Artificial promiscuous t helper cell epitopes as immune stimulators for synthetic peptidic immunogens

Abstract

To provide novel artificial promiscuous T helper cell epitopes (Th epitopes) designed to provide optimum immunogenicity of a target antigen site.SOLUTION: The target antigenic site may include a B cell epitope, a CTL epitope, a peptide hapten, a non-peptide hapten, or any immunologically reactive analog thereof. The disclosed Th epitopes, when covalently linked to a target antigenic site in a peptide immunogen construct, elicit a strong B cell antibody response or an effector T cell response to the target antigenic site. The Th epitopes are immunosilent on their own, i.e., little, if any, of the antibodies generated by the peptide immunogen constructs will be directed towards the Th epitope, thus allowing a very focused immune response directed to the targeted antigenic site. The promiscuous artificial Th epitopes provide effective and safe peptide immunogens that do not generate inflammatory anti-self cell immune responses following administration.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] This application is a PCT international application claiming the benefit of U.S. Provisional Patent Application No. 62 / 782,253, filed December 19, 2018, which is incorporated herein by reference in its entirety.

[0002] Immune responses require cooperative interactions between antigen-presenting cells and T-helper (Th) cells. Induction of an effective antibody response requires that antigen-presenting cells recognize target antigenic sites on the subject immunogen and that T-helper cells recognize T-helper cell epitopes. Generally, the T-helper epitopes on the subject immunogen are distinct from its B-cell epitope(s) or associated effector T-cell (e.g., cytotoxic T lymphocyte or CTL) epitope(s). B-cell and associated effector T-cell epitopes are sites on the desired target immunogen that are recognized by B-cells and associated cells, resulting in the production of antibodies or cytokines against the desired target site. The natural conformation of the target determines the site to which the antibody or associated effector T-cell will directly bind. Induction of a Th-cell response requires Th-cell receptors that recognize a complex on the membrane of the antigen-presenting cell that is formed between a processed peptide fragment of the target protein and the associated class II major histocompatibility complex (MHC). Thus, peptide processing and three-way recognition of target proteins are required for Th cell responses. The three-part complex is difficult to define because 1) key MHC class II contact residues are variably located within various MHC-binding peptides (Th epitopes), 2) different MHC-binding peptides have variable lengths and different amino acid sequences, and 3) MHC class II molecules can be highly diverse depending on the genetic makeup of the host. The immune response to a particular Th epitope is determined, in part, by the host's MHC genes, and the reactivity of Th epitopes varies among individuals in a population. Th epitopes that are reactive among species and individuals within a single species (i.e., promiscuous Th epitopes) are difficult to identify.

[0003] Multiple factors are required for each component step of T cell recognition, such as proper peptide processing by antigen-processing cells, presentation of the peptide by genetically determined class II MHC molecules, and recognition of the MHC molecule and peptide complex by receptors on Th cells. The requirements for promiscuous Th epitope recognition to provide broad responsiveness can be difficult to determine.

[0004] It is clear that for the induction of antibodies and related cytokines for immune response, immunogens must contain both B cell epitopes / effector T cell epitopes and Th cell determinants(s). Generally, carrier proteins (e.g., keyhole limpet hemocyanin; KLH) are bound to target immunogens to obtain Th responses in order to increase the immunogenicity of the target immunogens. However, there are many disadvantages to using large carrier proteins to enhance the immunogenicity of target immunogens. In particular, it is difficult to produce well-defined, safe and effective peptide-carrier protein conjugates because (a) chemical conjugation involves reactions that can result in heterogeneity in size and composition, such as conjugation with glutaraldehyde (Borras-Cuesta et al., Eur J Immunol, 1987; 17: 1213-1215); (b) carrier proteins introduce the potential for undesired immune responses, such as allergic and autoimmune reactions (Bixler et al., WO89 / 06974); (c) large peptide carrier proteins induce irrelevant immune responses that are primarily misdirected to the carrier protein rather than the target site (Cease et al., Proc Natl Acad Sci USA, 1987; 84: 4249-4253); and (d) carrier proteins also introduce the potential for epitope suppression in hosts previously immunized with immunogens containing the same carrier protein. When the host is subsequently immunized with another immunogen in which the same carrier protein is coupled to a different hapten, the resulting immune response is enhanced in the case of the carrier protein and suppressed in the case of the hapten (Schutze et al., J Immunol, 1985; 135: 2319-2322).

[0005] To avoid the above risks, it is desirable to induce T cell help without the use of conventional carrier proteins. Summary of the Invention

[0006] The present disclosure provides promiscuous artificial T helper cell (Th) epitopes for stimulating functional site-specific antibodies against target antigens for prophylactic and therapeutic use. The present disclosure also relates to peptide immunogenic constructs containing the Th epitopes, compositions containing the Th epitopes, methods of making and using the Th epitopes, and antibodies made with the peptide immunogenic constructs containing the Th epitopes.

[0007] The disclosed artificial T helper cell (Th) epitopes can be linked to B cell epitope(s) and / or effector T cell epitope(s) ("target antigenic site(s)") via any spacer to generate peptide immunogen constructs. The disclosed Th epitopes confer to peptide immunogens the ability to induce strong T helper cell-mediated immune responses with production of high levels of antibodies and / or cellular responses against the target antigenic site. The disclosed peptide immunogen constructs provide advantageous replacement of large carrier proteins and pathogen-derived T helper cell sites in peptide immunogens with the disclosed artificial Th epitopes that are specifically designed to improve the immunogenicity of the target antigenic site. Relatively short peptide immunogen constructs containing the disclosed Th epitopes induce high levels of antibodies and / or effector cell-associated cytokines against a particular target antigenic site without eliciting a significant inflammatory or immune response against the Th epitope.

[0008] The immune response induced by the peptide immunogen construct (antibody titer, C maxThe response time (including the onset of antibody production, duration of response, etc.) can be modulated by varying: (a) the choice of Th epitope that is chemically linked to the B cell epitope, (b) the length of the B cell epitope, (c) the adjuvant used in the formulation containing the peptide immunogen construct, and / or (d) the dosing regimen including the dosage per immunization and the time of priming and boosting for each immunization. Thus, specific immune responses against target antigenic sites can be engineered using the disclosed Th epitopes, which may facilitate tailoring personalized medicine to the individual characteristics of any patient or subject.

[0009] The peptide immunogen constructs containing the disclosed artificial Th epitopes of the present invention can be represented by the following formula: (A) n -(Target antigen site)-(B) o -(Th) m -(A) n -X or (A) n -(Th) m -(B) o -(Target antigen site)-(A) n -X or (A) n -(Th) m -(B) o -(Target antigen site)-(B) o -(Th) m -(A) n -X or {(A) n -(Th) p -(B) o -(Target antigen site)-(B) o -(Th) p -(A) n -X} m (In the formula, each A is independently an amino acid; each B is independently a heterologous spacer; each Th is independently an artificial Th epitope; the target antigenic site is a B cell epitope, a CTL epitope, a peptide hapten, a non-peptide hapten, or an immunologically reactive analog thereof; X is an amino acid, α-COOH, or α-CONH2; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 1, 2, 3, or 4; o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 0, 1, 2, 3, or 4).

[0010] An example of a peptide hapten as a target antigenic site is the beta amyloid (Aβ) protein (Aβ 1-14 ) (SEQ ID NO: 56). Examples of non-peptide haptens include tumor-associated carbohydrate antigens (TACAs) or small molecule drugs.

[0011] The present disclosure also relates to compositions comprising peptide immunogen constructs containing artificial Th epitopes. Such compositions can induce antibody responses in immunized hosts against desired target antigenic sites. Target antigenic sites may be derived from pathogenic organisms, normally immune-silent self-antigens, or tumor-associated targets.

[0012] The disclosed compositions are useful in many diverse medical and veterinary applications, including vaccines that provide protective immunity against infectious diseases, immunotherapies for the treatment of disorders resulting from dysfunction of normal physiological processes, immunotherapies for the treatment of cancer, and as agents that desirably intervene and modify normal physiological processes.

[0013] Some of the target antigens that may be covalently linked to the Th epitopes of the present invention include, by way of example only, beta-amyloid (Aβ) for the treatment of Alzheimer's disease, alpha-synuclein (α-Syn) for the treatment of Parkinson's disease, the outer membrane proximal domain of membrane-bound IgE (or IgE EMPD) for the treatment of allergic diseases, Tau for the treatment of tauopathies, including Alzheimer's disease, and portions of interleukin 31 (IL-31) for the treatment of atopic dermatitis. More specifically, target antigens include Aβ 1-14 (described in U.S. Patent No. 9,102,752), α-Syn 111-132 (described in International PCT Application No. PCT / US2018 / 037938), IgE EMPD 1-39 (described in International PCT Application No. PCT / US2017 / 069174), Tau 379-408 (described in International PCT Application No. PCT / US2018 / 057840), and IL-31 97-144 (described in International PCT Application No. PCT / US2018 / 065025), as well as the target antigenic sites described in Tables 3A and 3B.

[0014] The present disclosure also provides methods for preventing and / or treating a disease or condition in a subject by administering to a subject in need thereof a peptide immunogen construct (comprising the disclosed artificial Th epitopes and antigen-presenting epitopes). In some embodiments, the peptide immunogen construct produces an immunogenic inflammatory response in the subject that is at least about 3-fold lower than the immunogenic inflammatory response of a positive control, as shown in Example 12.

[0015] The present disclosure also relates to antibodies produced by peptide immunogen constructs containing the disclosed artificial Th epitopes. The antibodies produced by the peptide immunogen constructs are highly specific for the target antigenic site, rather than for the artificial Th epitopes.

[0016] References: Each patent, publication, and non-patent literature cited in this application is incorporated herein by reference in its entirety, as if each was individually incorporated by reference. 1. BORRAS-CUESTA, F., et al., “Engineering of immunogenic peptides by co‐linear synthesis of determinants recognized by B and T cells”, Eur. J. Immunol., 17:1213-1215 (1987). 2. CEASE, K.B., et al., “Helper T-cell antigenic site identification in the acquired immunodeficiency syndrome virus gp120 envelope protein and induction of immunity in mice to the native protein using a 16-residue synthetic peptide”, Proc. Natl. Acad. Sci. USA, 84:4249-4253 (1987). 3. CHIANG, H-L., et al., “A novel synthetic bipartite carrier protein for developing glycotope-based vaccines”, Vaccine, 30(52):7573-7581 (2012). 4. DANISHEFSKY, S.J., et al., “Development of Globo-H Cancer Vaccine”, Acc. Chem. Res.48(3):643-652 (2015). 5. HUANG, C.Y., et al., “Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen”, Proc. Natl. Acad. Sci. USA 103(1):15-20 (2006). 6. JACQUES, S., et al., “Chemoenzymatic synthesis of GM3 and GM2 gangliosides containing a truncated ceramide functionalized for glycoconjugate synthesis and solid phase applications”, J. Am. Chem. Soc., 134(10):4521-4 (2012). 7. KATIAL, R.K., et al., “Cytokine Production in Cell Culture by Peripheral Blood Mononuclear Cells from Immunocompetent Hosts”, Clin. Diag. Lab. Immunol., 5(1):78-81 (1998). 8. KUDUK, S.D., et al., “Synthetic and Immunological Studies on Clustered Modes of Mucin-Related Tn and TF O-Linked Antigens: The Preparation of a Glycopeptide-Based Vaccine for Clinical Trials against Prostate Cancer”; J. Am. Chem. Soc., 120(48):12474-85 (1998). 9. KUZNIK, G., et al., “Chemical and enzymatic synthesis of high-affinity selectin ligands”, Bioorganic & Medicinal Chemistry Letters, 7(5):577-580 (1997). 10. MEISTER, G.E., et al., “Two novel T cell epitope prediction algorithms based on MHC-binding motifs; comparison of predicted and published epitopes from Mycobacterium tuberculosis and HIV protein sequences”, Vaccine, 13(6):581-591 (1995). 11. PARTIDOS, C.D., et al., “Immune responses in mice following immunization with chimeric synthetic peptides representing B and T cell epitopes of measles virus proteins”, J. of Gen. Virology, 72:1293-1299 (1991). 12. ROTHBARD, J.B., et al., “A sequence pattern common to T cell epitopes”, EMBO J., 7(1):93-101 (1988). 13. SCHUTZE, M.P., et al., “Carrier-induced epitopic suppression, a major issue for future synthetic vaccines”, J. Immunol., 135(4):2319-2322 (1985). 14. TOYOKUNI, T., et al., “Synthetic carbohydrate vaccines: synthesis and immunogenicity of Tn antigen conjugates”, Bioorg. Med. Chem., 11:1119-32 (1994). 15. WO1989 / 06974 by BIXLER et al., “T-Cell Epitope As Carriers Molecule For Conjugate Vaccines” (1989-08-10). 16. WANG et al., WO1995 / 011998, “Structured Synthetic Antigen Libraries As Diagnostics, Vaccines And Therapeutics” (1995-05-04). 17. WO1999 / 066957, “Artificial T Helper Cell Epitopes As Immune Stimulators For Synthetic Peptide Immunogens” (December 29, 1999) and corresponding U.S. Patent No. 6,713,301 (March 30, 2004) by WANG. 18. WANG, U.S. Patent No. 5,912,176, “Antibodies against a host cell antigen complex for pre and post exposure protection from infection by HIV” (1999-06-15) and corresponding U.S. Patent Nos. 5,961,976 (1999-10-05) and 6,090,388 (2000-07-18). 19. WANG, U.S. Patent No. 6,025,468, “Artificial T helper cell epitopes as immune stimulators for synthetic peptide immunogens including immunogenic LHRH peptides” (2000-02-15) and corresponding U.S. Patent Nos. 6,228,987 (2001-05-08) and 6,559,282 (2003-05-06). 20. WANG et al., U.S. Patent No. 6,048,538, “Peptides derived from the non-structural proteins of foot and mouth disease virus as diagnostic reagents” (2000-04-11) and corresponding U.S. Patent No. 6,107,021 (2000-08-22). 21. WANG et al., U.S. Patent No. 6,811,782, “Peptide composition as immunogen for the treatment of allergy” (2004-11-02) and corresponding U.S. Patent No. 7,648,701 (2010-01-19). 22. WANG, U.S. Patent No. 6,906,169, “Immunogenic peptide composition comprising measles viral Fprotein Thelper cell epitope (MUFThl-16) and N-terminus of β-amyloid peptide” (2005-06-14) and corresponding U.S. Patent Nos. 7,951,909 (2011-05-31) and 8,232,373 (2012-07-31). 23. WANG, U.S. Patent No. 9,102,752, “Peptide vaccine for prevention and immunotherapy of dementia of the Alzheimer's type” (2015-08-11). 24. WANG et al., U.S. Publication No. 2013 / 0236487, “Designer Peptide-Based PCV2 Vaccine” (2013-09-12). 25. WANG, U.S. Publication No. 2014 / 0335118, “Synthetic Peptide-Based Marker Vaccine And Diagnostic System For Effective Control Of Porcine Reproductive And Respiratory Syndrome (PRRS)” (2014-11-13). 26. WANG, U.S. Publication No. 2015 / 0306203, “Synthetic Peptide-Based Emergency Vaccine Against Foot And Mouth Disease (FMD)” (October 29, 2015). 27. WANG et al., U.S. Publication No. 2017 / 0216418, “Immunogenic LHRH Composition and Use Thereof In Pigs” (2017-08-03). 28. International PCT application PCT / US2017 / 069174 by WANG, “Peptide Immunogens and Formulations Thereof Targeting Membrane-Bound IgE for Treatment of IgE Mediated Allergic Diseases” (filed 2017-12-31). 29. International PCT Application PCT / US2018 / 037938 by WANG, “Peptide Immunogens From the C-Terminal End of Alpha-Synuclein Protein and Formulations Thereof for Treatment of Synucleinopathies” (filed 2018-06-15) 30. International PCT application PCT / US2018 / 057840 by WANG, “Tau Peptide Immunogen Constructs” (filed 2018-10-26). 31. International PCT application PCT / US2018 / 065025 by WANG, “Peptide Immunogens of IL-31 and Formulations Thereof for the Treatment and / or Prevention of Atopic Dermatitis” (filed 2018-12-11). [Brief description of the drawings]

[0017] [Figure 1] Schematic diagrams showing exemplary formulations and features of the T helper cell epitope platform described herein: (A) is a schematic diagram summarizing components that may be included in a formulation with a peptide immunogen containing a Th epitope carrier, including UBITh®; (B) summarizes some features and technical advantages of the T helper cell epitope platform described herein, including UBITh®. [Diagram 2] Graph showing theoretical results that can be obtained using the T helper cell epitope platform described herein. [Diagram 3] Enhancement of immunogenicity of peptide immunogen constructs with selected individual Th epitope peptides, including short non-immunogenic B epitope peptides derived from alpha synuclein (top panel) and IgE EMPD (bottom panel). Numbers under the bars for alpha synuclein (top panel) correspond to the peptide immunogen constructs listed in Table 5, and numbers under the bars for IgE EMPD (bottom panel) correspond to the peptide immunogen constructs listed in Table 6. [Figure 4A] 4A-4D are graphs showing anti-α-synuclein antibody titers by ELISA obtained after immunization of guinea pigs with a mixture of three separate peptide immunogen constructs containing α-Syn (G111-G132), IgE-EMPD (G1-C39), and IL-6 (C73-C83) covalently linked to individual Th epitopes as shown in Table 8. The specific α-synuclein peptide immunogen constructs evaluated in these graphs are summarized in Table 5. Figure 4A contains the antibody titer data for all of the peptide immunogen constructs evaluated. [Figure 4B] 4A-4C are graphs showing anti-α-synuclein antibody titers by ELISA obtained after immunization of guinea pigs with a mixture of three separate peptide immunogen constructs containing α-Syn (G111-G132), IgE-EMPD (G1-C39), and IL-6 (C73-C83) covalently linked to individual Th epitopes shown in Table 8. The specific α-synuclein peptide immunogen constructs evaluated in these graphs are summarized in Table 5. FIG. 4B contains a subset of the data shown in FIG. 4A to highlight that peptide immunogen constructs can reach similar Cmax values ​​at different rates. [Diagram 5] Graphs showing anti-IgE EMPD antibody titers by ELISA obtained after immunization of guinea pigs with a mixture of three separate peptide immunogen constructs containing α-Syn (G111-G132), IgE-EMPD (G1-C39), and IL-6 (C73-C83) covalently linked to individual Th epitopes as shown in Table 8. The specific IgE EMPD peptide immunogen constructs evaluated in these graphs are summarized in Table 6. [Figure 6] Graphs showing anti-IL-6 antibody titers by ELISA obtained after immunization of guinea pigs with a mixture of three separate peptide immunogen constructs containing α-Syn (G111-G132), IgE-EMPD (G1-C39), and IL-6 (C73-C83) covalently linked to individual Th epitopes as shown in Table 8. The specific IL-6 peptide immunogen constructs evaluated in these graphs are summarized in Table 7. [Figure 7] Graph showing anti-DPR (dipeptide repeat) antibody titers by ELISA obtained after immunization of guinea pigs with peptide immunogen constructs shown in Table 9. [Figure 8] Graph showing anti-Aβ1-28 titers by ELISA obtained after immunization of guinea pigs with different amounts of Aβ vaccine (UB-311) containing two peptide immunogens Aβ1-14-εK-KKK-MvF5 Th (SEQ ID NO: 67) and Aβ1-14-εK-HBsAg3 Th (SEQ ID NO: 68). [Figure 9]Graph showing anti-Aβ1-28 titers by ELISA obtained after immunization of guinea pigs with different priming and boosting doses of Aβ vaccine (UB-311) containing two peptide immunogens Aβ1-14-εK-KKK-MvF5 Th (SEQ ID NO: 67) and Aβ1-14-εK-HBsAg3 Th (SEQ ID NO: 68). [Figure 10] Graphs showing anti-Aβ1-28 antibody titers by ELISA obtained after immunization of human subjects with Aβ vaccine (UB-311) during a 3-month boost (top panel) or with Aβ vaccine (UB-311) during a 6-month boost (bottom panel). The boxed portion of each graph highlights the average titer for all human subjects in the study. [Figure 11] Graphs showing anti-LHRH antibody titers and testosterone concentrations obtained after immunization of pigs with different amounts of a mixture of LHRH peptide immunogen constructs (SEQ ID NOs: 239-241) shown in Table 10 using MONTANIDE ISA50V as adjuvant. Figure 11A shows antibody titers and testosterone concentrations obtained after immunization of rats with a 100 μg amount of LHRH formulation. Figure 11B shows antibody titers and testosterone concentrations obtained after immunization of rats with a 300 μg amount of LHRH formulation. [Figure 12] Graph showing anti-IL-6 antibody titers by ELISA obtained after immunization of rats with different amounts of IL-6 peptide immunogen construct of SEQ ID NO: 243 or a placebo control in formulations containing different adjuvants (i.e., MONTANIDE ISA51 or ADJUPHOS). [Figure 13] Graphs showing anti-IgE-EMPD antibody titers obtained after immunizing macaques with different amounts of the IgE-EMPD peptide immunogen construct of SEQ ID NO: 178. Figure 13A shows antibody titers obtained using ADJUPHOS as an adjuvant, formulated as a stabilized immunostimulating complex with CpG3. Figure 13B shows antibody titers obtained using MONTANIDE ISA51 as an adjuvant, formulated as a stabilized immunostimulating complex with CpG3. [Figure 14]Graphs showing anti-LHRH antibody titers and testosterone concentrations obtained after immunizing pigs with different amounts of a mixture of LHRH peptide immunogen constructs (SEQ ID NOs: 239-241) shown in Table 10 using different adjuvants. Figure 14A shows antibody titers obtained using Emulsigen D as an adjuvant. Figure 14B shows antibody titers obtained using MONTANIDE ISA50V as an adjuvant. [Figure 15] Detection of promiscuous and artificial Th peptide-responsive T cells in naive peripheral blood mononuclear cells of normal donors. [Figure 16] Structures of tumor-associated carbohydrate antigens (TACA): GD3, GD2, Globo-H, GM2, fucosyl GM1, PSA, Ley, Lex, SLex, SLea, and STn. [Figure 17] Exemplary stepwise synthesis of glycan-linked UBITh® peptides by solid-phase peptide synthesis scheme. [Figure 18] Exemplary stepwise synthesis of glycan-linked UBITh® peptides by solid-phase peptide synthesis scheme. [Figure 19] Exemplary stepwise synthesis of glycan-linked UBITh® peptides by solid-phase peptide synthesis scheme. [Figure 20] Exemplary stepwise synthesis of glycan-linked UBITh® peptides by solid-phase peptide synthesis scheme. [Figure 21] Exemplary stepwise synthesis of glycan-linked UBITh® peptides by solid-phase peptide synthesis scheme. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present disclosure provides promiscuous artificial T helper cell (Th) epitopes for stimulating functional site-specific antibodies against target antigens for prophylactic and therapeutic use. The present disclosure also relates to peptide immunogenic constructs containing the Th epitopes, compositions containing the Th epitopes, methods of making and using the Th epitopes, and antibodies generated by the peptide immunogenic constructs containing the Th epitopes.

[0019] The disclosed artificial T helper cell (Th) epitopes can be linked to B cell epitopes and / or effector T cell epitopes (e.g., cytotoxic T cells; CTLs) ("target antigenic site(s)") via any spacer to generate peptide immunogen constructs. The disclosed Th epitopes confer to peptide immunogens the ability to induce a strong T helper cell-mediated immune response with the production of high levels of antibodies and / or cellular responses (e.g., cytokines) against the target antigenic site for therapeutic efficacy. The disclosed peptide immunogen constructs provide advantageous replacement of large carrier proteins and pathogen-derived T helper cell sites in peptide immunogens with the disclosed artificial Th epitopes that are specifically designed to improve the immunogenicity of the target antigenic site. Relatively short peptide immunogen constructs containing the disclosed Th epitopes induce high levels of antibodies and / or effector cell-associated cytokines against a particular target antigenic site without eliciting a significant inflammatory or immune response against the Th epitope.

[0020] The immune response induced by the peptide immunogen construct (antibody titer, C max The response time (including the onset of antibody production, duration of response, etc.) can be modulated by varying: (a) the choice of Th epitope that is chemically linked to the B cell epitope, (b) the length of the B cell epitope, (c) the adjuvant used in the formulation containing the peptide immunogen construct, and / or (d) the dosing regimen including the dosage per immunization and the time of priming and boosting for each immunization. Thus, specific immune responses against target antigenic sites can be engineered using the disclosed Th epitopes, which may facilitate tailoring personalized medicine to the individual characteristics of any patient or subject.

[0021] The disclosed peptide immunogen constructs containing artificial Th epitopes can elicit antibody and / or cytokine responses against a desired target antigenic site in an immunized host. The target antigenic site can be any amino acid sequence derived from a specific protein, a cancer antigen-associated carbohydrate, a small molecule drug compound, or any target peptide or protein. In some embodiments, the present disclosure describes promiscuous artificial Th epitopes that can be used to provide peptide immunogens that elicit antibodies targeted to amyloid beta (Aβ), foot and mouth disease (FMD) capsid protein, glycoprotein from porcine reproductive and respiratory syndrome virus (PRRSV), luteinizing hormone releasing hormone (LHRH), and any other peptide or protein sequence.

[0022] In certain embodiments, the target antigenic site is taken from a normally immune-silent self-antigen or tumor-associated neo-antigen target (e.g., Aβ, Tau, alpha-synuclein, dipeptide protein, IgE EMPD, IL-6, CGRP, amylin, IL-31, neo-antigen, etc.). Non-limiting representative sequences of self-antigen and tumor-associated neo-antigen sites are shown in Table 3A. In other embodiments, the target antigenic site is taken from a pathogenic organism (e.g., FMDV, PRRSV, CSFV, HIV, HSV, etc.). Non-limiting representative sequences of pathogenic antigenic sites are shown in Table 3B.

[0023] The peptides or target antigenic sites of the present invention can be useful in medical and veterinary applications. For example, peptide immunogen constructs containing the disclosed artificial Th epitopes can be used in vaccine compositions that provide protective immunity against infectious diseases or neurodegenerative diseases, or in pharmaceutical compositions that treat disorders resulting from the malfunction of normal physiological processes, in immunotherapy to treat cancer, type 2 diabetes, or as drugs that intervene in normal physiological processes.

[0024] general The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references or portions of references cited in this application are expressly incorporated herein by reference in their entirety for any purpose.

[0025] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Thus, "comprising A or B" means including A, or B, or A and B. It should be further understood that all amino acid sizes and all molecular weight or molecular mass values ​​given for polypeptides are approximate and are provided for illustration purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0026] Peptide immunogen constructs The term "peptide immunogen" or "peptide immunogen construct" as used herein refers to a molecule that contains an artificial Th epitope that is covalently linked to a target antigen site through a covalent bond (e.g., a conventional peptide bond or a thioester), with or without a heterologous spacer, to form a single larger peptide. Typically, a peptide immunogen construct contains (a) a heterologous promiscuous artificial Th epitope; (b) a target antigen site, such as a B cell epitope or an effector T cell epitope (e.g., CTL); and (c) an optional heterologous spacer.

[0027] The presence of promiscuous artificial Th epitopes in peptide immunogens can induce strong Th cell-mediated immune responses and high levels of antibodies against target antigenic sites in animals after immunization with the peptide immunogen. The disclosed peptide immunogen constructs provide advantageous replacement of large carrier proteins and pathogen-derived T helper cell sites in peptide immunogens with the disclosed artificial Th epitopes that are specifically designed to improve the immunogenicity of the target antigenic sites. Relatively short peptide immunogen constructs containing the disclosed Th epitopes induce high levels of antibodies and / or effector cell-associated cytokines against specific target antigenic sites without eliciting significant inflammatory or immune responses against the Th epitopes.

[0028] The peptide immunogen constructs containing the disclosed artificial Th epitopes of the present invention can be represented by the following formula: (A) n -(Target antigen site)-(B) o -(Th) m -(A) n -X or (A) n -(Th) m -(B) o -(Target antigen site)-(A) n -X or (A) n -(Th) m -(B) o -(Target antigen site)-(B) o -(Th) m -(A) n -X or {(A) n -(Th) p -(B) o -(Target antigen site)-(B) o -(Th) p -(A) n -X} m (In the formula, each A is independently an amino acid; each B is independently a heterologous spacer; each Th is independently an artificial Th epitope; the target antigenic site is a B cell epitope, a CTL epitope, a peptide hapten, a non-peptide hapten, or an immunologically reactive analog thereof; X is an amino acid, α-COOH, or α-CONH2; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 1, 2, 3, or 4; o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 0, 1, 2, 3, or 4).

[0029] Peptide immunogens of the disclosure can comprise from about 20 to about 100 amino acids. In some embodiments, the peptide immunogen construct contains about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acid residues.

[0030] The various components of the disclosed IL-31 peptide immunogen constructs are described below.

[0031] A-amino acids Each A in the immunogenic peptides of the present disclosure is independently a heterologous amino acid.

[0032] The term "heterologous" as used herein refers to an amino acid sequence that is not part of or is not homologous to the wild-type amino acid sequence of the target antigenic site (e.g., B-cell epitope). Thus, the heterologous amino acid sequence of A contains an amino acid sequence that is not naturally found in the protein or peptide of the target antigenic site. Because the sequence of component A is heterologous to the target antigenic site, the native amino acid sequence of the target antigenic site is not extended in either the N- or C-terminal direction when component A is covalently attached to the target antigenic site.

[0033] In some embodiments, each A is independently an unnatural or natural amino acid.

[0034] Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0035] Unnatural amino acids include, but are not limited to, ε-N-lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitrotyrosine, pyroglutamic acid, and the like.

[0036] In some embodiments, n is 0, indicating that no amino acid is added at that position in the formula. In other embodiments, n is 1, and is selected from any natural or unnatural amino acid. In certain embodiments, n is greater than 1, and each A is independently the same amino acid. In other embodiments, n is greater than 1, and each A is independently a different amino acid.

[0037] B - any heterologous spacer Each B in the immunogenic peptides of the present disclosure is an optional heterologous spacer. The optional heterologous spacer of component B is independently an amino acid, -NHCH(X)CHSCHCO-, -NHCH(X)CHSCHCO(εN)Lys-, -NHCH(X)CHS-succinimidyl(εN)Lys-, -NHCH(X)CHS-(succinimidyl)-, and / or any combination thereof. The spacer may contain one or more natural or unnatural amino acid residues as described above for component A.

[0038] As mentioned above, the term "heterologous" refers to an amino acid that is not part of or is not homologous to the wild-type amino acid sequence of the target antigenic site (e.g., B cell epitope). Thus, when the spacer is an amino acid, the spacer contains an amino acid sequence that is not naturally found in the protein or peptide of the target antigenic site. Because the sequence of component B is heterologous to the target antigenic site, the native amino acid sequence of the target antigenic site is not extended in either the N-terminal or C-terminal direction when component B is covalently attached to the target antigenic site.

[0039] The spacer can be a flexible hinge spacer that enhances the separation of the Th epitope and the target antigen site. In some embodiments, the flexible hinge sequence can be proline-rich. In certain embodiments, the flexible hinge has the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 55), which is modeled from the flexible hinge region found in immunoglobulin heavy chains. Wherein Xaa can be any amino acid. In some embodiments, Xaa is aspartic acid. In some embodiments, the conformational spacing provided by the spacer can allow for more efficient interaction between the presented peptide immunogen and the appropriate Th and B cells. The immune response to the Th epitope can be enhanced to improve immune reactivity.

[0040] When o>1, each B is independently the same or different. In some embodiments, B is Gly-Gly, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:55), εNLys, εNLys-Lys-Lys-Lys (SEQ ID NO:53), Lys-Lys-Lys-εNLys (SEQ ID NO:54), Lys-Lys-Lys, -NHCH(X)CHSCHCO-, -NHCH(X)CHSCHCO(εNLys)-, -NHCH(X)CHS-succinimidyl-εNLys-, or -NHCH(X)CHS-(succinimidyl)-, and / or any combination thereof. Exemplary heterologous spacers are shown in Table 2.

[0041] target antigen site Target antigenic sites may include any amino acid sequence from any target peptide or protein, including foreign or self peptides or proteins, B cell epitopes, CTL epitopes, peptide haptens, non-peptide haptens, or immunologically reactive analogs thereof. Target antigenic sites may be specific proteins, cancer antigen-associated carbohydrates, small molecule drug compounds, or any amino acid sequence from any target peptide or protein. In some embodiments, the present disclosure describes promiscuous artificial Th epitopes that can be used to provide peptide immunogens that elicit antibodies targeted to amyloid beta (Aβ), foot and mouth disease (FMD) capsid protein, glycoprotein from porcine reproductive and respiratory syndrome virus (PRRSV), luteinizing hormone releasing hormone (LHRH), and any other peptide or protein sequence.

[0042] In certain embodiments, the target antigenic site is taken from a normally immune-silent self-antigen or tumor-associated neo-antigen target (e.g., Aβ, Tau, alpha-synuclein, dipeptide protein, IgE EMPD, IL-6, CGRP, amylin, IL-31, neo-antigen, etc.). Non-limiting representative sequences of self-antigen and tumor-associated neo-antigen sites are shown in Table 3A. In other embodiments, the target antigenic site is taken from a pathogenic organism (e.g., FMDV, PRRSV, CSFV, HIV, HSV, etc.). Non-limiting representative sequences of pathogenic antigenic sites are shown in Table 3B.

[0043] In certain embodiments, the target antigenic site is selected from the group consisting of luteinizing hormone releasing hormone (LHRH) (e.g., U.S. Pat. Nos. 6,025,468, 6,228,987, 6,559,282, and U.S. Patent Publication No. US2017 / 0216418); amyloid beta (Aβ) (e.g., U.S. Pat. Nos. 6,906,169, 7,951,909, 8,232,373, and 9,102,752); foot and mouth disease capsid protein (e.g., U.S. Pat. Nos. 6,048,538, 6,107,021, and U.S. Patent Publication No. 2015 / 0306203); HIV virion endonucleases for the prevention and treatment of HIV infection. pitopes (e.g., U.S. Patent Nos. 5,912,176, 5,961,976, and 6,090,388); capsid protein from porcine circovirus type 2 (PCV2) (e.g., U.S. Patent Publication No. 2013 / 0236487), glycoprotein from porcine reproductive and respiratory syndrome virus (PRRSV) (e.g., U.S. Patent Publication No. 2014 / 0335118), IgE (e.g., U.S. Patent Nos. 7,648,701 and 6,811,782), alpha-synuclein (α-Syn) (International PCT Application No. PCT / US2018 / 037938), the extracellular membrane proximal domain of membrane-bound IgE (or IgE EMPD) (International PCT Application No. PCT / US2017 / 069174), Tau (International PCT Application No. PCT / US2018 / 057840), and Interleukin-31 (IL-31) (International PCT Application No. PCT / US2018 / 065025), Plasmodium CS antigen for the prevention of malaria; CETP for the prevention and treatment of arteriosclerosis; IAPP (Amylin) for the prevention and treatment of type 2 diabetes, and any other peptide or protein sequence derived from a portion thereof. All patents and patent publications are incorporated herein by reference in their entirety.

[0044] In other embodiments, the target antigenic site is a non-peptide hapten, including tumor-associated carbohydrate antigens (TACAs) and small molecule drug compounds. Examples of TACAS include GD3, GD2, Globo-H, GM2, Fucosyl GM1, GM2, PSA, Le, as further discussed in Example 11 and Figures 16 and 17. y , Lex , SLe x , SLe a , Tn, TF, and STn.

[0045] Th-T helper epitopes Promiscuous artificial T helper cell (Th) epitopes in peptide immunogen constructs enhance the immunogenicity of target antigenic sites, thereby promoting the production of specific, high-titer antibodies against rationally designed, optimized target B cell epitopes.

[0046] The term "promiscuous" as used herein refers to Th epitopes that are reactive between species and among individuals of a single species.

[0047] The term "artificial" used in relation to Th epitopes refers to amino acid sequences that are not found in nature. Thus, the artificial Th epitopes of the present disclosure have heterologous sequences to target antigen sites. As mentioned above, the term "heterologous" refers to amino acid sequences that are derived from amino acid sequences that are not part of or homologous to the wild-type sequence of the target antigen site. Thus, a heterologous Th epitope is a Th epitope that is derived from an amino acid sequence that is not naturally found at the target antigen site. Since the Th epitope is heterologous to the target antigen site, the natural amino acid sequence of the target antigen site is not extended in either the N-terminal or C-terminal direction when the heterologous Th epitope is covalently attached to the target antigen site.

[0048] The Th epitope may have an amino acid sequence derived from any species (e.g., human, porcine, bovine, canine, rat, mouse, guinea pig, etc.). The Th epitope may also have promiscuous binding motifs to MHC class II molecules of multiple species. In certain embodiments, the Th epitope contains multiple promiscuous MHC class II binding motifs that allow for maximum activation of T helper cells leading to the initiation and regulation of immune responses. The Th epitope is preferably itself immunosilent, i.e., antibodies generated by the peptide immunogen construct are rarely, if ever, directed to the Th epitope, thereby allowing for a highly focused immune response directed to the target antigen site.

[0049] Epitopes may range in size from about 15 to about 50 amino acid residues. In some embodiments, Th epitopes may have about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acid residues. Epitopes may share common structural features and specific landmark sequences. In some embodiments, Th epitopes have an amphipathic helix, i.e., an alpha-helical structure with hydrophobic amino acid residues occupying one face of the helix and charged polar residues occupying the surrounding faces.

[0050] The Th epitopes and disclosures of WO1999 / 066957, and corresponding US Pat. No. 6,713,301, are incorporated herein by reference in their entireties.

[0051] Promiscuous Th determinants can be effective in boosting poorly immunogenic peptides. Well-designed promiscuous Th / B cell epitope chimeric peptides can induce Th responses accompanied by antibody responses targeted to B cell sites in most members of a genetically diverse population. In some embodiments, Th cells can be fed to target antigen peptides by covalently linking a peptide carrier to a well-characterized promiscuous Th determinant.

[0052] Promiscuous Th epitopes may contain additional primary amino acid patterns. In some embodiments, promiscuous Th epitopes may contain a Rothbard sequence, where the promiscuous Th epitopes contain a charged residue (e.g., -Gly-), followed by 2-3 hydrophobic residues, followed by a charged or polar residue (Rothbard and Taylor, EMBO J, 1988; 7:93-101). Promiscuous Th epitopes may follow the 1, 4, 5, 8 rule, where a positively charged residue is followed by hydrophobic residues at positions 4, 5, and 8, conforming to an amphipathic helix with positions 1, 4, 5, and 8 located on the same face. In some embodiments, the 1, 4, 5, 8 pattern of hydrophobic and charged and polar amino acids may be repeated within a single Th epitope. In some embodiments, the promiscuous T cell epitope may contain at least one of the Rothbard sequence or epitopes following the 1, 4, 5, 8 rule. In other embodiments, the Th epitope contains multiple Rothbard sequences.

[0053] Promiscuous Th epitopes derived from pathogens include the hepatitis B surface Th cell epitope (HBsAg Th), hepatitis B core antigen Th cell epitope (HBc Th), pertussis toxin Th cell epitope (PT Th), tetanus toxin Th cell epitope (TT Th), measles virus F protein Th cell epitope (MVF Th), Chlamydia trachomatis major outer membrane protein Th cell epitope (CT Th), diphtheria toxin Th cell epitope (DT Th), Plasmodium falciparum peri-sporozoite Th cell epitope (PF Th), Schistosoma mansoni triosephosphate isomerase Th cell epitope (SM Th), and Escherichia coli TraT Th cell epitope (TraT Th), Clostridium tetani, Bordetella pertussis, cholera toxin, influenza MP1, influenza NSP1, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV). Examples of Th epitopes used in this disclosure are shown in Table 1.

[0054] In some embodiments, the Th epitopes of the present disclosure may be combinatorial Th epitopes that contain a mixture of peptides containing similar amino acid sequences. Structured synthetic antigen libraries (SSALs), also referred to as combinatorial artificial Th epitopes, contain a large number of Th epitopes with amino acid sequences organized around a structural framework of invariant residues substituted at specific positions. The sequence of the SSAL epitopes is determined by retaining relatively invariant residues and varying other residues to obtain recognition of diverse MHC restriction elements. The sequence of the SSAL epitopes can be determined by aligning the primary amino acid sequences of promiscuous Th, selecting and retaining residues that are involved in the unique structure of the Th peptide as a scaffold framework, and varying the remaining residues according to known MHC restriction elements. The invariant and variable positions with preferred amino acids of the MHC restriction elements can be used to obtain MHC binding motifs, which can be used to design SSALs of Th epitopes.

[0055] Heterologous Th epitope peptides presented as combinatorial sequences contain a mixture of amino acid residues represented at specific positions within a peptide framework based on the variable residues of that particular peptide's homologs. In some embodiments, Th epitope library sequences are designed to maintain the structural motifs of promiscuous Th epitopes and accommodate reactivity to a broad range of haplotypes. In some embodiments, members of SSAL can be degenerate Th epitopes SSAL1 Th1, modeled from promiscuous epitopes (e.g., SEQ ID NOs: 1-5) obtained from the F protein of measles virus. In other embodiments, members of SSAL can be degenerate Th epitopes SSAL2 Th2, modeled from promiscuous epitopes (e.g., SEQ ID NOs: 19-24) obtained from HBsAg1.

[0056] The total number of peptides present in the mixture of combinatorial artificial Th epitopes (or SSALs) after synthesis can be calculated by multiplying the number of choices available at each variable position. For example, SEQ ID NO: 16 contains 5 variable positions, each with 2 different residue choices, thus representing 32 different peptide combinations (i.e., 2x2x2x2x2=2). 5 Similarly, SEQ ID NO:5 represents 524,288 different peptide combinations (i.e., 2x4x2x4x2x4x4x4x4x2x4x2x4=2 5 x4 7 =524,288). The combinatorial artificial Th epitope arrays include (a) a mixture of all peptides encompassed by the variable sequences, and (b) each individual peptide that contains a single sequence within the combination.

[0057] In some embodiments, the charged residues Glu or Asp can be added at position 1 to increase the charge surrounding the hydrophobic face of Th. In some embodiments, the hydrophobic face of the amphipathic helix can be maintained by hydrophobic residues 2, 5, 8, 9, 10, 13, and 16. In some embodiments, amino acid residues 2, 5, 8, 9, 10, and 13 can be altered to provide a facade capable of binding to a broad range of MHC restriction elements. In some embodiments, the alteration of amino acid residues can expand the range of immune responsiveness of the artificial Th epitope.

[0058] An artificial Th epitope may incorporate all the properties and characteristics of known promiscuous Th epitopes. In some embodiments, the artificial Th epitope is a member of the SSAL. In some embodiments, the artificial Th site may be combined with peptide sequences obtained from self- and foreign antigens to provide enhanced antibody responses to site-specific targets. In some embodiments, the artificial Th epitope immunogen may provide an effective and safe antibody response, exhibit high immune potency, and exhibit broad reactive responses.

[0059] Idealized artificial Th epitopes are also provided. These idealized artificial Th epitopes are modeled on two known natural Th epitopes and the SSAL peptide prototype disclosed in WO95 / 11998. SSALS incorporate combinatorial MHC molecule binding motifs (Meister et al., 1995) that are intended to induce broad immune responses among members of a genetically diverse population. SSAL peptide prototypes were designed based on Th epitopes of measles virus and hepatitis B virus antigens and modified by introducing multiple MHC binding motifs. The design of other Th epitopes was modeled from other known Th epitopes by simplifying, adding, and / or modifying multiple MHC binding motifs to generate a series of novel artificial Th epitopes. Promiscuous artificial Th moieties were incorporated into synthetic peptide immunogens bearing various target antigen sites. The resulting chimeric peptides were able to stimulate effective antibody responses against the target antigen sites.

[0060] The prototypical artificial helper T cell (Th) epitope, a mixture of four peptides (SEQ ID NOs: 1-4), designated "SSAL1 Th1" in Table 1, is an idealized Th epitope modeled after the promiscuous Th epitope of the F protein of measles virus (Partidos et al. 1991). The model Th epitope, designated "MVF Th(UBITh®5)" (SEQ ID NO: 6) in Table 1, corresponds to residues 288-302 of the measles virus F protein. Following the "Rothbard rules", MVF Th (SEQ ID NO: 6) was modified to the SSAL1 Th1 prototype (SEQ ID NOs: 1-4) by adding a charged residue Glu / Asp at position 1 to increase the charge surrounding the hydrophobic face of the epitope; adding or retaining charged residues or Gly at positions 4, 6, 12, and 14; and adding or retaining charged residues or Gly at positions 7 and 11. The hydrophobic face of the Th epitope includes residues at positions 2, 5, 8, 9, 10, 13, and 16. Hydrophobic residues commonly associated with promiscuous epitopes were substituted at these positions to obtain the combinatorial Th SSAL epitope, SSAL1 Th1 (SEQ ID NO: 1-4). Another important feature of the prototype SSAL1 Th1 (SEQ ID NO: 1-4) is that positions 1 and 4 are imperfectly repeated as palindromes on either side of position 9 to mimic an MHC binding motif. Furthermore, this "1, 4, 9" palindrome pattern of SSAL1 Th1 was modified in SEQ ID NO: 2 (Table 1) to more closely reflect the sequence of the original MvF model Th (SEQ ID NO: 6).

[0061] Combinatorial artificial Th epitopes can be simplified to provide a series of single sequence epitopes. For example, the combinatorial sequence of SEQ ID NO: 5 can be simplified to single sequence Th epitopes represented by SEQ ID NOs: 1-4. These single sequence Th epitopes can be bound to target antigen sites to provide enhanced immunogenicity.

[0062] In some embodiments, the immunogenicity of the Th epitope may be improved by extending the N-terminus with non-polar and polar uncharged amino acids, e.g., Ile and Ser, and the C-terminus with charged and hydrophobic amino acids, e.g., Lys and Phe. In addition, the addition of a lysine residue or multiple lysine residues (e.g., KKK) to the Th epitope may improve the solubility of the peptide in water. Further modifications included substitution of the C-terminus with the common MHC binding motif AxTxIL (Meister et al., 1995).

[0063] The artificial Th epitopes can be known natural Th epitopes or SSAL peptide prototypes. In some embodiments, SSAL-derived Th epitopes can incorporate combinatorial MHC molecule binding motifs intended to induce broad immune responses among members of a genetically diverse population. In some embodiments, SSAL peptide prototypes modified by introducing multiple MHC binding motifs can be designed based on Th epitopes of measles virus and hepatitis B virus antigens. In some embodiments, the artificial Th epitopes can be simplified, added, and / or modified to multiple MHC binding motifs to generate a series of novel artificial Th epitopes. In some embodiments, the newly adapted promiscuous artificial Th sites can be incorporated into synthetic peptide immunogens with various target antigen sites. In some embodiments, the resulting chimeric peptides can stimulate effective antibody responses against the target antigen sites.

[0064] The artificial Th epitopes of the present disclosure can be a contiguous sequence of natural or non-natural amino acids that comprises the binding site of class II MHC molecules. In some embodiments, the artificial Th epitopes can enhance or stimulate antibody responses to target antigen sites. In some embodiments, the Th epitopes can be composed of contiguous or discontinuous amino acid segments. In some embodiments, not all amino acids of the Th epitopes are involved in MHC recognition. In some embodiments, the Th epitopes of the present invention can include immunologically functional homologs, such as immune enhancing homologs, cross-reactive homologs, and segments thereof. In some embodiments, the functional Th homologs can further include conservative substitutions, additions, deletions, and insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues to provide the Th stimulating function of the Th epitope.

[0065] The epitope can be directly attached to the target site. In some embodiments, the Th epitope can be attached to the target site via any heterologous spacer, for example, a peptide spacer such as Gly-Gly or (ε-N)Lys. The spacer can physically separate the Th epitope from the B cell epitope and disrupt the formation of any artificial secondary structure created by attaching the Th epitope or a functional homologue to the target antigen site, eliminating interference with Th and / or B cell responses.

[0066] As shown in Table 1, Th epitopes include idealized artificial Th epitopes and combinatorial idealized artificial Th epitopes. In some embodiments, the Th epitopes are promiscuous Th cell epitopes of SEQ ID NOs: 1-52, any homologs thereof, and / or any immunological analogs thereof. Th epitopes also include immunological analogs of Th epitopes. Immunological Th analogs include immune enhancing analogs, cross-reactive analogs, and segments of any of these Th epitopes sufficient to enhance or stimulate an immune response to a target antigen site.

[0067] Functional immunological analogs of Th epitope peptides are also useful and are included as part of the present invention. Functional immunological Th analogs may include conservative substitutions, additions, deletions, and insertions of one to about five amino acid residues in the Th epitope that do not essentially alter the Th stimulatory function of the Th epitope. Conservative substitutions, additions, and insertions can be accomplished using natural or non-natural amino acids, as described above for the target antigen site. Table 1 identifies additional variations of functional analogs to Th epitope peptides. In particular, SEQ ID NOs: 6 and 7 of MvF1 and MvF2 Th are functional analogs of SEQ ID NOs: 16 and 17 of MvF4 and MvF5 in that the amino acid frames differ due to the deletion (SEQ ID NOs: 6 and 7) or incorporation (SEQ ID NOs: 16 and 17) of two amino acids at the N-terminus and C-terminus, respectively. The differences between these two sets of analog sequences do not affect the function of the Th epitopes contained within these sequences. Thus, functional immunological Th analogs include several versions of Th epitopes derived from the measles virus fusion protein MvF1-4 Th (SEQ ID NOs: 6-18) and the hepatitis surface protein HBsAg 1-3 Th (SEQ ID NOs: 19-31).

[0068] The Th epitope in the peptide immunogen construct can be covalently linked to either the N-terminus or C-terminus of the target antigen site to produce a chimeric Th / B cell site peptide immunogen. In some embodiments, the Th epitope can be covalently linked to the target antigen site by chemical coupling or direct synthesis. In some embodiments, the Th epitope is covalently linked to the N-terminus of the target antigen site. In other embodiments, the Th epitope is covalently linked to the C-terminus of the target antigen site. In certain embodiments, multiple Th epitopes are covalently linked to the target antigen site. When multiple Th epitopes are linked to the target antigen site, each Th epitope can have the same amino acid sequence or different amino acid sequences. In addition, when multiple Th epitopes are linked to the target antigen site, the Th epitopes can be arranged in any order. For example, the Th epitopes can be contiguously linked to the N-terminus of the target antigenic site, or contiguously linked to the C-terminus of the target antigenic site, or a Th epitope can be covalently linked to the N-terminus of the target antigenic site, while another Th epitope is covalently linked to the C-terminus of the target antigenic site. There is no restriction on the arrangement of the Th epitopes with respect to the target antigenic site.

[0069] In some embodiments, the Th epitope is covalently linked directly to the target antigenic site, while in other embodiments, the Th epitope is covalently linked to the target antigenic site via a heterologous spacer, which is described in more detail below.

[0070] Synthesis method The peptide immunogen of the present disclosure can be synthesized using chemical methods.In some embodiments, the peptide immunogen of the present disclosure can be synthesized using solid-phase peptide synthesis.In some embodiments, the peptide of the present invention is synthesized using automated Merrifield peptide solid-phase synthesis, using t-Boc or Fmoc to protect α-NH2 or side chain amino acid.

[0071] Heterologous Th epitope peptides presented as combinatorial sequences contain a mixture of amino acid residues represented at specific positions within the peptide framework based on the variable residues of the homologues of that particular peptide. A collection of combinatorial peptides can be synthesized in one process by adding a mixture of designated protected amino acids in place of one specific amino acid at a designated position during the synthesis process. Such combinatorial heterologous Th epitope peptide assemblies can allow broad Th epitope coverage of animals with diverse genetic backgrounds. Representative combinatorial sequences of heterologous Th epitope peptides include SEQ ID NOs: 5, 10, 13, 16, 24, and 27 shown in Table 1. The epitope peptides of the present invention provide broad reactivity and immunogenicity to animals and patients from genetically diverse populations.

[0072] Interestingly, inconsistencies and / or errors that may be introduced during synthesis of peptide immunogen constructs containing Th epitopes, B cell epitopes, and / or Th epitopes and B cell epitopes do not in most cases prevent or block the desired immune response of treated animals. In fact, inconsistencies / errors that may be introduced during peptide synthesis generate multiple peptide analogs along with target peptide synthesis. These analogs may include amino acid insertions, deletions, substitutions, and premature terminations. As mentioned above, such peptide analogs are suitable for peptide preparations as contributors to antigenicity and immunogenicity when used in immunological applications, such as solid-phase antigens for immunodiagnosis or as immunogens for vaccination.

[0073] Peptide immunogen constructs containing Th epitopes are simultaneously generated in parallel with the target antigen site in a single solid-phase peptide synthesis. Th epitopes also include immunological analogs of Th epitopes. Immunological Th analogs include immune enhancing analogs, cross-reactive analogs, and segments of any of these Th epitopes sufficient to enhance or stimulate the immune response to the target antigen site.

[0074] After complete assembly of the desired peptide immunogen, the solid phase resin can be treated to cleave the peptide from the resin and remove the functional groups on the amino acid side chains. The free peptide can be purified by HPLC and characterized biochemically. In some embodiments, the free peptide is characterized biochemically using amino acid analysis. In some embodiments, the free peptide is characterized using peptide sequence. In some embodiments, the free peptide is characterized using mass spectrometry.

[0075] The peptide immunogens of the invention can be synthesized using haloacetylated and cysteinylated peptides through the formation of a thioether bond. In some embodiments, a cysteine ​​can be added to the C-terminus of a Th-containing peptide, and the thiol group of the cysteine ​​residue can be N α It can be used to form covalent bonds to electrophilic groups such as chloroacetyl modified groups or maleimide-derivatized α- or ε-NH2 groups of lysine residues. The resulting synthetic intermediates can be coupled to the N-terminus of target antigenic site peptides.

[0076] Longer synthetic peptide conjugates can be synthesized using nucleic acid cloning techniques. In some embodiments, the Th epitopes of the invention can be synthesized by expressing recombinant DNA and RNA. To construct a gene expressing the Th / target antigen site peptide of the invention, the amino acid sequence can be reverse translated into a nucleic acid sequence. In some embodiments, the amino acid sequence is reverse translated into a nucleic acid sequence using codons optimized for the organism in which the gene is expressed. A gene encoding the peptide can be created. In some embodiments, a gene encoding the peptide can be made by synthesizing overlapping oligonucleotides that code for the peptide and the necessary regulatory elements. The synthetic gene can be assembled and inserted into a desired expression vector.

[0077] The synthetic nucleic acid sequences of the present disclosure may include nucleic acid sequences encoding the Th epitopes of the present invention, peptides containing Th epitopes, their epitope-functional homologs, and nucleic acid constructs characterized by changes in the non-coding sequences that do not alter the immunological properties of the peptides or encoded Th epitopes. The synthetic genes can be inserted into a suitable cloning vector, recombinants can be obtained, and characterized. The Th epitopes and peptides containing Th epitopes can then be expressed under conditions appropriate for the selected expression system and host. The Th epitopes or peptides can be purified and characterized.

[0078] Pharmaceutical Compositions The present disclosure also describes pharmaceutical compositions comprising the peptide immunogens of the present disclosure.In some embodiments, the pharmaceutical compositions of the present disclosure can be used as a pharma- ceutical acceptable delivery system for the administration of peptide immunogens.In some embodiments, the pharmaceutical compositions of the present disclosure can comprise an immunologically effective amount of one or more peptide immunogens.

[0079] The peptide immunogens of the present invention can be formulated as immunogenic compositions. In some embodiments, the immunogenic compositions can include adjuvants, emulsifiers, pharma- ceutically acceptable carriers, or other components routinely provided in vaccine compositions. Adjuvants or emulsifiers that can be used in the present invention include alum, incomplete Freund's adjuvant (IFA), liposin, saponin, squalene, L121, EMULSIGEN, monophosphoryl lipid A (MPL), dimethyldioctadecylammonium bromide (DDA), QS21, and ISA720, ISA51, ISA35, ISA206, and other effective adjuvants and emulsifiers. In some embodiments, the compositions of the present invention can be formulated for immediate release. In some embodiments, the compositions of the present invention can be formulated for sustained release.

[0080] Adjuvants used in the pharmaceutical compositions may include oils, aluminum salts, virosomes, aluminum phosphate (e.g., ADJU-PHOS®), aluminum hydroxide (e.g., ALHYDROGEL®), liposyn, saponin, squalene, L121, Emulsigen®, monophosphoryl lipid A (MPL), QS21, ISA35, ISA206, ISA50V, ISA51, ISA720, and other adjuvants and emulsifiers.

[0081] In some embodiments, the pharmaceutical composition contains MONTANIDE™ ISA51 (an oil adjuvant composition consisting of vegetable oil and mannide oleate to generate a water-in-oil emulsion), TWEEN® 80 (also known as polysorbate 80 or polyoxyethylene (20) sorbitan monooleate), CpG oligonucleotides, and / or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w / o / w) emulsion that includes EMULSIGEN or EMULSIGEN D as an adjuvant.

[0082] FIG. 1A is a schematic diagram summarizing the components that may be included in a formulation having a peptide immunogen containing a Th epitope carrier, including UBITh®. The exemplary formulation shown in FIG. 1A includes two separate peptide immunogen constructs. Each peptide immunogen construct contains (a) a custom functional B cell epitope designed to elicit highly targeted antibodies against the desired epitope, (b) a linker that serves to optimize the presentation of the B cell epitope to the immune system to enhance immunogenicity, and (c) a Th epitope that is itself immunosilent but serves to promote a strong response against the B cell epitope. The formulation also contains a pharma- ceutically acceptable adjuvant or carrier that is appropriate for the particular application for which the composition is to be used. Additionally, the composition can be formulated into a stabilized immune stimulatory complex using CpG oligonucleotides (discussed further below).

[0083] FIG. 1B summarizes some features and technical advantages of the T helper cell epitope platform described herein, including UBITh®. For example, the Th epitope platform described herein is long-acting but reversible without boosting. The Th epitope platform generates highly specific antibodies against B cell epitopes, with few, if any, antibodies generated against the linker or Th epitope sequence. Furthermore, the Th epitope platform can be used with a variety of B cell epitopes, allowing for limitless combinations of peptide immunogen constructs.

[0084] In some embodiments, the composition is formulated for use as a vaccine. The vaccine composition can be administered by any convenient route, including subcutaneous, oral, intramuscular, intraperitoneal, parenteral, or enteral administration. In some embodiments, the immunogen is administered in a single dose. In some embodiments, the immunogen is administered over multiple doses.

[0085] The pharmaceutical composition can be prepared as an injectable, either as a liquid solution or suspension. A liquid vehicle containing the tau peptide immunogen construct can also be prepared prior to injection. The pharmaceutical composition can be administered in any suitable application mode, such as id, iv, ip, im, intranasal, oral, subcutaneous, etc., and in a suitable delivery device. In certain embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other modes of administration, including oral and intranasal application, can also be prepared.

[0086] The compositions of the invention may comprise an effective amount of one or more peptide immunogens and a pharma- ceutically acceptable carrier. In some embodiments, a suitable dosage unit form of the composition may contain about 0.5 μg to about 1 mg of peptide immunogen per kg of subject body weight. In some embodiments, a suitable dosage unit form of the composition may contain about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 200 μg, about 300 μg, about 400 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, or about 1000 μg of peptide immunogen per kg of subject body weight. In some embodiments, a composition in a suitable dosage form may contain about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, or about 500 μg of peptide immunogen per kg of subject body weight. In some embodiments, a composition in a suitable dosage unit form may contain about 0.5 μg to about 1 mg of peptide immunogen per kg of subject body weight. In some embodiments, a composition in a suitable dosage unit form may contain about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 200 μg, about 300 μg, about 400 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, or about 1000 μg of peptide immunogen. In some embodiments, a composition in a suitable dosage form may contain about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, or about 500 μg of peptide immunogen.

[0087] When delivered in multiple doses, the composition can be divided into appropriate amounts for each administration. In some embodiments, the dose is about 0.2 mg to about 2.5 mg. In some embodiments, the dose is about 1 mg. In some embodiments, the dose is about 1 mg and is administered by injection. In some embodiments, the dose is about 1 mg and is administered intramuscularly. In some embodiments, the administration can be followed by repeated (booster) administrations. Dosage can be optimized according to the age, weight, and overall health of the subject.

[0088] Vaccines containing a mixture of peptide immunogens may provide enhanced immune efficacy in a broader population. In some embodiments, the mixture of peptide immunogens includes Th sites derived from MVF Th and HBsAg Th. In some embodiments, vaccines containing a mixture of peptide immunogens may provide improved immune responses to target antigenic sites.

[0089] The immune response to the Th / target antigen site conjugate can be improved by delivery by entrapment in or on biodegradable microparticles. In some embodiments, peptide immunogens can be encapsulated with or without adjuvants, and such microparticles can carry immune stimulating adjuvants. In some embodiments, the microparticles can be co-administered with peptide immunogens to enhance the immune response.

[0090] immune stimulating complex The present disclosure also relates to pharmaceutical compositions containing tau peptide immunogen constructs in the form of immune stimulating complexes with CpG oligonucleotides. Such immune stimulating complexes are specially adapted to function as adjuvants and peptide immunogen stabilizers. The immune stimulating complexes are in the form of microparticles that can efficiently present tau peptide immunogens to cells of the immune system to generate an immune response. The immune stimulating complexes may be formulated as suspensions for parenteral administration. The immune stimulating complexes may also be formulated in the form of w / o emulsions, as suspensions combined with inorganic salts or in-situ gelling polymers for efficient delivery of tau peptide immunogens to cells of the host's immune system after oral administration.

[0091] The stabilized immunostimulatory complex can be formed by complexing the tau peptide immunogen construct with an anionic molecule, an oligonucleotide, a polynucleotide, or a combination thereof via electrostatic binding. The stabilized immunostimulatory complex can be incorporated into a pharmaceutical composition as an immunogen delivery system.

[0092] In certain embodiments, the tau peptide immunogen construct is designed to contain cationic moieties that are positively charged at a pH ranging from 5.0 to 8.0. The net charge of the cationic moieties of a tau peptide immunogen construct, or mixture of constructs, is calculated by assigning a charge of +1 to each lysine (K), arginine (R), or histidine (H), a charge of -1 to each aspartic acid (D) or glutamic acid (E), and a charge of 0 to other amino acids in the sequence. The charges are summed within the cationic moieties of the tau peptide immunogen construct and expressed as a net average charge. Suitable peptide immunogens have cationic moieties with a net average positive charge of +1. Preferably, the peptide immunogens have a net positive charge in the range of greater than +2. In some embodiments, the cationic moieties of the tau peptide immunogen construct are heterologous spacers. In certain embodiments, the cationic moiety of the tau peptide immunogen construct has a charge of +4 when the spacer sequence is (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 53), or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 54).

[0093] As used herein, an "anionic molecule" refers to any molecule that has a negative charge at a pH ranging from 5.0 to 8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge of the oligomer or polymer is calculated by assigning a -1 charge to each phosphodiester or phosphorothioate group of the oligomer. Suitable anionic oligonucleotides are single-stranded DNA molecules having 8 to 64 nucleotide bases, and the number of repeats of the CpG motif ranges from 1 to 10. Preferably, the CpG immunostimulatory single-stranded DNA molecule contains 18 to 48 nucleotide bases, and the number of repeats of the CpG motif ranges from 3 to 8.

[0094] More preferably, the anionic oligonucleotide has the formula: 5'X 1 CGX 2 3' (wherein C and G are unmethylated, X1 is selected from the group consisting of A (adenine), G (guanine), and T (thymine), and X2 is C (cytosine) or T (thymine). Alternatively, the anionic oligonucleotide is represented by the formula: 5'(X 3 )2CG(X 4 ) 23' (wherein C and G are unmethylated and X 3 is selected from the group consisting of A, T, or G; 4 is C or T). In certain embodiments, the CpG oligonucleotide can be CpG1 (SEQ ID NO: 146), CpG2 (SEQ ID NO: 147), or CpG3 (SEQ ID NO: 148).

[0095] The resulting immunostimulating complex is usually in the form of particles with sizes ranging from 1 to 50 microns, which is a function of many factors including the relative charge stoichiometry and molecular weight of the interacting species. Micronized immunostimulating complexes have the advantage of providing adjuvant and upregulation of specific immune responses in vivo. Furthermore, stabilized immunostimulating complexes are suitable for preparation of pharmaceutical compositions by a variety of processes including water-in-oil emulsions, inorganic salt suspensions, and polymer gels.

[0096] Purpose Peptide immunogens containing the artificial Th epitopes of the present disclosure may be useful in medical and veterinary applications. In some embodiments, the peptide immunogens can be used as vaccines to obtain protective immunity against infectious diseases, immunotherapies to treat disorders resulting from the malfunction of normal physiological processes, immunotherapies to treat cancer, and drugs to intervene in or modify normal physiological processes.

[0097] The artificial Th epitope of the present disclosure can induce immune response when combined with the target B cell epitopes of various microorganisms, proteins or peptides.In some embodiments, the artificial Th epitope of the present disclosure can bind to one target antigen site.In some embodiments, the artificial Th epitope of the present disclosure can bind to two target antigen sites.

[0098] The artificial Th epitopes of the present disclosure can bind to target antigenic sites to prevent and / or treat various diseases and conditions. In some embodiments, the compositions of the present invention can be used to prevent and / or treat neurodegenerative diseases, infectious diseases, arteriosclerosis, prostate cancer, boar taint, animal immunocastration, endometriosis, breast cancer and other gynecological cancers influenced by gonadal steroid hormones, and for male and female contraception. For example, the artificial Th epitopes can bind to antigenic sites in the following proteins: A somatostatin, which promotes growth in livestock. b. IgE to treat allergic diseases. c. The CD4 receptor on Th cells to treat and / or prevent human immunodeficiency virus (HIV) infection and immune disorders. d. Foot and mouth disease (FMD) viral capsid protein to prevent FMD. e. HIV virion epitopes for preventing and treating HIV infection. f. Circumsporozoite antigens of Plasmodium falciparum to prevent and treat malaria. g. CETP to prevent and treat arteriosclerosis. h. Aβ to treat or vaccinate against Alzheimer's disease. i. Alpha-synuclein to treat or vaccinate against Parkinson's disease. j. Tau to treat and vaccinate against tauopathies, including Alzheimer's disease. k. IL-31 to treat atopic dermatitis. l. CGRP to prevent and treat migraines. m.IAPP (amylin) to prevent and treat type 2 diabetes.

[0099] The use of heterologous artificial Th epitopes has been found to be particularly important for targeting proteins involved in neurodegenerative diseases (e.g., Aβ, alpha-synuclein, Tau).Specifically, when administered to a subject, peptide immunogens containing the endogenous Th epitopes of targeted neurodegenerative proteins can cause brain inflammation.In contrast, peptide immunogen constructs containing heterologous artificial Th epitopes that bind to the antigenic site of neurodegenerative proteins do not cause brain inflammation.

[0100] FIG. 2 is a graph showing theoretical results that can be obtained using the Th epitope platform described in this application. The graph shows that peptide immunogen constructs containing B cell epitopes conjugated to Th epitopes have a fast onset time and a C maxに It indicates quick arrival. C max The following examples demonstrate the therapeutic range of Th100 and Th200 peptide immunogens by varying the Th epitope, dosage of peptide immunogen construct, and adjuvant used. max , duration of action, onset time, and t max indicates that all can be controlled and / or adjusted.

[0101] Amyloid beta Aβ peptides are thought to be central to the development and progression of Alzheimer's disease. Toxic forms of Aβ oligomers and Aβ fibrils are suggested to be involved in the death of synapses and neurons that lead to the pathology of Alzheimer's disease and dementia. A successful disease-modifying therapy for Alzheimer's disease may include products that affect the disposition of Aβ in the brain.

[0102] The peptide immunogens of the present disclosure may include a Th cell epitope and an Aβ targeting peptide. In some embodiments, the Th cell epitope is Th1 or Th2. In some embodiments, the peptide immunogens may include Th1 and Th2. The Aβ targeting peptide, or the B cell epitope, may be Aβ. 1-14 , Aβ 1-16 , Aβ1-28 , Aβ 17-42 , or Aβ 1-42 In some embodiments, the Aβ targeting peptide can be 1-14 As used herein, Aβ x-y The term refers to the Aβ sequence from amino acid x to amino acid y of the full-length wild-type Aβ protein.

[0103] The peptide immunogens of the present disclosure may include multiple Aβ targeting peptides. In some embodiments, the peptide immunogens may include two Aβ targeting peptides. In some embodiments, the peptide immunogens may include one Aβ targeting peptide. 1-14 and one Aβ 1-42 In some embodiments, the peptide immunogen may comprise two Aβ 1-14 In some embodiments, the peptide immunogen may comprise a targeting peptide. 1-14 The antibodies can contain targeting peptides, each binding to a different Th cell epitope as a chimeric peptide.

[0104] This disclosure provides two Aβ 1-14 Aβ containing targeting peptides 1-14 Peptide vaccines are also provided, each linked to a different Th cell epitope as a chimeric peptide. 1-14 The peptide can be formulated in a Th1-biased delivery system to minimize T cell inflammatory reactivity. 1-14 The peptides can be formulated in a Th2-biased delivery system to minimize T cell inflammatory reactivity.

[0105] Specific Embodiments (1) A promiscuous artificial T helper cell (Th) epitope selected from the group consisting of SEQ ID NOs: 32 to 52.

[0106] (2) A peptide immunogen construct having the formula: (A) n -(Target antigen site)-(B) o -(Th)m -(A) n -X or (A) n -(Th) m -(B) o -(Target antigen site)-(A) n -X or (A) n -(Th) m -(B) o -(Target antigen site)-(B) o -(Th) m -(A) n -X or {(A) n -(Th) p -(B) o -(Target antigen site)-(B) o -(Th) p -(A) n -X} m (In the formula, each A is independently an amino acid; each B is independently a heterologous spacer; Each Th is independently a promiscuous artificial Th epitope according to (1); the target antigenic site is a B cell epitope derived from a foreign antigenic protein, a self antigenic protein, or an immunologically reactive analog thereof; X is an amino acid, α-COOH, or α-CONH2; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 1, 2, 3, or 4; o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 0, 1, 2, 3, or 4).

[0107] (3) The peptide immunogen construct described in (2), wherein the target antigen site is a B cell epitope derived from a foreign antigen protein selected from the group consisting of foot-and-mouth disease (FMD) capsid protein, glycoprotein derived from porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), human immunodeficiency virus (HIV), and herpes simplex virus (HSV).

[0108] (4) The target antigen site is (a) an Aβ peptide having the amino acid sequence of SEQ ID NO: 56, 57, 58, 59, or 60; (b) alpha-synpeptide having the amino acid sequence of SEQ ID NO: 61; (c) an IgE EMPD peptide having the amino acid sequence of SEQ ID NO: 62; (d) a Tau peptide having the amino acid sequence of SEQ ID NO: 63, 69, 70, or 71; (e) an IL-31 peptide having the amino acid sequence of SEQ ID NO: 64 or 72, and (f) an IL-6 peptide having the amino acid sequence of SEQ ID NO: 145 The peptide immunogen construct according to (2), wherein the B cell epitope is derived from an autoantigen protein selected from the group consisting of:

[0109] (5) The peptide immunogen construct of (2), wherein the heterologous spacer of component B is selected from the group consisting of amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 53), Lys-Lys-Lys-εNLys (SEQ ID NO: 54), Gly-Gly, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 55), and any combination thereof.

[0110] (6) The peptide immunogen construct of (2), wherein the heterologous spacer is selected from the group consisting of (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 53), and Lys-Lys-Lys-εNLys (SEQ ID NO: 54).

[0111] (7) A pharmaceutical composition comprising the peptide immunogen construct according to (2).

[0112] (8) A method for preventing and / or treating a disease, condition, or illness in a subject, comprising administering to the subject a pharma- ceutical effective amount of the pharmaceutical composition according to (7).

[0113] (9) The method according to (8), wherein the target antigen site is a B cell epitope derived from a foreign antigen protein selected from the group consisting of foot-and-mouth disease (FMD) capsid protein, glycoprotein derived from porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), human immunodeficiency virus (HIV), and herpes simplex virus (HSV).

[0114] (10) The target antigen site is (a) an Aβ peptide having the amino acid sequence of SEQ ID NO: 56, 57, 58, 59, or 60; (b) alpha-synpeptide having the amino acid sequence of SEQ ID NO: 61; (c) an IgE EMPD peptide having the amino acid sequence of SEQ ID NO: 62; (d) a Tau peptide having the amino acid sequence of SEQ ID NO: 63, 69, 70, or 71; (e) an IL-31 peptide having the amino acid sequence of SEQ ID NO: 64 or 72, and (f) an IL-6 peptide having the amino acid sequence of SEQ ID NO: 145 The method according to (8), wherein the B cell epitope is derived from an autoantigen protein selected from the group consisting of:

[0115] (11) A peptide immunogen construct represented by the following formula: (A) n -(Target antigen site)-(B) o -(Th) m -(A) n -X or (A) n -(Th) m -(B) o -(Target antigen site)-(A) n -X or (A) n -(Th) m -(B) o -(Target antigen site)-(B) o -(Th) m -(A) n -X or {(A) n -(Th) p -(B) o -(Target antigen site)-(B) o -(Th) p -(A) n -X} m (In the formula, each A is independently an amino acid; each B is independently a heterologous spacer; each Th is independently a promiscuous artificial Th epitope selected from the group consisting of SEQ ID NOs: 1 to 52; the target antigenic site is a CTL epitope, a tumor-associated carbohydrate antigen (TACA), a B-cell epitope derived from a neoantigen, a small molecule drug, or an immunologically reactive analog thereof; X is an amino acid, α-COOH, or α-CONH2; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 1, 2, 3, or 4; o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 0, 1, 2, 3, or 4).

[0116] (12) The peptide immunogen according to (11), wherein the target antigen site is a CTL epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 76 to 144.

[0117] (13) The peptide immunogen according to (12), wherein the target antigen site is an HIV-derived CTL epitope selected from the group consisting of SEQ ID NOs: 76 to 82.

[0118] (14) The peptide immunogen according to (12), wherein the target antigen site is an HSV-derived CTL epitope selected from the group consisting of SEQ ID NOs: 83 to 106.

[0119] (15) The peptide immunogen according to (12), wherein the target antigen site is a CTL epitope derived from FMDV selected from the group consisting of SEQ ID NOs: 107 to 123.

[0120] (16) The peptide immunogen according to (12), wherein the target antigen site is a CTL epitope derived from PRRSV selected from the group consisting of SEQ ID NOs: 124 to 142.

[0121] (17) The peptide immunogen according to (12), wherein the target antigen site is a CSFV-derived CTL epitope selected from the group consisting of SEQ ID NOs: 143 to 144.

[0122] (18) The target antigen site is GD3, GD2, Globo-H, GM2, Fucosyl GM1, GM2, PSA, Le y , Le x , SLe x , SLe a , Tn, TF, and STn.

[0123] (19) The peptide immunogen according to (11), wherein the target antigen site is a B cell epitope derived from a neoantigen selected from the group consisting of SEQ ID NOs: 73 to 75.

[0124] (20) The peptide immunogen according to (11), wherein the target antigen site is a small molecule drug.

[0125] (21) The peptide immunogen construct according to (11), wherein the heterologous spacer of component B is selected from the group consisting of amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 53), Lys-Lys-Lys-εNLys (SEQ ID NO: 54), Gly-Gly, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 55), and any combination thereof.

[0126] (22) The peptide immunogen construct according to (11), wherein the heterologous spacer is selected from the group consisting of (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 53), and Lys-Lys-Lys-εNLys (SEQ ID NO: 54).

[0127] (23) A pharmaceutical composition comprising the peptide immunogen construct according to (11).

[0128] (24) A method for preventing and / or treating a disease, condition, or illness in a subject, comprising administering to the subject a pharma- ceutical effective amount of the pharmaceutical composition according to (23).

[0129] (25) The method according to (24), wherein the disease, condition, or illness is HIV, and the target antigen site is an HIV-derived CTL epitope selected from the group consisting of SEQ ID NOs: 76 to 82.

[0130] (26) The method according to (24), wherein the disease, condition, or illness is HSV, and the target antigen site is an HSV-derived CTL epitope selected from the group consisting of SEQ ID NOs: 83 to 106.

[0131] (27) The method according to (24), wherein the disease, condition, or illness is FMDV, and the target antigen site is an FMDV-derived CTL epitope selected from the group consisting of SEQ ID NOs: 107 to 123.

[0132] (28) The method according to (24), wherein the disease, condition, or illness is PRRSV, and the target antigen site is a PRRSV-derived CTL epitope selected from the group consisting of SEQ ID NOs: 124 to 142.

[0133] (29) The method according to (24), wherein the disease, condition, or illness is CSFV, and the target antigen site is a CSFV-derived CTL epitope selected from the group consisting of SEQ ID NOs: 143 to 144.

[0134] (30) The method according to (24), wherein the disease, condition, or illness is CSFV, and the target antigen site is a CSFV-derived CTL epitope selected from the group consisting of SEQ ID NOs: 143 to 144.

[0135] (31) The method according to (24), wherein the disease, condition, or condition is cancer, and the target antigen site is a TACA selected from the group consisting of GD3, GD2, Globo-H, GM2, Fucosyl GM1, GM2, PSA, Ley, Lex, SLex, SLea, Tn, TF, and STn.

[0136] (32) The method according to (24), wherein the disease, condition, or illness is cancer, and the target antigenic site is a B cell epitope derived from a neoantigen selected from the group consisting of SEQ ID NOs: 73 to 75.

[0137] (33) A method for modulating an immune response in a subject, comprising: (a) preparing a plurality of peptide immunogen constructs according to (11), wherein the target antigenic site remains constant and the Th epitope on each peptide immunogen construct is different; (b) preparing a plurality of pharmaceutical compositions, each of which comprises one of the peptide immunogen constructs prepared in (a) and a pharma- ceutically acceptable adjuvant or carrier; (c) administering to a different subject each of the pharmaceutical compositions prepared in (b); (d) monitoring the immune response of each of said subjects; and (e) selecting said pharmaceutical composition that generates a desired immune response; The method comprising:

[0138] (34) The method according to (33), wherein the pharma- ceutically acceptable adjuvant or carrier in each of the pharmaceutical compositions is the same. (35) The method according to (33), wherein the pharma- ceutically acceptable adjuvants or carriers in each of the pharmaceutical compositions are different. EXAMPLES

[0139] Example 1 Preparation of peptides and peptide immunogen constructs Peptides, including the peptide immunogen constructs, were synthesized using automated solid-phase synthesis, purified by preparative HPLC, and characterized by matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry, amino acid analysis, and reverse-phase HPLC.

[0140] The Aβ vaccine (UB-311) contains two peptide immunogens, each of which targets the N-terminal Aβ 1-14 The peptide immunogens are synthetically linked via amino acid spacers to different Th cell epitope peptides (UBITh® epitopes) derived from two pathogen proteins, hepatitis B surface antigen and measles virus fusion protein. Specifically, the peptide immunogens linked to the measles virus fusion protein are Aβ 1-14 -εK-KKK-MvF5 Th (SEQ ID NO: 67), and the peptide immunogen bound to the hepatitis B surface antigen is Aβ 1-14 -εK-HBsAg3Th (SEQ ID NO: 68).

[0141] UB-311 was formulated in an alum-containing Th2-biased delivery system and administered at an equimolar ratio to the peptide Aβ. 1-14 -εK-HBsAg3 and Aβ 1-14-εK-KKK-MvF5 Th. The two Aβ immunogens were mixed with polyanionic CpG oligodeoxynucleotides (ODN) to form stable immune stimulatory complexes in micron-sized particles. Aluminum inorganic salt (ADJU-PHOS®) was added to the final formulation along with sodium chloride for isotonicity and 0.25% 2-phenoxyethanol as a preservative.

[0142] The sequences of some exemplary target antigenic sites (B cell epitopes and CTL epitopes) are shown in Tables 3A and 3B, respectively. Aβ is a target antigenic site that is covalently linked to a Th epitope. 1-14 , rat IL-6 72-82 , and IgE-EMPD 1-39 The sequences of some exemplary peptide immunogen constructs containing: α-synuclein covalently linked to various Th epitopes are shown in Table 4. 111-132 The sequences of peptide immunogen constructs containing IgE-EMPD covalently linked to various Th epitopes are shown in Table 5. G1-C39 The sequences of peptide immunogen constructs containing human IL-6 covalently linked to various Th epitopes are shown in Table 6. 73-83 The sequences of peptide immunogen constructs containing a dipeptide repeat (DPR) sequence covalently linked to UBITh®1 are shown in Table 7. The sequences of peptide immunogen constructs containing LHRH covalently linked to various Th epitopes are shown in Table 10.

[0143] Example 2 Evaluation of heterologous T helper epitopes derived from pathogens and alpha-synuclein enhancing the immunogenicity of selected B epitope peptides 111-132 , IgE EMPD 1-39 , and IL-6 73-83 Their incorporation into the design of peptide immunogen constructs a. Peptide immunogen synthesis Three short B cell epitope peptides from alpha-synuclein (AA 111-132; SEQ ID NO: 61); IgE EMPD (AA 1-39; SEQ ID NO: 62); and IL-6 (AA 73-83; SEQ ID NO: 145), which have been extensively characterized for their functional properties, were used as representative target antigenic sites. These three B cell epitopes were made into peptide immunogen constructs according to the formula shown below to evaluate the ability of representative promiscuous artificial Th epitopes (selected from SEQ ID NOs: 1-52) to render the three individual target antigenic sites immunogenic. (Th) m -(B) o -(Target antigen site)-X (In the formula, Th is selected from the artificial Th epitopes disclosed herein, m is 1; (B) o is a spacer having the amino acid sequence of SEQ ID NO: 53 or 54, the target antigenic site is a B cell epitope of SEQ ID NO: 61, 62, or 145; X was the amino acid -CONH2).

[0144] The amino acid sequences of the generated α-synuclein, IgE EMPD, and IL-6 peptide immunogen constructs are shown in Tables 5, 6, and 7, respectively.

[0145] b. Formulations Containing Peptide Immunogen Constructs and Immunization Representative immunogenicity studies were performed in guinea pigs to evaluate the relative efficacy of each of the heterologous T helper epitopes shown in Table 1. After the various α-synuclein, IgE EMPD, and IL-6 peptide immunogens were generated, constructs containing the same Th epitope sequence were mixed together in a 1:1:1 ratio as shown in Table 8. For example, to prepare Formulation No. 1 shown in Table 8, α-synuclein, IgE EMPD, and IL-6 constructs containing the UBITH® 1 epitope (SEQ ID NOs: 149, 178, and 207) were mixed together. The mixture of α-synuclein, IgE EMPD, and IL-6 peptide immunogen constructs was mixed with the adjuvant MONTANIDE ISA50V2 and then formulated as a stabilized immune stimulatory complex using CpG3 oligonucleotides. Each of the 29 formulations shown in Table 8 contained a total of 135 μg of peptide (45 μg per peptide) in a volume of 0.5 mL.

[0146] The formulations were administered to guinea pigs (3 per group) at 0, 3, and 6 weeks post-primary immunization (wpi) by intramuscular (im) injection. Serum samples were collected at 0, 3, 6, and 8 wpi to assess antibody titer levels.

[0147] C. Immunogenicity results Results obtained 8 weeks after primary immunization (8 wpi) were used to evaluate different α-synuclein ( FIG. 3 top panel), IgE ( FIG. 3 bottom panel), and IL-6 (Table 15) peptide immunogen constructs. Graphs containing immunogenicity data obtained throughout the α-synuclein study are shown in FIG. 4A; IgE-EMPD is shown in FIG. 5; IL-6 is shown in FIG. 6.

[0148] All Th epitopes were able to enhance the immunogenicity of three short B cell epitope peptides to various degrees. Specifically, Th epitopes: KKKMvF3 Th (SEQ ID NO: 13), Clostridium tetani TT2 Th (SEQ ID NO: 36), EBV EBNA-1 Th (SEQ ID NO: 42), MvF5 Th;UBITh®1 (SEQ ID NO: 17), EBV BHRF1 Th (SEQ ID NO: 41), MvF4 Th;UBITh®3 (SEQ ID NO: 16), and cholera toxin Th (SEQ ID NO: 33) enhanced the immunogenicity of α-synuclein peptide (SEQ ID NO: 61) more than other Th epitopes (Figure 3 upper panel and Figure 4A). These peptide immunogen constructs are represented in Table 5 as formulation numbers 13, 22, 21, 01, 19, 03, and 11, respectively.

[0149] For the IgE EMPD peptide (SEQ ID NO:62), the Clostridium tetani TT4 Th (SEQ ID NO:38), UBITh®1 (SEQ ID NO:17), UBITh®3 (SEQ ID NO:16), HBsAg1 Th;SSAL2 Th2 (SEQ ID NO:24), KKKMvF3 Th (SEQ ID NO:13), Clostridium tetani TT2 Th (SEQ ID NO:36), cholera toxin Th (SEQ ID NO:33), EBV BHRF1 Th (SEQ ID NO:41), and HBsAg3 Th;UBITH®2 (SEQ ID NO:28) Th epitopes enhanced the immunogenicity of IgE EMPD over other Th epitopes (Figure 3, bottom panel and Figure 5). These peptide immunogen constructs are represented in Table 6 as formulation numbers 24, 01, 03, 14, 13, 22, 11, 19, and 02, respectively.

[0150] IL-6 73-83Of the cyclic peptides (SEQ ID NO: 145), the Th epitopes of HBsAg3 Th; UBITH®2 (SEQ ID NO: 28), UBITh®1 (SEQ ID NO: 17), UBITh®3 (SEQ ID NO: 16), Clostridium tetani TT1 Th (SEQ ID NO: 34), and Clostridium tetani TT4 Th (SEQ ID NO: 38) were found to be the most potent in enhancing the resulting immunogenicity of IL-6 (Table 15 and Figure 6). These peptide immunogen constructs are represented in Table 7 as formulation numbers 02, 01, 03, 20, and 24, respectively.

[0151] These results indicate that different immunogenicity against a single target B cell epitope can be obtained when using different artificial Th epitopes as disclosed herein. Careful calibration of the immunogenicity of each peptide immunogen construct in different species, including primates, is necessary to ensure success in the selection of the final Th peptide and development of the final vaccine formulation.

[0152] cC max Speed ​​up to Further analysis of immunogenicity data for α-synuclein peptide immunogen constructs covalently linked to different Th epitopes revealed that specific C max It becomes clear that the C-terminal end of the guinea pigs can be achieved at different rates. Figure 4B contains a subset of the immunogenicity data reported in Figure 4A. Specifically, guinea pigs immunized with formulations no. 13 and 21, which contain Th epitopes of KKKMvF3 Th (SEQ ID NO: 13) and EBV EBNA-1 Th (SEQ ID NO: 42) covalently linked to an α-synuclein peptide (SEQ ID NO: 61), achieved the same C-terminal end of the guinea pigs up to 8 wpi. max In particular, α-synuclein peptide immunogen constructs containing KKKMvF3 Th (SEQ ID NO: 13) achieve a faster C than α-synuclein peptide immunogen constructs containing EBV EBNA-1Th (SEQ ID NO: 42). maxSimilarly, α-synuclein peptide immunogen constructs containing UBITh®1 (SEQ ID NO: 17) reached a C faster than α-synuclein peptide immunogen constructs containing EBV BHRF1 Th (SEQ ID NO: 41). max the α-synuclein peptide immunogen construct containing Clostridium tetani TT1 Th (SEQ ID NO: 34) reached C faster than the α-synuclein peptide immunogen construct containing influenza MP1_1 Th (SEQ ID NO: 48). max The results shown in Figure 4B indicate that the selection of Th epitopes leads to increased antibody titers. max This shows that the rate at which the

[0153] d.Summary The results of this experiment showed that the immune response (antibody titers, C max These results indicate that the response time (including response time, onset of antibody production, duration of response, etc.) can be modulated by the choice of Th epitope that is chemically linked to the B cell epitope. Thus, specific immune responses to target antigenic sites can be engineered by varying the Th epitope that is conjugated to the B cell epitope in a peptide immunogen construct, which may facilitate tailoring personalized medicine to the individual characteristics of any patient or subject.

[0154] Example 3 The immunogenicity of peptide immunogen constructs may vary depending on the length of the B cell epitope sequence. Immunization and evaluation of three dipeptide repeat (DPR) peptide immunogen constructs is described in detail below.

[0155] A. Vaccination and serum collection Three DPR peptide immunogen constructs were generated having amino acid sequences of SEQ ID NOs: 236, 237, and 238, as shown in Table 9. Each peptide immunogen was formulated with MONTANIDE™ ISA51 and CpG and immunized into guinea pigs (3 guinea pigs per group) at a dose of 400 μg / ml for the primary immunization and at booster doses of 100 μg / ml at 3, 6, 9, and 12 weeks post-injection (WPI).

[0156] b. Assessment of antibody titers An ELISA assay was performed to evaluate the immunogenicity of the designer DPR peptide immunogen constructs. The DPR B cell epitope peptide or peptide immunogen constructs were used to coat plate wells that served as target peptides. Guinea pig immune serum was diluted from 1:100 to 1:100,000 in a 10-fold serial dilution. Cutoff A 450 The titers of the tested sera, expressed as Log10, were calculated by linear regression analysis of A450nm with A set to 0.5. All peptide immunogens induced strong immunogenic titers against the B epitope peptide coated on the plate wells.

[0157] c. Peptide-based ELISA test for antibody specificity analysis An ELISA assay for evaluating immune serum samples was developed as follows.

[0158] Wells of a 96-well plate were individually coated with 100 μL of the same DPR peptide immunogen construct (i.e., SEQ ID NO: 236, 237, or 238) used to immunize the animals at 2 μg / mL in 10 mM NaHCO3 buffer pH 9.5 for 1 hour at 37°C.

[0159] d. Evaluation of antibody reactivity against DPR by ELISA test The peptide-coated wells were incubated with 250 μL of 3% gelatin by weight in PBS at 37° C. for 1 hour to block non-specific protein binding sites, followed by washing three times with PBS containing 0.05% TWEEN® 20 by volume and drying. The serum to be analyzed was diluted 1:20 (unless otherwise stated) with PBS containing 20% ​​normal goat serum by volume, 1% gelatin by weight, and 0.05% TWEEN® 20 by volume. One hundred microliters (100 μL) of the diluted specimen (e.g., serum, plasma) was added to each well and reacted at 37° C. for 60 minutes. The wells were then washed six times with 0.05% TWEEN® 20 by volume in PBS to remove unbound antibodies. Horseradish peroxidase (HRP)-conjugated species (e.g., mouse, guinea pig, or human)-specific goat anti-IgG was used as a labeled tracer to bind to the antibody / peptide antigen complex formed in the positive wells. One hundred microliters of peroxidase-labeled goat anti-IgG in 1% normal goat serum with 0.05% TWEEN® 20 (volume) in PBS at a pre-titrated optimal dilution was added to each well and incubated for an additional 30 minutes at 37°C. The wells were washed six times with 0.05% TWEEN® 20 (volume) in PBS to remove unbound antibody and reacted with 100 μL of a substrate mixture containing 0.04% by weight 3',3',5',5'-tetramethylbenzidine (TMB) and 0.12% by volume hydrogen peroxide in sodium citrate buffer for an additional 15 minutes. This substrate mixture was used to detect the peroxidase label by forming a colored product. The reaction was stopped by adding 100 μL of 1.0 M H2SO4 and the absorbance at 450 nm (A450) was determined. For the determination of antibody titers of immunized animals receiving various DPR-derived peptide immunogens, 10-fold serial dilutions of sera from 1:100 to 1:10,000 were tested and the titers of the test sera, expressed as Log10, were calculated by linear regression analysis of A450 with a cutoff A450 set at 0.5.

[0160] e. Immunogenicity assessment Pre-immune and immune serum samples from animals were collected according to the experimental immunization protocol and heated at 56°C for 30 minutes to inactivate serum complement factors. After administration of pharmaceutical compositions containing the DPR peptide immunogen constructs, blood samples were obtained according to the protocol to assess immunogenicity against the specific target site(s). Serially diluted sera were tested and positive titers were calculated as the Log of the inverse dilution. 10 The immunogenicity of certain pharmaceutical compositions was evaluated for their ability to elicit high titer B cell antibody responses against a desired epitope specificity within the target antigen while maintaining low to negligible antibody reactivity against the Th epitopes utilized to provide the desired enhanced B cell response.

[0161] f. Immunoassay of DPR levels in immune serum of mice Serum DPR levels of mice administered DPR-derived peptide immunogens were measured by sandwich ELISA (Cloud-clon, SEB222Mu) using anti-DPR antibody as capture antibody and biotin-labeled anti-DPR antibody as detection antibody. Briefly, antibodies were immobilized on 96-well plates at 100ng / well in coating buffer (15mM Na2CO3, 35mM NaHCO3, pH9.6) and incubated overnight at 4°C. The coated wells were blocked with 200μL / well of assay diluent (0.5% BSA, 0.05% TWEEN®-20, 0.02% ProClin300 in PBS) for 1 hour at room temperature. The plates were washed three times with 200μL / well of washing buffer (PBS with 0.05% TWEEN®-20). Purified recombinant DPR was used to generate a standard curve (ranging from 156 to 1,250 ng / mL by two-fold serial dilution) in assay diluent containing 5% mouse serum. Fifty microliters (50 μL) of diluted serum (1:20) and standards were added to the coated wells. Incubation was carried out at room temperature for 1 h. All wells were aspirated and washed six times with 200 μL / well of wash buffer. Captured DPR was incubated with 100 μL of detection antibody solution (biotin-labeled HP6029 at 50 ng / ml in assay diluent) for 1 h at room temperature. Bound biotin-HP6029 was then detected using streptavidin poly-HRP (1:10,000 dilution, Thermo Pierce) for 1 h (100 μL / well). All wells were aspirated and washed six times with 200 μL / well wash buffer, and the reaction was stopped by adding 100 μL / well 1M H2SO4. A standard curve was generated by using SoftMax Pro software (Molecular Devices) generating a four parameter logistic curve fit and used to calculate the concentration of DPR in all tested samples. Data were compared using Student's t-test by using Prism software.

[0162] g. Purification of anti-DPR antibody Anti-DPR antibodies were purified from serum collected 3 to 15 weeks after injection (WPI) of guinea pigs or mice immunized with DPR peptide immunogen constructs containing peptides of different sequences (SEQ ID NO: 236, 237, or 238) by using an affinity column (Thermo Scientific, Rockford). Briefly, after equilibration with buffer (0.1 M phosphate and 0.15 M sodium chloride, pH 7.2), 400 μL of serum was added to a Nab Protein G spin column, mixed end-over-end for 10 min, and centrifuged at 5,800 x g for 1 min. The column was washed three times with binding buffer (400 μL). Then, elution buffer (400 μL, 0.1 M glycine pH 2.0) was added to the spin column to elute the antibodies after centrifugation at 5,800 x g for 1 min. The eluted antibodies were mixed with neutralization buffer (400 μL, 0.1 M Tris pH 8.0) and the concentration of these purified antibodies was measured by using Nan-Drop at OD280 with BSA (bovine serum albumin) as a standard.

[0163] h.Results Immunogenicity titers to the DPR peptide or peptide immunogens from immunized guinea pig sera were assessed by ELISA.

[0164] Figure 7 shows the profile of antisera over a 15-week period from guinea pigs immunized with three different DPR peptide immunogen constructs. Antisera from guinea pigs at 0, 3, 6, 9, 12, and 15 wpi were diluted in 10-fold serial dilutions. ELISA plates were coated with DPR peptide or peptide immunogen. Cutoff A 450 Linear regression analysis of A450nm with A set to 0.5 gave Log 10 The titers of the test sera, expressed as .times. ...

[0165] Next, the ELISA data of the DPR peptide immunogen constructs containing polyGA peptides (SEQ ID NOs: 236, 237, and 238) were plotted as a graph shown in FIG. 7, where the same peptide immunogens used to immunize the animals were bound to the ELISA plate for analysis. All DPR immunogen constructs were highly immunogenic to the corresponding DPR peptide or peptide immunogen. The ELISA results showed that no detectable antibody titers were observed in each group prior to immunization at week 0. The data indicate that the length of the dipeptide repeat in the DPR peptide immunogen constructs may have a moderate effect on antibody titers. Specifically, as shown in FIG. 7, the polyGA 10, 15, and 25 repeat constructs (SEQ ID NOs: 236, 237, and 238, respectively) have different immunogenicity compared to each other.

[0166] Interestingly, the length of the B cell epitope peptide can have an impact on the immunogenic profile of the peptide immunogen construct. 10 While the immunogenicity of the peptide immunogen construct (SEQ ID NO: 236) steadily increases over time with regular boosts, FIG. 7 (center graph) shows that the immunogenicity of the peptide immunogen construct (SEQ ID NO: 236) steadily increases over time with regular boosts, 15 FIG. 7 (right graph) shows that the immunogenicity of the peptide immunogen construct (SEQ ID NO: 237) peaks at approximately 9 wpi before declining. 25 1 shows that the immunogenicity of a peptide immunogen construct (SEQ ID NO: 238) increases steadily over time with periodic boosting.

[0167] The results in FIG. 7 show that immune responses can be influenced depending on the length of the B cell epitope used in the peptide immunogen construct.

[0168] i. Overview The results of this study demonstrated that the immune response (antibody titers, C maxThese results show that the length of the B cell epitope used in the peptide immunogen construct (including the length of the B cell epitope, the onset of antibody production, the duration of the response, etc.) can be regulated by the length of the B cell epitope used in the peptide immunogen construct. Thus, a specific immune response to a target antigenic site can be engineered by varying the length of the B cell epitope in the peptide immunogen construct, which may facilitate tailoring personalized medicine to the individual characteristics of any patient or subject.

[0169] Example 4 The immunogenicity of peptide immunogen constructs may vary depending on the amount of peptide administered and the administration regimen. Immunization and evaluation of various doses, as well as administration schedules of the peptide immunogen constructs, are described in detail below.

[0170] a.UB-311 vaccine (Aβ peptide immunogen) The Aβ vaccine (UB-311) contains two peptide immunogens, each of which targets the N-terminal Aβ 1-14 The peptide immunogens are synthetically linked via amino acid spacers to different Th cell epitope peptides (UBITh® epitopes) derived from two pathogen proteins, hepatitis B surface antigen and measles virus fusion protein. Specifically, the peptide immunogens linked to the measles virus fusion protein are Aβ 1-14 -εK-KKK-MvF5 Th (SEQ ID NO: 67), and the peptide immunogen bound to the hepatitis B surface antigen is Aβ 1-14 -εK-HBsAg3Th (SEQ ID NO: 68).

[0171] UB-311 was formulated in an alum-containing Th2-biased delivery system and administered at an equimolar ratio to the peptide Aβ. 1-14 -εK-HBsAg3 and Aβ 1-14-εK-KKK-MvF5 Th. The two Aβ immunogens were mixed with polyanionic CpG oligodeoxynucleotides (ODN) to form stable immune stimulatory complexes in micron-sized particles. Aluminum inorganic salt (ADJU-PHOS®) was added to the final formulation along with sodium chloride for isotonicity and 0.25% 2-phenoxyethanol as a preservative.

[0172] b. Different doses of UB-311 in guinea pigs Guinea pigs were administered the Aβ vaccine (UB-311) at doses containing 0, 1, 3, 10, 30, 100, 300, 600, and 1,000 μg of total peptide immunogen construct at 0, 3, and 6 wpi. Serum samples were collected at 0, 3, 5, 7, and 9 wpi to assess antibody titers.

[0173] Figure 8 is a graph showing the antibody titer results obtained for each immunization dose. The results show that the amount of peptide used for immunization can have a significant impact on the antibody titer, but the optimal technical effect should be evaluated for each dose. Specifically, Figure 8 shows that increasing the dose of administered peptide from 0 μg to 600 μg directly corresponds to an increase in immunogenicity. However, when 1,000 μg of peptide immunogen is administered, the immunogenicity can be substantially reduced compared to lower doses of peptide immunogen constructs (300 μg and 600 μg compared to 1,000 μg). Thus, the optimal immunogenicity obtained by peptide immunogen constructs is not linear and should be carefully evaluated for each peptide immunogen construct.

[0174] c. Different dosing schedules of UB-311 in guinea pigs can affect immunogenicity The dosing regimen of the Aβ vaccine (UB-311) was evaluated to determine whether the amount of peptide immunogen construct administered as the priming and boosting doses could affect the overall immunogenicity of the composition.

[0175] The UB-311 vaccine was administered to guinea pigs at 0, 3, and 6 wpi with different prime and boost doses administered. Specifically, a first group of animals was primed with a 100 μg dose of UB-311 at 0 weeks wpi and boosted with two doses of 400 μg of UB-311, while a second group of animals was primed with a 400 μg dose of UB-311 at 0 weeks wpi and boosted with two doses of 100 μg of UB-311. The results of this experiment are shown in FIG. 9.

[0176] FIG. 9 shows that the dosing regimen improved the immunogenicity (C max Specifically, animals primed with a low dose (100 μg UB-311) and boosted with a high dose (400 μg UB-311) showed a higher and longer C than animals primed with a high dose (400 μg UB-311) and boosted with a low dose (100 μg UB-311). max was achieved.

[0177] The results of this study indicate that the dosing regimen can affect the immunogenicity of peptide immunogen constructs.

[0178] Figure 10 provides a further example where dosing regimens can affect the immunogenicity of peptide immunogen constructs. Specifically, Figure 10 (top panel) shows anti-Aβ by ELISA obtained after immunization of human subjects with 300 μg of Aβ vaccine (UB-311) in a 3-month boosting regimen (top panel) or a 6-month boosting regimen (bottom panel) with Aβ vaccine (UB-311). 1-28 Antibody titers are shown, with the boxed portion of each graph highlighting the average titer for all human subjects in the study.

[0179] The results in FIG. 10 show that different immunogenicity profiles can be achieved depending on the dosing regimen provided to a subject.

[0180] d. Different administration schedules of LHRH peptide immunogen constructs in rats Dosage regimens of formulations containing different amounts of LHRH peptide immunogen constructs were evaluated to determine whether the total amount of peptide immunogen construct may affect the immunogenicity of the formulation.

[0181] Specifically, three rats were immunized with an LHRH composition containing the three peptides shown in Table 10. A first group of rats was immunized with 100 μg of the three LHRH peptide immunogens, while a second group of rats was immunized with 300 μg of the three LHRH peptide immunogens. Immunogenicity and testosterone concentrations were evaluated and are reported in Figures 11A and 11B.

[0182] Figure 11A shows the antibody titers and testosterone concentrations obtained after immunizing rats with a 100 μg amount of the LHRH formulation. Figure 11B shows the antibody titers and testosterone concentrations obtained after immunizing rats with a 300 μg amount of the LHRH formulation. Figures 11A and 11B show that for the LHRH peptide immunogen construct, the high dose of 300 μg results in higher anti-LHRH titers and a higher reduction in testosterone concentrations compared to the low dose of the LHRH peptide immunogen construct.

[0183] Thus, the results in Figures 11A and 11B demonstrate that there is a direct correlation between the dosage level of the peptide immunogen construct and the technical effect achieved.

[0184] e.Overview The results of this experiment showed that the immune response (antibody titers, C max These results indicate that the response time (including the onset of antibody production, duration of response, etc.) can be modulated by using different dosing regimens. Thus, a specific immune response to a target antigenic site can be engineered by varying the patient's or subject's dosing regimen, which may facilitate tailoring personalized medicine to the individual characteristics of any patient or subject.

[0185] Example 5 The immunogenicity of peptide immunogen constructs may vary depending on the adjuvant used Immunization and evaluation of IL-6, IgE EMPD, and LHRH peptide immunogen constructs formulated with different adjuvants were evaluated as described below.

[0186] a. IL-6 peptide immunogen constructs The immunogenicity of formulations containing IL-6 peptide immunogen constructs utilizing different adjuvants was evaluated. POC studies in CIA rats demonstrated the designed peptide immunogen constructs with high immunogenicity and therapeutic efficacy against IL-6-induced pathogenesis implying potential immunotherapeutic applications in rheumatoid arthritis and other autoimmune diseases. The following studies focused on optimizing the peptide immunogen constructs and selecting adjuvants as well as dose determination in CIA Lewis rats.

[0187] The same peptide immunogen (SEQ ID NO: 243) and MONTANIDE ISA51 and ADJU-PHOS as different adjuvants, each formulated with CpG, were evaluated in a rat CIA immunization study. Five rats were assigned to each of the five groups, receiving one of the two adjuvant formulations, for a total of 10 groups receiving these two different adjuvants. All animals in the treatment groups were injected with different doses of 5, 15, 45, 150 μg in 0.5 ml by im route on days -7, 7, 14, 21, and 28 for priming and boosting, and clinical observations were performed until day 35. Two different adjuvant placebo groups without peptide immunogen were injected with only the adjuvant vehicle in the formulation.

[0188] Anti-IL-6 titers were measured by ELISA against rat IL-6 recombinant protein coated in plate wells. Results showed that none of the two placebo groups injected with the two different adjuvant vehicles had detectable anti-IL-6 antibody titers, while all treatment groups immunized with the IL-6 immunogen construct (SEQ ID NO: 243) in both adjuvant formulations developed antibodies against rat IL-6 by ELISA. Generally speaking, results showed that a dose-dependent pattern was observed, especially for the group with the ISA 51 formulation (Figure 12). The ISA 51 formulation induced a higher immune response in immunized rats than the ADJUPHOS formulation, with the immunogenicity of the ISA 51 formulation having a log10 value of more than 4 from all doses, respectively, compared to immunogenicity of 4 (log10) or less for the formulation prepared with ADJUPHOS adjuvant.

[0189] Figure 12 shows the kinetics of antibody responses over 43 days in rats immunized with different doses of SEQ ID NO:243 formulated in either ISA51 / CpG or ADJU-PHOS / CpG. ELISA plates were coated with recombinant rat IL-6. Sera were diluted 4-fold from 1:100 to 1:4.19x10 8 The four-parameter logistic curve for each serum sample was fitted with a nonlinear regression with a cutoff of 0.45 to obtain the Log 10 The titers of the tested sera, expressed as t, were calculated.

[0190] The results of this experiment indicate that the choice of adjuvant can have important technical impact on the immunogenicity of a peptide immunogen construct.

[0191] b. IgE EMPD peptide immunogen constructs The immunogenicity of formulations containing IgE EMPD peptide immunogen constructs utilizing different adjuvants was evaluated. POC studies in macaques demonstrated that designed peptide immunogen constructs with high immunogenicity and therapeutic efficacy against IgE EMPD induced pathology implying potential immunotherapy applications. The following studies focused on optimizing the peptide immunogen constructs and selecting adjuvants using the IgE EMPD peptide immunogen constructs.

[0192] The same IgE EMPD peptide immunogen (SEQ ID NO: 178) and different adjuvants, ADJU-PHOS and MONTANIDE ISA 51, formulated with CpG, were evaluated in a macaque immunization study.

[0193] Figures 13A and 13B show graphs depicting anti-IgE-EMPD antibody titers obtained after immunization of macaques with various amounts of the IgE-EMPD peptide immunogen construct of SEQ ID NO: 178 in different adjuvants. Figure 13A shows antibody titers obtained using ADJUPHOS as an adjuvant formulated as an immune stimulating complex stabilized with CpG3, while Figure 13B shows antibody titers obtained using MONTANIDE ISA51 as an adjuvant formulated as an immune stimulating complex stabilized with CpG3.

[0194] The results of this experiment show that IgE EMPD peptide immunogen constructs formulated with different adjuvants are more immunogenic when combined with MONTANTIDE ISA51 compared to formulations containing ADJUPHOS (Figures 13A and 13B). Therefore, the use of different adjuvants may result in different technical effects (immunogenicity) of the peptide immunogen constructs.

[0195] c.LHRH peptide immunogen construct The immunogenicity of formulations containing LHRH peptide immunogen constructs utilizing different adjuvants was evaluated. POC studies in pigs demonstrated that the designed peptide immunogen constructs had high immunogenicity and therapeutic efficacy against LHRH induced pathology, implying potential immunotherapy applications. The following studies focused on optimizing the peptide immunogen constructs and selecting adjuvants using the LHRH peptide immunogen constructs.

[0196] Emulsigen D and MONTANIDE ISA50V as different adjuvants formulated with the same LHRH peptide immunogen (SEQ ID NOs: 239-241) at the same concentration were evaluated in a pig immunization study.

[0197] Figures 14A and 14B show graphs depicting anti-LHRH antibody titers obtained after immunizing pigs with various amounts of LHRH peptide immunogen constructs of SEQ ID NOs: 239-241 in different adjuvants. Figure 14A shows antibody titers obtained using Emulsigen D as the adjuvant, while Figure 14B shows antibody titers obtained using MONTANIDE ISA50V as the adjuvant.

[0198] The results of this experiment show that LHRH peptide immunogen constructs formulated with different adjuvants have a different technical effect (reduced testosterone concentration) when formulated with MONTANTIDE ISA50V compared to a formulation containing Emulsigen D (Figures 15A and 14B). Therefore, the use of different adjuvants may result in a different technical effect (duration of effect, i.e., immunocastration in this case) of the peptide immunogen constructs.

[0199] d.Summary The results of this study demonstrated that the immune response (antibody titers, C maxThis shows that the response time (including response time, onset of antibody production, duration of response, etc.) can be modulated by the choice of adjuvant used in formulations containing the same concentration of peptide immunogen constructs. Thus, a specific immune response against a target antigen site can be engineered by varying either the Th epitope chemically linked to the B cell epitope or the adjuvant used in the formulation, which may facilitate tailoring personalized medicine to the individual characteristics of any patient or subject.

[0200] Example 6 Exclusive immunogenicity of peptide immunogen constructs in guinea pigs targeting Aβ peptides but not Th epitopes At weeks 0 and 4, Aβ was co-formulated in equimolar ratios with peptide immunoconjugates. 1-14 -εK-KKK-MvF5 (SEQ ID NO: 67) and Aβ 1-14 Six guinea pigs were immunized with -εK-HBsAg3 (SEQ ID NO: 68). At week 8, the animals were bled and serum samples were taken to measure anti-Aβ peptide and anti-Th epitope antibody titers (log 10 The antibody responses of all six guinea pigs were directed against Aβ but not against the two artificial Th epitopes (MvF5 Th and HBsAg3 Th), as shown in Table 11. 1-42 The peptide was specifically targeted.

[0201] Example 7 Cellular immune responses in peripheral blood mononuclear cell (PBMC) cultures of baboons and macaques derived from animals immunized with UB-311 vaccine Peripheral blood mononuclear cells (PBMCs) from UB-311-immunized baboons and cynomolgus monkeys were separated by Ficoll-hypaque gradient centrifugation. For peptide-induced proliferation and cytokine production, cells (2 × 10 per well) were cultured at 10 °C for 1 h. 5 ) alone or with added individual peptide domains (Aβ 1-14 , Aβ 1-42, UBITh®, and unrelated peptides. Mitogens (PHA, PWM, Con A) were used as positive controls (10 μg / mL in 1% v / v culture medium). On day 6, 1 μCi of 3H-thymidine (3H-TdR) was added to each of three replicate culture wells. After 18 hours of incubation, cells were harvested and incorporation of 3H-TdR was measured. Stimulation index (SI) represents cpm in the presence of antigen divided by cpm in the absence of antigen; SI>3.0 was considered significant.

[0202] Cytokine analysis (IL-2, IL-6, IL-10, IL-13, TNFα, IFNγ) from cynomolgus PMBC cultures was performed on aliquots of medium alone or in the presence of peptide domains or mitogens. Concentrations of individual cytokines were determined using monkey-specific cytokine sandwich ELISA kits (U-CyTech Biosciences, Utrecht, The Netherlands) according to the kit instructions.

[0203] PMBCs were isolated from whole blood collected from macaques at weeks 15, 21, and 25.5 of immunized animals. 1-14 and Aβ 1-42 Isolated PBMCs were cultured in the presence of

[0204] Aβ 1-14 When the peptide was added to the culture medium, no proliferative response was observed by lymphocytes. 1-42 When the peptide was added to PBMC cultures, a positive proliferative response was observed.

[0205] PBMC samples collected at weeks 15, 21, and 25.5 were also tested for cytokine secretion in the presence of Aβ peptide or PHA mitogen. As shown in Table 12, three cytokines (IL-2, IL-6, TNFα) were upregulated by Aβ peptide or PHA mitogen. 1-14 Full-length Aβ, not peptides 1-42No detectable secretion was detected in UBITh® AD vaccine treated samples compared to placebo vaccine samples in response to peptide, and the other three cytokines tested in the presence of Aβ peptide (IL-10, IL-13, IFNγ) were below the assay detection limit in all PBMC cultures.

[0206] Aβ 17-42 N-terminal Aβ lacking a peptide domain and containing a foreign T-helper epitope 1-14 Macaques were immunized with the UB-311 vaccine, which contained only the peptide immunogen. 1-42 It is shown that the positive proliferation results seen in PBMC cultures in the presence of peptide were not associated with a UB-311 vaccine response, but rather a background response to native full-length Aβ.

[0207] These results suggest that Aβ 1-14 These results support the safety of the UB-311 vaccine, which contains only the native and foreign T-helper epitopes, and shows that it does not elicit a potential inflammatory anti-self cell-mediated immune response against native full-length Aβ peptide in normal macaques. In contrast, adverse events associated with encephalitis in clinical trial studies of the AN-1792 vaccine were due, in part, to the vaccine's monomeric or fibrillar / aggregated Aβ. 1-42 This was due to the inclusion of a T cell epitope within the immunogen.

[0208] Example 8 Lymphoproliferation and cytokine analysis of PBMCs from Alzheimer's disease patients immunized with UB311 vaccine. Peripheral blood mononuclear cells (PBMCs) from Alzheimer's disease patients were isolated by Ficoll-hypaque gradient centrifugation. For peptide-induced proliferation and cytokine production, cells (2.5x105 per well) were cultured in triplicate alone or with the addition of individual peptide domains (at a final concentration of 10 μg / mL), including Aβ1-14 (SEQ ID NO:56), Aβ1-16 (SEQ ID NO:57), Aβ1-28 (SEQ ID NO:59), Aβ17-42 (SEQ ID NO:58), Aβ1-42 (SEQ ID NO:60), and an unrelated 38mer peptide (p1412). Cultures were incubated at 37°C with 5% CO2 for 72 hours, then 100 μL of supernatant was removed from each well and frozen at -70°C for cytokine analysis. 10 μl of medium containing 0.5 μCi of 3H-thymidine (3H-TdR, Amersham, Cat. No. TRK637) was added to each well and incubated for 18 h, followed by detection of radioisotope incorporation by liquid scintillation counting. The mitogen phytohemagglutinin (PHA) was used as a positive control for lymphocyte proliferation. Cells cultured alone without Aβ peptide or PHA mitogen were used as negative and positive controls. Stimulation index (SI) was calculated as the mean counts per minute (cpm) of triplicate experimental cultures containing Aβ peptide divided by the mean cpm of triplicate negative control cultures, and an SI>3.0 was considered a significant proliferative response.

[0209] A. Proliferation assay Peripheral blood mononuclear cell samples were isolated from whole blood collected at week 0 (baseline) and week 16 (4 weeks after the third dose) from Alzheimer's disease patients vaccinated with the UB-311 vaccine and then cultured in the absence or presence of various Aβ peptides. As shown in Table 13, Aβ 1-14 No significant proliferative response by lymphocytes was observed when other Aβ peptides, or p1412 (an unrelated control peptide) were added to the culture medium. As expected, a positive proliferative response was demonstrated when the PHA mitogen was added to the culture medium. The observation of similar responses to PHA before and after UB-311 immunization (P=0.87) does not suggest a significant change in the immune function of the study subjects (Table 13).

[0210] Statistical analysis. Differences in lymphocyte proliferation between weeks 0 and 16 were examined by paired t-test. Statistical significance levels were determined by two-tailed tests (p<0.05). R version 2.14.1 was used for all statistical analyses.

[0211] b. Cytokine analysis Cytokine analysis (IL-2, IL-6, IL-10, TNF-α, IFN-γ) of PBMC cultures was performed on aliquots of cultures containing cells alone or in the presence of Aβ peptide domains or PHA. Concentrations (pg / mL) of individual cytokines were determined using human-specific cytokine sandwich ELISA kits (U-CyTech Biosciences, Utrecht, The Netherlands) according to the manufacturer's instructions (Clin Diag Lab Immunol. 5(1):78-81 (1998)).

[0212] PBMC samples taken from Alzheimer's disease patients administered the UB-311 vaccine at weeks 0 and 16 were also tested for cytokine secretion, either containing cells alone (negative control) after 3 days of culture or in the presence of Aβ peptide, p1412 (unrelated peptide) or PHA mitogen (positive control). The quantifiable range of the kit is 5-320 pg / mL. Any measured concentrations below 5 pg / mL or above 320 pg / mL were designated below the limit of quantification (BQL) or above the limit of quantification (AQL), ​​respectively. However, for statistical considerations, the BQL or AQL was replaced by the lower limit of quantification (5 pg / mL) or upper limit of quantification (320 pg / mL), respectively. The mean concentrations of each cytokine at weeks 0 and 16 are shown in Table 14. As expected, cytokine production was significantly increased in the presence of the positive control, PHA, except for IL-2. Aβ 1-14 Cytokine production in response to stimulation with Aβ or other Aβ peptides was observed at baseline (week 0) and at week 16, with most values ​​being similar to the corresponding negative controls (cells alone).

[0213] To assess changes in cellular immune responses after immunization, changes in mean cytokine concentrations from baseline at week 16 compared with negative controls were examined using paired Wilcoxon signed-rank tests. Four cytokines (IFN-γ, IL-6, IL-10, and TNF-α) were significantly associated with increased cellular immunity compared with full-length Aβ. 1-42 showed a marked increase in secretion in response to the peptide; this observation was consistent with the 1-42 This may be due to conformational epitopes of the aggregates. 1-14 or other Aβ peptides.

[0214] c.Summary The UB-311 vaccine contains two peptide immunogens, each of which synthetically binds the N-terminal Aβ1-14 peptide to the MvF5 Th and HBsAg3 Th epitopes, respectively. In vitro lymphocyte proliferation and cytokine analysis was used to evaluate the impact of UB-311 vaccine immunization on cellular immune responses. As shown in Table 13, no proliferative response by lymphocytes was observed when Aβ1-14 peptide or other Aβ peptides were added to the culture medium. Upregulation of cytokine secretion by lymphocytes of patients immunized with the UB-311 vaccine was not detected upon treatment with other Aβ peptides, except Aβ1-14 and Aβ1-42, which induced a substantial increase in four cytokines (IFN-γ, IL-6, IL-10, TNF-α) after UB-311 immunization at week 16 compared to week 0 levels before treatment (Table 14). The increased cytokine release due to Th2-type T cell responses is likely unrelated to the UB-311 vaccine response, since no upregulation was detected with Aβ1-14 alone. The response to Aβ1-42 is speculated to be a background response to native Aβ that may be associated with native T-helper epitopes identified in Aβ1-42. The lack of IL-2 production observed in response to PHA is consistent with the results reported by Katial RK,et al.in Clin Diagn Lab Immunol 1998;5:78-81 under similar experimental conditions using normal human PBMC. In conclusion, these results indicate that the UB-311 vaccine did not generate a potentially inflammatory anti-autologous cellular immune response in mild to moderate Alzheimer's disease patients participating in a phase I clinical trial, thereby further indicating the safety of the UB-311 vaccine.

[0215] Example 9 Promiscuous artificial Th-responsive cells can be detected in naive peripheral blood mononuclear cells (PMBCs) from normal blood donors with moderate immunogenic inflammatory responses compared to negative controls Using an ELISpot assay, we detected promiscuous artificial Th-responsive cells in naive peripheral blood mononuclear cells from normal blood donors and assessed their ability to induce an inflammatory response in comparison with the potent mitogen phytohemagglutinin (PHA) and a negative control.

[0216] The ELISpot assay uses a sandwich enzyme-linked immunosorbent assay (ELISA) technique. For the detection of T cell activation, IFN-γ or related cytokines were detected as analytes. Either monoclonal or polyclonal antibodies specific for the selected analyte were precoated on PVDF (vinylidene difluoride)-backed microplates. Appropriately stimulated cells were pipetted into the wells and the microplates were placed in a humidified 37°C CO2 incubator for the designated period of time. During this incubation period, the immobilized antibodies in the immediate vicinity of the secreting cells bound to the secreted analyte. After washing away any cells and unbound material, biotinylated polyclonal antibodies specific for the selected analyte were added to the wells. After washing away any unbound biotinylated antibodies, alkaline phosphatase conjugated to streptavidin was added. Subsequently, unbound enzyme was removed by washing and a substrate solution (BCIP / NBT) was added. A blue-black precipitate formed and appeared as a spot at the site of cytokine localization. Each individual spot represents an individual cell secreting the analyte. Spots were counted with an automated ELISpot reader system or manually using a stereomicroscope.

[0217] In the in vitro studies carried out, PHA at 10 μg / mL of culture was used as a positive control. UBITh®1 (SEQ ID NO: 17) and UBITh®5 (SEQ ID NO: 6) peptides were tested for the number of responder cells present in peripheral blood mononuclear cells of normal normal blood donors. A mixture of promiscuous artificial Th epitope peptides of SEQ ID NO: 33-52 was prepared as another positive control. In standard T cell stimulator cell culture conditions, medium alone was used as a negative control. In summary, 100 μL / well of PBMCs (2x10 ) stimulated with mitogen (PHA at 10 μg / mL) or Th antigen (UBITh®1, UBITh®5, or multi-Th mixture at 10 μg / mL) was cultured at 100 μL / well. 5 The wells / plates were incubated at 37° C. in a CO2 incubator for 48 hours. The supernatants of the wells / plates were collected. The cells on the plates were washed and processed for detection of the target analyte IFN-γ.

[0218] As shown in FIG. 15, representative donors 1, 2, and 3 were tested for responsive cells to random artificial UBITh®1 or UBITh®5 epitope peptides. Overwhelming IFN-γ ELISPOT counts were always detected in naive donors (PBMCs cultured with PHA; too many to count), while PBMCs cultured with control medium gave background IFN-γ ELISPOT counts of 5-50. Moderate ELISPOT counts of 20-120 were detected for naive donor PBMCs cultured with UBITh®1 or UBITh®5. A mixture of multiple Th peptides with SEQ ID NOs: 33-52 was also cultured with naive donor PBMCs for comparison with ELISPOT counts of 20-300 as expected. Such stimulatory responses elicited by UBITh®1 or UBITh®5 peptides are about 3-5 times higher compared to the negative control.

[0219] In summary, promiscuous artificial Th response cells can be easily detected in naive donor PBMCs that are primed to initiate immune responses that favor B cell antibody production and corresponding effector T cell responses by secreting signature cytokines. Herein, IFN-γ is used as an example to illustrate this stimulatory nature of these Th epitope peptides. However, such stimulatory inflammatory responses are moderate enough to initiate suitable effector cell responses (B cells for antibody production, cytotoxic T cells for killing target antigen cells) so as not to cause undesirable inflammatory pathophysiological responses during the vaccination process.

[0220] Example 10 Neoepitope-based peptide immunogen constructs and formulations thereof for personalized cancer immunotherapy The inventors are in the midst of a revolution in T cells in cancer treatment. In recent years, new therapies such as immune checkpoint inhibitors (ICIs) and adoptive cell therapies such as chimeric antigen receptor-T (CAR-T) have brought new hope to millions of people with cancer. ICI drugs help overcome natural barriers created by cancer that prevent the patient's own immune system, and in particular, the patient's own T cells, from fighting the cancer. For example, CAR-T therapy manipulates a patient's T cells to mount an immune response against specific tumor cells. These new therapies will lead to future therapies designed to "educate" T cells to attack tumors more precisely.

[0221] There is growing excitement about personalized, or neoantigen, cancer vaccines as a way to provide new hope to millions of people with cancer. Neoantigens are individualized tumor mutations that are viewed as foreign by most individuals' immune systems. Personalized vaccines therefore target these neoantigens, which educate the immune system to find and kill tumors.

[0222] Bioinformatics, proteomics, cell-based assays, and non-standard methods have been widely applied to efficiently identify bona fide neoantigens. RNA frameshift (FS) variants formed by INDELs, short insertions and deletions, in microsatellites and exon mis-splicing are a rich source of highly immunogenic neoantigens.

[0223] Arrays containing all possible (e.g., 400K) FS (frameshift) tumor-derived peptides can be applied to detect antibody reactivity from a single drop of a patient's blood, thereby providing an excellent tool for cancer diagnosis and identification of neo-antigenic CTL epitopes.

[0224] MHC typing can be performed using RNAseq, while NetMHC and NetMHCpan can be used to predict neo-antigen binding affinities.

[0225] In an in vitro HLA-agnostic assay, polypeptides representing each identified mutation from the patient's tumor are individually delivered to their own antigen-presenting cells (APCs), which then process and present the peptides on the cell surface where they are recognized by a variety of effector T cells. If the T cells recognize and bind the peptides, a cytokine response will be elicited. The true antigens are measured and determined to be "good" (i.e., stimulatory) or "bad" (i.e., inhibitory). The patient's T cells, i.e., CD4 + (helper T cells) and CD8 + The actual antigens to which both IL-1 and IL-2 (killer T cells) respond can be identified and selected as neoepitopes for the design of neoepitope peptide immunogen constructs.

[0226] Therefore, personalized cancer vaccines are developed in which the patient's immune system is already primed by including experimentally validated neo-antigens to which the patient has a pre-existing response.

[0227] In summary, neoepitope peptides derived from specific experimentally confirmed neoantigens containing B or CTL neoepitopes using the design principles described in this disclosure can be rendered highly immunogenic. By involving promiscuous artificial Th epitopes covalently linked to selected neoepitopes, such neoepitope peptide immunogen constructs can promote the induction and maintenance of antibodies against neoantigens and the induction of effector CTL function, as well as the generation of B and CTL memory cells that lead to sustained B and CTL responses and potent antitumor immunity.

[0228] Representative known neoantigens with selected target epitope sequences for melanoma neoantigen, histone 3 variant H3.3K27M for glioma, and KRAS (D to G mutant) for colorectal cancer are shown as SEQ ID NOs: 73, 74, and 75, respectively, in Table 3A.

[0229] The designed neoepitope peptide immunogen constructs and formulations thereof of the present disclosure can be further evaluated for safety, immunogenicity, and efficacy in clinical trials, which consist of three parts: 1) Safety and immunogenicity studies as monotherapy in cancer patients with no evidence of disease but at high risk of recurrence. 2) A study of the safety, immunogenicity, and efficacy of a neoepitope vaccine in combination with an FDA-approved immune checkpoint inhibitor in patients with advanced or metastatic solid tumors. 3) Studying the safety, immunogenicity, and efficacy of the neoepitope vaccine as monotherapy in patients with recurrent or refractory solid tumors who have failed to respond or whose cancer has progressed following treatment with immune checkpoint inhibitors.

[0230] Eligible patients who have completed treatment (e.g., surgical resection, neoadjuvant and / or adjuvant chemotherapy, and / or radiation therapy) for cutaneous melanoma, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), or urothelial carcinoma and have no evidence of disease on CT or MRI may participate in these cancer immunotherapies.

[0231] Example 11 Tumor-associated carbohydrate antigen (TACA)-B epitope immunogen constructs and their formulations for cancer immunotherapy Tumor cells are characterized by abnormal glycosylation patterns resulting in heterogeneity, truncation, and overexpression of surface oligosaccharides. Three major categories of sugar structures are GD3, GD2, Globo-H, GM2, Fucosyl GM1, PSA, and Le, as shown in Figure 16. y , Le x , SLe x , SLe a , and have been identified as potential tumor-associated carbohydrate antigens (TACAs), designated as STn. (1) Mucin-associated o-glycans: Tn, TF, and STn (2) Sphingolipids including gangliosides GM3, GM2, GD2, GD3, fucosyl GM1, and neutral globoside globol H (3) Blood type antigen: SLe x , Le y Le x , SLe a , and Le y

[0232] Globol H is expressed on breast cancer cell surfaces as a glycolipid and is an attractive tumor marker. Using a glycal assembly approach to oligosaccharide synthesis, Globol H hexasaccharide was synthesized. Globa H can be functionalized at the reducing end to function as a B-hapten and allow for covalent immobilization with Th helper peptides shown in Table 2 to form immunogenic peptide constructs. The use of the disclosed Th epitopes in this type of application is much more versatile than other approaches using KLH or other conventional carrier proteins, as previously described by Danishefsky and Livingston (website: glycopedia.eu / Hetero-TACA-vaccines-based-on-protein-carriers).

[0233] Primary amines at the N-terminus of each polypeptide or at the side chains of lysine (K) residues of proteins are available as targets for N-hydroxysuccinimide (NHS)-type cross-linker reagents at pH 7-9 to form stable amide bonds with the release of an N-hydroxysuccinimide leaving group. In addition, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (HL Chiang, et al. Vaccine. 2012 30(52), 7573-7581), p-nitrophenyl ester (PNP) (SJ Danishefsky, et al. Acc. Chem. Res. 2015, 48(3), 643-652), along with linkers of different chain lengths, can also function as important TACA conjugate linkers to Th helper peptides shown in Table 2.

[0234] The artificial Th epitopes of the present disclosure can be used in cancer vaccine compositions, including: (a) an immunogenic composition essentially comprising the glycans of Globol H or its immunogenic fragments; (b) an immunogenic fragment covalently linked to a promiscuous artificial T helper epitope peptide shown in Table 2. The epitope peptides shown in Table 2 (e.g., UBITh®) are individually prepared stepwise by solid phase peptide synthesis (SPPS) and Fmoc chemistry with C-terminal to N-terminal deprotection / coupling. The target peptide sequences were constructed accordingly.

[0235] Glycans can be coupled to artificial Th epitope peptides with or without a spacer by forming an amide bond directly on the solid-phase resin, and the coupling reaction is monitored by the Kaiser test. Two strategies can be used for this coupling: (1) preparation of a glycan derivative with an activated leaving group, followed by coupling of the resin-bound spacer-bound Th peptide (the N-terminus has a free amine), or (2) conversion of the resin-bound N-terminal amine group to an activated ester, followed by coupling reaction with the glycan. Direct coupling of the glycan to the resin-bound and spacer Th peptide would allow a more efficient coupling reaction with an activated group on the resin-bound free amine group, followed by release of the coupled glycan peptide from the resin by a standard resin-free cleavage reaction. Steric hindrance between two free large molecules, such as polysaccharides and long-chain peptides, makes the glycan-peptide coupling reaction more difficult, which leads to lower efficiency and lower yields.

[0236] In some embodiments, the B-hapten conjugated T helper carrier is a spacer-conjugated Th-peptide as set forth in Table 2.

[0237] In some embodiments, the linker is a p-nitrophenyl linker, an N-hydroxysuccinimide linker, a p-nitrophenyl ester (PNP ester), or an N-hydroxysuccinimide ester (NHS ester), which can react with primary amines at pH 7-9 to form a stable amide bond with release of an N-hydroxysuccinimide leaving group.

[0238] The following abbreviations are used in this example: PNP: p-nitrophenyl ester; NPC: N-nitrophenyl chloroformate; DSS (disuccinimidyl suberate); and NHS: N-hydroxysuccinimide; MBS: m-maleimidobenzoyl-N-hydroxysuccinimide ester.

[0239] The following steps of chemical reactions and preparations are included to demonstrate various embodiments (Figures 17-21).

[0240] 1. Preparation of Activated UBITh® Bearing p-Nitrophenyl (PNP) Groups Artificial Th epitope (e.g., UBITh®) carriers can be synthesized by automated solid-phase synthesis and Fmoc chemistry. After Fmoc deprotection of the N-terminal amino acid on the growing peptide chain, the free amino group can be converted to an activated 4-nitrophenyl group by treatment with 4-nitrophenyl chloroformate (NPC, 10 equivalents) in a DMF solution containing 10% triethylamine. The resulting peptide-resin mixture can be washed with a DCM solution to remove reagents and 4-nitrophenol residues. The desired activated UBITh® with a p-nitrophenyl group can be obtained for further conjugation with glycans.

[0241] 2. Preparation of Activated UBITh® Bearing N-Hydroxysuccinimide (NHS) Groups UBITh® peptide carriers can be synthesized using automated solid-phase synthesis and Fmoc chemistry. After Fmoc deprotection of the N-terminal amino acid on the growing peptide chain, the free amino group can be converted to an activated N-hydroxysuccinimide by treatment with DSS in DMF solution. The resulting peptide-resin can be washed with DCM solution to remove reagent residues. The desired activated UBITh® with N-hydroxysuccinimide groups can be obtained for further conjugation with glycans.

[0242] 3. Synthesis of Globol H Conjugated UBITh® Peptides The preparation of Globol H hexasaccharide analogue (2) with a terminal amine group can be carried out according to a one-pot synthesis strategy (CY Huang, et al., Proc. Natl Acad Sci USA 2006, 103, 15-20). In summary, a solution of Globol H can be introduced into activated UBITh® resin, followed by gentle mixing for 3 hours. After cleavage from the resin and complete deprotection, the crude Globol H conjugated UBITh® peptide can be purified by preparative high performance liquid chromatography (HPLC) and characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and reversed-phase HPLC analysis.

[0243] 4. Preparation of Globol H Activated Ester Globol H hexylamine (2) can be dissolved in anhydrous DMF solution. Then, p-nitrophenyl adipate diester can be added and stirred at room temperature for 2-4 hours. The reaction can be monitored by TLC and Kaiser test to confirm the disappearance of the free amine group. The DMF solvent can be removed under reduced pressure without heating, and then the resulting residue can be extracted three times with dichloromethane and water containing 0.5% acetic acid. The resulting aqueous solution can be concentrated and purified by reverse phase column (RP-C18) chromatography (isocratic elution with MeOH / H2O containing 1% acetic acid).

[0244] 5. Preparation of GM3 Active Ester The synthesis and purification of GM3 ganglioside analogues has been described previously (Jacques S, et al., J. Am. Chem. Soc., 2012 134 (10): 4521-4). GM3 analogue amine X (4.5 mg; 5.2 μmol) can be dissolved in dimethylformamide (1.5 mL) and triethylamine (3.0 equiv.) can be added. Then, p-nitrophenyl adipate diester (10.0 equiv.) can be added. The reaction can be monitored by TLC (CHCl-MeOH-HO-AcOH; 4:5:1:0.5) and allowed to go to completion. The pH of the reaction can be adjusted to 5.0 with acetic acid, followed by coevaporation with toluene (3×). The residue can be concentrated and the resulting residue can be purified by HPLC (Beckman C18-silica semi-preparative column) using a gradient of MeOH-HO containing 1% acetic acid. 1H NMR spectra can be obtained on CD3OD.

[0245] 6. Preparation of Tn Active Ester The saccharide Tn can be synthesized based on previous reports (T. Toyokuni, et al., Bioorg Med Chem. 1994, 11, 1119-32; and S. D. Scott, et al., J. Am. Chem. Soc., 1998, 120 (48), 12474-85). Tn analog 6 (mmol), NHS (160 mg, 1.39 mmol), and EDC (268 mg, 1.40 mmol) in dry CHCl (25 mL) can be stirred at room temperature for 1 h. The mixture can be washed with pre-cooled H0 (3×30 mL), dried (NaSO), and concentrated to obtain the succinimide-ester derivative (7) as a colorless syrup.

[0246] 7. Sialyl Lewis x (sLe x ) Preparation of active ester (9) sLe xThe preparation of tetrasaccharide derivative (8) can be achieved by published synthetic strategy (G. Kuznik, Bioorganic & Medicinal Chemistry Letters, 7 (5): 577-580, 1997). Compound (8) added to a solution of NPC (2 eq.) in DMF / DCM is warmed to room temperature and stirred for an additional 30 min. The concentrated crude mixture can be washed with DCM and aqueous solution. The resulting aqueous solution can be worked up under reduced pressure and purified on a RP C18 column.

[0247] 8. Basic Steps for Producing Glycoconjugates Standard coupling reactions of individual glycans onto individual resin-bound spacer-incorporated Th peptides follow standard peptide bond formation coupling procedures carried out by standard solid phase peptide synthesizer(s) via carbodiimide coupling reactions.

[0248] The resin-bound glycan-peptide can be removed from the resin, recovered, precipitated, and lyophilized according to standard peptide synthesis procedures.

[0249] T cell peptide epitope (e.g., UBITh®) carriers can be synthesized by automated solid-phase synthesis and Fmoc chemistry. After Fmoc deprotection of the N-terminal amino acid on the growing peptide chain, a free amino group can be made available for further conjugation reactions. Globo H, TN, GM3, SLe with activated leaving group modification x Synthetic sugar analogs such as can be dissolved in DMF solution and added to the SPPS system for reaction with the N-terminal amine of the UBITh® peptide. The reaction can be monitored by Kaiser test. After cleavage from the resin and complete deprotection, the sugar-conjugated UBITh® peptide can be purified by preparative high performance liquid chromatography (HPLC) and characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and reverse-phase HPLC analysis.

[0250] In summary, this example details the effective conjugation of individual glycans to individual Th helper epitope peptides shown in Table 2, allowing such carbohydrate-peptide immunogen constructs to be efficiently prepared for subsequent vaccine formulation. These carbohydrate vaccine formulations using artificial Th epitopes can provide focused antibody responses against targeted carbohydrate antigens normally present on cancer cells to enable immunotherapy of cancer patients in clinical protocols.

[0251] Example 12 Enhancement of effector T cell function by conjugation of promiscuous artificial T helper epitope(s) with effector cell epitopes (e.g., CTL epitopes) for the development of a universal T cell vaccine against viral infections and formulations thereof Introduction T helper cells carry the surface marker CD4 and express a surface receptor known as the T cell receptor, which is composed of a polypeptide heterodimer (e.g., designated α / β). T helper cells recognize viral peptides in association with class II MHC proteins, usually on the surface of antigen-presenting cells (APCs). These interactions result in the activation, proliferation, and differentiation of T helper cells that provide sufficiently high binding affinity.

[0252] T helper cells (CD4 + CD4+ T cells provide soluble mediators and receptor-ligand interactions to cells of both the innate and adaptive immune systems that trigger and regulate effector functions. These cells are a heterogeneous population, and to date, several subsets have been characterized, including Th1, Th2, Th17, and T follicular helper (Tfh) cells. Regulatory CD4+ T cells suppress the growth and function of T cell helper and cytotoxic subsets. + There are also T cells (Tregs).

[0253] Each type of effector T cell is regulated by key transcriptional regulators, expresses a distinct array of cell surface molecules, and secretes "signature" cytokines, which together promote a specific role of that T cell subset within an arm of the immune system.

[0254] Tfh cells are distinguished from other helper subsets by their unique ability to target B cell follicles and provide support to antigen-specific B cells that have undergone somatic hypermutation (SHM) of Ig V region genes and have changed affinity for antigen. Tfh cells secrete the cytokine IL-21, which is essential for B cell differentiation and for generating high affinity isotype-switched antibody responses against viruses. Tfh-mediated signals ensure the selection of B cells with high affinity for immunizing antigens, which can then differentiate to become long-lived plasma cells or memory B cells. Due to the potential emergence of autoreactive B cell clones through the process of SHM and the longevity of selected clones, it is paramount that stringent tolerance mechanisms exist to control the delivery of positive selection signals from Tfh cells to B cells.

[0255] Th1 cells are primarily responsible for enhancing the cytotoxic response. These cells promote the cellular response to viral infections by stimulating the maturation of cytotoxic T cell precursors, in part through the secretion of the cytokines IL-2 and IFN-γ. Th1 cells also secrete tumor necrosis factor (TNF), mediating delayed-type hypersensitivity reactions and promoting the production of IgG2a antibodies. Th1 cells greatly enhance the immune response by activating macrophages and other T cells at the site of viral infection. This response is the basis of the delayed-type hypersensitivity reaction, which is a recognized part of the pathogenesis of many viral infections.

[0256] Other Th cells, including Th2 and Th17 cells, also contribute to the immune response to viral infections by promoting inflammation or the production of certain antibody isotypes.

[0257] Some T cells can downregulate other T cell and / or B cell responses. + Distinct subsets of T cells. There are two basic types of T-regs: (1) tTregs, which are generated in the thymus during negative selection and are thought to be primarily involved in the control of autoimmune diseases, and (2) iTregs, which are induced during an immune response and are involved in terminating the immune response and restoring the immune system to homeostasis. T-reg cells may also help maintain the balance between defense and immune-mediated pathology.

[0258] The effect of helper T cells in peptide vaccination is enormous. + T helper cells generate strong and sustainable CD8 + In view of the local immunoregulatory function of CD4 T cells, it is preferable to activate cognate help in the target tissue from targeted viral or tumor antigens. Foreign antigens, as well as tumor-associated proteins, often contain immunogenic stretches (Th epitopes) that function as hot spots in the immune system. Through the covalent attachment of selected promiscuous artificial Th epitopes (e.g., CTL epitopes) that target B or effector T cells, the peptide immunogen constructs designed and described in this invention can promote the close interaction of APC, CD4, and CD8 T cells for the induction of optimal and protective CD8 T cell responses.

[0259] Examples of CTL epitope peptides as target antigenic sites for incorporation into virus-specific universal T cell vaccines 1. HIV CTL Vaccine Components: Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir, primarily in resting memory CD4+ T cells. This reservoir presents a major barrier to curing HIV-1 infection. To clear the reservoir, pharmacological reactivation of latent HIV-1 has been tested both in vitro and in vivo. A key question that remains is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can eliminate infected cells in ART-treated patients after latency is reversed. After extensive data mining, we identified CTLs that can recognize epitopes from unmutated latent HIV-1 in every chronically infected patient tested, using specific peptides (SEQ ID NOs: 76-82) representative of CTL epitopes shown in Table 3B, which will be incorporated into the design of a universal HIV T-cell vaccine. Chronically infected patients retain a broad-spectrum virus-specific CTL response. Appropriate boosting of this response through this HIV universal T cell vaccine incorporating these CTL epitope peptides individually covalently linked to the promiscuous artificial Th epitopes of the present invention (SEQ ID NOs: 1-52) is expected to result in elimination of the latent reservoir.

[0260] 2. HSV CTL Vaccine Components Herpes simplex viruses infect a high percentage of the world's population and establish a latent infection in which the viral genome is retained in sensory neurons, but no virions are produced. Periodic resurgence of the virus in this latent state results in lesions that can affect the mucosal surfaces of the mouth and lips, the genital tract, and the cornea of ​​the eye, and less frequently, the skin and brain. HSV-2 can be fatal to newborns who contract it from the birth canal; corneal HSV-1 infection is the leading source of blindness; and cerebral HSV-1 infection accounts for approximately one-quarter of cases of potentially fatal viral encephalitis. HSV-1 vaccines that have progressed into clinical trials have been primarily designed for Ab production and have been largely ineffective. Evidence supports the role of CD8 in controlling HSV infection in both mice and humans. + This suggests an important role for T cells.

[0261] HSV type 1 (HSV-1) expresses genes in the order of immediate early (α), early (β), leaky late (γ1), and true late (γ2) genes, and viral DNA synthesis is an absolute prerequisite for γ2 gene expression only. The γ1 protein glycoprotein B (gB) is expressed by CD8 in acutely infected trigeminal ganglia (TG). + Effector T cells and CD8 in latently infected TG + Strongly immunodominant CD8 recognized by 50% of both memory T cells + T cell epitope (gB 498-505 ) is contained.

[0262] Extensive data mining and thorough data analysis revealed that HSV-specific CD8 + The entire T cell repertoire was included in the consideration for HSV CTL vaccine design. Furthermore, different sets of HSV-1 gB epitopes were expressed in the CD4 + It has been shown that gB is recognized by T cells. 166-180 , gB 661-675 , and gB 666-680 CD4 lyses LCLs infected with autologous HSV-1 and vaccinia virus (expressing gB [VVgB]). + Targeted by CTL. 166-180 and gB 666-680 CD4 IgG from HSV-1-seropositive healthy "asymptomatic" individuals + gB appeared to be preferentially recognized by T cells. 661-675 CD4 from severely ill "symptomatic" individuals + It appears that gB is preferentially recognized by T cells. An effective immunotherapeutic herpes vaccine could target potentially "symptomatic" gB 661-675 In addition, three VP11 / 12 CD8 epitopes highly recognized in asymptomatic individuals + They also identified an epitope that was shown to elicit strong protective immunity in a "humanized" HLA-A*02:01 transgenic mouse model of ocular herpes.

[0263] A series of HSV CTL epitopes are identified using specific peptides representative of the CTL epitopes shown in Table 3B (SEQ ID NOs: 83-106) that are incorporated into this inventive design for the development of a universal multi-epitope-based HSV T cell vaccine.

[0264] 3. Universal T-cell vaccine for FMDV, PRRSV and CSFV in the swine industry Foot and mouth disease virus (FMDV), porcine reproductive and respiratory syndrome virus (PRRSV), and classical swine fever virus (CSFV) are debilitating pathogens in the swine industry. The development of effective vaccines against these pathogens is of substantial importance to the swine industry.

[0265] Neutralizing antibodies induced during vaccination are very effective in controlling disease and viral transmission, but do not confer cross-subtype protection and may become ineffective due to antigenic changes. Cellular immune responses, especially the production of cytotoxic T lymphocytes (CTLs), have attracted much attention due to their potential in developing efficient and cross-protective peptide vaccines against various viruses. For example, CTL epitope peptides can be used to develop cross-protective human influenza vaccines, including recombinant viral vectors and peptide vaccines, and the CTL epitope peptides identified for FMDV serotype O were cross-reactive to other FMDV serotypes. However, most analyses were limited to specific viral proteins and could only identify very few CTL epitopes.

[0266] After extensive data mining, design, synthesis, labor-intensive and time-consuming immunogenicity and functional assay procedures, evaluation of a large set of designer CTL peptides has allowed validation of selected virus-specific CTL epitopes derived from various FMDV, PRRSV, and CSFV viral proteins. Selected CTL peptides representative of these epitopes are shown in Table 3B, including SEQ ID NOs: 107-145.

[0267] In our selection and identification process, a bioinformatics pipeline was integrated for the analysis of porcine virus sequences to solve some of the following problems: (1) genetic variation, (2) incomplete screening from specific surface proteins, and (3) inappropriate prediction based on non-porcine leukocyte antigens. The incorporation of these CTL epitope peptides with appropriate binding of promiscuous artificial Th epitope peptides resulting in this peptide immunogen construct of the present invention will lead to the development of T cell vaccines with sustained memory and long-lasting CTL responses. The commercial development of such highly accurate and effective peptide-based porcine vaccines against FMDV, PRRSV, CSFV, and other viral infections that frequently ravage the porcine industry will be of paramount importance to the livestock industry.

[0268] [Table 1]

[0269] [Table 2]

[0270] [Table 3]

[0271] [Table 4]

[0272] [Table 5]

[0273] [Table 6]

[0274] [Table 7]

[0275]

Table 8

[0276]

Table 9

[0277]

Table 10

[0278]

Table 11

[0279]

Table 12

[0280]

Table 13

[0281]

Table 14

[0282]

Table 15

[0283]

Table 16

[0284]

Table 17

[0285]

Table 18

[0286]

Table 19

[0287]

Table 20

Claims

1. A promiscuous artificial T helper cell (Th) epitope selected from the group consisting of SEQ ID NOs: 32-52.

2. A peptide immunogen construct, represented by the formula: (A) ｎ - (target antigen site) - (B) ｏ - (Th) ｍ - (A) ｎ -X or (A) ｎ - (Th) ｍ - (B) ｏ - (target antigen site) - (A) ｎ -X or (A) ｎ - (Th) ｍ - (B) ｏ - (target antigen site) - (B) ｏ - (Th) ｍ - (A) ｎ -X or {(A) ｎ - (Th) ｐ - (B) ｏ - (target antigen site) - (B) ｏ - (Th) ｐ - (A) ｎ -X} ｍ (In the formula, Each A is independently an amino acid; each B is independently a heterologous spacer; Each Th is independently a promiscuous artificial Th epitope according to claim 1; the target antigenic site is a B cell epitope derived from a foreign antigenic protein, an autoantigenic protein, or an immunologically reactive analog thereof; X is an amino acid, α-COOH, or α-CONH ２ and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 1, 2, 3, or 4; o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 0, 1, 2, 3, or 4).

3. 3. The peptide immunogen construct of claim 2, wherein the target antigenic site is a B cell epitope derived from a foreign antigenic protein selected from the group consisting of foot and mouth disease (FMD) capsid protein, glycoprotein from porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), human immunodeficiency virus (HIV), and herpes simplex virus (HSV).

4. The target antigen site is (a) an Aβ peptide having the amino acid sequence of SEQ ID NO: 56, 57, 58, 59, or 60; (b) alpha-syn peptide having the amino acid sequence of SEQ ID NO:61; (c) an IgE EMPD peptide having the amino acid sequence of SEQ ID NO:62; (d) a Tau peptide having the amino acid sequence of SEQ ID NO: 63, 69, 70, or 71; (e) an IL-31 peptide having the amino acid sequence of SEQ ID NO: 64 or 72, and (f) an IL-6 peptide having the amino acid sequence of SEQ ID NO:

145.

3. The peptide immunogen construct of claim 2, wherein the B cell epitope is derived from an autoantigenic protein selected from the group consisting of:

5. 3. The peptide immunogen construct of claim 2, wherein said heterologous spacer of component B is selected from the group consisting of the amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO:53), Lys-Lys-Lys-εNLys (SEQ ID NO:54), Gly-Gly, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:55), and any combination thereof.

6. 3. The peptide immunogen construct of claim 2, wherein the heterologous spacer is selected from the group consisting of (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO:53), and Lys-Lys-Lys-εNLys (SEQ ID NO:54).

7. A pharmaceutical composition comprising the peptide immunogen construct of claim 2.

8. 10. A method of preventing and / or treating a disease, condition, or illness in a subject comprising administering to the subject a pharma- ceutically effective amount of the pharmaceutical composition of claim 7.

9. 9. The method of claim 8, wherein the target antigenic site is a B cell epitope derived from a foreign antigenic protein selected from the group consisting of foot and mouth disease (FMD) capsid protein, glycoprotein from porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), human immunodeficiency virus (HIV), and herpes simplex virus (HSV).

10. The target antigen site is (a) an Aβ peptide having the amino acid sequence of SEQ ID NO: 56, 57, 58, 59, or 60; (b) alpha-syn peptide having the amino acid sequence of SEQ ID NO:61; (c) an IgE EMPD peptide having the amino acid sequence of SEQ ID NO:62; (d) a Tau peptide having the amino acid sequence of SEQ ID NO: 63, 69, 70, or 71; (e) an IL-31 peptide having the amino acid sequence of SEQ ID NO: 64 or 72, and (f) an IL-6 peptide having the amino acid sequence of SEQ ID NO:

145. The method of claim 8, wherein the B cell epitope is derived from an autoantigen protein selected from the group consisting of:

11. A peptide immunogen construct, represented by the formula: (A) ｎ - (target antigen site) - (B) ｏ - (Th) ｍ - (A) ｎ -X or (A) ｎ - (Th) ｍ - (B) ｏ - (target antigen site) - (A) ｎ -X or (A) ｎ - (Th) ｍ - (B) ｏ - (target antigen site) - (B) ｏ - (Th) ｍ - (A) ｎ -X or {(A) ｎ - (Th) ｐ - (B) ｏ - (target antigen site) - (B) ｏ - (Th) ｐ - (A) ｎ -X} ｍ (In the formula, Each A is independently an amino acid; each B is independently a heterologous spacer; Each Th is independently a promiscuous artificial Th epitope selected from the group consisting of SEQ ID NOs: 1-52; the target antigenic site is a CTL epitope, a tumor-associated carbohydrate antigen (TACA), a B-cell epitope derived from a neoantigen, a small molecule drug, or an immunologically reactive analog thereof; X is an amino acid, α-COOH, or α-CONH ２ and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 1, 2, 3, or 4; o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 0, 1, 2, 3, or 4).

12. The peptide immunogen of claim 11, wherein the target antigen site is a CTL epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 76-144.

13. The peptide immunogen of claim 12, wherein the target antigenic site is a CTL epitope derived from HIV selected from the group consisting of SEQ ID NOs: 76-82.

14. The peptide immunogen of claim 12, wherein the target antigenic site is a CTL epitope derived from HSV selected from the group consisting of SEQ ID NOs: 83-106.

15. The peptide immunogen of claim 12, wherein the target antigenic site is a CTL epitope derived from FMDV selected from the group consisting of SEQ ID NOs: 107-123.

16. The peptide immunogen of claim 12, wherein the target antigenic site is a CTL epitope derived from PRRSV selected from the group consisting of SEQ ID NOs: 124-142.

17. The peptide immunogen of claim 12, wherein the target antigenic site is a CTL epitope derived from CSFV selected from the group consisting of SEQ ID NOs: 143-144.

18. The target antigen sites are GD3, GD2, Globo-H, GM2, Fucosyl GM1, GM2, PSA, Le ｙ , Le ｘ , S.L.e. ｘ , S.L.e. ａ 12. The peptide immunogen of claim 11, which is a TACA selected from the group consisting of Tn, TF, and STn.

19. 12. The peptide immunogen of claim 11, wherein the target antigenic site is a B cell epitope derived from a neoantigen selected from the group consisting of SEQ ID NOs: 73-75.

20. The peptide immunogen of claim 11 , wherein the target antigenic site is a small molecule drug.

21. 12. The peptide immunogen construct of claim 11, wherein said heterologous spacer of component B is selected from the group consisting of the amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO:53), Lys-Lys-Lys-εNLys (SEQ ID NO:54), Gly-Gly, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:55), and any combination thereof.

22. 12. The peptide immunogen construct of claim 11, wherein the heterologous spacer is selected from the group consisting of (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO:53), and Lys-Lys-Lys-εNLys (SEQ ID NO:54).

23. A pharmaceutical composition comprising the peptide immunogen construct of claim 11.

24. 24. A method of preventing and / or treating a disease, condition, or illness in a subject comprising administering to the subject a pharma- ceutically effective amount of the pharmaceutical composition of claim 23.

25. 25. The method of claim 24, wherein the disease, condition, or condition is HIV and the target antigenic site is a CTL epitope from HIV selected from the group consisting of SEQ ID NOs: 76-82.

26. 25. The method of claim 24, wherein the disease, condition, or illness is HSV and the target antigenic site is a CTL epitope from HSV selected from the group consisting of SEQ ID NOs: 83-106.

27. 25. The method of claim 24, wherein the disease, condition, or illness is FMDV and the target antigenic site is a CTL epitope from FMDV selected from the group consisting of SEQ ID NOs: 107-123.

28. 25. The method of claim 24, wherein the disease, condition, or illness is PRRSV and the target antigenic site is a CTL epitope from PRRSV selected from the group consisting of SEQ ID NOs: 124-142.

29. 25. The method of claim 24, wherein the disease, condition, or condition is CSFV and the target antigenic site is a CTL epitope from CSFV selected from the group consisting of SEQ ID NOs: 143-144.

30. 25. The method of claim 24, wherein the disease, condition, or condition is CSFV and the target antigenic site is a CTL epitope from CSFV selected from the group consisting of SEQ ID NOs: 143-144.

31. 25. The method of claim 24, wherein the disease, condition, or disorder is cancer and the target antigenic site is a TACA selected from the group consisting of GD3, GD2, Globo-H, GM2, Fucosyl GM1, GM2, PSA, Ley, Lex, SLex, SLea, Tn, TF, and STn.

32. 25. The method of claim 24, wherein the disease, condition, or illness is cancer and the target antigenic site is a B cell epitope derived from a neoantigen selected from the group consisting of SEQ ID NOs: 73-75.

33. 1. A method for modulating an immune response in a subject, comprising: (a) preparing a plurality of peptide immunogen constructs according to claim 11, wherein the target antigenic site remains constant and the Th epitope on each peptide immunogen construct is different; (b) preparing a plurality of pharmaceutical compositions, each of which comprises one of the peptide immunogen constructs prepared in (a) and a pharma- ceutically acceptable adjuvant or carrier; (c) administering to different subjects each of the pharmaceutical compositions prepared in (b); (d) monitoring the immune response of each of said subjects; and (e) selecting said pharmaceutical composition that produces a desired immune response; The method comprising:

34. 34. The method of claim 33, wherein the pharma- ceutically acceptable adjuvant or carrier in each of the pharmaceutical compositions is the same.

35. 34. The method of claim 33, wherein the pharma- ceutically acceptable adjuvant or carrier in each of the pharmaceutical compositions is different.

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