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。2003年，欧洲发生了高致病性禽流感H7N7病毒人感染大爆发。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，从中国、俄罗斯、哈萨克斯坦、蒙古和西藏的候鸟和野生鸟类中分离到H5N1病毒。自2003年12月以来，印度尼西亚、越南、泰国和柬埔寨报告了人感染H5N1病例，截至2005年8月5日，共有112例病例和57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)是一种由内部基因突变决定的具有冷适应、温度敏感(Ts)和减毒(ATT)表型的减毒活疫苗种子病毒。重新组合的H1N1和H3N2型人流感病毒与AA-ca病毒的六个内部基因片段已被反复证明具有这些表型，并且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。 目前尚不清楚携带禽流感HA和NA基因的候选疫苗病毒对人类是否会过度减毒，或者是否会与一些残留毒力有关。减毒活疫苗必须能够复制到能够引起保护性免疫反应的水平，而不会在宿主中引起疾病，因此必须在减毒和传染性之间取得平衡。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，以最大限度地延迟预防感染，而大流行准备的一个重要方法是在甲型流感实际传播之前产生和评估被认为具有大流行潜力的甲型流感候选疫苗。 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。候选H9N2疫苗病毒显示了AA CA疫苗供体病毒的ts、ca和att表型。H9n2aa疫苗病毒在小鼠中具有免疫原性，并保护小鼠免受同源和异种h9n2病毒的后续攻击。该疫苗已在临床上大量生产，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年在亚洲引起人感染的h5n1病毒的候选疫苗，我们应用了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h5n1流感病毒的血凝素和神经氨酸酶基因以及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。候选大流行疫苗的ts和ca表型将在体外进行评估，并将在小鼠身上进行临床前研究，以确定疫苗的减毒水平、免疫原性和对抗攻击的有效性。H5N1疫苗的临床试验将集中在候选流感疫苗的安全性、传染性和免疫原性上。

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. In 2003, a large outbreak of human infections with a highly pathogenic avian influenza H7N7 virus occurred in Europe. 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 China, Russia, Kazakhstan, Mongolia and Tibet. Human cases of H5N1 infection have been reported since December 2003 in Indonesia, Vietnam, Thailand and Cambodia with a total of 112 cases and 57 deaths as of August 5, 2005. 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 internal 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 these 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. It is not known whether the candidate vaccine viruses bearing avian influenza HA and NA genes will be over-attenuated for humans or will be associated with some residual virulence. 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 and infectivity 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 spread. 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. The candidate H9N2 vaccine virus demonstrated the ts, ca and att phenotypes of the AA ca vaccine donor virus. The H9N2 AA ca vaccine virus was immunogenic in mice and protected mice from subsequent challenge with homologous and heterologous H9N2 viruses. 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, we 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 ts and ca phenotypes of the candidate pandemic vaccines will be evaluated in vitro and preclinical studies will be carried out in mice to determine the level of attenuation, immunogenicity and efficacy against challenge. Clinical trials of the H5N1 vaccines will focus on safety, infectivity and immunogenicity of the candidate influenza vaccines.