EP0770131A1 - Immunodominante polypeptiden - Google Patents

Immunodominante polypeptiden

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Publication number
EP0770131A1
EP0770131A1 EP95923485A EP95923485A EP0770131A1 EP 0770131 A1 EP0770131 A1 EP 0770131A1 EP 95923485 A EP95923485 A EP 95923485A EP 95923485 A EP95923485 A EP 95923485A EP 0770131 A1 EP0770131 A1 EP 0770131A1
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EP
European Patent Office
Prior art keywords
seq
poiypeptide
amino acid
polypeptides
protein
Prior art date
Legal status (The legal status 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 status listed.)
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Application number
EP95923485A
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English (en)
French (fr)
Inventor
Guy Timothy British Biotech LAYTON
Mercedes British GARCIA-VALCARCEL MUNOZ-REPISO
Wendy Jane Fowler
David Richard The Medical College HARPER
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Vernalis R&D Ltd
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British Biotech Pharmaceuticals Ltd
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Publication of EP0770131A1 publication Critical patent/EP0770131A1/de
Withdrawn legal-status Critical Current

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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
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to the identification and utilisation of isolated polypeptides containing the important immunodominant epitopes on the VZV gE glycoprotein for vaccine design and diagnosis purposes.
  • Varicella Zoster virus is the highly infectious etiologic agent of two distinct clinical syndromes, chickenpox (varicella), resulting from primary infection, and zoster (shingles) following reactivation of latent virus. Following initial infection, the virus replicates in the respiratory epithelium and then produces a primary viraemia in the first week following infection. Secondary viraemia follows as a result of infection of the liver, spleen and other organs. A vesicular rash appears by days 14-17 in almost all cases. In temperate countries primary infection usually occurs in children and is commonly a mild disease.
  • VZV Reactivation of VZV involves spread within the ganglia followed by migration down the neurone, causing a characteristic rash across the area of skin enervated from that ganglion (the dermatome). Zoster typically occurs in later life, and unlike herpes simplex virus, a single episode is common.
  • Reactivation of VZV is strongly associated with immunosuppression, which can arise from a number of causes, including the decrease of effective cell-mediated immunity with ageing (Miller (1980) Neurology 30:582-587; and Berger et al . (1981) Immunology 32:24-27), immunosuppressive drugs or treatments (Patel et al. (1979) Journal of Paediatrics. 94:223-230; Meyers et al. (1980) Journal of Infectious Diseases 141 :479-487; Arvin et al. (1980) Journal of Clinical Investigations 65:869-878; and Arvin et al.
  • shingles is a common occurrence appearing 1 -2 years after initiation of chemotherapy/irradiation treatment.
  • chemotherapy/irradiation treatment between 5-35% will reactivate VZV to develop herpes zoster.
  • VZV congenital varicella syndrome
  • Post-herpetic neuralgia is unresponsive to antiviral therapy and therefore is likely to be independent of continued viral replication.
  • Varicella vaccine there is currently no Varicella vaccine available for the mass market.
  • a live attenuated strain (Oka/ Biken) has been licensed in Japan since 1987 (Takehashi et al. (1974) Lancet 2:1288-1290).
  • a live varicella vaccine (VARIVAX/VARILRIX) derived from the same master seed of Oka strain virus has been developed by Merck, Sharp and Dohme Research Laboratories and SmithKline Beecham and evaluated in clinical trials since 1987.
  • the vaccine provides good protection against chickenpox although for effective immunisation of immunocompromised children, the vaccine must be given in the remission stage and anti-cancer chemotherapy and steroid treatments must be suspended 2 weeks before and after vaccination (Kangro (1990) Review of Medical Microbiology1 :205-212).
  • the vaccine is not effective in children under six months of age.
  • the Oka vaccine was derived from a Japanese isolate of VZV by passage at reduced temperature and in caviid cells.
  • the molecular basis of its attenuation is not known (described in Kangro (1990) Review of Medical Microbiology 1 :205-212).
  • Acyclovir (Zovirax) has been used as an effective chemotherapy treatment to halt the progression of VZV disease and to shorten the time of healing. Treatment within 24 hours of rash onset results in fewer lesions and shorter duration of fever. However, in trials conducted in the USA in 1990 and 1991 , children treated with acyclovir did not return to school any more rapidly than those who received the placebo (Gershon, A et al. (1992) Journal of Infectious Diseases. 166 suppl 1 :S63-8. ). Famciclovir (Famvir), a related drug, has recently been licensed for use against shingles. The question of who should receive the drugs is controversial as both are relatively expensive, $20 for a five-day course in a 10-kg child, and of limited clinical benefit. Several novel anti-VZV drugs are in development or in clinical trials, but are not yet licensed.
  • VZV is highly infectious and worldwide estimates of infection are 57 million cases in children and 3 million in adults per annum, with 5 million cases of shingles.
  • An effective childhood vaccination program could substantially reduce the annual incidence of chickenpox.
  • the current live attentuated vaccine is unsuitable for such a program due to concerns over batch variability, re-infection, re-activation and long-term efficacy.
  • the VZV genome encodes six glycoproteins which have recently been assigned new designations to correspond to their herpes simplex virus (HSV) homologues.
  • HSV herpes simplex virus
  • the previous nomenclature named the glycoproteins as gpl-VI whereas the corresponding nomenclature based on the HSV homologues designates the glycoproteins as, respectively, gE, gB, gH, gi, gC and gL.
  • VZV homologue of HSV gD There is no VZV homologue of HSV gD.
  • the VZV glycoproteins are highly immunogenic, in particular gE, gB and gH.
  • gE is complexed with gl
  • gH is complexed with gL.
  • the gE/gl complex forms a weak Fc receptor, while gH appears to be associated with cell-to-cell spread.
  • the major immunoreactive proteins in Westem blot analysis of sera from patients with varicella and zoster are the glycoproteins, gE and gB, and the assembly proteins. Although gH is highly immunogenic, it is denaturation sensitive and poorly detected by Western blotting. Up to thirty virion proteins can be detected by Westem blotting or radioimmune precipitation (Harper et al. (1988) J.Med Virol. 25:387-398). There is now good evidence to show that envelope proteins are immunogenic, eliciting neutralising antibodies and both helper and cytotoxic T- cell responses (Arvin et al. (1991) J.lmmunol. 146:257-264; Vafai (1993) Vaccine. 11 :937-940).
  • the gE protein sequence contains at least three distinct immunogenic domains which have not been mapped.
  • the first contains two complement-dependent neutralising epitopes.
  • the second contains five complement-dependent, overlapping epitopes and one non-neutralising epitope.
  • the third contains a complement-enhanced epitope (Forghani, B. Dupuis, KW and Schmidt (1990) Journal of Clinical Microbiology. 28(ll):2500-2506).
  • Hayward et al. (1989, Viral Immunology 2:175-184) isolated blood mononuclear cells from VZV-infected individuals and stimulated with VZV in the form of live cell- associated virus. Some of the CD4+ lines isolated with specificity for VZV also proliferated in culture with VZV gE. Additionally, most of the VZV-specific CD4+ T cell lines provided antigen-specific help to B cells for IgG antibody production. This data indicates that T-helper cell epitopes are present in gE. A CD8+-mediated cytotoxic function was demonstrated against autologous VZV-infected lymphoblastoid cell targets by depletion of the CD4+ T lymphocytes.
  • gE induces neutralising antibodies, T-helper cell responses and also contains epitopes recognised by CD4+ and CD8+ cytotoxic lymphocytes from VZV-infected individuals.
  • recombinant vaccinia viruses expressing gE or gl are capable of inducing VZV-neutralising antibodies and the secreted N-terminus protein fragment (511 amino acids) of the gE protein was capable of eliciting complement-dependent VZV neutralising antibodies in rabbits (Vafai (1993), Vaccine. 11:937-940).
  • Sub-unit vaccines are therefore in theory, generally accepted as potential alternatives to that of the live attenuated Oka virus for use in vaccine treatment, particularly in immunosuppressed patients, especially children.
  • Sub-unit vaccines could also be of use in boosting the anti-VZV immune responses in the elderly, who are more susceptible to reactivation of the latent virus.
  • the identification of a poiypeptide of VZVgE containing a neutralising epitope identifiable with sera raised in a mammal other than human, and wherein human sera do not recognise said epitope, may be useful in sub-unit vaccine design for administration to children under the age of 6 months since their maternal antibodies will not recognise the epitope, however, antibodies raised by the child against this epitope could confer protection from viral infection.
  • an isolated poiypeptide sub-unit of the VZVgE glycoprotein consisting of amino acids 1 - 161 (SEQ ID NO:1), or a functional variant thereof.
  • This poiypeptide contains the major VZVgE immunodominant epitopes.
  • a poiypeptide is considered to comprise 4 or more linked amino acids.
  • Immunodominant as used throughout this specification is defined as the recognition by antibodies, either neutralising monoclonal antibodies or sera from humans infected with VZV, when tested against either fragments of VZVgE or short linear peptides. "Immunodominant” therefore refers to the most antigenic components of VZVgE.
  • variants as used herein in relation to an isolated poiypeptide of the invention, is meant an amino acid sequence which is substantially homologous to that of the poiypeptide.
  • a variant also includes sequences which are not subsequences of wild-type VZVgE but which include a poiypeptide of the invention.
  • a variant of an isolated VZVgE poiypeptide of the invention which is substantially homologous to that poiypeptide may retain at least 66%, and preferably at least 70%, 80%, 90% or 95% homology with that poiypeptide.
  • nucleic acid encoding such variant poiypeptide molecule may hybridise with nucleic acid coding for the natural poiypeptide (or would do so but for the degeneracy of the genetic code), for example under stringent conditions (such as at 35 ⁇ >C to 65°C in a salt solution of approximately 0.9M).
  • functional variant is meant a variant whose altered amino acid sequence does not substantially diminish the antigenic or immunogenic properties of the poiypeptide. Functional variants may also tolerate conservative amino acid substitutions within the epitope region.
  • conservative amino acid substitutions is meant the localised replacement of individual amino acids possessing a certain charge with another amino acid possessing a similar charge
  • conservative changes are inter alia : alanine to glycine, valine, leucine or isoleucine; tyrosine to phenylalanine or tryptophan; and lysine to arginine or histidine.
  • an isolated poiypeptide sub-unit of the VZVgE glycoprotein consisting of amino acids 1 - 134 (SEQ ID NO: 2), or a functional variant thereof.
  • an isolated poiypeptide of the VZVgE glycoprotein consisting of amino acids 101 - 161 (SEQ ID NO: 3), or a functional variant thereof.
  • polypeptides having amino acid sequences present in SEQ ID NO: 1, which contain the antigenic and/or immunogenic epitopes present in the N-terminus of the VZVgE glycoprotein, said polypeptides having the following amino acid sequences:
  • amino acid polypeptides having amino acid sequences present within the polypeptides of SEQ ID NOs: 5-15 supra, which contain the important antigenic and/or immunogenic epitopes within the N-terminus of the VZVgE glycoprotein, possessing the following amino acid sequences:
  • YEPYYHSDHA (SEQ ID NO:16) ⁇ ) ESSWVNRGES (SEQ ID NO: 17) iii) SRKAYDHNSP (SEQ ID NO: 18) iv) YIWPRNDYDG (SEQ ID NO:19) v) HEHHGVYNQG (SEQ ID NO:20) vi) RGIDSGERLM (SEQ ID NO:21) vii) LGDDTGIHVI (SEQ ID NO:22) viii) RQYGDVFKGD (SEQ ID NO:23)
  • B-cell epitopes are mostly between 4 and 6 amino acids in length
  • functional variants of the above 10-mers may possess up to six amino acid differences, either amino acid substitutions or deletions.
  • amino acid polypeptides having amino acid sequences present within the polypeptides of SEQ ID NOs: 5 - 15 supra, which contain the important antigenic epitopes within the N-terminus of the VZVgE glycoprotein, possessing the following amino acid sequences:
  • B-cell epitopes are mostly between 4 and 6 amino acids in length
  • functional variants of the above 6-mers may possess up to two amino acid differences, either amino acid substitutions or deletions.
  • polypeptides of this invention can be synthesised chemically. For example, by the Merryfield technique (Journal of American Chemical Society 85:2149-2154, 1968).
  • the immunodominant polypeptides of the invention can be produced from a DNA sequence using recombinant DNA technology.
  • the DNA can be synthesised chemically, or isolated by one of several approaches known to the artisan, for example using Polymerase Chain Reaction (PCR) or by cloning from a genomic library.
  • PCR Polymerase Chain Reaction
  • nucleic acid sequence encoding the desired immunodominant poiypeptide once isolated or synthesised, can be cloned into any suitable expression vector using convenient restriction sites.
  • Expression vectors usually include an origin of replication, a promoter, a translation initiation site, optionally a signal peptide, a polyadenylation site, and a transcription termination site. These vectors also usually contain an antibiotic marker gene for selection. Suitable expression vectors may be plasmids, cosmids, viruses including retroviruses.
  • the coding sequence for the poiypeptide is placed under the control of an appropriate promoter, control elements and a transcriptional terminator so that the DNA sequence encoding the poiypeptide is transcribed into RNA in the host cell transformed by the expression vector construct.
  • the coding sequence may or may not contain a signal peptide or leader sequence for secretion of the poiypeptide out of the host cell.
  • nucleic acid may be injected directly into animals to induce an immune response and that such nucleic acid vaccines can be protective (Tang et al. (1992) Nature. 356:152; Ulmer et al. (1993) Science. 259:1745; WO 90/11092 (Vical); herein incorporated by reference).
  • DNA encoding the polypeptides of the invention may require incorporation into an expression construct or vector so that the poiypeptide is expressed from a mammalian promoter.
  • Ribonucleotides encoding the polypeptides of the invention can also be used in RNA-based vaccines (Martinon et al. (1993) Eur. J. Immunol.
  • VZVgE epitopes in vivo after immunisation.
  • mRNA encoding the desired poiypeptide may require encapsulation within, for example, liposomes.
  • the nucleic acid sequence encoding a desired immunodominant poiypeptide of VZVgE can therefore also be formulated for therapeutic use by direct nucleic acid injection into the patient.
  • nucleic acid encoding a desired immunodominant poiypeptide of the invention, in the preparation of a pharmaceutical composition for inducing or stimulating an immune response against VZV infection.
  • compositions for inducing or stimulating an immune response against VZV infection which composition comprises nucleic acid encoding a desired immunodominant poiypeptide of the invention, and one or more pharmaceutically acceptable carriers.
  • Another aspect of the invention provides for the use of the polypeptides of the invention for the preparation of a pharmaceutical composition for inducing or stimulating an immune response against VZV infection.
  • the immune response stimulated may be antibody responses or T-cell responses.
  • These T-cell responses may be T-helper (Th) or cytotoxic T-lymphocyte (CTL) responses.
  • Th T-helper
  • CTL cytotoxic T-lymphocyte
  • compositions for inducing or stimulating an immune response against VZV infection which composition comprises a poiypeptide of the invention, and one or more pharmaceutically acceptable carriers.
  • polypeptides containing the VZVgE epitopes identified herein can be used to stimulate an immune response in vivo.
  • Either the polypeptides, or longer sequences containing them, can be used alone, or in conjunction with a suitable adjuvant.
  • lipid tails can be added to the polypeptides, a means that has been shown to enhance the induction of CTL responses in vivo (Deres et al. (1989) Nature. 342:561 -563).
  • polypeptides of this invention containing these important epitopes, is of great importance in making available rational sub-unit vaccines and represent an improvement over the sub-units presently available for vaccine compositions.
  • inclusion of several of the polypeptides (and epitopes) or multiple copies of polypeptides of this invention within an antigen presenting vehicle results in increased antibody titers, and the inclusion of several different polypeptides enables an enhanced and broader immune response to be raised.
  • the polypeptides are likely to be present in longer sequences.
  • non homologous amino acids flanking the poiypeptide of the invention may be present. These may serve to enhance poiypeptide presentation to B-cells or antigen presenting cells, for example by presentation of the poiypeptide in a loop structure. Loop structures may be generated by incorporation of cysteine residues in the flanking sequences.
  • the non homologous sequences may comprise T cell epitopes which enhance antibody responses to the polypeptides of the invention, for example the promiscuous tetanus toxin and mycobacterial tuberculosis T-helper epitopes.
  • sub-units of the poiypeptide consisting of amino acids 1 - 161 (SEQ ID NO:1) which include any or all of the 6-mer (SEQ ID NOs:24 - 28), 10-mer (SEQ ID NOs:16 - 23), 20-mer (SEQ ID NOs:5 - 14) or 21-mer (SEQ ID NO:15) polypeptides of the invention, or variants of these polypeptides.
  • amino acid sequences which are not as a whole a sub-sequence of wild-type VZVgE glycoprotein but which includes as a sub-unit a poiypeptide of the invention.
  • one or more polypeptides or multiple copies of the same poiypeptide of the invention is fused at the N- or C-terminus or internally within a carrier protein.
  • Ty p1 means full length or self-assembling truncated variants of the self-assembling protein derived from the yeast retrotransposon TYA gene.
  • a suitable carrier protein is the self-assembling Ty virus-like particle antigen presentation system (WO-A-8803562 and WO-A-8803563, herein incorporated by reference).
  • Polypeptides of the invention containing the important VZV antigenic and/or immunogenic epitopes could be introduced, either individually or in combination, into the virus-like particles by fusing the polypeptides at the Ty p1 C- terminus, or at internal positions of the Ty p1 self-assembling protein (WO 94/14969, herein incorporated by reference). This permits multiple copies of the antigenic and/or immunogenic VZVgE epitopes to be incorporated into each particle.
  • Virus ⁇ like particles from other viral proteins including those from hepatitis-B virus (refer to EP-A-0175261), human papilloma virus or bluetongue virus, may also be used as carriers
  • virus-derived particles based on retroviral GAG proteins such as those of WO-A-8803562 and WO-A-8803563 can be used to present the immunogenic polypeptides to the immune system.
  • the large fragments (SEQ ID NOs: 1 - 4) or the important small molecular weight antigenic polypeptides (SEQ ID NOs: 5 - 28) can be incorporated into liposomes, ISCOMS, cochleates or live vaccine vectors; for example bacterial or viral vectors.
  • any suitable antigen presentation system known to those skilled in the art could be used (reviewed in Vaccine Design: The subunit and adjuvant approach: Eds M.F. Powell and M.J. Newman, Plenum Press, New York, 1995), and the small size of the polypeptides means that numerous copies of a single epitope, or of multiple epitopes can be incorporated into the same carrier molecule or vector.
  • a particulate antigen presentation composition purified following the expression, self-assembly and particle formation in a suitable host such as Saccharomyces cerevisiae or Escherichia coli of a hybrid protein comprising a poiypeptide of the invention fused to the C-terminus of the self-assembling Ty p1 protein (from WO-A-8803563).
  • a purified particulate antigen presentation composition comprising a poiypeptide of the invention fused to the C- terminus of the self-assembling Ty p1 protein (from WO-A-8803563) and also comprising one or more polypeptides of the invention inserted at internal positions within the Ty p1 protein (according to WO 94/14969).
  • An embodiment of the invention provides for a purified particulate antigen presentation composition
  • a purified particulate antigen presentation composition comprising a poiypeptide of (SEQ ID NO:2) or (SEQ ID NO:3), fused to the C-terminus of the self-assembling Ty p1 protein (from WO-A- 8803563) and comprising one or more polypeptides of (SEQ ID NOs:5-28) inserted at internal positions within the Ty p1 protein (see WO 94/14969).
  • the preferred Ty p1 intemal position is the C2 position (as disclosed in WO 94/14969) situated at amino acids 132 - 133 of the Ty p1 protein.
  • the Ty particle forming protein sequence (p1) is disclosed in Dobson et al., (1994) EMBO J. 3:1115 (herein incorporated by reference).
  • a preferred Ty-VLP construct comprises the insertion of the neutralising epitope containing poiypeptide of SEQ ID NO: 15 at the C2 position of the Ty p1 protein (according to WO 94/14969, herein incorporated by reference) and the poiypeptide of SEQ ID NO:2 fused at the C-terminus of the Ty p1 protein.
  • Another preferred Ty-VLP construct comprises the insertion of the neutralising epitope containing poiypeptide of SEQ ID NO:9 at the C2 position of the Ty p1 protein (according to WO 94/14969, herein incorporated by reference) and the poiypeptide of SEQ ID NO:3 fused at the C-terminus of the Ty p1 protein.
  • Another preferred Ty-VLP construct comprises the insertion of the neutralising epitope containing poiypeptide of SEQ ID NO:15 at the C2 position of the Ty p1 protein (according to WO 94/14969, herein incorporated by reference) and the poiypeptide of SEQ ID NO:3 fused at the C-terminus of the Ty p1 protein.
  • injectable compositions of the invention will typically comprise sterile water, or physiological saline, although other ingredients to aid solubility or for preservation purposes may be included.
  • One or more appropriate adjuvants may also be present. Examples of suitable adjuvants are inter alia : Aluminium hydroxide, muramyl dipeptide and saponin.
  • immunodominant polypeptides of this invention can be directly expressed in humans by means of appropriate live viral expression vectors such as inter alia : adeno, influenza, vaccinia or herpes simplex, and also by means of live bacterial vectors such as inter alia : E.coli, Lactobaccillus, Salmonella or BCG.
  • live viral expression vectors such as inter alia : adeno, influenza, vaccinia or herpes simplex
  • live bacterial vectors such as inter alia : E.coli, Lactobaccillus, Salmonella or BCG.
  • the antigenic nature of the polypeptides of this invention it is likely that it will be easier to produce antibodies than with conventional antigens currently available.
  • the invention thus further provides for antibodies raised against the polypeptides of this invention.
  • the antibodies may be polyclonal, obtained for example by injecting the polypeptides into a selected mammal (inter alia rabbit, mouse, goat, or horse), and later collecting the immunised serum from the animal, and treating this according to procedures known in the art.
  • the antibodies may be monoclonal, produced by hybridoma cells, phage display libraries or other methodology.
  • Monoclonal antibodies may be rat, mouse or human derived, and rodent antibodies may be humanised using recombinant DNA technology according to techniques known in the art.
  • the poiypeptide antigens of this invention may also enhance the production of human antibodies for therapeutic use. These would be useful in the therapeutic control of varicella or zoster, by passive immunisation. Passive immunisation here refers to the administering to a patient of preformed antibodies. Passive immunisation treatments using varicella-zoster immune globulin prepared from plasma with a high anti-VZV antibody titre are currently performed, and have been found to be effective in modifying VZV in high-risk varicella-susceptible persons. Antigens and antibodies of the invention also find use in diagnostic applications.
  • Such reagents can be incorporated into standard immunoassay formats; competitive binding assays, non-competitive 'sandwich' assays, radioimmunoassays (RIA), enzyme immunoassay (EIA) and enzyme-linked immunosorbant assay (ELISA) as well known to those skilled in the art.
  • the polypeptides and antibodies of this invention may be used unlabelled, or may be directly labelled by joining, either covalently or non-covalently to a substance that provides for a detectable signal.
  • a method for detecting the presence of anti-VZVgE antibodies comprises coating a support surface, such as a multi-well plate, with polypeptides or VZVgE:Ty-VLPs of the invention, and testing serum samples by adding the sample to the coated support surface and allowing any anti-VZVgE antibodies to bind. After washing, a labelled anti-species antibody conjugate is added, incubated to allow attachment to bound VZV antibody, and washed off. Finally, a reagent for detecting the label on the labelled antibody is added, washed off and detected.
  • a suitable labelled antibody for use in the above method is an anti-human immunoglobulin peroxidase conjugated antibody, and the detection reagent a peroxidase enzyme substrate.
  • kits for detecting the presence of anti- VZVgE antibodies in human sera which kit comprises a support surface coated with the polypeptides or VZVgE:Ty-VLPs of the invention, a labelled antibody cross- reactive with human antibodies and a reagent for detecting the label on the labelled antibody.
  • Eschericia co/V strain DH5f 3 [(!380d/acZ ⁇ M15 recA1 endlk ⁇ gyrA96 thi-1 7s R17 ( ⁇ K-, m ⁇ +) supE ⁇ re/A1] was used for plasmid manipulation. Maintenance and growth was in 2 x YT media. Plasmid DNA was used to transform Saccharomyces cerevisiae strain MC2 (mating type a, leu 2-3, leu 2-112, frpl , ura 3-52, p/ ⁇ >1-1122, pepA-3, prd -407). MC2 was maintained and grown in SC-glc media (6.7% (w/v) yeast nitrogen base, 1% (w/v) glucose).
  • Hybrid VLPs were produced essentially according to the method of example 3 of WO-A-8803562, and example 1 of WO-A-9320840. Seven PCR fragments spanning the VZVgE open reading frame (ORF) were generated from VZV genomic DNA derived from VZV isolate H-551. The VZVgE fragments contained (encoded) amino acids 1 - 134, 101 - 161 ,161 - 233, 201 - 333, 303 - 435, 402 - 536 and 506 - 623.
  • PCR oligonucleotide primers used were designed (based on the published sequence in Davidson and Scott (1986) ibid.) to incorporate an 8 base pair "stuffer fragment" (non-hybridisable sequence) at the 5'- end followed by either BglU or SamHI restriction endonuclease sites.
  • C-terminal Ty:gE fusions were produced by ligating the SoTII /SamHI cut VZVgE PCR generated fragments to the truncated TYA gene at the unique Bam HI site of the yeast expression vector pOGS40 (see WO-A-8803562 and WO-A-9320840).
  • the hybrid particles produced were purified by fractionation on a 35 - 60% sucrose gradient and gel exclusion chromatography ready for the antigenicity and immunogenicity studies
  • Fragments 1 , 2 (3' and 5') and 3 produced Ty:gE fusion proteins that assembled into hybrid VLPs, fragments 4 and 5 did not produce particulate protein and fragment 6 produced only low levels of VLPs.
  • the Ty-gE plasmid constructs and their corresponding amino acid numbers are described in Table 1.
  • VLP expression The levels of VLP expression are indicated as follows; high (+++), good (++), low (+), equivocal (+/-) and negative (-).
  • gE(1-134):VLP, gE(101-161):VLP, gE(161-233):VLP and gE(201 -333):VLPs and the Ty:gE(303-435),Ty:gE(402-536) and Ty:gE(506-623) fusion proteins were selected for antigenicity and immunogenicity studies.
  • VZV Varicella-Zoster virus
  • the polyclonal PZ6 sera (pooled sera from 6 individuals with zoster), chickenpox and zoster donor sera were obtained from St. Bartholomew's Hospital (London).
  • the monoclonal antibody SG1A is an affinity-purified neutralising murine MAb (lgG1) and was obtained from VIRO Research Inc. Both IF-B9 and 3B3 neutralising monoclonal antibodies have been produced in ascitic fluid and have been characterised as lgG2a.
  • IF-B9 was obtained from Dr. J. Taylor-Weideman (Cambridge University, U.K.) and 3B3 was obtained from Dr. C. Grose (University of Iowa, U.S.A.).
  • VZVgE:VLPs The antigenicity of the VZVgE:VLPs were analysed by Westem blot and ELISA.
  • Table 2 shows the Western blot and ELISA data produced using the polyclonal zoster pooled sera, the combined results from six varicella (chickenpox) sera and the anti-VZVgE neutralising monoclonal antibodies: SG1A, 3B3 and IF-B9.
  • the antibody responses were determined against the following VZV gE:VLPs: gE(1- 134):VLP (OGS1208), gE(101 -161):VLP (OGS1207), gE(161-233):VLP (OGS1209) and gE(201-333):VLP (OGS1210).
  • the highest antibody responses were obtained with gE (1-134):VLP for both the polyclonal anti-VZV sera and the monoclonal antibodies SG-1A and IF-B9.
  • the next highest was gE(101-161):VLP to which the 3B3 and SG-1A neutralising monoclonal antibodies also reacted.
  • polypeptides Twenty-nine synthetic polypeptides were synthesised by the standard solid phase method (supplied by CRB ZENECA or GENOSYS Biotechnologies Inc.). The polypeptides covered the 1-161 gE VZV sequence of SEQ ID NO:1. All the polypeptides, except poiypeptide 29 (21-mer), were 20-mers and most overlapped by 15 amino acids. The polypeptides corresponded to the following amino acid sequence positions of SEQ ID NO:1 :
  • a standard ELISA was performed. Briefly, the polypeptides were dissolved at 30-40 ⁇ g/ml in "bicarbonate coating buffer” (carbonate/bicarbonate buffer, 0.05 M, pH 9.6) and ELISA plates were immediately coated and incubated overnight at 4°C. The sera were tested in quadruplicate against the polypeptides. Colorimetric detection of antibody binding was then performed using anti-mouse or anti-human IgG/peroxidase conjugates, followed by peroxidase substrate.
  • bicarbonate coating buffer carbonate/bicarbonate buffer, 0.05 M, pH 9.6
  • each poiypeptide as used in examples 3a overlaps with the neighbouring poiypeptide usually by 15 amino acids, a single B-cell epitope will be present in, and identified by 2 or 3 neighbouring polypeptides. It follows from scrutiny of the PEPSCANTM data that the human VZVgE linear immunodominant B-cell epitopes are predicted to lie within the ten amino acid sequences of SEQ ID NOs:16 - 23.
  • polypeptides containing the immunodominant epitopes are:
  • LGDDTGIHVI SEQ ID NO:22
  • 6-mer polypeptides overlapping by 2 amino acids were prepared by the standard solid phase method (supplied by CRB ZENECA) as in example 3a. These polypeptides were used to further delineate the linear VZVgE Human and monoclonal antibody defined B-cell epitopes within amino acids 1 - 134 of SEQ ID NO:1, using an ELISA inhibition assay. Sera or monoclonal antibodies were pre- incubated overnight at 4°C, with the 6-mer polypeptides prior to testing in the ELISA described in example 3a using the 20- and 21-mer polypeptide-coated plates. The 6-mer polypeptides used in the ELISA inhibition assay corresponded to the following amino acid sequence positions of SEQ ID NO:1:
  • 6-mer polypeptides containing the immunodominant epitopes are:
  • Guinea pig and mouse sera were pooled separately and tested by direct ELISA using VZV lysate antigen (5 ⁇ g/ml) to coat microtitre plates, or neutralisation assay (described below). Any sera that was positive was tested by PEPSCANTM analysis using the methodology as described in examples 3a and 3b. All the results are shown in Table 3. VZV neutralisation assay.
  • VZV varicella-zoster virus
  • the positive sera generated above was heated at 56°C for 30 minutes to inactivate endogenous complement and bovine serum.
  • the viral inocula (prepared above) was pre-incubated for 30 minutes at 37°C with appropriate dilutions of test sera. Where complement-dependent neutralisation is assayed, six-haemolytic units of rabbit complement serum was added.
  • the test neutralisation mixture was: 40 ⁇ l of sera dilution (in complete medium), 20 ⁇ l of complement or complete medium, 100 ⁇ l of cell-free VZV.
  • Neutralisation is defined as a reduction in the number of plaques to 50% of the mean value seen without sera present.
  • polypeptides containing the immunodominant epitopes are:
  • DEDKLDTNSVYEPYYHSDHA (SEQ ID NO: 5)
  • VYNQGRGIDSGERLMQPTQM (SEQ ID NO:12)
  • QPTQMSAQEDLGDDTGIHVI (SEQ ID NO:13)
  • DDRHKIVNVDQRQYGDVFKGD (SEQ ID NO:15) Guinea pig.
  • HSDHAESSWVNRGESSRKAY (SEQ ID NO: 7)
  • LGDDTGIHVI SEQ ID NO:22
  • LGDDTGIHVI SEQ ID NO:22
  • lymphocyte proliferation assay of this example was performed according to standard methodology known to one skilled in the art and as found in "Practical Immunology- third edition” By Hudson and Hay, published by Blackwell Scientific publications.
  • mice Ten animals from four different strains of inbred mice (BALB/c, C57BLJ6, CBA and B10.G), with H-2 haplotypes d, b, k and q, respectively, were immunised subcutaneously with either VZVgE(1-134):VLPs (100 ⁇ g/mouse in aluminium hydroxide) or VZVgE(101-161):VLPs (100 ⁇ g/mouse in aluminium hydroxide). Seven to fourteen days later lymph nodes were removed and the lymph node cells restimulated in vitro for 6 days with either VZV lysate or mock lysate at 10 ⁇ g/ml. Lymphocyte proliferation was measured by tritiated thymidine incorporation. The stimulation indices (S.I.) for these four strains are indicated in Tables 4 and 5.
  • VZVgE (1 - 134):VLPs and VZVgE(101-161):VLPs may be useful in stimulating VZV T-cell immunity in the outbred human population.
  • hybrid VLPs comprising Tv p1 polypeptides containing internal and C-terminal VZVoE epitopes.
  • Hybrid VLPs were produced essentially according to the method of example 3 of WO-A-8803562, example 1 of WO-A-9320840, and example 4 of WO-A- 9414969 (herein incorporated by reference).
  • the C2 insertion site used in this example and as identified in WO-A-9414969, is located at amino acids 132 -133 of the Ty protein.
  • the Ty particle forming protein sequence is disclosed in Dobson et al., (1994) EMBO J. 3:1115.
  • Double-stranded DNA encoding the amino acids of poiypeptide 29 (comprising SEQ ID NO: 15) was prepared by annealing together complimentary synthetic oligonucleotides (SEQ ID NO: 29 and 30), encoding the amino acids of SEQ ID NO:15 and possessing single overhanging ends complimentary to Nhe ⁇ restriction sites.
  • This double-stranded VZVgE encoding DNA fragment was ligated into Nhe ⁇ digested pOGS813 DNA (constructed according to example 4 of WO-A- 9414969).
  • the resultant plasmid vector was called pOGS1226.
  • This plasmid was subsequently digested with SamHII (located at the C-terminus of the p1 protein encoded DNA) and the BamHMBglW cut 1 - 134 VZVgE PCR fragment was ligated into this location as described in example 1 (supra).
  • the resultant plasmid was designated pOGS1229.
  • the p1 particle-forming poiypeptide encoded by this vector possesses the 1 - 134 amino acid sequence of VZVgE (SEQ ID NO:2) at its C-terminus, and the VZVgE epitope comprised in SEQ ID NO:15 located at the surface exposed C2 position within the p1 protein.
  • Plasmids pOGS1223 and pOGS1228 were also constructed according to the same method.
  • SEQ ID NO:31 and 32 comprise the complimentary oligonucleotides which when annealed encode the poiypeptide of SEQ ID NO: 9, and possess Nhe compatible ends for cloning into the C2 position of the Ty p1 protein. This yielded plasmid pOGS1223.
  • the VZVgE 101 - 161 poiypeptide fragment (SEQ ID NO:3) was then cloned into the C-terminus of TyA gene yielding pOGS1228.
  • the particle- forming hybrid Ty p1 polypeptides derived from the expression in yeast of this plasmid possess amino acids 101 - 161 of VZVgE at the Ty p1 C-terminus and amino acids comprising SEQ ID NO:9 located at the C2 position within Ty p1.
  • the VZVgE 101 - 161 poiypeptide fragment (SEQ ID NO:3) was also cloned into the C-terminus of TyA gene of pOGS1226, yielding pOGS1230.
  • the particle-forming hybrid Ty p1 polypeptides derived from the expression in yeast of this plasmid possess amino acids 101 - 161 of VZVgE at the Ty p1 C-terminus and amino acids comprising SEQ ID NO: 15 located at the surface exposed C2 position within the p1 protein.
  • Particles from the expression in yeast of pOGS1226, pOGS1229, pOGS1223 and pOGS1228, were purified as described before.
  • HSDHAESSWVNRGESSRKAY (SEQ ID NO: 7)
  • VYNQGRGIDSGERLMQPTQM (SEQ ID NO:12)
  • LGDDTGIHVI SEQ ID NO:22

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