Treatment of Inflammatory Diseases

Abstract

The present disclosure provides a method for treating inflammatory diseases, particularly age-related inflammatory diseases, in a mammalian subject in need thereof, comprising an effective amount of a virus-like particle comprising viral structural proteins and a galectin-3 antigen, and a composition or vaccine comprising same for that purpose.

Description

[Technical field]

[0001] The present disclosure relates to the treatment of inflammatory diseases in a subject, particularly inflammatory diseases associated with aging. [Background technology]

[0002] Aging is the primary risk factor for most neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. The most common types of neurodegenerative diseases occur primarily in older individuals. In fact, one in ten people over the age of 65 is expected to suffer from a neurodegenerative disease, with the likelihood increasing exponentially with age.

[0003] Neurodegenerative diseases of the central nervous system (CNS) are defined by the progressive and irreversible loss of neurons and are accompanied by behavioral disorders, including loss of motor and / or cognitive function. Given the strong association between neurodegenerative diseases and aging, neurodegeneration is often considered part of the brain aging process.

[0004] Few or no effective treatments are available for neurodegenerative diseases, and most focus on alleviating symptoms rather than targeting the root cause of the disease. Thus, addressing and directly slowing the brain aging process may offer a better strategy to reduce the onset and burden of neurodegenerative diseases.

[0005] Neurodegenerative diseases encompass a wide variety of severe and debilitating conditions, including Parkinson's disease, amyotrophic lateral sclerosis (ALS, "Lou Gehrig's disease"), Huntington's disease, Alzheimer's disease, etc. These diseases are characterized by a gradual but relentless worsening of the patient's condition over time.

[0006] Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common neurodegenerative diseases. Alzheimer's disease (AD) is a multifactorial progressive neurodegenerative disease characterized by memory loss and cognitive impairment. Currently, available therapies for Alzheimer's disease (AD) have shown limited efficacy, but to date there is no true cure.

[0007] Parkinson's disease (PD) is characterized by progressive degeneration of dopamine (DA) neurons that project from the substantia nigra pars compacta (SNpc) to the dorsal striatum. The resulting loss of dopamine in the striatum leads to debilitating motor dysfunction, including muscular rigidity, resting tremor, impaired postural reflexes, and bradykinesia. Currently, available therapies for Parkinson's disease (PD) provide relief from motor symptoms but are insufficient to offer the neuroprotective effects necessary to prevent the progressive degeneration of dopamine (DA) neurons. Furthermore, no therapy simultaneously targets the multiple debilitating non-motor symptoms of PD.

[0008] Neurodegenerative diseases exhibit chronic, abnormal inflammation.

[0009] Galectins are members of the lectin family that show high affinity for β-galactosides. In mammals, about 15 galectins have been discovered that are encoded by LGALS genes and are numbered consecutively. Currently, only galectins-1, -2, -3, -4, -7, -7B, -8, -9, -9B, -9C, -10, -12, -13, -14 and -16 have been identified in humans.

[0010] Galectin-3 (Gal-3) is a member of a highly conserved family of animal lectins that bind to β-galactoside-containing glycoconjugates (glycoproteins or glycolipids) (Henderson NC 2006; Mourad-Zeidan AA 2008). Gal-3 is unique among other galectin family proteins in its structure, which consists of two domains: a carboxyl-terminal domain that contains the carbohydrate-binding region and an amino-terminal domain that is mainly composed of tandem repeats of nine amino acids that bridge carbohydrate and non-carbohydrate ligands (Barondes SH 1994). Gal-3 is secreted by various types of cells, including monocytes, macrophages, and epithelial cells, but mainly by macrophages (Reynolds 2005; MacKinnon AC 2008). Secreted extracellular Gal-3 forms homodimers or pentamers, which are important for biological function. The N-terminal domain of galectin-3 has been demonstrated to be important for the formation of protein oligomers (Non-Patent Document 6: Dumic J 2006, Non-Patent Document 7: Kuklinski S 1998, Non-Patent Document 8: Lepur A 2012). The released protein can function as an extracellular molecule to activate cells, mediate cell-cell and cell-ECM interactions, induce migration of various types of cells, and negatively regulate T cell receptor signaling (Non-Patent Document 9: Yang RY 1996). Gal-3 has been shown to be increased in various models of fibrotic diseases and patients, including pulmonary fibrosis, liver fibrosis, systemic sclerosis, and myocardial fibrosis (Non-Patent Document 10: Henderson NC 2008, Non-Patent Document 11: Barman, et al. 2019, Non-Patent Document 12: Nishi Y 2007, Non-Patent Document 13: De Boer 2010). This suggests that Gal-3 may be both an important mediator of tissue fibrosis and an effective therapeutic target for tissue fibrosis.To date, preclinical and clinical studies of investigational galectin-3 inhibitors have shown protective effects against fibrotic disorders (Non-Patent Document 14: Nikhil Hirani 2017, Non-Patent Document 15: Yu L 2013).

[0011] It has been reported that plasma galectin-3 levels are important as a biomarker and therapeutic agent in neurodegenerative diseases such as idiopathic Parkinson's disease (IPD), neuroprotective and neurorepair treatment options (Non-Patent Document 16: Yazar HO, et al., J Clinical Neuroscience 2019). Galectin-3 was found to be highly upregulated in the brains of AD patients and 5xFAD (familial Alzheimer's disease) mice, and specifically expressed in microglia associated with Aβ plaques (Non-Patent Document 17: Boza-Serrano A et al. Acta Neuropathology, 2019). Galectin-3 levels in serum and CSF samples were higher in AD and ALS patients compared to controls, potentially suggesting a critical association between serum and CSF levels of galectin-3 and cognitive status in AD and ALS patients and healthy controls (Non-Patent Document 18: Ashraf GM et.al. Front Neuroscience, 2018).

[0012] One of the major success stories in medicine over the past 100-150 years has been vaccines targeting various infectious diseases. Vaccines, together with antibiotics, have likely been more important to human health than any other part of the human body. The success of vaccines has led to increased interest in using vaccine technology to treat non-infectious diseases such as allergy, autoimmunity, and cancer. However, the targets of these diseases are generally self-antigens, which can cause problems with efficacy. Inducing strong antibody responses against self-antigens compared to non-self-antigen bacterial, viral, or parasitic proteins is considerably more difficult due to resistance mechanisms (Hellman 2008; Falk Saupe 2015).

[0013] Alphaviruses comprise a series of genetically, structurally, and serologically related mosquito-borne viruses in the family Togaviridae. Alphaviruses include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus, Mucambo virus, Pixuna virus, Western equine encephalitis virus (WEEV), Sindbis virus, Semliki forest virus, Middleburg virus, Chikungunya virus (CHIKV), O'nyong-nyong virus, Ross River virus, Barmah forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Babanki virus, Kiziragachi virus, Highland J virus, Fort Morgan virus, Nudum virus, and Boggy Creek virus. The capsid, a structural subunit containing a single viral protein, associates with the RNA genome in an icosahedral nucleocapsid. In virions, the capsid is surrounded by a lipid envelope that is covered with a regular array of transmembrane protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, E1 and E2.

[0014] Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of standard native viruses but lack the viral genome, and thus may provide safer and cheaper vaccine candidates. A handful of VLP-based prophylactic vaccines are currently commercially available worldwide: GlaxoSmithKline's Engerix® (Hepatitis B virus) and Cervarix® (Human Papillomavirus), and Merck and Co., Inc.'s Recombivax HB® (Hepatitis B virus) and Gardasil® (Human Papillomavirus) are some examples. Other VLP-based vaccine candidates are in clinical trials or undergoing preclinical evaluation, such as influenza virus, parvovirus, Norwalk virus, and various chimeric VLPs. Despite their success in preclinical trials, many others are still limited to small-scale platform studies. The implications of large-scale VLP production are discussed in relation to process control, monitoring, and optimization. The main upstream and downstream technical challenges are identified and discussed accordingly. Successful breakthroughs of VLP-based vaccines are briefly presented along with the latest results of clinical trials and recent developments in chimeric VLP-based technologies directed towards either therapeutic or prophylactic vaccination.

[0015] To date, VLP-based vaccines have been produced against over 30 different viruses that infect humans and other animals, including AAV (adeno-associated virus), H5N3 (avian influenza), BFDV (budgie fledgling disease virus), BTV (bluetongue virus), Ebola, Enterovirus 71, GHPV (goose hemorrhagic polyomavirus), HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis delta virus), HEV (hepatitis E virus), HIV, HPV (human papilloma virus), IBDV (infectious bursal disease virus), influenza A, influenza A H1N1, influenza A, influenza B ... Examples of such viruses include H3N2, JC polyomavirus, Marburg, MS2, IPCV (Indian peanut cramp virus), NDV (Newcastle disease virus), No (Norovirus), Nv (Norwalk virus), PhMV (ground cherry mottle virus), polyomavirus, PPV (porcine parvovirus), RHDV (rabbit hemorrhagic disease virus), rotavirus, SARS, SIV (simian immunodeficiency virus), SV40 (simian virus 40), SVDV (swine vesicular disease virus), etc. (Non-Patent Document 21: Expert Rev. Vaccines 9(10), 1149-1176, 2010).

[0016] US Patent No. 9,353,353 discloses virus-like particles (VLPs) containing one or more chikungunya virus structural proteins, which are useful for formulating vaccines or antigenic compositions against chikungunya that induce immunity against infection or at least one symptom thereof. US Patent No. 9,487,563 discloses modified alphavirus or flavivirus virus-like particles (VLPs) and methods for enhancing the production of modified VLPs for use in preventing or treating alphavirus and flavivirus-mediated diseases. US Patent No. 9,249,191 discloses virus-like particles of chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV), where the virus-like particles contain at least one antigen inserted into the E2 envelope protein to form a fusion protein. U.S. Patent No. 9,969,986 discloses an alphavirus virus-like particle, which comprises alphavirus viral structural proteins including envelope protein E3, which has been modified to contain at least one foreign antigen inserted into its furin cleavage site (these cited references are incorporated herein by reference). [Prior art documents] [Patent documents]

[0017] [Patent Document 1] U.S. Patent No. 9,353,353 [Patent Document 2] U.S. Patent No. 9,487,563 [Patent Document 3] U.S. Patent No. 9,249,191 [Patent Document 4] U.S. Patent No. 9,969,986 [Patent Document 5] US Patent Application Publication No. 2005 / 0118191 [Patent Document 6] WO2022 / 225057 [Non-patent literature]

[0018] [Non-Patent Document 1] Henderson NC, Mackinnon AC, Farnworth SL, Poirier F, Russo FP, Iredale JP, Haslett C, Simpson KJ, Sethi T. 2006. "Galectin-3 regulates myofibroblast activation and hepatic fibrosis." Proc Natl Acad Sci USA 103(13):5060-5. [Non-Patent Document 2] Mourad-Zeidan AA, Melnikova VO, Wang H, Raz A, and Bar-Eli M. 2008. "Expression profiling of Galectin-3-depleted melanoma cells reveals its major role in melanoma cell plasticity and vasculogenic mimicry." Am J Pathol 173:1839-1852. [Non-Patent Document 3] Barondes SH, Cooper DN, Gitt MA, Leffler H. 1994. "Galectins. Structure and function of a large family of animal lectins." J Biol Chem 269:20807-20810. [Non-Patent Document 4] Herbert Y. Reynolds, "Lung Inflammation and Fibrosis, An Alveolar Macrophage-centered Perspective from the 1970s to 1980s" Am J Respir Crit Care Med Vol 171. pp 98-102, 2005 [Non-Patent Document 5] MacKinnon AC, Farnworth SL, Hodkinson PS, Henderson NC, Atkinson KM, Leffler H, Nilsson UJ, Haslett C, Forbes SJ, and Sethi T. 2008. "Regulation of alternative macrophage activation by galectin-3." J Immunol 180:2650-2658. [Non-Patent Document 6] Dumic J, Dabelic S, Flougel M. 2006. "Galectin-3: An open-ended story." BBA-Gen Subjects 1760:616-635. [Non-Patent Document 7] Kuklinski S, Probstmeier R. 1998. "Homophilic binding properties of galectin-3: Involvement of the carbohydrate recognition domain." J Neurochem 70:814-823. [Non-Patent Document 8] Lepur A, Salomonsson E, Nilsson UJ, Leffler H. 2012. "Ligand induced galectin-3 protein self-association." J Biol Chem 287:21751-21756. [Non-Patent Document 9] Yang RY, Hsu DK, and Liu FT. 1996. "Expression of galectin-3 modulates T-cell growth and apoptosis." Proc Natl Acad Sci USA 93:6737-6742. [Non-Patent Document 10] Henderson NC, Mackinnon AC, Farnworth SL, Kipari T, Haslett C, Iredale JP, Liu FT, Hughes J, Sethi T. 2008. "Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis." Am J Pathol. 172(2):288-9

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[0019] The present disclosure relates to the treatment of inflammatory diseases. In particular, the present disclosure provides methods for treating inflammatory diseases, especially age-related inflammatory diseases, in a mammalian subject. [Means for solving the problem]

[0020] The method is for treating inflammatory diseases, particularly age-related inflammatory diseases, comprising administering to a subject in need thereof an effective amount of a virus-like particle comprising viral structural proteins and a galectin-3 antigen.

[0021] The present disclosure also provides a virus-like particle comprising viral structural proteins and a galectin-3 antigen for use in treating inflammatory diseases in a mammalian subject, particularly age-related inflammatory diseases.

[0022] The present disclosure also provides a pharmaceutical composition for the treatment of an inflammatory disease, particularly an age-related inflammatory disease, in a mammalian subject, comprising a virus-like particle comprising a viral structural protein and a galectin-3 antigen.

[0023] The present disclosure also provides the use of a virus-like particle comprising viral structural proteins and a Galectin-3 antigen for the manufacture of a medicament for the treatment of an inflammatory disease in a mammalian subject, particularly an inflammatory disease associated with aging. [Brief description of the drawings]

[0024] [Figure 1] Anti-mouse galectin-3 (Gal-3) antibodies in the serum of mice immunized with VEEV-Gal-3-VLP. VEEV-Gal-3 VLP was administered intramuscularly to one group of mice (group 2) and saline to the other group (group 1). Mice were administered at the start of the experiment, 2 weeks and 4 weeks after the first administration. Six weeks after the start of the experiment, blood samples were obtained from each mouse and Gal-3 in the serum was detected by using ELISA. [Diagram 2] Results of step-through passive avoidance test in 5×FAD mice. Five months after the start of the experiment, the step-through passive avoidance test was performed, and the latency 24 hours after the electrical stimulation was measured. [Diagram 3] Gal-3 levels in the hippocampus of 5xFAD mice immunized with VEEV-Gal-3-VLP. After the study shown in Fig. 2, mouse brains were harvested and the levels of Gal-3 and β-amyloid relative to β-actin were measured in brain lysates. [Figure 4] Effect of VEEV-Gal-3-VLP on survival of aged C57BL / 6J mice. C57BL / 6J mice were divided into two groups. VEEV-Gal-3 VLP was intramuscularly administered to one group of mice (group B6-V) and saline to the other group (group B6-N). [Diagram 5] Effect of VEEV-Gal-3-VLP on survival of 5xFAD mice. 5xFAD mice were divided into two groups. VEEV-Gal-3 VLP was administered intramuscularly to one group of mice (group FV) and saline to the other group (group FN). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] As used herein, the term "galectin-3 antigen" refers to any antigenic structure derived from galectin-3 protein that can be recognized by the immune system and / or stimulates a cellular immune response and / or stimulates the production of antigen-specific antibodies. The galectin-3 epitope peptide may be a fragment of naturally occurring galectin-3 protein, or a fragment of naturally occurring galectin protein with some modifications. The naturally occurring galectin protein is preferably a human galectin protein, and more preferably a human galectin-3 protein. In one embodiment, the modified fragment has at least 80%, 85%, 90%, 95%, or 98% amino acid sequence identity with the fragment of naturally occurring galectin-3 protein. In one embodiment, the modified peptide fragment is a mutant in which at most 10% of amino acids are deleted, substituted, and / or added to the fragment of naturally occurring galectin-3 protein.

[0026] Galectin-3 [Human (Homo sapiens)] (GenBank accession number: AAB86584.1) MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI (SEQ ID NO: 1)

[0027] Examples of galectin-3 antigens, i.e., galectin-3 epitope peptides, include the following: ADNFSLHDALSGSGNPNPQGWPGAWGNQPA (SEQ ID NO: 2) YPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGA (SEQ ID NO: 3) YPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGA (SEQ ID NO: 4) ADSFSLNDALAGSGNPNPQGYPGAWGNQPA (SEQ ID NO:5)

[0028] Gal-3 epitope peptide SEQ ID NO:2 consists of a human N-terminal Gal-3 epitope peptide. Gal-3 epitope peptides SEQ ID NO:3 and 4 consist of human Gal-3 N-terminal repeat epitope peptides, with SEQ ID NO:4 having a longer repeat than SEQ ID NO:3. SEQ ID NO:5 encodes a mouse Gal-3 N-terminal peptide.

[0029] In WO2022 / 225057 (the contents of which are incorporated herein by reference), VLPs carrying the above-mentioned Gal-3 epitope peptides were prepared and the immunogenicity of those VLPs was confirmed.

[0030] Alphaviruses As used herein, "alphavirus" refers to an RNA-containing virus belonging to the Togaviridae family of viruses. Exemplary Togaviridae viruses include, but are not limited to, Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Everglades Virus, Mucambo Virus, Pixuna Virus, Western Equine Encephalitis Virus (WEEV), Sindbis Virus, Semliki Forest Virus, Middleburg Virus, Chikungunya Virus (CHIKV), O'Nyong-Nyong Virus, Ross River Virus, Barmah Forest Virus, Getah Virus, Sagiyama Virus, Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus, Wataroa Virus, Babanki Virus, Kiziragati Virus, Highland J Virus, Fort Morgan Virus, Ndum Virus, Boggy Creek Virus, and Okelbo Virus.

[0031] By "viral structural protein" is meant a polypeptide or fragment thereof having at least about 80% amino acid sequence identity to a naturally occurring viral capsid or envelope protein and having immunogenic activity in a mammal. In some embodiments, the alphavirus structural protein has at least about 85%, 90%, 95% or more amino acid sequence identity to the alphaviruses discussed above.

[0032] The viral structural protein and the Galectin-3 antigen may be directly or indirectly fused, in one embodiment, one or two linkers may be interposed between the N-terminal residue of the antigen and the viral structural protein and / or between the C-terminal residue of the antigen and the viral structural protein.

[0033] The antigen or viral structural protein can be cut and replaced by a short linker. In some embodiments, the antigen or viral structural protein comprises one or more peptide linkers. Typically, the linker is composed of 2 to 25 amino acids (e.g., 2, 3, 4, 5 or 6 amino acids). Usually, the linker is 2 to 15 amino acids long, but in certain circumstances, the linker may be only one, such as a single glycine (G) residue.

[0034] In one embodiment, a nucleic acid molecule in which a polynucleotide encoding a viral structural protein is genetically fused to a polynucleotide encoding an antigen is expressed in a host cell (e.g., a mammalian cell (e.g., 293F cell) such that the first and second attachment sites are linked through a peptide bond. In this case, the viral structural protein and the antigen are linked through a peptide bond. For this embodiment, the first and / or second attachment sites can be genetically modified from the original protein or antigen. For example, the first attachment site is modified from the viral structural protein such that the protein is conjugated to the antigen through a linker peptide, including SG, GS, SGG, GGS, and SGSG. When the viral structural protein is chemically conjugated to the antigen, the first and second attachment sites can be linked through a chemical cross-linker. Examples of cross-linkers include, but are not limited to, SMPH, sulfo-MBS, sulfo-EMCS, sulfo-GMBS, sulfo-SIAB, sulfo-SMPB, sulfo-SMCC, SVSB, SIA and other cross-linkers available from Pierce Chemical Company.

[0035] Preferably, the antigen may be linked to a Chikungunya virus structural protein or a Venezuelan Equine Encephalitis virus structural protein as a genetically engineered fusion protein.

[0036] A Chikungunya virus structural protein or Venezuelan Equine Encephalitis virus structural protein as used in this application may be a Chikungunya virus envelope protein or a Venezuelan Equine Encephalitis virus envelope protein, may be a capsid, may be a complex of one or more envelope proteins, and / or may be a capsid protein.

[0037] Examples of Chikungunya viruses include, but are not limited to, strain 37997 and strain LR2006 OPY-1. Examples of Venezuelan equine encephalitis viruses include, but are not limited to, strain TC-83.

[0038] The chikungunya virus structural protein or Venezuelan equine encephalitis virus structural protein used in this application may be a naturally occurring viral structural protein or a modified protein thereof. The modified protein may be a fragment of a naturally occurring viral structural protein. In one embodiment, the modified protein has at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity with a naturally occurring viral capsid and / or envelope protein. In one embodiment, the modified protein is a mutant in which at most 10% of amino acids are deleted, substituted and / or added to the naturally occurring viral capsid and / or envelope protein. For example, a K64A or K64N mutation can be introduced into the capsid of the Venezuelan equine encephalitis virus structural protein used in this application.

[0039] The Chikungunya virus structural proteins or Venezuelan Equine Encephalitis virus structural proteins may consist of or include the capsid, E3, E2, and E1 proteins. The E3 and E2 proteins may be expressed together such that E2 and E3 can form one protein.

[0040] Examples of Chikungunya virus structural proteins include, but are not limited to, capsid-E3-E2-E1 of Chikungunya virus strain 37997, and capsid-E3-E2-E1 of Chikungunya virus LR2006 OPY-1.

[0041] Examples of Venezuelan Equine Encephalitis virus structural proteins include, but are not limited to, capsid-E3-E2-E1 of Venezuelan Equine Encephalitis virus strain TC-83.

[0042] An exemplary Chikungunya virus structural protein sequence is provided in Genbank Accession No. ABX40006.1 and is set forth below (SEQ ID NO:6):

[0043] [ka]

[0044] Another exemplary Chikungunya virus structural protein sequence is provided in Genbank Accession No. ABX40011.1 and is set forth below (SEQ ID NO:7):

[0045] [ka]

[0046] An exemplary Venezuelan Equine Encephalitis virus structural protein is set forth below (SEQ ID NO:8):

[0047] [ka] In one embodiment, the first attachment site comprises an amino group, preferably the amino group of a lysine residue, hi one embodiment, the second attachment site comprises a sulfhydryl group, preferably the sulfhydryl group of a cysteine.

[0048] According to the present application, there is provided a chikungunya virus-like particle or Venezuelan equine encephalitis virus-like particle, which comprises a chikungunya virus structural protein or Venezuelan equine encephalitis virus structural protein and at least one galectin-3 antigen, wherein the at least one galectin-3 antigen is inserted into E3 of the viral structural protein, and the chikungunya virus structural protein or Venezuelan equine encephalitis virus structural protein and the galectin-3 antigen are expressed as a fusion protein. The galectin-3 antigen may be directly or indirectly inserted into E3 of the viral structural protein.

[0049] The viral structural proteins of chikungunya virus and Venezuelan equine encephalitis consist of E1, E2, 6K, and E3. 6K is naturally cleaved during the assembly process and removed from the VLP. Mature VLP consists of capsid, E1, and E2. In this specification and claims, the term "viral structural protein" refers not only to those having 6K, but also to those after 6K has been removed.

[0050] The 6K sequences of CHIKV and VEEV used in the working example are as follows: CHIKV OPY-1 strain, 6K: 749-809aa of SEQ ID NO:6 atyqeaaiylwneqqplfwlqaliplaalivlcnclrllpcccktlaflavmsvgahtvsa (SEQ ID NO: 9) CHIKV strain 37997, 6K: 749-809aa of SEQ ID NO:7 atyyeaaaylwneqqplfwlqaliplaalivlcnclkllpcccktlaflavmsigahtvsa (SEQ ID NO: 10) VEEV TC-83 strain, 6K: 758-813aa of SEQ ID NO:8 ettwesldhlwnnnqqmfwiqlliplaalivvtrllrcvccvvpflvmagaagaga (SEQ ID NO:11)

[0051] For Chikungunya virus structural proteins, at least one Galectin-3 antigen can be inserted in place of the Furin site (RKRR) from 322R to 325R of SEQ ID NO: 6 or 7. For example, for Chikungunya virus structural proteins, at least one Galectin-3 antigen is inserted between residue H at position 321 and residue S at position 326 of SEQ ID NO: 6 or 7; between residue P at position 320 and residue S at position 326 of SEQ ID NO: 6 or 7; or between residue S at position 319 and residue S at position 326 of SEQ ID NO: 6 or 7. The VLP_CHI 0.56 vector can be used to prepare Chikungunya virus-like particles in which a Galectin-3 antigen is inserted between residues 321 and 326 of SEQ ID NO: 6 or 7. When a galectin-3 antigen is inserted between residues 321 and 326 of SEQ ID NO: 6 or 7, the virus-like particle provided by the present application can be a chikungunya virus-like particle consisting of an E2 and E3 complex, a capsid and E1, in which case at least one galectin-3 antigen is inserted into the E3 region.

[0052] For Venezuelan equine encephalitis virus structural proteins, at least one galectin-3 antigen can be inserted in place of the furin site (RKRR) from 331R to 334R of SEQ ID NO: 8. For example, for Venezuelan equine encephalitis virus structural proteins, at least one galectin-3 antigen is inserted between G at position 330 and S at position 335 of SEQ ID NO: 8; between P at position 329 and S at position 335 of SEQ ID NO: 8; or between C at position 328 and S at position 335 of SEQ ID NO: 8. The VLP_VEEV 0.66 vector can be used to prepare Venezuelan equine encephalitis virus-like particles in which an antigen is inserted between residues 330 and 335 of SEQ ID NO: 8. When an antigen is inserted between residues 330 and 335 of SEQ ID NO:8, the virus-like particle provided by the present application can be a Venezuelan Equine Encephalitis virus-like particle consisting of an E2 and E3 complex, a capsid and E1, in which case at least one galectin-3 antigen is inserted in the E3 region.

[0053] "Alteration" refers to a change in an amino acid or nucleotide at a specific position with respect to a polypeptide or polynucleotide sequence. As used herein, alteration includes substitution, deletion, or insertion of an amino acid or nucleotide at a specific position of a polypeptide or polynucleotide. In some embodiments, an alteration of an alphavirus capsid protein nuclear localization signal includes substitution of a charged amino acid (e.g., lysine or arginine) with an uncharged amino acid (e.g., alanine or asparagine, or any amino acid except basic charged amino acids such as lysine or arginine).

[0054] "Analog" refers to a molecule that is not identical but has similar functional or structural properties. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide, but has certain biochemical modifications that enhance the function of the analog compared to the naturally occurring polypeptide. Such biochemical modifications may, for example, increase the protease resistance, membrane permeability, or half-life of the analog without altering ligand binding. Analogs may also include unnatural amino acids.

[0055] In this disclosure, "comprises," "comprising," "containing," "having," and the like, may have the meaning ascribed to them in U.S. patent law and may mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise have the meaning ascribed to them in U.S. patent law, but the terms are open-ended, thus permitting the presence of more than what is described, but excluding prior art embodiments, so long as the basic or novel characteristics of what is described are not altered by the presence of more than what is described.

[0056] "Detecting" refers to identifying the presence, absence, or amount of the analyte to be detected.

[0057] By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0058] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

[0059] "Effective amount" refers to the amount of agent required to ameliorate the symptoms of a disease compared to untreated patients. The effective amount of active compound used to practice the methods disclosed herein for preventing or treating a disease varies according to the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

[0060] By "ameliorate" is meant to lessen, suppress, attenuate, alleviate, arrest or stabilize the occurrence or progression of a disease or its symptoms.

[0061] The present disclosure provides several targets that are useful for the development of highly specific drugs for treating or preventing the diseases described herein.In addition, the methods disclosed herein provide an easy means for identifying therapies that are safe to use in subjects.In addition, the methods of the present invention provide a route for analyzing virtually any number of compounds for their effects on the diseases described herein with high-volume throughput, high sensitivity and low complexity.

[0062] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0063] By "isolated polynucleotide" is meant a nucleic acid molecule (e.g., DNA) that is free of the genes that flank it in the naturally occurring genome of the organism from which it is derived. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector; that is incorporated into an autonomously replicating plasmid or virus; or that is incorporated into the genomic DNA of a prokaryote or eukaryote; or that exists as a molecule separated independently from other sequences (e.g., cDNA, or fragments of a genome or cDNA produced by PCR or restriction enzyme digestion). In addition, the term includes RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0064] By "isolated polypeptide" is meant a polypeptide that has been separated from components that naturally accompany it. Ordinarily, a polypeptide is isolated when it is at least 60% by weight free from proteins and naturally occurring organic molecules that naturally accompany it. Preferably, a preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0065] By "marker" is meant any protein or polynucleotide that has an alteration in expression levels or activity that is associated with a disease or disorder.

[0066] As used herein, "nuclear localization signal" or "NLS" refers to an amino acid sequence that, when present on the surface of a polypeptide, directs the polypeptide into the nucleus of a target cell. NLS sequences are known in the art. For example, see Goldfarb, D., and N. Michaud (1991) Trends Cell Biol. 1, 20-24; Gorlich, D., and IW Mattaj (1996) Science 271, 1513-1518). In one embodiment, the NLS comprises one or more short sequences of positively charged amino acids, such as lysine or arginine. Consensus sequences for NLS include KK / RXK / R (Schneider, J. et al. (1988) Cell 54, 117-125) and two clusters of basic amino acids separated by a spacer of about 10 amino acids, e.g., KR[PAATKKAGQA]KKKK (Dingwall et al., Cell Biol. 107 (3): 841-9). With respect to the alphavirus amino acid sequences disclosed herein, the NLS is present at amino acids 67-70 (KRKK) of the EEEV capsid protein; at amino acids 67-70 (KKKK) of the WEEV capsid protein; at amino acids 64-68 (KKPKK) of the VEEV capsid protein; at amino acids 62-69 (RRNRKNKK) of the CHIKV capsid protein; at amino acids 71-74 (RKKK) of the Ross River virus capsid protein; and at amino acids 64-68 (KKPKK) of the Barmah Forest virus capsid protein.

[0067] As used herein, "obtaining," as in "obtaining an agent," includes synthesizing, purchasing, or otherwise acquiring the agent.

[0068] By "reduce" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0069] "Reference" refers to a standard or control condition.

[0070] A "reference sequence" is a defined sequence used as a standard for sequence comparison. A reference sequence may be a subset or the entirety of a particular sequence; for example, it may be a segment of a full-length cDNA sequence or gene sequence, or it may be a complete cDNA sequence or gene sequence. For polypeptides, the length of a reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides, or any integer number therebetween or thereabout.

[0071] By "specifically binds" is meant a compound or antibody that recognizes and binds to a polypeptide of the invention, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, that naturally contains the polypeptide of the invention.

[0072] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule that encodes a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will usually exhibit substantial identity. A polynucleotide with "substantial identity" to an endogenous sequence can usually hybridize with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule that encodes a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will usually exhibit substantial identity. A polynucleotide with "substantial identity" to an endogenous sequence can usually hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to a pairing that forms a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or between parts of them under various stringency conditions.

[0073] For example, stringent salt concentrations will usually be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvents, such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions will usually include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent, such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. By combining these various conditions as necessary, various levels of stringency are achieved. In a preferred embodiment, hybridization will be performed in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In a more preferred embodiment, hybridization will be performed in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In a most preferred embodiment, hybridization will be performed in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations of these conditions will be readily apparent to one of skill in the art.

[0074] In most applications, the washing steps following hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, washing stringency can be increased by lowering salt concentration or by increasing temperature. For example, stringent salt concentrations for washing steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for washing steps will usually include a temperature of at least about 25°C, more preferably at least about 42°C, even more preferably at least about 68°C. In a preferred embodiment, washing steps will be performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washing steps will be performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will be performed at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.

[0075] Additional variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity with a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0076] Sequence identity is usually measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions usually include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary approach to determining the degree of identity can use the BLAST program, where closely related sequences are indicated by a probability score between e<”3” and e<”100”.

[0077] "Subject" means a mammal, including, but not limited to, a human or a non-human mammal, such as a cow, horse, dog, sheep, or cat.

[0078] Ranges provided herein are understood to be shorthand for all values ​​within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0079] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and / or its associated symptoms. Although not intended to prevent, it will be understood that treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0080] As used herein, unless specifically stated otherwise or clear from the context, the term "or" is understood to be inclusive.

[0081] Unless specifically stated otherwise or clear from the context, as used herein, the terms "a," "an," and "the" are understood to be in the singular or in the plural.

[0082] Unless specifically stated or clear from the context, the term "about" as used herein is understood to be within the range normally accepted in the art, for example, within 2 standard deviations of the mean value. About can be understood as within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, within 0.5%, within 0.1%, within 0.05%, or within 0.01% of the stated value. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term about.

[0083] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0084] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0085] Alphavirus Polynucleotides Overall, the disclosure includes any nucleic acid sequence encoding a VLP having structural protein modifications that enhance VLP expression in mammalian cells. In one embodiment, the alphavirus polypeptide comprises at least one alphavirus E2 protein or capsid protein NLS that includes a modification that enhances VLP expression in mammalian cells. In one embodiment, the alphavirus E2 protein has a non-lysine residue (e.g., asparagine) at an amino acid position corresponding to amino acid 234 of the CHIKV E2 protein, and / or a modification at an amino acid position corresponding to amino acid 251 of the CHIKV E2 protein that destabilizes the E2 protein during viral budding. In another embodiment, the alphavirus polypeptide comprises at least one alphavirus capsid protein having a non-lysine residue (e.g., alanine or asparagine) at an amino acid position corresponding to a lysine residue of the alphavirus capsid protein NLS, and / or having a non-arginine residue (e.g., alanine or asparagine) at an amino acid position corresponding to an arginine residue of the alphavirus capsid protein NLS. In certain embodiments, the alphavirus capsid protein is a WEEV CBA87 strain capsid protein having one or more of the following modifications: K67N, K68N, and / or K69N. In certain embodiments, the alphavirus capsid protein is a VEEV TC83 strain capsid protein having one or more of the following modifications: K64N, K65A, K65N, K67A, and / or K67N. In some embodiments, the alphavirus capsid protein is an EEEV PE-6 strain capsid protein having a K67N modification. In certain embodiments, the alphavirus capsid protein is a CHIKV (strain 37997) strain capsid protein having one or more of the following modifications: R62A, R63A, R65A, K66A, K68A, and / or K69A. In a specific embodiment, the alphavirus capsid protein is a Ross River Virus capsid protein having one or more of the following alterations: R71N, K72N, K73N, and / or K74N.In a specific embodiment, the alphavirus capsid protein is a Barmah Forest Virus capsid protein having one or more of the following modifications: K64A, K64N, K65A, K65N, K67A, K67N, K68A, and / or K68N. An isolated nucleic acid molecule can be manipulated by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector for which the 5' and 3' restriction sites are known or for which the polymerase chain reaction (PCR) primer sequences are disclosed is considered isolated, but a nucleic acid sequence that exists in its natural state in its natural host is not considered isolated. In certain exemplary embodiments, the vector comprises a nucleic acid segment of CHIKV 37997 or WEEV 71V-1658, or a fragment thereof. The vector can further comprise a CMV / R promoter. The vector can also comprise a capsid protein, or a fragment thereof.

[0086] In other exemplary embodiments, in addition to the E2 protein, the vector includes another envelope protein selected from the group consisting of E3, 6K, and E1. In certain examples, the vaccine can include capsid, E3, E2, 6K, and E1. In other examples, the vaccine can include E3, E2, 6K, and E1.

[0087] In certain embodiments, a nucleic acid molecule set forth in the sequences disclosed herein comprises a nucleotide sequence encoding a polypeptide having at least about 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more identity (e.g., when compared to the entire length of the amino acid sequence) to a polypeptide encoding a protein selected from alphavirus capsid, E3, E2, 6K and El, including capsid, E3, E2, 6K and El of CHIKV or VEEV.

[0088] In some embodiments of the present invention, the protein may contain mutations that produce silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded protein or the way in which the protein is made. Nucleotide variants may be created for a variety of reasons, such as to optimize codon expression for a particular host, see U.S. Patent Application Publication No. 2005 / 0118191, which is incorporated herein by reference in its entirety for all purposes.

[0089] In addition, the nucleotides can be sequenced to ensure that the correct coding region has been cloned and does not contain any unwanted mutations. The nucleotides can be subcloned into an expression vector (e.g., baculovirus) for expression in any cell. Those skilled in the art will appreciate that a variety of subcloning methods are available and possible.

[0090] Isolated nucleic acid can be, but need not be, substantially purified.For example, a nucleic acid that is isolated in a cloning vector or an expression vector is not pure in that it may only comprise a small percentage of the material in the cell in which it resides.Such a nucleic acid is isolated as the term is used herein because it can be easily manipulated by standard techniques known to those skilled in the art.

[0091] Expression of Polypeptides Generally, VLPs containing one or more alphavirus polypeptides of the present invention can be produced by transforming a suitable host cell with all or a portion of a nucleic acid molecule encoding the polypeptide, or a fragment thereof, in a suitable expression vehicle.

[0092] Those skilled in the art of molecular biology will appreciate that any of a wide variety of expression systems can be used to provide recombinant proteins. The precise host cell used is not critical to the invention. The polypeptides of the invention can be produced in prokaryotic hosts (e.g., E. coli) or eukaryotic hosts (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, COS cells). Such cells are available from a wide range of sources (e.g., American Type Culture Collection, Rockland, MD; see also, e.g., Ausubel et al., supra). Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells, e.g., Sf9, Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells. Examples of fungal (including yeast) host cells are S. cerevisiae, Kluyveromyces lactis (K. lactis), Candida species including C. albicans and C. glabrata, Aspergillus nidulans, Shizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica. Examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, HeLa cells, MDCK cells, Vero cells and Hep-2 cells. Xenopus laevis ovary cells or other cells of amphibian origin can also be used.Prokaryotic host cells include bacterial cells, such as E. coli, B. subtilis, and Mycobacterium.

[0093] Methods for cloning said proteins are known in the art. Genes encoding alphavirus proteins, such as structural proteins of certain CHIKV, WEEV, EEEV, VEEV, Ross River virus, or Barmah Forest virus, can be isolated by RT-PCR from polyadenylated mRNA extracted from cells infected with said viruses. The resulting product genes can be cloned into vectors as DNA inserts. The term "vector" refers to a vehicle by which nucleic acid can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, etc., which can replicate autonomously or integrate into the chromosome of a host cell. Vectors can also be naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same strand, poly-lysine conjugated DNA or RNA, peptide conjugated DNA or RNA, liposome conjugated DNA, etc., which do not replicate autonomously. In many, but not all, common embodiments, the vectors of the invention are plasmids or bacmids.

[0094] The present invention further provides nucleotides encoding proteins, including chimeric molecules, cloned into expression vectors capable of expression in cells resulting in the formation of VLPs. An "expression vector" is a vector, such as a plasmid, that is capable of promoting the expression as well as replication of a nucleic acid incorporated therein.

[0095] Typically, the nucleic acid molecule to be expressed is "operably linked" to a promoter and / or enhancer and is under the transcriptional regulatory control of the promoter and / or enhancer.

[0096] There are various expression systems for producing the polypeptide of the present invention.Expression vectors useful for producing such polypeptides include, but are not limited to, chromosomal, episomal and viral vectors, such as bacterial plasmid-derived, bacteriophage-derived, transposon-derived, yeast episome-derived, insertion element-derived, yeast chromosomal element-derived, baculovirus, papovavirus, e.g. SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus-derived vectors, and combinations thereof.

[0097] The constructs and / or vectors provided herein include alphavirus polynucleotides encoding structural polypeptides, including envelope or capsid proteins or portions thereof described herein. The vectors can be, for example, phage, plasmid, viral, or retroviral vectors. The constructs and / or vectors containing the nucleotides are required to be operably linked to a suitable promoter, such as, but not limited to, the CMV promoter, the phage lambda PL promoter, the E. coli lac, phoA, and tac promoters, the SV40 early and late promoters, and the promoters of retroviral LTRs. Other suitable promoters are known to those skilled in the art depending on the host cell and / or the desired expression rate. The expression constructs further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcript expressed by the construct preferably includes a translation initiation codon at the beginning of the polypeptide to be translated and a termination codon appropriately arranged at the end of the polypeptide to be translated.

[0098] It will be preferred that the expression vector contains at least one selection marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Preferred among the vectors are viral vectors such as baculovirus, poxvirus (e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., canine adenovirus), herpes virus and retrovirus. Other vectors that can be used with the present invention include vectors for use in bacteria, including pQE70, pQE60 and pQE-9, pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Among the preferred eukaryotic vectors are pFastBacl, pWINEO, pSV2CAT, pOG44, pXTl and pSG, pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors will be readily apparent to those skilled in the art.

[0099] Recombinant constructs can be prepared and used to transfect, infect or transform eukaryotic and / or prokaryotic cells and to express viral proteins, including those described herein. Thus, the present invention provides a host cell that contains a nucleic acid encoding an alphavirus structural gene, including capsid, E3, E2, 6K, and El or portions thereof, and / or any of the above-described chimeric molecules, and that comprises a vector (or vectors) that permits expression of the alphavirus structural gene, including capsid, E3, E2, 6K, and El or portions thereof, and / or any of the above-described chimeric molecules in said host cell under conditions that permit formation of a VLP.

[0100] In one embodiment, the vector is a recombinant baculovirus. In another embodiment, the recombinant baculovirus is transfected into an insect cell. In a preferred embodiment, the cell is an insect cell. In another embodiment, the insect cell is an Sf9 cell.

[0101] In another embodiment, the vector and / or host cell comprises nucleotides encoding alphavirus genes, including capsid, E3, E2, 6K, and E1, or portions thereof, as described.

[0102] In another embodiment, the vector and / or host cell consists essentially of the alphavirus capsid, E3, E2, 6K, and E1, or portions thereof, as described herein. In a further embodiment, the vector and / or host cell consists of the alphavirus proteins including the capsid, E3, E2, 6K, and E1, or portions thereof, as described herein. These vectors and / or host cells contain the alphavirus core, E3, E2, 6K, and E1, or portions thereof, as described herein, and may contain additional cellular components such as cellulVLProteins, baculovirus proteins, lipids, carbohydrates, etc.

[0103] One particular bacterial expression system for polypeptide production is the E. coli pET expression system (Novagen, Inc., Madison, Wis.). According to this expression system, the DNA encoding the polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of T7 regulatory signals, expression of the polypeptide is achieved by inducing expression of T7 RNA polymerase in the host cell. This is usually achieved using a host strain that expresses T7 RNA polymerase in response to IPTG induction. Once produced, the recombinant polypeptide is then isolated according to standard methods known in the art, for example, the methods described herein.

[0104] Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system uses a GST gene fusion system designed to express genes or gene fragments as fusion proteins at high levels, allowing rapid purification and recovery of the functional gene product. The protein of interest is fused to the carboxyl terminus of glutathione S-protein transferase from Schistosoma japonicum and is easily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. The fusion protein can be recovered by elution with glutathione under mild conditions. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of a recognition site for a site-specific protease upstream of this domain. For example, proteins expressed in pGEX-2T plasmids can be cleaved by thrombin; those expressed in pGEX-3X can be cleaved by factor Xa.

[0105] Alphavirus Polypeptides and Analogs The present invention provides VLPs comprising one or more alphavirus polypeptides. The present invention also includes VLPs comprising one or more alphavirus polypeptides or fragments thereof that have been modified to enhance or not inhibit their ability to modulate immune responses. In one embodiment, the present invention provides a method for optimizing the amino acid or nucleic acid sequence of an alphavirus by making modifications. Such modifications may include certain mutations, deletions, insertions, or post-translational modifications. The present invention further includes analogs of any naturally occurring polypeptide of the present invention. Analogs may differ from naturally occurring polypeptides of the present invention by differences in amino acid sequence, by post-translational modifications, or both. Analogs of the present invention will, overall, exhibit at least 85%, more preferably 90%, most preferably 95% or even 99% identity with all or a portion of a naturally occurring amino acid sequence of the present invention. The length of sequence comparison is at least 10, 13, 15 amino acid residues, preferably at least 25 amino acid residues, more preferably more than 35 amino acid residues.

[0106] Alterations of alphavirus polypeptides include, but are not limited to, site-specific, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil-containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, and the like. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like. Mutagenesis involving, for example, chimeric constructs is also included in the present invention. In one embodiment, mutagenesis can be guided by known information about naturally occurring molecules or altered or mutated naturally occurring molecules, such as sequences, sequence comparisons, physical properties, crystal structures, and the like.

[0107] In one embodiment, the present invention provides a polypeptide variant that differs from a reference polypeptide. The term "variant" refers to an amino acid sequence that is altered by one or more amino acids relative to a reference sequence. A variant can have "conservative" changes, where the substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. Alternatively, a variant can have "non-conservative" changes, e.g., replacement of glycine with tryptophan. Similar minor variations can also include amino acid deletions or insertions, or both. Guidance on determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, e.g., DNASTAR software. Desirably, the variant exhibits substantial biological activity. In one embodiment, the protein variant forms VLPs and elicits an antibody response when administered to a subject.

[0108] Natural variants can occur due to mutations in proteins. These mutations can lead to antigenic variations within individual groups of infectious agents. Thus, humans infected with a particular strain develop antibodies against that virus, and when a new virus strain appears, the antibodies against the older strain no longer recognize the new virus, and reinfection can occur. The present invention encompasses all antigenic and genetic variations of proteins from infectious agents to generate VLPs.

[0109] In addition to full-length polypeptides, the present invention also includes fragments of any one of the polypeptides of the present invention. As used herein, the term "fragment" means at least 5, 10, 13, or 15. In other embodiments, the fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments, at least 60-80 or more contiguous amino acids. Fragments of the present invention may be generated by methods known to those of skill in the art, or may result from normal protein processing (e.g., removal of amino acids from a nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing events or alternative protein processing events).

[0110] Non-protein analogs having chemical structures designed to mimic the functional activity of an alphavirus VLP or one or more alphavirus polypeptides can be administered in accordance with the methods of the invention. Alphavirus analogs may exceed the physiological activity of a native alphavirus.

[0111] Methods for analog design are well known in the art, and analogs can be synthesized according to such methods by modifying the chemical structure such that the resulting analog exhibits the immunomodulatory activity of a native alphavirus polypeptide, including, but not limited to, substituting alternative R groups and altering the degree of saturation at specific carbon atoms of the native alphavirus molecule.

[0112] Preferably, the analogs are relatively resistant to degradation in vivo and provide a longer lasting therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0113] Pharmaceutical Compositions and Administration The invention features pharmaceutical compositions comprising VLPs comprising viral structural proteins of an alphavirus and a galectin-3 antigen as described herein. Pharmaceutical compositions useful herein contain the VLPs of the invention and a pharmaceutical carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to the vertebrate receiving the composition and which can be administered without undue toxicity. As used herein, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in mammals, more particularly in humans. These compositions can be useful as vaccine and / or antigen compositions for inducing a protective immune response in vertebrates.

[0114] Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. A thorough discussion of pharma-ceutically acceptable carriers, diluents, and other excipients is published in Remington's Pharmaceutical Sciences (Mack Pub. Co. NJ, current edition). The formulation should be suitable for the mode of administration. In a preferred embodiment, the formulation is suitable for administration to humans and is preferably sterile, non-particulate, and / or non-pyrogenic.

[0115] If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.The composition can be in the form of a solid, such as lyophilized powder suitable for reconstitution, or in the form of a liquid, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.Oral preparations can contain standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0116] In certain embodiments, the VLP compositions are supplied in liquid form, eg, in a hermetically sealed container indicating the quantity and concentration of the VLP composition.

[0117] Preferably, the VLP composition in liquid form is supplied at at least about 50 μg / ml, more preferably at least about 100 μg / ml, at least about 200 μg / ml, at least 500 μg / ml, or at least 1 mg / ml of the sealed container.

[0118] Alternatively, the vaccine formulation is administered intranasally, either by instillation, large particle aerosol (greater than about 10 microns) or spray to the upper respiratory tract, or small particle aerosol (less than 10 microns) or spray to the lower respiratory tract.While any of the above delivery routes will result in an immune response, intranasal administration offers the added advantage of eliciting mucosal immunity at the site of entry for many viruses, including alphaviruses, e.g., CHIKV or VEEV.

[0119] Thus, the present invention also includes a method for formulating a vaccine or antigenic composition for inducing immunity against an infection in a mammal or at least one symptom thereof, comprising adding an effective dose of a VLP, such as an alphavirus (e.g., CHIKV or VEEV), to the formulation.

[0120] In certain cases, a single dose of immune stimulation is preferred, but additional dosages can be administered by the same or different route to achieve the desired effect. In neonates and young children, for example, multiple doses may be required to induce a sufficient level of immunity. Administration may continue at intervals throughout childhood as necessary to maintain a sufficient level or protection.

[0121] Similarly, adults who are particularly susceptible to repeated or severe infections, such as, for example, health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary or immune systems, may require multiple immunizations to establish and / or maintain a protective immune response. The level of induced immunity can be monitored, for example, by measuring the amount of neutralizing secretory and serum antibodies, and dosages can be adjusted or vaccinations repeated, as necessary, to induce and maintain the desired level of protection.

[0122] The dosage of the pharmaceutical preparation can be easily determined by those skilled in the art, for example, by first identifying the effective dose to induce a preventive or therapeutic immune response, for example, by measuring the serum titer of virus-specific immunoglobulin, or by measuring the inhibition rate of antibodies in serum samples or urine samples or mucosal secretions. The dosage can be determined from animal studies. A non-limiting list of animals used to study the effectiveness of vaccines includes guinea pigs, hamsters, ferrets, chinchillas, mice and cotton rats, as well as non-human primates. Although most animals are not natural hosts for infectious agents, they can still be useful in studying various aspects of disease. For example, any of the above animals can be dosed with a vaccine candidate, for example, the VLPs of the present invention, to partially characterize the induced immune response and / or determine whether any neutralizing antibodies are produced. For example, many studies have been performed in mouse models, since mice are small and their low cost allows researchers to carry out studies on a large scale.

[0123] In addition, human clinical studies can be carried out by those skilled in the art to determine the preferred effective dose for humans. Such clinical studies are routine and well known in the art. The exact dose to be used will also depend on the route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal test systems.

[0124] Also, as is well known in the art, the immunogenicity of a particular composition can be enhanced by the use of non-specific stimulators of immune response, known as adjuvants. As used herein, the term "adjuvant" refers to a compound that, when used in combination with a particular immunogen in a formulation, will enhance, alter or modify the resulting immune response. Adjuvants have been used empirically to promote a general boost in immunity to unknown antigens. Adjuvants have been used in immunization protocols to boost responses for many years, and thus are well known to those skilled in the art. Some adjuvants affect the manner in which antigens are presented. For example, immune responses are boosted when protein antigens are precipitated by alum. Emulsification of antigens also extends the period of antigen presentation. The inclusion of any adjuvants described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)," which is incorporated herein by reference in its entirety for all purposes, is contemplated within the scope of the present invention.

[0125] Exemplary adjuvants include complete Freund's adjuvant (a non-specific stimulator of immune response that contains killed Mycobacterium tuberculosis), incomplete Freund's adjuvant and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene / Tween-80 emulsion, is also contemplated. MF-59, Novasomes®, MHC antigens can also be used.

[0126] The VLPs of the invention can also be formulated with "immunostimulants". These are the body's own chemical messengers (cytokines) to enhance the immune system's response. Immunostimulants include, but are not limited to, various cytokines, lymphokines and chemokines with immunostimulatory, immune enhancing and proinflammatory activity, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors [e.g., granulocyte macrophage (GM) colony stimulating factor (CSF)]; and other immunostimulatory molecules such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc. Immunostimulatory molecules may be administered in the same formulation as the VLPs or separately. Either the protein or an expression vector encoding the protein can be administered to generate an immunostimulatory effect. Thus, in one embodiment, the invention includes antigen and vaccine formulations that include adjuvants and / or immunostimulants.

[0127] Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, multiple sclerosis and Huntington's disease, exhibit chronic abnormal inflammation.

[0128] According to the present disclosure, the composition was shown to reduce Gal-3 levels in the brain and improve cognitive function, which strongly suggests that it may be useful in treating not only neurodegenerative diseases, but also inflammatory diseases in general.

[0129] Examples of inflammatory diseases include those of various organs, such as the lungs, joints, eyes, intestines, skin, heart, kidneys, and central nervous system (CNS). Lung diseases include asthma, adult respiratory distress syndrome, bronchitis, and cystic fibrosis (which may additionally or alternatively involve the intestines or other tissues). Joint diseases include rheumatoid arthritis, rheumatoid spondylitis, arthritis, gouty arthritis, and other joint diseases. Inflammatory eye diseases include uveitis (including iritis) and conjunctivitis. Inflammatory bowel diseases include Crohn's disease, ulcerative colitis, and peripheral proctitis. Skin diseases include those involving cell proliferation, such as psoriasis, eczema, and dermatitis (whether allergic in origin or not). Cardiac diseases include coronary infarction injury. Kidney diseases include chronic kidney disease (CKD), such as glomerulonephritis (with or without nephrotic syndrome, including, for example, idiopathic nephrotic syndrome or minimal change nephropathy). Other inflammatory diseases and immune disorders include tissue necrosis in chronic inflammation, endotoxic shock, smooth muscle proliferation disorders (eg, restenosis following angioplasty), and tissue rejection following transplant surgery.

[0130] Inflammation in the CNS is part of the pathogenesis of neurodegenerative diseases. Neurodegenerative diseases include, but are not limited to, Parkinson's disease, amyotrophic lateral sclerosis (ALS, "Lou Gehrig's disease"), Huntington's disease, Alzheimer's disease, Creutzfeldt-Jakob disease, and general neurodegenerative diseases.

[0131] According to the present disclosure, the compositions or methods of the present invention are useful for the treatment of inflammatory diseases, particularly inflammatory diseases associated with aging, thereby enhancing biological functionality associated with systemic health and disease progression, promoting longevity and healthspan, and / or delaying or inhibiting the cellular aging process in a subject.

[0132] The present application will now be described in detail with reference to the following examples, which are not intended to limit the scope of the present application. [Example 1]

[0133] (1) Preparation of VLPs carrying Gal-3 antigen Galectin-3 (Gal-3) vaccine epitopes were designed by using the protein amino acid sequences and 3D structures of human and mouse Gal-3 proteins (Table 1). Linkers SGG (N-terminus) and GGS (C-terminus) were attached to the epitope peptide SEQ ID NO:5. For the generation of mammalian expression vector, pVEEV Gal-3 plasmid DNA vector, a synthetic dsDNA fragment of Gal-3 epitope peptide sequence (gBlocks, IDT) was inserted into a specific position (between G at position 330 and S at position 335) of DNA encoding VEEV E3 envelope protein in pVEEV vector to obtain VEEV-Gal-3 VLPs according to the procedure disclosed in WO2022 / 225057.

[0134] [Table 1]

[0135] FreeStyle 293F cells (Thermo Fisher Scientific, #R790-07) were cultured in suspension with FreeStyle 293 Expression medium (Thermo Fisher Scientific, #12338-018) at 37°C with 8% CO2 in a shaking incubator. The pVEEV Gal-3 VLP plasmid DNA expression vector was transfected into 293F cells with PEI (polyethyleneimine, Polysciences, #23966) at 0.8 μg / 10 DNA. 6 cells, DNA:PEI=1:3 (w / w), and 1.25 × 10 6 The cells were transfected at 1 mL of culture medium. Culture supernatants were harvested 4 days after transfection and clarified by centrifugation (3,000 rpm, 10 min) followed by filtration through a 0.45 μm PES membrane filter system (VWR #10040-470).

[0136] The VLP-containing culture supernatant was layered on top of 1.5 mL of OptiPrep (60% w / v) (Iodixanol, Accurate Chemical, #AN1114542) and centrifuged at 52,000 × g for 1.5 h in an SW-28 rotor (Beckmann). After removing the supernatant to leave 1.5 mL above the interface, the two layers were mixed to make a 50% OptiPrep solution and centrifuged at 360,000 × g for 2.5 h in an NVT100 rotor (Beckman) to form a density gradient.

[0137] Crude VLPs were collected and purified by size-exclusion chromatography using a Biologic Duo-Flow FPLC system (Bio-Rad) equipped with a HiPrep 16 / 60 Sephacryl S-500 HR column (GE, #28-9356-06) and phosphate-buffered saline (PBS). The VLP-containing fraction was concentrated with an Amicon Ultra-15 centrifugal filter unit (EMD Millipore, #UFC910024) and filtered through a 0.20 μm PES membrane.

[0138] Total protein concentration was measured by BCA Protein Assay (Pierce, #23225) according to the manufacturer's instructions. Purity of VLP samples was confirmed by SDS-PAGE analysis (Any kD Mini-PROTEAN TGX Precast Protein Gel, Bio-Rad, #456-9035) followed by Coomassie dye-based staining using QC Colloidal Coomassie Stain (Bio-Rad, #1610803). Sucrose and EDTA were added to the VLP solution to a final concentration of 250 mM and 5 mM, respectively. Protein concentration of VLP samples was adjusted to 0.4 mg / mL and stored at -80°C.

[0139] The molecular size and purity of VEEV VLPs containing Gal-3 epitope peptide were analyzed by SDS-PAGE under denaturing conditions. The obtained VLPs were consistent with the theoretical molecular weight and had a purity of more than 90%. A protein band related to the Gal-3 peptide fusion protein with VEEV viral structural proteins was confirmed.

[0140] (2) Detection of anti-mouse galectin-3 (Gal-3) antibodies in immunized mice 5xFAD mice were used as Alzheimer's disease model mice. The mice were divided into two groups. VEEV-Gal-3 VLPs were mixed with RIBI adjuvant and intramuscularly administered to one group of mice (group 2), and saline was intramuscularly administered to the other group (group 1). The concentration of VLPs was 0.2mg / ml, and 50 micrograms of VLPs were administered to each mouse. The mice were administered at the start of the experiment, 2 weeks and 4 weeks after the first administration. Six weeks after the start of the experiment, blood samples were obtained from each mouse, and serum was prepared. The produced anti-mouse Gal-3 antibodies were detected using ELISA in which Gal-3 protein was coated on an ELISA plate. The results showed that virus-like particles containing mouse Gal-3 derived polypeptides induced anti-mouse Gal-3 antibodies in group 2 mice (see Figure 1).

[0141] (3) Venezuelan equine encephalitis virus-like particles containing viral structural polypeptides prolonged the latency of a step-through passive avoidance test in 5×FAD mice.

[0142] Five months after the start of the experiment, a step-through passive avoidance test was performed. The passive avoidance test is commonly used as a method for testing avoidance memory retention. Passive avoidance responses were measured using a "step-through" apparatus. The apparatus is divided into two chambers. On the first day, the mouse was placed in the light chamber. When the mouse moved from the light area to the dark chamber, an electric shock was delivered for 3 seconds (0.3 mA). On the second day, the mouse was placed in the light chamber, and the time it took for the mouse to enter the dark chamber was recorded as the "retention time." The results showed that the latency was prolonged in the VLP-Gal-3 injected group, suggesting memory retention in the VLP-Gal-3 injected group (see FIG. 2).

[0143] (4) VEEV-Gal-3-VLP reduced Gal-3 in the hippocampus of 5×FAD mice. After the study, mouse brains were harvested in microtubes and homogenized with lysis buffer. The lysates were centrifuged for 10 min at 4°C, and the supernatants were separated on NUPAGE 12%, Bis-Tris gels and transferred to PVDF membranes. The membranes were blocked with Bullet Blocking One (nacalai tesque) and incubated overnight at 4°C with primary antibodies. The primary antibodies were anti-Gal-3 (1:1000, R&D) and anti-β-amyloid (1:1000, BioLegend). Following this incubation, the membranes were incubated with anti-rat secondary antibodies (1:3000, JacksonImmuno Research) or anti-mouse secondary antibodies (1:5000, JacksonImmuno Research). The blots were developed by chemiluminescence, and images were scanned using a chemiluminescence detector (LAS500, GE Healthcare). β-actin was used as a loading control. As a result, Gal-3 expression levels in the hippocampus were significantly decreased in the VLP-Gal-3 injected group (see FIG. 3).

[0144] (5) VEEV-Gal-3-VLP extended the lifespan of aged C57BL / 6J mice. C57BL / 6J mice were divided into two groups: VEEV-Gal-3 VLPs were administered intramuscularly to one group of mice (group B6-V) and saline to the other group (group B6-N).

[0145] VLPs were administered three times at the age of 101-102 weeks. After about 5 weeks, some mice in group B6-N gradually died. On the other hand, no mice in group B6-V died (see FIG. 4).

[0146] (6) VEEV-Gal-3-VLP extended the lifespan of 5×FAD mice. 5xFAD mice were divided into two groups: VEEV-Gal-3 VLPs were administered intramuscularly to one group of mice (group FV) and saline to the other group (group FN).

[0147] VLPs were administered four times at 28, 30, 32 and 42 weeks of age. Some mice in group FN gradually died after 50 weeks of age. On the other hand, none of the mice in group B6-V died until 54 weeks of age (see Figure 5).

Claims

1. Viral-like particles (VLPs) containing viral structural proteins and galectin-3 antigen for use in the treatment of inflammatory diseases.

2. The VLP according to claim 1, wherein the inflammatory disease is an age-related inflammatory disease.

3. The VLP according to claim 1, wherein the inflammatory disease is inflammation in the central nervous system (CNS).

4. The VLP according to claim 3, wherein the inflammation in the CNS is a neurodegenerative disease.

5. The method according to claim 4, wherein the neurodegenerative disease is Parkinson's disease, amyotrophic lateral sclerosis (ALS, "Lou Gehrig's disease"), Huntington's disease, Alzheimer's disease, Creutzfeldt-Jakob disease, or a general neurodegenerative disease.

6. The VLP according to any one of claims 1 to 5, wherein the viral structural protein is derived from an alphavirus or a flavivirus.

7. The VLP according to claim 6, wherein the viral structural protein is derived from chikungunya virus or Venezuelan encephalitis virus.

8. The VLP according to claim 7, wherein the viral structural protein is derived from Chikungunya virus strain 37997 or strain OPY-1, or Venezuelan encephalitis virus strain TC-83.

9. The VLP according to claim 7, wherein at least one galectin-3 antigen is inserted into the envelope protein E3 of the viral structural protein.

10. The VLP according to claim 7, wherein the galectin-3 antigen is selected from the peptides of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:

5.

11. The VLP according to claim 7, wherein the galectin-3 antigen is the peptide of SEQ ID NO:

12.

12. The VLP according to claim 7, wherein at least one galectin-3 antigen is inserted between residues corresponding to positions 321 and 326 of SEQ ID NO: 6, positions 321 and 326 of SEQ ID NO: 7, or positions 330 and 335 of SEQ ID NO:

8.

13. A pharmaceutical or vaccine composition for the treatment of inflammatory diseases in mammals, comprising an effective amount of virus-like particles containing viral structural proteins and galectin-3 antigen.

14. Use of virus-like particles containing viral structural proteins and galectin-3 antigens for the manufacture of pharmaceuticals for the treatment of inflammatory diseases in mammals.

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