炭疽杆菌致死因子LF253的制备及活性分析

目的 :制备具有生物学活性的重组致死因子253(lethal factor 253,LF253)抗原,获得纯化的目的蛋白,检测其与全分子致死因子(lethal focfor,LF)蛋白竞争性结合保护性抗原(protective antigen,PA)的能力。方法:PCR扩增致死因子LF253片段的DNA,将目的基因插入p ET-28a(+)表达载体中,利用大肠杆菌BL21(DE3)作为宿主菌,IPTG诱导重组蛋白表达,通过His标签亲和层析柱获得目的蛋白,Western blot和ELISA法检测蛋白抗原性,Biacore T-100测定重组蛋白与保护性抗原PA结合的亲和力,细胞毒性实验检测其生物学活性。结果:成功构建原核表达载体p ET-28a/LF253,诱导获得重组蛋白s LF253的表达。Western blot和ELISA检测结果证实,该重组蛋白具有良好抗原特异性;细胞毒实验结果表明,重组蛋白可在体内外中和致死毒素引起的生物学效应。结论:本研究制备的融合蛋白s LF253能够与保护性抗原PA结合,可竞争性抑制LF全分子蛋白与PA的聚合,阻断炭疽毒素的致死作用,为今后炭疽疫苗等药物的研发奠定了基础。     

国内外炭疽研究文献计量与可视化分析

目的：探索炭疽研究领域的发展情况、研究趋势及动态前沿,以期为我国炭疽研究提供信息支持,为生物反恐提供策略思路。方法基于Web of Knowledge文献数据平台（ SCI）,综合应用Pajek、VOSviewer、Bibexcel软件;基于中国科学引文数据库（ CSCD）数据平台,使用Excel软件。结果通过文献计量可视化分析显示,炭疽研究主要分布在欧美国家,美国炭疽研究整体实力最强,军事医学科学院、中国科学院等机构为我国炭疽主要科研力量,但我国与国际相关研究机构尚存在一定差距。结论我国应加强炭疽研究,提高整体研究实力,为应对可能发生的生物恐怖事件提供必要的保障。     

Evaluation of anthrax vaccine safety in 18 to 20 year olds: A first step towards age de-escalation studies in adolescents

Background/objectivesAnthrax vaccine adsorbed (AVA, BioThrax^®) is recommended for post-exposure prophylaxis administration for the US population in response to large-scale Bacillus anthracis spore exposure. However, no information exists on AVA use in children and ethical barriers exist to performing pre-event pediatric AVA studies. A Presidential Ethics Commission proposed a potential pathway for such studies utilizing an age de-escalation process comparing safety and immunogenicity data from 18 to 20 year-olds to older adults and if acceptable proceeding to evaluations in younger adolescents. We conducted exploratory summary re-analyses of existing databases from 18 to 20 year-olds (n = 74) compared to adults aged 21 to 29 years (n = 243) who participated in four previous US government funded AVA studies.MethodsData extracted from studies included elicited local injection-site and systemic adverse events (AEs) following AVA doses given subcutaneously at 0, 2, and 4 weeks. Additionally, proportions of subjects with ≥4-fold antibody rises from baseline to post-second and post-third AVA doses (seroresponse) were obtained.ResultsRates of any elicited local AEs were not significantly different between younger and older age groups for local events (79.2% vs. 83.8%, P = 0.120) or systemic events (45.4% vs. 50.5%, P = 0.188). Robust and similar proportions of seroresponses to vaccination were observed in both age groups.ConclusionsAVA was safe and immunogenic in 18 to 20 year-olds compared to 21 to 29 year-olds. These results provide initial information to anthrax and pediatric specialists if AVA studies in adolescents are required.     

Sporadic human cutaneous anthrax outbreak in Shaanxi Province,China: report of two cases from 2018

China’s compulsory annual livestock anthrax vaccination policy has remarkably reduced but not completely eradicated human anthrax infections. Herein we describe a sporadic human cutaneous anthrax outbreak involving two cases in 2018 in Shaanxi Province, both involving herdsman who dealt with unvaccinated and potentially sick cattle. Both patients showed Bacillus anthracis-positive blister smear and blood culture. Treatment with penicillin was followed by uneventful recovery for both. The prompt performance of the prophylactic measures successfully interrupted the further transmission of this sporadic human cutaneous anthrax outbreak.     

Epitope-focused peptide immunogens in human use adjuvants protect rabbits from experimental inhalation anthrax

BackgroundAnthrax represents a formidable bioterrorism threat for which new, optimized vaccines are required. We previously demonstrated that epitope-focused multiple antigenic peptides or a recombinant protein in Freund's adjuvant can elicit Ab against the loop neutralizing determinant (LND), a cryptic linear neutralizing epitope in the 2ß2–2ß3 loop of protective antigen from Bacillus anthracis, which mediated protection of rabbits from inhalation challenge with B. anthracis Ames strain. However, demonstration of efficacy using human-use adjuvants is required before proceeding with further development of an LND vaccine for testing in non-human primates and humans.MethodsTo optimize the LND immunogen, we first evaluated the protective efficacy and immune correlates associated with immunization of rabbits with mixtures containing two molecular variants of multiple antigenic peptides in Freunds adjuvant, termed BT-LND(2) and TB-LND(2). TB-LND(2) was then further evaluated for protective efficacy in rabbits employing human-use adjuvants.ResultsImmunization of rabbits with TB-LND(2) in human-use adjuvants elicited protection from Ames strain spore challenge which was statistically indistinguishable from that elicited through immunization with protective antigen. All TB-LND(2) rabbits with any detectable serum neutralization prior to challenge were protected from aerosolized spore exposure. Remarkably, rabbits immunized with TB-LND(2) in Alhydrogel/CpG had significant anamnestic increases in post-challenge LND-specific Ab and neutralization titers despite little evidence of spore germination in these rabbits.ConclusionsAn LND-specific epitope-focused vaccine may complement PA-based vaccines and may represent a complementary stand-alone vaccine for anthrax.     

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg''s native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.     

The NLRP1 and CARD8 inflammasomes

Inflammasomes are multiprotein complexes that activate inflammatory cytokines and induce pyroptosis in response to intracellular danger-associated signals. NLRP1 and CARD8 are related germline-encoded pattern recognition receptors that form inflammasomes, but their activation mechanisms and biological purposes have not yet been fully established. Both NLRP1 and CARD8 undergo post-translational autoproteolysis to generate two non-covalently associated polypeptide chains. NLRP1 and CARD8 activators induce the proteasome-mediated destruction of the N-terminal fragment, liberating the C-terminal fragment to form an inflammasome. Here, we review the danger-associated stimuli that have been reported to activate NLRP1 and/or CARD8, including anthrax lethal toxin, Toxoplasma gondii, Shigella flexneri and the small molecule DPP8/9 inhibitor Val-boroPro, focusing on recent mechanistic insights and highlighting unresolved questions. In addition, we discuss the recently identified disease-associated mutations in NLRP1 and CARD8, the potential role that DPP9’s protein structure plays in inflammasome regulation, and the emerging link between NLRP1 and metabolism. Finally, we summarize all of this latest research and consider the possible biological purposes of these enigmatic inflammasomes.     

Special Considerations for Prophylaxis for and Treatment of Anthrax in Pregnant and Postpartum Women

In August 2012, the Centers for Disease Control and Prevention, in partnership with the Association of Maternal and Child Health Programs, convened a meeting of national subject matter experts to review key clinical elements of anthrax prevention and treatment for pregnant, postpartum, and lactating (P/PP/L) women. National experts in infectious disease, obstetrics, maternal fetal medicine, neonatology, pediatrics, and pharmacy attended the meeting, as did representatives from professional organizations and national, federal, state, and local agencies. The meeting addressed general principles of prevention and treatment for P/PP/L women, vaccines, antimicrobial prophylaxis and treatment, clinical considerations and critical care issues, antitoxin, delivery concerns, infection control measures, and communication. The purpose of this meeting summary is to provide updated clinical information to health care providers and public health professionals caring for P/PP/L women in the setting of a bioterrorist event involving anthrax.     

Exoproteome analysis of a novel strain of Bacillus cereus implicated in disease resembling cutaneous anthrax

Bacillus cereus belongs to B. cereus sensu lato group, shared by six other related species including Bacillus anthracis. B. anthracis is the causative agent for serious illness affecting a wide range of animals as well as humans and is a category A Biological and Toxin Warfare (BTW) agent. Recent studies indicate that a Bacillus species other than B. anthracis can cause anthrax-like disease and role of anthrax virulence plasmids (pXO1 and pXO2) on the pathogenicity of B. cereus has been documented. B. cereus strain TF5 was isolated from the tissue fluid of cutaneous anthrax-like skin lesions of a human patient from an anthrax endemic area in India. The strain harboured a PA gene, however, presence of pXO1 or pXO2-like plasmids could not be ascertained using reported primers. Abundant exoproteome of the strain in the early stationary phase was elucidated using a 2-DE MS approach and compared with that from a reference B. cereus strain. Analysis of proteins showing qualitative and quantitative differences between the two strains indicated an altered regulatory mechanism and putative role of S-layer protein and sphingomyelinase in the pathogenesis of strain TF5. Phylogenetic analysis of the S-layer protein indicated close affiliation of the strain with anthracis-like B. cereus strains such as B. cereus var. anthracis strain CI; whereas sphingomyelinase exhibited specific relationship with all the strains of B. anthracis apart from that with anthracis-like B. cereus strains.