The mucosal immunity plays an important role against African swine fever virus (ASFV) infection and the efficacy of mucosal vaccination is highly dependent on the adjuvant. However, the mucosal adjuvant for ASFV vaccination is poorly studied. Toll-like receptor (TLR) ligands such as the FlaB flagellin from Vibrio vulnificus and the heat shock protein 70 from Mycobacterium tuberculosis (mHsp70) hold a great promise as novel vaccine adjuvant. However, the mucosal adjuvanticities of such TLR ligands have not been studied in pigs. In this study, three recombinant Adenovirus (rAd) vectors, namely rAd-F1, rAd-FlaB-F1 and rAd-F1-Hsp70, were constructed by fusing the FlaB or mHsp70 to ASFV CD2v-p30-p54 fusion antigen. Western blotting showed that the three fusion proteins expressed in rAd-infected cells reacted positively with ASFV antibodies. After intranasal immunization of pigs with the three rAd vectors, the antigen-specific IgG antibodies were detectable from day 7 after primary immunization, which were significantly boosted by the secondary immunization. Strong Th1/Th2 cytokine responses were detected in the peripheral blood mononuclear cells. Compared to immunization with the control rAd-F1, significantly higher levels of the antigen-specific IgA antibodies were detected in the nasal fluids, tracheal washes and lung lavages.¹ Compared to immunization with rAd-Flab-F1, immunization with rAd-F1-Hsp70 induced significantly stronger mucosal IgA antibody response. Cytokine detection of the pig lung lavages showed that the elevated IgA antibody responses were correlated mainly with IL-4, IL-10 and IFN-α, which were confirmed by the significantly increased antigen-recall cytokine expression in the porcine alveolar macrophages. These data suggest that mHsp70 has potent mucosal adjuvanticity in pigs, and the fusion rAd vector can be used for ASFV mucosal vaccine development.

黏膜免疫对非洲猪瘟病毒（ASFV）感染具有重要作用，黏膜疫苗接种的效果很大程度上取决于佐剂。然而，用于 ASFV 疫苗接种的粘膜佐剂研究很少。Toll 样受体 (TLR) 配体，例如来自创伤弧菌的 FlaB 鞭毛蛋白和来自结核分枝杆菌的热休克蛋白 70(mHsp70) 作为新型疫苗佐剂具有很大的前景。然而，尚未在猪中研究此类 TLR 配体的粘膜佐剂性。本研究通过将 FlaB 或 mHsp70 与 ASFV CD2v-p30-p54 融合抗原融合构建了三种重组腺病毒 (rAd) 载体，即 rAd-F1、rAd-FlaB-F1 和 rAd-F1-Hsp70。Western印迹显示，在rAd感染的细胞中表达的三种融合蛋白与ASFV抗体呈阳性反应。用三种rAd载体对猪进行鼻内免疫后，从初次免疫后第7天起可检测到抗原特异性IgG抗体，二次免疫显着增强了这种抗体。在外周血单个核细胞中检测到强烈的 Th1/Th2 细胞因子反应。与用对照 rAd-F1 免疫相比，¹与 rAd-Flab-F1 免疫相比，rAd-F1-Hsp70 免疫诱导显着更强的粘膜 IgA 抗体反应。猪肺灌洗液的细胞因子检测表明，升高的 IgA 抗体反应主要与 IL-4、IL-10 和 IFN-α 相关，这可以通过猪肺泡巨噬细胞中抗原召回细胞因子表达的显着增加来证实。这些数据表明，mHsp70在猪的黏膜佐剂性很强，融合rAd载体可用于ASFV黏膜疫苗的开发。