CN117295516A - 用于治疗hsv的疫苗组合物和方法 - Google Patents
用于治疗hsv的疫苗组合物和方法 Download PDFInfo
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- CN117295516A CN117295516A CN202280034408.3A CN202280034408A CN117295516A CN 117295516 A CN117295516 A CN 117295516A CN 202280034408 A CN202280034408 A CN 202280034408A CN 117295516 A CN117295516 A CN 117295516A
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Abstract
本发明涉及一种用于治疗HSV或针对HSV疫苗接种的疫苗组合物,所述疫苗组合物包含一种或多种编码单纯疱疹病毒(HSV)结构蛋白或其免疫原性片段的mRNA。
Description
相关申请的交叉引用
本申请要求于2021年3月11日向欧洲专利局提交的欧洲专利申请21162170的优先权,该申请的全部内容并入本文用于全部目的。
序列表
本申请包含计算机可读形式的序列表,其通过引用并入本文。
技术领域
本发明涉及一种用于治疗HSV或针对HSV疫苗接种的疫苗组合物,所述疫苗组合物包含一种或多种编码单纯疱疹病毒(HSV)结构蛋白或其免疫原性片段的mRNA。
背景技术
单纯疱疹病毒是被称为疱疹病毒科这一病毒科的病毒属。感染人类的物种通常称为单纯疱疹病毒1(HSV-1)和单纯疱疹病毒2(HSV-2),其中它们的正式名称分别是人疱疹病毒1(HHV-1)和人疱疹病毒2(HHV-2)。HSV-1的最初感染通常发生在儿童期或青春期,并持续终生。在世界范围内HSV-1的感染率在40%与80%之间,在社会经济地位较低的人群中更高。在许多情况下,暴露于HSV-1的人表现出无症状的血清转化。然而,最初感染也可能是严重的,引起广泛的1至2mm水疱,其与干扰进食和饮水至脱水点的严重不适相关,持续10至14天,并且在接种后1至26天发生。复发性唇疱疹影响大约三分之一的美国人口,并且这些患者通常每年经历1至6次发作。红斑基底上的丘疹在几小时内变成水泡,随后在72至96小时内发展为溃疡、结痂和愈合阶段(Cernik等人,2008,Arch Intern Med.,第168卷,第1137-1144页)。2003年全球估计认为,16.2%的人口感染了HSV-2,是生殖器疱疹的主要原因。病毒通过进入称为潜伏期的非复制状态而成功避免被免疫系统清除的能力导致终生感染。自潜伏期的周期性再激活是可能的,并且导致病毒从最初感染部位脱落。由疱疹导致的生殖器损伤通常非常疼痛,并且可导致显著的心理病态。病毒也可以在出生期间从母亲传给孩子。没有治疗,80%的患有播散性疾病的婴儿死亡,并且存活的那些婴儿通常脑损伤。此外,生殖器疱疹与两至三倍的HIV获得、高达五倍的每次性行为基础上的HIV传播的风险增加相关,并且可能占高HSV-2流行人群中新HIV感染的40-60%(Looker等人,2008,Bulletin ofthe World Health Organization,第86卷,第805-812页)。
目前,阿昔洛韦,一种合成的无环嘌呤-核苷类似物,是HSV感染的标准疗法,并且已极大地帮助控制了症状。前体药物伐昔洛韦(转化为阿昔洛韦)和泛昔洛韦(转化为喷昔洛韦)已经得到许可,并且分别比阿昔洛韦和喷昔洛韦具有更好的口服生物利用度。可用的药物具有极好的安全裕度,因为它们仅在病毒感染的细胞内部被病毒胸苷激酶转化为活性药物。然而,HSV可以通过产生胸苷激酶缺陷的突变体或通过选择其胸苷激酶不能磷酸化阿昔洛韦的突变体而经由编码胸苷激酶的病毒基因的突变,产生对阿昔洛韦的抗性。大多数抗阿昔洛韦的临床HSV分离株在胸苷激酶中有缺陷,尽管在一些中已经检测到改变的DNA聚合酶。由于HSV能够在变得活跃前潜伏在神经元中数月或数年,这种疗法可用于治疗由HSV引起的症状,但不能避免病毒的周期性再激活。
因此,最有效和经济的抗HSV途径是预防病毒的最初感染和/或周期性再激活的疫苗。在过去的几十年里,在这种疫苗的开发中已经投入了许多努力。然而,迄今为止开发强效HSV疫苗的尝试集中于在临床试验中显示低性能的有限数量的抗原。因此,迫切需要抗HSV的疫苗。最近已经尝试开发基于HSV糖蛋白的核苷修饰mRNA的HSV疫苗(US2020/0276300),然而这些仍然处于开发早期阶段。仍然需要进一步的HSV疫苗接种。
发明内容
本发明满足了这种需要,并提供了新的疫苗组合物,所述疫苗组合物包含一种或多种mRNA,其中所述mRNA中的每一种编码选自UL48;UL48和UL49;UL11、UL16和UL21;或UL31和UL34的单纯疱疹病毒(HSV)结构蛋白或其免疫原性片段。具体而言,在本发明的疫苗组合物中,所述mRNA编码具有与SEQ ID NO:6的氨基酸序列具有80%或更高同一性的氨基酸序列的UL48、具有与SEQ ID NO:7的氨基酸序列具有62%或更高同一性的氨基酸序列的UL49、具有与SEQ ID NO:1的氨基酸序列具有75%或更高同一性的氨基酸序列的UL11、具有与SEQID NO:2的氨基酸序列具有72%或更高同一性的氨基酸序列的UL16、具有与SEQ ID NO:3的氨基酸序列具有80%或更高同一性的氨基酸序列的UL21、具有与SEQ ID NO:8的氨基酸序列具有85%或更高同一性的氨基酸序列的UL31、和具有与SEQ ID NO:8的氨基酸序列具有70%或更高同一性的氨基酸序列的UL34。
优选地,当以疫苗组合物的形式施用于受试者时,本发明疫苗组合物中的所述HSVmRNA中的每一种能够引发免疫应答。
此外,本发明的疫苗组合物可进一步包含一种或多种编码单纯疱疹病毒(HSV)糖蛋白的mRNA,所述HSV糖蛋白选自a)具有与SEQ ID NO:11的氨基酸序列具有70%或更高同一性的氨基酸序列的HSV糖蛋白D(gD)或其免疫原性片段,b)具有与SEQ ID NO:10的氨基酸序列具有70%或更高同一性的氨基酸序列的HSV糖蛋白B(gB)或其免疫原性片段,和c)具有与SEQ ID NO:4的氨基酸序列具有70%或更高同一性或与SEQ ID NO:5的氨基酸序列具有80%或更高同一性的氨基酸序列的HSV糖蛋白E(gE)或其免疫原性片段,或其任何组合。
特别优选的疫苗组合物包含结构蛋白UL48与糖蛋白gD和/或gB一起;结构蛋白UL48和UL49与糖蛋白gE一起,结构蛋白UL11、UL16和UL21与糖蛋白gE、gD和/或gB一起,结构蛋白UL31和UL34与糖蛋白gD和/或gB一起。
本发明的疫苗组合物可任选地包含编码单纯疱疹病毒(HSV)糖蛋白的mRNA,所述mRNA是包含一个或多个假尿苷残基的核苷修饰的mRNA,优选地其中所述一个或多个假尿苷残基包含m1Ψ(1-甲基假尿苷);m1acp3Ψ(1-甲基-3-(3-氨基-5-羧基丙基)假尿苷)、Ψm(2'-0-甲基假尿苷)、m5D(5-甲基二氢尿苷)、m3Ψ(3-甲基假尿苷)或其任何组合。
在所述疫苗组合物的这些具体实施方案中,编码选自以下的所述糖蛋白的免疫原性片段的核苷修饰的mRNA:
(i)HSV gD,包含来自HSV-2株333的氨基酸26-331或来自另一种HSV株的同源序列,优选地其中所述核苷修饰的mRNA的核酸序列如SEQ ID NO:12所示;和/或
(ii)HSV gE,包含来自HSV-2株2.12的氨基酸24-405或来自另一种HSV株的同源序列,优选地其中所述核苷修饰的mRNA的核酸序列如SEQ ID NO:13所示。
此外,本发明疫苗组合物中的mRNA可以编码HSV-1多肽、HSV-2多肽或其混合物。
此外,所述疫苗组合物中的mRNA中的每一种可以进一步包含poly-A尾、m7GpppG帽、3'-0-甲基-m7GpppG帽或抗反向帽类似物、帽非依赖性翻译增强子、和/或增强翻译的5'和3'非翻译区和/或是密码子优化(例如SEQ ID NOs:25-30)。
此外,在本发明的疫苗组合物中,所述mRNA可以被包封在纳米颗粒、脂质、聚合物、胆固醇或细胞穿透肽中,优选在脂质体纳米颗粒中。
本发明的疫苗组合物可用于治疗或预防受试者中的单纯疱疹病毒(HSV)感染。所述HSV感染可以选自HSV-1感染、HSV-2感染、原发性HSV感染、原发性HSV感染后的爆发(flare)、复发或HSV唇(labialis)、潜伏性HSV感染的再激活、HSV脑炎、HSV新生儿感染、生殖器HSV感染或口腔HSV感染。
本发明的疫苗组合物可以配制用于肌内施用、皮下施用、皮内施用、鼻内、阴道内、直肠内施用或局部施用,优选地其中所述组合物是注射用疫苗,任选地包含药学上可接受的注射用载体或佐剂。
本发明的疫苗组合物可用作药物和/或用于治疗。
本发明的疫苗组合物可用于治疗和/或预防单纯疱疹病毒(HSV)感染的方法中。
序列表概述
SEQ ID NO:1是HSV-2的UL11蛋白的示例性氨基酸序列。
SEQ ID NO:2是HSV-2的UL16蛋白的示例性氨基酸序列。
SEQ ID NO:3是HSV-2的UL21蛋白的示例性氨基酸序列。
SEQ ID NO:4是HSV-2的gE蛋白的示例性氨基酸序列。
SEQ ID NO:5是HSV-2的gE蛋白的胞质尾的示例性氨基酸序列。
SEQ ID NO:6是HSV-2的UL48蛋白的示例性氨基酸序列。
SEQ ID NO:7是HSV-2的UL49蛋白的示例性氨基酸序列。
SEQ ID NO:8是HSV-2的UL31蛋白的示例性氨基酸序列。
SEQ ID NO:9是HSV-2的UL34蛋白的示例性氨基酸序列。
SEQ ID NO:10是HSV-2的gB蛋白的示例性氨基酸序列。
SEQ ID NO:11是HSV-2的gD蛋白的示例性氨基酸序列。
SEQ ID NO:12是核苷修饰的HSV-2的示例性gD RNA核苷酸序列片段(所有尿苷残基都是1-甲基-假尿苷)。
SEQ ID NO:13是核苷修饰的HSV-2的示例性gE RNA核苷酸序列片段(所有尿苷残基都是1-甲基-假尿苷)。
SEQ ID NO:14是HSV-2的UL48的示例性RNA序列。
SEQ ID NO:15是HSV-2的UL49的示例性RNA序列。
SEQ ID NO:16是HSV-2的UL11的示例性RNA序列。
SEQ ID NO:17是HSV-2的UL16的示例性RNA序列。
SEQ ID NO:18是HSV-2的UL21的示例性RNA序列。
SEQ ID NO:19是HSV-2的UL31的示例性RNA序列。
SEQ ID NO:20是HSV-2的UL34的示例性RNA序列。
SEQ ID NO:21是HSV-2的gE蛋白的胞质尾的示例性RNA序列。
SEQ ID NO:22是HSV-2的gD的示例性RNA序列。
SEQ ID NO:23是HSV-2的gB的示例性RNA序列。
SEQ ID NO:24是HSV-2的gE的示例性RNA序列。
SEQ ID NO:25是HSV-2的UL48的包括示例性UTR和示例性polyA尾的示例性密码子优化的RNA序列,所有尿苷残基都是1-甲基-假尿苷。
SEQ ID NO:26是HSV-2的UL11的包括示例性UTR和示例性polyA尾的示例性密码子优化的RNA序列,所有尿苷残基都是1-甲基-假尿苷。
SEQ ID NO:27是HSV-2的UL16的包括示例性UTR和示例性polyA尾的示例性修饰(1-甲基-假尿苷)的密码子优化的RNA序列,所有尿苷残基都是1-甲基-假尿苷。
SEQ ID NO:28是HSV-2的UL21的包括示例性UTR和示例性polyA尾的示例性修饰(1-甲基-假尿苷)的密码子优化的RNA序列,所有尿苷残基都是1-甲基-假尿苷。
SEQ ID NO:29是HSV-2的gD的包括示例性UTR和示例性polyA尾的示例性修饰(1-甲基-假尿苷)的密码子优化的RNA序列,所有尿苷残基都是1-甲基-假尿苷。
SEQ ID NO:30是HSV-2的ICP4的包括示例性UTR和示例性polyA尾的示例性修饰(1-甲基-假尿苷)的密码子优化的RNA序列,所有尿苷残基都是1-甲基-假尿苷。
SEQ ID NO:31是HSV-2的ICP4蛋白的示例性氨基酸序列(GenBank登录号QIH12398.1)。
附图说明
图1示出了具有以下蛋白相应SEQ ID NOs的示例性氨基酸序列:HSV-2的UL11蛋白,HSV-2的UL16蛋白,HSV-2的UL21蛋白,HSV-2的gE蛋白,HSV-2的gE蛋白的胞质尾,HSV-2的UL48蛋白,HSV-2的UL49蛋白,HSV-2的UL31蛋白,HSV-2的UL34蛋白,HSV-2的gB蛋白,HSV-2的gD蛋白。
图2示出了具有以下核苷酸相应SEQ ID NOs的示例性核苷酸序列:核苷修饰的HSV-2的gD RNA核苷酸序列片段,核苷修饰的HSV-2的gE RNA核苷酸序列片段,HSV-2RNA序列的UL48,HSV-2RNA序列的UL49,HSV-2RNA序列的UL11,HSV-2RNA序列的UL16,HSV-2RNA序列的UL21,HSV-2RNA序列的UL31,HSV-2RNA序列的UL34,HSV-2RNA序列的gE蛋白的胞质尾,HSV-2RNA序列的gD,HSV-2RNA序列的gB,HSV-2RNA序列的gE,包括UTR和polyA的HSV-2RNA序列的密码子优化的UL48,包括UTR和polyA的HSV-2RNA序列的密码子优化的UL11,包括UTR和polyA的HSV-2RNA序列的密码子优化的UL16,包括UTR和polyA的HSV-2RNA序列的密码子优化的UL21,包括UTR和poly A的HSV-2RNA序列的密码子优化的gD,包括UTR和poly A的HSV-2RNA序列的密码子优化的ICP4。
图3示出了源自mRNA UL48(SEQ ID NO:25)的示例性蛋白质的Western Blot分析。Western印迹布局(L-R):梯子,阳性对照,阴性对照(第2和6天收集),来自用UL48假尿苷(1-甲基-假尿苷)mRNA转染的细胞的样品(第1、2和6天收集),来自用UL48尿苷mRNA转染的细胞的样品(第1、2和6天收集)。
图4示出了源自mRNA UL11、UL16和UL21(SEQ ID NOs:26、27和28)的示例性蛋白质的Western Blot分析。Western印迹布局(L-R):阳性对照,阴性对照(第2和6天收集),来自用UL11、UL16和UL21假尿苷(1-甲基-假尿苷)mRNA转染的细胞的样品(第1和2天收集)。
图5示出了在与示例性UL48修饰的mRNA(SEQ ID NO:25)孵育后使用ELISA测量IFNγ释放。来自IFNγELISA的OD450读数。图的布局(顶部-底部):来自无样品ELISA的空白读数,阴性对照样品(未转染的PBMC),来自用UL48假尿苷(1-甲基-假尿苷)mRNA转染的PBMC的样品(第3天收集)。误差条表示标准偏差。
图6示出了在与示例性ICP4和gD修饰的mRNA(SEQ ID NOs:30和29)孵育后使用ELISA测量IFNγ释放。来自IFNγELISA的OD450读数。图的布局(顶部-底部):来自无样品ELISA的空白读数,阴性对照样品(未转染的PBMC),空转染(无mRNA转染的PBMC),来自用ICP4假尿苷mRNA转染的PBMC的样品(第3天收集),来自用gD假尿苷(1-甲基-假尿苷)mRNA转染的PBMC的样品(第3天收集)。误差条表示标准偏差。
发明详述
如上所述,本发明提供了新的疫苗组合物,所述疫苗组合物包含一种或多种mRNA,其中所述mRNA中的每一种编码选自UL48;UL48和UL49;UL11、UL16和UL21;或UL31和UL34的单纯疱疹病毒(HSV)结构蛋白或其免疫原性片段。尽管研究集中于使用糖蛋白诸如gE、gC和gD作为抗原(参见US2020/0276300,例如其中的SEQ ID NOs:4和16,对应于本文的SEQ IDNO:12和13),但本发明人惊奇地发现对编码结构HSV蛋白的mRNA的免疫反应是相当地强的。此外,由于结构蛋白通常不糖基化,因此不必修饰所用mRNA中的核苷。
术语“mRNA”是指信使核糖核酸。通常,这种mRNA编码多肽并在靶细胞中翻译成其编码的蛋白质。为了增强这种翻译,所述mRNA可以进一步包含poly-A尾、m7GpppG帽、3'-0-甲基-m7GpppG帽或抗反向帽类似物、帽非依赖性翻译增强子和/或增强翻译的5'和3'非翻译区(例如,如本文中SEQ ID NOs:25-30所示)。
“多肽”是指包含通过肽键连接在一起的氨基酸聚合物的分子。所述术语在本文中不意指分子的特定长度,因此在本文中与术语“蛋白质”可互换使用。当在本文中使用时,术语“多肽”或“蛋白质”还包括由表达盒或载体表达的或者可以从本发明的宿主细胞分离的“目的多肽”或“目的蛋白质”。多肽包含氨基酸序列,因此有时包含氨基酸序列的多肽在本文中称为“包含多肽序列的多肽”。因此,本文中术语“多肽序列”与术语“氨基酸序列”可互换使用。
术语“氨基酸”或“aa”是指天然存在的和合成的氨基酸,以及以与天然存在的氨基酸相似的方式起作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸,以及后来被修饰的那些氨基酸,例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指具有与天然存在的氨基酸相同的基本化学结构的化合物,即与氢、羧基、氨基和R基团结合的碳,例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。这些类似物具有修饰的R基团(例如正亮氨酸)或修饰的肽骨架,但保留了与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指具有与氨基酸的一般化学结构不同的结构、但以与天然存在的氨基酸相似的方式起作用的化合物。
术语“单纯疱疹病毒”和“HSV”在本文中可互换使用,并且通常是指单纯疱疹病毒属的病毒,即,猿(Ateline)疱疹病毒1、牛疱疹病毒2、猴(Cercopithecine)疱疹病毒1、猴疱疹病毒2、猴疱疹病毒16、人疱疹病毒1、人疱疹病毒2、袋鼠(Macropodid)疱疹病毒1、袋鼠疱疹病毒2、猿猴(Saimiriine)疱疹病毒1。优选的单纯疱疹病毒属的病毒种是感染人的病毒。甚至更优选的病毒种是单纯疱疹病毒1(HSV-1)和单纯疱疹病毒2(HSV-2),它们也分别称为人疱疹病毒1和2(HHV-1和HHV-2)。
如本文中所用的术语“疫苗组合物”涉及包含本发明mRNA的组合物,所述组合物可以用于预防或治疗受试者中与HSV相关的病理状况。“疫苗组合物”可以包括或不包括一种或多种增强活性成分免疫活性的其它成分,或诸如缓冲液、还原剂、稳定剂、螯合剂、填充剂、渗透平衡剂(张度剂);表面活性剂、多元醇、抗氧化剂;冻干保护剂;消泡剂;防腐剂;和着色剂、去污剂、钠盐和/或抗微生物剂等。所述疫苗组合物可另外包含药物组合物典型的其它组分。本发明的疫苗优选地用于人和/或兽医用途。所述疫苗组合物可以是无菌的和/或无热原的。所述疫苗组合物可以是对人等渗的。
所述疫苗组合物优选地包含治疗有效量的本发明的mRNA。
本发明疫苗组合物的编码HSV多肽UL48的mRNA优选地编码与SEQ ID NO:6的氨基酸序列具有80%或更高同一性的氨基酸序列,其中所述HSV UL48 mRNA在以疫苗组合物的形式施用于受试者时能够引发免疫应答。优选地,所述mRNA与SEQ ID NO:14或其至少200个核苷酸长的片段具有至少80%同一性。
术语“UL48”在本文中使用时涉及HSV的包膜(tegument)蛋白VP16。SEQ ID NO:6示例性地描述了HSV-2UL48的氨基酸序列,其也以登录号AHG54712.1保藏于NCBI GenBank。然而,术语“UL48”还涵盖具有与SEQ ID NO:6所示氨基酸序列共有一定程度同一性的氨基酸序列的UL48多肽,并且还涵盖相对于如本文中所述SEQ ID NO:6所示参考序列具有突变的多肽。相应地,术语“UL48”涵盖与SEQ ID NO:6的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、79%、78%、77%、76%、75%或优选80%或更高的氨基酸序列同一性的多肽,或与SEQID NO:6的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123或优选98个氨基酸的取代、插入和/或缺失的多肽。从本发明mRNA翻译的优选的UL48蛋白可与UL49形成二聚体,或可与UL49和gE或gE的胞质尾形成三聚体。
本发明疫苗组合物的编码HSV多肽UL49的mRNA优选地编码与SEQ ID NO:7的氨基酸序列具有62%或更高同一性的氨基酸序列,其中所述编码HSV多肽UL49的mRNA在以疫苗组合物的形式施用于受试者时能够引发免疫应答。优选地,所述mRNA与SEQ ID NO:15或其至少200个核苷酸长的片段具有至少80%同一性。
术语“UL49”在本文中使用时涉及HSV的包膜蛋白VP22。SEQ ID NO:7示例性地描述了HSV-2UL49的氨基酸序列,其也以登录号AKC42813.1保藏于NCBI GenBank。然而,术语“UL49”还涵盖具有与SEQ ID NO:7所示氨基酸序列共有一定程度同一性的氨基酸序列的UL49多肽,并且还涵盖相对于如本文中所述SEQ ID NO:7所示参考序列具有突变的多肽。相应地,术语“UL49”涵盖与SEQ ID NO:2的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、80%、79%、78%、77%、76%、74%、73%、72%、71%、69%、68%、67%、66%、65%、64%、63%、61%、60%、59%、58%、57%或优选62%或更高的氨基酸序列同一性的多肽,或与SEQ IDNO:7的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130或优选115个氨基酸的取代、插入和/或缺失的多肽。从本发明mRNA翻译的优选的UL49蛋白可与UL48和/或gE或gE的胞质尾形成复合物。相应地,优选的UL49蛋白可与UL48或gE或gE的胞质尾形成二聚体,或可与UL48和gE或gE的胞质尾形成三聚体。
在本发明进一步优选的实施方案中,编码包含HSV多肽UL48、UL49的多聚体复合物的蛋白质的mRNA包含在本发明的疫苗组合物中。这些也可以编码包含HSV多肽gE的胞质结构域的三聚体。在这种情况下,从本发明的mRNA翻译的多聚体复合物包含HSV多肽UL48、UL49和gE的胞质结构域。
“序列同一性”或“%同一性”是指使用标准化算法比对的至少两个多肽序列之间的残基匹配百分比。这种算法可以以标准化和可再现的方式在被比较的序列中插入缺口,以便优化两个序列之间的比对,并因此实现两个序列的更有意义的比较。为了本发明的目的,使用NCBI BLAST程序2.3.0版本(Jan-13-2016)(Altschul等人,Nucleic Acids Res.(1997)25:3389-3402)确定两个氨基酸序列之间的序列同一性。两个氨基酸序列的序列同一性可以用以以下参数设置的blastp来确定:矩阵:BLOSUM62;字长:3;期望值:10;缺口成本:存在=11;扩展=1;组成调整:条件组合分数矩阵调整。
术语“免疫应答”是指优选但不仅在体内诱导体液和/或细胞介导的免疫应答的能力。体液免疫应答包括B细胞介导的抗体应答。细胞介导的免疫包括T细胞介导的免疫应答,包括但不限于CD4+T细胞和CD8+T细胞。抗原引发免疫应答的能力被称为免疫原性,其可以是体液和/或细胞介导的免疫应答。本发明的免疫应答优选地是针对HSV的免疫应答,甚至更优选地是在受试者中针对HSV感染的免疫应答。
体内诱导体液和/或细胞介导的免疫应答的能力可以使用生殖器HSV-2感染的豚鼠模型来确定,该模型准确地反映人的疾病,并代表检查候选抗病毒化合物和疫苗的发病机理和治疗效力的系统。它也是解决生殖器固有和神经组织固有免疫记忆的性质的理想系统。豚鼠的生殖器感染导致自限性外阴阴道炎,其神经病学表现反映了在人疾病中发现的那些。雌性豚鼠的原发性疾病涉及生殖器上皮细胞中的病毒复制,其通常限于八天。在此期间,病毒到达感觉神经末梢,并通过逆向转运被转运至感觉神经节中的细胞体和脊髓中的自主神经元。在该位点短期急性复制后,免疫系统通常在接种后第15天时消除急性病毒复制,病毒维持终生、潜伏的感觉神经元感染。在从原发性HSV-2生殖器感染恢复后,豚鼠经历发作性自发性复发性感染和疾病。HSV-2复发可能表现为会阴红斑和/或水疱损伤的临床明显疾病,或表现为以从生殖道脱落病毒为特征的无症状复发。疫苗功效可以例如使用豚鼠生殖器感染模型来评估。动物可以用5x101 PFU、5x102 PFU、5x103 PFU、5x104 PFU、5x106PFU、5x107 PFU、5x108 PFU或5x109 PFU并且优选5x105 PFU的HSV-2(例如MS株)阴道内感染。动物可在感染前或感染后免疫一次、两次、三次、四次、五次或更多次。优选地,在感染后第15天,以15天的间隔免疫动物两次。通常,任何合适的施用途径都可用于免疫。然而,动物优选地进行肌内免疫。可能的对照组或者用仅佐剂(例如CpG 100μg/Alum 150μg)或者用PBS(均为阴性对照)或者用HSV-2dI5-29突变病毒株(阳性对照)模拟免疫。用与佐剂组合的候选疫苗免疫的组在每轮免疫中可接受0.1μg、0.5μg、1μg、2μg、3μg、4μg、5μg、10μg、15μg、25μg、30μg、35μg、40μg、50μg、60μg、70μg、80μg、90μg、100μg、150μg、200μg和优选20μg剂量的各自mRNA。作为读出,可以收集阴道拭子用于评估病毒反复脱落的频率和程度,例如从感染后第0天至第200天,感染后第1天至第180天,感染后第3天至第160天,感染后第5天至第140天,感染后第7天至第120天,感染后第10天至第100天,感染后第12天至第90天。可以每1、2、3、4、5、6、7、8、9或10天收集阴道拭子。优选地,每天收集阴道拭子,从感染后第15天至第85天。在相同的时间间隔内,每天对复发性生殖器疱疹性病变的严重性(评分0至4)和持续时间进行评分。优选地,在研究结束时,测定抗体应答以及CD4+和CD8+T细胞应答。
多种途径可用于本发明疫苗组合物的施用,包括但不限于口服、局部、经皮、皮下、静脉内、腹膜内、肌内或眼内。然而,如果需要,本领域技术人员可以容易地选择任何其它途径。
施用给受试者的本发明疫苗组合物的确切剂量可取决于治疗目的(例如治疗急性疾病vs.预防性接种)、施用途径、年龄、体重、一般健康状况、性别、饮食、施用时间、药物相互作用和病症的严重程度,并且可由本领域技术人员通过常规实验确定。施用剂量优选为有效剂量,即有效引发免疫应答。在一个优选的实施方案中,所述疫苗组合物以50-150μg、优选100μg的两个剂量施用,每次间隔14-42天,优选间隔28天。
本发明的疫苗组合物可一次或多次施用给受试者,诸如2、3、4、5、6、7、8、9、10次或更多次。
如本文中所用的“受试者”涉及动物,优选哺乳动物,其可以是例如小鼠、大鼠、豚鼠、仓鼠、兔、狗、猫或灵长类动物。优选地,所述受试者是人。然而,术语“受试者”还包括细胞,优选哺乳动物细胞,甚至更优选人细胞。这样的细胞可以是免疫细胞,优选淋巴细胞。
本发明疫苗组合物的编码HSV多肽UL11的mRNA优选地编码与SEQ ID NO:1的氨基酸序列具有75%或更高同一性的氨基酸序列,其中所述HSV UL11 mRNA在以疫苗组合物的形式施用于受试者时能够引发免疫应答。优选地,所述mRNA与SEQ ID NO:16或其至少200个核苷酸长的片段具有至少80%同一性。
术语“UL11”在本文中使用时涉及HSV的包膜蛋白。SEQ ID NO:1示例性地描述了HSV-2UL11的氨基酸序列,其也以登录号AHG54674.1保藏于NCBI GenBank。然而,术语“UL11”还涵盖具有与SEQ ID NO:1所示氨基酸序列共有一定程度同一性的氨基酸序列的UL11多肽,并且还涵盖相对于如本文中所述SEQ ID NO:1所示参考序列具有突变的多肽。相应地,术语“UL11”涵盖与SEQ ID NO:1的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、80%、79%、78%、77%、76%、74%、73%、72%、71%、70%或优选75%或更高的氨基酸序列同一性的多肽,或与SEQ ID NO:1的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、25、26、27、28、29或优选24个氨基酸的取代、插入和/或缺失的多肽。从本发明mRNA翻译的优选的UL11蛋白可与UL16、UL21和/或gE或gE的胞质尾形成复合物。相应地,从本发明mRNA翻译的优选的UL11蛋白可与UL16或gE或gE的胞质尾形成二聚体,可与UL16和UL21或与UL16和gE或gE的胞质尾形成三聚体,和/或可与UL16、UL21和gE或gE的胞质尾形成三聚体。
本发明疫苗组合物的编码HSV多肽UL16的mRNA优选地编码与SEQ ID NO:2的氨基酸序列具有75%或更高同一性的氨基酸序列,其中所述编码HSV多肽UL16的mRNA在以疫苗组合物的形式施用于受试者时能够引发免疫应答。优选地,所述mRNA与SEQ ID NO:17或其至少200个核苷酸长的片段具有至少80%同一性。
术语“UL16”在本文中使用时涉及HSV的包膜蛋白。SEQ ID NO:2示例性地描述了HSV-2UL16的氨基酸序列,其也以登录号AHG54679.1保藏于NCBI GenBank。然而,术语“UL16”还涵盖具有与SEQ ID NO:2所示氨基酸序列共有一定程度同一性的氨基酸序列的UL16多肽,并且还涵盖相对于如本文中所述SEQ ID NO:2所示参考序列具有突变的多肽。相应地,术语“UL16”涵盖与SEQ ID NO:2的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、80%、79%、78%、77%、76%、75%、74%、73%、71%、70%、69%、68%、67%或优选72%或更高的氨基酸序列同一性的多肽,或与SEQ ID NO:2的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123或优选104个氨基酸的取代、插入和/或缺失的多肽。从本发明mRNA翻译的优选的UL16蛋白可与UL11、UL21和/或gE或gE的胞质尾形成复合物。相应地,从本发明mRNA翻译的优选的UL16蛋白可与UL21或UL11形成二聚体,可与UL11和UL21形成三聚体,和/或可与UL11、UL21和gE或gE的胞质尾形成四聚体。
本发明疫苗组合物的编码HSV多肽UL21的mRNA优选地编码与SEQ ID NO:3的氨基酸序列具有80%或更高同一性的氨基酸序列,其中所述编码HSV多肽UL21的mRNA在以疫苗组合物的形式施用于受试者时能够引发免疫应答。优选地,所述mRNA与SEQ ID NO:18或其至少200个核苷酸长的片段具有至少80%同一性。
术语“UL21”在本文中使用时涉及HSV的包膜蛋白。SEQ ID NO:3示例性地描述了HSV-2UL21的氨基酸序列,其也以登录号AHG54684.1保藏于NCBI GenBank。然而,术语“UL21”还涵盖具有与SEQ ID NO:3所示氨基酸序列共有一定程度同一性的氨基酸序列的UL21多肽,并且还涵盖相对于如本文中所述SEQ ID NO:3所示参考序列具有突变的多肽。相应地,术语“UL21”涵盖与SEQ ID NO:3的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、79%、78%、77%、76%、75%或优选80%或更高的氨基酸序列同一性的多肽,或与SEQ ID NO:3的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166或优选134个氨基酸的取代、插入和/或缺失的多肽。从本发明mRNA翻译的优选的UL21蛋白可与UL11、UL16和/或gE或gE的胞质尾形成复合物。相应地,优选的UL21蛋白可与UL16形成二聚体,可与UL11和UL16形成三聚体,和/或可与UL11、UL16和gE或gE的胞质尾形成四聚体。
如本文中所述,编码包含HSV多肽UL11、UL16、UL21的多聚体复合物的蛋白质的mRNA可以进一步包含编码HSV糖蛋白gE的mRNA。在这种情况下,从本发明mRNA翻译的多聚体复合物包含HSV多肽UL11、UL16、UL21和gE。
本发明疫苗组合物的mRNA编码的HSV多肽UL31优选地包含与SEQ ID NO:8的氨基酸序列具有85%或更高同一性的氨基酸序列,其中所述编码HSV多肽UL31的mRNA在以疫苗组合物的形式施用于受试者时能够引发免疫应答。优选地,所述mRNA与SEQ ID NO:19或其至少200个核苷酸长的片段具有至少80%同一性。
术语“UL31”在本文中使用时涉及HSV的病毒粒出口蛋白(virion egressprotein)。SEQ ID NO:8示例性地描述了HSV-2UL31的氨基酸序列,其也以登录号AHG54695.1保藏于NCBI GenBank。然而,术语“UL31”还涵盖具有与SEQ ID NO:8所示氨基酸序列共有一定程度同一性的氨基酸序列的UL31多肽,并且还涵盖相对于如本文中所述SEQID NO:8所示参考序列具有突变的多肽。相应地,术语“UL31”涵盖与SEQ ID NO:1的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、84%、83%、82%、81%、80%或优选85%或更高的氨基酸序列同一性的多肽,或与SEQID NO:8的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、56、57、58、59、60、61或优选46个氨基酸的取代、插入和/或缺失的多肽。从本发明mRNA翻译的优选的UL31蛋白可与UL34形成二聚体。
本发明疫苗组合物的mRNA编码的HSV多肽UL34优选地包含与SEQ ID NO:9的氨基酸序列具有70%或更高同一性的氨基酸序列,其中所述编码多肽UL34的HSV mRNA在以疫苗组合物的形式施用于受试者时能够引发免疫应答。优选地,所述mRNA与SEQ ID NO:20或其至少200个核苷酸长的片段具有至少80%同一性。
术语“UL34”在本文中使用时涉及HSV的病毒粒出口蛋白。SEQ ID NO:9示例性地描述了HSV-2UL34的氨基酸序列,其也以登录号AHG54698.1保藏于NCBI GenBank。然而,术语“UL34”还涵盖具有与SEQ ID NO:9所示氨基酸序列共有一定程度同一性的氨基酸序列的UL34多肽,并且还涵盖相对于如本文中所述SEQ ID NO:9所示参考序列具有突变的多肽。相应地,术语“UL34”涵盖与SEQ ID NO:2的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、80%、79%、78%、77%、76%、75%74%、73%、72%、71%、69%、68%、67%、66%、65%或优选70%或更高的氨基酸序列同一性的多肽,或与SEQ ID NO:9的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、76、77、78、79、80、81、82、83、84、85、86、87、88或优选75个氨基酸的取代、插入和/或缺失的多肽。从本发明mRNA翻译的优选的UL34蛋白可与UL31形成二聚体。
如所述,本发明的每种mRNA可编码含有突变的蛋白质,所述突变诸如相对于SEQID NO:1(UL11)、SEQ ID NO:2(UL16)、SEQ ID NO:3(UL21)、SEQ ID NO:4(gE)、SEQ ID NO:5(gE的胞质结构域)、SEQ ID NO:6(UL48)、SEQ ID NO:7(UL49)、SEQ ID NO:8(UL31)和SEQID NO:9(UL34)所示参考序列的插入、缺失和取代。
在本发明进一步优选的实施方案中,包含编码上文所述结构HSV多肽的mRNA的疫苗组合物也可以编码一种或几种HSV糖蛋白。优选的糖蛋白是gE、gB和gD。
本发明疫苗组合物的编码HSV糖蛋白gE的mRNA优选地编码与SEQ ID NO:4的氨基酸序列具有70%或更高同一性的氨基酸或其免疫原性片段。优选地,所述mRNA与SEQ IDNO:24或其至少200个核苷酸长的片段具有至少80%同一性。
术语“ICP4”在本文中使用时涉及HSV的主要病毒转录因子4,例如以登录号QIH12398.1(2020年3月8日版)保藏于NCBI GenBank且具有本文中的SEQ ID NO:31。然而,术语“ICP4”还涵盖具有与SEQ ID NO:31所示氨基酸序列共有一定程度同一性的氨基酸序列的ICP4多肽,并且还涵盖相对于如本文中所述SEQ ID NO:31所示参考序列具有突变的多肽。相应地,术语“ICP4”涵盖与SEQ ID NO:31的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、80%、79%、78%、77%、76%、75%、74%、73%、72%、71%、69%、68%、67%、66%、65%或优选70%或更高的氨基酸序列同一性的多肽,或与SEQ ID NO:31的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122 123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180或优选165个氨基酸的取代、插入和/或缺失的多肽。优选的ICP4蛋白是从ICP4mRNA(例如SEQ ID NO:30)翻译的。编码ICP4的mRNA可以是SEQ ID NO:30。优选地,ICP4mRNA与SEQ ID NO:30或其至少200个核苷酸长的片段具有至少80%同一性。在一些方面,本发明的疫苗组合物包含至少一种编码单纯疱疹病毒(HSV)糖蛋白的mRNA,所述HSV糖蛋白选自a)具有与SEQ ID NO:11的氨基酸序列具有70%或更高同一性的氨基酸序列的HSV糖蛋白D(gD)或其免疫原性片段,b)具有与SEQ ID NO:10的氨基酸序列具有70%或更高同一性的氨基酸序列的HSV糖蛋白B(gB)或其免疫原性片段,和c)具有与SEQ ID NO:4的氨基酸序列具有70%或更高同一性或与SEQ ID NO:5的氨基酸序列具有80%或更高同一性的氨基酸序列的HSV糖蛋白E(gE)或其免疫原性片段,或其任何组合;任选地,d)具有与SEQ ID NO:31的氨基酸序列具有70%或更高同一性的氨基酸序列的HSV ICP4或其免疫原性片段,或其任意组合。在一些进一步的方面,本发明的疫苗组合物包含:(i)UL48和gD和/或gB,任选的ICP4(例如,如上);(ii)UL48和UL49与gE;(iii)UL11、UL16和UL21与gE、gD和/或gB;或(iv)UL31和UL34与gD和/或gB。
术语“gE”在本文中使用时可有时称为“糖蛋白E”。SEQ ID NO:4示例性地描述了HSV-2gE的氨基酸序列,其也以登录号AHG54732.1保藏于NCBI GenBank。然而,术语“gE”还涵盖具有与SEQ ID NO:4所示氨基酸序列共有一定程度同一性的氨基酸序列的gE多肽,并且还涵盖相对于如本文中所述SEQ ID NO:4所示参考序列具有突变的多肽。相应地,术语“gE”涵盖与SEQ ID NO:4的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、80%、79%、78%、77%、76%、75%、74%、73%、72%、71%、69%、68%、67%、66%、65%或优选70%或更高的氨基酸序列同一性的多肽,或与SEQ ID NO:4的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122 123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180或优选165个氨基酸的取代、插入和/或缺失的多肽。从本发明mRNA翻译的优选的gE蛋白可与UL48形成二聚体,可与UL31和UL34形成三聚体,和可与UL11、UL16和UL21形成四聚体。
所述编码gE的mRNA也可以由HSV多肽gE的胞质结构域组成。优选地,所述gE mRNA与SEQ ID NO:21或其至少200个核苷酸长的片段具有至少80%同一性。优选地,所述mRNA与SEQ ID NO:21或其至少200个核苷酸长的片段具有至少80%同一性。
本发明疫苗组合物的mRNA编码的gE的胞质结构域优选地包含如SEQ ID NO:5所示的氨基酸序列。然而,本文中还设想gE的胞质结构域包含与SEQ ID NO:5的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、79%、78%、77%、76%、75%或优选80%或更高的序列同一性的氨基酸序列,或与SEQ ID NO:5的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、24、25、26、27或优选23个氨基酸的取代、插入和/或缺失的多肽。从本发明mRNA翻译的优选的gE的胞质结构域可与UL48形成二聚体,可与UL31和UL34形成三聚体,和可与UL11、UL16和UL21形成四聚体。
本发明疫苗组合物的编码HSV糖蛋白gE的mRNA优选地编码与SEQ ID NO:11的氨基酸序列具有70%或更高同一性的氨基酸或其免疫原性片段。优选地,所述mRNA与SEQ IDNO:22或其至少200个核苷酸长的片段具有至少80%同一性。
术语“gD”在本文中使用时可有时称为“糖蛋白D”。SEQ ID NO:11示例性地描述了HSV-2gD的氨基酸序列。然而,术语“gD”还涵盖具有与SEQ ID NO:11所示氨基酸序列共有一定程度同一性的氨基酸序列的gD多肽,并且还涵盖相对于如本文中所述SEQ ID NO:11所示参考序列具有突变的多肽。相应地,术语“gD”涵盖与SEQ ID NO:11的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、80%、79%、78%、77%、76%、75%、74%、73%、72%、71%、69%、68%、67%、66%、65%或优选70%或更高的氨基酸序列同一性的多肽,或与SEQ ID NO:11的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122 123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180或优选165个氨基酸的取代、插入和/或缺失的多肽。从所述疫苗组合物的mRNA翻译的优选的gD蛋白可与从所述疫苗组合物的mRNA翻译的UL11、UL16和UL21蛋白形成复合物。所得优选的gD蛋白可与UL48形成二聚体,可与UL31和UL34形成三聚体,和可与UL11、UL16和UL21形成四聚体。
本发明疫苗组合物的编码HSV糖蛋白gB的mRNA优选地编码与SEQ ID NO:10的氨基酸序列具有70%或更高同一性的氨基酸或其免疫原性片段。优选地,所述mRNA与SEQ IDNO:23或其至少200个核苷酸长的片段具有至少80%同一性。
术语“gB”在本文中使用时可有时称为“糖蛋白B”。SEQ ID NO:10示例性地描述了HSV-2gB的氨基酸序列。然而,术语“gB”还涵盖具有与SEQ ID NO:10所示氨基酸序列共有一定程度同一性的氨基酸序列的gB多肽,并且还涵盖相对于如本文中所述SEQ ID NO:10所示参考序列具有突变的多肽。相应地,术语“gB”涵盖与SEQ ID NO:10的氨基酸序列相比具有99%、98%、97%、96%、95%、94%、93%、92%、91%、90%、89%、88%、87%、86%、85%、84%、83%、82%、81%、80%、79%、78%、77%、76%、75%、74%、73%、72%、71%、69%、68%、67%、66%、65%或优选70%或更高的氨基酸序列同一性的多肽,或与SEQ ID NO:10的氨基酸序列相比具有至多1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122 123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180或优选165个氨基酸的取代、插入和/或缺失的多肽。从所述疫苗组合物的mRNA翻译的优选的gB蛋白可与从所述疫苗组合物的mRNA翻译的UL11、UL16和UL21形成复合物。所得优选的gB蛋白可与UL48形成二聚体,可与UL31和UL34形成三聚体,和可与UL11、UL16和UL21形成四聚体。
还优选的是编码gE、gB或gD或其免疫原性片段的核苷修饰的mRNA。优选的片段描述于US2020/0276300,并且涵盖假尿苷残基,优选m1Ψ(1-甲基假尿苷);m1acp3Ψ(1-甲基-3-(3-氨基-5-羧基丙基)假尿苷、Ψm(2'-0-甲基假尿苷)、m5D(5-甲基二氢尿苷)、m3Ψ(3-甲基假尿苷)或其任何组合。具体地,SEQ ID NO:12和13的mRNA分别是这样的核苷修饰的gD和gE mRNA。假尿苷修饰的序列的其它实例示于SEQ ID NOs:25-30。
如所述,本发明的每种mRNA可编码含有突变的蛋白质,所述突变诸如相对于SEQID NO:1(UL11)、SEQ ID NO:2(UL16)、SEQ ID NO:3(UL21)、SEQ ID NO:6(UL48)、SEQ IDNO:7(UL49)、SEQ ID NO:8(UL31)、SEQ ID NO:9(UL34)、SEQ ID NO:4(gE)、SEQ ID NO:5(gE的胞质结构域)、SEQ ID NO:10(gB)和SEQ ID NO:11(gD)所示参考序列的插入、缺失和取代。
在一方面,本发明的mRNA编码UL48蛋白,单独或与编码选自gD或gB的糖蛋白的mRNA组合。
在本发明进一步优选的实施方案中,疫苗组合物中的mRNA编码在翻译后形成多聚体复合物的两个或三个结构多肽。另外,所述疫苗组合物中可包含一种或多种编码糖蛋白gE、gB和/或gD或其免疫原性片段的mRNA。
具体地,本发明的疫苗组合物可包含编码UL48和UL49的mRNA,当其翻译时可形成复合物。或者,本发明的疫苗组合物可包含编码UL48、UL49和糖蛋白gE的mRNA,当所有这些翻译时可形成复合物。
在另一个实施方案中,本发明的疫苗组合物可包含编码UL11、UL16和UL21的mRNA,当其翻译时可形成复合物。包含编码UL11、UL16和UL21的mRNA的本发明疫苗组合物可进一步包含一种或多种编码糖蛋白gE、gB或gD的mRNA。
或者,本发明的疫苗组合物可包含编码UL31和UL34的mRNA,当其翻译时可形成复合物。包含编码UL31和UL34的mRNA的本发明疫苗组合物可进一步包含一种或多种编码糖蛋白gB或gD的mRNA。
所述疫苗组合物中的mRNA可编码HSV-1多肽、HSV-2多肽或其混合物。
本发明的疫苗组合物可进一步包含药学上可接受的载体或佐剂。
术语“载体”和“赋形剂”在本文中可互换使用。药学上可接受的载体包括但不限于稀释剂(填充剂(filler)、增量剂(bulking agent),例如乳糖、微晶纤维素)、崩解剂(例如淀粉羟乙酸钠、交联羧甲基纤维素钠)、粘合剂(例如PVP、HPMC)、润滑剂(例如硬脂酸镁)、助流剂(例如胶体SiO2)、溶剂/助溶剂(例如水性媒介物、丙二醇、甘油)、缓冲剂(例如柠檬酸盐、葡糖酸盐、乳酸盐)、防腐剂(例如苯甲酸钠、对羟基苯甲酸酯类(Me、Pr和Bu)、BKC)、抗氧化剂(例如BHT、BHA、抗坏血酸)、润湿剂(例如聚山梨醇酯、脱水山梨醇酯)、消泡剂(例如西甲硅油)、增稠剂(例如甲基纤维素或羟乙基纤维素)、甜味剂(例如山梨糖醇、糖精、阿斯巴甜、乙酰磺胺)、调味剂(例如薄荷、柠檬油、奶油硬糖(butterscotch)等)、湿润剂(例如丙二醇、乙二醇、甘油、山梨糖醇)。另外的药学上可接受的载体是(生物可降解的)脂质体;由生物可降解聚合物聚(D,L)-乳酸-共乙醇酸(PLGA)制成的微球,白蛋白微球;合成聚合物(可溶的);纳米纤维,蛋白质-DNA复合物;蛋白质缀合物;红细胞;或病毒体。各种基于载体的剂型包括固体脂质纳米颗粒(SLN)、聚合物纳米颗粒、陶瓷纳米颗粒、水凝胶纳米颗粒、共聚肽纳米颗粒、纳米晶体和纳米悬浮液、纳米晶体、纳米管和纳米线、功能化纳米载体、纳米球、纳米胶囊、脂质体、脂质乳剂、脂质微管/微圆柱、脂质微泡、脂球、脂多丛、反相脂质胶束、树状聚合物、醇质体(ethosome)、多组分超薄胶囊、水质体(aquasome)、药质体(pharmacosome)、胶体(colloidosome)、囊泡(niosome)、盘状体(discome)、原核生物质体(proniosome)、微球、微乳液和聚合物胶束。其它合适的药学上可接受的赋形剂尤其描述于Remington's Pharmaceutical Sciences,15th Ed.,Mack Publishing Co.,New Jersey(1991)和Bauer等人,Pharmazeutische Technologie,5th Ed.,Govi-Verlag Frankfurt(1997)。本领域技术人员将能够容易地根据例如药物组合物的制剂和给药途径选择合适的药学上可接受的载体。
如本文中所用的术语“佐剂”是指增强、增加或加强宿主对抗原或其片段的(抗体和/或细胞介导的)免疫应答的物质。根据本发明使用的示例性佐剂包括无机化合物,诸如明矾、氢氧化铝、磷酸铝、碱式磷酸钙、TLR9激动剂CpG寡脱氧核苷酸、TLR4激动剂单磷酰脂质(MPL)、TLR4激动剂吡喃葡萄糖脂质(GLA)、油包水乳液Montanide ISA 51和720、矿物油诸如石蜡油、病毒体、细菌产物诸如灭活的百日咳博德特氏菌(Bordetella pertussis)、牛分枝杆菌、类毒素、非细菌有机物诸如角鲨烯、硫柳汞、去污剂(Quil A)、细胞因子诸如IL-1、IL-2、IL-10和IL-12、和复合组合物诸如弗氏完全佐剂和弗氏不完全佐剂。通常,根据本发明使用的佐剂优选地强化对本发明的多聚体复合物的免疫应答和/或将其调节至期望的免疫应答。
术语“药学上可接受的”意指不干扰根据本发明的多聚体复合物的生物学活性的有效性的无毒材料。
疫苗组合物的用途
本发明还涉及所述疫苗组合物在受试者中诱导针对HSV的免疫应答的方法中的用途。
在本发明的优选实施方案中,所述疫苗组合物用于治疗、预防或改善HSV感染或预防HSV再激活。HSV感染可以选自HSV-1感染、HSV-2感染、原发性HSV感染、原发性HSV感染后的爆发(flare)、复发或HSV唇(labialis)、潜伏性HSV感染的再激活、HSV脑炎、HSV新生儿感染、生殖器HSV感染和口腔HSV感染。
因此,所述疫苗组合物可用于对抗由HSV引起的疾病和/或相关症状。还设想本发明的疫苗组合物可用于清除受试者中的病毒,即治疗后,使用本领域普通技术人员已知的合适方法例如PCR、ELISA等,在从受试者获得的合适样品中无法检测到HSV。因此,本发明的疫苗组合物可用于阻断原发性感染、阻止原发性疾病、阻断病毒再激活和再感染,以及阻断潜伏。
为了减少生殖器疱疹的机会,需要预防性疫苗来预防母亲的第一次HSV感染,而在母亲诊断为患有活动性HSV感染的情况下需要有效的治疗。本发明的多聚体复合物可以用作预防性疫苗,例如用于待产妇或儿童,或用作血清阳性女性的治疗性疫苗,以在分娩时防止亚临床再激活。
在本发明进一步优选的实施方案中,所述疫苗组合物在用于在受试者中诱导针对HSV-1或HSV-2的免疫应答的方法中使用。
术语“多核苷酸”、“核苷酸序列”或“核酸分子”在本文中可互换使用,是指通常从一个脱氧核糖或核糖连接到另一个的核苷酸聚合体形式。术语“多核苷酸”优选地包括单链和双链形式的DNA或RNA。本发明的核酸分子可以包括RNA的有义和反义链(含有核糖核苷酸)两者、cDNA、基因组DNA和上述的合成形式和混合聚合物。它们可以被化学或生物化学修饰,或者可以含有非天然或衍生的核苷酸碱基,这是本领域技术人员容易理解的。这些修饰包括例如标记、甲基化、用类似物取代一个或多个天然存在的核苷酸、核苷酸间修饰诸如不带电荷的键(例如膦酸甲酯、磷酸三酯、氨基磷酸酯、氨基甲酸酯等)、带电荷的键(例如硫代磷酸酯、二硫代磷酸酯等)、悬垂部分(例如多肽)、嵌入剂(例如吖啶、补骨脂素等)、螯合剂、烷化剂和修饰的键(例如α异头核酸等)。还包括在其经由氢键合和其它化学相互作用结合指定序列的能力方面模拟多核苷酸的合成分子。这样的分子是本领域已知的,包括例如其中肽键取代分子骨架中的磷酸酯键的那些。
本发明的疫苗组合物可用于初免-加强方案(prime boost regimen)。在初免-加强方案中,使用由两种或更多种类型的疫苗组成的初免/加强疫苗,包括初次免疫(初免)中使用的疫苗和加强免疫(加强)中使用的疫苗。初次免疫中使用的疫苗和加强免疫中使用的疫苗可以彼此不同。初次免疫和加强免疫可以顺序进行,然而这不是强制性的。初免/加强方案包括但不限于例如mRNA初免/蛋白质加强。然而,加强组合物也可用作初免组合物,而所述初免组合物用作加强组合物。
需要指出的是,如本文中所用,除非上下文另有明确指示,单数形式“一个(a)”、“一种(an)”和“所述/该(the)”包括所提及的复数。因此,例如,提及的“一种表达盒”包括本文中公开的表达盒中的一种或多种,提及的“所述/该方法”包括提及的本领域普通技术人员已知的可修饰或替代本文所述的方法的等效步骤和方法。
本说明书及其后的权利要求书全文中,除非上下文有其它要求,词语“包括/包含(comprise)”以及变形词语诸如“包括/包含(comprises)”和“包括/包含(comprising)”将被理解为意指包括陈述的整数或步骤或整数或步骤的集合,其并不排除任何其它的整数或步骤或整数或步骤的集合。当在本文中使用时,术语“包括/包含(comprising)”能够以术语“含有(containing)”替代或当在本文中使用时用术语“具有(having)”替代。
当在本文中使用时,“由……组成”排除了未在权利要求要素中规定的任何要素、步骤或成分。当在本文中使用时,“本质上由……组成”并不排除实质上不影响权利要求的基本特征和新特征的材料或步骤。本文的每一个实例中,术语“包括/包含”、“本质上由……组成”和“由……组成”中任意一个可被其它两个术语中的任意一个替代。
如本文中所用的术语“约(about)”或“大约(approximately)”的含义是在给定值或范围的20%内,优选地10%内,和更优选地5%内。其还包括具体数目,例如约20包括20。
除非本文中另外定义,否则结合本发明使用的科技术语应当具有本领域普通技术人员通常所理解的含义。另外,除非上下文另外规定,否则单数形式的术语应当包括复数形式并且复数形式的术语应当包括单数形式。通常根据本领域熟知的常规方法实施本发明的方法和技术。总体而言,与本文所描述的生物化学、酶学、分子和细胞生物学、微生物学、遗传学以及蛋白质和核酸化学及杂交学的技术结合使用的命名法是本领域熟知并且常用的那些。
除非另外指示,否则本发明的方法和技术一般是根据本领域熟知的常规方法并且如本说明书全篇所引用和论述的各种通用和更具体参考文献中所描述来实施。参见例如Sambrook等人,Molecular Cloning:ALaboratory Manual,3rd ed.,Cold Spring HarborLaboratory Press,Cold Spring Harbor,N.Y.(2001);Ausubel等人,Current Protocolsin Molecular Biology,J,Greene Publishing Associates(1992,and Supplements to2002);Handbook of Biochemistry:Section A Proteins,Vol I 1976CRC Press;Handbook of Biochemistry:Section A Proteins,Vol II 1976CRC Press。与本文所描述的分子和细胞生物学、蛋白质生物化学、酶学和医药化学结合使用的命名法及其实验程序和技术是本领域熟知并且常用的那些。
实施例
以下假设的实施例说明了本发明,但不应解释为限制本发明的范围。
实施例1:
将来自四个HSV-2感染个体和两个未感染个体的PBMC解冻并静置过夜。将细胞以5×105个细胞/孔接种到平板上,随后用5μg/mL HSV-2UL48mRNA单独或与5μg/mL UL49 mRNA刺激48小时。然后收集上清液,用Luminex仪器分析IFN-γ的分泌。从每个孔中减去背景信号(由缓冲液刺激的细胞产生),结果表示为pg/ml。
实施例2:
将来自HSV-2感染豚鼠和对照豚鼠的脾细胞(1×105个细胞)与10μg/mL HSV-2UL31 mRNA和10μg/mL UL34 mRNA混合。然后将细胞转移到ELISPOT抗干扰素γ(IFN-γ)抗体包被的平板(Multiscreen HTS平板;Millipore)上并孵育20小时。此后根据标准ELISPOT方案将平板显色,并使用自动读数器将IFN-γ分泌细胞定量为斑点。未刺激的细胞和20μg/mL PHA分别用作阴性和阳性对照。
实施例3:
将来自十四个HSV-2感染个体和六个未感染个体的PBMC解冻并静置过夜。将细胞以2×105个细胞/孔铺到ELISPOT抗干扰素γ(IFN-γ)抗体包被的平板上。随后用5μg/mLHSV-2UL31 mRNA和5μg/mL HSV-2UL34 mRNA刺激细胞48小时。此后根据制造商的说明书将平板显色,并使用自动读数器将IFN-γ分泌细胞计数为斑点。从每个孔中减去背景信号(由缓冲液刺激的细胞产生),结果表示为每2×105个PBMC的SFU(斑点形成单位)。
实施例4:
将来自四个HSV-2感染个体和两个未感染个体的PBMC解冻并静置过夜。将细胞以2×105个细胞/孔铺到ELISPOT抗干扰素γ(IFN-γ)抗体包被的平板上。随后细胞用5μg/mLHSV-2UL48 mRNA单独或与5μg/mL UL49mRNA刺激48小时。此后根据制造商的说明书将平板显色,并使用自动读数器将IFN-γ分泌细胞计数为斑点。从每个孔中减去背景信号(由缓冲液刺激的细胞产生),结果表示为每2×105个PBMC的SFU(斑点形成单位)。
实施例5:
将来自十四个HSV-2感染个体和六个未感染个体的PBMC解冻并静置过夜。将细胞以2×105个细胞/孔铺到ELISPOT抗干扰素γ(IFN-γ)抗体包被的平板上。随后细胞用5μg/mL HSV-2UL48 mRNA单独或与5μg/mL UL49mRNA组合刺激48小时。此后根据制造商的说明书将平板显色,并使用自动读数器将IFN-γ分泌细胞计数为斑点。从每个孔中减去背景信号(由缓冲液刺激的细胞产生),结果表示为每2×105个PBMC的SFU(斑点形成单位)。
实施例6:
将来自四个HSV-2感染个体和两个未感染个体的PBMC解冻并静置过夜。将细胞以2×105个细胞/孔铺到ELISPOT抗干扰素γ(IFN-γ)抗体包被的平板上。随后细胞用5μg/mLHSV-2UL11 mRNA、5μg/mL UL16 mRNA和5μg/mL UL21 mRNA或编码UL11、UL16或UL21的相应的mRNA(标准化为该组合中单个蛋白的量)刺激48小时。此后根据制造商的说明书将平板显色,并使用自动读数器将IFN-γ分泌细胞计数为斑点。从每个孔中减去背景信号(由缓冲液刺激的细胞产生),结果表示为每2×105个PBMC的SFU(斑点形成单位)。
实施例7:
将来自十四个HSV-2感染个体和六个未感染个体的PBMC解冻并静置过夜。将细胞以2×105个细胞/孔铺到ELISPOT抗干扰素γ(IFN-γ)抗体包被的平板上。随后细胞用5μg/mL HSV-2UL11 mRNA、5μg/mL UL16 mRNA和5μg/mL UL21 mRNA刺激48小时。此后根据制造商的说明书将平板显色,并使用自动读数器将IFN-γ分泌细胞计数为斑点。从每个孔中减去背景信号(由缓冲液刺激的细胞产生),结果表示为每2×105个PBMC的SFU(斑点形成单位)。
实施例8:
将来自四个HSV-2感染个体和两个未感染个体的PBMC解冻并静置过夜。将细胞以5×105个细胞/孔接种到平板上,随后用5μg/mL HSV-2UL31mRNA和5μg/mL HSV-2UL34 mRNA刺激48小时。然后收集上清液,用Luminex仪器分析IFN-γ的分泌。从每个孔中减去背景信号(由缓冲液刺激的细胞产生),结果表示为pg/ml。
实施例9:
将来自四个HSV-2感染个体和两个未感染个体的PBMC解冻并静置过夜。将细胞以5×105个细胞/孔接种到平板上,随后用5μg/mL HSV-2UL11mRNA、5μg/mL UL16 mRNA和5μg/mL UL21 mRNA刺激48小时。然后收集上清液,用Luminex仪器分析IFN-γ的分泌。从每个孔中减去背景信号(由缓冲液刺激的细胞产生),结果表示为pg/ml。
实施例10:mRNA转染和Western印迹
方法:
将HEK 293T细胞以0.4×106个/ml的浓度接种在含有RPMI培养基和10%FBS的12孔板中,并在37℃和5% CO2下孵育。第二天,使用Invitrogen LipofectamineMessegerMAX Transfection试剂盒转染细胞。每孔加入2-4.5μl1μg/μl mRNA(SEQ ID NO:25和SEQ ID NOs:26、27和28)。空转染孔仅添加转染试剂,阴性对照孔没有添加任何物质。
在随后的几天中收获细胞。为了做到这一点,从孔中除去培养基,并将70μl冷却的Thermo Scientific RIPA裂解和提取缓冲液连同10μl 7x cOmplete(不含EDTA的蛋白酶抑制剂混合物)加入到每个孔中。将平板在4℃下孵育2-3分钟,然后使用细胞刮刀分离细胞。将细胞-缓冲液混合物转移到1.5ml Eppendorf管中并在冰上孵育。将70μl含有50mM DTT的2x Biorad Laemli缓冲液加入到每个管中,并将样品在90℃下煮沸5分钟。对于阳性对照,将重组蛋白样品加入RIPA缓冲液和蛋白酶中,并以与其它样品相同的方式处理。
在将样品加载在凝胶上之前,向每个样品中加入1μl用于细胞裂解的ThermoScientific Pierce Universal Nuclease。然后将20μl的每种样品上样到InvitrogenBolt 4-12% Bis-Tris Plus凝胶上,在90V运行40分钟。此后,使用iBlot 2系统和iBlot 2微型PVDF Transfer Stacks转移样品。所用的设置是:20V 1分钟,23V 4分钟,25V 90秒。
然后在4℃下在含有0.1% Tween-20的5% BSA TBS中将膜封闭过夜。将商业购买或内部生产的第一抗体以1:1000的浓度加入封闭缓冲液中,并在室温下孵育,同时轻轻摇动1小时。然后用含0.1% Tween-20的TBS洗膜3次。之后,将第二抗体以1:5000的浓度加入封闭缓冲液中,并在室温下孵育,同时轻轻摇动1小时。再次用含0.1% Tween-20的BST洗膜3次。
为了进行成像,将300μl SuperSignal West Femto Maximum SensitivitySubstrate施加于膜上,并使用具有100mm F/2.8透镜的全帧照相机和暗箱对其进行成像。
结果:
图3(源自mRNA UL48-SEQ ID NO:25的蛋白质的Western Blot)清楚地显示在mRNA构建体转染后UL48蛋白质的表达。最高表达在转染后1天是明显的,然后随时间下降。这表明由于应用我们的UL48 mRNA构建体(例如SEQ ID NO:25)而产生强有力的蛋白表达。
图4(源自mRNA UL11、UL16和UL21-SEQ ID NOs:26、27和28的蛋白质的WesternBlot)清楚地显示在我们的mRNA构建体共转染后UL11、UL16和UL21的表达。如对于UL48所观察到的,转染后一天的表达最高。
实施例11:使用PBMC的免疫原性数据
方法:
PBMC增殖和IFNγELISA方案
将从HSV-2+供体收集的PBMC解冻,并使其在12孔板中以1×106个细胞/ml的浓度在含有10% FBS的RPMI中生长过夜。第二天,使用Invitrogen LipofectamineMessegerMAX Transfection试剂盒转染细胞。每孔加入1-2μl1μg/μl mRNA(SEQ ID NO:25、SEQ ID NO:2和SEQ ID NO:30)。空转染孔仅添加转染试剂,阴性对照孔没有添加任何物质。
转染后3天收获样品。将上清液以500RCF离心6分钟。然后,使用Invitrogen HumanIFN Gamma Uncoated ELISA试剂盒和F96 Maxisorp Nunc-Immuno板评估IFNγ水平。在Tecan Infinite M Plex平板读数器中进行OD450测量。
结果:
ELISA结果显示在来自HSV2+供体的PBMC中由假尿苷UL48、gD和ICP4 mRNA触发的IFNγ分泌。这些数据表明,特异性免疫应答是由所应用的HSV-2mRNA的表达触发的。阴性对照孔中不加入mRNA或转染试剂。对于空白孔,在ELISA期间不加入生物学样品。
图5显示在与UL48修饰的mRNA孵育后,使用ELISA测量的IFNγ释放,在阴性对照孔中不加入mRNA或转染试剂,对于空白孔,在ELISA期间不加入生物学样品。
图6显示在与ICP4和gD修饰的mRNA孵育后,使用ELISA测量的IFNγ释放,空转染孔仅加入转染试剂,阴性对照孔没有加入任何物质。对于空白孔,在ELISA期间不加入生物学样品。
结论:
进行Western印迹和PBMC实验以评估本发明mRNA构建体的稳定性和功能性。用于疫苗组合物的mRNA的设计是关键的,因此已经进行了评价。关于稳定性,本发明的疫苗mRNA包含优化的5'帽、5'和3'UTR、和polyA尾。Western印迹分析(图3和4)清楚地显示UL48、UL11、UL16和UL21mRNA的强表达,并表明构建体是稳定的,这对于包含所述mRNA的疫苗的效力是根本的。
除了稳定性之外,还已证实免疫应答可以由该疫苗组分触发。在该背景下,使用具有1-甲基-假尿苷的修饰残基优化了疫苗mRNA以降低先天性非特异性免疫应答。IFNγELISA结果确实表明在与功能性UL48、gD和ICP4mRNA孵育后该免疫因子的特异性释放(图5和图6)。
本领域技术人员将容易地理解,本发明非常适于实现所述目的并获得所述结果和优点,以及其中固有的那些。此外,对于本领域技术人员来说显而易见的是,在不背离本发明的范围和精神的情况下,可以对本文中公开的发明进行各种替换和修改。本文中所述的组合物、方法、程序、治疗、分子和具体化合物目前代表了示例性的某些实施方案,并且不旨在作为对本发明范围的限制。本领域技术人员可以想到其中的变化和其它用途,这些变化和用途涵盖在由权利要求的范围限定的本发明的精神内。本说明书中对先前公开的文献的列举或讨论不应必然地被认为是承认该文献是现有技术的一部分或是公知常识。
在没有任何元件或限制的情况下,可以适当地实施本文中示例性描述的本发明,所述元件或限制没有在本文中具体公开。因此,例如,术语“包括”、“包含”、“含有”等应被广义地而非限制性地理解。另外,本文中采用的术语和表达方式已经用作描述性术语而非限制性术语,并且在使用这些术语和表达方式时,无意排除所示和所述特征或其部分的任何等同物,而是应认识到在所要求保护的本发明的范围内,各种修改是可能的。因此,应当理解,尽管通过示例性实施方案和任选的特征具体公开了本发明,但本领域技术人员可以采用本文中实施的发明的修改和变化,并且这样的修改和变化被认为在本发明的范围内。
本发明在本文中已经被广泛和一般地描述。落入一般公开内容的每个较窄的种类和亚属分组也形成本发明的一部分。这包括本发明的一般性描述,附带条件或负面限制从该属中除去任何主题,而不管所去除的材料是否在本文中具体叙述。本文所引用的所有文献,包括专利申请和科学出版物,均以引用方式并入本文。
其它实施方案在以下权利要求书内。另外,在本发明的特征或方面以马库什组的形式描述的情况下,本领域技术人员将认识到本发明也由此以马库什组的任何单独成员或成员亚组的形式描述。
序列表
<110> 雷德生物科技股份有限公司
<120> 用于治疗HSV的疫苗组合物和方法
<130> LC23310022P
<150> EP21162170
<151> 2021-03-11
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Tyr Arg Glu Cys Lys Phe Leu Ser Thr Leu Pro Ser Asp Val Ile Asp
100 105 110
Trp Gly Asp Ala His Val Pro Glu Arg Ser Pro Ile Asp Ile Arg Ala
115 120 125
His Gly Asp Val Ala Phe Pro Thr Leu Pro Ala Thr Arg Asp Glu Leu
130 135 140
Pro Ser Tyr Tyr Glu Ala Met Ala Gln Phe Phe Arg Gly Glu Leu Arg
145 150 155 160
Ala Arg Glu Glu Ser Tyr Arg Thr Val Leu Ala Asn Phe Cys Ser Ala
165 170 175
Leu Tyr Arg Tyr Leu Arg Ala Ser Val Arg Gln Leu His Arg Gln Ala
180 185 190
His Met Arg Gly Arg Asn Arg Asp Leu Arg Glu Met Leu Arg Thr Thr
195 200 205
Ile Ala Asp Arg Tyr Tyr Arg Glu Thr Ala Arg Leu Ala Arg Val Leu
210 215 220
Phe Leu His Leu Tyr Leu Phe Leu Ser Arg Glu Ile Leu Trp Ala Ala
225 230 235 240
Tyr Ala Glu Gln Met Met Arg Pro Asp Leu Phe Asp Gly Leu Cys Cys
245 250 255
Asp Leu Glu Ser Trp Arg Gln Leu Ala Cys Leu Phe Gln Pro Leu Met
260 265 270
Phe Ile Asn Gly Ser Leu Thr Val Arg Gly Val Pro Val Glu Ala Arg
275 280 285
Arg Leu Arg Glu Leu Asn His Ile Arg Glu His Leu Asn Leu Pro Leu
290 295 300
Val Arg Ser Ala Ala Ala Glu Glu Pro Gly Ala Pro Leu Thr Thr Pro
305 310 315 320
Pro Val Leu Gln Gly Asn Gln Ala Arg Ser Ser Gly Tyr Phe Met Leu
325 330 335
Leu Ile Arg Ala Lys Leu Asp Ser Tyr Ser Ser Val Ala Thr Ser Glu
340 345 350
Gly Glu Ser Val Met Arg Glu His Ala Tyr Ser Arg Gly Arg Thr Arg
355 360 365
Asn Asn Tyr Gly Ser Thr Ile Glu Gly Leu Leu Asp Leu Pro Asp Asp
370 375 380
Asp Asp Ala Pro Ala Glu Ala Gly Leu Val Ala Pro Arg Met Ser Phe
385 390 395 400
Leu Ser Ala Gly Gln Arg Pro Arg Arg Leu Ser Thr Thr Ala Pro Ile
405 410 415
Thr Asp Val Ser Leu Gly Asp Glu Leu Arg Leu Asp Gly Glu Glu Val
420 425 430
Asp Met Thr Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Glu Met Leu
435 440 445
Gly Asp Val Glu Ser Pro Ser Pro Gly Met Thr His Asp Pro Val Ser
450 455 460
Tyr Gly Ala Leu Asp Val Asp Asp Phe Glu Phe Glu Gln Met Phe Thr
465 470 475 480
Asp Ala Met Gly Ile Asp Asp Phe Gly Gly
485 490
<210> 7
<211> 302
<212> PRT
<213> 人疱疹病毒2
<400> 7
Met Thr Ser Arg Arg Ser Val Lys Ser Cys Pro Arg Glu Ala Pro Arg
1 5 10 15
Gly Thr His Glu Glu Leu Tyr Tyr Gly Pro Val Ser Pro Ala Asp Pro
20 25 30
Glu Ser Pro Arg Asp Asp Phe Arg Arg Gly Ala Gly Pro Met Arg Ala
35 40 45
Arg Pro Arg Gly Glu Val Arg Phe Leu His Tyr Asp Glu Ala Gly Tyr
50 55 60
Ala Leu Tyr Arg Asp Ser Ser Ser Ser Glu Asp Asn Asp Glu Ser Arg
65 70 75 80
Asp Thr Ala Arg Pro Arg Arg Ser Ala Ser Val Ala Gly Ser His Gly
85 90 95
Pro Gly Pro Ala Arg Ala Pro Pro Pro Pro Gly Gly Pro Val Gly Ala
100 105 110
Gly Gly Arg Ser His Ala Pro Pro Ala Arg Thr Pro Lys Met Thr Arg
115 120 125
Gly Ala Pro Lys Ala Pro Ala Thr Pro Ala Thr Asp Pro Ala Arg Gly
130 135 140
Arg Arg Pro Ala Gln Ala Asp Ser Ala Val Leu Leu Asp Ala Pro Ala
145 150 155 160
Pro Thr Ala Ser Gly Arg Thr Lys Thr Pro Ala Gln Gly Leu Ala Lys
165 170 175
Lys Leu His Phe Ser Thr Ala Pro Pro Ser Pro Thr Ala Pro Trp Thr
180 185 190
Pro Arg Val Ala Gly Phe Asn Lys Arg Val Phe Cys Ala Ala Val Gly
195 200 205
Arg Leu Ala Ala Thr His Ala Arg Leu Ala Ala Val Gln Leu Trp Asp
210 215 220
Met Ser Arg Pro His Thr Asp Glu Asp Leu Asn Glu Leu Leu Asp Leu
225 230 235 240
Thr Thr Ile Arg Val Thr Val Cys Glu Gly Lys Asn Leu Leu Gln Arg
245 250 255
Ala Asn Glu Leu Val Asn Pro Asp Ala Ala Gln Asp Val Asp Ala Thr
260 265 270
Ala Ala Ala Arg Gly Arg Pro Ala Gly Arg Ala Ala Ala Thr Ala Arg
275 280 285
Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro Leu Glu
290 295 300
<210> 8
<211> 305
<212> PRT
<213> 人疱疹病毒2
<400> 8
Met Tyr Asp Ile Ala Pro Arg Arg Ser Gly Ser Arg Pro Gly Pro Gly
1 5 10 15
Arg Asp Lys Thr Arg Arg Arg Ser Arg Phe Ser Ala Ala Gly Asn Pro
20 25 30
Gly Val Glu Arg Arg Ala Ser Arg Lys Ser Leu Pro Ser His Ala Arg
35 40 45
Arg Leu Glu Leu Cys Leu His Glu Arg Arg Arg Tyr Arg Gly Phe Phe
50 55 60
Ala Ala Leu Ala Gln Thr Pro Ser Glu Glu Ile Ala Ile Val Arg Ser
65 70 75 80
Leu Ser Val Pro Leu Val Lys Thr Thr Pro Val Ser Leu Pro Phe Ser
85 90 95
Leu Asp Gln Thr Val Ala Asp Asn Cys Leu Thr Leu Ser Gly Met Gly
100 105 110
Tyr Tyr Leu Gly Ile Gly Gly Cys Cys Pro Ala Cys Ser Ala Gly Asp
115 120 125
Gly Arg Leu Ala Thr Val Ser Arg Glu Ala Leu Ile Leu Ala Phe Val
130 135 140
Gln Gln Ile Asn Thr Ile Phe Glu His Arg Thr Phe Leu Ala Ser Leu
145 150 155 160
Val Val Leu Ala Asp Arg His Ser Thr Pro Leu Gln Asp Leu Leu Ala
165 170 175
Asp Thr Leu Gly Gln Pro Glu Leu Phe Phe Val His Thr Ile Leu Arg
180 185 190
Gly Gly Gly Ala Cys Asp Pro Arg Phe Leu Phe Tyr Pro Asp Pro Thr
195 200 205
Tyr Gly Gly His Met Leu Tyr Val Ile Phe Pro Gly Thr Ser Ala His
210 215 220
Leu His Tyr Arg Leu Ile Asp Arg Met Leu Thr Ala Cys Pro Gly Tyr
225 230 235 240
Arg Phe Ala Ala His Val Trp Gln Ser Thr Phe Val Leu Val Val Arg
245 250 255
Arg Asn Ala Glu Lys Pro Ala Asp Ala Glu Ile Pro Thr Val Ser Ala
260 265 270
Ala Asp Ile Tyr Cys Lys Met Arg Asp Ile Ser Phe Asp Gly Gly Leu
275 280 285
Met Leu Glu Tyr Gln Arg Leu Tyr Ala Thr Phe Asp Glu Phe Pro Pro
290 295 300
Pro
305
<210> 9
<211> 250
<212> PRT
<213> 人疱疹病毒2
<400> 9
Met Ala Gly Met Gly Lys Pro Tyr Gly Gly Arg Pro Gly Asp Ala Phe
1 5 10 15
Glu Gly Leu Val Gln Arg Ile Arg Leu Ile Val Pro Thr Thr Leu Arg
20 25 30
Gly Gly Gly Gly Glu Ser Gly Pro Tyr Ser Pro Ser Asn Pro Pro Ser
35 40 45
Arg Cys Ala Phe Gln Phe His Gly Gln Asp Gly Ser Asp Glu Ala Phe
50 55 60
Pro Ile Glu Tyr Val Leu Arg Leu Met Asn Asp Trp Ala Asp Val Pro
65 70 75 80
Cys Asn Pro Tyr Leu Arg Val Gln Asn Thr Gly Val Ser Val Leu Phe
85 90 95
Gln Gly Phe Phe Asn Arg Pro His Gly Ala Pro Gly Gly Ala Ile Thr
100 105 110
Ala Glu Gln Thr Asn Val Ile Leu His Ser Thr Glu Thr Thr Gly Leu
115 120 125
Ser Leu Gly Asp Leu Asp Asp Val Lys Gly Arg Leu Gly Leu Asp Ala
130 135 140
Arg Pro Met Met Ala Ser Met Trp Ile Ser Cys Phe Val Arg Met Pro
145 150 155 160
Arg Val Gln Leu Ala Phe Arg Phe Met Gly Pro Glu Asp Ala Val Arg
165 170 175
Thr Arg Arg Ile Leu Cys Arg Ala Ala Glu Gln Ala Leu Ala Arg Arg
180 185 190
Arg Arg Ser Arg Arg Ser Gln Asp Asp Tyr Gly Ala Val Ala Val Ala
195 200 205
Ala Ala His His Ser Ser Gly Ala Pro Gly Pro Gly Val Ala Ala Ser
210 215 220
Gly Pro Pro Ala Pro Pro Gly Arg Gly Pro Ala Arg Pro Trp His Gln
225 230 235 240
Ala Val Gln Leu Phe Arg Ala Pro Arg Pro
245 250
<210> 10
<211> 904
<212> PRT
<213> 人疱疹病毒2
<400> 10
Met Arg Gly Gly Gly Leu Ile Cys Ala Leu Val Val Gly Ala Leu Val
1 5 10 15
Ala Ala Val Ala Ser Ala Ala Pro Ala Ala Pro Ala Ala Pro Arg Ala
20 25 30
Ser Gly Gly Val Ala Ala Thr Val Ala Ala Asn Gly Gly Pro Ala Ser
35 40 45
Arg Pro Pro Pro Val Pro Ser Pro Ala Thr Thr Lys Ala Arg Lys Arg
50 55 60
Lys Thr Lys Lys Pro Pro Lys Arg Pro Glu Ala Thr Pro Pro Pro Asp
65 70 75 80
Ala Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Ala His Leu
85 90 95
Arg Glu Ile Lys Val Glu Asn Ala Asp Ala Gln Phe Tyr Val Cys Pro
100 105 110
Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg Cys
115 120 125
Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala Val Val
130 135 140
Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys Ala Thr Met Tyr Tyr
145 150 155 160
Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly His Arg Tyr Ser Gln
165 170 175
Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val Pro Phe Glu Glu Val
180 185 190
Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser Thr Ala Lys Tyr
195 200 205
Val Arg Asn Asn Met Glu Thr Thr Ala Phe His Arg Asp Asp His Glu
210 215 220
Thr Asp Met Glu Leu Lys Pro Ala Lys Val Ala Thr Arg Thr Ser Arg
225 230 235 240
Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala
245 250 255
Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp
260 265 270
Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp
275 280 285
Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr
290 295 300
Glu His Thr Ser Tyr Ala Ala Asp Arg Phe Lys Gln Val Asp Gly Phe
305 310 315 320
Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala Thr Ser Pro Thr Thr
325 330 335
Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala Trp Asp Trp Val
340 345 350
Pro Lys Arg Pro Ala Val Cys Thr Met Thr Lys Trp Gln Glu Val Asp
355 360 365
Glu Met Leu Arg Ala Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp
370 375 380
Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Gln Tyr Ser Leu Ser
385 390 395 400
Arg Val Asp Leu Gly Asp Cys Ile Gly Arg Asp Ala Arg Glu Ala Ile
405 410 415
Asp Arg Met Phe Ala Arg Lys Tyr Asn Ala Thr His Ile Lys Val Gly
420 425 430
Gln Pro Gln Tyr Tyr Leu Ala Thr Gly Gly Phe Leu Ile Ala Tyr Gln
435 440 445
Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr Val Arg Glu Tyr Met
450 455 460
Arg Glu Gln Asp Arg Lys Pro Arg Asn Ala Thr Pro Ala Pro Leu Arg
465 470 475 480
Glu Ala Pro Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr Thr Ser
485 490 495
Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Arg
500 505 510
His Val Asn Asp Met Leu Gly Arg Ile Ala Val Ala Trp Cys Glu Leu
515 520 525
Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro
530 535 540
Asn Ala Ile Ala Ser Ala Thr Val Gly Arg Arg Val Ser Ala Arg Met
545 550 555 560
Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val Ala Pro Asp
565 570 575
Asn Val Ile Val Gln Asn Ser Met Arg Val Ser Ser Arg Pro Gly Thr
580 585 590
Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp Gln Gly Pro
595 600 605
Leu Ile Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg Leu Thr Arg
610 615 620
Asp Ala Leu Glu Pro Cys Thr Val Gly His Arg Arg Tyr Phe Ile Phe
625 630 635 640
Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser His Gln Leu
645 650 655
Ser Arg Ala Asp Val Thr Thr Val Ser Thr Phe Ile Asp Leu Asn Ile
660 665 670
Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg
675 680 685
His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg
690 695 700
Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp Thr Val Ile
705 710 715 720
Arg Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Cys Ala Phe Phe
725 730 735
Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly Lys Val Val Met Gly
740 745 750
Val Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser Ser Phe Met
755 760 765
Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val Leu Ala Gly
770 775 780
Leu Val Ala Ala Phe Phe Ala Phe Arg Tyr Val Leu Gln Leu Gln Arg
785 790 795 800
Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu Leu Lys Thr
805 810 815
Ser Asp Pro Gly Gly Val Gly Gly Glu Gly Glu Glu Gly Ala Glu Gly
820 825 830
Gly Gly Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg
835 840 845
Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala Arg
850 855 860
Lys Lys Gly Thr Ser Ala Leu Leu Ser Ser Lys Val Thr Asn Met Val
865 870 875 880
Leu Arg Lys Arg Asn Lys Ala Arg Tyr Ser Pro Leu His Asn Glu Asp
885 890 895
Glu Ala Gly Asp Glu Asp Glu Leu
900
<210> 11
<211> 393
<212> PRT
<213> 人疱疹病毒2
<400> 11
Met Gly Arg Leu Thr Ser Gly Val Gly Thr Ala Ala Leu Leu Val Val
1 5 10 15
Ala Val Gly Leu Arg Val Val Cys Ala Lys Tyr Ala Leu Ala Asp Pro
20 25 30
Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asn Leu Pro
35 40 45
Val Leu Asp Arg Leu Thr Asp Pro Pro Gly Val Lys Arg Val Tyr His
50 55 60
Ile Gln Pro Ser Leu Glu Asp Pro Phe Gln Pro Pro Ser Ile Pro Ile
65 70 75 80
Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu
85 90 95
His Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Asp Glu
100 105 110
Ala Arg Lys His Thr Tyr Asn Leu Thr Ile Ala Trp Tyr Arg Met Gly
115 120 125
Asp Asn Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Pro
130 135 140
Tyr Asn Lys Ser Leu Gly Val Cys Pro Ile Arg Thr Gln Pro Arg Trp
145 150 155 160
Ser Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu Asp Asn Leu Gly Phe
165 170 175
Leu Met His Ala Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu
180 185 190
Val Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His
195 200 205
Arg Ala Arg Ala Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro
210 215 220
Ala Ala Cys Leu Thr Ser Lys Ala Tyr Gln Gln Gly Val Thr Val Asp
225 230 235 240
Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu Asn Gln Arg Thr Val
245 250 255
Ala Leu Tyr Ser Leu Lys Ile Ala Gly Trp His Gly Pro Lys Pro Pro
260 265 270
Tyr Thr Ser Thr Leu Leu Pro Pro Glu Leu Ser Asp Thr Thr Asn Ala
275 280 285
Thr Gln Pro Glu Leu Val Pro Glu Asp Pro Glu Asp Ser Ala Leu Leu
290 295 300
Glu Asp Pro Ala Gly Thr Val Ser Ser Gln Ile Pro Pro Asn Trp His
305 310 315 320
Ile Pro Ser Ile Gln Asp Val Ala Pro His His Ala Pro Ala Ala Pro
325 330 335
Ser Asn Pro Gly Leu Ile Ile Gly Ala Leu Ala Gly Ser Thr Leu Ala
340 345 350
Val Leu Val Ile Gly Gly Ile Ala Phe Trp Val Arg Arg Arg Ala Gln
355 360 365
Met Ala Pro Lys Arg Leu Arg Leu Pro His Ile Arg Asp Asp Asp Ala
370 375 380
Pro Pro Ser His Gln Pro Leu Phe Tyr
385 390
<210> 12
<211> 1386
<212> RNA
<213> 人工序列
<220>
<223> 所有尿苷残基都是1-甲基-假尿苷 - 编码
来自单纯疱疹病毒-2的糖蛋白D
<400> 12
ggaauaaaag ucucaacaca acauauacaa aacaaacgaa ucucaagcaa ucaagcauuc 60
uacuucuauu gcagcaauuu aaaucauuuc uuuuaaagca aaagcaauuu ucugaaaauu 120
uucaccauuu acgaacgaua gcaugacccg ccugaccgug cuggcccugc uggccggccu 180
gcuggccucc ucccgcgcca aguacgcccu ggccgacccc ucccugaaga uggccgaccc 240
caaccgcuuc cgcggcaaga accugcccgu gcuggaccag cugaccgacc cccccggcgu 300
gaagcgcgug uaccacaucc agcccucccu ggaggacccc uuccagcccc ccuccauccc 360
caucaccgug uacuacgccg ugcuggagcg cgccugccgc uccgugcugc ugcacgcccc 420
cuccgaggcc ccccagaucg ugcgcggcgc cuccgacgag gcccgcaagc acaccuacaa 480
ccugaccauc gccugguacc gcaugggcga caacugcgcc auccccauca ccgugaugga 540
guacaccgag ugccccuaca acaagucccu gggcgugugc cccauccgca cccagccccg 600
cugguccuac uacgacuccu ucuccgccgu guccgaggac aaccugggcu uccugaugca 660
cgcccccgcc uucgagaccg ccggcaccua ccugcgccug gugaagauca acgacuggac 720
cgagaucacc caguucaucc uggagcaccg cgcccgcgcc uccugcaagu acgcccugcc 780
ccugcgcauc ccccccgccg ccugccugac cuccaaggcc uaccagcagg gcgugaccgu 840
ggacuccauc ggcaugcugc cccgcuucau ccccgagaac cagcgcaccg uggcccugua 900
cucccugaag aucgccggcu ggcacggccc caagcccccc uacaccucca cccugcugcc 960
ccccgagcug uccgacacca ccaacgccac ccagcccgag cuggugcccg aggaccccga 1020
ggacuccgcc cugcuggagg accccgccgg caccgugucc ucccagaucc cccccaacug 1080
gcacaucccc uccauccagg acguggcccc ccaccacuaa cuaguaguga cugacuagga 1140
ucugguuacc acuaaaccag ccucaagaac acccgaaugg agucucuaag cuacauaaua 1200
ccaacuuaca cuuacaaaau guuguccccc aaaauguagc cauucguauc ugcuccuaau 1260
aaaaagaaag uuucuucaca uucuaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380
aaaaac 1386
<210> 13
<211> 1614
<212> RNA
<213> 人工序列
<220>
<223> 所有尿苷残基都是1-甲基-假尿苷 - 编码
来自单纯疱疹病毒-2的糖蛋白E
<400> 13
ggaauaaaag ucucaacaca acauauacaa aacaaacgaa ucucaagcaa ucaagcauuc 60
uacuucuauu gcagcaauuu aaaucauuuc uuuuaaagca aaagcaauuu ucugaaaauu 120
uucaccauuu acgaacgaua gcaugcgcau gcagcugcug cugcugaucg cccugucccu 180
ggcccuggug accaacuccc gcaccuccug gaagcgcgug accuccggcg aggacguggu 240
gcugcugccc gcccccgccg gccccgagga gcgcacccgc gcccacaagc ugcugugggc 300
cgccgagccc cuggacgccu gcggcccccu gcgccccucc uggguggccc uguggccccc 360
ccgccgcgug cuggagaccg ugguggacgc cgccugcaug cgcgcccccg agccccuggc 420
caucgccuac uccccccccu uccccgccgg cgacgagggc cuguacuccg agcuggccug 480
gcgcgaccgc guggccgugg ugaacgaguc ccuggugauc uacggcgccc uggagaccga 540
cuccggccug uacacccugu ccgugguggg ccuguccgac gaggcccgcc agguggccuc 600
cguggugcug gugguggagc ccgcccccgu gcccaccccc acccccgacg acuacgacga 660
ggaggacgac gccggcgugu ccgagcgcac ccccgugucc gugccccccc ccaccccccc 720
ccgccgcccc cccguggccc cccccaccca cccccgcgug auccccgagg ugucccacgu 780
gcgcggcgug accgugcaca uggagacccc cgaggccauc cuguucgccc ccggcgagac 840
cuucggcacc aacgugucca uccacgccau cgcccacgac gacggccccu acgccaugga 900
cguggugugg augcgcuucg acgugcccuc cuccugcgcc gagaugcgca ucuacgaggc 960
cugccuguac cacccccagc ugcccgagug ccuguccccc gccgacgccc ccugcgccgu 1020
guccuccugg gccuaccgcc uggccgugcg cuccuacgcc ggcugcuccc gcaccacccc 1080
ccccccccgc ugcuucgccg aggcccgcau ggagcccgug cccggccugg ccuggcuggc 1140
cuccaccgug aaccuggagu uccagcacgc cuccccccag cacgccggcc uguaccugug 1200
cgugguguac guggacgacc acauccacgc cuggggccac augaccaucu ccaccgccgc 1260
ccaguaccgc aacgccgugg uggagcagca ccugccccag cgccagcccg agcccgugga 1320
gcccacccgc ccccacgugc gcgccuaacu aguagugacu gacuaggauc ugguuaccac 1380
uaaaccagcc ucaagaacac ccgaauggag ucucuaagcu acauaauacc aacuuacacu 1440
uacaaaaugu ugucccccaa aauguagcca uucguaucug cuccuaauaa aaagaaaguu 1500
ucuucacauu cuaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaac 1614
<210> 14
<211> 1470
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL48的RNA序列
<400> 14
auggaucugc ugguggauga ucuguuugcg gaugcggaug gcgugagccc gccgccgccg 60
cgcccggcgg gcggcccgaa aaacaccccg gcggcgccgc cgcuguaugc gaccggccgc 120
cugagccagg cgcagcugau gccgagcccg ccgaugccgg ugccgccggc ggcgcuguuu 180
aaccgccugc uggaugaucu gggcuuuagc gcgggcccgg cgcugugcac caugcuggau 240
accuggaacg aagaucuguu uagcggcuuu ccgaccaacg cggauaugua ucgcgaaugc 300
aaauuucuga gcacccugcc gagcgaugug auugauuggg gcgaugcgca ugugccggaa 360
cgcagcccga uugauauucg cgcgcauggc gauguggcgu uuccgacccu gccggcgacc 420
cgcgaugaac ugccgagcua uuaugaagcg auggcgcagu uuuuucgcgg cgaacugcgc 480
gcgcgcgaag aaagcuaucg caccgugcug gcgaacuuuu gcagcgcgcu guaucgcuau 540
cugcgcgcga gcgugcgcca gcugcaucgc caggcgcaua ugcgcggccg caaccgcgau 600
cugcgcgaaa ugcugcgcac caccauugcg gaucgcuauu aucgcgaaac cgcgcgccug 660
gcgcgcgugc uguuucugca ucuguaucug uuucugagcc gcgaaauucu gugggcggcg 720
uaugcggaac agaugaugcg cccggaucug uuugauggcc ugugcugcga ucuggaaagc 780
uggcgccagc uggcgugccu guuucagccg cugauguuua uuaacggcag ccugaccgug 840
cgcggcgugc cgguggaagc gcgccgccug cgcgaacuga accauauucg cgaacaucug 900
aaccugccgc uggugcgcag cgcggcggcg gaagaaccgg gcgcgccgcu gaccaccccg 960
ccggugcugc agggcaacca ggcgcgcagc agcggcuauu uuaugcugcu gauucgcgcg 1020
aaacuggaua gcuauagcag cguggcgacc agcgaaggcg aaagcgugau gcgcgaacau 1080
gcguauagcc gcggccgcac ccgcaacaac uauggcagca ccauugaagg ccugcuggau 1140
cugccggaug augaugaugc gccggcggaa gcgggccugg uggcgccgcg caugagcuuu 1200
cugagcgcgg gccagcgccc gcgccgccug agcaccaccg cgccgauuac cgaugugagc 1260
cugggcgaug aacugcgccu ggauggcgaa gaaguggaua ugaccccggc ggaugcgcug 1320
gaugauuuug aucuggaaau gcugggcgau guggaaagcc cgagcccggg caugacccau 1380
gauccgguga gcuauggcgc gcuggaugug gaugauuuug aauuugaaca gauguuuacc 1440
gaugcgaugg gcauugauga uuuuggcggc 1470
<210> 15
<211> 906
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL49的RNA序列
<400> 15
augaccucuc gccgcuccgu caagucgugu ccgcgggaag cgccgcgcgg gacccacgag 60
gagcuguacu auggcccggu cuccccggcg gauccagaga guccgcgcga cgacuuccgc 120
cgcggcgcug gcccgaugcg cgcgcgccca aggggcgagg uucgcuuucu ccauuaugac 180
gaggcugggu augcccucua ccgggacucg ucgucguccg aagacaacga cgagucccgg 240
gauaccgcgc gaccgcgucg uucggcgucc gucgcgggcu cucacggccc cggccccgcg 300
cgcgcuccuc caccccccgg gggccccgug ggcgccggcg ggcgcucgca cgccccuccc 360
gcgcggaccc ccaaaaugac gcgcggggcg ccuaaggccc ccgcgacccc ggcgaccgac 420
ccugcccgcg gcaggcgacc cgcccaggcc gacuccgccg ugcuccuaga cgcccccgcu 480
cccacggccu cgggaagaac caagacaccc gcccagggac uggccaagaa gcugcacuuc 540
agcaccgccc caccgagccc cacggcgccg uggacccccc ggguggccgg guucaacaag 600
cgcgucuucu gcgccgcggu cgggcgccug gcggccacgc acgcccggcu ggcggcggua 660
cagcuguggg acaugucgcg gccgcacacc gacgaagacc ucaacgagcu ccucgaccuc 720
accaccauuc gcgugacggu cugcgagggc aagaaccucc ugcagcgcgc gaacgaguug 780
gugaaucccg acgcggcgca ggacgucgac gcgaccgcgg ccgcccgggg ccgccccgcg 840
gggcgugccg ccgcgaccgc acgggccccc gcccgcucgg cuucccgucc ccgccgcccc 900
cucgag 906
<210> 16
<211> 291
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL11的RNA序列
<400> 16
augggccucg cguucuccgg ggcgcggccc ugcugcugcc gacacaacgu caucaucacc 60
gacggcgggg aggucgucuc gcugaccgcc cacgaauuug acgucgugga caucgagucc 120
gaagaagagg guaacuucua cgugcccccc gacaugcgcg uggucacccg ggcgccgggg 180
ccccaguacc ggcgcgcauc ggacccccca ucgcgucaca ccagacggcg ggaccccgac 240
guggcccgcc cuccugccac gcucacgccc ccacucucug acagcgaaua a 291
<210> 17
<211> 1119
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL16的RNA序列
<400> 17
auggcacagc gggcacucug gcguccccag gcgacgccag gccccccggg agccgcggcc 60
ccgccggguc accggggcgc cccccccgac gcgcgcgccc ccgacccggg gcccgaagcc 120
gaccucgucg cgcgcaucgc caacucuguc uucguguggc gcgucguucg gggggacgag 180
cggcuuaaga uuuuucggug ccucacgguc cucaccgagc cgcugugcca gguggcccuu 240
ccggaccccg accccgagcg ugcccuuuuc ugcgagaucu uccuguaccu gacgcggccc 300
aaggcgcugc gccugccuuc gaacacguuc uuugccauuu ucuucuuuaa ccgcgagcgc 360
cgcuacugcg ccaccgucca ccuccgcagc guaacgcacc cccggacccc gcugcugugc 420
acccuagccu ucggccaccu ggaagcugcc ucccccccgg aggaaacccc cgaccccgcc 480
gccgaacagc ucgcggacga gcccguggcc cacgagcucg acggcgccua ccugguaccc 540
acggaaccgc ccccgaaucc gggggcgugc ugugcccugg guccgggggc cugguggcac 600
cuccccggcg gccgcauaua cugcugggcc auggaugaug accugggguc gcucuguccc 660
ccgggcagcc gggcccgcca uuuaggaugg cugcuaucca ggauuaccga ccccccggga 720
ggaggcggcg ccugcgcccc gacggcccac aucgauuccg ccaacgcgcu guggcgcgcc 780
cccgccgugg cggaggccug ucccugcguc gccccgugca ugugguccaa uauggcccag 840
cgcacccugg ccguccgggg ggacgcgagc cugugucagc uccucuucgg gcaccccgug 900
gacgcgguua uccugcggca ggcgacccgc cgcccgcgca ucacggccca ccugcacgag 960
gucgucgugg gccgggacgg cgcggaaagc gucauccgcc cgaccagcgc cggguggcgc 1020
cucugcguuc ugucaucgua uaccagccgc cuguuugcaa cgagcugccc cgcggucgcg 1080
cgggccgugg ccagagccuc aucguccgau uacaaauaa 1119
<210> 18
<211> 1599
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL21的RNA序列
<400> 18
auggagcuca gcuaugccac cacccugcac caccgggacg uuguguuuua cgucacggca 60
gacagaaacc gcgccuacuu ugugugcggg ggguccguuu auuccguagg gcggccucgg 120
gauucucagc cgggggaaau ugccaaguuu ggccuggugg uccgggggac aggccccaaa 180
gaccgcaugg ucgccaacua cguacgaagc gagcuccgcc agcgcggccu gcgggacgug 240
cggcccgugg gggaggacga gguguuccug gacagcgugu gucugcuaaa cccgaacgug 300
agcuccgagc gagacgugau uaauaccaac gacguugaag ugcuggacga augccuggcc 360
gaauacugca ccucgcugcg aaccagcccg ggggugcugg ugaccggggu gcgcgugcgc 420
gcgcgagaca gggucaucga gcuauuugag cacccggcga ucgucaacau uuccucgcgc 480
uucgcguaca cccccucccc cuacguauuc gcccuggccc aggcgcaccu cccccggcuc 540
ccgagcucgc uggagccccu ggugagcggc cuguuugacg gcauucccgc cccgcgccag 600
ccccuggacg cccgcgaccg gcgcacggau gucgugauca cgggcacccg cgcccccaga 660
ccgauggccg ggaccggggc cgggggcgcg ggggccaagc gggccaccgu cagcgaguuc 720
gugcaaguga agcacaucga ccguguugug uccccgagcg ucucuuccgc ccccccgccg 780
agcgcccccg acgcgagucu gccgcccccg gggcuccagg aggccgcccc gccgggcccc 840
ccgcucaggg agcuguggug gguguucuac gccggcgacc gggcgcugga ggagccccac 900
gccgagucgg gauugacgcg cgaggagguc cgcgccgugc auggguuccg ggagcaggcg 960
uggaagcugu uugggucggu gggggcuccg cgggcguuuc ucggggccgc gcuggcccug 1020
agcccgaccc aaaagcucgc cgucuacuac uaucucaucc accgggagcg gcgcaugucc 1080
cccuuccccg cgcucgugcg gcucgucggu cgguacaucc agcgccacgg ccuguacguu 1140
cccgcgcccg acgaaccgac guuggccgau gccaugaacg ggcuguuccg cgacgcgcug 1200
gcggccggga ccguggccga gcagcuccuc auguucgacc uccucccgcc caaggacgug 1260
ccggugggga gcgacgcgcg ggccgacagc gccgcccugc ugcgcuuugu ggacucgcaa 1320
cgccugaccc cggggggguc cgucucgccc gagcacguca uguaccucgg cgcguuccug 1380
ggcguguugu acgccggcca cggacgccug gccgcggcca cgcauaccgc gcgccugacg 1440
ggcgugacgu cccugguccu gaccgugggg gacgucgacc ggauguccgc guuugaccgc 1500
gggccggcgg gggcggcugg ccgcacgcga accgccgggu accuggacgc gcugcuuacc 1560
guuugccugg cucgcgccca gcacggccag ucuguguga 1599
<210> 19
<211> 918
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL31的RNA序列
<400> 19
auguaugaca ucgccccccg ucgcuccggc ucccggcccg ggcccggccg cgacaagacc 60
cggcggcggu cgcgcuuuuc cgccgccggg aaccccggcg uggagcgccg ggccucccgc 120
aagagccugc cgucgcacgc gcgcaggcug gagcucugcc ugcacgagcg ccgucgcuac 180
cgcggcuucu ucgccgcccu cgcccagacg cccuccgagg agaucgccau cgugcgcucg 240
cucuccgugc cccuggugaa gacgaccccc gugucgcucc ccuucagccu ggaccagacc 300
guggccgaca acugccugac ccucucgggc augggcuacu accuggggau cgggggcugc 360
ugccccgcgu gcagcgcggg cgacgggcgg cuggccaccg ucagccgcga ggcccucauc 420
cuggccuucg ugcagcagau caauacgauc uuugagcacc gcaccuuucu ggccucccug 480
gucgugcugg ccgaccgcca cagcaccccc cuccaggacc uccuggccga uacgcucggc 540
cagcccgagc ucuuuuucgu gcacacgauc cugcgcggag ggggggcgug cgacccccgg 600
uuucuuuuuu acccggaccc cacguacggg ggccacaugc uguacgucau cuuccccggc 660
acgucggccc accugcacua ccggcucauc gaucggaugc ugaccgcgug ccccggcuac 720
cgguucgccg cccacgugug gcagagcacg uuugugcucg uggucagacg caacgcagag 780
aaaccagcgg acgcggaaau uccgaccgug ucggccgcag acauuuauug caagaugcgg 840
gacaucagcu ucgacggggg gcucaugcua gaguaucaaa ggcucuaugc aacauucgac 900
gaguuuccuc cgccguag 918
<210> 20
<211> 753
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL34的RNA序列
<400> 20
auggcgggga uggggaagcc cuacggcggc cgcccggggg acgcguucga gggucucguu 60
cagcgcauca ggcucauugu ucccaccacg cugcgcggcg ggggugggga gucgggcccc 120
uacucgccau ccaacccgcc cucgagaugu gccuuccagu uccacggcca ggaugggucc 180
gacgaggccu ucccgaucga guacguccug cggcucauga acgacugggc cgaugugccc 240
ugcaaccccu accugcgcgu gcagaacacc ggcguuucgg ugcuguuuca gggguuuuuu 300
aaccggcccc acggcgcccc ggggggcgcg aucacggcgg agcagaccaa cgugauucug 360
cacuccaccg agacgacggg acugucccuc ggagaccugg acgacgucaa ggggcgccuc 420
ggccuggacg cccggccgau gauggccagc auguggauca gcugcuuugu gcgcaugccc 480
cgggugcagc ucgcguuucg guucaugggc cccgaggacg ccguucgcac gcggcggauc 540
cugugucgcg ccgccgagca ggcccucgcc cgucgccgcc gguccaggcg gucccaggau 600
gacuacgggg cgguggcggu ggcggcggcg caccacucuu ccggagcgcc cgggccgggg 660
gucgccgccu cgggcccgcc agcgccgccc ggacggggac cggcccgucc guggcaucag 720
gccgugcagu uguuccgggc cccgcguccg uga 753
<210> 21
<211> 321
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的gE蛋白的胞质尾的RNA序列
<400> 21
cgcaggcgcu ccuggcgggc gguuaaaagc cgggccucgg cgacgggccc cacuuacauu 60
cgcguggcgg acagcgagcu guacgcggac uggaguucgg acagcgaggg ggagcgcgac 120
gggucccugu ggcaggaccc uccggagaga cccgacucuc ccuccacaaa uggauccggc 180
uuugagaucu uaucaccaac ggcuccgucu guauaccccc auagcgaggg gcguaaaucu 240
cgccgcccgc ucaccaccuu ugguucggga agcccgggcc gucgucacuc ccaggccucc 300
uauucguccg uccucuggua a 321
<210> 22
<211> 1182
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的gD的RNA序列
<400> 22
auggggcguu ugaccuccgg cgucgggacg gcggcccugc uaguugucgc ggugggacuc 60
cgcgucgucu gcgccaaaua cgccuuagca gaccccucgc uuaagauggc cgaucccaau 120
cgauuucgcg ggaagaaccu uccgguuuug gaccagcuga ccgacccccc cggggugaag 180
cguguuuacc acauucagcc gagccuggag gacccguucc agccccccag caucccgauc 240
acuguguacu acgcagugcu ggaacgugcc ugccgcagcg ugcuccuaca ugccccaucg 300
gaggcccccc agaucgugcg cggggcuucg gacgaggccc gaaagcacac guacaaccug 360
accaucgccu gguaucgcau gggagacaau ugcgcuaucc ccaucacggu uauggaauac 420
accgagugcc ccuacaacaa gucguugggg gucugcccca uccgaacgca gccccgcugg 480
agcuacuaug acagcuuuag cgccgucagc gaggauaacc ugggauuccu gaugcacgcc 540
cccgccuucg agaccgcggg uacguaccug cggcuaguga agauaaacga cuggacggag 600
aucacacaau uuauccugga gcaccgggcc cgcgccuccu gcaaguacgc ucucccccug 660
cgcauccccc cggcagcgug ccucaccucg aaggccuacc aacagggcgu gacggucgac 720
agcaucggga uguuaccccg cuuuaucccc gaaaaccagc gcaccgucgc ccuauacagc 780
uuaaaaaucg ccggguggca cggccccaag cccccguaca ccagcacccu gcugccgccg 840
gagcuguccg acaccaccaa cgccacgcaa cccgaacucg uuccggaaga ccccgaggac 900
ucggcccucu uagaggaucc cgccgggacg gugucuucgc agaucccccc aaacuggcac 960
aucccgucga uccaggacgu cgcgccgcac cacgcccccg ccgcccccag caacccgggc 1020
cugaucaucg gcgcgcuggc cggcaguacc cuggcggcgc uggucaucgg cgguauugcg 1080
uuuuggguac gccgccgcgc ucagauggcc cccaagcgcc uacgucuccc ccacauccgg 1140
gaugacgacg cgccccccuc gcaccagcca uuguuuuacu ag 1182
<210> 23
<211> 2192
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的gB的RNA序列
<400> 23
augcgcggug gcggauugau cugcgcccug guggugggug cacugguggc ugcugucgcu 60
ucagcugcuc cugcugcucc ugcagcuccu cgcgcuagug guggcguugc ugccaccgug 120
gcagcgaacg gagguccagc uagccguccu cccccagucc cgucuccugc aaccacuaag 180
gcacgcaagc guaaaaccaa gaaaccgccu aagaggccug aagcaacucc cccaccggac 240
gcaaacgcua cuguggcugc uggucacgcu acucucaggg cucauuugag agagaucaag 300
guugaaaacg cugacgccca guucuacgug ugcccucccc caacaggugc aacuguugug 360
caguucgaac agccucgcag guguccuacc aggccugagg gucaaaacua cacugaaggc 420
aucgcugucg uuuucaagga gaacauugcc ccauacaagu ucaaagcaac cauguacuac 480
aaagacguga cugucucgca agugugguuc ggacaccguu acucccaauu cauggguauc 540
uucgaggaca gggcucccgu uccauucgag gaagugaucg auaagauuaa cgccaaaggc 600
gugugcaggu ccacagcaaa guacgucaga aacaacaugg agacaacggc uuuccaccgc 660
gacgaucaug agaccgacau ggaauugaag ccugcgaaag uggcuacaag gacgucgaga 720
ggcuggcaca ccacugaucu gaaguacaac cccuccaggg uugaagccuu ccauagauac 780
ggaacaacgg ugaacuguau cguugaggaa guggaugccc gcucagugua ccccuacgac 840
gaguucgucc uggcuaccgg agauuucguu uacaugaguc cauucuacgg auacagggag 900
gguucgcaca ccgaacauac uuccuacgca gcggacagau ucaagcaggu ggaugguuuc 960
uacgcucgcg accugaccac uaaagcacgu gcgacuucuc cuacaacgag gaaccugcuc 1020
accacuccca aguucacagu cgcuugggac uggguuccaa agaggccggc cgucugcacc 1080
augacuaaau ggcaggaggu ugacgaaaug cugagagcug aauacggcgg aagcuuccgc 1140
uucuccagcg augccaucuc uacaacguuc accacuaacc ugacucaaua cucccucagc 1200
cguguggacc ugggugacug uaucggacgc gaugcccgug aggcaauuga ccgcauguuc 1260
gcgcguaagu acaacgcuac acacaucaaa gugggccagc cacaauacua ccucgcgacg 1320
gguggcuucu ugauugcuua ccagccuuug cugucaaaca cucucgcuga auuguacguc 1380
agggaguaca ugagagaaca agacaggaag ccaagaaacg cgacaccggc uccucugaga 1440
gaggcuccgu cugcuaacgc cucaguggaa cgcaucaaga caaccuccuc cauugaguuc 1500
gcucgucucc aguucaccua caaccacauc caacgucaug ugaacgacau gcugggaagg 1560
auugcagucg cguggugcga gcuccagaac cacgaacuga cccucuggaa cgaggccagg 1620
aagcucaacc caaacgcuau cgccucagca acugucggca ggagaguuag ugcgagaaug 1680
uugggagacg ucauggcugu uagcaccugc guuccugugg cccccgauaa cgucauuguu 1740
caaaacucua ugcgcgucag uucgcgucca ggaacuuguu acucccgccc gcucguuagc 1800
uuccguuacg aggaccaggg uccacugauc gaaggucaac ucggcgagaa caacgaauug 1860
cgccugacac gugaugcucu ggagccuugu acggucgguc accgccguua cuucauuuuc 1920
ggagguggcu acguguacuu cgaggaauac gcauacaguc accaguuguc gcgcgcugac 1980
gugaccacug ucuccacauu caucgacuug aacauuacga ugcuggagga ucacgaauuc 2040
gugccccugg gugucuacac acgucaugaa aucaaggaca gcggccucuu ggauuacacc 2100
gagguccaga ggagaaacca acugcacgac cucagauucg cugacaucga uacugugauu 2160
cgcgcggaug cuaacgcugc cauguucgcu gg 2192
<210> 24
<211> 1251
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的gE的RNA序列
<400> 24
auggccaggg gcgccggccu gguguucuuc gugggcgugu ggguggugag cugccuggcc 60
gccgccccca ggaccagcug gaagagggug accagcggcg aggacguggu gcugcugccc 120
gcccccgccg gccccgagga gaggaccagg gcccacaagc ugcugugggc cgccgagccc 180
cuggacgccu gcggcccccu gaggcccagc uggguggccc uguggccccc caggagggug 240
cuggagaccg ugguggacgc cgccugcaug agggcccccg agccccuggc caucgccuac 300
agcccccccu uccccgccgg cgacgagggc cuguacagcg agcuggccug gagggacagg 360
guggccgugg ugaacgagag ccuggugauc uacggcgccc uggagaccga cagcggccug 420
uacacccuga gcgugguggg ccugagcgac gaggccaggc agguggccag cguggugcug 480
gugguggagc ccgcccccgu gcccaccccc acccccgacg acuacgacga ggaggacgac 540
gccggcguga gcgagaggac ccccgugagc gugccccccc ccaccccccc caggaggccc 600
cccguggccc cccccaccca ccccagggug auccccgagg ugagccacgu gaggggcgug 660
accgugcaca uggagacccc cgaggccauc cuguucgccc ccggcgagac cuucggcacc 720
aacgugagca uccacgccau cgcccacgac gacggccccu acgccaugga cguggugugg 780
augagguucg acgugcccag cagcugcgcc gagaugagga ucuacgaggc cugccuguac 840
cacccccagc ugcccgagug ccugagcccc gccgacgccc ccugcgccgu gagcagcugg 900
gccuacaggc uggccgugag gagcuacgcc ggcugcagca ggaccacccc cccccccagg 960
ugcuucgccg aggccaggau ggagcccgug cccggccugg ccuggcuggc cagcaccgug 1020
aaccuggagu uccagcacgc cagcccccag cacgccggcc uguaccugug cgugguguac 1080
guggacgacc acauccacgc cuggggccac augaccauca gcaccgccgc ccaguacagg 1140
aacgccgugg uggagcagca ccugccccag aggcagcccg agcccgugga gcccaccagg 1200
ccccacguga gggccccccc ccccgccccc agcgccaggg gcccccugag g 1251
<210> 25
<211> 1692
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL48的包括UTR和polyA的密码子优化的RNA序列,
所有尿苷残基都是1-甲基-假尿苷
<400> 25
uaauacgacu cacuauaagg acucacuauu uguuuucgcg cccaguugca aaaagugucg 60
ucaccaugga ccugcucguu gacgauuugu ucgcugacgc agauggugug agcccucccc 120
caccgaggcc agcugguggc ccuaagaaca ccccugcugc cccuccccug uacgcaacug 180
gcagauuguc acaggcgcaa cugaugccua guccaccgau gccugugccu cccgcagcuc 240
ucuucaacag guugcuggac gauuugggcu ucagcgcagg acccgcguug ugcacaaugc 300
uggauacgug gaacgaggac cuguucucug gcuuccccac aaacgcagac auguaccgcg 360
aguguaaauu ccugucuacg cucccaucag acgucaucga uuggggcgac gcccacguuc 420
ccgaaagguc uccaaucgac auuagagcac auggagaugu cgcguuccca acccugccgg 480
cuacucgcga cgagcuccca ucauacuacg aagcgauggc ucaguucuuc aggggagagu 540
ugcgcgcccg ugaagaaagu uacagaacag ugcuggcuaa cuucugcucg gcccucuacc 600
gcuacuugcg ugcauccguc aggcagcugc acagacaagc gcauaugagg gguagaaacc 660
gcgaccugcg cgaaaugcuc cguaccacua ucgcugauag guacuacaga gagacugcuc 720
gccuggcccg ugucuuguuc cugcaccucu acuuguuccu gucacgcgaa auucucuggg 780
cugccuacgc cgagcaaaug augcguccag auuuguucga cggccugugc ugugaccucg 840
agucguggcg ccaguuggcu ugucuguucc aaccauugau guucaucaac ggaucccuga 900
ccguucgugg ugugccugug gaggcacgcc gucuccgcga auugaaccac auucgugagc 960
aucucaacuu gccucugguu cguucugcag cugcugagga accuggagcu ccccucacaa 1020
cgccaccggu guugcagggc aaccaagccc gcuccagcgg auacuucaug cucuugaucc 1080
gugcuaagcu ggacuccuac ucuucagugg ccaccuccga gggugaaucg gucaugcgcg 1140
aacacgcuua cucacguggc aggacaagaa acaacuacgg uaguacgauu gaaggccugc 1200
ucgaccugcc cgacgaugac gaugcaccag cagaggcugg acugguggcu ccucgcaugu 1260
ccuuccucag cgccggucag agaccgagga gacucagcac cacugccccc aucaccgaug 1320
ucucuuuggg ugacgagcuc cguuuggaug gcgaggaagu ugacaugacu ccugcugaug 1380
cccucgacga uuucgaccug gaaaugcucg gagaugucga gaguccuucg cccgguauga 1440
cacacgaccc cguuuccuac ggugcucugg auguggacga uuucgaguuc gaacaaaugu 1500
ucacggacgc aaugggcauc gacgauuucg gagguggcgc gggauccgga gguggcggaa 1560
gcgguggcgg agguucucac caccaucauc accaucacca uuaaaaaaaa aaaaaaaaaa 1620
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680
aaaaaaaaaa aa 1692
<210> 26
<211> 607
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL11的包括UTR和polyA的密码子优化的RNA序列,
所有尿苷残基都是1-甲基-假尿苷
<400> 26
uaauacgacu cacuauaagg acucacuauu uguuuucgcg cccaguugca aaaagugucg 60
ucaccauggg uuuggcuuuu aguggagcua ggccuuguug uuguaggcau aaugugauua 120
uuacugaugg uggcgaagug gugucacuca cugcucauga guucgauguu guugauauag 180
aaagugagga agaagggaau uucuaugucc cgccagauau gcggguagug acuagagccc 240
ccggcccaca auauagaaga gccagcgauc cacccucucg acauacucga cgacgagauc 300
cagauguagc acggccccca gcgaccuuga ccccaccccu guccgauucc gagugagaga 360
gcucgcuuuc uugcugucca auuucuauua aagguuccuu uguucccuaa guccaacuac 420
uaaacugggg gauauuauga agggccuuga gcaucuggau ucugccuaau aaaaaacauu 480
uauuuucauu gcugcgucca aucaaccucu ggauuacaaa aaaaaaaaaa aaaaaaaaaa 540
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600
aaaaaaa 607
<210> 27
<211> 1435
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL16的包括UTR和polyA的密码子优化的RNA序列,
所有尿苷残基都是1-甲基-假尿苷
<400> 27
uaauacgacu cacuauaagg acucacuauu uguuuucgcg cccaguugca aaaagugucg 60
ucaccauggc ucaaagagcc cuguggagac cacaagccac cccuggacca cccggcgcug 120
ccgcuccacc uggacauaga ggugcucccc cagaugcuag agcuccugau ccagguccgg 180
aagcagauuu gguugccaga auugcgaaua gcguguuugu cuggagaguu gugcgcggug 240
augaaaggcu gaaaaucuuc cgcugucuga cuguacuuac ggaacccuua ugucaagucg 300
cauuacccga uccugauccu gaaagagcuc uguuuuguga aaucuuucug uaucucaccc 360
gcccgaaagc ucuccggcuc ccgucuaaua ccuucuucgc gaucuucuuc uucaauagag 420
aaaggcggua uugugcaaca gugcaucuuc ggucuguuac ccauccgaga acaccacucc 480
ucuguacacu ggcuuuugga caucucgagg cggcuucucc acccgaagag acuccugauc 540
cagcagcuga gcaacuggca gaugaaccag uugcacauga acuugauggg gcauaucucg 600
uuccaacuga gccaccacca aacccaggug ccuguugcgc guugggacca ggagcuuggu 660
ggcaucugcc aggugggcgg aucuauuguu gggcuaugga cgacgaucuc gguagucugu 720
gcccacccgg gagucgugcu agacaccucg gcuggcuucu uucacgcauc acggauccac 780
ccgggggggg cggggcaugu gcucccacag cucauauuga cagugcuaau gcccuuuggc 840
gagcuccagc aguugcugaa gcuugcccgu guguggcucc uuguaugugg agcaacaugg 900
cgcaacggac acuugcugug cgcggugaug ccucacucug ccaauugcug uuuggucauc 960
cugucgaugc ugugauacuu cgucaagcca cuagacgucc caggauuaca gcacaucucc 1020
augaaguggu gguaggaagg gauggagcag agagugugau uaggcccacg uccgcuggcu 1080
ggagacugug uguguuaagc uccuacacaa guaggcuuuu cgcuacuagu uguccggccg 1140
uugcaagagc ugucgcaagg gcauccaguu cugacuauaa guaggagagc ucgcuuucuu 1200
gcuguccaau uucuauuaaa gguuccuuug uucccuaagu ccaacuacua aacuggggga 1260
uauuaugaag ggccuugagc aucuggauuc ugccuaauaa aaaacauuua uuuucauugc 1320
ugcguccaau caaccucugg auuacaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1435
<210> 28
<211> 1915
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的UL21的包括UTR和polyA的密码子优化的RNA序列,
所有尿苷残基都是1-甲基-假尿苷
<400> 28
uaauacgacu cacuauaagg acucacuauu uguuuucgcg cccaguugca aaaagugucg 60
ucaccaugga acuguccuac gcaacaacuc uucaucauag agauguaguc uucuauguga 120
ccgccgaucg gaauagggca uauuucgucu guggcgguag ugucuacucu gugggcagac 180
cgagggacag ccaaccuggc gagaucgcua aauucgggcu cgucgugagg gguaccggac 240
caaaggauag aaugguggca aauuaugugc gguccgaauu gaggcaaaga ggucuccgcg 300
auguuagacc uguuggcgaa gaugaaguau uucucgauuc cguuugcuug uugaauccaa 360
auguuuccuc agaacgcgau guaaucaaca cuaaugaugu cgaggucuug gaugaguguc 420
ucgcugagua uuguacaagc cucaggacga gucccggugu uuuagucaca ggaguuaggg 480
ucagagcacg cgauagagug auugaauuau ucgaacaucc cgcuauugua aauaucucua 540
gccgauuugc auauacgccu ucuccauaug uguuugcucu cgcucaagcc caucugccac 600
gucugccauc cucacucgaa ccgcucguau cuggguuguu cgaugggaua ccggcaccaa 660
gacaaccgcu cgaugcucga gaucgccgua ccgauguggu uauaaccggg acaagagcac 720
cacggccuau ggcgggaaca ggcgcaggcg gagccggcgc uaaacgcgcu acaguguccg 780
aauuuguaca ggucaaacau auagauagag uggucucacc uaguguaagc agugcgccac 840
ccccuagugc gccugaugcu ucacuucccc cgcccggacu gcaagaagcg gcaccaccag 900
gaccaccccu gagagaauua uggugggucu uuuaugcugg agauagggcu cuugaagaac 960
cucaugcaga aagcggccug accagggaag aagugagggc uguccacggu uuuagagaac 1020
aagccuggaa auuauucggc ucuguuggag cccccagggc cuuccugggc gcagcauuag 1080
cacucucucc cacacagaaa cuggcagugu auuauuacuu gauacaucgc gaaaggagaa 1140
ugucuccguu uccggcccuu guacgcuugg ugggaagaua uauucaaagg cauggacucu 1200
augugccugc cccagaugag cccacccucg cugacgcuau gaauggucug uuucgggaug 1260
cucuugcugc aggcacaguu gcagaacaac uguuaauguu ugauuuguua cccccuaaag 1320
auguaccagu uggaucugau gcaagagcug auagugcugc acuccucaga uucgucgaua 1380
gccagcggcu cacacccggu ggcagcguga gcccagaaca ugugauguau cucggggcau 1440
uucuuggugu ucuuuaugcu gggcauggca gacuugcugc cgcgacucac acugcucggu 1500
ugacaggugu aacaagccuc guuuugacag uuggcgaugu agauaggaug ucagccuucg 1560
aucgaggucc agccggcgca gcaggacgaa cuagaacugc aggauaucuc gaugcucucu 1620
uaacagugug ucuugcccgg gcucaacaug ggcaauccgu uuaggagagc ucgcuuucuu 1680
gcuguccaau uucuauuaaa gguuccuuug uucccuaagu ccaacuacua aacuggggga 1740
uauuaugaag ggccuugagc aucuggauuc ugccuaauaa aaaacauuua uuuucauugc 1800
ugcguccaau caaccucugg auuacaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1915
<210> 29
<211> 1339
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的gD的包括UTR和polyA的密码子优化的RNA序列,
所有尿苷残基都是1-甲基-假尿苷
<400> 29
uaauacgacu cacuauaagg acucacuauu uguuuucgcg cccaguugca aaaagugucg 60
ucaccauggg ccgccucacu agugggguug guacugcugc guuacugguc guggcugucg 120
ggcuuagagu gguuugugcu aaguaugcac uugcugaucc aucacugaaa auggcagacc 180
caaaccgcuu cagggguaag aauuugcccg ugcuggauca auugacagau ccaccgggcg 240
uuaaaagagu cuaucauauc caacccaguc ucgaagaucc uuuucaaccc ccuuccauuc 300
ccauuaccgu auauuaugcc guauuagagc gggcuugucg uuccguacuu uugcacgcgc 360
cauccgaagc accucaaauu gucagaggag cauccgauga agcuagaaaa cauaccuaua 420
aucucacaau agcuugguac agaaugggcg auaacugugc cauuccaauu acagucaugg 480
aguauacgga auguccuuau aauaaaaguc ucggaguaug uccaauuaga acccaaccac 540
gguggucaua uuacgauucc uucagugcgg uuucugaaga caaucucggg uuucucaugc 600
augcuccagc uuuugaaacu gcaggcacau aucuuagacu gguuaaaauu aaugauugga 660
ccgaaauaac ccaguucauu uuggaacauc gagcuagggc aagcuguaaa uaugcccugc 720
cucuccggau uccaccugcc gcuuguuuga cuagcaaagc uuaucagcaa ggaguaaccg 780
uagauucuau ugguaugcug ccuagguuca uuccagagaa ucaacggacu guugcucucu 840
auucccugaa gauugcggga uggcaugggc cuaaaccacc cuauacauca acucuuuuac 900
cgcccgaacu cucugauacg acaaaugcua cacagccaga gcuggugccc gaggauccag 960
aagauagugc ucugcuggaa gacccggcug gcacagucuc cagccaaauu ccacccaauu 1020
ggcauauucc cucaauucaa gauguggccc cucaucaugc uccugcugca ccgaguaauc 1080
cagguugaga gagcucgcuu ucuugcuguc caauuucuau uaaagguucc uuuguucccu 1140
aaguccaacu acuaaacugg gggauauuau gaagggccuu gagcaucugg auucugccua 1200
auaaaaaaca uuuauuuuca uugcugcguc caaucaaccu cuggauuaca aaaaaaaaaa 1260
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320
aaaaaaaaaa aaaaaaaaa 1339
<210> 30
<211> 1474
<212> RNA
<213> 人工序列
<220>
<223> HSV-2的ICP4的包括UTR和polyA的密码子优化的RNA序列,
所有尿苷残基都是1-甲基-假尿苷
<400> 30
uaauacgacu cacuauaagg acucacuauu uguuuucgcg cccaguugca aaaagugucg 60
ucaccauggc aguucgcaga gcuggccggc aaccaccucg cccucuuggc ggugccagag 120
gcggcggcgg ugccgguccu guaagagguc ugggucggcc uggagccaga guaggggcua 180
gagcaggccg cagaggagcg gcuguaagac ccgaccaugc agcggcagug caugcuggga 240
gaggugggga uggaguugca ccagaaccag cucgcggggc cagaggacgc ggugcagguc 300
cuggucucuu gccugacuua ggucgcgggg cccagcaaca auuacugcau uuacgccagc 360
gcggagccgg aagggcccca ccuggaguga gacacggugg ugggccucuc cgauugggac 420
ccggcgccag ggguggacga agaggacaug aacccccuuu gcgaccacgu gcggaagggu 480
ugcccgccga ucaaccugca ccaaggcuga gagcuccugc uggggcccgc gagagaggag 540
cugauagagg ugcgaaucca aggagaagac ggcgccgcca accacccaga agaaggcggc 600
ggccucggga agcucgacgu agaagaaggc cuguugcagu ggggggggga guugccgguc 660
gcagagcucg aggggcacgc cgacugagac guaggggcgg agccagacgu cccggcgcac 720
cugaacgaag ggcucgauug agagcugguc ggggacggcg aaggcgacgc cguagacgcc 780
guaggcgacg augguggcgc agaccugccc gcggaggagg cccuagaggg agaggcguuc 840
cgggccguuu gccgcgggau ccaggugggg caggaggagg gcuuaggcgc aggccuggcg 900
gacgggcagg ggcagggcgg ucaccagcga ggagagcacc ugccccuggc gccagaggca 960
gagggcggcc agcagcaagg cgaagggccc cgccugccag guuagccgca agggcugcgg 1020
ugagagcccg aagggccggc gccgaugcac cagcccgcgg ccccgcaaga ggccgaaggc 1080
aaaggggccg acgaggaagg cgcgcccgua gagaaccggg caggcggggc ccugguccgg 1140
gcgcugcagc agaaccagca ccagcagaac uucgacggag aagaaggaga gggccugcgc 1200
ucccugagcc agaacccgcc uaggagagcu cgcuuucuug cuguccaauu ucuauuaaag 1260
guuccuuugu ucccuaaguc caacuacuaa acugggggau auuaugaagg gccuugagca 1320
ucuggauucu gccuaauaaa aaacauuuau uuucauugcu gcguccaauc aaccucugga 1380
uuacaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1474
<210> 31
<211> 1320
<212> PRT
<213> 人疱疹病毒2
<400> 31
Met Ser Ala Glu Gln Arg Lys Lys Lys Thr Thr Thr Thr Thr Gln Gly
1 5 10 15
Arg Gly Ala Glu Val Thr Met Ala Asp Glu Asp Gly Gly Arg Leu Arg
20 25 30
Ala Ala Ala Glu Thr Thr Gly Gly Pro Gly Ser Pro Asp Pro Ala Asp
35 40 45
Gly Pro Pro Pro Thr Pro Asn Pro Asp Arg Arg Pro Ala Ala Arg Pro
50 55 60
Gly Phe Gly Trp His Gly Gly Pro Glu Glu Asn Glu Asp Glu Asp Asp
65 70 75 80
Asp Ala Ala Ala Asp Ala Asp Glu Ala Ala Pro Ala Ser Gly Glu Ala
85 90 95
Val Asp Glu Pro Ala Ala Asp Gly Val Val Ser Pro Arg Gln Leu Ala
100 105 110
Leu Leu Ala Ser Met Val Asp Glu Ala Val Arg Thr Ile Pro Ser Pro
115 120 125
Pro Pro Glu Arg Asp Gly Ala Glu Glu Glu Ala Ala Arg Ser Pro Ser
130 135 140
Pro Pro Arg Thr Pro Ser Met Arg Ala Asp Tyr Gly Glu Glu Asn Asp
145 150 155 160
Asp Asp Asp Asp Asp Asp Asp Asp Asp Arg Asp Ala Gly Arg Trp Val
165 170 175
Arg Gly Pro Glu Thr Thr Ser Ala Val Arg Gly Ala Tyr Pro Asp Pro
180 185 190
Met Ala Ser Leu Ser Pro Arg Pro Pro Ala Pro Arg Arg His His His
195 200 205
Arg His His Arg Arg Arg Arg Ala Pro Arg Arg Arg Ser Ala Ala Ser
210 215 220
Asp Ser Ser Lys Ser Gly Ser Ser Ser Ser Ala Ser Ser Ala Ser Ser
225 230 235 240
Ser Ala Ser Ser Ser Ser Ser Ala Ser Ala Ser Ser Ser Asp Asp Asp
245 250 255
Asp Asp Asp Asp Ala Ala Ala Arg Ala Pro Ala Ser Ala Ala Asp His
260 265 270
Ala Ala Gly Gly Thr Leu Gly Ala Asp Asp Glu Glu Ala Gly Val Pro
275 280 285
Ala Arg Ala Pro Gly Ala Ala Pro Arg Pro Ser Pro Pro Arg Ala Glu
290 295 300
Pro Ala Pro Gly Ala Gly Ala Gly Ser Ala Arg Thr Pro Ala Ala Thr
305 310 315 320
Ala Gly Arg Leu Glu Arg Arg Arg Ala Arg Ala Ala Val Ala Gly Arg
325 330 335
Asp Ala Thr Gly Arg Phe Thr Ala Gly Arg Pro Arg Arg Val Glu Leu
340 345 350
Asp Ala Asp Ala Ala Ser Gly Ala Phe Tyr Ala Arg Tyr Arg Asp Gly
355 360 365
Tyr Val Ser Gly Glu Pro Trp Pro Gly Ala Gly Pro Pro Pro Pro Gly
370 375 380
Arg Val Leu Tyr Gly Gly Leu Gly Asp Ser Arg Pro Gly Leu Trp Gly
385 390 395 400
Ala Pro Glu Ala Glu Glu Ala Arg Ala Arg Phe Glu Ala Ser Gly Ala
405 410 415
Pro Ala Pro Val Trp Ala Pro Glu Leu Gly Asp Ala Ala Gln Gln Tyr
420 425 430
Ala Leu Ile Thr Arg Leu Leu Tyr Thr Pro Asp Ala Glu Ala Met Gly
435 440 445
Trp Leu Gln Asn Pro Arg Val Ala Pro Gly Asp Val Ala Leu Asp Gln
450 455 460
Ala Cys Phe Arg Ile Ser Gly Ala Ala Arg Asn Ser Ser Ser Phe Ile
465 470 475 480
Ser Gly Ser Val Ala Arg Ala Val Pro His Leu Gly Tyr Ala Met Ala
485 490 495
Ala Gly Arg Phe Gly Trp Gly Leu Ala His Val Ala Ala Ala Val Ala
500 505 510
Met Ser Arg Arg Tyr Asp Arg Ala Gln Lys Gly Phe Leu Leu Thr Ser
515 520 525
Leu Arg Arg Ala Tyr Ala Pro Leu Leu Ala Arg Glu Asn Ala Ala Leu
530 535 540
Thr Gly Ala Arg Thr Pro Asp Asp Gly Gly Asp Ala Asn Arg His Asp
545 550 555 560
Gly Asp Asp Ala Arg Gly Lys Pro Ala Ala Ala Ala Ala Pro Leu Pro
565 570 575
Ser Ala Ala Ala Ser Pro Ala Asp Glu Arg Ala Val Pro Ala Gly Tyr
580 585 590
Gly Ala Ala Gly Val Leu Ala Ala Leu Gly Arg Leu Ser Ala Ala Pro
595 600 605
Ala Ser Ala Pro Ala Gly Ala Asp Asp Asp Asp Asp Asp Asp Gly Pro
610 615 620
Gly Gly Gly Gly Gly Gly Arg Arg Ala Glu Ala Gly Arg Val Ala Val
625 630 635 640
Glu Cys Leu Ala Ala Cys Arg Gly Ile Leu Glu Ala Leu Ala Glu Gly
645 650 655
Phe Asp Gly Asp Leu Ala Ala Val Pro Gly Leu Ala Gly Ala Arg Pro
660 665 670
Ala Ala Pro Pro Arg Pro Gly Pro Ala Gly Ala Ala Ala Pro Pro His
675 680 685
Ala Asp Ala Pro Arg Leu Arg Ala Trp Leu Arg Glu Leu Arg Phe Val
690 695 700
Arg Asp Ala Leu Val Leu Met Arg Leu Arg Gly Asp Leu Arg Val Ala
705 710 715 720
Gly Gly Ser Glu Ala Ala Val Ala Ala Val Arg Ala Val Ser Leu Val
725 730 735
Ala Gly Ala Leu Gly Pro Ala Leu Pro Arg Ser Pro Arg Leu Leu Ser
740 745 750
Ser Ala Ala Ala Ala Ala Ala Asp Leu Leu Phe Gln Asn Gln Ser Leu
755 760 765
Arg Pro Leu Leu Ala Asp Thr Val Ala Ala Ala Asp Ser Leu Ala Ala
770 775 780
Pro Ala Ser Ala Pro Arg Glu Ala Arg Lys Arg Lys Ser Pro Ala Pro
785 790 795 800
Ala Arg Ala Pro Pro Gly Gly Ala Pro Arg Pro Pro Lys Lys Ser Arg
805 810 815
Ala Asp Ala Pro Arg Pro Ala Ala Ala Pro Pro Ala Gly Ala Ala Pro
820 825 830
Pro Ala Pro Pro Thr Pro Pro Pro Arg Pro Pro Arg Pro Ala Ala Leu
835 840 845
Thr Arg Arg Pro Ala Glu Gly Pro Asp Pro Gln Gly Gly Trp Arg Arg
850 855 860
Gln Pro Pro Gly Pro Ser His Thr Pro Ala Pro Ser Ala Ala Ala Leu
865 870 875 880
Glu Ala Tyr Cys Ala Pro Arg Ala Val Ala Glu Leu Thr Asp His Pro
885 890 895
Leu Phe Pro Ala Pro Trp Arg Pro Ala Leu Met Phe Asp Pro Arg Ala
900 905 910
Leu Ala Ser Leu Ala Ala Arg Cys Ala Ala Pro Pro Pro Gly Gly Ala
915 920 925
Pro Ala Ala Phe Gly Pro Leu Arg Ala Ser Gly Pro Leu Arg Arg Ala
930 935 940
Ala Ala Trp Met Arg Gln Val Pro Asp Pro Glu Asp Val Arg Val Val
945 950 955 960
Ile Leu Tyr Ser Pro Leu Pro Gly Glu Asp Leu Ala Ala Gly Arg Ala
965 970 975
Gly Gly Gly Pro Pro Pro Glu Trp Ser Ala Glu Arg Gly Gly Leu Ser
980 985 990
Cys Leu Leu Ala Ala Leu Gly Asn Arg Leu Cys Gly Pro Ala Thr Ala
995 1000 1005
Ala Trp Ala Gly Asn Trp Thr Gly Ala Pro Asp Val Ser Ala Leu
1010 1015 1020
Gly Ala Gln Gly Val Leu Leu Leu Ser Thr Arg Asp Leu Ala Phe
1025 1030 1035
Ala Gly Ala Val Glu Phe Leu Gly Leu Leu Ala Gly Ala Cys Asp
1040 1045 1050
Arg Arg Leu Ile Val Val Asn Ala Val Arg Ala Ala Asp Trp Pro
1055 1060 1065
Ala Asp Gly Pro Val Val Ser Arg Gln His Ala Tyr Leu Ala Cys
1070 1075 1080
Glu Val Leu Pro Ala Val Gln Cys Ala Val Arg Trp Pro Ala Ala
1085 1090 1095
Arg Asp Leu Arg Arg Thr Val Leu Ala Ser Gly Arg Val Phe Gly
1100 1105 1110
Pro Gly Val Phe Ala Arg Val Glu Ala Ala His Ala Arg Leu Tyr
1115 1120 1125
Pro Asp Ala Pro Pro Leu Arg Leu Cys Arg Gly Ala Asn Val Arg
1130 1135 1140
Tyr Arg Val Arg Thr Arg Phe Gly Pro Asp Thr Leu Val Pro Met
1145 1150 1155
Ser Pro Arg Glu Tyr Arg Arg Ala Val Leu Pro Ala Leu Asp Gly
1160 1165 1170
Arg Ala Ala Ala Ser Gly Ala Gly Asp Ala Met Ala Pro Gly Ala
1175 1180 1185
Pro Asp Phe Cys Glu Asp Glu Ala His Ser His Arg Ala Cys Ala
1190 1195 1200
Arg Trp Gly Leu Gly Ala Pro Leu Arg Pro Val Tyr Val Ala Leu
1205 1210 1215
Gly Arg Asp Ala Val Arg Gly Gly Pro Ala Glu Leu Arg Gly Pro
1220 1225 1230
Arg Arg Glu Phe Cys Ala Arg Ala Leu Leu Glu Pro Asp Gly Asp
1235 1240 1245
Ala Pro Pro Leu Val Leu Arg Asp Asp Ala Asp Ala Gly Pro Pro
1250 1255 1260
Pro Gln Ile Arg Trp Ala Ser Ala Ala Gly Arg Ala Gly Thr Val
1265 1270 1275
Leu Ala Ala Ala Gly Gly Gly Val Glu Val Val Gly Thr Ala Ala
1280 1285 1290
Gly Leu Ala Thr Pro Pro Arg Arg Glu Pro Val Asp Met Asp Ala
1295 1300 1305
Glu Leu Glu Asp Asp Asp Asp Gly Leu Phe Gly Glu
1310 1315 1320
Claims (15)
1.一种疫苗组合物,其包含一种或多种mRNA,其中所述mRNA中的每一种编码选自以下的单纯疱疹病毒(HSV)结构蛋白或其免疫原性片段:
(i)UL48;
(ii)UL48和UL49;
(iii)UL11、UL16和UL21;或
(iv)UL31和UL34。
2.根据权利要求1所述的疫苗组合物,其中UL48具有与SEQ ID NO:6的氨基酸序列具有80%或更高同一性的氨基酸序列,UL49具有与SEQ ID NO:7的氨基酸序列具有62%或更高同一性的氨基酸序列,UL11具有与SEQ ID NO:1的氨基酸序列具有75%或更高同一性的氨基酸序列,UL16具有与SEQ ID NO:2的氨基酸序列具有72%或更高同一性的氨基酸序列,UL21具有与SEQ ID NO:3的氨基酸序列具有80%或更高同一性的氨基酸序列,UL31具有与SEQ ID NO:8的氨基酸序列具有85%或更高同一性的氨基酸序列,UL34具有与SEQ ID NO:8的氨基酸序列具有70%或更高同一性的氨基酸序列。
3.根据权利要求1或2所述的疫苗组合物,其中当以疫苗组合物的形式施用于受试者时,所述HSV mRNA中的每一种能够引发免疫应答。
4.根据权利要求1至3中任一项所述的疫苗组合物,其进一步包含至少一种编码单纯疱疹病毒(HSV)糖蛋白的mRNA,所述HSV糖蛋白选自a)具有与SEQ ID NO:11的氨基酸序列具有70%或更高同一性的氨基酸序列的HSV糖蛋白D(gD)或其免疫原性片段,b)具有与SEQ IDNO:10的氨基酸序列具有70%或更高同一性的氨基酸序列的HSV糖蛋白B(gB)或其免疫原性片段,和c)具有与SEQ ID NO:4的氨基酸序列具有70%或更高同一性或与SEQ ID NO:5的氨基酸序列具有80%或更高同一性的氨基酸序列的HSV糖蛋白E(gE)或其免疫原性片段,或其任何组合。
5.根据权利要求4所述的疫苗组合物,其包含:
(i)UL48和gD和/或gB,任选的ICP4;
(ii)UL48和UL49与gE;
(iii)UL11、UL16和UL21与gE、gD和/或gB;或
(iv)UL31和UL34与gD和/或gB。
6.根据权利要求4或5所述的疫苗组合物,其中所述至少一种编码单纯疱疹病毒(HSV)糖蛋白的mRNA是包含一个或多个假尿苷残基的核苷修饰的mRNA,优选地其中所述一个或多个假尿苷残基包含m1Ψ(1-甲基假尿苷);m1acp3Ψ(1-甲基-3-(3-氨基-5-羧基丙基)假尿苷)、Ψm(2'-0-甲基假尿苷)、m5D(5-甲基二氢尿苷)、m3Ψ(3-甲基假尿苷)或其任何组合。
7.根据前述权利要求中任一项所述的疫苗组合物,其中所述至少一种mRNA编码HSV-1多肽。
8.根据前述权利要求中任一项所述的疫苗组合物,其中所述至少一种mRNA编码HSV-2多肽。
9.根据权利要求6所述的疫苗组合物,其中
(i)编码HSV gD的所述免疫原性片段的所述核苷修饰的mRNA包含来自HSV-2株333的氨基酸26-331或来自另一种HSV株的同源序列,优选地其中所述核苷修饰的mRNA的核酸序列如SEQ ID NO:12所示;和/或
(ii)编码HSV gE的所述免疫原性片段的所述核苷修饰的mRNA包含来自HSV-2株2.12的氨基酸24-405或来自另一种HSV株的同源序列,优选地其中所述核苷修饰的mRNA的核酸序列如SEQ ID NO:13所示。
10.根据权利要求1-9中任一项所述的疫苗组合物,其中所述mRNA中的一种或多种还包含;
(i)poly-A尾,优选地根据SEQ ID NOs:25-30中的一个或多个;
(ii)m7GpppG帽、3'-0-甲基-m7GpppG帽或抗反向帽类似物;
(iii)帽非依赖性翻译增强子;和/或
(iv)增强翻译的5'和3'非翻译区,优选地根据SEQ ID NOs:25-30中的一个或多个。
11.根据权利要求1-10中任一项所述的疫苗组合物,其中所述mRNA中的一种或多种被包封在纳米颗粒、脂质、聚合物、胆固醇或细胞穿透肽中,优选地在脂质体纳米颗粒中。
12.根据权利要求1-11中任一项所述的疫苗组合物,其用于治疗或预防受试者中的单纯疱疹病毒(HSV)感染。
13.根据权利要求12所述用途的疫苗组合物,其中所述HSV感染选自HSV-1感染、HSV-2感染、原发性HSV感染、原发性HSV感染后的爆发、复发或HSV唇、潜伏性HSV感染的再激活、HSV脑炎、HSV新生儿感染、生殖器HSV感染或口腔HSV感染。
14.根据权利要求13或14所述用途的疫苗组合物,其中所述疫苗组合物用于肌内施用、皮下施用、皮内施用、鼻内、阴道内、直肠内施用或局部施用,优选地其中所述组合物是用于注射的疫苗。
15.根据前述权利要求中任一项所述的疫苗组合物,其还包含用于注射的药学上可接受的载体或佐剂。
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CA3002822A1 (en) * | 2015-10-22 | 2017-04-27 | Modernatx, Inc. | Herpes simplex virus vaccine |
KR102457556B1 (ko) * | 2016-03-14 | 2022-10-21 | 레드바이오텍 아게 | Hsv 치료를 위한 수단 및 방법 |
CN111246854A (zh) * | 2017-08-17 | 2020-06-05 | 宾夕法尼亚大学理事会 | 编码单纯疱疹病毒糖蛋白的修饰mrna疫苗及其用途 |
BR112021010611A2 (pt) * | 2018-12-14 | 2021-08-24 | Glaxosmithkline Biologicals S.A. | Composições e métodos de vacina de reforço (prime boost) heterólogo |
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2022
- 2022-03-11 CN CN202280034408.3A patent/CN117295516A/zh active Pending
- 2022-03-11 BR BR112023018282A patent/BR112023018282A2/pt unknown
- 2022-03-11 JP JP2023555311A patent/JP2024512394A/ja active Pending
- 2022-03-11 CA CA3211277A patent/CA3211277A1/en active Pending
- 2022-03-11 AU AU2022233957A patent/AU2022233957A1/en active Pending
- 2022-03-11 EP EP22714389.8A patent/EP4304641A1/en active Pending
- 2022-03-11 KR KR1020237034532A patent/KR20230156744A/ko unknown
- 2022-03-11 US US18/280,908 patent/US20240156951A1/en active Pending
- 2022-03-11 WO PCT/EP2022/056345 patent/WO2022189634A1/en active Application Filing
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AU2022233957A1 (en) | 2023-10-12 |
AU2022233957A9 (en) | 2023-10-26 |
EP4304641A1 (en) | 2024-01-17 |
KR20230156744A (ko) | 2023-11-14 |
BR112023018282A2 (pt) | 2023-12-12 |
JP2024512394A (ja) | 2024-03-19 |
CA3211277A1 (en) | 2022-09-15 |
WO2022189634A1 (en) | 2022-09-15 |
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