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Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA

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Chinese Medical Journal 2024年第6期137卷 729-736页

作者： Lin Yuan Zhiying Liu Xin Zhang Feili Wei Shan Guo Na Guo Lifeng Liu Zhenglai Ma Yunxia Ji Rui Wang Xiaofan Lu Zhen Li Wei Xia Hao Wu Tong Zhang Bin Su Beijing key Laboratory for HIV/AIDS Research Clinical and Research Center for Infectious DiseasesBeijing Youan HospitalCapital Medical UniversityBeijing 100069China Beijing Institute of Hepatology Beijing Youan HospitalCapital Medical UniversityBeijing 100069China

Background:Total human immunodeficiency virus(HIV)DNA and integrated HIV DNA are widely used markers of HIV *** digital polymerase chain reaction(ddPCR)can be used for absolute quantification without needing a standard ***,we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV ***:The limit of detection,dynamic ranges,sensitivity,and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat(LTR)and human CD3 gene(for total HIV DNA)and ACH-2 cells(for integrated HIV DNA).Forty-two cases on stable suppressive antiretroviral therapy(ART)were assayed in total HIV DNA and integrated HIV *** coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive(CD4^(+))T-cell counts,CD8^(+)T-cell counts and CD4/CD8 T-cell ratio,*** assay linear dynamic range and lower limit of detection(LLOD)were also ***:The assay could detect the presence of HIV-1 copies 100%at concentrations of 6.3 copies/reaction,and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction(95%confidence intervals[CI]:3.6-6.5 copies/reaction)with linearity over a 5-log_(10)-unit range in total HIV DNA *** the integrated HIV DNA assay,the LLOD was 8.0 copies/reaction(95%CI:5.8-16.6 copies/reaction)with linearity over a 3-log 10-unit *** HIV DNA in CD4^(+)T cells was positively associated with integrated HIV DNA(r=0.76,P<0.0001).Meanwhile,both total HIV DNA and integrated HIV DNA in CD4^(+)T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8^(+)T-cell ***:This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and *** can be readily adapted for measuring HIV DNA with non-B clades,and it could be beneficial for testing in clinical trials.

关键词： Human immunodeficiency virus HIV Integrated HIV-1 DNA Total HIV DNA Droplet digital polymerase chain reaction HIV reservoir Antiretroviral therapy

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Secondary analysis of malaria rapid diagnostic tests from rounds 5–8 of WHO product testing with a focus on false-negative results

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Military Medical Research 2022年第3期9卷 379-381页

作者： Biao Xu Bo Tu Fang Chu Mohamed Jalloh Jin-Song Mu Jun-Jie Zheng Wei-Wei Chen Chinese Military Medical Expert Group in Sierra Leone the Fifth Medical Center of Chinese PLA General HospitalBeijing100039China The 34Th Military Hospital Wilberforce BarracksFreetownSierra Leone

Despite the widespread use of malaria rapid diagnostic test(RDT)in clinical practice,there are a lot of *** conducted a secondary analysis of 129 malaria RDT data from rounds 5–8 of the World Health Organization(WHO)product testing summary and discuss the causes of false-negative(FN)results with a focus on low parasite density,improper RDT storage,operation and interpretation,and Plasmodium falciparum with a pfhrp2/3 gene *** results demonstrated that the malaria RDTs currently commercially available might cause FN results in practice.

关键词： Malaria Rapid diagnostic tests False-negative results WHO product testing

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Allogeneic gene-edited HIV-specific CAR-T cells secreting PD-1 blocking scFv enhance specific cytotoxic activity against HIV Envt cells in vivo

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Virologica Sinica 2023年第2期38卷 285-295页

作者： Hanyu Pan Xinyi Yang Jing Wang Huitong Liang Zhengtao Jiang Lin Zhao Yanan Wang Zhiming Liang Xiaoting Shen Qinru Lin Yue Liang Jinglong Yang Panpan Lu Yuqi Zhu Min Li Pengfei Wang Jianqing Xu Hongzhou Lu Huanzhang Zhu State key Laboratory of Genetic Engineering And Key Laboratory of Medical Molecular Virology of Ministry of Education/HealthSchool of Life SciencesFudan UniversityShanghai200438China Shanghai Institute of infectious disease and Biosecurity School of Life SciencesFudan UniversityShanghai200438China Department of infectious disease Key Laboratory of Medical Molecular Virology of Ministry of Education/HealthSchool of Basic Medical Sciences and Shanghai Public Health Clinical CenterFudan UniversityShanghai201508China Scientific Research Center Shanghai Public Health Clinical CenterFudan UniversityShanghai201508China Department of infectious diseases and Immunology Shanghai Public Health Clinical CenterFudan UniversityShanghai201508China Department of infectious diseases and Nursing Research Institution National Clinical Research Center for Infectious DiseasesThe Third People's Hospital of ShenzhenShenzhen518112China

HIV-specific chimeric antigen receptor(CAR)T-cells have been developed to target HIV-1 infected CD4t T-cells that express HIV Env ***,T cell exhaustion and the patient-specific autologous paradigm of CAR-T cell hurdled clinical ***,we created HIV-specific CAR-T cells using human peripheral blood mononuclear cells and a 3BNC117-E27(3BE)CAR construct that enabled the expression of programmed cell death protein(PD-1)-blocking scFv E27 and the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody 3BNC117 to target native HIV *** with T cells expressing 3BNC117-CAR alone,3BE CAR-T cells showed greater cytotoxic activity against HIV Envt cells with stronger proliferation capability,higher killing efficiency,and enhanced cytokine secretion in the presence of HIV Env-expressing ***,we manufactured TCR-deficient 3BE CAR-T cells through gene editing and demonstrated that these CAR-T cells could effectively kill HIV Env^(+) cells in vivo without the occurrence of severe graft-versus-host disease(GvHD)in NSG *** data suggest that we have provided a feasible approach to the generation of“off-theshelf”anti-HIV CAR-T cells in combination with PD-1 checkpoint blockade immunotherapy,which can be a powerful therapeutic candidate for the functional cure of HIV.

关键词： Anti-HIV CAR-T PD-1 blocking TCR disruption Cellular immunotherapy

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Relationship between the genetic variation in interleukin 28B and response to antiviral therapy in patients with chronic hepatitis C

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Chinese Medical Journal 2012年第13期125卷 2334-2338页

作者： XIE Jun-qiang GUO Xiao-yan ZHANG Xiao-hong LIN Bing-liang XIE Dong-ying GAO Zhi-liang WANG Gen-shu ZHAO Zhi-xin Department of infectious diseases Third Affiliated Hospital Sun Yat-sen University Guangzhou Guangdong 510630 China Department of Liver Transplantation Third Affiliated Hospital Sun Yat-sen University Guangzhou Guangdong 510630 China

Background Genetic variations at the interleukin 28B （IL-28B） locus are important in predicting outcome following therapy for chronic hepatitis C virus （HCV） infection. The aim of this research was to evaluate the role of IL-28B single nucleotide polymorphism （SNP） variations in Chinese patients undergoing pegylated interferon-a plus ribavirin （PEG-IFN-a/RBV） treatment. Methods To determine the effect of IL-28B variation on the response to HCV therapy, these variants were genotyped in a cohort of 220 patients who were chronically infected with HCV and received combined PEG-IFN-a/RBV therapy. Results The proportions of rs12979860 CC, CT, and TT genotypes were 71.4%, 25.0%, and 3.6% respectively, in the sustained virological response （SVR） group; 15.8%, 60.5%, and 23.7% respectively, in the null virological response （NVR） group; and 38.1%, 52.4%, and 9.5% respectively, in the relapse （Rel） group （P 〈0.05）. Logistic regression analysis showed that, compared to those having the CC genotype, CT heterozygotes had an increased risk of NVR and Rel （OR=10.95, 95% CI =4.12-29.11, P=1.5×10-7 and OR=3.93, 95% CI =1.86-8.32, P=-2.1×10-4 respectively）. The RNA quantification assay showed that patients with genotype CC exhibited much higher levels of IL-28 expression than those with genotype CT or TT （P 〈0.001）. Conclusions The IL-28B SNP rs12979860 genotype was related to the effectiveness of HCV therapy： patients with the CC rs12979860 genotype had higher rates of SVR than those with the CT or TT genotype, and the CC genotype revealed a significantly higher level of IL-28 mRNA expression.

关键词： hepatitis C virus interleukin 28B genetic variation sustained virological response relapse

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Risk Factors for Severity and Mortality in Adult Patients Confirmed with COVID-19 in Sierra Leone: A Retrospective Study

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infectious diseases & Immunity 2022年第2期2卷 83-92页

作者： Bo Tu Sulaiman Lakoh Biao Xu Marta Lado Reginald Cole Fang Chu Susan Hastings-Spaine Mohamed Bole Jalloh Junjie Zheng Weiwei Chen Stephen Sevalie Fifth Medical Center of Chinese PLA General Hospital Beijing 100039China College of Medicine and Allied Health Sciences University of Sierra LeoneFreetownSierra Leone Joint Medical Unit Republic of Sierra Leone Armed Forces34 Military Hospital Wilberforce FreetownFreetownSierra Leone Partners In Health Sierra Leone

Background:The coronavirus disease 2019(COVID-19)is a highly infectious respiratory *** is no recommended antiviral treatment approved for COVID-19 in Sierra Leone,and supportive care and protection of vital organ function are performed for the *** study summarized the clinical characteristics,drug treatments,and risk factors for the severity and prognosis of COVID-19 in Sierra Leone to provide evidence for the treatment of ***:Data of 180 adult COVID-19 patients from the 34th Military Hospital in Freetown Sierra Leone between March 31,2020 and August 11,2020 were retrospectively *** with severe and critically ill are classified in the severe group,while patients that presented asymptomatic,mild,and moderate disease were grouped in the non-severe *** clinical and laboratory information was retrospectively collected to assess the risk factors and treatment strategies for severe *** information,travel history,clinical symptoms and signs,laboratory detection results,chest examination findings,therapeutics,and clinical outcomes were collected from each case *** logistic analysis was adopted to identify the risk factors for ***,the clinical efficacy of dexamethasone treatment was ***:Seventy-six(42.22%)cases were confirmed with severe COVID-19,while 104 patients(57.78%)were divided into the non-severe ***(56.67%,102/180)and cough(50.00%,90/180)were the common symptoms of *** death rate was 18.89%(34/180),and severe pneumonia(44.12%,15/34)and septic shock(23.53%,8/34)represented the leading reasons for *** older age population,a combination of hypertension and diabetes,the presence of pneumonia,and high levels of inflammatory markers were significantly associated with severity of COVID-19 development(P<0.05 for all).Altered level of consciousness[odds ratio(OR)=56.574,95%confidence interval(CI)5.645–566.940,P=0.001],high levels of n

关键词： COVID-19 Clinical type Dexamethasone Risk factor

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Precision Methylome and In Vivo Methylation Kinetics Characterization of Klebsiella pneumoniae

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Genomics, Proteomics & Bioinformatics 2022年第2期20卷 418-434页

作者： Jing Fu Ju Zhang Li Yang Nan Ding Liya Yue Xiangli Zhang Dandan Lu Xinmiao Jia Cuidan Li Chongye Guo Zhe Yin Xiaoyuan Jiang Yongliang Zhao Fei Chen Dongsheng Zhou CAS key Laboratory of Genome Sciences and Information Beijing Institute of GenomicsChinese Academy of Sciences and China National Center for BioinformationBeijing 100101China Department of Oncology Henan Provincial People’s HospitalPeople’s Hospital of Zhengzhou UniversityPeople’s Hospital of Henan UniversityZhengzhou 450001China University of Chinese Academy of Sciences Beijing 100049China Department of Medical Research Center Peking Union Medical College HospitalPeking Union Medical College&Chinese Academy of Medical SciencesBeijing 100730China State key Laboratory of Pathogen and Biosecurity Beijing Institute of Microbiology and EpidemiologyBeijing 100071China CAS key Laboratory of Genomic and Precision Medicine Beijing Institute of GenomicsChinese Academy of Sciences and China National Center for BioinformationBeijing 100101China

Klebsiella pneumoniae(***)is an important pathogen that can cause severe hospital-and community-acquired *** systematically investigate its methylation features,we determined the whole-genome sequences of 14 *** strains covering varying serotypes,multilocus sequence types,clonal groups,viscosity/virulence,and drug *** methylomes were further characterized using Pacific Biosciences single-molecule real-time and bisulfite *** identified 15 methylation motifs[13 N6-methyladenine(6mA)and two 5-methylcytosine(5mC)motifs],among which eight were *** corresponding DNA methyltransferases were also ***,we analyzed the genomic distribution of GATC and CCWGG methylation motifs shared by all strains,and identified differential distribution patterns of some hemi-/un-methylated GATC motifs,which tend to be located within intergenic regions(IGRs).Specifically,we characterized the in vivo methylation kinetics at single-base resolution on a genome-wide scale by simulating the dynamic processes of replication-mediated passive demethylation and MTase-catalyzed *** slow methylation of the GATC motifs in the replication origin(oriC)regions and IGRs implicates the epigenetic regulation of replication initiation and *** findings illustrate the first comprehensive dynamic methylome map of *** at single-base resolution,and provide a useful reference to better understand epigenetic regulation in this and other bacterial species.

关键词： DNA methylation Epigenomics 6mA 5mC Epigenetic regulation

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Animal models for COVID-19:advances,gaps and perspectives

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Signal Transduction and Targeted Therapy 2022年第8期7卷 2817-2840页

作者： Changfa Fan Yong Wu Xiong Rui Yuansong Yang Chen Ling Susu Liu Shunan Liu Youchun Wang Division of Animal Model Research Institute for Laboratory Animal ResourcesNational Institutes for Food and Drug Control(NIFDC)National Rodent Laboratory Animal Resources CenterBeijing 102629China Department of Microbiology&infectious disease Center School of Basic Medical SciencesPeking University Health Science CenterBeijing 100083China College of Life Sciences Northwest UniversityProvincial Key Laboratory of Biotechnology of Shaanxi ProvinceNorthwest UniversityXi’an 710069China Division of HIV/AIDS and Sexually Transmitted Virus Vaccines Institute for Biological Product ControlNational Institutes for Food and Drug Control(NIFDC)BeijingChina

COVID-19,caused by SARS-CoV-2,is the most consequential pandemic of this *** the outbreak in late 2019,animal models have been playing crucial roles in aiding the rapid development of vaccines/drugs for prevention and therapy,as well as understanding the pathogenesis of SARS-CoV-2 infection and immune responses of ***,the current animal models have some deficits and there is an urgent need for novel models to evaluate the virulence of variants of concerns(VOC),antibodydependent enhancement(ADE),and various comorbidities of *** review summarizes the clinical features of COVID-19 in different populations,and the characteristics of the major animal models of SARS-CoV-2,including those naturally susceptible animals,such as non-human primates,Syrian hamster,ferret,minks,poultry,livestock,and mouse models sensitized by genetically modified,AAV/adenoviral transduced,mouse-adapted strain of SARS-CoV-2,and by engraftment of human tissues or *** understanding the host receptors and proteases is essential for designing advanced genetically modified animal models,successful studies on receptors and proteases are also *** improved alternatives for future mouse models are proposed,including the reselection of alternative receptor genes or multiple gene combinations,the use of transgenic or knock-in method,and different strains for establishing the next generation of genetically modified mice.

关键词： prevention drugs establishing

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HLA-DR expression on regulatory T cells is closely associated with the global immune activation in HIV-1 infected subjects naive to antiretroviral therapy

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Chinese Medical Journal 2011年第15期124卷 2340-2346页

作者： XIAO Jian QIAN Ke-lei CAO Qing-hua QIU Chen-li QIU Cao XUE Yi-le ZHANG Xiao-yan ZHONG Ping XU Jian-qing LI Ming-yuan WANG Ying Department of Microbiology West China School of preclimcal and Forensic Medicine Sichuan University Chengdu Sichuan 610041 China Department of AIDS/STD Shanghai Municipal Center for Disease Control and Prevention Shanghai 200336 China Shanghai Public Health Clinical Center Institutes of Biomedical Sciences key Laboratory of Medical Molecular Virology of Ministry of Education/Health Shanghai Medical College Fudan University Shanghai 201508 China

Background The frequencies of regulatory T cells （Tregs） increased over the HIV infection but its counts actually decreased. We proposed that the decrease of Treg counts may cause the reduction of inhibitory effect and thereby account for the over-activation of Tregs during HIV infection. However, it remains unknown whether Tregs are also over-activated and thereafter the activation induced death may lead to the decrease of Tregs. Methods Tregs were defined as CD4＋CD25＋CD127lo/-T cells. Eighty-one HIV-1 infected patients were enrolled in our study, and twenty-two HIV-1 seronegative donors were recruited as the control. The levels of HLA-DR on Tregs were determined by FACSAria flow cytometer. Results Compared to HIV-1 seronegative donors, the levels of HLA-DR on CD4＋CD25＋CD127lo/- Tregs were significantly increased in HIV-1 infected patients, and its increase was positively associated with viral loads （r=0.3163, P=-0.004） and negatively with CD4 T-cell counts （r=-0.4153, P 〈0.0001）. In addition, significant associations between HLA-DR expression on CD4＋CD25＋CD127lo/- Tregs and the percentages of HLA-DR, CD38, Ki67 expressing CD4＋ and CD8＋ T cells were also identified. Conclusion HLA-DR on Tregs is a good marker for viral replication and disease progression. The over-activation of Tregs might result in the decrease of Tregs.

关键词： human immunodeficiency virus type 1 T-lymphocytes, regulatory HLA-DR antigens immune activation

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Activity of superior interferon α against HIV-1 in severe combined immunodeficient mice reconstituted with human peripheral blood leukocytes

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Chinese Medical Journal 2011年第3期124卷 396-400页

作者： ZHANG Wei TONG Xiao Tadashi Nakasone YUE Xue-tian Naoki Yamamoto LIU Xin-yuan YANG Rong-ge Laboratory of HIV Molecular Epidemiology and Virology State Key Laboratory of Virology Wuhan Institution of VirologyChinese Academy of Sciences Wuhan Hubei 430071 China AIDS Research Center National Institute of Infectious Diseases1-23-1 Toyama Shinjuku-ku Tokyo 162-8640 Japan Zhejiang Sci-Tech University Hangzhou Zhejiang 310018 China Zhejiang Sci-Tech University Hangzhou Zhejiang 310018 China Laboratory of Molecular Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological SciencesChinese Academy of Sciences Shanghai 200031 China

Background Interferon （IFN） can inhibit human immunodeficiency virus type 1 （HIV-1） replication in vitro and in ***, IFN therapy for HIV infection was limited by its moderate antiviral efficacy and its frequent adverse effects. In the present study we evaluated the anti-HIV efficacy of a novel synthesized superior interferon α （slFNα）.Methods We performed in vitro experiments with HIV-1 IIB infected MT4 cells, and evaluated in vivo anti-HIV efficacy of slFNα in severe combined immunodeficient （SClD） mice reconstituted with human peripheral blood leukocytes （hu-PBL-SClD mice）.Results We found that the 50% effective concentrations （EC5o） of slFNα against the replication of HIV-1 in MT4 cells was 0.06 ng/ml, representing stronger antiviral activity than interferon-α in vitro. In the hu-PBL-SCID mice, a dose-dependent protection pattern was observed： with 0.45 μg and 1.35 μg slFNα daily treatments, parts of SCID mice were protected from HIV infection, whereas 2.25 μg sIFNα daily treatments resulted in a terminally complete *** slFNα shows good anti-HIV activity both in vitro and in SCID mice, may be a promising anti-HIV agent deserving clinical investigation, especially considering the potential of IFN-α to inhibit HIV replication in patients infected with drug-resistant variants or co-infected with hepatitis C virus （HCV）.

关键词： human immunodeficiency virus type 1 severe combined immunodeficient mouse interferon-α

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The Establishment and Application of a Kraken Classifier for Salmonella Plasmid Sequence Prediction

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china CDC weekly 2022年第49期4卷 1110-1116页

作者： Zhenpeng Li Bo Pang Xin Lu Biao Kan State key Laboratory of infectious disease Prevention and Control National Institute for Communicable Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing MunicipalityChina School of Public Health Shandong UniversityJinan CityChina.

Introduction:Salmonella is a key intestinal pathogen of foodborne disease,and the plasmids in Salmonella are related to many biological characteristics,including virulence and drug resistance.A large number of plasmid contigs have been sequenced in bacterial draft genomes,however,these are often difficult to distinguish from chromosomal ***:In this study,three different customized Kraken databases were used to build three different Kraken *** genome benchmark datasets and simulated draft genome benchmark datasets were ***-fold cross-validation was used to evaluate the performance of the three different Kraken classifiers by two benchmark ***:The predictive performance of the classifier based on all national Center for Biotechnology Information plasmids and Salmonella complete genomes was *** optimal Kraken classifier was performed with Salmonella isolated in *** plasmid carrying rate of Salmonella in china is 91.01%,and it was found that the Kraken classifier could find more plasmid contigs and antibiotic resistance genes(ARGs)than results derived from a plasmid replicon-based method(PlasmidFinder).Moreover,it was found that in the strains carrying ARGs,plasmids carried more ARGs[three,95%confidence interval(CI):1–14]than chromosomes(one,95%CI:1–7).Discussion:We found building a high-quality customized database as a Kraken classifier to be ideal for the prediction of Salmonella plasmid sequences from bacterial draft *** the future,the Kraken classifier established in this study will play a significant role in ARG monitoring.

关键词： classifier database Application

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