Vaccine composition containing adjuvants

The present invention provides vaccine compositions comprising 3 De-O-acylated monophosphoryl lipid A and QS21. The vaccines compositions are potent inducers of CTL and .gamma. IFN responses.

The present invention relates to novel vaccine formulations, to methods for 
their production and to their use in medicine. In particular, the present 
invention relates to vaccines containing QS21, an Hplc purified non-toxic 
fraction derived from the bark of Quillaja Saponaria Molina, and 3 
De-O-acylated monophosphoryl lipid A (3 D-MPL). 
3 De-O-acylated monophosphoryl lipid A is known from GB2220 211 (Ribi). 
Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 
5 or 6 acylated chains and is manufactured by Ribi Immunochem Montana. 
QS21 is a Hplc purified non-toxic fraction of a saponin from the bark of 
the South American tree Quillaja saponaria molina and a method for its 
production is disclosed (as QA21) in U.S. Pat. No. 5,057,540. 
The present invention is based on the surprising discovery that 
formulations containing combinations of QS21 and 3 D-MPL synergistically 
enhance immune responses to a given antigen. 
For example, a vaccine formulation of the malarial antigen, RTS, S in 
combination with 3D-MPL and QS21 results in a powerful synergistic 
induction of CS protein-specific cytotoxic T lymphocyte (CTL) response in 
the spleen. 
RTS is a hybrid protein comprising substantially all the C-terminal portion 
of the circumsporozoite (CS) protein of P. falciparum linked, via four 
amino acids of the preS.sub.2 portion of Hepatitis B surface antigen, to 
the surface (S) antigen of hepatitis B virus. Its full structure is 
disclosed in co-pending International Patent Application No. 
PCT/EP92/02591, published under Number WO 93/10152 claiming priority from 
UK patent application No.9124390.7. When expressed in yeast RTS is 
produced as a lipoprotein particle, and when it is co-expressed with the S 
antigen from HBV it produces a mixed particle known as RTS,S. 
The observation that it is possible to induce strong cytolytic T lymphocyte 
responses is significant as these responses, have been shown to induce 
protection against disease in certain animal models. 
The present inventors have shown that the combination of the two adjuvants 
QS21 and 3D-MPL with the recombinant particulate antigen RTS,S results in 
a powerful induction of CS protein-specific CTL in the spleen. QS21 also 
enhances induction of CTL on its own, while 3D-MPL does not. The 
combination can be said to act in a synergistic way, because it has an 
effect that is larger than the sum of the separate effects of each 
adjuvant. The synergy between these two adjuvants for CTL induction is a 
surprising observation which has important implications for the use of 
recombinant molecules as vaccines for induction of CTL-mediated immunity. 
Induction of CTL is easily seen when the target antigen is synthesised 
intracellularly (e.g. in infections by viruses, intracellular bacteria, or 
in tumours), because peptides generated by proteolytic breakdown of the 
antigen can enter the appropriate processing pathway, leading to 
presentation in association with class I molecules on the cell membrane. 
However, in general, pre-formed soluble antigen does not reach this 
processing and presentation pathway, and does not elicit class I 
restricted CTL. Therefore conventional non-living vaccines, while 
eliciting antibody and T helper responses, do not generally induce 
CTL-mediated Immunity. The combination of the two adjuvants QS21 and 
3D-MPL can overcome this serious limitation of vaccines based on 
recombinant proteins, and induce a wider spectrum of immune responses. 
CTLS specific for CS protein have been shown to protect from malaria in 
mouse model systems (Romero et al. Nature 341:323 (1989)). In human trials 
where volunteers were immunised using irradiated sporozoites of P. 
falciparum, and shown to be protected against subsequent malaria 
challenge, induction of CTL specific for CS epitopes was demonstrated 
Malik et al. Proc. Natl. Acad. Sci. U.S.A. 88:3300 (1991)),. 
The ability to induce CTLs specific for an antigen administered as a 
recombinant molecule is relevant to malaia vaccine development, since the 
use of irradiated sporozoites would be impractical, on the grounds of 
production and the nature of the immune response. 
In addition to malaria vaccines, the ability to induce CTL responses would 
benefit vaccines against herpes simplex virus, cytomegalovirus, human 
Immunodeficiency virus, and generally all cases where the pathogen has an 
intracellular life stage. 
Likewise, CTL specific for known tumour antigens could be induced by a 
combination of a recombinant tumour antigen and the two adjuvants. This 
would allow the development of anti cancer vaccines. 
In certain systems, the combination of 3D-MPL and QS21 have been able to 
synergistically enhance interferon .gamma. production. The present 
inventors have demonstrated the synergistic potential of 3D-MPL and QS21 
by utilising a herpes simplex antigen known as gD.sub.2 t. gD.sub.2 t is a 
soluble truncated glycoprotein D from HSV-2 and is produced in CHO cells 
according to the methodology Berman et al. Science 222 524-527(1983). 
IFN-.gamma. secretion is associated with protective responses against 
intracellular pathogens, including parasites, bacteria and viruses. 
Activation of macrophages by IFN-.gamma. enhances intracellular killing of 
microbes and increases expression of Fc receptors. Direct cytotoxicity may 
also occur, especially in synergism with lymphotoxin (another product of 
TH1 cells). IFN-.gamma. is also both an inducer and a product of NK cells, 
which are major innate effectors of protection. TH1 type responses, either 
through IFN-.gamma. or other mechanisms, provide preferential help for 
IgG2a immunoglobulin isotypes. 
Glycoprotein D is located on the viral envelope, 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 it is probably the best characterized (Cohen et al. J. 
Virology 60 157-166). It is known to play a central role in viral 
attachment to cell membranes in vivo. Moreover, glycoprotein D has been 
shown to be able to elict neutralizing 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 neutralizing antibody titre in the patients' sera. It is therefore 
apparent that the ability to induce neutralizing 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 neutralizing antibody, but also cellular immunity 
mediated through T-cells, particularly cytotoxic T-cells. 
In this instance the gD.sub.2 t is HSV2 glycoprotein D of 308 amino acids 
which comprises amino acids 1 though 306 of the naturally occurring 
glycoprotein with the addition of Asparagine and Glutamine at the C 
terminal end of the truncated protein. 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 truncated glycoprotein D (rgD.sub.2 t) or equivalent proteins 
secreted from mammalian cells, is preferably used in the vaccine 
formulations of the present invention. 
The formulations of the present invention are very effective in inducing 
protective immunity in a genital herpes model in guinea pigs. Even with 
very low doses of antigen (e.g. as low as 5 .mu.g rgD.sub.2 t) the 
formulations protect guinea pigs against primary infection and also 
stimulate specific neutralising antibody responses. The inventors, 
utilising formulation of the present invention, have also demonstrated 
Effector cell mediated responses of the TH1 type in mice. 
Accordingly, the present invention provides a vaccine or pharmaceutical 
formulation comprising an antigen in conjunction with 3 Deacylated 
monophosphoryl lipid A and QS21. Such a formulation is suitable for a 
broad range of monovalent or polyvalent vaccines. 
Preferably the vaccine formulations will contain an antigen or antigenic 
composition capable of eliciting an immune response against a human or 
animal pathogen, which antigen or antigenic composition is derived from 
HIV-1, (such as gpl20 or gp160), any Feline Immunodeficiency virus, human 
or animal herpes viruses, such as gD or derivatives thereof or Immediate 
Early protein such as ICP27 from HSV-1 or HSV-2, cytomegalovirus (esp 
Human, such as gB or derivatives thereof), Varicella Zoster Virus (such as 
gpI, II or III), or from a hepatitis virus such as hepatitis B virus for 
example Hepatitis B Surface antigen or a derivative thereof, hepatitis A 
virus, hepatitis C virus and hepatitis E virus, or from other viral 
pathogens, such as Respiratory Syncytial virus, human papilloma virus or 
Influenza virus, or derived from bacterial pathogens such as Salmonella, 
Neisseria, Borrelia (for example OspA or OspB or derivatives thereof), or 
Chlamydia, or Bordetella for example P.69, PT and FHA, or derived from 
parasites such as plasmodium or Toxoplasma. 
The formulations may also contain an anti-tumour antigen and be useful for 
immunotherapeutically treating cancers. 
The formulation may also be useful for utilising with herpetic light 
particles such as described in International Patent Application No. 
PCT/GB92/00824 and, International Patent Application No. PCT/GB92/00179. 
Derivatives of Hepatitis B Surface antigen are well known in the art and 
include, inter alia, those PreS.sub.1, PreS.sub.2 S antigens set forth 
described in European Patent applications EP-A-414 374; EP-A-0304 578, and 
EP 198-474. 
In a further aspect of the present invention there is provided a vaccine as 
herein described for use in medicine. 
The ratio of QS21:3D-MPL will typically be in the order of 1:10 to 10:1; 
preferably 1:5 to 5:1 and often substantially 1:1. The preferred range for 
optimal synergy is 2.5:1 to 1:1 3D-MPL: QS21. Typically for human 
administration QS21 and 3-D MPL will be present in a vaccine in the range 
1 .mu.g-100 .mu.g, preferably 10 .mu.g-50 .mu.g per dose. Often the 
vaccine will not require any specific carrier and be formulated in an 
aqueous or other pharmaceutically acceptable buffer. In some cases it may 
be advantageous that the vaccines of the present invention will further 
contain alum or be presented in an oil in water emulsion, or other 
suitable vehicle, such as for example, liposomes, microspheres or 
encapsulated antigen particles. 
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 and how it is presented. 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 appropriate immune responses in subjects. Following an 
initial vaccination, subjects may receive one or several booster 
immunisation adequately spaced. 
The formulations of the present invention maybe used for both prophylatic 
and therapeutic purposes. 
Accordingly in one aspect, the invention provides a method of treatment 
comprising administering an effective amount of a vaccine of the present 
invention to a patient.

EXAMPLES 
1.0 Synergy between 3D-MPL and QS21 for induction of Interferon .gamma. 
secretion. 
In order to test the ability of 3D-MPL and QS21 based adjuvant formulations 
of rgD.sub.2 t, to induce effector cell mediated immune responses, groups 
of Balb/c mice were vaccinated, and their draining lymph node cells tested 
for IFN-.gamma. secretion as described below. 
1.1 rgD2t formulations 
This experiment compared three adjuvant formulations: 
i) rgD.sub.2 t in 3D-MPL 
ii) rgD.sub.2 t in QS21 
iii) rgD.sub.2 t in 3D-MPL/QS21 
These formulations were made up as follows. rgD.sub.2 t was produced in CHO 
cells and corresponds to the mature 1-283 amino acids of HSV-2 gD and is 
produced according to the methodology of Berman (supra) and EP 0139417. 
* rgD.sub.2 t/3D-MPL 
5 .mu.g of rgD.sub.2 t/dose are incubated 1 h, under agitation, at room 
temperature, then mixed with a 3D-MPL suspension (25 .mu.g/dose). The 
volume is adjusted to 70 .mu.l/dose using a sodium chloride solution (5M, 
pH 6.5.+-.0.5) and water for injection to obtain a final concentration of 
0.15M sodium chloride. pH is kept at 6.5.+-.0.5. 
* rgD.sub.2 t/QS21 
5 .mu.g rgD.sub.2 t/dose are incubated 1 h at room temperature under 
agitation The volume is adjusted using sodium chloride solution (5M, pH 
6.5.+-.0.5) and water for injection to 70 .mu.l. QS21 (10 .mu.g/dose) is 
then added. pH is kept at 6.5.+-.0.5.and sodium chloride final 
concentration at 0.15M. 
* rgD.sub.2 t/3D-MPL/QS21. 
5 .mu.g rgD.sub.2 t/dose are incubated 1 h at room temperature under 
agitation. 3D-MPL (25 .mu.l/dose) is added as an aqueous suspension. The 
final volume of 70 .mu.l is completed by addition of an aqueous solution 
of QS21 (10 .mu.g/dose) and the pH kept at 6.5.+-.0.5 and the sodium 
chloride concentration at 0.15M. 
1.2 IMMUNISATION 
Mice were injected into the hind footpads with 35 .mu.L/footpad of 
formulation. Thus each mouse received 70 .mu.L. Immunisation were on days 
0, and 14. Animals were sacrificed on day 21. 
1.3 INTERFERON .gamma. ASSAYS 
Popliteal lymph node cells from immnunised mice were stimulated in vitro 
using rgD.sub.2 t at 10, 1, 0.1, 0 .mu.g/ml. Triplicate cultures (200 
.mu.l volumes) were set up in round bottom 96-well microtiter plates, 
using 2.times.10.sup.5 responder cells and 2.times.10.sup.5 irradiated 
(3000 rad) syngeneic naive spleen cells. Culture medium was RPMI 1640 with 
10% foetal calf serum. Aliquots of 100 .mu.l of culture medium from each 
replicate were harvested and pooled for IFN-.gamma.determinations. 
Cultures were assayed at 72 hours. For all assays, a control group using 
ConA (Boehringer Mannheim) at 5 .mu.g/mL was included. This was always 
positive. 
Secretion of IFN-.gamma. was determined using a commercial ELISA assay 
manufactured by Holland Biotechnology (distributed by Gibco). Assays were 
carried out on 100 .mu.l of pooled supernatant from triplicate wells. 
Secretion of IFN-.gamma. above the assay background of 50 pg/.mu.l was 
observed in all three formulation groups (see Table). In addition, a 
synergistic effect between QS21 and 3D-MPL was observed. While each 
adjuvant on its own induced cells capable of secreting IFN-.gamma. in 
response to rgD.sub.2 t, their combination induced more than twice the sum 
of individual responses. 
1.4 Results 
Synergy between QS21 and 3D-MPL for induction of IFN-.gamma. secretion. 
______________________________________ 
QS21/3D-MPL QS21 3D-MPL 
Immunization: rgD2t rgD2t rgD2t 
______________________________________ 
In vitro 10.0 1351 1105 515 
stimulation 
1.0 914 116 192 
(.mu.g/mL gD2t): 
0.1 335 &lt;50 143 
0.0 101 &lt;50 139 
______________________________________ 
IFN-.gamma. is expressed in pg/mL. 
The table clearly shows that the combined vaccine induces 
IFN-.gamma.-secretion in a synergistic manner. 
2.0 Synergy Between 3D-MPL and QS21 for the induction of CTLs 
In order to test the ability of RTS,S particles in 3D-MPL and QS21 based 
adjuvant formulations to induce CTLs, groups of B10 .BR mice were 
immunised and their spleen cells stimulated in vitro and tested in 
cytotoxicity assays on L cells expressing the CS protein. 
2.1 Formulation of RTS,S particles. 
RTS,S particles were formulated in three different compositions: 
1. RTS,S particles ((10.mu.g) with QS21 (10.mu.g) and 3D-MPL (254.mu.g); 
2. RTS,S particles ((10.mu.g) with QS21 (10.mu.g); 
3. RTS,S particles ((10.mu.g) with 3D-MPL (25.mu.g); 
The formulations were made up as follows: 
RTS, S/3 D-MPL 
10 .mu.g of RTS,S particles/dose was incubated at room temperature under 
agitation then mixed with a 3D MPL aqueous suspension (25 .mu.g/dose). The 
volume is then adjusted to 70 .mu.l/dose using water for injections and a 
sodium chloride solution (5N, pH 6.5.+-.0.5) to reach a final 
concentration of 0.15M sodium chloride (pH is kept at 6.5.+-.0.5). 
RTS,S/QS21 
10 .mu.g of RTS,S particles/dose incubated 1 h. at room temperature under 
agitation. The volume is adjusted using water for injection and a sodium 
chloride solution (5N, pH 6.5.+-.05) and completed to a final volume of 70 
.mu. l/ dose with an aqueous solution of QS21 (10.mu.g/dose). pH is kept 
at 6.5.+-.0.5 and sodium chloride final concentration at 0.15M. 
RTS,S /3D-MPL/QS21 
10 .mu.g of RTS,S particles/dose are incubated 1 h. at room teperature 
under agitation then mixed with a 3D-MPL (aqueous suspension (25 
.mu.g/dose) The volume is then adjusted with water for injection and a 
sodium chloride solution (5D pH 6.5.+-.0.5). The final volume is completed 
by addition of an aqueous solution of QS21 (10.mu.g/dose). pH is kept at 
6.5.+-.0.5, and sodium chloride final concentration at 0.15 M. 
2.2 Immunisation of mice with RTS,S particles 
Four to six week old female mice of the strain B10.BR (H-2.sup.k) were 
purchased from IFFA CREDO (France). Groups of 3 animals were immunised by 
intra foot-pad injection of 35 .mu.L of antigen formulation into each hind 
limb. The animals were boosted with a second equal dose of antigen 
injected two weeks later. 
2.3. In vitro stimulation on anti CS CTL 
Two weeks after the boost, spleen cells were harvested and stimulated in 
vitro using syngeneic fibroblasts transfected with the P. falciparum 
circumsporozoite protein gene (7G8 clone). These CS-transfectant cells 
have been described in the paper by Kumar, S. et al. (1988), Nature 
334:258-260. 
The cultures were established in RPMI 1640 medium supplemented with 10% of 
heat inactivated foetal calf serum and usual additives, in conditions well 
known to those of skill in the art. 
Responder cells were cultured at a concentration of 10.sup.6 cells/mL in 
the presence of 10.sup.5 CS-transfectants per mL. To prevent proliferation 
of CS-transfectant cells, these were irradiated using a dose of 
2.times.10.sup.4 rad. The cultures were fed by replacing 1/2 of culture 
medium on day 3 and 6, and tested for cytolytic activity on day 7. 
2.4. Cytotoxicity assay for anti-CS CTL 
Responder cell cultures were harvested, washed, and mixed at ratios varying 
from 100:1 to 0.3:1 with a constant number of 2000 target cells, in 
volumes of 200 .mu.L of medium in V-bottom 96-well plates. Target cells 
were syngeneic fibroblast cells that had been labelled with .sup.51 Cr. 
Two different types of target cells were used: 
1. L cells 
2. CS transfected L cells 
These are described in: Kumar, S. et al. (1988), Nature 334:258-260. 
The assay was incubated for 6 hours at 37.degree. C., then the amount of 
radioactivity released into the supernatant by lysis of target cells was 
determined. Cytolytic activity is expressed as % specific lysis: 
Results: 
______________________________________ 
% Specific lysis by formulation: 
Effector: 
1. RTS,S/ 
target QS21/ 2. RTS,S/ 
3. RTS,S/ 
Target cells: 
ratio 3D-MPL QS21/ 3D-MPL 
______________________________________ 
CS transfected L 
100 58 17 1 
cells 30 53 10 0 
10 47 5 1 
3 27 1 0 
1 11 0 0 
0.3 2 -2 -1 
L cell 100 3 -2 5 
30 -2 1 4 
10 0 -1 2 
3 0 3 4 
1 -1 4 2 
0.3 3 1 2 
______________________________________ 
Immunisation of B10.BR mice with RTS,S adjuvanted with QS21 and 3D-MPL 
(formulation #1) induced in the spleen high levels of CTL specific for the 
circumsporozoite component of RTS,S. Immunisation with RTS,S particles 
adjuvanted with QS21 (formulation #2) also induced CTL in the spleen, but 
only at about 1/30 th of the levels given by formulation #1. RTS,S with 
3D-MPL (formulation #3) did not induce CTL. 
Since the target cells used in this assay do not express MHC class II 
molecules, the effector cells can be assumed to be CD8.sup.+, class I 
restricted CTL. 
3. Other formulation 
Hepatitis B Surface Antigen, Alum 3D-MPL and QS21. 
The preparation B Surface antigen (HBsAg) is well documented. See for 
example Harford et al Develop. Biol. Standard 54 p125 (1983), Gregg et al 
Biotechnology 5 p479 (1987) EP-A-O 226 846 and EP-A-299 108 and references 
therein. 3D-MPL was obtained from Ribi Immunochem, QS21 was obtained from 
Cambridge Biotech, and Aluminium hydroxide was obtained from Superfos 
(Alhydrogel). 
A number of different formulations were made up for studies of cell 
mediated immunity in mice and for studies in Rhesus monkeys. 
3.1 Formulation 1 was made up in phosphate buffer (pH 6.8) to comprise the 
following per 60 .mu.l dose. 
______________________________________ 
20 .mu.g HBsAg 
30 .mu.g Al(OH).sub.3 
30 .mu.g 3D-MPL 
10 .mu.g QS21 
10 mM PO.sub.4.sup.3- 
0.15M NaCl 
______________________________________ 
The formulation was made up in the following manner. 20ptg HBsAg/dose was 
incubated with A1(OH).sub.3 for one hour at room temperature with gentle 
shaking. 3D-MPL was added as an aqueous suspension, and the formulation 
completed by the addition of QS21, phosphate buffer and sodium chloride 
and incubated for one hour at room temperature. The final formulation had 
a pH of between 6.5 and 7.0 and used for foot pad studies in mice. 
3.2 Formulation 2 was made up in a phosphate buffer (pH6.8) to comprise the 
following per 200 .mu.l dose. 
______________________________________ 
1 .mu.g HBsAg 
100 .mu.g Al(OH).sub.3 
50 .mu.g 3D-MPL 
20 .mu.g QS21 
10 mM PO.sub.4.sup.3- 
0.15M NaCl 
______________________________________ 
The formulation was made up in the following manner. HBsAg and A1(OH.sub.3) 
were incubated together for one hour at room temperature with gentle 
shaking. The formulation was completed by the addition of A1(OH).sub.3, 
3D-MPL as an aqueous suspension and QS21, with phosphate buffer and sodium 
chloride solution and incubated again for thirty minutes. The pH of the 
formulation was kept between 6.5 and 7.0 and used for humoral immunity 
studies in mice. 
3.3 Formulation 3 was made up in a similar manner, in a phosphate buffer 
(pH6.5-7.0) to contain the following per 1 ml dose: 
______________________________________ 
10 .mu.g HBsAg 
500 .mu.g Al(OH).sub.3 
50 .mu.g 3D-MPL 
10 .mu.g QS21 
______________________________________ 
The formulation was used for monkey studies. 
4. Conclusions 
The combination of the two adjuvants QS21 and 3D-MPL with the recombinant 
particulate antigen RTS,S resulted in a powerful induction of CS protein 
specific CTL in the spleen. QS21 enhances induction of CTL on its own, 
while 3D-MPL does not. The combination can be said to act in a synergistic 
way, because it has an effect that is larger than the sum of the separate 
effects of each adjuvant. The synergy between these two adjuvants for CTL 
induction is a surprising observation which supports our observation of 
synergy between QS21 and 3D-MPL for induction of T cells capable of 
secreting IFN-.gamma. in response to stimulation with the soluble 
recombinant protein rgD.sub.2 t. This finding has important implications 
for the use of recombinant molecules as vaccines for induction of CTL 
mediated immunity, since the combination of the two adjuvants QS21 and 
3D-MPL can overcome this serious limitation of vaccines based on 
recombinant proteins, and induce a wider spectrum of immune responses than 
hitherto. 
The mouse cell mediated immunogenicity data show that QS21 based 
formulations of rgD.sub.2 t induce a significant synergistic TH 1 type T 
cell response (IFN-.gamma. secretion). 
Such TH1 type T cells have been shown to be involved in induction of 
delayed type hypersensitivity responses in mice. Our own data in 
prophylaxis of HSV disease show that concomitant induction of neutralizing 
antibody titers and antigen specific DTH responses affords the best 
protection against herpes simplex disease. 
Taken together, these data suggested that QS21 formulations of rgD.sub.2 t 
may be effective in inducing a protective response against HSV disease. 
The data presented show an unexpected synergistic effect between 3D 
Monophosphoryl lipid A and QS21, in inducing IFN-.gamma. secreting antigen 
specific T cells. Such a synergy may translate in improved ability to 
induce a protective response against HSV disease, and indeed these 
formulations are effective in protecting against disease in guinea pigs.