The present invention relates to nanoparticle vaccines comprised of a carrier, particularly polymerized lipids, having multiple copies of an antigen or combinations of different antigens displayed on the carrier. Such antigen-displaying nanoparticles may also display a targeting molecule on its surface in order to direct it to a specific site or cell type to optimize a desired immune response. The present invention also relates to encapsulating an antigen or combinations of different antigens within such nanoparticles, with or without a targeting molecule displayed on its surface. The antigens used in this invention are effective to produce an immune response against a variety of pathological conditions.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that nanoparticle vaccines having multivalent surface antigens (presented on the exterior or interior or the particle) or encapsulated antigens elicit significantly increased immune responses (Guan, et al., "Liposomal Formulations of Synthetic MUC1 Peptides: Effects of Encapsulation Versus Surface Display of Peptides on Immune Responses," Bioconj. Chem. 9:451458 (1998), which is hereby incorporated by reference in its entirety). Additionally, co-display of targeting molecule(s) on the polymerized liposome nanoparticle for purposes of directing the vaccine to a specific in vivo location increases the efficiency and effectiveness of the desired immune response.

Polymerization of the membrane greatly "freezes" the positions of the items displayed on the particle surface. As presentation of antigenic elements in a polyvalent array is believed to be an important contributor toward promoting an immunological response (Chackerian, et al., "Induction of Autoantibodies to Mouse CCR5 with Recombinant Papillomavirus Particles," Proc. Natl. Acad. Sci. USA 96(5):2373-2378 (1999), which is hereby incorporated by reference in its entirety), a fixed surface-displayed rigid array is likely to be a more successful antigenic presenter than a fluid surface. Once the polymerized particle is prepared and assayed for vaccine effectiveness surface changes which may alter its activity or toxicity are unlikely to occur.

In the present invention, antigens may also be contained inside the nanoparticle, with or without surface displayed antigens and/or targeting molecules, depending upon the specific disease application. The present invention provides compositions and methods for use in various pharmaceutical applications, including vaccinating a subject for protection against infection by a pathogenic agent or for vaccination of a subject for resolution of a chronic infectious disease. Such subjects may include humans and wild or domestic animal populations such as bison, elk, cows, horses, sheep, goats, pigs, fowl, cats and dogs, although this invention may be applied to other species as well. Administration of the vaccine of this invention may be carried out orally, intradermally, intermuscularly, intraperitoneally, intravenously, subcutaneously, intranasally, sublingually, buccally, vaginally, or rectally.

In a preferred embodiment, the present invention relates to a nanoparticle that comprises a carrier, and polymerized liposome carriers are preferred, although various other carriers known to persons skilled in the art also would be appropriate. The polymerized liposome carrier may be either phospholipid or non-phospholipid based. The carrier preferably carries or displays (on the interior or exterior) multiple copies of antigen or combination of different antigens and targeting molecules. In another preferred embodiment the antigen-displaying carrier does not include targeting molecule(s). In a third preferred embodiment, the carrier displays antigen or a combination of different antigens and a targeting molecule on its surface, and encapsulates antigen or a combination of antigens within the nanoparticle. In another preferred embodiment, the antigen-displaying carrier encapsulates antigen(s) but does not display targeting molecule(s). In yet another preferred embodiment, the carrier displays targeting molecule(s) without antigen on its surface and encapsulates antigen or a combination of antigens within the nanoparticle.

According to the methods and compositions of the present invention, surface exposed antigen(s) and/or targeting molecule(s) may be attached to the nanoparticles by any means known in the art. Conjugation methods of this invention include chemical complexing, which may be either ionic or non-ionic in nature, or covalent binding. Such conjugation of antigen or targeting molecule may occur to reactive head groups of individual lipid monomers, or a collection of lipid monomers prior to assembly of the nanoparticle. Alternatively the antigen or targeting molecule can be attached to reactive head groups after the polymerized nanoparticle is formed.

The antigen or antigens of the present invention that are displayed on or within the nanoparticle induce an immune response against onset of disease caused by a variety of pathogenic conditions. In a preferred embodiment, the antigen may be derived from, but are not limited to, pathogenic bacterial, fungal, or viral organisms, Streptococcus species, Candida species, Brucella species, Salmonella species, Shigella species, Pseudomonas species, Bordetella species, Clostridium species, Norwalk virus, Bacillus anthracis, Mycobacterium tuberculosis, human immunodeficiency virus (UV), Chlamydia species, human Papillomaviruses, Influenza virus, Paramyxovirus species, Herpes virus, Cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus, Hepatitis viruses, Plasmodium species, Trichomonas species, sexually transmitted disease agents, viral encephalitis agents, protozoan disease agents, fungal disease agents, bacterial disease agents, cancer cells, or mixtures thereof Other preferred embodiments include self-antigens for the treatment or prevention of autoimmune diseases. Another preferred embodiment includes adhesins or surface exposed cell signaling receptors or ligands. In still another preferred embodiment, the targeting agent or molecule directs the vaccine to a mucosal membrane. In yet another preferred embodiment, adjuvant(s) may be incorporated in the vaccine.

The nanoparticle vaccines of the present invention are superior to other platforms for several reasons: the spheroid assemblies are simple and inexpensive to synthesize and are very stable; the structures are polymerized to be "rigid", not suffering from folding uncertainties, unlike conventional bilayer liposomes, they are inert with regard to random fusion with themselves or cell membranes; and the surface character and displayed molecular orientation is easily manipulated because the polymer backbone tolerates nearly any appended molecule in a wide range of ratios. Therefore, it is an object of the present invention to provide new methods and compositions for a superior nanoparticle-based multivalent vaccine against a broad range of diseases and disorders.

DETAILED DESCRIPTION OF THE INVENTION

I. General Description

The prevention of microbial infections and pathogenic processes via the use of vaccines is considered one of the most effective and desirable procedures to combat illness. In this art, antigens or immunogens are introduced into an organism in a manner that stimulates an immune response in the host organism in advance of an infection or disease. As used herein, the term "antigen" or "immunogen" means a molecule that is specifically recognized and bound by an antibody. The molecule may be a protein or peptide of bacterial, fungal, protozoan, or viral origin, or a fragment derived from these antigens, a carbohydrate, or a carbohydrate mimetic peptide. The antigenic molecule(s) may also include self-antigens for the treatment of autoimmune diseases. Additionally, the antigenic molecule(s) may also include carbohydrates, nucleic acids, small organic molecules, or conjugates of any of these compounds. The specific portion of the antigen that is bound by the antibody is termed the "epitope".

The induction of an immune response depends on many factors, among which are believed to be the chemical composition and configuration of the antigen, the immunogenic constitution of the challenged organism, and the manner and period of administration of the antigen. An immune response has many facets, some of which are exhibited by the cells of the immune system (e.g., B-lymphocytes, T-lymphocytes, macrophages, and plasma cells). Immune system cells may participate in the immune response through interaction with an antigen or other cells of the immune system, the release of cytokines and reactivity to those cytokines. Immune response is conveniently (but arbitrarily) divided into two main categories--humoral and cell-mediated. The humoral component of the immune response includes production of antibodies specific for an antigen. The cell-mediated component includes the generation of delayed-type hypersensitivity and cytotoxic effector cells against the antigen.

Polymerized nanoparticles can be readily used in conjunction with synthetic vaccines. Individual "small" antigens, especially small peptides or carbohydrates, are difficult to administer and generally fail to elicit an effective immune response due to the hapten-related size issues. Thus, combining multiple copies of an antigen into a multivalent display enhances the immuno-recognition by the host, particularly human beings and commercially important livestock and other animals.

In addition, immunizations with multivalently displayed antigens can be improved by including targeting molecules or adhesins to direct the nanoparticle to the appropriate immune cell or location. A necessary step in the successful colonization and, ultimately, production of disease by microbial pathogens is the ability to adhere to host surfaces. This fundamental idea has led to an enormous amount of research over the last two decades that deals with understanding how pathogens target and adhere to host cells (Finlay, et al., "Common Themes in Microbial Pathogenicity Revisited," Micro. Molec. Biology Rev. 61(2): 136-169 (1997), which is hereby incorporated by reference in its entirety). Examples of such molecules which target mucosal epithelium include the tetanus toxoid; P pili of E. coli; type IV pili of Pseudomonas aeruginosa, Neisseria species, Moraxella species, EPEC, or Vibrio cholerae; fimbrial genes and several a fimbrial adhesins, including FHA, pertactin, pertussis toxin and BrkA of Bordetella pertussis; and SipB-D of Salmonella typhimurium (Finlay, et al., "Common Themes in Microbial Pathogenicity Revisited," Micro. Molec. Biology Rev. 61(2): 136-169 (1997), which is hereby incorporated by reference in its entirety); and the adenovirus adhesin (Gallichan, et al., "Mucosal Immunity and Protection after Intranasal Immunization with Recombinant Adenovirus Expressing Herpes Simplex Virus Glycopritein B," J. Infect. Dis. 168:622-629 (1993), which is hereby incorporated by reference in its entirety); or the Reovirus sigma-1 protein which targets the M-cell (Wu, et al., "M Cell-Targeted DNA Vaccination," PNAS 98(16):9318-9323 (2001), which is hereby incorporated by reference in its entirety); among other targeting molecules or adhesins.

The majority of the infections are caused by pathogens that first contact and then either colonize or cross mucosal surfaces to infect the host. It is possible to prevent the initial infection at mucosal surfaces by stimulating production of secretory IgA (S-IgA) antibodies directed against relevant virulence factors. S-IgA may prevent the initial interaction of the pathogen with the mucosal surface by blocking attachment and/or colonization, neutralizing surface acting toxins, or even inactivating invading viruses inside of epithelial cells.

Mucosal immunization may be an effective means of inducing not only S-IgA but also systemic antibody and cell-mediated immunity (Ghee, et al., "New Perspectives in Vaccine Development: Mucosal Immunity to Infections," Infect. Agents Dis. 2(2): 55-73 (1993) and Cardenas, et al., "Oral Immunization Using Live Attenuated Salmonella spp. as Carriers of Foreign Antigens," Clin. Microbiol. Rev. 5(3):328-342 (1992), which are hereby incorporated by reference in their entirety). While mucosal vaccination is attractive for inducing a variety of immune responses, mucosally administered antigens are frequently not immunogenic and require an adjuvant. E. coli heat-labile enterotoxin holotoxin (LT) and Vibrio cholerae enterotoxin (CT) represent promising mucosal adjuvants (Holmgren, et al., "Cholera as a Model for Research on Mucosal Immunity and Development of Oral Vaccines," Curr. Opin. Immunol. 4(4):387-391 (1992), which is hereby incorporated by reference in its entirety). These adjuvants can be used to promote the production of serum and/or mucosal antibodies as well as cell-mediated immune responses against co-administered antigens.

Derived from the cell wall of Salmonella Minnesota, MPL adjuvant has proven ability to boost the potency of modern vaccines. This adjuvant may be a key component of vaccines using technologies such as recombinant and synthetic antigens. While vaccines incorporating these antigens are safer than previous attenuated or killed whole-cell vaccines, many of them are poorly immunogenic in the absence of a potent adjuvant. MPL adjuvant has demonstrated utility with peptide, bacterial sub-unit and synthetic polysaccharide antigens. Humoral, cell mediated and mucosal immunity can be stimulated by altering formulations and delivery routes.

Incorporation of the above mentioned adjuvants into the nanoparticle surface array, intercalation into the nanoparticle wall or encapsulation into the nanoparticle interior may provide an effective means of delivering to and stimulating the mucosal immune system to produce either or both humoral or cellular mucosal immunity to nanoparticle delivered antigens. As used herein the term "adjuvant" means any material which modulates to enhance the humoral and/or cellular immune response.

As used herein, the terms "displayed" or "surface exposed" are considered to be synonyms, and refer to antigens or other molecules that are present (e.g., accessible to immune site recognition) at the external surface of a structure such as a nanoparticle. From the targeted nanoparticle vaccines, we can expect highly intense, anamnestic and long-lasting immune responses (several years). Thus, the nanoparticle multivalency and targeting enhance the antigen concentration and promote delivery that favors the formation of high-affinity Th/B cell collaborations needed for optimal induction of the antibody response.

Phage display library technology is currently being used to discover many interesting peptide ligands that have immunogenic properties. However, a limitation of that technology is in recreating the conformational characteristics of the identified peptide to be similar to the viral capsid display platform. In many cases, the single, monomeric, synthetic peptide sequence fails to fold in the three-dimensional architecture or recreate a multi-peptide strand conformation that was present on the multivalent phage display. However, reassembling them in multivalent form amidst specific matrix lipid formulations on polymerized nanoparticle vaccines, such as in the present invention, often can restore the immunological activity of such peptides that have been isolated from the phage library.

Nanoparticle vaccines of the present invention are important new forms of drugs and drug delivery systems because the presentation of multivalent or aggregated antigens on the nanoparticle surface can enhance the desired immune response of a treated host. As used herein, the term "multivalent" means that more than one copy or type of antigen is displayed on a nanoparticle, preferably via linkers attached to component lipid monomers. Moreover, the one or more copies or types of antigen may be attached to the nanoparticle through two separate linkers, or may be attached to the nanoparticle via a common linker.

Arranging multiple copies of an antigen on a carrier and presenting them spatially is often more stimulatory than dispersed or solute molecules. The displayed antigens are able to bind more effectively to immune sites in the living body, thereby engaging more cell surface molecules on the specialized cells and antigen processing receptors involved in generating immune responses. As used herein, the term "antigen processing receptor" refers to receptors that mediate the uptake and processing of antigens, and then present the antigens for the development of immunity. Such receptors may be found on, for example, M-cells, dendritic cells and macrophages. Multivalent antigens have the advantage of increasing the desired immune response. Additionally, combinations of different antigens can be displayed on the same nanoparticle for purposes of eliciting a stronger immune response against one pathogen or against multiple pathogens at one time. It is envisioned that the specific display parameters important for protective efficacy against a specific disease or pathogen may vary.

Appropriate antigens for use with this vaccine technology may be derived from, but not limited to, pathogenic bacterial, fungal, or viral organisms, Streptococcus species, Candida species, Brucella species, Salmonella species, Shigella species, Pseudomonas species, Bordetella species, Clostridium species, Norwalk virus, Bacillus anthracis, Mycobacterium tuberculosis, human immunodeficiency virus (HIV), Chlamydia species, human Papillomaviruses, Influenza virus, Paramyxovirus species, Herpes virus, Cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus, Hepatitis viruses, Plasmodium species, Trichomonas species, sexually transmitted disease agents, viral encephalitis agents, protozoan disease agents, fungal disease agents, cancer cells, or mixtures thereof Other appropriate molecules incorporated in the nanoparticle vaccines may include self-antigens, adhesins, or surface exposed cell signaling receptors or ligands. A variety of diseases and disorders may be treated by such nanoparticle vaccine constructs or assemblies, including: inflammatory diseases, infectious diseases, cancer, genetic disorders, organ transplant rejection, autoimmune diseases and immunological disorders.

T-cell activating molecules and/or adjuvants may be co-displayed or encapsulated with antigen(s) to direct the nanoparticle vaccine to a particular in vivo location or to enhance a certain desired immune response. Similarly, the addition of a targeting agent or agents to such nanoparticles provides the ability to direct such vaccines to a specific in vivo location, which increases the efficiency and effectiveness of a desired immune response. Targeted delivery to a specific site maximizes vaccine response and efficiency and minimizes potential side effects.

As used herein, the term "liposome" is defined as an aqueous or aqueous-buffered compartment enclosed by a lipid bilayer (Stryer, Biochemistry, 2nd Edition, W. H. Freeman & Co., p. 213 (1981), which is hereby incorporated by reference in its entirety). In general, liposomes can be prepared by a thin film hydration technique followed by a few freeze-thaw cycles. Liposomal suspensions can also be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 to Eppstein, et al., which is hereby incorporated by reference in its entirety.

As used herein, the term "nanoparticle" means a polymer sphere or spheroid that can be formulated to have a regular arrayed surface of defined, linked molecules in the nanometer size range (about 20 nm to 500 nm). Preferably, self-assembling monomers are utilized to form the nanoparticles.

In the nanoparticle vaccine constructs of the present invention, antigen(s) and/or targeting molecule(s) may be conjugated to individual monomeric lipid units and combined into self-assembling spheroid particles of a predetermined size. The nanoparticle chemistry allows nearly any type of immunogenic antigen to be attached to the particle surface, including proteins, peptides, carbohydrates, nucleic acids, small organic molecules, self-antigens, or conjugates of any of these compounds. The lipid monomer and a displayed molecule are conjugated, either covalently (via a tether or other linker moiety) or by complexing (either ionic or non-ionic), depending on the nature of the molecule being displayed.

Alternatively, conjugating molecules to the surface of a preformed nanoparticle is also encompassed by this invention. A linker or spacer molecule may also be used in conjugating antigen or other molecules to the nanoparticle. As used herein, the terms "linker" or "spacer" mean the chemical groups that are interposed between the nanoparticle and the surface exposed molecule(s). Preferably, linkers are conjugated to the surface molecule at one end and at their other end to the nanoparticle.

The hollow interior of the nanoparticles of this invention can be used to deliver antigen or antigens to the cells or tissues of interest. The release rate of such encapsulated antigen(s) can be modulated, for example, by varying the degree of polymerization of a liposome, by synthesizing the nanoparticle with some proportion of enzymatically degradable lipids, or by other means of altering the "leakiness" of the nanoparticle, as is known in the art.

The lipid monomers are typically selected from the group consisting of fatty acids containing 8-30 carbon atoms in a saturated, monounsaturated, or multiply unsaturated form. Furthermore, the lipid monomers may be acylated derivatives of polyamino, polyhydroxy, or mixed aminohydroxy compounds; glycosylacylglycerols; sphingolipids; steroids; terpenes; prostaglandins; non-saponified lipids; and mixtures thereof. The lipid monomers can also be diacetylene containing compounds.

The lipid monomers are polymerized, according to techniques known in the art, in order to provide stability and a certain rigidity to the constructs. As used herein, the term "polymerized" or "polymerization" encompass any process that results in the conversion of small molecular monomers into larger molecules consisting of repeated units. Typically, polymerization involves chemical crosslinking of molecular monomers to one another by exposure to UV light or other polymer-promoting catalysts.

As used herein, the term "polymerized liposome" means a liposome in which the constituent lipids are covalently bonded to each other by intermolecular interactions. The lipids can be bound together within a single layer of the lipid bilayer (the leaflets) and/or bound together between the two layers of the bilayer. Polymerizing the bilayer structure makes the assembly dramatically more resistant to enzymatic breakdown by acids, bile salts or enzymes present in the gastrointestinal tract compared to conventional, phosphotidylcholine-based liposomes. Similarly, the macromolecular nature of the nanoparticles covered with surface targeting or other small molecules can retard some of the physiological degradative pathways which would ordinarily degrade such molecules.

Non-polymerized liposomes have been used to change the pharmacodynamics of therapeutic substances either encapsulated inside their structures or displayed on their surfaces. Entrapment of sensitive molecules within the nanoparticle can shield the material from such degradative processes. This is an important aspect of the present invention when considered in its antigen-delivery embodiments. The demonstration of this principal has been described and is known in the art with regard to conventional bilayer liposomes. The escape rate of the encapsulated drug is largely controlled by the lipophilicity of the drug or its solubility in the lipid membrane.

Polymerized liposome nanoparticles, on the other hand, can be formulated with a defined "leakiness" by having pores of an optimal size, by making the nanoparticles with specific ratios of enzymatically degragdable lipids. In this way, engineering the encapsulating nanoparticle can modulate the optimal escape rate of any antigen at immune uptake sites, and techniques to modulate leakiness and escape or release rates also are known in the art.

II. Specific Embodiments

The vaccine system of the present invention is versatile, as the presentation of multiple and different antigens provides for immunization for several different and distinct infective stimuli. For example, a single vaccine prepared in accordance herewith may present antigens for more than one bacterial, viral, or fungal species to elicit immune responses to each of these distinct stimuli. Additionally, T-cell directing peptides along with carbohydrates or peptides as antigens can be incorporated into the particle to facilitate humoral and cellular immunity to such antigens.

The spheroid assembly of the nanoparticle vaccine carrier of the present invention is easy to construct and functionalize. It is polymerized to be "rigid", not suffering from the folding uncertainties associated with soluble linear or branched chain polymers, and unlike conventional liposomes, they are inert toward random fusion with themselves or other membranes. These carriers resemble a very simplified bilayer surface and as such, allow the recognition elements to be varied and investigated systematically.

In general, it will be readily appreciated that the practice of this invention is not critically dependent on the chemical details of the composition. The practitioner is free to assemble the composition according to a number of different approaches. Variations in polymerization chemistry and the conjugation of antigens, adjuvants, and/or targeting molecules are permitted and included in the scope of this invention. Additionally, combining the techniques described herein to create a combination nanoparticle vaccines with molecules both encapsulated and surface displayed (exterior or interior) is included in the scope of this invention.

Designing particular linkages between the displayed molecules and lipid monomers also is well within the skill of the ordinary practitioner. The optimization of such linkages and compounds may be achieved by routine adjustment and following the effects of adjustment on immune response in one of many techniques established in the art.

The following description and examples are provided merely as an illustration of possible approaches and preferred embodiments. Persons skilled in the art will readily understand that various modifications may be made according to the teachings herein.

Preparation of Nanoparticle Vaccines Having Surface-exposed Molecule(s) and/or Targeting Molecule(s):

When assembling nanoparticles according to embodiments of the present invention, which have surface displayed antigen or types of antigen and/or targeting molecules, two strategies are employed to display virtually any molecule or protein multivalently. Depending upon the kind of molecule and its sensitivity to nanoparticle formulation conditions, either the antigen(s) and/or target molecules are preconjugated to a polymerizable lipid or the nanoparticle is pre-formed and conjugation of the surface-exposed molecules is conducted as a final step. While it is not critical that particular surface exposed molecules always be chosen with respect to particular receptors, it is important that at least one molecule type specifically interacts with (or binds to) a receptor that leads to antigen processing, and that at least one molecule type is therefore capable of eliciting a protective immune response.

A certain proportion of the lipid monomers in the nanoparticle are attached to the antigen and a distinct proportion of the lipids in the nanoparticle are attached to a second type of antigen or targeting molecule that is different from the first molecule type. It is important to note that the different types of molecules are displayed in a randomly generated regular array on the nanoparticle carrier. In effect, the antigen processing receptor(s) or targeting molecule binding receptor(s) readily accept those multivalently displayed units formed by first and second (or more) displayed molecule pairs that have the optimal spacing and charge/hydrophobicity characteristics. The preferred embodiments of the invention are produced according to the methods described herein, in which the relative amounts and respective ratios of the lipid monomers bearing different display molecules as well as spacer monomers are determined empirically.

Surface-exposed molecules (antigens and/or targeting molecules) may be conjugated or complexed to the nanoparticle using any means known in the art. The term "conjugated" refers to molecules that are covalently bound to each other through one or more linker molecules; whereas the term "complexed" refers to molecules that are non-covalently bound to each other through one or more linker molecule.

For instance, surface-exposed molecules may be conjugated to a lipid using an appropriate linker. The term "linker" refers to a compound that is capable of covalently binding two molecules together. Linking may be performed with either homo- or heterobifunctional agents, i.e., SPDP, DSS, SIAB.

Methods for linking are disclosed in PCT/DK00/00531 (WO 01/22995) to deJongh, et al., which is hereby incorporated by reference in its entirety. Such methods may generally include the steps of:

a) reacting an antigen or immunogen with a reactive linker end thereby producing a mixture of linker derivatives of the antigen(s);

b) isolating the antigen derivatized with a single linker residue,

c) activating the isolated linker derivative of the antigen,

d) reacting the activated linker derivative of the antigen with the lipid thereby producing conjugates between the antigen and the lipid monomer.

Note that the above steps may be conducted with the addition of the targeting molecule(s) attached to a lipid in the cases where targeted vaccines are desired.

In one embodiment, the first linker is a bifunctional linker (i.e., with two functional groups), preferably a heterobifunctional linker (i.e., with two different functional groups). In a further embodiment, the linker is selected from the non-limiting group consisting of N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-3-(2-pyridylthio)propionate (SPDP), N-succinimidyl S-acetylthioacetate (SATA), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and N-g-maleimidobutyryloxy-succinimide ester (GMBS). In a further embodiment, the linker is Traut's Reagent 2-iminothiolane in combination with SPDP. In still a further embodiment the linker is succinmidyl dicarbonyl pentane or disuccinimidyl suberate. In a further embodiment, the linker is selected among those disclosed in The Pierce Products Catalogue (Pierce Chemical Company, USA) and the Double Agents.TM. Cross-Linking Reagents Selection Guide (Pierce Chemical Company), which are herein incorporated by reference.

In the general method presented above, any suitable method may be used to purify the linker derivatized attachment molecule. For instance, the linked attachment molecule may be purified by preparative reverse phase HPLC (RP-HPLC). In another embodiment, the linked attachment molecule may be purified by membrane filtration, such as ultrafiltration or diafiltration. Unreacted linker may be removed by size exclusion chromatography, such as gel filtration, or equilibrium dialysis. The final conjugate may also be purified using any suitable means, including for instance gel filtration, membrane filtration, such as ultrafiltration, or ion exchange chromatography, or a combination thereof.

Molar ratios to be used in linking methodology may be readily optimized by those of skill in the art, but generally will vary between about 1:1 to about 5:1 linker to attachment molecule depending on the linker and the efficiency of linking reaction. The ratio of linked antigen(s) to lipid may also be readily optimized by those of skill in the art, but will generally range from about 1:1 to about 10:1 lipid to antigen.

Alternatively, surface-exposed molecules may be complexed with a lipid using an appropriate complexing agent. The term "complexing agent" refers to a compound that is capable of non-covalently binding two molecules together. Complexes may be formed between a 6.times. His tag on one molecule and a nitrilotriacetic acid-metal ion complex on the other molecule.

Additionally, peptide and protein antigens may be expressed as fusion proteins operably linked to the lipid. Fusion proteins are known in the art, such as those disclosed in Yu et al., "The Biologic Effects of Growth Factor-Toxin Conjugates in Models of Vascular Injury Depend on Dose, Mode of Delivery, and Animal Species," J. Pharm. Sci., 87(11):1300-4 (1998); McDonald et al., "Large-Scale Purification and Characterization of Recombinant Fibroblast Growth Factor-Saporin Mitotoxin," Protein Expr. Purif., 8(1):97-108 (1996); Lappi et al., "Expression and Activities of a Recombinant Basic Fibroblast Growth Factor-Saporin Fusion Protein," J. Biol. Chem., 269(17):12552-8 (1994); Wu et al., "Gene Transfer Facilitated by a Cellular Targeting Molecule, Reovirus Protein .sigma. 1," Gene Therapy, 7:61-69 (2000); and Prieto et al., "Expression and Characterization of a Basic Fibroblast Growth Factor-Saporin Fusion Protein in Escherichia coli," Ann. N Y Acad. Sci. 638:434-7 (1991), all of which are hereby incorporated by reference in their entirety. By way of example, fusion-derived immunogen conjugates include K99 fimbrial protein from bovine enterotoxigenic E. coli fused to protein .sigma. 1, colonization factor antigen 1 fimbrial protein from human enterotoxigenic E. coli fused to protein .sigma. 1, or myelin basic protein fused to protein .sigma. 1.

FIG. 1 (see Original Patent) depicts several of the conjugations scenarios which may be used to attach a variety of molecular classes to the nanoparticle vaccine carriers of this invention. By incorporating a water-soluble linker between lipid and antigen or target molecule, a great range of flexibility is allowed for the tethered linker. Such linker length may range from one atom to several hundred.

Once the antigen(s) and/or target molecule(s) are conjugated to lipid, the nanoparticle can be formed by mixing the lipid with other polymerizable spacer or matrix lipids. By controlling the amounts of target lipid to matrix lipid incorporated into the nanoparticle formulation, the density of the surface array of molecules can be varied. In this way, the optimal density of antigen(s) and/or target molecules leading to the highest biological activity can be determined. Similarly, the effectiveness of a drug or toxin may also be optimized.

The matrix lipid can be charged or uncharged to impart a surface of electronically neutral or specific charge around the target molecule. Charged lipids can be readily synthesized by attaching functional moieties, such as an SO.sub.4 molecule to the head group, which would, in this case, result in a negative charge. Methods of synthesizing variously charged lipids are further described in U.S. Pat. No. 6,235,309 to Nagy, et al. and Bruehl, et al., "Polymerized Liposome Assemblies: Bifunctional Macromolecular Selectin Inhibitors Mimicking Physiological Selectin Ligands," Biochem. 40:5964-5974 (2001), which are hereby incorporated by reference in their entirety. The surrounding charge, positive or negative (or neutral) can play a dramatic role in the overall biological activity of the nanoparticle carrier.

Methods for polymerized nanoparticle preparation are disclosed in U.S. Pat. No. 6,235,309 to Nagy, et al., which is hereby incorporated by reference in its entirety. Such methods may generally include the steps of

a) mixing a desired molar percentage of antigen-lipid (and/or targeting-lipid) with a desired molar percentage of matrix lipid;

b) evaporating the stock solvent(s) under vacuum and adding a desired amount of water or buffer to create a desired concentration of lipid suspension;

c) agitating the suspension by probe sonication or other sonication method to promote the lipids to self-assemble into uni- or multi-lammelar liposome structures. Alternatively, the lipid suspension can be pressed through a membrane or orifice of defined pore size to produce extruded liposomes of a desired size range;

d) the pre-polymerized liposomes are cooled and caused to polymerize by UV light exposure or other polymer-promoting catalysts.

By varying the amount of crosslinking of the lipid monomers during the polymerization process, different degrees of polymerization, and therefore different degrees of rigidity, may be obtained. This may be useful for specific display characteristics (i.e., spatial orientation) desired for presented antigen(s) and/or target molecule(s) or to allow for resistance or susceptibility of the nanoparticle to enzymatic or other degradative pathways or processes. The degree of crosslinking in the polymerized liposomes preferably ranges from about 30 to 100 percent (i.e., up to 100 percent of the available bonds are made).

Alternatively, the nanoparticle can be formed with reactive conjugation groups decorating the surface. This technique will obviously be successful only if the conjugation groups are compatible with the polymerization conditions in an aqueous environment. If the groups are unaffected by polymerized nanoparticle formation, simple incubation with the reactive antigen or target molecule will affect the conjugation, as schematically depicted in FIG. 3 (see Original Patent).

Preparation of Nanoparticle Vaccines Having Encapsulated Antigen(s):

To encapsulate antigen the nanoparticle is constructed in a media that contains a suitably high concentration of the soluble antigen. Because the nanoparticle has an aqueous compartment fully surrounded by a polymer membrane the resulting antigen concentration on the inside of the nanoparticle will be equal to that of the bulk media surrounding the nanoparticle. The non-encapsulated antigen can be removed from the nanoparticle solution by either size exclusion chromatography or equilibrium dialysis. Once the non-encapsulated antigen is removed, the polymerized nanoparticles can be prepared and purified by any suitable method discussed in the above example, with the resulting nanoparticle as depicted in FIG. 4 (see Original Patent).

Claim 1 of 12 Claims

1. A conjugated system comprising: polymerized liposomes produced from lipid monomers which do not contain phosphate groups and which are cross-linkable and an antigen conjugated to the polymerized liposomes so that the antigen is surface exposed on the polymerized liposomes, wherein the antigen elicits an immune response.
 

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