TW202408543A - 改良疫苗設計發展 - Google Patents
改良疫苗設計發展 Download PDFInfo
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Abstract
本文中提供單離的多核苷酸,其編碼包含與訊號序列及跨膜域以及任選地鐵蛋白融合之抗原蛋白的多肽。本文中亦提供單離的多核苷酸,其編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4以及多肽,該多肽係包含與訊號序列及跨膜域及任選地鐵蛋白融合之抗原蛋白。該抗原可為流行性感冒。如RNA的該多核苷酸可用作對抗流行性感冒感染的疫苗。
Description
本揭露總體係關於一種新穎疫苗設計領域及治療及/或免疫抗原的方法及組成物。具體而言,本揭露係關於一種流行性感冒疫苗。
流行性感冒(influenza;流感)係由流行性感冒病毒引起的具傳染性的呼吸疾病,其感染鼻子、喉嚨及肺部。某些人(如老年人、幼兒及帶有特定健康狀況的人)罹患嚴重流感併發症的風險較高。流行性感冒(流感)病毒主要有兩型:A型及B型。該經常於人群中傳播的A型及B型流行性感冒病毒(人類流行性感冒病毒)為每年季節性流感大流行的禍首。
預防流感第一個也是最重要的步驟為每年施打流感疫苗。流感疫苗已經顯示能減少流感相關疾病且減少導致住院甚至死亡的嚴重流感併發症的風險。
有不同類型的流感疫苗可供使用,其包含:四價流感疫苗,其防範四種不同流感病毒;高劑量流感疫苗,高劑量疫苗所含有的抗原(疫苗中幫助人體建立對流感防護力的部分)量是常規流感疫苗的4倍且特別許可給65歲以上的人群;細胞型流感疫苗,細胞型疫苗係生長於源自哺乳動物之培養細胞而非母雞的蛋中;鼻腔噴霧流感疫苗,其減毒流行性感冒
活疫苗(LAIV)係以鼻腔噴霧形式施予;藉由噴射注射器(jet injector)接種的流感疫苗,其核可給18至64歲的人群使用;佐劑疫苗,佐劑流感疫苗是在疫苗中添加一種成分製成之有助於產生較強免疫反應,並且特別許可給65歲以上的人群;重組流感疫苗,其重組流感疫苗係利用一種不需要雞蛋培養疫苗病毒之方法生產(https://www.cdc.gov/flu/about/index.html)。有關疫苗有效性(VE)的數據來自CDC 3月發病率及死亡率週報。VE係利用2021年10月至2022年2月期間在美國七個不同研究中心加入美國流行性感冒疫苗有效性網路之3,363名患有急性呼吸道感染(ARI)之小孩及成人的數據計算得出。觀察到的VE僅16%的2021-22季節性流感疫苗可保護美國人口免受當前流行的最常見流行性感冒病毒A(H3N2)感染。更具體而言,在接受醫療照護的門診患者中,對該流行性感冒A(H3N2)型病毒引起的輕微至中等ARI的VE為16%,效果並不顯著。進一步,VE對接受治療的流行性感冒A型病毒引起的ARI門診病患的VE為效果更不顯著的14%。該流行的最常見之流行性感冒株的低疫苗有效性尤其讓人擔憂(https://www.clinicaltrialsarena.com/comment/us-flu-vaccine-efficacy/)。
預計將有更多長期高效性之流行性感冒疫苗。
本揭露係關於一種能誘發針對抗原的保護之新穎抗原活化(antigenically-active)蛋白/多肽。本文中所揭露的該蛋白/多肽包含與訊號序列及跨膜域以及任選地鐵蛋白(ferritin)融合的抗原蛋白。
在另一態樣中,本揭露係關於一種新穎的多核苷酸,其係編碼上述能誘發針對抗原的保護之新穎的抗原活化蛋白/多肽。
在另一態樣中,本揭露係關於一種可表現上述抗原活化蛋白/多肽的新穎α病毒(alphavirus)複製子(replicon)(自我放大RNA;saRNA)。該α病毒複製子包含編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4之如RNA的多核苷酸及編碼上述抗原活化蛋白/多肽作為目標基因(gene of interest)的多核苷酸。
在又另一態樣中,本揭露係關於一種包含上述多肽或多核苷酸的疫苗。具體而言,本揭露提供一種包含編碼多肽的多核苷酸之疫苗,該多肽包含與訊號序列及跨膜域以及任選地鐵蛋白融合的抗原蛋白。該疫苗較佳是包含saRNA,該saRNA係包含編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4、以及多肽的多核苷酸,該多肽包含與訊號序列及跨膜域以及任選地鐵蛋白融合的抗原蛋白。在一較佳態樣中,該抗原係流行性感冒抗原。該疫苗可用於預防及/或治療個體之流行性感冒感染。
在又另一態樣中,本揭露係關於一種針對抗原的免疫反應之誘發及/或加強方法。在一較佳實施態樣中,該個體之流行性感冒免疫、預防或治療的方法係包含對需要的個體投予有效量的上述多肽或多核苷酸,例如該saRNA。
在還另一態樣中,本揭露係關於一種上述多肽或多核苷酸於製備藥物的用途。
在進一步的態樣中,本揭露係關於一種新穎多核苷酸,其係編碼包含與訊號序列及跨膜域以及任選地鐵蛋白融合的抗原蛋白之多肽。
該多核苷酸可為saRNA,該saRNA係包含編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4、以及多肽的多核苷酸,該多肽係包含與訊號序列及跨膜域以及任選地鐵蛋白融合的抗原蛋白。
圖1為本發明揭露之saRNA的代表性構築體(construct)。
圖2為F1、F2、F4及F5構築體的示意圖。
圖3為轉染(transfect)該saRNA的細胞溶胞產物(lysate)之西方轉漬(Western blotting)結果。
圖4為轉染該saRNA的細胞所表現的HA抗原FACS分析。
如本文中所述,「流行性感冒(influenza)」意指正黏液病毒科(family Orthomyxoviridae;RNA病毒的一種)。流行性感冒病毒分類為A、B、C及D型。這些主要類型大體產生相似症狀,但抗原性完全無關,因此感染一型無法賦予對其他類型的免疫力。A型病毒造成大型流行性感冒流行,B型病毒造成較小的局部爆發。C型病毒僅引起人類中輕微的呼吸道疾病。目前尚不清楚流行性感冒D型病毒會感染人類,僅在豬及牛中觀察到。
流行性感冒A型病毒依亞型分類,流行性感冒B型及流行性感冒A型的亞型皆進一步分為病毒株(strain)。流行性感冒A型的亞型主
要依據血球凝集素(hemagglutinin;H)及神經胺糖酸苷酶(neuraminidase;N)之兩種表面抗原(外源(foreign)蛋白)來區分。流行性感冒A型亞型的實例包含H1N1、H5N1及H3N2。流行性感冒B型病毒進一步區分為B/Yamagata及B/Victoria兩種主要譜系。流行性感冒B型病毒株及流行性感冒A型亞型病毒株係通過基因序列的變異進一步鑑別。
本文中所述之「流行性感冒結構蛋白」可為自然產生的病毒結構蛋白或其修飾蛋白。該修飾蛋白可為自然產生病毒結構蛋白的片段。在一實施態樣中,該修飾蛋白具有至少70%、75%、80%、85%、90%、95%或98%的自然產生病毒結構蛋白或其片段之胺基酸序列同一性。在一實施態樣中,該修飾蛋白為基於自然產生的病毒套膜(envelope)蛋白或其片段至多10%胺基酸缺失、取代及/或添加的突變蛋白。
如本文中所述,「跨膜域(transmembrane domain;TM)」為衍生自自然或合成來源的蛋白。在來源為自然下,在某些態樣中該域係衍生自任何膜結合(membrane-bound)或跨膜蛋白。在一態樣中,該膜結合或跨膜蛋白為流行性感冒的異源(heterologous)蛋白。該膜結合或跨膜蛋白的實例可包含T細胞受體的α、β及ζ鏈、CD28、CD3ε、CD45、CD4、CD5、CDS、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154;如人類的TLR1-TLR10及小鼠的TLR1-TLR9及TLR11-TLR13等類鐸受體(toll-like receptor;TLR);如IL-1-28受體、RANTES受體(CCR1、CCR3、CCR5)、MIP-1受體、PF4受體、M-CSF受體及屬於GPCR趨化介素受體之NAP-2受體等白血球介素(interleukin;IL)受體;
以及血球凝集素(HA)。在另一態樣中,該膜結合或跨膜蛋白係衍生自流行性感冒病毒的蛋白。
該跨膜蛋白的實例亦可包含下列:5-脂氧化酶(Lipoxygenase)活化蛋白、ABC轉運蛋白(Transporters)、ACBP、β類澱粉蛋白(Amyloid beta)(A4)、Bcl-2抑制蛋白(Inhibitors)、BNIPs、CAAX蛋白酶、細胞色素(Cytochromes)P450、E-NPPs、EPHA1、EPHA2、EPHA3、EPHA4、脂肪酸去飽和酶(Desaturases)、γ分泌酶(Gamma secretase)、葡萄糖轉運蛋白、血型醣蛋白(Glycophorins)、GPCR、HER2/ErbB2、HER3/ErbB3、HER4/ErbB4、HSD-11β、缺氧誘發蛋白、免疫球蛋白、胰島素受體、整合素(Integrins)、離子通道、MAPEG、MFS、MinK家族、MPPs、肽酶AD、肽酶家族M48、肽酶MA、蛋白Jagged、受體型激酶、SNARE複合體、硫酸酯酶(sulfatase)、TNF受體、跨膜蛋白14、轉運蛋白、TROBP、VEGF受體、醛去氫酶(Aldehyde Dehydrogenases)、氨及脲轉運蛋白、連接FMN氧化還原酶(Oxidoreductases)、含白胺酸重複序列(LRR)跨膜蛋白、白三烯(Leukotriene)C4合成酶、溶小體(Lysosome)相關膜醣蛋白、主要內生蛋白(MIP)/FNT超家族、微粒體***素E(Microsomal prostaglandin E)合成酶、類N去氧核糖基轉移酶((deoxy)ribosyltransferase)膜蛋白、中性/鹼性神經醯胺酶(Ceramidases)、寡糖轉移酶(Oligosaccharyl transferase)、五體配體門控(Pentameric Ligand-gated)離子通道、類視紫質(Rhodopsin)受體及幫浦、單股螺旋ATP酶調節蛋白、鯊烯/八氫茄紅素(Squalene/phytoene)合成酶、硬脂醯基-CoA去飽和酶1、斯特寧(Stannin;SNN)膜蛋白、T細胞表面醣蛋白CD3 ζ鏈、三角形四肽
重複(TPR)α螺旋重複蛋白(Tetratricopeptide repeat(TPR)Alpha-Helical Repeat Proteins)以及有NAD(P)結合羅士曼摺疊(Rossmann-fold)域的跨膜蛋白。
此外,附於脂雙層或其他膜主體蛋白(integral proteins)及胜肽的膜單側/膜周邊蛋白(monotopic/peripheral proteins)亦可作為跨膜蛋白使用。實例可包含:α/β水解酶(Alpha/Beta-Hydrolase)、膜連蛋白(Annexins)、類Bet V1蛋白、含C1域蛋白、含C2域蛋白、依靠CoA醯基轉移酶(CoA-Dependent Acyltransferases)、含CRAL-TRIO域蛋白、類DNA水解酶I蛋白、纖維蛋白原(Fibrinogen)、FYVE/PHD鋅指蛋白、類半乳糖結合域蛋白、醣脂轉移蛋白(Glycolipid Transfer Protein)、類免疫球蛋白超家族(E組)蛋白、脂質載運蛋白(Lipocalin)、脂氧化酶(Lipoxygenase)、PGBD超家族、類PH域蛋白、磷脂酸肌醇3-/4-激酶(Phosphatidylinositol 3-/4-Kinase)、類PLC磷酸二酯酶(Phosphodiesterase)、磷酸酪胺酸蛋白磷酸酶II(Phosphotyrosine Protein Phosphatases II)、含P環核苷三磷酸水解酶(P-Loop Containing Nucleoside Triphosphate Hydrolase)、蛋白激酶超家族、含PX域蛋白、蛋白皂角素(Saposin)、突觸核蛋白(Synuclein)以及轉錄因子tubby。
如本文中所述,「鐵蛋白(ferritin)」指衍生自哺乳類的鐵蛋白、衍生自兩棲類的鐵蛋白、衍生自細菌的鐵蛋白或衍生自植物的鐵蛋白之任一種或至少兩種的組合。較佳為衍生自哺乳類的鐵蛋白或衍生自細菌的鐵蛋白。較佳的衍生自哺乳類的鐵蛋白包含衍生自人類的鐵蛋白、衍生自鼠類的鐵蛋白或馬脾臟的鐵蛋白的任一種或至少兩種之組合。較佳的衍生自
兩棲類鐵蛋白包含牛蛙。較佳的衍生自細菌的鐵蛋白包含幽門螺旋桿菌(Helicobacter pylori)鐵蛋白或大腸桿菌鐵蛋白。該鐵蛋白較佳的來源包含自然萃取產物、人工合成產物或基因工程科技產物的任一種或至少兩種之組合。在一實施態樣中,可使用幽門螺旋桿菌(H.pylori)-牛蛙雜交鐵蛋白,例如,通過將牛蛙(Rana catesbeiana)鐵蛋白下亞基(lower subunit)(UniProt:P07797且具有N8Q突變以消除潛在的N醣化位點)之2-9殘基與具有I7E突變的幽門螺旋桿菌非血基質鐵蛋白(UniProt:Q9ZLI1,3-167殘基)融合而構成之幽門螺旋桿菌-牛蛙雜交鐵蛋白,以保存在人與牛蛙鐵蛋白中發現的的保守鹽橋(在人類輕鏈(light chain)及牛蛙下亞基鐵蛋白皆有6R及14E)與牛蛙鐵蛋白的6R(Kanekiyo et al.,Cell.2015 Aug 27;162(5):1090-1100)。
如本文中所述,「核苷(nucleoside)」指由鳥嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)、尿核苷(U)、胞核苷(C)或其修飾核苷所構成的分子。
修飾核苷包含但不限於:假尿核苷(pseudouridine)、N1-甲基-假尿核苷、5-甲基-尿核苷、假胞核苷、N1-甲基-假胞核苷及5-甲基-胞核苷。
假尿核苷或假胞核苷為尿核苷或胞核苷的異構物,其中該尿核苷或該胞核苷藉由碳-碳鍵而非氮-碳醣苷鍵連接。
在一實施態樣中,該修飾核苷獨立地選自N1-甲基-假尿核苷或5-甲基-胞核苷。在一實施態樣中,該mRNA或該saRNA實質地包含100%修飾胞核苷(例如,5-甲基-胞核苷)及100%修飾尿核苷(例如,N1-甲
基-假尿核苷)、100%修飾胞核苷及80%修飾尿核苷以及50%修飾胞核苷及50%修飾尿核苷。在一實施態樣中,saRNA包含少於100%的修飾尿核苷。
本揭露所述術語「跨膜域(transmembrane domain)」包含至少該膜結合或跨膜蛋白的跨膜區域。此外,該跨膜域亦可包含該膜結合或跨膜蛋白的近膜域(juxtamembrane domain,JDM)及/或細胞質尾部(cytoplasmic tail)。
或者是,在某些實施態樣中該跨膜域為合成的。在某些態樣中,該合成跨膜域主要包含如白胺酸及纈胺酸的疏水性殘基。在某些態樣中,在合成跨膜域的各端皆會發現***酸、色胺酸及纈胺酸的三聯體(triplet)。
較佳的跨膜域可為由流行性感冒病毒血球凝集素(HA)、CD80及類鐸受體4(TLR4)所衍生的。具體的實例可包含由流行性感冒病毒血球凝集素的跨膜域及細胞質尾部所構成的蛋白「HA(TM/CT)」;由人類CD80的跨膜域及細胞質尾部所構成的蛋白;由跨膜域(TM)及類鐸/白血球介素-1受體域(TIR)所構成的蛋白;以及由近膜域(JDM)所構成的蛋白。
如本文中所述,依據上下文「訊號序列(signal sequence)」(有時稱為訊號肽、靶向訊號(targeting signal)、定位訊號(localization signal)、定位序列、轉運肽(transit peptide)、前導序列(leader sequence)或前導肽)為多核苷酸或多肽。訊號序列係由長約9至200個核苷酸或3至70個胺基酸形成,且任選地併入編碼區域或蛋白的5’端或N端。某些訊號序列在蛋白運輸至所欲位點後,由例如訊號肽酶將其從該蛋白裂解。
該訊號序列不受限且可選自各種序列。在某些實施態樣中,該訊號序列可為該α病毒複製子所表現的目標蛋白。
在某些實施態樣中,可使用流行性感冒HA、COVID-19或IL-2為該訊號序列,特別是流行性感冒HA。
該流行性感冒結構蛋白、該跨膜域及/或該訊號序列可為直接或間接融合。在一實施態樣中,一個或兩個連接子(linker)可***該等之間。
該流行性感冒結構蛋白、該跨膜域及/或該訊號序列亦可為截短並由短連接子取代的。在某些實施態樣中,該病毒結構蛋白、該跨膜域及/或該訊號序列包含一個以上的胜肽連接子。
該抗原蛋白、該訊號序列、該跨膜域及任選地該鐵蛋白可進一步與T細胞表位(epitope)融合。「T細胞表位」可為CD4+ T細胞靶向表位、CD8+ T細胞靶向表位或Pan-DR表位(PADRE)。如本文中所述,T細胞表位可衍生自欲治療的病毒。
在本揭露所述術語「PADRE」指Pan HLA DR結合表位,一種普遍地活化抗原特異CD4+ T細胞的胜肽。該PADRE的胺基酸序列為AKFVAAWTLKAAA。
該病毒結構蛋白、該訊號序列、該跨膜域及任選地該鐵蛋白以及至少一個如PADRE的普遍表位可直接或間接地融合。在一實施態樣中,一個或兩個連接子可***該等之間。
該病毒結構蛋白、該訊號序列、該跨膜域及任選地該鐵蛋白以及至少一個以上的T細胞靶向表位亦可為截短並由短連接子取代的。在
某些實施態樣中,該病毒結構蛋白、該跨膜域及/或該訊號序列包含一個以上的胜肽連接子。
一個短連接子的實例係由2至25個胺基酸(例如,2、3、4、5或6個胺基酸)所構成。通常其長度為2至15個胺基酸,如SG、GS、SGG、GGS、SGSG及TRGGS。在特定情境下,該連接子可僅由如甘胺酸(G)、絲胺酸(S)及半胱胺酸(C)等一種胺基酸所構成。
當該流行性感冒結構蛋白通過化學交聯子化學性地接合至該跨膜域及/或該訊號序列時,該交聯子的實例包含但不限於:SMPH、磺基(sulfo)-MBS、磺基-EMCS、磺基-GMBS、磺基-SIAB、磺基-SMPB、磺基-SMCC、SVSB及SIA。亦可使用市面上的化學交聯子。
IgG的衍生物質亦可作為連接子使用。IgG的衍生物質的實例包含含有(i)完整(鉸鏈(hinge)-CH2CH3)、(ii)一半(鉸鏈-CH3)及(iii)短(僅12個胺基酸鉸鏈)之IgG1至IgG4。較佳的實例為IgG4-CH3。
所述之「α病毒結構蛋白(alphavirus structural protein)」指具有至少與自然產生的病毒殼體(capsid)或套膜蛋白有約80%胺基酸序列同一性的多肽或其片段。在一實施方案中,該α病毒結構蛋白具有與東方馬腦炎病毒(Eastern Equine Encephalitis Virus;EEEV)、委內瑞拉馬腦炎病毒(Venezuelan Equine Encephalitis Virus;VEEV)、沼澤地病毒(Everglades Virus)、穆坎布病毒(Mucambo Virus)、皮春納病毒(Pixuna Virus)、西方馬腦炎病毒(Western Equine Encephalitis Virus;WEEV)、辛德比病毒(Sindbis Virus)、塞姆利基森林病毒(Semliki Forest Virus)、米德爾堡病毒(Middleburg Virus)、屈公病毒(Chikungunya Virus;CHIKV)、奧
-奈氏病毒(O'nyong-nyong Virus)、羅斯河病毒(Ross River Virus)、巴馬森林病毒(Barmah Forest Virus)、蓋塔病毒(Getah Virus)、鷺山病毒(Sagiyama Virus)、貝巴魯病毒(Bebaru Virus)、馬雅羅病毒(Mayaro Virus)、尤納病毒(Una Virus)、奧拉病毒(Aura Virus)、瓦塔羅阿病毒(Whataroa Virus)、巴班肯病毒(Babanki Virus)、孜拉加奇病毒(Kyzylagach Virus)、高地J病毒(Highlands J virus)、摩根堡病毒(Fort Morgan Virus)、恩杜穆病毒(Ndumu Virus)或博吉河病毒(Buggy Creek Virus)有至少85%、90%、95%或更高的胺基酸同一性。α病毒結構蛋白野生型胺基酸序列可由GenBank取得。
在特定實施態樣中,該α病毒為CHIKV,例如CHIKV 37997株或LR2006 OPY-1株。在其他實施態樣中,該α病毒為VEEV,例如VEEV TC-83株。
所述之「α病毒複製子(alphavirus replicon)」指可在生體內(in vivo)於目標細胞內引導其自身擴增的RNA分子。該複製子編碼催化RNA擴增的聚合酶(nsp1、nsp2、nsp3及nsp4)並含有由該被編碼的聚合酶所辨識及使用之複製所需的順式(cis)RNA序列。α病毒複製子通常含有下列元件:5’UTR、編碼α病毒非結構蛋白(nsp1、nsp2、nsp3及nsp4)的序列、3’UTR及多腺核苷酸(poly A)訊號。α病毒複製子亦含有一個以上的引導目標基因的表現之病毒性次基因體(sub-genomic)啟動子。先前技術參考文獻中教示此等序列可具有一個以上的突變。
本揭露所提供的該α病毒複製子可具有如圖1所示的構築體。
在本揭露中,「包含(comprises/comprsing)」、「含有(containing)」及「具有(having)」等可具有美國專利法賦予它們的含義且可指「包含(includes/including)」等;「實質上由...所構成(consisting essentially of/consists essentially)」同樣具有美國專利法所賦予的含義,並且該術語係開放式的,允許存在比所引用的更多的內容,只要所引用的基本或新穎的特徵不因所引用的更多的內容的存在而改變,但排除先前技術實施態樣。
所述之「片段(fragment)」指多肽或核酸分子的一部分。此部分較佳為含有參考核酸分子或多肽全長的至少10%、20%、30%、40%、50%、60%、70%、80%或90%。片段可含有10、20、30、40、50、60、70、80、90或100、200、300、400、500、600、700、800、900或1000個核苷酸或胺基酸。
所述之「參考(reference)」指標準或控制條件。
所述之「參考序列(reference sequence)」係用作序列比較基礎之明確序列。參考序列可為特定序列之次集合或整體;例如,全長cDNA或基因序列的一段,或該完整的cDNA或基因序列。對多肽而言,該參考多肽序列的長度通常為至少約16個胺基酸,較佳為至少約20個胺基酸,更佳為至少約25個胺基酸,及又更佳為約35個胺基酸、約50個胺基酸或約100個胺基酸。對核酸而言,該參考核酸序列的長度通常為至少約50個核苷酸,較佳為至少約60個核苷酸,更佳為至少約75個核苷酸,及又更佳為約100個核苷酸或約300個核苷酸,或其附近或其間之任何整數。
序列同一性通常利用序列分析軟體(例如,Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center,1710 University Avenue,Madison,Wis.53705、BLAST、BESTFIT、GAP或PILEUP/PRETTYBOX程式)測量。該軟體藉由指定各種取代、缺失及/或其他修飾分配同源度(degrees of homology)來匹配相同或相似序列。保守性取代通常包含下列群組內的取代:甘胺酸及丙胺酸;纈胺酸、異白胺酸及白胺酸;天冬胺酸、麩胺酸、天冬醯胺酸及麩醯胺酸;絲胺酸及蘇胺酸;離胺酸及精胺酸;以及***酸及酪胺酸。在確定同一性程度的例示性方法中,可利用BLAST程式,可能性分數(probability score)介於e<"3>及e<"100>之間表示密切相關序列。
所述之「有效量(effective amount)」指相對於未治療的病患,改善疾病症狀所需的製劑量。用於實踐本發明以預防或治療疾病的該活性化合物之有效量係依據投予的方式、年齡、體重及個體的整體健康狀況而有不同。最終,由主治醫師或獸醫決定合適的量及給藥方案。該量係為「有效」的量。
通過全身投予可獲得滿意的效果,例如:肌肉內投予、皮下投予或靜脈內投予1-4次,每次103-1010感染單位(Infectious Unit;IU)或0.01-500μg的量,較佳為每次105-1010IU或0.1-100μg,例如每一次107-109IU或1-50μg。該複製子可較佳地配製於適合以常規方式投予的疫苗組成物中。
所述之「個體(subject)」指哺乳動物包含但不限於,人類或非人類哺乳動物,例如:牛、馬、犬、羊或貓。
如本文中所述,術語「治療(treat/treating/treatment)」等意指降低或改善與其相關的病症及/或症狀。應理解雖未排除,治療病症或病況並不需要完全地消除與其相關的該病症、病況或症狀。
如本文中所述,術語「預防(prevent/preventing/prevention)」及「預防性治療(prophylactic treatment)」等意指降低不具有但有產生病症或病況的風險或易產生病症或病況之個體中產生病症或病況的機率。
除非特別說明或由上下文顯而易見,如本文中所述,術語「或」應理解為包括在內。
除非特別說明或由上下文顯而易見,如本文中所述,術語「一個」、「一種」及「該」應理解為單數或複數。
在本說明書及專利範圍中,術語「約」涵蓋相關數值±20%、±10%或±5%的值。
所屬技術領域應理解說明書及專利範圍中所述之多核苷酸序列將在代表DNA的序列中引用「T」,但當該序列代表RNA時,該「T」將被「U」取代。
本文中所提供的任何疫苗組成物或方法可與一個以上任何其他本文中所提供的疫苗組成物或方法併用。
術語「載體(vector)」指核酸序列可在生物體、細胞或細胞組.分(cellular components)間繁衍及/或轉移的手段。載體包含自主複製或可嵌入宿主細胞染色體之質體、病毒、噬菌體、原病毒(pro-viruses)、噬菌粒
(phagemids)、轉位子(transposons)及人工染色體等。載體亦可為不會自主複製之裸露的(naked)RNA多核苷酸、裸露的DNA多核苷酸、在同一股中由DNA及RNA二者組成的多核苷酸、多離胺酸接合的DNA或RNA、胜肽接合的DNA或RNA、脂質體(liposome)接合的DNA等。在許多但非全部常見的實施態樣中,本發明的載體為質體或桿粒(bacmids)。
通常,待表現的核酸分子係與啟動子及/或強化子「可操作地連接(operably linked)」,並且受到該啟動子及/或強化子的轉錄調控控制。
轉染方法及表現載具的選擇將取決於所選擇的宿主系統。例如,Ausubel等人(同上)描述了轉染方法;表現載具可選自由如Cloning Vectors:A Laboratory Manual(P.H.Pouwels等人,1985,Supp.1987)所提供者。本段中所引用的文獻係通過引用而併入本文中。
存在多種用於產生本發明之構築體的表現系統。用於生產該構築體的表現載體包含但不限於,衍生自染色體、游離基因組(episomal)及病毒的載體,例如,衍生自細菌質體、衍生自噬菌體、衍生自轉位子,衍生自酵母菌游離基因組、衍生自***子(insertion elements)、衍生自酵母菌染色體單元(chromosomal elements)、衍生自如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、如SV40的乳突多瘤空泡病毒(papova viruses)、痘瘡病毒(vaccinia viruses)、腺病毒、雞痘病毒(fowl pox viruses)、假性狂犬病病毒(pseudorabies viruses)及反轉錄病毒等病毒的載體及衍生自其組合的載體。。
本文所使用的構築體及/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、以及編碼包含抗原之多肽的目標
基因,該抗原係例如與訊號序列及跨膜域融合之病毒結構蛋白,如上所述。該構築體或載體的具體實例如圖1所示。
該載體可為例如噬菌體、質體、病毒或反轉錄病毒載體。該包含核苷酸的構築體及/或載體應可操作地連接至合適的啟動子,如非限制性實例之CMV啟動子、噬菌體λ PL啟動子、大腸桿菌lac、phoA及tac啟動子、SV40早期或晚期啟動子及反轉錄病毒LTRs啟動子。依據宿主細胞及/或所需的表現速率,其他合適的啟動子為所屬技術領域具有通常知識者所習知的。該表現構築體將進一步包含用於轉錄起始、終結以及在轉錄區中用於轉譯之核糖體結合位點的位點。由構築體所表現的轉錄物的編碼部分較佳為包含位於起始處的轉譯起始密碼子以及適當地位於待轉譯之多肽末端的終止密碼子。
載體較佳為包含至少一個選擇標記(selectable marker)。該標記包含用於真核細胞培養的二氫葉酸還原酶(dihydrofolate reductase)、G418或新黴素(neomycin)抗性,以及用於大腸桿菌及其他細菌培養的四環素、康黴素或安比西林抗性基因。較佳的載體為如桿狀病毒、痘病毒(poxvirus)(例如,痘瘡病毒、鳥類痘(avipox)病毒、金絲雀痘(canarypox)病毒、雞痘病毒、浣熊痘(raccoonpox)病毒、豬痘病毒等)、腺病毒(例如,犬腺病毒)、帶狀皰疹病毒(herpesvirus)及反轉錄病毒的病毒載體。可與本發明使用的其他載體包含使用於細菌的載體,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。較佳的真核載體為pFastBacl pWINEO、pSV2CAT、pOG44、pXTl及pSG、pSVK3、
pBPV、pMSG及pSVL。所屬技術領域具有通常知識者可輕易推及其他合適的載體。
重組構築體可被製備並可用於將包含本文中所述的病毒蛋白轉染、表達至原核細胞或真核細胞。因此,在一實施態樣中,本揭露提供一種宿主細胞,該宿主細胞在允許α病毒複製子顆粒形成的條件下,包含含有編碼α病毒結構蛋白(包含殼體、E3、E2、6K及El或其部分)之核酸的載體(或載體們),以及包含編碼α病毒nsp1、nsp2、nsp3及nsp4的核酸及至少一個病毒目標基因的載體。
在一實施態樣中,該載體為重組桿狀病毒。在另一實施態樣中,該重組桿狀病毒係轉染至昆蟲細胞。在較佳的實施態樣中,該細胞為昆蟲細胞。在另一實施態樣中,該昆蟲細胞為Sf9細胞。
一種用於多肽生產之特定的細菌表現系統係大腸桿菌pET表現系統(Novagen,Inc.,Madison,Wis)。依據此表現系統,編碼多肽的DNA以設定為允許表現的方向***至pET載體。由於編碼該多肽的基因在T7調控訊號的控制下,因此藉由誘發該宿主細胞T7 RNA聚合酶的表現來獲得該多肽的表現。通常由利用響應IPTG誘發而表現T7 RNA聚合酶的宿主株來獲得。一旦生產,重組多肽接著依據所屬技術領域具有通常知識者習知的標準方法,例如本文中所述者單離。
依據選擇的載體及宿主細胞,在該重組蛋白表現且該α病毒複製子生成的條件下,培養經該載體轉染的宿主細胞來產生該構築體,並且將含有α病毒複製子的構築體與α病毒結構蛋白顆粒一起包裝形成。在一實施態樣中,本揭露包含構築體的製造方法,其涉及共轉染一種載體,
其包含編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4的多核苷酸,以及至少一個目標基因,該目標基因編碼包含與訊號序列及/或跨膜域融合之病毒結構蛋白的多肽;以及至少一種載體,其各自編碼至少一個α病毒結構蛋白,進入合適的宿主細胞,並在允許構築體形成的條件下表現該α病毒結構蛋白。在另一實施態樣中,該真核細胞係選自酵母菌、昆蟲、兩棲類、鳥類或哺乳類細胞所構成的群組。所屬技術領域或所屬技術領域具有通常知識者周知如何選擇合適的生長環境。
培養產生本發明之α病毒複製子顆粒之細胞的方法包含但不限於,批次式(batch)、補料分批式(batch-fed)、連續式(continuous)及灌注式(perfusion)細胞培養技術。在一實施態樣中,以編碼α病毒複製子的載體及包含編碼殼體之多肽的載體,以及包含編碼如衍生自CHIKV或VEEV的套膜蛋白的多核苷酸之載體共轉染細胞,將該細胞培養在生物反應器或發酵槽中,在其中進行細胞繁殖及表現蛋白(例如,重組蛋白)以供純化及單離。通常,細胞培養在無菌且溫度及大氣條件控制下執行。生物反應器為用於培養細胞環境條件如溫度、大氣、震盪及/或pH可被監控的槽。在一實施態樣中,該生物反應器為不銹鋼槽。在另一實施態樣中,該生物反應器為預滅菌的塑膠袋(例如,Cellbag.RTM.,Wave Biotech,Bridgewater,N.J.,該引用文件的內容依引用併入本文中)。在其他實施態樣中,該預滅菌塑膠袋為約10L至1000L的袋子。
在另一實施態樣中,如α病毒複製子的RNA分子可由模板DNA序列依所屬技術領域常規流程生成。生體外轉錄(in vitro transcription,IVT)方法允許模板引導合成RNA分子。IVT方法允許合成
大量RNA轉錄本。一般而言,IVT使用包含目標序列上游的啟動子序列的DNA模板。該啟動子序列最常見的是源自噬菌體,例如T7、T3或SP6啟動子序列,但也允許許多其他啟動子序列,包含重新(de novo)設計者。該DNA模板的轉錄本通常最佳由利用對應特定噬菌體啟動子序列的RNA聚合酶來獲得。例示性的RNA聚合酶包含但不限於T7 RNA聚合酶、T3 RNA聚合酶或SP6 RNA聚合酶與其他。IVT通常起始於dsDNA但也可在單股上進行。用於體外轉錄的套組如T7轉錄套組(RiboMaxTM Express Large Scale RNA production System,Promega(WI USA))。
如本文中所述,術語「醫藥上可接受之載劑」意指一種以上相容的固態或液態填料、稀釋劑或封裝物質,其適合投予至人類或其他脊椎動物,包含所屬技術領域具有通常知識者習知的任何及所有的水性溶劑(例如,水、酒精/水性(alcoholic/aqueous)溶液、食鹽水溶液、如氯化鈉的注射用載具(parenteral vehicles)及Ringer氏葡萄糖)、非水性溶劑(例如,丙二醇、聚乙二醇、植物油及如油酸乙酯(ethyloleate)的可注射的有機酯)、分散介質、塗層、介面活性劑、抗氧化劑、防腐劑(例如,抗菌或抗黴劑、抗氧化劑、螯合劑及惰性氣體)、等張劑、吸收延遲劑、鹽類、藥物、藥物穩定劑、凝膠、黏合劑、賦形劑、崩散劑、潤滑劑、甜味劑、調味劑、色劑、液體及營養補充劑等其他相似材料及其組合。疫苗組成物中各種組份的pH及準確濃度依據習知參數調整。
封裝物質指包裝該多核苷酸或載體的輸送載具,如複製子顆粒(例如,美國專利公告號2019-0185822中所述之α病毒複製子顆粒,該文件內容依引用併入)及脂質輸送系統(例如,脂質體(liposome))。
在某些實施態樣中,本揭露的該疫苗組成物或配方包含脂質輸送系統,如脂質體、脂複合體(lipoplexe)、脂質奈米顆粒、或其任意組合。本文中所述的如α病毒複製子之多核苷酸可利用一種以上的脂質體、脂複合體或脂質奈米顆粒配製。可使用脂質體、脂複合體或脂質奈米顆粒可用於提升該多核苷酸導引的蛋白生產效力,因為這些配方可增加該多核苷酸之細胞轉染;及/或增加編碼蛋白的轉譯。亦可使用該脂質體、脂複合體或脂質奈米顆粒以增加該多核苷酸的穩定度。
脂質體為人工製備的囊泡(vesicle),其主要可由脂雙層構成且可作為投予醫藥配方的輸送載具使用。脂質體可為不同的尺寸。多層囊泡(multilamellar vesicle;MLV)直徑可為數百奈米,且可含有一系列由狹窄水性隔間區隔的同心圓雙層膜。小單細胞囊泡(small unicellular vesicle;SUV)直徑可小於50nm,而大單層囊泡(large unilamellar vesicle;LUV)直徑可介於50至500間。脂質體的設計包含但不限於,改善脂質體附著至不健康組織或活化如但不限於胞吞(endocytosis)事件的助噬素(opsonins)或配體。脂質體可含有高或低pH值以改良該醫藥配方的輸送。
該脂質體的形成可取決於包埋的醫藥配方及脂質體成分、分散脂質囊泡的基質性質、包埋物質的有效濃度及其潛在毒性、在應用及/或輸送該囊泡過程中涉及的任何其他流程、最佳尺寸、所欲應用的囊泡多分散性(polydispersity)及保存期限、以及批次間再現性及安全有效率之脂質體產物製程放大等等。
在某些實施態樣中,本文中所述的如α病毒複製子之多核苷酸係可由該脂質體封裝及/或其可被包含在水性核心中,然後由該脂質體封裝。
在某些實施態樣中,本文中所述的如α病毒複製子之多核苷酸係可配製在陽離子型水包油(oil-in-water)乳化劑,其乳化劑顆粒包含可與該多核苷酸交互作用錨定該分子至乳化劑顆粒的油核心及陽離子型脂質。在某些實施態樣中,該本文中所述之多核苷酸可配製為包含親水相分散於連續性疏水相的油包水(water-in-oil)乳化劑。
在某些實施態樣中,本文中所述的如α病毒複製子之多核苷酸可配製為脂質-聚陽離子(lipid-polycation)複合物。作為非限制性實例,該聚陽離子可包含陽離子型胜肽或多肽,例如但不限於聚離胺酸、聚鳥胺酸及/或聚精胺酸以及陽離子肽。
在某些實施態樣中,本文中所述的如α病毒複製子之多核苷酸可配製在脂質奈米顆粒中(LNP)。
脂質奈米顆粒配方通常包含一種以上的脂質。在某些實施態樣中,該脂質為陽離子型或可離子化的脂質。在某些實施態樣中,脂質奈米顆粒配方進一步包含其他組分,包含磷脂、結構性脂質、四級銨化合物及能夠降低顆粒聚集的分子,例如PEG或PEG修飾脂質。在某些實施態樣中,該陽離子型且可離子化的脂質在該脂質化合物中的量之範圍為從約0.01mol%至約99mol%。
LNP包含吸引陰離子性核酸之pH敏感的可離子化陽離子型脂質以形成的自我組裝奈米顆粒核心以確保高封裝度。在生理性pH下,LNPs係中性,消除了永久陽離子型分子觀察到的毒理機制。
同樣的pH敏感脂質負責對胞吞體(endosome)的酸性環境產生反應並觸發該胞吞體的擾動並釋放該核酸至該細胞中。
此基於複製子的疫苗技術係疫苗的獨特平台技術,因為RNA可自我放大以產生疫苗抗原並輸送至細胞器官。進一步,此基於複製子的疫苗技術克服通常與基於DNA的疫苗相關的挑戰,如基因體嵌入(genome integration)或投予所需的高劑量及如電穿孔的設備;且基於該自我複製系統相比於mRNA技術,預期最小劑量有較高免疫原性。
依據本發明,新穎的抗原活化蛋白/多肽亦可用於製造診斷用及保護抗原之抗體,並且最小化ADE的可能性。本文中所揭露之蛋白/多肽包含編碼與訊號序列及/或跨膜域(TMD)序列融合的RBD之最短序列,旨在最大化免疫原性並最小化ADE。
參考以下實施例以詳細地描述本發明,惟其非旨在限制本發明之範疇。
實施例1
編碼下列所示之構築體1-12的各基因均由Integrated DNA Technologies,Inc.(https://www.idtdna.com/pages)合成。
1.構築體1
2.構築體2
3.構築體3
4.構築體4
5.構築體5
6.構築體6
7.構築體7
構築體8至14
構築體8至14分別對應於構築體1至7並進一步於C端融合PADRE(T表位)。(SEQ ID NO:8-14)
流行性感冒A型病毒HA1:
流行性感冒A型病毒HA2:
COVID-19訊號序列:MFVFLVLLPLVSS(SEQ ID NO:17)
流行性感冒A型病毒HA1訊號序列:
MKAILVVLLYTFATANA(SEQ ID NO:18)
流行性感冒A型病毒HA1頭部:
sg:連接子
衍生自流行性感冒A型病毒的TM/CT(A/波多黎各/8/1934(H1N1)):
GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:20)
或
衍生自流行性感冒A型病毒的TM/CT(A/加利福尼亞/07/2009(H1N1)):
GVKLESTRIYQILAIYSTVASSLVIVVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:21)
鐵蛋白(幽門螺旋桿菌-牛蛙雜交鐵蛋白):
PADRE:AKFVAAWTLKAAA(SEQ ID NO:23)
實施例2
[製備複製子載體]
α病毒複製子之構築體示意圖顯示於圖1。圖1的啟動子係使用T7啟動子。
以WO 2019/124441所揭露之流程製備VEEV全長複製子質體載體。實施例1中製備的構築體1至14各作為目標基因使用。將編碼該構築體的核苷酸選殖(clone)至在SG啟動子控制下的該VEEV複製子載體。藉由***AscI及SbfI限制位(restriction sites)來創造編碼各片段的VEEV複製子質體,以獲得全長VEEV TC-83複製子質體。
SG啟動子、5’UTR、3’UTR及多腺核苷酸尾的核苷酸序列如下。RNA序列由使用DNA序列作為模板取得。
SG啟動子:cctgaatggactacgacatagtctagtccgccaag(SEQ ID NO:24)
5’UTR:
ataggcggcgcatgagagaagcccagaccaattacctacccaaa(SEQ ID NO:25)
3’UTR:
gcgatcgcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattttgtttttaatatttc(SEQ ID NO:26)
多腺核苷酸尾:
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa(SEQ ID NO:27)
VEEV TC-83複製子nsP1-4胺基酸序列如下。
加底線者為對應nsp3的胺基酸序列。
在此實施例中,對應SEQ ID NO:28中從1330-1886殘基的nsp3的胺基酸序列由以下所示序列取代。該加底線的序列不同於SEQ ID NO:28。
實施例3
[製備自我放大RNA(saRNA)]
藉由使用Nrul或BspQ1限制酶(restriction enzymes)在37℃或50℃切割3小時,而將在T7啟動子下游具有流行性感冒HA變體(variant)序列的質體線性化。該線性化質體接著利用Wizard Plus SV Miniprep DNA Purification System(Promega)純化,且saRNA利用T7 RiboMAX Express Large-Scale RNA Production System(Promega)進行體外轉錄。在DNA水解酶處理後,該saRNA以RNeasy midi套組(Qiagen)純化,且接著利用NEB(New England Biolabs)端帽(cap)程序(NEB,M20280)以Vaccinia Capping System(NEB)加入7-甲基鳥核苷(7-methylguanosine)端帽來修飾。該端帽化的saRNA利用Monarch套組(NEB)純化。
[西方轉漬法]
HEK293T細胞利用lipofectamine(Promega)以0.5的saRNA轉染。收集轉染後18小時的細胞,在細胞裂解緩衝液(Cell Signaling Technology)中裂解並以SDS-PAGE分部(fractionated)(Any kD acrylamide gel,Bio-Rad)。利用抗IAV H1N1(A/加利福尼亞/07/2009)血球凝集素、兔多株抗體(1:5000稀釋;Sino Biological)及辣根過氧化物酶接合小鼠抗兔IgG(1:2000;Santa Cruz Biotechnology)並以西方轉漬法偵測蛋白質。該蛋白質條帶係利用ChemiDocTM XRS+(Bio-Rad)及Image LabTM軟體(Bio-Rad)加強化學發光而可視化。
具有構築體6、13、3及10作為目標基因的質體載體的結果示於圖3。如圖2所示,該等構築體分別對應於F01、F02、F04及F05。該西方轉漬試驗結果表明該以saRNA-流感變體轉染的細胞表現具有預期分子量的流行性感冒抗原。
[流式細胞儀分析]
為了分析細胞表面蛋白,收集該轉染的HEK293T細胞並以PBS洗滌,及以抗IAV H1N1(A/加利福尼亞/07/2009)血球凝集素、兔多株抗體及驢抗兔IgG二級PE(1:200稀釋;BioLegend)染色。利用Attune聲學集中(acoustic focusing)細胞計數儀(cytometer)(applied biosystems)評估表面蛋白的水平。使用未轉染的HEK293T細胞作為對照組。結果顯示於圖4。在圖4中,R3部分含有抗原陽性的細胞,而R5部分含有在R3部分之中抗原高度陽性的細胞。
流式細胞儀分析表明流行性感冒抗原表現在經saRNA-流感變體轉染的細胞表面。在先前的研究中,R5部分細胞的比例與在動物模型試驗中對抗原的免疫原性相關。
實施例4
[製備α病毒複製子顆粒]
轉染該在實施例2製備的全長複製子質體10μg、VEEV Env表現質體1μg及VEEV殼體NLS突變株1μg(或VEEV殼體表現質體1μg)至HEK293T細胞。轉染後48至95小時收集上清液。利用離子交換管柱純化該複製子顆粒。以該純化顆粒的製品稀釋液感染HEK293T或Vero細胞以判定感染效價(titer)。使用該純化的複製子顆粒以產生診斷及免疫用抗原。
實施例5
[製備封裝於脂質奈米顆粒(LNP)中的mRNA或自我放大RNA(saRNA)]
使用該在實施例2製備包含編碼構築體1至14作為目標基因的DNA序列質體載體。線性化該質體載體並作為模板使用。基於T7轉錄套組(RiboMaxTM Express Large Scale RNA production System,Promega,(WI USA))提供的程序執行T7體外轉錄。該線性DNA模板與T7酵素及rNTP混合以合成RNA。對於合成含有修飾核苷酸的RNA,加入如5-甲基-胞核苷三磷酸及N1-甲基-假尿核苷三磷酸之修飾的NTP至該體外轉錄反應混合物。純化的RNA產物利用牛痘加帽酶(vaccinia capping enzyme)加帽以生成自我放大RNA。
在脂質奈米顆粒中封裝取得的mRNA或saRNA以生成mRNA或saRNA顆粒。
TW202408543A_112120150_SEQL.xml
Claims (20)
- 一種單離的多核苷酸,其係編碼包含與訊號序列及跨膜域以及任選地鐵蛋白融合之抗原蛋白的多肽。
- 如請求項1所述之多核苷酸,其中,該抗原蛋白為流行性感冒蛋白。
- 如請求項1所述之多核苷酸,其中,該流行性感冒蛋白為流行性感冒病毒血球凝集素1(HA1)及/或流行性感冒病毒血球凝集素2(HA2)。
- 如請求項1所述之多核苷酸,其中,該流行性感冒蛋白係HA1的頭部區域。
- 如請求項1至4中任一項所述之多核苷酸,其中,該鐵蛋白係衍生自幽門螺旋桿菌或幽門螺旋桿菌-牛蛙雜交鐵蛋白。
- 如請求項1至5中任一項所述之多核苷酸,其進一步編碼α病毒非結構蛋白nsp1、nsp2、nsp3及nsp4。
- 如請求項1至6中任一項所述之多核苷酸,其中,該跨膜域及/或訊號序列係藉由連接子而與該抗原蛋白融合。
- 如請求項1至7中任一項所述之多核苷酸,其中,該多核苷酸為RNA。
- 如請求項1至7中任一項所述之多核苷酸,其中,該多核苷酸為DNA。
- 一種載體,其係包含請求項1至10中任一項所述之多核苷酸。
- 如請求項10所述之載體,其係包含啟動子、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、編碼包含與訊號序列及跨膜域融合之抗原蛋白的多肽之目標基因、3’UTR以及多腺核苷酸尾。
- 一種疫苗組成物,其係包含請求項1至11中任一項所述之多核苷酸或載體及醫藥上可接受的載劑。
- 如請求項12所述之疫苗組成物,其中,該醫藥上可接受的載劑為輸送載具。
- 如請求項13所述之疫苗組成物,其中,該輸送載具係由一個以上α病毒結構蛋白或脂質輸送系統所構成之顆粒。
- 一種請求項1至11中任一項所述之多核苷酸或載體之用途,其係用以製備藥物。
- 如請求項15所述之用途,其中,該藥物係用以誘發個體中的免疫調節。
- 如請求項15所述之用途,其中,該藥物係用以治療或預防個體中由感染引起的病症。
- 一種個體中的免疫調節之誘發方法,其係包含對需要的個體投予免疫上有效量的請求項12至14中任一項所述之疫苗組成物。
- 一種個體體內抗原之治療、預防及/或免疫方法,其係包含對需要的個體投予有效量的請求項12至14中任一項所述之疫苗組成物。
- 一種多肽,其係包含與訊號序列及跨膜域、以及任選地鐵蛋白融合之抗原蛋白。
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