Herpes simplex vaccine comprising HSV glycoprotein GD and 3 deacylated monophosphoryl lipid A

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

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
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. 
2. Description of the Prior Art 
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. et 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 et al J. 
Virology 60 157-166). 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 et 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. 
The ability to induce neutralising antibody alone is insufficient to 
adequately control the disease. In 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. 
SUMMARY OF THE INVENTION 
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 10 .mu.g-100 .mu.g preferably 25-50 .mu.g per 
dose wherein the antigen will typically be present in a range 2-50 .mu.g 
per dose. 
3D-MPL may be obtained according to the methods described in U.S. Pat. No. 
4,912,094 (Ribi).

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
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 is well known in the art, see for example 
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 rgD.sub.2 t. 
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 sulfilydryl groups 
located on the surface of the Hepatitis B surface antigen. The covalent 
coupling of glycoprotein D of HSV-2 to a particulate carrier is described 
in U.K. Patent Application No. 9027623.9 as follows: 
Covalent coupling of the glycoprotein D of Herpes Simplex 2 Virus to a 
particulate carrier 
Introduction 
The glycoprotein D of HSV 2 (gD.sub.2 t) expressed in CHO cells (Lasky and 
Dowbenko DNA, 1984, 3(1), 23-29) is covalently coupled to a recombinant 
HBsAg particle containing free SH groups. 
Materials and Methods 
a. Agents 
5,5'Dithiobis 2-nitrobenzoic acid (Ellman's reagent or DTNB) and 
N-succinimidyl(4-iodoacetyl)- aminobenzoate (SIAB) were purchased from 
PIERCE. 
2,4,6-Trinitrobenzenesulfonic acid (TNBS) was obtained from SERVA. 
Recombinant gD.sub.2 t was expressed in CHO cells and purified by 
SmithKline Biologicals. 
gD.sub.2 t was iodinated by the enzymobeads method of PIERCE. 
HBsAg particles were produced by SmithKline Biologicals. 
b. Methods 
b. 1. Characterization of gD.sub.2 t 
b. 1.1 Quantitative determination of sulfhydryl groups: 
To 150 ml of gD.sub.2 t (23 mM in Na.sub.2 HPO.sub.4 0.02 M pH 7) 1 ml of 
DTNB (3.28 mM in Na.sub.2 HPO.sub.4 0.04 M pH 8) is added. 
After 5 min, the optical density at 412 nm is determined against a blank 
lacking protein. An extinction coefficient of 1.3.times.10.sup.4 M-.sup.1 
cm-.sup.1 is used to calculate the concentration of sulfhydryl groups 
reacting (Ellman, Arch. Biochem. Biophys., 1959, 82, 70). 
b. 1.2 Quantitative determination of lysine residues: 
50 ml of TNBS/H.sub.2 O 24.5 mM are added to 50 ml of gD.sub.2 t (58 mM in 
Na.sub.2 HPO.sub.4 0.02 M pH 7) diluted in 200 ml of borate buffer (0.05 M 
Na.sub.2 B.sub.4 O.sub.7 adjusted to pH 9.5 with 0.05 M NaOH). 
After 3 hours in the dark at room temperature the change in absorbance at 
367 nm is followed against a blank without protein. 
The extent of trinitrophenylation is calculated on the basis of an 
e.sub.367 nm=1.1.times.10.sup.4 M-.sup.1 cm-.sup.1 (Plapp et al., J. Biol. 
Chem. 1971, 246 (4), 939-945). 
b.2. Activation of gD.sub.2 t with SIAB 
100 ml of gD.sub.2 t (23 mM in Na.sub.2 HPO.sub.4 0.02 M pH 7)+100 ml 
gD.sub.2 t I.sup.125 are incubated for 30 min at 37.degree. C. with 2 ml 
SIAB (25 mM in DMSO) which corresponds to a molar ratio succinimide/lysine 
of 2. The excess of cross-linker is eliminated by dialysis (2 hours 
against Na.sub.2 HPO.sub.4 0.02 M pH 8) and the reaction mixture is 
concentrated to 100 ml by ultrafiltration on an YM 10 centricon. 
b.3. Coupling to HBsAg particle 
gD.sub.2 t (100 ml), concentrated (1 mg/ml) and SIAB-activated, is 
incubated with 53 ml of HBsAg particles (1 mg/ml in Na.sub.2 HPO.sub.4 10 
mM pH 7.2, NaCl 150 mM) for various times at 37.degree. C. 
The initial molar ratio gD.sub.2 /S monomer is 1/1. 
The particulate gD.sub.2 t is purified by a 1.5 M CsCl gradient (45 hours, 
65000 rpm in a 70.1 Ti rotor). 
b.4. Quantification of gD.sub.2 t coupled per particle 
50 ml of water are added to a vial of enzymobeads. After one hour, 50 ml of 
Na.sub.2 HPO.sub.4 0.2 M pH 7.2, 25 ml gD.sub.2 t (1 mg/ml), 0.5 mCi 
NaI.sup.125 (Amersham) and 25 ml 1% b-D-Glucose are added. 
After 20 min at room temperature, the reaction is completed and the 
iodinated protein is separated from free iodine by chromatography on DOWEX 
Ag 1.times.8 resin saturated by BSA 1%. 
The specific activity of the gD.sub.2 t involved in the coupling may be 
determined by the radioactivity detected in the mixture of labeled and 
non-labeled gD.sub.2 t. The amount of gD.sub.2 t coupled to particles may 
be determined by this specific activity. 
a. Characterization of gD.sub.2 t 
a. 1. Quantitative determination of sulfhydryl groups 
No free thiol is detected on the gD.sub.2 t by DTNB. This result fits the 
aminoacid sequence of the protein. The truncated gD.sub.2 t molecule used 
(283 aa) contains 6 cysteine residues, each involved in disulfide bridges 
that constitute discontinuous epitopes. Therefore, gD.sub.2 t is an ideal 
molecule for the activation step with a heterobifunctional cross-linker 
without risk of homopolymerisation. 
a.2. Quantitative determination of lysine residues 
The number of free amino groups are detected by TNBS either on the native 
or on the SIAB activated gD.sub.2 t. The number of free lysines decreases 
as a function of the excess of SIAB. 
With a molar ratio SIAB/lysine of 2, four residues are activated. 
The number of detected lysines on the native protein (10) is close to the 
number determined in the amino acid sequence (11). 
b. Activation of gD.sub.2 t by SIAB 
After an activation of 30 min at 37.degree. C. with a molar ratio of 
SIAB/lysine of 2, the absence of homopolymers of gD.sub.2 t is checked by 
gel filtration. 
On a TSK 3000 column, the homopolymers elute in the void volume (8 min) and 
the monomeric gD.sub.2 t has a retention time of 15 min. 
Despite the absence of cysteine residues in the protein, formation of 
homopolymers is observed when gD.sub.2 t is activated at a concentration 
of 2.5 mg/ml. An aspecific reactivity of the halogen in SIAB for lysine, 
methionine or histidine residues may explain this phenomenon (see Means 
and Feeney, Chemical Modifications of Proteins; Holden Day publ., 1971, 
page 107). 
Initial protein concentration is a decisive factor in homopolymerisation 
events. If gD.sub.2 t is activated at a concentration of 0.5 mg/ml, the 
formation of homopolymers decreases from 50 to 10%. 
c. Coupling to HBsAg particle 
The HBsAg-gD.sub.2 t conjugate obtained after a 30 min, 2 hrs or over night 
incubation at 37.degree. C. is purified by CsCl gradient. The gD.sub.2 t 
homopolymers have a different density to the carrier and do not 
contaminate the conjugate. The yield of coupling increases with time as 
shown in Table 2. 0.2 gD.sub.2 t molecules are coupled per S monomer 
(twenty per particle) as calculated by radioactivity detected in the 
particle's density area. 
Influence of incubation on the yield of coupling: 
______________________________________ 
Time gD.sub.2 t/S monomer 
______________________________________ 
30 min 0.08 
2 hrs 0.14 
15 hrs 0.20 
______________________________________ 
The formulations of the present invention are very effective in inducing 
protective immunity, even with very low doses of antigen (e.g. as low as 5 
.mu.g rgD.sub.2 t). 
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 an 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, 
Md., U.S.A 1978. Encapsulation within liposomes is described, for example, 
by Fullerton, U.S. Pat. No. 4,235,877. Conjugation of proteins to 
macromolecules is disclosed, for example, by Likhite, U.S. Pat. No. 
4,372,945 and by Armor et al., U.S. Pat. No. 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 .mu.g of protein, preferably 2-100 .mu.g, most 
preferably 4-40 .mu.g. 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 treat, 
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. 
Comprison of Adjuvant Efficacy of a Recombinant Herpes Simpex 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.sub.2 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 the Antigen 
As it is well known in the art, HSV rgD.sub.2 t is a genetically engineered 
recombinant truncated glycoprotein produced in transfected Chinese hamster 
ovary (CHO) cells (see for example European Patent No. 0189417). 
2. Anitgen-Adjuvant Preparations and Immunization Schedules 
Two separate experiments were performed to evaluate the protective immunity 
of several rgD.sub.2 t formulations in the guinea pig model. In the first 
experiment, groups of guinea pigs were immunized three times with a low 
antigen dose (5 .mu.g of rgD.sub.2 t) 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. 
2.1. Antigen-Adjuvant Preparations 
In the first experiment, guinea pigs were immunized with the following 
adjuvant preparations. Each dose (5 .mu.g) 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 .mu.g of purified rgD.sub.2 t was adsorbed overnight at 4.degree. C. 
on aluminium hydroxide (alum) corresponding to 0.25 mg equivalents 
Al.sup.3+ in 0.25 ml of 150 mM NaCl 10 mM phosphate buffer pH 6.8. 
2.1.2. gD2t/Aluminium Hyroxidie plus 3D-MPL 
3D-MPL was obtained from Ribi Immunochem Research, Inc. After an overnight 
adsorption of 5 .mu.g gD.sub.2 t 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 .mu.g 3D-MPL was then added to 
the alum-bound rgD.sub.2 t. 
For both rgD.sub.2 t/Alum preparations, more than 98% of the rgD.sub.2 t 
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 .mu.g rgD.sub.2 t in aqueous 
phase, yielding a 1% oil in water emulsion containing 100 .mu.g 3D-MPL. 
Similar adjuvant formulations prepared as above but containing different 
amounts of rgD.sub.2 t 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. 
rgD.sub.2 t/Alum: Five or 20 .mu.g rgD.sub.2 t; 0.5 mg equivalents 
Al.sup.3+ per 0.5 ml dose. 
rgD2t/Alum plus 3D-MPL: Five or 20 .mu.g rgD.sub.2 t; 0.5 mg equivalents 
Al.sup.3+ ; 50 .mu.g 3D-MPL per 0.5 ml dose. 
rgD2t/3D-MPL in o/w emulsion (R): Five or 20 .mu.g rgD.sub.2 t were 
formulated in an 1% o/w emulsion as described above (2.1.3). A 0.5 ml dose 
contained 5 .mu.g or 20 .mu.g rgD.sub.2 t, 50 .mu.g 3D-MPL in a 1% o/w 
emulsion. 
rgD2t/3D-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. 50 .mu.g of 3D-MPL was then added to the emulsion. 
One volume of this emulsion, containing 3D-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, 50 .mu.g 3D-Mpl and 5 .mu.g or 
20 .mu.g rgD2t 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 .mu.g rgD.sub.2 t 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 .mu.g rgD.sub.2 t/3D-MPL (100 .mu.g) in o/w emulsion (R) 
Group 2 (n=4): 5 .mu.g rgD.sub.2 t/Alum plus 3D-MPL (100 .mu.g) 
Group 3 (n=4): 5 .mu.g rgD.sub.2 t/Alum 
Group 4 (n=5): Alum alone 
Group 5 (n=5): 3D-MPL (100 .mu.g) alone 
Group 6 (n=8): untreated 
Animals were bled every 2 weeks for antibody determinations by ELISA 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 rgD.sub.2 t 
formulations are described below. 
In order to compare the protective immunity induced by the rgD.sub.2 t 
formulations, all the guinea pigs were challenged intravaginally with 
10.sup.5 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 rgD.sub.2 t 
formulations was evaluated in larger animal groups. Two antigen doses were 
compared (5 and 20 .mu.g) and different adjuvant composition were tested. 
A dose of 50 .mu.g 3 DMPL was used and its effects compared to the 100 
.mu.g 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): 20 .mu.g rgD.sub.2 t/3DMPL (50 .mu.g) o/w emulsion (R) 
Group II (n=8): 5 .mu.g rgD.sub.2 t/3DMPL (50 .mu.g) o/w emulsion (R) 
Group III (n=10): 20 .mu.g rgD.sub.2 t/3DMPL (50 .mu.g) o/w emulsion (S) 
Group IV (n=10): 5 .mu.g rgD.sub.2 t/3DMPL (50 .mu.g) o/w emulsion (S) 
Group V (n=10): 20 .mu.g rgD.sub.2 t/Alum+3DMPL (50 .mu.g) 
Group VI (n=10): 5 .mu.g rgD.sub.2 t/Alum+3DMPL (50 .mu.g) 
Group VII (n=4): Alum+3DMPL (50 .mu.g) alone 
Group VIII (n=4): 3DMPL (50 .mu.g) o/w emulsion (R) alone 
Group IX (n=8): untreated 
Immunizations were given in a 0.5 ml dose. Control groups were immunized 
according to the same protocol with adjuvant alone (Groups VII and VII) or 
were intreated (Group IX). 
A last group (Group X) was immunized with a gD.sub.2 t Alum+3D-MPL 
formulation containing 100 .mu.g 3D-MPL in a 0.25 ml dose, according to 
the protocol described in the first prophylactic experiment: 
Group X (n=10): 5 .mu.g rgD.sub.2 t/Alum plus 3DMPL (100 mg). 
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.sub.2 t (anti-gD.sub.2 t antibodies 
of lgG 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. Read-outs 
Several read-outs were set up to evaluate the specific antibody and cell 
mediated responses induced by vaccination with rgD.sub.2 t 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 rgD.sub.2 t as the coating 
antigen. 
3.1.1. Detection of IgG Antibodies Specific for rgD2t in sera 
Antigen and antibody solutions were used at 50 .mu.l per well. Antigen was 
diluted to a final concentration of 1 .mu.g/ml in PBS and was adsorbed 
overnight at 4.degree. 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.degree. 
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 rgD.sub.2 
t-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.degree. 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.degree. C. Plates were washed as above and 
incubated with a solution of o-phenylenediaiine 
(Sigma) 0.04% H.sub.2 O.sub.2 0.03% in 0.1 M citrate buffer at pH 4.5. 
Color reaction was stopped after 15 min by the addition of H.sub.2 SO.sub.4 
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 linear regression analysis 
using a computer program. 
3.1.2. Detection of IgG Antibodies Specific for rgD2t in Vaginal Washings 
Vaginal washings were first calibrated for their total IgG content by ELISA 
as follows. Maxisorp Immuno-plates were coated overnight at 4.degree. C. 
with 1 .mu.g/ml (50 .mu.l 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 IgG2 (Serotec, Sopar Biochem, Belgium) diluted 1/1000 in saturation 
buffer was added to each well and incubated for 1 h 30 min at 37.degree. 
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 rgD.sub.2 t 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 .mu.g/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.degree. C. One hundred 
microliters of BHK 21 cell suspension at 4.10.sup.5 cells/ml were then 
added to each well (4.10.sup.4 cells/well). The plates were centrifuged 
for 5 minutes at 1000 rpm and incubated for five days at 37.degree. C. in 
the presence of 7% CO.sub.2. 
After this period, the culture medium was gently removed and 100 .mu.l of a 
solution of cristal violet (10% methanol, 90% H.sub.2 O, 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. 
3.3. Delay-Type Hypersensitivity (DTH) 
The different rgD.sub.2 t 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 .mu.g of rgD.sub.2 t 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 .mu.g rgD.sub.2 t 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 rgD.sub.2 t. 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 L R 
Stanberry et al (J. of Infectious Diseases 1982, 146:397-403; 
Intervirology 1985, 24:226-231). 
Briefly, 2 weeks after the last immunization, the guinea pigs were 
challenged with 10.sup.5 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 or redness; 
0.5=redness; 1=vesicle; 1.5=.gtoreq.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). 
The degree of protection provided by the different rgD.sub.2 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 (.gtoreq.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 
.gtoreq.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 &gt;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 .mu.g/ml streptomycin, 100 .mu.g/ml 
gentamycin and 1 .mu.g/ml amphotericin B (swab medium). 
Each swab was broken and put into a sterile 12.times.75 mm 5 ml polyallomer 
tube containing 1 ml of swab medium. The tubes were then vortexed in order 
to take the virus out and frozen until use. For the titration itself, 6 
wells culture plates containing 5.10.sup.5 cells/well were incubated 
overnight at 37.degree. 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 .mu.l of each samples dilution were transferred 
in duplicate on the cell monolayers and kept for one hour at 37.degree. 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.degree. C. After this incubation period, the medium was gently removed 
and 1 ml of a solution of cristal violet (10% methanol, 90% H.sub.2 O, 
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. Results 
In a first set of experiments, groups of guinea pigs were immunized with a 
low antigen dose (5 .mu.g rgD.sub.2 t) formulated in 4 different 
formulations. This suboptimal antigen dose was chosen in order to select 
the more potent rgD.sub.2 t 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 rgD.sub.2 t formulations 
containing 3D-MPL as immunostimulant showed higher ELISA and neutralizing 
titers in their sera than the group immunized with the rgD.sub.2 t/Alum 
vaccine. Good mean neutralizing titers were induced after 3 immunizations 
with rgD.sub.2 t 3D-MPL o/w (R) or rgD.sub.2 t Alum 3D-MPL. 
4.2. Induction of Effector T Cell Response (DTH) 
Skin test results (Table 2) showed that rgD.sub.2 t formulated in 3D-MPL 
o/w emulsion induced the strongest DTH response. A specific DTH response 
was also induced by rgD.sub.2 t Alum 3D-MPL. Similar experiments conducted 
in mice also revealed that rgD.sub.2 t combined with Alum plus 3D-MPL was 
very potent in inducing an in vivo effector T cell response, in contrast 
to rgD.sub.2 t 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 FIG. 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 rgD.sub.2 t 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 
rgD.sub.2 t/Alum 3D-MPL. This group never developed herpetic vesicles 
during the observation period (lesion score&lt;1). Moreover, very low viral 
replication could be detected in the vaginal swabs collected. In contrast 
animals rgD.sub.2 t adsorbed on alum were poorly protected (75% skin 
lesion incident). 
4.4. Effect of Vaccination on HSV Recurrent Disease 
Results are illustrated in FIG. 1 and summarized in Table 4. 
Vaccination with rgD.sub.2 t 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 rgD.sub.2 t Alum treated groups. 
In order to further evaluate the factors influencing the efficacy of 
prophylactic rgD.sub.2 t vaccines containing 3DMPL, a second set of 
experiments was initiated on larger guinea pig numbers. 
Two antigen doses were compared (5 and 20 .mu.g) and different adjuvant 
compositions were tested. Three immunizations were administered at days 0, 
28 and 84. Animals were bled every two weeks for individual antibody 
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.sub.2 t. 
Induction of Humoral Immunity 
Results (Table 5) indicated that all the rgD.sub.2 t formulations 
containing 3D-MPL 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 rgD.sub.2 t 
formulation containing either 5 .mu.g or 20 .mu.g gD.sub.2 t. There was no 
significant difference in the humoral response measured in the groups 
immunized with a rgD.sub.2 t Alum vaccine containing either 50 .mu.g 
3D-MPL (Group VI) or 100 mg 3D-MPL (Group X). 
It is interesting to note that systemic anti-rgD2t antibodies (lgG class) 
could be detected in the vaginal washings of all vaccinated groups. This 
mucosally located anti-rgD.sub.2 t 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 5 .mu.g rgD.sub.2 t 
Alum 3D-MPL formulation containing either 50 .mu.g or 100 .mu.g 3D-MPL 
(Groups VI and X) showed significantly (p&lt;0.05) reduced skin lesion 
severity as well as reduction of skin lesions incidence. 
Very similar results were observed in the group vaccinated with 5 .mu.g 
rgD.sub.2 t in a 3D-MPL o/w emulsion (Group III). In the three vaccinated 
groups, very low viral replication could be detected in the vaginal swabs 
collected 5 days after the challenge. 
Effect 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&gt;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 
rgD.sub.2 t 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.sub.2 t 
3D-MPL 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.sub.2 t (5 .mu.g). 
6. Immunogenicity of gD2t Formulations in Primates 
6.1 Comparative Immunogenicity of rgD.sub.2 t/Alum and rgD.sub.2 t/Alum 
3D-MPL Form 
The immunogenicity of rgD.sub.2 t/Alum and rgD.sub.2 t/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 20 mg rgD.sub.2 t and 0.5 mg equivalents 
AL.sup.3+ /dose. A dose of 50 .mu.g 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 0.5 ml dose (20 rgD.sub.2 
t). 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 intradermally on the belly different rgD.sub.2 t doses (20, 5 and 1 
.mu.g) 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). As 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 rgD.sub.2 t/Alum 
vaccinated monkeys. In contrast, monkeys receiving a third immunization 
with rgD.sub.2 t/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 rgD.sub.2 t combined with Alum plus 
3D-MPL was very potent in inducing an in vivo effector T cell response, in 
contrast to the rgD.sub.2 t Alum formulation. A strong DTH response was 
observed in all rgD.sub.2 t Alum 3D-MPL vaccinated animals skin tested 
with 20 mg rgD.sub.2 t. Specific DTH responses were also measured with the 
lower gD.sub.2 t concentrations (5 and 1g) in the majority of the monkeys 
(3/4 for the 5 .mu.g dose and 2/4 for the 1 .mu.g dose). These rgD.sub.2 t 
doses induced weaker skin test responses than the 20 mg rgD.sub.2 t 
concentration. 
6.2. Immunogenicity of rgD.sub.2 t/Alum 3D-MPL Formulations in Rhesus 
Monkeys 
The immunogenicity of rgD.sub.2 t/Alum 3D-MPL vaccines containing different 
rgD.sub.2 t doses (100 .mu.g, 10 .mu.g, or 5 .mu.g) was compared in rhesus 
monkeys. 
6.2.1. Experimental Procedure 
Each formulation contained 0.5 .mu.g equivalents Al.sup.3+ and 50 .mu.g 
3D-MPL per dose. Three groups of rhesus monkeys (4 monkeys/group) were 
immunized three times at days 0, 28 and 77, as follows: 
Group 1: 100 .mu.g rgD.sub.2 t Alum plus 3D-MPL (50 .mu.g) 
Group 2: 20 .mu.g rgD.sub.2 t Alum plus 3D-MPL (50 .mu.g) 
Group 3: 5 .mu.g rgD.sub.2 t Alum plus 3D-MPL (50 .mu.g) 
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 anti-HSV2 antibody 
activity. Good ELISA and neutralizing titers were observed in the three 
vaccinated groups receiving either 100, 20 and 5 mg gD.sub.2 t in 
Alum+3D-MPL. (Data not shown). 
6.3. Conclusions 
Results obtained in cercopithecus aethiops dearly indicate that a rgD.sub.2 
t 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 rgD.sub.2 t 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 rgD.sub.2 t Alum+3D-MPL 
formulation is very effective in inducing a specific humoral response, 
even with low doses of antigen (5 .mu.g or 20 .mu.g rgD.sub.2 t). 
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 .mu.g rgD.sub.2 t). Protection data also show that these 
rgD.sub.2 t 3D-MPL formulations are more potent in providing protection. 
Such 3D-MPL formulations are able to improve specific humoral 
(neutralizing antibodies) and effector cell mediated (DTH) immune 
responses. 
Furthermore, the rgD.sub.2 t 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. 
TABLE 1 
__________________________________________________________________________ 
Anti-HSV antibody response in sera of guinea pigs iminunized 
with rgD.sub.2 t formulations before and after viral challenge. 
Vaccine.sup.(1) Pre-challenge.sup.(2) 
Post-challenge.sup.(3) 
Group 
Antigen 
Adjuvant 
ELISA titer 
Neutralizing titer 
ELISA titer 
Neutralizing titer 
__________________________________________________________________________ 
1 rgD.sub.2 t 
3D-MPL o/w (R) 
81291 .+-. 20822 
1600 68720 .+-. 24648 
2200 .+-. 765 
2 rgD.sub.2 t 
Alum 3D-MPL 
39897 .+-. 30165 
2000 .+-. 800 
27224 .+-. 13093 
1800 .+-. 765 
3 rgD.sub.2 t 
Alum 20346 .+-. 23704 
600 .+-. 400 
28622 .+-. 24024 
1333 .+-. 461 
4 -- Alum &lt;100 &lt;50 737 .+-. 878 
142 .+-. 85 
5 -- 3D-MPL &lt;100 &lt;50 259 .+-. 244 
1275 .+-. 1304 
6 untreated 
-- &lt;100 &lt;50 225 .+-. 194 
119 .+-. 141 
__________________________________________________________________________ 
.sup.(1) rgD.sub.2 t dose = 5 .mu.g. Animals were immunized three times a 
days 0, 28 and 95. They were challenged 2 weeks later with 10.sup.5 pfu 
HSV2. 
.sup.(2) Sera collected the day before challenge (= 14 days after the 
third immunization) 
.sup.(3) Sera collected 2 weeks after challenge. 
Values are given as arithmetic mean titers .+-. SD. 
TABLE 2 
______________________________________ 
Skin Test Results (DTH) in guinea pigs vaccinated 
with rgD.sub.2 t formulations. 
Guinea 
Pig 24 hr reading 
48 hr reading 
Formulation # E (mm) E (mm) 
E (mm) 
I (mm) 
______________________________________ 
rgD.sub.2 t 3D-MPL o/w (R) 
1 20 15 14 10 (N) 
2 15 10 10 3 
3 20 17 (N) 
15 12 (N) 
rgD.sub.2 t Alum 3D-MPL 
1 10 8 10 4 
2 15 12 12 3 
3 11 9 12 0 
Alum 3D-MPL 1 0 0 0 0 
2 0 0 0 0 
3 0 0 0 0 
untreated 1 0 0 0 0 
2 0 0 0 0 
______________________________________ 
Guinea pigs were immunized at days 0 and 21 with 5 .mu.g rgD.sub.2 t 
formulation (given intramuscularly). They were given intradermally 5 .mu. 
rgD.sub.2 t in saline at day 29. Skin test was read at 24 h and 48 h. 
E = erythema at site of ID injection in millimeters. 
I = induration at site of ID injection in millimeters. 
N = necrosis at skin test site. 
TABLE 3 
__________________________________________________________________________ 
Effect of immunization with rgD.sub.2 t formulations on the clinical 
and virological course of primary HSV2 infection in guinea pigs. 
Vaccine.sup.(1) Incidence of 
Skin Lesion 
Vaginal Virus 
Group 
Antigen 
Adjuvant 
Skin Lesions.sup.(2) 
Severity.sup.(3) 
Titers.sup.(4) 
__________________________________________________________________________ 
1 rgD.sub.2 t 
3D-MPL o/w (R) 
0/4 0.1 .+-. 0.3 
0 
2 rgD.sub.2 t 
Alum 3D-MPL 
0/4 1 .+-. 0.4 
6.25 .+-. 12.4 
3 rgD.sub.2 t 
Alum 3/4 4.4 .+-. 2.7 
3575 .+-. 6010 
4 -- Alum 5/5 6.2 .+-. 2.6 
5216 .+-. 6295 
5 -- 3D-MPL 4/5 5.1 .+-. 3.6 
3298 .+-. 4475 
6 untreated 
-- 7/8 7.3 .+-. 4.7 
2214 .+-. 4519 
__________________________________________________________________________ 
.sup.(1) rgD.sub.2 t dose = 5 .mu.g. Animals were immunized three times a 
days 0, 28 and 95. They were challenged 2 weeks later with 10.sup.5 pfu 
HSV2. 
.sup.(2) Number animals showing a lesion score .gtoreq.1 during the 12 
days observation period 
.sup.(3) Sum of the lesion scores (days 1-12), arithmetic mean .+-. SD 
.sup.(4) Peak HSV titer (pfu/ml) in vaginal swabs collected 5 days post 
challenge. 
TABLE 4 
__________________________________________________________________________ 
Effect of immunization with rgD.sub.2 t fomulations on the recurrent 
genital HSV2 disease in guinea pigs. 
Incidence of 
Episodes of 
Vaccine (1) Skin recurrent 
Recurrence.sup.(4) 
Group 
Antigen 
Adjuvant 
Lesions.sup.(2) 
disease.sup.(3) 
days Numbers 
__________________________________________________________________________ 
1 rgD.sub.2 t 
3D-MPL o/w (R) 
1/4 1 .+-. 2 
0.7 .+-. 1.5 
2 rgD.sub.2 t 
Alum 3D-MPL 
2/4 1 .+-. 0.8 
1.7 .+-. 3.5 
3 rgD.sub.2 t 
Alum 3/3 4.3 .+-. 1.5 
8.3 .+-. 5 
4 -- Alum 4/5 3.8 .+-. 3.3 
7.6 .+-. 6.5 
5 -- 3D-MPL 5/5 2.6 .+-. 1.1 
6 .+-. 4.4 
6 untreated 6/8 3.5 .+-. 2.2 
9.9 .+-. 6 
__________________________________________________________________________ 
.sup.(1) rgD.sub.2 t dose 5 .mu.g. Animals were immunized three times at 
days 0, 28 and 95. They were challenged 2 weeks later with 10.sup.5 pfu 
HSV2. 
.sup.(2) Number animals showing a lesion score .gtoreq.1 during the 
observation period (days 13-60) 
.sup.(3) One recurrent episode is preceded and followed by a day without 
lesion and characterised by at least two days with erythema (score = 0.5) 
or one day with vesicle(s) (lesion score .gtoreq.1). Results expressed aa 
arithmetic mean .+-. SD (observation period: days 13-60). 
.sup.(4) Total days animals experienced a recurrent herpetic episode, 
arithmetic mean .+-. SD (observation period: days 13-39). 
TABLE 5 
__________________________________________________________________________ 
COMISON OF THE EFFECT OF DIFFERENT ADJUVANT 
FORMULATIONS ON THE IMMUNOGENICITY OF rgD2t IN GUINEA PIGS 
Anti HSV antibody response 
Anti-HSV antibody response 
after three immunizations - 
after two immunisations 
prechallenge titer.sup.(3) 
rgD2t VACCINE.sup.(1) 
IN SERA IN VAGINAL.sup.(5) 
IN SERA.sup.(4) 
GROUP 
dose 
Adjuvant Elisa titer 
Neutral titer 
WASHINGS 
Elisa titer 
Neutral titer 
__________________________________________________________________________ 
I 20 .mu.g 
3DMPL (50 .mu.g) o/w (R) 
31462 .+-. 9087 
850 .+-. 396 
0.780 .+-. 0.376 
19958 .+-. 10171 
3400 .+-. 1994 
II 5 .mu.g 
3DMPL (50 .mu.g) o/w (R) 
35015 .+-. 14395 
412 .+-. 264 
1.000 .+-. 0.177 
51688 .+-. 40120 
4342 .+-. 2879 
III 20 .mu.g 
3DMPL (50 .mu.g) o/w (S) 
16720 .+-. 12641 
1380 .+-. 758 
0.700 .+-. 0.232 
36647 .+-. 24126 
4080 .+-. 2883 
IV 5 .mu.g 
3DMPL (50 .mu.g) o/w (S) 
14992 .+-. 9885 
840 .+-. 571 
0.570 .+-. 0.200 
45082 .+-. 24221 
4560 .+-. 2502 
V 20 .mu.g 
Alum 3DMPL (50 .mu.g) 
14452 .+-. 7476 
740 .+-. 499 
0.620 .+-. 0.175 
16015 .+-. 7846 
3280 .+-. 2276 
VI 5 .mu.g 
Alum 3DMPL (50 .mu.g) 
10174 .+-. 4219 
420 .+-. 301 
0.520 .+-. 0.175 
20488 .+-. 9562 
2640 .+-. 1510 
VII -- Alum 3DMPL (50 .mu.g) 
&lt;100 &lt;50 &lt;0.020 &lt;100 &lt;50 
VlII 
-- 3DMPL (50 .mu.g) o/w (R) 
&lt;100 &lt;50 &lt;0.020 &lt;100 &lt;50 
IX -- untreated &lt;100 &lt;50 &lt;0.020 &lt;100 &lt;50 
X 5 .mu.g 
Alum 3DMPL (100 .mu.g) 
4602 .+-. 3953 
163 .+-. 151 
0.671 .+-. 1.187 
16588 .+-. 6945 
2560 .+-. 1678 
__________________________________________________________________________ 
.sup.(1) Animals were immunized three times at days 0, 28 and 64. They 
were challenged 2 weeks later with 10.sup.5 pfu HSV2. 
.sup.(2) Sera and vaginal washings collected 14 days after the second 
immunization. 
.sup.(3) Sera collected the day before challenge (= 14 days after the 
third immunization). 
.sup.(4) Values are given as arithmetic mean titers .+-. SD 
.sup.(5) Corresponds to the optical density (at 492 nm) per 0.5 .mu.g/ml 
total IgG measured in the standard antigD2t ELISA assay, arithmetic mean 
.+-. SD. 
TABLE 6 
__________________________________________________________________________ 
EFFECT OF IMMUNIZATION WITH rgD2t FORMULATIONS ON THE CLINICAL AND 
VIROLOGICAL COURSE OF HSV2 INFECTION IN GUINEA PIGS 
VACCINE 
5 .mu.pg rgD2t 
5 .mu.g rgD2t 
5 .mu.g rgD2t 
CONTROLS 
3DMPL o/w (s) 
Alum 3DMPL (50 .mu.g) 
Alum 3DMPL (100 .mu.g) 
Groups VII - VIII - 
Group III 
Group VI Group X IX 
__________________________________________________________________________ 
PRIMARY HSV2 INFFECTION 
Incidence of skin lesions (%) 
1/9 11% 
1/10 10% 0/10 0% 12/14 86% 
Skin lesion severity 
1.2 .+-. 1 
0.7 .+-. 0.7 
0.9 .+-. 1 8.6 .+-. 5.1 
Vaginal virus titers (pfu/ml) 
0 0 1.5 .+-. 4.7 
1077 .+-. 1682 
RECURRENT HSV2 INFFCTION 
Incidence of skin lesions (%) 
2/9 22% 
2/10 20% 3/10 30% 11/14 79% 
Recurrence day number 
` 1 .+-. 2.3 
1.6 .+-. 2.1 
1.6 .+-. 2.7 
7.3 .+-. 6 
Recurrence episode number 
0.2 .+-. 0.4 
0.6 .+-. 0.7 
0.5 .+-. 0.8 
1.9 .+-. 1.2 
__________________________________________________________________________ 
Experimental schedule: 3 immunizations at days 0, 28 and 84. 
Challenge 2 weeks after the last immunization with 10.sup.5 pfu HSV2. 
Primary HSV2 infection: 
(observation period days 4 to 12 post challenge) 
Incidence of skin lesions (%): 
number of animals with vesicle(s) (lesion score .gtoreq.1) 
Skin lesion severity: 
sum of the lesion scores (for the days 4 to 12), arithmetic 
mean .+-. SD 
Vaginal virus tiiers: 
virus titers (pfu/ml) in vaginal swabs collected 5 days 
after the challenge 
Recurrent HSV2 infection: 
(observation period days 13 to 39 post challenge) 
Incidence of skin lesions (%): 
number (%) of animals with vesicle(s) (lesion score 
.gtoreq.1) 
Recurrence day number: 
total days animals experienced a recurrent herpetic disease, 
arithmetic mean .+-. SD. 
Animals were scored positive for recurrent disease either if 
a 0.5 lesion score 
(erythema) was recorded for 2 successive days at least or if 
a lesion score .gtoreq.1 (vesicle(s)) 
was observed at any day. 
Recurrence episode number: 
arithmetic mean, .+-. SD 
TABLE 7 
__________________________________________________________________________ 
DTH RESULTS IN AFRICAN GREEN MONKEYS VACCINATED 
WITH GD2T ALUM OR GD2T ALUM 3D MPL 
24 h reading 48 h reading 
MONKEY gD2t 
gD2t 
gD2t gD2t 
gD2t 
gD2t 
VACCINE 
NB PBS 
1 .mu.g 
5 .mu.g 
20 .mu.g 
PBS 
1 .mu.g 
5 .mu.g 
20 .mu.g 
__________________________________________________________________________ 
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 0 and 28 with 20 .mu.g 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 
TABLE 8 
__________________________________________________________________________ 
COMATIVE IMMUNOGENICITY OF GD2T ALUM AND GD2T ALUM 3D MPL 
FORMULATIONS IN AFRICAN GREEN MONKEYS: SEROLOGICAL RESPONSES 
Post II Post III 
14 days 28 days 
56 days 14 days 
MONKEY ELISA NEUT ELISA ELISA NEUT ELISA NEUT 
VACCINE* 
NB TITER TITER 
TITER TITER TITER 
TITER TITER 
__________________________________________________________________________ 
gD2t ALUM 
JO358 1388 400 6572 1135 200 2050 400 
JO359 4731 400 3232 1866 100 2110 200 
JO363 1376 200 2316 1300 50 1205 50 
JO364 5914 1600 5275 3740 400 6323 800 
JO366 21104 400 3696 2550 200 2302 200 
Arit mean .+-. SD 
6902 .+-. 8190 
600 .+-. 565 
4218 .+-. 1697 
2118 .+-. 1062 
190 .+-. 134 
2796 .+-. 2915 
330 .+-. 290 
gD2t ALUM/ 
JO348 7120 200 10175 4490 200 11082 400 
3DMPL JO349 14437 1600 15409 7361 800 15848 1600 
JO375 7990 800 5170 2953 800 6797 1600 
JO515 6515 200 7246 3660 100 6497 200 
Arit mean .+-. SD 
9015 .+-. 3664 
700 .+-. 663 
9500 .+-. 4442 
4616 .+-. 1934 
475 .+-. 377 
10056 .+-. 4392 
950 .+-. 754 
__________________________________________________________________________ 
*Each vaccine dose contains 20 pg gD2t 
ELISA titer = midpoint titer 
NEUT titer = reciprocal of the highest serum dilution giving 100% 
protection against the cytopathogen effect