目的：Sabin脊髓灰质炎疫苗的成功应用使全球脊髓灰质炎发病率大幅下降，但截止2025年4月巴基斯坦、阿富汗等国家仍有1型野毒株（Wild poliovirus 1，WPV1）的流行。另外，脊灰口服减毒活疫苗（Oral live attenuated poliomyelities vaccine，OPV）在罕见的情况下能发生回复突变，恢复神经毒力，导致循环型疫苗衍生脊髓灰质炎病毒（Circulating vaccine-derived polioviruses，cVDPVs）疫情的发生。尽管灭活疫苗（Inactived polio vaccine，IPV）安全性较好，但不能激活初始的肠道黏膜免疫，在人群中短时间阻断肠道复制和传播方面能力不及OPV。为应对突发的WPV1和cVDPVs的问题，迫切需要开发更加安全的新型减毒疫苗（Novel oral poliovirus vaccine，nOPV）作为战略储备。本研究旨在基于对脊灰病毒毒力决定因素及进化机制的认识，建立一种遗传稳定性更好的新型减毒脊髓灰质炎疫苗株。旨在保留其抗原性和免疫原性的同时，增强衰减表型的遗传稳定性，降低回归毒力和环境传播风险。

方法：本研究首先构建了Sabin Ⅰ型脊灰的感染性克隆，在这一技术平台的支持下，对Sabin Ⅰ的重要毒力位点进行编辑以进一步降低毒力、提高稳定性，初步获得nOPV1，并做初步鉴定。编辑部位包括脊灰病毒翻译的核糖体结合位点5′ 非翻译区（5′ untranslated region，5′-UTR）区、复制相关位点2C的顺式复制元件（Cis-acting replication element，cre）区域、以及脊灰复制需要的聚合酶编辑区3D。另外，根据目前报道的上市nOPV2常发生的突变位点，构建突变的nOPV1，检验突变位点对nOPV1的影响。

结果：（1）成功构建了脊灰Sabin Ⅰ型感染性克隆，并对其生长特性和遗传稳定性与Sabin I母株进行比较鉴定；（2）在已构建的Sabin Ⅰ型感染性克隆基础上进行四个位点突变：5′-UTR内的两种修饰（重新定位的cre和domV），降低翻译效率并防止编辑的domV区域在复制过程中发生重组被替换；2C编码区cre区域8个核苷酸位置的同义突变以减少重组事件的概率；3D聚合酶中的一个突变（K38R），降低复制效率并提高保真度，获得构建的nOPV 1疫苗候选株感染性克隆；（3）在已获得构建的nOPV 1疫苗候选株感染性克隆基础上，根据nOPV 2接种以后全球收集的流行病学数据，从健康nOPV2疫苗接种者中发现的常见基因突变筛选潜在减毒株，在病毒基因组的5'-UTR cre区域共统计到8种不同组合的突变，将这些突变通过定点突变引入到nOPV 1疫苗候选株上，一共获得C121U、U123C、G179A、U123C+A181G、G179A+A181G、G196del、U123C+G196del、A181G、C121U+G179A共9种潜在疫苗株（编号4.1～4.9）；（4）在细胞水平上对这些候选疫苗株进行筛选，测定连续传代病毒基因组的遗传稳定性，绘制生长曲线、温度敏感性曲线。测序结果显示，疫苗候选株发生显著突变的位点不高于Sabin株，且候选株具有与Sabin株相似的生长曲线，并且通过一系列的基因修饰，成功降低了疫苗株的复制能力，限制其适应性。

结论：本研究基于Sabin Ⅰ型脊髓灰质炎病毒成功构建感染性克隆并进行多位点遗传改造，系统评估了各候选疫苗株的生长特性与遗传稳定性。通过整合多种稳定性增强策略，包括5′-UTR区结构调整、编码区保真度增强突变及全球流行数据中筛选的典型突变，有效提升了候选疫苗株的遗传稳定性与安全性。多株候选nOPV1病毒样本在细胞水平上展现出与Sabin株相当的生长能力与热稳定性，同时复制能力受控，突变率降低，具备作为下一代口服脊髓灰质炎减毒疫苗的研发潜力。研究结果为新型更安全、更稳定的脊灰疫苗设计提供了理论依据和实验基础，为全球根除脊灰战略提供有力支持。

Objective : The successful deployment of the Sabin oral poliovirus vaccine (OPV) has led to a dramatic global decline in polio incidence. However, as of March 2025, outbreaks of wild poliovirus type 1 (WPV1) persist in countries such as Pakistan and Nigeria. Moreover, although rare, OPV can undergo genetic reversion, restoring its neurovirulence and causing circulating vaccine-derived poliovirus (cVDPV) outbreaks. In contrast, the inactivated poliovirus vaccine (IPV), while exhibiting a superior safety profile, does not induce primary intestinal mucosal immunity and is less effective than OPV in temporarily blocking intestinal replication and transmission within communities.

To address ongoing WPV1 and cVDPV outbreaks, there is an urgent need to develop novel, safer attenuated vaccines (nOPV) for strategic stockpiling. This study aims to develop a novel attenuated poliovirus vaccine strain with enhanced genetic stability, informed by current insights into the virulence determinants and evolutionary mechanisms of poliovirus. The goal is to maintain the antigenicity and immunogenicity of the vaccine strain while minimizing the risk of reversion to virulence and subsequent environmental transmission.

Methods : In this study, we first constructed an infectious clone of the Sabin Ⅰ poliovirus. Utilizing this technological platform, we introduced targeted edits at key virulence-associated sites to further attenuate the virus and enhance its genetic stability, resulting in the initial development and characterization of a novel oral poliovirus vaccine candidate (nOPV1). The edited regions included the 5′ untranslated region (5′-UTR) involved in ribosome binding for viral translation, the cis-acting replication element (cre) within the replication-associated 2C coding region, and the 3D polymerase region essential for viral replication fidelity. In addition, based on mutation hotspots previously reported in licensed nOPV strains, we engineered a series of mutant nOPV1 constructs to evaluate the impact of these mutations on the stability and attenuation profile of the candidate vaccine.

Results : (1) Successfully constructed an infectious clone of the Sabin I poliovirus and conducted comparative analyses of its growth characteristics and genetic stability relative to the parental Sabin I strain. (2) Based on the constructed infectious clone, four site-specific mutations were introduced to generate a candidate nOPV1 strain: two modifications in the 5′-UTR—repositioning of the cre and alteration of the domain V (domV)—aimed at reducing translational efficiency and preventing recombination-mediated replacement of the modified domV; a set of eight synonymous mutations in the cre region within the 2C coding sequence to reduce the likelihood of recombination events; and a K38R substitution in the 3D polymerase to lower replication efficiency and enhance fidelity. (3) Building upon this nOPV1 candidate infectious clone, and guided by global epidemiological data collected following nOPV2 vaccination, we further screened for potential attenuating mutations based on frequently observed sequence variants in healthy vaccine recipients. A total of eight mutation sites in various combinations within the 5′-UTR and cre region were identified and introduced into the nOPV1 backbone via site-directed mutagenesis. This yielded nine candidate strains (designated No. 4.1–4.9), carrying the following mutations or combinations thereof: C121U, U123C, G179A, U123C+A181G, G179A+A181G, G196del, U123C+G196del, A181G, and C121U+G179A. (4) These candidate strains were evaluated at the cellular level by assessing their growth kinetics, thermal sensitivity, and genetic stability across serial passages. Sequencing results showed that the vaccine candidate strain had no more significant mutation sites than the Sabin strain. The candidate strain has a growth curve similar to that of the Sabin strain, and through a series of genetic modifications, we successfully reduced the replication ability of the vaccine strain and limited its adaptability.

Clnclusions: In this study, an infectious clone based on the Sabin type I poliovirus was successfully constructed and genetically modified at multiple sites. The growth characteristics and genetic stability of various vaccine candidates were systematically evaluated. By integrating multiple stability-enhancing strategies—including structural rearrangements in the 5′-UTR, fidelity-enhancing mutations in the coding region, and incorporation of typical mutations identified from global epidemiological data following nOPV2 vaccination—the genetic stability and safety of the candidate strains were effectively improved. Several nOPV1 candidates demonstrated growth characteristics and thermotolerance comparable to the Sabin strain, while exhibiting controlled replication capacity and reduced mutation rates. These results highlight the potential of the constructed strains as next-generation novel oral poliovirus vaccine type 1 (nOPV1) candidates and provide a solid theoretical and experimental foundation for the development of safer and more stable polio vaccines, supporting global polio eradication efforts.