WO1993015749A1

WO1993015749A1 - Vaccines produced from herpes simplex virus light particles

Info

Publication number: WO1993015749A1
Application number: PCT/EP1993/000300
Authority: WO
Inventors: Myriam Francotte; Moncef Slaoui; Nathalie Marie-Josephe Claude Garcon-Johnson; Jean-Paul Prieels
Original assignee: Smithkline Beecham Biologicals (S.A.)
Priority date: 1992-02-12
Filing date: 1993-02-09
Publication date: 1993-08-19
Also published as: GB9202933D0; MX9300744A; AU3495993A; ZA93919B

Abstract

Therapeutic potential of Herpes Simplex Light particles are disclosed. In addition novel formulations of such Light particles are also disclosed which provide enhanced efficacy in both prophylactic and therapeutic experiments.

Description

VACCINES PRODUCED FROM HERPES SIMPLEX VIRUS LIGHT PARTICLES .

The present invention relates to vaccines, in particular to vaccines against herpes infections and to their use in both the treatment and prophylaxis of herpes infections.

Herpes Simplex Virus type 1 (HSV-1) and type 2 (HSV-2) are human pathogens which infect more than 80% of the population causing a broad spectrum of diseases ranging from relatively benign cutaneous lesions to fatal viral encephalitis. In the USA, it has been estimated that annually approximately 500,000 people have first genital herpetic infections, that 10 million people have recurring genital infections, that there are about 500,000 episodes of herpes kerato conjunctivitis, and that about 720,000 cesarean sections are performed aimed at preventing transmission of HSV to the newborn. Notwithstanding the amount of Caesarian sections, at least 1,800 cases of HSV infections in newborns occur each year (Roizman Reviews of Infections diseases 1991 (Suppl 11) : S892-894).

Initially HSV infects mucous membranes, and replicates at the port of entry. Following replication the virus enters the peripheral neurons where active replication is switched off in an unknown manner and latent infection in neurons is established for the life of the host. This virus periodically can reactivate to establish a clinical infection. Considerable research has been undertaken into the biology of Herpetic viruses and also into vaccines and anti viral drugs to combat the effects of Herpes Simplex infections. Recently, Szilagyi and Cunningham (J.

General Virology (1991) 72 661-668) have reported the identification and characterisation of non-infectious herpes simplex virus-related particles, which they called Light (or L-) particles. L particles although resembHng virions in appearance lacked the viral nucleocapside and viral DNA and were found to be non infectious.

Given the scale of the HSV health problem a safe and effective vaccine is clearly needed. The present inventors have found that HSV - L - particles not only show a prophylatic effect, but also a therapeutic effect and thus maybe utilised as a therapeutic vaccine. Accordingly, in a first aspect there is provided a herpetic Light particle for use in the manufacture of a medicament for treating a subject suffering from Herpetic infections. The invention also provides a method of treating a patient suffering from a herpetic infection the treatment comprising administering an effective amount of a herpetic L - particle.

The L particles for use in the invention are free of viral nucleic acid and therefore are non-infectious, however they are able to fuse with cell membranes and as such have the potential to induce cytolytic T cells (CTLs). Such CTL responses play an important role in modifying herpetic diseases. Moreover as L - particles contain a large number of antigens, this allows for a pluripotent immune response. They contain

glycoproteins which are involved in the induction of neutralising

antibodies as well as viral proteins which are potential targets of cytotoxic T cells in humans such as ICP4, ICP0 and Vmw 65.

The present invention further provides novel vaccine formulations of HSV - Light particles which in relevant animal models show enhanced prophylatic activity and also show a beneficial therapeutic effect. In particular, the vaccines of the present invention show a significant decrease in the number of recurrent episodes, the recurrence day numbers and also a significant decrease in the severity of the recurrent episode in both prophylatic and therapeutic situations.

Accordingly the present invention provides a formulation comprising a herpetic Light particle in conjunction with 3D - Monophosphoryl Lipid A (3D-MPL) and a suitable carrier. Preferably the carrier will be an oil in water emulsion or alum. Typically, 3D-MPL will be present in a dose range 10μg - 100μg preferably 25-75 per dose, whereas the light particle will typically be present in a range 2-50μg. Alum will typically be present in a standard concentration of 0.5 mg/dose. As an alternative to 3D-MPL, QS21 may be provided, but best results have been achieved with 3D-MPL.

3D-MPL may be obtained according to the methods described in British patent No. 2,220,211. Chemically it is a mixture of 3 de - 0 - acylated monophosphryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem Montana USA. QS21 on the other hand, is an hplc non- toxic fraction of a saponin from the bark of the South American tree

Quillaja saponaria molina and its method of production is disclosed (as

QA21) in US patent No. 5,057,540.

Preferably the LP preparations are treated with inactivating agents such as formaldehyde to kill any residual remaining live virus, optionally the preparations may additionally be irradiated with UV radiation. In a preferred embodiment of the invention the L particles are derived from attenuated HSV viruses which lack neurovirulence. For example a deletion variant of HSV- 2 strain HG52, termed JH2604 is avirulent (Tatia et al J. Gen Virol. 70, 3073-3078) and has a 1488 base pair deletion within both copies of the long repeat region of the genome [ie terminal Long repeat (TR_L) and internal inverted long repeat (IR_L) regions]. HSV-1 Glasgow strain modified in the terminal portion of R_L lacks

neurovirulence and therefore are preferably used to derive the Light particles for the present invention. In particular such viruses are described in copending UK application No. 9102126.1. published as PCT Application W092/13943. For example HSV strain 1716 .

Preferably the viruses are further modified by introducing a temperature sensitive mutation into the UL26 gene which encodes a protease involved in virion packaging and which at the non-permissive temperature results in the relative overproduction of Light particles.

Due to their non-pathogenic nature, these viruses are exceptional candidates for further modification. For example they may be further modified so as to carry heterologous antigens. Thus HSV-1 viruses can be engineered so as to express antigens from HSV-2, such as HSV-2 gD,

ICPO, ICP4 and Vmw65. Light particles may be derived from such a virus and the resulting light particle elicits both antibody and CTL responses to both type 1 and type 2 virus and, moreover, enhances the overall immune response. Similarly other envelope antigens from the other pathogens may be expressed. For example, gene products from HCMV, VZV, EBV, HHV6, HHV7, and HIV as well as other envelope viruses may be expressed on the light particle. For example an HSV-1 1716 strain has been modified to express HSV-2 gD and also modified to contain a temperature sensitive mutation in its

UL26 gene; this mutant produces L particles containing heterologous

HSV -- gD.

In addition to the above-mentioned modification or in the alternative, the virus from which the light particles are derived may be further modified by introducing a deletion which renders the LAT promoter ineffective.

Such a mutation adds a further level of safety, reducing both the frequency and rate of reactivation from latency of any potential remaining virion.

The Light particles utilised in the invention may be prepared by the method of Szilagyi & Cunningham (supra) and Rixon et al, J Gen Virol (1992) 23. p277-284 (for temperature sensitive mutants). Briefly cells are infected at 5 pfu/cell at the non-permissive temperature (npt) 38.5°C and the supernatant virus harvested at 30 K overnight in normal cell growth medium (Eagles) and the Light particles isolated utilising density gradient centrifugation. The production of Light Particles is also disclosed in International Patent Application No. PCT/GB 92/00824.

The mode of administration of the vaccine of the invention may be any suitable route which delivers an immunoprotective or therapeutic amount of the Light particle to the subject. However, the vaccine is preferably administered parenterally via the intramuscular or deep subcutaneous routes. Other modes of administration may also be employed, where desired, such as oral administration or via other parenteral routes, ie, intradermally, intranasally, or intravenously. The appropriate immunoprotective or therapeutic and non-toxic dose of the vaccine can be determined readily by those skilled in the art, ie, the appropriate immunoprotective and non-toxic amount of the Light particle contained in the vaccine of this invention may be in the range of the effective amounts of antigen in conventional whole virus vaccines. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the age, general health, sex, and diet of the patient; the time of administration; the route of administration; synergistic effects with any other drugs being administered; and the degree of protection being sought. Of course, the administration can be repeated at suitable intervals if necessary.

In the following examples two HSV L-Particle preparations were utilised and derived from HSV 1 strain 1716 infected cells or from HSVl 1760 (a Ts 1201 mutant strain) in the manner described by Szilagyi and

Cunningham J.G. Virol 1991 72 p661-668, and Rixon et al (Supra) and International Patent Application No. PCT/GB/92/00824. Example 1

1. Evaluation of the prophylactic and therapeutic potential of HSV light particles (LP) in the intra vaginal model of HSV2 infection

Several experiments were carried out to evaluate the immunogenicity and protective efficacy of different HSV LP vaccine formulations in guinea pigs. 1.1. Antigen-Adjuvant preparations

1.1.1. Prophylactic experiment

This experiment was performed with purified LP from 1716 mutant. The LP preparation was treated with formaldehyde before formulation. This resulted in complete inactivation of any contaminating live virus. Two formulations were prepared as decribed below. Each dose (5 μg HSV LP) was administrated in a 0.5 ml volume. * HSV 1716 LP / aluminium hydroxide

Aluminium hydroxide (Al(OH)3) was obtained from Superfos (Alhydrogel Superfos, Denmark).

5 μg of HSV 1716 LP preparation was adsorbed overnight at 4°C under agitation on 0.5 mg Al(OH)3 in 0.5 ml of 150 mM NaCl 10 mM phosphate buffer pH 6.8. One dose contained 5 μg HSV 1716 LP and 0.5 mg Al(OH)3.

* HSV 1716 LP / Al(OH)3 / 3D-MPL 5 μg of HSV 1716 LP preparation was adsorbed on Al(OH)3 as described before. The adjuvant preparation was centrifuged and its supernatant removed. 50 μg 3D-MPL was then added to the formulation. Volume was completed to 0.5 ml with the adsorption buffer. One dose contained 5 μg HSV 1716 LP, 0.5 mg Al(OH)3 and 50 μg 3D-MPL.

1.1.2. Therapeutic experiments Experiments were carried out either with purified LP produced by 1716 mutant or with purified LP from 1760 strain (1716 with tsl201 mutation), in combination with several adjuvants. The preparation of the HSV LP vaccines is described below. Each dose (40 μg HSV LP) was administrated in a 0.5 ml volume.

* HSV 1716 LP / Al(OH)3 / 3D-MPL

Preparation of this formulation was similar as described above. 40 μg of HSV LP were used instead of 5 μg. One dose contained 40 μg HSV 1716 LP, 0.5 mg Al(OH)3 and 50 μg 3D-MPL

* HSV 1716 LP / 3D-MPL in oil in water emulsion The vehicle was prepared as follows : to phosphate buffered sahne (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 comprised only submicron particles. To 0.25 ml of this 2 fold concentrated emulsion were added 50 μg of 3D-MPL and 40 μg of HSV

1716 LP. Volume was completed to 0.5 ml with PBS. The final preparation consisted of 0.2% Tween 80, 2.5% Pluronic 121, 5% squalane, 50 μg 3D- MPL and 40 μg HSV 1716 LP in a 0.5 ml dose. * HSV 1716 LP / Al(OH)3 / QS21

40 μg of HSV 1716 LP preparation was adsorbed overnight at 4°C under agitation on 0.5 mg Al(OH)3 in 0.5 ml of 150 mM NaCl pH 6.5. After centrifugation and removal of the supernatant, 20 μg QS21 was added to the formulation and volume was completed to 0.5 ml with the adsorption buffer. One dose contained 40 μg HSV 1716 LP, 0.5 mg Al(OH)3 and 20 μg

QS21.

* HSV 1716 LP / QS21

40 μg of HSV 1716 LP were mixed with 20 μg QS21 in 0.5 ml of 150 mM NaCl pH 6.5. One dose contained 40 μg HSV 1716 LP and 20 μg QS21.

* HSV 1760 LP / Al(OH)3 / 3D-MPL

This formulation was prepared as decribed above. One dose contained 40 μg HSV 1760 LP, 0.5 mg Al(OH)3 and 50 μg 3D-MPL.

Measurements

Several measurements were taken to evaluate the specific antibody response induced by vaccination with HSV LP vaccines. The protective value of these vaccines was assessed in the guinea pig intravaginal model. Anti-gD2t antibody titers and anti-HSV2 neutralizing activity were determined according the methods described in International patent application no. W0 92/16231. Briefly these were carried out as follows:

ELISA

An ELISA was designed to detect and quantify gD-specific antibodies in guinea pig sera using rgD₂t as the coating antigen. Antigen and antibody solutions were used at 50 μl per well. Antigen was diluted to a final concentration of 1 μg/ml in PBS and was adsorbed 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% (wash 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 (IgG1 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 2 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 non-linear regression analysis using a computer program.

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 4.10⁵ cells/ml were then added to each well (4.10⁴ 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 μl 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 observed in the control wells. Guinea pig intra vaginal challenge model

The guinea pig model for HSV genital infection has been described by L. Stanberry et al (J. of Infectious Diseases 1982,146: 397-403; Intervirology 1985, 24 : 226-231). Briefly, in prophylactic experiments, the guinea pigs were challenged intravaginally with 105 pfu of HSV2 strain MS, two weeks after the last vaccination. The clinical course of primary infection was monitored by daily observation of the incidence and severity of external genital skin lesions during the 4-12 day post challenge period. Animals were then examined daily for evidence of recurrent herpetic lesions from days 13 to 39. In therapeutic experiments, guinea pigs were challenged at day 0 with 10⁵ pfu HSV2 strain MS. After recovery from primary infection, they were evaluated daily for recurrent herpetic disease (days 13 to 21) and were then randomized according to their primary and recurrent scores (providing an equivalent distribution of animals with mild to severe infection in each group) to receive either no treatment or vaccination. Vaccines were administrated on days 21 and ±42 after challenge. The pattern of recurrent disease was generally observed until day ± 80 post challenge. The herpetic lesions on the external genital skin were quantitated by using a lesion score scale ranging from 0 to 4 (0 = no lesion 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 of vesicles increase; 3.5 = lesions covering all the surface of the genital skin; 4 = ulcerated lesions with maceration).

Clinical symptoms were defined as :

● Lesion severity : sum of the daily scores measured during the primary infection (days 4-12);

● Lesion incidences during the primary infection : % of animals with vesicle(s) (score≥1) between days 4 and 12 post challenge; ● Number of recurrent episodes : one recurrent episode is preceded and followed by a day without lesion and characterized by at least two days with vesicle(s) Gesion score≥1);

● Recurrence day number : total days animals experienced recurrent herpetic episodes during the observation period;

● Recurrence severity : sum of lesion scores during the observation

period

● Lesion incidences during the recurrent disease : % of animals with

vesicle(s) during the observation period.

1.3. Prophylactic efficacy of HSV LP vaccines

The purpose of this study was to evaluate the immunogenicity and protective efficacy of HSV 1716 LP formulations, as compared to rgD2t formulations as positive controls. rgD2t is a truncated glycoprotein which has been implicated in the prophylaxis of HSV infections (WO 92/16231).

1.3.1. Immunization schedule Groups of 8 female Hartley guinea pigs were vaccinated three times at days 0, 28 and 84 with the following vaccines:

● 5 μg HSV 1716 LP / Al(OH)3

● 5 μg HSV 1716 LP / Al(OH)3 + 3D-MPL

● 5 μg recombinant gD2t protein (untreated) / Al(OH)3 + 3D-MPL

● 5 μg recombinant gD2t protein (treated with formaldehyde) / Al(OH)3 + 3D-MPL.

The light particles preparation was treated with formaldehyde before formulation. This resulted in complete inactivation of the contaminating live virus. As positive controls, two guinea pigs groups were vaccinated with 5 μg gD2t combined with Al(OH)3 (0.5 mg / dose) and 3D-MPL (50 μg / dose), using either formaldehyde-treated gD2t or untreated gD2t.

Immunisations were given subcutaneously in a 0.5 ml dose. One control group was untreated. Animals were bled every two weeks for individual antibody determination by ELISA (anti-gD antibody response) and neutralization assays, as described above. In order to evaluate the protective immunity induced by the different vaccines, all the guinea pigs were challenged intravaginally with 10° 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) as well as for evidence of recurrent herpetic disease (days 13 to 39 post challenge).

1.3.2. Results

Results are given in Tables 1 and 2 and illustrated in Figure 1.

As shown in table 1, both vaccinated groups produced high neutralizing titers after the third immunization.

Effect of vaccination on HSV primary disease is shown in Table 1 - Figure 1. As compared to the control group that became infected and experienced acute primary disease, all vaccinated animals showed significantly reduced skin lesion severity, as well as reduction of skin lesions incidence.

Effect of vaccination on HSV recurrent disease is shown in Table 2 - Figure 1. The recurrence day number and the incidence of skin lesions were reduced in all vaccinated groups. The light particles formulated with aluminium hydroxide + 3D-MPL were more effective in providing protection from recurrent HSV2 disease than the light particles

aluminium hydroxide formulation.

1.3.3. Conclusions

HSV1716 light particles formulated with aluminium hydroxide were very potent in providing protection against primary and recurrent HSV2 disease when administrated to guinea pigs prior to intravaginal challenge. The protection level was improved by addition of 3D-MPL to this vaccine.

1.4. Therapeutic experiments

The aim of these experiments was to investigate the immunotherapeutical potential of several HSV LP formulations on the recurrent herpetic disease in guinea pigs with established HSV2 infection. Three different therapeutic experiments were performed with purified HSV LP produced either by 1716 or 1760 strains.

1.4.1. Experimental procedure

Guinea pigs were inoculated intravaginally at day 0 with 10⁵ pfu HSV2. After recovery from the initial infection, animals were evaluated daily for recurrent herpetic disease (days13 to 21). They were randomised as described above to receive either no treatment or HSV LP vaccines, as follows:

Experiment 1

Group 1 (n=15) : 40 μg HSV 1716 LP / Al(OH)3 / 3D-MPL

Group 2 (n=15) : 40 μg HSV 1716 LP / 3D-MPL in oil in water (o/w) emulsion

Group 3 (n=7) : untreated

The light particles preparation was treated with formalin before formulation. Vaccines were administered subcutaneously on days 21 and 47 after challenge. The pattern of recurrent disease was observed daily until day 82 post challenge.

Experiment 2

Group 1 (n=8) : 40 μg HSV 1716 LP / Al(OH)3 / 3D-MPL

Group 2 (n=9 ) : 40 μg HSV 1716 LP / Al(OH)3 / QS21

Group 3 (n=9) : 40 μg HSV 1716 LP / QS21

Group 4 (n=10 ) : 20 μg gD2t / Al(OH)3 / QS21

Group 5 (n=9) : Al(OH)3 QS21 alone

Group 6 (n=9 ) : untreated The light particles preparation was subjected to UV and formaldehyde inactivation before formulation. Guinea pigs were vaccinated on days 21 and 42 post HSV2 challenge. They were scored daily in the manner described above until day 65 post challenge.

Experiment 3

Group 1 (n=21) : 40 μg HSV 1760 LP / Al(OH)3 / 3D-MPL

Group 2 (n=22) : Al(OH)3 / 3 D-MPL alone

The LP preparation was UV and formaldehyde treated before formulation. Vaccinations were given on days 21 and 41 post infection. Animals were examined daily for recurrent herpetic disease until day 76 post HSV2 challenge.

1.4.2. Results

The results of the experiments are shown on Tables III (Experiment 1), IV (Experiment 2) and V (Experiment 3). The data are expressed as the number of recurrent episodes, the number of recurrent days and as the severity of recurrences. As shown in the three different experiments, immunization with the HSV LP vaccines significantly aitered the recurrent herpetic disease in guinea pigs with established HSV2 infection, as compared to control groups.

1.4.3. Conclusions

The results demonstrate that therapeutic treatment of guinea pigs with HSV LP in different adjuvant formulations reproducibily interfere with ongoing HSV2 recurrent disease by decreasing disease severity, recurrence day number and recurrence episode number. This effect is enhanced when 3D-MPL is utilised in the formulation 2. Evaluation of HSV light particles. Human cell mediated

immunity in vitro

2.1. Sensitization of human cells to lysis by autologous HSV2 specific CTL

A cytolytic assay was carried out to test the ability of purified LP from 1760 strain to sensitize targets cells for lysis by human HSV2 specific cytotoxic T cells (CTL).

2.1.1. Experimental procedure The LP 1760 preparation used in these experiments was treated with UV to inactivate any potential existing virion.

Peripheral blood mononuclear cells (PBMC) were isolated from a patient with herpetic genital lesions (patient 106). This patient was previously shown to have a frequency of HSV2 specific cytotoxic cells (CTL) of 1/6000; these CTLs were able to recognize the following target antigens: ICP27, Vmw65 and gD of HSV2 expressed in autologous lymphoblastoids cells by recombinant vaccinia virus. The PBMC were stimulated twice by co-culture with autologous HSV2 (strain HG52) infected lymphoblasts, in the presence of human recombinant IL2 and supernatant from PHA

stimulated human lymphoblasts as a source of growth factors. They were then tested for lysis of chromium 51 labelled autologous EBV transformed target cells infected with HSV2 (positive control) or with vaccinia virus psCll.W (negative controlXl hour at 37 C; m.o.i =10) or incubated with purified HSV 1760 LP for 16 hours at 37 C, at three different LP / cell ratios (100, 1000 and 10000 LP / cell). Effector and target cells were mixed at different ratios and coincubated for 4 hours at 37 C. Released 51Cr in the supernatant was then counted. 2.1.2 Results

As shown in Figure 2, human targets incubated with HSV 1760 LP were lyzed by human HSV specific CTL induced by in vitro stimulation of PBMC with HSV2 infected cells. These results indicate that 1760 LP can sensitize target cells for lysis by human effector cells specific for HSV.

Lysis of target cells treated with HSV 1760 LP correlated with the ratio of

LP per target cell. Only background lysis was seen on negative control target.

2.2. In vitro stimulation of human HSV T cell proliferative responses by LP and measure of cytokine secretion Human T cells stimulation by HSV 1760 LP was evaluated in a

proliferative assay. The phenotype of the stimulated T cells was analysed by measuring the production of interleukin 2 (IL2), interleukin 4 (IL4) and interferon gamma (IFNγ) 2.2.1 Experimental procedure

PBMC were isolated from a patient with herpetic genital lesions (patient I31). They were stimulated in 96-well plate cultures (2×105 cells / well) with the following antigens:-

● soluble recombinant gD2t at different concentrations (5, 0.5, 0.05, 0.005 and 0.0005 μg/ml)

● purified HSV 1760 LP at three different LP / cell ratios (100, 1000 and 10000 LP / cell)

● whole UV inactivated HSV2 virus (strain HG52) at the equivalent of m.o.i 1 and 10 before irradiation

● tetanus toxoid as positive control antigen (at 1 μg / ml)

● T cell mitogen as positive control (PHA at 4 μg / ml)

● medium alone as negative control

Proliferation assay

After 3 days incubation at 37 C, tritiated thymidine was added to the cultures for 18 hours. Proliferation (expressed as incorporation of 3H) was measured by harvesting the cells onto filters and beta emission counting.

IFNγ and IL2 secretion assays After 3 days culture at 37 C, supematants from replicate stimulated cultures were harvested, pooled and assayed for IFNγ and IL2 using specific ELISA kits marketed by Holland Biotechnology and British

Biotechnology, respectively. The amount of cytokine produced was determined by reference to a standard curve. The sensitivity of these kits is 20 pg / ml for IFNγ and 55 pg / ml for IL2.

IL4 secretion assay IL4 quantification was evaluated in culture supematants after 2 days stimulation, using an ELISA kit from British Biotechnology. The sensitivity of this assay is 5 pg / ml

2.2.2. Results

As shown in Table VI, HSV 1760 LP were very potent in stimulating human PBM cell proliferative responses in vitro. These stimulated cells produced high levels of IFNγ. IL2 and IL4 were below the sensitivity level of the ELISA assays. Similar cytokine secretion profile was observed after stimulation with HSV2 virus. In contrast, gD2t stimulated PBM cells produced IL2 and IFNγ (but at lower levels as compared to HSV2 or LP stimulated T cells) and no IL4. These data suggest that HSV 1760 LP and HSV2 virus stimulated cells with a functional phenotype (CTL or NK) different from cells stimulated by soluble gD2t (TH1 secretion pattern).

2.3. Conclusions

In a manner similar to HSV2 virus, purified HSV 1760 LP are shown to be able to sensitize target cells for recognition by human HSV specific CTL and to stimulate human T cell proliferative responses and IFNγ secretion. TABLE I PROPHYLACTIC POTENTIAL OFHSV1716 LP FORMULATIONS

IMMUNOGENICITY AND PROTECTION AGAINST PRIMARY DISEASE

Vaccine(1) Adjuvant gD Elisa Incidence Skin

Ab titers of skin Neutr. Ab lesions lesions (2) titers severity

(3) _gD2t 5 μg Al(OH)3 + 3 3D- 11650 ± 5249 10% 2400 ± 1619 0.4 ± 0.6 formaldehyde MPL gD2t 5 μg Al(OH)3+ 3D- 9200 ± 7030 20% 2400 ± 1718 1.3 ± 1.5 untreated MPL

HSV 1716LP5μg Al(OH)3 1610 ± 2306 30% 1175 ± 1153 1.3 ± 1.8

HSV1716LP 5μg Al(OH)3 + 3D- 3425 ± 2821 10% 2720 ± 2205 0.7 ± 0.9

MPL

untreated - <100 92% <50 9.3 ± 4.3 controls

(1) Groups of 8 animals were vaccinated on days 0,28 and 84.

(2) Is the % of animals with vesicles (score≥ 1) between days 4 and 12 post infection.

(3) Is the sum of lesion between days 4 and 12 post infection: arithmetic mean ± SD.

TABLE II

PROPHYLACTIC POTENTIAL OF HSV 1716 LP FORMULATIONS

PROTECTION AGAINST RECURRENT DISEASE

Vaccine Adjuvant Incidence of Recurrence days

recurrent number (b)

disease (a) gD2t 5 μg Al(OH)3+ 3D- 30% 1 ± 1.6

formaldehyde MPL gD2t 5 μg Al(OH)3 + 3D- 40% 2.9 ± 4

MPL

untreated

HSV1716 Al(OH)3 40% 2.9 ± 4.4

LP5μg

HSV1716 Al(OH)3 + 3D- 20% 0.8 ± 1.7

LP5μg MPL imtreated Al(OH)3 60% 5.1 ± 6.2

controls

(a) animals with vesicles (lesion score≥ 1) during the observation period (days 13→ 39)

(b) average number of days with recurrent disease during the observation period (days 13→ 39).

Claims

1. A herpetic light particle for use in the manufacture of a medicament for treating a subject suffering from herpetic infections.

2. A vaccine formulation comprising a herpetic light particle and 3

Deacylated monophosphoryl lipid A or QS21.

3. A vaccine formulation as claimed in claim 2 wherein the 3 Deacylated monophosphoryl lipid A is present in a dose range of 10 μg - 100 μg per dose.

4. A vaccine formulation as claimed in claim 2 or 3 wherein the L-particles are derived from Herpes Simplex virus (HSV).

5. A vaccine formulation as claimed in claim 4 wherein the L-particles are derived from HSV-1.

6. A vaccine formulation as claimed in any of claims 2 to 6 wherein the L particle expresses a heterologous antigen.

7. A vaccine formulation as claimed in claim 6 wherein the heterologous antigen is selected from HSV-2 gD, HSV-2 ICPO, HSV-2 ICP4, HSV-2 Vmw65 and the L-Particles is derived from HSV- 1.

8. A vaccine formulation as claimed in 6 wherein the heterlogous antigen is selected from an HCMV, VZV, EBV, HHV6, HHV7 or HIV gene product.