDNA followed by translation of mRNA resulting from said transcription.

17. Proteins of claim 16 weighing about 130,000 daltons.

18. Proteins of claim 16 weighing about 155,000 daltons.

19. A method of raising antibodies to P_^ falciparum merozoite glycophorin binding protein comprising immunizing a subject animal with a cell or cells containing cDNA for said protein so as to elicit formation of said antibodies.

20. Method of claim 19, where said cells are E. coli transformed by vectors containing said cDNA.

21. Method of claim 20, where said cells are JM103 induced with IPTG.

22. Antibodies produced by the process of claim 19.

23. Substantially pure P^ falciparum merozoite protein which binds to glycophorin and has a molecular weight of about 130,000 daltons.

24. Substantially pure P^_ falciparum merozoite protein which binds to glycophorin and has a molecular weight of about 155,000 daltons.

25. The protein of claim 23 or 24, comprising an essentially repeating unit of 50 amino acids.

26. The protein of claim 23 or 24, where said protein contains the amino acid sequence: Leu Thr Ser Ala Asp Pro Glu Gly Gin lie Met Arg Glu Tyr Ala

Ala Asp Pro Glu Tyr Arg Lys Asp Leu Glu lie Phe His Lys lie

Leu Thr Asn Thr Asp Pro Asn Asp Glu Val Glu Arg Arg Asn Ala Asp Asn Lys Glu Ala.

27. A method for inducing antibody formation comprising immunizing an animal subject with P_^ falciparum merozoite protein which binds to glycophorin, said immunization causing formation of antibodies.

28. The antibodies produced by the process of claim 20.

29. Fusion protein produced by E^_ coli transformed by cDNA expressing P__;_ falciparum merozoite protein binding to glycophorin.

O