TW202338090A - 高效疫苗 - Google Patents
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Abstract
本文提供一種單離之多核苷酸,其編碼非結構性蛋白nsp1、nsp2、nsp3及nsp4以及包含抗原蛋白的多肽,該抗原蛋白融合至訊號序列、穿膜域及選自CD4+ T細胞表位及CD8+ T細胞表位之至少一種肽。該多核苷酸可用於製造針對病毒感染,尤其是COVID-19感染的疫苗,治療癌症及/或發炎性疾病。
Description
本揭露一般係關於包含α病毒複製子及抗原之高效疫苗領域,且係關於針對抗原用於治療及/或免疫化的方法及組成物。特定而言,本揭露係關於用於冠狀病毒諸如SARS-CoV-2(COVID-19)之疫苗。
冠狀病毒係一大類病毒,其通常引起輕度至中度以上的呼吸道疾病,如普通感冒。惟,在過去二十年間,三種新型冠狀病毒業經從動物宿主出現,其引起重度及廣泛分佈之疾病及死亡。
存在數百種冠狀病毒,其大多數在諸如豬、駱駝、蝙蝠及貓之類的動物中傳播。有時,彼等病毒跳轉至人類,亦即所謂溢出事件,並可引起疾病。影響人類的七種已知冠狀病毒中之四種僅引起輕度至中度疾病。三種可造成更為嚴重的,甚至致死之疾病。SARS冠狀病毒(SARS-CoV)於2002年11月出現,並引起嚴重急性呼吸道症候群(SARS)。該病毒於2004年消失。中東呼吸道症候群(MERS)由MERS冠狀病毒(MERS-CoV)引起。MERS於2012年9月經鑑定,其自駱駝中之動物宿主傳輸並持續以引起散發的及局部的疫情。本世紀出現的第三種新型冠狀病毒稱為SARS-CoV-2。其造成新冠病毒肺炎(COVID-19),其在
2019年12月於中國出現,並於2020年3月11日由世界衛生組織宣佈為全球大流行。(冠狀病毒:https://www.niaid.nih.gov/diseases-conditions/coronaviruses)
感染SARS-CoV-2病毒的人大多數將經歷輕度至中度的呼吸道疾病,且無需進行特殊治療即可康復。老年人及彼等具有基礎醫療問題如心血管疾病、糖尿病、慢性呼吸道疾病及癌症者更可能發展為重症疾病。任何人、在任何年齡皆可能感染COVID-19,並變為重症疾病或死亡。(新冠病毒肺炎(COVID-19):https://www.who.int/health-topics/coronavirus#tab=tab_1)
奧密克戎(o)(B.1.1.529變異株)於2021年11約24日由南非作為新SARS-CoV-2變異株報告給WHO。
初步證據表明,相較於其他相關變異株,被奧密克戎再次感染的風險增加(亦即,先前業經患有COVID-19的人將會變成更容易被奧密克戎再次感染)。(關於奧密克戎的最新進展:https://www.who.int/news/item/28-11-2021-update-on-omicron)
目前存在數種針對COVID-19的獲批疫苗。特定而言,mRNA疫苗顯示超過95%之效力。惟,在全世界範圍內需要提供更多疫苗以阻止此大流行。為了提供甚至對於全世界範圍內之任何變異株皆有效之疫苗,需要進行更多疫苗研發,特定而言,基於能夠在短時間內對任何變異株產生疫苗有效性的技術的疫苗。
[專利文獻]
[PTL 1]
WO2021/21068
本揭露之目的為提供新設計概念,用於研發針對諸如病毒抗原、癌症抗原及其他之抗原的疫苗,該疫苗可有效預防或治療感染性疾病或癌症。
本揭露係關於新穎的抗原活性蛋白質/多肽,其能夠誘導針對抗原的免疫反應。本文所揭露之蛋白質/多肽包括抗原蛋白,該抗原蛋白融合至訊號序列、穿膜域以及選自CD4+ T細胞表位及CD8+ T細胞表位的至少一種肽。該抗原蛋白可係衍生自冠狀病毒結構蛋白的蛋白質或其片段。
冠狀病毒結構蛋白可包含棘(S)蛋白、核殼體(N)蛋白、膜(M)蛋白及小包膜蛋白(E)。抗原蛋白可衍生自該等結構蛋白中之任一者或其組合。該等抗原蛋白之具體實例可包括棘蛋白之S1及/或S2次單元,且尤其是S1次單元之受體結合域(RBD)。
另一方面,本揭露係關於一種編碼上述新穎抗原活性蛋白/多肽的新穎多核苷酸,該多核苷酸能夠誘導針對抗原蛋白的保護。
另一方面,本揭露係關於一種新穎的α病毒複製子,該複製子可表現上述抗原活性蛋白/多肽。該α病毒複製子包括諸如編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4的RNA之多核苷酸,以及編碼上述抗原活性蛋白/多肽的多核苷酸,作為目標基因。
又一方面,本揭露係關於一種包含上述多肽或多核苷酸之疫苗。本揭露尤其提供一種疫苗,該疫苗包含多核苷酸,該多核苷酸編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4以及包含抗原蛋白的多肽,該抗原蛋白融合至訊號序列、穿膜域以及選自CD4+ T細胞表位及CD8+ T細胞表位的至少一種
肽。該CD4+ T細胞表位可係萬用表位。在該抗原蛋白質為衍生自病毒結構蛋白的蛋白質的情況下,該疫苗可用於預防及/或治療受試者以使其免於病毒感染。
又一方面,本揭露係關於一種用於免疫化、預防或治療受試者以使其免於病毒感染的方法,包含向有此需要之受試者投予有效量的上述多肽或多核苷酸。
再一方面,本揭露係關於上述多肽或多核苷酸用於製造藥物的用途。
另一方面,本揭露係關於新穎的多核苷酸,該多核苷酸編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4以及包含抗原肽之多肽,其中該多核苷酸包含經修飾之核苷(例如,N1-甲基假尿苷或5-甲基胞苷或兩者)。
這一高效疫苗設計亦應用於開發用於治療癌症或發炎性疾病的疫苗。
圖1係α病毒複製子之構建體。
圖2係針對SARS-CoV-2變異株之RBD蛋白的ELISA。
圖3係ACE抑制檢定。
圖4係具有經修飾之核苷的多核苷酸在先天性免疫中之效應。
圖5係疫苗組成物對於攻毒COVID-19 WT株或伽馬(γ)變異株之倉鼠之體重的效應。
圖6係疫苗組成物對於攻毒COV-2 WT株或伽馬變異株之倉鼠中之SARS-CoV-2的定量RT-PCR檢定的效應。
圖7係疫苗組成物對於攻毒SARS-COV-2 WT株或伽馬變異株之倉鼠之肺的組織病理學分析的效應。
如本文所用,「抗原」指代能夠被抗體或T細胞受體結合的分子。另外,抗原能夠被免疫系統辨識且/或能夠誘導體液免疫反應及/或細胞免疫反應,導致B及/或T淋巴細胞之活化。如本文所用,抗原包括但不限於病毒、過敏原、自體抗原、半抗原、癌抗原(亦即,腫瘤抗原)及傳染病抗原諸如結核分枝桿菌(Mycobacterium tuberculosis)以及有機小分子諸如濫用的藥物(如尼古丁)及其片段及衍生物。此外,用於本揭露的抗原可係肽、蛋白質、域、碳水化合物、生物鹼、脂質或小分子諸如類固醇激素及其片段及衍生物、自體抗體及細胞激素自身。如本文所用,「抗原肽」指代可用作抗原的蛋白質或肽。
如本文所用,「病毒」可係嚴重急性呼吸道症候群相關冠狀病毒(SARS)、嚴重急性呼吸道症候群冠狀病毒2(SARS-CoV-2)、Ebola、HIV、B型肝炎病毒(HBV)、流感病毒、C型肝炎病毒(HCV)、人類乳突病毒(HPV)、巨細胞病毒(CMV)、屈公病毒、呼吸道融合病毒(RSV)、登革熱病毒或原黏液病毒(orthymyxoviridae)科病毒,但不限於此。
如本文作用,「冠狀病毒」意為指代屬於冠狀病毒科的單股、正義RNA病毒。示例性冠狀病毒科病毒包括但不限於SARS-Cov、MERS-Cov及SARS-CoV-2(COVID-19)。SARS-CoV-2(COVID-19)可包括已知及未知的突變
株。已知突變株可包括SARS-CoV-2 E484K_N501Y_K417T突變株(伽馬,巴西變異株)、T478K_L452R(德爾塔(δ),印度變異株)、E484K_N501Y_K417N突變株(貝塔(β),南非變異株)、E484K突變株及K417N_S477N_T478K_E484A_Q493K_G446S_N501Y_Y505H_G496S_Q498R_N440K_G339D_S375F_S373P_S371IL突變株(奧密克戎)。冠狀病毒基因體編碼大量非結構蛋白及四種主要結構蛋白,該等主要結構蛋白包括棘(S)、核鞘(N)、膜(M)及小包膜(E)蛋白。棘(S)蛋白係一種大的包膜糖蛋白,由S1及S2次單元構成。「受體結合域(RBD)」位於S1次單元內。本文所使用的抗原蛋白之較佳實例為冠狀病毒RBD、其片段或其突變株。棘蛋白、棘蛋白之S1及S2次單元以及COVID-19及其突變株之RBD業經鑑定並公開(Wrapp et al.,Science Vol.367,issue 6483(2020)pp.1260-1263 10.1126/science.abb2507;Z.Wang et al.,Nature Vol 592(2021)pp616-622,所引述之檔案之內容藉由引用併入本文)。
本文所使用的「冠狀病毒結構蛋白」可係天然存在的病毒結構蛋白或其經修飾之蛋白質。經修飾之蛋白質可係天然存在的病毒結構蛋白或其突變體之片段。於一個態樣中,經修飾之蛋白質與天然存在的病毒結構蛋白或其片段具有至少70%、75%、80%、85%、90%、95%、98%胺基酸序列同一性。於一個態樣中,經修飾之蛋白質為突變體,其中基於天然存在的病毒包膜蛋白或其片段,至多10%的胺基酸係經缺失、取代及/或添加。
如本文所用,「穿膜域(TM)」為衍生自天然或合成來源的蛋白質。若該來源為天然者,則在一些方面,該域係衍生自任何膜結合或穿膜蛋白。一方面,膜結合或穿膜蛋白為與抗原肽之起源異源的蛋白質。膜結合或穿膜蛋白之實例可包括以下各項之α、β或ζ鏈:T細胞受體、CD28、CD3ε、CD45、CD4、
CD5、CDS、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、toll樣受體(TLR)諸如人類中之TLRI-TLRIO以及小鼠中之TLRI-TLR9及TLRI1-TLR13、間白素(IL)受體諸如IL-1-28受體、RANTES受體(CCRI、CCR3、CCR5)、MIP-I受體、PF4受體;屬於GPCR細胞激素受體的M-CSF受體及NAP-2受體;血球凝集素(HA)。另一方面,膜結合或穿膜蛋白為衍生自COVID-19的蛋白質,諸如COVID-19棘蛋白。
穿膜蛋白之實例亦可包括下列者:5-脂肪加氧酶活化蛋白、ABC運輸蛋白、ACBP、類澱粉蛋白β(A4)、Bcl-2抑制劑、BNIP、CAAX蛋白酶、細胞色素P450、E-NPP、EPHAI、EPHA2、EPHA3、EPHA4、脂肪酸去飽和酶、γ分泌酶、葡萄糖運輸蛋白、血型醣蛋白、GPCR、HER2/ErbB2、HER3/ErbB3、HER4/ErbB4、HSD-11β、缺氧誘導蛋白、免疫球蛋白、胰島素受體、整聯蛋白、離子通道、MAPEG、MFS、MinK家族、MPP、肽酶AD、肽酶家族M48、肽酶MA、蛋白質Jagged、受體型激酶、SNARE複合體、硫酸酯酶、TNF受體、穿膜蛋白14、運輸蛋白、TROBP、VEGF受體、醛脫氫酶、氨及脲運輸蛋白、FMN聯結氧化還原酶、含有富白胺酸重複序列(LRR)之穿膜蛋白、白三烯C4合成酶、溶體相關膜醣蛋白、主要內在蛋白(MIP)/FNT超家族、微粒體前列腺素E合成酶、N-(去氧)核糖基轉移酶樣膜蛋白、中性/鹼性神經醯胺酶、寡醣基轉移酶、五聚物配體-門控離子通道、視紫質樣受體及泵、單螺旋ATPase調節物、鯊烯/八氫番茄紅素合成酶、硬脂醯基-CoA去飽和酶1、Stannin(SNN)膜蛋白、T細胞表面醣蛋白CD3 ζ鏈、三角形四肽(Tetratricopeptide)重複序列(TPR)α-螺旋重複序列蛋白、具有NAD(P)-結合Rossmann折疊域之穿膜蛋白。
此外,接附至脂質雙層或其他整合蛋白及肽的單型/膜周邊蛋白亦可用作穿膜蛋白。實例可包括α/β-水解酶、Annexin、Bet V1樣蛋白、含C1域之蛋白質、含C2域之蛋白質、CoA依賴性醯基轉移酶、含CRAL-TRIO域之蛋白質、DNA酶I樣蛋白質、纖維蛋白原、FYVE/PHD鋅指蛋白、半乳糖結合域樣蛋白質、醣脂轉移蛋白、免疫球蛋白樣超家族(E組)蛋白、脂質運載蛋白、脂肪加氧酶、PGBD超家族、PH域樣蛋白質、磷脂酸肌醇3-/4-激酶、PLC樣磷酸二酯酶、磷酸酪胺酸蛋白磷酸酶II、含P環之核苷三磷酸酯水解酶、蛋白激酶超家族、含PX域之蛋白質、皂素(Saposin)、突觸核蛋白及轉錄因子tubby。
於本揭露之一方面,穿膜域至少包括膜結合或穿膜蛋白之穿膜區域。此外,穿膜域亦可包括膜結合或穿膜蛋白之近膜域(JMD)及/或細胞質尾。
替代性地,於一些態樣中,穿膜域為合成的。於一些態樣中,合成的穿膜域主要包含疏水殘基諸如白胺酸及纈胺酸。於一些態樣中,苯丙胺酸、色胺酸及纈胺酸之三聯體將見於合成的穿膜域之各端。
較佳之穿膜域可係彼等衍生自流感病毒血球凝集素(HA)、CD80、Toll樣受體4(TLR4)或COVID-19棘蛋白。具體實例可包括包含流感病毒血球凝集素之柔性近膜區域或柔性鏈接子、穿膜域及細胞質尾的蛋白質「HA(柔性-TM-cyt)」;由人類CD80之穿膜域及細胞質尾組成的蛋白質;由穿膜域(TM)及Toll/間白素-1受體域(TIR)組成的蛋白質;及由COVID-19棘(S)蛋白之近膜域(JMD)及穿膜域組成的蛋白質。
如本文所用,「核苷」指代由鳥嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)、尿苷(U)、胞苷(C)或其經修飾之核苷組成的分子。
經修飾之核苷包括但不限於,假尿苷、N1-甲基-假尿苷、5-甲基-尿苷、假尿苷、N1-甲基-假胞苷及5-甲基-胞苷。
假尿苷或假胞苷為尿苷或胞苷之異構物,其中尿嘧啶或胞嘧啶係經由碳-碳而非氮-碳糖苷鍵接附。
於一個態樣中,經修飾之核苷係獨立地選自N1-甲基-假尿苷或5-甲基-胞苷。於一個態樣中,mRNA或saRNA中所包括的實質上100%之胞苷及尿苷分別為經修飾之胞苷(例如,5-甲基-胞苷)及經修飾之尿苷(例如,N1-甲基-假尿苷)。於一個態樣中,100%之胞苷為經修飾之胞苷,且80%之尿苷為經修飾之尿苷;而於另一態樣中,50%之胞苷為經修飾之胞苷,且50%之尿苷為經修飾之尿苷。於再一態樣中,saRNA中的少於100%之尿苷為經修飾之尿苷。
如本文所用,「訊號序列」(有時指代為訊號肽、靶向訊號、定位訊號、定位序列、轉運肽、前導序列或前導肽)為多核苷酸或多肽,取決於語境。訊號序列之長度為約9至200個核苷酸或3至70個胺基酸,其係視需要併入編碼區域或蛋白質之5'或N-端。在將蛋白質轉運至所欲位點後,一些訊號序列從蛋白質裂解,例如,藉由訊號肽酶。
於一些態樣中,可採用IL-2,尤其是人類IL-2之訊號序列。於另一態樣中,可採用COVID-19棘蛋白之訊號序列。
如本文所用,CD4+ T-細胞表位為肽,其結合至II類主要組織相容性複合體(MHC)分子(MHC-II)並觸發CD4+ T細胞免疫反應。CD4+ T細胞表位可係衍生自病毒的肽,該抗原蛋白質係衍生自該病毒。衍生自病毒蛋白的CD4+ T細胞表位可藉由已知表位映射程序進行鑑定。於本揭露中,由藉由鏈接
子諸如「AA」連結的多個CD4+ T細胞表位組成的肽可用作「CD4+ T細胞表位」。替代性地,CD4+ T細胞表位可係泛DR表位(PADRE)。
本揭露中使用的表達「PADRE」意指Pan HLA DR結合表位或萬用表位,其活化幾乎全部CD4+ T細胞,與HLA單倍型無關。多種肽業經鑑定為PADRE,且已知PADRE中之任一者可用於本揭露中。PADRE之一個實例為AKFVAAWTLKAAA(SEQ ID NO:1)。
如本文所用,CD8+ T細胞表位為肽,其結合至I類主要組織相容性複合體(MHC)分子(MHC-I)且觸發CD8+ T細胞免疫反應。該CD8+ T細胞表位可係衍生自病毒的肽,該抗原蛋白係衍生自該病毒。衍生自病毒蛋白的CD8+ T細胞表位可藉由已知表位作圖(mapping)程序進行鑑定。於本揭露中,由藉由鏈接子諸如AA連結的多個CD8+ T細胞表位可用作「CD8+ T細胞表位」。
抗原蛋白、訊號序列、穿膜域及選自CD4+ T細胞表位及CD8+ T細胞表位之至少一種肽可直接或間接地融合。於一個態樣中,一個或兩個鏈接子可經插置於其間。
又,冠狀病毒結構蛋白、訊號序列、穿膜域及選自CD4+ T細胞表位及CD8+ T細胞表位之至少一種肽可經截短並藉由短鏈接子替換。於一些態樣中,冠狀病毒結構蛋白、穿膜域及/或訊號序列包括一個或多個肽鏈接子。
短鏈接子之實例由2至25個胺基酸(例如,2、3、4、5或6個胺基酸)組成。通常,其長度為2至15個胺基酸,諸如SG、GS、SGG、GGS SGSG及TRGGS。於某些情況下,鏈接子可由僅一個胺基酸諸如甘胺酸(G)、絲胺酸(S)及半胱胺酸(C)組成。
當冠狀病毒結構蛋白係透過化學交聯鏈接子化學地接合至訊號序列、穿膜域及選自CD4+ T細胞表位及CD8+ T細胞表位之至少一種肽,該化學交聯鏈接子之實例包括但不限於,SMPH、Sulfo-MBS、Sulfo-EMCS、Sulfo-GMBS、Sulfo-SIAB、Sulfo-SMPB、Sulfo-SMCC、SVSB及SIA。亦可採用市場上可獲得之化學交聯鏈接子。
IgG衍生之物質亦可用作鏈接子。IgG衍生之物質之實例包括IgG1至IgG4,包含(i)全(鉸鏈-CH2CH3)、(ii)半(鉸鏈-CH3)及(iii)短(僅12aa鉸鏈)。較佳之實例為IgG4-CH3。
如本文所用,「α病毒」意為指代含RNA之病毒,其屬於披膜病毒(Togaviridae)科之病毒。示例性披膜病毒科病毒包括但不限於東方馬腦炎病毒(EEEV)、委內瑞拉馬腦炎病毒(VEEV)、沼澤地(Everglades)病毒、米坎波(Mucambo)病毒、皮舒納(Pixuna)病毒、西方馬腦炎病毒(WEEV)、辛得比斯(Sindbis)病毒、塞姆利基森林(Semliki Forest)病毒、米德堡(Middleburg)病毒、屈公(Chikungunya)病毒(CHIKV)、阿尼昂-尼昂(O'nyong-nyong)病毒、羅氏河病毒、巴馬森林(Barmah Forest)病毒、蓋他(Getah)病毒、鷺山(Sagiyama)病毒、貝巴魯(Bebaru)病毒、馬雅羅(Mayaro)病毒、烏那(Una)病毒、奧拉(Aura)病毒、沃他羅阿(Whataroa)病毒、巴班肯(Babanki)病毒、克孜拉格其(Kyzylagach)病毒、高地(Highlands)J病毒、福特摩根(Fort Morgan)病毒、恩杜姆(Ndumu)病毒、柏吉河(Buggy Creek)病毒及歐克波(Ockelbo)病毒。
「α病毒結構蛋白」意為一種多肽或其片段,其與天然存在的病毒殼體或包膜蛋白具有至少約80%胺基酸序列同一性。於一個態樣中,α病毒結構蛋白與以下病毒具有至少約85%、90%、95%或更大的胺基酸序列同一性:
東方馬腦炎病毒(EEEV)、委內瑞拉馬腦炎病毒(VEEV)、沼澤地病毒、米坎波病毒、皮舒納病毒、西方馬腦炎病毒(WEEV)、辛得比斯病毒、塞姆利基森林病毒、米德堡病毒、屈公病毒(CHIKV)、阿尼昂-尼昂病毒、羅氏河病毒、巴馬森林病毒、蓋他病毒、鷺山病毒、貝巴魯病毒、馬雅羅病毒、烏那病毒、奧拉病毒、沃他羅阿病毒、巴班肯病毒、克孜拉格其病毒、高地J病毒、福特摩根病毒、恩杜姆病毒或柏吉河病毒。α病毒結構蛋白之野生型胺基酸序列可自GenBank獲得。
於具體態樣中,α病毒為CHIKV,例如,CHIKV株37997或LR2006 OPY-I。於其他態樣中,α病毒為VEEV,例如,VEEV株TC-83。
「α病毒複製子」意為一種RNA分子,其可在標靶細胞中引導其自己的活體內擴增。複製子編碼催化RNA擴增的聚合酶(nsp1、nsp2、nsp3、nsp4)且含有複製所需的順式RNA序列,該等序列係由所編碼之聚合酶辨識並利用。α病毒複製子典型含有以下順序之元件:5'UTR,編碼α病毒非結構蛋白之序列(nsp1、nsp2、nsp3、nsp4),3' UTR,及poly A尾。α病毒複製子亦含有一個或多個病毒亞基因體(SG)啟動子,其引導目標基因之表現。彼等序列可具有先前技藝中所教示之一個或多個突變。
本揭露所提供的α病毒複製子可具有圖1中顯示之構建體。
於本揭露中,「包含」、「含有」及「具有」等可具有美國專利法中所賦予其等之意義,且可以為「包括」等;「實質上由...組成」或「實質由...組成」同源具有美國專利法中所賦予之意義其該術語係開放性者,允許超過其所引述者之存在,只要其所引述者之基本及新穎特徵未被超過其所引述者之存在改變即可,但排除先前技藝態樣。
「片段」意為蛋白質、多肽或多核苷酸之一部分。這一部分含有,較佳地,參考核酸分子或多肽之完整長度的至少10%、20%、30%、40%、50%、60%、70%、80%、90%。片段可含有10、20、30、40、50、60、70、80、90個,或100、200、300、400、500、600、700、800、900或1000個核苷酸或胺基酸。
「參考」意為標準或對照條件。
「參考序列」係用作序列對比之基準的界定序列。參考序列可係特定序列之子集或整體,例如,全長cDNA或基因序列之鏈段,或完全cDNA或基因序列。對於多肽,參考多肽序列之長度通常為至少約16個胺基酸,較佳至少約20個胺基酸,更佳至少約25個胺基酸,且甚至更佳約35個胺基酸、約50個胺基酸或約100個胺基酸。對於核酸,參考核酸序列之長度通常為至少約50個核苷酸,較佳至少約60個核苷酸,更佳至少約75個核苷酸,且甚至更佳約100個核苷酸或約300個核苷酸,或接近該等數值或該等數值之間的任意整數。
序列同一性典型係使用序列分析軟體(例如,威斯康辛大學生物技術中心遺傳性計算機小組之序列分析軟體包,1710 University Avenue,Madison,Wis.53705;BLAST、BESTFIT、GAP或PILEUP/PRETTYBOX程式)。此軟體藉由分配同源程度至各種取代、缺失及/或其他修飾來匹配相同或相似之序列。保守取代典型包括下列組內之取代:甘胺酸、丙胺酸;纈胺酸、異白胺酸、白胺酸;天冬胺酸、麩胺酸、天冬醯胺酸、麩醯胺酸;絲胺酸、蘇胺酸;離胺酸、精胺酸;以及苯丙胺酸、酪胺酸。於測定同一性程度之示例性方法中,可使用BLAST程式,其中介於e<"3>與e<"100>之間的機率評分指示密切相關之序列。
「有效量」意為相對於未經治療之患者減輕疾病之症候所需的藥劑之量。用來實踐本揭露之用於預防或治療疾病的活性化合物之有效量依據投
予方式以及受試者之年齡、體重及一般健康而變。最終,主治醫生或獸醫將決定適當的量及給藥方案。此量係指代為「有效」量。
令人滿意之效應可藉由全身性投予獲得,例如,以肌肉內投予、皮下投予或靜脈內投予1至4次,投予之量為每次103至1010感染單位(Infectious Unit)(IU)或0.01至500μg,較佳為每次105至1010IU或0.1至100μg,例如,每一次107至109IU或1至50μg。複製子可較佳地配製於適用於以習用方式投予之疫苗組成物中。本揭露提供一種疫苗組成物,其包含本文所揭露的α病毒複製子。
「受試者」意為哺乳動物,包括但不限於,人類或非人類哺乳動物,諸如牛、馬、狗、羊或貓。
如本文所用,術語「治療」等指代降低或減輕病患及/或與其相關之症候。應知悉,儘管不妨礙,但治療疾患或病況不需要該疾患、病況或與其相關之症候經完全消除。
如本文所用,「預防」、「阻止」、「預防性治療」等指代降低在受試者中發展為疾患或病況的可能性,該受試者未患有疾患或病況但處於發展為該疾患或病況風險下或易於發展為該疾患或病況。
如本文所用,除非具體制定或從語境中明顯可見,否則術語「或」係理解為開放性者。
如本文所用,除非具體制定或從語境中明顯可見,否則術語「一」及「該」係理解為單數或複數。
本領域將知曉,說明書及申請專利範圍中所揭示之多核苷酸序列將在代表性DNA序列中引述「T」,但在表示RNA之序列中,「T」將會取代為「U」。
本文所提供的任何疫苗組成物或方法可與本文所提供的任何其他疫苗組成物及方法中之一者或多者合用。
術語「載體」指代核酸序列可藉以在器官、細胞或細胞室之間傳播及/或轉移的機構。載體包括質體、病毒、噬菌體(bacteriophage)、前病毒、噬菌質體(phagemid)、轉位子、人工染色體等,其自發地複製或可整合至宿主細胞之染色體內。載體亦可為裸RNA多核苷酸、裸DNA多核苷酸、由相同股內之DNA及RNA兩者構成的多核苷酸、多離胺酸接合之DNA或RNA、肽接合之DNA或RNA、脂質體接合之DNA等,其不會自發地複製。於很多但非全部普通態樣中,本揭露之載體為質體或穿梭載體(bacmid)。
典型地,待表現之核酸分子係「可操作地鏈接」至啟動子及/或增強子,且受到啟動子及/或增強子的轉錄調節控制。
轉染之方法及表現媒介物之選擇將取決於所選擇的宿主系統。轉染方法係揭示於例如Ausubel et al.(同上文)中,表現媒介物可選自彼等提供於例如Cloning Vectors:A Laboratory Manual(P.H.Pouwels et al.,1985,Supp.1987)中者。本段落中所引述之參考文獻藉由引用併入本文。
存在多種表現系統用於產生本揭露之構建體。可用於產生該等構建體之表現載體包括而不限於,染色體型、附加型及病毒衍生之載體,例如,衍生自細菌質體、噬菌體、轉位子、酵母附加體(yeast episome)、插入元件、酵母染色體元件、病毒諸如α病毒(例如,屈公病毒(CHIKV)及委內瑞拉馬腦炎病毒
(VEEV))、桿狀病毒、乳多空病毒諸如SV40、牛痘病毒、腺病毒、禽痘病毒、假狂犬病病毒及反轉錄病毒的載體,以及衍生自其組合的載體。
本文所使用的構建體及/或載體包含編碼非結構蛋白nsp1、nsp2、nsp3及nsp4的α病毒多核苷酸及編碼多肽的目標基因,該多肽包含融合至上述訊號序列及/或穿膜域的冠狀病毒結構蛋白。
載體可係例如噬菌體、質體、病毒或反轉錄病毒載體。包含該等核苷酸的構建體及/或載體應可操作地鏈接至適當的啟動子,諸如CMV啟動子,噬菌體λ PL啟動子,大腸桿菌lac、phoA及tac啟動子,SV40早期及完全啟動子,以及反轉錄病毒LTR之啟動子為非限制性實例。其他合適之啟動子將係熟練技術人員所已知者,取決於宿主細胞及/或所欲之表現率。表現構建體將復含有用於轉錄起始、終止之位點,以及,在所轉錄之區域內,用於轉譯的核糖體結合位點。由該等構建體表現的轉錄本之編碼部分較佳將包括在開始處之轉錄起始密碼子及大致位於待轉譯之多肽末端處的終止密碼子。
載體較佳將包括至少一個可選擇之標記。此類標記包括,對於真核細胞培養為,二氫葉酸還原酶、G418或新黴素抗性;以及,對於在大腸桿菌及其他細菌中培養為,四環素、康黴素或胺苄青黴素。載體中之較佳載體為病毒載體,諸如桿狀病毒、痘病毒(例如,牛痘病毒、鳥類痘病毒、金絲雀痘病毒、禽痘病毒、洗熊痘病毒、天鵝痘病毒等)、腺病毒(例如,狗腺病毒)、皰疹病毒及反轉錄病毒。可與本揭露合用之其他載體包含用於細菌中之載體,其包含pQE70、pQE60及pQE-9、pBluescript載體、Phagescript載體、pNH8A、pNH16a、pNH18A、pNH46A、ptrc99a、pKK223-3、pKK233-3、pDR540、pRIT5。較佳之
真核載體為pFastBacl pWINEO、pSV2CAT、pOG44、pXT1及pSG、pSVK3、pBPV、pMSG及pSVL。其他合適的載體對於熟練技術人員為顯而易見者。
可製備重組構建體並用於將病毒蛋白(包括本文所揭示之彼等)轉染至真核細胞及/或原核細胞中,可在真核細胞及/或原核細胞中表現該等病毒蛋白。因此,於一個態樣中,本揭露提供宿主細胞,其包含一種或多種含有核酸的載體,該核酸編碼α病毒結構蛋白,包括殼體、E3、E2、6K及E1或其部分,及包含核酸及目標基因的載體,該等核酸編碼α病毒nsp1、nsp2、nsp3及nsp4,且該目標基因編碼多肽,該多肽包含訊號序列、冠狀病毒RBD、穿膜域及選自CD4+ T細胞表位及CD8+ T細胞表位之至少一種肽,在允許形成α病毒複製子顆粒的條件下進行。
於一個態樣中,該載體為重組桿狀病毒。於另一態樣中,該重組桿狀病毒經轉染至昆蟲細胞內。於較佳態樣中,該細胞為昆蟲細胞。於另一態樣中,該昆蟲細胞為Sf9細胞。
用於多肽產生的一種特定細菌表現系統為大腸桿菌pET表現系統(Novagen,Inc.,Madison,Wis)。根據該表現系統,將編碼多肽的DNA以經設計為允許表現的取向插入至pET載體中。由於表現此多肽的基因係處於T7調節訊號之控制下,多肽之表現係藉由誘導T7 RNA聚合酶在宿主細胞內之表現而達成。這典型係藉由使用因應於IPTG誘導而表現T7 RNA聚合酶的宿主株而達成。一旦產生,即根據本領域中已知之標準方法,例如本文所揭示之彼等,將重組多肽單離。
取決於所選擇的載體及宿主細胞,該等構建體係藉由下述產生:使由該等載體轉染的宿主細胞在一定條件下生長,從而表現重組蛋白並生成α
病毒複製子,並且形成含有用該α病毒結構蛋白之顆粒封裝的α病毒複製子的構建體。於一個態樣中,提供一種產生構建體之方法,且涉及將包含編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4的多核苷酸及至少一種編碼包含冠狀病毒結構蛋白的多肽的目標基因的載體,該冠狀病毒結構蛋白融合至訊號序列及/或穿膜域,以及各自編碼至少一種α病毒結構蛋白的至少一種載體共轉染至適合的宿主細胞;以及在允許構建體形成的條件下表現該α病毒結構蛋白。於另一態樣中,真核細胞係選自由酵母、昆蟲、兩棲動物、鳥類或哺乳動物細胞所成群組。適當生長條件的選擇處於具有本領域通常知識之人士的技能範圍內。
使產生本揭露之α病毒複製子顆粒的細胞生長的方法包括,但不限於,批式、分批饋料式、連續式及灌注式細胞培養技術。於一個態樣中,使得用編碼α病毒複製子的載體及包含編碼殼體之多肽的載體以及包含編碼包膜蛋白的載體(諸如彼等衍生自CHIKV或VEEV者)共轉染的細胞在生物反應器或醱酵腔室中生長,其中細胞增殖並表現蛋白質(例如,重組蛋白)以進行純化及單離。典型地,細胞培養係於無菌、控溫及氣氛條件下執行。生物反應器為用來培養細胞之腔室,可監測其中之環境添加諸如溫度、氣氛、攪拌及/或pH。於一個態樣中,生物反應器為不鏽鋼腔室。於另一態樣中,該生物反應器為預滅菌之塑膠袋(例如,Cellbag.RTM.,Wave Biotech,Bridgewater,N.J.,所引述之檔案的內容藉由引用併入本文)。於其他態樣中,該預滅菌之塑膠袋為約10L至1000L之袋。
於另一態樣中,RNA分子諸如α病毒複製子可藉由本領域已知之習用程序自模板DNA序列生成。活體外轉錄(IVT)方法允許進行RNA分子的模板引導之合成。IVT方法允許進行RNA轉錄本的大量合成。通常,IVT利用包含位於目標序列上游之啟動子序列的DNA模板。最常見之啟動子序列係起源
於噬菌體,諸如T7、T3或SP6啟動子序列,但可容忍很多其他啟動子序列,包括彼等從頭設計者。DNA模板之轉錄典型係藉由使用對應於特定噬菌體啟動子序列之RNA聚合酶而最佳達成。示例性RNA聚合酶包括但不限於T7 RNA聚合酶、T3 RNA聚合酶或SP6 RNA聚合酶等。IVT通常以dsDNA起始,但可在單股上前行。用於活體外轉錄之套組諸如T7轉錄套組(RiboMaxTM Express Large Scale RNA production System,Promega(WI USA))。
如本文所用,術語「醫藥上可接受之載劑」意為一種或多種可相容之固體或液體填充物、稀釋劑或囊封物質,其等係適用於向人類或其他脊椎動物投予,包括任何及全部水性溶劑(例如,水、醇/水性溶液、鹽水溶液、腸胃外媒介物,諸如氯化鈉及林格氏右旋糖(Ringer's dextrose))、非水性溶劑(例如,丙二醇、聚乙二醇、植物油及可注射有機酯諸如油酸乙酯)、分散介質、塗料、界面活性劑、抗氧化劑、防腐劑(例如,抗細菌或抗真菌劑、抗氧化劑、螯合劑及惰性氣體)、等張劑、吸收延遲劑、鹽、藥物、藥物安定劑、凝膠、接著劑、賦形劑、崩解劑、潤滑劑、甜味劑、矯味劑、染劑、流體及營養物質補充劑、類似材料及其組合,如具有本領域通常技藝者所已知者。疫苗組成物中各種組分之pH及確切濃度係根據已知參數予以調整。
囊封物質指代將多核苷酸或載體封裝於其內的遞送媒介物,諸如複製子顆粒(例如,美國專利公開第2019/0185822號中所揭示之α病毒複製子顆粒,該檔案之內容藉由引用併入本文)及脂質遞送系統(例如,脂質體)。
於一些態樣中,本揭露之疫苗組成物或製劑包含脂質遞送系統,例如,脂質體、脂複合物(lioplexes)、脂質奈米顆粒或其任何組合。多核苷酸諸如本文所揭示之α病毒複製子可使用一種或多種脂質體、脂複合物(lipoplexes)
或脂質奈米顆粒配製。脂質體、脂複合物(lipoplexes)或脂質奈米顆粒可用於改進該等多核苷酸引導之蛋白質產生的效力,蓋因此等製劑可增加由該多核苷酸所致的細胞轉染;及/或增加所編碼之蛋白質的轉譯。脂質體、脂複合物(lipoplexes)或脂質奈米顆粒亦可用於增加多核苷酸之安定性。
脂質體為經人工製備之媒介物,其可主要由脂質雙層構成且可用作遞送媒介物以進行醫藥製劑之投予。脂質體可係不同尺寸。多層媒介物(MLV)可具有數百奈米之直徑,且可含有藉由窄水性隔室分隔的一系列同心雙層。小的單層媒介物(SUV)可具有小於50nm之直徑,而大的單層媒介物(LUV)可具有介於50nm與500nm之間的直徑。脂質體設計可包括但不限於調理素或配體,以改進脂質體對不健康組織之接附或激活事件諸如但不限於胞吞作用。脂質體可含有低或高pH值,以便改進醫藥製劑之遞送。
脂質體之形成可取決於所入陷之醫藥製劑及脂質體成分;該脂質媒介物於其內分散的介質之屬性;所入陷之物質的有效濃度及其潛在毒性、於媒介物之應用及/或遞送期間所涉及的任何額外過程;用於預期應用的最優尺寸、多分散度及保質期;以及安全且高效之脂質體產物的批次間可再現性及放大生產等。
於一些態樣中,本文所揭示之多核苷酸諸如α病毒複製子可藉由脂質體予以囊封,且/或其可包含在水性核心內,而該水性核心可隨後藉由脂質體予以囊封。
於一些態樣中,本文所揭示之多核苷酸諸如α病毒複製子可經配製於陽離子性水包油乳液中,其中該乳液顆粒包含油核心及陽離子脂質,該陽離子脂質可與將該分子錨定至乳液顆粒的多核苷酸交互作用。於一些態樣中,本文
所揭示之多核苷酸可經配製於油包水乳液中,該乳液包含連續之疏水相,親水相分散於該疏水相中。
於一些態樣中,本文所揭示之多核苷酸諸如α病毒複製子可經配製於脂質-聚陽離子複合體中。作為非限制性實例,該聚陽離子可包括陽離子性肽或多肽,諸如但不限於,聚離胺酸、聚鳥胺酸及/或聚精胺酸及該等陽離子肽。
於一些態樣中,本文所揭示之多核苷酸諸如α病毒複製子可經配製於脂質奈米顆粒(LNP)中。
脂質奈米顆粒製劑典型包含一種或多種脂質。於一些態樣中,該脂質為陽離子或可離子化之脂質。於一些態樣中,脂質奈米顆粒製劑復包含其他組分,包括磷脂質、結構脂質、四級胺化合物及能夠降低顆粒集聚之分子,例如,經PEG或PEG修飾之脂質。於一些態樣中,脂質組成物中之陽離子及可離子化之脂質的量在約0.01mol%至約99mol%範圍內。
LNP含有pH敏感的可離子化之陽離子脂質,其吸引陰離子核酸以形成自組裝奈米顆粒之核心,從而確保高囊封。於生理pH,LNP係中性,消除了使用永久陽離子分子時所見的毒性之機制。
此等相同的pH敏感脂質導致對胞內體之酸性環境做出反應並觸發胞內體之破壞且將核酸釋放到細胞內。
這一基於複製子的疫苗技術為一種獨特的疫苗接種平台技術,蓋因RNA可自我擴增以產生疫苗抗原並將其遞送至細胞器(cellular organ)內。此外,這一基於複製子的疫苗技術克服了與基於DNA之疫苗相關的共同挑戰,諸如基因體整合之風險或投予所需之高劑量及裝置例如電穿孔,且預期基於自我複製系統的免疫劑量獲得高於mRNA技術的免疫原性。又,我們設計基於受體
結合域(RBD)的抗原,其具有訊號序列、穿膜域及選自CD4+ T細胞表位及CD8+ T細胞表位之至少一種肽。較佳態樣包括用於CD4+ T細胞之萬用表位諸如PADRE,以增強免疫原性。
於一個態樣中,疫苗組成物可係單價,其僅含有一種類型的α病毒複製子。於另一態樣中,疫苗組成物為二價,其包含兩種類型的α病毒複製子,各自編碼衍生自不同株或變異株的抗原蛋白。
根據本揭露,新穎抗原活性蛋白/多肽亦有用於產生用於診斷的抗體及針對冠狀病毒進行保護,同時最小化ADE之可能性。本文所揭露之蛋白質/多肽包括編碼冠狀病毒RBD的最小序列,該冠狀病毒RBD融合至訊號序列、穿膜域(TMD)以及選自CD4+ T細胞表位及CD8+ T細胞表位之至少一種肽,旨在最大化免疫原性且最小化ADE。
這一高效疫苗設計亦可應用於針對癌症或發炎性疾病的疫苗之開發。
本發明將參考下列實施例予以詳細揭示,惟,該等實施例並非意圖限制本申請之範疇。
[實施例1]
編碼下示構建體1的基因係由Integrated DNA Technologies,Inc.合成(https://www.idtdna.com/pages)。
構建體1
構建體1之全胺基酸如下:
訊號序列COVID19(1-13):
MFVFLVLLPLVSS(SEQ ID NO:3)
COVID-19-RBD(巴西(伽馬)變異株):
HA鏈接子:
GVKLESMGIY(SEQ ID NO:5)
HA TM:
QILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:6)
PADRE:
AKFVAAWTLKAAA(SEQ ID NO:1)
[實施例2]
編碼下文所述構建體2及3的基因由Integrated DNA Technologies,Inc.(https://www.idtdna.com/pages)合成。
全胺基酸:
訊號序列:
MFVFLVLLPLVSS(SEQ ID NO:3)
COVID-19-RBD(奧密克戎變異株):
HA鏈接子:
GVKLESMGIY(SEQ ID NO:5)
HA TM:
QILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:6)
PADRE:
AKFVAAWTLKAAA(SEQ ID NO:1)
全胺基酸:
鏈接子:
gaa
CD8表位:
[實施例3]
複製子載體之製備
α病毒複製子之構建體示意圖係顯示於圖1中。
於亞基因體(SG)啟動子之控制下,將實施例1中製備的目標DNA之基因經選殖至VEEV複製子載體內。編碼各片段之VEEV複製子質體係藉由
在AscI及SbfI限制位點進行插入而創建,以獲得全長VEEV TC-83複製子構建體。
SG啟動子、5'UTR、3'UTR及Poly A尾之核苷酸序列如下。RNA序列係藉由使用彼等DNA序列作為模板而獲得。
SG啟動子:cctgaatggactacgacatagtctagtccgccaag(SEQ ID NO:11)
5'UTR:ataggcggcgcatgagagaagcccagaccaattacctacccaaa(SEQ ID NO:12)
3' UTR:
Poly A尾:
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa(SEQ ID NO:14)
VEEV TC-83複製子nsP1-4胺基酸序列如下。
對應於nsp3之胺基酸序列係加下劃線。
[實施例4]
囊封於脂質奈米顆粒(LNP)中之自我擴增RNA(saRNA)的製備
使用載體,該等載體包含編碼於實施例1及2中製備之構建體1至3的DNA序列。該DNA經線性化且用作模板。T7活體外轉錄係基於由T7轉錄套組(RiboMaxTM Express Large Scale RNAproduction system,Promega,(WI USA))所提供之方案來執行:將線性DNA模板與T7酶及rNTP混合以合成RNA。對於含有經修飾之核苷酸的RNA之合成,將經修飾之NTP諸如5-甲基-胞苷及三磷酸Nl-甲基-假尿苷添加至活體外轉錄混合物中。經純化之RNA產物係使用疫苗封端酶進行封端,以給出自我擴增RNA。
以如上之相同模式,合成saRNA,其包含編碼對照構建體「RBD(武漢)-TM」之目標基因,該構建體由COVIDI9訊號序列、武漢株之COVID-19-
RBD、HA鏈接子及HA TM組成;且亦合成saRNA,其包含編碼構建體「RBD(巴西)-TM」之目標基因,該構建體由COVID19訊號序列、巴西(伽馬)變異株之COVID-19-RBD、HA鏈接子及HA TM組成。
「RBD(武漢)-TM」之胺基酸序列如下:
上文中,武漢株之COVID-19-RBD的胺基酸序列如下:
所獲得之saRNA經封裝於脂質奈米顆粒中以給出saRNA顆粒。所獲得之saRNA顆粒用於以下實施例中。
[實施例5]
免疫作用
方法:於第0週及第4週,用10ug的saRNA LNP(其表現SARS-CoV-2棘RBD(武漢株)與HA穿膜(RBD(武漢)-TM)或RBD巴西(伽馬)與HA穿膜(RBD(巴西)-TM))、實施例1之構建體1(亦即,RBD(巴西(伽馬))與HA穿膜加PADRE(RBD(巴西)-TM-PADRE)、PBS(安慰劑)經肌肉內對黃金敘利亞倉鼠(n為12隻
每組)進行兩次免疫。在第2次免疫後四週(第55天),藉由針對武漢株之SARS-Cov-2RBD蛋白的ELISA來評估來自經免疫之倉鼠的血清之抗體效價。巴西變異株、印度-德爾塔變異株、印度-德爾塔加變異株及哥倫比亞-謬(μ)變異株。結果係顯示於圖2中。
結果:相較於用RBD(巴西(伽馬))-TM免疫之血清中的抗體,用RBD(巴西(伽馬))-TM-PADRE免疫之血清中的抗體針對每種RBD變異株誘導了高出超過3倍的抗體(IC50效價)。
[實施例6]
ACE2抑制作用檢定
方法:使用可商購之套組(Genscript L00847)檢視獲自用實施例4中製備的疫苗免疫之倉鼠的血清對於RBD變異株與ACE2間之結合的抑制效應。血清係與共軛至辣根過氧化物酶之RBD變異株於37℃一起孵育30分鐘。將混合物添加至ACE2塗覆之96孔盤,並於37℃孵育15分鐘。洗滌該平盤後,添加TMB受質並於室溫孵育15分鐘。在微量平盤讀出器中量測於450nm之吸光度。根據下式,自該吸光度計算抑制。
抑制(%)=(1-樣品之吸光度/陰性對照之平均吸光度)x 100
結果係顯示於圖3中。
結果:全部疫苗皆具有針對SARS-CoV-2奧密克戎BA.2變異株以及武漢株的抑制效應,且其中巴西伽馬PADRE疫苗具有最高之抑制效應。
[實施例7]
經修飾之核苷於先天性免疫中之效應
方法:使用THP-I雙重細胞(InvivoGen),其中所分泌之螢光素酶(Luca gene)
的特徵為,在干擾素刺激之基因(ISG)54的最小啟動子的控制下與IFN刺激之反應元件(ISRE)共軛。檢視實施例1之構建體1(樣品1)、及使用5-甲基-胞苷代替胞苷的實施例1之構建體1(樣品2)的IFN調節因子(IRF)誘導效應。收穫經傳代之THP-I雙重細胞,並使用培養基(RPMI-1640+10% FBS)製備5.6 x 105個細胞/mL的細胞懸浮液。將180μL之該細胞懸浮液種植在96孔培養盤之每個孔中。然後,向每孔中添加20μL的磷酸鹽緩衝鹽水(對照)或者Lipofectamine(LipofectamineTM RNAiMAX轉染試劑)與樣品1或樣品2的混合物。細胞係於37℃在5% CO2下孵育24小時。
將20μL之培養上清液以等分小樣添加至另一96孔白色盤中,向每個孔中添加50μL之發泡試劑(QUANTI-Luc,InvivoGen)溶液,並於添加後立即量測發光強度。
根據下式,自該發光強度計算每個樣品之倍數增加並用作IRF誘導之係數。
倍數增加=(樣品之發光強度)/(對照之發光強度)
結果係顯示於圖4中。
相較於樣品1(使用胞苷),樣品2(使用5-甲基-胞苷,經修飾之核苷)之IRF誘導較低,顯示先天性免疫刺激效應的明確之降低。
[實施例8]
動物研究
用10ug之實施例4中製備的saRNA對黃金敘利亞倉鼠(u=6隻每組)經肌肉內注射兩次。本實施例中使用的saRNA為RBD(武漢)-TM、RBD(巴西)-TM及
RBD(巴西)-TM-PADRE,於0及4週使用。於第56天,用活SARS CoV-2武漢株(WT)或巴西(伽馬)變異株接種倉鼠。
體重量測
方法:
從研究第56天至研究第64天終止,在接種後每天記錄體重,以及體重改變之百分比的均值。
結果:
結果係顯示於圖5中。用RBD(伽馬)-TM-PADRE免疫化之組阻止倉鼠之體重減輕,其水準與WT接種之倉鼠的經RBD(伽馬)-TM或RBD(WT)-TM免疫化之組類似(左圖)。另一方面,於經伽馬接種之動物中未觀察到RBD(伽馬)-TM-PADRE組中的體重降低,而其他兩組皆表現出輕度降低(右圖)。
SARS-CoV-2之定量RT-PCR檢定
方法:
於選擇的接種後時間點,自經麻醉之動物收集口腔拭片,並測試每mL中的SARS-CoV2 RNA複本之量。本檢定之偵檢下限為50 RNA複本/mL。
結果:
結果係顯示於圖6中。相較於在接受WT(左圖)或伽馬(右圖)之後經RBD(伽馬)-TM或RBD(WT)-TM免疫化之組,在經RBD(伽馬)-TM-PADRE免疫化之組中觀察到口腔拭片病毒RNA載量的較大程度之降低。
組織學
方法:
在屍檢時,收集左肺並置於10%中性緩衝福爾馬林中以進行組織病理學分
析。組織切片經裁切並處理為蘇木精及伊紅(H&E)染色之載片。
結果:
結果係顯示於圖7中。用RBD(伽馬)-TM-PADRE免疫化之組阻止在接種WT(左起第三列,上)或伽馬(左起第三列,下)後的左肺中之病理學檢查所見者的發展。
TW202338090A_111148556_SEQL.xml
Claims (26)
- 一種單離之多核苷酸,其編碼α病毒非結構性蛋白nsp1、nsp2、nsp3及nsp4以及包含抗原蛋白的多肽,該抗原蛋白融合至訊號序列、穿膜域及選自CD4+ T細胞表位及CD8+ T細胞表位之至少一種肽。
- 如請求項1所述之多核苷酸,其中該抗原蛋白係融合至CD4+ T細胞表位,其中該CD4+ T細胞表位為Pan-DR表位(PADRE)。
- 如請求項1所述之多核苷酸,其中該抗原蛋白為衍生自病毒、癌症或細胞激素之蛋白質。
- 如請求項1所述之多核苷酸,其中該抗原蛋白為衍生自選自由重度急性呼吸道症候群相關冠狀病毒(SARS)、重度急性呼吸道症候群冠狀病毒2(SARS-CoV-2)、Ebola病毒、HIV、B型肝炎病毒(HBV)、流感病毒、C型肝炎病毒(HCV)、人類乳突病毒(HPV)、巨細胞病毒(CMV)、屈公病毒、呼吸道融合病毒(RSV)、登革熱病毒、原黏液病毒科病毒及結核分枝桿菌所組成之群組之病毒或細菌的蛋白質。
- 如請求項4所述之多核苷酸,其中該抗原蛋白進一步融合至CD8+ T細胞表位,以及,其中該CD8+ T細胞表位為衍生自病毒的肽,該抗原蛋白係衍生自該病毒。
- 如請求項4或5所述之多核苷酸,其中該抗原蛋白係衍生自冠狀病毒。
- 如請求項6所述之多核苷酸,其中該抗原蛋白係衍生自冠狀病毒棘(S)蛋白。
- 如請求項7所述之多核苷酸,其中該抗原蛋白為冠狀病毒棘(S) 蛋白中之S1及/或S2次單元。
- 如請求項8所述之多核苷酸,其中該抗原蛋白為冠狀病毒棘(s)蛋白中之S1次單元。
- 如請求項9所述之多核苷酸,其中該抗原蛋白為冠狀病毒S1次單元之接受體結合域(RBD)。
- 如請求項1所述之多核苷酸,其中該穿膜域係衍生自流感病毒血球凝集素(HA)、CD80或經修飾的衍生自該抗原蛋白之穿膜域。
- 如請求項11所述之多核苷酸,其中該穿膜域係衍生自流感病毒血球凝集素(HA)。
- 如請求項11所述之多核苷酸,其中該經修飾的穿膜域包含COVID-19棘(S)蛋白之近膜域及穿膜域。
- 如請求項1至13中任一項所述之多核苷酸,其中該穿膜域及/或訊號序列係透過鏈接子融合至該抗原蛋白。
- 如請求項6所述之多核苷酸,其中該冠狀病毒為COVID-19。
- 如請求項1所述之多核苷酸,其中該多核苷酸為RNA。
- 一種載體,其包含如請求項1所述之多核苷酸。
- 如請求項17所述之載體,其包含啟動子;5' UTR;編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4的多核苷酸;SG啟動子;編碼包含抗原蛋白之多肽的目標基因,該抗原蛋白融合至訊號序列、穿膜域及選自CD4+ T細胞表位及CD8+ T細胞表位之至少一種肽;3' UTR;及poly A尾。
- 一種疫苗組成物,其包含:如請求項1所述之多核苷酸或包含如請求項1之多核苷酸的載體,及醫藥上可接受之遞送媒介物。
- 如請求項18所述之疫苗組成物,其中該遞送媒介物為由一種或多種α病毒結構蛋白組成的顆粒或為脂質遞送系統。
- 一種針對受試者中之抗原進行治療、預防及/或免疫化的方法,其包含向有此需要之受試者投予有效量的如請求項19所述之疫苗。
- 一種單離之多核苷酸,該多核苷酸編碼α病毒非結構性蛋白nsp1、nsp2、nsp3及nsp4以及包含抗原蛋白的多肽,該抗原蛋白融合至訊號序列、穿膜域及選自CD4+ T細胞表位及CD8+ T細胞表位之至少一種肽,其中該多核苷酸包含經修飾之核苷。
- 如請求項22所述之多核苷酸,其中該經修飾之核苷為經修飾之胞苷及/或經修飾之尿苷。
- 如請求項23所述之多核苷酸,其中該經修飾之胞苷為5-甲基-胞苷,且該經修飾之尿苷為Nl-甲基-假尿苷。
- 如請求項22所述之多核苷酸,其中該多核苷酸中之實質上100%的胞苷為經修飾之胞苷。
- 如請求項22所述之多核苷酸,其中該多核苷酸中之少於100%的尿苷為經修飾之尿苷。
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US10906944B2 (en) * | 2020-06-29 | 2021-02-02 | The Scripps Research Institute | Stabilized coronavirus spike (S) protein immunogens and related vaccines |
CN111961138B (zh) * | 2020-10-20 | 2021-03-30 | 苏州茂行生物科技有限公司 | 疫苗融合蛋白 |
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