Synthetic nanocarrier combination vaccines

(57) Dosage forms and related methods are disclosed that comprise a first population of synthetic nanocarriers having one or more first antigens attached thereto, one or more second antigens that are not attached to synthetic nanocarriers, and a pharmaceutically acceptable excipient.

023397 Β1

Related Applications

This application claims priority according to 35 I..8.C. §119 of provisional applications US 61/348713, filed May 26, 2010, 61/348717, filed May 26, 2010, 61/348728, filed May 26, 2010, and 61/358 635, filed June 25, 2010. , the full contents of each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Both to minimize the number of childhood vaccinations and / or to provide a broader immune defense against different strains of a given pathogen, there is often a need to combine multiple antigens in a single dosage form, the resulting vaccine is called a multivalent vaccine. The amount of antigens that can be combined into a single dosage form can be limited by the amount of each antigen necessary to elicit the necessary immune response and the solubility of the antigen in water.

At some point, the total fluid volume of the dosage form becomes too large to conveniently and / or safely administer the vaccine intramuscularly and / or subcutaneously. This limitation is particularly noticeable in the case of a conjugate of multivalent vaccines, such as Rgeupag ™, where each excellent oligosaccharide antigen is conjugated to a protein carrier (for example, with 7 or 13 oligosaccharide antigens conjugated to SCM197, a non-toxic mutated diphtheria toxin); or tetravalent meningococcal vaccines, where antigens are also conjugated to SCM197 or other detoxified forms of diphtheria toxin.

Another limitation of existing vaccine formulations is their limited coverage or their physical incompatibility with each other, which may preclude the usual mixing of two existing vaccines to create a new, combined vaccine. For example, vaccines may consist of virus-like particles consisting of one or more antigens that self-assemble or bind to self-assembled proteins. Examples include Seguaph ™ and SagyayaP ™, which are vaccines against human papillomavirus (NRU). Both of these vaccines target antigens that are derived from protein L1 of a limited number of NRU strains. These vaccines do not provide protection against all strains of NRU. To expand the coverage of the strains of these vaccines, it is desirable to be able to mix in additional viral antigens that are compatible with existing vaccine formulations, which provides wider coverage, and therefore creates a new expanded multivalent vaccine. Under certain circumstances, it may not be possible to simply interfere with additional traditionally obtained antigens to an existing vaccine due to undesirable interactions between additional traditionally obtained antigens and an existing vaccine (which may lead to precipitation, aggregation, etc.).

Therefore, compositions and methods are needed that can solve the above problems associated with vaccines.

A brief description of the present invention

In one aspect, a dosage form is provided comprising (1) a first population of synthetic nanocarriers having one or more first antigens attached thereto, (2) one or more second antigens that are not attached to synthetic nanocarriers, and (3) a pharmaceutically acceptable excipient .

In one embodiment, any of the provided dosage forms further comprises one or more adjuvants that are associated with synthetic nanocarriers of the first synthetic nanocarrier population. In another embodiment, one or more adjuvants attached include any of the adjuvants provided herein. In one embodiment, one or more adjuvants include P1igoshs® block copolymers, specifically modified or prepared peptides, muramyl dipeptide, aminoalkyl glucosaminide 4 phosphates, KC529, bacterial toxoids, toxin fragments, To11-like receptor agonists 2, 3, 4, 5, 7, 8, 9 and / or combinations thereof; adenine derivatives; immunostimulatory DNA; immunostimulatory RNA; imidazoquinolinamines, imidazopyridinamines, 6,7-fused cycloalkylimidazopyridinamines, imidazoquinolinamines with bridging bonds in position 1 and 2; imiquimod; resimvod; type I interferons; poly 1: C; bacterial lipopolysaccharide (L8); U8U-O; NMOV-1; flagellin or part or derivatives thereof; or immunostimulatory DNA molecules containing CfO. In another embodiment, one or more attached adjuvants include an agonist of a 1-11-like receptor 2, 3, 4, 7, 8, or 9. In yet another embodiment, one or more adjuvants attached include imidazoquinoline or oxoadenine. In a further embodiment, imidazoquinoline comprises resiquimod or imiquimod.

In another embodiment, any of the dosage forms provided below includes one or more adjuvants that are not associated with synthetic nanocarriers of the first population of synthetic carriers. In one embodiment, one or more non-adjuvanted adjuvants include stimulators or agonists of image recognition receptors, mineral salts, alum, alum combined with enterobacteria monophosphoryl lipid A (MPB), MPB®

- 1 023397 (A804), A815, saponins, O8-21 / OC1-A, 18COM, 18COMATC1X ™, MP59 ™, Moyashbe® 18A 51, Mop1atbe® Ι8Α 720, A802, liposomes and liposome formulations, A801, synthesized or specifically prepared microparticles and microcarriers, bubbles of the outer membrane of bacterial origin from Ν. dyoggyeog Syuatafa Tgasiotike, chitosan particles, depot forming agents, Iigots® block copolymers, specifically modified or prepared peptides, muramyl dipeptide, aminoalkyl glucosaminide-4-phosphates, KC529, bacterial toxoids, fragments of toxins-3, 4, 5, 2 agonists 7, 8, 9 and / or combinations thereof; adenine derivatives; immunostimulatory DNA; immunostimulatory RNA; imidazoquinolinamines, imidazopyridinamines, 6,7 condensed cycloalkylimidazopyridinamines, imidazoquinolinamines with bridging in position 1 and 2; imiquimod; resimvod; agonist for the surface molecule OS SE40; type I interferons; poly 1: C; bacterial lipopolysaccharide (L8); U8U-O; NMSV-1; flagellin or part or derivatives thereof; immunostimulatory DNA molecules containing CfO; pro-inflammatory stimuli released from necrotic cells; urate crystals; activated components of the complement reaction cascade; activated components of immune complexes; complement receptor agonists; cytokines or agonists of the cytokine receptor. In another embodiment, one or more non-adjuvanted adjuvants include alum, A801, A802, A804, A815, MPB, 08-21. saponin or an immunostimulatory nucleic acid containing CfO.

In yet another embodiment, any of the provided dosage forms, one or more first antigens are identical to one or more second antigens.

In a further embodiment, any of the dosage forms further includes a second population of synthetic nanocarriers having one or more third antigens attached thereto; where the first and third antigens are not identical.

In a further embodiment, one or more of the first antigens of any of the dosage forms comprises an antigen for a B cell or an antigen for a T cell. In one embodiment, the antigen for a T cell is a universal antigen for a T cell or an antigen for a helper cell. In another embodiment, one or more of the first antigens includes an antigen for a B cell or an antigen for a T cell and a universal antigen for a T cell or an antigen for a T helper cell. In yet another embodiment, the antigen for the T helper cell includes a peptide that is derived from or derived from ovalbumin. In yet another embodiment, a peptide that is derived from or derived from ovalbumin includes the sequence set to 8E0 ΙΌ ΝΟ: 1. In an embodiment of any of the dosage forms, a universal antigen for a T cell or an antigen for a T helper cell is coupled by encapsulation. In another embodiment, one or more second antigens of any of the dosage forms comprises an antigen for a B cell or an antigen for a T cell.

In one embodiment, any of the provided dosage forms includes a vaccine that contains a second antigen that is not attached to synthetic nanocarriers. In another embodiment, the vaccine comprises a hapten-carrier conjugate, a virus-like particle, a synthetic nanocarrier vaccine, a protein subunit vaccine, or an attenuated virus. In yet another embodiment, a vaccine is any vaccine provided herein. In yet another embodiment, the vaccine is intended against any infectious agent provided herein. In yet another embodiment, an anthrax vaccine; diphtheria, tetanus and / or whooping cough; hemophilic influenza type B; hepatitis B; hepatitis A; hepatitis C; herpes zoster (depriving); human papillomavirus (NRU); flu Japanese encephalitis; tick-borne encephalitis; measles, mumps and / or rubella; meningococcal infection; pneumococcal infection; poliomyelitis; rabies; rotavirus; typhoid fever; chicken pox; vaccines (smallpox) or yellow fever. In a further embodiment, the vaccine comprises V1OTNKAH, □ Arthas IΝΡΆΝΚIΧ, TN1RE1) 1A TK1N1V1T, ΚΙΝΚΙΧ, REP1AK1H, REETASE, REPUAHN1V, ASTN1V, N1VEK1H, Soma NAUK1H, UAOTA, EΝOEΚIΧ-Β, KESOMV1UAH HB, ΤΑΙΝΗΙΧ, 2O8TAUAH, OAKPA81, SEKUAK1H, RIAMKH, RIU1KZH, EEE / OXT / RIEEUAE, AEJYYA, AOMRY, RYM18T, DE-UAH, 1X1AKO, M-M-K II, PKO0A1T ME ^ MIEE, MEOSE, MEUEUKEU, PEXU, REXU, REXU, REXU, REX KAVAUEKT, KOTATEO, KOTAK1X, OESAUAS, VOO8TK1X, AOACE, TURN1M U1, U1UOT1R VEKT, UAK1UAH, ASAM2000 or ΥΡ-UAAH.

In another embodiment, one or more first antigens and / or one or more second antigens are obtained or derived from any of the infectious agents provided herein. In one embodiment, the infectious agent is a virus of the Abeyoshushbay, Ryogiaoutbay, Negrekutbay, Nerabiautbay, P1autushbay, Keyousbay, Obyoshuhousbay, Ragatuhoutbay, Rar Shotaushbay, Klabousbaye, Todautbasg or Viruses. In yet another embodiment, one or more of the first antigens and / or one or more of the second antigens is derived or derived from adenovirus, Coxsackie virus, hepatitis A virus, poliovirus, rhinovirus, herpes simplex virus, chickenpox virus, Epstein-Barr virus, human cytomegalovirus ,

- 2 023397 human herpesvirus, hepatitis B virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, Ηΐν, influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, human papillomavirus rabies virus, rubella virus, human bokavirus or parvovirus B19. In yet another embodiment, one or more of the first antigens and / or one or more of the second antigens are derived or derived from bacteria of the genus VogbsUP. Votteya, Vtiseya, Satru1oyasTet, and SYashuFa SYashuyoryya, S1okytyshsh, Sogupsas1sg1it, EpTetosossik, EksyeysYa, Rtaps1ke11a, Naeshoryyik, NeysoyasTet, efoieya, Ysr1okrya, YkTeya, MusoyasTetsht, Musor1akta, №1kkeya, Rkeiyotoiak, ShskeTSha, 8a1tope11a, 8Y§e11a, 8Taryu1osossik, 8TterTosossik , Tteropes νίΒ ^ or Uetysha. In an additional embodiment, one or more of the first antigens and / or one or more of the second antigens are obtained or are derived from VotyeTeya Reykik, Votteya VitgyotGey, Vtiseya aVoyik, Vtiseya sash, Vtiseya sheyTeyk1k, Vtiseya kshk, Sashru1oyasTet e). SyashuFa reyishoshaye, Syashufa yasyoshayk, Syashuyory Paariyasts S1okTyisht Yoiytiyit, S1okytysh yyysye, S1okytish retGtgisty8, S1okytosytassettettettettettetstettettettettettatettettatettetstettettetstatettatettettatettettettettettettatettetstatettasettettetstatettettettettettettettettettettetstatettetstatettetstatettetstatettetstatettetstatettetstatettetstatettetstatettetstatettetstatettetstatettetstatettetstatettetstatettetstatettetstatettest Rtaishkeya TYatepyk, Naeshoryyik 1pDiep / ac NeysoyasTet ru1oy, e§yupeya rpeishoryya, erTokrya shTetto§ai8, PkTeya shoposuTo§epek, MusoasYutshsh 1ertae, MusoyasTeyish TietsYoyk, MusoyasTeyish i1setai5, Musor1aksha rieishoshae, №ykspa §opottyoeae, №ykspa tei1p§1yyek, Rkeiyotoiak aetifioca, KGskeyyyskeTTki, 8a1hop1111 Gury, 8a1hop11a Turyshishish, 8Ге11а коппец 8Garyu1ossossik aiteik, 8Тарюу1ossossky sryspysyk, 8Taryu1ossossik kartyoroseyesseris8, 8 Tteropesh ra1yyish, νίΒ ^ syo1etae or Uetysha rsk11k. In another embodiment, one or more of the first antigens and / or one or more of the second antigens are obtained or are derived from a fungus of the genus SapYya, AkretfPik, SturTosossik, H1kTor1aksha, Rieishosukyk or 8Tasyuyoyuk. In a further embodiment, one or more of the first antigens and / or one or more of the second antigens are derived or derived from C. a1Ysai8, Akrstfpik GitfaShk, Akretfpik yauik, SturTosossik peoGotshaiz, SturTosossik 1aiteyts SturTosossik a1Yyik, SturTosossik §ay, ShkTor1aksha sarki1aTit, Riyesosyukyuyukuyut 8.

In yet another embodiment, one or more first antigens and / or one or more second antigens include or are derived from or derived from any of the antigens provided herein. In one embodiment, the antigen includes VI, VII, E1A, E319K, 52K, νπ, surface antigen, BA protein, capsid protein, nucleocapsid, surface outgrowth, transmembrane proteins, bb, b18, bb5, bb8, bb19, early antigen, capsid antigen , PP65,₈B, p52, potential nuclear antigen-1, N83, envelope protein, domain of the envelope protein E2, §р120, р24, lipopeptides Оа§ (17-35), Оа§ (253-284), ΝεΓ (66-97), ΝεΓ (116-145), Po1 (325-355), neuraminidase, nucleocapsid protein, matrix protein, phosphoprotein, fusion protein, hemagglutinin, hemagglutinin neuraminidase, glycoprotein, E6, E7, enveloped lipoprotein or non-structural protein (N8). In another embodiment, one or more of the first antigens and / or one or more of the second antigens include or is derived from or derived from pertussis toxin (PT), filament hemagglutinin (PHA), pertactin (RCC), fimbrin (ΡΙΜ 2/3), νΗΕ; EVRA, OkrA, Sha, PtrA, M1tA, E7 / L12, Ό15, 0187, νύΤ, My, AGiA, E7 / L12, extramembrane protein, L8, antigen type A, antigen type B, antigen type C, antigen type C, antigen type Ό, antigen type E, Pc, ΡΙίΌ, C \\ p84, alpha-toxin, tetatoxin, fructose-1,6-bisphosphate-aldolase (RBA), glyceraldehyde-3-phosphate dehydrogenase (SRE), pyruvate: ferredoxine oxidoreductase (RROK), factor -O elongation (EP-O), hypothetical protein (HP), toxin T, toxoid antigen, capsular polysaccharide, protein Ό, M1p, nucleoprotein (No.), ΚΌ1, PE35, PPE68, ExxA, ExxB, ΚΌ9, ΕxxV, Ncr70, lipopolysaccharide, surface antigen, 8p1, 8p2, 8p3, glycerophosphodiester of phosphodiesterase, outer membrane protein, protein of chaperone-switzerland) V is a protein. In yet another embodiment, one or more of the first antigens and / or one or more of the second antigens include or is derived from or derived from a surface antigen, capsular glycoprotein, Urk3P, Nkr60, the main surface protein, MkgS1, MkgC3, MkgC8, Mk§s9 or 8sN834.

In one embodiment of any of the provided dosage forms, one or more first antigens and / or one or more second antigens comprise or is derived from or derived from one or more human papillomavirus proteins. In another embodiment of any of the dosage forms provided, one or more of the first antigens comprise or derives from or originates from human papillomavirus protein b1, and one or more second antigens is derived from or originates from human papillomavirus protein b2. In yet another embodiment of any of the dosage forms provided, one or more of the first antigens comprises or derives from or originates from human papillomavirus protein b2 and one or more second antigens that is derived from or derived from human papillomavirus protein b1. In another embodiment of any of the provided dosage forms, one or more first antigens and / or one or more second antigens comprise or is derived from or derived from one or more hepatitis B virus proteins. In another embodiment, any of the provided dosage forms is one or more first antigens and / or one or more second antigens

- 3 023397 include or receive or derive from the hepatitis B surface antigen (ΗΒδΑ§). In one embodiment, the HBcAd out strain produced in §assyagotus8 ssgsuyas. In another embodiment, when one or more of the first antigens is derived or derived from hepatitis B virus, one or more second antigens are included or derived or derived from one or more human papillomavirus proteins. In a further embodiment, when one or more second antigens are derived or derived from hepatitis B virus, one or more first antigens are included or derived or derived from one or more human papillomavirus proteins. In one embodiment, one or more of the human papillomavirus proteins is human b1 and / or b2 human papillomavirus protein. In another embodiment of any of the provided dosage forms, one or more first antigens and / or one or more second antigens include or are derived from or derived from one or more proteins of the influenza virus. In one embodiment, the influenza virus is influenza A virus, avian influenza virus Η5Ν1, or influenza virus Α Η1Ν1. In another embodiment of any of the provided dosage forms, one or more of the first antigens is derived or derived from the influenza A virus M2 protein, and one or more second antigens are obtained or derived from the avian influenza virus hemagglutinin г5Ν1. In a further embodiment of any of the dosage forms provided, one or more of the first avian influenza virus hemagglutinin antigens Η5Ν1 and one or more second antigens are derived or derived from the M2 protein of influenza A virus. In yet another embodiment of any of the provided dosage forms, one or more of the first antigens is derived or derived from the influenza A virus M2 protein, and one or more second antigens are obtained or derived from the beta-propiolactone inactivated influenza virus Α Η1Ν1. In a further embodiment of any of the provided dosage forms, one or more of the first antigens is derived or derived from betaapropiolactone inactivated influenza virus Α Η1Ν1, and one or more second antigens are obtained or derived from influenza A virus protein M2.

In one embodiment, any of the provided dosage forms, a pharmaceutically acceptable excipient comprises a preservative, a buffer, saline, phosphate-buffered saline, a coloring agent, or a stabilizer.

In another embodiment of any of the dosage forms provided, the first synthetic nanocarriers include lipid-based nanoparticles, polymer nanoparticles, metal nanoparticles, surfactant emulsions, dendrimers, buckyballs, nanowires, virus-like particles, peptide or protein based particles, lipid polymer nanoparticles, spheroidal nanoparticles, cuboidal nanoparticles, pyramidal nanoparticles, elongated nanoparticles, cylindrical nanoparticles or toroidal nanoparticles. In one embodiment, each of the populations of synthetic nanocarriers includes one or more polymers. In another embodiment, one or more polymers include a polyester. In yet another embodiment, one or more polymers comprise or further comprise a polyester coupled to a hydrophilic polymer. In yet another embodiment, the polyester comprises poly (lactic acid), poly (glycolic acid), a copolymer (lactic and glycolic acid), or polycaprolactone. In a further embodiment, the hydrophilic polymer comprises a polyether. In yet a further embodiment, the polyester comprises polyethylene glycol.

In another aspect, a method is provided comprising administering any of the provided compositions to a subject. In one embodiment, the subject has or is at risk of infection or an infectious disease. In another embodiment, the subject has or is at risk of cancer.

In one embodiment of any of the methods provided, the dosage form is administered by oral, subcutaneous, pulmonary, intranasal, intracutaneous or intramuscular administration. In yet another aspect, any of the dosage forms is provided for use in therapy or prophylaxis. In another aspect, provide any of the dosage forms for use in any of the provided methods. In yet another aspect, any of the dosage forms for use in a method of treating or preventing cancer is provided. In a further aspect, any of the dosage forms are provided for use in a method for treating or preventing an infection or infectious disease. In one embodiment, any of the dosage forms, the method comprises administering the dosage form by oral, subcutaneous, pulmonary, intranasal, intracutaneous or intramuscular administration. In another aspect, the use of any of the dosage forms for the manufacture of a medicament for use in any of the methods is provided.

Brief Description of the Figures

In FIG. Figure 1 shows antibody titers in mice immunized with a combination of Νϋ-Μ2ε and free hemagglutinin from a bird flu virus strain Η5Ν1 (Vietnam).

In FIG. Figure 2 shows antibody titers in mice immunized with a combination of ΝΟ-Μ2ε and free hemagglutinin from a bird flu virus strain Η5Ν1 (Vietnam) mixed with 80 μg of alum.

In FIG. Figure 3 shows antibody titers in mice immunized with a combination of ΝΟ-Μ2ε and beta-propiolactone inactivated with the influenza virus Α Η1Ν1 (Ν1Ν1 New Caledonia / 20/99 / 1UK 116) mixed with 80 μg of alum.

In FIG. Figure 4 shows antibody titers in mice immunized with a combination of ΝΟ ^ κε ^ 2 and HBcAγ strain au№, produced in Zassnatusheus cetewuay yeast yeast mixed with 80 μg of alum.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is understood that the present invention is not limited in particular to the illustrated materials or process parameters, since these may, of course, vary. It is also understood that the terminology used herein is for the purpose of describing specific embodiments of the present invention only, and is not intended to limit the use of alternative terminology to describe the present invention.

All publications, patents and patent applications cited in this document, both above and below, are hereby incorporated by reference in their entirety for all purposes.

Used in the present description and the accompanying claims, the singular include references to the plural, unless the content clearly indicates otherwise. For example, a reference to a polymer includes a mixture of two or more such molecules, a reference to a solvent includes a mixture of two or more such solvents, a reference to an adhesive includes mixtures of two or more such materials, and the like.

Introduction

The inventors have unexpectedly and unexpectedly discovered that the problems and limitations set forth above can be overcome by putting into practice the present invention disclosed herein. In particular, the inventors unexpectedly discovered that it is possible to provide compositions, and related methods, that comprise a dosage form comprising: a first population of synthetic nanocarriers having one or more first antigens attached thereto, one or more second antigens that do not attach to synthetic nanocarriers, and a pharmaceutically acceptable excipient.

In embodiments, synthetic nanocarrier populations can be combined with one or more second antigens (which can be included in a wide variety of ways) to form the dosage forms of the present invention. One or more second antigens can be provided in the form of solutions, in the form of a suspension, in the form of a powder, and the like. and can be provided in the form of a vaccine composition. For example, in an embodiment, one or more second antigens can be provided in the form of a vaccine formulation of a hapten-carrier protein or live attenuated virus, and a population of synthetic nanocarriers mixed with vaccine formulations of a hapten-protein carrier or live attenuated virus forms a multivalent vaccine dosage form (or increases the valency of the hapten-carrier protein vaccine or live attenuated virus). In another embodiment, a population of synthetic nanocarriers can be combined with proteins taken from an infectious agent to formulate the multivalent vaccine of the present invention. In another embodiment, a population of synthetic nanocarriers can be added to another population of synthetic nanocarriers that include one or more second antigens to formulate a multivalent vaccine with a synthetic nanocarrier. In other embodiments, a population of synthetic nanocarriers can be combined with protein antigens in the form of virus-like particles to formulate the multivalent vaccine of the present invention. In other embodiments, additional antigens other than one or more of the first and / or second antigens can be included in the dosage form (by mixing and other techniques disclosed herein or traditionally known).

In an embodiment, synthetic nanocarriers comprising one or more first antigens and optionally a universal antigen for a T cell or an antigen for a T helper and / or an adjuvant can be added to one or more second antigens (eg, an existing vaccine) to create a combination vaccine with extended coverage of antigen.

For example, Oatyaky® and Stoaph®® vaccines for protection against NRU include epitopes of the protein antigen from the protein of the main structural protein L1, which comes from 4 and 2 sets of NRU strains, respectively. Vaccines are known with the antigen from peptide L1 from no more than 9 different strains of NRU. Such a vaccine with multiple peptide antigens will potentially protect the individual from most, but not all, NRU strains. If a population of synthetic nanocarriers, including one or several peptide epitopes from another structural protein of NRU, L2, is added to an existing vaccine based on L1 protein, broader protection will be obtained from repeated administration of NRU with the possible creation of a universal vaccine against NRU. This population of water-dispersed synthetic nanocarriers of b2 peptide can simply be mixed with existing aqueous vaccine formulations, just as an excipient or solvent is added to the composition. This simple method for expanding coverage dispenses with the introduction of the L2 peptide into the form of a recombinant protein antigen, as is traditionally done. This is illustrated in the following examples 1 and 2, which together illustrate the composition of the combined vaccine against NRU containing the conventional CagDaky® extended with synthetic nanocarriers comprising a peptide that is derived from protein L2.

An inventive approach to a vaccine with a combination of synthetic nanocarriers can be generalized to include prophylactic or therapeutic vaccines against another infectious disease with less than 100% protection against various strains of the infectious agent. It can also be used for prophylactic and / or therapeutic vaccines directed against targets not related to an infectious disease, such as cancer or low molecular weight agents. In embodiments, inventive compositions are provided for combinations of synthetic nanocarriers with existing conventional vaccines that can be simply formulated without limiting the solubility of the protein antigen at higher concentrations. This can reduce multivalent vaccine volumes and increase ease of formulation.

Examples 3-6 and 8-11 show various embodiments of the present invention. Examples 3 and 4 illustrate a combination vaccine from conventional hepatitis B vaccines expanded with synthetic nanocarriers, which include surface-adsorbed heparin as the first antigen. Examples 5 and 6 illustrate an oral combination vaccine of a conventional antirotavirus vaccine extended with synthetic nanoparticles, which include peptides derived from L2 protein of the NRU. Examples 8 and 9 illustrate a combination vaccine from free hemagglutinin against the bird flu strain Η5Ν1 (Vietnam), expanded with synthetic nanocarriers, which include M2e, the peptide for the T-helper OP-К and K.848 without or with an adjuvant, respectively. Example 10 illustrates the combination of an inactivated vaccine against the influenza virus Α Ν1Ν1 and extended synthetic nanocarriers, which include M2e, the peptide for T-helper OP-ΙΙ and adjuvant K848 with mixed alum. Example 11 illustrates a combination of a recombinant hepatitis B surface antigen expanded with synthetic nanocarriers, which include peptide L2, peptide for T-helper OP-L and K848 with mixed alum. The compositions provided in the examples also provide herein, as well as methods for their administration to a subject.

The present invention will now be described in more detail below.

Definitions

Adjuvant means an agent that does not constitute a specific antigen, but increases the strength and duration of the immune response to a co-administered antigen, preferably an antigen present in a dosage form together with an antigen, and more preferably to a concomitant administered antigen. Such adjuvants may include, but are not limited to, stimulators of image recognition receptors, such as To11-like receptors, ΚΙΟ-1 and ΝΟΌ-like receptors (ΝΕΚ), mineral salts such as alum, alum combined with monophosphoryl lipid (MPB) A enterobacteria such as Exbepa soy, 8a1shoeie 11a pppps501a. 8a1shoeie 11a Turyshigshsh or 8Y1§e11a Lschisp. or, in particular, with MPB® (Α804), MPB A of the aforementioned bacteria separately, saponins, such as 0821, Ουΐΐ-Α, 18COM, 18COMATK1X ™, emulsions such as MP59 ™, MoShashDe® Ι8Α 51 and Ι8Α 720, A802 ( 0821 + squalene + MPB®), Α815, liposomes and liposome formulations, such as Α801, synthesized or specifically prepared microparticles and microcarriers, such as vesicles of the outer membrane of bacterial origin (WMD) from Ν. § ioiggyeaa, Syufafa gaoboshi8 and others, or chitosan particles, depot forming agents, such as P1igoshs® block copolymers, specifically modified or prepared peptides, such as muramyl dipeptide, aminoalkyl glucosaminide-4-phosphates, such as KC529, or proteins or fragments of toxins.

In embodiments, adjuvants include agonists for pattern recognition receptors (RKKs), including but not limited to To11-like receptors (TBK), namely TBK 2, 3, 4, 5, 7, 8, 9, and / or combinations thereof. In other embodiments, adjuvants include To11-like receptor agonists 3, To11-like receptor agonists 7 and 8, or To11-like receptor agonists 9; preferably, the listed adjuvants include imidazoquinolines; such as K848 (resiquimod); derivatives of adenine, such as those disclosed in US Pat. No. 6,329,381 (8shoshno Rhasseiysa 1 Soschraiu), published US patent application 2010/0075995, W ^ aXx c1 a1., or AO 2010/018132, Sashrock c1 a1 .; immunostimulatory DNA or immunostimulatory RNA. In particular embodiments, synthetic nanocarriers include, as adjuvants, compounds that are agonists for 1-11-like receptors (TBK) 7 and 8 (TBK agonists 7/8). Among the generally accepted TK 7/8 agonist compounds disclosed in US Pat. No. 6,696,076, Tosha 1 al. include imiquimod and resiquimod. In particular embodiments, the adjuvant may be an agonist for the surface molecule of ES SE40. In certain embodiments

- 6 023397 implementation to stimulate immunity, and not resistance, the synthetic nanocarrier includes an adjuvant that promotes ES maturation (necessary for priming naive T cells) and the production of cytokines, such as type I interferons, which promote immune responses with antibodies. In embodiments, adjuvants may also include immunostimulatory RNA molecules, such as, without limitation, dsRNA or poly 1: poly C12I (available as Ashrégie®, or poly 1: C, or poly 1: polyC12I, known as TK3 stimulants) and / or uncovered in R. Ney with a1 a1., 8 reexeδρεείίΐε Keso§p1Yop оГ 8ш§1е-8йайей ΡΝΑ У1а Той-йке ГсссрГг 7 apy 8 δείεηεε 303 (5663), 1526-1529 (2004); one. Uoyteg et A1., Immune modulation using chemically modified ribonucleosides and oligoribonucleotides (1 type typoiayop bj sjetjuyu tuyu puojs1eoh1yekh apy ochshbogshs1eoyeyeh) \ UO 2008033432 A2; BUT. Rhoghacn eT a1., Immunostimulatory oligoribonucleotides containing a specific motif (s) of sequence and targeting the pathway of a JP-like receptor 8 ) \ UO 2007062107 A2; E. ITEP ET A1., Modified oligoribonucleotide analogues with enhanced immunostimulatory activity (MY WAY оп опис опис опис опис ап й й й й й ап ап ап ап ас))))))))) US Patent Application Publication No. 2006241076; C. YrGogy et A1., Immunostimulatory viral RNA oligonucleotides and their use for the treatment of cancer and infections FROM. Irgy et al., Immunostimulatory C, I-containing oligoribonucleotides, compositions and screening methods (1). In some embodiments, the adjuvant may be a TK-4 agonist, such as a bacterial lipopolysaccharide (L8), νδν-C and / or NMSV-1. In some embodiments, adjuvants may include TK-5 agonists, such as flagellin, or parts or derivatives thereof, including, but not limited to, US Pat. Nos. 6,130,082, 6,585,980, and 7192,725. In particular embodiments, synthetic nanocarriers include a ligand for a 1-11-like receptor (TB) -9, such as immunostimulatory DNA molecules containing CpC, which induce the production of type I interferon and stimulate the activation of T and Cells, leading to increased production of antibodies and cytotoxic T -cellular responses (Kpe§ eT a1., CPC shoyGh sh BasTeya1 ΌΝΑ ytg§egeg yesT In this asyuyop. No. Tsigs. 1995. 374: 546-549; Syu eT a1. СрС ойгойеохупис1еоййех ас! ah ahishuap Thx ΙιαΙ X№1Tsy op T eereg 1 (TH1) 1ttyyu. T exp. May. 1997.186: 16231631; YrGogy et A1. CpC-sopTsshp huShayeys oggopisel1eoyyekh rgotoTe In apy sutoTohyu This gehropkheh Togo rgΤείη apypep: and ps \ u s1axh oG uassche aishchuapTkh. Eig. T 1ttypo1. 1997.27: 2340-2344; Cotap et A1. 1TypokhT1ti1aTogu ΌΝΑ hssssspssx Gipsyop ah T eereg-1-rgotoyp§ aishchuapTkh. ΝαΙ. May. 1997. 3: 849-854; Eau1X εΐ a1. СрС ΌΝΑ ΐδ а dig a lot of money ОГ хрссШс 1ттипюу ш туе ипшты / с χνίΐΗ hessotЫпапт ксраОбх In HigGas upgep. T 1ttypo1. 1998. 160: 870-876; YrGogy εΐ a1., Vas1spa1 ΌΝΑ ah 1tipe this asYuAtog. Tepeykh MugoOto 1. 1998.6: 496-500; U.S. Patent No. 6,207,646, Κτ еε§ ET A1; US patent No. 7223398, TIS et A1; US patent No. 72504 03, νΑη ΝεχΤ ET A1; or U.S. Patent No. 7,566,703, Κτίε§ eT a1.).

In some embodiments, the adjuvants may be pro-inflammatory stimuli released from necrotic cells (e.g., urate crystals). In some embodiments, the adjuvants may be activated components of the complement reaction cascade (e.g., SI21, SI35, etc.). In some embodiments, the adjuvants may be activated components of the immune complexes. Adjuvants also include complement receptor agonists, such as molecules that bind to SI21 or SI35. In some embodiments, a complement receptor agonist induces endogenous opsonization of a synthetic nanocarrier with complement. In some embodiments, the adjuvants are cytokines, which are small proteins or biological factors (in the range of 5 kDa-20 kDa) that are released by the cells and specifically affect cell-cell interaction, communication and the behavior of other cells. In some embodiments, the cytokine receptor agonist is a small molecule, an antibody, a fusion protein, or an aptamer.

In embodiments, at least a portion of the adjuvant dose can be attached to synthetic nanocarriers, preferably the entire dose of adjuvant is attached to synthetic nanocarriers. In other embodiments, at least a portion of the adjuvant dose is not coupled to synthetic nanocarriers. In embodiments, the adjuvant dose includes two or more types of adjuvants. For example, and without limitation, adjuvants that act on various TK receptors can be combined. As an example, in an embodiment, a TK 7/8 agonist can be combined with a TK 9 agonist. In another embodiment, a TK 7/8 agonist can be combined with a TK 4 agonist. In yet another embodiment, a TK 9 agonist can be combined with a TK 3 agonist.

To administer or to administer means to provide a dosage form to a subject in a manner that is pharmacologically applicable.

An effective amount is any amount of a composition giving one or more desired immune responses. This amount may be for w uygo or w u1yo appointments. For ιη prescription purposes, the amount may be such that, as the general practitioner believes, may have a clinical result for a subject in need of an immune response with antibodies specific for one or more antigens. In embodiments, the implementation, therefore, the effective amount is such that, as the physician will believe, can generate an immune response with antibodies against the antigen (s) of the inventive compositions provided herein. Effective amounts can be controlled by conventional methods. An amount that is effective for generating one or more of the desired immune responses may also be the amount of a composition provided herein that gives the desired therapeutic expected result or the desired therapeutic result. Therefore, in other embodiments, the amount is effective such that the clinician will consider that it will provide therapeutic utility (including prophylactic utility) to the subject presented herein. Such subjects include those who have or are at risk of cancer, infection or infectious disease.

Amounts effective will depend, of course, on the particular subject to be 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); a specific route of administration and similar factors within the knowledge and practical experience of a general practitioner. These factors are well known to those skilled in the art and may involve no more than traditional research. In general, it is preferable that the maximum dose be used, i.e. the highest safe dose, in accordance with the results of a thorough medical assessment. Those skilled in the art will understand, however, that a patient may insist on a lower dose or tolerated dose for medical reasons, psychological reasons, or essentially any other reason. The antigen (s) of any of the inventive compositions provided herein may, in embodiments, be in an effective amount.

Antigen means an antigen for a B cell or an antigen for a T cell. In embodiments, antigens are attached to synthetic nanocarriers. In other embodiments, antigens are not attached to synthetic nanocarriers. In embodiments, the dosage forms of the present invention comprise one or more antigens, for example, one or more first antigens, one or more second antigens, one or more third antigens, one or more fourth antigens, one or more additional antigens. In embodiments, antigens are administered in conjunction with synthetic nanocarriers. In other embodiments, antigens are not administered together with synthetic nanocarriers. Antigen type (s) means molecules that have the same or substantially the same antigenic characteristics.

At least a portion of the dose means at least some portion of the dose, varying to include the entire dose.

A subject at risk is one that the general practitioner believes has a chance to undergo the disease or condition described in this document, including but not limited to infection, an infectious disease, or cancer.

An antigen for a B cell means any antigen that is either recognized and triggers an immune response in a B cell (for example, an antigen that, in particular, is recognized by a B cell receptor on a Cell). In some embodiments, an antigen that is an antigen for a T cell is also an antigen for a B cell. In other embodiments, the T cell antigen is also not a B cell antigen. Antigens for B cells include, without limitation, proteins, peptides, small molecules, carbohydrates. In some embodiments, the B cell antigen comprises a non-protein antigen (i.e., a non-protein or peptide antigen). In some embodiments, the B cell antigen comprises a carbohydrate associated with an infectious agent. In some embodiments, the B cell antigen comprises a glycoprotein or a glycopeptide associated with an infectious agent.

An infectious agent may be a bacterium, virus, fungus, protozoa, parasite or prion. In some embodiments, the B cell antigen comprises a weakly immunogenic antigen. In some embodiments, the B cell antigen comprises an abused substance or a portion thereof. In some embodiments, the B cell antigen comprises a dependency causing substance, or a portion thereof. Addictive substances include, but are not limited to, nicotine, a narcotic drug, a cough suppressant, a tranquilizer, and a sedative. In some embodiments, the B cell antigen comprises a toxin, such as a toxin from a chemical weapon or a natural source, or a contaminant. The antigen for B cells may also include a harmful exogenous agent. In some embodiments, the B cell antigen comprises an autoantigen. In other embodiments, the B cell antigen includes an alloantigen, an allergen, a contact allergen, a degenerative disease antigen, a hapten, an infectious disease antigen, a cancer antigen, an atopic disease antigen, an autoimmune disease antigen, an addictive substance, a xenoantigen or an enzyme of a metabolic disease or its enzymatic product.

- 8 023397

Attach or attach or attaches (etc.) refers to the chemical association of one structural unit (eg, fragment) with another. In some embodiments, the attachment is covalent, meaning that the attachment takes place in the sense of the presence of a covalent bond between two structural units. In non-covalent embodiments, non-covalent attachment is mediated by non-covalent interactions, including, without limitation, charge interactions, affinity interactions, metal coordination, physical adsorption, guest-host interactions, hydrophobic interactions, TT stacking interactions, interactions with hydrogen bonds, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions and / or combinations thereof. In embodiments, encapsulation is a form of compound. In embodiments, the synthetic nanocarrier populations possess one or more antigens and / or adjuvants attached to them, that is, the plurality, preferably the majority, of the synthetic nanocarriers within the population have one or more antigens and / or adjuvants attached to them that are similar to each other . In other embodiments, inventive dosage forms may contain antigens and / or adjuvants that are not attached to synthetic nanocarriers within a population of synthetic nanocarriers.

Occurs means taken from the source and subjected to significant modification. For example, a peptide or nucleic acid with a sequence with only 50% identity with a natural peptide or nucleic acid, preferably a natural consensus peptide or nucleic acid, will be indicated as derived from a natural peptide or nucleic acid. A significant modification is a modification that substantially affects the chemical or immunological properties of the material in question. Peptides and nucleic acids that occur may also include those having a sequence with greater than 50% identity with a natural peptide or nucleic acid sequence if said peptides and nucleic acids that occur have altered chemical or immunological properties compared to natural peptide or nucleic acid. Such chemical or immunological properties include hydrophilicity, stability, affinity, and ability to bind to a carrier, such as a synthetic nanocarrier.

Dosage form means pharmacologically and / or immunologically active material in a medium, carrier, diluent or device suitable for administration to a subject.

Encapsulating means enclosing inside a synthetic nanocarrier, preferably enclosing completely inside a synthetic nanocarrier. Most or all of the substance that is encapsulated is not accessible to the local environment external to the synthetic nanocarrier.

Encapsulation is different from absorption, in which most or all of the substance is placed on the surface of the synthetic nanocarrier, and the substance remains accessible to the local environment external to the synthetic nanocarrier.

Identical means that a substance shares one or more common chemical and / or immunological characteristics with another substance. For example, one or more antigens are identical when both sets of antigens share one or more common chemical or immunological characteristics. Substances, such as antigens, are not identical when they do not meet the criteria that they are identical. Certain biologically active macromolecules can be described as having a percent identity with respect to each other, which is a measure of the coincidence of their sequences, as is traditionally known in the art. Such biologically active macromolecules are identical in the framework of the present invention when they share more than 20% identity, preferably more than 30% identity, preferably more than 40% identity, preferably more than 50% identity, preferably more than 60% identity, preferably more than 70% identity, preferably more than 80% identity, or preferably more than 90% identity with each other.

An infection or infectious disease is any condition or disease caused by a microorganism, pathogen or other agent, such as a bacterium, fungus, prion or virus.

Isolated nucleic acid means a nucleic acid that is separated from its native environment and is present in sufficient quantity to permit its identification or use. An isolated nucleic acid is one that is (ΐ) amplified by νίίτο through, for example, a polymerase chain reaction (CSC);

(ίί) produced recombinantly by cloning;

(ίίί) purified, such as by cleavage and gel separation; or (ίν) is synthesized by, for example, chemical synthesis.

An isolated nucleic acid is one that is easy to manipulate using recombinant DNA techniques well known in the art. Thus, the nucleotide sequence contained in a vector in which 5 ′ and 3 ′ restriction sites are known, or for which sequences of polymerase chain reaction primers (CSCs) have been disclosed, is considered to be isolated, on the other hand, the nucleic acid sequence existing in its native state in its natural host is not considered. Isolated nucleic acid can be substantially purified, but this is not necessary. For example, a nucleic acid that is isolated in a cloning or expression vector is not pure, in the sense that it can only make up a negligible fraction of the material in the cell in which it is located. Such a nucleic acid is isolated, however, in the role of the expression used in this document, since it is readily used for standard techniques known to those skilled in the art. Any of the nucleic acids provided herein may be isolated. In some embodiments, the antigens in the compositions provided herein are present in the form of an isolated nucleic acid, such as an isolated nucleic acid, which encodes an antigenic peptide, polypeptide or protein.

An isolated peptide, polypeptide or protein means that the polypeptide (or peptide or protein) is separated from its native environment, and it is present in sufficient quantity to allow its identification or use. This means, for example, that the polypeptide (or peptide or protein) can be (v) selectively produced by expression cloning or (ϋ) purified, such as by chromatography or electrophoresis. Isolated peptides, proteins or polypeptides may be, but are not necessarily, substantially pure. Since an isolated peptide, polypeptide or protein can be mixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the polypeptide (or peptide or protein) can comprise only a small percentage of the weight of the preparation. The polypeptide (or peptide or protein) is nevertheless isolated, in the sense that it is separated from substances with which it could be associated in living systems, i.e. isolated from other proteins (or peptides or polypeptides). Any of the peptides, polypeptides or proteins provided herein may be isolated. In some embodiments, the antigens in the compositions provided herein are peptides, polypeptides, or proteins.

The maximum dimension of a synthetic nanocarrier means the largest dimension of a nanocarrier measured along any axis of the synthetic nanocarrier. A minimum synthetic nanocarrier measurement means the smallest synthetic nanocarrier measurement, measured along any axis of the synthetic nanocarrier. For example, for a spheroidal synthetic nanocarrier, the maximum and minimum dimensions of the synthetic nanocarrier will be almost identical, and will be the size of its diameter. Similarly, for a cuboidal synthetic nanocarrier, the minimum dimension of a synthetic nanocarrier will be the smallest of its height, width or length, while the maximum dimension of a synthetic nanocarrier will be the largest of its height, width or length. In an embodiment, the minimum measurement is 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, greater than 100 nm. In an embodiment, the maximum measurement is 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 measurement 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 110 nm, more preferably greater than 120 nm, more preferably greater than 130 nm and even more preferably greater than 150 nm. Aspect ratios of the maximum and minimum measurements of inventive synthetic nanocarriers may vary, depending on the embodiment. For example, the aspect ratio of maximum to minimum measurements of synthetic nanocarriers may vary from 1: 1 to 1,000,000: 1, preferably from 1: 1 to 100,000: 1, more preferably from 1: 1 to 1000: 1, even more preferably from 1: 1 to 100 : 1 and even more preferably from 1: 1 to 10: 1. Preferably, the maximum measurement 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 microns, more preferably equal to or less than 2 microns 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, the maximum measurement is 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. The measurement of the size of the synthetic nanocarrier is obtained by suspending the synthetic nanocarriers in a liquid (usually aqueous) medium and using dynamic light scattering (for example, using the Biooxaep 2e1aBa§ apparatus).

Receive means taken from the source without significant modification. A significant modification is a modification that substantially affects the chemical or immunological properties of the material in question. For example, by way of non-limiting example, a peptide or nucleic acid having a sequence with more than 90%, preferably more than 95%, preferably more than 97%, preferably more than 98%, preferably more than 99%, preferably 100% identity with natural a peptide or nucleotide sequence, preferably a natural consensus peptide or nucleotide sequence, and chemical and / or immunological properties, which do not differ significantly from the natural peptide or nucleic acid will be indicated as derived from a natural peptide or nucleotide sequence. Such chemical or immunological properties include hydrophilicity, stability, affinity, and ability to bind to a carrier, such as a synthetic nanocarrier.

Pharmaceutically acceptable carrier (s) or excipient (s) means materials that are contained within the dosage form, but do not substantially contribute to the primary pharmacological activity of the dosage form. In embodiments, the materials are pharmacologically inactive. In embodiments, pharmaceutically acceptable excipients include preservatives, buffers, saline or phosphate buffered saline, a coloring agent, or stabilizers. Pharmaceutically acceptable excipients include a number of materials known in the art, including without limitation saccharides (such as glucose, lactose, etc.), preservatives such as antimicrobials, reducing agents, coloring agents, saline (such as phosphate buffered saline) and buffers.

A population means a certain group of synthetic nanocarriers, united by one or more common physical or chemical characteristics. General physical or chemical characteristics may include the presence of a common set of surface antigens, common adjuvant (s) attached, common materials that make up the ready-to-use nanocarrier, general shape, total particle size, etc. Multiple populations of synthetic nanocarriers can be identified, for example, a first population, a second population, a third population, a fourth population, and the like.

Subject means animals, including warm-blooded mammals, such as humans and primates; birds domestic pets or farm animals such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish reptiles; zoos and wild animals, etc.

Synthetic (e) nanocarrier (s) means a discrete object that is not found in nature and which has at least one dimension that is less than or equal to 5 microns in size. Albumin nanoparticles are usually included in synthetic nanocarriers, however, in certain embodiments, synthetic nanocarriers do not include albumin nanoparticles. In embodiments, the inventive synthetic nanocarriers do not include chitosan.

A synthetic nanocarrier may be, without limitation, one or a plurality of 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, metal nanoparticles, surfactant emulsions, dendrimers, buckyballs, nanowires, virus-like particles (i.e., particles that are mainly are made up of viral structural proteins, but are not infectious or have low infectivity) peptide particles or protein-based particles (also referred to herein as protein particles, i.e. particles where most of the material constituting their structure is peptides or proteins) (such as albumin nanoparticles) and / or nanoparticles that develop using a combination of nanomaterials, such as lipid-polymer nanoparticles. Synthetic nanocarriers can be of a number of various forms, including without limitation spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, etc. The synthetic nanocarriers of the present invention include one or more surfaces, including without limitation internal surfaces (surfaces generally facing the inside of the synthetic nanocarrier) and external surfaces (surfaces generally facing the external environment of the synthetic nanocarrier). Illustrative synthetic nanocarriers that can be adapted for use in practice of the present invention include: (1) biodegradable nanoparticles disclosed in U.S. Patent No. 5,543,158, Oge £ s1 a1., (2) polymer nanoparticles from published U.S. Patent Application No. 20060002852, 5>; · ι11ζιη; πι εΐ a1., (3) lithographic nanoparticles by the method of published US patent application No. 20090028910, OsEipops s1 a1., (4) disclosure νθ 2009/051837, νοη Aibyai εΐ a1., (5) nanoparticles disclosed in published US patent application No. 2008/0145441, Reiabek s1 a1. , (6) protein nanoparticles, disclosed in U.S. Patent Application Publication No. 20090226525, 1θ8 Κίοδ εΐ a1., (7) virus-like particles disclosed in U.S. Published Patent Application No. 20060222652, §ЬЬе1 с1 a1., (8) virus-like particles linked to nucleic acid 11 023397 disclosed in U.S. Published Patent Application No. 20060251677, Washington, A1 al., (9) virus-like particles disclosed in νθ 2010047839A1 or νθ 2009106999A2, or (10) nanoprecipitated nanoparticles disclosed in P. RaoPssSh s1 a1., BshTase-tobSheb RsA-bakeb YaporaShskk Sha! Sap EgayepIu AkkoaaTe apb Όεΐίνετ Utk-bke Ratys1e8 Yapotebeksh. 5 (6): 843-853 (2010). In embodiments, the synthetic nanocarriers may have an aspect ratio of greater than 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7, or greater than 1:10.

The synthetic nanocarriers of the present invention, which have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not contain a surface with hydroxyl groups that activate complement, or alternatively contain a surface that consists mainly of fragments that are not complement activating hydroxyl groups. In a preferred embodiment, the synthetic nanocarriers of the present invention, having 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 significantly activates complement or alternatively contains a surface that consists mainly of fragments that do not substantially activate complement. In a more preferred embodiment, the synthetic nanocarriers of the present invention, which 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 activates complement or alternatively contains a surface that consists mainly of fragments that do not activate complement . In embodiments, synthetic nanocarriers do not include virus-like particles. In embodiments where the synthetic nanocarriers include virus-like particles, the virus-like particles contain an unnatural adjuvant (meaning that URN contain an adjuvant other than naturally occurring RNA formed during URR production). In embodiments, the synthetic nanocarriers may have an aspect ratio of greater than 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7, or greater than 1:10.

Antigen for a T cell means any antigen that is recognized and triggers an immune response in a T cell (for example, an antigen that, in particular, is recognized by a T cell receptor on a T cell or презентации cell by presenting the antigen or part of it associated with the molecule histocompatibility complex (MHC) class I or class II, or associated with the CE1 complex.In some embodiments, the antigen, which is an antigen for a T cell, is also an antigen for a B cell. In other embodiments, the T cell antigen is also not an antigen for the Cell. Antigens for T cells are typically proteins or peptides. T cell antigens can be an antigen that stimulates a CE8 + T cell response, a CE4 + T cell response, or both. Nanocarriers, therefore, in some embodiments, can effectively stimulate both types of responses.

In some embodiments, an antigen for a T cell is a universal antigen for a T cell, or an antigen for a memory T cell (i.e., one to which the subject has preexisting memory and which can be used to stimulate T cell assistance in relation to an unrelated antigen, for example, an unrelated antigen for a B cell). Universal antigens for T cells include tetanus toxoid, also one or more peptides derived from tetanus toxoid, Epstein-Barr virus or influenza virus. Universal antigens for T cells also include components of the influenza virus, such as hemagglutinin, neuraminidase or nuclear protein, or one or more peptides derived from them. In some embodiments, the universal antigen for T cells is not one that is presented in combination with the MHC molecule. In some embodiments, the universal antigen for T cells is not complexed with the MHC molecule for presentation to the T helper cell. Therefore, in some embodiments, a universal antigen for T cells is not an antigen for T helper cells. However, in other embodiments, the universal antigen for T cells is an antigen for T helper cells.

In embodiments, the T-helper cell antigen may include 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 RAER-peptide (known from the work §eye C1 A1., US patent No. 7202351). In other embodiments, the T-helper cell antigen may include ovalbumin or a peptide derived from or derived from it. Preferably, ovalbumin includes the amino acid sequence set forth in accession No. AAB59956, W_990483.1. AAA48998 or CAA2371. In other embodiments, a peptide derived or derived from ovalbumin comprises the following amino acid sequence: H-11e-8etC1p-A1a-Ua1-H18-A1a-A1a-H18-A1a-C1i-11e-A8p-C1i-A1a-C1u-At §-OH (8EO ΙΌ ΝΟ: 1). In other embodiments, the antigen for the T helper cell may include one or more lipids or glycolipids, including, but not limited to: α-galactosylceramide (α-Ca1Cet), glycosphingolipids with α-linkage (from 8ββββαβ8 8 crp.), Galactosyl diacylglycerol (β BotGSP), lipophosphoglycan (from B-Sntash bodeno-vaη ^) and phosphatidylinositol-tetramannoside (P1M4) (from Musoacanthyloptera). For

- 12 023397 additional lipids and / or glycolipids, useful as an antigen for a T helper cell, see V. Settsjyo1o1 a1., Natpe88shd shuapai ΝΚΤ ce118 sh ussaiop 51ga1 sschs5. No. Tsigs Ksu 1ttype. 9: 28-38 (2009).

In embodiments, antigens for CE4 + T cells can be derived from antigens for CE4 + T cells that are derived from a source, such as a natural source. In such embodiments, antigen sequences for CE4 + T cells, for example, those peptides that bind to MHC II, may have at least 70, 80, 90, or 95% identity with the antigen obtained from the source. In embodiments, an antigen for a T cell, preferably a universal antigen for a T cell or an antigen for a T helper cell, can be attached to, or disconnected from, a synthetic nanocarrier. In some embodiments, a universal antigen for T cells or an antigen for a T helper cell is encapsulated in the synthetic nanocarriers of the inventive compositions.

Vaccine means a composition of a substance that improves the immune response to a particular pathogen or disease. The vaccine typically contains factors that stimulate the subject's immune system to recognize a specific antigen as foreign and remove it from the subject's body. The vaccine also creates an immunological 'memory', so the antigen will be quickly recognized and a response will be generated if a person undergoes repeated antigenic stimulation. Vaccines can be prophylactic (for example, to prevent future infection by any pathogen) or therapeutic (for example, a vaccine against tumor-specific antigens for treating cancer). In embodiments, the vaccine may include dosage forms of the present invention. In other embodiments, an inventive dosage form may include a vaccine containing a second antigen that is not attached to synthetic nanocarriers. Vaccines of the present invention may include a hapten-carrier conjugate, a virus-like particle, a synthetic nanocarrier vaccine, a protein subunit vaccine, or an attenuated virus. In some embodiments, the vaccine includes any of the vaccines, including the commercially available vaccines described herein.

Inventive compositions

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 implementation of the synthetic nanocarriers flat or lamellar form. In some embodiments, the synthetic nanocarriers are cubes or cuboidal. In some embodiments, the synthetic nanocarriers are ovals or ellipses. In some embodiments, the synthetic nanocarriers are cylinders, cones, or pyramids.

In some embodiments, it is desirable to use a population of synthetic nanocarriers that are relatively uniform in size, shape, and / or composition, so 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, can have a minimum dimension or a maximum dimension within 5, 10, or 20% of the average diameter or average measurement of synthetic nanocarriers. In some embodiments, the implementation of the population of synthetic nanocarriers may be heterogeneous in relation to size, shape and / or composition.

Synthetic nanocarriers may be monolithic or hollow and may include one or more layers. In some embodiments, each layer has a unique composition and unique properties relative to the other layer (s). Just one example, synthetic nanocarriers can have a core / shell structure, where the core is a single layer (e.g., a polymer core), and the shell is a second layer (e.g., a lipid bilayer or monolayer). Synthetic nanocarriers may contain many different layers.

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

In some embodiments, synthetic nanocarriers may include one or more polymers. In some embodiments, such a polymer may be surrounded by a coating layer (e.g., a liposome, a lipid monolayer, a micelle, etc.). In some embodiments

- 13 023397 implementation of the various elements of synthetic nanocarriers can be combined with the polymer.

In some embodiments, an immunoreactive surface, a targeting fragment, an antigen, an adjuvant, and / or an oligonucleotide can be covalently associated with a polymer matrix. In some embodiments, the covalent association is mediated by a linker. In some embodiments, the immunoreactive surface, targeting fragment, antigen, adjuvant and / or oligonucleotide can be non-covalently associated with a polymer matrix. For example, in some embodiments, the immunoreactive surface, targeting fragment, antigen, adjuvant and / or oligonucleotide can be adsorbed onto, encapsulated inwardly, surrounded and / or dispersed throughout the thickness of the polymer matrix. Alternatively or additionally, the immunoreactive surface, targeting fragment, antigen, adjuvant and / or nucleotide can be associated with the polymer matrix through hydrophobic interactions, interaction of charges, van der Waals forces, etc.

A wide variety of polymers and methods for forming polymer matrices from them are traditionally known. Basically, the polymer matrix includes one or more polymers. The polymers can be natural or unnatural (synthetic) polymers. The polymers can be homopolymers or copolymers containing two or more monomers. With respect to the sequence, the copolymers may be random, block or may contain a combination of random and block sequences. Typically, the polymers in accordance with the present invention are organic polymers.

Examples of polymers suitable for use in the present invention include, without limitation, 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, lactide-glycolide copolymer, polycaprolactone, polyhydroxy acid (e.g. polyphyloxyalkonoate)), poly (orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, polyamines, polylysine, polylysine and PEO copolymers, as well as poly (ethyleneimine), poly (ethyleneimine) and PEO copolymers.

In some embodiments, the polymers of the present invention include polymers that have been approved for human use by the US Food and Drug Administration (ΡΌΑ) based on 21 C.P.K. § 177.2600, including without limitation polyesters (e.g. polylactic acid, copolymer (lactic and glycolic acid), polycaprolactone, for example, poly (1,3-dioxan-2-one)); polyvalerolactone; polyanhydrides (e.g. poly (sebacic anhydride)); polyethers (e.g. polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates and polycyanoacrylates.

In some embodiments, the polymers may be hydrophilic. For example, polymers may contain anionic groups (for example, a phosphate group, a sulfate group, a carboxylate group); cationic groups (e.g., quaternary amine group) or polar groups (e.g., hydroxyl group, thiol group, amine group). In some embodiments, the implementation of a synthetic nanocarrier containing a hydrophilic polymer matrix, produces a hydrophilic environment within the synthetic nanocarrier. In some embodiments, the polymers may be hydrophobic. In some embodiments, the implementation of a synthetic nanocarrier containing a hydrophobic polymer matrix, produces a hydrophobic environment within the synthetic nanocarrier. The choice of hydrophilicity or hydrophobicity of the polymer may have an effect on the nature of the materials that are incorporated (eg, combined) into the synthetic nanocarrier.

In some embodiments, the polymers can be modified with one or more fragments and / or functional groups. In accordance with the present invention, a number of fragments or functional groups can be used. In some embodiments, the polymers can be modified using polyethylene glycol (PEO), using carbohydrate and / or using acyclic polyacetals derived from polysaccharides (Pap18OU, 2001, AC8 8utro8shsh 8sps5. 786: 301). Certain implementation options can be performed using the basic ideas of US patent No. 5543158, Oge £ s (a1., Or publication of international application ^ 02009/051837, Wup Apyap s (a1.

In some embodiments, the polymers can be modified with a lipid or fatty acid group. In some embodiments, the fatty acid group may be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachinic, behenic, or lignoceric acid. In some embodiments, the fatty acid group may be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic or erucic acid.

In some embodiments, the polymers can be polyesters, including copolymers containing units of lactic acid and glycolic acid, such as a copolymer of lactic acid and glycolic acid and a copolymer of lactide and glycolide, collectively referred to herein as POBA; and homopolymers containing glycolic acid units referred to herein as POA and lactic acid units such as poly-L-lactic acid, poly-E-lactic acid, poly-E, L-lactic acid, poly-L-lactide , poly-E-lactide and poly-Ό, Lactide, collectively referred to herein as PBA. In some embodiments, exemplary polyesters include, for example, polyhydroxy acids; copolymers of REO and copolymers of lactide and glycolide (for example, copolymers of PBA and PEO, copolymers of PAO and PEO, copolymers of POBA and PEO and their derivatives. In some embodiments, the implementation of the polyesters include, for example, poly (caprolactone), copolymers of poly (caprolactone) and PEO, copolymers of L-lactide and Lysine, poly (serine ester), poly (4-hydroxy-L-proline ester), poly [a- (4aminobutyl) -B-glycolic acid] and their derivatives.

In some embodiments, the polymer may be POBA. PHOA is a biocompatible and biodegradable copolymer of lactic acid and glycolic acid, and various forms of PHOA are characterized by their ratio of lactic acid: glycolic acid. Lactic acid may be L-lactic acid, Ό-lactic acid or Ό, Ό-lactic acid. The disintegration rate of POA can be controlled by changing the ratio of lactic acid: glycolic acid. In some embodiments, the POAA used in accordance with the present 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.

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

In some embodiments, the polymers may be cationic polymers. Basically, cationic polymers are capable of condensing and / or protecting negatively charged chains of nucleic acids (e.g., DNA or its derivatives). Amino-containing polymers such as poly (lysine) (/ ayeet c1 a1., 1998, Abu. Ogid Όεΐ. Keu., 30:97 and KaBaioou c1 a1., 1995, VyusoishchdeTe Syem., 6: 7), poly (ethyleneimine) (PE1; Voikk! S1 a1., 1995, Rtos. Yab. Asab. 8c1., IZA, 1995, 92: 7297), and poly (amidoamine) dendrimers (Kikotekka-LaiIo s1 a1., 1996, RTOS. ΝαΝ. Asab. Zek, IZA, 93: 4897; Tapd s1 a1., 1996, VyusooshchadTe Skst .. 7: 703; and Naepk1et s1 a1., 1993, VyusoshchadTe Skst .. 4: 372) are positively charged at physiological pH, form ion pairs with nucleic acids and mediate transfection in a number of cell lines. In embodiments, inventive synthetic nanocarriers may not (may exclude) cationic polymers.

In some embodiments, the polymers can be degradable polyesters carrying cationic side chains (Pu1iash s1 a1., 1999, Masto1esi1ek, 32: 3658; Watteta s1 a1., 1993, 1. Am. Syem. 8oc, 115: 11010; K \ uop s1 a1., 1989, Mastomoeci, 22: 3250; NT c1 ai., 1999, 1. Am. Syem. 8oc, 121: 5633; apb 2yoi c1 ai., 1990, Mastomoeci, 23: 3399). Examples of these polyesters include a copolymer of L-lactide and L-lysine (Wattet C1 al., 1993, 1. At. Siet. 8oc, 115: 11010), poly (serine ester) (2oy C1 al., 1990, Masto1esi1ek, 23: 3399), poly (4-hydroxy-L-proline ester) (Pu1iam s1 a1., 1999, Masto1esi1ek, 32: 3658; apb ct s1 a1., 1999, 1. Am. Syet. 8oc, 121: 5633) and poly (4-hydroxy-b-proline ester) (Pu1iam s1 a1., 1999, Mastomoecie1, 32: 3658 ; apb Όιμ e! a1., 1999, 1. Am. Siet. 8os, 121: 5633).

The properties of these and other polymers and methods for their preparation are well known in the art (see, for example, U.S. Pat. ; 5010167; 4806621; 4638045 and 4946929; \ Wapd s1 a1., 2001, 1. At. Syet. 8oc, 123: 9480; Ht s1 a1., 2001, 1. At. Syet. 8oc, 123: 2460; 2000, Ass. Syet. Ke8., 33:94; Yeahit, 1999, 1. Soy1to1. Ke1eake, 62: 7; and Ipsy s1 a1., 1999, Syem. Keu., 99: 3181). In general, a number of methods for synthesizing certain suitable polymers are described in Communication of Jesus Christ! Ro1umet Zsheise apb RoKtssp Apipsk apb Ammoyim Zayk, Eb. Lü Oöläyäk, Retdamop Rtekk, 1980; Rg1ps1r1sk about! RoKtsp / Aiop Ob1ai, 1st \ UPU & Zopk, Roipy Ebyyui, 2004; Soyemrotatu Ro1umet Syemyu, Aysosk s1 a1., Rteyue-NaI, 1981; Ospipe c1 a1., 1997, No. 1ite, 390: 386 and U.S. Patent Nos. 6506577, 6632922, 6686446 and 6818732.

In some embodiments, the polymers may be linear or branched polymers. In some embodiments, the polymers may be dendrimers. In some embodiments, the polymers can be substantially cross-linked. In some embodiments, the polymers may be substantially free of cross-linking. In some embodiments, the polymers can be used in accordance with the present invention without going through a crosslinking step. It is further understood that inventive synthetic nanocarriers may include block copolymers, grafted copolymers, combinations, mixtures and / or adducts from any of the above and other polymers. Those skilled in the art will appreciate that the polymers listed herein are an illustrative, non-exhaustive list of polymers that can be used in accordance with the present invention.

In some embodiments, synthetic nanocarriers comprise one or more polymers. Polymer synthetic nanocarriers, therefore, can also include those described in the publication of the international application \ UO 2009/051837, Wop Lypai s1 a1., Including, without limitation, those with one or more hydrophilic components. Preferably, one or more polymers comprise a polyester such as poly (lactic acid), poly (glycolic acid), a copolymer of lactic and glycolic acid, or polycaprolactone. More preferably, one or more polymers include or further comprise a polyester coupled to a hydrophilic polymer, such as a polyester. In embodiments, the polyether comprises polyethylene glycol. Even more preferably, one or more polymers comprise a polyester and a polyester coupled to a hydrophilic polymer, such as a polyester. In other embodiments, one or more polymers are combined with one or more antigens and / or one or more adjuvants. In embodiments, at least some of the polymers are combined with antigen (s) and / or at least some of the polymers are combined with adjuvant (s). Preferably, when more than one type of polymer is present, one of the types of polymer being combined with antigen (s). In embodiments, one of the other types of polymer is combined with adjuvant (s). For example, in embodiments where the nanocarriers comprise a polyester and a polyester bonded to a hydrophilic polymer such as a polyester, the polyester bonded to an adjuvant, while the polyester bonded to a hydrophilic polymer such as a polyester is bonded to antigen (s). In embodiments where the nanocarriers comprise an antigen for a T helper cell, wherein the antigen for the T helper cell can be encapsulated in a nano-carrier.

In some embodiments, the implementation of synthetic nanocarriers do not contain a polymer component. In some embodiments, the implementation of synthetic nanocarriers may contain metal particles, quantum dots, ceramic particles, etc. In some embodiments, the non-polymeric synthetic nanocarrier is a collection of non-polymer components, such as a collection of metal atoms (e.g., gold atoms).

In some embodiments, synthetic nanocarriers may optionally contain one or more amphiphilic structural units. In some embodiments, the implementation of the amphiphilic structural unit may facilitate the production of synthetic nanocarriers with increased stability, improved uniformity, or increased viscosity. In some embodiments, amphiphilic structural units can be associated with the inner surface of the lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Many amphiphilic structural units known in the art are suitable for use in the preparation of synthetic nanocarriers in accordance with the present invention. Such amphiphilic structural units include, but are not limited to, phosphoglycerides; phosphatidylcholines; dipalmitoylphosphatidylcholine (ERRS); dioleylphosphatidylethanolamine (PORE); dioleloxypropyltriethylammonium (ΌΟΤΜΆ); dioleylphosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (ERRO); hexanecanol; fatty alcohols such as polyethylene glycol (PEO); polyoxyethylene-9-lauryl ether; surface active fatty acid such as palmitic acid or oleic acid; fatty acid; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; sorbitan trioleate (§rai®85) glycocholate; sorbitan monolaurate (§rai®20); polysorbate 20 (T \ usp®20); polysorbate 60 (T \ usp®60); polysorbate 65 (T \ usp®65); polysorbate 80 (T \ usp®80); polysorbate 85 (T \ usp®85); polyoxyethylene monostearate; surfactin; poloxamer; ester of sorbitan and fatty acids such as sorbitan trioleate; lecithin; lysolecithin; phosphatidyl series; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid; cerebrosides; dicetyl phosphate; dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine; hexadecylamine; 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 the properties of a highly active surfactant; deoxycholates; cyclodextrins; chaotropic salts; ion pairing agents; and combinations thereof. The component amphiphilic structural unit may be a mixture of various amphiphilic structural units. Those skilled in the art will appreciate that this is an illustrative, non-exhaustive list of substances with surfactant activity. Any amphiphilic structural unit can be used in the production of synthetic nanocarriers that are used in accordance with the present invention.

In some embodiments, synthetic nanocarriers may optionally contain one or more carbohydrates. Carbohydrates can be natural or synthetic. Carbohydrate may be a naturally occurring carbohydrate derivative. In certain 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, glucoronic acid, galacturonic acid, glucosuronic acid, mannuronic acid, glucosuronic acid, glucosuronic acid galactosamine and neuraminic acid. In certain embodiments, the carbohydrate is a polysaccharide including, but not limited to, pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methyl cellulose (HPMC), hydroxycellulose (HC), methyl cellulose (MS), dextran, cyclodextran, glycogen, starch, hydroxyethylcra, hydroxyethylcra, hydroxyethylcra, hydroxyethylcra, hydroxyethylcra, hydroxyethylcra, hydroxyethylcra, hydroxyethyl chitosan, Ν, Ο carboxylmethylchitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucomannan, pustulan, heparin, hyaluronic acid, Kurdlan and Xanthan. In embodiments, the inventive synthetic nanocarriers do not contain (or in particular exclude) carbohydrates such as a polysaccharide. In certain embodiments, the carbohydrate may include a carbohydrate derivative such as a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.

The compositions of the present invention include inventive synthetic nanocarriers in combination with pharmaceutically acceptable excipients such as preservatives, buffers, saline or phosphate-buffered saline. Compositions can be prepared using conventional pharmaceutical manufacturing techniques and compounds to achieve the applicable dosage forms. Typical inventive compositions may contain inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate or citrate) and pH adjusters (e.g., hydrochloric acid, sodium hydroxide or potassium hydroxide, salts of citrate or acetate, amino acids and their salts), antioxidants (e.g. ascorbic acid, alpha-tocopherol), surfactants (e.g. polysorbate 20, polysorbate 80, polyoxyethylene 9-10-nonylphenol, sodium deoxycholate), solution stabilizers and / or cryo / lyostabilizers (e.g. sucrose, lactose, mannitol, trehalose), osmotic pressure regulators (e.g. salt or sugar), antibacterial agents (e.g. benzoic acid, phenol, gentamicin), anti-foaming agents agents (e.g. polydimethylsilosone), preservatives (e.g. thimerosal, 2-phenoxyethanol, ΕΌΤΑ), polymer stabilizers and viscosity adjusters (e.g. polyvinylpyrrolidone, poloxamer 488, carboxymethyl cellulose) and cosolvents (e.g. glycerin, polyethylene glycol, ethanol). In an embodiment, inventive synthetic nanocarriers are suspended in a sterile injectable saline solution with a preservative. In embodiments where synthetic nanocarriers are prepared as carriers for antigens and / or adjuvants for use in vaccines, methods for attaching antigens or adjuvants to synthetic nanocarriers may be applicable. If the antigens or adjuvant is a small molecule, it may be advantageous to attach the antigens or adjuvant to the polymer prior to the assembly of synthetic nanocarriers. In embodiments, it may also be advantageous to produce synthetic nanocarriers with surface groups that are used to attach antigens or an adjuvant to a synthetic nanocarrier by using these surface groups rather than attaching antigens or an adjuvant to the polymer, and then using this polymer conjugate to construct synthetic nanocarriers .

In certain embodiments, the attachment may be a covalent linker. In embodiments, the peptides of the present invention can be covalently attached to the outer surface via a 1,2,3-triazole linker formed by the reaction of 1,3-dipolar cycloaddition of azido groups on the surface of a nanocarrier with an antigen and / or adjuvant containing an alkynyl group, or reaction 1, 3-dipolar cycloaddition of alkynes on the surface of a nanocarrier with antigens or adjuvants containing an azido group. Such cycloaddition reactions are preferably carried out in the presence of a Cu (1) catalyst together with a suitable Cu (1) ligand and a reducing agent to reduce the Cu (11) compound to the catalytically active Cu (1) compound. This Cu (1) -catalyzed azide-alkyne cycloaddition (CuAAS) can also be called a click reaction.

Additionally, the covalent attachment may include a covalent linker, which 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, and an amine linker.

An amide linker is formed through an amide bond between an amine on one component, such as an antigen or adjuvant, with a carboxylic acid group of a second component, such as a nanocarrier. The amide bond in the linker can be prepared using any of the conventional amide bond formation reactions with properly protected amino acids or antigens or adjuvants and activated carboxylic acids, such as, for example, Ν-hydroxysuccinimide-activated ester.

A disulfide linker is formed through the formation of a disulfide (δ-δ) bond between two sulfur atoms in the form of, for example, Κ ^ δ-δ-Κ ^ A disulfide bond can be formed by a thiol exchange on an antigen or adjuvant containing a thiol / mercaptan group (-3H) with another activated thiol group on a polymer or nanocarrier, or a nanocarrier containing thiol / mercaptan groups with an antigen or adjuvants containing an activated thiol group.

Triazole linker, in particular 1,2,3-triazole in the form of

<img file="EA023397B1_D0001.tif" />

where is k! and K₂ can be any chemical structural unit, is formed by the reaction of 1,3-dipolar cycloaddition of an azide attached to the first component, such as a nanocarrier, with terminal alkine attached to the second component, such as a peptide. The 1,3-dipolar cycloaddition reaction is carried out with or without a catalyst, preferably with a Cu (1) catalyst, which binds the two components through a 1,2,3-triazole functional group. This chemistry is described in detail by Zbp1e88 s1 a1., Apgete. Eat. Ιηί. Εά. 41 (14), 2596, (2002) and MeMa1, c1 a1, Cb. Κεν., 2008, 108 (8), 2952-3015 and is often referred to as a click reaction or CuAAS.

In embodiments, a polymer containing an azide or alkynyl group terminated with respect to the polymer chain is prepared. This polymer is then used to produce a synthetic nanocarrier in such a way that several alkynic or azide groups are located on the surface of such a nanocarrier. Alternatively, a synthetic nanocarrier can be obtained in another way and subsequently functionalized with alkynyl or azide groups. An antigen or adjuvant is prepared with the presence of either an alkyno (if the polymer contains azide) or azide (if the polymer contains alkyne) groups. The antigen or adjuvant is then allowed to react with the nanocarrier through a 1,3-dipolar cycloaddition reaction with or without a catalyst that covalently attaches the antigen or adjuvant to the particle via a 1,4-disubstituted 1,2,3-triazole linker.

The thioether linker is obtained by the formation of a sulfur-carbon (thioether) bond in the form, for example, Κι-δ-Κ₂. The thioether can be prepared either by alkylating a thiol / mercaptan (-3H) group on one component, such as an antigen or adjuvant with an alkylating group, such as a halide or epoxide, on a second component, such as a nanocarrier. Thioether linkers can also be formed by Michael attaching a thiol / mercaptan group on one component, such as an antigen or adjuvant, to an electron-deficient alkene group on the second component, such as a polymer containing a maleimide group or a vinyl sulfone group as a Michael acceptor. Alternatively, thioether linkers can be prepared by radical thiolene reaction of a thiol / mercaptan group on one component, such as an antigen or adjuvant, with an alkene group on the second component, such as a polymer or nanocarrier.

A hydrazone linker is obtained by reacting a hydrazide group on one component, such as an antigen or adjuvant, with an aldehyde / ketone chemical group on a second component, such as a nanocarrier.

The hydrazide linker is formed by the reaction of a hydrazine group on one component, such as an antigen or adjuvant, with a carboxylic acid group on a second component, such as a nanocarrier. Such a reaction is usually carried out using chemistry similar to the formation of an amide bond, where the carboxylic acid is activated by an activating reagent.

The imine or oxime linker is formed by the reaction of an amino group or an alkoxy amino group (or aminooxy group) on one component, such as an antigen or adjuvant, with an aldehyde or ketone group on the second component, such as a nanocarrier.

A urea or thiourea linker is obtained by reacting an amino group on one component, such as an antigen or adjuvant, with an isocyanate or thioisocyanate group on a second component, such as a nanocarrier.

The amidine linker is obtained by reacting an amine group on one component, such as an antigen or adjuvant, with an ester imidogroup on a second component, such as a nanocarrier.

The amine linker is prepared by an alkylation reaction of an amine group on one component, such as an antigen or adjuvant, by an alkylating group, such as a halide, epoxy or sulfonate ester group on a second component, such as a nanocarrier. Alternatively, an amine linker can also be obtained by reductive amination of an amine group on one component, such as an antigen or adjuvant, with an aldehyde or ketone group on

- 18 023397 a second component, such as a nanocarrier, with a suitable reducing agent, such as sodium cyanoborohydride or sodium triacetoxyborohydride.

The sulfonamide linker is obtained by reacting an amine group on one component, such as an antigen or adjuvant, with a sulfonyl halide (such as sulfonyl chloride) group on a second component, such as a nanocarrier.

The sulfone linker is obtained by Michael attachment of a nucleophile to a vinyl sulfone. Either vinyl sulfone or a nucleophile can be on the surface of a nanoparticle or attach to an antigen or adjuvant.

The antigen or adjuvant can also be conjugated to a nanocarrier using non-covalent conjugation methods. For example, a negatively charged antigen or adjuvant can be conjugated to a positively charged nanocarrier by electrostatic adsorption. An antigen or adjuvant containing a ligand to a metal can also be conjugated to a nanocarrier containing a metal complex through a metal-ligand complex.

In embodiments, an antigen or adjuvant can be attached to a polymer, for example, a lactic acid-polyethylene glycol block polymer, prior to assembly of the synthetic nanocarrier, or the synthetic nanocarrier can be formed with reactive or activation groups on its surface. In the latter case, the antigen or adjuvant can be obtained with a group that is compatible with the chemistry of addition determined by the surface of synthetic nanocarriers. In other embodiments, the peptide antigen can be attached to the URP or liposomes using a suitable linker. A linker is a compound or reagent that is capable of connecting two molecules together. In an embodiment, the linker may be a homobifunctional or heterobifunctional reagent described in Netshaikoi 2008. For example, an URP or a liposome synthetic nanocarrier containing a carboxyl group on the surface can be treated with a homobifunctional linker, adipic acid dihydrazide (ΛΌΗ), in the presence of an EES to form the corresponding synthetic nanocarrier with ΛΌΗlinker. The resulting ΛΌΗ-linked synthetic nanocarrier is then conjugated to the peptide antigen containing the acid group through the other end of the ΛΌΗ-linker to Νί '.' to obtain the corresponding conjugate URP or liposome peptide.

For detailed descriptions of the available conjugation methods, see Netshaiko O T Vusop) id TssNpishsk, 2ib ηοη, Rybikeb Ub Asabeshu Rtekk, 1ps., 2008. In addition to covalent attachment, the antigen or adjuvant can be coupled by absorption or by absorption to the pre-formed nanosized can be attached by encapsulation during the formation of a synthetic nanocarrier.

Methods for the preparation and use of inventive dosage forms and methods associated with them

Synthetic nanocarriers can be prepared using a wide variety of methods known in the art. For example, synthetic nanocarriers can be formed using methods such as nanoprecipitation, focusing a stream using liquid channels, spray drying, solvent evaporation from a single and double emulsion, solvent extraction, phase separation, milling, microemulsion techniques, microprocessing, nanoprocessing, protective layers, simple and complex coacervation and other methods well known to those skilled in the art. Alternatively or additionally, the syntheses of aqueous and organic solvents for monodispersed semiconductor, conductive, magnetic , organic and other nanomaterials have been described (ReIedbio e1 a1., 2005, §sha11, 1:48; Mstau e! A1., 2000, App. Keu. Ma1. 8ce, 30: 545 and Tbpbabe e! A1., 2001, SNes. Ma!., 13: 3843). Additional methods have been described in the literature (see, for example, Ooibslou, H., Mutosarki1kb apb No.poratbs1ek w Mebushu apb Rnatshasu, SKS Rtekk, Vosa Ka1op, 1992; Masho ^ / e1 a1., 1987, 1. Co1to1. Ke1eake, 5:13; MacShutob: e1 a1., 1987, Keasbue Rhoetzhetk, 6: 275 and Masho ^ I / e1 a1., 1988, 1. Arr1. Rooket δει., 35: 755, as well as US patents Nos. 5578325 and 6007845 ; R. Raoesses e1 a1., 'Biggass-tobbyub ROBA-bakeb No.pobyuyek 1Na1 sap Ebbaepbu Akkos1a1e apb Όεΐίνετ Uiik-bke Ratbs1ek Mshotsbybyu. 5 (6): 843-853 (2010)).

Various materials can be encapsulated in synthetic nanocarriers, as desired, using a number of methods including, without limitation, C. Ak1e1e e1 a1., Уп б б юк / / / / / / / / Г Г Ь Ь О О 1. 1. 1. 1. 1. 1. 1. В В В ю ю. 8s1. Rousset Ebi, Wo1. 17, no. 3, pp. 247-289 (2006); K. A ^ oik1ak1k Redu1a1eb Po1u (Basbbe) apb Ro1u (Basbbe-So-01usoIbe) #Poratbos1ek: Rratabop, Rtoretbek apb Rokbys Arrbsabopk ΐη Aegis OSP \ 'stu Sitgep! Aegis of OSGS'stu 1: 321-333 (2004); C. Keb e1 a1., '^ Apoepsarca1abop I. Msbyubk Got rterabopop oG btid-1oabeb robshebs paporatbs1ek Mshotsbybyu 2: 8-21 (2006); R. Raoesses e1 a1., 'Bhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh. 5 (6): 843-853 (2010). Other methods suitable for encapsulating materials, such as oligonucleotides, in synthetic nanocarriers may be used, including, without limitation, the methods disclosed in United States Patent No. 6,632,671 to Idet, October 14, 2003.

In certain embodiments, synthetic nanocarriers are prepared using a nanoprecipitation process or spray drying. The conditions used to obtain synthetic nanocarriers can be changed to yield particles of the desired size or properties (for example, hydrophobicity, hydrophilicity, external morphology, stickiness, shape, etc.). The method for producing synthetic nanocarriers and applicable conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) may depend on the materials that need to be attached to the synthetic nanocarriers and / or the composition of the polymer matrix.

If the particles obtained using any of the above methods have a size that varies outside the desired range, the particles can be sorted by size, for example, using a sieve.

Elements of inventive synthetic nanocarriers (such as the fragments that make up the immunoreactive surface, targeting fragments, polymer matrices, antigens, adjuvants, etc.) can be attached to a complete synthetic nanocarrier, for example, via one or more covalent bonds, or can be combined with using one or more linkers. Additional methods for functionalizing synthetic nanocarriers can be adapted from published U.S. Patent Application No. 2006/0002852, §a1tap C1 A1., U.S. Published Patent Application No. 2009/0028910, EsBpnops C1 A1., Or published international patent application ^ Θ / 2008/127532 A1, Μιιτίΐιγ s1 a1.

Alternatively or additionally, synthetic nanocarriers can be attached to the immunoreactive surface, targeting fragments, adjuvants, various antigens and / or other elements directly or indirectly by non-covalent interactions. In non-covalent embodiments, non-covalent attachment is mediated by non-covalent interactions, including without limitation charge interactions, affinity interactions, metal coordination, physical adsorption, guest-host interactions, hydrophobic interactions, TT stacking interactions, interactions with hydrogen bonds, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions and / or combinations thereof. Such attachments can be arranged so that they are on the outer surface or inner surface of the inventive synthetic nanocarrier. In embodiments, encapsulation and / or adsorption is a form of attachment.

A wide variety of one or more second antigens (or additional antigens that do not attach to a population of synthetic nanocarriers) can be included in the dosage form, and can be included in a wide variety of ways. The types of one or more second antigens (or additional antigens that do not attach to a population of synthetic nanocarriers) suitable for use with the present invention have been discussed elsewhere in this document.

There is a wide variety of methods for incorporating one or more of the first or several second antigens (or additional antigens that do not attach to a population of synthetic nanocarriers) in an inventive dosage form. In an embodiment, one or more second antigens can be mixed into a dosage form together with a population of synthetic nanocarriers. For example, in an embodiment, a vaccine that contains one or more second antigens can be mixed with a population of synthetic nanocarriers to form inventive dosage forms. In embodiments, inventive synthetic nanocarriers can be included in inventive dosage forms together with one or more of the first antigens that are different, similar or identical, as well as with one or more second antigens, in a wide variety of ways, including without limitation: with or without an adjuvant, using or not using another delivery medium, administered separately at different times and / or at a different location on the body and / or a different way of immunization.

In embodiments, synthetic nanocarrier populations can be combined with one or more second antigens (which can be included in a wide variety of ways) to form the dosage forms of the present invention. One or more second antigens can be provided in the form of a solution, the form of a suspension, the form of a powder, etc., and can be provided as a vaccine composition. For example, in an embodiment, one or more second antigens can be provided in the form of a hapten-carrier protein, oligosaccharide, oligosaccharide complex, fusion of the oligosaccharide-carrier protein, a vaccine composition with a live attenuated or recombinant virus, and a population of synthetic nanocarriers mixed with a hapten protein -carrier, oligosaccharide, oligosaccharide complex, fusion oligosaccharide-carrier protein, vaccine composition with live attenuated or recombinant virus, for the formation of a multivalent vaccine dosage form (or increase the valency of the vaccine composition with a hapten carrier protein or with a live attenuated virus). In embodiments, one or more second antigens may be included in anthrax vaccine; diphtheria, tetanus and / or whooping cough; hemophilic influenza type B; hepatitis B; hepatitis A; hepatitis C; herpes zoster (depriving); human papillomavirus (NRU); flu Japanese encephalitis; tick-borne encephalitis; measles, mumps and / or rubella; meningococcal infection; pneumococcal infection; poliomyelitis; rabies; ro-20 023397 tavirus; typhoid fever; chickenpox; smallpox (smallpox animals) or yellow fever. In other embodiments, one or more of the second antigens is included in a commercially available vaccine, including but not limited to ΒΙΟΤΗΚΑΧ, OARTLECET ΙΝΕΛΝΚΙΧ, ΤΚΙΡΕΏΙΆ, ΤΕΙΗΙΒΙΤ, ΚΙΝΕΙΧ, ΡΕΏΙΑΚΙΧ, REOTACEE, ΡΕΏνΑΧΗΙΒ, ASTH1B, ΗΙΒΕΕΙΧ, COMAH, NAUCH, VΑ ^ ΤΑ, ΕΝΘΕΕΙΧ -Β, ΕΕСΟΜΒIVΑΧ ΗΒ, ΤνίΝΕΙΧ, ΖΟδΤΑVΑΧ, ОАЕЭАЕЕЕ CE ^ АШХ, ЕЕЦЫХ, ЕШνίΕΙΝ, ΕΕυΖΟΝΕ, Е ^ υ ^ ΑVΑ ^, АЕЕиЫА, АО1ЕЬи, ΕΕΈΜΙδΤ, 1Е ^ АХ, 1Х1АЕО, М, II-ЕО, М ^, ΜΕΝΟΜυΝΕ, METSTEA, ΜΕΝνΕΟ, ΡΝΕυМΟVΑΧ 23, ΡΕΕVΝΑΕ, RSU13, 1ROP, IM IVΑΧ ΕΑΒIΕδ, ΕΑΒΑVΕΕΤ, ΕΟΤΑΤΕ ^, ΕΟΤΑΕIΧ, ^ ΕСΑVΑС, ΒΟΟδΤΕΙΧ, AOACEE, ΤΥΡΗIΜ VI, νΐνΟΤΙΕ ΒΕΕ ^, VΑΕIVΑΧ, АСАМ2000 or УЕ ^ АХ.

In another embodiment, the synthetic nanocarrier population can be combined with proteins taken from an infectious organism, such as human influenza A virus protein HA, either in protein form or in virus-like particles, to form the multivalent vaccine dosage form of the present invention. In another embodiment, a synthetic nanocarrier population can be added to another synthetic nanocarrier population that contains one or more second antigens to form a multivalent vaccine dosage form with synthetic nanocarriers. In other embodiments, additional antigens other than one or more of the first and / or second antigens (by mixing and other techniques disclosed herein or traditionally known) can be incorporated into a dosage form. In embodiments, inventive compositions provided herein comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or more different antigens.

In embodiments, one or more of the first antigens and / or one or more of the second antigens include or is derived from or derived from a virus from the virus family shown in the table below. 1. In another embodiment, one or more of the first antigens and / or one or more of the second antigens include or receive or come from a virus from the species shown in table. one. In yet another embodiment, one or more of the first antigens and / or one or more of the second antigens include or are derived from or derived from the antigen shown in Table 1. one.

Table 1. Viral Infectious Agents

<td>Family</td><td>Illustrative view</td><td>Illustrative antigens</td>

<td>ΑάθηονίΓίάβθ</td><td>adenovirus</td><td>VI, VII, E1A, EZ-19K, 52K</td>

<td rowspan="4">ΡίοοΓηβνίΓΐάββ</td><td>coxsackie virus</td><td>νρι</td>

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

<td>virus polio</td><td>protein FOR, capsid protein</td>

<td>rhinovirus (e.g. type sixteen)</td><td>nucleocapsid outgrowth surface and transmembrane proteins</td>

<td rowspan="5">Negresuhgtäe</td><td>herpes simplex (type 1 and type 2)</td><td>capsid proteins (e.g., ib, b18, bb5, bb8 and bb)</td>

<td>chickenpox smallpox</td><td>early antigen</td>

<td>Epstein virus Barra</td><td>early capsid antigen antigen</td>

<td>cytomegalovirus of man</td><td>Rrb5, dB, p52</td>

<td>herpes virus person (e.g. type 8)</td><td>latent nuclear antigen-1</td>

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

<td>ΓΙδνίνίΓίάββ</td><td>hepatitis C virus yellow virus fever virus dengue virus West Nile</td><td>N33, shell protein (e.g. E2 domain)</td>

<td>KeRgoU1g0.s1ae</td><td>ΗΡΥ</td><td>dr12O, p24 and lipopeptides Garden (17-35), Garden (253284), £ £ (66-97), £ £ (116-145) and Po1 (325355); see Kobegrz e £ a!.,</td>

- 21 023397

<td></td><td></td><td>SG, Ιιοιηυηοΐ. MeBoOz, 365 (1-2): 27-37, 2011</td>

<td>ΟΓίΗοηγχονίΓίάββ</td><td>flu virus</td><td>neuraminidase surface antigen</td>

<td rowspan="5">RagapuhoU1G1s1ae</td><td>measles virus</td><td rowspan="5">nucleocapsid protein matrix protein phosphoprotein, protein fusions, hemagglutinin, hemagglutinin- neuraminidase glycoprotein</td>

<td>mumps virus</td>

<td>parainfluenza virus</td>

<td>respiratory syncytial virus</td>

<td>Metapneumovirus of man</td>

<td>ΡθρίΙΙοπ ^ νίΓΪάθθ</td><td>papillomavirus of man (e.g. type 16 and 18)</td><td>EB, E7, capsid antigen</td>

<td>KNaBo νί g 1s1a e</td><td>rabies virus</td><td>membrane lipoprotein</td>

<td>Todauge</td><td>rubella virus</td><td>capsid protein</td>

<td>ΡθΓονίΓίάθβ</td><td>bokavirus person parvovirus B19</td><td>capsid protein non-structural protein (N3)</td>

In embodiments, one or more of the first antigens and / or one or more of the second antigens include or are derived from, or derived from, a bacterium of the bacterial genus shown in Table 1 below. 2. In another embodiment, one or more of the first antigens and / or one or more of the second antigens include or are derived from or derived from the bacterial species shown in table. 2. In yet another embodiment, one or more of the first antigens and / or one or more of the second antigens include or are derived from or derived from the antigen shown in Table 1. 2.

Table 2. Bacterial Infectious Agents

<td>Pathogenic used aterial ROD</td><td>Illustrative view</td><td>Illustrative antigens</td>

<td>VoglebeYa</td><td>Vogs1eBe11a regT 115515</td><td>pertussis toxin (RT), filament hemagglutinin (RIA), pertakgin (ΡΚΝ) and Fimbria (PM 2/3)</td>

<td>Wogge</td><td>Wogge Biggs</td><td>νίδΕ; OPA and ΟδρΑ</td>

<td rowspan="4">Vgise11a</td><td>Bruise</td><td>NLA, PrgrA, M1CA, L7 / P2, P15, 0187, νΐτσ, Mb, A £ aA</td>

<td>VgiseSap Sap15</td><td>B7 / b2</td>

<td>Wow TEOOEPZHS</td><td>extramembrane proteins such like Oshr28</td>

<td>High School</td><td></td>

<td>Satru1obbac1: er</td><td>Satru1obac £ eg Zesipy</td><td>Bp3, 100-kDa antigen</td>

<td rowspan="3">Syatusia and SL2ashus (ORLYa</td><td>SMATUS LA rpeiyayupgay</td><td rowspan="3">See Chxbag eO a1., B 1n Ы Ы ои 5 5 GLgeasez. 181: 3521 (2000)</td>

<td>SATUSL Notes 5</td>

<td>SYATUSLRKYa rzYёas!</td>

<td rowspan="2">C1oz £ r1cIip1</td><td>SYSGZS-SLIT Boobs</td><td>types of antigens A, B, C, ϋ And E</td>

<td>С1о5ёгАсЛит ZYSYSE</td><td>GUS, Γΐΐϋ and Smr84</td>

- 22 023397

<td rowspan="2"></td><td>01θ5ϋΓΪάίιΐΓΠ rugbghpdepz</td><td>alpha toxin, theta toxin, fructose-1, b-bisphosphate- aldolase (GVA), glyceraldehyde-3-phosphate dehydrogenase (6Ρϋ), pyruvate: ferredoxin oxidoreductase (RGOE), elongation factor-0 (ΕΕ-Θ) and hypothetical protein (HP)</td>

<td>S1o5bg1b1itp bebap1</td><td>T-toxin</td>

<td>Sogupasbeghit</td><td>SogupEasbegs.it SNRBNeghe</td><td>toxoid antigen</td>

<td>Epbegosossie</td><td>Epbegosossie TaesaI5 Epbegosossis £ aes1it</td><td>capsular polysaccharides</td>

<td>Exodus</td><td>E5cbc1c? 11a</td><td>See Mog1e1 eb a1., ΡΝΑ5 107 (20): 9072-9077 (2010)</td>

<td>Ggaps1ze11a</td><td>Egaps15e11a £ i1agep215</td><td>See Nau1agoua eb a1., Rgobeopeis 2 (7): 657-867, 2002</td>

<td>NaetorM1i5</td><td>NayporNiz 1n £ 1</td><td>capsular polysaccharides, protein ϋ</td>

<td>NECESSARY</td><td>NeoBac1: er ru1og1</td><td>See Vitapp eb a1., Rgobeoshkhse 4 (10): 2843- 2643, 2004</td>

<td>Bd1ope11a</td><td>Bd1ope11a rpeitor</td><td>Μίρ</td>

<td>Yerbozrhg</td><td>Yerbozrhga hpbeggodapz</td><td>See Woip eb a1., 1n £ eSb 1ti 59 (5): 1772-1777, 1991</td>

<td>Yzbega</td><td>Y5beg1a toposubodepez</td><td>nucleoprotein (ΝΡ)</td>

- 23 023397

<td rowspan="3">Musoasbeghit *</td><td>MusoRasGeggit 1st place</td><td></td>

<td>MusoBas £ eίίυπ Boobsi1o515</td><td>KSH, CUT, PPE68, Exxa, ExxB, U9 and ExxU</td>

<td>Musoasbeggit and 1segapz</td><td></td>

<td>Garbage</td><td>Garbage! Aeta rpeitopgai</td><td>Nzr70</td>

<td rowspan="2">ΝβίΒεθΓΪβ</td><td>Ebezegha auxiliary</td><td></td>

<td>Bj155g1a ιαβηίηςϊίίάίδ</td><td>See, Ybb eb a1., L. 1pTesOiz from Obzease 190 (8): 1488-1497, 2004</td>

<td>Rzeisktopaa</td><td>Rzeysknpopaa Aegidum 1 pose</td><td>lipopolysaccharides</td>

<td>Iskebbzt</td><td>P1ske £$ $ 1a ghske ^ bbI</td><td>surface antigen</td>

<td rowspan="2">8a1top11a</td><td>5a1top11a drills</td><td></td>

<td>5a1top11a YurYtikhit</td><td></td>

<td>5Yde11a</td><td>Zide11a zoppeb</td><td></td>

<td rowspan="3">ЗбарЬу1ossossis</td><td>5karNu1ossossi5 aigeya</td><td>See UubUbzka eb a1., Rgobeotbs 2 (5): 580-590, 2002; Ebg eb a1., ΡΝΑ3 99 (10): 6573-6578, - 2002</td>

<td>5thar1ossoss θρίάβπαίάίε</td><td></td>

<td>5thar1ossoss Zagorububsiz</td><td></td>

<td rowspan="3">Zbberbossossis</td><td>Zbberbossossis adaasb! ae</td><td></td>

<td>Zbberbossossis ppeitoShaye</td><td>5р1, 5р2, ЗрЗ</td>

<td>Zbberbossossis roodepez</td><td>Bb eb a1., D. 1p1esb1oiz Schzease 189 (1): 79-89, 2004</td>

<td>Theropeta</td><td>Theropeta rashyit</td><td>glycerophosphodiester phphosphesterase a</td>

<td>νίΡτΐο</td><td>νίΡΓίο сьо1еге</td><td>outer membrane protein such as OTRK</td>

<td>WagsFpFa</td><td>Uegztpba resbbz</td><td>Chaperone Doorman protein capsule protein (E1) and ν'- protein</td>

In other embodiments, the implementation of one or more first antigens and / or one or more second antigens include or receive or come from a fungus from the genus of fungi, shown below in table. 3. In another embodiment, one or more of the first antigens and / or one or more of the second antigens include or are derived from or derived from the fungal species shown in Table. 3. In yet another embodiment, one or more of the first antigens and / or one or more of the second antigens include or are derived from or derived from the antigen shown in Table 1. 3.

- 24 023397

Table 3. Fungal Infectious Agents

<td>Kind</td><td>Illustrative view</td><td>Illustrative antigens</td>

<td>SapscDa</td><td>S. a1Ysapz</td><td>surface antigens see also TIotaz eb a1., RGTeotsz 6 (22): 6033- 6041, 2006</td>

<td>AzregdShiv</td><td>AzregdSchiz GitZdzSiz and AzregdSchiz £ 1 out</td><td>ZTeuepz ET A1., MesIsa1 Musoodu 49 (5irr1, 1): 3170-3176, 2011</td>

<td>SgurTosossis</td><td>SgurEossoss peobogtapps, SgurEossoss 1aigepTI and</td><td>capsular glycoproteins</td>

<td></td><td>SgurTosossis A1YDiz SgurSossoss DARI</td><td></td>

<td>H18bor1azta</td><td>Sz1: op1a5ta sarzi1aTiyu</td><td>UrzZr, NzrbO</td>

<td>Rpielshsuz Iz</td><td>RpeitosuzM 5 3 ΐΓονθοϋ</td><td>major surface proteins (Mzd) such as MzdS1, MzdSZ, MzdS8 and MzdS9</td>

<td>ZTasBuoTguz</td><td>Z'BasNuoTguz sabagit</td><td>Ssb334</td>

Combinations of a population of synthetic nanocarriers and one or more second antigens can be carried out using conventional pharmaceutical mixing methods. These include liquid-liquid mixing, in which two or more suspensions containing a synthetic nanocarrier population or one or more second antigens are directly combined or combined through one or more diluent containing vessels. Since synthetic nanocarriers can also be obtained or stored in powder form, dry powder-to-powder mixing can be carried out if one or more second antigens are available in powder form, and two or more powders can be resuspended in a common medium. Depending on the properties and interaction potential of synthetic nanocarriers and one or more second antigens, one or another way of mixing may be preferred. Techniques suitable for applying the practice of the present invention can be found in &amp; lt; RTI ID = 0.0 &amp; gt; &amp; lt; / RTI &amp; gt; &amp; lt; / RTI &amp; gt; EDTAGD. Rai1, Uyuyug Α. SHEET-OBSGSCH, AID 8i / APPS M. Kgek1a, 2004 Doni ADSu &amp; 8opk, 1ps .; and RNaggaaseiysk: TNs 8 above about £ Show Rogsh Oskscp, 2D ED. Units M. Ε. Αυΐεη, 2001, SNIGSYP Uut§51ope. In an embodiment, inventive synthetic nanocarriers are suspended in a sterile injectable saline solution with a preservative.

Doses of dosage forms contain varying numbers of populations of synthetic nanocarriers and varying amounts of one or more second antigens of the present invention. The amount of synthetic nanocarriers and / or one or more second antigens present in the inventive dosage forms may vary in accordance with the nature of the antigens, the therapeutic utility to be achieved, and other such parameters. In embodiments, studies can be conducted to determine the optimal dose to determine the optimal therapeutic amount of a population of synthetic nanocarriers and the amount of one or more second antigens that must be present in the dosage form. In embodiments, a population of synthetic nanocarriers and one or more second antigens are present in a dosage form in an amount effective to elicit an immune response to one or more first antigens and one or more second antigens

- 25 023397 when administered to a subject. It is possible to determine the amounts of the first, second and / or subsequent antigens effective for generating an immune response using traditional studies and techniques to determine the optimal dose in subjects.

In embodiments, inventive synthetic nanocarriers can be formulated by mixing non-adjuvanted adjuvants in the same diluent or delivery system as the synthetic nanocarrier population and one or more second antigens. Such adjuvants may include, but are not limited to, mineral salts such as alum, alum combined with monophosphoryl lipid (MPB) A enterobacteria, such as Escherichia coli, 8a-1a11aaaaaaaaaaaaaaaaaaa. 8a1tope11a 1uRtigst or 8iGe11a yeehiegt, or in particular with MPB® (A804), MPB A of the aforementioned bacteria separately, saponins, such as 08-21, -A, 18COM, 18COMATK1X ™, emulsions, such as MP59 ™, Moyatie® 18A 51 and Ι8Α 720, A802 (0821 + squalene + MPB®), A815, liposomes and liposome formulations, such as A801, synthesized or specifically prepared microparticles and microcarriers, such as vesicles of the outer membrane of bacterial origin (WMD) Ν. § ioiggyeae, Syatufa yasiotaya and others, or chitosan particles, depot forming agents, such as P1igots® block copolymers, specifically modified or prepared peptides, such as muramyl dipeptide, aminoalkyl glucosaminide-4-phosphates, such as KC529, or protein and or fragments of toxins. Dosages of such other adjuvants can be determined using conventional studies to determine the optimal dose. In embodiments, the adjuvant that is not attached to the listed populations of synthetic nanocarriers may be the same or different from the adjuvant that is attached to synthetic nanocarriers.

Typical inventive compositions containing synthetic nanocarriers may contain inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate or citrate) and pH adjusters (e.g., hydrochloric acid, sodium hydroxide or potassium hydroxide, salts of citrate or acetate, amino acids and their salts), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., Polysorbate 20, Polysorbate 80, polyoxyethylene 9-10-nonylphenol, sodium deoxycholate), solution stabilizers and / or cryo / lyostabilizers (e.g. sucrose, lactose, mannitol, trehalose), osmotic pressure regulating agents (e.g. salt or sugar), antibacterial agents (e.g. benzoic acid, phenol, gentamicin), anti-foaming agents (e.g. polydimethylsilosone), preservatives (e.g. thimerosal, 2-phenoxyethanol, EETA), polymer stabilizers and viscosity adjusting agents (e.g. polyvinylpyrrolidone, poloxamer 488, carboxymethyl cellulose), and cosolvents (e.g. glycerin, polyethylene glycol, ethanol).

The compositions of the present invention contain inventive synthetic nanocarriers in combination with pharmaceutically acceptable excipients. Compositions can be prepared using conventional pharmaceutical manufacturing techniques and compounds to achieve the applicable dosage forms. Techniques suitable for applying the practice of the present invention can be found in Clinox about £ 1puy8yta1 M1xx§: 8c1epse apy Prgase, E8. Come on b. Rai1, Uyuyug A. Ayeto-Oyep§, 8i / aiis M. Kgsla, 2004 1oyp \ UPSU &amp; 8op8, 1ps .; and Rähtasseiyes: Thb 8c1epse o £ Ooaagc Rogt Όεδί ^ η, 2ηά Her., Her. M.E. Ai1si, 2001; In an embodiment, inventive synthetic nanocarriers are suspended in a sterile injectable saline solution with a preservative.

It should be understood that the compositions of the present invention can be made by any suitable method, and the present invention is in no way limited to compositions that can be obtained using the methods described herein. Choosing an acceptable method may require attention to the properties of specific fragments that are associated.

In some embodiments, inventive synthetic nanocarriers are manufactured under sterile conditions or are sterilized at the end. This can ensure that the resulting composition is sterile and non-infectious, thus increasing safety compared to non-sterile compositions. This provides a valuable safety measure, especially when subjects receiving synthetic nanocarriers have immune defects, are suffering from infection and / or are susceptible to infection. In some embodiments, inventive synthetic nanocarriers can be lyophilized and stored in suspension or as a lyophilized powder, depending on the formulation technique over long periods without loss of activity.

Inventive compositions can be administered by a number of routes of administration, including, without limitation, subcutaneous, intramuscular, intradermal, oral; intranasal, transmucosal, sublingual, rectal, ophthalmic, transdermal, transdermal, or using a combination of these pathways.

Dosages of dosage forms contain varying amounts of synthetic nanocarriers or their populations and varying amounts of antigens and / or adjuvants of the present invention. The number of synthetic nanocarriers and / or antigens and / or adjuvants present in the inventive dosage forms may vary in accordance with the nature of the antigens, the therapeutic utility to be achieved, and other such parameters. In embodiments, studies can be conducted to determine the optimal dose to determine the optimal therapeutic amount of synthetic nanocarriers or their population and the number of antigens and / or adjuvants that must be present in the dosage form. In embodiments, synthetic nanocarriers and antigens and / or adjuvants are present in a dosage form in an amount effective to elicit an immune response to antigens when administered to a subject. It is possible to determine the amounts of antigens and / or adjuvants effective to generate an immune response using traditional studies and techniques to determine the optimal dose in subjects.

Inventive dosage forms can be administered at a number of frequencies. In a preferred embodiment, at least one administration of the dosage form is sufficient to produce a pharmacologically significant 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 significant response.

The compositions and methods described herein can be used to induce, enhance, suppress, modulate, direct, or redirect an immune response. The compositions and methods described herein can be used in the diagnosis, prevention and / or treatment of conditions such as malignant tumors, infectious diseases, metabolic diseases, degenerative diseases, autoimmune diseases, inflammatory diseases, immunological diseases or other disorders 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, exogenous toxin, or other harmful agent.

Subjects referred to in this document have or are at risk of cancer. Malignant tumors include, but are not limited to, breast cancer; bile duct cancer; bladder cancer; brain cancer, including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal carcinoma; stomach cancer; hematologic neoplasms, including acute lymphoblastic and myeloid leukemia, for example, B-cell Cb; T-cell acute lymphoblastic leukemia / lymphoma; Bernard histiolymphocytosis; chronic myeloid leukemia, multiple myeloma; AIDS-associated leukemia and adult T-cell leukemia / lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease, liver cancer; lung cancer; lymphomas, including Hodgkin's disease and lymphoblastic lymphomas; neuroblastomas; oral cancer, including squamous cell cancer; ovarian cancer, including arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer; sarcomas, 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 an embryonic cell tumor, such as seminoma, neseminoma (teratomas, choriocarcinoma), stromal tumors and germ cell tumors; thyroid cancer, including adenocarcinoma and medullary thyroid carcinoma, as well as kidney cancer, including adenocarcinoma and Wilms tumor.

The subjects provided herein have or are at risk of infection or infectious disease. Infections or infectious diseases include, but are not limited to, viral infectious diseases such as AIDS, chickenpox (chickenpox), runny nose, cytomegalovirus infection, Colorado fever, dengue fever, Ebola hemorrhagic fever, vesicular stomatitis, hepatitis, herpes simplex, herpes zoster, herpes zoster, herpes zoster, herpes zoster (influenza), Lassa fever, measles, cercopithecus hemorrhagic marburg-virus fever, infectious mononucleosis, mumps, norovirus infection, poliomyelitis, progressive multifocal leukoencephalopathy, rabies, rubella, 8AK8, smallpox (smallpox animals), viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, West Nile fever 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, listeriemosis, borreliosis, borreliosis attack, MK8A infection, nocardiosis, whooping cough (convulsive cough), plague, pneumococcal pneumonia, ornithosis, O-fever, Rocky Mountain spotted fever (KM8R), salmonellosis, scarlet fever, shigellosis, syphilis, tetanus, trachoma, tuberculosis, tularemia, typhoid fever, typhus and urinary tract infections; parasitic infectious diseases such as African trypanosomiasis, amoebiasis, ascariasis, babesiosis, Chagas disease, clonorchiasis, cryptosporidiosis, cysticercosis, diphyllobothriasis, dracunculiasis, echinococcosis, enterobiasis, fascioliasis, fasciolopsideosis

- 27 023397 filariasis, infections caused by free-living amoeba, giardiasis, gnatostomiasis, hymenolepidosis, coccidiosis, Indian visceral leishmaniasis, leishmaniasis, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis, toxiosis, trichosis, shchitozozloz, shade, trizochozloza, shade, triple stenosis, triple stenosis trichiuriasis, trichomoniasis and trypanosomiasis; fungal infections such as aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, foot epidermophytosis (dermatophytosis) and inguinal epidermophytosis; prion infectious diseases such as Alpers syndrome, malignant hereditary insomnia, Gerstmann-Streussler-Sheinker syndrome, Kuru and a variant of Creutzfeldt-Jakob disease

Examples

Example 1. Synthetic nanocarriers with covalently attached adjuvant.

Nanocarriers, including PbOA-K848, PbA-PEO-I3 and the ovalbumin peptide, were prepared using the double emulsion method, where the ovalbumin peptide is encapsulated in nanocarriers.

Polyvinyl alcohol (M \\ = 11 KDA - 31 KDA, 87-89% partially hydrolyzed) was purchased from TT Vaket. The ovalbumin peptide 323-339 was obtained from Vaset Ashepsak 1ps. (3132 Kakta 6ΐτεεΐ, Tottaps CA 90505. Product No. 4065609). Conjugates POBA-K848 and PBA-PEO-I3 were synthesized and purified.

The above materials were used to prepare the following solutions:

one. conjugate POBA-K848 at the rate of 100 mg / ml in methylene chloride;

2. RIA-RES-Sh conjugate at the rate of 100 mg / ml in methylene chloride;

3. ovalbumin peptide 323-339 at the rate of 70 mg / ml in 0.13n. HC1;

4. polyvinyl alcohol at the rate of 50 mg / ml in 100 mm phosphate buffer, pH 8.

Solution 1 (0.75 ml) and solution 2 (0.25 ml) were combined and solution 3 (0.1 ml) or 0.13 N. HC1 (0.1 ml) was poured into a small vessel and the mixture was sonicated at an amplitude of 50% for 40 s using Wadcop Cl1 Zajet 250. Solution 4 (2.0 ml) was poured into this emulsion and sonicated at an amplitude of 30 % for 40 s with the use of Vtkopop Osch-1 Zoshjet 250 formed a second emulsion. It was poured into a beaker with a stirrer containing 70 mM phosphate-buffered saline, pH 8 (30 ml), and this mixture was stirred at room temperature for 2 hours to form nanocarriers.

To wash the nanocarriers, a portion of the dispersion of nanoparticles (26.5 ml) was transferred to a 50 ml centrifuge tube and rotated at 9500 rpm (13800 d) for one hour at 4 ° С, the supernatant was removed, and the sediment in the tube was resuspended in 26, 5 ml of phosphate-saline buffer. The centrifugation procedure was repeated and the in vitro pellet was resuspended in 8.3 g of phosphate-salt buffer for a final nanocarrier dispersion of about 10 mg / ml.

To a suspension of synthetic nanocarriers (10 mg / ml in PBH (buffer at pH 7.4), 5 ml containing about 12.5 mg (M \ U: 20,000; 0,000625 mmol) PBA-PEO-S3) was added with weak while stirring, the peptide δΕφ ΙΌ N0: 2); which comes from b2, where X is a linker group including an acetylene linker (33 mg). Sodium ascorbate solution (100 mM in N) was added.₂O, 0.3 ml), and then a solution of CuZO₄ (10 mM in water, 0.6 ml). The resulting light yellow suspension was stirred at 20 ° C for 15 hours, and an additional CuZO solution was added.₄ (0.3 ml) and sodium ascorbate solution (0.15 ml). The suspension was stirred for 5 hours at 20 ° C and diluted with RVP buffer (pH 7.4) to 10 ml and centrifuged to remove the supernatant. Residual nanocarrier sediments were washed twice with RVZ buffer. The washed nanocarriers were then resuspended in 5 ml of PBZ buffer and stored frozen. Conjugation of the L2 peptide on the surface of synthetic nanocarriers was confirmed by HPLC analysis of split nanocarriers and bioanalysis.

Example 2. Composition with synthetic nanocarriers and non-attached antigens (possible).

A portion of a suspension of synthetic nanocarrier 4 ml from Example 1 containing 8 mg of substituted b2 nanocarriers is centrifuged to precipitate particles. The supernatant is removed and 0.5 ml of Oatyaky® suspension is added, a tetravalent human papillomavirus vaccine (types 6, 11, 16 and 18) containing purified virus-like particles (UBP) of the main capsid (L1) protein of the HPA types 6, 11, 16 and eighteen. The combination vaccine is shaken to resuspend the nanocarriers and the resulting suspension is stored at -20 ° C until use.

Example 3. Synthetic nanocarriers with non-covalently attached adjuvant [possible].

The cationic disulfide nanocarriers of RCCT containing DNA are prepared by the method described in the patent application OsZipops. ^ 02008118861. Example 16, except that the ssDNA fluorescein from Example 16 is replaced with the modified CpO 7909 DNA phosphothioate. After isolation, the cationic nanocarriers are suspended in 1.0 ml of a solution of PBH containing 10 mg / ml of heparin. After stirring at room temperature for 2 hours, the nanocarriers are isolated by centrifugation and washed twice with RW during centrifugation and decantation. Nanocarriers containing CPO 7909 with surface-adsorbed heparin are resuspended in 1.0 ml of PBH and stored at -20 ° C until use.

- 28 023397

Example 4. Composition with synthetic nanocarriers and an unadjoint antigen (possible).

A portion of a suspension of synthetic nanocarriers 1 ml from example 3 containing 10 mg of heparin-substituted nanocarriers is centrifuged to precipitate particles. The supernatant is removed and 1 ml of a suspension of vaccines ResotLAax HB® or Epgepch-B®, vaccines against human hepatitis B virus (HBV) containing purified protein-like particles consisting of the protein of the main surface antigen (HBzA) of HBI is added. The combination vaccine is shaken to resuspend the nanocarriers, and the resulting suspension is stored at −20 ° C. until use. A similar method is used to combine the heparin-substituted nanocarriers from Example 3 with 1 ml of a suspension of a bivalent vaccine against hepatitis A and B viruses (Τ ^ ΐητΐχ®) consisting of purified HBcA and inactivated human hepatitis A virus.

Example 5. Synthetic nanocarriers with covalently attached adjuvant [possible].

Example 5A Obtaining 4848 covalently attached to a thiol

<img file="EA023397B1_D0002.tif" />

3,3'-Dithio-bis-propionic acid (catalog number 109010) is purchased from AlpnCet1c1 Sotrapu. 4848 are synthesized in 8e1ec1a VuzOepsez. Solution of 3,3'-dithio-bis-propionic acid (2.10 mg, 1.0x10 '² mol) and NVTi (15.2 g, 4x10 ')² mol) in ЕУАс (450 ml) was stirred at room temperature under argon for 45 min. Compound 4848 (6.28 g, 2x10 ') was added.² mol), and then B1REA (20.9 ml, 1.2 x 10¹ mole). The mixture was stirred at room temperature for 6 hours and then at 50-55 ° C for 15 hours. After cooling, the mixture was washed with 1% citric acid solution (2x40 ml), water (40 ml) and brine solution (40 ml). The solution is dried over Να₂8Ο. | (10 g), and after filtration, ethyl acetate was removed in vacuo. The product was recrystallized from 2-methoxyethanol to provide 6.5 mg (78%) of a white solid.

The above disulfide (5.0 mg) was dissolved in chloroform (200 ml) and the solution was treated with dithiothreitol (1.0 mg). After stirring at room temperature for 2 hours, the solution in chloroform is washed with water (100 ml) and then dried over sodium sulfate. After filtration to remove the drying agent, chloroform is removed in vacuo and the remaining solid is purified by silica gel chromatography using 10% methanol in methylene chloride as eluent. Fractions containing thiol-4848 conjugate were combined and evaporated to give 3.5 mg of thiol-4848 conjugate (70%) as a white solid.

Example 5B Obtaining nanocarriers.

Synthetic gold nanocarriers are prepared as described in Example (a) of US Patent Application 2009 2009 0104268 A1 MiA1bTbNTb, except that the BC11 peptide is replaced by the thiol4848 conjugate from Example 5A above, and the oligosaccharide antigens are replaced by a peptide that originates from b2, H A1a-Tyt-O1n-Ley-Tut-Luz-Tyt-Suz-Luz-O1n-A1a-O1y-Tyt-Suz-Rto-Rto-Azr-11e-11e-Rto-LuzUa1-X (8Er 1B N0: 2) ; where X is a linker group comprising a cysteine ​​residue. After washing and concentration, as described in the application MiAlb, a suspension of particles of 1.0 mg, is used as described in example 6.

Example 6. Composition with synthetic nanocarriers and non-attached antigens (possible).

A part of the gold nanocarriers 1.0 mg from Example 5 was added to 1 ml of an oral suspension of the live recombinant antirotavirus vaccine 4o1apx® against gastroenteritis caused by rotavirus type O1 and types not related to O1 (03, 04 and 09). The combined oral vaccine is shaken to resuspend the nanocarriers, and the resulting suspension is stored at −20 ° C. until used as the combined oral vaccine.

Example 7. Synthetic nanocarriers with antigen for T-helper and adjuvant.

The ovate salt of the amide of the ovalbumin peptide 323-339 was purchased from Vaset Atepsaz 1ps. (3132 Kazyta 81tee1, Tottaps CA 90505. Product code 4065609.) PHOA-4848, a copolymer of B / L-lactide and glycolide, 4-amino -2- (ethoxymethyl) -a, a-dimethyl -1H-imidazo [4,5 -c] quinoline-1-ethanolamide approximately

- 29 023397

7000 Yes, obtained from PbOA with a ratio of lactide to glycolide of 3: 1 and having an approximate content of conjugated resiquimod of 8.5% wt./weight, custom-made for RppssupOlObA1 8up111C515 (300 Oeogde Rayegkoi иνε # 206, Bp51o1, PA 19007.) PPA -PEC-C6-I3, a block copolymer consisting of a block of poly-O / L-lactide (PBA) of approximately 23,000 Da and a block of polyethylene glycol (PEO) of approximately 2000 Da, which ends with an amide-conjugated C6H12 linker to an azide, were synthesized by conjugation of IO-PEO-SOOI to amino-C6H12-azide and then the formation of the PBA block by ring opening polymerization of the resulting IO-PEO-Sb-III with β-lactide. Polyvinyl alcohol РНЕиг, И8Р (85-89% hydrolyzed, viscosity 3.4-4.6 mPa-s) was purchased from ΕΜΌ Syetual L 1ps. (480 §0th! Estosga! Koai 0 | L51otep, W 08027. Product number 4-88).

The solutions were prepared as follows:

Solution 1: ovalbumin peptide 323-339 at the rate of 20 mg / ml was prepared in 0.13 N. Η0 at room temperature.

Solution 2: PbOA-K848 at a rate of 50 mg / ml and PbA-PEO-C6-No at a rate of 50 mg / ml in dichloromethane was prepared by dissolving each separately at 100 mg / ml in dichloromethane, followed by combining in equal parts by volume.

Solution 3: polyvinyl alcohol at the rate of 50 mg / ml in 100 mm in 100 mm phosphate buffer, pH 8

Solution 4: 70 mM phosphate buffer, pH 8.

Initially, a primary emulsion (\ M1 / ​​0) was prepared using solution 1 and solution 2. Solution 1 (0.2 ml) and solution 2 (1.0 ml) were combined in a small glass tube for pressure work and sonicated at an amplitude of 50 % for 40 s with the use of Vgaizoi ΌίβίΗιΙ ZoshPsg 250. A secondary emulsion (A1 / O / A2) was then formed by adding solution 3 (2.0 ml) to the primary emulsion, vortexing to form a coarse dispersion, and then sonicating at an amplitude of 30% for 40 s using Vgapkop ΌίβίΡιΙ ZoshPsg 250 .

The secondary emulsion was poured into an open 50 ml beaker containing a 70 mM phosphate buffer solution (30 ml) and stirred at room temperature for 2 hours to allow the evaporation of dichloromethane and the formation of a suspension of nanocarriers. A part of the suspended nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 21,000 ° C for 45 min, removing the supernatant and resuspending the precipitate in phosphate-buffered saline. This washing procedure was repeated, and then the precipitate was resuspended in phosphate-buffered saline to provide a nanocarrier suspension having a nominal concentration of 10 mg / ml polymer based. Two identical batches were obtained and then combined to form a single homogeneous suspension, which was stored frozen at -20 ° C until further use.

Table 4. Characterization of nanocarrier

<td>Nanocarrier</td><td>Effective diameter (nm)</td><td>TK agonist, % wt./weight.</td><td>Antigen, % wt./weight.</td>

<td></td><td> 209</td><td>K848, 4.2</td><td>Ovalbumin peptide 323-339, 2.4</td>

Example 8. Immunization with synthetic nanocarriers with attached antigen and free protein without admixed adjuvant.

Materials and methods.

(1) Nanocarriers with surface REO-C6-No, containing PbOA-K848 and Oua-peptide, prepared as described above in Example 7, 7 mg / ml suspension in Pb§.

(2) Peptide M2e modified with an alkyn linker attached to the C-terminal O1y; С8 Vyu So, catalog number С84956, series: Н308, MV 2650, TPA salt; sequence:

Η-Μеΐ-δег- ^ е- ^ е-ΤЬг-О1и-Vа1-О1и-Ьг-Ргο-ЬЬг-Αг§-Α8η-О1и-Τгρ-О1и-Su8-Αг§-Su8-δeg-Α8ρ-О1уΟγ ΝΗί'Ή2ί.'ί'Ή (5ЕО ΙΌ ^: 3).

(3) Catalysts: Cu8O₄, 100 mM in ΌΙ-water; TNRTA ligand, 200 mM in ΌΙ-water; freshly prepared sodium ascorbate, 200 mM in ΌΙ-water.

(4) Buffer PB§ at pH 7.4.

A suspension of HC (7 mg / ml, 4 ml) was concentrated to a volume of approximately 1 ml in a centrifuge. A solution of M2e peptide (20 mg) in 2 ml of PB§ buffer was added. A pre-mixed solution of 0.2 ml Cu8O was added.₄ (100 mmol) and 0.2 ml of TNPTA ligand (200 mmol), followed by 0.4 ml of sodium ascorbate (200 mmol). The resulting light yellow suspension was stirred in the dark at ambient room temperature for 18 hours. The suspension was then diluted with PB§ buffer to 10 ml and centrifuged to remove the supernatant. The ΝΟ-Μ2ε conjugates in the form of a precipitate were additionally washed twice with 10 ml of PB§ buffer and resuspended in buffer, pH 7.4 to a final concentration of approximately 6 mg / ml (about 4 ml) and stored at 4 ° C.

Results.

Antibody titers were measured in mice immunized with a combination of ΝΟ-Μ2ε and free hemagglutinin from a bird flu virus strain Η5Ν1 (Vietnam). ΝΟ-Μ2ε contained T helper

- 30 023397 peptide ΟΡ-ΙΙ (2.4%) and adjuvant K848 (4.2%). Each bar represents a titer to the antigen. Five animals per group were immunized with SC 120 mcg Νί '.' and 10 μg of hemagglutinin H5 per injection, 2 times at 3 weekly intervals. Titres for day 33 after the first immunization are shown (E18A to PBA-PEOM2e and H5 hemagglutinin, respectively).

The results show that immunization with a combination of Νί carrying an antigen mixed with a free protein without a mixed adjuvant leads to the production of antibodies to both the antigen carried by Νί and the free protein. When Νί containing the surface peptide M2e from influenza A virus (ectodomain of the matrix protein M2, amino acids 2-27) was mixed with the free hemagglutinin protein of influenza A virus and used to immunize animals, a strong humoral response was induced in all animals to both peptides M2e and hemagglutinin ( Fig. 1). No reactivity was detected in the serum of mice before immunization.

Example 9. Immunization with synthetic nanocarriers with attached antigen and free protein with admixed adjuvant.

Materials and methods.

(1) Nanocarriers with surface RES-S6-I3. containing PHOA-K848 and Ota-peptide, prepared as described above in example 7, 7 mg / ml suspension in RVB.

(2) M2e peptide modified with an alkyn linker attached to the C-terminal Οΐγ; С8 Βίο Со, catalog number С84956, series: Н308, MV 2650, TPA salt; sequence:

H-Meΐ-δеt- ^ е- ^ е-ТЬτ-Ο1и-Vа1-Ο1и-ТТ-Ρτο-ТТТ-АТ§-А8η-Ο1и-Тτρ-Ο1и-Сγ8-АТ§-Сγ8-δеτ-A8ρ-Ο1НССНу 81T) GO N0: 3).

(3) Catalysts: Cu8O₄, 100 mM in ΌΙ-water; TNRTA ligand, 200 mM in ΌΙ-water; freshly prepared sodium ascorbate, 200 mM in ΌΙ-water.

(4) Buffer PB8, pH 7.4.

A suspension of HC (7 mg / ml, 4 ml) was concentrated to a volume of approximately 1 ml in a centrifuge. A solution of M2e peptide (20 mg) in 2 ml of PB8 buffer was added. A pre-mixed solution of 0.2 ml CuB0 was added.₄ (100 mmol) and 0.2 ml of TNPTA ligand (200 mmol), followed by 0.4 ml of sodium ascorbate (200 mmol). The resulting light yellow suspension was stirred in the dark at ambient room temperature for 18 hours. The suspension was then diluted with PB8 buffer to 10 ml and centrifuged to remove the supernatant. The NS-M2e conjugates as a precipitate were additionally washed twice with 10 ml of PB8 buffer and resuspended in buffer, pH 7.4, to a final concentration of about 6 mg / ml (about 4 ml) and stored at 4 ° C.

Results.

Antibody titers were measured in mice immunized with a combination of NС-М2е and free hemagglutinin from the bird flu virus strain Η5Ν1 (Vietnam) mixed with 80 μg of alum. NC-M2e contained the T-helper peptide OP-11 (2.4%) and the K848 adjuvant (4.2%). Each bar represents a titer to the antigen. Five animals per group were immunized with a dose of 120 μg HC and 10 μg H5 hemagglutinin per injection, 2 times at 3-week intervals. The titers for day 33 after the first immunization are shown (E18A to PBA-PEO-M2e and H5 hemagglutinin, respectively).

The results show that immunization with a combination of ΝΟ carrying an antigen mixed with a second antigen (free protein) with an adjuvant mixed with it leads to the development of antibodies to both the antigen carried by ΝΟ and the second antigen. ΝΟ containing the surface peptide M2e from influenza A virus (ectodomain of the matrix protein M2, amino acids 2-27) was mixed with the free hemagglutinin protein of influenza A virus and used to immunize animals by mixing with alum (1pt_ | cc1 A1ish, P1etse), a strong humoral response was induced in all animals both to the M2e peptide and to hemagglutinin (Fig. 2). No reactivity was detected in the serum of mice before immunization.

Example 10. Immunization with synthetic nanocarriers with attached antigen and viral vaccine and adjuvant Materials and methods.

(1) Nanocarriers with surface REO-C6-No, containing PbOA-K.848 and Ota peptide, prepared as described above in Example 7, 7 mg / ml suspension in PB8.

(2) M2e peptide modified with an alkyn linker attached to the C-terminal Οΐγ; С8 Vyu So, catalog number С84956, series: Н308, MV 2650, TPA salt; sequence:

Η-Meΐ-δеt- ^ е- ^ е-ТТτ-Ο1и-Vа1-Ο1и-ТТ-Ρτο-ТТТ-АТ§-А8η-Ο1и-Тτρ-Ο1и-Сγ8-АТ§-Сγ8-δеτ-A8ρ-Ο1НССН 81T) ΙΌ ΝΟ: 3).

(3) Catalysts: Cu8O₄, 100 mM in ΌΙ-water; TNRTA ligand, 200 mM in ΌΙ-water; freshly prepared sodium ascorbate, 200 mM in ΌΙ-water.

(4) Buffer PB8, pH 7.4.

A suspension of HC (7 mg / ml, 4 ml) was concentrated to a volume of approximately 1 ml in a centrifuge. A solution of M2e peptide (20 mg) in 2 ml of PB8 buffer was added. A pre-mixed solution of 0.2 ml Cu8O was added.₄ (100 mmol) and 0.2 ml of TNPTA ligand (200 mmol), followed by 0.4 ml of sodium ascorbate (200 mmol). The resulting light yellow suspension was stirred in the dark at ambient room temperature for 18 hours. The suspension was then diluted with PB8 buffer to 10 ml and centrifuged to remove the supernatant. NC-M2e conjugates in the form of a precipitate were additionally washed twice with 10 ml of ΡΒδ buffer and resuspended in buffer, pH 7.4, to a final concentration of about 6 mg / ml (about 4 ml) and stored at 4 ° C.

Results.

Antibody titers were measured in mice immunized with a combination of ΝΟ-Μ2ε and β-propiolactone inactivated with influenza A virus Η1Ν1 (Η1Ν1 New Caledonia / 20/99 / ΐνΕ 116) mixed with 80 μg of alum. ΝΟ-Μ2ε contained the T-helper peptide ΟΡ-II (2.4%) and the E848 adjuvant (4.2%). Each bar represents a titer to the antigen. Five animals per group were immunized with SC 120 μg ΝС and 1 μg of thimerosal-containing inactivated Η1Ν1 New Caledonia injection, 2 times at 3-week intervals. The titers for day 33 after the first immunization are shown (EYEA to RLA-RES-M2e and Η1Ν1 New Caledonia, respectively).

The results show that immunization with a combination of N0 carrying an antigen mixed with an inactivated viral vaccine and an adjuvant leads to the production of antibodies to both the перенос tolerated antigen and the inactivated virus. When ΝΌ containing the surface peptide M2e from the influenza A virus (ectodomain of the matrix protein M2, amino acids 2-27) was mixed with the inactivated influenza A virus NSh1i was used to immunize animals, mixed with alum (1uyes1 A1ish, P1egse), a strong humoral response was induced in all animals to both the M2e peptide and the inactivated influenza A virus Η1Η1 (Fig. 3). No reactivity was detected in the serum of mice before immunization.

Example 11. Immunization with synthetic nanocarriers with attached antigen and recombinant vaccine with adjuvant.

Materials and methods.

(1) Nanocarriers with surface RES-S6-I3. containing PBCA-E848 and ΟνΑ-peptide. prepared as described above in example 7, 7 mg / ml suspension in ΡΒδ.

(2) Peptide ΗΓνίΓ), modified with an alkynyl linker attached to the C-terminal L-amino acid; Β; κ1ιαη Ashepsak, 1ps, series B06055, ΜΒ 2595 salt ΤЕΑ; sequence:

Η-Α1а-ΤЬг-С1η- ^ еи-Τуг- ^ ук-ЬЬг-Сук- ^ ук-С1η-Α1а-С1у-ЬЬг-Сук-Ρгο-Ρгο-Αкρ-I1е-I1е-Ρгο- ^ ук-Vа1- ^ yk (5hexinoyl) ^ Yu (with Suk-Suk-disulfide bond) (δΕΟ ΙΌ ΝΟ: 2).

(3) Catalysts: ^ δΟ ^ 100 mM in ΌΙ-water; TNRTA ligand, 200 mM in ΌΙ-water; freshly prepared sodium ascorbate, 200 mM in ΌΙ-water.

(4) Buffer ΡΒδ, pH 7.4.

A suspension of HC (7 mg / ml, 4 ml) was concentrated to a volume of approximately 1 ml in a centrifuge. A solution of peptide L2 (20 mg) in 2 ml of ΡΒδ buffer was added. A pre-mixed solution of 0.2 ml of &amp; ιδΟ. · Ι (100 mmol) and 0.2 ml of TNPTA ligand (200 mmol) were added, followed by 0.4 ml of sodium ascorbate (200 mmol). The resulting light yellow suspension was stirred in the dark at ambient room temperature for 18 hours. Then the suspension was diluted with ΡΒδ buffer to 10 ml and centrifuged to remove the supernatant. Conjugates G '.'-B2 in the form of a precipitate were additionally washed twice with 10 ml of buffer ΡΒδ and resuspended in buffer, pH 7.4 to a final concentration of about 6 mg / ml (about 4 ml) and stored at 4 ° C.

Results.

Antibody titers were measured in mice immunized with a combination of NС-peptide L2 and ΗΒ ^ β of the out strain obtained in the fungus EassBagoshusek segetzhae mixed with 80 μg of alum. The HC2 peptide L2 contained the T-helper peptide β-II (2.4%) and the E848 adjuvant (4.2%). Each bar represents a titer to the antigen. Five animals per group were immunized with SC 120 μg HC and 0.6 μg recombinant ΗΒ ^ β per injection, 2 times at 3-week intervals. The titers for day 33 after the first immunization are shown (EYEA to RLA-RES-M2e and ZhkAd out, respectively).

The results show that immunization with a combination of ΝΌ carrying an antigen mixed with a recombinant vaccine and an adjuvant leads to the production of antibodies to both the antigen tolerated by ΝΌ and the inactivated virus. When ΝΌ containing the surface peptide L2 from the minor capsid protein L2 of the ΗΡV-16 virus (amino acids 17-36) was mixed with the recombinant hepatitis B surface antigen (ZhkAd) and used to immunize animals, mixed with alum (1uyes1 A1ish, P1egse), strong the humoral response was induced in all animals to both the L2 peptide and the recombinant β ^ β (Fig. 4). No reactivity was detected in the serum of mice before immunization.