WO1996001900A1 - Immunodominant polypeptides - Google Patents

Abstract

Identification and utilisation of isolated polypeptides containing the important immunodominant epitopes on the Varicella Zoster virus gE protein.

Description

IMMUNODOMINANT POLYPEPTIDES.

Field of the Invention.

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.

Background of the invention.

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. Thereafter the virus enters into a latent state in neuronal cells and also in satellite cells of ganglia, especially of the dorsal root ganglia of the spinal column (reviewed by Grose, C. In "Human herpesvirus infections- Clinical aspects." (R. Glaser and T. Gotlieb-Stemasky eds.), Marcel Dekker. Inc. New York.). 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. (1986) Journal of infectious diseases 154:422-429.) and other infections, notably with HIV (Glesby et al. (1993) Journal of Infectious Diseases 168:1264-1268; and Wallace et al. (1994) Southern Medical Journal 87:74-76).

In immunocompromised children and adults with leukaemia or lymphoma, shingles is a common occurrence appearing 1 -2 years after initiation of chemotherapy/irradiation treatment. In renal transplant recipients, between 5-35% will reactivate VZV to develop herpes zoster.

Pregnant women who contract chickenpox sometimes develop a rare condition called congenital varicella syndrome. As VZV is a neurotropic virus, it can also cause a range of rare neurological complications of poorly understood pathogenesis (Kennedy (1987) In: Infections of the Nervous System. pp177-208. Edited by P.G.E. Kennedy and R.T. Johnson, London. Butterworths.). One example is post-herpetic neuralgia in the form of long-lasting, severe dermatomal pain subsequent to shingles, this becomes more common with age, and may be associated with virus- induced damage to neuronal tissue.

Post-herpetic neuralgia is unresponsive to antiviral therapy and therefore is likely to be independent of continued viral replication.

Vaccines and Treatments

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). A three year study (1987-1989) of 140 healthy OKA/Merck vaccine recipients was initiated in Ohio (Johnson, C et al. (1989) Pediatrics 84(3) :418-421). The persistence of anti-varicella antibodies were measured and clinical re-infections were examined three years after vaccination. Suspected varicella cases were confirmed by a fourfold increase in titre. They found that the vaccination resulted in antibody persistence for two years and the few re-infections that did occur were greatly attenuated. Out of 133 children, 7 became reinfected; all had <70 vesicles (in natural varicella the average number of lesions is approximately 500) and 6 out of 7 were afebrile.

Although the vaccine appears to be effective in children there are a number of disadvantages with this vaccine;

1) The vaccine apparently has little effect on the treatment of shingles (Gershon (1987) Annual Review of Medicine. 38:41-50). By definition, the people most at risk of shingles are immunosuppressed and are therefore the least suitable for inoculation of a live herpesvirus vaccine.

2) There is concern over the duration and magnitide of immunity since chickenpox delayed until adulthood may result in more serious disease and complications. Sera taken from 35 children with cancer who had been vaccinated with the live varicella vaccine were assayed and it was found that the levels of immunity observed after vaccination were substantially weaker and more variable than those detected following natural VZV infection in otherwise healthy individuals (Harper et al. (1990) Journal of Medical Virology 30:61-67).

3) Subclinical re-infection of individuals can re-occur and there is also evidence that the vaccine virus can reactivate to cause zoster, particularly in immunocompromised children (Kangro (1990) Review of Medical Microbiology1:205-212).

4) The vaccine is not effective in children under six months of age.

5) There are serious difficulties with producing and distributing large quantities of vaccine for a larger market particularly as the virus vaccine shows batch- to- batch variation, is unstable and expensive. Recent enhancements in vaccine production systems by Merck have resulted in significant improvements in this area, but the basic vaccine formulation remains unchanged (Bennet et al. (1992) Developments in Biological Standardisation 74:215-221 ).

6) 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).

7) A mass vaccination programme of healthy children and adults with attenuated Oka vaccine would be costly. The cost of a single vaccine in 1985 was estimated to be $15 (Preblud et al. (1985) Postgraduate Medical Journal 61 (suppl 4):17-22). As inflation and the costs of healthcare both in the U.S.A. and Europe have significantly increased such a programme would be very expensive. However, this expense must be balanced against the costs of healthcare treatments, loss of wages and other expenses associated with incidence of annual Varicella-zoster virus infections. This study excluded the vaccination of immunocompromised patients who are most at risk from complications resulting from varicella-zoster virus infections.

There are other forms of treatment for varicella infections. Passive immunisation using immunoglobulin is effective for prevention of severe varicella in immune compromised children. However, it is not recommended for healthy children. The treatment is expensive, in short supply and is only recommended where VZV infection has been confirmed. Research to produce human or humanised monoclonal antibodies is currently under way, but this treatment is not yet in clinical trials.

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. However, 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. 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.

There is no VZV homologue of HSV gD. As would be expected, the VZV glycoproteins are highly immunogenic, in particular gE, gB and gH. In the virion, gE is complexed with gl, while 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.

Immune Response to VZV Infection Envelope proteins

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 complete DNA sequence of the VZV genome, identifying the open reading frames and the translated amino acid sequences, is published in the Journal of General Virology 67:1759-1816, by Davidson and Scott (1986), and where references are made herein to the numbering of amino acids in the gE sequence, that numbering refers to the 68*h open reading frame (glycoprotein gp1 ; starting at nucleotide position 115808) therein.

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. CTL recognition of two VZV proteins, IE62 (immediately early or tegument protein) and gE was demonstrated in limiting dilution cultures of T lymphocytes obtained from immune donors. It was shown that antiviral CTL activity against targets expressing VZV proteins was mediated equally well by T lymphocytes of the CD4+ or CD8+ phenotype (Hayward et al. (1989) Viral Immunology. 2:175-184; Arvin et al. (1991), J.lmmunol. 146:257- As detailed above, there is direct evidence that gE induces neutralising antibodies, T-helper cell responses and also contains epitopes recognised by CD4+ and CD8+ cytotoxic lymphocytes from VZV-infected individuals. Furthermore, 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).

Presentation of the most immunodominant polypeptides or peptides of viral proteins to the immune system should serve to stimulate the immune system sufficiently to prevent subsequent infection by VZV.

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.

Such sub-unit vaccines have been previously proposed, utilising the most immunogenic proteins, especially the glycoproteins, or portions thereof, for use in sub-unit vaccines and EP 192 902 claims the use of a 0.9Kb nucleic acid sequence of VZVgC (now gE) coding for a large poiypeptide for use in a sub-unit composition.

Previously, it has not been possible to successfully vaccinate children under the age of 6 months and this is thought to be due to the presence of maternal antibodies which may clear the vaccine before it can stimulate the childs immune system.

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. Brief description of the invention

Large sub-units are however, not as useful in sub-unit design as would be smaller molecular weight polypeptides containing only the specific antigenic epitopes responsible for eliciting the immunogenic responses. The inventors, realising this, have suceeded in identifying those epitopes within the gE protein important for generating immune responses. Polypeptides containing these epitopes can, because of their small molecular weight, be easily incorporated into carrier proteins or other suitable vehicles for presentation to the immune system. Following administration to the subject, an immune response raised against the most important antigenic epitopes can provide protection against subsequent infection.

Detailed description of the invention

According to the first aspect of the invention there is provided 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.

For the purpose of this invention 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.

By the term "variant" 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). By "functional variant", however, 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. By 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, examples of conservative changes are inter alia : alanine to glycine, valine, leucine or isoleucine; tyrosine to phenylalanine or tryptophan; and lysine to arginine or histidine.

According to a second aspect of the invention there is provided an isolated poiypeptide sub-unit of the VZVgE glycoprotein consisting of amino acids 1 - 134 (SEQ ID NO: 2), or a functional variant thereof.

According to another aspect of the invention there is provided an isolated poiypeptide of the VZVgE glycoprotein consisting of amino acids 101 - 161 (SEQ ID NO: 3), or a functional variant thereof.

According to another aspect of the invention there is provided a series of 20 or 21 amino acid polypeptides, or functional variants thereof, 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:

i) DEDKLDTNSVYEPYYHSDHA aa's 41 - 60, (SEQ ID NO: 5) ii) YEPYYHSDHAESSWVNRGES aa's 51 - 70, (SEQ ID NO: 6) iii) HSDHAESSWVNRGESSRKAY aa's 56 - 75, (SEQ ID NO: 7) iv) NRGESSRKAYDHNSPYIWPR aa's 66 - 85, (SEQ ID NO: 8) v) SRKAYDHNSPYIWPRNDYDG aa's 71 - 90, (SEQ ID NO: 9) vi) YIWPRNDYDGFLENAHEHHG aa's 81 - 100, (SEQ ID NO: 10) vii) NDYDGFLENAHEHHGVYNQG aa's 86 - 105, (SEQ ID NO.-11) viii) VYNQGRGIDSGERLMQPTQM aa's 101 - 120, (SEQ ID NO:12) ix) QPTQMSAQEDLGDDTGIHVI aa's 116 - 135, (SEQ ID NO:13) x) GIHVIPTLNGDDRHKIVNVD aa's 131 - 150, (SEQ ID NO: 14) xi) DDRHKIVNVDQRQYGDVFKGD aa's 141 - 161, (SEQ ID NO: 15)

It can be seen from comparisons between SEQ ID NO:1 and SEQ ID NOs: 5 to 15 that some of these 20 or 21 amino acid polypeptides overlap each other in the wild- type VZVgE protein. Each of these individual 20 or 21 amino acid polypeptides possess antigenic properties, and therefore, a poiypeptide containing all of the amino acids of SEQ ID NOs: 5 to 12 in sequence is therefore highly antigenic. Therefore, according to another aspect of the invention there is provided an isolated poiypeptide of the VZVgE glycoprotein consisting of amino acids 41 - 120 (SEQ ID NO: 4), or a functional variant thereof.

From comparisons of the antigenicity of neighbouring 20 or 21-mer amino acid polypeptides spanning the first 161 amino acids of the VZVgE protein, and overlapping by 14 to 16 amino acids, usually 15 amino acids, with each adjacent poiypeptide the inventors were able to establish the positions of most of the antigenic epitopes.

According to another aspect of the invention there is provided a series of 10 amino acid polypeptides, or functional variants thereof, 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:

i) YEPYYHSDHA (SEQ ID NO:16) ϋ) 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)

Since it is known that 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.

Using ELISA inhibition assays, synthetic 6-mer polypeptides spanning the regions of the antigenic 20-mers enabled the epitopes to be delineated further.

According to another aspect of the invention there is provided a series of 6 amino acid polypeptides, or functional variants thereof, 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:

i) WVNRGE (SEQ ID NO:24) ii) RKAYDH (SEQ ID NO:25) iii) WPRNDY (SEQ ID NO:26) iv) HHGVYN (SEQ ID NO:27) v) TGIHVI (SEQ ID NO:28)

Since it is known that 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.

The polypeptides of this invention can be synthesised chemically. For example, by the Merryfield technique (Journal of American Chemical Society 85:2149-2154, 1968). Alternatively, 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.

Nucleic acid sequences encoding the polypeptides SEQ ID NOs: 1 to 28 supra, or functional variants, also form part of the invention.

The 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. Numerous expression vectors and systems are known, both for prokaryotes and eukaryotes, and the selection of an appropriate system is a matter of choice. Expression and purification of the polyprotein product of the invention can be easily performed by one skilled in the art. See, Sambrook et al.. "Molecular cloning-A Laboratory Manual, second edition".

Recent studies have shown that 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). With DNA-based vaccines, 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. 23:1719-1722) to express the VZVgE epitopes in vivo after immunisation. In order to prime a CTL response 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.

Accordingly, there is provided for the use of 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.

There is also provided for a pharmaceutical composition 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.

There is also provided for a pharmaceutical composition 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.

There are several means by which the 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. Alternatively, 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).

The small size and molecular weight of the 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.

For vaccine purposes, the polypeptides are likely to be present in longer sequences. For example, 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. Alternatively, 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. Accordingly, also included within the scope of the invention are 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. Also included within the scope of the invention are 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.

In one preferred type of composition, 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. As used herein, 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

Similarly, 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.

Alternatively, 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. Indeed, 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.

Thus, in a further aspect of the invention there is provided 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).

In a further aspect of the invention there is provided 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 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.

The 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.

Because of 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. Alternatively, 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.

In a further aspect of the invention there is provided a method for detecting the presence of anti-VZVgE antibodies which method 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.

Another aspect of the invention provides for a kit 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.

The following examples describe the invention for illustrative purposes only. They are not intended to limit the scope of the invention in any way. Examples

The techniques of genetic manipulation used in the following examples are well known to those skilled in the art of genetic engineering and Cell Biology. A description of the techniques used can be found in "Molecular cloning-A laboratory manual, second edition" By J. Sambrook, E.F. Fritsch and T. Maniatis published by Cold Spring Harbor Laboratory, Box 100, New York. A description of standard immunological techniques, known to those skilled in the art, can be found in "Practical Immunology- third edition" By Hudson and Hay, published by Blackwell Scientific publications.

Example 1.

Strains and media: Eschericia co/V strain DH5f 3 [(!380d/acZΔM15 recA1 endlkλ gyrA96 thi-1 7s R17 (ΓK-, mκ+) supE Λ 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. The 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.

TABLE 1. Expression of VZV gE-VLPs.

Plasmid gE Amino Acid # VLP formation fragment

pOGS1208 1 1 - 134 + + + pOGS1207 2-5' end 101 - 161 + + +

pOGS1209 2-3' end 161 - 233 + + + pOGS1210 3 201 - 333 + +

pOGS1211 4 303 - 435 - pOGS1212 5 402 - 536 + /- pOGS1213 6 506 - 623 +

Key: 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.

Example 2.

Antigenicity of hybrid Tv:αE VLPs.

Virus and cell culture. Varicella-Zoster virus (VZV) was obtained from a clinical isolate (H-551) at St. Bartholomew's Hospital, London. The virus was cultured in MRC5 cells as described in Harper et al. (1988; J. Med. Virol. 25:387-398).

Antisera and MAb. 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.).

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.

The human sera were also tested by Westem blot against yeast cell lysates containing Ty p1 fusion proteins carrying VZVgE sequences 303-435, 402-536 and 506-623. No specific reactivity was seen against the fusion proteins. TABLE 2. Antigenicity of VZV gE : VLPs

The antibody responses are indicated as follows; very strong (+++), strong (++), good (+), weak (+/-) and negative (-).ND = Not determined.

Example 3a.

Epitope mapping of human sera and monoclonal antibodies using synthetic overlapping polypeptides (PEPSCAN™).

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 :

Polypept de 1 amino acids 1-20. Polypept de 2 amino acids 6-25. Polypepti de 3 amino acids 11-30. Polypept de 4 amino acids 16-35. Polypept de 5 amino acids 21-40. Polypept de 6 amino acids 26-45. Polypept de 7 amino acids 31-50. Polypept de 8 amino acids 36-55. Polypepti de 9 amino acids 41-60. Polypept de 10 amino acids 46-65. Polypept de 11 amino acids 51-70. Polypepti de 12 amino acids 56-75. Polypept de 13 amino acids 62-81. Polypept de 14 amino acids 66-85. Polypept de 15 amino acids 71-90. Polypept de 16 amino acids 76-95. Polypept de 17 amino acids 81-100. Polypept de 18 amino acids 86-105. Polypept de 19 amino acids 91-110. Polypept de 20 amino acids 96-115. Polypepti de 21 amino acids 101-120. Polypepti de 22 amino acids 106-125. Poiypeptide 23 amino acids 111 -130. Poiypeptide 24 amino acids 116-135. Poiypeptide 25 amino acids 121-140. Poiypeptide 26 amino acids 126-145. Poiypeptide 27 amino acids 131-150. Poiypeptide 28 amino acids 136-155. Poiypeptide 29 amino acids 141-161

A standard ELISA was performed. Briefly, the polypeptides were dissolved at 30-40 μg/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.

Human zoster sera, human varicella sera and monoclonal antibodies, identified polypeptides according to SEQ ID NOs: 4 - 11 , as containing the linear immunodominant B-cell epitopes of VZVgE(1 - 161).

Because 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 PEPSCAN™ 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.

List of polypeptides containing linear immunodominant human B-cell epitopes and neutralising monoclonal antibody defined epitopes.

The polypeptides containing the immunodominant epitopes are:

DEDKLDTNSVYEPYYHSDHA aa's 41 - 60, (SEQ ID NO: 5)

YEPYYHSDHAESSWVNRGES aa's 51 - 70, (SEQ ID NO: 6) HSDHAESSWVNRGESSRKAY aa's 56 - 75, (SEQ ID NO: 7)

NRGESSRKAYDHNSPYIWPR aa's 66 - 85, (SEQ ID NO: 8)

SRKAYDHNSPYIWPRNDYDG aa's 71 - 90, (SEQ ID NO: 9)

YIWPRNDYDGFLENAHEHHG aa's 81 - 100, (SEQ ID NO: 10)

NDYDGFLENAHEHHGVYNQG aa's 86 - 105, (SEQ ID NO:11)

VYNQGRGIDSGERLMQPTQM aa's 101 - 120, (SEQ ID NO:12)

QPTQMSAQEDLGDDTGIHVI aa's 116 - 135, (SEQ ID NO:13)

GIHVIPTLNGDDRHKIVNVD aa's 131 - 150, (SEQ ID NO:14)

DDRHKIVNVDQRQYGDVFKGD aa's 141 - 161, (SEQ ID NO:15)

YEPYYHSDHA (SEQ ID NO:16)

ESSWVNRGES (SEQ ID NO:17)

SRKAYDHNSP (SEQ ID NO:18)

YIWPRNDYDG (SEQ ID NO:19)

HEHHGVYNQG (SEQ ID NO:20)

RGIDSGERLM (SEQ ID NO:21)

LGDDTGIHVI (SEQ ID NO:22)

RQYGDVFKGD (SEQ ID NO:23)

Example 3b

ELISA inhibition assay

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:

Amino acids 56-81 :

Poiypeptide 30 HSDHAE

Poiypeptide 31 DHAESS

Poiypeptide 32 AESSWV

Poiypeptide 33 SSWVNR

Poiypeptide 34 WVNRGE

Poiypeptide 35 NRGESS

Poiypeptide 36 GESSRK

Poiypeptide 37 SSRKAY

Poiypeptide 38 RKAYDH

Poiypeptide 39 AYDHNS

Poiypeptide 40 DHNSPY

Amino acids: 71-90.

Poiypeptide 41 YIWPRN

Poiypeptide 42 WPRNDY

Poiypeptide 43 RNDYDG

Amino acids 96-107:

Poiypeptide 44 HEHHGV

Poiypeptide 45 HHGVYN

Poiypeptide 46 GVYNQG

Poiypeptide 47 YNQGRG

Amino acids 126-135:

Poiypeptide 48 LGDDTG

Poiypeptide 49 DDTGIH

Poiypeptide 50 TGIHVI List of polypeptides found to contain linear immunodominant human B-cell and neutralising monoclonal antibody defined epitopes.

The 6-mer polypeptides containing the immunodominant epitopes are:

WVNRGE (SEQ ID NO:24)

RKAYDH (SEQ ID NO:25)

WPRNDY (SEQ ID NO:26)

HHGVYN (SEQ ID NO:27)

TGIHVI (SEQ ID NO:28)

Example 4a.

Immunogenicity studies of VZV:VLPs in mice and guinea pigs.

Outbred swiss white mice and Hartley guinea pigs were immunised intramuscularly with VZVgE(1-134):VLPs or VZVgE(101-161):VLPs (100 μg/mouse or 240 μg/guinea pig in aluminium hydroxide [aluminium hydroxide:protein ratio 5:1 ; Alhydrogel 85, Superfos Ltd.] or complete Freund's adjuvant) on days 0 and 28 for mice or days 0, 30 and 60 for guinea pigs. Two weeks later they were bled. Guinea pig and mouse sera were pooled separately and tested by direct ELISA using VZV lysate antigen (5 μg/ml) to coat microtitre plates, or neutralisation assay (described below). Any sera that was positive was tested by PEPSCAN™ analysis using the methodology as described in examples 3a and 3b. All the results are shown in Table 3.

VZV neutralisation assay.

Cell-free varicella-zoster virus (VZV) was prepared by scraping VZV-infected MRC5 fibroblasts 2-3 days after infection when cytopathic effect is 10-30%. The material was sonicated 3 x 2 minutes on ice and centrifuged at 1500G for 15 minutes in a melanoma cells, harvested at 50% of maximum cytopathic effect (cell death).

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: 40μl of sera dilution (in complete medium), 20μl of complement or complete medium, 100μl of cell-free VZV.

Confluent monolayers of human melanoma cells (Mewo line) were used to minimise complement toxicity. Neutralisation mixtures were added to the Mewo cells for one hour. After absorption, virus was removed and the cells rinsed in PBS and overlaid with maintenance medium solidified with 0.15% agarose. Cultures were incubated at 32°C, and the assay read at 8 days post-infection after plaque staining with crystal violet.

Neutralisation is defined as a reduction in the number of plaques to 50% of the mean value seen without sera present.

List of polypeptides containing linear immunodominant mouse and guinea pig B-cell epitopes.

The polypeptides containing the immunodominant epitopes are:

Mouse.

DEDKLDTNSVYEPYYHSDHA (SEQ ID NO: 5) VYNQGRGIDSGERLMQPTQM (SEQ ID NO:12) QPTQMSAQEDLGDDTGIHVI (SEQ ID NO:13) DDRHKIVNVDQRQYGDVFKGD (SEQ ID NO:15) Guinea pig.

DEDKLDTNSVYEPYYHSDHA (SEQ ID NO: 5)

HSDHAESSWVNRGESSRKAY (SEQ ID NO: 7)

SRKAYDHNSPYIWPRNDYDG (SEQ ID NO: 9)

NDYDGFLENAHEHHGVYNQG (SEQ ID NO:11)

QPTQMSAQEDLGDDTGIHVI (SEQ ID NO:13)

GIHVIPTLNGDDRHKIVNVD (SEQ ID NO:14)

Example 4b

Because each poiypeptide as used in examples 4a 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 PEPSCAN™ data that the mouse and guinea pig VZVgE linear immunodominant B- cell epitopes are predicted to lie within the following ten amino acid sequences:

Mouse.

YEPYYHSDHA (SEQ ID NO:16)

RGIDSGERLM (SEQ ID NO:21)

LGDDTGIHVI (SEQ ID NO:22)

RQYGDVFKGD (SEQ ID NO:23)

Guinea piα.

YEPYYHSDHA (SEQ ID NO:16)

ESSWVNRGES (SEQ ID NO:17)

YIWPRNDYDG (SEQ ID NO:19)

HEHHGVYNQG (SEQ ID NO:20)

LGDDTGIHVI (SEQ ID NO:22) Example 5.

The 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.

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 μg/mouse in aluminium hydroxide) or VZVgE(101-161):VLPs (100 μg/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 μg/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.

Table 4. VZVgE(1-134):VLP immunised lysate or mock lysate stimulation indices.

Mouse strain VZV lysate Mock lysate

BALB/c 21.5 3.2

C57BL/6 59.3 2.5

CBA 10.5 2.9

B10.G 4.4 1.9

Table 5. VZVαE(101-161ϊ:VLP immunised lysate or mock lysate stimulation indices.

Mouse strain VZV lysate Mock lysate

BALB/c 5.4 0.7

C57BL/6 8.5 1.3

CBA 10.9 0.5

B10.G 2.8 1.1

Proliferative T cell responses to VZV lysate are observed in all four strains. This indicates that VZVgE (1 - 134):VLPs and VZVgE(101-161):VLPs may be useful in stimulating VZV T-cell immunity in the outbred human population. Example 6.

Construction of 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. Westem blot analysis using the two neutralising monoclonal antibodies 3B3 (recognising the VZVgE epitope within SEQ ID NO: 15, amino acids 141 - 161) and IF-B9 (recognising the VZVgE epitope within SEQ ID NO: 9, amino acids 71 - 90) confirmed the presence of the two different VZVgE antigenic fragments in the two sets of particles.

SEQ ID NO:1

1 MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV

51 YEPYYHSDHA ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG

101 VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD

151 QRQYGDVFKG D (161)

SEQ ID NO:2

1 MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV

51 YEPYYHSDHA ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG

101 VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHV (134)

SEQ ID NO:3

101 VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD

151 QRQYGDVFKG D (161)

SEQ ID NO:4

41 DEDKLDTNSV YEPYYHSDHA ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM (120) SEQ ID NOs: 5 to 28

DEDKLDTNSVYEPYYHSDHA (SEQ ID NO: 5)

YEPYYHSDHAESSWVNRGES (SEQ ID NO: 6)

HSDHAESSWVNRGESSRKAY (SEQ ID NO: 7)

NRGESSRKAYDHNSPYIWPR (SEQ ID NO: 8)

SRKAYDHNSPYIWPRNDYDG (SEQ ID NO: 9)

YIWPRNDYDGFLENAHEHHG (SEQ ID NO: 10)

NDYDGFLENAHEHHGVYNQG (SEQ ID NO:11)

VYNQGRGIDSGERLMQPTQM (SEQ ID NO:12)

QPTQMSAQEDLGDDTGIHVI (SEQ ID NO:13)

GIHVIPTLNGDDRHKIVNVD (SEQ ID NO:14)

DDRHKIVNVDQRQYGDVFKGD (SEQ ID NO:15)

YEPYYHSDHA (SEQ ID NO:16)

ESSWVNRGES (SEQ ID NO:17)

SRKAYDHNSP (SEQ ID NO:18)

YIWPRNDYDG (SEQ ID NO:19)

HEHHGVYNQG (SEQ ID NO:20)

RGIDSGERLM (SEQ ID NO:21)

LGDDTGIHVI (SEQ ID NO:22)

RQYGDVFKGD (SEQ ID NO:23)

WVNRGE (SEQ ID NO:24)

RKAYDH (SEQ ID NO:25)

WPRNDY (SEQ ID NO:26)

HHGVYN (SEQ ID NO:27)

TGIHVI (SEQ ID NO:28)

5' - CTAGCGATGATAGACACAAGATTGTTAACGTTGATCAAAGAC AATACGGTGATGTTTTCAAGGGTGATG-3' (SEQ ID NO:29) 5' - CTAGCATCACCCTTGAAAACATCACCGTATTGTCTTTGATCA ACGTTAACAATCTTGTGTCTATCATCG-3' (SEQ ID NO:30)

5' - CTAGCTCTAGAAAGGCTTACGATCACAACTCTCCATACATTT GGCCAAGAAACGATTACGATGGTG -3' (SEQ ID NO:31 )

5' - CTAGCACCATCGTAATCGTTTCTTGGCCAAATGTATGGAGAGT TGTGATCGTAAGCCTTTCTAGAG-3' (SEQ ID NO:32)

Claims

Claims.

1. An isolated poiypeptide having the amino acid sequence as defined by SEQ ID NO:1.

2. An isolated poiypeptide having an amino acid sequence which is present in SEQ ID NO:1 , said poiypeptide having the amino acid sequence as defined by SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

3. An isolated poiypeptide, having an amino acid sequence which is present in SEQ ID NOs:1 , 2, 3 or 4, referred to in claims 1 and 2, said poiypeptide having one of the following amino acid sequences:

i) DEDKLDTNSVYEPYYHSDHA (SEQ ID NO: 5) ii) YEPYYHSDHAESSWVNRGES (SEQ ID NO: 6) iii) HSDHAESSWVNRGESSRKAY (SEQ ID NO: 7) iv) NRGESSRKAYDHNSPYIWPR (SEQ ID NO: 8) v) SRKAYDHNSPYIWPRNDYDG (SEQ ID NO: 9) vi) YIWPRNDYDGFLENAHEHHG (SEQ ID NO: 10) vii) NDYDGFLENAHEHHGVYNQG (SEQ ID NO.11) viii) VYNQGRGIDSGERLMQPTQM (SEQ ID NO:12) ix) QPTQMSAQEDLGDDTGIHVI (SEQ ID NO:13) x) GIHVIPTLNGDDRHKIVNVD (SEQ ID NO:14) xi) DDRHKIVNVDQRQYGDVFKGD (SEQ ID NO:15) xii) YEPYYHSDHA (SEQ ID NO:16) xiii) ESSWVNRGES (SEQ ID NO:17) xiv) SRKAYDHNSP (SEQ ID NO:18) xv) YIWPRNDYDG (SEQ ID NO:19) xvi) HEHHGVYNQG (SEQ ID NO:20) xvii) RGIDSGERLM (SEQ ID N0.21) xviii) LGDDTGIHVI (SEQ ID NO:22) xix) RQYGDVFKGD (SEQ ID NO:23) xx) WVNRGE (SEQ ID NO:24) xxi) RKAYDH (SEQ ID NO:25) xxii) WPRNDY (SEQ ID NO:26) xxiii) HHGVYN (SEQ ID NO:28)

4. A functional variant of any of the polypeptides as claimed in claims 1 to 3.

5. A poiypeptide sub-unit of the poiypeptide of claim 1 which includes any of the polypeptides claimed in claims 2, 3, or 4.

6. An amino acid sequence which is not as a whole a subsequence of native VZVgE glycoprotein but which includes as a sub-unit a poiypeptide having the sequence defined in any of claims 1 to 5.

7. A poiypeptide as claimed in any of claims 1 to 5 or the amino acid sequence as claimed in claim 6 having an N-terminus or C-terminus carrying a lipid tail.

8. Nucleic acid coding for a poiypeptide as claimed in any of claims 1 to 5, or the amino acid sequence as claimed in claim 6.

9. The use of nucleic acid as claimed in claim 8 for the preparation of a pharmaceutical composition for inducing or stimulating an immune response against VZV infection.

10. A pharmaceutical composition for inducing or stimulating an immune response against VZV infection, which composition comprises nucleic acid as claimed in claims 8, and one or more pharmaceutically acceptable carriers.

11. The use of one or more isolated poiypeptide as claimed in any of claims 1 to 5 or claim 7, or the amino acid sequence as claimed in claim 6, for the preparation of a pharmaceutical composition for inducing or stimulating an immune response against VZV infection.

12. A pharmaceutical composition for inducing or stimulating an immune response against VZV infection, which composition comprises one or more isolated polypeptides as claimed in any of claims 1 to 5 or claim 7, or the amino acid sequence as claimed in claim 6 and one or more pharmaceutically acceptable carriers.

13. A pharmaceutical composition as claimed in claim 12 wherein the poiypeptide is, polypeptides are, or the amino acid sequence is fused to a carrier molecule.

14. A pharmaceutical composition as claimed in claim 13 where the carrier molecule is a particle forming protein derived from the p1 protein of a yeast Ty retrotransposon, and in said composition said fused molecules are assembled into virus-like particles.

15. A pharmaceutical composition as claimed in claim 14 where one of the polypeptides is SEQ ID NO:2 or SEQ ID NO:3.

16. A pharmaceutical composition as claimed in claim 15 wherein the poiypeptide of SEQ ID NO:2 is fused to the C-terminus of Ty p1 protein and the poiypeptide of SEQ ID NO: 15 is inserted within the Ty p1 protein at amino acid positions 132-133.

17. A pharmaceutical composition as claimed in claim 15 wherein the poiypeptide of SEQ ID NO:3 is fused to the C-terminus of Ty p1 protein and the poiypeptide ofSEQ ID NO: 9 is inserted within the Ty p1 protein at amino acid positions 132-133.

18. A pharmaceutical composition as claimed in claim 15 wherein the poiypeptide of SEQ ID NO:3 is fused to the C-terminus of Ty p1 protein and the poiypeptide of SEQ ID NO: 15 is inserted within the Ty p1 protein at amino acid positions 132-133.

19. A pharmaceutical composition as claimed in claim 12 wherein the poiypeptide is, or polypeptides are, presented by a live vector.

20. An antibody raised against an isolated poiypeptide as claimed in any of claims 1 to 5 or claim 7.

21. The use of an antibody as claimed in claim 20 for passive immunisation treatment.

22. An isolated poiypeptide as claimed in any of claims 1 to 5 or claim 7, the amino acid sequence as claimed in claim 6 or a virus-like particle as claimed in any of claims 14 - 18, for use in diagnosis of VZV infection.

23. A method for determining the presence of anti-VZVgE antibodies in a sample by incubating said sample with an isolated poiypeptide as claimed in any of claims 1 to 5 or claim 7, and detecting immunocomplex.

24. A kit for determining the presence of anti-VZVgE antibodies in a human sample, said kit containing a support surface coated with the polypeptides as claimed in any of claims 1 to 5 or claim 7, or virus-like particles as claimed in any of claims 14 - 18, a labelled antibody cross-reactive with human antibodies and a reagent for detecting the label on the labelled antibody.