Methods and compositions for reducing non-tumor disease

Abstract

Provided herein are compositions and methods related to improving general health in a subject and inhibiting age-related disease in a subject, such as chronic inflammatory diseases, by administering vaccines that include one or more REDN peptides.

Description

CALV.050WO PATENT METHODS AND COMPOSITIONS FOR REDUCING NON-TUMOR DISEASE INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 627,933, filed February 1, 2024, which is hereby incorporated by reference in its entirety. Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57. REFERENCE TO SEQUENCE LISTING

[0002] The present application is filed with a Sequence Listing in Electronic format. The Sequence Listing is provided as a file entitled “SEQLISTING_CALV050WO,” created January 23, 2025, which is approximately 28.2 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. BACKGROUND Field

[0003] The present application relates generally to compositions and methods for improving general health and / or reducing age-related disease, and methods of making and using such compositions. More particularly, the compositions include vaccines having one or more RNA error derived neoantigen (REDN) peptides that improve health in a subject and / or reduce age- related disorders. Also provided herein are compositions for treating chronic diseases, and methods of using the compositions for treating chronic diseases, including, for example, arthritis, dementia, or heart disease. Also provided herein are methods and compositions to increase longevity and healthspan. Description of the Related Art

[0004] There are multiple theories about what causes aging and deaths. A common theme among several of these theories is that inflammation is a primary cause. For example, the “bad cell” concept is that senescent cells that are not removed by the immune system cause inflammation that inhibits stem cells, causing aging. It has been shown in mouse models thatremoving these old stem cells reduces chronic diseases, and increases lifespan and healthspan. Tumors also elicit inflammatory responses. SUMMARY

[0005] Some embodiments provided herein relate to vaccines. In an aspect, disclosed herein are vaccines to reduce the incidence of death and extend healthy life in a subject. In some embodiments, the vaccine includes a RNA error derived neoantigen (REDN) peptide or a nucleic acid encoding a REDN peptide. In some embodiments, the vaccine is administered to reduce the incidence of a non-tumor age-related disease in the subject.

[0006] In some embodiments, the vaccine is suitable for administration in humans. In some embodiments, the vaccine is suitable for administration in a companion animal. In some embodiments, the companion animal is a dog.

[0007] In some embodiments, the vaccine comprises a sequence of at least 8 amino acids having at least 95% sequence identity to a sequence of at least 8 amino as set forth in any of SEQ ID NOs: 1-31, or a nucleic acid encoding a sequence of at least 8 amino as set forth in any of SEQ ID NOs: 1-31. In some embodiments, the REDN peptide has a sequence comprising at least 8 amino acids of the sequences set forth in any of SEQ ID NOs: 1-31.

[0008] In some embodiments, the REDN peptide has a sequence set forth in any of SEQ ID NOs: 1-31. In some embodiment, the vaccines further include an adjuvant. In some embodiments, the adjuvant includes ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), Hiltonol, IL-12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, oil- in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), Pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex adjuvant formulation (SAF), synthetic polynucleotides (poly IC / poly AU), TiterMax Tomatine, Vaxfectin, XtendIII, or Zymosan.

[0009] Some embodiments provided herein relate to methods of prolonging life in a subject. In some embodiments, the method includes administering the vaccine as described herein. Further disclosed herein are methods of inhibiting non-tumor age-related disease in a subject. In some embodiments, the method includes administering the vaccine as described herein. In some embodiments, the disease is a chronic inflammatory disease. In some embodiments, the disease is heart disease, Alzheimer’s disease, or diabetes.

[0010] Some embodiments provided herein relate to methods of treating, ameliorating, or inhibiting a chronic disease in a subject. In some embodiments, the methods include administering any vaccine as described herein, or any vaccine prepared by the methods of making vaccines described herein. In some embodiments, the chronic disease is arthritis, asthma, chronic obstructive pulmonary disease, dementia, diabetes, fatty liver, epilepsy, heart disease, hypertension, pain, or stroke.

[0011] In some embodiments, disclosed herein are methods of improving general health in a subject. In some embodiments, the method includes administering a vaccine as described herein.

[0012] Some embodiments provided herein relate to methods of preparing a vaccine. In some embodiments, the method includes obtaining a biological sample from a subject with advanced age, contacting the biological sample with a peptide array including one or more REDN peptides, detecting binding of the biological sample to a REDN peptide of the peptide array, and preparing a vaccine including the REDN peptide. In some embodiments, the REDN peptide is detected as being bound by the biological sample.

[0013] In another aspect, disclosed herein are methods of preparing a vaccine against one or more age-related diseases. In some embodiments, the method includes obtaining a first biological sample from a young subject; injecting senescent cells from a subject with advanced age into the young subject; obtaining a second biological sample from the young subject; applying the first biological sample to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides; measuring immune response of the first biological sample to the peptide array to obtain a first REDN signature; applying the second biological sample to a peptide array comprising a plurality of REDN peptides; measuring immune response of the second biological sample to the peptide array to obtain a second REDN signature; comparing the first REDN signature and the second REDN signature to identify at least one REDN peptide of theplurality of REDN peptides which provokes a greater immune response with the second biological sample; and preparing a vaccine comprising the one or more REDN peptides identified.

[0014] In another aspect, disclosed herein are methods of preparing a vaccine against one or more age-related diseases, the method including obtaining one or more biological samples from one or more young subjects; obtaining one or more biological samples from one or more subjects of first advanced age; applying each of the biological samples obtained in steps a) and b) to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides; measuring immune response of each of the biological samples to the peptide array to obtain a REDN signature for each of the biological samples; comparing the REDN signatures for each of the biological samples to identify one or more REDN peptides of the plurality of REDN peptides; and preparing a vaccine comprising the one or more REDN peptides identified. In some embodiments, the method further includes obtaining one or more biological samples from one or more subjects of a second advanced age.

[0015] In some embodiments, the REDN peptides of the peptide array included a peptide having a sequence as set forth in any of SEQ ID NOs: 1-31.

[0016] In some embodiments, the vaccines further include an adjuvant. In some embodiments, the adjuvant includes ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), Hiltonol, IL-12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, oil- in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), Pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex adjuvant formulation (SAF), synthetic polynucleotides (poly IC / poly AU), TiterMax Tomatine, Vaxfectin, XtendIII, or Zymosan.

[0017] Disclosed herein are methods of prolonging life and / or healthspan in a subject. Further disclosed herein are methods of inhibiting non-tumor age-related disease in a subject. Further disclosed herein are methods of improving general health in a subject. In someembodiments, the methods include administering the vaccine prepared by the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0019] Figure 1 illustrates exemplary IFN-γ enzyme-linked immunosorbent spot (ELISPOT) T-cell responses to individual Vaccination Against Canine Cancer Study (VACCS) peptides or VACCS peptide pool in VACCS vaccinated dogs who are still living or have died of non–cancer related causes.

[0020] Figure 2 is a graph depicting CD4 IFN-γ production six months after VACCS pool stimulation measured by ICS.

[0021] Figure 3 is a graph depicting CD4 TNFα production six months after VACCS pool stimulation measured by ICS.

[0022] Figure 4 depicts REDNs included in an embodiment of a DNA vaccine and which were included in an embodiment of a peptide vaccine.

[0023] Figure 5 illustrates a graph depicting non-cancer related deaths in dogs enrolled in the VACCS vaccine trial, and a comparison of VACCS vaccinated vs placebo vaccinated dogs.

[0024] Figure 6 schematically illustrates a first exemplary method to develop anti- aging vaccines.

[0025] Figure 7 schematically illustrates a second exemplary method to develop anti- aging vaccines. DETAILED DESCRIPTION

[0026] Throughout the description, including in the Summary and Drawings, reference is made to particular features of the disclosure. It is to be understood that the disclosure in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the disclosure,or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments of the disclosure, and in the disclosure generally.

[0027] Embodiments provided herein relate generally to compositions and methods related to improving health in a subject and inhibiting age-related disease in a subject, such as chronic inflammatory diseases, by administering vaccines that include one or more REDN peptides.

[0028] There are numerous and often related causes of aging ( López-Otín, Cell 186, 2023). Most of these, particularly those causing inflammation, are also often causes of chronic diseases (Furman, Nat Med 25, 2019; He, Cell 169, 2017). One of the most studied causes of both aging and chronic disease are senescent cells (SC). Embodiments of the present disclosure relate to compositions and methods for creating vaccines to eliminate, reduce, or inhibit SCs with the aim of improving healthy longevity and reducing the incidence of chronic diseases.

[0029] Experimental evidence in multiple organisms supports focusing on SCs. In mice, transfer of SCs from old mice to young mice, increases chronic ailments and shortens life span in the young mice (Xu, Nat Med 24, 2018). On the other hand, treating older mice with drugs (senolytics) that kill SCs increases longevity and decreases the incidence of specific chronic diseases (Xu, Nat Med 24, 2018; Kirkland, EBioMed 21, 2017; Zhu, Aging Cell 21, 2017). SCs effectively fulfill Koch’s postulates for being a causative agent in aging.

[0030] Based on these types of results, there is considerable effort to explore using senolytic drugs to decrease the incidence of particular chronic diseases and, hopefully, contribute to lifespan in humans (Chaib, Nat Med 28, 2022). However, several senolytics are associated with acute and long-term side effects (Naqvi, Cancer 126, 2020; Suvarna, Eur J Pharmacol 862, 2019). Suda et al. have demonstrated an interesting alternative approach (Suda, Nature Aging 1, 2021). They found that glycoprotein nonmetastatic melanoma protein B (GPNMB) was preferentially expressed on SCs in mice. By vaccinating older or diseased mice with this protein, they showed positive effects both on longevity and disease severity. The implication is that vaccination against SCs improves normal and pathological age-related phenotypes and increases lifespan in mice. This concept is further demonstrated by two murine studies that tested chimeric antigen receptor (CAR) T cells directed against the urokinase plasminogen activator receptor (uPAR) (Amor, Nat 583, 2020) and Natural Killer Group 2 Member D (NKG2D) ligands (Yang, Sci Transl Med, 2023),both overexpressed in SCs. However, GPNMB, uPAR and NKG2D ligands are self-antigens. In the cancer field, self-antigens that worked in mouse models as vaccines did not translate to humans (Zhong, BMC Genom 21, 2020).

[0031] Embodiments of the present disclosure relate to compositions and methods for reducing SCs using vaccines, thereby reducing and / or preventing age-related and chronic diseases. Previous studies have demonstrated that tumors in mice, dogs, and humans create frameshift neoantigens through RNA misprocessing that are shared across tumor types and subjects (Shen, Sci Rep 9, 2019). Previous studies have also shown that these neoantigens (referred to herein as RNA error derived neoantigens (REDNs)) confer both therapeutic and prophylactic protection in mouse models (Peterson, BMC Immunol 21, 2020). Previous studies have further found that these REDNs are protective as a preventative cancer vaccine in dogs (unpublished). In some embodiments, these REDNs are capable of provoking an immune response in many different individuals (rather than being specific to an individual), allowing for the production of off-the- shelf, preventative vaccines. Previous work has developed a photolithography system (Shen, RSC Adv 10, 2020) to create peptide arrays displaying all the REDNs that can be produced in a tumor. This allows a simple blood-based screen for antibodies elicited by the tumor produced REDNs (Shen, J Transl Med 21, 2023).

[0032] Recently, it has been shown that SCs, like tumors, also have a significant increase in RNA misprocessing (Harries, FEBS J 290, 2023; Wang, Sci Rep 8, 2018). It has been proposed that this misprocessing is a hallmark of SCs (Harries, FEBS J 290, 2023). Though it has not been shown that misprocessing results in the formation of REDNs, it seems highly likely. This raises the possibility of creating a REDN based vaccine to eliminate SCs. There are about 2 million different REDNs that are bioinformatically predicted that could theoretically be produced by various mechanisms in humans, and approximately 1.4 million in dogs). These mechanisms include, for example, exon skipping, mis-initiation of translation (Shen, Sci Rep 9, 2019), or tryptophan pausing (Bartok, Nat 590, 2021). To find those made by SCs, the RNA of SCs could be sequenced, or existing databases can be searched. However, this would not reveal which were made as REDNs or elicited an immune response.

[0033] Embodiments provided herein relate to methods of determining which REDNs elicit an immune response. In some embodiments, REDN peptide arrays are used to determine which REDNs elicit an immune response. In some embodiments, SCs from old mice are injectedinto young mice and assayed for new antibodies against the SCs. In some embodiments, biological samples from old and young animals (such as people or dogs) may be applied to REDN peptide arrays to survey for differences in reactivity to the REDNs. In some embodiments, candidate REDNs that have mouse homologs are tested as SC vaccines in old mice. There is approximately a 30% overlap of predicted REDNs between species. Previous studies have shown REDNs produced by dogs can serve as cancer protective vaccines in mice (Zhang, Sci Rep 8, 2018).

[0034] As used herein, the term “vaccine” means a composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example, a bacterial or viral pathogen, or to a cellular constituent correlated with a pathological condition, such as an age-related and / or chronic inflammatory disease. A vaccine may include a polynucleotide, a peptide or polypeptide, a virus, a bacterium, a cell or one or more cellular constituents. In some cases, the virus, bacteria or cell may be inactivated or attenuated to prevent or reduce the likelihood of infection, while maintaining the immunogenicity of the vaccine constituent. The immunogenic material may include live- attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides or DNA derived from them. In some cases, the vaccine is a subunit vaccine, which is an immunizing agent that has been treated to remove traces of nucleic acid (such as viral nucleic acid) so that only protein subunits remain. The subunits have less risk of causing adverse reactions. The vaccine can also be a live vaccine, which is a vaccine prepared from living attenuated organisms or from viruses that have been attenuated but can still replicate in the cells of the host organism. The immunogenic material for an age-related vaccine may include, for example, a protein or peptide expressed by a senescent cell. Vaccines may elicit both prophylactic (preventative) and therapeutic responses.

[0035] As used herein, the term “vector” means a virus, bacterium, or other microbe, or a nucleic acid, used to deliver an antigen or a gene for an antigen, as part of a vaccine. A nucleic acid vector is a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other geneticelements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.

[0036] As used herein, the term “nucleic acid” refers to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (for example, A, T, G, C), this also includes an RNA sequence (for example, A, U, G, C) in which “U” replaces “T.” ”Nucleotide” includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

[0037] Conventional notation is used herein to describe nucleotide sequences: the left- hand end of a single- stranded nucleotide sequence is the 5 ‘-end; the left-hand direction of a double- stranded nucleotide sequence is referred to as the 5’-direction. The direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand;” sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5’ to the 5’- end of the RNA transcript are referred to as “upstream sequences;” sequences on the DNA strand having the same sequence as the RNA and which are 3’ to the 3’ end of the coding RNA transcript are referred to as ”downstream sequences.” “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

[0038] As used herein, “encode(s)” or “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (for example, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non- coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0039] As used herein, “inhibit,” “inhibiting,” and “inhibition” refer to decreasing an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0040] The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof.

[0041] As used herein, the term “antibody” encompasses, but is not limited to, whole immunoglobulin (for example, an intact antibody) of any class. Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains. Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domainof the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), for example, IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0042] The term “variable” is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each include four FR regions, largely adopting a beta sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A. et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity.

[0043] As used herein, the term “antibody” or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab′)2, Fab′, Fab, sFv, scFv, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain FS 1-78, FS 6-21, FS SMC1A binding activity are included within the meaning of the term “antibody or fragment thereof” Such antibodies andfragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

[0044] Also included within the meaning of “antibody or fragments thereof” are conjugates of antibody fragments and antigen binding proteins (single chain antibodies).

[0045] The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove / add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment.

[0046] The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, for example, the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

[0047] The term “sequence identity” as used herein refers to the similarity between amino acid sequences is expressed in terms of the similarity between the sequences. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); thehigher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

[0048] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math.2:682, 1981; Needleman and Wunsch, J. Mol. Biol.48:643, 1970; Higgins and Sharp, Gene 73:437, 1988; Higgins and Sharp, CAB IOS 5:351, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:319, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0049] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 415:603, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0050] Homologs and variants of a polypeptide are typically characterized by possession of at least 75%, for example at least 80%, sequence identity counted over the full- length alignment with the amino acid sequence using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

[0051] The term “healthspan” refers to the period of an individual’s life during which the individual remains generally healthy and free from serious illness or disability. In some embodiments, healthspan is a concept that goes beyond the traditional measure of lifespan, which focuses solely on the length of time a person lives. In some embodiments, healthspan relates to the quality of life and overall health, encompassing physical, mental, and / or social well-being of the individual.

[0052] The term “companion animal” refers to a domesticated animal that is kept primarily for a person's company, entertainment, or as a source of emotional support, and may include, for example, dogs, cats, birds, rabbits, and small rodents. Vaccines

[0053] Tumor cells produce many frameshift variants through aberrant RNA processing. These RNA error derived neoantigen (REDN) peptides produce an immune response to the tumor cells. In some embodiments, vaccines against REDNs reduce the incidence of nascent tumors and therefore reduce tumor induced inflammation. In some embodiments, the vaccines provided herein that include REDNs were administered to dogs in a trial to investigate use of the vaccines in treating cancer. Surprisingly and unexpectedly, administration of an REDN vaccine reduced non-tumor deaths and may have increased energy and activity, compared to the control animals. Thus, in addition to targeting tumor cells, in some embodiments, the vaccines provided herein may inhibit, reduce, and / or eliminate senescent cells, thereby reducing non-tumor deaths associated with aging. In some embodiments, the vaccine may reduce micro tumors, therefore generally reducing inflammation and non-tumor chronic diseases.

[0054] Some embodiments provided herein relate to vaccines that include RNA error derived neoantigen (REDN) peptides generated by RNA mis-processing. As used herein, RNA mis-processing may include insertions or deletions that arise during transcription and mis-splicing or trans-splicing of an RNA transcript. RNA mis-processing may also include mis-initiation at exon 1 by the ribosome, frameshifting by the ribosome at tryptophan codons or translation of non- coding RNA. In some instances, transcription insertions / deletions (“indels”) arise through transcription of microsatellite repeat areas. Such RNA mis-processing errors may cause a frameshift in the RNA, creating an mRNA variant transcript and, following translation, a RNA error derived neoantigen (REDN) peptide.

[0055] In some embodiments, the vaccines reduce the incidence of death and / or extend healthy life in a subject. As used herein, the term “subject” refers an animal, typically a mammal, including, for example humans, non-human primates, farm animals such as cattle, horses, sheep, goats, or swine, or domestic animals such as rabbits, dogs, and cats, or laboratory animals such as rats, mice, or guinea pigs. As used herein, “patient” means a human subject. In some embodiments, the vaccines reduce the incidence of death and / or extend healthy life in humans. In some embodiments, the vaccines reduce the incidence of death and extend healthy life in dogs.

[0056] In some embodiments, the vaccines include a REDN peptide having a sequence set forth in any of SEQ ID NOs: 1-31 or a nucleic acid (such as DNA or RNA) encoding a sequence set forth in any of SEQ ID NOs: 1-31. SEQ ID NOs: 1-31 relate to sequence of REDN peptides for administration to dogs. In some embodiments, the vaccines include a REDN peptide corresponding to the human homolog of any of the sequences set forth in SEQ ID NOs: 1-31, or a nucleic acid encoding a REDN peptide corresponding to the human homolog of any of the sequences set forth in SEQ ID NOs: 1-31. In some embodiments, the vaccines include a peptide having a sequence set forth in any of SEQ ID NOs: 1-31 (or their equivalents in humans). In some embodiments, the vaccines include a nucleic acid encoding a peptide having a sequence set forth in any of SEQ ID NOs: 1-31 (or their equivalents in humans).

[0057] In some embodiments, the vaccine is suitable for administration in a subject. In some embodiments, the vaccine is suitable for administration in humans. In some embodiments, the vaccine is suitable for administration in dogs. In some embodiments, the vaccine is suitable for administration in mice.

[0058] In some embodiments, the vaccine comprises a sequence of at least 8 amino acids having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence of at least 8 amino as set forth in any of SEQ ID NOs: 1-31. In some embodiments, the vaccine comprises a sequence of 8 to 15 amino acids having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence of the same length (between 8 to 15 amino acids) as set forth in any of SEQ ID NOs: 1-31. Without being bound by theory, eight amino acids is the minimal length of a peptide for MHC I presentation. Accordingly, a vaccine may be prepared which includes a REDN peptide fragment that is at least 8 amino acids in length.

[0059] In some embodiments, the REDN peptide has a sequence comprising at least 8 amino acids of the sequences set forth in any of SEQ ID NOs: 1-31, or a nucleic acid encoding at least 8 amino acids of the sequences set forth in any of SEQ ID NOs: 1-31. In some embodiments, the REDN peptide has a sequence comprising 8 to 15 amino acids of the sequences set forth in any of SEQ ID NOs: 1-31. In some embodiments, the vaccine includes a nucleic acid encoding 8 to 15 amino acids of the sequences set forth in any of SEQ ID NOs: 1-31.

[0060] In some embodiments, the vaccines further include an adjuvant. As used herein, the term “adjuvant” means a vehicle used to enhance antigenicity; such as a suspension of minerals (alum, aluminum hydroxide, aluminum phosphate) on which antigen is adsorbed; or water-in- oil emulsion in which antigen solution is emulsified in oil (MF-59, Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund’s complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and / or causes influx of macrophages). Adjuvants also include immunostimulatory molecules, such as cytokines, costimulatory molecules, and for example, immunostimulatory DNA or RNA (for example, Hiltonol®) molecules.

[0061] The vaccines also can be formulated to contain an adjuvant in order to enhance the immunological response. Suitable adjuvants include, but are not limited to, lysolecithin, pluronic polyols, polyanions, other peptides, oil emulsions, and potentially useful human adjuvants such as Bacillus Calmette Guerin (BCG) and Corynebacterium parvum. Adjuvants for inclusion in the inventive vaccines desirably are safe, well tolerated, such as QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-1, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59 (as described in, for example, Kim et al., Vaccine, 18: 597 (2000)). Other adjuvants that can be included in the vaccines include lectins, growth factors, cytokines, and lymphokines (for example, alpha- interferon, gamma-interferon, platelet derived growth factor (PDGF), gCSF, gMCSF, TNF, epidermal growth factor (EGF), IL- 1, IL-2, IL-4, IL-6, IL-8, IL-10, and IL- 12).ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, Alum, Aluminum phosphate, Aluminum potassium sulfate, Bordetella pertussis, Calcitriol, Chitosan, Cholera toxin, CpG, Dibutyl phthalate, Dimethyldioctadecylammonium bromide (DDA), Freund’s adjuvant, Freund’s complete, Freund’s incomplete (IF A), GM-CSF, GMDP, Gamma Inulin, Glycerol, HBSS (Hank’s Balanced Salt Solution), Hiltonol, Imiquimod, Interferon-Gamma, ISCOM, Lipid Core Peptide(LCP), Lipofectin, Lipopolysaccharide (LPS), Liposomes, MF59, MLP+TDM, Monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, Oil-in-water emulsion, P1005 (non- ionic copolymer), Pam3Cys (lipoprotein), Pertussis toxin, Poloxamer, QS21, RaLPS, Ribi, Saponin, Seppic ISA 720, Soybean Oil, Squalene, Syntex Adjuvant Formulation (SAF), Synthetic polynucleotides (poly IC / poly AU), TiterMax Tomatine, Vaxfectin, Xtendlll, and Zymosan.

[0062] Cosolvents may be added to a vaccine. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.

[0063] Supplementary compounds (for example, preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral, and antifungal agents) can also be incorporated into the vaccines. Vaccines may therefore include preservatives, antioxidants, and antimicrobial agents.

[0064] Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelf life of the vaccine. Suitable preservatives are known in the art and include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate. Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.

[0065] In certain embodiments, the methods and vaccines disclosed herein may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers of use are conventional. Remington’s Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, vaccines to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Methods Including Vaccine Administration

[0066] Further disclosed herein are methods of prolonging life in a subject. In some embodiments, prolonging life means causing the subject to live beyond the life expectancy of the subject. The term “life expectancy” refers to the number of years a subject can expect to live. By definition, life expectancy is based on an estimate of the average age that members of a particular population group will be when they die. In some embodiments, the methods include administering a vaccine as described herein to a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a dog.

[0067] Further disclosed are methods of inhibiting non-tumor age-related disease in a subject. Age-related diseases include cardiovascular disease, cancer, arthritis, dementia, cataract, osteoporosis, diabetes, hypertension, and Alzheimer's disease. A non-tumor age-related disease refers to any age-related disease that is not cancer. In some embodiments, the methods include administering a vaccine as described herein to a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a dog.

[0068] In some embodiments, the disease is a chronic inflammatory disease. Examples of chronic inflammatory diseases include heart disease, rheumatoid arthritis, Alzheimer’s disease, cognitive diseases, metabolic diseases, and diabetes.

[0069] Further disclosed herein are methods of improving general health and the length of time the of better health, such as healthspan, in a subject. Improving general health may include improving subjective well-being and improving energy levels in a subject. In some embodiments, the methods include administering a vaccine as described herein to a subject.

[0070] Some embodiments provided herein relate to methods of reducing, delaying, preventing, treating, inhibiting, or ameliorating a chronic disease in a subject. In some embodiments, the methods include administering a vaccine described herein to a subject. As usedherein, the term “chronic disease” has its ordinary meaning as understood in light of the specification, and refers to a disease or condition that usually lasts for more than three months and may get worse over time. A chronic disease may often be controlled, but not cured.

[0071] As used herein, the terms “treating,” “treatment,” “therapeutic,” or “therapy” do not necessarily mean total cure or abolition of the disease or condition.

[0072] As used herein, the term “inhibit” refers to the reduction or prevention of a disease or disorder. The reduction can be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values. As used herein, the term “delay” refers to a slowing, postponement, or deferment of an event, such as a disease or disorder, to a time which is later than would otherwise be expected. The delay can be a delay of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay are not to be construed as necessarily indicating a 100% inhibition or delay. A partial inhibition or delay may be realized.

[0073] As disclosed herein, “amelioration” has its ordinary meaning as understood in light of the specification, and is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the pathological condition being treated. In some embodiments, the method can completely inhibit, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the pathological condition, or at least one or more of the symptoms that characterize the pathological condition. In some embodiments, the method can delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

[0074] As used herein, a “subject” refers to an animal that is the object of treatment, inhibition, or amelioration, observation, or experiment. “Animal” includes cold- and warm- blooded vertebrates and / or invertebrates such as fish, shellfish, or reptiles and, in particular, mammals. “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and / or apes, and, in particular, humans. In some embodiments, the subject is human.

[0075] Some embodiments disclosed herein related to selecting a subject or patient in need. In some embodiments, a patient is selected who is in need of treatment of a chronic disease or an age-related disease or disorder, such as heart disease, arthritis, diabetes, Alzheimer’s disease,dementia, cardiovascular disease, or any diseases described herein. In some embodiments, a patient is selected who has previously been treated for a disease or disorder described herein. In some embodiments, a patient is selected who has previously been treated for being at risk of a disease or disorder described herein. In some embodiments, a patient is selected who has developed a recurrence of a disease or disorder described herein. In some embodiments, a patient is selected who has developed resistance to a disease or disorder described herein. In some embodiments, a patient is selected who may have any combination of the aforementioned selection criteria.

[0076] In some embodiments, a booster vaccine is administered to the subject after an amount of time has passed since initial administration of the vaccine. In some embodiments, the amount of time is less than a year, about a year, about two years, or a time frame therebetween.

[0077] Any route of administration can be used to deliver the vaccine to the subject. Indeed, although more than one route can be used to administer the vaccine, a particular route can provide a more immediate and more effective reaction than another route. Exemplary routes of administration for contact or in vivo delivery which a vaccine can optionally be formulated include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, ophthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, or intralymphatic.

[0078] Formulations suitable for parenteral administration include aqueous and non- aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable, or synthetic oils.

[0079] In some examples, the vaccine is administered via intramuscular injection, for example, using a syringe or needleless delivery device. In this respect, this disclosure also provides a syringe or a needleless delivery device including the vaccine. The vaccine also can be applied or instilled into body cavities, absorbed through the skin (for example, via a transdermal patch), inhaled, ingested, topically applied to tissue, or administered parenterally via, for instance, intravenous, peritoneal, or intraarterial administration.

[0080] The vaccine can be administered in or on a device that allows controlled or sustained release, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant. Implants, devices, such as an implantable device, for example, a mechanical reservoir or an implant or a device made of a polymeric composition, are particularly useful for administration of the vaccine. The vaccine also can be administered in the form of a sustained-release formulation including, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), and / or a polylactic-glycolic acid. It can also be administered using a gene gun via microparticles.

[0081] The dose of the vaccine administered will depend on a number of factors, including the size of a target tissue, the extent of any side-effects, the particular route of administration, and the like. The dose ideally includes an “effective amount” of the vaccine, for example, a dose which provokes a desired immune response in the subject. As used herein, the term “effective amount” includes an amount of agent, such as an agent that is sufficient to generate a desired response, such an immune response. In some examples, an “effective amount” is one that treats (including prophylaxis) one or more symptoms and / or underlying causes of any of a disorder or disease, for example to treat and / or reduce an age-related and / or chronic inflammatory disease in a subject. In one example, an effective amount is a therapeutically effective amount. In one example, an effective amount is an amount that reduces one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with an age-related and / or chronic inflammatory disease. The desired immune response can entail production of antibodies, protection upon subsequent challenge, immune tolerance, immune cell activation, and the like. One dose or multiple doses of the vaccine can be administered to a mammal to elicit an immune response with desired characteristics, including the production of specific antibodies, or the production of functional T cells.

[0082] The term “therapeutically effective amount” is used to indicate an amount of an active compound, or pharmaceutical agent, that elicits the biological or medicinal response indicated. For example, a therapeutically effective amount of compound can be the amount needed to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being administered the therapy. This response may occur in a tissue, system, animal, or human and includes alleviation of the signs or symptoms of the disease being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, in view ofthe disclosure provided herein. The therapeutically effective amount of the compounds disclosed herein required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.

[0083] Effective dosages and schedules for administering the vaccines may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the vaccines are those large enough to produce the desired effect in which the symptoms / disorder are / is affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counter indications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 mg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above. Following administration of a disclosed vaccine, for treating, inhibiting, or reducing a an age-related and / or chronic inflammatory disease, the efficacy of the therapy or prophylaxis can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a vaccine disclosed herein is efficacious in treating, inhibiting, or reducing a an age-related and / or chronic inflammatory disease in a subject by observing that the composition reduces symptoms and other metrics related to the age related and / or chronic inflammatory disease. Methods of preparing vaccines

[0084] Further disclosed herein are methods of preparing a vaccine.

[0085] Embodiments provided herein relate to methods of preparing a vaccine against one or more age-related diseases. In some embodiments methods include a) obtaining a first biological sample from a young subject. In some embodiments, the subject is a test animal. In some embodiments, the subject is an inbred test animal. In some embodiments, the method further includes b) injecting senescent cells from a subject with advanced age into the young subject, c) obtaining a second biological sample from the young subject, d) applying the first biological sample to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, e) measuring immune response of the first biological sample to the peptide array to obtain a first REDN signature, f) applying the second biological sample to a peptide array comprising a plurality of REDN peptides, g) measuring immune response of the second biological sample to the peptide array to obtain a second REDN signature, h) comparing the first REDN signature and the second REDN signature to identify at least one REDN peptide of the plurality of REDN peptides which provokes a greater immune response with the second biological sample, and i) preparing a vaccine comprising the one or more REDN peptides identified in step h).

[0086] In some embodiments, the prepared vaccines may be used for testing effects on the healthspan effects in a subject, such as a test animal. In some embodiments, peptides may be determined from the vaccine which have a positive effect on a test animal, and for such positive REDNs, the homolog REDNs in a human and or companion animal may be determined and a second vaccine may be prepared comprising those homolog REDNs. For example, the method may further include j) testing the REDN vaccine for anti-aging effects in one or more subjects, k) finding the homologs for the positive effector REDNs, l) using these homologs in a vaccine for a person or companion animal such as a dog.

[0087] Some embodiments provided herein relate to methods of preparing a vaccine against one or more age-related diseases. In some embodiments, the methods include a) obtaining one or more biological samples from one or more young subjects, b) obtaining one or more biological samples from one or more subjects of first advanced age, c) applying each of the biological samples obtained in steps a) and b) to a peptide array comprising a plurality of REDN peptides, d) measuring immune response of each of the biological samples to the peptide array to obtain a REDN signature for each of the biological samples, e) comparing the REDN signature for each of the biological samples to identify one or more REDN peptides of the plurality of REDNpeptides, and f) preparing a vaccine comprising the one or more REDN peptides identified in step e).

[0088] In some embodiments, step b) further includes obtaining one or more biological samples from one or more subjects of a second advanced age. In some embodiments, a subject of the second advanced age is at least three years, at least five years, at least ten years, at least twenty years, or more, older than a subject of the first advanced age of step b). In some embodiments the subject of the first advanced age is an adult, fully mature subject. In some embodiments, the subject of the second advanced age is a subject of a late stage of life. For example, in the case of a human subject, a subject of the second advanced age may be a subject that is more than 70 years old, more than 80 years old, or more than 90 years old.

[0089] In some embodiments the REDNs that are bioinformatically predicted based on sequence information (such as proximity to tryptophan codons) or detected by RNA sequencing could be screened in animal models for aging or chronic disease directly without screening on a REDN array or other immunological screen.

[0090] In some embodiments, the methods include obtaining a biological sample from a subject. In some embodiments, the biological sample includes blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof. In some embodiments, the biological sample includes antibodies. In some embodiments, the biological sample is blood or serum.

[0091] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig. In some embodiments, the subject is of an advanced age. In some embodiments, the subject has an age- related disease. In some embodiments, the subject does not have an age-related disease. In some embodiments, the subject is a human. In some embodiments, when the subject is a human, advanced age is age above 55 years. In some embodiments, the subject is a dog. In some embodiments, when the subject is a dog, advanced age is over 5 years.

[0092] In some embodiments, the subject has a chronic inflammatory disease. In some embodiments, the subject has been diagnosed with a chronic inflammatory disease. In some embodiments, the inflammatory disease is an age-related chronic inflammatory disease. Examples of chronic inflammatory diseases related to aging include heart disease, rheumatoid arthritis, Alzheimer’s disease, and Type II diabetes. In some embodiments, the disease is heart disease, Alzheimer’s disease, or diabetes.

[0093] In some embodiments, the methods of preparing a vaccine include contacting the biological sample with a peptide array including one or more REDN peptides. As used herein, the term “contacting” includes placement in direct physical association, including solid or liquid forms. In some embodiments, the REDN peptides of the peptide array include a peptide having a sequence as set forth in any of SEQ ID NOs: 1-31 (or their equivalents in humans).

[0094] As used herein, the term “array” refers to an arrangement of molecules, such as biological macromolecules (such as peptides), in addressable locations on or in a substrate. A “microarray” is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis. The array of molecules (“features”) makes it possible to carry out a very large number of analyses on a sample at one time. Within an array, each arrayed sample is addressable, in that its location can be reliably and consistently determined within at least two dimensions of the array. The feature application location on an array can assume different shapes. For example, the array can be regular (such as arranged in uniform rows and columns) or irregular. Thus, in ordered arrays the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position. Often, ordered arrays are arranged in a symmetrical grid pattern, but samples can be arranged in other patterns (such as in radially distributed lines, spiral lines, or ordered clusters). Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (such as hybridization or binding data, including for instance signal intensity). In some examples of computer readable formats, the subject features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer. In some cases, the methods provided herein involve multiplexed arrays in which a plurality of peptides or polypeptides attached to a solid support are contacted to a biological sample (for example, blood or other bodily tissue obtained from a subject).

[0095] In certain embodiments, the peptide array is a plurality of short linear peptides immobilized on a solid surface (for example, a polystyrene or other solid substrate). As used herein, the terms “peptide” and “polypeptide” refer to a polymer in which the monomers are alpha amino acids joined together through amide bonds. Peptides are two or often more amino acid monomers long. Standard abbreviations for amino acids are used herein (see Stryer, 1988, Biochemistry, Third Ed., incorporated herein by reference). In certain embodiments, random-sequence peptide arrays are used. As used herein, the term “substrate” refers to any type of solid support to which the peptides are immobilized. Examples of substrates include, but are not limited to, microarrays; beads; columns; optical fibers; wipes; nitrocellulose; nylon; glass; quartz; diazotized membranes (paper or nylon); silicones; polyformaldehyde; cellulose; cellulose acetate; paper; ceramics; metals; metalloids; semiconductive materials; coated beads; magnetic particles; plastics such as polyethylene, polypropylene, and polystyrene; gel-forming materials; silicates; agarose; polyacrylamides; methylmethacrylate polymers; sol gels; porous polymer hydrogels; nanostructured surfaces; nanotubes (such as carbon nanotubes); and nanoparticles (such as gold nanoparticles or quantum dots). When bound to a substrate, the peptides can be directly linked to the support, or attached to the surface via a linker. Thus, the solid substrate and / or the peptides can be derivatized using methods known in the art to facilitate binding of the peptides to the solid support, so long as the derivatization does not eliminate detection of binding between the peptides and antibodies in the sera.

[0096] In some embodiments, the arrays include about 100, about 200, about 300, about 500, about 1,000, about 2,000, about 3,000, about 5,000, about 7,500, about 10,000, about 12,500, about 15,000, about 17,500, about 20,000, about 22,500, about 25,000, about 27,500, about 30,000, about 32,500, about 35,000, about 37,500, about 40,000, about 50,000, about 100,000, about 200,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, about 1,000,000, about 1,500,000, about 2,000,000, or about 2,500,000, about 3,000,000, about 3,500,000, about 4,000,000, about 4,500,000, or about 5,000,000 REDN peptides.

[0097] In some embodiments the REDN peptides are in-situ synthesized on the array. As used herein, “in-situ synthesis” refers to synthesis of peptides or polypeptides in situ on an array. This could be done with photoactivatable amino acids as done by Nimble Therapeutics (maskless photolithography), PEPperPRINT, a standard mask-based system (much like Intel uses to lay down circuits), BOC or FMOC peptide chemistry, or other synthesis methods known in the art.

[0098] In some embodiments, the REDN peptides of the array are fixed on a substrate. In some embodiments, the substrate includes glass, silica, composite, resin, or combination thereof. In some embodiments, the array is configured to detect binding by at least one offluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, electro-interference sensing, and surface plasmon resonance.

[0099] In some embodiments, the methods of preparing a vaccine further include detecting binding of the biological sample to a REDN peptide of the peptide array. As used herein, “binding” refers to an association between two substances or molecules, such as the association of an antibody with a peptide. Binding can be detected by any procedure known to one skilled in the art, such as by physical or functional properties of the formed complexes, such as a target / antibody complex. As used herein, the term “detect,” “detection,” “detectable,” or “detecting” is understood both on a quantitative and a qualitative level, as well as a combination thereof.

[0100] The methods provided herein are sensitive and involve small quantities of biological samples from a subject. In some embodiments, biological samples from a subject are too concentrated and require a dilution prior to being contacted with an array of the invention. A plurality of dilutions can be applied to a biological sample prior to contacting the sample with an array of the invention. A dilution can be a serial dilution, which can result in a geometric progression of the concentration in a logarithmic fashion. For example, a ten-fold serial dilution can be 1 M, 0.01 M, 0.001 M, and a geometric progression thereof. A dilution can be, for example, a one-fold dilution, a two-fold dilution, a three-fold dilution, a four-fold dilution, a five-fold dilution, a six-fold dilution, a seven-fold dilution, an eight-fold dilution, a nine-fold dilution, a ten- fold dilution, a sixteen-fold dilution, a twenty-five-fold dilution, a thirty -two-fold dilution, a sixty- four-fold dilution, and / or a one-hundred-and-twenty-five-fold dilution.

[0101] The binding of a molecule to an array in accordance with certain embodiments of the methodology disclosed herein creates a pattern of binding that can be associated with a condition. The affinity of binding of a molecule to a peptide in the array can be mathematically associated with a condition. The off-target binding pattern of an antibody to a plurality of different peptides of the invention can be mathematically associated with a condition. The avidity of binding of a molecule to a plurality of different peptides can be mathematically associated with a condition.

[0102] The peptide array can be contacted with the biological sample (for example, sera) under any suitable conditions to promote binding of antibodies in the sample to peptides immobilized on the array. Thus, the methods presented herein are not limited by any specific type of binding conditions employed. Such conditions will vary depending on the array being used, the type of substrate, the density of the peptides arrayed on the substrate, desired stringency of thebinding interaction, and nature of the competing materials in the binding solution. In certain embodiments, the conditions include a step to remove unbound antibodies from the addressable array.

[0103] Similarly, any suitable detection technique can be used in the methods provided herein to detect binding of antibodies (“antibody reactivity”) in the biological sample to peptides on the array. Such reactivity may be measured or estimated in any operable way, such as, for example, by ELISA or by microarray assay. In one embodiment, any type of detectable label can be used to label peptides on the array, including but not limited to radioisotope labels, fluorescent labels, luminescent labels, and electrochemical labels (for example, ligand labels with different electrode mid-point potential, where detection includes detecting electric potential of the label). Alternatively, bound antibodies can be detected, for example, using a detectably labeled secondary antibody. In some embodiments, binding is detected by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, electro-interference, Bio-Layer Interferometry, electro-interference sensing, and surface plasmon resonance.

[0104] In some embodiments, the methods further include preparing a vaccine including a REDN peptide (REDN) that was detected as being bound by the biological sample. In some embodiments, the REDN was detected as being bound by the biological sample as described above. In some embodiments, the method is performed using sample(s) from one or more subjects that have a chronic inflammatory and / or age-related disease. In some embodiments, the method is performed with sample(s) from one or more subjects that do not have a chronic inflammatory and / or age-related disease (healthy subject). In some embodiments, the REDN peptide was detected as being bound by the biological sample in a diseased subject but not a healthy subject. In some embodiments, the REDN peptide was detected as being bound by the biological sample in a diseased group but not a healthy group.

[0105] In some embodiments, the vaccine includes a REDN peptide(s) having a sequence set forth in any of SEQ ID NOs: 1-31 (or their equivalents in humans). In some embodiments, the vaccine includes a peptide having a sequence set forth in any of SEQ ID NOs: 1-31 (or their equivalents in humans). In some embodiments, the vaccine includes a nucleic acid encoding a peptide having a sequence set forth in any of SEQ ID NOs: 1-31 (or their equivalents in humans).

[0106] Vaccines may be prepared according to methods known to those skilled in the art. For example, in some embodiments, the vaccines further include an adjuvant. In some embodiments, the adjuvant is ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), Hiltonol, IL-12, IL-2, imiquimod, interferon- gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, oil-in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), Pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex adjuvant formulation (SAF), synthetic polynucleotides (poly IC / poly AU), TiterMax Tomatine, Vaxfectin, XtendIII, or Zymosan.

[0107] Described herein are methods of prolonging life and / or healthspan in a subject. Further provided herein are methods of inhibiting non-tumor age-related disease in a subject. Further provided herein are methods of improving general health in a subject. In some embodiments, the methods include administering a vaccine as described herein, such as a vaccine prepared as described above.

[0108] Additional variations than those described herein will be apparent from this disclosure. For example, depending on the embodiment, certain acts, events, or functions of any of the compositions, systems, or methods described herein can be performed in a different sequence, can be added, merged, or left out altogether. Moreover, in certain embodiments, acts or events can be performed concurrently. In addition, different tasks or processes can be performed by different machines and / or computing systems that can function together.

[0109] The foregoing description and examples has been set forth merely to illustrate the disclosure and are not intended as being limiting. Each of the disclosed aspects and embodiments of the present disclosure may be considered individually or in combination with other aspects, embodiments, and variations of the disclosure. In addition, unless otherwise specified, none of the steps of the methods of the present disclosure are confined to any particular order of performance. Modifications of the disclosed embodiments incorporating the spirit andsubstance of the disclosure may occur to persons skilled in the art and such modifications are within the scope of the present disclosure.

[0110] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0111] Although the embodiments are described in considerable detail with reference to certain methods and materials, one skilled in the art will appreciate that the disclosure herein can be practiced by other than the described embodiments, which have been presented for purposes of illustration and not of limitation. Therefore, the scope of the appended claims should not be limited to the description of the embodiments contained herein.

[0112] Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation,’ ‘including but not limited to,’ or the like; the term ‘comprising’ as used herein is synonymous with ‘including,’ ‘containing,’ or ‘characterized by,’ and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term ‘having’ should be interpreted as ‘having at least;’ the term ‘includes’ should be interpreted as ‘includes but is not limited to;’ the term ‘example’ is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; and use of terms like ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words of similar meaning should not be understood as implying that certain features are critical, essential, or even important to the structure or function, but instead as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment. In addition, the term “comprising” is to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps but may include additional steps. When used in the context of a compound, composition or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components. Likewise, a group of items linked with the conjunction ‘and’ should not be read as requiring that each and every oneof those items be present in the grouping, but rather should be read as ‘and / or’ unless expressly stated otherwise. Similarly, a group of items linked with the conjunction ‘or’ should not be read as requiring mutual exclusivity among that group, but rather should be read as ‘and / or’ unless expressly stated otherwise.

[0113] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0114] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0115] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The indefinite article “a” or “an” does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0116] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0117] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0118] Some embodiments provided herein are provided as set forth in the following enumerated alternatives.

[0119] 1. A vaccine to reduce the incidence of death and extend healthy life in a subject, wherein the vaccine comprises a RNA error derived neoantigen (REDN) peptide or a nucleic acid encoding a REDN peptide, wherein the vaccine is administered to reduce the incidence of a non-tumor age-related disease in the subject.

[0120] 2. The vaccine of alternative 1, wherein the vaccine is suitable for administration in humans.

[0121] 3. The vaccine of alternative 1, wherein the vaccine is suitable for administration in a companion animal.

[0122] 4. The vaccine of alternative 3, wherein the companion animal is a dog.

[0123] 5. The vaccine of any of alternatives 1-4, wherein the vaccine comprises a sequence of at least 8 amino acids having at least 95% sequence identity to a sequence of at least 8 amino acids as set forth in any of SEQ ID NOs: 1-31, or a nucleic acid encoding a sequence of at least 8 amino acids having at least 95% sequence identity to a sequence of at least 8 amino acids as set forth in any of SEQ ID NOs: 1-31.

[0124] 6. The vaccine of any of alternatives 1-5, wherein the REDN peptide has a sequence comprising at least 8 amino acids of the sequences set forth in any of SEQ ID NOs: 1- 31.

[0125] 7. The vaccine of any of alternatives 1-5, further comprising an adjuvant.

[0126] 8. The vaccine of alternative 7, wherein the adjuvant comprises ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), Hiltonol, IL-12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, MontanideISA-50, nor-MDP, oil-in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), Pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex adjuvant formulation (SAF), synthetic polynucleotides (poly IC / poly AU), TiterMax Tomatine, Vaxfectin, XtendIII, or Zymosan.

[0127] 9. The vaccine of any of alternatives 1-8, wherein the non-tumor age-related disease comprises heart disease, Alzheimer’s disease, or diabetes.

[0128] 10. The vaccine of any of alternatives 1-8, wherein the non-tumor age-related disease comprises a chronic inflammatory disease.

[0129] 11. A method of prolonging life and / or healthspan in a subject, comprising administering the vaccine of any of alternatives 1-8.

[0130] 12. A method of inhibiting non-tumor age-related disease in a subject, comprising administering the vaccine of any of alternatives 1-8.

[0131] 13. The method of alternative 12, wherein the disease is a chronic inflammatory disease.

[0132] 14. The method of alternative 12, wherein the disease is heart disease, Alzheimer’s disease, or diabetes.

[0133] 15. A method of improving general health in a subject, comprising administering the vaccine of any of alternatives 1-8.

[0134] 16. A method of treating, ameliorating, or inhibiting a chronic disease in a subject, comprising administering the vaccine of any one of alternatives 1-8.

[0135] 17. The method of alternative 16, wherein the chronic disease is arthritis, asthma, chronic obstructive pulmonary disease, dementia, diabetes, fatty liver, epilepsy, heart disease, hypertension, pain, or stroke.

[0136] 18. A method of preparing a vaccine, comprising: a) obtaining a biological sample from a subject with advanced age, b) contacting the biological sample with a peptide array comprising one or more RNA error derived neoantigen (REDN) peptides, c) detecting binding of the biological sample to a REDN peptide of the peptide array, and d) preparing a vaccine comprising a REDN peptide, wherein the REDN peptide is detected as being bound by the biological sample in step c).

[0137] 19. The method of alternative 18, wherein the REDN peptides of the peptide array comprise a peptide having a sequence as set forth in any of SEQ ID NOs: 1-31.

[0138] 20. The method of alternative 18, wherein the vaccine further comprises an adjuvant.

[0139] 21. The method of alternative 20, wherein the adjuvant comprises ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), Hiltonol, IL-12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, oil-in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), Pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex adjuvant formulation (SAF), synthetic polynucleotides (poly IC / poly AU), TiterMax Tomatine, Vaxfectin, XtendIII, or Zymosan.

[0140] 22. A method of preparing a vaccine against one or more age-related diseases, the method comprising: a) obtaining a first biological sample from a young subject; b) injecting senescent cells from a subject with advanced age into the young subject; c) obtaining a second biological sample from the young subject; d) applying the first biological sample to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides; e) measuring immune response of the first biological sample to the peptide array to obtain a first REDN signature; f) applying the second biological sample to a peptide array comprising a plurality of REDN peptides; g) measuring immune response of the second biological sample to the peptide array to obtain a second REDN signature; h) comparing the first REDN signature and the second REDN signature to identify at least one REDN peptide of the plurality of REDN peptides which provokes a greater immune response with the second biological sample; and i) preparing a vaccine comprising the one or more REDN peptides identified in step h).

[0141] 23. A method of preparing a vaccine against one or more age-related diseases, the method comprising: a) obtaining one or more biological samples from one or more young subjects; b) obtaining one or more biological samples from one or more subjects of first advanced age; c) applying each of the biological samples obtained in steps a) and b) to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides; d) measuring immuneresponse of each of the biological samples to the peptide array to obtain a REDN signature for each of the biological samples; e) comparing the REDN signatures for each of the biological samples to identify one or more REDN peptides of the plurality of REDN peptides; and f) preparing a vaccine comprising the one or more REDN peptides identified in step e).

[0142] 24. The method of alternative 23, wherein step b) further comprises obtaining one or more biological samples from one or more subjects of a second advanced age.

[0143] 25. A method of prolonging life in a subject, comprising administering the vaccine prepared by the method of any of alternatives 18-24.

[0144] 26. A method of inhibiting non-tumor age-related disease in a subject, comprising administering the vaccine prepared by the method of any of alternatives 18-24.

[0145] 27. A method of improving general health in a subject, comprising administering the vaccine prepared by the method of any of alternatives 18-24.

[0146] 28. A method of treating, ameliorating, or inhibiting a chronic disease in a subject, comprising administering the vaccine prepare by the method of any one of alternatives 18-24. EXAMPLES

[0147] Embodiments described herein are further defined in the following Examples. It should be understood that these Examples are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the disclosure to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the disclosure, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosure of each reference set forth herein is incorporated herein by reference in its entirety. Example 1

[0148] Sera from dogs diagnosed with 8 different types of tumors was collected. The sera were applied to arrays bearing 400,000 peptides that are 15 amino acids in length, representing 220,000 REDNs. These REDNs were produced by exon-mis-splicing, exon 1 mis-initiation oftranslation and INDELs created in microsatellites by transcription. Without being bound by theory, the inventors of the present application reasoned that if a tumor is producing a particular REDN it will be recognized by the antibody binding to the corresponding peptide on the array. The detection is with a labelled secondary antibody.

[0149] 127 REDN peptides were identified which were reactive in more than 10% of the samples. Of these, 31 were chosen for the vaccine, and 31 were delivered as a prime as DNA and 21 were delivered as 30mer peptides. The list of peptides used is presented in Table 1. Figure 4 summarizes which peptides were included in a DNA vaccine, and which were also included in a peptide vaccine.nic / iiiIg gen gti eti eti eti eti eti eti eti eti eti eti eti etiicn g icn n icici llellellellellellellellllllllllllepililil lptat t t t t ty p p ps sasasasasasasetasetaetaetaetaetat s-- o o o o o o o osososososos s-s s- snr r r r r r r r r r r r rSi isiciciciciciciciciciciciciciFm m martm m m m m m m m m m m m mni.teslorueo doseseseseseseseP m m Y Y Y Y Y Y Y 4 M 7 B 2 2 1 2 4 2 e CR-fr5F A M 0 0 A1N4 I H 1 o 5 2 3 B 4 H B 2 9 1 1 1 7 N P maC 5 3 D 2 4 K 5 6 2 V O A N M A H M S L S D B H4 C HFZ R C L S CIC E S C PE R N HP E L S L P D P E S C A B S O W SseYseYseYseYseYFsesese“Y Y Y”;ledo mo o oA / A / A / A / A / A / A / A / A / A / A / es83 N N N N N N N N N N N N N N uo mleleleniD D D n oitcT_ T_ Aet9 8 _ 7orp“12 9 2 7sn 1 3 3 3 64 93 95 75 65 64 15 83 94 15aee emtillti elltillgn gn g g g g g g g g g”l ”.etasetaeticilicn ilicn ilicn ilicn ilicn ilicn iicn iicn iicn iic ei ddeoosariosp p p p p plplplplplp im mricrocrcis-sis-sis-sis-sis-sis-sis-sis-sis-sis-sis-siefn m m m m m m m m m m m m m msu o oCm encin.t eu orqPeS 3“C:“s sn A na3 1eK H 3 A 91 P 4 1 N o 2itmRTN 3IR 4 D A 6 2 5 2 B 1 M T M C L 1 P C 1 R C E A L A P A D G N LaiG v”dL N A N C P EL H 6 A Aerb’fn S H G U L S T P C M G T ACIP C E T T S N L P b o A C

[0150] 810 owner dogs were enrolled in the trial, approximately 400 received the vaccine and approximately 400 received a mock vaccine. Dogs were between 5 and 11 years old on enrollment. They were examined every 6 months and given a peptide boost yearly.

[0151] The primary clinical goal was to reduce the incidence of malignant tumors. Preliminary analysis indicates that the vaccine may be having such an effect. The trial is slated to run until May 2024. Multiple types of data are being collected on the dogs in the trial. This included clinical data and information collected from the owners.

[0152] The dogs in the trial are older so, as expected there have been both tumor and non-tumor deaths (NTDs) registered. A surprising result is that there was a reduction in NTDs in the vaccine relative to control arms. Dogs have died for a number of reasons, including euthanasia due to ambulatory impairment, dementia and heart disease. Table 2 is a list of dogs that have died and the cause when known.de ,taaidePyvmvlLaeM leerto A aseesC W S o P M M r orfetnfiothotaarbeudegisHe eu elrd?elusao esrn Bldinfuloitaai ,deyrn w osarw d osyiratmeln n nf cib arwoziontaalaeh n tikis enpegrh g Sioisren Rviorma ilfsscan rtkn PLhtub R w UuselLhcnio PniU A u Emaenicp cau orV G B A A A A B B S C C 33 64 79 28 09 50 11 A 0 0 0 1 1 0 0 VDI02 02 02 02 02 04 04tr sx 1202 / 8 / 9seYtnt to no no M M M lanid otytiy ld ti eni,en pe e i elinilSziu dseboziboacce .nerain.na ,u oyrhtsiaoissm nabhtm d de ediapvityhisaN / otusesaiymiu g ni eziziviss , ev karetavanaaialE. ranhehtx uatilil e-sni nanaeryt iticenpo uastbsmapaarahtb d u oneiulsc hse thutgeiuorx n n gneo d ngeley B E A m pem HiDeE pa c aD m B A A B B B A 81 72 44 9 8 4 50 5 7 8 00 00 00 00 0 0 14 4 4 4 04 04 04l ssisor )elLcs (M M M n.oihttn aate ?oitali sd DatDul rVali sueC?Dulcivlncn n u vlr o wonacoitccio tsaVd kn nrtVoefsad GnaU NnIGnaB A B B 69 40 80 6 1 3 0 2 0 0 0 0 4 4 6 06x 2202 / 42 / / 01 / seY818 4ht6 6 6n k kht1oeeeenht2o nhto no M W W M M M dnoldlaateearunasr sy s u d ouehe spad,d s,dip oor sua eunll loaidenioz oi l sn ntaaoiloiet rs cn ndesszinwcd nins artalayal aw wiy wketraoisna se b ydrabcirerauc nao onnatilionnuccir ufht rgetrno mfis taoci suhtnk knhtbo kn U Aepfeu Ega evpsul solibrcm u E n u U u E m U B C D C C C C D 47 8 0 3 3 8 3 40 02 01 4 1 1 2 30 0 0 10 20 0 0 06 2 4 4 4 06 06 06

[0153] As evident in Table 3, there were significantly fewer NTD in the vaccine group (C,D). The difference in tumor incidence between the vaccine and control groups (A,B) increased after year two in the trial. As seen in Table 3, this was also true for the NTDs. This is consistent with the idea that the difference in NTDs is related to the vaccine. Figure 5 illustrates a graph depicting non-cancer related deaths in dogs enrolled in the VACCS vaccine trial, and a comparison of VACCS vaccinated vs placebo vaccinated dogs. Table 3 Control Vaccine Chi-square (p=) Total 20 8 52 (002)[ ] o a e ogs a were vacc na e eve ope an mmune response o the vaccine. If the vaccine caused the differences in NTDs, then one would predict that the dogs without a vaccine response would be enriched in the NTDs relative to the vaccinees that had not died. Figure 1 illustrates IFN-γ ELISPOT T cell responses to individual VACCS peptides or VACCS peptide pool in VACCS vaccinated dogs who are still living or have died of non-cancer related causes. Figure 2 compares CD4 IFN-γ production and Figure 3 compares CD4 TNFα production six months after the vaccine for living and dead groups. 7.5% of the dogs that were vaccinated and did not have a tumor or die, did not have an immune response six months after the vaccine. As shown in Table 1, two of the four dogs that could be assessed (50%) did not have an immune response. Though the numbers are low, this finding is consistent with the idea that the vaccine is causing the differences in NTDs.

[0155] It would be desirable if one could extend not only life but health. As a measure of this, the owners were asked to describe how often their dogs were more active after completing the vaccine. The owners completed surveys every six months. As shown in Table 4, dogs in the vaccine group had a greater number of “more active” entries than the control group as reported at the month 24 assessment. Table 4 Vaccine Control Chi-Square (p=)

[0156] This data surprisingly and unexpectedly provides evidence that REDNs derived from mis-processing of RNA confer protection as vaccines against death from disease in dogs, by broadly reducing chronic disease, and extending life. The same concept is applicable to humans and other animals. The REDNs in the current vaccine were chosen originally to protect against developing tumors. The 31 REDNs were chosen from 220,000 possible REDNs. Tumors can produce approximately 1.4 million possible REDNs in dogs and approximately 2.1 million in humans. Therefore, it is likely that more and better REDNs with activity specifically against chronic inflammatory disease could be chosen.

[0157] The REDNs in the current vaccine were chosen based on analysis of subjects with tumors. If immune activity against senescent cells is the basis for the reduction in deaths, then it should be possible to develop aging vaccines with more focused components. Example 2

[0158] Sera is collected from older subjects with specific chronic diseases, including heart disease, Alzheimer’s disease, diabetes, etc. These sera are assayed on the discovery REDN arrays bearing all the possible RNA generated REDNs for humans. Peptides are chosen that represent all the chronic diseases assayed. These peptides are encoded in a nucleic acid vaccine (mRNA, DNA, saRNA, etc.). As many as 50 REDNs can be encoded in the vaccine.

[0159] The vaccine is administered to a person at age 55, when chronic diseases become more prevalent. The person receives a booster every two years. The person experiences a reduction in chronic inflammatory disease, as compared to a person who did not receive the vaccine. The person lives longer than expected based on population statistics for the person. The person experiences greater energy and quality of life compared people of similar age who did not receive the vaccine. Example 3

[0160] Sera is collected from dogs with specific chronic diseases, including heart disease, Alzheimer’s disease, diabetes, etc. These sera are assayed on the discovery REDN arrays bearing all the possible RNA generated REDNs for dogs. Peptides are chosen that represent all the chronic diseases assayed. These peptides are encoded in a nucleic acid vaccine (mRNA, DNA, saRNA, etc.). As many as 50 REDNs can be encoded in the vaccine.

[0161] The vaccine is administered to a dog at age 5, when chronic diseases become more prevalent. The dog receives a booster every two years. The dog experiences a reduction in chronic inflammatory disease, as compared to a dog who did not receive the vaccine. The dog lives longer than expected based on population statistics for the dog. The dog experiences greater energy and quality of life compared dogs of similar age who did not receive the vaccine. Example 4

[0162] A REDN vaccine is administered to older dogs and the immune response is assessed, including ability to kill autologous SCs in vitro. The dogs are monitored over 3-5 years for safety, inflammation markers, incidence of chronic diseases, death and activity markers. Example 5

[0163] Figure 6 schematically illustrates an exemplary method to develop a vaccine against one or more age-related diseases. As shown in Figure 6, senescent cells (SC) from old mice are injected into young mice, who develop an immune response to the REDNs. Blood from the young mice, pre and post-injection, is applied to the REDN peptide arrays. The signatures are compared to determine which signatures are specific to aged REDN SC peptides. These peptides are tested directly as vaccines in old mice for extending life and improving health. Homologs for these REDNs in dogs and humans are similarly discovered and developed for use in anti-aging vaccines for dogs and humans, respectively. Example 6

[0164] Figure 7 schematically illustrates a further exemplary method to develop a vaccine against one or more age-related diseases. As depicted in Figure 7, sera from a number of young people, old people, and centenarians are collected and applied to the REDN peptide arrays. The signatures across each of the classes are compared to identify REDNs that associate with the long lived, healthy individuals. As an example, these may include REDNs that are identified in young and centenarians but not in old individuals. As another example, these may include REDNs unique to centenarians. Vaccines are prepared including the anti-aging associated REDNs as identified using the REDN peptide arrays, and the vaccines are administered to subjects such as dogs, mice, and / or people.

[0165] As used herein, the section headings are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose, including the disclosures specifically referenced herein. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein.

[0166] Although this disclosure has been described in the context of certain embodiments and examples, those skilled in the art will understand that the embodiments provided herein extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the disclosure and obvious modifications and equivalents thereof. In addition, while several variations have been shown and described in detail, other modifications, which are within the scope of this disclosure, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the disclosure. It should be understood that various features and aspects of the disclosed embodiments can be combined with, or substituted for, one another in order to form varying modes or embodiments described herein. Thus, it is intended that the scope of the disclosure should not be limited by the particular disclosed embodiments described above.

[0167] It should be understood, however, that this detailed description, while indicating preferred embodiments described herein, is given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art.

[0168] The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive manner. Rather, the terminology is simply being utilized in conjunction with a detailed description of embodiments of the systems, methods and related components. Furthermore, embodiments may include several novel features, no single one ofwhich is solely responsible for its desirable attributes or is believed to be essential to practicing the embodiments herein described.

Claims

WHAT IS CLAIMED IS:

1. A vaccine to reduce the incidence of death and extend healthy life in a subject, wherein the vaccine comprises an RNA error derived neoantigen (REDN) peptide or a nucleic acid encoding a REDN peptide, wherein the vaccine is administered to reduce the incidence of a non-tumor age-related disease in the subject.

2. The vaccine of claim 1, wherein the vaccine is suitable for administration in humans.

3. The vaccine of claim 1, wherein the vaccine is suitable for administration in a companion animal.

4. The vaccine of claim 3, wherein the companion animal is a dog.

5. The vaccine of claim 1, wherein the vaccine comprises a sequence of at least 8 amino acids having at least 95% sequence identity to a sequence of at least 8 amino acids as set forth in any of SEQ ID NOs: 1-31, or a nucleic acid encoding a sequence of at least 8 amino acids having at least 95% sequence identity to a sequence of at least 8 amino acids as set forth in any of SEQ ID NOs: 1-31.

6. The vaccine of claim 1, wherein the REDN peptide has a sequence comprising at least 8 amino acids of the sequences set forth in any of SEQ ID NOs: 1-31.

7. The vaccine of claim 1, further comprising an adjuvant.

8. The vaccine of claim 7, wherein the adjuvant comprises ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), Hiltonol, IL-12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, oil-in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), Pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin,Seppic ISA 720, soybean oil, squalene, Syntex adjuvant formulation (SAF), synthetic polynucleotides (poly IC / poly AU), TiterMax Tomatine, Vaxfectin, XtendIII, or Zymosan.

9. The vaccine of claim 1, wherein the non-tumor age-related disease comprises heart disease, Alzheimer’s disease, or diabetes.

10. The vaccine of claim 1, wherein the non-tumor age-related disease comprises a chronic inflammatory disease.

11. A method of prolonging life and / or healthspan in a subject, comprising administering the vaccine of claim 1.

12. A method of inhibiting non-tumor age-related disease in a subject, comprising administering the vaccine of claim 1.

13. The method of claim 12, wherein the disease is a chronic inflammatory disease.

14. The method of claim 12, wherein the disease is heart disease, Alzheimer’s disease, or diabetes.

15. A method of improving general health in a subject, comprising administering the vaccine of claim 1.

16. A method of treating, ameliorating, or inhibiting a chronic disease in a subject, comprising administering the vaccine of claim 1.

17. The method of claim 16, wherein the chronic disease is arthritis, asthma, chronic obstructive pulmonary disease, dementia, diabetes, fatty liver, epilepsy, heart disease, hypertension, pain, or stroke.

18. A method of preparing a vaccine, comprising: a) obtaining a biological sample from a subject with advanced age, b) contacting the biological sample with a peptide array comprising one or more RNA error derived neoantigen (REDN) peptides, c) detecting binding of the biological sample to a REDN peptide of the peptide array, and d) preparing a vaccine comprising a REDN peptide, wherein the REDN peptide is detected as being bound by the biological sample in step c).

19. The method of claim 18, wherein the REDN peptides of the peptide array comprise a peptide having a sequence as set forth in any of SEQ ID NOs: 1-31.

20. The method of claim 18, wherein the vaccine further comprises an adjuvant.

21. The method of claim 20, wherein the adjuvant comprises ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), Hiltonol, IL-12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, oil-in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), Pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex adjuvant formulation (SAF), synthetic polynucleotides (poly IC / poly AU), TiterMax Tomatine, Vaxfectin, XtendIII, or Zymosan.

22. A method of preparing a vaccine against one or more age-related diseases, the method comprising: a) obtaining a first biological sample from a young subject; b) injecting senescent cells from a subject with advanced age into the young subject; c) obtaining a second biological sample from the young subject; d) applying the first biological sample to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides; e) measuring immune response of the first biological sample to the peptide array to obtain a first REDN signature; f) applying the second biological sample to a peptide array comprising a plurality of REDN peptides; g) measuring immune response of the second biological sample to the peptide array to obtain a second REDN signature; h) comparing the first REDN signature and the second REDN signature to identify at least one REDN peptide of the plurality of REDN peptides which provokes a greater immune response with the second biological sample; andi) preparing a vaccine comprising the one or more REDN peptides identified in step h).

23. A method of preparing a vaccine against one or more age-related diseases, the method comprising: a) obtaining one or more biological samples from one or more young subjects; b) obtaining one or more biological samples from one or more subjects of first advanced age; c) applying each of the biological samples obtained in steps a) and b) to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides; d) measuring immune response of each of the biological samples to the peptide array to obtain a REDN signature for each of the biological samples; e) comparing the REDN signatures for each of the biological samples to identify one or more REDN peptides of the plurality of REDN peptides; and f) preparing a vaccine comprising the one or more REDN peptides identified in step e).

24. The method of claim 23, wherein step b) further comprises obtaining one or more biological samples from one or more subjects of a second advanced age.

25. A method of prolonging life in a subject, comprising administering the vaccine prepared by the method of claim 18.

26. A method of inhibiting non-tumor age-related disease in a subject, comprising administering the vaccine prepared by the method of claim 18.

27. A method of improving general health in a subject, comprising administering the vaccine prepared by the method of claim 18.

28. A method of treating, ameliorating, or inhibiting a chronic disease in a subject, comprising administering the vaccine prepare by the method of claim 18.

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