TWI654993B - Use of cationic biodegradable polyceramic microparticles for vaccine delivery - Google Patents

Use of cationic biodegradable polyceramic microparticles for vaccine delivery Download PDF

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TWI654993B
TWI654993B TW106137957A TW106137957A TWI654993B TW I654993 B TWI654993 B TW I654993B TW 106137957 A TW106137957 A TW 106137957A TW 106137957 A TW106137957 A TW 106137957A TW I654993 B TWI654993 B TW I654993B
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protein
hydrogen phosphate
calcium hydrogen
antigen
microparticles
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TW201828983A (en
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Chien Hsiung Pan
潘建雄
Guo Chung Dong
董國忠
Hsin Wei Chen
陳信偉
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National Health Research Institutes
財團法人國家衛生研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1611—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

本發明係關於一種以陽離子型生物可降解性陶瓷聚合物微粒子做為遞送疫苗之載體之用途，該陽離子型生物可降解性陶瓷聚合物微粒子係以磷酸氫鈣(calcium hydrogenphosphate；CHP)修飾其表面，於本發明中，以此磷酸氫鈣修飾之陶瓷聚合物微粒子所製備之疫苗，顯現出較低的毒性，並可延長抗原之停留時間且增強免疫反應。 The invention relates to the use of cationic biodegradable ceramic polymer microparticles as a carrier for delivering vaccines. The cationic biodegradable ceramic polymer microparticles are modified with calcium hydrogen phosphate (CHP) on the surface thereof. In the present invention, the vaccine prepared with the calcium hydrogen phosphate-modified ceramic polymer microparticles exhibits lower toxicity, can extend the residence time of the antigen, and enhances the immune response.

Description

陽離子型生物可降解性陶瓷聚合物微粒子用以遞送疫苗之用途    Use of cationic biodegradable ceramic polymer particles for delivery of vaccine   

本發明係關於一種以陽離子型生物可降解性陶瓷聚合物微粒子所製備之蛋白質疫苗，尤其特指一種以表面經陽離子修飾之磷酸氫鈣(calcium hydrogenphosphate；CHP)微粒子所製備之次單元疫苗。 The present invention relates to a protein vaccine prepared with cationic biodegradable ceramic polymer microparticles, and particularly to a subunit vaccine prepared with cation-modified calcium hydrogen phosphate (CHP) microparticles.

接種疫苗之安全性係當前越來越受關注之議題，因此，開發新穎技術以製備更安全且更有效之疫苗用以預防新興傳染病乃當務之急；蛋白質疫苗(protein vaccine)係一類對於人類來說具備安全性之優良疫苗，然而，蛋白質疫苗尚具有需要額外的佐劑或者透過新的免疫途徑來彌補其免疫力低於其他種類疫苗等缺點，並仍需克服其缺乏誘導CD8 T淋巴細胞免疫反應之問題，同時仍需持續開發具有安全性、有效性及足夠的免疫原性的蛋白質疫苗。 The safety of vaccination is an issue of increasing concern. Therefore, it is urgent to develop new technologies to prepare safer and more effective vaccines to prevent emerging infectious diseases. Protein vaccines are a type of Excellent vaccine with safety, however, protein vaccines still need additional adjuvants or new immunization pathways to make up for their shortcomings. Compared with other types of vaccines, they still need to overcome their lack of induction of CD8 T lymphocyte immune response. However, there is still a need to continue to develop protein vaccines that are safe, effective, and adequately immunogenic.

聚乳酸-甘醇酸共聚物(poly lactic-co-glycolic acid,PLGA)係聚乳酸(poly lactic acid,PLA)和聚乙醇酸(poly glycolic acid,PGA)的共聚物，其用於藥物遞送之設計及成效已被明確定義，由於其具有長期於臨床之使用經驗、良好的降解特性和持續給藥的可能性，聚乳酸-甘醇酸共聚物在各種適用於生物之可降解聚合物中最受青睞，近期的文獻亦揭示聚乳酸-甘醇酸共聚物之降解可以用於以期望的劑量持續釋放藥物而無需進行外科手術，然而，用於製備聚乳酸-甘醇酸共聚物的陽離子表面活性劑仍存在生物毒性的問題。 Polylactic acid-co-glycolic acid (PLGA) is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA), which is used for drug delivery. The design and effectiveness have been clearly defined. Due to its long-term clinical experience, good degradation characteristics, and the possibility of continuous administration, polylactic acid-glycolic acid copolymers are among the most suitable biodegradable polymers. Popular, recent literature also reveals that degradation of polylactic acid-glycolic acid copolymers can be used for sustained release of drugs at desired doses without the need for surgery. However, cationic surfaces used to prepare polylactic acid-glycolic acid copolymers Active agents still have problems with biological toxicity.

美國專利公開案US 2004/0013742 A1揭示了一種用於成骨細胞或骨髓基質細胞生長之生物可降解陶瓷，其具備骨傳導和骨誘導性質，該新穎之陶瓷包含以六亞甲基二異氰酸酯(hexamethylene diisocyanate,HMDI)修飾之磷酸氫鈣(calcium hydrogenphosphate,CaHPO4)，六亞甲基二異氰酸酯係透過共價鍵被接枝到磷酸氫鈣上，因而使磷酸氫鈣之表面帶有正電荷，且其生物毒性低於習知用於藥物遞送之帶正電荷的CTAB-PLGA。 U.S. Patent Publication US 2004/0013742 A1 discloses a biodegradable ceramic for osteoblast or bone marrow stromal cell growth, which has osteoconductive and osteoinductive properties. The novel ceramic contains hexamethylene diisocyanate ( hexamethylene diisocyanate (HMDI) modified calcium hydrogen phosphate (CaHPO 4 ), hexamethylene diisocyanate is grafted onto calcium hydrogen phosphate through a covalent bond, so that the surface of calcium hydrogen phosphate has a positive charge, And its biological toxicity is lower than conventionally charged CTAB-PLGA known for drug delivery.

在先前的研究中，我們使用修飾磷酸氫鈣的生物陶瓷作為載體，將骨碎補(Gu-Sui-Bu)運送到骨細胞培養系統中，並評估骨碎補固著於磷酸氫鈣(Gu-Sui-Bu-immobilized modified calcium hydrogenphosphate,GI-MCHP)作用於骨細胞之活性(Biomaterials 24(2003)873-882)，據此，本發明進一步探討疫苗製劑之相關應用。 In previous studies, we used modified calcium hydrogen phosphate bioceramics as a carrier to transport bone fragmentation (Gu-Sui-Bu) to an osteocyte culture system, and evaluated the fixation of bone fragmentation to calcium hydrogen phosphate (Gu -Sui-Bu-immobilized modified calcium hydrogen phosphate (GI-MCHP) acts on the activity of bone cells ( Biomaterials 24 (2003) 873-882). Based on this, the present invention further explores the related applications of vaccine preparations.

基於上述之目的，本發明提供一種陽離子型生物可降解性陶瓷聚合物，用以做為蛋白質疫苗中的抗原載體。 Based on the above objectives, the present invention provides a cationic biodegradable ceramic polymer for use as an antigen carrier in a protein vaccine.

據此，於一方面，本發明係有關於一種蛋白質疫苗組成物，其係包含一蛋白質或一胜肽抗原，以及一陽離子型生物可降解性陶瓷聚合物 微粒子，所述之陶瓷聚合物微粒子係用以將該蛋白質或該胜肽抗原吸附於於其陽離子表面。 Accordingly, in one aspect, the present invention relates to a protein vaccine composition comprising a protein or a peptide antigen, and a cationic biodegradable ceramic polymer particle, said ceramic polymer particle system It is used to adsorb the protein or the peptide antigen to its cationic surface.

於一實施例中，該陽離子型生物可降解性陶瓷聚合物微粒子係經表面修飾之一磷酸氫鈣(calcium hydrogenphosphate,MCHP)修飾微粒子。 In one embodiment, the cationic biodegradable ceramic polymer microparticles are surface-modified calcium hydrogen phosphate (MCHP) modified microparticles.

於一實施例中，該磷酸氫鈣修飾微粒子之粒徑係介於0.1至10微米(μm)。 In one embodiment, the particle size of the calcium hydrogen phosphate modified particles is between 0.1 and 10 micrometers (μm).

於一實施例中，該磷酸氫鈣修飾微粒子之表面電位係介於+1至+50毫伏(mV)，較佳者，係介於+1至+40毫伏(mV)。 In one embodiment, the surface potential of the calcium hydrogen phosphate modified particles is between +1 and +50 millivolts (mV), and more preferably between +1 and +40 millivolts (mV).

於一實施例中，該陽離子型生物可降解性陶瓷聚合物微粒子之抗原吸附率(antigen adsorption rate)係每毫克(mg)之該陽離子型生物可降解性陶瓷聚合物微粒子吸附0.1至100微克(μg)之該蛋白質或該胜肽抗原。 In one embodiment, the antigen adsorption rate of the cationic biodegradable ceramic polymer particles is 0.1 to 100 micrograms per milligram (mg) of the cationic biodegradable ceramic polymer particles. μg) of the protein or the peptide antigen.

於一實施例中，該蛋白質或該胜肽抗原係包含至少一腫瘤抗原。 In one embodiment, the protein or the peptide antigen system comprises at least one tumor antigen.

於另一實施例中，該蛋白質或該胜肽抗原係包含至少一病毒抗原；於本文中，「胜肽」(peptide)及「多胜肽」(polypeptide)為相互通用之名詞。 In another embodiment, the protein or the peptide antigen system comprises at least one viral antigen; herein, "peptide" and "polypeptide" are mutually common terms.

於一實施例中，該蛋白質或該胜肽抗原係包含至少一登革熱病毒次單元；於另一實施例中，該蛋白質或該胜肽抗原係包含登革熱病毒套膜結構域III(ED3)胜肽，較佳者，係第二型登革熱(dengue-2)之登革熱病毒套膜結構域III(ED3)胜肽。 In one embodiment, the protein or peptide antigen system comprises at least one dengue virus subunit; in another embodiment, the protein or peptide antigen system comprises dengue virus envelope membrane domain III (ED3) peptide Preferably, it is the dengue virus envelope membrane domain III (ED3) peptide of dengue-2.

於一實施例中，該蛋白質或該胜肽抗原係包含至少一細菌抗 原；於另一實施例中，該蛋白質或該胜肽抗原係包含至少一真菌抗原。 In one embodiment, the protein or peptide antigen system comprises at least one bacterial antigen; in another embodiment, the protein or peptide antigen system comprises at least one fungal antigen.

於另一方面，本發明係關於一種以表面修飾之磷酸氫鈣(modified calcium hydrogenphosphate；MCHP)作為抗原載體以製備蛋白質疫苗之用途。 In another aspect, the present invention relates to the use of surface modified modified calcium hydrogen phosphate (MCHP) as an antigen carrier to prepare a protein vaccine.

圖1係以表面修飾磷酸氫鈣(modified calcium hydrogenphosphate；MCHP)微粒子製備次單位疫苗(subunit vaccine)之示意圖。 FIG. 1 is a schematic diagram of preparing a subunit vaccine using surface modified calcium hydrogen phosphate (MCHP) microparticles.

圖2係說明表面修飾磷酸氫鈣微粒子製備之蛋白疫苗於活體外之蛋白質吸附特性(protein adsorption)；磷酸氫鈣-卵清白蛋白(ovabumin,OVA)疫苗係以0.1%之氫氧化鈉(NaOH)及2%之十二烷基硫酸鈉(Sodium dodecyl sulfate,SDS)處理以沖提所吸附之卵清白蛋白，並以蛋白質定量套組(micro-BCA kit)進行卵清白蛋白定量；卵清白蛋白定量圖係分別顯示吸附於聚乳酸-甘醇酸共聚物(PLGA(+))或磷酸氫鈣之卵清白蛋白，其定量後之平均值及標準差。 Figure 2 illustrates the protein adsorption characteristics of protein vaccine prepared by surface modified calcium hydrogen phosphate microparticles in vitro; calcium hydrogen phosphate-ovalbumin (OVA) vaccine uses 0.1% sodium hydroxide (NaOH) And 2% Sodium dodecyl sulfate (SDS) treatment to extract the adsorbed ovalbumin, and use a micro-BCA kit to perform ovalbumin quantification; ovalbumin quantification The graph shows the quantified average and standard deviation of ovalbumin albumin adsorbed on a polylactic acid-glycolic acid copolymer (PLGA (+)) or calcium hydrogen phosphate, respectively.

圖3係說明控制表面修飾磷酸氫鈣製備之蛋白疫苗於活體內之釋放動態；BALB/c小鼠係分別以皮下注射2組樣本，分別為螢光染劑(Alexa Fluor® 647)標記之溶解態卵清白蛋白，或螢光染劑(Alexa Fluor® 647)標記之磷酸氫鈣-卵清白蛋白疫苗，並透過活體影像系統(IVIS)監測螢光衰退，圖3A及圖3B係分別顯示平均螢光強度及活體影像。 Figure 3 illustrates the release of protein vaccine prepared by controlling the surface modified calcium hydrogen phosphate in vivo; BALB / c mice were injected subcutaneously with two groups of samples, each of which was labeled with a fluorescent dye (Alexa Fluor ® 647) for dissolution. Ovalbumin, or a calcium hydrogen phosphate-ovalbumin vaccine labeled with a fluorescent dye (Alexa Fluor ® 647), and the fluorescence decay was monitored by the in-vivo imaging system (IVIS). Figure 3A and Figure 3B respectively show the average fluorescence Light intensity and live image.

圖4係說明磷酸氫鈣濃度之最佳化以強化蛋白疫苗之免疫原性 (immunogenicity)；C57BL/6小鼠(n=4)係分別以皮下注射4組樣本，分別為(1)0.5微克(μg)之卵清白蛋白、(2)10微克之磷酸氫鈣-0.5微克之卵清白蛋白疫苗、(3)1微克之磷酸氫鈣-0.5微克之卵清白蛋白疫苗及(4)0.25微克之磷酸氫鈣-0.5微克之卵清白蛋白疫苗；圖4A係顯示以酶聯免疫斑點法(ELISPOT)分析對卵清白蛋白具專一性之T淋巴細胞反應；圖4B係顯示以酵素結合免疫吸附分析法(ELISA)分析對卵清白蛋白具專一性之免疫球蛋白(IgG)效價；分析結果係將各別從三隻小鼠所獲得之數值計算過後，以平均值及標準差呈現，並以two-way ANOVA進行統計分析，p值小於0.05者係表示具顯著性差異。 Figure 4 illustrates the optimization of the calcium hydrogen phosphate concentration to enhance the immunogenicity of the protein vaccine; C57BL / 6 mice (n = 4) were injected subcutaneously into four groups of samples, respectively (1) 0.5 μg (μg) ovalbumin, (2) 10 micrograms of calcium hydrogen phosphate-0.5 micrograms of ovalbumin vaccine, (3) 1 microgram of calcium hydrogen phosphate-0.5 micrograms of ovalbumin vaccine and (4) 0.25 micrograms of Calcium hydrogen phosphate-0.5 microgram ovalbumin vaccine; Figure 4A shows the analysis of T lymphocyte response specific to ovalbumin by enzyme-linked immunospot assay (ELISPOT); Figure 4B shows the analysis by enzyme-linked immunosorbent assay (ELISA) analysis of immunoglobulin (IgG) titers specific to ovalbumin; the analysis results are calculated from the values obtained from three mice, and presented as the average and standard deviation, and presented as two -way ANOVA was used for statistical analysis. A p- value less than 0.05 indicates a significant difference.

圖5係說明表面修飾磷酸氫鈣製備之蛋白疫苗確實誘發增強免疫原性及毒殺型T淋巴細胞反應；C57BL/6小鼠係分別以皮下注射2組樣本以進行評估，分別為溶解的卵清白蛋白，或磷酸氫鈣-卵清白蛋白疫苗；圖5A係顯示以酶聯免疫斑點法(ELISPOT)分析對卵清白蛋白具專一性之產生干擾素-γ(interferon-γ；IFN-γ)之T淋巴細胞反應；圖5B係顯示以酵素結合免疫吸附分析法(ELISA)分析對卵清白蛋白具專一性之免疫球蛋白(IgG)產量；分析結果係取各小鼠所獲得之數值並以Student’s test進行統計分析，p值小於0.05者係表示具顯著性差異。 Figure 5 illustrates that the protein vaccine prepared with surface modified calcium hydrogen phosphate did induce enhanced immunogenicity and cytotoxic T lymphocyte response; C57BL / 6 mice were injected subcutaneously in two groups of samples for evaluation, respectively, as dissolved egg whites Protein, or calcium hydrogen phosphate-ovalbumin vaccine; Figure 5A shows the analysis of T-interferon-γ (IFN-γ) specific to ovalbumin using an enzyme-linked immunospot assay (ELISPOT) analysis. Lymphocyte response; Figure 5B shows the analysis of the production of ovalbumin-specific immunoglobulin (IgG) by enzyme-linked immunosorbent assay (ELISA); the analysis results are based on the values obtained by each mouse and measured by Student ’s test Statistical analysis was performed, and those with a p- value of less than 0.05 indicated significant differences.

圖6係說明表面修飾磷酸氫鈣製備之第二型登革熱(dengue-2)次單元疫苗確實增強專一性T淋巴細胞反應；圖6A及圖6B係分別顯示產生干擾素-γ及產生介白素-4(interleukin-4；IL-4)之T淋巴細胞反應，其係將BALB/c小鼠(n=4)以皮下注射3組樣本，分別為(1)磷酸緩衝溶液(PBS)、(2)10微克已溶解的第二型登革熱病毒(D2)套膜結構域III(ED3)重組蛋白或(3)磷 酸氫鈣製備-二型登革熱病毒套膜結構域III(MCHP-D2)抗原疫苗，並以酶聯免疫斑點法進行分析；實驗結果係以two-way ANOVA進行統計分析，p值小於0.05者係表示具顯著性差異。 Figure 6 illustrates that the dengue-2 subunit vaccine prepared by surface-modified calcium hydrogen phosphate indeed enhances the specific T lymphocyte response; Figures 6A and 6B show the production of interferon-γ and interleukin, respectively -4 (interleukin-4; IL-4) T lymphocyte response, which is a subcutaneous injection of BALB / c mice (n = 4) into 3 groups of samples, respectively (1) phosphate buffer solution (PBS), ( 2) 10 micrograms of solubilized type 2 dengue virus (D2) mantle domain III (ED3) recombinant protein or (3) calcium hydrogen phosphate preparation-type 2 dengue virus mantle domain III (MCHP-D2) antigen vaccine The results were analyzed statistically by two-way ANOVA. Those with a p value of less than 0.05 indicated a significant difference.

圖7係說明表面修飾磷酸氫鈣製備之第二型登革熱(dengue-2)次單元疫苗確實增強抗體反應；實驗係以酵素結合免疫吸附分析法分析對第二型登革熱病毒(D2)具有專一性之免疫球蛋白(IgG)效價，實驗結果係以one-way ANOVA進行統計分析。 Figure 7 illustrates that the dengue-2 subunit vaccine prepared by surface-modified calcium hydrogen phosphate indeed enhances the antibody response; the experiment is based on enzyme-linked immunosorbent analysis to be specific for type 2 dengue virus (D2) The immunoglobulin (IgG) titer, the experimental results are statistical analysis using one-way ANOVA.

圖8係說明表面修飾磷酸氫鈣製備之第二型登革熱(dengue-2)次單元疫苗確實增強病毒清除率；實驗係於最後一次免疫接種四週後以第二型登革熱病毒攻毒，而後取得病毒血症(viremia)的血液以溶斑試驗(plaque assay)定量登革熱病毒；實驗結果係以two-way ANOVA進行統計分析。 Figure 8 illustrates that the dengue-2 subunit vaccine prepared by surface-modified calcium hydrogen phosphate did enhance virus clearance; the experiment was performed four weeks after the last immunization with the second type of dengue virus, and then the virus was obtained The blood of viremia was quantified by dengue virus by plaque assay; the experimental results were statistically analyzed by two-way ANOVA.

為了闡明本發明之特點，以下結合圖式以說明本發明之較佳實施例，而非用以限制本發明之技術，在不脫離本發明之新穎概念的精神和範圍的情況下，其中的變化和修改均被包含於本發明之範圍內。 In order to clarify the characteristics of the present invention, the following describes the preferred embodiments of the present invention with reference to the drawings, rather than limiting the technology of the present invention, without departing from the spirit and scope of the novel concept of the present invention. And modifications are included in the scope of the present invention.

實施例1、以表面修飾磷酸氫鈣(MCHP)製備登革熱蛋白疫苗Example 1. Preparation of dengue protein vaccine with surface modified calcium hydrogen phosphate (MCHP)

表面修飾磷酸氫鈣之微粒子經秤重後轉移到1.5毫升(ml)微量管中，以冰磷酸緩衝溶液(PBS)沖洗後再重新懸浮於冰磷酸緩衝溶液中，每一劑量係將10-0.25毫克(mg)表面修飾磷酸氫鈣微粒子懸浮於200微升(μl)冰磷酸緩衝溶液中，接著再取10-1微克(μg)第二型登革熱病毒套膜結構域III(D2-ED3)重組蛋白加入前述表面修飾磷酸氫鈣微粒子懸浮液中，於4℃下以垂直旋轉的方式隔夜混合以進行蛋白質吸附，經蛋白質吸 附之流程後，即獲得磷酸氫鈣-第二型登革熱病毒套膜結構域III(D2-ED3)蛋白疫苗；於本實施例中，較佳者，係以10微克第二型登革熱病毒套膜結構域III重組蛋白及10毫克表面修飾磷酸氫鈣微粒子於200微升磷酸緩衝溶液中製備該登革熱蛋白疫苗。 The surface-modified calcium hydrogen phosphate microparticles are weighed and transferred to a 1.5 ml (ml) microtube, rinsed with ice phosphate buffer solution (PBS), and resuspended in ice phosphate buffer solution. Each dose is 10-0.25 Micrograms (mg) of surface-modified calcium hydrogen phosphate microparticles were suspended in 200 microliters (μl) of ice phosphate buffer solution, and then 10-1 micrograms (μg) of type 2 dengue virus envelope membrane domain III (D2-ED3) were reconstituted The protein was added to the aforementioned surface-modified calcium hydrogen phosphate microparticle suspension, and mixed overnight by vertical rotation at 4 ° C for protein adsorption. After the attached procedure, a calcium hydrogen phosphate-type 2 dengue virus envelope membrane domain III (D2-ED3) protein vaccine is obtained; in this embodiment, preferably, 10 micrograms of type 2 dengue virus envelope Recombinant domain III protein and 10 mg of surface modified calcium hydrogen phosphate microparticles were used to prepare the dengue vaccine in 200 microliters of phosphate buffer solution.

圖1係顯示以表面修飾磷酸氫鈣(MCHP)微粒子遞送疫苗之示意圖；簡言之，具備生物可降解性之陽離子型陶瓷聚合物微粒子與次單元抗原反應後即可形成磷酸氫鈣蛋白疫苗(例如：卵清白蛋白；OVA)，由於表面修飾磷酸氫鈣微粒子之粒徑僅介於5-0.5毫米(mm)，因此磷酸氫鈣製備之疫苗將優先被巨噬細胞或樹突細胞(dendritic cells；DC)吞噬並活化CD80/86的表現，在經蛋白裂解及交叉呈現(cross-presentation)後，胜肽將被呈現給具專一性之T淋巴細胞並誘發適應性免疫反應(adaptive immune response)。 Figure 1 is a schematic diagram showing the delivery of vaccines with surface-modified calcium dicalcium phosphate (MCHP) microparticles; in short, biodegradable cationic ceramic polymer microparticles react with subunit antigens to form a dicalcium phosphate protein vaccine ( For example: ovalbumin; OVA), because the particle size of the surface modified calcium hydrogen phosphate particles is only between 5-0.5 millimeters (mm), the vaccine prepared by calcium hydrogen phosphate will be preferentially treated by macrophages or dendritic cells (dendritic cells). (DC) Phagocytosis and activation of CD80 / 86. After proteolysis and cross-presentation, the peptide will be presented to specific T lymphocytes and induce an adaptive immune response. .

實施例2、磷酸氫鈣蛋白疫苗於活體外之蛋白質吸附特性(protein adsorption)以及於活體內之控制釋放Example 2. Protein adsorption of dicalcium phosphate protein vaccine in vitro and controlled release in vivo

蛋白吸附率測試Protein adsorption rate test

實驗係取10毫克陽離子型聚乳酸-甘醇酸共聚物微粒子或10毫克磷酸氫鈣微粒子，在4℃下分別於300微升磷酸緩衝溶液中與100微克卵清白蛋白(ovalbumin；OVA)隔夜旋轉混合，而後去除未吸附之卵清白蛋白，再以300微升磷酸緩衝溶液清洗微粒子，接著將微粒子沉降後，以100微升沖提緩衝液(包含0.2N NaOH及1% SDS)置於旋轉器上隔夜沖提，以獲得吸附在微粒子之卵清白蛋白，接著，經離心後以BCA protein assay定量上清液中卵清白蛋白之濃度。 In the experiment, 10 milligrams of cationic polylactic acid-glycolic acid copolymer particles or 10 milligrams of calcium hydrogen phosphate microparticles were rotated overnight at 100C with 100 micrograms of ovalbumin (OVA) in 300 microliters of phosphate buffer solution at 4 ° C. Mix, then remove the unadsorbed ovalbumin, then wash the microparticles with 300 microliters of phosphate buffer solution, and then settle the microparticles, and then add 100 microliters of buffer (containing 0.2N NaOH and 1% SDS) to the spinner It was washed overnight to obtain ovalbumin adsorbed on the microparticles. Then, the concentration of ovalbumin in the supernatant was quantified by BCA protein assay after centrifugation.

圖2係顯示卵清白蛋白定量後之平均值及標準差，所述之卵清白蛋白係分別吸附於聚乳酸-甘醇酸共聚物微粒子(PLGA(+))或磷酸氫鈣微粒子，由結果可以得知，聚乳酸-甘醇酸共聚物微粒子及磷酸氫鈣微粒子之蛋白質吸附率幾乎相同，其蛋白質吸附率分別為每毫克聚乳酸-甘醇酸共聚物微粒子吸附2.81±0.71微克卵清白蛋白，以及每毫克磷酸氫鈣微粒子吸附2.47±0.41微克卵清白蛋白。 Figure 2 shows the average and standard deviation of ovalbumin after quantification. The ovalbumin is adsorbed on polylactic acid-glycolic acid copolymer microparticles (PLGA (+)) or calcium hydrogen phosphate microparticles. It is known that the protein adsorption rates of polylactic acid-glycolic acid copolymer particles and calcium hydrogen phosphate microparticles are almost the same, and their protein adsorption rates are 2.81 ± 0.71 microgram ovalbumin per mg of polylactic acid-glycolic acid copolymer particles, And 2.47 ± 0.41 micrograms of ovalbumin were adsorbed per milligram of calcium hydrogen phosphate particles.

疫苗於活體內之釋放動態Vaccine release in vivo

實驗係分為2組，每組包含3隻BALB/c小鼠，分別以皮下注射不同樣本，其中一組是以50微克螢光染劑(Alexa Fluor® 647)標記之卵清白蛋白並溶於200微升磷酸緩衝溶液中，另一組則是螢光染劑(Alexa Fluor® 647)標記之磷酸氫鈣-卵清白蛋白疫苗並溶於200微升磷酸緩衝溶液中，接著以活體影像系統拍攝活體影像並計算螢光強度，將標計螢光染劑之卵清白蛋白定量後，係於圖3A顯示每一區域之螢光強度，且於圖3B以縱向編排影像以顯示螢光衰減程度。 The experimental department was divided into 2 groups, each group contained 3 BALB / c mice, and different samples were injected subcutaneously. One group was 50 micrograms of fluorescent dye (Alexa Fluor ® 647) labeled ovalbumin and dissolved in In 200 microliters of phosphate buffer solution, the other group is a fluorescent dye (Alexa Fluor ® 647) -labeled calcium hydrogen phosphate-ovalbumin vaccine and dissolved in 200 microliters of phosphate buffer solution, and then shot with a live imaging system In vivo images were calculated and fluorescence intensity was calculated. After quantifying the ovalbumin of the standard fluorescent dye, the fluorescence intensity of each area was shown in FIG. 3A, and the image was arranged in a vertical direction in FIG. 3B to show the degree of fluorescence attenuation.

由圖3A及圖3B之結果指出，於注射樣本48小時後，磷酸氫鈣-卵清白蛋白疫苗之蛋白量仍維持95%，相反地，未吸附於磷酸氫鈣之溶解態卵清白蛋白，其蛋白量在注射樣本48小時內已急遽下降至18%，且於注射樣本72小時後已無法偵測到螢光訊號；由此些結果可以得知，利用表面修飾磷酸氫鈣所製備之蛋白疫苗，能夠延長抗原維持於被免疫動物體內的時間，並可控制抗原之釋放。 The results of FIGS. 3A and 3B indicate that the protein content of the calcium hydrogen phosphate-ovalbumin vaccine remained at 95% 48 hours after the injection of the sample. On the contrary, the dissolved ovalbumin that was not adsorbed to the calcium hydrogen phosphate had The amount of protein has dropped sharply to 18% within 48 hours of the injection of the sample, and the fluorescent signal cannot be detected after 72 hours of injection. From these results, it can be known that the protein vaccine prepared by surface modification of calcium hydrogen phosphate It can prolong the time that the antigen is maintained in the immunized animal, and can control the release of the antigen.

實施例3、優化表面修飾磷酸氫鈣微粒子之使用劑量以增強疫苗之免疫原性Example 3: Optimizing the dosage of surface modified calcium hydrogen phosphate microparticles to enhance the immunogenicity of the vaccine

實驗係將C57BL/6小鼠分別以皮下注射4組不同樣本，分別為(1)0.5微克(μg)之卵清白蛋白、(2)10微克之磷酸氫鈣-0.5微克之卵清白蛋白疫苗、(3)1微克之磷酸氫鈣-0.5微克之卵清白蛋白疫苗及(4)0.25微克之磷酸氫鈣-0.5微克之卵清白蛋白疫苗，樣本皆溶於200微升磷酸緩衝溶液中，並於小鼠注射樣本兩週後接種相同的疫苗，於接種疫苗一週後犧牲小鼠並取得脾臟細胞，接著以酶聯免疫斑點法(ELISPOT)分析脾臟細胞中對卵清白蛋白具專一性之T淋巴細胞反應，並分別分析專一性T淋巴細胞產生干擾素-γ及介白素-4之產量，對干擾素-γ及介白素-4具專一性之斑點形成細胞(spot-forming cells；SFC)數量係扣除培養基背景值後計算而獲得，分析結果係計算各別從三隻小鼠所獲得之數值後，以平均值及標準差呈現，並以two-way ANOVA進行統計分析，p值小於0.05者係表示具顯著性差異。 In the experiment, C57BL / 6 mice were subcutaneously injected with 4 different samples, respectively (1) 0.5 microgram (μg) ovalbumin, (2) 10 microgram calcium hydrogen phosphate-0.5 microgram ovalbumin vaccine, (3) 1 microgram of calcium hydrogen phosphate-0.5 microgram of ovalbumin vaccine and (4) 0.25 microgram of calcium hydrogen phosphate-0.5 microgram of ovalbumin vaccine, the samples were dissolved in 200 microliters of phosphate buffer solution, and The mice were vaccinated with the same vaccine two weeks after the injection of the samples. The mice were sacrificed and the spleen cells were obtained one week after the vaccination, and then the T lymphocytes specific for ovalbumin in the spleen cells were analyzed by ELISPOT. Response and analysis of the production of interferon-γ and interleukin-4 by specific T lymphocytes, and spot-forming cells (SFC) specific for interferon-γ and interleukin-4 Quantities were obtained after deducting the background value of the culture medium. The analysis results were calculated by calculating the values obtained from the three mice, and presented as the average and standard deviation. The two-way ANOVA was used for statistical analysis. The p value was less than 0.05. This means that there is a significant difference.

結果如圖4A所示，相較於10微克表面修飾磷酸氫鈣微粒子製備之卵清白蛋白疫苗，以0.25微克及1微克表面修飾磷酸氫鈣微粒子製備之卵清白蛋白疫苗具有較佳之增強免疫原性之效果。 The results are shown in FIG. 4A. Compared with the ovalbumin vaccine prepared with 10 micrograms of surface modified calcium hydrogen phosphate microparticles, the ovalbumin vaccine prepared with 0.25 micrograms and 1 microgram of surface modified calcium hydrogen phosphate microparticles has better immunogenicity. The effect.

此外，由磷酸氫鈣-卵清白蛋白疫苗所誘導之抗體反應係透過分析對卵清白蛋白具專一性之免疫球蛋白(IgG)效價來評估，實驗係以酵素結合免疫吸附分析法(ELISA)分析，且分析結果係以各別從三隻小鼠所獲得之數值之平均值及標準差呈現，並以two-way ANOVA進行統計分析，p值小於0.05者係表示具顯著性差異；結果如圖4B所示，相較於以10微克表面修飾磷酸氫鈣微粒子製備之卵清白蛋白疫苗，以0.25微克及1微克 表面修飾磷酸氫鈣微粒子製備之卵清白蛋白疫苗可誘發產生較高效價之免疫球蛋白；由上述結果可以得知，以0.25微克至1微克表面修飾磷酸氫鈣微粒子製備蛋白疫苗，可達到增強疫苗免疫原性之效果，較佳者，係以1微克表面修飾磷酸氫鈣微粒子與0.5微克卵清白蛋白混合以製備成磷酸氫鈣卵清白蛋白疫苗。 In addition, the antibody response induced by the calcium hydrogen phosphate-ovalbumin vaccine was evaluated by analyzing the immunoglobulin (IgG) titer specific to ovalbumin. The experimental system was enzyme-linked immunosorbent assay (ELISA) Analysis, and the analysis results are presented as the average and standard deviation of the values obtained from the three mice, and statistical analysis was performed by two-way ANOVA. A p value less than 0.05 indicates a significant difference; the results are as follows: As shown in FIG. 4B, compared to an ovalbumin vaccine prepared with 10 micrograms of surface modified calcium hydrogen phosphate microparticles, an ovalbumin vaccine prepared with 0.25 micrograms and 1 microgram of surface modified calcium hydrogen phosphate microparticles can induce higher titer immunity Globulin; from the above results, it can be known that the protein vaccine prepared with 0.25 micrograms to 1 micrograms of surface modified calcium hydrogen phosphate microparticles can achieve the effect of enhancing the immunogenicity of the vaccine, preferably, 1 micrograms of surface modified calcium hydrogen phosphate microparticles It is mixed with 0.5 micrograms of ovalbumin to prepare a calcium hydrogen phosphate ovalbumin vaccine.

實施例4、表面修飾磷酸氫鈣微粒子製備之蛋白疫苗可增強免疫原性及毒殺型T淋巴細胞反應Example 4: A protein vaccine prepared with surface modified calcium hydrogen phosphate microparticles can enhance immunogenicity and cytotoxic T lymphocyte response

磷酸氫鈣蛋白疫苗誘發T淋巴細胞反應Calciphosphate vaccine induces T lymphocyte response

實驗係將C57BL/6小鼠以皮下注射2組不同樣本以進行評估，分別為0.5微克已溶解的卵清白蛋白，或磷酸氫鈣-卵清白蛋白疫苗，樣本皆溶於200微升磷酸緩衝溶液中，並於小鼠注射樣本兩週後接種相同的疫苗，於接種疫苗一週後犧牲小鼠並取得脾臟細胞，接著以酶聯免疫斑點法(ELISPOT)分析脾臟細胞中產生干擾素-γ之專一性T淋巴細胞反應，其係扣除培養基之背景值後，分別分析對卵清白蛋白具專一性、對CD-8之OT-1胜肽具專一性或對CD-4之OT-2胜肽具專一性之斑點形成細胞數量，分析結果係以各別從三隻小鼠所獲得之數值之平均值及標準差呈現，並以two-way ANOVA進行統計分析，p值小於0.05者係表示具顯著性差異。 C57BL / 6 mice were subcutaneously injected with two different samples for evaluation, 0.5 micrograms of dissolved ovalbumin or calcium hydrogen phosphate-ovalbumin vaccine. The samples were dissolved in 200 microliters of phosphate buffer solution. The mice were vaccinated with the same vaccine two weeks after the injection of the samples. The mice were sacrificed and the spleen cells were obtained one week after the vaccination. Then, the specificity of interferon-γ production in the spleen cells was analyzed by ELISPOT. T lymphocyte response, after subtracting the background value of the culture medium, is analyzed for specificity for ovalbumin, specificity for OT-1 peptide of CD-8, or specificity for OT-2 peptide of CD-4. The number of specific spot-forming cells, the analysis results are presented as the average and standard deviation of the values obtained from the three mice, respectively, and statistical analysis was performed by two-way ANOVA. A p value less than 0.05 indicates a significant Sexual difference.

結果如圖5A所示，表面修飾磷酸氫鈣微粒子製備之蛋白疫苗誘發干擾素-γ反應之效果顯著優於卵清白蛋白，此外，注射磷酸氫鈣-卵清白蛋白疫苗之組別亦可測得對CD-8之OT-1胜肽具專一性之毒殺型T淋巴細胞反應，而注射卵清白蛋白之組別則未觀察到T淋巴細胞反應。 The results are shown in FIG. 5A. The effect of the protein vaccine prepared by surface-modified calcium hydrogen phosphate microparticles on eliciting an interferon-γ response is significantly better than that of ovalbumin. In addition, the group injected with calcium hydrogen phosphate-ovalbumin vaccine can also be measured A specific cytotoxic T lymphocyte response to OT-1 peptide of CD-8, while no T lymphocyte response was observed in the group injected with ovalbumin.

磷酸氫鈣蛋白疫苗誘發抗體反應Calcineurin vaccine induces antibody response

實驗係將C57BL/6小鼠以皮下注射2組不同樣本以進行評估，分別為0.5微克已溶解的卵清白蛋白，或磷酸氫鈣卵清白蛋白疫苗，樣本皆溶於200微升磷酸緩衝溶液中，並於小鼠注射樣本間隔兩週後接種相同的疫苗兩次，於小鼠接受免疫六週後取其血清，接著以酵素結合免疫吸附分析法(ELISA)分析對卵清白蛋白具專一性之免疫球蛋白(IgG)效價，分析結果係取各小鼠所獲得之數值並以Student’s test進行統計分析，p值小於0.05者係表示具顯著性差異。 In the experiment, C57BL / 6 mice were subcutaneously injected with two different samples for evaluation, 0.5 micrograms of dissolved ovalbumin, or calcium hydrogen phosphate ovalbumin vaccine. The samples were dissolved in 200 microliters of phosphate buffer solution. The mice were vaccinated twice with the same vaccine two weeks after the injection of the samples, and the serum was taken six weeks after the mice were immunized. The enzyme-linked immunosorbent assay (ELISA) was used to analyze the specificity of ovalbumin. The immunoglobulin (IgG) titer. The analysis result is the value obtained by each mouse and statistical analysis is performed by Student's test. A p value less than 0.05 indicates a significant difference.

結果如圖5B所示，由磷酸氫鈣蛋白疫苗所誘發之免疫球蛋白產量顯著高於卵清白蛋白，且其所誘發之免疫球蛋白產量為卵清白蛋白誘發之免疫球蛋白產量的十倍。 The results are shown in FIG. 5B. The immunoglobulin production induced by the calcineurin vaccine was significantly higher than that of ovalbumin, and the immunoglobulin production induced by it was ten times that of ovalbumin-induced immunoglobulin production.

實施例5、表面修飾磷酸氫鈣微粒子製備之第二型登革熱(dengue-2)次單元疫苗增強具抗原專一性之T淋巴細胞反應Example 5: Dengue-2 Subunit Vaccine Prepared by Surface Modified Calcium Hydrogen Phosphate Microparticles Enhances Antigen-Specific T Lymphocyte Response

磷酸氫鈣-登革熱蛋白疫苗誘發T淋巴細胞反應Calcium hydrogen phosphate-dengue protein vaccine induces T lymphocyte response

實驗係以皮下注射的方式給予BALB/c小鼠(n=4)樣本，並分為3組不同的樣本，分別為(1)磷酸緩衝溶液(PBS)、(2)10微克已溶解的第二型登革熱病毒(D2)套膜結構域III(ED3)重組蛋白或(3)磷酸氫鈣-第二型登革熱病毒套膜結構域III(MCHP-D2)抗原疫苗，樣本皆溶於磷酸緩衝溶液中，並於注射樣本兩週後接種相同疫苗兩次，於最後一次接種的一週後犧牲小鼠並取得脾臟細胞，接著以酶聯免疫斑點法(ELISPOT)分析產生干擾素-γ及產生介白素-4(interleukin-4；IL-4)之T淋巴細胞反應，其係扣除培養基之背景值後，分別分析對第一型至第四型登革熱病毒套模結構域III(ED3)胜肽混合物或含有CD8毒殺性T細胞可辨識的登革熱抗原第2-6 號胜肽具專一性之斑點形成細胞數量，實驗結果係顯示自各小鼠取得數值後計算所得之平均值及標準差，並以two-way ANOVA進行統計分析，p值小於0.05者係表示具顯著性差異。 The experiment was performed by subcutaneous injection of BALB / c mice (n = 4) samples and divided into 3 different samples, respectively (1) phosphate buffer solution (PBS), (2) 10 micrograms of dissolved Recombinant protein of type 2 dengue virus (D2) mantle domain III (ED3) or (3) calcium hydrogen phosphate-type 2 dengue virus mantle domain domain III (MCHP-D2) antigen vaccine, samples are dissolved in phosphate buffered solution After two weeks of injection, the same vaccine was vaccinated twice. The mice were sacrificed and spleen cells were obtained one week after the last vaccination. Then, the production of interferon-γ and interleukin was analyzed by ELISPOT. T-lymphocyte response of interleukin-4 (IL-4), after deducting the background value of the culture medium, the peptide mixtures of dengue virus type III (ED3) mantle domains of type 1 to type 4 were analyzed respectively Or the number of specific spot-forming cells containing dengue antigens No. 2-6, which is recognizable by CD8 toxin-killing T cells, and the experimental results show the average value and standard deviation calculated from the values obtained by each mouse, and two -way ANOVA statistical analysis, p = less than 0.05 represents a donor line having significant Difference.

結果由圖6A及圖6B指出，磷酸氫鈣-第二型登革熱病毒套膜結構域III(D2 ED3)抗原疫苗顯著誘發對第二型登革熱抗原具專一性之干擾素-γ及介白素-4之T淋巴細胞反應，且其誘發之效果顯著高於登革熱病毒套模結構域III重組蛋白，而無法測得對其他血清型之登革熱抗原具專一性之T淋巴細胞反應，此外，結果亦顯示磷酸氫鈣-第二型登革熱病毒套膜結構域III(D2 ED3)抗原疫苗可誘發對CD-8之登革熱抗原D2-6具專一性之毒殺型T細胞反應。 The results are shown in Figures 6A and 6B. Calcium hydrogen phosphate-type 2 dengue virus envelope membrane domain III (D2 ED3) antigen vaccine significantly induces interferon-γ and interleukin- 2 specific for type 2 dengue antigen. 4 T lymphocyte response, and its induction effect is significantly higher than that of dengue virus overmold domain III recombinant protein, and T lymphocyte responses specific to dengue antigens of other serotypes cannot be measured. In addition, the results also show Calcium hydrogen phosphate-type 2 dengue virus envelope membrane domain III (D2 ED3) antigen vaccine can elicit a cytotoxic T cell response specific to CD-8 dengue antigen D2-6.

實施例6、表面修飾磷酸氫鈣微粒子製備之第二型登革熱(dengue-2)次單元疫苗增強抗體反應以及病毒清除率Example 6: Dengue-2 subunit vaccine prepared from surface modified calcium hydrogen phosphate microparticles enhances antibody response and virus clearance

磷酸氫鈣-登革熱蛋白疫苗誘發抗體反應Calcium hydrogen phosphate-dengue protein vaccine induces antibody response

實驗係以實施例3所述之方法對BALB/c小鼠(n=4或8)進行免疫接種，於接種六週後取得小鼠之血清，並以酵素結合免疫吸附分析法(ELISA)分析血清中對登革熱病毒抗原D2 ED3具專一性之免疫球蛋白之效價，實驗結果係以每一隻小鼠之血清所測得的免疫球蛋白效價之平均值及標準差來顯示，並以one-way ANOVA進行統計分析，p值小於0.05者係表示具顯著性差異。 In the experiment, BALB / c mice (n = 4 or 8) were immunized by the method described in Example 3. Six weeks after inoculation, the serum of the mice was obtained and analyzed by enzyme-linked immunosorbent assay (ELISA). The titer of immunoglobulin specific for dengue virus antigen D2 ED3 in the serum. The experimental results are shown by the average and standard deviation of the immunoglobulin titer measured in the serum of each mouse, and displayed as One-way ANOVA was used for statistical analysis. A p- value less than 0.05 indicates a significant difference.

受磷酸氫鈣登革熱蛋白疫苗免疫接種之小鼠，以第二型登革熱病毒攻毒後形成之病毒血症(viremia)Viremia of mice immunized with calcium hydrogen phosphate dengue protein vaccine and challenged with type 2 dengue virus

實驗係以實施例3所述之方法對BALB/c小鼠(n=4)進行免疫接種，並於最後一次免疫後以第二型登革熱病毒攻毒，其係預先以第二型登革熱病毒感染K562細胞，再取5 x 107個感染後細胞進行腹腔內接種，而後於不同時間點取得小鼠之血漿，再以螢光集落分析法(fluorescent focus assay)量測每毫升血漿中所包含的集落形成單位(focus forming units；Ffu)以評估病毒血症形成的情形，實驗結果係以平均值及標準差顯示。 The experiment was performed by immunizing BALB / c mice (n = 4) by the method described in Example 3, and after the last immunization, they were challenged with type 2 dengue virus, which was previously infected with type 2 dengue virus For K562 cells, 5 x 10 7 infected cells were inoculated intraperitoneally, and then mouse plasma was obtained at different time points, and then measured by fluorescence focus assay Focus forming units (Ffu) are used to assess the formation of viremia. The experimental results are shown as mean and standard deviation.

結果如圖8所示，以磷酸氫鈣-第二型登革熱病毒套膜結構域III(D2 ED3)抗原疫苗進行免疫接種之小鼠，在攻毒之後其體內形成的病毒血症低於登革熱病毒D2 ED3重組蛋白之組別及控制組，以此結果證實了，在小鼠模式中，磷酸氫鈣登革熱蛋白疫苗確實可提供較佳的保護效果以對抗登革熱病毒感染。 The results are shown in Fig. 8. After immunized mice with calcium hydrogen phosphate-type 2 dengue virus envelope membrane domain III (D2 ED3) antigen vaccine, the viremia formed in vivo after challenge was lower than that of dengue virus. The D2 ED3 recombinant protein group and control group confirmed that in the mouse mode, the calcium hydrogen phosphate dengue protein vaccine can indeed provide better protection against dengue virus infection.

綜合上述，本發明所提供之陶瓷聚合物微粒子，由於其表面帶有陽離子，使得蛋白質抗原得以有效地吸附於其表面，進一步而言，本發明之一實施例係以表面修飾磷酸氫鈣之陶瓷聚合物微粒子作為抗原載體，得以藉以控制抗原之釋放，並能在接受免疫接種之動物體內維持並持續釋放長達六個月，藉由磷酸氫鈣修飾之陶瓷聚合物微粒子而達到延長抗原於動物體內的滯留時間，而能夠延長抗體產生的時程，並有效誘導毒殺型T淋巴反應，因此，本發明所提供之以表面修飾磷酸氫鈣之陶瓷聚合物微粒子所製備之蛋白疫苗，適用於長期疫苗(long-term vaccines)以及抗腫瘤疫苗之開發。 To sum up, the ceramic polymer microparticles provided by the present invention have cations on the surface, so that the protein antigen can be effectively adsorbed on the surface. Further, one embodiment of the present invention is a ceramic with surface modified calcium hydrogen phosphate Polymer microparticles can be used as antigen carriers to control the release of antigens, and can be sustained and sustained for up to six months in immunized animals. The ceramic polymer microparticles modified with calcium hydrogen phosphate can be used to extend the antigen to animals. The retention time in the body can prolong the time course of antibody production and effectively induce a toxic killer T-lymph response. Therefore, the protein vaccine prepared by the ceramic polymer microparticles with surface modified calcium hydrogen phosphate provided by the present invention is suitable for long-term use. Development of long-term vaccines and anti-tumor vaccines.

Claims (11)

一種蛋白質疫苗組成物，其係包含：一蛋白質或一胜肽抗原；以及一陽離子型生物可降解性陶瓷聚合物微粒子，用以將該蛋白質或該胜肽抗原吸附於於其陽離子表面，其中該陽離子型生物可降解性陶瓷聚合物微粒子係一表面經修飾之磷酸氫鈣(calcium hydrogenphosphate,CHP)微粒子。A protein vaccine composition comprising: a protein or a peptide antigen; and a cationic biodegradable ceramic polymer particle for adsorbing the protein or the peptide antigen on a cationic surface thereof, wherein the The cationic biodegradable ceramic polymer particles are a calcium hydrogen phosphate (CHP) particle with a modified surface. 如申請專利範圍第1項所述之蛋白質疫苗組成物，其中該表面經修飾之磷酸氫鈣微粒子之粒徑係介於0.1至10微米(μm)。The protein vaccine composition according to item 1 of the patent application scope, wherein the particle size of the surface-modified calcium hydrogen phosphate microparticles is between 0.1 and 10 micrometers (μm). 如申請專利範圍第1項所述之蛋白質疫苗組成物，其中該表面經修飾之磷酸氫鈣微粒子之表面電位係介於+1至+40毫伏(mV)。The protein vaccine composition according to item 1 of the patent application range, wherein the surface potential of the surface-modified calcium hydrogen phosphate microparticles is between +1 and +40 millivolts (mV). 如申請專利範圍第1項所述之蛋白質疫苗組成物，其中該陽離子型生物可降解性陶瓷聚合物微粒子之抗原吸附率(antigen adsorption rate)係每毫克(mg)之該陽離子型生物可降解性陶瓷聚合物微粒子吸附0.1至100微克(μg)之該蛋白質或該胜肽抗原。The protein vaccine composition according to item 1 of the scope of patent application, wherein the antigen adsorption rate of the cationic biodegradable ceramic polymer microparticles is the cationic biodegradability per milligram (mg) The ceramic polymer microparticles adsorb 0.1 to 100 micrograms (μg) of the protein or the peptide antigen. 如申請專利範圍第1項所述之蛋白質疫苗組成物，其中該蛋白質或該胜肽抗原係包含至少一腫瘤抗原。The protein vaccine composition according to item 1 of the patent application scope, wherein the protein or the peptide antigen system comprises at least one tumor antigen. 如申請專利範圍第1項所述之蛋白質疫苗組成物，其中該蛋白質或該胜肽抗原係包含至少一病毒抗原。The protein vaccine composition according to item 1 of the application, wherein the protein or the peptide antigen system comprises at least one viral antigen. 如申請專利範圍第1項所述之蛋白質疫苗組成物，其中該蛋白質或該胜肽抗原係包含至少一登革熱病毒次單元。The protein vaccine composition according to item 1 of the patent application scope, wherein the protein or the peptide antigen system comprises at least one dengue virus subunit. 如申請專利範圍第7項所述之蛋白質疫苗組成物，其中該蛋白質或該胜肽抗原係包含登革熱病毒套膜結構域III(envelope domain III；ED3)胜肽。The protein vaccine composition according to item 7 of the patent application scope, wherein the protein or the peptide antigen system comprises a dengue virus envelope domain III (ED3) peptide. 如申請專利範圍第1項所述之蛋白質疫苗組成物，其中該蛋白質或該胜肽抗原係包含至少一細菌抗原。The protein vaccine composition according to item 1 of the patent application scope, wherein the protein or the peptide antigen system comprises at least one bacterial antigen. 如申請專利範圍第1項所述之蛋白質疫苗組成物，其中該蛋白質或該胜肽抗原係包含至少一真菌抗原。The protein vaccine composition according to item 1 of the patent application scope, wherein the protein or the peptide antigen system comprises at least one fungal antigen. 一種以表面經修飾之磷酸氫鈣(modified calcium hydrogenphosphate,MCHP)微粒子作為抗原載體以製備蛋白質疫苗之用途。 The invention uses a modified calcium hydrogen phosphate (MCHP) microparticles on the surface as an antigen carrier to prepare a protein vaccine.
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