Novel Vaccine Immunogens for Pandemic H5 Influenza

描述（由申请人提供）：当流行病毒的组成突然变化导致个体对该毒株几乎没有或没有免疫力时，就会发生流感大流行。20世纪世纪发生的三次大流行爆发（1918年、1957年和1968年）分别代表了甲型流感病毒的三种不同抗原亚型：H1N1、H2N2和H3N2。对未来大流行病的关切使人们把注意力集中在禽H5N1病毒上，这种病毒不仅显示出感染人类的可能性，而且还显示出很高的病死率。给人群接种疫苗以保护他们免受未来大流行病毒的侵害是一个明显的问题。联合国禽流感和人类流感高级协调员大卫纳巴罗说：“在大流行病毒出现之前，我们无法获得针对大流行病毒的疫苗，然后可能会有六个月的间隔，直到我们有合理数量的疫苗。“目前正在努力研制针对H5N1病毒的大流行前疫苗，这种疫苗可以对不同的毒株提供广泛的保护。然而，高致病性H5N1病毒分化成不同的进化枝增加了开发针对多种H5N1亚型的预防性疫苗的复杂性。我们在这里提出了一种新的方法来解决这个问题，利用定向分子进化的力量。该技术包括用于产生新的蛋白质序列的方法以及筛选蛋白质变体的手段。体外同源DNA重组可以从编码疫苗所需蛋白质的病原体基因中创建高质量功能多样性文库。然后可以筛选变体蛋白质的文库以改善免疫原性。唯一的主要相关保护免受流感是抗体的表面糖蛋白，主要是血凝素（HA）。我们假设可以产生比野生型蛋白更具免疫原性的H5 HA蛋白的变体，并且将诱导具有潜在广泛中和效力的免疫血清。将通过野生型H5 HA编码基因的体外DNA重组来产生编码变体以检验该假设的基因。这项工作所针对的具体产品是一种基于HA的疫苗，对许多H5N1病毒具有广泛的中和活性。尽管如此，一旦出现大流行毒株，变异株和由它们诱导的血清储备的建立将提供一种快速选择制备疫苗所需的免疫原的手段。目前的建议将评估这一想法的可行性。国会预算办公室的一项研究估计，严重大流行的后果可能包括仅在美国就有2亿人感染，9000万人临床患病，200万人死亡。如果大流行是由H5N1引起的，鉴于观察到的这种病毒感染的病死率>50%，死亡人数可能会高得多。这里提出的这种战略如果成功，可以提供一种预防大流行性H5流感毒株的措施，并有可能挽救数百万人的生命。公共卫生相关性：当个人对病毒的保护不力时，流感大流行就会发生。我们建议建立一个候选疫苗的集合，可以针对新出现的大流行病进行快速测试。这里提出的这种战略可以提供针对大流行性流感病毒的保护，挽救数百万人的生命。

DESCRIPTION (provided by applicant): Influenza pandemics occur when an abrupt change in the composition of the circulating viruses results in a strain to which individuals have little or no immunity. The three pandemic outbreaks that occurred in the 20th century in 1918, 1957, and 1968 represent three different antigenic subtypes of influenza A virus: H1N1, H2N2, and H3N2, respectively. The concern of a future pandemic has focused attention on the avian H5N1 virus, which has demonstrated not only the possibility of infecting humans but to do so with a high case-fatality rate. Vaccinating populations to protect them from a future pandemic virus poses an obvious problem. David Nabarro, UN senior coordinator for avian and human influenza, stated: "We cannot have a vaccine against a pandemic virus until the pandemic virus appears and then there will be an interval of perhaps six months until we have a reasonable quantity of vaccine." Efforts are underway to create pre-pandemic vaccines to H5N1 viruses that can confer broad protection against different strains. However, the divergence of highly pathogenic H5N1 viruses into distinct clades has increased the complexity of developing a prophylactic vaccine against multiple H5N1 subtypes. We propose here a novel way to approach this problem, using the power of directed molecular evolution. This technology encompasses a method for creating novel protein sequences coupled with a means of screening the protein variants. In vitro homologous DNA recombination can create libraries of high-quality functional diversity from pathogen genes encoding the proteins needed to create a vaccine. Libraries of the variant proteins can then be screened for improved immunogenicity. The only major correlate of protection against influenza is antibody to its surface glycoproteins, primarily the hemagglutinin (HA). We hypothesize that variants of the H5 HA protein can be created that are more immunogenic than the wild-type proteins, and will induce immune sera with potentially broad neutralization potency. Genes encoding the variants to test this hypothesis will be created by in vitro DNA recombination of wildtype H5 HA-encoding genes. The specific product that is being targeted by this work is an HA-based vaccine with broad neutralization activity against many H5N1 viruses. The creation of a stockpile of variants and the sera induced by them will nonetheless provide a means to rapidly choose the immunogen needed to prepare a vaccine once a pandemic strain has appeared. The current proposal will assess the feasibility of this idea. A study by the Congressional Budget Office estimates that the consequences of a severe pandemic could include 200 million people infected, 90 million clinically ill, and 2 million dead in the United States alone. If a pandemic is caused by H5N1, the numbers of deaths could be considerably higher given the observed >50% case-fatality rate for infection by this virus. A strategy such as that proposed here could - if successful - provide a measure of protection against a pandemic H5 influenza strain and potentially save millions of lives. PUBLIC HEALTH RELEVANCE: Influenza pandemics occur when individuals are poorly protected from the virus. We propose to create a collection of vaccine candidates that can rapidly be tested against an emerging pandemic. A strategy such as that proposed here could provide protection against a pandemic flu virus and save millions of lives.