Treatment of inflammatory conditions

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to the treatment of inflammatory conditions, particularly age-related inflammatory conditions, in a subject. Background Art

[0002] Aging is a major risk factor for most neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. The most common types of neurodegenerative diseases occur primarily in older individuals. In fact, it is estimated that 1 in 10 individuals over the age of 65 will suffer from a neurodegenerative condition, and this likelihood increases exponentially with age.

[0003] Neurodegenerative diseases of the central nervous system (CNS) are defined as the progressive and irreversible loss of neuronal cells and are associated with behavioral impairments, including loss of motor and / or cognitive functions. Given the strong link between neurodegenerative diseases and aging, neurodegeneration is often considered part of the aging process of the brain.

[0004] There are few or no effective treatments available for neurodegenerative conditions, and most focus on alleviating symptoms rather than targeting the root cause of the disease. Therefore, directly addressing the brain's aging process to slow its progression may provide a better strategy to reduce the onset and burden of neurodegenerative diseases.

[0005] Neurodegenerative diseases include a range of severely debilitating conditions, including Parkinson's disease, amyotrophic lateral sclerosis (ALS, "Lou Gehrig's disease"), Huntington's disease, Alzheimer's disease, etc. These conditions are characterized by a gradual but relentless deterioration 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 deficits. Currently available therapies for Alzheimer's disease (AD) show limited efficacy, and there is still no real cure.

[0007] Parkinson's disease (PD) is characterized by progressive degeneration of dopamine (DA) neurons projecting 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 rigidity, resting tremor, postural instability, and bradykinesia. Currently available therapies for Parkinson's disease (PD) provide relief of motor symptoms, but fall short of demonstrating the neuroprotective effects required to prevent progressive degeneration of dopamine (DA) neurons. In addition, no therapy simultaneously targets multiple debilitating non-motor symptoms of PD.

[0008] Neurodegenerative diseases show chronic abnormal inflammation.

[0009] Galectins are members of the lectin family that show high affinity for β-galactosides. About 15 galectins encoded by the LGALS gene have been found in mammals and are numbered in a sequential manner. 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 the highly conserved animal lectin family that binds to glycoconjugates (glycoproteins or glycolipids) containing β-galactoside (Non-Patent Document 1: Henderson NC 2006, Non-Patent Document 2: Mourad-Zeidan AA 2008). Gal-3 is different from other galectin family proteins in that its structure consists of two domains: a carboxyl terminal domain containing a carbohydrate binding region and an amino terminal domain mainly composed of tandem repeats of 9 amino acids for cross-linking carbohydrate and non-carbohydrate ligands (Non-Patent Document 3: Barondes SH 1994). It is secreted by various types of cells (including monocytes, macrophages, and epithelial cells), but is mainly secreted by macrophages (Non-Patent Document 4: Reynolds 2005, Non-Patent Document 5: MacKinnon AC 2008). Secreted extracellular Gal-3 forms homodimers or pentamers, and is important for biological functions. The N-terminal domain of galectin-3 has been shown to be important for the formation of protein oligomers (non-patent literature 6: Dumic J 2006, non-patent literature 7: Kuklinski S1998, non-patent literature 8: Lepur A2012). The released protein can act as an extracellular molecule to activate cells, mediate cell-cell and cell-ECM interactions, induce the migration of various types of cells, and negatively regulate T cell receptor signaling (non-patent literature 9: Yang RY 1996). Gal-3 is shown to increase in various fibrotic disease models and patients (including pulmonary fibrosis, liver fibrosis, systemic sclerosis and cardiac fibrosis) (non-patent literature 10: Henderson NC 2008, non-patent literature 11: Barman et al. 2019, non-patent literature 12: Nishi Y 2007, non-patent literature 13: De Boer 2010). This suggests that Gal-3 may be an important mediator and effective therapeutic target for tissue fibrosis. To date, preclinical and clinical studies of investigational galectin-3 inhibitors have shown protection against fibrotic conditions (Non-Patent Document 14: Nikhil Hirani 2017, Non-Patent Document 15: Yu L 2013).

[0011] It is reported that plasma galectin-3 levels will become important as biomarkers and therapeutic agents in neuroprotective and neurorepair treatments for neurodegenerative diseases such as idiopathic Parkinson's disease (IPD) (Non-patent document 16: Yazar HO et al., J Clinical Neuroscience 2019). Galectin-3 is highly upregulated in the brains of AD patients and 5×FAD (familial Alzheimer's disease) mice and is specifically expressed in microglia associated with Aβ plaques (Non-patent document 17: Boza-Serrano A et al. Acta Neuropathology, 2019). Compared with controls, galectin-3 is higher in serum and CSF samples of AD and ALS patients, potentially indicating a clear association between serum and CSF levels of Gal-3 and cognitive status in AD and ALS patients and healthy individuals (controls) (Non-patent document 18: Ashraf GM et al. Front Neuroscience, 2018).

[0012] During the past 100-150 years, one of the major success stories in medicine is vaccines targeting various infectious diseases. Vaccines, together with antibiotics, are probably more important to human health than any other part of the human race. Due to the success of vaccines, there is an increasing interest in using vaccine technology to treat non-infectious diseases such as allergies, autoimmunity, and cancer. However, the targets for these diseases are usually self-antigens, which can cause efficacy problems. Due to tolerance mechanisms, it is quite difficult to induce a strong antibody response to self-antigens compared to non-self-antigens-bacteria, viruses, or parasite proteins (non-patent literature 19: Hellman 2008, non-patent literature 20: Falk Saupe 2015).

[0013] Alphaviruses comprise a group of genetically, structurally, and serologically related mosquito-borne viruses of 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, Whataroa Virus, Babanki Virus, and Virus, Kyzylagach Virus, Highlands J virus, Fort Morgan Virus, Ndumu Virus, and Buggy Creek Virus. The structural subunit capsid containing a single viral protein is associated with the RNA genome in an icosahedral nucleocapsid. In the virion, the capsid is surrounded by a lipid envelope 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 (VLP) are polyprotein structures that imitate the organization and conformation of real natural viruses but lack viral genomes, which may produce safer and cheaper candidate vaccines. At present, several preventive vaccines based on VLP are commercialized worldwide: EngerixR (hepatitis B virus) and CervarixR (human papillomavirus) of GlaxoSmithKline and Recombivax HBR (hepatitis B virus) and GardasilR (human papillomavirus) of Merck and Co., Inc. are some examples. Other candidate vaccines based on VLP are in clinical trials or are experiencing preclinical evaluations, such as influenza virus, parvovirus, Norwalk (Norwalk) and various chimeric VLPs. Many others are still limited to small-scale basic research, although they have been successful in preclinical testing. The impact of large-scale VLP production has been discussed under the background of process control, monitoring and optimization. Correspondingly identified and discussed major upstream and downstream technical challenges. Successful VLP-based vaccine blockbusters are briefly presented along with the latest results from clinical trials and the latest developments in chimeric VLP-based technologies for therapeutic or prophylactic vaccination.

[0015] To date, VLP-based vaccines have been produced for more than 30 different viruses that infect humans and other animals. Examples include AAV (adeno-associated virus), H5N3 (avian influenza), BFDV (Budgerigarfledgling disease virus), BTV (Bluetongue virus), Ebola, Enterovirus 71, GHPV (Goose hemorrhagic polyoma virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis delta virus), HEV (hepatitis E virus), HIV, HPV (human papillomavirus), IBDV (Infectious bursal disease virus), influenza A, influenza A H1N1, influenza A H3N2, JC polyomavirus, Marburg, MS2, IPCV (Indian peanut clump virus), virus), NDV (Newcastle disease virus), No (Norovirus), Nv (Norwalk virus), PhMV (Physalis 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 1: Expert Rev. Vaccines 9 (10), 1149-1176, 2010).

[0016] U.S. Patent No. 9,353,353 discloses virus-like particles (VLPs) comprising one or more Chikungunya virus structural proteins, which can be used to formulate vaccines or antigenic compositions of Chikungunya that induce immunity to infection or at least one symptom thereof. U.S. Patent No. 9,487,563 discloses modified alphavirus or flavivirus virus-like particles (VLPs) and methods for enhancing the production of modified VLPs for preventing or treating alphavirus and flavivirus-mediated diseases. U.S. Patent No. 9,249,191 discloses Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) virus-like particles, wherein 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 alphavirus virus-like particles, wherein the virus-like particles contain alphavirus structural proteins containing envelope protein E3, wherein the envelope protein E3 is modified to contain at least one foreign antigen inserted into its furin protease cleavage site (these cited references are incorporated herein by reference). Reference List

[0017] Patent Literature [PTL 1] U.S. Patent No. 9,353,353 [PTL 2] U.S. Patent No. 9,487,563 [PTL 3] U.S. Patent No. 9,249,191 [PTL 4] U.S. Patent No. 9,969,986 [PTL 5] U.S. Patent Publication No. 2005 / 0118191 [PTL 6] WO 2022 / 225057

[0018] [Non-patent literature] [NPL 1] Henderson NC, Mackinnon AC, Farnworth SL, Poirier F, Russo FP, Iredale JP, Haslett C, Simpson KJ, Sethi T. 2006. "Galectin-3regulatesmyofibroblast activation and hepatic fibrosis." Proc Natl Acad Sci USA 103(13):5060-5. [NPL 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. [NPL 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. [NPL 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 [NPL 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. [NPL 6]Dumic J,Dabelic S,Flougel M. 2006. "Galectin-3:An open-ended story." BBA-Gen Subjects 1760:616-635. [NPL 7]Kuklinski S, Probstmeier R. 1998. "Homophilic binding properties of galectin-3: Involvement of the carbohydrate recognition domain." J Neurochem 70:814-823. [NPL 8]Lepur A, Salomonsson E, Nilsson UJ, Leffler H. 2012. "Ligand-induced galectin-3 protein self-association." J Biol Chem 287:21751-21756. [NPL 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. [NPL 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):288-98. [NPL 11]Barman, Scott A, Xueyi Li, Stephen Haigh, Dmitry Kondrikov, Keyvan Mahboubi, Zsuzsanna Bordan and Stepp W David. 2019. "Galectin-3 is Expressed in Vascular Smooth Muscle Cells and Promotes Pulmonary Hypertension through changes in Proliferation, Apoptosis and Fibrosis." Epub ahead of print. [NPL 12]Nishi Y,Sano H,Kawashima T,Okada T,Kuroda T,Kikkawa K,Kawashima S,Tanabe M,Goto T,Matsuzawa Y,Matsumura R,Tomioka H,Liu FT,ShiraiK.2007."Role of Galectin-3in Human Pulmonary Fibrosis"Allergol Int.56(1):57-65. [NPL 13]De Boer,R.A.,Yu,L.和van Veldhuisen,D.J.2010."Galectin-3incardiac remodeling and heart failure."Curr.Heart Fail.Rep.7,1-8. [NPL 14]Nikhil Hirani,Alison Mackinnon,Lisa Nicol,Jeremy Walker,PaulFord,Hans Schambye,Anders Pederson,Ulf Nilsson,Hakon Leffler,Tracy Thomas,Danielle Francombe,John Simpson,Michael Gibbons,Toby M.Maher.2017."TD139,ANovel Inhaled Galectin-3 Inhibitor for the Treatment of Idiopathic PulmonaryFibrosis(IPF).Results from the First in(IPF)Patients Study."American Journalof Respiratory and Critical Care Medicine 195:A7560. [NPL 15] Yu L, Ruifrok WP, Meissner M, Bos EM, van Goor H, Sanjabi B, vander Harst P, Pitt B, Goldstein IJ, Koerts JA, et al. 2013. "Genetic and pharmacological inhibition of galectin-3 prevents cardiac remodeling by interfering with myocardial fibrogenesis." Circ Heart Fail 6:107-117. [NPL 16] H.O. Yazar et al. "A preliminary data: Evaluation of serum Galectin-3 levels in patients with Idiopathic Parkinson’s Disease" Journal of Clinical Neuroscience 70(2019)164-168 [NPL 17] Boza-Serrano A et al. "Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates infammatory response in Alzheimer’s disease" Acta Neuropathologica(2019)138:251-273 [NPL 18] Ghulam Md Ashraf and Saleh S Baeesa "Investigation of Gal-3 Expression Pattern in Serum and Cerebrospinal Fluid of Patients Suffering From Neurodegenerative Disorders" Frontiers in Neuroscience 2018 Vol. 12, article 430 [NPL 19] Hellman, L. 2008. "Therapeutic vaccines against IgE-mediated allergies." Expert Rev Vaccines 7(2):193-208. [NPL 20]Falk Saupe, Elisabeth J.M. Huijbers, Tobias Hein, Julia Femel, Jessica Cedervall, Anna-Karin Olsson and Lars Hellman. 2015. "Vaccines targeting self-antigens: mechanisms and efficacy-determining parameters." FASEB J 29(8):3253-62. [NPL 21]Anto'nio [NPL 22]Maria Candida M, Mellado, Leda R, Castilho, Manuel J.T. Carrondo and Paula M. Alves "Virus-like particles in vaccine development" Expert Rev. Vaccines 9(10), 1149-1176, 2010 [NPL 23]Goldfarb, D. and N. Michaud (1991) "Pathways for the nuclear transport of proteins and RNAs" Trends Cell Biol. 1, 20-24 [NPL 24]Gorlich, D. and I.W. Mattaj (1996) "Nucleocytoplasmic Transport" Science 271, 1513-1518 [NPL 25]Schneider, J. et al. (1988) "A Mutant SV40 Large T Antigen Interferes with Nuclear Localization of a Heterologous Protein" Cell 54, 117-125 [NPL 26]Dingwall et al. (1988) "The Nucleoplasmin Nuclear Location Sequence Is Larger and More Complex than That of SV-40 Large T Antigen" J. Cell Biol. 107(3):841-9 Summary of the Invention

[0019] Note: There seems to be a mistake in the original text where [NPL 25] in line is misspelled as [NPL 26] in the translation. It should be corrected to [NPL 25] in the translation for accuracy.The present disclosure relates to the treatment of inflammatory conditions. In particular, the present disclosure provides a method for treating inflammatory conditions, especially age-related inflammatory conditions, in a mammalian subject, comprising administering to a subject in need thereof an effective amount of a virus-like particle comprising a viral structural protein and a galectin-3 antigen.

[0020] The present disclosure also provides a virus-like particle comprising a viral structural protein and a galectin-3 antigen for use in treating inflammatory conditions, particularly age-related inflammatory conditions, in a mammalian subject.

[0021] The present disclosure also provides a pharmaceutical composition for treating inflammatory conditions, especially age-related inflammatory conditions, in a mammalian subject, comprising a virus-like particle containing a viral structural protein and a galectin-3 antigen.

[0022] The present disclosure also provides the use of a virus-like particle comprising a viral structural protein and a galectin-3 antigen for the manufacture of a medicament for treating an inflammatory condition, particularly an age-related inflammatory condition, in a mammalian subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] [ 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 physiological saline was administered intramuscularly to another group (Group 1). The mice were administered at the beginning of the experiment, 2 weeks after the first administration, 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.

[0024] [ Figure 2 ] Results of the shuttle passive avoidance test in 5×FAD mice. Five months after the start of the experiment, the shuttle passive avoidance test was performed and the latency after electrical stimulation was measured 24 h.

[0025] [ Figure 3 ]The amount of Gal-3 in the hippocampus of 5×FAD mice immunized with VEEV-Gal-3-VLP. Figure 2 Following the indicated tests, mouse brains were collected and the amounts of Gal-3 and β-amyloid compared to β-actin in brain lysates were measured.

[0026] [ Figure 4 Effect of VEEV-Gal-3-VLP on the 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 (B6-V group) and normal saline was intramuscularly administered to the other group (B6-N group).

[0027] [ Figure 5 Effect of VEEV-Gal-3-VLP on the survival of 5×FAD mice. 5×FAD mice were divided into two groups. VEEV-Gal-3 VLP was intramuscularly administered to one group of mice (FV group) and physiological saline was intramuscularly administered to the other group (FN group). DETAILED DESCRIPTION

[0028] As used herein, the term "Galectin-3 antigen" refers to any antigenic structure derived from a Galectin-3 protein that can be recognized by the immune system and / or stimulate a cell-mediated immune response and / or stimulate the production of antibodies specific for the antigen. The Galectin-3 epitope peptide may be a fragment of a naturally occurring Galectin-3 protein, or a fragment of a naturally occurring Galectin protein with some modifications. The naturally occurring Galectin-3 protein may preferably be a human Galectin protein, and in particular 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 the naturally occurring Galectin-3 protein. In one embodiment, the modified peptide fragment is a mutant in which up to 10% of the amino acids are deleted, substituted and / or added based on the fragment of the naturally occurring Galectin-3 protein.

[0029] Galectin-3 [Homo sapiens] (GenBank accession number: AAB86584.1)

[0030] Examples of Galectin-3 antigens, i.e., Galectin-3 epitope peptides may include the following:

[0031] Gal-3 epitope peptide SEQ ID NO: 2 consists of 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 and SEQ ID NO: 4 has a longer repeat sequence than SEQ ID NO: 3. SEQ ID NO: 5 encodes mouse Gal-3 N-terminal peptide.

[0032] In WO 2022 / 225057 (the content of this document is incorporated herein by reference), VLPs carrying the above Gal-3 epitope peptides were prepared and the immunogenicity of these VLPs was confirmed.

[0033] Alphavirus As used herein, "alphavirus" means 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, Mukambu Virus, Pixona Virus, Western Equine Encephalitis Virus (WEEV), Sindbis Virus, Semliki Forest Virus, Middelburg Virus, Chikungunya Virus (CHIKV), Anyang-Nyang Virus, Ross River Virus, Bama Forest Virus, Geta Virus, Lushan Virus, Bebaru Virus, Mayaro Virus, Una Virus, Ora Virus, Vodaro River Virus, Babanken Virus, Kuzilagachi Virus, Highland J Virus, Fort Morgan Virus, Ndumau Virus, Bogi River Virus, Ockelbo Virus.

[0034] "Viral structural protein" means a polypeptide or fragment thereof that has at least about 80% amino acid sequence identity with a naturally occurring viral capsid or envelope protein and has immunogenic activity in a mammal. In one embodiment, the alphavirus structural protein has at least about 85%, 90%, 95% or greater amino acid sequence identity with the alphavirus discussed above.

[0035] The viral structural protein and the galectin-3 antigen may be fused directly or indirectly. In one embodiment, one or two linkers may be 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.

[0036] Antigen or viral structural protein can be truncated and replaced by short linker.In some embodiments, antigen or viral structural protein comprises one or more peptide linkers.Usually, linker is made up of 2-25 amino acid (for example 2,3,4,5 or 6 amino acid).Usually, its length is 2-15 amino acid, although in some cases, it can be only one, such as single glycine residue.

[0037] 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)) so that the first attachment site and the second attachment site are connected by a peptide bond. In this case, the viral structural protein and the antigen are connected by a peptide bond. For this embodiment, the first attachment site and / or the second attachment site can be genetically modified from the initial protein or antigen. For example, the first attachment site is modified from the viral structural protein so 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 attachment site and the second attachment site can be connected by a chemical cross-linking agent as a compound. Examples of cross-linking agents include, but are not limited to, SMPH, sulfo-MBS, sulfo-EMCS, sulfo-GMBS, sulfo-SIAB, sulfo-SMPB, sulfo-SMCC, SVSB, SIA, and other cross-linking agents available from Pierce Chemical Company.

[0038] Preferably, the antigen may be linked to a Chikungunya virus structural protein or a Venezuelan equine encephalitis virus structural protein as a fusion protein produced by genetic engineering.

[0039] The Chikungunya virus structural protein or Venezuelan equine encephalitis virus structural protein used in the present application may be a Chikungunya virus or Venezuelan equine encephalitis virus envelope protein or capsid or a complex of one or more envelope proteins and / or capsid proteins.

[0040] Examples of Chikungunya viruses include, but are not limited to, strain 37997 and strain LR2006 OPY-1.

[0041] Examples of Venezuelan equine encephalitis viruses include, but are not limited to, strain TC-83.

[0042] The chikungunya virus structural protein or Venezuelan equine encephalitis virus structural protein used in the present 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 up to 10% of amino acids are deleted, substituted and / or added based on a naturally occurring viral capsid and / or envelope protein. For example, a K64A or K64N mutation may be introduced into the capsid of the Venezuelan equine encephalitis virus structural protein used in the present application.

[0043] The Chikungunya virus structural protein or the Venezuelan equine encephalitis virus structural protein may consist of or include capsid, E3, E2 and E1 proteins. The E3 and E2 proteins may be expressed together so that E2 and E3 may form one protein.

[0044] 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.

[0045] 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.

[0046] An exemplary Chikungunya virus structural protein sequence is provided as Genbank Accession No. ABX40006.1, which is described below (SEQ ID NO: 6): [Chemical formula 1]

[0047] Another exemplary Chikungunya virus structural protein sequence is provided as Genbank Accession No. ABX40011.1, which is described below (SEQ ID NO:7): [Chemical formula 2]

[0048] An exemplary Venezuelan equine encephalitis virus structural protein is described below (SEQ ID NO: 8): [Chemical formula 3] In one embodiment, the first attachment site comprises an amino group, preferably an amino group of a lysine residue. In one embodiment, the second attachment site comprises a sulfhydryl group, preferably a sulfhydryl group of a cysteine ​​residue.

[0049] According to the present application, the Chikungunya virus-like particle or the Venezuelan equine encephalitis virus-like particle comprises a Chikungunya virus structural protein or a Venezuelan equine encephalitis virus structural protein and at least one galectin-3 antigen, wherein at least one galectin-3 antigen is inserted into E3 of the virus structural protein, and the Chikungunya virus structural protein or the Venezuelan equine encephalitis virus structural protein and the galectin-3 antigen are expressed as a fusion protein that can be provided. The galectin-3 antigen can be directly or indirectly inserted into E3 of the virus structural protein.

[0050] The viral structural proteins of Chikungunya virus and Venezuelan equine encephalitis are composed of E1, E2, 6K and E3. 6K is naturally cut and removed from VLP during the assembly process. Mature VLP consists of capsid, E1 and E2. In this specification and claims, "viral structural proteins" refer not only to those with 6K, but also to those after 6K is removed.

[0051] The 6K sequences of CHIKV and VEEV used in the working examples are as follows: CHIKV strain OPY-1, 6K: 749-809aa of SEQ ID NO: 6 atyqeaaiylwneqqplfwlqaliplaalivlcnclrlllpcccktlaflavmsvgahtvsa(SEQ IDNO:6) CHIKV strain 37997, 6K: 749-809aa of SEQ ID NO: 7 atyyeaaaylwneqqplfwlqaliplaalivlcnclkllpcccktlaflavmsigahtvsa(SEQ IDNO:7) VEEV strain TC-83, 6K: 758-813aa of SEQ ID NO: 8 ettwesldhlwnnnqqmfwiqlliplaalivvtrllrcvccvvpflvmagaagaga (SEQ ID NO:8)

[0052] For the Chikungunya virus structural protein, at least one galectin-3 antigen can be inserted to replace the furin protease site (RKRR) from 322R to 325R of SEQ ID NO: 6 or 7. For example, for the Chikungunya virus structural protein, at least one galectin-3 antigen is inserted between residues H at position 321 and S at position 326 of SEQ ID NO: 6 or 7; between P at position 320 and S at position 326 of SEQ ID NO: 6 or 7; or between S at position 319 and 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, wherein the galectin-3 antigen is inserted between residues 321 and 326 of SEQ ID NO: 6 or 7. When the 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 may be a Chikungunya virus-like particle, which is composed of a complex of E2 and E3, a capsid and E1, and wherein at least one galectin-3 antigen is inserted into the E3 region.

[0053] Venezuelan equine encephalitis virus structural protein, at least one galectin-3 antigen can be inserted to replace the furin protease site (RKRR) from 331R to 334R of SEQ ID NO: 8. For example, for Venezuelan equine encephalitis virus structural protein, at least one galectin-3 antigen is inserted between 330 G and 335 S of SEQ ID NO: 8; between 329 P and 335 S of SEQ ID NO: 8; or between 328 C and 335 S of SEQ ID NO: 8. The VLP_VEEV 0.66 vector can be used to prepare Venezuelan equine encephalitis virus-like particles, wherein the antigen is inserted between residues 330 and 335 of SEQ ID NO: 8. When the antigen is inserted between residues 330 and 335 of SEQ ID NO: 8, the virus-like particles provided by the present application can be Venezuelan equine encephalitis virus-like particles, which are composed of a complex of E2 and E3, a capsid and E1, and wherein at least one galectin-3 antigen is inserted into the E3 region.

[0054] In terms of polypeptide sequence or polynucleotide sequence, "change" means the change of amino acid or nucleotide at the specified position. As used herein, the change includes the replacement, deletion or insertion of amino acid or nucleotide at the specified position of polypeptide or polynucleotide. In some embodiments, the change of alphavirus capsid protein nuclear localization signal includes replacing charged amino acid (e.g., lysine or arginine) with uncharged amino acid (e.g., alanine or asparagine, or any amino acid except basic charged amino acid such as lysine or arginine).

[0055] "Analog" means a molecule that is not identical but has similar functional or structural features. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide while having certain biochemical modifications relative to the naturally occurring polypeptide that enhance the function of the analog. Such biochemical modifications may increase the protease resistance, membrane permeability, or half-life of the analog without altering, for example, ligand binding. Analogs may include non-natural amino acids.

[0056] In the present disclosure, “comprises,” “comprising,” “containing,” and “having,” etc. may have the meanings ascribed to them in U.S. patent law, and may mean “includes,” “including,” etc.; “consisting essentially of” or “consists essentially of” also have the meanings ascribed to them in U.S. patent law, and the term is open-ended, allowing for more than what is listed, as long as the basic or new features listed are not varied beyond what is listed, but excluding prior art embodiments.

[0057] "Detecting" means identifying the presence, absence or amount of an analyte to be detected.

[0058] "Disease" means any condition or disorder that damages or interferes with the normal function of cells, tissues, or organs.

[0059] "Agent" means any small molecule compound, antibody, nucleic acid molecule or polypeptide or fragment thereof.

[0060] "Effective amount" means the amount of a medicament required to improve the symptoms of a disease relative to an untreated patient. The effective amount of an active compound used to practice the methods disclosed herein for preventing or treating a disease varies depending on 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.

[0061] "Ameliorate" means to reduce, inhibit, attenuate, diminish, arrest or stabilize the development or progression of a disease or a symptom thereof.

[0062] The present disclosure provides a number of targets that can be used to develop drugs that are highly specific for treating or preventing the diseases described herein. In addition, the methods disclosed herein provide an easy way to identify therapies that are safe for use in subjects. In addition, these methods provide a way to analyze the effects of almost any number of compounds on the diseases described herein with high throughput, high sensitivity and low complexity.

[0063] 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 entire length of a reference nucleic acid molecule or polypeptide. A fragment may 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.

[0064] "Isolated polynucleotide" means a nucleic acid molecule (e.g., DNA) that is free of genes that flank the gene in the naturally occurring genome of the organism from which the nucleic acid molecule is derived. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector; incorporated into an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryotic or eukaryotic organism; or exists as a separate molecule (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene encoding another polypeptide sequence.

[0065] "Isolated polypeptide" means a polypeptide that has been separated from components that naturally accompany it. Generally, a polypeptide is isolated when it is at least 60% by weight free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the polypeptide of the present 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.

[0066] "Marker" means any protein or polynucleotide having an altered expression level or activity that is associated with a disease or disorder.

[0067] As used herein, "nuclear localization signal" or "NLS" is an amino acid sequence that targets a polypeptide to the nucleus when present on the surface of a polypeptide. NLS sequences are known in the art. See, for example, Non-Patent Literature 22: Goldfarb, D. and N. Michaud (1991) Trends Cell Biol. 1, 20-24; Non-Patent Literature 23: Gorlich, D. and IW Mattaj (1996) Science 271, 1513-1518). In one embodiment, the NLS includes a short sequence of one or more positively charged amino acids (such as lysine or arginine). The consensus sequence of NLS includes KK / RXK / R (Non-patent document 24: Schneider, J. et al. (1988) Cell 54, 117-125) and two basic amino acid clusters separated by a spacer of about 10 amino acids, for example KR[PAATKKAGQA]KKKK (Non-patent document 25: 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 of the EEEV capsid protein (KRKK); amino acids 67-70 of the WEEV capsid protein (KKKK); amino acids 64-68 of the VEEV capsid protein (KKPKK); amino acids 62-69 of the CHIKV capsid protein (RRNRKNKK); amino acids 71-74 of the Ross River virus capsid protein (RKKK); and amino acids 64-68 of the Bama Forest virus capsid protein (KKPKK).

[0068] As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing or otherwise acquiring an agent.

[0069] By "reduced" is meant a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0070] "Reference" means a standard or control condition.

[0071] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the 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, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the 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, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer therebetween.

[0072] "Specifically binds" means a compound or antibody that recognizes and binds to the polypeptide of the present invention but does not substantially recognize and bind to other molecules in a sample (eg, a biological sample) that naturally includes the polypeptide of the present invention.

[0073] Nucleic acid molecules that can be used in the methods of the present invention include any nucleic acid molecules encoding polypeptides of the present invention or fragments thereof. Such nucleic acid molecules do not need to be 100% identical to endogenous nucleic acid sequences, but generally will show basic identity. Polynucleotides having "basic identity" to endogenous sequences are generally capable of hybridizing with at least one chain of a double-stranded nucleic acid molecule. Nucleic acid molecules that can be used in the methods of the present invention include any nucleic acid molecules encoding polypeptides of the present invention or fragments thereof. Such nucleic acid molecules do not need to be 100% identical to endogenous nucleic acid sequences, but generally will show basic identity. Polynucleotides having "basic identity" to endogenous sequences are generally capable of hybridizing with at least one chain of a double-stranded nucleic acid molecule. "Hybridization" means pairing between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form double-stranded molecules under various stringency conditions.

[0074] For example, stringent salt concentrations will typically be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent (e.g., formamide), while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will typically include a temperature of at least about 30° C., more preferably at least about 37° C., and most preferably at least about 42° C. Different additional parameters, such as hybridization time, concentration of detergent (e.g., sodium dodecyl sulfate (SDS)), and inclusion or non-inclusion of carrier DNA, are well known to those skilled in the art. Various stringency levels are achieved by combining these different conditions as desired. In a preferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur 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 occur 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 those skilled in the art.

[0075] For most applications, the stringency of the washing step after hybridization will also be different. Washing stringency conditions can be defined by salt concentration and by temperature. As above, washing stringency can be increased by reducing salt concentration or by increasing temperature. For example, the stringent salt concentration for the washing step will preferably be less than about 30mM NaCl and 3mM trisodium citrate, and most preferably less than about 15mM NaCl and 1.5mM trisodium citrate. The stringent temperature conditions for the washing step will generally include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the washing step will occur at 25°C in 30mM NaCl, 3mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 42°C in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 68°C in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS.

[0076] 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" means a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0077] Sequence identity is generally measured using sequence analysis software (e.g., the sequence analysis software package of the Genetics Computer Group at the 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 generally include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, wherein probability scores between e<"3> and e<"100> indicate closely related sequences.

[0078] "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.

[0079] The ranges provided herein are to be understood as shorthand for all values ​​within the range. For example, a range of 1-50 is to be understood to include any number, combination of numbers, or subrange selected 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.

[0080] As used herein, the terms "treat," "treating," "treatment," and the like refer to the alleviation or amelioration of a disorder and / or symptoms associated therewith. It should be appreciated that, although not excluded, treating a disorder or condition does not require complete elimination of the disorder, condition, or symptoms associated therewith.

[0081] Unless specifically stated or obvious from the context, as used herein, the term "or" is to be understood as inclusive.

[0082] As used herein, the terms "a", "an" and "the" are to be understood as being in the singular or plural, unless specifically stated or clear from the context.

[0083] Unless specifically stated or apparent from the context, as used herein, the term "about" is understood to be within the normal range allowed in the art, such as within 2 standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0084] The enumeration of a list of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of the enumerated groups. The enumeration of embodiments for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0085] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0086] Alphavirus polynucleotide Generally, the disclosure includes any nucleic acid sequence encoding VLP, and the VLP has a change in the structural protein that enhances the expression of VLP in mammalian cells. In one embodiment, the alphavirus polypeptide includes at least one alphavirus E2 protein or capsid protein NLS, which includes a change that increases the expression of VLP in mammalian cells. In one embodiment, the alphavirus E2 protein has a non-lysine residue (e.g., asparagine) at the amino acid position corresponding to amino acid 234 in CHIKV E2 protein and / or has a modification that makes E2 protein unstable during virus budding at the amino acid position corresponding to amino acid 251 in CHIKV E2 protein. In another embodiment, the alphavirus polypeptide includes at least one alphavirus capsid protein, which has a non-lysine residue (e.g., alanine or asparagine) at the amino acid position corresponding to a lysine residue in the alphavirus capsid protein NLS and / or has a non-arginine residue (e.g., alanine or asparagine) at the amino acid position corresponding to an arginine residue in the alphavirus capsid protein NLS. In a particular embodiment, the alphavirus capsid protein is a WEEV strain CBA87 capsid protein, which has one or more changes in K67N, K68N and / or K69N. In certain embodiments, the alphavirus capsid protein is a VEEV strain TC83 capsid protein, which has one or more changes in K64N, K65A, K65N, K67A and / or K67N. In some embodiments, the alphavirus capsid protein is an EEEV strain PE-6 capsid protein, which has a change in K67N. In a particular embodiment, the alphavirus capsid protein is a CHIKV (strain 37997) strain capsid protein, which has one or more changes in R62A, R63A, R65A, K66A, K68A and / or K69A. In a particular embodiment, the alphavirus capsid protein is a Ross River virus capsid protein, which has one or more changes in R71N, K72N, K73N and / or K74N. In a specific embodiment, the alphavirus capsid protein is a Bama Forest virus capsid protein, which has one or more changes in K64A, K64N, K65A, K65N, K67A, K67N, K68A and / or K68N. The isolated nucleic acid molecule can be manipulated by recombinant DNA techniques well known in the art. Therefore, the nucleotide sequence contained in the vector in which the 5' and 3' restriction sites are known or the polymerase chain reaction (PCR) primer sequence is disclosed is considered to be isolated, but the nucleic acid sequence present in its natural state in its natural host is not. In certain exemplary embodiments, the vector comprises CHIKV 37997 or WEEV 71V-1658 nucleic acid segment or a fragment thereof. The vector may further comprise a CMV / R promoter. The vector may also comprise a capsid protein or a fragment thereof.

[0087] In other exemplary embodiments, in addition to the E2 protein, the vector further comprises another envelope protein selected from E3, 6K and E1. In certain examples, the vaccine may comprise capsid, E3, E2, 6K and E1. In other examples, the vaccine may comprise E3, E2, 6K and E1.

[0088] In a particular embodiment, the nucleic acid molecule shown 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 total length of the amino acid sequence) to a polypeptide encoding a protein selected from alphavirus capsid, E3, E2, 6K and E1 (including CHIKV or VEEV capsid, E3, E2, 6K and E1).

[0089] In some embodiments of the invention, the protein may comprise a mutation containing substitutions, additions or deletions that produce silence but do not alter the properties or activity of the encoded protein or changes in how the protein is prepared. Nucleotide variants may be produced for a variety of reasons, such as optimizing codon expression for a particular host, see U.S. Patent Publication 2005 / 0118191, which is incorporated herein by reference in its entirety for all purposes.

[0090] In addition, the nucleotides can be sequenced to ensure that the correct coding region is 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 understand that various subcloning methods are available and possible.

[0091] An isolated nucleic acid may be substantially purified, but not necessarily. For example, an isolated nucleic acid within a cloning or expression vector is not pure because it may comprise only a minor percentage of the material in the cell in which it is located. Such a nucleic acid is isolated as the term is used herein because it can be readily manipulated by standard techniques known to those of ordinary skill in the art.

[0092] Peptide expression Generally, VLPs comprising one or more alphavirus polypeptides herein can be produced by transforming a suitable host cell with all or part of a nucleic acid molecule encoding the polypeptide, or a fragment thereof, in an appropriate expression vector.

[0093] Those skilled in the art of molecular biology will appreciate that any of a variety of expression systems can be used to provide recombinant proteins. The exact host cell used is not critical to the present invention. The polypeptide of the present invention can be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, such as Sf21 cells, or mammalian cells, such as NIH 3T3, HeLa, COS cells). Such cells can be obtained 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, such as Sf9, Sf21, Trichoplusiani cells, such as High Five cells and Drosophila S2 cells. Examples of fungal (including yeast) host cells are Saccharomyces cerevisiae, Kluyveromyces lactis (K lactis), Candida species, including C. albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomyces 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 and Hep-2 cells. Xenopus laevis oocytes or other cells of amphibian origin may also be used. Prokaryotic host cells include bacterial cells, such as E. coli, B. subtilis, and mycobacteria.

[0094] Methods for cloning the protein are known in the art. For example, genes encoding specific alphavirus proteins (e.g., CHIKV, WEEV, EEEV, VEEV, Ross River virus, or Bama Forest virus structural proteins) can be isolated by RT-PCR from polyadenylated mRNA extracted from cells infected with the virus. The resulting product gene can be cloned into a vector as a DNA insert. The term "vector" refers to a tool by which nucleic acid can be propagated and / or transferred between organisms, cells, or cell components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, etc., which replicate autonomously or can be integrated into the chromosome of a host cell. The vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA in the same chain, a polylysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, etc., which cannot replicate autonomously. In many but not all common embodiments, the vector of the present invention is a plasmid or a bacmid.

[0095] The present invention further provides nucleic acids encoding proteins (including chimeric molecules) cloned into expression vectors that can be expressed in cells that provide for VLP formation.An "expression vector" is a vector, such as a plasmid, that is capable of promoting the expression and replication of a nucleic acid incorporated therein.

[0096] Typically, a nucleic acid molecule to be expressed is "operably linked" to a promoter and / or enhancer and is under transcriptional regulatory control by the promoter and / or enhancer.

[0097] There are a variety of expression systems for producing the polypeptides of the present invention. Expression vectors that can be used to produce such polypeptides include, but are not limited to, chromosomal, episomal, and viral-derived vectors, such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculovirus, papovavirus such as SV40, vaccinia virus, adenovirus, fowl pox virus, pseudorabies virus, and retroviruses, as well as vectors derived from combinations thereof.

[0098] Constructs and / or vectors provided herein include alphavirus polynucleotides encoding structural polypeptides, and the structural polypeptides include envelope proteins or capsid proteins or parts thereof as described herein. The vector may be, for example, a phage, a plasmid, a virus or a retroviral vector. Constructs and / or vectors comprising nucleotides should be operably connected to appropriate promoters, such as CMV promoters, bacteriophage λPL promoters, Escherichia coli lac, phoA and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs are non-limiting examples. Depending on the host cell and / or the desired expression rate, other suitable promoters are known to the technician. The expression construct will further contain sites for transcription initiation and termination, and contain a ribosome binding site for translation in the transcription region. The coding portion of the transcript expressed by the construct will preferably include a translation initiation codon at the beginning of the polypeptide to be translated and a termination codon appropriately positioned at the end of the polypeptide to be translated.

[0099] The expression vector will preferably include at least one selective marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for cultivation in Escherichia coli and other bacteria. Preferred in the vector are viral vectors, such as baculovirus, poxvirus (e.g., vaccinia virus, fowlpox virus, canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., canine adenovirus), herpesvirus and retrovirus. Other vectors that can be used with the present invention include vectors for bacteria, including pQE70, pQE60 and pQE-9, pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Preferred eukaryotic vectors are pFastBaclpWINEO, pSV2CAT, pOG44, pXT1 and pSG, pSVK3, pBPV, pMSG and pSVL. Other suitable vectors will be readily apparent to the skilled person.

[0100] Recombinant constructs can be prepared and used for transfection, infection or transformation, and viral proteins (including those described herein) can be expressed into eukaryotic cells and / or prokaryotic cells. Therefore, the present invention provides host cells comprising one or more vectors containing nucleic acids encoding the alphavirus structural genes described above (including capsids, E3, E2, 6K and E1 or portions thereof) and / or any chimeric molecules, and allowing the alphavirus structural genes described above (including capsids E3, E2, 6K and E1 or portions thereof), and / or any chimeric molecules to be expressed in the host cells under conditions that allow the formation of VLPs.

[0101] 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.

[0102] In another embodiment, the vector and / or host cell comprises nucleotides encoding alphavirus genes as described, including capsid, E3, E2, 6K and E1 or portions thereof.

[0103] In another embodiment, the vector and / or host cell consists essentially of alphavirus capsid, E3, E2, 6K and E1 or portions thereof as described herein. In another embodiment, the vector and / or host cell consists of alphavirus proteins comprising capsid, E3, E2, 6K and E1 or portions thereof. These vectors and / or host cells contain alphavirus core, E3, E2, 6K and E1 or portions thereof as described herein, and may contain additional cellular components, such as cellular VL proteins, baculovirus proteins, lipids, carbohydrates, etc.

[0104] A specific bacterial expression system for polypeptide production is the Escherichia coli pET expression system (Novagen, Inc., Madison, Wis.). According to this expression system, the DNA encoding the polypeptide is inserted into the pET vector in an orientation designed to allow expression. Since the gene encoding this polypeptide is under the control of the T7 regulatory signal, the expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is generally achieved using a host strain expressing T7 RNA polymerase in response to IPTG induction. Once produced, the recombinant polypeptide is then separated according to standard methods known in the art, such as those described herein.

[0105] Another bacterial expression system for polypeptide production is pGEX expression system (Pharmacia). This system adopts GST gene fusion system, and it is designed for high-level expression as gene or gene fragment of fusion protein and rapid purification and recovery of functional gene product. The protein of interest is fused to the carboxyl terminus of glutathione S-transferase protein from Schistosoma japonicum, and easily purified from bacterial lysate by affinity chromatography using Glutathione Sepharose 4B. Fusion protein can be recovered by eluting with glutathione under mild conditions. The glutathione S-transferase domain is promoted from the cutting of fusion protein by the presence of recognition site for site-specific protease in the domain upstream. For example, the protein expressed in pGEX-2T plasmid can be cut with thrombin; those expressed in pGEX-3X can be cut with factor Xa.

[0106] Alphavirus peptides and analogs The present invention provides VLPs comprising one or more alphavirus polypeptides. Also included in the present invention are VLPs comprising one or more alphavirus polypeptides or fragments thereof, which are modified in a manner that enhances or does not inhibit their ability to regulate an immune response. In one embodiment, the present invention provides a method for optimizing an alphavirus amino acid sequence or nucleic acid sequence by producing changes. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The present invention further includes analogs of any naturally occurring polypeptides of the present invention. Analogs may differ from naturally occurring polypeptides of the present invention by amino acid sequence differences, post-translational modifications, or both. Analogs of the present invention typically exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity to all or part of the naturally occurring amino acid sequence of the present invention. The length of the sequence comparison is at least 10, 13, 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably more than 35 amino acid residues.

[0107] The alteration of alphavirus polypeptides includes, but is not limited to, site-directed mutagenesis, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using templates containing uracil, oligonucleotide-mediated mutagenesis, phosphothioate-modified DNA mutagenesis, mutagenesis using duplex DNA with gaps, and the like. Other 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, such as mutagenesis involving chimeric constructs, is also included in the present invention. In one embodiment, mutagenesis can be guided by known information of naturally occurring molecules or altered or mutated naturally occurring molecules, such as sequence, sequence comparison, physical properties, crystal structure, and the like.

[0108] In one embodiment, the invention provides polypeptide variants that are different from reference polypeptides.The term "variant" refers to an amino acid sequence in which one or more amino acids are changed relative to a reference sequence.The variant may have a "conservative" change, in which the substituted amino acid has a similar structure or chemical properties, such as replacing leucine with isoleucine.Alternatively, the variant may have a "non-conservative" change, such as replacing glycine with tryptophan.Similar minor changes may also include amino acid deletions or insertions, or both.Computer programs well known in the art (e.g., DNASTAR software) can be used to find guidance for determining which amino acid residues can be substituted, inserted or deleted without eliminating biological or immunological activity.Desirably, the variant shows significant biological activity.In one embodiment, the protein variant forms VLPs and triggers an antibody response when administered to a subject.

[0109] Natural variants can occur due to mutations in proteins. These mutations can lead to antigenic variability within the individual group of infectious agents. Therefore, people infected with a specific strain will develop antibodies against the virus, and when newer viral strains appear, antibodies against the previous strains no longer recognize the newer virus and reinfection may occur. The present invention includes all antigenic and genetic variability of proteins from infectious agents for the preparation of VLPs.

[0110] In addition to the full-length polypeptide, the present invention also includes any fragment of the polypeptide 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 continuous amino acids, at least 30 continuous amino acids or at least 50 continuous amino acids, and in other embodiments is at least 60 to 80 or more continuous amino acids. Fragments of the present invention can be produced by methods known to those skilled in the art, or can be produced by normal protein processing (e.g., removing amino acids that are not needed for biological activity from nascent polypeptides, or removing amino acids by selective mRNA splicing or selective protein processing events).

[0111] Non-protein analogs having a chemical structure designed to mimic the functional activity of one or more alphavirus polypeptides of an alphavirus VLP can be administered according to the methods of the invention. Alphavirus analogs can exceed the physiological activity of native alphaviruses.

[0112] Analog design methods are well known in the art, and analog synthesis can be performed by modifying the chemical structure so that the resulting analog exhibits the immunomodulatory activity of the native alphavirus polypeptide. These chemical modifications include, but are not limited to, replacing alternative R groups and changing the degree of saturation of specific carbon atoms of the native alphavirus molecule.

[0113] Preferably, the analogs are relatively resistant to degradation in vivo, resulting in a longer lasting therapeutic effect when administered.Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0114] Pharmaceutical compositions and administration The present invention is characterized by a pharmaceutical composition comprising VLPs, the VLPs comprising viral structural proteins of alphaviruses as described herein and galectin-3 antigens. Pharmaceutical compositions useful herein contain a pharmaceutically acceptable carrier and VLPs of the present invention, the pharmaceutically acceptable carrier comprising any suitable diluent or excipient, including any medicinal substance that does not itself induce an immune response harmful to the vertebrate receiving the composition, and which can be administered without excessive toxicity. As used herein, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency, or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopeias, for mammals, and more particularly for humans. These compositions can be used as vaccines and / or antigenic compositions for inducing protective immune responses in vertebrates.

[0115] 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 pharmaceutically acceptable carriers, diluents and other excipients is presented 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, preferably sterile, non-particulate and / or pyrogen-free.

[0116] If necessary, the composition may also contain a small amount of a wetting or emulsifying agent, or a pH buffer. The composition may be in solid form, such as a lyophilized powder suitable for reconstitution, a liquid solution, a suspension, an emulsion, a tablet, a pill, a capsule, a sustained release formulation, or a powder. Oral formulations may include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0117] In certain embodiments, the VLP composition is provided in liquid form, eg, in a sealed container indicating the amount and concentration of the VLP composition.

[0118] Preferably, the liquid form of the VLP composition is provided in a hermetically sealed container 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.

[0119] Alternatively, the vaccine formulation is administered intranasally by drops, large particle aerosol (greater than about 10 microns) or spray into the upper respiratory tract or small particle aerosol (less than 10 microns) or spray into the lower respiratory tract. Although any of the above delivery routes will produce an immune response, intranasal administration confers the additional benefit of eliciting mucosal immunity at the site of entry of many viruses (including alphaviruses, such as CHIKV or VEEV).

[0120] Thus, the present invention also includes a method of formulating a vaccine or antigenic composition for inducing immunity in a mammal to an infection or at least one symptom thereof, comprising adding to the formulation an effective dose of a VLP, such as an alphavirus (eg, CHIKV or VEEV).

[0121] In some cases, it is preferred to stimulate immunity with a single dose, however, additional doses may be administered by the same or different routes to achieve the desired effect. For example, in newborns and infants, multiple administrations may be required to induce adequate levels of immunity. If necessary, administration may be continued at intervals throughout childhood to maintain adequate levels or protection.

[0122] Similarly, adults who are particularly susceptible to repeated or severe infections, such as health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary function 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 the dose adjusted or the vaccination repeated if necessary to induce and maintain the desired level of protection.

[0123] The technical staff can easily determine the dosage of the pharmaceutical preparation, for example, by first identifying the dosage that effectively causes preventive or therapeutic immune response, for example, by measuring the serum titer of virus-specific immunoglobulin or by measuring the inhibition ratio of antibody in serum sample or urine sample or mucosal secretion. The dosage can be determined by animal research. The non-limiting list of animals used to study vaccine efficacy includes guinea pigs, hamsters, ferrets, chinchillas, mice and cotton rats and non-human primates. Most animals are not the natural hosts of infectious pathogens, but can still be used for the research of disease in all aspects. For example, candidate vaccines, for example VLP of the present invention, can be given to any above animal, to partially characterize the immune response of induction, and / or determine whether any neutralizing antibodies have been produced. For example, many studies have been carried out in mouse models, because mice are small in size, and their low cost allows researchers to study on a large scale.

[0124] In addition, human clinical studies can be performed by technicians 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. The effective dose can be inferred from a dose-response curve derived from an in vitro or animal test system.

[0125] As is also well known in the art, the immunogenicity of a particular composition can be enhanced by using a non-specific stimulant (called an adjuvant) of the immune response. As used herein, the term "adjuvant" means a compound that, when used in combination with a specific immunogen in a formulation, will enhance, change or modify the resulting immune response. Adjuvants have been used experimentally to promote a general increase in immunity to unknown antigens. Immunization protocols have used adjuvants to stimulate reactions for many years, and therefore, adjuvants are well known to those of ordinary skill in the art. Some adjuvants affect the way antigens are presented. For example, when protein antigens are precipitated by aluminum adjuvants (alum), immune responses increase. Emulsification of antigens also prolongs the duration of antigen presentation. It is intended that any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)" is included within the scope of the present invention, and the document is incorporated herein by reference in its entirety for all purposes.

[0126] Exemplary adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing 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). Also considered is RIBI in a 2% squalene / Tween-80 emulsion, which contains three components extracted from bacteria, namely MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS). MF-59, Novasomes.RTM., MHC antigens can also be used.

[0127] VLP of the present invention can also be formulated with "immunostimulant". These "immunostimulants" are the body's own chemical messengers (cytokines) that increase the immune system response. Immunostimulants include, but are not limited to, various cytokines, lymphokines and chemokines with immunostimulation, immunoenhancement 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 factors, Flt3 ligand, B7.1, B7.2, etc. Immunostimulatory molecules can be applied in the same preparation as VLP, or can be applied separately. Protein or protein-encoding expression vectors can be applied to produce immunostimulatory effects. Therefore, in one embodiment, the present invention comprises antigens and vaccine preparations containing adjuvants and / or immunostimulants.

[0128] Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and Huntington's disease, display chronic, abnormal inflammation.

[0129] According to the present disclosure, the composition shows a reduction in Gal-3 levels in the brain and an improvement in cognitive function, which strongly suggests that it is useful not only for treating neurodegenerative diseases, but also for general inflammatory conditions.

[0130] Examples of inflammatory conditions are various organs, such as inflammatory conditions of the lungs, joints, eyes, intestines, skin, heart, kidneys, and central nervous system (CNS). The condition of the lungs includes asthma, adult respiratory distress syndrome, bronchitis, and cystic fibrosis (which may additionally or alternatively involve the intestines or other tissues). The condition of the joints includes rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, and other arthritis conditions. Inflammatory eye diseases include uveitis (including iritis) and conjunctivitis. Inflammatory bowel disease includes Crohn's disease, ulcerative colitis, and distal proctitis. Skin diseases include those associated with cell proliferation, such as psoriasis, eczema, and dermatitis (whether or not from allergies). The condition of the heart includes coronary artery infarction injury. The condition of the kidneys includes chronic kidney disease (CKD), such as glomerulonephritis (with and without nephrotic syndrome, for example, including idiopathic nephrotic syndrome or minimal change nephropathy). Other inflammatory conditions and immune disorders include tissue necrosis in chronic inflammation, endotoxin shock, smooth muscle proliferative disorders (eg, restenosis after angioplasty), and tissue rejection after transplant surgery.

[0131] 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 conditions.

[0132] According to the present disclosure, the composition or method can be used to treat inflammatory conditions, especially age-related inflammatory conditions, thereby enhancing biological functions related to overall health and disease progression, promoting longevity and healthy lifespan, and / or delaying or inhibiting the cellular aging process in a subject.

[0133] The present application will be described in detail with reference to the following examples, however, these examples are not intended to limit the scope of the present application.

[0134] Example 1 (1) Preparation of VLPs with Gal-3 antigen The galectin-3 (Gal-3) vaccine epitope was designed by using the protein amino acid sequence and 3D structure of human and mouse Gal-3 proteins (Table 1). The linkers SGG (N-terminus) and GGS (C-terminus) were attached to the epitope peptide SEQ ID NO: 5. To produce the mammalian expression vector pVEEV Gal-3 plasmid DNA vector, the dsDNA fragment (gBlocks, IDT) of the synthetic Gal-3 epitope peptide sequence was inserted into the specific position (between G at position 330 and S at position 335) of the DNA encoding the VEEV E3 envelope protein in the pVEEV vector according to the procedure disclosed in WO 2022 / 225057 to obtain VEEV-Gal-3 VLP.

[0135] [Table 1] Gal-3 epitope peptide

[0136] FreeStyle 293F cells (Thermo Fisher Scientific, #R790-07) were cultured in suspension in a shaking incubator at 37°C and 8% CO2 using FreeStyle 293 Expression Medium (ThermoFisher Scientific, #12338-018). 6 cells, DNA:PEI=1:3 (w / w) and 1.25×10 6 Under the condition of 10 cells / mL culture medium, 293F cells were transfected with pVEEV Gal-3 VLP plasmid DNA expression vector by PEI (polyethyleneimine, Polysciences, #23966). The culture supernatant was harvested 4 days after transfection and clarified by centrifugation (3,000 rpm, 10 min), and then filtered with a 0.45 μm PES membrane filtration system (VWR #10040-470).

[0137] The culture supernatant containing VLPs was layered onto 1.5 mL of OptiPrep (60% (w / v) iodixanol, Accuratechemical, #AN1114542) and centrifuged at 52,000 × g for 1.5 hr in a SW-28 rotor (Beckman). After removing the supernatant to leave 1.5 mL above the interface, the two layers were mixed to prepare a 50% OptiPrep solution and centrifuged at 360,000 × g for 2.5 hr in a NVT100 rotor (Beckman) to form a density gradient.

[0138] Crude VLPs were collected and purified by size exclusion chromatography using a Biologic Duo-Flow FPLC system (Bio-Rad) using a HiPrep 16 / 60 Sephacryl S-500HR column (GE, #28-9356-06) and phosphate buffered saline (PBS). The fractions containing VLPs were concentrated by an Amicon Ultra-15 centrifugal filter device (EMD Millipore, #UFC910024) and filtered through a 0.20 μm PES membrane.

[0139] According to the manufacturer's instructions, the total protein concentration was measured by BCA protein assay (Pierce, #23225). The purity of VLPs was confirmed by SDS-PAGE analysis (Any kD Mini-PROTEAN TGX precast protein gel, Bio-Rad, #456-9035), followed by staining based on Coomassie dye using QC colloidal Coomassie dye (Bio-Rad, #1610803). Sucrose and EDTA were added to the VLP solution at a final concentration of 250mM and 5mM, respectively. The protein concentration of the VLP samples was adjusted to 0.4mg / mL and stored at -80°C.

[0140] The molecular size and purity of VEEV VLPs containing the Gal-3 epitope peptide were analyzed by SDS-PAGE under denaturing conditions. The obtained VLPs matched the theoretical molecular weight and the purity was over 90%. Protein bands for the fusion protein Gal-3 peptide and VEEV viral structural proteins were confirmed.

[0141] (2) Detection of anti-mouse galectin-3 (Gal-3) antibodies in immunized mice 5×FAD 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 administered intramuscularly to one group of mice (Group 2), and normal saline was administered intramuscularly to the other group (Group 1). The concentration of VLPs was 0.2 mg / ml and 50 micrograms of VLPs were administered to each mouse. The mice were administered at the beginning of the experiment, 2 weeks and 4 weeks after the first administration. 6 weeks after the start of the experiment, blood samples were obtained from each mouse and serum was prepared. The anti-mouse Gal-3 antibodies produced were detected using ELISA, in which the 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 ).

[0142] (3) Venezuelan equine encephalitis virus-like particles containing viral structural polypeptides increase the latency of the shuttle passive avoidance test in 5×FAD mice.

[0143] 5 months after the experiment started, a shuttle passive avoidance test was carried out. The passive avoidance test is commonly used as a method for testing avoidance memory retention. The passive avoidance reaction is measured using a "shuttle" device. The device is divided into two compartments. On the first day, mice were placed in an illuminated compartment. When mice moved from the illuminated area to the dark compartment, a 3s electric shock (0.3mA) was released. On the second day, mice were placed in an illuminated compartment and the time when mice entered the dark compartment was recorded as "residence time".

[0144] The results showed that the VLP-Gal-3 injected group increased the latency, which indicates the memory retention in the VLP-Gal-3 injected group (see Figure 2 ).

[0145] (4) VEEV-Gal-3-VLP reduces Gal-3 in the hippocampus of 5×FAD mice. After testing, mouse brains were collected in microtubes and homogenized with lysis buffer. The lysates were centrifuged at 4°C for 10 min and the supernatants were resolved on NUPAGE 12%, Bis-Tris gels and transferred to PVDF membranes. The membranes were blocked with BulletBlocking 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 (1:000, BioLegend). After this incubation, the membranes were incubated with anti-rat secondary antibodies (1:3000, Jackson ImmunoResearch) or anti-mouse secondary antibodies (1:5000, Jackson ImmunoResearch). The blots were developed by chemiluminescence and the images were scanned using a chemiluminescence detector (LAS500; GE Healthcare). β-actin was used for loading control. As a result, the expression level of Gal-3 in the hippocampus was significantly decreased in the VLP-Gal-3 injected group (see Figure 3 ).

[0146] (5) VEEV-Gal-3-VLPs extend the lifespan of aged C57BL / 6J mice. C57BL / 6J mice were divided into two groups. VEEV-Gal-3 VLPs were intramuscularly administered to one group of mice (B6-V group) and physiological saline was intramuscularly administered to the other group (B6-N group).

[0147] VLPs were administered three times at 101-102 weeks of age. After about 5 weeks, some mice in the B6-N group gradually died. On the other hand, no mice in the B6-V group died (see Figure 4 ).

[0148] (6) VEEV-Gal-3-VLP prolongs the lifespan of 5×FAD mice. 5×FAD mice were divided into two groups. VEEV-Gal-3 VLPs were intramuscularly administered to one group of mice (FV group) and physiological saline was intramuscularly administered to the other group (FN group).

[0149] VLPs were administered 4 times at 28, 30, 32, and 42 weeks of age. Some mice in the FN group gradually died after 50 weeks of age. On the other hand, mice in the B6-V group did not die at all before 54 weeks of age (see Figure 5 ).

Claims

1. A virus-like particle (VLP) comprising viral structural proteins and a galectin-3 antigen for use in treating inflammatory conditions.

2. The VLP of claim 1, wherein the inflammatory condition is an age-related inflammatory condition.

3. The VLP of claim 1, wherein the inflammatory condition is inflammation in the central nervous system (CNS).

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

5. The method of 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 condition.

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

7. The VLP of claim 6, wherein the viral structural protein is derived from Chikungunya virus or Venezuelan equine encephalitis virus.

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

9. The VLP according to any one of claims 1 to 8, wherein at least one Galectin-3 antigen is inserted into the envelope protein E3 of the viral structural proteins.

10. The VLP according to any one of claims 1 to 9, 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 any one of claims 1 to 9, wherein the Galectin-3 antigen is a peptide of SEQ ID NO:

12.

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

8.

13. A pharmaceutical composition or vaccine composition for treating an inflammatory condition in a mammalian subject, comprising an effective amount of a virus-like particle comprising a viral structural protein and a galectin-3 antigen.

14. Use of a virus-like particle comprising a viral structural protein and a galectin-3 antigen for the manufacture of a medicament for treating an inflammatory condition in a mammalian subject.

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