CN101888854A - 用流感病毒血凝素假型包装的慢病毒和使用方法 - Google Patents
用流感病毒血凝素假型包装的慢病毒和使用方法 Download PDFInfo
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Abstract
本发明涉及细胞培养方法，试剂盒和细胞系，用于在降低水平的一或多种污染物的存在下制备重组产物，例如治疗蛋白和抗体，本发明还涉及纯化所述产物的方法。
Description
本发明涉及用来自其他类型病毒的病毒蛋白例如流感病毒血凝素(HA)、神经氨酸酶(NA)或M2蛋白假型包装的慢病毒(pseudotypedlentivirus)。本发明还涉及修饰的慢病毒载体和基因传递系统；使用假型包装的慢病毒的抗原、免疫原或疫苗；使用假型包装的慢病毒诱导免疫或检测病毒产物的方法；筛选具有抗病毒活性或具有阻断病毒进入宿主细胞能力的分子的基于假型包装的慢病毒的方法。
当病毒正常表达的包膜蛋白被替换为来自不同病毒的外源包膜蛋白或嵌合包膜蛋白或混合包膜蛋白时，病毒被假型包装。假型包装赋予病毒新的属性，例如改变其结合宿主细胞的能力，修饰其天然的宿主范围或允许其将附加的遗传信息转移到宿主细胞内。
慢病毒属代表反转录病毒科的一个属。它们的基本结构包括核心内的RNA基因组，结合受体的包膜蛋白排列于核心上或贯穿核心进行排列。改造的慢病毒载体表现出慢病毒的部分或全部特点，但可包括修饰天然慢病毒功能特点的慢病毒结构改变，其中改造的慢病毒载体源自于天然慢病毒。例如，慢病毒载体的RNA基因组可被修饰以包含用于整合到靶宿主细胞内的外源多核苷酸序列或转基因。通过以外源病毒的包膜蛋白进行替换，修饰慢病毒载体的宿主范围，天然慢病毒的包膜蛋白可被假型包装。
慢病毒载体可为复制型或非复制型。复制型载体编码感染宿主细胞和复制自身所需的全部物质，而非复制型载体则不能。出于生物安全性考虑，而且相比于复制型载体其一般具有携带更多外源遗传物质的更高容量，非复制型载体可为优选的。
制备慢病毒载体、假型包装慢病毒包膜蛋白和使用这类载体转导多核苷酸序列的方法为本领域知晓，并通过引用下列出版物并入本文。
Kingsman等人和美国专利6,669,936描述了缺乏功能性慢病毒辅助基因产物并具有感染和转导能力的慢病毒载体。
Leboulch等人和美国专利6,365,150描述了产生重组慢病毒的慢病毒包装细胞，其中通过几乎消除导致复制型辅助病毒产生的可能性，重组慢病毒提供了更高的安全性。
Marasco等人和美国专利6,830,892描述了可用于筛选靶分子的慢病毒载体。
Marasco等人和美国专利7,078,031描述了采用这些载体的假型包装的慢病毒载体和基因传递。
Spencer等人和美国专利7,090,837描述了采用慢病毒载体的慢病毒包装构建体、包装系统和基因转导。
McKay等人Gene Ther.13：715(2006)描述了采用来自鸡瘟病毒(FPV，H7/Rostok)的流感病毒血凝素(HA)的基于慢病毒的基因传递。
Matrosovich等人(25)表明NA在病毒感染早期发挥重要作用。
这类载体及其使用方法通过引用Current Protocols in Molecular Biology，卷1(2006年11月20日)也被并入本文，特别参看第9章“Introduction of DNAinto Mammalian Cells”或参考上文引用的文献或下文的参考文献部分。
慢病毒载体还可包括一种或多种报道基因，例如编码绿色荧光蛋白(GFP)的多核苷酸。适宜的报道基因、报道基因整合到慢病毒载体上的方法和检测报道基因活性的方法为本领域所熟知，并通过引用Current Protocols in Molecular Biology，卷1(2006年11月20日)并入本文，特别参看第9章Part II，“Uses of fusion genes in mammalian transtection”。
以来自其他病毒的包膜蛋白假型包装的慢病毒载体为各种基础科学和临床应用提供了强大的工具。首先，作为基因传递系统，它可指导基因体外和体内转移进入所需组织和细胞(1，2)。其次，作为基础研究的工具，它可用于揭示包膜蛋白介导的细胞进入的分子机制。第三，作为免疫原，它可用于抗传染病和癌症的疫苗开发(4，5)。第四，作为抗原，它已被用于开发新的中和测定，以测量感染和接种过程中的抗体应答。最后，作为细胞进入的载体，它可用于开发筛选进入阻断剂的高通量系统，从而帮助开发新的抗病毒药物(3)。
来自水泡性口膜炎病毒的G糖蛋白(VSV-G)广泛用于假型包装慢病毒载体，这是因为其可高效进行慢病毒载体的假型包装，以将基因转移靶向到范围宽泛的细胞和组织(8-10)。但是，一些细胞和组织例如极化上皮的顶膜或粘膜组织可抵抗以VSV-G假型包装的慢病毒载体(1，11)。
为了克服这一限制，已采用来自其他病毒例如线状病毒(1，2)、正粘病毒(11，12)、副粘病毒(13)、丙型肝炎病毒(14)和其他反转录病毒(12，15)的包膜蛋白对病毒进行假型包装。
流感病毒是RNA病毒正粘病毒科的成员。流行性感冒俗称流感是鸟类和哺乳类的传染病。人类的常见流感症状是发热、咽痛、肌肉疼痛、严重头痛、咳嗽、虚弱及全身不适。更严重情况下流感可引起肺炎，肺炎是致死疾病，特别是对幼儿和老年人。
流感分为三种类型，分别称为A型流感、B型流感和C型流感(流感家族内的其他两个成员Dhori病毒和Thorgoto病毒通过蜱传播，极少见到)。A型流感病毒(包括禽流感病毒)在人类引起最严重的疾病，但B型流感也经常引起爆发。
A型、B型和C型的命名最初指对病毒的抗体应答的大致分类，现在已知还涉及三种病毒类型相应的M1(衣壳蛋白或基质蛋白)或核蛋白(NP)的遗传差异。这些病毒的遗传差异的研究表明它们曾在某个时候具有共同的祖先。H5N1禽流感病毒属于A型流感病毒。
类型(A、B或C)是流感病毒名称的首个重要部分。其次是亚型，得名于突出于病毒包膜的血凝素(HA)或神经氨酸酶(NA)表面蛋白的大致分类。有16种HA亚型(名为H1-H16)和9种NA亚型(名为N1-N9)。这些A型流感病毒亚型的所有可能组合可感染鸟类，但只有含有H1、H2、H3、H5、H7和H9以及N1、N2和N7表面蛋白的A型流感亚型可感染人类，其中到目前为止仅有H1、H2、H3与N1和N2以不同程度感染过人类。H5亚型被认为是具有广泛人传染性的候选新亚型。由于该亚型对于全球大多数人的免疫系统都是“新的”，如果该亚型产生广泛的人传染性，有可能导致大流行，即在全球各地引发传染。
因此，A型流感病毒的全称同时包括型和亚型，例如流感病毒A/H5N1或流感病毒A/H3N2；书写时可使用括号代替斜杠，即A(H3N2)等。在本申请中，由于讨论的所有流感病毒型均为A型，因此只使用了亚型组合即H3N2来表示病毒。
三种重要的流感病毒蛋白是血凝素(HA)、神经氨酸酶(NA)和M2，它们是流感病毒表面的包膜蛋白。
成熟的流感病毒体表面上的HA突起是HA1和HA2异型二聚体的三聚体复合物(16，17)。它结合到靶细胞表面上含有唾液酸的受体上，并通过介导内吞病毒的膜与内体的膜的融合，负责病毒穿透进入细胞质(18，19)。
HA最初在膜结合核糖体上合成，并以单条多肽前体HA0转运到内质网腔内，随后被切割为两条由二硫键连接的链HA1和HA2。
一种形式的HA0在HA1的羧基末端具有多个碱性氨基酸，它被位于反面高尔基体网络(TGN)的细胞内肽酶切割。第二种形式的HA0在HA1的羧基末端不具有多个碱性氨基酸，它被两组蛋白酶中的其中一组体内切割：纤溶酶(一种凝血因子X样蛋白酶)和类胰蛋白酶Clara(分化的呼吸上皮细胞的产物(22-24))。
NA以同型四聚体存在于成熟流感病毒体的表面上。它催化末端唾液酸和邻近D-半乳糖或D-半乳糖胺之间α-酮苷键的裂解。NA的一个功能是除去HA、NA和细胞表面上的唾液酸(26)。它还可允许运输病毒通过呼吸道上的粘蛋白层，使得病毒可靶向上皮细胞(27)。一些禽流感病毒的NA蛋白还具有引起血细胞凝集的受体结合位点，但仍不清楚该受体结合功能在流感病毒生命周期中的作用。近来发现NA在病毒感染早期也发挥了重要作用(29)。
大约20到60个M2蛋白分子以同型四聚体表达在成熟病毒体的表面。它们起着离子通道的作用，允许离子在病毒脱壳期间进入病毒体，同时还可作为离子通道，调节TGN和转运囊泡的pH。有趣的是，到目前为止禽流感病毒株A/鸡/德国/34(H7N1)鸡瘟病毒(FPV)Rostock是其HA依赖M2在TGN和转运囊泡中的离子通道活性以维持其生物发生过程中正确构象的唯一一种病毒株。没有M2时，FPV H7HA在ER上表达，极少到达细胞表面(11，32)。
FPV H7HA已被用于假型包装反转录病毒和基于EIAV或HIV-1的慢病毒载体(11，33)。近来McKay等人(11)报道M2显著提高FPV H7HA对慢病毒载体的假型包装。此外，他们证实，采用可溶的细菌NA或共表达编码NA的cDNA来处理产生FPVH7HA/M2假型包装的慢病毒的细胞会增加假病毒体从生产细胞的释放。最后，他们证实这种FPV H7HA/M2假型包装的慢病毒有效地转导了离体极化小鼠气管培养以及体内小鼠气管上皮。
正如上文指出，已在鸟类鉴定出16种HA亚型。其中来自血清型1、2和3的HA已传播给人类，并在人际间扩散；而来自血清型5、7和9的HA也已传播给人类，但目前尚未报道人际传播(34)，但在人气道内发现被SAα2，6半乳糖中断的唾液酸寡糖和被SAα2，3半乳糖中断的唾液酸寡糖(35)。
如上所述，结合SAα2，3半乳糖的FPV H7HA具有在生物发生期间依赖M2的独特特点(31，32)。在本发明之前，不清楚以来自其他病毒株的HA假型包装的慢病毒载体是否需要M2和NA。
因此发明人研究各种HA亚型导入到慢病毒时的效应以及NA和M2的效应，旨在改进假型包装的慢病毒和源自这些假型包装的慢病毒的载体。
具体而言本发明涉及以下列蛋白假型包装的慢病毒载体：
包含HA表位或HA细胞附着配体的流感病毒HA蛋白或含HA蛋白片段的蛋白，
其中所述HA蛋白不是鸡瘟病毒H7。
正如本文描述，这种假型包装的慢病毒载体具有许多有益属性，具体而言发明人已发现单纯以HA假型包装的慢病毒载体可允许所需多核苷酸序列或转基因转导进入HA结合的靶细胞。
优选情况下，HA蛋白选自由上文定义的(即不是鸡瘟病毒H7)H1、H2、H5和H7组成的组，更优选自由H1、H2和H5组成的组。具体而言：
-H1蛋白可包含SEQ ID NO：3的肽序列；相应的核酸编码序列是SEQID NO：16；
-H2蛋白可包含SEQ ID NO：1的肽序列；相应的核酸编码序列是SEQID NO：18；或
-H5蛋白可包含SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ IDNO：7的肽序列；相应的核苷酸编码序列是SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21。
还可使用其他可表达上述蛋白但稍有不同的核酸，并可通过已知方法从所述蛋白推演出这些核酸。
HA蛋白是流感病毒毒力和靶特异性的一个决定因素，因此已对HA蛋白进行了广泛而长期的研究。到目前为止分离的各种形式的HA每个都重要，因此具有本发明这类HA分子的新假型包装的慢病毒物质提供了本申请书详述的各种优点。
优选情况下，慢病毒还包含NA。具体而言NA蛋白可包含SEQ IDNO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11的肽序列；相应的核苷酸编码序列是SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ IDNO：25。
虽然假型包装的慢病毒结合到靶细胞和转导其遗传物质时只需要HA，但发明人已发现当假型病毒除HA外还整合NA时，转导效率明显提高。
优选情况下慢病毒载体包含来自H5N1禽流感病毒株的NA。
优选情况下慢病毒载体还包含NA和M2。假型病毒包含NA和M2以及HA时，可实现转导效率的进一步提高。具体而言，M2蛋白由SEQ IDNO：2的核苷酸编码序列编码。
单纯使用NA(而非M2)与H5HA共转染导致H5NA在细胞表面的表达升高，转导效率也显著提高(4到5个对数级)。当HA和NA构建体的比例位于4∶1和8∶1之间时，得到了最佳的转导效率。此外，H5H1、NA和M2的共转染提供了转导效率的额外适度增加(2到3倍)。
发明人业已发现NA可显著增强H5HA假型病毒的转导效率，但M2不能。H5HA/NA/M2假型病毒模拟了流感病毒的早期感染步骤，这使得它们适合于开发高通量测定，以评估对禽流感病毒的中和抗体应答和筛选禽流感病毒的进入阻断剂。
与表达H5的野生型禽流感病毒一样，H5HA/NA/M2假型包装的慢病毒通过受体介导的胞吞作用进入细胞，而且细胞进入被H5HA特异的免疫血清有效地中和。具体而言，H5HA/NA/M2假型病毒的进入可被H5HA特异的小鼠免疫血清以及来自感染H5N1但病愈的人患者的恢复期血清中和。
H5HA/NA/M2假型病毒将遗传物质转导到广泛范围的细胞内，其效率类似于VSV-G假型病毒，这使得假型包装的慢病毒载体具有成为新型转染试剂的巨大潜力。
这些结果至少在两个方面与近来McKay等人报道的慢病毒载体的PFVH7HA假型包装具有极大的差异(11)。首先，在他们的报道中，已证实PFVH7HA在细胞表面的表达需要M2。没有M2的共转染时，仅能在细胞内部检测到PFV H7HA，提示PFV H7HA经分泌途径的运输受损。
相比之下，发明人业已发现M2的表达不是得到其他HA蛋白如H5在包装细胞表面的表达所必需的。
不受任何具体解释的约束，H5HA和PFV H7HA在包装细胞的表面表达对M2依赖性的差异与先前了解的HA的生物发生是一致的(27)。到目前为止，H7HA是其生物发生依赖M2在TGN和转运囊泡中的离子通道活性以维持正确构象的唯一HA(27)。
其次，McKay等人表明M2和NA协同增加(约750倍)PFV H7HA假型包装的慢病毒载体的转导效率。但是，在本发明中，发现共转染NA，而非M2，使得H5HA假型包装的慢病毒载体的转导效率增加了4到5个对数级(图2)。
同样，采用最佳比例的HA和NA来共转染M2，导致转导效率的进一步适度(2到3倍)增加(图3)。M2在我们研究中带来的2到3倍的增加比既往研究中的30倍增加要低得多(11)。这种差异同样可以用PFV H7HA和H5HA在包装细胞中的生物发生过程中对M2的依赖性来解释。但是，不清楚相比于既往研究(例如25倍)NA在本发明中大幅提高HA假型包装的慢病毒载体的转导效率(接近4个对数级)的原因。在他们的研究中，大多数NA效应是使用来自霍乱弧菌的可溶细菌NA蛋白发现的，虽然在某些实验中也检测了来自A/PR/8/34的流感病毒NA的共转染，但没有观察到本发明中的效应。
NA基因源于H5N1禽流感病毒株，其密码子已被优化。不受特定作用机制的约束，因此一种可能的解释是NA对H5HA假型包装的慢病毒载体转导效率的大幅提高是源自高致病性禽流感病毒株的NA的独特性。
具体而言，具有这种增强转导效率的H5HA/NA/M2假型病毒将具有许多基础科学和临床领域的应用。
首先，作为基因传递系统，它可有效地经顶膜转导上皮细胞(11)。因此，它有可能用于直接将基因体内导入到粘膜上皮内。
其次，它可用作基础研究的工具，揭示病毒进入的分子机制，从而揭示潜在发病机制。
最后，该研究所显示的H5HA/NA/M2假型病毒上保存良好的受体结合位点和抗原决定簇(图5和图6)显示，H5HA/NA/M2假型病毒可用于开发高通量测定来全面研究接种H5N1和感染H5N1个体的免疫状态和筛选进入阻断剂，从而开发出新抗病毒药物。
优选情况下，慢病毒载体还包含转基因。
如果它是适宜的报道基因，例如各种形式的绿色荧光蛋白(GFP)或荧光素酶中的其中一种，这种转基因可用作附加标记。或者，转基因可用作选择标记，例如赋予对特定物质如卡那霉素的抗性的选择标记。或者这类转基因可为期望导入到靶细胞内的目的基因产物。具体而言，这可为抗病毒基因，以期研究其对假型包装的慢病毒的效应。
优选情况下，表达HA的多核苷酸的密码子已按靶细胞或宿主细胞进行优化。
这类密码子优化的HA确保在靶细胞或宿主细胞内的最佳表达水平。不同生物具有特定的密码子偏好性，即最常用于指定特定肽的各种氨基酸残基的密码子。通过修饰编码序列，使得它使用靶细胞或宿主细胞的偏好密码子，确保更好和更一致的表达水平。
优选情况下，编码HA和NA以及M2(若有)的多核苷酸的密码子已按靶细胞或宿主细胞进行优化，这些多核苷酸可与本文附录的序列表中列出的核酸序列稍有不同，只要它们能够表达相关蛋白。
在本发明中，假型包装的慢病毒载体不局限于具体的HA、NA和M2蛋白序列，相反发明人已证实来自相同或不同病毒分离物的HA、NA和M2蛋白的组合或实际来自不同HA或NA组的分离物在用于单个假型包装的慢病毒载体时，可具有所需的生物属性和免疫属性。
优选情况下HA蛋白由来自不同HA同源物的至少两个部分组成。
优选情况下NA蛋白由来自不同NA同源物的至少两个部分组成。
发明人业已发现使用包含至少两个天然蛋白的部分的蛋白时，这些嵌合的HA或NA蛋白均具有类似于原蛋白的免疫原性水平，并具有生物活性。发明人业已发现联合被保守残基分隔开的不同HA或NA蛋白的部分或结构域可做到这一点，参考本文的图14和图15即可得到鉴定。
还提供了一种包含本发明慢病毒载体和药用赋形剂、载体和/或免疫学佐剂的组合物。
还提供了一种慢病毒载体的包装系统，其包含：
至少一种表达HA的包装载体，和
转移载体构建体，其包含产生序列和包装序列、表达Gag和Pol慢病毒蛋白的序列并任选包含转基因，
其中所述HA蛋白不是鸡瘟病毒H7HA。
还提供了一种慢病毒载体的包装系统，其包含：
至少一种表达HA的包装载体，
表达Gag和Pol慢病毒蛋白的辅助构建体，和
转移载体构建体，其包含产生序列和包装序列并任选包含转基因；
其中所述HA蛋白不是鸡瘟病毒H7HA。
涉及含有至少一种表达HA的包装载体和转移载体构建体的慢病毒载体包装系统，其中转移载体构建体包含产生序列和包装序列，表达Gag和Pol慢病毒蛋白的序列并任选包含转基因。这类包装系统还可含有至少一种表达HA的包装载体，表达Gag和Pol慢病毒蛋白的辅助构建体，包含产生序列和包装序列和任选转基因的转移载体构建体。优选情况下，所述载体不表达鸡瘟病毒H7HA。还涉及以上述慢病毒载体转染的靶细胞或宿主细胞，其中以本发明的慢病毒载体转染的靶细胞或宿主细胞具有掺入到染色体DNA上的转基因。可将多核苷酸序列或转基因与本发明的慢病毒载体接触，使其转导进入细胞。还提供了一种诱导免疫应答的方法，包括向受试者施用上文定义的慢病毒载体，其量足以诱发针对所述载体的免疫应答。
本发明的另一实施方式是一种诱导免疫应答的方法，其包括向受试者施用上述的慢病毒载体(或以其转染的宿主细胞)，其量足以诱导免疫应答。这种免疫应答可为抗假型包装的慢病毒例如抗HA组分的细胞应答或体液应答。
还提供了一种鉴定中和抗体的方法，其包括：
将上文定义的慢病毒载体与抗体接触，所述接触的时间和条件适合抗体结合到慢病毒载体上，和
测定所述接触对所述慢病毒载体结合或感染宿主细胞的能力的影响。
发明人已开发出具有这种高效率的H5HA/NA/M2假型病毒，用于高通量测定以评估中和抗体应答和筛选H5N1禽流感病毒的进入阻断剂。
该技术的具体应用包括以下应用。以包含HA表位或HA细胞附着配体的流感病毒HA蛋白或含有HA蛋白片段的蛋白假型包装的慢病毒载体。优选情况下，HA(或其他基因，例如NA或M2的基因)的密码子已按特定靶细胞或宿主细胞优化。密码子优化为分子生物学领域所熟知。最优选情况下，慢病毒载体没有以鸡瘟病毒H7HA假型包装，而且HA蛋白是H1、H2、H5或H7，优选是H1、H2或H5。但是，也可用NA例如来自H5N1禽流感病毒株的NA来假型包装这类慢病毒载体。优选情况下，可使用已发现可更有效地假型包装慢病毒载体的同源流感病毒HA和NA对来假型包装所述载体。所述载体还可同时包括NA和M2以及HA。以流感病毒HA蛋白或HA蛋白片段假型包装的慢病毒载体可包括转基因。假型包装的慢病毒载体可混合于或混悬于适宜的缓冲液或培养基中，或与载体或免疫学佐剂混合。促进免疫应答的佐剂例如明矾、弗氏不完全佐剂或弗氏完全佐剂、Ribi佐剂或其他佐剂为免疫学领域所熟知。
还提供了以本发明慢病毒载体转染的靶细胞或宿主细胞。
还提供了一种包含本发明靶细胞或宿主细胞、药用赋形剂、载体和/或免疫学佐剂的组合物。
优选情况下以本发明慢病毒载体转染的靶细胞或宿主细胞，其中所述转基因已被掺入到所述细胞的染色体DNA上。
还提供了一种将多核苷酸序列或转基因转导到细胞内的方法，其包括使细胞与本发明的慢病毒载体在足以发生转导的时间和条件下接触。
还提供了一种鉴定调节病毒对细胞的结合或调节病毒对细胞的感染的分子的方法，其包括：
使细胞与候选分子和本发明假型包装的慢病毒接触，和
测定所述候选分子调节病毒对细胞的结合或抑制病毒对细胞的感染的能力。
通过将慢病毒载体与抗体在适宜抗体结合到慢病毒载体的条件和时间下接触，并测定所述接触对所述慢病毒载体结合或感染宿主细胞能力的影响，可使用上述假型包装的慢病毒载体鉴定或表征中和抗体。本发明的另一方面涉及调节例如增加或降低病毒对细胞的结合的分子或降低(或在某些场合下促进)病毒感染的分子的鉴定或表征，
这类方法包括下列步骤：
使细胞与候选分子和本发明的假型包装的慢病毒接触，随后测定候选分子调节病毒对所述细胞的结合或抑制病毒对细胞的感染的能力。这类方法待检测的分子包括但不限于非蛋白药物、非抗体的肽或多肽、抗体或抗体片段、碳水化合物、脂质和其他药理物质和药物，包括有机剂和无机剂。优选情况下所述分子是非蛋白药物。
或者所述分子是非抗体的肽或多肽。
最优选情况下所述分子是抗体。
最优选情况下所述分子包含碳水化合物或脂质。
为了更好理解本发明和显示本发明是如何实施的，现在仅以例举方式参照下列附图以具体实施方式、依据本发明的方法和过程来显示本发明，其中
图1：显示了表达H5HA、NA和M2的转移载体、包装载体以及DNA构建体的示意图。H5HA在293T包装细胞上的细胞表面表达，其中293T包装细胞分别以转染试剂对照(阴性对照)、H5HA(1A)、H5HA和M2(1B)、H5HA和NA(1C)、以及H5HA、NA和M2(1D)转染。转染细胞先后以H5HA特异的混合免疫血清和结合FITC的山羊抗小鼠IgG抗体进行染色。
图2：显示了以源自293T细胞的上清转导的MDCK细胞的相对荧光素酶活性(RLA)(2A)，其中293T细胞分别以转染试剂对照、含或不含各种标明量的NA假型病毒的H5HA转染；以源自293T细胞的上清转导的MDCK细胞的RLA(2B)，其中293T细胞分别以转染试剂对照、含或不含各种标明量的M2假型病毒的H5HA转染。
图3：显示了以源自293T细胞上清转导的MDCK细胞的相对荧光素酶活性(RLA)，其中293T细胞以含或不含各种标明量的M2的4∶1的H5HA和NA转染。
图4：显示了以转染试剂对照或含有H5HA/NA/M2或相当于10ngHIV-1gag p24的VSV-G假型病毒的上清转导的CHO、MDCK、293T、HeLa、Vero、Caco2、HT29和CEMss细胞的相对荧光素酶活性(RLA)。转染期间，293T细胞以8∶2∶1最佳比例的H5HA、NA和M2转染。
图5：显示了MDCK细胞的相对荧光素酶活性(RLA)，其中MDCK细胞先以巴佛洛霉素A1(5a)或NH4Cl(5b)预处理，后以含有H5HA/NA/M2假型病毒的上清转导。转染期间，293T细胞以8∶2∶1最佳比例的H5HA、NA和M2转染。
图6：显示了以H5HA特异的混合免疫前血清样品或混合免疫后血清样品预处理的H5HA/NA/M2或VSV-G假型病毒的转导效率的抑制百分数。
图7：显示了慢病毒载体的荧光素酶活性结果，其中慢病毒载体以源自几种亚型的禽流感病毒和人流感病毒的流感病毒HA和NA假型包装。同源的流感病毒HA和NA对对慢病毒载体的假型包装更有效，该结果对于流感病毒的进一步研究和利用具有意义。
图8显示了单独以H5HA(进行或不进行外源NA处理)转染或以H5HA/NA共转染的细胞的上清和细胞裂解物的蛋白印记。
图9显示了单独以H5HA(进行或不进行外源NA处理)转染或以H5HA/NA共转染的细胞的分离组分的蛋白印记，其中嵌图A是未转染细胞，嵌图B是单独以H5HA转染的细胞，嵌图C是以H5HA/NA共转染的细胞，嵌图D是以H5HA转染并以外源NA处理的细胞。
图10显示了H5HA的系统发育及H5HA的各种亚进化枝(subclade)。
图11显示了抗H5HA(亚进化枝1.1)的小鼠血清和抗H5N1的恢复期人血清(亚进化枝2.3)的HI(图11A)和微量中和测定(图11B)结果，以作为与新H5HA/NA测定的比较。
图12显示了抗HA(亚进化枝1.1)小鼠血清的新H5HA/NA测定的结果和抗H5N1(亚进化枝2.3)恢复期人血清的新H5HA/NA测定的结果。
图13显示了经HPLC自几种中草药分离的两种化合物的EC50(13A和13C)和CC50(13B和13D)(化合物1：图13A和B；化合物2：图13C和D)。
图14显示了具有多碱性氨基酸位点突变的H1HA序列和不同病毒株的H5MA序列的肽序列比较。
图15显示了不同NA序列的肽序列比较。
本发明还涉及本文附录的序列表列出的和下文总结的许多序列：
SEQ ID NO：1是H2HA的肽序列
SEQ ID NO：2是M基因的核苷酸编码序列
SEQ ID NO：3是H1HA WSN的肽序列
SEQ ID NO：4是H5HA 2004泰国的肽序列
SEQ ID NO：5是H5HA 2005柬埔寨的肽序列
SEQ ID NO：6是H5HA 2006柬埔寨的肽序列
SEQ ID NO：7是H5HA 2006上海的肽序列
SEQ ID NO：8是H5N1 NA 2004泰国的肽序列
SEQ ID NO：9是H5N1 NA 2005 Combiant的肽序列
SEQ ID NO：10是H5N1 NA 2006上海的肽序列
SEQ ID NO：11是T-NA(WU)-2的肽序列
SEQ ID NO：12是H151HA的肽序列
SEQ ID NO：13是H515HA的肽序列
SEQ ID NO：14是H151HA的核苷酸编码序列
SEQ ID NO：15是H515HA的核苷酸编码序列
SEQ ID NO：16是H1HA WSN的核苷酸编码序列
SEQ ID NO：17是H5HA 2004泰国的核苷酸编码序列
SEQ ID NO：18是H2HA的核苷酸编码序列
SEQ ID NO：19是H5HA 2005柬埔寨的核苷酸编码序列
SEQ ID NO：20是H5HA 2006柬埔寨的核苷酸编码序列
SEQ ID NO：21是H5HA 2006上海的核苷酸编码序列
SEQ ID NO：22是H5N1 NA 2004泰国的核苷酸编码序列
SEQ ID NO：23是H5N1 NA Combiant的核苷酸编码序列
SEQ ID NO：24是H5N1 NA上海的核苷酸编码序列
SEQ ID NO：25是T-NA(WU)-2的核苷酸编码序列
现在以例举方式通过发明人预见的具体实施方式描述本发明。下列描述中阐明了许多具体细节以提供透彻的理解。但对于本领域技术人员显而易见的是，本发明实施时可不受这些具体细节的限制。其他情况下，没有描述熟知的方法和结果，以免不必要地模糊描述内容。
实施例1：材料和方法
使用以下材料和方法得到下述数据。
细胞系。包装细胞系293T培养在含有0.5mg/ml G418的DMEM完全培养基中[即添加10％FBS、2mM L-谷氨酰胺、1mM丙酮酸钠、青霉素(100U/ml)，)和链霉素(100μg/ml)的高糖DMEM；Invitrogen Life Technologies]。HeLa、Vero、人CD4T细胞系CEMss、CHO和MDCK细胞系培养在DMEM完全培养基中。购买人上皮细胞系HT29和Caco-2，以DMEM完全培养基培养。
表达密码子优化的H5HA、NA和M2的转移载体、包装载体和DNA质粒。转移载体pHR’CMV-Luc和包装载体pCMVΔR8.2最初由Naldini等人开发(36)。(36).采用GCG Package(Genetic Computer Group，Inc.Madison，WI)测定H5N1禽流感病毒株A/泰国/1(KAN-1)2004的密码子优化的H5HA、NA和M2序列，并按前人描述方法经递归PCR生成这些序列，并将这些序列分别插入到源自pNGVL-3的哺乳动物表达载体CMV/R(38)。形成的质粒构建体分别命名为CMV/R-H5HA、CMV/R-NA和CMV/R-M2(图1A)。
假型病毒的生成。为了产生HIV-1载体的假型病毒，采用磷酸钙沉淀方法以14μg pHR’CMV-Luc、14μg pCMVΔR8.2和编码密码子优化的H5HA(见上文)的2μg DNA质粒(加入或不加入编码密码子优化的NA和M2的各种标明量的DNA质粒)共转染4.5×106个293T包装细胞。作为对照，293T细胞也以HIV-1-荧光素酶转移载体和编码VSV-G的DNA质粒进行共转染。过夜温育后，细胞以HBSS洗涤一次，随后培养在添加100μM丁酸钠的10ml DMEM完全培养基中。8小时后，细胞以HBSS洗涤一次，随后培养在10ml的DMEM完全培养基中。16-20小时时，收集含有假型病毒的上清，按照前人描述方法以ELISA测量293FT包装细胞的上清和/或细胞裂解物中的HIV-1gag p24的量(39)。
假型病毒的转导。在测量假型病毒转导效率的单循环测定中，在1μg/ml聚凝胺的存在下以各种量的含有假型病毒的上清过夜转导1×105个MDCK(ATCC CCL-34)、CEMss(Southwest Foundation for Biomedical Research，sanAntonio，Texas的Jon Allan博士提供的一种人CD4T细胞系)、CHO(ATCCCRL-11398)、293T(ATCC CRL-1573)、HeLa(ATCC CCL-2)、Vero(ATCCCCL-81)、HT-29(ATcc HTB-38)和Caco2细胞(ATCC HTB-37)。细胞随后以HBSS洗涤两次，继而在DMEM完全培养基中培养两天。随后收集细胞，HBSS(无酚红)洗涤一次，重新悬浮于200μl的HBSS(无酚红)中。按照厂家说明(Promega)以BrightGlo荧光素酶测定测量50μl细胞混悬液中的荧光素酶活性。
以编码H5HA的质粒DNA免疫小鼠。每3周向6周龄的雌性BALB/c小鼠(每组5只小鼠)分别(肌内)注射100μg的CMV/R质粒DNA表达载体、CMV/R-HA、CMV/R-HA-mutant-1或CMV/R-HA-mutant-2，一共注射三次。分别于首次免疫前7天和第三次免疫后2周取免疫前血清和免疫后血清。以ELISA和中和测定(见下文)测定抗H5HA的抗体应答。
FACS分析。为了研究H5HA的细胞表面表达，分别以转染试剂对照、H5HA、H5HA/NA、H5/M2和H5HA/NA/M2转导的1X106个293T细胞以免疫H5HA质粒DNA的小鼠血清冰上温育40分钟。细胞随后以FACS缓冲液(含1％BSA和0.02％NaN3的PBS)洗涤两次，继而与结合FITC的山羊抗小鼠IgG Ab冰上温育40分钟。细胞随后以FACS缓冲液洗涤两次，继而以含1％甲醛的0.5ml FACS缓冲液固定。在FACScan(Becton Dickinson，Mountain View，CA)上进行FACS分析。
中和测定。为了测定H5HA/NA/M2假型病毒的转导是否可以被源自H5HA免疫小鼠的血清中和。分别在加入或不加入热灭活的免疫前血清样品和热灭活的免疫后血清样品的逐级5倍稀释液的情况下温育100μl上述产生的H5HA/NA/M2假型病毒和VSV-G假型病毒1小时。混合物随后加入到24孔平板中的MDCK细胞上。过夜温育后，除去含有病毒的上清，替换为新鲜完全培养基。转导48小时后，测量上述转导细胞的荧光素酶活性的量，测定转导效率。中和活性表示为与免疫前血清样品相比，每个免疫后血清样品稀释倍数的转导抑制百分数(荧光素酶活性)。％抑制＝{1-[免疫后血清样品中的荧光素酶/免疫前血清样品中的荧光素酶]}x100。效价按赋予50或90％抑制(IC50或IC90)的血清稀释倍数的倒数计算。
H5HA/NA/M2假型病毒进入的药理学抑制。为了测定H5HA/NA/M2假型病毒是否通过受体介导的胞吞作用进入细胞，使用亲溶酶体剂氯化铵(NH4Cl)和囊泡H+-ATP酶抑制剂巴佛洛霉素A1(BafA1)(Sigma，St.Louis，MO)在H5HA/NA/M2假型病毒转导前和转导期间处理靶细胞。以二甲基亚砜(DMSO)制备BafA1的工作液，-20℃储存。以蒸馏水制备NH4Cl的原液，以0.22μm滤膜除菌。
为了评测BafA1和NH4Cl对H5HA/NA/M2假型病毒进入的效应，24孔平板上每孔接种2×104个MDCK细胞，转导前加入或不加入各种标明量的BafA1和NH4Cl预处理细胞1小时。转导期间，每孔加入含有H5HA/NA/M2假型病毒的100μl上清，并在相同药物的存在下37℃过夜温育。随后除去上清，替换为新鲜的完全培养基。转导48小时后，收集细胞，按上述方法测量转导细胞的荧光素酶活性。
嵌合H151 HA和H515 HA的构建。通过两个HA蛋白特别是H1HA(WSN)SEQ ID NO：3位置72和294处的两个保守半胱氨酸残基之间的结构域交换，制备嵌合H151 HA和H515 HA。为了在第一和第二PCR反应中制备嵌合分子H151的HA开放阅读框，从含有H1HA(WSN)开放阅读框即SEQ ID NO：16的质粒分离第一和第三结构域。在第三反应中，经PCR从含有H5 HA 2004泰国开放阅读框即SEQ ID NO：17的质粒分离第二结构域。在含有上述PCR反应所有混合产物并采用识别和退火到第一结构域的最5’部分和第三结构域的最3’部分的引物的终反应中，进行终反应，通过凝胶纯化得到全长的PCR产物，测定DNA序列，以确保它们是正确的产物并具有正确的DNA序列。形成的H151 HA核酸编码序列是SEQ IDNO：14，它被亚克隆到上述的慢病毒载体上，以备进一步使用和研究。PCR循环条件采用了标准的酶和模板，并使用标准技术设计引物。
为了在第一和第二PCR反应中制备嵌合分子H515的HA开放阅读框，从含有H5HA泰国开放阅读框即SEQ ID NO：17的质粒分离第一和第三结构域。在第三反应中，经PCR从含有H1HA(WSN)开放阅读框即SEQ IDNO：16的质粒分离第二结构域。在含有上述PCR反应所有混合产物并采用识别和退火到第一结构域的最5’部分和第三结构域的最3’部分的引物的终反应中，进行终反应，通过凝胶纯化得到全长的PCR产物，测定DNA序列，以确保它们是正确的产物。形成的H515 HA核酸编码序列是SEQ IDNO：15，它被亚克隆到上述的慢病毒载体上，以备进一步使用和研究。
实施例2：NA的共转染，而非M2单独转染，增强了H5HA在包装细胞上
的细胞表面表达
为了测定编码NA或M2的DNA质粒共转染对H5HA细胞表面表达的效应，在加入或不加入各种量的编码NA或M2的DNA质粒的情况下，以慢病毒转移载体pHR’CMV-Luc和包转载体pCMVΔR8.2以及编码H5HA的DNA质粒转染293T包装细胞(图1A)。转染48小时后，293T包装细胞以抗H5HA特异的免疫血清染色。
相比于之前对FPV H7HA的报道(11)，没有NA和/或M2的共转染时，单纯H5HA的确在包装细胞的细胞表面表达(图1A)，而H5HA和M2的共转染不增强H5HA的细胞表面表达(图1B)。
H5HA和NA的共转染或H5HA、NA和M2的共转染增强了H5HA的细胞表面表达。但是，H5HA在以H5HA和NA共转染的细胞以及以H5HA、NA和M2共转染的细胞中的表达水平非常类似，表明NA而非M2增强了H5HA在包装细胞上的细胞表面表达(图1C和1D)。
此外，发现从以H5HA和NA共转染的细胞和以H5HA、NA和M2共转染的细胞所收集的上清含有类似量的p24，其量比从单独以H5HA转染的细胞收集的上清要高出2倍。
实施例3：NA的共转染，而非M2单独转染，显著增强了H5HA假型包装
的慢病毒载体的转导效率
为了测定编码NA或M2的DNA质粒的共转染对慢病毒载体转导效率的效应，在加入或不加入各种量的编码NA或M2的DNA质粒的情况下，以HIV-1-荧光素酶转移载体和编码H5HA的DNA质粒转染293FT包装细胞。收集含有重组假型病毒的上清，以具有相同量的HIV-1gag p24的上清转导MDCK细胞。转导48小时后，按相对荧光素酶活性测量转导效率。
正如图2A所示，其中没有编码NA或M2的质粒DNA的共转染，在单独以H5HA假型包装的慢病毒载体中检测出极低但可测量的相对荧光素酶活性(RLA大于1000)。但是，H5HA与编码NA的质粒DNA的共转染显著提高了转导效率(RLA范围为500,000-7,000,000)，取决于共转染的NA质粒DNA的量。
当HA和NA构建体的比例为4∶1或8∶1时，得到最佳的转导效率。相比之下，H5HA与只编码各种量的M2的质粒DNA的共转染根本没有转导效率(RLA低于100)(图2B)。实验重复5次以上，得到类似结果。因此，这些结果清楚表明H5HA与NA而非M2的共转染显著增强(接近4个对数级)H5HA假型包装的慢病毒载体的转导效率。
实施例4：M2对H5HA/NA假型包装的慢病毒载体转导效率的效应
发明人继而研究了M2对以H5HA和NA共转染的慢病毒载体的转导效率的效应。为了实现这一目的，在加入或不加入各种量的M2的情况下，以最佳比例(4∶1)的H5HA和NA共转染293T包装细胞。收集含有假型病毒的上清，用于转导MDCK细胞。转导48小时后，按相对荧光素酶活性测量转导效率。
图3显示在各种量的M2的共转染的存在或不存在下，以H5HA/NA假型病毒转导的MDCK细胞的RLA。与H5HA和NA共转染进行假型包装的慢病毒载体所转导的细胞相比(RLA 7,000,000)，M2的共转染导致转导效率适度(约2-3倍)升高。此外，发明人还发现在亚最佳H5HA和NA比例(1∶1.25或16∶1)时，M2的共转染导致转导效率的最小升高甚至下降(数据未显示)。因此，似乎M2带来的适度(2到3倍)升高不仅依赖于M2剂量，还依赖于H5HA和NA的适宜比例。而且，这种升高比之前报道的以FPVH7HA、NA和M2进行假型包装的慢病毒载体的升高要低得多(11)。
实施例5：H5HA/NA/M2假型包装的慢病毒载体的宿主范围
发明人还比较了H5HA/NA/M2假型包装的慢病毒载体和VSV-G假型包装的慢病毒载体在8个不同细胞系CHO、MDCK、293T、HeLa、Vero、Caco2、HT29和CEMss中的转导效率。为了实现这一目的，在聚凝胺的存在下以含有H5HA/NA/M2假型包装的慢病毒载体或VSV-G假型包装的慢病毒载体(相当于10ng的HIV-1gag p24)的上清转导细胞。转导48小时后，按上述方法测量转导细胞的荧光素酶活性。
图4显示了这8个细胞系中每1个检测到的相对荧光素酶活性。除Vero细胞以外，H5HA/NA/M2假型病毒转导在其他所有细胞系中的转导效率与VSV-G假型病毒转导类似或更高。
实施例6：H5HA/NA/M2假型包装的慢病毒载体的细胞进入是通过受体介
导的胞吞作用
接下来测定H5HA/NA/M2假型病毒是否通过受体介导的胞吞作用进入细胞。为了实现这一目的，MDCK靶细胞以各种剂量的巴佛洛霉素A1或NH4Cl进行预处理。由于巴佛洛霉素A1以DMSO溶解，MDCK靶细胞还以相同量的DMSO进行预处理，作为对照。预处理后，细胞以含有H5HA/NA/M2假型病毒的上清转导。转导48小时后测量荧光素酶活性。
图5显示了接受或未接受巴佛洛霉素A1或NH4Cl预处理的MDCK细胞的相对荧光素酶活性。在两种预处理中，RLA均呈现剂量依赖性下降。10nM巴佛洛霉素A1预处理细胞导致转导效率下降1个对数级。50和100nM巴佛洛霉素A1预处理细胞导致转导效率下降2个或更多的对数级(图5A)。1MM NH4Cl预处理细胞导致转导效率下降50％。10MM NH4Cl预处理细胞导致转导效率下降1个对数级(图5B)。因此，发明人确定5HA/NA/M2假型病毒通过受体介导的胞吞作用进入细胞。
实施例7：H5HA特异的免疫血清，而非免疫前血清，中和H5HA/NA/M2
假型病毒的细胞进入
发明人测定了H5HA/NA/M2假型病毒是否可被H5HA特异抗体中和。为了检测这一点，含有H5HA/NA/M假型病毒或VSV-G假型病毒的100μl上清以各种稀释倍数的H5HA特异免疫前血清或免疫后血清在37℃下温育1小时(参看材料和方法，了解详情)。温育后，将假型病毒和血清混合物加入到MDCK靶细胞上进行转导。转导48小时后测量荧光素酶活性。由于VSV-G包膜与细胞质膜的脂质双层的脂质部分相互作用，VSV-G假型病毒绕开了HA包膜与其含唾液酸受体需相互作用以进入细胞的要求。因此，它们被用作阴性对照。免疫后血清样品的中和活性表示为与免疫前血清样品相比，每个免疫后血清样品稀释倍数时的转导抑制百分数(荧光素酶活性)：％抑制＝{1-[免疫后血清样品中的荧光素酶/免疫前血清样品中的荧光素酶]}x100。效价按赋予50或90％抑制(IC50或IC90)的血清稀释倍数的倒数计算。
图6显示了混合免疫后血清样品的抑制百分数，其中血清样品来自以含H5HA突变体的质粒DNA免疫的小鼠(参看材料和方法)。1∶250稀释时，抑制几乎达到100％。1∶500稀释时，抑制为90％。1∶1000稀释时，抑制接近62％。1∶2000稀释时，抑制为50％。但在相同的稀释倍数时，没有检测到对VSV-G假型病毒的抑制(图6)。
实施例8：NA增强H5HA的机制的进一步研究
为了测定NA增强的机制，发明人在以H5HA单独转染(加或不加外源NA处理)或以H5HA/NA共转染的细胞中，比较了HA和HIV-1gag蛋白在转染细胞、浓缩上清和假病毒颗粒的量。
发现尽管H5HA具有多碱性氨基酸切割位点，但只有50％HA0被切割为HA1和HA2，而HA0、切割的HA和HIV-1gag的量是相似的，不论细胞是以H5HA单独转染还是以H5HA/NA共转染，参见图8的细胞裂解嵌图。
但是，在H5HA/NA共转染的细胞的浓缩上清中检测到比单独转染H5HA的细胞多得多的切割HA和HIV-1gag以及更少的HA0，参见图8的上清嵌图。
而且，发现在H5HA/NA共转染的细胞所产生的假病毒颗粒中检测到比H5HA单独转染以及外源NA处理的细胞多得多的H5HA，参见图9。
因此，在H5HA/NA假型包装的慢病毒载体中，共转染的NA使用了两种以前未知的机制来增强假型病毒的转导效率，即优先释放含切割的H5HA的假型病毒和掺入更多量的H5HA到假病毒颗粒中。
实施例9：新的抗HA抗体的中和活性测定
目前测量抗HA抗体、免疫个体血清以及感染个体血清的中和活性的标准测定有：1)血凝抑制(HI)测定和2)微量中和测定。
HI测定是替代(surrogate)测定，可能不能反映抗体真实的中和活性。该测试利用了血凝素凝集红细胞(红细胞，RBC)这一原理来鉴定病毒。当加入抗血凝素抗原的抗体时，它们将结合到HA分子的抗原位点，抑制HA与RBC上受体的结合。因此，当存在HA时，抗体将结合到HA上，阻止RBC的血细胞凝集。
微量中和测定利用了野生型病毒和更精确测量抗HA抗体的中和活性的基于细胞的测定。但是，该测定的示值读数是在显微镜下观察的细胞病变效应(CPE)。因此，它是一种主观测量，难以定量和开发成样品和使用者之间可重复的系统。此外，由于该测定使用了野生型病毒进行感染，如果使用了高致病性流感病毒例如H5N1，它仅可在3级生物防护水平或以上的实验室安全进行。
因此，发明人根据本专利申请所描述的工作，旨在开发一种新的基于H5HA/NA假型包装的慢病毒载体的中和测定，据信它将最终取代目前用于测量抗HA抗体、免疫个体血清以及感染血清的中和活性的测定，因为这种基于H5HA/NA假型包装的慢病毒载体的中和测定提供了客观和定量的数据。由于使用假型包装而非野生型病毒，它只需要2级生物防护水平。
为了实现这一目的，发明人已产生一组涵盖H5HA主要亚进化枝的H5HA/NA假型病毒(参见图10)。采用特异抗H5HA(亚进化枝1.1)的小鼠免疫血清和针对感染H5N1(亚进化枝2.3)的人个体的恢复期血清，发明人进行了平行中和研究。
发明人比较了HI测定(参见图11A)、微量中和测定(参见图11B)和基于H5HA/NA假型病毒的新中和测定(参见图12)的中和效价。
该比较的结果显示，基于假型病毒的中和测定不仅给出了与微量中和测定类似的结果，而且它还更加敏感(至少多出两个对数级)和更易于定量。
更重要的是，发明人发现尽管亚进化枝1.1和2.3之间有一定程度的交叉反应性，抗同源亚进化枝的中和效价要远高于抗异源亚进化枝的中和效价，凸显了使用一组涵盖H5HA全部主要亚进化枝的假型病毒以用于接种或感染个体的中和活性的任何血清学调查的重要性(参见图10到12)。
因此，发明人已清楚表明，这种基于H5HA/NA假型包装的慢病毒载体的新中和测定比微量中和测定更加敏感和更易于定量。此外，得益于这种新测定使用的材料，它可由经验更少的人员在更大范围的实验室内安全进行。
实施例10：H5HA/NA假型包装的慢病毒在筛选抗病毒化合物的新方法中
的应用
发明人还使用H5HA/NA假型包装的慢病毒载体来开发基于细胞的测定，以筛选抗病毒药物。
采用该测定筛选经HPLC从某些中草药分离的小分子文库，鉴定出两个EC50约为5μM(图13A和13C)且SI分别＞25和9.12的两个化合物(1和2)(参见图13)。CC5O是导致50％细胞毒性的化合物剂量；EC50是导致病毒转导或感染50％被抑制的化合物剂量。SI即安全指数按下式计算：SI＝CC50/EC50。通常具有高SI的化合物更为安全(细胞毒性更低)。同样重要的是，测试开发期间，发明人通过将转导、细胞培养和荧光素酶活性测量结合到1个孔内，还成功地简化了96孔方式的测定步骤。
实施例11：其他的假型病毒
发明人继续拓展了HA/NA假型病毒的范围，除了上述主要的H5HA亚进化枝以外，还产生了表达H1HA、H2HA和H7HA的HA/NA假型病毒。此外，他们还制备了H1HA和H5HA的嵌合HA(表1)，并证实它们具有生物活性和高度的免疫原性。
这些表达嵌合HA的新假型病毒将是剖析HA分子抗原性和免疫原性极为有用的工具，而这又反过来促进开发新的更好候选疫苗。
表I
| 对照 | 63 | 71 |
| H1/N1 | 1064645 | 1237576 |
| H5/N1(Th) | 2911097 | 3045658 |
| H151/N1(Th) | 1577439 | 1765842 |
| H515/N1(Th) | 1653841 | 1762159 |
在表I中，Th是指源自A/泰国/(KAN-1)/2004H5N1禽流感病毒株的蛋白。
表I显示了以表达H1HA/N1NA、H5HA/N1NA、嵌合H151HA/N1NA或嵌合H515HA/N1NA的假型病毒转导的MDCK细胞的相对荧光素酶活性。通过H1HA(SEQ ID NO：3)和H5HA(SEQ ID NO：4)在SEQ ID NO：3上位于位置72和294处的保守半胱氨酸的结构域交换，构建嵌合肽H151HA(SEQ ID NO：12)和嵌合肽H515HA(SEQ ID NO：13)。因此，H151HA(SEQID NO：12)包含来自H1HA(SEQ ID NO：3)的残基1-72、来自H5HA(SEQ IDNO：4)的残基72-293和来自H1HA(SEQ ID NO：3)的残基295-569。同样，H515HA(SEQ ID NO：13)包含来自H5HA(SEQ ID NO：4)的残基1-71、来自H1HA(SEQ ID NO：3)的残基73-294和来自H5HA(SEQ ID NO：4)的残基294-568。以同样的方式通过在保守残基之间交换结构域，可生成两个或更多HA蛋白结构域的部分的其他组合。本文附带的图14和图15的HA蛋白和NA蛋白的比对显示了贯穿排列在这两个蛋白上的几个保守残基。这点再加上这些各异的HA蛋白和NA蛋白的完整肽序列为将这些蛋白的结构域的各种组合生成新的嵌合形式提供了基础。
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序列表
<110>上海巴斯德研究所(Institut Pasteur of Shanghai)
ZHOU Paul
TSAI Cheguo
TOYADA Tetsuya
BUCHY Philippe
<120>流感病毒血凝素假型包装的慢病毒和使用方法
<130>F226-154PCT
<160>25
<170>PatentIn version 3.3
<210>1
<211>566
<212>PRT
<213>Influenza virus
<400>1
Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp
1 5 10 15
Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp
20 25 30
Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu
35 40 45
Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro
50 55 60
Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro
65 70 75 80
Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met Glu
85 90 95
Lys Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Ash Asp
100 105 110
Tyr Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys
115 120 125
Val Lys Ile Leu Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly
130 135 140
Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn
145 150 155 160
Met Val Trp Leu Thr Lys Lys Gly Ser Asp Tyr Pro Val Ala Lys Gly
165 170 175
Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val
180 185 190
His His Pro Asn Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val
195 200 205
Gly Thr Tyr Val Ser Val Ser Thr Ser Thr Leu Asn Lys Arg Ser Thr
210 215 220
Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Leu Gly Ser Arg Met
225 230 235 240
Glu Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu
245 250 255
Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys
260 265 270
Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys
275 280 285
Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro
290 295 300
Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val
305 310 315 320
Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln
325 330 335
Ile Glu Ser Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly
340 345 350
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr
355 360 365
His His Ser Asn Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser
370 375 380
Thr Gln Lys Ala Phe Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile
385 390 395 400
Glu Lys Met Asn Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Gly Asn
405 410 415
Leu Glu Arg Arg Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe
420 425 430
Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn
435 440 445
Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp
450 455 460
Lys Val Arg Met Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly
465 470 475 480
Cys Phe Glu Phe Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val
485 490 495
Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu
500 505 510
Asn Arg Asn Glu Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr
515 520 525
Gln Ile Leu Ala Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala
530 535 540
Ile Met Met Ala Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu
545 550 555 560
Gln Cys Arg Ile Cys Ile
565
<210>2
<211>1039
<212>DNA
<213>Influenza virus
<400>2
agcaaaagca ggtagatgtt gaaagatgag tcttctaacc gaggtcgaaa cgtacgttct 60
ctctatcatc ccgtcaggcc ccctcaaagc cgagatcgcg cagaaacttg aagatgtctt 120
tgcaggaaag aacaccgatc tcgaggctct catggagtgg ctaaagacaa gaccaatcct 180
gtcacctctg actaaaggga ttttgggatt tgtattcacg ctcaccgtgc ccagtgagcg 240
aggactgcag cgtagacgct ttgtccagaa tgccctaaat ggaaatggag atccaaataa 300
tatggataga gcagtcaagc tatataagaa gctgaaaaga gaaataacat tccatggggc 360
taaggaggtc gcactcagct actcaaccgg tgcacttgcc agttgcatgg gtctcatata 420
caacaggatg ggaacggtga ctacggaagt ggcttttggc ctagtgtgtg ccacttgtga 480
gcagattgca gattcacagc atcggtctca cagacagatg gcaactacca ccaacccact 540
aatcagacat gagaacagaa tggtgctggc cagcactaca gctaaggcta tggagcagat 600
ggcaggatca agtgagcagg cagcggaagc catggagatc gctaatcagg ctaggcagat 660
ggtgcaggca atgaggacaa ttgggactca tcctaactct agtgctggtc tgagagataa 720
tcttcttgaa aatttgcagg cctaccagaa acgaatggga gtgcagatgc agcgattcaa 780
gtgatcctat tgttgttgcc gcaaatatca ttgggatctt gcacttgata ttgtggattc 840
ttgatcgtct tttcttcaaa tgcatttatc gtcgccttaa atacggtttg aaaagagggc 900
ctgctacggc aggggtacct gagtctatga gggaagagta ccggcaggaa cagcagagtg 960
ctgtggatgt tgacgatggt cattttgtca acatagaatt ggagtaaaaa actaccttgt 1020
ttctactaat acggaagac 1039
<210>3
<211>569
<212>PRT
<213>Influenza virus
<400>3
Met Lys Ala Lys Leu Leu Val Leu Leu Tyr Ala Phe Val Ala Thr Asp
1 5 10 15
Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr
20 25 30
Val Asp Thr Ile Phe Glu Lys Asn Val Ala Val Thr His Ser Val Asn
35 40 45
Leu Leu Glu Asp Arg His Asn Gly Lys Leu Cys Lys Leu Lys Gly Lys
50 55 60
Ala Pro Leu Gln Leu Gly Lys Cys Ser Ile Thr Gly Trp Leu Leu Gly
65 70 75 80
Asn Pro Glu Cys Asp Ser Leu Leu Pro Ala Arg Ser Trn Ser Tyr Ile
85 90 95
Val Glu Thr Pro Asn Ser Glu Asn Gly Ala Cys Tyr Pro Gly Asp Phe
100 105 110
Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Leu
115 120 125
Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr
130 135 140
Phe Asn Gly Val Thr Val Ser Cys Ser His Arg Gly Lys Ser Ser Phe
145 150 155 160
Tyr Arg Asn Leu Leu Trp Leu Thr Lys Glu Gly Asp Ser Tyr Pro Lys
165 170 175
Leu Thr Asn Ser Tyr Val Asn Asn Lys Gly Lys Glu Val Leu Val Leu
180 185 190
Trp Gly Val His His Pro Ser Ser Ser Asp Glu Gln Gln Ser Leu Tyr
195 200 205
Ser Asn Gly Asn Ala Tyr Val Ser Val Ala Ser Ser Asn Tyr Asn Arg
210 215 220
Arg Phe Thr Pro Glu Ile Ala Ala Arg Pro Lys Val Lys Asp Gln His
225 230 235 240
Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile
245 250 255
Ile Phe Glu Ala Thr Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala
260 265 270
Leu Ser Arg Gly Phe Glu Ser Gly Ile Ile Thr Ser Asn Ala Ser Met
275 280 285
His Glu Cys Asn Thr Lys Cys Gln Thr Pro Gln Gly Ser Ile Asn Ser
290 295 300
Asn Leu Pro Phe Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro
305 310 315 320
Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Val Thr Gly Leu Arg Asn
325 330 335
Ile Pro Ser Ile Gln Tyr Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Ile Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Asn Leu Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Leu Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met
485 490 495
Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu
530 535 540
Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn
545 550 555 560
Gly Ser Leu Lys Cys Arg Ile Cys Ile
565
<210>4
<211>568
<212>PRT
<213>Influenza yirus
<400>4
Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser
1 5 10 15
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asn Ile
35 40 45
Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys
50 55 60
Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
65 70 75 80
Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val
85 90 95
Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn
100 105 110
Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu
115 120 125
Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser
130 135 140
Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Arg Lys Ser Ser Phe Phe
145 150 155 160
Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile
165 170 175
Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp
180 185 190
Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln
195 200 205
Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg
210 215 220
Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly
225 230 235 240
Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn
245 250 255
Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile
260 265 270
Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly
275 280 285
Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser
290 295 300
Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys
305 310 315 320
Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335
Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350
Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
355 360 365
Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys
370 375 380
Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser
385 390 395 400
Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415
Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp
420 425 430
Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met
435 440 445
Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly
465 470 475 480
Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu
485 490 495
Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala
500 505 510
Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly
515 520 525
Ile Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala
530 535 540
Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly
545 550 555 560
Ser Leu Gln Cys Arg Ile Cys Ile
565
<210>5
<211>568
<212>PRT
<213>Influenza yirus
<400>5
Met Glu Lys Ile Val Leu Leu Phe Ala Val Val Ser Leu Val Lys Ser
1 5 10 15
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45
Leu Glu Arg Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys
50 55 60
Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
65 70 75 80
Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val
85 90 95
Glu Arg Ala Asn Pro Val Asn Gly Leu Cys Tyr Pro Gly Asp Phe Asn
100 105 110
Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu
115 120 125
Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser
130 135 140
Leu Gly Val Ser Ser Ala Cys Pro Tyr Leu Gly Lys Pro Ser Phe Phe
145 150 155 160
Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile
165 170 175
Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Met Trp
180 185 190
Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln
195 200 205
Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg
210 215 220
Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly
225 230 235 240
Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn
245 250 255
Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile
260 265 270
Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly
275 280 285
Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser
290 295 300
Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys
305 310 315 320
Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335
Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350
Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
355 360 365
Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys
370 375 380
Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser
385 390 395 400
Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415
Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp
420 425 430
Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met
435 440 445
Gly Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly
465 470 475 480
Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu
485 490 495
Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala
500 505 510
Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly
515 520 525
Ile Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala
530 535 540
Leu Ala Ile Met Ile Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly
545 550 555 560
Ser Leu Gln Cys Arg Ile Cys Ile
565
<210>6
<211>567
<212>PRT
<213>Influenza virus
<400>6
Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser
1 5 10 15
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45
Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys
50 55 60
Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
65 70 75 80
Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val
85 90 95
Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn
100 105 110
Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu
115 120 125
Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser
130 135 140
Leu Gly Val Ser Ala Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe
145 150 155 160
Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile
165 170 175
Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Met Trp
180 185 190
Gly Ile His His Pro Asn Asp Glu Ala Glu Gln Thr Lys Leu Tyr Gln
195 200 205
Asn Pro Ile Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg
210 215 220
Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly
225 230 235 240
Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn
245 250 255
Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile
260 265 270
Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly
275 280 285
Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser
290 295 300
Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys
305 310 315 320
Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335
Pro Gln Arg Glu Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala
340 345 350
Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly
355 360 365
Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu
370 375 380
Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile
385 390 395 400
Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn
405 410 415
Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly
420 425 430
Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu
435 440 445
Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr
450 455 460
Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn
465 470 475 480
Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser
485 490 495
Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg
500 505 510
Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Ile
515 520 525
Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu
530 535 540
Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser
545 550 555 560
Leu Gln Cys Arg Ile Cys Ile
565
<210>7
<211>567
<212>PRT
<213>Influenza virus
<400>7
Met Glu Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser
1 5 10 15
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45
Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys
50 55 60
Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
65 70 75 80
Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val
85 90 95
Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110
Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu
115 120 125
Lys Ile Pro Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser
130 135 140
Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Thr Pro Ser Phe Phe
145 150 155 160
Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile
165 170 175
Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Ile Leu Trp
180 185 190
Gly Ile His His Ser Asn Asn Ala Ala Glu Gln Thr Lys Leu Tyr Gln
195 200 205
Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Leu Arg
210 215 220
Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly
225 230 235 240
Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn
245 250 255
Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile
260 265 270
Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Val Glu Tyr Gly
275 280 285
Asn Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser
290 295 300
Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys
305 310 315 320
Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335
Pro Leu Arg Glu Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala Ile Ala
340 345 350
Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly
355 360 365
Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu
370 375 380
Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile
385 390 395 400
Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn
405 410 415
Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly
420 425 430
Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu
435 440 445
Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr
450 455 460
Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn
465 470 475 480
Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser
485 490 495
Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg
500 505 510
Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr
515 520 525
Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu
530 535 540
Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser
545 550 555 560
Leu Gln Cys Arg Ile Cys Ile
565
<210>8
<211>449
<212>PRT
<213>Influenza virus
<400>8
Met Asn Pro Asn Lys Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Val
1 5 10 15
Thr Gly Met Val Ser Leu Met Leu Gln Ile Gly Asn Leu Ile Ser Ile
20 25 30
Trp Val Ser His Ser Ile His Thr Gly Asn Gln His Lys Ala Glu Pro
35 40 45
Ile Ser Asn Thr Asn Phe Leu Thr Glu Lys Ala Val Ala Ser Val Lys
50 55 60
Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Asn Gly Trp Ala Val Tyr
65 70 75 80
Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val
85 90 95
Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe
100 105 110
Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr
115 120 125
Val Lys Asp Arg Ser Pro His Arg Thr Leu Met Ser Cys Pro Val Gly
130 135 140
Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser
145 150 155 160
Ala Ser Ala Cys His Asp Gly Thr Ser Trp Leu Thr Ile Gly Ile Ser
165 170 175
Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile
180 185 190
Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu
195 200 205
Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp
210 215 220
Gly Pro Ser Asn Gly Gln Ala Ser His Lys Ile Phe Lys Met Glu Lys
225 230 235 240
Gly Lys Val Val Lys Ser Val Glu Leu Asp Ala Pro Asn Tyr His Tyr
245 250 255
Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile Thr Cys Val Cys
260 265 270
Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gln
275 280 285
Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp
290 295 300
Asn Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser Ser
305 310 315 320
Asn Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly
325 330 335
Val Trp Ile Gly Arg Thr Lys Ser Thr Asn Ser Arg Ser Gly Phe Glu
340 345 350
Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Ser Phe Ser
355 360 365
Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly
370 375 380
Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro
385 390 395 400
Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Ser Thr Ile
405 410 415
Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr
420 425 430
Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp
435 440 445
Lys
<210>9
<211>449
<212>PRT
<213>Influenza virus
<400>9
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Ile
1 5 10 15
Thr Gly Met Val Ser Leu Met Leu Gln Val Gly Asn Met Ile Ser Ile
20 25 30
Trp Ile Ser His Ser Ile His Thr Gly Asn Gln His Gln Ala Glu Pro
35 40 45
Ile Ser Asn Ala Asn Phe Leu Thr Lys Arg Ala Val Ala Ala Val Lys
50 55 60
Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Asn Gly Trp Ala Val Tyr
65 70 75 80
Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val
85 90 95
Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe
100 105 110
Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr
115 120 125
Ala Lys Asp Arg Ser Pro His Arg Thr Leu Met Ser Cys Pro Val Gly
130 135 140
Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser
145 150 155 160
Ala Ser Ala Cys His Asp Gly Thr Ser Trp Leu Thr Ile Gly Ile Ser
165 170 175
Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile
180 185 190
Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu
195 200 205
Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp
210 215 220
Gly Pro Ser Asn Gly Gln Ala Ser His Lys Ile Phe Lys Met Glu Lys
225 230 235 240
Gly Lys Val Val Lys Ser Val Glu Leu Asp Ala Pro Asn Tyr His Tyr
245 250 255
Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile Thr Cys Val Cys
260 265 270
Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gln
275 280 285
Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp
290 295 300
Asn Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser Ser
305 310 315 320
Asn Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly
325 330 335
Val Trp Ile Gly Arg Thr Lys Ser Thr Asn Ser Arg Ser Gly Phe Glu
340 345 350
Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Ser Phe Ser
355 360 365
Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly
370 375 380
Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro
385 390 395 400
Cys Phe Trp Ile Glu Leu Ile Arg Gly Arg Pro Lys Glu Ser Thr Ile
405 410 415
Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr
420 425 430
Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp
435 440 445
Lys
<210>10
<211>449
<212>PRT
<213>Influenza virus
<400>10
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Val
1 5 10 15
Ile Gly Ile Val Ser Leu Met Leu Gln Ile Gly Asn Met Val Ser Leu
20 25 30
Trp Val Ser His Ser Ile Gln Thr Gly Asn Gln His Gln Val Glu Pro
35 40 45
Ile Ser Asn Thr Asn Phe Leu Thr Glu Lys Ala Val Ala Ser Val Thr
50 55 60
Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly Trp Ala Val His
65 70 75 80
Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val
85 90 95
Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe
100 105 110
Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr
115 120 125
Val Lys Asp Arg Ser Pro His Arg Thr Leu Met Ser Cys Pro Val Gly
130 135 140
Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser
145 150 155 160
Ala Ser Ala Cys His Asp Gly Thr Ser Trp Leu Thr Ile Gly Ile Ser
165 170 175
Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Met Ile
180 185 190
Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu
195 200 205
Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp
210 215 220
Gly Pro Ser Asn G1y Gln Ala Ser Tyr Lys Ile Phe Lys Met Glu Lys
225 230 235 240
Gly Lys Val Val Lys Ser Val Glu Leu Asp Ala Pro Asn Tyr His Tyr
245 250 255
Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile Thr Cys Val Cys
260 265 270
Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gln
275 280 285
Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp
290 295 300
Asn Pro Arg Pro Asn Gly Gly Thr Gly Ser Cys Gly Pro Val Ser Pro
305 310 315 320
Asn Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly
325 330 335
Val Trp Ile Gly Arg Thr Lys Ser Pro Ser Ser Arg Ser Gly Phe Glu
340 345 350
Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Ser Phe Ser
355 360 365
Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly
370 375 380
Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro
385 390 395 400
Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Ser Thr Ile
405 410 415
Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr
420 425 430
Val Ser Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp
435 440 445
Lys
<210>11
<211>449
<212>PRT
<213>Influenza virus
<400>11
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Val
1 5 10 15
Ile Gly Ile Val Ser Leu Met Leu Gln Ile Gly Asn Met Ile Ser Ile
20 25 30
Trp Val Ser His Ser Ile Gln Thr Gly Asn Gln His Gln Ala Glu Pro
35 40 45
Ile Ser Asn Thr Asn Phe Leu Thr Glu Asn Ala Val Ala Ser Val Thr
50 55 60
Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly Trp Ala Val His
65 70 75 80
Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val
85 90 95
Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe
100 105 110
Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr
115 120 125
Val Lys Asp Arg Ser Pro His Gly Thr Leu Met Ser Cys Pro Met Gly
130 135 140
Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser
145 150 155 160
Ala Ser Ala Cys His Asp Gly Thr Ser Trp Leu Thr Ile Gly Ile Ser
165 170 175
Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile
180 185 190
Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu
195 200 205
Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp
210 215 220
Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe Lys Met Glu Lys
225 230 235 240
Gly Lys Val Val Lys Ser Val Glu Leu Asn Ala Pro Asn Tyr His Tyr
245 250 255
Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile Ile Cys Val Cys
260 265 270
Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gln
275 280 285
Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp
290 295 300
Asn Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser Pro
305 310 315 320
Asn Gly Ala Tyr Gly Ile Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly
325 330 335
Val Trp Ile Gly Arg Thr Lys Ser Thr Asn Ser Arg Ser Gly Phe Glu
340 345 350
Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp Ser Asp Phe Ser
355 360 365
Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly
370 375 380
Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro
385 390 395 400
Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Ser Thr Ile
405 410 415
Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr
420 425 430
Val Ser Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp
435 440 445
Lys
<210>12
<211>569
<212>PRT
<213>Artificial
<220>
<223>Chimeric HA peptide
<400>12
Met Lys Ala Lys Leu Leu Val Leu Leu Tyr Ala Phe Val Ala Thr Asp
1 5 10 15
Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr
20 25 30
Val Asp Thr Ile Phe Glu Lys Asn Val Ala Val Thr His Ser Val Asn
35 40 45
Leu Leu Glu Asp Arg His Asn Gly Lys Leu Cys Lys Leu Lys Gly Lys
50 55 60
Ala Pro Leu Gln Leu Gly Lys Cys Ser Val Ala Gly Trp Leu Leu Gly
65 70 75 80
Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile
85 90 95
Val Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe
100 105 110
Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe
115 120 125
Glu Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala
130 135 140
Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Arg Lys Ser Ser Phe
145 150 155 160
Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr
165 170 175
Ile Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu
180 185 190
Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr
195 200 205
Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln
210 215 220
Arg Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser
225 230 235 240
Gly Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile
245 250 255
Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys
260 265 270
Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr
275 280 285
Gly Asn Cys Asn Thr Lys Cys Gln Thr Pro Gln Gly Ser Ile Asn Ser
290 295 300
Asn Leu Pro Phe Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro
305 310 315 320
Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Val Thr Gly Leu Arg Asn
325 330 335
Ile Pro Ser Ile Gln Tyr Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Ile Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Asn Leu Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Leu Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met
485 490 495
Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu
530 535 540
Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn
545 550 555 560
Gly Ser Leu Lys Cys Arg Ile Cys Ile
565
<210>13
<211>568
<212>PRT
<213>Artificial
<220>
<223>Chimeric HA peptide
<400>13
Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser
1 5 10 15
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45
Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys
50 55 60
Pro Leu Ile Leu Arg Asp Cys Ser Ile Thr Gly Trp Leu Leu Gly Asn
65 70 75 80
Pro Glu Cys Asp Ser Leu Leu Pro Ala Arg Ser Trp Ser Tyr Ile Val
85 90 95
Glu Thr Pro Asn Ser Glu Asn Gly Ala Cys Tyr Pro Gly Asp Phe Ile
100 105 110
Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Leu Glu
115 120 125
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr Phe
130 135 140
Asn Gly Val Thr Val Ser Cys Ser His Arg Gly Lys Ser Ser Phe Tyr
145 150 155 160
Arg Asn Leu Leu Trp Leu Thr Lys Glu Gly Asp Ser Tyr Pro Lys Leu
165 170 175
Thr Asn Ser Tyr Val Asn Asn Lys Gly Lys Glu Val Leu Val Leu Trp
180 185 190
Gly Val His His Pro Ser Ser Ser Asp Glu Gln Gln Ser Leu Tyr Ser
195 200 205
Asn Gly Asn Ala Tyr Val Ser Val Ala Ser Ser Asn Tyr Asn Arg Arg
210 215 220
Phe Thr Pro Glu Ile Ala Ala Arg Pro Lys Val Lys Asp Gln His Gly
225 230 235 240
Arg Met Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile Ile
245 250 255
Phe Glu Ala Thr Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala Leu
260 265 270
Ser Arg Gly Phe Glu Ser Gly Ile Ile Thr Ser Asn Ala Ser Met His
275 280 285
Glu Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser
290 295 300
Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys
305 310 315 320
Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335
Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350
Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
355 360 365
Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys
370 375 380
Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser
385 390 395 400
Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415
Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp
420 425 430
Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met
435 440 445
Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly
465 470 475 480
Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu
485 490 495
Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala
500 505 510
Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly
515 520 525
Ile Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala
530 535 540
Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly
545 550 555 560
Ser Leu Gln Cys Arg Ile Cys Ile
565
<210>14
<211>1707
<212>DNA
<213>Artificial
<223>Coding Seq for H151HA
<400>14
atgaaggcca agctgctggt gctgctgtac gccttcgtgg ccaccgacgc cgacaccatc 60
tgcatcggct accacgccaa caacagcacc gacaccgtgg acaccatctt cgagaagaac 120
gtggccgtga cccacagcgt gaacctgctg gaggacagac acaacggcaa gctgtgcaag 180
ctgaagggca aggcccccct gcagctgggc aagtgcagcg tggccggctg gctgctgggc 240
aaccccatgt gcgacgagtt catcaacgtg cccgagtgga gctacatcgt ggagaaggcc 300
aaccccgtga acgacctgtg ctaccccggc gacttcaacg actacgagga gctgaagcac 360
ctgctgagca gaatcaacca cttcgagaag atccagatca tccccaagag cagctggagc 420
agccacgagg ccagcctggg cgtgagcagc gcctgcccct accagagaaa gagcagcttc 480
ttcagaaacg tggtgtggct gatcaagaag aacagcacct accccaccat caagagaagc 540
tacaacaaca ccaaccagga ggacctgctg gtgctgtggg gcatccacca ccccaacgac 600
gccgccgagc agaccaagct gtaccagaac cccaccacct acatcagcgt gggcaccagc 660
accctgaacc agagactggt gcccagaatc gccaccagaa gcaaggtgaa cggccagagc 720
ggcagaatgg agttcttctg gaccatcctg aagcccaacg acgccatcaa cttcgagagc 780
aacggcaact tcatcgcccc cgagtacgcc tacaagatcg tgaagaaggg cgacagcacc 840
atcatgaaga gcgagctgga gtacggcaac tgcaacacca agtgccagac cccccagggc 900
agcatcaaca gcaacctgcc cttccagaac atccaccccg tgaccatcgg cgagtgcccc 960
aagtacgtga gaagcaccaa gctgagaatg gtgaccggcc tgagaaacat ccccagcatc 1020
cagtacagaa gaaagaagag aggcctgttc ggcgccatcg ccggcttcat cgagggcggc 1080
tggaccggca tgatcgacgg ctggtacggc taccaccacc agaacgagca gggcagcggc 1140
tacgccgccg accagaagag cacccagaac gccatcaacg gcatcaccaa caaggtgaac 1200
agcatcatcg agaagatgaa cacccagttc accgccgtgg gcaaggagtt caacaacctg 1260
gagaagagaa tggagaacct gaacaagaag gtggacgacg gcttcctgga catctggacc 1320
tacaacgccg agctgctggt gctgctggag aacgagagaa ccctggactt ccacgacctg 1380
aacgtgaaga acctgtacga gaaggtgaag agccagctga agaacaacgc caaggagatc 1440
ggcaacggct gcttcgagtt ctaccacaag tgcgacaacg agtgcatgga gagcgtgaga 1500
aacggcacct acgactaccc caagtacagc gaggagagca agctgaacag agagaagatc 1560
gacggcgtga agctggagag catgggcgtg taccagatcc tggccatcta cagcaccgtg 1620
gccagcagcc tggtgctgct ggtgagcctg ggcgccatca gcttctggat gtgcagcaac 1680
ggcagcctga agtgcagaat ctgcatc 1707
<210>15
<211>1704
<212>DNA
<213>Artificial
<220>
<223>H515HA DNA Coding SEQ
<400>15
atggagaaga tcgtgctgct gttcgccatc gtgagcctgg tgaagagcga ccagatctgc 60
atcggctacc acgccaacaa cagcaccgag caggtggaca ccatcatgga gaagaacgtg 120
accgtgaccc acgcccagga catcctggag aagacccaca acggcaagct gtgcgacctg 180
gacggcgtga agcccctgat cctgagagac tgcagcatca ccggctggct gctgggcaac 240
cccgagtgcg acagcctgct gcccgccaga agctggagct acatcgtgga gacccccaac 300
agcgagaacg gcgcctgcta ccccggcgac ttcatcgact acgaggagct gagagagcag 360
ctgagcagcg tgagcagcct ggagagattc gagatcttcc ccaaggagag cagctggccc 420
aaccacacct tcaacggcgt gaccgtgagc tgcagccaca gaggcaagag cagcttctac 480
agaaacctgc tgtggctgac caaggagggc gacagctacc ccaagctgac caacagctac 540
gtgaacaaca agggcaagga ggtgctggtg ctgtggggcg tgcaccaccc cagcagcagc 600
gacgagcagc agagcctgta cagcaacggc aacgcctacg tgagcgtggc cagcagcaac 660
tacaacagaa gattcacccc cgagatcgcc gccagaccca aggtgaagga ccagcacggc 720
agaatgaact actactggac cctgctggag cccggcgaca ccatcatctt cgaggccacc 780
ggcaacctga tcgccccctg gtacgccttc gccctgagca gaggcttcga gagcggcatc 840
atcaccagca acgccagcat gcacgagtgc aacaccaagt gccagacccc catgggcgcc 900
atcaacagca gcatgccctt ccacaacatc caccccctga ccatcggcga gtgccccaag 960
tacgtgaaga gcaacagact ggtgctggcc accggcctga gaaacagccc ccagagagag 1020
agaagaagaa agaagagagg cctgttcggc gccatcgccg gcttcatcga gggcggctgg 1080
cagggcatgg tggacggctg gtacggctac caccacagca acgagcaggg cagcggctac 1140
gccgccgaca aggagagcac ccagaaggcc atcgacggcg tgaccaacaa ggtgaacagc 1200
atcatcgaca agatgaacac ccagttcgag gccgtgggca gagagttcaa caacctggag 1260
agaagaatcg agaacctgaa caagaagatg gaggacggct tcctggacgt gtggacctac 1320
aacgccgagc tgctggtgct gatggagaac gagagaaccc tggacttcca cgacagcaac 1380
gtgaagaacc tgtacgacaa ggtgagactg cagctgagag acaacgccaa ggagctgggc 1440
aacggctgct tcgagttcta ccacaagtgc gacaacgagt gcatggagag cgtgagaaac 1500
ggcacctacg actaccccca gtacagcgag gaggccagac tgaagagaga ggagatcagc 1560
ggcgtgaagc tggagagcat cggcatctac cagatcctga gcatctacag caccgtggcc 1620
agcagcctgg ccctggccat catggtggcc ggcctgagcc tgtggatgtg cagcaacggc 1680
agcctgcagt gcagaatctg catc 1704
<210>16
<211>1707
<212>DNA
<213>Influenza yirus
<400>16
atgaaggcca agctgctggt gctgctgtac gccttcgtgg ccaccgacgc cgacaccatc 60
tgcatcggct accacgccaa caacagcacc gacaccgtgg acaccatctt cgagaagaac 120
gtggccgtga cccacagcgt gaacctgctg gaggacagac acaacggcaa gctgtgcaag 180
ctgaagggca aggcccccct gcagctgggc aagtgcagca tcaccggctg gctgctgggc 240
aaccccgagt gcgacagcct gctgcccgcc agaagctgga gctacatcgt ggagaccccc 300
aacagcgaga acggcgcctg ctaccccggc gacttcatcg actacgagga gctgagagag 360
cagctgagca gcgtgagcag cctggagaga ttcgagatct tccccaagga gagcagctgg 420
cccaaccaca ccttcaacgg cgtgaccgtg agctgcagcc acagaggcaa gagcagcttc 480
tacagaaacc tgctgtggct gaccaaggag ggcgacagct accccaagct gaccaacagc 540
tacgtgaaca acaagggcaa ggaggtgctg gtgctgtggg gcgtgcacca ccccagcagc 600
agcgacgagc agcagagcct gtacagcaac ggcaacgcct acgtgagcgt ggccagcagc 660
aactacaaca gaagattcac ccccgagatc gccgccagac ccaaggtgaa ggaccagcac 720
ggcagaatga actactactg gaccctgctg gagcccggcg acaccatcat cttcgaggcc 780
accggcaacc tgatcgcccc ctggtacgcc ttcgccctga gcagaggctt cgagagcggc 840
atcatcacca gcaacgccag catgcacgag tgcaacacca agtgccagac cccccagggc 900
agcatcaaca gcaacctgcc cttccagaac atccaccccg tgaccatcgg cgagtgcccc 960
aagtacgtga gaagcaccaa gctgagaatg gtgaccggcc tgagaaacat ccccagcatc 1020
cagtacagaa gaaagaagag aggcctgttc ggcgccatcg ccggcttcat cgagggcggc 1080
tggaccggca tgatcgacgg ctggtacggc taccaccacc agaacgagca gggcagcggc 1140
tacgccgccg accagaagag cacccagaac gccatcaacg gcatcaccaa caaggtgaac 1200
agcatcatcg agaagatgaa cacccagttc accgccgtgg gcaaggagtt caacaacctg 1260
gagaagagaa tggagaacct gaacaagaag gtggacgacg gcttcctgga catctggacc 1320
tacaacgccg agctgctggt gctgctggag aacgagagaa ccctggactt ccacgacctg 1380
aacgtgaaga acctgtacga gaaggtgaag agccagctga agaacaacgc caaggagatc 1440
ggcaacggct gcttcgagtt ctaccacaag tgcgacaacg agtgcatgga gagcgtgaga 1500
aacggcacct acgactaccc caagtacagc gaggagagca agctgaacag agagaagatc 1560
gacggcgtga agctggagag catgggcgtg taccagatcc tggccatcta cagcaccgtg 1620
gccagcagcc tggtgctgct ggtgagcctg ggcgccatca gcttctggat gtgcagcaac 1680
ggcagcctga agtgcagaat ctgcatc 1707
<210>17
<211>1704
<212>DNA
<213>Influenza yirus
<400>17
atggagaaga tcgtgctgct gttcgccatc gtgagcctgg tgaagagcga ccagatctgc 60
atcggctacc acgccaacaa cagcaccgag caggtggaca ccatcatgga gaagaacgtg 120
accgtgaccc acgcccagga catcctggag aagacccaca acggcaagct gtgcgacctg 180
gacggcgtga agcccctgat cctgagagac tgcagcgtgg ccggctggct gctgggcaac 240
cccatgtgcg acgagttcat caacgtgccc gagtggagct acatcgtgga gaaggccaac 300
cccgtgaacg acctgtgcta ccccggcgac ttcaacgact acgaggagct gaagcacctg 360
ctgagcagaa tcaaccactt cgagaagatc cagatcatcc ccaagagcag ctggagcagc 420
cacgaggcca gcctgggcgt gagcagcgcc tgcccctacc agagaaagag cagcttcttc 480
agaaacgtgg tgtggctgat caagaagaac agcacctacc ccaccatcaa gagaagctac 540
aacaacacca accaggagga cctgctggtg ctgtggggca tccaccaccc caacgacgcc 600
gccgagcaga ccaagctgta ccagaacccc accacctaca tcagcgtggg caccagcacc 660
ctgaaccaga gactggtgcc cagaatcgcc accagaagca aggtgaacgg ccagagcggc 720
agaatggagt tcttctggac catcctgaag cccaacgacg ccatcaactt cgagagcaac 780
ggcaacttca tcgcccccga gtacgcctac aagatcgtga agaagggcga cagcaccatc 840
atgaagagcg agctggagta cggcaactgc aacaccaagt gccagacccc catgggcgcc 900
atcaacagca gcatgccctt ccacaacatc caccccctga ccatcggcga gtgccccaag 960
tacgtgaaga gcaacagact ggtgctggcc accggcctga gaaacagccc ccagagagag 1020
agaagaagaa agaagagagg cctgttcggc gccatcgccg gcttcatcga gggcggctgg 1080
cagggcatgg tggacggctg gtacggctac caccacagca acgagcaggg cagcggctac 1140
gccgccgaca aggagagcac ccagaaggcc atcgacggcg tgaccaacaa ggtgaacagc 1200
atcatcgaca agatgaacac ccagttcgag gccgtgggca gagagttcaa caacctggag 1260
agaagaatcg agaacctgaa caagaagatg gaggacggct tcctggacgt gtggacctac 1320
aacgccgagc tgctggtgct gatggagaac gagagaaccc tggacttcca cgacagcaac 1380
gtgaagaacc tgtacgacaa ggtgagactg cagctgagag acaacgccaa ggagctgggc 1440
aacggctgct tcgagttcta ccacaagtgc gacaacgagt gcatggagag cgtgagaaac 1500
ggcacctacg actaccccca gtacagcgag gaggccagac tgaagagaga ggagatcagc 1560
ggcgtgaagc tggagagcat cggcatctac cagatcctga gcatctacag caccgtggcc 1620
agcagcctgg ccctggccat catggtggcc ggcctgagcc tgtggatgtg cagcaacggc 1680
agcctgcagt gcagaatctg catc 1704
<210>18
<211>1698
<212>DNA
<213>Influenza virus
<400>18
atggccatca tctacctgat cctgctgttc accgccgtga gaggcgacca gatctgcatc 60
ggctaccacg ccaacaacag caccgagaag gtggacacca tcctggagag aaacgtgacc 120
gtgacccacg ccaaggacat cctggagaag acccacaacg gcaagctgtg caagctgaac 180
ggcatccccc ccctggagct gggcgactgc agcatcgccg gctggctgct gggcaacccc 240
gagtgcgaca gactgctgag cgtgcccgag tggagctaca tcatggagaa ggagaacccc 300
agagacggcc tgtgctaccc cggcagcttc aacgactacg aggagctgaa gcacctgctg 360
agcagcgtga agcacttcga gaaggtgaag atcctgccca aggacagatg gacccagcac 420
accaccaccg gcggcagcag agcctgcgcc gtgagcggca accccagctt cttcagaaac 480
atggtgtggc tgaccaagaa gggcagcgac taccccgtgg ccaagggcag ctacaacaac 540
accagcggcg agcagatgct gatcatctgg ggcgtgcacc accccaacga cgagaccgag 600
cagagaaccc tgtaccagaa cgtgggcacc tacgtgagcg tgagcaccag caccctgaac 660
aagagaagca cccccgagat cgccaccaga cccaaggtga acggcctggg cagcagaatg 720
gagttcagct ggaccctgct ggacatgtgg gacaccatca acttcgagag caccggcaac 780
ctgatcgccc ccgagtacgg cttcaagatc agcaagagag gcagcagcgg catcatgaag 840
accgagggca ccctggagaa ctgcgagacc aagtgccaga cccccctggg cgccatcaac 900
accaccctgc ccttccacaa cgtgcacccc ctgaccatcg gcgagtgccc caagtacgtg 960
aagagcgaga agctggtgct ggccaccggc ctgagaaacg tgccccagat cgagagcaga 1020
agaaagaaga gaggcctgtt cggcgccatc gccggcttca tcgagggcgg ctggcagggc 1080
atggtggacg gctggtacgg ctaccaccac agcaacgacc agggcagcgg ctacgccgcc 1140
gacaaggaga gcacccagaa ggccttcgac ggcatcacca acaaggtgaa cagcgtgatc 1200
gagaagatga acacccagtt cgaggccgtg ggcaaggagt tcggcaacct ggagagaaga 1260
ctggagaacc tgaacaagaa gatggaggac ggcttcctgg acgtgtggac ctacaacgcc 1320
gagctgctgg tgctgatgga gaacgagaga accctggact tccacgacag caacgtgaag 1380
aacctgtacg acaaggtgag aatgcagctg agagacaacg tgaaggagct gggcaacggc 1440
tgcttcgagt tctaccacaa gtgcgacgac gagtgcatga acagcgtgaa gaacggcacc 1500
tacgactacc ccaagtacga ggaggagagc aagctgaaca gaaacgagat caagggcgtg 1560
aagctgagca gcatgggcgt gtaccagatc ctggccatct acgccaccgt ggccggcagc 1620
ctgagcctgg ccatcatgat ggccggcatc agcttctgga tgtgcagcaa cggcagcctg 1680
cagtgcagaa tctgcatc 1698
<210>19
<211>1704
<212>DNA
<213>Influenza virus
<400>19
atggagaaga tcgtgctgct gttcgccgtg gtgagcctgg tgaagagcga ccagatctgc 60
atcggctacc acgccaacaa cagcaccgag caggtggaca ccatcatgga gaagaacgtg 120
accgtgaccc acgcccagga catcctggag agaacccaca acggcaagct gtgcgacctg 180
gacggcgtga agcccctgat cctgagagac tgcagcgtgg ccggctggct gctgggcaac 240
cccatgtgcg acgagttcat caacgtgccc gagtggagct acatcgtgga gagagccaac 300
cccgtgaacg gcctgtgcta ccccggcgac ttcaacgact acgaggagct gaagcacctg 360
ctgagcagaa tcaaccactt cgagaagatc cagatcatcc ccaagagcag ctggagcagc 420
cacgaggcca gcctgggcgt gagcagcgcc tgcccctacc tgggcaagcc cagcttcttc 480
agaaacgtgg tgtggctgat caagaagaac agcacctacc ccaccatcaa gagaagctac 540
aacaacacca accaggagga cctgctggtg atgtggggca tccaccaccc caacgacgcc 600
gccgagcaga ccaagctgta ccagaacccc accacctaca tcagcgtggg caccagcacc 660
ctgaaccaga gactggtgcc cagaatcgcc accagaagca aggtgaacgg ccagagcggc 720
agaatggagt tcttctggac catcctgaag cccaacgacg ccatcaactt cgagagcaac 780
ggcaacttca tcgcccccga gtacgcctac aagatcgtga agaagggcga cagcaccatc 840
atgaagagcg agctggagta cggcaactgc aacaccaagt gccagacccc catgggcgcc 900
atcaacagca gcatgccctt ccacaacatc caccccctga ccatcggcga gtgccccaag 960
tacgtgaaga gcaacagact ggtgctggcc accggcctga gaaacagccc ccagagagag 1020
agaagaagaa agaagagagg cctgttcggc gccatcgccg gcttcatcga gggcggctgg 1080
cagggcatgg tggacggctg gtacggctac caccacagca acgagcaggg cagcggctac 1140
gccgccgaca aggagagcac ccagaaggcc atcgacggcg tgaccaacaa ggtgaacagc 1200
atcatcgaca agatgaacac ccagttcgag gccgtgggca gagagttcaa caacctggag 1260
agaagaatcg agaacctgaa caagaagatg gaggacggct tcctggacgt gtggacctac 1320
aacgccgagc tgctggtgct gatgggcaac gagagaaccc tggacttcca cgacagcaac 1380
gtgaagaacc tgtacgacaa ggtgagactg cagctgagag acaacgccaa ggagctgggc 1440
aacggctgct tcgagttcta ccacaagtgc gacaacgagt gcatggagag cgtgagaaac 1500
ggcacctacg actaccccca gtacagcgag gaggccagac tgaagagaga ggagatcagc 1560
ggcgtgaagc tggagagcat cggcatctac cagatcctga gcatctacag caccgtggcc 1620
agcagcctgg ccctggccat catgatcgcc ggcctgagcc tgtggatgtg cagcaacggc 1680
agcctgcagt gcagaatctg catc 1704
<210>20
<211>1701
<212>DNA
<213>Influenza virus
<400>20
atggagaaga tcgtgctgct gttcgccatc gtgagcctgg tgaagagcga ccagatctgc 60
atcggctacc acgccaacaa cagcaccgag caggtggaca ccatcatgga gaagaacgtg 120
accgtgaccc acgcccagga catcctggag aagacccaca acggcaagct gtgcgacctg 180
gacggcgtga agcccctgat cctgagagac tgcagcgtgg ccggctggct gctgggcaac 240
cccatgtgcg acgagttcat caacgtgccc gagtggagct acatcgtgga gaaggccaac 300
cccgtgaacg acctgtgcta ccccggcgac ttcaacgact acgaggagct gaagcacctg 360
ctgagcagaa tcaaccactt cgagaagatc cagatcatcc ccaagagcag ctggagcagc 420
cacgaggcca gcctgggcgt gagcgccgcc tgcccctacc agggcaagag cagcttcttc 480
agaaacgtgg tgtggctgat caagaagaac agcacctacc ccaccatcaa gagaagctac 540
aacaacacca accaggagga cctgctggtg atgtggggca tccaccaccc caacgacgag 600
gccgagcaga ccaagctgta ccagaacccc atcacctaca tcagcgtggg caccagcacc 660
ctgaaccaga gactggtgcc cagaatcgcc accagaagca aggtgaacgg ccagagcggc 720
agaatggagt tcttctggac catcctgaag cccaacgacg ccatcaactt cgagagcaac 780
ggcaacttca tcgcccccga gtacgcctac aagatcgtga agaagggcga cagcaccatc 840
atgaagagcg agctggagta cggcaactgc aacaccaagt gccagacccc catgggcgcc 900
atcaacagca gcatgccctt ccacaacatc caccccctga ccatcggcga gtgccccaag 960
tacgtgaaga gcaacagact ggtgctggcc accggcctga gaaacagccc ccagagagag 1020
agaagaaaga agagaggcct gttcggcgcc atcgccggct tcatcgaggg cggctggcag 1080
ggcatggtgg acggctggta cggctaccac cacagcaacg agcagggcag cggctacgcc 1140
gccgacaagg agagcaccca gaaggccatc gacggcgtga ccaacaaggt gaacagcatc 1200
atcgacaaga tgaacaccca gttcgaggcc gtgggcagag agttcaacaa cctggagaga 1260
agaatcgaga acctgaacaa gaagatggag gacggcttcc tggacgtgtg gacctacaac 1320
gccgagctgc tggtgctgat ggagaacgag agaaccctgg acttccacga cagcaacgtg 1380
aagaacctgt acgacaaggt gagactgcag ctgagagaca acgccaagga gctgggcaac 1440
ggctgcttcg agttctacca caagtgcgac aacgagtgca tggagagcgt gagaaacggc 1500
acctacgact acccccagta cagcgaggag gccagactga agagagagga gatcagcggc 1560
gtgaagctgg agagcatcgg catctaccag atcctgagca tctacagcac cgtggccagc 1620
agcctggccc tggccatcat ggtggccggc ctgagcctgt ggatgtgcag caacggcagc 1680
ctgcagtgca gaatctgcat c 1701
<210>21
<211>1701
<212>DNA
<213>Influenza virus
<400>21
atggagaaga tcgtgctgct gctggccatc gtgagcctgg tgaagagcga ccagatctgc 60
atcggctacc acgccaacaa cagcaccgag caggtggaca ccatcatgga gaagaacgtg 120
accgtgaccc acgcccagga catcctggag aagacccaca acggcaagct gtgcgacctg 180
gacggcgtga agcccctgat cctgagagac tgcagcgtgg ccggctggct gctgggcaac 240
cccatgtgcg acgagttcat caacgtgccc gagtggagct acatcgtgga gaaggccaac 300
cccgccaacg acctgtgcta ccccggcaac ttcaacgact acgaggagct gaagcacctg 360
ctgagcagaa tcaaccactt cgagaagatc cccatcatcc ccaagagcag ctggagcgac 420
cacgaggcca gcagcggcgt gagcagcgcc tgcccctacc agggcacccc cagcttcttc 480
agaaacgtgg tgtggctgat caagaagaac aacacctacc ccaccatcaa gagaagctac 540
aacaacacca accaggagga cctgctgatc ctgtggggca tccaccacag caacaacgcc 600
gccgagcaga ccaagctgta ccagaacccc accacctaca tcagcgtggg caccagcacc 660
ctgaacctga gactggtgcc caagatcgcc accagaagca aggtgaacgg ccagagcggc 720
agaatggact tcttctggac catcctgaag cccaacgacg ccatcaactt cgagagcaac 780
ggcaacttca tcgcccccga gtacgcctac aagatcgtga agaagggcga cagcgccatc 840
atgaagagcg aggtggagta cggcaactgc aacaccaagt gccagacccc catcggcgcc 900
atcaacagca gcatgccctt ccacaacatc caccccctga ccatcggcga gtgccccaag 960
tacgtgaaga gcaacaagct ggtgctggcc accggcctga gaaacagccc cctgagagag 1020
agaagaagaa agagaggcct gttcggcgcc atcgccggct tcatcgaggg cggctggcag 1080
ggcatggtgg acggctggta cggctaccac cacagcaacg agcagggcag cggctacgcc 1140
gccgacaagg agagcaccca gaaggccatc gacggcgtga ccaacaaggt gaacagcatc 1200
atcgacaaga tgaacaccca gttcgaggcc gtgggcagag agttcaacaa cctggagaga 1260
agaatcgaga acctgaacaa gaagatggag gacggcttcc tggacgtgtg gacctacaac 1320
gccgagctgc tggtgctgat ggagaacgag agaaccctgg acttccacga cagcaacgtg 1380
aagaacctgt acgacaaggt gagactgcag ctgagagaca acgccaagga gctgggcaac 1440
ggctgcttcg agttctacca caagtgcgac aacgagtgca tggagagcgt gagaaacggc 1500
acctacgact acccccagta cagcgaggag gccagactga agagagagga gatcagcggc 1560
gtgaagctgg agagcatcgg cacctaccag atcctgagca tctacagcac cgtggccagc 1620
agcctggccc tggccatcat ggtggccggc ctgagcctgt ggatgtgcag caacggcagc 1680
ctgcagtgca gaatctgcat c 1701
<210>22
<211>1347
<212>DNA
<213>Influenza virus
<400>22
atgaacccca acaagaagat catcaccatc ggcagcatct gcatggtgac cggcatggtg 60
agcctgatgc tgcagatcgg caacctgatc agcatctggg tgagccacag catccacacc 120
ggcaaccagc acaaggccga gcccatcagc aacaccaact tcctgaccga gaaggccgtg 180
gccagcgtga agctggccgg caacagcagc ctgtgcccca tcaacggctg ggccgtgtac 240
agcaaggaca acagcatcag aatcggcagc aagggcgacg tgttcgtgat cagagagccc 300
ttcatcagct gcagccacct ggagtgcaga accttcttcc tgacccaggg cgccctgctg 360
aacgacaagc acagcaacgg caccgtgaag gacagaagcc cccacagaac cctgatgagc 420
tgccccgtgg gcgaggcccc cagcccctac aacagcagat tcgagagcgt ggcctggagc 480
gccagcgcct gccacgacgg caccagctgg ctgaccatcg gcatcagcgg ccccgacaac 540
ggcgccgtgg ccgtgctgaa gtacaacggc atcatcaccg acaccatcaa gagctggaga 600
aacaacatcc tgagaaccca ggagagcgag tgcgcctgcg tgaacggcag ctgcttcacc 660
gtgatgaccg acggccccag caacggccag gccagccaca agatcttcaa gatggagaag 720
ggcaaggtgg tgaagagcgt ggagctggac gcccccaact accactacga ggagtgcagc 780
tgctaccccg acgccggcga gatcacctgc gtgtgcagag acaactggca cggcagcaac 840
agaccctggg tgagcttcaa ccagaacctg gagtaccaga tcggctacat ctgcagcggc 900
gtgttcggcg acaaccccag acccaacgac ggcaccggca gctgcggccc cgtgagcagc 960
aacggcgcct acggcgtgaa gggcttcagc ttcaagtacg gcaacggcgt gtggatcggc 1020
agaaccaaga gcaccaacag cagaagcggc ttcgagatga tctgggaccc caacggctgg 1080
accgagaccg acagcagctt cagcgtgaag caggacatcg tggccatcac cgactggagc 1140
ggctacagcg gcagcttcgt gcagcacccc gagctgaccg gcctggactg catcagaccc 1200
tgcttctggg tggagctgat cagaggcaga cccaaggaga gcaccatctg gaccagcggc 1260
agcagcatca gcttctgcgg cgtgaacagc gacaccgtgg gctggagctg gcccgacggc 1320
gccgagctgc ccttcaccat cgacaag 1347
<210>23
<211>1347
<212>DNA
<213>Influenza virus
<400>23
atgaacccca accagaagat catcaccatc ggcagcatct gcatgatcac cggcatggtg 60
agcctgatgc tgcaggtggg caacatgatc agcatctgga tcagccacag catccacacc 120
ggcaaccagc accaggccga gcccatcagc aacgccaact tcctgaccaa gagagccgtg 180
gccgccgtga agctggccgg caacagcagc ctgtgcccca tcaacggctg ggccgtgtac 240
agcaaggaca acagcatcag aatcggcagc aagggcgacg tgttcgtgat cagagagccc 300
ttcatcagct gcagccacct ggagtgcaga accttcttcc tgacccaggg cgccctgctg 360
aacgacaagc acagcaacgg caccgccaag gacagaagcc cccacagaac cctgatgagc 420
tgccccgtgg gcgaggcccc cagcccctac aacagcagat tcgagagcgt ggcctggagc 480
gccagcgcct gccacgacgg caccagctgg ctgaccatcg gcatcagcgg ccccgacaac 540
ggcgccgtgg ccgtgctgaa gtacaacggc atcatcaccg acaccatcaa gagctggaga 600
aacaacatcc tgagaaccca ggagagcgag tgcgcctgcg tgaacggcag ctgcttcacc 660
gtgatgaccg acggccccag caacggccag gccagccaca agatcttcaa gatggagaag 720
ggcaaggtgg tgaagagcgt ggagctggac gcccccaact accactacga ggagtgcagc 780
tgctaccccg acgccggcga gatcacctgc gtgtgcagag acaactggca cggcagcaac 840
agaccctggg tgagcttcaa ccagaacctg gagtaccaga tcggctacat ctgcagcggc 900
gtgttcggcg acaaccccag acccaacgac ggcaccggca gctgcggccc cgtgagcagc 960
aacggcgcct acggcgtgaa gggcttcagc ttcaagtacg gcaacggcgt gtggatcggc 1020
agaaccaaga gcaccaacag cagaagcggc ttcgagatga tctgggaccc caacggctgg 1080
accgagaccg acagcagctt cagcgtgaag caggacatcg tggccatcac cgactggagc 1140
ggctacagcg gcagcttcgt gcagcacccc gagctgaccg gcctggactg catcagaccc 1200
tgcttctgga tcgagctgat cagaggcaga cccaaggaga gcaccatctg gaccagcggc 1260
agcagcatca gcttctgcgg cgtgaacagc gacaccgtgg gctggagctg gcccgacggc 1320
gccgagctgc ccttcaccat cgacaag 1347
<210>24
<211>1347
<212>DNA
<213>Influenza virus
<400>24
atgaacccca accagaagat catcaccatc ggcagcatct gcatggtgat cggcatcgtg 60
agcctgatgc tgcagatcgg caacatggtg agcctgtggg tgagccacag catccagacc 120
ggcaaccagc accaggtgga gcccatcagc aacaccaact tcctgaccga gaaggccgtg 180
gccagcgtga ccctggccgg caacagcagc ctgtgcccca tcagaggctg ggccgtgcac 240
agcaaggaca acagcatcag aatcggcagc aagggcgacg tgttcgtgat cagagagccc 300
ttcatcagct gcagccacct ggagtgcaga accttcttcc tgacccaggg cgccctgctg 360
aacgacaagc acagcaacgg caccgtgaag gacagaagcc cccacagaac cctgatgagc 420
tgccccgtgg gcgaggcccc cagcccctac aacagcagat tcgagagcgt ggcctggagc 480
gccagcgcct gccacgacgg caccagctgg ctgaccatcg gcatcagcgg ccccgacaac 540
ggcgccgtgg ccgtgctgaa gtacaacggc atgatcaccg acaccatcaa gagctggaga 600
aacaacatcc tgagaaccca ggagagcgag tgcgcctgcg tgaacggcag ctgcttcacc 660
gtgatgaccg acggccccag caacggccag gccagctaca agatcttcaa gatggagaag 720
ggcaaggtgg tgaagagcgt ggagctggac gcccccaact accactacga ggagtgcagc 780
tgctaccccg acgccggcga gatcacctgc gtgtgcagag acaactggca cggcagcaac 840
agaccctggg tgagcttcaa ccagaacctg gagtaccaga tcggctacat ctgcagcggc 900
gtgttcggcg acaaccccag acccaacggc ggcaccggca gctgcggccc cgtgagcccc 960
aacggcgcct acggcgtgaa gggcttcagc ttcaagtacg gcaacggcgt gtggatcggc 1020
agaaccaaga gccccagcag cagaagcggc ttcgagatga tctgggaccc caacggctgg 1080
accgagaccg acagcagctt cagcgtgaag caggacatcg tggccatcac cgactggagc 1140
ggctacagcg gcagcttcgt gcagcacccc gagctgaccg gcctggactg catcagaccc 1200
tgcttctggg tggagctgat cagaggcaga cccaaggaga gcaccatctg gaccagcggc 1260
agcagcatca gcttctgcgg cgtgaacagc gacaccgtga gctggagctg gcccgacggc 1320
gccgagctgc ccttcaccat cgacaag 1347
<210>25
<211>1347
<212>DNA
<213>Influenza virus
<400>25
atgaacccca accagaagat catcaccatc ggcagcatct gcatggtgat cggcatcgtg 60
agcctgatgc tgcagatcgg caacatgatc agcatctggg tgagccacag catccagacc 120
ggcaaccagc accaggccga gcccatcagc aacaccaact tcctgaccga gaacgccgtg 180
gccagcgtga ccctggccgg caacagcagc ctgtgcccca tcagaggctg ggccgtgcac 240
agcaaggaca acagcatcag aatcggcagc aagggcgacg tgttcgtgat cagagagccc 300
ttcatcagct gcagccacct ggagtgcaga accttcttcc tgacccaggg cgccctgctg 360
aacgacaagc acagcaacgg caccgtgaag gacagaagcc cccacggcac cctgatgagc 420
tgccccatgg gcgaggcccc cagcccctac aacagcagat tcgagagcgt ggcctggagc 480
gccagcgcct gccacgacgg caccagctgg ctgaccatcg gcatcagcgg ccccgacaac 540
ggcgccgtgg ccgtgctgaa gtacaacggc atcatcaccg acaccatcaa gagctggaga 600
aacaacatcc tgagaaccca ggagagcgag tgcgcctgcg tgaacggcag ctgcttcacc 660
gtgatgaccg acggccccag caacggccag gccagctaca agatcttcaa gatggagaag 720
ggcaaggtgg tgaagagcgt ggagctgaac gcccccaact accactacga ggagtgcagc 780
tgctaccccg acgccggcga gatcatctgc gtgtgcagag acaactggca cggcagcaac 840
agaccctggg tgagcttcaa ccagaacctg gagtaccaga tcggctacat ctgcagcggc 900
gtgttcggcg acaaccccag acccaacgac ggcaccggca gctgcggccc cgtgagcccc 960
aacggcgcct acggcatcaa gggcttcagc ttcaagtacg gcaacggcgt gtggatcggc 1020
agaaccaaga gcaccaacag cagaagcggc ttcgagatga tctgggaccc caacggctgg 1080
accggcaccg acagcgactt cagcgtgaag caggacatcg tggccatcac cgactggagc 1140
ggctacagcg gcagcttcgt gcagcacccc gagctgaccg gcctggactg catcagaccc 1200
tgcttctggg tggagctgat cagaggcaga cccaaggaga gcaccatctg gaccagcggc 1260
agcagcatca gcttctgcgg cgtgaacagc gacaccgtga gctggagctg gcccgacggc 1320
gccgagctgc ccttcaccat cgacaag 1347
Claims (27)
1.一种由以下蛋白假型包装的慢病毒载体：
包含HA表位或HA细胞附着配体的流感病毒HA蛋白或含有HA蛋白片段的蛋白，
其中所述HA蛋白不是鸡瘟病毒H7HA。
2.如权利要求1所述的慢病毒载体，其中所述HA蛋白选自于由H1、H2、H5和H7组成的组。
3.如权利要求1或2所述的慢病毒载体，其中所述HA蛋白选自于由SEQ ID NO：1、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：12、SEQ ID NO：13组成的组。
4.如权利要求1、2或3所述的慢病毒载体，其还包含NA。
5.如权利要求1、2、3或4所述的慢病毒载体，其还包含来自H5N1禽流感病毒株的NA。
6.如权利要求4或5所述的慢病毒载体，其中所述NA蛋白选自于由SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11组成的组。
7.如权利要求1到6任一项所述的慢病毒载体，其还包含NA和M2。
8.如权利要求1到7任一项所述的慢病毒载体，其还包含转基因。
9.如权利要求1到8任一项所述的慢病毒载体，其中表达HA的多核苷酸已针对靶细胞或宿主细胞进行密码子优化。
10.如权利要求1到9任一项所述的慢病毒载体，其中表达HA以及NA和M2(如果存在的话)的所述多核苷酸已针对靶细胞或宿主细胞进行密码子优化。
11.如权利要求1到10任一项所述的慢病毒载体，其中所述HA蛋白由来自不同HA同源物的至少两个部分组成。
12.如权利要求3到10任一项所述的慢病毒载体，其中所述NA蛋白由来自不同NA同源物的至少两个部分组成。
13.一种组合物，其包含权利要求1到12任一项所述的慢病毒载体、药用赋形剂、载体和/或免疫学佐剂。
14.一种慢病毒载体包装系统，其包括：
至少一种表达HA的包装载体，和
转移载体构建体，其包含产生序列和包装序列、表达Gag和Pol慢病毒蛋白的序列，并任选包含转基因，
其中所述HA蛋白不是H7HA。
15.一种慢病毒载体包装系统，其包括：
至少一种表达HA的包装载体，
表达Gag和Pol慢病毒蛋白的辅助构建体，和
转移载体构建体，其包含产生序列和包装序列，并任选包含转基因；
其中所述HA蛋白不是H7HA。
16.一种诱导免疫应答的方法，其包括向受试者施用权利要求1到12任一项所述的慢病毒载体，其量足以诱导对所述载体的免疫应答。
17.一种鉴定中和抗体的方法，其包括：
使权利要求1到12任一项所述的慢病毒载体与抗体接触，接触的时间和条件适合所述抗体结合到所述慢病毒载体上，和
测定所述接触对所述慢病毒载体结合或感染宿主细胞的能力的影响。
18.一种用权利要求1到12任一项所述的慢病毒载体转染的靶细胞或宿主细胞。
19.一种组合物，其包含权利要求18所述的靶细胞或宿主细胞、药用赋形剂、载体和/或免疫学佐剂。
20.一种以权利要求8所述的慢病毒载体转染的靶细胞或宿主细胞，其中所述转基因已被掺入到所述细胞的染色体DNA上。
21.一种将多核苷酸序列或转基因转导到细胞内的方法，其包括使细胞与权利要求8所述的慢病毒载体在足以发生转导的时间和条件下接触。
22.一种鉴定调节病毒对细胞的结合或调节病毒对细胞的感染的分子的方法，其包括：
使细胞与候选分子和权利要求1到12任一项所述的假型包装的慢病毒接触，和
测定所述候选分子调节病毒对所述细胞的结合或抑制病毒对所述细胞的感染的能力。
23.如权利要求22所述的方法，其中所述分子是非蛋白药物。
24.如权利要求22所述的方法，其中所述分子是非抗体的肽或多肽。
25.如权利要求22所述的方法，其中所述分子是抗体。
26.如权利要求22所述的方法，其中所述分子包括碳水化合物或脂质。
27.一种使用权利要求8所述的慢病毒载体来转染至少一种靶细胞的方法，其中所述HA、NA和M2使用的比例是8∶2∶1。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
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| US88270006P | 2006-12-29 | 2006-12-29 | |
| US60/882,700 | 2006-12-29 | ||
| PCT/IB2008/001097 WO2008087563A2 (en) | 2006-12-29 | 2008-01-02 | Lentivirus pseudotyped with influenza hemagglutinin and methods of use |
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| CN101888854A true CN101888854A (zh) | 2010-11-17 |
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| EP (1) | EP2111233A2 (zh) |
| JP (1) | JP2010514439A (zh) |
| CN (1) | CN101888854A (zh) |
| BR (1) | BRPI0806336A2 (zh) |
| CA (1) | CA2673994A1 (zh) |
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| WO (1) | WO2008087563A2 (zh) |
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| WO2009036063A1 (en) * | 2007-09-11 | 2009-03-19 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Pseudotyped retroviral vectors and methods of making and using them |
| US8298820B2 (en) | 2010-01-26 | 2012-10-30 | The Trustees Of The University Of Pennsylvania | Influenza nucleic acid molecules and vaccines made therefrom |
| AU2015202267B2 (en) * | 2010-01-26 | 2017-03-23 | The Trustees Of The University Of Pennsylvania | Influenza nucleic acid molecules and vaccines made therefrom |
| SG189236A1 (en) * | 2010-10-04 | 2013-05-31 | Massachusetts Inst Technology | Hemagglutinin polypeptides, and reagents and methods relating thereto |
| EP2670430B1 (en) | 2011-01-31 | 2015-04-01 | Baxter International Inc | Recombinant viral vectors and methods for inducing a heterosubtypic immune response to influenza a viruses |
| US9441019B2 (en) | 2011-09-23 | 2016-09-13 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Influenza hemagglutinin protein-based vaccines |
| WO2015054639A1 (en) | 2013-10-11 | 2015-04-16 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Epstein-barr virus vaccines |
| US20160251680A1 (en) | 2013-11-05 | 2016-09-01 | Clontech Laboratories, Inc. | Dry transfection compositions and methods for making and using the same |
| AU2015373928B2 (en) | 2014-12-31 | 2019-10-17 | The Usa, As Represented By The Secretary, Dept. Of Health And Human Services | Novel multivalent nanoparticle-based vaccines |
| SG10201912944QA (en) | 2016-09-02 | 2020-02-27 | The Usa As Represented By The Secretary | Stabilized group 2 influenza hemagglutinin stem region trimers and uses thereof |
| CN115960842B (zh) * | 2022-11-26 | 2023-09-01 | 哈尔滨维科生物技术有限公司 | 一种提高重组禽流感病毒感染鸡胚能力的方法 |
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| GB0202569D0 (en) * | 2002-02-04 | 2002-03-20 | Oxford Biomedica Ltd | Delivery means |
| EP1398041A1 (en) * | 2002-09-13 | 2004-03-17 | Université de Nantes | Recombinant lentiviral vector pseudotyped with the hemagglutinin protein for gene transfer into the retina |
| CA2541872A1 (en) * | 2006-04-26 | 2007-10-26 | Institut Pasteur | H5 pseudotyped viruses and uses thereof |
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2008
- 2008-01-02 EP EP08737581A patent/EP2111233A2/en not_active Withdrawn
- 2008-01-02 JP JP2009543569A patent/JP2010514439A/ja active Pending
- 2008-01-02 CN CN2008800066143A patent/CN101888854A/zh active Pending
- 2008-01-02 WO PCT/IB2008/001097 patent/WO2008087563A2/en active Application Filing
- 2008-01-02 MX MX2009007106A patent/MX2009007106A/es not_active Application Discontinuation
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| WO2008087563A2 (en) | 2008-07-24 |
| MX2009007106A (es) | 2010-04-30 |
| WO2008087563A3 (en) | 2008-11-27 |
| EP2111233A2 (en) | 2009-10-28 |
| CA2673994A1 (en) | 2008-07-24 |
| US20100137412A1 (en) | 2010-06-03 |
| JP2010514439A (ja) | 2010-05-06 |
| BRPI0806336A2 (pt) | 2011-09-06 |
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