WO1992000323A2 - HUMAN CYTOMEGALOVIRUS (HCMV) ANTIGEN pp150 - Google Patents

Abstract

This 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.

Description

HUMAN CYTOMEGALOVIRUS (HCMV) ANTIGEN ppl50

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 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.

Like other herpes viruses, HCMV has a propensity to persist in the body after initial infection, and may reactivate at a later date to give clinical disease. Although the site of latenc /persistence has not been clearly defined for HCMV, numerous studies have suggested 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. 15,, 402-9) and infection may be transmitted by infected blood products (Wilhelm J.A., et al (1986) J. Infec. Dis 154, 169-171). Serological screening of blood donors for HCMV allows establishment of a reserve of HCMV negative blood for 'at risk' patients such as transplant recipients. Serodiagnosis of HCMV is also of importance in the clinical virology laboratory particularly in monitoring and matching transplant recipients and donors and will assume a wider significance as new antiviral therapies become available.

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)). and 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)). Western blot analysis with human sera indicates that pp 150 is highly immunogenic, apparently more so than any other of the HCMV structural proteins. It has been disclosed by Jahn et al (Jahn, G., e_t al.. J.Gen.Virol. , 68. 1327-1337, (1987) that sera will react very strongly with the phosphorylated matrix protein, ppl50. Although previous studies did not discriminate between the antibody responses against pplϋO and the nucleocapsid protein of similar size, Jahn et al. demonstrated by reproducible separation of the two proteins in 'high-bis' gels that the stronger reactivity of all sera tested was directed against the phosphorylated matrix protein. The immune reaction against this polypeptide persists longer than those against other HCMV polypeptides after convalescence. (Landini, M.P. , et al Med.Virol, 12, 303-311, (1985)).

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)). Since 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. In particular 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. It will be appreciated that 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. Conveniently, 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. Advantageously 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. For a description of the construction of 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.

There is in a further aspect provided 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;

(ii) determining whether the said ppl50 has bound to any said antibodies.

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.

(i) contacting a solid phase to which are immobilised antibodies reactive with ppl50 expressed in insect cells with a test sample;

(ii) determining whether the said antibodies have bound to any said HCMV antigen.

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.

A 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.

A variety of assay formats may be employed. 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. In addition 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).

Alternatively 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. 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. For example, 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 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. Alternatively 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.

Those types of assay in which an antigen is used to label an already captured antibody require some form of labelling of the antigen which will allow it to be detected.

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. In particular 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.

For those assays in which the antigen is used to provide a measure of HCMV antigen in a sample, 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.

In general in homogeneous assays 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. 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. After washing, bound enzyme is detected using a specific substrate involving a colour change. A test kit for use in such an EIA comprises:

(1) ppl50, labelled with an enzyme;

(2) a substrate for the enzyme;

(3) means providing a surface on which pρl50 is immobilised; and

(4) optionally, washing solutions and/or buffers.

The following Examples Illustrate the present invention.

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 (A and B) 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.

Example 1: Construction of Transfer Vector pAc36C

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 Anglian 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:

GAATAATCCGGGATATTTA ^

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 pρl50 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 (50μg/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).

From published literature (Jahn, K. , supra) it is known that the ppl50 gene resides within the 8.5kb Bam HI K fragment of the CMV genome. With reference to the disclosure of Jahn, et al. , as shown in the nucleotide sequence, a Bam HI fragment contains the pρl50 fragment and this Bam HI fragment is contained within a Hind D3 fragment as described in the disclosure (map units 0.16-0.186). Therefore after electrophoresis of the Bam HI digested genomic fragments through 0.7% agarose, bands in the region of 8.5 kb were excised and subcloned into the vector pSP65 using standard techniques. Recombinant plasmids were analysed by restriction analysis to confirm the presence of the correct 8.5kb Bam HI K fragment.

Expression of entire pp!50 gene in baculovirus

Expression of 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.

5' oligo: AAG GAT CCC ATG AGT TTG CAG TTT A >

5' ppl50 3'

3' oligo: AA GGA TCC CTA TTC CTC CGT GTT CT >

5' ppl50 3'

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^OC,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.

EXAMPLE 3 : Generation of Recombinant baculovirus

To generate recombinant baculovirus lμg recombinant plasmid was cotransfected into insect cells together with 2μg 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.

Supernatants were then plated out in plaque assays and inclusion negative reco binants identified by usual screening using a Nikon microscope. Recombinant viruses were purified by three rounds of plaque purification and expression of recombinant antigen from

35 purified plaques analysed by SDS-PAGE, Western blot, S methionine and ELISA.

EXAMPLE 4 : Analysis of expression product

To analyse expression 2x10 insect cells in 3cm petri dishes were infected at an M01 of 3 with purified recombinant virus. After incubation for lh at 28 C to allow for virus adsorption the inoculum was aspirated and cells refed. At 24,48 and 72h cells were harvested into SDS-PAGE buffer, run on 10% polyacrylamide gels and either stained with Coomassie blue (Fig 2) or Western blotted onto nitrocellulose and probed with CMV positive human sera. Peak expression was seen on day 2 with the expected 150 kDa of recombinant protein accounting for approximately 25% of the total cellular protein (Fig.2). Lesser intensity recombinant protein bands were also seen at -HOkDa and 90kDa. Western blotting indicated that the 150 kDa product was highly immunoreactive with CMV positive human sera. The HOkDa and 90kDa bands were also immunoreactive. It is unclear whether these varying molecular weight species (plus the ~60kDa immunoreactive band) reflect degradation products of the full ppl50 species or variations in processing e.g. phosphorylation, glycosylation.

Additional cultures, infected as above, were labelled from 22-23hpi

35 with 20μCi/ml S methionine in methionine free TC100 and the products similarly analysed by SDS-PAGE. Four molecular weight species of 150,

110, 90 and 60 kDa were identified suggesting that if processing or proteolytic cleavage occurs it is an early event.

ELISA

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 (lOOμl/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).

Combined antigens in ELISA

Although immunoreactivity of ppl50 alone showed good concordance with whole virus lysate the effect of co-coating ppl50 with other cloned, baculovirus expressed antigens notably gB and IE-I, was investigated. In most instances no improvement was seen over and above ppl50 alone. However, with a small number of sera, enhanced reactivity was seen by co-coating ppl50 antigens with gB (See results in Table 1 below)

Claims

1. A process for producing ppl50 of HCMV, which comprises

(i) inserting DNA encoding ppl50 into a baculovirus transfer vector;

(ii) transforming insect cells with a baculovirus and the resulting transfer vector;

(iii) culturing transformants thus obtained; and

(iv) recovering ppl50 of HCMV.

2. A process according to claim 1 wherein the DNA sequence has more than 90% homology with the DNA sequence disclosed in Figure 5.

3. A process according to claim 3 wherein the transfer vector is pAc36C.

4. A process according to claim 1 wherein the baculovirus transfer vector is derived from Autoerapha californica nuclear polyhedrosis virus.

5. A process according to claim 1 wherein the insect cells are isolated from Spodoptera frugiperda.

6. A process according to claim 1 wherein ppl50 is recovered by lysis of infected cells in the presence of a detergent.

7. A method for 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 produced by the process according to claim 1 with a test sample; (ii) determining whether the said ppl50 has bound to any said antibodies.

8. A method for determining the presence of HCMV antigen in a human body fluid comprising:

(i) contacting a solid phase to which are immobilised antibodies reactive with ρpl50 produced by the process according to claim 1 with a test sample;

(ii) determining whether the said antibodies have bound to any said HCMV antigen.

9. A test kit suitable for use in determining the presence of HCMV antibodies, which kit comprises:

(i) ppl50 produced by the process according to claim 1 labelled with a revealing label;

(ii) means for detecting the revealing label; and

(iii) means providing a surface on which ppl50 produced by the process according to claim 1 is immobilised.

0 A test kit suitable for use in determining the presence of HCMV antigen, which kit comprises:

(i) HCMV antibodies reactive with ppl50 produced by the process according to claim 1;

(ii) HCMV antibodies reactive with ppl50 produced by the process according to claim 1 labelled with a revealing label;

(iii) means for detecting the revealing label; and (iv) means providing a surface on which HCMV antibodies reactive with ppl50 produced by the process according to claim 1 are immobilised.

11. A vaccine for conferring immunity to HCMV, which comprises ppl50 produced accorc^" -g to the process of claim 1 in association with a pharmaceutically acceptable carrier.

12. Use of ppl50 of HCMV produced by the process according to claim 1 in medicine.

13. Use of ppl50 of HCMV produced by the process according to claim 1 in the manufacture of a vaccine for conferring immunity to HCMV infection.

14. A method of conferring immunity to HCMV infection in man comprising administering an effective amount of a vaccine according to claim 11.