Tao T, Skiadopoulos M H, Davoodi F, Riggs J M, Collins P L, Murphy B R
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Virol. 2000 Jul;74(14):6448-58. doi: 10.1128/jvi.74.14.6448-6458.2000.
We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated cp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955-2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503-510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the cp45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.
我们试图利用先前用于快速生产减毒活1型副流感病毒（PIV1）疫苗候选株的反向遗传技术，开发一种用于婴幼儿的减毒活2型副流感病毒（PIV2）疫苗株。PIV1疫苗候选株命名为rPIV3-1cp45，是通过将减毒的cp45 PIV3疫苗候选株中的PIV1全长血凝素神经氨酸酶（HN）和融合蛋白（F）替换为PIV3的相应蛋白而产生的（T. Tao等人，《病毒学杂志》72:2955 - 2961，1998；M. H. Skiadopoulos等人，《疫苗》18:503 - 510，1999）。然而，采用相同策略，我们未能获得在野生型PIV3骨架中携带全长PIV2糖蛋白的重组嵌合PIV3 - PIV2分离株。当使用嵌合的HN和F开放阅读框（ORF）而非完整的PIV2 F和HN ORF构建全长互补DNA（cDNA）时，获得了有活力的PIV3 - PIV2嵌合体。获得的病毒命名为rPIV3 - 2CT（其中PIV2胞外结构域和跨膜结构域与PIV3胞质结构域融合）和rPIV3 - 2TM（其中PIV2胞外结构域与PIV3跨膜和胞质尾结构域融合），它们在体外和体内具有相似的表型。因此，似乎PIV3的HN或F糖蛋白仅胞质尾对于成功获得PIV3 - PIV2嵌合体是必需的。尽管rPIV3 - 2CT和rPIV3 - 2TM在体外能高效复制，但它们在仓鼠、非洲绿猴（AGM）和黑猩猩的呼吸道中复制时表现为中度至高度减毒。这一意外发现表明，PIV2和PIV3的HN和F蛋白嵌合本身在体内决定了一种减毒表型。尽管有这种减毒作用，但这些病毒具有高度免疫原性，并且能保护仓鼠和AGM免受野生型PIV2的攻击，它们是作为PIV2疫苗进行临床评估的有前景的候选株。通过添加位于HN和F基因之外的12个PIV3cp45突变，这些嵌合病毒进一步减毒。这些突变的减毒作用与嵌合作用相加，因此如果需要，包含全部或部分cp45突变提供了一种进一步减毒PIV3 - PIV2嵌合疫苗候选株的方法。
