Field of the Invention

[0001] The present invention relates generally to a synthetic chromatin vaccine (SCV) that can be used as an immunostimulatory agent, particularly as a vaccine or adjuvant or both, in a treatment and/or prophylactic setting.

Background

[0002] Vaccines have been produced traditionally using live or attenuated

microorganisms, or various preparations of their components; e.g., cell wall fractions or isolated or recombinantly produced peptides. However, it is generally recognized that the production of vaccines using whole organisms and/or their components has a number of disadvantages. For example, immunization using whole cells of pathogens can produce long lasting immunity but carries, among other risks, major safety risks related to causing autoimmune or strong allergic responses ¹ . Whole protein preparations also suffer from the potential to generate autoimmunity, as well as problems associated with protein purity and stability, large scale protein expression difficulties, difficulties with the introduction of desired post-translational modification {e.g. glycosylation) into recombinant proteins and poor or undesired immune responses (inflammation, autoimmunity, etc.) ¹ .

[0003] More recently, fully synthetic peptide-based vaccines have been developed that have the potential to provide therapeutic molecules and methods which may overcome some of the disadvantages of traditional vaccines.

[0004] A chemically defined peptide vaccine has several advantages over

conventional vaccines including better thermo-stability, cost effectiveness, safety and minimal side effects due to the targeted immune response ^{2 4} . However, vaccines based on short peptides are rapidly degraded once administered, and the effectiveness of the vaccination is transient and limited due to poor solubility and immunogenicity ⁵ . For example, the use of minimal antigenic epitopes to stimulate the immune system (e.g., for immunization) frequently results in a poor to insufficient immune response ¹ .

[0005] Accordingly, there is a need in the art for new vaccines that will overcome at least some of the weakness of vaccines based on short peptides and/or that will at least provide the public with a useful choice. Summary of Invention

[0006] In one aspect the invention relates to a synthetic chromatin vaccine

comprising nucleosomes comprising an amino acid sequence of at least one histone protein fused to an amino acid sequence of at least one peptide antigen.

[0007] In another aspect the invention relates to a composition comprising a

synthetic chromatin vaccine as described herein and a carrier, diluent or excipient.

[0008] In another aspect the invention relates to a method of stimulating an immune response in a subject comprising administering a synthetic chromatin vaccine as described herein or a composition comprising a synthetic chromatin vaccine as described herein to the subject.

[0009] In another aspect the invention relates to the use of a synthetic chromatin vaccine as described herein in the manufacture of a medicament for stimulating an immune response in a subject.

[0010] In another aspect the invention relates to a synthetic chromatin vaccine or composition comprising a synthetic chromatin vaccine as described herein for stimulating an immune response in a subject.

[0011] Various embodiments of the different aspects of the invention as discussed above are also set out below in the detailed description of the invention, but the invention is not limited thereto. Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.

[0012] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

Brief Description of Drawings

[0013] The invention will now be described by way of example only and with

reference to the drawings in which:

[0014] [Fig. l] provides a schematic illustration of a synthetic chromatin vaccine as described herein and its characterization. A. Following assembly with pUC19/16x601, individual histones were incorporated in the multiple nucleosome array at the interval of approximately 200 base pairs of DNA. Individual mononucleosomes displayed each N- terminal tail of histones outside of the core wherein fused peptides of interest are connected and exposed on the surface. B. Illustration of chromatin assembly and study in animals. A model peptide derived from Ras oncogene (G12V) was connected to the N- terminus of individual histones proteins and the recombinant histones were purified in E. coli and reconstituted as histone octamers. Resultant histone octamers were used to prepa re the G12V peptide-tagged chromatin using plasmid DNA. Properly assembled chromatin was confirmed by micrococcal nuclease (MNase) assay and transmission electron microscopy (TEM) . The synthetic chromatin vaccine was administered to mice as described herein to measure antibody and cytokine responses.

[0015] [Fig 2] illustrates the preparation of octameric histones displaying G12V

peptide. A. Individual histones translationally fused with G12V peptide were purified to homogeneity and examined by immunoblot using anti-G12V specific antibody. B. The octameric histones were purified by Superdex 200 pg gel filtration chromatography from the mixture of H3, H4, H2A and H2B. The chromatograms were shown for wild type histone octamer (arrow head on the top panel) and hybrid octamer (arrow head on the bottom panel) . C. The purified histone octamers were analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie brilliant blue staining

[0016] [Fig . 3] illustrates the analysis of assembled synthetic chromatin vaccine by micrococcal nuclease (M Nase) assay. 500 ng of assembled synthetic chromatin vaccines from either wild type or G12V-tagged histones, were stored for the indicated period and digested by incubation with 4 mU and 16 mU of MNase for 10 min at 30°C. Purified DNA was analyzed by 1.2% agarose gel electrophoresis.

[0017] [Fig . 4] shows transmission electron microscopic (TEM) images of G12V

peptide-tagged chromatin . A. Chromatin was fixed by 3.7% formaldehyde and examined by TEM after uranyl acetate and phosphotungstic acid staining. Four representative images are shown. B. G12V peptide-displayed chromatin was visualized by the same procedure as in the panel A.

[0018] [Fig . 5] shows the antibody responses to the G12V peptide displayed on

chromatin (i .e., a synthetic chromatin vaccine as described herein) in vaccinated mice.

A. Immunization schedule of synthetic chromatin vaccines. B. Antibody response in mice administered vaccines without added adjuvant (Trial 1). Groups of mice (n=6) were vaccinated with DNA, WT chromatin, G12V histone, G12V chromatin (SCV). Antibody responses were measured in sera using histone-coated ELISA plates. C. Anti-histone antibody responses in mice administered with vaccines co-formulated with adj uvants DDA and MPL (Trial 2). Groups of mice (n = 6) were vaccinated with DDA/MPL alone, G12V histone mixed with DDA/M PL, G12 chromatin mixed with DDA/M PL. Antibody responses were measured in sera using histone-coated ELISA plates. D. Anti-G12V peptide antibody response of vaccines co-formulated with adjuvants, DDA and MPL (Trial 2). Antibody responses were measured using G12V peptide-coated ELISA plates. Results are shown as OD450 values using a sera dilution of 1 : 100. ^^statistically different P<0.05, ** statistically different P<0.01. n.s. not statistically significant.

[0019] [Fig . 6] illustrates that vaccination with G12V-tagged chromatin does not induce cytokine responses. Splenocytes from the sacrificed mice in trial 2 were stimulated by addition of PBS (control) or G12V histones. Secretion of cytokines were measured by cytometric bead array. Groups of mice (n = 6) were vaccinated with DDA/M PL adj uvant alone, G12V histone mixed with DDA/MPL, G12 chromatin mixed with DDA/M PL. Panels A to Panel F: IFN-g, TNF, IL-2, IL-6, IL-10, IL- 17A) .

Detailed Description of the Invention

[0020] Definitions:

[0021] The following definitions are presented to better define the present invention and as a guide for those of ordinary skill in the art in the practice of the present invention.

[0022] Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains. Examples of definitions of common terms in microbiology, molecular biology, pharmacology and biochemistry can be found in (Meyers, 1995, Lewin et al. , 2011, Madigan et al. , 2009, Singleton and Sainsbury, 2006, Lederberg, 2000, Reddy, 2007) .

[0023] It is also believed that practice of the present invention can be performed using standard microbiological, molecular biology, pharmacology and biochemistry protocols and procedures as known in the art, and as described, for example in (Burtis et al. , 2015, Lewin et al., 2011, Whitby and Whitby, 1993, Reddy, 2007, Sambrook and Russell, 2001) and other commonly available reference materials relevant in the art to which this disclosure pertains, and which are all incorporated by reference herein in their entireties.

[0024] The term "synthetic" and grammatical variations thereof as used herein to define a synthetic chromatin vaccine means that the chromatin in the array, which comprises proteins and DNA, has been produced synthetically in vitro by a self-assembly process from biological components that have been isolated, purified or chemically synthesized, and not obtained as a chromatin fiber from nature or produced as isolated and purified chromatin fibers from a natural source. Although the DNA and proteins comprised in the synthetic chromatin vaccine described herein may comprise certain features that may be found together in nature, the DNA and proteins comprised in the synthetic chromatin vaccine described herein as a whole are not found together in nature.

[0025] The term "chromatin" is used herein as known in the art and refers to a

complex of DNA and associated proteins including histones, which contains a repeating nucleosome structure.

[0026] The terms "nucleosome" and "nucleosome structure" and grammatical

variations of such are used herein mean a structure that is produced by the assembly of DNA and four types of histone proteins [H3, H4, H2A, H2B] in vitro, displaying a "beads on a string" structure. A nucleosome or nucleosome structure in the context of this disclosure includes not only multi-nucleosome units but also monomeric nucleosomes.

[0027] The phrase "stimulate an immune response" and grammatical variations thereof as used herein means any measurable or observable increase in an amount or level of immune stimulation as measured by an increase in antibody production and/or cytokine production by the subject that is attributable to the SCV vaccination relative to the level of immune stimulation (i.e., antibody or cytokine production) in an appropriate control subject; e.g., placebo or non-active agent.

[0028] A "suitable control subject" as used herein is an appropriate control subject (untreated or treated with a defined control treatment) according to art-accepted criteria for monitoring a disease or condition. In preferred embodiments the stimulation of the immune system following administration of a synthetic chromatin vaccine as described herein is a statistically significant stimulation relative to an appropriate control subject (untreated or treated with a defined control treatment) according to art-accepted criteria for monitoring a disease or condition.

[0029] The term "statistically significant" as used herein describes a mathematical measure of difference between groups. The difference is said to be statistically significant if it is greater than what might be expected to happen by chance alone.

[0030] The term "antigen" refers to a molecule that contains one or more epitopes (linear, overlapping, conformational or a combination of these) that, upon exposure to a subject, can induce an immune response in the subject, that is specific for that antigen.

[0031] The term "epitope" refers to an antigenic determinant, i.e. a point of

interaction on the antigen for specific antibodies or an antigenic determinant, for example, that is presented on an MHC molecule and recognized by a T-cell receptor. An antigen may contain more than one antigenic determinant.

[0032] A peptide antigen as described herein can be any number of amino acid

residues that can be expressed at the end of a fusion protein comprising the amino acid sequence encoding at least one histone protein, wherein upon expression, the histone protein-peptide antigen fusion protein forms histone octamers that form nucleosomes that form chromatin.

[0033] A peptide antigen may be derived from a protein which is a suitable target for prophylactic or therapeutic vaccines. A peptide antigen "derived from" a target protein is to be understood herein as to comprise a contiguous amino acid sequence selected from the target protein, which, while preserving its antigenic properties, may be modified by deletion or substitution of one or more amino acids, by extension at the N- and/or C- terminus with additional amino acids or functional groups.

[0034] The terms "cancer associated antigen" and "cancer specific antigen" when used in reference to an antigen as described herein refers to a cancer antigen that is known in the art to cause or be associated with the production, by the body's immune system, of an antibody that specifically or selectively binds to at least one epitope of the cancer antigen.

[0035] The terms "cancer associated antigen" and "cancer specific antigen" as used herein mean a synthetic peptide antigen that comprises the amino acid sequence of at least one epitope of a protein expressed from a cancer associated or cancer specific gene.

[0036] The term "pharmaceutical composition" as used herein encompasses a

product comprising one or more active agents, and pharmaceutically acceptable excipients comprising inert ingredients, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. In general,

pharmaceutical compositions are prepared by bringing the active agent into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. Said compositions are prepared according to conventional mixing, granulating, or coating methods, respectively, and contain a percentage (%) of the active ingredient and can be determined by a skilled worker in view of the art. By "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" it is meant that the excipient or carrier must be compatible with the other ingredients of the formulation and not harmful to the subject to whom the composition is administered.

[0037] Pharmaceutically acceptable excipients have been approved by relevant

government regulatory agencies. Excipients include but are not limited to sterile liquids such as water and oils, including animal, vegetable, synthetic or petroleum oils, saline solutions, aqueous dextrose and glycerol solutions, starch glucose, lactose, sucrose, gelatin, sodium stearate, glycerol monostearate, sodium chloride, propylene glycol, ethanol, wetting agents, emulsifying agents, binders, dispersants, thickeners, lubricants, pH adjusters, solubilizers, softening agents, surfactants and the like. The compositions of the invention can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders and sustained-release formulations. Examples of suitable

pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 18 ^th Ed., Gennaro, ed. (Mack Publishing Co. 1990). The pharmaceutically acceptable excipient is present in a composition as described herein in an amount that does not impair the activity of the SCV as described herein, or of a composition comprising a synthetic chromatin vaccine as described herein.

[0038] The term "effective amount" refers to a sufficient quantity of the synthetic chromatin vaccine, in a suitable composition, and in a suitable dosage form to stimulate the immune system to obtain a measurable or observable result as compared to a suitable control subject. In one example, the measurable or observable result is an increase in antibody production by the subject in response to the administration of the synthetic chromatin vaccine. A "therapeutically effective amount" will typically stimulate the immune system to provide at least some level of treatment and/or prophylaxis of the desired disease conditions in the subject in addition to providing for the measurable or observable result discussed above.

[0039] A person skilled in the art will be able to formulate a synthetic chromatin vaccines described herein as a composition, particularly a pharmaceutical composition, by determining an appropriate mode of use, application and/or administration of the composition with reference to the literature and as described herein, and then formulating the composition for such mode with reference to the literature and as described herein.

[0040] A person skilled in the art will also be able to choose the appropriate mode of administration of the medicament with reference to the literature and as described herein. By way of non-limiting example, a systemic application would be preferred for stimulation of the immune system generally. The terms "administering" or

"administration" refer to placement of the composition or compound of the invention into a subject by a method appropriate to result in an immune response. The dosage form is selected and used as appropriate depending on the therapeutic purpose and the subject. The dose of the composition of the invention may be selected depending on the therapeutic purpose and the cha racteristics of the subject including their age, sex, general health and disease progression. In general, the compound of the invention may be administered in a dose of 0.01 to 100 mg, preferably 0.1 to 50 mg per day, per kg of body weight, either once or divided over several administrations.

[0041] The term "about" when used in connection with a referenced numeric

indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, "a bout 100" means from 90 to 110 and "about six" means from 5.4 to 6.6.

[0042] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting statements in this specification that include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

[0043] The term "consisting essentially of" as used herein means the specified

materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0044] The term "consisting of" as used herein means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim.

[0045] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein a re hereby expressly disclosed . These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0046] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. In the disclosure and the claims, "and/or" means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.

[0047] Detailed Description :

[0048] A nucleosome is a basic repeating unit of chromatin in the euka ryotic

genome. The single nucleosome pa rticle consists of DNA of 147 base pairs wrapping around a histone octameric core containing two copies of four different histones, H3, H4, H2A and H2B ⁶ ' ⁷ . Each nucleosome particle is connected by a linker DNA, which results in a 10 nanometer (nm) fiber in diameter with the appearance of "beads-on-a-string".

Since the length of linker DNA is up to about 80 base pairs, the nucleosome array shows repeated nucleosome particles at every 200 base pairs of DNA.

[0049] The N-terminal histone tails are rich in basic a mino acids, such as lysine and arginine, and exposed outside of the nucleosome core pa rticle ⁸ ' ⁹ . The intrinsic flexibility and surface exposure from the 10 nm chromatin fiber make the histone tails subjected to efficient modifications by diverse chromatin remodeling and modifying enzymes in cells. Once assembled, the nucleosome array is naturally stable for several months ¹⁰ . All N-terminal tails of histones, except H2B, are dispensable for the chromatin assembly, suggesting that the N- terminal tails can be significantly altered in the nucleosomal array ¹¹ . This has also been demonstrated using common peptide tags such as Flag or HA, which have been added to the histone tails in the nucleosome structure ^{12 3} . Without wishing to be bound by theory, the inventor believes that due to their polymeric nature, a large repertoire of differently altered histones would be randomly incorporated into the nucleosomes of the SCV as described herein. This random incorporation likely results in an SCV that displays various types of diverse arrays of antigenic peptides and/or repeated peptide epitopes from the same peptide.

[0050] The supercoiled DNA for the nucleosomal assembly can be any type of DNA.

However, strong nucleosome positioning sequences, derived from the artificial 601 widom repeat or 5S rRNA gene have been shown to enhance added stability and regularity to the nucleosome array ¹⁴ . Recombinant histone prepa rations from E. coli inclusion bodies and chromatin self- assembly using salt gradient dialysis method have been well-established previously ¹⁰⁴⁵ . In contrast, the use of entirely synthetic chromatins assembled in vitro has been limited to academic investigations for elucidating chromatin structure and for studying epigenetic chromatin modifications and their effects on cells.

[0051] To explore the feasibility of the 10 nm chromatin fiber as a peptide carrier for effective adjuvant or vaccine preparation, we prepared "synthetic chromatin" using plasmid DNA and G12V peptide-tagged histones. The "synthetic chromatin" of the invention and as described herein comprises a peptide segment of 17 amino acids derived from ras oncogene (G12V) displayed on the surface of chromatin. The ras gene was chosen because the codon 12 mutation accounts for the most frequent oncogenic ras mutations in human cancers and the mutated peptides derived from ras oncogene are known to be presented on both MHC class I and II molecules as a tumor-specific antigen ^16-19 . This makes mutant ras peptides attractive antigens for a therapeutic cancer vaccine and several mutant ras peptide vaccines have been clinically tested to treat advanced cancer patients ^17,19 .

[0052] A synthetic chromatin vaccine as described herein induces a stronger

antibody response against the peptide than those produced from G12V-tagged histones alone (Fig. 5D). Importantly and quite surprisingly, the results disclosed herein suggest that chromatin display of an appropriate peptide can re-direct what would be an expected T cell-dependent immune response against the peptide antigen into a T cell- independent immune response (Fig. 6). This redirection is reflected in higher specific IgG antibody responses (Fig. 5D), not commonly observed from T cell-independent immune response.

[0053] Without wishing to be bound by theory, the inventors believe that a synthetic chromatin vaccine as described herein is useful for stimulating the immune system in a T-cell independent manner by engaging and activating B-cells (Fig. 7). The inventors also believe that the synthetic chromatin vaccine described herein is useful for the manufacture of a medicament for stimulating the immune system, particularly in a T-cell independent manner.

[0054] Example 1 details the preparation of a synthetic chromatin vaccine and the use of the synthetic chromatin vaccine as an immunostimulatory agent in mice.

[0055] Based on the findings described herein the inventors believe that a synthetic chromatin vaccine as described herein can be designed to display a number of different peptides, including peptide antigens. The choice of peptide is believed to be within the skill of those in the art when considering the use to which the synthetic chromatin vaccine as described herein is to be put.

[0056] Exemplified herein is a single peptide, a portion of a ras oncogene. Flowever, the invention is not so limited. The inventor believes that a skilled person can chose an appropriate peptide as known in the art for expression and incorporation into a synthetic chromatin vaccine as described herein. [0057] Accordingly, in one aspect the invention relates to a synthetic chromati n vaccine comprising nucleosomes comprising an amino acid sequence of at least one histone protein fused to the amino acid sequence of at least one peptide antigen .

[0058] In one embodiment the at least one histone is translationally fused to the amino acid sequence of the at least one peptide antigen.

[0059] In one embodiment the peptide antigen comprises from 2 to 100 amino acid residues, preferably from 5 to 75, from 10 to 50, from 15 to 25, preferably from 15 to 20 amino acid residues.

[0060] In one embodiment the peptide antigen comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75, 80,. 85, 90, 95 or 100 amino acids.

[0061] In one embodiment the peptide antigen comprises more than 100 amino acid residues.

[0062] In one embodiment the peptide antigen comprises 10 to 30 amino acid

residues.

[0063] In one embodiment the peptide antigen comprises 12 to 25 amino acid

residues.

[0064] In one embodiment the peptide antigen comprises 15 to 20 amino acid

residues.

[0065] In one embodiment the peptide antigen comprises 16 to 18, preferably 17 amino acid residues.

[0066] In one embodiment the peptide antigen is a cancer-associated antigen.

[0067] In one embodiment the peptide antigen is a cancer-specific antigen.

[0068] In one embodiment the cancer-associated or cancer-specific antigen is a ras antigen .

[0069] In one embodiment the ras antigen comprises the amino acid sequence KLVVVGAVGVGKSALTI (SEQ ID NO: 1).

[0070] In one embodiment the ras antigen consists essentially of, or consists of SEQ ID NO: 1.

[0071] In one embodiment the at least one histone protein is H3, H4, H2A or H2B.

[0072] In one embodiment the synthetic chromatin vaccine comprises histone

octamers. [0073] In one embodiment the histone octamers comprise a mixture of H3, H4, H2A and H2B.

[0074] In one embodiment the histone octamers are hybrid octamers comprising at least one wild type histone and at least one G12V-tagged histone.

[0075] In one embodiment the wild type histones are H3, H4 or a combination of both.

[0076] In one embodiment the at least one G12V-tagged histone is G12V-tagged H2A (G12V-H2A) or G12V-tagged H2B (G12V-H2B).

[0077] In one embodiment the hybrid octamers comprise wild type histones H3 and H4 and G12V-tagged histones G12V-H2A and G12V-H2B.

[0078] In another aspect the invention relates to a composition comprising a

synthetic chromatin vaccine as described herein and a ca rrier, diluent or excipient.

[0079] In one embodiment, the composition is a pharmaceutical composition .

[0080] In one embodiment the composition or pharmaceutical composition comprises the synthetic chromatin vaccine and an adjuvant.

[0081] In one embodiment the adjuvant stimulates a T cell independent immune response of the synthetic chromatin vaccine.

[0082] In one embodiment the adjuvant is diethyldioctodecylammonium bromide (DDA) or monophosphoryl lipid A (MPL).

[0083] In one embodiment the carrier, diluent or excipient is a pharmaceutically acceptable carrier, diluent or excipient.

[0084] In one embodiment the composition or pharmaceutical composition comprises an effective amount of the synthetic chromatin, preferably a prophylactically and/or therapeutically effective amount of the synthetic chromatin . In one embodiment the composition or pharmaceutical composition consists essentially of an effective amount of the synthetic chromatin, preferably a prophylactically and/or therapeutically effective amount of the synthetic chromatin.

[0085] In one embodiment the effective amount is an a mount of the synthetic

chromatin vaccine that, when administered to a subject, stimulates the immune system by at least 5%, preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, preferably at least 99% as compared to a suitable control subject. [0086] An acceptable carrier, diluent, or excipient, particularly a pharmaceutically acceptable carrier, diluent or excipient may be selected as known in the art, in view of a planned manner of use, application and/or administration.

[0087] In another aspect the invention relates to a method of stimulating an immune response in a subject comprising administering a synthetic chromatin vaccine as described herein or a composition comprising a synthetic chromatin vaccine as described herein to the subject.

[0088] In one embodiment the subject is identified as being a subject that would benefit from stimulation of an immune response.

[0089] In one embodiment the synthetic chromatin vaccine is administered in

combination with an additional immunostimulatory agent.

[0090] In one embodiment the synthetic chromatin vaccine is administered

separately, simultaneously or sequentially with the immunostimulatory agent.

[0091] In one embodiment, the additional immunostimulatory agent is an antigen that stimulates an immune response.

[0092] In one embodiment the antigen stimulates a specific immune response to the antigen.

[0093] In one embodiment the antigen stimulates an immune response in a subject that has a T cell immunodeficiency.

[0094] In one embodiment the cause of the T cell immunodeficiency is selected from the group consisting of treatment of the subject for cancer by chemotherapy or radiotherapy or both, acquired immune deficiency syndrome (AIDS), and Inherited T- Cell Deficiency Disorders.

[0095] In one embodiment the additional immunostimulatory agent is an adjuvant.

[0096] In one embodiment the additional immunostimulatory agent is an adjuvant that stimulates the T cell independent immune response of the synthetic chromatin vaccine.

[0097] In one embodiment the adjuvant stimulates a general or non-specific immune response.

[0098] In one embodiment the adjuvant is diethyldioctodecylammonium bromide (DDA) or monophosphoryl lipid A (MPL).

[0099] In one embodiment the immune response to the SCV is a T cell independent response. [0100] In one embodiment the T cell independent response is a T cell independent antibody response.

[0101] In one embodiment the immune response to the SCV is a B cell response.

[0102] In one embodiment stimulating an immune response is stimulating a T cell independent immune response.

[0103] In one embodiment stimulating a T cell independent immune response is stimulating a T cell independent antibody response.

[0104] In one embodiment administration is systemic or local administration.

[0105] In one embodiment administration is parenteral administration.

[0106] In one embodiment, parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration.

[0107] A particular and effective dosage regime according to a method of stimulating an immune response according to the invention will be dependent on the desired level of stimulation to be achieved during the course of treatment, and on the responsiveness of the treated subject to the course of treatment. An effective treatment may last from several hours to several days to several months, or until an acceptable therapeutic outcome is affected or assured or until an acceptable stimulation of the immune response in the subject is observed.

[0108] An optimal dosing schedule (s) may be calculated from drug accumulation as measured in the body of a treated subject. It is believed to be within the skill of persons in the art to be able to easily determine optimum and/or suitable dosages, dosage formulations and dosage regimes. Of course, the optimum dosages may vary depending on the relative potency of the antibacterial combination or composition as described herein, but will be estimable from an EC50s found to be effective in suitable cells in vitro and in an appropriate in vivo animal model. In general, dosage is from 0.00001 g to 99 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, but not limited thereto.

[0109] In one embodiment the subject is an animal, preferably a mammal.

[0110] In one embodiment the mammal is selected from the group consisting of canines, felines, bovines, ovines, cervines, caprines, porcines, lagomorphs, rodents, camelids and hominids. [0111] In one embodiment the mammal is selected from the group consisting of cats, dogs, rats, stoats, ferrets, possums, guinea pigs, mice, hamsters, zebra, elephants, lions, tigers, cheetah, monkeys, apes, macaques, tarsiers, lemurs, giraffes, prairie dogs, meerkats, bears, otters, tapiers, cows, horses, pigs, sheep, goats, deer, minks, hippopotami and humans.

[0112] In one embodiment the mammal is a human .

[0113] In one embodiment, the method comprises administering the synthetic

chromatin vaccine in a heterologous prime-boost regimen.

[0114] In one embodiment, the heterologous prime-boost regimen comprises

sequential administration of the synthetic chromatin vaccine and a second vaccine that targets the same antigen as the peptide antigen comprised in the synthetic chromatin vaccine.

[0115] In one embodiment the second vaccine is a different vaccine that stimulates a T cell response.

[0116] In one embodiment, the synthetic chromatin vaccine is administered before the second vaccine.

[0117] In one embodiment the second vaccine is administered before the synthetic chromatin vaccine.

[0118] In one embodiment the heterologous prime boost regime enhances an

immune response compared to the immune response measured or observed when the synthetic chromatin vaccine is administered alone.

[0119] In another aspect the invention relates to the use of a synthetic chromatin vaccine as described herein in the manufacture of a medicament for stimulating the immune system in a subject.

[0120] In one embodiment the medicament comprises an effective amount of the synthetic chromatin vaccine. In one embodiment the effective amount is a

therapeutically effective amount.

[0121] In one embodiment the effective amount comprises an amount of a synthetic chromatin vaccine as described herein that is effective at stimulating an immune response in a subject, preferably in a T cell independent manner.

[0122] In one embodiment the medicament comprises about 100 pg/ml, preferably about 90 pg/mL, about 80 pg/mL, about 70 pg/mL, about 60 pg/mL, about 50 pg/mL, about 40 pg/mL, about 30 pg/mL, about 20 pg/mL, about 10 pg/mL, about 5 pg/mL, preferably about 1 pg/mL of the synthetic chromatin vaccine. [0123] In one embodiment the medicament comprises at least one additional immunostimulatory agent.

[0124] In one embodiment the medicament comprises an effective amount of the additional immunostimulatory agent.

[0125] In one embodiment the effective amount of the additional immunostimulatory agent is a therapeutically effective amount.

[0126] In one embodiment the additional immunostimulatory agent is an antigen or adj uvant as described herein.

[0127] In one embodiment the medicament comprises about 100 pg/ml, preferably about 90 pg/mL, about 80 pg/mL, about 70 pg/mL, about 60 pg/mL, about 50 pg/mL, about 40 pg/mL, about 30 pg/mL, about 20 pg/mL, about 10 pg/mL, about 5 pg/mL, preferably about 1 pg/mL of the additional immunostimulatory agent.

[0128] In one embodiment the medicament consists essentially of an effective

amount of the combination of the synthetic chromatin vaccine and the additional immunostimulatory agent.

[0129] In one embodiment the effective amount of the synthetic chromatin vaccine and of the additional immunostimulatory agent is a therapeutically effective amount.

[0130] In one embodiment the medicament is formulated for administration, or is in a form for administration, to a subject in need thereof.

[0131] In one embodiment the medicament is in a form for, or is formulated for parenteral administration.

[0132] In one embodiment pa renteral administration is selected from the g roup consisting of di rect application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration.

[0133] In one embodiment the medicament is for, is formulated for, or is in a form for administration separately, simultaneously or sequentially with the additional immunostimulatory agent.

[0134] In one embodiment the medicament comprises a synthetic chromatin vaccine as described herein and an additional antigen or adjuvant, wherein the medicament is for, is formulated for, or is in a form for separate, simultaneous or sequential administration of the components in the combination to a subject.

[0135] In one embodiment the medicament comprises a synthetic chromatin vaccine as described herein and an antigen or adjuvant, wherein the medicament is for, is formulated for, or is in a form for administration to a subject that has shown a nonresponse or reduced response to a prior treatment.

[0136] In another aspect the invention relates a synthetic chromatin vaccine or composition comprising a synthetic chromatin vaccine as described herein for stimulating an immune response in a subject.

[0137] Specifically contemplated as embodiments of these aspects of the invention relating to the use of a synthetic chromatin vaccine as described herein for stimulating an immune response in a subject and the use of a synthetic chromatin vaccine in the manufacture of a medicament for stimulating an immune response in a subject, are all of the embodiments set out herein relating to the aspect the invention that is a method of stimulating an immune response in a subject as described herein, including all the embodiments encompassed within this method aspect, and including all of the embodiments of the synthetic chromatin vaccine and compositions comprising the synthetic chromatin vaccine that are comprised therein.

[0138] Various aspects of the invention will now be illustrated in non-limiting ways by reference to the following examples.

Examples

Example 1

Material and Methods

[0139] Purification of histone octamers from inclusion bodies

[0140] The pET3 expression vectors to express Xenopus laevis histones have been extensively used previously for wild type histone expression and purification ²⁰ . Addition of mutant G12V ras peptide (K ⁵ LVVVGAV ¹² GVGKSALTI ²¹ ) (SEQ ID NO: 1) to the second codon of histone genes was performed using PCR-based mutagenesis and confirmed by DNA sequencing.

[0141] Alternatively, the modified histone genes can be prepared by a gene synthesis (GenScript Biotech Corp) from the sequences as shown in Table 1. The mutant G12V ras peptide is shown capital letters. Insoluble inclusion bodies were obtained by expressing each histone gene in BL21 (DE3) CodonPlus stain (Stratagene) using twice concentrated Luria broth (sigma) supplemented with 0.5% glucose (w/v). Induction conditions of histone expression, inclusion body preparation and two-step column purification procedures using Sephacryl S200 (GE Healthcare) and SP sepharose Fast Flow (GE Healthcare) were described previously ¹¹ " ²⁰ ' ²¹ . Purified recombinant core histones and their G12V peptide tagged variants (G12V-histones) were dissolved in the unfolding buffer (8M Guanidine- HCI, 20 mM sodium acetate, pH 5.2, 10 mM DTT) and reconstituted to histone octamers containing wild type, G12V histones or a hybrid of wild type and G12V histones. Refolding and purification of histone octamers were performed as described previously using Superdex 200 gel filtration chromatography (HighLoad 16/600, GE Heathcare) ¹¹ ' ²⁰ ' ²¹

[0142] Table 1

[0143] Salt gradient chromatin assembly from DNA and purified octamers

[0144] DNA containing 16 repeats of widom 601 sequence (pUC19/16x601) was previously described ¹⁰ ' ²² . Supercoiled plasmid DNA was purified using conventional CsCI gradient ultracentrifugation. To obtain physiologically spaced nucleosome particles on the synthetic chromatin fiber, various histones to DNA mass ratios were screened using microscale salt dilution procedure ²³ . Large-scale assembly of up to 60 pg of chromatin was done in the dialysis membrane using a step-gradient dialysis as described previously ²⁴ . The quality of this synthetic nucleosome array was assessed by Micrococcal

Nuclease (MNase) assay as described previously using the assembled chromatin ^25-27 .

[0145] Visualizing chromatin fiber using Transmission Electron Microscopy (TEM)

[0146] Synthetic chromatin was dialyzed against a low salt buffer (10 mM Tris-HCI, pH 7.5, 1 mM EDTA, 2.5 mM NaCI, 10 mM b-merca ptoethanol) and adjusted to a final concentration of 10 ng/mI with distilled water. The chromatin fiber was fixed with 3.7% formaldehyde in 10 mM NaC03 buffer (pH 8.5) for 10 min and dialyzed against distilled water containing 10 mM b-mercaptoethanol. A formvar treated, carbon coated TEM grid was impregnated with the chromatin sample for 5 min and rinsed with water. After sequential staining with 2% uranyl acetate in water and 1% phosphotungstic acid in 70% ethanol, the grid was air dried and examined by FEI Tecnai G2 Biotwin

transmission electron microscope.

[0147] Immunization of mice and immunological assays

[0148] All animal experiments were approved by the AgResearch Grasslands Animal Ethics Committee (Palmerston North, New Zealand). C57/BL6 mice were purchased from a commercial small animal supplier and immunized at between 6 - 8 weeks of age.

[0149] Trial 1 without adjuvants: Groups of mice ( = 6) were vaccinated by intramuscular injection three times at 2-week intervals with either plasmid DNA (2 pg), wild type chromatin (2 pg of each DNA and histone octamer), G12V- tagged histone octamer (2 pg) or G12V-tagged synthetic chromatin (2 pg of each DNA and hybrid histone octamer). Prior to vaccination, mice were anaesthetised using sub-cutaneous injection of ketamine and medetomidine. Immediately after vaccination, the anaethesia was reversed using atipamezole.

[0150] Trial 2 with adjuvants: Groups of mice (n = 6) were vaccinated by the intramuscular route three times at 2-week intervals with DDA/MPL adj uvant alone, G12V histone (2pg of each DNA and histone octamer) mixed with the adjuvants

diethyldioctodecylammonium bromide (DDA) (Sigma, 250 pg/dose) and monophosphoryl lipid A (MPL) (Sigma, 25 pg/dose), G12V chromatin (2 pg of each DNA and hybrid histone octamer) mixed with DDA/MPL. MPL was prepared as a stock solution at 1 mg/ml in 0.2% triethyla mine, with repeated heating to 70°C to aid solubilization ²⁷ . The same anaethesia protocol was used as in Trial 1.

[0151] Two weeks after the final immunisation, mice were anaesthetised using

ketamine/medetomadine and blood was collected from each mouse by cardiac puncture. The blood was allowed to clot and centrifuged prior to collecting serum. Mice were euthanised and spleens removed for culture of splenocytes using methods previously reported ²⁸ . Splenocytes were cultured at 37°C a nd 10% C02 in air and stimulated with phosphate buffered sali ne (PBS, control), Concanavlin A (positive control, Sigma), G12V peptides, or G12V histones, each with a final concentration of 5 pg/ml .

[0152] Measurement of serum antibody

[0153] Specific antibody responses to G12V-histone a nd G12V peptides in sera were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). High binding ELISA plates (Greiner Bio-One) were coated overnight at 4°C with either 100 pi of 5 pg/ml of G12V histones in antigen coating buffer (0.1 M sodium carbonate, pH 9.6) or 20 pg/ml of G12V peptide with 20 pg/ml of bovine serum albumin (BSA) carrier. Formaldehyde (36.5%, Sigma) was added at 1% final concentration immediately before applying the peptide coating solution to the plate ³⁰ . After antigen coating, the wells were washed with 200 pi wash buffer (0.1% Tween 20, 50 mM Tris- Cl, pH 7.6, 150 mM NaCI) three times each with 5 min incubation at room temperature.

[0154] Two hundred pi of blocking buffer (2% BSA in wash buffer) was dispensed into each well and incubated for 1 h at 37°C followed by three washes with wash buffer. Sera were diluted 1 : 100 in blocking buffer and 100 mI added to each well. Following incubation at 37°C for 1 h, the wells were washed three times with wash buffer and 100 mI of 1 : 10,000 diluted a-mouse IgG HRP secondary antibody (Santa Cruz Biotechnology) in blocking buffer was added . Following incubation for 1 h at room temperature, the plates were washed three times with wash buffer and TMB (3,3 ,5,5 ^' - tetramethylbenzidine) substrate kit (Thermo Scientific) was used according to the manufacturer's instruction. The color development was measured by a FLUOsta r Galaxy (BMG Labtech, Germany) at 450 nm wavelength. The raw values of optical density at 450 nm were normalized by subtracting the values from blank controls (no addition of primary antibody). Triplicate data was presented as mean ± standard deviation.

[0155] Measurement of cytokines

[0156] Splenocyte culture supernatants were removed after 3 days incubation and frozen at -20°C until assayed. Levels of IFN-g, IL-2, IL-4, IL-6, IL-10, IL-17A and TNF were measured using a cytometric bead array (CBA; mouse Th l-Th2 cytokine kit: BD Biosciences) according to the manufacturer's instructions. Fluorescence was measured using a FACSVerse flow cytometer (BD Biosciences) and analysed using FCAP array softwa re (BD Biosciences) . Results for all cytokines were calculated as the cytokine value of the antigen-stimulated sample minus that of the PBS-stimulated sample.

[0157] Statistical analyses

[0158] Analysis of the cytokine and antibody responses were performed using t test.

The level of significance was set at a P value of <0.05.

Results and discussion

Experimental design and histone octamer preparation

[0159] To test the initial feasibility and immune response of ras-derived peptide on the synthetic chromatin, the mutated region of ras oncogene (glycine to valine at the 12th residue: G12V) was selected. Codon-optimized G12V primers starting from methionine were designed to add 17 amino acids of G12V peptide

(K ⁵ LVVVGAV ¹² GVGKSALTI ²¹ ) (SEQ ID NO: 1) to the second codon of each histone gene. The employed peptide sequence is basic at the isoelectric point 10.8 and expected to be hydrophobic in nature with poor solubility in water (peptide solubility calculator by pepcalc.com) and a poor antigen profile (1.8 out of 5 in antigen profiler peptide tool, Thermofisher) . Given the intrinsic structure of N-terminal tails of histones in the nucleosome, translationally fused hydrophobic G12V peptides were expected to protrude outside from the interna l octameric histone core of each nucleosome repeat (Figure 1A). The overall procedure of vaccine prepa ration and characterization is illustrated in Figure IB.

[0160] Individual histones were purified from insoluble inclusion bodies from E. coli BL21 cells and checked for purity and relative amounts by SDS-PAGE with Coomassie brilliant blue staining and immunoblot using anti-G12V specific antibody (Fig . 2A) .

Reconstituted octamers containing wild type histones were purified using Superdex 200 gel filtration chromatog raphy at the elution volume, 64 ml (Fig . 2B, upper panel).

Flowever, recombinant histone octamers containing all four G12V-tagged histones resulted in heavy precipitation after refolding procedure and poor yield after gel filtration step (data not shown) . To overcome this problem, hybrid octamers were prepa red by mixing wild type histones (FH3 and H4) and G12V-tagged histones (G12V-FI2A and G12V- H2B) . The elution volume of hybrid octamer was 70 ml, indicating that the hybrid octamer forms more compact octamers, probably resulting from hydrophobic G12V peptide sequence. Purified octamers were adj usted to 1 mg/ml of final concentration and analyzed the staining intensity using Coomassie brilliant blue G250 before chromatin assembly (Fig . 2C).

Characterization of G12V-chromatin fiber

[0161] To compa re the quality of chromatins, MNase digestion assays were

performed to determine the regularity of nucleosome spacing on the chromatin fiber.

The MNase digestion pattern with 200 base pair of DNA ladder was clearly observed from the chromatin from hybrid histone octamer of G12V-histones (Fig . 3, lanes 3 and 4), which is compa rable to the wild type chromatin (Fig . 3, lanes 1 and 2). To compare relative stability of G12V-tagged chromatin and wild type chromatin, the assembled chromatins were incubated at 37°C for two weeks (Fig . 3, lanes 5 to 12) or stored at 4°C for four months (Fig . 3, lanes 13 to 16) . The M Nase digestion patterns of wild type and G12V-tagged chromatins were all comparable rega rdless of the different storage periods and conditions, indicating that the synthetic peptide display on the chromatin did not alter the intrinsic stability of 10 nm chromatin fiber.

[0162] To compa re 10 nm chromatin fibers using transmission electron microscopy, formaldehyde- fixed chromatins were dual stained using uranyl acetate (positive stain) and phosphotungstic acid (negative stain) and visualized for their beads-on-string structure (Fig. 4). Given that supercoiled plasmid DNA was used for the chromatin assembly, it is not surprising that a majority of chromatin is intertwined and visualized like a linear chromatin. However, a few circular chromatin images show that the whole DNA backbone is not evenly saturated by nucleosomes where they are locally positioned at the saturation level on approximately two third of DNA backbone (Fig. 4A, image 4, Figure 4B, image 4). The result is consistent with a previous observation that nucleosome assembly on the supercoiled plasmid DNA is a cooperative process through initiating nucleosome assembly preferentially from the previous positioned

nucleosomes ³⁰ .

[0163] Collectively, our results indicate that the chromatin fibers prepared either from wild type or hybrid octamers are comparable in terms of MNase digestion pattern, their stability and TEM images.

Antibody immune response of G12V-chromatin vaccines

[0164] To study immune responses, mice were immunized from the vaccines

containing G12V-tagged chromatin (Fig. 5A). The vaccines were well tolerated and did not cause any abnormal skin reaction or behavior. The mice were euthanized after the third vaccination and spleens were removed for culture of splenocytes to measure cellular immune responses. Sera were collected for measurement of antigen-specific antibody responses. Since DNA has intrinsic adjuvant activity derived from

unmethylated CpG motifs in the sequence, the immunogenicity of G12V- tagged chromatin without formulation with additional adjuvant was first examined ³² ' ³³ . Mice were vaccinated with either plasmid DNA, wild type chromatin (DNA and wild type histone octamer assembly), G12V-tagged histone octamer or G12V-tagged chromatin (DNA and hybrid histone octamer assembly). Serum total IgG antibody responses were measured against G12V-peptide (data not shown) and G12V-histones (Fig. 5B). In this trial, antibody responses observed were somewhat low, suggesting that the SCV in the absence of added adjuvant did not stimulate antigen-specific antibodies as well as desired (data not shown). Studies of cell-mediated immune responses, measurement of lymphocyte proliferation or IFN-g release from splenocytes re-stimulated with G12V peptide also showed lower cellular responses (data not shown).

[0165] From these results, we concluded that a desirable formulation of a synthetic chromatin vaccine as described herein should include an added adjuvant in order to enhance the antigen-specific immune response.

[0166] Several studies have shown that cell-free fragmented DNAs are found in blood where a majority of DNA exists as oligonucleosomes ³⁴ ' ³⁵ . Given that nuclear DNA and fragmented nucleosome are constantly found in healthy people's blood, DNA and nucleosomes seem to be considered a self-antigen unless there are immunological disorders such as autoimmune pathogenesis. Even though circulating nucleosomes are suggested as one of the main causative agents i n autoimmune diseases such as lupus erythematosus ³⁶ , our studies demonstrate that highly purified synthetic chromatin fibers consisting of foreign DNA and four core histones originated from Xenopus has little immunogenicity in mice. ³⁷

[0167] In a second trial, the SCV was formulated with a n adjuvant system consisting of the cationic liposome DDA and TLR4 agonist MPL to study immune responses against the G12V peptide. Mice were vaccinated DDA/MPL adj uvant alone, G12V histone mixed with the adj uvants or G12V chromatin/adjuvant.

[0168] Total IgG antibody responses were measured in the sera using ELISA plates coated with G12V- histones (Fig . 5C). Formulation with DDA/MPL adjuvants stimulated specific antibody responses against histones in mice vaccinated with G12V-tagged histones and G12V-tagged chromatin (t-test, p < 0.01, n = 6) . IgG titres of G12V- chromatin vaccine group were comparable with titres from the G12V-histone group (t- test, p- 0.3, n = 6) . To measure antibody response against G12V peptide, ELISA plate coated with the peptide was used to measure a titer of anti-G12V peptide antibodies in the serum. Interestingly, vaccination with G12V-tagged chromatin appeared to elicit a higher IgG antibody response in comparison with DDA/MPL control group (p < 0.05, n = 6) whereas vaccine containing G12V histone did not induce a significant antibody response (p= 0.76, n = 6). In connection, histone and peptide-specific IgM responses in the vaccination of G12V-tagged chromatin were not statistically different to those in the DDA/M PL and G12V histone vaccine groups (data not shown).

[0169] Without wishing to be bound by theory, the inventors believe that a synthetic chromatin as described herein comprising G12V chromatin formulated as a vaccine with either DDA or MPL induces a better antibody response specific to the displayed peptide. These results suggest that the synthetic peptide display on chromatin provide a surprisingly effective means of eliciting a peptide-specific antibody response.

Cytokine immune response of G12V-chromatin vaccines

[0170] To measure the T cell mediated immune response, splenocytes were prepared from immunized mice and stimulated in vitro with G12V peptide or histones. A cytometric bead array was used to measure the responsiveness of T cells (Thl, Th2 and Thl7) as an indicator of cell-mediated immune response. No detectable cytokine release was observed upon stimulation with G12V peptide in vitro, suggesting that none of the peptide displays, either as a fusion histone or chromatin fiber are effective at stimulating peptide-specific T cell response at the vaccine concentrations used in the trial (data not shown). However, when G12V histones were used as the recall antigen, the mice vaccinated with G12V histones showed a high response for each of the cytokines including a major inflammatory cytokine (Fig. 6). In contrast, the increased expression of these cytokines was not observed in splenocytes from mice vaccinated with G12V chromatin as their cytokine responses were comparable to the DDA/MPL control mice.

[0171] The results depicted in Figures 5 and 6 suggest that the vaccine containing the peptide-tagged chromatin and an adjuvant can stimulate antibody response specifically to the displayed peptide without a significant help of T cell immune responses. In contrast, peptide tagged histone alone can stimulate both T cell and B cell immune responses as expected but did not stimulate statistically significant antibody responses against the displayed peptide. The data suggests that peptide display on chromatin is a better stimulator of peptide-specific antibody responses even without significant T cell stimulation.

[0172] Collectively, the results in this study suggest that a synthetic chromatin

vaccine as described herein comprising a 10 nm chromatin fiber displaying a defined peptide antigen, can be used as a vaccine to elicit an efficient TI antibody response. Without wishing to be bound by theory, the inventors believe that a synthetic chromatin vaccine as described herein is useful for delivering a number of immunostimulatory agents, pa rticularly peptides, of interest, that are poorly or insoluble, have lower than desired immunogenicity by themselves, or a combination of both.

[0173] The chromatin- based peptide display described herein also provides a

platform for vaccine development for immune deficient patients with defective T cell functions or suffering from T cell depletion after anticancer chemotherapy.

[0174] The synthetic chromatin vaccine and compositions of the invention and as disclosed herein find use as immunostimulatory agents and vaccines.

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