MXPA01003737A - Adjuvant systems and vaccines - Google Patents

Abstract

The present invention provides vaccine and adjuvant formulation comprising an immunostimulant and a metal salt. The immunostimulant is adsorbed onto a particle of metal salt and the resulting particle is essentially devoid of antigen.

Description

AUXILIARY SYSTEMS AND VACU NAS The present invention relates to improved vaccines, auxiliary systems and processes for the preparation of such vaccines and auxiliary systems. In particular, the vaccines and auxiliary systems of the present invention comprise additional metal salts and immunostimulants, such as, monophosphoryl lipid A, or derivatives thereof, Quil A or derivatives thereof, or immunostimulatory oligonucleotides, such as CpG. Aluminum salts are well known in the art as they provide a safe excipient with auxiliary activity. It is thought that the mechanism of action of these adjuvants includes the formation of an antigen deposit, so that the antigen can remain at the site of injection for up to 3 weeks after administration, and also the formation of antigen / salt complexes. metallic, which are more easily captured by cells that present antigens. In addition to aluminum, other metal salts have been used to adsorb antigens, including zinc, calcium, cerium, chromium, iron and beryllium salts. The aluminum hydroxide and phosphate salts are the most common. Formulations of vaccines containing aluminum salts, antigen and additional immunostimulant are known in the art. Such formulations induced greater immune responses in comparison with those stimulated by aluminum salts and sun antigen. The formulation of these vaccine preparations had previously involved a specific manufacturing procedure, because it was believed that in order for the optimal immune responses, the antigen had to be adsorbed on the same aluminum salt particle as the immunostimulant. In this way, when the antigen is taken up by a cell presenting antigen, the co-adsorbed immunostimulant exerts its stimulatory activity directly on that same antigen presenting cell. Aluminum-based vaccine formulations, wherein the antigen and the 3-de-O-acylated monophosphoryl lipid A (3D-MPL) immunostimulant, are adsorbed on the same particle are described in EP 0 576 478 B 1, EP 0 689 454 B1 and EP 0 633 784 B1. In these cases, the antigen is adsorbed first on the aluminum salt, followed by the adsorption of the 3D-MPL immunostimulant on the same aluminum salt particles. Such processes first involve the suspension of 3D-MPL by sonication in a water bath, until the particles reach a particle size between 80 and 500 nm. The antigen is normally adsorbed on the aluminum salt for one hour at room temperature under agitation. The 3D-MPL suspension is then added to the adsorbed antigen and the formulation is incubated at room temperature for 1 hour, and then maintained at 4 ° C until use. The prior art formulation processes provide immunologically potent vaccines, however, they contain several commercial disadvantages. In order for a vaccine to be suitable for human administration, the process must be uniform and subject to a control of Good Manufacturing Practices (GM P) and Quality Control (QC). In some cases, the processes of the prior art provide a vaccine, wherein all the antigen or antigens are adsorbed on the same metal salt particle. The process is then complicated by the requirement that 3D-MPL be adsorbed on the same metal particle. This can be particularly problematic in the case of combination vaccines containing multiple antigens (the adsorption of which may be dependent on the affinity of each antigen to the particular metal salt at a given pH). The processes of the prior art can have problems, depending on which antigens are present, in reproducibility and QC of vaccine. Additionally, if anything unwanted occurs with the QC of a particular antigen, or an occurrence that may result in contamination of the vaccineThis may result in the disposal of all individual components, and not just the particular antigen in which the problem occurred. Moreover, in some circumstances, combination vaccines may require the sequential addition of antigens, such as a time-consuming and expensive process. Accordingly, the prior art processes can be complex, difficult to control and costly. Surprisingly, the present inventors have discovered that it is not necessary to adsorb the antigen and the immunostimulant on the same particle. In contrast to the accepted thinking in the art, it has been found that good vaccines can be produced when the antigen is adsorbed on particles. of particular metallic salts, the which are discrete of those metal salt particles that are associated with the immunostimulant. The improved process comprises the adsorption of immunostimulant, on a metal salt particle, followed by the adsorption of the antigen on another metal salt particle, followed by the mixing of the discrete metal particles to form a vaccine. The present invention also provides an auxiliary composition, an immunostimulant, adsorbed on a metal salt particle, characterized in that the metal salt particle is substantially free of another antigen. Additionally, vaccines are provided by the present invention and are characterized in that the immunostimulant is adsorbed onto metal salt particles, which are substantially free of another antigen, and because the metal salt particles that are adsorbed to the antigen are substantially free of another immunostimulant. Accordingly, the present invention provides an auxiliary formulation comprising immunostimulant, which has been adsorbed on a metal salt particle, characterized in that the composition is substantially free of another antigen. Moreover, this auxiliary formulation is an intermediate that is required during the process of the present invention for the manufacture of a vaccine. Accordingly, a process for the manufacture of a vaccine comprising mixing the auxiliary composition of the invention with an antigen is provided. Preferably, the antigen has been pre-adsorbed onto a metal salt. Said metal salt can be identical or similar to the metal salt, which is adsorbed on the immunostimulant. The present invention further provides a vaccine composition comprising immunostimulant adsorbed on a first particle of a metal salt and antigen adsorbed on a metal salt, characterized in that the first and second metal salt particles are different. Alternatively, the vaccines that form part of the present invention comprise two major populations of complexes, a first complex comprising (a) immunostimulant adsorbed on a metal salt particle, characterized in that said metal salt particle is substantially free of antigen.; and a second complex comprising (b) antigen adsorbed on a metal salt particle. In addition, the vaccine composition can comprise two major populations of complexes, a first complex comprising (a) a stimulant inm adsorbed on a metal salt particle, characterized in that said metal salt particle is substantially free of antigen; and a second complex comprising (b) antigen adsorbed on a metal salt particle, characterized in that said metal salt particle is substantially free of immunostimulant. The metal salts present in these two major populations of complexes may be identical or different. Additionally, in the case of a combination vaccine, where a plurality of different antigens may be present, the second complex (described above) pM & amp; to understand a plurality of antigens adsorbed on different metal particles. The definition of substantially free of other antigens, in relation to this invention, is where not more than 20% by mass of the total material capable of being adsorbed on the metal salt particle is a different antigen, preferably not more than 10%, and most preferably no more than 5%. Alternatively, substantially free in immunostimulant, in relation to this invention, is where not more than 20% by mass of the total material capable of being adsorbed to the metal salt particle is immunostimulant, preferably not more than 10% and most preferably no more than 5% Routine tests, evident to the man skilled in the art, could be used to determine whether the antigen and immunostimulant, are adsorbed on different discrete particles, including but not limited to the separation of the vaccine in different fractions by the free flow of the formulation within an electric field, or techniques such as sedimentation rate analysis, which are particularly suitable for non-particulate antigens, followed by assay of the immunostimulant or antigen in the fractions. Also provided in the present invention is an assembly comprising a container having an immunostimulant adsorbed on a metal salt; and a second container having antigen, said antigen being adsorbed, preferably, on a metal salt. The process of the present invention is especially useful when commercial scale quantities of combination vaccines are required. Combination vaccines are single dose vaccines containing more «EEjAjMíiqfrftgfti of an antigen of more than one pathogen. Such vaccines can reduce the number of vaccinations required to induce protection against many pathogens and diseases. For example, if a vaccine comprises AIOH3, 3D-MPL and the antigens V, X, W, Y, Z, previous processes involve formulating antigens and 3D-MPL on the same particle of AIOH3. Such processes of the prior art require that V, W, X, Y, Z be adsorbed on the AIOH3, followed by the addition of free 3D-MPL on each of the pre-adsorbed antigen complexes. In contrast, in the formulation process of the present invention, antigens V, W, X, Y, Z are individually adsorbed on separate particles of AIOH3 in separate containers. 3D-MPL is also adsorbed on AIOH3 in another vessel. The vaccine is then formed by simple mixing of material taken from each of the separate containers. In the case of the AIOH3 particles, which are associated with the 3D-MPL, they can be discrete of the AIOH3 particles, which are associated with the antigens. Alternatively, the present invention provides a process for making a vaccine comprising an immunostimulant, an antigen and a metal salt, comprising: 1. Adsorb antigen to a first particle of metal salt; 2. Adsorb the immunostimulant to a second particle of a metal salt, and 3. Mix the products of steps 1 and 2 above.

The present invention provides a process for the manufacture of vaccines, which overcomes the problems present in the prior art. Each individual antigen-metal salt complex may be subject to GMP controls, and if any one is contaminated by a particular metal antigen-salt preparation, then the integrity of other antigens and immunostimulant will not be compromised. Surprisingly, and in contrast to the accepted thinking in the art, the vaccines produced by the process of the present invention are as potent as those prepared using the prior art process. The definition of immunostimulant within the meaning of this invention, can be described as a natural or synthetic compound, which has known auxiliary activity, said auxiliary activity is derived from the direct or indirect stimulatory effect of the compound on the cells of the immune system. by itself and not through other non-stimulatory effects, such as, a deposit effect or focused on the immune system. Examples of such immunostimulants are described in a chapter in "Vaccine Design - The subunit and adjuvant approach" (Edited by Powell, M F. and Newman, MJ. 995), Pharmaceutical Biotechnology (Plenum Press, New York and London, ISBN 0-306-44867-X) entitled "Compendium of vaccine adjuvants and excipients", by Powell, MF and Newman M These immunostimulants include those within the present invention bacterially derived compounds, such as monofosfopl * J & amp; & amp? 9m? * X?? Ué? »Ji? lipid A or derivatives thereof; saponins derived from plants or derivatives thereof, for example Quil A; or immunostimulatory oligonucleotides, such as, CpG, block copolymers, cholera toxin, immunostimulatory cytokines, such as, GM-CSF and I L-1, poliriboA and poliriboU and muramyl tripeptide (MTP). Monophosphoryl lipid A is a bacterially derived compound with helper activity, and is a preferred immunostimulant for use in the present invention. This toxic compound has been altered to form less toxic derivatives, one such derivative is 3-de-O-acylated monophosphoryl lipid (called 3D-MPL or d3-MPL, to indicate that the 3-position of the reducing end glucosamine is de-O-acylated). For the preparation of 3D-M PL, see GB 2 220 21 1 A. Quimically, it is a mixture of monophosphoryl lipid to 3-deacylated with 3, 4, 5 or 6 acylated chains. Preferably, in the compositions of the present invention, a small particle of MPL is used. The small particle of MPL has a particle size so that it can be sterilized by filtering through a 0.22 μm filter. Such preparations are described in the international patent application no. WO 94/21 292. Further improvements are described in GB 9807933.8, which discloses stable 3D-MPL preparations consisting of the tri and tetra acyl lo congeners. GB 2 220 21 1 A mentions that the endotoxicity of the previously used enterobacterial lipopolysaccharides (LPS) is reduced while the immunogenic properties are conserved. However, GB 2 220 21 1 cites these findings simply in relation to bacterial (gram negative) systems. Another preferred immunostimulant for use in the present invention is Quil A and its derivatives. Quil A is a saponin preparation isolated from the South American tree Quilaja Saponaria Molina and was first described by Dalsgaard et al. in 1 974 ("Saponin adjuvants" (Auxiliary of saponin), Archiv. Für die gesamte Virusforschung, Vol. 44, Srpinger Verlag, Berlin, p243-254) for having auxiliary activity. The purified fragments of Quil A have been isolated by H PLC, which retain auxiliary activity without the toxicity associated with Quil A (EP 0 362 278), for example QS7 and QS21 (also known as QA7 and QA21). The particular formulations of QS21 have been described, which are particularly preferred, these formulations further comprise a sterol (WO96 / 33739). CpG is an immunostimulatory oligonucleotide without known auxiliary properties (WO 96/02555). Preferred CpG sequences within the context of this invention are: (TCC ATG AGC TTC CTG ACG TT, Krieg 1 826), (TCT CCC AGC GTG CGC CAT, Krieg 1 758) and TCG TCG TTT TGT CGT TTT GTC GTT. The present invention relates to the particular formulation process and characteristics of the auxiliary, and thus, can be used with a wide variety of antigens. The vaccines of the present invention can be used for initiation and booster doses, and used for the induction of immune responses to, and infection protection mediated by, a wide variety of antigens. In addition, the present invention provides a method for eliciting an immune response to an antigen comprising the use of a vaccine comprising a metal salt, non-stimulant and antigen, wherein the immunostimulant is adsorbed onto metal salt particles, which are discrete. of those metal salt particles, which are adsorbed to the antigen. Some of the pathogens and antigens are listed below. Viral hepatitis, caused by hepatitis A, B, C, D viruses, and E, it is a common viral disease. Via viruses B and C, in particular, is also responsible for many cases of liver cancer. In this way, the development of effective vaccines is critical and, despite notable successes, remains an ongoing task. A review of modern hepatitis vaccines, including a variety of key references, can be found in Lancet, May 1, 1990 at page 1 142 ff (Prof. A. L. W. F. Eddleston). See also, "Viral Hepatitis and Liver Disease" (Vyas, BN, Dienstag, J. L., and Hoofnagle, J. H., Eds, Gruà andne and Stratton, Inc. (1 984). ) and "Viral Hepatitis and Liver Disease" (Proceedings of the 1 990 International Symposium, eds FB Hollinger, S. M. Lemon and H. Margo s, published by Williams and Wilkins). used in the expression "hepatis B antigen" is used to refer to any antigenic material derived from a hepatitis B virus, which can be used to induce immunity to the virus in humans .. Infection with hepatitis B virus (H BV) It is a widespread problem, but vaccines are now available that can be used for mass immunization, for example, the product "Engerix-B" (SmithKIíne Beecham pie), which is obtained by genetic engineering techniques. .? s «t. \ "Eí £ a,,., - ZZíi¿¿: 7 £ ¡..: - ^ ¿¿¿., - The preparation of Hepatitis B surface antigen (HBsAg) is well documented. See, for example, Harford et al in Develop Biol Standard 54, page 1 25 (1 983), Gregg et al in Biotechnology, 5, page 479 (1 987), EP-A-0 226 846, EP-A-0 299 1 08 and references therein As used herein, the term "surface antigen of Hepatitis B "or" HBsAg "includes any HBsAg antigen or fragment of the same that exhibits the antigenicity of H BV surface antigen. It will be understood that in addition to the 226 amino acid sequence of the HBsAg S antigen (see Tiollais et al, Nature, 31 7, 489 (1985) and references therein), HBsAg as described herein may contain, if desired, all or part of a pre-S sequence as described in the above references and in EP-A -0 278 940 In particular, the H BsAg may comprise a polypeptide comprising an amino acid sequence comprising residues 1 2-52 followed by residues 1 33- 145, followed by residues 1 75-400 of the L protein of HBsAg in relation to the open reading frame in a Hepatitis B virus serotype ad (this polypeptide is referred to as L *, see EP 0 41 4 374) H BsAg within the scope of the invention may also include the preS1 polypeptide -preS2- S described in EP 0 1 98 474 (Endotronics) or the like thereof, such as those described in EP 0 304 578 (Me Cormick and Jones) HBsAg, as described herein, may also refer to mutants, for example, the "escape mutant" described in WO 91/14703 or publication European Patent Application No. 0 51 1 855 A1, especially HBsAg wherein the amino acid substitution at position 1 45 is glycine arginine Normally, the HBsAg will be in the form of a particle. The particles may comprise, for example, S protein alone or may be composite particles, for example, (L *, S), where L * is as defined above and S denotes the S protein of HBsAg. Said particle is advantageously in the form in which it is expressed in yeast. The component that provides protection against Hepatitis A is, preferably, the product known as "Havrix" (SmithKine Beecham Biologicals), which is a killed attenuated vaccine derived from HAV strain M-1 75 of HAV [see "Inactivated Candidiate Vaccines" for Hepatitis A "(inactivated candidate vaccines for Hepatitis A), by FE Andre, A. Hepburn and E. D'Hondt (1980), Prog. Med. Virol. Vol 37, pages 72-95 and the monograph of the product "Havrix" published by Smith KIine Beecham Biologicals (1 991). Thus, in a preferred embodiment of the present invention, a combination vaccine comprising H BsAg antigen and Hepatitis A is provided. In addition, a process for the production of a hepatitis A and B combination vaccine, and a product derived from that process, is provided in the present invention. Other combination vaccines are available in the market, including the l nfanrixM R range, made by SmithKine Beecham Biologicals. Such vaccines are based on a combination of "nucleus" of Diphtheria toxin, Tetanus toxin and B. pertussis antigens. This vaccine comprises a component of pertussis (either B. pertussis dead cell or acellular pertussis, which usually consists of two antigens - PT and FHA, and often 69 kDa, optionally with one or both agglutinogen 2 or agglutinogen 3) . Such vaccines are frequently referred to as DTPw (whole cell) or DTPa (acellular). Particular combination vaccines within the scope of the invention include: Diphtheria-Tetanus-Pertussis-Hepatitis B (DTP-HB) Diphtheria-Tetanus-Hepatitis B (DT-H B) Hib-Hepatitis B DTP-Hib-Hepatitis BI PV ( inactivated polio vaccine) -DTP-Hib-Hepatitis B The pertussis component is suitably a whole-cell pertussis vaccine or an acellular pertussis vaccine containing partially or highly purified antigens. The above combinations may optionally include a component that is protective against Hepatitis A. Preferably, the Hepatitis A component is inactivated HM-1 75 formalin. Advantageously, HM-1 75 is purified by treating H M-1 75 cultured with trypsin, separating the intact virus from small protein digested with protease by permeation chromatography and inactivating with formalin. Advantageously, the Hepatitis B vaccine is a pediatric vaccine. Other combination vaccines of the present invention are described in GB 98051 05.5 (Smith KIine Beecham Biologicals, s. A.), Such combination vaccines being especially beneficial for vaccines for adolescents. Preferred combinations are based around a "core" combination of a Hepatitis B antigen (Hep B) and a Herpes Sim plex antigen (HSV). Optionally, one or more antigens derived from the following group can be added to this "nucleus": Epstein Barr virus antigen (EBV), Hepatitis A antigen (Hep A), human papilloma virus (HPV) antigen. The combination may further comprise Varicella Zoster virus antigens (VZV), human cytomegalovirus (HCMV) or toxoplasma Preferably, the vaccine formulations of the present invention contain an antigen or antigenic composition capable of eliciting an immune response against a human pathogen. , said antigen or antigenic composition is derived from H IV-1, (such as, tat, nef, gp120 or gp1 60), human herpes viruses, such as gD or derivatives thereof or Immediate Early protein, such as ICP27 from HSV1 or HSV2, cytomegalovirus ((human) (such as g B or derivatives thereof), rotavirus (including attenuated live virus), Epstein barr virus (such as, gp350 or derivatives thereof), Varicella zost virus er (such as gpl, II and I E63), or of a hepatitis virus, such as hepatitis B virus (eg, hepatitis B surface antigen or a derivative thereof), hepatitis A virus, hepatitis virus C and hepatitis E virus, or other viral pathogens, such as, paramyxoviruses: respiratory syncytial virus (such as, F and G proteins or derivatives thereof), parainfluenza virus, measles virus, mumps virus, virus human papilloma (e.g., HPV6, 11, 16 and 18), flavivirus (e.g., yellow fever virus, dengue virus, mite-borne encephalitis virus, Japanese encephalitis virus) or influenza virus, or derivatives of bacterial pathogens, such as, Neisseria spp, including N. gonorrhea and N. meningitidis (eg, capsular polysaccharides and conjugates thereof), transferrin binding proteins, lactoferrin binding proteins, PílC, adhesins); Streptococcus spp, including S. pneumoniae (e.g., capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline binding proteins), S. pyogenes (e.g., M proteins or fragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S. mutans; Haemophilus spp, including H. influenzae type B (eg, PRP and conjugates thereof), non-typeable H. influenzae (eg, OMP26, high molecular weight adhesins, P5, P6, lipoprotein D), H. ducreyi; Moraxella spp, including M catarrhalis, also known as Branhamella catarrhalis (for example, adhesins and invasins of high and low molecular weight); Bordetella spp, including B. pertussis (eg, pertactin, pertussis toxin or derivatives thereof, filamentous haemagglutinin, cyclic adenylate, fimbriae), ß. parapertussis and B bronchiseptica; Mycobacterium spp, including M. tuberculosis (eg, ESAT6, antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli (eg, colonization factors, heat-labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof), enterohemorrhagic E. coli, enteropathogenic E. coli (e.g. toxin similar to shiga toxin or derivatives thereof); Vibrio spp, including V. cholera (for example, cholera toxin or derivatives of m ism a); Shigella spp, including S. sonnei, S dysenteriae, S. flexnerii; Yersmia spp, including Y enterocolitic (eg, a Yop protein), Y. Pestis, and Pseudotuberculosis, Campylobacter spp, including C. jejuni (eg, toxins, adhesins and wrVasins) and C. coli; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp. , including L. monocytogenes; Helicobacter spp, including H. pylori (for example, urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus spp., Including S. aureus, S. epidermidis; Enterococcus spp., Including E. faecalis, E. faecium; Clostridum spp. , including C. tetani (e.g., tetanus toxin and derivative thereof), C. botulinum (e.g., botulinum toxin and derivative thereof), C. difficile (e.g. Clostridium toxins A or B and derivatives thereof); Bacillus spp., Including B. anthracis (for example, botulinum toxin and derivatives thereof); Corynebacterium spp. , including C. diphtheriae (for example, toxin of dteria and derivatives thereof); Borrelia spp., Including B. burgdorferi (for example, OspA, OspC, DbpA, DbpB), B. garinii (for example, OspA, OspC, DbpA, DbpB), fí. afzelii (for example, OspA, OspC, DbpA, DbpB), fí. Anderson (for example, OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia spp. , including E. equi and the agent of human granulocytic ehrlichiosis; Rickettsia spp. , including R. rickettsii; Chalmydia spp., Including C. trachomatis (e.g., MOMP, heparin binding proteins), C. pneumoniae (e.g. MOMP, heparin-binding proteins), C. psittaci; Leptospira spp. , including L. interrogans; Treponema spp., Including T. pallidum (e.g., rare outer membrane proteins), T. denticola, T. hyodysenteriae; or derivatives of parasites such as, Plasmodium spp., including P. falciparum, Toxoplasma spp. , including T gondii (for example, SAG2, SAG3, Tg34), Entamoeba spp, including E histolytica, Babesia spp., including f. microti; Trypanosoma spp., Including T. cruzi; Giardia spp., Including G. lamblia; Leshmania spp., Including L. major; Pneumocystis spp. , including P. carinii; Trichomonas spp., Including T. vaginalis, Schisostoma spp., Including S. mansoni, or yeast derivatives, such as, Candida spp., Including C. albicans; Cryptococcus spp., Including C. neoformans. In a preferred aspect, the vaccine formulation of the invention comprises the HIV-1 antigen, gp1 20, especially when expressed in CHO cells. In a further embodiment, the vaccine formulation of the invention comprises gD2t as defined hereinbefore. In a preferred embodiment of the present invention, the vaccines containing the claimed adjuvant comprise the HPV viruses considered to be responsible for genital warts, (HPV 6 or HPV 1 1 and others), and the HPV viruses responsible for cervical cancer ( HPV16, H PV 1 8 and others). Particularly preferred forms of vaccine comprise L1 particles or capsomeres, and fusion proteins comprising one or more antigens selected from the H PV6 and H PV1 1 E6, E7, L1 and L2 proteins. The most preferred forms of fusion protein are L2E7 as described in GB 95 1 5478.7, and protein D (1/3) -E7 described in GB 971 7953 5 (WO99 / 1 0375). The vaccines of the present invention further comprise antigens derived from parasites that cause Malaria. For example, the preferred antigens of Plasmodia falciparum include RTS. S and TRAP RTS is a hybrid protein that substantially comprises the entire C-term in portion of the cyclosporozoite (CS) protein of P falciparum linked via four amino acids from the preS2 portion of the Hepatitis B surface antigen to the antigen. of surface area (S) of hepatitis B virus. Its complete structure is described in the international patent application no. PCT / EP92 / 02591, published under the number WO 93/10152 claiming priority of the UK patent application no. 9124390.7. When expressed in yeast, RTS is produced as a lipoprotein particle, and when co-expressed with the S antigen from HV, it produces a mixed particle known as RTS.S. The TRAP antigens are described in the international patent application no. PCT / GB89 / 00895, published under WO 90/01496. A preferred embodiment of the present invention is a Malaria vaccine, wherein the antigen preparation comprises a combination of the RTS.S and TRAP antigens. Other plasmodia antigens that are potential candidates for components of a multi-stage Malaria vaccine are P. faciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMPI, Pf332, LSA1, LSA3, STARP, SALSA , PfEXPI, Pfs25, Pfs28, PFS27 / 25, Pfs16, Pfs48 / 45, Pfs230 and their analogs in Plasmodium spp. The formulations may also contain an anti-tumor antigen and may be useful for cancer with immunotherapeutic treatment. For example, the auxiliary formulation finds utility with tumor rejection antigens, such as those for prostate, breast, colorectal, pulmonary, pancreatic, renal or melanoma cancers. Exemplary antigens include MAGE 1 and MAGE 3 or other Mage antigens for the treatment of melanoma, PRAME, BAGE or GAGE (Robbins and Kawakami, 1996, Current Opinions m Immunology 8, pp. 628-636, Van den Eynde et al., I nternational Journal of Clinical & Laboratory Research (presented in 1 997); Corréale et al. (1997), Journal of the National Cancer I nstitute 89, p293. In fact, these antigens are expressed in a wide range of tumor types, such as melanoma, lung carcinoma, sarcoma and bladder carcinoma. Other tumor specific antigens are suitable for use with adjuvant of the present invention and include, but are not restricted to, prostate specific antigen (PSA) or Her-2 / neu, KSA (GA733), MUC-1 and carcinoembryonic antigen ( CEA). Other antigens have been suggested as pan-cancer therapeutic antigens including Tyrosinase and Survivin. In accordance with one aspect of the present invention, a vaccine comprising an adjuvant composition according to the invention and a tumor rejection antigen is provided. It is anticipated that the compositions of the present invention will be used to formulate vaccines containing antigens derived from Borrelia sp. For example, the antigens may include nucleic acid, antigen derived from pathogens or antigenic preparations, recombinantly produced protein or peptides, and chimeric fusion proteins. In particular, the antigen is OspA. The OspA can be a mature protein comette in a virtue of the lipidated form of the host cell (E. coli) called (Lipo-OspA) or a non-lipidated derivative. Such non-lipid derivatives include the non-lipidated NS 1-OspA fusion protein, which has the first N-terminal 81 amino acids of the non-structural protein (NS 1) of the influenza virus, and the complete OspA protein and another, M DP-OspA is a non-ionized form of OspA that carries 3 additional N-terminal inoperates. The vaccines of the present invention can be used for prophylaxis or allergy therapy. Such vaccines would comprise allergen-specific antigens (e.g., Der p1, and pollen-related antigens) and non-allergen-specific antigens (e.g., stanworth decapeptide). The amount of antigen in each vaccine dose is selected as an amount, which induces an immunoprotective response without significant adverse side effects in the normal vaccinates. Such amount will vary depending on the specific immunogen employed and how it is presented. In general, it is expected that each dose will comprise 1-1000 μg of antigen, preferably 1 -500 μg, preferably 1 -100 μg, most preferably 1 to 50 μg. An optimal amount for a particular vaccine can be assessed by standard studies that involve the observation of appropriate immune responses in subjects. Following an initial vaccination, the subjects may receive one or several reinforcement inoculations spaced adequately.

Typically for human administration, the immunostimulant will be present in the range of 1 μg-1000 μg, preferably 10 μg-500 μg, more preferably 20-200 μg per dose, more preferably 20-1 00 μg per dose, and very much. preferably 1 0-50 μg per dose. The present invention further provides the adjuvants and vaccines of the present invention for use in medicine, specifically as a method for treating a mammal suffering from, or susceptible to, a pathogenic infection, or cancer or allergy. of auxiliaries and vaccines of the present invention in the manufacture of an immunoprophylactic and immunotherapeutic treatment of infe @ f | pf? is viral, bacterial, parasitic, allergic or cancer. The formulations of the present invention can be used both for prophylactic and therapeutic purposes. The vaccine preparation is generally described in "Vaccine Design - the subunit and adjuvant approach", edited by Powell, M. F. and Newman, M.J.; 1995, Pharmaceutical Biotechnology (Plenum Press, New York and London, ISBN 0-306-44867-X). The present invention is illustrated by, but not limited to, the following examples.

Example 1, Materials and methods Serology The quantification of anti-H Bs antibody was performed by ELISA using H Bs (Hep 286) as coating antigen. The antigen and antibody solutions were used at 50 μl per well. The antigen was diluted to a final concentration of 1 μg / ml in PBS and adsorbed overnight at 4 ° C to the wells of 96-well microtiter plates (Maxisorb I mmuno plate, Nunc, Denmark). The plates were then incubated for 1 h at 37 ° C with PBS containing 1% bovine serum albumin and 0 1% TWEEN 20 (saturation buffer, 1 00 μl / well). Two-fold dilutions of sera (starting in the ^ - ^ ¿^^^ aSj ^^^^ aaj & i ^ & dilution 1/100) in the saturation buffer, were added to the plates coated with HBs and incubated for 1 h 30 min at 37 ° C. The plates were washed four times with PBS 0.1% TWEEN 20 and IgG1, IgG2a, IgG2b or anti-mouse Ig conjugated with biotin (Amersham, UK) diluted 1/1000 in buffer was added to each well and incubated for 1 h 30 min. at 37 ° C. After a washing step, streptavidin-biotinylated peroxidase complex (Amersham, UK) diluted 1/5000 in saturation buffer was added for an additional 30 min at 37 ° C. The plates were washed as before and incubated for 20 min with a solution of o-phenylenediamine (Sigma) 0.04% H2O2 0.3% in 0.1% TWEEN 20, 0.05M citrate buffer pH 4.5. The reaction was stopped with 2N H2SO4 and read at 490/630 nm. The ELISA titers were calculated from a reference by SoftmaxPro (using a four parameter equation) and expressed in EU / μl.

Proliferation of T cells 2 weeks after the second immunization, the mice were killed, the spleens were aseptically removed in deposits. Cell suspensions were prepared in RPM I 1 640 medium (GI BCO) containing 2 μM L-glutamine, antibiotics, 5 × 1 0"5 M 2-mercaptoethanol and 1% normal syngeneic mouse serum. final concentration of 2x1 06 cells / ml in 200 μl in 96-well round-bottom plates with different concentrations (1-0-0 03 μg / ml) of antigen H Bs Each test was performed in quadruplicate After 96 h of culture a 37 ° C under 5% CO2, the cells were pulsed for 1 8 h with 3H-thymidine (Amersham, UK, 5Ci / mmol) at 0.5 μCi / well and then collected on Unifilter plates (Packard) with a cell harvester. The radioactivity incorporated in a scintillation counter (Topcount, Packard) was measured. The results were expressed in cpm (average cpm in cavities per quadruplicate) or as stimulation indices (average cpm in cultures of cells with average antigen / cpm in cell cultures without antigen).

Production of cytokines 2 weeks after the second immunization, mice were killed, spleens were removed aseptically in tanks (3 reservoirs per group). Cell suspensions were prepared in RPMI 1640 medium (GI BCO) containing 2 μM L-glutamma, antibiotics, 5 × 1 0 5 M 2-mercaptoethanol and 55 fetal calf serum. Cells were cultured at a final concentration of 5x1 06 cells. / ml in 1 ml, in 24-well round-bottomed plates with different concentrations (1-0-0.1 μg / ml) of H Bs antigen. The supernatants were collected 96 h later and frozen until the presence was tested of I FN? e I L-5 by ELISA.

IFN production? The quantification of I FN? was performed by ELI SA using Genzyme reagents. Samples and antibody solutions were used at 50 μl per well. Plots of 96-well microtiter plates (Maxisorb I mmuno plate, Nunc, Denmark) were coated overnight at 4 ° C with 50 μl of IFN? anti-mouse hamster diluted at 1.5 μg / ml in carbonate buffer pH 9.5. The plates were then incubated for 1 h at 37 ° C with 1 00 μl of PBS containing 1% bovine serum albumin and 0.1% TWEEN 20 (saturation buffer). Two-fold dilutions of stimulation supernatant in vitro (starting in 1/2) in saturation buffer were added to the plates coated with anti-I FN? and incubated for 1 h 30 min at 37 ° C. The plates were washed 4 times with PBS TWEEN 0.1% (wash buffer) and IFN? goat anti-mouse conjugate with biotin diluted in saturation buffer at a final concentration of 0.5 μg / ml to each well and incubated for 1 h at 37 ° C After a washing step, the AMDEX conjugate (Amersham) diluted 1 / 1 0000 in saturation buffer was added for 30 min at 37 ° C. The plates were washed as before and incubated with 50 μl of TMB (Biorad) for 10 min. The reaction was stopped with 0.4N H2SO4 and read at 450/630 nm. Concentrations were calculated using a standard curve (standard I mouse FNα) by SoftmaxPro (four parameter equation) and expressed in pg / ml.

Production of IL-5 The quantification of I L-5 was performed by ELI SA using Pharmingen reagents. Antibody samples and solutions were used at 50 μl per well. 96-well microtiter plates (M asixorb plate I mm one, Nunc, Denmark) were coated overnight at 4 ° C with 50 μl of anti I L-5. -rat rat diluted to 1 μg / ml in carbonate buffer pH 9.5. The plates were then incubated for 1 h at 37 ° C with 100 μl of PBS containing 1% bovine serum albumin and 0.1% TWEEN 20 (saturation buffer). Two-fold dilutions of stimulation supernatant in vitro (starting at 1/2) in saturation buffer were added to the plates coated with anti-I FN? and were incubated for 1 h at 30 min at 37 ° C. The plates were washed 4 times with PBS TWEEN 0.1% (wash buffer) and rat anti-mouse L-5 conjugated with biotin, diluted in saturation buffer, was added. at a final concentration of 1 μg / ml to each well and incubated for 1 h at 37 ° C. After a wash step, AMDEX conjugate (Amersham) diluted 1 / 10,000 in saturation buffer for 30 mm at 37 ° C was added. . The plates were washed as before and incubated with 50 μl of TMB (BioRad) for 1 5 min. The reaction was stopped with 0 4N H2SO4 and read at 450/630 nm The concentrations were calculated using a standard curve (I L-5 of recombinant mouse) by SoftmaxPro (four parameter equation) and expressed in pg / ml Example 2. Immunogenicity studies in mice In order to test the concept of M PL in a pathogen-free solid particulate carrier, an immunogenicity study was carried out in Balb / C mice using several sequences of vaccine formulations HABM PL ^ s *** ^ ** ^^ 7 Table 1, Formulations of vaccines Group Formulation (HB-AIPO4) + 3D-MPL + (HA-AIOH) 3) (3D-MPL-AI (OH) 3) + (HA-AI (OH) 3) + (HB- AIPO4) (3D-MPL-AIPO4) + (HA-AI (OH) 3) + (HB-AIPO4) Description of the formulation process: Group 1. The formulation process of the prior art. The antigen is first adsorbed on the metal salt followed by the addition of free 3D-MPL, resulting in the adsorption of the 3D-MPL on the same metal salt particle as the antigen. Groups 2 and 3. The formulation process of the present invention. 3D-MPL is adsorbed on a metallic salt particle, the antigens are adsorbed on separate particles of metallic salt, followed by the mixing of the pre-adsorbed complexes Immunization scheme Groups of 10 mice were immunized subcutaneously twice in a range of 4 weeks with HAB-based formulations (1/10 human dose, ie HAV 72 ELU, HBs 2 μg, MPL 5 μg). On day 14 post-II, the lymphoproliferative response and cytokine production (I L5 / I FN?) Were analyzed after in vitro restimulation of spleen cells with H Bs and HAV. The blood was taken from the retro-orbital sinus in On day 35, the antibody response to H Bs and HAV and the isotypic profile (H Bs only) were monitored by ELI SA Results The humoral responses (Ig and isotypes) were measured by ELISA using HBs as a coating antigen for HBV and using the * • Behring set for HAV. Only the bleeding of 14 days after I I was analyzed Figure 1 shows antibody responses of anti-H Bs Ig measured in individual sera and represented as GMT. Figure 2 shows the isotypic distribution (IgG 1, IgG2a and IgG2b) calculated from analysis in deposited sera.

No differences were observed in antibody titers between the group 1 and the novel formulations (groups 2 and 3) In addition, the novel formulations (groups 2 and 3) stimulate similar proportions of isotypes lgG 1 and lgG 2a / b, such as those stimulated by the formulations of the prior art (group 1).

Cell-mediated immune responses Cell-mediated immune responses (lymphoproliferation and production of I FN? / I L-5) were measured at 14 days post II after in vitro restimulation of spleen cells with H Bs or HA antigens For each group of mice, 5 animals were sacrificed and the spleens were deposited for in vitro tests. Figure 3 shows the lymphoproliferation monitored in spleen cells restimulated with HBs. _ "Figure 4 shows the monitoring of cytokine monitored in spleen cells restimulated with HBs.

No lymphoproliferative responses were observed between the formulations. Strong responses of I FN-? (+/- 1000 pg / ml) with all the groups, moreover, no difference was observed in the production of I L-5 (below 60 pg / ml) between the groups.

Conclusions No significant differences were observed in humoral and cell-mediated immune responses, at H BsAg between the HABMPL formu- lation sequences.

Example 3, HSV Vaccination of guinea pigs The previous example demonstrated the efficacy of the new formulations and processes, with respect to Hepatitis antigens. This example investigated the immunogenicity and protective efficacy of Herpes Simplex virus g D vaccines formulated with alum and 3D-MP L in the classical process, compared to the process of the present invention. The two vaccines were compared in the intravaginal protection model. of the HSV I ndies ^ j *% * »A. sa ^^ - ^ SSi ^ S? í? ^ Experimental protocol Two groups of 1 2 Hartley guinea pigs, hembaras, were immunized twice on days 0 and 28. On day 57, the animals were challenged intra-vaginally with 105 pfu of the HSV2 MS strain (1 00 μl). . After the challenge, the animals were monitored daily for clinical signs of the primary disease from day 4 to 1 2. The blood was taken from the retro-orbital sinus on days 14 and 28, after the second immunization and the antibody response Anti-gD (IgG) was monitored by ELISA.

Formulation process gD2t was produced from HSv2 according to the techniques described in WO 92/16231. 3D-MPL was purchased from Ribi I mmunoChem I nc, Montana, US AIOH3 was purchased from Superfos. The formulations were prepared 15 days before the first injection All incubations were performed at room temperature with shaking Group 4 Formulations based on AI (OH) 3 (250 μl / dose) classic form gD2t (5 μg) was adsorbed on 1 25 μg of AI (OH) 3 for 1 5 min before the addition of M PL (1 2 5 μg) Thirty minutes later, the formulation was buffered with a 10-fold concentrated PBS solution pH 7.4. After 15 min, 500 μg / ml phenoxyethanol was added as preservative.

H2O + AI (OH) 3 + Ag-15m-MPL-30m-10xPBSpH7.4-15m-2 phenoxy Group 5. Formulations based on AI (OH) 3 (250 μl / dose): new form gD2t (5 μg) was adsorbed in 1 00 μg of AI (OH) 3 for 1 5 min and stored as a concentrated monomass. On the other hand, MPL (1 2.5 μg) was adsorbed in 25 μg of AI (OH) 3 for 30 min and stored as another concentrated monomass. For the final formulation, the g D2t adsorbed in H2O and PBS concentrated 1 0 times pH 7.4 were diluted. Fifteen minutes later, M PL adsorbed was added before the addition of phenoxyethanol as preservative.

AI (OH) 3 + Ag AI (OH) 3 + MPL H2O + 1 0xPBS pH 7.4 + Ads g D2t-1 5m-Ads M PL-15m-2 phenoxy Quantification of samples Quantitation of anti-g D antibody was performed by ELISA using gD 43B31 8 as coating antigen Antigen and antibody solutions were used at 50 μl per well The antigen was diluted to a final concentration of 1 μg / ml in PBS and adsorbed overnight at 4 ° C to 96-well microtiter plate wells (Maxisorb Immuno plate, Nunc, Denmark). The plates were then incubated for 1 h at 37 ° C with PBS containing 1% bovine serum albumin and 0.1% Tween 20 (saturation buffer). The two-fold dilutions of sera in the saturation buffer were added to the gD coated plates and incubated for 1 h 30 min at 37 ° C. Plates were washed four times with PBS 0.1% Tween 20 and anti-guinea pig IgG conjugated with biotin (Amersham, UK) diluted 1 / 10,000 in saturation buffer was added to each cavity, and incubated for 1 h 30 min at 37 ° C. After a washing step, the streptavidin-biotinylated peroxidase complex (Amersham, UK) diluted 1/1000 in saturation buffer was added for an additional 30 min at 37 ° C. Plates were washed as before and incubated for 20 min with a solution of o-phenylenediamine (Sigma) 0.04% H2O2 0.03% in 0.1% Tween 20 citrate buffer 0 05M pH 4.5. The reaction was stopped with 2N H2SO4 and read at 490/630 nm. The ELISA titers were calculated from a reference by SoftmaxPro (using a four parameter equation) and expressed in EU / ml.

Statistical analysis Statistical analyzes were performed on serology data using U N I STAT- The protocol applied for an analysis of variance of a pathway can be described briefly as follows 1) Logarithmic transformation of data ^^^^ '^ «^ 2) Test of Kolmogorov Smírnov in each population (group), in order to verify normality 3) Tests of Hartley and Cochran, in order to verify the homogeneity of the vapanza between the different populations (groups) 4) Vapanza analysis on the selected data data of 14 days post II or 28 days post II Results Serology Figure 5 shows the anti-gD IgG antibody responses measured in post II in individual sera No striking difference in antibody titers between formulations was observed on day 14 post II (1 7090-18508 EU / ml for GMT) or 28 days post II (10227-1 1965 EU / ml for GMT) A vapanza one-way analysis was performed separately on high anti-gD IgG titers either by vaccine formulation, from both points in the time after the logarithmic transformation of the data No statistically significant differences were detected between the formulations (values p = 0 7397 and 0 5078 for data of 14 days post II and 28 days post II, respectively) Protection of the disease The protection against the primary disease was evaluated between 4 to 1 2 days post challenge, to compare several parameters in vaccinated and untreated animals. vxik-. - i • The percentage of animals or no or no injuries (vaginal or external).

• The primary infection index (Pl) calculated for the group as follows:? (maximum record x incidence Repressed in%). 7? ~ • The sum of injury records (day 4 to 1 2) expressed as the average and the number of animals showing injuries (N). • The average cumulative records calculated for each group between days 4 and 12.

Table 2 Summary of injury parameters * Sum of injury records for days 4 to 1 2 post-infection (animals without injuries were not considered). Injury records: no lesion (0), vaginal lesions (0.5 or 1), external skin vesicles (2, 4, 8 or 1 6). ** index of primary infection = (record max I) x (% incidence); with l = 0, 0.5, 1, 2, 4, 8 or 16.

Figure 6 shows the cumulative lesion registration curves after HSV challenge. A high percentage of vaccinated animals did not develop any lesion (66% to 83%) or developed vaginal lesions. In comparison, 89% of the animals in the control group showed external lesions. A strong reduction in the primary infection rate was observed in vaccinated animals (97% to 99%). This was accompanied by a very low severity of injury for the vaccinated groups (mean = 0.5 or 1), compared to the untreated group (mean = 28). As shown by the cumulative record curves, both groups (4 and 5) gave a very good and comparable level of protection against the primary disease.

Conclusion Old and new processes for the formulation of HSV vaccine were compared. No difference was observed statistically Significant between the two processes either in the IgG titers or in the protection against the primary disease.

Example 4 Vaccination of mice with HPV Several formulations sequences were compared (based on AIOH or AIPO4) of E7 antigen of human papilloma virus and 3D-MPL, with respect to its ability to induce antigen-specific humoral responses. Comparable Ig titers are obtained with formulations with mixed adsorption of 3D-MPL and protein D 1 73 -E7 in the same carrier (form 1) and formulations where 3D-MPL is adsorbed by separated in an antigen-free carrier (form a 2) Protein D 1/3 E7 , ^^^. ^ ^^^^^^^^^ was prepared according to the procedure of WO 99/1 0375. The formulations of antigen and either based on AIOH or based on AIPO4. The antigen and 3D-MPL were adsorbed sequentially to the same aluminum salt particles (form 1), or adsorption was done separately before mixing (form 2). Groups of 10 mice were immunized using the following formulations (description in Material and Methods) - The mice were immunized dlii times in a range of 21 days by the intramuscular route. Sera were collected on day 35 (14 days post II) and analyzed for the presence of E7 specific antibodies (see Material and Methods). prepared 5 days before the first injection All incubations were performed at room temperature with shaking I. Formulations based on Al (50 μl / dose): classical form (form 1) PD1 / 3E7 (5 μg) was adsorbed in 50 μg of AI (OH) 3 or AIPO4 for 30 min before the addition of MPL (5 μg) Thirty minutes later, the formulation was buffered with a solution of PO4, NaCl pH 6 8 concentrated 1 0 times After 1 5 min, 50 μg / ml thiomersal was added as preservative H2O + Al + Ag-30m-MPL-30m-10xPNpH6 8-15m-T? II. Formulations based on Al (50 μl / dose): new form (form 2) PD1 / 3E7 (5 μg) was adsorbed in 10 μg of AI (OH) 3 or AIPO4 for 30 min and stored as concentrated monomases. Part, MPL (5 μg) was adsorbed in 20 μg of AI (OH) 3 or AIPO4 for 30 min and stored as other concentrated monomases. For the final formulations, the adsorbed antigen was diluted in H2O and PO4 solution, NaCl pH 6 8 concentrated 1 0 times, before the addition of the adsorbed M PL and the rest of Al (20 μg) Thirteen minutes later, 50 μg / ml of thiomersa l was added as preservative Al + Ag Z »&afci¿-ü?;. '.

Al + MPL - * H2O + 10xPN pH 6.8 + Ads PD1 / 3E7 + Ads MPL + AI-30m-Tio SEROLOGY ^ * The quantification of anti-E7 antibody was carried out by Elisa, using E7 (Bollen) as coating antigen. Antigen and antibody solutions were used at 50 μl per well. The antigen was diluted to a final concentration of 3 μg / ml in carbonate buffer pH 9.5 and adsorbed overnight at 4 ° C to 96-well microtiter plate wells (Maxisorb Immuno plate, Nunc, Denmark). The plates were incubated for 1 h at 37 ° C with PBS containing 1% bovine serum albumin and 0 1% Tween 20 (saturation buffer). Two-fold dilutions of sera (starting at 1/00 dilution) in the saturation buffer were added to the plates coated with E7 and incubated for 1 h 30 min at 37 ° C. The plates were washed 3 times with PBS 0 1 % Tween 20 and anti-mouse IgGtot (lgG 1, lgG2a or lgG2b) conjugated with biotin (Amersham, UK), diluted 1/5000 in saturation buffer to each cavity and incubated for 1 h 30 min at 37 ° C was added. . After a washing step, the streptavidin-biotinylated peroxidase complex (Amersham, UK) diluted 1/5000 in saturation buffer was added for an additional 30 min at 37 ° C. The plates were washed as before and incubated for 10 m in with TM B (tetra-methyl-benzidine). The reaction was stopped with 4N H2SO4 and read at 450 nm. Midpoint dilutions were calculated by SoftmaxPro (using a four parameter equation) Results The anti-E7 Ig titres measured in sera deposited by ELISA, expressed in EU / ml are as follows The comparable titles are obtained when comparing formulations based on AIOH or formulations based on AIPO4. When MPL is added to the AIOH or AIPO4 formulations, the titres reached are above 1 0,000 EU / ml, as compared to less than 5,000 EU / ml for Al formulations Comparable titres are obtained with both formulation sequences Several sequences of formulations (based on AIOH or AIPO4) of antigen and MPL were compared, with respect to their ability to induce antibody production antigen-specific: All formulations containing MPL induce higher levels of E7-specific Eg than alum formulations. The comparable Ig titres are obtained with formulations with mixed adsorption of MPL and pD 1/3-E7 in the same carrier (form 1) and formulations where M PL is adsorbed separately in an antigen-free carrier (form a 2) twenty í & j fes * & ¿

Claims (30)

1. REIVINDU ClONES 1 . An auxiliary composition comprising an immunostimulant adsorbed on a metal salt particle, characterized in that the metal salt particle is its > tesfantly free of another antigen, and because the immunostimulant is not a saponin derived from the bark of Quillaja Saponaria Molina.
2. 2. An auxiliary composition as claimed in claim 1, wherein the metal salt particle is a salt of aluminum, zinc, calcium, cerium, chromium, iron or beryllium. 3. An auxiliary composition as claimed in claims 1 or 2, wherein the metal salt is a phosphate or hydroxide. 4. An auxiliary composition as claimed in any of claims 1 to 3, wherein the metal salt is aluminum hydroxide or aluminum phosphate. 5. An auxiliary composition as claimed in any of claims 1 to 4, wherein the immunostimulant is monophosphoryl lipid A or a derivative thereof. 6. An auxiliary composition as claimed in re-excitation 5, wherein the monophosphoryl lipid derivative A is 3-de-O-acylated monophosphoryl lipid A. 7. An auxiliary composition as claimed in any of claims 1 to 4, wherein the immunostimulant is oligonucleotide containing CpG. aaaa ^^ aa ^^^, ^^ 8. A process for the manufacture of a vaccine composition, comprising the mixture of a) an auxiliary composition comprising an immunostimulant adsorbed on a metal salt particle, characterized in that the metal salt particle is substantially 5 free of another antigen, and b) an antigen. 9. A process for the manufacture of a vaccine composition as claimed in claim 8, characterized in that the antigen is adsorbed on a metal salt particle. 10. A process as claimed in any of claims 8-10 or 9, wherein the antigen is selected from the group comprising: antigens derived from human immunodeficiency virus, Varicella Zoster virus, Herpes Simplex virus type 1, virus of Herpes Simplex type 2, human cytomegalovirus, Dengue virus, Hepatitis A virus, B, C or E, respiratory syncytial virus, human papilloma virus, human 15 influenza, Hib, Meningitis virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bortdetella, Plasmodium or Toxoplasma, IgE peptides, Der p1, pollen-related antigens; or antigens associated with tumor (TAA), MAGE, BAGE, GAGE, MUC-1, Her-2 neu, LnRH (GnRH), CEA, PSA, KSA or PRAME. 20 1 1. A vaccine composition comprising an auxiliary composition according to claims 1 to 7, further comprising an antigen. 1 2. A vaccine produced according to the process claimed in any of claims 8 to 10.
3. 3. A vaccine that buffers a saponin adsorbed on a metal salt particle, wherein the vaccine comprises an antigen, characterized in that the metal salt particle is substantially free of another antigen. 5 14. A vaccine according to the 1 3 re-excitation, where the saponin is QS21. 5. A vaccine composition comprising two major populations of complexes, a first complex comprising (a) an immunostimulant adsorbed on a metal salt particle, characterized in that said metal salt particle is substantially free of antigen; and a second complex comprising (b) antigen adsorbed on a metal salt particle 16 A vaccine composition comprising two major populations of complexes, a first complex comprising (a) a 15 immunostimulant adsorbed on a metal salt particle, characterized in that said metal salt particle is substantially free of antigen; and a second complex comprising (b) antigen adsorbed on a metal salt particle, characterized in that said metal salt particle is substantially free of monophosphoryl lipid 20 A or derivative thereof. 1 A vaccine composition as claimed in claims 1 5 or 1 6, wherein the metal salt present in the first and second complexes is identical. 1 8 A vaccine composition as claimed in any of the 25 claims 1 5 to 1 7, wherein the second complex comprises a ^^^^^ iTMs- ^ g ^ & i ^ i ^ &T ^^ plurality of sub-complexes, each sub-complex comprising a different antigen adsorbed on a metal particle. 19. A vaccine composition as claimed in any of claims 13 to 18, wherein the metal salt is a salt of aluminum, zinc, calcium, cerium, chromium, iron or beryllium. 20. A vaccine composition as claimed in claim 19, wherein the metal salt is a phosphate or hydroxide. 21. A vaccine composition as claimed in claim 20, wherein the metal salt is aluminum hydroxide or aluminum phosphate. 22. A vaccine composition as claimed in any of claims 11-12 and 15 to 21, wherein the immunostimulant is 3-de-O-acylated monophosphoryl lipid A. 23. A vaccine composition as claimed in any of claims 11 to 18, wherein the immunostimulant is CpG. A vaccine composition as claimed in any of claims 11 to 21, wherein the antigen is selected from the group comprising: human immunodeficiency virus, Varicella Zoster virus, Herpes Simplex virus type 1, Herpes Simplex virus type 2, human cytomegalovirus, Dengue virus, Hepatitis A, B, C or E virus, respiratory syncytial virus, human papilloma virus, influenza virus, Hib, meningitis virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium or toxoplasma, decapeptide stanworth, Der p1, antigens related to pollen; or antigens associated with cancer, MAGE, BAGE, GAGE, MUC-1, Her-2 neu, LnRH (GnRH), CEA, PSA, tyrosmase, Survivm, KSA or PRAME 25. A vaccine composition as claimed in claim 24, wherein the antigen is a bmbination of Hepatitis A antigen and Hepatitis B antigen. 26. A c-cAM vaccine composition is claimed in claim 24, wherein the antigen Plasmodium is one or more antigens selected from the following group: RTS.S and TRAP. 27. A vaccine composition as claimed in claim 24, for use in medicine. 28. The use of the vaccine composition as claimed in claim 24, for the manufacture of a medicament suitable for the prophylaxis or treatment of viral, bacterial, parasitic, allergic or cancer infections. 29. A method of treating a mammal suffering from, or susceptible to, a pathogenic infection, or cancer, or allergy, comprising administering a safe and effective amount of a vaccine composition according to claim 24. An assembly comprising two containers, a container having liposid monophosphoryl A, or derivative thereof, adsorbed on a metal salt; and the second container having antigen adsorbed on a metal salt. ^^ Ar ^ g ^ a? Bba ^. ^^ 1