Data Sheet 1_Circular mRNA-LNP vaccine encoding self-assembled E2-TMD-mi3 nanoparticles licit enhanced CSFV-specific immunity over commercial subunit vaccine.docx

The E2 subunit vaccine is crucial for eliminating Classical Swine Fever Virus (CSFV) due to its favorable biosafety and Differentiating Infected from Vaccinated Animals (DIVA) capability. However, low immunogenicity and high costs limit its broader application. To overcome these bottlenecks, we leveraged mRNA-LNP technology to design next-generation E2 glycoprotein vaccines with enhanced immunogenicity and cost-effectiveness. We designed different E2 glycoprotein coding sequences incorporating CD154 adjuvants and mi3 self-assembled nanoparticles, delivered via cmRNA-LNP formulations in murine immunogenicity testing. Among these, E2-TMD-mi3 cmRNA-LNP vaccine induced high-titer antibodies with a 78.25% ± 1.32% blocking rate at day 14 post-booster, significantly higher than the commercial subunit vaccine (39.74% ± 3.30%, p<0.01). To further optimize vaccine performance, we compared cmRNA-LNP formulations incorporating with different cationic lipids. Notably, AX4-LNP formulation induced superior cellular and humoral immunity compared to other cationic lipids. In mice, this vaccine induced robust humoral immunity, achieving a mean blocking rate of 80.55% ± 2.06% by day 14 post-booster, alongside potent cellular immunity (IFN-γ ELISpot, 319.60 ± 45.23 SFC/105 cell, 5.6-fold higher than that of the commercial vaccine). In swine, the CSFV-specific antibody blocking rate remained at 54.76% ± 3.21% at 120 days post-primary vaccination. In contrast, the antibody blocking rates in other cmRNA-LNP vaccine groups and the commercial vaccine group were below the positivity threshold (<40%, set according to the manufacturer’s technical specifications), outperforming commercial subunit vaccines. Moreover, this vaccine does not affect the body weight gain of immunized pigs and does not cause inflammatory reactions at the immunization site. Ultimately, we successfully developed a cmRNA-LNP vaccine incorporating the E2-TMD-Mi3 coding sequence and AX4-LNP, which demonstrated superior immunogenicity compared to commercial subunit vaccines. This study establishes a modular cmRNA-LNP platform combining mi3 nanoparticles, overcoming traditional subunit vaccine limitations for porcine viral pathogens.

E2亚单位疫苗因具备优异的生物安全性与区分感染与免疫动物（Differentiating Infected from Vaccinated Animals, DIVA）能力，对于猪瘟病毒（Classical Swine Fever Virus, CSFV）的根除至关重要。然而其免疫原性不足且成本高昂，限制了其更广泛的应用。为突破上述瓶颈，本研究借助mRNA-LNP（脂质纳米粒包裹的信使核糖核酸）技术，设计了新一代E2糖蛋白疫苗，以提升免疫原性并优化成本效益。我们设计了多种整合CD154佐剂与mi3自组装纳米粒的E2糖蛋白编码序列，并通过cmRNA-LNP（编码信使RNA-脂质纳米粒）制剂在小鼠体内开展免疫原性评价试验。在上述候选疫苗中，E2-TMD-mi3 cmRNA-LNP疫苗在加强免疫后第14天即可诱导出高滴度特异性抗体，阻断率达78.25% ±1.32%，显著高于商业亚单位疫苗的39.74% ±3.30%（p<0.01）。为进一步优化疫苗性能，我们对比了搭载不同阳离子脂质的cmRNA-LNP制剂。值得注意的是，相较于其他阳离子脂质制剂，AX4-LNP制剂可诱导更优异的细胞免疫与体液免疫应答。在小鼠模型中，该疫苗可诱导强烈的体液免疫，加强免疫后第14天的平均抗体阻断率可达80.55% ±2.06%，同时伴随强效的细胞免疫反应（干扰素-γ酶联免疫斑点试验：319.60 ±45.23斑点形成细胞/10^5个细胞，较商业疫苗提升5.6倍）。在猪体内，初次免疫后120天，猪瘟病毒特异性抗体阻断率仍维持在54.76% ±3.21%。与之相对，其余cmRNA-LNP疫苗组与商业疫苗组的抗体阻断率均低于阳性阈值（<40%，依据厂商技术规范设定），该疫苗的免疫效果显著优于商业亚单位疫苗。此外，该疫苗不会影响免疫仔猪的体重增长，也不会在免疫部位引发炎症反应。本研究成功开发了搭载E2-TMD-Mi3编码序列与AX4-LNP的cmRNA-LNP疫苗，其免疫原性显著优于商业亚单位疫苗。本研究构建了一套整合mi3纳米粒的模块化cmRNA-LNP平台，突破了传统亚单位疫苗在猪源病毒性病原体防控中的应用局限。