Artificial promiscuous t helper cell epitopes that facilitate targeted antibody production with limited t cell inflammatory response

Abstract

To provide a method of treating a condition of producing an immunogenic inflammatory response.SOLUTION: Provided is a method of treating a condition comprising administering a peptide to a subject in need thereof, the peptide comprising a T helper cell epitope and an antigen-presenting epitope, and the peptide producing an immunogenic inflammatory response that is at least about 3- fold lower than an immunogenic inflammatory response of a positive control. The T helper cell epitopes are novel, heterologous, promiscuous and artificial T helper cell epitopes designed to provide optimal immunogenicity of a target antigenic site. The disclosed T helper cell epitopes, when covalently linked to a B cell epitope in a peptide immunogen construct, elicit a strong antibody response against the B cell epitope of the target antigenic site.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This disclosure is a PCT international application which claims the benefit of U.S. Provisional Application No. 62 / 667,123, filed May 4, 2018, which is incorporated by reference in its entirety. [Background technology]

[0002] Immune responses require coordinated interactions between antigen-presenting cells and helper T cells. To elicit an effective antibody response, antigen-presenting cells must recognize the target antigenic site of the immunogen of interest, and T helper cells must recognize the T helper cell epitope. Generally, the T helper epitope of the immunogen of interest is different from its B cell epitope(s). B cell epitopes are sites on the desired target that are recognized by B cells, resulting in the production of antibodies against the desired target site. The natural conformation of the target determines the site to which the antibody directly binds. Eliciting a Th cell response requires a Th cell receptor that recognizes 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, a Th cell response requires peptide processing and three-way recognition of the target protein. Defining the three-part complex is difficult because 1) key MHC class II contact residues are variably located within different MHC-binding peptides (Th epitopes), 2) different MHC-binding peptides are of variable length and have different amino acid sequences, and 3) MHC class II molecules can be highly diverse depending on the genetic makeup of the host. Immune responsiveness 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. It is difficult to identify promiscuous Th epitopes, i.e., Th epitopes that are reactive across species and across individuals within a single species.

[0003] Each component step of T cell recognition requires multiple factors, including proper peptide processing by antigen-processing cells, presentation of the peptide by genetically determined class II MHC molecules, and recognition of the MHC molecule or peptide complex by receptors on Th cells. Determining the requirements for promiscuous Th epitope recognition to provide broad responsiveness can be difficult.

[0004] It is clear that for the induction of antibodies, an immunogen must contain both B cell determinants and Th cell determinants(s). Generally, to enhance the immunogenicity of a target, a Th response is provided by coupling the target to a carrier protein. The drawbacks of this approach are many. It is difficult to produce a well-defined, safe and effective peptide-carrier protein conjugate for the following reasons:

[0005] a. Chemical coupling is a random reaction that introduces heterogeneity in size and composition, for example, conjugation with glutathialdehyde (Borras-Cuesta et al., Eur J Immunol, 1987; 17: 1213-1215).

[0006] b. Carrier proteins induce the potential for unwanted immune responses, such as allergic and autoimmune reactions (Bixler et al., WO 89 / 06974).

[0007] c. Large peptide carrier proteins induce irrelevant immune responses that are misdirected primarily to the carrier protein rather than to the target site (Cease et al., Proc Natl Acad Sci USA, 1987; 84: 4249-4253); and

[0008] d. Carrier proteins also introduce the possibility of epitope suppression in a host previously immunized with an immunogen containing the same carrier protein: if the host is subsequently immunized with another immunogen in which the same carrier protein is linked to a different hapten, the resulting immune response is enhanced by the carrier protein but inhibited by the hapten (Schutze et al., J Immunol, 1985; 135: 2319-2322).

[0009] To avoid the above risks, it is desirable to elicit T cell help without the use of conventional carrier proteins.

[0010] Incorporation by Reference 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. [Brief description of the drawings]

[0011] [Figure 1] Figure 1: Detection of promiscuous and artificial Th peptide-responsive T cells in naive peripheral blood mononuclear cells of normal donors. Summary of the Invention

[0012] The present disclosure provides promiscuous artificial T helper cell (Th) epitopes that can be used to generate peptide immunogens that can stimulate functional site-specific antibody responses for therapeutic effect. The disclosed artificial Th epitopes can be linked to synthetic peptide B cell epitopes ("target antigenic sites") via optional spacers to generate immunogenic peptides. The immunogenic peptides can also include other components, including general immune stimulatory sequences.

[0013] Artificial Th epitopes confer the peptide immunogens the ability to induce a strong T helper cell-mediated immune response, producing high levels of antibodies against the "target antigenic site". The present invention further provides for the advantageous replacement of carrier proteins and pathogen-derived T helper cell sites in established peptide immunogens with artificial Th epitopes specifically designed to improve their immunogenicity. Short peptide immunogens bearing artificial Th epitopes of the present invention induce high levels of antibodies targeting specific target antigenic site B cell epitopes without causing significant inflammatory responses.

[0014] The artificial Th epitopes of the present invention can be linked to a target antigenic site and, optionally, to an immunostimulatory sequence. The immunogenic peptides of the present invention can be represented by the following formula: (A) n -(Target antigenic site)-(B) o -(Th) m -X or (A) n -(B) o -(Th) m -(B) o -(Target antigenic site)-X or (A) n -(Th) m -(B) o -(Target antigenic site)-X or (Target antigenic site)-(B) o -(Th) m -(A) n -X or (Th) m -(B) o -(Target antigenic site)-(A) n -X where: each A is independently an amino acid; each B is independently an amino acid, -NHCH(X)CHSCHCO-, -NHCH(X)CHSCHCO(εN)Lys-, -NHCH(X)CHS-succinimidyl(εN)Lys-, or -NHCH(X)CHS-(succinimidyl)-; each Th is independently an artificial Th cell epitope, analog or segment thereof; The target antigenic site is a B cell epitope, a peptide hapten thereof, or an immunologically reactive analog thereof; X is an amino acid, α-COOH, or -CONH2; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 1, 2, 3, or 4; and o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0015] An example of a peptide hapten as a target antigenic site is the amino acid sequence of the beta amyloid (Aβ) protein. 1-14 (Aβ 1-14 ) (SEQ ID NO:56).

[0016] The compositions of the invention comprise peptides capable of eliciting an antibody response in an immunized host against a desired target antigenic site, which may be derived from pathogenic organisms, normally immune-silent self-antigens, and tumor-associated targets.

[0017] The compositions of the invention are therefore useful in many diverse medical and veterinary applications, including vaccines to provide protective immunity against infectious diseases, immunotherapy for the treatment of disorders resulting from the dysfunction of normal physiological processes, immunotherapy for the treatment of cancer, and as agents that desirably intervene and modify normal physiological processes.

[0018] Some target antigens that may be covalently linked to the Th epitopes of the present invention include the following moieties: beta amyloid (Aβ) for the treatment of Alzheimer's disease, alpha synuclein (α-Syn) for the treatment of Parkinson's disease, the extracellular 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 interleukin-31 (IL-31) for the treatment of atopic dermatitis, to name a few. More specifically, Aβ1-14 (described in U.S. Patent No. 9,102,752), α-Syn 126-135 (described in U.S. Provisional Application No. 62 / 521,287), IgE EMPD 1-39 (described in International PCT Application No. PCT / US2017 / 069174), Tau 379-408 (described in U.S. Provisional Application No. 62 / 578,124), and IL-31 97-144 (described in U.S. Provisional Application No. 62 / 597,130).

[0019] In some embodiments, the present invention provides a method of treating a condition comprising administering to a subject in need thereof a peptide, the peptide comprising a T helper cell epitope and an antigen-presenting epitope, the peptide generating an immunogenic inflammatory response that is at least about 3-fold lower than the immunogenic inflammatory response of a positive control.

[0020] The present disclosure provides promiscuous artificial T helper cell (Th) epitopes that can be used to generate peptide immunogens that can stimulate functional site-specific antibody responses for therapeutic effect. The disclosed artificial Th epitopes can be linked, via optional spacers, to synthetic peptide B cell epitopes ("target antigenic sites") to generate immunogenic peptides.

[0021] The artificial Th epitopes confer the peptide immunogens the ability to induce a strong T helper cell-mediated immune response, producing high levels of antibodies against the "target antigenic site". The present invention further provides for the advantageous replacement of carrier proteins and pathogen-derived T helper cell sites in established peptide immunogens with artificial Th epitopes specifically designed to improve their immunogenicity. The short peptide immunogens bearing the artificial Th epitopes of the present invention induce high levels of antibodies targeting specific target antigenic site B cell epitopes without causing significant inflammatory responses.

[0022] The peptide immunogens of the present disclosure can induce an antibody response in an immunized host against a desired target antigenic site. In some embodiments, the antigenic site is taken from a pathogenic organism (e.g., FMDV VP1, PRRSV GP5, etc.). In some embodiments, the antigenic site is taken from a normally immune-silent self-antigen or tumor-associated target (e.g., Aβ, Tau, alpha-synuclein, IgE EMPD, IL-31, etc.).

[0023] The present disclosure describes artificial Th epitopes that can be used to provide peptide immunogens that induce antibodies targeting specific proteins. The target antigenic site can include any amino acid sequence from any target peptide or protein. In some embodiments, the present disclosure describes artificial Th epitopes that can be used to provide peptide immunogens that induce antibodies targeting 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.

[0024] The peptides of the invention may be useful in medical and veterinary applications, in some embodiments, the peptides of the invention may be used as vaccines to provide protective immunity against infectious diseases or neurodegenerative diseases, as vaccines to treat disorders resulting from dysfunction of normal physiological processes, as immunotherapies to treat cancer, and as drugs to intervene in normal physiological processes.

[0025] Peptide Immunogens As used herein, the term "peptide immunogen" refers to a molecule that contains a Th epitope covalently linked to a target antigenic site via a traditional peptide bond to form a single larger peptide, or via other forms of covalent linkages, such as thioesters.

[0026] The present disclosure provides peptide immunogens and compositions comprising the peptide immunogens. In some embodiments, the immunogenic peptide comprises an artificial heterologous Th epitope, a target antigenic site comprising a B cell epitope, and an optional heterologous spacer.

[0027] The presence of artificial Th epitopes in peptide immunogens can induce strong Th cell-mediated immune responses. In some embodiments, the presence of artificial heterologous Th epitopes in immunogenic peptides can generate high levels of antibodies directed to target antigenic sites. In some embodiments, the present disclosure describes the advantageous replacement of carrier proteins and pathogen-derived Th cell sites in established peptide immunogens with artificial heterologous Th cell epitopes designed to improve immunogenicity. In some embodiments, peptide immunogens with artificial Th epitopes can induce high levels of antibody production targeting B cell epitopes (e.g., Aβ, tau, alpha synuclein, IgE EMPD, IL-31, etc.).

[0028] In some embodiments, an immunogenic peptide of the present disclosure may be represented by the formula: (A) n -(Target antigenic site)-(B) o -(Th) m -X or (A) n -(B) o -(Th) m -(B) o -(Target antigenic site)-X or (A) n -(Th) m -(B) o -(Target antigenic site)-X or (Target antigenic site)-(B) o -(Th) m -(A) n -X or (Th) m -(B) o -(Target antigenic site)-(A) n -X During the ceremony: each A is independently an amino acid; each B is independently an amino acid, -NHCH(X)CHSCHCO-, -NHCH(X)CHSCHCO(εN)Lys-, -NHCH(X)CHS-succinimidyl(εN)Lys-, or -NHCH(X)CHS-(succinimidyl)-; each Th is independently an artificial Th cell epitope, analog or segment thereof; The target antigenic site is a B cell epitope, a peptide hapten thereof, or an immunologically reactive analog thereof; X is an amino acid, α-COOH, or -CONH2; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 1, 2, 3, or 4; and o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0029] Peptide immunogens of the disclosure may comprise 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. In some embodiments, peptide immunogens of the disclosure may comprise about 20, about 30, about 40, about 50, about 60, about 70, or about 80 amino acid residues.

[0030] A-amino acids Each A in the immunogenic peptides of the present disclosure is independently a heterologous amino acid sequence.

[0031] The term "heterologous" as used herein refers to an amino acid sequence that is not a part of or is not homologous to the wild-type amino acid sequence of the target antigenic site (B-cell epitope). Thus, the heterologous amino acid sequence of A includes an amino acid sequence that is not naturally found in the protein or peptide of the target antigenic site. The sequence of component A is heterologous to the target antigenic site, such that when component A is covalently linked 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.

[0032] In some embodiments, each A is independently a non-naturally occurring or naturally occurring amino acid.

[0033] 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.

[0034] Non-naturally occurring 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.

[0035] In some embodiments, n is greater than 1 and each A is independently the same amino acid. In some embodiments, n is greater than 1 and each A is independently a different amino acid.

[0036] B - Optional heterologous spacer Each B in the immunogenic peptides of the present disclosure is any heterologous spacer.

[0037] As stated 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 (B cell epitope). Thus, when the spacer is an amino acid, the spacer comprises an amino acid sequence that is not naturally found in the protein or peptide of the target antigenic site. The sequence of component B is heterologous to the target antigenic site, so that when component B is covalently linked 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.

[0038] 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 comprise one or more naturally or non-naturally occurring amino acid residues, as described above for component A.

[0039] The spacer may be a flexible hinge spacer to enhance separation of the Th epitope and the target antigenic site. In some embodiments, the flexible hinge sequence may be proline-rich. In certain embodiments, the flexible hinge has the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 55), modeled from the flexible hinge region found in immunoglobulin heavy chains, where Xaa can be any amino acid. In some embodiments, Xaa is aspartic acid. In some embodiments, the conformational separation provided by the spacer may allow for more efficient interaction between the presented peptide immunogen and the appropriate Th and B cells. The immune response to the Th epitope may 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, -NHCH(X)CHSCHCO-, -NHCH(X)CHSCHCO(εNLys)-, -NHCH(X)CHS-succinimidyl-εNLys-, or -NHCH(X)CHS-(succinimidyl)-, and / or any combination thereof.

[0041] Exemplary heterologous spacers are shown in Table 2.

[0042] target antigenic site The present disclosure describes artificial Th epitopes that can be used to provide peptide immunogens that elicit antibodies that target specific proteins. The target antigenic site may comprise any amino acid sequence from any target peptide or protein, including foreign or self peptides or proteins.

[0043] In some embodiments, the present disclosure describes artificial Th epitopes that may be used to provide peptide immunogens that elicit antibodies targeting luteinizing hormone releasing hormone (LHRH) (e.g., U.S. Pat. Nos. 6,025,468, 6,228,987, 6,559,282, and U.S. Patent Application Publication No. 2017 / 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 Application Publication No. 2015 / 0216418); No. 306203; HIV virion epitopes for the prevention and treatment of HIV infection (e.g., U.S. Patent Nos. 5,912,176, 5,961,976, and 6,090,388); capsid proteins from porcine circovirus type 2 (PCV2) (e.g., U.S. Publication No. 2013 / 0236487), glycoproteins from porcine reproductive and respiratory syndrome virus (PRRSV) (e.g., U.S. Publication Nos. 2014 / 0335118), IgE (e.g., U.S. Publication Nos. 7648701 and 6811782), alpha-synuclein (α-Syn) (U.S. Provisional Application No. 62 / 521,287), membrane-bound IgE (or IgE the extracellular membrane proximal domain of EMPD (International PCT Application No. PCT / US2017 / 069174), Tau (U.S. Provisional Application No. 62 / 578,124), and interleukin-31 (IL-31) (U.S. Provisional Application No. 62 / 597,130), Plasmodium CS antigen for the prevention of malaria; CETP for the prevention and treatment of arteriosclerosis; and any other peptide or protein sequence. All patents and patent publications are incorporated herein by reference in their entirety.

[0044] Exemplary target antigenic sites are shown in Table 3.

[0045] Th-T helper epitopes The Th epitopes of the peptide immunogen constructs enhance the immunogenicity of the targeted antigenic site and, through rational design, promote the production of specific, high titer antibodies against the optimized targeted B cell epitopes.

[0046] In some embodiments, Th epitope is a heterologous sequence.As mentioned above, the term "heterologous" refers to an amino acid sequence derived from an amino acid sequence that is not part of or homologous to the wild-type sequence of target antigenic site.Therefore, a heterologous Th epitope is a Th epitope derived from an amino acid sequence that is not naturally found in target antigenic site.Because Th epitope is heterologous to target antigenic site, when heterologous Th epitope is covalently bound to target antigenic site, the natural amino acid sequence of target antigenic site is not extended in either N-terminal or C-terminal direction.

[0047] 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 to allow maximum activation of T helper cells that lead to the initiation and regulation of immune responses. The Th epitope is preferably immune silent by itself, i.e., very few, if any, of the antibodies generated by the peptide immunogen construct are directed against the Th epitope, allowing a highly focused immune response to be directed to the target antigenic site.

[0048] The size of the epitope may range from about 15 to about 50 amino acid residues. In some embodiments, the Th epitope may have about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acid residues. The Th epitopes may share common structural features and certain landmark sequences. In some embodiments, the Th epitope has an amphipathic helix, i.e., an alpha-helical structure in which hydrophobic amino acid residues dominate one face of the helix and charged and polar residues dominate the surrounding faces.

[0049] The Th epitopes and disclosures of WO1999 / 066957, and corresponding US Pat. No. 6,713,301, are hereby incorporated by reference in their entireties.

[0050] Promiscuous Th determinants can be effective in boosting poorly immunogenic peptides. Well-designed promiscuous Th / B cell epitope chimeric peptides can elicit Th responses with antibody responses that target B cell sites in most members of a genetically diverse population. In some embodiments, Th cells can be delivered to target antigen peptides by covalently linking a peptide carrier to a well-characterized promiscuous Th determinant.

[0051] Promiscuous Th epitopes may include additional primary amino acid patterns. In some embodiments, promiscuous Th epitopes may include a Rothbard sequence, where the promiscuous Th epitopes include a charged residue (e.g., -Gly-), followed by 2 to 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, and 8 rule, where a positively charged residue is followed by hydrophobic residues at positions 4, 5, and 8, which corresponds 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, charged, and polar amino acids may be repeated within a single Th epitope. In some embodiments, promiscuous T cell epitopes may include at least one of a Rothbard sequence or an epitope following the 1, 4, 5, 8 rule. In other embodiments, the Th epitope comprises two or more Rothbard sequences.

[0052] Promiscuous Th epitopes derived from pathogens include, but are not limited to, 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 circumsporozoite Th cell epitope (PF Th), Schistosoma mansoni triosephosphate isomerase Th cell epitope (SM Th), and Escherichia coli TraT Th cell epitopes (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.

[0053] In some embodiments, the Th epitopes of the present disclosure may be combinatorial Th epitopes, which include a mixture of peptides with similar amino acid sequences. Structured synthetic antigen libraries (SSALs), also called combinatorial artificial Th epitopes, contain a large number of Th epitopes with amino acid sequences organized around a structural framework of invariant residues with substitutions at specific positions. The sequence of the SSAL epitopes is determined by retaining relatively invariant residues and varying other residues to provide 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 responsible for the unique structure of the Th peptide as a scaffold framework, and varying the remaining residues according to known MHC restriction elements. Using the invariant and variable positions with preferred amino acids of the MHC restriction elements, it is possible to obtain MHC binding motifs that can be used to design SSALs of Th epitopes.

[0054] 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, the Th epitope library sequences are designed to maintain the structural motifs of promiscuous Th epitopes and accommodate reactivity to a broader range of haplotypes. In some embodiments, the members of the SSAL can be a degenerate Th epitope SSAL1 Th1 modeled on a promiscuous epitope taken from the F protein of the measles virus (e.g., SEQ ID NOs: 1-5). In other embodiments, the members of the SSAL can be a degenerate Th epitope SSAL2 Th2 modeled on a promiscuous epitope taken from HBsAg1 (e.g., SEQ ID NOs: 19-24).

[0055] 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 options available at each variable position. For example, SEQ ID NO: 16 contains 5 variable positions, and each variable position has two different residue options (i.e., 2x2x2x2x2=2). 5 =32), which corresponds to 32 different peptide combinations. Similarly, SEQ ID NO:5 corresponds to 524,288 different peptide combinations (i.e., 2x4x2x4x2x4x4x4x4x2x4x2x4=2 5 x4 7 = 524,288). The combinatorial artificial Th epitope array includes (a) a mixture of all peptides contained in the variable sequence, and (b) each individual peptide that comprises a single sequence within the combination.

[0056] In some embodiments, the charged residues Glu or Asp may 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 may 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 may be altered to provide a facade with the ability to bind a broad range of MHC restriction elements. In some embodiments, variation of amino acid residues may expand the range of immune responsiveness of the artificial Th epitope.

[0057] 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 taken 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, may show high immune potency, and may show broad reactivity.

[0058] 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) aimed at eliciting broad immune responses among members of genetically diverse populations. The SSAL peptide prototypes were designed based on Th epitopes of measles virus and hepatitis B virus antigens that were modified by introducing multiple MHC binding motifs. The design of other Th epitopes was modeled on 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 sites were incorporated into synthetic peptide immunogens bearing a variety of target antigenic sites. The resulting chimeric peptides were able to stimulate effective antibody responses against the target antigenic sites.

[0059] The prototypical artificial helper T cell (Th) epitope, a mixture of four peptides (SEQ ID NOs: 1-4), shown in Table 1 as "SSAL1TH1", is an ideal Th epitope modeled after the promiscuous Th epitope of the F protein of measles virus (Partidos et al. 1991). The model Th epitope, shown in Table 1 as "MVF Th(UBITh®5)" (SEQ ID NO: 6), corresponds to residues 288-302 of the measles virus F protein. MVF Th (SEQ ID NO: 6) was modified relative 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; by adding or retaining charged residues or Gly at positions 4, 6, 12, and 14; and by adding or retaining charged residues or Gly at positions 7 and 11 according to the "Rothbard rules". 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 ThSSAL 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 the MHC binding motif. This "1, 4, 9" palindrome pattern of SSAL1 Th1 was further modified in SEQ ID NO: 2 (Table 1) to more closely reflect the sequence of the original MvF model Th (SEQ ID NO: 6).

[0060] Combinatorial artificial Th epitopes may be simplified to provide a series of single sequence epitopes. For example, the combinatorial sequence of SEQ ID NO: 5 may be simplified to single sequence Th epitopes represented by SEQ ID NOs: 1-4. These single sequence Th epitopes may bind to target antigenic sites to enhance immunogenicity.

[0061] 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. Furthermore, the addition of a lysine residue or multiple lysine residues (e.g., KKK) to the Th epitope may improve the aqueous solubility of the peptide. Further modifications included substitution of the C-terminus with the general MHC binding motif AxTxIL (Meister et al, 1995).

[0062] 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 aimed at eliciting broad immune responses among members of genetically diverse populations. In some embodiments, SSAL peptide prototypes can be designed based on Th epitopes of measles virus and hepatitis B virus antigens modified by introducing multiple MHC binding motifs. In some embodiments, the artificial Th epitopes may simplify, add, or and / or modify 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 bearing various target antigenic sites. In some embodiments, the resulting chimeric peptides can stimulate effective antibody responses against the target antigenic sites.

[0063] The artificial Th epitopes of the present disclosure may be contiguous sequences of natural or non-natural amino acids that comprise class II MHC molecule binding sites. In some embodiments, the artificial Th epitopes may enhance or stimulate antibody responses to target antigenic sites. In some embodiments, the Th epitopes may consist 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 may include immunologically functional homologs, such as immune enhancing homologs, cross-reactive homologs, and segments thereof. In some embodiments, the functional Th homologs may further include conservative substitutions, additions, deletions, and insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues and provide the Th stimulatory function of the Th epitope.

[0064] The epitope may be attached directly to the target site. In some embodiments, the Th epitope may be attached to the target site through any heterologous spacer, for example, a peptide spacer such as Gly-Gly or (ε-N)Lys. The spacer may physically separate the Th epitope from the B cell epitope and disrupt the formation of any artificial secondary structure created by the binding of the Th epitope or functional homologue to the target antigenic site, thereby eliminating interference with Th and / or B cell responses.

[0065] Th epitopes include idealized artificial Th epitopes and combinatorial idealized artificial Th epitopes, as shown in Table 1. 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 antigenic site.

[0066] 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 1 to about 5 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 may be accomplished using natural or non-natural amino acids as described above for the target antigenic site. Table 1 identifies additional variations of functional analogs of 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 by deletion (SEQ ID NOs: 6 and 7) or by the inclusion of two amino acids at the N-terminus and C-terminus, respectively (SEQ ID NOs: 16 and 17). The differences between these two series of analog sequences do not affect the function of the Th epitopes contained in 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).

[0067] The Th epitopes of the peptide immunogen construct may be covalently linked at either the N-terminus or C-terminus of the target antigenic site to generate chimeric Th / B cell site peptide immunogens. In some embodiments, the Th epitopes may be covalently linked to the target antigenic site via chemical coupling or via direct synthesis. In some embodiments, the Th epitopes are covalently linked to the N-terminus of the target antigenic site. In other embodiments, the Th epitopes are covalently linked to the C-terminus of the target antigenic site. In certain embodiments, two or more Th epitopes are covalently linked to the target antigenic site. When two or more Th epitopes are linked to the target antigenic site, each Th epitope may have the same amino acid sequence or different amino acid sequences. Furthermore, when two or more Th epitopes are linked to the target antigenic site, the Th epitopes may be arranged in any order. For example, the Th epitopes may 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 may be covalently linked to the N-terminus of the target antigenic site while another Th epitope remains covalently linked to the C-terminus of the target antigenic site. There is no restriction on the arrangement of the Th epitopes relative to the target antigenic site.

[0068] 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 through a heterologous spacer, which is described in more detail below.

[0069] Method of synthesis The peptide immunogens of the present disclosure may be synthesized using chemical methods. In some embodiments, the peptide immunogens of the present disclosure may be synthesized using solid-phase peptide synthesis. In some embodiments, the peptides of the present invention are synthesized using automated Merrifield solid-phase peptide synthesis using t-Boc or Fmoc to protect α-NH2 or side chain amino acids.

[0070] 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 homologs of that particular peptide. The assembly of combinatorial peptides can be synthesized in one process by adding a mixture of designated protected amino acids, rather than a specific one, to designated positions during the synthesis process. Such combinatorial heterologous Th epitope peptide assemblies can allow broad Th epitope coverage in 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 Th epitope peptides of the present invention provide broad reactivity and immunogenicity in animals and patients from genetically diverse populations.

[0071] Interestingly, inconsistencies and / or errors that may be introduced during the synthesis of peptide immunogen constructs containing Th epitopes, B cell epitopes, and / or Th and B cell epitopes, in most cases, do not interfere with or prevent the desired immune response in treated animals. In fact, inconsistencies / errors that may be introduced during peptide synthesis generate multiple peptide analogs along with the 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, either as solid-phase antigens for immunodiagnostic purposes or as immunogens for vaccination purposes.

[0072] Peptide immunogen constructs containing Th epitopes are simultaneously generated in parallel with the target antigenic 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 antigenic site.

[0073] After complete assembly of the desired peptide immunogen, the solid phase resin may be treated to cleave the peptide from the resin and remove functional groups on the amino acid side chains. The free peptide may be purified by HPLC and biochemically characterized. In some embodiments, the free peptide is biochemically characterized 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.

[0074] The peptide immunogens of the invention may be synthesized using haloacetylated and cysteinylated peptides through the formation of a thioether bond. In some embodiments, a cysteine ​​may be added to the C-terminus of the Th-containing peptide, and the thiol group of the cysteine ​​residue may be N α It can be used to form a covalent bond to electrophilic groups such as chloroacetyl modified groups or maleimide derivatized α- or ε-NH2 groups of lysine residues. The resulting synthetic intermediate can be coupled to the N-terminus of a target antigenic site peptide.

[0075] Longer synthetic peptide conjugates may be synthesized using nucleic acid cloning techniques. In some embodiments, the Th epitopes of the invention may be synthesized by expressing recombinant DNA and RNA. To construct a gene expressing the Th / target antigenic site peptide of the invention, the amino acid sequence may 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 may be made. In some embodiments, a gene encoding the peptide may be made by synthesizing overlapping oligonucleotides that code for the peptide and the necessary regulatory elements. The synthetic gene may be assembled and inserted into a desired expression vector.

[0076] The synthetic nucleic acid sequences of the present disclosure may include nucleic acid sequences encoding the Th epitopes of the present invention, peptides containing the Th epitopes, their immunologically functional homologs, and nucleic acid constructs characterized by changes in non-coding sequences or encoded Th epitopes that do not alter the immunogenicity of the peptides. The synthetic genes may be inserted into a suitable cloning vector, and recombinants may be obtained and characterized. The Th epitopes and peptides containing the Th epitopes may then be expressed under conditions appropriate for the selected expression system and host. The Th epitopes or peptides may be purified and characterized.

[0077] 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.

[0078] The peptide immunogens of the present invention may be formulated as immunogenic compositions. In some embodiments, the immunogenic compositions may include adjuvants, emulsifiers, pharma- ceutically acceptable carriers, or other components that are conventionally 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 may be formulated for immediate release. In some embodiments, the compositions of the present invention may be formulated for sustained release.

[0079] 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.

[0080] In some embodiments, the pharmaceutical composition comprises Montanide™ ISA51 (an oil adjuvant composition consisting of vegetable oil and mannide oleate for the production of 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 with Emulsigen or Emulsigen D as the adjuvant.

[0081] In some embodiments, the composition is formulated for use as a vaccine. The vaccine composition may 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.

[0082] The pharmaceutical composition may be prepared as an injection, either in a liquid solution or suspension. A liquid vehicle containing the tau peptide immunogen construct may also be prepared prior to injection. The pharmaceutical composition may be administered by any suitable application mode, such as id, iv, ip, im, intranasal, oral, subcutaneous, etc., and with any 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, may also be prepared.

[0083] 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 unit dosage form of the composition may comprise about 0.5 μg to about 1 mg of peptide immunogen per kg of subject body weight. In some embodiments, a suitable unit dosage form of the composition may comprise 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 unit dosage 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 unit dosage 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.

[0084] When delivered in multiple doses, the composition may 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 may be followed by repeated (booster) doses. The dosage may be optimized according to the age, weight, and general health of the subject.

[0085] Vaccines containing a mixture of peptide immunogens may improve 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.

[0086] The immune response to the Th / target antigenic site conjugate can be improved by being delivered entrapped in or on a biodegradable microparticle. In some embodiments, the peptide immunogen can be encapsulated with or without an adjuvant, and such microparticles can carry immunostimulatory adjuvants. In some embodiments, the microparticles can be co-administered with the peptide immunogen to enhance the immune response.

[0087] immune stimulating complex The present disclosure also relates to pharmaceutical compositions comprising tau peptide immunogen constructs in the form of immune stimulating complexes with CpG oligonucleotides. Exemplary CpG oligonucleotides are shown in Table 5. Such immune stimulating complexes are particularly adapted to act as adjuvants and peptide immunogen stabilizers. The immune stimulating complexes are particulate and can efficiently present tau peptide immunogens to cells of the immune system to generate an immune response. The immune stimulating complexes can be formulated as suspensions for parenteral administration. The immune stimulating complexes can also be formulated in the form of w / o emulsions, as suspensions combined with mineral salts or in situ gelling polymers for efficient delivery of tau peptide immunogens to cells of the host's immune system after parenteral administration.

[0088] The stabilized immune stimulating complex may be formed by complexing the tau peptide immunogen construct with an anionic molecule, an oligonucleotide, a polynucleotide, or a combination thereof via electrostatic association. The stabilized immune stimulating complex may be incorporated into a pharmaceutical composition as an immunogen delivery system.

[0089] In certain embodiments, the tau peptide immunogen construct is designed to include a cationic moiety that is positively charged at a pH ranging from 5.0 to 8.0. The net charge of the cationic moiety of the 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 moiety of the tau peptide immunogen construct and expressed as a net average charge. A suitable peptide immunogen has a cationic moiety with a net average positive charge of +1. Preferably, the peptide immunogen has a net positive charge in the range of greater than +2. In some embodiments, the cationic moiety of the tau peptide immunogen construct is a heterologous spacer. 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 or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO:53).

[0090] As used herein, an "anionic molecule" refers to any molecule that is negatively charged 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 charge of -1 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.

[0091] More preferably, the anionic oligonucleotide has the formula: 5'X 1 CGX 2 3', where C and G are unmethylated, X1 is selected from the group consisting of A (adenine), G (guanine), and T (thymine), and X 2is C (cytosine) or T (thymine). Alternatively, the anionic oligonucleotide has the formula: 5'(X 3 )2CG(X 4 ) 23', where C and G are unmethylated and X 3 is selected from the group consisting of A, T, or G; 4 is C or T.

[0092] The resulting immunostimulating complex is usually in the form of particles ranging in size from 1 to 50 microns, which is a function of many factors including the relative charge stoichiometry and molecular weight of the interacting species. Particulate immunostimulating complexes have the advantage of providing adjuvant effects and upregulation of specific immune responses in vivo. Furthermore, the 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.

[0093] Purpose The peptides of the invention may be useful in medical and veterinary applications. In some embodiments, the peptides of the invention may be used as vaccines to provide protective immunity against infectious diseases, immunotherapies to treat disorders resulting from dysfunction of normal physiological processes, immunotherapies to treat cancer, and as drugs to intervene in or modify normal physiological processes.

[0094] 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 be linked to one target antigenic site.In some embodiments, the artificial Th epitope of the present disclosure can be linked to two target antigenic sites.

[0095] The artificial Th epitopes of the present disclosure may be linked to target antigenic sites to prevent and / or treat various diseases and conditions. In some embodiments, the compositions of the present invention may be used for the prevention and / or treatment of neurodegenerative diseases, infectious diseases, arteriosclerosis, prostate cancer, boar odour prevention, immunological castration of animals, treatment of endometriosis, prevention and / or treatment of breast cancer and other gynecological cancers influenced by gonadal steroid hormones, and for male and female contraception. For example, the artificial Th epitopes may be linked to antigenic sites of the following proteins: A somatostatin, which promotes growth in livestock. b. IgE to treat allergic diseases. c. The CD4 receptor on Th cells for treating and / or preventing human immunodeficiency virus (HIV) infection and immune disorders. d. Foot and mouth disease (FMD) virus capsid protein to prevent FMD. e. HIV virion epitopes for preventing and treating HIV infection. f. Plasmodium falciparum circumsporozoite antigens for preventing and treating malaria. g. CETP for preventing and treating arteriosclerosis. h. Aβ for treating or vaccinating 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 for treating atopic dermatitis.

[0096] 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, peptide immunogens containing endogenous Th epitopes of target neurodegenerative proteins can cause brain inflammation when administered to a subject. In contrast, peptide immunogen constructs containing heterologous artificial Th epitopes linked to antigenic sites of neurodegenerative proteins do not cause brain inflammation.

[0097] Amyloid beta Aβ peptides are believed to be key in the development and progression of Alzheimer's disease. Toxic forms of Aβ oligomers and Aβ fibrils have been suggested to be responsible for the death of synapses and neurons that lead to the pathology of Alzheimer's disease and dementia. Successful disease-modifying therapies for Alzheimer's disease may involve products that affect the disposition of Aβ in the brain.

[0098] 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 , Aβ 1-28 , Aβ 17-42 , or Aβ 1-42 In some embodiments, the Aβ targeting peptide can be 1-14 As used herein, the term Aβ x-y denotes the Aβ sequence from amino acid x to amino acid y of the full-length wild-type Aβ protein.

[0099] The peptide immunogens of the present disclosure may comprise multiple Aβ targeting peptides. In some embodiments, the peptide immunogens may comprise two Aβ targeting peptides. In some embodiments, the peptide immunogens may comprise one Aβ targeting peptide. 1-14 and one Aβ 1-42 In some embodiments, the peptide immunogen comprises two Aβ 1-14 In some embodiments, the peptide immunogen comprises two Aβ antigens, each linked to a different Th cell epitope as a chimeric peptide. 1-14 It may also include a targeting peptide.

[0100] The present disclosure also provides two Aβ peptides, each linked to a different Th cell epitope as a chimeric peptide. 1-14Aβ containing targeting peptides 1-14 In some embodiments, a peptide vaccine is provided. 1-14 The peptide may be formulated in a Th1-biased delivery system to minimize T cell inflammatory reactivity. In some embodiments, the chimeric Aβ 1-14 The peptides may be formulated in a Th2-biased delivery system to minimize T cell inflammatory reactivity.

[0101] 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 all purposes.

[0102] 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 is further understood that all amino acid sizes and all molecular weight or molecular weight values ​​given for polypeptides are approximate and are provided for descriptive purposes. Although methods and materials similar or equivalent to those described herein may 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 illustrative only and not intended to be limiting. EXAMPLES

[0103] Example 1 Preparation of peptides and peptide immunogenic constructs Peptides, including 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.

[0104] The Aβ vaccine (UB-311) contains two peptide immunogens, each of which encodes an N-terminal Aβ 1-14 The peptide immunogens are synthetically linked through 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 target Aβ 1-14 -εK-KKK-MvF5Th (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).

[0105] UB-311 is formulated in a Th2-biased delivery system that contains alum and inhibits the peptide Aβ 1-14 -εK-HBsAg3 and Aβ 1-14 The antibody contained an equimolar ratio of Aβ-εK-KKK-MvF5Th. The two Aβ immunogens were mixed with polyanionic CpG oligodeoxynucleotides (ODN) to form stable immunostimulatory complexes in micron-sized particles. Aluminum mineral salt (ADJU-PHOS®) was added to the final formulation along with sodium chloride for tonicity and 0.25% 2-phenoxyethanol as a preservative.

[0106] The sequences of some exemplary target antigenic sites (B cell epitopes) are shown in Table 3. Aβ as a target antigenic site covalently linked to a Th epitope 1-14 The sequences of some exemplary peptide immunogen constructs, including:

[0107] Example 2 Exclusive immunogenicity of peptide immunogenic constructs in guinea pigs targeting Aβ peptides but not Th epitopes Six guinea pigs were treated at weeks 0 and 4 with peptide immunogen constructs Aβ formulated together in an equimolar ratio. 1-14 -εK-KKK-MvF5 (SEQ ID NO: 67) and Aβ 1-14 At week 8, animals were bled and serum samples were collected to measure anti-Aβ peptide and anti-Th epitope antibody titers (log 10 As shown in Table 6, the antibody responses of all six guinea pigs were 1-42 The peptide was specifically targeted and two artificial Th epitopes (MvF5Th and HBsAg3Th) were not targeted.

[0108] Example 3 Cellular immune responses in peripheral blood mononuclear cell (PBMC) cultures from baboons and cynomolgus monkeys (macaques) Peripheral blood mononuclear cells (PBMCs) from baboons and Cynomolgus macaques were isolated by Ficoll-hypaque gradient centrifugation. For peptide-induced proliferation and cytokine production, cells (2 × 10 per well) were cultured in a 5% CO2-free medium. 5 ) alone or as individual peptide domains (Aβ 1-14 , Aβ 1-42 , UBITh®, and an irrelevant peptide). Mitogens (PHA, PWM, Con A) were used as positive controls (10 μg / mL in 1% v / v of 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 3H-TdR incorporation 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.

[0109] Cytokine analysis (IL2, IL6, IL10, IL13, TNFα, IFNγ) from cynomolgus monkey PBMC cultures was performed on aliquots of culture 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.

[0110] PBMCs were isolated from whole blood collected from macaques at weeks 15, 21, and 25.5. The isolated PBMCs were transfected with various Aβ peptides (Aβ 1-14 and Aβ 1-42 ) were cultured in the presence of

[0111] Aβ 1-14 When the peptide was added to the culture medium, no proliferative response by lymphocytes was observed. 1-42 When the peptide was added to PBMC cultures, a positive proliferative response was observed.

[0112] 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 7, three cytokines (IL2, IL6, TNFα) were upregulated by full-length Aβ peptide or PHA mitogen. 1-42 showed detectable secretion in response to peptides, but not Aβ 1-14 No response was observed to the peptide. No upregulation of cytokine secretion was detected in UBITh® AD vaccine treated samples when compared to placebo vaccine samples. 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.

[0113] Macaques are known to express Aβ 17-42 N-terminal Aβ with foreign T-helper epitopes that do not contain peptide domains 1-14Immunized with UB-311 vaccine containing only peptide immunogen, Aβ 1-42 The positive proliferation results noted in PBMC cultures in the presence of peptide were not associated with a UB-311 vaccine response, indicating that they were a background response to native Aβ.

[0114] These results suggest that Aβ 1-14 This supports the safety of the UB-311 vaccine, which contains only Aβ and foreign T-helper epitopes, and has been shown not to generate potentially inflammatory anti-autologous cellular immune responses to Aβ peptides in normal cynomolgus monkeys. In contrast, adverse events related to encephalitis in clinical trial studies of the AN-1792 vaccine were associated with the vaccine's fibrillar / aggregated Aβ. 1-42 This was due in part to the inclusion of T cell epitopes within the immunogen.

[0115] Example 4 Lymphocyte proliferation and cytokine analysis 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 (2.5 × 10 per well) were cultured in 100 wells. 5 ) were cultured alone or in triplicate with Aβ 1-14 (SEQ ID NO:56), Aβ 1-16 (SEQ ID NO:57), Aβ 1-28 (SEQ ID NO:59), Aβ 17-42 (SEQ ID NO:58), Aβ 1-42 Individual peptide domains, including (SEQ ID NO: 60) and an unrelated 38-mer peptide (p1412), were added (at a final concentration of 10 μg / mL). Cultures were incubated at 37° C. with 5% CO2 for 72 hours, after which 100 μL of supernatant was removed from each well and frozen at −70° C. for cytokine analysis. 0.5 μCi 3 H-thymidine ( 310 μL of culture medium containing H-TdR (Amersham, Cat. No. TRK637) was added to each well and after 18 h of incubation, radioisotope incorporation was detected 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. The stimulation index (SI) was calculated as the mean counts per minute (cpm) of triplicate experimental cultures with Aβ peptide divided by the mean cpm of triplicate negative control cultures. An SI > 3.0 was considered a significant proliferative response.

[0116] 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) and then cultured in the presence or absence of various Aβ peptides. As shown in Table 8, Aβ 1-14 No significant proliferative response by lymphocytes was observed when other Aβ peptides or p1412 (an irrelevant control peptide) were added to the culture medium. As expected, a positive proliferative response was observed when the PHA mitogen was added to the culture medium. The observation of a similar response to PHA before and after UB 311 immunization (p=0.87) suggests no significant changes in the immune function of the studied subjects (Table 8).

[0117] Statistical analysis. Differences in lymphocyte proliferation at weeks 0 and 16 were examined by paired t-test. Statistical significance level was determined by two-tailed tests (p<0.05). R version 2.14.1 was used for all statistical analyses.

[0118] b. Cytokine analysis Cytokine analysis (IL-2, IL-6, IL-10, TNF-α, IFN-γ) from PBMC cultures was performed on aliquots of culture medium 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)).

[0119] PBMC samples collected at weeks 0 and 16 were also tested for cytokine secretion after 3 days of culture in the presence of cells alone (negative control) or Aβ peptide, p1412 (irrelevant peptide) or PHA mitogen (positive control). The quantification range of the kit is 5–320 pg / mL. Any measured concentrations below 5 pg / mL or above 320 pg / mL were indicated as 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 (5 pg / mL) or upper (320 pg / mL) quantifiable limit, respectively. The mean concentrations of each cytokine at weeks 0 and 16 are shown in Table 9. As expected, cytokine production was significantly increased in the presence of the positive control, PHA, except for IL-2. Aβ 1-14 or other Aβ peptides was observed at baseline (week 0) and at week 16, although most values ​​appeared similar to the corresponding negative controls (cells only).

[0120] To assess changes in cellular immune responses after immunization, changes in mean cytokine concentrations from baseline to week 16 were compared with those in the negative control group 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 This observation was consistent with previous findings that Aβ secretion was significantly increased in response to the peptide. 1-42This may be due to conformational epitopes of the aggregates. 1-14 or other Aβ peptides.

[0121] c.Summary The UB-311 vaccine contains N-terminal Aβ synthetically linked to the MvF5Th and HBsAg3Th epitopes, respectively. 1-14 Two peptide immunogens carrying peptides were included. To evaluate the effect of UB-311 vaccine immunization on cellular immune responses, in vitro lymphocyte proliferation and cytokine analysis were used. As shown in Table 8, Aβ 1-14 No proliferative response by lymphocytes was observed when the Aβ peptide or any other Aβ peptide was added to the culture medium. Upregulation of cytokine secretion by lymphocytes of UB-311 vaccinated patients induced a considerable increase in four cytokines (IFN-γ, IL-6, IL-10, TNF-α) after UB-311 immunization at week 16, when compared with the levels at week 0 before treatment (Table 9). 1-14 and Aβ 1-42 The increase in cytokine release due to Th2-type T cell responses was not detected during treatment with any other Aβ peptide except for Aβ. 1-14 However, this is likely unrelated to the UB-311 vaccine response, as no upregulation was detected in Aβ alone. 1-42 The response to Aβ 1-42 It is suspected to be a background response to native Aβ that may be related to native T-helper epitopes identified in. The lack of IL-2 production in response to PHA was observed, which is consistent with the findings reported by Katial RK, et al. Clin Diagn Lab Immunol 1998; 5: 78-81 under similar experimental conditions with normal human PBMCs. In conclusion, these results show that the UB-311 vaccine did not generate a potentially inflammatory anti-autologous cellular immune response in patients with mild to moderate Alzheimer's disease who participated in a phase I clinical trial, thus further demonstrating the safety of the UB-311 vaccine.

[0122] Example 5 Promiscuous artificial Th-responsive cells could be detected in naive peripheral blood mononuclear cells (PMBCs) of normal blood donors with a moderate immunogenic inflammatory response when compared to negative controls An ELISpot assay was used to detect promiscuous artificial Th-responsive cells in naive peripheral blood mononuclear cells from normal blood donors and to assess their efficacy in inducing an inflammatory response compared with the potent mitogen phytohemagglutinin (PHA) and a negative control.

[0123] The ELISpot assay employs a sandwich enzyme-linked immunosorbent assay (ELISA) technique. To detect T cell activation, IFN-γ or related cytokines were detected as analytes. Either monoclonal or polyclonal antibodies specific for the selected analyte were precoated onto a PVDF (polyvinylidene fluoride)-backed microplate. Appropriately stimulated cells were pipetted into the wells, and the microplate was placed in a humidified 37°C CO2 incubator for the specified time. During this incubation period, the immobilized antibodies in close proximity to the secreting cells bound to the secreted analyte. After washing away the cells and unbound material, a biotinylated polyclonal antibody specific for the selected analyte was added to the well. After washing to remove unbound biotinylated antibody, 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 spots at the sites of cytokine localization, with each individual spot representing an individual analyte-secreting cell. Spots were counted using an automated ELISpot reader system or manually using a stereomicroscope.

[0124] In the in vitro studies performed, PHA at 10 μg / mL 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 responsive cells present in peripheral blood mononuclear cells of normal normal blood donors. A mixture of promiscuous artificial Th epitope peptides with 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. Briefly, 100 μL / well of PBMCs (2x10 5 The wells / plates were incubated at 37° C. in a CO2 incubator for 48 hours. The supernatants from the wells / plates were collected. The cells on the plates were washed and processed for detection of the target analyte IFN-γ.

[0125] As shown in Figure 1, representative donors 1, 2, and 3 were tested for responsive cells to promiscuous 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 showed background IFN-γ ELISPOT counts of 5-50. Moderate ELISPOT counts were detected for naive donor PBMCs cultured with UBITh®1 or UBITh®5, ranging from 20 to about 120. A mixture of multi-Th peptides of SEQ ID NOs: 33-52 was also cultured with naive donor PBMCs, resulting in ELISPOT counts, as expected, ranging from 20 to about 300. Such stimulatory responses elicited by UBITh®1 or UBITh®5 peptides are approximately 3-5-fold higher compared to the negative control.

[0126] In summary, promiscuous artificial Th-responsive cells can be easily detected in naive donor PBMCs that are ready to initiate immune responses to aid B cell antibody production and corresponding effector T cell responses by secreting signature cytokines. Here, we use IFN-γ as one example to illustrate this stimulatory nature of these Th epitope peptides. However, such stimulatory inflammatory responses are mild enough to initiate appropriate effector cell responses (B cells for antibody production, cytotoxic T cells for killing of target antigen cells) so as not to cause harmful inflammatory pathophysiological responses during the vaccination process.

[0127] [Table 1]

[0128] [Table 2]

[0129] [Table 3]

[0130] [Table 4]

[0131] [Table 5]

[0132] [Table 6]

[0133] [Table 7]

[0134]

Table 8

[0135]

Table 9

[0136]

Table 10

Claims

1. A composition for use in producing an antibody against a B cell epitope of a target antigen and for use in suppressing an inflammatory response in a patient in need thereof, comprising: the composition comprising an effective amount of a peptide immunogen for administration to the patient; the peptide immunogen having a B cell epitope covalently linked, either directly or via a heterologous spacer, to a heterologous T helper cell epitope; the heterologous T helper cell epitope is SEQ ID NO: 17 or SEQ ID NO: 6, the heterologous spacer is Gly-Gly, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:55) (wherein Xaa is any amino acid), εNLys, εNLys-Lys-Lys-Lys (SEQ ID NO:53), Lys-Lys-Lys-εNLys (SEQ ID NO:54), Lys-Lys-Lys, -NHCH(X)CH 2 SCH 2 CO-, -NHCH(X)CH 2 SCH 2 CO(εNLys)-, -NHCH(X)CH 2 S-succinimidyl-εNLys-, or -NHCH(X)CH 2 S-(succinimidyl)-, or any combination thereof; The composition, wherein the composition does not have a T helper cell epitope of any one of SEQ ID NOs: 33-52.

2. The composition described in claim 1, wherein the composition produces an inflammatory response that is at least three times lower than the inflammatory response to a positive control, the positive control being a phytohemagglutinin mitogen, and the inflammatory response being an increase in cytokine concentrations measured in peripheral blood mononuclear cells.

3. The composition described in claim 2, wherein the increase in cytokine concentration is an increase in the concentration of IFN-γ.

4. The composition of claim 2, wherein the inflammatory response is an increase in cytokine concentration, and the increase in cytokine concentration is an increase in the concentration of IL-6, IL-10, IFN-γ, and / or TNF-α measured in peripheral blood mononuclear cells of the patient.

5. The composition of claim 1, wherein the composition produces an inflammatory response that is at least three times higher than the inflammatory response to a negative control, and the inflammatory response is an increase in IFN-γ concentrations measured in peripheral blood mononuclear cells.

6. The composition of claim 1, wherein the composition does not have a carrier protein.

7. A composition described in any one of claims 1 to 6, wherein the T helper cell epitope is sequence number 6.

8. A composition described in any one of claims 1 to 6, wherein the T helper cell epitope is sequence number 17.

9. The composition described in any one of claims 2 to 7, wherein the B cell epitope is derived from beta amyloid (Aβ).

10. The composition of claim 9, wherein the B cell epitope is Aβ 1-14 of SEQ ID NO:

56.

11. The composition described in any one of claims 1 to 8, wherein the B cell epitope is not derived from beta amyloid (Aβ).

12. The composition of claim 11, wherein the B cell epitope is derived from a self-peptide or self-protein.

13. The composition of claim 12, wherein the B cell epitope is derived from alpha synuclein (α-Syn), tau (Tau), IgE EMPD, or IL-31.

14. The composition described in claim 13, wherein the B cell epitope is any one of SEQ ID NOs: 61 to 64.

15. The composition of any one of claims 1 to 8, wherein the T helper cell epitope is linked to the B cell epitope via a heterologous spacer.

16. The composition of claim 15, wherein the heterologous spacer is (εN)Lys, εNLys-Lys-Lys-Lys (SEQ ID NO: 53), or Lys-Lys-Lys-εNLys (SEQ ID NO: 54).

17. The composition of any one of claims 1 to 8, wherein the composition is for intravenous administration, subcutaneous administration, intradermal administration, or intramuscular administration.

18. The composition of claim 17, wherein the composition comprises 100 μg to 1000 μg of peptide immunogen.

19. The composition of claim 17, wherein the composition comprises 150 μg to 750 μg of peptide immunogen.

20. The peptide immunogen of claim 1, wherein A is an amino acid or an immunostimulatory sequence: B is a heterologous spacer, Th is a T helper cell epitope; the target antigenic site is a B cell epitope; X is the α-COOH of an amino acid, -CONH 2 ; The composition according to any one of claims 1 to 8, wherein n is 1 to 10, m is 1, and o is 1: (A) n - (target antigenic site) - (B) o - (Th) m -X, (A) n - (B) o - (Th) m - (B) o - (target antigenic site) -X, (A) n - (Th) m - (B) o - (target antigenic site) -X, (target antigenic site)-(B) o -(Th) m -(A) n -X, or (Th) m - (B) o - (target antigenic site) - (A) n -X.

21. The B cell epitope is Aβ 1-14 of SEQ ID NO:56, said T helper cell epitope linked to said B cell epitope via said heterologous spacer; the heterologous spacer is (εN)Lys, εNLys-Lys-Lys-Lys (SEQ ID NO:53), or Lys-Lys-Lys-εNLys (SEQ ID NO:54); the composition is for intramuscular administration, the composition having 100 μg to 300 μg of the peptide immunogen; The composition of claim 1, wherein the composition produces an inflammatory response that is at least three times lower than the inflammatory response to a positive control, the positive control being a phytohemagglutinin mitogen, and the inflammatory response being an increase in the concentration of IFN-γ measured in peripheral blood mononuclear cells.

22. The composition of claim 21, wherein the composition produces an inflammatory response that is at least three times greater than the inflammatory response to a negative control, and the inflammatory response is an increase in the concentration of IFN-γ measured in peripheral blood mononuclear cells.

23. The method of claim 22, wherein the B cell epitope is not derived from beta amyloid (Aβ), and the B cell epitope does not include an endogenous T helper cell epitope of the target antigen; said T helper cell epitope linked to said B cell epitope via said heterologous spacer; the heterologous spacer is (εN)Lys, εNLys-Lys-Lys-Lys (SEQ ID NO:53), or Lys-Lys-Lys-εNLys (SEQ ID NO:54); the composition is for intramuscular administration, the composition having 100 μg to 300 μg of the peptide immunogen; The composition of claim 1, wherein the composition produces an inflammatory response that is at least three times lower than the inflammatory response to a positive control, the positive control being a phytohemagglutinin mitogen, and the inflammatory response being an increase in the concentration of IFN-γ measured in peripheral blood mononuclear cells.

24. The composition described in claim 23, wherein the B cell epitope is any one of SEQ ID NOs: 61 to 64.

25. The composition described in claim 23, wherein the composition produces an inflammatory response that is at least three times higher than the inflammatory response to a negative control, and the inflammatory response is an increase in the concentration of IFN-γ measured in peripheral blood mononuclear cells.

26. A composition for use in producing an antibody against a B cell epitope of a target antigen, the composition not inducing an inflammatory pathophysiological response in a patient in need thereof, comprising: the composition comprising an effective amount of a peptide immunogen for administration to the patient; the peptide immunogen has a B cell epitope covalently linked to a heterologous T helper cell epitope via a heterologous spacer; the B cell epitope is Aβ 1-14 of SEQ ID NO:56; the heterologous T helper cell epitope is SEQ ID NO: 17 or SEQ ID NO: 6, the heterologous spacer is εNLys-Lys-Lys-Lys (SEQ ID NO: 53) or Lys-Lys-Lys-εNLys (SEQ ID NO: 54); the composition does not have a T helper cell epitope of any one of SEQ ID NOs: 33-52; the composition produces an inflammatory response that is at least three times lower than the inflammatory response to a positive control, the positive control being a phytohemagglutinin mitogen, and the inflammatory response being an increase in the concentration of IFN-γ measured in peripheral blood mononuclear cells; The composition, wherein the composition produces an inflammatory response that is at least three times greater than the inflammatory response to a negative control, the inflammatory response being an increase in the concentration of IFN-γ measured in peripheral blood mononuclear cells.

27. The composition of claim 26, wherein the composition does not have a carrier protein.

28. A composition for use in producing an antibody against a B cell epitope of a target antigen, the composition not inducing an inflammatory pathophysiological response in a patient in need thereof, comprising: the composition comprising an effective amount of a peptide immunogen for administration to the patient; the peptide immunogen having a B cell epitope covalently linked to a heterologous T helper cell epitope via a heterologous spacer; the B cell epitope is derived from a self-peptide or self-protein, the B cell epitope is not derived from β-amyloid (Aβ), and the B cell epitope does not include an endogenous T helper cell epitope of the target antigen; the T helper cell epitope is SEQ ID NO: 17 or SEQ ID NO: 6, the heterologous spacer is Gly-Gly, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:55) (wherein Xaa is any amino acid), εNLys, εNLys-Lys-Lys-Lys (SEQ ID NO:53), Lys-Lys-Lys-εNLys (SEQ ID NO:54), Lys-Lys-Lys, -NHCH(X)CH 2 SCH 2 CO-, -NHCH(X)CH 2 SCH 2 CO(εNLys)-, -NHCH(X)CH 2 S-succinimidyl-εNLys-, or -NHCH(X)CH 2 S-(succinimidyl)-; The composition, wherein the composition does not have a T helper cell epitope of any one of SEQ ID NOs: 33-52.

29. The composition of claim 28, wherein the heterologous spacer is εNLys, εNLys-Lys-Lys-Lys (sequence number 53) or Lys-Lys-Lys-εNLys (sequence number 54).

30. The composition described in claim 28, wherein the B cell epitope is any one of SEQ ID NOs: 61 to 64.

31. The composition described in claim 28, wherein the composition produces an inflammatory response that is at least three times lower than the inflammatory response to a positive control, the positive control being a phytohemagglutinin mitogen, and the inflammatory response being an increase in cytokine concentrations measured in peripheral blood mononuclear cells.

32. The composition of claim 28, wherein the composition does not have a carrier protein.

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