Immunogenicity and vaccine efficacy of <i>Actinobacillus pleuropneumoniae</i>-derived extracellular vesicles as a novel vaccine candidate

<i>Actinobacillus pleuropneumoniae</i> (APP) is a significant pathogen in the swine industry, leading to substantial economic losses and highlighting the need for effective vaccines. This study evaluates the potential of APP-derived extracellular vesicles (APP-EVs) as a vaccine candidate compared to the commercial Coglapix vaccine. APP-EVs, isolated using tangential flow filtration (TFF) and cushioned ultracentrifugation, exhibited an average size of 105 nm and a zeta potential of −17.4 mV. These EVs demonstrated stability under external stressors, such as pH changes and enzymatic exposure and were found to contain 86 major metabolites. Additionally, APP-EVs induced dendritic cell (DC) maturation in a Toll-like receptor 4 (TLR4)-dependent manner without cytotoxicity. APP-EVs predominantly elicited Th1-mediated IgG responses in immunized mice without significant liver and kidney toxicity. Contrarily, unlike Coglapix, which induced stronger Th2-mediated responses and notable toxicity. In addition, APP-EVs triggered APP-specific Th1, Th17, and cytotoxic T lymphocyte (CTL) responses and promoted the activation of multifunctional T-cells. Notably, APP-EV immunization enhanced macrophage phagocytosis and improved survival rates in mice challenged with APP infection compared to those treated with Coglapix. These findings suggest that APP-EVs are promising vaccine candidates, capable of inducing potent APP-specific T-cell responses, particularly Th1, Th17, CTL, and multifunctional T-cells, thereby enhancing the protective immune response against APP infection.胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，以下简称APP）是养猪产业中的重要病原菌，可造成显著的经济损失，凸显了开发高效疫苗的迫切需求。本研究对比商用Coglapix疫苗，评估了APP来源的细胞外囊泡（APP-derived extracellular vesicles，APP-EVs）作为疫苗候选株的潜力。采用切向流过滤（tangential flow filtration，TFF）结合衬垫式超速离心分离得到的APP-EVs，平均粒径为105 nm，ζ电位（zeta potential）为-17.4 mV。这些细胞外囊泡在pH变化、酶解暴露等外界胁迫下表现出良好稳定性，且携带86种主要代谢产物。此外，APP-EVs可通过Toll样受体4（Toll-like receptor 4，TLR4）依赖的方式诱导树突状细胞（dendritic cell，DC）成熟，且无细胞毒性。APP-EVs在免疫小鼠中主要诱导Th1介导的IgG免疫应答，未引发明显的肝肾毒性；与之相反，Coglapix可诱导更强的Th2介导的免疫应答，并伴随显著毒性。此外，APP-EVs可触发APP特异性的Th1、Th17及细胞毒性T淋巴细胞（cytotoxic T lymphocyte，CTL）应答，并促进多功能性T细胞的活化。值得注意的是，与Coglapix处理组相比，APP-EV免疫可增强小鼠的巨噬细胞吞噬功能，并在APP感染攻毒后提升小鼠存活率。上述结果表明，APP-EVs是极具潜力的疫苗候选株，可诱导强效的APP特异性T细胞应答，尤其是Th1、Th17、CTL及多功能性T细胞应答，从而增强针对APP感染的保护性免疫反应。