Methods and compositions of quaternary immunomodulatory nanoparticles for medical applications and immunotherapy

Abstract

The present disclosure provides a method of enhancing an immune response in a subject in need thereof comprising administering to the subject an effective amount of an antigen and an immunomodulator, where the immunomodulator is a quaternary immunomodulatory nanoparticle (QIN). The invention also provides the use of said QIN in the manufacture of chimeric nanoparticles, pharmaceutical compositions and medicaments for the treatment of subjects suffering from cancer or viral infection.

Description

Field of the Invention

[0001] The present disclosure relates to quaternary immunomodulatory nanoparticles (QIN) derived from hepatitis E virus nanoparticles, and in particular to the use of QIN in enhancing the immune response of a subject and in the manufacture of chimeric nanoparticles, pharmaceutical compositions and drugs. Background Art

[0002] Hepatitis E is an enterically transmitted virus that causes acute liver inflammation in humans. The HEV-derived quaternary immunomodulatory nanoparticles (QINs) display icosahedral cages that are stable in acidic and proteolytic environments, features retained from their natural transmission pathways.

[0003] Hepatitis E virus is composed of an unenveloped icosahedral capsid containing a 7.2 kilobase single-stranded RNA genome and is an enterically transmitted virus that can cause acute liver inflammation in humans. The major capsid protein is extremely important for viral assembly, immunogenicity and host interaction. The recombinant capsid protein can self-assemble into virus-like particles when expressed in insect cells after deleting 111 amino acids at the N-terminus and 52 amino acids at the C-terminus. The QIN displays an icosahedral cage that is stable in acidic and proteolytic environments and retains the characteristics of its natural transmission pathway. QIN is composed of 60 subunits, forming an icosahedral nanoparticle. QIN is stable in acidic environments and resistant to proteolytic digestion, so it has great advantages as an oral delivery vehicle.

[0004] Ganoderma, also known as Lingzhi, is a natural fungal medicine derived from traditional Chinese medicine and is one of the most famous types of traditional Chinese medicine. For centuries, it has been widely used in China, the United States, Japan, South Korea, and other Asian countries to treat various diseases. Its immunomodulatory proteins and polysaccharides have been identified as key bioactive factors in cancer immunotherapy and NF-kB and MAPK pathways. Therefore, even in modern medical practice, Ganoderma is used to treat chronic bronchitis, bronchial asthma, leukopenia, coronary heart disease, arrhythmia, and acute infectious hepatitis. However, Ganoderma is mainly used as a complementary treatment outside of current clinical first-line treatment. Many studies have shown that Ganoderma has anti-tumor effects caused by regulating the immune system, which is due to its fungal immunomodulation proteins (FIPs), polysaccharides, and triterpenoids. Among these components, the important immunomodulatory protein GMI cloned from Ganadorma microsporum has been intensively studied. In vitro experiments conducted by Lin et al. found that GMI inhibited EGF-induced phosphorylation and activation of EGFR and AKT pathway kinases in a dose-dependent manner. In vivo studies also suggested that oral administration of GMI could inhibit tumor growth and induce autophagy in mouse models.

[0005] Porcine circovirus type 2 (PCV2) is a small, non-enveloped DNA virus that is considered one of the most important swine pathogens of porcine circovirus disease (PCVD), causing economic losses and reduced productivity in all major swine producing countries worldwide. Vaccination against PCV2 is now routine practice in the swine industry in most developed countries. CIRCOQ, a commercially available PCV2 subunit vaccine, has been developed by Rebold Genetics Co., Ltd. (Taipei, Taiwan) for the protection against the newly emerged PCV2. Summary of the invention

[0006] The present invention provides medical applications of quaternary immunomodulatory nanoparticles (QINs), in particular, the use of quaternary immunomodulatory nanoparticles (QINs) in enhancing the immune response of subjects suffering from cancer or viral infection and in the manufacture of chimeric nanoparticles, pharmaceutical compositions and drugs.

[0007] In one aspect of the present invention, a method of enhancing an immune response in a subject in need thereof is provided, and the method comprises administering to the subject an effective amount of an antigen and an immunomodulatory agent, wherein the immunomodulatory agent is a quaternary immunomodulatory nanoparticle (QIN).

[0008] Preferably, QIN is further coupled to an antigenic determinant of a Ganoderma microsporum immunomodulatory (GMI) protein.

[0009] Preferably, the antigenic determinant of the GMI protein is coupled to QIN by being inserted into the capsid subunit of QIN.

[0010] Preferably, the antigenic determinant of the GMI protein has the sequence shown in SEQ ID NO:1.

[0011] Preferably, the antigen is selected from the group consisting of a tumor antigen, a viral antigen, a bacterial antigen, a self antigen and a fungal antigen.

[0012] Preferably, the viral antigen is a PCV2 antigen.

[0013] In another aspect of the present invention, a chimeric nanoparticle comprising QIN coupled to an antigenic determinant of a GMI protein is provided.

[0014] In another aspect of the present invention, a pharmaceutical composition comprising QIN and a pharmaceutically acceptable carrier, excipient or diluent is provided.

[0015] In another aspect of the present invention, there is provided a use of the above pharmaceutical composition in the manufacture of a medicament for treating a subject suffering from cancer or viral infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1a Illustrated is the expression of 22 selected genes affected by QIN in qPCR analysis using QIAGEN plates.

[0017] Figure 1b Illustrated is the expression of four selected genes affected by QIN in separate qPCR analyses.

[0018] Figure 2 The generated protein-protein interaction network illustrates the interactions of the 13 selected genes with each other among all the selected gene expressions affected by QIN.

[0019] Figure 3 Description from Figure 2 Expanded protein-protein interaction network of 13 genes stimulated by QIN in the .

[0020] Figure 4a Illustration of the expression of 11 selected genes affected by QIN and QIN-GMI in qPCR analysis using QIAGEN plates.

[0021] Figure 4bIllustrated are the expressions of three selected genes affected by QIN and QIN-GMI in separate qPCR analyses.

[0022] Figure 5 Illustrated is the expression of 22 selected genes affected by GMI protein, QIN and QIN-GMI in qPCR gene analysis using QIAGEN plates.

[0023] Figure 6 Describes the results of animal testing using QIN as an immunomodulator (QIN-IM) in pigs vaccinated with CIRCOQ. DETAILED DESCRIPTION

[0024] The following representative examples illustrate various features and embodiments of the present disclosure, which are intended to be illustrative rather than limiting. One of ordinary skill in the art will readily appreciate that the specific embodiments are merely illustrative of the present invention, as more fully described in the appended claims. Each embodiment and feature described in this application should be understood to be interchangeable and combinable with each embodiment contained therein.

[0025] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All publications cited herein are incorporated herein by reference.

[0026] In one embodiment, a method of enhancing an immune response in a subject in need thereof comprises administering to the subject an effective amount of an antigen and an immunomodulatory agent, wherein the immunomodulatory agent is a quaternary immunomodulatory nanoparticle (QIN).

[0027] One embodiment provides chimeric nanoparticles comprising QIN coupled to an antigenic determinant of a GMI protein.

[0028] One embodiment provides a pharmaceutical composition comprising QIN and a pharmaceutically acceptable carrier, excipient, or diluent.

[0029] In one embodiment, the above-described pharmaceutical composition is provided for the manufacture of a medicament for treating a subject suffering from cancer or a viral infection.

[0030] In one embodiment, the QIN is coupled to an antigenic determinant of a Ganoderma microsporum immunomodulatory (GMI) protein. In one embodiment, the QIN can be administered orally to a subject. In another embodiment, the subject can be an animal species selected from the group consisting of mice, rats, rabbits, dogs, pigs, sheep, cattle, and primates. In another embodiment, the antigen is selected from the group consisting of a tumor antigen, a viral antigen, a bacterial antigen, a self antigen, and a fungal antigen.

[0031] Example

[0032] Materials and Methods

[0033] Cell culture:

[0034] THP-1 cells were purchased from the American Type Culture Collection (ATCC). The cells were cultured in RPMI-1640 medium supplemented with 10% FBS in a 6% CO2, 37°C incubator. 5 Cells were passaged at a concentration of 100 cells / ml the day before treatment. Cells were harvested 48 hours after treatment for RNA extraction, and their supernatants were used for ELISA analysis of cytokines.

[0035] RNA and cDNA:

[0036] RNA was purified from the treated cells using RNeasy Mini Kit (Qiagen, Model 74104). TM 0.5 μg of RNA was reverse transcribed using III First-Strand Synthesis SuperMix for qRT-PCR (Thermofisher, model 11752250) to obtain cDNA for RT-PCR.

[0037] RT-PCR:

[0038] Use pre-designed species-specific and gene-specific KiCqStart TM Primers. RT-PCR was performed with Luminaris Color HiGreen qPCR Master Mix (Thermofisher, Model K0392). The final concentration of each forward and reverse primer was 0.3 mM, and the reaction volume was 20 ml. A three-step cycle program was followed. The reaction started at 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of the following: 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. PCR was performed using the CFX96 Real-Time PCR Detection System from Bio-Rad.

[0039] Enzyme-linked immunosorbent assay (ELISA):

[0040] Human inflammatory cytokines in the supernatant were analyzed using the Multi-Analyte ELISArray kit (Qiagen, Model MEH-004A). The cytokines measured in this analysis were IL1a, IL1b, IL2, IL4, IL6, IL8, IL10, IL12, IL17A, IFNg, TNFa, and GM-CSF. A panel of target cytokine-specific antibodies was coated on a 96-well ELISA plate, with each antibody coated in a row of 8-well strips. Cytokines in each sample were detected according to standard ELISA protocols. Positive and negative controls were included in this assay.

[0041] Building QIN's Architecture

[0042] The coding sequence dORF2 of QIN (obtained from, for example, but not limited to, crystal structure analysis of capsid protein [Hepatitis E virus (Myanmar strain)], sequence ID: 2ZZQ_A (SEQ ID NO: 1); other known sequences of the second open reading frame (ORF2) can also be used herein, such as the sequence described in U.S. Patent Publication No. US20190031720A1) was modified to the sequence shown in SEQ ID NO: 2 to meet the optimal expression efficiency in insect cells, and then synthesized by Integrated DNA Technologies (Skokie, Illinois, USA). According to pFastBac1-dORF2 was generated using the protocol of the Baculovirus Expression System Kit (Life Technologies) and used to generate the recombinant baculovirus AcBactoBac-dORF2. Similarly, the recombinant transfer vector plasmid pVL1392-dORF2 was prepared and then linearized with BestBac TM The recombinant baculovirus AcBestBac-dORF2 was generated by transfection with 2.0 (Δv-cath / chiA) parental baculovirus DNA (Expression Systems, Davis, CA, USA). Both AcBactoBac-dORF2 and AcBestBac-dORF2 were routinely amplified in Sf-9 cells grown in ESF921 serum-free medium (Expression Systems, Davis, CA, USA) at 27°C. Virus titers were determined using an anti-gp64 antibody.

[0043] General parameters for inoculation (with AcBactoBac-dORF2 or AcBestBac-dORF2) and harvesting of cell culture supernatant. During the optimization phase, 12 parameters were tested and performed as follows. Day 1: 1×106 Four 500mL High Five cell cultures were inoculated into ESF921 medium at a concentration of 10 cells / mL. The next day: HEV-573C (BestBac) was infected with one culture at a multiplicity of infection (MOI) = 0.1, and another culture was infected with MOI = 3.0. HEV-573C (Bacmid) was infected with one culture at MOI = 0.1, and another culture was infected with MOI = 3.0. The culture was shaken and cultured for two hours. For each culture bottle, 100mL of culture was transferred to each of four 250mL flasks. The viable cell count and activity percentage were determined every day. Cells were stained for gp64 expression 1 day and 2 days after infection (days post-infection, dpi). At 5, 6 and 7dpi, 100mL of cells under each culture condition were precipitated, and the supernatant of each culture was aseptically transferred to two 50mL test tubes. The precipitate was stored at -80°C. Supernatant samples were stored at 4°C.

[0044] Subsequently, 2×10 6 High Five cells / mL. At 6 dpi, cell supernatants were collected for purification. QIN expressed from ORF2 construct variants was purified by ultracentrifugation. QIN was collected and purified by multiple centrifugation steps including CsCl equilibrium density gradient ultracentrifugation. Purified QIN was resuspended in 10 mM potassium-MES buffer (pH 6.2) and stored at 4°C. TM The spectrophotometer was based on ultraviolet spectroscopy (A 280 ) Determine the protein concentration and purity of the fractions showing QIN positive signal in the ELISA test.

[0045] Construction of chimeric nanoparticle QIN-GMI constructs:

[0046] The GMI antigenic determinant MSDTALIFTLAWNVK (SEQ ID NO: 3) was inserted into QIN, dORF2 (i.e., the capsid subunit of QIN herein) within the 498-506 amino acid positions (dORF2 was obtained from, for example, but not limited to, the crystal structure analysis of the capsid protein [Hepatitis E virus (Myanmar strain)], sequence ID: 2ZZQ_A (SEQ ID NO: 1); other known sequences of ORF2 can also be used herein, such as the sequence described in U.S. Patent Publication No. US20190031720A1) to obtain QIN-GMI as shown in SEQ ID NO: 4, which was synthesized by Integrated DNA Technologies (IDT) after optimization for optimal expression in insect cells. The following process is similar to the above-mentioned process for constructing the QIN construct. Briefly, the synthesized DNA fragment was cloned into the BestBac Bacmid vector (Expression Systems, Davis, California, USA). The mutant ORF2 protein was expressed in High Five cells at MOI=3 and collected at 6 dpi, followed by multi-step density gradient ultracentrifugation (see Li et al., 2005, J Virol 79(20):12999-13006) [2] The purified chimeric nanoparticles QIN-GMI were resuspended in 10 mM potassium-MES buffer (pH 6.2) and stored at 4°C.

[0047] Transmission Electron Microscope:

[0048] Purified QIN and QIN-GMI were loaded onto glow-discharged, carbon-coated electron microscope grids and stained with 2% uranyl acetate and examined under a JOEL JEM-1230 transmission electron microscope at a magnification of 30,000 times. Images were recorded with a CCD camera (TVIPS Gauting, Germany).

[0049] Table 1: Amino acid sequence listing

[0050]

[0051]

[0052] Animal experiments (see Do et al., 2021, Can J Vet Res 85(2):93-100) [2] :

[0053] The animal experimental protocol performed in this study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ruibao Gene Co., Ltd., and this study was conducted in the animal facilities of the GMP facility of Ruibao Gene Co., Ltd. Healthy pigs aged 21 days (+ / - 1 day) that tested negative for PCV2, porcine reproductive and respiratory syndrome virus (PRRSV), and classical swine fever virus (CSFV) were selected for this study.

[0054] The study was divided into three experimental groups: Group A, Group B and Group C. Group A: 4 piglets as placebo group; Group B: 5 piglets vaccinated with CIRCOQ at 3 to 6 weeks of age and oral administration of QIN at a dose of 20 μg / kg daily; and Group C: 5 piglets vaccinated with CIRCOQ only. Four weeks later (i.e. 10 weeks of age), all piglets were challenged with PCV2. The immune response was analyzed with the increase in body weight as a factor of immunomodulator. The increase in body weight, IgG and IgA levels were measured and statistically analyzed.

[0055] All experimental pigs were ear-tagged and could be tracked individually by clicking on the number. Subsequently, the pigs in each group were housed in different pens in the same pig house and cared for in the same way. Clinical signs, including respiratory failure, dermatitis, growth retardation and mortality, were monitored daily. Productivity indicators, including average weight gain (AWG) and average daily weight gain (ADWG), were calculated based on the time from the start of the experiment at 21 days of age to the end of 182 days of age.

[0056] Thirty serum samples were randomly collected from each group at 0, 14, 28, 56, 84, 140, and 161 days after vaccination, and the antibody response and the extent of PCV2 viremia were determined using a commercially available PCV2 enzyme-linked immunosorbent assay (ELISA) kit (SK105; BioChek, Reeuwijk, The Netherlands). According to the manufacturer's instructions, serum samples were considered positive for PCV2 antibodies if the sample-to-positive (S / P) ratio was higher than 0.4.

[0057] Statistical analysis

[0058] One-way analysis of variance (ANOVA) was used to analyze continuous data (Ct values ​​of PCV2 qRT-PCR, serological IgG and IgA), followed by Tukey's multiple comparison test at each time point. Fisher's exact test and chi-square test were used for discrete data (clinical symptoms such as respiratory failure, growth retardation, dermatitis, and death). P < 0.05 was considered significant.

[0059] All of the above-cited patents and publications are expressly and entirely incorporated herein by reference.

[0060] Genes selected in this example:

[0061] Innate immunity: TLR8 / TLR7, LY96, TLR4, CD14

[0062] Toll-like receptors (TLRs) are important pathogen recognition receptors (PRRs) in the host and are key molecules in the innate immune system, inflammatory response and acquired immunity. Among them, TLR8 and TLR7 are highly similar in structure and can recognize viral single-stranded RNA (ssRNA). Another Toll-like receptor, TLR5, is considered to have therapeutic potential for the treatment of Alzheimer's disease (AD) because of its innate immunity.

[0063] Lymphocyte antigen 96 (LY96), also known as "myeloid differentiation factor 2 (MD-2)", is associated with TLR4 and the LPS-LY96 (MD2)-TLR4 pathway of innate immunity. LY96, together with Toll-like receptor 4 (TLR4), binds lipopolysaccharide (LPS) with high affinity, inducing the formation of activated homodimers LPS-LY96 (MD2)-TLR4, which in turn activates downstream signaling of cytokine and chemokine production, as well as the initiation of inflammatory and immune responses.

[0064] CD14 is a glycosylphosphatidylinositol (GPI)-anchored receptor known to act as a co-receptor with TLR4 and promote cellular responses to low doses of lipopolysaccharide (LPS).

[0065] Multiple immune-destructive mechanisms contribute to the development of chronic infection and cancer, such as the production of anti-inflammatory cytokines, the induction of regulatory T (Treg) cells, and the expression of immune checkpoint molecules, including CTLA-4 and PD-1. CTLA-4 is expressed on T cells and interacts with CD80 / CD86, thereby limiting T cell activation.

[0066] Tumor immunity: ICAM1, ITGAM, NFKBIA

[0067] Intracellular adhesion molecule 1 (ICAM1) is associated with a variety of immune and inflammatory responses in the tumor microenvironment and the formation of tertiary lymphoid structures (TLS). Variation in the ITGAM gene, which encodes the CD11b chain of the αMβ2 (Mac-1; CD11b / CD18) integrin, is one of the strongest genetic risk factors for autoimmune diseases such as systemic lupus erythematosus (SLE). NFKBIA, which encodes the nuclear factor of B-cell inhibitor-α-kappa-light polypeptide gene enhancer, has been proposed as an inhibitor of the EGFR signaling pathway associated with tumorigenesis.

[0068] Cytokines / humoral immunity / inflammatory response: C3, MX1, CCL2, CXCL10, CXCL8, CCL5, CD40, CD4

[0069] Complement protein C3 is required for the efficient induction of humoral and cellular acquired immune responses to either M2e-based or HA-based vaccination. MX1 is a GTPase that is part of the antiviral response induced by interferons (type I and III) in infected hosts. Viral proteins, such as polymerase basic protein 2 (PB2) and nucleoprotein (NP), are considered direct targets of MX1. Inhibition of PB2-NP interaction is an active process that requires enzymatic activation of MX1. The chemokine CCL2, also known as “monocyte chemoattractant protein-1” (MCP-1), and its major chemokine receptor CCR2 have been implicated in the pathogenesis of a variety of different disease processes, including vascular permeability and attraction of immune cells during metastasis, several different neurological diseases, autoimmune diseases, obesity, and atherosclerosis. The involvement of CCL2 signaling in a variety of diseases makes it an attractive therapeutic target. The antimicrobial gene CXCL-10 encodes a ligand for the CXC subfamily of chemokines and receptors CXCR3. Binding of CXCL-10 to CXCR3 produces pleiotropic effects, including stimulation of monocyte, natural killer, and T cell migration, and regulation of adhesion molecule expression. The ligand CXCL8 and its chemokine receptors CXCR1 / 2 are critical for the activation and trafficking of inflammatory mediators, as well as tumor progression and metastasis. The CXCL8-CXCR1 / 2 signaling axis is involved in the pathogenesis of multiple diseases, including chronic obstructive pulmonary diseases (COPD), asthma, cystic fibrosis, and cancer.

[0070] CCL5 is a cytotoxic CD8 + T cells are considered to be one of the main groups of effector immune cells in anti-tumor immunity; however, the lack of CD8+ T cells in the central tumor area has become a major obstacle to immunotherapy of solid tumors, especially colorectal cancer (CRC). Previous studies have shown that CCL5 deficiency promotes CD8 + T cells accumulate at tumor sites in a CRC mouse model to delay tumor growth and metastasis.

[0071] CD40 is a potential target for cancer immunotherapy. CD40 agonists have shown some anti-tumor activity and manageable toxicity profiles in patients with advanced solid malignancies.

[0072] CD4 +T cells are primarily considered to be coordinators of immune responses and are able to differentiate into different helper T cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions.

[0073] Interleukin 1β (IL1B) is a proinflammatory cytokine that is important for both normal immune responses and chronic inflammatory diseases.

[0074] Cytotoxic T cell (CTL) responses are determined by the peptide repertoire presented by the subject's HLA class I molecules. During aging, senescent cells accumulate in human tissues and contribute to age-related pathologies. It has been proposed that HLA-E expression is induced by proinflammatory cytokines associated with a senescence-associated secretory phenotype and is regulated in vitro by p38 MAP kinase signaling.

[0075] STAT1, STAT2 and IRF9 are interferon-stimulated genes responsible for signal transduction downstream of type I and type III IFN receptors. Transcriptional regulation of their expression represents an important feedback loop regulating IFN signaling.

[0076] Heme catabolism: HMOX1

[0077] Heme oxygenase is an essential enzyme in heme catabolism that cleaves heme to form biliverdin (which is subsequently converted to bilirubin by biliverdin reductase) and carbon monoxide (a putative neurotransmitter). Heme oxygenase occurs in the form of two isozymes, one is the inducible heme oxygenase-1 (HMOX1) and the other is the constitutive heme oxygenase-2 (HMOX2).

[0078] Intestinal immunity: CCR6 is inhibited by QIN

[0079] The intestinal lamina propria (LP) contains antigen-presenting cells with characteristics of dendritic cells and macrophages, collectively referred to as mononuclear phagocytes (MNPs). The association of MNPs with epithelial cells is thought to play an important role in multiple aspects of intestinal immunity, including imprinting the ability of MNPs to induce IgA production, inducing the expression of intestinal homing molecules on T cells, and promoting the capture of luminal antigens and microorganisms and subsequent immune responses in the mesenteric lymph nodes (MLN). CCR6 is the only known CCL20 receptor, and MNP binding to epithelial cells requires CCR6. These findings identify a key role for CCR6 in promoting LP-MNP binding to steady-state intestinal epithelial cells to perform multiple functions that promote intestinal immune homeostasis.

[0080] AQP1: This gene encodes a small integral membrane protein with six double-layer spanning domains that functions as a water channel protein. This protein allows for the passive transport of water along an osmotic gradient. This gene may be involved in diseases associated with imbalances in ocular fluid movement.

[0081] IL6: Interleukin 6 (IL-6) has a wide range of effects on cells of the innate and adaptive immune systems as well as non-immune systems, and often expresses a hormone-like signature that influences homeostatic processes. IL-6 has context-dependent pro-inflammatory and anti-inflammatory properties and is now considered an important target for clinical intervention.

[0082] IL23A: Risankizumab is a fully human monoclonal antibody that selectively targets interleukin (IL)-23A, interfering with the IL-23 / 17 axis, which plays a key role in keratinocyte proliferation. Risankizumab was approved globally in 2019 for the treatment of moderate to severe psoriasis. Drugs that block the IL-23 pathway are the most recently approved drugs for the treatment of psoriasis, and risankizumab appears to be the most effective of the three approved IL-23 blockers.

[0083] DDX58: Interestingly, several innate immune genes associated with psoriasis have recently been identified among the interferon and antiviral response genes. These genes are DDX58 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 58), which encodes the protein RIG-I, and IFIH1, which encodes the protein MDA5. Both RIG-I and MDA5 are innate immune sensors within cells that are extremely important for the detection of double-stranded RNA. RIG-I specifically recognizes shorter RNAs containing a 5' triphosphate and a 5' blunt-ended base pair, while MDA5 is thought to recognize longer RNA structures. Both are important for the recognition of dsRNA viruses or viruses that utilize dsRNA replication intermediates and the subsequent initiation of a type I interferon response.

[0084] Experimental Results

[0085] See also Figure 1a and 1b ,in Figure 1a Illustrates the expression of 22 selected genes affected by QIN in qPCR analysis using QIAGEN plates. Figure 1b The expression of four selected genes affected by QIN in separate qPCR analyses is illustrated. It can be found that most of the 26 genes in total showed a somewhat consistent pattern, where except for CCR6 and AQP1 which were slightly inhibited in THP-1 monocytes, the remaining genes STAT1, HLA-E, UL1B, HLA-A, CD40, CD4, NFKBIA, CCL5, CD80, MX1, CXCL8, TLR7, CCL2, TLR5, C3, CD86, ITGAM, ICAM1, CD14, LY96, TLR8, TLR4, HMOX1, and CXCL10 were all stimulated / activated by QIN.

[0086] Protein-protein interaction analysis

[0087] See also Figure 2 , which illustrates the interaction relationship between the 13 selected genes shown in the generated protein-protein interaction network among all selected gene expressions affected by QIN. The protein-protein interaction (PPI) map of the 13 highly expressed genes is shown in Figure 2. Figure 1a and 1bAs shown, it was generated using the STRING toolkit (https: / / version11.string-db.org / cgi / input.pl?sessionId=uoELhwcyqUyX&input_page_show_search=on) for the analysis of genes stimulated by QIN. Different colors represent clustering (grouping) of interaction similarities. According to the protein-protein interaction map, 10 genes including HMOX1 are closely related to each other.

[0088] Please read further Figure 3 , which is described from Figure 2 Using existing knowledge of protein-protein interactions, we extended the protein-protein interaction network of 13 genes stimulated by QIN in Figure 2 The 13 genes shown to be stimulated by QIN were further expanded to other related genes, and another map of 113 interactors was generated. Among them, 19 genes are related to positive regulation of RNA polymerase II promoter transcription; 16 genes are related to signal transduction; 14 genes are related to apoptotic processes; 14 genes are related to transcription and DNA templates; and 12 genes are related to innate immune responses. For details, please refer to Table 2 below.

[0089] Table 2: Gene ontology (GO) annotations of the QIN-GMI network of 13 highly expressed genes

[0090] Biological Process count ％ P-value Positive regulation of transcription from RNA polymerase II promoters 19 23.5 5.44E-08 Signal transduction 16 19.8 5.91E-05 Inflammatory response 15 18.5 3.52E-10 Cell apoptosis process 14 17.3 4.41E-07 Transcription, DNA template 14 17.3 5.33E-02 Innate immune response 12 14.8 1.31E-06 Positive regulation of transcription, DNA template 11 13.6 4.52E-05 Positive regulation of gene expression 10 12.4 1.20E-06 Negative regulation of transcription from RNA polymerase II promoters 10 12.4 2.64E-03 Cellular response to lipopolysaccharide 9 11.1 1.95E-08 Virus infection process 9 11.1 3.00E-05 Negative regulation of apoptosis 9 11.1 5.37E-04 LPS-mediated signal transduction pathway 8 9.9 4.49E-11 Signal transduction pathways of cell surface receptors 8 9.9 1.30E-04 Positive regulation of GTPase activity 8 9.9 8.37E-03

[0091] QIN vs QIN-GMI

[0092] See also Figure 4a and 4b ,in Figure 4a The expression of 11 selected genes affected by QIN and QIN-GMI in qPCR analysis using QIAGEN plates is illustrated; Figure 4b The expression of 3 selected genes affected by QIN and QIN-GMI in separate qPCR analyses is illustrated. It can be seen that, overall, the genes stimulated by QIN-GMI were similar to those stimulated by QIN alone, and all 14 selected genes met the criteria. Of these, 13 genes (50% of the 26 genes in the QIN list) in the QIN-GMI treatment condition were in the same direction as the QIN treatment condition. One of the genes (TLR7) was in a slightly opposite direction.

[0093] GMI vs QIN vs QIN-GMI

[0094] Further reading Figure 5, which illustrates the expression of 22 selected genes affected by GMI, QIN and QIN-GMI in qPCR gene analysis using QIAGEN plates. It can be found that, in general, the trends of genes stimulated by GMI, QIN and QIN-GMI are similar, and all 22 selected genes meet this criterion. Among them, 19 genes (80% of the genes) under GMI treatment conditions have the same trend as those under QIN and QIN-GMI treatment conditions. Three genes, CD4, CD80 and CD3, were affected in opposite directions. Surprisingly, QIN alone showed a similar gene profile to GMI and QIN-GMI, which shows the potential role of QIN as an immunomodulator.

[0095] Animal test results:

[0096] Based on the experimental results, QIN was further used as an oral immunomodulator for piglets vaccinated with PCV2 sub-unit vaccine, which improved immune response and weight gain.

[0097] See Table 3 and Figure 6 It can be found that the average weight gain (AWG) or average daily weight gain (ADWG) of pigs in group B, which were vaccinated with PCV2 subunit vaccine (CIRCOQ) and oral QIN daily, was the highest, while that of pigs in group C, which were vaccinated with PCV2 subunit vaccine (CIRCOQ) only, was the second highest, and that of pigs in group A, which were given placebo, was the lowest (Table 3). The same trend was found in the serum PCV2-specific IgG response ( Figure 6 ). In contrast, there were no differences in IgA measurements (data not shown).

[0098] Table 3: Growth and body weight of pigs treated with placebo, CIRCOQ plus oral QIN, and CIRCOQ alone

[0099]

[0100] discuss

[0101] The original purpose of this study was to use QIN as an oral delivery vehicle for the immunomodulator GMI. In addition to QIN-GMI, stimulation with QIN alone in HTP-1 cells resulted in a gene profile similar to that of GMI. From the protein-protein interaction network generated from the 13 highly expressed genes stimulated by QIN, only 9 of the 113 genes affected genes related to the viral infection process (Table 2). This finding may indicate that QIN alone may be a potential immunomodulator without triggering an actual viral immune response. However, a similar gene profile with a more significant stimulation by QIN with an inserted GMI peptide (i.e., QIN-GMI) may be explained by a multivalent QIN that contains 60 copies of the GMI protein peptide on its surface. A stronger stimulation of the gene profile associated with the innate / acquired immune response can only indicate the dose of QIN. A lower amount of QIN-GMI than GMI alone is required to exert the effect of an immunomodulator. Animal trials on piglets vaccinated with a PCV2 subunit vaccine (CIRCOQ) further validated the immunomodulatory effects of oral QIN, showing better average weight gain and higher IgG titers.

[0102] in conclusion

[0103] As mentioned above, the chimeric QIN-GMI stimulated a higher gene expression profile than that obtained with QIN alone and GMI protein alone, without stimulating a strong viral immune response in THP-1 monocytes. Surprisingly, QIN alone also showed a smaller but similar trend than QIN-GMI in the genetic analysis. These results show that QIN alone, as an oral immunomodulator, showed better average weight gain and higher IgG titers in animal experiments in piglets vaccinated with PCV2 subunit vaccine (CIRCOQ). Therefore, the positive impact of QIN as an immunomodulator (QIN-IM) and even QIN-GMI on PCV2 subunit vaccine can be demonstrated.

[0104] Without further elaboration, it is believed that one of ordinary skill in the art can utilize the present invention to its fullest extent based on the above description.Therefore, the above specific embodiments should be interpreted as merely illustrative and not limiting the rest of the present disclosure in any way.

[0105] References

[0106] 1. Li TC, Takeda N, Miyamura T, Matsuura Y, Wang JCY, Engvall H, Hammar L, Xing L, Cheng RH: Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus. Journal of Virology, 2005, 79(20): 12999-13006.

[0107] 2. Do DT, Tran KDV, Quach AT, Lee D, Chang FC, Wu CP, Tat TN, Chae C: A comparative efficacy test of 1 versus 2 doses of CIRCOQ PCV2 subunit vaccine against naturally occurring PCV2-type d in piglets with high maternally derived antibodies (MDAs) on a Vietnamese swine farm. Canadian Journal of Veterinary Research, 2021, 85(2): 93-100.

Claims

1. A method for enhancing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of an antigen and an immunomodulator, wherein the immunomodulator is a quaternary immunomodulatory nanoparticle (QIN).

2. The method according to claim 1, wherein the QIN is further coupled to an antigenic determinant of a Ganoderma microsporum immunomodulatory (GMI) protein.

3. The method of claim 2, wherein the antigenic determinant of the GMI protein is coupled to the QIN by being inserted into the capsid subunit of the QIN. The method according to claim 2 , wherein the antigenic determinant of the GMI protein has a sequence as shown in SEQ ID NO:

1.

5. The method of claim 1, wherein the QIN is administered to the subject orally.

6. The method of claim 1, wherein the subject comprises an animal species selected from the group consisting of mice, rats, rabbits, dogs, pigs, sheep, cattle, and primates.

7. The method of claim 1, wherein the antigen is selected from the group consisting of a tumor antigen, a viral antigen, a bacterial antigen, a self antigen, and a fungal antigen.

8. The method of claim 7, wherein the antigen is a viral antigen.

9. The method of claim 8, wherein the viral antigen is a porcine circovirus type 2 (PCV2) antigen.

10. The method of claim 9, wherein the PCV2 antigen is administered to the subject via a PCV2 vaccine.

11. A chimeric nanoparticle comprising a quaternary immunomodulatory nanoparticle (QIN) coupled to an antigenic determinant of a Ganoderma microsporum immunomodulatory (GMI) protein.

12. The chimeric nanoparticle of claim 11, wherein the antigenic determinant of the GMI protein is coupled to the QIN by being inserted into the capsid subunit of the QIN.

13. The chimeric nanoparticle of claim 11, wherein the antigenic determinant of the GMI protein has a sequence as shown in SEQ ID NO:

1.

14. A pharmaceutical composition comprising quaternary immunomodulatory nanoparticles (QIN) and a pharmaceutically acceptable carrier, excipient or diluent.

15. The pharmaceutical composition according to claim 14, wherein the QIN is further coupled to an antigenic determinant of the GMI protein.

16. The pharmaceutical composition according to claim 15, wherein the antigenic determinant of the GMI protein is coupled to the QIN by being inserted into the capsid subunit of the QIN. 17 . The pharmaceutical composition according to claim 15 , wherein the antigenic determinant of the GMI protein has a sequence as shown in SEQ ID NO:

1.

18. Use of the pharmaceutical composition according to claim 15 in the manufacture of a medicament for treating a subject suffering from cancer or viral infection.

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