Wu Qing, Jin Yuhan, Cao Weiyue, Ren Zeheng, Li Xinyu, Li Zhitong, Han Jiachi, Shi Chunxu, Gao Rui, Yan Min, Zhu Shengrui, Guan Wenpeng, Shen Xinyuan, Bai Lin, Ren Guiping
Biopharmaceutical Lab, College of Life Science, Northeast Agricultural University, Harbin, 150030, China.
Research Center of Genetic Engineering of Pharmaceuticals of Heilongjiang Province, Northeast Agricultural University, Harbin, 150030, China.
Vet Res Commun. 2025 Sep 5;49(5):298. doi: 10.1007/s11259-025-10877-8.
Canine parvovirus (CPV) poses a severe threat to canine health, necessitating the development of safer and more effective vaccines. While traditional vaccines carry risks of virulence reversion and environmental contamination, subunit vaccines-especially neutralizing epitope vaccines-offer promising alternatives by eliciting targeted immune responses with enhanced safety.
We employed bacterial display technology to express 11 overlapping CPV VP2 gene fragments on the periplasmic membrane of E. coli. Key neutralizing antigenic epitopes of CPV VP2 were mapped using four neutralizing single-chain antibodies in combination with FCM. The identified epitopes were concatenated via GS linkers and expressed as a recombinant VP2D protein. Then, animal experiments were conducted to evaluate the immunogenicity of the epitope-based vaccine in mice.
The data shows that the antigenic epitopes of the four single-chain antibodies are located in V2-V3, V8, V10 and V11 respectively. After linking five antigenic epitope fragments, we constructed a novel chimeric antigen protein, VP2D, and achieved the efficient expression of VP2D. Animal immunization evaluation results demonstrated that the VP2D vaccine exhibited superior immunological properties compared to both the VP2 vaccine and the commercial vaccine. The VP2D vaccine significantly enhanced the host's capacity to mount both humoral and cellular immune responses. Notably, the antibody titers in the VP2D vaccine group were consistently 1.2- to 1.5-fold higher than those in the VP2 vaccine group throughout the experimental period. Furthermore, the proportions of CD4⁺ T cells and F4/80⁺ cells were increased by up to 12% and 8%, respectively, in the VP2D group. Additionally, both the protein levels and mRNA transcription of inflammatory cytokines were elevated in the VP2D vaccine group relative to both the VP2 and commercial vaccine groups, indicating a consistently enhanced inflammatory response.
Identified CPV-VP2 neutralizing antigenic epitopes (70-150, 410-440, 510-585 aa), and an effective candidate vaccine for the prevention of CPV was provided.
犬细小病毒（CPV）对犬类健康构成严重威胁，因此需要研发更安全、更有效的疫苗。传统疫苗存在毒力回复和环境污染的风险，而亚单位疫苗——尤其是中和表位疫苗——通过引发靶向免疫反应并提高安全性，提供了有前景的替代方案。
我们利用细菌展示技术在大肠杆菌的周质膜上表达11个重叠的CPV VP2基因片段。使用四种中和单链抗体结合流式细胞术（FCM）对CPV VP₂的关键中和抗原表位进行定位。通过GS接头连接鉴定出的表位，并表达为重组VP2D蛋白。然后，进行动物实验以评估基于表位的疫苗在小鼠中的免疫原性。
数据表明，四种单链抗体的抗原表位分别位于V2-V3、V8、V10和V11。连接五个抗原表位片段后，我们构建了一种新型嵌合抗原蛋白VP2D，并实现了VP2D的高效表达。动物免疫评估结果表明，与VP2疫苗和商业疫苗相比，VP2D疫苗表现出优异的免疫学特性。VP2D疫苗显著增强了宿主产生体液免疫和细胞免疫反应的能力。值得注意的是，在整个实验期间，VP2D疫苗组的抗体滴度始终比VP2疫苗组高1.2至1.5倍。此外，VP2D组中CD4⁺ T细胞和F4/80⁺细胞的比例分别增加了高达12%和8%。此外，相对于VP2和商业疫苗组，VP2D疫苗组中炎性细胞因子的蛋白水平和mRNA转录均升高，表明炎症反应持续增强。
鉴定出CPV-VP2中和抗原表位（70-150、410-440、510-585氨基酸），并提供了一种预防CPV的有效候选疫苗。
