Novel Vaccine Immunogens for Pandemic H5 Influenza

描述（由申请人提供）：当流行病毒的成分突然发生变化，导致个体很少或没有免疫力的毒株时，就会发生流感大流行。发生在20世纪的1918年、1957年和1968年的三次大流行暴发分别代表了甲型流感病毒的三种不同抗原亚型：H1N1、H2N2和H3N2。对未来大流行的担忧使人们把注意力集中在H5N1禽流感病毒上，该病毒已证明不仅有可能感染人类，而且有很高的病死率。为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David Nabarro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，然后可能需要6个月的时间才能获得合理数量的疫苗。”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病毒分化为不同的进化支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。该技术包括一种创建新蛋白质序列的方法，以及一种筛选蛋白质变体的方法。在体外，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的功能多样性文库。然后，可以对变异蛋白文库进行筛选，以提高免疫原性。预防流感的唯一主要相关因素是对其表面糖蛋白的抗体，主要是血凝素（HA）。我们假设可以创建比野生型蛋白更具免疫原性的H5 HA蛋白变体，并将诱导具有潜在广泛中和效力的免疫血清。编码变异的基因将通过野生型H5 ha编码基因的体外DNA重组来创建，以测试这一假设。这项工作针对的特定产品是一种以ha为基础的疫苗，对许多H5N1病毒具有广泛的中和活性。尽管如此，一旦大流行毒株出现，建立变种和由它们诱导的血清储备将提供一种快速选择所需免疫原以制备疫苗的手段。目前的提案将评估这一想法的可行性。美国国会预算办公室（Congressional Budget Office）的一项研究估计，仅在美国，一场严重流行病的后果可能包括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，鉴于观察到该病毒感染的50%以上病死率，死亡人数可能会高得多。这里提出的这种策略——如果成功的话——可以提供一种针对H5流感大流行毒株的保护措施，并有可能挽救数百万人的生命。公共卫生相关性：当个人对病毒防护不力时，就会发生流感大流行。我们建议建立一个候选疫苗集合，可以对新出现的大流行进行快速测试。这里提出的这种策略可以预防流感大流行，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.