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WO1992000323A2 - pp150 DU CYTOMEGALOVIRUS HUMAIN - Google Patents

pp150 DU CYTOMEGALOVIRUS HUMAIN Download PDF

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Publication number
WO1992000323A2
WO1992000323A2 PCT/GB1991/001064 GB9101064W WO9200323A2 WO 1992000323 A2 WO1992000323 A2 WO 1992000323A2 GB 9101064 W GB9101064 W GB 9101064W WO 9200323 A2 WO9200323 A2 WO 9200323A2
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WIPO (PCT)
Prior art keywords
ppl50
hcmv
process according
produced
antigen
Prior art date
Application number
PCT/GB1991/001064
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English (en)
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WO1992000323A3 (fr
Inventor
Brian Colin Rodgers
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The Wellcome Foundation Limited
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Filing date
Publication date
Application filed by The Wellcome Foundation Limited filed Critical The Wellcome Foundation Limited
Publication of WO1992000323A2 publication Critical patent/WO1992000323A2/fr
Publication of WO1992000323A3 publication Critical patent/WO1992000323A3/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
    • 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
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a process for producing human cytomegalovirus (HCMV) protein in insect cells and to vaccines and immunoassays containing the protein obtained by the process.
  • HCMV human cytomegalovirus
  • HCMV belongs to the herpes virus group and is encountered by over 50% of the adult population as judged by serological reactivity. Infection in normal individuals is mild or inapparent, but in immunocompromised hosts such as transplant recipients and AIDS patients HCMV may cause serious clinical problems and significant mortality. HCMV infection is also a common problem during pregnancy giving rise to infections of the foetus and newborn with a spectrum of associated syndromes and developmental abnormalities.
  • HCMV has a propensity to persist in the body after initial infection, and may reactivate at a later date to give clinical disease.
  • lymphocytes or macrophages as probable reservoirs of virus (Einhorn, L. Ost. (1984) J. Infec. Dis. 149, 207-214; Rice G.P.A., et al (1984) Proc.Nat.Acad.Sci. 81, 6134-8; St. Jeor. S, et al (1977) Infect, and Immn.
  • HCMV is a double stranded DNA virus, approximately 240kB in length, and contains two prominent large proteins with apparent molecular weights of about 150kDa.
  • One of these is assumed to be the major nucleocapsid protein (Gibson, . , Virology, 128, 391-406, (1983)).
  • the other polypeptide is a basic phosphorylated matrix-tegument protein, which was designated as basic phospho protein or pp 150 and is distinguished from the nucleocapsid protein, (Gibson, W. , (1983) , supra.: Roby, C. , and Gibson, . , J.Virol., 5£, 715-727, (1986)).
  • ppl50 The DNA sequence encoding ppl50 has been mapped by screening a bacteriophage lambda gtll cDNA expression library with monospecific rabbit antisera (Jahn, G. et al. , J. of Virology, pl358-1367, May, (1987)). Several defined regions of ppl50 have been expressed in E.coli as 3-galactosidase fusion proteins and have been tested for their immunoreactivity with human sera (Scholl, B-C, et al.. J.Gen.Virol. , 69, 1195-1204, (1988)).
  • ppl50 is outstanding among all virion constituents in eliciting a humoral immune response and is remarkably immunogenic, is the ideal candidate for use in the development of new diagnostic reagents. However the entire ppl50 has never before been successfully expressed in a substantially undegraded form in an expression system.
  • the present invention provides a process for producing ppl50 of HCMV in a substantially undegraded form, which comprises expressing DNA encoding ppl50 in insect cells.
  • the DNA sequence is that sequence disclosed by G. Jahn, et al (supra.. see Example 2 and Figure 5) or has at least 90%, preferably 95%, more preferably 98% homology with that sequence.
  • the DNA sequence may correspond to the naturally-occurring sequence, or it may be related to that sequence by mutation, including single or multiple base substitutions, deletions, insertions and inversions, provided that the resulting DNA enclodes a protein which has the equivalent biological and immunological activity of ppl50 of HCMV.
  • the DNA encoding ppl50 is inserted into the insect cells using a baculovirus transfer vector, in particular a vector derived from the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) .
  • a recombinant transfer vector normally contains the polyhedrin promoter which is used to drive expression of the foreign DNA.
  • the vector incorporates a restriction site into which the foreign DNA is inserted a short distance downstream of the N-terminus of the polyhedrin gene product.
  • the natural ATG translation start codon of the polyhedrin gene is mutated such that the polyhedrin sequence prior to the restriction site is transcribed but not translated.
  • a preferred transfer vector is pAc36C, which may be constructed from the transfer vector pAc360 (Summers and Smith, 1987, "A Manual of methods for Baculovirus vectors and Insect cell culture procedure") by site directed mutagenesis.
  • pAc36C see EPA-88307970.9 (Publication No. 0 340 359).
  • the transfer vector containing the DNA encoding ppl50 and a baculovirus are normally used to co-transfect insect cells susceptible to baculovirus infection. Homologous recombination occurs within the cells resulting in recombinant insect cells containing the foreign DNA. The recombinant insect cells may then be cultured to express ppl50.
  • ppl50 may be isolated and purified using conventional techniques and procedures available in the art. One procedure is to extract ppl50 by lysis of Infected cells in a 0.25 NP40. ppl50 can also be analysed by methods well-known in the art, such as SDS-PAGE, Western Blotting and ELISA.
  • the invention further provides a vaccine containing ppl50 made by a process according to the present invention in association with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers in this instance are liquid media suitable for use as vehicles to introduce ppl50 into the patient.
  • An example of such a carrier is saline solution.
  • ppl50 may be In solution or suspended as a solid in the carrier, or it may be solubilised by the addition of pharmaceutically acceptable detergent.
  • the vaccine may also comprise an adjuvant for stimulating the immune response and thereby enhancing the effect of the vaccine.
  • a convenient adjuvant for use in the present invention is aluminium hydroxide.
  • a vaccine may also comprise ppl50 and other CMV antigens such as gB.
  • a method for inducing immunity to HCMV in susceptible vertebrate hosts comprising the administration of an effective amount of a vaccine, as herebefore defined, to the host.
  • the vaccines may be administered by any conventional method for the administration of vaccines including oral and parenteral (eg. subcutaneous or intramuscular) injection.
  • the treatment may consist of a single dose of vaccine or a plurality of doses over a period of time.
  • the present invention further relates to a method of determining the presence of antibodies to HCMV in a human body fluid comprising: (i) contacting a solid phase to which is immobilised ppl50 of HCMV which is expressed in insect cells with a test sample;
  • An immunoassay for carrying out such a detection method may comprise ppl50 produced by the process described herein for contacting with the bodily sample and means for detecting HCMV-specific antibodies that bind to ppl50.
  • the present invention also relates to a method for determining the presence of HCMV antigen in a human body fluid comprising.
  • An immunoassay for carrying out such a detection method may comprise HCMV antibodies raised against ppl50 produced by the process described herein and means for detecting HCMV antigens that bind to the antibodies.
  • test sample of any appropriate physiological fluid may be used in the assay, for example urine, plasma, blood, serum, semen, tears, saliva or cerebrospinal fluid.
  • the antigen can be used to capture selectively antibody against HCMV from solution, to label selectively such antibody already captured, or to both capture and label.
  • the antigen may be used in a variety of homogeneous assay formats in which the antibodies which react with the antigen are detected in solution with no separation of phases.
  • the antigen can also be used for HCMV antigen detection.
  • the type of assay in which the antigen is used to capture antibodies from solution involve immobilisation of the antigen onto a solid surface.
  • This surface should be capable of being washed in some way.
  • the sort of surfaces which may be used are polymers of various types (moulder into microtitre wells; beads; dipsticks of various types; aspiration tips; electrodes; and optical devices), particles (for example latex; stabilised blood, bacterial or fungal cells; spores; gold or other metallic sols; and proteinaceous colloids; with the usual size of the particle being from 0.1 to 5 microns), membranes (for example nitrocellulose; paper; cellulose acetate; and high porosity/high surface area membranes of an organic or inorganic material) .
  • the attachment of the antigen to the surface can be by passive adsorption from a solution of optimum composition which may include surfactants, solvents, salts, chaotropes; or by active chemical bonding.
  • Active bonding may be through a variety of reactive or activatible functional groups which may be attached to the surface (for example condensing agents; active esters, halides, anhydrides; amino, hydroxyl, or carboxyl groups; sulphydryl groups; carbonyl groups; diazo groups; unsaturated groups).
  • the active bonding may be through a polypeptide (itself attached to the surface passively or through active bonding) or through a carrier protein such as albumin or casein, to which the antigen may be chemically bonded by any of a variety of methods and which may confer advantages because of isoelectric point, charge, hydrophilicity or other physico-chemical property.
  • the antigen may also be attached to the surface (usually but not necessarily a membrane) following electrophoretic separation of a reaction mixture e.g. an immune precipitation.
  • the captured antibody After contacting (reacting) the surface bearing the antigen with a test sample and removing the excess of the sample where necessary by any of a variety of means (washing, centrifugation, filtration, magnetism, capilliary action) , the captured antibody is detected by a revealing label, any means which will give a detectable signal.
  • this may be achieved by use of a labelled molecule or particle as defined above which will react with the captured antibody (for example protein A or protein G and the like; anti-species or anti-immunoglobulin-sub-type; rheumatoid factor; antibody to the antigen used in a competitive or blocking fashion; or any molecule containing an epitope of the antigen including the antigen itself and other proteins and peptides derived directly or indirectly from HCMV)
  • the captured antibody for example protein A or protein G and the like; anti-species or anti-immunoglobulin-sub-type; rheumatoid factor; antibody to the antigen used in a competitive or blocking fashion; or any molecule containing an epitope of the antigen including the antigen itself and other proteins and peptides derived directly or indirectly from HCMV
  • the detectable signal may be optical or radio-active or physico- chemical, provided by directly labelling the molecule referred to with for example a dye, radiolabel, electroactive species, magnetically resonant species or fluorophore; or indirectly by labelling the molecule or particle with an enzyme itself capable of giving rise to a measurable change of any sort.
  • the detectable signal may be due to, for example, agglutination, diffraction effect or birefringent effect occurring if any of the surfaces referred to are particles.
  • the labelling can be direct, by chemically or passively attaching for example a radio-, magnetic resonant-, particle or enzyme label to the antigen; or indirect by attaching any form of label to a molecule which will itself react with the antigen e.g. antibody to the antigen; with subsequent reaction of the labelled molecule with the antigen.
  • the chemistry of bonding a label can be directly through a moiety already present in the antigen such as an amino group or through an inserted group such as a maleimide.
  • Capture of the antibody may be on any of the surfaces already mentioned, by any reagent, including passive or activated adsorption, which will result in specific antibody or immune complexes being bound.
  • capture of the antibody could be by anti-species or anti-immunoglobulin-sub-type, by rheumatoid factor, proteins A, G and the like, or by any molecule containing the epitope making up the antigen as described above.
  • the antigen may be labelled in any of the ways described above, and used in either a competitive binding fashion so that its binding by any specific molecule on any of the surfaces exemplified above is blocked by antigen in the sample, or in a non-competitive fashion when antigen in the sample is bound specifically or non-specifically to any of the surfaces above, in turn binds a specific bi- or poly-valent molecule (e.g. an antibody) and the remaining valencies of the molecule are used to capture the labelled antigen.
  • a specific bi- or poly-valent molecule e.g. an antibody
  • the antigen and an antibody are labelled, so that, when the antibody reacts with the antigen in free solution, the two labels interact, for example to allow non-radiative transfer of energy captured by one label to the other label, with appropriate detection of the excited second label or quenched first label (e.g. by fluorimetry, magnetic resonance or enzyme measurement).
  • Addition of either antigen or antibody in a sample results in restriction of the interaction of the labelled pair, and so to a different level of signal in the detector.
  • a suitable assay format for detecting HCMV antibody is the direct sandwich enzyme immunoassay (EIA) format.
  • EIA direct sandwich enzyme immunoassay
  • ppl50 is coated onto microtitre wells.
  • a test sample and ppl50 to which an enzyme is coupled (conjugated protein) are added simultaneously. Any specific antibody binds both to the ppl50 coating the well and to the conjugated antigen. Typically the same antigen is used on both sides of the sandwich.
  • a test kit for use in such an EIA comprises:
  • washing solutions and/or buffers optionally, washing solutions and/or buffers.
  • Figure 1 shows the Bam HI ppl50 digested fragment cloned into the baculovirus vector pAc36C.
  • Figure 2 shows the characterisation of baculovirus ppl50 recombinants using polyacrylamide gel analysis and SDS-PAGE and Coomassie blue staining.
  • Figure 3 shows the results of CMV sera assayed in a standard anti-human ELISA format using ppl50 and whole viruse lysate.
  • Figure 4 shows the results of CMV sera assayed in an anti-human ELISA format using ppl50 and other cloned baculovirus expressed antigens.
  • Figure 5 shows the nucleotide sequence of the 6,360 - base pair DNA segment of the HCMV gene. Vertical arrows mark the 5' and 3' termini of the ppl50 sequence.
  • the transfer vector pAc36C was derived from pAc360 (Summers and Smith, 1987, "A Manual of methods for baculovirus vectors and insect cell culture procedures") by site directed mutatgenesis using kits obtained from Yalen Biotech and Amersham International.
  • An 852 bp cDNA for human gamma interferon was subcloned as a BamHI fragment into the BamHI site of pAc360. From this a lkb Dral fragment which extends from about 700 bp upstream of the polyhedrin ATG translation codon to 300 bp inside the 5' end of the gamma interferon cDNA insert was subcloned into the Smal site of M13K19 (Anglian Biotech) .
  • the RF form of the construct was used to confirm the litigation and single strand template was derived and mutated with the 19 mer:
  • the underlined G had the effect of converting the ATG of the polyhedrin translation codon to ATC.
  • the mutation was confirmed by DNA sequencing and a 136 bp EcoRV-BamHI fragment encompassing the mutation was used to replace the same fragment in pAc360 with the mutagenised fragment to derive pAc36C.
  • EXAMPLE 2 Cloning of PD150 and construction of recombinant transfer vector derived from pAc36C.
  • CMV p ⁇ l50 was cloned from a genomic library of CMV strain AD169 (A.T.C.C.).
  • Virus was grown in MRC-5 diploid human fibroblasts and supernatant fluid collected from 4-7 d.p.i.
  • Virus was pelleted at 70000g for lh, the virus resuspended in PBS, and enveloped and naked capsids separated by centrifugation through a glycerol-tartrate gradient.
  • Viral DNA was extracted by proteinase K digestion (50 ⁇ g/ml for 2h at 55 C) followed by two extractions with phenol/chloroform. DNA was ethanol precipitated and digested with Bam HI (70u for 3h at 37°C).
  • ppl50 was achieved by PCR amplification using oligonucleotides homologous to the 5' and 3' termini of the ppl50 gene (Jahn, et al. , supra) and including Bam HI restriction sites to facilitate cloning into the baculovirus vector pAc36C.
  • 3' oligo AA GGA TCC CTA TTC CTC CGT GTT CT >
  • the entire ppl50 gene was amplified from 50ng target Bam HI K/PSP65 plasmid and 200 ng of each of the above oligonucleotides using 30 cycles of a replication (1.5'x94°C,2'x55 O C,9'x70°C) .
  • the - 3.1kb product was resolved on a 1% agarose gel, and the band excised, extracted twice with phenol/chloroform and Bam HI digested.
  • the Bam HI digested fragment was then cloned into the baculovirus vector pAc36C (see Fig.l) by standard ligation/transformation procedures.
  • Recombinant plasmids were analysed by restriction digests to orientate the ppl50 insert and stocks of DNA prepared by centriguation through CsCl gradients.
  • baculovirus l ⁇ g recombinant plasmid was cotransfected into insect cells together with 2 ⁇ g wild type AcNPV using calcium phosphate coprecipitation (transfection buffer: 140mM NaCl, 25mM Hepes pH7.5, 120mM CaCl.) . After 4h at 28°C monolayers were washed with TC100, refed and incubated for 3 days at 28 C.
  • Recombinant ppl50 was extracted by lysis of infected cells in 0.25 NP40 and coated onto microtitre plates by overnight incubation in 50mM bicarbonate buffer pH9.5.
  • a panel of 220 human sera of known CMV status was assayed in a standard anti-human ELISA format. Sera diluted 1:10 were incubated (lOO ⁇ l/well) at 37 C for 30' , plates washed 3 x in Tween-saline, and further incubated with peroxidase conjugated anti-human Ig. After further washing in Tween-saline immunoreactivity was visualised by the addition of TMB. Concordance of ppl50 reactivity with whole virus lysate reactivity was good (see Fig 3).

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Abstract

Y@invention se rapporte à un procédé destiné à produire une protéine de cytomégalovirus humain (HCMV) dans des cellules d'insectes et à des vaccins et des analyses immunologiques contenant la protéine obtenue au moyen de ce procédé.
PCT/GB1991/001064 1990-07-02 1991-07-01 pp150 DU CYTOMEGALOVIRUS HUMAIN WO1992000323A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB909014652A GB9014652D0 (en) 1990-07-02 1990-07-02 Antigen
GB9014652.3 1990-07-02

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WO1992000323A2 true WO1992000323A2 (fr) 1992-01-09
WO1992000323A3 WO1992000323A3 (fr) 1992-02-06

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GB (1) GB9014652D0 (fr)
IE (1) IE912291A1 (fr)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998002746A1 (fr) * 1996-07-12 1998-01-22 Akzo Nobel N.V. Reactif de peptides permettant la detection de cytomegalovirus (cmv) humain
WO1998007033A1 (fr) * 1996-08-16 1998-02-19 Abbott Laboratories Western blot test ameliore servant a l'identification d'anticorps specifiques du virus cmvh
US6448389B1 (en) 1996-04-23 2002-09-10 The Wistar Institute Of Anatomy And Biology Human cytomegalovirus DNA constructs and uses therefor

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3782867D1 (en) * 1986-06-12 1993-01-14 Behringwerke Ag, 3550 Marburg, De Human cytomegalovirus phospho-protein
GB8810808D0 (en) * 1988-05-06 1988-06-08 Wellcome Found Vectors

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6448389B1 (en) 1996-04-23 2002-09-10 The Wistar Institute Of Anatomy And Biology Human cytomegalovirus DNA constructs and uses therefor
WO1998002746A1 (fr) * 1996-07-12 1998-01-22 Akzo Nobel N.V. Reactif de peptides permettant la detection de cytomegalovirus (cmv) humain
US6936251B2 (en) 1996-07-12 2005-08-30 Biomerieux, B.V. Peptide reagent for the detection of human cytomegalovirus (CMV)
WO1998007033A1 (fr) * 1996-08-16 1998-02-19 Abbott Laboratories Western blot test ameliore servant a l'identification d'anticorps specifiques du virus cmvh
JP2001501457A (ja) * 1996-08-16 2001-02-06 アボツト・ラボラトリーズ Ｈｃｍｖウイルスに対して特異的な抗体を同定するための改良されたウェスタンブロット試験
US6287760B1 (en) 1996-08-16 2001-09-11 Abbott Laboratories Western blot test for the identification of antibodies specific against the HCMV virus

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AU8204991A (en) 1992-01-23
WO1992000323A3 (fr) 1992-02-06
IE912291A1 (en) 1992-01-15

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