Developing a Paramyxovirus-based H5N1 Vaccine

描述(申请人提供)：H5N1对人类健康构成迫在眉睫的威胁。安全有效的疫苗是防止人类人口大规模暴发的最好方法。副流感病毒5(PIV5)是一种副粘病毒，会引起狗的犬舍咳嗽，但尚不知道会在人类身上引起任何疾病。PIV5可以在许多细胞中产生高滴度(高达8x108个空斑形成单位(PFU)/毫升)，包括世界卫生组织批准用于疫苗生产的Vero细胞。PIV5可以感染人类细胞系，也可以感染原代人类细胞。在我们的初步研究中，一次剂量为104pfu的表达H3亚型HA基因的活重组PIV5在小鼠中提供了对A3型流感病毒感染的免疫力。我们假设PIV5是疫苗开发的良好载体，我们建议测试PIV5作为H5N1疫苗的载体。我们将专注于以下具体目标：1.检测表达H5N1血凝素的重组PIV5(PIV5-H5)作为H5N1疫苗；2.检测以PIV5为抗原表达的其他H5N1蛋白作为疫苗的潜力；3.测试突变的PIV5病毒作为疫苗载体的可能性。 与公共卫生的相关性：甲型流感病毒每年都会导致严重的发病率和死亡率。目前在人类中流行的毒株(即H1N1、H1N2和H3N2型)感染了高达15%的世界人口，并在美国导致平均36,000人死亡和226,000人住院(28)，以及全球数百万人死亡。在过去的一个世纪里，大流行性流感的零星暴发造成了大量死亡，最著名的是1918年的西班牙流感，并在全球范围内造成了5000多万人死亡。地平线上即将出现另一种可能大流行的H5N1流感毒株。自2003年以来，这种禽流感病毒最明显地出现在东南亚，导致数百万禽类死亡，309人感染，187人死亡(世卫组织，2007年5月31日向世卫组织报告的甲型H5N1禽流感确诊人间病例累计数量)，并有可能成为下一次大流行。 目前，FDA批准的唯一针对H5N1的疫苗具有严重的局限性，特别是因为它必须接种两次，而且与传统流感疫苗相比，需要相当高的疫苗浓度才能达到中等水平的疗效。使用H5N1病毒HA和NA的传统疫苗免疫原性差，存在安全和生产问题。通过反向遗传学产生了H5N1减毒活疫苗，但产生重新组合的风险在大多数情况下禁止使用这种疫苗。灭活病毒疫苗也是通过反向遗传学获得并大量生产的，但NIAID临床试验的初步结果表明，要想有效，需要多次免疫和标准流感病毒抗原剂量的6倍，即90ug而不是15ug的抗原，同时只对一小部分(~50%)接种的人提供保护。尽管如此，FDA最近已批准H5N1灭活疫苗用于18岁至之间的人群，这一年龄段不是最容易受到流感病毒感染的年龄段。因此，有理由需要新的疫苗策略，以提供更高的免疫原性和安全性。 副流感病毒5(PIV5)是一种副粘病毒，会引起狗的犬舍咳嗽，但尚不知道会在人类身上引起任何疾病。PIV5可以在许多细胞中产生高滴度(高达8x108个空斑形成单位(PFU)/毫升)，包括世界卫生组织批准用于疫苗生产的Vero细胞。PIV5可以感染人类细胞系，也可以感染原代人类细胞。在我们的初步研究中，一次剂量为104pfu的表达H3亚型HA基因的活重组PIV5在小鼠中提供了对A3型流感病毒感染的免疫力。在这个建议中，我们假设PIV5是一个很好的疫苗开发载体，我们建议测试PIV5作为H5N1疫苗的载体。

DESCRIPTION (provided by applicant): H5N1 is a looming threat to human health. A safe and effective vaccine is the best way to prevent large-scale outbreaks in the human population. Parainfluenza virus 5 (PIV5), a paramyxovirus, causes kennel cough in dogs, but is not known to cause any illness in humans. PIV5 can be produced in high titers (up to 8x108 plaque forming units (PFU)/ml) in many cells including Vero cells, which are WHO approved for vaccine production. PIV5 can infect human cell lines as well as primary human cells. In our preliminary studies, a single dosage of 104 PFU of a live recombinant PIV5 expressing a HA gene from the H3 subtype provided immunity against influenza A virus subtype 3 infection in mice. We hypothesize that PIV5 is a good vector for vaccine development and we propose to test PIV5 as a vector for H5N1 vaccine. We will focus our efforts on following specific aims: 1. Testing recombinant PIV5 expressing HA of H5N1 (PIV5-H5) as a vaccine for H5N1; 2. Testing the potential of other H5N1 proteins expressed using PIV5 as antigens for vaccine; and 3. Testing mutant PIV5 viruses as vaccine vectors. PUBLIC HEALTH RELEVANCE: Influenza A virus causes significant morbidity and mortality each year. Strains currently circulating in humans (i.e. H1N1, H1N2, and H3N2) infect up to 15% of the world population and cause an average of 36,000 deaths and 226,000 hospitalizations in the United States (28), as well as millions deaths world wide. Sporadic outbreaks of pandemic influenza have caused significant mortality over the past century, most notably the Spanish flu of 1918, and have caused over 50 million deaths world wide. On the horizon is another potentially pandemic strain of influenza, H5N1. This avian influenza virus has most notably emerged in Southeast Asia and resulted in the destruction of millions of birds, infected 309 people, caused 187 human fatalities since 2003 (WHO, Cumulative Number of Confirmed Human Cases of Avian Influenza A/(H5N1) Reported to WHO, 31 May 2007), and threatens to become the next pandemic. Currently, the only FDA-approved vaccine against H5N1 has serious limitations, particularly as it has to be given twice and requires substantial higher concentrations of the vaccine to achieve a moderate level of efficacy compared to conventional influenza vaccines. Conventional vaccines utilizing the HA and NA of H5N1 viruses have been poorly immunogenic and have safety and production issues. A live-attenuated H5N1 vaccine has been generated by reverse genetics, but the risk of generating a reassortant prohibits use of this vaccine in most instances. Inactivated virus vaccines have also been derived by reverse genetics and produced in large quantities, but preliminary results from NIAID clinical trials suggest that efficacy will require both multiple immunizations and 6 times the standard influenza virus antigen dose, i.e. 90 ug instead of 15 ug of antigen while only providing protection in a subset (~50%) of vaccinated individuals. Nonetheless, FDA has recently approved the inactivated H5N1 vaccine for use in people between age 18 and 64, an age group that is not the most vulnerable to influenza virus infection. Thus, there is a rationale need for new vaccine strategies that provide increased immunogenicity and safety. Parainfluenza virus 5 (PIV5), a paramyxovirus, causes kennel cough in dogs, but is not known to cause any illness in humans. PIV5 can be produced in high titers (up to 8x108 plaque forming units (PFU)/ml) in many cells including Vero cells, which are WHO-approved for vaccine production. PIV5 can infect human cell lines as well as primary human cells. In our preliminary studies, a single dosage of 104 PFU of a live recombinant PIV5 expressing a HA gene from the H3 subtype provided immunity against influenza A virus subtype 3 infection in mice. In this proposal, we hypothesize that PIV5 is a good vector for vaccine development and we propose to test PIV5 as a vector for H5N1 vaccine.