Compositions and methods of use for targeting apoptosis-related spot-like proteins with caspase activation and recruitment domain (ASC)

Abstract

Immunogens include an immunogenic carrier and an antigenic apoptosis-related spot-like protein containing a caspase activation and recruitment domain (ASC) peptide linked to the immunogenic carrier. In one or more embodiments, the immunogenic vector is a Q [beta] virus-like particle (VLP). The immunogen may be formulated as a composition useful in the treatment of inflammatory medical conditions.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 402,601, filed Aug. 31, 2022, which is incorporated herein by reference in its entirety.

[0003] Government Support

[0004] This invention was made with government support under Grant No. NS083704 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] Sequence Listing

[0006] This application contains a Sequence Listing that has been submitted electronically via the Patent Center to the United States Patent and Trademark Office, which is in the form of an.xml file named “0310000175WO01.xml”, is 21 kilobytes in size, and was created on Aug. 30, 2023. The information contained in the Sequence Listing is incorporated herein by reference. Summary of the Invention

[0007] The present disclosure, in one aspect, describes an immunogen that includes an immunogenic carrier and an antigenic apoptosis - associated speck - like protein containing a caspase - activation and recruitment domain (ASC) peptide linked to the immunogenic carrier. In one or more embodiments, the immunogenic carrier is a Qβ virus - like particle (VLP).

[0008] In one or more embodiments, the ASC peptide comprises the amino acid sequence of SEQ ID NO: 1 or an antigenic fragment thereof. In one or more of these embodiments, the antigenic fragment of SEQ ID NO: 1 comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0009] In one or more embodiments, the immunogenic carrier is linked to the ASC peptide via a 6 - [β - maleimidopropionamido] hexanoic acid N - hydroxysuccinimide ester (SMPH) cross - linking molecule.

[0010] In one or more embodiments, the immunogen further comprises a second antigenic ASC peptide. In one or more of these embodiments, both ASC peptides are displayed on the same VLP.

[0011] In another aspect, the present disclosure describes a composition comprising an immunogen, the immunogen comprising an immunogenic carrier and an ASC peptide linked to the immunogenic carrier.

[0012] In one or more embodiments, the composition can comprise a first group of VLPs displaying a first antigenic ASC peptide and a second group of VLPs displaying a second antigenic ASC peptide.

[0013] In one or more embodiments, the composition can further comprise an adjuvant.

[0014] In one or more embodiments, the composition is a vaccine.

[0015] In another aspect, the present disclosure describes a method of treating an inflammatory disorder in a subject. Generally, the method comprises administering to the subject a therapeutically effective amount of a composition, wherein the composition comprises an immunogen, the immunogen comprising an immunogenic carrier and an antigenic ASC peptide linked to the immunogenic carrier.

[0016] In one or more embodiments, the method further comprises administering to the individual at least one additional therapeutic agent for treating the inflammatory disorder. In one or more of these embodiments, the immunogen comprises a second antigenic ASC peptide. In one or more of these embodiments, both antigenic ASC peptides are linked to a single carrier.

[0017] In one or more embodiments, the composition comprises a first group of immunogens and a second group of immunogens. The first group of immunogens comprises a first group of immunogenic carriers and a first antigenic ASC peptide linked to the first group of immunogenic carriers. The second group of immunogens comprises a second group of immunogenic carriers and a second ASC peptide linked to the second group of immunogenic carriers.

[0018] In one or more embodiments, at least one antigenic ASC peptide comprises the amino acid sequence of SEQ ID NO: 1 or an antigenic fragment thereof. In one or more of these embodiments, the antigenic fragment of SEQ ID NO: 1 comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0019] In one or more embodiments, the composition is administered to the individual before the individual exhibits symptoms or clinical signs of the inflammatory disorder.

[0020] In another aspect, the present disclosure describes a nucleic acid encoding an immunogen, the immunogen comprising an immunogenic carrier and an antigenic ASC peptide linked to the immunogenic carrier.

[0021] In another aspect, the present disclosure describes an expression vector comprising a nucleic acid encoding an immunogen, the immunogen comprising an immunogenic carrier and an antigenic ASC peptide linked to the immunogenic carrier.

[0022] In another aspect, the present disclosure describes a host cell comprising an expression vector, the expression vector comprising a nucleic acid encoding an immunogen, the immunogen comprising an immunogenic carrier and an antigenic ASC peptide linked to the immunogenic carrier.

[0023] The foregoing summary is not intended to describe every disclosed embodiment or every implementation of the present invention. The following description more specifically exemplifies illustrative embodiments. Throughout the application, guidance is provided through lists of examples, which may be used in various combinations. In each case, the listed lists are only representative groups and should not be construed as exclusive lists. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 . Structure of apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) protein. ASC comprises three domains: a pyrin domain (pyr), a flexible semi-structured linker region (linker), and a caspase activation and recruitment domain (CARD).

[0025] Figure 2 . Peptide sequences of ASC epitopes developed for VLPs. Sequences are shown in the direction from the amino (N) terminus to the carboxyl (C) terminus. The positions of the ASC epitopes are shown relative to the overall ASC protein structure. H1-6 represent individual alpha helices within the PYD and CARD regions of the ASC protein. The N-terminal cysteine residue shown in the ASC linker peptide sequence (SEQ ID NO: 3) is an artifact of the conjugation method, where the terminal cysteine residue is required for the conjugation reaction to occur. Other ASC epitopes include the native terminal cysteine residues of the protein. The remainder of the amino acid sequence shown in the immunogenic portion of SEQ ID NO: 1 (amino acids 92-106 of SEQ ID NO: 1). The amino acid sequences of the immunogenic ASC peptide of ASC helix 4 (SEQ ID NO: 4) and the immunogenic ASC peptide of the ASC C-terminus (SEQ ID NO: 5) are shown.

[0026] Figure 3.The coat protein of bacteriophage Qβ spontaneously assembles into virus-like particles (VLPs). 6-[β-maleimidopropionamido]hexanoic acid N-hydroxysuccinimide ester (SMPH) is a bifunctional crosslinker that can chemically conjugate the ASC peptide to surface-exposed lysine residues on the Qβ monomer. The final product is a Qβ VLP that displays the ASC peptide in a highly repetitive multivalent manner.

[0027] Figure 4 .Mobility shift gel electrophoresis of Qβ and Qβ conjugated with the ASC linker, helix 4, and C-terminal peptide indicates the success of the conjugation reaction for VLP synthesis.

[0028] Figure 5 .Schematic of the experimental timeline for all preliminary data. Animals were inoculated with either the Qβ control vaccine or the Qβ-ASC VLP vaccine at two months of age, with two doses administered three weeks apart. Three weeks after the second inoculation, blood samples were collected for antibody titer analysis and used for antibody reactivity experiments with human and mouse brain tissues. Safety monitoring was conducted for an additional 2.5 months. At six months of age, terminal blood samples were collected for blood cell counts and blood biochemical profiling. The animals were then injected with 5 mg / kg lipopolysaccharide to induce a systemic inflammatory response. Nine hours later, the animals were sacrificed and serum and brain tissues were collected to analyze inflammasome activation.

[0029] Figure 6 .Dilution curves of immune sera from mice inoculated with the Qβ ASC-linker construct, indicating an antibody response was generated.

[0030] Figure 7 .Dilution curves of immune sera from mice inoculated with the Qβ ASC-helix 4 construct, indicating an antibody response was generated.

[0031] Figure 8 .Dilution curves of immune sera from mice inoculated with the Qβ ASC-C-terminal construct, indicating an antibody response was generated.

[0032] Figure 9 .Total body weights of animals inoculated with Qβ control and Qβ-ASC VLPs over a 4-month period. There was no difference in animal body weight between the treatment and control groups at any time point, indicating that Qβ-ASC VLP inoculation was well tolerated.

[0033] Figure 10. Complete blood count and blood chemistry panel of Qβ-ASC VLP-inoculated mice at four months post-inoculation with contour boxes relative to Qβ control, indicating statistically significant differences (p<0.05, one-way ANOVA) from the Qβ control, and individual graphs of all statistically significant findings are shown with a dashed line indicating the normal reference range. Overall, the Qβ-ASC VLP vaccine was well tolerated. There were no differences in immune cell counts / percentage between the treatment group and the Qβ control group. The Qβ-ASC conjugate vaccine was associated with mild anemia, but there were no differences in red blood cell parameters between the other Qβ-ASC VLP-inoculated groups and the control group. There were no differences in platelet characteristics between the Qβ-ASC VLP-inoculated groups and the control group. Multiple organ function markers evaluated by the basic blood chemistry panel were unchanged. Qβ control-inoculated mice showed hyperglycemic levels indicating diabetes, while Qβ-ASC VLP-inoculated mice maintained normal blood glucose levels.

[0034] Figure 11 . Immunohistochemical micrographs of human hippocampal brain tissue from Alzheimer's disease patients stained with immune sera from animals inoculated with anti-ASC antibody (AdipoGen AL177) or Qβ-control or Qβ-ASC VLP at a 1:500 dilution. Qβ-ASC VLP induced antibody binding to human ASC protein in human Alzheimer's disease brain tissue, with strong staining in a pattern similar to that of the commercially available anti-ASC antibody. Qβ-control immune sera showed only low levels of non-specific background staining.

[0035] Figure 12 . Western blots of mouse and human hippocampal lysates stained with anti-ASC antibody (AdipoGen AL177, 1:2000 dilution) or immune sera from animals inoculated with Qβ-control or Qβ-ASC VLP (1:1000 dilution). Qβ-ASC VLP inoculation elicited a highly specific antibody response with less off-target binding compared to the commercially available antibody. Antibodies elicited by the Qβ-ASC helix 4 vaccine showed no binding to denatured proteins, indicating that the antibodies may be specific for the native protein conformation. Immune sera from Qβ-control inoculation showed substantial non-specific antibody binding, with binding molecular weights overlapping with the non-specific binding of the commercially available AL177 antibody. Antibodies elicited by ASC-VLP appeared to preferentially bind to human proteins rather than mouse proteins.

[0036] Figure 13. An indirect sandwich ELISA was performed on human and mouse hippocampal lysates using an anti-ASC antibody (AdipoGen AL177, diluted 1:100) as the capture antibody and Qβ-control or Qβ-ASC VLP immune sera (diluted 1:1000) as the detection antibody. HRP-conjugated goat anti-mouse antibody was used to detect the presence of vaccine-induced antibodies using a 3,3′,5,5′-tetramethylbenzidine chromogenic substrate detected at 450 nm. Antibodies elicited by Qβ-ASC VLP preferentially bind to human ASC protein rather than mouse ASC protein, indicating a high degree of specificity of the antibody response and limited protein cross-reactivity. The high detection of Qβ-control antibodies may be due to overlap of non-specific protein binding with the AdipoGen AL177 antibody used as the protein capture antibody (see Figure 12 ). ****p < 0.001, **p < 0.01, *p < 0.05. One-way ANOVA.

[0037] Figure 14 . ELISA-based quantification of serum interleukin-1β (IL-1β) levels in Qβ-control and Qβ-ASC VLP-inoculated mice injected with lipopolysaccharide (LPS) to induce inflammasome responses. Although the antibodies elicited by ASC VLP have a lower affinity for mouse ASC protein, there is a trend towards decreased levels of mature IL-1β in the sera of mice inoculated with Qβ-ASC linker and Qβ-ASC helix 4, suggesting that circulating Qβ-ASC VLP-elicited antibodies may neutralize endogenous mouse ASC protein in the serum.

[0038] Figure 15 . Western blot of hippocampal brain lysates from Qβ-control and Qβ-ASC VLP-inoculated mice after stimulation with lipopolysaccharide (LPS) to induce inflammasome activation. The downstream effector protein caspase-1 of the inflammasome was significantly reduced in all Qβ-ASC VLP treatment groups compared to the Qβ-control group. **p < 0.01, ***p < 0.005. One-way ANOVA.

[0039] Figure 16 . The microglial NLRP3 inflammasome is triggered and activated by AD protein aggregates. Description of the cellular pathways involved in activating the inflammasome.

[0040] Figure 17 . Description of the ASC-dependent mechanism by which the inflammasome propagates inflammation and the spread of misfolded protein aggregates. Specific embodiments

[0041] The present disclosure describes compositions and methods for targeting apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), which protein is sometimes also referred to in the scientific literature by the gene name PYCARD encoding the protein. The compositions can be used to treat inflammatory disorders by targeting inflammasomes.

[0042] An inflammasome is an innate immune complex that is capable of sensing a range of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). The classical inflammasome initiates an inflammatory signaling cascade mediated by the maturation of interleukin-1β and interleukin-18 by caspase-1. Inflammasomes are associated with many disease states and often lead to a long-term cytotoxic inflammatory state in many diseases characterized by chronic inflammation, including Alzheimer's disease. Although there are several different inflammasome complexes that respond to different PAMPs and DAMPs, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) protein is a common component of most inflammasomes and is a component of ASC specks, which are large protein complexes that can act as signal amplification platforms to enhance inflammasome activity.

[0043] Alzheimer's disease (AD) is a leading cause of death worldwide. Currently, no treatment has been proven to prevent or cure AD. Four pathological features of AD in the brain include the accumulation of extracellular amyloid-β protein aggregates, the accumulation of intracellular tau protein aggregates, low-level chronic neuroinflammation, and extensive neuronal degeneration leading to memory loss and cognitive impairment. Therapeutic approaches have largely ignored the role of chronic neuroinflammation in disease progression. The NLRP3 inflammasome / ASC speck signaling complex, which originates from inflammatory microglia, is a mediator of the chronic inflammatory state in the AD brain. Gene knockout of NLRP3 or ASC genes reduces AD disease pathology in animal models.

[0044] The present disclosure describes novel vaccine compositions directed against the NLRP3 inflammasome / ASC speck signaling complex. The compositions described herein generate a neutralizing antibody response, thereby reducing neuroinflammation and inhibiting the disease progression of AD. The compositions include a virus-like particle (VLP) vaccine platform (e.g., a Qβ-based VLP platform) for antigen presentation of short peptide sequences from apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) protein, which ASC protein is a component of the NLRP3 inflammasome / ASC speck.

[0045] The amino acid sequence of human ASC protein is shown in SEQ ID NO: 1. Human ASC protein includes a pyrin domain (amino acids 1-91 of SEQ ID NO: 1) and a caspase activation and recruitment domain (CARD) domain (amino acids 107-195 of SEQ ID NO: 1). The amino acid sequence of mouse ASC protein is shown in SEQ ID NO: 2. Mouse ASC protein includes a pyrin domain (amino acids 1-91 of SEQ ID NO: 2) and a caspase activation and recruitment domain (CARD) (amino acids 105-193 of SEQ ID NO: 2). Human ASC protein exists in at least three isoforms. Isoform 1 is shown in SEQ ID NO: 1 and has a canonical form with 195 amino acid residues. Isoform 2 has 176 amino acid residues: amino acids 1-92 and 112-195 of SEQ ID NO: 1 - i.e., SEQ ID NO: 1 with amino acids 93-111 excised. Isoform 3 has 135 amino acid residues: amino acids 1-25 and 86-195 - i.e., SEQ ID NO: 1 with amino acids 26-85 excised. Isoform 1 and isoform 2 are considered the active isoforms of ASC. Isoform 3 binds to caspase-1 and has a certain degree of inhibitory function.

[0046] Each vaccine contains an ASC peptide conjugated to Qβ phage virus-like particles (VLPs). In one vaccine, herein referred to as the "ASC linker" vaccine, the ASC peptide includes amino acids 92-106 of SEQ ID NO: 1 and targets the linker region between the pyrin domain and the CARD domain ( Figure 2 ). In a second vaccine, herein referred to as the "ASC helix 4" vaccine, the ASC peptide includes amino acids 155-173 of SEQ ID NO: 1 and targets helix 4 of the CARD domain. In a third vaccine, herein referred to as the "ASC C-terminal" vaccine, the ASC peptide includes amino acids 173-195 and targets the C-terminal of the CARD domain.

[0047] In safety studies, animals received a two - injection vaccination protocol starting at two months of age and a booster inoculation three weeks after the first injection. Vaccine conditions included three different ASC peptide sequences conjugated to Qβ phage VLP or non - conjugated Qβ VLP sham treatment, for a total of four treatment groups of five mice each. Compared to sham - inoculated mice, IgG titers against ASC peptides were significantly increased in each ASC - VLP group. There were no differences in survival rate or changes in animal body weight between the ASC - inoculated and sham - inoculated groups up to four months after inoculation. A complete blood count and blood chemistry panel were also performed on each mouse, and no significant differences indicating severe disease or increased infection were shown. These findings indicate that these three vaccines are safe for therapeutic intervention of inflammasome - mediated cytokine signaling in various mouse disease models, including Alzheimer's disease, in pre - clinical trials.

[0048] Excessive interleukin - 1 signaling leads to inflammatory diseases

[0049] Interleukin 1 (IL - 1) is an inflammatory cytokine that plays multiple roles in health and disease as a major signaling component of the innate immune system. It acts as a major regulator of inflammation through several innate immune processes, including serving as a leukocyte pyrogen, leukocytic endogenous mediator, and activator, an activator of the acute - phase response to infection and injury, and a mediator of fever. Although IL - 1 plays a protective role in acute responses to infection and tissue injury, dysregulated IL - 1 signaling can lead to various disease states, including auto - inflammatory diseases (e.g., cryopyrin - associated periodic syndromes, familial Mediterranean fever, etc.), metabolic syndromes (e.g., type 2 diabetes), excessive acute inflammation (e.g., sepsis), chronic inflammatory diseases (e.g., rheumatoid arthritis, chronic obstructive pulmonary disease, gout, Alzheimer's disease, etc.), and malignancies (e.g., HER2 - negative breast cancer). The US Food and Drug Administration has approved several drugs that directly target IL - 1β signaling for the treatment of various inflammatory diseases. While directly targeting IL - 1 signaling is sometimes effective in controlling several diseases, targeting the upstream production of IL - 1 may be more effective.

[0050] Inflammasomes are mediators of IL - 1 signaling

[0051] Inflammasomes are innate immune signaling complexes that catalyze the maturation of IL - 1 cytokines for inflammatory signaling. The NLRP3 inflammasome is a multi - protein oligomeric complex that includes the nod - like receptor family pyrin domain - containing 3 (NLRP3) protein, the apoptosis - associated speck - like protein containing a caspase - activation and recruitment domain (ASC) protein, and caspase - 1 ( Figure 16)。The ASC protein is a common component of multiple inflammasome types, including NLRP1, NLRP3, NLRP6, NLRP12, NLRC4, and AIM2 inflammasomes. After inflammasomes are activated, ASC can polymerize into large fibrils, which can aggregate together to form a large supramolecular complex called an ASC speck. ASC specks are protein complexes approximately 1 micron in diameter that can serve as signal amplification platforms to enhance IL-1 maturation.

[0052] NLRP3 inflammasome-mediated interleukin-1β signaling is associated with Alzheimer's disease and other neurodegenerative diseases and may drive pathology.

[0053] Reactive microglia are innate immune cells of the brain that drive tau pathology in animal models of Alzheimer's disease in an IL-1-dependent manner. This IL-1 signaling is mainly driven by the NLRP3 inflammasome / ASC speck activity of microglia. Thus, therapeutic agents that target inflammasome / ASC speck activity within the central nervous system may provide benefits for the treatment of Alzheimer's disease and other neurodegenerative disorders. Previous immunotherapies for Alzheimer's disease have involved vaccines using a virus-like particle (VLP) platform to target the tau protein. These VLPs generate a strong antibody response against pathological tau protein aggregates and clear these aggregates from the brain, thereby improving memory and cognition in rodent models of Alzheimer's disease. VLPs have advantages over monoclonal antibody immunotherapies because they are inexpensive and easy to develop and do not require frequent re-administration throughout an individual's lifetime. This disclosure describes a similar technique using VLP technology that targets the inflammasome / ASC speck to treat inflammatory diseases, including Alzheimer's disease.

[0054] VLP display

[0055] Many viral structural proteins have the intrinsic ability to self-assemble into virus-like particles (VLPs), which are structurally similar to the viruses from which they are derived, but because they lack viral genomes, they are absolutely not infectious. VLPs can not only serve as independent vaccines, but also because their particulate properties and multivalent structure stimulate a strong immune response, so they can be used as a platform to enhance the immunogenicity of heterologous antigen targets. For example, when short immunogenic peptides are displayed on VLPs in a highly repetitive, multivalent manner, peptide-specific B cells are strongly activated, thereby producing high-titer, long-lasting antibody responses. VLPs from different viral types can be used as an effective platform for antigen display. The immunogens described herein are based on VLPs from related single-stranded RNA phage families (including MS2, PP7, AP205 and Qβ). These VLPs can be produced by expressing a single viral structural protein called an outer shell from a bacterial plasmid. Peptides can be displayed on VLPs by using a bioconjugation technology using cross-linked molecules. In one or more embodiments, peptides can be displayed on VLPs by conjugating the peptides to VLPs via 6-[β-maleimidopropionamido]hexanoic acid succinimidyl ester (SMPH) cross-linking molecules. This technology allows VLPs to display target peptides at high cost, typically 180-360 peptides per VLP, and confers strong immunogenicity to the displayed immunogenic peptides.

[0056] VLP-based vaccines targeting inflammasome-mediated inflammatory diseases

[0057] This vaccine technology has therapeutic potential for treating a variety of inflammatory diseases, including but not limited to autoinflammatory diseases, metabolic syndrome, acute inflammation, chronic inflammatory diseases, malignancies, and neurodegenerative diseases such as Alzheimer's disease.

[0058] Exemplary embodiments of ASC-targeted vaccines target epitopes on the ASC protein ( Figure 1 ). Epitopes that have been targeted include flexible linker sequences, α Helix 4 and contains the CARD region α The C-terminal region of helices 5 and 6 ( Figure 2 In one or more alternative exemplary embodiments, other epitopes can be targeted. The Qβ phage coat protein that spontaneously self-assembles into VLPs is used as an antigen display platform, and 6-[β-maleimidopropionamido] hexanoic acid succinimidyl ester (SMPH) is used as a chemical cross-linking agent to chemically conjugate with the ASC epitope peptide sequence ( Figure 3 The final product is Qβ VLPs that display ASC peptide epitopes in a multivalent, highly repetitive manner, which can induce a strong B cell response, thereby generating high-affinity antibodies against the ASC epitope antigen.

[0059] althoughFigure 3 Illustrative embodiments are described where VLPs display a single ASC peptide in a multivalent manner (i.e., the VLPs display multiple copies of a single ASC peptide), but the vaccine can be designed such that the VLPs display more than one ASC peptide. Thus, in one or more embodiments, the VLPs can display one, two, three, or more different ASC peptides. Any conventional method (e.g., mobility shift gel electrophoresis) can be used to evaluate the efficiency of peptide conjugation on the VLPs. Figure 4 Shows the mobility shift of Qβ to higher molecular weights depending on the number of ASC peptides successfully conjugated to the Qβ monomer.

[0060] Once generated, the ASC-VLP compositions can be used to immunize a subject. Any conventional method (e.g., enzyme-linked immunosorbent assay (ELISA)) can be used to evaluate the immunogenicity of a given ASC-VLP vaccine composition. The ASC linker, ASC helix 4, and ASC C-terminus VLPs are immunogenic and elicit a high-titer IgG antibody response. Figures 6 - 8 Shows data demonstrating the immunogenicity of the ASC linker ( Figure 6 ), ASC helix 4 ( Figure 7 ), and ASC C-terminus ( Figure 8 ) VLPs.

[0061] The safety of a given ASC-VLP vaccine composition can be evaluated by monitoring the adverse health effects in immunized subjects (e.g., over a six-month period). Subjects can be monitored using, for example, a complete blood count profile, a basic blood chemistry panel, weight changes, and survival rates. Figure 5 A schematic of the experimental timeline for all the preliminary data is provided. Figure 5 The experimental timeline in

[0062] shows that safety monitoring was conducted for 2.5 months after the VLP vaccine injection dose and the first blood draw. Figure 9 For example, Figure 10 the data shown demonstrate that the ASC linker vaccine, ASC helix 4 vaccine, and ASC C-terminus vaccine are well-tolerated and none of the vaccines caused a significant weight loss compared to sham inoculation with Qβ VLPs lacking any ASC peptides. Similarly, Figure 10 the complete blood count profile is shown, demonstrating that all the vaccines are well-tolerated and only the ASC linker vaccine showed mild anemia (e.g., low hemoglobin and hematocrit).

[0063] Any suitable inflammasome activity measurement can be used to evaluate the efficacy of the ASC-VLP vaccine composition. In an illustrative context of Alzheimer's disease, an illustrative model designed to measure cognitive ability can be used. For example, brain lysates from human AD patients or a PBS vehicle can be injected into the hippocampus of healthy C57B16 / J mice. The animals can then be further subdivided into groups that receive ASC-VLP vaccination and groups that receive sham vaccine treatment. After a predetermined period of time (e.g., two months in some illustrative models), the animals can be subjected to a series of cognitive behavioral tasks and then sacrificed for histological and biochemical analysis of disease pathology and inflammatory markers.

[0064] Metabolic disorders such as type 2 diabetes are associated with inflammasome activity as a potential risk factor. Figure 10 It was demonstrated that mice receiving sham vaccination with Qβ VLP lacking any ASC peptides developed hyperglycemic levels indicative of diabetes, while animals vaccinated with ASC-VLP maintained healthy blood glucose levels. This is an exploratory finding that indicates another possible illustrative model for measuring the efficacy of ASC-VLP vaccination under inflammasome-mediated disease conditions.

[0065] Accordingly, the present disclosure describes compositions and methods that provide state-of-the-art immunotherapeutic approaches targeting proteins that cause damaging inflammation. The compositions described herein target inflammasome-related proteins and inflammatory cytokines. In one or more embodiments, the compositions and methods described herein induce antibody production while minimally inducing a severe inflammatory response. Accordingly, the compositions and methods may be suitable for treating inflammatory disorders, including but not limited to Alzheimer's disease. Vaccines targeting ASC can reduce neuroinflammation and disease pathology in animal AD models. The NLRP3 inflammasome promotes the pathological tau aggregation process by regulating kinase / phosphatase activity of inflammatory signals and can be targeted to reduce the ptau burden.

[0066] The composition comprises a VLP-based immunogen, said immunogen comprising an antigenic ASC peptide (also referred to herein as "ASC targeting peptide"), such as the amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or an immunogenic fragment thereof. Exemplary immunogenic ASC peptide fragments include, but are not limited to, any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or an immunogenic fragment of any of the foregoing. Additionally, the immunogen may comprise a VLP displaying more than one population of antigenic ASC peptides, said populations of antigenic ASC peptides including, for example, a first population of antigenic ASC peptides comprising the amino acids of a first ASC immunogenic peptide and a second population of antigenic ASC peptides comprising a second immunogenic ASC peptide. Thus, the immunogen can be designed to display one, two, three, four, five, six, or more antigenic ASC peptides.

[0067] In another aspect, the immunogenic composition can comprise more than one population of VLPs. For example, the immunogenic composition can comprise a first population of VLPs displaying a first antigenic ASC peptide and a second population of VLPs displaying a second antigenic ASC peptide. Further, the immunogenic composition can comprise a first population of VLPs displaying one or more antigenic ASC peptides and a second population of VLPs displaying one or more antigenic ASC peptides, wherein the number and identity of the antigenic peptides displayed by the VLPs in the second population are independent of the number and identity of the antigenic peptides displayed by the first population of VLPs.

[0068] As used herein, an antigenic ASC peptide can refer to any ASC amino acid sequence that elicits an ASC-targeted immune response when introduced into an immunocompetent subject. Exemplary antigenic ASC peptides include peptides having an amino acid sequence containing any one or more of the following: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, any immunogenic fragment of any of the foregoing peptides, or any peptide structurally similar to any of the foregoing peptides or immunogenic fragments.

[0069] As used herein, a peptide is "structurally similar" to a reference polypeptide if the amino acid sequence of the peptide has a specified amount of identity as compared to the reference peptide. The structural similarity of two peptides can be determined by aligning the residues of the two peptides (e.g., a candidate polypeptide and any one of SEQ ID NOs: 1-21) to optimize the number of amino acids that are identical along their sequence lengths; gaps are permitted to be present in either or both of the sequences being aligned in order to optimize the number of identical amino acids, provided that the amino acids in each sequence must still maintain their correct order. A candidate peptide is a peptide that is compared to a reference peptide (e.g., any one of SEQ ID NOs: 1-21). A candidate peptide can be isolated from, for example, an animal, or can be produced using recombinant techniques, or by chemical or enzymatic synthesis.

[0070] Pairwise comparison analysis of amino acid sequences can be performed using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI). Alternatively, the Blastp program of the BLAST 2 search algorithm can be used to compare peptides, as described by Tatiana et al. (FEMS Microbiol Lett, 174, 247-250 (1999)), and is available on the website of the National Center for Biotechnology Information (NCBI). The default values of all BLAST 2 search parameters can be used, including matrix=BLOSUM62; open gap penalty=11, extend gap penalty=1, gap x_dropoff=50, expect=10, word size=3, and filter on.

[0071] The antigenic ASC peptide may include amino acids other than any of SEQ ID NOs: 1-21, provided that the additional amino acids do not abolish the immunogenicity to ASC. For example, the antigenic ASC peptide may have a linker region containing the amino acids GGGC (SEQ ID NO: 22) or CGGG (SEQ ID NO: 23).

[0072] In the comparison of two amino acid sequences, the structural similarity can be referred to as the percentage of "identity" or the percentage of "similarity". "Identity" refers to the presence of identical amino acids. "Similarity" refers not only to the presence of identical amino acids but also includes the presence of conservative substitutions. Conservative substitutions of amino acids in the immunogenic peptides as described herein can be selected from other members of the amino acid class to which the amino acid belongs. For example, in the field of protein biochemistry, it is well known that an amino acid belonging to an amino acid group having a specific size or property (such as charge, hydrophobicity, and hydrophilicity) can replace another amino acid without changing the activity of the protein, especially in a protein region not directly related to biological activity. For example, non-polar (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. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys substituting for Arg and vice versa to maintain a positive charge; Glu substituting for Asp and vice versa to maintain a negative charge; Ser substituting for Thr to maintain a free -OH; and Gln substituting for Asn to maintain a free -NH2. Similarly, bioactive analogs of polypeptides containing deletions or additions of one or more contiguous or non-contiguous amino acids but not abolishing the functional activity of the peptide are also contemplated.

[0073] Table 1 provides exemplary ASC targeting peptides.

[0074] Table 1.

[0075]

[0076]

[0077] Thus, in one or more embodiments, the ASC targeting peptides described herein can include peptides (or peptide domains - i.e., a portion of a larger amino acid sequence) having at least 66%, at least 70%, at least 75%, at least 79%, at least 81%, at least 83%, at least 84%, at least 87%, at least 90%, at least 93% or at least 96% sequence similarity to the amino acid sequence of any one of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21. Thus, in one or more embodiments, compared to a reference ASC peptide sequence, an ASC targeting polypeptide comprising a domain corresponding to any one of SEQ ID NOs: 3-21, or a portion of an ASC peptide corresponding to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 can include a total of no more than six, no more than five, no more than four, no more than three, no more than two or no more than one amino acid deletion, amino acid addition, and / or non-conservative amino acid substitution.

[0078] In one or more embodiments, the ASC targeting peptides described herein can include a peptide (or peptide domain - i.e., a portion of a larger amino acid sequence) having at least 66%, at least 70%, at least 75%, at least 79%, at least 81%, at least 83%, at least 84%, at least 87%, at least 90%, at least 93% or at least 96% sequence identity to the amino acid sequence of any one of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21. Thus, in one or more embodiments, compared to a reference ASC peptide sequence, an ASC targeting polypeptide comprising a domain corresponding to any one of SEQ ID NOs: 3 - 21, an ASC peptide portion corresponding to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 can include a total of no more than six, no more than five, no more than four, no more than three, no more than two or no more than one amino acid deletion and / or amino acid addition.

[0079] In one or more embodiments, the ASC targeting peptides described herein can be designed to provide additional sequences, e.g., added C - terminal or N - terminal amino acids, which can facilitate purification, for example, by capture on a column or using an antibody. Such tags include, for example, histidine - rich tags, which allow purification of polypeptides on nickel columns. Such genetic modification techniques and suitable additional sequences are well known in the field of molecular biology. Alternatively or additionally, the additional amino acid sequence can facilitate conjugating or otherwise linking the ASC targeting peptide to a VLP.

[0080] Virus-like particles (VLPs) can include any particles containing viral proteins assembled into structures that are structurally similar to the virus from which they are derived, but lacking sufficient viral genome such that they are non-replicative and thus non-infectious. Thus, VLPs may contain at least some viral genome, but the viral genome is genetically modified such that the viral genes responsible for infectivity and / or replication are inactivated. Exemplary VLPs include, but are not limited to, VLPs of Qβ, MS2, PP7, AP205, and other bacteriophage coat proteins, capsid and core proteins of hepatitis B virus, measles virus, Sindbis virus, rotavirus, foot-and-mouth disease virus, Norwalk virus, retroviral GAG proteins, retrotransposon Ty protein p1, surface proteins of hepatitis B virus, human papillomavirus, human polyomavirus, RNA bacteriophages, Ty, fr bacteriophages, GA-bacteriophages, AP 205-bacteriophages, particularly Qβ-bacteriophages, cowpea chlorotic mottle virus, cowpea mosaic virus, human papillomavirus (HPV), bovine papillomavirus, porcine parvovirus, parvoviruses such as B19, porcine parvovirus (PPV), and canine parvovirus (CPV), 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).

[0081] Antigenic ASC peptides can be conjugated to an immunogenic carrier by chemical conjugation or by expressing a genetically engineered fusion construct. The conjugation does not necessarily need to be direct, but can be through a linker sequence. More generally, in cases where an antigenic peptide is fused, conjugated, or otherwise linked to an immunogenic carrier, a spacer sequence or linker sequence is typically added at one or both ends of the antigenic peptide. Such linker sequences typically contain sequences recognized by proteasomes, proteases of endosomes, or other vesicular compartments of the cell.

[0082] In one embodiment, the antigenic ASC peptide can be displayed as a fusion protein together with a subunit of an immunogenic carrier. The fusion of the peptide can be achieved by inserting the amino acid sequence of the ASC antigenic peptide into the primary sequence of the immunogenic carrier, or by fusing it to the N-terminus or C-terminus of the immunogenic carrier.

[0083] When the immunogenic carrier is a VLP, the chimeric antigen peptide-VLP subunit can self-assemble into a VLP. The VLP that displays the epitope fused to its subunit is also referred to herein as a chimeric VLP. For example, European application No. EP90310264A (European patent No. EP0421635 B1) describes the use of chimeric hepadnavirus core antigen particles to present foreign peptide sequences in virus-like particles.

[0084] Flanking amino acid residues can be added to either end of the antigen ASC targeting peptide sequence for fusion to either end of the VLP subunit sequence, or for internal insertion of such peptide sequences into the VLP subunit sequence. Glycine and serine residues are particularly advantageous amino acids for adding to the flanking sequences of the peptide to be fused. Glycine residues confer additional flexibility, which may reduce the potential destabilizing effects of fusing foreign sequences to the VLP subunit sequence.

[0085] In one or more embodiments, the immunogenic carrier is a VLP of an RNA bacteriophage (e.g., Qβ). The major coat protein of the RNA bacteriophage spontaneously assembles into a VLP when expressed in bacteria (e.g., Escherichia coli (E. coli)). Fusion protein constructs have been described in which the antigen peptide has been fused to the C-terminus of a truncated form of the A1 protein of Qβ, or inserted within the A1 protein (Kozlovska et al., 1996, Intervirology 39:9-15). Assembly of Qβ particles that display the fusion epitope generally involves A1 protein-antigen fusion and the presence of wild-type coat protein to form mosaic particles. However, embodiments are contemplated that involve VLPs, particularly VLPs of the RNA bacteriophage Qβ coat protein, that consist solely of VLP subunits with antigen peptides fused thereto.

[0086] The production of mosaic particles can be achieved in a variety of ways. In one illustrative method, efficient display of the fusion epitope on the VLP is mediated by expressing in an E. coli strain a plasmid encoding a Qβ A1 protein fusion having a UGA stop codon between the coat protein and the coat protein extension, the E. coli strain containing a plasmid encoding a cloned UGA suppressor tRNA, resulting in translation of the UGA codon as Trp (pISM3001 plasmid). In a second illustrative method, the coat protein gene stop codon is modified to UAA and a second plasmid expressing the A1 protein-antigen fusion is co-transformed. The second plasmid encodes a different antibiotic resistance and an origin of replication compatible with the first plasmid. In a third illustrative method, the Qβ coat protein and the A1 protein-antigen fusion are encoded in a bicistronic manner, operably linked to a promoter (e.g., the Trp promoter).

[0087] Other VLPs suitable for antigen or epitope fusions are described, for example, in International Patent Application No. PCT / IB2002 / 004132 (International Publication No. WO 03 / 024481A2) and include bacteriophage fr, RNA phage MS-2, capsid proteins of papillomaviruses, retrotransposon Ty, yeast, and retrovirus-like particles, HIV2 Gag, cowpea mosaic virus, parvovirus VP2 VLPs, HBsAg (U.S. Patent No. 4,722,840). Examples of chimeric VLPs suitable as immunogenic carriers include those described in Kozlovska et al., 1996, Intervirology 39:9-15. Other examples of VLPs suitable as immunogenic carriers include, but are not limited to, HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, HIVGAG, tobacco mosaic virus, virus-like particles of SV-40, polyomavirus, adenovirus, herpes simplex virus, rotavirus, and Norwalk virus.

[0088] In one or more embodiments, vaccine constructs containing ASC peptides having the amino acid sequences of SEQ ID NO3, SEQ ID NO:4, or SEQ ID NO:5 are synthesized by conjugating the peptides to Qβ phage VLPs using a bifunctional crosslinker (SMPH). The ASC peptides can be modified to contain a linker peptide at the C-terminus (e.g., GGGC linker sequence; SEQ ID NO:6) or at the N-terminus (e.g., CGGG linker sequence; SEQ ID NO:7). The SMPH crosslinker conjugates the free amine on the surface of the Qβ VLP to the cysteine residue of the linker peptide. In an exemplary synthetic method, the Qβ VLP is purified from the free, unconjugated crosslinker and then reacted with the ASC peptide at a molar ratio of approximately 10 peptides:1 VLP.

[0089] For any recombinantly expressed antigenic ASC peptide (whether conjugated to an immunogenic carrier or not) described herein, the present disclosure describes an isolated nucleic acid sequence encoding any embodiment of an antigenic ASC targeting peptide, or any compositional fragment of an antigenic ASC targeting peptide, having the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, or any fragment thereof. In some embodiments, the isolated nucleic acid encodes an antigenic ASC targeting peptide that comprises the amino acids of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. For any amino acid sequence of an antigenic ASC targeting peptide, or one or more compositional fragments of an antigenic ASC targeting peptide, one of ordinary skill in the art can use routine methods to determine the full range of polynucleotides encoding that amino acid sequence.

[0090] As used herein, the terms “nucleic acid” or “oligonucleotide” refer 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, as well as recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, nucleic acid conjugates, and oligonucleotides. Nucleic acids can be single-stranded, double-stranded, linear, or covalently circular closed molecules. Nucleic acids can be isolated. The term “isolated nucleic acid” means: (i) a nucleic acid amplified in vitro, such as by polymerase chain reaction (PCR); (ii) a nucleic acid produced recombinantly by cloning; (iii) a purified nucleic acid isolated, for example, by cleavage and gel electrophoresis; (iv) a nucleic acid synthesized, for example, by chemical synthesis; or (vi) a nucleic acid extracted from a sample. Nucleic acids can be introduced (i.e., transfected) into cells. When transfecting cells with RNA, the RNA can be modified by stabilization modifications, capping, or polyadenylation.

[0091] As used herein, "amplified DNA" or "PCR product" refers to a DNA amplification fragment of a defined size. There are a variety of well-known techniques in the art for detecting PCR products. PCR product detection methods include, but are not limited to, gel electrophoresis, which uses agarose or polyacrylamide gels and addition of ethidium bromide staining (DNA intercalator), labeled probes (radioactive or non-radioactive labels, Southern blotting), labeled deoxynucleotides (for direct incorporation of radioactive or non-radioactive labels), or silver staining to directly visualize the amplified PCR product; restriction endonuclease digestion, which relies on agarose gel electrophoresis, polyacrylamide gel electrophoresis, or high performance liquid chromatography (HPLC); dot blotting, which uses hybridization of amplified DNA with a specific labeled probe (radioactive or non-radioactive label); high performance liquid chromatography using ultraviolet detection; electrochemiluminescence coupled with voltage-triggered chemical reactions / photon detection; and direct sequencing using radioactive or fluorescently labeled deoxynucleotides to determine the precise sequence of nucleotides of the DNA fragment of interest, oligonucleotide ligation assay (OLA), PCR, qPCR, DNA sequencing, fluorescence, gel electrophoresis, magnetic beads, allele-specific primer extension (ASPE), and / or direct hybridization.

[0092] Generally, nucleic acids can be extracted, isolated, amplified, or analyzed by a variety of techniques, such as those described in Molecular Cloning: A Laboratory Manual (Fourth Edition), by Green and Sambrook, 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 interactions. Sequencing can be performed by any method known in the art. DNA sequencing techniques include classical dideoxy sequencing reactions (Sanger method), which use labeled terminators or primers and gel separation in a 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 with a labeled oligonucleotide probe library, sequencing by synthesis using allele-specific hybridization with a labeled clone library followed by ligation, real-time monitoring of the incorporation of labeled nucleotides during the polymerization step, polymerase cloning sequencing, and SOLiD sequencing. Isolated molecules can be sequenced by sequential or single extension reactions using polymerase or ligase, or by single or sequential differential hybridization with a probe library.

[0093] In another aspect, the present disclosure describes host cells comprising any of the isolated nucleic acid sequences and / or proteins described herein. Accordingly, the present disclosure encompasses host cells that translate nucleic acids (e.g., mRNA) to produce ASC-VLP subunits.

[0094] The nucleic acid constructs of the invention can be introduced into host cells to be modified such that ASC-VLP subunit peptides are expressed intracellularly, thereby generating genetically engineered cells. A variety of methods are known in the art for introducing nucleic acids into cells, including virus- and non-virus-mediated techniques. Examples of typical non-virus-mediated techniques include, but are not limited to, electroporation, calcium phosphate-mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome-mediated transfer, microinjection, biolistic-mediated transfer (nanoparticles), cationic polymer-mediated transfer (DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), etc.) or cell fusion. Other transfection methods 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).

[0095] The nucleic acid constructs described herein can be introduced into host cells to be modified, allowing for intracellular expression of the nucleic acid-encoded protein. A variety of host cells are known in the art to be suitable for protein expression. Examples of typical cells for transfection and protein expression include, but are not limited to, bacterial cells, eukaryotic cells, yeast cells, insect cells or plant cells, such as Escherichia coli, Bacillus spp., Streptomyces spp., Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e.g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, and 293F.

[0096] In one or more embodiments, the antigenic ASC peptide can be chemically conjugated to an immunogenic carrier using techniques well known in the art. The conjugation can be achieved by single-point conjugation (e.g., N-terminus or C-terminus) or a locked structure where both ends of the peptide are conjugated to an immunogenic carrier protein or scaffold structure (e.g., VLP) to allow free movement of the peptide. The conjugation is carried out by conjugation chemistry known to those skilled in the art, such as through cysteine residues, lysine residues, or another carboxyl moiety. Thus, for example, for direct covalent conjugation, carbodiimide, glutaraldehyde, or N-[γ-maleimidobutyryloxy] succinimide ester may be used, using common commercially available heterobifunctional linkers such as 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or succinimidyl 3-(2-pyridyldithio)propionate (SPDP).

[0097] For example, examples of the conjugation of peptides (especially cyclic peptides) to protein carriers through hydrazide peptide derivatives are described in International Patent Application No. PCT / EP2003 / 004551 (International Publication No. WO 2003 / 092714A1). After the coupling reaction, the immunogen can be easily separated and purified, for example, by dialysis, high performance liquid chromatography, gel filtration, fractionation, etc. Peptides terminated with cysteine residues (preferably with a linker outside the cyclized region) can be conveniently conjugated to carrier proteins by maleimide chemistry.

[0098] When the immunogenic carrier is a VLP, several antigenic peptides having the same or different amino acid sequences can be conjugated to a single VLP particle, thus preferably resulting in a repetitive and ordered structure presenting several antigenic determinants in a directed manner, as described in International Patent Applications PCT / IB1999 / 001925 (International Publication No. WO 00 / 032227), PCT / IB2002 / 004132 (International Publication No. WO 2003 / 024481), PCT / IB2002 / 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 can be the same as or different from the antigenic peptide displayed by a second VLP subunit in the same VLP. In other embodiments, one or several antigen molecules can be linked to one VLP subunit. Thus, a particular feature of the VLP of the RNA phage coat protein (especially the Qβ coat protein VLP) is that several antigens can be conjugated to each subunit. This allows the generation of dense antigen arrays.

[0099] Another feature of VLPs derived from RNA phages is that their expression yield in bacteria is very high, allowing large amounts of material to be produced at an affordable cost. In addition, the use of VLPs as carriers allows the formation of robust antigen arrays and conjugates with variable antigen densities, respectively. Specifically, the use of VLPs of RNA phages, especially VLPs of the RNA phage Qβ coat protein, allows for very high antigen densities to be achieved.

[0100] ASC-targeted VLPs can be used to treat subjects at risk of developing or having developed an inflammatory disorder that involves, at least in part, inflammasome assembly and / or inflammatory cytokines.

[0101] As used herein, "treatment" or variations thereof refers to reducing, limiting progression, ameliorating, or eliminating symptoms or signs associated with a disorder to any extent. A "sign" or "clinical sign" is an objective physical finding related to a particular disorder that can be detected by someone other than the subject. A "symptom" is any subjective evidence of a disease or disorder of the subject.

[0102] "Treatment" can be therapeutic or prophylactic. "Therapeutic" and variations thereof refer to treatment that ameliorates one or more existing symptoms or clinical signs associated with a disorder. "Prophylactic" and variations thereof refer to treatment that limits the development and / or appearance of disease symptoms or clinical signs to any extent. Generally, "therapeutic" treatment is initiated after the subject manifests the disorder, while "prophylactic" treatment is initiated before the subject manifests the disorder.

[0103] Prophylactic treatment (e.g., initiated before the subject manifests symptoms or clinical signs of the disorder (e.g., when the inflammatory disorder remains subclinical)) is referred to herein as treatment of a subject "at risk" of having the disorder. The term "at risk" as used herein means that the subject may or may not actually have the risk. Thus, for example, a subject "at risk" of developing a disorder is a subject having one or more risk factors associated with the disorder, such as genetic susceptibility, ancestry, age, gender, geographic location, lifestyle, or medical history. Thus, ASC-targeted VLPs can be administered prophylactically before the subject manifests symptoms or clinical signs of an inflammatory disorder.

[0104] Accordingly, the composition can be administered before, during, or after a subject first manifests symptoms or clinical signs of an inflammatory disorder. Treatment initiated before a subject first manifests symptoms or clinical signs associated with an inflammatory disorder may result in a reduced likelihood that the subject will experience clinical evidence of the inflammatory disorder, a reduced severity of the symptoms and / or clinical signs of the disorder, and / or complete resolution of the inflammatory disorder, compared to a subject to whom the composition is not administered. Treatment initiated after a subject first manifests symptoms or clinical signs associated with an inflammatory disorder may result in a reduced severity of the symptoms and / or clinical signs of the inflammatory disorder and / or complete resolution of the inflammatory disorder, compared to a subject to whom the composition is not administered.

[0105] Accordingly, the method includes administering to a subject having or at risk of having an inflammatory disorder involving inflammasome assembly and / or inflammatory cytokines an effective amount of the composition. In this regard, an "effective amount" is an amount capable of effectively reducing, limiting progression, improving, or resolving symptoms or clinical signs associated with the inflammatory disorder.

[0106] Accordingly, the ASC-targeting Qβ VLPs described herein can be formulated with a pharmaceutically acceptable carrier. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Such media and agents are contemplated for use in therapeutic compositions, except insofar as any conventional media or agent is incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the composition. As used herein, "pharmaceutically acceptable" means that the material is non-biological or otherwise undesirable, i.e., the material can be administered to an individual together with the ASC-targeting Qβ VLPs without causing an undesirable biological effect or interacting in a harmful manner with any of the other components of the pharmaceutical composition containing it.

[0107] Accordingly, the ASC-targeting Qβ VLPs can be formulated into a pharmaceutical composition. The pharmaceutical composition can be formulated into a variety of forms suitable for the preferred route of administration. Accordingly, the composition can be administered by known routes, including, for example, orally, parenterally (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.) or topically (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, rectal, etc.). The pharmaceutical composition can be administered to mucosal surfaces, for example, by administration to the nasal mucosa or respiratory mucosa (e.g., by spraying or aerosolization). The composition can also be administered by sustained release or delayed release.

[0108] Accordingly, the ASC-targeted Qβ VLPs can be provided in any suitable form, including but not limited to solutions, suspensions, emulsions, sprays, aerosols, or any form of mixtures. The compositions can be formulated with any pharmaceutically acceptable excipient, carrier, or vehicle for delivery. For example, the formulations can be delivered in conventional topical dosage forms, such as creams, ointments, aerosol formulations, non-aerosol sprays, gels, lotions, etc. The formulations can also include one or more additives, including, for example, adjuvants (whether ADVAX adjuvant or other adjuvants), skin penetration enhancers, coloring agents, fragrances, flavoring agents, humectants, thickening agents, etc.

[0109] The formulations can conveniently be in unit dosage form and can be prepared by methods well known in the pharmaceutical art. The methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the ASC-targeted Qβ VLPs into association with a carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions can be prepared by uniformly and / or intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.

[0110] The amount of ASC-targeted Qβ VLPs administered can vary depending on various factors, including but not limited to the inflammatory disorder being treated, the weight, physical condition, and / or age of the subject, and / or the route of administration. Accordingly, the absolute weight of ASC-targeted Qβ VLPs contained in a given unit dosage form can vary widely and depends on factors such as the species, age, weight, and physical condition of the subject, and / or the method of administration, etc. Thus, it is impractical to specify generally an amount of ASC-targeted Qβ VLPs that is effective for all possible applications. However, a person of ordinary skill in the art can readily determine the appropriate amount, taking such factors fully into consideration.

[0111] In one or more embodiments, the method can include administering an amount of ASC-targeted Qβ VLPs sufficient to provide a dose, for example, of about 50 ng / kg to about 1 mg / kg to a subject, but in one or more embodiments, the method can be carried out by administering a dose of ASC-targeted Qβ VLPs outside of this range.

[0112] Administration

[0113] In one or more embodiments, the method comprises administering sufficient ASC-targeted Qβ VLPs to provide a minimum dose of at least 50 ng / kg, such as 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 μg / kg, at least 2 μg / kg, at least 5 μg / kg, at least 10 μg / kg, at least 20 μg / kg, at least 50 μg / kg, at least 100 μg / kg, at least 200 μg / kg, or at least 500 μg / kg.

[0114] In one or more embodiments, the method comprises administering sufficient ASC-targeted Qβ VLPs to provide a maximum dose of no more than 1 mg / kg, no more than 500 μg / kg, no more than 250 μg / kg, no more than 200 μg / kg, no more than 150 μg / kg, no more than 100 μg / kg, no more than 50 μg / kg, no more than 25 μg / kg, no more than 10 μg / kg, no more than 5 μg / kg, no more than 2 μg / kg, no more than 1 μg / 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.

[0115] In one or more embodiments, the method comprises administering sufficient ASC-targeted Qβ VLPs to provide a dose that falls within a range having endpoints of any of the foregoing minimum doses and any of the foregoing maximum doses greater than the minimum dose. For example, in one or more embodiments, the method can comprise administering sufficient ASC-targeted Qβ VLPs to provide a subject with a dose of from 200 ng / kg to about 10 μg / kg, such as a dose of from about 700 ng / kg to about 5 μg / kg.

[0116] In one or more embodiments, a single dose up to multiple doses of ASC-targeted Qβ VLPs can be administered, for example, weekly, but in one or more embodiments, the method can be carried out by administering ASC-targeted Qβ VLPs at a frequency outside of that range. When multiple doses are used over a period of time, the amount of each dose can be the same or different. For example, a daily dose of 1 mg can be administered as a single dose of 1 mg, two doses of 0.5 mg each, or as a first dose of 0.75 mg followed by a second dose of 0.25 mg. In addition, when multiple doses are used over a period of time, the intervals between doses can be the same or different.

[0117] Administration frequency

[0118] In certain embodiments, the ASC-targeted Qβ VLPs can be administered at a minimum frequency of at least once a year, such as 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 a month, or at least once every two weeks.

[0119] In certain embodiments, the ASC-targeted Qβ VLPs can be administered at a maximum frequency of no more than once a week, such as no more than once every two weeks, no more than once a month, no more than once every two months, no more than once every three months, no more than once every six months, or once a year.

[0120] In one or more embodiments, the ASC-targeted Qβ VLPs can be administered at a frequency defined by a range having as endpoints any of the minimum frequencies listed above and any of the maximum frequencies listed above that are more frequent than the minimum frequency.

[0121] Duration of treatment

[0122] The duration of administration of the antigenic ASC peptides described herein (e.g., the time period during which the antigenic ASC peptides are administered) can vary depending on any of a variety of factors (e.g., patient response, etc.). For example, the antigenic ASC peptides can be administered for a time period within the range of about one day to about one week, about two weeks to about four weeks, about one month to about two months, about two months to about four months, about four months to about six months, about six months to about eight months, about eight months to about one year, about one year to about two years, or about two years to about four years or longer. In one or more embodiments, the ASC-targeted Qβ VLPs can be administered as a single treatment. In other embodiments, the subject can be administered the ASC-targeted Qβ VLPs for life. In certain embodiments, the ASC-targeted Qβ VLPs can be administered monthly (e.g., every four weeks) until effective.

[0123] In some cases, the ASC-targeted Qβ VLPs can be administered at an initial frequency for an initial period and then at a lower frequency. For example, the dosing regimen can include administering three doses of the ASC-targeted Qβ VLPs at a monthly frequency (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.

[0124] When the ASC-targeted Qβ VLP composition is used for prophylactic treatment, a priming dose and / or a booster dose can generally be administered. The booster dose should be administered at an appropriate interval (e.g., once a year) to increase the circulating antibody levels that have dropped below the desired level. The booster dose may include the ASC-targeted peptide with or without the original immunogenic carrier. The booster composition can include an alternative immunogenic carrier or may be without any carrier. In addition, the booster composition can be formulated with or without an adjuvant.

[0125] In an illustrative embodiment, the ASC-VLP vaccine can be administered in a three-dose regimen, where the three doses are administered at intervals ranging from one to six months or one to twelve months. Additional boosters can be administered at an appropriate frequency (e.g., every twelve months) as needed. In an illustrative embodiment, the treatment regimen can include a first dose of 100 μg VLP or in the range of 50 μg to 300 μg VLP and repeat booster doses of 50 μg to 300 μg VLP (e.g., 100 μg VLP).

[0126] In one or more embodiments, the ASC-VLP vaccine can be combined with one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered before, after, and / or simultaneously with the ASC-VLP vaccine. The ASC-VLP vaccine and the additional therapeutic agents can be co-administered. As used herein, "co-administered" means administering two or more components of a combination such that the combined therapeutic or prophylactic effect can be greater than the therapeutic or prophylactic effect of any one of the components administered alone. The two components can be co-administered simultaneously or sequentially. The components for simultaneous co-administration can be provided in one or more pharmaceutical compositions. The sequential co-administration of two or more components includes the situation where each component can be present at the treatment site simultaneously. Alternatively, the sequential co-administration of two components can include the situation where at least one of the components has been cleared from the treatment site, but at least one cellular effect of administering that component (e.g., inactivation of inflammasomes, inhibition of one or more inflammatory cytokines, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, in certain cases, the co-administered combination includes components that never exist together as a chemical mixture. In one or more alternative embodiments, the ASC-VLP vaccine and the additional therapeutic agent can be administered as part of a mixture or admixture. In some aspects, administering the ASC-VLP vaccine can achieve the effectiveness of a lower dose of other therapeutic modalities compared to administering one or more other therapeutic agents alone, thereby reducing the likelihood, severity, and / or extent of toxicity observed when administering a higher dose of one or more other therapeutic agents.

[0127] Exemplary additional therapeutic agents include, but are not limited to, anti-ASC antibody agents. Exemplary anti-ASC antibody agents include, but are not limited to, antibodies against full-length ASC (e.g., mAb, 04-147, clone 2EI-7, 1:1,000 dilution, MilliporeSigma, Burlington, MA), antibodies against the CARD region (e.g., mAb, 653902, clone TMS-1, 1:500 dilution, BioLegend, San Diego, CA; pAb, sc-22514-R, clone N-15R, 1:200 dilution, Santa Cruz Biotechnology, Inc., Dallas, TX)

[0128] In the foregoing specification and the appended 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 should 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 recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0129] In the foregoing description, for clarity, specific embodiments may be described separately. Throughout this specification, references to "one embodiment", "an embodiment", "certain embodiments" or "one or more embodiments", etc. mean that a particular feature, configuration, composition or property described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases throughout this specification do not necessarily refer to the same embodiment of the present disclosure. Moreover, in one or more embodiments, the particular features, configurations, compositions or properties may be combined in any suitable manner. Additionally, in one or more embodiments, the particular features, configurations, compositions or properties may be combined in any suitable manner. 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 mutually exclusive.

[0130] For any method disclosed herein that includes discrete steps, these steps may be performed in any feasible order. And, as needed, any combination of two or more steps may be performed simultaneously.

[0131] As used herein, the terms "preferred" and "preferably" mean that certain embodiments of the invention may provide certain benefits in certain circumstances. However, in the same or other circumstances, other embodiments may also be preferred. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not available, and is not intended to exclude other embodiments from the scope of the invention.

[0132] The present invention is illustrated by the following examples. It should be understood that specific examples, materials, amounts, and procedures should be construed broadly in accordance with the scope and spirit of the invention as shown herein.

[0133] Examples

[0134] Qβ-VLPs were produced in Escherichia coli (E. coli) using the method described previously (Crossey, E. et al. Vaccine. 2015). The ASC peptide was synthesized (GenScript Biotech Corp., Piscataway, NJ) and stored as a lyophilized powder at -20 °C. The ASC peptide was conjugated to lysine residues exposed on the upper surface of the assembled VLPs using the bifunctional crosslinker 6-[(β-maleimidopropionamido)hexanoic acid N-hydroxysuccinimide ester] (SMPH; Thermo Fisher Scientific, Inc., Waltham, MA) ( Figure 3 ). The conjugation efficiency was confirmed by mobility shift gel electrophoresis on a 10% SDS denaturing polyacrylamide gel ( Figure 4 ).

[0135] C57B1 / 6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were 2 months old. Mice were randomly assigned to Qβ control sham-treatment, Qβ ASC-linker, Qβ ASC-helix 4, or Qβ ASC-C-terminal vaccine treatment groups (n = 5 mice / group) according to sex. Vaccines were administered by intramuscular injection into the right hind limb at a dose of 5 μg VLP (10 μg for the ASC-C-terminal vaccine due to poor conjugation efficiency) suspended in 50 μL of sterile water (concentration 100 - 200 ng / μL) at 2 months of age, and a booster vaccine injection was given 3 weeks later. ( Figure 5 )

[0136] Three weeks after the second injection, plasma samples were collected by retro-orbital capillary and the antibody titers against the target peptides used for the synthetic vaccine were evaluated using an indirect enzyme-linked immunosorbent assay (ELISA). The immune sera of the inoculated mice were used as the primary antibody at multiple dilutions, and detection was performed using horseradish peroxidase-conjugated goat anti-mouse antibody with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, where the absorbance was measured at 450 nm( Figures 6 - 8 ).

[0137] The inoculated animals were observed weekly for signs of deteriorating health, including poor grooming behavior, lethargy, dermatitis, masses, open wounds, or other evidence of infection. Animal weights were collected at four time points, including at two months, three months, five months, and six months of age( Figure 9 ). At six months of age, blood samples were obtained by retro-orbital capillary collection into tubes coated with ethylenediaminetetraacetic acid (EDTA) and heparin, and then evaluated using a complete blood count( Figure 10 ) on an Abaxis Vetscan HM5 and a mouse blood chemistry panel( Figure 10 ) on an Abaxis Vetscan VS2.

[0138] The inoculated animals were then intraperitoneally injected with 5 mg / kg lipopolysaccharide (LPS; from Escherichia coli 055:B5, MilliporeSigma, Burlington, MA) and sacrificed nine hours after injection. Terminal blood collection was performed by cardiac puncture. The animals were then perfused through the heart with ice-cold 0.125 M phosphate buffer, and the brains were microdissected and frozen on dry ice. The blood samples were allowed to clot on ice for 30 minutes and then centrifuged twice at 5000 RPM for five minutes to isolate the immune sera, which were stored at -80 °C. Serum interleukin-1β levels were evaluated using a mouse IL-1β DuoSet ELISA kit (R&D systems, catalog number DY401) according to the manufacturer's instructions, where the serum was diluted 50% in reagent diluent (R&D Systems, Inc., Minneapolis, MN)( Figure 14 ).

[0139] Homogenize hippocampal brain tissue samples in 10% weight / volume tissue protein extraction reagent (T-PER, Thermo Fisher Scientific, Inc., Waltham, MA) and 1% protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, Inc., Waltham, MA). Dissolve the cells in 1x LDS / RA buffer (Thermo Fisher Scientific), sonicate for 30 seconds, and boil at 95 °C for 15 minutes. Split the lysates by SDS-PAGE on a 4-12% Bis-Tris gradient gel and perform immunoblotting with caspase-1 p20 (AdipoGen Life Sciences, Inc., San Diego, CA) and GAPDH (MilliporeSigma, Burlington, MA) Figure 15 ).

[0140] Perform immunohistochemical analysis of ASC-VLP immunoreactivity using formalin-fixed and paraffin-embedded human Alzheimer's disease hippocampal brain sections (n = 1). First, hydrate the sections successively in xylene, ethanol, and PBS with 0.1% Tween (PBST), then incubate in 10 mM sodium citrate buffer (pH 6.0) at 95 °C for 30 minutes for antigen retrieval, wash in PBS with 0.1% Tween (PBST), and quench with 0.3% H2O2 in PBST for 20 minutes. Block the sections with 5% normal goat serum for one hour at room temperature. Incubate the sections with rabbit polyclonal anti-ASC antibody (1:500, AdipoGen Life Sciences, Inc., San Diego, CA) or immune sera from Qβ control or Qβ-ASC VLP-inoculated animals (1:500). After washing in PBST, incubate the sections with biotinylated secondary antibody (1:250, Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Then incubate the sections with ABC (Vector Laboratories, Inc., Newark, CA) reagent for 30 minutes at room temperature. Visualize the immunoreactivity signals by developing the sections in SIGMAFAST 3,3′-diaminobenzidine (DAB) tablets (MilliporeSigma, Burlington, MA). Dehydrate the slides in ethanol and xylene, then cover with a coverslip and image with a brightfield microscope Figure 11 ).

[0141] Uninoculated C57Bl6 / J mice (n = 4) injected with 5 mg / kg LPS and sacrificed after 9 hours (as described above), ASC - / - transgenic mice (n = 1) and hippocampal brain lysates from human Alzheimer's disease patients (n = 4) were used to evaluate the immune serum antibody cross-reactivity between human and mouse ASC. Hippocampal brain lysates were resolved by SDS-PAGE as described above and immunoblotted using rabbit polyclonal anti-ASC antibody (1:2000, AdipoGen Life Sciences, Inc., San Diego, CA) or immune serum from Qβ control or Qβ-ASC VLP immune serum (1:1000) Figure 12 ). The indirect sandwich ELISA method was performed as follows: Rabbit polyclonal anti-ASC antibody (1:200, AdipoGen Life Sciences, Inc., San Diego, CA) was used as the capture antibody and incubated with hippocampal brain lysates from mice and humans for one hour at room temperature, and Qβ control or Qβ-ASC VLP immune serum was used as the detection antibody, followed by HRP-conjugated secondary antibody (1:10,000, Jackson ImmunoResearch Laboratories Inc., West Grove, PA), and the bound anti-ASC antibody was detected using the absorbance of TMB substrate at 450 nm Figure 13 ).

[0142] The complete disclosures of all patents, patent applications, and publications cited herein and electronically available materials (including, for example, nucleotide sequence submissions in GenBank and RefSeq, and amino acid sequence submissions in SwissProt, PIR, PRF, PDB, and the translations of the annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. If there are any inconsistencies between the disclosure of the present application and the disclosure of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples are given for clarity of understanding only. It should not be construed as imposing unnecessary limitations. The present invention is not limited to the exact details shown and described, and variations that are obvious to those skilled in the art will be included within the scope of the invention as defined by the claims.

[0143] Unless otherwise indicated, all numerical values expressing quantities of ingredients, molecular weights, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless 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 application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0144] While 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. Any numerical value, however, inherently contains a range necessarily resulting from the standard deviation found in its 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 following the heading unless otherwise specified.

[0146] Sequence Listing Independent Text

[0147] SEQ ID NO: 1 - Human ASC Sequence

[0148]

[0149] Pyrin Domain: 1 - 91

[0150] CARD Domain: 107 - 195

[0151] SEQ ID NO: 2 - Mouse ASC Sequence

[0152]

[0153] Pyrin Domain: 1 - 91

[0154] CARD Domain: 105 - 193

[0155] SEQ ID NO: 3 - ASC Linker Peptide

[0156]

[0157] SEQ ID NO: 4 - ASC Helix 4 Peptide

[0158]

[0159] SEQ ID NO: 5

[0160]

[0161] SEQ ID NO: 6 - Human ACS Peptide Sequence

[0162]

[0163] SEQ ID NO: 7 - Human ACS Peptide Sequence

[0164]

[0165] SEQ ID NO: 8 - Human ACS Peptide Sequence

[0166]

[0167] SEQ ID NO: 9 - Human ACS Peptide Sequence

[0168]

[0169] SEQ ID NO: 10 - Human ACS Peptide Sequence

[0170]

[0171] SEQ ID NO: 11 - Human ACS Peptide Sequence

[0172]

[0173] SEQ ID NO: 12 - Human ACS Peptide Sequence

[0174]

[0175] SEQ ID NO: 13 - Mouse ACS Peptide Sequence

[0176]

[0177] SEQ ID NO: 14 - Mouse ACS Peptide Sequence

[0178]

[0179] SEQ ID NO: 15 - Mouse ACS Peptide Sequence

[0180]

[0181] SEQ ID NO: 16 - Mouse ACS Peptide Sequence

[0182]

[0183] SEQ ID NO: 17 - Mouse ACS Peptide Sequence

[0184]

[0185] SEQ ID NO: 18 - Mouse ACS Peptide Sequence

[0186]

[0187] SEQ ID NO: 19 - Mouse ACS Peptide Sequence

[0188]

[0189] SEQ ID NO: 20 - Mouse ACS Peptide Sequence

[0190]

[0191] SEQ ID NO: 21 - Mouse ACS Peptide Sequence

[0192]

[0193] SEQ ID NO: 22 - Linker

[0194]

[0195] SEQ ID NO: 23 - Linker

[0196]

Claims

1. An immunogen, the immunogen comprising: An immunogenic carrier comprising Qβ phage virus-like particles (VLPs); and An antigenic apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) peptide linked to the immunogenic carrier.

2. The immunogen according to claim 1, wherein the ASC peptide comprises the amino acid sequence of SEQ ID NO: 1 or an antigenic fragment thereof.

3. The immunogen according to claim 2, wherein the fragment of SEQ ID NO: 1 comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:

12.

4. The immunogen according to any one of the preceding claims, wherein the immunogenic carrier is linked to the ASC peptide via a 6-[β-maleimidopropionamido]hexanoic acid N-hydroxysuccinimide ester (SMPH) crosslinking molecule.

5. The immunogen according to any one of the preceding claims, the immunogen further comprising a second antigenic ASC peptide.

6. The immunogen according to claim 5, wherein both ASC peptides are displayed on a single VLP.

7. A composition, the composition comprising the immunogen according to any one of the preceding claims.

8. The composition according to claim 7, the composition comprising: A first population of VLPs displaying a first antigenic ASC peptide; and A second population of VLPs displaying a second antigenic ASC peptide.

9. The composition according to claim 7 or 8, the composition further comprising an adjuvant.

10. A method of treating an inflammatory disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition, the composition comprising an immunogen, the immunogen comprising: An immunogenic carrier comprising Qβ phage virus-like particles (VLPs); and An antigenic apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) peptide linked to the immunogenic carrier.

11. The method according to claim 10, wherein the method further comprises administering to the subject at least one additional therapeutic agent for treating the inflammatory disorder.

12. The method according to claim 10 or 11, wherein the immunogen further comprises a second antigenic ASC peptide.

13. The method according to claim 12, wherein both antigenic ASC peptides are linked to a single carrier.

14. The method according to claim 12, wherein the composition comprises: A first population of immunogens, which comprises: A first population of immunogenic carriers; and A first antigenic ASC peptide linked to the first population of immunogenic carriers; and A second population of immunogens, which comprises: A second population of immunogenic carriers; and The second ASC peptide linked to the second population of immunogenic carriers.

15. The method according to any one of claims 10-14, wherein at least one antigenic ASC peptide comprises the amino acid sequence of SEQ ID NO: 1 or an antigenic fragment thereof.

16. The method according to claim 15, wherein the fragment of SEQ ID NO: 1 comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:

12.

17. The method according to any one of claims 10-16, wherein the composition is administered to the subject before the subject exhibits symptoms or clinical signs of an inflammatory disorder.

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

19. An expression vector comprising the nucleic acid of claim 18.

20. A host cell comprising the expression vector of claim 19.

21. A vaccine comprising the composition according to any one of claims 1-9.

22. A method of treating an inflammatory disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine of claim 21.

23. The method according to claim 22, wherein the method further comprises administering to the subject at least one additional therapeutic agent for treating the inflammatory disorder.

24. The method according to claim 22 or 23, wherein the vaccine is administered to the subject before the subject exhibits symptoms or clinical signs of inflammation.

25. The method according to any one of claims 22-24, wherein the method further comprises administering to the subject at least one additional therapeutic agent for treating the inflammatory disorder.

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