基于杆状病毒表达系统制备的诺如病毒GⅡ.17型病毒样颗粒体液免疫原性研究

Evaluation of humoral immunogenicity of recombinant virus-like particles of Norovirus GⅡ.17 produced based on baculovirus expression system

目的初步评价基于杆状病毒表达系统制备的诺如病毒(Norovirus,No V)GⅡ.17型病毒样颗粒(virus-like particles,VLPs)的免疫原性。方法制备并纯化No V GⅡ.17 VLPs,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecylsulfate-polyacrylamide gel electrophroesis,SDS-PAGE)、免疫印迹法(Western blot)、透射电镜和粒径检测方法对其进行鉴定。GⅡ.17型VLPs采用肌内免疫途径,免疫Balb/C小鼠。研究Al(OH)_(3)佐剂组4个免疫剂量(0.4、2、10、50μg)及不使用佐剂组3个剂量(0.4、2、10μg)的免疫应答。另外,设PBS、PBS加佐剂2个对照组。将每组的5只SPF级雌性健康Balb/c小鼠(6~8周龄)分别于0、3周进行初次免疫和加强免疫。于免疫前(第0天),初次免疫后3周(第21天),加强免疫后1周(第28天)、3周(第42天)分别眼眶采血。采用ELISA检测VLP-特异性Ig G抗体,组织血型抗原(histo-blood group antigen,HBGA)受体-VLPs结合阻断试验检测50%阻断滴度(50%of block‐ing titer,BT_(50))。组间特异性Ig G抗体水平和BT_(50)水平进行对数转换后,采用Two-way ANOVA法进行统计学处理。结果成功制备No V GⅡ.17 VLPs。经SDS-PAGE检测,No V GⅡ.17 VLPs纯度为95%,Western blot分析可见特异性条带,经透射电镜和粒径分析颗粒形态完整,粒径均一。2针肌内免疫后,疫苗不同剂量组(0.4、2、10μg)产生VLP-特异性Ig G抗体水平分别为4222、67559、135118;BT_(50)抗体水平分别为12.5、57.4、174.1,Al(OH)_(3)作为佐剂的不同剂量组(0.4、2、10μg)产生VLP-特异性Ig G抗体水平分别为12800、117627、409600;BT_(50)抗体水平分别为19.0、100.0、229.7,抗体水平明显高于无佐剂组(P均<0.05)。结论Al(OH)_(3)佐剂具有显著的免疫增强作用,肌内注射作为免疫途径,2针免疫,剂量高于2μg可产生较高的抗体Ig G和BT_(50)水平。应用含铝佐剂的GⅡ.17型VLPs免疫小鼠,诱生了较好的HBGA受体结合阻断抗体,可作为诺如病毒疫苗的组分抗原。

Objective To evaluate the humoral immunogenicity of virus-like particles(VLPs)of Norovirus(NoV)GⅡ.17 genotype assembled based on recombinant baculovirus expression system.Methods The NoV GⅡ.17 VLPs in the culture medium were purified.The purity and integrity of GⅡ.17 VLPs were determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE),Western blot,transmission electron microscopy and particle size analysis.The NoV GⅡ.17 VLPs were administered via intramuscular injection to immu‐nize 6—8 weeks old female Balb/C mice at 4 doses(0.4,2,10,50μg)with adjuvant Al(OH)_(3) and 3 doses(0.4,2,10μg)without adjuvant Al(OH)_(3).Two control groups were established,one given PBS(pH7.4)and the other given PBS plus Al(OH)_(3).The BALB/c mice(5 per/group)were immunized twice,in week 0 and week 3,respec‐tively.Retro-orbital blood was collected before the first immunization(on Day 0),and on Day 21,Day 28,and Day 42 after immunization.VLP-specific IgG antibody level was assessed using enzyme-linked immunosorbent assay,and 50%blocking titer(BT_(50))using histo-blood group antigen(HBGA)-VLP binding blockade test.The IgG and BT_(50) levels were log transformed and compared across groups using two-way analysis of variance(ANOVA).Results NoV GⅡ.17 VLPs were successfully prepared.SDS-PAGE analysis indicated that the purity was up to 95%.Western blot showed specific doublet band with expected sizes.Transmission electron microscopy and particle size analysis showed that the purified NoV GⅡ.17 VLPs were morphologically intact with uniform size.After two in‐tramuscular injections,the levels of VLP-specific IgG antibody produced by the doses of 0.4,2,and 10μg were 4222,67559,and 135118,respectively;the levels of BT_(50) antibody were 12.5,57.4 and 174.1,respectively.Among the groups immunized with VLPs plus Al(OH)_(3) as adjuvant,the levels of VLP-specific IgG antibody pro‐duced by the doses of 0.4,2 and 10μg were 12800,117627 and 409600,respectively;the antibody levels of BT_(50) were 19.0,100.0 and 229.7,respectively.At the same dose,the levels of VLP-specific IgG antibody and BT_(50) in the groups with Al(OH)_(3) as adjuvant were significantly higher than those in the groups without adjuvant(P<0.05).Conclusions The Al(OH)_(3) adjuvant has a significant immune-enhancing effect.High titers of VLP-specific IgG antibody and BT_(50) may be produced after two intramuscular injections at doses higher than 2μg.The NoV GⅡ.17 VLPs in combination with aluminum adjuvant can induce high titers of HBGA receptor blocking anti‐bodies in mice,suggesting that it could be used as a candidate antigen for vaccine against norovirus.