CN104918635A - 影响病毒粒体解组装的黄病毒包膜蛋白突变 - Google Patents
影响病毒粒体解组装的黄病毒包膜蛋白突变 Download PDFInfo
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Abstract
制成了黄病毒包膜蛋白中心单体接触界面中的突变,其调节所述黄病毒的感染性。所述突变降低包膜二聚体蛋白解离的能力。
Description
对相关申请的交叉引用
本申请基于并要求2012年11月7日提交的美国非临时申请流水号13/671,111的优先权,其内容通过提述并入本文。
发明背景
黄病毒属包含超过60种紧密相关的病毒,包括几种具有全球和地域性传染病学重要意义的人类病原体。病毒粒体由三种结构蛋白构成,命名为衣壳(capsid)(“C”),被膜(membrane)(“M”)以及包膜(envelop)(“E”)。在被感染的细胞中发现的未成熟的黄病毒粒体含有前被膜(“prM”)蛋白,其为M蛋白的前体。未成熟的病毒粒体含有prM-E异质二聚体,构成病毒粒体包膜。prM蛋白作为缓慢折叠的E的伴侣蛋白,阻止E在运输过程中pH介导的不可逆重排,并在病毒粒体释放前被切割。黄病毒感染的细胞释放非感染性的亚病毒颗粒,其仅包含包膜蛋白prM和E。这些可以通过表达黄病毒prM-E基因盒产生。其组装途径-细胞内运输、碳水化合物处理、成熟、prM切割以及分泌-与感染性病毒粒体类似。
E蛋白包含一段长的胞外域,紧跟着一段茎-锚区。对于蜱传脑炎和登革热病毒的E蛋白,其黄病毒E蛋白胞外域(约400个氨基酸,不包括羧基末端的茎和跨膜域)以及其二聚和三聚形式无论在融合前和融合后构象的三维结构已经解析。见Bressanelli等,Structure of a flavivirus envelopeglycoprotein in its low-pH-induced membrane fusion conformation,EMBO J 121-12(2004);Modis等,A ligand-binding pocket in the dengue virus envelopeglycoprotein,Proc Natl Acad Sci USA 100 6986-6991(2003)Epub May 20,2003;Modis等,Structure of the dengue virus envelope protein after membrane fusion,Nature 427 313-319(2004);Modis等,Variable surface epitopes in the crystalstructure of dengue virus type 3envelope glycoprotein,J Virol 79 1223-1231(2005);以及Rey等,The envelope glycoprotein from tick-borne encephalitisvirus at 2A resolution,Nature 375 291-298(1995),其通过提述并入本文。胞外域形成方向平行于病毒被膜的细长二聚体(见图1,通过同源建模得到的NY99E400模型的顶视图)。在头-尾二聚体中,每一个单体由结构域I、II和III构成。二聚体中的单体接触不是沿着分子的整个长度连续的。在二聚体轴中存在两个孔占据切割的prM的位置(见Rey等,The envelopeglycoprotein from tick-borne encephalitis virus at 2A resolution,Nature 375291-298(1995))。除了结构域II中的两个短的α螺旋外,整个分子中β链是主要的。
每一个位于中心的N端结构域I含有两个二硫键,并带有单个碳水化合物侧链,其屏蔽位于结构域II尖端的融合肽,并有助于所述二聚体的整体稳定性(见Rey等,The envelope glycoprotein from tick-borne encephalitis virus at2A resolution,Nature 375 291-298(1995))。结构域II,或二聚化结构域,具有细长的指状结构,并在两个不同的位点参与单体与单体的相互作用。远端环通过三个二硫键稳定,并形成容纳融合肽的尖端,其纳入由第二个单体的结构域I和III提供的疏水口袋中。这一接触很大程度上是非极性的,并由来自一个亚基上的结构域I和III的残基和另一个亚基上的结构域II的尖端组成。在中心处的接触,其中可以看到两个突出的α螺旋,主要只涉及结构域II的疏水侧链。结构域III含有C端,并在病毒粒体中连接到茎,随后是将单体锚定在被膜中的跨膜区。
尽管在不同的黄病毒中E蛋白氨基酸序列的差异,12个半胱氨酸残基在物种之间绝对保守。在西尼罗病毒(West Nile virus)E蛋白中,这些半胱氨酸残基形成六个二硫键(见Nowak等,Analysis of disulfides present in themembrane proteins of the West Nile flavivirus,Virology 156 127-137(1987)),并在所有目前已确定的E蛋白的X射线结构中,在预期的位置被发现。这有力的支持了对于所有黄病毒E蛋白,其整体结构构造和折叠是相似的这一目前理解。
暴露于酸性pH导致病毒粒体构造的大量重排,伴随着生物学活性例如感染性、膜结合和融合的失活。在病毒进入期间,诱导的改变是融合过程的关键组分。见Corver等,Membrane fusion activity of tick-borne encephalitisvirus and recombinant subviral particles in a liposomal model system,Virology269 37-46(2000);Heinz等,The machinery for flavivirus fusion with host cellmembranes,Curr Opin Microbiol 4 450-455(2001);和Stiasny等,Membraneinteractions of the tick-borne encephalitis virus fusion protein E at low pH,JVirol 76 3784-3790(2002)。E蛋白介导的pH诱导的融合机制涉及E从二聚体到三聚体形式的重排(见Stiasny等,Structural requirements forlow-pH-induced rearrangements in the envelope glycoprotein of tick-borneencephalitis virus,J Virol 70 8142-8147(1996))。融合(fusogenic)E三聚体的形成是一个两步的过程,其中二聚体首先在低pH的影响下解离,然后重新联合形成三聚体。所述两步模型首先在E-400胞外域的研究中获得。在茎-锚区不存在时,暴露于酸性pH导致二聚体的可逆解离,其不导致三聚化。进一步的研究揭示了茎-锚区(约氨基酸400-449)对于溶液中低pH诱导的二聚体向三聚体的不可逆转化的功能(见Allison等,Mapping of functionalelements in the stem-anchor region of tick-borne encephalitis virus envelopeprotein E,J Virol 73 5605-5612(1999))。低pH诱导的从二聚体到三聚体的不可逆改变提示在病毒粒体中,E作为亚稳定的二聚体存在,并当施加适当的触发时(在这种情况下是低pH)改变为更稳定的三聚体。已证明E的三聚体形式对热变性比二聚体形式更稳定。然而,与I类融合蛋白不同,这种向更稳定的构象的转变不能通过热处理诱导,其只能导致E的变性(见Stiasny等,Role of metastability and acidic pH in membrane fusion by tick-borneencephalitis virus,J Virol 75 7392-7398(2001))。这表明天然E二聚体的质子化对于能量上更稳定的最终三聚体形式的形成所需的单体中间结构的产生是不可缺少的(见Heinz等,Flavivirus structure and membrane fusion,AdvVirus Res 59 63-97(2003))。
对于许多黄病毒,神经毒性和神经侵袭表型与包膜蛋白有关。见Cecilia等,Nucleotide changes responsible for loss of neuroinvasiveness in Japaneseencephalitis virus neutralization-resistant mutants,Virology 181 70-71(1991);Chambers等,Yellow fever/Japanese encephalitis chimeric viruses:constructionand biological properties,J Virol 73 3095-3101(1999);Gualano等,Identification of a major determinant of mouse neurovirulence of dengue virustype 2using stably cloned genomic-length cDNA,J Gen Virol 79 437-446(1998);Hasegawa等,Mutations in the envelope protein of Japanese encephalitis virusaffect entry into cultured cells and virulence in mice,Virology 191 158-165(1992);Holzmann等,A single amino acid substitution in envelope protein E oftick-borne encephalitis virus leads to attenuation in the mouse model,J Virol 645156-5159(1990);Holzmann等,Characterization of monoclonalantibody-escape mutants of tick-borne encephalitis virus with reducedneuroinvasiveness in mice,J Gen Virol 78 31-37(1997);Jiang等,Single aminoacid codon changes detected in louping ill virus antibody-resistant mutants withreduced neurovirulence,J Gen Virol 74 931-935(1993);McMinn,The molecularbasis of virulence of the encephalitogenic flaviviruses,J Gen Virol 78 2711-2722(1997);Pletnev等,Construction and characterization of chimeric tick-borneencephalitis/dengue type 4viruses,Proc Natl Acad Sci USA 89 10532-10536(1992);以及Pletnev等,Chimeric tick-borne encephalitis and dengue type 4viruses:effects of mutations on neurovirulence in mice,J Virol 67 4956-4963(1993),其全部通过提述并入本文。然而,基因组其它部分中的突变也涉及神经毒性的缺失/获得。见Butrapet等,Attenuation markers of a candidatedengue type 2vaccine virus,strain 16681(PDK-53),are defined by mutations inthe 5'noncoding region and nonstructural proteins 1and 3,J Virol 74 3011-3019(2000);Duarte dos Santos等,Determinants in the Envelope E Protein and ViralRNA Helicase NS3That Influence the Induction of Apoptosis in Response toInfection with Dengue Type 1Virus,Virology 274 292-308(2000);Dunster等,Molecular and biological changes associated with HeLa cell attenuation ofwild-type yellow fever virus,Virology 261 309-318(1999);Muylaert等,Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1protein:effects on virus replication and mouse neurovirulence,Virology 222159-168(1996);Ni等,Molecular basis of attenuation of neurovirulence ofwild-type Japanese encephalitis virus strain SA14,J Gen Virol 76 409-413(1995);和Xie等,Yellow fever 17D vaccine virus isolated from healthyvaccinees accumulates very few mutations,Virus Res 55 93-99(1998),其全部通过提述并入本文。蛋白E中的突变导致的减毒最广泛的研究使用活减毒JE疫苗SA14-14-2,其中已经鉴定了E蛋白中区别减毒疫苗病毒和其毒性亲本SA14的9个氨基酸的差异。占主导地位的减毒效应与定位在结构域I和II界面处的被称为“铰链”区的E138K突变相关联(见Rey等,The envelopeglycoprotein from tick-borne encephalitis virus at 2A resolution,Nature 375291-298(1995))。铰链位点被认为在与病毒进入相关的E蛋白二聚体向三聚体的转化中扮演关键角色。在这个区域中的修饰调节黄病毒在小鼠中的毒性(见Cecilia等,Nucleotide changes responsible for loss of neuroinvasiveness inJapanese encephalitis virus neutralization-resistant mutants,Virology 181 70-71(1991);Gualano等,Identification of a major determinant of mouseneurovirulence of dengue virus type 2using stably cloned genomic-length cDNA,J Gen Virol 79 437-446(1998);Hasegawa等,Mutations in the envelope proteinof Japanese encephalitis virus affect entry into cultured cells and virulence inmice,Virology 191 158-165(1992);Hurrelbrink等,Attenuation of Murray Valleyencephalitis virus by site-directed mutagenesis of the hinge and putativereceptor-binding regions of the envelope protein,J Virol 75 7692-7702(2001);McMinn等,Murray valley encephalitis virus envelope protein antigenic variantswith altered hemagglutination properties and reduced neuroinvasiveness in mice,Virology 211 10-20(1995);和Sumiyoshi等,Characterization of a highlyattenuated Japanese encephalitis virus generated from molecularly cloned cDNA,J Infect Dis 171 1144-1151(1995),其全部通过提述并入本文)。对于毒性的减轻或回复重要的其它位点鉴定为在E中的位置176/177和264/279,其也存在于SA14-14-2中。前者定位于中心结构域,其在病毒进入期间在酸介导的E蛋白重排成融合活性三聚体期间经历改变(见Bressanelli等,Structure ofa flavivirus envelope glycoprotein in its low-pH-induced membrane fusionconformation,EMBO J 12 1-12(2004))。后一位置也定位于铰链区,并可能功能上与通过E138K突变确定的位点协作,由于涉及附近位置的突变损害E蛋白的血凝和融合特性,并降低在小鼠中的神经侵袭性(见Hurrelbrink等,Attenuation of Murray Valley encephalitis virus by site-directed mutagenesis ofthe hinge and putative receptor-binding regions of the envelope protein,J Virol75 7692-7702(2001)和McMinn等,Murray valley encephalitis virus envelopeprotein antigenic variants with altered hemagglutination properties and reducedneuroinvasiveness in mice,Virology 211 10-20(1995))。存在于SA14-14-2中最后的突变集群定位于E蛋白的结构域III和茎-锚区,其对于病毒附着细胞和与prM相互作用重要。位置315周围的突变导致改变的病毒趋向性和毒性的变化(见Jennings等,Analysis of a yellow fever virus isolated from a fatal caseof vaccine-associated human encephalitis,J Infect Dis 169 512-518(1994);Jiang等,Single amino acid codon changes detected in louping ill virusantibody-resistant mutants with reduced neurovirulence,J Gen Virol 74 931-935(1993);Ni等,Attenuation of Japanese encephalitis virus by selection of itsmouse brain membrane receptor preparation escape variants,Virology 24130-36(1998);和Ryman等,Mutation in a 17D-204vaccine substrain-specificenvelope protein epitope alters the pathogenesis of yellow fever virus in mice,Virology 244 59-65(1998))。茎-锚区的完整性对于prM-E异质二聚体的稳定性也是必须的(见Allison等,Mapping of functional elements in the stem-anchorregion of tick-borne encephalitis virus envelope protein E,J Virol 73 5605-5612(1999))。SA14-14-2中在远端单体接触界面发现的唯一氨基酸改变(见图1)涉及高度保守的融合环(氨基酸98-110)中的L107F取代。在绝大多数的黄病毒中,这一位置被Leu占据,除了在TBE病毒的毒性大大降低的美洲亲属波瓦森(Powassan)和鹿蜱黄病毒中出现Phe的仅有的两个已知例外。将这一突变回复为Leu与神经毒性表型的仅部分回复相关联(见Arroyo等,Molecular basis for attenuation of neurovirulence of a yellow feverVirus/Japanese encephalitis virus chimera vaccine(ChimeriVax-JE),J Virol 75934-942(2001)),表明在该位点仅承受轻微的减毒改变。在远端或中心接触界面其它已知突变的缺乏表明存在针对影响二聚体形成改变的强大选择压力。这与这一接触界面对于病毒感染周期结束时病毒粒体组装/成熟,以及在下一个繁殖周期的初始阶段病毒粒体的功能性解组装的重要性一致。
发明简述
本发明涉及黄病毒包膜蛋白中心单体接触界面中的突变,其调节所述黄病毒的传染性。所述突变显著的降低了病毒感染宿主细胞的能力,导致病毒传播的延迟或抑制。与以前描述的突变不同,这些突变典型的不影响病毒复制或组装以及从感染的细胞释放传染性病毒粒体。相对地,组装和释放的病毒粒体在向前-融合(pre-fusogenic)状态的转变中被抑制,该转变对于许多的(如果不是全部的)包膜病毒是在作为融合介导的病毒进入宿主细胞之前特征性的中间状态。产生的病毒保持了高免疫原性,然而由于其不能建立高效的病毒血症而很大程度上更安全。该方法打开了合理设计保留与亲本病原体相似的免疫原性的安全减毒疫苗的可能性。
因此,在一个方面,本发明涉及能够沿着高度保守的中心单体接触界面形成二聚体的黄病毒包膜单体蛋白中的一个或多个突变。所述黄病毒的中心单体接触界面对应于西尼罗病毒包膜蛋白的氨基酸256至260。所述包膜蛋白具有中心单体接触界面中一个或多个突变,其降低二聚体的解离。在另一个方面,所述黄病毒包膜单体蛋白中的一个或多个突变导致在中心单体接触界面的至少两个盐桥。在一个示例性的方面,对于蚊媒病毒,对应于西尼罗病毒包膜蛋白位置256处发现的甘氨酸的黄病毒氨基酸被碱性氨基酸取代,例如赖氨酸或精氨酸。在另一个示例性的方面,对于蜱媒病毒,对应于西尼罗病毒包膜蛋白位置260处发现的甘氨酸的黄病毒氨基酸被碱性氨基酸取代,例如赖氨酸或精氨酸。
另一个方面,本发明涉及黄病毒包膜单体蛋白,其具有包含选自下组序列的中心单体接触界面:RSQEG(SEQ ID NO:1)、RNQEG(SEQ ID NO:2)、GDQTR(SEQ ID NO:3)、KSQEG(SEQ ID NO:4)、KNQEG(SEQ ID NO:5)、和GDQTK(SEQ ID NO:6)。
在优选的方面,具有所述创造性突变的黄病毒选自西尼罗病毒、昆津病毒(Kunjin virus)、日本脑炎病毒(Japanese encephalitis virus)、墨累谷脑炎病毒(Murray Valley encephalitis virus)、登革热血清型1病毒(dengue serotype1virus),登革热血清型2病毒(dengue serotype 2virus),登革热血清型3病毒(dengue serotype 3virus),登革热血清型4病毒(dengue serotype 4virus),黄热病病毒(yellow fever virus)、蜱媒脑炎病毒(tick-borne encephalitisvirus)、波瓦森病毒(Powassan virus)以及鄂木斯克出血热病毒(Omskhemorrhagic fever virus)。
在另一个方面,本发明涉及编码具有创造性突变的黄病毒包膜单体蛋白的多核苷酸。本发明也涉及包含所述多核苷酸的载体,以及具有此类载体的宿主细胞。
再一个方面,本发明涉及活减毒黄病毒,其编码具有本文描述的创造性突变的黄病毒包膜二聚体蛋白。在优选的方面,所述黄病毒选自西尼罗病毒、昆津病毒、日本脑炎病毒、墨累谷脑炎病毒、登革热血清型1病毒,登革热血清型2病毒,登革热血清型3病毒,登革热血清型4病毒,黄热病病毒、蜱媒脑炎病毒、波瓦森病毒以及鄂木斯克出血热病毒。另一个方面,所述创造性突变在活嵌合黄病毒中。所述病毒(嵌合或非嵌合)可选的具有一个或多个包膜蛋白突变,例如对应于西尼罗病毒包膜蛋白选自下组氨基酸的氨基酸残基中的突变:氨基酸107、138、176、224、264、280、316和440。
再一个方面,本发明涉及包含编码具有本文描述的创造性突变的黄病毒包膜二聚体蛋白的减毒黄病毒的免疫原性组合物。此类组合物可以用于施用于患者,包括人。施用的示例性途径包括静脉内、肌肉内、腹膜内,或皮下。
在另一个方面,本发明涉及编码具有本文描述的创造性突变的包膜二聚体蛋白的黄病毒的重组遗传构建体。所述重组遗传构建体可以包含载体例如质粒。在一个方面,所述重组遗传构建体是编码真核启动子控制下的感染性(+)RNA分子的感染性DNA。此类重组遗传构建体可以编码对应的重组蛋白。在另一个方面,本发明涉及稳定或瞬时转染有所述重组遗传构建体的宿主细胞。
再一个方面,本发明涉及包含编码具有本文描述的创造性突变的黄病毒包膜二聚体蛋白的重组遗传构建体的免疫原性组合物。再一次,此类组合物可以用于施用于患者,包括人。施用的示例性途径包括静脉内、肌肉内、腹膜内,或皮下。
本发明的另外的方面,其附属的优点和新颖特征于此一起,将部分阐述于随后的说明书中,并且部分的将通过检查下文对于本领域的技术人员变得显而易见,或从本发明的实施中了解。本发明的目的和优点可以通过随附的权利要求中具体指出的手段和联合实现。
附图简述
图1是NY99E蛋白胞外域的三维模型。所述模型由同源建模制成。所述结构域的边界通过椭圆形和相邻的数字指示。
图2是NY99E蛋白胞外域的预融合二聚体形式。A组是所述二聚体的顶视图,其蛋白骨架显示为实心色带。B组和C组显示了A组中的中心接触界面的展开图。上面链的N→C方向为从左上前方到右上后方;下面链的N→C方向为从右下前方到左下后方。接触界面处的氨基酸以示意图示出(B组),或以空间填充表示(C组)。为了清楚,后者的序列轮廓不完全遵循多肽骨架。也就是说,氨基酸字母的位置并不遵循多肽骨架,其通过相邻氨基酸的-αC-N-αC-N-αC-原子相连来追踪,因为并不是骨架中的所有氨基酸在空间填充表示中都清晰可见。
图3是本发明使用的感染性DNA构建体的示意图。δ─肝炎δ核酶序列;BG-牛生长素转录终止和多聚腺甘酸化信号序列;CMV-巨细胞病毒启动子/增强子序列;bla-氨苄青霉素抗性基因;ori-pBR322复制起点;i2383和i358/393标记内含子的位置。单个元件没有按照比例绘制。
图4是带有Ser257Arg(A组)和Ser257Lys(B组)突变的NY99E蛋白胞外域的预融合二聚体形式的三维模型。同源建模使用SwissModel和坐标文件PDB#1OAN和PDB#1UZG进行。
图5是带有Gly256Arg(A组)和Gly256Lys(B组)突变的NY99E蛋白胞外域的预融合二聚体形式的三维模型。同源建模使用SwissModel和坐标文件PDB#1OAN和PDB#1UZG进行。
图6是野生型和突变病毒的感染特性的模型。玻璃盖玻片上的BHK细胞使用相应的感染性DNA转染,细胞在所示的时间固定并使用WN特异性抗血清随后抗小鼠IgG-FITC缀合物染色。
图7图表显示了在脑内接种1μg修饰的感染性DNA pCMVNY99后的动物存活。
发明详述
本发明涉及黄病毒包膜蛋白具有在二聚体形成期间提供中心单体接触界面的高度保守序列的发现。干扰二聚体的解离理论上干扰病毒的传播。因此,本发明涉及黄病毒包膜蛋白的中心单体接触界面处的氨基酸修饰,其调节二聚体的解离。所述氨基酸修饰可以包括在所述中心单体接触界面中取代、插入和/或删除多肽序列。
本发明涉及能够沿着中心单体接触界面形成二聚体的黄病毒包膜单体蛋白,所述中心单体接触界面在对应于西尼罗病毒包膜蛋白的氨基酸256至260的黄病毒包膜蛋白氨基酸处,使得所述包膜蛋白具有一个或多个降低所述二聚体解离的突变。在示例性的实施方案中,对于蚊媒病毒,对应于西尼罗病毒包膜蛋白的氨基酸256(例如对应WN的G256)的氨基酸使用碱性氨基酸取代,例如精氨酸或赖氨酸。在256位置的氨基酸突变仍然优选的在二聚体形成期间提供对称或近似对称的界面。在另一个示例性的方面,对于蜱媒病毒,对应于西尼罗病毒包膜蛋白的氨基酸260(例如对应WN的G260)的氨基酸使用碱性氨基酸取代,例如精氨酸或赖氨酸。再一次,在260位置的氨基酸突变仍然优选的在二聚体形成期间提供对称或近似对称的界面。
所述黄病毒可以具有包含选自下组序列的野生型中心单体接触界面:GSQEG(SEQ ID NO:7)、GNQEG(SEQ ID NO:8)、GDQTG(SEQ ID NO:9)、和GDQTA(SEQ ID NO:10)。因此,在本发明的一个方面中,所述中心单体接触界面包含Gly至Arg的取代,使得所述中心单体接触界面具有选自下组的序列:RSQEG(SEQ ID NO:1)、RNQEG(SEQ ID NO:2)、GDQTR(SEQ IDNO:3)、KSQEG(SEQ ID NO:4)、KNQEG(SEQ ID NO:5)、和GDQTK(SEQID NO:6)。
如表1一般所示,几乎所有的蚊媒黄病毒在中心单体接触界面处具有GSQEG(SEQ ID NO:7)或GNQEG(SEQ ID NO:8)序列。丝氨酸和苏氨酸都是含有羟基的氨基酸,天冬氨酸和谷氨酸都是带负电荷的氨基酸,而甘氨酸和丙氨酸都是疏水的,非极性氨基酸。在西尼罗和其它蚊媒黄病毒中,对应一个包膜蛋白单体中G256的氨基酸临近于模型中对应另一个单体中E259的氨基酸。作为结果,对应本文描述的G256位置的碱性氨基酸的氨基酸突变导致此类病毒两个盐桥的形成((+)与(-))。
如表1所示,几乎所有的蜱媒黄病毒在中心单体接触界面处具有GDQTG(SEQ ID NO:9)或GDQTA(SEQ ID NO:10)序列。在蜱媒病毒中,对应一个包膜蛋白单体D257的氨基酸临近于模型中对应另一个单体中G260(或A260)的氨基酸。作为结果,对应本文描述的G260位置的碱性氨基酸的氨基酸突变导致此类病毒两个盐桥的形成((+)与(-))。
确定所给黄病毒中哪个氨基酸对应另一个黄病毒的氨基酸可以通过标准氨基酸序列比对来实行,如本领域的技术人员所熟知的。对应于西尼罗病毒的黄病毒序列的例子示于下面的表1中。因此,应当理解本发明包含在中心单体接触界面的突变,其降低任意黄病毒物种或株系中二聚体的解离。
在一个方面,所述黄病毒可以是蜱媒病毒、蚊媒病毒,或无节肢动物媒介物的病毒。所述黄病毒可以是哺乳动物蜱媒病毒,例如Alkhurma病毒("ALKV")、鹿蜱病毒("DT")、加德格兹谷病毒(Gadgets Gully virus)("GGYV")、Kadam病毒("KADV")、Karshi病毒、科萨努森林病病毒(Kyasanur Forest disease virus)("KFDV")、兰加特病毒(Langat virus)("LGTV")、跳跃病(Louping ill)病毒("LIV")、鄂木斯克出血热病毒("OHFV")、波瓦森病毒("POWV"),皇家农场病毒(Royal Farm virus)("RFV"),蜱媒脑炎病毒("TBEV"),或土耳其绵羊脑炎病毒(Turkish sheepencephalitis virus)("TSE")。所述黄病毒可以是海鸟蜱传病毒,例如Meaban病毒("MEAV"),索马里兹暗礁病毒(Saumarez Reef virus)("SREV"),或Tyuleniy病毒("TYUV")。所述黄病毒可以是蚊媒病毒,例如Calbertado病毒或Duck tembusu病毒。所述黄病毒可以具有未知的脊髓动物宿主,例如Aedes黄病毒,Calbertado病毒,细胞融合剂病毒(Cell fusion agent virus),Culex黄病毒,Culex theileri黄病毒,Kamiti River病毒,Nakiwogo病毒,或Quang Binh病毒。所述黄病毒可以选自下组:Aroa病毒("AROAV"),Dengue病毒("DENV"),Kedougou病毒("KEDV"),Bussuquara病毒,Cacipacore病毒("CPCV"),Koutango病毒("KOUV"),Ilheus病毒("ILHV"),日本脑炎病毒("JEV"),墨累谷脑炎病毒("MVEV"),Rocio病毒("ROCV"),圣路易斯脑炎病毒("SLEV"),乌苏图(Usutu)病毒("USUV"),西尼罗病毒("WNV"),Yaounde病毒("YAOV"),Kokobera病毒("KOKV"),Bagaza病毒("BAGV"),Ilheus病毒("ILHV"),Israel turkey脑膜脑脊髓炎病毒("ITV"),Ntaya病毒("NTAV"),Tembusu病毒("TMUV"),Spondweni病毒组,Zika病毒("ZIKV"),Banzi病毒("BANV"),Bouboui病毒("BOUV"),Edge Hill病毒("EHV"),Jugra病毒("JUGV"),Saboya病毒("SABV"),Sepik病毒("SEPV"),Uganda S病毒("UGSV"),Wesselsbron病毒("WESSV"),或黄热病病毒("YFV")。具有未知节肢动物媒介物的病毒的例子包括Entebbe bat(蝙蝠)病毒("ENTV"),Yokose病毒("YOKV"),Apoi病毒("APOIV"),Cowbone Ridge病毒("CRV"),Jutiapa病毒("JUTV"),Modoc病毒("MODV"),Sal Vieja病毒("SVV"),San Perlita病毒("SPV"),Bukalasa蝙蝠病毒("BBV"),Carey Island病毒("CIV"),Dakar蝙蝠病毒("DBV"),Montana myotis白质脑炎病毒("MMLV"),Phnom Penh蝙蝠病毒("PPBV"),和Rio Bravo病毒("RBV")。在一个方面,所述黄病毒选自下组:西尼罗病毒、昆津病毒、日本脑炎病毒、墨累谷脑炎病毒、登革热血清型1病毒、登革热血清型2病毒、登革热血清型3病毒、登革热血清型4病毒、黄热病病毒、蜱媒脑炎病毒、波瓦森病毒和鄂木斯克出血热病毒。
除了以上列出的病毒,在中心单体接触界面中包括创造性突变的嵌合黄病毒在本发明的范围内。一般来说,嵌合黄病毒包含具有来自于两种或更多不同黄病毒(包括不同的黄病毒株)的序列的基因组的病毒。例如,这些嵌合体可以包含以下黄病毒(例如骨架黄病毒),其中结构蛋白被第二种病毒的对应结构蛋白代替。例如,所述嵌合体可以组成为骨架黄病毒(例如黄热病毒),其中黄病毒的prM和E蛋白被第二种病毒(例如登革热病毒(血清型1-4)、日本脑炎病毒、西尼罗病毒或其它病毒,例如本文中提到的那些)的prM和E蛋白代替。所述嵌合体病毒可以从任何病毒或其株的组合制造。示例性的嵌合体描述于Yamshchikov,美国专利号7,455,832和Guirakhoo等,美国专利申请号2007/0269458,其通过提述并入本文。
本发明的黄病毒包膜蛋白对于制备减毒黄病毒株是有用的。除了此类用途,所述蛋白作为补充工具来揭示黄病毒包膜蛋白的行为和功能的机制是有用的。例如,所述蛋白可以用于筛选调节当前所述(instant)蛋白活性的分子(能够治疗黄病毒诱导的感染)。在一个方面,本发明的黄病毒包膜蛋白可以是分离的和/或纯化的。
在另一个方面,本发明涉及编码如本文描述的蛋白的核苷酸序列,包括由于遗传密码简并,编码这些蛋白的所有可能的核苷酸序列的例子。本发明的核酸可以通过重组DNA技术和/或化学DNA合成的熟知方法得到。本发明还提供包含编码所述当前所述蛋白的多核苷酸的重组构建体,或编码此类蛋白的减毒黄病毒株。所述构建体可以是通过本发明的载体转化的原核或真核宿主细胞中的载体的形式。
因此,在另一个方面,本发明涉及活减毒黄病毒,其中黄病毒包膜单体蛋白能够沿着中心单体接触界面形成二聚体,所述中心单体接触界面在对应于西尼罗病毒包膜蛋白氨基酸256至260的对应氨基酸处,并且所述黄病毒包膜单体蛋白具有一个或多个降低所述二聚体解离的突变。
本发明的活减毒黄病毒可以在体内使用感染性DNA的方法产生,例如描述于Yamshchikov,美国专利号7,459,163和Yamshchikov,美国专利号7,455,832,其通过提述并入本文。本领域的技术人员应当理解,本发明的感染性DNA可以使用任何适合的载体形成。一般来说,载体是核酸分子(典型的,DNA或RNA),其用于将乘客核酸序列(即DNA或RNA)转移到宿主细胞中。三种常见类型的载体包括质粒、噬菌体和病毒。优选的,所述载体是质粒。也就是说,本发明的感染性DNA疫苗包含作为质粒产生的DNA,其可以被引入动物组织并在其中被动物细胞表达来产生黄病毒基因组大小的信使核糖核酸(“mRNA”)分子,其翻译产生病毒多聚蛋白,被细胞体系处理提供全套黄病毒蛋白,其能够起始上述初级RNA转录物的复制,因此起始在引入上述DNA质粒的动物组织中的病毒复制周期。
用于常规DNA疫苗的适合的和示例性的质粒载体包括但不限于pBR322(ATCC#31344);pUC19(ATCC#37254);pcDNA3.1(Invitrogen,Carlsbad,CA;Cat.NO.V385-20;DNA序列可获得于http://www.invitrogen.com/vectordata/index.html);pNGVL(National GeneVector Laboratory,University of Michigan,MI);p414cyc(ATCC#87380)、p414GALS(ATCC#87344)、pBAD18(ATCC#87393)、pBLCAT5(ATCC#77412)、pBluescriptIIKS(ATCC#87047)、pBSL130(ATCC#87145)、pCM182(ATCC#87656)、pCMVtkLUC(ATCC#87633)、pECV25(ATCC#77187)、pGEM-7zf(ATCC#87048)、pGEX-KN(ATCC#77332)、pJC20(ATCC#87113)、pUB110(ATCC#37015)、pUB18(ATCC#37253)。
本发明的感染性DNA也优选的处于适合的启动子的控制下。对于真核表达,适合的启动子包括巨细胞病毒(“CMV”)早期启动子,或者Rous肉瘤病毒(“RSV”)LTR启动子和SV40启动子。
本发明的免疫原性组合物中活减毒病毒或重组构建体的量优选为治疗有效量。例如,在WN重组构建体的情况下,质粒的治疗有效量一般是使得编码WN病毒的核苷酸序列在所述组合物施用的宿主中实行其免疫功能而不导致过度的负面影响的必需量。要使用的质粒以及要施用的组合物/疫苗的确切量将根据不同的因素而不同,例如所使用的转录启动子的强度,所治疗的病症的种类,施用的模式,以及在所述组合物中的其它成分。优选的,所述组合物或疫苗制剂由约10ng到约1μg质粒构成。要注意非复制DNA疫苗通常需要更大的质粒DNA量(通常为10到100μg)。
本发明的疫苗和药物组合物也可以包括药学上可接受的载体。载体包括稀释剂、佐剂、赋形剂或媒介物,减毒活病毒或感染性DNA与其一同施用。此类药物载体可以是无菌的液体,例如水或油,包括石油,动物、植物或合成来源的,例如花生油、豆油、矿物油、芝麻油等等。适合的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、二醇、水、乙醇等等。所述组合物,如果需要,也可以含有少量的润湿剂或乳化剂,或pH缓冲剂。这些组合物可以采取溶液、悬浮液、乳剂、片剂、丸剂、胶囊、粉剂、缓释制剂等等的形式。所述组合物可以配置为栓剂,使用传统的组合剂和载体例如甘油三酯。口服制剂可以包括标准的载体例如药物级甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。适合的药物载体的例子描述于E.W.Martin的“Remington's Pharmaceutical Sciences”中。载体可以包括助剂,其包括但不限于稀释剂、稳定剂(例如糖和氨基酸)、防腐剂、润湿剂、乳化剂、pH缓冲剂、粘度增强剂、颜色等等。载体包括但不限于稳定剂、防腐剂和缓冲剂。适合的稳定剂为例如SPGA、吐温组合物(例如可从A.G.Scientific,Inc.,San Diego,CA得到的)、碳水化合物(例如山梨糖醇、甘露糖醇、淀粉、蔗糖、葡聚糖、谷氨酸盐、或葡萄糖)、蛋白(例如干燥乳清、白蛋白或酪蛋白),或其降解产物。适合的缓冲剂的非限制性例子包括碱金属磷酸盐。适合的防腐剂为硫汞撒(thimerosal)、硫柳汞(merthiolate)和庆大霉素(gentamicin)。稀释剂包括水、水性缓冲液(例如缓冲盐水),醇和多元醇(例如甘油)。所述组合物可以包括alpha-干扰素、beta-干扰素、gamma-干扰素、粒细胞巨噬细胞集落刺激因子(“GM-CSF”)、巨噬细胞集落刺激因子(“M-CSF”)、白介素2(“IL-2”)、白介素12(“IL-12”)和/或CpG寡核苷酸。对于制备此类组合物,可以使用本领域熟知的方法。根据本发明的不同实施方案的疫苗和免疫原型组合物可以以液体、冷冻悬浮液或冻干的形式制备和/或销售。典型的,本发明的疫苗和/或药物组合物含有通常用于此类组合物的药学上可接受的载体。药物组合物和疫苗制剂的例子描述于Sterner等,美国专利号8,048,429,其通过提述并入。
皮下注射、皮内引入、通过皮肤按压(impression),以及其它施用方式例如腹膜内、静脉内、口服、或吸入递送也是适合的。例如,含有本发明的感染性DNA的载体可以通过本领域已知的方法引入所需的宿主中,例如,转染、电穿孔、显微注射、微粒、微胶囊、转导、细胞融合、DEAE葡聚糖、磷酸钙沉淀、脂转染(脂质体融合)、使用基因枪(粒子轰击)或DNA载体转运蛋白。
施用可以是单次或多次的(例如单剂量或包括增强剂)。此类施用可以是单独的或与其它针对黄病毒的活性治疗试剂联合。
疫苗纯化、其它疫苗组分、疫苗制备以及疫苗的施用一般描述于Wicker等,美国公开专利申请号2009/0117149和Brown等,美国公开专利申请号2011/0236421,其通过提述并入。
本发明还涉及用于引起针对病原性黄病毒株,包括高病原性的NY99病毒株的免疫应答或保护性免疫的重组构建体或药物组合物。根据相关的方面,本发明涉及用于预防和/或治疗黄病毒相关疾病的疫苗。
术语“一个”或“一种”可以表示一个(一种)或多个(多种)。如在本文权利要求中所使用的,当与词语“包含”一起使用时,所述词语“一个”或“一种”可以表示一个(一种)或多于一个(一种)。
术语“约”用于表示值包括所采用的用来确定所述值的设备和方法的误差的固有变差,或存在于研究受试者之间的变差。
术语“编码”关于核酸用来使发明对技术人员容易理解;然而,这一术语可以与“包含”替换使用。
术语“或”的使用用于表示“和/或”,除非明确指明只指代某项,或所述项是相互排斥的,虽然本公开支持只指代某项和“和/或”的定义。如本文所使用的“另一个”可以表示至少第二个或更多。
术语“肽”、“寡肽”、“多肽”、“多聚蛋白”和“蛋白”在本文中可替换使用,并指代任何长度的多聚形式的氨基酸,其可以包括编码的和非编码的氨基酸,化学或生物化学修饰或衍生的氨基酸,以及具有修饰的肽骨架的多肽。
术语“治疗”指代黄病毒的病毒复制症状或相关疾病被抑制、改善或完全消除的过程。如本文使用的,术语“预防/阻止”指代黄病毒病毒复制或相关疾病被阻挠或延迟的过程。
术语“重组”如本文所使用的,表示具体的序列是克隆、限制性、和/或连接步骤的各种组合产生的具有与自然体系中发现的同源序列可区分的结构编码序列的构建体的产物。例如,编码所述结构编码序列的DNA序列可以从cDNA片段和短的寡核苷酸连接序列,或一系列寡核苷酸组装,来提供能够在重组转录单元中表达的合成基因。此类序列可以以不被内部非翻译序列或通常存在于真核基因中的内含子打断的开放阅读框的形式提供。相反的,为了稳定的目的,此类序列可以以开放阅读框的形式提供,其被插入的人工非翻译序列或通常不存在于病毒基因的内含子打断。还可以使用包含相关序列的基因组DNA。除了内含子外的非翻译DNA序列也可以存在于所述开放阅读框的5’或3’,其中此类序列不干扰编码区的操作或表达。因此,例如,术语“重组”多核苷酸或核酸指代非自然发生的,或通过人工组合两个原本解离的序列片段制造的。这种人工组合通常通过化学合成方法或通过人工操作分离的核酸片段例如通过基因工程技术完成。这种通常是为了用编码相同或保守氨基酸的多余密码子代替密码子,而通常引入或移除序列识别位点。或者,其用来将期望功能的核酸片段连接到一起来产生所需的功能组合。
术语“构建体”一般指代重组核酸,一般是重组DNA,其为了表达特定核苷酸序列的目的被产生,或用于其它重组核苷酸序列的构建。
相似的,术语“重组蛋白”指代非自然发生的多肽或多聚蛋白,或通过人工组合两个原本分离的氨基酸序列片段制造的。这种人工组合可以通过重组DNA技术的标准技术完成,例如上述的,例如重组蛋白可以由重组多核苷酸编码。因此,重组蛋白是由全部或部分重组多核苷酸编码的氨基酸序列。
术语“免疫活性”或“免疫原性”指代天然的、重组的或合成的病毒或肽,或编码此类病毒或肽的天然的、重组的或合成的核酸,当接种到患者中时诱导特异性的体液和/或细胞免疫应答的能力。
因此,术语“免疫应答”指代T细胞反应或针对抗原的抗体增加的血清水平,或指代针对抗原的中和性抗体的存在,例如黄病毒蛋白。
术语“保护”或“保护性免疫”本文中指代免疫期间诱导的血清抗体或T细胞应答针对黄病毒导致的疾病或死亡进行保护(部分或完全)的能力。
本发明的术语“受试者”或“患者”优选的为鸟类,例如鸡、乌鸦、鹰、鹦鹉、鹅、火烈鸟等,或哺乳类,例如小鼠、牛、猪、马、鸡、猫、狗等,优选的为人类。
术语“治疗有效剂量”或“治疗有效量”表示对于所施用者产生所需的效果的剂量或量。确切的剂量将依赖于治疗的目的,通过本领域的技术人员使用已知技术将是可确定的。
术语“药学上可接受的”表示由联邦政府或州政府的监管机构批准,或列于美国药典或其它一般公认药典用于动物,更具体的用于人。因此,如本文所使用的,术语“药学上可接受的载体”表示但不限于,含有本发明的DNA构建体或减毒活病毒的媒介物,其可以接种于哺乳动物宿主中而没有不良影响。
虽然本发明描述的是中心单体接触界面中的突变,但应当理解本发明的病毒还可以包括一个或多个另外的突变。例如,在西尼罗病毒(或其它黄病毒)的情况下,此类另外的突变可以在西尼罗病毒包膜蛋白的位置197(例如L突变为F)、316(例如A突变为V)或440(例如K突变为R)的区域中(或其组合)。因此所述突变可以是在西尼罗包膜蛋白的氨基酸102-112、138(例如E突变为K)、176(例如Y突变为V)、177(例如T突变为A)、244(例如E突变为G)、264(例如Q突变为H)、280(例如K突变为M)、311-321和/或435-445的一处或多处。作为具体的例子,使用西尼罗病毒株NY99-火烈鸟382-99的序列(GenBank登录号AF196835)作为参考,位置107的赖氨酸可以使用苯基丙氨酸取代,位置316的丙氨酸可以使用缬氨酸取代,和/或位置440的赖氨酸可以使用精氨酸取代。可以突变的氨基酸的其它组合的例子包括以下:176、177和280;176、177、244、264和280;和138、176、177和280。此外,这些突变可以存在于其它黄病毒的对应氨基酸中,如本文所述。
以下实施例用来说明本发明的示例性实施方案。本领域的技术人员应当理解实施例中公开的技术遵循本发明人发现的在本发明的实施中发挥良好的代表技术,并且可以认为构成其实施的优选方式。然而,根据本公开,本领域的技术人员应当理解不脱离本发明的精神和范围,在公开的具体的实施方案中可以做出许多变化,并仍然得到相似或类似的结果。
实施例1:WN NY99包膜蛋白的同源性建模和单体接触界面的分析
DEN2(PDB#1OAN,(见Modis等,A ligand-binding pocket in the denguevirus envelope glycoprotein,Proc Natl Acad Sci USA 100 6986-6991[Epub May20 2003](2003))和DEN3(PDB#1UZG,(见Modis等,Variable surface epitopesin the crystal structure of dengue virus type 3envelope glycoprotein,J Virol 791223-1231(2005))E同二聚体的坐标文件用于用Swiss Model的同源性建模(见Guex等,SWISS-MODEL and the Swiss-PdbViewer:an environment forcomparative protein modeling,Electrophoresis 18 2714-2723(1997)和Schwede等,SWISS-MODEL:an automated protein homology-modeling server,NuclAcids Res 31 3381-3385(2003))来建立NY99胞外域二聚体的模型;所述模型使用VectorNTI软件包(Invitrogen)的3D Molecule Viewer来展示、探索和操作。如图2所示,两个单体之间的接触界面不是连续的,在prM的位置有两个孔(见Rey等,The envelope glycoprotein from tick-borne encephalitisvirus at 2A resolution[see comments],Nature 375 291-298(1995))。紧密接触的区域在二聚体的中心处及在其任一远端部分对称形成,在图2A中分别通过一个框和两个椭圆标注。远端接触大部分涉及非极性氨基酸,其中cd融合环适合由结构域I和III形成的疏水口袋中。
与此相反,中心接触界面(在图2B和2C中详细示出)主要包括亲水和极性氨基酸。组成由各条链提供的两个相同α螺旋的氨基酸看起来不形成紧密接触。两个单体的多肽链在二聚体中的这个位点交叉成锐角,像“X”(如果从图像平面的上部相应的侧面看),所述螺旋占据“X”排列的左上和右上角。然而,交叉周围的氨基酸(GSQEG,示意图示于图2B,空间填充如图2C)看上去确实形成紧密接触。如表1所示,实际上序列不仅在同种的株之间,也在该属的成员之间高度保守。尤其是,蜱媒病毒的整个组在位置257和259带有相互交换(S→D和E→T),因此在这些位置维持了带负电荷和含羟基的氨基酸的组合。在这一序列的空间填充表示中(图2C),各条链中的Ser-257的羟基侧链并列。羟基基团对同一链的Gln-258或Glu-259的骨架NH同样接近(3.11对3.15A),并可能形成氢键。一条链中的Glu-259侧链的氧原子与相对链中的Ser-257的骨架NH接近(3.06-3.14A),以形成氢键,其可能有助于二聚体的稳定性。保守的Gln-258的侧链在图2C中图平面的背侧的两条链中都暴露,并且看起来不与该结构的其它元件靠近。对位置256和260的小氨基酸(Gly或Ala)的选择性压力可能通过接触界面处高度对称排列的空间限制施加。
表1.对应西尼罗病毒位置256至260的中心单体接触界面处的氨基酸序列比对
为了清楚,中心单体接触界面两侧侧翼的两个氨基酸也提供于表1中。
实施例2:WN黄病毒的感染性克隆
WN系2病毒的稳定感染性克隆的组装(pSP6WN956)已经报道。见Yamshchikov等,An infectious clone of the West Nile flavivirus,Virology 281294-304(2001)。这一克隆在SP6启动子的转录控制下组装。为了简化操作和稳定所述感染性克隆,使用为了JE感染性DNA的稳定所开发的方案将它转变为感染性DNA(iDNA)形式(见Yamshchikov等,A new strategy in designof+RNA virus infectious clones enabling their stable propagation in E.coli,Virology 281 272-280(2001))。pSP6WN956构建体中的SP6启动子使用CMV启动子取代,并且一个132bp的人工内含子被插入到位置358(在衣壳基因的末端)来增加在大肠杆菌(E.coli)中增殖期间所述构建体的稳定性。将后面有牛生长激素转录终止信号(“BG”)的反义链肝炎δ核酶工程化到WNcDNA的末端来实现增加的3’末端形成的保真度,产生最终构建体pCMVWN956(i358)δBG(进一步称为pCMVWN956;图3)。在使用此质粒转染后24小时,病毒复制焦点可用间接免疫荧光法容易检测到(图4)。WNiDNA的特异性感染性(specific infectivity)在Vero细胞中为1-5×106pfu/μgDNA。
隔离群(isolate)385-99是以Vero 1代由R.Tesh(Galveston,TX)提供的。所述病毒从死于1999年8月纽约布朗克斯动物园(Bronx Zoo)的雪白猫头鹰中回收。由于分离的地理位置和时间,其非常可能代表了NY99株的一个独立隔离群。在Vero 2代测定的385-99基因组的全部核苷酸序列(GenBank#DQ211652)与之前提交的同一隔离群的核苷酸序列(GenBank#AY842931)的不同之处在于在位置630处的一个沉默A→G取代。385-99隔离群在8个核苷酸中与原型NY99隔离群382-99不同(GenBank#AF196835),并且在E167的一个氨基酸中与原型NY99隔离群382-99不同(Phe→Leu)。与pSP6WN956不同,相似的pSP6NY99构建体非常不稳定并表现出自发重排的高倾向。所述iDNA形式通过插入短的内含子允许此类不稳定的构建体稳定,阻止了在大肠杆菌中增殖期间问题区域的表达(见Mishin et al.,A'minimal'approach in design of flavivirus infectious DNA,Virus Res 81 113-123(2001)and Yamshchikov et al.,A new strategy in design of+RNA virus infectiousclones enabling their stable propagation in E.coli,Virology 281 272-280(2001));在转染易感性细胞后,内含子被真核转录体系精确移除,从而恢复了病毒ORF。在位置393和2384处内含子的插入允许相对稳定的NY99感染性克隆的组装。所述pCMVNY99(i393i2384)δBG构建体(进一步称为pCMVNY99,图3)在转染后24小时产生抗原阳性焦点,在转染后40小时产生几乎完全感染的单层。特异性感染性为5-8×106pfu/μg;从此iDNA回收的病毒具有与亲本385-99相同的生物学特性(结果未示出)。以上两种iDNA质粒用于创造带有结构蛋白基因相互交换的嵌合构建体。图3中显示了此类嵌合物之一pCMV[CprMENY99]WN956,其衍生物被发明人用在正在进行的人减毒西尼罗疫苗的开发中。其携带385-99的所有结构蛋白,取代WN956的那些,并将前者的高免疫原性和后者的减毒表型组合。
实施例3:影响病毒感染性的突变的鉴定
如上所述,高度保守的序列GSQEG形成E二聚体中基本回文的中心单体接触界面。在建模实验中,选择了几个可能增强在这一接触界面处的相互作用,并影响对病毒感染性至关重要的pH介导的二聚体解离的突变。
基本原理:选择可能增强单体和单体相互作用的突变。中心接触界面的回文性质起源于所述二聚体的二次对称性,其可以在图2A中的顶视图中看到。图2B和2C的展开图显示在野生型蛋白中,GSQEG序列形成高度对称的接触。这一对称的保存作为重要的因素包括在我们的建模实验中。也就是说,无法采用侧链构象来形成对称或近似对称界面的氨基酸被排除。通常来说,未研究将在此接触界面干扰单体-单体相互作用的突变。预期此类突变有害于病毒粒体的组装是合理的。与此相反,加强两个单体之间相互作用的突变可能具有对病毒粒体组装可以忽略的影响,但对所述二聚体的解离产生不利影响。接触界面的对称性意味着GSQEG序列中的单个突变由于各条链的贡献将导致界面处的双重效果。
高度保守的带负电氨基酸的存在(对于蚊媒黄病毒为E而对于蜱媒亚组为D)促进了关于接触界面处的单体相互作用是否能够通过引入能够与其形成盐桥的带正电荷的氨基酸来增强的研究。如上所述,Gln-258侧链暴露于图2图平面的背侧,其潜在的相互作用伴侣没有鉴定。然而,一条链中的Gly-256和Ser-257两者似乎与另一条链中的Glu-259的侧链紧密靠近,促使对这些位置的突变的探究。对Ser-257被Arg或Lys取代的E突变蛋白建模示于图4A和4B中。显然Arg-257或Lys-257都不能形成对称的界面。为了改善侧链的对称性,通过操作扭转角(使用SwissProt DeepView 3.7)在两条链处的折叠由于原子之间的多冲突而没有成功(结果未示出)。虽然Arg-257可以稍微更好的纳入,产生的排列表明各个链中的Arg-257侧链似乎与同一条链的Glu-259侧链靠近。因此,所期待的两条链之间的潜在盐桥不能形成。
使用Arg或Lys取代Gly-256的E突变蛋白建模产生了较令人鼓舞的结果。产生的RSQEG(图5A)和KSQEG(图5B)似乎形成近似对称的接触界面,扭转角的操作产生了几种具有改善的对称性,没有原子之间冲突的旋转异构体(结果未示出)。最重要的是,一条链中的Arg-256和Lys-256的侧链似乎都靠近另一条链中的Glu-258侧链,因此能够在链之间形成所需的盐桥。如上所述,单个突变将导致在回文接触界面任一端的两个盐桥的形成。由于这个原因,选择Gly256Arg和Gly256Lys突变来探究其在NY99E蛋白二聚体的形成和行为,以及NY99病毒的生物学特性上的影响。
实施例4:突变构建体回复的设计和突变病毒的生物学特性
为了检查所述修饰的减毒效果,Gly256Arg和Gly256Lys突变如图3所示引入pCMVNY99感染性DNA构建体中。作为对比,GSQEG序列(发现于蚊媒病毒中)改变为蜱媒病毒特征性的GDQTG。所述pCMVNY99质粒当转染哺乳动物细胞时和直接接种小鼠后,通常均产生高感染性和高毒性的NY99病毒。后者在通过任意途径接种少至1pg的感染性DNA后(i.m.,i.d.,i.c.),导致动物100%的死亡率。
如图6所示,两种加强二聚体的突变对病毒的感染性有显著的影响,导致病毒在感染的哺乳动物细胞中与产生于野生型构建体的NY99病毒相比传播延迟。于此相反,蜱媒黄病毒构造的插入对病毒的感染性没有明显影响。
突变衍生物的毒性在成年小鼠脑内接种模型中测试。由6只5-6周龄的小鼠的组i.c接种pCMVNY99的1μg G256R、G256K或(S257D,E259T)衍生物,并对于死亡率观察动物21天。垂死的动物使其安乐死并算作死于感染,使用病毒特异性RT-PCR通过病毒在脑中的存在确定。与感染试验一致,(S257D,E259T)突变体为高毒性,在接种后5天杀死了所有动物。与之相反,在G256R、G256K组仅各有两只小鼠发现垂死并使其安乐死;然而,通过RT-PCR对病毒在脑中存在的揭示没有进行。不过假设动物死亡是由感染引起的,在脑内接种这样一个严格的测试中67%的存活率表明两种突变病毒的高减毒水平。所有存活的动物表现出高水平的NY-99特异性抗体,其终点稀释滴度超过1:2560,表明所述动物确实暴露于感染性DNA。
从上述将可以看出本发明很好的适用于本文以上所述的所有目的和目标,以及其它对于本发明是固有的和显而易见的优点。由于不脱离本发明的范围,可以做出许多可能的实施方案,应当理解本文所述的或示于附图的所有事项都被解释为说明性的,而不具有限制意义。虽然示出了和讨论了具体的实施方案,但当然可能进行各种修改,并且本发明不限于本文所述部分和步骤的具体形式或排列,除了包括于以下的权利要求中的相关限制。此外,应当理解某些特征和子组合是有用的,并且在不参考其它特征和子组合下可以采用。这被考虑并在权利要求的范围内。
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Claims (33)
1.能够沿着中心单体接触界面形成二聚体的黄病毒包膜单体蛋白,所述黄病毒包膜蛋白具有对应于西尼罗病毒包膜蛋白氨基酸256至260的中心单体接触界面,并且其中所述黄病毒包膜蛋白在所述中心接触界面中具有突变,该突变减少所述二聚体的解离。
2.权利要求1的蛋白,其中所述突变导致在所述中心单体接触界面处两个盐桥的形成。
3.权利要求1的蛋白,其中所述黄病毒包膜单体蛋白来自于蚊媒病毒,并且其中所述突变包含对应于西尼罗病毒包膜蛋白氨基酸256的黄病毒包膜单体蛋白氨基酸被碱性氨基酸取代的突变。
4.权利要求3的蛋白,其中所述碱性氨基酸选自赖氨酸和精氨酸。
5.权利要求1的蛋白,其中所述黄病毒包膜单体蛋白来自于蜱媒病毒,并且其中所述突变包含对应于西尼罗病毒包膜蛋白氨基酸260的氨基酸被碱性氨基酸取代的突变。
6.权利要求5的蛋白,其中所述碱性氨基酸选自赖氨酸和精氨酸。
7.权利要求1的蛋白,其中所述中心单体接触界面具有选自下组的野生型序列:GSQEG(SEQ ID NO:7)、GNQEG(SEQ ID NO:8)、GDQTG(SEQID NO:9)、和GDQTA (SEQ ID NO:10)。
8.权利要求1的蛋白,其中所述黄病毒包膜单体蛋白具有包含选自下组的序列的中心单体接触界面:RSQEG(SEQ ID NO:1)、RNQEG(SEQ IDNO:2)、GDQTR(SEQ ID NO:3)、KSQEG(SEQ ID NO:4)、KNQEG(SEQ IDNO:5)、和GDQTK(SEQ ID NO:6)。
9.权利要求1的蛋白,其中所述黄病毒包膜单体蛋白来自于选自下组的黄病毒:西尼罗病毒、昆津病毒(Kunjin virus)、日本脑炎病毒、墨累河谷脑炎病毒(Murray Valley encephalitis virus)、登革热血清型1病毒、登革热血清型2病毒、登革热血清型3病毒、登革热血清型4病毒、黄热病病毒、蜱媒脑炎病毒、波瓦森病毒(Powassan virus)和鄂木斯克出血热病毒(Omskhemorrhagic fever virus)。
10.权利要求1的蛋白,其中所述黄病毒包膜单体蛋白来自于西尼罗病毒。
11.编码权利要求1的黄病毒包膜单体蛋白的多核苷酸。
12.包含权利要求11的多核苷酸的载体。
13.包含权利要求12的载体的宿主细胞。
14.编码权利要求1的黄病毒包膜单体蛋白的减毒黄病毒。
15.权利要求14的减毒黄病毒,其中所述黄病毒选自下组:西尼罗病毒、昆津病毒、日本脑炎病毒、墨累河谷脑炎病毒、登革热血清型1病毒、登革热血清型2病毒、登革热血清型3病毒、登革热血清型4病毒、黄热病病毒、蜱媒脑炎病毒、波瓦森病毒和鄂木斯克出血热病毒。
16.权利要求14的减毒黄病毒,其为嵌合黄病毒。
17.权利要求14的减毒黄病毒,其中所述黄病毒在对应于选自氨基酸107、138、176、177、224、264、280、316和440的西尼罗病毒包膜蛋白氨基酸的氨基酸残基中具有一个或多个包膜蛋白突变。
18.包含权利要求14的减毒黄病毒的免疫原性组合物。
19.权利要求18的免疫原性组合物,其进一步包含佐剂。
20.用于在患者中诱导免疫应答的方法,其包括:
得到权利要求18的免疫原性组合物;
将所述免疫原性组合物施用于所述患者。
21.权利要求20的方法,其中所述患者是人类。
22.权利要求20的方法,其中所述施用是静脉内、肌肉内、腹膜内或皮下。
23.编码权利要求14的黄病毒的重组遗传构建体。
24.权利要求23的重组遗传构建体,其进一步包含载体。
25.权利要求24的重组遗传构建体,其中所述载体是质粒。
26.权利要求25的重组遗传构建体,其中所述质粒包含在真核启动子控制下的编码感染性(+)RNA分子的DNA。
27.权利要求26的重组遗传构建体,其中所述真核启动子包含CMV启动子。
28.用权利要求23的重组遗传构建体稳定或瞬时转染的宿主细胞。
29.包含权利要求23的重组遗传构建体的免疫原性组合物。
30.权利要求29的免疫原性组合物,其进一步包括佐剂。
31.用于在患者中诱导免疫应答的方法,其包含:
得到权利要求29的免疫原性组合物;
将所述免疫原性组合物施用于所述患者。
32.权利要求31的方法,其中所述患者是人类。
33.权利要求31的方法,其中所述施用是静脉内、肌肉内、腹膜内或皮下。
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US11007260B2 (en) | 2015-07-21 | 2021-05-18 | Modernatx, Inc. | Infectious disease vaccines |
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AU2016335120A1 (en) | 2015-10-07 | 2018-05-24 | The University Of North Carolina At Chapel Hill | Methods and compositions for dengue virus vaccines and diagnostistics |
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