Metapneumovirus Biology and Vaccine Development

人偏肺病毒（HMPV）于2001年首次报道，并迅速被认为是全球呼吸道疾病的重要病原体，特别是在儿科人群、免疫功能低下的个体和体弱的老年人中。我们正在使用重组DNA方法来表征病毒的分子生物学和发病机制，并开发HMPV的减毒衍生物，用作活的鼻内儿科疫苗。 HMPV是一种包膜病毒，其基因组为约13.3 kb的RNA的单负义链。我们先前确定了两个遗传亚组A和B中每一个的第一个完整的HMPV基因组序列。HMPV编码9种蛋白质：通过与人呼吸道合胞病毒（其更熟知的亲戚）类似，HMPV蛋白是：N，核蛋白; P，磷蛋白; M，基质蛋白; F，融合蛋白; M2-1和M2-2推定的RNA合成因子; SH，小疏水蛋白; G，附着糖蛋白;和L，病毒聚合酶。 我们以前还开发了一种HMPV的反向遗传系统，从而可以在细胞培养中完全从克隆的cDNA产生完全的感染性病毒。这为基础研究和疫苗设计提供了基因组工程方法。我们发现，四个病毒基因可以单独删除，并在各种组合很少或没有影响病毒复制在体外，即：G，SH，M2-1和M2-2。对这些病毒在仓鼠和非洲绿色猴中的减毒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病毒是有希望成为针对HMPV的减毒活疫苗的候选者。 通过将HMPV的N或P开放阅读框替换为其来自密切相关的禽偏肺病毒（AMPV）C亚组的对应物，产生了额外的候选疫苗。我们发现，由于自然宿主范围的限制，AMPV在灵长类动物中是减毒的，并且希望将AMPV基因引入HMPV中将赋予这种减毒表型。在仓鼠和非洲绿色猴中的评价表明，情况确实如此，P-替代病毒（HMPV-Pa）尤其是有希望的候选疫苗，而HMPV-Na病毒的减毒不充分，需要输入进一步的减毒突变。HMPV-Pa也是一种有吸引力的候选疫苗，因为它在Vero细胞中的复制效率比HMPV高25倍，这是一种也用AMPV观察到的表型，并将促进疫苗生产（因为Vero细胞是疫苗生产的底物）。 我们建立了野生型HMPV和HMPV-Pa和del-M2-2病毒的种子库用于临床试验。在我们的实验室中，以现行药品生产质量管理规范为指导，在适合人用产品的条件下生成病毒。使用合同设施，我们已经生成了野生型病毒和HMPV-Pa衍生物的临床试验材料; del-M2-2病毒正在进行中。将在血清阳性成人的临床试验中分析野生型病毒，以建立复制基准，据此可以判断减毒衍生物。减毒候选疫苗将在血清阳性成人、血清阳性儿童、血清阴性儿童和HMPV初治婴儿的I期临床试验中连续评价。

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 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-nave infants.