TW202306972A - 用於治療嚴重急性呼吸道症候群冠狀病毒2(sars-cov-2)感染之方法 - Google Patents
用於治療嚴重急性呼吸道症候群冠狀病毒2(sars-cov-2)感染之方法 Download PDFInfo
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- C07K16/1002—Coronaviridae
- C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
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Abstract
本發明提供一種在個體中治療SARS-CoV-2感染的方法,其包含對該個體投予一治療有效量之胜肽,其為人類表面蛋白D的一重組片段。本發明亦提供一種用於治療SARS-CoV-2感染之醫藥組合物,其包含一治療有效量之人類表面蛋白D的一重組片段與一醫藥上可接受載體。
Description
本發明係有關一種人類表面蛋白D的重組片段(rfhSP-D),其可發展用於治療嚴重急性呼吸道症候群冠狀病毒2(SARS-COV-2)之感染。
COVID-19為由嚴重急性呼吸道症候群冠狀病毒2(SARS-CoV-2)造成的感染性疾病,其導致輕度至重度呼吸道症候群,其中平均的案例致死率為2%。相較於其他類型的冠狀病毒,SARS-CoV-2與SARS-CoV具有76-95%的蛋白相似度,且與MERSCoV具有29-46%的蛋白相似度。儘管SARS-CoV-2與SARS-CoV之間具有驚人相似度,但後者的案例致死率為10%。
SARS冠狀病毒2(SARS-CoV-2)為一種有套膜的冠狀病毒,其屬於冠狀病毒科(Coronaviridae family)病毒,且在遺傳上接近SARS-CoV(約80%序列相似度)與蝙蝠冠狀病毒RaTG13(96.2%)。SARS-CoV-2之套膜由刺突(S)醣蛋白、小型套膜(E)醣蛋白、膜(M)醣蛋白、核鞘(N)蛋白及數個推定之輔助蛋白被覆。SARS-CoV-2藉由使用S醣蛋白之S1次單元與血管張力素轉化酶2(ACE2)受體結合而介導其進入宿主細胞。然而,病毒進入宿主細胞不僅需要與ACE2受體結合,還需要跨膜蛋白酶絲胺酸2(TMPRSS2)經由在S1/S2位點切割S蛋白而誘發S蛋白。此切割對於病毒-宿主細胞膜融合及細胞進入非常關鍵。在病毒複製、組裝及釋放後,感染之宿主細胞發生焦亡(pyroptosis),從而釋放損傷相關之分子模式(DAMP)。隨後,DAMP被周圍的巨噬細胞與單核細胞識別,彼等藉由誘導細胞激素風暴以響應病毒感染。然而,在一些情況下,亦可出現免疫反應受損或失調,導致急性呼吸道窘迫症候群(ARDS)。
由於全球COVID-19大流行,重點在於開發,迫切需要開發針對SARS-CoV-2感染的新治療策略。
本發明意外發現,人類表面蛋白D的一重組片段(以下稱為「rfhSP-D」)可干擾SARS-CoV-2 S1與血管張力素轉化酶2(ACE-2)受體的結合。此外,藉由使用表現SARS-CoV-2 S1蛋白之假型慢病毒顆粒的測試,證實胜肽rfhSP-D能抑制SARS-CoV-2感染。據此,本發明提供一種用於治療SARS-CoV-2感染之新方法。
在一方面,本發明提供一種在個體中治療SARS-CoV-2感染的方法,其包含對該個體投予一治療有效量之人類表面蛋白D(SP-D)的一重組片段(rfhSP-D)。
在一特定具體實施例中,胜肽rfhSP-D具有如SEQ ID NO: 3所示之胺基酸序列。
根據本發明之一特定實例,胜肽rfhSP-D由如SEQ ID NO: 3所示之序列所組成。
根據本發明之一特定具體實施例,胜肽rfhSP-D係以有效抑制SARS-CoV-2進入該個體之宿主細胞中的量投予。
另一方面,本發明提供一種用於治療SARS-CoV-2感染之醫藥組合物,其包含一治療有效量之rfhSP-D胜肽,以及一醫藥上可接受載體。
在本發明之一特定具體實施例中,胜肽rfhSP-D具有如SEQ ID NO: 3所示之胺基酸序列。
根據本發明之一特定實例,胜肽rfhSP-D由如SEQ ID NO: 3所示之序列所組成。
根據本發明之特定具體實施例,治療有效量為可有效抑制SARS-CoV-2進入個體之宿主細胞中的量。
又一方面,本發明提供胜肽rfhSP-D在製備用於治療SARS-CoV-2感染之藥物之用途。
根據本發明之特定具體實施例,該藥物包含一治療有效量之胜肽rfhSP-D,以及一醫藥上可接受載體。在一些具體實施例中,治療有效量為可有效抑制SARS-CoV-2進入個體之宿主細胞中的量。
在本發明之一特定具體實施例中,胜肽rfhSP-D包含如SEQ ID NO: 3所示之序列。
在本發明之一特定實例中,胜肽rfhSP-D由如SEQ ID NO: 3所示之胺基酸序列所組成。
應當理解,上述一般描述及下列詳細描述皆僅用於示例及說明,而不限制本發明。
上述發明內容將參考下列實例之具體實施例而進一步說明。然而,其不應理解為本發明之內容僅侷限於下列具體實施例,且基於本發明上述內容之所有發明皆屬於本發明之範疇。
除非另有定義,否則本文中使用的所有技術和科學術語具有與本發明所屬領域之技術人員通常理解的相同含義。
如本文中所用,單數形式「一」、「一者」及「該」包括複數個參考物,除非上下文另有明確規定。因此,例如,提及「一樣本」包括複數個此類樣本及本領域技術人員已知之其等同物。
本發明提供一種在個體中治療SARS-CoV-2感染的方法,其包含對該個體投予一治療有效量之人類表面蛋白D的一重組片段(rfhSP-D)。本文亦提供一種用於治療SARS-CoV-2感染之醫藥組合物,其包含一治療有效量之該胜肽rfhSP-D,以及一醫藥上可接受載體。
本文中使用的「表面蛋白D」或「SP-D」等詞意指含膠原蛋白之C型凝集素,且為膠凝素(collectin)家族之成員,已知參與肺表面恆定與免疫[1]。SP-D主要由第II型肺泡細胞與克氏(Clara)細胞合成並分泌至肺的氣室中[2, 3]。其主要結構分為四個區域:富含半胱胺酸之N端、由Gly-X-Y三聯重複體組成之三螺旋膠原蛋白區域、α-螺旋盤繞頸部區域及C端C型凝集素或碳水化合物識別結構域(CRD)[1]。作為一種多樣的先天免疫分子,SP-D可與多種病原體交互作用,觸發針對病毒、細菌及真菌以及凋亡細胞的清除機制[4]。根據本發明,天然(野生型)之SP-D經鑑定具有如SEQ ID NO: 1所示之胺基酸序列。
本文中使用的「人類表面蛋白D的一重組片段」或「重組人類表面蛋白D之片段」等詞意指重組人類SP-D之片段,以下稱為胜肽「rfhSP-D」,其包含天然人類SP-D之胺基酸殘基(aa)199至375,其中在aa 200處之脯胺酸(P)突變為絲胺酸(S)(亦即,SEQ ID No: 3之胜肽的aa 2)。在一特定實例中,胜肽rfhSP-D由如SEQ ID NO: 3所示之胺基酸序列所組成。
本文中使用的「個體」乙詞包括人類及/或非人類動物,如陪伴動物(例如,狗、貓等)、農場動物(例如,牛、綿羊、豬、馬等)或實驗動物(例如,大鼠、小鼠、天竺鼠等)。
本文中使用的「治療」、「處理」或「處置」等詞意指將包括一或多種活性劑之組成物施加或投予至具有疾病、疾病之症狀或病況或疾病之進展的個體,其目的在於治療、治癒、減輕、緩解、改變、補救、改善、改進或影響疾病、疾病之症狀或病況、由疾病引起之殘疾或疾病之進展。
本文中使用的「治療有效量」乙詞意指相較於未接受一藥劑量之相應個體,一造成疾病、失調或副作用、或疾病或失調之進展速度下降的治療、治癒、預防或改善效果的量。本術語亦包括在其範圍內有效增進正常生理功能的量。
針對治療用途,治療有效量之rfhSP-D係配製成用於投予的醫藥組合物。據此,本發明進一步提供一種醫藥組合物,其包含一治療有效量之rfhSP-D,以及一或多種醫藥上可接受載體。
針對投遞與吸收之目的,本發明之一治療有效量之SP-D可與醫藥上可接受載體配製成適用形式之醫藥組合物。
本文中使用的「醫藥上可接受載體」乙詞意指在與製劑之其他成分相容且對欲以醫藥組合物投予之個體無害的意義上可接受之載體、稀釋劑或賦形劑。取決於藥學製劑之需求,本領域通常已知或使用之任何載體、稀釋劑或賦形劑皆可用於本發明。該載體可為活性成分之稀釋劑、載劑、賦形劑或基質。合適之賦形劑的一些實例包括乳糖、右旋糖、蔗糖、山梨糖、甘露糖、澱粉、阿拉伯膠、磷酸鈣、藻酸鹽、 黃蓍膠、明膠、矽酸鈣、微晶纖維素、聚乙烯吡咯烷酮、纖維素、無菌水、糖漿及甲基纖維素。所述組成物可額外包含潤滑劑,如滑石、硬脂酸鎂及礦物油;潤濕劑;乳化劑與懸浮劑;防腐劑,如羥基苯甲酸甲酯與羥基苯甲酸丙酯;甜味劑;以及香味劑。
本發明之組成物在投予患者後可提供胜肽rfhSP-D之快速、持續或延遲釋放效果。根據本發明,醫藥組合物可適於藉由任何合適途徑投予,包括但不侷限於,口服、直腸、鼻腔、局部、陰道或非經口途徑(如肌肉、靜脈、皮下及腹腔)、經皮、栓劑及鼻內方法。
藉由以下實例進一步說明本發明,其以說明之目的而非限制之目的提供。
實例
材料與方法
1. rfhSP-D
之表現與純化
編碼人類SP-D之膠原蛋白區域、α-螺旋盤繞頸部及CRD區域的8個Gly-X-Y重複體DNA序列(SEQ ID NO: 2)在T7啟動子之下進行選殖,並使用構築體pUK-D1在大腸桿菌BL21(lDE3)pLysS中表現(25, 26)。初級細菌接種體(25 ml)在37℃下之含有34 mg/ml氯黴素與100 mg/ml安比西林(Sigma-Aldrich)的Luria-Bertani(LB)培養基(500 ml)中生長,直到OD600達到0.6。在異丙基 β-D-硫半乳糖苷(IPTG)(0.5 mM)誘導後,轉形之大腸桿菌細胞在37℃下之搖晃器上另外生長3小時。藉由離心(5000 rpm,4℃,10分鐘)收取細菌細胞,並將細胞沉澱物重新懸浮在4℃下之含有50 mM Tris-HCl(pH 7.5)、200 mM NaCl、5 mM EDTA、0.1% v/v Triton X-100、0.1 mM苯甲基磺醯氟(PMSF)(Sigma-Aldrich)及50 mg溶菌酶/ml(Sigma-Aldrich)的裂解緩衝液中1小時。隨後,使用Soniprep 150(MSE,London,UK),裂解之細胞溶解產物在60 Hz下以2分鐘(12個循環)之間隔進行30秒超音波處理,接著離心(12,000 rpm,15分鐘)。使用含有0.5 M Tris-HCl、0.1 M NaCl(pH7.5)及8 M尿素之緩衝液(50 ml),在4℃下將包涵體進行變性1小時。將可溶性分液針對含不同濃度尿素(4 M、2 M、1 M、0 M)之相同緩衝液各自透析2小時。隨後,在4℃下將重新摺疊之材料針對親和力緩衝液(50 mM Tris-HCl(pH7.5)、100 mM NaCl、10 mM CaCl
2)透析2小時。隨後,將親和力緩衝液透析之上清液填載至麥芽糖-瓊脂糖管柱(5 ml)(Sigma-Aldrich)上;使用含有50 mM Tris-HCl、100 mM NaCl及10 mM EDTA之溶析緩衝液溶析結合的rfhSP-D。純化之rfhSP-D在SDS-PAGE上運行以評估其純度。使用內毒素移除樹脂(Sigma-Aldrich)移除LPS。使用QCL-1000美洲鱟變形細胞裂解系統(Lonza)測定LPS含量,發現其含量為每毫克rfhSP-D小於5皮克。rfhSP-D經鑑定具有SEQ ID NO: 3之胺基酸序列,其包含天然人類SP-D之胺基酸殘基(aa)199至375,其中在aa 200處之脯胺酸(P)突變為絲胺酸(S)(亦即,SEQ ID No: 3之胜肽的aa 2)。
2. ELISA
聚苯乙烯微量滴定盤(Sigma-Aldrich)在4℃下以SARS-CoV-2刺突S1蛋白(NativeAntigen S1,NCBI登錄號YP_009724390.1 AA1-674,在HEK 293細胞中產生;Acro,AA Val 16 - Arg 685,登錄# QHD43416.1,在HEK 293細胞中產生)或RBD(Acro,Arg319-Phe541,登錄#QHD43416.1,在HEK 293細胞中產生)(27)(5 μg/ml,100 μl/孔)塗覆過夜,其使用碳酸鹽/重碳酸鹽(CBC)緩衝液(pH 9.6)(Sigma-Aldrich)進行。翌日,微量滴定孔以含有0.05% v/v Tween 20(Sigma-Aldrich)與5mM CaCl
2(Thermo Fisher Scientific)之Tris緩衝之鹽水-Tween(TBST,pH 7.2-7.4)洗滌三次。隨後,各孔藉由含有1% w/v BSA與5mM CaCl
2之TBS阻斷1小時。在以TBST洗滌三次後,各孔在4℃下以含有兩倍稀釋之rfhSP-D或重組人類全長SP-D(hFL-SP-D,R&D,1920-SP,在HEK 293細胞中產生)蛋白(100ml/孔)的阻斷緩衝液培養過夜。翌日,洗滌各孔,接著在室溫下以生物素化之小鼠抗人類SP-D檢測抗體(1:180)(R&D Systems)培養2小時。在洗滌後,各孔以卵白素山葵過氧化酶(HRP)-共軛體(1:40;R&D System)培養20分鐘,接著洗滌三次。將TMB受質(100 ml/孔;Thermo Fisher Scientific)添加至各孔中,並使用1M H
2SO
4(50 ml/孔;Sigma-Aldrich)停止反應。藉由VersaMax™ ELISA微量盤讀儀測量450nm處的吸光度。
3.
競爭性ELISA
聚苯乙烯微量滴定盤在4℃下以2 μg/ml rfhSP-D(100 μl/孔)塗覆過夜,其使用CBC緩衝液進行,並以含有0.05% v/v Tween 20與5mM CaCl
2之TBS緩衝液洗滌三次。各孔以含有1% BSA與5mM CaCl
2之TBS阻斷1小時。隨後,各孔洗滌三次,並在4℃下分別以含SARS-CoV-2刺突S1蛋白(綿羊-IgG標籤)或RBD(His-標籤)(2.5或5 μg/ml,100 μl/孔)之阻斷緩衝液(含有10mM 麥芽糖與10mM EDTA)培養過夜。翌日,洗滌各孔,接著以抗綿羊IgG-HRP抗體(GeneTex,GTX27111,0.5 μg/ml,100 μl/孔)(1:2000)或抗His抗體(GeneTex,GTX628914,0.5 μg/ml,100 μl/孔)(1:2000)培養2小時。針對RBD結合之檢測,各孔進一步以抗小鼠IgG抗體(Abcam,ab6728,0.5 μg/ml,100 μl/孔)(1:2000)培養2小時。在洗滌後,各盤以TMB受質(100 μl/孔)培養,接著以1M H
2SO
4(50 μl/孔)淬滅。藉由VersaMax™ ELISA微量盤讀儀記錄450nm處的吸光度。
4.
西方墨點法
HEK293T與HEK293T-ACE2細胞(0.5 × 10
5個)在冰上藉由含蛋白酶抑制劑(AMRESCO VWR life sciences)之RIPA緩衝液(Thermo Fisher Scientific)裂解15分鐘,接著進行離心(13000 rpm,4℃,15分鐘)。將30 μg樣品重新懸浮在Laemmli樣品緩衝液(10 μl)中,並在100℃下加熱10分鐘。將樣品填載至SDS-PAGE(8% v/v)凝膠中,接著在轉移緩衝液 [25mM Tris-HCl(pH 7.5),190 mM甘胺酸(Sigma-Aldrich)及20% v/v甲醇(Thermo Fisher Scientific)]中以電泳方式轉移至PVDF薄膜(320 mA,2小時)(Sigma-Aldrich)上。薄膜在室溫下藉由稀釋在TBS+ 0.05% v/v Tween 20(TBST)中之5% w/v奶粉(Sigma-Aldrich)阻斷1小時,並在4℃下以抗SARS-CoV-2(COVID-19)刺突抗體(GeneTex,GTX135356;1:1000)或抗ACE2抗體[SN0754](GeneTex,GTX01160;1:1000)培養過夜。將薄膜洗滌三次,並在室溫下以二次山羊抗兔IgG山葵過氧化酶(HRP)-共軛體(1:10000;Fisher Scientific)探測1小時。在TBST洗滌後,藉由Western Lightning Plus ECL(PerkinElmer)測量蛋白表現,並使用FluorChem R系統(ProteinSimple,San Jose,CA,USA)進行化學發光檢測。
5.
細胞培養與處理
將過度表現ACE2受體的人類胚胎腎(HEK)293T或HEK293T細胞(HEK293T-ACE2)培養在完全Gibco Dulbecco’s Modified Eagle Medium(DMEM)中,其中補充10% v/v胎牛血清(FBS)、100 U/ml青黴素(Sigma-Aldrich)及100 μg/ml鏈黴素(Sigma-Aldrich),並在繼代前於37℃之5% v/v CO
2存在下生長約48小時。藉由殺稻瘟菌素S HCl(Thermo Fisher Scientific)篩選穩定表現ACE2的HEK-293T細胞。由於HEK293T細胞會貼附,因此在37℃下使用2×胰蛋白酶-EDTA(0.5%)(Thermo Fisher Scientific)進行10分鐘的分離。隨後,細胞在1,500 rpm下離心5分鐘,接著重新懸浮於完全DMEM培養基中。為了確定細胞計數與存活力,將等體積之細胞懸浮液與台盼藍(0.4% w/v)(Thermo Fisher Scientific)溶液進行渦旋,接著使用具有Neubauer刻度的血球計(Sigma-Aldrich)進行細胞計數。隨後,將細胞重新懸浮於完全DMEM中以供進一步使用。
6.
穩定表現ACE2之產生
HEK-293T細胞人類ACE2基因係藉由使用Kapa HiFi PCR kit(Kapa Biosystems)從MGC基因庫(cDNA殖株MGC:47598)中擴增,並藉由使用GenBuilder
TM選殖套組(GeneScript
®)次選殖至pLAS2w.Pbsd(來自台灣中央研究院RNA核心設施的慢病毒轉移載體)之NheI與EcoRI位點。為了產生攜帶人類ACE2基因之VSVG假型慢病毒,藉由使用TransIT
®-LT1轉染試劑(Mirus),將三種質體(pCMV-DR8.91、pLAS2w.ACE2.Pbsd及pMD.G)瞬時轉染至HEK-293T細胞中。收取培養基以感染HEK-293T細胞,接著以5 mg/ml殺稻瘟菌素篩選感染細胞一週以產生HEK-293T-ACE2穩定細胞。
7.
流式細胞法
使用流式細胞法評估過度表現ACE2受體之HEK293T細胞(HEK293T-ACE2)與單獨之HEK293T細胞之間的ACE2表現。簡言之,在室溫下將轉染和未轉染ACE2之HEK293T細胞(1x10
5個細胞)與ACE2抗體 [N1N2,N-term(GeneTex,GTX101395),(1:250)]培養1小時。在PBS洗滌後,細胞在室溫之閉光下以連接Alexa Fluor 647之山羊抗兔IgG(H+L)交叉吸附二次抗體(Thermo Fisher Scientific)(每管0.6 μl/100 μl)探測1小時。在以PBS洗滌後,將細胞重新懸浮於FACS緩衝液(含有2% FBS之PBS)中,並進行流式細胞法。
針對使用rfhSP-D之結合實驗,含有C端His-標籤之SARS-CoV-2 S1蛋白(Acro;S1N-C52H3)(5 μg/ml)在4℃下以抗His抗體(Genetex;GT359)(1:100)進行標記1小時,接著在4℃下以一系列兩倍稀釋之rfhSP-D(10 μg/ml)或空白組(僅培養基)預培養1小時。在37℃下將HEK293T-ACE2細胞(1x10
5個細胞)與SARS-CoV-2 S1蛋白、抗His抗體及rfhSP-D之混合物培養於DMEM不完全培養基中2小時。收集細胞並以FACS緩衝液洗滌兩次,並以抗小鼠IgG-PE共軛體(GeneTex,GTX25881)(1:100)培養30分鐘且洗滌三次。從FSC與SSC點圖進行活細胞門控,以藉由CytoFLEX確定在其等表面上之含有S1的PE陽性細胞。
8.
螢光顯微鏡
HEK293T與HEK293T-ACE2細胞(0.5 × 10
5個)在標準培養條件下之完全DMEM培養基中的蓋玻片上生長過夜,如上所述。翌日,細胞以PBS洗滌三次,蓋玻片以4% v/v聚甲醛(Sigma-Aldrich)固定15分鐘,接著洗滌兩次。蓋玻片以0.25% v/v Triton-100(Sigma-Aldrich)滲透15分鐘。在洗滌後,蓋玻片以2% w/v BSA阻斷1小時,並以ACE2抗體[SN0754(1:250)(GeneTex,GTX01160)]培養,接著在室溫避光下以山羊抗兔IgG(H+L)交叉吸附二次抗體(1:500)(Thermo Fisher Scientific)培養1小時。在以二次抗體培養後,細胞以PBS洗滌兩次,並安裝在含有DAPI(Abcam)之培養基中的載玻片上,以在直立式螢光顯微鏡(BX51;Olympus)下觀察。
9. SARS-CoV-2
假型慢
病毒之產生
藉由以pCMV-DR8.91、pLAS2w.Fluc.Ppuro及pcDNA3.1-nCoVSD18(合成其C端具有54個核苷酸缺失之SARS-CoV-2刺突基因並選殖至pcDNA3.1表現載體中)瞬時轉染HEK293T細胞,產生攜帶SARS-CoV-2刺突蛋白之假型慢病毒。在前一天種植HEK293T細胞,接著使用TransIT
®-LT1轉染試劑(Mirus)轉染指定之質體。在16小時後補充培養基,並在轉染後48小時與72小時收取。藉由以4,000 xg離心10分鐘移除細胞碎片,並使上清液通過0.45-mm注射過濾器(Pall Corporation)。將假型慢病毒進行等分並儲存在-80℃直至進一步使用。使用細胞存活力試驗估計SARS-CoV-2假型慢病毒之轉導單位(TU),以響應慢病毒之有限稀釋。簡言之,在慢病毒轉導前一天,將穩定表現人類ACE2之HEK293T細胞分盤在96孔培養盤上。在滴定方面,將不同量之慢病毒顆粒添加至含有聚凝胺(最終濃度為8 mg/ml)的培養基中。在37℃下之96孔培養盤中以1,100 x g進行30分鐘的自旋感染。
細胞在37℃下培養16小時後,將含有病毒顆粒與聚凝胺之培養基移除,並更換成含有2.5 mg/ml嘌黴素的新鮮完全DMEM。在以嘌黴素處理48小時後,移除培養基,並根據製造商的說明,使用10% AlarmaBlue 試劑評估細胞存活。將未感染細胞(未處理嘌黴素)之存活率設定為100%。藉由繪製細胞存活率與稀釋病毒劑量之關係圖,確定病毒顆粒滴度(TU)。
10.
假型病毒
中和試驗
以pCMVDR8.91、pcDNA nCoV-SD18及pLAS2w.FLuc.Ppuro質體(分別為5、2、8 μg)轉染10 cm Petri培養盤中的HEK293T細胞。翌日,細胞以PBS輕輕洗滌,並更換成10 ml之新鮮培養基(含有10% FBS之RPMI)。收集48與72小時的培養基並儲存在-80℃以供將來使用。HEK293T-ACE2細胞(過度表現ACE2受體之HEK293T細胞)(0.5x10
5個細胞)以rfhSP-D(0、5、10及20 μg/ml)預處理24小時,接著以PBS洗滌兩次。將含有培養基(500 μl/孔)之SARS-CoV-2假型慢病毒顆粒添加至細胞中,接著在標準培養條件之37℃下培養。在2小時後,將新鮮的完全DMEM培養基(500 μl)添加至細胞中並在37℃下培養。在培養72小時後,細胞以PBS洗滌兩次,並在37℃下以裂解緩衝液培養10分鐘。使用ONEGlo™螢光素酶檢測系統(Promega)與FlexStation測量螢火蟲螢光素酶活性(RLU)。
11.
統計分析
使用GraphPad Prism 6.0軟體產生所有圖形。以未配對t檢定用於統計分析。根據*p<0.05,在處理與未處理rfhSP-D的條件之間判斷顯著差異值。誤差槓顯示SEM(圖示說明)。
結果
1. rfhSP-D
和重組人類全長SP-D(hFL-SP-D)與S1蛋白及其RBD之交互作用
在SARS-CoV中,S-蛋白為宿主先天免疫系統識別的主要表面醣蛋白。SARS-CoV-2之S蛋白與SARS-CoV的具有近76%一致性。先前的研究指出,SP-D與SARS-CoV之S蛋白的結合需要Ca
2+;所述結合由麥芽糖抑制。因此,本研究之第一部分旨在使用直接結合ELISA檢查無LPS之rfhSP-D和hFL-SP-D與刺突蛋白(S1)的交互作用。結果發現,rfhSP-D/hFL-SP-D以劑量依賴方式結合SARS-CoV-2 S1蛋白(圖1A);此交互作用由麥芽糖與EDTA抑制(圖2A)。在測試的不同rfhSP-D濃度中,在10 μg/ml觀察到rfhSP-D與SARS-CoV-2 S1(5 μg/ml)的強烈與最大結合。SARS-CoV-2與其細胞受體ACE2之結合係由S蛋白之RBD區域介導。據報導,相較於SARS-CoV,SARS-CoV-2之RBD與ACE2受體的結合親和力更高。此外,SARS-CoV-2之RBD已知在刺突蛋白誘導之病毒附著、融合及進入宿主細胞中具有關鍵作用。在此情況下,本研究亦旨在確定rfhSP-D/hFL-SP-D經由直接ELISA與SARS-CoV-2之RBD結合的能力(圖1B)(塗覆RBD,以兩倍稀釋之rfhSP-D(100 μg/ml)培養,並以抗SP-D抗體探測;R&D Systems)。rfhSP-D以劑量依賴性方式結合RBD。其降低麥芽糖的結合親和力,但由EDTA螯合Ca
2+未顯著影響rfhSP-D與RBD區域之間的交互作用(圖2B)。在不存在RBD之情況下未觀察到rfhSP-D結合,指出本試驗中缺乏非特異性交互作用。為了進一步評估麥芽糖與EDTA(5、10及20 mM)之劑量反應,塗覆rfhSP-D並以S1與RBD(2.5與5 μg/ml)探測(圖2B)。該等結果顯示,rfhSP-D之CRD區域與S蛋白之RBD區域之間可能以鈣非依賴性方式發生蛋白-蛋白交互作用。
2. rfhSP-D
抑制SARS-CoV-2 S1與HEK293T細胞膜上表現之ACE2的交互作用
SARS-CoV-2之S1刺突蛋白含有RBD,其可識別並與其細胞受體血管張力素轉化酶2(ACE2)交互作用,從而介導病毒進入宿主細胞中。由於發現rfhSP-D在蛋白層次上與刺突蛋白及其RBD交互作用,發明人亦測試rfhSP-D與過度表現ACE2受體之HEK293T細胞交互作用的能力。經由免疫螢光顯微法(圖3A)、流式細胞法(圖3B)及西方墨點法(圖3C)測量ACE2受體之表現量,驗證ACE2受體基因成功轉染至HEK293T細胞中。使用ACE2抗體(SN0754)之ACE2受體的定量與定性分析顯示,當相較於單獨的HEK293T細胞時,HEK293T-ACE2細胞上的ACE2訊號更高(圖3A、B)。本研究亦聚焦在檢查rfhSP-D治療是否可抑制SARS-CoV-2 S1與HEK293T細胞上的ACE2受體之間的交互作用(圖4)。結果發現,SARS-CoV-2 S1蛋白(2 μg/ml)與不同rfhSP-D濃度(0.625-10 μg/ml)之預培養以劑量依賴性方式降低S1與過度表現ACE2受體之HEK293T細胞的結合(圖4)。相較於對照組(S1 + 0 μg/ml rfhSP-D),發現10 μg/ml之rfhSP-D可將S1與HEK293T細胞上之ACE2 受體的結合降低約7.95%(圖4)。
3. rfhSP-D
作為SARS-CoV-2感染之進入抑制劑
在確認rfhSP-D能防止SARS-CoV-2 S1蛋白與過度表現ACE2受體之HEK293T細胞之間的交互作用後,發明人使用螢光素酶報導子試驗及表現SARS-CoV-2 S1蛋白之假型慢病毒顆粒研究rfhSP-D是否調節病毒進入(圖5)。生產SARS-CoV-2假型慢病毒顆粒為一種安全策略,用於研究S1醣蛋白在不同rfhSP-D濃度下識別與中和病毒的作用。藉由以含有所示pcDNA3.1-nCoV-SD18(SARS-CoV-2刺突基因)、pLAS2w.Fluc.Ppuro及pCMV-DR8.91之編碼序列的質體共轉染HEK293T細胞,生產具有套膜蛋白S1假型化之慢病毒顆粒。經由西方墨點法,分析了在48與72小時收取之純化的假型顆粒與細胞溶解產物,並使用抗SARS-CoV-2(COVID-19)刺突多株抗體測定SARS-CoV-2刺突蛋白的表現量(圖5A)。相較於細胞+SARS-CoV-2(1.5 × 10
5RLU),預培養rfhSP-D(5與10 μg/ml)之細胞顯示螢光素酶活性(1.0 × 10
5RLU)明顯降低約0.5 RLU倍(圖5B)。在以rfhSP-D處理後,螢光素酶活性降低,指出rfhSP-D與SARS-CoV-2 S1蛋白之間的交互作用干擾了含有S1之病毒顆粒與ACE2的結合,從而防止病毒進入HEK323T-ACE2細胞(圖5)。
4.
結論
在本發明中,證實了胜肽rfhSP-D之能力為在模擬人類SARS-CoV-2感染之過度表現hACE-2的HEK293T細胞中可作為表現SARS-CoV-2 S1蛋白之假型慢病毒顆粒的進入抑制劑。本發明發現,胜肽rfgSP-D可與SARS-CoV之S蛋白交互作用,導致增進吞噬作用。鑑於本發明之發現,胜肽rfhSP-D作為SARS-CoV-2感染的進入抑制劑,且為存在於肺表面之潛在先天免疫分子,胜肽rfhSP-D預期在COVID-19之發病機制中扮演重要保護作用。
本發明證實,tge親和力純化及無LPS之rfhSP-D與SARS-CoV-2之S1蛋白及其受體結合結構域(RBD)以類似於重組hFL-SP-D之劑量依賴性方式交互作用。在藉由EDTA或麥芽糖抑制rfhSP-D與S蛋白結合的實例中,顯示rfhSP-D與SARS-CoV-2之S蛋白上的碳水化合物部分結合。亦檢查了處理rfhSP-D是否可抑制SARS-CoV-2 S1與HEK293T細胞上之ACE2受體的交互作用。以不同的rfhSP-D濃度(0.625-10 μg/ml)預培養,SARSCoV-2 S1蛋白(5 μg/ml)顯示以劑量依賴性方式降低與過度表現ACE2受體之HEK293T細胞的結合。
靶向病毒進入宿主細胞為設計與開發抗病毒療法的一種新興方法,係因病毒傳播可在病毒週期的早期階段受限制或阻斷,因此降低了由所釋放病毒顆粒造成的抗藥性。在本發明中,藉由螢光素酶報導子試驗檢查rfhSP-D對SARS-CoV-2的進入抑制劑作用。產生假型慢病毒顆粒,以作為一種模擬SARSCoV-2結構表面的安全替代方法,並測試rfhSP-D治療是否可促進或防止病毒進入宿主細胞。相較於未處理之樣品(1 RLU倍;細胞 + SARSCoV-2),以rfhSP-D(5或10 μg/ml)處理的RLU減少約0.5倍。在rfhSP-D處理後的發光訊號顯著降低指出,rfhSP-D與SARS-CoV-2-S1的交互作用限制病毒的結合與進入,顯示rfhSP-D對SARSCoV-2感染的進入抑制作用。
SARS-CoV-2介導之肺損傷與瀰漫性肺泡損傷和氣室水腫相關,因此,伴隨發炎細胞之間質浸潤、觸發凝血及纖維蛋白沈積。在SARS感染期間需考慮之潛在生物標記包括發炎血漿標記、凝血及纖維蛋白分解的程度升高。肺泡上皮屏障損傷為急性呼吸道窘迫症候群(ARDS)與急性肺損傷(ALI)的特徵;血漿表面蛋白(如SP-A與SP-D)的含量可具有一預測值。因此,本研究促使進一步調查肺表面在COVID-19中的作用。
總之,含有同源三聚體頸部與CRD區域之胜肽rfhSP-D藉由限制病毒進入過度表現ACE2受體之HEK293T細胞,作為SARS-CoV-2感染之進入抑制劑。將有關rfhSP-D參與及其相關抗病毒效果之知識往前推進以開發針對多個細胞傳訊途徑之新穎治療方法的時機已成熟。由於細胞類型與推定之受體的不同效果與變異,使rfhSP-D觸發抗病毒效果的機制具有病毒特異性。rfhSP-D對SARS-CoV-2具有明顯的治療潛力。
上文中引用的所有出版物、專利及專利文獻在此皆併入本案以作為參考資料,如同單獨之併入以作為參考資料。
已參照各種具體及較佳具體實施例與技術描述本發明。然而,本領域技術人員將理解,在維持在本發明之精神與範疇內的情況下可進行許多變化與修改。
無
配合附圖閱讀將能更佳地理解本發明如上之概述以及如後之詳細描述在。為了說明本發明,在圖式中顯示現有較佳之具體實施例。
在圖式中:
圖1提供了經由直接ELISA確定人類SP-D的一重組片段(rfhSP-D)和重組人類全長SP-D(hFL-SP-D)與SARS-CoV-2之刺突(S1)(A)及其RBD(B)的結合。微量滴定孔在4℃下以含有SARS-CoV-2刺突S1蛋白(5 μg/ml)(HEK 293細胞)或RBD(5 μg/ml)(HEK 293細胞)之碳酸鹽-重碳酸鹽緩衝液(pH 9.6)塗覆過夜。翌日,各孔以含有1% BSA與5mM CaCl
2之Tris緩衝鹽水(TBS)緩衝液(pH 7.2-7.4)阻斷。各孔在以TBS洗滌後,在4℃下以含有一系列兩倍稀釋之rfhSP-D或hFL-SP-D蛋白的阻斷緩衝液培養過夜。使用生物素化之小鼠抗人類SP-D檢測抗體(1:180)檢測S1蛋白與rfhSP-D之間的結合,接著以卵白素山葵過氧化酶(HRP)-共軛體(1:40)進行探測。數據以三次獨立實驗之三重複的平均值±SEM表示。使用非配對t檢定統計分析確定顯著性。誤差槓顯示SEM。將對照組、麥芽糖組及EDTA組與含有2.5 μg或5 μg S1(RBD)之CaCl
2相比較。
圖2提供了競爭性ELISA,其顯示麥芽糖與EDTA對rfhSP-D與S1(A)及其RBD(B)之結合的影響。聚苯乙烯微量滴定盤以2μg/ml rfhSP-D塗覆,並以SARS-CoV-2刺突S1蛋白(2.5與5 μg/ml)(綿羊-IgG標籤)或RBD(His-標籤)(2.5與5 μg/ml)培養。所述結合使用抗綿羊IgG HRP抗體(1:2000)或抗His抗體(1:2000)檢測。藉由VersaMax™ ELISA微量盤讀儀紀錄450nm處之吸光度。使用未配對t檢定統計分析確定顯著性。誤差槓顯示SEM。所有組別皆與含有RBD之CaCl
2相比較(*p < 0.05;**p < 0.01;***p < 0.001;****p < 0.0001)。
圖3提供了藉由免疫螢光顯微法(A)、流式細胞法(B)及西方墨點法(C)之ACE2受體在HEK293T細胞上的表現。(A)將HEK293T(0.5x10
5個細胞)與HEK293T-ACE2細胞(0.5x10
5個細胞)種植在蓋玻片上,接著在37℃之標準培養條件下培養。細胞在以PBS洗滌兩次後,使用ACE2抗體[SN0754](1:250)檢測兩個細胞株中之ACE2表現,接著在室溫下培養1小時。在PBS洗滌後,添加山羊抗兔IgG(H+L)交叉吸附二次抗體(1:500)。在PBS洗滌後,將蓋玻片安裝在帶有DAPI之培養基中的顯微鏡載玻片上,並在螢光顯微鏡(Olympus)下觀察。(B)ACE2表現的流式細胞法分析係藉由使用ACE2抗體[N1N2]、N-term(GeneTex)(1:250)的螢光強度變化確定。藉由CytoFLEX檢測ACE2表現。(C)ACE2表現係藉由使用ACE2抗體[SN0754](GeneTex)(1:1000)的西方墨點法檢查。
圖4顯示處理rfhSP-D抑制SARS-CoV-2 S1與HEK293T細胞上之ACE2受體之間的交互作用。藉由以抗His抗體(10ug/ml)標記SARS-CoV-2 S1蛋白(5 ug/ml)製造蛋白複合物,接著在室溫下以rfhSP-D(0.625、1.25、2.5、5或10 μg/ml)培養2小時。在37℃下將此複合物(S1+抗His+rfhSP-D)添加在HEK293T-ACE2細胞(1x10
5個細胞)上2小時。收集細胞,並以FACS緩衝液洗滌兩次,並以抗小鼠IgG PE共軛體(GeneTex,GTX25881)(1:100)培養30分鐘並洗滌三次。藉由CytoFLEX檢測S1染色的細胞。使用未配對t檢定統計分析確定顯著性。所有組別皆與S1組相比較。誤差槓顯示SEM。M=空白組(*p < 0.05;**p < 0.01;****p < 0.0001)(n = 3)。
圖5顯示rfhSP-D作為SARS-CoV-2感染之進入抑制劑。(A)藉由西方墨點法確定SARS-CoV-2假型慢病毒顆粒與含培養基之假型慢病毒顆粒的S1表現。(B)以SARS-CoV-2 S1假型慢病毒顆粒轉導之rfhSP-D處理之HEK293T細胞(過度表現ACE2受體)的螢光素酶報導子活性。使用未配對t檢定統計分析確定顯著性。所有組別皆與VSV-S1相比較。誤差槓顯示SEM。M=培養基(**p<0.01;***p<0.001;****p<0.0001)(n = 3)。
無
序列表 <![CDATA[<110> 王志堯]]> <![CDATA[<120> 用於治療嚴重急性呼吸道症候群冠狀病毒2 (SARS-COV-2)感染之方法]]> <![CDATA[<130> WJY0004US]]> <![CDATA[<160> 3 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![CDATA[<211> 375]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 1]]> Met Leu Leu Phe Leu Leu Ser Ala Leu Val Leu Leu Thr Gln Pro Leu 1 5 10 15 Gly Tyr Leu Glu Ala Glu Met Lys Thr Tyr Ser His Arg Thr Met Pro 20 25 30 Ser Ala Cys Thr Leu Val Met Cys Ser Ser Val Glu Ser Gly Leu Pro 35 40 45 Gly Arg Asp Gly Arg Asp Gly Arg Glu Gly Pro Arg Gly Glu Lys Gly 50 55 60 Asp Pro Gly Leu Pro Gly Ala Ala Gly Gln Ala Gly Met Pro Gly Gln 65 70 75 80 Ala Gly Pro Val Gly Pro Lys Gly Asp Asn Gly Ser Val Gly Glu Pro 85 90 95 Gly Pro Lys Gly Asp Thr Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly 100 105 110 Val Pro Gly Pro Ala Gly Arg Glu Gly Pro Leu Gly Lys Gln Gly Asn 115 120 125 Ile Gly Pro Gln Gly Lys Pro Gly Pro Lys Gly Glu Ala Gly Pro Lys 130 135 140 Gly Glu Val Gly Ala Pro Gly Met Gln Gly Ser Ala Gly Ala Arg Gly 145 150 155 160 Leu Ala Gly Pro Lys Gly Glu Arg Gly Val Pro Gly Glu Arg Gly Val 165 170 175 Pro Gly Asn Thr Gly Ala Ala Gly Ser Ala Gly Ala Met Gly Pro Gln 180 185 190 Gly Ser Pro Gly Ala Arg Gly Pro Pro Gly Leu Lys Gly Asp Lys Gly 195 200 205 Ile Pro Gly Asp Lys Gly Ala Lys Gly Glu Ser Gly Leu Pro Asp Val 210 215 220 Ala Ser Leu Arg Gln Gln Val Glu Ala Leu Gln Gly Gln Val Gln His 225 230 235 240 Leu Gln Ala Ala Phe Ser Gln Tyr Lys Lys Val Glu Leu Phe Pro Asn 245 250 255 Gly Gln Ser Val Gly Glu Lys Ile Phe Lys Thr Ala Gly Phe Val Lys 260 265 270 Pro Phe Thr Glu Ala Gln Leu Leu Cys Thr Gln Ala Gly Gly Gln Leu 275 280 285 Ala Ser Pro Arg Ser Ala Ala Glu Asn Ala Ala Leu Gln Gln Leu Val 290 295 300 Val Ala Lys Asn Glu Ala Ala Phe Leu Ser Met Thr Asp Ser Lys Thr 305 310 315 320 Glu Gly Lys Phe Thr Tyr Pro Thr Gly Glu Ser Leu Val Tyr Ser Asn 325 330 335 Trp Ala Pro Gly Glu Pro Asn Asp Asp Gly Gly Ser Glu Asp Cys Val 340 345 350 Glu Ile Phe Thr Asn Gly Lys Trp Asn Asp Arg Ala Cys Gly Glu Lys 355 360 365 Arg Leu Val Val Cys Glu Phe 370 375 <![CDATA[<210> 2]]> <![CDATA[<211> 522]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 2]]> ggatccccgg gattgaaggg ggacaaaggc attcctggag acaaaggagc aaagggagaa 60 agtgggcttc cagatgttgc ttctctgagg cagcaggttg aggccttaca gggacaagta 120 cagcacctcc aggctgcttt ctctcagtat aagaaagttg agctcttccc aaatggccaa 180 agtgtcgggg agaagatttt caagacagca ggctttgtaa aaccatttac ggaggcacag 240 ctgctgtgca cacaggctgg tggacagttg gcctctccac gctctgccgc tgagaatgcc 300 gccttgcaac agctggtcgt agctaagaac gaggctgctt tcctgagcat gactgattcc 360 aagacagagg gcaagttcac ctaccccaca ggagagtccc tggtctattc caactgggcc 420 ccaggggagc ccaacgatga tggcgggtca gaggactgtg tggagatctt caccaatggc 480 aagtggaatg acagggcttg tggagaaaag cgtcttgtgg tc 522 <![CDATA[<210> 3]]> <![CDATA[<211> 177]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 3]]> Gly Ser Pro Gly Leu Lys Gly Asp Lys Gly Ile Pro Gly Asp Lys Gly 1 5 10 15 Ala Lys Gly Glu Ser Gly Leu Pro Asp Val Ala Ser Leu Arg Gln Gln 20 25 30 Val Glu Ala Leu Gln Gly Gln Val Gln His Leu Gln Ala Ala Phe Ser 35 40 45 Gln Tyr Lys Lys Val Glu Leu Phe Pro Asn Gly Gln Ser Val Gly Glu 50 55 60 Lys Ile Phe Lys Thr Ala Gly Phe Val Lys Pro Phe Thr Glu Ala Gln 65 70 75 80 Leu Leu Cys Thr Gln Ala Gly Gly Gln Leu Ala Ser Pro Arg Ser Ala 85 90 95 Ala Glu Asn Ala Ala Leu Gln Gln Leu Val Val Ala Lys Asn Glu Ala 100 105 110 Ala Phe Leu Ser Met Thr Asp Ser Lys Thr Glu Gly Lys Phe Thr Tyr 115 120 125 Pro Thr Gly Glu Ser Leu Val Tyr Ser Asn Trp Ala Pro Gly Glu Pro 130 135 140 Asn Asp Asp Gly Gly Ser Glu Asp Cys Val Glu Ile Phe Thr Asn Gly 145 150 155 160 Lys Trp Asn Asp Arg Ala Cys Gly Glu Lys Arg Leu Val Val Cys Glu 165 170 175 Phe
Claims (7)
- 一種在個體中治療嚴重急性呼吸道症候群冠狀病毒2(SARS-CoV-2)感染之方法,其包含對該個體投予一治療有效量之胜肽,其為人類表面蛋白D的一重組片段(rfhSP-D)。
- 如請求項1之方法,其中該胜肽係以有效抑制SARS-CoV-2進入該個體之宿主細胞中的量投予。
- 如請求項1之方法,其中該胜肽包含如SEQ ID NO: 3所示之序列。
- 如請求項1之方法,其中該胜肽由如SEQ ID NO: 3所示之序列所組成。
- 一種用於治療SARS-CoV-2感染之醫藥組合物,其包含一治療有效量之具有如SEQ ID NO: 3所示序列之胜肽,以及一醫藥上可接受載體。
- 如請求項4之醫藥組合物,其中該治療有效量為有效抑制SARS-CoV-2進入個體之宿主細胞中的量。
- 如請求項4之醫藥組合物,其中該胜肽由如SEQ ID NO: 3所示之序列所組成。
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