固有淋巴细胞(innate lymphoid cells,ILCs)是新发现的淋巴系统家族成员,在介导免疫反应、调节组织稳态和炎症方面发挥着重要作用.根据效应功能、所分泌的细胞因子及转录因子的差异,其可分为几种不同的亚群:Ⅰ型ILCs、Ⅱ型ILCs、Ⅲ型ILCs.ILCs在组织中分布广泛,并可能是病毒感染期间,率先产生免疫应答的细胞之一.本文主要就ILCs发育、分型及其在严重急性呼吸综合征冠状病毒2、呼吸道合胞病毒、肠道病毒等病毒感染过程中引发的免疫应答等方面的最新研究进展进行综述,旨在利用ILCs免疫机制,为新型抗病毒方法的开发提供思路.

目的 探索柯萨奇病毒A组16型(coxackievirus A 16,CA16)纯化液在小试条件下的最适超滤浓缩参数,并验证其在放大规模工艺中的适用性.方法 制备CA16灭活纯化液,在小试条件下,对CA16纯化液进行超滤浓缩,比较采用不同截留分子量(10、30及100 KD)的超滤膜,以不同加液方法(恒体积及补液法)、浓缩倍数(10、20、30及50倍)、上样浓度(原倍、2及4倍稀释)及洗膜次数(1～5次)条件下浓缩样品的抗原回收率、蛋白含量和膜通量,筛选最适参数;应用此参数进行放大规模的3批CA16纯化样品超滤浓缩,验证其适用性.结果 在小试条件下,截留分子量100 KD的超滤膜较30、10 KD的抗原回收率及蛋白含量均相近,但膜通量提升16％和100％;恒体积法较补液法浓缩样品的杂蛋白去除率提升约2倍,膜通量提高58％;10倍浓缩的样品回收率较30及50倍浓缩样品高10％～15％,蛋白含量较其他浓缩倍数低2～4倍;稀释上样与原倍上样的浓缩样品相比,抗原回收率和膜通量均无明显变化;膜表面多次洗涤可显著提高抗原回收率,但洗涤3次后即达峰值.在放大规模条件下,3批CA16纯化液抗原回收率均值为92.3％,蛋白含量均值为86.54 μg/mL,膜通量均值为141.5 L/(m2·h).结论 在小试条件下,采用截留分子量为100 KD的超滤膜,以恒体积法对原倍CA16病毒纯化液进行10倍浓缩,同时洗膜表面3次,可获得更高通量及抗原回收率且较低蛋白含量的浓缩样品;此条件在放大规模条件下亦适用.

Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are recognized as the major pathogens responsible for human hand-foot-mouth disease. To develop a bivalent EV71-CA16 vaccine, rhesus macaques immunized with two doses of this vaccine via the intradermal route were challenged with EV71 or CA16, and their clinical symptoms, viral shedding, neutralizing antibodies, IFN-γ-specific ELISpots, and tissue viral load were examined longitudinally. Specific immunity against EV71 and CA16 was observed in the macaques, which exhibited controlled proliferation of the EV71 and CA16 viruses and upregulated expression of immune-related genes compared with the controls. Furthermore, broad protection against EV71 and CA16 challenge without immunopathological effects was observed in all the immunized macaques. These studies suggest that the bivalent EV71-CA16 inactivated vaccine was effective against wild-type EV71 or CA16 viral challenge in rhesus macaques.

目的 探索氢氧化铝佐剂、N,O-CMCS纳米粒佐剂在不同免疫方式、不同免疫剂量时对柯萨奇病毒A组16型(CA16)灭活疫苗诱导小鼠体液免疫应答的影响.方法 将ICR小鼠随机分为阴性对照组,CA16灭活疫苗高、低剂量(200、40 U)组;氢氧化铝佐剂组,氢氧化铝佐剂CA16灭活疫苗高、低(200、40 U)剂量组;N,O-CMCS纳米粒佐剂组,N,O-CMCS纳米粒佐剂CA16灭活疫苗高、低(200、40 U)剂量组,共9个大组.每个大组按不同免疫方式(im、ip、id)随机分为3个小组,共计27个小组,每个小组6只小鼠(雌雄各半),注射量为200μL/只,共免疫2次,间隔4周.分别在初次免疫后第28天、加强免疫后的第14、28天采集小鼠尾静脉血,采用微量中和试验法检测小鼠血清抗体水平,计算中和抗体几何平均滴度(GMT),中和抗体滴度≥1∶8判定为阳性,中和抗体滴度＜1∶8判定为阴性,计算抗体阳转率.结果 氢氧化铝佐剂CA16灭活疫苗的抗体GMT值和中和抗体阳转率最高,无佐剂疫苗组其次,N,O-CMCS佐剂疫苗组最低;CA16灭活疫苗组的高剂量组的抗体GMT值和中和抗体阳转率均明显高于低剂量;在氢氧化铝佐剂CA16灭活疫苗组中比较中和抗体阳转率,id优于im.结论 选用氢氧化铝佐剂、CA16灭活疫苗高剂量、id免疫方式可显著增强CA16灭活疫苗诱导小鼠的体液免疫应答.

Objective To verify the consistency of the ELISA kit prepared in original manufacturing site for the HAV antigen detection after manufacturing site change of inactivated hepatitis A vaccine (human diploid cell) .Methods The ELISA kit was prepared with the anti-HAV polyclonal antibody-coated plate and the horseradish peroxidase-labeled anti-HAV antibody prepared in original manufacturing site , and its linearity , accuracy , precision , specificity and stability were verified . Meanwhile ,ELISA kit was used to detect HAV antigen concentrations in the intermediate products of inactivated hepatitis A vaccine .Results In new manufacturing site ,the linear range of the ELISA kit was 0 .2-6 .4 IU /ml ,and the linear determination coefficient was > 0 .99 .The precision of the ELISA kit was good ,and the coefficients of variation were all < 15％ .The recovery rates of samples with different HAV antigen concentrations were 84％ -104％ .The ELISA kit specifically detected HAV antigen ,and did not react with non-HAV and cell matrix for HAV culture .There was no effect on the detection results when the antibody-coated plate was placed at 37 ℃ for 7 d .The detection results showed that the average antigen contents of HAV harvests ,HAV purified liquid and final bulk of vaccine were 213 .25 , 146 .70 and 48 .91 IU /ml ,respectively ,meeting with decline pattern of the HAV antigen content in the process .Conclusion The ELISA kit prepared in original manufacturing site can be used to detect the inactivated hepatitis A vaccine (human diploid cell) prepared in new manufacturing site .

目的 探讨肠道病毒71型(enterovirus 71,EV71)和柯萨奇病毒A组16型(coxsakievirus A 16,CA16)VP1蛋白在人支气管上皮细胞中对天然免疫相关信号分子的表达及T细胞免疫反应的影响.方法 经RT-PCR法扩增EV71和CA16的VP1序列,插入至载体pcDNA3.1(+),构建重组真核表达质粒pcDNA-EV7 1-VP1和pcDNA-CA16-VP1.分别转染人支气管上皮细胞16HBE,于转染后24、48和72 h收集样本,进行细胞内天然免疫相关信号分子表达的检测和刺激T细胞增殖的检测.结果 经双酶切及测序鉴定,重组真核表达质粒pcDNA-EV71-VP1和pcDNA-CA16-VP1构建正确.与阴性对照组比较,EV71-VP1组IFNγ、OX40L、RANKL、TNF-α和IL-2的水平上调,而CA16-VP1组的上调幅度较小.EV71-VP1及CA16-VP1组16HBE细胞的胞内外提取物均可明显刺激分泌IFNγ的T细胞增殖(P＜0.05)结论 EV71和CA16 VP1蛋白在人上皮细胞16HBE中表达后,可差异性诱导细胞中天然免疫信号分子表达及相似的非特异性刺激T细胞增殖能力.

Human diploid cells are the cell matrix for safer vaccine production approved by WHO,and have become the first choice for vaccine production in the world.The diploid 2BS and KMB17 strains independently developed in China are derived from human embryonic lung cells.Because they have no carcinogenicity,no xenobiotic pollutants and relatively stable cell characteristics,they are used more and more often in human vaccine development and production.Since 2BS and KMB17 cells are sensitive to a variety of viruses,they have also been used as cell substrates in the development of many viral vaccines.The vaccines produced by these two kinds of human diploid cells have been widely marketed in China,including hepatitis A vaccine,poliovirus vaccine,and inactivated enterovirus 71 vaccine.

Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are the major pathogens of human hand, foot, and mouth disease (HFMD). In our previous study, intramuscular immunization with the inactivated EV71 vaccine elicited effective immunity, while immunization with the inactivated CA16 vaccine did not. In this report, we focused on innate immune responses elicited by inactivated EV71 and CA16 antigens administered intradermally or intramuscularly. The distributions of the EV71 and CA16 antigens administered intradermally or intramuscularly were not obviously different, but the antigens were detected for a shorter period of time when administered intradermally. The expression levels of NF-κB pathway signaling molecules, which were identified as being capable of activating DCs, ILCs, and T cells, were higher in the intradermal group than in the intramuscular group. Antibodies for the EV71 and CA16 antigens colocalized with ILCs and DCs in skin and muscle tissues under fluorescence microscopy. Interestingly, ILC colocalization decreased over time, while DC colocalization increased over time. ELISpot analysis showed that coordination between DCs and ILCs contributed to successful adaptive immunity against vaccine antigens in the skin. EV71 and/or CA16 antigen immunization via the intradermal route was more capable of significantly increasing neutralizing antibody titers and activating specific T cell responses than immunization via the intramuscular route. Furthermore, neonatal mice born to mothers immunized with the EV71 and CA16 antigens were 100% protected against wild-type EV71 or CA16 viral challenge. Together, our results provide new insights into the development of vaccines for HFMD.

目的 建立第一代国家柯萨奇病毒A组16型(CV-A16)抗原标准品,用于CV-A16疫苗的抗原含量检测.方法 由中国食品药品检定研究院(中检院)牵头,联合我国主要的CV-A16灭活疫苗研发单位,共计6个实验室,采用协标试剂盒,分别对两个相同、编盲设置的候选CV-A16抗原标准品(A、B)进行CV-A16抗原含量协作标定.并且分别采用各实验室建立的CV-A16抗原检测试剂盒和各自工艺制备的CV-A16疫苗原液,以候选标准品为标准进行适用性研究.结果 各实验室A/B相对抗原含量比为0.978～1.082 (CV:6.2％～12.4％),表明各实验室检测结果准确、可靠.故将A、B结果合并分析.候选标准品(A、B)的几何均值为2 194.5 kU/ml,几何变异系数(geometric coefficient of variation,GCV)为11.0％,且协标结果呈正态分布,符合标准品研制要求.将候选标准品的抗原含量暂定为2 000 U/ml进行适用性研究,共3家实验室完成该研究,结果显示3个采用各自工艺制备的CV-A16原液,在各自建立的CV-A16检测试剂盒上均与候选标准品具有良好的线性和平行性,适用性良好.结论 建立的候选标准品可作为CV-A16抗原标准品,赋值为2 000 U/ml,用于CV-A16疫苗的抗原含量检测.

柯萨奇病毒A16型(coxsakievirus A16,CA16)是引起手足口病的主要病原,但其发病及相关免疫机制尚不清楚,导致其疫苗研发面临诸多困难.众多研究表明,呼吸道黏膜上皮细胞及其相关免疫细胞尤其是天然淋巴细胞(innate lymphoid cell,ILC),在很大程度上促进病毒感染中天然免疫和适应性免疫反应的激活.然而,ILC在CA16感染诱导的免疫反应中如何发挥作用仍未知晓.为揭示CA16感染过程中ILC与病原的相互关系及发挥的作用,首先利用定量反转录-聚合酶链反应(quantitative reverse transcription-polymerase chain reaction,qRT-PCR)检测CA16感染的人支气管上皮细胞16HBE和以特别配制的佐剂导入CA16灭活抗原的小鼠支气管上皮细胞CP-M175中对ILC具有激活效应的重要基因表达情况.然后,将特别配制的CA16灭活疫苗通过鼻腔免疫BALB/c小鼠,分析呼吸道黏膜组织中ILC与病毒抗原之间的相互关系.体内外实验结果均显示,CA16活病毒和灭活的病毒抗原能诱导ILC活化所需信号通路分子的表达增加,且ILC分泌的细胞因子表达增加.免疫荧光和共聚焦显微镜观察结果亦证实,病毒抗原与ILC1/ILC3分别存在共定位现象.比较4种途径/程序免疫的小鼠体内抗CA16中和抗体和特异性细胞免疫应答情况,发现采用特别配制的CA16灭活疫苗免疫小鼠能诱导产生较好的抗病毒免疫应答.结果提示,CA16抗原能通过激活ILC及其相关信号通路诱导产生有效的免疫反应.

Coxsackievirus A16 (CV-A16) causes human hand, foot and mouth disease, but its pathogenesis is unclear. In rhesus macaques, CV-A16 infection causes characteristic vesicles in the oral mucosa and limbs as well as viremia and positive viral loads in the tissues, suggesting that these animals reflect the pathologic process of the infection. An immunologic analysis indicated a defective immune response, which included undetectable neutralizing antibodies and IFN-γ-specific memory T-cells in macaques infected with CV-A16. Furthermore, existing neutralizing antibodies in macaques immunized with the inactivated vaccine were surprisingly unable to protect against a viral challenge despite the presence of a positive T-cell memory response against viral antigens. The virus was capable of infecting pre-conventional dendritic cells and replicating within them, which may correlate with the immunological characteristics observed in the animals.

目的 研究生产场地变更后,原生产工艺制备的甲型肝炎灭活疫苗(人二倍体细胞)的抗原特性.方法 采用双抗体夹心ELISA法检测在新场地制备的甲肝灭活疫苗病毒浓缩液和疫苗原液的甲型肝炎病毒(hepatitis A virus,HAV)抗原含量.SDS-PAGE和Western blot分析二者的蛋白组分和特异性;疫苗成品(成人剂量)稀释为不同剂量(640、320、160、80和40 EU),免疫ICR小鼠(并设铝佐剂对照组),采用竞争ELISA法检测小鼠血清中HAV抗体效价;同时称量免疫后不同时间小鼠体重.结果 病毒浓缩液和疫苗原液的HAV抗原含量分别为12 800和3 200 EU/ml.疫苗原液中仅含HAV结构蛋白组分,且可与HAV抗体特异性结合.初免后28 d,640、320和160 EU剂量疫苗组小鼠血清抗体阳转率为100％,80和40 EU剂量疫苗组二免后血清抗体阳转率达100％和90％,所有免疫组小鼠血清抗体效价二免后14 d较初免28 d上升约2倍;疫苗免疫组小鼠体重及体重增幅与佐剂对照组相比,均无药物作用引起的异常变化(P＞0.05).结论 在新场地采用原生产工艺制备的甲型肝炎灭活疫苗原液有单纯的抗原蛋白组分和免疫学特异性,且与前期研究结果相比,其免疫原性及安全性具有一致性.

During the development of enterovirus 71 (EV71) inactivated vaccine for preventing human hand, foot and mouth diseases (HFMD) by EV71 infection, an effective animal model is presumed to be significant and necessary. Our previous study demonstrated that the vesicles in oral regions and limbs potentially associated with viremia, which are the typical manifestations of HFMD, and remarkable pathologic changes were identified in various tissues of neonatal rhesus macaque during EV71 infection. Although an immune response in terms of neutralizing antibody and T cell memory was observed in animals infected by the virus or stimulated by viral antigen, whether such a response could be considered as an indicator to justify the immune response in individuals vaccinated or infected in a pandemic needs to be investigated. Here, a comparative analysis of the neutralizing antibody response and IFN-γ-specific T cell response in vaccinated neonatal rhesus macaques and a human clinical trial with an EV71 inactivated vaccine was performed, and the results showed the identical tendency and increased level of neutralizing antibody and the IFN-γ-specific T cell response stimulated by the EV71 antigen peptide. Importantly, the clinical protective efficacy against virus infection by the elicited immune response in the immunized population compared with the placebo control and the up-modulated gene profile associated with immune activation were similar to those in infected macaques. Further safety verification of this vaccine in neonatal rhesus macaques and children confirmed the potential use of the macaque as a reliable model for the evaluation of an EV71 candidate vaccine.

To investigate the long-term effects on immunity of an inactivated enterovirus 71 (EV71) vaccine and its protective efficacy.

Objectives: To investigate the biological characteristics of the two types of virion fractions of Coxsackievirus A 16 (CA16), which include the real virion fraction and pseudo-virion fraction in their structure, pathogenicity and immunogenicity. Methods: We obtained the two CA16 virion fractions by density gradient centrifugation. The morphology of virion fractions was analyzed by electron microscopy, while the antigenic characteristics and immunogenicity of two virion fractions were determined by ELISA, SDS-PAGE, Western blot, qRT-PCR, and the mouse model of immune response. Results: The two virion fractions contained the major viral antigen components in their structures, showed similar pathogenicity in a neonatal murine model and were capable of inducing an effective primary immune response in adult mice, regardless of the essential distinction between the two virion fractions, which was the cleavage of VP0 to VP2 and VP4. Conclusions: The two CA16 virion fractions showed antigenicity and immunogenicity with inducing a specific immune response in animals.

Objective To analyze the two entervirus 71(EV71) virion fractions and their immune characteristics at various time points in dynamic process of viral proliferation and provide a reference for development and quality control of vaccine.Methods Two EV71 virion fractions were obtained by OptiPrep density gradient centrifugation and observed for morphology by electron microscopy,determined for infectious titer by microtitration,for antigen content by BA-ELISA,for viral RNA content by QRT-PCR,and identified by SDS-PAGE and Western blot.The obtained capsid and solid virus particles were inactivated with formaldehyde,mixed with aluminium hydroxide as an adjuvant,and injected s.c.to mice in severalsites.Intravenous blood samples of mice were collected 28 d after the first immunization and 7 and 14 d after the second immunization,from which sera were separated and determined for neutralizing antibody against EV71 by microneutralization test.The male mice 14 d after the second immunization were mated with the female ones,of which the suckling mice 24 h after birth were injected i.c.with EV71 FY-23 and observed for survival rate within 10 d.Results Two virus bands with different sedimentation coefficients were obtained by OptiPrep density gradient centrifugation.The virion fraction in upper layer was mainly consisted of empty particles,while that in lower layer with solid particles.However,the virion fraction in lower layer consisted of empty and solid particles,both of which were recognized by specific antisera against EV71.The infectious titer of virion fraction in lower layer during virus infection was 2 ～ 3 log higher than that in upper layer.Both the titers of two virion fractions showed increasing tendencies,while that in lower layer showed a significantly increasing tendency.The RNA contents of two virion fractions in upper and lower layers were different.The VP0 in lower layer was cleaved into VP2 and VP4,while the virion fraction in upper layer was mainly consisted of VPO.Both the virion fractions induced the positive conversion of antibody in mice 28 d after the first immunization and 7 and 14 d after the second immunization.However,the titer and increasing rate of neutralizing antibody induced by the virion fraction in upper layer were significantly lower than those in lower layer (each P ＜ 0.001).Both the protective rates of suckling mice induced by immunization of adult mice with two virion fractions were 100％.Conclusion Both the EV71 virion fractions may be used as the vaccine antigen for immunization.

The coxsackie A16 virus (CA16), along with enterovirus 71 (EV71), is a primary pathogen that causes hand, foot, and mouth disease (HFMD). To control HFMD, CA16, and EV71 vaccines are needed. In this study, an experimental inactivated CA16 vaccine was prepared using human diploid cells, and the vaccine's immunogenicity was analyzed in mice and rhesus monkeys. The results showed that the neutralizing antibody was developed in a dose-dependent manner, and was sustained for 70 days with an average GMT (geometric mean titer) level of 80 to 90 in immunized mouse and for 56 days with GMT of higher than 300 in monkeys. The neutralizing antibody had a cross-neutralizing activity against different viral strains (genotype A and B), and the specific IFN-γ-secreting cell response was activated by these virus strains in an ELISPOT assay. This study provides evidence for the potential use of inactivated CA16 as a candidate for use in vaccines.

Objective To evaluate the intra- and inter-reproducibility of test method for neutralizing antibody against coxsacievirus A16(CA16)in various laboratories. Methods The test method and experimental standard for neutralizing antibody against CA16 were developed according to the WHO requirements for test method for neutralizing antibody against poliovirus. Twenty-two serum samples were collected and tested for neutralizing antibody against CA16 in strain G10 of genotype A and strains W190,33,203,P11 and CS02 of genotype B separately to evaluate the intra- and interreproducibility of the method in laboratories A ~ G. The influences of these strains on test result of neutralizing antibody against CA16 were compared in laboratories A ~ E. Results The intra-coefficients of variation(CVs) of test results of17 of the 22 samples were 0 ~ 11. 4%,indicating good intra-reproducibility of the method. However,except three samples with low titers and one with high titter,the difference of GMTs of other serum samples was within 3. 0 folds among seven laboratories,which was acceptable. The test results of strains W190,33,203,P11 and CS02 showed similar tendencies to that of strain G10. The test results of strains W190 and 33 in the same laboratory showed no significant difference with that of strain G10. However,the test results of strains 203,P11 and CS02 were significantly lower than that of G10,indicating a certain difference in biological activities of strains of genotypes A and B. Conclusion The test method for neutralizing antibody against CA16 of strain G10 were used effectively in the cooperative laboratories,which was beneficial to the standardization of CA16 neutralizing antibody. Howerer, the applicabilidy of the method needs to be further assessed and confirmed.

BACKGROUND:Enterovirus 71 (EV71) is a major cause of hand, foot, and mouth disease in children and may be fatal. A vaccine against EV71 is needed.METHODS:We conducted a randomized, double-blind, placebo-controlled phase 3 trial involving healthy children 6 to 71 months of age in Guangxi Zhuang Autonomous Region, China. Two doses of an inactivated EV71 vaccine or placebo were administered intramuscularly, with a 4-week interval between doses, and children were monitored for up to 11 months. The primary end point was protection against hand, foot, and mouth disease caused by EV71.RESULTS:A total of 12,000 children were randomly assigned to receive vaccine or placebo. Serum neutralizing antibodies were assessed in 549 children who received the vaccine. The seroconversion rate was 100% 4 weeks after the two vaccinations, with a geometric mean titer of 170.6. Over the course of two epidemic seasons, the vaccine efficacy was 97.4% (95% confidence interval [CI], 92.9 to 99.0) according to the intention-to-treat analysis and 97.3% (95% CI, 92.6 to 99.0) according to the per-protocol analysis. Adverse events, such as fever (which occurred in 41.6% of the participants who received vaccine vs. 35.2% of those who received placebo), were significantly more common in the week after vaccination among children who received the vaccine than among those who received placebo.CONCLUSIONS:The inactivated EV71 vaccine elicited EV71-specific immune responses and protection against EV71-associated hand, foot, and mouth disease. (Funded by the National Basic Research Program and others; ClinicalTrials.gov number, NCT01569581.).

During HSV-1 infection, the viral UL31 protein forms a complex with the UL34 protein at the cellular nuclear membrane, where both proteins play important roles in the envelopment of viral nucleocapsids and their egress into the cytoplasm. To characterize the mechanism of HSV-1 nucleocapsid egress, we screened host proteins to identify proteins that interacted with UL31 via yeast two-hybrid analysis. Transmembrane protein 140 (TMEM140), was identified and confirmed to bind to and co-localize with UL31 during viral infection. Further studies indicated that TMEM140 inhibits HSV-1 proliferation through selectively blocking viral nucleocapsid egress during the viral assembly process. The blockage function of TMEM140 is mediated by impeding the formation of the UL31-UL34 complex due to competitive binding to UL31. Collectively, these data suggest the essentiality of the UL31-UL34 interaction in the viral nucleocapsid egress process and provide a new anti-HSV-1 strategy in viral assembly process of nucleocapsid egress.