Human respiratory syncytial virus (RSV) is the leading etiologic agent of lower respiratory tract infections in children, but no licensed vaccine exists. Previously, we developed two parainfluenza virus 5 (PIV5)-based RSV vaccine candidates that protect mice against RSV challenge. PIV5 was engineered to express either the RSV fusion protein (F) or the RSV major attachment glycoprotein (G) between the hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L) genes of the PIV5 genome [PIV5-RSV-F (HN-L) and PIV5-RSV-G (HN-L), respectively]. To investigate the stability of the vaccine candidates in vitro, they were passaged in Vero cells at high and low multiplicities of infection (MOIs) for 11 generations and the genome sequences, growth kinetics, and protein expression of the resulting viruses were compared with those of the parent viruses. Sporadic mutations were detected in the consensus sequences of the viruses after high-MOI passages, and mutation rates increased under low-MOI-passage conditions. None of the mutations abolished antigen expression. Increased numbers of mutations correlated with increased growth rates in vitro, indicating that the viruses evolved through the course of serial passages. We also examined the in vivo stability of the vaccine candidates after a single passage in African green monkeys. No mutations were detected in the consensus sequences of viruses collected from the bronchoalveolar lavage (BAL) fluid of the animals. In vivo, mutations in RSV G and PIV5 L were found in individual isolates of PIV5-RSV-G (HN-L), but plaque isolates of PIV5-RSV-F (HN-L) had no mutations. To improve upon the PIV5-RSV-F (HN-L) candidate, additional vaccine candidates were generated in which the gene for RSV F was inserted into earlier positions in the PIV5 genome. These insertions did not negatively impact the sequence stability of the vaccine candidates. The results suggest that the RSV F and G gene insertions are stable in the PIV5 genome. However, the function of the foreign gene insertion may need to be considered when designing PIV5-based vaccines.

IMPORTANCE The genetic stability of live viral vaccines is important for safety and efficacy. PIV5 is a promising live viral vector and has been used to develop vaccines. In this work, we examined the genetic stability of a PIV5-based RSV vaccine in vitro and in vivo. We found that insertions of foreign genes, such as the RSV F and G genes, were stably maintained in the PIV5 genome and there was no mutation that abolished the expression of RSV F or G. Interestingly, the function of the inserted gene may have an impact on PIV5 genome stability.

人呼吸道合胞病毒（RSV）是儿童下呼吸道感染的主要病因，但尚无许可的疫苗。以前，我们开发了两种基于副流感病毒5（PIV5）的RSV候选疫苗，可以保护小鼠免受RSV攻击。PIV5经过改造，可以表达PIV5基因组[PIV5-RSV-F]的血凝素神经氨酸酶（HN）和RNA依赖性RNA聚合酶（L）基因之间的RSV融合蛋白（F）或RSV主要附着糖蛋白（G）。 （HN-L）和PIV5-RSV-G（HN-L）]。研究候选疫苗的体外稳定性，它们在高和低感染复数（MOI）的Vero细胞中传代了11代，并将所得病毒的基因组序列，生长动力学和蛋白质表达与亲本病毒进行了比较。在高MOI传代后，在病毒的共有序列中检测到零星突变，并且在低MOI传代条件下，突变率增加。没有一个突变消除了抗原表达。突变数量的增加与体外生长速率的提高有关，表明病毒是通过连续传代的过程进化而来的。我们还检查了体内在非洲绿猴中单次传代后，候选疫苗的稳定性。从动物的支气管肺泡灌洗液（BAL）收集的病毒的共有序列中未检测到突变。体内在单独的PIV5-RSV-G（HN-L）分离株中发现了RSV G和PIV5 L的突变，但PIV5-RSV-F（HN-L）的噬菌斑分离株没有突变。为了改进PIV5-RSV-F（HN-L）候选物，生成了其他疫苗候选物，其中RSV F的基因插入了PIV5基因组的较早位置。这些插入没有负面影响候选疫苗的序列稳定性。结果表明RSV F和G基因插入在PIV5基因组中是稳定的。但是，在设计基于PIV5的疫苗时，可能需要考虑外源基因插入的功能。

重要事项活病毒疫苗的遗传稳定性对于安全性和有效性至关重要。PIV5是一种有前途的活病毒载体，已被用于开发疫苗。在这项工作中，我们在体外和体内研究了基于PIV5的RSV疫苗的遗传稳定性。我们发现，PIV5基因组中稳定插入了外源基因（例如RSV F和G基因）的插入，并且没有突变消除了RSV F或G的表达。有趣的是，插入基因的功能可能具有对PIV5基因组稳定性的影响。