Treatment of obesity

Abstract

The present disclosure provides a method for treating or preventing obesity in a mammalian subject which needs a treatment or prevention of obesity, the method including administering an effective amount of virus-like particles containing a viral structural protein and a galectin-3 antigen to the subject.

Description

Obesity Treatment

[0001] The present disclosure relates to methods for treating obesity in a mammalian subject in need thereof. The present disclosure also relates to methods for reducing visceral fat or inhibiting visceral fat gain in a mammalian subject.

[0002] Overweight and obesity are defined as abnormal or excessive fat accumulation that poses a health risk. A body mass index (BMI) above 25 is considered overweight, and a BMI above 30 is considered obese. The problem has grown to epidemic proportions, with over 4 million people dying per year as a result of being overweight or obese in 2017, according to the Global Burden of Disease Study.

[0003] The prevalence of overweight and obesity continues to grow among adults and children. Between 1975 and 2016, the prevalence of overweight or obesity among children and adolescents aged 5-19 increased more than four-fold globally, from 4% to 18%.

[0004] Obesity is one aspect of the double burden of malnutrition, and today there are more obese people than underweight people in every region except sub-Saharan Africa and Asia. Once thought to be a problem only in high-income countries, overweight and obesity are now increasing dramatically in low- and middle-income countries, particularly in urban settings. The vast majority of overweight or obese children live in developing countries, where the rate of increase in obese children is more than 30% higher than the rate in developed countries.

[0005] Galectins are members of the lectin family that exhibit high affinity for β-galactosides. Approximately 15 galectins, encoded by the LGALS gene, have been discovered in mammals and are consecutively numbered. Currently, only galectins-1, -2, -3, -4, -7, -7B, -8, -9, -9B, -9C, -10, -12, -13, -14, and -16 have been identified in humans.

[0006] Galectin-3 (Gal-3) is a member of a highly conserved family of animal lectins that bind to glycoconjugates (glycoproteins or glycolipids) containing β-galactosidase (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 carboxy-terminal domain containing the carbohydrate-binding region and an amino-terminal domain consisting of tandem repeats of nine amino acids that primarily crosslink carbohydrate and non-carbohydrate ligands (Barondes SH 1994). Gal-3 is secreted by various cell types, including monocytes, macrophages, and epithelial cells, but primarily 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 multimers (Dumic J 2006; Kuklinski S 1998; 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 cell types, and negatively regulate T cell receptor signaling (Yang RY 1996). Gal-3 has been shown to be elevated in various fibrotic disease and patient models, including pulmonary fibrosis, liver fibrosis, systemic sclerosis, and myocardial fibrosis (Henderson NC 2008; Barman et al. 2019; Nishi Y 2007; De Boer 2010). This suggests that Gal-3 may be an important mediator of tissue fibrosis and an effective therapeutic target.To date, preclinical and clinical studies of investigational galectin-3 inhibitors have shown protective effects against fibrotic disorders (Nikhil Hirani 2017, Yu L 2013).

[0007] One of the major success stories in medicine over the past 100-150 years has been vaccines targeting various infectious diseases. Along with antibiotics, vaccines are arguably 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 allergies, autoimmunity, and cancer. However, the targets of these diseases are typically self-antigens, which can pose efficacy challenges. Due to immune tolerance mechanisms, it is significantly more difficult to generate strong immune responses against self-antigens compared to non-self-antigens, such as bacterial, viral, or parasitic proteins (Hellman 2008; Falk Saupe 2015).

[0008] Alphaviruses include a group of genetically, structurally, and serologically related mosquito-borne viruses in the Togaviridae family. 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, assembles with the RNA genome to form an icosahedral nucleocapsid. In the virion, the capsid is surrounded by a lipid envelope covered with regularly arranged transmembrane protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, E1 and E2.

[0009] Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of standard natural viruses but lack the viral genome, potentially resulting in safer and less expensive 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 & Co., Inc.'s Recombivax HB® (hepatitis B virus) and Gardasil® (human papillomavirus) are some examples. Other VLP-based vaccine candidates, such as influenza virus, parvovirus, Norwalk virus, and various chimeric VLPs, are in clinical trials or undergoing preclinical evaluation. Despite their success in preclinical trials, many others are still limited to small-scale basic research. The implications of large-scale VLP production are discussed in the context of process control, monitoring, and optimization. Major 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 for either therapeutic or prophylactic vaccination.

[0010] To date, VLP-based vaccines have been produced against over 30 different viruses that infect humans and other animals. Examples include AAV (adeno-associated virus), H5N3 (avian influenza), BFDV (budgie flu 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 papillomavirus), IBDV (infectious bursal disease virus), influenza A, influenza A H1N1, influenza A These include H3N2, JC polyomavirus, Marburg, MS2, IPCV (Indian peanut crump 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), and SVDV (swine vesicular disease virus) (Non-Patent Document 18: Expert Rev. Vaccines 9(10), 1149-1176, 2010).

[0011] U.S. Patent No. 9,353,353 discloses virus-like particles (VLPs) comprising 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. 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 use in the prevention or treatment of 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, which 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, which comprise alphavirus structural proteins including envelope protein E3, which has been modified to contain at least one foreign antigen inserted into its furin cleavage site (these references are incorporated herein by reference).

[0012] U.S. Patent No. 9,353,353 U.S. Patent No. 9,487,563 U.S. Patent No. 9,249,191 U.S. Patent No. 9,969,986 U.S. Patent Application Publication No. 2005 / 0118191 WO2022 / 225057

[0013] 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 U S A 103(13):5060-5.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.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.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, 2005MacKinnon 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.Dumic J, Dabelic S, Flougel M. 2006. "Galectin-3: An open-ended story." BBA-Gen Subjects 1760:616-635.Kuklinski S, Probstmeier R. 1998. "Homophilic binding properties of galectin-3: Involvement of the carbohydrate recognition domain." J Neurochem 70:814-823.Lepur A, Salomonsson E, Nilsson UJ, Leffler H. 2012. "Ligand induced galectin-3 protein self-association." J Biol Chem 287:21751-21756.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.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.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.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, Shirai K. 2007. "Role of Galectin-3 in Human Pulmonary Fibrosis" Allergol Int. 56(1):57-65.De Boer, R. A., Yu, L., and van Veldhuisen, D. J. 2010. "Galectin-3 in cardiac remodeling and heart failure." Curr. Heart Fail. Rep. 7, 1-8.Nikhil Hirani, Alison Mackinnon , Lisa Nicol , Jeremy Walker , Paul Ford , Hans Schambye , Anders Pederson, Ulf Nilsson, Hakon Leffler , Tracy Thomas , Danielle Francombe , John Simpson , Michael Gibbons , Toby M. Maher. 2017. "TD139, A Novel Inhaled Galectin-3 Inhibitor for the Treatment of Idiopathic Pulmonary Fibrosis (IPF). Results from the First in (IPF) Patients Study." American Journal of Respiratory and Critical Care Medicine 195:A7560.Yu L, Ruifrok WP, Meissner M, Bos EM, van Goor H, Sanjabi B, van der 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.Hellman, L. 2008. "Therapeutic vaccines against IgE-mediated allergies." Expert Rev Vaccines 7(2):193-208.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.Antonio Roldao, Maria Candida M, Mellado, Leda R, Castilho, Manuel JT Carrondo and Paula M Alves "Virus-like particles in vaccine development" Expert Rev. Vaccines 9(10), 1149-1176, 2010Goldfarb, D., and N. Michaud (1991) "Pathways for the nuclear transport of proteins and RNAs" Trends Cell Biol. 1, 20-24Gorlich, D., and I. W.Mattaj (1996) "Nucleocytoplasmic Transport" Science 271, 1513-1518Schneider, J. et al. (1988) "A Mutant SV40 Large T Antigen Interferes with Nuclear Localization of a Heterologous Protein" Cell 54,117-125Dingwall et al. (1988) "The Nucleoplasmin Nuclear Location Sequence Is Larger and More Complex than That of SV-40 Large T Antigen" Cell Biol. 107 (3): 841-9.

[0014] An object of the present disclosure is to provide a method for treating or preventing obesity in a mammalian subject in need thereof. Another object of the present disclosure is to provide a method for reducing visceral fat or inhibiting visceral fat gain in a mammalian subject in need thereof. A further object of the present disclosure is to provide a pharmaceutical composition or use for use in the treatment or prevention of obesity.

[0015] Thus, the present disclosure provides a method for treating or preventing obesity in a mammalian subject in need thereof, the method comprising administering to the subject an effective amount of a virus-like particle comprising a viral structural protein and a galectin-3 antigen. The present disclosure also provides a method for reducing visceral fat or inhibiting visceral fat gain in a mammalian subject in need thereof, the method comprising administering to the subject an effective amount of a virus-like particle comprising a viral structural protein and a galectin-3 antigen.

[0016] The present disclosure also provides a pharmaceutical composition for treating or preventing obesity, comprising a virus-like particle comprising a viral structural protein and galectin-3.

[0017] The present disclosure also provides the use of a virus-like particle comprising a viral structural protein and galectin-3 for the manufacture of a medicament for treating or preventing obesity.

[0018] Anti-mouse galectin-3 (Gal-3) antibodies in the serum of VEEV-Gal-3-VLP-immunized mice.

[0019] Effect of VEEV-Gal-3-VLP on body weight of C57BL / 6J mice.

[0020] Effect of VEEV-Gal-3-VLP on visceral fat in C57BL / 6J mice.

[0021] Anti-human galectin-3 (Gal-3) antibodies in the serum of VEEV-Gal-3-VLP-immunized mice.

[0022] As used herein, the term "galectin-3 antigen" refers to any antigenic structure derived from the galectin-3 protein that can be recognized by the immune system and / or stimulate a cellular immune response and / or stimulate the production of antibodies specific to said antigen. A galectin-3 epitope peptide may be a fragment of a native galectin-3 protein, or a fragment of a native galectin protein with some modifications. The native galectin-3 protein may preferably be a human galectin protein, and particularly may be a human galectin-3 protein. In certain embodiments, the modified fragment has at least 80%, 85%, 90%, 95%, or 98% amino acid sequence identity with a fragment of a native galectin-3 protein. In certain embodiments, the modified peptide fragment is a variant in which up to 10% of amino acids have been deleted, substituted, and / or added relative to a fragment of a native galectin-3 protein.

[0023] Galectin-3 [Human (Homo sapiens)] (GenBank accession number: AAB86584.1) MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI (SEQ ID NO: 1)

[0024] Examples of galectin-3 antigens, i.e., galectin-3 epitope peptides, may include the following: ADNFSLHDALSGSGNPNPQGWPGAWGNQPA (SEQ ID NO: 2) YPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGA (SEQ ID NO: 3) YPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGA (SEQ ID NO: 4) ADSFSLNDALAGSGNPNPQGYPGAWGNQPA (SEQ ID NO: 5)

[0025] Gal-3 epitope peptide SEQ ID NO: 2 consists of a human N-terminal Gal-3 epitope peptide. Gal-3 epitope peptides SEQ ID NOs: 3 and 4 consist of N-terminal repeat sequence epitope peptides of human Gal-3, with SEQ ID NO: 4 having a longer repeat sequence than the repeat sequence of SEQ ID NO: 3. SEQ ID NO: 5 encodes the N-terminal peptide of mouse Gal-3.

[0026] In WO2022 / 225057 (the contents of which are incorporated herein by reference), VLPs containing the Gal-3 epitope peptide were prepared and the immunogenicity of those VLPs was confirmed.

[0027] Alphaviruses As used herein, "alphavirus" is intended to refer to an RNA-containing virus belonging to the Togaviridae family. 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, Kiziragachi virus, Highland J virus, Fort Morgan virus, Ndum virus, Boggy Creek virus, and Okelbo virus.

[0028] "Viral structural protein" refers to a polypeptide or fragment thereof that has at least about 80% amino acid sequence identity to a native viral capsid or envelope protein and that is immunogenic in a mammal. In certain embodiments, the alphavirus structural protein has at least about 85%, 90%, 95%, or more amino acid sequence identity to the above-mentioned alphavirus.

[0029] The viral structural protein and the galectin-3 antigen may be fused directly or indirectly, and in certain embodiments, 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.

[0030] The antigen or viral structural protein can be truncated and replaced with a short linker. In some embodiments, the antigen or viral structural protein includes one or more peptide linkers. Typically, the linker consists 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 in length, although in certain cases, the linker can be only one amino acid, such as a single glycine residue.

[0031] In some embodiments, a nucleic acid molecule in which a polynucleotide encoding a viral structural protein and a polynucleotide encoding an antigen are genetically fused is expressed in a host cell (e.g., a mammalian cell (e.g., 293F cell)) such that the first and second binding sites are linked via a peptide bond. In this case, the viral structural protein and the antigen are linked via a peptide bond. In this embodiment, the first and / or second binding sites may be genetically modified from the original protein or antigen. For example, the first binding site is modified from the viral structural protein so that the viral structural protein is conjugated to the antigen via 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 binding sites may be linked via 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.

[0032] 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 techniques.

[0033] As used herein, a chikungunya virus structural protein or Venezuelan equine encephalitis virus structural protein may be an envelope protein or a capsid, or one or more envelope proteins and / or one or more capsid proteins, of a chikungunya virus or a Venezuelan equine encephalitis virus.

[0034] Examples of chikungunya viruses include, but are not limited to, strains 37997 and LR2006 OPY-1. Examples of Venezuelan equine encephalitis viruses include, but are not limited to, strain TC-83.

[0035] The chikungunya virus structural proteins or Venezuelan equine encephalitis virus structural proteins used herein may be native viral structural proteins or modified proteins thereof. The modified proteins may be fragments of native viral structural proteins. In some embodiments, the modified proteins may have at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% amino acid sequence identity to native viral capsid and / or envelope proteins. In some embodiments, the modified proteins are mutants in which up to 10% of amino acids have been deleted, substituted, and / or added relative to the native viral capsid and / or envelope proteins. For example, a K64A or K64N mutation may be introduced into the capsid of the Venezuelan equine encephalitis virus structural proteins used herein.

[0036] Chikungunya virus or Venezuelan Equine Encephalitis virus structural proteins 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 can form one protein.

[0037] 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 strain LR2006 OPY-1.

[0038] 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.

[0039] The sequence of an exemplary Chikungunya virus structural protein is provided in GenBank Accession No. ABX40006.1 and is set forth below (SEQ ID NO: 6):

[0040] The sequence of another exemplary Chikungunya virus structural protein is provided in GenBank Accession No. ABX40011.1 and is set forth below (SEQ ID NO:7):

[0041] An exemplary Venezuelan Equine Encephalitis virus structural protein is set forth below (SEQ ID NO:8):

[0042] In certain embodiments, the first binding site comprises an amino group, preferably the amino group of a lysine residue, hi certain embodiments, the second binding site comprises a sulfhydryl group, preferably the sulfhydryl group of a cysteine.

[0043] According to the present application, there may be provided a chikungunya virus-like particle or Venezuelan equine encephalitis virus-like particle comprising 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 inserted directly or indirectly into E3 of the viral structural protein.

[0044] The viral structural proteins of chikungunya virus and Venezuelan equine encephalitis virus consist of E1, E2, 6K, and E3. 6K is naturally cleaved and removed from the VLP during assembly. Mature VLPs consist of capsid, E1, and E2. In this specification and claims, "viral structural protein" refers not only to the protein containing 6K, but also to the protein after 6K has been removed.

[0045] The CHIKV and VEEV 6K sequences used in the examples are as follows: CHIKV OPY-1 strain, 6K: 749-809aa of SEQ ID NO: 6 atyqeaaiylwneqqplfwlqaliplaalivlcnclrllpcccktlaflavmsvgahtvsa (SEQ ID NO: 9) CHIKV 37997 strain, 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)

[0046] With respect to the chikungunya virus structural proteins, at least one galectin-3 antigen may be inserted in place of the furin site (RKRR) from 322R to 325R of SEQ ID NO: 6 or 7. For example, with respect to the chikungunya virus structural proteins, at least one galectin-3 antigen is inserted between the H residue at position 321 and the S residue at position 326 of SEQ ID NO: 6 or 7; between the P residue at position 320 and the S residue at position 326 of SEQ ID NO: 6 or 7; or between the S residue at position 319 and the S residue at position 326 of SEQ ID NO: 6 or 7. The VLP_CHI 0.56 vector may 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 may be a chikungunya virus-like particle consisting of an E2 and E3 complex, a capsid, and E1, and the at least one galectin-3 antigen is inserted into the E3 region.

[0047] With respect to Venezuelan equine encephalitis virus, at least one galectin-3 antigen may be inserted in place of the furin site (RKRR) from 331R to 334R of SEQ ID NO: 8. For example, with respect to Venezuelan equine encephalitis virus structural protein, 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 may be used to prepare Venezuelan equine encephalitis virus-like particles in which 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 particle provided herein may be a Venezuelan equine encephalitis virus-like particle consisting of an E2 and E3 complex, a capsid, and E1, and wherein the at least one galectin-3 particle is inserted in the E3 region.

[0048] "Alteration" refers to a change in an amino acid or nucleotide at a specified position in a polypeptide or polynucleotide sequence. As used herein, alteration includes an amino acid or nucleotide substitution, deletion, or insertion at a specified position in a polypeptide or polynucleotide. In some embodiments, an alteration in an alphavirus capsid protein nuclear localization signal includes the 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 a basic charged amino acid such as lysine or arginine).

[0049] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding native polypeptide while having certain biochemical modifications that enhance the function of the analog compared to the native polypeptide. Such biochemical modifications may improve the analog's protease resistance, membrane permeability, or half-life, for example, without altering its binding to a ligand. Analogs may also contain unnatural amino acids.

[0050] In this disclosure, "comprise," "comprising," "containing," and "having," etc., may have the meaning ascribed to them in U.S. patent law and may mean "include," "including," etc.; "consisting essentially of" or "consisting essentially," etc., have the meaning ascribed to them in U.S. patent law, and the terms are open-ended, allowing for the presence of other things than what is described, but excluding prior art embodiments, so long as the basic or novel characteristics of what is described are not changed by the presence of other things than what is described.

[0051] "Detecting" refers to determining the presence, absence, or amount of the analyte to be detected.

[0052] "Disease" means any condition or disorder that impairs or interferes with the normal function of a cell, tissue, or organ.

[0053] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0054] "Effective amount" refers to the amount of agent required to alleviate the symptoms of disease compared with untreated patients.The effective amount of active compound used to carry out the method disclosed herein for preventing or treating disease varies depending on the mode of administration, the age, weight and overall health of the subject.Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen.This amount is referred to as an "effective" amount.

[0055] By "ameliorate" is meant to lessen, inhibit, attenuate, relieve, arrest, or stabilize the onset or progression of a disease or its symptoms.

[0056] The present disclosure provides numerous targets that are useful for the development of highly specific drugs to treat or prevent the diseases described herein. In addition, the methods disclosed herein provide a simple means for identifying safe treatments for use in subjects. In addition, the methods provide a high volumetric throughput, high sensitivity, and low complexity approach to analyze virtually any number of compounds effective against the diseases described herein.

[0057] "Fragment" refers to a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the 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.

[0058] An "isolated polynucleotide" refers to 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; an autonomously replicating plasmid or virus; or a recombinant DNA that is incorporated into the genomic DNA of a prokaryote or eukaryote; or a recombinant DNA that exists independently from other sequences as an isolated molecule (e.g., cDNA, or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion). Additionally, the term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene encoding additional polynucleotide sequences.

[0059] "Isolated polypeptide" refers to a polypeptide separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60% free, by weight, from the proteins and naturally occurring biomolecules that naturally accompany it. Preferably, at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the preparation is a polypeptide of the present disclosure. Isolated polypeptides may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemical protein synthesis. Purity can be measured by any appropriate method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0060] "Marker" means any protein or polynucleotide whose expression level or activity is altered in association with a disease or disorder.

[0061] As used herein, a "nuclear localization signal" or "NLS" is an amino acid sequence that, when present on the surface of a polypeptide, targets the polypeptide to the nucleus of a cell. NLS sequences are known in the art. See, for example, Goldfarb, D., and N. Michaud (1991) Trends Cell Biol. 1, 20-24; Gorlich, D., and I. W. Mattaj (1996) Science 271, 1513-1518. In certain embodiments, the NLS comprises one or more short sequences of positively charged amino acids, such as lysine and arginine. Consensus sequences for NLS include K-K / R-X-K / R (Schneider, J. et al. (1988) Cell 54, 117-125) and a sequence in which two clusters of basic amino acids are separated by a spacer of approximately 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, NLSs are 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 Barmah Forest virus capsid protein (KKPKK).

[0062] As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise obtaining the agent.

[0063] By "reduce" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0064] "Reference" means a standard or control condition.

[0065] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA 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 at least 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 therebetween or thereabout.

[0066] By "specifically binds" is meant a compound or antibody that recognizes and binds to a polypeptide of the present disclosure, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, that naturally contains a polypeptide of the present disclosure.

[0067] Nucleic acid molecules useful in the methods of the present disclosure include any nucleic acid molecule encoding a polypeptide or fragment thereof of the present disclosure. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence can typically hybridize to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present disclosure include any nucleic acid molecule encoding a polypeptide or fragment thereof of the present disclosure. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence can typically hybridize to at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to the pairing of complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form a double-stranded molecule under various stringency conditions.

[0068] For example, stringent salt concentrations would 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 achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, 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. Varying additional parameters, such as hybridization time, detergent concentration, e.g., sodium dodecyl sulfate (SDS), and the presence or absence of carrier DNA, is well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization will be carried out at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will be carried out at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will be carried out at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those of skill in the art.

[0069] For most applications, post-hybridization washing steps will also vary in stringency. Wash stringency conditions can be determined by salt concentration and temperature. As described above, wash stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for the wash 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 the wash steps will usually include temperatures 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 wash step will be performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash steps will be performed in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 42° C. In a more preferred embodiment, the wash steps will be performed in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68° C.

[0070] 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 to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or a reference nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such sequences are at least 60%, more preferably 80% or 85%, more preferably 90%, 95%, or 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0071] Sequence identity is typically 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 typically 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 approach to determining the degree of identity, a BLAST program may be used, where a probability score between e<"3"> and e<"100"> indicates closely related sequences.

[0072] "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.

[0073] Ranges provided herein are understood to be shorthand for all numbers 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.

[0074] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or alleviating a disorder and / or its associated symptoms. It will be understood that treating a disorder or condition does not require that the disorder, condition, or its associated symptoms be completely eliminated, although not prevented.

[0075] Unless specifically stated otherwise or clear from context, the term "or" as used herein is understood to be inclusive.

[0076] Unless specifically stated otherwise or clear from the context, the terms "a," "an," and "the," as used herein, are understood to be singular or plural.

[0077] Unless specifically stated otherwise or clear from the context, the term "about" as used herein is understood to mean within a range normally accepted in the art, e.g., within two standard deviations of the mean. About may be understood as 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 clearly contradicted by the context, all numerical values ​​provided herein are modified by the term about.

[0078] 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 among the listed groups or combination of 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.

[0079] Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

[0080] Alphavirus Polynucleotides Generally, the present disclosure encompasses any nucleic acid sequence encoding a VLP having an alteration in a structural protein that enhances expression of the VLP in mammalian cells. In certain embodiments, the alphavirus polypeptide comprises at least an alphavirus E2 protein or capsid protein NLS that comprises an alteration that increases expression of the VLP in mammalian cells. In certain embodiments, the alphavirus E2 protein has a non-lysine residue (e.g., asparagine) at the amino acid position corresponding to amino acid 234 of the CHIKV E2 protein and / or has a modification at the 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 an alphavirus capsid protein having a non-lysine residue (e.g., alanine or asparagine) at the amino acid position corresponding to the lysine residue of the NLS of the alphavirus capsid protein and / or a non-arginine residue (e.g., alanine or asparagine) at the amino acid position corresponding to the arginine residue of the NLS of the alphavirus capsid protein. In certain embodiments, the alphavirus capsid protein is a capsid protein of the WEEV strain CBA87 having one or more of the K67N, K68N, and / or K69N alterations. In certain embodiments, the alphavirus capsid protein is a capsid protein of the VEEV strain TC83 having one or more of the K64N, K65A, K65N, K67A, and / or K67N alterations. In some embodiments, the alphavirus capsid protein is a capsid protein of the EEEV strain PE-6 having the K67N alteration. In certain embodiments, the alphavirus capsid protein is a capsid protein of CHIKV (strain 37997) having one or more of the R62A, R63A, R65A, K66A, K68A, and / or K69A alterations. In certain embodiments, 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 certain embodiments, the alphavirus capsid protein is a Barmah Forest Virus capsid protein having one or more of the following alterations: K64A, K64N, K65A, K65N, K67A, K67N, K68A, and / or K68N. Isolated nucleic acid molecules may be manipulated by recombinant DNA techniques well known in the art. That is, 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, whereas a nucleic acid sequence existing in its natural state in a natural host is not considered isolated. In certain exemplary embodiments, the vector comprises a 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.

[0081] In other exemplary embodiments, in addition to the E2 protein, the vector comprises another envelope protein selected from the group consisting of E3, 6K, and E1. In particular examples, the vaccine may comprise capsid, E3, E2, 6K, and E1. In other examples, the vaccine may comprise E3, E2, 6K, and E1.

[0082] In certain embodiments, a nucleic acid molecule represented by a sequence 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 full length of the amino acid sequence) to the polypeptide sequence of a protein selected from alphavirus capsid, E3, E2, 6K, and E1, including capsid, E3, E2, 6K, and E1 of CHIKV or VEEV.

[0083] In some embodiments of the present disclosure, proteins may contain mutations that result in silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded protein or how the protein is made. Nucleotide variants can be produced for various 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.

[0084] Additionally, the nucleotides can be sequenced to ensure that the correct coding region has been replicated 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.

[0085] An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid isolated within a cloning or expression vector is not pure, in that it may represent only a small percentage of the material in the cell in which it resides. Such nucleic acids are readily manipulated by standard techniques known to those skilled in the art and, as the term is used herein, are isolated.

[0086] Polypeptide Expression Generally, VLPs comprising one or more alphavirus polypeptides of the present disclosure may 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 a suitable expression vehicle.

[0087] Those skilled in the field of molecular biology will understand that any of a variety of expression systems may be used to provide recombinant proteins. The particular host cell used is not critical to the present invention. Polypeptides of the present disclosure may be produced in prokaryotic hosts (e.g., Escherichia coli) or eukaryotic hosts (e.g., Saccharomyces cerevisiae), insect cells such as Sf21 cells, or mammalian cells such as NIH3T3, HeLa, or COS cells). Such cells are available from a wide range of sources (e.g., American Type Culture Collection, Rockland, Md.; see also 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 include S. cerevisiae, Kluyveromyces lactis (K. lactis), C. albicans, and C. Examples of mammalian cells include Candida species, including C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica. Examples of mammalian cells include 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 ovarian cells or other cells of amphibian origin may also be used.Prokaryotic host cells include bacterial cells such as E. coli, B. subtilis, and Mycobacterium.

[0088] Methods for cloning such proteins are known in the art. For example, genes encoding specific alphavirus proteins, such as viral structural proteins of 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 the virus. The resulting genes can be cloned as DNA inserts into vectors. The term "vector" refers to any means by which nucleic acids can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, and the like, which can self-replicate or integrate into a host cell chromosome. 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, and the like, which do not self-replicate. In many, but not all, common embodiments, the vectors of the invention are plasmids or bacmids.

[0089] The present invention further provides nucleotides encoding proteins comprising 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 and replication of a nucleic acid incorporated therein.

[0090] Typically, the nucleic acid molecule to be expressed is "operably linked" to and is under the transcriptional regulatory control of a promoter and / or enhancer.

[0091] A variety of expression systems exist for producing the polypeptides of the present disclosure. Expression vectors useful for producing such polypeptides include, but are not limited to, chromosomal, episomal, and virally derived vectors, such as vectors derived from bacterial plasmids, bacteriophage, transposon, yeast episomes, insertional elements, yeast chromosomal elements, viruses such as baculovirus, papovaviruses such as SV40, vaccinia virus, adenovirus, fowlpox, pseudorabies, and retroviruses, and combinations thereof.

[0092] The constructs and / or vectors provided herein contain an alphavirus polynucleotide encoding a structural polypeptide, including an envelope or capsid protein or portion thereof, as described herein. The vector may be, for example, a phage vector, a plasmid vector, a viral vector, or a retroviral vector. The construct and / or vector containing the nucleotide should be operably linked to an appropriate promoter, non-limiting examples of which include the CMV promoter, the phage lambda PL promoter, the E. coli lac, phoA, and tac promoters, the SV40 early and late promoters, and promoters of retroviral LTRs. Other suitable promoters will be known to those of skill in the art depending on the host cell and / or desired expression rate. The expression construct will further contain sites for transcription initiation, termination, and, within the transcribed region, a ribosome binding site for translation. 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.

[0093] Expression vectors will preferably contain at least one selectable marker. Such markers include dihydrofolate reductase, G418 resistance, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin, or ampicillin resistance genes for culturing in E. coli and other bacteria. Preferred 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), herpesvirus, and retrovirus. Other vectors that can be used with the present invention include bacterial vectors, including pQE70, pQE60, and pQE-9, pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5. Preferred eukaryotic vectors include 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.

[0094] Recombinant constructs may be prepared and used for transfection, infection, or transformation of eukaryotic and / or prokaryotic cells, and for expression of viral proteins, including those described herein, in eukaryotic and / or prokaryotic cells. That is, the present invention provides a host cell comprising a vector (or vectors) containing nucleic acid encoding alphavirus structural genes, including capsid, E3, E2, 6K, and E1, or portions thereof, and / or any of the chimeric molecules described above, and allowing expression of the alphavirus structural genes, including capsid, E3, E2, 6K, and E1, or portions thereof, and / or any of the chimeric molecules described above, under conditions allowing for the formation of VLPs in said host cell.

[0095] 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.

[0096] 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.

[0097] In another embodiment, the vector and / or host cell consists essentially of the alphavirus capsid, E3, E2, 6K, and E1, or portions thereof, described herein. In a further embodiment, the vector and / or host cell consists of alphavirus proteins, including the capsid, E3, E2, 6K, and E1, or portions thereof, described herein. These vectors and / or host cells contain the alphavirus core, E3, E2, 6K, and E1, or portions thereof, described herein, and may contain additional cellular components such as cellular VL proteins, baculovirus proteins, lipids, carbohydrates, etc.

[0098] One particular bacterial expression system for polypeptide production is the E. coli pET expression system (Novagen, Inc., Madison, Wis.). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to enable expression. Because the gene encoding such a polypeptide is under the control of a T7 regulatory signal, expression of the polypeptide is achieved by inducing expression of T7 RNA polymerase in the host cell. This is typically 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, such as those described herein.

[0099] Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system designed for high-level expression of genes or gene fragments as fusion proteins and rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxy terminus of the glutathione-S-transferase protein from Schistosoma japonicum and easily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. The fusion protein can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione-S-transferase domain from the fusion protein is facilitated by the presence of a site-specific protease recognition site upstream of this domain. For example, proteins expressed in the pGEX-2T plasmid can be cleaved with thrombin, and proteins expressed in the pGEX-3X plasmid can be cleaved with factor Xa.

[0100] 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 in a manner that enhances or does not inhibit their ability to modulate an immune response. In certain embodiments, the present invention provides methods for optimizing the amino acid or nucleic acid sequence of an alphavirus by making modifications. Such modifications may include specific mutations, deletions, insertions, or post-translational modifications. The present invention also includes analogs of any native polypeptide of the present disclosure. Analogs differ from the native polypeptide of the present disclosure by differences in amino acid sequence, post-translational modifications, or both. Analogs of the present disclosure will generally exhibit at least 85%, more preferably 90%, more preferably 95%, and even more preferably 99% identity to all or part of a native amino acid sequence of the present disclosure. The length of sequence comparison is at least 10, 13, or 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably greater than 35 amino acid residues.

[0101] Alterations to alphavirus polypeptides include, but are not limited to, site-directed, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil-containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, natural 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 that results in, for example, chimeric constructs is also encompassed by the present invention. In certain embodiments, mutagenesis can be guided by known information, such as sequence, sequence comparison, physical properties, crystal structure, etc., of the native molecule or the altered or mutated native molecule.

[0102] In some embodiments, the present invention provides polypeptide variants that differ from a reference polypeptide. The term "variant" refers to an amino acid sequence in which one or more amino acids have been altered relative to the reference sequence. A variant may have "conservative" changes, such as the substitution of leucine with isoleucine, where the substituted amino acid has similar structural or chemical properties. Alternatively, a variant may have "non-conservative" changes, such as the substitution of glycine with tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance on which amino acid residues can be substituted, inserted, or deleted without impairing biological or immunological activity can be obtained using computer programs well known in the art, such as DNASTAR software. Desirably, the variant exhibits substantial biological activity. In some embodiments, the protein variant forms VLPs and elicits an antibody response when administered to a subject.

[0103] Natural variants can occur due to protein mutations. These mutations can lead to antigenic variability within individual groups of infectious pathogens. That is, humans infected with a particular strain will produce antibodies against that virus, but if a new viral strain emerges, antibodies against the older strain will not recognize the new virus, and reinfection may occur. The present invention encompasses all antigenic and genetic variability of proteins from infectious pathogens for creating VLPs.

[0104] In addition to full-length polypeptides, the present invention also includes fragments of any of the polypeptides of the present disclosure. As used herein, the term "fragment" means at least 5, 10, 13, or 15 contiguous amino acids. In other embodiments, fragments are 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 to 80 or more contiguous amino acids. Fragments of the present disclosure 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 or alternative protein processing events).

[0105] Non-protein analogs having chemical structures designed to mimic the functional activity of alphavirus VLPs or one or more alphavirus polypeptides may be administered in accordance with the methods of the present disclosure. Alphavirus analogs may also exceed the biological activity of the native alphavirus.

[0106] Methods for designing analogs 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 the native alphavirus polypeptide. These chemical modifications include, but are not limited to, substituting alternative R groups and changing the degree of saturation of particular carbon atoms of the native alphavirus molecule.

[0107] Preferably, the analogs are relatively resistant to in vivo degradation and provide a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0108] Pharmaceutical Compositions and Administration The present invention features pharmaceutical compositions comprising VLPs comprising the alphavirus viral structural proteins and galectin-3 antigens described herein. Pharmaceutical compositions useful herein contain a pharmaceutically acceptable carrier, including a suitable diluent or excipient, which includes any pharmaceutical that does not itself provoke an immune response harmful to the vertebrate receiving the composition and which may be administered without undue toxicity, and a VLP of the present disclosure. As used herein, the term "pharmaceutically acceptable" means one approved by a federal or state regulatory agency for use in mammals, more specifically humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias. These compositions may be useful as vaccines and / or antigenic compositions for eliciting a protective immune response in vertebrates.

[0109] 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 found 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 human administration and is preferably sterile, particulate-free, and / or non-pyrogenic.

[0110] If necessary, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition may be in the form of a solid, such as a lyophilized powder suitable for reconstitution, or a solution, suspension, emulsion, tablet, pill, capsule, sustained-release formulation, or powder. Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0111] In certain embodiments, the VLP composition is supplied in liquid form, eg, in a hermetically sealed container indicating the quantity and concentration of the VLP composition.

[0112] Preferably, the liquid form of the VLP composition is supplied in a 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.

[0113] Alternatively, vaccine formulations are administered intranasally, either by instillation, large particle aerosol (greater than about 10 microns), or spray to the upper respiratory tract, or by small particle aerosol (less than 10 microns) or spray to the lower respiratory tract. While both of these delivery routes result in an immune response, intranasal administration has the added advantage of eliciting mucosal immunity at the site of entry for many viruses, including alphaviruses such as CHIKV or VEEV.

[0114] Thus, the present invention also includes a method for formulating a vaccine or antigenic composition that induces immunity to an infection in a mammal or at least one symptom thereof, comprising adding to the formulation an effective dose of a VLP, such as an alphavirus (e.g., CHIKV or VEEV).

[0115] In certain cases, a single dose of the vaccine is preferred to stimulate immunity, but additional doses can be administered by the same or different routes to achieve the desired effect. For example, in newborns and infants, multiple doses may be required to induce a sufficient level of immunity. Administration can continue throughout childhood as needed to maintain a sufficient level of protection.

[0116] 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 those 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 secretory and serum neutralizing antibodies, and dosages can be adjusted or vaccinations repeated as necessary to elicit and maintain the desired level of protection.

[0117] The dosage of a pharmaceutical preparation can be easily determined by those skilled in the art by first identifying an effective dose for eliciting a preventive or therapeutic immune response, for example, by measuring serum titers of virus-specific immunoglobulins or measuring the inhibition rate of antibodies in serum samples, urine samples, or mucosal secretions. The dosage can be determined through animal tests. A non-limiting list of animals used to study vaccine efficacy includes guinea pigs, hamsters, ferrets, chinchillas, mice, cotton rats, and non-human primates. Although most animals are not natural hosts of infectious pathogens, they can still be useful for studying various aspects of disease. For example, any of the above animals can be administered with a vaccine candidate, such as the VLPs of the present disclosure, to partially characterize the induced immune response and / or determine whether any neutralizing antibodies are produced. For example, many studies have been conducted in mouse models because the small size and low cost of mice allow researchers to conduct studies on a large scale.

[0118] In addition, human clinical trials can be conducted by those skilled in the art to determine preferred effective doses for humans. Such clinical trials are routine and well known in the art. The precise dose to be employed will also depend on the route of administration. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal test systems.

[0119] As is well known in the art, the immunogenicity of a particular composition can be enhanced by the use of nonspecific stimulators of the immune response, known as adjuvants. As used herein, the term "adjuvant" refers to a compound that, when used in combination with a specific immunogen in a formulation, augments, modulates, or modulates the resulting immune response. Adjuvants have been used empirically to promote overall immunity to unknown antigens. Adjuvants have been used for many years in immunization protocols to stimulate responses, 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 enhanced when protein antigens are precipitated with alum. Emulsification of antigens also extends the duration of antigen presentation. It is contemplated within the scope of the present invention to include any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)," incorporated herein by reference in its entirety for all purposes.

[0120] Exemplary adjuvants include complete Freund's adjuvant (a nonspecific 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). 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®, and MHC antigens may also be used.

[0121] The VLPs of the present disclosure may be formulated with "immunostimulatory substances." These are the body's own chemical messengers (cytokines) to enhance the immune system's response. Immunostimulatory substances include, but are not limited to, various cytokines, lymphokines, and chemokines with immunostimulatory, immunoenhancing, and proinflammatory activities, 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, and B7.2. The immunostimulatory molecules may be administered in the same formulation as the VLP or separately. Either the protein or an expression vector encoding the protein may be administered to produce an immunostimulatory effect. Thus, in certain embodiments, the present invention encompasses antigenic and vaccine formulations that include adjuvants and / or immunostimulatory substances.

[0122] Obesity increases the risk of various diseases such as hyperlipidemia, ischemic heart disease, cerebrovascular disease, diabetes, dementia, etc. Therefore, the VLPs of the present disclosure can be used for the treatment, prevention, or risk reduction of these diseases.

[0123] 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.

[0124] (1) Preparation of VEEV VLPs Carrying the Gal-3 Antigen (VEEV-Gal-3 VLPs) Using the amino acid sequences and 3D structures of human and mouse Gal-3 proteins, a galectin-3 (Gal-3) vaccine epitope was designed (Table 1). Linkers SGG (N-terminus) and GGS (C-terminus) were attached to the epitope peptide SEQ ID NO:5. To generate the mammalian expression vector pVEEV Gal-3 plasmid DNA vector, a synthetic dsDNA fragment (gBlocks, IDT) of the Gal-3 epitope peptide sequence was inserted into a 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 VLPs.

[0125] FreeStyle 293F cells (Thermo Fisher Scientific, #R790-07) were cultured in a shaking incubator under 8% CO ２ The 293F cells were cultured in suspension in FreeStyle 293 Expression medium (Thermo Fisher Scientific, #12338-018) at 37°C in the presence of 0.8 μg / 10 μg of DNA. The pVEEV Gal-3 VLP plasmid DNA expression vector was transfected into the 293F cells using PEI (polyethyleneimine, Polysciences, #23966). ６ Cells, DNA:PEI=1:3 (w / w), and 1.25 × 10 ６ Transfection was performed at a ratio of 1 mL of cells / culture medium. Culture supernatants were collected 4 days after transfection and clarified by centrifugation (3,000 rpm, 10 minutes) followed by filtration through a 0.45 μm PES membrane filter system (VWR #10040-470).

[0126] The VLP-containing culture supernatant was layered on 1.5 mL of OptiPrep (60% w / v) (iodixanol, Accurate Chemical, #AN1114542) and centrifuged at 52,000 × g for 1.5 hours in an SW-28 rotor (Beckman). After removing the supernatant so that 1.5 mL remained above the interface, the two layers were mixed to create a 50% Optiprep solution, which was then centrifuged at 360,000 × g for 2.5 hours in an NVT100 rotor (Beckman) to form a density gradient.

[0127] 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 fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (EMD Millipore, #UFC910024) and filtered through a 0.20 μm PES membrane.

[0128] Total protein concentration was measured by BCA Protein Assay (Pierce, #23225) according to the manufacturer's instructions. VLP purity 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 final concentrations of 250 mM and 5 mM, respectively. The protein concentration of the VLP samples was adjusted to 0.4 mg / mL and stored at -80°C.

[0129] The molecular size and purity of VEEV VLPs containing the Gal-3 epitope peptide were analyzed by SDS-PAGE under denaturing conditions. The resulting VLPs were consistent with the theoretical molecular weight and had a purity of over 90%. A protein band corresponding to the fusion protein between the Gal-3 peptide and a VEEV viral structural protein was confirmed. In subsequent studies, the resulting VLPs were referred to as "VEEV-Gal-3 VLPs."

[0130] (2) Detection of anti-mouse galectin-3 (Gal-3) antibodies in the serum of VEEV-Gal-3 VLP-immunized mice. Twelve-month-old C57BL / 6J mice were used as an animal model of increased visceral fat. The 12-month-old mice were divided into two groups. VEEV-Gal-3 VLPs were intramuscularly administered to one group of mice (Group 2), and normal saline was administered to the other group (Group 1). Mice were administered VLPs or saline at the start of the experiment, 2 weeks, 4 weeks, and 3 months after the initial administration. Six weeks after the start of the experiment, blood samples were obtained from each mouse, and serum was prepared. Anti-mouse Gal-3 antibodies in the serum were detected using ELISA, in which mouse Gal-3 protein was coated on an ELISA plate. The results show that VEEV-Gal-3 VLPs induce anti-mouse Gal-3 antibodies in mice from group 2 (see FIG. 1).

[0131] (3) Effect of VEEV-Gal-3 VLP on Body Weight and Visceral Fat in C57BL / 6J Mice Three and six months after the start of the experiment, the animals' body weights were measured. The results showed a decrease in body weight in the VLP-Gal-3-treated group (Figure 2). The mice were then sacrificed, and the heart, part of the liver, and kidneys were excised. After the skin incision was closed, visceral fat was measured by magnetic resonance imaging (MRI). The results showed a decrease in visceral fat in the VEEV-Gal-3 VLP-treated group (Figure 3).

[0132] (4) Detection of anti-human galectin-3 (Gal-3) antibodies in the serum of VEEV-Gal-3 VLP-immunized mice Six weeks after the start of the experiment, blood samples were obtained from each of the VEEV-Gal-3 VLP-immunized mice (Group 2), and serum was prepared. Anti-human Gal-3 antibodies in the serum were detected using ELISA in which human Gal-3 protein was coated on an ELISA plate. The results show that VEEV-Gal-3 VLPs induce not only anti-mouse Gal-3 antibodies but also anti-human Gal-3 antibodies.

Claims

1. A pharmaceutical composition for treating or preventing obesity in a mammalian subject comprising an effective amount of a virus-like particle comprising a viral structural protein and a galectin-3 antigen.

2. The pharmaceutical composition of claim 1, wherein the viral structural protein is derived from an alphavirus or a flavivirus.

3. The pharmaceutical composition of claim 2, wherein the viral structural proteins are derived from chikungunya virus or Venezuelan equine encephalitis virus.

4. The pharmaceutical composition of claim 3, wherein the viral structural proteins are derived from Chikungunya virus strain 37997 or OPY-1, or Venezuelan equine encephalitis virus strain TC-83.

5. A pharmaceutical composition according to any one of claims 1 to 4, wherein the galectin-3 antigen is inserted into the envelope protein E3 of the viral structural proteins.

6. The pharmaceutical composition according to any one of claims 1 to 5, 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.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the galectin-3 antigen is a peptide of SEQ ID NO:

12.

8. A pharmaceutical composition according to any one of claims 5 to 7, wherein the galectin-3 antigen is inserted between residues corresponding to residues 321 and 326 of SEQ ID NO:1, residues 321 and 326 of SEQ ID NO:2, or residues 330 and 335 of SEQ ID NO:

3.

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

10. A method for treating or preventing obesity in a mammalian subject in need thereof, comprising administering to the subject an effective amount of a virus-like particle comprising a viral structural protein and a galectin-3 antigen.

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