WO1996017938A1

WO1996017938A1 - Glycoproteins from herpes virus and vaccines containing them

Info

Publication number: WO1996017938A1
Application number: PCT/EP1994/004058
Authority: WO
Inventors: Roberto Manservigi; Penelope Mavromara; Anna Boero; Rafaela Argnani; Silvia Zucchini; Silvia Sabbioni; Vivi Miriagou; Enzo Cassai
Original assignee: Bruschettini S.R.L.
Priority date: 1994-12-06
Filing date: 1994-12-06
Publication date: 1996-06-13
Also published as: EP0796333A1; AU1273495A

Abstract

The invention refers to the expression of glycoproteins D and G of herpes simplex virus type 2 and of glycoprotein E of herpes simplex virus type 1 from human cells. The invention refers also to vaccine formulation containing said glycoproteins.

Description

GLYCOPROTEINS FROM HERPES VIRDS AND VACCINES CONTAINING THEM

The present invention refers to the expression of a Herpes simplex virus type 2 (HSV-2) glycoprotein D and G and of a Herpes simplex virus type 1 (HSV-1) glycoprotein E by human cells, to their preparation and purification and to pharmaceutical compositions comprising the aforementioned proteins, useful for the prophylaxis and for the therapy of patients affected with recurrent herpetic infections from HSV-1 and/or HSV-2. The Herpes simplex virus type I and type II are ubiquitous in the human population. Generally, HSV-1 are responsible for primary gengivomastitis, recurrent perilabial Herpes, herpetic keratitis, encephalitis and cutaneous infection of the supra-umbilical region. HSV-2 support the infections localized at the female and male genital organs and at the skin of the sub- umbilical region. After the primary infection the virus remains in the body in a latency condition and it is subjected to periodical reactivations which may result in the asymptomatic release or in the recurrent infection.

The frequency of recurrences is very high: according to different epidemiological studies, from 16 to 45% cf adults of general population suffer from at least one herpetic relapse episode in the course of life. Only in the USA, 5-20 millions people suffer from genital herpetic recurrence and the incidence of the disease is continuously increasing also in other parts of the world; the genital pathology is particularly serious also because it makes the development of other sexually-transmitted diseases or the neonatal infection easier. The neonatal Herpes may be caused by both types of virus. The interest for the neonatal disease derives from epidemiological and clinical arguments. In fact, it is reported an increase in the frequency of genital infection and of neonatal Herpes. Moreover, the perinatal infection gives an anatomo-clinical pattern which, differently from what occurs for the primary post-natal infection and for the subsequent recurrences, involves an high death-rate.

It is therefore of high medical interest the availability of a safe and effective vaccine which can protect both from HSV-1 and HSV-2 , reducing thereby the number of recurrences and therefore the spread of pathology.

Several attempts have been made and a particularly promising approach involved the use of viral glycoproteins, whose genes have been cloned in suitable expression vectors and the corresponding products produced in bacteria, baculovirus, yeasts and mammalian cells. The immunization of test animals with these recombinant products and a number of different vaccine formulations turned out to protect from acute infection; however, no effect on the latent infection and on the recurrence frequency was in several cases noticed or the results turned out to be not reproducible on experimental models in monkeys and humans. Probably, since recurrences are caused by reactivation of endogenous latent virus rather than by exogenous re-infection, it is particularly important to stimulate an immune response similar to that occurring after natural infection. It is therefore evident the importance of the availability of recombinant glycoproteins produced in human cells.

The expression of the cloned genes in human cells provides, in fact, correct post-translational processes and the production of a protein as similar as possible to that expressed in the human viral infection. Particularly, glycosylation may influence the immunogenic properties of a protein.

The carbohydrates may directly confer antigenicity to viral glycoprotein by contributing specific antigenic determinants. They may affect availability of proteins epitopes by steric hindrance or lead to charge alterations. They may also influence the overall folding of the protein and therefore its three- dimensional conformation as a consequence of allosteric changes.

This confor ational effect of glucosylation can be critical for gB-1, since it contains discontinuous epitopes in the N-terminal domain. The proteins produced by cells of heterologous mammals are glycosylated in the correct sites, but differ in the oligosaccharides of the side-chains and in the fine structure; it is also known that the glycosidic compositions of the HSV-1 glycoproteins depend on the species of infected cells.

We have now focused our attention on the glycoproteins B, D, G and E as herpetic antigens for the preparation of the vaccine.

This interest is due to their crucial role in the initial step of infection, in the spread of the virus; moreover gB, gD and gE can induce a high neutralization titer, killer cell activity and protection against lethal challenge.

Glycoproteins B, D, G and E have already been obtained by recombinant DNA techniques from bacteria, baculovirus, yeast and CHO cells and their use has already been proposed (US 5149529, US 5171568, WO

85/04587, WO 88/02634) .

It has been also reported the expression in human cells of a type B glycoprotein lacking the transmembrane hydrophobic sequences as well as its protective activity against the HSV infection in mice

(J. Virol., 1990, 64, 431-436).

It has now been found that glycoproteins of the B, D, G and E type, lacking the transmembrane hydrophobic domains expressed by human cells transformed with suitable vectors have, with respect to the HSV glycoproteins up to now studied, remarkable advantages in the preparation of vaccines.

The glycoproteins type D and G from HSV-2 and type E from HSV-1 lacking transmembrane and intracytoplasmatic domains expressed by human cells are new and are a first object of the invention.

A further object of the invention is provided by a vaccine for human use, useful for prophylactic and therapeutic purposes, containing at least one of the glycoproteins D, G and E as defined above, in combination with a glycoprotein B lacking transmembrane hydrophobic sequences expressed in transformed human cells.

DEFINITIONS The abbreviation gB-ls refers to a recombinant glycoprotein wherein the transmembrane hydrophobic sequences (639 bp) have been excised by Espl digestion and the gene for gB-ls was reconstructed in frame with carboxy terminal sequences. The secreted protein consists of 690 aa.

The abbreviation gD-2s refers to a recombinant glycoprotein wherein the carboxy terminal region coding for transmembrane and cytoplasmatic hydrophobic domains has been deleted. The secreted protein consists of 343 aa.

The abbreviation gG-2s refers to a recombinant protein wherein the carboxy terminal region coding for the transmembrane and cytoplasmatic hydrophobic domains has been deleted. The secreted protein consists of 688 aa.

The abbreviation gE-ls refers to a recombinant glycoprotein lacking the last 144 aa in the carboxy terminal region but mantaining most of the extracellular domain. The secreted protein consists of 406 aa.

The abbreviations pRP-RSV and pRPneoCMV refer to the vectors used to express the recombinant glycoproteins.

These vectors comprise the replication origin and the early region of the human papovavirus BK. The presence of the BK sequences allows the vector to persist episomally and replicate in human cells.

The number of copies of the vector may change according to the cloned gene and generally the quantity of product is related to the number of copies and is highly dependent on cellular factors interacting with the replication origin of BK. Two potent eukaryotic promotors, such as LTR sequences of Rous sarcoma virus and the early cytomegalovirus promoter, and genes allowing the selection of transformed cells, such as the mouse dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate and the neo gene conferring resistance to the antibiotic G-418, have been inserted in these vectors. Since, viral sequences can be amplified using DHFR vectors, the treatment of the transformed clone with high concentration of methotrexate, increases the expression of the recombinant gB-ls of about 10-100 times.

The 293 line derives from human embryo kidney cells transformed with the type 5 Adenovirus DNA and costitutively expresses the early products of the viral genes E1A and ElB. The 293 cells are the best recipients for the expression of vectors based on BK since cellular factors enhance the vector amplification acting on the replication origin sequences of BK and E1A and ElB antigens are able to transactivate various viral promoters, such as those of Rous sarcoma and cytomegalovirus. Construction of the Recombinant Vectors

The HSV genome consists of two segments, named L (long) and S (short). Each segment consists of unique sequences (UL and US) enclosed between repeated and inverted sequences (TR and IR).

The genome of HSV-1 strain F was used to construct a library of BamHI fragments cloned in pBR322 and pBK-1 plasmids, while the genome of HSV-2 strain 333 was used to construct a library of Bglll fragments cloned in the pkC7 plasmid. The gene of the glycoprotein B type 1 (gB-1) is contained in the BamHI G fragment, the gene of the glycoprotein E type 1 (gE-1) in the BamHI J fragment , whereas the genes of glycoproteins D and G type 2 (gD-2 and gG-2) are contained in the fragment Bglll L (Figures 1, 3, 5 and 7).

Construction of a vector for the expression of gB-ls The DNA of HSV-1 strain F was digested with BamHI and the obtained fragments were separated on 0.8% agarose gel. The BamG fragment, containing the gB-1 gene, was then cloned in the pBK-1 plasmid to obtain a vector (pBK-MT-gBl) for the expression of the full length gB-1.

The gene coding for gB-ls was derived from pBK-MT- gBl changing after digestion with Espl (Figure 1). This enzyme recognizes two restriction sites in correspondence to the carboxy terminal anchor region of the glycoprotein, allowing the excision of 639 nucleotide fragment coding for the transmembrane region. The vector was then self-ligated to yield the gB-ls gene, coding for the extramembrane domains (amino-terminal) and intracytoplasmic (carboxy- terminal) .

The gB-ls gene was excised from pRP-MT-gBl by a double digestion with Xhol and Clal and inserted in the BamHI of the pRP-RSV vector. Since the gB-ls and vector ends are not compatible for ligation, they were blunt-ended by filling-in using the Klenow fragment of DNA polymerase I. The gene coding for gB-ls is found in the recombinant plasmid under the control of the Rous sarcoma promoter LTR and of the polyadenylation signals of the TK gene of HSV-1. The vector further comprises the replication origin and the early region of the papovavirus BK, allowing the vector to replicate and to persist episomally in human cells. The DHFR gene expressing a mutant enzyme resistant to methotrexate is also present in the vector, which can be used to amplify the recombinant vector in transformed cellular clones. Analysis of the gB-ls expression and isolation of stable clones

Figure 2A shows the results obtained from the short-term transfection experiments carried out on human cells HeLa and 293 and on monkey cells COS-7 , to analyze the ability of pRP-RSV-gBS to express the secreted glycoprotein. Sub-confluent cellular monolayers were transfected with the pRP-RSV-gBs DNA and after 48 hours the presence of gB-ls in the culture medium was assayed, by i munoprecipitation with monoclonal antibody 1-144 anti gB-1. As positive control 293 cells were infected with the HSV-1 strain F. The electrophoretic profile of the immunoprecipitate, compared with that of standard proteins, showed in HSV-1 (F) infected 293 cells a product at about 120 KD, corresponding to gB-1; a product of about 90 KD, corresponding to gB-ls, was observed in the 293 cells transfected with the recombinant plasmid. Expression of gB-ls was very low in COS-7 and absent in HeLa cells. Densitometric analyses showed that more than 95% of the product is secreted in the culture medium of 293 cells and that the amount of gB-ls is comparable to that of gB-1 synthesized in the 293 cells litycally infected by HSV-1 (F).

In order to obtain stable cellular clones constitutively expressing gB-ls, 293 cells were co- transfected with the pRP-RSV-gBs and p5V2-neo vectors; the latter confers resistance to G418 and allows the biochemical selection of the transformed clones.

Different stable cellular clones were isolated in the presence of G418 (300 μg/ml), expanded in massive cultures and analyzed by immunoprecipitation with the monoclonal antibody 1-144 anti-gB-1. As it is observed in Figure 2B, it is possible to detect in the surnatant of stable clones 9, 13 and 20 the product of 90 KD corresponding to gB-ls. A kinetic analysis of the production of gB-ls in the clone 20 (Figure 2 C) has shown that secretion starts between the first and the sixth hour after labelling with ³⁵S-methionine, reaches a peak at 24 hours to remain then constant up to 72 hours. From a direct electrophoretic analysis (without immunoprecipitation) of the culture media of the different stable clones, it was observed that gB-ls is the only protein regularly detected in considerable amounts. The amount of produced gB-ls was then detected by densitometic analysis of the protein bands obtained in gel polyacrylamide. The different clones expressed gB-ls in concentrations ranging from 0.5 to 0.25 μg/ml/10⁶ cells in 24 hours. In order to increase the expression of gB-ls, the clones expressing the recombinant antigen were propagated in the presence of methotrexate. It was in fact reported that cloned sequences in a vector containing the DHFR gene may be amplified in the presence of the drug. After different passages in the presence of increasing concentrations of methotrexate (0.6-6 μM) , the production of gB-ls increased by 10-100 times (Figure 2D).

Construction of a vector for the expression of gD-2s

The HSV-2 strain 333 was digested with Bglll and the obtained fragments were separated by 4% agarose gel electrophoresis. The Bglll L fragment, containing the gD-2 and gG-2 genes, was then eluted from the gel and cloned in the pKC7 plasmid (Gene, ]_, 79-82, 1979). The new recombinant vector pBglllL was then digested with Hindlll and Xbal and the 4.5 Kb fragment, containing the gD-2 gene, was cloned into the pUC19 vector. The obtained intermediate vector, named pgD2 , was finally used to obtain the sequences coding for the gD-2 secreted (gD-2s) of 343 aa .

The pgD2 vector was digested with Spel and BssHI and the 1.07 Kb fragment, containing gG-2s, was cloned in the Xhol site of the expression vector pRP-RSV (Figure 3) .

The obtained vector, named pRP-RSV gD2s was then used to transfect human cells of the 293 line and to obtain stable clones able to constitutively express gD- 2s. Analysis of the gP-2s expression and isolation of stable clones

In order to evaluate the ability of the pRP-RSV gD2s vector to express the secreted protein, short term transfection tests were carried out in human 293 cells, using 10 μg of the vector DNA. After 36 hours from transfection, the culture medium of the transfected cells was substituted with a serum-free medium and after 12 hours incubation the medium was recovered and concentrated 10 times by ultracentrifugation. The concentrated medium was contacted with the monoclonal antibody anti-gD2 III 174. The immunoconjugate was then eluted on polyacrylamide gel and the gD-2 expression was evaluated by immunoblotting. The results of the short term transfection were positive and showed that using pRp-RSV gD2s vector it is possible to obtain the expression of secreted gD-2.

In order to obtain stable cellular clones constitutively expressing gD-2s, 293 cells were co- transfected with the pRP-RSV gD2s (10 μg) and pSV2 neo (1 μg) vectors; the latter confers resistance to G418 and allows the selection of the transformed clones. Different stable cellular clones were isolated in the presence of G418 (300 μg/ml), expanded in massive cultures and analyzed by immunoprecipitation with the anti gD-2 monoclonal antibody III 174.

As shown in Figure 4. the 52 KD product corresponding to gD-2s may be found in the supernatant of clones 1, 2, 4, 5, 7 and 9. The amount of produced gD-2s was measured by densitometric analysis of the protein bands obtained in polyacrylamide SDS gel. The different clones expressed gD-2s in concentrations ranging from 0.1 to 0.5 μg/ml/10⁶ cells in 24 hours. The gD-2s expression increases by about 10-100 times propagating the cells in the presence of increasing methotrexate concentrations.

Construction of a vector for the expression of gG-2s

The pBglllL plasmid, containing the cloned Bgllll fragment of HSV-2, was digested with BamHI and the 4.5 Kb fragment obtained was cloned in the BamH site of the pUC19 vector. Starting from this intermediate vector, named pgG2, the expression vectors for membrane gG-2 (gG-2) and secreted glycoprotein (gG-2s) were constructed.

In the first instance, the pgG2 vector was digested with BstYl and a 4686 bp fragment was isolated, to which the linkers for Xhol were added. After Xhol digestion, a 2506 bp fragment, corresponding to the structural gene gG-2, was obtained. This fragment was cloned in the single Xhol site of the expression vector pRPneoC V.

To obtain the vector comprising the sequence coding for gG-2s an intermediate molecule, named pXgG2 , was constructed deriving from the pgG2 vector after digestion with Xbal and deletion of a 2.0 Kb fragment. This recombinant molecule was then digested with BstYI and Hindlll and the 2160 bp fragment coding for gG-2s was eluted. This fragment, after addition of the linkers for XChoI , was cloned in the single Xhol site of the pRPneoCMV expression vector (Figure 5). The recombinant plasmid named pRPneoC VgG-2s was used to transfect 293 cells and to obtain stable clones able to constitutively express gG-2s.

Analysis of the gG-2s expression and isolation of stable clones

To evaluate the expression of gG-2s , short term transfection tests were carried out in human 293 cells, using 10 μg of the vector DNA. After 36 hours from transfection, the culture medium of the transfected cells was substituted with a serum-free medium and after 12 hours incubation the medium was recovered and concentrated 10 times by ultracentrifugation.

The pRPneoCMVgG-2 transfected cells were collected in a suitable buffer and sonicated. The concentrated medium and the cellular lysate so obtained were contacted with the anti gG-2 monoclonal antibody H1206- 3. The immunoconjugate was then eluted on polyacrylamide gel and the gG-2s expression was evaluated by immunoblotting using the same antibody.

The results of this short-term transfection were positive and showed that it is possible to obtain with both vectors a good expression of the glycoprotein in

293 cells in both the secreted and membrane form thereof.

To obtain stable cellular clones constitutively expressing gG-2s, 293 cells were transfected with 5 μg of DNA of the pRPneoCMV vector and G418 resistant clones were selectioned and selection was mantained for

15-20 days.

Drug resistant cellular clones were isolated from the mono-layer in degeneration, transferred into micro- wells and finally expanded in T25 flasks.

The stable clones were then analyzed for the gG-2s expression, in immunoblotting tests using the H1206-3 antibody. As control, the lysate of HSV-2 strain G infected cells was immunoprecipitated, so as to compare the molecular weight of the gG-2 produced during the viral infection with that obtained from the stable clones (Figure 6). 3 out of 20 analyzed clones expressed gG-2s. The amount of secreted gG-2s was evaluated by means of densito etric analysis or polyacrylamide gel stained with the silver stain technique. The different clones were able to secrete from 0.10 to 0.20 μg/ml of gG-2s for 10⁶ cells in 24 hours. Construction of a vector for the expression of gE-ls

The coding sequences for the HSV-1 gE (aa 1 to aa 501) were taken from plasmid ρRB123 which contains the BamHI J fragment of the HSV-1 (F) DNA cloned into the BamHI site of pBR322 (Post et al., 1980). Plasmid pHPI400 was constructed by cloning the 2.1 Kb Nrul- Ba HI fragment from pRB123 carrying most of the gE coding sequences into the HincII-BamHI site of pGEM- 3Zf⁺ (Promega). The 1.3Kb Apal-Apal fragment from pHPI400 was treated with T4 DNA polymerase and blunt end ligated into the Smal site of pUC19 to yield pHPI402 (Figure 7). This Apal-Apal fragment, relative to the transcriptional initiation site for the gE-1 gene, extends from nucleotide -40 to +1291. For the expression of the gE-1 in E. coli the pMAL-C2 (New England Biolabs) expression vector was used to construct plasmid pHPI427. The 980 bp Sphl-SphI fragment from pHPI402 containing the gE-1 DNA sequences from nucleotide +338 to +1291, relative to the transcriptional initiation site for gE-1, was given blunt ends with T4 polymerase and ligated into the XmnI site of the pMAL-C2 vector. For expression of gE in eukaryotic cells the episomal replicating vector pRP- RSV (Manservigi et al., J. Virol. 1990, 6±, 431-436) was used to construct plasmid pHPI404. The 1.3Kb Apal- Apal fragment from pHPI400 was ligated into the BamHI cloning site of the pRP-RSV vector. Both fragment and vector were given blunt ends before ligation. The resulting pHPl404 plasmid contains a truncated gE-1 gene lacking the last 430 nucleotides.

Analysis of the gE-ls expression and isolation of stable clones

The 239 gE-ls stable cell lines were obtained by cotransfection of 239 cells with pHPl404 (20 μg) and pSV2neo (2 μg) plasmid DNA using the calcium phosphate- DNA coprecipitation method. Forty-eight hours post- transfection the cells were split at 1:10 dilution in DMEM medium containing 10% fetal calf serum and G-418 at a final concentration of 400 μg/ml. After 10 days, individual neo ycin resistant clones were isolated by the serial dilution technique and tested for expression of gE-ls protein by immunoprecipitation with V3 polyclonal antiserum as described below. The ability of the pHPI404 plasmid to express gE- ls was initially checked in a transient assay with 293 cells and the synthesis of gE-ls was detected by Western blot analysis 40h after transfection (data not shown). To obtain stable expression of gE-ls, plasmids pHPI404 and pSV2-neo were cotransfected into the 293 cells and stable transformants were selected based on the neo⁺ phenotype. Several individual G418 resistant clones were isolated and tested for constitutive expression of the gE-ls by immunoprecipitation. Specifically, cells were labeled for 2h with ^->S- methionine and the proteins from cell lysates and culture medium were immunoprecipitated with the anti- gE-ls fusion protein antiserum (V3). As shown in figure 8, a protein of 58 kDa in size was present in the culture medium of clones A' 3-9 and A' 4-15 and reacted specifically and very strongly with the antibody. This protein was present only in trace amount in the cell lysates and its relative mobility on PAGE was higher than that predicted for the amino acid sequence, indicating that the secreted gE-ls was modified during its transport through the exocytic pathway. In addition, a second band of approximately 45 kDa was consistently present in the immunoprecipitate derived from cell clone lysates but was absent in the mock- treated cells (figure 8). This band most likely represents a cell-associated immature form of the gE-ls protein. The gE-ls was the only protein detected in considerable quantities in the serum-free culture media of the above cell line when were analyzed by Coomassie blue staining. The A' 4-15 clone, that expressed the highest amount of gE-ls, was chosen for further studies. Preparation and purification of the vaccine.

The cellular clones n. 20, 9, 8 and A' 4-15, respectively expressing the largest amounts of gB-ls, gD-2s, gG-2s and gE-ls, were grown on multilayers culture vessels with Iscove medium added with 10% foetal bovine serum.

When cells reached confluence, the culture medium was substituted with serum-free medium (production medium) which was collected after 48-72 hours of incubation at 34-37^βC. A specific production of about 200 ng/10⁶ cells in 24 hours was obtained. The concentration of the four glycoproteins has been determined by ELISA technique, using suitable standards and monoclonal and polyclonal antibodies anti gB-ls, gD-2s, gG-2s and gE-ls.

The recombinant cellular clones were also adapted to grow in a bioreactor at a cellular concentration of about 6 x 10 cells/ml using serum-free medium. A specific production of about 500 ng/10⁶ cells in 24 hours was obtained. The glycoproteins were purified from the culture medium by means of sequential passages on chromatographic columns, e.g. lenthyl lectine and affinity columns and concentrated by ultrafiltration.

For example, about 2 litres of medium added with protease inhibitors (PMSF and aprotinine) were eluted through a 30 ml column of lenthyl lectine or of sepharose-4B conjugated concanavalin A with a flow of 50 ml/h. After a number of washings, the glycoproteins were eluted with PBS containing 0.5M NaCl-0.5M a- methylmannoside-0,1% Triton and protease inhibitors. The presence of glycoproteins in the fractions was analyzed by immunoenzymatic methods such as ELISA and Western blotting. The pooled fractions containing the desired glycoprotein were further purified by passage through an immunoaffinity column, constituted by monoclonal antibodies bound to activated sepharose-4b. After various washings of the column with suitable buffers, the glycoproteins have been eluted with 3 M ammonium thyocyanate, pH 7.5, and analyzed by ELISA and Western. The fractions containing the desired glycoprotein were finally concentrated by ultrafiltration with an Amicon PM 10" membrane. The total glycoprotein concentration was evaluated by electrophoresis in the presence of preset amounts of albumine and/or using kit for the protein analysis (Bio-Rad).

The so obtained glycoproteins turned out to be remarkably active in protecting animals infected by HSV.

It is particularly surprising that the above described proteins show an activity, when administered associated to an adjuvant such as aluminium hydroxide or phosphate, substantially comparable to that obtainable using more effective adjuvants having however toxicity problems or anyhow not accepted for clinical use such as muramyldipeptide.

The invention provides therefore evident applicative advantages in comparison to the prior art. The vaccine of the invention may contain: suitable adjuvant substances, preferably aluminium hydroxide, aluminium phosphate or other adsorbent approved for human administration according to different pharmacopoeias, in doses ranging from

0.05 to 1.25 g aluminium/dose; - suitable preserving agents, e.g. thimerosal 0.05 mg/ l; possible conventional excipients. The subcutaneous administration is preferred to the oral and to the parental route.

The precise dosage will depend on patient's characteristics (weight, age, health), pathology (spread, seriousness) and on the used formulation. Generally, an effective dose of glycoproteins above defined for an adult weighing 70 kg ranges from 0.01 μg/dose to about 0,1 mg/dose, preferably from 0.1 to 100 μg/dose. The composition may be administered in different steps, so as to induce a suitable immune response. Protection tests in animals

It has already been shown (Table 1) that gB-ls effectively protects rabbits from herpetic keratitis not only when administered emulsified with an equal volume of Freund complete adjuvant but also absorbed on aluminium hydroxide, a by far less effective adjuvant which proved to be insufficient in most instances (J. Immunol. Methods 124(1): 95-102, 1989; Science 227: 1490-1492, 1985; J. Immunol. 141(5): 1720-1707, 1988; Abstract n. 266 in: "Program and Abstracts of the ll^tn International Herpesvirus Workshop. University of Leeds, Leeds, U.K., 1986; Infect Immun. 41: 556:562, 1983; Antiviral Research 11: 203-214, 1989; 17^th International Herpesvirus Workshop, August 1-7, 1992 Heriot-Watt University Conference Centre - Edimburgh Lothian Region Scotland, p. 388).

This finding is particularly relevant since the aluminium compounds are the only adjuvants approved for human use.

The same results were obtained repeating the tests of protection from lethal challenge with HSV-1 in mice (Table 2). 2 Month old female Balb/c mice were immunized on day 1 by intraperitoneal injection of 10 μg of gB-ls + Freund complete adjuvant (group 1) or 10 μ of gB-ls + alum (group 2); on day 25, immunization was repeated with the same amount of gB-ls formulated in incomplete Freund adjuvant (group 1) or with alum (group 2). Control animals were injected i.p. with culture medium of normal 293 cells in the same way as animals receiving gB-ls (group 3) or a non-lethal dose (1/10 of LD₅₀) of HSV-1 LV into the footpad on day 1 (group 4).

On day 50 lethal challenges of HSV-1 strain 13 were injected i.p. and death-rate was observed for 20 days.

The mice immunized with both preparations of gB-ls achieved complete protection, identical to that obtained with viable virus, whereas no animal of group 3 survived. In a further test, the activity of the glycoproteins expressed by human cells was compared to that of glycoproteins expressed by bacteria, therefore lacking the post-translational changes playing a major role in human infections. In particular, gE-ls was tested in comparison to a form of the glycoprotein E expressed in E. coli (bgE-ls) in the protection of mice from HSV-1 lethal challenge. The immunogenic activity on the humoral immune response was also evaluated by measuring the serum neutralizing activity. Mice were immunized with two i.p. injections of 5 μg of protein with complete Freund adjuvant on day 1 and with incomplete adjuvant on day 25. The controls were treated analogously with the culture medium of 293 normal cells or with a single administration of a non- lethal dose of HSV-1 (F) in the footpad. One week before challenge, the blood was sampled for the dosage of specific antibodies by i_n vitro neutralization test (HSV-1 F, Vero cells): the antibody titers (50% plaque reduction) were expressed as the reciprocal of serum dilutions. The results are summarized in Table 3 and show that glycoproteins expressed by human cells gave an almost complete protection (only one dead animal in the gE-ls group), similar to that provided by the alive virus, whereas bgE-ls, expressed in bacteria, gave only a partial protection.

In a test of therapeutical vaccination, adult female outbred Hartley guinea-pigs, weighing 350-400 g, were infected intravaginally with 5x10 pfu of HSV-2 virus MS strain (ATCC VR-540) on day 0. The infection was treated with 5 mg/kg of acyclovir, in the drinking water, from Dl to D10. Death-rate, severity and persistance of primary lesions were then recorded and the infected animals were randomly subdivided into 4 groups of 12 animals each which were subjected to the following treatments: group 1: no treatment; group 2: culture medium of non-transformed 293 cells; group 3: gB-ls + gD-2o + gG- 2s + gE-ls; group 4: as group 3 plus alum. The dosage was 0.3 μg of protein subcutaneously on days 21, 28, 35, 42, 49, 63, 77 and 91. An observer unaware of the treatment recorded daily from day 21 to day 120 the presence of any recurrences (defined as episodes preceded and followed by at least one day without lesions), the site, number and seriousness of the lesions (0 ■ no symptoms; 1 - swelling and/or erythema; 2 - few small vesicles; 3 = many large vesicles; 4 _* ulcerated and soaked lesions).

The results are shown in Table 4 as N^β of animals with recurrences, number of recurrences after 99 days of observation, total score of the severity of recurrences (sum of the 99 days scores) and show that the vaccination remarkably improved the recurrence pathology. Experiments on the immune response in human cells

Immunogenicity of gB-ls was analysed by testing its ability to stimulate cell-mediated immune response of immune donors. The medium used was RPMI 1640 (Gibco, Paisley, Scotland) supplemented with 2mM L-glutamine, 1% nonessential amino acids, ImM sodium pyruvate, 50 μg/ml gentamycin and 5% human serum (RPMI-HS) or 10% fetal calf serum. For TCC growth, RPMI-HS was supplemented with human recombinant interleukin 2. Results, shown in Table 5, demonstrate the ability of gB-ls to induce high levels of proliferation of peripheral blood lymphocytes (PBL). CD4+, HSV-specific , HLA class II-restricted T cell clones (TCC) were generated from immune donors which had been previously stimulated with gB-ls. The specificity of TCC was defined as the capacity to proliferate in response to autologous, irradiated, Epstein-Barr virus induced lymphoblastoid cell lines pulsed with gB-ls. The marmoset cell line B95.8 (ATCC), which produces EBV, was grown in RPMI-FCS. The virus containing supernatant of overgrown cultures was collected, filtered through a 0.2 μm membrane filter and stored at -80*C, until use. PBL were incubated with EBV containing supernatant (1/4) in the presence of 600 ng/ l cyclosporin A (CSA) in RPMI-FCS. When the wells contained large aggregates of EBV-transformed B cells, they were pooled and mantained by serial passages in RPMI-FCS.

TABLE 1

Keratitis severity days after infection

10 12 18

controls 2.0 3.0 4.8 jτ. gB-ls + CFA 0.6 0.5 0.2 0.0 0.0 0.0 0.0 gB-ls + alum 0.8 0.4 0.3 0.0 0.0 0.0 0.0 r

Keratitis severity was scored from 0 to 5 according to

0 - clear cornea, no fluorescein dying

1 - spotted keratitis

2 -. dendriditic keratitis affecting <25% of the corneal surface

3 = lesions affecting 25-50% of the cornea

4 =^■ lesions affecting 50-75% of the cornea

5

Th reported scores are the mean value for each group.

_0- means CNS involvment with paralysis and death within 3-4 days.

TABLE 2

Group Antigen N^β deaths/N^β treated % protection

1 gB-ls + CFA 0/14 100 2 gB-ls + alum 0/15 100 en 3 cell. 293, culture medium 13/13 0 4 HSV-1 LV 0/14 100

TABLE 3

Antigen In vitro No. dead/ Protection [%] neutralization No. treated

gE-ls 970 1/12 92 bgE-ls 120 8/12 34

HSV-1 [F] >1280 0/12 100

293 cell CM <20 12/12 0

TABLE 4

Group No. of No. of % of animals Total animals/ recurrences with recurrences score group [rnean±sd] [mean±sd]

1 12 10.4±1.9 90% 55.5±5.8

2 11 11.Oil.5 87% 50.7±4.9

3 11 7.5±2.1 61% 22.8±3.4

4 12 5.6il.8 57% 18±2.4

TABLE 5 a) Proliferative response of PBL obtained from 10 healthy HSV-1 immune donors [ID] and 3 healthy nonimmune donors [NID], to gB-ls and heat-inactivated HSV-1 [7-day culture].

Results are expressed as the mean cpm of triplicate culture.

Donor Medium gB-ls HSV-1

ID-1 759 16,310 14,634

ID-2 672 34,217 31,286

ID-3 598 10,738 93,852

ID-4 936 1,704 35,431

ID-5 1,096 14,325 33,514

ID-6 851 22,764 15,884

ID-7 951 36,909 25,839

ID-8 576 40,085 28,751

ID-9 826 45,429 23,210

ID-10 748 16,532 36,285

NID-1 823 806 784

NID-2 227 1,651 850

NID-3 448 418 975

b) Total number of TCC obtained from PBL of 4 HSV-1 immune donors [ID] stimulated in vitro vith gB-ls and number of TCC able to proliferate in the presence of gB-ls-pulsed autologous EBV-LCL (gB-ls-specific TCC). For each donor, TCC were achieved by seeding a total number of 100 cells in 300 wells of Terasaky trays [0.3 cell/well]. ID Total Specific

PC 48 25 DM 23 5 FB 64 32 KT 38 26

Claims

1. Recombinant glycoprotein D of Herpes simplex virus type 2 expressed by human cells and lacking the transmembrane and the intracytoplasmic domains.

2. Recombinant glycoprotein G of Herpes simplex virus type 2 expressed by human cells and lacking the transmembrane and the intracytoplasmic domains.

3. Recombinant glycoprotein E of Herpes simplex virus type 1 expressed by human cells and lacking the transmembrane and the intracytoplasmic domains.

4. Recombinant glycoproteins according to claims 1-3 obtainable from 293 cells transformed by the pRP- RSVgD2s or pRP-neoCMVgG-2s vectors.

5. A therapeutical and prophylactic vaccine comprising a glycoprotein of claims 1-4 optionally in combination with the recombinant glycoprotein B of Herpes simplex virus type 1 expressed by human cells and lacking the transmembrane and the intracytoplasmic domains.

6. A vaccine according to claim 5 comprising aluminium hydroxide or phosphate as adjuvant.