 COMPOSITIONS AND USES
专利摘要:
The present invention relates to immunogenic compositions and methods of production thereof, and especially to immunogenic compositions comprising an oxoadenine adjuvant-crosslinked protein antigen. 公开号:BE1024148B1 申请号:E2016/5178 申请日:2016-03-10 公开日:2017-11-22 发明作者:David Burkhart；Michael Cochran；Daniel Larocque；Helene G. Bazin-Lee；Julien St-Jean；Christopher W. Cluff 申请人:Glaxosmithkline Biologicals S.A.； IPC主号:
专利说明:
COMPOSITIONS AND USES TECHNICAL FIELD OF THE INVENTION The present invention relates to immunogenic compositions and methods of creating self-adjuvanting vaccines and self-adjuvant immunotherapies comprising antigen-adjuvant conjugates and related methods of production. More particularly, the invention relates to an immunogenic composition comprising an oxoadenine adjuvant-crosslinked protein antigen. GOVERNMENT SUPPORT CLAUSE Aspects of the present invention have been made with the United States Government under contract # HHSN272200900036C with the NIH; the government may have certain rights over the invention. CONTEXT Effective and safe adjuvants are used to make subunit vaccines and sufficiently immunogenic immunotherapies. The simple admixture of an adjuvant with subunit vaccines or immunotherapies often requires the addition of adjuvant amounts that can lead to undesirable side effects. Such a mixture may require the development of a very demanding and expensive process and the characterization of the product may lead to a short shelf life, or may result in an inability to freeze or lyophilize the final product. In addition, the adjuvant dosage requirements can lead to a recovery of undesirable side effects. As a result, there is a need to improve the efficacy of subunit and adjuvant immunotherapies and vaccines. For optimal presentation of antigens to T cells, antigens and adjuvants must not only be absorbed by the same antigen presenting cell, but also be absorbed by the same phagosome (Medzhitoy, Nature 2006, Vol 440). Conventional antigen / adjuvant mixtures often require large amounts of adjuvant to ensure that both are phagocytosed simultaneously. An excess of adjuvant leads to an open reactogenicity which limits the utility of these adjuvant systems. SUMMARY OF THE INVENTION In a first aspect, the invention relates to an immunogenic composition comprising an oxoadenine compound bound to an antigen. The oxoadenine compound is suitably a 1-methyl-butoxy-oxoadenine having a piperidinyl substituent. The oxoadenine compounds of the present invention and the methods for the synthesis of oxoadenine compounds are described in WO2010 / 018134. In one aspect, the oxoadenine compound comprises formula (1): in which ; R1 is a branched or saturated C1-6 alkoxy group; R2 is a group having the structure: n is an integer having a value of 1 to 6; and Het is a 6-membered saturated heterocycle containing a nitrogen atom, in which Het is attached to the - (CH 2) n- moiety at any carbon atom of the heterocycle; or a pharmaceutically acceptable salt thereof. In another embodiment, R1 is n-butyloxy. In another embodiment, R1 is a (1S) -1-methylbutoxy group In another embodiment, n is 1, 2, 3, 4, 5 or 6. 4) In one aspect of the invention, the oxoadenine molecule is compound A. In one aspect of the invention, the oxoadenine molecule is compound B. In one aspect, the invention relates to an immunogenic composition comprising an oxoadenine compound bound to an antigen wherein the oxoadenine compound is bound to the antigen via a crosslinking agent. In one aspect of the invention, the oxoadenine-linked crosslinking agent is a hydrophilic compound. In another aspect of the invention, the addition of the oxoadenine crosslinking agent increases the aqueous solubility of oxoadenine compared to the solubility in the absence of the crosslinking agent. In another aspect of the invention, active oxoadenine is more soluble in water than inactive oxoadenine. In another aspect of the invention, the increased aqueous solubility of the active oxoadenine provided by the crosslinking agent decreases the amount of undesired aggregate in the antigen-adjuvant conjugate composition compared to the active oxoadenine exhibiting no increased solubility. In one aspect, the oxoadenine-linked crosslinking agent is a charged compound. In one aspect, the crosslinking agent is an amine-to-sulfhydryl crosslinking agent. In one aspect, the crosslinking agent contains an N-hydroxysuccinimide ester (NHS ester) and maleimide reactive groups at opposite ends of the crosslinking agent. In one aspect, the oxoadenine-linked crosslinking agent is GMBS, (N-gamma-maleimidobutyryl-oxysuccinimide ester) Hetero-bifunctional crosslinking agent, GMBS In one aspect, the GMBS crosslinking agent is initially reacted with oxoadenine and activates it. In one aspect, the GMBS crosslinking agent is initially reacted with the antigen and activates it. In another aspect, the crosslinking agent comprises Traut reagent (2-iminothiolane). Traut crosslinking reagent (2-iminothiolane HCl) In one aspect, the GMBS crosslinking agent is bound to oxoadenine via Traut's reagent. In a particular aspect, the crosslinking agent has the following structure: In one aspect, the antigen is directly conjugated to GMBS and the adjuvant is directly conjugated to Traut's reagent (referred to herein as "T-chemistry") B + 2-iminothiolane compound In another aspect, the adjuvant is directly conjugated to GMBS while the antigen is directly conjugated to Traut's reagent (referred to herein as "G-chemistry") When cross-linked to form the antigen-adjuvant conjugate, the conjugate can be described as having the following G or T chemistries: Chemistry G Chemistry T In one aspect of the invention, the antigen is conjugated to oxoadenine via the crosslinking agent. In the context of the present invention, the antigen conjugated to the adjuvant by the crosslinking agent induces a specific antigen response when administered to a subject, and in a particularly suitable aspect of the invention, the immunogenic composition induces a specific immune response in the absence of other additional adjuvant (eg, alum), in one aspect, a clinically significant specific immune response In one aspect of the invention, the antigen is an antigen of diphtheria toxin, and in a particular aspect of the invention it is "CRM" 197 (Cross Reactive Material 197). In another aspect of the invention, the antigen is a cytomegalovirus ("CMV") antigen, and in a particular aspect of the invention, a CMV gB antigen. In another aspect of the invention, the antigen is a cytomegalovirus ("CMV") antigen, and in a particular aspect of the invention, a CMV LVL759 gB antigen. In another aspect of the invention, the antigen is a hepatitis B antigen, and in a particular aspect of the invention a hepatitis B surface antigen ("HBsAg"). In another aspect of the invention, the antigen is a varicella or chickenpox and zoster ("VZV") antigen, and in a particular aspect of the invention a VZV gE antigen. In another aspect of the invention, the antigen is a tumor antigen, and in a particular aspect of the invention, a MAGE antigen or a BRAME antigen. In another aspect of the invention, the antigen is an influenza virus antigen, and in a particular aspect of the invention, an HA antigen. In another aspect of the invention, the antigen is an HIV antigen, and in a particular aspect of the invention, an HIV gag antigen or HIV env antigen. In another aspect of the invention, the oxoadenine molecule is bound to the antigen via a crosslinking agent and wherein the crosslinking agent is a hydrophilic compound and preferably thereby increases the aqueous solubility of the crosslinking agent. oxoadenine compared to solubility in the absence of the crosslinking agent. In another aspect, the invention comprises an active oxoadenine for crosslinking purposes in which the active oxoadenine is more soluble in water than the inactive oxoadenine. In another aspect of the invention, an active oxoadenine having increased aqueous solubility provided by the crosslinking agent thus reduces the amount of undesired aggregate in the immunogenic composition, compared to active oxoadenine having no increased solubility. . In another aspect of the invention, the number of antigen-conjugated adjuvant molecules (the "copy number") for the population of antigen-adjuvant conjugates in the immunogenic composition is in a desired range, particularly between 3 and 6 adjuvant adjuvant molecules to an antigen, where the number of copies represents the "approximate average" of the true number of immunoeffectors per molecule of antigen of a formulation. In another aspect of the invention, a first immunogenic composition comprising the antigen-adjuvant conjugate induces an increased immune response compared to a second immunogenic composition having equal amounts of antigen and adjuvant relative to the first immunogenic composition, but wherein the adjuvant and the adjuvant are not conjugated to each other (for example, adjuvant and antigen are mixed). The terms "aspects" of the invention and "embodiments" of the invention have the same meaning, are used interchangeably herein and are intended to mean a further but not restrictive description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Process flow diagram of T chemistry, an activated antigen with GMBS conjugated to an adjuvant. Figure 2. MALDI-TOF MS of antigen (1), GMBS-conjugated antigen (2) and adjuvant-conjugated antigen (3). The molecular weight offsets indicate an average of 11 copies of GMBS per antigen and an average of 3 copies of adjuvant per antigen. Figure 3. SDS-PAGE of antigen and antigen-adjuvant conjugate using a 10% Bis-Tris gene. The gel shows an increase in the molecular weight of the antigen-adjuvant conjugate in lane 3 compared to the unconjugated antigen in lane 2. Figure 4. RP-HPLC chromatogram showing control antigen (high), antigen-adjuvant conjugate prior to anion exchange purification (medium), and final antigen-adjuvant conjugate after purification (low). Small amounts of free unconjugated adjuvant were observed with a retention time of 10.2 minutes in the sample before the exchange of anions (medium) which were removed after the anion exchange (low) . Figure 5. SEC-HPLC chromatogram of antigen (high) and antigen-adjuvant conjugate (low) showing a small amount of dimer in the conjugate (<5%). FIG. 6: data showing RP-HPLC of fractions collected during ion exchange chromatography of a conjugate of compound B. Figure 7: TLR7 activation at very low doses by low copy number CRM-compound A conjugates and high copy number CRM-compound A using the TLR7 / NF-kB / SEAP stable reporter cell line by Imgenex . CRM-compound A conjugates (lower copy number or higher copy number) activate the human TLR7 pathway as efficiently as compound A alone. FIG. 8: dose-determination study of the A-CRM compound demonstrating that a dose-response in terms of CRM-specific IgG is observed with the CRM-compound A conjugates in BALB / c mice of 2 different origins in using T chemistry and G chemistry. Figure 9: Proof of concept study in mice demonstrating that antigen-adjuvant conjugates induce a significant humoral response based on CRM197 specific antibody. Figure 10a: Proof of concept study in mice demonstrating that CRM-compound B fusion conjugates are more effective even with 10x less adjuvant to mount a significant humoral response based on CRM197 specific antibody. FIG. 10b: the evaluation of the activation of T lymphocytes of immunized mouse splenocytes shows that the CRM-compound B fusion conjugate triggers a greater activation of CRM197 specific T lymphocytes compared to the CRM + compound B mixture at a concentration 1 X or 10 X. Figure 11a and 11b: levels of antibodies specific to CRM 7 days after the 2nd and 3rd injection; immunogenicity shows that CRM-oxoadenine is a potent antigen-adjuvant conjugate. Figure 12a: IgG specific IgG (humoral response) assay from gB conjugate vs. unadjuvanted gB control in mice. Figure 12b: Cell-mediated immunity (CMI) analysis to evaluate the CD4-IFNy response in mice. Figure 13: Dose determination in breeding pigs to test two types of CRM-compound A (G) conjugates vs. CRM-A (T) compound. Figure 14: Test of the CRM-specific humoral (antibody) response of three different TLR7 / 8-ligand conjugates in Yorkshire-Landrace-Duroc breeding pigs using the intramuscular route for immunization. Figure 15: Antibody response triggered by fused conjugates vs unadjuvanted controls after intradermal immunization in breeding pigs (Yorkshire-Landrace). Figure 16: Evaluation of the immunogenicity of HBsAg conjugated to compound B vs a mixture using the intradermal immunization route in the intradermal model of Yorkshire-Landrace Duroc breeding pigs. A comparison is also made with the intramuscular route (HBsAg + alum). DETAILED DESCRIPTION The present invention is described in terms known and appreciated by those skilled in the art. For ease of reference, certain terms will be defined below. However, the fact that some are defined will not be considered to indicate that defined terms are used inconsistently with their ordinary meaning or, alternatively, that undefined terms are undefined or not used in their ordinary and accepted meaning. On the other hand, all terms used herein are intended to describe the invention so that one skilled in the art will appreciate the scope of the present invention. The following definitions are intended to clarify, in a non restrictive manner, the defined terms. References to an "alkyl" group include references to straight chain and branched chain aliphatic isomers of the corresponding alkyl group, containing up to 8 carbon atoms, for example up to 6 carbon atoms, or up to four carbon atoms, or up to two carbon atoms, or one carbon atom. These references to the "alkyl" group are also applicable when an alkyl group is part of another group, for example an alkylamino group or an alkoxy group. Examples of such alkyl groups and alkyl group-containing groups are C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkylamino, and C 1-6 alkoxy. References to a "heterocycle" or a "heterocyclyl" group refer to a monocyclic, saturated heterocyclic aliphatic ring containing carbon atoms and a heteroatom, said heteroatom being a nitrogen atom. Such a heterocyclic ring is a piperidine or piperidinyl group. In a first aspect, the invention relates to an immunogenic composition comprising an oxoadenine compound bound to an antigen, wherein the oxoadenine is substituted at the C9 position with a piperidinylalkyl moiety. The oxoadenine compounds of the present invention and methods for their synthesis are described in WO2010 / 018134. A compound of formula 1 in which ; R1 is a C1-C8 alkoxy group, branched or saturated; R2 is a group having the structure: n is an integer having a value of 1 to 6; and Het is a 6 membered saturated heterocycle containing a nitrogen atom, in which Het is attached to the - (CH 2) n - moiety at any heterocycle atom; or pharmaceutically acceptable salts thereof. 4) Examples of oxoadenine compounds include: In a particular embodiment, the oxoadenine compound is the compound A In another particular embodiment, the oxoadenine compound is compound B. Conjugation Oxoadenine compounds are known to act as immunopotentiators and / or adjuvants. These oxoadenine compounds are covalently coupled to antigens such as, for example, CRM197 (inactivated by the 197 mutation in diphtheria toxin) or recombinant gB (human cytomegalovirus). Many chemical reactions are available to crosslink amines and molecules containing thiol groups (see Hermanson 2nd Edition). Several other classes of heterobifunctional crosslinking agents can be used to control the position of the TLR agonists on the antigen based on the crosslinking site and the length / composition of the linker. The use of different conjugation strategies can produce more or less effective responses; the conjugation strategy must be rigorously selected to provide the desired activity. Alternative strategies are described in this document. In a strategy, referred to as the G chemistry process, the antigenic protein fragment is reacted with a crosslinking nucleophile (e.g., a linker, e.g., Traut's reagent). The oxoadenine compound is activated with a complementary crosslinking electrophile (e.g., GMBS) and then added to the active antigen to terminate the conjugation. In a strategy variant, the T chemistry process, the TLR agonist (e.g., COMPOUND A) is reacted with a crosslinking reagent or linker (e.g., Traut's reagent). Then, the TLR and the fixed crosslinking material are reacted with the active antigen with GMBS. More specifically, in the chemistry process T, representing an embodiment of the present invention, In one aspect, the GMBS binds to the antigen via an amide linkage and the antigen-GMBS conjugate is then purified to remove any unconjugated GMBS. The number of GMBS copies per antigen is then determined using mass spectrometry techniques. Then, the GMBS-activated antigen is conjugated to the adjuvant via the free thiol group on the adjuvant. The free thiol group may be part of the oxoadenine or the modified oxoadenine structure. Alternatively, the free thiol group may also be added to the oxoadenine ring, for example by the use of a crosslinking reagent (eg, Traut's reagent). The number of copies of adjuvant molecules crosslinked to the antigen is determined and the composition is purified and characterized. Figure 1 (diagram). More particularly, the T chemistry may involve: (i) adjusting the pH of the antigenic protein fragment with a buffer such as a Good buffer (e.g., bicine) at pH greater than 7, (ii) l activating the buffered antigenic protein fragment having a pH greater than 7 with a crosslinking agent (e.g., GMBS); (iii) removing the excess crosslinking agent (GMBS) by means of centrifugal purification ultrafiltration; (iv) conjugation with an excess of adjuvant-crosslinking material (eg, compound A-Traut); (v) removing the excess TLR-crosslinking material by centrifugal ultrafiltration purification and / or ion exchange chromatography; (vi) filtration with a PVDF syringe filter to produce a sterile conjugated protein of the invention. In addition, protecting groups may be required to prevent dimerization of the activated peptide, for example, capping free thiol groups on the antigen when the process uses thiol-conjugation. Purification by ion exchange chromatography: In cases where the limited solubility of the adjuvant or adjuvant-linker (Traut) prevents the complete purification of the unconjugated adjuvant by ultrafiltration, it may be necessary to use ion exchange chromatography to remove the additive. In yet another aspect, a protein antigen is conjugated to an oxoadenine (e.g., COMPOUND A) according to "T chemistry" by performing the following steps: Step 1: pH adjustment of the protein to 7.4 Step 2: Pre-dissolution of GMBS in DMSO at ~ 1 mg / mL Step 3: Equilibrate Protein at 25 ° C Step 4: Add necessary GMBS Equivalents to Protein Step 5: Vortex Submission for 10 seconds to mix Step 6: Allow activation to take place for 0.5 h at 25 ° C Step 7: Transfer the Protein into a 15 mL Amicon Centrifuge Tube with a 30 kD MWCO Step 8: Wash the 3 X protein with 15 mL each time using 20 mM bicine, pH = 7.4, 0.15 M NaCl buffer Step 9: reconstitution of the 0.5 mg / mL protein in a washing buffer and taking an aliquot for MALDI-TOF MS. Step 10: Preparation of Traut A-reagent compound by dissolving compound A in DMF / H2O (9/1) at 25 mg / mL and adding 1 equivalent of 2-imminothiolane dissolved in DMF / H 2 O (9/1 ) at 25 mg / mL and 2.5 equivalents of diisopropylethylamine. The reaction was allowed to incubate at 25 ° C for 45 minutes and tested by LCMS to monitor the reaction. Step 11: Add Equivalents of A-Traut Compound to Activated Protein and Vortex Submission for 10 Seconds to Mix Step 12: Allow conjugation to take place at room temperature. Step 13: At t = 2 hr, take an aliquot and check it with MALDI-TOF MS to determine how many copies of Compound A should be added to the protein. Step 14: The number of copies of Compound A being sufficient, transfer the reaction solution to a 15 mL Amicon centrifuge tube with a 30 kD MWCO Step 15: Wash the X protein with 15 mL each time using 20 mM bicine, pH = 7.4, 0.15 M NaCl buffer Step 16: Reconstitution of the protein 0.5 mg / mL in wash buffer and aliquot taken by MALDI-TOF MS and HPLC analysis Step 17: Conjugate sterilization by filtration using a 0.22 micron PVDF filter Step 18: Store the conjugate at 2-8 ° C In the process, free thiol groups can cause dimer and trimer formation. To control the occurrence of free thiols and thereby minimize the formation of dimers and trimers, the free thiol groups are capped with IAA or NHM as follows: 1. Styling of free thiol groups in the antigen (e.g. gB) by treatment for 10 minutes with IAA, or NHM then purification by ultrafiltration to remove excess. 2. Activation with 20 to 40 equivalents of GMBS, reacted for 30 min at RT 3. Purification of excess GMBS by centrifugal ultrafiltration with a MWCO fr 30 kD 4. Conjugation with 20 molar excess of 668-Traut for 2 to 12 hours 5. Purification by centrifugal ultrafiltration with a MWCO of 30 kD to remove the excess of compound A-Traut 6. Sterilization by filtration with a 0.22 μm PVDF syringe filter 7. Characterization of Conjugates by MALDI-TOF, RP and SEC-HPLC, LAL, and BCA The reaction conditions (time, stoichiometry, base) may vary depending on the conjugated adjuvant and the reactivity of the primary / secondary amine, HCl salt vs free base, solubility, etc. Reaction conditions and purification methods may also vary depending on the properties of the antigen including sequence, stability, size, solubility, etc. Number of copies In one embodiment, an oxoadenine compound is covalently linked to the CRM197 molecule with three oxoadenine molecules per CRM antigen molecule. The copy number of conjugated oxoadenine can be controlled by multiple variables. For example, in the case of cross-linking by GMBS, some of the variables that control copy number are: excess of GMBS used to react with the antigen, the reaction temperature, the type of buffer, the reaction concentration, the reaction pH and finally, the excess oxoadenine activated during the final addition step. The stoichiometric relationship of the adjuvant molecules with the antigen is determined using techniques known in the art and in one aspect of the invention by mass spectrometry. The number of adjuvant molecules conjugated to an antigen will typically vary for any given number of molecules. In one aspect of the invention, the number of adjuvant molecules conjugated to the antigen will be present as a Gaussian distribution. In another aspect of the invention, in a majority of antigen-adjuvant conjugates in a composition, the ratio of antigen to adjuvant molecules is the number of copies. Therefore, the term "copy number" and the stoichiometric relationship of the immunostimulatory / immunopotentiating compounds with the antigen as measured by mass spectrometry or other methods represents an average of the true number of immunoeffectors per molecule of antigen. a formulation. In one aspect of the invention, the average number of copies is 1, 2, 3, 4, 5 or 6 or between 1 and 6, 2 and 5, and 3 and 4 oxoadenine molecules per molecule of antigen. . ANTIGENS The immunogenic formulations of the present invention comprise one or more antigens. In some aspects of the invention, an immunogenic composition may comprise more than one type of antigen, wherein all of the antigens are conjugated to an adjuvant while in another aspect, an immunogenic composition may include more than one type of antigen. an antigen in which at least one type of antigen is conjugated to an adjuvant, but another type of antigen may not be conjugated. In one aspect of the invention, these antigens may be selected from the group consisting of: diphtheria, hepatitis B surface antigen, cytomegalovirus b (gB) glycoprotein (CMV) and glycoprotein E (gE) Varicella and Herpes Zoster Virus (VZV) When administered, the antigens will typically be present in a final concentration of at least 1 μg / mL each, eg 1 μg / mL or in the range of 1 to 1 μg / mL. 20 μg / mL, 2 to 15 μg / mL, 2.5 to 10 μg / mL, 3 to 8 μg / mL, or 4 to 6 μg / mL or 5 μg / mL. In another aspect of the invention, the antigens may be present in a wider range of final concentrations, for example from 1 to 200 μg / ml and from 1 to 50 μg / ml, In general, the concentration of any antigen will be sufficient. to elicit an immune response against this antigen It is preferred that the protective efficacy of the individual antigens is not suppressed by combining them, although the immunogenicity itself (eg, ELISA titers) may be reduced. While at least one of the antigens in the immunogenic composition is conjugated to an adjuvant according to the present invention, in another embodiment of the invention other additional antigens are not so conjugated. For example, in an immunogenic composition of the present invention, a first antigen is conjugated to an oxoadenine adjuvant, while a second, different antigen in the immunogenic composition is not conjugated to an oxoadenine adjuvant. In these embodiments, the additional antigen may be free or, for example, may be adsorbed to a particle or vector, for example adsorbed to an aluminum salt, such as aluminum hydroxide. or aluminum phosphate or a mixture of aluminum hydroxide and aluminum phosphate. The antigens of the present invention will typically be protein antigens. Antigens in the immunogenic compositions of the invention will be present in "immunologically effective amounts", i.e., administration of that amount to an individual, in a single dose or as part of a series of doses, is effective for the treatment or prevention of a disease. The dosage may be a single-dose regimen or a multiple-dose regimen (e.g., including booster doses). Fragment (s) of the antigen. Fragments of the antigen (abbreviated as "Ag" herein) used in the present invention typically have certain properties useful in conjugation. In one aspect, the fragments of the antigen are proteins that are stable in solution and not susceptible to substantial aggregation. In another aspect, the fragment of the antigen contains a minimum of three available amines at which conjugation can occur and is less than 500 kD in size. In another aspect, preferred features or properties include: a solubility of the antigen fragment greater than 1 mg / mL, a monomer or oligomer (preferably a monomer), and a size of less than 150 kD. These fragments are readily available commercially and / or can be readily prepared by those skilled in the art. Fragments of the antigen that can be used in the invention include, for example, those set forth in Table 1, below. Table 1 * before detox: store at 20 ° C in glycerol; after detox: one month at 4 ° C. In certain aspects of the invention, the antigen candidates may include pertussis antigens, tetanus antigens, rhinovirus antigens, TB antigens, allergic antigens, and malaria antigens. CRM197 The present invention may comprise a diphtheria antigen, for example a diphtheria toxoid. The preparation of diphtheria toxoid (DT) is well documented. Any suitable diphtheria toxoid can be used. For example, DT may be produced by purifying the toxin from a Corynebacterium diphtheriae culture followed by chemical detoxification, but is alternatively performed by purifying a recombinant, or genetically detoxified analog of the toxin (e.g., CRM197 , or other mutants as described in US 4,709,017, US 5,843,711, US 5,601,827, and US 5,917,017). In one aspect of the invention, CRM197 is a non-toxic form of diphtheria toxin but is not immunologically distinguishable from diphtheria toxin. CRM197 is produced by C. diphtheriae infected with non-toxigenic beta phage beta-created by nitrosoguanidine mutagenesis of toxigenic corynephage (Uchida et al., Nature New Biology 233; 8-11). The CRM197 protein has the same molecular weight as diphtheria toxin but differs from it by a simple base change in the structural gene. This leads to a modification of glycine glycine amino acids at position 52 which renders fragment A incapable of binding to NAD and therefore non-toxic (Pappenheimer 1977, Ann Rev, Biochem 46, 69-94, Rappuoli Applied and Environmental Microbiology Sept 1983 p560-564). CMV gB In one aspect of the present invention, the antigen is derived from a cytomegalovirus (CMV). In a particular embodiment, the antigen is a gB polypeptide that suitably comprises at least a portion of an extracellular domain of the gB protein. The extracellular domain may further comprise a fusion loop domain 1 (FL1) and a fusion loop domain 2 (FL2), wherein at least one of the FL1 and FL2 domains comprises at least one deletion or substitution of amino acids. In another embodiment, the antigen is the truncated recombinant gB protein, deleted from a portion of the transmembrane domain, the cytoplasmic domain, as well as a portion of the leader sequence, optionally in a truncated form having mutations. additional in the FL1 and FL2 fusion loops of gB. It has been observed that the prior art gB polypeptides, such as the CMV gB polypeptide lacking a transmembrane domain, show heterogeneity in the N-terminal portion of the polypeptide. A homologous population should be understood as a population consisting primarily of a single mature polypeptide, the polypeptide invariably starting with a given amino acid at the N-terminus. In the present invention, a "mature" polypeptide refers to a polypeptide in which the signal sequence has been deleted by cleavage. In the present invention, in an N-terminal homogeneous population, more than 30%, suitably at least 80%, more suitably 80% to 90%, more suitably at least 99% mature polypeptides produced after cleavage of the signal sequence begin with the same amino acid at the N-terminus. Accordingly, in one embodiment, the invention relates to a preparation comprising a population of mature CMV gB polypeptides produced after cleavage of the signal sequence, wherein at least 30%, at least 80%, of 80% to 90% are present. % of mature gB polypeptides comprise the same amino acid at the N-terminus. In particular, the mature polypeptides of the invention suitably start with serine or histidine at the N-terminal position. HBsAg The envelope of the hepatitis B virus consists of a protein known as the hepatitis B surface antigen, and contains two other antigens known as pre-S1 and pre-S2 antigens. The heart of the virus contains two other proteins that act as antigens - the "e" antigen and the capsid antigen. In a particular embodiment, the antigen is a hepatitis B antigen, preferably the hepatitis B surface antigen (HBsAg). The preparation of hepatitis B surface antigen (HBsAg) is well documented. See, for example, Harford et al. in Develop. Biol. Standard 54, page 125 (1983), Gregg et al. in Biotechnology, 5, page 479 ¢ 1987), EP-A-0226846, EP-A-0299108 and references cited therein. As used herein, the term " hepatitis B surface antigen " or " HBsAg " includes any hepatitis B surface antigen or fragment thereof exhibiting the antigenicity of the hepatitis B surface antigen. surface antigen of HBV. It will be appreciated that in addition to the 226 amino acid sequence of the HBsAg S antigen (see Tiollais et al., Nature, 317, 489 (1985) and the references therein cited), HBsAg as described in US Pat. This document may, if desired, contain all or part of a pre-S sequence as described in the references above and in EP-A-0278940. HBsAg as described herein may also refer to variants, for example the "escape mutant" described in WO 91/14703. In another aspect, the HBsAg may comprise a protein described as SL * in European Patent Application No. 0 414 374, in other words a protein whose amino acid sequence consists of parts of the acid sequence amino acids of the large protein (L) of the hepatitis B virus (subtype ad or ay), characterized in that the amino acid sequence of the protein consists of: (a) residues 12 to 52, followed by residues 133 to 145, followed by residues 175 to 400 of said L protein; or (b) residue 12, followed by residues 14 to 52, followed by residues 133 to 145, followed by residues 175 to 400 of said protein L. HBsAg may also refer to the polypeptides described in EP0198474 or in EP0304578. In particular, the HBsAg may comprise a polypeptide comprising an amino acid sequence comprising residues 133 to 145 followed by residues 175 to 400 of the HBsAg L protein relative to the open reading frame on a hepatitis B virus. serotype Ad (this polypeptide is referred to as L *, see EP 0414374). HBsAg within the scope of the invention may also include the preS1-preS2-S polypeptide described in EP0198474 (Endotronics) or analogs thereof as described in EP0304578 (McCormick and Jones). HBsAg as described herein may also refer to mutants, for example the "escape mutant" described in WO 91/14703 or EP0511855A1, especially HBsAg wherein the acid substitution amine at position 145 is a glycine to arginine substitution. The preparation of hepatitis B surface antigen (HBsAg) is well documented. See, for example, Hartford et al., 1983, Develop. Biol. 54: 125, Gregg et al., 1987, Biotechnology 5: 479, EP0226846, EP0299108. It can be prepared in the following way. One method involves the purification of the particulate antigen from the plasma of chronic hepatitis B carriers, since large amounts of HBsAg are synthesized in the liver and released into the bloodstream during infection with the hepatitis B virus. HBV. Another method involves the expression of the protein by methods using recombinant DNA. HBsAg can be prepared by expression in yeast Saccharomyces cerevisiae, Pichia, insect cells (e.g., H15) or mammalian cells. HBsAg may be inserted into a plasmid, and its expression of the plasmid may be controlled by a promoter such as the "GAPDH" promoter (of the glyceraldehyde-3-phosphate dehydrogenase gene). The yeast can be grown in a synthetic medium. HBsAg can then be purified by a process involving steps such as precipitation, ion exchange chromatography, and ultrafiltration. After purification, HBsAg may be dialyzed (e.g., with cysteine). HBsAg can be used in particulate form. HBsAg can be in the form of particles. The particles may comprise, for example, protein S alone or may be composite particles, for example L *, S) where L * is as defined above and S is HBsAg S protein. Said particle is advantageously in the form in which it is expressed in yeast. In one embodiment, HBsAg is present in an amount of 1 to 20 μg, 5 to 20 μg, 8 to 15 μg or approximately or exactly 10 μg per 0.5 ml dose. In one embodiment, HBsAg is the antigen used in EngerixB ™ (GlaxoSmithKline Biologicals S.A.), which is further described in WO 93/24148. VZV gE The VZV antigen is the glycoprotein gE of VZV (also known as gpl) or its immunogenic derivative. The wild-type or full-length gE protein is composed of 623 amino acids comprising a signal peptide, the main portion of the protein, a hydrophobic anchoring region (residues 546 to 558) and a C-tail. terminal. In one aspect, a C-terminal truncation of gE (referred to as truncated gE or truncation of gE) is used whereby truncation removes 4-20% of the total amino acid residues at the end. carboxy. In another aspect, truncated gE does not possess the carboxy terminal anchoring region (suitably approximately amino acids 547-62 of the wild-type sequence). In a further aspect, gE is a truncated gE having the sequence of SEQ ID NO: 1. The gE antigen, the non-anchored derivatives thereof (which are also immunogenic derivatives) and their production are described in EP0405867 and in references cited therein [see also Vafai A. Antibody binding sites on truncated forms of varicella-zoster virus gpl (gE) glycoprotein Vaccine 1994 12: 1265-9]. EP192902 also describes gE and its production. truncated gE having the sequence of SEQ ID NO: 1, is also described by Haumont et al. Virus Research (1996) Vol 40, p99-204, fully incorporated herein by reference. Leroux-Roels I. et al. (JID 2012: 206 1280-1290) reported a Phase I / II clinical trial evaluating a truncated VZV gE subunit vaccine with ASOI adjuvant. Adj uants As explained herein, in one aspect of the present invention, "self-adjuvant" antigen-adjuvant conjugates are capable of inducing a specific immune response in the absence of additional antigen or adjuvant. However, the use of these antigen-adjuvant conjugates in an immunogenic composition of the present invention does not preclude the use of other antigens or adjuvants in the immunogenic composition. The immunogenic compositions of the present invention may further comprise a pharmaceutically acceptable excipient, such as a suitable adjuvant. Suitable adjuvants include an aluminum salt such as aluminum hydroxide or aluminum phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, or may be cationically or anionically derivatized saccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives (e.g., of reduced toxicity), 3-0-deacylated MPL synthetic mimetics of MPL such as aminoalkyl glucosamide, (AGP) A, saponin, QS21, tocol (EP 0382271), incomplete Freund's adjuvant (Difco Laboratories, Detroit, MI), adjuvant 65 of Merck (Merck and Company, Inc., Rahway, NJ), AS-2, AS-3, AS-4 (Smith-Kline Beecham, Philadelphia, PA), MF59 (Novartis), CpG oligonucleotides, bioadhesives and mucoadhesives, microparticles, liposomes, ether-based formulations, polyoxyethylene, polyoxyethylene ester-based formulations, muramyl peptides or imidazoquinolone compounds (e.g. imiquamode and its homologs) and oxoadenines. Human immunomodulators suitable for use as adjuvants in the invention include cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5). , IL-6, IL-7, IL-12, etc.) / macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), colony stimulating factor granulocytes and macrophages (GM-CSF) can also be used as adjuvants. Non-immunological components The immunogenic compositions of the present invention will typically include, in addition to the aforementioned antigenic components and adjuvants, one or more pharmaceutically acceptable carriers or excipients, which include any excipient which does not itself induce the production of harmful antibodies to the recipient individual. the composition. Suitable excipients are typically large, slowly metabolized macromolecules such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al., 2001, Vaccine, 19: 2118), trehalose (WO 00/56365), lactose and lipid aggregates (such as oil droplets or liposomes). These vectors are well known to those skilled in the art. The compositions may also contain diluents, such as water, saline, glycerol, etc. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. A physiological saline solution, phosphate buffered, pyrogen-free and sterile is a typical vector. A detailed discussion of pharmaceutically acceptable excipients is available in Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th Edition, ISBN: 0683306472. Compositions utilizing conjugates of the invention may be more readily freeze-dried or spray-drying than their mixed counterparts or may be in aqueous form, i.e. some form of solutions or suspensions. In some cases, liquid formulations comprising an antigen-adjuvant conjugate as described allow the compositions to be administered directly from their packaged form, without the need for reconstitution in an aqueous medium, and thus are ideal for injection. The compositions may be in vials or in pre-filled syringes. Syringes may or may not have needles. A syringe will include a single dose of the composition, while a vial may comprise a single dose or multiple doses (e.g., 2 doses). In one embodiment, the dose is for humans. In another embodiment, the dose is for an adult, a teenager, a young child, an infant or a human being younger than one year, and may be administered by injection. The improved adjuvant effect of the conjugates and compositions comprising the conjugates may allow the manufacture of immunogenic compositions having a reduced antigen content but giving an equivalent immunogenic effect, compared to a similar antigen and adjuvant that are not conjugated. A formulation using the new conjugate can thus provide a beneficial effect of reducing the required doses. An improved reactogenicity profile of the conjugates and compositions comprising the conjugates may allow the manufacture of effective immunogenic compositions with reduced reactogenicity compared to compositions comprising a similar antigen and adjuvant, but which are not conjugated. For example, the reactogenicity profile of the antigen-adjuvant conjugate can be improved over that of the mixed antigen and adjuvant. Similarly, the manufacture of immunogenic compositions having improved efficacy with substantially equivalent reactogenicity compared to compositions comprising a similar antigen and adjuvant, but which are not conjugated. For example, the effectiveness of the antigen-adjuvant conjugate can be improved over that of the mixed antigen and adjuvant. A formulation using the novel conjugate can thus provide an increased therapeutic window. The immunogenic compositions of the invention can be packaged as a single dose or as multiple doses (eg, 2 doses). For multiple dose forms, vials are preferred to pre-filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of the injection composition has a volume of 0.5 mL. In one embodiment, the immunogenic compositions of the invention have a pH of between 6.0 and 8.0, in another embodiment, the immunogenic compositions of the invention have a pH between 6.3 and 6. , 9, for example, 6.6 ± 0.2. The compositions can be buffered at this pH. The pH can be maintained by the use of a buffer. If a composition comprises an aluminum hydroxide salt, a histidine buffer can be used (WO 03/009869). The composition must be sterile and / or pyrogen-free. The immunogenic compositions of the invention may be isotonic with humans. The immunogenic compositions of the invention may comprise an antimicrobial. Preservatives are preferably present at low levels. A preservative may be exogenously added and / or may be a component of the bulk antigens that are mixed to form the composition (eg, present as a preservative in the antigens). The immunogenic compositions of the present invention may comprise a detergent, for example Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels, eg, <0.01%. The immunogenic compositions of the invention may include sodium salts (e.g., sodium chloride) to impart tonicity. The composition may comprise sodium chloride. In one embodiment, the concentration of sodium chloride in the composition of the invention is in the range of 0.1 to 100 mg / mL (for example, 1 to 50 mg / mL, 2 to 20 mg / mL). mL, 5 to 15 mg / mL) and in another embodiment, the sodium chloride concentration is 10 ± 2 mg / mL NaCl, e.g., about 9 mg / mL. The immunogenic compositions of the invention will generally comprise a buffer. A phosphate or histidine buffer is conventional. The immunogenic compositions of the present invention may comprise free phosphate ions in solution (for example, by the use of a phosphate buffer) so as to promote non-adsorption of the antigens. In one embodiment, the immunogenic compositions of the invention are formulated for in vivo administration to the host, such that the individual components of the composition are formulated so that the immunogenicity of the individual components is not affected. not substantially modified by the other individual components of the composition. By substantially modified step is meant that during immunization, an antibody titer against each component is obtained which is greater than 60%, 70%, 80% or 90%, or 95 to 100% of the title obtained when antigen is administered alone. Thus, in preferred embodiments, no (significantly) harmful effect occurs on the other components (in terms of protective efficacy) in the combination compared to their administration alone. In one aspect of the invention, the immunogenic composition comprising the antigen-adjuvant conjugates is "self adjuvant". The term "self-adjuvant" in this context means that the administration of the immunogenic composition comprising the antigen-adjuvant conjugate is sufficient to induce a specific immune response effective against the antigen, in the absence of other pharmacologically active compounds ( for example, other antigens or adjuvants). For example, in one aspect, a self-adjuvant composition comprising an adjuvant-conjugated antigen provides a better specific antibody response as measured by serum titers and preferably a specific antibody response, clinically significant against antigen such as measured by functional antibody titers and / or cell-mediated immunity as measured by T-cell activation. Examples of disease states in which the immunogenic compositions of the present invention have potentially beneficial effects include allergic diseases, inflammatory conditions, immune-mediated disorders, infectious diseases, and cancer. The compounds of the present invention may also be potentially used as vaccines comprising adjuvant-conjugated antigens. As modulators of the immune response, the antigen-adjuvant conjugates of the present invention may be useful, alone or in combination with other compositions, in the treatment and / or prevention of immune disorders, including but not limited to inflammatory or allergic diseases such as asthma, allergic rhinitis and rhinoconjunctivitis, food allergy, hypersensitivity-related lung diseases, eosinophilic pneumonitis, delayed hypersensitivity disorders, diabetes, multiple sclerosis, atherosclerosis, pancreatitis, gastritis, colitis, osteoarthritis, psoriasis, sarcoidosis, pulmonary fibrosis, respiratory distress syndrome, bronchiolitis, chronic obstructive pulmonary disease, sinusitis, cystic fibrosis, actinic keratosis, cutaneous dysplasia, chronic urticaria, eczema and all types of dermatitis. The immunogenic compositions of the present invention may also be useful in the treatment of infectious diseases including, but not limited to, those caused by hepatitis viruses (e.g., hepatitis B virus, hepatitis C), human immunodeficiency virus, papillomavirus, herpesvirus, respiratory viruses (eg, influenza virus, respiratory syncytial virus, rhinovirus, metapneumovirus, parainfluenzavirus, SARS), and the virus West Nile. The compositions of the present invention may also be useful in the treatment of microbial infections caused, for example, by bacteria, fungi, or protozoa. These include, but are not limited to, tuberculosis, bacterial pneumonia, aspergillosis, histoplasmosis, candidiasis, pneumocystosis, leprosy, chlamydial infection, cryptococcal disease, cryptosporidosis, toxoplasmosis, leishmaniasis, malaria, Ebola and trypanosomiasis. The immunogenic compositions of the present invention may also be useful in the treatment of various cancers, in particular the treatment of cancers which are known to respond to immunotherapy and which include, but are not limited to, renal cell carcinoma, cancer lung, breast cancer, colorectal cancer, bladder cancer, melanoma, leukemia, lymphoma and ovarian cancer. Those skilled in the art will appreciate that the references herein to treatment or therapy may, depending on the condition, extend to prophylaxis and treatment of established conditions. Thus, the antigen-adjuvant conjugates of the present invention may be useful in vaccines and useful as therapeutic agents. The immunogenic composition of the present invention may, for example, be formulated for oral, topical, inhaled, intranasal, oral, parenteral (e.g. intravenous, intradermal, or intramuscular) or rectal administration. In one aspect, the compounds of the present invention are formulated for oral administration. In another aspect, the compounds of the present invention are formulated for topical administration, for example intranasal or inhalation administration. formulations In one embodiment, the immunogenic compositions of the invention are formulated as a vaccine for in vivo administration to the host, such that they confer a higher antibody titer than the seroprotection criterion. for each antigenic component for an acceptable percentage of human subjects. This is an important test in evaluating the effectiveness of a vaccine on a population. Antigens with an associated antibody titer above which a host is considered seroconverted against the antigen are well known, and these are published by organizations such as WHO. In one embodiment, more than 80% of a statistically significant sample of subjects are seroconverted, in another embodiment more than 90% of a statistically significant sample of subjects are seroconverted, in yet another embodiment, more than 93% of a statistically significant sample of subjects are seroconverted and in another embodiment 96 to 100% of a statistically significant sample of subjects are seroconverted. The amount of antigen in each vaccine dose is selected as an amount that induces an immunoprotective response without significant adverse side effects in typical vaccines. Such an amount will vary depending on the specific antigens that are used. Each dose is generally expected to comprise from 1 to 1000 μg of total antigen, or from 1 to 100 μg, or from 1 to 40 μg, or 1 to 5 μg. An optimal amount of a particular vaccine can be verified by studies involving observation of antibody titers and other responses in the subject. The vaccines of the present invention may be packaged in different types of containers, for example, in vials, syringes, etc. A multi-dose vial will typically include a resealable plastic orifice through which a sterile needle may be inserted to withdraw a dose of vaccine, said orifice closing once the needle has been removed. The vaccine can be provided in different containers (for example, 2 or 3). The contents of the containers may be extemporaneously mixed before being administered to a host in a single injection or may be administered concomitantly at different sites. The dose of the vaccine, or each vaccine in the case of a kit, which is administered concomitantly (in at least two containers) will typically be 0.5 mL. The invention relates to a method for developing an immune response in a mammal, comprising the step of administering an effective amount of an immunogenic composition of the invention. The compositions may be administered prophylactically (i.e., to prevent infection) as with a vaccine. The immune response is preferably protective and preferably involves antibodies. The method can induce an immune response following the booster. After initial administration, subjects may receive one or more (subsequent) booster immunizations spaced appropriately. The dosage regimen may be a single dose regimen or a multiple dose regimen. Multiple doses may be used in a primary immunization regimen and / or a booster immunization regimen. The administration of the primary dose may be followed by the administration of a booster dose. An appropriate interval between sensitization doses and between sensitization and booster may be routinely determined. The immunogenic compositions of the present invention may be used to protect or treat a susceptible mammal by administering said composition directly to a patient. Direct administration may be achieved by parenteral administration (including, but not limited to: intramuscular, intraperitoneal, intradermal, intravenous, or interstitial space of tissue); or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, atrial, pulmonary or other mucosal administration. In one embodiment, administration is by intramuscular injection into the thigh or forearm. The injection can be performed via a needle (for example, a hypodermic needle), but needle-free injection can alternatively be used. In one embodiment, an intramuscular dose is 0.5 mL. The terms "comprising", "understand" and "include" used herein are understood by the inventors as optionally substitutable by the terms "consisting of", "consisting of" and "consisting of", respectively, for each occurrence. This does not alter the normal meaning of these terms, and is only intended to provide a basis for substitution, not to make two terms of equivalent meaning. EXAMPLE 1 Synthesis of oxoadenines Oxoadenines and methods for the preparation of oxoadenines such as compounds A and C are known in the art and are in particular described in WO 2010/018134. The synthesis of compound B oxoadenine is carried out according to Scheme 1 and as described below. Compounds 1, 2, 40, 41, 42 and 43 exposed in the synthesis below are further described in WO 2010/018134 (using the same numbering as in patent application WO 2010/018134). 4-Bromopyridine hydrochloride A (2.5 g) was separated between 1N sodium hydroxide (20 mL) and ethyl acetate (3 x 20 mL). The organic layer was separated, dried over Na 2 SO 4 and concentrated in vacuo. The resulting oil was dissolved in TEA (2.6 M) and degassed under nitrogen. 4-pentyn-1-ol (1.1 eq) was added followed by bis (triphenylphosphine) palladium (II) chloride (0.01 eq) and copper (I) iodide (0). , 02 eq.) And then the reaction mixture was allowed to stir at reflux for 20 minutes. Aqueous treatment (ethyl acetate / water) and purification by silica gel chromatography (0 to 30% gradient of ethyl acetate in heptane) gave B in 82% yield. B was dissolved in acetic acid (0.05M) and the solution was hydrogenated using a H-Cube® continuous flow hydrogenation reactor (ThalesNano) (20% Pd (OH) cartridge). 2 / C, 100 bar H2, 90 ° C, 1 mL / min). After the completion of the hydrogenation, the reaction mixture was concentrated and dried under vacuum. The crude product obtained was dissolved in CH 2 Cl 2 (0.4M) and then reacted with Et 3 N (1.5 eq) and di-t-butyl bicarbonate (1, 2 eq.) At room temperature for 30 minutes. After an aqueous treatment (CH 2 Cl 2 / H 2 O) and the purification by chromatography on silica gel (gradient of 0 to 30% of ethyl acetate in heptane), compound C was isolated in a yield of 80%: NMR ΧΗ (400 MHz, CDCl3) d 4.06 (s, 2H), 3.64 (t, 2H), 2.66 (t, 2H), 1, 54-1.66 (m, 5H), 1, 45 (s, 9H), 1.24-1.39 (m, 8H), 1.08 (m, 2H), CBr4 (1.6 eq) and PPI13 (1.2 eq) were slowly added. (exothermic reaction) to a solution of C in CH2Cl2 (0.45M) at 0 ° C. After 5 minutes the reaction mixture was allowed to warm to room temperature, allowed to stir at room temperature for 45 minutes, concentrated and directly purified by silica gel chromatography (gradient 0 to 30% d ethyl acetate in heptane) to give D in 92% yield. K2CO3 (325 mesh, 3.0 eq.) Was added to a solution of 43 in DMF (0.25M) and the reaction mixture was sonicated for several seconds to obtain a fine suspension which was then left stirring at 60 ° C for 1 hour. After cooling to 50 ° C, D (1.2 eq) was added to the reaction mixture and then allowed to stir overnight at 50 ° C. After cooling to room temperature and aqueous treatment (ethyl acetate / water), the resulting crude product was purified by silica gel chromatography (gradient from 0 to 10% methanol in chloroform). Purified product E was dissolved in methanol (0.1 M) and reacted with 4N HCl in dioxane (6.0 eq) at room temperature for 1 hour. The reaction mixture was concentrated and dried in vacuo, the residue was purified by silica gel chromatography (0 to 100% CHCl 3 / CH 3 OH / H 2 O 90/10 / 0.5 in CHCl 3 / CH 3 OH / H 2 O 85 / 15 / 1.0) to obtain F in 64% yield (2 steps). 1 H NMR (400 MHz, CD3OD) gamma 5.14 (m, 1H), 3.81 (t, 2H), 3.36 / 3.32 (m, 4H), 2.97 (d of t, 2H) , 1.92 (m, 2H), 1.75 (p, 2H), 1.72 (m, 1H), 1.57 (m, 2H) 1.5-1.3 (m, 14H), , 95 (t, 3H); ES TOF-MS positive calculated for [M + H] 391.28222, found 391.0843. SCHEME 1 EXAMPLE 2 Example: HBsAg-compound B conjugation method (chemistry G) 1) The adjuvant was activated with GMBS by addition of 3 equivalents of GMBS and 1.5 of TEA to 1 equivalent of adjuvant in DMF / H2O 80% / 20%. The reaction was incubated at room temperature for 20 minutes. 2) The sample was diluted with acetonitrile and 10mM ammonium acetate pH = 6 and purified by RP-HPLC to remove unreacted GMBS and all dimer adjuvant. 3) Fractions containing activated adjuvant with GMBS (tested by CLSM) were collected and dried by Speed Vac. GMBS-activated dry adjuvant was stored at -20 ° C until use. 4) The antigen was activated by adding 40 equivalents of 2-iminothiolane (Traut's reagent) to the protein solution dropwise while gently mixing the solution. The solution was incubated at room temperature for 1.5 hours. The solution was purified by ultrafiltration using 15 mM Amicon ultrafiltration tubes with a 30 kDa MWCO and then washed 7 times with 100 mM phosphate buffer. 1) containing 10 mM EDTA and pH = 7.2. (A sample aliquot was retained for MALDI-TOF testing). 2) 15 equivalents of GMBS-activated purified adjuvant were added to purified activated antigen with Traut's reagent and incubated at room temperature for 2 hours. The number of copies was determined by MALDI-TOF. 3) The sample was centrifuged for 5 minutes and the supernatant was filtered with a 0.1 μm filter to remove insoluble particles. 4) The filtrate was purified by ultrafiltration using a 15 mL Amicon ultrafiltration tube with a 100 kDa MWCO. The sample was washed with 100 mM phosphate buffer pH = 6.8 with 150 mM NaCl. 5) The purified conjugate was sterilized by filtration and characterized by RP-HPLC, SEC-HPLC, MALDI-TOF, SDS-PAGE, BOA and LAL. Example: gE-compound B conjugation (Chemistry T) 1) The antigen was activated with GMBS by dropwise addition to the antigen of 42 equivalents of GMBS dissolved in DMSO, while subjecting to a delicate vortex, and incubated at room temperature for 30 minutes. 2) Unreacted GMBS was purified by ultrafiltration using 15 ml Amicon ultrafiltration tubes with a 30 kDa MWCO and then washed 5 times with 100 mM phosphate buffer, pH = 6.8. An aliquot was retained for MALDI-TOF assay. 3) The adjuvant was activated with Traut's reagent by addition of 0.95 equivalents of Traut's reagent and 2.5 equivalents of Hunig's base (N, N-diisopropylethylamine) in DMF / H2O 90% / 10% then incubated at room temperature for ~ 1 hour. An aliquot was retained for RP-HPLC testing. 4) 20 equivalents of activated adjuvant with Traut's reagent were added dropwise to purified antigen activated with GMBS while subject to a delicate vortex and incubated at room temperature for 2 hours. 5) The conjugate was purified by ultrafiltration using 15 ml Amicon ultrafiltration tubes with a 30 kDa MWCO and then washed 8 X with 10 mM phosphate buffer, pH = 6.8. 6) The remaining unconjugated adjuvant of the conjugate was removed by anion exchange. The conjugate was loaded onto a prewashed, strong Pierce anion exchange column and then washed with 10 mM phosphate buffer, pH 6.8, containing increasing concentrations of NaCl (up to 500 mM). The fractions were collected and analyzed by RP-HPLC to determine the concentration of protein and free adjuvant. Fractions containing conjugate without free adjuvant were combined and desalted by ultrafiltration using an ultrafiltration tube. Amicon 15 mL with a 10 kDa MWCO and then washed with phosphate buffer, pH = 6.8 7) The conjugate was sterilized by filtration and characterized by RP-HPLC, SEC-HPLC, SDS PAGE, MALDI-TOF, BCA and LAL. EXAMPLE 3 Analytical characterization of conjugates MALDI-TOF mass spectrometry was used to characterize the mass of the antigen (gB), the GMBS conjugated antigen and the adjuvant conjugated antigen (a TLR2 agonist). The difference in molecular weight between each group was used to calculate the average number of GMBS and adjuvant molecules attached to each antigen. The antigen was protonated with an equal volume of 2% TFA before addition of a molar excess of the 2,5-dihydroxyacetophenone matrix compound. The mixture was then added to a ground steel plate for co-crystallization of the antigen in the matrix. Samples were then analyzed by MALDI-TOF MS with Bruker Microflex. (Figure 2) The size of antigen and antigen / adjuvant conjugates were confirmed by SDS-PAGE in conjunction with a relative concentration of monomer in higher molecular weight aggregates. A Novex NuPAGE SDS-PAGE gel system was used with reducing conditions and prefabricated gels were selected based on the size of the antigen. (FIG. 3) In FIG. 3, the results for the antigen (1), the TLR-7/8 agonist conjugate (2), the TLR-2 agonist conjugated antigen (FIG. 3), GMBS antigen-adjuvanted antigen (4), TLR-7/8 agonist-conjugated antigen (5), TLR-7/8 agonist conjugated agonist and TLR- 2 (6). The increase in the molecular weight of the conjugates is shown by the upward shift of the gel bands relative to the unconjugated antigen. The gel also demonstrates that the dimer and trimer are stabilized by the conjugation procedure. Reverse phase HPLC was used to confirm that all of the unconjugated adjuvant was removed from the conjugate by the purification process. HPLC with a PDA detector and a Jupiter C4 column with a flow rate of 1 mL / min and a gradient of 3% acetonitrile and 97% aqueous trifluoroacetic acid solution (trifluoric acid) were used. 0.1% luoroacetic acid in water for HPLC) at 90% acetonitrile and 10% trifluoroacetic acid at 0.1%. Absorbance was measured for wavelengths of 215 to 320 nm. (Figure 4) The RP-HPLC chromatogram of the buffer only (1), the antigen only (2) and the antigen / adjuvant conjugate (3) showed a slight shift in retention time for the conjugate and without unconjugated adjuvant . HPLC was also used to confirm the conjugation of the adjuvant to the antigen and to determine the percentage of monomer of the conjugates. HPLC equipment with a UV detector and a TSK gel column (G3000SWXL) was used with a flow rate of 0.8 mL / min 10 mM phosphate buffer. Absorbance was measured for wavelengths of 215 and 280 nm. (Figure 5) The SEC-HPLC chromatogram of the buffer only (1), the antigen only (2) and the antigen / adjuvant conjugate (3) showed a retention time lag for the conjugate and without unconjugated adjuvant. A Thermo Scientific Pierce BCA Protein Assay kit was used to quantify the concentration of the antigen after purification using BSA as a standard protein. A Lonza Kinetic QCL Assay kit was used to quantify endotoxin levels in purified, sterilized conjugates by filtration. In cases where the limited solubility of the adjuvant or adjuvant-linker (Traut) prevents the complete purification of the unconjugated adjuvant by ultrafiltration, it may be necessary to use ion exchange chromatography to remove the remaining adjuvant. For example, following the conjugation of Traut B-reagent to CRM-GMBS and purification by ultrafiltration, approximately 0.9 μg / ml of non-conjugated Traut B-reactive compound remained in the sample and did not could be purified by further ultrafiltration. For ion exchange purification, the conjugate was loaded onto a washed anion exchange column where the negative charge protein associated with the positive charge column. The unconjugated B-Traut compound was removed by washing with 10 mM phosphate buffer. The purified conjugate was then eluted from the column by washing with 10 mM phosphate buffer containing increasing amounts of NaCl. The fractions were collected and examined by RP-HPLC then fractions containing the CRM-compound B conjugate were removed without the non-conjugated Traut B-reactant and desalted by ultrafiltration. The final purified compound B conjugate had less than 0.1 μg / ml of non-conjugated Traut B-reactive compound. (Figure 6). RP-HPLC fractions collected during ion exchange chromatography of a conjugate of compound B. The column was first washed with 1M NaCl to remove impurities from the column with a retention time of about 3.5 minutes. The column was washed with 10 mM phosphate buffer before loading the conjugate. With loading and additional washes with 10 mM phosphate buffer, the unconjugated Traut B-reactive compound was removed with a retention time of about 10.2 minutes while the protein conjugate remained on the column until eluting with 400 mM NaCl. EXAMPLE 4 The ability of CRM-Compound A conjugates having a lower copy number (four copies of Compound A) and a higher copy number (six copies of Compound A) to modulate TLR7 activation was then evaluated (FIG. 7). For this experiment, compound A was used as a reference and the conjugated compounds were diluted so that the copy number of compound A was the same at each dilution (same molecular ratio relative to compound A). The results obtained from this analysis demonstrate that the conjugated compounds of the present invention are effective as a ligand alone, compound A, to activate the TLR7 pathway. This also suggests that the compounds of the conjugate, although much larger than the ligand itself, do not generate steric hindrance having a significant impact on their receptor. EXAMPLE 5 Dose determination studies have identified the optimal dose of conjugates to be used in subsequent experiments. TLR7 / 8 oxoadenine (Compound A) was conjugated with Model CRM197 antigen to test its immunogenic potential in a dose-finding study. Method: Anti-CRM197 and anti-gB antibodies were quantified in mouse serum by ELISA. At a terminal time point, whole mouse blood was collected and centrifuged on a Vacutainer blood collection tube containing gel for serum separation. Serum samples were stored at -80 ° C. NUNC Maxisorp plates were first coated with the CRM197 or gB protein at 5 or 4 μg / mL, using a 50 mM sodium carbonate buffer, respectively, overnight at 4 ° C. The ELISA plates were then washed using 0.05% PBS / Tween 20. Super Block (ScyTek laboratories) was added to the plates and incubated at 37 ° C for at least one hour. The serum samples and standards used here were mixtures of mouse antisera prequantized against CRM197 or gB. Calibrators and sera were plated in each plate and serially diluted 1/2 in the plates before incubating at 37 ° C for 2 hours. After washing, AffiniPure anti-mouse goat IgG, diluted with peroxidase and specific for the Fcy fragment (Jackson ImmunoResearch Laboratories Inc.) was added for 1 hour at 37 ° C. A final wash was performed before adding the TMB Substrate Reagent (BD OptEIA ™, BD Biosciences) for 30 minutes at RT. Plates were immediately quenched using 2N sulfuric acid and read at 450 nm using a SpectraMax microplate reader (Molecular Devices, Inc.). The formulations were made on the day of the injections. The injection volume was 50 μL per mouse. A typical formulation contains: 20 μg to 25 μg antigen diluted with H 2 O and PBS pH 7.4 for isotonicity. Dose determination studies were conducted to identify the optimal dose of conjugates for use in subsequent experiments. For reasons related to the chemistry and reagent availability, we started with the ligand TLR7 / 8 (compound A) as an adjuvant in the CRM197 model antigen conjugate to test their immunogenic potential in a determination study. doses. Different log 10 doses of CRM-compound A conjugates were evaluated from 0.01 to 10 μg (based on total protein content). The results (FIG. 7) demonstrated that 10 μg is an optimal dose to obtain a significantly higher humoral response specific to CRM197. This has been demonstrated for conjugates having G or T chemistry as explained above. (Figure 8) EXAMPLE 6 Antigen-Adjuvant Conjugate Compared to Mixed Adjuvant A proof-of-concept study was conducted on BALB / c mice. Mice were injected with various immunogenic compositions (50 μl volume in PBS buffer per mouse) using the intramuscular route at days 0-21 and 42 and serum was collected and analyzed on day 49 by ELLSA. CRM197 specific as described in Example 5. The following vaccine formulations were tested: 1 - CRM alone (purified CRM197 protein) at 10 μg per mouse 2 - Conjugate: CRM-compound A at a total dose of 10 μg per mouse. The dose of CRM-bound compound A in this composition is 0.15 μg CRM197 purified protein at 10 μg + 0.15 μg of compound A described as 1 X mixture CRM197 purified protein at 10 μg + 1.5 μg of compound A described as 10 X mixture CRM197 purified protein at 10 μg + 15 μg of compound A described as 100 X mixture As shown in Figure 9, CRM-compound A conjugate is more immunogenic than simple mixtures of CRM and compound A, even when the amount of compound A is 100X greater than that present in the conjugate. The amount of CRM197 used in the assays was kept constant (10 μg CRM197). Only the amount of the compound A molecule has been modified in the Ad-Mix formulation. In particular, 0.15 μg of compound A adjuvant conjugated to 10 μg of CRM197 triggered a significant response of antibodies against CRM197. This contrasts with AdMix, which did not show a significant immunogenic response with 0.15 μg of compound A. Taken together, these results demonstrate that conjugation of adjuvant to antigen increases immunogenicity while using less adjuvant per dose compared to the admixture of adjuvant and antigen. We therefore performed another proof-of-concept study in BALB / c mice using a CRM-compound B conjugate. Mice were injected with different CRM / compound B compositions (50 μL volume in PBS buffer per ml). mice using the intramuscular route at days 0, 21 and 42). Sera were collected and analyzed on day 49 by a CRM197 specific ELISA as described in Example 5. The following formulations were tested: 1 - PBS alone 2 - CRM alone (purified CRM197 protein) at 10 pg per mouse 3 - Conjugate: CRM-compound B at a total dose of 10 μg per mouse. The dose of CRM-bound compound A in this composition is 0.2 μg 4 - CRM197 purified protein at 10 μg + 0.2 μg of compound B described as 1 x 5 mixture - CRM197 purified protein at 10 μg + 2 μg of the compound B described as 10 x 6 - purified CRM197 protein at 10 μg + 20 μg of compound B described as 100 x 7 - purified CRM197 protein at 10 μg + adjuvant containing TLR4 (AS01E) previously described herein (2.5 pg MPL, 2.5 μg QS21 in a liposomal formulation). As demonstrated in Figure 10a, the CRM-compound B conjugate is more immunogenic than simple mixtures of CRM and compound B (p-value less than 0.01), even when the amount of compound B is 10 X greater than the amount present in the conjugate. The amount of CRM197 used in the assays was kept constant (10 μg CRM197). Only the amount of CRX-compound B molecule has been modified in the Ad-Mix formulation. The results demonstrate that the CRM-compound B conjugate allows a factor of 10 reduction of the adjuvant for equivalent immunogenicity. In addition, CRM-compound B is equivalent to AS01E + CRM for the antibody response. Stoichiometric calculation prior to injection demonstrates that 10 μg of CRM protein contained 0.2 μg of compound B. Each CRM-containing composition contained an equal amount of CRM antigen (10 μg per mouse). Therefore, for an appropriate dose comparison, the 1X mixture of CRM + Compound B contains 10 μg of CRM protein and 0.2 μg of compound B mixed prior to injection into the mice. The 10 X mixture (10 μg of CRM protein and 2 μg of compound B), and the 100 X mixture (10 μg of CRM protein and 20 μg of compound B) were also compared. AS01E, which was used as a reference, (2.5 μg of MPL (TLR4 agonist), 2.5 μg of QS21 in a liposomal formulation) by injection. We found that the CRM-compound B conjugate exhibits a CRM-specific antibody response similar to the CRM + AS01E reference, suggesting that the TLR7 agonist as the compound at a very low dose, for example 0.2 μg, when It is covalently bound to an antigen could promote a similar antibody response to that promoted by reference adjuvants containing TLR, such as AS01E. Again, the conjugation of the adjuvant to an antigen has a beneficial effect on the immune response compared to the admixture of an adjuvant and an antigen. In addition, we demonstrated that the CRM-compound B conjugate triggers an immune response of IgG to CRM197 similar to that of the powerful adjuvant containing TLR4 called AS01E + CRM197. The response of T cells in splenocytes was measured Mouse spleens were harvested and single-cell suspensions were generated in 10% RPMI + FBS using 100 micron cell filters. The splenocytes were plated in triplicate (1.5 x 107 / mL, 200 μL / well) in a 96-well round bottom plate. CRM protein (1 μg / ml), anti-mouse CD28 (1 μg / ml) and anti-mouse CD49d were added to stimulate CRM-specific cells. Unstimulated cells were used as a negative control. A BD PMA cocktail (1 μL / well) was used for the positive control. The plate was incubated at 37 ° overnight and then for 6 hours with Brefeldin A (10 μg / ml). The cells were stained on the surface with CD3, CD4 & CD8, then intracellular staining was performed with TNF and IFNy using a BD Fix / Perm buffer system. The cells were acquired on an LSRII using FACSDiva software. Primary selection was performed on CD3 + / CD4 + cells for analysis. The data is presented in Figure 10b. The data show a greater number of CRM-specific CD4 T cells producing INFY in mouse splenocytes immunized with CRM-compound B conjugate compared to the 1X or 10X mix formulation, suggesting that conjugation of compound B with CRM197 leads to activation of CD4 T cells in vivo. The analyzes show that Thl CMI (CD4-IFNg + cells) were more numerous in the case of CRM-compound B vs. CRM alone (FIG. 10b). The data could be extracted from the MIC analysis on the Thl CMI (CD4-IFNg + cells) were more numerous in the conjugate vs. CRM alone or compound A + CRM mixed 1 X or 10 X. In addition, CRM-compound B has a tendency for 3 out of 4 mice to be better for CD4-IFNg + cells. These data suggest that the CRM-compound B conjugate can trigger T-cell activation and provide better vaccine efficacy, even using a lower amount of adjuvants. EXAMPLE 7 Three different CRM197 antigen model-conjugated TLR7 / 8 compounds were tested for their immunogenicity in mice, two oxoadenines and one imidazoquinoline, Compound C CRM alone (CRM197 purified protein) was included as a control without adjuvant. Mice (BALB / c CR) were injected with different compositions (50 μL volume in PBS buffer per mouse) using the intramuscular route at days 0, 21 and 42. The sera were collected and analyzed for days 28 and 49 by a CRM197-specific ELISA method, as described in Example 5. The CRM-specific antibody level 7 days after the second and third injections demonstrates that CRM-oxoadenine conjugates are potent conjugates antigen-adjuvant (Figures 11a and 11b). The CRM-Compound B conjugate exhibits a higher CRM197 specific antibody response among the TLR7 / 8 agonists tested herein. EXAMPLE 8 A recombinant antigen (CMV gB) from a eukaryotic expression system in CHO cells was conjugated to an adjuvant and evaluated. BALB / c mice (5 per group) were immunized intramuscularly (quadriceps) with gB or gB-compound A conjugate (0.001, 0.01, 0.1, 1 or 10 μg diluted in saline). days 0, 20, and 41. Mice were drawn for serum collection on days 31 (post 2) and 52 (post 3). The data is presented in Figure 12a. Mouse spleens were harvested and single-cell suspensions were generated in 10% RPMI + FBS using 100 micron cell filters. The splenocytes were plated in triplicate (1.5 x 107 / mL, 200 μL / well) in a 96-well round bottom plate. GB (4 μg / ml), anti-mouse CD28 (1 μg / ml) and anti-mouse CD49d were added to stimulate specific gB cells. Unstimulated cells were used as a negative control. A BD PMA cocktail (1 μL / well) was used as a positive control. The plate was incubated at 37 ° overnight and then for 6 hours with Brefeldin A (10 μg / ml). The cells were stained on the surface with CD3, CD4 & CD8 followed by intracellular staining with IL-2, IL-4, TNF and IFNy using a BD Fix / Perm buffer system. The cells were acquired on an LSRII using FACS Diva software. Primary gating was performed on CD3 + / CD4 + cells for analysis. The data is shown in Figure 12b. Immunogenicity data demonstrated that a gB-compound A conjugate used at a dose of 0.01 μg promoted a specific antibody response of 100 fold (2 × log 10) greater than the response of gB compared to gB alone, without adjuvant. It is interesting to note that the benefit of the gB-compound A conjugate over gB alone has not been clearly demonstrated at higher doses. Although IgG data are not dose-dependent, cell-mediated immunity (CMI) data suggest that Thl CMI (CD4-IFNg + cells) were more numerous for gB-compound A vs. gB alone (Figure 12b) page 46. Thl CMI (CD4-IFNg + cells) were more numerous for gB-compound A vs. gB alone. Taken together, the antibody and T cell activation data suggest that small amounts of gB-compound A conjugate trigger T-cell activation and potentially provide a functional response of the neutralizing antibodies to the antigen of the 1 'antigen. herpesvirus such as hCMV gB. EXAMPLE 9 A pig vaccine dose-finding study (genetic background: Yorkshire-Duroc-Landrace cross section) was conducted to identify the appropriate dose for subsequent studies in larger animals. Both types of chiral chemical reactions described above, G and T conjugation, and three vaccine concentrations (1, 10 and 100 μg) were tested. Pigs were immunized with 1 mL of vaccine on days 0 and 21 and serum was collected on day 31 (10 days after secondary injection). An ELISA as described in Example 5 was performed except that the HRP-conjugated secondary antibody was an anti-pig goat IgG (Bethyl Laboratories). As described in Figure 13, CRM-Compound A triggers a CRM197-specific IgG response close to a dose-determining manner. All animals (5/5) receiving the 100 μg dose of CRM (= 1.5 μg of compound A) responded to CRM-compound A. Therefore, the 100 μg dose of CRM was chosen to other studies in farmed pigs. A brief visual assessment of injection site adverse events was performed on two randomly selected pigs by testing an additional dose of 150 μg compound A alone. No adverse visual effects at the injection site were observed, suggesting that Compound A molecules have a good safety profile. EXAMPLE 10 To better identify the most promising LRT conjugate for CRM197 using the 100 μg dose, three adjuvant conjugates TLR7 / 8-antigen: CRM-compound A were prepared; CRM-compound C; and CRM-compound B. In addition, an adjuvant conjugate TLR2-antigen was tested. (Data not shown). CRM197 alone was used without adjuvant as a control. Yorkshire-Landrace breeding pigs were immunized on days 0, 21 and 42 and the final sera were collected on day 52. The pig specific ELISA was performed as described in Example 5. The Results highlighting an anti-CRM IgG response in the final samples are shown in Figure 14. The anti-CRM197 IgG response in CRM-compound B suggests that compound B is active and immunogenic in cultured pigs. EXAMPLE 11. Intradermal immunization of pork We evaluated the intradermal immunization route using the CRM-Compound B conjugate. Four-month-old Yorkshire-Landrace-Cambrio pigs were immunized in the dermis of the flank on days 0 and 23 with 100 μl of different vaccines. The sera were taken on day 37 and a CRM197 IgG-specific ELISA was performed as previously described in Example 5. The groups of animals were as follows: The anti-CRM197 specific IgG measurements demonstrate that CRM-compound B conjugate at 100 μg is considerably more immunogenic than the antigen-only control, without adjuvant using the intradermal voice. These antibody titers in the range of 10,000 to 110,000 ng / mL CRM-compound B conjugate suggest that the intradermal immunization route is also suitable for vaccination with TLR7 / 8-antigen conjugates. (Figure 15) EXAMPLE 12 Anti-HBs antigens were quantified in the serum of Yorkshire-Landrace breeding pigs using an ELISA. The pigs were immunized on day 1, day 28 and the sera were collected on day 58 (end time point). Whole blood was collected and centrifuged on a blood collection tube Vacutainer containing gel for the separation of serum. Serum samples were stored at -80 ° C. The Maxisorp 96-well flat-bottomed plates (cat # Nunc: 439454) were coated with 100 μl per well of hepatitis B surface antigen diluted to 2 μg / ml in carbonate-bicarbonate coating buffer. 50 mM, pH 9.6. For standard curves, 100 μl per well of anti-pork goat IgG capture antibody (cat.-ethyl: A100-104A) diluted 1/100 in carbonate-bicarbonate coating buffer was dispensed. Plates were incubated for 1 hour at room temperature. Plates were washed (4 x 250 μL) using wash buffer (TBS containing 0.05% Tween-20) and then blocked with 200 μL per well of blocking solution (TBS containing 0 μl). , 05% Tween-20 and 1% BSA (cat Sigma: A7030)). Plates were incubated for 30 minutes at room temperature. The blocking solution was discarded and 100 μL per well of blocking solution was dispensed into the plates. 100 μl of reference porcine serum (Bethyl: RS 10-107) diluted to 1/6,600 or samples (previously pre-diluted if necessary) were added to the first row of plates, followed by dilution. serially twice for a final volume in each well of 100 μL. Plates were incubated for 1 hour at room temperature. The plates were washed and 100 μl per well of HPR-conjugated anti-swine IgG (Bethyl cat: A100-104P) diluted 1/75000 in blocking solution was dispensed. Plates were then incubated for 30 minutes at room temperature. The plates were washed and 100 μl per well of TMB substrate (cat .: 555214) was added to the plates, protected from light, for 30 minutes at room temperature. The reaction was terminated using 100 μL per well of stop solution (1 M H 2 SO 4). The plate was read immediately on a 450 nm microplate reader using a SpectraMax microtiter plate reader (Molecular Devices, Inc.). The results were analyzed with a Soft Max Pro matrix according to the following criterion:% CV less than 30% between a minimum of 2 concentrations calculated for the final quantification. The result of the antibody response (FIG. 16) shows that the HBsAg-compound B conjugate using the intradermal route is considerably superior to the standard HBsAg-alum intramuscular vaccine (unpaired t-test P = 0.0034). These results suggest that TLR7-conjugated vaccines are highly immunogenic compared to standard intramuscular vaccine formulations, for example with alum. Legend of figures Figure 1. Process Flow Diagram Figure 2, MALDI-TOF MS of antigen (1), GMBS-conjugated antigen (2) and adjuvant-conjugated antigen (3). The molecular weight offsets indicate an average of 11 copies of GMBS per antigen and an average of 3 copies of adjuvant per antigen. Figure 3, SDS-PAGE of an antigen and antigen / adjuvant conjugates using a 3-8% tris-acetate gel. Antigen (1), TLR-7/8 (2) agonist-conjugated antigen, TLR-2 agonist-conjugated antigen (3), cross-linked non-adjuvanted GMBS antigen (4), agonist-conjugated antigen TLR-7/8 (5), an antigen conjugated to TLR-7/8 and TLR-2 (6) agonists. The increase in the molecular weight of the conjugates is evidenced by the band shift in the gel relative to the unconjugated antigen. The gel also demonstrates that the dimer and trimer are stabilized by the conjugation procedure. Figure 4 The chromatogram of RP-HPLC buffer only (1), antigen only (2) and antigen / adjuvant conjugate (3) shows a small retention time shift for the conjugate and without unconjugated adjuvant. Figure 5. The SEC-HPLC chromatogram of buffer only (1), antigen only (2) and antigen / adjuvant conjugate (3) showed a small retention time shift for the conjugate and without unconjugated adjuvant. Figure 6. Data showing RP-HPLC of fractions collected during ion exchange chromatography of a CRM-compound B conjugate. Figure 7. Activation of TLR7 at very low doses by low copy number CRM-compound A conjugates and high copy number CRM-compound A using the TLR7 / NF-kB / SEAP stable reporter cell line (Imgenex) corp.). CRM-compound A conjugates (low copy number or high copy number) activate the human TLR7 pathway as efficiently as compound A alone. Figure 8. Dose determination study of the A-CRM compound showing that a dose-response in terms of CRM-specific IgG is observed with CRM-compound A conjugates in BALB / c mice using the T-configuration vs G. Figure 9. Proof-of-concept study in mice demonstrating that CRM antigen-adjuvant conjugates-A compounds (fusion) induce a significant humoral response based on a CRM197 specific antibody level. Anti-CRM specific IgG titers in serum at day 49 (7 days post 3). Figure 10a. Proof-of-concept study in mice demonstrating that CRM-B compounds (FUSION) fused conjugates are more effective even with 10x less adjuvant to mount a significant humoral response based on the CRM-197 specific antibody. Figure 10b. Evaluation of T cell activation of immunized mouse splenocytes demonstrates that the CRM-compound B fusion conjugate triggers greater activation of CRM 197-specific T cells compared to CRM + Compound B at 1 X concentration. or lOx. Figure 11a. Level of CRM-specific antibodies 7 days after the 2nd injection; immunogenicity demonstrates that CRM-compound B conjugate is more immunogenic than CRM-compound A and CRM-compound C constructs in mice. Figure 11b. Level of CRM-specific antibodies 7 days after the 3rd injection; immunogenicity demonstrates that CRM-compound B and CRM-compound A conjugates are immunogenic constructs in mice. Figure 12a. IgG-specific IgG (humoral response) assay with FUSION vs. gB control in adjuvant in mice. Figure 12b. Cell-mediated immunity (CMI) analysis to evaluate the CD4-IFNy response in mice. Figure 13. Dose determination in farmed pigs to test two types of conjugates; CRM-Compound A (G) vs. CRM-Compound A (T). Figure 14. Test of the CRM-specific humoral (antibody) response of three different TLR7 / 8-ligand conjugates in Yorshire-Landrace-Duroc breeding pigs using the intramuscular route for immunization. Figure 15. Antibody response elicited by fused conjugates vs unadjuvanted controls following intradermal immunization in breeding pigs (Yorkshire-Landrace). Figure 16. HBsAG specific IgG in serum 28 days post intradermal secondary immunization. BPGSKL0047BE00 BE Sequence Listing LIST OF SEQUENCES <110> GlaxosmithKline Biologicals SA <120> COMPOSITIONS AND USES <130> VR65753 WO <150> US 61/130 812 <151> 03-10-2015 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 546 <212> PRT <213> vi rus <400 > 1 Met Gly Thr Asn Val Lys Pro Val Val Gly Leu Val Met Gly Phe Gly 15 10 15 Island Thr Island Gly Thr Leu Arg Island Thr Asn Pro Val Arg Ala ser Val 20 25 30 Leu Arg Tyr Asp Asp Phe His Asp Asp Asp Asp Asp Leu Asp Asp Asp 35 40 45 Ser Val Tyr Glu Pro Tire Tyr His Ser Asp His Ala Glu Ser Ser Trp 50 55 60 Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr As His Asp Ser Asn Ser 65 70 75 80 Tyr Isle Trp Pro Arg Asn Asp Asp Asp Gly Phe Leu Glu Asn Ala His 85 go 95 Glu His His Gly Valley Tyr Asn Gin Gly Arg Gly Island Asp ser Gly Glu 100 105 110 Arg Leu Met Gin Pro Thr Gin Met Ser Ala Gin Asp Glu Leu Gly Asp 115 120 125 Asp Thr Gly Island His Val Island Pro Thr Leu Asn Gly Asp Asp Arg His 130 135 140 Lys Island Val Asn Val Asp Gin Arg Gin Tyr Gly Asp Val Phe Lys Gly 145 150 155 160 Asp Leu Asn Pro Lys Pro Gin Gly Gin Arg Leu Isle Glu Val Ser Val 165 170 175 Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Gin Island Arg Island Tyr 180 185 190 BPGSKL0047BE00 Sequence Listing □ Gly Val Arg Tyr Thr Glu Thr Trp Ser Phe Leu Pro Ser Leu Thr cys 195 200 205 Thr Gly Asp Al has Al a Pro Al has Gin Island His island cys Leu Lys His Thr 210 215 220 Thr Cys Phe Gin Asp val val Val Asp Val Asp cys Al a Glu Asn Thr 225 230 235 240 Lys Glu Asp Gin Leu Al Glu Isle Ser Tyr Arg Phe Gin Gly Lys Lys 245. 250,255 Glu Al has Asp Gin Pro Trp Val Val Asn Island Thr Thr Thr Leu Phe Asp 260 265 270 Glu Leu Glu Leu Asp Pro Pro Glu Island Glu Pro Gly Val Leu Lys Val 275 280 285 Leu Arg Thr Glu Lys Gin Tyr Leu Gly Val Tyr Trp Asn Met Arg 290 295 300 Gly ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu val Thr Trp Lys 305 310 315 320 Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gin Pro 325 330 335 Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His val Phe Ser 340 345 350 Val Gly Asp Thr Phe Ser Ala Leu Met His Leu Gin Tyr Lys Island His 355 360 365 Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Island Asp 370 375 v 380 Pro Thr Cys Gin Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro 385 390 395 40o Asn Ala Pro Gin cys Leu Ser His Met Asn ser Gly cys Thr phe Thr 405 4i 415 Ser Pro His Leu Ala Gin Arg Val Ala Ser Thr Val Tyr Gin Asn Cys 420 425 430 Glu His Ala Asp Asn Tire Thr Ala Tyr cys Leu Gly Island Ser His Met 435 440 445 Glu Pro ser Phe Gly Leu Leu Leu asd Gly Gly Thr Thr Leu Lys 450 455 460 BPGSKL0047BE0O Listing Sequences B Phe Val Asp Thr Pro Glu Ser Leu Gly Leu Tyr Val Phe Valley Val 465 470 475 480 Tyr Phe Asn Gly His Val Glu Al a Val Al a Tyr Thr val Val Ser Thr 485 490 495 val Asp His Phe val Asn Al a Glu Glu Island Arg Gly Phe Pro Pro Thr 500 505 510 Ala Gly Pro Gin Pro Ala Thr Thr Lys Pro Lys Glu Thr Island Pro Val 515 520 525 Asn Pro Gly Thr Ser Pro Leu Isle Arg Tyr Ala Ala Thr Trp Gly Gly 530 535 540 Leu Ala 545
权利要求:
Claims (29) [1] An immunogenic composition comprising an oxoadenine having a structure comprising formula 1 in which ; R 1 is a branched or saturated C 1-6 alkoxy group; R2 is a group having the structure: n is an integer having a value of 1 to 6; and Het is a 6 membered saturated heterocycle containing a nitrogen atom, in which Het is attached to the - (CH 2) n - moiety at any carbon atom of the heterocycle; or pharmaceutically acceptable salts thereof, said oxoadenine being covalently bound to an antigen. [2] 2. The immunogenic composition according to claim 1, wherein the number of oxoadenine molecules per molecule of antigen is 2, 3, 4, 5, 6 or between 1 and 8, 2 and 5 and 3 and 4. [3] The immunogenic composition of claim 1 or claim 2, wherein the oxoadenine has a structure selected from the group of [4] The immunogenic composition of claim 1 or claim 2, wherein the structure of oxoadenine is Compound A. [5] The immunogenic composition of claim 1 or claim 2, wherein the structure of oxoadenine is Compound B. [6] An immunogenic composition according to any one of claims 1 to 5, wherein the oxoadenine molecule is linked to an antigen via a crosslinking agent and wherein the crosslinking agent is a hydrophilic compound. [7] An immunogenic composition according to any one of claims 1 to 6, wherein the oxoadenine crosslinking agent increases the aqueous solubility of oxoadenine compared to the solubility in the absence of the crosslinking agent. [8] 8. An immunogenic composition according to any one of claims 1 to 7, wherein the active oxoadenine is more water-soluble than the inactive oxoadenine. [9] An immunogenic composition according to any one of claims 1 to 8, wherein the increased aqueous solubility of the active oxoadenine provided by the crosslinking agent decreases the amount of undesired aggregates in the antigen-adjuvant conjugate composition compared to active oxoadenine with no increased solubility. [10] 10. An immunogenic composition according to one of the oxoadenine-linked crosslinking agent is a charged compound. [11] 11. The immunogenic composition of claim 9, wherein the oxoadenine-related crosslinking agent is Traut's reagent or GMBS. [12] 12. An immunogenic composition according to claim 10, wherein the oxoadenine-linked crosslinking agent is Traut reagent reacted with GMBS. [13] An immunogenic composition according to any one of claims 1 to 5, wherein the crosslinking agent is a heterobifunctional crosslinking agent. [14] 14. An immunogenic composition according to any one of claims 1 to 13, further comprising a "carrier protein" in which the antigen is bound to the vector and the oxoadenine molecule is bound to the carrier protein. [15] 15. An immunogenic composition according to any one of claims 1 to 13, wherein the antigen is an antigen selected from the group consisting of: CRM-197, tetanus toxoid, HA, CMV gB, preF, Hash- 2, HPV E7, pertussis toxoid (PT), HBs (eg S antigen), ovalbumin, HIV (eg, gag, env), PRAME antigen and MAGE antigen (e.g. MAGE A3). [16] 16. The immunogenic composition of claim 1, wherein the antigen is conjugated to oxoadenine by the crosslinking agent and the antigen facilitates a specific immune response associated with the antigen in the absence of additional antigens. [17] 17. The immunogenic composition of claim 1, wherein the antigen is a diphtheria toxin antigen. [18] 18. The immunogenic composition of claim 17, wherein the diphtheria toxin is "CRM" 197 (Cross Reactive Material 197). [19] 19. The immunogenic composition according to any one of claims 1 to 13, wherein the antigen is a hepatitis B antigen. [20] 20. The immunogenic composition according to any one of claims 1 to 13, wherein the antigen is a hepatitis B surface antigen ("HBsAg"). [21] 21. The immunogenic composition according to any one of claims 1 to 13, wherein the antigen is a varicella or varicella zoster antigen ("VZV"). [22] 22. The immunogenic composition of claim 21 wherein the antigen is a VZV gE antigen. [23] 23. The immunogenic composition of claim 21 wherein the VZE gE antigen is truncated and comprises SEQ ID NO: 1. [24] 24. An immunogenic composition comprising a population of antigen-adjuvant conjugates, wherein the average number of adjuvant molecules conjugated to each antigen is between 1 and 5, or 2 and 4 or is 3 or 4. [25] 25. A process for producing an antigen-adjuvant conjugate comprising the steps of Figure 1. [26] 26. The immunogenic composition according to any one of the preceding claims, in an increased amount, in other words an antibody response higher than an equal dose or a dose up to 100 times that of the same unmixed antigens and adjuvants. covalently. [27] 27. The immunogenic composition of any one of the preceding claims, wherein the conjugate elicits an increased immune response, i.e., an antibody response greater than an equal dose or a dose up to 100 times that of same antigens and adjuvant not covalently mixed. [28] 28. The immunogenic composition of claim 1 for use in a medicament. [29] 29. The immunogenic composition of claim 1 for use in eliciting an immune response.
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同族专利:
公开号 | 公开日 US10646566B2|2020-05-12| US20180036403A1|2018-02-08| EP3268036A1|2018-01-17| US20200345841A1|2020-11-05| US11207404B2|2021-12-28| WO2016142880A1|2016-09-15| BE1024148A1|2017-11-21|
引用文献:
公开号 | 申请日 | 公开日 | 申请人 | 专利标题 WO2011017611A1|2009-08-07|2011-02-10|Glaxosmithkline Biologicals Sa|Lipidated oxoadenine derivatives| WO2010018134A1|2008-08-11|2010-02-18|Smithkline Beecham Corporation|Novel adenine derivatives| BR112017009648A2|2014-11-13|2017-12-19|Glaxosmithkline Biologicals Sa|compound, methods for treating allergic diseases or other inflammatory conditions or preventing disease, allergic rhinitis or asthma, composition, and use of a compound.| WO2016142880A1|2015-03-10|2016-09-15|Glaxosmithkline Biologicals Sa|Compositions and uses|WO2016142880A1|2015-03-10|2016-09-15|Glaxosmithkline Biologicals Sa|Compositions and uses| US20210371440A1|2018-04-13|2021-12-02|Glaxosmithkline Biologicals Sa|Tlr7 and / or tlr8 agonists| CN110018253A|2019-04-15|2019-07-16|艾美卫信生物药业（浙江）有限公司|The high-efficient liquid phase determining method of Free protein content in a kind of biological products|
法律状态:
2018-02-12| FG| Patent granted|Effective date: 20171122 |
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