Multivalent synthetic nanocarrier vaccines

Description

BACKGROUND OF THE INVENTION

[0001] Polyvalent vaccines are a useful method of generating an immune response to certain foreign substances that would not otherwise be desirable. For example, vaccination against multiple strains of the virus may provide more robust cross-protection against multiple strains of this virus compared to vaccination with a monovalent vaccine.

[0002] However, current polyvalent vaccines and the methods for their preparation require improvement. For example, current approaches to conjugating antigens to protein carriers are complex and provide low yields. Furthermore, it is necessary to develop new techniques for conjugating new antigens to carrier proteins as conventional techniques may fail due to the relative fragility of conventional carrier proteins.

[0003] Compositions and methods are needed that provide improved polyvalent vaccines.

SUMMARY OF THE INVENTION

[0004] In one aspect, the invention provides compositions as defined in the appended claims.

[0005] In another aspect, the invention provides a method as defined in the appended claims.

SHORT DESCRIPTION OF THE FIGURES

[0006]

Figure 1 shows anti-nicotine (dark gray bars) and anti-ovalbumin (light gray bars) titers in unimmunized mice and mice injected with NC-Nic and NC-OVA (5 animals / group, sc, 100 μg of each NC per injection, 2 times at 3-week intervals).

Fig. 2 shows the titers of anti-nicotine, anti-ovalbumin and anti-L2 peptide antibodies in unimmunized mice and mice injected with NC-Nic-OVA and NC-L2 (5 animals / group, sc, 100 μg each NC per injection, 2 times per injection). intervals of 3 weeks).

Fig. 3 shows the titers of anti-nicotine, anti-ovalbumin, anti-M2e and anti-L2 peptide antibody titers in unimmunized mice and mice injected with NC-Nic-OVA and NC-M2e-L2 (5 animals / group, sc, 100 μg of each NC per injection, 2 times at 3-week intervals).

Fig. 4 shows antibody titers against M2e peptide and L2 peptide in unimmunized mice and mice injected with M2e and NC-L2

-2 (5 animals / group, sc, 100 µg of each NC per injection, 2 times at 3 week intervals).

Figure 5 shows antibody titers against HA5 protein and ovalbumin in unimmunized mice and mice injected with NC-HA5 and NC-OVA (5 animals / group, sc, 100 µg of each NC per injection, 2 times at 3-week intervals).

Fig. 6 shows antibody titers anti-HA, anti-ovalbumin, anti-M2e and anti-L2 peptide in unimmunized mice and mice injected with NC-HA5, NC-OVA and NC-M2e-L2 (5 animals / group, sc, 100 μg each NC per injection, 2 times at 3-week intervals).

Figure 7 shows the antibody titers in mice immunized with the combination of NC-M2e, NC-L2 peptide and NC-nicotinovalbumin.

Fig. 8 shows the antibody titers in mice immunized with the combination of NC-3'nicotine and NC-1'-nicotine.

DETAILED DESCRIPTION OF THE INVENTION

[0007] Before describing the invention in detail, it is to be understood that the invention is not limited to specific exemplary materials or method parameters as such values ​​may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments of the invention only, and is not intended to limit the use of alternative terminology to describe the invention.

[0008] As used in the specification and claims, the singular form includes plural references unless the content clearly dictates otherwise. For example, reference to "polymer" includes a mixture of two or more such molecules, reference to "solvent" includes a mixture of two or more such solvents, reference to "adhesive" includes mixtures of two or more such materials, and so on.

INTRODUCTION

[0009] The inventors have surprisingly and unexpectedly found that the problems and limitations noted above can be overcome by using the invention disclosed herein. In particular, the inventors have surprisingly found that it is possible to provide the compositions of the invention and related methods that address the problems and limitations in the art by providing a composition comprising a dosage form comprising: a first population of synthetic nanocarriers that contain a first set of surface antigens; a second population of synthetic nanocarriers that contain a second set of surface antigens; and a pharmaceutically acceptable excipient; wherein the first set of surface antigens and the second set of surface antigens are structurally different.

[0010] In another aspect, the invention provides a composition comprising: a dosage form comprising: a first population of synthetic nanocarriers that include a first

-3set of surface antigens; a second population of synthetic nanocarriers that contain a second set of surface antigens; and a pharmaceutically acceptable excipient; wherein the first set of surface antigens and the second set of surface antigens are immunologically distinct.

[0011] In one aspect, the invention provides a composition comprising: a dosage form comprising: a first synthetic nanocarrier agent for presenting a first set of surface antigens; a second synthetic nanocarrier for displaying a second set of surface antigens; and a pharmaceutically acceptable excipient; wherein the first set of surface antigens and the second set of surface antigens are structurally different.

[0012] In one aspect, the invention provides a composition comprising: a dosage form comprising: a first synthetic nanocarrier agent for presenting a first set of surface antigens; a second synthetic nanocarrier for displaying a second set of surface antigens; and a pharmaceutically acceptable excipient; wherein the first set of surface antigens and the second set of surface antigens are immunologically distinct.

[0013] In another aspect, the invention provides a method, comprising: preparing a first population of synthetic nanocarriers that contain a first set of surface antigens; obtaining a second population of synthetic nanocarriers that contain a second set of surface antigens; and combining the first and second populations of synthetic nanocarriers into a dosage form; wherein the first set of surface antigens and the second set of surface antigens are structurally different.

[0014] In yet another aspect, the invention provides a method comprising: obtaining a first population of synthetic nanocarriers that comprises a first set of surface antigens; obtaining a second population of synthetic nanocarriers that contain a second set of surface antigens; and combining the first and second populations of synthetic nanocarriers into a dosage form; wherein the first set of surface antigens and the second set of surface antigens are immunologically distinct.

[0015] It has been found possible to produce first and second populations of synthetic nanocarriers that contain first and second sets of surface antigens, respectively, that can be combined with a pharmaceutically acceptable excipient to produce a dosage form. This dosage form may, in some embodiments, be useful as a polyvalent vaccine. The inventors have further appreciated some advantages in formulating the dosage forms of the invention, especially with respect to conventional polyvalent vaccines. These include, but are not limited to, minimizing vaccine volume, which is a problem with conventional polyvalent vaccines, and minimizing protein interactions

The protein present in conventional polyvalent vaccines is the hapten protein carrier that can lead to non-specific binding and precipitation.

[0016] A further advantage of the invention is that combining different populations of synthetic nanocarriers that contain sets of surface antigens allows different methods to be used to combine different sets of surface antigens with different populations of synthetic nanocarriers. This can be a significant advantage in embodiments where incompatible conjugation methods are required to couple sets of surface antigens to populations of synthetic nanocarriers. As an example, Streptococcus pneumonia vaccines (US Patent No. 6,132,723 of the Alberta Research Council and WO 2008/143709 Wyeth) contain multiple antigens. Since the chemical attachment conditions are not the same for all polysaccharide antigens (WO 2008/143709), conjugation methods that attach all surface antigens to a single population of synthetic nanocarriers in a single conjugation environment would be undesirable. The practice of examples of the invention in which different populations of synthetic nanocarriers are first conjugated to certain sets of surface antigens and then combined could alleviate the problems noted in the art.

[0017] Another example of polyvalent vaccines that could benefit from this embodiment of the invention include N. meningitides vaccines that are polysaccharide-based and polyvalent. Such embodiments may target either N. meningitidis groups A and C (bivalent) or groups A, C, W135 and Y (tetravalent).

[0018] The examples show certain embodiments of the invention wherein peptides, polysaccharides, small molecules etc. are coupled to a first population of synthetic nanocarriers and / or a second population of synthetic nanocarriers. These populations are then combined to produce the compositions of the invention.

[0019] The invention will now be described in more detail.

DEFINITION

[0020] A "substance of abuse" is any substance ingested by an individual (eg, a human) for purposes other than those for which it is indicated, or in a manner or amount other than as prescribed by a physician. The substance abused, in some embodiments, is an addictive substance. In some embodiments, the substance to be abused for inclusion in the nanocarrier is a complete molecule, analog, or part thereof. An "addictive substance" is a substance that causes obsession, compulsion, or physical or psychological dependence. In some embodiments, the addictive substance for inclusion in the nanocarrier is a complete molecule, analog, or a portion thereof.

[0021] An "adjuvant" is an agent that is not a specific antigen, but increases the strength and longevity of the immune response to an administered antigen (eg, a co-administered antigen). Such adjuvants may include, but are not limited to

5 stimulators of pattern recognition receptors such as Toll-like receptors, RIG-1 and NOD-like receptors ( NOD-like Receptor - NLR), mineral salts such as alum, alum combined with monophosphoryl lipid (MPL) A from enterobacteria such as Escherihia coli, Salmonella minnesota, Salmonella typhimurium or Shigella flexneri or specifically with MPL® (AS04), MPL And the above-mentioned bacteria separately, saponins such as QS21, Quil-A, ISCOM, ISCOMATRIX ™, emulsions such as MF59 ™, Montanide® ISA 51 and ISA 720, AS02 (QS21 + squalene + MPL®), liposomes and liposomal formulations, such as AS01, AS15, synthesized or specially prepared microparticles and microcarriers, such as vesicles derived from the outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others or chitosan molecules, depot (sustained release) formers such as Pluronic® block copolymers, specifically modified or prepared peptides such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates such as RC529 or proteins such as such as bacterial toxoids or fragments of toxins.

[0022] In embodiments, adjuvants include agonists for pattern recognition receptors (PRRs), including, but not limited to, Toll-like receptors (TLRs), especially TLRs 2, 3, 4, 5, 7, 8, 9 and / or their combinations. In other examples, adjuvants made include agonists for Toll-like 3 receptors, agonists for Toll-like 7 and 8 receptors, or agonists for Toll-like 9 receptors; preferably said adjuvants include imidazoquinolines; such as R848; adenine derivatives such as those described in US Patent 6,329,381 (Sumitomo Pharmaceutical Company), Published Patent Application US 2010/0075995 by Biggadike et al. or WO 2010/018132 Camposa et al; immunostimulatory DNA; or immunostimulatory RNAs.

[0023] In specific embodiments, the synthetic nanocarriers contain as adjuvants compounds that are agonists for Toll-like receptors (TLRs) 7 and 8 ("7/8 TLR agonists"). Useful are TLR 7/8 agonists described in US Patent 6,696,076 to Tomai et al, including, but not limited to, imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2-bridged imidazoquinoline amines. Preferred adjuvants include imiquimod and resiquimod (also known as R848). In certain embodiments, synthetic nanocarriers contain a ligand for a Toll-like receptor (TLR) -9, such as CpG, that induce type I interferon production and stimulate T and B cell activation leading to increased antibody production and cytotoxic T cell responses (Krieg and et al., CpG motifs in bacterial DNA trigger direct B cell activation. Nature. 1995. 374: 546-549; Chu et al. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. J. Exp. Med. 1997. 186: 1623-1631; Lipford et al. CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants. Eur. J. Immunol. 1997. 27: 2340-2344; Roman et al. Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat. Med. 1997. 3: 849-854; Davis et al. CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen. J. Immunol. 1998. 160: 870-876; Lipford et al., Bacterial DNA as immune cell activator.

-6Trends Microbiol. 1998. 6: 496-500; US patent 6,207,646 by Krieg et al; US patent 7,223,398 by Tuck et al; U.S. Patent 7,250,403 by Van Nest et al; or U.S. Patent 7,566,703 by Krieg et al.).

[0024] In specific embodiments, the adjuvant may be an agonist for a CD40 DC surface molecule. In some embodiments, to stimulate immunity rather than tolerance, the synthetic nanocarrier comprises an adjuvant that stimulates DC maturation (needed to stimulate naive T cells) and the production of cytokines such as type I interferons that stimulate antibody immune responses and antiviral immunity. In embodiments, adjuvants can also include immunostimulatory RNA molecules such as, but not limited to, dsRNA, ssRNA, poly I: C, or poly I: poly C12U (available as Ampligen®, both poly I: C and poly I: polyC12U). are known as TLR3 stimulants) and / or those disclosed in F. Heil et al., "Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8" Science 303 (5663), 1526-1529 (2004) ; J. Vollmer et al., "Immune modulation by chemically modified ribonucleosides and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al., "Immunostimulatory oligoribonucleotides containing specific sequence motif (s) and targeting the Tolllike receptor 8 pathway" WO 2007062107 A2; E. Uhlmann et al., "Modified oligcoribonucleotide analogs with enhanced immunostimulatory activity"; Patent application publication US 2006241076; G. Lipford et al., "Immunostimulatory viral RNA oligonucleotides and use for treating cancer and infections" WO 2005097993 A2; G. Lipford et al., "Immunostimulatory G, U-containing oligoribonucleotides, compositions, and screening methods" WO 2003086280 A2.

[0025] In some embodiments, the adjuvant may be a TLR-4 agonist such as bacterial lipopolysaccharide (LPS), VSV-G, and / or HMGB-1. In some embodiments, adjuvants can include TLR-5 agonists such as flagellin or parts or derivatives thereof, including, but not limited to, those disclosed in US Patent Nos. 6,130,082, 6,585,980, and 7,192,725.

[0026] In some embodiments, adjuvants can be pro-inflammatory stimuli released from necrotic cells (eg, urate crystals). In some embodiments, adjuvants can be activated components of the complement cascade (e.g., CD21, CD35, etc.). In some embodiments, adjuvants can be activated components of immune complexes. Adjuvants also include complement receptor agonists such as the CD21 or CD35 binding molecule. In some embodiments, a complement receptor agonist induces endogenous complement opsonization of a synthetic nanocarrier. In some embodiments, the adjuvants are cytokines, which are small proteins or biological agents (in the 5 kD - 20 kD range) that are released by cells and have specific effects on cell-cell interaction, communication, and other cell behavior. In some embodiments, the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer.

[0027] In embodiments, at least a portion of the adjuvant dose may be coupled to synthetic nanocarriers, preferably the entire dose of adjuvant is coupled to

-7with synthetic nanocarriers. In other embodiments, at least a portion of the adjuvant dose is not coupled to synthetic nanocarriers. In embodiments, an adjuvant dose comprises two or more types of adjuvants. For example and without limitation, adjuvants that act on different TLRs can be combined. For example, in an embodiment, a 7/8 TLR agonist can be combined with a TLR9 agonist. In another embodiment, a 7/8 TLR agonist can be combined with a TLR4 agonist. In yet another embodiment, a TLR 9 agonist may be combined with a TLR3 agonist.

[0028] "Administering" or "administering" means delivering a substance to the subject in a manner that is pharmacologically useful.

[0029] "Effective amount" means any amount of the composition that produces one or more of the desired immune responses. The amount may be for in vitro or in vivo purposes. For in vivo purposes, the amount can be such as the physician would consider likely to be of clinical benefit to a subject in need of an antibody response specific for one or more antigens. "Antibody response" means any immune response that results in the production or stimulation of B cells and / or the production of antibodies. Therefore, in embodiments, an effective amount is one that, in the physician's judgment, can generate an antibody response against the surface antigen (s) of the compositions of the invention provided herein. Effective amounts can be monitored by routine means. The amount that is effective in generating one or more of the desired immune responses can also be the amount of the composition provided herein that produces the desired therapeutic endpoint or desired therapeutic result. Therefore, in other embodiments, an effective amount is one that a physician would consider providing a therapeutic benefit (including prophylactic benefit) when delivered to a subject. Such individuals include those who have or are at risk of developing cancer, infection, or an infectious disease.

[0030] The antigen (s) of any of the compositions provided herein may, in embodiments, be in an effective amount. In some embodiments, an effective amount is one at which the clinician judges can generate antibody titers against the sets of surface antigens of the compositions provided herein. "Antibody titer" means the production of a measurable level of antibodies. Preferably, the antibody response or the production of the antibody titer is in a human. In some embodiments, the antibodies are isotype specific antibodies, such as IgG or a subclass thereof. Methods for measuring antibody titers are known in the art and include an enzyme linked immunosorbent assay (ELISA). Methods of measuring antibody responses are also described in detail in the Examples. Preferably, the antibody response or antibody titer is specific to a set of surface antigens. In some embodiments where synthetic nanocarriers also contain a universal antigen in addition to a set of surface antigens against which a specific immune response occurs, such as an antibody response or antibody titer, the immune response is specific to the set of surface antigens but not to the universal antigen.

[0031] Effective amounts will, of course, depend on the particular subject being treated; the severity of the condition, disease or disorder; individual patient parameters, including age, physical condition, size and weight; duration of treatment; the nature of concomitant therapy (if any); the particular route of administration and similar factors within the knowledge and experience of the practitioner. These factors are well known to those skilled in the art and can only be solved by routine experimentation. It is generally recommended that the "maximum dose" be used, that is, the highest safe dose based on rational medical judgment. It will be understood by those skilled in the art that a patient may insist on a lower dose or a tolerated dose for medical, psychological, or virtually any other reason.

[0032] "Antigen" means B cell antigen or T cell antigen. In embodiments, the antigens are coupled to synthetic nanocarriers. In other embodiments, the antigens are not coupled to synthetic nanocarriers. In embodiments, the antigens are co-administered with synthetic nanocarriers. In other embodiments, the antigens are not co-administered with synthetic nanocarriers. "Antigen type (s)" means molecules that have the same or substantially the same antigenic properties.

[0033] "At least a fraction of a dose" means at least a fraction of a dose, up to and including the total dose.

[0034] A "at risk" subject is one for whom a physician believes there is a chance of the disease or condition specified herein, including, but not limited to, infection, infectious disease, cancer, or addiction.

[0035] "B cell antigen" means any antigen that is recognized by and triggers an immune response in a B cell (eg, an antigen that is specifically recognized by a B cell receptor in a B cell). In some embodiments, the T cell antigen is also a B cell antigen. In other embodiments, the T cell antigen is also not a B cell antigen. B cell antigens include, but are not limited to, proteins, peptides, small molecules, carbohydrates, oligosaccharides, and polysaccharides. In some embodiments, the B cell antigen comprises a non-protein antigen (ie, a non-protein or peptide antigen). In some embodiments, the B cell antigen comprises a carbohydrate, oligosaccharide, or polysaccharide associated with an infectious agent. In some embodiments, the B cell antigen comprises a glycoprotein or a glycopeptide associated with an infectious agent. The infectious agent can be a bacterium, virus, fungus, protozoan or parasite. In some embodiments, the B cell antigen comprises a weakly immunogenic antigen. In some embodiments, the B cell antigen comprises an abusive or addictive substance or a portion or analog thereof. Addictive substances include, but are not limited to, nicotine, narcotic, cough suppressant, sedative, and anesthetic. In some embodiments, the B cell antigen includes a toxin such as a toxin from chemical weapons or natural sources. The B cell antigen may also include a hazardous environmental factor. In some examples

The B cell antigen includes its own antigen. In other embodiments, the B cell antigen includes an alloantigen, an allergen, a contact sensitiser, a degenerative disease antigen, a hapten, an infectious disease antigen, a tumor antigen, an atopic disease antigen, an autoimmune disease antigen, an addictive substance, a xenoantigen, or a metabolic disease enzyme or an enzyme product thereof. .

[0036] "Pair" or "conjugated" or "pairs" (and the like) means one unit (eg moiety) chemically bonded to another. In some embodiments, the coupling is covalent, meaning that the coupling occurs in the context of the presence of a covalent bond between two entities. In non-covalent embodiments, non-covalent conjugation is mediated by non-covalent interactions, including, but not limited to, charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stack interactions, hydrogen bonding interactions. van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and / or combinations thereof. In embodiments, the encapsulation is in the form of a coupling.

[0037] "Derived" means an adapted or modified from the original source. For example, as a non-limiting example, a peptide antigen derived from an infectious strain may have several unnatural amino acid residues substituted for the natural amino acid residues found in the original antigen found in the infectious strain. Adaptations or modifications may arise for a number of reasons, including, but not limited to, increased specificity, easier processing of the antigen, or improved safety.

[0038] In embodiments, a peptide or nucleic acid with a sequence with only 50% identity to a natural peptide or nucleic acid, preferably a natural consensus peptide or nucleic acid, will be derived from a natural peptide or nucleic acid. In other embodiments, the material is substantially modified. A substantially modified material means a material that is modified in such a way that the modification significantly affects the chemical or immunological properties of the material in question. Derivative peptides and nucleic acids may also include those with a sequence identity of more than 50% to that of a natural peptide or nucleic acid, if said derivative peptides and nucleic acids have altered chemical or immunological properties compared to the natural peptide or nucleic acid. These chemical or immunological properties include hydrophilicity, stability, affinity, and the ability to couple to a carrier, such as a synthetic nanocarrier.

[0039] "Dosage form" means a pharmacologically and / or immunologically active material, such as a vaccine, in a vehicle, carrier, carrier, or article suitable for administration to a subject.

[0040] To "encapsulate" or "encapsulate" means to enclose in a synthetic nanocarrier, preferably completely encapsulate in a synthetic nanocarrier. Most or all of the encapsulated material is not exposed to a local environment external to the synthetic nanocarrier. Encapsulation is distinct from adsorption, which places most or all of the substance on the surface of a synthetic nanocarrier and leaves the substance exposed to a local environment external to the synthetic nanocarrier.

[0041] "Immunologically different" refers to the difference between some surface antigens that can be seen if the sera generated by immunization generate a distinct antibody response spectrum for each of the surface antigens. The antigen-specific surface antibodies recognize only a specific set of surface antigens and will bind to distinguishable binding patterns with other sets of surface antigens. For example, in the case of immunization with set of surface antigens A, the produced antiserum will bind to set of surface antigens A, but not set of surface antigens B. If two or more surface antigens are combined in a single synthetic nanocarrier, a washout test can be designed. panning assay, which distinguishes the binding patterns of sera against two sets of surface antigens. In embodiments, the first set of surface antigens and the second set of surface antigens are immunologically different. In other embodiments, the first set of surface antigens, the second set of surface antigens, and the third set of surface antigens are immunologically distinct.

[0042] "Infection" or "infectious disease" means any condition or disease caused by a microorganism, pathogen or other agent such as bacteria, fungus, prion or virus. The surface antigens in the composition of the invention provided herein may be obtained or may be derived from any infectious agent, such as those which may cause the infections or infectious diseases set forth herein.

[0043] "Infectious genus" means the genus that includes organisms capable of infecting an individual. In embodiments, the surface antigens may be or may be derived from the first infectious type or obtained or obtained from the second infectious type. In the embodiments, the first infectious type and the second infectious type are the same. In other embodiments, the first infectious type and the second infectious type are different.

[0044] "Infectious species" means species that contain organisms capable of infecting an individual. In embodiments, the surface antigens may or may be derived from the first infectious species or obtained or obtained from a second infectious species. In embodiments, the first infectious species and the second infectious species are of the same genus. In other embodiments, the first infectious species and the second infectious species are also the same. In some embodiments, the first

-11 infectious species and second infectious species are different but of the same genus. In other embodiments, different infectious species are of different types.

[0045] "Infectious strain" means a strain that contains organisms capable of infecting an individual. In embodiments, the surface antigens may be or may be derived from the first infectious strain or obtained or obtained from a second infectious strain. In embodiments, the first infectious strain and the second infectious strain are of the same species. In other embodiments, the first infectious strain and the second infectious strain are also the same. In still other embodiments, they are of the same species but of a different strain. In some embodiments, the first infectious strain and the second infectious strain are of different species but of the same genus.

[0046] "Isolated nucleic acid" means a nucleic acid that is separate from its native environment and is present in an amount sufficient to enable it to be identified or used. An isolated nucleic acid can be one that is (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, such as by cleavage and gel separation; or (iv) synthesized for example by chemical synthesis. An isolated nucleic acid is one that can be readily manipulated with recombinant DNA techniques well known in the art. Accordingly, a nucleotide sequence contained in a vector where 5 'and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated, but an existing nucleic acid sequence is not considered to be. in its native state with its natural host. The isolated nucleic acid can be substantially purified, but need not be. For example, a nucleic acid that is isolated in a cloning or expression vector is not pure as it may only contain a small percentage of the material in the cell in which it is. However, such a nucleic acid is isolated because, as used herein, it can be easily manipulated by standard techniques known to those skilled in the art. Any of the nucleic acids described herein can be isolated. In some embodiments, the antigens in the compositions provided herein are in the form of an isolated nucleic acid, such as an isolated nucleic acid that encodes an antigenic peptide, polypeptide, or protein.

[0047] "Isolated peptide, polypeptide or protein" means a peptide, polypeptide or protein separated from its native environment and present in an amount sufficient to allow its identification or use. This means, for example, that the peptide, polypeptide or protein can be (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. The isolated peptides, polypeptides, or proteins may or may not be substantially pure. Since the isolated peptide, polypeptide or protein may be mixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the peptide, polypeptide or protein may contain only a small percentage by weight of the preparation. However, a peptide, polypeptide or protein is separated because it has been separated from substances with which it can be associated in living systems,

-12 i.e. isolated from other peptides, polypeptides or proteins. Any of the peptides, polypeptides, or proteins described herein can be isolated. In some embodiments, the antigens in the compositions provided herein are peptides, polypeptides or proteins.

[0048] "Maximum dimension of synthetic nanocarrier" means the largest dimension of the nanocarrier measured along any axis of the synthetic nanocarrier. "Minimum dimension of synthetic nanocarrier" means the smallest dimension of a synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, for a spheroidal synthetic nanocarrier, the maximum and minimum dimensions of the synthetic nanocarrier would be substantially identical and would be the size of its diameter. Similarly, in the case of a rectangular synthetic nanocarrier, the minimum size of the synthetic nanocarrier would be the smallest of its height, width, or length, while the maximum dimension of the synthetic nanocarrier would be the largest of its height, width, or length. In an embodiment, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample, based on the total number of synthetic nanocarriers in the sample, is greater than 100 nm. In an embodiment, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample, based on the total number of synthetic nanocarriers in the sample, is equal to or less than 5 µm. Preferably a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 110 nm, more preferably equal to or greater than 120 nm , more preferably equal to or greater than 130 nm, and even more preferably equal to or greater than 150 nm. The maximum and minimum size ratios of the synthetic nanocarriers of the invention may vary depending on the embodiment. For example, the maximum to minimum size ratios of synthetic nanocarriers can range from 1: 1 to 1,000,000: 1, preferably from 1: 1 to 100,000: 1, more preferably from 1: 1 to 1,000: 1, even more preferably from 1: 1 to 100: 1 and even more preferably from 1: 1 to 10: 1. Preferably, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample, based on the total number of synthetic nanocarriers in the sample, is equal to or less than 3 μm, more preferably equal to or less than 2 μm. , more preferably equal to or less than 1 µm, more preferably equal to or less than 800 nm, more preferably equal to or less than 600 nm and even more preferably equal to or less than 500 nm. In preferred embodiments, a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm, more preferably equal to or greater than 120 nm, more preferably equal to or greater than 130 nm, more preferably equal to or greater than 140 nm, and even more preferably equal to or greater than 150 nm. Measurement of synthetic nanocarrier sizes is obtained by suspending the synthetic nanocarriers in a liquid (usually aqueous) medium and using dynamic light scattering (e.g. using the Brookhaven ZetaPALS instrument).

[0049] "Molecular weight less than 10,000" means a molecular weight calculated on the basis of the molecular structure of the molecule less than 10,000.

[0050] "Obtained" means taken from a source without significant modification. A fundamental modification is a modification that significantly affects the chemical or immunological properties of the material. For example, as a non-limiting example, a peptide or nucleic acid with a sequence of more than 90%, preferably more than 95%, preferably more than 97%, preferably more than 98%, preferably more than 99%, preferably 100% identity with the natural peptide or a nucleotide sequence, preferably a natural consensus peptide or a nucleotide sequence, with chemical and / or immunological properties that do not differ significantly from the natural peptide or nucleic acid, can be defined as derived from a natural peptide or a nucleotide sequence. These chemical or immunological properties include hydrophilicity, stability, affinity, and the ability to couple to a carrier, such as a synthetic nanocarrier. In exemplary embodiments, the material obtained is from the original source and has not been adapted or modified. For example, in embodiments, the antigens obtained from the source may include the original sequence of amino acid residues from that source. In other embodiments, for example, antigens obtained from a source may contain the original molecular structure with that source.

[0051] "Oligosaccharide (s)" means a saccharide polymer containing a small number (usually from two to twenty) of saccharide units linked by glycosidic bonds. Where there is a large number of saccharide units, the oligosaccharides may contain polysaccharides.

[0052] "Peptide (s)" means compounds having amino acid residues connected primarily by peptide bonds between carboxyl and amino groups of adjacent amino acid residues and having 100 amino acid residues or less. Certain peptide bonds in a peptide may be replaced with other types of bonds for various purposes, such as stabilization or conjugation.

[0053] "Pharmaceutically acceptable excipient (or carrier)" means a pharmacologically inactive material used together with the cited synthetic nanocarriers to formulate the compositions of the invention. Pharmacologically inactive materials may be added to the dosage forms of the invention to further facilitate administration of the compositions. Pharmaceutically acceptable excipients include a variety of materials known in the art including, but not limited to, saccharides (such as glucose, lactose, and the like), preservatives such as antimicrobials, dissolution aids, dyes, saline (such as such as phosphate buffered saline) and buffers. Examples, without limitation, of pharmaceutically acceptable excipients include calcium carbonate, calcium phosphate, various diluents, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, preservatives, various pharmaceutical carriers, sterile saline, lyophilization stabilizers, and the like. The compositions can be prepared using conventional manufacturing and compounding techniques

-14 useful dosage forms. In one embodiment, the synthetic nanocarriers of the invention are suspended in a sterile saline injection together with a preservative.

[0054] "Polysaccharide (s)" means a saccharide polymer made of a plurality of saccharide units linked by glycosidic bonds. With a low number of saccharide units, the polysaccharides may include oligosaccharides.

[0055] "Population" means a specific group of synthetic nanocarriers that share one or more physical or chemical characteristics. Common physical or chemical properties may include common set of surface antigens, common conjugated adjuvant (s), common nano-volume forming materials, common shape, common particle size, and the like. Multiple populations of synthetic nanocarriers can be identified, for example, the first population, the second population, the third population, the fourth population, and the like. In an embodiment, three or more populations of synthetic nanocarriers may be present, preferably wherein each population of synthetic nanocarriers comprises a set of surface antigens; and wherein each set of surface antigens is structurally or immunologically different from one another.

[0056] "Protein (s)" means compounds, typically with a molecular weight greater than 1000 Daltons, containing amino acid residues primarily linked by peptide bonds between carboxyl and amino groups of adjacent amino acid residues. The proteins may also contain additional binding structures such as secondary structures, tertiary structures, and the like. Certain peptide bonds in proteins can be replaced with other types of bonds for various purposes, such as stabilization or conjugation.

[0057] "Monovalent surface antigens set" means a set of surface antigens in which the surface antigens are not different, preferably not structurally and / or immunologically different. In embodiments, a set of monovalent surface antigens consists of multiple copies of one type of surface antigen that is not structurally or immunologically different (i.e., multiple copies of the same antigen). Multiple copies of the same antigen may, in some embodiments, be linked together, as shown in US 2003/0223938. A set of monovalent surface antigens that consists of multiple copies of a single type of surface antigen that is not structurally or immunologically different is not a set of oligovalent (or polyvalent) surface antigens.

[0058] "Set of oligovalent (or polyvalent) surface antigens" means a set of surface antigens in which there are a limited number that is greater than one different types of surface antigens, the difference preferably being a structural difference and / or an immunological difference. In preferred embodiments, a limited number of surface antigens

The kit comprises 2 to 15 types of surface antigens, preferably 2 to 10 types of surface antigens, more preferably 2 to 8 types of surface antigens, more preferably 2 to 7 types of surface antigens, more preferably 2 to 6 types of surface antigens, more preferably 2 to 5 types of surface antigens , more preferably 2 to 4 types of surface antigens, more preferably 2 to 3 types of surface antigens, and even more preferably 2 types of surface antigens. In other embodiments, the set of oligovalent (or polyvalent) surface antigens includes at least 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. or more types of surface antigens.

[0059] "Set of surface antigens" means a group of surface antigens which are identified, preferably identified by measurement and / or prediction, on the basis of their properties, preferably their structural and / or their immunological properties. The set of surface antigens can be identified, partially or wholly, based on prediction using the chemical synthesis methods used for the synthesis, together with the chemical methods used for conjugation, the set of surface antigens, and / or the population of synthetic nanocarriers that make up the set of surface antigens. Multiple sets of surface antigens can be identified; for example, the first set, the second set, the third set, and so on.

[0060] "Structurally different" or "structural difference" means displaying different molecular structures for interaction with the B-cell receptor. In the embodiments, this difference can be expressed by comparing the frequency and types of displayed antigens in a set of surface antigens with the frequency and types of the displayed antigens. in a different set of surface antigens. If the frequency of occurrence and / or the types of antigens presented are different in the different sets, it can be considered that the sets of surface antigens are structurally different. In the embodiments, the difference in the frequency and / or types of antigens presented can be determined by comparing the chemical synthesis strategy and the formulation strategy used to generate the surface antigens and / or couple the surface antigens to the surface of the synthetic nanocarriers. For example, in embodiments, if the set of surface antigens has been generated using a particular chemical or compounds and a different set of surface antigens has been generated using a different chemical or compounds, then it can be determined that the two sets of surface antigens are different. In another embodiment, if the surface antigens were generated using three chemicals to produce a set of three surface antigens, and other surface antigens were generated using the two chemicals to produce a set of two surface antigens, then the two sets of surface antigens can be said to be different . In yet another embodiment, when different chemical synthesis and formulation strategies are used (including the use of different amounts of materials - with experimental error - in the strategies), two sets of

- 16 surface antigens and (respectively) couplings the two sets of surface antigens to the surfaces of synthetic nanocarriers and the two sets of surface antigens share the same conformation and orientation, then the two sets of surface antigens are unlikely to be structurally different. In embodiments, the structural difference between the first set of surface antigens and the second set of surface antigens comprises nondifferent sets of molecules that are shown in orientations that differ between the first and second set of surface antigens. In embodiments, the structural difference between the first set of surface antigens and the second set of surface antigens comprises nondifferent sets of molecules that are shown in orientations that differ between the first and second set of surface antigens. In embodiments, the structural difference between the first set of surface antigens and the second set of surface antigens comprises sets of molecules whose molecular structure is different between the first and second set of surface antigens.

[0061] "Subject" means animals, including warm-blooded mammals such as humans and primates; birds; domestic animals or farm animals such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals such as mice, rats, and guinea pigs; fish; reptiles; zoo and wild animals; and the like.

[0062] "Surface antigen (s)" means an antigen on or around the surface of a synthetic nanocarrier. In preferred embodiments, the surface antigens include B-cell antigens. In embodiments, the surface antigens are coupled to the surface of the synthetic nanocarriers.

[0063] "Synthetic nanocarrier (s)" means a discrete object that does not occur in nature and that has at least one dimension that is less than or equal to 5 microns. Albumin nanoparticles are generally included as synthetic nanocarriers, however, in some embodiments, synthetic nanocarriers do not include albumin nanoparticles. In embodiments, the synthetic nanocarriers of the invention do not contain chitosan.

[0064] A synthetic nanocarrier may be, but is not limited to, one or more lipid-based nanoparticles (e.g., liposomes) (also referred to herein as lipid nanoparticles, i.e. nanoparticles where most of the material that makes up their structure is lipids), polymer nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles (i.e. (molecules which are mainly composed of viral structural proteins but which are not infectious or low infectivity), peptide or protein based molecules (also referred to herein as protein molecules, i.e. molecules in which most of the material that makes up their structure, are peptides or proteins) (such as albumin nanoparticles) and / or nanoparticles that are developed using a combination of nanomaterials such as lipid-polymer nanoparticles. Synthetic nanocarriers can be of many different

- 17 shapes including, but not limited to, spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like. The synthetic nanocarriers of the invention contain one or more surfaces. Examples of synthetic nanocarriers that can be adapted for use in the practice of the invention include: (1) biodegradable nanoparticles described in Gref et al. US Patent 5,543,158, (2) polymer nanoparticles from Saltzman et al. Published patent application US 20060002852, (3) lithographically constructed nanoparticles published US patent application 20090028910 by DeSimone et al., (4) disclosure WO 2009/051837 von Andriana et al. (5) nanoparticles described in published patent application US 2008/0145441 by Penadesa et al., (6) protein nanoparticles disclosed in published patent application US 20090226525 to de los Rios et al. (7) virus-like particles disclosed in published patent application US 20060222652 by Sebbel et al., (8) nucleic acid-conjugated virus-like particles disclosed in published patent application US 20060251677 by Bachmann et al., (9) virus-like particles disclosed in WO2010047839A1 or WO2009106999A 10) nanoprecipitated nanoparticles disclosed in P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5 (6): 843-853 (2010). In exemplary embodiments, the synthetic nanocarriers can have an aspect ratio greater than 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7, or greater than 1:10.

[0065] Synthetic nanocarriers of the invention that have a minimum dimension equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not contain surfaces with hydroxyl groups that activate complement, or alternatively contain a surface that consists essentially of moieties, which are not hydroxyl groups that activate complement. In a preferred embodiment, synthetic nanocarriers of the invention that have a minimum dimension equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not contain a surface that substantially activates complement, or alternatively includes a surface that consists essentially of moieties, which do not substantially activate complement. In a more preferred embodiment, the synthetic nanocarriers of the invention that have a minimum dimension equal to or less than about 100 nm, preferably equal to or less than 100 nm, contain no complement activating surface or alternatively contain a surface that consists essentially of non-complement activating moieties. . In embodiments, synthetic nanocarriers exclude virus-like particles. In embodiments, when the synthetic nanocarriers contain virus-like particles, the virus-like particles contain an unnatural adjuvant (that is, the VLPs contain an adjuvant other than the naturally occurring RNA produced during the production of the VLP). In exemplary embodiments, the synthetic nanocarriers can have an aspect ratio greater than 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7, or greater than 1:10.

[0066] "T cell antigen" means any antigen which is recognized by and triggers an immune response in the T cell (e.g., an antigen that is specifically

Recognized by a T cell receptor on a T cell or NKT cell by the presentation of an antigen or a portion thereof associated with a Class I or Class II major histocompatibility complex (MHC) molecule or associated with the CD1 complex. In some embodiments, the T cell antigen is also a B cell antigen. In other embodiments, the T cell antigen is also not a B cell antigen. T cell antigens are generally proteins or peptides. The T cell antigens may be an antigen that stimulates a CD8 + T cell response, a CD4 + T cell response, or both. Nanocarriers, therefore, in some embodiments can be effective in stimulating both types of response.

[0067] In some embodiments, the T cell antigen is a "universal" T cell antigen or T cell memory antigen (ie, one for which the patient has pre-existing memory and which can be used to enhance T cell support against an unrelated antigen, for example unrelated B-cell antigen). Universal T cell antigens include tetanus toxoid as well as one or more peptides derived from tetanus toxoid, Epstein-Barr virus, or influenza virus. Universal T cell antigens also include influenza virus components such as hemagglutinin, neuraminidase, or a nuclear protein or one or more peptide derivatives thereof. In some embodiments, the universal T cell antigen is not one that is complexed with the MHC molecule. In some embodiments, the universal T cell antigen is not complexed with an MHC molecule for presentation to a T helper cell. Accordingly, in some embodiments, the universal T cell antigen is not a helper T cell antigen. However, in other embodiments, the universal T cell antigen is a helper T cell antigen.

[0068] In embodiments, the helper T cell antigen may comprise one or more peptides derived from or derived from tetanus toxoid, Epstein-Barr virus, influenza virus, respiratory syncytial virus, measles virus, mumps virus, rubella virus, cytomegalovirus, adenovirus, diphtheria toxoid or PADRE peptide (known from Sette et al. US Patent 7,202,351). In other embodiments, the T-helper antigen may include ovalbumin or a peptide derived from or derived therefrom. Preferably, the ovalbumin comprises the amino acid sequence set forth in Accession No. AAB59956, NP_990483.1, AAA48998 or CAA2371. In other embodiments, the ovalbumin derived or derived peptide comprises the following amino acid sequence: H-Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-GluAla-Gly- Arg-OH (SEQ ID NO: 1). In other embodiments, the helper T cell antigen may comprise one or more lipids or glycolipids including, but not limited to: α-galactosylceramide (α-GalCer), α-linked glycosphingolipids (from Sphingomonas spp.), Galactosyl diacylglycerols (from Borrelia burgdorferi) ), lipophosphoglycan (from Leishmania donovani) and phosphatidylinositol tetramannoside (PIM4) (from Mycobacterium leprae). For additional lipids and / or glycolipids useful as T helper antigen, see V. Cerundolo et al., "Harnessing invariant NKT cells in vaccination strategies." Nature Rev Immun, 9: 28-38 (2009).

[0069] In embodiments, the CD4 + T cell antigens may be derived from a CD4 + T cell antigen that is obtained from a source, such as a natural source. In such embodiments, the antigenic sequences of CD4 + T cells, such as peptides that bind to MHC II, may be at least 70%, 80%, 90%, or 95% identical to the antigen obtained from the source. In embodiments, the T cell antigen, preferably a universal T cell antigen or a helper T cell antigen, may be coupled to or detached from the synthetic nanocarrier. In some embodiments, a universal T cell antigen or T helper antigen is encapsulated in the synthetic nanocarriers of the compositions of the invention.

[0070] "Types of surface antigens", "types of surface antigens", etc. means a defined group of surface antigens that share one or more chemical and / or immunological characteristics.

[0071] "Vaccine" means a composition of substances that improves the immune response to a specific pathogen or disease. The vaccine usually contains factors which stimulate the patient's immune system to recognize the specific antigen as foreign and eliminate it from the subject's body. The vaccine also creates an immune 'memory' so that the antigen will be quickly recognized and reacted upon if a person is re-exposed. Vaccines may be prophylactic (for example, to prevent future infection with any pathogen) or therapeutic (for example, a tumor specific antigen vaccine to treat cancer). In embodiments, the vaccine may contain the dosage forms of the invention.

CARRIER POPULATIONS AND SURFACE ANTIGEN SETS

[0072] In embodiments, the populations of synthetic nanocarriers share common physical or chemical characteristics. In embodiments, such common physical or chemical characteristics may include a common set of surface antigens, a common conjugated adjuvant (s), common materials forming the volume of a nanocarrier, common shape, common particle size, common surface charge, and the like. The types of adjuvants, materials, shapes, and particle sizes are discussed in this application.

[0073] In embodiments, a population may share a set of common surface antigens. These common surface antigens may be grouped together based on common physical or chemical characteristics, such as, but not limited to, structural or immunological properties. In embodiments, the common features may include a common orientation or conformation, or sets of molecules having a common molecular structure, or all of the above. In embodiments, the common surface antigens may include those with a molecular weight of less than 10,000. In other embodiments, the common surface antigens may include peptides, proteins, oligosaccharides, polysaccharides, or small molecules. In yet other embodiments, the common surface antigens may include those of mass

20 molecular weight less than 10,000 and including common peptides, proteins, oligosaccharides, polysaccharides or small molecules. In other embodiments, the common surface antigens may be grouped based on the infectious organism from which they are obtained or derived; and would be classified as sharing a common genus, species and / or strain. In embodiments where the surface antigens have a molecular weight of less than 10,000, common surface antigens can be grouped together based on molecular classes such as chemical warfare agents, environmental toxins, addictive or abused substances, and physiologically endogenous molecules including, but not limited to hormones, lipids, and neurotransmitters. In embodiments, the sets of common surface antigens can be determined by the strength of their ability to induce an antibody response in vivo. For example, one set of surface antigens may have the ability to induce the production of high affinity antibodies in vivo, while another set of common surface antigens may induce the production of low affinity antibodies in vivo.

[0074] In embodiments, the set of surface antigens (e.g., the first and / or second set of surface antigens) may include antigens obtained or derived from an infectious agent. In some embodiments, the infectious agent is a bacterium, fungus, virus, protozoan, or parasite. In other embodiments, the virus is pox virus, pox virus, Ebola virus, Marburg virus, measles virus (in embodiments, the antigen may or may be derived from a hemagglutinin protein, a hemagglutinin epitope, hemagglutinating amino acids 106-114, and / or 519-550). etc.), dengue virus, influenza virus, influenza A virus (in embodiments, the antigen may or may be derived from HA protein, M2e protein, etc.), H5N1 influenza virus, H1N1 influenza virus, salmon infectious anemia virus, parainfluenza virus, respiratory syncytial virus, rubella virus, human immunodeficiency virus, human papillomavirus, varicella zoster virus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, rotin virus, JC, rhovirus , adenovirus, papillomavirus (in embodiments, the antigen may or may be derived from the L1 or L2 protein), parvovirus, picornavirus, poliovirus, mumps virus, rabies virus, reovirus, rubella virus, togavirus, orthomyxovirus, retrovirus, hepadnavirus, coxsackie virus, equine encephalitis virus, tick-borne encephalitis virus, Japanese encephalitis virus, yellow fever virus, rift valley fever virus hepatitis A, hepatitis B, hepatitis C, hepatitis D virus or hepatitis E virus.

[0075] In embodiments, the set of surface antigens (e.g., the first and / or second set of surface antigens) comprises or is derived from, or is derived from, a virus of the family of viruses shown below in Table 1. In another embodiment, the set of surface antigens (e.g., the first and / or / or second set of surface antigens) comprises or is derived from or derived from a species virus

Shown in Table 1. In yet another embodiment, the set of surface antigens (e.g., the first and / or second set of surface antigens) comprises or is derived from, or is derived from, the antigen set forth in Table 1.

Table 1: Viral Infectious Agents ____________________________________________

<td>Family</td><td>Sample species</td><td>Exemplary antigens</td>

<td>Adenoviridae</td><td>adenovirus</td><td>VI, VII, E1A, E3-19K, 52K</td>

<td rowspan="4">Picornaviridae</td><td>Coxsackie virus,</td><td>VP1</td>

<td>hepatitis A virus</td><td>surface antigen</td>

<td>polio virus,</td><td>protein 3A, capsid protein</td>

<td>Rhinovirus (e.g. type 16)</td><td>nucleocapsid, surface projection and transmembrane proteins</td>

<td rowspan="5">Herpesviridae</td><td>Herpes simplex (type 1 and type 2)</td><td>capsid proteins (e.g. UL6, UL18, UL35, UL38, and UL19)</td>

<td>Varicella-zoster virus</td><td>early antigen</td>

<td>Epstein-Barr virus</td><td>early antigen, capsid antigen</td>

<td>Human cytomegalovirus</td><td>Pp65, gB, p52</td>

<td>Human herpesvirus (e.g. type 8)</td><td>latent nuclear antigen 1</td>

<td>Hepadnaviridae</td><td>hepatitis virus B</td><td>surface antigen</td>

<td>Flaviviridae</td><td>hepatitis C virus, yellow fever virus, dengue virus, West Nile virus</td><td>NS3, envelope protein (e.g. E2 domain)</td>

<td>Retroviridae</td><td>HIV</td><td>gp120, p24, and the lipopeptides Gag (17-35), Gag (253-284), Nef (66-97), Nef (116-145) and Pol (325-355); see Roberts et al., J. Immunol. Methods, 365 (1-2): 27-37, 2011</td>

<td>Orthomyxoviridae</td><td>flu virus</td><td>neuraminidase, a surface antigen.</td>

<td rowspan="3"></td><td>measles virus</td><td rowspan="3">nucleocapsid protein, matrix protein, phosphoprotein, fusion protein, haemagglutinin, haemagglutinin- neuraminidase, glycoprotein,</td>

<td>Mumps virus</td>

<td>Parainfluenza virus</td>

<td>Paramyxoviridae</td><td>Respiratory syncytial virus</td><td></td>

<td></td><td>Human metapneumovirus</td><td></td>

<td>Papillomaviridae</td><td>Papillomavirus human (e.g. type 16 and 18)</td><td>E6, E7, capsid antigen</td>

<td>Rhabdoviridae</td><td>Rabies virus</td><td>capsular lipoprotein</td>

<td>Togaviridae</td><td>Rubella virus</td><td>capsid protein</td>

<td>Paroviridae</td><td>Human bokavirus, parvovirus B19</td><td>capsid protein, non-structural protein (NS)</td>

[0076] In other embodiments, the set of surface antigens (e.g. first and / or second sets of surface antigens) may include antigens obtained or obtained from bacterial organisms such as Borrelia species, Bacillus anthracis, Borrelia burgdorferi, Bordetella pertussis, Bordetella Parapertussis, Camphylobacter jejuni, Chlamydia, Chlamydial psittacomatium, Chlamydial species Clostachomatium, Chlamydial species Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Corynebacterium diphtheriae, Coxiella, Enterococcus, Erlichia, Escherichia coli, Francisella tularensis, Haemophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Lactobacillus species, Legionella species, Legionella pneumophila, Leptospirosis interrogans, Listeria species, Listeria monocytogenes, Mycobacterium species, Mycobacterium tuberculosis species, Mycobacterium species, Mycobacterium tuberculosis, Mycobacterium, Mycobacterium species, Mycobacterium, Mycobacterium Neumyculosis, Mycobacterium, Mycobacterium, Mycobacterium, Mycobacterium, Mycobacterium. Neisseria gonorrhoeae, Pneumococcus species (e.g. type 6A, 6B, 3, 4, 14, 19F, etc.), Pseudomonas species, Pseudomonas aeruginosa, Salmonella species, Salmonella typhi, Salmonella enterica, Rickettsia species, Rickettsia ricketsii, Rickettsia typhi, Shigella species, Staphylococcus species, Staphylococcus aureus, Streptococcus species, Streptococcus pnuemoniae, Streptococcus pyrogenes, Streptococcus mutans, Treponema species, Treponema pallidum, Vibrio species, Vibrio cholerae, Yersinia pestis and the like.

[0077] In embodiments, the set of surface antigens (e.g., the first and / or second set of surface antigens) comprises or is derived from or is derived from bacteria of the types of bacteria set forth below in Table 2. In another embodiment, the set of surface antigens (e.g., the first and / or or the second set of surface antigens) comprises or is derived from or derived from the bacterial species shown in Table 2. In yet another embodiment, the set of surface antigens (e.g., the first and / or second set of surface antigens) comprises or is derived from, or is derived from, the antigen set forth in Table 2.

<td>Table 2: Bakers</td><td colspan="2">infectious agents</td>

<td>Pathogenic bacterial agents</td><td>Examples Type</td><td>Exemplary antigens</td>

<td>Bordetella</td><td>Bordetella pertussis</td><td>whooping cough toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN) and fimbriae (FIM 2/3)</td>

<td>Borrelia</td><td>Borrelia burgdorferi</td><td>VlsE; DbpA and OspA</td>

<td rowspan="4">Brucella</td><td>Brucella abortus</td><td>Hia, PrpA, MltA, L7 / L12, D15, 0187, VirJ, Mdh, AfuA</td>

<td>Brucella canis</td><td>L7 / L12</td>

<td>Brucella melitensis</td><td>extra-membrane proteins such as Omp28</td>

<td>Brucella suis</td><td></td>

<td>Campylobacter</td><td>Campylobacter jejuni;</td><td>LPS, 100-kD antigen</td>

<td>Chlamydia and Chlamydophila</td><td>Chlamydia pneumoniae Chlamydia trachomatis Chlamydophila psittaci</td><td>see Richard et al., J. Infectious Diseases. 181: S521 (2000)</td>

<td rowspan="4">Clostridium</td><td>Clostridium botulinum</td><td>types of antigens A, B, C, D and E</td>

<td>Clostridium difficile</td><td>FliC, FliD and Cwp84</td>

<td>Clostridium perfringens</td><td>alpha toxin, theta toxin, fructose 1,6-diphosphate aldolase (FBA), glyceraldehyde-3- dehydrogenase phosphate (GPD), oxidoreductase pyruvate: ferredoxin (PFOR) factor G-elongation (EF-G) and hypothetical protein (HP)</td>

<td>Clostridium tetani</td><td>toxin T.</td>

<td>Corynebacterium</td><td>C oryneb acterium</td><td>toxoid antigen</td>

<td></td><td>diphtheriae</td><td></td>

<td rowspan="2">Enterococcus</td><td>Enterococcus faecalis</td><td rowspan="2">capsular polysaccharides</td>

<td>Enterococcus faecium</td>

<td>Escherichia</td><td>Escherichia coli</td><td>see Moriel et al., PNAS 107 (20): 9072-9077 (2010)</td>

<td>Francisella</td><td>Francisella tularensis</td><td>see Havlasova et al., Proteomics 2 (7): 857-867, 2002</td>

<td>Haemophilus</td><td>Haemophilus influenzae</td><td>capsular polysaccharides, protein D,</td>

<td>Helicobacter</td><td>Helicobacter pylori</td><td>see Bumann et al., Proteomics 4 (10): 2843-2843, 2004</td>

<td>Legionella</td><td>Legionella pneumophila</td><td>Mip</td>

<td>Leptospire</td><td>Leptospira interrogans</td><td>see Brown et al., Infect Immu 59 (5): 1772-1777,1991</td>

<td>Listeria</td><td>Listeria monocytogenes</td><td>nucleoprotein (NP)</td>

<td rowspan="3">Mycob acterium *</td><td>Mycobacterium leprae</td><td></td>

<td>Mycobacterium tuberculosis</td><td>RD1, PE35, PPE68, EsxA, EsxB, RD9 and EsxV</td>

<td>Mycobacterium ulcerans</td><td></td>

<td>Mycoplasma</td><td>Mycoplasma pneumoniae</td><td>Hsp70</td>

<td rowspan="2">Neisseria</td><td>Neisseria gonorrhoeae</td><td></td>

<td>Neisseria meningitidis</td><td>see Litt et al., J. Infectious Disease 190 (8): 1488-1497, 2004</td>

<td>Pseudomonas</td><td>Pseudomonas aeruginosa</td><td>lipopolysaccharides</td>

<td>Rickettsia</td><td>Rickettsia rickettsii</td><td>surface antigen</td>

<td rowspan="2">Salmonella</td><td>Salmonella typhi</td><td></td>

<td>Salmonella typhimurium</td><td></td>

<td>Shigella</td><td>Shigella sonnei</td><td></td>

<td>Staphylococcus</td><td>Staphylococcus aureus</td><td>see Vytvtska et al., Proteomics 2 (5): 580-590,2002; Etz et al., PNAS 99 (10): 6573-6578; 2002</td>

<td rowspan="2"></td><td>Staphylococcus epidermidis</td><td></td>

<td>Staphylococcus saprophyticus</td><td></td>

<td rowspan="3">Streptococcus</td><td>Streptococcus agalactiae</td><td></td>

<td>Streptococcus pneumoniae</td><td>Sp1, Sp2, Sp3</td>

<td>Streptococcus pyogenes</td><td>Lei et al., J. Infectious Disease 189 (1): 79-89, 2004</td>

<td>Treponema</td><td>Treponema pallidum</td><td>glycerophosphodiesterase, phosphodiesterase</td>

<td>Vibrio</td><td>Vibrio cholerae</td><td>outer membrane proteins such as OmpK</td>

<td>Yersinia</td><td>Yersinia pestis</td><td>chaperone-usher protein, envelope protein (F1) and protein V</td>

[0078] In yet other embodiments, the set of surface antigens (e.g. first and / or second sets of surface antigens) may include antigens obtained or obtained from antigens of fungi, protozoa and / or parasitic organisms such as Aspergillus species, Candida species, Candida albicans, Candida tropicalis, Cryptococcus, Cryptococcus neoformans species, amoebiasis, Histoplasma capsulatum, Leishmania species, Nocardia asteroides, Plasmodium falciparum, Toxoplasma gondii, Trichomonas vaginalis, Toxoplasma species, Trypanosoma brucei, Schistosoma mansoni and the like.

In yet other embodiments, the set of surface antigens (e.g., the first and / or second set of surface antigens) may include toxin derived or derived antigens, such as O-Alkyl (&lt;C10, including cycloalkyl) alkyl (Me , Et, n-Pr or i-Pr) -fluorophosphonates (e.g. Sarin: O-isopropyl methylfluorophosphonate, Soman: O-pinacolyl methylfluorophosphonate), O-Alkyl (C10, including cycloalkyl) N, N-dialkyl (Me, Et , n-Pr or i-Pr) cyanophosphates (e.g. tabun: O-ethyl N, N-dimethylamidocyanidophosphate), O-Alkyl (H or &lt;C10, including cycloalkyl) S-2-dialkyl (Me, Et, n-Pr or i-Pr) -aminoethylalkyl (Me, Et, n -Pr or i-Pr) phosphonothiolates and corresponding alkylated or protonated salts (e.g. VX: S-2-diisopropylaminoethyl O-ethyl methylphosphonothiolate), sulfur mustard: 2-chloroethylchloromethyl sulfide, mustard gas: bis (2-chloroethyl) sulfide, bis (2-chloroethylthio) methane, Sesquimustard: 1,2-bisethyl (2-chloro) ethane, 1,3-bis (2-chloroethylthio) n-propane, 1,4-bis (2-chloroethylthio) -n-butane, 1,5-bis (2-chloroethylthio) -n-pentane, bis (2-chloroethylthiomethyl) ether, O-mustard: Bis (2-chloroethylthioethyl) ether, Lewisite: Lewisite 1: 2-chlorovinyl dichloroarsine, Lewisite 2: Bis (2-chlorovinyl) chloroarsine, Lewisite 3: Tris (2-chlorovinyl) arsine, nitrogen mustards: HN1: Bis (2-chloroethyl) ethylamine, HN2: Bis (2-chloroethyl) methylamine, HN3: Tris (2-chloroethyl) amine, saxitoxin, ricin, amiton: O, O-diethyl S (2- (diethylamino) ethyl) phosphorothiolate and corresponding alkylated and protonated salts, PFIB: 1,1,3,3,3-pentafluoro-2- (trifluoromethyl) -1-propene, 3-quinuclidine benzenesulfate (BZ), phosgene: Carbonyl dichloride, cyanogen chloride, hydrogen cyanide and chloropicrin: trichloronitromethane.

[0080] In other embodiments, the set of surface antigens (e.g., the first and / or second set of surface antigens) comprises or is derived from or derived from a fungus of the fungal genera shown below in Table 3. In another embodiment, the set of surface antigens (e.g. and / or the second set of surface antigens) comprises or is obtained from or derived from the fungal species shown in Table 3. In yet another embodiment, the set of surface antigens (e.g., the first and / or second set of surface antigens) comprises or is derived from or derived from the antigen set forth in Table 3.

Table 3: Fungal infectious agents ________________________________________________

<td>Types</td><td>Sample species</td><td>Exemplary antigens</td>

<td>Candida</td><td>C. albicans</td><td>surface antigens, see also: Thomas et al., Proteomics 6 (22): 6033-6041, 2006</td>

<td>Aspergillus</td><td>Aspergillus fumigatus and Aspergillus flavus.</td><td>Stevens et al., Medical Mycology 49 (Suppl. 1): S170-S176, 2011</td>

<td>Cryptococcus</td><td>Cryptococcus neoformans, Cryptococcus laurentii i Cryptococcus albidus, Cryptococcus gattii</td><td>envelope glycoproteins,</td>

<td>Histoplasma</td><td>Histoplasma capsulatum</td><td>Yps3P, Hsp60</td>

<td>Pneumocystis</td><td>Pneumocystis</td><td>major surface proteins (Msg) such as</td>

<td></td><td>jirovecii</td><td>MsgC1, MsgC3, MsgC8 and MsgC9</td>

<td>Stachybotrys</td><td>Stachybotrys chartarum</td><td>SchS34,</td>

[0081] In yet other embodiments, the set of surface antigens (e.g., the first and / or second set of surface antigens) may include antigens derived or derived from an abusive or addictive substance. In some embodiments, the abused or addictive substance is a drug, such as an illegal drug, an over-the-counter drug, or a prescription drug. In other embodiments, the abused or addictive substance has a mood altering effect and thus includes inhalants and solvents. In other embodiments, the abused or addictive substance is a substance that has neither mood altering properties nor intoxicating properties, and thus includes anabolic steroids. Abused or addictive substances include, but are not limited to, cannabinoids (e.g., Hashish, marijuana), depressants (e.g. Barbytes, benodiazepines, flunitrazepam (Rohypnol), GHB, methaqualone (quaaludes)), dissociative anesthetics (e.g. Ketamine, PCP), hallucinogens (e.g. LSD, mescaline, psilocybin), opioids and morphine derivatives, heroine phentanylodeine , morphine, opium), stimulants (amphetamines, cocaine, ecstasy (MDMA), methamphetamine, methylphenidate (Ritalin), nicotine), anabolic steroids and inhalants. In embodiments, the antigen comprises a cocaine analog such as norkocaine. In other embodiments, the antigen comprises cotinine.

[0082] In embodiments of the invention, different populations of synthetic nanocarriers may be combined, each containing a set of surface antigens. The difference between the populations is based on the differences between the sets of surface antigens.

[0083] In certain example embodiments, these differences may include differences in physical or chemical characteristics such as, but not limited to, structural or immunological properties. In embodiments, the differences may include differences in the orientation of the surface antigen or conformation, or differences in the molecular structure between the sets of surface antigens. In yet other embodiments, the difference in surface antigens may be based on the infectious organism from which they are obtained or derived; and will be classified as derived from a different genus, species and / or strain. In embodiments where the surface antigens have a molecular weight of less than 10,000, the surface antigens can vary based on chemical classes such as chemical warfare agents, addictive substances, or abused and endogenous molecules including, but not limited to, hormones. lipids and neurotransmitters.

[0084] In embodiments, the differences may include differences in the orientation of the surface antigen or conformation. For example, different points of attachment of the surface antigen to the synthetic nanocarrier would result in different

-28 representations of this surface antigen. These different presentations can generate antibodies that recognize different epitopes of the surface antigen. The surface antigens can be presented in a variety of conformations and can be synthesized or modified to achieve these conformations. For example, truncation of peptides or proteins can be performed, resulting in modified conformational changes in the peptide or protein antigen of interest. Alternatively, amino acids or chemical linkers may be added to increase the length or stabilize a particular orientation which changes the exposure of the peptide or protein antigen. Likewise, antigens, such as those having a molecular weight of less than 10,000, or oligosaccharides or polysaccharides, may be altered by adding a chemical linker or by chemical modification.

[0085] In other embodiments, differences between the populations of synthetic nanocarriers can be based on differences between the sets of surface antigens based on different molecular structures and / or the frequency of the antigens. In some embodiments, the difference may include a difference in the frequency of one or more types of surface antigens between the sets.

[0086] In embodiments where a population comprises a monovalent set of surface antigens, the molecular structure, preferably the antigen type, of that set of surface antigens may differ from the molecular structure of the set of monovalent surface antigens of another population or of multiple other populations. In some embodiments where a population comprises a monovalent set of surface antigens, the frequency of the surface antigens making up its set of surface antigens may be different from the frequency of the surface antigens making up the set of monovalent surface antigens of another population or many others. population.

[0087] In embodiments where the population of synthetic nanocarriers comprises a set of oligovalent (or polyvalent) surface antigens, different types of antigens at different frequencies may be combined into a joint to form combinations of such types of surface antigens. Accordingly, in embodiments where the at least one population of synthetic nanocarriers comprises a set of oligovalent (or polyvalent) surface antigens, the molecular structure of a set of oligovalent (or polyvalent) surface antigens (which can be expressed as a function of both the molecular structure of each antigen type along with their occurrence within the set) may differ from the molecular structure of the set of surface antigens of another population or other populations. In embodiments, this may be because another population contains a monovalent set of surface antigens (the sets of surface antigens will by definition be different) or because another population comprises a set of oligovalent (or polyvalent) surface antigens in which the molecular structures of the two sets antigens

The surfaces (expressed as the molecular structure of each antigen type and / or the frequency of each antigen type within the kit) are different.

[0088] For example, sets of surface antigens may consist of a set of enantiomers such as nicotine (R) and (S). Enantiomers may be present in equal amounts on the same or different nanocarriers, and may be present in unequal amounts on the same or different populations of synthetic nanocarriers. In another embodiment, the set of surface antigens may contain two structurally different but related molecules such as cotinine and nicotine, optically pure or racemic. Cotinine and nicotine may be present in equal amounts on the same or different populations of synthetic nanocarriers, and may be unequally present in the same or different populations of synthetic nanocarriers. In addition, sets of surface antigens may be composed of antigens from a single organism composed of several serotypes such as polysaccharides of envelope antigens from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F of Streptococcus Pneumoniae. Different antigens may be present in equal amounts on the same or different populations of synthetic nanocarriers, and may be unequally present in the same or different populations of synthetic nanocarriers. In embodiments, the sets of surface antigens may comprise a family of different antigens from a single organism, such as the human papillomavirus L1 and L2 capsid proteins. Sets of surface antigens may be present in equal amounts on the same or different populations of synthetic nanocarriers, and may be unequally present in the same or different populations of synthetic nanocarriers. In embodiments, the sets of surface antigens may contain several small molecules with different structures, such as VX war gases, sarin, and soman. Different compounds may be present in equal amounts on the same or different populations of synthetic nanocarriers, and may be present in unequal amounts in the same or different populations of synthetic nanocarriers. For example, in an embodiment, one set of surface antigens may include 50% VX and 50% sarin, while another set of surface antigens may include 80% VX and 20% sarin, where the percentage of the surface antigens may be weight percent or mole percent based on the total mass or total number of moles of surface antigens.

[0089] In embodiments, differences in the sets of surface antigens may include providing a population of synthetic nanocarriers that comprises a set of surface antigens of a genus or types, such as with a molecular weight of less than 10,000 and / or peptides, proteins, oligosaccharides. polysaccharides or small molecules; and then providing a different population of synthetic nanoparticles containing a different set of surface antigens of a genus or types, such as having a molecular weight of less than 10,000 and / or being peptides, proteins, oligosaccharides, polysaccharides or small molecules.

[0090] In embodiments, where the first set of surface antigens comprises surface antigens with a molecular weight less than 10,000, the second

The set of surface antigens includes peptides, proteins, oligosaccharides, polysaccharides, or small molecules (provided the sets are structurally or immunologically different). In embodiments, where the first set of surface antigens comprises surface antigens including peptides, the second set of surface antigens comprises surface antigens including those with a molecular weight of less than 10,000 and / or including proteins, oligosaccharides, polysaccharides or small molecules. In embodiments, where the first set of surface antigens comprises surface antigens including proteins, the second set of surface antigens comprises surface antigens including those with a molecular weight of less than 10,000 and / or including peptides, oligosaccharides, polysaccharides or small molecules. In embodiments where the first set of surface antigens comprises surface antigens including oligosaccharides, the second set of surface antigens comprises surface antigens including those with a molecular weight of less than 10,000 and / or including peptides, proteins, polysaccharides or small molecules. In embodiments, when the first set of surface antigens comprises surface antigens including polysaccharides, the second set of surface antigens comprises surface antigens including those with a molecular weight of less than 10,000 and / or including peptides, proteins, oligosaccharides or small molecules. In embodiments where the first set of surface antigens includes small molecule surface antigens, the second set of surface antigens includes surface antigens including those with a molecular weight of less than 10,000 and / or including peptides, proteins, oligosaccharides or polysaccharides (as long as the sets differ structurally or immunologically).

[0091] In embodiments, where the second set of surface antigens comprises surface antigens with a molecular weight less than 10,000, the first set of surface antigens includes peptides, proteins, oligosaccharides, polysaccharides or small molecules (provided the sets are structurally or immunologically different) . In embodiments, when the second set of surface antigens comprises surface antigens including peptides, the first set of surface antigens comprises surface antigens including those with a molecular weight of less than 10,000 and / or including proteins, oligosaccharides, polysaccharides or small molecules. In embodiments where the second set of surface antigens comprises surface antigens including proteins, the first set of surface antigens comprises surface antigens including those with a molecular weight of less than 10,000 and / or including peptides, oligosaccharides, polysaccharides or small molecules. In embodiments, where the second set of surface antigens comprises oligosaccharide-containing surface antigens, the first set of surface antigens comprises surface antigens including those with a molecular weight of less than 10,000 and / or containing peptides, proteins, polysaccharides or small molecules. In embodiments where the second set of surface antigens comprises polysaccharide-containing surface antigens, the first set of surface antigens comprises surface antigens including those of molecular weight

-31 of less than 10,000 and / or containing peptides, proteins, oligosaccharides or small molecules. In embodiments, where the second set of surface antigens comprises small molecule surface antigens, the first set of surface antigens includes surface antigens including those with a molecular weight of less than 10,000 and / or including peptides, proteins, oligosaccharides or polysaccharides (as long as the sets differ structurally or immunologically).

[0092] Other differences between the populations of synthetic nanocarriers may be based on differences between the sets of surface antigens based on the source of the antigens. For example, in an embodiment, such differences may be based on differences in infectious genera, species, and / or strains from which the surface antigens were obtained or derived. For example, one population of synthetic nanocarriers may contain a set of surface antigens obtained or derived from a bacterial source such as E. coli, Mycobacterium tuberculosis, Clostridium tetani, or Bacillus anthracis, while another population may contain a set of surface antigens obtained or derived from a viral source. such as influenza virus, hepatitis B virus, hepatitis C virus and human herpes virus. In embodiments, the synthetic nanocarrier population may contain a set of surface antigens obtained or derived from a bacterial source, such as those listed above, while another population may contain a set of surface antigens obtained or derived from fungi, such as Candida albicans, or Pneumocystis jiroveci. In other embodiments, one population of synthetic nanocarriers may contain a set of surface antigens obtained or derived from a viral source, while another population may contain a set of surface antigens obtained or derived from parasites, such as Plasmodium falciparum. The other combinations and component combinations shown in the above figures are considered to be within the scope of the invention.

[0093] In other embodiments, different sets of surface antigens can be obtained or obtained from infectious genera, species, or strains that are not dissimilar. In embodiments, these differences may result from the selection of different antigens within the infectious genus, species, or strain. For example, one set of surface antigens may be obtained or may be derived from one viral envelope protein, while a different set of surface antigens may be obtained or obtained from different epitopes of the same or a different viral coat protein of the same virus. In one embodiment, different sets of surface antigens may be obtained or may be derived from one viral protein, for example cytomegalovirus (CMV) capsid protein or other CMV proteins, which may contain several different epitopes. Likewise, different sets of surface antigens may be obtained or may be derived from different epitopes of diphtheria or tetanus toxin.

[0094] In other embodiments, differences between the populations of synthetic nanocarriers may be based on differences between the sets of surface antigens based on different chemical classes of antigens. For example, in the case of

For molecules with a molecular weight less than 10,000, these differences may be based on differences in the chemical backbone or the overall molecular structure or activity exhibited by such molecules. For example, sets of different surface antigens can be obtained or can be derived from sets of surface antigens with different structures but similar activities, such as opiates such as morphine and heroin. In other embodiments, the different sets of surface antigens may be composed of molecules with similar structures but with different activities as exemplified by enantiomers such as Ritalin (R) and (S) or nicotine (R) and (S). In embodiments, the difference between the sets of surface antigens may include compounds and their metabolites such as terfenadine and fexofenadine or astemazole and norastemazole. The differences between the sets of surface antigens may also be based on the unrelated structure of compounds such as nicotine and methamphetamine.

[0095] In other embodiments, differences between the populations of synthetic nanocarriers may be based on differences between the sets of surface antigens based on immunological differences between the sets of surface antigens. In embodiments, sets of surface antigens can be defined by their ability to elicit an immune response in vivo. For example, one set of surface antigens may be capable of inducing a high level of production of high affinity antibodies for an antigen of interest in vivo, while a second set of surface antigens may not induce high levels of production of high affinity antibody in vivo to that antigen. As another example, the first set of surface antigens may be capable of generating antigen-specific antibody titers from the first set of surface antigens, while the second set of surface antigens may be capable of generating antigen-specific antibody titers of the second set of surface antigens. In embodiments, the second set of surface antigens generate antibody titers specific for the antigens of the second set of surface antigens, but not for the antigens of the first set.

COMPOSITIONS OF THE INVENTION AND RELATED METHODS

[0096] Synthetic nanocarriers can be made using a wide variety of methods known in the art. For example, synthetic nanocarriers can be produced by methods such as nanoprecipitation, concentration of the flow through flow channels, spray drying, single and dual emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, microprocessing, nanoprocessing, loss layers, simple and complex coacervation and other methods well known to those skilled in the art. Alternatively or additionally, the synthesis of aqueous and organic solvents for monodisperse semiconductor, conductive, magnetic, organic and other nanomaterials has been described (Pellegrino et al., 2005, Small, 1:48, Murray et al., 2000, Ann. Rev. Mat. Sci. , 30: 545;

-33i Trindade et al., 2001, Chem. Matt. 13: 3843). Additional methods have been described in the literature (see, e.g., Doubrow, Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6: 275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35: 755, as well as US Patents 5,578,325 and 6,007,845; P. Paolicelli et al., "Surfacemodified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5 (6): 843-853 (2010)).

[0097] In embodiments, the invention comprises synthetic nanocarriers for displaying sets of surface antigens, preferably sets of monovalent or oligovalent surface antigens. A particular embodiment of the invention comprises a first synthetic nanocarrier agent for presenting a first set of surface antigens, preferably a first set of monovalent or oligovalent surface antigens; and a second synthetic nanocarrier for displaying a second set of surface antigens; preferably a second set of monovalent or oligovalent surface antigens. Such synthetic nanocarriers for displaying surface antigens are disclosed throughout the disclosure and include the embodiments disclosed herein.

[0098] Various materials can be encapsulated in synthetic nanocarriers as desired using a variety of methods, including, but not limited to, C. Astete et al., "Synthesis and characterization of PLGA nanoparticles," J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K. Avgoustakis "Pegylated Poly (Lactide) and Poly (Lactide-CoGlycolide) Nanoparticles: Preparation, Properties and Possible Applications in Drug Delivery" Current Drug Delivery 1: 321-333 (2004); C. Reis et al., "Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles," Nanomedicine 2: 8-21 (2006); P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5 (6): 843-853 (2010). Other methods suitable for encapsulating materials such as oligonucleotides into synthetic nanocarriers can be used, including, but not limited to, the methods described in United States Patent 6,632,671 to Unger on October 14, 2003.

[0099] In some embodiments, the synthetic nanocarriers are made by a nanoprecipitation or spray-drying process. The conditions used in the preparation of synthetic nanocarriers can be varied to provide particles of the desired size or properties (eg, hydrophobicity, hydrophilicity, external morphology, "stickiness", shape, etc.). The method of obtaining synthetic nanocarriers and the conditions used (e.g. solvent, temperature, concentration, air flow rate etc.) May depend on the materials to be coupled with the synthetic nanocarriers and / or the polymer matrix composition.

[0100] If the particles produced by any of the above methods have a size range outside the desired range, the particles can be sized by, for example, using a sieve, differential centrifugation, or sedimentation.

The elements of the synthetic nanocarriers of the invention, such as the moieties of the immunogenic surface, targeting molecules, polymer matrices, antigens, adjuvants, and the like, may be coupled to a synthetic nanocarrier, e.g., one or more covalent bonds or they may be linked using one or more linkers. Additional methods of functionalizing synthetic nanocarriers can be adapted from published patent application US 2006/0002852 Salt-zman et al., Published patent application US 2009/0028910 of DeSimone et al. Or published international patent application WO / 2008/127532 A1 by Murthy et al.

[0102] Alternatively or additionally, synthetic nanocarriers may be coupled to moieties that include the immunogenic surface, targeting moieties, adjuvants, antigens, and / or other elements directly or indirectly through non-covalent interactions. In non-covalent embodiments, non-covalent conjugation is mediated by non-covalent interactions, including, but not limited to, charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stack interactions, hydrogen bonding interactions. van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and / or combinations thereof. Such couplings may be located on the outer surface or inner surface of the synthetic nanocarrier of the invention. In embodiments, the encapsulation and / or absorption is a conjugation form.

[0103] A wide variety of synthetic nanocarriers can be used in the present invention. In some embodiments, the synthetic nanocarriers are spheres or spheroids. In some embodiments, the synthetic nano-carriers are flat or plate-shaped. In some embodiments, the synthetic nanocarriers are cubes or cuboid. In some embodiments, the synthetic nanocarriers are ovals or ellipses. In some embodiments, the synthetic nanocarriers are cylinders, cones, or pyramids.

[0104] In some embodiments, it is desirable to use a population of synthetic nanocarriers that is relatively uniform in size, shape, and / or composition such that each synthetic nanocarrier has similar properties. For example, at least 80%, at least 90%, or at least 95% of synthetic nanocarriers, based on the total number of synthetic nanocarriers, may have a minimum size or a maximum dimension that is within 5%, 10%, or 20% of the average diameter or the average size of synthetic nanocarriers. In some embodiments, the population of synthetic nanocarriers can be heterogeneous in size, shape, and / or composition.

[0105] Synthetic nanocarriers can be solid or hollow and can include one or more layers. In some embodiments, each layer has a unique composition and unique properties with respect to the other layer (s). Giving just one

For example, synthetic nanocarriers can have a core / sheath structure, the core being one layer (e.g., polymer core) and the shell being the second layer (e.g., lipid bilayer or monolayer). Synthetic nanocarriers can include many different layers.

[0106] In some embodiments, synthetic nanocarriers may optionally include one or more lipids. In some embodiments, the synthetic nanocarrier may include a liposome. In some embodiments, the synthetic nanocarrier may include a lipid bilayer. In some embodiments, a synthetic nanocarrier may include a lipid monolayer. In some embodiments, the synthetic nanocarrier may contain micelles. In some embodiments, a synthetic nanocarrier may comprise a core comprising a polymer matrix surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In some embodiments, a synthetic nanocarrier may comprise a non-polymeric core (e.g., metal particle, quantum dot, ceramic particle, bone particle, viral particle, proteins, nucleic acids, carbohydrates, etc.) surrounded by a lipid layer (e.g. lipid bilayer, lipid monolayer, etc.).

[0107] The synthetic nanocarriers in the first and second populations, and optionally in each additional population, include one or more polymers. In some embodiments, such a polymer may be surrounded by a coating layer (e.g., liposome, lipid monolayer, micelle, etc.). In some embodiments, various elements of the synthetic nanocarriers can be coupled to the polymer.

[0108] In some embodiments, the immunoglobulin surface, targeting molecule, antigens, adjuvants, and / or the oligonucleotide may be covalently linked to a polymer matrix. In some embodiments, the covalent bond is mediated by a linker. In some embodiments, the immunogenic surface, targeting moiety, antigens, adjuvants, and / or the oligonucleotide may be non-covalently associated with the polymer matrix. For example, in some embodiments, the immunogenic surface, targeting moiety, antigens, adjuvants, and / or oligonucleotides may be encapsulated, surrounded, and / or dispersed within the polymer matrix. Alternatively or additionally, the immunoglobulin surface, targeting moiety, antigens, adjuvants, and / or nucleotides may be bound to the polymer matrix through hydrophobic interactions, charge interactions, van der Waals forces, etc.

[0109] A wide variety of polymers and methods of making polymer matrices therefrom are conventionally known. Generally, the polymer matrix comprises one or more polymers. The polymers can be natural or non-natural (synthetic) polymers. The polymers can be homopolymers or copolymers containing two or more monomers. In terms of sequence, the copolymers can be random, block or contain a combination of random and block sequences. Typically the polymers of the invention are organic polymers.

Examples of polymers suitable for use in the invention include, but are not limited to, polyethylenes, polycarbonates (e.g., poly (1,3-dioxan-2-one)), polyanhydrides (e.g., poly (sebacic anhydride)), polypropyl fumarates , polyamides (e.g. polycaprolactam), polyacetals, polyethers, polyesters (e.g. polylactide, polyglycolide, polylactide-co-glycolide, polycaprolactone, polyhydroxy acids (e.g. poly (P-hydroxyalkanoate)), poly (orthoesters), polycyanoacrylates, poly (vinyl alcohols), polyurethanes polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, polyamines, polylysines, polylysine-PEG polymers, poly (ethyleneimine), poly - ethyleneimine PEG-copolymers and polyphosphazines.

[0111] In some embodiments, the polymers of the invention include polymers that have been approved for human use by the U.S. Food and Drug Administration (FDA) under 21 CFR § 177 2600, including, but not limited to, polyesters (e.g. polylactic acid, poly (lactic-co-glycolic acid), polycaprolactone, polyolactone, poly (1,3-dioxan-2-one)); polyanhydrides (e.g. poly (sebacic anhydride)); polyethers (e.g. poly (ethylene glycol)); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.

[0112] In some embodiments, the polymers may be hydrophilic. For example, the polymers may contain anionic groups (e.g., a phosphate group, a sulfate group, a carboxyl group); cationic groups (e.g., quaternary amine); or polar groups (e.g., hydroxyl, thiol, amino). In some embodiments, a synthetic nano-carrier comprising a hydrophilic polymer matrix creates a hydrophilic environment within the synthetic nano-carrier. In some embodiments, the polymers may be hydrophobic. In some embodiments, a synthetic nano-carrier comprising a hydrophobic polymer matrix creates a hydrophobic environment in the synthetic nano-carrier. The choice of the hydrophilicity or hydrophobicity of the polymer can affect the nature of the materials that are incorporated (e.g., coupled) into the synthetic nanocarrier.

[0113] In some embodiments, the polymers may be modified with one or more residues and / or functional groups. Various moieties or functional groups may be used in the invention. In some embodiments, the polymers can be modified with polyethylene glycol (PEG), carbohydrate, and / or acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786: 301). Certain embodiments can be made using the general information of US Patent 5,543,158 to Gref et al. Or WO2009 / 051837 of Von Andriana et al.

[0114] In some embodiments, the polymers can be modified with a lipid group or a fatty acid. In some embodiments, the fatty acid group can be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid. In some embodiments, the fatty acid group can be one or more of palmitic acid, oleic acid, vaccenic acid, linoleic acid, alpha-linoleic acid, gamma-linoleic acid, arachidonic acid,

-37gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic or erucic.

[0115] In some embodiments, the polymers can be polyesters, including copolymers containing lactic acid and glycolic acid units such as poly (lactic acid-co-glycolic acid) and poly (lactide-co-glycolide), collectively referred to herein as "PLGA "; and homopolymers containing glycolic acid units, referred to herein as "PGA", and lactic acid units such as poly-L-lactic acid, poly-D-lactic acid, poly-D, L-lactic acid, poly-L-lactide, poly-D -lactide and poly-D, L-lactide, collectively referred to herein as "PLA". In some embodiments, exemplary polyesters include, for example, polyhydroxy acids; PEG copolymers and copolymers of lactide and glycolide (e.g. PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG copolymers and their derivatives). In some embodiments, the polyesters include, for example, poly (caprolactone), poly (caprolactone) -PEG copolymers, poly (L-lactide-co-L-lysine), poly (serine ester), poly (4-hydroxy-L ester) -proline), poly [α (4-aminobutyl) -L-glycolic acid] and their derivatives.

[0116] In some embodiments, the polymer may be PLGA. PLGA is a biocompatible and biodegradable copolymer of lactic acid and glycolic acid, and the various forms of PLGA are characterized by their lactic acid: glycolic acid ratio. The lactic acid can be L-lactic acid, D-lactic acid, or D, L-lactic acid. The rate of degradation of PLGA can be controlled by changing the ratio of lactic acid to glycolic acid. In some embodiments, the PLGA for use according to the invention is characterized by a lactic acid: glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60. approximately 25:75 or approximately 15:85.

[0117] In some embodiments, the polymers can be one or more acrylic polymers. In some embodiments, acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly (acrylic acid), poly (methacrylic acid), alkylamide (methacrylic acid, poly (methacrylic acid) copolymer. methyl methacrylate), poly (methacrylic acid anhydride), methyl methacrylate, polymethacrylate, poly (methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations containing one or more of the above polymers. The acrylic polymer may contain fully polymerized copolymers of acrylic and methacrylic esters with a low content of quaternary ammonium groups.

[0118] In some embodiments, the polymers may be cationic polymers. Generally, cationic polymers are capable of condensing and / or protecting negatively charged strands of nucleic acids (e.g., DNA or derivatives thereof). Amine-containing polymers such as poly (lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6: 7), poly (ethyleneamine) ) (PEI; Boussif et al., 1995, Proc.

-38Natl. Acad. Sci., US, 1995, 92: 7297) and poly (amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., US, 93: 4897; Tang et al., 1996, Bioconjugate Chem., 7: 703; and Haensler et al., 1993, Bioconjugate Chem., 4: 372) are positively charged at physiological pH, form ion pairs with nucleic acids, and mediate transfection in various cell lines. In embodiments, the synthetic nanocarriers of the invention may be free (or may exclude) cationic polymers.

[0119] In some embodiments, the polymers can be degradable polyesters having cationic side chains (Putnam et al., 1999, Macromolecules, 32: 3658, Barrera et al., 1993, J. Am. Chem. Soc., 115: 11010 ; Kwon et al., 1989,

Macromolecules, 22: 3250; Lim et al., 1999, J. Am. Chem. Soc., 121: 5633; and Zhou et al., 1990, Macromolecules, 23: 3399). Examples of these polyesters include poly (L-lactide-co-lysine) (Barrera et al., 1993, J. Am. Chem. Soc., 115: 11010), poly (serine ester) (Zhou et al., 1990, Macromolecules , 23: 3399), poly (4-hydroxy-L-proline) ester (Putnam et al., 1999, Macromolecules, 32: 3658 and Lim et al., 1999, J. Am. Chem. Soc., 121: 5633) and poly (4-hydroxy-L-proline) ester (Putnam et al., 1999, Macromolecules, 32: 3658 and Lim et al., 1999, J. Am. Chem. Soc., 121 : 5633).

[0120] The properties of these and other polymers and their methods of making them are well known in the art (see, for example, U.S. Patents 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,66500; 5,399,665; 5,399,175; 5,514,378; 5,512,66500; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123: 9480; Lim et al., 2001, J. Am. Chem. Soc., 123: 2460 ; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62: 7; and Uhrich et al., 1999, Chem. Rev. 99: 3181). More generally, various methods of synthesizing certain suitable polymers are described in the Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Red. Goethals, Pergamon Press, 1980; Principles of Polymerization by Odiana, John Wiley &amp; Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390: 386; and in U.S. Patent Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

[0121] In some embodiments, the polymers can be linear or branched polymers. In some embodiments, the polymers may be dendrimers. In some embodiments, the polymers may be substantially cross-linked. In some embodiments, the polymers may be substantially crosslink free. In some embodiments, polymers can be used according to the invention without going through a cross-linking step. It should further be understood that the synthetic nanocarriers of the invention may include block copolymers, graft copolymers, blends, mixtures and / or adducts of any of the foregoing and other polymers. Skilled artisans will appreciate that the listed polymers are an exemplary, non-exhaustive list of polymers that may be useful in the present invention.

[0122] In some embodiments, synthetic nanocarriers include one or more polymers. Polymeric synthetic nanocarriers can therefore also include these

As described in WO2009 / 051837 of Von Andriana et al. Including, but not limited to, one or more hydrophilic components. Preferably, the one or more polymers include a polyester, such as poly (lactic acid), poly (glycolic acid), poly (lactic acid-glycolic acid), or polycaprolactone. More preferably, the one or more polymers comprises or additionally comprises a polyester conjugated with a hydrophilic polymer such as a polyether. In embodiments, the polyether comprises poly (ethylene glycol). Even more preferably, the one or more polymers include polyester and a polyester conjugated with a hydrophilic polymer such as a polyether. In other embodiments, the one or more polymers are conjugated with one or more antigens and / or one or more adjuvants. In embodiments, at least a portion of the polymers are conjugated to the antigen (s), and / or at least a portion of the polymers are conjugated to the adjuvant (s). Preferably, when more than one type of polymer is present, one type of polymer is conjugated to the antigen (s). In embodiments, one of the other types of polymer is conjugated with the adjuvant (s). For example, in embodiments where the nanocarriers include polyester and polyester coupled to a hydrophilic polymer such as polyether, the polyester is coupled to an adjuvant, while the polyester coupled to a hydrophilic polymer such as polyether is coupled to the antigen (s). In embodiments where the nanocarriers contain a universal T cell antigen, such as a helper T cell antigen, the universal T cell antigen may be encapsulated in the nanocarrier.

[0123] In some embodiments, the synthetic nanocarriers do not contain a polymer component. In some embodiments, synthetic nanocarriers can include metal particles, quantum dots, ceramic particles, and the like. In some embodiments, the non-polymeric synthetic nanocarrier is an aggregate of non-polymeric components, such as an aggregate of metal atoms (e.g., gold atoms).

[0124] In some embodiments, synthetic nanocarriers may optionally contain one or more amphiphilic units. In some embodiments, the amphiphilic unit can promote the production of synthetic nanocarriers with increased stability, improved homogeneity, or increased viscosity. In some embodiments, the amphiphilic units can be bonded to the inner surface of a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Many amphiphilic units known in the art are suitable for use in making the synthetic nanocarriers of the invention. Such amphiphilic units include, but are not limited to, phosphoglycerides; phosphatidylcholine;

dipalmitoylphosphatidylcholine (DPPC); dioleylphosphytidylethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanodecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; diglycerides of fatty acids; fatty acid amides; sorbitan trioleate (Span®85); glycocholate; sorbitan monolaurate (Span®20); polysorbate 20 (Tween®20);

-40 Polysorbate 60 (Tween®60); polysorbate 65 (Tween® 65); polysorbate 80 (Tween®80); polysorbate 85 (Tween®85); polyoxyethylene monostearate; surfactin; poloxomer; a sorbitan fatty acid ester such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol, sphingomyelin; phosphatidylethanolamine (kephalin); cardiolipin; phosphatidic acid; cerebrosides; diketylphosphate;

dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine; hexadecyl amine; acetyl palmitate; glycerol ricinoleate; hexadecyl stearate; isopropyl myristate; tyloxapol; poly (ethylene glycol) 5000-phosphatidylethanolamine; poly (ethylene glycol) 400 monostearate; phospholipids; synthetic and / or natural detergents with high surfactant properties; deoxycholates; cyclodextrins; chaotropic salts; ion evaporation agents; and their combinations. The component of the amphiphilic unit may be a mixture of different amphiphilic units. Skilled artisans will appreciate that this is an exemplary, non-exhaustive list of substances with surfactant activity. Each amphiphile unit can be used to produce synthetic nanocarriers for use in the present invention.

[0125] In some embodiments, synthetic nanocarriers may optionally include one or more carbohydrates. Carbohydrates can be natural or synthetic. The carbohydrate may be a derivatized natural carbohydrate. In some embodiments, the carbohydrate comprises a monosaccharide or disaccharide, including, but not limited to, glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellobiose, mannose, xylose, arabinose, glucuronic acid, glucuronic acid, galacturonic acid. . , galatosamine and neuraminic acid. In some embodiments, the carbohydrate is a polysaccharide, including, but not limited to, pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxy cellulose (HC), methyl cellulose (MC), dextran, cyclodextran, glycogen, glycogen, glycogen, glycerolemoscopic , chitosan, N, carboxylmethylchitosan, alginine and alginic acid, starch, chitin, inulin, konjac ("weirdo"), glucan, pustulan, heparin, hyaluronic acid, curdlan and xanthan. In embodiments, the synthetic nanocarriers of the invention do not contain (or specifically exclude) carbohydrates such as a polysaccharide. In some embodiments, the carbohydrate may contain a carbohydrate derivative such as a sugar alcohol including, but not limited to, mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.

[0126] The compositions of the invention contain the synthetic nanocarriers of the invention, in combination with pharmaceutically acceptable excipients such as preservatives, buffers, saline or phosphate buffered saline. The compositions can be prepared using conventional manufacturing and mixing techniques to provide useful dosage forms. In one embodiment, the synthetic nanocarriers of the invention are suspended in a sterile saline injection together with a preservative.

[0127] In embodiments, when preparing synthetic nanocarriers as carriers for antigens and / or adjuvants for use in vaccines, methods for conjugating antigens and / or adjuvants to synthetic nanocarriers may be useful. If the antigen and / or adjuvant is a small molecule, it may be advantageous to attach the antigen and / or adjuvant to the polymer prior to assembling the synthetic nanocarriers. In embodiments, it may also be advantageous to produce synthetic nanocarriers with surface groups that are used to couple the antigen and / or adjuvant to the synthetic nanocarrier by using these surface groups instead of attaching the antigen and / or adjuvant to the polymer, and then using this polymer conjugate in construction of synthetic nanocarriers.

[0128] Surface antigens can be coupled to synthetic nanocarriers in a variety of ways. In embodiments, the surface antigen is covalently or non-covalently coupled to the outer surface of the synthetic nanocarrier.

[0129] In some embodiments, the coupling may be through a covalent linker. In embodiments, the surface antigens and / or adjuvants of the invention can be covalently coupled to the outer surface using a 1,2,3-triazole linker formed by a 1,3-dipolar cycloaddition reaction of azide groups on the nanocarrier surface with the surface antigens and / or adjuvants containing the group alkyne or 1,3-dipolar cycloaddition reaction of alkynes on the nanocarrier surface with surface antigens and / or azide-containing adjuvants. Such cycloaddition reactions are preferably performed in the presence of a Cu (I) catalyst together with a suitable Cu (I) ligand and a reducing agent to reduce the Cu (II) compound to the active catalytic Cu (I) compound. This Cu (I) catalyzed azide-alkyne cycloaddition (CuAAC) may also be referred to as a "click" reaction.

[0130] Additionally, the covalent coupling may include a covalent linker that includes an amide linker, a disulfide linker, a thioether linker, a hydrazone linker, a hydrazide linker, an imine or oxime linker, a urea or thiourea linker, an amidine linker, an amine linker, and a sulfonamide linker.

[0131] The amide linker is formed by an amide bond between an amine on one component, such as a peptide, and a carboxylic acid group on a second component, such as a nanocarrier. The amide bond in the linker can be formed using any of the conventional amide bond formation reactions with suitably protected amino acids or peptides and an activated carboxylic acid such as an N-hydroxysuccinimide activated ester.

[0132] The disulfide linker is produced by forming a disulfide (SS) bond between two sulfur atoms in the form of, for example, R1-SS-R2. A disulfide bond can be formed by thiol exchange of thiol / mercaptan (-SH) containing surface antigens and / or adjuvants with another activated thiol group on

- A group of polymer or nano-carrier or nano-carrier containing thiol / mercaptan groups with an antigen and / or adjuvants containing an activated thiol group.

[0133] Triazole linker, specifically the 1,2,3-form triazole

<img file="PL2575876T3_D0001.tif" />

wherein R 1 and R 2 may be any chemical unit resulting from a 1,3-dipolar cycloaddition reaction of an azide attached to a first component such as a nanocarrier with a terminal alkyne attached to a second component such as an antigen and / or adjuvant. The 1,3-dipolar cycloaddition reaction is carried out with or without a catalyst, preferably a Cu (I) catalyst, which combines the two components via the 1,2,3-triazole function. This chemistry has been described in detail by Sharpless et al., Angew. Chem. Int. Ed. 41 (14), 2596, (2002) and Meldal, et al., Chem. Rev., 2008, 108 (8), 2952-3015 and is often referred to as the click reaction or CuAAC.

[0134] In embodiments, a polymer is prepared containing an azide or alkyne group terminal to the polymer chain. This polymer is then used to prepare a synthetic nanocarrier such that multiple alkyne or azide groups are disposed on the surface of the nanocarrier. Alternatively, the synthetic nanocarrier may be prepared by some other route and then functionalized with alkyne or azide groups. The antigen and / or adjuvant is prepared in the presence of an alkyne (if the polymer contains an azide) or an azide (if the polymer contains an alkyne group). The antigen and / or adjuvant is then reacted with the nanocarrier via a 1,3-dipolar cycloaddition reaction with or without a catalyst that covalently binds the antigen and / or adjuvant to the molecule via a 1,4-disubstituted 1,2,3-triazole linker.

[0135] The thioether linker is prepared by forming a sulfur-carbon bond (thioether) in the form of, for example, R1-S-R2. The thioether can be prepared either by alkylating a thiol / mercaptan (-SH) group on one component, such as an antigen and / or an adjuvant, with an alkylating group, such as a halide or epoxide, on a second component, such as a nanocarrier. Thioether linkers can also be made by Michael addition of a thiol / mercaptan group on one component, such as an antigen and / or adjuvant, to an electron-deficient alkene group on a second component, such as a polymer containing a maleimide group or a vinyl sulfone group, as a Mchael acceptor. Alternatively, thioether linkers may be made by radically reacting a thiol / mercaptan group on one component, such as an antigen and / or an adjuvant with an alkene group on the other component, such as a polymer or nanocarrier.

[0136] The hydrazone linker is formed by reacting a hydrazide group on one component, such as an antigen and / or an adjuvant, with an aldehyde / ketone group on another component, such as a nanocarrier.

[0137] A hydrazide linker is formed by reacting a hydrazine group on one component, such as an antigen and / or an adjuvant, with a carboxylic acid group on another component, such as a nanocarrier. This reaction is generally performed using chemistry similar to amide bond formation in which the carboxylic acid is activated with an activating reagent.

[0138] An imine or oxime linker is formed by reacting an amino or Nalkoxyamino (or aminooxy) group on one component such as an antigen and / or an adjuvant with an aldehyde or ketone group on a second component such as a nanocarrier.

[0139] The urea or thiourea linker is prepared by reacting an amino group on one component such as an antigen and / or adjuvant with an isocyanate or thioisocyanate group on a second component such as a nanocarrier.

[0140] The amidine linker is formed by reacting an amino group on one component such as an antigen and / or an adjuvant with an imide ester group on another component such as a nanocarrier.

[0141] The amine linker is formed by an alkylation reaction of an amino group on one component, such as an antigen and / or an adjuvant, with an alkylating group, such as a halide, epoxy, or sulfonate group, on a second component, such as a nanocarrier. Alternatively, the amine linker can also be prepared by reductive amination of an amine group on one component, such as an antigen and / or an adjuvant with an aldehyde or ketone group on a second component, such as a nanocarrier, with a suitable reducing reagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.

[0142] The sulfonamide linker is prepared by reacting an amino group on one component, such as an antigen and / or adjuvant, with a sulfonyl halide (such as sulfonyl chloride or sulfonyl fluoride) on a second component, such as a nanocarrier. The sulfone linker is produced by Michael addition of a nucleophile to a vinyl sulfone. The vinyl sulfone or nucleophile can be on the surface of the nanoparticle or attached to an antigen or adjuvant.

[0143] The antigen or adjuvant can also be coupled to the nanocarrier using non-covalent conjugation methods. For example, a negatively charged antigen or adjuvant can be coupled to a positively charged nanocarrier by electrostatic adsorption. A metal ligand-containing antigen or adjuvant can also be coupled to a metal complex-containing nanocarrier using a metalligand complex.

[0144] In embodiments, the antigen or adjuvant may be attached to the polymer, for example poly (lactic glycol) -block-poly (ethylene glycol), prior to the composition of the synthetic nanocarrier, or the synthetic nanocarrier may be formed with reactive or activated groups on its surface. . In the latter case, the antigen or adjuvant can be prepared with a group that is compatible with the attachment chemistry that is presented by the surface of the synthetic nanocarriers. In other embodiments, the peptide antigen may be attached to VLPs or liposomes

-44 using the appropriate linker. A linker is a compound or reagent capable of coupling two molecules together. In an embodiment, the linker may be a homobifunctional or heterobifunctional reagent as described in Hermanson 2008. For example, a synthetic VLP or liposomal nanocarrier having a carboxyl group on the surface can be treated with a homodunctional linker, adipic dihydrazide (ADH), in the presence of EDC, to form the corresponding synthetic nanocarrier with an ADH linker. The resulting ADH-linked synthetic nanocarrier is then coupled to a peptide antigen and / or an acid group-containing adjuvant through the other end of the ADH linker on the NC to form the appropriate VLP peptide or liposome conjugate.

[0145] For detailed descriptions of the available conjugation methods, see Hermanson GT "Bioconjugate Techniques", 2nd Edition, Academic Press, Inc., 2008. In addition to covalent binding, the antigen and / or adjuvant may be coupled by adsorption to a previously prepared synthetic nanocarrier or may be coupled to it. by encapsulation during the formation of a synthetic nanocarrier.

[0146] In embodiments, surface antigens can be non-covalently coupled to synthetic nanocarriers using a variety of non-covalent interactions including, but not limited to, charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, and interactions. stacking TT, hydrogen interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipoldipole interactions and / or combinations thereof. In embodiments, the encapsulation is in the form of a coupling. When conjugating charged surface antigens, synthetic nanocarriers can be generated in the presence of surfactants that adsorb to the surfaces of the synthetic nanocarrier and thereby charge the synthetic nanocarrier. The charged surface antigens can then be non-covalently attached to the charged synthetic nanocarrier via a charge-charge interaction (see for example OHagen WO2000006123A1).

[0147] In embodiments, the synthetic nanocarriers of the invention can be combined with one or more adjuvants by mixing in the same carrier or delivery system. Such adjuvants may include, but are not limited to, an adjuvant as provided herein, such as alum, alum combined with monophosphoryl lipid (MPL) A from enterobacteria such as Escherihia coli, Salmonella minnesota, Salmonella typhimurium or Shigella flexneri, or specifically with MPL® (AS04) , MPL A of the above-mentioned bacteria separately, saponins such as QS-21, Quil-A, ISCOM, ISCOMATRIX ™, emulsions such as MF59 ™, Montanide® ISA 51 and ISA 720, AS02 (QS21 + squalene + MPL®), liposomes and liposomal formulations such as AS01, AS15, synthesized or specially prepared microparticles and microcarriers, such as vesicles derived from the outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, or chitosan molecules, depot forming (sustained release) forms such as Pluronic® block copolymers, specifically modified or prepared peptides such as muramyl dipeptide, 4-phosphates

-45aminoalkylglucosaminides such as RC529 or proteins such as bacterial toxoids or fragments of toxins. The dosages of such other adjuvants can be determined using conventional dose testing.

[0148] In embodiments, the synthetic nanocarriers of the invention can be combined with other antigens different, similar or identical to those coupled to the nanocarrier (with or without adjuvant, with or without the use of a different delivery vehicle) administered separately at a different point. temporarily and / or elsewhere in the body and / or by other immunization route or with another antigen and / or synthetic carrier carrying the adjuvant, administered separately at a different time point and / or elsewhere in the body and / or by another route of immunization.

[0149] Populations of synthetic nanocarriers can be combined to form the dosage forms of the invention using conventional pharmaceutical mixing methods. These include liquid-liquid mixing in which two or more suspensions, each containing one or more subset of nanocarriers, are directly brought together or brought into contact with each other via one or more diluent containing vessels. Since synthetic nanocarriers can also be made or stored in powder form, a dry powder-powder mixing can be performed, just as two or more powders can be re-suspended in a common base. Depending on the properties of the nanocarriers and their interaction potentials, there may be benefits attributed to one or the other blending route.

[0150] Typical compositions of the invention containing synthetic nanocarriers may contain inorganic or organic buffers (e.g. sodium or potassium salts of phosphate, carbonate, acetate or citrate) and pH adjusting agents (e.g. hydrochloric acid, sodium or potassium hydroxide, citrate or acetate salts). , amino acids and their salts), antioxidants (e.g. ascorbic acid, alpha-tocopherol), surfactants (e.g. polysorbate 20, polysorbate 80, polyoxyethylene-9-10-nonylphenyl, sodium deoxycholate), solution stabilizers and / or cryo / ly stabilizers (e.g. sucrose, lactose, mannitol, trehalose), osmotic regulators (e.g. salts or sugars), antibacterial agents (e.g. benzoic acid, phenol, gentamicin), antifoam agents (e.g. polydimethylsilosone), preservatives (e.g. thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity regulators (e.g. polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g. glycerol, polyethylene glycol, ethanol).

[0151] The compositions of the invention contain synthetic nanocarriers of the invention in combination with pharmaceutically acceptable excipients or carriers. The compositions can be prepared using conventional manufacturing and mixing techniques to provide useful dosage forms. Suitable techniques for use in practicing the invention can be found in the Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley &amp; Sons, Inc .; and Pharmaceutics: The Science of Dosage Form Design, 2nd Edition Edited by ME Auten, 2001, Churchill Livingstone. In one example

In embodiments, the synthetic nanocarriers of the invention are suspended in a sterile saline injection solution together with a preservative.

[0152] Dosage form doses contain varying amounts of the synthetic nanocarrier population of the invention. The amount of synthetic nanocarriers present in the dosage forms of the invention may be varied according to the nature of the sets of surface antigens, the therapeutic benefit to be achieved, and other such parameters. In embodiments, dose ranging studies can be performed to determine the optimal therapeutic amount of synthetic nanocarriers to be present in the dosage form. In embodiments, the first and second populations are present in an amount effective to generate an immune response to the first set of surface antigens and the second set of surface antigens when administered to the subject. It may be possible to determine the amount of the first, second and / or subsequent populations effective to generate an immune response using conventional research and dosing techniques in subjects. The dosage forms of the invention can be administered at various frequencies. In a preferred embodiment, at least one administration of the dosage form is sufficient to generate a pharmacologically appropriate response. In a more preferred embodiment, at least two administrations, at least three administrations or at least four administrations of the dosage form are used to provide a pharmacologically appropriate response.

[0153] It should be understood that the compositions of the invention may be prepared by any suitable means, and that the invention is by no means limited to the compositions which can be prepared using the methods described. The selection of an appropriate method may require attention to the properties of the particular linked moieties. In embodiments, the production methods comprise preparing a first population of synthetic nanocarriers that include a first set of surface antigens; obtaining a second population of synthetic nanocarriers that contain a second set of surface antigens; and combining the first and second populations of synthetic nanocarriers into a pharmaceutical dosage form; wherein the first set of surface antigens and the second set of surface antigens are structurally or immunologically different.

[0154] In some embodiments, the synthetic nanocarriers of the invention are made under sterile conditions or are finally sterilized. This can ensure that the resulting composition is sterile and non-infectious, which improves safety compared to non-sterile compositions. This provides a valuable safener, especially when people receiving synthetic nanocarriers have immunity defects, suffer from infection, and / or are susceptible to infection. In some embodiments, the synthetic nanocarriers of the invention can be lyophilized and stored in suspension or lyophilized powder, depending on the formulation strategy, for an extended period of time without loss of activity.

[0155] The compositions of the invention can be administered by a variety of routes of administration, including, but not limited to, parenteral (such as subcutaneous, intramuscular, intravenous or intradermal); orally; transdermal, intranasal, transmucosal, sublingual, rectal, ophthalmic, transdermal, transdermal, or a combination of these routes.

[0156] The compositions and methods described herein can be used to induce, enhance, modulate, stimulate, suppress, direct, or redirect an immune response. The compositions and methods described can be used in the diagnosis, prevention and / or treatment of diseases such as cancer, infectious diseases, metabolic diseases, degenerative diseases, autoimmune diseases, inflammatory diseases, diseases of the immune system or other diseases and / or conditions. The compositions and methods described herein can also be used to prevent or treat addiction, such as nicotine or drug addiction. The compositions and methods described herein can also be used to prevent and / or treat a condition resulting from exposure to a toxin, hazardous substance, environmental toxin, or other noxious agent.

[0157] The subjects listed herein may be addicted to, or be at risk of becoming addicted to, an abusive or addictive substance.

[0158] The subjects set forth herein may have or be at risk for cancer. Cancers include, but are not limited to, breast cancer; bile duct cancer; bladder cancer; a malignant tumor of the brain including gliomas and medulloblastomas; cervical cancer; chorionicoma; colorectal cancer; endometrial cancer; esophageal cancer; stomach cancer; haematological malignancies including acute lymphocytic and myeloid leukemia, e.g. CLL from B cells; acute lymphoblastic leukemia / T cell lymphoma; hairy cell leukemia; chronic myeloid leukemia, multiple myeloma; AIDS-related leukemias and adult T-cell leukemia / lymphoma; intraepithelial cancers including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including those derived from epithelial cells, stromal cells, germ cells, and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcoma, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancer, including germ cell tumors such as seminoma, non-seminoma (teratomas, chorionic carcinoma), stromal tumors, and germinal tumors; thyroid cancer including thyroid adenocarcinoma and medullary cancer; and renal cancer, including adenocarcinoma and Wilms' tumor.

[0159] The subjects set forth herein may have an infection or be at risk of infection or an infectious disease. Infections or infectious diseases include, but are not limited to, viral infectious diseases such as AIDS, chicken pox (Varicella), common cold, cytomegaly, Colorado tick fever, dengue fever, Ebola hemorrhagic fever, hand, foot and mouth disease, hepatitis , herpes, shingles, HPV, influenza, Lassa fever, measles, Marburg virus, haemorrhagic fever, infectious mononucleosis, mumps,

-48Norovirus, polio, progressive multifocal leukoencephalopathy, rabies, rubella, SARS, smallpox, viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, West Nile disease and yellow fever; bacterial infectious diseases such as anthrax, bacterial meningitis, botulism, brucellosis, campylobacteriosis, cat scratch disease, cholera, diphtheria, typhus, gonorrhea, impetigo, legionellosis, leprosy (Hansen's disease), leptospirosis, listeriosis, Lyme disease, melioidosis, rheumatic fever, MRSA infection, nocardiosis, whooping cough (whooping cough), plague, pneumococcal pneumonia, parrot disease, Q fever, Rocky Mountain spotted fever (RMSF), salmonellosis, scarlet fever, Shigella infection, syphilis, tetanus, trachoma, tuberculosis, tularemia, typhoid fever, typhus and urinary tract infections; infectious parasitic diseases such as African trypanosomiasis, amoebiasis, ascariasis, babesiosis, Chagas disease, clonorchosis, cryptosporidiosis, cysticercosis, diphyllobothriosis, dracunculosis, echinococcosis, enterobiosis, fascioliosis, fascioliosis, gagiasis, amoebiasis, amoebiasis , kala-azar, leishmaniasis, malaria, metagonimosis, myiasis, onchocercosis, lice, pinworm infection, scabies, schistosomiasis, tapeworm, toxocarosis, toxoplasmosis, trichinosis, trichinosis, trichuriasis, trichomoniasis and trypanosomiasis; fungal infectious diseases such as aspergillosis, blastomycosis, candidiasis, cryptococcosis, coccidioidomycosis, histoplasmosis, tinea pedis and tinea pedis; infectious diseases of prions such as Alpers disease, Fatal Familial Insomnia, Gerstmann Straussler-Scheinker syndrome, Kuru disease and variant Creutzfeldt-Jakob disease.

EXAMPLES

[0160] The invention will be more readily understood by reference to the following examples, which are included merely to illustrate certain aspects and embodiments of the invention, and not as limitations.

[0161] It will be understood by those skilled in the art that various adaptations and modifications to the embodiments just described can be configured without departing from the scope and spirit of the invention. Other suitable techniques and methods known in the art can be used in many specific agents by those skilled in the art and in light of the description of the disclosure described herein.

[0162] Accordingly, it should be understood that the invention may be practiced otherwise than as described. The above description is exemplary and not limiting. Many other embodiments will be apparent to those skilled in the art from reading the above description. The scope of the invention should therefore be determined with reference to the appended claims.

Example 1: Formulation for the first nanocarrier population (prognostic)

[0163] Synthetic nanocarriers containing the PLGA-R848 conjugate (adjuvant), the PLA-PEG-N3 conjugate (peptide antigen linker) and the OVA peptide (T cell antigen) are prepared by a double emulsion process, the OVA peptide being encapsulated in synthetic nanocarriers. For suspension of synthetic nanocarriers (10 mg / ml in PBS (pH 7.4 buffer), 5 ml, containing approximately 12.5 mg (MW: 20,000; 0.000625 mmol)

-49PLA-PEG-N3), the HPV L1 peptide containing an acetylene linker (33 mg) is added with gentle mixing. Sodium ascorbate solution (100 mM in H 2 O, 0.3 mL) is added followed by a CuSO 4 solution (10 mM in water, 0.6 mL). The resulting light yellow suspension is stirred at 20 ° C for 15 hours and additional CuSO4 solution (0.3 ml) and sodium ascorbate solution (0.15 ml) are added. The suspension is stirred for 5 hours at 20 ° C and diluted to 10 ml with PBS (pH 7.4) and centrifuged to remove the supernatant. Residual nanocarrier pellets are washed twice with PBS buffer. The washed NCs (nanocarriers) are then resuspended in 5 ml PBS buffer and stored frozen. Coupling of the L1 peptide on the surface of the synthetic nanocarriers is confirmed by HPLC analysis of the digested synthetic nanocarriers and a bioassay.

Example 2: Formulation for the second (prognostic) nanocarrier population

[0164] Using the general outlines of the procedures of Example 1 above, synthetic nanocarriers containing PLA-R848, PLA-PEG-N3, and an encapsulated OVA peptide are prepared and coupled to the HPV L2 peptide to provide L2-peptide-coupled synthetic nanocarriers.

Example 3: Formulation linking the first and second nanocarrier populations (prognostic)

[0165] The synthetic nanocarrier formulations of Examples 1 and 2 above are thawed and diluted in PBS to a final concentration of 5 mg nanocarriers per milliliter. Equal aliquots of each (0.5 ml) are pooled to provide a population of nanocarriers that contain both the HPV L1 and L2 peptides.

Example 4: Preparation of nanocarriers

Preparation of NC-Nic-OVA

[0166] PLGA-R848, poly-D / L-lactide-co-glycolide, 4-amino-2- (ethoxymethyl) -α, α-dimethyl-1H-imidazo [4,5-c] quinoline-1-ethanol amide approximately 7,000 Da made from PLGA in a 3: 1 ratio of lactide to glycolide and having approximately 8.5% w / w. conjugated resiquimod contents were custom-made from Princeton Global Synthesis (300 George Patterson Drive # 206, Bristol, PA 19007). PLA-PEG-Nicotine (S642), Poly-D / L-block-poly (ethylene glycol) - (±) -trans-3'-hydroxymethylnicotinic ether with a PEG block of about 5,000 Da and a PLA block of about 21,000 Da has been produced on request from Princeton Global Synthesis (300 George Patterson Drive # 206, Bristol, PA 19007). PL-PEG-Maleimide, a block copolymer consisting of an approximately 22,000 Da poly-D / L-lactide (PLA) block and an approximately 2,900 Da poly (ethylene glycol) (PEG) block that is maleimide-functional terminated, synthesized from commercial starting materials by generating a PLA block by ring-opening polymerization of dl-lactide with HO-PEG-maleimide. Polyvinyl alcohol PhEur, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027. Part Number 4-88).

[0167] The solutions were prepared as follows:

-50 Solution 1: 0.13N HCl in Purified Water

Solution 2: PLGA-R848 @ 50 mg / ml, PLA-PEG-nicotine @ 25 mg / ml and PLA-PEGmaleimide 25 mg / ml in dichloromethane was prepared by dissolving each polymer separately in dichloromethane at 100 mg / ml and then combining the 2 parts of the PLGA-R848 solution to 1 part of the PLA-PEG-nicotine solution and the PLA-PEGmaleimide solution.

Solution 3: polyvinyl alcohol @ 50 mg / ml in 100 mM in 100 mM phosphate buffer, pH 8.

[0168] Solution 4: 70 mM phosphate buffer, pH 8.

[0169] A first emulsion (W1 / O) was made first using solution 1 and solution 2. Solution 1 (0.2 ml) and solution 2 (1.0 ml) were combined in a small pressure glass tube and sonicated at 50% amplitude for 40 seconds with the Branson Digital Sonifier 250. A secondary emulsion (W1 / O / W2) was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, centrifugation to form a coarse dispersion, then sonicated at 30% amplitude for 40 seconds using a Branson Digital Sonifier 250.

[0170] The secondary emulsion was added to an open 50 ml beaker containing a 70 mM phosphate buffer (30 ml) and stirred at room temperature for 2 hours, allowing the dichloromethane to evaporate and form the nanocarriers in suspension. A portion of the suspended nanocarriers were washed by transferring the nanocarrier suspension to a centrifuge tube, centrifugation at 21,000 rcf for 45 minutes, removing the supernatant and resuspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was resuspended in phosphate buffered saline to obtain a nanocarrier suspension with a nominal concentration of 10 mg / ml based on polymer. The nanocarrier suspension was stored frozen at -20 ° C until further use.

Table 4: NC-Nic-OVA characteristics

<td>Nanocarrier</td><td>Efficient^(nm)</td><td>diameter</td><td>TLR agonist,% w / w</td><td>Agonist w / w</td><td>cells</td><td>T,</td><td> %</td>

<td>NC-Nic-OVA</td><td colspan="2"> 215</td><td>R848, 4.2</td><td colspan="4">Lack</td>

(1) NC with PEG-nicotine and PEG-MAL on the surface, prepared as above;

6.5 mg / ml suspension in PBS buffer.

(2) OVA Protein (Egg White Ovalbumin): Worthington, Lot # POK12101, MW: 46,000.

(3) Traut's reagent (2-iminothiolane.HCl): MP Biomedical, Lot # 8830KA, MW: 137.6 (4) pH 8 buffer (sodium phosphate, 20 mM with 0.5 mM EDTA).

(5) pH 7 1x PBS buffer.

OVA protein (10 mg) was dissolved in 1 ml of pH 8 buffer. Freshly prepared Traut's reagent solution in pH 8 buffer (0.25 ml, 2 mg / ml) was added to the oval protein solution. The resulting solution was stirred under argon in the dark for 1.5 hours. The solution was diafiltered with a MWCO 3K diafiltration tube and washed twice with a pH 8 buffer. The resulting thiol-modified OVA was dissolved in 1 ml of a pH 8 buffer under argon. The NC suspension (3 ml, 6.5 mg / ml) was centrifuged to remove the supernatant. The modified OVA solution was then mixed with the NC granules. The resulting suspension was stirred at room temperature under argon in the dark for 12 hours. The NC suspension was then diluted to 10 ml pH 7 with PBS and centrifuged. The resulting NC pellet was washed with 2x10 ml pH 7 PBS. The NC-Nic-OVA conjugates were then resuspended in PBS at pH 7 (approximately 6 mg / ml, 3 ml) stored at 4 ° C.

Obtaining NC-OVA

[0171] PLGA-R848, poly-D / L-lactide-co-glycolide, 4-amino-2- (ethoxymethyl) -α, α-dimethyl-1H-imidazo [4,5-c] quinoline-1-ethanol amide approximately 7,000 Da made from PLGA in a 3: 1 ratio of lactide to glycolide and having approximately 8.5% w / w. conjugated resiquimod contents were custom-made from Princeton Global Synthesis (300 George Patterson Drive # 206, Bristol, PA 19007). PL-PEG-Maleimide, a block copolymer consisting of an approximately 22,000 Da poly-D / L-lactide (PLA) block and an approximately 2,900 Da polyethylene glycol (PEG) block that is maleimide-functional terminated, was synthesized from commercial starting materials by preparation of a PLA block by ring-opening polymerization of dl-lactide with HOPEG-maleimide. PhEur polyvinyl alcohol, USP (85-89% hydrolyzed, viscosity 3.44.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027. Part Number 4-88).

[0172] The solutions were prepared as follows:

1: 0.13N HCl solution in purified water

Solution 2: PLGA-R848 @ 50 mg / ml and PLA-PEG-maleimide 50 mg / ml in dichloromethane was prepared by dissolving each polymer separately in dichloromethane at 100 mg / ml, then combining 1 part of PLGA-R848 solution to 1 part of the solution PLA-PEG-maleimide.

Solution 3: Polyvinyl alcohol @ 50 mg / ml in 100 mM in 100 mM phosphate buffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

[0173] A first emulsion (W1 / O) was made first using Solution 1 and Solution 2. Solution 1 (0.2 ml) and solution 2 (1.0 ml) were combined in a small glass pressure tube and sonicated at 50% amplitude. for 40 seconds using the Branson Digital Sonifier 250. Then a secondary emulsion (W1 / O / W2) was formed by adding Solution 3

-52 (2.0 ml) to original emulsion, centrifugation to produce a coarse dispersion followed by sonication at 30% amplitude for 40 seconds using the Branson Digital Sonifier

250.

[0174] The secondary emulsion was added to an open 50 ml beaker containing 70 mM phosphate buffer (30 ml) and stirred at room temperature for 2 hours, allowing the dichloromethane to evaporate and form the nanocarriers in suspension. A portion of the suspended nanocarriers were washed by transferring the suspension of nanocarriers to a centrifuge tube, centrifugation at 21,000 rcf for 45 minutes, removing the supernatant and resuspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was resuspended in phosphate buffered saline to obtain a nanocarrier suspension with a nominal concentration of 10 mg / ml based on polymer. The nanocarrier suspension was stored frozen at -20 ° C until further use.

Table 5 NC-OVA characteristics

<td>Nanocarrier</td><td>Effective ^(nm)</td><td>diameter</td><td>TLR agonist, weight / weight</td><td> %</td><td>T cell agonist,% w / w</td>

<td>NC-OVA</td><td colspan="2"> 208</td><td colspan="2">R848 4.3</td><td>Lack</td>

(1) NC with PEG-MAL on the surface, prepared as above; 6 mg / ml suspension in PBS buffer.

(2) OVA Protein (Egg White Ovalbumin): Worthington, Lot # POK12101, MW: 46,000.

(3) Traut's reagent (2-iminothiolano.HCl): MP Biomedical, Lot # 8830KA, MW: 137.6.

(4) pH 8 buffer (sodium phosphate, 20mM with 0.5mM EDTA).

(5) pH 7 1x PBS buffer.

[0175] OVA protein (20 mg) was dissolved in 1 ml of pH 8 buffer. Freshly prepared Traut's reagent solution in pH 8 buffer (0.5 ml, 2 mg / ml) was added to the ovine protein solution. The resulting solution was stirred under argon in the dark for 1.5 hours. The solution was diafiltered with a MWCO 3K diafiltration tube and washed twice with a pH 8 buffer. The resulting thiol-modified OVA was dissolved in 1 ml of a pH 8 buffer under argon. The NC suspension (4 ml, 6 mg / ml) was centrifuged to remove the supernatant. The modified OVA solution was then mixed with the NC pellets. The resulting suspension was stirred at room temperature under argon in the dark for 12 hours. The NC suspension was then diluted to 10 ml pH 7 with PBS and centrifuged. The resulting NC pellet was washed with 2x10 ml pH 7 PBS. The NC-OVA conjugates were then resuspended in pH 7 with PBS (approximately 6 mg / ml, 4 ml) stored at 4 ° C.

Preparation of NC-HA5

[0176] The peptide 323-339 amide acetate salt of ovalbumin was purchased from Bachem Americas Inc.

(3132 Kashiwa Street, Torrance CA 90505 Product Code 4065609.) PLGA-R848, poly-D / Lactide-co-glycolide, 4-amino-2- (ethoxymethyl) -α, α-dimethyl-1H-imidazo amide [4, 5c] quinoline-1-ethanol at approximately 7,000 Da made from PLGA in a 3: 1 ratio of lactide to glycolide and having approximately 8.5% w / w. conjugated resiquimod contents were custom-made from Princeton Global Synthesis (300 George Patterson Drive # 206, Bristol, PA 19007). PL-PEG-maleimide, a block copolymer consisting of an approximately 22,000 Da polyD / L-lactide (PLA) block and an approximately 2,900 Da polyethylene glycol (PEG) block that is maleimide-functional terminated, was synthesized from commercial starting materials by making the block PLA by dl-lactide ring-opening polymerization with HO-PEG-maleimide. poly (vinyl alcohol) PhEur, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027. Part Number 4-88).

[0177] The solutions were prepared as follows:

Solution 1: 323-339 ovalbumin peptide @ 20 mg / ml was prepared in 0.13N HCl at room temperature.

Solution 2: PLGA-R848 @ 50 mg / ml and PLA-PEG-maleimide 50 mg / ml in dichloromethane was prepared by dissolving each polymer separately in dichloromethane at 100 mg / ml, then combining 1 part of PLGA-R848 solution to 1 part of the solution PLA-PEG-maleimide.

Solution 3: polyvinyl alcohol @ 50 mg / ml in 100 mM in 100 mM phosphate buffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

[0178] A first emulsion (W1 / O) was made first using Solution 1 and Solution 2. Solution 1 (0.2 ml) and solution 2 (1.0 ml) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds with the Branson Digital Sonifier 250. A secondary emulsion (W1 / O / W2) was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, centrifugation to form a coarse dispersion, then sonicated at 30% amplitude for 40 seconds using a Branson Digital Sonifier 250.

[0179] The secondary emulsion was added to an open 50 ml beaker containing 70 mM phosphate buffer (30 ml) and stirred at room temperature for 2 hours, allowing the dichloromethane to evaporate and form the nanocarriers in suspension. A portion of the suspended nanocarriers were washed by transferring the suspension of nanocarriers to a centrifuge tube, centrifugation at 21,000 rcf for 45 minutes, removing the supernatant and resuspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was resuspended in phosphate buffered saline to obtain a nanocarrier suspension at nominal concentration.

- 5,410 mg / ml based on polymer. The nanocarrier suspension was stored frozen at -20 ° C until further use.

<td>Table 6: C</td><td colspan="3">Characteristics of NC-HA5</td>

<td>Nanocarrier</td><td>Effective diameter^(nm)</td><td>TLR agonist,% weight / weight</td><td>T cell agonist,% weight / weight</td>

<td>NC-HA5</td><td> 216</td><td>R848, 3.6</td><td>Peptides OVA 323-339, 2.0</td>

(1) NC with PEG-MAL on the surface, prepared as above; 6.7 mg / ml suspension in PBS buffer.

(2) HA5 protein: Recombinant Haemagglutinin, A / Vietnam / 1203/2004, MW: 72,000, provided as a solution in PBS-Tween pH 7 (0.55 mg / ml).

(3) Traut's reagent (2-iminothiolano.HCl): MP Biomedical, Lot # 8830KA, MW: 137.6.

(4) pH 8 buffer (sodium phosphate, 20mM with 0.5mM EDTA).

(5) pH 7 1x PBS buffer.

[0180] HA5 protein (0.21 g in 0.38 ml pH 7.1 in PBS-tween buffer) was diluted to 0.5 ml with pH 8 buffer. Freshly prepared Traut's reagent solution in pH 8 buffer (0.02 ml, 2 mg / ml) was added to the HA5 protein solution. The resulting solution was stirred under argon in the dark for 1.5 hours. The solution was diafiltered with a MWCO 3K diafiltration tube and washed twice with a pH 8 buffer. The obtained modified HA5 protein with a thiol group was dissolved in 0.5 ml of a pH 8 buffer under an argon atmosphere. The NC suspension (3 ml, 6.7 mg / ml) was centrifuged to remove the supernatant. The modified HA5 solution was then mixed with the NC pellets. The resulting suspension was stirred at room temperature under argon in the dark for 12 hours. The NC suspension was then diluted to 10 ml pH 7 with PBS and centrifuged. The resulting NC pellet was washed with 2x10 ml pH 7 PBS. The NC-HA5 conjugates were then resuspended in pH 7 with PBS (approximately 6 mg / ml, 4 ml) stored at 4 ° C.

Preparation of NC-L2, NC-M2e or NC-M2e-L2

[0181] The peptide 323-339 amide acetate salt of ovalbumin was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505 Product code 4065609.) PLGA-R848, poly-D / Lactide-co-glycolide, 4-amino-2- (ethoxymethyl) -α, α-dimethyl-1H-imidazo amide [4, 5-c] quinoline-1-ethanol at approximately 7,000 Da was prepared from PLGA in a 3: 1 ratio of lactide to glycolide and having approximately 8.5% w / w. conjugated resiquimod contents were custom-made from Princeton Global Synthesis (300 George Patterson Drive # 206, Bristol, PA 19007). PLA-PEG-C6-N3, a block copolymer consisting of an approximately 23,000 Da poly D / L-lactide (PLA) block and an approximately 2,000 Da polyethylene glycol (PEG) block which is terminated by an amide-coupled azide linker C6H12, synthesized by coupling HO-PEG-COOH with amino azide-C6H12, and then generated a PLA block by ring-opening polymerization of the resulting

-55HO-PEG-C6-N3 with dl-lactide. PhEur polyvinyl alcohol, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South

Democrat Road Gibbstown, NJ 08027. Part number 4-88).

[0182] The solutions were prepared as follows:

Solution 1: 323-339 ovalbumin peptide @ 20 mg / ml was prepared in 0.13N HCl at room temperature.

Solution 2: PLGA-R848 @ 50 mg / ml and PLA-PEG-C6-N3 @ 50 mg / ml in dichloromethane was prepared by dissolving each separately at 100 mg / ml in dichloromethane and then combining in equal parts by volume.

Solution 3: Poly (vinyl alcohol) @ 50 mg / ml in 100 mM in 100 mM phosphate buffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

[0183] A first emulsion (W1 / O) was made first using Solution 1 and Solution 2. Solution 1 (0.2 ml) and solution 2 (1.0 ml) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds with the Branson Digital Sonifier 250. A secondary emulsion (W1 / O / W2) was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, centrifugation to form a coarse dispersion, then sonicated at 30% amplitude for 40 seconds using a Branson Digital Sonifier 250.

[0184] The secondary emulsion was added to an open 50 ml beaker containing 70 mM phosphate buffer (30 ml) and stirred at room temperature for 2 hours, allowing the dichloromethane to evaporate and form the nanocarriers in suspension. A portion of the suspended nanocarriers were washed by transferring the nanocarrier suspension to a centrifuge tube, centrifugation at 21,000 rcf for 45 minutes, removing the supernatant and resuspending the phosphate buffered saline pellets. This washing procedure was repeated and then the pellet was resuspended in phosphate buffered saline to obtain a nanocarrier suspension with a nominal concentration of 10 mg / ml based on polymer. Two identical batches were prepared and then combined to form a homogeneous suspension for frozen storage at -20 ° C for further use.

<td>Table 7: Characteristics of N</td><td colspan="4">C-L2, NC-M2e or NC-M2e-L2</td>

<td>Nanocarrier</td><td>Effective diameter ^(nm)</td><td>TLR agonist, weight / weight</td><td> %</td><td>Antigen,% weight / weight</td>

<td>NC-L2, NC-M2e or NC- M2e-L2</td><td> 209</td><td colspan="2">R848, 4.2</td><td>Ova Peptide 323339, 2.4</td>

(1) PEG-C6-N3 surface nanocarriers containing PLGA-R848 and Ova peptide, prepared as above, 7 mg / ml suspension in PBS.

(2) HPV16 L2 peptide modified with an alkyne linker attached to the C-terminal amino group of Lys; Bachem Americas, Inc, Lot B06055, MW 2595, salt

TFA; Sequence:

H-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-ProPro-Asp-Ile-Ile-Pro-Lys-Val-Lys (5-hexynoyl) -NH2 (with a Cys-Cys disulfide bond).

(3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200 mM in DI water; sodium ascorbate, 200 mM in freshly prepared DI water.

(4) PBS buffer, pH 7.4.

[0185] The NC suspension (7 mg / ml, 4 ml) was concentrated to 1 ml volume by centrifugation. A solution of L2 peptide (20 mg) in 2 ml of PBS buffer was added. A pre-mixed solution of 0.2 ml of CuSO4 (100 mM) and 0.2 ml of THPTA ligand (200 mM) were added followed by 0.4 ml of sodium ascorbate (200 mM). The resulting light yellow suspension was stirred in the dark at room temperature for 18 hours. The suspension was then diluted to 10 ml with PBS buffer and centrifuged to remove the supernatant. The NC-L2 conjugate pellets were further washed twice with 10 ml of PBS buffer and resuspended in pH 7.4 buffer at a final concentration of approx. 6 mg / ml (approx. 4 ml) and stored at 4 ° C.

(1) PEG-C6-N3 surface nanocarriers containing PLGA-R848 and Ova peptide, prepared as above, 7 mg / ml suspension in PBS.

(2) M2e peptide modified with an alkyne linker attached to the C-terminus of Gly; CS Bio Co, Catalog No. CS4956, Batch: H308, MW 2650, TFA salt; Sequence:

H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thy-Arg-Asn-Glu-Trp-GluCys-Arg-Cys-Ser-Asp-Gly-Gly-NHCH2CCH.

(3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200 mM in DI water; sodium ascorbate, 200 mM in freshly prepared DI water.

(4) PBS buffer, pH 7.4.

[0186] The NC suspension (7 mg / ml, 4 ml) was concentrated to 1 ml volume by centrifugation. A solution of M2e peptide (20 mg) in 2 ml of PBS buffer was added. A pre-mixed solution of 0.2 ml of CuSO4 (100 mM) and 0.2 ml of THPTA ligand (200 mM) were added followed by 0.4 ml of sodium ascorbate (200 mM). The resulting light yellow suspension was stirred in the dark at room temperature for 18 hours. The suspension was then diluted to 10 ml with PBS buffer and centrifuged to remove the supernatant. The NC-M2e conjugate pellets were further washed twice with 10 ml of PBS buffer and resuspended in pH 7.4 buffer at a final concentration of approx. 6 mg / ml (approx. 4 ml) and stored at 4 ° C.

(1) PEG-C6-N3 surface nanocarriers containing PLGA-R848 and Ova peptide, prepared as above, 7 mg / ml suspension in PBS.

(2) HPV16 L2 peptide modified with an alkyne linker attached to the C-terminal amino group of Lys; Bachem Americas, Inc, Lot B06055, MW 2595, salt

TFA; Sequence:

H-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-Pro-ProAsp-Ile-Ile-Pro-Lys-Val-Lys (5-hexynoyl) -NH2 (with a Cys-Cys disulfide bond).

(3) M2e peptide modified with an alkyne linker attached to the C-terminus of Gly; CS Bio Co, Catalog No. CS4956, Batch: H308, MW 2650, TFA salt; Sequence:

H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thy-Arg-Asn-Glu-Trp-GluCys-Arg-Cys-Ser-Asp-Gly-Gly-NHCH2CCH.

(4) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200 mM in DI water; sodium ascorbate, 200 mM in freshly prepared DI water.

(5) PBS buffer, pH 7.4.

[0187] The NC suspension (7 mg / ml, 2 ml) was concentrated to 0.5 ml volume by centrifugation. A mixture of L2 peptide (5 mg) and M2e peptide (5 mg) in 1 ml PBS buffer was added. A pre-mixed solution of 0.2 ml of CuSO4 (100 mM) and 0.2 ml of THPTA ligand (200 mM) were added followed by 0.4 ml of sodium ascorbate (200 mM). The resulting light yellow suspension was stirred in the dark at room temperature for 18 hours. The suspension was then diluted to 10 ml with PBS buffer and centrifuged to remove the supernatant. The NCM2e-L2 conjugate pellets were further washed twice with 10 ml PBS buffer and resuspended in pH 7.4 buffer at a final concentration of approx. 6 mg / ml (approx. 2 ml) and stored at 4 ° C.

Example 5: Immunization with two monovalent antigenic nanocarriers leads to an immune response to both antigens

Anti-nicotine (dark gray bars) and anti-ovalbumin (light gray bars) titers were measured in unimmunized mice and mice injected with NC-Nic and NC-OVA (as prepared in Example 4) (5 animals / group, sc, 100 μg of each NC per injection, 2 times at 3-week intervals). The titers on day 33 after NC immunization are shown in Figure 1 (ELISA against polylysine-nicotine or ovalbumin) (group 1: unimmunized, group 2: immunized with NC-Nic and NC-OVA).

[0189] Mice were injected with 100 µg of NC-Nic (nanocarrier displaying nicotine on the outer surface and containing the helper peptide OP-II and adjuvant R848 in NC) and 100 µg of NC-OVA (nanocarrier displaying ovalbumin on the outer surface and containing the helper peptide OP-II) and R848 adjuvant in NC) (subcutaneous, hind limbs) at 3-week intervals (days 0 and 21). Anti-nicotine and anti-ovalbumin antibody titers were measured on day 33 after NC immunization. Antibody titers against nicotine and ovalbumin (EC50) as measured by ELISA against polylysine-nicotine or ovalbumin protein (Fig. 1) are shown. Also shown are titers for

-58 unimmunized control mice. The results show that mice immunized with the combination of two monovalent antigenic nanocarriers (NC-Nic and NC-OVA) produce antibodies against both antigens.

Example 6: Immunization with monovalent and bivalent antigen nanocarriers leads to an immune response to all three antigens

[0190] Anti-nicotine, anti-ovalbumin and anti-L2 peptide titers were measured in unimmunized mice and mice injected with NCNic-OVA and NC-L2 (as prepared in Example 4) (5 animals / group, sc, 100 μg of each NC per injection, 2 times at 3-week intervals). The titers on day 33 after NC immunization are shown in Fig. 2 (ELISA against polylysine-nicotine, ovalbumin protein or PLA-PEG-L2 peptide) (group 1: unimmunized, group 2: immunized with NC-Nic-OVA and NC-L2).

[0191] Mice were injected with 100 µg NC-Nic-OVA (nanocarrier displaying nicotine on the outer surface and containing OP-II helper peptide and R848 adjuvant in NC) and 100 µg NC-L2 (HPV L2 peptide (aa17-36) peptide nanocarrier on the outer surface and containing the helper peptide OP-II and adjuvant R848 in NC) (subcutaneously, hind limbs) at 3-week intervals (days 0 and 21). Titers of anti-nicotine, anti-ovalbumin, and anti-L2 peptide titers were measured at 33 weeks. the day after NC immunization. Shown are anti-nicotine, anti-ovalbumin, and anti-L2 peptide (EC50) titers measured by ELISA against polylysine-nicotine or the protein of ovalbumin and L2 peptide (Fig. 2). Also shown are the titers for unimmunized control mice. The results show that mice immunized with a combination of one monovalent and one bivalent antigen nanocarrier (NC-Nic-OVA and NCL2) produce antibodies to all three antigens.

Example 7: Immunization with two bivalent antigenic carriers leads to an immune response to all four antigens

Anti-nicotine, anti-ovalbumin, anti-M2e and anti- L2 peptide antibody titers were measured in unimmunized mice and mice injected with NC-Nic-OVA and NC-M2e-L2 (as prepared in Example 4) (5 animals / group, sc, 100 µg of each NC per injection, 2 times at 3-week intervals). The titers on day 33 after NC immunization are shown in Fig. 2 (ELISA against polylysine-nicotine, ovalbumin protein, PLA-PEG-M2e peptide or PLA-PEG-L2 peptide) (group 1: unimmunized, group 2: immunized with NC-Nic-OVA and NCM2e-L2).

[0193] Mice were injected with 100 µg NC-Nic-OVA (nanocarrier displaying nicotine and ovalbumin on the outer surface and containing the OP-II helper peptide and R848 adjuvant in NC) and 100 µg NC-M2e-L2 (nanocarrier displaying the influenza M2e (aa2 -27) and HVP L2 peptide (aa17-36) on the outer surface and containing the helper peptide OP-II and adjuvant R848 in NC) (subcutaneously, hind limbs) at 3-week intervals (days 0 and 21). Antibody titers against nicotine, against ovalbumin, against

M2e and anti-L2 peptide were measured on day 33 after NC immunization. The titers of anti-nicotine, anti-ovalbumin, anti-M2e and anti-L2 peptide (EC50) antibody titers measured by ELISA against polylysine-nicotine or ovalbumin protein, M2e peptide and L2 peptide are shown (Fig. 3). Also shown are the titers for unimmunized control mice. The results show that mice immunized with the combination of two bivalent antigen nanocarriers (NC-NicOVA and NC-M2e-L2) produce antibodies to all three antigens.

Example 8: Immunization with two monovalent antigenic peptide nanocarriers leads to an immune response to both peptide antigens

[0194] Antibody titers against M2e peptide and anti L2 peptide were measured in unimmunized mice and mice injected with NC-M2e and NC-L2 (as prepared in Example 4) (5 animals / group, sc, 100 μg of each NC per injection, 2 times at 3-week intervals). The titers on day 33 after NC immunization are shown in Figure 4 (ELISA against PLA-PEG-M2e peptide or PLA-PEG-L2 peptide) (group 1: unimmunized, group 2: immunized with NC-M2e and NC-L2).

[0195] Mice were injected with 100 µg NC-M2e (nanocarrier displaying the influenza M2e (aa2-27) peptide on the outer surface and containing the helper peptide OP-II and adjuvant R848 in NC) and 100 µg NC-L2 (nanocarrier displaying the HPV L2 peptide ( aa17-36) on the outer surface and containing the helper peptide OP-II and adjuvant R848 in NC) (subcutaneously, hind limbs) at 3-week intervals (days 0 and 21). Antibody titers against M2e peptide and L2 peptide in serum were measured at 33. the day after NC immunization. Antibody titers against M2e peptide, against L2 peptide (EC50) measured by ELISA against M2e peptide and L2 peptide are shown (Fig. 4). Also shown are the titers for unimmunized control mice. The results show that mice immunized with a combination of two monovalent peptide antigens (NC-M2e and NC-L2) produce antibodies against both antigens.

Example 9: Immunization with two monovalent antigenic peptide carriers leads to an immune response to both protein antigens

[0196] Antibody titers against HA5 protein and ovalbumin protein were measured in unimmunized mice and mice injected with NC-HA5 and NC-OVA (as prepared in Example 4) (5 animals / group, sc, 100 µg of each NC per injection, 2 times at 3-week intervals). The titers on day 33 after NC immunization are shown in Figure 5 (ELISA against HA protein or ovalbumin protein) (group 1: unimmunized, group 2: immunized with NC-HA5 and NC-OVA).

[0197] Mice were injected with 100 µg of NC-HA5c protein (nanocarrier displaying the influenza H5N1 HA protein on the outer surface and containing the OP-II helper peptide and R848 adjuvant in NC) and 100 µg of NC-OVA (nanocarrier displaying ovalbumin on the outer surface and containing the peptide). accessory OP-II and R848 adjuvant in NC) (subcutaneously, hind limbs) at 3-week intervals (days 0 and 21). Anti-HA5 and anti-ovalbumin serum titers were measured on day 33 after NC immunization.

-60 The antibody titers against HA5 and ovalbumin (EC50) measured by the method are shown

ELISA against HA H5N1 protein and ovalbumin protein (Figure 5). Also shown are the titers for unimmunized control mice. The results show that mice immunized with a combination of two monovalent nanocarrier protein antigens (NC-HA5 and NCOVA) produce antibodies against both antigens.

Example 10: Immunization with two monovalent and one bivalent antigenic carrier leads to an immune response to all four antigens

[0198] Antibody titers against HA, against ovalbumin, against M2e peptide and against L2 peptide were measured in unimmunized mice and mice injected with NC-HA5, NC-OVA and NC-M2e-L2 (as prepared in Example 4) (5 animals / group, sc, 100 µg of each NC per injection, 2 times at 3-week intervals). The titers on day 33 after NC immunization are shown in Fig. 6 (ELISA against HA protein, ovalbumin protein, PLA-PEG-M2e peptide or PLA-PEGL2 peptide) (group 1: unimmunized, group 2: immunized with NC-HA5, NC-OVA and NC-M2eL2).

[0199] Mice were injected with 100 µg NC-HA5 protein (nanocarrier displaying influenza H5N1 HA protein on the outer surface and containing the OP-II helper peptide and R848 adjuvant in NC), 100 μg NC-OVA (nanocarrier displaying ovalbumin on the outer surface and containing the helper peptide OP-II and adjuvant R848 in NC) and 100 μg NCMe-L2 (nanocarrier displaying influenza M2e peptide (aa2-27) and HPV L2 peptide (aa17-36) on the outer surface and containing the helper peptide OP-II and adjuvant R848 in NC) (subcutaneously, hind limbs) at 3-week intervals (days 0 and 21). Antibody titers against HA, and against ovalbumin and against L2 peptide were measured at 33. the day after NC immunization. The titers of anti-HA, anti-ovalbumin, anti-M2e peptide and anti-L2 peptide (EC50) titers measured by ELISA against HA protein, ovalbumin protein, M2e peptide and L2 peptide are shown (Fig. 6). Also shown are the titers for unimmunized control mice. These results show that mice immunized with the combination of two monovalent and one bivalent antigen nanocarrier (NC-HA5, NC-OVA and NC-M2e-L2) produce antibodies to all four antigens.

Example 11: Immunization with two monovalent and one bivalent antigen nanocarriers leads to an immune response to all four antigens

[0200] Antibody titers were measured in mice immunized with the combination of NC-M2e, NC-L2 peptide and NC-nicotine-ovalbumin (as prepared in Example 4). The NCM2e and NC-L2 peptide contained the T-helper OP-II peptide (2.0% and 2.4%, respectively) and the R848 adjuvant (3.6% and 4.3%, respectively); NC-nicotine-ovalbumin contained adjuvant R848 (4.2%). Each bar in Fig. 7 shows the anti-antigen titer. Five animals per group were immunized sc 120 μg of each NC per injection, 2 times at 3-61 weekly intervals. The titers on day 33 after the first immunization are shown (ELISA against PLA-PEG-M2e, PLA-PEG-L2, ovalbumin and polylysine-nicotine, respectively).

[0201] These results show that immunization with a combination of two NCs each containing a different peptide antigen together with an NC carrying the two other antigens results in the production of antibodies to all four NC borne antigens. When identical amounts of the three NCs, the first containing the M2e surface peptide from influenza A virus (ectodomain of the matrix protein M2, amino acids 2-27), the second containing the L2 surface peptide from the HPV virus (amino acids 17-36 of the HPV-16 capsid protein) and the third carrying nicotine and ovalbumin protein - used to immunize animals, all animals induced a strong humoral response against all four NC-conjugated antigens (Fig. 7). No reactivity was detected in the sera of mice before immunization.

Example 12: Immunization with two monovalent nanocarriers with the antigen in different steric orientations leads to an immune response in both orientations of the NC-3'-nicotine preparation

[0202] The peptide 323-339 amide acetate salt of ovalbumin was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505 Product code 4065609.) PLGA-R848, poly-D / Lactide-co-glycolide, 4-amino-2- (ethoxymethyl) -α, α-dimethyl-1H-imidazo amide [4, 5-c] quinoline-1-ethanol at approximately 7,000 Da was prepared from PLGA in a 3: 1 ratio of lactide to glycolide and having approximately 8.5% w / w. conjugated resiquimod contents were custom-made from Princeton Global Synthesis (300 George Patterson Drive # 206, Bristol, PA 19007). PLA-PEG-Nicotine (S-642), Poly-D / L-block-poly (ethylene glycol) ether - (±) -trans-3'-hydroxymethylnicotinic with a PEG block of around 5000 Da and a PLA block of around 21,000 Da has been produced on request from Princeton Global Synthesis (300 George Patterson Drive # 206, Bristol, PA 19007). PhEur polyvinyl alcohol, USP (8589% hydrolyzed, viscosity 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027. Part Number 4-88).

[0203] The solutions were prepared as follows:

Solution 1: 323-339 ovalbumin peptide @ 20 mg / ml was prepared in 0.13N HCl at room temperature.

Solution 2: PLGA-R848 @ 50 mg / ml, PLA-PEG-nicotine @ 25 mg / ml and PLA @ 25 mg / ml in dichloromethane was prepared by dissolving each polymer separately in dichloromethane at 100 mg / ml and then combining the 2 parts PLGA-R848 solution to 1 part PLA-PEG-nicotine solution and PLA solution.

Solution 3: Polyvinyl alcohol @ 50 mg / ml in 100 mM in 100 mM phosphate buffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

[0204] A first emulsion (W1 / O) was made first using Solution 1 and Solution 2. Solution 1 (0.2 ml) and solution 2 (1.0 ml) were combined in a small glass tube

And sonicated at 50% amplitude for 40 seconds using a Branson Digital

Sonifier 250. A secondary emulsion (W1 / O / W2) was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, centrifugation to form a coarse dispersion, then sonicated at 30% amplitude for 40 seconds using the Branson Digital Sonifier

250.

[0205] The secondary emulsion was added to an open 50 ml beaker containing 70 mM phosphate buffer (30 ml) and stirred at room temperature for 2 hours, allowing the dichloromethane to evaporate and form the nanocarriers in suspension. A portion of the suspended nanocarriers were washed by transferring the suspension of nanocarriers to a centrifuge tube, centrifugation at 21,000 rcf for 45 minutes, removing the supernatant and resuspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was resuspended in phosphate buffered saline to obtain a nanocarrier suspension with a nominal concentration of 10 mg / ml based on polymer. The nanocarrier suspension was stored frozen at -20 ° C until further use.

Table 8: Characteristics of NC-3'-nicotine

<td>Nanocarrier</td><td>Effective diameter^(nm)</td><td>TLR agonist,% weight / weight</td><td>T cell agonist,% weight / weight</td>

<td>NC-3'- Nicotine</td><td> 193</td><td>R848, 4.2</td><td>Ova Peptide 323-339, 2.1</td>

Obtaining NC-1'-Nicotine

[0206] The peptide 323-339 amide acetate salt of ovalbumin was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505 Product code 4065609.) PLGA-R848, poly-D / Lactide-co-glycolide, 4-amino-2- (ethoxymethyl) -α, α-dimethyl-1H-imidazo amide [4, 5-c] quinoline-1-ethanol at approximately 7,000 Da was prepared from PLGA in a 3: 1 ratio of lactide to glycolide and having approximately 8.5% w / w. conjugated resiquimod contents were custom-made from Princeton Global Synthesis (300 George Patterson Drive # 206, Bristol, PA 19007). PLA-PEG-1'-Nic, a block copolymer consisting of a polyD / L-lactide (PLA) block of approximately 23,000 Da and a polyethylene glycol (PEG) block of approximately 2,000 Da, which is conjugated to nicotine via a 4-carbon bond with 1 'amino group on nicotine. Briefly, nicotine with a 1 'butyl alcohol linker was converted to HO-PEG-1'-Nic by polymerization with ethylene oxide, and then an extension of PLA was produced by polymerization of HO-PEG-1'-Nic by ring opening with dllactide. PLA with an intrinsic viscosity of 0.22 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A.) PhEur Polyvinyl Alcohol, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027. Part Number 4-88).

[0207] The solutions were prepared as follows:

-63 Solution 1: 323-339 ovalbumin peptide @ 20 mg / ml was prepared in 0.13N HCl at room temperature.

Solution 2: PLGA-R848 @ 50 mg / ml, PLA-PEG-1'-Nic @ 25 mg / ml and PLA @ 25 mg / ml in dichloromethane was prepared by dissolving each polymer separately in dichloromethane at 100 mg / ml and then combining 2 parts of PLGA-R848 solution into 1 part of PLA-PEG-1'-Nic solution and PLA solution.

Solution 3: Polyvinyl alcohol @ 50 mg / ml in 100 mM in 100 mM phosphate buffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

[0208] A first emulsion (W1 / O) was made first using Solution 1 and Solution 2. Solution 1 (0.2 ml) and solution 2 (1.0 ml) were combined in a small pressure glass tube and sonicated at 50% amplitude for 40 seconds with the Branson Digital Sonifier 250. A secondary emulsion (W1 / O / W2) was then formed by adding Solution 3 (2.0 ml) to the primary emulsion, centrifugation to form a coarse dispersion, then sonicated at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250.

[0209] The secondary emulsion was added to an open 50 ml beaker containing 70 mM phosphate buffer (30 ml) and stirred at room temperature for 2 hours, allowing the dichloromethane to evaporate and form the nanocarriers in suspension. A portion of the suspended nanocarriers were washed by transferring the suspension of nanocarriers to a centrifuge tube, centrifugation at 21,000 rcf for 45 minutes, removing the supernatant and resuspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was resuspended in phosphate buffered saline to obtain a nanocarrier suspension with a nominal concentration of 10 mg / ml based on polymer. The nanocarrier suspension was stored frozen at -20 ° C until further use.

Table 9: Characteristics of NC-1'-nicotine

<td>Nanocarrier</td><td>Effective diameter^(nm)</td><td>TLR agonist,% weight / weight</td><td>T cell agonist,% weight / weight</td>

<td>NC-1'- Nicotine</td><td> 238</td><td>R848, 3.9</td><td>Owa peptide 323-339, 2.8</td>

Immunization and outcomes

[0210] Antibody titers were measured in mice immunized with the combination of NC-3'-nicotine and NC-1'-nicotine. NC-3'-nicotine and NC-1'-nicotine contained the T II OP-II helper peptide (2.1%) and R848 adjuvant (4.2%). Each bar in Fig. 8 represents the anti-antigen titer. Five animals per group were immunized sc with 120 µg of each NC per injection, 2 times at 3-week intervals. The titers at day 33 after the first immunization are shown (ELISA made against polylysinnicotine respectively).

[0211] These results show that immunization with a combination of two NCs each containing the same antigen but in different steric orientations results in the generation of antibodies against both of these different orientations of the same antigen. When identical amounts of two NCs, the first having surface nicotine linked to the NC at the 3 'position and the second having the surface nicotine linked to the NC at the 1' position were used to immunize the animals, a strong humoral response was induced in all animals against both nicotine orientations (Fig. 8). No reactivity was detected in the sera of mice before immunization.

Example 13: Preparations of polymers and nanocarriers

Preparation of PLGA-R848

[0212] PLGA-R848 was prepared by reacting the PLGA polymer having an acid end group with R848 in the presence of a coupling agent such as HBTU as follows. A mixture of PLGA (Lakeshores Polymers, MW -5000, 7525DLG1A, acid number 0.7 mmol / g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) in anhydrous EtOAc (160 mL) was stirred at under argon for 50 minutes. Compound R848 (2.2 g, 7 mmol) was added followed by diisopropylethylamine (DIPEA) (5 ml, 28 mmol). The mixture was stirred at room temperature for 6 hours and then at 50-55 ° C overnight (about 16 hours). After cooling, the mixture was diluted with EtOAc (200 mL) and washed with saturated NH4Cl solution (2 x 40 mL), water (40 mL), and brine solution (40 mL). The solution was dried over Na2SO4 (20 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA) (300 mL) was then added and the polymer conjugate precipitated from solution. The polymer was then washed with IPA (4 x 50 ml) to remove residual reagents and dried under vacuum at 35-40 ° C for 3 days as a white powder (10.26 g, GPC MW was 5200, R848 loading was 12% by HPLC).

[0213] In a similar manner, PLA-R848 was prepared by reacting PLA-CO2H (polylactide with an acid end group) with R848.

Preparation of PLA-PEG-CO2H

[0214] A mixture of HO-PEG-CO2H (MW: 2000, 1.0 g, 0.5 mmol), dl-lactide (10.8 g, 75 mmol) and Na2SO4 (15 g) in a 100 mL round bottom flask was dried in under vacuum at 60 ° C for 2 days. Anhydrous toluene (30 ml) was added and the mixture was refluxed under argon. Sn (Oct) 2 (0.162 ml, 0.5 mmol) was added. The mixture was refluxed under argon overnight and cooled to room temperature. The mixture was diluted with CH2Cl2 (200 mL) and filtered through a pad of celite. The filtrate was concentrated to a thick sticky residue. 10% MeOH in diethyl ether (200 mL) was added to precipitate the polymer with vigorous stirring. The polymer was then washed with 10% MeOH in ether (100 mL) and dried under vacuum at 30 ° C to give the PLA-PEG-CO2H copolymer as an off-white foam solid (10.0 g, H NMR in CDC13 showed the polymer was MW 21,000).

Preparation of PLA-PEG-NH2

A mixture of HO-PEG-NH2.HCl (MW: 3500, 1.0 g, 0.28 mmol), dl-lactide (6.1 g, 42 mmol) and Na2SO4 (10 g) in 100 ml the round bottom flask was dried under vacuum at 60 ° C for 1 day. Anhydrous toluene (30 ml) was added and the mixture was heated to 90 ° C under an argon atmosphere. Sn (Oct) 2 (0.1 mL, 0.28 mmol) was added. The mixture was refluxed under argon overnight and cooled to room temperature. The mixture was diluted with ethyl acetate (200 ml) and filtered through a pad of celite. The filtrate was concentrated to a thick sticky residue. 10% MeOH in t-butyl methyl ether (MTBE) (200 mL) was added to precipitate the polymer with vigorous stirring. The polymer was then washed with 5% MeOH in MTBE (50 ml) and MTBE (50 ml) and dried under vacuum at 30 ° C to give the PLA-PEG-NH2.HC1 copolymer as an off-white foam solid (5.0 g, H NMR in CDC13 showed the polymer to have a MW of 18,000).

Preparation of PLA-PEG-PEG3-N3

[0216] PLA-PEG-N3 polymer was prepared by ring-opening polymerization of HO-PEG azide with dl-lactide in the presence of a catalyst such as Sn (Oct) 2 as follows. HO-PEG-CO2H (MW 3500, 1.33 g, 0.38 mmol) was treated with NH2-PEG3-N3 (MW 218.2, 0.1 g, 0.458 mmol) in the presence of DCC (MW 206, 0.117 g, 0 , 57 mmol) and NHS (MW 115, 0.066 g, 0.57 mmol) in dry DCM (10 mL) overnight. After filtration to remove the insoluble by-product (DCC-urea), the solution was concentrated and then diluted with ether to precipitate the polymer, HO-PEG-PEG3-N3 (1.17 g). After drying, HO-PEG-PEG3-N3 (MW 3700, 1.17 g, 0.32 mmol) was mixed with dl-lactide (recrystallized from EtOAc, MW 144, 6.83 g, 47.4 mmol) and Na2SO4 (10 g) in a 100 ml flask. The solid mixture was dried under vacuum at 45 ° C overnight and dry toluene (30 ml) was added. The resulting suspension was heated to 110 ° C under argon and Sn (Oct) 2 (MW 405, 0.1 mL, 0.32 mmol) was added. The mixture was refluxed for 18 hours and then cooled to room temperature. The mixture was diluted with DCM (50 ml) and filtered. After concentration, MTBE (200 ml) was added to the oily residue to precipitate the polymer, which was washed once with 100 ml of 10% MeOH in MTBE and 50 ml of MTBE. After drying, PLA-PEG-PEG3-N3 was obtained as a white foam (7.2 g, average MW: 23,700 by H NMR).

Preparation of PLA-PEG-C6-N3

HO-PEG-CO2H (MW 3500, 1.00 g, 0.29 mmol) was treated with 6-azido-1hexylamine (H2N-C6-N3) (MW 142, 0.081 g, 0.57 mmol) in the presence of DCC (MW 206, 0.118 g, 0.57 mmol) and NHS (MW 115, 0.066 g, 0.57 mmol) in dry DCM (10 mL) overnight. After filtration to remove the insoluble by-product (DCC-urea), the solution was concentrated and then diluted with MTBE to precipitate the polymer, which was then washed twice with MTBE and dried under vacuum at 30 ° C overnight to give the polymer HO-PEG-C6-N3 (1.1 g). HO-PEG-C6-N3 polymer (1.1 g, 0.29 mmol) and dl-lactide (6.5 g, 45 mmol) were mixed in dry toluene (60 ml). The mixture was heated to reflux, while 30 ml of toluene was removed by azeotropic distillation. The resulting solution was cooled to 100 ° C and Sn (Oct) 2 (0.095 ml,

-660.29 mmol). The solution was refluxed under argon overnight and then cooled to room temperature. The solution was then added to 150 ml of 2-propanol to precipitate the polymer, which was washed with 2-propanol (100 ml) and dried under vacuum at 30 ° C for 2 days, yielding the PLA-PEG-C6-N3 copolymer as an off-colored solid. white (6.8 g, the MW by GPC is 27,000 at DPI 1.5).

Preparation of PLA-PEG (5K) -CONH2NH2

[0218] HO-PEG (5k) -CO2H mixture (JenKem Technology, US) (MW: 5000, 1.0 g,

0.2 mmol), tert-butyl carbazate (Boc-hydrazide) (MW: 132, 0.053 g, 0.4 mmol), DCC (MW 206, 0.083 g, 0.4 mmol), and N-hydroxysuccinimide (NHS) ( MW 115, 0.05 g, 0.4 mmol) in dry DCM (15 ml) was stirred at room temperature for 25 h. Insoluble DCC-urea was removed by filtration and the filtrate was concentrated. The residue was added to 50 ml of MTBE to precipitate the polymer, which was washed twice with 40 ml of MTBE and dried under vacuum for 2 days, yielding HO-PEG (5k) -CONHNHtBoc as a white powder (1.07 g). HO-PEG (5k) -CONHNHtBoc polymer (1.07 g, 0.20 mmol) and dl-lactide (4.32 g, 30 mmol) were mixed in dry toluene (70 ml). The mixture was heated to reflux, while 50 ml of toluene was removed by azeotropic distillation. The resulting solution was cooled to 100 ° C and Sn (Oct) 2 (0.065 mL, 0.20 mmol) was added. The solution was refluxed under argon for 22 hours and cooled to room temperature. The solution was then added to 150 ml of 2-propanol to precipitate the polymer, which was washed with 2-propanol (60 ml) and dried under vacuum at 30 ° C for 2 days to give the PLA-PEG (5k) -CONHNHtBoc copolymer as a white body. The polymer was dissolved in 50 ml of dry DCM and cooled with ice water. Trifluoroacetic acid (TFA) (15 ml) was added and the resulting solution was stirred at room temperature overnight. The yellowish solution was concentrated to dryness. The residue was added to 200 ml of 2-propanol to precipitate the polymer which was washed with 100 ml of 2-propanol. The polymer was dried at 30 ° C under vacuum to give the desired polymer as PLA-PEG (5k) CONHNH2 (3.4 g, MW by NMR: 24,000)

Preparation of PLA-PEG-MAL

HO-PEG (3K) -maleimide (HO-PEG-MAL) (Laysan Bio, Inc) (MW: 3000, 0.6 g, 0.2 mmol) was mixed with dl-lactide (recrystallized from EtOAc, MW 144, 4.32 g, 30 mmol) and Na2SO4 (4 g) in a 100 mL flask. The solid mixture was dried under vacuum at 60 ° C overnight and dry toluene (20 ml) was added. The resulting suspension was heated to 110 ° C under argon and Sn (Oct) 2 (MW 405, 0.065 ml, 0.2 mmol) was added. The mixture was refluxed for 20 hours and then cooled to room temperature. The mixture was diluted with DCM (50 ml) and filtered. After concentration, 10% MeOH in diethyl ether (80 ml) was added to the oily residue to precipitate the polymer which was washed once with 80 ml of 10% MeOH in ether and 60 ml of ether. After drying at 30 ° C under vacuum overnight, PLA-PEG (3K) -MAL was obtained as a white foam (3.26 g, average weight: 24,000 by H NMR).

-67 Receiving PLA-PEG-SH (Predictive)

[0220] PLA-PEG-SH copolymer is prepared according to the literature (Nisha C. Kalarickal et al., Macromolecules 2007, 40: 1874-1880). Briefly, the following steps are performed.

[0221] Step-1. Preparation of tBuS-PEG: Anhydrous THF (22 ml), potassium naphthalene (0.2 M solution in THF, 12 ml) and tBu-SH (0.54 ml, 4.8 mmol) are introduced into a sealed 100 ml round bottom flask. . The ingredients are mixed for at least 15 min to ensure thiolate formation, at which point liquid ethylene oxide (EO) (11.5 mL, 0.230 mol) is added using a double-ended needle. The polymerization reaction is carried out for 48 hours and the product is recovered by precipitation in cold diethyl ether. The MW of the polymer by GPC is about 2100.

[0222] Step-2. Preparation of (PEG-S) 2: tBu-S-PEG from step 1 (1.0 g) was dissolved in DMSO (19 ml) and then TFA (106 ml, 15/85 v / v) was added to the final concentration of polymer 8 mg / ml. The reaction mixture is stirred for 20 minutes, then the TFA is removed on the rotary evaporator. The residue is then precipitated twice in cold diethyl ether to recover crude PEG disulfide. The crude (PEG-S) 2 is further purified by fractionated precipitation. Thus, the polymer (1.0 g) was dissolved in dichloromethane (100 mL) and then cold diethyl ether was added gradually with stirring until a precipitate appeared. The solution was further stirred for 30 minutes, and the precipitated mass was isolated by filtration and dried under vacuum. The recovery of PEG disulfide (PEG-S) 2 at the end of the two to three fractions is in the range 55-60%.

Step-3. Preparation of (PLA-b-PEG-S) 2 by ring opening polymerization of dl-lactide: (PEG-S) 2 (0.4 g, 0.10 mmol) and dl-lactide (4.32 g, 30 mmol) stirred in dry toluene (70 ml). The mixture is heated to reflux and 50 ml of toluene are removed by azeotropic distillation. The resulting solution was cooled to 100 ° C and Sn (Oct) 2 (0.065 mL, 0.20 mmol) was added. The solution is refluxed under argon for 18-20 hours and cooled to room temperature. The solution is then added to 150 ml of 2-propanol to precipitate the polymer which is washed with 2-propanol (60 ml) and ether (60 ml) and dried under vacuum at 30 ° C for 2 days to give (PLA-PEG-S) 2 (about 4.0 g, MW: 46,000)

[0224] Step-4. Preparation of PLA-PEG-SH by reduction of (PLA-PEG-S) 2: (PLAPEG-S) 2 from step 3 (3.2 g, 0.07 mmol) was dissolved in deoxygenated THF (25 ml) and Bu3P ( 1.7 ml, 7.0 mmol, 100 eq based on the disulfide units). The reaction mixture is stirred under argon at room temperature overnight. The reduced thiolated polymer is recovered by precipitation in cold diethyl ether, then filtered under argon and further dried under vacuum to yield PLAPEG-SH as an off-white solid body (about 3.0 MW: 23,000)

Preparation of nanocarriers with a PEG-X surface containing encapsulated Ova peptide

[0225] Nanocarriers containing PLGA-R848, PLA-PEG-X (where X = carboxylic acid (CO2H), amine (NH2), C6-azide (C6-N3) or PEG3 azide (PEG3-N3), hydrazide

-68 (CONHNH2), maleimide (MAL), thiol (SH) and nitrilotriacetic acid (NTA)) containing the ova peptide were obtained by a double emulsion process, the ova peptide being encapsulated in nanocarriers. Polyvinyl alcohol (Mw = 11 KD - 31 KD, 87-89% partially hydrolyzed) was purchased from JT Baker. Ovalbumin Peptide 323-339 (sequence: H-Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-ArgNH2 acetate salt, Lot # B06395 ) obtained from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505), acid-terminated PLA (100DL2A) was obtained from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211); PLGA-R848 and PLA-PEG-X conjugates were prepared as described above in the same example.

[0226] The above materials were used to prepare the following solutions:

1. PLGA-R848 conjugate in methylene chloride @ 100mg / ml,

2. PLA-PEG-X in methylene chloride @ 100 mg / ml,

3. PLA (100DL2A) in methylene chloride @ 100mg / ml,

4. Ovalbumin peptide 323 - 339 in 0.13N HCl @ 70 mg / ml i

5. Polyvinyl alcohol in 100 mM phosphate buffer, pH 8 @ 50 mg / ml.

[0227] Solution # 1 (0.50 ml), solution # 2 (0.25 ml) and solution # 3 (0.25 ml) were combined and solution # 4 in 0.13N HCl (0.1 ml) was added to the solution. a small vessel and the mixture was sonicated at 50% amplitude for 40 seconds using the Branson Digital Sonifier 250. Solution # 5 (2.0 ml) was added to this emulsion and sonicated at 30% amplitude for 40 seconds using the Branson Digital Sonifier 250 on the second emulsion. Then, added to a beaker containing 70 mM phosphate buffer pH 8 (30 ml), and this mixture was stirred at room temperature for 2 hours to form nanocarriers.

[0228] To wash the nanocarriers, a portion of the nanocarrier dispersion (26.5 ml) was transferred to a 50 ml centrifuge tube and centrifuged at 9,500 rpm (rpm - 13,800 g) for one hour at 4 ° C. The supernatant was removed and the pellet was resuspended in 26.5 ml of phosphate buffered saline. The centrifugation procedure was repeated and the pellet was resuspended in 8.3 g of phosphate buffered saline for a final nanocarrier dispersion of approximately 10 mg / ml containing the encapsulated ova peptide.

Preparation of nanocarriers with PEG-X surface without encapsulated ova peptide

[0229] In a similar manner to the procedure described directly above, nanocarriers without the ova peptide were produced where solution # 4 was eliminated in the formulation.

Example 14: Nanocarriers with the derived antigen (prognostic)

[0230] PTH ​​Nanocarriers: PEG-CONHNH2 surface hydrazide nanocarriers are prepared as described above in Example 13. The PTH (parathyroid hormone) protein is acylated by the lysine amino group with 4-formylbenzoic acid in the presence of EDC. HCl and NHS are used to generate PTH containing benzaldehyde groups. After purification by dialfiltration using a MWCO 1K filter, the modified PTH is coupled to NCs containing hydrazide on the surface in a buffer

-69PBS (pH 8-9). After purification by washing the pellet with PBS buffer, the resulting NC-modified PTH conjugate is suspended in pH 7.4 buffer.

[0231] PTH-modified nanocarriers: PEGCO2H surface group nanocarriers are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer at pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS. Modified PTH dissolved in the same PBS buffer is then added to the resulting NC suspension. Coupling can be performed at 4 ° C overnight. After washing the pellet with PBS buffer, the resulting NC-modified PTH conjugate was suspended in PBS buffer pH 7.4.

[0232] Equal parts of the two nanocarriers can then be combined to form an NC suspension for further study.

Example 15: Monovalent and bivalent nanocarriers with antigens of the same kind of infectious agents (prognostic)

[0233] Nanocarriers with surface PEG-CO2H groups are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. The influenza A HA protein trimer and M2e HA protein dissolved in pH 6.0 buffer are then added to the resulting NC suspension. Coupling can be performed at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained trimer NC-HA / M2e protein conjugate is suspended in PBS buffer at pH 7.4.

[0234] In the same manner, a monomeric NC-HA protein conjugate is prepared using the monomeric HA protein of human influenza A virus.

[0235] The nanocarriers can then be combined to form an NC suspension for further studies.

Example 16: Monovalent and bivalent nanocarriers with antigens from other types of infectious agents (prognostic)

[0236] Nanocarriers with surface PEG-CO2H groups are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. The influenza A HA protein trimer and M2e HA protein dissolved in pH 6.0 buffer are then added to the resulting NC suspension. Coupling can be performed at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained trimer NC-HA / M2e protein conjugate is suspended in PBS buffer at pH 7.4.

[0237] In the same manner, an infectious salmon anemia virus conjugate is prepared using an inactivated infectious salmon anemia virus.

[0238] The nanocarriers can then be combined to form an NC suspension for further study.

-70 Example 17: Monovalent nanocarriers with antigens of the same species of the infectious agent (prognostic)

[0239] Nanocarriers with surface PEG-CO2H groups are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. Measles hemagglutinin antigen (recombinant fragment containing measles hemagglutinin immunodominant regions, amino acids 106-114 and 519-550) is dissolved in pH 6.0 buffer and then added to the resulting NC suspension. Coupling can be performed at 4 ° C overnight. After washing the pellet with PBS buffer, the resulting measles Nc-hemagglutinin conjugate is resuspended in PBS buffer pH 7.4.

[0240] In the same manner, an NC-measles fusion antigen conjugate is prepared using a fragment of a measles fusion protein (a recombinant fragment corresponding to amino acids 399-525 of a large measles fusion protein).

[0241] The nanocarriers can then be combined to form an NC suspension for further study.

Example 18: Monovalent nanocarriers with antigens of different species of the infectious agent (prognostic)

[0242] Nanocarriers with surface PEG-CO2H groups are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. Human influenza A HA protein trimer dissolved in pH 6.0 buffer is then added to the resulting NC suspension. Coupling can be performed at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained NC-HA protein conjugate trimer was suspended in PBS buffer pH 7.4 for further testing.

[0243] Streptococcus pneumonia (PnPs) 6B polysaccharide was selected as a representative of the PnPs serotype. Purified native (ie no size reduction after purification) PnPs-6B was dissolved in 2M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) is added (CDAP / PnPs ratio: 1.0 mg / mg). The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, the resulting activated PnPs6B solution is added to NC with PEG-CONHNH2 (PEG-hydrazide) surface groups prepared as described above in Example 13 in pH 9 buffer. The resulting NC and PnPs-6B suspension is shaken for 1 hour and quench with 2 M glycine solution. After washing the pellet with PBS buffer, the obtained NC-PnPs-6B conjugate was suspended in PBS buffer pH 7.4.

[0244] The nanocarriers can then be combined to form an NC suspension for further study.

Example 19: Monovalent nanocarriers with antigens from the same strain of an infectious agent (prognostic)

[0245] PEG-X nanocarriers on the surface are prepared as follows. Monodisperse PRINT nanocarriers (PRINT NC) containing PLGA-R848, PLA-PEG-X (where X = carboxylic acid (CO2H), amine (NH2), C6-azide (C6-N3) or PEG3-azide (PEG3-N3), hydrazide (CONHNH2), maleimide (MAL) and thiol (SH)) containing the ova peptide are obtained by the replication method of molecules in dry templates. Particle Replication in Non-wetting Templates (PRINT) as described in the literature ((1) "Direct Fabrication and Harvesting of Monodisperse, Shape Specific Nano-Biomaterials"; Rolland, JP; Maynor, BW; Euliss, LE; Exner, AE; Denison, GM; DeSimone, J. M J. Am. Chem. Soc. 2005, 127, 10096; (2) "The Complex Role of Multivalency in Nanoparticles Targeting the Transferrin Receptor for Cancer Therapies" Jin Wang, Shaomin Tian, ​​Robby A Petros, Mary E. Napier, and Joseph M. DeSimone, J. Am. Chem. Soc., 2010, 132 (32), pp. 11306-11313). PRINT-NC with surface PEG-CO2H groups was activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. Pneumococcal surface protein A (PspA) dissolved in pH 6.0 buffer is then added to the resulting NC suspension. Coupling can be performed at 4 ° C overnight. After washing the pellet with PBS buffer, the resulting NC-PsPA conjugate is resuspended in PBS buffer pH 7.4.

[0246] Purified native PnPs-6B was dissolved in 2M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) is added (CDAP / PnPs ratio: 1.0 mg / mg). The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, the resulting activated PnPs-6B solution is added to PRINT NC with PEGCONHNH2 (PEG-hydrazide) surface groups prepared as described above in pH 9 buffer. The resulting NC and PnPs-6B suspension is shaken for 1 hour and quenched with 2M glycine solution. After washing the pellet with PBS buffer, the obtained NC-PnPs-6B conjugates were suspended in PBS buffer pH 7.4.

[0247] The nanocarriers can then be combined to form an NC slurry for further studies.

Example 20: Monovalent nanocarriers with antigens from different strains of the infectious agent (prognostic)

[0248] Purified native PnPs-6B was dissolved in 2M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) is added (CDAP / PnPs ratio: 1.0 mg / mg). The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, the resulting activated PnPs-6B solution is added to NC with PEGCONHNH2 (PEG-hydrazide) surface groups prepared as described above in Example 13 in pH 9 buffer. The resulting NC and PnPs-6B suspension is shaken for 1 hour and quenched with 2M glycine solution. After washing the pellet with PBS buffer, the obtained NC-PnPs6B conjugates were suspended in PBS buffer pH 7.4.

[0249] Purified native PnPs14 was dissolved in 2M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) is added (CDAP / PnPs ratio: 1.0 mg / mg). The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, the resulting activated PnPs14 solution is added to PRINT NC with PEGCONHNH2 (PEG-hydrazide) surface groups prepared as described above in pH 9 buffer. The resulting PRINT NC and PnPs14 suspension is shaken for 1 hour and quenched with 2M glycine solution. After washing the pellet with PBS buffer, the obtained PRINT NCPnPs14 conjugates were suspended in PBS buffer pH 7.4.

[0250] Gold NC with a PEG-X surface (where X = carboxylic acid (CO2H), amine (NH2), azide (N3), hydrazide (CONHNH2) and aldehyde (CHO)) is prepared as follows.

Step-1. Formation of gold NC (AuNC): An aqueous solution of 500 ml of 1 mM HAuC14 is heated to reflux for 10 minutes with vigorous stirring in a 1 L round bottom flask equipped with a condenser. A solution of 50 ml of 40 mM trisodium citrate is then quickly added to the stirred solution. The resulting deep wine red solution is refluxed for 25-30 minutes, the heat is withdrawn and the solution is cooled to room temperature. The solution is then filtered through a 0.8 µm membrane filter to obtain an AuNC solution. AuNC is characterized by visible light spectroscopy and transmission electron microscopy. AuNCs have a diameter of approx. 20 nm limited by citrate with an absorption peak at 520 nm.

Step-2. AuNCs functionalized with PEG-X with HS-PEG-X: AuNCs are functionalized with HS-PEG-X (MW range: 1500-5000) (where X = carboxylic acid (CO2H), amine (NH2), azide ( N3), hydrazide (CONHNH2), and aldehyde (CHO)) as follows. A solution of 150 ml of HS-PEG-X (10 μM in 10 mM carbonate buffer, pH 9.0) is added to 1 ml of citrate-limited gold nanocarriers with a diameter of 20 nm (1.16 nM) to produce a molar ratio of thiol to gold 2500: 1. The mixture is stirred at room temperature under argon for 1 hour to allow complete replacement of the thiol with citrate on the gold nanocarriers. The PEG-X AuNC on the surface is then purified by centrifugation at 12,000 g for 30 minutes. The supernatant was decanted and the AuNC-PEG-X pellet was resuspended in appropriate PBS buffer for further bioconjugation with biomolecules. Purified native PnPs-19F was dissolved in 2M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) is added (CDAP / PnPs ratio: 1.0 mg / mg). The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, the resulting activated PnPs19F solution is added to AuNC with PEG-CONHNH2 (PEG-hydrazide) surface groups prepared as described above in pH 9 buffer. The resulting suspension of AuNC and PnPs19F is shaken for 1 hour and quenched with 2M glycine solution. After washing

-73pellet with PBS buffer, the obtained AuNC-PnPs-19F conjugates were suspended in PBS buffer at pH

7,4.

[0253] The nanocarriers can then be combined to form an NC slurry for further study.

Example 21: Monovalent nanocarriers with the same antigen but different orientation (prognostic)

[0254] PRINT NC with a nicotine surface analog linked via the 3 'position is prepared from a PLA-PEG-3-HO-MeNic copolymer derived from trans-3'hydroxymethylnicotine (3-HO-MeNic), PLGA-R848 and ova peptide as described above. The resulting PRINT NC containing the 3'-surface nicotine analogue is suspended in a buffer at pH 7.4.

[0255] In a similar manner, PRINT NC with a nicotine surface analog linked via the 1 'position is prepared from a PLA-PEG-1-butyl-Nic copolymer obtained from 1'-butyl-nicotine (1-butyl-Nic), PLGA-R848 and the OVA peptide as described above. The resulting PRINT NC containing the 1'-surface nicotine analogue is suspended in a buffer at pH 7.4.

[0256] The nanocarriers can then be combined to form an NC slurry for further studies.

Example 22: Monovalent nanocarriers with the same antigen but different conformation (prognostic)

[0257] Nanocarriers with surface PEG-CO2H groups are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. Measles virus hemagglutinin loop epitope (HNE, H379-410, intact disulfide) dissolved in pH 6.0 buffer is then added to the resulting NC suspension. Coupling can be performed at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained NC-HNE conjugates were suspended in PBS buffer pH 7.4.

[0258] The highly conserved measles virus hemagglutinin loop epitope (HNE, H379-410) contains three cysteine ​​residues, two of which (Cys386 and Cys394) form a disulfide bridge. The HNE peptide containing the disulfide bridge is reduced with dithiothreitol (DTT) in PBS buffer to obtain reduced HNE. NCs with PEG-CO2H surface groups are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. The reduced HNE dissolved in pH 6.0 buffer is then added to the resulting NC suspension under argon in the presence of DTT. Conjugation can be carried out at 4 ° C

Overnight under argon. After washing the pellet with PBS buffer, the resulting NC-reduced HNE conjugates are resuspended in PBS pH 7.4.

[0259] The nanocarriers can then be combined to form an NC slurry for further studies.

Example 23: Monovalent and bivalent nanocarriers with low molecular weight antigens of different structures (Prognostic)

[0260] AuNCs with PEG-CO2H surface groups are obtained as described above. The AuNCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. Trans-3'aminomethylnicotine prepared from commercially available 4-cotininecarboxylic acid (US patent application: US2007 / 0129551 Al) in a buffer at pH 6.0 is added to the activated AuNC. Coupling can be performed at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained AuNC-nicotine conjugates are suspended in PBS buffer pH 7.4.

[0261] VLPs with surface functional groups such as azide or alkyne for CuAAC click chemistry are prepared as described in the literature ("Surface Functionalization of Virus-Like Particles by Direct Conjugation Using Azide-Alkyne Click Chemistry", Kedar G Patel and James R. Swartz; Bioconjugate Chem. 2011, 22 (3), pp. 376387). A cocaine analog containing an alkyne or azide linker and a methamphetamine analog containing an alkyne or azide linker are prepared according to literature procedures as B-cell surface antigen epitopes. The molar equivalent of a mixture of cocaine analog and methamphetamine analog with azide linker is treated with a VLP containing a surface alkyne group under standard conditions CuAAC to form VLP-cocaine-methamphetamine conjugates.

[0262] The nanocarriers can then be combined to form an NC slurry for further studies.

Example 24: Bivalent nanocarriers with structured oligosaccharide antigens (prognostic)

[0263] Purified PnPs-6B are size reduced with dilute acid or under sonication conditions to give oligomeric PnPs-6B which is dissolved in 2 M NaCl. Similarly, purified PnPs-3 are size reduced with dilute acid or under sonication conditions to give oligomeric PnPs-3 which is dissolved in 2M NaCl. A molar-equilibrated mixed solution of oligomeric PnPs-6B and PnPs-3 is prepared from these solutions. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) (CDAP / PnPs ratio: 1.5 mg / mg) is added to the stirred PnPs solution. The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, the resulting activated PnPs-6B / PnPs-3 oligomeric solution is added to AuNC with PEGCONHNH2 (PEG-hydrazide) surface groups prepared as described above in pH 9 buffer.

The resulting AuNC and the activated PnPs-6B / PnPs-3 suspension are shaken for 1 hour and the reaction is quenched with 2M glycine solution. After washing the pellet with PBS buffer, the obtained AuNC-PnPs-6B / 3 conjugates were suspended in PBS buffer at pH 7.4.

[0264] VLPs containing carboxylic acid (CO2H) groups on the surface are activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated VLP pellets were then washed with pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in pH 6.0 buffer. Purified PnPs-4 is size reduced with dilute acid or under sonication conditions to give oligomeric PnPs-4 which is dissolved in 2M NaCl. Likewise, purified PnPs-19F is size reduced with dilute acid or under sonication conditions to give oligomeric PnPs-19F which is dissolved in 2M NaCl. A molar-equilibrated mixed solution of oligomeric PnPs-4 and PnPs-19F is prepared from these solutions. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) (CDAP / PnPs ratio: 1.5 mg / mg) is added to the stirred PnPs solution. The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, a solution of adipic acid dihydrazide (ADH) linker in pH 9 buffer is added to the activated mixed PnPs-4 / 19F solution. The resulting solution is stirred for 1 hour and quenched with 2M glycine solution and purified by dialysis. Purified oligomeric PnPs-4 / 19F with ADH linker in pH 6.0 buffer is then added to activated VLPs in pH 6.0 buffer and the resulting suspension is stirred at 4 ° C overnight and purified by dialysis or pellet washing. producing VLP-PnP-4 / 19F conjugates for further research.

[0265] The nanocarriers can then be combined to form an NC slurry for further study.

Example 25: Bivalent nanocarriers with structured polysaccharide antigens (prognostic)

[0266] Purified native PnPs-6B was dissolved in 2M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) is added (CDAP / PnPs ratio: 1.0 mg / mg). The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, a solution of adipic acid dihydrazide (ADH) linker in pH 9 buffer is added to the activated mixed PnPs-6B solution. The resulting solution is stirred for 1 hour and purified by dialysis. Purified PnPs-6B with ADH linker is dissolved in pH 6.0 buffer for NC coupling.

[0267] Purified N. meningitidis meningococcal polysaccharide serogroup A (NmA) is dissolved in 1 M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) (CDAP / NmA ratio: 1.5 mg / mg) is added. The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, adipic acid (ADH) dihydrazide linker solution in pH 9 buffer is added to the activated mixed NmA solution.

The resulting solution is stirred for 1-2 hours and purified by dialysis. Cleaned up

NmA with ADH linker is dissolved in pH 6.0 buffer for NC coupling.

[0268] Nanocarriers with surface PEG-CO2H groups are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. An equimolar mixed solution of PnPs-6B with ADH linker and NmA with ADH linker in pH 6.0 buffer is added to the activated NC solution and the resulting suspension is stirred at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained NC-PnPs6B / NmA conjugates were suspended in PBS buffer pH 7.4.

[0269] Purified native PnPs-19F was dissolved in 2M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) is added (CDAP / PnPs ratio: 1.0 mg / mg). The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, a solution of adipic acid dihydrazide (ADH) linker in pH 9 buffer is added to the activated mixed PnPs-19F solution. The resulting solution is stirred for 1 hour and purified by dialysis. Purified PnPs-19F with ADH linker is dissolved in pH 6.0 buffer for NC coupling.

[0270] Purified N. meningitidis meningococcal polysaccharide serogroup C (NmC) is dissolved in 1 M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) (CDAP / NmC ratio: 1.5 mg / mg) is added. The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, a solution of adipic acid dihydrazide (ADH) linker in pH 9 buffer is added to the activated mixed NmC solution. The resulting solution is stirred for 1-2 hours and purified by dialysis. Purified NmC with ADH linker is dissolved in pH 6.0 buffer for NC coupling.

[0271] Nanocarriers with surface PEG-CO2H groups are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. An equimolar mixed solution of PnPs-19F with ADH linker and NmC with ADH linker in pH 6.0 buffer is added to the activated NC solution and the resulting suspension is stirred at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained NC-PnPs-19F / NmC conjugates were suspended in PBS buffer pH 7.4.

[0272] The nanocarriers can then be combined to form an NC slurry for further study.

Example 26: Bivalent and monovalent nanocarriers with small molecule antigens in different orientations (prognostic)

[0273] PEG-CONHNH2 (PEG-hydrazide) surface nanocarriers are prepared as described above in Example 13 and suspended in a pH 6.0 buffer at 4 ° C.

-77 The cocaine analog GNC (6- (2R, 3S) -3- (benzoyloxy) -8-methyl-8-azabicyclo [3.2.1] octane-2-carbonyloxy-hexanoic acid) is prepared according to the described procedure ("Cocaine Analog

Coupled to Disrupted Adenovirus: A Vaccine Strategy to Evoke High-titer Immunity Against

Addictive Drugs ”Martin J Hicks et al., Mol Ther 2011, 19: 612-619). This compound was activated by EDC / NHS in DMF and the activated GNC-NHS ester was isolated and purified for NC conjugation. Another cocaine analogue, All, is prepared according to the reported procedure ("Positional linker effects in haptens for cocaine immunopharmacotherapy", Akira Ino, Tobin J. Dickerson, and Kim D. Janda; Bioorganic &amp; Medicinal Chemistry Letters 17 (2007) 42804283) and activates by EDC / NHS as above. An equimolar portion of each activated cocaine analog in excess to NC surface hydrazide PEG is mixed with NC in a pH 6.0 buffer. The resulting suspension is stirred at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained NC-GNC / cocaine Al1 conjugates were suspended in PBS buffer pH 7.4.

[0274] Norkocaine is treated with succinic anhydride to prepare cocaine containing the succinic acid linker, SNC, and then activates EDC / NHS according to the reported procedure (Fox BS, Kantak KM, Edwards MA et al. Efficacy of a therapeutic cocaine vaccine in rodent models. Nat. Med. 2 (10), 1129-1132 (1996) NC with surface PEG-CONHNH2 (PEG-hydrazide) is prepared as described above in Example 13 and suspended in pH 6.0 buffer at 4 ° C. An excess amount of activated cocaine analog, SNC, is added to the NC. The resulting suspension is stirred at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained NC-SNC cocaine conjugates were suspended in PBS buffer pH 7.4.

[0275] The nanocarriers can then be combined to form an NC slurry for further studies.

Example 27: Bivalent and monovalent nanocarriers with peptide antigens with various additives (prognostic)

[0276] Nanocarriers with surface PEG-azide (PEG-N3) are prepared according to Example 13 and suspended in pH 7 degassed buffer with argon. Ovalbumin (325-336) peptide with C-terminal propargylamide group (C-alkyne group) is obtained by standard solid phase peptide synthesis and the obtained purified Ova (325-336) -C-alkyne peptide is dissolved in pH 7 buffer in an argon atmosphere. Ovalbumin (325-336) peptide with N-terminal amine acylated with 5-hexanoic acid (N-terminal alkyne group) is obtained by standard solid phase peptide synthesis and the obtained purified OVA (325-336) -N-alkyne peptide is dissolved in a pH 7 buffer under an argon atmosphere. NC with surface PEG-N3 is mixed with an equimolar amount of each of the owa-C-alkyne and N-alkyne peptides in a buffer at pH 7 under argon, and the resulting suspension is subjected to a CUAAC click reaction according to the reported protocol ("Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation ”, Hong V, Presolski SI, Ma C, Finn MG .; Angew Chem Int Ed Engl. 2009; 48 (52): 9879-83). Obtained NC-ova-C-linked peptide / peptide conjugates

The -78ova-N-linked are purified by washing the pellet with pH 7 buffer and resuspended in pH 7 buffer.

[0277] Recombinant virus-like particles (VLPs) are prepared according to standard procedure. In particular, VLP from rabbit haemorrhagic disease virus is obtained and conjugated to the ova (323-339) peptide via a heterobifunctional linker such as sulfosuccinimidyl 4- (Nmaleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) as described by Matthew Peacey et al. ((1) Peacey M, Wilson S, Baird MA, Ward VK. "Versatile RHDV virus-like particles: incorporation of antigens by genetic modification and chemical conjugation" Biotechnol Bioeng; 2007; 98: 968-77; (2) Peacey M, Wilson S, Perret R, Ronchese F, Ward VK, Young V, Young S, Baird, MA. "Virus-like particles from rabbit hemorrhagic disease virus can induce ananti-tumor response" Vaccine; 2008; 26: 5334-5337). The resulting VLP-ova peptide conjugates are purified and resuspended in pH 7 buffer.

[0278] The nanocarriers can then be combined to form an NC slurry for further study.

Example 28: Monovalent nanocarriers with protein antigens coupled at different attachment points on the protein (rs protein tag activated) (prognostic)

[0279] Nanocarriers with surface PEG-CO2H groups are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. Measles hemagglutinin protein (MHP) dissolved in pH 6.0 buffer is then added to the resulting NC suspension. Coupling can be performed at 4 ° C overnight. After washing the pellet with PBS buffer, the resulting NCMHP conjugate was suspended in PBS buffer pH 7.4.

[0280] PEG-NTA surface nanocarriers for the Ni-His tag complex are prepared as described in Example 13. NC are then treated with NiCl2 solution in binding buffer (50 mM phosphate buffer system, 300 mM NaCl, 10 mM imidazole, pH 8 0) to generate NC with the NTA-Ni surface complex. After washing the pellet with PBS buffer, the resulting NC is suspended in a binding buffer under an argon atmosphere. A solution of His6-tagged recombinant measles haemagglutinin protein in binding buffer is added to the NC suspension and the suspension is incubated at 4 ° C overnight under argon. The resulting NC-NTA-His6-MHP conjugate pellets were washed with pH 7 buffer and resuspended in PBS buffer.

[0281] The nanocarriers can then be combined to form an NC slurry for further study.

Example 29: Monovalent nanocarriers with conjugated oligosaccharide antigens at different attachment points (activated hydroxyl group and linker) (prognostic)

[0282] Nanocarriers with surface groups of PEG-CONHNH2 (PEG-hydrazide) were prepared as described in Example 13 and suspended in a pH 9 buffer under an argon atmosphere. Purified PnPs-6B is reduced in size by dilute acid or by sonication, yielding oligomeric PnPs-6B which is dissolved in 2M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) (CDAP / PnPs ratio: 1.5 mg / mg) is added to the PnPs-6B solution. The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, the resulting activated PnPs-6B oligomeric solution is added to the NC with PEG-CONHNH2 (PEGhydrazide) surface groups. The resulting NC and the activated PnPs-6B suspension are shaken for 1 hour and quenched with 2M glycine solution. After washing the pellet with PBS buffer, the obtained NC-PnPs-6B conjugates were suspended in PBS buffer pH 7.4.

[0283] Nanocarriers with surface PEG-CO2H groups are prepared as described above in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. Oligo PnPs-6B with a 3-aminopropyl linker is prepared according to the reported method ("Synthetic 6 B Di-, Tri-, and Tetrasaccharide-Protein Conjugates Pneumococcal Type 6A and 6B Common and 6B- Specific Epitopes That Elicit Protective Antibodies in Mice", Jansen WTM et al. Infect Immun. 2001; 69 (2): 787-793). Oligomeric PnPs-6B-3-propylamine in pH 6 buffer is added to the activated NC. The resulting suspension is stirred at 4 ° C overnight under argon. After washing the pellet with PBS buffer, the NC-PnPs-6B conjugates were suspended in PBS buffer pH 7.4.

[0284] The nanocarriers can then be combined to form an NC slurry for further study.

Example 30: Monovalent nanocarriers with conjugated polysaccharide antigens at different attachment points on the polysaccharide (prognostic)

[0285] NmA is attached through the CDAP-activated hydroxyl groups to NC via multiple attachment points. Purified N. meningitidis meningococcal polysaccharide serogroup A (NmA) is dissolved in 1 M NaCl. A solution of 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) in CH3CN (100 mg / ml) (CDAP / NmA ratio: 1.5 mg / mg) is added. The pH of the resulting solution was adjusted to 9 with 0.2 M aqueous Et3N or dilute NaOH solution. After 3-4 minutes, adipic acid (ADH) dihydrazide linker solution in pH 9 buffer is added to the activated mixed NmA solution. The resulting solution is stirred for 1-2 hours and purified by dialysis. Purified NmA with ADH linker is dissolved in pH 6.0 buffer for NC coupling.

[0286] NC with PEG-CO2H surface groups are prepared as described in Example 13. NC are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer

-80 to remove unreacted EDC / NHS and resuspend in pH 6.0 buffer. Solution

NmA with ADH linkers in pH 6.0 buffer is added to the activated NC solution and the resulting suspension is stirred at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained NC-NmA conjugates were suspended in PBS buffer pH 7.4.

[0287] NmA becomes attached to the NC via the terminal amino group. NCs with PEG-CO2H surface groups are prepared as described in Example 13. NCs are then activated with excess EDC / NHS in PBS buffer pH 6 at 4 ° C for 1-2 hours. The activated NC pellets are then washed with a pH 6.0 buffer to remove unreacted EDC / NHS and resuspended in a pH 6.0 buffer. Purified NmA is subjected to reductive amination with NH4Cl and sodium cyanoborohydride (NaCNBH3) in a pH 7 buffer to produce amino-NmA according to the reported procedure ("Development and phase 1 clinical testing of a conjugate vaccine against meningococcus A and C", Costantino P , Viti S, Podda A, Velmonte MA, Nencioni L, Rappuoli R. Vaccine. 1992; 10 (10): 691-8). Amino-NmA is then added to the slurry of activated NC and the resulting slurry stirred at 4 ° C overnight. After washing the pellet with PBS buffer, the obtained NC-NmA conjugates were suspended in PBS buffer pH 7.4.

[0288] The nanocarriers can then be combined to form an NC slurry for further studies.

SEQUENCE LIST

&amp; Lt; 110 &amp; gt; Selecta Biosciences, Inc.

<120> SYNTHETIC POLYVALENT VACCINES

CARRIER <130> S1681.70014WO00 <150> US 61 / 348.713 <151> 2010-05-26 <150> US 61 / 348.717 <151> 2010-05-26 <150> US 61 / 348.728 <151> 2010-05 -26 <150> US 61 / 358,635 <151> 2010-06-25 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 17

-81 <212> PRT <213> G. gallus <400> 1

Ile Cheese Gin Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly 15 10 15

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