AU660528B2 - Process for the expression of herpes simplex virus type 1 glycoprotein E and methods of use - Google Patents

Description

66 528 S F Ref: 233090

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFCATION FOR A STANDARD PATENT

ORIGINAL

S* Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Cedars-Sinai Medical Center 8700 Beverly Boulevard Los Angeles California 90048-1865 UNITED STATES OF AMERICA Anthony Bart Nesburn, Steven Lewis Wechsler and Homayon Ghiasi Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Process for the Expression of Herpes Simplex Virus Type 1 Glycoprotein E and Methods of Use e oooo The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845/3 Process For The Expression Of Herpes simplex Virus Type 1 Glycoprotein E And Methods of Use Field Of The Invention The present invention is in the fields of infectious diseases and molecular biology.

Background of The Invention A. Herpes Simplex Virus Type 1 (HSV-1) Glycoprotein E Glycoprotein E (gE) is one of ten documented Herpes simplex virus type 1 (HSV-1) glycoproteins. In HSV-1 infected cells, gE is synthesised as an unglycosylated peptide having a molecular weight of 59 kDa. The unglycosylated polypeptide is cleaved and partially glycosylated to produce a precursor gE (pgE) of approximately 65 kDa, which is then further glycosylated to the mature form of gE with a molecular weight of about kDa.

The ten HSV glycoproteins are located on the surface of the virus, some of which are reported to be the primary inducers and targets of both humoral (antibody) and cellmediated immune responses to HSV-1 infection. In one study, Blacklaws et al. have shown glycoproteins B and D to induce high neutralising antibody titers and to protect from latent herpes infection They also found, however, that vaccinia expressed glycoproteins G, H and I produced no protective response in immunised mice and that vaccinia expressed gE demonstrated only a very weak neutralising antibody response against HSV. Blacklaws et al. also found that vaccination with gE expressed by vaccinia virus did not protect against the establishment of latent infections, nor did it protect mice against lethal HSV-1 challenge In another study, Para et al. found that, only in the presence of complement, antibodies against immunoaffinity purified gE neutralised HSV-1 infectivity. But even then, the neutralisation titers were low, and the extent of the anti-gE S 25 antibodies' role in neutralisation was not determined In contrast to these reports, we have expressed in a baculovirus system, gE that is capable of eliciting a strong protective immune response against HSV-1 infection. In fact, vaccination with our recombinant baculovinis expressed gE induced high neutralising antibody titers, a DTH response and protected against lethal HSV-1 challenge in mice. The neutralising antibody titers we obtained were much higher than titers obtained by either Para et al. against their immunoaffinity purified gE, or Blacklaws et al. against their vaccinia expressed gE In addition, our neutralising antibody titers against baculovirus expressed gE were only partially complement dependent, which in contrast to Para's S results required the presence of complement with their immunoaffinity purified gE for even low neutralising activity. This ability to produce large quantities of high quality bioactive gE, is critical in the development of an effective vaccine against HSV.

B. DNA Technology Recombinant DNA and associated technologies can be applied to effectively provide the large quantities of high quality bioactive HSV glycoprotein E required for a therapeutic IPrv1100016:GSA 1 of 16 or prophylactic HSV vaccine.

DNA technology involves in part, producing a replicable expressio vehicle or transplacement vector by the DNA recombination of an origin of replication, one or more phenotypic selection characteristics, an expression promoter, a heterologous gene insert and remainder vector. The resulting expression vehicle is introduced into cells by transformation and large quantities of the recombinant vehicle obtained by growing the transformant. Where the gene is properly inserted with reference to portions which govern the transcription and translation of the encoded DNA message, the expression vehicle may produce the polypeptide sequence for which the inserted gene codes. This process of producing the polypeptide is called "expression." The resulting product may be obtained by lysing the host cell, and recovering the product by appropriate purification.

A wide range of host cells can be used, including prokaryotic and eukaryotic organisms. In addition to microorganisms, cultures of cells derived from multicellular organisms, whether vertebrate or invertebrate, may also be used as hosts. Our system involved use of baculovirus, the polyhedrin promoter system and insect cells as host cells to produce high quantities of bioactive gE. To our knowledge, we are the first to express gE in this system.

The references cited herein are all incorporated by reference.

Summary Of The Invention The present invention relates to the production of HSV-1 gE, by recombinant DNA techniques, and its use as an immunogen in a vaccine to protect against HSV-1 and/or HSV-2 infections. Vaccines made from genetically engineered immunogens should be safer than conventional vaccines made from attenuated virus because there is no risk of infection to the recipient; and specifically with the herpes virus, there should be no risk of 25 cervical cancer. Alternatively, the genetically engineered glycoprotein or protein product may be used to produce antibodies for use in passive immunotherapy. The invention also relates to the transformed cell line, which contains the subject transplacement vector, and its cultures which produce HSV-1 gE.

To this end, we constructed a recombinant baculovirus expressing high levels of HSV-1 gE in Sf9 cells. We unexpectedly discovered, however, that vaccination of mice with our expressed gE, induced high neutralising antibody titers, a DTH response, and protection against lethal HSV-1 challenge. Methods and compositions are therefore provided for the cloning and expression of HSV gE gene in single-cell aust organisms.

Also described are methods for culturing these novel single-cell organisms to produce the HSV gE gene product as well as methods for the purification of the gene product.

A human host is then preferably inoculated with a vaccine comprising an immunity inducing dose of gE alone or with one or more HSV glycoproteins or proteins by the systemic route, the enteric route or by the ocular route. The vaccine may also comprise one or more adjuvants administered with, before or after the glycoprotein component of IPriv1100015:GSA 2 of the vaccine.

The vaccine of the invention may be conveniently utilised in liquid form, freezedried, spray dried or lyophilised form, in combination with one or more suitable preservatives and protective agents to protect the glycoproteins or proteins during processing.

A. Antigen The baculovirus expressed gE migrated on gels as a doublet band with apparent molecular weights of 68 and 70 kDa. The recombinant gE was glycosylated, as demonstrated by its susceptibility to tunicamycin treatment. Indirect immunofluorescence also demonstrated that it was transported to the membrane of Sf9 cells. Mice vaccinated with our expressed gE developed high serum titers of HSV-1 neutralising antibodies, which was demonstrated by plaque reduction assays. Glycoprotein E also induced a strong delayed type hypersensitivity (DTH) response to HSV-1, and mice vaccinated with the recombinant gE were protected from both intraperitoneal and ocular lethal HSV-1 challenge.

B. Adjuvants Vaccines are often administered in an emulsion with various adjuvants. The adjuvants aid in attaining a more durable and higher level of immunity using smaller amounts of antigen in fewer doses than if the immunogen were administered alone. The adjuvants for use in the present invention include but are not limited to alum, Freund's, MTP-PE and ISCOMs (Quil In addition, the vaccine may comprise a lipoqome or :."other membrane bound vesicle comprising one or more HSV-1 glycoproteins administered with or without one or more adjuvants to induce the cell mediated immune response.

C. Immunisation Routes And Dosages 25 1he vaccine can be administered by the systemic route, the ocular route either alone or in combination with systemic vaccination, or the enteric route. The systemic route includes but is not limited to subcutaneous, intramuscular or intravenous injection in one or multiple doses. The ocular route includes but is not limited to subconjunctival injection, Ssurface drops, a slow-release device such as a collagen shield, a hydrogel contact lens or an ALZA "Ocusert" in one or multiple doses.

Doses to be administered are variable and depend on the desired effect and on the chosen administration route, with 1 to 3 doses generally comprising the vaccination.

However, inoculation doses to humans by injection vary from about 10ptg to 500itg. For ocular vaccination, the human dosages vary from about lig to 500pg; whereas for enteric 35 vaccination, the human dosages vary from about lpg to 800itg.

It is therefore a general object of the present invention to express high levels of HSV-1 gE.

It is an object of the present invention to express high levels of HSV-1 gE from one virus strain in a single vector system.

IPrlv 100016:GSA 3 of 16 It is also an object of the present invention to express high levels of bioactive HSV-1 gE from cells which have been infected or transformed with a recombinant baculovirus.

It is another object of the present invention to obtain a transformed cell line which produces HSV-1 gE.

It is a further object of the present invention to develop an effective vaccine for the treatment or prevention of HSV in a host.

These and other objects will become readily apparent to those skilled in the art from the following description and appended claims.

Brief Description Of The Drawings The present invention will be described in connection with the accompanying drawing in which: FIGURE 1 is a schematic diagram of the construction of the pAc-gEl recombinant baculovirus transfer vector containing the HSV-1 gE gene. Panel A: Details of preparation of the recombinant vector containing the gE gene of HSV-1 strain KOS are given in the Detailed Description below. Panel B: The thick solid black line indicates the extent of the 1,822 nucleotide isolated gE fragment that was inserted into the baculovirus vector. The arrow head indicates the 3' end of the gene. The dashed lines show regions removed by enzymatic digestions as indicated. The gI and US9 designations show the location of the adjacent ends of these flanking genes that have been removed. The numbers indicate the 20 number of nucleotides in the regions indicated by brackets. The gE initiating ATG codon is 27 nucleotides from the start of the gE fragment, which in turn is 203 nucleotides from the 3' end of gI. The gE TAA termination codon is 141 nucleotides from the end of the gE fragment, which in turn is 278 nucleotides from the start of the US9 gene. Thus, the final gE construct contains no HSV-1 genes except gE and cannot produce any HSV-1 proteins 25 other than gE. Panel C: The polyhedrin gene promoter sequence near the start of the gE gene in the final vector is followed by the modified BamH I/Bcl I site (underlined), 27 noncoding nucleotides of the gE gene, and the start of the gE coding sequence (ATG).

FIGURE 2 is a Western blot analysis and coomassie blue staining of baculovirus-gE.

Western blot analysis of baculovirus-gE expression and glycosylation. Insect cells were infected with the baculovirus-gE recombinant (vAc-gEl) (MOI of 10 PFU/cell for 72 hr). Vero cells were infected with HSV-1 strain KOS (MOI of 10 PFU/cell for 20 hr).

Total cell lysates were analysed by Western blots using 1BA10 monoclonal antibody Lanes: M, molecular weight markers; 1, 4 x 105 HSV-1 infected Vero cells; 2, 4 x 103 vAc-gEl infected Sf9 cells; 3, tunicamycin treated vAc-gEl infected cells; 4, wild-type AcNPV infected insect cells; 5, mock infected Sf9 cells. All lanes shown are from the same exposure of the same gel. For the purpose of photography, lanes 4 and 5 were moved from a different location on the autoradiogram. Detection of expressed gE by coomassie blue staining. Total cell lysates from recombinant gE or wild-type baculovirus pfu/cell, 72 hr post infection) were run on 10% SDS-PAGE and stained with IPriv 100015:GSA 4 of 16 coomassie brilliant blue. Lanes: M, molecular weight markers; B, wild-type baculovirus infected cells; gE, vAc-gEl infected cells 72 hr post infection. The arrows indicate the positions of expressed gE (gE) and the wild-type polyhedrin protein bands.

FIGURE 3 shows the neutralisation of HSV-1 infectivity with anti-baculovirus-gE sera. Serial dilutions of heat-inactivated pooled sera from baculovirus-gE, mockvaccinated (wild type baculovirus), or HSV-1 (strain KOS) inoculated mice were incubated with 100 PFU of HSV-1 strain KOS in the presence of fresh or heat-inactivated guinea pig complement as described in the text and the remaining infectious virus was titrated.

Detailed Description The present invention utilises recombinant DNA techniques to insert a DNA sequence coding for HSV-1 gE or a portion thereof, into a DNA transplacement vector, such that the vector is capable of replicating and directing expression of the gE gene in a foreign host. The resulting recombinant DNA molecule is introduced into insect cells to enable high production of gE, or a portion or molecular variant thereof by the host cells.

The gE produced is then isolated and purified for use in immunotherapy against both HSV type 1 and/or type 2.

The Examples set forth below describe use of baculovirus, the polyhedrin promoter system and insect cells as host cells. However, it would be well within the skill of the art to use analogous techniques to construct expression vectors for expression of desired gE 20 and gE products in alternative host cell cultures. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims.

A. Viruses and Cells The E2 strain of Autographa californica nuclear polyhedrosis virus (AcNPV) and 25 Spodoptera frugiperda clone 9 (Sf9) cells were grown using TNM-FH containing fetal bovine serum as previously described Plaque purified HSV-1 (strains McKrae and KOS) and Vero cells were grown as previously described i' B. Construction of the AcNPV recombinant transfer vector.

As shown in FIGURE 1, plasmid pSG25 containing the EcoRI H restriction fragment from HSV-1 strain KOS was digested with Dde I and a fragment containing the complete coding region of HSV-1 gE was isolated. The linearized fragment was treated with Bal 31 and then recut with Sal I. After addition of Bcl I linker the complete gE structural gene was inserted into the BamH I site of the vector pAcYMl under control of the baculovirus polyhedrin gene promoter. The bottom of the figure shows the polyhedrin gene promoter sequence of the final vector in the area of the start of the gE gene. This is followed by the modified BamH I/Bcl I site (underlined), 27 noncoding nucleotides of tl, gE gene, and the start of the gE coding sequence (ATG).

C. Transfection and selection of recombinant viruses Sf9 cells were cotransfected with purified infectious baculovirus (AcNPV) DNA and IPriv1100016:GSA 6 of pAc-gEl plasmid DNA as described Following three cycles of plaque purification, three polyhedrin-negative recombinant viruses were selected. The recombinant baculoviruses all expressed gE with similar properties. One was chosen arbitrarily for further study and was designated vAc-gEl.

D. Tunicamycin treatment Infected cells (10 PFU/cell) were incubated in 4 ug/mL tunicamycin in TNM-FH media Biosciences, Lenexa, KS) from 0-48 hr post infection and harvested for SDS-PAGE as described E. Western blots Western immunoblot analyses were carried out under denaturing conditions, as previously described The nitrocellulose blots were reacted with one of the three antigE monoclonal antibodies Fdl72 7-8 (Chatterjee et al., unpublished), 1BA10 or total HSV-1 antibody (made in rabbit against purified HSV-1 virions) for 1 hour at 4 C.

The blots were then incubated with 12 5I-protein A for 1 hour at 250C and autoradiographed.

F. Immunisation of mice Twenty Balb/c mice (6-8 weeks old) were vaccinated with freeze-thawed whole cell lysates of Sf9 cells infected with baculovirus expressing gE. Lysates from 1 x 106 Sf9 cells were injected subcutaneously with Freund's complete adjuvant on day 0 or with Freund's 20 incomplete adjuvant on days 21 and 42. Intraperitoneal injections were given concurrently using lysates from 1 x 106 infected Sf9 cells in PBS. We estimate from coomassie blue staining that extract from i x 106 baculovirus-gE infected cells contains approximately 30 lg of gE. Eighteen mock vaccinated mice were inoculated with Sf9 cells infected with wild type bacalovirus using the same regimen. A positive control group of 25 eleven mice was immunised three times i.p. with 2 x 105 PFU of the nonvirulent HSV-1 strain KOS. Sera were collected 3 weeks after the final vaccination and pooled for each group.

For HSV-1 ocular protection studies, 5 mice/group were vaccinated twice in a similar manner using freeze-thawed whole cell lysates from 1 x 106 Sf9 cells infected with baculovirus expressing gE. Injections were done subcutaneously with Freund's complete adjuvant on day 0 and with Freund's incomplete adjuvant on day 21. Intraperitoneal injections were given concurrently using lysates from 1 x 106 infected Sf9 cells in PBS.

Mock vaccinated mice were inoculated with Sf9 cells infected with wild type baculovir"' using the same regimen. Mice in the positive control group were immunised at the same time i.p. with 2 x 105 PFU of the nonvirulent HSV-1 st-in KOS.

6. Serum neutralisation assay For in vitro seum neutralisation assays, heat inactivated pooled sera were diluted in MEM, mixed with 100 PFU of HSV-1 strain KOS, and incubated for 30 min at 37 0

C.

Two and one half percent fresh or heat-inactivated guinea pig complement was added and IPrlv1100015:GSA 6ol the mixture was incubated for an additional 30 min. Duplicate samples were added to CV-1 cells in 24-well microtiter plates and residual HSV-1 infectivity was assayed. The plates were incubated at 37 0 C for 72 hr, stained with 1% crystal violet, and the plaques were counted. The experiment was repeated twice and the means of the antibody titers plaque reduction) were expressed as the reciprocal of the serum dilution.

H. Delayed type hypersensitivity (DTH) To study the DTH response to gE, mice were vaccinated three times as described above. Twenty one days after the final vaccination, mice were injected with 2 x 106 PFU of uv-inactivated HSV-1 (strain McKrae) in 10gl of MEM, in the dorsal side of the right ear. Ear thickness was measured just prior to injection and 24h, 48h, and 72h after injection using a micrometer (Mitutoyo, Tokyo, Japan) and recorded as net swelling (postchallenge minus pre-challenge ear thickness) Controls included HSV-1 (strain KOS) vaccinated mice (positive control) and wild type baculovirus (mock) vaccinated mice (negative control).

I. Viral challenge Three weeks after the final vaccination, mice were challenged i.p. with 2 x 10 6

PFU

(4 LD50 by the i.p. route) or ocularly with 2 x 105 PFU/eye (10 LD50 by the ocular route) of the virulent HSV-1 strain Mckrae. Challenged mice were monitored for two weeks.

20 Results Construction of recombinant baculoviruses expressing gE The strategy for the construction of the baculovirus transfer vector (pAc-gEl) containing the complete gE open reading frame from HSV-1 strain KOS is described above and shown in FIGURE 1A. FIGURE 1B illustrates in more detail the extent of the gE 25 gene present in the final baculovirus vector. The entire gE structural gene is present, with minimal additional 5' and 3' sequences. The 5' DdeI cut and subsequent Bal 31 digestion resulted in the inclusion of only 27 HSV-i nucleotides upstream of the initial gE ATG codon. The SalI restriction cut at the 3' end resulted in the inclusion of only 141 HSV-1 nucleotides downstream of the gE TAA termination codon. The 5' end of the gE fragment begins 203 nucleotides from gI, the nearest upstream gene. The 3' end of the gE fragment ends 278 nucleotides from US9, the nearest downstream gene. Thus, the gE-baculovirus construct contains the entire structural gE gene with no additional HSV-1 genes (or portions of HSV-1 genes). No HSV-1 proteins other than gE can be expressed from this gE-baculovirus. Following cotransfection, incorporation of the vector into baculovirus, and isolation of a recombinant, the presence of HSV-1 gE DNA in the recombinant baculovirus was verified by Southern blot hybridisation.

B. Identification of expressed gE in insect cells To analyse the size of baculovirus expressed gE in insect cells, total protein extracts from HSV-1 infected Vero cells or the baculovirus-gE recombinant (vAc-gEl) infected Sf9 IPriv110001 6:GSA 7 of 16 cells were run on 10% SDS-PAGE and analysed by Western blotting using anti-gE monoclonal antibody IBA10 This is illustrated in FIGURE 2a. Glycoprotein E from HSV-1 infected Vero cells had an apparent molecular weight of 80-85 kDa (FIGURE 2a, lane 1) in agreement with the previously reported apparent molecular weight of gE Two bands from the baculovirus-gE extract with apparent molecular weights of 68 and kDa reacted strongly with the gE specific antibody (FIGURE 2a, lane Similar results were seen with total HSV-1 antibody and anti-gE monoclonal antibodies Fdl72 and 7-8.

The level of gE expression (accumulation) seen by Western analysis was similar at 48, 72, and 96 hr post infection.

Monoclonal antibody 1BA10 (lane 2) and total HSV-1 antibody (not shown) also reacted slightly with a smaller band with an apparent molecular weight of 50 kDa (faintly seen in FIGURE 2a, lane Monoclonal antibodies Fdl72 and 7-8 did not react with this band. The identity of this band is not clear, but it may represent a gE breakdown product.

Anti-gE anti serum did not react with any bands from wild type baculovirus infected cells (lane 4) or uninfected Sf9 cells (lane C. Glycosylation and cellular localisation of baculovirus expressed gE To determine if the baculovirus expressed gE underwent N-glycosylation, S recombinant baculovirus infected cells were treated with tunicamycin from 0-48 hr post infection. Following tunicamycin treatment most of the 68 and 70 kDa species were 20 replaced by two bands with apparent molecular weights of 64 and 66 kDa (FIGURE 2a, lane This indicates that the 68 and 70 kDa polypeptides both contained N-linked sugars. The change in gE mobility is consistent with the presence of 2 potential N-linked glycosylation sites in the gE polypeptide as determined by sequence analysis The tunicamycin treated gE size is also compatible with the reported molecular weight of 66 S 25 kDa for in vitro translated gE (12).

Indirect immunofluorescence staining of gE-baculovirus infected cells was done using Fdl72 anti-gE monoclonal antibody. Strong cell surface fluorescence was seen similar to authentic HSV-1 gE suggesting that the expressed gE was transported to the cell surface.

D. HSV-1 neutralisation by serum from gE vaccinated mice The immunogenicity of the recombinant gE was studied by immunising mice with lysates from whole insect cells infected with baculovirus-gE as described above. These mice were also used in the i.p. challenge study below. Three weeks after the final vaccination (and prior to the i.p. HSV-1 challenge described below), mice were bled and their sera were tested for HSV-1 neutralising activity. Pooled sera were heat-inactivated and then incubated with HSV-1 in the presence of fresh or heat-inactivated guinea pig complement. Sera from immunised mice was capable of high HSV-1 neutralising activity in vitro (FIGURE 3 and Table As illustrated in Table I below, neutralising activity was higher in the presence of fresh complement (solid circles) than heat-inactivated [Priv110001:OGSA 8 of 16 complement (open circles). In addition, on Western blots, sera from baculovirus-gE immunised mice reacted with authentic gE from HSV-1 infected Vero cells. Thus, our baculovirus expressed gE appeared able to induce an immune response in mice that was directed against authentic gE.

Table I. Induction of neutralising antibody and DTH in mice immunised with a recombinant baculovirus expressing HSV-I gE.

____Increased ear thicknessa (mm x 10- 2 Neut. Antibody Immunisation 24 hr 48 hr 72 hr C' C' vAc-gE1 7.0 7.4 14.8 >320 247 KOS 8.3 14.7 15.5 e >320 >320 mock (AcNPV) 4.8 5.6 8.1 <10 aMean S.D. of six mice/group.

bNeutralisation titers are expressed as the reciprocal geometric means of the dilution that produce a 50% reduction in plaque numbers.

Clncreased ear thickness of the vAc-gEl and KOS vaccinated mice at 72 hr. were statistically different from mock vaccinated mice by the Student t-test (p<0.001).

E. Induction of DTH response in gE vaccinated mice To study the delayed type hypersensitivity (DTH) response to gE glycoprotein, groups of 6 mice were vaccinated three times as above and DTH responses determined as described above. Both gE and KOS vaccinated mice developed OTH responses. Ear swelling peaked at 72 hr post challenge (Table followed by a decline in swelling after Sday 3. At 72 hr the DTH response (ear swelling) in gE vaccinated mice was similar to that in HSV-1 vaccinated mice and significantly higher than the swelling in wild-type baculovirus mice (p<0.001).

F. Protection of mice from HSV-1 challenge by vaccination with gE Vaccinated mice were challenged by i.p. injection of HSV-1 strain McKrae (2 x 106 PFU) three weeks after the third inoculation. As illustrated in Table II below, 95% of Smice vaccinated with expressed gE (19 of 20) survived the lethal challenge, whereas only 39% of mock-vaccinated mice (7 of 18) survived. In addition, 100% of mice immunised with KOS were protected (11 of 11). Hence, the level of protection by the expressed gE was similar to that of KOS, and was significantly higher than the mock-vaccinated group (p<0.01).

Table II. Lethal intraperitoneal and ocular challenge of mice immunised with a recombinant baculovirus expressing HSV-1 gE.

Si.p. challengea Ocular challengeb Immunisation Survival/Total Survival/Total baculovirus-gE 19/20C 5/5 100%) KOS 11/11 C (100%) 5/5c (100%) mock (AcNPV) 7/18 0/5 [Priv1)00016:GSA 9 of 16 aMice were vaccinated three times as described above and challenged i.p. with 2 x 106 PFU!mouse. Numbers in parenthesis are percent survival.

bMice were vaccinated twice and challenged ocularly with 2 x 105 PFU/eye.

CSurvival rates (protection) of the baculovirus gE and KOS vaccinated mice were significantly different from the mock vaccinated survival rate (Fisher's Exact test, p 0.01).

To determine whether systematic vaccination with expressed gE could also provide protection against ocular infection, mice vaccinated twice were challenged ocularly with 2 x 105 PFU/eye of HSV-1 strain Mckrae three weeks after the last vaccination. The mice were monitored for ten days. Five of 5 mock vaccinated mice (100%) developed significant eye disease and died following HSV-1 challenge. In contrast, all 5 of the gE and all 5 of the HSV-1 vaccinated mice survived and developed no apparent eye disease (see Table II). These results suggest that immunisation with baculovirus expressed gE can protect mice against both i.p. and ocular lethal HSV-1 challenge.

In summary, the present invention involves the high level expression of gE in a baculovirus expression system. The gE expressed in this system was glycosylated and transported to the cell surface. Antibodies raised in mice against the expressed gE neutralised the infectivity of HSV-1 in a partially complement dependent manner, and mice vaccinated with gE developed a DTH response to HSV-1. More importantly, mice systematically vaccinated with gE were protected from lethal intraperitoneal and lethal 20 ocular HSV-1 challenge, making gE a useful and important component in any subunit vaccine against HSV-1.

G. Purification Of gE The baculovirus expressed. E of the present invention may be purified for human use according to standard techniques including but not limited to, immunoaffinity 25 chromatography (15) and collection of secreted truncated gE from the supernatant medium of cell cultures (16).

1. Immunoaffinity Chromatography The procedures for immunoaffinity chromatography are as set forth in Essentially, the gE protein was purified in roller bottles by sequential steps of lentil lectin chromatography, immunoaffinity chromatography and concentration by ultrafiltration.

For the first step, 2 litres of conditioned medium was supplemented with ImM PMSF and 0.5% iprotinin and then loaded onto a 30mL column of lentil lectin-Sepharose-4B (Sigma Chemical Co., St. Louis, Mo.) at a flow-rate of 50mL/h. The column was washed sequentially with 100mL of PBS and 100mL of PBS containing 0.5 M NaC1. The bound fraction was eluted with PBS containing 0.5M NaC1, 0.5 M a-methylmannoside, 0.1% Triton X-100, and 0.5% aprotinin, and fractions were assayed for gE by enzyme-linked immunosorbent assay (ELISA).

The peak column fractions were pooled and applied to a 10mL immunoaffinity column prepared by linking 70mg of a rabbit anti-gE polyclonal antibody to cyanoger IPriv110001 6:GSA 10 of bromide-activated Sepharose 4B. The gE-specific rabbit antiserum was raised against gE protein, which was purified by preparative SDS-polyacrylamide gel electrophoresis from HSV-l infected Vero cell lysates. Prior to coupling, an IgG-enriched fraction was prepared from the gE-specific rabbit antiserum by precipitation with 33% saturated ammonium sulfate. Following application of the !ectin column eluate to the immunoaffinity column, the column was washed consecutively with 20mL of 10mM Tris hydrochloride, pH 7.5, and lOmL of LB without SDS and BSA and then with 30mL of Tris hydrochloride, pH 7.5-0.5M NaC1. The bound fraction was eluted with 3M ammonium thiocyanate, pH 7.5, and the gE protein peak was detected by ELISA and Western analysis. The peak fractions were concentrated and equilibrated in storage buffer (100mM NaCl, 10mM Tris hydrochloride, pH 7.5, 1mM EDTA, 7.5% glycerol) by ultrafiltration with a PM10 membrane (Amicon Corp., Danvers, Mass.). To remove protein absorbed to the membrane surface, the membrane was washed with storage buffer plus 0.1% Triton X-100, and this wash was then combined with the initial concentrated fraction.

2. Collection of the secreted truncated gE Procedures for the collection of the secreted truncated gE are described in (16) and will n -t be repeated here. However, basically, the fragment encoding the transmembrane anchor sequence was excised from the gE gene. The deleted gE gene was then reconstructed by self-ligation to put in frame sequences coding for the extramembrane ard C-terminal intracytoplasmic domains. The product was detected after transfection by immuno-precipitation of the supernatant medium of cell cultures with anti-gE monoclonal antibody.

H. Pharmaceutical Compositions The gE of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions in almixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described for example in Remington's Pharmaceutical Sciences by E.W. Martin. These compositions will contain an effective amount of gE together with a suitable amount of vehicle in order to prepare pharmaceutically acceptable compositions suitable for effective administration to the host.

For purely descriptive and not limiting purposes, an example of a pharmaceutical preparation of gE for parenteral administration prepared according to the present invention is described.

The vaccine may be supplied as a single dose vial of lyophilised baculovirus expressed HSV-1 gE, alone or in combination with one or more immunogenic HSV-1 glycoproteins, and a vial of diluent with alum. Alternatively, the vaccine may be supplied in a multidose vial, and a vial of diluent with alum.

The invention being described, it is clear that these methods can be modified, which IPrIv1100015:GSA 11 of modifications do not diverge from the spirit and purpose of the invention and which would be apparent to one skilled in the art. It is therefore understood that the present invention is not to be construed as limited to such, but rather to the lawful scope of the appended claims.

References 1. BLACKLAWS, KRISHNA, MINSON, A.C. and NASH, A.A.

Immunogenicity of Herpes simplex virus type 1 glycoproteins expressed in vaccinia virus recombinants. Virology, 177: 727-736 (1990).

2. PARA, BAUCKE, R.B. and SPEAR, P.G. Glycoprotein gE of Herpes simplex Virus Type 1: Effects of anti-gE on virion infectivity and on virus induced Fcbinding receptors. J. Virol., 41: 129-136 (1982).

3. DUBIN, FRANK, and FRIEDMAN, H.M. Herpes simplex virus type 1 encodes two Fc receptors which have different binding characteristics for monomeric immunoglobulin G (IgG) and IgG complexes. J. Virol., 64: 2725-2731 (1990).

4. SUMMERS, M.D. and SMITH, G.E. A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin #1555 (1987).

5. ROCK, NESBURN, GHIASI, ONG, LEWIS, T.L., LOKENSGARD, J.R. and WECHSLER, S.L. Detection of latency related viral RNAs In 20 trigeminal ganglia of rabbits latently Infected with Herpes simplex virus type 1. J. Virol., 61: 3820-3826 (1987).

6. GOLDIN, SANDRI-GOLDIN, LEVINE, M. and GLORIOSO, J.C. Cloning of Herpes simplex virus type 1 sequences representing the whole genome.

J. Virol., 38: 50-58 (1981).

25 7. LASKY, DOWBENKO, SIMONSEN, C.C. and BERMAN, P.W.

Protection of mice from lethal Herpes simplex virus infection by vaccination with a secreted form of cloned glycoprotein D. BioTech., 4: 527-532 (1984).

8. GHIASI, KAIWAR, NESBURN, A.B. and WECHSLER, S.L.

S Immunoselection of recombinant baculoviruses expressing high levels of biologically active Herpes simplex virus type 1 glycoprotein D. Arch. Vir., In press (1991a).

9. CHATTERJEE, S. KOGA, J. and WHITLEY, R.J. A role for Herpes simplex virus type 1 glycoprotein E in induction of cell fusion. J. Gen. Virol., 70: 2157-2162 (1989).

NASH, FIELD, H.J. and QUARTEY-PAPAFIO, R. Cell-mediated immunity in Herpes simplex virus infected mice: Induction, characterisation and antiviral effects of delayed type hypersensitivity. J. Gen. Virol., 48: 351-357 (1980).

11. MCGEOCH, DOLAN, DONALD, S. and RIXON, F.J. (1985).

aequence determination and genetic content of the short unique region in the genome of Herpes simplex Virus Type 1. J. Mol. Biol., 181: 1-13 (1985).

IPriv1100015:GSA 12 of 16 13 12. LEE, PARA, M.F. and SPEAR, P.G. Location of the structural genes for glycoproteins gD and gE and for other polypeptides in the S component of Herpes simplex Virus Type 1 DNA. J. Virol., 43: 41-49 (1982).

13. BELL, CRANAGE, BORYSIEWICZ, L. and MINSON, T. Induction of immunoglobulin G Fc receptors by recombinant vaccinia viruses expressing glycoproteins and I of Herpes simplex virus type 1. J. Virol., 64: 2181-2186 (1990).

14. MANIATIS, FRITSCH, E.F. and SAMBROOK, J. Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

PACHL, BURKE, STUVE, SANCHEZ-PESCADOR, L., VAN NEST, MASIARZ, F. and DINA, D. Expression of Cell-Associated and Secreted Forms of Herpes simplex Virus Type 1 Glycoprotein gB in Mammalian Cells.

Jnl. Virol., 61(2):315-325 (1987).

16. MANSERVIGI, GROSSI, GUALANDRI, BALBONI, P.G., MARCHINI, ROTALA, RIMESSI, DI LUCA, CASSAI, E. and BARBANTI-BRODANO, G. Protection from Herpes simplex Virus Type 1 Lethal and Latent Infections by Secreted Recombinant Glycoprotein B Constitutively Expressed in Human Cells with a BK Virus Episomal Vector. Jnl. Virol., 64(1):431-436 (1990).

S

oooo« *ooo IPrlv1100016:GSA 13 of 16 INFORMATION FOR SEQ 11D NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1822 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATGGATCGCG GGGCGGTGGT GGGGTTTCTT CTCGGTGTTT 0..0

GGAACGCCCA

GCTCCGGGGC

GATGGGTGCG

GAGACGGTCG

CCCCCGGCCC

GCCGTGGTTA

ACCCTGTCCG

GTGCAACCGG

AATGACGAGG

CCGCCTCCCC

GGGGTGACCG

AGCACGAACG

GTCTGGTTGA

AAACGTCCTG

CTACGGGGCG

GCCCCTTACA

TGGATGCGGC

CATCTGCGA.C

ACCGGAGTCT

TGGGCGACAT

CCCCAGTTCC

GCGAGGACGA

ccGccccccc

TGCGTATGGA

TCTCCATCCA

GGTTCGACGT

GAGACGGGTG

CGGCCCGACC

CCCGTCGTGG

GTGCATGCGC

CGGGGGTCTA

GGTTATTCAC

AAAGGACCCG

GACCCCACCC

AAGTCTCGCC

GAGGTCTTGG

GACTCCGGAA

TGCCATCGCC

GCCGACCTCG

AGTGTCGGCG

CAGAAACTAC

GTCTCGCTGA

GCTCCGGTCC

CGAhCGGACT

GGGGTCCGAG

GCTCGCCAAG

CCGACCCCAG

GGCACTCCCG

CCCAGCGCCC

GCTATCCTGT

CACGACGACC

TGTGCCGAGA

GTGTTGTATC

AGGACGTTTC

TATGGGCCGT

TGCCCCCCAA

CGCTGGCGA4-

TCGT',TGGCA

AGACGGACAG

TGGCCTCGGT

CCGATTACGA

CCAGCGGGAC

CCGAAGTCTC

TTTCCCCCGG

AGACCTACTC

TGCGAATATA

GTGCTTGGCG

GTTGCTTCCA

GGAACCCCTG

GCAGGTGCCC

GGCGTACGCC

GGAGCGCGCG

CGGCCTGTAT

GGTCCTGGTG

CGAGGATGAC

CCCCCGGCTC

ACATGTGCGT

GGAGACGTTC

CATGGACGTC

CGAATCGTGT

[Privi 1000 1 6:GSA 1 f1 14 of 18

CTGTATCACC

ACGTGGACGT

CCGCGCTGTT

TCCGTCAATC

GTGTACGTCA

TACCGGAACG

ACCCACCCGC

GGGGCGGTGA

ATGACCTGTT

CCCACGTACA

GGAGAACGCG

AATGG;ATCCG

GGGCATCAAT

TCCCAGGCCT

CTCC(,'CCGAAC

CGACGAACGC

GCCACACCCG

CGCAGCTCCC

CTCGCCTGGC

CGGCCGAGGC

TGGAGTTCCG

ACGACCATAT

CGGTGGTGGA

ACGTCGGGGC

TGGGGGCCGC

GGCGCAGGCG

TTCGCGTGGC

ACCAGGTCCC

GCTTTGAGAT

CTCGCCGCCA

CCGATTCGTC

TGGGCGACCG

GGACCCCCCC

CGACCCCCGG

AGAATGTCTG

CGTCCGCAGC

TCACATGGAG

GGACGCGTCC

TCACGCCTGG

ACAGCCCCTC

CCCTCCCCAC

CCTGCTGCTG

TGCCTGCCGG

CGACAGCGAG

GTGGCTGGCC

CTTATCACCA

GCTCACAACC

CGTCTTCTGG

CC(3GCGAGGT

GRCATGACCG

GC

TCCCCGGCCG

TACGCGGGGT

CCCGTCCCGG

CCACAACACT

GGCCACATTA

CCACAGCGCG

GCGCCCCCAA

TCTGCACTGG

CCGGTTAAAA

CTGTACGCGG

CCCCCGGAGA

ACGGCTCCGT

TTTGGATCCG

TAAGGCGCCC

GGACGTCGGA

CCCGCCCCTC

ACGCGCCGTG

GTTCCAGAAC

GGCTGGCGTG

CCGGCCTGTA

CCATCAGCAC

GCGCGGATTT

CCCACGGCGC

GGMTTCGGT

GCAGGGCCTC

ACTGGAGCTC

GACCCGACTC

CTGTATACCC

GAAGGCCCGA

CATCCCGAGG

GACGAGCTAA

GCCACGTCGA

CGCCGCGAGT

AAACCCCCCA

GCAGGCGGCC

TCTGTGTGTG

CGC(;GCGCAG

GGCCGAGCCC

CCTGCGGTTA

GTGGGCGTGT

GGGTAAGGGG

GGACP%.GCGAG

TCCCTCCACC

CCGTAGCGAT

TCGCCGTTAC

CCCCACGTCG

TCGCGATTTC

CCGCGCCCTC

900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1822 te.

55

S

S. 55 S S [PrivlIOO016:GSA 1 f1 16 of

Claims (16)

1. A recombinant baculovirus wherein the genetic construct comprises: a) a gene sequence encoding HSV-1 gE or a biologically active derivative thereof; and b) a polyhedrin gene promoter sequence wherein said gE gene sequence is functionally linked to the regulatory elements of said polyhedrin promoter.

2. A recombinant baculovirus according to claim 1 which is capable of expressing HSV-1 gE by cell infection in a culture medium.

3. A recombinant baculovirus substantially as hereinbefore described with reference to any one of the Examples.

4. A recombinant baculovirus transplacement vector containing the genetic construct as characterised in claims 1 or 2 and being capable of introducing it into the baculovirus genome. A recombinant baculovirus transplacement vector which is pAc-gEl.

6. A recombinant baculovirus transplacement vector substantially as hereinbefore described with reference to any one of the Examples.

7. A process for the preparation of a recombinant baculovirus according to any one of claims 1 to 3, comprising introducing a recombinant baculovirus transplacement vector according to any one of claims 4 to 6 and a receptor baculovirus into a host cell, allowing a transplacement of -aid genetic construct from said recombinant baculovirus transplacement vector to said receptor baculovirus, and isolating the recombinant baculovirus of any one of claims 1 or 2.

8. A process for the preparation of a recombinant baculovirus substantially as hereinbefore described with reference to any one of the Examples.

9. A recombinant insect cell containing a recombinant baculovirus according to any one of claims 1 to 3 and being capable of expressing said HSV-1 gE.

10. The recombinant insect cell according to claim 9 which is derived from Spodoptera Frugiperda.

11. A process for obtaining biologically active HSV-1 gE, comprising culturing a to recombinant cell according to claims 9 or 10 and recovering said HSV-1 gE from the culture.

12. The process of claim 11 wherein said HSV-1 gE is obtained without a contamination by other products or proteins.

13. A biologically active HSV-1 gE obtained according to the process of claims 11 or 12 and being free from other products or proteins.

14. A pharmaceutical preparation for the treatment or prevention of HSV infection containing a biologically active HSV-1 gE according to claim 13. A pharmaceutical preparation according to claim 14 for administration to humans or animals.

16. A pharmaceutical preparation according to claim 15 which can be administered [Priv 100016:GSA 16 o 18 17 by the parenteral route.

17. A pharmaceutical preparation according to claim 15 which can be administered by the enteral route.

18. A pharmaceutical preparation according to claim 15 which can be administered by the ocular route. Dated 3 March, 1993 Cedars-Sinai Medical Center Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON *too see#0 .06 *$a (PrIvI1tOOOI6:GSA 1 ~1 17of IS Process for the Expression of Herpes simplex Virus Type I Glycoprotein E and Methods of Use Abstract A process for obtaining Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) from cells which have been infected or transformed with a recombinant Baculovirus is disclosed. The gE produced is then isolated and purified for use in immunotherapy against HSV infections. In the recombinant Baculovirus of the invention, the genetic construct comprises a recombinant baculovirus wherein the genetic construct comprises: a) a gene sequence encoding HSV-1 gE or a biologically active derivative thereof; and b) a polyhedrin gene promoter sequence wherein said gE gene sequence is functionally linked to the regulatory elements of said polyhedrin promoter. ee e e o S