AU676166B2 - Immediate early HSV-2 viral protein ICP27 as vaccine - Google Patents

Description

(W"I DATE 24/01/94 AOJP DATE 14/04/94 PCT NUMBER PCT/LP93/101525 111III iiI111111111Hl l AU9343264 (51) International Patent Classification C12N 15' 8, C07K 13/00 C12N 15/o2, 15/86, 7/04 A61 K391245, C1 2N 15/44 (11) International Publication Number: Al (43) International Publication Date: W~O 94/00575 6 January 1994 (06,01.94)1 (21) International Application Number: (22) International Filing Date: PCTwE P93 '01525 IS June 1993 (15.06.93) Priority data: 9213559.9 25 June 1992 (25.06.92) Applicant (for all designated States except LUS): SMITH- KLINE BEECHAM BIOLOGICALS [BE/BE]: 89, rue de H'nstitut, B-1330 Rixensart (BE).

(72) Inventors; and (7S) Inventors/Applicants tfor V'S onli-) SLAQUI, Moncer [N4C/BE]; PALA, Pietro IiTPIIE]; KOUTSOUKOS, Marguerite IBE.!BE]; SmithiKline Beecham Biologicals 89, rue de llInstitut, B-1330 Rixensart (BE).

(74) Agent: DALTON, Marcus, Jonathan, William; SmithKline Beecham, Corporate Patents, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey KT18 5XQ (GB).

(81) Designated States: AT. AU. 1B. BO0, BR. CA, C11, CZ, DEi, DK, ES, Fl. 01). HU, JP., KP, KR. KZ., LK, LU, MG, MN. MW, NL. NO, NZ, PL, PT, RO, RU, SD, SE, SK, UA, US, VN, European patent (AT, BE, C11, DE, DK, ES, FR, GB, GR. III, IT, LUJ, MC, NL, PT, SE), QAPI patent (BW, 113. CF. CG, Cl, CM, GA, GN, ML, MR, NE, SN, TD, TG).

Published Wth international searchi report.

676 1 66 (54)Title: IMMEDIATE EARLY HSV-2 VIRAL PROTEIN ICP27 AS VACCINE (57) Abstract Immediate early HSV-2 viral protein ICP27 recognised by cytolytic T-lymphocyte (CTL) cells in humans, mcthodq for preparation thercof and use in vaccine, pSCII/ICP27 Ma ICP27 (HSV2) 5563 bp 91.91 bp Xhal ATONwI p

SAMHI

dig est Ntcl- EtoR 1 t-in)I digestNeill- X bat (till-in) Ligat'efn tWb) Ncot SarnHI IJCo

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WO 94/00575 PCT/EP93/01525 IMMEDIATE EARLY HSV-2 VIRAL PROTEIN ICP27 AS VACCINE The present invention relates to therapeutic and prophylatic vaccines, novel antigens for use in such vaccine(s), methods for their preparation and their use in human medicine. In particular the present invention relates to antigens from Herpes Simplex (HSV) capable of stimulating a cytotoxic T lymphocyte response.

HSV causes lifelong infection and recurrent disease in man. There are two closely related serotypes of HSV, these are known as HSV-1 and HSV-2 respectively.

In primary infections, after replication at a skin or mucosal site, the virus moves to the dorsal root ganglia and usually enters a latent phase. Reactivations then occur after appropriate stimuli, resulting in vesicles and ulcers at the mucocutaneous sites innervated by the ganglia. While neutralizing antibodies are shown to protect against primary infection and disease, their presence has no effect on the course or frequency of recurrent herpetic disease. T cell mediated immune responses, particularly of the delayed type hypersensitivity (DTH) or cytolytic (CTL) effector types have also been shown to protect against primary disease in mouse animal models. Furthermore, individuals with compromised T cell functions may undergo severe and sometimes life-threatening herpetic disease. These observations suggest a central role for effector T cell functions in control of herpes virus infections in man.

The major surface glycoproteins of Herpes Simplex Virus, gD and gC have been suggested for use in vaccines (EP 139 417 Genentech). These primarily stimulate a neutralising antibody response.

Since the mechanism of antigen recognition by 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 T cell mediated responses. However since the HSV 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 HSV 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 seems therefore desirable to induce not just an antibody response, but also CTL. An effective vaccine should prime CTL capable of acting as early as possible as soon as signs of reactivation of latent virus appear.

Previous studies have identified human CTL responses to various herpes -1- SUBSTITUTE SHEET

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WO 94/00575 PCT/EP93/01525 simplex structural components such as glycoproteins gD, gB (Zarling et al. 1986), but the relevance of these CTL for virus clearance is not known. Moreover, such CTL were HLA class II restricted, and although expression of class II molecules is induced in keratinocytes during HSV replication, it may occur too late to prevent the appearance of lesions.

In order to identify the most important CTL target antigens for prophylatic or therapeutic vaccine purposes, the present inventors have taken into consideration the HSV replicative cycle. After primary infection and during reactivation from a latent state in neuronal ganglia, HSV is mostly intracellular, with minimal exposure to neutralizing antibodies. However, the beginning of viral protein synthesis inside a cell that harbours viral genome will generate viral protein fragments that will be presented by MHC molecules on the surface of the cell, making it a target for CTL of the appropriate specificity. The replication cycle of HSV lasts about 18-20 hours and involves an ordered expression of a or immediate early (IE) P or ec.rly and y or late gene products. Therefore early CTL attack and consequent lysis of the infected cells prior to late structural gene expression could prevent new virions being made and therefore prevent spread of the virus to neighbouring cells. In order to be most useful, CTL should detect the very first viral proteins that appear inside the cell after infection and reactivation.

We have analyzed the specificity of human HSV specific CTL towards immediate early viral protein ICP27. First, we investigated the CTL response in peripheral blood mononuclear cells (PBMC) from patients with herpetic genital lesions of varying clinical severity. We used autologous HSV-2 infected lymphoblasts as stimulators to induce HSV-2 specific HLA restricted CTL in mass and limiting dilution cultures. Strong responses were found in PBMC samples obtained days to weeks after the occurrence of lesions. The frequency of HSV-2 specific CTL ranged between 1/10000 and 1/36000.

Using vaccinia virus recombinant ICP27.V the gene product was expressed in EBV transformed lymphoblastoid target cells for cytotoxicity assays. The recombinant infected target cells were recognized by a fraction of HSV-2 specific CTL induced by in vitro restimulation with HSV-2 infected lymphoblasts. This IE protein constitutes therefore a candidate component for HSV vaccines aimed at inducing CTL mediated immunity.

The present invention is therefore, directed towards an immediate early HSV- 2-viral protein ICP27 that is recognised by cytolytic T lymphocyte (CTL) in humans.

In particular an ICP 27 having substantially the sequence as shown in ID Sequence No.1 (protein sequence). The term substantially means at least 85% homologous, preferably 90 to 95% homologous, more preferably greater than 95% homologous.

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Il~ac-- r WO 94/00575 PCr/EP93/01525 Accordingly, the present invention provides a vaccine composition, for therapeutically or prophylactically treating HSV infections, comprising HSV-2, immediate early protein ICP27 or an immunologically active fragment thereof. The ICP27 protein may be expressed as a fusion protein or on a carrier such as a Hepatitis B surface antigen, or presented by a live bacterial carrier, such as listeria, shigella, BCG or Salmonella. Alt.rnatively, the protein may be presented as in a live viral vector, such as vaccina, adenovirus or poliovirus. Alternatively the protein may be incorporated into an HSV light particle, as described in British patent application No.

91147140.0 and 9109763.4. (published: WO 92/13943 and PC GB92/00824).

Such forms of presentation of ICP27 form part of the invention. A preferred embodiment of the invention is a vaccinia recombinant which expresses an HSV-2 ICP27 protein or an immunologically active fragment thereof.

This is the first medical use ascribed to this protein, and accordingly in one aspect of the invention there is provided HSV-2 ICP27 for use in medicine.

ICP27 is an immediate early protein and its function in the virus is poorly understood, but it is known to be essential for viral replication and involved in virus genome transactivation [McCarthy, McMahan, L. Schaffer, P.A. (1989).

Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient J. Virol. 63:18-27.; Rice, Su, L, Knipe, D.M. (1989). Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities. J. Virol. 83:3899-3407].

As used herein, an immunological fragment of ICP27 is a portion of the protein which is capable of eliciting a functional immunological response.

In animal models we have shown that inoculation with Recombinant vaccinia viruses expressing ICP27 after primary challenge with virulent virus, substantially reduces the frequency and severity of recurrent episodes of the disease.

A further aspect of the invention provides a process for the preparation of the ICP 27 HSV-2 protein or an immunogenic derivative thereof, which process comprises expressing DNA encoding said protein or derivative thereof in a recombinant host cell and recovering the product, and thereafter, optionally, preparing a derivative thereof.

A DNA molecule comprising such coding sequence eg as shown in ID Sequence No.2 or a fragment thereof forms a further aspect of the invention and can be synthesized by standard DNA synthesis techniques, such as by enzymatic ligation as described by D.M. Roberts t al in Biochemistry 1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymatic polymerization, or by a combination of these techniques. With reference to ID sequence 2, the coding sequence for the mature protein ends at base no.1536.

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WO 94/00575 PCT/EP93/01525 Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 100-37oC, generally in a volume of 50l or less. Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 0.01M MgCl 2 0.01M dithiothreitol, ImM spermidine, ImM ATP and 0.1mg/ml bovine serum albumin, at a temperature of 4 0 C to ambient, generally in a volume of 501l or less. The chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fragments A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982),or in other scientific publications, for example M.J. Gait, H.W.D.

Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, 6243; B.S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M.D.

Matteucci and M.H Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D. Matteucci and M.H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S.P. Adams et al., Journal of the American Chemical Society,1983, 105, 661; N.D.

Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H.W.D. Matthes et al., EMBO Journal, 1984, 3, 801.

Alternatively, the coding sequence can be derived from HSV-2 mRNA, using known techniques reverse transcription of mRNA to generate a complementary cDNA strand), and commercially available cDNA kits.

The invention is not limited to the specifically disclosed sequence, but includes all molecules coding for the protein or an immunogenic derivative thereof, as described above.

DNA polymers which encodes mutants of the protein of the invention may be prepared by site-directed mutagenesis of the cDNA which codes for the protein by conventional methods such as those described by G. Winter et a in Nature 1982, 299, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., 10, 6487-6500, or deletion mutagenesis such as described by Chan and Smith in Nucl. Acids Res., 1984, 12, 2407-2419 or by G. Winter er al in Biochem. Soc. Trans., 1984, 12, 224-225.

The process of the invention may be performed by conventional recombinant techniques such as described in Maniatis et. al., Molecular Cloning A Laboratory Manual; Cold Spring Harbor, 1982-1989.

In particular, the process may comprise the steps of: i) preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide -4- SUBSTITUTE SHEET

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WO 94/00575 PCT/EP93/01525 sequence that encodes said HSV-2 ICP 27 protein or an immunogenic derivative thereof; ii) transforming a host cell with said vector, iii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said protein; and iv) recovering said protein.

The term 'transforming' is used herein to mean the introduction of foreign DNA into a host cell by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S.M. Kingsman and AJ. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988. The term 'transformed' or 'transformant' will hereafter apply to the resulting host cell containing and expressing the foreign gene of interest.

The expression vector is novel and also forms part of the invention.

The replicable expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, such as the DNA polymer encoding the 16 kDa protein, or fragments thereof, under ligating conditions.

Thus, the DNA polymer may be preformed or formed during the construction of the vector, as desired.

The choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses.

The preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis et al cited above.

The recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Maniatis t al cited above, or "DNA Cloning" Vol. II, D.M.

Glover ed., IRL Press Ltd, 1985.

The choice of transforming conditions is determined by the host cell. Thus, a bacterial host such as E. coli may be treated with a solution of CaCl 2 (Cohen et al, Proc. Nat Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbC1, MnC1 2 potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol. Mammalian cells in SUBSTITUTE SHEET

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WO 94/00575 PCT/EP93/01525 culture may be transformed by calcium co-precipitation of the vector DNA onto the cells. The invention also extends to a host cell transformed with a replicable expression vector of the invention.

Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis et al and "DNA Cloning" cited above. Thus, preferably the cell is supplied with nutrient and cultured at a temperature: below 45 0

C.

The product is recovered by conventional mnethods according to the host cell. Thus, where the host cell is bacterial, such as E. coli it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate. Where the host cell is mammalian, the product may generally be isolated from the nutrient medium or from cell free extracts. Conventional protein isolation techniques include selective precipitation, absorption chromatography, and affinity chromatography including a monoclonal antibody affinity column.

Alternatively, the expression may be carried out in insect cells using a suitable vector such as the Baculovirus. In a particular aspect of this invention, the protein is expressed in Lepidoptera cells to produce immunogenic polypeptides. For expression of the protein in Lepidoptera cells, use of a baculovirus expression system is preferred. In such system, an expression cassette comprising the protein coding sequence, operatively linked to a baculovirus promoter, typically is placed into a shuttle vector. Such vector contains a sufficient amount of bacterial DNA to propagate the shuttle vector in E. coli or some other suitable prokaryotic host. Such shuttle vector also contains a sufficient amount of baculovirus DNA flanking the desired protein coding sequence so as to permit recombination between a wild-type baculovirus and the heterologous gene. The recombinant vector is then cotransfected into Lepidoptera cells with DNA from a wild-type baculovirus. The recombinant baculoviruses arising from homologous recombination are then selected and plaque purified by standard techniques. See Summers et al., TAES Bull (Texas Agricultural Experimental Station Bulletin) NR 1555, May, 1987.

A process for expressing the CS protein in insect cells is described in detail in USSN 287,934 of SmithKline RIT (WO/US 89/05550).

Production in insect cells can also be accomplished by infecting insect larvae.

For example, the protein can be produced in Heliothis virescens caterpillars by feeding the recombinant baculovirus of the invention along with traces of wild type baculovirus and then extracting the protein from the hemolymph after about two days.

See, for example, Miller et al., PCT/W088/02030.

The novel protein of the invention may also be expressed in yeast cells as described for the CS protein in EP-A-0 278 941.

-6- SUBSTITUTE SHEET WO 94/00575 PCT/EP93/01525 Vaccina constructs can be made by methods well known in the art, see for example European Patent Application EP-083-286 Health Research Inc., Inventors Paoletti and Panicali. The construction of such a vaccinia construct is presented in more detail in the examples.

ICP27 has been shown by the present inventors, to be recognised by human HSV specific CTL induced by in vitro stimulation of PBMC (peripheral blood mononuclear cells) with HSV-2 infected cells. By using infected cells, as stimulator cells in vitro, viral epitopes which are synthesized in the cytoplasm, are preferentially presented by class I molecules. Thus the spectrum of effector cells stimulated in vitro by this approach will include both class I and class II restricted T cells.

This is in contrast with stimulation of primed PBMC using inactivated free virus, known to preferentially induce class II restricted effector CTLs, as the virus enters antigen presenting cells by endocytosis and is processed by the class II pathway. Neo-synthesis of antigen does not occur, and class I restricted presentation is less likely to occur.

The antigenic specificity of human CTL responses to HSV is highly relevant for an effective subunit vaccine, since HSV infection is characterised by the ability to establish latency and reactivate periodically. During latency and reactivation there is minimal exposure of free virus to antibodies as the virus is mainly maintained intracellularly.

In order to maximise the protective ability of a vaccine according to the invention, the vaccine may also preferably contain one or more other HSV proteins, other immediate early, early or late proteins capable of stimulating a CTL response in humans, such as gD or gC Vmw65, RR 2 ICPO or ICP 4 In particular, the vaccine may advantageously contain a truncated gD derivative from HSV-2 as described in EP 139 417 B. Also the vaccine may contain HSV-1 proteins or cocktails of variants of the same proteins where they exist.

Also the vaccine may contain HSV-1 proteins or cocktails of variants of the same proteins where they exist.

The vaccine of the present invention will preferably be adjuvanted. Known adjuvants will include alum (aluminium hydroxide) mycobacterium derived antigens such as Freunds complete or incomplete adjuvants, and muramyldipeptide (MDP) and derivatives, saponin type adjuvants such as QS21 (US Patent No 5057540) and the like. A particularly preferred adjuvant preparation is monophosphoryl lipid A (3D-MPL) which is commercially available from Ribi Immunochem and may be prepared according to the method of GB 2220211, or QS21 commercially available from Cambridge Biotech.

In such cases 3D-MPL and/or QS21 will be present in the range 10g -7- SUBSTITUTE

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WO 94/00575 PCT/EP93/01525 100p.g, and preferably 25 50 gg per dose. The vaccine containing 3D-MPL or QS21 will typically be presented on alum or in an oil in water emulsion.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S.

Patent 4,474,757.

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 ihamunogen is employed and how it is presented. Generally, it is expected that each dose will comprise 1-1000 gg of protein, preferably 2-100 gig, most preferably 4-40 p.g. 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 immunisation adequately spaced.

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

The rationale for immunotherapeutic use of the invention is that the frequency of HSV specific CTL, that exert an immune surveillance function against the virus, may physiologically decline with time after the last antigen-triggered expansion.

Alternatively virus infection may not trigger a strong enough CTL response.

When low numbers of such CTL exist in the body, a reactivating HSV infection will have more chances to go through more rounds of viral replication before being detected by HSV specific T cells, resulting in larger clinically apparent herpetic lesions. However, if CTL levels are maintained at a given level by a suitable protocol of therapeutic vaccination, the time during which reactivating virus replicates unchecked will be kept to a minimum. This will have a beneficial effect in HSV infected individuals, eliminating or reducing the severity of clinically detectable recurrent lesions. This effect will be in addition to, and non exclusive of, the advantage provided by the specificity of CTL for an immediate early antigen, as referred to above.

A suitable protocol of therapeutic vaccination may be defined as a pharmacologically acceptable amount of vaccine preparation administered at regular -8- SUBSTITUTE SHEET WO 94/00575 PCT/EP93/01525 time intervals in HSV infected individuals, which results in elimination or reduced severity of previously occurring recurrent herpetic disease.

EXAMPLE 1 HSV2 ICP27 EXPRESSION IN VACCINIA VIRUS 1. INSERTION OF A SYNTHETIC POLYLINKER IN A VACCINIA EXPRESSION VECTOR pRit13389 was obtained by insertion of a synthetic polylinker: SC111 5' GGG AAA ACC ATG GAT CCA TGG CAG GTA CTA GTG TCG ACT AAC TAA CTA A 3' SC112 3' CCC TIT TGG TAC CTA GGT ACC GTC CAT GAT CAC AGC TGA TTG AT GAT T in the unique SmaI site of the PSC11 vaccinia virus expression vector Mackett M. et al., 1985. DNA cloning vol II, chap 7, a practical approach, Ed. DM Glover, ISBN 0- 947946-19-5). See Figure 1.

2. CONSTRUCTION FOR THE EXPRESSION OF HSV2 GENE ICP27 A large part of the ICP27'S open reading frame (1481 bp) was cloned as a fragment of 1666 bps Xhol/Mlul T4 polymerase treated. This fragment contains 185 bp downstream of the gene.

The missing 3' terminal region was reconstituted by a synthetic oligonucleotide: UL541 5' CAT GGC TAC CGA CAT TGA TAT GCT AAT CGA CCT AGG ATT GGA CCT GTC CGA CAG CGA GC 3' UL542 3' CG ATG GCT GTA ACT ATA CGA TIA GCT GGA TCC TAA CCT GGA CAG GCT GTC GCT CGA GCT These two fragments were ligated together in pRitl3 389 between the Ncol and Spel T4 polymerase treated sites. This construction is called pRitl3 395, see figure 2.

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r~ WO 94/00575 I(/07 I )3/0l525 3. LXPRESSION IN VACCINIA VIRUS The expression cassette was used to traisform vaccinia virus following the M.

MACKETT et al. protocol except that 20 pg of plasmid DNA was used by experiment (Mackett M. a al., 1985. ibid). CVI cells (ATCC CCL70) and WR vaccinia virus strain were used for the transfection. Selection of recombinants was realized in rat 2 cells (ATCC CRL 1764) following Mackett et al. 8-galactosidase activity was detected as described in Chakrabarti S. et al., Mol. Cell. Biol. 1985 1:3403-3409.

4. RECOMBINANTS VACCINIA VIRUS Expression of recombinant genes products in vaccinia virus was tested by Western blot. The antibodies used for protein detections were obtained by immunizing rabbits with synthetic peptides (ICP27 aa2-12 ATDIDMLIDLG coupled to BSA carrier)].

Rabbit sera were diluted to 1/1000 to perform the Western blot.

EXAMPLE 2 Expression of ICP27 (HSV2) in E. coli Introduction: the Eschcrichia coli expression system used was plasmid pMG81, which is a derivative of pMG27N developed for the efficient production of proteins in E. coli (reference 1 below).

Plasmid pMG27N is a derivative of the vector pAS which has been used to synthesize large quantities of numerous foreign proteins (ref. The pMG27N plasmid, as pAS1, utilizes signals from 1 phage DNA to drive the transcription ar.J translation of inserted foreign genes. The plasmid contains the 1 promoter PL; operator OL1 two utilization sites (Nutt and NutR) to relieve transcriptional polarity effects when N protein is provided; the clI ribosome binding site including the clI translation initiation codon incorporated in an Ndel restriction site (ref. Plasmid pMG1 has been constructed by inserting the 81 first amino acids of the NSI coding region from influenza strain A/PR/8/34 cleaved from plasmid pAS1 EH/801 (ref. 4) by BamHI and Ncol into pMG27N digested by BamHI and Sad. A synthetic DNA linker resulting from the ligation of two synthetic oligonuclcotides (5'ATCCCGGGATAAAAACAACCAAGGTAATGGACA3'and

GTTGGTTCCATTACCTGTT

3 was introduced between the Ncol and the SacI sites. pMG81 is a derivative ofpMGl. pMG1 has been digested with Bglil-PstI to remove the ampicillin resistance gene. The kanamycin resistance gene from transposon Tn903 (13-15) was isolated from plasmid pOTS207 (16) by a EcoRI-PstI digestion and ligated into pMG1 along with a synthetic DNA linker resulting from the ligation of two synthetic oligonucleotides (5'AATTCGTACCTA 3 SUBSTITUTE SHEET I I- WO 94/0575 PcT/EP93/0152S and 5'GCATGGATCTAG 3 The AR58 bacterial lysogcn usce ior the production of the NS1-ICP27 protein is a derivative of the standard NIH E. coli K12 strain N99 (Fsu-galK2 lacZ- thr-). It contains a defective phage lambda lysogen (galE::Tn 10, 1 Kilc1857 HI) which is Kil- prevents the shut-off of host macromolecular synthesis), has a c1857 mutation (a temperature sensitive lesion in the cI repressor protein), and has the HI deletion which removes the lambda phaL: right operon and the host bio, uvr3, and chlA loci (ref. 17). The AR58 strain was generated by transduction of N99 with a Pi phage stock previously grown on an SA500 (galE::TnlO, 1 Kil- c1857 HI) derivative. The introduction of the defective lambda lysogen into N99 was selected with tetracycline by virtue of the presence of a TnO transposon coding for tetracycline resistance in the adjacent galE gene. N99 and SA500 are E. coli K12 strains derived from Dr. Martin Rosenberg's laboratory at the National Institute of Health.

An important feature of this system is that plasmids containing the PL promoter are introduced into an E. coli lysogenic host to stabilize the plasmid DNA (ref. Cloning into lysogens also precludes the synthesis of proteins that may be toxic to the cells For these purposes, defective lambda phage lysogens are employed so that no phage production ever occurs. The integrated lambda phage DNA in the host genome directs the synthesis of a cI repressor protein which binds to the 01 operator on the plasmid and prevents the binding of RNA polymerase to the PL promoter. The inserted gene is therefore transcriptionally silent and no synthesis of the recombinant protein can occur. It is for this reason that turing the cloning and growth of cells, there is no expression. However, if the defective lambda gene carries a temperature sensitive mutation in the ci gene (ref. 11), PL directed transcription can be regulated. Bacteria are grown in the absence of expression (30°C repressor is active) and then shifted to 420C to inactivate the repressor and turn on the synthesis of the desired gene product Construction of plasmid pMG81/ICP27Plasmid pMG81/IC?27, that expresses NS1-ICP27, has been constructed as follows (figure 3).

A 1.7 kb fragment containing the ICP27 gene has been prepared by digesting plasmid pSC11/ICP27 with EcoR I, filling the protruding ends using the T4 DNA polymerase, and finally digesting the linear fragment with Nco I, Upon isolation on agarose gel and electroelution, this fragment has been ligated with plasmid pMG81 previously digested with Xba I, treated with the T4 DNA polymerase and then digested with No I. The ligation mixture has been transformed into Escherichia coli strain AR58. The transformants were selected onto solid medium containing kanamycin.

Expression of NSI/ICP27 A strain AR58(pMG81/ICP27) has been incubated in 20 ml LB medium containing kanamycin, at 30°C up to an optical density (620 nm) of 0.6. The culture has then been incubated at 42°C for 3 hours.

-11- SUBSTITUTE SHEET WO 94/00575 PCT/EP93/01525 Five ml of the culture have been taken at time 0, 30 min, 1 hour, 2 hours and 3 hours after induction. These samples have been submitted to an SDS-polyacrylamide gel electrophoresis. The proteins were then blotted onto a nitrocellulose membrane by electrotranfer. The ICP27-specific proteins were detected using rabbit ca-ICP27 antibodies. Western blot analysis indicated the presence of a sharp band whose size was in agreement with the expected size of NS1-ICP27. Smaller ICP27-specific polypeptides were also detected with the rabbit antiserum: they probably represent degraded or incomplete product of the NS1-ICP27 gene.

References 1. Gross et al. 1985. Mol. Cell.Biol. 5:1015 2. Rosenberg et al. 1983. Methods Enzymol. 101:123 3. Shatzman et al. 1983 in Experimental manipulation of gene expression (Inouya, M. pp. 1-14. Academic New York.

4. Young et al. 1983. Proc.Natl.Acad.Sci.USA 80:6105 Yamada et al. 1985. J.Exp.Med. 162:663 6. Ferguson et al. 1984. Science 224:1343 7. Watt et al. 1985. Mol. Cell.Biol. 5:448 8. Devara et al. 1984. Cell 36:43 9. Ferguson et al. 1985. J.Biol.Chem. 260:3652 Shimataka, H. and Rosenberg, M. 1981. Nature (London) 292:128 11. Sussman, R. and Jacob, F. 1962. C.R.Hebd.Seances Acad.Sci.Ser.A 254:1517 12. Wallich et al. 1989. Nucleic Acids Res. 17:8864 13. Oka, A. et al. 1981. J.Mol.Biol. 147:217 14. Berg, D.E. et al. 1978. in Microbiology (Sclessinger, ed.) pp. 13-15. American Society for Microbiology, Washington, D.C.

Nomura et al. 1978. in The single-stranded DNA phages (Denhardt et al., eds.)pp. 467-472. Cold Spring Harbor Laboratory, New York.

16. Shatzman, A. and Rosenberg, M. 1987. Methods Enzymol. 152:661.

17. Castellazzi et al. 1982. Molec.Gen.Genet. 117:211 EXAMPLE 3: Recombinant Vaccinia Virus Immunizations Induction of CTL specific for ICP27 by immunisation with recombinant vaccinia virus expressing ICP27 of HSV-1 in Balb/c mice has been described by Banks et al (1991) J. Virol. 65: 3185 -3191.

Similarly, vaccination with vaccinia recombinants expressing ICP27 of HSV- 2 induces in Balb/c mice a specific CTL response and protects them against zosteriform spread of Wild type HSV n or HSV 2.

-12- SUBSTITUTE SHEET WO 94/00575 WO 940575PTEP93/01525 Table 1 C VACCINATION wrIT WVICP27 (HSVI OR HSV2) PROTECTS BALB/C MICE AGAINST ZOSTERIFORM SPREAD OF WILD TYPE HSV1 OR HSV2

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VACCINATION VIRUS DOSE LESIONS DEATHS CTL WVICP27 HSVI17+ 0 0 0 (HSV1) 1(40 0 I-SV2 MS 150 0 0 0 WVICP27 HSV1 17+ 105 0 0 (1ISV2) 1(40 0 130 0 HSV2 MS 150 0 140 0 103___ 0 0 CONTROL HSV1 17+ 105 100 100 VV tk- 14 100 100 13 100 100 HSV2 MS 10 5 100 100 104 100 100 103__ 100 j 100 Groups of Balb/c mice (15 mice/group) were immunized with 10 pfu of ICP27.VV in the footpad and challenged 2 weeks later with wild type HSV- 1 or HSV-2 The occurrence of zosteriform lesions and death was then recorded.

Balb/c mice were immunized with 10 pfu of ICP27.VV in the footpad.

Draining popliteal lymph nodes were removed 5 days later for CTL assays, without in vitro restimulation, in order to evaluate the primary response.

EXAMPLE 3: Recognition of ICP 27 by human CTL Materials and methods: Patients Blood samples from patients attending the sexually transmitted disease cOinic of the Topical Medicine Institute, Antwerp, were collected by venipuncture into 13 SUBSTITUTE SHEET WO 94/00575 PCT/EP93/01525 heparinised tubes. Patients had genital herpetic lesions of varying clinical severity and differed in recurrent disease patterns. One asymptomatic sexual partner of a patient with recurrent disease was included in the study.

Viruses Herpes simplex virus. The HG52 strain of herpes simplex virus type 2(HSV-2) used in these experiments was kindly provided by Prof. Subak-Sharpe (MRC, Glasgow, The virus was grown in BHK21 cells infected at a multiplicity of infection of 0.003 plaque forming units per cell. The cells were harvested at 5-7 da: 's after infection, disrupted by freezing/thawing and sonicated. The virus titre was determined by plaque assay on BHK21 cells.

ICP27 vaccinia recombinant was produced as herein described.

Medium PBMC cultures were grown in RPMI 1640 (Gibco, Ghent, Belgium) supplemented with 10% heat inactivated foetal calf serum (FCS)(Flow laboratories, Irwine, Scotland), 2 x 10 3 M L-glutamine, 100 IU/mL penicillin, 100 tg/mL streptomycin, 5 x 10 5 M mercaptoethanol, 1% MEM non-essential amino acids (Gibco), 1 x 10 3 M sodium pyruvate MEM (Gibco).

Cells. Peripheral blood mononuclear cells (PBMC) were obtained from blood by separation on a Lymphoprep (Nycomed, Oslo, Norway) density gradient (Boyum, A.

(1968) Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77). PBMC were frozen in DMSO 90% FCS and thawed just before use as responder cells.

An aliquot of PBMC from each patient was used to derive lymphoblastoid cell lines (LCL) by transformation with Epstein-Barr virus (EBV obtained from culture supematants of the persistently infected marmoset cell line B-95.8, as described (Walls, E.V. and Crawford, D.H. (1987)) Generation of human B lymphoblastoid cell lines using Epstein Barr virus. In Lymphocytes, a practical approach, Klaus, G.G.B. (editor) pp.149 162. The LCL were used as target cells in cytotoxicity assays.

PHA-activated lymphoblasts for use as stimulator cells were prepared by culturing 5 x 106 PBMC with 4 I.g/mL PHA-P (Sigma) for 72 hours at 37 0 C and with 5 U.mL rlL2 (Boehringer) for 7 more days at 370C. The lymphoblasts were then infected with HSV-2 10) for 16 hours at 37 0 C and treated with 1% formaldehyde in PBS for 20 min at Mass cultures PBMC were thawed and stimulated in 24-well plate cultures containing 2 x 106 responder cells and 5 x 105 stimulator cells, in the presence of 1 U/mL human recombinant IL-2 (rIL-2, Boehringer Mannheim) and 5% (v/v) supernatant from PHA activated lymphoblasts. Cultures were fed every 3-4 days with medium supplemented with 5 U/mL rIL-2 and 5% PHA-blast superatant. Cultures were re-stimulated on day 10 and tested on day 20 for cytolytic activity.

14- SUBSTITUTE SHEET WO 94/00575 PCTI/EP93/01525 Limiting dilution cultures. PBMC were thawed and distributed into 96-well round-bottom plates. The number of responder cells per well ranged between 10 3 and 4 x 104 and 24 to 32 wells were set up for each input cell concentration.

Autologous stimulator cells (5 x 10 4 .well) were added to all wells. Control wells without responder cells were included. Cultures received 1 U/ml rIL-2 and 5% (v/v) PHA-blast supernatant at the onset, and were fed with 5U/ml rIL-2 and 5% (v/v) PHA-blast supernatant every 4-6 days. Equal aliquots from each individual culture were tested on day 14-21 in a chromium release assay against 3 different target cell types; autologous LCL infected with HSV-2, psCI 1.V and ICP27.VV.

Cytotoxicity assays LCL target cells were infected with HSV-2, psCll.VV or ICP27.V for 1 hour at 37 0 C, washed and labelled with 500 Ci of 1 Cr (Medgenix, Fleurus, Belgium) for 1 hour at 37oC. Target cells were then washed twice, incubated on ice for 30 min, washed once and 2 x 103 cells per well were distributed into the wells containing responder cells and control wells containing medium or Triton X-100 3% in water (spontaneous release and maximum release, respectively). Effector and target cell mixtures were incubated for 4 hours at 37 0 C in a total of 200 pL, then 100 pL of supernatant were harvested and released 5 1 Cr counted. Results were expressed as specific lysis according to the formula: (experimental spontaneous release) specific lysis x 100 (total spontaneous release) CTL frequency determinations and scatter plots. Responder frequencies were calculated using the maximum likelihood method described by Fazekas de St.

Groth (Fazekas de St. Groth, S. (1982). The evaluation of limiting dilution assays. J.

Imm. Meth. 49:R11-R23). For each target cell type, wells were scored as positive if the specific lysis was higher than the cut-off value defined as the average of control wells without responder cells 3 standard deviations. The frequency estimates of HSV-2 and ICP27 specific CTL were obtained after exclusion of any wells that scored positive on control targets (psC11.VV infected).

Scatter plots were used to evaluate the specificity of cytolytic activity in limiting dilution cultures. Individual wells were split into equal volumes and tested for lysis of target cells infected with HSV-2, psC11.VV, or ICP27.VV. Each well thus produced multiple values of specific lysis. Paired values for selected target cell types were used as coordinates to represent each well in a 2-dimensional cartesian plot. By plotting the cut-off values for each target cell type, the plot was partitioned into 4 sectors: wells negative on both targets, positive on both, and exclusively positive on one but not the other target.

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WO 94/00575 I'CT/EP93/01S25 Results Mass culture induction of HSV-2 specific CTL. PBMC from 10 patients with various clinical symptoms of genital herpes were stimulated in mass cultures as described in materials and methods. The cultures were then tested for HSV-2 specific cytolytic activity in 5 1 Cr release assays, using autologous and heterologous LCL target cells as HLA restriction controls.

Representative results are shown in Table 2. Six patients like 102 had HSV-2 specific HLA-restricted effectors, while in four patients, like 109, no such activity could be demonstrated. All patients where CTL could not be induced had active lesions at the time when the blood sample was taken. Only one patient with ongoing lesions had detectable CTL specific for HSV-2 (table 2).

Frequency of HSV-2 specific CTL. In order to estimate the frequency of HSV-2 specific CTL, varying numbers of patient PBMC were cultured in the presence of a constant number of autologous stimulator cells (HSV-2 infected PHAblasts prepared as described in materials and methods). Each well was tested for lysis of autologous LCL infected with HSV-2 or psCl 1.VV as negative control. Out of 14 patients tested in this way, 3 (08, 114 and K01) had high frequencies of effectors that lysed psC11.VV infected target cells, and were therefore not considered. In 7 patients (107,109, 111, 114,116,118, 120) the frequency of HSV-2 specific CTL ranged between 1/10000 and 1/36000 (see for an example patient 111, table while the remaining 3 other patients (101, 110, 112,) had frequencies lower than 1/89000 (table The correlation that was seen with mass cultures between detectable CTL activity and time of appearance of lesions is no longer evident: patients that were negative for CTL in mass cultures show CTL detectable in limiting dilution cultures. No obvious correlation seems to exist between number of recurrences per year and frequency of CTL. Detection of CTL activity in limiting dilution cultures appeared more sensitive than in mass cultures. For instance, patient 109 appeared negative in mass cultures, but HSV-2 specific CTL were found in limiting dilution cultures (compare table 2 and table 3).

Viral antigens recognized by HSV-2 specific CTL. In order to evaluate the role of ICP27 in CTL recognition, limiting dilution cultures of patient PBMC stimulated with HSV-2 infected stimulators were split 4-ways and 3 aliquots from each well tested on autologous LCL infected with HSV-2, psC11 vaccina virus.VV or ICP27.VV. (Table 4) Out of 7 patients with high frequencies of HSV-2 specific CTL, two (116 and 118) had ICP27 specific CTL (frequencies of 1/22000 and 1/50000). In patient 118, ICP27 seems to be the main HSV-2 antigen recognized by CTL. Intermediate frequencies of CTL specific for ICP27 antigen occurred in patients like 107. Patient 109 had very low frequencies of CTL specific for ICP27.

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Table 2.

Correlation between detection of HSV-2 specific CTL in mass cultures and time since appearance of lesions.

Patient Patient history* HSV-2 specific

CTL

Time since last episode Recurrences per year_____ 102 8 days primary yes infection 107 ongoing 8 no*** 108 ongoing 7 yes 109 ongoing 3 no*** 112 1 month 6 yes 114 1 year 1 yes 116 asymptomatic** none yes 117 ongoing 12 no*** 118 ongoing 6 no*** K01 10 days >10 yes Patient PBMC were stimulated in mass cultures with HSV-2 infected stimulator cells and tested for cytolytic activity on uninfected or HSV-2 infected autologous or heterologous LCL targets.

Disease history was reported by patients. The number of recurrences per year is normalized to one year on the basis of the number of recurrences in variable length intervals that the patient reported.

This patient is the asymptomatic sexual partner of a patient with recurrent lesions.

These patients had detectable CTL in limiting dilution cultures.

17- SUBSTITUTE SHEET WO 94/00575 PCT/EP93/01525 Table 3.

Frequency of HSV-2 specific CTL Patient Frequency of CTL Patient history* recognizing target cells infected by: psC11.VV HSV-2 Time since Recurrences last per year episode 101 36000 107000 6 months 12 107 73000 23000 ongoing 8 108 13000 10000 ongoing 7 109 141000 36000 ongoing 3 110 43000 too low ongoing persistent lesions 111 68000 22000 2 weeks 112 41000 89000 1 month 6 114 14000 45000 1 year 1 116 too low 24000 asymptomatic 118 48000 18000 ongoing 6 120 67000 28000 1 month primary infection K01 20000 7000 10 days PBMC were stimulated in limiting dilution cultures with autologous HSV-2 infected lymphoblasts.

Individual wells were split and tested on target cells infected with HSV-2 or psC 11.VV.

Disease history was reported by patients. The number of recurrences per year is normalized to one year on the basis of the number of recurrences in variable length intervals that the patient reported.

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_LII _I WO 94/00575 PCT/EP93/01525 Table 4.

Frequency of ICP27 specific CTL compared to that of CTL recognizing HSV-2.

Patient Frequency of CTL Patient history recognizing target cells infected by: HSV-2 ICP27.VV Time since last Recurrences per episode year 107 23000 70000 ongoing 8 109 36000 194000 ongoing 3 111 22000 179000 2 weeks 116 24000 5000 no lesions none 117 10000 108000 ongoing 12 118 18000 22000 ongoing 6 120 28000 198000 1 month primary lesion PBMC from patients with good responses to H" V-2 (CTL frequencies between 1/10000 and 1/36000) were evaluated for recognition of ICP27. The frequencies were calculated after exclusion of wells with lytic activity on control target cells infected with psC11.W.

Disease history was reported by patients. The number of recurrences per year is normalized to one year on the basis of the number of recurrences in variable length intervals that the patient reported.

We show herein for the first time that ICP27 HSV-2 is recognized by human HSV specific CTL induced by in vitro stimulation of PBMC with HSV-2 infected cells. ICP27 is a 63 Kdalton polypeptide coded by one of the five alpha genes that are expressed first upon infection, and reaches peak synthesis at 2-4 hours. ICP27 has regulatory functions and is probably essential for expression of late genes. In a subset of 7 patients with frequencies of HSV-2 specific CTL ranging between 1/10000 and 1/36000, two patients had frequencies of ICP27 specific CTL of 1/22000 and 1/5000.

These frequencies are calculated after exclusion of all cultures scoring positive on control target cells, and constitute therefore minimal estimates.

These results show that an important share of the human response to herpes simplex virus is directed against a non-virion polypeptide. For each of the patients that did recognize this antigen, the response constituted a major component of the total response to HSV-2, although we cannot exclude that underrepresentation of late antigens may have occurred in the conditions used to infect target cells with HSV-2.

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Claims (7)

1. Isolated immediate early HSV-2 viral protein ICP27 that is recognised by cytolytic T lymphocyte (CTL) in humans having substantially the sequence shown in Seq. ID 1 or an immunologically or antigenically equivalent derivative or fragment thereof, wherein the derivative is expressed as a fusion protein.

2. Immediate early HSV-2 viral protein ICP27 that is recognised by cytolytic T lymphocyte (CTL) in humans having substantially the sequence shown in Seq. ID 1 or an immunologically or antigenically equivalent derivative or fragment thereof, wherein the protein, derivative or fragment is presented by a live bacterial vector or a viral vector or is incorporated into an HSV light particle.

3. A protein according to claim 2 wherein the viral vector is vaccinia.

4. A process for the preparation of a protein, derivative or fragment, according to claim 1 which comprises: preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes said HSV-2 ICP27 protein, derivative or fragment thereof; (ii) transforming a host cell with said vector; (iii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said protein, derivative or fragment; and (iv) recovering said protein derivative or fragment. A vaccine composition for therapeutically or prophylactically treating HSV infections, comprising an immediate early HSV-2 viral protein ICP27 that is recognised by cytolytic T lymphocyte (CTL) in humans having substantially the V sequence shown in Seq.

ID 1 or an immunologically or antigenically equivalent derivative or fragment thereof in admixture with a suitable carrier.

6. A vaccine composition according to claim 5 which comprises 3D-MPL or QS21. 9607 10,p:\pcrjnjs,43264-93.cN20 -21

7. A method of treating a human or animal susceptible to HSV infections comprising administering an effective amount of a vaccine as defined in claim or 6. Dated this 20th day of September, 1995 SmithKline Beecham Biologicals By its Patent Attorneys Davies Collison Cave t 7 4U~L A -r0 Y 950920,p\oper\jms,43264-93.cla, 2 1 WO 94/00575 PCTr/EP93/O 1525 Sequence No. 1 1 51 101 151 201 251 301 351 401 451 5*01 MATDIDMLI D MFDPCGDGGA TTGVWSRLGT YGPGGADSTP DVSGGPRPRG AVLRSISERA GGFDAETRRV EALASADETL YLKARGLCGL TVGVGAGETM VHGKYFYCNS LGLDLSDSEL EEDALERDEE GRRDDPESDS SGECSSSDED E-AIDAAIPKG RRSASPREPH KPRRRVSRNA TRQAPPPLtMA AVERISESFG SWETLVAHGP AWCKMCIHHN DDLCSRRRLS HFYIPGACMA LF PPARPEDAGT PEASTPRPAA RRGADDPPPA GGKVARIQPP STKAP HPRGG RRGRRRGRGR HI4QGGRHE'AS ARTDGPGATH GEARRGGEQL LSLTPPHADG RAPVPERKAP SADTIDPAVR RSALVMQDPF GGMPFPAANS PWAPVLATQA SLYRTFAANP RAASTAKAMR DCVLRQENLI LPLRPQDPII GTAAAVLENL ATRLRPFLQC DIIWIASFVL I: ARI.ANRV ERGVSEIDYT GLIEILDTHR QECSSRVCEL TASHTIAPLY 22 SUBSTITUTE SHEET WO 94/00575 PCT/EP93/01525 Sequence No. 2 1 ATGGCTA.CCG ACATTGATAT GCTAATCGAC CTAGGATTGG ACCTGTCCGA 51 101 151 201 2 c' 301 351 401 451 501 551 601 CA': aAGCTC ACGACCCCGA ATGGAPLGACC TCCCAAAGGT CGACGCCTCG ACCACCGGCG GGAACCGCAC CACCGCATCC TACGGCCCCG CAGAAACGCC ACGGCCCCGG GACGTCTCCG GAGGAGGACG GTCCGACAGC CCTGCGGAGA CCCCCGGCCC CCCGGCPLGCG TGTGGTCGCG GGGGGGAAGG CCGAGGCGGG GCGC7CGCCGA CACAACCAAG CGCCACCCAC GGGGCCCG.CG, CTCTGGAGCG AGCGGGGAGT CGGAGGGGCG GCCCCGAGGA CGGCGGGGAG CCTCGGGACC TGGCCCGCAT CGGCGAGG1TC GGACGAGGAG GTTCCTCGTC GAGGCCATCG CGCCGGCACC CCGACGATCC AGGCGGTCGG CCAACCCCCG GCCGCCGGGG GGCCGCCGCG GGACGAGGAC ACGCGGCGAT CCCGAAGCCT GCCACCCGCG CTTCCCCCCG TCGACCAAGG CCGGGGTCGA CTCCACACCA AAACCCCGCC GGCGCGTCTC GGGGTCGCCA GGCGAGGCGC GCCACGAGGC CCCCGCGTCG GGCGCGGAGG ACGCGCCAGG GCGCGGACGG GGAGCAGCTC CCCCCCCTCC 651 701 751 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 GCTGATGGCG CTGTCCCTGA CCCCCCCGCA CGCGGACGGC TCCCGGAGCG AAAGGCGCCC TCTGCCGACA CCATCGACCC GCGGTTCTGC GATCCATATC CGAGCGCGCG GCG7GTCGAGC AAGCTT-TGGA CGCAGTGCCC TGGTCATGCA AGACCCr.TTT .,GTTTCCCCC CGCGAACAGC CCCTGGGCTC CCGTGCTGGC GGGGGGTTTG ACGCCGAGAC CCGTCGGGTT TCCTGGGAAA TCACGGCCCG AGCCTCTACC GCACATTCGC AGCCJACCCG CGACAGCCAA GGCCATGCGC GACTGCGTGC TGCGCCAGGA GAGGCCCTGG CGTCCGCGGA TGAGACGCTG GCGEGGTGCP TCACCACAAT CTGCCGCTCC GCCCCCAGGA CCCTATCATC CCGCCGTGCT GGAAAACCTC GCCACGCGCC TGCGCCCCT'. TACCTGAAGG CCCGAGGCCT GTGCGGGCTG GACGACCTG* ACGCCTGTCG GALCATTAAGG ATATTGCCTC CTTTGTGTT CCCGCCTCGC CAP.CCGCGTC GAGCGCGGCG TGTCGGAGA ACCGTGGGGG TTGGGGCCGG CGAGACGATG CACTTTTAC CGCGCCCCGG CGCCGTTCGG GCATCAGCC& GGCGGGATGC CACCCAAGCG CCCTGGTCGC CGGGCCGCGT AAATCTCATC AG.NTGTGCAT GGAACGGCGG r~ TCTGCAGTGC r GCTCGCGGCG G GTCATCCTGG T CGACTACACG A TCCCGGGGGC 23 SUBSTITUTE SHPET WO 94/00575 PICT/EP'93/01525 Sequence No. 2 con td. 1401 1451 1501 1551 1601 1651 1701 CTGCATGGCG CCAGTCGCGT GTGCACGGCA CATATTGACG GCGGGTGGGG GGCGATG1ACC GCGCGTCCAG GGTCTCATTG AAATACTGGA CACGCACCGC CAGGAGTGTT GTGCGAGCTG ACGGCCAGTC ACACTATCGC CCCCWTATAT AATACTTCTA CTGCAACTCC CTATTTTAGG CAAGhATAAA TCJ\ACCChAG TGGTTCCGTG TGIkTGTTCTT GGCGCGCGCG CGGAGh~CTCC GGGGCGATGC CGGCGTGCGC GTGGGAGGAG CACCGGATMA ATGTGGGGCC CCGGCCCGGC CCGCTTCIATP GAACTCACGG CAGIACGCGT -24.- SUBSTITUTE SHEET