WO1992005193A1

WO1992005193A1 - Plasmodium liver stage antigens

Info

Publication number: WO1992005193A1
Application number: PCT/GB1991/001605
Authority: WO
Inventors: Robert Edward Sinden; Andreas Suhrbier; Larry Aldon Winger
Original assignee: Imperial College Of Science, Technology And Medicine
Priority date: 1990-09-19
Filing date: 1991-09-19
Publication date: 1992-04-02
Also published as: GB9020438D0; JPH06500996A; EP0549662A1; AU8611491A

Abstract

Plasmodium liver stage antigens are disclosed as well as cDNA sequences encoding such antigens of P.berghei and including homologous sequences of P.vivax and P.Falciparum.

Description

PLASMQDIUM LIVER STAGE ANTIGENS

This invention relates to the isolation and production of aπtigenic proteinaceous material from parasites of the genus Plasmodium, the production of monoclonal antibodies responsive thereto, and anti-malarial vaccines containing them.

The search for an effective anti-malarial vaccine has been hampered by two main factors: the low immunogenicity of the malarial parasite and its extremely varied and complex life cycle.

Following the bite of the infective mosquito the mammalian malarial sporozoite invades the hepatocyte wherein it multiplies for the next 2-15 days (dependant on the parasite species). Thereafter liver merozoites are released into the blood stream in which the intra-erthrocytic sexual and pathogenic asexual stages then proliferate initiating the blood stage of the disease responsible for the symptoms and pathology of malaria. Malaria vaccines based on antigens from the sporozoite (Herrington, et al., 1987), asexual blood stages (Patarroyo, et al., 1988) and sexual stages (Carter, et al., 1988) are currently being evaluated. In contrast liver stages antigens have been little studied as possible vaccine candidates because these intracellular stages were considered to be inaccessible to the immune system of the host (Cox, 1988). It is now clear from studies in rodent models (P. berghei and P. yoelii), however, that a substantial part of the protection afforded by immunising with irradiated sporozoites results from CD8+ T cell activity directed against the infected hepatocyte rather than from antibodies directed against the circumsporozoite proteins (CSP) on the circulating sporozoites (Weiss, 1987; Schofield, et al., 1987; Hoffman, et al., 1989). Processed CSP associated with the class 1 major histocompatability complex on the surface of the infected liver cell appears to be one target for such cytotoxic T cells (Kumar, et al., 1988; Sadoff, et al., 1988 ; Hoffman, et al., 1989a; Romero, et al., 1989). The single CD8+ T cell epitope of P. falciparum CSP shows a high degree of polymorphism (Loc yer, et al., 1989) and the response to it may also be geneticaDy restricted (Good, et al., 1988). It is still unclear to what extent these phenomena compromise the use of the CSP as the basis for a synthetic vaccine (De Groot, et al., 1989; Hoffman, et al., 1989b; De la Cruz, et al., 1988). The large number of blood stage antigens also expressed in the liver stage (Danforth, Orjih <k Nussenzweig, 1978; Szarfman, et al., 1987, Suhrbier, et al., 1989a) and those antigens specific to the liver stage (Druihle, et al., 1984; Guerin-Marshand, et al., 1987) may all be potential targets for protective CD8+ T cells (Townsend ic Bodmer, 1989). Studies which have shown that the liver stage parasite can be killed by cytokines (principally gamma interferon) (Mazier, et al., 1988; Scholfield, et al., 1987) and phagocytes (Shortt & Garnham, 1984; Terzakis, et al., 1979; Meis, et al., 1987) further highlight the need to identify the antigens relevant to liver stage immunity.

In accordance with the present invention a liver stage antigen (Pbl 1) has been identified and isolated from cultures of Plasmodium berghei and a monoclonal antibody (anti-Pbl 1) produced specific to that antigen.

In its broadest aspects, therefore, the present invention contemplates:

1. A purified isolate comprising the P. berghei liver stage antigen Pbl 1, and Pbl 1 homologues from the genus Plasmodium, especially P. falciparum and P. vivax (referred to herein as homologous Pbl 1).

2. Liver stage antigens according to 1 obtained by the expression of genomic or cDNA encoding therefor in an expression vehicle.

3. Monoclonal antibodies specific to Plasmodium liver stage antigens according to 1 or 2.

4. Anti-malarial vaccines comprising a Plasmodium liver stage antigen according to 1 or 2, or a monoclonal antibody according to 3.

5. Genomic or cDNA fragments encoding Plasmodium liver stage antigens according to 1.

6. Transfection vectors containing a genomic or cDNA fragment according to 5 and capable of expressing Pbl 1 or homologous Pbl 1 in a host cell.

7. Transfected cells containing an expression vector containing a genomic or cDNA fragment according to 5 and capable, when cultured, of expressing Pbl 1 or homologous Pbl 1. 8. A hybridoma producing monoclonal antibody according to 3.

In a more specific aspect, the present invention provides:

1.1 A purified isolate comprising the liver stage antigen of P.berghei identified herein as Pbl 1.

3.1 The monoclonal antibody: anti-Pbl 1 (23.46.78).

5.1 A genomic or cDNA fragment encoding P.berghei liver stage antigen Pbl 1. According to preliminary analysis this sequence has the following properties.

It is 314 base pairs in length; it has no stop codons; it was in open reading frame with B-galactosidase; it has an Eco Rl site at one end; it is very AT rich (a characteristic of malarial DNA); it codes for 103 amino-acids with a combined M.W. of 12,082, i.e. it is only a fraction

(ca 1/3-1/4) of the native molecule; the sequence has no homology with any other sequence in the available gene database.

The sequence described thus far is set out in the appendix hereto as

SEQ. ID. No. 1.

6.1 An expression vector containing a genomic or cDNA fragment according to 5.1.

7.1 A host bacterium transfeeted with a vector according to 6.1 and capable when cultured of producing Pbl 1.

7.2 E. coli clone Eco.Pbl 1.

8.1 A bybridoma producing monoclonal antibody 23.46.7 (mono¬ clonal anti-Pbl 1.)

The P. berghei liver stage antigen identified herein as Pbl 1 has been identified in vivo as localised to the parasitophorous vacuole, i.e. the space surrounding the infecting parasite in the infected hepatocyte, and on the surface of the parasitophorous vacuole membrane . Molecular weight studies on the isolated antigen indicate a molecular weight (Mr) of 35 + 5 kDA. The antigen is specifically recognised by monoclonal antibody 23.46.7 (hereinafter described). The antigen is expressed throughout the liver stage of the infection by P. berghei from 3-5 hours post-invasion by the infecting sporozoites to the release of the merzoites from the liver into the blood stream.

As already indicated, the present invention also extends to Pbl 1 homologues from parasites of the genus Plasmodium, and more especially the human parasites P. falciparum and P. vivax.

As referred to herein, homologous DNA refers to equivalent linear sequences of DNA from organisms of the same genus, or of the same species, and which are identical, or substantially identical, differing one from the other merely by the substitution of equivalent alleles or by virtue of the degeneracy of the genetic code, and which encode identical or substantially identical proteins in two or more species within that genus, or two or more strains within that species, such proteins, referred to as homologous proteins or antigenic homologues, having substantially the same structures and properties and performing substantially the same or equivalent biological functions in each of the strains or species referred to, the terms homology, homologous, homologue etc, being construed accordingly. Experimental

Production of anti-Pbl 1 monoclonal antibody

In the initial stage, 48-hour liver stage schizonts of P. berghei were cultured in human HepG2 cells using the techniques of Hollingdale and Suhrbier (see Hollingdale, 1983; and Suhrbier, et al., 1988, listed hereinafter).

In a separate stage BALB/c mice were immunized with HepG2 cells alone and were then treated with cyclophosphamide to suppress the subsequent response against the HepG2 cells. After the three week recovery period the animals were immunised three times with freeze- thawed 48-hour EE schizonts grown as above (approximately 2.5 x lO*-- cells with a 10-20% infection rate) given at 2 week intervals. Assay of the serum by indirect immunofluorescence on acetone-fixed cultured EE schizonts in HepG2 cells showed that although some HepG2 background reactivity occured, individual mice exhibited significant antibody reactivity to the parasite. After 5 months the mice were innoculated intravenously with 10⁴ sporozoites on each of two succeeding days. On day 6 and/or 7 after sporozoite inoculation, when a Giemsa-stained blood smear showed positive blood parasitemia, the mouse was sacrificed and the spleen removed for fusion. The fusion protocol was performed as described by Winger, et al (1987). The supernatants of the resulting hybridomas were screened by indirect immunofluorescence on live and acetone fixed cultured liver schizonts, washed parasitized blood smears and smeared air-dried sporozoites. Ascites fluid of selected monoclonal antibodies, which had immunofluorescent titres of between 1/100,000 and 1/500,000 on liver parasites, were used at 1/50 dilutions on thick films of highly parasitized blood to verify the liver specificity of the monoclonal antibodies.

PASSIVE IMMUNISATION TRIALS

Monoclonal antibodies 17.9.15 (specific to an ookinete surface antigen Winger, et al., 1987), 17.6.1 (binding to an integral antigen of liver merozoites and all blood stage parasites; Suhrbier, et al., 1989b), 23.8.1 (an anti-cicumsporozoite protien antibody, data not shown) and anti-Pbl 1 (Plasmodium berghei liver 1 obtained as above) were purified from ascites fluid (Reik, et al 1987). All the antibodies were of the IgG 1 subclass. Protein levels were determined using a Biorad protein standard and by spectrophotometric analysis at 280nm. Using an ELISA for murine gamma interferon, no gamma interferon was detected in these preparations of levels of 97pcg or 1U per mg IgG. In one experiment the antibodies were further purified on a Protein G affinity column (Biorad). In the series of six experiments, lmg of purified monoclonal antibody of PBS was injected intravenously into groups of 3 or 4 mice 24 hours after infection with equal numbers of sporozoites (approx. 5x10^s) and blood parasitemias were monitored on days 5, 6, 7 and 8 post infection. The experiments were performed in a double blind manner and Giesma stained smears were randomised and coded prior to reading (lO^rbc/slide).

13mm coverslips with a confluent monolayer of HepG2 cells were infected with approximately 2x10^ sporozoites in the presence of lmg/ml of purified anti-Pbl 1 or the same concentration of control antibody 17.6.1. The parasites were then cultured for 48 hours in the presence of antibodies before being fixed and parasite numbers, size and maturity determined by light microscopy.

Results

The fusion produced a total of 34 clones, of which 16 reacted with both liver and blood stages, 5 with sporozoites, 12 with HepG2 cells and 1, which reacted specifically with the liver stage. This monoclonal antibody, designated anti-Pbll (Plasmodium berghei liver 1), showed no cross-reaction, using indirect i munofluorescent staining, with blood stage parasites, sporozoites or ookinetes of P. berghei. Liver stages of the closely related rodent .parasite P. yoelii cultured in primary mouse hepatocytes did not react with anti-Pbl 1. Liver stages of the human parasite P.vivax cultured in HepG2 cells (Hollingdale, 1988) also failed to react with anti-Pbl 1.

Pbl 1 could first be clearly detected by indirect immunofluorescent antibody staining on intracellular P. berghei liver stage trophozoites 3 hours after invasion. As the trophozoites and schizont developed a peripheral staining pattern was observed, which was often associated with protrusions into the cytoplasm of the host cell. These protrusions frequently had the appearance of chains of small vesicles and whose presence correlated with the growth (presumably trophic) phase of parasite development. Parasites grown in W138 cells, a less satisfactory host cell, in which parasites are smaller and take longer to mature, showed an abundance of such structures.

When maturing the parasite segments, first to form cytomeres and subsequently merzoites, antigens associated with the parasite plasmalemma and/or parasotiphorous vacuole are invaginated. During this period the peripheral staining pattern of anti-Pbl 1 was maintained indicating that Pbl 1 is associated with the parasitophorous vacuole and its limiting membrane (PVM), which does not invaginate during segmentation of the parasite. Preliminary immunoelectron microscopy studies have confirmed this localisation. The pattern seen in segmenters would be consistent with localisation on the PVM, which has by this stage been disrupted by the emerging merozoites. No staining was detected on the surface of the infected cells or on the free liver merozoites, which had broken away from the segmenters.

Pbl 1 was also expressed on intra-hepatic trophozoites derived from irradiated sporozoites.

When anti-Pbl 1 monoclonal antibodies were passively transferred to mice given a sporozoite inoculum 24 hours previously, the course of the sebsequent parasitemia appeared to be slightly delayed by approximately 12 hours when compared to control hosts inoculated with heterologous monoclonal antibodies or phosphate buffered saline (PBS).

Pbl 1 expression in E. coli.

1. Construction P. berghei DNA Library

20 mice were infected with P. berghei ANKA strain, by blood passage. At 20-50% parasitaemia the mice were bled and the total of 25 mis of heparinized blood was filtered through a Whatman CF11 column to remove white blood cells. The blood was centrifuged at 1,500-3,000 g for 10 minutes and the cells resuspended in phosphate buffered saline (PBS) to a haematocrit of 50% and passed through the column to achieve a maximal final white blood cell/parasite ratio of 1:10,000. The filtrate was collected and spun again as described above to achieve a final packed preparation containing the parasites. DNA was extracted from the pelleted parasites as follows: To the pellet 30 ml of lysis buffer (50mm Tris-HCl pH 9, 100mm EDTA, 200 mm NaCl) was added; to this mixture proteinase K was added to a final concentration of lOOμg/ml, and the mixture incubated at 65°C for 30 mins. This was then followed by a second incubation at 37 °C for 1.5 hours. After this incubation, nucleic acids were extracted by the standard method of phenol/chloroform extraction (Maniatis et al.; 1982 Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory, New York p. 458). The purified nucleic acids were ethanol precipitated and the RNA was digested from this precipitate by the addition of RNAse enzyme to a concentration of lOOμg/ml. Following this digestion, the phenol/chloroform extraction and ethanol precipitation were repeated to give a final DNA extract hereafter referred to as P. berghei DNA.

A P. berghei genomic library was constructed from the P. berghei DNA as follows: lOμg of P. berghei DNA was digested with EcoRl using excess EcoRl enzyme (50U/μg P. berghei DNA) in the presence of 20mM Tris-KCl pH 8.5, 2mM MgCl₂ and BSA (lOμg/ml) at 370°C for 22 hours (Polisky, B., et al, 1975, Proc. Nat. Acad. Sci. USA, 72, 3310-3314). The DNA was then used to construct a igtll genomic DNA library as described by Young, R.A. όc Davis, R.W. (1983, Proc. Nat. Acad. Sci. USA, 80, 1194- 1198). The conditions and reagents used to construct the library were as described by the manufacturers in the Amersham* cDNA yl gtll cloning kit (code No. RPN 1280).

* Amersham International, Amersham, Bucks

2. Screening of the library with monoclonal antibody and selection of positive clone.

The library was screened with monoclonal antibody 23.46.7 using established protocols (Huynh, T.V., et al 1985 in DNA Cloning, a practical approach, edited by D.M. Glover, IRC Press, Oxford, Vol. 1, p. 49-108).

The λgtll library was screened as plaques on a lawn of E. coli Y1090 cells. The library was plated out and incubated at 42 °C for 3 hours or until pin-prick plaques were seen. Nitrocellulous filter discs, with marked orientation, impregnated with 10m M IPTG were placed on the lawn and incubated for 2 hours at 37°C. The nitrocellulous filter was then removed and non-specific binding sites blocked with 5% milk powder in 0.1% Tween 20 in phosphate buffered saline. The filters were then incubated in 10 mis of monoclonal antibody 23.46.78 at 50μg/ml in 0.1% Tween in PBS. This incubation was for 1.5 hours at room temperature. The filters were then washed in 0.1% Tween 20/PBS for 20 mins.; this wash was repeated 3 times. Horseradish peroxidase -labeled goat-anti mouse IgG (Biorad) was used at a dilution of 1/1,000 in wash buffer to bind to the first antibody, by incubation at room temperature for 1.5 hours. The filters were then washed as above and incubated in diaminobenzidine substrate at a final concentration of 500μg/ml and 20μl of 30% hydrogen peroxide in 100 mis phosphate buffered saline. Positive plaques were then aligned with the original plates to identify and select positive colonies. These were then rescreened repeatedly using the above method until a pure stock of the clone was obtained.

3. Amplification by PCR transfer of DNA insert into N13 NP10

The positive clone descibed above, now identified as EcoPBl.l in gtll was amplified using standard PCR methods using the protocols and reagents provided in the Perkin-Elmer Cetus GeneAmp amplification reagent kit with AmpliTaq™ DNA polymerase (Cat. No. N. 801-0055, Perkin-Elmer Cetus). The reaction was set up under mineral oil according to their protocol, namely: 10 x reaction buffer; dNTP's mixture with 1.25mM of each dATP, dCTP, dGTP, dTTP; EcoPbl 1, Phage stock, freeze thawed; Forward primer; reverse primer, and Taq polymerase.

Amplification involved the usual 3 steps of denaturation at 94°C for 1 min., annealing of primers at 50°C for 1 min., and extension using Taq polymerase at 72°C for 2 mins. This cycle was repeated for a total time of 5 hours. The primers (forward and reverse) were obtained from New England Biolabs, (Cat. No. 1218 and 1222).

4. Insertion of EcoPbl 1 into M13mpl0 and Puc 13

The PCR product obtained as above was frozen at -20°C and the mineral oil removed from the surface. The DNA was then phenol/chloroform extracted once and 50μl of the aqueous phase was passed down a Sephadex CL6B column. The overhanging ends of the amplified DNA were filled using 2.5 U/μl E. coli DNA polymerase Klenow fragment, obtained from Amersham International, (Cat. No. T21412), using standard methods, (Sambrook, Fritsch, Maniatus, Molecular Cloning, A lab manual, 2nd edition, Chapter 5, p 5.42). This reaction was at room temperature for 15 mins. and was stopped by heating at 65 °C for ID mins.

The amplified DNA was then passed through a Sepharose CL6B column spun at 1,500 rpm for 3 mins. T4 polynucleotide kinase (Amersham International, Cat. No. 2020Y) was then added (10 U/μl) and incubated in the presence of ligase buffer (200mM Tris HC1, pH 6, 50mM MgCl2, 50mM dithiothreitol, 500mg/ml BSA, lOmM ATP) at 37°C for 2 hours, the reaction being stopped by heat treatment at 65 °C for 10 mins. (Sambrook, Fritsch, Maniatis, Molecular Cloning, A lab manual, 2nd edition, chapter 5, p 5.68). The PCR amplified DNA was taken for ligation to the vectors M13mpl0 and Pucl3.

The following ligation was set up using M13mpl0 (Amersham N4531) and Puel3 (Pharmacia 27-4975-01). Both vectors were cut with Smal, and phosphatase treated. The PCR insert was then mixed with 10 x ligase buffer; vector (lOng/μl); T4 DNA ligase (Amersham Cat No. T2050Y) the volume made up with H2O, and the mixture incubated overnight at 4°C.

The ligation mix was then diluted 1/100 and 5μl was used to transform DH5σ(F^* host cells for M13 and DH5 host cells for Pucl3 using the protocol described by Hanahan, D. (1986, in DNA Cloning, a practical approach, edited by D.M. Glover, IRC Press, Oxford, Vol 1, p 109-135). (DH5° nd DH5e⁽F^* cells from Gibco BRL Cat. Nos. 530 8265SA and 530 8264SA respectively). Transformed cells were plated out in the presence of lOOμm IPGT and 2% X-gal and incubated overnight at 37°C. Reeombinants were identified by their coloration. Blue plaques were parentals, M13 reeombinants were colourless and Pucl3 reeombinants were white.

5. Ml 3 miniprep procedure for the preparation of single stranded DNA

M13 reeombinants were picked and inoculated into 1.5ml 1/100 dilution of JM101 cells (Pharmacia, Cat. No. 27 1509 01) in 2 x TY medium (16 gms Bactotryptone, lOg yeast extract , 5g NaCl per litre) and grown at 37°C for 6 hours. 1 ml of clear supernatent from the plaque preparation (spun cells 2 x in Eppendorf centrifuge) was added to 250μl of PEG NaCl (20% PEG 6,000; 2.5mM NaCl) and then mixed and incubated at room temperature for 15 mins.

This preparation was then spun for 5 mins. in a microfuge, decanted and the white viral pellet (containing M13 phage) was then resuspended in lOOμl of TE buffer pH 8, (10m M Tris HCljlmM EDTA) and phenol/- chloroform was used to extract the DNA as described above. DNA was removed from the aqueous phase by sodium acetate precipitation at a final concentration of 0.3 M at pH 5.35 and subsequent addition of 2 1/2 vols. of absolute ethanol. DNA was later collected by centrifugation, washed in 70% ethanol, dried under vacuum and then resuspended in 25μl TE buffer. Reeombinants were confirmed by running mini preps of the DNA on agarose gel against non-recombinant M13 single stranded DNA. Identification of reeombinants was based on the difference in size of the DNA from the two preparations. Positive clones obtained in M13 mini DNA preparations were sequenced using a Sequenase kit (USA Biochemical Corporation). The chain termination method (Sanger, F., Miklen, S. <5c Coulson, A.R., 1977, Proc. Nat. Acad. Sci. USA 74, 5463-5467) was used. This technique (Tabor, S. & Richardson, CO., 1987, Proc. Nat. Acad. Sci. USA, 84, No. 14, 4767-4771) required the synthesis of a DNA strand by a modified bacteriphage T7 DNA polymerase (Sequenase) using a single stranded DNA template from M13mpl0 and recombinant clones as described in the manufactures instructions ("Sequenase: Step-by-step protocols for DNA sequencing with Sequenase" 5th edition 1989, United States Biochemical Corporation).

Isolation of P. falciparum and P. vivax Homologues

The insert Pbl 1, already cloned into the M13 vector, may be PCR amplified (see above) using synthetic oligonucleotides based on the above insert sequence. The PCR amplified insert can then be hexalabelled using ³²P (Feinberg and Vogelstein, 1984, Analytical Biochemistry, 137, 266-267) and then used to screen a P. falciparum gtll library and other available libraries (Sambrook, et al., 1989, (see above ref.) chapter 2, p 2.108-2.111 and p 2.114-2.117) constructed in the same way as the P. berghei library described above. Alternatively the labelled insert may be used to screen southern blots of P. falciparum and P. vivax restriction digested DNA using standard protocols (Sambrook, et al., 1989 (See above ref.) chapter 9, p 9.31-9.40 and p 9.52-9.55) to identify any possible homology between these strains and P_. berghei. Following identification of the P. falciparum and P. vivax homologues by the procedures outlined above, these homologues may be isolated and subeloned utilising the procedures described above for P_^ berghei to obtain finally the desired homologous Pbl 1 antigens of j\ falciparum and P. vivax.

Deposit Details

E. coli clone Eco.Pbl 1

Deposited under the provisions of the Budapest Treaty on 30th August 1990 with the National Collection of Industrial and Marine Bacteria (NCIMB), Aberdeen, Scotland, Deposit No. NCIMB 40314

Hybridoma cell line (23.46.78)

Deposited under the provisions of the Budapest Treaty on 18th September 1990 with the European Collection of Animal Cell Cultures ECACC, Porton Down, Deposit No. 90091801

Finally, when considering the scope of the present invention, it is to be taken into account that it is well known that various fragments of an antigen or antibody will, depending on the size and the location of that fragment, retain sufficient antigenic/specific binding (antibody) capacity that, as a practical matter, that fragment can be considered as substantially equivalent to the antigen/antibody in question. The terms antigen/antibody are therefore to be construed as covering such functionally equivalent fragments. Likewise references to DNA sequences are to be taken to include fragments, allelic and degenerative variants thereof retaining for practical purposes the functionality of the complete sequence. APPENDIX

SEO ID No . 1

Sequence type : Nucl eotide

Sequence length: 314 base pairs

Strandedness: Single

Topology: Linear

Molecule type: Genomic DNA

Original source organism: P.berghei

Immediate experimental source: E.coli clone Eco.Pbl 1 (NCIMBL 40714)

Features: from bp - 1 to -17 ) contiguous nucleotide sequences of and bp 315 to 323: ) the λgt 11 vector

bp 1 to 314 putative peptide encoding sequence.

SEO ID No. 1

5' cccgtca gtatcggcgg AATTTTTTTG TGTAGATGGA GATTCTAGCT 30

GTTCTTGTTT TTTTGTTTCT GACGATGATG 60

ATGTTGATGA TTCTTGTTGT CGTGCTAATT 90

CACCAGAAAA ATATATAGAA GAATCTCATA 120 AGCACTACAC AAAAGGGTTT CTAGAACAAT 150

ATACAGAGCC CAAACCAAGC GATCATACAT 180

ACAGTTATAC CCCCACTGAA GAAGCATATA 211

ACACACATTA TATGGCATCA GATACTCATG 240

AAGATTATGG CAAACTATTT ACAGATGAAC 270 ATAAGGACGA AATAAATGAT AATATAGTTT 300

ATCACGATGA ATTC cagctgagc 3' 314

Claims

CLAIMS :

1. Purified isolates comprising the P.berghei liver stage antigen Pbl 1 and homologues thereof from other parasites of the genus Plasmodium.

2. Homologous Pbl 1 from parasites of the species P.falciparum and P.vivax.

3. Liver stage antigens according to claim 1 or 2 obtained by the expression of a genomic or cDNA fragment encoding therefor in an expression vehicle.

4. Liver stage antigens according to claim 3 wherein said genomic or cDNA fragment comprises the sequence SEQ ID No. 1 or a substantially (at least 90%) homologous sequence.

5. A purified isolate of P.berghei liver stage antigen Pbl 1.

6. Cloned P.berghei liver stage antigen Pbl 1.

7. Monoclonal antibodies specific to Plasmodium liver stage antigens as claimed in any one of claims 1 to 6.

8. Monoclonal antibody 23.46.78.

9. An anti-malarial vaccine comprising a liver stage antigen according to any one of claims 1 to 6 or a monoclonal antibodv according to claim 7 or 8.

10. A genomic or cDNA fragment encoding a Plasmodium liver stage antigen as claimed in any one of claims 1 to 6.

11. A genomic or cDNA fragment encoding P.berghei liver stage antigen Pbl 1.

12. A genomic or cDNA fragment according to claim 11 wherein said fragment comprises the sequence SEQ ID No. 1 or a substantially (at least 90%) homologous sequence.

13. A transfection vector containing a genomic or cDNA fragment according to claim 10, 11 or 12.

14. A vector according to claim 13 comprising said genomic or cDNA fragment inserted into λgtll phage, M13 and Puc.

15. Transfected cells containing an expression vector according to claims 13 or 14 and capable when cultured of expressing the Pbl or homologous Pbl 1.

16. Transfected cells according to claim 15 which are bacterial cells.

17. E.coli. Pbl 1( CIMB 40314)

18. A hybridoma cell line capable of producing monoclonal antibody according to claims 7 or 8.

19. Hybridoma cell line 23.46.78 (ECACC Deposit No. 90091801).