FR2730936A1 - Increasing immunogenicity of alum-contg. vaccine - Google Patents

Abstract

Vaccine contg. an antigen absorbed on alum or formulated in an alum-contg. compsn. and having greater immunogenicity than known vaccines contains 2-120 mu g/kg body wt. or 0.1-6 mg/dose of a muramyl peptide of formula (I) or MDP-Lys-GDP (II, NAc-Mur-L- Lys-D-isoGln-glyceryl-sn-dipalmitoyl). R = H or Me; X = L-Ala or L-Thr, and R1,R2 = OH, NH2 or 1-4C alkoxy, \-1 must be alkoxy when X = L-Ala. Also claimed is a method for the prepn. of a vaccine as above.

Description

VACCINE WITH INCREASED IMMUNOGENICITY
The present invention relates to an improvement to vaccines containing alum in their compositions and wherein the antigen can be partially or completely absorbed on alum. The improvement consists in the combination in the constitutive composition of these vaccines of 2 to 120 μg per kg of body weight or 0.1 to 6 mg per dose, of muramyl peptide; these vaccines thus improved have the particular advantage of conferring increased immunity so that the number of injections necessary for immunization is reduced. The only immunoadjuvants currently used in human vaccines are aluminum-based compounds such as aluminum hydroxide or phosphates (alum).

This is the case in particular with different vaccines whose active principle consists of an antigen obtained by in vitro genetic recombination such as Havrix, vaccine against hepatitis A whose active principle is an antigen obtained from a strain of the virus. hepatitis A cultivated in cell culture, and containing aluminum hydroxide at a rate of 1.5 mg per dose (0.5 mg of aluminum) and marketed by the company Smith Kline & BR <
French (6, Esplanade Charles De Gaulle, 92731 Nanterre Cedex) under the trade name of Havrix. Two administrations carried out at one month intervals allow to induce seroconversion in 98% of individuals.

Three recombinant vaccines against hepatitis B virus are currently marketed, these are:
- Genhevac B of Pasteur Mérieux Serums and Vaccines (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 listed above are only effective after at least three injections at 0, 30 and 60 or 180 days followed by a mandatory booster at the end of one year; in addition a number of subjects are few answering or non-answering. In the total population they represent about 5% and the ability to obtain a seroconversion depends very much on the age of the subject, it is already significantly weaker from 30-35 years. In addition there are particularly unresponsive groups, these are hemodialysis (30 to 45% of this population) and kidney transplant recipients, 75 to 95% of whom do not respond to vaccines.Different studies have studied this resistance to cancer. immunization with hepatitis B vaccines: the work of
Stevens et al (1) Zachoval et al (2) Grob PJ et al (3). This problem of non-response to a vaccine against hepatitis B becomes extremely serious in countries where the endemic viral infection is important, and where about 5% of the population represents a considerable number of individuals, it is also particularly critical in populations at risk, such as hemodialysis, which are precisely subject to numerous blood transfusions with the subsequent risk of viral contamination. Finally having to practice at least three injections followed by a booster to immunize the responder patients , the fourth injection coming one year after the first, represents a particularly difficult and risky follow-up.

 The problem of developing a vaccine that can, on the one hand, give existing non-responders immunization against infection with the hepatitis virus and, on the other hand, make it possible to reduce the number of vaccinating doses in normal responders allowing much more rigorous monitoring and prophylaxis in endemic areas or at-risk populations is a medical necessity.

 Another problem for the prophylaxis of hepatitis in endemic areas is the constitution of a mixed vaccine against different hepatitis, including hepatitis A and B. Today, the double vaccination against hepatitis A and B requires six injections, four of which for hepatitis B and two for hepatitis A, also the prophylaxis of hepatitis would be advantageously improved by: - the improvement of the immunogenic power of recombinant vaccines against hepatitis B, - the existence of a mixed hepatitis vaccine B + hepatitis A allowing a minimum of injections to obtain immunization against infection with these two viruses which today is impossible given the immunizing power of current vaccines against hepatitis B, - the possibility of to immunize non-responders, the possibility of prophylaxis after 1 or 2 injections in normal responders, the possibility of prophylaxis or mmunization when the active ingredient of the vaccine is a hapten or more generally a molecule hardly immunogenic.

dans laquelle le groupe R est un hydrogène ou un groupe méthyl ; X est un résidu L-alanyl ou L-thréonyl, et R1 et R2 sont indépendamment l'un de l'autre, des groupes hydroxyle, amino, 0(CH2)xH avec @ = 1, 2, 3 ou 4, étant entendu que, lorsque X est un résidu L-alanyle, I'un au moins de ces deux groupes R1 et R2 est toujours un groupe 0(CH2)xH tel que précédemment défini. Plus particulièrement encore le muramyl peptide de l'invention est l'un de ceux entrant dans la formule ci-dessus dans laquelle le groupe R est un groupe méthyl et le groupe R2 est un groupe
NH2.Some muramyl peptides like 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 efficacy and acute toxicity, subacute and chronic toxicity tests as well as in phase 1 trials in humans; excellent tolerance has been demonstrated following administration to more than 200 subjects for Murabutide and 50 subjects for Murametide.To solve the problem posed above to improve vaccines including those existing against hepatitis B or to create a mixed vaccine against hepatitis with increased immunogenicity to reduce the number of injections on the one hand and to protect against infection those subjects who were previously unresponsive with conventional vaccines on the other hand, the inventors have had the idea of associating muramyl peptides with existing vaccines whose antigens are formulated in the presence of alum. The adjuvant doses contain an amount of between 2 and 120 pu per kg of body weight, or 0.1 to 6 mg per kg of body weight. unit dose of muramyl water-soluble peptide of formula:

wherein the R group 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, O (CH2) xH with @ = 1, 2, 3 or 4, being understood that when X is an L-alanyl residue, at least one of these two groups R1 and R2 is always a group O (CH2) xH as previously defined. Even more particularly, the muramyl peptide of the invention is one of those falling within the above formula in which the R group is a methyl group and the R2 group is a group
NH2.

In this formula the preferred MDP derivatives are murabutide, murametide, muradimetide or MDP-threonyl. Another derivative of the MDP is also preferred is MDP-L-Lys-GDP (Nac
Wall-L-Lys-D-iso Gln glyceryl-sn-dipalmitoyl) which has the property of being water-soluble.

 The preferred adjuvant doses are 6 to 60 μg per kg body weight or, for a 50 kg person, 0.3 to 3 mg per vaccinating dose.

The combination of a derivative of MDP as described above with a hepatitis B vaccine containing antigens obtained by genetic recombination and absorbed on aluminum hydroxide (Alum) is particularly interesting:
- to obtain a recombinant hepatitis B vaccine that is effective in two injections instead of the three or four required in existing vaccines,
- to formulate a mixed vaccine including hepatitis A + hepatitis B, to vaccinate a population with a limited number of injections,
- or to immunize populations that previously consisted of non-responders to the existing vaccine, including hemodialysis or kidney transplant.

The invention therefore relates to hepatitis vaccines absorbed on alum and improved by the combination of 2 to 120 μg per kg of body weight or 0.1 to 6 mg per vaccinating dose, preferably 6 to 60 μg per kg or 0, 3 to 3 mg per dose of either a compound of formula (I) or the MDP-Lys
GDP.

The vaccines of the invention may in particular be improved by a derivative of formula (I) in which
the group R is a methyl group, and
the group R2 is an NH2 group, and more particularly 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; it could be:
- a hepatitis B vaccine
- a mixed hepatitis A + B vaccine
- any other combination vaccine, provided that it contains the recombinant antigen for hepatitis B.

 The hepatitis B vaccines of the invention preferentially contain as vaccinating antigen the S region and all or part of the pre-S region, particularly the pre-S 2 region of the virus surface antigen.

The invention also relates to a method of manufacturing a vaccine having an increased immunogenicity, characterized in that said vaccine consists of antigens absorbed on alum or formulated in a composition containing alum, and in which are added 2 at 120 μg per kg of body weight or 0.1 to 6 mg per vaccinating dose, preferably between 6 and 60 μg per kg or 0.3 to 3 mg per vaccinating dose of muramyl peptide of formula (I) above or MDP -Lys
GDP.

 Advantageously, the derivative of the muramyl peptide is chosen from murametide, murabutide, muradimetide and threonyl MDP. The method of the invention makes it possible to manufacture improved vaccinating compositions against hepatitis B, and also makes it possible to manufacture mixed vaccinating compositions making it possible to vaccinate a population at a time against the hepatitis B virus or any mixed vaccine provided that it contains the recombinant antigen against hepatitis B and in particular a mixed vaccine hepatitis A + B or hepatitis A + B + C, since their formulation contains alum.

 Finally, the invention relates to the use of 2 to 120 μg per kg of body weight or 0.1 to 6 mg per dose and preferably 6 to 60 μg per kg of weight or 0.3 to 3 mg per dose vaccinating a muramyl peptide of formula (1) above or MDP-Lys-GDP, to improve the immunogenicity of a vaccine composed of recombinant antigens absorbed on alum or formulated in a composition containing alum.

This use is particularly applicable to the recombinant vaccine against hepatitis B, or to a mixed vaccine provided that it contains the recombinant antigen against hepatitis B and in particular a mixed vaccine hepatitis
A + B or hepatitis A + B + C, since their formulation contains alum. In this use, murabutide, murametide, muradimetide and threonyl MDP are particularly preferred.

Vaccination against small molecules, such as haptens, is problematic because of their low immunogenicity.

In order to stimulate the latter, the hapten can be coupled to a carrier molecule (carrier in English); but the risk is to provoke a response against the carrier molecule to the detriment of the hapten.

The use of 0.1 to 6 mg per vaccinating dose, and preferably 0.3 to 3 mg of muramylpeptides of formula (1) or of MDP-Lys-GDP in the manufacture of a vaccine comprising at least one hapten as Vaccine antigen is also part of the invention, as well as vaccines containing as immunogen at least one hapten coupled or not to a carrier molecule.

 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 by the nonlimiting examples below and the figures, the combination of a derivative of formula (I) or of MDP-Lys-GDP with alum d in a vaccinating composition has the effect of inducing:
1) Specific T Cells It is known that T cells capable of proliferating in the presence of the antigen which have therefore been sensitized specifically thereto are the cells responsible for the establishment of immune memory. It is thanks to them that the vaccinated individual can immediately respond to an attack by the virus against which he has sought to acquire protection. It is therefore essential to propose a vaccine capable not only of inducing antibodies durably, but also memory T cells. This allows vaccinated individuals to benefit from two lines of immune defenses.

 2) an increased synthesis of specific antibodies: This increased synthesis is detrimental to the synthesis of IgE which are known for the risks they cause to cause reactions of an allergic nature. The following examples illustrate the ability of the various muramyl peptides to synergize with alum.

 3) to induce a specific response against a hapten used as an immunogen in a vaccine.

In all the above, these effects are more particularly induced when the weight / weight ratio between the alum and the muramyl peptides remains at
within a certain range which is from 0.15 to 35 and preferably 1 and 10. This ratio is calculated on the following quantities per vaccinating dose:
-1 to 3.5 mg of alum,
-0.1 to 6 mg of muramylpeptide.

 The figure and the examples below illustrate the improvement provided by the invention without limiting it.

 Figure 1 shows the proliferation of T cells measured by incorporation of tritiated thymidine, as a function of increasing amount of hepatitis antigens.

Example 1: Increase of Specific T Cells
The surprising effect obtained, and in particular that of rendering immunogenic a currently non-immunogenic vaccine in certain categories of the population may be linked to the specific increase in a proliferative response of 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 that has been induced. This is illustrated by the following experiment:
Mice were sensitized to hepatitis B virus surface antigen, T cell proliferation was measured by tritiated thymidine incorporation after incubation with the antigen present in the culture medium. response in four cases with respect to the adjuvant used: (a) no adjuvant (white squares); b) Alum alone at a dose of 40 μg (black triangles); (c) murabutide alone at a dose of 100 μg (white circles) and d)
The combination of murabutide at 100 μg and alum at 40 μg (black squares).

The results indicated in this figure show that the alum alone is capable of increasing the proliferation of T cells, but that the combination with murabutide induces a response 3 times higher than that obtained in the presence of alum alone as shown by the measurement. the incorporation of tritiated thymidine (45,000 CPM versus 15,000).

 The protocol of this experiment is as follows: Balb / C mice received 5 μg 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, T cells were cultured in PBS or with increasing doses of HBS antigens as indicated on the abscissa.

After five days the proliferative response was measured by incorporation of tritiated thymidine. The results of Figure (I) are indicated as the average number of strokes per minute in triplicates
EXAMPLE 2 Effect of Murabutides on the Symptom of Serum Antibodies
The effect of the addition of a CDM derivative of formula (I) or MDP
Lys-GDP in the presence of alum can also be demonstrated by a relative increase in the response to specific immunoglobulins following the vaccinating effect. A recombinant Hepatitis B vaccine absorbed on alum was used. Different experiments were carried out either on Swiss mice or on Balb / C mice; they consistently demonstrate the effect of the addition of murabutide on hepatitis B vaccines absorbed on alum.Furthermore, these experiments have shown reproducibly that the level of IgG antibodies is increased while that of IgE antibodies is increased. decreased indicating that the use of muramyl peptide has reduced the risks of the allergic type to the vaccine.

a) Two groups of 40 mice were immunized with two injections of vaccine at 1 month intervals in the presence or absence of
Murabutide. 70% of the animals treated with murabutide have antibody titers (measured using the EIA AUSAB kit, Laboratories
Abbot) greater than 50 mIU and this for more than 4 months, while only 25% of the witnesses show this title, which moreover is transitory because it is already collapsed after 2 months.

 b) Table 1 represents the cumulative results obtained in 6 similar experiments. Titers of anti-HBS antibodies are expressed in milli-units per ml as a function of time. A control group received two vaccinations, a second control group received three vaccinations and an experimental group received two injections of the vaccine supplemented with murabutide.

Table 1

<tb> Immunization <SEP> Antibody <SEP> Antibody <SEP> Antibody <SEP> Antibodies
<tb><SEP> J20 <SEP> J40 <SEP> J70 <SEP> J140
<tb> HBS + alum <SEP> 30 <SEP> 120 <SEP> 170 <SEP> 250
<tb> 2 <SEP> injections
<tb> HBS <SEP> + <SEP> alum <SEP> 30 <SEP> 120 <SEP> 950 <SEP> 1200
<tb> 3 <SEP> injections
<tb> HBS <SEP> + <SEP> alum <SEP> + <SEP> Murabutide <SEP> 40 <SEP> 300 <SEQ> 1500 <SEQ> 2600
<tb> 2 <SEP> injections
<Tb>
The swiss mice (8 per group) received by subcutaneous injection 1 μg of HBS antigen and 135 μg of alum with or without 100 μg of murabutide at day 0. They receive a booster injection at 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 clear that the animals having received only two vaccinations with murabutide have an antibody titre higher than that of the animals having received three injections of the vaccine alone, as for the controls having received two injections, the titre of their antibodies remains modest and decreases rapidly. .

These experiments show that the improvement of
Existing hepatitis B by the addition of a muramyl peptide as defined in formula (I) and more particularly murametide, murabutide, muradimetide, or MDP-Lys-GDP can solve a problem as important as to vaccinate responders in one or two injections and to vaccinate populations currently resistant to vaccination either naturally or following treatments such as hemodialysis or transplantation.

Example 3 Effect of Murametide on the Synthesis of Specific Antibodies
Similar results were obtained using murametide and muradimetide as shown by the following experiments:
BalblC mice (8 per group) received by subcutaneous injection 1 μg of HBS antigen and 135 μg of alum with or without 100 μg of murametide on days 0 and 30. The antibodies were measured by ELISA on days 28 and 54 .

Table 2

<tb> Immunization <SEP> Antibody <SEP> J28 <SEP> Antibody <SEP> J54
<tb> HBS <SEP> + <SEP> alum <SEP> (Control) <SEP> 35 <SEP> 520
<tb> HBS <SEP> + <SEP> alum <SEP> + <SEP> 245 <SEP> 2700
<tb> Muramétide
<Tb>
Example 4 Effects of Muradimetide and MDP-Lvs-GDP on the Synthesis of Specific Antibodies
In a separate experiment performed in Swiss mice receiving the same doses of vaccine and adjuvant as those indicated in Example 3, muradimetide and MDP-Lys-GDP were able to induce primary and secondary responses 3 to 4 times higher than the controls.

Table 3

<tb> Immunization <SEP> Antibody <SEP> J21 <SEP> Antibody <SEP> J80
<tb> HBS <SEP> + <SEP> alum <SEP> (Control) <SEP> 10 <SEP> 700
<tb> HBS <SEP> + <SEP> alum <SEP> + <SEP> Muradimetide <SEP> 60 <SEP> 2250
<tb> HBS <SEP> + <SEP> alum <SEP> + MDP-Lys-GDP <SEP> 35 <SEP> 2600
<Tb>
The invention can also be applied to other antigens as shown in Example 5.

EXAMPLE 5 Efficacy of the combination of Murabutide and alum opur to improve the humoral response to a BHCG vaccine (BHCG conjugated with tetanus toxoid)
The antigen is a conjugate containing ss chain of choriogonatrope hormone (ssHCG) coupled to tetanus toxoid (TT) (20% ssHCG in the conjugate). This conjugate is intended to be used as a contraceptive vaccine because anti-HCG antibodies block the implantation and development of the egg.

Balb / C mice (groups of 8 females) received subcutaneously 10 g of ssHCG conjugated to the toxoid. The antigen was administered either alone or in a Freund's Incomplete emulsion (FIA) or with alum (200 μg) or with Murabutide (100 μg) or with alum and Murabutide.

Antibodies combining with HCG were measured by ELISA at day 20.

The ssHCG-TT conjugate is administered absorbed on the alum, and the results obtained shown in Table 4 below.

Table 4:
Efficacy of the combination of Murabutide and alum to increase the humoral response to a ssHCG vaccine (ssHCG conjugated with tetanus toxoid)

<tb> Adjuvant <SEP> Response <SEP> antibody <SEP> at <SEP> day <SEP> 20
<tb> None <SEP> (Witness) <SEP> 12,500
<tb> FIA <SEP> (oil) <SEP> 26,000
<tb> Alum <SEP> 15,500
<tb> Murabutide <SEP> 53,000
<tb> Alum <SEP> + <SEP> Murabutide <SEP> 200,000
<Tb>
It is clear that after a single injection of vaccine absorbed on alum and adjuvanted with Murabutide one obtains very high titres of antibodies which recognize the hormone HCG. Separate experiments have shown that these antibodies are biologically active and neutralize HCG activity.

The data obtained in Example 5 are very much enhanced by those described in Example 6 below:
Example 6: hapten-carrier system where muramyl peptide / alum ormet combination to obtain high anticorns titers but especially biolopipuously active antibodies. essential result for the success of a vaccination
In Example 6, the purpose 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 not immunogenic. Even after coupling to a carrier molecule, it is unable after several injections to induce the production of a significant level of antibody.

The hormone called Luteinizing Hormone - Releasing
Hormone or LH-RH is synthesized centrally in the hypothalamus and following a cascade of interactions, it completely controls the synthesis of sex hormones in the male or female and in particular the production of testosterone. Consequently, LH-RH controls the development and functioning of the testes, prostate and seminal vesicles.If the level of LH-RH is low but especially for what interests us, if its activity is neutralized by antibodies biologically active, there is a decrease in the rate of circulating testosterone and involution of the sexual organs. It has been shown by numerous authors and in all the tested species (mouse, rat and guinea pig, cattle), that a conjugate LH-RH on tetanus toxoid (LH-RH-TT) does not make it possible to obtain antibodies biologically active (i.e., capable of neutralizing LH-RH activity) only when administered in combination with a very potent adjuvant preparation. The commonly used preparation is Freund's complete adjuvant (FCA), which is also well known to have significant side effects that prevent human or veterinary use. Or there are cancers (prostate cancer) whose development is linked to the secretion of testosterone. It is therefore important if one wishes to develop an immunological treatment by vaccination of this type of cancer, to find an effective formulation of LH-RH compatible with a clinical administration. The same need exists if one wants to use in the animal an anti-LH-RH vaccine in order to cause a castration effect.

The combination muramylpeptidelalum meets these requirements as demonstrated by the following example.

In the experiment described, Sprague-Dawley male rats weighing 250 g received at 30 days intervals three conjugate injections.
LH-RH-TT (50cil). This conjugate was administered: a) alone, b) in an FCA emulsion, c) mixed with 100 μl muramétide, d) absorbed on alum (400 μl), e) absorbed on alum and added with muramétide.

Anti-LH-RH antibodies are measured at various times during the experiment as well as blood testosterone levels. After 130 days, the animals are sacrificed and their sexual organs removed for histological study. The results of a typical experiment are recorded in the following Table 5. They show that the highest antibody levels were obtained in the group receiving the FCA but also in the last group (alum / muramétide). Even more interestingly, only in these two groups is there a considerable drop in testosterone and an involution of the sexual organs.

The results of the Muramétide-Alum combination on the production of biologically active anti-LH-RH antibodies are shown in Table 5 below: TABLE 5

Title <SEP> antibodies <SEP> anti-LR-RH <SEP><SEP><SEP> @ es @ osteone <SEP><SEP> Histology <SEP> rate <SEP> organs <SEP>#
<tb> ob @ enus <SEP> by <SEP> Elisa <SEP> at <SEP> days <SEP> ng / ml <SEP> serum <SEP> at <SEP> days <SEP> Test <SEP> Prostate <SEP > Seminal Vesicles <SEP>
<tb> IMMUNIZATION <SEP> Weight <SEP> Number <SEP> Size <SEP> Number <SEP> Size <SEP> Number
<tb> 20 <SEP> 40 <SEP> 70 <SEP> 130 <SEP> 20 <SEP> 40 <SEP> 70 <SEP> 130 <SEP> average <SEP> of animals <SEP> mean <SEP> d animals <SEP> average <SEP> of animals
<tb> (g) <SEP> affected <SEP> (mm) <SEP> achieved <SEP> (mm) <SEP>
<tb> LH-RH-TT <SEP><1000<SEP> 1200 <SEP> 3800 <SEP> 3200 <SEP> 3.5 <SEP> 1.85 <SEP> 1.50 <SEP> 1.10 <MS> 2 <SEP> 0/6 <SEP> 2.67 <SEP> 0/6 <SEP> 6.5 <SEP> 0/6
<tb> LH-RH-TT + PCA <SEP> 1000 <SEP> 7500 <SEP> 25000 <SEP> 22000 <SEP> 2.80 <SEP> 0.60 <SEP> 0.20 <SEP> 0.10 <SEP> 0.4 <SEP> 5/6 <SEP> NT @ <SEP> NT
<tb> LH-RH-TT + murametic acid <SEP><1000<SEP> 1100 <SEP> 3500 <SEP> 3500 <SEP> 2.95 <SEP> 1.50 <SEP> 1.35 <SEP> 1, 5 <SEP> 1.95 <SEP> 0/6 <SEP> NT <SEP> NT
<tb> LH-RH-TT + Alum <SEP> 1000 <SEP> 3450 <SEP> 13000 <SEP> 7100 <SEP> 2.70 <SEP> 0.70 <SEP> 0.70 <SEP> 0.80 <SEP> 1.55 <SEP> 1/6 <SEP> 2.8 <SEP> 1/6 <SEP> 5.6 <SEP> 1/6
<tb> LH-RH-TT <SEP> + <SEP> Alum <SEP> + <SEP> 1300 <SEQ> 9800 <SEP> 28000 <SEW> 15000 <SEW> 2.55 <SEW> 0.50 <SEP > 0.07 <SEP> 0.14 <SEP> 0.5 <SEP> 5/6 <SEP> 2.08 <SEP> 5/6 <SEP> 3.6 <SEP> 5/6
<tb> Muramétide
<tb># The organs were sampled and then weighed or measured. After having undergone an adequate preparation, they were subjected to histopathological examinations. The lesions observed in affected animals are characteristic of a lack of stimulation by testosterone.

  NT = not tested.

 Similar results were obtained using murabutide or MDP-lys-GDP associated with alum.

Examples 5 and 6 described above show that the combination of muramyl peptides with antigens absorbed on alum is particularly advantageous in the case where the vaccinating principle is a hapten requiring coupling to a carrier molecule. This case may arise when the vaccinating principle is a peptide, a low immunogenic protein or a sugar.

On the basis of these results, one skilled in the art can easily envisage the production of polyvalent vaccines containing, besides a haptene, another vaccinating antigen, from the moment when one of the vaccinating molecules is absorbed on alum and where the conjunction The vaccinating agent contains a derivative of the MDP according to the invention.

REFERENCES (1) Stevens, CE, Goodman, Al, Szmuness, W., Weseley, SA, Fotino,
M. Hepatitis B. vaccinia: immune responses in haemodialysis patients.

The Lancet 1980, ii; 1211-1213.

(2) Zachoval, R., Frosner, G., Deinhardt, F. Impfung gegen Hepatitis B.

Ergehnisse einer Immunogenitàtsstudie. Münchner medizinische
Wochenschrift 1981, 123: 1506-1508.

(3) Grob, PJ, Binswanger, U., Zaruba, K., Joller-Jemelka, HI, Schmid,
M., Hadji, W., Blumberg, A., Abplanalp, A., Herwig, W., Iselin, H.

And immunodeficiency of hepatitis B subunit vaccine in hemodialysis and in renal transplant recipients. Antiviral Research 1983, 3: 43-52.

Claims (23)

claims  1. Vaccines composed of antigens absorbed on alum or formulated in a composition containing alum having an increased immunogenicity compared to that of existing vaccines and characterized in that they are associated with 2 to 120 μg per kg of body weight or 0.1 to 6 mg per vaccinating dose:  or a muramyl peptide of formula:

 wherein the R group is a hydrogen or a methyl group; X is L-alanyl or L-threonyl, and R1 and R2 are independently of each other, hydroxyl, amino, O (CH2) xH with @ = 1, 2, 3 or 4, with the proviso that that when X is an L-alanyl residue, at least one of these two groups R1 and R2 is always O (CH2) xH as previously defined.  - or the MDP-Lys-GDP - (II)  2. Vaccines according to claim 1 characterized in that the vaccinating dose contains 6 to 60 μg per kg of body weight or 0.3 to 3 mg per vaccinating dose of a compound of formula (I) or (II).  3. Vaccines according to one of claims 1 or 2 characterized in that the muramyl peptide is one of those falling within the formula of claim 1 wherein:  the group R is a methyl group, and  the group R2 is an NH 2 group  4. Vaccine according to one of claims 1 to 3 characterized in that the muramyl peptide is selected from murabutide, murametide, muradimetide or MDP-threonine.  5. Vaccine according to one of claims 1 to 4 characterized in that the vaccine allows the simultaneous prophylaxis of hepatitis A, B and C.  6. Vaccine according to one of claims 1 to 4 characterized in that the vaccine allows the simultaneous prophylaxis of hepatitis A and B.  7. Vaccine according to one of claims 1 to 4 characterized in that the vaccine allows prophylaxis of hepatitis B, including hemodialysis, kidney transplant or other poor responders to conventional vaccinations.  8. Vaccine according to claim 7 characterized in that the antigen consists of the S region and all or part of the pre-S region of the surface antigen of the hepatitis B virus.  9. Vaccine according to any preceding claim characterized in that the weight ratio of alum relative to the weight of the derivative of muramylpeptide is between 0.15 and 35.  10. Vaccine according to claim 9 characterized in that the weight ratio of alum relative to the weight of the derivative of muramylpeptide is between 1 and 10.  11. Process. for producing a vaccine having increased immunogenicity, said vaccine consisting of antigens absorbed on alum or formulated in a composition containing alum, characterized in that the composition further contains 2 to 120 μg per kg of body weight or 0.1 to 6 mg per vaccinating dose: either of a muramyl peptide of formula (I)

 or MDP-Lys-GDP (II) in which the R group is a hydrogen or a methyl group; X is L-alanyl or L-threonyl, and R1 and R2 are independently of each other, hydroxyl, amino, O (CH2) xH with @ = 1, 2, 3 or 4, with the proviso that that when X is an L-alanyl residue, at least one of these two groups R1 and R2 is always a group O (CH2) xH as previously defined.  12. The method of claim 11 characterized in that the vaccinating dose contains 6 to 60 pg per kg or 0.3 to 3 mg per dose of a compound of formula (I) or (II).  13. The method of claim 11 or 12, characterized in that the muramyl peptide is one of those falling within the formula of the claim wherein:  the group R is a methyl group, and  the group R2 is an NH 2 group  14. The method of claim 11 to 13 characterized in that muramyl peptide is selected from murabutide, murametide, muradimetide or MDP-threonine.  15. Method according to one of claims 11 to 14 characterized in that the vaccine is chosen from:  - an hepatitis B vaccine,  - a mixed vaccine containing at least one antigen specific for the hepatitis B virus,  - A mixed vaccine containing at least one hapten coupled or not to a carrier molecule.  16. The method of claim 14 characterized in that the specific antigen of hepatitis B virus is a recombinant antigen.  17. Use of 2 to 120 per kg of body weight or 0.1 to 6 mg per vaccinating dose of a muramyl peptide of formula (I) or (II) in the manufacture of a vaccine composed of antigens absorbed on Valum or formulated in a composition containing alum.  18. Use of 2 to 120 per kg of body weight or 0.1 to 6 mg per vaccinating dose of a muramyl peptide of formula (I) or (II) in the manufacture of a vaccine containing at least one coupled hapten or no to a carrier molecule absorbed on alum or formulated in a composition containing alum.  19. Use according to claim 18 characterized in that said vaccine contains at least one hapten and recombinant antigen.  20. Use according to claims 17 to 19, characterized in that the muramyl peptide is one of those falling within the formula (I) in which:  the group R is a methyl group, and  the group R2 is an NH 2 group  21. Use according to one of claims 17 to 17, characterized in that the muramyl peptide is chosen from murametide, murabutide, muradimetide or MDP-threonine.  22. Use according to one of claims 17 to 20 characterized in that the vaccine allows prophylaxis of hepatitis in non-responders or poor responders to the usual vaccines, especially in hemodialized or renal transplant patients.  23. Use according to one of claims 17 to 20 characterized in that the vaccine allows prophylaxis after one or two administrations instead of three in normal responders.