CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority of Provisional Application No. 62/441 ,682, filed on January 3, 2017, the content of which is incorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

[0002] The present invention relates to a composition and a method for modulation of immune responses. Particularly, the present invention relates to a pharmaceutical composition and a method for enhancement of immune responses using an antigen fusion protein containing an antigen and an antagonist of an Fc gamma receptor.

2. The Prior Art

[0003] Immunogenicity of vaccine antigens is a major concern for vaccine development because the potential of a vaccine to prevent or treat diseases highly depends on the ability of the vaccine antigens to effectively induce immune responses. One strategy commonly used to increase immunogenicity of vaccines is addition of adjuvants into the vaccine formulations. Adjuvants are immune-augmenting substances of heterogeneous types, and they act through a variety of mechanisms, including prolonging the release time of antigens from the injection site, providing a vehicle for antigen transport throughout the lymphatic system, and acting as immunostimulants. Though adjuvants effectively enhance immune responses, many of them may cause unwanted hypersensitivity reactions.

[0004] An alternative to efficiently induce immune responses is to directly stimulate antigen-presenting cells. Professional antigen-presenting cells capture and process antigens in the peripheral tissue, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs, and secrete cytokines to initiate immune responses. Dendritic cells, the most potent antigen-presenting cells, play a critical role during the initiation of adaptive immune responses, leading to differentiation of naive T cells (CD4 ⁺ or CD8 ⁺ T cells) into effector cells (helper or cytotoxic T cells) and further regulation of humoral immune responses. These characteristics have made dendritic cells as prime targets for immune-modulation strategies.

[0005] Antigen-presenting cells express various Fc gamma receptors (FcyRs), which mediate internalization of antigen- antibody complexes (also termed immune complexes) and regulate immune responses. FcyRs are divided into activating receptors, such as FcyRI, FcyRIIA, FcyRIII, and FcyRIV, and inhibitory receptors, such as FcyRIIB, according to the function of FcyRs. Previous reports have shown that immune complexes are efficient in induction of immune responses through the engagement of FcyRs on dendritic cells. However, it is costly to employ the immune complexes in vaccine development since preparation of antibodies and the associated immune complexes is expensive and complicated.

[0006] Accordingly, there is a need to develop a novel strategy to enhance antigen- specific immune responses by targeting the antigen to antigen-presenting cells without the use of immune complexes.

SUMMARY OF THE INVENTION

[0007] As a result, the present invention provides a pharmaceutical composition, including an antigen fusion protein which includes an antigen and an antagonist of an Fc gamma receptor (FcyR).

[0008] In another aspect, the present invention provides a method of enhancing immunogenicity of an antigen, including conjugating the antigen with an antagonist of an Fc gamma receptor to form an antigen fusion protein.

[0009] In one further aspect, the present invention provides a method of enhancing an immune response to an antigen in a subject, including administering to the subject an effective amount of an antigen fusion protein, wherein the antigen fusion protein includes the antigen and an antagonist of an Fc gamma receptor.

[0010] In one embodiment of the present invention, the antagonist of the Fc gamma receptor is a formyl peptide receptor-like 1 inhibitory protein (FLIPr) or a FLIPr-like protein.

[0011] In another embodiment of the present invention, the antigen is a polypeptide derived from a cancer cell or a virus. The antigen may be selected from the group consisting of survivin, mesothelin, and Zika virus envelope protein domain III.

[0012] In yet another embodiment of the present invention, the enhanced immune response includes an increase in CD4 ⁺ T cells, CD8 ⁺ T cells, or combinations thereof, the secretion of a cytokine selected from the group consisting of interferon gamma (IFN-γ), interleukin-2 (IL-2), interleukin-5 (IL-5), interleukin-17A (IL-17A), and combinations thereof, and the production of antigen-specific immunoglobulin G (IgG) antibodies.

[0013] The pharmaceutical composition of the present invention utilizes the antigen fusion protein which targets antigens to antigen-presenting cells via binding to FcyRs to augment antigen-specific immune responses in a subject, leading to increasing numbers of antigen-specific CD4 ⁺ or CD8 ⁺ T cells, and elevated levels of proinflammatory cytokines and antigen-specific antibodies. The provided methods of enhancing immunogenicity of an antigen and enhancing immune responses to an antigen in a subject are simple and may be applied in the development of potent vaccines, such as antitumor and antivirus vaccines, without the use of additional adjuvant or immune complexes. Since the present invention increases the efficacy of vaccine antigens, it provides a strategy to elicit efficient immune responses in subjects with compromised immunity, such as elderly patients.

[0014] The present invention is further explained in the following drawings and examples. It is understood that the examples given below do not limit the scope of the invention, and it will be evident to those skilled in the art that modifications can be made without departing from the scope of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be apparent to those skilled in the art from the following detailed description of the preferred embodiments, with reference to the attached drawings, in which:

[0016] FIG. 1A shows an image of a polyacrylamide gel displaying purified recombinant proteins, including an ovalbumin (OVA) and two OVA fusion proteins fusing OVA with formyl peptide receptor-like 1 inhibitory protein (OVA-FLIPr) or with FLIPr-like protein (OVA-FLIPr-like), respectively;

[0017] FIG. IB shows detection of the purified recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein, by western blotting using an anti-His-tag antibody;

[0018] FIG. 2A shows the binding of FcyRI to the recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein;

[0019] FIG. 2B shows the binding of FcyRIIa-H131 to the recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein;

[0020] FIG. 2C shows the binding of FcyRIIb to the recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein;

[0021] FIG. 2D shows the binding of FcyRIIIa-V158 to the recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein;

[0022] FIG. 2E shows the binding of FcyRIIIa-F158 to the recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein;

[0023] FIG. 3A shows the frequencies of IFN-γ- secreting CD4 ⁺ T cells in the peptide-pulsed splenocytes from mice immunized twice with the OVA, the OVA-FLIPr fusion protein, the OVA-FLIPr-like fusion protein, or phosphate buffered saline (PBS);

[0024] FIG. 3B shows the frequencies of IFN-γ- secreting CD8 ⁺ T cells in the peptide-pulsed splenocytes from mice immunized twice with the OVA, the OVA-FLIPr fusion protein, the OVA-FLIPr-like fusion protein, or PBS;

[0025] FIG. 4A shows carboxyfluorescein succinimidyl ester (CFSE) profiles of the OT- 1 peptide-pulsed splenocytes labeled with high concentration of CFSE from mice immunized twice with the OVA, the OVA-FLIPr fusion protein, the OVA-FLIPr-like fusion protein, or PBS; the percentage shown in each panel represents the percent specific killing in one experiment;

[0026] FIG. 4B shows the percent specific killing of the OT-1 peptide-pulsed splenocytes from mice immunized twice with the OVA, the OVA-FLIPr fusion protein, the OVA-FLIPr-like fusion protein, or PBS;

[0027] FIG. 5 shows the estimated tumor volume in mice immunized twice with the OVA, the OVA-FLIPr fusion protein, the OVA-FLIPr-like fusion protein, or PBS, and then inoculated with EG7 tumor cells;

[0028] FIG. 6A shows an image of a polyacrylamide gel displaying a purified recombinant survivin;

[0029] FIG. 6B shows an image of a polyacrylamide gel displaying a purified recombinant survivin-FLIPr (Sur-FLIPr) fusion protein;

[0030] FIG. 6C shows detection of the purified recombinant survivin by western blotting using the anti-His-tag antibody;

[0031] FIG. 6D shows detection of the purified recombinant Sur-FLIPr fusion protein by western blotting using the anti-His-tag antibody;

[0032] FIG. 7A shows the frequencies of IFN-γ- secreting CD8 ⁺ T cells in the medium-pulsed splenocytes from mice immunized twice with the survivin, Sur-FLIPr fusion protein, or PBS;

[0033] FIG. 7B shows the frequencies of IFN-γ- secreting CD8 ⁺ T cells in the RAH peptide-pulsed splenocytes from mice immunized twice with the survivin, Sur-FLIPr fusion protein, or PBS;

[0034] FIG. 7C shows the frequencies of IFN-γ- secreting CD8 ⁺ T cells in the survivin21-29 peptide-pulsed splenocytes from mice immunized twice with the survivin, Sur-FLIPr fusion protein, or PBS;

[0035] FIG. 7D shows the frequencies of IFN-γ- secreting CD8 ⁺ T cells in the survivin57-64 peptide-pulsed splenocytes from mice immunized twice with the survivin, Sur-FLIPr fusion protein, or PBS;

[0036] FIG. 8 shows the estimated tumor volume in mice inoculated with EG7 tumor cells and then immunized twice with the survivin, the Sur-FLIPr fusion protein, or PBS;

[0037] FIG. 9 shows the levels of proinflammatory cytokines secreted by helper T cells in the mesothelin-pulsed splenocytes from mice immunized twice with a recombinant mesothelin, a recombinant mesothelin-FLIPr fusion protein, or PBS; the cytokines include IFN-γ, IL-2, IL-5, and IL-17A;

[0038] FIG. 10A shows an image of a polyacrylamide gel displaying a purified recombinant protein of Zika virus envelope protein domain III (ZE3);

[0039] FIG. 10B shows an image of a polyacrylamide gel displaying a purified recombinant ZE3-FLIPr fusion protein;

[0040] FIG. IOC shows detection of the purified recombinant ZE3 by western blotting using the anti-His-tag antibody;

[0041] FIG. 10D shows detection of the purified recombinant ZE3-FLIPr fusion protein by western blotting using the anti-His-tag antibody;

[0042] FIG. 11 shows the binding of FcyRIIA to the recombinant proteins, including the ZE3 and the ZE3-FLIPr fusion protein; and

[0043] FIG. 12 shows the titers of ZE3-specific IgG antibodies in the sera from mice immunized twice with the ZE3 or the ZE3-FLIPr fusion protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definition

[0044] Numerical quantities given herein are approximate, and experimental values may vary within 20 percent, preferably within 10 percent, and most preferably within 5 percent. Thus, the terms "about" and "approximately" refer to within 20 percent, preferably within 10 percent, and most preferably within 5 percent of a given value or range.

[0045] As used herein, the term "immunogenicity" refers to the ability of an antigen to elicit or induce an immune response. An antigen which causes a greater immune response is of higher immunogenicity.

[0046] The term "an effective amount" refers to the amount of an active agent that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on routes of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.

[0047] The present invention relates to a pharmaceutical composition, which contains an antigen fusion protein including an antigen and an antagonist of an Fc gamma receptor, and a method of enhancing immune responses to an antigen in a subject, including administering to the subject an effective amount of the antigen fusion protein for immunization. In the following examples, ovalbumin (OVA), survivin (Sur), mesothelin, and Zika virus envelope protein domain III (ZE3) were used as exemplary antigens for investigation of the immune-augmenting effects of the antigen fusion protein of the present invention. The formyl peptide receptor-like 1 inhibitory protein (FLIPr; SEQ ID NO: l) and its homolog FLIPr-like (SEQ ID NO:2), which are potent FcyR antagonists secreted by Staphylococcus aureus to evade FcyR-mediated host immunity, are the exemplary antagonists of the FcyRs for preparation of the FLIPr- or FLIPr-like-containing fusion proteins.

Methods and Materials

Cloning and expression of the antigen fusion proteins

[0048] The FLIPr or the FLIPr-like segment of the antigen fusion protein was preferably conjugated to the carboxyl terminus of an antigen, such as OVA, survivin, and ZE3, via a peptide linker composed of three repeats of four glycine residues and one serine residue. The recombinant antigen or antigen fusion protein may further include a short peptide of histidine residues (His-tag) for convenient purification using metal-chelating affinity chromatography. According to the amino acid sequences of the recombinant OVA (SEQ ID NO:3), OVA-FLIPr fusion protein (SEQ ID NO:4), OVA-FLIPr-like fusion protein (SEQ ID NO:5), survivin (SEQ ID NO:6), survivin-FLIPr (Sur-FLIPr) fusion protein (SEQ ID NO:7), mesothelin (SEQ ID NO:8), mesothelin-FLIPr fusion protein (SEQ ID NO:9), ZE3 (SEQ ID NO: 10), and ZE3-FLIPr fusion protein (SEQ ID NO:ll), the corresponding nucleotide sequences were determined based on Escherichia coli codon usage and the DNA with each of these nucleotide sequences were fully synthesized. The synthesized DNA was then amplified by polymerase chain reaction (PCR). The PCR products were cloned into the Ndel and Xhol sites of an expression vector pET-22b(+) (Novagen) to generate expression plasmids of the antigens or the antigen fusion proteins. These plasmids were used to produce the recombinant antigens and antigen fusion proteins.

[0049] For preparation of the recombinant proteins, E. coli BL21 (DE3) was transformed with each of the abovementioned expression plasmids. The transformed cells were cultured at 37°C overnight, and protein expression was induced by adding 1 mM isopropyl β-D-l-thiogalactopyranoside (IPTG), followed by incubation at 37°C for 3 hours. Next, the transformed cells were lysed by a French press (Constant Systems, Daventry, UK) at 27 Kpsi in a homogenization buffer (20 mM Tris (pH 8.0), 50 mM sucrose, 500 mM NaCl, 10% glycerol). The cell lysate were then centrifuged at 80,000 xg for 40 minutes to obtain a cell pellet containing inclusion bodies. Most of the recombinant proteins were present in inclusion bodies and were solubilized with an extraction buffer (50 mM NaH ₂ P0 ₄ , 5 mM ethylenediaminetetraacetic acid (EDTA), 200 mM NaCl, 0.5 M urea, 1 % Triton X-100, pH 6.0). The recombinant proteins were purified by loading the extracted fraction onto an immobilized metal affinity chromatography column (QIagen, Hilden, Germany).

Enzyme-linked immunosorbent assay (ELISA) for FcyR binding

[0050] To detect the binding of the recombinant antigens or antigen fusion proteins to FcyR subclasses, 96-well plates were coated overnight at 4°C with 0.1 mL recombinant proteins at 1 μg/mL in phosphate buffered saline (PBS; sodium chloride 137 mM, potassium chloride 2.7 mM, sodium hydrogen phosphate 10 mM, and potassium dihydrogen phosphate 1.8 mM, pH 7.4). The plates were washed three times with PBS supplemented with 0.05% v/v Tween 20 and incubated with a serial dilution of various biotin-conjugated recombinant FcyR proteins, including FcyRI, FcyRIIa-H131, FcyRIIb, FcyRIIIa-V158, FcyRIIIa-F158, and FcyRIIA. After incubation at room temperature for 2 hours, the plates were washed and incubated for 30 min with horseradish peroxidase (HRP)-conjugated streptavidin. For detection of the FcyR binding, 3, 3', 5, 5'-tetramethylbenzidine (TMB) was added to the plates and the absorbance at 450nm was measured with an ELISA reader.

Animal studies

[0051] Female C57BL/6 mice were purchased from the National Laboratory Animal Center. All the mice were housed at the Laboratory Animal Center of the National Health Research Institutes (Taiwan). All the animal studies were approved and were performed in compliance with the guidelines of the Animal Committee of the National Health Research Institutes. For immunization, mice were arbitrarily assigned to groups and subcutaneously administered twice with the recombinant antigens or antigen fusion proteins at a two-week interval.

Enzyme-linked immunospot (ELISPOT) assay

[0052] The number of IFN-y-producing cells was determined using mouse IFN-γ ELISPOT kits (eBio science). Briefly, 96-well plates with polyvinylidene difluoride (PVDF) membranes were first coated with a capture antibody and incubated at 4°C for 18 hours. The plates were washed twice and blocked with RPMI medium supplemented with 10% fetal bovine serum (FBS) for one hour to prevent nonspecific binding in later steps. Splenocytes from the immunized mice were seeded at a density of 5xl0 ⁵ cells/well and stimulated with one of the indicated peptides.

[0053] The splenocytes from the mice immunized with the OVA or the OVA fusion proteins were stimulated with 10 μg/mL of an OVA peptide termed OT-1 (SIINFEKL; SEQ ID NO: 12), another OVA peptide termed OT-2 (ISQAVHAAHAEINEAGR; SEQ ID NO:13), a control peptide for OT-1 peptide (RAHYNIVTF; SEQ ID NO: 14), or a control peptide for OT-2 peptide (GRLIT VNPIVTEKDS ; SEQ ID NO:15) and incubated for two days.

[0054] The splenocytes from the mice immunized with the survivin or the survivin fusion protein were stimulated with 10 μg/mL of a survivin peptide termed survivin21-29 (TFKNWPFLE; SEQ ID NO:16), another survivin peptide termed survivin57-64 (CFFCFKEL; SEQ ID NO: 17), or a control RAH peptide (SEQ ID NO:14) and incubated for three days.

[0055] After incubation, the splenocytes were removed from the plates by washing three times with 0.05% (w/v) Tween 20 in PBS. A 100 aliquot of biotinylated detection antibody was added to each well. The plates were incubated at 37 °C for 2 hours. The washing steps were repeated as above, and after a 45 -minute incubation at room temperature with the avidin-HRP complex reagent, the plates were washed three times with 0.05% (w/v) Tween 20 in PBS and then three times with PBS alone. A 100 aliquot of 3-amine-9-ethyl carbazole (Sigma-Aldrich) staining solution was added to each well to develop the spots. The reaction was stopped after one hour by placing the plates under tap water. The spots were counted using an ELISPOT reader (Cellular Technology Ltd.)

In vivo cytotoxicity assay

[0056] Splenocytes from naive C57BL/6 mice were divided into two populations. One population was pulsed with 10 μΜ OT-1 peptide at 37 °C for 90 min. These cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) at a final concentration of 10 μΜ (CFSE ^high ) at 37 °C for 15 min. In parallel, the other population was pulsed with a control peptide for OT-1 (SEQ ID NO: 14) and labeled with 1 μΜ CFSE (CFSE ^low ). The two populations were mixed equally and 2xl0 ⁷ cells were injected intravenously into the immunized mice. After 24 hours, single cells from spleen were isolated and CFSE intensities were analyzed using FACSCalibur flow cytometer and CellQuest Pro software. The percentage of specific killing was calculated as follows: % specific killing = [l-(% CFSE ^high /% CFSE ^high before injection)/(% CFSE ^low /% CFSE ^low before injection)].

EG7 tumor model

[0057] Tumor-bearing mice were established by inoculating EG7 cells into mice via subcutaneous injection before or after immunization with the antigen fusion protein. The EG7 tumor cells are derived from EL4 cells, a mouse lymphoma cell line. The presence or absence of tumor was assessed by visual inspection and palpation. Tumor size was measured three times per week with a caliper, and mice were sacrificed when tumor volume reached 3000 mm ³ . Tumor volume was estimated as follows: tumor volume = tumor width X tumor length X (tumor width + tumor length)/2.

EXAMPLE 1

Preparation and FcyR binding of the OVA fusion proteins

[0058] The DNA encoding OVA, OVA-FLIPr fusion protein, or OVA-FLIPr-like fusion protein was synthesized, amplified by PCR, and cloned into a pET-22b-based vector to generate the expression plasmid pOVA, pOVA-FLIPr, or pOVA-FLIPr-like. E. coli were then transformed with each of the plasmids for protein expression. After purification with immobilized metal affinity chromatography, the successful production of the recombinant proteins was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting.

[0059] As shown in FIG. 1A, the recombinant OVA, OVA-FLIPr fusion protein, and OVA-FLIPr-like fusion protein were visualized on the polyacrylamide gel stained with Coomassie brilliant blue. As shown in FIG. IB, the recombinant OVA, OVA-FLIPr fusion protein, and OVA-FLIPr-like fusion protein were detected by western blotting using an anti-His-tag antibody (BioRad CatNo:MCA1396P).

[0060] To analyze the functional activity of the recombinant OVA-FLIPr fusion protein and OVA-FLIPr-like fusion protein, a capture ELISA was performed to confirm that both of the OVA fusion proteins directly interact with FcyR isoforms. As shown in FIGs. 2A-2E, the recombinant OVA-FLIPr fusion protein and OVA-FLIPr-like fusion protein bound all the tested FcyR isoforms, including FcyRI (FIG. 2A), FcyRIIa-H131 (FIG. 2B), FcyRIIb (FIG. 2C), FcyRIIIa-V158 (FIG. 2D), and FcyRIIIa-F158 (FIG. 2E). In contrast, there was no interaction between the recombinant OVA and the FcyR isoforms. The results were mean + standard error (SEM) of duplicate wells. These results indicate that the antigen fusion protein of the present invention can directly bind to different FcyR isoforms.

EXAMPLE 2

Immunization with the OVA fusion protein enhances OVA-specific T-cell responses

[0061] To ascertain whether the FcyR binding activity of the antigen fusion protein of the present invention associates with the immune responses in vivo, C57BL/6 mice were arbitrarily assigned to groups (6 mice per group) and subcutaneously administered twice with 10 μg of the OVA, the OVA-FLIPr fusion protein, or the OVA-FLIPr-like fusion protein at a two-week interval. The mice administered with PBS alone served as negative controls. One week after the second immunization, the splenocytes of the immunized mice were harvested and examined for the number of IFN-y- secreting CD4 ⁺ and CD8 ⁺ T cells by ELISPOT assay.

[0062] As shown in FIGs. 3A-3B, background levels of IFN-y- secreting cells were detected from all of the splenocytes without stimulation (medium alone) or stimulated with control peptides. After stimulation with an OT-2 peptide (a CD4 ⁺ T cell epitope; FIG. 3A) or an OT-1 peptide (a CD8 ⁺ T cell epitope; FIG. 3B), low frequencies of the IFN-γ- secreting cells were observed in the splenocytes of OVA immunized mice. In contrast, the mice immunized with the OVA-FLIPr fusion protein or the OVA-FLIPr-like fusion protein induced high frequencies of IFN-γ- secreting cells. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antigen- specific T-cell responses.

EXAMPLE 3

Immunization with the OVA fusion protein stimulates cytotoxic immunity

[0063] To investigate whether the antigen fusion protein of the present invention stimulates cytotoxic immunity, an in vivo cytotoxicity assay was performed. C57BL/6 mice were arbitrarily assigned to groups (7-8 mice per group) and administered subcutaneously with 30 μg of the OVA, the OVA-FLIPr fusion protein, or the OVA-FLIPr-like fusion protein two times at a two-week interval. The mice administered with PBS alone served as negative controls. An equal mixture of OT-1 peptide-pulsed splenocytes labeled with high concentration of CFSE (CFSE ^hlgh ) and control peptide-pulsed splenocytes labeled with lower concentration of CFSE (CFSE ^low ) were then injected into the immunized mice via an intravenous route. The immunized mice were sacrificed after 24 hours and the killing of the peptide-pulsed splenocytes in spleen by cytotoxic T lymphocytes was analyzed using flow cytometry.

[0064] FIG. 4A shows CFSE profiles of the peptide-pulsed splenocytes from each group of the immunized mice. FIG. 4B shows the percent specific killing of the peptide-pulsed splenocytes from each group of the immunized mice. According to FIGs. 4A-4B, less specific killing of the peptide-pulsed splenocytes was observed for the OVA immunized mice. In contrast, the mice immunized with the OVA-FLIPr fusion protein or the OVA-FLIPr-like fusion protein exhibited significantly more killing of the peptide-pulsed splenocytes. These results indicate that immunization with the antigen fusion protein of the present invention can effectively stimulate antigen- specific cytotoxic immunity.

EXAMPLE 4

Immunization with the OVA fusion protein induces antitumor responses

[0065] To examine whether the antigen fusion protein of the present invention induces antitumor responses in vivo, C57BL/6 mice were arbitrarily assigned to groups (6 mice per group) and administered twice with 10 μg of the OVA, the OVA-FLIPr fusion protein, or the OVA-FLIPr-like fusion protein at a two-week interval. The mice administered with PBS alone served as negative controls. One week after the second immunization, mice were inoculated subcutaneously on the left flank with 5xl0 ⁵ EG7 tumor cells transfected with an OVA gene and producing OVA constitutively, followed by measurement of the Tumor volume. [0066] As shown in FIG. 5, tumor growth was inhibited in the mice immunized with the OVA-FLIPr fusion protein or the OVA-FLIPr-like fusion protein. In contrast, the mice immunized with the OVA did not show significant reduction of tumor volume when compared with the mice immunized with PBS. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antigen- specific antitumor responses in the tumor-bearing subjects.

EXAMPLE 5

Preparation of the survivin fusion protein

[0067] According to the similar procedures described in Example 1, recombinant proteins of survivin, an inhibitor protein to apoptosis, and Sur-FLIPr fusion protein were prepared and analyzed by SDS-PAGE and western blotting.

[0068] As shown in FIGs. 6A-6B, the recombinant survivin and Sur-FLIPr fusion protein were visualized on the polyacrylamide gel stained with Coomassie brilliant blue. As shown in FIGs. 6C-6D, the recombinant survivin and Sur-FLIPr fusion protein were detected by western blotting using the anti-His-tag antibody.

EXAMPLE 6

Immunization with the survivin fusion protein enhances survivin-specific T-cell responses

[0069] C57BL/6 mice were arbitrarily assigned to groups (6 mice per group) and subcutaneously administered twice with 30 μg of the survivin or the Sur-FLIPr fusion protein at a two-week interval. The mice administered with PBS alone served as negative controls. One week after the second immunization, the splenocytes of the immunized mice were harvested and examined for the number of IFN-y-secreting CD8 ⁺ T cells by ELISPOT assay.

[0070] As shown in FIGs. 7A-7D, the background levels of IFN-γ- secreting cells were detected from all of the splenocytes without stimulation (medium alone) or stimulated with a control RAH peptide. After stimulation with a survivin21-29 peptide or a survivin57-64 peptide (CD8 ⁺ T cell epitopes), low frequencies of the IFN-γ- secreting cells were observed in the splenocytes of survivin immunized mice. In contrast, the mice immunized with the Sur-FLIPr fusion protein induced high frequencies of IFN-γ- secreting CD8 ⁺ T cells. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antigen- specific T-cell responses.

EXAMPLE 7

Immunization with the survivin fusion protein induces antitumor responses

[0071] C57BL/6 mice subcutaneously inoculated with 5xl0 ⁴ EG7 tumor cells were arbitrarily assigned to groups (9-11 mice per group) and administered twice with 30 μg of the survivin or the Sur-FLIPr fusion protein on day 3 and 10 after tumor inoculation. The mice administered with PBS alone served as negative controls. Tumor volume was measured to assess the antitumor activity of the Sur-FLIPr fusion protein. [0072] As shown in FIG. 8, tumor growth was inhibited in the mice immunized with the Sur-FLIPr fusion protein. In contrast, the mice immunized with the survivin did not show significant reduction of tumor volume when compared with the mice immunized with PBS. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antigen- specific antitumor responses.

EXAMPLE 8

Immunization with the mesothehn fusion protein enhances mesothelin-specific T-cell responses

[0073] Recombinant proteins of mesothehn, a protein reported to be overexpressed in tumors such as mesothelioma and ovarian and pancreatic adenocarcinoma, and mesothelin-FLIPr fusion protein were prepared according to the similar procedures described in Example 1 (data not shown). C57BL/6 mice were arbitrarily assigned to groups (3 mice per group) and subcutaneously administered twice with 10 μg of the mesothelin or the mesothelin-FLIPr fusion protein at a one-week interval. The mice administered with PBS alone served as negative controls. One week after the second immunization, the splenocytes of the immunized mice were isolated and stimulated with 10 μg/mL of the mesothelin for five days, and then the levels of IFN-γ, IL-2, IL-5, and IL-17A secreted into the culture medium were measured using ELISA kits (eBioscience) according to the manufacturer's instructions. [0074] As shown in FIG. 9, high levels of proinflammatory cytokines, including IFN-γ, IL-2, IL-5, and IL-17, were secreted by helper T cells in the stimulated splenocytes from the mice immunized with the mesothelin-FLIPr fusion protein. In contrast, small amounts of these cytokines were detected in the splenocytes from the mesothelin or PBS-immunized mice. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antigen- specific helper T-cell responses.

EXAMPLE 9

Preparation and FcyR binding of the ZE3 fusion protein

[0075] According to the similar procedures described in Example 1, recombinant proteins of ZE3 and ZE3-FLIPr fusion protein were prepared and analyzed by SDS-PAGE and western blotting.

[0076] As shown in FIGs. 10A-10B, the recombinant ZE3 and ZE3-FLIPr fusion protein were visualized on the polyacrylamide gel stained with Coomassie brilliant blue. As shown in FIGs. 10C-10D, the recombinant ZE3 and ZE3-FLIPr fusion protein were detected by western blotting using the anti-His-tag antibody.

[0077] To analyze the functional activity of the recombinant ZE3-FLIPr fusion protein, a capture ELISA was performed to confirm the interaction between FcyRIIA and the ZE3-FLIPr fusion protein. As shown in FIG. 11, the binding of FcyRIIA to the recombinant ZE3-FLIPr fusion protein was detected. In contrast, there was weak interaction between the recombinant ZE3 and FcyRIIA. This result demonstrates that the antigen fusion protein of the present invention can directly bind to FcyR.

EXAMPLE 10

Immunization with the ZE3 fusion protein enhances antibody production

[0078] C57BL/6 mice were arbitrarily assigned to groups (4 mice per group) and subcutaneously administered twice with 10 μg of the ZE3 or ZE3-FLIPr fusion protein at a two-week interval. Four weeks after the first immunization, the sera of the immunized mice were collected for determination of the titers of anti-ZE3 IgG antibodies using ELISA. In brief, a 3-fold serial dilution (starting at 1:33) of the serum sample was prepared. The recombinant ZE3 was coated onto 96-well plates. Bound IgG was detected with HRP-conjugated goat anti-mouse IgG Fc antibody (ICN Cappel). After washing, TMB was added for color development. The absorbance was measured with an ELISA reader at 450 nm. The ELISA end-point titer was defined as the serum dilution that produced an OD450 value of 0.2. All titers were obtained from the titration curve by interpolation, unless the OD value was less than 0.2 at the starting dilution (1 :33).

[0079] As shown in FIG. 12, superior production of the ZE3-specific IgG antibodies were observed in the mice immunized with the ZE3-FLIPr fusion protein. Comparatively, fewer ZE3-specific IgG were produced by the ZE3-immunized mice. This result indicates that immunization with the antigen fusion protein of the present invention can effectively induce antigen- specific antibody production.

[0080] In conclusion, the antigen fusion protein of the present invention can induce higher antigen-specific immune responses in a subject than the antigen itself. Therefore, a pharmaceutical composition which contains such antigen fusion protein may be employed in the development of potent vaccines, such as antitumor and antivirus vaccines. The examples of the present invention also demonstrate a simple and direct method of enhancing immunogenicity of an antigen and a method of enhancing immune responses to an antigen in a subject. These methods may increase the efficacy of vaccine antigens and elicit efficient immune responses in subjects with compromised immunity.