Role of a noncanonical disulfide bond in the stability,affinity, and flexibility of a VHH specific for the Listeria virulence factor InlB

A distinguishing feature of camel (Camelus dromedarius) VHH domains are noncanonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding. To examine the role of a noncanonical disulfide bond on antigen binding and the biophysical properties of a VHH domain, we have used the VHH R303, which binds the Listeria virulence factor InlB as a model. Using site directed mutagenesis, we produced a double mutant of R303 (C33A/C102A) to remove the extra disulfide bond of the VHH R303. Antigen binding was not affected by loss of the disulfide bond, however the mutant VHH displayed reduced thermal stability (T_m = 12°C lower than wild‐type), and a loss of the ability to fold reversibly due to heat induced aggregation. X‐ray structures of the mutant alone and in complex with InlB showed no major changes in the structure. B‐factor analysis of the structures suggested that the loss of the disulfide bond elicited no major change on the flexibility of the CDR loops, and revealed no evidence of loop immobilization upon antigen binding. These results suggest that the noncanonical disulfide bond found in camel VHH may have evolved to stabilize the biophysical properties of the domain, rather than playing a significant role in antigen binding.     

Misannotations of the genes encoding sugar N‐formyltransferases

Tens of thousands of bacterial genome sequences are now known due to the development of rapid and inexpensive sequencing technologies. An important key in utilizing these vast amounts of data in a biologically meaningful way is to infer the function of the proteins encoded in the genomes via bioinformatics techniques. Whereas these approaches are absolutely critical to the annotation of gene function, there are still issues of misidentifications, which must be experimentally corrected. For example, many of the bacterial DNA sequences encoding sugar N‐formyltransferases have been annotated as l ‐methionyl‐tRNA transferases in the databases. These mistakes may be due in part to the fact that until recently the structures and functions of these enzymes were not well known. Herein we describe the misannotation of two genes, WP_088211966.1 and WP_096244125.1, from Shewanella spp. and Pseudomonas congelans, respectively. Although the proteins encoded by these genes were originally suggested to function as l ‐methionyl‐tRNA transferases, we demonstrate that they actually catalyze the conversion of dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose to dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose utilizing N¹⁰‐formyltetrahydrofolate as the carbon source. For this analysis, the genes encoding these enzymes were cloned and the corresponding proteins purified. X‐ray structures of the two proteins were determined to high resolution and kinetic analyses were conducted. Both enzymes display classical Michaelis–Menten kinetics and adopt the characteristic three‐dimensional structural fold previously observed for other sugar N‐formyltransferases. The results presented herein will aid in the future annotation of these fascinating enzymes.     

HIV-1 Env三聚体抗原改造研究进展

HIV-1疫苗研发是当前艾滋病研究的一大热点,其病毒表面包膜糖蛋白Env三聚体介导病毒与细胞融合,是HIV-1疫苗研究的重要靶点。因此,设计并在体外表达类天然Env三聚体对HIV-1疫苗的研发具有重要的意义。近年来,Env三聚体研究取得了显著的进展。SOSIP、NFL2P、UFO等抗原改造方法实现了类天然Env三聚体的体外表达,逐步解决了改造抗原产量低、结构不稳定等问题,且表达的Env三聚体抗原能在动物免疫中诱导机体产生较高的中和抗体水平。Env三聚体改造方法促进了HIV-1疫苗的研究。文中综述了SOSIP、NFL2P、UFO三种HIV-1 Env三聚体抗原改造方法,对比各个改造方法优缺点,并结合自身工作提出相关建议,为后续HIV-1抗原的相关设计提供指导。     

Overview of the diversity of erythrocyte blood group antigens

During the differentiation and maturation of erythrocytes, the surface molecules of erythrocytes are gradually expressed and stabilized. These molecules are to be antigenic in addition to their functions of maintaining cell membrane structural stability, material transport and exchange of cells and signal transmission between cells. The antigenic molecules on the erythrocyte surface are called erythrocyte blood group antigens. The blood group antigens and their corresponding blood group antibodies in vivo are important indicators for clinical blood transfusion and organ transplantation, and also form the basis for research on blood group related diseases. Three hundred and sixty-eight erythrocyte blood group antigens have been confirmed so far, which are classified into 39 blood group systems, 5 blood group collections and 2 blood group series. Based on the diversity of blood group antigens and their composition of glycolipids, glycoproteins and other molecules, this study mainly reviews the classification, molecular structure, antibody response and gene regulation of blood group antigens, and explains the main reasons for the diversity of blood group antigens.     

Identification and characterization of a new 34 kDa MORN motif-containing sporozoite surface-exposed protein,Cp-P34, unique to Cryptosporidium

Despite the public health impact of childhood diarrhea caused by Cryptosporidium, effective drugs and vaccines against this parasite are unavailable. Efforts to identify vaccine targets have focused on critical externally exposed virulence factors expressed in the parasite s invasive stages. However, no single surface antigen has yet been found that can elicit a significant protective immune response and it is likely that pooling multiple immune targets will be necessary. Discovery of surface proteins on Cryptosporidium sporozoites is therefore vital to this effort to develop a multi-antigenic vaccine. In this study we applied a novel single-domain camelid antibody (VHH) selection method to identify immunogenic proteins expressed on the surface of Cryptosporidium parvum sporozoites. By this approach, VHHs were identified that recognize two sporozoite surface-exposed antigens, the previously identified gp900 and an unrecognized immunogenic protein, Cp-P34. This Cp-P34 antigen, which contains multiple Membrane Occupation and Recognition Nexus (MORN) repeats, is found in excysted sporozoites as well as in the parasite s intracellular stages. Cp-P34 appears to accumulate inside the parasite and transiently appears on the surface of sporozoites to be shed in trails. Identical or nearly identical orthologs of Cp-P34 are found in the Cryptosporidium hominis and Cryptosporidium tyzzeri genomes. Except for the conserved MORN motifs, the Cp-P34 gene shares no significant homology with genes of other protozoans and thus appears to be unique to Cryptosporidium spp. Cp-P34 elicits immune responses in naturally exposed alpacas and warrants further investigation as a potential vaccine candidate.     

Incorporation of the Tat cell‐penetrating peptide into nanofibers improves the respective antitumor immune response

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant‐free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)‐restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell‐penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor‐specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat_12.5 nanofiber) increased antigen cross‐presentation by bone marrow‐derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat_12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat_12.5 nanofibers exhibited increased presentation of the H2kb‐epitope (reminiscent for cross‐presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat_12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat_12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor‐infiltrating IFN‐γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber‐based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.     

磁共振成像检查联合血清HE4、TK1、CA199检测在卵巢癌诊断中的应用价值

摘要 目的：研究磁共振成像（MRI）检查联合血清人附睾蛋白4（HE4）、细胞质胸苷激酶（TK1）、糖类抗原199（CA199）检测在卵巢癌诊断中的应用价值。方法：将2017年4月～2019年12月我院收治的卵巢癌患者54例作为卵巢癌组,卵巢良性肿瘤患者49例作为卵巢良性肿瘤组,所有受试者均进行MRI检测,并采集受试者血清检测HE4、TK1、CA199水平,以受试者工作特征（ROC）曲线分析MRI检查联合血清HE4、TK1、CA199检测诊断卵巢癌的效能。结果：卵巢癌MRI特点包括以下几点：①囊实性肿块;②形态不规则;③呈长T2/T1等信号;④病灶边界模糊;⑤增强扫描结果呈病灶显著强化;⑥腹膜转移者存在显著腹腔积液以及散在分布的结节状种植病灶。卵巢良性肿瘤MRI特点包括以下几点：①囊壁光滑;②呈长T1以及长T2信;③病灶边界清晰;④增强扫描结果提示病灶无强化/稍有强化。卵巢癌组血清HE4、TK1、CA199水平分别为（87.13±15.32）pmol/L、（2.15±0.13）pmol/L、（95.39±15.25）U/mL,明显高于卵巢良性肿瘤组的（66.42±10.19）pmol/L、（0.85±0.20）pmol/L、（37.94±1.05）U/mL（均P＜0.05）。经ROC曲线分析可得：MRI检查联合血清HE4、TK1、CA199检测诊断卵巢癌的灵敏度、特异度以及曲线下面积均高于上述各项单独诊断。结论：卵巢癌患者血清HE4、TK1、CA199水平较高,MRI检查联合血清HE4、TK1、CA199检测诊断卵巢癌的价值较高,具有一定的临床应用价值。     

苄非他明人工抗原合成

目的: 通过化学方法对苄非他明进行结构修饰并保留抗原决定簇,将结构改造后的产物与载体偶联合成苄非他明抗原。方法: 苄非他明经化学修饰后,增加活性基团连接上一类可用的经化学修饰的连接臂,使用碳二亚胺法与载体蛋白偶联成苄非他明人工合成抗原。该抗原通过紫外吸收光光谱扫描技术、SDS-PAGE电泳法及胶体金免疫层析法进行偶联效果和抗原活性的鉴定。结果: 苄非他明半抗原结构与载体偶联成功,该抗原具有较高的纯度和活性,与苄非他明抗体反应表现出较高的特异性。结论: 该方法合成的苄非他明抗原可用于免疫检测方法,也可作免疫原制备相关抗体。     

抗原抗体复合物型治疗性疫苗(乙克)临床研究中患者血清乙型肝炎病毒表面抗原下降的分析与启示

近年来全球慢性乙型肝炎(chronic hepatitis B,CHB)防治指南提出了“功能性治愈”(functional cure)的概念,即患者经过治疗达到血清乙型肝炎病毒表面抗原(hepatitis B virus surface antigen,HBsAg)消失,但现有抗病毒治疗很难实现这一目标。本研究对既往临床试验中经抗原抗体复合物型治疗性疫苗(乙克)治疗后的CHB患者HBsAg下降情况进行了归纳分析,结果显示,经乙克治疗随访后达到乙型肝炎e抗原(hepatitis B e antigen,HBeAg)血清学转换者的HBsAg下降高达0.95log₁₀IU/mL,显著高于未达到HBeAg血清学转换者的0.32log₁₀IU/mL(P<0.01),而经氢氧化铝佐剂治疗随访后发生HBeAg血清学转换(0.49log₁₀IU/mL)者与未发生HBeAg血清学转换者(0.36log₁₀IU/mL)之间HBsAg下降无统计学差异。乙克组治疗过程中,丙氨酸氨基转移酶(alanine aminotransferase,ALT)骤升(ALT flare)在HBsAg下降>1.0log₁₀IU/mL者中较多见,氢氧化铝组未观察到此现象。回归分析显示,乙克治疗后HBsAg下降的影响因素有患者出现HBeAg血清学转换、感染的HBV为B基因型、治疗过程中ALT出现10倍增高,以及基线血清HBsAg为高水平。结果提示,乙克诱导的特异性免疫对降低CHB患者血清HBsAg水平有一定效果,采用“抗病毒药物治疗＋针对HBsAg的中和性抗体被动免疫＋乙克主动免疫”的“三明治”治疗策略可能会提高“功能性治愈”率。     

乙型肝炎病毒e抗原定量检测的性能验证和临床应用探索

乙型肝炎病毒e抗原(hepatitis B e antigen, HBeAg)的定量检测对乙型肝炎临床诊疗具有一定的重要性,但其定量检测还未成为常规检验项目。本研究对HBeAg定量检测系统进行性能验证,比较HBeAg定量和定性检测的相关性和一致性,分析HBeAg定量结果和乙型肝炎病毒DNA(hepatitis B virus DNA, HBV DNA)的关系,为HBeAg定量检测在临床诊疗的应用提供依据。通过收集710例2019年3月至5月于复旦大学附属华山医院就诊的慢性乙型肝炎患者血清样本,参照美国临床实验室标准化协会(The Clinical & Laboratory Standards Institute, CLSI)相关文件的要求,对雅培ARCHITECTi4000SR全自动免疫分析仪检测的HBeAg定量试剂的精密度、分析灵敏度、线性范围/可报告范围、携带污染率进行验证和评价;采用化学发光微粒子免疫检测技术(chemiluminescence microparticle immuno assay, CMIA)对618例患者进行HBeAg定性和定量检测;采用荧光定量PCR对慢性乙型肝炎患者进行HBV DNA检测,比较HBV DNA和HBeAg定量结果的相关性。本研究证实HBeAg定量试剂检测性能验证结果良好;HBeAg定量和定性检测相关性良好;126例同时有HBeAg定量检测和HBV DNA定量检测的结果显示,两种方法呈正相关且一致性良好。HBeAg定量检测可用于常规实验室检测来辅助HBV感染的临床诊疗。