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Comprehensive Flow Cytometry Analysis of PEI-Based Transfections for Virus-Like Particle Production

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research 2020年第1期2020卷 50-59页

作者： Daniel J.Blackstock Alvenne Goh Shamitha Shetty Giulia Fabozzi Rong Yang Vera BIvleva Richard Schwartz Joseph Horwitz vaccine Production Program Lab Vaccine Research CenterNIAIDNIHGaithersburgMDUSA

The generation of stable clones for biomolecule production is a common but lengthy and labor-intensive *** complex molecules,such as viruses or virus-like particles(VLPs),the timeline becomes even more ***,in the early stages of development,transient production methods serve as a reasonable alternative to stable clone *** this work,an investigation of a polyethylenimine-(PEI-)based transfection method for the transient production of Chikungunya(Chik)VLPs,a vaccine candidate molecule,was *** effort focuses on tracking cell population responses during transfection,understanding how process changes affect these responses,and monitoring patterns in cell performance over the culture *** labeling and VLP staining were employed to comprehensively track cells via flow cytometry and to draw correlations between plasmid DNA(pDNA)uptake and the resulting VLP *** method detected high transfection efficiency(≥97%)in all samples tested and demonstrated the capability to track kinetics of plasmid-cell *** varied transfection cell concentrations,the pDNA binding kinetics are altered and saturation binding is observed in the lowest cell concentration sample tested in less than 3 hours of ***,in all samples,the flow cytometry analysis of relative pDNA amount versus VLP expression staining showed that cells which contained fewer pDNA complexes resulted in the highest levels of VLP ***,to determine the potential breadth of our observations,we compared daily expression patterns of ChikVLP with a reporter,monomeric GFP *** similarities detected suggest the interpretations presented here to likely be more broadly informative and applicable to PEI-based transient production of additional biological products as well.

关键词： transient kinetics likely

Protectivity of Freeze Dried Inactivated Rift Valley Fever vaccine

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Open Journal of Veterinary Medicine 2024年第2期14卷 21-37页

作者： Diana M. Abulmagd Mohamed Hassan Atwa Noha Ezz Aldin Marwa Yehia Hammad Taradi Abdel Fattah Said Rift Valley Fever vaccine research Department Veterinary Serum and Vaccine Research Institute Agriculture Research Center (ARC) Cairo Egypt

Background: Vaccinations for animals are crucial for food production, animal welfare, public health, and animal health. They are an affordable way to stop animal sickness, increase food production efficiency, and lessen or stop the spread of zoonotic diseases to humans. Animal vaccines that are both safe and efficacious are vital to modern culture. The vaccine should induce a strong, protective and prolonged immune response against the antigenic factor. In order to achieve these goals, novel vaccination techniques and an efficient adjuvant are required to render the vaccine immunogenically protective and trigger a strong immune response. Aim: Our study aims to promote and enhance the immunogenicity against RVF virus disease through lyophilized inactivated RVF vaccine through induction of early cellular, high and prolonged humeral immunity in vaccinated animals using cabopol as stabilizer and Saponin or normal saline as a diluent at time of vaccination. Moreover, manufacturing of these vaccines is easy to be done. Results: The gained results revealed that RVF freeze-dried vaccine with Carbopol that reconstituted using Saponin elicited better immune response than that reconstituted using normal saline (NaCl). The cell mediated immune response as represented by lymphocyte blastogenesis and phagocytic activity were markedly increased with high levels when we used Saponin as a diluent than that in group vaccinated with vaccine diluted with NaCl, on the other side the humeral immune response in group vaccinated using the Saponin as diluent is more detected and stayed within the protective level till the end of 11th month post vaccination (1.5 TCID50) while the immune response induced after using normal saline as a diluent stayed within the protective level till the end of 10th month post vaccination (1.8 TCID50). Conclusion: The use of Saponin as a diluent for reconstitution of the freeze dried RVF vaccine is preferable than the

关键词： Rift Valley Fever Saponin Cabopol Binary Ethylenemine Serum Neutralization Test

Mucosal epithelial cells: the initial sentinels and responders controlling and regulating immune responses to viral infections

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Cellular & Molecular Immunology 2021年第7期18卷 1628-1630页

作者： Jingyi Yang Huimin Yan vaccine and Immunology research center Translational Medical Research InstituteShanghai Public Health Clinical CenterFudan UniversityShanghaiChina

In the context of host defense,immune protection is mostly provided by hematopoietic immune cells,including neutrophils,macrophages,T cells,B cells,***,in recent decades,emerging evidence has identified many additional diverse roles of nonhematopoietic cells in mammalian immunity,although most of these studied focused on a single-cell *** July 2020,Krausgruber et *** the laboratory of Christoph Bock published the paper“Structural cells are key regulators of organ-specific immune responses”in *** authors used multiomics and systematically investigated the regulation of immune-associated genes in three major types of structural cells(epithelial cells,endothelial cells,and fibroblasts)in 12 different organs in mice.

关键词： immunity organs mostly

Fighting Ebola with ZMapp: spotlight on plant-made antibody

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Science China(Life Sciences) 2014年第10期57卷 987-988页

作者： ZHANG YunFang LI DaPeng JIN Xia HUANG Zhong vaccine research center Institut Pasteur of Shanghai Chinese Academy of Sciences

Currently,a large Ebola endemic is ongoing in West *** to the World Health Organization,as of August19,the viral outbreak has led to 2240 suspected and confirmed cases and 1229 deaths in four countries:Guinea,Liberia,... 详细信息

关键词：聚光灯疾病预防控制中心战斗抗体制造植物世界卫生组织利比里亚

Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration(hALR), Expression and Purification of hALR

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Chemical research in Chinese Universities 2006年第2期22卷 201-204页

作者： SUN Tian-xu WU Yong-ge YU Xiang-hui JIANG Chun-lai JIN Ying-hua CHENG Yue KONG Wei vaccine research center College of Life Science Jilin University Changchun 130012 P. R. China

Experimental evidence has been presented to suggest that the human augmenter of liver regeneration （hALR） serves as a hepatotruphic growth factor during liver regeneration and as a generalized growth factor during pancreas transplant/regeneration. A prokaryotic expression plasmid, pRSET/6his-c-myc-hALR was constructed, by cloning synthesized hALR cDNA into pRSET/6his-c-myc that was improved on the basis of pRSET B by the group. As a result, the protein was highly expressed in E. coli BL21. The recombinant hALR was over 60% of the total protein in E. coli. Its validity was confirmed by means of Western Blotting. The protein was purified by Ni-NTA affinity chrumatography and this FAD-dependent sulthydryl oxidase activity was measured.

关键词： Augmenter of liver regeneration Prokayotic expression plasmid FAD-dependent sulthydryl oxidase

Montanide ISA-720 and Naloxone in HBsAg vaccine Formulation:Cytokine Profiling and Monitoring of Long-Lasting Humoral Immune Responses

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Biomedical and Environmental Sciences 2022年第9期35卷 792-803页

作者： Mina Mirzaee Setareh Haghighat Bahareh Golkaran Fatemeh Asgarhalvaei Rayhaneh Mirzaee Morteza Taghizadeh Mohammad Ali Savoji Behzad Esfandiari Mehdi Mahdavi Department of Microbiology Faculty of Advanced Sciences&TechnologyTehran Medical SciencesIslamic Azad University(IAUPS)TehranIran Department of Biotechnology Faculty of Advanced Sciences&TechnologyPharmaceutical Sciences BranchIslamic Azad UniversityTehran-Iran(IAUPS) Department of Medical vaccine Razi Vaccine and Serum Research InstituteAgricultural ResearchEducation and Extension Organization(AREEO)KarajIran Department of Epidemiology and Biostatistics Pasteur Institute of IranTehranIran Department of Laboratory Animal Science Pasteur Institute of IranKarajIran Advanced Therapy Medicinal Product(ATMP)Department Breast Cancer Research CenterMotamed Cancer InstituteAcademic Center for EducationCulture and Research(ACECR)TehranIran Recombinant vaccine research center Tehran University of Medical SciencesTehranIran Department of Immunology Pasteur Institute of IranTehranIran Immunotherapy Group The Institute of Pharmaceutical Sciences(TIPS)Tehran University of Medical SciencesTehranIran

Objective This study aimed to investigate the effects of Montanide ISA-720 and Naloxone(NLX)in Hepatitis B surface antigen(HBsAg)vaccine formulation on cytokine and long-lasting antibody *** First,the HBsAg was formulated in Montanide ISA-720 adjuvant and Naloxone at 5 and 10mg/*** experimental mice were immunized three times at a 2-week interval,and then IL-4,IL-2,TNF-α,and IFN-γcytokines;long-lasting IgG antibody responses 220 days after the last shot;and IgG1/IgG2a isotypes were assessed by *** The HBsAg-Alum group exhibited the highest IL-4 cytokine response among the experimental groups,whereas NLX in HBsAg-MON720 vaccine formulation did not affect cytokine *** addition,NLX in Alum-based vaccine suppressed IL-4 cytokine response and increased the IL-2/IL-4 cytokine ***,HBsAg-MON720 was more potent than HBsAg-Alum in the induction of antibody responses,and NLX in Alum-and MON720-based vaccines induced long-lasting antibody *** NLX in Alum-based vaccine decreased IL-4 cytokine response,increased IL-2/IL-4 cytokine ratio,and improved long-lasting humoral immune responses in both vaccine ***,the adjuvant activity of NLX in the vaccine formulation depends on the type of adjuvant and the nature of the antigen in the vaccine formulation.

关键词： HBsAg vaccine Naloxone Montanide ISA-720 Long-lasting humoral response

Construction of Recombinant Modified Vaccinia Ankara(MVA) Expressing Hepatitis B Virus Surface Antigen

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Chemical research in Chinese Universities 2006年第2期22卷 213-216页

作者： CHEN Yu YANG Xiao-song YU Xiang-hui JIANG Chun-lai WU Yong-ge JIN Ying-hua CHEN Yue CHEN Yan LI Wei KONG Wei vaccine research center College of Life Science Jilin University Changchun 130012 P. R. China

The T lymphocyte response has been shown to be the determinant in the clearance of many viral infections. Hence, therapeutic vaccine candidates against HBV are designed to enhance this response of the immune system. Vaccinia virus vector-based vaccines have been proposed as excellent candidates to elicit long-term and strong T lymphocyte mediated immune responses. In this study, the recombinant MVA expressing HBV surface antigen has been constructed, which can elicit a potent T cell mediated response. The ELISA results for the surface protein in the medium of the recombinant MVA, strongly indicate that the recombinant virus has been successfully obtained.

关键词： Recombinant MVA Therapeutic vaccine T lymphocyte response

Establishment of a Tumor-bearing Mouse Model Stably Expressing EGFP Labeled Human MUC1 VNTRs

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Chemical research in Chinese Universities 2008年第4期24卷 505-510页

作者： ZHANG Shu-zi ZHANG Hai-hong ZHANG Wa SHI He-liang YU Xiang-hui KONG Wei LI Wei vaccine research center College of Life Science Jilin University Changchun 130012 P R. China

Two eukaryotic vectors expressing 9 tandem repeats of human MUCI（VNTR）, VR1012-VNTR, and pEGFP-VNTR, were constructed by cloning VNTR gene into VR1012 and pEGFP, respectively. VNTR stably expressing murine Lewis lung carcinoma（LLC） cell line（VNTR^＋ LLC） was established by Lipofectamine-mediated transfection of pEGFP-VNTR into LLC cells. The EGFP expression was observed under a fluorescent microscope and VNTR expression in VNTR^＋ LLC cells was confirmed by means of Western blotting. A syngenic graft tumor model was generated by subcutaneous injection of VNTR^＋ LLC cells into C57/BL6 mice and tumor size increased rapidly with time and in a cell qumber dependent manner. VNTR mRNA expression in the tumor formed was confirmed by RT-PCR. After the third immunization mice were challenged subcutaneously with 5×10^5 VNTR^＋ LLC cells, a significant reduction of subcutaneous tumor growth was observed in the groups immunized with VNTR plasmid DNA compared with that in the groups immunized with the vector DNA alone. Thus, the suppression of subcutaneous tumor was antigen-specific. This model is useful for the development of tumor vaccines targeting MUCI VNTRs.

关键词： MUCI VNTR Tumor antigen Tumor model Tumor vaccine

Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

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Chemical research in Chinese Universities 2008年第6期24卷 767-770页

作者： ZHANG Hai-hong ZHANG Xi-zhen ZHAO Dong-hai SHI He-liang YU Yong-hui WU Yong-ge YU Xiang-hui KONG Wei vaccine research center College of Life Science Jilin University Changchun 130012 P R. China

Survivin, a novel member of inhibitor of apoptosis（IAP） protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in *** BL21, and the relative molecule mass（Mr） of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni^2＋ affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.

关键词： Clone Survivin Expression Polyclonal antibody