WO1992003554A1

WO1992003554A1 - Infectious laryngotracheitis virus vaccine

Info

Publication number: WO1992003554A1
Application number: PCT/AU1991/000383
Authority: WO
Inventors: Michael George Sheppard; Christopher Prideaux; Michael Johnson; Kevin John Fahey; Jennifer Joy York; Kritaya Kongsuwan
Original assignee: Arthur Webster Pty. Ltd.
Priority date: 1990-08-24
Filing date: 1991-08-23
Publication date: 1992-03-05

Abstract

A non-infectious subunit vaccine for use against infectious laryngotracheitis virus (ILTV) comprises as active immunogen at least one glycoprotein of ILTV, or an immunogenic peptide derived therefrom, and optionally an adjuvant. Production of polypeptides displaying the antigenicity of ILTV glycoproteins by recombinant DNA means is disclosed. The disclosure extends to recombinant live virus vaccines containing gene sequences for ILTV glycoproteins, to recombinant ITL virus having heterologous DNA inserted into a non-essential region and to the use of ILTV promoter regions in recombinant virus.

Description

"INFECTIOUS LARYNGOTRACHEITIS VIRUS VACCINE"

This invention relates to the use of specific glycoproteins of infectious laryngotracheitis virus (ILTV), which are major immunogens in chickens, in vaccines against infectious laryngotracheitis (ILT). The invention also relates to the use of ILTV as a delivery vector for heterologous genes inserted into the glycoprotein or other region(s) of the ILTV genome where they are expressed by the homologous ILTV promoters or by heterologous promoters such as other herpesvirus promoters.

Infectious laryngotracheitis virus is a herpesvirus belonging to the alphaherpesvirinae subfamily (Gallid herpesvirus 1; Roizman et.al, 1981) which causes an acute upper respiratory tract infection in chickens. The disease is found worldwide and sporadic outbreaks occur in which the severity of clinical symptoms may vary considerably. As outbreaks can result in mortalities of 10-40% and reduced egg production, the disease is of considerable importance to the intensive poultry industry. In recent years, a milder form of infectious laryngotracheitis has become widespread in England (Curtis and Wallis, 1983). In Australia, both the severe and mild forms of the disease are prevalent (Beveridge, 1981), but are controlled by the use of an attenuated live vaccine delivered in the drinking water or by eyedrop. This vaccine is produced from a field isolate of low virulence and retains some pathogenicity, particularly in younger chickens. A subunit vaccine or a recombinant viral vector-based vaccine expressing the protective immunogen(s) of the virus would be safer and could possibly be used in the eradication of the disease in certain regions of the world.

Both antibody and cell-mediated immune mechanisms are thought to play a role in immunity to herpesvirus infections; antibody in preventing infection and the establishment of latent infections, and cell-mediated immunity in recovery from an easting infection. The glycoproteins of herpesviruses are important targets of both the humoral and cell-mediated arms of the host immune response (reviewed in Marsden, 1987). Furthermore vaccination with either purified glycoproteins or recombinant virus vectors expressing herpesvirus glycoproteins protects experimental animals against lethal challenge infection with herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) or pseudorabies virus. In ILT, cell-mediated mechanisms are known to be important in immunity following vaccination. Naive chickens can be protected against challenge infection by the transfer of immune lymphoid cells (Fahey et.al, 1984), while bursectomised chickens that are unable to synthesise specific antibodies are protected against a challenge infection by vaccination (Fahey and York, 1990).

Development of a subunit or recombinant vaccine for ILT requires knowledge of the protective immunogens of the virus. Two families of ILTV glycoproteins have been described (York et.al, 1987), but little is known of other antigens of the virus, or of the significance of the immune response to the viral glycoprotein.

The viral glycoproteins produced in cells infected with either vaccine strain or virulent isolates of ILTV have been identified by in vitro labelling using [¹⁴C] glucosamine and [¹⁴C] mannose (York et.al, 1987). Chicken antisera to the vaccine strain and to a virulent isolate, and rabbit antisera to the vaccine strain, immunoprecipitated four major viral glycoproteins of 205, 115, 90 and 60K moLwt. Additional glycoprotein bands were recognised by immune chicken and rabbit sera in Western blotting using a glycoprotein fraction purified from extracts of virus infected cells. Two antigenically distinct families of ILT viral glycoproteins have been defined by monoclonal antibodies; the 205 K complex of glycoproteins (205, 115 and 90K glycoproteins) and the 60K glycoproteins.

It has now been found that these major viral glycoproteins of ILTV are able to induce both antibody and cell-mediated immune responses in chickens, and furthermore that vaccination of chickens with preparations of ILT viral glycoproteins protect the chickens against clinical ILT and also against replication of the challenge virus.

Accordingly, in one aspect of the present invention, there is provided a non- infectious subunit vaccine against ILTV, which comprises as active immunogen at least one glycoprotein of ILTV, or an immunogenic peptide derived therefrom, together with, if desired, an adjuvant.

In one embodiment die vaccine may comprise a non-infectious subunit vaccine containing one or more glycoproteins of ILTV which have been obtained by isolation from virus-infected cells, or by synthetic methods, particularly by recombinant DNA techniques, or from transformed cell cultures. Alternatively, the vaccine may be in the form of a recombinant live virus vector having inserted therein a nucleotide sequence coding for at least one glycoprotein of ILTV or an immunogenic peptide derived therefrom. Preferably, the active immimogen in the vaccine is selected from die group consisting of the 205K complex of glycoproteins and the 60K glycoprotein of ILTV. In a related aspect, the invention provides a method for protecting chickens and other poultry against ILTV, which method comprises administering die vaccine described above to said chickens or other poultry As described in detail below, it has been shown tiiat when affinity purified ILTV glycoproteins were formulated into subunit vaccines they protect chickens against both clinical disease and also against replication of die challenge virus. Furthermore, a subunit vaccine containing essentially the glycoproteins of the 205K complex protects 100% of the chickens.

The vaccine according to this invention may comprise an immunogenic peptide derived from a glycoprotein of ILTV, for example, by recombinant DNA techniques or chemical synthesis. A suitable immunogenic peptide may be derived so tiiat it comprises all or at least the major immunogenic determinants of a glycoprotein of ILTV and dius exhibits the same or similar immunogenicity. If required, the glycoprotein(s) may also be coupled to a carrier molecule to increase immunogenicity and hence efficacy as a vaccine.

Preferably, the non-infectious subunit vaccine of this invention comprises an adjuvant. The vaccine may, for example, be delivered in an aqueous-mineral oil emulsion, such as an emulsion achieved by using an oil-phase emulsifier (e.g. Tween 80). Additional adjuvants may also be included if required, for example Al OH₃, saponin or a derivative of muramyl dipeptide. As described above, die vaccine of this invention may be in the form of a live recombinant viral vaccine which contains the nucleotide sequence (or sequences) coding for one or more of the immunogenic ILTV glycoproteins disclosed herein, or an immunogenic peptide derived therefrom. Inoculation with such a live recombinant vaccine will induce protective immunity against ILT in avian species. By way of example, such a live recombinant vaccine may comprise fowlpox virus, avian adenovirus or other avian virus expressing one or more nucleotide sequences for ILTV glycoproteins. Because of the protective response obtained when purified ILTV glycoproteins were formulated into subunit vaccines, the genes for these glycoproteins have now been cloned and characterised. In particular, restriction endonuclease maps of the ILTV genome have now been produced, and die locations of the two glycoprotein genes (gp60 and gp205) identified. In addition, clones encoding gp60 and gp205 have been sequenced and relevant promoter regions identified. As a result of the identification of tiiese genes, production of die glycoproteins as recombinant products for use in subunit vaccines, as well as production of live recombinant viral vaccines expressing these glycoproteins, maybe achieved using methods well known to persons skilled in die art.

In further work using the λgt11 vector system which allows the expression of inserted DNA fragments as β-galactosidase fusion proteins and using monoclonal antibodies to die 205K complex of glycoproteins to screen the library of recombinant phages, recombinant phages encoding die desired epitope have been identified and die nucleotide sequence of die gene encoding tiiis 205K complex of glycoproteins determined.

Accordingly, there is now disclosed herein means for the production of die 205K complex of glycoproteins (also referred to herein as ILTV "gpB") as a recombinant product for use in subunit vaccines, as well as in the production of the live recombinant viral vaccines expressing this complex.

Accordingly, in another aspect of the present invention, there is provided a recombinant DNA molecule comprising a nucleotide sequence capable of being expressed as all or at least a substantial portion of the 205K complex of glycoproteins or the 60K glycoprotein of ILTV, or an immunogenic peptide derived therefrom. The nucleotide sequence may have expression control sequences operatively linked tiiereto, such control sequences being derived from a homologous or heterologous source. In this aspect, the invention also provides a recombinant DNA cloning vehicle (such as a plasmid or bacteriophage) comprising an expression control sequence operatively linked to a nucleotide sequence capable of being expressed as all or a substantial portion of the 205K complex of glycoproteins or the 60K glycoprotein of ILT, or an immunogenic peptide derived therefrom, as well as a host organism (such as a bacterium or yeast) containing such a cloning vehicle.

The present invention also extends to syntiietic polypeptides displaying the antigenicity of the ILTV glycoproteins discussed above. Such syntiietic polypeptides may comprise fusion polypeptides wherein die sequence displaying die desired antigenicity is fused to an additional heterologous polypeptide sequence. As used herein, die term "synthetic" means that the polypeptides have been produced by chemical or biological synthesis. In yet another aspect of the present invention, a number of genes of infectious laryngotracheitis virus (ILTV) have been identified as non-essential regions (providing insertion sites of foreign genes). - These genes include (1) die ILTV glycoprotein gp60 gene which encodes a protein of 995 amino acids; (2) the Kprl/YL fragment ORF3 gene encoding a protein of 298 amino acids; and (3) die ILTV homologue of the HSV protein kinase gene.

Disease prevention is of major economic concern in the poultry industry. The development of techniques for the insertion of foreign DNA into virus vectors has let to the expression of many foreign antigens in chimeric viruses, and the possibility of using these as live recombinant vaccines. Besides fowlpox virus (FPV), whose suitability as a live vaccine vector in poultry has been demonstrated several other virus families (eg Adenoviridae, Herpesviridae) are potential candidates for use as viral vectors. Among these other viruses, infectious laryngotracheitis virus, which belongs to the alpha herpesvirus class, presents an additional advantage when used as a viral vector in chickens. It can be administered in an aerosol form, tiius permitting an easy and inexpensive delivery system suitable for the highly intensive poultry industry. There are several reports on the use of herpesviruses to carry and express genes from other viruses or other species. Viruses used in such a way are known as "vectors" and genes, other than their own, expressed in such a way are referred to as "foreign genes". In order to develop a vector system based on herpesviruses it is necessary to identify suitable promoters for die expression of foreign genes and "non-essential" regions of herpesviruses into which a foreign gene can be inserted without disrupting an essential function of the virus. The term "non-essential" in this context means non-essential for growth under at least some conditions in which die virus can be grown in vitro and under at least some conditions in which it survives in vivo.

In this aspect, therefore, the present invention provides a recombinant ILT virus, characterised in that heterologous DNA is inserted into a non-essential region of the ILTV genome. Preferably, the region of the ILTV genome into which the heterologous DNA is inserted is the region corresponding to the gp60 gene.

Further investigation of the ILTV genome has also revealed tiiat the promoter regions in the ILTV genome, particularly those for the glycoprotein genes gp60, gp205 (gpB) and ORF3, are major promoter regions. Accordingly, knowledge of the sequences of these regions, and particularly the gp60 promoter region, enables these promoter regions to be used for the expression of heterologous genes, either in ILTV or in a foreign host cell or organism.

In this aspect, the invention further provides a recombinant virus, particularly ILTV, characterised in that heterologous DNA is inserted into a non-essential region of the host virus genome. Where the host virus is ILTV, expression of said heterologous DNA is controlled by an ILTV promoter region br by a heterologous promoter. Further details of die various aspects of the present invention are set out in the following Examples.

EXAMPLE 1

This example demonstrates die specificity of the serum antibody response of chickens to glycoprotein antigens of ILTV as determined by Western blotting, and the ability of ILTV glycoproteins to elicit a cell-mediated immune response.

SA-2, the vaccine strain of ILTV used in Australia, was propagated and assayed in monolayer cultures of primary chicken kidney (CK) cells (Fahey et.al, 1983).

Detergent extracts of virus-infected cells were prepared at 18-20 h post infection using 1% (v/v) Nonidet P40 and 1% (w/v) sodium deoxycholate (York et.al., 1987). The glycoprotein fraction of die detergent extract was obtained by affinity chromatography on a lentil lectin Sepharose 4B column (York et.al, 1987).

For Western blotting, detergent extracts and glycoprotein fractions were separated by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) under reducing conditions at an acrylamide concentration of 8%, and transferred to nitrocellulose membrane (York et.al, 1987). The membrane was cut into 5 mm strips and die binding of immune chicken serum was detected using anti-chicken IgG and ¹²⁵I- labelled Protein A

Immune chicken serum was collected 8 to 12 weeks after eyedrop vaccination of 6-week-old specified patiiogen-free (SPF) White Leghorn chickens (CSIRO SPF Poultry Unit, Maribyrnong) with approximately 10⁵ PFU of SA-2 ILT vaccine (Arthur Webster Pty.Ltd., Castle Hill). For a time course study of the development of antibodies to ILTV, two 8-week-old chickens were vaccinated witii SA-2 by eyedrop. Blood was collected on days 0, 7, 14, 21 and 28 days post vaccination.

ILTV antigens obtained by immunoprecipitation of detergent extracts with monoclonal antibodies (Mabs) to ILTV were tested for their ability to elicit a delayed-type hypersensitivity (DTH) reaction in the wattle of cockerels vaccinated witii SA-2 by eyedrop 4 weeks earlier. The ILTV-specific Mabs have been described previously (York et.al, 1987). In brief, Mab 39-2 (Group I) recognises a single glycoprotein of 60K molecular weight in Western blotting, while Mabs 22- 37, 131-6 and 12-1 (Group II) recognise a complex of glycoproteins of 205, 160, 115, 90 and 85K molecular weight. Immunoprecipitations were carried out using Protein A-Sepharose beads as described previously. The antigen-antibody complexes were dissociated by incubation with 1% SDS for 10 min at room temperature as preliminary experiments had shown tiiat the recovery of antigens in a form able to elicit DTH reactions was maximal when 1% SDS was used to dissociate the antigen-antibody complexes, ratiier than 1 M propionic acid, 3 M potassium thiocyanate or 8 M urea (data not shown). The thickness of each wattle was measured at time zero (Ag₀, C₀) and a 0.1 ml volume of antigen (Ag) was injected subcutaneously into the right wattle. For each test antigen, a control antigen (C) was prepared by an identical treatment of uninfected cells and 0.1 ml of the control preparation was injected into the left wattle. At 24 h after injection the thickness of both wattles was measured (Ag₂₄, C₂₄) and die DTH index was calculated as the difference between the increase in thickness of the test wattle and die increase in the control wattie, i.e. (Ag₂₄-Ag₀)-(C₂₄-C₀). A DTH index of greater than 0.4 was considered positive. The differences between group mean indices were analysed by die non-parametric Kruskal-Wallis Q test as the variance of treatment groups was strongly heterogenous.

The reactivity of sera from 21 chickens, which had been inoculated witii SA-2 strain of ILTV, with a detergent extract of CK cells infected witii SA-2 was characterised using Western blotting. A total of 24 bands ranging in molecular weight from 205 to 26K were recognised by immune chicken sera (Table 1), however not all sera reacted with all bands and die intensity of the reaction with particular bands also varied from chicken to chicken. In general, bands with molecular weights of 205, 160, 115, 90, 67, 34 and 26K were recognised strongly. More than 75% of the sera from chickens vaccinated witii SA-2 reacted witii bands of 205, 160, 115, 90, 67, 60, 52, 34 and 26K (Table 1). Most sera showed litde or no reactivity with detergent extracts of uninfected cells.

When 11 of the SA-2 antisera were reacted in Western blotting against the lentil lectin affinity purified glycoproteins obtained from a detergent extract of SA-2-infected cells, a much simpler pattern was obtained. Seven bands witii molecular weights of 205, 160, 135, 115, 90, 85 and 60K were most commonly recognised, with bands of 74, 67 and 50K also being detected. Again, the intensity of these reactions varied between antisera but generally they reacted most strongly with the 205, 115, 90 and 60K bands. Immune sera showed very little reactivity with the glycoprotein fraction of uninfected cells.

Of the bands in the detergent extract that were most commonly and strongly recognised by immune sera (205, 160, 115, 90, 67, 60, 52, 34 and 26K), all except two (34 and 26K) were also recognised in the purified ILTV glycoprotein fraction. Thus the viral glycoproteins are the major immunogens of ILTV recognised by antibody. Testing of weekly sequential bleeds in Western blotting assays against the purified glycoprotein fraction, showed tiiat antibody was detectable by day 14 post vaccination. At this time there was a strong response to the 205K band, but responses were also present to other glycoproteins, albeit weaker responses (data not shown). Therefore there was no evidence of a sequential response to the different ILTV glycoproteins. Nor was it possible to determine whetiier glycoprotein or protein antigens were recognised first as there was a considerable difference in the response of the two chickens when the sequential sera were Western blotted against the total detergent extract (data not shown). In order to determine whether the glycoproteins of ILTV were also able to elicit a cell-mediated immune response, ILTV glycoproteins immunoprecipitated by Group I or Group II Mabs were tested for their ability to elicit a DTH reaction in 12-week-old cockerels which had been vaccinated 4 weeks earlier. When tested in die DTH assay, the complete detergent extract of virus-infected cells elicited a strong reaction (Table 2). The glycoproteins precipitated by Mabs 39-2, 22-37 and 131-6 also elicited positive DTH reactions in most chickens showing that both tiiese groups of glycoproteins were able to elicit cell-mediated immune responses to ILTV. The response to the antigens precipitated by tiiese Mabs was significantly different (p < 0.05) to the response to antigens precipitated by normal mouse serum (Table 2). However none of the chickens injected witii glycoproteins precipitated by Mab 12-1 (Group II) showed a positive DTH reaction. Most of the glycoproteins precipitated by Mab 12-1 appeared similar by Western blotting to those precipitated by the other Group II Mabs except that the 90 and 85K bands were either absent or at a concentration too low to be detected by the immune chicken serum. It has been noted previously that the reaction of Mab 12-1 with the 90 and 85K bands in Western blotting is variable and tiiat it only precipitates the 115 and 90K bands weakly (York et.al, 1987). The inability of the Mab 12-1 precipitate to elicit a positive DTH response could be explained if an epitope critical for eliciting the DTH response is present on the 90 or 85K glycoprotein which were absent or in low amounts in the immunoprecipitate. The ability of both groups of ILTV glycoproteins to induce antibody and cell- mediated immune responses suggests that tiiey could prove to be important protective immunogens of the virus and tiierefore are prime candidates for inclusion in a subunit or recombinant vaccine. EXAMPLE 2

This example demonstrates the vaccination of chickens with ILTV glycoproteins purified by lentil lectin affinity chromatography. Immune responses to the vaccines were measured by a virus neutralisation assay and a delayed-type hypersensitivity (DTH) assay and cprrelated with protection. The efficacy of the glycoprotein vaccines in providing protection against a challenge infection was assessed by both clinical signs and by detecting viral antigen in tracheal scrapings by ELISA

Figure 1 is a Western blot of glycoprotein vaccines used in Experiment 1. The glycoprotein preparations used for the primary (lanes 1,3) and secondary (lanes 2, 4) vaccination were reacted in Western blotting with chicken antiserum to ILTV (lanes 1,2) or normal chicken serum (NCS; lanes 3, 4) diluted 1:400. The molecular weights (kilodaltons) of the glycoproteins are indicated on the left hand side. Figure 2 is a Western blot of glycoprotein preparations for Experiment 2. The glycoprotein fractions use- to prepare the primary vaccines were probed with chicken antiserum to ILTV diluted 1:400. Lane 1 shows the original glycoprotein fraction, and lanes 2 and 3 the glycoprotein fraction after treatment once (lane 2) or twice (lane 3) with Mab 10-2. The material that bound to the antibody- coated Protein A-Sepharose beads after one (lane 4) or two (lane 5) treatments with Mab 10-2 is shown in lanes 4 and 5. The molecular weights of the glycoproteins are indicated on die left hand side. Figure 3 is a Western blot of sera from chickens in Experiment 2 that were protected against challenge. Sera were diluted 1:50 and reacted in Western blotting with a detergent extract of virus-infected cells. Panels a and b show 5 representative sera from chickens that were protected following vaccination with total glycoprotein vaccine (a, lanes 1-5) and with depleted glycoprotein vaccine (b, lanes 1-5), respectively. The molecular weights of the glycoproteins are indicated at the centre.

MATERIALS AND METHODS

Experimental animals

Specifϊed-paihogen-free White Leghorn chickens of the CSIRO-W line produced by t he CSIRO SPF Poultry Unit, Maribrynong, Victoria were boused in flexible plastic isolators and fed fumigated feed and acidified water.

Cells and Viruses

SA-2, the vaccine strain of ELTV used in Australia, and CSW-1, a virulent isolate used for challenge infections, have been described previously (York et al, 1987). ILTV strains were propagated in monolayer cultures of chicken kidney (CK) cells which were grown in Eagle's basal medium supplemented with 10% tryptose phosphate broth, 5% newborn calf serum, Hepes buffer (0.015 M), araphotericin B (2.5 μg/ml), penicillin (0.06 mg/ml) and streptomycin (0. 1 rag/ml). Virus stocks were prepared by harvesting virus-infected cells at 24 hr post infection (York et al., 1987).

Detergent extracts of virus-Infected cells

Extracts were prepared at 18-20 hr post infection, using 1% Nonidct P40 and 1% sodium deoxycholate (York et al, 1987), from cells that had been infected at a multiplicity of approximately 5 plaque-forming units (PFU) per cell.

Affinity purification of glycoproteins from detergent extracts

The glycoprotein fraction of the detergent extract was obtained by affinity chromatography on a lentil lectin Sepharose 4B column (Pharmacia (Australia) Pty Ltd). The extract was allowed to bind to the column for 1 hr, then the column was washed with 5 column volumes of equilibration buffer (0.05 M Tris-HCl, pH 8.0, 0.15 M Nad, 0.1% Nonidet P40). The bound glycoproteiis were eluted with 0.2 M methyl glucoside in equilibration buffer. Fractions were concentrated to the original volume by membrane filtration using a YM10 membrane (Amicon Corporation, Danvers, MA.) and phenylmethylsulfonylfluoride was added to a final concentration of 1 mM.

Preparation of glycoprotein subunit vaccines

The glycoprotein fraction was depleted of the 60K glycoprotein by reacting it with monoclonal antibody (Mab) 10-2 (York et al., 1987) bound to protein A-Sepharose beads coated with rabbit anti-mouse lg, using the immunoprecipitation protocol described previously (York et al., 1987). The depleted preparation was treated a second time with the monoclonal antibody to ensure that all material reacting with the antibody was removed. The 60K glycoprotein (and also the antibody) was removed from the beads by treatment with 1% sodium dodecyl sulphate (SDS) for 15 min at room temperature.

All glycoprotein preparations were inactivated by treatment with β-propiolactone. The glycoprotein preparation (18 parts) was mixed with 0.5 M Na₂HPO₄ pH 8.0 (1 part) and the mixture was added to 2% (v/v) β-propiolactone. After thorough mixing, the tube was incubaied for 1 hr at 37º. The preparation was transferred to a fresh glass tube, incubated as before and then incubated at 4º overnight. Infectious virus was not detected in any inactivated glycoprotein preparation after three passages in CK cells. Protein concentrations were determined using the bicincboninic acid (Pierce Chemical Co., Rockford, IL, USA) assay (Smith et.al., 1985; Redinbaugh and Turley, 1986).

Vaccination of chickens

Experiment 1. Three groups of 4-week-old chickens were used. One group of 16 chickens was vaccinated by eyedrop with approximately 10⁵ PFU of commercial live ILT vaccine (Arthur Webster Pty Ltd, Sydney) and a second group of 17 chickens was vaccinated intraperitoneally with 70 μ% of affinity purified glycoproteins emulsified in an equal volume of Freund's complete adjuvant (FCA), in a total volume of 1 ml. A third group of 16 chickens were held as unvaccinated controls. At 4 weeks after vaccination all chickens were bled and the group receiving the glycoprotein vaccine were given a second 1 ml intraperitoneal injection of 200 μ% of glycoproteins emulsified in an equal volume of Freund's incomplete adjuvant (FIA).. Six weeks after primary vaccination the DTH response was measured in six 10-week-old cockerels from each group, as only the cockerels bad sufficiently developed wattles at this age. At 8 weeks after primary vaccination all chickens were bled and then challenged by intratracbeal inoculation of 10⁵ PFU of the CSW-1 isolate of ILTV in a 200 μl volume. Clinical signs of gasping respiration or death were noted at day 5 after challenge when the chickens were euthanased Tracheal scrapings were collected and the presence of ILTV antigen was determined by ELISA Protection was assessed at day 5 after challenge as a compromise between detecting clinical signs of disease and detecting viral antigen in the trachea. Considerably higher levels of morbidity and mortality would have been expected at days 6-8 after challenge (Fahey et.al., 1983), but antigen is not detectable after day 6 post challenge (York and Fahey, 1988). Experiment 2. Five groups of twelve 4-week-old chickens were used. One group was vaccinated by eyedrop with commercial ELT vaccine. Groups 2 and 3 were vaccinated twice intraperitoneally with different glycoprotein vaccines which were emulsified in FCA for the primary vaccination and in F1A for the secondary vaccination. Group 2 received doses of 350 μg of the complete glycoprotein vaccine in the primary vaccination and 160 μg in the secondary vaccination. Group 3 were vaccinated with 260 μg of the depleted glycoprotein preparation in the primary vaccination and 56 μg in the secondary vaccination. Group 4 were vaccinated with the immunoprecipitated 60K glycoprotein. A fifth group was held as unvaccinated controls. Groups 2, 3 and 4 were revacdnated intrapcritoneally at 4 weeks after primary vaccination with the appropriate antigen in FIA The DTH reaction of 6 cockerels from each group was tested 6 weeks after primary vaccination. All chickens were bled and then challenged intratracheally with 2.5 × 10⁵ PFU CSW-1 in a 200 μl volume at 8 weeks after primary vaccination. At day 3 after challenge the chickens were eutbanased and tracbeal scrapings were collected for examination for ELTV antigen by ELISA.

Delayed-type hypersensltivlty assay

A detergent extract was prepared from virus-infected and uninfected CK cells and dialysed overnight against phosphate-buffered saline (PBS). The thickness of each wattle was measured at time zero and a 0.1 ml volume of the antigen extracted from infected cells was injected subcutaneously into the right wattle. The same volume of the control antigen prepared from uninfected cells was injected into the left wattle. At 24 hr after injection the thickness of both wattles was measured and the DTH index was calculated as the difference between the increase in thickness of the test wattle and the increase in thickness of the control wattle. A DTH index of greater than 0.5 was considered positive. The differences between group mean indices were analysed by the non-parametric Kruskal-Wallis Q test as the variance of treatment groups was strongly heterogeneous. Virus neutralization

Neutralizing activity in serum was assayedby a plaque reduction test asdescribed by York et al.

(1989). The neutralizing titre was taken as the dilution which produced a 70% reduction in the number of plaques compared to controls.

Detection of viral antigen

Viral antigen present in tracheal scrapings was assayed by ELISA as described previously (York and Fahey, 1988). In brief, an equal volume of PBS containing 1% Nonidet P40 was added to the scrapings and the sample was vortexed for 30 sec. Debris was removed by centrifugation in a microfuge at 12.000 rpm for 1 min. The supernatant fluid was added to polyvinyl chloride microtitre Plates coated with rabbit IgG antibody to ILTV. Mab 131-24 culture supemate, diluted 1/50 in 2% non-dairy whiteher in PBS, was then added followed by affinity purified goal anti-mouse Ig coojugated to horseradish peroxidase (T ago Inc., Burlingame, CA) diluted 1:1000 in non-dairy whitener. Finally 5-aminosalicylic add was added as substrate. An absorbance of greater than 0.1 above background was considered positive.

Western blotting

Detergen, extracts were separated by SDS polyacrylamide gel elearophorcsis under reducing conditions using 8% acrylantide and trcnsferred to nitrocellulose membrane (Burnette. 1981) The membrane was incubated in Tris-buffered saline (TBS) containing 5% skin, milk powder (Blotto) for 1 hr at 37º. or beld at 4º until required, and then cut into 5 mm strips which were abated for 1 hr at room temperature with chicken serum diiuted in Blorto. After two 15 min washes in TBS containing 0.05% Tween 20, anti-chicken IgG (1/1000 in Blotto; Cappel Laboratories, Cochranville, PA. USA) was added and incubated for 1 hr at room temperature. The strips were washed as before and incabated for 30 min a, room temperature with ¹²⁵I - Iabelled ptotein A (0.05 uci/ml in Blotto; Ametsham (Australia) Pty Ltd Sydney.) Aftter washing the strips were exposed to Fuji X-ray film at - 70º for 1-4 days. Chicken anllserunm

Antiserum to the SA-2 strain of ILTV was a pool of serum collected from 5 chickens 12 weeks after eyedrop ino∞lalion of approximately 10⁵ PFU of commercial ILT vaccine.

RESULTS

Composition of glycoprotein vaccines

Experiment 1. The glycoprotein preparations used for the primary and secondary immunizations in Experiment 1 are shown in Fig. 1 after Western bloning with chicken antiserum. Major glycoprotein bands of 205, 115, 90. 85, 74, 60 and 50K, plus a minor band of 160K were present in both preparations ie. glycoproteins of the two major families of ILTV glycoproteins previously defined by monocional antibodies, the 205K complex and the MK glycoprotein, plus glycoproteins of 74 and 50K molecular weight.

Experiment 2. The effect of depletion of the glycoprotein preparation by Mab 10-2 is show, in Fig. 2. The various glycoprotein preparations used for the preparation of the primary vaccines were reacted in Western bloaing wi.h chicken antiserum. The original glycoprotein fraction contained the same glycopro.eins as in Experiment 1 (lane 1) . After one treatment with Mab 10-2 there was a considerable reduction in the intensity of the band at MK (lane 2) and a strongly reading hand of 60K was present in the material eluted from the protein A-Sepharose beads (lane 4). No material that reacted with chicken anriserum was eluted from the beads aftet a second treament of the depleted glycoprolein preparation with a fresh balch of Mab 10-2 on protein A-Sepharose beads (lane 5). Ahhough .here was no further reduction in the intensity of the band at MK that was recognised by the chicken antiserum (lane 3), no material which reacted with Mab 10-2 by Western bloning remained in the depleted preparation after the second precision with Mab 10-2 (da.a not shown). Similar results were obtained with the glycoprotein preparations used for the secondary vaccinations (daia not shown). Three glycoprotein preparations were used for vaccination: the original glycoprotein preparation, the preparation that was treated twice with Mab 10-2 and the 60K glycoprotein recovered from the protein A-Sepharose beads following the first immunoprecipitation with Mab 10-2.

Vaccination studies

Experiment 1. Only 1 of 17 birds that received glycoprotein vaccine bad virus neutralizing antibody by 4 weeks after the primary vaccination, but 16 of the 17 birds were positive by 4 weeks after the second immunization with ILTV glycoproteins, although the mean litre of the group vaccinated with the glycoprotein vaccine (1/43) was considerably lower than that of the birds vaccinated with live vaccine (1/153). The one chicken that did not produce neutralizing antibody after 2 doses of glycoprotein vaccine had a high titrc of antibody to ILTV which was detectable by ELISA. All chickens from the two vaccinated groups that were tested bad positive DTH responses (Table 3).

After challenge with the virulent CSW-1 isolate of ILTV, 31% of the unvaccinated chickens showed clinical signs of ILT by day 5 after challenge (Table 3). At this time, viral antigen was absent in the trachea of all chickens vaccinated with live vaccine and 71% (12/17) of those that received glycoprotein vaccine (Table 3). Of the 5 chickens that were not protected, all bad neutralizing antibody in their semm at the time of challenge. Two of these chickens also bad positive ETTH responses 2 weeks before challenge (remaining 3 were not tested). The one chicken in the glycoprotein vaccine group that did not produce neutralizing antibody still resisted the challenge infection as evidenced by the absence of viral antigen in the trachea. All chickens that were protected by vaccination with ELTV glycoproteins produced good Western blotting antibody responses, including the one chicken that did not produce virus neutralizing antibody. When sera from these birds was Western blotted against detergent extract of virus-infected CK cells they were found to contain antibodies which recognised bands of 205, 160, 115, 90, 85, 67, 60, and 50K (data not shown). Three of the 5 birds that were not protected also produced good Western blotting antibody responses to the vaccine (data not shown), while the 2 birds that did not produce any antibody detectable by Western blotting had neutralizing antibody titres of 1/40 and 1/10 respectively. Experiment 2. All chickens vaccinated with either the total glycoprotein extract or the glycoprotein extract depleted of the 60K glycoprotein produced virus neutralizing antibody after two immunizations (Table 4). None of the chickens immunized with the 60K glycoprotein produced neutralizing antibody, nor did they produce ILTV antibody detectable by ELISA. However, 2 weeks prior to challenge (6 weeks after primary immunization) 4 of the chickens immunized with the 60K glycoprotein had low DTH responses, although the mean DTH index of this group was not significantly different to that of the unvaccinated group. The mean DTH indices of chickens vaccinated with either the live vaccine, the total glycoprotein preparation or the depleted glycoprotein preparation were significantly greater than that of the unvaccinated controls.

In this experiment a slightly higher dose of challenge virus was used and protection was assessed at day 3 after challenge to maximize the detection of viral antigen. Following intratracheal challenge there was a total absence of viral antigen in the tracbeas of 83% (10/12) of the chickens vaccinated with the total glycoprotein extract, and 100% of the group that received the depleted glycoprotein vaccine (Table 4). Only one of the chickens vaccinated with the 60K glycoprotein did not have viral antigen in the trachea 3 days after the challenge infection.

Figures 3a and 3b show the reactivity in Western biorting of 5 representative sera obtained immediately before challenge from chickens receiving the glycoprotein vaccine and 5 representative sera from the group that received the depleted glycoprotein vaccine. Sera from both groups reacted with bands of 205, 160, 135, 115, 90, 85, 67, 60 and 50K. Only sera from some of the birds from each group recognised the 135K band: 8 of 12 from the total glycoprotein vaccine group and 5 of 12 of the depleted glycoprotein vaccine group. Sera from the chickens that were immunized with the depleted glycoprotein preparation showed Western blotting reactivity to bands in the 60K region. The 2 chickens in the total glycoprotein vaccine group that were not protected had virus neutralizing antibody titres of 1/40 and 1/160 respectively, and also produced antibody detectable by Western blotting. The one chicken immunized with the glycoprotein vaccine that was negative for DTH 2 weeks prior to challenge was protected against infection.

DISCUSSION

To determine whether the glycoproteins were indeed protective immunogens of ILTV they were purified by lectin affinity chromatography and formulated into glycoprotein subunit vaccines. Vaccination with various preparations of affinity purified ELTV glycoproteins protected up to 100% of chickens. They were protected not only against clinical disease, but also against replication of the challenge virus in the trachea. As far as we are aware, this is the only reported instance of this degree of protection against a herpesvirus infection following vaccination with a subunit glycoprotein vaccine. In other herpesvirus diseases, vaccination with either individual glycoproteins or mixtures of glycoproteins can be shown to prevent or reduce clinical disease but does not prevent recovery of the challenge virus from the vaccinated animals (Chan, 1983; Platt, 1984; Babiuk et.al., 1987; Marchioli et.al., 1987; Meignier et.al., 1987; Stanberry et al., 1987). The only reports of prevention of replication of challenge virus in the tissues of the natural host are the vaccination of pigs against PRV (Maes and Scbutz, 1983), and of calves against BHV-1 (Lupton and Reed, 1980). In both cases the vaccine was a erode Nonidet P40 extract of virus-infected cells containing both protein and glycoprotein antigens and high doses of antigen (5-6 mg of protein per dose) were used. More recent vaccination studies on PRV and BHV-1, which used either mixtures of glycoproteins or individual glycoproteins, failed to prevent replication of the challenge virus (Platt, 1984; Babiuk et.al., 1987; Marchioli et.al., 1987; Israel et al., 1988).

Vaccination with HSV glycoproteins has also been shown to reduce the incidence of recurrent (Stanberry et al ., 1987; Wachsman et.al, 1987) and latcnt infections (Cremer et.al., 1985) in mice and guinea pigs. It is possible therefore that vaccination with ILTV glycoproteins could decrease the occurrence of latent virus, but this question was not addressed in the present study.

By using Mab 10-2 to lemove the 60K glycoprotein from the total glycoprotein preparation, a glycoprotein prtparation confining largely glycoproteins of the 205K complex (205, 160, 115, 90 and 85K) was produced, without the need to use the denaturing conditions required for the elution of viral glycoproteins from Mab affinity columns. Depletion of the 60K glycoprotein from the total glycoprotein preparation did not reduce its efficacy as a vaccine. If anything the efficacy of this deplete preparation was slightly enhanced as the vaccinated chickens had higher neutralizing antibody titres and 100% were protected against challenge. However, it must be noted that birds vaccinated with the glycoprotein preparation depleled of the 60K glycoprotein still produced antibody to a 60K antigen. This may be a co-migrating antigen unrelated to the 60K glycoprotein recognised by Mab 10-2, or the antibodies may be to 60K glycoprotein in which the epitope recognised by Mab 10-2 was destroyed or denatured. mre could be a number of reasons for the lack of efficacy of the 60K glycoprotein as a vaccine. Firstly, the dose of the glycoprotein may have been insufficient Secondly, the presence of 1% SDS in the vaccine preparation may have affected the conformation and hence the immunogenicity of if 60K glycopro.ein, panicularly as no antibody responses were detected Alternatively, the glycoprotein may have remained bound to the Mab used for depletion which may have interfered with its recognition by the immune system.

In these studies, neither antibody nor DTΗ responses of the chickens vaccinated with glyco- protein preparations correlated with protection against challenge infection. The group which received the depleted glycoprotein fraction had the higbest neutralizing antibody response and every chicken in this group was protected against challenge with virulent ILTV. However, 2 chickens that were not protected by vaccination with the complete glycoprotein vaccine in

Experiment 2 bad serum neutralizing antibody, while the one chicken in Experiment 1 without detectable neutralizing antibody was protected. Immunization studies with glycoproteins of other herpesviruses have also shown that protection does not always correlate with the presence of neutralizing antibody responses in serum. Animals can be protected in the absence of neutralizing antibody (Zweerink et.al, 1981; Schrier et.al., 1983; Chan et al, 1985; Wacbsman et al., 1987) while the presence of neutralizing antibody does not ensure protection (Israel et al.,

1988).

As was the case for neutralizing antibody responses, the presence of antibody reactive in

Western blotting did not ensure protection, and no difference was readily apparent in the specificity of the response of chickens that were protected or were susceptible. Although the

160 and 135K glycoproteins were absent or poorly represented in the vaccines, sera from many vaccinated chickens recognised these bands in Western blotting, particularly the 160K glycoprotein, suggesting that either these two antigens are related to one or more of the other glycoproteins present in tbe detergent extract, or they are very strong immunogens.

Overall, 6 chickens with positive DTH indices were not protected, while one DTH-negative chicken was protected. For HSV, a strong correlation was found between DTH reactivity and protection induced by infectious virus (Nash et.al., 1980) or glycoprotein C (Schrier et al., 1983). In contrast, no DTH responses were detected in mice protected by immunization with glycoprotein B (Chan et al., 1985). In tbe latter study, protection was attributed to the T-belper cell population (Chan et al., 1985). Both T-belper cells and cytotoxic T-cells have been shown to play an important role in recovery from HSV infections in experimental animals (Nash et al., 1981; Larsen et al, 1983; Sethi et al, 1983). It may be that measures of cell-mediated immune responses other than DTH, such as the cytotoxic T-cell response or tbe generation of T-helper cells, or alternatively, tbe magnitude of local immune responses would provide a better correlation of protection against ILT than either serum neutralizing antibody or DTH responses. Studies with other viruses causing respiratory infections have shown a correlation between local mucosal antibody, in the form of virus-specific IgA, and protection from disease (Perkins et.al, 1969; Ogra et.al, 1971; Liew et al., 1984). However this docs not seem to be the case for ILT as bursectomized chickens without mucosal antibody were able to resolve a primary infection as effectively as intact chickens, and there was still no replication of challenge virus in vaccinated-bursectomized chickens (Fahey and York, 1990). In the present studies, vaccination with ILTV glycoproteins by tbe intraperitoneal route was able to stimulate a proteαive immune response in the respiratory tract, in accordance with the considerable body of evidence that now suggests that such immunization protocols can evoke immunity at a distant mucosal site (Mestecky, 1987), although these mechanisms have not been formally demonstrated in tbe chicken.

EXAMPLE 3

This example details the analysis of the genome of ILTV and construction of a restriction enzyme map thereof.

MATERIALS AND METHODS

Virus and cells.

SA-2 the vaccine strain of ILTV used in Australia, was propagated in monolayer cultures of chicken kidney (CK) cells.

Preparation of viral DNA

Confluent monolayers of CK cells were infected with virus at a MOI of 1:100 and incubated at 37 ºC until more than 90% of the cells showed CPE. Virus was isolated from both cytoplasmic and cell free fractions and DNA extracted essentially according to the method of Wilkie (1973) by treatment with 2% SDS,

NTE-saturated phenol (NTE: 10mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM

EDTA) and chloroform/isoamylalcohol (24:1). the DNA was precipitated in 70% ethanol and resuspended in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). Cloning of virus DNA

A cosmid library was prepared by ligating partially digested Sau3A ILTV DNA with BamHI digested pHC79 (Collins and Hohns, 1978; Hohns and Collins, 1980) and packaged by standard techniques (Sambrook et.al, 1989). A set of EcoRI clones was prepared in a similar way using sonicated ILTV treated with T4 polymerase, EcoRI linkers attached and finally digested with EcoRI before ligation with EcoRI digested pHC79 and packaged. The transforming strain was E.coli MB406 and transformants were screened using colony blot hybridisation. Whole virus DNA was also digested with SmaI, KpnI or EcoRI, or alternatively restriction fragments were isolated from low melting point agarose gels and ligated into the plasmid pUC18 (Yanisch-Perron et.al, 1985) transformed into E.coli JM101 bacteria and selected by standard methods. Plasmid DNA was obtained by alkali lysis technique (Ish-Horowitz and Burke, 1981).

DNA analysis.

To map the genome, ILTV DNA was digested with SmaI, KpnI or EcoRI and the fragments separated through 0.5% agarose gels, stained with ethidium bromide and photographed. After photography, DNA was transferred and fixed to nylon membranes (Southern, 1975). The relative amount of DNA in each band was estimated from photographic negatives of gels scanned with a laser microdensitometer. SmaI, KpnI, NotI or EcoRI ILTV fragments cut from low melting point agarose gels, or plasmids containing ILTV fragments, were radiolabelled using a random hexamer priming kit (BRESATEC) (Feinberg and Vogelstein, 1983). These radiolabelled fragments were hybridised to the Southern blots (50% formamide, 5 X SSC (750 mM NaCl, 75 mM sodium citrate), 1 X Denhardt's reagent (Denhardt, 1966) (0.2% BSA, 0.2% polyvinylpyrrolidine, 0.2% ficoll),

20 mM sodium phosphate (pH 6.5), sonicated denatured salmon sperm DNA (100 μg/ml), at 42 °C for 16 hrs. Filters were washed for about 1 hour using three changes of 2 X SSC, 0.1% SDS at 42ºC and finally for 15 mins with 0.1 X SSC, 1%. SDS at 68 ºC. Filters were then wrapped in Glad- Wrap and exposed to X-ray film at -80°C for 2-5 days.

Terminal fragments.

The terminii of ILTV genome were tentatively identified by end-labelling of viral DNA with dATP (³²P), then confirmed by Exonuclease III digestion. Labelled DNA was then digested with NoS or SmaI and hybridised to corresponding digests of ILTV DNA.

Figure 4 shows restriction maps of SA-2 ILTV DNA for the enzymes EccRI, KpnI and SmaI. The filled rectangles above the maps indicate the gp205 (gp200) gene and the gp60 gene, respectively. The 155 kpb of ILTV genome is represented as two unique sequences (U_L, U_S) and 2 large inverted repeat sequences (IR_s, TR_s) flanking U_s. The scale below shows fractional genome length. EXAMPLE 4

This example shows the cloning and sequencing of the ILTV gp60, Kpn K/ORF3 and PK genes.

MATERIALS AND METHODS 1. Virus strain

A mild isolate SA-2 vaccine strain of ILTV was used to obtain DNA for cloning, SA-2 is used commercially as a vaccine throughout Australia and was obtained from the National Biological Standards Laboratory, Parkville, Australia and used at passage levels 5 and 20.

2. Tissue culture medium

ILTV was propagated in monolayer cultures of primary chicken kidney (CK) cells. CK cells were grown in Eagle's basal medium (Commonwealth Serυm Laboratories) supplemented with 5% newborn calf serum (Flow Laboratories Australasia Pty Ltd), 2μg/ml fungizone, 100μg/ml streptomycin and 100 IU/ml penicillin.

3. Virus DNA isolation

SA-2 ILT virus was inoculated on to confluent monolayers of CK cells at a multiplicity of infection of approximately 0.5 plaque forming unit (pfu) per cell. Cells were pre-washed in PBS (phosphate saline buffer), and the virus inoculum was added to the cells in 1ml of serum-free medium per 90 mm x 14 mm petri dish. After 1 incubation at 37°C to allow the virus to adsorb to the cells, 5 mis of medium containing 5% newborn calf serum was added. After 24 b when 80-100% of the cell monolayers showed cytopathic effeα, the cell monolayers were harvested. This was done by scraping the cells from tissue culture dishes and centrifuge for 10 minutes at 2500 φm. The cell pellet was resuspended in 10 mM KCL, 15 mM MgCl₂ and 10 mM Tris-HCl pH7.5. To extract the cell associated virus, the cells were disrupted by repeated fxceze-thawing in a solution of 0.5% NP40. The supernatant containing virus was pooled and treated with RNase A (100μg/ml), extracted twice with phenol-chlorpform-isoamyl alcohol (50:48:2 v/v/v, saturated with 10 mM TrisHCl pH7.5, 1 mM EDTA, 10 mM NaCL) and then with ether. The viral DNA was precipitated by addition of 50% volume of isopropanol. Viral DNA was finally resuspended in 10 mM Tris-HCl pH7.5, 1 mM EDTA (TE) or in deionised H₂O. 4. Construction and screening of λgtII library

Approximately 2 ug of ILTV DNA was sheared by sonication; made flush-ended with T4 DNA polymerase; methylated by EcoRI methyltransferase; ligated to EcoRI linkers (Biolabs); and digested with EcoRI fragments. Fragments of 0.5-2 kilobases were isolated by agarose gel electrophoresis and ligated to λgt11 arms previously digested with EcoRI and dephosphatased with calf intestine alkaline phosphatase (Amersham). After ligation, the lambda DNA was packaged using Packagene extract (Promega). The result was a library of 100,000 phages. The library was plated on lawn of E. coli Y1090 and screened using the Super immunoscreening system of Amersham according to the instructions of the supplier. Positive plaques were picked, plaque purified and rescreened uniformly. High titre stocks of each positive phage and phage DNA were prepared by making plate lysate on E. coli Y1090.

5. Cloning of viral DNA into plasmid vectors

1 μg of ILTV DNA was cut with the restriction enzyme BamHl and Kpnl (Pharmacia) and the resulting DNA fragments separated by agarose gel electrophoreses and transferred to Hybond membranes (Amersham). The filters were then probed with EcoRI inserts from λgt11-ILTV recombinant phages (X24-4, X27-1) which were positive with gp60 monoclonal antibodies. The DNA fragments from these two phages were labelled with deoxyadenosine [α³²P] triphosphate by using a random hexamer priming method. Hybridization was in 5xSSC, 0.5% sodium dedocyl sulphate (SDS), 5xDenhardt's solution and 100μg/ml denatured herring sperm DNA at 65°C. Washes were 0.5 × SSC, 0.1% SDS at 65ºC

Hybridization results showed that the DNA cloned into the X24-4 and X27-1 mapped to the 670 bp, the 5.3 Kbp KpnI fragments and BamHI 4.5 Kbp fragment. These fragments are located between 0.7-0.8 map units on ILTV genome. To identify and characterize the gp60 coding sequence, the three restriction fragments were cloned into KpnI - cut and BamHl - cut phosphatased - treated pUC18 plasmid and resulting recombinant plasmids were called pKpn0.6, pKpnl/K and pBam4.5 respectively. DNA sequencing

DNA sequencing was carried out by the dideoxynucleotide termination method with [³⁵S]dATP. The DNA synthesis reaction was primed with either a 17-mer residue that hybridized to pUC or M13 sequences adjacent to the insert DNA or with custom 20-mer oligonucleotides complementary to a specific region of the insert DNA. Specific oligonucleotides were synthesized using Pharmacia LKB Gene Assembler Plus.

DNA sequence reading and analyses were done using HIBIO DNAsis software package

(Hitachi America, Ltd) and with the programs available through the Australian National Genomic Information Service (ANGIS). Searches of protein data bases and comparison of homologous sequences were performed with the Fast A program of Pearson and

Upman, 1988 (Pro. Natl. Acad. Sci. USA 85, 2444-2448).

RESULTS

Figure 5 shows the genome structure of ILTV showing the KpnI site distribution and the location of the KpnI/K fragment that has been sequenced. The latter has been expanded in the lower part of the figure to show the overlapping BamHI 4.5 Kbp fragment We noted two KpnI restriction enzyme sites which had not been previously identified in the restriction map of the SA-2 strain. Open reading frames (ORFs) are depicted as open boxes with the direction of transcription indicated by the arrows. The unique long region, U_L, the unique short region, U_s;IRs = the short internal repeat and TR_s = the short terminal repeat. B, BamHI and K, KpnI restriction enzyme sites. Figure 6 shows the nucleotide and predicted amino acid sequence of the ILTV gp60 gene with the numbering referring to the nucleotides and amino acids. The EcoRI inserts of λ27 and λ24-4 were labelled with ³²P dATP, and used to probe the KpnI and BamHl digests of ILTV DNA. Hybridization resuhs showed that the DNA cloned into the two recombinant phages mapped to the small 678 bp Kpnl fragment (cross-hatched), the KpnI/K fragment and to the 4.5 Kbp BamHI fragment (Figures). Nucleotide analysis of the 5.3 Kbp Kpnl/K and the 4,557

Kbp BamHI fragment revealed several potential open reading frames (data not shown). One ORF encodes the C-terminal part of the gene referred to as the KpnK/ORF3 and this ORF is described in Figure7 . A second ORF specifying the FLTV gp60 gene product and has a size of 2985 bp. The predicted translated protein is 995 amino acids. The gp60 coding region contains an average base composition of 24%A, 22%T, 26%G and 28%C. with the G+C content of 54%, which is higher than the estimate of 45% for total ILTV DNA as determined from buoyant density measurements (Plummer, G. et al., 1969) The deduced amino acid sequence of the gp60 gene has several features of a glycoprolein. These include 19 hydrophobic amino acid residues at the N- terminus which may commpond to the signal sequence. A second region of hydrophobic amino acids (positions 960 to 989) at the C-terminus could function as a transmembrane anchor sequence. There are nine potential N-linked glycosylation sites on the ILTV gp60 protein (underlined). One of these sites at residue 677, may not be active due to the presence of a proline residue within the N-X-S/T signal.

The DNA sequences upstream and downstream of this open reading frame were analysed for putative transcriptional control elements. The sequence

TAATAAA" at positions 294-301 could be a potential TATA boo element for this gene. There is no obvious polyadcnylation signal downstream for the stop codon.

Analysis of the sequence showed that there are repeated sequences within the ILTV gp60 coding region (overlined and numbered). When the repeat sequences were examined closely it is observed that they are not perfectly conserved but several copies had diverged so that only 4 amino acids out of 7 were conserved in all 13 copies of the repeats. When the predicted amino acid sequence of the gp60 protein was compared with the PIR protein sequence database using FASTA, there is no significant homology between the protein and any sequenced berpesviπis proteins in the database.

The predicted molecular weight of the coded amino acid sequence is 107,503 daltons which is significantly larger than the apparent mass of the 63Kd polypeptide detected by monoclonal antibodies on Western bot. It is noted that there is a potential internal proteolytic cleavage for the gp60 precursor polypeptide. A potential cleavage site (R-R-S) is present at residues 679 to 681. The predicted (cleaved) polypeptide product of this gene would have unglycosylated M_r value of approximately 70Kd which is close to the apparent mass of 63 Kd. Fϊgure 7 shows the nucleotide and predicted amino acid sequence of the SA-2 strain of ILTV KpnK/ORF3 gene.

KpnK/ORF3 is 298 amino acids long and is located 5' of the gp60 gene. It has a predicted M_r of 32309 daltons and contains four potential glycosylation sites (underlined). The protein has an N-terminal hydrophobic region, which might be a signal sequence for translation on membrane-bound ribosomes.

The DNA sequences upstream and downstream of this open reading frame have common features in eukaryotic transcription initiation and termination signals. A consensus "TATA" box (5'-TATAAA-3'), characteristic of many eukaryotic and also herpesviral promoters is present 81 nucleotides upstream from the proposed start codon. The sequences 5'-GGCTCCATA-3' which resides 25 nucleotides upstream from the "TATA" box exhibits similarities to the "CAT" box consensus sequence (5'-GGOTCAATCT-3'). Regarding 3' elements, two potential polyadcnylatioπ signals 5'-AATAAA-3' are found at the 3' end of the gene. Also a conserved sequence 5'-YGTGTTYY-3' (GT-rich region - double underlined) located approximately 17 bp downstream from the poly A signal. This sequence is believed to be required for efficient formation of a mRNA 3' terminus and polyadenylation is predicted to occur between these 2 conserved signal sequences.

A search for amino acid sequence similarities between ORF3 and the herpesvirus database did not produce convincing results. However by comparing the predicted amino acid sequence of ORF3 with short amino acid sequences derived from other herpesvirus ORFs, it appears that ORF3 between residues 135-186 has some homology to PRV gX gene (residues 129-180). Figure 8 shows the DNA sequence of part of the protein kinase (PK) gene of ILTV. The sequence starts from the left end of KpnI/K fragment and ends 656 bp from the KpnI. Alignment of the amino acid sequence with analogous sequences published for herpesvirus genes clearly identifies the gene as a member of the herpesvirus protein kinase (PK) gene family. The protein contains most of the characteristic motifs that are highly conserved in the catalytic domain of eukaryotic protein kinases (Hanks, S.T.1988). The conserved amino acid residues located within the catalytic domain were identified and shown in circles. Roman numerals denote subdomains of the catalytic region defined by Hank et al The sequences at position 98-100 (SPE, in squares) in subdomain VIII was reported to be highly conserved, and has been shown to be essential for phosphorylation. The presence of a threonine residue 5 bp upstream of the SPE sequence suggest that ILTV PK would be phosphorylated serine/threonine and not tyrosine. EXAMPLE 5

This example shows the expression of inserted DNA fragments as β-galactosidase fusion proteins, and determination of the nucleotide sequence encoding the 205K complex of glycoproteins. Figure 9 shows mapping of recombinant phage λ26-2 DNA on the ILTV genome. ELTV DNA was digested with EcoRI, resulting DNA fragments were separated by agarose gel electrophoresis and transferred to Hybond membrane. One set of fragments was hybridised with ³²P-labelled EcoRI insert from A26-2 (lane 2), while the other set was hybridised with 32P-labelled ILTV DNA (lane 1). The letters denote the assignments for each hybridising ILTV- EcoRI restriction enzyme fragment based on the previous restriction endonuclease cleavage maps of ILTV genomic DNA. Figure 10 she a physical map of EcoRI restriction sites for ILTV DNA and an expanded restriction map of the EcoRI "U" fragment. The EcoRI restriction cleavage map is that described above. U_L and U_s, long and short unique regions of the genome, respectively; TR and IR terminal and inverted repeat regions of the genome, respectively. Restriction sites are denoted: P = PstI, B = BglII, S = SalI, X=XbaI. The position and direction of the ILTV "gB" transcript is shown below the expanded map of the EcoRI "U". Nucleotide sequence strategy is indicated by arrows. Figure 11 shows the nucleotide sequence of the EcoRI "U" fragment encoding the "gB" homologue of ILTV and the deduced amino acid sequence. The putative TATA box of the promoter is boxed. The polyadenylation site, AATAAA is double underlined. Broken lines indicate GC-rich regions. The presumed signal sequence at the N-terminus and the membrane-spanning region at the C-terminus are indicated by italics and bracketed ([]). The putative N-linked glycosylation sites of the consensus N-X-S/T are underlined. Brackets ({}) indicate the beginning and end of the insert present in the recombinant λ26-2. Numbers at right indicate positions of nucleotides, and the predicted amino acids. Figure 12 shows expression of ILTV "gB" gene. Total cytoplasmic RNAs from ILTV infected (lane 1) and mock infected (lane 2) were fractionated on an agarose /formaldehyde gel and transferred onto Hybond-N membrane. The membrane was hybridised to the BglII- PstI fragment (Fig.10). Arrowheads indicate the location of chicken ribosomal RNAs 28S and 18S which were used as size standard and were estimated to be 4.2kb and 1.6kb respectively.

Figure 13 is a hydropathy plot of the predicted ILTV "gB" amino acid sequence. The plot was based on the algorithm of Kyte and Doolittle (1982) by using a 11-amino acid window. The two most hydrophobic regions in the N- and C-termini are predicted to represent the signal sequence and the transmembrane anchor region, respectively, of the glycoprotein molecule.

Figure 14 shows homology of ILTV "gB" with those of other herpesviruses. Multiple alignment of the amino acid sequences predicted for the "gB"-like proteins of 10 different herpesviruses. Sequences were aligned using CLUSTAL program (Higgins and Sharp, 1988). Asterisks indicate identical amino acids and dots represent conserved amino acid substitutions. Putative N-linked glycosylation sites are shown in bold and underlined. The signal sequences are double underlined and the triple transmembrane domains are boxed. Conserved cysteine and proline residues are shown by■ and≡ respectively.

MATERIALS AND METHODS

Cells, viruses and viral DNA isolation.

The strain used in the present study is the SA-2 vaccine strain of ILTV used in Australia. ILTV was propagated in monolayer cultures of primary chicken kidney (CK) cells grown in Eagle's basal medium (Commonwealth Serum Laboratories) supplemented with 5% newborn calf serum (Flow Laboratories Australasia Pty.Ltd.). Cell associated viral DNA was prepared using the slightly modified method of Whalley et.al. (1981) from infected cell cultures at 24hr post infection. Infected cells were harvested by scraping the cells from tissue culture dishes and centrifuged for 10 min at 7000 rpm. The cell pellet was then resuspended in CAV buffer (10 mM KCl, 15 mM MgCl₂ and 10 mM Tris-HCl pH7.5) and disrupted by repeated freeze-thawing in a solution of 0.5% NP40. The supernatant was pooled and extracted with phenolchloroform and incubated with RNase A (10 ug/ml). DNA was precipitated by addition of 50% volume of isopropanol. Enzymes

Enzymes were purchased from Pharmacia, or Promega and used as specified by the manufacturers.

Construction and screening of λgt11 library

Approximately 2 ug of ILTV DNA was sheared by sonication, made flush-ended with T4 DNA polymerase, methylated by EcoRI methyltransferase, ligated to ϋcσRI linkers (Biolabs) and digested with EcoRI endonuclease. Fragments of 0.5-2 kb were isolated by agarose gel electrophoresis and ligated to λgt11 arms previously digested with EcoRI and dephosphorylated with calf intestine alkaline phosphatase (Amersham). After ligation, the DNA was packaged using Packagene extract (Promega), transfected and resulted in a library of 100,000 phages. The library was plated on a lawn of E. coli Y1090 and screened using the Super immunoscreening system of Amersham according to the instructions of the supplier. Positive plaques were picked, plaque purified and rescreened uniformly. High titre stocks of each positive phage and phage DNA were prepared by making plate lysate (Maniatis et.al., 1982) on E coli Y1090.

Cloning methods, Southern hybridization and DNA sequencing

All the subcloning into plasmid and M13 vectors was performed using standard methods (Maniatis et al., 1982).

For hybridization, ILTV or plasmid DNAs were digested with appropriate restriction enzymes and the resulting DNA fragments separated by agarose gel electrophoresis and transferred to Hybond membranes (Amersham). The filters were then probed with DNA fragments which were labeled with deoxyadenosine [α³²P] triphosphate using a random hexamer priming kit (Bresatec, South Australia). Hybridization was in 5 X SSC, 0.5% sodium dedocyl sulphate (SDS), 5 X Denhardt's solution and 100 ug/ml denatured herring sperm DNA at 65°C. Washes were performed using 0.5 X SSC, 0.1% SDS at 65°C.

DNA sequencing was carried out by the dideoxynucleotide chain termination method using the Sequeπase sequencing kit (United States Biochemical). The DNA synthesis reactions were primed with either a 17-mer oligonucleotide residue that hybridized to pUC or M13 sequences adjacent to the insert DNA or with custom 20-mer oligonucleotides complementary to a specific region of the insert DNA. Specific oligonucleotides were synthesized using Pharmacia LKB Gene Assembler Plus.

DNA sequence analysis

DNA sequence reading and analyses were done using a HIBIO DNAsis software package (Hitachi America, Ltd.) and with the programs available through the Australian National Sequence Analysis Facility (ANSAF). Searches of protein databases and comparison of homologous sequences were performed with the FASTN/P program of Lipman and Pearson (1985).

Northern blot analysis

The guanidium thiocyanate technique was used to isolate cytoplasmic RNA from ILTV-infected or uninfected control CK cells (Maniatis et.al., 1982). About 15 ug of total RNA was denatured and separated on 1% agarose/formaldehyde gels and transferred onto Hybond-N membrane. Blots were probed with a ³²P-labcled 632 base pairs (bp) PstI-BglII fragment (see Fig. 10).

RESULTS

Isolation of λgt11 recombinant clones expressing ILTV glycoprotein genes

A λgt11 library of randomly generated 500 bp to 2 kb ILTV DNA fragments inserted into the EcoRI site of the lacZ gene was constructed. In this library the translational products of inserted open reading frames are expected to be part of a β-galactosidasc fusion protein (Young and Davis, 1983). The library was grown on E. coli Y1090; fusion proteins were induced by isopropyl-)β-D-thiogalactopyranoside (IPTG) and screened with a mixture of appropriately diluted monoclonal antibodies (kindly provided by Dr Jenny York). Monoclonal antibodies (MAbs) used were 10-1, 10-2, 39-2 (group 1) which immunoprecipitated the 60K glycoprotein and 12-1, 22-7, 23-1, 131-6 (group 2) which reacted with the 205K glycoprotein complex in Western blot (York and Fahey, 1988). From among 50,000 plaques screened, thirteen positive clones were identified. Ten clones reacted with group 1 MAbs and the other three reacted with group 2 MAbs. Of the three recombinant phages which were positive with group 2 MAbs, one phage designated 26-2, was plaque-purified and DNA prepared for further studies.

The EcoRI insert fragments of 26-2 phage were labeled with ³²P dATP, and used to crobe EcoRI digests of ILTV DNA Hybridization results showed that the DNA cloned into this phage mapped to the EcoRI fragment "U" (Fig. 9). This fragment is located between 0.23-0.25 map units on ILTV genome with reference to the previously published physical map . To identify and characterize the gp205K coding sequences, we determined tbe nucleotide sequence of both strands of this 3 kb fragment. Generation of subclones of the EcoRI fragment "U" in both M13 and pUC was accomplished using the restriction sites on the 3 kb segment of DNA as shown in Fig .10 Gaps were filled in using custom oligonucleotide primers.

DNA sequence of the EcoRl "U" fragment

A restriction map for EcoRI sites in the ILTV genome and a more detailed map for the region spanning 0.23-0.25 map units are shown in Fig. 10. The complete nucleotide sequence of the region containing the putative gp205K coding sequence is shown in Fig 11. There is a single large open reading frame within this region extending from the ATG codon beginning 185 bp 3' of the EcoRI site (Fig.11 to a TAA termination codon, starting at nucleotide 2804, (Fig. 11). Translation of this 2619 bp would produce a polypeptide of 873 amino acids. There are two other possible initiation codons in the vicinity of the assigned ATG, one at position 155 to 157 and the other at nucleotides 200 to 202 (see Fig. lD.Both of these codons are in frame with the above mentioned open reading frame. However, the assigned initiation codon ATG at 185 bp resides within the sequence GACATGG which conforms well to the consensus sequence (A/G)CCATGG (Kozak, 1984). It has a purine (G) at position -3, C at-1, and a G at +4 which is considered to be the most strongly conserved features of the flanking sequence of the initiation codon of eukaryotic mRNAs.

Searching for upstream cis-regulatory sequence, two potential TATA box homologues were found. They are located 45 and 148 bp upstream of the putative start codon (at 140 bp and 38 bp in Fig. 11. We suggest that the TATA box position at bp 38-41 (Fig.11 in box) is the functional TATA box of this gene for two reasons: (i) its local sequence TATATTT has some features proposed for the consensus TATA box sequence TATA (A/T)A(A/T) (Corden et.al., 1980), and (ii) SI nuclease mapping indicated that the potential RNA polymerase initiation site of this gene mapped at about 144 nucleotides upstream of the ATG (C. T. Prideaux, unpublished data). Other putative cis-regulatory elements found are the GC-rich regions (Fig .11 indicated by broken lines) which are potential binding sites for the promoter-specific transcription factor Spl (Briggs et al, 1986).

A potential polyadenylation signal AATAAA, was found 20 bp downstream from the termination codon (Fig. 1 1).The G+C content of the sequence is 44.4%, which is close to the estimate of 45% for total ILTV DNA as determined from buoyant density measurements (Plummet et al, 1969).

Size of the gp205 RNA transcript

Northern blot analysis (Fig. I2)detected a single transcript 2.9-3 kb in size from cells infected with ELTV. The probe used for hybridization was derived from the 632 bp BglII-PstI fragment (Fig. 10). Using the same probe, a similar result (not shown) was obtained with the poly (A+) RNA indicating that the RNA is polyadenylated. Allowing 100 bp for polyadenylation, this transcript size is consistent with the predicted 5' and 3' ends of the mRNA (see above).

Structure of the predicted polypeptide

The deduced amino acid sequence for the polypeptide encoded by the 2619 bp open reading frame is shown above the DNA sequence in Fig. 11. The molecular mass of the 873 amino acids primary translation product is 98,895 daltons. The predicted protein has features common to other membrane-spanning glycoproteins. A hydrophobicity plot (Fig. 13)identified a sequence of 16 hydrophobic amino acids at the extreme NH₂ end (Figs. 1 land 13 which may function as the signal peptide. Applying the weight-matrices criteria of von Heijne (1986) for the prediction of the cleavage site, the cleavage might occur at the isoleucine residues 14. A broad hydrophobic domain at amino acids 690 to 761 near the C-terminus (Figs.11 and 13) represents a membrane anchor sequence. A targe extracellular domain (amino acid. 17 to 689) contains nine potential N-linked glycosylation sites (underlined in Fig. 11).C-terminal amino acids 762 to 873 have net positive charge and may function as the cytoplasmic domain. gp205 shares significant amino acid bomology with gB-like glycoproteins of other rerpesviruses

To obtain the identity of the gP205 predicted translated product, we searched the Swiss, NBR F nnd GenBank protein databases for homoiogous sequences. The resuits indicated high homology with the gB family of proteins in herpesviruses. Identities are scattered in the central portion of the proteins with little or no identities a, the N and C termini. Identities were scored as follows:

33 and 34% with HSV-! and Marek's disease virus (MDV) gBs respectively, 36 to 39% with pseudorabies virus (PRV) gO, varicella-zoster virus (VZV) gpπ and equine herpes virus type 1 ( EHV - 1 ) gB . comparison to gam maherpes virus gBs indicated identities of 31% with gB analog of Epstein-Barr virus (EBV) (BALF2) and of 29% with herpesvirus saimiri (HVS) gB. Comparison with gB of human cytomegaiovirus (HCMV) indicated identity of 29%.

Muitiple alignments of ten herpesvirus gBs (Fig. 14)have highlighted several charaaeristics of conserved sequence. The common structural features of the gB-like proteins shown in Fig. 14 are: (i) the conservation of tea cysteine (C) residues which were perfectly aligned in gB of ail ten viruses. This accounts for all cysteines of ILTV protein except the two which occur in srgnal sequence. This observation indicates that the proteins are conserved in their secondary and tertiary structures since C-C disulfide bonds are important determinants of the tertiary structure of the protein, (ii) Six sites of prolines occur at conserved positions (Fig. 14) (iii) The triple hydrophobic transmembrane regions which were found in similar positions and consisted of three distinct peaks of hydrophobicity (positions 851 to 918 in Fig.14).This structure is believed to enable the protein to traverse the membrane three times (Pellett et.al., 1985a). (iv) Some of the putative N-linked glycosylation sites exist in similar positions but are not strictly conserved (at positions 184, 298, 457, 486, 652 and 793 in Fig.14). The motif CYSRP at positions 702-706 which was noted by Ross et.al. (1989) to be conserved in mammalian herpesviruses and in MDV, has the sequence CYTRS in ILTV.

EXAMPLE 6

This example shows the identification of various ILTV promoters and use of the ILTV glycoprotein B (GbP) gene promoter in the regulation and expression of foreign vaccine antigens in ILTV viral vectors.

Figure 15 shows the nucleotide sequence of the ILTV glycoprotein B promoter.

Binding positions of the oligonucleotides used to isolate the fragment by the polymerase chain reaction are underlined. The ATG translation initiation codon is in bold, and possible TATA and CAAT consensus signal sequences are boxed.

Figure 16 shows the construction of pPgB-CAT. The ILTV glycoprotein B promoter was isolated using the polymerase chain reaction. Restriction enzyme sites, Pstl and Xbal, engineered into the oligonucleotides used for the polymerase chain reaction were digested, facilitating the cloning of the fragment into pUC and subcloning into pCAT-BASIC

Figure 17 shows the nucleotide sequence of die 5' non-coding regions of the ILTV ORF3 and gp60 genes isolated by polymerase chain reaction. Binding positions of the oligonucleotides used to isolate the fragments by use of the polymerase chain reaction are underlined. The ATG translation initiation codon is in bold, and possible TATA and CAAT consensus signals are boxed. MATERIALS AND METHODS

Cells and Viruses

Primary chicken kidney ( CK ) cells were prepared by trypsinization of kidneys isolated from two to four week old specific pathogen free ( CSIRO, SPF Poultry Unit, Maribyrnong, Victoria ) chickens as described by Fahey et al. ( 1983 ). ILTV SA2 vaccine strain was obtained from Arthur Websters Pty. Ltd (Northmead, 2152, Australia ). ILTV was grown on CK cells in Eagle's Basal medium ( Gibco Laboratories ) supplemented with 5% bovine calf serum ( BCS) and lOmM Hepes. Virus stocks were frozen ( -70 °C ) and thawed three times prior to infection to release ILTV from cells.

Infection of Cells Monolayers of primary CK cells were prepared in 50 mm petri-dishes ( IxlO⁶ cells) and infected with ILTV at a multiplicity of 2-5 plaque forming units (p.f.u.) per cell. Prior to addition of ILTV, the growth medium was removed from cells and the monolayer washed twice with phosphate buffered saline ( PBS ). After 2 hr of absorption at 39 °C, the monolayers were washed and growth medium added.

Enzymes and Chemicals

Restriction enzymes and other DNA modifying enzymes were obtained from various sources and used according to the manufacturers' instructions or as oudinedbyManiatis et al. ( 1982 ). Cellphect transfection and dideoxy sequencing kits were from Pharmacia. Chloramphenicol acetyl transferase ( CAT ) assay system was from Promega. Isolation of ILTV Genomic DNA

Confluent monolayers of CK cells were infected with ILTV at a level of 0.1 plaque forming units per cell and incubated at 39 °C until cellular cytopathic effects were greater than 90%. Virus was isolated from both cytoplasmic and cell free fractions and DNA extracted essentially according to the method of Wilkie ( 1973 ) by treatment with 2% SDS, NTE-saturated phenol ( NTE: 10 mM Tris- HCl, pH8.0, 100 mM NaCl, 1 mM EDTA ) and chlorophorm/isoamylalcohol (24:1 ). The DNA was precipitated in 70% ethanol and resuspended in TE buffer ( 10 mM Tris-HCl, pH7.5, 1 mM EDTA ).

Amplification of ILTV DNA Fragments Using PCR

Amplification of the 5' non-coding region from the ILTV gpB gene was performed using the polymerase chain reaction ( PCR ). Reactions were performed in 50 ul volumes comprising 50 ng of ILTV DNA, 3 ng of each oligonucleotide primer, 50 mM KC1, 10 mM Tris-HCl, pH8.4, 2.5 mM MgCl₂, 200 ug/ml BSA, 200 uM dATP, 200 uM dCTP, 200 uM dGTP, 200 uM dTTP and 1 unit of Taq polymerase ( Cetus ). Reaction mixtures were overlayed with an equal volume of paraffin oil and heated to 94 ºC for 1 min, cooled to 55 °C for 2 min and heated to 72 °C for 5 min. This cycle was repeated 35 times using a Perkin Elmer Cetus DNA Thermal Cycler.

Introduction of Plasmid DNA Into CK Cells

Monolayers of CK cells prepared in 50mm petri-dishes ( 1×10⁶ cells ) were transfected with CsCl₂ purified promoter-CAT plasmid constructs using a Pharmacia Cellphect transfection kit as detailed by the manufacturer. Briefly 1-10ug of plasmid DNA in 1ul of isotonic Tris-HCl, pH7.5, was mixed with an equal volume of DEAE-Dextran solution ( end cone. 0.5mg/ml ). The DNA/dextran solution was added drop-wise to monolayers of CK cells which had been washed twice with isotonic Tris-HCl, pH7.5, and incubated at room temperature. After 15mins the DNA/dextran solution was carefully removed, the monolayers washed with isotonic Tris-HCl, and complete growth medium added.

Assay of CAT Activity Estimations of CAT activity in CK monolayers were made using a Promega CAT enzyme assay system. Briefly monolayers of transfected CK cells were scraped into the medium witii the use of a rubber policeman, pelleted at 1,000 φm for 5mins at 4ºC, and washed once with PBS and once with TEN buffer ( 40mM Tris-HCl pH7.5, ImM EDTA, 15mM Nacl ). The pelleted cells were resuspended in 100ul of 0.25M Tris-HCl pH8.0, and subjected to three cycles of freeze thawing ( -70 °C to 37 °C ), with vortexing after each cycle. The extract was then heated to 60 ºC for 10min and clarified by microfuging for lOmins. Standard CAT assays contained 55ul of cell extract, 5ul of 14C-chloramphenicol ( Amersham Int. ) and 2.5ul n- butryl CoenzymeA ( 5mg/ml; Promega ). Reactions were carried out at 37 °C for 12hrs, and terminated by extraction with 150ul of mixed xylenes (Aldrich Chemical Co. ). The xylene phase containing the 14C n-butyryl chloramphenicol reaction products was isolated by microfugation for 3mins ( upper phase ) and purified by back extraction with 150ul 0.25M Tris-HCl pH8.0. Fifty microlitre aliquots were added to 1ml Econofluor scintillation fluid ( NEN Research Products ) and counted in a LKB Wallac 1209 Rackbeta scintillation counter.

RESULTS

Isolation of the ILTV Glycoprotein B Promoter

We have previously reported the nucleotide sequence of a region of the ILTV genome containing the gpB gene. Based on this sequence oligonucleotides were designed to isolate the 5' noncoding region from the ILTV gpB gene ( PgB ), by use of the polymerase chain reaction ( Figure 15 ). To facilitate the cloning of the PCR products, the oligonucleotides were designed to contain unique restriction enzyme sites; the oligonucleotide furthest 5' to the open reading frame contained an internal Pstl site, while the oligonucleotide immediately 5' to the open reading frame contained an Xbal site. Oligonucleotide priming positions are given in figure 15.

Products of PCR reactions were separated on agarose gels, revealing a unique species ( results not shown ) corresponding to that predicted by sequence data. The fragment was isolated from the gel, cut with restriction enzymes Pstl and Xbal and purified by gene cleaning ( BRESA, Australia ). The resulting fragment was cloned into pUC18 ( Yanish-Perron et al., 1985 ), which had previously been restriction enzyme digested with Pstl and Xbal prior to phosphatase treatment. Clones were screened for the presence of promoter fragments by standard procedures. A number of clones containing the correct size fragments were further characterised by double stranded sequencing to confirm the cloning of promoter fragment ( pUC-PgB ).

Construction of pPgB-CAT Vector

The promoter fragment was sub-cloned from the pUC-PgB construct into the pCAT-Basic plasmid ( Promega ), to form pPgB-CAT ( Figure 16 ). The pCAT-Basic plasmid contains the chloramphenicol acetlytransferase ( CAT ) gene without any transcription regulation signals, and was designed for the specific purpose of assaying DNA fragments for promoter activity. The CAT gene product is readily assayed, allowing accurate qualification of promoter activity. The orientation, and correct cloning of the promoter fragment adjacent to the CAT gene was confirmed by double stranded sequencing ( results not shown ) using a 15mer oligonucleotide which bound specifically to a region of the CAT gene 25bp 3' to the translation initiation signal. The promoter fragment was cloned adjacent to the CAT gene in the same orientation as it was with respect to the ILTV gpB open reading frame.

Assay of Promoter Activity

Monolayers of CK cells, a minimum of 2 petri-dishes per assay, were transfected with 1-10ug of pPgB-CAT. To determine if promoter activity was up-regulated by infection with ILTV, transfected cells were infected with ILTV, or mock-infected with PBS 14 hr post-transfection, and assayed for CAT activity 48hrs post-infection ( Table 5 ). From Table 5 it can be seen that pPgB-CAT expresses CAT activity at levels substantially higher than back-ground CK cell levels. The level of CAT activity observed was seen to be affected by the level of input plasmid DNA, but not in a linear ratio. Infection of pPgB-CAT transfected cells (1ug/plate ) with ILTV resulted in a 14.5 fold increase in the level of CAT activity present in cells. Levels of CAT activity above back-ground were not detected in culture media from transfected and infected cells ( results not shown ). DISCUSSION

The polymerase chain reaction was used to isolate a 573bp fragment of ILTV DNA extending upstream from the first nucleotide 5' to the open reading frame of the ILTV gpB gene. The ATG translation initiation codon of the gpB gene was not included in the promoter fragment, thus eliminating the production of fusion proteins and the need to align open reading frames, when expressing foreign genes. When the ILTV gpB promter was aligned adjacent to the marker gene CAT, and transfected into CK cells, levels of CAT activity observed were significantly above background. The activity observed was shown to be dependent on the level of pPgB-CAT transfected into cells. The infection of cells containing the pPgB-CAT construct resulted in an increased level of CAT expression. This increased level of CAT activity is typical of herpesvirus promoters where trans-activating factors encoded by the virus serve to increase promter activity ( reviewed Roizman and Sears, 1990 ).

In addition to the gpB promoter we have identified and cloned the 5' non-coding regions of the ILTV glycoprotein 60 and ORF3 genes ( Figure 17 ), the sequences of which are given elsewhere. These other additional regions may also be suitable promoters for the expression of foreign vaccine antigens in ILTV recombinants.

EXAMPLE 7

This example shows the construction of a fowlpox virus (FPV) recombinant expressing the ELTV glycoprotein B gene, and its ability to protect chickens from infection with viruuent ILTV. Figure 18 shows the structure of recombinant plasmid for insertion of the LacZ, Ecogpt and ILTV glycoprotein B gene into the FPV TK gene. Figure 18A is a schematic representation (not drawn to scale) of the genes inserted into the FPV recombinant. In addition to these genes pAF09-gpB also contains the ampicillin resistance gene and an E.coli origin of replication. Figure 18B shows the junction region between the FPV E/L promoter and the ILTV gpB gene. The ATG of the E/L promoter is in phase with two ATG codons of the gpB gene.

MATERIALS AND METHODS

Virus and Cells:

FPV (mild vaccine strain, Arthur Websters Pty. Ltd., Northmead, 2152, Australia) was propagated on monolayers of chicken embryo skin (CES) cells as previously described (Prideaux and Boyle, 1987). Primary CES cells were prepared as described by Silim et al. (1982) with the modification the collagenase at 100μg/ml (Sigma C2139) was used to digest the skin of 13-day-old specific pathogen free embryos (CSIRO, SPF Poultry Isolation Unit, Maribyrnong, Victoria, Australia) in place of trypsin.

Primary chicken kidney (CK) cells were prepared by trypsinisation of kidneys isolated from two to four week old specific pathogen free chickens as described by Fahey et.al. (1983). ILTV SA2 vaccine strain was obtained from Arthur Websters Pty. Ltd. ILTV was grown on CK cells in Eagle's Basal Medium (Gibco Laboratories) supplemented with 5% bovine calf serum and 10 mM Hepes. Virus stocks were frozen (-70 °C) and thawed three times prior to infection to release ILTV from cells. Enzymes and Chemicals:

Restriction enzymes and other DNA modifying enzymes were obtained from various sources and used according to the manufacturers instructions or as outlined by Maniatis et.al. (1982). 5-Bromo-4-chloro-3-indoyl-β-D galactopyranoside (X-Gal) was obtained from Sigma Chemical

Company.

Isolation of Genomic DNA:

Confluent monolayers of CK cells were infected with ILTV at a level of 0.1 plaque forming units (p.f.u.) per cell and incubated at 39 °C until cellular cytopathic effects were greater than 90%. Virus was isolated from both cytoplasmic and cell free fractions. DNA was extracted essentially according to the method of Wilkie (1973) by treatment with 2% SDS, NTE-saturated phenol (NTE: lOmM Tris-HCl. pH 8.0, 100mM NaCl, ImM EDTA) and chloroform/isoamylalcohol (24:1). The DNA was precipitated in 70% ethanol and resuspended in TE buffer (lOmM Tris-HCl, pH7.5, ImM EDTA). Isolation of the ILTV Glycoprotein B Gene:

Isolation of the ILTV glycoprotein B (gpB) gene was performed using the polymerase chain reaction (PCR). Reactions were carried out in 50μl volumes comprising 50ng of ILTV genomic DNA, 3ng of each oligonucleotide primer, 50mM KCl, 10mM Tris- HCl, pH8.4, 2.5mM MgCl₂, 200μg/ml BSA, 200mM dATP, 200μM dCTP, 200μM dGTP, 200μM dTTP and 1 unit of Taq polymerase (Cetus). Reaction mbctures were overlayed with an equal volume of paraffin oil and heated to 94 °C for 1 min, cooled to 55 °C for 2 min and heated to 72 °C for 5 min. This cycle was repeated 35 times using a Perkin Elmer Cetus DNA Thermal Cycler.

Plasmid Constructs:

Specific oligonucleotides were designed to isolate the gpB gene by use of the PCR, based on sequence data presented elsewhere in this patent. The oligonucleotides bound to the ILTV gpB gene at nucleotides 140-172 and 3000-3030 as presented in this patent. To facilitate the cloning of the PCR product BamHl restriction enzyme sites were engineered into the oligonucleotides. The isolated gpB gene was inserted into FPV using the plasmid vehicle pAF09 (provided by Dr. David Boyle, CSIR0, Australian Animal Health Lab., Geelong, Australia). This plasmid vehicle is suitable for the insertion of foreign genes into the FPV thymidine kinase (TK) gene, and contains the E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene under the transcriptional control of the W P7.5 promoter, acting as a co-expressed selectable marker as previously described by Boyle and Coupar (1988a). In addition pAF09 also contains the E.coli LacZ gene under the transcription control of the FPV late promoter (Kumar and Boyle, 1990) allowing rapid identification of FPV recombinants (Prideaux et.al. 1990).

The ILTV gpB gene was introduced into the BamHI site of pAF09 (Figure 18A; pAF09-gpB), 3' to the FPV E/L promoter (Kumar and Boyle, 1990). The gpB gene and FPV promoter were aligned in such away that the gpB gene translation initiation codon was in frame with initiation codon of the E/L promoter, producing a fusion gene with 4 FPV amino acids upstream of the ILTV gpB gene.

Construction and Isolation of Recombinant Virus:

The FPV recombinant (FPV-gpB) was constructed using the protocol described previously by Boyle and Coupar (1988b) utilising the co-expressed Ecogpt gene for recombinant virus selection. The co-expression of the Ecogpt gene enables recombinant virus to replicate in medium containing MXHAT (MXHAT: 2μg/ml mycophenolic acid, 250μg/ml hypoxanthine, 0.4μM aminopterin and 320μM thymidine) selective conditions (Boyle and Coupar, 1988a).

Recombinant virus expressing β-galactosidase were selected by plaquing under non-selective conditions and staining with X-Gal (200μ/ml) in growth medium containing 1% agar. Recombinants expressing β-galactostdase produced characteristic b.ue plaques (Chakrabarti et.al., 1985; Panicali et.al, 1986). Recombinant virus was plaque purified three times prior to the production of working stocks.

Test Animals, Vaccination and Challenge Protocols:

Groups of 12 White Leghorn chickens from the CS1RO SPF Poultry Unit housed in positive pressure isolators, were vaccinated and challenged at 8 weeks of age as se, out in Table 6. Sera was obtained from all birds prior to vaccination, and assayed for ILTV antibody by ELISA as described elsewhere in this patent. ILTV SA2 and FPV M vaccines were obtained from Arthur Websters Pty. Ltd. and used according to the manufacturers instructions. The PPV-gpB recombinan, was dilu,ed 1 in 10 in vaccine diluen, (Arthur Websters Pty. Ltd, end titre appro, 1X10⁶p.f.u./ml) and administered to the wing web using a bificated needle, as for FPV M. Birds were challenged by intratracheal inoculation of ILTV CSW-1 (Bagus, et.al., 1986). Post-Mortem Evaluation of vaccines:

Three days after challenge birds were killed, trachea removed and tracheal exudate collected and assayed for ILTV antigen by ELISA,

RESULTS

ELISA assays of serum taken from birds prior to vaccination showed all birds to be free of anti-ILTV antibodies.

Results of ILTV antigen ELISA's from tracheal exudates of all birds are given in Table 7 as percentage of birds protected against challenge with ILTV CSW-1. From Table 7 it can be seen that all birds vaccinated with the commercial ILTV SA2 vaccine were protected from challenge with ILTV CSW-1. The unvaccinated, and FPV M vaccinated birds both showed a protection level of 17%. This protection may be the result of insufficient challenge, or failure of virus to enter the trachea. The FPV-gpB recombinant protected 58% of birds challenged, significantly (Fisher's exact test) higher than the non-ILTV vaccinated birds. Table 1 ILTV proteir recognised by Immune chicken sera using Western blotting of a detergent extract of SA-2-lnfected cells separated by SDS-PAGE

Approximate

molecular % sera positive weight

(K)

205 100

160* 100

135 52

125 24

115* 86

105 43

90* 91

85 43

74 29

70 24

67* 91

60 100

56 57

54 24

52 95

50 10

46 57

41 67

40 24

36 24

34* 100

31 38

29 38

26* 76

* Bands strongly recognised Table 2. OTH reaction elicited In vaccinated cockerels by ILTV glycoproteins Immunoprecipitated by monoclonal antibodies.

Antibody used for Mean No. +ve^b/ lmmunoprecipitatlon DTH index^a No. tested

39-2 (Group I) 0.72 ± 0.15^d 5/6

22-37 (Group II) 0.92 ± 0.17^d 5/6

131-6 (Group II) 0.76 ± 0.20^d 4/6

12-1 (Group II) 0.06 ± 0.04 0/6

Normal mouse 0.11 ± 0.07 0/6 serum

Detergent extract^c 1.02 ± 0.44^d 3/4

a Mean + standard error. b DTH index of >0.4 was considered positive. c Whole detergent extract of virus-infected cells. d Significantly different to normal mouse serum (p<0.05). TABLE 3

EXPERIMENT 1. IMMUNE RESPONSES AND PROTECTION AGAINST INTRATRΛCHEΛL CHALLENGE

AFTER VACCINATION OF CHICKENS WITH ILTV GLYCOPROTEINS

DTH

Clinical

Vaccine VN^a DTH index^b No . +ve/ signs(%)^d Protection(%)^e

No. tested^c

Unvaccinated 0 0.07 ± 0.07 0/6 31 0

Total

glycoprotein 43 1.61 ± 0.24^f 6/6 0 71

Live virus 153 2.49 ± 0.429 6/6 0 100 ^a Geometric mean virus neutralization titre (reciprocal) of groups of 16-17 chickens.

b Mean DTH index ± standard error of 6 cockerels measured at 2 weeks after secondary vaccination. ^c DTH index of >0.5 was considered positive.

d Clinical signs (gasping respiration and death) observed at day 5 after challenge with 10⁵ PFU of CSW-1.

e Protection was assessed 4 weeks after secondary vaccination by the absence of viral antigen in the trachea at day 5 after intratracheal challenge.

f Significantly different to controls, p<0.05.

9 Significantly different to controls, p<0.01.

TABLE 5

-ILTV +ILTV

CK CELLS 130 140

PpGB-CAT

1ug 350 5,050

10ug 900 2,950

Table 5: ILTV glycoprotein B promoter expression of the marker gene CAT. The ILTV gpB promoter was linked to the CAT gene and 1 or 10ug of plasmid DNA transfected into CK cells. Enzymes activity was determined for a minimum of two plates for each assay, following infection with ILTV (+ ILTV), or mock infection (-ILTV). Results are expressed as counts per minute.

TABLE 6

Schedule for the vaccination of chickens with

ILTV SA2, FPV M, PBS or FPV-gpB.

Day 0

(i) Pre-bleeded birds from wing vein

(ii) Vaccinate birds

(A) 12 birds 50ul PBS intraocular

(B) 12 birds ILTV SA2 50ul intraocular

(C) 12 birds FPV M bificated needle

(D) 12 birds FPV-gpB bificated needle. Day 10

Groups C and D revaccinated, in opposite wing web, as for Day O.

Day 20

All groups challenged with ILTV CSW-1 by intra-trachea inoculation

(1×10⁵ p.f.u. in 200 ul).

Day 23

Trachea removed from all chickens and scraped to isolate ILTV antigen.

TABLE 7

Protection of vaccinated chickens against challenge with ILTV CSW-1

Percentage Protected

Control 17

ILTV SA2 100

FPV M 17 FPV-gpB 58

Chickens were challenged with 10⁵ p.f.u. .ILTV CSW-1 by intra-tracheal inoculation.

Protection was assessed by the absence of viral antigen in the trachea as detected by ELISA.

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Claims

1. A non-infectious subunit vaccine for use against infectious laryngotracheitis virus (ILTV), which comprises as active immunogen at least one glycoprotein of ILTV, or an immimogenic peptide derived therefrom, and optionally an adjuvant.

2. A vaccine according to claim 1, wherein said active immunogen is selected from the group consisting of the 205K complex of glycoproteins and the 60K glycoprotein of ILTV.

3. A vaccine according to claim 1, wherein said active immunogen is an immunogenic peptide comprising all or at least the major antigenic determinants of an ILTV glycoprotein and exhibits the immunogenicity of said DLTV glycoprotein.

4. A vaccine according to claim 1, wherein said active immunogen is coupled to a carrier molecule to increase its immunogenicity.

5. A vaccine according to any one of claims 1 to 4, wherein said adjuvant is an aqueous-mineral oil emulsion.

6. A vaccine according to claim 5, wherein said adjuvant further comprises AlOH₃, saponin or a muramyl dipeptide derivative.

7. A vaccine for use against ILTV, which comprises as active immunogen a recombinant live virus vector having inserted therein a nucleotide sequence coding for at least one glycoprotein of ILTV, or an immunogenic peptide derived therefrom.

8. A vaccine according to claim 7, wherein said nucleotide sequence codes for peptide selected from the 205K complex of glycoproteins and the 60K glycoprotein of ILTV, or an immunogenic peptide derived therefrom.

9. A vaccine according to claim 7, wherein said live virus vector comprises fowlpox virus or another avian virus.

10. A vaccine according to claim 9, wherein said live virus vector comprises avian adenovirus.

11. A method for protecting chickens or other poultry against ILTV, which method comprises to said chickens or other poultry an effective amount of a vaccine according to any of claims 1 to 10.

12. A recombinant DNA molecule comprising a nucleotide sequence capable of being expressed as all or at least a substantial portion of the 205K complex of glycoproteins or the 60K glycoprotein of ILTV, or an immimogenic peptide derived therefrom

13. A recombinant DNA molecule according to claim 12 comprising a nucleotide sequence encoding the 205K complex of glycoprotein of ILTV substantially corresponding to the sequence shown in Figure 11, or degenerate variants thereof, or a fragment of said sequence.

14. A recombinant DNA molecule according to claim 12 comprising a nucleotide sequence encoding the 60K glycoprotein of ILTV substantially corresponding to the sequence shown in Figure 6, or degenerate variants thereof, or a fragment of said sequence.

15. A recombinant DNA molecule according to any of claims 12 to 14, including an expression control sequence operatively linked to said nucleotide sequence or fragment thereof.

16. A recombinant DNA cloning vehicle comprising an expression control sequence operatively linked to a nucleotide sequence capable of being expressed as all or a substantial portion of the 205k complex of glycoproteins or the 60K glycoprotein of ILTV, or an immunogenic peptide derived therefrom.

17. A host cell transformed with a recombinant DNA cloning vehicle according to claim 16.

18. A synthetic, including recombinant, polypeptide displaying the antigenicity of the 205K complex of glycoproteins or the 60K glycoprotein of ILTV.

19. A synthetic polypeptide according to claim 18, comprising a fusion polypeptide wherein the sequence displaying the desired antigenicity is fused to an additional heterologous polypeptide sequence.

20. A recombinant ILT virus, characterised in that heterologous DNA is inserted into a non-essential region of the ILTV genome.

21. A recombinant virus according to claim 20 characterised in that the heterologous DNA is inserted into a region of the ILTV genome corresponding to (1) the ILTV glycoprotein gp60 gene, (2) the Kpn I/K fragment ORF 3 gene, or (3) the ILTV homologue of the HSV protein kinase gene.

22. A recombinant DNA molecule comprising an ILTV promoter region operatively linked to a heterologous DNA sequence.

23. A recombinant DNA molecule according to claim 22, wherein said ILTV promoter region is the region corresponding to (1) the gp60 promoter region, (2) the gp205 (gpB) promoter region, or (3) the ORF3 promoter region.

24. A recombinant virus, characterised in that heterologous DNA is inserted into a non-essential region of the host virus genome and expression of said heterologous DNA is controlled by an ILTV promoter region.

25. A recombinant virus according to claim 24, wherein said ILTV promoter region is the region corresponding to (1) the gp60 promoter region, (2) the gp205

(gpB) promoter region, or (3) the ORF 3 promoter region.

26. A recombinant virus according to claim 24 or claim 25, wherein said host virus is ILTV.