AP298A - Herpes simplex vaccine comprising HSV Glycoprotein gD and 3 deacylated mono-phosphoryl lipid A. - Google Patents

Abstract

Novel herpes simplex (HSV) vaccine formulations are provided. These comprise HSV glycoprotein gD or immunological fragments in conjunction with 3 Deacylated monophosphoryl lipid A.

Description

Vaccine

The present invention relates to novel vaccine formulations, methods for preparing them and to their use in therapy. In particular, the present invention relates to novel formulations for treating Herpes Simplex Virus infections, more particularly Herpes Simplex virus 2 (HSV-2) infections.

HSV-2 is the primary etiological agent of herpes genitalis and together , with HSV-1 (the causative agent of herpes labialis) are characterised by their ability to induce both acute diseases and to establish a latent infection, primarily in neuronal ganglia cells.

Genital herpes is estimated to occur in about 5 million people in the U.S.A. alone with 500,000 clinical cases recorded every year (primary and recurrent infection),. Primary infection typically occurs after puberty and is characterised by the localised appearance of painful skin lesions, which persist for a period of between 2 to 3 weeks. Within the following six months after primary infection 50% of patients will experience a recurrence of the disease. About 25% of patients may experience between 10-15 recurrent episodes of the disease each year. In immunocompromised patients the incidence of high frequence recurrence is statistically higher than in the normal patient population.

Both HSV-1 and HSV-2 virus have a number of glycoprotein components located on the surface of the virus. These are known as gA, gB, gC, gD and gE etc.

Glycoprotein D is located on the viral membrane, and is also found in the cytoplasm of infected cells (Eisenberg R.J. gfc al; J of Virol 1980 35 428- * 435). It comprises 393 amino acids including a signal peptide and has a molecular weight of approximately 60 kD. Of all the HSV envelope glycoproteins this is probably the best characterised (Cohen gl al J· Virology fiQ 157-166). In vivo it is known to play a central role in viral attachment to cell membranes. Moreover, glycoprotein D has been shown to be able to elicit neutralising antibodies in vivo (Eing gl al J. Med. Virology 127: 59-65). However, latent HSV-2 virus can still be reactivated and induce recurrence of the disease despite the presence of high neutralising antibodies titre in the patients sera.

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- 2 The ability to induce neutralising antibody alone is insufficient to adequately control the disease. Ia order to prevent recurrence of the disease, any vaccine will need to stimulate not only neutralising antibody, but also cellular immunity mediated through T-cells. The present invention achieves these aims.

The present invention provides a vaccine comprising HSV glycoprotein D or an immunological fragment thereof in conjunction with 3-o-deacylated monophosphoryl lipid A (3D-MPL) a deacylated derivative of monophosphoryl lipid A, and a suitable carrier. Typically the glycoprotein D will be from HSV-2. The carrier may be an oil in water emulsion, or alum, 3D-MPL will be present in the range of 10ng - 100μ^ preferably 2550pg per dose wherein the antigen will typically be present in a range 250pg per dose.

3D-MPL may be obtained according to the methods described in British patent No. 2220211 (RIBI).

An embodiment of the invention is a truncated HSV-2 glycoprotein D of 308 amino acids which comprises amino acids 1 through 306 naturally occuring glycoprotein with the addition Asparagine and Glutamine at the C terminal end of the truncated protein devoid of its membrane anchor region. This form of the protein includes the signal peptide which is cleaved to yield a mature 283 amino acid protein. The production of such a protein in Chinese Hamster ovary cells has been described in Genentech's European patent EP-B-139 417.

The mature truncate preferably is used in the vaccine formulations of the present invention as is designated rgD2t.

The HSV antigen may be chemically or otherwise conjugated to a particulate carrier. A particularly preferred approach is to chemically conjugate to particulate Hepatitis B surface antigen through free sulfhydryl groups located on the surface of the Hepatitis B surface antigen. See copending U.K. Patent application No. 9027623.9.

The formulations of the present invention are very effective in inducing

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-3protective immunity, even with very low doses of antigen (e.g. as low as 5 gg rgD2t).

They provide excellent protection against primary Infection and stimulate, advantageously both specific humoral (neutralising antibodies) and also effector cell mediated (DTH) immune responses.

Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. ; squalane or squalene, a emulsifier, e.g. Tween 80, in am aqueous carrier. The aqueous carrier may be for example, phosphate buffered saline.

The present invention in a further aspect provides a vaccine formulation as herein described for use in medical therapy, particularly for use in the treatment or prophylaxis of Herpes Simplex viral infections.

The vaccine of the present invention will contain an immunoprotective quantity of HSV gD or immunological fragment thereof and this maybe prepared by conventional techniques.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.

The amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon ' which specific immunogen is employed. Generally, it is expected that each dose will comprise 1-1000 gg of protein, preferably 2-100 gg, most preferably 4-40 gg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects may receive a boost in about 4 weeks.

In addition to vaccination of persons susceptible to HSV infections, the pharmaceutical compositions of the present invention may be used to

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-4treat, immunotherapeutically, patients suffering from HSV infections.

In a further aspect of the present invention there is provided a method of manufacture as herein described, wherein the method comprises mixing HSV-2 glycoprotein D or an immunological fragment with a carrier, e.g. an oil in water emulsion or alum, and 3D-MPL.

Comparison of adjuvant efficacy of a recombinant Heroes Simplex Virus

Glycoprotein D Subunit Vaccine

In this study, the ability of several adjuvants to improve the protective immunity of a recombinant glycoprotein D from Herpes Simplex Virus (HSV) type 2 (rgD£t) was evaluated in a guinea pig model. Adjuvants tested were aluminium hydroxide, aluminium hydroxide in combination with 3 Deacyl-Monophosphoryl Lipid A, and 3 Deacyl-Monophosphoryl Lipid A delivered in an oil in water emulsion.

1. Description of tire antigen

HSV rgD£t is a genetically engineered recombinant truncated glycoprotein produced in transfected Chinese hamster ovary (CHO) cells (European Patent No. 0 139 417).

2. Antigen-Adjuvant preparations and immunization schedules

Two separate experiments were performed to evaluate the protective immunity of several rgDgt formulations in the guinea pig model. In the first experiment, groups of guinea pigs were immunized three times with a low antigen dose (5 yg of rgD2t) in 4 adjuvant formulations prepared as described below. Two weeks after the last immunization, they were challenged intravaginally with HSV type 2 and were monitored daily for the development of primary and recurrent HSV2 disease. In the second experiment, these formulations were further evaluated on larger animal groups. Factors influencing efficacy of these formulations were also tested such as antigen dose and adjuvant composition.

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-52.1. Antigen-Adjuvant preparations

In the first experiment, guinea pigs were immunized with the following adjuvant preparations. Each dose (5 gg) was administered in a 0.25 ml volume.

2.1.1. rgD2t /Alum (Aluminium Hydroxide)

Alum was obtained from Superfos (Alhydrogel, (Boehimte) Superfos, Denmark). Five gg of purified rgD2t was adsorbed overnight at 4°C on aluminium hydroxide (alum) corresponding to 0.25 mg equivalents Al^+ in 0.25 ml of 150 mM NaCl 10 mM phosphate buffer pH 6.8.

2.1.2. rgD2t/ Aluminium Hydroxide plus 3D-MPL

D-MPL was obtained from Ribi Immunochem Research, Inc. After an overnight adsorption of 5 gg gD2t on alum as described in 2.1.1., the adjuvant preparation was centrifuged and its supernatant removed. An equal volume of adsorption buffer containing 100 gg 3P-MPL was then added to the alum-bound rgD2t.

For both rgD2t/Alum preparations, more than 98% of the rgD2t was found to be incorporated in aluminium hydroxide adjuvant.

2.1.3. rgD2t/3D-MPL in an oil in water emulsion (R)

The oil in water emulsion was prepared using 12% w/v lecithin added to Squalene oil and 0.08% Tween 80. 3D-MPL was added at a concentration 100 fold higher than the final desired concentration. 1% of this preparation was then mixed in a 0.25 ml volume to 5 gg rgD2t in aqueous phase, yielding a 1% oil in water emulsion containing 100 gg 3DMPL.

Similar adjuvant formulations prepared as above but containing different amounts of rgD2t and/or immunostimulator were used in the second experiment. They were administered in a total volume of 0.5 ml. These formulations are described below.

rgP2t/Alum: Five or 20 gg rgP2t; 0.5 mg equivalents Al^+ per 0.5

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-6ml dose.

rgD2t/Alum plus 3Ρ-ΜΡζ: Five or 20 gg rgP2t; 0.5 mg equivalents Al3+; 50 gg 3P-MPL per 0.5 ml dose.

rgP2t/3P-MPL in o/w emulsion (R): Five or 20 gg rgP2t were formulated in an 1% o/w emulsion as described above (2.1.3). A 0.5 ml dose contained 5 gg or 20 gg rgP2t, 50 gg 3P-MPL in a 1% o/w emulsion.

rgP2t/3P-MPL in o/w emulsion (S): The vehicle was prepared as follows: To phosphate buffered saline (PBS) containing 0.4% (v/v) Tween 80 are added 5% (v/v) Pluronic L121 and 10% squalane and the resulting mixture microfluidized ten times through a microfluidizer (Model M/110 Microfluidics Corp.,) such that the resulting emulsion comprises only submicron particles. 50gg of 3P-MPL was then added to the emulsion. One volume of this emulsion, containing 3P-MPL was mixed with an equal volume of twice concentrated antigen and vortexed briefly to ensure complete mixing of the components. The final preparation consisted of 0.2% Tween 80, 2.5% Pluronic L121, 5% Squalane, 50gg 3P-Mpl and 5 gg or 20 gg rgP2t in a 0.5 ml dose.

2.2. Immunization schedule

Groups of female Hartley guinea pigs (200-250 gr) were immunized three times at day 0, 28 and 95 with 5 gg rgP2t formulated in 4 different adjuvant formulations.

Immunizations were done subcutaneously with injection volume of 0.25 ml. Control animals were injected according to the same protocol with adjuvant alone or were untreated.

The different groups were immunized as follows:

Group 1 (n = 4): 5 gg rgP2t/3P-MPL (100 gg) in o/w emulsion (R)

Group 2 (n = 4): 5 gg rgP2t/Alum plus 3P-MPL (100 gg)

Group 3 (n = 4): 5 gg rgP2t/Alum

Group 4 (n = 5): Alum alone

Group 5 (n = 5): 3P-MPL (100 gg) alone

Group 6 (n = 8): untreated

Animals were bled every 2 weeks for antibody determinations by

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-7ELISA and neutralization assays as described below.

The different formulations were also tested for their ability to induce T cell mediated immunity, as measured by the induction of delayed-type hypersensitivity responses. The read-outs applied for evaluation of the humoral and cellular immune responses induced by the different rgD2t formulations are described below.

In order to compare the protective immunity induced by the rgD2t formulations, all the guinea pigs were challenged intravaginally with 10$ plaque-forming units (pfu) of HSV2, strain MS, 2 weeks after the last immunization. They were monitored daily for clinical signs of acute infection as well as for evidence of recurrent herpetic diseases. Vaginal swab samples were collected on day 5 after viral challenge and titered for infectious virus.

A detailed description of the guinea pig intravaginal model is given below.

In the second experiment, the immunogenicity of the following rgD2t formulations was evaluated in larger animal groups. Two antigen doses were compared (5 and 20pg) and different adjuvant composition were tested. A dose of 50|Xg 3 DMPL was used and its effects compared to the lOOpg dose previously used.

Groups of female Hartley guinea pigs were immunized three times at days 1, 28 and 84, as follows:

Group I	(n = 8)
Group II	(n = 8)
Group III	(n = 10)
Group IV	(n = 10)
Group V	(n= 10)
Group VI	(n = 10)
Group VII	(n = 4)
Group VIII	(n = 4)
Group IX	(n = 8)

20gg rgD2t/3DMPL (50gg) o/w emulsion (R) 5pg rgD2t/3DMPL (50pg) o/w emulsion (R) 20pg rgD2t/3DMPL (50pg) o/w emulsion (S) 5pg rgD2t/3DMPL (50pg) o/w emulsion (S) 20pg rgD2t/Alum + 3DMPL (50|ig)

5pg rgD2t/Alum + 3DMPL (50pg)

Alum + 3DMPL (50pg) alone

3DMPL (50pg) o/w emulsion (R) alone untreated

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-8Immunizations were given in a 0.5 ml dose. Control groups were immunized according to the same protocol with adjuvant alone (Groups VII and VIII) or were intreated (Group IX).

A last group (Group X) was immunized with a gD£t Alum + 3DMPL formulation containing lOOgg 3D-MPL in a 0.25 ml dose, according to the protocol described in the first prophylactic experiment:

Group X (n = 10) : 5gg rgD2t/Alum plus 3DMPL (lOOmg).

Animals were bled every two weeks for individual antibody determinations by ELISA and neutralization assays, as described below. Vaginal washings were collected after the second immunization and were assayed for the presence of systemic antibodies specific for gD£t (antigD2t antibodies of IgG class). Guinea pigs were challenged intravaginally with 105 pfu HSV2 (strain MS) 2 weeks after the last immunization.

After challenge, they were monitored daily for clinical signs of acute infection (days 4 to 12 post challenge) as well as for evidence of recurrent herpetic disease (days 13 to 39 post challenge).

3. Bead-outs

Several read-outs were set up to evaluate the specific antibody and cell mediated responses induced by vaccination with rgD2t formulations. The protective value of these formulations was assessed in the guinea pig intravaginal model.

3.1. ELISA

An ELISA was designed to detect and quantify gD-specific antibodies in guinea pig sera and vaginal washings, using rgD2t as the coating antigen.

3.1.1. Detection of IgG antibodies specific for rgD2t in sera

Antigen and antibody solutions were used at 50 μΐ per well. Antigen was diluted to a final concentration of 1 gg/ml in PBS and was

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-9adsorbed overnight at 4°C to the wells of 96 wells microtitre plate (Maxisorp Immuno-plate, Nunc, Denmark). The wells were then washed 5 times with PBS Tween 0.1% (wqph buffer) and incubated for 1 hour at 37°C with PBS containing 1% bovine serum albumin, 4% newborn calf serum and 0.1% Tween (saturation buffer). Three-fold dilutions of sera (starting at 1/100 dilution) in the saturation buffer were added to the rgD2t-coated wells and incubated for 2 hrs at room temperature. The plates were washed as above and biotin-conjugated sheep anti-guinea pig IgG (IgGl and IgG2 specific, Serotec, Sopar Biochem., Belgium) diluted 1/3000 in saturation buffer was added to each well and incubated for 1 h.30 min. at 37°C. After a washing step, streptavidin-biotinylated peroxidase complex (Amersham, UK) diluted 1/1000 in saturation buffer was added and incubated for 30 min. at 37°C. Plates were washed as above and incubated with a solution of o-phenylenediamine (Sigma) 0.04% H2O2 0.03% in 0.1 M citrate buffer at pH 4.5.

Color reaction was stopped after 15 min by the addition of H2SO4

M and the absorbance was readed at 492 nm.

ELISA titer was defined as the reciprocal of serum dilution which produced an absorbance (optical density measured at 492 nm equal to 50% of the maximal absorbance value (midpoint titer).

ELISA titers were calculated by a 4 parameter Unear regression analysis using a computer program.

3.1.2. Detection of IeG antibodies specific for rgD2t in vaginal washings

Vaginal washings were first calibrated for their total IgG content by ELISA as foUows. Maxisorp Immuno-plates were coated overnight at . 4°C with 1 gg/ml (50 μΐ per well) of purified goat anti-guinea pig IgG (Sigma, Belgium) diluted in PBS. The plates were washed and incubated with saturation buffer as above. Vaginal washings were diluted serially with two-fold dilutions (starting at a 1/100 dilution) in the saturation buffer and added to the plates. A standard curve of purified guinea pig IgG (Sigma, Belgium) was included (two fold dilution starting at a 100 ng/ml concentration) in each plate.

After a 2 hrs incubation at room temperature, the plates were washed as above and biotin-conjugated sheep antibodies specific for guinea pig IgGl and IgG 2 (Serotec, Sopar Biochem, Belgium) diluted

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- 10171000 in saturation buffer was added to each well and incubated for 1 h 30 min at 37°C. Next steps (addition of streptavidin-biotinylated peroxidase complex and color revelation) were as described above (3.1.1.).

The concentration of total IgG present in the vaginal washings was determined from the IgG standard curve, by a 4 parameters monlinear regression analysis using a computer program.

After calibration of their total IgG content, vaginal washings were tested for the presence of IgG antibodies specific for rgD2t using the same ELISA as described for anti-gD antibody sera quantifications. Results were expressed as optical densities measured at 492 nm per 0.5 pg/ml total IgG.

3.2. Neutralization assay

A 96 well format neutralization assay was set up as follows:

Serial two-fold dilutions of the samples to be tested were prepared directly in the 96 W plates (25 ul/well of each serum dilutions, duplicates). Fifty microliters of a mixture containing 4000 pfu of virus HG52 and complement (1/100 final dilution in the well) were added to each well. The plates were incubated for 1 hour at 37°C. One hundred microliters of BHK 21 cell suspension at 4.10^ cells/ml were then added to each well (4.10^ cells/well). The plates were centrifuged for 5 minutes at 1000 rpm and incubated for five days at 37°C in the presence of 7% CO2.

After this period, the culture medium was gently removed and 100 μΐ of a solution of cristal violet (10% methanol, 90% H2O, 0.3% cristal violet) were added to each well and incubated for 20 min. at room temperature. The plates were then abundantly washed with tapwater. The presence of plaques can easily be monitored by microscopic examination.

The neutralizing titer was defined as the reciprocal of the highest serum dilution at which no viral plaque was observed (100% protection of cytopathogen effect). It is important to note that at this time point, a complete cytopathogen effect (100% lysis of the cell monolayer) was

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- 11 observed in the control wells.

3.3. Delayed-Type Hypersensitivity (DTH)

The different rgD2t formulations were also tested for their ability to induce a T cell specific immune response as measured by the induction of delayed-type hypersensitivity responses.

The adjuvant formulations prepared for the first experiment were used in this study. These preparations contained 5 pg of rgD2t per 0.25 ml dose. The immunization schedule was as follows: primary immunization: 0.25 ml of vaccine formulation given intramuscularly; booster immunization: 0.25 ml of vaccine formulation given intramuscularly 21 days later; skin test: 5 pg rgD2t given intradermally (in saline) 8 days later. All guinea pigs were skin tested with saline as control.

In addition, control guinea pigs (non immunized animals) were skin tested with rgD2t. Erythema and induration at site of intradermal injection were monitored 24 and 48 hrs later.

3.4. Guinea-pig intravaginal model

The guinea pig model for HSV genital infection has been described by LR Stanberry et al (J. of Infectious Diseases 1982,146:397403; Intervirology 1985, 24:226-231).

Briefly, 2 weeks after the last immunization, the guinea pigs were challenged with 10^ pfu of HSV2 strain MS by intravaginal instillation. The clinical course of the primary infection was monitored by daily observation of the incidence and severity of external genital skin lesions during the 12-day post-challenge period.

Vaginal swabs were collected on day 5 after viral challenge and titered for infectious HSV2 by plaque assay, as described below. Animals were then examined daily for evidence of recurrent herpetic lesions from days 13 to 60. The herpetic lesions on the external genital skin were quantitated by using a lesion score scale ranging from 0 to 4 (0 = no lesion

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- 12 or redness; 0.5 = redness; 1 = vesicle; 1.5 = > 4 small vesicles; 2 = larger vesicles; 2.5 = several large vesicles resulting from the fusion of vesicles as in score 2; 3 = size and number ofyesicles increase; 3.5 = lesions covering all the surface of the genital skin; 4 = ulcerated lesions with maceration).

The degree of protection provided by the different rgD£t vaccines was evaluated according to the criteria defined below.

Protection against primary disease (days 0 - 12)

The animal was considered to be not protected if the following lesions were recorded:

- more than one red area at any time,

- one red area persisting in the same area for at least 3 successive days (0.5 lesion score),

- one or several vesicles (> 1 lesion score).

Protection against recurrent disease (days 13 - 60)

The animal was scored positive for recurrent disease either if a 0.5 lesion score was recorded for 2 successive days at least or if a lesion score > 1 was observed at any day. An episode of recurrent disease was preceded and followed by a day without any lesions or redness.

The lesion severity for an animal is calculated as the sum of the scores measured during the primary infection (days 1 - 12). The lesion incidence represents the number of animals showing a lesion of > 1 during the observation period (days 1-12 [primary disease] or days 13 - 60 [recurrent diseases]).

3.5. Virus titration in vaginal swabs

Vaginal swabs were collected at day 5 after viral challenge. The vaginal vault was swabbed with a calcium alginate tipped swab premoistered in Basal Eagle's medium supplemented with 2% fetal calf serum, 2 mM L glutamine, 100 U/ml penicillin, 100 gg/ml streptomycin, 100 gg/ml gentamycin and 1 pg/ml amphotericin B (swab medium).

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- 13Each swab was broken and put into a sterile 12 x 75 mm 5 ml polyallomer tube containing 1 ml of swab medium. The tubes were then vortexed in order to take the viru^ out and frozen until use. For the titration itself, 6 wells culture plates containing 5.10^ cells /well were incubated overnight at 37°C. The tubes were thawed and serial dilutions of the samples in swab medium were prepared. After removal of the culture medium in the 6 wells, 200 μΐ of each samples dilution were transferred in duplicate on the cell monolayers and kept for one hour at 37°C. Four ml of a culture medium containing 1.5% carboxymethylcellulose were added to each well. The plates were then incubated for 2 days at 37°C. After this incubation period, the medium was gently removed and 1 ml of a solution of cristal violet (10% methanol, 90% H2O, 0.3% cristal violet) was added to each well for 15 min. The plates were then thoroughly rinsed and the plaques were counted. HSV2 titer was expressed in pfu/ml.

4. EfiSidtS

In a first set of experiments, groups of guinea pigs were immunized with a low antigen dose (5 gg rgD2t) formulated in 4 different formulations. This suboptimal antigen dose was chosen in order to select the more potent rgD2t adjuvant combination that could provide protection against primary and recurrent HSV disease when administered to guinea pigs prior to intravaginal HSV2 inoculation (prophylactic trials).

4.1. Induction of humoral immunity

As shown in Table 1. groups vaccinated with rgD2t formulations containing 3D-MPL as immunostimulant showed higher ELISA and neutralizing titers in their sera than the group immunized with the rgD2t/Alum vaccine. Good mean neutralizing titers were induced after 3 immunizations with rgD2t 3D-MPL o/w (R) or rgD2t Alum 3D-MFL.

4.2. Induction of effector T cell response (DTH)

Skin test results (Table 2) showed that rgD2t formulated in 3DMPL o/w emulsion induced the strongest DTH response. A specific DTH response was also induced by rgD2t Alum 3D-MPL. Similar experiments

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- 14conducted in mice also revealed that rgD2t combined with Alum plus 3DMPL was very potent in inducing an in vivo effector T cell response, in contrast to rgD2t Alum formulation.

4.3. Effect of vaccination on HSV primary disease

Two weeks after the third immunization, guinea pigs were challenged intravaginally with HSV2. The effect of vaccination on the clinical and virological course of primary HSV2 infection is illustrated in Figure 1 and summarized in Table 3. As compared to the control groups (Groups 4 to 6) that became infected and experienced acute primary disease, 100% of the animals vaccinated with the rgD2t 3D-MPL o/w formulation showed no evidence of herpetic disease, as monitored by skin lesion incidence and severity. Moreover, these animals did not show any viral replication in the vaginal tract as determined by vaginal virus titration at day 5 post challenge. Very similar results were obtained in the group vaccinated with rgD2t/Alum 3D-MPL. This group never developed herpetic vesicles during the observation period Gesion score <

1). Moreover, very low viral replication could be detected in the vaginal swabs collected. In contrast animals rgD2t adsorbed on alum were poorly protected (75% skin lesion incident).

4.4. Effect of vaccination on HSV recurrent disease

Results are illustrated in Figure 1 and summarized in Table 4.

Vaccination with rgD2t formulations containing 3D-MPL (Groups 1 and 2) significantly altered the development of recurrent herpetic diseases. Two groups had significantly fewer recurrent episodes and recurrent day numbers than control or rgD2t Alum treated groups.

In order to further evaluate the factors influencing the efficacy of prophylactic rgD2t vaccines containing 3DMPL, a second set of experiments was initiated on larger guinea pig numbers.

Two antigen doses were compared (5 and 20pg) and different adjuvant compositions were tested. Three immunizations were administered at days 0, 28 and 84. Animals were bled every two weeks for individual

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- 15antibody determination by ELISA and neutralization assays. Vaginal washings were collected after the second immunization and were tested for the presence of systemic antibodies specific for rgD£t.

Induction of humoral immunity

Results (Table 5) indicated that all the rgD£t formulations containing 3DMPL were able to stimulate high ELISA and neutralizing titers in the guinea pig sera.

The mean ELISA and neutralizing titers induced after three immunizations were very similar in the sera of groups vaccinated with a rgD2t formulation containing either 5pg or 20pg gD2t. There was no significant difference in the humoral response measured in the groups immunized with a rgD2t Alum vaccine containing either 50pg 3D-MPL (Group VI) or lOOmg 3D-MPL (Group X).

It is interesting to note that systemic anti-rgD2t antibodies (IgG class) could be detected in the vaginal washings of all vaccinated groups. This mucosally located anti-rgD2t antibody response may play an important protective role by decreasing the load of infectious virus in the genital tract during primary infection.

Effect of vaccination on HSV primary disease

Two weeks after the third immunization, guinea pigs were challenged intravaginally with HSV2. The effect of vaccination on the clinical and virological course of primary HSV2 infection is summarized in Table 6. As compared to the controls, animals vaccinated with a 5pg rgD2t Alum 3DMPL formulation containing either 50gg or lOOpg 3D-MPL (Groups VI and X) showed significantly (p<0.05) reduced skin lesion severity as well as reduction of skin lesions incidence.

Very similar results were observed in the group vaccinated with 5pg rgD2t in a 3D-MPL o/w emulsion (Group ΠΙ). In the three vaccinated groups, very low viral replication could be detected in the vaginal swabs collected 5 days after the challenge.

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- 16Effect of vaccination on HSV -recurrent disease

Results are given in Table 6. As compared to the control groups, the incidence of skin lesions and the recurrence day number were significantly (p>0.05) reduced in the three vaccinated groups. These groups had also fewer recurrent episodes than control groups.

5. Conclusions

Results obtained in guinea pigs clearly show that vaccination with a rgD2t formulation containing 3D-MPL delivered in an oil in water emulsion or combined with aluminium hydroxyde is very effective in providing protection against primary and recurrent HSV2 disease when administered to guinea pigs prior to HSV2 inoculation. Such rgD£t 3DMPL formulations are able to improve specific humoral (neutralizing antibodies) and effector cell mediated (DTH) immune responses. These results are obtained using a low dose of rgD£t (5pg).

6. Immunogenicity of gD2t formulations in primates

6.1 Comparative immunogenicity of reDgt/Alum and lyDot/Alum 3D-MPL fonm

The immunogenicity of rgD2t/Alum and rgD2t/Alum 3D-MPL vaccines were evaluated in cercopithecus aethiops (African Green Monkeys, AGM).

Three immunizations were given at 0, 1 and 3 months. Specific humoral (ELISA and neutralizing titers) and effector cell mediated (DTH) immune responses were measured.

6.1.1. Experimental procedure

Each formulation contained 20mg rgD2t and 0.5mg equivalents AL³⁺/dose. A dose of 50pg 3D-MPL was used. Groups of cercopithecus aethiops (AGM) were immunized 3 times at days 0, 28 and 84.

Immunizations were given intramuscularly in a 0.5ml dose (20 rgD2t).

Animals were bled every ± 2 weeks for antibody determination by ELISA and neutralization assays. The two formulations were also tested for their ability to induce T cell mediated immunity, as measured by the induction of delayed-type hypersensitivity (DTH) responses. Monkeys were given

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- 17intradermally on the belly different rgD£t doses (20, 5 and lug) in saline 13 days after the second immunization. They were also skin tested with saline alone as control. Erythema and induration at site of intradermal injection were monitored 24 hrs and 48 hrs later.

6.1.2. Results

a) Induction of humoral immunity

Before vaccination, none of the monkey sera showed any anti-HSV2 antibody activity (data not shown). Aa shown in table 7, both vaccines induced good ELISA and neutralizing titers after the second immunization. This antibody response was not boosted with a third immunization in the rgD2t/Alum vaccinated monkeys. In contrast, monkeys receiving a third immunization with rgD2t/Alum 3D-MPL produced increased ELISA and neutralizing antibody responses (mean ELISA titer: 10056; mean neutralizing titer: 950).

b) Induction of effector T cell response (DTH)

Skin test results (table 8) showed that rgD2t combined with Alum plus 3D-MPL was very potent in inducing an in vivo effector T cell response, in contrast to the rgD2t Alum formulation. A strong DTH response was observed in all rgD2t Alum 3D-MPL vaccinated animals skin tested with 20mg rgD2t. Specific DTH responses were also measured with the lower gD2t concentrations (5 and lug) in the majority of the monkeys (3/4 for the 5|ig dose and 2/4 for the lgg dose). These rgD2t doses induced weaker skin test responses than the 20mg rgD2t concentration.

6.2. Immunogenicitv of reD^i/Alum 3D-MPL formulations in rhesus monkeys

The immunogenicity of rgD2t/Alum 3D-MPL vaccines containing different rgD2t doses (100|ig, 10gg, or 5gg) was compared in rhesus monkeys.

6.2.1. Experimental procedure

Each formulation contained 0.5pg equivalents Al³⁺ and 50pg 3DMPL per dose. Three groups of rhesus monkeys (4 monkeys/group) were immunized three times at days 0, 28 and 77, as follows:

B45003

- 18 Group 1 Group 2 Group 3 lOOgg rgD£t Alum plus 3D-MPL (50gg) 20gg rgD2t Alum plus 3D-MPL (50gg) 5gg rgD2t Alum plus 3D-MPL (50gg)

Immunizations were given intramuscularly in a 1 ml dose. Animals were bled every ± 2 weeks for antibody determination by ELISA and neutralization assays.

6.2.2. Induction of humoral immunity

Before vaccination, none of the monkey sera showed any antiHSV2 antibody activity. Good ELISA and neutralizing titers were observed in the three vaccinated groups receiving either 100, 20 and 5mg gD2t in Alum + 3D-MPL. (Data not shown).

6.3. Conclusions

Results obtained in cercopithecus aethiops clearly indicate that a rgD2t vaccine containing a combination of Alum with 3D-MPL significantly improve humoral (neutralizing antibodies) and effector cell mediated (DTH) specific immune responses. As compared to this vaccine, a rgD2t Alum formulation is less potent in inducing neutralizing antibodies and is unable to induce an in vivo DTH response.

Results obtained in rhesus monkeys also show that a rgD2t Alum + 3DMPL formulation is very effective in inducing a specific humoral response, even with low doses of antigen (5gg or 20gg rgD2t).

7. General Conclusions

Results obtained in guinea pigs clearly indicate that adjuvant formulations containing either 3D-MPL delivered in an oil in water emulsion or combined with aluminium hydroxide are very effective in inducing a protective immune response with a recombinant HSV glycoprotein vaccine in the intravaginal guinea pig challenge animal model, even with very low doses of antigen (5 gg rgD2t). Protection data also show that these rgD2t 3D-MPL formulations are more potent in

B45003

AP Ο Ο Ο 2 9 8

- 19providing protection. Such 3D-MPL formulations are able to improve specific humoral (neutralizing antibodies) and effector cell mediated (DTH) immune responses.

Furthermore, the rgD2t Alum 3D-MPL formulation was shown to also improve immunogenicity at the antibody level and to induce an effector T cell response in primates, suggesting that this adjuvant effect is not restricted to small animal species.

- -('20 <3A

TAR I.E 1 : Anti-HSV antibody response in sera of guinea pige immunized with rgD2t formulation· before and after viral challenge.

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ELISA titer	81291± 20822	39897 ± 30165	20346 ± 23704	< 100	<100	<100
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TABLE 7

DTH RESULTS IN AFRICAN GREEN MONKEYS VACCINATED WITH GD2T ALUM OR GD2T ALUM 3D MPL

	24 h reading	48 h reading
VACCINE	MONKEY	PBS	gD2t	gD2t	gD2t	PBS	flD2t	gD2t	gD2t
	NB		1M	5 Mg	20pfl		1 MS	5 MS	20 pg
gD2t	JO358		ND				ND
ALUM	JO359	-	ND	-	-	-	ND	-	-
	JO363	-	ND	-	-	-	ND	-	-
	JO364	-	ND	-	-	-	ND	-	-
	JO366	-	ND	-	-	—	ND	-	—
gD2t	JO348			E	I 2-4			I	I
ALUM	JO349	-	E 1-2	I 5-8	cm	-	E	I	I
3D MPL	JO375	-	mm	mm	E 7-9	-	E	I	I
	JO515	-	E 1-2	I 3-4	mm	-	-	-	Eweak
			mm	mm	I 4-6
			-	-	mm
					E
CONTROLS	JO320
	JS110	-	-	-	-	-	-	-	-

Monkeys were immunized at days O and 28 with 20 pg gD2t formulation (given intramuscularly). They were given intradermally in the belly different gD2t doses in saline 13 days later. Skin test was read at 24 h and 48 h.

E : erythema at site of ID injection

I : induration at site of ID injection

ND = not done

BAD ORIGINAL d

AP 0 0 0 2 9 8

COMPARATIVE IMMUNOGENICITY OF GD2T ALUM AND GD2T ALUM 3D MPL FORMULATIONS IN AFRICAN GREEN MONKEYS: SEROLOGICAL RESPONSES

Claims (10)

1. Claim

   1. A vaccine formulation comprising an HSV glycoprotein D or an immunological fragment thereof in conjunction with 3Deacylated monophosphoryl lipid A and a suitable carrier.
2. 2. A vaccine formulation as claimed in claim 1 wherein the carrier is alum.
3. 3. A vaccine formulation as claimed in claim 2 wherein the carrier is an oil in water emulsion.
4. 4. A vaccine formulation as claimed in any of claims 1 to 3 wherein the glycoprotein D is an HSV-2 glycoprotein D or immunological framgment thereof.
5. 5. A vaccine formulation as claimed in claim 1 to 4 wherein the glycoprotein D is a truncated protein.
6. 6. A vaccine formulation as claimed in claim 5 wherein the truncated protein is HSVgD2 and is devoid of the C terminal anchor region.
7. 7. A vaccine formulation as claimed herein wherein the glycoprotein D is conjuncted to a particulate carrier.
8. 8. A vaccine formulation as claimed herein wherein 3Deacylated monophosphoryl lipid A is present is the range of lOgg - lOOgg per dose.
9. 9. A vaccine formulation as claimed herein for use in medicine.
10. 10. Use of HSV glycoprotein gD or an immunological fragment thereof in conjunction with 3Deacylated monophosphoryl lipid A in the manufacture of a medicament for the prophylaxis or treatment of HSV infections.

    1/. A method of treating a~huinan subject suffet ing from or susceptible to Herpes-Simplex infections comprising admnnsWnig an effective

    B45003/C1

    AP Ο Ο Ο 2 9 8

    -2<4 · ¥£. A method of producing a vaccine according to any of claims 1 to 8 wherein the method comprises mixing HSV glycoprotein D or immunological fragment with a carrier and 3Deacylated monophosphoryl lipid A.