Methods and Compositions of Quaternary Immunomodulatory Nanoparticles for Medical Use 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 together with an immunomodulatory agent, wherein the immunomodulatory agent is a quaternary immunomodulatory nanoparticle (QIN). The present disclosure also provides the use of QIN in the manufacture of a chimeric nanoparticle, a pharmaceutical composition and a medicament for treating a subject suffering from cancer or a viral infection.

Description

Technical Field

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

Background Art

[0002] Hepatitis E is an enteric virus that causes acute liver inflammation in humans. Quaternary immunomodulatory nanoparticles (QINs), derivatives thereof based on hepatitis E virus nanoparticles, exhibit a stable icosahedral cage that is characteristic of being retained from its natural transmission pathway in an acidic and proteolytic environment.

[0003] The hepatitis E virus is composed of a non-enveloped icosahedral capsid that encapsulates a 7.2 kilobase single-stranded RNA genome and is an enteric virus that causes acute liver inflammation in humans. The major capsid protein is essential for virus assembly, immunogenicity, and host interaction. Recombinant capsid protein can self-assemble into virus-like particles when expressed in insect cells after deletion of 111 amino acids from the N-terminus and 52 amino acids from the C-terminus. QINs are stable in acidic and proteolytic environments and exhibit an icosahedral cage with characteristics retained from its natural transmission pathway. QINs consist of 60 subunits and form icosahedral nanoparticles. QINs are stable in acidic environments and resistant to proteolytic digestion, thus providing a great advantage as an oral delivery vehicle.

[0004] A natural fungal medicine derived from Ganoderma, also known as Lingzhi in traditional Chinese medicine, is one of the most well-known traditional medicinal species in China. It has been widely used for centuries to treat various diseases in China, the United States, Japan, South Korea, and other Asian countries. Its immunomodulatory proteins and polysaccharides have been identified as important bioactive factors in cancer immunotherapy and the NF-kB and MAPK pathways. Therefore, even in modern medical practice, Ganoderma has been used to treat chronic bronchitis, bronchial asthma, leukopenia, coronary heart disease, arrhythmia, and acute infectious hepatitis. However, in the current clinical setting, it is mainly used as an adjuvant therapy in addition to primary treatment. Many studies have shown that Ganoderma has antitumor effects caused by modulating the immune system due to its fungal immunomodulatory protein (FIP), polysaccharides, and triterpenoids. Among them, GMI, an important immunomodulatory protein cloned from Ganadorma microsporum, has been extensively studied. In in vitro tests conducted by Lin et al., it was found that GMI dose-dependently inhibits EGF-induced phosphorylation and activation of EGFR and AKT pathway kinases. In vivo studies also suggested that oral administration of GMI inhibits tumor growth and induces autophagy in a mouse model.

[0005] Porcine circovirus type 2 (PCV2) is a small, non-enveloped DNA virus and is recognized as one of the most important porcine pathogens of porcine circovirus disease (PCVD), causing economic losses and reduced productivity in all major pig-producing countries worldwide. Vaccination against PCV2 is currently routinely carried out in the pig farming industry in most developed countries. CIRCOQ, a commercially available PCV2 subunit vaccine, has been developed by Reber Genetics Company (Taipei City, Taiwan) to protect against emerging PCV2. SUMMARY OF THE INVENTION

[0006] The present invention provides for the medical applications of quaternary immunomodulatory nanoparticles (QINs), in particular, the enhancement of immune responses in subjects suffering from cancer or viral infections, as well as the use of QINs in the manufacture of chimeric nanoparticles, pharmaceutical compositions, and medicaments.

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

[0008] Preferably, the QIN further binds to an epitope of a Ganoderma microsporum immunomodulatory (GMI) protein.

[0009] Preferably, the epitope of the GMI protein binds to the QIN by insertion into the capsid subunit of the QIN.

[0010] Preferably, the epitope of the GMI protein has the sequence represented by SEQ ID NO: 1.

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

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

[0013] In another aspect of the present invention, there is provided a chimeric nanoparticle comprising a QIN bound to an epitope of a GMI protein.

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

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

[0016]

Fig. 1a

[0017]

Fig. 1b

[0018]

Fig. 2

[0019]

Fig. 3

[0020]

Fig. 4a

[0021]

Fig. 4b

[0022]

Fig. 5

[0023]

Fig. 6

[0024] The following representative examples illustrate various features and embodiments of the present disclosure and are intended to be exemplary and not limiting. Those skilled in the art will readily understand that specific examples are merely illustrative of the invention more fully described in the claims that follow. It should be understood that all embodiments and features described in this application are interchangeable and combinable with all embodiments included herein.

[0025] Unless otherwise defined, 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 this invention belongs. All publications cited herein are incorporated 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 together with an immunomodulator, wherein the immunomodulator is a quaternary immunomodulatory nanoparticle (QIN).

[0027] One embodiment provides a chimeric nanoparticle comprising QIN conjugated to an epitope of a GMI protein.

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

[0029] In one embodiment, the pharmaceutical composition described above is provided for manufacturing a medicament for treating a cancer or virus-infected subject.

[0030] QIN binds to an epitope of the Ganoderma microsporum immunomodulatory (GMI) protein. In one embodiment, QIN can be orally administered 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, cows, and primates. In another embodiment, the antigen can be selected from the group consisting of tumor antigens, viral antigens, bacterial antigens, self-antigens, and fungal antigens.

[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 an incubator at 6% CO2 and 37°C. The cells were passaged at a concentration of 5×10 5 / ml the day before treatment. Cells were harvested 48 hours after treatment for RNA extraction, and the supernatant was harvested for cytokine ELISA analysis.

[0035] RNA and cDNA:

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

[0037] RT-PCR:

[0038] Pre-designed species-specific and gene-specific KiCqStart™ primers from Sigma were used. RT-PCR was performed using Luminaris Color HiGreen qPCR Master Mix (Thermofisher K0392). The final concentration of each forward and reverse primer was 0.3 mM, and the reaction volume was 20 μl. A three-step cycling protocol was followed. The reaction was initiated at 50 °C for 2 minutes, 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds. PCR was performed using a CFX96 Real-Time PCR Detection System from Bio-Rad.

[0039] ELISA:

[0040] Human inflammatory cytokines in the supernatant were analyzed using the Multi-Analyte ELISArray Kit (Qiagen MEH-004A). The cytokines represented by this array are IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12, IL17A, IFNγ, TNFα, and GM-CSF. 96-well ELISA plates were coated with a panel of cytokine-target specific antibodies, one per 8-well strip. Cytokines in each sample were detected following standard ELISA procedures. Positive and negative controls were included in the assay.

[0041] Construction of the QIN construct

[0042] The dORF2, which is the coding sequence of QIN (for example, but not limited to, obtained from the crystal structure analysis of the capsid protein [Hepatitis E virus (Myanmar strain)] sequence ID: 2ZZQ_A (SEQ ID NO: 1), and other known sequences of the second open reading frame (ORF2) described in, for example, US Patent No. 20190031720 can also be used herein) was modified to be like the sequence represented by SEQ ID NO: 2 to meet the best expression efficiency in insect cells and then synthesized by Integrated DNA Technologies (Coralville, Iowa). pFastBac1-dORF2 was produced and used to generate the recombinant baculovirus AcBactoBac-dORF2 according to the Bac-to-Bac® baculovirus expression system kit protocol (Life Technologies). Similarly, the recombinant transfer vector plasmid pVL1392-dORF2 was produced and then transfected with linearized BestBac™ 2.0 (Δv-cath / chiA) parental baculovirus DNA (Expression Systems LLC, Davis, California) to generate the recombinant baculovirus AcBestBac-dORF2. Both AcBactoBac-dORF2 and AcBestBac-dORF2 were routinely amplified in Sf-9 cells grown at 27 °C in ESF921 serum-free medium (Expression Systems LLC, Davis, California). The virus titer was determined using an antibody against gp64.

[0043] (Common parameters used for inoculation (with AcBactoBac-dORF2 or AcBestBac-dORF2) and collection of cell culture supernatants. During the optimization phase, 12 parameters were tested and executed as follows. Day 1: 1×10 6Seed four 500 mL cultures of High Five cells in ESF921 at cells / mL. Day 2: Infect the cultures with multiplicity of infection (MOI) = 0.1 and MOI = 3.0 with HEV-573C (BestBac). Infect the cultures with MOI = 0.1 and MOI = 3.0 with HEV-573C (Bacmid). Incubate the cultures with shaking for 2 hours. For each bottle, transfer 100 mL of the culture to each of four 250 mL flasks. Examine the viable cell count and viability daily. Stain the cells for gp64 expression at 1 and 2 days post-infection (dpi). At 5, 6, and 7 dpi, pellet the cells from 100 mL of each culture condition, sterilize the supernatant for each culture and transfer it to two 50 mL tubes. Store the pellet at -80 °C. Store the supernatant samples at 4 °C.

[0044] Subsequently, High Five cells at 2 million cells / mL were inoculated at a multiplicity of infection of 3 viruses per cell (MOI = 3). At 6 dpi, the cell supernatant was harvested for purification. Ultracentrifugation purification of QIN expressed from variants of the ORF2 construct was performed. QIN was recovered and purified through multiple centrifugation steps including CsCl equilibrium density gradient ultracentrifugation. The purified QIN was resuspended in 10 mM potassium-MES buffer (pH 6.2) and stored at 4 °C. The protein concentration and purity of the fraction showing a positive signal for QIN in the ELISA test were determined by UV spectrophotometry (A 280 ) using a NanoDrop™ spectrophotometer.

[0045] Construction of the chimeric nanoparticle QIN-GMI construct:

[0046] The GMI epitope MSDTALIFTLAWNVK (SEQ ID NO: 3) was inserted at the positions 498 - 506 aa of dORF2 which is QIN (i.e., the capsid subunit of QIN in this specification) (for example, but not limited to, obtained from the crystal structure analysis of the capsid protein [Hepatitis E virus (Myanmar strain)] SEQ ID: 2ZZQ_A (SEQ ID NO: 1), and other known sequences of ORF2, such as the sequences described in US Patent No. 20190031720 can also be used in this specification) to obtain QIN - GMI represented by SEQ ID NO: 4, which was synthesized by Integrated DNA Technologies, Inc. (IDT) after optimizing the sequence for best expression in insect cells. The following procedure is the same as the procedure described above for the construction of the QIN construct. Briefly, the synthesized DNA fragment was cloned into the BestBac Bacmid vector (Expression Systems, LLC, Davis, California, USA). The mutated ORF2 protein was expressed in High Five cells at an MOI = 3 and harvested at 6 dpi, followed by multiple steps of 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 microscopy:

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

[0049]

Table 1

[0050] Animal experiment (see Do et al., 2021, Can J Vet Res 85(2):93-100) [2] :

[0051] The protocol of the animal experiment conducted in this study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Reber Genetics, Co., Ltd., and this study was conducted in the animal facility of the GMP plant of Reber Genetics, Co., Ltd. Healthy pigs at 21 days of age (+ / -1 day) that tested negative for Porcine circovirus 2 (PCV2), Porcine reproductive and respiratory syndrome virus (PRRSV), and Classical swine fever virus (CSFV) were selected for this study.

[0052] This study had three experimental groups, namely Group A, Group B, and Group C. In Group A, four piglets were used as the placebo group. In Group B, five piglets were vaccinated with CIRCOQ and orally administered QIN at a dose of 20 μg / kg daily from 3 weeks of age to 6 weeks of age. In Group C, five piglets were vaccinated with CIRCOQ only. Then, after 4 weeks (i.e., at 10 weeks of age), all piglets were challenged with PCV2 virus. The immune response and weight gain were analyzed as factors of the immunomodulator. The measurement of weight gain, IgG, and IgA levels, as well as statistical analysis, were performed.

[0053] All experimental pigs were tagged in the ear and individually tracked by clicking on the number. Then, the pigs in each group were housed in different pens in the same shed and raised in the same way. Clinical signs including respiratory failure, dermatitis, stunted growth, and death were monitored daily. Productivity indicators including average weight gain (AWG) and average daily weight gain (ADWG) were calculated based on the time of the experiment starting at 21 days of age and ending at 182 days of age.

[0054] Thirty serum samples were randomly collected from each group on days 0, 14, 28, 56, 84, 140, and 161 after vaccination and tested using a commercially available porcine circovirus type 2 (PCV2) enzyme-linked immunosorbent assay (ELISA) kit (SK105, BioChek, Reeuwijk, the Netherlands) to examine antibody responses and the levels of PCV2 viremia. According to the manufacturer's instructions, serum samples were considered positive for PCV2 antibodies when the sample-to-positive (S / P) ratio was higher than 0.4.

[0055] Statistical analysis

[0056] Continuous data (PCV2 qRT-PCR, Ct values of serum IgG and IgA) were analyzed using one-way analysis of variance (ANOVA), 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 signs such as respiratory failure, stunting, dermatitis, and death). A value of P < 0.05 was considered significant.

[0057] All patents and literature cited above are hereby expressly and fully incorporated by reference into this specification.

[0058] Selected genes in this example:

[0059] Innate immunity: TLR8 / TLR7, LY96, TLR4, CD14

[0060] Toll-like receptors (TLRs), which act as important pathogen recognition receptors (PRRs) in the host, are important molecules for the innate immune system, inflammatory responses, and adaptive immunity. Among these, TLR8 and TLR7, which share high structural similarity, recognize viral single-stranded RNA (ssRNA). Another toll-like receptor, TLR5, is thought to have potential for treating Alzheimer's disease (AD) through its innate immunity.

[0061] Lymphocyte antigen 96 (LY96), also known as "myeloid differentiation factor 2 (MD-2)", and TLR4 are associated with the LPS-LY96 (MD2)-TLR4 pathway of innate immunity. LY96 binds to lipopolysaccharide (LPS) with high affinity together with Toll-like receptor 4 (TLR4), induces the formation of the activated homodimer LPS-LY96 (MD2)-TLR4, and leads to the activation of downstream signal transduction of cytokine and chemokine production, as well as the initiation of inflammatory and immune responses.

[0062] CD14 is a glycosylphosphatidylinositol (GPI)-anchored receptor that is known to function as a co-receptor that works together with TLR4 and promotes the cellular response to low-dose lipopolysaccharide (LPS).

[0063] The development of chronic infections and cancer is promoted by various immune destruction mechanisms, such as the production of anti-inflammatory cytokines, the induction of regulatory T (Treg) cells, and the expression of immune checkpoint molecules such as CTLA-4 and PD-1. CTLA-4 expressed on T cells interacts with CD80 / CD86, thereby restricting T cell activation.

[0064] Tumor immunity: ICAM1, ITGAM, NFKBIA

[0065] Intracellular adhesion molecule 1 (ICAM1) is involved in various immune and inflammatory responses, as well as the formation of tertiary lymphoid-like structures (TLS) in the tumor microenvironment. Mutations in the ITGAM gene encoding the CD11b chain of αMβ2 (Mac-1, CD11b / CD18) integrin are one of the strongest genetic risk factors for autoimmune diseases such as systemic lupus erythematosus (SLE). NFKBIA, which encodes the nuclear factor of the κ light chain polypeptide gene enhancer in B cell inhibitor-α, has been proposed as an inhibitor of the EGFR signaling pathway associated with tumor formation.

[0066] Cytokine / humoral immunity / inflammatory response: C3, MX1, CCL2, CXCL10, CXCL8, CCL5, CD40, CD4

[0067] Complement protein C3 is necessary for the effective induction of humoral and cellular adaptive immune responses to vaccination with M2e-based or HA-based vaccines. MX1 is a GTPase and is, in part, an antiviral response of infected hosts induced by interferons (type I and type III). Viral proteins such as polymerase basic protein 2 (PB2) and nucleoprotein (NP) have been suggested to be direct targets of MX1. Inhibition of the PB2-NP interaction is an active process that requires enzymatically active MX1. Chemokine CCL2, also known as “monocyte chemoattractant protein-1” (MCP-1), and its main chemokine receptor CCR2 are involved in the etiology of several different disease processes such as vascular permeability and the attraction of immune cells during metastasis, several different neuropathies, autoimmune diseases, obesity, and atherosclerosis. The involvement of CCL2 signaling in multiple diseases makes it an attractive therapeutic target. CXCL-10, an antimicrobial gene, encodes a ligand for chemokines of the CXC subfamily and the receptor CXCR3. The binding of CXCL-10 to CXCR3 has multifaceted effects such as stimulation of monocyte, natural killer, and T cell migration, and regulation of adhesion molecule expression. Ligand CXCL8 and its chemokine receptor CXCR1 / 2 are essential for the activation and transport of inflammatory mediators and the progression and metastasis of tumors. The CXCL8-CXCR1 / 2 signaling axis is involved in the etiology of several diseases such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, and cancer.

[0068] CCL5 is a cytotoxic CD8 + T cell, which is considered one of the major populations of effector immune cells in anti-tumor immunity, but the absence of CD8 + T cells in the central tumor region is a major obstacle to solid tumor immunotherapy, especially colorectal cancer (CRC). Previous studies have demonstrated that CCL5 deficiency delays tumor growth and metastasis by promoting the accumulation of CD8 + T cells at the tumor site.

[0069] CD40 is a promising therapeutic target for cancer immunotherapy. CD40 agonists have shown some antitumor activity and a manageable toxicity profile in patients with advanced solid malignancies.

[0070] CD4 + T cells are mainly recognized as orchestrators of the immune response and can differentiate into different T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions.

[0071] Interleukin 1β (IL1B) is a pro-inflammatory cytokine important for both normal immune responses and chronic inflammatory diseases.

[0072] The cytotoxic T lymphocyte (CTL) response is determined by the peptide repertoire presented by an individual's HLA class I molecules. Senescent cells accumulate in human tissues during aging and contribute to age-related pathologies. HLA-E expression has been proposed to be induced by cytokines associated with the senescence-associated secretory phenotype and regulated by p38 MAP kinase signaling in vitro.

[0073] STAT1 is an interferon-stimulated gene that, together with STAT2 and IRF9, is responsible for signal transduction downstream of both type I IFN receptors and type III IFN receptors. The transcriptional regulation of their expression is an important feedback loop that regulates IFN signaling.

[0074] Heme catabolism: HMOX1

[0075] Heme oxygenase, an enzyme essential for heme catabolism, cleaves heme to form biliverdin, which is then converted to bilirubin by biliverdin reductase and carbon monoxide, a neurotransmitter candidate. Heme oxygenase exists as two isozymes, inducible heme oxygenase-1 (HMOX1) and constitutive heme oxygenase-2 (HMOX2).

[0076] Intestinal immunity: CCR6 is inhibited by QIN

[0077] The intestinal lamina propria (LP) contains antigen-presenting cells with the characteristics of dendritic cells and macrophages, collectively called mononuclear phagocytes (MNP). The association between MNP and the epithelium plays an essential role in multiple aspects of intestinal immunity, such as imprinting of MNP with the ability to induce IgA production, induction of the expression of gut-homing molecules on T cells, promotion of the capture of luminal antigens and microorganisms, and subsequent immune responses in mesenteric lymph nodes (MLN). CCR6, the only known receptor for CCL20, was required for MNP to associate with the epithelium. These findings revealed that CCR6 plays an important role in promoting LP-MNP to associate with the intestinal epithelium and perform multiple functions that promote intestinal immune homeostasis under steady-state conditions.

[0078] AQP1: This gene encodes a small integral membrane protein with six transmembrane domains that functions as a water channel protein. This protein enables passive transport of water along an osmotic gradient. This gene is a candidate for disorders associated with imbalance of aqueous humor movement.

[0079] IL6: Interleukin 6 (IL-6) has broad effects on cells of the innate and adaptive immune systems as well as cells that are not immune system cells and often exhibits hormone-like properties that affect homeostasis processes. IL-6 has context-dependent pro-inflammatory and anti-inflammatory properties and is currently regarded as a prominent target in clinical interventions.

[0080] IL23A: Risankizumab is a fully human monoclonal antibody that selectively targets interleukin (IL)-23A and interferes with the IL-23 / 17 axis, which plays an important role in keratinocyte proliferation. In 2019, risankizumab was globally approved for the treatment of moderate to severe psoriasis. Drugs that block the IL-23 pathway are a recently approved treatment for psoriasis, and risankizumab appears to be the most effective among the three approved IL-23 blocking drugs.

[0081] DDX58: Interestingly, some of the more recent associations of innate immune genes with psoriasis have been found in 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 intracellular innate immune sensors important for the detection of double - stranded RNA. RIG - I specifically recognizes shorter RNAs containing a 5' triphosphate moiety and a blunt - ended base pair at the 5' end, while MDA5 is thought to recognize longer RNA structures. Both are important for the recognition of dsRNA viruses or viruses that utilize replication dsRNA intermediates and for the subsequent induction of the type I interferon response.

[0082] Experimental results

[0083] See FIGS. 1a and 1b. FIG. 1a shows the expression of 22 selected genes affected by QIN in qPCR analysis using QIAGEN plates, and FIG. 1b shows the expression of 4 selected genes affected by QIN in individual qPCR analyses. Most of the total 26 genes showed a somewhat consistent pattern, and with the exception that CCR6 and AQP1 were slightly suppressed in THP - 1 monocytes, the remaining genes of 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 found to be stimulated / activated by QIN.

[0084] Protein - protein interaction analysis

[0085] Please refer to Figure 2. Figure 2 shows the relationships of 13 selected genes that interact with each other as shown in the generated protein-protein interaction network among all the selected gene expressions affected by QIN. The protein-protein interaction (PPI) map of the 13 highly expressed genes shown in Figures 1a and 1b was created using the STRING tool kit (https: / / version11.string-db.org / cgi / input.pl?sessionId=uoELhwcyqUyX&input_page_show_search=on), and the genes stimulated by QIN were analyzed. Different colors indicated clustering (grouping) for similarity in interactions. According to the protein-protein interaction map, 10 genes including HMOX1 are closely related to each other.

[0086] Please refer to Figure 3. Figure 3 shows an expanded protein-protein interaction network from the 13 genes stimulated by QIN in Figure 2. The 13 genes stimulated by QIN shown in Figure 2 were further expanded to other related genes using the current knowledge of protein-protein interactions, generating another map with a total of 113 interacting factors. Among them, 19 genes are related to the positive regulation of transcription from RNA polymerase II promoters, 16 genes are related to signal transduction, 14 genes are related to the apoptosis process, 14 genes are related to transcription, DNA template, and 12 are related to the innate immune response. For details, please refer to Table 2 below.

[0087]

Table 2

[0088] QIN vs QIN-GMI

[0089] Please refer to FIGS. 4a and 4b. FIG. 4a shows the expression of 11 selected genes affected by QIN and QIN-GMI in qPCR analysis using a QIAGEN plate, and FIG. 4b shows the expression of 3 selected genes affected by QIN and QIN-GMI in individual qPCR analyses. Generally, the genes stimulated by QIN-GMI are similar to QIN alone, and it can be found that all 14 selected genes conform to this criterion. Among these, 13 genes (50% of the 26 genes in the QIN list) under the QIN-GMI treatment condition were in the same direction as the QIN treatment condition. One gene, TLR7, was slightly in the opposite direction.

[0090] GMI vs QIN vs QIN-GMI

[0091] Please refer to FIG. 5. FIG. 5 shows the expression of 22 selected genes affected by GMI, QIN, and QIN-GMI in qPCR gene analysis using a QIAGEN plate. Generally, it can be found that all 22 selected genes conform to the criterion that genes stimulated by GMI, QIN, and QIN-GMI show a similar trend. Among these, 19 genes (80% of the genes) under the GMI treatment condition were in the same trend as the QIN and QIN-GMI treatment conditions. Three genes, including CD4, CD80, and CD3, were affected in the opposite direction. Surprisingly, QIN alone showed a gene profile similar to GMI and QIN-GMI, suggesting its potential role as an immunomodulator.

[0092] Animal test results:

[0093] Based on the experimental results, QIN was further used as an oral immunomodulator in PCV2 subunit vaccine-inoculated piglets with improved immune response and weight gain.

[0094] Please refer to Table 3 and Figure 6. It can be found that the average weight gain (AWG) or average daily weight gain (ADWG) of Group B pigs inoculated with CIRCOQ, a PCV2 subunit vaccine, and orally administered QIN daily was the highest, followed by Group C pigs inoculated only with CIRCOQ, and finally Group A pigs inoculated with placebo were the lowest (Table 3). The same trend was found in the serum PCV2-specific IgG response (Figure 6). In comparison, there was no difference in IgA measurement (data not shown).

[0095]

Table 3

[0096] Discussion

[0097] The original theoretical basis of this study was to use QIN as an oral delivery carrier for the immunomodulatory factor GMI. In addition to QIN-GMI, QIN alone can stimulate a gene profile similar to GMI in HTP-1 cells. From the protein-protein interaction network generated from 13 highly expressed genes stimulated by QIN, only 9 out of 113 genes related to the viral process were affected (Table 2). This finding may indicate that QIN alone could be a candidate immunomodulatory agent that does not elicit a robust viral immune response. However, a similar gene profile with a more vital stimulation by the GMI peptide inserted with QIN (i.e., QIN-GMI) could be explained by the multivalent QIN containing 60 copies of the GMI peptide on its surface. A stronger stimulation of the gene profile related to the innate / adaptive immune response could only indicate the dose of QIN. QIN-GMI, which is lower than GMI alone, is necessary to function as an immunomodulatory agent. The immunomodulatory effect of orally administered QIN was further verified by animal tests on piglets inoculated with CIRCOQ, a PCV2 subunit vaccine, showing better average weight gain and higher IgG titers.

[0098] Conclusion

[0099] As described above, chimeric QIN-GMI can stimulate a higher gene expression profile than QIN alone and GMI alone without stimulating a strong viral immune response in THP-1 monocytes. Surprisingly, QIN alone also showed a similar trend, although less than QIN-GMI, in gene analysis. These results indicate that QIN alone was used as an oral immunomodulator in an animal study on piglets vaccinated with the PCV2 subunit vaccine CIRCOQ, which showed better average weight gain and higher IgG titers. Therefore, it can be demonstrated that QIN (QIN-IM) and even QIN-GMI as immunomodulators have a positive effect on the PCV2 subunit vaccine.

[0100] Without further elaboration, those skilled in the art will, based on the above description, be able to utilize the present invention to its fullest extent. Therefore, the above specific examples should be construed as merely illustrative and in no way limiting the remainder of the present disclosure. References 1.Li T-C,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.J Virol 2005,79(20):12999-13006. 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.Can J Vet Res 2021,85(2):93-100.

Claims

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

2. The QIN further binds to an epitope of a Ganoderma microsporum immunomodulatory (GMI) protein. The method according to claim 1.

3. The epitope of the GMI protein binds to the QIN by insertion into the capsid subunit of the QIN. The method according to claim 2.

4. The epitope of the GMI protein has the sequence represented by SEQ ID NO:

1. The method according to claim 2.

5. The QIN is orally administered to the subject. The method according to claim 1.

6. The subject includes an animal species selected from the group consisting of mice, rats, rabbits, dogs, pigs, sheep, cows, and primates. The method according to claim 1.

7. The antigen is selected from the group consisting of tumor antigens, viral antigens, bacterial antigens, self-antigens, and fungal antigens. The method according to claim 1.

8. The antigen is a viral antigen. The method according to claim 7.

9. The viral antigen is a porcine circovirus type 2 (PCV2) antigen. The method according to claim 8.

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

11. A chimeric nanoparticle comprising a bound QIN and an epitope of a GMI protein.

12. The epitope of the GMI protein binds to the QIN by insertion into the capsid subunit of the QIN. The chimeric nanoparticle according to claim 11.

13. The epitope of the GMI protein has the sequence represented by SEQ ID NO:

1. The chimeric nanoparticle according to claim 11.

14. A pharmaceutical composition comprising QIN and a pharmaceutically acceptable carrier, vehicle, or diluent Pharmaceutical composition.

15. The QIN further binds to an epitope of a GMI protein. The pharmaceutical composition according to claim 14.

16. The epitope of the GMI protein binds to the QIN by insertion into the capsid subunit of the QIN. The pharmaceutical composition according to claim 15.

17. The epitope of the GMI protein has the sequence represented by SEQ ID NO:

1. The pharmaceutical composition according to claim 15.

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

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