人类副流感病毒3型（HPIV3）是一种主要的儿科呼吸道病原体，缺乏可用的疫苗或抗病毒药物。我们通过密码子对去优化(CPD)产生了活的减毒HPIV3疫苗候选物。HPIV3开放阅读框（ORFs）编码核蛋白（N），磷蛋白（P），矩阵(M)，融合（F），血凝素-神经氨酸酶（HN），和聚合酶（L）被单独或组合修饰以产生12种病毒，命名为Min-N，Min-P,Min-M,Min-FHN,Min-L,Min-NP,Min-NPM,最小不良贷款，Min-PM,Min-PFHN,Min-MFHN,和Min-PMFHN。N或L的CPD严重降低了体外生长，没有进一步评估。P或M的CPD与体外干扰素（IFN）反应增加和减少有关，分别,但对病毒复制影响不大。在Vero细胞中,F和HN延迟病毒复制的CPD，但最终滴度与野生型（wt）HPIV3相当。在人肺上皮A549细胞中，CPDF和HN诱导更强的IFN应答，病毒滴度降低了100倍，F和HN蛋白的表达显着降低，而不影响N或P或蛋白质在病毒体中的相对包装。仓鼠鼻内感染后，对于携带CPDF和HN的病毒，鼻甲和肺中的复制倾向于减少最多，最大减少约10倍。尽管体内复制减少（体外CPDF和HN的表达降低），所有病毒均诱导与wt相似的血清HPIV3中和抗体滴度，并提供针对HPIV3攻击的完全保护。总之,HPIV3的CPD产生了适合进一步开发的有希望的疫苗候选物。
Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.
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