WO1987001386A1 - Recombinant virus - Google Patents

Recombinant virus Download PDF

Info

Publication number
WO1987001386A1
WO1987001386A1 PCT/AU1986/000256 AU8600256W WO8701386A1 WO 1987001386 A1 WO1987001386 A1 WO 1987001386A1 AU 8600256 W AU8600256 W AU 8600256W WO 8701386 A1 WO8701386 A1 WO 8701386A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
virus
antigen
recombinant
vaccinia virus
Prior art date
Application number
PCT/AU1986/000256
Other languages
English (en)
French (fr)
Inventor
Christopher John Langford
Stirling John Edwards
Original Assignee
The Walter And Eliza Hall Institute Of Medical Res
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by The Walter And Eliza Hall Institute Of Medical Res filed Critical The Walter And Eliza Hall Institute Of Medical Res
Publication of WO1987001386A1 publication Critical patent/WO1987001386A1/en
Priority to KR1019870700378A priority Critical patent/KR880700073A/ko
Priority to NO871737A priority patent/NO871737L/no
Priority to FI871885A priority patent/FI871885A/fi
Priority to DK219187A priority patent/DK219187D0/da

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • 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
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to a recombinant virus, and in particular it relates to a recombinant vaccinia virus which has been modified to optimize the immunogenicity of foreign immunogenic polypeptides 5 expressed thereby.
  • recombinant virus denotes infective virus which has been genetically modified by incorporation of 10 foreign genes or genetic material into the virus genome. The modified virus then expresses the foreign gene in the form of a "foreign" polypeptide on infection of a cell by the recombinant virus.
  • recombinant vaccinia virus has a corresponding meaning.
  • the foreign viral antigen which is now expressed by the vaccinia virus is in a near normal situation and its processing, modification, transport and final localization on the surface of the infected cell may be very similar to that in a normal infection.
  • herpes simplex glycoprotein D (Paoletti et al, 1984; Cremer et al, 1985) , hepatitis B surface antigen (Smith et al, 1983; Moss et al, 1984) , vesicular stomatitis virus glycoprotein G, and influenza virus hemagglutinin (Smith et al, 1983; Panicali et al, 1983) genes are inserted into recombinant vaccinia virus, live recombinant viruses can be used to immunize animals against infection.
  • the present invention provides in one aspect, a recombinant virus, characterised in that it includes a coding sequence for a hybrid polypeptide, said hybrid polypeptide comprising _at least one immunogenic polypeptide segment which is foreign to the virus or virus infected cells in association with a surface or membrane-associated polypeptide segment to locate said hybrid polypeptide on or at the surface of virus infected cells.
  • this invention provides a recombinant vaccinia virus, characterised in that it includes a coding sequence for a hybrid polypeptide, said hybrid polypeptide comprising at least one immunogenic polypeptide segment which is foreign to vaccinia virus or vaccinia virus infected cells, in association with a surface or membrane-associated polypeptide segment to locate said hybrid polypeptide on or at the surface of vaccinia virus infected cells.
  • this invention provides a DNA molecule comprising a coding sequence for a hybrid polypeptide, said hybrid polypeptide comprising at least one immunogenic polypeptide segment which is foreign to vaccinia virus or vaccinia virus infected cells in association with a surface or membrane-associated polypeptide segment to locate said hybrid polypeptide on or at the surface of vaccinia virus infected cells.
  • hybrid polypeptide comprising at least one immunogenic polypeptide segment which is foreign to vaccinia virus or vaccinia virus infected cells in association with a surface or membrane-associated polypeptide segment to locate said hybrid polypeptide on or at the surface of vaccinia virus infected cells.
  • the present invention is illustrated by way of example by the expression of a hybrid polypeptide based on the secreted repetitive plasmodial antigen (the S-antigen) in a recombinant vaccinia virus.
  • the S-antigen proteins of Plasmodiu falciparum are secreted by the parasite into the space which separates the limiting membrane of the dividing parasites and the inner membrane of the red blood cell initially formed during the invagination process accompanying the parasite invasion of the red blood cell and subsequently elaborated during parasite growth.
  • Immunogold electromicroscopy indicates that this space, known as the parasitophorous vacuole, is filled with
  • a primary aim in the work leading to the present invention was to investigate the use of the live recombinant vaccine virus in the delivery of plasmodial blood-stage antigens to immunize animals and to compare the immune responses to those obtained using peptides generated by recombinant DNA techniques or synthesized chemically.
  • a theoretical advantage of the live viral delivery approach is that it should better stimulate the cellular arm of the immune system which may be of prime importance in the efficacy of anti-parasitic vaccines.
  • the mouse im unoglobulin gene has been used to provide the sequences necessary for the expression of the immunogenic polypeptide on the infected cell surface.
  • Figure 1 illustrates the construction of transfection plasmids containing the deleted S-antigen gene of the FCQ27/PNG (FC27) isolate of P.falciparum.
  • (a) shows the structure of the genomic copy of the FC27 S-antigen gene with sequences encoding a signal peptide (dark shading) and approximately 100 copies of the 11 amino acid repeating peptide sequence shown beneath the gene.
  • the S-antigen gene is located immediately downstream from the vaccinia virus 7.5K gene promoter and is flanked on both sides by vaccinia virus TK gene sequences as shown in (c) .
  • a hybrid S-antigen gene containing an immunoglobulin transmembrane sequence (hatched) and intracellular domain (dotted) was constructed from pV8 to generate the plasmid pVA20 which is shown in part in (d) .
  • Figure 2 is a Western blot analysis of S-antigen produced by BSC1 cells infected with the recombinant vaccinia virus V8 (lanes 2 and 3) compared with that produced by E.coli under the control of the p ⁇ " C9 ⁇ -galactosidase promoter (lane 1) .
  • Lanes 2 and 3 show the relative amounts of S-antigen associated with the virus infected cells and the culture medium at 48 hours after infection. The culture medium was centrifuged at 12,000g for 3mins prior to analysis. Filters were probed with a rabbit antisera recognizing only the 11 amino acid repeat portion of the S-antigen.
  • Figure 3 shows the time course of the synthesis and secretion of S-antigen in vaccinia infected BSC1 cell monolayers.
  • Cells were infected for lhr at lpfu/cell. At this time the innoculum was removed and fresh medium was added.
  • a sample of the culture medium was taken at various times after infection and subjected to centrifugation at 12,000g for 3mins. The supernatent from this centrifugation was taken for analysis. The remaining cells and medium were scraped from the dishes and quantitatively transferred to a fresh tube. Following sonication a sample was taken and dissolved in SDS sample buffer for analysis by SDS/PAGE. Equal fractions of each sample were analysed. Filters were probed with a rabbit antisera which specifically recognized the 11 amino acid repeat of the S-antigen.
  • Figure 4 is a diagramatic representation of the steps used in the subcloning of the mouse membrane IgG transmembrane sequence into the Sphl site at the 3 ' end of the FC27 S-antigen gene.
  • a 186bp Haelll fragment encoding the transmembrane, intracellular domain and a portion of the hinge region was isolated from the ⁇ l cDNA clone described by Tyler et al, 1982.
  • Sphl linker DNA was added to the ends of this fragment which after Sphl digestion, was cloned into the Sphl site of the S-antigen gene clone pFC27 Aha2 to generate the new clone pA20.
  • the sequence at the junction of the S-antigen gene and the immunoglobulin gene is shown at the bottom and indicates the new amino acid sequence at the junction.
  • the amino acids alanine and proline, indicated with an asterisk, are not present in either of the parental proteins as they are generated by the Sphl linker DNA sequences.
  • Six amino acids of the extracellular domain of the immunoglobulin gene are present in the new hybrid protein.
  • Figure 5 shows that the S-antigen produced by cells infected with the VA20 recombinant virus is no longer secreted.
  • Samples of infected cells and culture medium were collected as described in Figure 3 48 hours after infection with either V8 or VA20 recombinant virus at lpfu/cell. The total amount of S-antigen appears to remain the same, however very little is secreted into the medium in the case of VA20 infected cells.
  • Westerns were probed with a rabbit anti-FC27 S-antigen repeat antisera.
  • Figure 6 BSC-1 cells infected for 48hrs with either the V8 _or VA20 recombinant virus were solubilized in 0.05% Triton X114 for lhr at 4°C.
  • Figure 7 shows indirect immunofluorescence of BSGl cells infected 18hrs earlier with recombinant virus VA20 (A and C) or V8 (B and D) .
  • Cells were either fixed prior to staining (A and B) to permeabilize the cells ' and allow detection of intracellular S-antigen or fixed after staining to detect S-antigen localized on the surface of the infected cells (C and D) .
  • Figure 8 sets out the antibody titres of mice sera assayed at 3 weeks after a single IP immunization with 1x10 PFU of the V8 or VA20 recombinant virus.
  • Ninety-six well microtitre trays were coated with a FC27 S-antigen repeat/3-galactosidase fusion polypeptide preparation at a predetermined optimal concentration of 3 ⁇ g/ml.
  • Pre-immune sera were serial diluted to determine the dilution at which half maximal absorbance was reached. Thse values are plotted for both the BALB/c.H-2 and 129/J strains of mice used.
  • Figure 9 is a plot of the absorbance values obtained in a typical ELISA assay of rabbit antisera taken at one to five weeks after a single ID injection g of 10 PFU of live recombinant virus VA20.
  • Sera were J assay for both anti S-antigen antibodies as described in Figure 8 at a standard dilution of 1:320 of the sera (dotted line) or for anti-vaccinia antibodies using plates coated with BPL inactivated vaccinia virus at a standard dilution of 1:2580 of the serum (solid lines).
  • - (B) shows the absorbance values obtained in a ELISA assay in which individual rabbit antisera were assayed for anti S-antigen antibodies at 2 weeks after
  • Recombinants were selected in which the S-antigen gene was inserted in the correct orientation 3' to the vaccinia 7.5K protein early gene promotor and flanked on either side by the 5' and 3' ends of the vaccinia virus TK gene sequences of the plasmid vector.
  • This new construct, pV8, was used to transfect CV1 cells infected with wild type vaccinia virus giving rise to the recombinant vaccinia virus V8, containing the S-antigen gene.
  • the addition of the mouse membrane immunoglobulin transmembrane sequence to the S-antigen gene was used to transfect CV1 cells infected with wild type vaccinia virus giving rise to the recombinant vaccinia virus V8, containing the S-antigen gene.
  • a 186bp Haelll fragment containg sequences encoding six amino acids of the hinge region, 26 amino acids of the transmembrane domain and 28 amino acids of the intracellular domain of the mouse IgG, immunoglobulin was isolated from the ⁇ l cDNA clone described by Tyler et al (1982). Sphl linker DNA with the sequence 5'-CCGCATGCGG-3' was then ligate to the Haelll fragment, digested with Sphl and cloned into the unique Sphl site located 65bp from the .3' end of the S-antigen gene in the subclone pFC Aha2.
  • This plasmid DNA was used to transfect vaccinia infected CV1 cells to produce the recombinant vaccinia virus VA20.
  • Recombinant viruses con ⁇ taining the S-antigen genes were screened for the presence of DNA by dot blot analysis or for the production of S-antigen which was detected by a high titre polyclonal antisera, R210, raised by immunizing rabbits with a ⁇ -galactosidase fused polypeptide from clone Agl6 (Coppel et al, 1983) con taining 23 copies of the 11 amino acid FC27 S-antigen repeating polypeptid Expression of S-antigen in recombinant vaccinia infected cells.
  • Confluent monolayers of BSC-1 cells were routinely infected at lpfu/cell with purifi recombinant virus and allowed to incubate at 37°C for 18-48hrs at which ti the infected cells and/or the supernatant were harvested and dissolved in SDS, sample buffer and boiled. Samples were then analysed by immunoblotti 0; and probed with a rabbit anti S-antigen antisera, R210 which recognizes th repeating epitope of the S-antigen molecule.
  • Triton X114 partitioning Recombinant vaccinia infected cells were dissol in 0.5% Triton X114 in PBS for lhr at 4°C. Following centrifugation at 20 rpm to remove nuclei in the Triton X114 soluble material was layered over 5 cushion of 6% Triton in 0.06% sucrose/PBS and then the temperature was rai to 37°C. The cloudy suspension of insoluble material was removed by centr fugation at 37°C. This fraction which is referred to as the Triton X114 pellet, should contain the integral membrane proteins by virtue of the greater affinity of their hydrophobic transmembrane sequences for the Trit Q , X114 detergent which becomes insoluble at elevated temperature (Bordier, 1981).
  • BSC-1 cells were grown onto sterile glass covers!ips for a period of 6hrs after which they were infected at 0.5pfu/cell with either the V8 or VA20 recombinant viruses or TK nonreco binant virus as control. After 18hrs, coverslips were rinsed in cold PBS and then stained immediately with rabbit anti-S-antigen antisera followed by FITC conjugate sheep anti-rabbit antibodies. Cells were then post fixed in cold 95% ethanol:5% glacial acetic acid prior to mounting under glycerol and visualization by fluorescence microscopy.
  • mice within a few days of the first immunization, reaching a size of approximate 1 to 1.5cm in diameter. Occasionally these lesions ulcerated. Lesions were no longer apparent after two weeks. Rabbits were bled at weekly intervals and the sera analysed for anti-S-antigen or anti-vaccinia antibodies in an ELISA assay. Age and weight-matched inbred mice of various strains were
  • FC27 S-antigen gene of P.falciparum is expressed in recombinant vaccinia
  • the recombinant virus was indeed the correct length (data not shown).
  • abnormal SDS binding characteristics result in this aberrant MW determination on SDS/PAGE.
  • S-antigen appears to be synthesized in recombinant vaccinia infected cells under the control of vaccinia promotor elements.
  • the S-antigen is secreted from vaccinia infected cells
  • Monolayers of BSC1 cells were infected with purified recombinant virus V8. After Bit, the virus innoculum was replaced with fresh medium and then at various times the cells and culture medium were harvested, separated by centrifugation and subjected to analysis by immunoblotting. Detectable
  • Triton X114 partition experiments (Bordier, 1981) were performed to test if, by this criteria, the hybrid S-antigen containing the transmembran segment behaved as a typical integral membrane protein. Indeed, whereas the V8 protein behaves exclusively as a hydrophilic soluble protein, the majority of the VA20 protein partitioned into the detergent phase ( Figure 6 indicating that the hydrophobic transmembrane sequence had converted the soluble S-antigen protein into a membrane-associated protein.
  • BSC-1 cells infected with either VA20 or V8 recombinant virus were subjected to indirec immunofluroescence 18hrs after infection.
  • Figure 9a shows that the anti S-antigen Ab titres in the VA20 immunized rabbits peaked two weeks after immunization despite the fact that anti- vaccinia antibody titres continued to climb over the next three week period The same was true of the rabbit responses to the V8 recombinant although here the responses were very small and difficult to measure.
  • Figure 9B shows that the anti S-antigen Ab titres in the VA20 immunized rabbits peaked two weeks after immunization despite the fact that anti- vaccinia antibody titres continued to climb over the next three week period The same was true of the rabbit responses to the V8 recombinant although here the responses were very small and difficult to measure.
  • Figure 9B shows that the anti S-antigen Ab titres in the VA20 immunized rabbits peaked two weeks after immunization despite the fact that anti- vaccinia antibody titres continued to climb over the next three week period The same was true of the rabbit responses to the V8 recombinant although here the responses were very small
  • Example 1 is a specific example of a more general method by which biologically important molecules which, although themselves not surface antigen molecules, can be redirected to the surface of recombinant vaccinia virus-infected cells. This example also shows how crucial this surface localization of the antigen is for the induction of good immune responses to the foreign introduced antigen.
  • the antigen chosen for Example 1, the malarial S-antigen has to date not been implicated as a potential vaccine candidate primarily because the immunodominant repeat portion of the molecule is remarkably variant. These repeating structures vary 5 enormously in their number, length and amino acid composition which greatly affects their immunological properties but not, it would seem, their behaviour as secreted proteins.
  • the present Example illustrates that it is possible 0 to replace the S-antigen repeating epitope with an unrelated sequence which is- of importance as a vaccine molecule.
  • These hybrid molecules containing in addition an appropriate trans-membrane anchoring sequence, should be, in many cases, as efficiently 5 transported to the surface of the recombinant virus-infected cell as is the hybrid S-antigen molecule described above.
  • NANP Asn-Ala-Asn-Pro
  • this Example is one example of an approach by which one can tailor an antigenic determinant into a "carrier" molecule designed to deliver this epitope to the surface of recombinant vaccinia virus infected cells.
  • a murine retrovirus is also able to express the product of the hybrid VA20 gene on the surface of virus-infected mouse cells in culture demonstrating that this approach may be of general applicability to a variety of antigenic epitopes expressed in any of a number of "carrier" epitopes in a variety of recombinant viral vector systems.
  • Figure 10 is a diagramatic representation of the manipulations required to delete the 33bp S-antigen repeating sequences from pVA20 and to replace them with sequences encoding 16, 32 and 48 copies of the 4 amino acid repeating epitope of the P.falciparum circumsporozoite coat protein.
  • Figure 11 is a schematic representation of the
  • P.falciparum circumsporozoite coat protein gene (at top) showing the sequence of the dominant 4 amino acid repeating unit, NANP.
  • sequences of the synthetic oligonucleotides used in the synthesis of the new insert and (at bottom) the sequence of the 5" and 3' junction regions between the BamHl cut plasmid pLK8 (S-antigen sequences) and the new insert sequences.
  • 6BP adaptor sequences shown in solid boxes at the 5' and 3' ends of the insert, the Sau3A sites flanking the insert and how a BamHl site is only regenerated at the 5' end of the insert.
  • 5' and 3' refer to the ends of the coding strand of the insert DNA.
  • Figure 12 shows double stranded DNA sequencing reactions of DNA from plasmids p6.44, p66.6 and p.666.34 containing 16, 32 and 48 copies, respectively, of the 12bp repeating sequence of the P.falciparum circumsporozoite protein. For clarity only the "A" ⁇ reactions are shown for the 3 constructs. At left is the sequence of the coding strand of the synthetic oligonucleotide used in the constructions with the sequence 5 '-AACVCCAACCC-3 ' . As can be seen, two pairs of A. doublets occur in each copy of the repeat.
  • the : sequencing primer was a 17 nucleotide homologous to coding strand sequences located 20bp 5 ' to the BamHl site.
  • Figure 13 is an immunoblot of proteins derived from recombinant virus-infected BSC-1 cells probed with an antisera (R516 anti NANP.,-K-LH) produced by immunizing rabbits with a 12 amino acid long synthetic peptide encoding 3 copies of the 4 amino acid squence NANP conjugated to KLH.
  • This antiserum recognises polypeptides produced by cells infected with recombinant virus (V6.44) containing the -6.44 hybrid gene described above but not produced in a parallel experiment with cells infected with a similar construct containing unrelated sequences inserted at the BamHl site (V-control) .
  • Molecular weights in Kdaltons are shown at right.
  • the plasmid pLK8 contains the S-antigen gene with less than one full copy of the 33bp repeating sequence and with a unique BamHl site located within this remaining partial repeat sequence. It is into this site that appropriately engineered sequences encoding antigenic determinants can be cloned, thus neatly replacing the S-antigen repeating epitope.
  • the incoming sequences in the situation described in this example would need to have BamHl "sticky ends" and to be engineered in such a way that the reading frame of the whole hybrid gene is maintained.
  • the total length of the insert DNA should be an equal multiple of 3 base pairs and that the reading frame be in phase with the GAT codon of the GGATCC BamHl site at the 5 1 end of the (coding strand of the) insert. It is also believed that the epitope to be expressed should be repeated as many times as possible to maximize its immunogenicity even if this epitope is represented only once in the native antigen molecule.
  • P.falciparum circumsporozoite coat protein has been chosen. It will be appreciated, however, that the same procedures could equally well be applied to the linear epitopes or the "mimotopes" of conformational epitopes of other antigen molecules.
  • 5 *-AACGCCAACCCC-3 ' and 5 • -GGTTGGCGTTGG-3 ' were made on an Applied Biosystems oligonucleotide synthesiser and purified by HPLC. These were then kinased prior to annealing and ligation in the standard way.
  • the oligonucleotides were designed so that the ends were complimentary in one orientation only, ensuring that only "head to tail” ligation of the double stranded monomers was possible.
  • the ligated fragments were then size fractionated on a low gelling temperature agarose 5; gel and DNA molcules in the size range from 180 to 600bp were isolated and purified from the agarose.
  • Recombinant bacterial clones containing the 12bp sequence were selected by colony hybridization using a
  • Plasmid DNA was isolated from the positive clones and digested with restriction enzymes to determine the clones which contained the longest inserts. A number of these were then sequenced using the double stranded DNA 5
  • the new 192b ⁇ insert in the hybrid gene p6.44 is flanked by Sau3A sites which allow the insert to be isolated and purified from the recombinant plasmid. However only one BamHl site at the 5 * of the coding j strand of the insert is regenerated in this hybrid (see figure 11) .
  • the recombinant plasmid can be linearised with BamHl, phosphatase treated and ligated with the isolated 192bp Sau3A fragment. Plasmid DNA was prepared from the transformed bacteria resulting from 0 this cloning and digested with restriction enzymes to select clones with double (or triple) inserts. These were then sequenced to determine which were in the correct orientation and to confirm the predicted sequence.
  • inserts containng 16(p6.44) , 32(p66.6) and 48(p666.34) copies of the 12bp repeat of the CSP gene have been poduced (see Q figures 10 and 12) .
  • FIG. 13 shows a Western blot of proteins produced by V6.44 virus-infected mammalian BSC-1 cells probed with a rabbit antisera raised against a 12 amino acid long synthetic peptide comprising 3 copies of the NANP peptide.
  • Isolate-specific S-antigen of Plasmodiu falciparum contains a repeated sequence of eleven amino acids. Nature 306, 751-756. Cremer, K.J. , Mackett, M. , Wohlenberg, C. , Notkins, A.L. and Moss, B. (1985 Vaccinia virus recombinant expressing herpes simplex virus type 1 glyc protein D prevents latent herpes in mice. Science 228, 737-740.
  • Vaccinia virus A selectable eukaryotic cloning and expression vector. Proc.Natl.Acad.Sci.USA 79, 7415-7419. Mackett, M. , Smith, G.L. and Moss, B. (1984). General method for production and selection of infectious vaccinia virus reco binants expressing foreign genes. J.Virol. 49, 857-864. Moss, B. , Smith, G.L. , Gerin, J.L. and Purcell , R.H. (1984). Live recombin vaccinia virus protects chimpanzees against hepatitis B. Nature 311, 67-69. Panicali, D. and Paoletti, E. (1982).
  • pox viruses as cloni vectors: Insertion of the thymidine kinase gene of herpes simplex viru into the DNA of infectious vaccinia virus. Proc.Natl.Acad.Sci.USA 79, 4927-4931.
  • liver vaccines using genetically engineered pox viruses Biological activity of vaccinia virus recombinants expres ing the hepatitis B surface antigen and the herpes simplex glycoprotei D. Proc.Natl.Acad.Sci.USA 81, 193-197.
  • mRNA for surface immunoglobulin ⁇ chains encodes a highly con served transmembrane sequence and a 28-residue intracellular domain. Proc.Natl.Acad.Sci.USA 79, 2008-2012. Valenzuela, P., Coit, D. , Medina-Selby, M.A. , Kuo, CH. , Van Nest, G. , Burk R.L. , Bull, P., Urdea, M.S. and Graves, P.V. (1985). Antigen engineer ing in yeast: Synthesis and assembly of hybrid hepatitis B surface antigen-herpes simplex 1 gD particles. Biotechnology 3, 323-326.
PCT/AU1986/000256 1985-08-29 1986-08-29 Recombinant virus WO1987001386A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
KR1019870700378A KR880700073A (ko) 1985-08-29 1987-04-20 재조합 바이러스
NO871737A NO871737L (no) 1985-08-29 1987-04-27 Rekombinant virus.
FI871885A FI871885A (fi) 1985-08-29 1987-04-29 Kombinationsvirus.
DK219187A DK219187D0 (da) 1985-08-29 1987-04-29 Rekombinant-virus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPH219885 1985-08-29
AUPH2198 1985-08-29

Publications (1)

Publication Number Publication Date
WO1987001386A1 true WO1987001386A1 (en) 1987-03-12

Family

ID=3771250

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1986/000256 WO1987001386A1 (en) 1985-08-29 1986-08-29 Recombinant virus

Country Status (11)

Country Link
EP (1) EP0244427A4 (ja)
JP (1) JPS63500637A (ja)
KR (1) KR880700073A (ja)
AU (1) AU594087B2 (ja)
DK (1) DK219187D0 (ja)
ES (1) ES2002128A6 (ja)
FI (1) FI871885A (ja)
IL (1) IL79880A0 (ja)
NZ (1) NZ217400A (ja)
WO (1) WO1987001386A1 (ja)
ZA (1) ZA866582B (ja)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5182211A (en) * 1987-08-07 1993-01-26 Institut Pasteur Plasmid vectors encoding a protein of a picornavirus
AU645274B2 (en) * 1989-09-29 1994-01-13 Institut National De La Recherche Agronomique Method for the preparation of an immunizing composition
US7641896B2 (en) 2002-07-05 2010-01-05 Folia Biotech Inc. Adjuvant viral particle
US8101189B2 (en) 2002-07-05 2012-01-24 Folia Biotech Inc. Vaccines and immunopotentiating compositions and methods for making and using them

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989007140A1 (en) * 1988-02-05 1989-08-10 Commonwealth Scientific And Industrial Research Or Gene expression system (particularly for rotavirus vp7 protein) involving a foreign signal peptide and optionally a transmembrane anchor sequence

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0012078A1 (fr) * 1978-11-27 1980-06-11 Institut Pasteur Vecteur permettant l'insertion d'un gène procaryote ou eucaryote, et l'excrétion de la protéine exprimée
AU1159683A (en) * 1982-01-11 1983-07-28 Animal Vaccine Research Corp. Virus with recombinant surface proteins
AU2342484A (en) * 1982-11-30 1984-06-18 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Process for producing poxvirus recombinants for expression offoreign genes
AU4233585A (en) * 1984-04-25 1985-11-15 Transgene S.A. Vaccine against rabies and preparation process thereof
EP0162738A1 (en) * 1984-04-09 1985-11-27 MOLECULAR GENETICS RESEARCH & DEVELOPMENT LIMITED PARTNERSHIP Production of pseudorabies virus subunit vaccines
US4603112A (en) * 1981-12-24 1986-07-29 Health Research, Incorporated Modified vaccinia virus

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU569722B2 (en) * 1983-01-28 1988-02-18 Saramane Pty Ltd Expression of plasmodium falciparum polypeptides from cloned cdna

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0012078A1 (fr) * 1978-11-27 1980-06-11 Institut Pasteur Vecteur permettant l'insertion d'un gène procaryote ou eucaryote, et l'excrétion de la protéine exprimée
US4603112A (en) * 1981-12-24 1986-07-29 Health Research, Incorporated Modified vaccinia virus
AU1159683A (en) * 1982-01-11 1983-07-28 Animal Vaccine Research Corp. Virus with recombinant surface proteins
AU2342484A (en) * 1982-11-30 1984-06-18 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Process for producing poxvirus recombinants for expression offoreign genes
EP0162738A1 (en) * 1984-04-09 1985-11-27 MOLECULAR GENETICS RESEARCH & DEVELOPMENT LIMITED PARTNERSHIP Production of pseudorabies virus subunit vaccines
AU4233585A (en) * 1984-04-25 1985-11-15 Transgene S.A. Vaccine against rabies and preparation process thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Immunology Today, Volume 6, No.8, issued 1985, August (Amsterdam), B. MOSS, 'Vaccinia Virus Expression Vector: A New Tool for Immunologists^, see pages 243-245. *
Modern Approaches Vaccines: Molecular Chemical Basis Virus Virulence Immunogenicity, (Pap.Conf.) 1983, issued 1984 (Cold Spring Harbor, N.Y.), E. PAOLETTI et al, 'Construction of Live Recombinant Vaccines Using Genetically Engineered Poxvirus', see pages 295-299. *
Science, Volume 224, issued 1984, April 27 (Washington D.C.), G.L. SMITH et al, 'Plasmodium Knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus', see pages 397-399. *
See also references of EP0244427A4 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5182211A (en) * 1987-08-07 1993-01-26 Institut Pasteur Plasmid vectors encoding a protein of a picornavirus
AU645274B2 (en) * 1989-09-29 1994-01-13 Institut National De La Recherche Agronomique Method for the preparation of an immunizing composition
US7641896B2 (en) 2002-07-05 2010-01-05 Folia Biotech Inc. Adjuvant viral particle
US8101189B2 (en) 2002-07-05 2012-01-24 Folia Biotech Inc. Vaccines and immunopotentiating compositions and methods for making and using them
US8282940B2 (en) 2002-07-05 2012-10-09 Folia Biotech Inc. Adjuvant viral particle
US9339535B2 (en) 2002-07-05 2016-05-17 Folia Biotech, Inc. Vaccines and immunopotentiating compositions and methods for making and using them

Also Published As

Publication number Publication date
DK219187A (da) 1987-04-29
AU6370186A (en) 1987-03-24
IL79880A0 (en) 1986-11-30
NZ217400A (en) 1989-09-27
FI871885A0 (fi) 1987-04-29
AU594087B2 (en) 1990-03-01
EP0244427A1 (en) 1987-11-11
KR880700073A (ko) 1988-02-15
ES2002128A6 (es) 1988-07-16
ZA866582B (en) 1987-04-29
JPS63500637A (ja) 1988-03-10
FI871885A (fi) 1987-04-29
DK219187D0 (da) 1987-04-29
EP0244427A4 (en) 1989-04-27

Similar Documents

Publication Publication Date Title
Langford et al. Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity
Barr et al. Recombinant Pfs25 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in experimental animals.
JP3051130B2 (ja) 組換えコクシジウム症ワクチン類
US6958235B1 (en) Recombinant protein containing a C-terminal fragment of plasmodium MSP-1
JPH08505768A (ja) 新規なタンパク質性粒子
US6551586B1 (en) Malaria vaccine based upon the addition of a MSA1 peptide
US5403581A (en) Coccidiosis vaccines
US6933130B1 (en) Recombinants process for preparing a complete malaria antigen, gp190/MSP1
AU594087B2 (en) Recombinant vaccinia virus sxpresses hybrid polypeptides on virus infected cell surface
PH27079A (en) Immunogenic recombinant yeast expression product and method for purifying it
US5217898A (en) Expression of the P. falciparum transmission-blocking antigen in yeast
WO1988002757A1 (en) Hybrid proteins or polypeptides
AU709479B2 (en) Recombinant attentuated canrypox ALVAC virus expression vectors containing heterologous DNA segments encoding malarial gene products
HUT51332A (en) Process for expressing p.falciparum cirkumsporozoita protein by yeast
CZ337897A3 (cs) Modifikované polypeptidy vykazující zvýšenou imunogenicitu
EP0474891A1 (en) Vectors for expression of malarial antigens on the surface of Salmonella vaccine strains
US20060127413A1 (en) Recombinant mva strains as potential vaccines against p. falciparum malaria
US5393523A (en) Plasmodium falciparum vaccine comprising a recombinant histidine-rich protein-HRP-II
US5853739A (en) Transmission-blocking vaccine against malaria
Yang et al. Addition of the MSA1 signal and anchor sequences to the malaria merozoite surface antigen 1 C-terminal region enhances immunogenicity when expressed by recombinant vaccinia virus
NO871737L (no) Rekombinant virus.
EP0250261A1 (en) Vaccines containing epitopes of both the CSP and a blood stage antigen of a plasmodium parasite
JPH10298104A (ja) ワクチン
AU8150687A (en) Hybrid proteins or polypeptides
Barr et al. Recombinant Pfs25 Protein of Plasmoch'um

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU DK FI JP KR NO US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE

WWE Wipo information: entry into national phase

Ref document number: 1986905164

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 871885

Country of ref document: FI

WWP Wipo information: published in national office

Ref document number: 1986905164

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1986905164

Country of ref document: EP