Vaccines for Pandemic Influenza

甲型流感病毒根据其表面糖蛋白、血凝素(HA)和神经氨酸酶(NA)的抗原性分为亚型，从禽类中分离出15个HA和9个NA亚型的流感病毒，但上个世纪只有H1N1、H2N2和H3N2亚型病毒在人类中广泛传播并引起流行病。水禽是新亚型甲型流感病毒进入人类种群的蓄水池。在过去10年中，已多次发现人感染H7、H5和H9三个亚型的禽流感病毒，每一例都伴随着家禽疾病的同期暴发。自1997年以来，亚洲曾多次出现由高致病性禽流感H5N1病毒引起的严重疫情。最近一次在家禽中爆发的H5N1疫情始于2003年底，影响到亚洲至少10个国家。最近的报告表明，已从亚洲、欧洲和非洲的候鸟和野生鸟类中分离出H5N1病毒。自2003年12月以来，已在5个国家报告了人感染H5N1病例，截至2006年8月1日，共有232例病例和134例死亡。 有几种潜在的策略可用于开发疫苗来保护人类免受流感病毒的侵袭，包括福尔马林灭活的全病毒或裂解病毒、HA亚单位和减毒活疫苗。减毒活疫苗通常会引起广泛的交叉反应保护，如果无法获得由实际大流行毒株产生的疫苗，这在大流行发生时可能是一个有用的特征。我们计划的目标是生产针对一系列具有大流行潜力的甲型流感亚型的候选减毒重组流感病毒疫苗，并在临床前研究和临床试验中对这些疫苗进行评估。疫苗病毒将包含一种具有大流行潜力的选定禽流感病毒的血凝素(HA)和神经氨酸酶(NA)基因，以及来自A/Ann Arbor/6/60冷适应(A/AA/6/60 ca)供体病毒的减毒基因。冷适应(CA)流感病毒A/Ann Arbor/6/60(AA)(H_2N_2)是一种具有冷适应性、温度敏感性(Ts)和减毒(Att)表型的减毒活疫苗种子病毒，其表型特征是编码非表面糖蛋白基因的基因突变，即编码内部蛋白基因的基因突变。重新组合的H1N1和H3N2型人流感病毒与AA-ca病毒的6个内部基因片段已被反复证明具有ts和att表型，在人类中的广泛评估已证明它们作为活病毒疫苗是减毒和安全的。这种方法已被许可用于大流行之间的甲型和乙型流感病毒感染。减毒活疫苗必须能够复制到能够在不引起宿主疾病的情况下产生保护性免疫反应的水平，因此必须在减毒、传染性和免疫原性之间取得平衡。在可能发生大流行的情况下，最佳公共卫生应对措施要求提供疫苗，以最小的延迟预防感染，而大流行准备的一个重要方法是在甲型流感大流行病毒实际出现之前，针对被认为具有大流行潜力的甲型流感亚型产生和评估候选疫苗。 我们通过基因重组产生了一个候选的h9n2流感疫苗；该疫苗株含有来自一种禽流感病毒的血凝素和神经氨酸酶基因，以及来自aa ca病毒的六个内部基因片段。基于在小鼠和雪貂身上有希望的临床前数据，该疫苗的临床批量已经产生，并在IND下进行了针对健康成年人的疫苗安全性和免疫原性的I期临床试验。临床试验的实验室结果分析正在进行中。 为了生产针对1997年、2003年和2004年在亚洲造成人类感染的H5N1病毒的候选疫苗，LID的科学家们与Medimmune，Inc.的科学家在CRADA下合作，并应用基于质粒的反向遗传学技术，即可以从与表达8个流感基因片段中的每一个片段的质粒共转染的细胞中恢复传染性病毒，以产生包含来自H5N1流感病毒的血凝素和神经氨酸酶基因以及来自AA CA病毒的6个内部基因片段的重组病毒。我们去除了高致病性H5N1流感病毒血凝素基因中与家禽致病相关的多个碱性氨基酸基序的毒力基序。候选H5N1疫苗病毒具有由AA-ca病毒内部蛋白基因指定的ts和att表型。更重要的是，候选疫苗在小鼠身上具有免疫原性。在接种单剂疫苗四周后，小鼠在受到1997年至2005年在亚洲分离的同源和抗原性截然不同的异源野生型H5N1病毒的攻击后，完全免于致死。在接种两剂疫苗四周后，小鼠和雪貂完全免受同源和异种wt H5N1病毒的肺部复制的影响。基于有希望的小鼠和雪貂的临床前数据，2004年H5N1 CA疫苗的临床批量已经产生，并在IND下进行了针对健康成年人的疫苗安全性和免疫原性的I期临床试验。临床试验正在进行中。

Influenza A viruses are divided into subtypes on the basis of the antigenicity of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA); influenza viruses bearing 15 HA and 9 NA subtypes have been isolated from birds, but only H1N1, H2N2, and H3N2 subtype viruses have circulated widely and caused epidemic disease in humans in the last century. Aquatic birds serve as a reservoir from which new subtypes of influenza A viruses enter the human population. In the last 10 years, human infections with avian influenza viruses (AIV) of three subtypes, H7, H5 and H9, have been detected on several occasions, accompanied in each case by contemporaneous outbreaks of disease in poultry. On several occasions since 1997, there have been serious outbreaks caused by a highly pathogenic avian influenza H5N1 viruses in Asia. The most recent H5N1 outbreak in poultry began in late 2003 and affected at least 10 countries in Asia. Recent reports indicate that H5N1 viruses have been isolated from migratory and wild birds in Asia, Europe and Africa. Human cases of H5N1 infection have been reported since December 2003 in 5 countries with a total of 232 cases and 134 deaths as of August 1, 2006. There are several potential strategies for the development of vaccines to protect humans against influenza viruses, including formalin inactivated whole or split virus, HA subunit, and live attenuated vaccines. Live attenuated vaccines generally induce broadly cross-reactive protection, which may be a useful feature in the event of a pandemic if a vaccine generated from the actual pandemic strain is not available. The goal of our program is to generate candidate live attenuated reassortant influenza virus vaccines against a range of influenza A subtypes that have pandemic potential and to evaluate these vaccines in preclinical studies and clinical trials. The vaccine viruses will contain the hemagglutinin (HA) and neuraminidase (NA) genes of a selected avian influenza virus with pandemic potential and the attenuating genes from the A/Ann Arbor/6/60 cold adapted (A/AA/6/60 ca) donor virus. The cold-adapted (ca) influenza virus A/Ann Arbor/6/60 (AA) (H2N2) has been developed as a live attenuated vaccine seed virus that exhibits cold-adaptation, temperature-sensitive (ts), and attenuation (att) phenotypes which are specified by mutations in the genes encoding the non-surface glycoprotein genes, i.e., the genes encoding the internal protein genes. Reassortant H1N1 and H3N2 human influenza A viruses with the six internal gene segments of the AA ca virus have been repeatedly demonstrated to bear the ts and att phenotypes and extensive evaluation in humans has proven them to be attenuated and safe as live virus vaccines. This approach has been licensed for general use for interpandemic influenza A and B virus infections. Live attenuated vaccines must be able to replicate to levels that elicit a protective immune response without causing disease in the host so a balance between attenuation, infectivity and immunogenicity must be achieved. An optimal public health response in the event of a potential pandemic requires that vaccines be available to prevent infection with minimum delay and an important approach to pandemic preparedness is to generate and evaluate candidate vaccines against influenza A subtypes that are recognized to have pandemic potential, prior to their actual emergence of a pandemic virus. We generated a candidate H9N2 influenza vaccine by genetic reassortment; the vaccine strain contains the hemagglutinin and neuraminidase genes from an avian H9N2 influenza virus and six internal gene segments from the AA ca virus. Based on promising preclinical data in mice and ferrets, a clinical lot of this vaccine was generated and a Phase I clinical trial of the safety and immunogenicity of the vaccine for healthy adults was undertaken under an IND. Analysis of laboratory results from the clinical trial is under way. In order to generate candidate vaccines against H5N1 viruses that have caused human infections in Asia in 1997, 2003 and 2004, LID scientists collaborated with scientists from Medimmune, Inc., under a CRADA and applied plasmid based reverse genetics, a technique in which infectious virus can be recovered from cells co-transfected with plasmids expressing each of the 8 influenza gene segments, to generate reassortant viruses that contain the hemagglutinin and neuraminidase genes from H5N1 influenza viruses and six internal gene segments from the AA ca virus. We removed the virulence motif of multiple basic amino acid motifs in the hemagglutinin gene of the highly pathogenic H5N1 influenza virus that are associated with pathogenicity in poultry. The candidate H5N1 vaccine viruses possessed the ts and att phenotypes specified by the internal protein genes of the AA ca virus. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of vaccine, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wild-type H5N1 viruses isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. Based on promising preclinical data in mice and ferrets, a clinical lot of the 2004 H5N1 ca vaccine was generated and a Phase I clinical trial of the safety and immunogenicity of the vaccine for healthy adults was undertaken under an IND. The clinical trial is in progress.