Metapneumovirus Biology and Vaccine Development

人类偏肺病毒(HMPV)于2001年首次报道，并迅速被认为是世界范围内呼吸道疾病的重要病原体，特别是在儿科人群、免疫功能低下的人和虚弱的老年人中。我们正在使用重组DNA方法来表征病毒的分子生物学和致病机制，并开发HMPV的减毒衍生物，用于儿童鼻内活疫苗。 HMPV是一种包膜病毒，其基因组是一条约13.3kb的单链负链RNA。HMPV编码9种蛋白质：与其近亲人类呼吸道合胞病毒相似，它们是：N，核蛋白；P，磷蛋白；M，基质蛋白；F，融合蛋白；M2-1和M2-2RNA合成因子；SH，小疏水蛋白；G，附着糖蛋白；和L，病毒聚合酶。 我们还开发了一种针对HMPV的反向遗传系统，利用该系统，可以在细胞培养中完全从克隆的cDNA中产生完全具有感染性的病毒。这为从事基础研究和设计疫苗的基因组工程提供了一种方法。我们发现，G、SH、M2-1和M2-2四个病毒基因可以单独或以不同的组合缺失，对病毒的体外复制几乎没有影响。对这些病毒在仓鼠和非洲绿猴体内的减毒作用和免疫原性的评价表明，del-G病毒(有或没有SH基因的额外缺失)和del-M2-2病毒是很有希望成为HMPV减毒活疫苗的候选毒株。 通过将HMPV的N或P开放阅读框替换为密切相关的禽偏肺炎病毒(AMPV)亚群C亚群的相应序列，产生了更多的候选疫苗。我们表明，由于自然宿主范围的限制，AMPV在灵长类动物中是弱化的，希望将AMPV基因导入HMPV将赋予这种减毒表型。对仓鼠和非洲绿猴的评估表明，情况确实如此，尤其是P-置换病毒(HMPV-PA)是一种有希望的候选疫苗，而HMPV-Na病毒的减毒效果不够好，需要引入进一步减毒的突变。HMPV-PA也是一个有吸引力的候选疫苗，因为它在Vero细胞中的复制效率是HMPV的25倍，这一表型也在AMPV中观察到，并将促进疫苗的制造(因为Vero细胞是疫苗制造的底物)。 我们建立了野生型HMPV、HMPV-PA和Del-M2-2病毒的种子库，用于临床试验。这些病毒是在我们实验室以当前良好制造规范为指导的条件下产生的，以便适合于人类使用的产品。利用合同设施，我们已经为野生型病毒和HMPV-PA衍生物产生了临床试验材料；del-M2-2病毒正在进行中。野生型病毒将在血清阳性成年人的临床试验中进行分析，以评估有HMPV经验的个人的再次感染，并建立一个复制基准，可以根据该基准来判断减毒衍生品。减毒疫苗候选疫苗将在第一阶段临床试验中相继在血清阳性成人、血清阳性儿童、血清阴性儿童和HMPV幼稚婴儿中进行评估。

Human metapneumovirus (HMPV) was first reported in 2001 and has quickly come to be recognized as a significant agent of respiratory tract disease worldwide, especially in the pediatric population, in immunocompromised individuals, and in the frail elderly. We are using recombinant DNA methods to characterize viral molecular biology and pathogenesis and to develop attenuated derivatives of HMPV for use as a live intranasal pediatric vaccine. HMPV is an enveloped virus with a genome that is a single negative-sense strand of RNA of approximately 13.3 kb. We previously determined the first complete HMPV genome sequences for each of the two genetic subgroups, A and B. HMPV encodes nine proteins: by analogy to human respiratory syncytial virus, its better-known relative, the HMPV proteins are: N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion protein; the M2-1 and M2-2 putative RNA synthesis factors; SH, small hydrophobic protein; G, attachment glycoprotein; and L, viral polymerase. We also previously developed a reverse genetic system for HMPV whereby complete infectious virus can be generated in cell culture entirely from cloned cDNAs. This provides a method for engineering the genome in pursuit of basic studies and for designing vaccines. We found that four viral genes could be deleted individually and in various combinations with little or no effect on viral replication in vitro, namely: G, SH, M2-1 and M2-2. Evaluation of the attenuation and immunogenicity of these viruses in hamsters and African green monkeys indicated that the del-G virus (with or without the additional deletion of the SH gene) and del-M2-2 virus are promising candidates to be live attenuated vaccines against HMPV. Additional vaccine candidates were generated by replacing the N or P open reading frame of HMPV with its counterpart from the closely related avian metapneumovirus (AMPV) subgroup C. We showed that AMPV is attenuated in primates due to a natural host range restriction, and it was hoped that the introduction of AMPV genes into HMPV would confer this attenuation phenotype. Evaluation in hamsters and African green monkeys showed that this indeed was the case, and the P-replacement virus (HMPV-Pa) in particular is a promising vaccine candidate whereas the HMPV-Na virus was insufficiently attenuated and would need importation of further attenuating mutations. HMPV-Pa also is an attractive vaccine candidate because it replicates 25-fold more efficiently than HMPV in Vero cells, a phenotype that also was observed with AMPV and will facilitate vaccine manufacture (since Vero cells are the substrate for vaccine manufacture). We created seed stocks of wild type HMPV and the HMPV-Pa and del-M2-2 viruses for clinical trials. The viruses were generated in our laboratory under conditions using Current Good Manufacturing Practices as a guide so as to be appropriate for products for human use. Using a contract facility, we have generated clinical trial material for the wild type virus and the HMPV-Pa derivative; the del-M2-2 virus is in progress. The wild type virus will be analyzed in clinical trials in seropositive adults to evaluate re-infection in HMPV-experienced individuals and to establish a benchmark for replication against which the attenuated derivatives can be judged. The attenuated vaccine candidates will be evaluated successively in Phase I clinical trials in seropositive adults, seropositive children, seronegative children, and HMPV-naive infants.