Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

Aim:

To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.

Methods:

The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F'' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting, indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.

Results:

A recombinant vector was constructed. The Fab was expressed in soluble form in E coli Top10F''. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL. The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).

Conclusion:

The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.     

Antibodies to cell surface proteins redirect intracellular trafficking pathways

Antibody-mediated intracellular delivery of therapeutic agents has been considered for treatment of a variety of diseases. These approaches involve targeting cell-surface receptor proteins expressed by tumors or viral proteins expressed on infected cells. We examined the intracellular trafficking of a viral cell-surface-expressed protein, rabies G, with or without binding a specific antibody, ARG1. We found that antibody binding shifts the native intracellular trafficking pathway of rabies G in an Fc-independent manner. Kinetic studies indicate that the ARG1/rabies G complex progressively co-localized with clathrin, early endosomes, late endosomes, and lysosomes after addition to cells. This pathway was different from that taken by rabies G without addition of antibody, which localized with recycling endosomes. Findings were recapitulated using a cellular receptor with a well-defined endogenous recycling pathway. We conclude that antibody binding to cell-surface proteins induces redirection of intracellular trafficking of unbound or ligand bound receptors to a specific degradation pathway. These findings have broad implications for future developments of antibody-based therapeutics.     

Multisynaptic projections from the ventrolateral prefrontal cortex to the dorsal premotor cortex in macaques – anatomical substrate for conditional visuomotor behavior

Lines of evidence indicate that both the ventrolateral prefrontal cortex (vlPFC) (areas 45/12) and dorsal premotor cortex (PMd) (rostral F2 in area 6) are crucially involved in conditional visuomotor behavior, in which it is required to determine an action based on an associated visual object. However, virtually no direct projections appear to exist between the vlPFC and PMd. In the present study, to elucidate possible multisynaptic networks linking the vlPFC to the PMd, we performed a series of neuroanatomical tract‐tracing experiments in macaque monkeys. First, we identified cortical areas that send projection fibers directly to the PMd by injecting Fast Blue into the PMd. Considerable retrograde labeling occurred in the dorsal prefrontal cortex (dPFC) (areas 46d/9/8B/8Ad), dorsomedial motor cortex (dmMC) (F7 and presupplementary motor area), rostral cingulate motor area, and ventral premotor cortex (F5 and area 44), whereas the vlPFC was virtually devoid of neuronal labeling. Second, we injected the rabies virus, a retrograde transneuronal tracer, into the PMd. At 3 days after the rabies injections, second‐order neurons were labeled in the vlPFC (mainly area 45), indicating that the vlPFC disynaptically projects to the PMd. Finally, to determine areas that connect the vlPFC to the PMd indirectly, we carried out an anterograde/retrograde dual‐labeling experiment in single monkeys. By examining the distribution of axon terminals labeled from the vlPFC and cell bodies labeled from the PMd, we found overlapping labels in the dPFC and dmMC. These results indicate that the vlPFC outflow is directed toward the PMd in a multisynaptic fashion through the dPFC and/or dmMC.     

平远县2001-2010年狂犬病流行病学分析

目的探讨平远县近10年来狂犬病流行病学特征及流行因素,为制订防控措施提供科学依据。方法收集2001-2010年平远县传染病疫情资料、狂犬病个案资料和狂犬病疫点调查处理资料,采用描述性流行病学方法进行分析。结果 2001-2010年平远县共报告狂犬病16例,年平均发病率为0.64/10万;死亡16例,病死率为100%;常年散发,秋季发病较多(占50%);发病以农村为主,农民和农村学生为主要发病人群;男性多于女性(性别比为5:3),40～70岁年龄组发病较多(占62.5%);头面部咬伤的潜伏期短,下肢咬伤的潜伏期较长;87.5%的病例未进行规范伤口处理,87.5%的病例未进行免疫接种。结论预防狂犬病须采取综合措施,加强犬类管理,加大健康教育力度,将农民和学生作为预防狂犬病的重点人群,规范暴露后伤口处理和提高狂犬疫苗接种率,才能有效降低发病率。     

A retrospective study of rabies in humans in Zimbabwe, between 1992 and 2003

This study aimed at examining the epidemiological features of rabies in humans in Zimbabwe. The data were taken from internal reports of the department of veterinary technical services at Harare covering the period 1st January 1992-31st December 2003 inclusive. Positive cases were examined in relation to age and sex of the victim, animal vector involved, season, and land-use categories. The majority of the confirmed positive human cases (85.7%) were recorded in communal areas. The 5-19 year age group and males constituted a highly vulnerable group. Over 90% of the cases were due to dog bites with jackals (Canis adustus and C. mesomelas), and honey badgers (Mellivora capensis) also contributing to the positive cases. Rabid cats and rabid wild animals had a high relative risk (RR) of biting humans. Animal-to-human transmission was highest during the dry months of July to November.     

人源抗狂犬病毒二硫键稳定性Fv抗体片段基因的构建与表达

目的 构建人源抗狂犬病毒二硫键稳定性Fv（dsFv）抗体基因,并在原核系统中实现高效表达,制备有活性的人源抗狂犬病毒dsFv抗体片段。方法采用PCR定点突变的方法,构建抗狂犬病毒dsFv抗体的重、轻链（VH、VL）突变基因,NdeⅠ和NotⅠ双酶切后分别插入pET-22b（＋）表达质粒中,转化E.coli BL21（DE3）,IPTG诱导表达。将VL、VH包涵体蛋白经盐酸胍变性,按比例混合在复性折叠缓冲液中,使二者折叠形成dsFv抗体片段。并以其母本抗体scFv作对照,对其抗原特异性和稳定性进行初步评价。结果 在VH的第44位氨基酸和VL的第100位氨基酸引入了半胱氨酸,成功构建了抗狂犬病毒dsFv抗体基因,并在原核系统中实现了对二者的高效表达,VH、VL蛋白在复性缓冲液中正确折叠形成了dsFv抗体片段。结论 对筛选获得的抗狂犬病毒scFv成功进行了稳定性改构,制备了有活性的dsFv抗体片段,改构以后的dsFv保持了对狂犬病毒的特异结合活性,而其在血清中的稳定性有明显改进,而且其热稳定性和抵抗尿素化学变性的能力亦大大改进。     

Magnetic resonance imaging abnormalities in encephalomyelitis due to paralytic rabies: A case report

The diagnosis of rabies, a potentially fatal neuroinfectious disease, should be strongly considered in all patients who develop encephalitis following an infected animal bite even when they have received post‐exposure prophylaxis. In the absence of confirmatory tests, typical magnetic resonance imaging findings help confirm the clinical diagnosis of rabies.     

湖南省20株狂犬病毒株与疫苗株N基因序列分析

目的 分析湖南省狂犬病毒株与疫苗株的N基因序列及遗传进化关系,从基因角度解决疫苗的选择问题,为狂犬病的防治提供科学依据.方法 采用KT-PCK法从市售家犬脑组织标本内获得N基因并进行测序,采用DNAStar软件包中的MegAlign软件,将所得结果与国内外发表的代表性疫苗株相应基因进行核苷酸、氨基酸序列同源性比对分析,并以Neighbor-Joining法构建系统发生树.结果 湖南省20株狂犬病毒与国内外13株疫苗代表的株核苷酸同源性在85.3%～94.2%(中位数88.6%).相应地,N基因编码的氨基酸序列同源性为95.4%～99.6%(中位数99.2%).其中,与中国疫苗株CTN、SRV-9,国外疫苗株CVS、RBE3-15、SADB19-1st及HEP-Fllury株的氨基酸序列同源性范围在99.2%～99.6%,并以与CTN疫苗株同源性中位数最高(90%).系统发生分为两大群.湖南省20株研究毒株与中国人用CTN株具有很高的亲缘性,构成第一群.其余的疫苗毒株构成第二基因群.结论 湖南省狂犬病毒株与中国人用疫苗株CTN同源性最高、亲缘关系最近,因此在湖南省使用CTN疫苗株效果可能较好.     

2021年邹城市钢山街道居民动物致伤及狂犬病疫苗 接种情况分析

摘 要：目的：了解邹城市钢山街道因动物致伤人群特征以及狂犬病疫苗、狂犬病免疫球蛋白接种情况，为预防和控制 狂犬病提供科学依据。方法：整理 2021年钢山街道居民因动物致伤者在当地犬伤门诊就诊的基本情况，采用χ2 检验和二元 logistic回归分析探讨因动物致伤者狂犬病疫苗全程接种率的影响因素。结果：2021年钢山街道居民因动物致伤就诊并接种狂 犬病疫苗者1 258例，其中，女性较多，共677例，占53.82%；年龄以0~14岁者为主，共452例，占35.93%；职业以学生为 主，共448例，占35.61%。1 258例就诊者中，全程接种狂犬病疫苗者1 153例，占91.65%；Ⅲ级暴露者982例，占78.06%， 其中，接种狂犬病免疫球蛋白者797例，占81.16%。致伤部位以上肢为主，共854例，占67.89%；致伤动物以家养犬为主， 共665例，占52.86%；致伤场所以户内为主，为1 173例，占93.24%。二元logistic回归分析结果显示，与农民相比，散居儿 童、学生、公务员、工人是狂犬病疫苗全程接种率的保护因素；与暴露分级Ⅱ级相比，暴露分级Ⅱ级是狂犬病疫苗全程接种 率的保护因素。结论：职业、暴露程度是影响狂犬病疫苗全程接种率的重要因素，应对不同职业、暴露分级人群开展狂犬病 防治知识个体化健康教育，提高狂犬病防治知识的普及率，同时强化犬只管理，提高豢养犬只人员安全意识。     

Rabies postexposure prophylaxis: What the U.S. emergency medicine provider needs to know

Approximately 55,000 patients per year in the United States are exposed to potentially rabid animals and receive rabies postexposure prophylaxis (PEP) and these patients commonly present to the emergency department (ED) for wound care and PEP. Despite the number of rabies exposures seen in EDs each year, there appears to be a knowledge gap among health care providers with regard to prescribing and administering rabies PEP. The following review aims to bridge that knowledge gap by discussing the importance of obtaining a comprehensive exposure history to determine the category of the encounter, the type of animal, and the location of the bite and of consulting outside expert resources to determine whether the rabies PEP series is indicated. In addition, this article will discuss dosing, administration, and schedule of the rabies vaccine and human rabies immune globulin to ensure patients are fully protected from developing rabies. Lastly, this article discusses the potential cost associated with rabies PEP and provides information on managing this barrier.