Developing a Paramyxovirus-based H5N1 Vaccine

描述（由申请人提供）：H5 N1是对人类健康的迫在眉睫的威胁。安全有效的疫苗是预防在人群中大规模爆发的最佳途径。副流感病毒5型（PIV 5）是一种副粘病毒，可引起狗的犬舍咳嗽，但目前尚不清楚是否会引起人类疾病。PIV 5可以在包括Vero细胞在内的许多细胞中以高滴度（高达8x 108空斑形成单位（PFU）/ml）生产，Vero细胞是WHO批准用于疫苗生产的细胞。PIV 5可以感染人细胞系以及原代人细胞。在我们的初步研究中，表达H3亚型HA基因的活重组PIV 5单次给药104 PFU可在小鼠中提供针对甲型流感病毒3亚型感染的免疫力。我们假设PIV 5是一个很好的疫苗开发载体，我们建议测试PIV 5作为H5 N1疫苗的载体。我们将致力于以下具体目标：1。测试表达H5 N1的HA的重组PIV 5（PIV 5-H5）作为H5 N1的疫苗; 2.测试使用PIV 5表达的其他H5 N1蛋白作为疫苗抗原的潜力;以及3.测试作为疫苗载体的突变体PIV 5病毒。 公共卫生相关性：甲型流感病毒每年都会导致严重的发病率和死亡率。目前在人类中传播的菌株（即H1N1，H1 N2和H3 N2）感染了多达15%的世界人口，在美国平均造成36，000人死亡和226，000人住院治疗（28），以及全世界数百万人死亡。在过去的世纪中，大流行性流感的零星爆发已经造成了显著的死亡率，最值得注意的是1918年的西班牙流感，并且已经在全世界造成了超过5000万人死亡。另一种潜在的大流行性流感病毒株H5 N1即将出现。这种禽流感病毒最显著地出现在东南亚，并导致数百万只禽类死亡，309人感染，自2003年以来造成187人死亡（WHO，向WHO报告的A/（H5 N1）禽流感确诊人间病例累积数，2007年5月31日），并有可能成为下一次大流行。 目前，FDA批准的唯一一种针对H5 N1的疫苗具有严重的局限性，特别是因为与常规流感疫苗相比，它必须给予两次，并且需要相当高的疫苗浓度才能达到中等水平的功效。利用H5 N1病毒的HA和NA的常规疫苗免疫原性差，并且具有安全性和生产问题。已经通过反向遗传学产生了减毒活H5 N1疫苗，但在大多数情况下，由于存在产生抗体的风险，因此禁止使用这种疫苗。灭活病毒疫苗也已通过反向遗传学获得并大量生产，但NIAID临床试验的初步结果表明，有效性将需要多次免疫和6倍标准流感病毒抗原剂量，即90 ug而不是15 ug抗原，同时仅在接种个体的一个子集（约50%）中提供保护。尽管如此，FDA最近已批准灭活H5 N1疫苗用于18至64岁的人群，这一年龄组并不是最容易感染流感病毒的人群。因此，有理由需要新的疫苗策略，提供增加的免疫原性和安全性。 副流感病毒5型（PIV 5）是一种副粘病毒，可引起狗的犬舍咳嗽，但目前尚不清楚是否会引起人类疾病。PIV 5可以在包括Vero细胞在内的许多细胞中以高滴度（高达8x 108空斑形成单位（PFU）/ml）生产，Vero细胞已被WHO批准用于疫苗生产。PIV 5可以感染人细胞系以及原代人细胞。在我们的初步研究中，表达H3亚型HA基因的活重组PIV 5单次给药104 PFU可在小鼠中提供针对甲型流感病毒3亚型感染的免疫力。在这个提议中，我们假设PIV 5是一个很好的疫苗开发载体，我们建议测试PIV 5作为H5 N1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.