IE85834B1 - Varicella-zoster virus antigen - Google Patents

Abstract

ABSTRACT The present invention relates to the construction of a recombinant plasmid which is capable of expressing a secretory truncated glycoprotein (T99) of Varicella-zoster virus (VZV) in mammalian cells.’ The secretory Tgp of the present invention contains at-1east~one epitope-capable of inducing antibody response; "The present inventionv contemplates the production and utilization of this secretory - The present invention is also directed towards the use of the secretory Tgp in diagnostic assays for detection of VZV.~ The present invention is also directed to first antibodies ' specific to secretory Tgp and to second antibodies specific to the first antibodies. These.second antibodies are also useful in diagnostic assays for VZV.

Description

VARICELLA-ZOSTER VIRUS ANTIGEN The present invention relates to the construction of a recombinant plasmid which is capable of expressing a secretory truncated glycoprotein (T99) of Varicella-zoster virus (VZV) in mammalian cells.’ The secretory Tgp of the present invention contains at-1east~one epitope-capable of inducing antibody response; "The present inventionv contemplates the production and utilization of this secretory - The present invention is also directed towards the use of the secretory Tgp in diagnostic assays for detection of VZV.~ The present invention is also directed to first antibodies ' specific to secretory Tgp and to second antibodies specific to the first antibodies. These.second antibodies are also useful in diagnostic assays for VZV.

Varicella-zoster virus is the causative agent of childhood chickenpox (varicella) and shingles tzoster), two distinct clinical manifestations. Varicella is the outcome of the primary encounter (infection) with VZV, whereas zoster is the result of VZV reactivation which occurs predominantly in aging and immunosuppressed individuals, including cancer and AIDS patients.

Tgp in a Vaccine against chickenpox and/or shingles.

There are 2.5 million estimated cases of chickenpox and 1.2 million cases of shingles per year in the united States. It is expected that the number of shingles patients will increase as the population ages. one of the most common complications of shingles includes postherpetic neuralgia which is characterized by interactable pain lasting for four weeks to several years after the onset of skin rash. other complications of VZV reactivation (shingles) include encephalitis, pneumonitis and disseminated zoster.

VZV is a member of the alpha herpesvirus family.

VZV contains a linear double—stranded DNA genome of approximately 125,000 base pairs and consists of the sequence of a long unique (U1)—inverted short repeat (IRs)—short unique (Us)rtermina1 short repeat (TRs). VZV DNA encodes five glycoproteins, designated gpl, gpII, gpIII;'gpIV and s mediated immune response in the infected individuals (Davison, et a1., supra, 1986). __ gpI or result in steric hindrances which influence the antigenic determinant recognized by anti—peptide antibodies.

An attenuated varicella—zoster virus vaccine has been used in Japan against chickenpox infection in leukemic children as well as for routine.vaccination in early childhood. This vaccine is currently being tested in the United States in children with leukemia and is expected to be used in healthy children and_for the-prevention of vzv ‘ ,reactivation (shingles) in the elderly population. .Although ; the attenuated varicella vaccine-hesfiheen shown to be safe , and effective.in inducing immunity again$t VZVminfe¢ti0n3 however, similar to_natural infection. attenuated varice11a_ vaccine becomes latent in human dorsal root ganglia and may- reactivate to produce shingles with its attendant neurolggic complications of postherpetic neuralgia and encephalitis.

Therefore, a subunit vaccine which would_avoid and . eliminate latency is desirable for immunization of children as well as for boosting immune response in the elderly who are more susceptible to VZV reactivation_(shing1es), Such subunit vaccine as contemplated by the present invention may be prepared by construction of recombinant viruses (e.g., vaccinia virus) expressing one or more VZV glycoproteins or, as particularly contemplated by the present invention, may be composed of secretory highly immunogenic VZV glycoprotein(s) which can be prepared and purified in large quantities and used for immunization and/or boosting the immune response against VZV infection. In addition, such highly purified VZV glycoproteins can be used as a diagnostic tool for the assessment of the immune status to VZV infection in immunosuppressed individuals (leukemic children, AIDS and cancer patients) as well as in vaccinated individuals and elderly population.

The gene for VZV gpl has been previously isolated, inserted into a plasmid and incorporated into a vaccinia virus expression system. Although gpI protein was produced by the vaccinia expression system, the product remained . within the cells and was therefore unsuitable for eliciting: an antigenic response in-gigg. . p p j The innovation of the present invention resides in" the construction of an expression vector which produces an truncated form of VZV gp which is secretedgfromamammaliand cells.

I. The applications of the present recombinant vaccinia viruses expressing secretoryrtruncated-VzV~ glycoproteins containing one or more highly immunogenic viral epitopes include: (1) using such recombinant viruses as subunit vaccines against Vzvtinfection; wherein secretion of ::; VZV glycoproteins following vaccination provides a stronger imune response to VZV glycoproteins as well as to VZV infection; (2) using large quantities of highly purified and immunogenic secretory VZV glyooproteins containing one or more epitopes as a subunit vaccine against primary VZV infection (chickenpox) in healthy children as well as in immunocompromised individuals and for boosting imunity against VZV reactivation (shingles) in the elderly; and (3) "using purified preparations of secretory truncated VZV glycoproteins in diagnostic kits as highly specific target antigens for the detection and assessment of antibody status to VZV glycoproteins.. since VZV reactivation is common in cancer and AIDS patients, there is also a need for the serological diagnosis of VZV infection in these patients. In addition, since VZV reactivation in the growing population of elderly individuals results in pain prior to the onset of clinical symptoms and may also result in encephalitis, pneumonitis and disseminated zoster, the only hope for an early treatment of these patients lies in a rapid means of diagnosis. Application of the present recombinantly prepared secretory VZV glycoproteins in diagnostic kits can provide a rapid and . inexpensive means for diagnosis of VZV infection.

A The ‘present invention is directed to an expression vector for secretory truncated VZV gp andvconstruction of said vector which permits extracellular secretion of the VZV protein.

More specifically, the present invention is directed to a recombinant DNA expression vector comprising a nucleotide sequence capable of expressing in an infected, transfected or -transformed host a mature polypeptide. said polypeptide being selected from the group consisting of: the 159 amino acid sequence of SEQ ID NO:1, the 511 amino acid sequence of SEQ" ID NO:2, a 276 amino acid sequence defined by an EcoPllEcoRI double digest of a DNA fragment spanning nucleotides ’115712,to 118181 of glycoprotein I of Varicella-zostervirus and containing gpl open reading frame, a 316 amino acid sequence defined by a 'BstE2/EcoRl double digest of a DNA fragment spanning nucleotides 115712 to 118181 of glycoprotein I of Varicella-zoster virus and containing gpl open reading frame, a 341 amino acid sequence defined by an Ndel/EcoR| doubt digest of a DNA fragment spanning nucleotides 115712 to 118181 of glycoprotein I of Variceila-zoster virus and containing gpl open reading frame, or a 408 amino acid sequence defined by an EcoR| single digest of a DNA fragment spanning nucleotides 115712 to 118181 of glycoprotein I of Varicella-zoster virus and containing gpl open reading frame, and wherein said polypeptide is capable of being secreted extraceilularly from said host and is capable of causing a VZV antibody response in mammais.

Another aspect of this invention contemplates a plasmid encoding a Variceila- zoster virus polypeptide said polypeptide being selected from the group’ set out above, and wherein said polypeptide is capable of being secreted extraoeiluiarly from a host and is capable of causing a VZV antibody response in a mammal.

A furthet aspect of the present invention is directed to a process for producing a secretory truncated Varicella-zoster virus glycoprotein wherein the glycoprotein is a polypeptide selected from the group set out above, and wherein said polypeptide is capable of causing a VZV antibody response in mammals, said process comprising the steps of:- a) providing a vector comprising a nucleotide sequence coding for said polypeptide, wherein the nucleotide sequence is capabie of being expressed by a host containing the vector; b) incorporating the vector into the host; and _ c) maintaining the host containing the vector under conditions suitabie for expression of the nucleotide sequence into said glycoprotein.

Yet another aspect of the present invention relates to a recombinant secretory truncated VZV gpl being a polypeptide selected from the group set out above, and wherein said polypeptides are secreted extracellularly from mammalian cells within which said polypeptides are produced and wherein said polypeptides include at least one antigenic epitope capable of eliciting an antibody response in a mammalian host.

Still another aspect of the present invention is the use of secretory truncated VZV gp being a polypeptide selected from the group set out above for the manufacture of a vaccine_ for stimulating an immune response to chickenpox and/or shingles. l ‘ Yet another aspect of this invention contemplates a method of diagnosing.

VZV comprising contacting serum, tissue or tissue extracts of an individual to be tested with an antibody against a secretory truncated VZV gp being a polypeptide selected from the group set out above for a time and under conditions necessary to form an antibody-antigen complex, and detecting any resultant antibody—antigen complex. g Fig. 1 is a schematic representation of the construction of a recombinant plasmid carrying a truncated VZV gpl gene having the e1 epitope. VZV gpl gene cloned in pGEM-'4 transcription vector (Vafai, et al., supra, 1988) was cleaved with Bgill (B) restriction enzyme downstream from e1 epitope and with l_E.c_>o'Rl (E) in the pGEM polyiinker. The truncated gpl gene was electroeluted, blunt-ended and cloned at the Smal (S) of vaccinia virus insertion vector pSC11 as described in detail in the Examples.

Fig. 2 demonstrates expression of a truncated VZV gpl by recombinant vaccinia virus. BSC-1 cells were infected with recombinant vaccinia virus carrying a truncated VZV gpl, as described in Fig. 2, (designated VV'l'gplBglll) and containing e1 epltope. After 22 hours, infected cells were labelled with [35S} methionine for 1 hour and cell Iysates were prepared as described in the Examples. Cell lysates were immunoprecipitated with the following monoclonal antibodies (MAbs) and a human serum and analysed by SDS-12% polyacrylamide PAGE:MAb79.7, directed against e1 epitope; human serum (H-serum) from a VZV seropositive individual; MAbCt, directed against VZV gpl and recognizing epitope(s) other than e1; MabG7, directed against \/ZV gpl and recognizing only the glycosylated form of et epitope; MAbG6, directed against VZV gplV; MAbF8, directed against VZV gpll: and MAbE10, directed against VZV gplll. The size (in kiiodaltons) of precursor and glycosylated form of the Tgp|Bg||l are shown on the left.

Fig. 3 demonstrates expression and secretion of Tgp|Bg|ll from the infected cells. In the left panel, cells were infected with VVTgplBg|l| and after 22 hours, infected cells were pulse-labelled with [358] methionine (300 poi/ml) for 10 min. Cells were either harvested or washed with serum-free medium and the label was chased for 1, 2, 3 and 7 hours. Uninfected cells (Un) were pulse-labelled for 10 min. and chased_,tor 7 hours. Cell lysates (CL) were prepared and imrnunoprecipitated with MAb79.7 (a) which is directed against VZV gpl e1 epitope and MADC1 (b) which is directed against VZV gpl but only recognizes epitope(s) other than e1. Right panel, tissue culture fluid (TCF) from uninfected (Un) and VVTgplBgIll-infected cell chased for '1, 2-, 3 and 7 hours, were immunoprecipitated with MAb79.7(a) and MAbC1 (b).

Samples were analysed by SDS-12% polyacrylamide PAGE as described in materials and methods. Apparent sizes (in kiiodaltons) of ‘precursor and glycosylated mature forms of Tgpl are shown.

Fig. 4 demonstrates expression of recombinant vaccinia virus carrying a full- size VZV gpl gene (designated Wgpl). Cells were infectedwith Wgpl (Cabirac, et al., 1988) and after 22 hours, cells were pulse-chased as described in Fig. 3 and cell Iysates"(CL) and tissue culture fluids (TCF) from uninfected (Un) and Wgpl-infected cells (vvgpl) were immunoprecipitated with MAb79.7 which is directed against VZV gpl e1 epitope. Samples were analysed by SDS-8% polyacrylamide PAGE as described in the Examples. The size-range of precursor-products of VZV gpl (Vafai, et al., 19:38) is indicated.

Fig. 5 demonstrates immunoprecipitation of VZV-infected cells with rabbit antibodies prepared against recombinant vaccinia virus carrying a truncated VZV gpl and containing e1 epitope (VVTgpl). Cells were infected with .VZV and after 48 hours, cells were pulse-labelled with [353] methionine (300 uci/ml) for 10 min. in the absence or presence of (15 pg/ml) tunicamycin (T M), which inhibits the addition of N- linked oligosaccharides to native VZV gpl. Cells were either harvested or washed and chased for 2 hours in the absence or presence of TM (15pg/ml). Cell lysates were prepared and immunoprecipitated with MADC1 (a) which recognizes both VZV gpl and gplv and rabbit anti-VVTgpl antibodies (FiAnti-VVTgp|). Samples were analysed by SDS-8% polyacrylamide PAGE as described in the Examples. Apparent sizes (in kilodaltons) or precursor-products of VZV gpl and gplv (Vafai, et al., supra, 1938) are indicated on the right. Lysozyme (14.3 kDa), [3-Iactoglobulin (18.4 kDa); oi- chymotrypsinogen (25.7 kDa), ovalbumin (43.0 kDa), bovine serum albumin (68.0 kDa), phosphorylase B (97.4 kDa), and myosin (200.0 kDa) were used as internal size markers. ' The present invention contemplates the construction of a recombinant plasmid having a truncated VZV gp (3, II, III, IV or V) gene carrying at least one epitope capable of inducing antibody response in mammalian cells. In particular, the present‘invention-relates to a vaccinia virus expression system capable of producing truncated VZV gp which can be secreted from mammalian cells into a host organism in mm) In one embodiment, the present invention contemplates the construction of a recombinant plasmid having a secretory truncated VZV gpl (referred to as TgplBglll or VV'l'gplBgl|l) carrying e1 epitope and production of said protein by the vaccinia virus expression vector in mammalian cells as described herein.

In another embodiment, the present invention contemplates the construction of a recombinant plasmid having a secretory truncated VZVgp| (referred to as Tgplxmalll or VVTgplXmalll) and production of said protein by the vaccinia virus expression vector in mammalian cells as described herein.

In another embodiment-, this invention contemplates the preparation and use of a vaccine compositon for the treatment of chickenpox and/or shingles.

Previously used vaccines have generally comprised (I) an attenuated live virus type of vaccine in which the virus has been rendered avirulent but not killed by some form of genetic attenuation; or (II) specific viral components isolated and purified from the virus and inactivated by formalin or some other chemical or physical treatment.

The present invention contemplates Type II vaccines, wherein the specific viral components isolated and purified from the virus and inactivated by formalin or other treatments are contemplated to be secretory truncated VZV gp. Unless otherwise specified in the Specification and Claims, VZV gp means VZY gpI, gpII, gpIII, gpIV or gpv. Furthermore, “truncatedW.as used in the specification and Claims is defined as a segment of indeterminate size of the VZV gp_(but' not the full-sized VZV 9P) wherein a substantial portion (or all) of the amino acid sequence.ceterminal of the region has~»» sbeen_deleted. .The present invention alsorcontemplates-theiap preparation of recombinant secretory truncated Vzvngp for use in a vaccine against VZV. ' 3 ' ' .In another embodiment, the present intention is directed to a Type II vaccine which contains secretory. truncated VZV gp.

By vaccine is meant an agent used to stimulate the ,immune system of a living organism so that immunological protection against future harm caused by an infectious.agent is provided. Administration.o£ a vaccine contemplated by the present invention to the patient (or animal) may be by any known or standard techniques. These include oral ingestion, intestinal intubation, or broncho-nasal spraying. other methods of administration, such as intravenous injection, that allow the carrier microbe to reach the human or animal's bloodstream may be acceptable when the carrier microbe is unable to reproduce.

In a further embodiment, the present invention contemplates a diagnostic assay for VZV, and additional1y,_a diagnostic kit for the detection of VZV antibody in various clinical manifestations of VZV infection and in vaccinated individuals.

The present invention represents a step forward from earlier attempts to obtain VZV gp protein. Previously, the known gene for VZV gpI was inserted into a plasmid and incorporated into a vaccinia virus expression system.

However, the VZV gpI produced by this expression system remained intracellular, i.e., within the mammalian ce1ls,' thus failing to permit antigenic activity (the production of antibodies). The discovery of_the present invention is an expression vector in vaccinia virus capable of producing, i.e.,_expressing a truncated VZV gp such as. for example, gpl,~gpII, gpIII, gpIV or.gpV, which is secreted fromf ; mammalian-cells. This.truncated gp further contains an epitope which causes the production-of neutralizing antibodies in invaded hosts. The VZV gp gene is cloned in pGEM transcription vector, cleaved with restriction enzymes. such as, for erample, EcoRI and Bgl II=restriction:enzymes (to remove part or substantially all of the C—termina1 region), blunt-ended and cloned at the Smal site of vaccinia virus insertion vector pSC11.as shown in Figure 1.

V_ In a_preferred embodiment, the present.invention contemplates the construction of a recombinant DNA expression vector as follows. A 592-bp DNA fragment containing 17 bp from pGEM-4 polylinker upstream from SmaI site and 575 nucleotides (spanning nucleotides 115712 to 116287 of the VZV genome) is cleaved with EcoRI and Bg1II, respectively, from PGEM recombinant carrying a blunt—ended VZV BglI DNA fragment (spanning nucleotides 115712 to 118181) containing gpI open reading frame (Davison and Scott, supra, p. 1800-1801, 1986, incorporated herein by reference). The truncated gpI (TgpI) DNA, encoding the N-terminal region of gpl with 159 amino acid residues and an estimated size of 17.5 kDa, is blunt- ended and cloned at the SmaI site of pSC11 plasmid vector (Fig. 1) and inserted into vaccinia genome as described in the.Eramp1es.

The truncated gpi (Tgpl / Bglll) contains el epitope located within 14 amino acid residues between residues 109 to-123 of the Predicted amino acid sequences of VZV gpl (Vafai, et al., supra, 1988; Vafai, et al., supra, 1989). The TgplBglll lacks the 464 amino acid residues at the C-terminal region of gpl, which apparently includes the membrane-anchoring region of gpl.

In another preferred embodiment, the present invention contemplates the construction of a recombinant expression vector as follows. A 1647-bp DNA fragment containing 17 bp from pGEM-4 polylinker upstream from the Smal site and 1630 VZV nucleotides (spanning nucleotides 115712 to 117342 of the VZV genome) is cleaved with EcoR1 and Xmalll, respectively, from pGEM recombinant carrying a blunt-ended VZV Bglli DNA fragment (spanning nucleotides 115712 to 118181) and containing gpl open reading frame (Davison and Scott, supra, p. 1800-1801, 1986).

The truncated gpl (T gplxmalll) DNA, encoding the N-terminal region of gpl with 511 amino acid residues, was blunt-ended and cloned at the Smal site of pSC11 plasmid vector and inserted into the vaocinia virus genome as described in the Examples.

The Tgplxmalll lacks the 112 amino acid residues at the C-terminal region of gpl, which apparently includes the membrane-anchoring region of gpl.

The present invention is directed to a recombinant DNA expression vector as described abovevwhich includes a nucleotide sequence capable of expressing‘. in an infected, transfected or transformed host a Tgp which is secreted extracellularly "from said host and which is capable of causing an antibody response in mammals, and preferably a mature polypeptide defined by the 159 amino acid sequence (Seq. id.

No. 1): Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met GIY P319 15 Gly Ile Ile Thr Gly Thr Leu Arg Ile Thr Asn Pro Val in-g Ala 30 Sr: Val Leu Arg Tyr Asp Asp Phe His Th: Asp Glu Asp Lys Leu 45 Asp Thr Asa Set Va}. '1’Y7-' 51“ 910 TY! TY?-' His 39‘ MP “is A13 60 Glu ser Ber '1‘r:p Val Asn Ax-9 Gly Glu ser. Set Arg I.-ys Ala Tyr 75 Asp His Ash sex Pro Tyr Ile Tr? P7-'0 fir‘! 35" “P TY’ “P 513' 9° Phe Leu cm Asn Ala His c1u His His G1? Val TY!‘ Mn '31“ 91? 1°5 1 M-9 c;1y_11e Asp Se: Gly Glu Arg Len Hat Gln Pro Thr Gln Bet 120 Set Ala cm Glu Asp Leu Gly 389 Asp Thr 61? I19 H15 "91 11° 135 Pro .'rtu5;x.eu nan Gly Asp Asp Ara His I-Y 11° V31 55“ “*1 “*9 15° Gln Arg Gln Tyr 611 Asp Val P118 1-Y5 159 or a mature polypeptide defined by the 511 amino acid sequence (Seq. Id. No. 2): The infected, transfected or transformed host contemplated by the present invention can be mammalian cells such as, for exampie, green monkey kidney celis (BSC-1), -13a- COS monkey cells, HeLa cells, hamster kidney cells and human fibrobtast ceils. in addition, any human tissue is contemplated as a suitable host. The present invention contemplates that the infected, transformed or transfected cell as described above can be caused to produce and secrete Tgp (truncated VZV glycoprotein) and preferably, TgplBglI| as defined by Seq. id. No. 1 or Tgplxmaill by the recombinant DNA expression vector of the present invention.

The present invention also contemplates other secretory Tgpls. Other Tgpls containing other immunogenic epitopes can be generated by cloning and expressing various truncated gpl genes. The other gpls can be obtained, for example, by utilising the following restriction enzymes: (21) EcoPl and EcoRI, wherein EcoPl cleaves at nucleotide 115751 and results in generation of a 276 amino acid sequence; (b) BstE2 and EcoRl, wherein BstE2 cleaves at nucleotide 116754 and results in generation of a 316 amino acid sequence; (:3) Nde1 and EcoRl, wherein Nde1 cleaves at nucleotide 116831 and results in generation of a 341 amino acid sequence; and (cl) EcoFiI and EcoRl wherein EcoRl cleaves at nucleotide 117034 and results in generation of a 408 amino acid sequence. _The various types of Tgpl contemplated by the present invention as described above are specifically constructed to eliminate that portion of the C-terminal region of the nucleotide sequence encoding the region of VZV gp which prevents the extracelluiar expression of that VZV gp (such as gpl, gpll, gplll, gpIV or gpV), i.e., apparently by eliminating the membrane anchoring region of the encoded protein. in preparing suitable expression vectors for such truncated secretory proteins, it is also necessary to maintain at least one epitope, e.g,, e1, so that the encoded Tgp (extracellularly secreted) elicits an antibody response in the host. Provided with the discovery-of the present invention one skilled in the art is able to construct various suitable expression vectors for use as vaccinesaand-detection systems, all contemplated by the present invention.W = The present invention also contemplates the modification of the recombinant DNA expression vector _described above in order to obtain other truncated VZV gps.

The discovery of the present invention is directed to deleting the native (naturally occurring) VZV DNA encoding the C—termina1 region of gpII, gpIII, gpIV or gpv, as described above for gpI, resulting in the production of TgpII, TgpIII, TgpIV or Tgpv, which can be secreted outside of mammalian cells and resulting in antigenic activity, in gigg, i.e., stimulating antibody formation.

The present invention also contemplates a process for producing a secretorv truncated Varicella-zoster virus gp. This process consists of the following steps: _sequence into said polypeptide. a) providing a vector comprising a nucleotide sequence coding for said polypeptide, wherein the nucleotide sequence is capable of being expressed by a host containing the vector, and wherein the nucleotide sequence is selected from the group of nucleic acids capable of encoding with a continuous nucleotide sequence a Varicella-zoster virus gp wherein a-substantial portion of VZV gp Cterminal region of the gp is genome coding for the deleted and wherein said gp is secreted extracellularly from said host and'includes at least one epitope effecting an antibody response in a mammalian host, and such as, for example, a polypeptide of the open wreading frame defined by the 159 amino acid sequence of Seq.

Id. No. I or a polypeptide of the open reading frame defined by the I59 amino acid sequence of Seq. Id. No. 2; incorporating the vector into the host; and maintaining the host containing the vector under conditions suitable for expression of the nucleotide This process can include a promoter operationally associated with said nucleotide sequence. This process further contemplates said nucleotide sequence including a region of nucleotides capable of encoding a leader sequence.

The expression of recombinant vaccinia virus carrying the TQP (VVTQP) can be analyzed by infection of, for example, BSC-1 cells with Tgp such as, for example, VVTgpI, and immunoprecipitation of cell lysates with a panel of MAbs prepared against VZV gpl, gpII, gpIII, gpIV and a VZV seropositive human serum or other conventional means known to those skilled in the art. "with respect to the fact that said Tgp is secreted from the infected cells, cells can be infected with Tgp such as, for example, VVTgpI, and radioactively-labeled.

Antibodies against Tgp such as, for example, VVTgpI, can be generated in, for example, rabbit (e.g., RAnti—VVTgpI as described in the Examples). These antibodies can be tested to determine whether Tgp such_as, for example, vVTgpI, is capable of inducing antibody response-which is recognized by VZV gp. Mammalian cells such as, for example, jBSC—l cells can be-infected with VZV and radioactive1y~’ "labeled in the absence or presence of tunicamycinicvafai} et 1 -31., 1989); The Tgp of the present invention is papab1e.o£' inducing antibody response which is recognized by native epitope (such as e1 as found in Tgpl defined by—Seq._Id.-No. 1) and is capable of neutralizing VZV infectivity in the presence of complement. ’ i Vaccines of the present invention may be administered parenterally (e.g., by intramuscular, _ subcutaneous, or intravenous injection). The amount required will vary with the antigenicity of the'ggne product and need only be an amount sufficient to induce an imune response typical of existing vaccines. Routine experimentation will easily establish the required amount. Typical initial dosages of vaccine could be about 0.001-100 mg antigen/kg body weight, with increasing amounts or multiple dosages used as needed to provide the desired level of protection.

The pharmaceutical carrier in which the vaccine is suspended or dissolved may be any solvent or solid that is non-toxic to the inoculated animal and compatible with the carrier organism or antigen gene product. Suitable _ pharmaceutical carriers include liquid carriers, such as normal saline and other non—toxic salts at or near physio- logical concentrations, and solid carriers, such as talc or sucrose. Adjuvants, such as Freund's adjuvant, complete or incomplete, may be added to enhance the antigenicity via the bronchial tubes, the vaccine is suitably present in the form of an aerosol. Booster immunizations may be repeated numerous times with beneficial results.

The present invention is also directed to a method for stimulating an immune response to chickenpox and shingles in a mammalian host. This method consists of administering an ef£eetive.amount.of secretory VZV Tgpw1e.g.,“TgpI,,TgpII, TgpIII,;TgpIV or Igpv) under conditions su£ficient_t6 cause the production of antibodies to said1Tgp,¢which’are'we1l recognized_by one of ordinary skill in the art. ‘The dosage effective amount is.0.001—100 mg of secretory VZV TQP/kg body weight.

The subject invention also encompasses antibodies, either monoclonal or polyclonal, which are useful in the therapeutic control of chickenpox and/or.shing1es- Said antibodies_can_be prepared as described above and by injecting mammalian species, e.g., human,.horse, rabbit, sheep, mice, etc. with inactivated Tgp or derivatives thereof ‘and then purifying said antibodies employing the detection systems contemplated and described in further detail below.

In another embodiment, the present invention relates to the development of specific human or other (e.g., African green monkey kidney cells, COS monkey cells, HeLa cells or Chinese hamster cells) polyclonal or monoclonal antibodies raised against secretory VZV Tgp (I, II, IIl, IV or V), as well as human-mouse chimeric polyclonal or monoclonal antibodies for administration in passive immunization against chickenpox and/or shingles.

Immunization refers to the process of inducing a continuing high antibody level in an organism i.e., in humans, which is directed against an antigen to which the organism has been previously exposed.

Eembodiment of“the?present ffiVenEion.’f””" gtechniques. .immune-system; Passive immunization, as defined herein, refers to resistance (e.g., temporary or sustained protection against infection) based on giving preformed antibodies to a patient from an in fiiyg or in'vitro source. The maih advantage of passive immunization is the prompt availability of large amounts of antibodies against Vzv as aes¢i:5aan:n*£5s ‘ human-mouse chimeric antibody is produced by oomhining the Fab.portion of the mouse imunoglobulin gene and £52-Fe portion of the human immunogiobulin gene by recombinant DNA ' The Production of human-mouse chimeric’ o'x antibodies is advantageous since large amounts of antibodies .can be produced by this system and human—mouse chimeric ‘antibodies can be recognized by‘cei1s of“the human?immune isystem whereas non4chimeric—antibodies would not be" i The present invention further contemplates the use of the above—described antibodies in a detection assay (immunoassay) for VZV.

A wide range of immunoassay techniques are available for utilization in the present invention as shown by reference to U.s. Patent Nos» 4,016,043; 4,424,279;- 4,018,653 and by Harlow. et al., supra. This, of course,* includes both single-site-and two—site, or "sandwich", assays of the non-competitive types, as well as in traditional Sandwich assays are among the A number of variations of the: technique exist, and all are intended to he the present invention. typical forward assay, an unlabeled antibody-' in a solid substrate and the sample to be‘ tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time ' sufficient to allow formation of an antibody-antigen binary invention. sandwich assay encompassed by In a complex, a second antibody, labeled with a reported molecule_' capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody—1abe1ed antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of the visible signal produced by the reported molecule. 'The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.

Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labeled antibody and sample to be tested are first combined, incubated and then added to the unlabeled surface bound antibody. These techniques are well known to those skilled in the art, and the possibility of minor variations will be readily apparent to those skilled in the art.

As used herein, “sandwich assay" is intended to encompass all variations on the basic two—site technique.

For example, these antibodies may be used to detect secretory Tgp, e.g., Tgpl, -'Tgp;II, TgpIII, TgpIV, Tg.pV or-more’: 1 . specifically, secretory gpI as defined byJSeq;:1d;vNo: 1, by- use of specific antigenic determinants Le;g.,=el epitope) as_ immobilized immunoadsorbants. .Serum is obtained from subjects to be tested and said serum contacted to the immobilized viral immunoadsorbants. If said serum contains antibodies to said imunoadsorbants, an antibody~adsorhant conjugate will result. After removing excess serum and non- bcund antibodies, a second antibody specific.to a first antibody, said first antibody-being capable of forming a conjugate with said immunoadsorbant, is added thus resulting in a double antibody-adsorbent conjugate. This double antibodyvadsorbant conjugate will only result if the test serum contains antibodies to the imunoadsorbant.

Consequently, standard detection techniques can be used to identify the conjugate.

E In another immunoassay, the competitive binding assay, a limiting amount of antibody specific for the molecule of interest (either an antigen or hapten) is combined with specific volumes of solutions containing an unknown amount of the molecule to be detected and a solution containing a detectably labeled known amount of the molecule to be detected or an analog thereof. Labeled and unlabeled molecules then compete for the available binding sites on the antibody. Phase separation of the free and antibody—bound molecules allows measurement of the amount of label present in each phase, thus indicating the amount of antigen or hapten in the sample being tested. A number of variations in this general competitive binding assay currently exist.

In any of the known immunoassays, for practical purposes, one of the antibodies to the antigen (secretory truncated VZV gp) will be typically bound to a solid phase".—»* and a second molecule, either theisecond antibody in a:. esandwich assay, or, in a competitive assay, the known amount" —‘ of antigen, will bear a detectable label or reporter molecule in order to allow visual detection of an antibody—antigen reaction. when two antibodies are employed, as in the sandwich assay, it is only necessary that one of the antibodies be specific for, e.g., secretory truncated VZV gpI, II, III, IV or V. The following description will relate to a discussion of a typical-forward sandwich assay; however, the general techniques are to be understood as being applicable to any of the contemplated imunoassays.* =In the typical forward sandwich assay, a first antibody having specificity for, e.g., secretory Tgpl, II, III, IV or V or the mature polypeptide as defined by Seq. Id.

No. 1 or its antigenic fragments is either covalently or passively bound to a solid surface. typically glass or a polymer, the most comonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. be in the form of tubes, beads, discs or microplates, or any The binding processes are well-known in the art and generally consist of cross—1inking, covalently binding, or physically adsorbing the molecule to the insoluble carrier. Following binding, the po1ymer—antibody complex is washed in preparation for the test sample. An aliquot of the sample to The solid surface is The solid supports may other surface suitable for conducting an immunoassay. be tested is then added to the solid phase complex and incubated at 25°C for a period of time sufficient to allow binding of any subunit present in the antibody. The ihcubation_period will vary, but will generally be in the range of about 2-40 minutes. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the haptenfivglhe second antibody is linked to a reporter‘ molecule ‘which-:_is used to indicate the bi___ndin‘g_,_ of the‘ second antibody to the hapteni . p dBy "reporter molecule", as used in the present specification and claims, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody.

Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuc1eotide— containing molecules. ’ In the case of an enzyme immunoassay (EIA), an enzyme is conjugated to the second antibody, generally by - means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta—galactosidase and alkaline phosphates, among others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; and for peroxidase conjugates; 1,2—phenylenediamine, 5-amino- salicyclic acid, or tolidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above.

The enzyme—1abeled antibody is added to the firstj antibody hapten complex, allowed to bind, and then excess" reagent is washed away. A solution containing the , appropriate substrate is then added to the ternary complex of antibodyeantigensantibody4- 1he:substrate will react with the ’ Aenzyme linked to the second antibodyfigiving afiqualitative visual signal, which may be further quantitated, usually‘ spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.. ' 'A1ternately, fluorescent compounds, such-as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. when activated by illumination with light of’a particular‘ wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the EIA, the fluorescent labeled antibody is allowed to bind to the first antibody—hapten complex.

After washing off the unbound reagent, the remaining ternary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. other reporter molecules, However, such as radioisotope, chemi- luminescent of bioluminescent molecules, may also be employed. It will be readily apparent to the skilled technician how to vary the procedure to suit the required purposes. it will also be apparent that the foregoing can be used to detect directly or indirectly (i.e.. via antibodies) VZV.

The present invention also contemplates a method of diagnosing VZV comprising contacting serum, tissue or tissue extracts of an individual to be tested with an antibody against secretory truncated VZV gp (such as, for example, ggpl, gpll, gplli, gplv or gpv) or an active fragment thereof, for a time and under conditions necessary to form an antibody-antigen complex, and then detecting any resultant antibody--antigen complex. Such conditions would be well recognized by an artisan of ordinary skill in the art.

In a further embodiment, the present invention also relates to a‘ kit for the detection of antibodies produced in response to secretory Tgp (such as, for example, Tgpl, Tgpll, Tgplll, Tgplv, TgpV or TgplBgl|l as defined by Seq. ld. No. .1 or TgplXrr_1a|ll as defined by Seq. id. No. 2) and its antigenic fragments (epitope(s)), the kit being compartmentalised to receive a first container adapted to contain an antibody having specificity for said antibody to Tgp or fragments thereof and a second container containing an antibody specific for first antibody and being labelled with a reporter molecule capable of giving a detectable signal. if the reporter molecule is an enzyme, then a third container, containing a substrate for said enzyme is provided.

The diagnostic kit of the present invention can be used to detect the antibody status of VZV glycoproteins. This kit represents a rapid method of diagnosis and detection of VZV in individuals exhibiting various clinical manifestations of VZV as well as in vaccinated individuals.

EXAMPLES 1) Materials And Methods ,.Varice11a—zoster virus (VZV) strain VZV86, originally isolated from a patient with zoster, was grown and propagated in African green monkey kidney cells (BSC-1) by the cocultivation method described in Vafai et al., Virus ~ ggg; lg: 319-336 (1989). Vaccinia virus (strain IHDJ) was grown in BSC-1 cells at’a multiplicity of infectioni(mto.i.) of 0.01 plaque forming units (PFU) as described in Cabirac, et al.V, Virus Res. _1g=2'o5-214 (1988).

) Preparation of Monoclonal and.Po1yc1ona1 Antibodies Monoclonal antibodies (MAbs) against VZV g1yco—- - described in Vafai et a1., Virus Res. 1:325-333 (1987).

) Construction of Recombinant Vaccinia Virus Fig. 1 illustrates the construction of the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982)), and ligated into the Smal site of the insertion vector pSC11(Chakrabarti, et al., , 5: 3403-3409 (1985)). The recombinant plasmid was designated pVVTgplBg|l'l (and is shown in Fig. 1). The recombinant vaccinia virus was generated by the method described by Mackett, et al. “The Construction and Characterisation of Vaccinia Virus Recombinants Expressing Foreign Genes” in (D. M. Glover, ed.), iRL Press, Oxford, p. 191-211 (1985) with modifications. 143B TK' cells (Mackett, et al., 1985) were grown overnight in the absence of bromodeoxyuridine (BudR), and were then infected with vaccinia virus (strain IHDJ) at a m.o.i. of 0.01 PFU and incubated at 37"C for 90 min. Infected cells were then transfected with 30 pg of pVVTgpl and 50 pg of Lipofectin reagent (BRL) according to the manufacturer's instructions. Cells were harvested 48 hours post-infection-transfection and the resulting virus stock was passaged twice in TK’ cells in the presence of 25 pg/ml BudR. Recombinant vaccinia virus was distinguished from spontaneous TK' virus by straining with bluo-gal (BRL) and was clonally isolated by three cycles of plaque purification.

In order to prepare a more immunogenic secretory truncated VZV gpl, the same methodology was employed with the modification that the recombinant plasmid (pGEM-4) containing the VZV gpl gene was cleaved with Xmalll at VZV nucleotide sequence 117342 and with EcoR1 (instead of Bgill and Eco R1). The truncated gpl gene, encoding the N—terminal region of gpl with 511 amino acid residues, was The recombinant plasmid was designated pVVTgplXmal|l; The recombinant vaccinia virus was generated as described above and designated V\/'l'gplXmalll. ) _ The expression of recombinant vaccinia virus carrying the truncated VZV gpl gene (designated VVTgp|Bglll and VVTgplXma|ll) were analysed by infection of BSC-1 cells with VVTgpl at a m.o.i. of 1.0 PFU. After 22-hour incubation at 37°C. infected cells were starved for methionine in methionine-free medium for 1 hour and were then labelled with [358] methionine (100 uci/ml) for 1 hour. Cells were then washed three times with cold phosphate-buffered saline and disrupted in 4 ml of lysis buffer (0.02 M sodium phosphate, pH 7.6, 0.1M NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS). Lysates were kept on ice for 2 hours and centrifuged at electroeluted, bIunt—ended and ligated into pSCll as described above. ,000 rpm in a Beckman SW60 rotor for 2 hours at 5°C. Supernatants were stored at -70°C until immunoprecipitation with monoclonal and poiyclonal antibodies prepared against VZV proteins. (See Fig:_. 2) .

The results shown in Fig. 2 demonstrate the expression of a primary translation product of 25 kDa by VVTgpIBgllI which was processed to a 32 kDa- polypeptide during 1 hour labelling period. Both 25 kDa and 32 koalproteins can be recognised by MAb79.7 which is directed against e1 epitope (Vafai, et al., 1988).

Human serum reacts with the 25 kDa species (Fig. 2, line 2) and longer exposures of the gels indicate a faint reactivity of human serum with mature form (32 kDa) of Tgpl.

The 32 kDa protein was also reacted with MAbG7 (Fig. 2, line 4) which recognises the fully glycosylated form of VZV gpl. However, MAbC1, which is directed against VZV gpl but only recognises epitope(s) other than e1, does not react with Tgpl (Fig. 2, line 3). MAbs directed against VZV gpIV (MAbG6), gpll (MAbF8) and gplll (MAbE10) do not also react with the precursor-product of Tgpl (Fig. 2, lines 5, 6, 7), indicating the specificity of Tgpl for MAb79.7.

These results indicate that the Tgpl expressed by VVTgplBg|li retains the native conformation necessary for recognition by MAb79.7 as we! as VZV seropositive human serum. The primary translation product of Tgpl of 25 kDa has been found to be larger than the expected 17.5 kDa deduced from 159 amino acid residues encoded by the Tgpl. I ) m BSC—1 cells were infected with V\fi'gpl8glll at a m.o.i. of 1.0 PFU and after '22—hour incubation period, cells were pulse—labelled with [358] methionine (300 uci/ml) for 10 min. Cells were either harvested or washed five times with serum—free medium and the label was chased for 1, 2, 3 and 7 hours. Uninfected cells were pulse-iabelled for 10 min. and chased for 7 hours. Cell iysates were prepared as described above and immunoprecipitated with MAb79.7 which is directed against VZV gplBg||l epitope (e1) (Vafai, et al., .L_‘s.Liml. 5212544-1551 (1988)) and MAbC1 which is directed against VZV gp|Bgll| but recognises epitope(s) other than e1.

Tissue culture fluids from uninfected and VVTgplBglI|-infected cells were coilected and centrifuged for 10 min. in a microfuge. Supematants were collected and after addition of equal volume of lysis buffer to each sample, the samples were centrifuged at 40,000 rpm in a Beckman SW60 rotor for 2 hours at 5°C. Supernatants were immunoprecipitated with MAb79.7 and MAbC1. (See Fig.3).

The results shown in Fig. 3 demonstrate that precursor TgplBglll (25 kDa) detected in the infected cells during a 10-min. pulse-labelling period isprocessed to a 32kDa protein species during 1 hour chase-period and that the intensity of both 25 kDa and 32 kDa bands decrease during 7 hours chase-period (Fig.3=, CL). The results also show that the mature glycosylated form of TgplBglll (32 kDa) appeared in tissue culture fluid of the infected cells within 1 hour chase-period and the intensity of the 32 kDa TgplBglll increases during a 7-hour chase-period (Fig. 3, TCF). These results indicate that the fully processed form of TgplBglll is released from the infected cells and is recognised by MAb79.7 but not MAbC1 which is directed only against gpl epitope(s) other than e1. In addition, western-blot analysis of the 32 kDa TgplBglll purified from the infected tissue culture fluids shows that secretory TgplBglll was recognised by MAb79.7.

Controls for these experiments were provided by infection of BSO-1 cells with a recombinant vaccinia virus carrying the untruncated (full-size) VZV gpl gene and expressing untruncated VZV gpl (Vvgpl) (Cabirac, et al., MiLI.is_Bes. 10: 205-214 (1988)). Pulse-chase experiments as above were performed. In contrast to V\/Tgpl, analysis of cell lysates and tissue culture fluid from Wgpl-infected cells indicated that native VZV gpl was not secreted from the infected cells (Fig. 4 ).

\“~. Antibodies to VVTgpl were generated in a rabbit by immunisation procedures described previously (Vafal, et al., Bflrusfles. Z: 325~333 (1987) and Mil:us_Res. 13: 319-336 (1989)). subcutaneously at multiple sites in the back and hind legs. After a preimmunisation bleed (for control sera) the animal received 1 x 107 PFU/ml of VVTgp|. This was followed by three weekly injections, each consisting of 1 x 10’ PFU/ml of VVTgp|.

A New Zeaiand white female rabbit was immunised V The animal was bled seven days after the last injection and the serum (designated RAnti-VVTgp|) was assayed by immunopreoipitation as described below. I (7) VZV was grown in BSC-1 cells by cocultivation and after 48 hours, infected cells were labelled with [355] methionine (300 pcilml) for 10 min. in'the absence or presence of tunicamycln (15 pg/ml). Cells were either harvested or washed three times with serum-free medium, and the label was chased in normal medium for’ 2‘ hours in the absence or presence of tunicamycin (15 pg/ml). Cells were "then washed three times with cold phosphate—buffered saline and cell lysates were prepared as described above and immunoprecipitated with RAnti-VVTgpI.

) Immunoprecipitation Cell lysates (400 ul) from uninfected, VVTgpI- Finally, 30 ul of a 10% Formalinéfixed S. aureus suspension- was added, and after 2 hours at 4°C, absorbed imune complexes were washed three times with lysis buffer and suspended in 20ul of THE buffer (50mM Tris, pH 7.4, 150 mM Nacl, 5mM EDTA). After addition of 10 ul of 3 x sample buffer (150 mM Tris, pH 7.0, 6%—SDS, 15% 2—mercaptoethanol, 0.03% bromophenol blue), the suspension was heated in boiling water for 4 min., cooled on ice, and analyzed by 8 to 12% polyacrylamide SDS—PAGE as described in Vafai, et a1., Q; viro1.~_5_g:953-959, l984 and J. Virol. g:2544—2551, 1_9'as.

(See Fig. 5.) I Cell lysates were imuunoprecipitated with MAbCl recognizes fiZ¥ gpl and gpIV and Ranti—VVTgpI B3111 and analysed by SDS-PAGE. As demonstrated in Fig.5A and B, the results show that similar to Hlbcl, RAnti-VVTgpI Bglll reacts with precursor-products of native VZV gpl in the presence or . . .= absence of tunicamycin. This shows that e1 epitope expressed by Vwrgpl Bglll induced antibody response which is-recognized by _' by VZV native gpI el epitope. 9) Neutralization Tests , .

Neutralization tests were performed by the constant virus-varying serum technique as described {Vafai, at al., 1987). Briefly, BSC-1 cells (108) were infected with VZV by cocultivation. After three days, when infected cultures showed 80-90% cytopathic effects,.cells were scraped into the tissue culture medium, centrifuged.at 2,000 x3g for 20 min. at 4°C and the cell pellet was resuspended in 4am1 of serum- free medium. The cell suspension was.dounce~homogenized (150 strokes) on ice in a Teflon—coated homogenizer. Thea homogenates were centrifuged at 800 x g for 10 min. and the supernatants were used for neutralization tests. -Aliquots (0.5 ml) containing 100-200 PFU were mixed with equal volumes-- of different dilutions {0,1:10, 1:50 and 1:100) of RAnti—' VVTgpI or rabbit anti-VZV antibodies (RAnti-VZV) prepared against VZV virions (Vafai, et al., 1987) or preimmune sera' and 0.25 ml guinea pig complement. The mixture were . incubated at 37°C for 2 hours and inoculated into 2 wells of BSC-1 cells grown in 6—we1l plates. After incubation at 37°C for 3 hours, the inoculum was removed, the cells were washed and overlaid with medium containing 2% fetal bovine serum (FBS) and incubated at 37°C for 5-7 days. Cells were then fixed with formaldehyde, stained with cresyl violet and plaques were counted as described (Vafai, et a1., 1984). i The results show a plaque reduction of, for example, 100% with 1:10 dilution of RAnti—VZV antibodies prepared against purified VZ virions (Vafai, et a1., 1987) in the presence or absence of complement and a plaque reduction of 50% with 1:10 dilution of RAnti-VVTgpI in the presence of complement. ’

Claims (2)

CLAIMS A recombinant DNA expression vector comprising a nucleotide sequence capable of expressing in an infected, transfected or transformed host a mature polypeptide, said polypeptide being selected from the group consisting of: the 159 amino acid sequence of SEQ ID N021. the 511 amino acid sequence of SEQ ID NO:2, a 276 amino -acid sequence defined by an EcoPl/EcoRl double digest of a DNA fragment spanning nucleotides 115712 to 118181 of glycoprotein I of Varicella-zoster virus and containing gpl open reading frame, a 316 amino acid sequence defined by a BstE2/EooRl double digest of a DNA fragment spanning nucleotides 115712 to 118181 of glycoprotein I of Varicella-zoster virus and containing gpl opening reading frame, a 341 amino acid sequence defined by an Ndei/EcoRl doubt digest of a DNA fragment spanning nucleotides 115712 to 118181 of giyccprotein I of Varicella-zoster virus and containing gpl open reading frame, or a 408 amino acid sequence defined by an EcoRl single digest of a DNA fragment spanning nucleotides 115712 to 118181 of glyooprotein I of Varice|la—zoster virus and containing gpl open reading frame, and wherein said polypeptide is capable of being secreted extracellularly from said host and is capable of causing a VZV antibody response in mammals. A recombinant DNA expression vector according to Claim 1, wherein said infected, transfected or transformed host is green monkey kidney cells (BSO-

1. ), CO8 monkey cells, HeLa cells, hamster kidney cells, human fibroblast cells, or human tissue celis. A cell transformed or transfected by the recombinant DNA expression vector according to Claim 1, wherein said cell is "caused to produce and secrete truncated VZV gpl by the vector. A plasmid encoding a Varicella-zoster virus polypeptide, said polypeptide being selected from the group set out in Claim 1, and wherein said polypeptide is capable of being secreted extracellularly from a host and is capable of causing a VZV antibody response in a mammal. A process for producing a secretory truncated Varicella-zoster virus glycoprctein wherein the glycoprotein is a polypeptide selected from the group - set out in Claim 1 and is capable of causing a VZV antibody response in . mammals, said process comprising the steps of:- a) providing a vector comprising a nucleotide sequence coding for said A polypeptide, wherein the nucleotide sequence is capable of being" expressed by a host containing the vector; b) incorporating the vector into the host; and c) maintaining the host containing the vector under conditions suitable _ for expression of the nucleotide sequence into said giycoprotein. A: process according to Claim 5, wherein said ‘vector includes a promoter operationally associated with said nucleotide sequence. A process according to Claim 5 or 6, wherein said host is a mammalian cell and wherein said nucleotide sequence further includes a region of nucleotides which encode a leader sequence. A process according to any of Claims 5 to 7, wherein said host cells are green monkey kidney cells (BSC-1), COS monkey cells, HeLa cells, hamster kidney cells, or human fibroblast cells or human tissue cells. A process according to Claims 5-8, wherein said expression vector is a plasmid. A recombinant secretory truncated VZV gpl being a polypeptide selected from the group set out in Claim 1, and wherein said polypeptides are secreted extracellularly from mammalian cells within which said polypeptides are produced and wherein said polypeptides include at least one antigenic epitope capable of eliciting an antibody response in a mammalian host. A vaccine for immunisation against chickenpox and/or shingles comprising an effective amount of a recombinant secretory truncated VZV gpi being a polypeptide selected from the group set out in Claim 1, and a conventional vaccine carrier. The use of secretory truncated VZV gp being a polypeptide selected from the group set out in Claim 1 for the manufacture of a vaccine for stimulating an immune response to chickenpox and/or shingles. A method of diagnosing VZV comprising contacting serum, tissue or tissue extracts of an individual to be tested with an antibody against a secretory truncated VZV gp being a polypeptide selected from the group set out in claim 1 for a time and under conditions necessary to form an antibody-antigen complex, and detecting any resultant antibody-antigen complex. Secretory truncated VZV gp consisting of SEQ ID NO: 1. ‘Secretory truncated VZV gp consisting of SEQ ID NO:

2. An expression vector substantially as hereinbefore described with reference to the Examples and Drawings. A cell substantially as hereinbefore described with reference to the Examples and Drawings. A plasmid substantially as hereinbefore described with reference to the Examples and Drawings. A process substantially as hereinbefore described with reference to the Examples and Drawings. A VZV glycoprotein whenever produced by a process as claimed in any of claims 8 to 14. A vaccine substantially as hereinbefore described with reference to the Examples and Drawings. The use of a VZV glycoprotein substantially as hereinbefore described with reference to the Examples and Drawings. ' A method substantially as hereinbefore described with reference to the Examples and Drawings.