壳寡糖的免疫佐剂效果及作为减毒活菌载体疫苗佐剂的可行性探索

Immunoadjuvant Effect of Chitosan Oligosaccharide and Its Feasibility of Being Used as an Adjuvant for Attenuated Live Bacteria Vector Vaccines

目的 探究壳寡糖(chitosan oligosaccharide, COS)的免疫佐剂效果,包括免疫激活作用和引发溶酶体逃逸作用,并探索其是否可以作为减毒活菌载体疫苗佐剂。方法 (1)以0(对照组)、0.1~4 mg/mL COS培养小鼠巨噬细胞RAW264.7细胞24 h,利用CCK8实验检测细胞活力的影响;以0(对照组)、1、2、4 mg/mL COS干预小鼠RAW264.7细胞24 h,通过RT-qPCR实验检测细胞因子IFN-γ、IL-10、TGF-β、TLR-4的mRNA表达水平。(2)RAW264.7细胞加入1 mL含有不同组分[钙黄绿素(Calcein)50μg/mL,COS 2 mg/mL,阻断剂bafilomycin A1 1μmol/mL]的PBS培养,分组为Calcein组、Calcein+COS组、Calcein+COS+Bafilomycin A1组。通过激光共聚焦显微镜观察RAW264.7细胞在有或无COS干预下对荧光染料Calcein的吞噬和胞内荧光分布情况,判断COS是否能够引发溶酶体逃逸。(3)用0.5、1、2、4、8 mg/mL COS包被减毒李斯特菌载体宫颈癌治疗性疫苗候选株LM?E6E7和LI?E6E7,测定Zeta电位变化,选择COS成功包被细菌的质量浓度。检测2 mg/mL COS包被LM?E6E7和LI?E6E7前后,RAW264.7细胞对疫苗菌株的吞噬率。结果 (1)CCK8结果显示,与对照组相比,不同质量浓度COS干预RAW264.7细胞24 h后,对细胞无毒性,并能够促进细胞增殖,差异有统计学意义(P<0.05);RT-qPCR结果表明,与对照组相比,COS干预能够上调RAW264.7细胞TLR-4和IFN-γ的mRNA水平,同时抑制TGF-β和IL-10的mRNA表达水平,以4 mg/mL COS组最为明显(P<0.05)。(2)激光共聚焦显微镜发现在COS干预下,Calcein+COS组比Calcein组有更多的荧光染料从溶酶体内释放进入胞内,而这个过程可以被bafilomycin A1阻断。(3)Zeta电位结果表明COS质量浓度达到2 mg/mL时,能成功包被到细菌表面。疫苗株被COS包被前、后,RAW264.7细胞对LM?E6E7的吞噬率分别为5.70%和22.00%,差异有统计学意义(P<0.05);RAW264.7细胞对LI?E6E7的吞噬率分别为1.55%和6.12%,差异有统计学意义(P<0.05)。结论 COS具有激活巨噬细胞免疫应答和引发溶酶体内物质逃逸溶酶体的作用,包被减毒活菌载体疫苗候选菌株可促进巨噬细胞对细菌的吞噬,可望进一步开发作为细菌载体类疫苗佐剂。

Objective To study the immunoadjuvant effects of chitosan oligosaccharide(COS),including the immune activation and the triggering of lysosomal escape,and to explore whether COS can be used as an adjuvant for attenuated live bacteria vector vaccines.Methods 1)Mouse macrophages RAW264.7 cells were cultured with COS at 0 mg/mL(the control group)and 0.1-4 mg/mL for 24 h and the effect on cell viability was measured by CCK8 assay.Mouse macrophages RAW264.7 were treated with COS at 0(the control group),1,2,and 4 mg/mL for 24 h.Then,the mRNA expression levels of the cytokines,including IFN-γ,IL-10,TGF-β,and TLR4,were determined by RT-qPCR assay.2)RAW264.7 cells were treated with 1 mL of PBS containing different components,including calcein at 50μg/mL,COS at 2 mg/mL,and bafilomycin A1,an inhibitor,at 1μmol/mL,for culturing.The cells were divided into the Calcein group,Calcein+COS group,and Calcein+COS+Bafilomycin A1 group accordingly.Laser scanning confocal microscopy was used to observe the phagocytosis and the intracellular fluorescence distribution of calcein,a fluorescent dye,in RAW264.7 cells in the presence or absence of COS intervention to determine whether COS was able to trigger lysosomal escape.3)LM∆E6E7 and LI∆E6E7,the attenuated Listeria vector candidate therapeutic vaccines for cervical cancer,were encapsulated with COS at the mass concentrations of 0.5 mg/mL,1 mg/mL,2 mg/mL,4 mg/mL,and 8 mg/mL.Then,the changes in zeta potential were measured to select the concentration of COS that successfully encapsulated the bacteria.Phagocytosis of the vaccine strains by RAW264.7 cells was measured before and after LM∆E6E7 and LI∆E6E7 were coated with COS at 2 mg/mL.Results 1)CCK8 assays showed that,compared with the findings for the control group,the intervention of RAW264.7 cells with COS at different concentrations for 24 h was not toxic to the cells and promoted cell proliferation,with the difference being statistically significant(P<0.05).According to the RT-qPCR results,compared with those of the control group,the COS intervention up-regulated the mRNA levels of TLR4 and IFN-γin RAW264.7 cells,while it inhibited the mRNA expression levels of TGF-βand IL-10,with the most prominent effect being observed in the 4 mg/mL COS group(P<0.05).2)Laser scanning confocal microscopy revealed that the amount of fluorescent dye released from lysosomes into the cells was greater in the Calcein+COS group than that in the Calcein group.In other words,a greater amount of fluorescent dye was released from lysosomes into the cells under COS intervention.Furthermore,this process could be blocked by bafilomycin A1.3)The zeta potential results showed that COS could successfully encapsulate the surface of bacteria when its mass concentration reached 2 mg/mL.Before and after the vaccine strain was encapsulated by COS,the phagocytosis of LM∆E6E7 by RAW264.7 cells was 5.70%and 22.00%,respectively,showing statistically significant differences(P<0.05);the phagocytosis of LI∆E6E7 by RAW264.7 cells was 1.55%and 6.12%,respectively,showing statistically significant differences(P<0.05).Conclusion COS has the effect of activating the immune response of macrophages and triggering lysosomal escape.The candidates strains of coated live attenuated bacterial vector vaccines can promote the phagocytosis of bacteria by macrophages.Further research is warranted to develop COS into an adjuvant for bacterial vector vaccine.