Addition of a missense mutation present in the L gene of respiratory syncytial virus (RSV) cpts530/1030 to RSV vaccine candidate cpts248/404 increases its attenuation and temperature sensitivity
Addition of a missense mutation present in the L gene of respiratory syncytial virus (RSV) cpts530/1030 to RSV vaccine candidate cpts248/404 increases its attenuation and temperature sensitivity
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DOI：
10.1128/jvi.73.2.871-877.1999
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发表时间：
1999-02-01
影响因子：
5.4
通讯作者：
Murphy, BR
中科院分区：
文献类型：
--
作者：
Whitehead, SS;Firestone, CY;Murphy, BR
呼吸道合胞病毒 (RSV) cpts530/1030 是一种减毒、温度敏感的 A 亚组候选疫苗，之前通过两轮连续化学诱变和生物选择从冷传代 RSV (cpRSV) 衍生而来。此处，cpts530/1030 在血清阴性黑猩猩的上呼吸道和下呼吸道中表现出高度减弱。然而，对血清阳性儿童的评估表明，它保留了足够的复制能力和毒力，无法直接用作减毒活疫苗。对cpts530/1030基因组的核苷酸序列分析表明，它获得了两个核苷酸取代(与其cpts530亲本相比)，这两个核苷酸取代都在L基因中：核苷酸位置8821(氨基酸108)处的沉默突变和核苷酸位置12458处的错义突变，导致氨基酸1321处酪氨酸变为天冬酰胺，本文称为作为1030突变。它还包含先前在 L 基因第 10060 位核苷酸处发现的 530 个错义突变。 cpts530/1030减毒的遗传基础是通过将530和1030突变引入cpRSV的cDNA克隆来定义的，从中衍生出重组RSV并进行分析以确定每个突变对cpts530/1030的温度敏感性（ts）和减毒（att）表型的贡献。源自 cpts530 的 530 突变此前已被证明与该病毒的 ts 和 aft 表型有关。在本研究中，1030突变被证明是cpts530/1030温度敏感性增加的原因。此外，1030突变被证明是导致小鼠上呼吸道和下呼吸道中cpts530/1030衰减水平增加的原因。 530 和 1030 突变对 ts 和 aft 表型的影响是相加的。通过使用反向遗传学，可以将 1030 突变（而不是 530 突变）引入对非常年幼的婴儿具有残留反应原性的减毒候选疫苗（即 cpts248/404）中。无法将530突变引入cpts248/404病毒被证明是由于其在L蛋白功能水平上与248错义突变不相容。由此产生的 rA2cp248/404/1030 突变病毒比 cpts248/404 亲代病毒对温度更敏感，减毒能力更强，使其成为通过使用反向遗传学改进现有疫苗病毒而创建的有前途的新型 RSV 候选疫苗。
Respiratory syncytial virus (RSV) cpts530/1030 is an attenuated, temperature-sensitive subgroup A vaccine candidate derived previously from cold-passaged RSV (cpRSV) by two sequential rounds of chemical mutagenesis and biological selection. Here, cpts530/1030 was shown to be highly attenuated in the upper and lower respiratory tracts of seronegative chimpanzees. However, evaluation in seropositive children showed that it retains sufficient replicative capacity and virulence to preclude its direct use as a live attenuated vaccine. Nucleotide sequence analysis of the genome of cpts530/1030 showed that it had acquired two nucleotide substitutions (compared to its cpts530 parent), both of which were in the L gene: a silent mutation at nucleotide position 8821 (amino acid 108) and a missense mutation at nucleotide position 12458 resulting in a tyrosine-to-asparagine change at amino acid 1321, herein referred to as the 1030 mutation. It also contained the previously identified 530 missense mutation at nucleotide 10060 in the L gene. The genetic basis of attenuation of cpts530/1030 was defined by the introduction of the 530 and 1030 mutations into a cDNA clone of cpRSV, from which recombinant RSV was derived and analyzed to determine the contribution of each mutation to the temperature sensitivity (ts) and attenuation (att) phenotypes of cpts530/1030. The 530 mutation, derived from cpts530, was previously shown to be responsible for the ts and aft phenotypes of that virus. In the present study, the 1030 mutation was shown to be responsible for the increased temperature sensitivity of cpts530/1030. In addition, the 1030 mutation was shown to be responsible for the increased level of attenuation of cpts530/1030 in the upper and lower respiratory tracts of mice. The 530 and 1030 mutations were additive in their effects on the ts and aft phenotypes. It was possible to introduce the 1030 mutation, but not the 530 mutation, into an attenuated vaccine candidate with residual reactogenicity in very young infants, namely, cpts248/404, by use of reverse genetics. The inability to introduce the 530 mutation into the cpts248/404 virus was shown to be due to its incompatibility with the 248 missense mutation at the level of L protein function. The resulting rA2cp248/404/1030 mutant virus was more temperature sensitive and more attenuated than the cpts248/404 parent virus, making it a promising new RSV vaccine candidate created by use of reverse genetics to improve upon an existing vaccine virus.