Compositions and methods targeting osteoactivin

Abstract

An immunogen includes an immunogenic carrier and an antigenic anti-osteoclavin peptide linked to the immunogenic carrier. The immunogenic carrier includes an RNA bacteriophage virus-like particle (VLP). In one or more embodiments, the RNA VLP is Qβ bacteriophage. In one or more embodiments, the anti-osteoclavin peptide includes a segment having at least 55% sequence similarity to an antigenic portion of SEQ ID NO:1. In one or more embodiments, the anti-osteoclavin peptide is conjugated to the VLP via an N-terminus linker sequence. In one or more embodiments, the anti-osteoclavin peptide is conjugated to the VLP via a C-terminus linker sequence.

Description

[0001] COMPOSITIONS AND METHODS TARGETING OSTEOACTIVIN

[0002] CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Patent Application No.

[0003] 63 / 532,140, filed August 11, 2023, which is incorporated herein by reference in its entirety

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing electronically submitted via Patent Center to the United States Patent and Trademark Office as an .xml file entitled “0310- 000184W001_Sequence Listing.xml” having a size of 4 kilobytes and created on August 6, 2024. The information contained in the Sequence Listing is incorporated by reference herein.

[0006] SUMMARY

[0007] This disclosure describes, in one aspect, an immunogen that includes an immunogenic carrier comprising an RNA bacteriophage virus-like particle (VLP) and an antigenic anti- osteoactivin peptide linked to the immunogenic carrier.

[0008] In one or more embodiments, the RNA VLP is Qf3 bacteriophage.

[0009] In one or more embodiments, the anti-osteoactivin peptide includes a segment having at least 55% sequence similarity to an antigenic portion of SEQ ID NO:1.

[0010] In one or more embodiments, the anti-osteoactivin peptide is conjugated to the VLP via an N-terminus linker sequence.

[0011] In one or more embodiments, the anti-osteoactivin peptide is conjugated to the VLP via a C -terminus linker sequence.

[0012] In another aspect, this disclosure describes a composition that includes an immunogen that includes an antigenic anti-osteoactivin peptide linked to an immunogenic RNA bacteriophage VLP carrier.

[0013] In one or more embodiments, the composition further includes a pharmaceutically acceptable carrier.

[0014] In one or more embodiments, the composition further includes an adjuvant In another aspect, this disclosure describes a method of inducing production of anti- osteoactivin antibodies in a subject. Generally, the method includes administering to the subject a composition that includes an antigenic anti-osteoactivin peptide linked to an immunogenic RNA bacteriophage VLP carrier.

[0015] In one or more embodiments, the anti-osteoactivin peptide includes a segment having at least 55% sequence similarity to SEQ ID NO: 1.

[0016] In one or more embodiments, the composition is administered in an amount effective to induce an immune response against atherosclerotic plaques in the subject

[0017] In another aspect, this disclosure describes a method of treating senescence in a subject. Generally, the method includes administering to the subject a composition that includes an antigenic anti-osteoactivin peptide linked to an immunogenic RNA bacteriophage VLP carrier.

[0018] In one or more embodiments, the anti-osteoactivin peptide includes a segment having at least 55% sequence similarity to SEQ ID NO: 1.

[0019] In one or more embodiments, the composition is administered in an amount effective to induce an immune response against atherosclerotic plaques in the subject

[0020] In one or more embodiments, the composition is administered in an amount effective to improve a cardiovascular indicium in the subject compared to a comparable control subject to which the composition is not administered. In one or more of these embodiments, the cardiovascular indicium is an increase in ejection fraction in the subject compared to a comparable control subject to which the composition is not administered.

[0021] In one or more embodiments, the composition is administered in an amount effective to improve a pulmonary indicium in the subject compared to a comparable control subject to which the composition is not administered. In one or more of these embodiments, the pulmonary indicium is an increase in breathing frequency in the subject compared to a comparable control subject to which the composition is not administered. In one or more embodiments, the pulmonary indicium is an increase in minute volume in the subject compared to a comparable control subject to which the composition is not administered.

[0022] In one or more embodiments, the composition is administered in an amount effective to increase survival compared to a comparable control subject to which the composition is not administered. In another aspect, this disclosure describes a method of treating a senopathy in a subject. Generally, the method includes administering to the subject a composition that includes an antigenic anti-osteoactivin peptide linked to an immunogenic RNA bacteriophage VLP carrier.

[0023] In one or more embodiments, the anti-osteoactivin peptide includes a segment having at least 55% sequence similarity to SEQ ID NO: 1.

[0024] In one or more embodiments, the composition is administered in an amount effective to induce an immune response against atherosclerotic plaques in the subject

[0025] In one or more embodiments, the composition is administered in an amount effective to improve a cardiovascular indicium in the subject compared to a comparable control subject to which the composition is not administered. In one or more of these embodiments, the cardiovascular indicium is an increase in ejection fraction in the subject compared to a comparable control subject to which the composition is not administered.

[0026] In one or more embodiments, the composition is administered in an amount effective to improve a pulmonary indicium in the subject compared to a comparable control subject to which the composition is not administered. In one or more of these embodiments, the pulmonary indicium is an increase in breathing frequency in the subject compared to a comparable control subject to which the composition is not administered. In one or more embodiments, the pulmonary indicium is an increase in minute volume in the subject compared to a comparable control subject to which the composition is not administered.

[0027] In one or more embodiments, the composition is administered in an amount effective to increase survival compared to a comparable control subject to which the composition is not administered.

[0028] In another aspect, this disclosure describes a nucleic acid that encodes an immunogen that includes an antigenic anti-osteoactivin peptide linked to an immunogenic RNA bacteriophage VLP carrier.

[0029] In another aspect, this disclosure describes an expression vector that includes a nucleic acid that encodes an antigenic anti-osteoactivin peptide linked to an immunogenic RNA bacteriophage VLP carrier.

[0030] In another aspect, this disclosure describes a cell that includes an expression vector that includes a nucleic acid that encodes an antigenic anti-osteoactivin peptide linked to an immunogenic RNA bacteriophage VLP carrier. In another aspect, this disclosure describes a vaccine that includes an antigenic anti- osteoactivin peptide linked to an immunogenic RNA bacteriophage VLP carrier.

[0031] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0032] BRIEF DESCRIPTION OF THE FIGURES

[0033] FIG. 1. Senescence is an irreversible, proinflammatory state of cell cycle arrest. Senescent cell accumulation can result from disruptions in cellular homeostasis such as DNA damage, oxidative stress, chromatin disruptions, telomere attrition, and others. The resultant pathway can activate a multitude of cyclin-dependent kinases such as pl 6, pl 9, and p21, which arrest the cell cycle and can eventually lead to senescence.

[0034] FIG. 2. Senescence can lead to atherosclerotic plaques. (A) Experimental schematic illustrating original vaccination strategy results. (B) Left: Oil red O staining visualizing atherosclerotic plaque within control vaccine (Cont vac) and osteoactivin vaccine (Gpnmb vac); Right: Atherosclerotic plaque area quantification. (C) Fluorescent images demonstrating senescent cell clearance between control (Cont vac) and therapeutic (Gpnmb vac) strategy.

[0035] FIG. 3. Ejection fraction illustrates cardiovascular effects of treatment with anti- osteoactivin VLP. Reductions in ejection fraction are associated with aging and with the development of atherosclerotic plaques. Senescent cells promote atherosclerotic plaque development and aging. Both the C-terminal and N-terminal configurations of the anti- osteoactivin VLP vaccines improved ejection fraction.

[0036] FIG. 4. Breathing frequency and minute volume. Breathing frequency and minute volume are associated with age and / or pathology (e.g., fibrosis). Senescent cells contribute to aging and fibrosis. Both the C-terminal and N-terminal configurations of the anti-osteoactivin VLP vaccine improved breathing frequency and minute volume. (A) Effects of anti-osteoactivin VLP vaccination on breathing frequency. (B) Effects of anti-osteoactivin VLP vaccination on minute volume. FIG. 5. Effect of treatment with anti-osteoactivin VLP on lifespan. Lifespan was observed to be significantly increased by the C-terminal VLP, but not the N-terminal.

[0037] FIG. 6. Effect of treatment with anti-osteoactivin VLP on occurrence of prolapse. Senescent cell accumulation is a possible pathophysiological driver of prolapse. (A) Pelvic organ prolapse can be graded by four stages (0-3). (B) Both C-terminal and N-terminal VLPs were effective at extending the time until Grade 2 rectal prolapse occurred.

[0038] FIG. 7. Alopecia and dermatitis comparison between C-terminal, N-terminal, and control VLPs. Senescent cell accumulation contributes to alopecia. Through visual hierarchy, C-terminal has the least amount of alopecia and dermatitis, followed by N-terminal, and then control.

[0039] FIG. 8. Comparison of current VLP strategy to previously published standard vaccination strategy. (A) Original lifespan analysis conducted in female mice as previously described (Suda et al., 2021 Nature Aging 1 : 1117-1 126) was not significant. (B) Vaccination with C-terminal configuration of anti-osteoactivin VLP provides statistically significant increase in lifespan.

[0040] FIG. 9. Comparison of antibody response using ELISA method on blood drawn from QP- vaccinated controls, C-terminal-VLP-vaccinated animals, and N-terminal-VLP -vaccinated animals after the full three-dose injection series (10 weeks after first injection). Custom plates were generated using the full length GPNMB peptide sequence in each well. Blood was drawn from each group and incubated in the wells. Three wells were repeated per animal. Fluorescence was read at an absorbance of 450 nm. The antibody responses in both the C-terminal-VLP- vaccinated animals and the N-terminal -VLP-vaccinated animals were different than the Q - vaccinated control.

[0041] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0042] This disclosure describes, in one aspect, immunogenic compositions that include an antigenic osteoactivin peptide linked to a virus-like particle (VLP) carrier. The compositions may be administered to a subject to elicit an immune response against osteoactivin. The compositions may be administered to a subject to treat one or more symptoms or clinical signs of senescence.

[0043] Osteoactivin, also known as glycoprotein nonmetastatic melanoma protein B (GPNMB), is a transmembrane protein that is associated with senescent cells in general, and, in particular, senescent endothelial cells that are partially responsible for development of atherosclerotic plaques. Eliminating senescent cells in the vasculature can limit progression and / or reverse development of atherosclerotic plaques. More generally, targeting osteoactivin can provide other clinical and / or therapeutic benefits.

[0044] Eliminating senescent cells can improve normal and pathological changes associated with aging. However, many agents that eliminate senescent cells inhibit antiapoptotic pathways, which can result in off target undesirable side effects in normal tissues. Osteoactivin has been identified as a molecular target for eliminating senescent cells that does not inhibit antiapoptotic pathways. Therefore, targeting osteoactivin reduces the likelihood and / or extent of undesirable side effects of conventional senolytic therapies.

[0045] Senescent cells accumulate in various tissues with aging or in response to metabolic stress. For example, senescent vascular endothelial cells are observed in human atherosclerotic plaques. Senescent vascular endothelial cells exhibit functional abnormalities that promote the development of atherosclerosis. Visceral adipose tissue develops senescence-like features in patients with type 2 diabetes and promotes insulin resistance. Senescence has been implicated in pelvic organ prolapse, and senescent lung cells can promote chronic obstructive pulmonary disorder. Thus, eliminating senescent cells (i.e., senolysis or senolytic therapy) could be an attractive strategy for anti senescence therapy.

[0046] Many existing senolytic agents improve age-associated pathologies and extend the healthy lifespan in aged mice. However, existing senolytic agents raise concerns over safety and specificity that cause undesirable off target side effects.

[0047] The osteoactivin peptide RRGDGRWKD (SEQ ID NO: 1) has been identified as an antigen that specifically targets therapy to senescent cells. This disclosure describes compositions in which the antigenic osteoactivin peptide RRGDGRWKD (SEQ ID NO:1) is presented on the surface of a virus-like particle. This disclosure further provides data showing that immunization against osteoactivin using the VLP platform provides effects that are not readily predictable from prior disclosures regarding immunizing against osteoactivin using the antigenic peptide RRGDGRWKD (SEQ ID NO: 1).

[0048] FIG. 1 provides a schematic illustration of metabolic pathways that lead to cell cycle arrest and senescence. The antigen peptide RRGDGRWKD (SEQ ID NO: 1) was conjugated to a VLP carrier via a linker peptide at either the N-terminal of the peptide or the C-terminal of the peptide: VLP-CGGGRRGDGRWKD (N-terminus conjugation, SEQ ID NO:2) or RRGDGRWKDGGGC-VLP (C-terminus conjugation, SEQ ID NO:3). Generally, naturally aged female mice were vaccinated intramuscularly, once every three weeks for three total doses, at a single vaccine concentration of 5 micrograms per 50 microliters, and then monitored every three weeks Vaccination with anti-osteoactivin VLP compositions have provided statistically significant cardiovascular effects, measured via echocardiogram and illustrated by ejection fraction (FIG. 3); pulmonary effects, measured via whole-body plethysmography (FIG. 4); extension of lifespan (FIG. 5); and reduced occurrence of rectal prolapse in a time-dependent manner in the mouse model (FIG. 6). Senescence has been implicated in prolapse onset and / or progression and is considered a marker of frailty in animal model lifespan studies. FIG. 6B, shows a time delay in the onset of Grade 2 pelvic organ prolapse.

[0049] Mice vaccinated with anti-osteoactivin VLP vaccine appear younger than untreated control mice, as reflected by a lesser degree of alopecia, dermatitis, and joint stiffness (FIG. 7). FIG. 8 shows compares lifespan data that reveals vaccination with the C-terminal configuration of the anti-osteoactivin VLP vaccine provided a statistically significant increase in lifespan compared to unvaccinated control mice (FIG. 8B). FIG. 8A shows that a previous non-VLP- based anti-osteoactivin vaccine (Suda et al., 2021 Nature Aging 1 :1117-1126) failed to provide a statistically significant increase in lifespan. Additionally, the previous report describing a non- VLP-based anti-osteoactivin vaccine provided no findings regarding cardiovascular effects, pulmonary effects, reduction in the occurrence of prolapse, or the incidence or extent of alopecia Finally, FIG. 9 shows that an antibody response exists when animals are vaccinated with the N- terminal-VLP or the C-terminal-VLP compared to animals vaccinated with a blank QP VLP. The differential physiological outcomes shown in the data of FIG. 8, FIG. 6B, and FIG. 7 suggest that the orientation of the peptide may influence vaccination strategy.

[0050] FIG. 2A shows a schematic illustration of senescent endothelium in vascular tissues and the use of immunotherapy to eliminate senescent endothelial cells As a result of the immunotherapy, mice exhibited a reduced degree of atherosclerotic plaques when receiving the osteoactivin vaccine compared to control mice that received a control vaccine (FIG. 2B).

[0051] VLP display

[0052] Many viral structural proteins have an intrinsic ability to self-assemble into virus-like particles (VLPs), which structurally resemble the virus from which they were derived but, because they lack viral genomes, they are noninfectious. VLPs not only can serve as stand-alone vaccines, but because their particulate nature and multivalent structure provoke strong immune responses, they can be used as platforms to enhance the immunogenicity of heterologous antigenic targets. For example, when short immunogenic peptides are displayed in a highly repetitive, multivalent fashion on VLPs, peptide-specific B cells are strongly activated, leading to high-titer, long-lasting antibody responses. VLPs derived from diverse virus types can serve as effective platforms for antigen display. The immunogens described herein are based on VLPs derived from a family of related single-stranded RNA bacteriophages, including MS2, PP7, AP205, and Q . These VLPs can be produced by expressing a single viral structural protein, called coat, from a plasmid in a bacterium. In one or more embodiments, the antigenic osteoactivin peptide may be displayed on a VLP by conjugation via a linker peptide such as, for example, amino acids 1-4 of SEQ ID NO:2. While described herein in the context of exemplary embodiments in which the linker peptide is amino acids 1-4 of SEQ ID NO:2, any suitable linker peptide may be used to conjugate the antigenic peptide of SEQ ID NO: 1 to the VLP. Suitable linker peptides are well known to those of ordinary skill in the art. Alternatively, the antigenic osteoactivin peptide may be displayed on a VLP by bioconjugation techniques using cross linker molecules. In one or more embodiments, a peptide may be displayed on a VLP by conjugating the peptide to the VLP through a succinimidyl-6-[P-maleimidopropionamido]hexanoate (SMPH) cross-linker molecule. This technique results in VLPs that display target peptides at high valency, usually 180-360 peptides per VLP, and confers strong immunogenicity to displayed immunogenic peptides.

[0053] While described herein in the context of exemplary embodiments using QP VLPs, the constructs and methods described herein can involve using any suitable VLP platform. The virus-like particle (VLP) can include any particle that includes viral proteins assembled to structurally resemble the virus from which they are derived but lack enough of the viral genome so that they are non-replicative and, therefore, noninfectious. A VLP may, therefore, include at least some of the viral genome, but the viral genome is genetically modified so that the viral genes responsible for infectivity and / or replication are inactivated. Exemplary VLPs include, but are not limited to, VLPs of QP, MS2, PP7, AP205, or other bacteriophage coat proteins, the capsid and core proteins of Hepatitis B virus, measles virus, Sindbis virus, rotavirus, foot-and- mouth-disease virus, Norwalk virus, the retroviral GAG protein, the retrotransposon Ty protein pl, the surface protein of Hepatitis B virus, human papilloma virus, human polyoma virus, RNA phages, Ty, frphage, GA-phage, AP 205-phage and, in particular, QP-phage, Cowpea chlorotic mottle virus, cowpea mosaic virus, human papilloma viruses (HPV), bovine papilloma viruses, porcine parvovirus, parvoviruses such as Bl 9, porcine (PPV) and canine (CPV) parvovirues, caliciviruses (e.g., Norwalk virus, rabbit hemorrhagic disease virus [RHDV]), animal hepadnavirus core Antigen VLPs, filamentous / rod-shaped plant viruses, including but not limited to Tobacco Mosaic Virus (TMV), Potato Virus X (PVX), Papaya Mosaic Virus (PapMV), Alfalfa Mosaic Virus (AIMV), and Johnson Grass Mosaic Virus (JGMV), insect viruses such as flock house virus (FHV) and tetraviruses, polyomaviruses such as Murine Polyomavirus (MPyV), Murine Pneumotropic Virus (MPtV), BK virus (BKV), and JC virus (JCV).

[0054] When bacteriophage MS2 coat protein is expressed from a plasmid in E. coll, it selfassembles into an icosahedral particle about 28 nm in diameter. Foreign peptide sequences genetically fused to coat protein end up on the VLP surface where they are displayed in highly immunogenic dense repetitive arrays. This disclosure describes an approach to displaying the osteoactivin on VLPs.

[0055] MS2 and PP7 coat proteins have highly similar three-dimensional structures. Each coat protein folds as a homodimer of two intimately intertwined subunits. Dimers self-assemble to produce the icosahedral VLP in which the N-termini of three different coat proteins are brought into proximity at each of the icosahedron’s three-fold symmetry axes. Since the C-termini are close to the N-termini, both ends are similarly arranged around the viral three-fold symmetry axes. In one or more embodiments, the VLPs described herein involve fusing the antigenic osteoactivin peptide (SEQ ID NO: 1) to the VLP through either terminus and exploiting the coat protein’s natural propensity for trimerization.

[0056] Construction and antigenicity of osteoactivin VLPs

[0057] QP VLPs that multivalently display the nine-amino-acid sequence (SEQ ID NO: 1) were prepared by chemically conjugating the antigenic peptide to the surface of VLP using a linker at either the N-terminal (SEQ ID NO:2) or the C-terminal (SEQ ID NO:3). While described below in the context of an exemplary embodiment in which the VLP platform used to present the antigenic anti-osteoactivin peptide is a QP VLP, the compositions and methods described herein can involve the use of any suitable VLP platform. Thus, as noted above, VLPs that present an antigenic anti-osteoactivin peptide can be derived from any one of a family of related singlestranded RNA bacteriophages including, but not limited to, MS2, PP7, AP205, or Q0. A similar strategy was used for N-terminal display (SEQ ID NO:2) and C-terminal display (SEQ ID NO:3), in which the peptide sequence was chemically linked to the QP via an SMPH linker. The resultant peptide sequences, with linker amino acids at either the N-terminal and C-terminal of the anti-osteoactivin peptide, were verified as conjugated by gel size electrophoresis.

[0058] The QP coat protein constructs were expressed in E. coli and self-assembled into VLPs displaying the osteoactivin peptide on their surface after expression and purification. VLP formation was confirmed by electron microscopy. Peptide display was quantified by mass spectrometry analysis of enzymatic digests of the purified VLPs.

[0059] Alternative designs

[0060] In one or more embodiments, the osteoactivin peptide may include one or more amino acid deletions, additions, substitutions, or a combination of such modifications to the amino acid sequence of SEQ ID NO: 1 . Alternative designs may better mimic the form of the osteoactivin peptide that naturally elicits antibodies. Regardless of the specific design, a vaccine directed against osteoactivin can provoke new immunity or strengthen and / or prolong a pre-existing immune response (e.g., from prior vaccination) against osteoactivin.

[0061] Compositions

[0062] This disclosure therefore describes VLP-based immunogens that include peptides of osteoactivin (e.g , SEQ ID NO: 1). or a structurally similar variant thereof. As used herein, a peptide or variant of a peptide is “structurally similar” to a reference peptide if the amino acid sequence of the peptide possesses a specified amount of identity compared to the reference peptide. Structural similarity of two peptides can be determined by aligning the residues of the two peptides (for example, a candidate peptide and SEQ ID NO: 1) to optimize the number of identical amino acids along the lengths of their sequences, gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate peptide is the peptide being compared to the reference peptide (e.g., SEQ ID NO: 1). A candidate peptide can be isolated, for example, from an animal, or can be produced using recombinant techniques, or chemically or enzymatically synthesized.

[0063] A pair-wise comparison analysis of amino acid sequences can be carried out using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI). Alternatively, peptides may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website. The default values for all BLAST 2 search parameters may be used, including matrix = BLOSUM62; open gap penalty = 11, extension gap penalty = 1, gap x dropoff = 50, expect = 10, wordsize = 3, and filter on.

[0064] An antigenic osteoactivin peptide can include amino acids in addition to SEQ ID NO: 1, so long as the additional amino acids do not eliminate immunogenicity toward osteoactivin. For example, an antigenic osteoactivin peptide may include a linker sequence such as, for example, amino acids 1-4 of SEQ ID NO:2 or any other suitable linker amino acid sequence known to those of skill in the art.

[0065] In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also includes the presence of conservative substitutions. A conservative substitution for an amino acid in an immunogenic peptide as described herein may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free -OH is maintained; and Gin for Asn to maintain a free -NH2. Likewise, biologically active analogs of a polypeptide containing deletions or additions of one or more contiguous or noncontiguous amino acids that do not eliminate a functional activity of the peptide are also contemplated.

[0066] In one or more embodiments, an osteoactivin peptide as described herein can include a peptide with at least 55%, at least 66%, at least 77% or at least 88% sequence similarity to the amino acids sequence of SEQ ID NO: 1. That is, an osteoactivin peptide can include a total of no more than four, no more than three, no more than two, or no more than one amino acid deletions and non-conservative amino acid substitutions compared to SEQ ID NO: 1 .

[0067] In one or more embodiments, an osteoactivin peptide as described herein can include a peptide with a segment having at least at least 55%, at least 66%, at least 77%, or at least 88% sequence identity to the amino acids sequence of SEQ ID NO: 1 . That is, an osteoactivin peptide can include a segment having a total of no more than four, no more than three, no more than two, or no more than one amino acid deletions and amino acid substitutions compared to SEQ ID NOT.

[0068] In one or more embodiments, an antigenic osteoactivin as described herein can be designed to provide additional sequences, such as, for example, the addition of added C-terminal or N-terminal amino acids. The additional amino acids can, for example, serve as a linker peptide, as exemplified in SEQ ID NO:2 and SEQ ID NOT. As another example, the additional amino acids can facilitate purification by trapping on columns or use of antibodies. Such tags include, for example, histidine-rich tags that allow purification of polypeptides on nickel columns. Such modification techniques and suitable additional sequences are well known in the molecular biology arts.

[0069] The virus-like particle (VLP) can include any particle that includes viral protein assembled to structurally resemble the virus from which they are derived but lack enough of the viral genome so that they are non-replicative and, therefore, noninfectious. A VLP may, therefore, include at least some of the viral genome, but the viral genome is genetically modified so that the viral genes responsible for infectivity and replication are inactivated. Exemplary VLPs include, but are not limited to, VLPs of Q0, MS2, PP7, AP205, or other bacteriophage coat proteins, the capsid and core proteins of Hepatitis B vims, measles vims, Sindbis vims, rotavims, foot-and-mouth-disease vims, Norwalk vims, the retroviral GAG protein, the retrotransposon Ty protein pl, the surface protein of Hepatitis B vims, human papilloma vims, human polyoma virus, RNA phages, Ty, frphage, GA-phage, AP 205-phage and, in particular, Q0-phage, Cowpea chlorotic mottle virus, cowpea mosaic virus, human papilloma viruses (HPV), bovine papilloma viruses, porcine parvovirus, parvoviruses such as Bl 9, porcine (PPV) and canine (CPV) parvovirues, caliciviruses (e g. Norwalk virus, rabbit hemorrhagic disease virus [RHDV]), animal hepadnavirus core Antigen VLPs, filamentous / rod-shaped plant viruses, including but not limited to Tobacco Mosaic Virus (TMV), Potato Virus X (PVX), Papaya Mosaic Virus (PapMV), Alfalfa Mosaic Virus (AIMV), and Johnson Grass Mosaic Virus (JGMV), insect viruses such as flock house virus (FHV) and tetraviruses, polyomaviruses such as Murine Polyomavirus (MPyV), Murine Pneumotropic Virus (MPtV), BK virus (BKV), and JC virus (JCV).

[0070] The antigenic osteoactivin peptide may be coupled to immunogenic carriers via chemical conjugation or by expression of genetically engineered fusion partners. In this context, the term “antigenic osteoactivin peptide” refers, collectively to the osteoactivin peptide of SEQ ID NO: 1 or any other peptide known to elicit an immunogenic response against osteoactivin.

[0071] The coupling does not necessarily need to be direct, but can occur through linker sequences, as expressly exemplified in SEQ ID NO:2 and SEQ ID NO:3. More generally, in the case that antigenic peptides either fused, conjugated, or otherwise attached to an immunogenic carrier, spacer sequence, or linker sequence are typically added at one or both ends of the antigenic peptides. If the VLP is designed to include more than one antigenic peptide, the coupling approach used for any one antigenic peptide may be selected independently of the coupling approach used for any other antigenic peptide.

[0072] In one embodiment, the antigenic peptide may be displayed as fusion protein with a subunit of the immunogenic carrier. Fusion of the peptide can be effected by inserting the antigenic peptide amino acid sequence into the immunogenic carrier primary sequence, or by fusion to either the N-terminus or C-terminus of the immunogenic carrier.

[0073] When the immunogenic carrier is a VLP, the chimeric antigenic peptide- VLP subunit can be capable of self-assembly into a VLP. VLP displaying epitopes fused to their subunits are also herein referred to as chimeric VLPs. For example, European Application No. EP903I0264A (European Patent No. EP0421635 Bl) describes the use of chimeric hepadnavirus core antigen particles to present foreign peptide sequences in a virus-like particle. Flanking amino acid residues may be added to either end of the sequence of the antigenic peptide to be fused to either end of the sequence of the subunit of a VLP, or for internal insertion of such peptide sequence into the sequence of the subunit of a VLP. Glycine and serine residues are particularly favored amino acids to be used in the flanking sequences added to the peptide to be fused. Glycine residues confer additional flexibility, which may diminish the potentially destabilizing effect of fusing a foreign sequence into the sequence of a VLP subunit.

[0074] In one or more embodiments, the immunogenic carrier is a VLP of a RNA phage, preferably Q . The major coat proteins of RNA phages spontaneously assemble into VLPs upon expression in bacteria such as, for example, E. coli.

[0075] Further VLPs suitable for fusion of antigens or antigenic determinants are described in, for example, International Patent Application No. PCT / TB2002 / 004132 (International Publication No. WO 03 / 024481 A2) and include bacteriophage fr, capsid protein of papillomavirus, retrotransposon Ty, yeast and also Retrovirus-like-particles, HIV2 Gag, Cowpea Mosaic Virus, parvovirus VP2 VLP, HBsAg (U.S. Patent No. 4,722,840). Examples of chimeric VLPs suitable for use as the immunogenic carrier include those described in Kozlovska et al., 1996, Intervirology 39:9-15. Further examples of VLPs suitable for use as the immunogenic carrier include, but are not limited to, HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, HIV GAG, Tobacco Mosaic Virus, Virus-like particles of SV-40, Polyomavirus, Adenovirus, Herpes Simplex Virus, Rotavirus, and Norwalk virus.

[0076] For any recombinantly expressed antigenic peptide described herein (whether or not coupled to an immunogenic carrier), this disclosure describes the nucleic acid that encodes the peptide or protein, an expression vector containing the nucleic acid, and a host cell containing the expression vector (autonomously or chromosomally inserted). This disclosure further describes a method of recombinantly producing the peptide or protein by expressing it in a host cell, with or without further isolating the immunogen.

[0077] Thus, this disclosure describes an isolated nucleic acid sequence that encodes any embodiment of an antigenic osteoactivin peptide as described herein. In one or more embodiments, the isolated nucleic acid encodes the antigenic peptide of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or a structurally similar variant of any of the foregoing. Given the amino acid sequence of any antigenic peptide, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods.

[0078] As used herein, the term “nucleic acid” or “oligonucleotide” refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, nucleic acids conjugates, and oligonucleotides. A nucleic acid may be single-stranded, double-stranded, linear, or covalently circularly closed molecule. A nucleic acid can be isolated. The term “isolated nucleic acid” means that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, (iv) was synthesized, for example, by chemical synthesis, or (vi) extracted from a sample. A nucleic might be introduced — i.e., transfected — into cells. When RNA is used to transfect cells, the RNA may be modified by stabilizing modifications, capping, or polyadenylation.

[0079] As used herein “amplified DNA” or “PCR product” refers to an amplified fragment of DNA of defined size. Various techniques are available and well known in the art to detect PCR products. PCR product detection methods include, but are not restricted to, gel electrophoresis using agarose or polyacrylamide gel and adding ethidium bromide staining (a DNA intercalant), labeled probes (radioactive or non-radioactive labels, southern blotting), labeled deoxyribonucleotides (for the direct incorporation of radioactive or non-radioactive labels) or silver staining for the direct visualization of the amplified PCR products; restriction endonuclease digestion, which relies on agarose gel electrophoresis, polyacrylamide gel electrophoresis, or high-performance liquid chromatography (HPLC); dot blots, using the hybridization of the amplified DNA on specific labeled probes (radioactive or non-radioactive labels); high-pressure liquid chromatography using ultraviolet detection; electrochemiluminescence coupled with voltage-initiated chemical reaction / photon detection; and direct sequencing using radioactive or fluorescently labeled deoxyribonucleotides for the determination of the precise order of nucleotides with a DNA fragment of interest, oligo ligation assay (OLA), PCR, qPCR, DNA sequencing, fluorescence, gel electrophoresis, magnetic beads, allele specific primer extension (ASPE) and / or direct hybridization. Generally, nucleic acid can be extracted, isolated, amplified, or analyzed by a variety of techniques such as those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 pages (2012); or as described in U.S. Patent No 7,957,913; U.S. Patent No. 7,776,616; U.S. Patent No. 5,234,809; and U.S. Patent No. 9,012,208. Examples of nucleic acid analysis include, but are not limited to, sequencing and DNA-protein interaction. Sequencing may be by any method known in the art. DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, and next generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina / Solexa sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing. Separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes.

[0080] This disclosure also describes a host cell including any of the isolated nucleic acid sequences and / or antigenic peptides described herein. Thus, this disclosure encompasses translation of a nucleic acid (e.g., an mRNA) by a host cell to produce any embodiment of antigenic peptide disclosed herein and / or any embodiment of VLP cribbed herein that displays any one or more antigenic peptide.

[0081] The nucleic acid constructs of the present invention may be introduced into a host cell to be altered, thus allowing expression within the cell of the antigenic peptide and / or the VLP displaying any one or more antigenic peptides, thereby generating a genetically engineered cell. A variety of methods are known in the art and suitable for introducing a nucleic acid into a cell, including viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Other methods of transfection include proprietary transfection reagents such as LIPOFECTAMINE (Thermo Fisher Scientific, Inc., Waltham, MA), HILYMAX (Dojindo Molecular Technologies, Inc., Rockville, MD), FUGENE (Promega Corp., Madison, WI), JETPEI (Polyplus Transfection, Illkirch, France), EFFECTENE (Qiagen, Hilden, Germany) and DreamFect (OZ Biosciences, Inc USA, San Diego, CA).

[0082] The nucleic acid constructs described herein may be introduced into a host cell to be altered, thus allowing expression within the cell of the protein encoded by the nucleic acid. A variety of host cells are known in the art and suitable for protein expression. Examples of typical cell used for transfection and protein expression include, but are not limited to, a bacterial cell, a eukaryotic cell, a yeast cell, an insect cell, or a plant cell such as, for example, E. coli. Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimuriuni , Drosophila S2, Spodoptera SJ9, CHO, COS (e g , COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F.

[0083] In one or more embodiments, the antigenic peptide can be chemically coupled to the immunogenic carrier using techniques well known in the art. Conjugation can occur to allow free movement of peptides via single point conjugation (e.g., either N-terminal or C-terminal point) or as a locked down structure where both ends of peptides are conjugated to either an immunogenic carrier protein or to a scaffold structure such as a VLP. Conjugation occurs via conjugation chemistry known to those skilled in the art such as via cysteine residues, lysine residues, or another carboxy moiety. Thus, for example, for direct covalent coupling, it is possible to use a carbodiimide, glutaraldehyde, or N-[y-maleimidobutyryloxy] succinimide ester, using common commercially available hetero-bifunctional linkers such as l-cyano-4- dimethylaminopyridinium tetrafluoroborate (CDAP) or succinimidyl 3-(2- pyridyldithio)propionate (SPDP).

[0084] Examples of conjugation of peptides, particularly cyclized peptides, to a protein carrier via acylhydrazine peptide derivatives are described in, for example, International Patent Application No. PCT / EP2003 / 004551 (International Publication No. WO 2003 / 092714 Al). After the coupling reaction, the immunogen can easily be isolated and purified using, for example, a dialysis method, a high-performance liquid chromatography method, a gel filtration method, a fractionation method, etc. Peptides terminating with a cysteine residue (preferably with a linker outside the cyclized region) may be conveniently conjugated to a carrier protein via maleimide chemistry. Several antigenic peptides, either having an identical amino acid sequence or different amino acid sequences, may be coupled to a single VLP particle, leading preferably to a repetitive and ordered structure presenting several antigenic determinants in an oriented manner as described in International Patent Applications PCT / TB 1999 / 001 25 (International Publication No. WO 00 / 032227), PCT / IB2002 / 004132 (International Publication No. WO 2003 / 024481), PCI7IB2002 / 000166 (International Publication No. WO 02 / 056905), and PCT / EP2003 / 007572 (International Publication No. WO 2004 / 007538). Thus, the antigenic peptide displayed by one VLP subunit in a VLP may the same or different than the antigenic peptide displayed by a second VLP subunit in the same VLP. In other embodiments, one or several antigen molecules can be attached to one VLP subunit. A specific feature of the VLP of the coat protein of RNA phages is thus the possibility to couple several antigens per subunit. This allows for the generation of a dense antigen array.

[0085] Another feature of VLPs derived from RNA phage is their high expression yield in bacteria that allows production of large quantities of material at affordable cost. Moreover, the use of the VLPs as carriers allows the formation of robust antigen arrays and conjugates, respectively, with variable antigen density. In particular, the use of VLPs of RNA phages allows a very high antigen density to be achieved.

[0086] Methods of Treatment

[0087] VLPs displaying one or more antigenic peptides may be used to treat a subject having, or at risk of having, any condition caused by the presence of senescent cells that express osteoactivin.

[0088] As used herein, “treat” or variations thereof refer to reducing, limiting progression, ameliorating, or resolving, to any extent, the symptoms or signs related to a condition. A “sign” or “clinical sign” refers to an objective physical finding relating to a particular condition capable of being found by one other than the patient. A “symptom” refers to any subjective evidence of disease or of a patient’s condition.

[0089] A “treatment” may be therapeutic or prophylactic. “Therapeutic” and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition. “Prophylactic” and variations thereof refer to a treatment that limits, to any extent, the development and / or appearance of a symptom or clinical sign of a condition. Generally, a “therapeutic” treatment is initiated after the condition manifests in a subject, while “prophylactic” treatment is initiated before a condition manifests in a subject.

[0090] Treatment that is prophylactic — e.g., initiated before a subject manifests a symptom or clinical sign of the condition such as, for example, while a tumor remains subclinical — is referred to herein as treatment of a subject that is “at risk” of having the condition. As used herein, the term “at risk” refers to a subject that may or may not actually possess the described risk. Thus, for example, a subject “at risk” of developing a condition is a subject possessing one or more risk factors associated with the condition such as, for example, genetic predisposition, ancestry, age, sex, geographical location, lifestyle, or medical history. Thus, the VLP displaying one or more antigenic osteoactivin peptides may be administered before a subject manifests a symptom or clinical sign of a condition caused by the presence of senescent cells that express osteoactivin.

[0091] Accordingly, a composition can be administered before, during, or after the subject first exhibits a symptom or clinical sign of a condition caused by the presence of senescent cells that express osteoactivin. Treatment initiated before the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the likelihood that the subject experiences clinical evidence of the condition compared to a subject to which the composition is not administered, decreasing the severity of symptoms and / or clinical signs of the condition, and / or completely resolving the condition. Treatment initiated after the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the severity of symptoms and / or clinical signs of the condition compared to a subject to which the composition is not administered, and / or completely resolving the condition.

[0092] Thus, the method includes administering an effective amount of the composition to a subject having, or at risk of having, a condition characterized, at least in part, by the presence of senescent cells that express osteoactivin. In this aspect, an “effective amount” is an amount effective to reduce, limit progression, ameliorate, or resolve, to any extent, a symptom or clinical sign related to the condition.

[0093] In one or more embodiments, and effective amount of the composition is an amount effective to reduce atherosclerotic plaques in a subject compared to a comparable control subject to which the composition has not been administered. In one or more embodiments, and effective amount of the composition is an amount effective to improve a cardiovascular indicium in a subject compared to a comparable control subject to which the composition has not been administered. In one or more of these embodiments, the cardiovascular indicium is an ejection fraction and an effective amount of the composition is an amount effective to increase ejection fraction compared to a comparable control subject to which the composition has not been administered.

[0094] In one or more embodiments, and effective amount of the composition is an amount effective to improve one or more pulmonary indicia in a subject compared to a comparable control subject to which the composition has not been administered. In one or more of these embodiments, the pulmonary indicium is breathing frequency and an effective amount of the composition is an amount effective to increase breathing frequency compared to a comparable control subject to which the composition has not been administered. In one or more alternative embodiments, the pulmonary indicium is minute volume and an effective amount of the composition is an amount effective to increase minute volume compared to a comparable control subject to which the composition has not been administered.

[0095] In one or more embodiments, and effective amount of the composition is an amount effective to prolong survival in a subject compared to a comparable control subject to which the composition has not been administered.

[0096] In one or more embodiments, and effective amount is an amount effective to reduce an indicium of frailty in a subject compared to a comparable control subject to which the composition has not been administered. In one or more of these embodiments, the indicium of frailty is prolapse and an effective amount of the composition is an amount effective to reduce the likelihood or incidence of prolapse compared to a comparable control subject to which the composition has not been administered.

[0097] Thus, any embodiment of the VLPs described herein may be formulated with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial, and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and / or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the VLP without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0098] The VLP may therefore be formulated into a pharmaceutical composition. The pharmaceutical composition may be formulated in a variety of forms adapted to a preferred route of administration. Thus, a composition can be administered via known routes including, for example, oral, parenteral (e.g, intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.). A pharmaceutical composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). A composition also can be administered via a sustained or delayed release.

[0099] Thus, a VLP may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The composition may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. The formulation may further include one or more additives including such as, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like.

[0100] A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the VLP into association with a carrier that constitutes one or more accessory ingredients. In general, a formulation may be prepared by uniformly and / or intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

[0101] The amount of VLP administered can vary depending on various factors including, but not limited to, the cancer being treated, the weight, physical condition, and / or age of the subject, and / or the route of administration. Thus, the absolute weight of VLP included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight, and physical condition of the subject, and / or the method of administration. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of VLP effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.

[0102] In one or more embodiments, the method can include administering sufficient VLP to provide a dose of, for example, from about 50 ng / kg to about 1 mg / kg to the subject, although in one or more embodiments the methods may be performed by administering VLP in a dose outside this range.

[0103] In one or more embodiments, the method includes administering sufficient VLP to provide a minimum dose of at least 50 ng / kg such as, for example, at least 100 ng / kg, at least 200 ng / kg, at least 300 ng / kg, at least 400 ng / kg, at least 500 ng / kg, at least 600 ng / kg, at least 700 ng / kg, at least 800 ng / kg, at least 900 ng / kg, at least 1 pg / kg, at least 2 pg / kg, at least 5 pg / kg, at least 10 pg / kg, at least 20 pg / kg, at least 50 pg / kg, at least 100 pg / kg, at least 200 pg / kg, or at least 500 pg / kg.

[0104] In one or more embodiments, the method includes administering sufficient VLP to provide a maximum dose of no more than 1 mg / kg, no more than 500 pg / kg, no more than 250 pg / kg, no more than 200 pg / kg, no more than 150 pg / kg, no more than 100 pg / kg, no more than 50 pg / kg, no more than 25 pg / kg, no more than 10 pg / kg, no more than 5 pg / kg, no more than 2 pg / kg, no more than 1 pg / kg, no more than 800 ng / kg, no more than 600 ng / kg, no more than 500 ng / kg, no more than 400 ng / kg, no more than 300 ng / kg, no more than 250 ng / kg, no more than 150 ng / kg, no more than 100 ng / kg, no more than 50 ng / kg, or no more than 25 ng / kg.

[0105] In one or more embodiments, the method includes administering sufficient VLP to provide a dose that falls within a range having as endpoints any minimum dose listed above and any maximum dose listed above that is greater than the minimum does. For example, in one or more embodiments, the method can includes administering sufficient VLP to provide a dose of from 200 ng / kg to about 10 pg / kg to the subject, for example, a dose of from about 700 ng / kg to about 5 pg / kg.

[0106] In one or more embodiments, VLP may be administered, for example, from a single dose to multiple doses per week, although in one or more embodiments the method can be performed by administering VLP at a frequency outside this range. When multiple doses are used within a certain period, the amount of each dose may be the same or different. For example, a dose of 1 mg per day may be administered as a single dose of 1 mg, two 0.5 mg doses, or as a first dose of 0.75 mg followed by a second dose of 0.25 mg. Also, when multiple doses are used within a certain period, the interval between doses may be the same or be different.

[0107] In certain embodiments, VLP may be administered at minimum frequency of at least once per year such as, for example, at least once every six months, at least once every four months, at least once every three months, at least once every two months, at least once per month, at least once every three weeks, or at least once every two weeks.

[0108] In certain embodiments, VLP may be administered at maximum frequency of no more than once per week such as, for example, no more than once every two weeks, no more than once per month, no more than once every two months, no more than once every three months, no more than once every six months, or once per year.

[0109] In one or more embodiments, VLP may be administered at a frequency defined by a range having as endpoints any minimum frequency listed above and any maximum frequency listed above that is more frequent than the minimum frequency.

[0110] The duration of administration of an antigenic VLP described herein, e.g., the period of time over which an antigenic VLP is administered, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, an antigenic VLP can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about one year, from about one year to about two years, or from about two years to about four years, or more. In one or more embodiments, the VLP may be administered as a once off treatment. In other embodiments, the VLP may be administered for the life of the subject. In certain embodiments, the VLP may be administered every three weeks until effective.

[0111] In one or more embodiments, the VLP may be administered at an initial frequency for an initial period and then administered at a lower frequency thereafter. For example, a dosing regimen may include administering three doses of the VLP at a frequency of once per month (i.e., an initial dose followed by a second dose one month after the initial dose) followed by an additional dose six months after the initial dose.

[0112] When a VLP composition is used for prophylactic treatment, it may be generally administered for priming and / or boosting doses. Boosting doses, when administered, are adequately spaced (e.g., yearly) to boost the level of circulating antibody that has fallen below a desired level. Boosting doses may include an antigenic peptide either with or in the absence of the original immunogenic carrier A booster composition may include an alternative immunogenic carrier or may be in the absence of any carrier. Moreover, a booster composition may be formulated either with or without adjuvant.

[0113] In some cases, the method can further include administering to the subject an additional therapeutic agent effective for treating the condition caused or characterized by the presence of senescent cells that express osteoactivin.

[0114] In one or more embodiments, the condition being treated can be a senopathy — i.e., a pathology, disease, or condition associated with senescence and / or senescent cells (e.g., senescent endothelial cells). Such conditions include, but are not limited to, cardiovascular diseases, metabolic disorders, musculoskeletal diseases, liver diseases, kidney diseases, neurodegenerative diseases, lung diseases, and / or inflammatory diseases.

[0115] Exemplary cardiovascular diseases include, but are not limited to, atherosclerosis (e.g., advanced atherosclerotic lesions, elastic fiber degradation, fibrous cap thinning, increased metalloprotease production, etc.) or hypertension (e.g., arterial stiffness, endothelial dysfunction, etc.).

[0116] Exemplary metabolic disorders include, but are not limited to, diabetes and related conditions (e.g., Type 1 diabetes, type 2 diabetes, insulin resistance, lipotoxicity, etc.) or obesity (e.g., adipose tissue inflammation, increased senescent P-galactosidase activity, etc.).

[0117] Exemplary musculoskeletal diseases include, but are not limited to, osteoporosis (e.g., bone mass loss, decreased bone strength, etc ), osteoarthritis (e.g., articular cartilage erosion, SASP-associated gene transcription, etc.), or sarcopenia (e.g., muscle atrophy, reduced muscle strength, etc.).

[0118] Exemplary liver diseases include, but are not limited to, liver fibrosis (e.g., steatosis, increased SASP production, etc.).

[0119] Exemplary kidney diseases include, but are not limited to, chronic kidney disease (e.g., decreased kidney function, increased Lamin Bl positive cells and IL-6 expression, etc.). Exemplary neurodegenerative diseases include, but are not limited to, Alzheimer’s disease (e.g., accumulation of tau proteins, synaptic dysfunction, cognitive decline, etc.), Parkinson’s disease (e.g., dopaminergic neurodegeneration, increased SA-0-gal expression, etc.), or amyotrophic lateral sclerosis (AES, e.g., motor neuron death, increased pl 6-positive nuclei, etc.).

[0120] Exemplary lung diseases include, but are not limited to, idiopathic pulmonary fibrosis (e.g., pulmonary fibrosis, increased SA-0-gal positive cells, increased mRNA levels of pl6INK4A, Bcl-2, IL-la, and / or IL-ip, etc.) or chromic obstructive pulmonary disease (COPD, e g., alveolar epithelial cell senescence, increased lung inflammation, etc ).

[0121] Exemplary inflammatory diseases include, but are not limited to, rheumatoid arthritis (e.g., joint inflammation, increased SASP factors, etc.) or atherosclerosis (e.g., senescent intimal foam cells, chronic inflammation, etc ).

[0122] In the preceding description and following claims, the term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended — i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0123] As used herein, “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” or the like are used in their open-ended inclusive sense, and generally mean “include, but not limited to,” “includes, but not limited to,” or “including, but not limited to.” Further, wherever embodiments are described herein with the language “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” and the like, otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. The term “consisting of’ means including, and limited to, that which follows the phrase “consisting of.” That is, “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of’ indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0124] As used herein, the word “exemplary” means to serve as an illustrative example and should not be construed as preferred or advantageous over other embodiments.

[0125] As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0126] In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.

[0127] In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0128] For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously.

[0129] EXAMPLES The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0130] Construction of osteoactivin VLPs

[0131] Peptide SEQ ID NO:2 and SEQ ID NOG were purchased from GenScript Biotech Corp. (Piscataway, NJ) and confirmed as greater than or equal to 95% purity via HPLC. The aminereactive arm of the heterobifunctional crosslinker SMPH was conjugated to surface-exposed lysine residues on Qp virus-like particles (VLPs). This was achieved by reacting the QP VLPs with SMPH at a 1 : 10 molar ratio. The QP VLP-SMPH conjugates were then purified by centrifugation using a filtration device (AMICON, Merck KGaA, Darmstadt, Germany). The QP VLP-SMPH conjugates were subsequently linked to either SEQ ID NO:2 peptide or SEQ ID NOG peptide via the sulfhydryl group on the C-terminal cysteine residue of the peptides. The peptide conjugation was performed by reacting QP VLP-SMPH with the peptides at a 1 : 10 molar ratio, followed by purification of the peptide-conjugated VLPs by centrifugation with the AMICON filter (Merck KGaA, Darmstadt, Germany). To assess the efficiency of conjugation, the peptide-conjugated VLPs were analyzed by SDS-PAGE using a 10% denaturing polyacrylamide gel.

[0132] Bacterial production of the osteoactivin VLPs

[0133] VLP Qp were produced by transforming a plasmid into E. coli. The plasmid contained an inducible T7 promoter upstream of an MS2 single chain dimer and antibiotic resistance gene (kanamycin) for bacterial selection after plating. Bacterial replication was scaled into Erlenmeyer flaks while using IPTG to induce the T7 promoter. Batch cultures were pelleted by centrifugation, supernatant removed, and pellets were resuspended in lysis buffer with addition sonication to ensure bacterial cell wall rupture. Extracted nucleic acids were digested and precipitated by ammonium sulfate before size-exclusion column separation. Fractional screening was performed from the size-exclusion column to determine VLP fraction. A second ammonium sulfate precipitation step was performed, dialyzed twice to remove salts, and the fraction was spun down in a filter device (AMICON, Merck KGaA, Darmstadt, Germany) to further concentrate VLPs. Animal studies

[0134] Fifteen Female C57BL / 6J mice were acquired at 18-months of age that had previously been placed in a retirement colony room. They were rescued and housed in an AAALAC facility, according to lACUC-approved methodologies, on a 14: 10 lightdark cycle with food and water ad libitum. For plethysmography, mice were placed in a mouse 4 site whole body plethysmography chamber (Data Sciences International, St. Paul, MN), linked to controller and software (BUXCO FINEPOINT, Data Sciences International, St. Paul, MN). Each chamber was calibrated before measurements were acquired, and each mouse was allowed a 20-minute acclimation period in the chamber before measurements began. Measurements were taken continuously for a 45-minute duration and normalized based on body weight. Data was analyzed and graphed using PRISM (v. 10.0.0) software (GraphPad Software, Inc., San Diego, CA. Echocardiography data was acquired using the Vevo 3100 (VISUALSONICS, software v 3.1.1.13103, Fujifilm Holdings Corp., Tokyo, Japan) and MX550D (VISUALSONICS, Fujifilm Holdings Corp., Tokyo, Japan). Mice were anesthetized with isoflurane in 100% O2 at a rate of IL / min. Fur was removed by application of a depilatory (NAIR, Church & Dwight Co., Inc., Ewing NJ), ultrasound transmission gel was applied (AQUASONIC, Parker laboratories, Inc., Fairfield, NJ), and mice were placed supine on a temperature-controlled platform (THM 150, VISUALSONICS, Fujifilm Holdings Corp., Tokyo, Japan). Heart rate was continuously monitored, and isoflurane was adjusted as needed for a targeted 400-500 bpm. Cardiac parameters (left ventricular dimensions, stroke volume, ejection fraction, wall thickness) were acquired on short axis M-mode. All echocardiography was performed the day of terminus for each cohort in a blinded fashion. Resultant digital images were downloaded and analyzed using Vevo Lab software (v. 5.6.1; Fujifilm Holdings Corp., Tokyo, Japan)). Rectal prolapse was assessed weekly during body mass measurements. Mice exhibiting Grade 2 or higher rectal prolapse were documented. Grade 0 - Grade 3 were based on the reference textbook Pelvic Floor Biomechanics From Animal Models, Chapter 6.

[0135] Peptide ELISA Protocol

[0136] Commercial streptavidin solution (Thermo Fisher Scientific, Inc., Waltham, MA) was prepared at a concentration of 50 pg / mL. ELISA microplates (Thermo Fisher Scientific, Inc., Waltham, MA) were prepared as follows: in one plate, 50 pL of streptavidin stock solution was added to 5 mL of PBS; in six plates, 80 pL of streptavidin stock solution (5 pg / pL) was diluted in 40 mL of PBS. Each well was labeled and filled with 50 pL of streptavidin solution using a multichannel pipette. Plates were incubated at 37 °C for two hours or at 4 °C overnight. Plates were washed four times with PBS to remove unbound streptavidin.

[0137] SMPH (succinimidyl 6-(beta-maleimidopropionarnido) hexanoate; Millpore Sigma, Burlington, MA) solution was diluted in DMSO to a concentration of 5 pg / pL. For example, 0.00005 g of SMPH was diluted in 100 pL of DMSO. A 5 mL SMPH solution was prepared for each plate by adding 5 pL of the SMPH stock solution to 5 mL of PBS. For six plates, 40 pL of SMPH stock solution was diluted in 40 mL of PBS. Each well was filled with 50 pL of SMPH solution using a multichannel pipette and incubated at room temperature for one hour. Plates were washed four times with PBS to remove unbound SMPH.

[0138] Peptide solution (Novus Biologicals, LLC, Centennial, CO) was prepared by adding 2.5 pL of a 10 pg / pL peptide stock solution to 5 mL of PBS, resulting in a final concentration of 250 ng / 50 pL per well. Each well was filled with 50 pL of the peptide solution and incubated overnight at 4 °C or for two hours at room temperature.

[0139] A 0.5% milk solution was prepared by dissolving 0.25 g of dry milk in 50 mL of PBS and stirred for 15 minutes or overnight. Plates were washed four times with PBS, then filled with 50 pL of the milk solution using a multichannel pipette. Plates were incubated for one hour on a gyroscopic mixer at room temperature and washed with PBS.

[0140] Sera were diluted starting with a 1 :40 dilution, followed by serial 1 :4 dilutions. For the first row, 2.5 pL of sera was added to 97.5 pL of milk solution. Subsequent rows received 75 pL of milk solution and 25 pL from the previous row. From the dilution plate, 50 pL from each well was transferred to the ELISA plate, avoiding contact with the well bottom. Plates were incubated for two hours on a gyroscopic mixer at room temperature and washed four times with PBS.

[0141] Goat anti-mouse HRP-conjugated secondary antibody was diluted 1 :5000 by mixing 1 pL of the antibody with 5 mL of milk solution per plate. Each well received 50 pL of the secondary antibody solution and was incubated for one hour at room temperature with rocking on a gyroscopic mixer. Plates were washed five times with PBS. To each well, 50 pL of TMB substrate was added and incubated for 5-20 minutes until the top three wells appeared greenish. The reaction was stopped by adding 50 pL of HO to each well. Absorbance was read using a plate reader.

[0142] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0143] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0144] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0145] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. Sequence Listing Free Text

[0146] SEQ ID NO: 1 - Osteoactivin peptide

[0147] RRGDGRWKD

[0148] SEQ ID NO:2 - Osteoactivin peptide, N-terminus conjugation

[0149] CGGGRRGDGRWKD

[0150] Linker: amino acids 1-4

[0151] SEQ ID NO:3 - Osteoactivin peptide, C-terminus conjugation

[0152] RRGDGRWKDG GGC Linker: amino acids 10-13

Claims

What is claimed is:

1. An immunogen comprising: an immunogenic carrier comprising an RNA bacteriophage virus-like particle (VLP); and an antigenic anti-osteoactivin peptide linked to the immunogenic carrier.

2. The immunogen of claim 1, wherein the RNA VLP comprises QP bacteriophage.

3. The immunogen of any preceding claim, wherein the anti-osteoactivin peptide comprises a segment having at least 55% sequence similarity to an antigenic portion of SEQ ID NO: 1.

4. The immunogen of any preceding claim, wherein the anti-osteoactivin peptide is conjugated to the VLP via an N-terminus linker sequence.

5. The immunogen of any preceding claim, wherein the anti-osteoactivin peptide is conjugated to the VLP via a C-terminus linker sequence.

6. A composition comprising the immunogen of any preceding claim.

7. The composition of claim 6, further comprising a pharmaceutically acceptable carrier.

8. The composition of claim 6 or claim 7, further comprising an adjuvant.

9. A method of inducing production of anti-osteoactivin antibodies in a subject, the method comprising administering to the subject the composition of any one of claims 6-8, the composition comprising: an immunogenic carrier comprising an RNA bacteriophage virus-like particle (VLP); and an antigenic anti-osteoactivin peptide linked to the immunogenic carrier.

10. The method of claim 9, wherein the anti-osteoactivin peptide comprises a segment having at least 55% sequence similarity to SEQ ID NOT.

11. The method of claim 9 or claim 10, wherein the composition is administered in an amount effective to induce an immune response against atherosclerotic plaques in the subject.

12. A method of treating senescence in a subject, the method comprising administering to the subject the composition of any one of claims 6-8, the composition comprising: an immunogenic carrier comprising an RNA bacteriophage virus-like particle (VLP); and an antigenic anti-osteoactivin peptide linked to the immunogenic carrier.

13. The method of claim 12, wherein the anti-osteoactivin peptide comprises a segment having at least 55% sequence similarity to SEQ ID NO:1.

14. The method of claim 12 or claim 13, wherein the composition is administered in an amount effective to induce an immune response against atherosclerotic plaques in the subject.

15. The method of any one of claims 12-14, wherein the composition is administered in an amount effective to improve a cardiovascular indicium in the subject compared to a comparable control subject to which the composition is not administered.

16. The method of claim 15, wherein the cardiovascular indicium comprises an increase in ejection fraction in the subject compared to a comparable control subject to which the composition is not administered.

17. The method of any one of claims 12-16, wherein the composition is administered in an amount effective to improve a pulmonary indicium in the subject compared to a comparable control subject to which the composition is not administered.

18. The method of claim 17, wherein the pulmonary indicium comprises an increase in breathing frequency in the subject compared to a comparable control subject to which the composition is not administered.

19. The method of claim 17, wherein the pulmonary indicium comprises an increase in minute volume in the subject compared to a comparable control subject to which the composition is not administered.

20. The method of any one of claims 12-19, wherein the composition is administered in an amount effective to increase survival compared to a comparable control subject to which the composition is not administered.

21. A method of treating a senopathy in a subject, the method comprising administering to the subject the composition of any one of claims 6-8, the composition comprising: an immunogenic carrier comprising an RNA bacteriophage virus-like particle (VLP); and an antigenic anti-osteoactivin peptide linked to the immunogenic carrier.

22. The method of claim 21, wherein the anti-osteoactivin peptide comprises a segment having at least 55% sequence similarity to SEQ ID NO: 1.

23. The method of claim 21 or claim 22, wherein the composition is administered in an amount effective to induce an immune response against atherosclerotic plaques in the subject.

24. The method of any one of claims 21-23, wherein the composition is administered in an amount effective to improve a cardiovascular indicium in the subject compared to a comparable control subject to which the composition is not administered.

25. The method of claim 24, wherein the cardiovascular indicium comprises an increase in ejection fraction in the subject compared to a comparable control subject to which the composition is not administered.

26. The method of any one of claims 21-25, wherein the composition is administered in an amount effective to improve a pulmonary indicium in the subject compared to a comparable control subject to which the composition is not administered.

27. The method of claim 26, wherein the pulmonary indicium comprises an increase in breathing frequency in the subject compared to a comparable control subject to which the composition is not administered.

28. The method of claim 26, wherein the pulmonary indicium comprises an increase in minute volume in the subject compared to a comparable control subject to which the composition is not administered.

29. The method of any one of claims 21-28, wherein the composition is administered in an amount effective to increase survival compared to a comparable control subject to which the composition is not administered.

30. A nucleic acid encoding the immunogen of claim 1.

31. An expression vector comprising the nucleic acid of claim 30.

32. A host cell comprising the expression vector of claim 31.

33. A vaccine comprising the immunogen of claim 1.

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