Mycobacterium bovis BCG as a Delivery System for the dtb Gene Antigen from Diphtheria Toxin

Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.

Plasmid instability when the hsp60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

The genetic modification of the live attenuated Mycobacterium bovis BCG to deliver a protective Corynebacterium diphtheriae antigen in vivo could be a safer and less costly alternative to the new and more expensive DTP vaccines available today, in particular to third world-countries. The stability of expression of heterologous antigens in BCG, however, is a major challenge to the use of live recombinant bacteria in vaccine development and appears to be dependent to a certain extent, on a genetic compatibility between the expression cassette within the plasmid construct and the mycobacterium host. In the quest for the best recombinant BCG transformant to express the dtb gene of C. diphtheriae we generated two new rBCG strains by transforming the Moreau substrain of BCG with the mycobacterial expression vectors pUS973 and pUS977, each one carrying a different promoter to drive the expression of the target antigen. After transformation recombinant BCG clones were selected on Middlebrook 7H10 kanamycin Agar plates, expanded in Middlebrook 7H9 kanamycin Broth and analyzed by agarose gel electrophoresis and immunoblotting. rBCGs transformed with the construct carrying the weak PAN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rBCGs transformed with the construct carrying the strong mycobacterium hsp60 promoter were unstable and consequently unfit for the expression of the C. diphtheriae gene.

Human liver chimeric mouse model based on diphtheria toxin-induced liver injury

AIM To establish an inducible liver injury mouse model and transplant human hepatocytes to obtain liverhumanized mice.METHODS We crossed three mouse strains,including albumin(Alb)-cre transgenic mice,inducible diphtheria toxin receptor(DTR) transgenic mice and severe combined immune deficient(SCID)-beige mice,to create Alb-cre/DTR/SCID-beige(ADSB) mice,which coincidentally harbor Alb-cre and DTR transgenes and are immunodeficient. As the Cre expression is driven by the liver-specific promoter Alb(encoding ALB),the DTR stop signal flanked by two lox P sites can be deleted in the ADSB mice,resulting in DTR expression in the liver. ADSB mice aged 8-10 wk were injected intraperitoneally(i.p.) with diphtheria toxin(DT) and liver damage was assessed by serum alanine aminotransferase(ALT) level. Two days later,mouse livers were sampled for histological analysis,and human hepatocytes were transplanted into the livers on the same day. A human ALB enzyme-linked immunosorbent assay was performed 7,14,21 and 28 d after transplantation. Human CD68 immunohistochemistry was performed 30 and 90 d after transplantation.RESULTS We crossed Alb-cre with DTR and SCID-beige mice to obtain ADSB mice. These mice were found to have liver damage 4 d after i.p. injection of 2.5 ng/g bodyweight DT. Bodyweight began to decrease on day 2,increased on day 7,and was lowest on day 4(range,10.5%-13.4%). Serum ALT activity began to increase on day 2 and reached a peak value of 289.7 ± 16.2 IU/m L on day 4,then returned to background values on day 7. After transplantation of human liver cells,peripheral blood human ALB level was 1580 ± 454.8 ng/m L(range,750.2-3064.9 ng/m L) after 28 d and Kupffer cells were present in the liver at 30 d in ADSB mice.CONCLUSION Human hepatocytes were successfully repopulated in the livers of ADSB mice. The inducible mouse model of humanized liver in ADSB mice may have functional applications,such as hepatocyte transplantation,hepatic regeneration and drug metabolism.

Secretory Expression of Recombinant Diphtheria ToxinMutants in B. Subtilis

Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118, but higher yield of 7. 1 mg/L was observed in SMS300 compared with 2. 1 mg/L in SMS118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be de-graded into two peptides of 37ku and 21ku.

Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study

Objective： This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor （DT388sBAFF） in Escherichia coli （E. coli） and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells （BALL-1）. Methods： A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction （PCR） and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）. The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, and then purified by Ni2＋-NTA affinity chromatography. The expression level of B cell-activating factor receptor （BAFF-R） on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide （MTT） assay. Results： The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion： The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

Diphtheria and Tetanus Antibody Persistence in Indian Pre-school Children and Response to a Booster Dose of DT Vaccine.

Despite effective vaccines, diphtheria (D) resurged recently in the former socialistic block, and tetanus (T) still occurs in less privileged countries. We studied the antibody persistence for D and T in Indian pre-school children who had received four doses of DTP vaccine and subsequently, the response to a booster dose. Anti-D and anti-T IgG antibodies prior to and one month after a DT vaccine were measured by ELISA in 223 healthy children of 4-6 years who had previously received four doses of the triple vaccine. Adverse reactions were monitored for one month. While 30% and 14% of subjects were susceptible to D and T, respectively, 98% and 100% of them attained seroprotection post-vaccination. Both responses were significant. Local, but not systemic reactions except fever were rather common. A high proportion of the Indian pre-school population is susceptible to D and T, despite of receiving four doses. The current policy of giving the fifth dose at this age is appropriate.

Specific killing effect of diphtheria toxin A fragment under control of DF3 promotor on human breast cancer cells

Objective: To study the effects of recombinant expression vector containing human breast cancer DF3 promotor and diphtheria toxin A fragment on human breast cancer cells. Methods: Constructing recombinant expression vector PGL3-DF3-DTA and transfecting it into human breast cancer cells of DF3 positive and negative. By means of RT-PCR to measure the expression of DTA in human breast cancer cells. MTT color-imetry was used to examine the effect of PGL3-DF3-DTA on growth of human breast cancer cells. By experiment on nude mice to observe the killing effect of PGL3-DF3-DTA on human breast cancer cells. Results: Recombinant expression vector PGL3-DF3-DTA was highly expressed in human breast cancer cell line of DF3 positive, and it could kill the human breast cancer cells not only in vitro but also in vivo. Conclusion: Recombinant expression vector PGL3-DF3-DTA could produce specific killing effect on human breast cancer cell line of DF3 positive.

Prognostic Significance of Electrocardiographic Changes in Diphtheria Myocarditis: A Cross-Sectional Study

Background: Diphtheria is still endemic in India due to inadequate immunization. The incidence of myocarditis is higher in these patients. Objectives: The objective of this study was to study clinical profile, clinical outcomes as well as immunization status of the patients diagnosed with diphtheria myocarditis in Indian scenario. Methodology: This prospective observational study was carried out in one of the tertiary care hospital of south India from August 2011 to December 2012. A total of 33 cases with clinically confirmed diagnosis of diphtheria myocarditis were enrolled depending upon the inclusion and exclusion criteria of the study. Electrocardiography and 2-dimensional echocardiography was done at the time of admission and repeated when required. Results: The most common age group affected is 5 - 10 years, with no sex difference in occurrence. Only 1 patient, out of 33 patients, was adequately immunized. Asymptomatic myocardial involvement (with only changes in electrocardiogram) was seen in 21 patients whereas 12 patients were symptomatic. The average duration of resolution of electrocardiographic changes was 4 - 6 weeks. It should be noted that out of 28 patients who developed conduction abnormalities, 24 patients died. Temporary pacemaker support was given for 8 patients, of whom only one patient recovered. The patient was followed till hospital discharge. Conclusion: The mortality associated with diphtheria myocarditis is higher in Indian population. As diphtheria can be prevented by adequate vaccination, efforts should be maximized for 90% coverage with three doses of diphtheria toxoid in children below one year of age and immunity towards it should be maintained by booster doses.

Seroimmunity to Tetanus and Diphtheria among Immunocompetent Adult Patients Admitted to a Tertiary Hospital for Any Reason

Ignoring the importance of receiving booster doses of vaccines in vast majority of adults could result in their lack of immunity against tetanus and diphtheria. In this prospective cohort study all immunocompetent adults ≥ 40 years old who were admitted for any reason during the time period of the survey were enrolled and antibody levels against tetanus and diphtheria were measured. 84 patients (48%) were 40 - 60 years old and 93 (52%) subjects aged older than 60 years. In general, anti-tetanus antibody titer was <0.1 IU/mL (non-immune range) in 83 people (46.9%). For anti-diphtheria antibody, 74 (41.8%) were considered non-immune. Of all people whose tetanus antibody level was <0.1, 19 had a complete history of childhood diphtheria and tetanus immunization and 64 had unknown vaccination history. Among people with diphtheria antibody levels < 0.1, 18 (24.3%) had complete history of childhood vaccination and 56 (75.6%) had unknown history.

Difference of the Pain during the DPT (Diphtheria-Pertussis-Tetanus) Vaccination

Background: The objective of this study is to determine whether analyze of the infant’s pain associated with diphtheria-pertussis-tetanus (DPT) immunization is useful for vaccination in children. It is not known whether the immunization pain can be prevented with the adequate choice of DPT vaccines among several manufacturers in Japan. Further, it is not clear whether the difference of the reaction during vaccination between gender and age. Design: Three manufacturer’s Japanese DPT vaccines were used in this study. The parents assessed their infant’s pain on a modified visual analogue scale (MVAS), the start of the crying and total crying time during the immunization. Results: The A manufacturer’s DPT vaccine was significantly lower on the proportion of crying, the duration of crying and MVAS score than the other two manufacturer’s DPT vaccines. The proportion of crying, the duration of crying and MVAS score was lower in the boy than in the girl. On the other hand, it had not found the difference of their reactions with age. Conclusions: Our studies have found that the adequate choice of DPT vaccine decreased vaccination pain. The studies also indicate that some tendencies with vaccinations were shown in children. To consider these tendencies was useful in performing less painful vaccination.

Comparative Pertussis Antibody Response of Nigerian Children to Diphtheria, Pertussis, Tetanus (DPT) and Pentavalent Vaccines

Background: In Nigeria Pentavalent vaccine had replaced Diphtheria-Pertussis- Tetanus [DPT] vaccine in the prevention of pertussis since 2012. Aims and Objectives: The aim of this study was to compare the anti-pertussis immunoglobin G (IgG) response of children who received DPT with those who received the pentavalent vaccine. Subjects and Methods: This study was carried out in Akpabuyo LGA of Cross River State from April to June 2016. It was a cross-sectional survey of anti-pertussis IgG levels in children aged 6 months to 5 years who received DPT and those who received pentavalent vaccine. IgG antibody levels were determined using enzyme-linked immunosorbent assay. The protective level was set at >11 DU according to manufacturer’s cut off point. Results: Seventy eight out of 230 children [33.9%] who had received DPT had protective levels of anti-pertussis IgG compared to 74 out of 192 children [38.5%] who had received pentavalent vaccine. The difference was not statistically significant [p = 0.61]. The median IgG antibody level in those who received DPT was 8.0 DU (interquartile range (IQR) 4.0 - 13.0) compared with 9.0 DU (IQR) 4.0 - 15.0 in those who received pentavalent vaccine [p = 0.18]. No single factor investigated predicted the development of protective levels of antibody in the multivariate analysis. Conclusion/Recommendation: There was no difference in the antipertussis antibody response between DPT and pentavalent vaccines recipients. Further study is needed to elucidate factors that could be responsible for low anti-pertussis antibody response in this population.

Assessing the impact of booster vaccination on diphtheria transmission:Mathematical modeling and risk zone mapping

The COVID-19 pandemic caused significant disruptions in the healthcare system,affecting vaccinations and the management of diphtheria cases.As a consequence of these disruptions,numerous countries have experienced a resurgence or an increase in diphtheria cases.West Java province in Indonesia is identified as one of the high-risk areas for diphtheria,experiencing an upward trend in cases from 2021 to 2023.To analyze the situation,we developed an SIR model,which integrated DPT and booster vaccinations to determine the basic reproduction number,an essential parameter for infectious diseases.Through spatial analysis of geo-referenced data,we identified hotspots and explained diffusion in diphtheria case clusters.The calculation of R0 resulted in an R0=1.17,indicating the potential for a diphtheria outbreak in West Java.To control the increasing cases,one possible approach is to raise the booster vaccination coverage from the current 64.84%to 75.15%,as suggested by simulation results.Furthermore,the spatial analysis revealed that hot spot clusters were present in the western,central,and southern regions,posing a high risk not only in densely populated areas but also in rural regions.The diffusion pattern of diphtheria clusters displayed an expansion-contagious pattern.Understanding the rising trend of diphtheria cases and their geographic distribution can offer crucial insights for government and health authorities to manage the number of diphtheria cases and make informed decisions regarding the best prevention and intervention strategies.

福建省3例输入性白喉棒状杆菌感染病例流行病学和病原学分析

目的对福建省发现的3例输入性白喉病例进行流行病学和病原学分析。方法对3例疑似皮肤白喉患者进行流行病学调查,采集患者皮肤破损的分泌物样品,进行白喉棒状杆菌分离培养。对纯培养的疑似白喉棒状杆菌进行生化鉴定和质谱分析,检测白喉棒状杆菌(Diphtheria toxin repressor,dtxR)、(Toxin A,toxA)、(Toxin B,toxB)等毒力相关基因,进行MLST序列分型。结果3例病例有典型的皮肤白喉临床表现,皮肤破损部位均分离到白喉棒状杆菌。2例病例分离到的2株白喉棒状杆菌毒力基因检测结果为dtxR(+)、toxA(-)、toxB(-),MLST型别均为ST-703;1例病例分离的白喉棒状杆菌菌株毒力基因检测结果为dtxR(+)、toxA(+)、toxB(+),MLST型别为ST-248。结论福建省存在白喉输入性风险,非毒原菌亦可引起皮肤白喉。

Cell-specific expression of the diphtheria toxin A-chain coding sequence induces cancer cell suicide

OBJECTIVE: To test whether the diphtheria toxin A (DT-A) chain coding sequence linked to murine immunoglobulin Kappa light chain (IgKappa) promoter and enhancer have selective cytocidal effects on IgKappa producing cells. METHODS: The diphtheria toxin A gene or beta galactosidase (beta-gal) gene were linked to a murine IgKappa promoter and enhancer to construct pcDNA3IgKappaDTA or pcDNA3IgKappaLacZ plasmids. These plasmids were transfected into IgKappa producing or non-producing cells by the liposome coated DNA method. Expression of beta-gal activity and effects on cell growth of transfected cells were assessed. RESULTS: The beta-gal gene, under the control of cytomegalovirus (CMV) promoter, can express in all cell lines. Expression of beta-gal under the control of the IgKappa promoter was detected only in the IgKappa producing cell line, CA46. Expression of beta-gal was greatly suppressed when cotransfected with pcDNA3IgKappaDTA in CA46 cells.Cell growth of CA46 cells transfected with pcDNA3IgKappaDTA plasmid was significantly inhibited compared with CA46 cells transfected with pcDNA3IgKappaLacZ. CONCLUSION: Selective killing of IgKappa producing cells can be attained by introducing the diphtheria toxin A gene under the control of IgKappa promoter and enhancer.

基于“人机料法环测”管理模式的疑似白喉在医疗机构中的防控策略

白喉是由白喉棒状杆菌引起的死亡率高的急性呼吸道传染病,是我国《传染病防治法》规定的乙类传染病。感染者咽喉部可出现白色伪膜,同时出现发热、声音嘶哑、犬吠样咳嗽、呼吸困难等症状,严重感染患者可能会产生全身性中毒症状,甚至诱发周围神经麻痹和心肌炎,曾严重危害人民健康。虽然现阶段白喉在我国属于罕见传染病,但国内仍有散发病例出现,与此同时,非产毒菌分离率呈上升趋势。本研究通过对西安市某三甲医院检出的疑似白喉患者的防控策略进行梳理,旨在有力、有序推进医疗机构内发现疑似白喉等罕见传染病的防控策略,为医疗卫生行业疑似白喉的医院感染防控提供先进成熟经验。

双联降脂与颈动脉支架成形术治疗颈动脉重度狭窄预防缺血性脑卒中发生的临床效果

目的对比双联降脂(阿托伐他汀钙片+普罗布考片)与颈动脉支架成形术(CAS)治疗颈动脉重度狭窄预防缺血性脑卒中发生的临床疗效及对载脂蛋白(Apo)A1/ApoB的影响。方法选取新乡市第二人民医院和郑州大学第五附属医院颈动脉重度狭窄患者共67例,药物组(口服阿托伐他汀钙片+普罗布考片)35例,手术组(CAS)32例。随访12个月,记录与狭窄血管有关供血区的缺血性脑卒中发生情况,以及载脂蛋白(Apo)A1/(Apo)B治疗前后的水平。结果随访6个月和12个月时药物组缺血性脑卒中发生率高于手术组,差异有统计学意义(P<0.05)。药物组治疗后ApoA1/ApoB水平升高,差异有统计学意义(P<0.05)。结论对于颈动脉重度狭窄患者,采用CAS较单纯使用药物治疗能够有效降低狭窄血管供血区的缺血性脑卒中发生率。但阿托伐他汀钙联合普罗布考治疗也能够提高ApoA1/ApoB比值,降低缺血性脑卒中发生风险。

Bacteremia complicated with pneumonia caused by Corynebacterium diphtheriae

Due to the use of vaccines,infections caused by Corynebacterium diphtheriae are now rare.Here we describe a case of bacteremia complicated with pneumonia caused by C.diphtheriae in a 69-year-old male patient.The patient presented at the emergency department with a 2 days history of fever of unknown origin.Subsequent examinations revealed pneumonia and bacteremia.Non-toxigenic C.diphtheriae strains were isolated from blood and sputum.The patient had antimicrobial therapy with good improvement.We highlight the important role of C.diphtheriae in causing bacteremia and pneumonia,and its accurate and timely diagnosis is needed to avoid poor visual outcomes.

Be Vigilant of Peripheral Circulatory Failure in Early Stage of Diphtheria Misdiagnosed as Myocarditis

The localized outbreaks of diphtheria still happened in some areas in recent years. The case—fatality rate Of diphtheria is rising and so is the case—fatality rate of toxic myocarditis. About 16 years ago, reviewing the clinical data concerning patients who had died of diphtheria in our hospital, we learned a lesson from our faults in the treatment of diphtheria for the pur-

血液灌流联合持续性血液滤过对敌草快中毒的疗效观察

目的探讨血液灌流联合持续性血液滤过在敌草快中毒中的临床应用效果。方法选取2020年1月至2021年12月郑州大学第一附属医院急诊医学部收治的84例敌草快中毒患者作为研究对象,根据治疗方式分为对照组和试验组。对照组接受血液灌流治疗,试验组给予血液灌流联合持续性血液滤过治疗,对比两组患者的肝肾功能、炎性细胞因子等,比较两组患者脏器功能障碍发生率、病死率及毒物清除时间的差异。结果试验组患者第7天谷丙转氨酶(ALT)、谷草转氨酶(AST)、血肌酐(Cr)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平较对照组患者均明显降低,差异均有统计学意义(P<0.05);而氧分压(PaO_(2))较对照组明显上升,差异有统计学意义(P<0.05)。两组患者血尿中毒物清除的时间比较,差异有统计学意义(P<0.05)。预后分析比较,试验组多脏器功能障碍综合征(MODS)的发生率(42.2%)与对照组(69.2%)比较,差异有统计学意义(P<0.05);两组病死率比较[22(48.9%)vs.24(61.5%)],差异无统计学意义(P>0.05)。结论早期血液灌流联合持续性血液滤过在一定程度上改善了患者的临床症状,降低了多脏器功能损伤发生率,在临床上值得应用。

白喉棒杆菌核酸检测试剂用国家参考品的研制

目的:研制白喉棒杆菌核酸检测试剂用国家参考品。方法:将6株白喉棒杆菌和10株非白喉棒杆菌代表性菌株在各自适宜的环境下培养,经菌株鉴定后,制备灭活菌液,对候选参考品进行均匀性和稳定性的评估以及准确性、特异性、重复性和最低检出限的验证研究,并组织3家实验室进行协作标定。结果:研制出白喉棒杆菌核酸检测试剂用候选参考品,由6个阳性参考品、10个阴性参考品、最低检出限参考品和重复性参考品组成。证实该候选参考品均匀性及稳定性良好,准确性、特异性及重复性好,最低检出限可达1.0×10~3~1.0×10~4个菌·m L^(-1)。协作标定结果也进一步表明阳性、阴性、重复性和最低检出限候选参考品均符合国家参考品的各项要求。结论:研制出的白喉棒杆菌核酸检测试剂参考品,可用于相关试剂盒的质量控制和评价。