WO2000043527A1 - Varicella-zoster virus vaccines - Google Patents

Abstract

This invention relates to a novel fusion protein comprising (1) a Varicella Zoster Virus gE protein or an immunologically active fragment thereof fused to (2) a different protein of Varicella Zoster Virus or an immunologically active fragment thereof, and to vaccine compositions comprising the fusion protein.

Description

VARICELLA-ZOSTER VIRUS VACCINES

The present invention relates to compositions for the treatment or prevention of Zoster in individuals infected with Varicella Zoster virus (VZN), and to the prevention or treatment of Varicella infections.

Varicella Zoster Virus is a human alpha herpes virus which causes two human diseases: on primary infection VZV causes childhood chicken pox (Varicella) thereafter the virus becomes latent and frequently reactivates (often decades later) to produce shingles (Zoster). During chicken-pox, the virus penetrates the peripheral nervous system where it remains latent until reactivates as the painful Zoster form. Whilst the virus is latent the expression of most viral genes are repressed. It is believed that cell mediated immunity plays a crucial role in the control of latency, since reactivation as Zoster (or shingles) is frequent in the elderly or in immunocompromised individuals.

VZV infection is characterized by minimal presence of free virus. During latency and reactivation virus is mainly intracellular. Accordingly, recurrent disease is not prevented even by high levels of neutralizing antibodies and virus control depends on cell mediated immunity. In order to obtain protection by vaccination, it is therefore desirable to induce not just an antibody response, but also a cytotoxic T lymphocyte (CTL) response. An effective vaccine should prime CTL capable of acting as early as possible as soon as signs of reactivation of latent virus appear.

As the mechanism of antigen recognition by cytotoxic T lymphocytes (CTL) involves breakdown of native antigen into peptides, binding of the proteolytic fragments to MHC molecules and export of the complex to the cell surface, any virus coded polypeptide not just those that are integral membrane proteins like the glycoproteins, can be a potential target of a T cell mediated response. However since the VZV genome codes for several non structural proteins and internal virion proteins, in addition to external glycoproteins, this results in a large number of potential CTL targets and it is not known which protein would be the most relevant. The genome of VZ Virus is composed of 71 open reading frames, encoding 68 proteins. The sequence of the virus DNA is known (Davison and Scott 1986). The Varicella Zoster Virus protein gE is encoded by the open reading frame 68 and is the most abundant and immunogenic protein amongst the 6 Varicella-Zoster Virus envelope glycoproteins. It is predominant in VZV-infected human cell membranes (Keller et al 1984; Montalvo and Grose 1986; Dubey et al 1988). Anti-gE antibodies have been detected in subjects with natural primary infection and in subjects vaccinated with the live OKA- VZV vaccine. It has been demonstrated that gE is more immunogenic than gB and gH, the two other predominant glycoproteins of VZV (Brunell et al 1987). A monoclonal antibody raised to gE is capable of neutralising infectious virus in vitro (Grose 1989).

The present invention provides a fusion protein comprising (1) a Varicella Zoster Virus gE protein or an immunologically active fragment thereof fused to

(2) a different protein of Varicella Zoster Virus or an immunologically active fragment thereof.

Different proteins of Varicella Zoster Virus include any other structural and non structural proteins from VZV, other immediate early or early proteins or proteins such as gB, gH, gC or gl.

In a further aspect of the invention there is provided recombinant DNA or RNA encoding the fusion protein of the invention. The recombinant DNA or RNA of the invention may form part of a vector, for example a plasmid, especially an expression plasmid from which the fusion protein may be expressed, or a recombinant live microorganism, such as a virus or bacterium. Such vectors also form part of the invention, as do host cells into which the vectors have been introduced.

In yet another aspect of the invention there is provided a vaccine composition comprising a fusion protein according to the invention, or comprising a nucleic acid encoding the fusion protein of the invention in combination with a pharmaceutically acceptable excipient.

In a preferred aspect the fusion protein of the invention comprises as component (2) the VZV immediate early protein IE 63 or an immunologically active fragment thereof.

The Varicella Zoster Virus immediate early protein IE 63 (also called iep34) is encoded by open reading frame 63. It has a predicted molecular mass of 30.5kDa (Debrus et al, J. Virology 69(5), 1995 p 3240) and is expressed in the early phases of the VZV infectious cycle. The gene starts at 1 10581 (START CODON) and goes through to 111414 (STOP CODON). Another copy of the gene in reverse orientation is found between base 119316 to 118483. The role of IE63 (also termed ie63) in the cell-mediated immune response has been demonstrated. Rather unusually the IE 63 protein has been shown to be expressed in a rat model, in neurons during latency. The protein has been expressed as a fusion protein in E.coli (Debrus et al).

It has now been discovered that VZV IE 63 protein is detected exclusively in the cytoplasm of neurons of latently infected human trigeminal and thoracic ganglia. This is the first identification of an alpha-herpesvirus protein expressed during latency in the human nervous system. The VZV IE63 protein is an important target for the immune system, and in particular is a T cell response target and thus is useful in the prevention and treatment of VZV infections, in particular, in the prevention of Zoster in patients already infected with VZV.

It has now been suφrisingly found that a vaccine composition comprising a fusion protein of VZV gE and VZV IE63 stimulates both humoral and cell-mediated responses against both gE and IE63. The level and quality of these responses is similar to that observed by immunisation with individual gE and IE63 proteins but the fusion protein gE-IE63 can be purified more easily than the proteins alone and thus shows a clear industrial advantage. Accordingly, the present invention preferably provides a fusion protein comprising

(1) a Varicella Zoster Virus gE protein or an immunologically active fragment thereof fused to

(2) a Varicella Zoster Virus IE63 protein or an immunologically active fragment thereof.

This is the first medical use for such a protein and thus the invention accordingly provides the fusion protein combining VZV gE and a structural or non-structural protein of VZV, in particular VZV IE63 for use in medicine.

In order to construct the DNA encoding a fusion protein according to the invention, cDNA containing the coding sequences of the proteins to be fused may be manipulated using standard techniques [see for example Maniatis T. et al Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y. (1982)].

In a further aspect of the invention there is provided a method of treating a human susceptible to or suffering from VZV infection, which comprises administering to a human, a safe and effective amount of a vaccine composition according to the invention.

The amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise 1-1000 μg of protein, preferably 2-100 μg, most preferably 4-40 μg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several booster immunisations adequately spaced.

In addition to vaccination of persons susceptible to VZV infections, the vaccine compositions of the present invention may be used to treat, immunotherapeutically, patients suffering from VZV infections, in order to prevent or significantly decrease recurrent disease, frequency, severity or duration of shingles episodes.

In a preferred vaccine of the invention, an aqueous solution of the fusion protein combining VZV gene gE and VZV gene IE63, can be used directly. Alternatively, the fusion protein, with or without prior lyophilization, can be mixed together or with any of the various known adjuvants. Such adjuvants include, but are not limited to, aluminium hydroxide, muramyl dipeptide and saponins such as Quil A, in particular QS21 or 3 Deacylated monophosphoryl lipid A (3D-MPL), and CpG oligonucleotides (see University of Iowa WO96/02555). Adjuvants or adjuvant systems that preferentially induce a TH1 response are preferred. Adjuvants which are capable of preferential stimulation of the TH1 cell response are described in International Patent Application Nos. WO 94/00153 and WO 95/17209.

A particular preferred adjuvant comprises QS21 and 3 De-O-acylated monophosphoryl lipid A (3D-MPL), optionally together with an oil in water emulsion.

3 De-O-acylated monophosphoryl lipid A is known from GB 222021 1 (Ribi). Chemically it is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in International Patent Application No. WO 92/16556.

QS21 is a Hplc purified non-toxic fraction of a saponin derived from the bark of the South American tree Quillaja Saponaria Molina and its method of its production is disclosed (as QA21) in US patent No. 5,057,540.

A preferred oil-in-water emulsion comprises a metabolisable oil. such as squalene, alpha tocopherol and tween 80. Additionally the oil in water emulsion may contain span 85 and/or lecithin. The ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10 : 1 ; preferably 1 : 5 to 5 : 1 and often substantially 1 : 1. The preferred range for optimal synergy is 2.5:1 to 1 :1 3D-MPL: QS21. Typically for human administration QS21 and 3D-MPL will be present in a vaccine in the range 1 μg - 100 μg, preferably 10 μg - 50 μg per dose. Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% Tween 80. Preferably the ratio of squalene: alpha tocopherol is equal or less than 1 as this provides a more stable emulsion. Span 85 may also be present at a level of 1%. In some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser.

As a further exemplary alternative, the fusion protein gE-IE63, can be encapsulated within microparticles such as liposomes. In yet another exemplary alternative, the VZV fusion gE-IE63 protein can be conjugated to an immunostimulating macromolecule, such as killed Bordatella or a tetanus toxoid.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, Voller et al. (eds), University Park Press, Baltimore, Maryland, 1978. Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, US Patent 4,372,954 and Armor et al, US Patent 4,474,757. Use of Quil A is disclosed by Dalsgaard et al., Acta Vet Scand, 18:349 (1977). 3D-MPL is available from Ribi immunochem, USA, and is disclosed in British Patent Application No. 2220211 and US Patent 4912094. QS21 is disclosed in US patent No. 5057540.

The use of the polynucleotide of the invention in genetic immunisation will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al, Hum Mol Genet 1992, 1 :363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNA complexed with specific protein carriers (Wu et al., J Biol Chem 1989: 264, 1985), co-precipitation of DNA with calcium phosphate

(Benvenisty & Reshef, PNAS, 1986:83, 9551), or with other facilitators, encapsulation of DNA in various forms of liposomes (Kaneda et al., Science 1989:243,375), particle bombardment (Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol 1993, 12:791) and in vivo infection using cloned retroviral vectors (Seegar et al, PNAS 1984:81,5849) or non-infectious, recombinant viral vectors, such as Semliki Forest helper 2 system (Berglund) et al Bio/Technology 1993, 1 1 :916-920. Suitable promoters for muscle transfection include CMV, RSV, Sra, actin, MCK, alpha globin, adenovirus and dihydrofolate reductase. A preferred way of delivering a polynucleotide in accordance with the invention utilitises gold particles delivered by means of a gene gun (US 5100792, US 5036006, and 4945050).

The polynucleotide compositions of the invention may be administered using the dosages and routes of administration described in WO 90/11092. It will be apparent however that the precise dosage will depend on factors such as the weight, sex, mode of administration, general health of the patient and the condition to be treated. Nonetheless for intramuscular use the dosages to be employed will typically be in the range of 0.05μg/kg to about 50 mg/kg, more typically from about 0.1 to 10 mg/kg. Subcutaneous, epidermal, intradermal or mucosal administration may be advantageous, due to the ability to induce high levels of CTL.

The vaccine of the present invention may additionally contain other antigenic components such as VZV gE, gB, gH, gl or gC or their truncated or anchorless derivatives or other immediate early proteins such as IE62 protein. In particular truncated gE, gB or gH as disclosed in European Patent application published under No. 0 405 867.

In a preferred embodiment of the present invention there is provided a vaccine composition comprising an anchorless VZV glycoprotein, especially gE, in combination with the fusion gE-IE63 protein.

By anchorless VZV glycoprotein is meant, a VZV glycoprotein derivative which is devoid of substantially all of the C-terminal anchor region and which allows for secretion when expressed in mammalian cells. Such proteins are described in EP-A-

0405867. In an alternative embodiment, a polynucleotide capable of expressing the gE-IE63 fusion protein may be used directly as a nucleic acid vaccine. Preferably the polynucleotide is DNA. In such cases, the polynucleotide will be under the control of a suitable promoter.

RNA immunisation may also be used. RNA in such cases, may be prepared in accordance with the methods of WO92/10578.

The invention will now be further described in the following Examples, which are illustrative only and do not in any way limit the scope of the invention.

EXAMPLES

Example 1

I. MATERIAL AND METHODS

1.1. Cloning and expression

I. La. Cloning and expression of amino extended ie63

The expression plasmid pNIV2094, encoding ie63, was constructed in 5 steps:

• A 4027 bp Sspl-Sspl DNA fragment was excised and recovered from the pUC19 plasmid carrying the genomic EcoRlA fragment of VZV, blunted and then inserted into the pUC 19 Hindi site, yielding the intermediate construct pNIV2032.

• A 832 bp Styl-Styl DNA fragment was recovered from pNIV2032, blunted and inserted into the pUC19 Hindi site, yielding the intermediate construct pNIV2052.

• A pair of synthetic oligonucleotides was inserted between Ncol and BamHl sites of pΝIN2052 to restore the 3' end of the ie63 coding sequence including the stop codon and to create a 3' BamHl site. This intermediate construct has been named pΝIV2037.

• A 856 bp Pstl-BamHl DNA piece was recovered from pNIV2037, blunted and inserted in the EcoRN site of the E.coli expression vector pMG81kn.

• The last step involved an excision by Ndel restriction followed by ligation to yield the final plasmid pNIV2094. The restriction map of pNIN2094 is shown in Figure 1 A. By construction, the protein when expressed via plasmid pΝIN2094 carries 10 excedentary Ν-terminal amino acids corresponding to the amino-terminal part of ΝS1 protein: MLTSTRWSKD [SEQ ID NO: 1 ].

For expression, plasmid pNIN2094 was used to transform E.coli strain AR58 [F^~ r⁺m⁺rpsL31 galk2 recA13 galE : Tnl0(Tet^R)Δ8(chlD-pgl)]. Recombinant bacteria were then grown in rich LB medium at 30°C up to and OD₆₂₀ of 0.35, then induced at 42°C for two hours to achieve expression and production of the protein.

Total proteins from induced and non-induced bacteria were separated onto SDS- PAGE; the data show that the induced bacteria produce a 43 kDa protein at about 5% of total proteins. This 43 kDa species is recognized in Western blot by a mouse polyclonal antiserum raised against an ie63 peptide (residues 70-86) and by a rabbit polyclonal antiserum raised against the complete ie63 protein.

I. b. Cloning and expression of authentic ie63 protein

The expression plasmid pΝIN4807, encoding the authentic ie63 protein, has been constructed as follows:

Plasmid pΝIV2094 was restricted with Aflll and Hpal to eliminate a DNA fragment comprising 18 nucleotides upstream to the ATG initiation codon and 66 nucleotides downstream to the ATG codon. It was replaced by a synthetic oligonucleotidic pair to reconstitute the missing part of the promoter and the 5' end of the ie63 coding sequence. The sequence of the oligonucleotides reads as follows :

5'TTAAGGAGGATATAACATATGTTTTGCACCTCACCGGCTACGCGGGGCG CCTCCTATATTGTATACAAAACGTGGAGTGGCCGATGCGCCCCGC

ACTCGTCCGAGTCAAAACCCGGGGCATCGGTTGATGTT 3 ' TGAGCAGGCTCAGTTTTGGGCCCCGTAGCCAACTACAA 5'

[SEQ ID NO: 2] For expression, plasmid pNIV4807 was used to transform E.coli strain AR58 as described above.

I. I.e. Cloning and expression of the DNA encoding the fusion protein gE-ie63. pNIV4801

• The starting material for this construction consisted 1) of pNIV2088 (a mammalian expression vector, pEE14 (Celltech), carrying in its Hindlll/Smal sites, a blunted Hindlll/Bcll DNA piece corresponding to the coding sequence of anchor-less gE protein), and 2) of pNIV2097, a plasmid carrying a gE-gB-ie63 fusion. The purpose of this plasmid is to provide convenient access to ie63 DNA for simple restrictions.

• Plasmid pNIV4801 was constructed by ligating four DNA fragments: - a 10.258 bp EcoRl/BstEll DNA piece excised from pNIV2088, covering the sequence of the pEE14 vector, an untranslated genomic sequence corresponding to the 56 bp preceding ORF68 in the VZV genome and the sequence coding for amino acids 1-316 of gE. a 674 bp BstElll Avail fragment also recovered from pNIV2088 and specifying amino acids 317 to 540 of gE. a Avall-Nspl 15/22 bp synthetic oligonucleotidic pair, coding for amino acids 541 to 546 of gE and followed by 3 bp allowing, via the Nspl site, the in frame fusion with the first ATG codon of ie63, which thus now encodes an internal methionine. 5' GACCGGAGGGCTTGCAGACATG 3' 3' GCCTCCCGAACGTCT 5' [SEQ ID NO: 3] a 841 bp NspI/EcoRI DNA fragment recovered from pNIN2097 and coding for amino acids 2 to 278 of ie63.

The final expression plasmid. pΝIN4801, thus, codes for a gE (signal", anchor^", aa 1- 546) x ie63(aal-aa278) fusion protein. (Fig. IB). The gE coding sequence is without anchor and contains ΝLS (nuclear localisation signal) of the transactivator ie63 protein also known as iep34. The full DNA sequence for pNIV4801 is given below as SEQ ID NO:4. SEQ ID NOS: 5 and 6 contain the DNA insert in the vector pEE14 and the fusion protein encoded by it. Note: pNIV4801, is also referred to as pRIT14583.

For expression, plasmid pNIV4801 was used to transfect CHOKl cells. After a 25 μM MSX selection, clones supernatants were tested in Western blot with a monoclonal antibody raised against gE.

1.2. Cultures

I.2.a. Fermentations

10 L fermentors were inoculated with precultures of recombinant E.coli bacteria carrying plasmids pNIV2094 or pNIV4807. The culture medium, Opti 3X-glucose, consists of 0.66% bactotryptone; 1.5% yeast extract; 1.5% NaCl; 0.3% KH₂P0₄; 1 % glucose and 20mg/L kanamycin. Fermentation proceeded at 30°C and pH 7.0. When the culture reached an OD₆₂₀ of 13, the temperature was shifted to 42°C and induction of ie63 expression was maintained for 2 hours. Bacteria were then harvested by centrifugation for 30 min at 12000 rpm and stored at -20°C.

I.2.b. Cultures in cell factories of a CHOKl recombinant cell line (gE-ie63 n°22) expressing the gE-ie63 fusion protein. The recombinant CHOKl cell line, gE-ie63 n°22, was cultivated in 6000 cm² cell factories (NUNC) in GMEM medium (Gibco) supplemented with 2% fetal calf serum (Gibco) and 2mM sodium butyrate. Medium harvesting (1.700ml) was performed every three days. Pools of four successive harvests, stored at -20°C before use, were engaged in the purification process (see below).

1.3. Purification

1.3. a. Purification of amino-extended ie63 protein Bacteria (60g), harvested from fermentors, were resuspended in buffer 20mM Tris- HC1 pH7.5, ImM aprotinin and ImM AEBSF, then lysed in a French pressure cell (2 passages at 15000 psi). Cell debris were eliminated by a first centrifugation (30 min at 15000 rpm, SS34 rotor) and the resulting supernatant was further clarified by ultracentrifugation (1 hour at 45000 rpm, rotor 60TI). The clear lysate was then applied onto a Q-sepharose fast flow column (15 x 2.6 cm Pharmacia) equilibrated in 20mM Tris-HCl pH 7.5 buffer. After extensive washing of the column with the same buffer, bound proteins were eluted stepwise from the column by increasing NaCl concentration in the buffer.

The ie63 protein elutes from the column at 400mM NaCl; the ie63 -containing fractions were immediately applied onto a Blue Trisacryl Plus LS column (7 x 2,6 cm, Biosepra), conditioned in 20mM Tris-HCl pH 7.5 buffer. After loading and washing the column extensively with the same buffer, the resin was eluted stepwise with increasing NaCl concentrations (500, 1000 and 2000 mM). The ie63 protein was recovered in fractions 1000 and 2000 mM NaCl. These were pooled and applied onto a Ni²⁺ chelate sepharose fast flow column (12 x 1 cm, Pharmacia), conditioned in PBS buffer, pH 7.5 containing 500mM NaCl. After washing of the column with starting buffer, proteins were eluted by increasing imidazole concentration in the column buffer. The ie63 protein was recovered from 20 and 30mM imidazole fractions and its purity estimated at more than 90%>.

I.3.b. Purification of gE-ie63 fusion protein

Spent culture medium (71, four harvests) was applied onto a Q-sepharose fast flow column (15 x 2,6 cm Pharmacia) conditioned in 20 mM Tris-HCl pH 7.5. After washing, proteins were released from the column by stepwise addition of NaCl in conditioning buffer. The gE-ie63 protein was recovered in the 1000 mM NaCl fraction. This material was then adjusted to 1000 mM (NH₄)₂S0₄ and applied onto a butyl TSK column (8 x 2,6 cm Tosohaas), conditioned in PBS buffer pH 7.5. 1000 mM (NH₄)₂S0₄.The column was first washed with PBS buffer supplemented with 200 mM (NH₄)₂S0₄ and then eluted with water to recover the gE-ie63 protein. The enriched fraction was further purified onto a Ni²⁺ chelate sepharose fast flow column (12x1 cm, Pharmacia) conditioned in PBS buffer, pH 7.5, 500mM NaCl. After washing of the column, bound proteins were eluted with increasing imidazole concentrations. The gE-ie63 protein eluted at 100 mM imidazole and was pure at more than 80%.

I.3.C. Purification of gE proteins

The purification protocol of the anchor-less gE protein, produced by engineered

CHOKl cells, has been published by us in Virus Research 40, 199-204 (1996).

1.4. Immunology

1.4. a. Animals and immunisations

8 weeks old female BalbC mice and 500 to 600 g female Dunkin Hartley guinea pigs were obtained from Iffa Credo.

First immunisation experiment

Four groups of 4 mice were twice immunised via footpads at 28 days interval.

Bleedings were performed on days 28 and 42. On day 42, mice were sacrificed to recover spleens and poplital lymph nodes. Groups 1 and 2 received 4 μg of amino- extended ie63 adjuvanted either with 3D-MPL + QS21 + an oil in water emulsion containing squalere, α-tocopherol and TweenδO (as described in W095/17210); or 3D-MPL + a less reactogenic form of QS21 (as described in W096/33739). Groups 3 and 4 received adjuvants only.

Second immunisation experiment

Five groups of 6 guinea pigs were twice immunised subcutaneously at 28 days interval. Bleedings occurred on days 28, 42 and 72. All antigens (8μg/guinea pig) were adjuvanted with an adjuvant composition "X" which is MPL 50 μg / QS21 50 μg / an oil in water emulsion of squalene + tocopherol + Tween 80, in a total of 500 μl. Group 1 received protein gE, group 2 received amino-extended ie63 protein, group 3, the fusion protein gE-ie63, group 4 gE and amino-extended ie63 separately, and group 5 the adjuvant.

Third immunisation experiment Five groups of 6 mice were twice immunised via footpads at 28 days interval. Bleedings occurred at days 28 and 42. On day 42, mice were sacrificed to recover spleen and poplital lymph nodes. All antigens (5μg/mouse) were adjuvanted with adjuvant X as described. The five groups are identical to those described in the second immunisation experiment.

I.4.b. ELISA tests a) IgG specific

Plates were coated with the gE protein (500ng/well), the gE-ie63 fusion protein

(200ng/well) or the amino-extended ie63 protein (20ng/well), for 16 hrs at 4°C. Plates were then washed 5 times with lOOμl per well of TBS-Tween (50mM Tris-HCl pH 7.5, 150mM NaCl, 0.1% Tween 80) and saturated for 1 hr at 37°C with 150μl of the same buffer supplemented with 1% BSA. 100 μl serial dilutions of sera to be tested were then incubated for 1 hr at 37°C. Plates were washed 5 times with TBS-Tween buffer and antigen-bound antibodies were detected with the second antibody (goat antimouse or goat antiguinea pig, Promega, USA) coupled to alkaline phosphatase (dilution 1/7500 in TBS-Tween buffer). The enzymatic activity was measured using the paranitrophenylphosphate substrate (Sigma) dissolved in diethanolamine buffer (pH 9.8). OD₄|_5nm was measured in a Biorad Novapath ELISA reader.

b) IgG 1 and IgG2a specific

Plates were coated and saturated as described above. lOOμl serial dilutions of sera to be tested were incubated for 1 hr at 37°C. Antigen-specific IgGl and IgG2a were detected with biotin-labelled antibodies against IgGl and IgG2a (rat antimouse, dilution 1/7000 in TBS-Tween buffer and 1% BSA, Biosource). Phosphatase alkaline- conjugated streptavidin (1/1000 dilution, Amersham) was then added to each well. Measure of the enzymatic activity proceeded as described above. 1.4. c. Neutralisation assays

VZV virus (Webster strain) was diluted up to a concentration of 4.10² pfu/ml in VZV buffer (PBS pH 7.1 ; 5% saccharose, 1% giutamate. 10% Fetal Calf serum). lOOμl of the suspension were incubated for 1 hr at 37°C with lOOμl of serial dilutions of sera to be tested (after complement inactivation) in VZV buffer. 2μl of guinea pig serum (Gibco-Grand Island) were added to each well. The mixture was then added onto confluent MRC5 cells and incubation proceeded for 2 hrs at room temperature in the dark. 800 μl of culture medium (BME, 2% FCS) were added and cells were allowed to grow for 7 days. At this time, spent medium was discarded and cells were fixed then stained with Coomassie blue for 10 minutes (0.5%o R250; 45% methanol, 10% acetic acid). The neutralising titre was expressed as the reciprocal of the serum dilution leading to a 50% reduction in the number of viral plaques.

1.4. d. Coupling gE and amino-extended ie63 to sepharose, depletion assays gE and ie63 proteins were coupled to activated CH4B sepharose (Pharmacia, Sweden). One milligram of gE or amino-extended ie63, prealably dialysed against the coupling buffer (0.1M NaHC0₃, 0.5M NaCl, pH 8) was incubated with 3ml of activated resin and 9ml of coupling buffer. The coupling efficiency was measured by dosing residual gE or ie63 proteins by ELISA. After coupling, residual reactive groups were blocked with 0.1M Tris-HCl, pH 8 and the gel was washed three times at pH 4 and pH 8, alternatively. For depletion assays, lOOμl of the serum to be tested were diluted five fold in PBS buffer and incubated with lOOμl of coupled gel for 16 hrs at 4°C. The supernatant was collected and the gel washed twice with 250μl of PBS. The negative control for depletion was achieved on BSA-coupled sepharose. After depletion, residual antibodies were undetectable in ELISA.

1.4. e. Proliferation assays

Lymphocytes were isolated from spleens and poplital lymph nodes. 4.10⁵ cells, in triplicate, were incubated with serial dilutions of antigens in 96 well plates (Nunc) for 4 days (10 base 2 dilutions of the antigen were tested, starting from a concentration of 25μg/ml). Cell proliferation was shown by measuring ³H-thymidine incorporation during 16 hours of proliferation. Negative controls were irrelevant antigens, obtained from bacterial or CHO sources and purified similarly as the antigens of interest.

OIL RESULTS AND DISCUSSION

ILL Cloning and expression of ie63

The expression level for authentic ie63 was of the same order of magnitude as the one measured for amino-extended ie63. For the immunological studies, the amino- extended ie63 protein was used since authentic ie63 was not available at that time.

II.2. Purifications of the proteins

II.2.a. Fermentation and purification of amino-extended ie63 protein

A fermentation protocol, suitable for the production of large quantities of recombinant ie63, has been developed starting from bacteria transformed with pNIV2094. Induction conditions for expression have been optimalised for 10L batch fermentations. Western blot analysis shows that the addition of lOg/L of glucose during the induction period extends bacterial growth and triples the expression level for ie63. Addition of 2g/L yeast extract had no such effect. pH regulation during induction appears crucial for maintaining ie63 in solution, preventing aggregation. At pH 7.0, 70% of ie63 protein remain soluble, whereas this percentage drops to 20%> in absence of pH regulation. The ie63 protein was purified in three steps; an anion exchange, a Blue trisacryl step and a metal chelate step. The final yield of the purification process was 250 μg ie63 per gram of packed bacteria. The molecular weight, estimated by SDS-PAGE, was 40 kDa. Lower Mw bands, also recognised by Western blot with anti ie63 polyclonal antibodies, represent degraded forms of the protein.

II.2.b. Purification of the fusion protein gE-ie63 The fusion protein gE-ie63 was purified in three steps: an anion exchange, an hydrophobic interaction and a metal chelate step. The protocol closely resembles the one used for gE, except that the fusion protein elutes from the Q-sepharose column at a higher NaCl concentration (1000 mM versus 300 mM). The final yield was 1 mg per liter of cell factory-derived spent medium. The fusion protein migrated on SDS-PAGE as a wide band of 180 kDa. together with finer 130, 85 and 75 kDa subspecies. All these products were recognized, in Western blot, by the monoclonal antibody βD6 raised against gE and by rabbit polyclonal antibodies raised against ie63. The amino terminal sequence of these 4 bands was determined by Edman degradation; all sequences proved identical and corresponded to the N-terminus of gE protein. Apparently, lower Mw products resulted from the degradation of the ie63 moeity of the fusion protein.

II.3. Immunology

II.3. a. First Experiment Groups of mice and immunisation schedule have been described in the Material and Methods section. It was found that the mean titer of specific IgGs was significantly lower for group 1 (ie63 + 3D-MPL + QS21 + an oil in water emulsion) than for group 2 (ie63 + 3D-MPL + QS21), i.e. 8864 versus 21148 (see figure 2). Control groups gave ELISA signals below the detection threshold. Mean titer for IgGl and IgG2a in sera of immunised animals were determined (see figure 3 and table 1 below).

Table 1

Again, the mean titer for IgGl and IgG2a in group 1 was lower than for group 2. However, the IgGl/IgG2a ratio is higher in group 1 than in group 2 (1.58 versus 1.05). which seems to indicate that the formulation of ie63 with 3D-MPL + QS21 + an oil in water emulsion biased the immune response towards a TH2 type, more clearly than with the other adjuvant. The lymphoproliferative response was measured after 3, 4 or 5 days incubation of the lymphocytes with the antigen. Responses were shown in Figure 4 (spleen cells derived from all 4 mice in each group were pooled as well as cells derived from poplital lymph nodes). Proliferation was observed only for group 1 (ie63 + 3D-MPL + QS21 + an oil in water emulsion).

At high concentrations of antigen, the proliferation index was around 10 for both spleens and poplital lymph nodes. Although 3 days are sufficient to read the response for spleen cells, experiments were generally performed after 5 days, the necessary time span for the read out of poplital lymph nodes. The failure to observe a lymphoproliferative response in group 2 appears to be due to a technical problem since the humoral response in this group was quite satisfactory.

In order to check the secretion of IFNγ and IL-5, two cytokines typical respectively of TH1 and TH2 responses, an aliquot of culture supernatants was tested. As seen in Figure 5, both IFNγ and IL-5 were found, in agreement with the presence of IgGl and IgG2a in mice sera. The highest concentrations for the two cytokines were obtained after 5 days of stimulation with ie63.

II.3.b. Second experiment

The humoral response, induced by the injection of proteins gE, ie63 or gE-ie63 was studied in guinea pigs. Groups and immunisation schedule have been detailed in the Materials and Methods section. ELISA titers and curves are shown in Table 2 and Figure 6.

As a rule, mean ELISA titers were lower at day 72 (44 days post 2^nd injection) than at day 42 (14 days post 2^nd injection). The humoral response appeared higher with the gE + ie63 formulation than with the gE formulation or the gE-ie63 fusion formulation when measured in gE ELISA. The same observation was done when comparing the gE + ie63 formulation to the others as measured in ie63 ELISA. However, in the fusion ELISA, the gE + ie63 formulation gave titers of the same order of magnitude than the gE-ie63 fusion formulation.

Neutralisation assays were performed with guinea pigs sera, for the three bleedings (see Material and Methods). The mean neutralising titers are shown in Figure 6.

The gE-ie63 fusion protein induced a titer in neutralising antibodies higher than those measured for gE or for gE + ie63, injected simultaneously. To the contrary of ELISA titers, neutralising titers did not decrease between days 42 and 72. Note that injection of ie63 did not induce detectable neutralising antibodies. This is also apparent when comparing neutralising titers induced by gE and gE + ie63, which for each bleeding are similar.

To check the significance of the difference between neutralising titers induced by gE and gE-ie63 fusion, sera from the 2^nd and 3^rd bleeding of guinea pigs immunised with the fusion protein were depleted on sepharose-immobilised gE or ie63. ELISA curves for sera from group 3, before and after depletion, are shown in Fig. 7 and 8. Depleted sera were then assayed for neutralisation, as shown in Table 3. Depletion on immobilised ie63 did not lead to any significant decrease of the neutralising titer whereas depletion on the gE sepharose column abolished the neutralising capacity. It can thus be concluded that the fusion between gE and ie63 did not create new epitopes.

II.3. c. Third experiment

Groups and immunisation schedule were identical than in the second experiment. The injected dose was however different (5μg) and the animals were mice instead of guinea pigs. Mean ELISA titers for specific IgGs were again measured. The gE + ie63 formulation did not induce a higher response (Figure 9) as was seen with guinea pigs. Whatever the ELISA assay, the titer for this formulation was the lowest of all. IgGl and IgG2a titers were similar in all three cases of formulation (Figure 9). Spleen cells and poplital lymph nodes were stimulated for 5 days with decreasing concentrations of gE, ie63 or gE-ie63 fusion.

For group 1 (gE), no significant proliferation occurred by in vitro stimulation with gE.

For group 2 (ie63), lymphocytes proliferated in function of the dose of ie63 (Figure 10). Using an irrelevant protein as negative stimulator, no proliferation was detected, indicating that the stimulation by ie63 was specific, confirming data obtained in the first experiment (II.3. a). The fusion protein induced a significant proliferation in only 1 out of 6 mice.

For group 3 (gE-ie63 fusion), no stimulation occurred with gE. However, ie63 induced a significant proliferation of spleen and poplital lymph nodes cells (Figure 1 1). This effect is specific since the injected fusion protein derived from CHO cells, whereas ie63 derives from bacteria.

Restimulation of lymphocytes with the fusion protein did not lead to interpretable results since the proliferative index was similar to that measured with an irrelevant protein also derived from CHO cells.

For group 4 (gE + ie63), results confirmed those obtained for groups 1, 2 and 3. gE did not induce proliferation, ie63 induced proliferation in 4 out of 6 mice. The data for gE-ie63 were not interpretable.

1III. GENERAL CONCLUSION

Immunisation of mice and guinea pigs with the gE-ie63 fusion protein stimulates both humoral and cell-mediated responses (against ie63 and gE).

The level of these responses is similar to that observed by immunisation with individual gE and ie63 proteins. Since gE-ie63 can be purified more easily than gE and ie63. it has an obvious industrial advantage. Example 2

Expression of gE-ie63 of VZV in CHO-K1, in suspension Transfection of CHO-K1 cells adapted to grow at suspension, serum free was performed using the same plasmid construct pEE14-gE-ie64 (but different DNA plasmid preparation).

New CHO-K1 clones growing in suspension, serum free (S/SF) conditions were generated. Their expression level was being evaluated by WB and Coomassie SDS- PAGE analysis.

1. Starting material

Plasmid DNA pNIV 4801 was used to transform E. coli strain DH5α. Small scale plasmid DNA preparation were made. EcoRI/Hindlll restriction analysis indicated presence of 2 populations of DNA plasmids as doublet bands at +/- 8900-9200 bp and +/- at 2200-2400 were detected instead of the expected 9200 bp, 1281 bp and 1258 bp bands. Therefore E. coli competent cells of strain HB101 and DH5α were transformed. Transformants in DH5α were obtained. Small scale DNA preparations were made and DNA digested with EcoRI/Hindlll. 3 candidate clones out of 12 contained the correct plasmid DNA's. Large plasmid DNA preparation was done using the CsCl-EtBr ultracentrifugation method (twice). Candidate plasmid DNA pEE14 gE-ie63 n° 17/2 was chosen and named pRIT15077 and used further for CHOKl transfections.

Several restriction enzyme analyses were done on the plasmid # 17/2 and the whole insert gE-ie64 and the flanking 5' and 3' regions were sequenced and found OK.

2. Transfection of CHO-K1 S/SF

Transfection of CHO-K1 cells derived from Master Cell Bank MCB CHO-K1 028W 1996/2 SHF P31 at p40 (suspension, serum free) was done with plasmid pEE14 gE- ie63 # 17/2 (also named pRIT 15077) using the classical DNA Ca-Phosphate co- precipitation technique. Cells were counted 48 hr post transfection and transferred into 96-well plates (Costar) at 5000 cells/well.

Transfectant clones were selected according to a modified procedure of the GS expression system described by Crockett et al. (Biotechnology 1990, vol. 8, p 662) and amplified in the presence of 30 μM methionine sulphoximine (MSX) in GMEM medium (Gibco) containing no glutamine and supplemented with additives (glutamate/asparagine/nucleosides) and 5 %> dialysed FBS (Foetal Bovine Serum, QA SBBio certified).

Cells were washed with fresh medium three times in the first week and two times in the second week after transfection. At the third wash, 20 % conditioned medium was added and then cells were washed each 3-4 days.

Following transfection, MSX resistant transfectant clones were transferred into 24- well plates after 3-6 weeks and the culture supernatant were harvested and tested for expression (secretion) of gE-ie64 fusion protein by WB analysis using a specific gE monoclonal βD6.

Expression of gE-ie64 fusion protein was detected in 10 out of 22 clones tested (derived from 2 transfections).

Clones were selected for their highest expression and good growth and viability (clones n° 3, 7 and 8) and were readapted to suspension serum free conditions and further evaluated and characterized.

Expression was evaluated at 37 °C and 33 °C in the presence or not of sodium butyrate (2 mM). Expression (1-5 mg/L) of secreted gE-ie64 protein could be detected by WB analysis (βD6). and by Coomassie staining. A major band at 66kD and a minor band at 85 kD could be detected by WB analysis (βD6). This band pattern was rather different from the 4-band pattern obtained with expression in adherent culture and serum.

An experiment was carried out in which clone # 3 growing S/SF was cultivated in presence of 1 % serum in GMEM medium. After WB analysis of the culture supernatant a heterogeneous band pattern with 2 major bands at 130 kD and 66 kD band could be detected. It is expected that the 130 kD band would be the normally secreted fusion protein gE-ie63 containing glycosylation modifications.

Another transfectant clone, # 27, grows slowly (+/- 3 weeks behind the other clones) showing one major strong band at 130 kD in the presence of 5 % serum (24 well).

3. MSX amplifications

In order to increase further the expression level of the CHO-Kl gE-ie64 clones various concentrations (100, 250 and 500 μM) of MSX was added to the cell culture of clones 3, 7 and 8. Expression analysis, for +/- serum, and +/- protease inhibitors was performed.

B45165

Neutralizing titer before and after depletion

Table 3

DNA and Protein Sequences

DNA sequence of pNIN4801 \SEQ ID NO: 4]

GAATTCATTGATCATTAATCAGCCATACCACATTTGTAGAGGTTTTACTTG CTTTAAAAAACCTCCCACACCTCCCCCCCTGAACCTGAAACATAAAATGA ATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAAT AAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATT CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCCT CTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTG CTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCAC TTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCGTG GCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCG GCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGA GTCGCATAAGGGAGAGCGTCGACCTCGGGCCGCGTTGCTGGCGTTTTTCC ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCA CGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCT TGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTG GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTG AAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTG CGCTCTGCTGAAG CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAAC CACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCA AAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCA ATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCC

TGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGG CCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATT TATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCC TGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTAC AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGG TTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAG CGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAG TGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC CATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCT GAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGG GATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAA ACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCA GTTCGATGTAACCC ACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTG GGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGC GACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAG CATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTA GAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC CTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGG CGTATCACGAGGCCCTGATGGCTCTTTGCGGCACCCATCGTTCGTAATGTT CCGTGGCACCGAGGACAACCCTCAAGAGAAAATGTAATCACACTGGCTCA CCTTCGGGTGGGCCTTTCTGCGTTTATAAGGAGACACTTTATGTTTAAGAA GGTTGGTAAATTCCTTGCGGCTTTGGCAGCCAAGCTAGAGATCCAGCTTTT TGCAAAAGCCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGC TCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCA GCCATGGGGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATG GGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCT TTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTTGC TGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGG GGACTTTCCACACCCTAACTGACACACATTCCACAGGGAAGCTAGCTTGG AATTAATTCCCCGCCCCCTTCCAATACAAAAACTAATTAGACTTTGAGTGA TCTTGAGCCTTTCCTAGTTTTTGTATTGGAAGGGCTCGTCGCCAGTCTCATT GAGAAGGCATGTGCGGACGATGGCTTCTGTCACTGCAAAGGGGTCACAAT TGGCAGAGGGGCG

GCGGTCTTCAAAGTAACCTTTCTTCTCCTGGCCGAGCCGAGAATGGGAGT AGAGCCGACTGCTTGATTCCCACACCAATCTCCTCGCCGCTCTCACTTCGC CTCGTTCTCGTGGCTCGTGGCCCTGTCCACCCCGTCCATCATCCCGCCGGC CACCGCTCAGAGCACCTTCCACCATGGCCACCTCAGCAAGTTCCCACTTG AACAAAAACATCAAGCAAATGTACTTGTGCCTGCCCCAGGGTGAGAAAGT CCAAGCCATGTATATCTGGGTTGATGGTACTGGAGAAGGACTGCGCTGCA AAACCCGCACCCTGGACTGTGAGCCCAAGTGTGTAGAAGAGTTACCTGAG TGGAATTTTGATGGCTCTAGTACCTTTCAGTCTGAGGGCTCCAACAGTGAC ATGTATCTCAGCCCTGTTGCCATGTTTCGGGACCCCTTCCGCAGAGATCCC AACAAGCTGGTGTTCTGTGAAGTTTTCAAGTACAACCGGAAGCCTGCAGA GACCAATTTAAGGCACTCGTGTAAACGGATAATGGACATGGTGAGCAACC AGCACCCCTGGTTTGGAATGGAACAGGAGTATACTCTGATGGGAACAGAT GGGCACCCTTTTGGTTGGCCTTCCAATGGCTTTCCTGGGCCCCAAGGTCCG TATTACTGTGGTGTGGGCGCAGACAAAGCCTATGGCAGGGATATCGTGGA GGCTCACTACCGCGCCTGCTTGTATGCTGGGGTCAAGATTACAGGAACAA ATGCTGAGGTCATGCCTGCCCAGTGGGAACTCCAAATAGGACCCTGTGAA GGAATCCGCATGGGAGATCATCTCTGGGTGGCCCGTTTCATCTTGCATCGA GTATGTGAAGACTTTGGGGTAATAGCAACCTTTGACCCCAAGCCCATTCCT GGGAACTGGAATGGTGCAGGCTGCCATACCAACTTTAGCACCAAGGCCAT GCGGGAGGAGAATGGTCTGAAGTAAGTAGCTTCCTCTGGAGCCATCTTTA TTCTCATGGGGTGGAA GGGCTTTGTGTTAGGGTTGGGAAAGTTGGACTTCTCACAAACTACATGCCA TGCTCTTCGTGTTTGTCATAACCTATCGTTTTGTACCCGTTGGAGAAGTGA CAGTACTCTAGGAATAGAATTACAGCTGTGATATGGCAAAGTTGTCACGT AGGTTCAAGCATTTAAAGGTCTTTAGTAAGAACTAAATACACATACAAGC AAGTGGGTGACTTAATTCTTACTGATGGGAAGAGGCCAGTGATGGGGGTC TTCCATCCAAAAGATAATTGGTATTACATGTTAGCGGACATGGTCTGAAGC ACTTAGCAGCACATAGTACACGGACAGACACGTCCGACTAACGTATTTAT TGGTTTCTTATAAGTCATCGTCCGTCTCCGCCCTgggggggggggggggggggggggg gggggggggggggAAGACTGTCTGAAGCATTGAGACATAGGTCACAAGGCAGA CACAGCCTCCATCAATATTTATTGTTTCTTGAACTCATGCCTGGCTCCTGCC CGTTGAAGGACAGGTTTCCTAGGTGACAAGGTCAGACCCTCACCTTTACT GCTTCCACCAGCCAGGCACATCGAGGAGGCCATGGAGAAACTAAGCAAG CGGCACCGGTACCACATTCGAGCCTACGATCCCAAGGCGGCCCTGGACAA TGCCCGTCGTCTGACTGGGTTCCACGAAACGTCCAACATCAACGCATTTTC TGCTGGTGTCGCCAAGTCGAATCGATCCGATTGCGAGAGAATTATTAAGA CGCGCCCTCTGCAATGTGACCTTGCAGTGACAGAAGCCATCGTCCGCACA TGCCTTCTCAATGAGACTGGCGACGGAGCCCTTCCAATACAAAAACTAAT TAGACTTTGAGTGATCTTGAGCCTTTCCTAGTTCATGCCACCCCGCCCCAG CTGTCTCATTGTAACTCAAAGGATGGAATATCAACGGTCTTTTTATTCCTC GTGCCCAGTTAATCCTTGCTTTTATTGGTCAGAATAGAGGAGTCAAGTTCT TAAT

GCCTATACACCAACCTCATTTCTTTTCTATTTAGCTTTCTACGTGGGGGTGG GAGGGGTAGGGAGGGGTAGGCGAAGGGAACGTAACCACATGCTTCATCT CATCAGGAATGCCATGTCCAGTAGGCAGAGCTGCCAAAGGTGTTATCTGA GAGCTTTCACgggggggTGCTAGTTCTTCAGGTAAACCAACTTTCTATTCCAA ATGGAAGTTAGGTGAGGAGTAGTGGAGGAGTTAATGCCCTCCATGAAGAC AGCTCAGTGTATCACCTGAGACAGATGGGTAGCCCTACTGTAAAAGAAGG AAAAGTTATTTCTGGGTCCTCCATTTATAACACAAAGCAGTAGTATTTTTA TATTTAAATGTAAAAACAAAAGTTATATATATGATATGTGGATATATGTGT ATTTCTAATTCAGAAACCATCCTAGTTACTGGGTTTGCCAAGTTTGAAGAG CTTGGTTAACAAGAAAGGATCTCTTGAGTAGAGGTGGGGGTGCAGTACCA GGAAAGTGGTTATCTGGGGCTCAGCCCTTTATTACTATGTGGGGTTTCCCT GCCCACTCTGCAGGAGCAGATGCTGGACAGGTAGCCAGGGTGGGACACA GTGCTTGCCACCACCTGTCCCTGTGCTTAGGCCTAAGATGCATATCTATCC ACACAGAGTTAGCAGGATGGAGTTGGCTGGTCAACTTGAACATTTGTTAC TGATAGGGGTGGTGGGTTTATTTTTTGGTGGCATAGCATGTCACATAAAGC AGGCCTTTGATATATTAAATTTTTTTAAAGCAAACATGTTCAGCTTTATCA CCTTGTAGGGTTTCTAACTTTACAGAATTGCCTGTTTGTTTCAGTGTCTCCA TCCACTTTGCTCTTGGAGGAACGGAGGACAGGCAGACCTGGAGTTAAAAC ATTTGTCATTTTGTGTCATAGTGTCTACTTTCTCCCAGCCAGAATATTCCTT TCCTTCTTAGGAGTCCTATGGAGTTTTGTTTTTGTTTTTTTTCTATTACGATA AACATA

CCCCACCTCCATTCTGGCTTGCCCTGCTGTTCTCTGGTTGTTTGTGTGCTGT CCGCAGCAGGCTGCCTGTGGTTTTCTCTTGCCATAGCAGCTTCTAATTGCC ATGTACGATATGTTCGATTAGATAACTCCTCATGTAAACAGACTGTAACTG CCAGAGCAGCGCTTATAAATCAACCTAACATTTATAAGATTTCCTCTTGAC TTGTTTCTTTGTGGTTGGCGGAGGAAGAAAAAAAAAAGCTGCAGTATTTTT TTGTTCCTTCATTTCCTATCAAAAGAAAGGGGAGTGGTTCTGTTTTGTTTAC TCGCAAAATAAGCTTATCTATTGGCTTTTCTTTTTTTTTTTTTTTTTAAACGG GCTTTTTCTTGTACCTAAATTTGGGGTAAGGTGTGAGAGTTTTTATAGTTTT TTGAGACAGGGTCTTGGTGTATACCCTTGGCTGGCCCTGGAGCTAACTATG TAGACTGGGCTAGCCTTTAACTTGCAGTTCTGGTTTCAATTAGGGTTTATA CATTTAGTCTTGGCAATTCCTAGGTTCCACGTTTAATCTCTTTTACATTTCA AAGCAGTGTTATCTGAAGAGTTCAGGCGggCAGAGTgggggAATTCAATAGA GTTACggAAAAAGCTAAAAAACAAGTTTTAAATACCAAgGTTATGTTGGgG GgGTGGCggCTTTTCACAGCTCCgTGAAGGTTCGAGTTTGAAACTGAATAAT CGAGGGGTGAGAAAGTCTTGATCTTATCCCCACAGTATGGCACCAAGCCT GGCTGTGCCTTCTAGCTTAGTCTGCCCTGTTGCTATTTAAGCACTTTCTTCA CTAGGCAAAAATAAAAGGAGCTTCCTCCTTTGCCATGGCGCTGTGCATGA TACGAAAAGGTAGCTATCTACTAGCATATTAACTCCACTGTTTTTGCTTTG TGTGTTTGGTTTTTGAGGAAGGGTCTCAACTGTGTATCCCTGGCTGGCCTG GCCGGATCTAGCTTCGTGTCAAGGACGGTGACTGCAGTGAATAATAA AATGTGTGTTTGTCCGAAATACGCGTTTTGAGATTTCTGTCGCCGACTAAA TTCATGTCGCGCGATAGTGGTGTTTATCGCCGATAGAGATGGCGATATTGG AAAAATCGATATTTGAAAATATGGCATATTGAAAATGTCGCCGATGTGAG TTTCTGTGTAACTGATATCGCCATTTTTCCAAAAGTGATTTTTGGGCATAC GCGATATCTGGCGATAGCGCTTATATCGTTTACGGGGGATGGCGATAGAC GACTTTGGTGACTTGGGCGATTCTGTGTGTCGCAAATATCGCAGTTTCGAT ATAGGTGACAGACGATATGAGGCTATATCGCCGATAGAGGCGACATCAAG CTGGCACATGGCCAATGCATATCGATCTATACATTGAATCAATATTGGCCA

TTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGG

CCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAG TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGA CTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGG CAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATG ACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTG ATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG GGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CCAAAATCAA

CGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGG CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGA ACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGA AGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAAC GCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATA GGCCCACCCCCTTGGCTTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTC TATACACCCCCGCTTCCTCATGTTATAGGTGATGGTATAGCTTAGCCTATA GGTGTGGGTTATTGACCATTATTGACCACTCCCCTATTGGTGACGATACTT TCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCTTTATTGGCTA TATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACA GGATGGGGTCTCATTTATTATTTACAAATTCACATATACAACACCACCGTC CCCAGTGCCCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAATC TCGGGTACGTGTTCCGGACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCT ACATCCGAGCCCTGCTCCCATGCCTCCAGCGACTCATGGTCGCTCGGCAG CTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGCACGATGCCCA CCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAA AATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAA GGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAG AGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTA GTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTG

ACAGACTAACAGA CTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCTTGACACGAAGCTT CGGGCGAATTGCGTGGTTTTAAGTGACTATATTCCGAGGGTCGCCTGTAAT ATGGGGACAGTTAATAAACCTGTGGTGGGGGTATTGATGGGGTTCGGAAT TATCACGGGAACGTTGCGTATAACGAATCCGGTCAGAGCATCCGTCTTGC GATACGATGATTTTCACACCGATGAAGACAAACTGGATACAAACTCCGTA TATGAGCCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGG GGAGAGTCTTCGCGAAAAGCGTACGATCATAACTCACCTTATATATGGCC ACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCATGGGG TGTATAATCAGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCC ACACAAATGTCTGCACAGGAGGATCTTGGGGACGATACGGGCATCCACGT TATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACC AACGTCAATACGGTGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAA GGCCAAAGACTCATTGAGGTGTCAGTGGAAGAAAATCACCCGTTTACTTT ACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGA GCTTTTTGCCGTCATTAACCTGTACGGGAGACGCAGCGCCCGCCATCCAGC ATATATGTTTAAAACATACAACATGCTTTCAAGACGTGGTGGTGGATGTG GATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCG TTTTCAAGGTAAGAAGGAAGCGGACCAACCGTGGATTGTTGTAAACACGA GCACACTGTTTGATGAACTCGAATTAGACCCCCCCGAGATTGAACCGGGT GTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTG GAACATGCGCGGCTCCGA

TGGTACGTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAA AAACAAGAAACCCTACGCCCGCAGTAACTCCTCAACCAAGAGGGGCTGA GTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTTT AGCTTGGCAATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTG CTGTTAGAGTGGTTGTATGTCCCCATCGATCCTACATGTCAACCAATGCGG TTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCATA TGAATTCCGGTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAA GCACAGTGTATCAAAATTGTGAACATGCAGATAACTACACCGCATATTGT CTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGG GGCACCACGTTAAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATA

CGTTTTTGTGGTGTATTTTAACGGGCATGTTGAAGCCGTAGCATACACTGT TGTATCCACAGTAGATCATTTTGTAAACGCAATTGAAGAGCGTGGATTTCC GCCAACGGCCGGTCAGCCACCGGCGACTACTAAACCCAAGGAAATTACCC CCGTAAACCCCGGAACGTCACCACTTCTACGATATGCCGCATGGACCGGA GGGCTTGCAtGACATGTTTTGCACCTCACCGGCTACGCGGGGCGACTCGTC CGAGTCAAAACCCGGGGCATCGGTTGATGTTAACGGAAAGATGGAATATG GATCTGCACCAGGACCCCTGAACGGCCGGGATACGTCGCGGGGCCCCGGC GCGTTTTGTACTCCGGGTTGGGAGATCCACCCGGCCAGGCTCGTTGAGGA CATCAACCGTGTTTTTTTATGTATTGCACAGTCGTCGGGACGCGTCACGCG AGATTCACGAAGATTGCGGCGCATATGCCTCGACTTTTATCTAATGGGTCG CACCAGACAGC

GTCCCACGTTAGCGTGCTGGGAGGAATTGTTACAGCTTCAACCCACCCAG ACGCAGTGCTTACGCGCTACTTTAATGGAAGTGTCCCATCGACCCCCTCGG GGGGAAGACGGGTTCATTGAGGCGCCGAATGTTCCTTTGCATAGGAGCGC ACTGGAATGTGACGTATCTGATGATGGTGGTGAAGACGATAGCGACGATG ATGGGTCTACGCC ATCGGATGTAATTGAATTTCGGGATTCCGACGCGGAA TCATCGGACGGGGAAGACTTTATAGTGGAAGAAGAATCAGAGGAGAGCA CCGATTCTTGTGAACCAGACGGGGTACCCGGCGATTGTTATCGAGACGGG GATGGGTGCAACACCCCGTCCCCAAAGAGACCCCAGCGTGCCATCGAGCG ATACGCGGGTGCAGAAACCGCGGAATATACAGCCGCGAAAGCGCTCACC GCGTTGGGCGAGGGGGGTGTAGATTGGAAGCGACGTCGACACGAAGCCC CGCGCCGGCATGATATACCGCCCCCCCATGGCGTGTAGTTGAAT

DNA Sequence of Plasmid insert [SEQ ID NO: 5]

The region in italics is the non-translated region. The underlined regions are the ATG start, the ATG of ie63 coding for an internal methionine and the stop.

1 AGCTTCGGG CGAATTGCGT GGTTTTAAGT GACTATATTC CGAGGGTCGC

51 CTGTAATAΎG GGGACAGTTA ATAAACCTGT GGTGGGGGTA TTGATGGGGT

101 TCGGAATTAT CACGGGAACG TTGCGTATAA CGAATCCGGT CAGAGCATCC 151 GTCTTGCGAT ACGATGATTT TCACACCGAT GAAGACAAAC TGGATACAAA 201 CTCCGTATAT GAGCCTTACT ACCATTCAGA TCATGCGGAG TCTTCATGGG

251 TAAATCGGGG AGAGTCTTCG CGAAAAGCGT ACGATCATAA CTCACCTTAT

301 ATATGGCCAC GTAATGATTA TGATGGATTT TTAGAGAACG CACACGAACA

351 CCATGGGGTG TATAATCAGG GCCGTGGTAT CGATAGCGGG GAACGGTTAA

401 TGCAACCCAC ACAAATGTCT GCACAGGAGG ATCTTGGGGA CGATACGGGC

451 ATCCACGTTA TCCCTACGTT AAACGGCGAT GACAGACATA AAATTGTAAA

501 TGTGGACCAA CGTCAATACG GTGACGTGTT TAAAGGAGAT CTTAATCCAA

551 AACCCCAAGG CCAAAGACTC ATTGAGGTGT CAGTGGAAGA AAATCACCCG

601 TTTACTTTAC GCGCACCGAT TCAGCGGATT TATGGAGTCC GGTACACCGA

651 GACTTGGAGC TTTTTGCCGT CATTAACCTG TACGGGAGAC GCAGCGCCCG

701 CCATCCAGCA TATATGTTTA AAACATACAA CATGCTTTCA AGACGTGGTG

751 GTGGATGTGG ATTGCGCGGA AAATACTAAA GAGGATCAGT TGGCCGAAAT

801 CAGTTACCGT TTTCAAGGTA AGAAGGAAGC GGACCAACCG TGGATTGTTG

851 TAAACACGAG CACACTGTTT GATGAACTCG AATTAGACCC CCCCGAGATT

901 GAACCGGGTG TCTTGAAAGT ACTTCGGACA GAAAAACAAT ACTTGGGTGT

951 GTACATTTGG AACATGCGCG GCTCCGATGG TACGTCTACC TACGCCACGT

1001 TTTTGGTCAC CTGGAAAGGG GATGAAAAAA CAAGAAACCC TACGCCCGCA

1051 GTAACTCCTC AACCAAGAGG GGCTGAGTTT CATATGTGGA ATTACCACTC

1 101 GCATGTATTT TCAGTTGGTG ATACGTTTAG CTTGGCAATG CATCTTCAGT

1 151 ATAAG ATACA TG AAGCGCCA TTTGATTTGC TGTTAGAGTG GTTGTATGTC

1201 CCCATCGATC CTACATGTCA ACCAATGCGG TTATATTCTA CGTGTTTGTA

1251 TCATCCCAAC GCACCCCAAT GCCTCTCTCA TATGAATTCC GGTTGTACAT

1301 TTACCTCGCC ACATTTAGCC CAGCGTGTTG CAAGCACAGT GTATCAAAAT

1351 TGTGAACATG CAG ATAACTA CACCGCATAT TGTCTGGG AA TATCTCATAT 1401 GGAGCCTAGC TTTGGTCTAA TCTTACACGA CGGGGGCACC ACGTTAAAGT

1451 TTGTAG ATAC ACCCG AGAGT TTGTCGGG AT TATACGTTTT TGTGGTGTAT

1501 TTTAACGGGC ATGTTGAAGC CGTAGCATAC ACTGTTGTAT CCACAGTAGA

1551 TCATTTTGTA AACGCAATTG AAGAGCGTGG ATTTCCGCCA ACGGCCGGTC

1601 AGCCACCGGC GACTACTAAA CCCAAGGAAA TTACCCCCGT AAACCCCGGA

1651 ACGTCACCAC TTCTACGATA TGCCGCATGG ACCGGAGGGC TTGCAGACAT

1701 GTTTTGCACC TCACCGGCTA CGCGGGGCGA CTCGTCCGAG TCAAAACCCG

1751 GGGC ATCGGT TG ATGTTAAC GG A A AG ATGG A ATATGG ATC TGC ACCAGG A

1801 CCCCTGAACG GCCGGGATAC GTCGCGGGGC CCCGGCGCGT TTTGTACTCC

1851 GGGTTGGGAG ATCCACCCGG CCAGGCTCGT TGAGGACATC AACCGTGTTT

1901 TTTTATGTAT TGCACAGTCG TCGGGACGCG TCACGCGAGA TTCACGAAGA

1951 TTGCGGCGCA TATGCCTCGA CTTTTATCTA ATGGGTCGCA CCAGACAGCG

2001 TCCCACGTTA GCGTGCTGGG AGGAATTGTT ACAGCTTCAA CCCACCCAGA

2051 CGCAGTGCTT ACGCGCTACT TTAATGGAAG TGTCCCATCG ACCCCCTCGG

2101 GGGG AAG ACG GGTTCATTG A GGCGCCG AAT GTTCCTTTGC ATAGG AGCGC

2151 ACTGGAATGT GACGTATCTG ATGATGGTGG TGAAGACGAT AGCGACGATG

2201 ATGGGTCTAC GCCATCGGAT GTAATTGAAT TTCGGGATTC CGACGCGGAA

2251 TC ATCGGACG GGG AAGACTT TATAGTGGAA GAAGAATCAG AGG AGAGCAC

2301 CGATTCTTGT GAACCAGACG GGGTACCCGG CGATTGTTAT CGAGACGGGG

2351 ATGGGTGCAA CACCCCGTCC CCAAAGAGAC CCCAGCGTGC CATCGAGCGA

2401 TACGCGGGTG CAGAAACCGC GGAATATACA GCCGCGAAAG CGCTCACCGC

2451 GTTGGGCGAG GGGGGTGTAG ATTGGAAGCG ACGTCGACAC GAAGCCCCGC

2501 GCCGGCATGA TATACCGCCC CCCCATGGCG TGT GTTGAA TG Sequence of fusion protein gE-ie63 encoded by PNIN4801 [SEQ ID NO: 6]

1 MGTV KPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV

51 YEPYYHSDHA ESSWVNRGES SR AYDHNSP YIWPRNDYDG FLENAHEHHG

101 VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD

1 1 QRQYGDVFKG DLNPKPQGQR LIEVS VEENH PFTLRAPIQR IYG VRYTETW

201 SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT KEDQLAEISY

251 RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI

301 WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV

351 FSVGDTFSLA MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP

401 NAPQCLSHMN SGCTFTSPHL AQRVASTVYQ NCEHADNYTA YCLGISHMEP

451 SFGLILHDGG TTLKFVDTPE SLSGLYVFVV YFNGHVEAVA YTVVSTVDHF

501 VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA WTGGLADMFC

551 TSPATRGDSS ESKPGASVDV NGKMEYGSAP GPLNGRDTSR GPGAFCTPGW

601 EIHPARLVED INRVFLCIAQ SSGRVTRDSR RLRR1CLDFY LMGRTRQRPT

651 LAC WEELLQL QPTQTQCLRA TLMEVSHRPP RGEDGFIEAP NVPLHRSALE

701 CDVSDDGGED DSDDDGSTPS DVIEFRDSDA ESSDGEDFIV EEESEESTDS

751 CEPDGVPGDC YRDGDGCNTP SPKRPQRAIE RYAGAETAEY TAAKALTALG

801 EGGVDWKRRR HEAPRRHDIP PPHGV*

Claims

1. A fusion protein comprising

(1) a Naricella Zoster Virus gE protein or an immunologically active fragment thereof fused to

(2) a different protein of Varicella Zoster Virus or an immunologically active fragment thereof.

2. A fusion protein as claimed in claim 1 comprising (1) a Varicella Zoster Virus gE protein or an immunologically active fragment thereof fused to

(2) a Varicella Zoster Virus IE63 protein or an immunologically active fragment thereof.

3. Recombinant DΝA or RΝA encoding a fusion protein as claimed in any one of the preceding claims.

4. An expression vector or a recombinant live microorganism comprising recombinant DΝA or RΝA according to claim 3.

5. A host transformed with a nucleic acid according to claim 3 or with a vector according to claim 4.

6. A vaccine composition comprising a fusion protein as claimed in claim 1 or claim 2 and a pharmaceutically acceptable excipient.

7. A vaccine composition comprising a nucleic acid encoding the fusion protein of claim 1 or claim 2 and a pharmaceutically acceptable excipient.

8. A fusion protein as claimed in claim 1 or claim 2 for use in medicine.

9. A process for the production of a fusion protein according to claim 1 or claim 2 which process comprises expressing recombinant DNA encoding said protein in a host cell and recovering the protein.

10. Use of a fusion protein as claimed in claim 1 or claim 2 for the manufacture of a medicament for the prevention or amelioration of Varicella or Zoster.

11. A vaccine composition as claimed in claim 6 or 7 additionally comprising other VZV antigens.

12. A vaccine composition as claimed in claim 11 wherein the further Varicella Zoster Virus protein is selected from the group, gE, gB, gH, gl, gC or IE62 or an immunologically functional derivative thereof.

13. A vaccine composition as claimed in claim 6, 7, 11 or 12 additionally comprising an adjuvant.

14. A vaccine composition as claimed in claim 13 wherein the adjuvant preferentially induces a TH1 response.

15. A vaccine composition as claimed in claim 13 wherein the adjuvant is selected from the group of adjuvants comprising: 3D-MPL, QS21, a mixture of QS21 and cholesterol, a CpG oligonucleotide, aluminium hydroxide, aluminium phosphate and tocopherol, and a water in oil emulsion or a combination of two or more of the said adjuvants.

16. A method of treating a patient suffering from or susceptible to Varicella Zoster Virus infection or recurrence, comprising administering to a patient a safe and effective amount of a vaccine composition as claimed in any one of claims 6,7,11, 12, 13, 14 or 15.

17. A method of producing a vaccine composition as claimed in any one of claims 6,7, 11 or 12, comprising mixing said fusion protein or nucleic acid with a pharmaceutically acceptable excipient.

18. A method as claimed in claim 17, wherein the excipient comprises an adjuvant as claimed in claim 13 - 15.