JP2002508748A - Use of virus-like particles as adjuvants - Google Patents

Abstract

  (57) [Summary] An adjuvant formulation comprising non-replicating virus-like particles (VLPs) is provided. VLPs can be from any of several viruses, and are useful for enhancing an immunological response to a selected antigen, where the antigen is different from the particle.

Description

DETAILED DESCRIPTION OF THE INVENTION                 Use of virus-like particles as adjuvants Technical field   The present invention relates generally to agents that enhance an immune response to a selected antigen. . In particular, the invention relates to virus-like particles as adjuvants and coadjuvants Use of (VLP), Formulations Containing VLPs, and Preparation and Use of the Compositions of the Invention For how to. background   Vaccine compositions often include cell-mediated and humoral immune responses. An immunological adjuvant to enhance is included. For example, the administered antigen Depot that can be deposited and / or precipitated and retain the antigen at the injection site Adjuvants are often used. A typical storage adjuvant is aluminum And water-in-oil emulsions. But storage adjuvant (But increasing antigenicity) when injected subcutaneously or intramuscularly It often stimulates severe and persistent local reactions such as granulomas, abscesses and scars. Other adjuvants such as lipopolysaccharide and muramyl dipeptide can be injected and / or Or lighter symptoms (flu-like symptoms, generalized joint discomfort, and sometimes Can provoke a febrile response in anterior uveitis, arthritis, and urethritis) .   Particle carriers with adsorbed or entrapped antigens bypass these problems Used in an attempt to Such carriers are selective for the immune system. Shows multiple copies of selected antigens and capture and capture of antigen in local lymph nodes And retention. The particles are precious by macrophages and Release can enhance antigen presentation. Examples of particle carriers include polymethyl meta Particle carriers derived from acrylate polymers, as well as poly (known as PEG) (Lactide) and poly (lactide-co-glycolide) derived microparticles. other Where they offer significant advantages over more toxic systems, antigen-containing PLG microparticles It suffers from several disadvantages. For example, production of microparticles is difficult and antigen Involves the use of harsh chemicals that can destroy and destroy their immunogenicity. Furthermore, anti The primary instability is due to the high shear forces used in the preparation of small particulates and Can occur due to interfacial effects in the emulsion.   Liposomes have also been used to overcome these problems. Liposo Is a lipid component, such as a phospholipid, used to entrap a drug. These are small vesicles formed from these. The use of liposomes as drug delivery systems Although mitigating some of the above problems, liposomes have low safety during storage and use. It is qualitative and large-scale production and production of liposomes is problematic.   The virions are capable of capturing or binding anti-virus to the host immune system. It can be used as a matrix for proper presentation of the original. For example, U.S. Patent No. No. 4,722,840 is a particle-forming fragment of a structural protein derived from a virus (eg, For example, hepatitis B virus (HBV) surface antigen fused to a heterologous polypeptide (HBsAg) (particle forming fragment). Tin dle et al., Virology (1994) 200: 547-557 describe human papillomavirus (HPV) type 16E 7 Production and Use of Chimeric HBV Core Antigen Particles Containing Epitopes for Transforming Protein Describe the use.   Adams et al., Nature (1987) 329: 68-70 describes a hybrid HIV gp120: T in yeast. y describes the recombinant production of VLP, Brown et al., Virology (1994) 198: 477-488 describe human Parvovirus B19 VP2 protein and human herpes simplex virus type 1 and Of chimeric protein consisting of epitopes derived from mouse and mouse hepatitis virus A59 Is described. Wagner et al., Virology (1994) 200: 162-175; Brand et al. Virol. Me th. (1995) 51: 153-168 and Wagner et al., Virology (1996) 220: 128-140 The assembly of La HIV-1 p55gag particles is described. U.S. Pat.No. 5,503,833 treats Of rotavirus VP6 sphere for encapsulation and delivery of agents Is described.   Regardless of the antigen presentation system described above, various pharmaceutical compositions and vaccines There is a continuing need for effective and safe adjuvants for use in medicine. Summary of the Invention   We surprisingly found that the combination of the selected antigen with VLP (where the anti- Differs from VLPs) in that it provides an enhanced immune response to the antigen in question. I came out.   In one embodiment, the invention then provides a VLP adjuvant, wherein the selected A vaccine composition comprising an antigen and a pharmaceutically acceptable excipient. Selected The different antigens are different from VLPs. In a particularly preferred embodiment, the VLP is a hepatitis surface. One or more particle forming polypeptides of the antigen, or papillomavirus L1 and / or Or an antigen derived from the L2 protein particle-forming polypeptide and selected Is a MenC / Hib oligosaccharide conjugate or HPV E7.   In another embodiment, the invention provides a VLP adjuvant and a selected antigen. Enhanced immune response in a vertebrate body, comprising administering to the vertebrate body To a method for producing Antigens are different from VLPs, and VLPs are Administered in an amount effective to elicit an enhanced immune response to the antigen in the body. You. VLPs can be administered to a subject before, after, or simultaneously with antigen administration.   In yet another embodiment, the invention relates to a process for providing a VLP Preparing an adjuvant formulation comprising combining an excipient with an acceptable excipient About how to.   These and other embodiments of the invention will be readily apparent to those skilled in the art in light of the present specification. Is done. BRIEF DESCRIPTION OF THE FIGURES   Figure 1 shows fetal baboon antibodies to a vaccine containing hepatitis B virus VLP (HBV) Is shown. Results are shown as the geometric mean of antibody concentration in IU / ml.   FIG. 2 shows the antibody response of fetal baboons to a vaccine containing HBV VLP. result Is shown as the geometric mean of the antibody concentrations in IU / ml.   FIG. 3 shows the fetal baboon anti-capsule antibody response to the vaccine containing HBV VLP. Show. Results are shown as the geometric mean of antibody concentration in IU / ml.   FIG. 4 shows the response of fetal baboon anti-capsule antibody to vaccine containing HBV VLP. Show. Results are shown as the geometric mean of antibody concentration in IU / ml.   5A and 5B show serum antibody responses of fetal baboons to a vaccine containing HBV VLP. FIG. 5A shows the IgG anti-capsule antibody response as determined by ELISA. FIG. 5B shows complementary mediated bacterial antibodies.   FIG. 6 shows the combination of HPV6b L1 VLP and E7 vaccine in rabbit humoral immune response. This shows the effect of convergence.                             Detailed description of the invention   The practice of the present invention is within the skill of the art unless otherwise indicated. Use traditional methods of science, chemistry, biochemistry, recombinant technology, immunology and pharmacology . Such techniques are explained fully in the literature. For example, Virology, 3rd edition , Volumes I and II (B.N. Fields and DM Knipe, eds., 1996); Remlngton's  Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Methods In Enzymology (ed. By S. Colowick and N. Kaplan, Acade. mic Press, Inc.); Handbook of Experimental Immunology, Volumes I-IV (D.M. . Weir and C.C. Blackwell Edition, 1986, Blackwell Scientific Publications); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); and DN See A Cloning: A Practical Approach, Volumes I and II (D. Glover, eds.) thing.   As used herein and in the appended claims, the singular forms "a", "an" And "the" are plural, unless the content clearly indicates otherwise including. A. Definition   In describing the present invention, the following terms will be used, and are defined as follows: Intended to be.   As used herein, the term "virus-like particle" or "VLP" Replicates derived from any of several viruses discussed further below Not an empty virus shell. VLPs generally contain one or more viral proteins. quality (Eg, capsid, coat, shell, surface and / or envelope proteins Including, but not limited to, proteins referred to as It is composed of particle-forming polypeptides derived from proteins. VLP is an appropriate launch In the current system, it can be formed spontaneously by recombinant expression of the protein. Specific VL Methods for producing P are known in the art and are discussed more fully below. Discussed. The presence of VLPs after recombinant expression of viral proteins is publicly known in the art. Detection using known conventional techniques (eg, electron microscopy, X-ray crystallography, etc.) I can do it. See, for example, Baker et al., Biophys. J. (1991) 60: 1445-1456; Hagensee et al. Virol (1994) 68: 4503-4505. For example, cryo-electron microscopy has revealed that the V Can be performed on vitrified aqueous samples of LP preparations and images are Recorded under exposure conditions.   By "particle-forming polypeptides" derived from specific viral proteins Full length or near full length viral proteins, and fragments thereof, Or a viral protein with an internal deletion (this is a condition that favors VLP formation) Under the ability to form VLPs). Thus, the polypeptide Reference molecule full length sequences, fragments, truncated and partial sequences, and analogs And precursor forms. Therefore, the term implies that a polypeptide Deletions, additions and substitutions to this sequence as long as it retains its ability to form You. Thus, the term includes natural changes in the particular polypeptide. Because Changes in the protein often occur during virus isolation. You. This term is also used as long as the protein retains the ability to form VLPs. Includes deletions, additions and substitutions that do not occur naturally in the protein.   Preferred substitutions are those that are naturally conserved (ie, at amino acid side chains). Substitutions that occur within a family of related amino acids). Specifically, amino Acids are generally divided into four families: (1) acid--aspartic acid and And glutamic acid: (2) basic--lysine, arginine, histidine; (3) non-polar Sex-alanine, valine, leucine, isoleucine, proline, phenylalanine And methionine, tryptophan; and (4) uncharged and polar--glycine, aspa Lagin, glutamine, cysteine, serine, threonine, tyrosine. Phenyla La Nin, tryptophan and tyrosine are sometimes classified as aromatic amino acids You. For example, lone replacement of leucine with isoleucine or valine, glutamic acid Or substitution of aspartic acid at serine, threonine at serine, or structure Similar conservative substitutions with similarly related amino acids have major effects on biological activity Not doing so is reasonably foreseeable. Therefore, it is substantially the same as the reference molecule. A small number of amino acids that have a amino acid sequence but do not substantially affect the immunogenicity of the protein Proteins that carry no acid substitutions are within the definition of a reference polypeptide.   Antigen is not captured in VLP and / or antigen and VLP are fusion proteins VLPs are "different" from the selected antigen if not co-expressed as But V LPs are selected by the selected antigen even if there is a sparse physical association between the antigen and the VLP. Are considered "different."   "Antigen" is a humoral and / or cellular antigen specific that stimulates the host immune system One or more epitopes (linear, conformational) Or both). This term is interchangeable with the term "immunogen" Used as possible. Typically, a B cell epitope comprises at least about 5 amino acids However, it can be as few as 3-4 amino acids. T cell epitopes (eg, C TL epitope) comprises at least about 7-9 amino acids and comprises a helper T cell. A vesicle epitope comprises at least about 12-20 amino acids. The term “antigen” refers to both Subunit antigen (ie, the whole organism to which the antigen is naturally related) Antigens separated and separated from), and killed, attenuated, or Inactivated means a bacterium, virus, fungus, parasite or other microorganism. Anti Idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes Antibodies such as (mimotope), which can mimic antigens or antigenic determinants, are also , Captured under the definition of antigen as used herein. Similarly, in vivo ( Expression of antigens or antigenic determinants in gene therapy and DNA immunization applications) Oligonucleotides or polynucleotides are also defined herein as antigens. Included in righteousness.   For the purposes of the present invention, the antigen may be any number as described more fully below. Derived from any of the known viruses, bacteria, parasites and fungi. This The term also contemplates any of a variety of tumor antigens. Further, for the purpose of the present invention Alternatively, an “antigen” is a protein that elicits an immune response, as defined herein. As long as it retains the ability to And conservative) modifications, such as deletions, additions and substitutions. This These modifications can be deliberate, such as by site-directed mutagenesis. Or may be accidental, such as by mutation of the host producing the antigen. You.   An “immunological response” to an antigen or composition is an antigen present in the composition of interest. Of a humoral and / or cellular immune response in a subject. For the purposes of the present invention, a “humoral immune response” is an immune response mediated by antibody molecules. Refers to an epidemiological response, while a "cellular immune response" refers to T lymphocytes and / or other leukemia. It is mediated by a sphere. One important aspect of cellular immunity is cytotoxicity. Includes antigen-specific responses by sex T cells ("CTLs"). CTL is a major histocompatibility gene Binds to proteins encoded by the complex (MHC) and expressed on the cell surface It has specificity for peptide antigens presented together. CTL is an intracellular microbe Induction and promotion of intracellular destruction of cells or lysis of cells infected with such microorganisms Help the progress. Another aspect of cell-mediated immunity involves the generation of antigen-specific responses by helper T cells. Including. Helper T cells may help stimulate the function of non-specific effector cells And the activity of non-specific effector cells on their surface to MHC molecules It acts to bind and concentrate on cells presenting the peptide antigen. "Fine The vesicular immune response also includes cytokines, chemokines, and activated T cells and And / or other such molecules produced by other leukocytes (CD4 + and CD8 + (Including those derived from T cells).   Compositions or vaccines that elicit a cellular immune response bind to MHC molecules on the cell surface. Presentation of the combined antigens can serve to sensitize a vertebrate animal subject. cell A mediated immune response is caused by fingers present at or near cells that present antigen on their surface. Turned In addition, antigen-specific T lymphocytes allow for future protection of the immunized host. Can be generated to work.   The ability of certain antigens to stimulate cell-mediated immunological responses is dependent on lymphoproliferation (lympho prollferation) (lymphocyte activation) assay, CTL cytotoxic cell assay, Or assay for antigen-specific T lymphocytes in sensitized subjects And may be determined by a number of assays. Such assays are known in the art. It is well known. See, for example, Erickson et al., J. Immunol. (1993) 151: 4189-4199; Doe et al., E ur.J.Immunol. (1994) 24: 2369-2376.   Thus, an immunological response as used herein is responsible for the production of CTLs and the And / or stimulate the production or activation of helper T cells. Eye The target antigen can also elicit an antibody-mediated immune response. Thus, the immunological response is It may include one or more of the following effects: production of antibodies by B cells; and / or Should be directed specifically to the antibodies present in the composition or vaccine of interest. T cell and / or γ^δActivation of T cells. These responses are infectious Sum and / or antibody-complement, ie, antibody-dependent cellular cytotoxicity (ADCC) It may serve to mediate and provide protection to the immunized host. Such a response Can be determined using standard immunoassays and neutralization assays well known in the art .   A vaccine composition comprising a selected antigen together with a VLP adjuvant of the invention, or Alternatively, a vaccine composition co-administered with a VLP adjuvant of the invention comprises a VLP adjuvant. Equivalent to an antigen administered without an adjuvant or coadjuvant A field capable of eliciting an immune response greater than the immune response elicited by the dose In this case, it presents “enhanced immunogenicity”. Thus, the vaccine composition is "enhanced" Enhanced immunogenicity ". Because antigens are more strongly immunogenic It is. Or, because the lower or lower dose of antigen is Is necessary to achieve an immune response in the subject to which it is administered. This Enhanced immunogenicity, such as administering VLP compositions and antigen controls to animals And standards such as radioimmunoassays and ELISAs well known in the art. Antibody titers and / or cell-mediated immunity against the two It can be determined by comparing the epidemics.   For the purposes of the present invention, an "effective amount" of a VLP adjuvant is co-administered. An amount that enhances the immunological response to the antigen.   By “vertebrate subject” is meant any member of the chordate phylum, These include humans and other primates (chimpanzees and other anuras (ape) and Including non-human primates, such as monkey species); cattle, sheep, pigs, goats, And farm animals such as horses; domestic animals such as dogs and cats; Laboratory animals, including rodents such as pigs and guinea pigs; chickens, turkeys, Domestic, wild, and hunting birds such as and other pheasant birds, ducks, cancer Including but not limited to birds. The term does not imply a specific age. Follow It is intended that both adult and newborn individuals be included. The above system is optional It is intended for use in any of the above vertebrate species. Because these vertebrates All immune systems function similarly.   "Pharmaceutically acceptable" or "pharmacologically acceptable" Causes an otherwise undesirable substance, i.e., some undesired biological effect. Interacts in a deleterious manner with or without any components of the composition containing the substance Rather, it refers to a substance that can be administered to an individual together with a VLP adjuvant formulation.   Depending on "physiological pH" or "physiological pH", about 7.2 or more and 8.0 or less It refers to a pH in the generic range, more typically in the range of about 7.2 to 7.6.   “Recombination” as used herein to describe a nucleic acid molecule refers to the following: Depending on the source or manipulation, polynucleotides of genomic, cDNA, semi-synthetic, or synthetic origin Otide means: (1) all or one of the naturally related polynucleotides Not related to the part; and / or (2) other than polynucleotides naturally linked To the polynucleotide. Used for proteins or polypeptides The term "recombinant" refers to a polynucleotide produced by the expression of a recombinant polynucleotide. Means a repeptide. Prokaryotic microorganisms cultured as single cell entities or "Recombinant host cell", "host cell", "cell", "cell line" indicating eukaryotic cell line "," Cell culture "and other such terms are used interchangeably and Can be used as a recipient for recombinant vectors or other transferred DNA Or the cells that have been used, and the first cells transfected Including progeny cells. The progeny of a single parent cell can be accidentally or intentionally mutated Therefore, morphologically with the original parent, or in the genomic or total DNA complement, It is understood that the components need not be exactly the same. Related properties (for example, Presence of a nucleotide sequence encoding the desired peptide) Progeny of a parent cell sufficiently similar to the parent to be included in the progeny intended by this definition , And are encompassed by the above terms.   As used herein, “treatment” refers to any of the following: (1) Prevention of infection or re-infection, as in traditional vaccines, (ii) reduction or Or (iii) substantial or complete elimination of the pathogen in question. The treatment is It can be effected prophylactically (before staining) or therapeutically (after infection). B. General method   At the heart of the invention is a vertebrate, where VLPs are administered with a selected antigen. For enhancing a humoral immune response and / or a cellular immune response in a subject It is a discovery that it can act as an adjuvant or a coadjuvant. Surprising Indeed, to produce enhanced immunogenicity, antigens are trapped within VLPs No need. Thus, the present invention requires the use of binders, harsh chemical treatments, etc. Absent. Because the antigen of interest is encapsulated or chemically conjugated to the VLP Because there is no. Further, the antigen size is not limited. Because this system is This is because it does not depend on the encapsulation of the antigen. Thus, the system of the present invention Useful with antigens and for preventing and / or treating multiple infections Provide powerful tools.   VLPs for use as adjuvants have the ability to form particles under appropriate conditions. Viable viral proteins, particles derived from viral proteins Ripeptide, or a combination of viral proteins or fragments thereof Can be formed from The requirements for particle-forming viral proteins are the details of the host cell. When particles are formed in the cytoplasm, proteins are likely to undergo the formation of virus-like structures. To the virus-like structure in the cells of the recombinant expression system used. If they are not sufficiently stable in the host cell in which they are expressed such that It is not. If this protein is secreted into the culture medium, VL Conditions can be adjusted so that P is formed. In addition, particle forming proteins Should not be cytotoxic in the current host and can replicate in the host where the VLP is used Should not be.   For example, some proteins autonomously become VLPs when the pH reaches the appropriate level. It is known that can be formed. Similarly, some proteins form VLPs Requires that a sufficient amount of protein be present.   Methods and suitable for forming particles from a wide range of viral proteins Conditions are well known in the art. For example, it is derived from the surface of hepatitis B virus (HBV). Proteins (e.g., surface antigen (sAg), and pre-surface sequences (pre-S1 and pre-S 2 (previously called pre-S), as well as any combination of these sequences)) Upon expression in a suitable host cell, for example, a mammal, insect, yeast, or Xeno It can form particles autonomously upon expression in pus cells. Therefore, the present invention HBV VLPs for use in sAg, pre-S1 or pre-S2, and Any combination (for example, sAg / pre-S1, sAg / pre-S2, sAg / pre-S1 / pre-S2, and And pre-S1 / pre-S2) particle forming proteins. For example, discussion of the HBV structure For the theory, see Macckett, M and Williamson, J.D., Human Vaccines and Vaccinat ion, "HBV vaccines--from laboratory to license: a case study," pages 159-176. And US Pat. No. 4,722,840 for a description of the recombinant production of various HBV particles. No. 5,098,704, No. 5,324,513, Beames et al., J. Virol. (1995) 69: 6833-683 8, Birnbau et al., J. Vlrol. (1990) 64: 3319-3330; Zhou et al., J. Virol. (1991) 65:54. See 57-5464.   In addition, particle-forming polypeptides derived from papillomavirus structural proteins L1 and L2 Tide is used herein alone as a VLP adjuvant and a coadjuvant. Or any use in combination. For use with the present invention The L1 and L2 proteins can be any of a variety of human and other animal papillomaviruses. , And its subgroups (HPV-1, HPV-2, HPV-5, HPV-6, HPV-8, HPV-11, HPV (Including, but not limited to, V-16, HPV-18, HPV-31, HPV-33, and HPV-45) It can be of origin. The sequences of L1 and L2 are known for various HPV types. example For example, for sequences of various HPV types, see Human Papillomaviruses: A Compilation an d Analysis of Nucleic Acid and Amino Acid Sequences (Myers, G. et al.), Los A See lamos National Laboratory, Los Alamos, New Mexico, 1994-1996.   From L1 and L2 to form VLPs either alone or in combination Methods are known, e.g., U.S. Pat.No. 5,437,951 (L1VLP in insect cells). WO 95/20659 (produced in eukaryotic cells using vaccinia virus vectors). WO93 / 02184 (Eukaryotic using vaccinia virus vector) Production of L1 / L2 VLPs in biological cells); Kirnbauer et al., J. Virol. (1993) 67: 6929-693. 6 (production of L1 and L1 / L2 VLPs in insect cells); Heino et al., Virology (1995) 214: 3. 49-359 (production of L1 and L1 / L2 VLPs in mammalian cells); Sasagawa et al., Virolog y (1995) 206: 126-135 (production of L1 and L1 / L2 VLPs in yeast cells); Zhou et al., Vir. ology (1991) 185: 251-257 (Vaccinia virus vector-infected epithelial cells L1 / L2 VLP production).   Similarly, some retroviral proteins form VLPs upon expression It is known. For example, rotavirus VP6 VLP is described in U.S. Patent No. 5,503,833. It can be produced as described. Generally, VP6 spherical particles contain recombinantly produced VP6. When the pH of the contained lysate becomes about pH 4.0 or more, it forms autonomously. Bovine rotavirus V P2 autonomously forms VLPs upon expression in the baculovirus system. For example See, Labbe et al., J. Virol. (1991) 65: 2946-2952.   Yeast retrotransposon Ty encodes a set of proteins Do. See, for example, Mellor et al., Nature (1985) 318: 583-586; Garfinkel et al., Cell (19 85) 42: 507-517; Adams et al., Cell (1987) 49: 111-119. Ty VLP is a yeast When expressed in the mother, the Ty element primary translation product p1, Combination of p1 and p3 (pre-Ty VLP), and particle-forming proteins of p1 and p3 It can be formed from degradation products. See, for example, Adams et al., Nature (1987) 329: 68-70. That.   VLPs for use in the present invention also include viruses derived from human parvovirus B19. Proteins (e.g., the viral proteins VP1 and And / or VP2 (see Brown et al., Virology (1994) 198: 477-488) and insects VP1, VP2 and / or VP3 proteins from polyomavirus expressed in cells Quality (Delos et al., Virology (1993) 194: 393-398; Forstova et al., Human Gene Therapy (19 95) 6: 297-306)). Retroviral gag proteins (e.g., Lau Derived from sarcoma virus (RSV) and Moloney murine leukemia virus (MLV) ) Are also autonomously forming VLPs when expressed in eukaryotic cells. As well as the p-specific core antigen p55gag, and deletion mutants thereof, herein Use is found. See, for example, Wagner et al., Arch. Virol. (1992) 127: 117-137; R et al., Virology (1994) 200: 162-175; Brand et al., J. Virol. Meth. (1995) 51: 153-168, And Wagner et al., Virology (1996) 220: 128-140.   As explained above, VLPs are derived from host cells for which the particle-forming polypeptide of interest is appropriate. When recombinantly expressed in cells, they can form autonomously. Therefore, in the present invention, VLPs for further use are conveniently prepared using recombinant techniques well known in the art. It is. In this regard, the gene encoding the particle forming polypeptide in question may be obtained by known techniques. Cells and tissues containing this gene using DNA surgery or from surgery Can be isolated directly from For example, a description of the technology used to obtain and isolate DNA. See Sambrook et al. (Supra) for listings. Encodes a particle-forming polypeptide Genes can also be produced synthetically, based on known sequences. Nucleoti Can be designed using codons appropriate for the particular amino acid sequence desired. one In general, one will select preferred codons for the intended host in which the sequence will be expressed. Perfect Sequences are generally derived from overlapping oligonucleotides prepared by standard methods. Build and build into a complete coding sequence. For example, Edge, Nature (1981) 292: 7 56; Nambair et al., Sclence (1984) 223: 1299; Jay et al., J. Biol. Chem (1984) 259: 6311. checking ...   Once the coding sequence for the desired particle forming polypeptide is isolated, Or, if synthesized, they may be any suitable vector or vector for expression. It can be cloned into Recon. Numerous cloning vectors are available to those skilled in the art. It is known. And the selection of an appropriate cloning vector is a matter of choice. See generally, Sambrook et al., Supra. The vector is then used to Transform a stubborn host cell. Suitable expression systems include bacteria known in the art, Mammals, baculoviruses / insects, vaccinia, semliki forest virus (SFV), Mammalian, yeast and Xenopus expression systems, including but not limited to . Particularly preferred expression systems are mammalian, vaccinia, insect, and yeast systems. .   For example, many mammalian cell lines are known in the art, and American Type Cult ure Collection (ATCC) available from immortalized cell lines (e.g., Chinese Ham) Star ovary (CHO) cells, HeLa cells, infant hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocarcinoma cells (e.g., Hep G2) and others). It is not limited to these. Similarly, E. coli, Bacillus subtilis, and Streptococ Bacterial hosts such as cus spp. find use in the expression constructs of the present invention. Departure Particularly useful yeast hosts in the field are Saccharomyces cerevisiae, Candid a albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragili s, Kluyveromyces lactis, Pichiaguillerimondii, Pichia pastoris, Schizosa ccharomyces pombe and Yarrowia lipolytica. Baculovirus Insect cells for use in expression vectors include, inter alia, Aedes aegypti, Autogra pha californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugip erda, and Trichoplusia ni. For example, Summer and Smith, Texa s See Agricultural Experiment Station Bulletin No. 1555 (1987).   Viral vectors (for example, pox family viruses (vaccinia virus and And avian poxviruses) are particles in eukaryotic cells. It can be used for offspring production. Further, Tomei et al., J. Virol. (1993) 67: 4017-40. 26 and Selby et al., J. Gen. Virol. (1993) 74: 1103-1113. Senior-based infection / transfection systems have also found use in the present invention. It is. In this system, cells are coated with bacteriophage T7 RNA polymerase. The transfected vaccinia virus is first transfected in vitro. This polymerase transcribes only the template with the T7 promoter In that it offers elaborate specificity. Following infection, cells will have a T7 promoter And transfected with the DNA of interest. Vaccinia wi The polymerase expressed in the cytoplasm from the Lus recombinant is then Transcribes transfected DNA into RNA that is translated into protein by a mechanism I do. This method involves high-level, transient, fine-scale production of large amounts of RNA and this translation product. Provides cytoplasmic production.   Depending on the expression system and host selected, VLPs are expressed by the particle-forming polypeptide. And transformed with the expression vector under conditions that allow the formation of VLPs Produced by growing host cells. Selection of appropriate growth conditions depends on the Within the field of technology. When VLPs are formed intracellularly, cells are chemically, Destroyed using physical or mechanical means. This means lyses the cells, , Leaving the VLP substantially intact. Such methods are known to those skilled in the art. Yes, and for example, Protein Purification Applications: A Practical Approa ch, (E.L.V. Harris and S. Angel, eds., 1990).   Then, gradient centrifugation (eg, cesium chloride (CsCl) and sucrose gradient) Methods for preserving its integrity, such as by Kirnbauer et al., J. Viro l. (1993) 67: 6926-6936), as well as standard purification techniques (eg, Ion exchange chromatography and gel filtration chromatography) Isolate the particles.   Once obtained, the VLP adjuvant of the present invention provides a vaccine set containing the desired antigen. At the same time as, immediately before, immediately before, or Can be administered separately, either: Vaccine compositions are used to treat infections And / or may be used both for prevention. Further, the adjuvant formulation of the present invention The activity of the antigen produced in vivo, i.e. in combination with DNA immunization Can be used to enhance sex.   VLP adjuvants can be used against or against one or more selected pathogens. Immunizing a vertebrate subject against a subunit antigen derived from Use in eliciting an immune response to one or several antigens. Can be used. Antigens that can be administered with a VLP adjuvant include proteins, poly Peptides, antigenic protein fragments, oligosaccharides, polysaccharides Including ids. Similarly, oligonucleotides or polynucleotides encoding the desired antigen The oligonucleotide can be administered with a VLP adjuvant for in vivo expression You.   Antigens are derived from a wide variety of viruses, bacteria, fungi, plants, protozoa, and other parasites Can be derived from For example, the present invention relates to the herpesvirus family, including: See applications for stimulating immune responses to a wide variety of proteins from Issue: Herpes simplex virus (HSV) type 1 and 2 derived proteins (eg, HSV-1 and HSV-2 gB, gD, gH, VP16, and VP22); varicella-zoster virus ( VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV) Derived antigens (including CMV gB and gH); and from other human herpes viruses Native antigens (eg, HHV6 and HHV7). (Eg, cytomegalovirus tan For an overview of the context of the protein code, see Chee et al., Cytomegaloviruses (JK McDou). gall, Springer-Verlag 1990) pp. 125-169; discussion of various HSV-1-encoded proteins For the theory, McGeoch et al. Gen. Virol. (1988) 69: 1531-1574; HSV-1 and HSV For a discussion of -2 gB and gD proteins and the genes that encode them, US Patent No. 5,171,568; Identification of Protein Coding Sequence in EBV Genome Davison, Baer et al., Nature (1984) 310: 207-211; and a review of VZV. And Scott, J .; Gen. Virol. (1986) 67: 1759-1816).   Furthermore, hepatitis family viruses (hepatitis A virus (HAV), hepatitis B virus). Ils (HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV), type E Immunity to antigens from hepatitis virus (HEV), including hepatitis G virus) The epidemic response can also be conveniently enhanced using a VLP adjuvant. For illustrative purposes, H The CV genome contains several viral proteins (E1 (also known as E) and E2 (also known as E2 / NSI), thereby providing (For a discussion of HCV proteins, including E1 and E2, see Houghto n et al., Hepatology (1991) 14: 381-388). Δ-antigen from HDV also Can be used with the VLP adjuvant system of the invention (eg, for the description of the δ-antigen See US Pat. No. 5,389,528).   Similarly, influenza viruses are another class of viruses for which the present invention is particularly useful. It is an example. Specifically, the influenza A envelope glycoproteins HA and NA Are particularly beneficial for generating an immune response. Many influenza A Many HA subtypes have been identified (Kawaoka et al., Virology (1990) 179: 759-767). Webster et al., “Antigenic Variation among type A influenza viruses” 127-16 Page 8; Palese and D.W. Kingsbury (eds.), Genetics of influenza viruses, Sp ringer-Verlag, New York). Thus, the immune response to any of these antigens Can be enhanced when they are administered with a VLP adjuvant of the invention. .   Another particularly useful antigen to be used in combination with the VLP adjuvant is human Antigen derived from papilloma virus (HPV) and polypeptide derived from the antigen (Eg, one or more of various early proteins, including E6 and E7), tick-borne Encephalitis virus, derived from HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.) And a polypeptide derived therefrom, wherein the antigen comprises the isolate HIV_IIIb , HIV_SF2, HIV_LAV, HIV_LAI, HIV_MNAntigens derived from, for example, gp120, gp41, gp160 , Gag, and pol, including, but not limited to, various HIV isolates Myers et al., Los Alamos Database, Lo s Alamos National Laboratory, Los Alamos, New Mexico (1992); Myers et al., Hum. an Retroviruses and Aids, 1990, Los Alamos, New Mexico: Los Alamos Natio nal Laboratory; and Modrow et al. Virol. (See (1987) 61: 570-578.) .   Proteins from other viruses also find use in the methods of the invention. And its proteins include, but are not limited to, for example, inter alia : Picornaviridae (eg, poliovirus); Caliciviridae; Togavirid ae (eg, rubella virus, dengue virus, etc.); Flaviviridae; Coronaviri dae; Reoviridae; Birnaviridae; Rhabodoviridae (for example, rabies virus Filovridae; Paramyxoviridae (eg, mumps virus, measles virus) Orthomyxoviridae (eg, influenza virus A) Type, type B and type C); Bunyaviridae; Arenaviridae; Retroviridae (eg For example, HTLV-1; HTLV-II; HIV-1; HIV-2); Simian immunodeficiency virus (SIV) family Proteins from Lee members. For example, these viruses and other viruses Virology, 3rd edition (W.K. Joklik, 1988); Fundamental V irology, 2nd edition (B.N. Fields and DM Knipe, eds., 1991)).   Particularly preferred bacterial antigens are diphtheria, tetanus, pertussis, meningitis, and other From an organism that causes a pathogenic condition, whose antigens include, but are not limited to: Not: Corynebacterium diphtheriae, Clostridium tetani, Bordetella pe rtusis, Neisseria meningitidis (Meningococcus serotypes A, B, C, Y, and And W135 (including MenA, B, C, Y, and W135), Haemophilus influenza B Type (Hib), and antigens from Helicobacter pylori. An example of a parasite antigen is Marari And antigens from organisms that cause Lyme disease.   Combinations of antigens from the above organisms can address multiple pathogens in a single vaccine. It can be conveniently used to induce immunity. For example, particularly preferred combinations Non-toxic mutant carriers derived from bacterial toxins (eg, CRM)₁₉₇Known as Bacterial surface from MenC and Hib bound to a non-toxic mutant of diphtheria toxin It is a combination of oligosaccharides. This binding prevents bacterial meningitis. And described in International Publication No. WO 96/14086 (published May 17, 1996). Will be posted.   In addition, the methods described herein provide a means for treating various malignant cancers. provide. For example, using the system of the present invention, specific proteins specific to the cancer in question may be used. Proteins (eg, activated oncogenes, fetal antigens, or activation markers) Can enhance both the humoral immune response and the cell-mediated immune response. like that Tumor antigens include any of a variety, including, inter alia: MAGE 1, 2, 3, 4, etc. MAGE (Melanoma-associated antigen E) (Boon, T. Scientifc American (March 1993): 82-89); any of a variety of tyrosinases; MART 1 (melano recognized by T cells Mutant ras; mutant p53; p97 melanoma antigen; CEA (carcinoembryonic antigen) .   The present invention is used to increase the immune response to a wide variety of antigens, thereby It is readily apparent that most diseases can be treated or prevented.   As explained above, the VLP formulation may include one or more antigens of interest, It may not be included. For example, the antigen can be used in the VLP composition Can be administered separately. In any event, the one or more selected antigens "Therapeutically effective" such that an immune response can be generated in the individual to which the antigen is administered. Amount "administered. The exact amount required will vary depending, inter alia, on the following factors: Become: subject to be treated; age and general condition of the subject to be treated; Immunization of a subject that synthesizes antibodies and / or increases a cell-mediated immune response The ability of the system; the degree of protection desired; the severity of the condition being treated; Antigen and its administration form. An appropriate effective amount can be readily determined by one skilled in the art. . Thus, a "therapeutically effective amount" is a relatively broad range that can be determined through routine trials. Settle in range. Generally, a "therapeutically effective" amount of the antigen is from about 0.1 μg to about 1000 μg, More preferably, the amount is on the order of about 1 μg to about 100 μg.   Similarly, VLP adjuvant is compared to administration of antigen alone without VLP adjuvant And the amount by which the antigen exhibits "enhanced immunogenicity" as defined above. Exists in. The amount effective to elicit an enhanced immune response will vary depending on the skilled artisan. Can be easily determined.   The composition may further comprise one or more "pharmaceutically acceptable excipients or vehicles." (Eg, water, saline, glycerol, ethanol, etc.). Further In addition, auxiliary substances, such as wetting or emulsifying agents, biological buffers, etc. May be in the vehicle. The biological buffer is pharmacologically acceptable, and Substantially provide the desired pH (ie, pH in the pharmacological range) to the adjuvant formulation Can be any solution. Examples of buffer solutions are saline, phosphate buffered saline Saline, Tris buffered saline, Hank's buffered saline, Eagle minimum essential medium ("MEM") and the like.   Antigens, by themselves, induce the production of antibodies harmful to the individual receiving the composition It is optionally combined with a carrier that is not a molecule. A good career is Typically, large, slowly metabolized macromolecules (eg, proteins, polysaccharides) Callide, polylactic acid, polyglycolic acid, polymerizable amino acid, amino acid copolymer , Lipid aggregates (eg, oily droplets or liposomes), and inert viruses Particles. Such carriers are well-known to those skilled in the art. In addition, these The carrier can function as a further immunostimulant. In addition, the antigen It is bound to bacterial toxoids such as toxoids from a, tetanus, cholera, etc. obtain.   In addition to the VLPs of the present invention, co-adjuvants also enhance the efficacy of pharmaceutical compositions Can be used to Such coadjuvants include, but are not limited to: But not limited to: (1) aluminum salts (eg, aluminum hydroxide, (2) Oil-in-water emulsion formulation (This may be due to other specific immunostimulants, such as muramyl peptide (see below). With or without bacterial cell wall components), such as (a) Model 110Y Microfluidizer (Microfluidizer) (Microfluidics, Newton, MA) 5% Squalene, 0.5 formulated into submicron particles using a rofluidizer % Tween 80, and 0.5% Span 85 (although not required, but necessary MF59 (International Publication Number) containing various amounts of MTP-PE (see below) WO 90/14837, (b) to produce emulsions of larger particle size Microfluidized or vortexed into submicron emulsion 10% Squalane, 0.4% Tween 80, 5% pluronic blocking -blocked) SAF, including polymer L121, and thr-MDP (see below) Rabi ni (c) 2% Squalene, 0.2% Tween 80, and monophospholipid A (MPL) , Trehalose dimycolate (TDM), and cell wall skeleton (CWS) One or more bacterial cell wall components, preferably MPL + CWS (Detox^TMIncluding Ribi^TMHorse mackerel (3) Saponin adjuvant (RAS) (Ribi Immunochem, Hamilton, MT); Bunt, for example, Stimulon^TM(Cambridge Bioscience, Worcester, MA) used Particles that can be or are produced from, eg, ISCOMs (immunostimulating complexes) (4) Freund's complete adjuvant (CFA) and incomplete Freund's adjuvant (5) cytokines (eg, interleukins (IL-1, IL-2, etc.) ), Macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.); ) Mucosal adjuvants (eg, cholera toxin (CT), pertussis toxin (PT), E. coli fever Adjuvants derived from stable toxins (LT) and variants thereof (eg, International Publication No. Nos. WO 95/17211, WO 93/13202, and WO 97/02348); and (7) )) Other substances that act as immunostimulants, which enhance the effectiveness of the composition. alum and MF59 is preferred.   Muramyl peptides include, but are not limited to: N-acetyl-mu Ramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl (normuramyl) -L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L- Alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glyce B-3-Hydroxyphosphoryloxy) -ethylamine (M TP-PE).   Once formulated, the compositions of the present invention can be administered parenterally, for example, by injection. Can be given. The composition can be injected either subcutaneously, intraperitoneally, intravenously, or intramuscularly. Can be done. Other modes of administration include oral and pulmonary administration, suppositories, mucosal and transdermal applications. Including. Dosage treatment may be a single dose schedule or a multiple dose schedule. You. Multi-dose schedules have a primary course of vaccination of 1 to 10 separate doses, followed by selection to maintain and / or enhance the immune response Other doses given at subsequent time intervals (eg, 1-4 doses for a second dose) Month, and optionally several months later). Medication regimen , At least in part, is determined by the needs of the subject and depends on the judgment of the practitioner. Exist. In addition, if prevention of the disease is desired, the vaccine will generally comprise the pathogen of interest. It is given before a temporary infection in the body. If treatment is desired, for example, symptoms or If a reduction in recurrence is desired, the vaccine is generally administered following the primary infection. You. C.Experiment   The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only. Provided only in a limited manner and in any manner limits the scope of the invention It is not intended.   Efforts are made to confirm accuracy with respect to numerical values used (eg, amounts, temperatures, etc.). However, some experimental errors and deviations should of course be tolerated.                                 Example 1   Hepatitis B virus (HBV) VLP and Haemophilus influenzae type b (Hib) and And / or vaccine containing Hib / Meningococcal C (MenC) conjugate in infant baboons The following studies were performed to evaluate immunogenicity.                              Materials and methods A.vaccine   The Hib / MenC conjugate vaccine and the Hib conjugate vaccine used in this study Park Carrier CRM₁₉₇Containing oligosaccharides independently conjugated to Produced as described in No. WO 96/14086 (published May 17, 1996).   The VLP used was the recombinant hepatitis B virus (HBV) pre-S2 / S antigen. VLP The Pre-S2 / sAg gene into an expression vector and use it to produce Using DG44 Chinese Hamster Ovary (CHO) cells (this is also the dhfr gene Was transfected). Cell proliferation and HB Essentially, expression of V particles was described by Michel et al., Bio / Technology (June 1985) 3: 561-566 and And Patzer et al., As described in Bio / Technology (July 1986) 4: 630-636. did.   The control vaccine formulation was HBV S antigen from yeast adsorbed on alum. Was included.   Reconstitute both complex polysaccharide vaccine and HBV VLP formulation on the day of vaccination And a dose of about 5 μg of each sugar, a total of 20 μg of CRM₁₉₇Protein, and 2.5 μg of H BV VLP provided. B. Vaccine group   At the start of the study, 1.5- to 4-month-old infant baboons were Groups were assigned. * N = 6 animals / group (1.5-4 months of age at the start of the study) Blood samples were collected before each immunization and two weeks after the third immunization.                                   result   Figures 1 and 2 show HBV VLP versus HBV VLP as determined by commercial E1A (Abbott). 1 shows the serum antibody response of an infant baboon. The result is the geometric mean of the antibody concentration in IU / ml. On a log scale of ± 95% C1, the vaccine containing HBV VLPs To indicate   As can be appreciated, HBV VLP alone given with MF59 was highly immunogenic. By nature, this is greater than 100 IU / ml after a single injection and 1000 IU / ml after a second injection. Reach a geometric mean titer of> 100,000 IU / ml after the third injection Done.   In contrast, the control vaccine given with alum is much lower It is immunogenic, does not respond to a single injection, and has There was a 1/10 response to the second and third injections. Not shown, MenC / Hib binding In group 5, where the body vaccine was given without VLP, the detectable hepatitis antibody response was Did not exist. These results were observed in clinical experiments in healthy adults. Corresponding to the difference in immunogenicity between these two vaccines.   Presence of Hib or Hib / MenC conjugate was compared to the response of the group receiving hepatitis B / MF59 alone. In comparison, the hepatitis antibody response was reduced. This may occur after the first or second injection. Was especially true. However, the third of the MF59 adjuvanted combination vaccines After injection of each, the respective antibody response to HBV was still 10 times higher than the response of vaccinated animals (geometric means 49,000 and 38,000 IU / ml vs. 5000 IU / ml for control HBV) (p <0.01).   Serum anti-capsular antibody response of infant baboons to Hib conjugate vaccine Determined using a combined assay. FIGS. 3 and 4 show that on a log scale in μg / ml, With Hib / MenC, HBV / Hib and M4F59 given with F59, and MF59 Mean antibody concentrations for the three groups given the vaccine containing HBV / Hib / MenC Indicates the degree.   All three groups showed strong antibody responses to Hib. Interestingly, H The presence of BV VLP appeared to increase the Hib antibody response. For example, the third injection Later, the geometric mean antibody concentration was 3 in the group receiving HBV with Hib / MenC and MF. 5 μg / ml versus 10.8 μg for the group receiving Hib / MenC / MF59 without HBV g / ml. The Hib response to HBV / Hib without MenC was even higher (1 00 μg / ml).   The serum antibody response of infant baboons to the MenC conjugate vaccine was also measured. The result 5A and 5B. FIG. 5A shows IgG anticapsules determined by ELISA. 3) shows the antibody response. FIG. 5B shows the complement-mediated bacterial antibody response. M including HBV VLP The antibody response to the F59-adjuvanted MenC combination vaccine was Hib with MF59. Responses were equal to or higher than those of animals receiving the / MenC vaccine alone. this Are IgG binding antibodies determined by ELISA and antibodies determined by bactericidal assay This was true for both functional activities.   Table 2 summarizes the geometric antibody response after the third injection for all groups. Reason As can be seen, control vaccine adsorbed to alum, or MF59 Animals receiving an experimental HBV VLP preparation with a response only to HBV. further , The magnitude of the response is more than 20 times higher than for the experimental MF59 adjuvanted vaccine It was higher.   HBV antibody response to Hib or two combination vaccines containing Hib and MenC , Approximately one-half of the response of animals receiving HBV / MF59 alone. I'm also concerned And they are still 8-10 times higher than the response observed with the control vaccine won. As already noted, H for two combination vaccines containing HBV VLPs The ib antibody response was greater than the response of animals given Hib / MenC / MF59 without HBV VLP (Group 5) it was high.   For MenC conjugates, there is also evidence that HBV VLP can enhance IgG MenC antibody response Also existed. ^*P = 0.07: + P ≦ 0.03                                 Example 2   Experiments also included human papillomavirus 6b (HPV 6b) L1 VLP and HPV E7. In order to evaluate the immunogenicity of the vaccine, it was performed in rabbits as follows.                              Materials and methods A.vaccine   The recombinant protein used in the vaccine of this study was HPV 6b as described. Cloned from Schwarz, EMBO J .; (1983) 2: 2341-2348. Virus-like particle (VLP) vaccines self-assemble into particles and perform ion exchange and Late stage from baculovirus purified via gel and gel filtration chromatography 1 (L1) protein. VLPs are approximately 50 nm in size, and HPV (Greer, J. Clin. Micro. (1995) 33: 2058-2063). Second The vaccine consisted of Early 7 (E7) protein from E. coli.   On the day of vaccination, the vaccine contains 17 μg of HPV 6 L1 VLP On the other hand, the E7 vaccine dose contains 50 μg of E7, and the L1 / E7 combination vaccine dose is It was prepared to contain 17 μg of VLP and 50 μg of E7. Total volume of all vaccines The volume was 0.5 ml and 0.25 ml was MF59 coadjuvant. B. Vaccine group   Young adult female New Zealand white rabbits are available in three possible (20 animals per group). Animals can be VLP, E7, or L1 or Received vaccines containing any of the E7 combinations. C.Immunization and blood collection schedule   Rabbits received intramuscular (hind leg) vaccinations during weeks 0, 3, and 6 . Blood samples were collected during weeks 0, 4, 5, 7, and 8. D.Determination of antigen-specific antibody response   Antibody response was measured using an antigen-specific ELISA. Titer, optical density 0.5 The dilution was determined for each serum.result   The rabbit serum titer results for the E7 and L111 antigens are summarized in FIG. Logarithm The results presented on the scale are the arithmetic mean of the antibody titers for the groups. MF59 Waku H Antibody titers generated from E7 alone with Significantly lower than VLPs (313 vs. 106,000; p <0.0001; and 1,400 vs. 65,000; p <0.0001). However, the anti-E7 average titer produced by the E7 / VLP combination showed that MF59 When injected with the combined E7 / L1 vaccine (and MF59) when compared to the associated E7 alone Seemed to be increased in those (825 vs. 313; 1,567 vs. 1,363). In addition, E7 / L1 The median titer was significantly higher at weeks 5 and 8 at the 5% level (respectively , P-values 0.0018 and 0.0125). In addition, the number of non-responders was E7 / L1, Was statistically significantly lower for (p-value 0.023, Fisher's exact test ( exact test)).   The data show that VLPs alone with MF59 are highly immunogenic, indicating that Potency of 482 after firing and 106,000 and 65,000 after the second and third injections I did it. However, anti-VLP levels for the combined VLP / E7 vaccine group after the second immunization The average titer decreased (106,000 vs. 89,000) and significantly decreased after 3 immunizations (65,00 0 vs. 35,000; p = 0.001).   Accordingly, novel VLP adjuvant compositions and methods of using and making them A method is disclosed. Although the preferred embodiments of the present invention have been described in some detail, Apparent modifications, as defined by the appended claims, are within the spirit and scope of this invention. It is understood that this can be done without departing from the scope and scope.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, L S, MW, SD, SZ, UG, ZW), EA (AM, AZ , BY, KG, KZ, MD, RU, TJ, TM), AL , AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, E E, ES, FI, GB, GE, GH, GM, GW, HU , ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, M D, MG, MK, MN, MW, MX, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, V N, YU, ZW (72) Inventor Greer, Catherine E.             United States California 94608,             Emilyville, Houghton Street             440 4560, Chiron Corporation             Inside (72) Inventors Van Nest, Gully A.             United States California 94608,             Emilyville, Houghton Street             440 4560, Chiron Corporation             Inside

Claims (1)

[Claims] 1. Virus-like particle (VLP) adjuvants, selected antigens, and pharmaceutically acceptable A vaccine composition comprising an acceptable excipient, wherein the selected antigen is the VLP. Different, vaccine compositions. 2. The vaccine composition according to claim 1, further comprising a coadjuvant. 3. 3. The vaccine composition according to claim 2, wherein said coadjuvant comprises MF59. 4. The VLP is formed from one or more particle forming polypeptides of a hepatitis surface antigen. A vaccine composition according to any of claims 1 to 3. 5. The particle-forming polypeptide is selected from the group consisting of sAg, pre-S1, and pre-S2. 5. The vaccine set according to claim 4, which is at least one hepatitis surface antigen selected. Adult. 6. 6. The vaccine set of claim 5, wherein said VLP is formed from sAg and pre-S2. Adult. 7. The VLP is a particle forming polypeptide of papillomavirus L1 and / or L2. The vaccine composition according to any one of claims 1 to 3, which is formed from: 8. The method according to claim 1, wherein the selected antigen is a MenC / Hib oligosaccharide conjugate. A vaccine composition according to any of the preceding claims. 9. The vaccine according to any one of claims 1 to 7, wherein the selected antigen is HPV E7. Chin composition. 10. Compositions useful for producing an enhanced immune response in a vertebrate subject Virus-like particle (VLP) adjuvant and a different selection from said VLP for the production of Use of the selected antigen. 11. The VLP is formed from one or more particle forming polypeptides of a hepatitis surface antigen. The use according to claim 10, wherein 12. Wherein the particle-forming polypeptide is a group consisting of sAg, pre-S1, and pre-S2. The use according to claim 11, which is at least one hepatitis surface antigen selected from: 13. 13. Use according to claim 12, wherein the VLP is formed from sAg and pre-S2. 14． The VLP is a papillomavirus L1 and / or L2 particle-forming polypeptide. The use according to claim 10, wherein the use is formed from a metal. 15. 2. The method of claim 1, wherein the selected antigen is a MenC / Hib oligosaccharide conjugate. 4. Use according to any of 4. 16. 15. The method of claim 10, wherein the selected antigen is HPV E7. Use of. 17． A method of preparing an adjuvant formulation, comprising: adding virus-like particles (VLPs) Providing and combining the VLP with a pharmaceutically acceptable excipient. Including, the method. 18. Combining the selected antigen with the VLP and the pharmaceutically acceptable excipient 18. The method of claim 17, further comprising the step of passing.