Antigenic fingerprinting of H5N1 avian influenza using convalescent sera and monoclonal antibodies reveals potential vaccine and diagnostic targets.
Antigenic fingerprinting of H5N1 avian influenza using convalescent sera and monoclonal antibodies reveals potential vaccine and diagnostic targets.
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DOI：
10.1371/journal.pmed.1000049
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发表时间：
2009-04-21
期刊：
影响因子：
15.8
通讯作者：
Golding H
中科院分区：
文献类型：
--
作者：
Khurana S;Suguitan AL Jr;Rivera Y;Simmons CP;Lanzavecchia A;Sallusto F;Manischewitz J;King LR;Subbarao K;Golding H
利用全基因组片段噬菌体展示文库，Hana Golding及其同事在感染H5N1禽流感后康复的人体内鉴定了血清抗体识别的病毒表位。高致病性H5N1禽流感病毒从家禽向人类的传播引起了人们对即将发生的流感大流行的担忧。正在作出协调一致的努力，准备有效的疫苗和疗法，包括针对H5N1的多克隆或单克隆抗体。由于缺乏针对禽流感的保护性免疫反应的信息，目前的努力受到阻碍。描述恢复期个体的B细胞反应可以帮助设计未来的疫苗和治疗方法。为了满足这一需求，我们建立了全基因组片段噬菌体展示文库（GFPDL），表达15-350个氨基酸片段，覆盖了A/Vietnam/1203/2004 （H5N1）的所有蛋白质。这些GFPDL用于分析5名H5N1感染康复个体的中和性人单克隆抗体和血清。这种方法导致了两种具有构象依赖表位的广泛中和的人单克隆抗体的定位。在H5N1恢复期血清中，我们在H5 HA[(-10)-223]、神经氨酸酶催化位点和M2外结构域中发现了几个潜在的保护性H5N1特异性人抗体表位。此外，据我们所知，我们首次在人类中发现了H5N1感染后对PB1-F2（一种假定的毒力因子）的强烈反应。重要的是，发现了新的表位，这些表位可被H5N1恢复期血清识别，但与对照组血清（H5N1 naïve、H1N1或H3N2血清阳性）不发生反应。据我们所知，这是首次描述H5N1感染后完整抗体库的研究。总的来说，这些数据将有助于合理的疫苗设计和新的h5n1特异性血清诊断监测工具。每年冬天，数百万人感染流感，这是一种呼吸道病毒感染。大多数人恢复得很快，但季节性流感爆发（流行病）每年造成约50万人死亡。这些流行病的发生是因为人体免疫系统对病毒蛋白（抗原）产生反应的微小但频繁的变化，这意味着一年通过感染或通过接种疫苗产生的免疫反应只能在明年提供部分预防流感的保护。流感病毒偶尔也会出现主要的抗原变化。人类对这些病毒（通常起源于动物或鸟类）很少或没有免疫力，因此它们可以引发致命的全球流行病（大流行）。令人担忧的是，上一次流感大流行发生在1968年，许多专家担心，另一次大流行现在已经姗姗来迟。他们认为，引发这种大流行的可能是H5N1禽流感病毒，这种病毒于1996年首次出现在中国的一只鹅身上。该名称表明病毒中存在的两种主要流感抗原的类型：H5N1携带5型血凝素和1型神经氨酸酶。在过去10年里，H5N1病毒已导致约400例确诊的人类流感病例和250多人死亡，但由于它不易在人与人之间传播，因此尚未引发人类大流行。然而，它有可能在任何时候获得这种能力，因此，优先开发既能预防H5N1大流行病毒株的疫苗，又能为未受疫苗接种保护的人开发基于抗体的抗病毒疗法（抗体是免疫系统产生的有助于抵抗感染的蛋白质；人们有时可以通过注射预先制备的抗体来保护自己免受感染）。要做到这一点，科学家需要知道人类免疫系统如何对H5N1病毒作出反应。特别是，他们需要知道免疫系统可以检测到病毒的哪些部分并产生抗体。因此，在这项研究中，研究人员描述了H5N1感染恢复期人群中发现的特异性抗体反应。研究人员制作了几个“基因组片段噬菌体展示文库”，收集细菌病毒（噬菌体），使每个噬菌体产生许多可能的非噬菌体蛋白短片段（多肽）中的一个。这种“文库”可用于研究来自特定来源的抗体识别哪些片段。在这种情况下，建立了若干文库，其中包含导致2004-2005年越南人类流感爆发的H5N1毒株的基因组片段（A/Vietnam/1203/2004）。研究人员利用这些文库分析了5名感染A/Vietnam/1203/2004病毒后康复的越南人产生的抗体。研究人员报告说，H5N1病毒恢复期的血液样本中含有能够识别几种病毒蛋白中的小区域（“表位”）的抗体，这些病毒蛋白包括血凝素、神经氨酸酶、一种名为M2的结构蛋白和一种名为PB1-F2的病毒蛋白，后者在一定程度上导致了H5N1感染的严重程度。从感染其他流感病毒的恢复期患者的血液中提取的抗体无法识别所发现的几个新表位。这组科学家还利用他们的噬菌体展示文库分析了从感染A/Vietnam/1203/2004病毒的患者身上产生的两种中和性人类单克隆抗体（中和抗体保护小鼠免受H5N1病毒通常致命的攻击；单克隆抗体是在实验室通过培养产生单一类型抗体的连续生长细胞系产生的）。重要的是，这两种中和性单克隆抗体都识别了“非连续构象依赖表位”——在蛋白质的多肽序列中彼此不相邻的蛋白质序列，但由于蛋白质折叠的方式，它们在空间上靠得很近。虽然在这一分析中可能遗漏了暴露于H5N1流感病毒的人体内产生的抗体库的某些方面，但这些发现提供了关于人类免疫系统如何对这种病毒感染作出反应的重要和详细的新信息。特别是，它们表明，从H5N1感染中恢复的人在至少6个月的时间里可以产生针对几种病毒蛋白的多种抗体，并识别出H5N1中可能特别善于刺激保护性免疫反应的特定部分。这些信息现在可用于帮助设计针对H5N1的疫苗和用于治疗H5N1感染的基于抗体的疗法，并开发用于监测人群中禽流感暴发的新工具。请通过本摘要的在线版本http://dx.doi.org/10.1371/journal.pmed.1000049访问这些网站。美国疾病控制和预防中心为患者和专业人员提供有关流感的信息，包括禽流感和大流行性流感的具体信息（以几种语言）。世界卫生组织提供有关流感（以几种语言）和H5N1禽流感（以几种语言）的信息。以及关于禽类和人类感染H5N1禽流感的全球时间表英国卫生保护局提供有关禽流感、大流行和流行（季节性）流感的信息。MedlinePlus提供有关流感和禽流感的其他信息的链接列表（英文和西班牙文）
Using whole-genome-fragment phage display libraries, Hana Golding and colleagues identify the viral epitopes recognized by serum antibodies in humans who have recovered from infection with H5N1 avian influenza. Transmission of highly pathogenic avian H5N1 viruses from poultry to humans have raised fears of an impending influenza pandemic. Concerted efforts are underway to prepare effective vaccines and therapies including polyclonal or monoclonal antibodies against H5N1. Current efforts are hampered by the paucity of information on protective immune responses against avian influenza. Characterizing the B cell responses in convalescent individuals could help in the design of future vaccines and therapeutics. To address this need, we generated whole-genome–fragment phage display libraries (GFPDL) expressing fragments of 15–350 amino acids covering all the proteins of A/Vietnam/1203/2004 (H5N1). These GFPDL were used to analyze neutralizing human monoclonal antibodies and sera of five individuals who had recovered from H5N1 infection. This approach led to the mapping of two broadly neutralizing human monoclonal antibodies with conformation-dependent epitopes. In H5N1 convalescent sera, we have identified several potentially protective H5N1-specific human antibody epitopes in H5 HA[(-10)-223], neuraminidase catalytic site, and M2 ectodomain. In addition, for the first time to our knowledge in humans, we identified strong reactivity against PB1-F2, a putative virulence factor, following H5N1 infection. Importantly, novel epitopes were identified, which were recognized by H5N1-convalescent sera but did not react with sera from control individuals (H5N1 naïve, H1N1 or H3N2 seropositive). This is the first study, to our knowledge, describing the complete antibody repertoire following H5N1 infection. Collectively, these data will contribute to rational vaccine design and new H5N1-specific serodiagnostic surveillance tools. Every winter, millions of people catch influenza, a viral infection of the airways. Most recover quickly but seasonal influenza outbreaks (epidemics) kill about half a million people annually. These epidemics occur because small but frequent changes in the viral proteins (antigens) to which the human immune system responds mean that an immune response produced one year by infection or through vaccination provides only partial protection against influenza the next year. Influenza viruses also occasionally appear that contain major antigenic changes. Human populations have little or no immunity to such viruses (which often originate in animals or birds), so they can start deadly global epidemics (pandemics ). Worryingly, the last influenza pandemic occurred in 1968 and many experts fear that another pandemic is now overdue. The trigger for such a pandemic, they think, could be the avian (bird) H5N1 influenza virus, which first appeared in 1996 in a goose in China. The name indicates the types of two major influenza antigens present in the virus: H5N1 carries type 5 hemagglutinin and type 1 neuraminidase. H5N1 has caused about 400 confirmed cases of human influenza and more than 250 deaths in the past decade but it has not started a human pandemic because it cannot pass easily between people. However, it could possibly acquire this ability at any time, so it is a priority to develop both vaccines that will provide protection against a pandemic H5N1 viral strain, as well as antibody-based antiviral therapies for people not protected by vaccination (antibodies are proteins produced by the immune system that help to fight infections; people can sometimes be protected from infection by injecting them with pre-prepared antibodies). To do this, scientists need to know how the human immune system responds to the H5N1 virus. In particular, they need to know which parts of the virus the immune system can detect and make antibodies against. In this study, therefore, the researchers characterize the specific antibody responses found in people recovering from infection with H5N1. The researchers made several “genome-fragment phage display libraries”, collections of bacterial viruses (phages) engineered so that each phage makes one of many possible short pieces (polypeptides) of a nonphage protein. Such “libraries” can be used to investigate which fragments are recognized by antibodies from a given source. In this case, several libraries were made that contained fragments of the genome of the H5N1 strain responsible for an outbreak of human influenza in Vietnam in 2004–2005 (A/Vietnam/1203/2004). The researchers used these libraries to analyze the antibodies made by five Vietnamese people recovering from infection with A/Vietnam/1203/2004. H5N1 convalescent blood samples, the researchers report, contained antibodies that recognized small regions (“epitopes”) in several viral proteins, including hemagglutinin, neuraminidase, a structural protein called M2, and a viral protein called PB1-F2 that is partly responsible for the severity of H5N1 infections. Several of the novel epitopes identified were not recognized by antibodies in blood taken from people recovering from infection with other influenza viruses. The researchers also used their phage display libraries to analyze two neutralizing human monoclonal antibodies generated from patients infected with A/Vietnam/1203/2004 (neutralizing antibodies protect mice against normally lethal challenge with H5N1; monoclonal antibodies are generated in the laboratory by creating continuously growing cell lines that produce a single type of antibody). Importantly, both of the neutralizing monoclonal antibodies recognized “noncontinuous conformation-dependent epitopes”—protein sequences that are not adjacent to one another in the polypeptide sequence of the protein, but that lie close together in space because of the way the protein is folded up. Although some aspects of the antibody repertoire produced in people exposed to the H5N1 influenza virus may have been missed in this analysis, these findings provide important and detailed new information about how the human immune system responds to infection with this virus. In particular, they show that people recovering from H5N1 infection make a diverse range of antibodies against several viral proteins for at least six months and identify specific parts of H5N1 that may be particularly good at stimulating a protective immune response. This information can now be used to help design vaccines against H5N1 and antibody-based therapies for the treatment of H5N1 infections, and to develop new tools for monitoring outbreaks of avian influenza in human populations. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000049. This study is further discussed in a PLoS Medicine Perspective by Malik Peiris The US Centers for Disease Control and Prevention provides information for about influenza for patients and professionals, including specific information on avian and pandemic influenza (in several languages) The World Health Organization provides information on influenza (in several languages) and on H5N1 avian influenza (in several languages), and a global timeline about H5N1 avian influenza infection in birds and people The UK Health Protection Agency provides information on avian, pandemic, and epidemic (seasonal) influenza MedlinePlus provides a list of links to other information about influenza and bird flu (in English and Spanish)
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影响因子：
3.1
作者：
Simmons, C. P.;Bernasconi, N. L.;Suguitan, A. L.
通讯作者：
Suguitan, A. L.
影响因子：
3.8
作者：
BLACK, RA;ROTA, PA;KENDAL, AP
通讯作者：
KENDAL, AP
影响因子：
82.9
作者：
Neirynck, S;Deroo, T;Fiers, W
通讯作者：
Fiers, W
DOI：
10.1097/01.qai.0000242465.50947.5f
发表时间：
2006-11-01
影响因子：
3.6
作者：
Khurana, Surender;Needham, James;Golding, Hana
通讯作者：
Golding, Hana
影响因子：
64.5
作者：
GREEN, N;ALEXANDER, H;LERNER, RA
通讯作者：
LERNER, RA