不同佐剂对人乳头瘤病毒16型L2E7E6蛋白免疫效果的影响

目的:研究CpG佐剂、弗氏佐剂、聚肌胞苷酸佐剂及左旋咪唑、西米替丁作为佐剂对人乳头瘤病毒16型L2E7E6融合蛋白在小鼠体内产生的免疫效果的影响。方法:以单独蛋白组、蛋白加各佐剂组分别肌肉注射免疫C57BL/6小鼠,检测不同佐剂诱发小鼠产生的体液免疫和细胞免疫应答水平,并观察其对小鼠肿瘤生长的抑制作用。结果:各免疫组均能检测到高滴度的抗L2、E7、E6蛋白IgG抗体(以IgG1为主),其中弗氏佐剂能显著提高E6蛋白的IgG和IgG1抗体水平和E7蛋白的IgG1抗体水平(P<0.05),CpG佐剂明显提高了E7蛋白的IgG2a抗体水平(P<0.01);而西米替丁佐剂则降低了E7抗原的IgG抗体水平(P<0.05);同时可以检测到CpG佐剂组能诱发小鼠产生针对E7、E6较强的细胞免疫反应,且能抑制70%的荷瘤小鼠肿瘤生长;此外弗氏佐剂与聚肌胞苷酸佐剂可产生较弱的针对E7肽的细胞免疫反应,能延缓荷瘤小鼠肿瘤形成时间,与单纯蛋白组相比差异显著(P<0.05)。结论:CpG佐剂、弗氏佐剂和聚肌胞苷酸佐剂都能提高人乳头瘤病毒16型L2E7E6融合蛋白的细胞免疫反应水平和抑制肿瘤生长能力,其中CpG佐剂效果较好,为促进该蛋白作为疫苗的研发提供了实验依据。     

超抗原SEA增强小鼠对HBV DNA 疫苗的免疫反应

观察超抗原SEA(D227A)的真核表达载体(pmSEA),对HBVDNA疫苗诱导Balbc小鼠(H2d)免疫应答的调节作用。肌内注射空载体pcDNA3、HBVDNA疫苗加pmSEA佐剂(pHBVS2S+pmSEA)或不加佐剂(pHBVS2S);ELISA法测定血清抗HBs;ELISPOT检测分泌IFNγ的脾淋巴细胞;4h51Cr释放法检测小鼠脾细胞CTL活性。HBVDNA佐剂组免疫小鼠抗HBsAg抗体滴度明显高于不加佐剂组,其IgG1IgG2a的比例不同于多肽免疫组,二者分别为0.282与10。HBVDNA佐剂组均能增强IgG1和IgG2a的产生,是不加佐剂组的1.36、1.73倍。佐剂组小鼠脾淋巴细胞IFNγ的分泌量是不加佐剂组2～3倍。CTL细胞杀伤活性(E:T=100)佐剂组与不加佐剂组分别为:69.77%±7.5%、42.81%±7.7%,差异显著(P<0.05)。HBVDNA疫苗具有较强的免疫原性,能够诱导机体产生特异性的抗体及CTL反应;pmSEA佐剂能够提高小鼠对DNA疫苗的免疫应答,有望成为DNA疫苗的免疫佐剂。     

H7N9灭活疫苗联合MF59佐剂在小鼠体内诱导的长效体液免疫应答

以BALB/c小鼠为模型,探讨H7N9流感病毒灭活疫苗免疫小鼠后所诱导的长效体液免疫应答的动态变化。不同剂量的流感H7N9全病毒灭活疫苗单独或辅以MF59佐剂肌肉注射免疫小鼠一次。连续采集免疫后小鼠15个月的血清,用ELISA方法检测特异性IgG抗体水平,血凝抑制(hemagglutination inhibition,HI)试验和微量中和(microneutralization,MN)试验检测第15个月时的HI抗体和中和抗体效价。实验结果发现,小鼠血清中的特异性IgG抗体水平随时间变化持续缓慢上升,第5个月时达到顶峰,随后略有下降但一直持续平稳状态;IgG抗体滴度与疫苗剂量成正相关,且添加佐剂能提高抗体滴度。HI及MN抗体检测表明,免疫后第15个月产生的抗体能有效中和病毒,且抗体跟疫苗剂量成正比。以上研究表明,H7N9流感病毒灭活疫苗免疫小鼠一次诱导产生的特异性抗体能在较长期内保持比较平稳的抗体滴度,为小鼠提供免疫保护;增加抗原剂量和添加MF59佐剂能增加疫苗特异性抗体水平。该研究为H7N9流感疫苗产生的长期保护效应提供了一定的数据积累和参考。     

不同佐剂条件下Aβ多肽B细胞表位疫苗诱导产生抗体的免疫反应特性分析

目的:研究阿尔茨海默病β淀粉样肽(Aβ)B细胞表位疫苗2Aβ1-15-PADRE(Aβ-T)诱导产生抗体的免疫反应特性,并探讨不同佐剂对该疫苗免疫反应效果的影响。方法:合成了含2个Aβ42的 B细胞表位—Aβ1-15及1个辅助T细胞表位—PADRE的多肽2Aβ1-15-PADRE。采用Al(OH)₃佐剂,弗氏佐剂,Abisco佐剂,MF59佐剂分别与多肽疫苗联合免疫小鼠,并另设3个对照组:无佐剂多肽免疫组(Mock),PBS免疫组(PBS),未免疫组(Native)。结果:5组多肽免疫组小鼠均产生了针对Aβ的特异性抗体,无佐剂多肽免疫组的IgG抗体滴度最低,Al(OH)₃佐剂组,MF59佐剂组,Abisco佐剂组小鼠IgG抗体滴度较高,弗氏佐剂组IgG抗体滴度最高。斑点杂交实验结果显示5组小鼠免疫后血清与Aβ42单体反应较弱,与寡聚体反应最明显,与纤维状Aβ42几乎不反应。结论:4种佐剂均能提高多肽疫苗的免疫反应,产生高水平抗Aβ的特异性抗体。5组免疫小鼠产生的抗体均与Aβ寡聚体反应较强,与纤维状Aβ42反应较弱,表明该多肽疫苗具有良好的应用前景。     

不同黏膜佐剂对猪丁型冠状病毒诱导小鼠免疫应答的影响

【目的】旨在为猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)灭活疫苗黏膜免疫筛选理想佐剂,降低疫苗副作用。利用小鼠模型评价不同佐剂制备的PDCoV灭活疫苗对体液免疫、细胞免疫和黏膜免疫应答的影响。【方法】将甘露聚糖肽(PA)、CpGODN2395、单磷酰脂质A(MPLA)佐剂分别与IMS 1313、GEL02佐剂联合制备PDCoV灭活疫苗,经鼻腔免疫BALB/c小鼠;将ISA201佐剂制备的PDCoV灭活疫苗经皮下免疫BALB/c小鼠,将PDCoV灭活抗原经鼻腔免疫BALB/c小鼠作为对照,间隔14 d加强免疫一次。用ELISA方法检测小鼠血清、支气管肺泡灌洗液(BALF)中的IgG、IgG1、IgG2a、IL-4、IFN-γ及粪便和BALF中sIgA表达水平;用MTT方法检测疫苗免疫后对小鼠脾淋巴细胞增殖的影响;观察并记录小鼠免疫后的临床表现,HE染色方法观察免疫小鼠主要器官组织的病理学变化,评价疫苗的安全性。【结果】ISA201组小鼠BALF和血清中的抗体(IgG、IgG1)及IL-4表达水平相对较高,但IgG2a、IFN-γ和粪便中sIgA表达水平...     

超抗原SEA增强小鼠对HBV DNA 疫苗的免疫反应

观察超抗原SEA(D227A)的真核表达载体（pmSEA）, 对HBV DNA 疫苗诱导Balb/c 小鼠(H2d）免疫应答的调节作用。 肌内注射空载体pcDNA3、HBV DNA 疫苗加pmSEA佐剂（pHBVS2S+pmSEA）或不加佐剂（pHBVS2S）; ELISA 法测定血清抗HBs; ELISPOT检测分泌IFN-γ的脾淋巴细胞;4 h51Cr 释放法检测小鼠脾细胞CTL 活性。HBV DNA佐剂组免疫小鼠抗HBsAg抗体滴度明显高于不加佐剂组,其IgG1/IgG2a的比例不同于多肽免疫组,二者分别为0.282与10。HBV DNA佐剂组均能增强IgG1和IgG2a的产生,是不加佐剂组的1.36、1.73倍。佐剂组小鼠脾淋巴细胞IFN-γ的分泌量是不加佐剂组2～3倍。CTL 细胞杀伤活性（E:T=100）佐剂组与不加佐剂组分别为：69.77%±7.5%、 42.81%±7.7%,差异显著(P<0.05)。HBV DNA 疫苗具有较强的免疫原性, 能够诱导机体产生特异性的抗体及CTL反应;pmSEA佐剂能够提高小鼠对DNA 疫苗的免疫应答,有望成为DNA 疫苗的免疫佐剂。     

人乳头瘤病毒16型E7变异株免疫原性研究

采用分子克隆技术,构建E7变异株重组质粒[pcDNA3.1-(by)E7]和E7标准株重组质粒[pcDNA3.1-(ys)-E7],并将两种质粒分别皮下免疫Balb/c小鼠,免疫后于不同时间提取小鼠血清和制备脾淋巴细胞悬液,分别用ELISA法和MTT比色法检测特异性抗体和特异性淋巴细胞增殖反应.基因免疫后,ELISA法显示,HPV16 E7变异株和标准株均能诱导特异性抗E7抗体;MTT比色法显示,E7标准株免疫组脾淋巴细胞在体外受到变异株E7蛋白的再次刺激后出现特异性淋巴细胞增殖反应,变异株E7免疫组脾淋巴细胞经过同样处理后,出现非特异性淋巴细胞增殖反应.结果表明HPV16E7变异株能诱导特异性体液免疫应答而不能诱导特异性细胞免疫应答,HPV16 E7变异株无论在结构还是免疫原性上均与标准株有差异.由此推测,HPV16E7变异可能导致其逃逸机体自然感染或疫苗诱导的免疫应答.用基因免疫方法研究E7变异株免疫原性也为其它不能或难以进行体外培养的病毒变异研究提供借鉴.     

HPV6L1/CtMOMP多表位融合基因在COS-7细胞的表达及其在小鼠的免疫效果观察

研究人乳头瘤病毒(human papillomavirus,HPV)6b结构蛋白L1(HPV6b L1)基因佐剂增强沙眼衣原体(Chlamydia trachomatis,Ct)主要外膜蛋白(MOMP)多表位(Ct MOMP168)DNA疫苗的免疫效果的可能性.构建pcDNA3.1(+)/HPV6b L1/Ct MOMP168融合重组质粒,转染COS-7细胞,用RT-PCR、激光共聚焦显微技术及Western印迹技术检测其表达.分别用pcDNA3.1(+)/HPV6b L1/Ct MOMP168、pcDNA3.1(+)/Ct MOMP168及pcDNA3.1(+)质粒肌肉免疫BALB/c小鼠,ELISA检测外周血中IgG及阴道分泌物中sIgA.结果表明,pcDNA3.1(+)/HPV6b L1/CtMOMP168可在COS-7细胞中表达;Ct MOMP168组和HPV6b L1/Ct MOMP168组均可刺激小鼠产生抗Ct MOMP特异性的抗体,抗体滴度随免疫次数增加而升高,且HPV6b L1/Ct MOMP168组小鼠产生的抗体滴度明显高于Ct MOMP168组(P0.05).结果提示,分子佐剂HPV6b L1与CtMOMP多表位基因融合能够显著增强Ct MOMP多表位DNA疫苗的体液免疫应答.     

阳离子脂质体DOTAP佐剂对H5N1流感病毒裂解疫苗免疫效果的影响

目的研究阳离子脂质体DOTAP佐剂对H5N1型流感病毒裂解疫苗免疫效果的影响。方法制备DOTAP阳离子脂质体流感病毒裂解疫苗(简称DOTAP流感裂解疫苗),检测其包封率。将BALB/c小鼠分为13组,分别用含0.1、1.0、10.0μg HA/只剂量以DOTAP、Al(OH)3、CPG-ODN为佐剂以及不含佐剂的流感裂解疫苗于0、21天皮下免疫,PBS作为对照组,用血凝抑制试验检测小鼠初次免疫后21、42天血清HI抗体滴度;用ELISA检测初次免疫后21、42天血清特异性IgG抗体、IgG1、IgG2a亚类抗体滴度,以及初次免疫后42天小鼠脾脏单个核细胞体外经抗原刺激后细胞因子IL-2、IL-4、IFN-γ的分泌水平。将BALB/c小鼠分为3组,分别用含不同DOTAP剂量(100、300、600μg/只)的DOTAP流感裂解疫苗于0、21天皮下免疫,检测初次免疫后21、42天小鼠血清HI抗体滴度和IgG抗体滴度。结果 DOTAP流感裂解疫苗粒径在300~400 nm,带正电荷,包封率在50%以上;DOTAP流感裂解疫苗诱导的HI抗体水平和特异性IgG抗体水平均高于流感裂解疫苗,而与铝佐剂和Cp G-ODN佐剂间差异无统计学意义;DOTAP流感裂解疫苗产生的抗体仍以IgG1亚类抗体为主,免疫后42天诱导的IgG2a亚类抗体水平高于流感裂解疫苗和铝佐剂,低于Cp G-ODN佐剂;DOTAP流感裂解疫苗免疫后既分泌高水平Th1型细胞因子IFN-γ,同时也分泌高水平Th2型细胞因子IL-4;不同DOTAP剂量的DOTAP流感裂解疫苗免疫后,其HI抗体滴度和IgG抗体滴度在低、中、高剂量组之间存在明显的量效关系。结论 DOTAP作为H5N1型流感病毒裂解疫苗的佐剂可显著提高流感裂解疫苗的免疫原性,其对体液免疫应答的增强作用不低于铝佐剂和Cp G-ODN佐剂,并具有诱导细胞免疫应答的能力。     

EV71与CA16双价灭活疫苗诱导小鼠免疫原性研究

目的评价EV71和CA16双价灭活疫苗免疫小鼠诱导的体液免疫和细胞免疫应答。方法分别应用EV71和CA16双价疫苗、EV71和CA16单价疫苗按单针、两针(0,14 d)的程序免疫小鼠,采用细胞病变法检测血清第1针免疫后21 d时中和抗体效价,应用LUMINEX液相芯片技术检测小鼠脾单个核细胞体外经抗原刺激分泌细胞因子的水平。结果双价疫苗与EV71单价疫苗单针、两针免疫相比,EV71中和抗体几何平均值相近(118.8∶84.0;159.5∶156.8,P0.05)。双价疫苗与CA16单价疫苗单针免疫诱导的CA16中和抗体均为阴性;两针免疫CA16中和抗体几何平均值一致(30.0∶26.9,P0.05)。加强免疫7 d后,小鼠脾MNC经EV71抗原刺激,双价疫苗较EV71单价疫苗诱导Th2类细胞因子IL-4、5、6、10和炎症因子TNF-α的分泌水平显著增高(P=0.020,P=0.027,P=0.038,P=0.019,P=0.026);MNC经CA16抗原刺激,双价疫苗与CA16单价疫苗相比诱导细胞因子水平差异无统计学意义(P0.05)。结论双价疫苗可诱导与单价疫苗相似的中和抗体应答水平,并可提高Th2类细胞因子应答,研究为EV71和CA16双价灭活疫苗的研发提供了实验依据。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.