Enhanced immunogenic vaccine

Vaccines, composed of antigens absorbed on alum or formulated in an alum-containing composition, have enhanced immunogenicity over existing vaccines. The vaccines of the invention are characterized by their combination with 2-120 .mu.g per kg or 0.1 to 6 mg by dose, preferentially 6-60 .mu.g per kg or 0.3 to 3 mg per dose, of a hydrosoluble muramyl peptide.

BACKGROUND OF THE INVENTION 
(1) Field of the Invention 
The present invention relates to an improvement to vaccines containing alum 
in their compositions and in which the antigen can be partially or 
completely absorbed on alum. The improvement consists in the combining in 
the composition constituting these vaccines of 2 to 120 .mu.g per kg body 
weight, or 0.1 to 6 mg per dose, of muramyl peptide; these vaccines thus 
improved have, in particular, the advantage of conferring an enhanced 
immunity, so that the number of injections needed for the immunization is 
reduced. The only immunoadjuvants currently used in human vaccines are 
aluminum-based compounds such as aluminum hydroxide or phosphates (alum). 
(2) Description of the Related Art 
This applies, in particular, to various vaccines in which the active 
principle consists of an antigen obtained by genetic recombination in 
vitro, such as Havrix, a hepatitis A vaccine in which the active principle 
is an antigen obtained from a hepatitis A virus strain cultured in cell 
culture, and containing aluminum hydroxide in the proportion of 1.5 mg per 
dose (0.5 mg of aluminum) and marketed by the company Smith Kline & French 
(6, Esplanade Charles De Gaulle, 92731 Nanterre Cedex), under the trade 
name Havrix. Two administrations performed at an interval of one month 
enable seroconversion to be induced in 98% of individuals. 
Three recombinant vaccines against the hepatitis B virus are currently on 
the market; they are: 
Genhevac B from Pasteur Merieux Serums et Vaccins (58, avenue Leclerc 69007 
LYON FRANCE), which vaccine contains not more than 3.5 mg of aluminum 
hydroxide per dose, 
Engerix B marketed by Smith, Kline & French, containing 1.9 mg of aluminum 
hydroxide per dose, 
Recombivax HB marketed by Merck, Sharp and Dhome, containing 1.5 mg of 
aluminum hydroxide per dose. 
The hepatitis B vaccines mentioned above are effective only after at least 
three injections at 0, 30 and 60 or 180 days, followed by an obligatory 
booster after one year; in addition, a number of subjects are poor 
responders or nonresponders. In the total population, they represent 
approximately 5%, and the capacity to obtain seroconversion depends 
greatly on the subject's age; it is already significantly smaller from 
30-35 years onwards. Furthermore, there are groups which are especially 
poor responders, namely hemodialysis patients (30 to 45% of that 
population) and renal transplant recipients, 75 to 95% of whom do not 
respond to vaccines. Various investigations have studied this resistance 
to immunization with hepatitis B vaccines: the investigations of Stevens 
et al. (1), Zachoval et al. (2) and Grob PJ et al. (3) should be 
mentioned. This problem of lack of response to a hepatitis B vaccine 
becomes extremely serious in countries in which this viral infection has 
substantial endemic character and where approximately 5% of the population 
represents a considerable number of individuals; it is, in addition, 
especially critical in at-risk populations such as hemodialysis patients, 
who are rightly driven to undergo a large number of blood transfusions 
with the subsequent risks of viral contamination. Lastly, the fact of 
having to perform not less than three injections followed by a booster in 
order to immunize responder patients, the fourth injection coming one year 
after the first, represents an especially difficult and risky follow-up 
situation. 
The problem of developing a vaccine which can, on the one hand, confer on 
existing nonresponders an immunization against a hepatitis virus 
infection, and, on the other hand, enable the number of vaccinating doses 
to be decreased in normal responders, permitting a follow up and a much 
more rigorous prophylaxis in regions where the infection is endemic or in 
at-risk populations, proves to be a medical necessity. 
Another problem arising for the prophylaxis of hepatitis in regions where 
the infection is endemic is to produce a mixed vaccine against the 
different kinds of hepatitis, and in particular hepatitis A and B. At the 
present time, double vaccination against hepatitis A and B necessitates 
six injections, four of them for hepatitis B and two for hepatitis A; 
thus, the prophylaxis of hepatitis would be advantageously improved by: 
improving the immunogenic power of recombinant hepatitis B vaccines, 
the existence of a mixed hepatitis B+hepatitis A vaccine enabling an 
immunization against infection by both of these viruses to be obtained in 
a minimum number of injections, which is impossible at the present time on 
account of the immunizing power of the current hepatitis B vaccines, 
the possibility of immunizing nonresponder subjects, 
the possibility of establishing a prophylaxis after 1 or 2 injections in 
normal responders, 
the possibility of establishing a prophylaxis or an immunization when the 
active principle of the vaccine is a hapten or, more generally speaking, a 
molecule which it is difficult to make immunogenic. 
BRIEF SUMMARY OF THE INVENTION 
Some muramyl peptides, such as murabutide or Nac-Mur-L-Ala-D-Gln n-methyl 
ester and murametide or Nac-Mur-L-Ala-D-Gln methyl ester have already been 
developed in preclinical trials of efficacy and of acute, subacute and 
chronic toxicity, as well as in phase 1 trials in man; excellent tolerance 
was demonstrated following an administration to more than 200 subjects for 
murabutide and 50 subjects for murametide. In order to solve the problem, 
set out above, of improving vaccines, in particular the existing ones 
against hepatitis B, or of creating a mixed hepatitis vaccine displaying 
enhanced immunogenicity, making it possible to reduce the number of 
injections on the one hand and to protect against infection subjects who 
were hitherto nonresponders with the traditional vaccines on the other 
hand, the inventors had the idea of combining muramyl peptides with 
existing vaccines in which the antigens are formulated in the presence of 
alum. The adjuvant doses contain an amount between 2 and 120 .mu.g per kg 
body weight, or 0.1 to 6 mg per single dose, of water-soluble muramyl 
peptide of formula: 
##STR1## 
in which the group R is a hydrogen or a methyl group, X is an L-alanyl or 
L-threonyl residue and R1 and R2 are, independently of one another, 
hydroxyl, amino or O(CH2).sub.x H groups with .sub.x =1, 2, 3 or 4, on the 
understanding that, when X is an L-alanyl residue, at least one of these 
two groups R1 and R2 is always an O(CH2).sub.x H group as defined above. 
Still more especially, the muramyl peptide of the invention is one of the 
ones corresponding to the above formula in which the group R is a methyl 
group and the group R2 is an NH2 group. 
In this formula, the preferred MDP derivatives are murabutide, murametide, 
muradimetide or MDP-threonyl. MDP an abbreviation of Muramyl-dipeptide. 
Another preferred MDP derivative is N.sup..alpha. 
(NAcMur)-L-Lys-D-isoGln-sn-glyceryl dipalmitoyl (abbreviated as 
MDP(L-Lys)-snGDP) which has the property of being water-soluble. 
The preferred adjuvant doses are 6 to 60 .mu.g per kg body weight, 
equivalent, for a person weighing 50 kg, to 0.3 to 3 mg per vaccinating 
dose.

DETAILED DESCRIPTION OF THE INVENTION 
The combination of an MDP derivative as described above with a hepatitis B 
vaccine containing antigens obtained by genetic recombination and absorbed 
on aluminum hydroxide (alum) is especially advantageous: 
either for obtaining a recombinant hepatitis B vaccine which is effective 
in two injections instead of the three or four which are necessary at the 
present time in the existing vaccines, 
or for formulating a mixed vaccine, in particular hepatitis A+hepatitis B, 
enabling a population to be vaccinated with a restricted number of 
injections, 
or for immunizing populations which hitherto consisted of subjects who were 
nonresponders to the existing vaccine, and in particular hemodialysis 
patients or renal transplant recipients. 
Hence the invention relates to hepatitis vaccines absorbed on alum and 
improved by the combining of 2 to 120 .mu.g per kg body weight, or 0.1 to 
6 mg per vaccinating dose, preferably of 6 to 60 .mu.g per kg or 0.3 to 3 
mg per dose, either of a compound of formula (I) or MDP-Lys-GDP. 
The vaccines of the invention can, in particular, be improved with a 
derivative of formula (I) in which 
the group R is a methyl group, and 
the group R.sub.2 is an NH.sub.2 group, and still more especially 
murabutide, murametide, muradimetide, MDP-threonine or MDP-Lys-GDP. 
A vaccine according to the invention is characterized in that it contains a 
recombinant hepatitis B antigen; the vaccine can be: 
either a hepatitis B vaccine 
or a mixed hepatitis A+B vaccine 
or any other mixed vaccine, provided it contains the recombinant hepatitis 
B antigen. 
The hepatitis B vaccines of the invention preferably contain as vaccinating 
antigen the S region and all or part of the pre-S region, especially the 
pre-S2 region, of the surface antigen of the virus. 
The invention also relates to a process for manufacturing a vaccine 
displaying enhanced immunogenicity, characterized in that said vaccine 
consists of antigens absorbed on alum or formulated in a composition 
containing alum, and to which are added 2 to 120 .mu.g per kg body weight, 
or 0.1 to 6 mg per vaccinating dose, preferably between 6 and 60 .mu.g per 
kg or 0.3 to 3 mg per vaccinating dose, of muramyl peptide of formula (I) 
above or MDP-Lys-GDP. 
Advantageously, the muramyl peptide derivative is chosen from murametide, 
murabutide, muradimetide and threonyl MDP. The process of the invention 
enables improved vaccinating compositions against hepatitis B to be 
manufactured, and also makes it possible to manufacture mixed vaccinating 
compositions enabling a population to be vaccinated both against the 
hepatitis B virus, or any mixed vaccine, provided it contains the 
recombinant hepatitis B antigen, and in particular a mixed hepatitis A+B 
or hepatitis A+B+C vaccine, provided their formulation contains alum. 
Lastly, the invention relates to the use of 2 to 120 .mu.g per kg body 
weight, or 0.1 to 6 mg per dose, and preferably of 6 to 60 .mu.g per kg 
weight or 0.3 to 3 mg per vaccinating dose, of a muramyl peptide of 
formula (1) above or of MDP-Lys-GDP, for improving the immunogenicity of a 
vaccine composed of recombinant antigens absorbed on alum or formulated in 
a composition containing alum. This use applies most especially to the 
recombinant hepatitis B vaccine, or to a mixed vaccine, provided it 
contains the recombinant hepatitis B antigen, and in particular a mixed 
hepatitis A+B or hepatitis A+B+C vaccine, provided their formulation 
contains alum. In this use, murabutide, murametide, muradimetide and 
threonyl MDP are more especially preferred. Vaccination against small 
molecules such as haptens is problematical in view of their low 
immunocenicity. In order to stimulate the latter, the hapten may be 
coupled to a carrier molecule; however, there is a risk of producing a 
response against the carrier molecule at the expense of the hapten. The 
use of 0.1 to 6 mg per vaccinating dose, and preferably 0.3 to 3 mg, of 
muramyl peptides of formula (1) or of MDP-Lys-GDP in the manufacture of a 
vaccine comprising at least one hapten as vaccinating antigen also forms 
part of the invention, as do the vaccines containing as immunogen at least 
one hapten coupled or not coupled to a carrier molecule. 
The vaccines comprising at least one hapten and at least one recombinant 
antigen, one of them being absorbed on alum, combined with a muramyl 
peptide derivative, form part of the invention. 
As shown in the nonlimiting examples below and the FIGURE, the combination 
of a derivative of formula (I) or of MDP-Lys-GDP with alum in a 
vaccinating composition has the effect of inducing: 
1) specific T cells: it is known that the T cells capable of proliferating 
in the presence of the antigen, which have hence been sensitized 
specifically to the latter, are the cells responsible for establishment of 
the immune memory. It is by virtue of these cells that the vaccinated 
individual can respond immediately to an attack by the virus against which 
he or she has sought to acquire protection. Hence it is of the utmost 
importance to provide a vaccine capable not only of inducing antibodies on 
a long-term basis, but also memory T cells. This enables the vaccinated 
individuals to benefit from two lines of immune defenses. 
2) an enhanced synthesis of specific antibodies: this enhanced synthesis 
takes place at the expense of the synthesis of IgE, which is known for the 
risk it runs of causing reactions of allergic nature. The examples which 
follow illustrate the capacity of the various muramyl peptides to give a 
synergistic effect with alum. 
3) of inducing a specific response against a hapten used as immunogen in a 
vaccine. In all of the foregoing, these effects are induced more 15 
especially when the weight/weight ratio of the alum to the muramyl 
peptides remains within a certain range, which is from 0.15 to 35 and 
preferably 1 and 10. This ratio is calculated on the following amounts per 
vaccinating dose: 
1 to 3.5 mg of alum, 
0.1 to 6 mg of muramyl peptide. 
The FIGURE and the examples below illustrate the improvement provided by 
the invention, without limiting the latter. 
EXAMPLE 1 
Increase in Specific T Cells 
The surprising effect obtained, and in particular that of rendering 
immunogenic a vaccine which is currently nonimmunogenic in some categories 
of the population, may be linked to the specific increase in a 
proliferative response of the T cells. This increase in the number of T 
cells sensitized to the antigen also makes it possible to evaluate the 
level of immunological memory which has been induced. This is illustrated 
by the following experiment: 
Mice were sensitized to the hepatitis B virus surface antigen; T cell 
proliferation was measured by incorporation of tritiated thymidine after 
incubation with the antigen present in the culture medium. FIG. 1 shows 
this response in four particular cases as regards the adjuvant used: a) no 
adjuvant (open squares); b) alum alone at a dose of 40 .mu.g (black 
triangles); c) murabutide alone at a dose of 100 .mu.g (open circles); and 
d) the combination of murabutide at 100 .mu.g and alum at 40 .mu.g (black 
squares). The results seen in this FIGURE show that alum alone is capable 
of increasing T cell proliferation, but that the combination with 
murabutide induces a response which is 3 times as high as that obtained in 
the presence of alum alone, as shown by measuring the incorporation of 
tritiated thymidine (45,000 CPM versus 15,000). 
The protocol of this experiment is as follows: Balb/C mice received 5 .mu.g 
of HBS antigens by subcutaneous injection at the base of the tail in 0.1 
ml of PBS buffer with or without adjuvants. Two weeks later, the T 
lymphocytes were cultured in PBS or with increasing doses of HBS antigens, 
as shown as abscissae. After five days, the proliferative response was 
measured by incorporation of tritiated thymidine. The results in FIG. (1) 
are shown as the average number of counts per minute in triplicate. 
EXAMPLE 2 
Effect of Murabutides on the Synthesis of Specific Antibodies 
The effect of adding an MDP derivative of formula (I) or MDP-Lys-GDP in the 
presence of alum can also be demonstrated by a relative increase in the 
response in terms of specific immunoglobulins following the vaccinating 
effect. A recombinant hepatitis B vaccine absorbed on alum was used. 
Various experiments were carried out, either on Swiss mice or on Balb/C 
mice; they demonstrate consistently the effect of adding murabutide on the 
hepatitis B vaccines absorbed on alum. Furthermore, these experiments 
showed reproducibly that the level of the IgG type antibodies is increased 
while that of the IgE antibodies is decreased, indicating that the use of 
the muramyl peptide enabled the allergic type risks of the vaccine to be 
reduced. 
a) Two groups of 40 mice were immunized with two injections of vaccine at 
an interval of 1 month in the presence or absence of murabutide. 70% of 
the animals treated with the murabutide have antibody titers (measured 
using the EIA AUSAB kit, Abbot Laboratories) above 50 mIU, this being the 
case for more than 4 months, whereas only 25% of the controls show this 
titer, which is, furthermore, transient since it has already slumped after 
2 months. 
b) Table 1 shows the aggregate of the results obtained in 6 similar 
experiments. The anti-HBS antibody titers are expressed in milliunits per 
ml as a function of time. One control group received two vaccinations, a 
second control group received three vaccinations and an experimental group 
received two injections of the vaccine with the addition of murabutide. 
TABLE 1 
______________________________________ 
Antibodies 
Antibodies 
Antibodies 
Antibodies 
Immunization D20 D40 D70 D140 
______________________________________ 
HBS + alum 
30 120 170 250 
2 injections 
HBS + alum 30 120 950 1200 
3 injections 
HBS + alum + 40 300 1500 2600 
murabutide 
2 injections 
______________________________________ 
The Swiss mice (8 per group) received by subcutaneous injection 1 .mu.g of 
HBS antigen and 135 .mu.g of alum with or without 100 .mu.g of murabutide 
on day 0. They receive a booster injection on day 30. On day 60, only the 
control group with alum and without murabutide receives a third injection. 
The antibodies are measured by EIA. 
It is clearly apparent that the animals which have received only two 
vaccinations with the murabutide have an antibody titer higher than that 
of the animals which have received three injections of the vaccine alone; 
as regards the controls which have received two injections, the titer of 
their antibodies remains modest and decreases rapidly. 
These experiments show that the improvement of the existing hepatitis B 
vaccines by adding a muramyl peptide as defined in the formula (I), and 
more especially murametide, murabutide, muradimetide or MDP-Lys-GDP, can 
make it possible to solve a problem of the magnitude of the one arising, 
namely to vaccinate responder individuals in 1 or 2 injections and to be 
able to vaccinate populations which are currently resistant to 
vaccination, either naturally or following treatments such as hemodialysis 
or a transplantation. 
EXAMPLE 3 
Effect of Murametide on the Synthesis of Specific Antibodies 
Similar results were obtained using murametide and muradimetide, as is 
shown by the following experiments: 
Balb/C mice (8 per group) received by subcutaneous injection 1 .mu.g of HBS 
antigen and 135 .mu.g of alum with or without 100 .mu.g of murametide on 
days 0 and 30. The antibodies are measured by ELISA on days 23 and 54. 
TABLE 2 
______________________________________ 
Immunization Antibodies D28 
Antibodies D54 
______________________________________ 
HBS + alum (Control) 
35 520 
HBS + alum + 245 2700 
murametide 
______________________________________ 
EXAMPLE 4 
Effects of Muradimetide and of MDP-Lys-GDP on the Synthesis of Specific 
Antibodies 
In a separate experiment performed on Swiss mice receiving the same doses 
of vaccine and of adjuvant as those mentioned in Example 3, muradimetide 
and MDP-Lys-GDP were capable of inducing primary and secondary responses 3 
to 4 times as high as those of the controls. 
TABLE 3 
______________________________________ 
Immunization Antibodies D21 
Antibodies D80 
______________________________________ 
HBS + alum (Control) 
10 700 
HBS + alum + muradimetide 60 2250 
HBS + alum + MDP-Lys-GDP 35 2600 
______________________________________ 
The invention may also be applied to other antigens as shown in Example 5. 
EXAMPLE 5 
Efficacy of the Combination of Murabutide and Alum for Increasing the 
Humoral Response to a .beta.HCG Vaccine (.beta.HCG Conjugated to Tetanus 
Toxoid) 
The antigen is a conjugate containing .beta. chain of chorionic gonatropic 
hormone (.beta.HCG) coupled to tetanus toxoid (TT) (20% of .beta.HCG in 
the conjugate). This conjugate is designed for use as a contraceptive 
vaccine, since anti-HCG antibodies block the implantation and development 
of the egg. 
Balb/C mice (groups of 8 females) received subcutaneously 10 .mu.g of 
.beta.HCG conjugated to the toxoid. The antigen was administered either 
alone or in an emulsion of Freund's incomplete (FIA) or with alum (200 
.mu.g) or with murabutide (100 .mu.g) or with alum and murabutide. The 
antibodies which combine with HCG were measured by ELISA on day 20. The 
.beta.HCG-TT conjugate is administered absorbed on alum, and the results 
obtained shown in Table 4 below. 
TABLE 4 
______________________________________ 
Efficacy of the combination of murabutide and alum for 
increasing the humoral response to a .beta.HCG vaccine 
(.beta.HCG conjugated to tetanus toxoid) 
Adjuvant Antibody response on day 20 
______________________________________ 
None (Control) 
12,500 
FIA (oil) 26,000 
Alum 15,500 
Murabutide 53,000 
Alum + murabutide 200,000 
______________________________________ 
It is clear that, after a single injection of vaccine absorbed on alum and 
with murabutide as adjuvant, very high titers of antibodies which 
recognize the HCG hormone are obtained. Separate experiments showed us 
that these antibodies are biologically active and neutralize the activity 
of HCG. The data obtained in Example 5 are strongly backed up by those 
described in Example 6 below: 
EXAMPLE 6 
Hapten-carrier System in Which the Muramyl Peptide/Alum Combination Enables 
High Antibody Titers but, Most Especially, Biologically Active Antibodies 
to be Obtained, an Essential Result for the Success of a Vaccination 
In Example 6, the objective of the vaccination is to obtain antibodies 
capable of neutralizing the biological activity of a hormone. This hormone 
is a decapeptide produced by the hypothalamus and is absolutely devoid of 
immunogenicity. Even after coupling to a "carrier" molecule, it is 
incapable after several injections of inducing the production of a 
significant level of antibodies. 
The hormone in question, called luteineizing hormone-releasing hormone or 
LH-RH, is synthesized at central level in the hypothalamus and, following 
a cascade of interactions, it controls completely the synthesis of the sex 
hormones in the male or the female, and especially the production of 
testosterone. Consequently, LH-RH controls the development and functioning 
of the testes, the prostate and the seminal vesicles. If the LH-RH level 
is low, but, most especially as far as we are concerned, if its activity 
is neutralized by biologically active antibodies, there is a decrease in 
the level of circulating testosterone and involution of the sex organs. It 
has been shown by many authors, and in all the species tested (mouse, rat 
and guinea pig, livestock), that an LH-RH conjugate with tetanus toxoid 
(LH-RH-TT) enables biologically active antibodies (that is to say ones 
capable of neutralizing LH-RH activity) to be obtained only if it is 
administered combined with a very potent adjuvant preparation. The 
preparation generally used is Freund's complete adjuvant (FCA), which is 
also well known to have substantial side effects that absolutely prohibit 
its use in human or veterinary medicine. Now, there are cancers (prostate 
cancers) whose development is linked to the secretion of testosterone. It 
is hence important, if it is desired to develop an immunological treatment 
by vaccination for this type of cancer, to find an effective formulation 
of LH-RH which is compatible with a clinical administration. The same need 
exists if it is desired to use an anti-LH-RH vaccine in order to produce a 
castration effect in animals. The muramyl peptide/alum combination meets 
these requirements, as demonstrated in the example which follows. 
In the experiment described, male Sprague-Dawley breed rats weighing 250 g 
received three injections of LH-RH-TT conjugate (50 .mu.g) at 30-day 
intervals. This conjugate was administered: 
a) alone, 
b) in an emulsion of FCA, 
c) mixed with 100 .mu.g of murametide, 
d) absorbed on alum (400 .mu.g), 
e) absorbed on alum and with the addition of murametide. 
The anti-LH-RH antibodies are measured at various times during the 
experiment, as are the blood testosterone levels. After 130 days, the 
animals are sacrificed and their sex organs removed for histological 
study. The results of a characteristic experiment are recorded in Table 5 
below. They show that the highest antibody levels were obtained in the 
group receiving FCA, but also in the last group (alum/murametide). Still 
more importantly, it is only in these two groups that a considerable fall 
in the testosterone level and an involution of the sex organs are 
observed. 
The results of the murametide/alum combination on the production of 
biologically active anti-LH-RH antibodies are shown in Table 5 below. 
TABLE 5 
__________________________________________________________________________ 
Histology of the organs .theta. 
Testes Prostate 
Seminal vesicles 
Number Number Number 
Titer of 
anti-LR-RH 
antibodies 
Testosterone 
level in 
Average of 
Average of 
Average of 
obtained by 
Elisa on days 
ng/ml serum on 
days weight 
animals size 
animals size 
animals 
IMMUNIZATION 
20 40 70 130 20 40 70 130 
(g) affected 
(mm) 
affected 
(mm) 
affected 
__________________________________________________________________________ 
LH-RH-TT &lt;1000 
1200 
3800 
3200 3.5 
1.85 
1.50 
1.10 
2 0/6 2.67 
0/6 6.5 0/6 
LH-RH-TT + 1000 7500 25,000 22,000 2.80 0.60 0.20 0.10 0.4 5/6 
NT.degree. NT 
FCA 
LH-RH-TT + &lt;1000 1100 3500 3500 2.95 1.50 1.35 1.5 1.95 0/6 NT NT 
murametide 
LH-RH-TT + 
1000 3450 
13,000 7100 
2.70 0.70 0.70 
0.80 1.55 1/6 
2.8 1/6 5.6 
1/6 
alum 
LH-RH-TT + 1300 9800 28,000 15,000 2.55 0.50 0.07 0.14 0.5 5/6 2.08 
5/6 3.6 5/6 
alum + 
murametide 
__________________________________________________________________________ 
.theta.The organs were removed, then weighed or measured. After undergoin 
appropriate preparation, they were subjected to anatomopathological 
examinations. The lesions observed in the affected animals are 
characteristic of an absence of stimulation by testosterone. 
.degree.NT = not tested. 
Similar results were obtained using murabutide or MDP-lys-GDP combined with 
alum. 
Examples 5 and 6 described above show that the combination of muramyl 
peptides with antigens absorbed on alum is especially advantageous in the 
case where the vaccinating principle is a hapten necessitating coupling to 
a carrier molecule. This case may arise when the vaccinating principle is 
a peptide, a poorly immunogenic protein or a sugar. 
On the basis of these results, a person skilled in the art can readily 
envisage the production of polyvalent vaccines containing, besides a 
hapten, another vaccinating antigen, provided that one of the vaccinating 
molecules is absorbed on alum and that the vaccinating combination 
contains an MDP derivative according to the invention. 
REFERENCES 
(1) Stevens, C. E., Goodman, A. I., Szmuness, W., Weseley, S. A., Fotino, 
M. Hepatitis B. vaccine: immune responses in haemodialysis patients The 
Lancet 1980, ii; 1211-1213. 
(2) Zachoval, R., Frosner, G., Deinhardt, F. Impfung gegen Hepatitis B. 
Ergehnisse einer Immunogenitatsstudie [Vaccination against hepatitis B. 
Results of an immunogenicity study]. Munchner medizinische Wochenschrift 
1981, 123: 1506-1508. 
(3) Grob, P. J., Binswanger, U., Zaruba, K., Joller-Jemelka, H. I., Schmid, 
M., Hacji, W., Blumberg, A., Abplanalp, A., Herwig, W., Iselin, H. 
Descoeudres, C. Immunogenicity of hepatitis B subunit vaccine in 
hemodialysis and in renal transplant recipients. Antiviral Research 1983, 
3: 43-52.