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目的:探讨OX40L作为H7N9全病毒灭活疫苗(whole-virion inactivated vaccine，WIV)免疫佐剂的保护效果。方法:将50只BALB/c小鼠随机分为5组进行肌肉免疫：未免疫组(对照组)、单独疫苗组(1.5 μg WIV)、佐剂疫苗组(1.5 μg WIV＋0.6/1.8/3.0 μg OX40L/Fc)。免疫后21 d，通过ELISA和血凝抑制(hemagglutination inhibition，HI)试验检测免疫小鼠血清中各类抗体水平，并使用50×半数致死量(median lethal dose，LDn 50)的H7N9流感病毒适应株攻击小鼠，记录小鼠体重变化及存活率，以评价OX40L/Fc与H7N9 WIV共免疫后的保护效果。免疫后7 d，通过流式细胞术探究OX40L佐剂效应的机制。n 结果:与单独疫苗组相比较，共免疫OX40L/Fc可使小鼠血清中的特异性IgG抗体水平显著升高，3组佐剂疫苗组(1.5 μg WIV＋0.6/1.8/3.0 μg OX40L/Fc)的抗体几何平均滴度(lgGMT)分别为3.79、4.40和4.20。共免疫显著增加了IgG1和IgG2a抗体的滴度，但对Th1/Th2免疫应答平衡没有显著影响。与单独疫苗组相比较，共免疫1.8 μg和3.0 μg OX40L/Fc小鼠的HI抗体水平分别为6.25±0.50和5.70±0.97，差异有统计学意义(n P<0.05)。共免疫1.8 μg和3.0 μg OX40L/Fc能够有效保护80%和75%的小鼠抵抗致死剂量病毒的攻击，损失的体重少于单独疫苗组，1.8 μg OX40L/Fc为最佳浓度。流式细胞术显示，与单独疫苗组相比，OX40L(0.6/1.8/3.0 μg)能够促进趋化因子受体CXCR5和程序性细胞死亡蛋白PD-1高效表达，增强H7N9 WIV免疫后淋巴结中滤泡辅助性T(Tfh)细胞的增殖(n P均<0.05)。n 结论:OX40L能够通过增殖Tfh细胞以增强H7N9 WIV诱导的抗体反应。“,”Objective:To analyze and evaluate the effect of OX40L as a potential adjuvant for H7N9 whole-virion inactivated vaccine (WIV).Methods:Fifty BALB/c mice were randomly divided into five groups and immunized intramuscularly with PBS (control group) and 1.5 μg WIV alone or in combination with 0.6, 1.8 or 3.0 μg Fc-fused OX40L (OX40L/Fc) adjuvant. Three weeks after immunization, IgG, IgG1 and IgG2 titers were measured by ELISA and hemagglutination inhibition (HI) assay. Moreover, the mice were challenged with 50×median lethal dose (LD n 50) of homologous virus and the changes in mouse body weight and survival rate were recorded to evaluate the effects of OX40L. Flow cytometry was used to analyze the mechanism of OX40L as an adjuvant 7 d after immunization.n Results:Compared with immunization with WIV alone, co-immunization of WIV with OX40L/Fc induced higher antigen-specific IgG in mice. The geometric mean titers (lgGMT) of antibodies induced by 0.6, 1.8 and 3.0 μg OX40L/Fc reached 3.79, 4.40 and 4.20, respectively. WIV combined with OX40L/Fc induced high levels of IgG1 and IgG2a without influencing Th1/Th2 balance. HI antibodies were also higher in WIV+ 1.8 μg OX40L/Fc and WIV+ 3.0 μg OX40L/Fc groups than in WIV group (6.25±0.50 and 5.70±0.97 vs 3.00±0.97, both n P<0.05). WIV combined with 1.8 or 3.0 μg OX40L/Fc could protect 80% or 75% of mice against lethal challenge with H7N9 and result in less weight loss as compared with WIV alone. The most effective dose of OX40L/Fc was 1.8 μg. Flow cytometry showed that WIV (0.6, 1.8, 3.0 μg) in combination with OX40L/Fc enhanced the proliferation of T follicular helper cells (Tfh) through promoting the expression of CXCR5 and PD-1 as compared with WIV alone (alln P<0.05).n Conclusions:This study suggested that OX40L was beneficial to potent antibody responses induced by H7N9 WIV through promoting Tfh cell proliferation.