WO2002098909A1 - Vaccins - Google Patents

Vaccins Download PDF

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Publication number
WO2002098909A1
WO2002098909A1 PCT/GB2002/002526 GB0202526W WO02098909A1 WO 2002098909 A1 WO2002098909 A1 WO 2002098909A1 GB 0202526 W GB0202526 W GB 0202526W WO 02098909 A1 WO02098909 A1 WO 02098909A1
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WIPO (PCT)
Prior art keywords
protein
nucleic acid
construct according
translocation
construct
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PCT/GB2002/002526
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English (en)
Inventor
Alice Marie Bennett
Stuart David Perkins
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The Secretary Of State For Defence
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Publication of WO2002098909A1 publication Critical patent/WO2002098909A1/fr

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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to novel vaccines, and in particular to modified nucleic acid vaccines which are useful in producing a prophylactic or therapeutic immune response in a host to which they are applied / which response is protective against a particular pathogen.
  • the invention further relates to pharmaceutical compositions containing the modified nucleic acid vaccines, and to methods of treatment using these.
  • Nucleic acid vaccines are known in the art and they represent a powerful approach to the generatipn of vaccines. In particular, they represent a radical change in the way that antigens are delivered, involving the introduction of plasmid DNA encoding an antigenic protein, which is then expressed in the cells of the vaccinated host. Nucleic acid vaccines have a number of advantages over conventional vaccines such as live attenuated, killed whole, or subunit vaccines. Although synthesised in the cells of the host like live-attenuated vaccines, nucleic acid vaccines are subunit vaccines which encode selected components of a pathogen, rather than the entire pathogen. Therefore, nucleic acid vaccines place the vaccinated host at no risk of infection.
  • nucleic acid vaccines can raise both humoral and cellular immune responses. This is due to the ability of proteins that are synthesised within cells to access pathways for presentation by both class I and class II major histocompatibility antigens. Nucleic acid vaccines are potentially simple and inexpensive to manufacture and produce. In addition, DNA is heat stable which would aid the transportation and subsequent storage of nucleic acid vaccines.
  • Gene gun technology gives improved DNA delivery compared with direct injection, but the DNA still only enters the bombarded cells, resulting in sub- optimal immune responses .
  • translocation proteins have the ability to translocate across cell membranes and so enter cells in an efficient manner. These proteins may also be. able to spread from cell to cell.
  • proteins include the tat protein from HIV, the homeodomain of antennapedia, or a functional fragment thereof, or the herpes simplex virus type I tegument protein VP22 or a functional fragment or homologue thereof.
  • translocation protein is the herpes simplex virus type I tegument protein VP22 which exhibits the remarkable property of efficient intracellular transport (Elliott and O'Hare, 1997 Cell . 88: 223-233). It is exported from the cells in which it is synthesised by a golgi-independent mechanism termed nonclassical secretion, despite, lacking a signal sequence. VP22 is then able to re-enter surrounding cells with very high efficiency. It has been shown that the protein VP22 can spread between different cell types and can also deliver a heterologous peptide into cells, following either endogenous synthesis and transport, or by application to the media and subsequent uptake. In addition, a 27kDa protein has been delivered into the nuclei of adjacent cells following endogenous synthesis.
  • VP22 has been used in the successful delivery of functional proteins.
  • the gene p53 which is mutated in a wide range of human malignancies, has been fused to VP22 and the chimeric proteins of approximately 90kDa have been used to establish intercellular transport (Phelan et al, Nat. Biotech. 1998 16(5): 440-443) .
  • the function of p53 was determined using a p53- negative cell line.
  • VP22 resides in the C-terminal domain and in particular in the 142 amino acids located at the C-terminal region.
  • a preferred fragment of VP22 for use in the constructs of the invention is the C-terminal region, comprising 142 amino acids.
  • VP22 is 301 amino acids in length and the preferred fragment corresponds to amino acids residue from 160- 301, in the published sequence.
  • VP22 in vaccines, where it is fused to a protein antigen is described in W098/55145.
  • Other types of fusion protein including VP22 are described in WO 98/32866.
  • a construct comprising a translocation protein linked to a nucleic acid, which encodes an antigen capable of generating a protective immune response in an animal to which it is administered.
  • construct includes complexes and conjugates, which include ionic or covalent bonds as well as other forms of interaction.
  • nucleic acid vaccines By harnessing translocation proteins to nucleic acid vaccines, improved delivery of the nucleic acid vaccine into target cells can be achieved.
  • the translocation protein is linked to the nucleic acid in such a way that neither component loses its full functional ability.
  • Suitable translocation proteins for use in the constructs of the invention include the tat protein from HIV, the homeodomain of antennapedia, or a functional fragment thereof, or the herpes simplex virus type I tegument protein VP22 or a functional ' fragment thereof.
  • the translocation protein is the herpes simplex virus type I tegument protein VP22 or homologue thereof; or a functional fragment of either of these.
  • VP22 refers to the protein VP22 of Herpes simplex virus (HSV) such as HSVl .
  • HSV Herpes simplex virus
  • the invention includes homologues thereof and fragments of any of these which are functionally active.
  • the VP22 protein has been fully characterised, for example as described in WO 97/05265 and WO 98/32866, the content of which is incorporated herein by reference.
  • homologues refers to sequences which share some level of sequence similarity as well as retaining translocation functionality.
  • homologues will have at least 60% identity, preferably 80% identity and more preferably 90% identity with VP22.
  • Homology is suitably assessed using one of the known algorithms, for example the similarity of a particular sequence to the VP22 sequence may be assessed using the multiple alignment method described by Lipman and Pearson, (Lipman, D.J. & Pearson, W.R. (1985) Rapid and
  • the sequences for which similarity is to be assessed should be used as the "test sequence” which means that the base sequence for the comparison, such as the sequence of VP22 should be entered first into the algorithm.
  • Particular homologues are derived from other herpes viruses including varicella zoster virus (VZV) , equine herpes virus (EHV) and bovine herpes virus (BHV) .
  • VZV varicella zoster virus
  • EHV equine herpes virus
  • BHV bovine herpes virus
  • Suitable fragments of HSV VP22 protein with transport activity include polypeptides corresponding to aminoacids 60-301 and 159- 301 of the full HSV1 VP22 sequence (1-301) .
  • Fusion polypeptides of one or more such fragments such as those described in W098/32886 may be used as the translocation protein in the constructs of the present invention.
  • the C-terminal domain of 142 amino acids is termed VP24 and is a particularly preferred translocation protein.
  • translocation protein is first prepared in pure form, and then mixed with plasmid DNA (NAV) in a suitable ratio and under suitable pH conditions, that a complex is formed.
  • NAV plasmid DNA
  • the sort of conditions at which this will occur will vary depending upon the type of plasmid DNA involved as well as the nature of the translocation protein used. However, in general pH conditions in the range of from 5 to 9 might be useful. These can be optimised using routine methods. Preferably, however, the pH conditions are in the range of 2 to 11, for example from 2.6 to 10.6. Most preferably it is carried out at pH conditions in the range of from 2 to 3 and preferably at about 2.6.
  • the reaction is suitably effected in a buffer to maintain the desired pH.
  • suitable buffers are well known in the art and include those buffers illustrated hereinafter.
  • the ratio of NAV to translocation protein again will be dependent upon the particular components employed. In general, however, an excess of translocation protein will be required, so that typical ratios of translocation protein to NAV will be from 1:2 to 1:500.
  • the preferred ratio of translocation protein to NAV will be from 1:1 to 1:10. It is most preferably a ratio of 1: 10. ,
  • the process will suitably be effected in a physiological buffer such as phosphate buffered saline.
  • the translocation protein such as VP22
  • the translocation protein is buffer exchanged with the appropriate buffer, for instance on an ion exchange column, and desalted, prior to mixing within the DNA.
  • the reaction is preferably effected in the presence of urea which assists in the binding.
  • Complex may be separated from the mixture after a suitable incubation period, for example of from 20-120 minutes and suitably at about 30 minutes. Separation may be effected using conventional methods Such as gel electrophoresis.
  • a DNA binding sequence is conjugated to the translocation protein.
  • Conjugation may be effected using any conventional methods, but preferably a nucleic acid encoding a fusion protein comprising the translocation protein and a DNA binding sequence is prepared. This may then be included in an expression vector using conventional methods. Transformation of an expression host, which may be a prokaryotic or eukaryotic cell, and in particular will be a prokaryotic cell such as E. coll. with the expression vector and culture of the cell using conventional DNA technology.
  • the translocation protein may be formed into a sphere with the nucleic acid entrapped within it. The sphere is preferably formed by mixing the translocation protein with an oligonucleotide. Fusion proteins of this type, nucleic acids encoding these fusion proteins, as well as vectors containing them and cells transformed with them form further aspects of the invention.
  • Suitable DNA binding sequences for use in the conjugates described above include the sequence KTPKKAK P (SEQ ID NO 1) which corresponds to residues 152-160 of human histone HI. This sequence has been shown in monomeric, di eric and trimeric forms to both interact with plasmid DNA and also to enhance transfection mediated by cationic lipids and PEI, both in vivo and in vitro (Schwartz et al, 1999 Gene Therapy 6: 282-292) .
  • DNA binding sequence There are other short peptide sequences of natural origin that can be used as DNA binding sequence and these include:
  • Protamine which is a small, naturally-occurring, arginine- rich DNA binding protein and can be used to avidly bind to DNA at physiological ionic conditions. It has been shown that a transferrin-protamine-DNA complex, formed by mixing of the transferrin-protamine conjugate and plasmid DNA, can bind an expression plasmid encoding the firefly luciferase gene (Wagner et al, Proc . Na tl . Acad. Sci , USA. 1990 87: 3410-3414). The positive charges of the protamine strongly interact with the negative charges of the phosphate backbone of DNA, resulting in neutral and stable NAV-protamine-VP24 complexes.
  • prota ine-derived peptides SRSRYYRQRQRSRRRRRR(SEQ ID NO 3) from Human protamine 1 (aa 11-28) and RRRLHRIHRRQHRSCRRRKRR (SEQ ID NO 4) from Human protamine 2 (aa 15-35) are also able to bind DNA in the way that the full length protamine is able to (Chen et al, Gene therapy. 1995 2: 116-123) .
  • GAL4 is a transcriptional activator of genes required for galactose catabolism in the yeast Sacchromyces cerevisiae and the full length protein is 881 amino acid in size. Within this there is a well-characterised GAL4 DNA-binding and dimerisation domain extending from amino acids 1-147 which has been expressed successfully and purified from E. coli, in functional form. This DNA-binding domain also harbours a natural nuclear localisation domain (Paul et al, 1997 Human Gene Therapy. 8: 1253-1262) . This GAL4 DNA-binding domain is expressed as a fusion with VP24 and purified.
  • the GAL4 target oligomer which may incorporated in the NAV is 5'-TCGACGGAGTACTGTCCTCCGC-3' (SEQ ID NO 5).
  • This fusion is mixed in varying proportions with a NAV in order to cross-link the fusion protein and NAV.
  • suitable conditions of pH and/or salt can be determined in order to create the construct.
  • the construct of the invention further comprises a sequence which functions as a nuclear accumulation signal and so may direct transport of proteins to the nucleus of infected cells.
  • sequences are found in the 10K proteins of Herpesvirus simiae (B virus) , Herpes simplex virus type 1, Pseudorabies virus, and Varicella- Zoster virus.
  • sequences are KIPIK (SEQ ID NO 6) from the Mat ⁇ 2 of Yeast; PKKKRKV (SEQ ID NO 7) from the large T antigen of SV40; AAFEDLRVLS (SEQ ID NO 8) from the nucleoprotein of Influenza virus; RKKRRQRRR (SEQ ID NO 9) from the Tat protein of HIV-1; RRRRRQTR (SEQ ID NO 10) from the 10K protein of B virus; RRRRRR (SEQ ID NO 11) from the 10K protein of HSV-1; RRRRR from the UK protein of PRZ and RKK from the UK protein of VZV.
  • sequences are preferably formed as part of the fusion protein described above.
  • a nucleic acid encoding a fusion protein including a translocation protein, a DNA binding sequence and a nuclear accumulation signal sequence is prepared and expressed in a suitable expression host using conventional methods.
  • the translocation protein is formed into a covalently bound conjugate with the nucleic acid using a coupling agent.
  • a coupling agent is 1- ethyl-3- (3-dimmethylaminopropyl) carbodiimide (Pierce).
  • the nucleic acid used in the construct of the invention is plasmid DNA which encodes an antigen. It is envisaged that any NAV can be used in the construct, for example :
  • NAV NAV encoding the protective H c domain of Botulinum neurotoxin type A, (Shyu et al., (2000) J. Biomed. Sci. 1_, 51-57); 2. NAV encoding P. falciparium circumsporozoite antigen (i.e. a malaria antigen), ( e et al., (2000) Vaccine 18_, 1893-1901);
  • the purified translocation protein is preferably buffer exchanged prior to mixing with the nucleic acid of the present invention. Binding in this way is based on charge or electrostatic interactions .
  • the protein will be buffer exchanged into either citrate phosphate buffer, carbonate-bicarbonate buffer or PBS or citrate phosphate buffer, carbonate-bicarbonate buffer and PBS each containing 8M urea.
  • the protein is then desalted out.
  • This protein for example the herpes simplex virus type I tegument protein VP22, or a functional fragment or homologue thereof, is then used to mix with a DNA vaccine to form complexes.
  • Constructs of the invention are suitably administered in the form of pharmaceutical compositions in which they are combined with pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carriers are liquid or solids, and preferably liquids such as saline.
  • the compositions of the invention are preferably formulated for parente,ral administration although other means of application are possible as described in the pharmaceutical literature, for example administration using a Gene Gun, (Bennett et al., (2000) Vaccine _18, 1893-1901).
  • DNA encoding the VP24 protein is cloned into a suitable expression vector (e.g. pET vector from Promega) .
  • the VP24 projtein is expressed in E. coli and purified.
  • Purified VP24 is mixed with plasmid DNA (NAV) in an optimised ratio, under optimised conditions of pH to form a VP24/NAV complex which can be delivered to target cells .
  • NAV plasmid DNA
  • DNA encoding the sequence KTPKKAKKP (SEQ ID NO 1) is fused to the N-terminus or the C-terminus of the VP24 gene in a suitable expression system (e.g. pET vector from Promega) to produce a peptide/VP24 fusion protein.
  • a suitable expression system e.g. pET vector from Promega
  • This fusion is mixed in varying proportions with a NAV in order to cross-link the fusion protein and NAV. Suitable conditions of pH and/or salt are used.
  • VP24 can be coupled to polylysine by the addition of l-ethyl-3- (3-dimmethylaminopropyl) carbodiimide resulting in a covalently bound conjugate.
  • the negatively charged plasmid DNA (NAV) noncovalently binds to the positively-charged polylysine in the presence of NaCl. This produces soluble complexes of VP24/ polylysine/NAV.
  • the interactions of the protein and the NAV can be studied by the techniques of Capillary gel electrophoresis and gel retardation.
  • the translocation patterns of the NAV and VP24 protein can be studied in vitro and tracked using specific antibodies and conjugation of the protein with AP or HRP.
  • VP22 protein was buffer exchanged into either citrate phosphate buffer, carbonate- bicarbonate buffer or PBS or citrate phosphate buffer, carbonate-bicarbonate buffer and PBS, each containing 8M urea, and desalted out. This protein was then mixed with a DNA vaccine to form complexes. When electrophoresed through an agarose gel, complexes of VP22 remained in the well of the gel. . Materials and Methods :
  • citrate phosphate buffer by addition of 891ml and 109ml respectively per litre.
  • the pH was 2.6.
  • 0.1M Sodium Carbonate and 0.1M Sodium Bicarbonate were prepared. These were used to form carbonate-bicarbonate buffer by addition of 900ml and 100ml respectively per litre.
  • the pH was 10.6.
  • Stocks of citrate phosphate buffer, carbonate-bicarbonate buffer and PBS were prepared, each containing 8M, 4M, 2M and 0M Urea.
  • Plasmid DNA was used at concentrations of 200ng and 400ng.
  • the protein was mixed in 1:1, 1:2, 1:5 and 1:10 DNA:VP22 ratios as shown in the following Table.
  • the method outlined above (without urea treatment) is used to see the effect these complexes have in mammalian cells.
  • the uptake of complexes of recombinant protein and DNA is studied in mammalian cells using tissue culture techniques i.e. it is an in vitro model.
  • the mammalian cells used are African Green Monkey Kidney Cells (COS-7) .
  • VP22 protein within the cells is studied using standard im unofluorescence techniques .
  • the protein is detected using a specific antibody.
  • a secondary antibody specific to the first and conjugated with FITC will allow detection of the protein by UV light via a confocal microscope. This allows determination of the uptake and localisation of the VP22 protein but does not give an indication of whether the DNA remains complexed.
  • complexes of VP22:DNA are added to cells and the DNA labelled with a dye.' This allows determination of uptake and localisation of the DNA.

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Abstract

Construction comprenant une protéine de translocation liée à un acide nucléique, qui code pour un antigène capable de générer une réaction immunitaire de protection chez un animal auquel elle est administrée. Ces constructions sont particulièrement utiles dans la formulation de vaccins à ADN nu, qui utilise un ADN de plasmide. L'invention décrit divers procédés de préparation de ces composés.
PCT/GB2002/002526 2001-06-05 2002-05-31 Vaccins WO2002098909A1 (fr)

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GBGB0113552.4A GB0113552D0 (en) 2001-06-05 2001-06-05 Vaccines

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992020316A2 (fr) * 1991-05-14 1992-11-26 University Of Connecticut Apport cible de genes codant des proteines immunogenes
US6017735A (en) * 1997-01-23 2000-01-25 Marie Curie Cancer Care Materials and methods for intracellular transport and their uses

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992020316A2 (fr) * 1991-05-14 1992-11-26 University Of Connecticut Apport cible de genes codant des proteines immunogenes
US6017735A (en) * 1997-01-23 2000-01-25 Marie Curie Cancer Care Materials and methods for intracellular transport and their uses

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HADDAD A ET AL: "TARGETED M CELL IMMUNIZATION FOR HIV-1 ENV DNA VACCINES", FASEB JOURNAL, FED. OF AMERICAN SOC. FOR EXPERIMENTAL BIOLOGY, BETHESDA, MD, US, vol. 14, no. 6, 20 April 2000 (2000-04-20), pages A1204, XP000995418, ISSN: 0892-6638 *
PAUL R W ET AL: "GENE TRANSFER USING A NOVEL FUSION PROTEIN, GAL4/INVASIN", HUMAN GENE THERAPY, XX, XX, vol. 8, no. 10, 1 July 1997 (1997-07-01), pages 1253 - 1262, XP000971829, ISSN: 1043-0342 *
STANKOVICS J ET AL: "Overexpression of human methylmalonyl CoA mutase in mice after in vivo gene transfer with asialoglycoprotein/polylysine/DNA complexes.", HUMAN GENE THERAPY. UNITED STATES SEP 1994, vol. 5, no. 9, September 1994 (1994-09-01), pages 1095 - 1104, XP001096336, ISSN: 1043-0342 *
WAGNER E: "TRANSFERRIN-POLYCATION CONJUGATES AS CARRIERS FOR DNA UPTAKE INTO CELLS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 87, no. 9, 1 May 1990 (1990-05-01), pages 3410 - 3414, XP000368690, ISSN: 0027-8424 *
WU Y ET AL: "GENE TRANSFER FACILITATED BY A CELLULAR TARGETING MOLECULE, REOVIRUS PROTEIN SIGMA1", GENE THERAPY, MACMILLAN PRESS LTD., BASINGSTOKE, GB, vol. 7, no. 1, January 2000 (2000-01-01), pages 61 - 69, XP000995427, ISSN: 0969-7128 *

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