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Induction of virus-neutralizing antibodies and T cell responses by dengue virus type 1 virus-like particles prepared from Pichia pastoris

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Chinese Medical Journal 2012年第11期125卷 1986-1992页

作者： TANG Yun-xia JIANG Li-fang ZHOU Jun-mei YIN Yue YANG Xiao-meng LIU Wen-quan FANG Dan-yun Department of Microbiology Zhongshan School of MedicineSun Yat-sen UniversityGuangzhouGuangdong 510080China

Background Dengue is currently a significant global health problem but no vaccines are available against the four dengue serotypes virus infections. The development of safe and effective vaccines has been hampered by the requirement of conferring complete protection against all four dengue serotypes and the lack of a convenient animal model. virus-like particles （VLPs） have emerged as a promising subunit vaccine candidate. One strategy of vaccine development is to produce a tetravalent dengue subunit vaccine by mixing recombinant VLPs, corresponding to all four dengue virus serotypes. Towards this end, this study aimed to establish a Pichia pastoris （P. pastoris） expression system for production of dengue virus type 1 （DENV-1） VLPs and evaluate the humoral and cellular immune response of this particle in mice. Methods A recombinant yeast P. pastoris clone containing prM and E genes of DENV-1 was constructed and DENV-1 VLPs expressed by this clone were analyzed by sucrose density gradient centrifugation, Western blotting, and transmission electron microscope. Groups of mice were immunized by these particles plus adjuvant formulations, then mice were tested by ELISA and neutralization assay for humoral immune response, and by lymphocyte proliferation and cytokine production assays for a cellular immune response. Results Our data demonstrated that recombinant DENV-1 VLPs consisting of prM and E protein were successfully expressed in the yeast P. pastoris. Sera of VLPs immunized mice were shown to contain a high-titer of antibodies and the neutralization assay suggested that those antibodies neutralized virus infection in vitro. Data from the T lymphocyte proliferation assay showed proliferation of T cell, and ELISA found elevated secretion levels of interferon IFN-γ and IL-4. Conclusions P. pastoris-expressed DENV-1 VLPs can induce virus neutralizing antibodies and T cell responses in immunized mice. Using P. pastoris to produce VLPs offers a promising and economic str

关键词： dengue virus virus-like particles Pichia pastoris

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The codon-optimized capsid gene of duck circovirus can be highly expressed in yeast and self-assemble into virus-like particles

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Journal of Integrative Agriculture 2017年第7期16卷 1601-1608页

作者： YANG Cui XU Yu JIA Ren-yong LIU Si-yang WANG Ming-shu ZHU De-kang CHEN Shun LIU Ma-feng ZHAO Xin-xin SUN Kun-feng JING Bo YIN Zhong-qiong CHENG An-chun Avian Disease Research Centre Sichuan Agricultural UniversityChengdu 611130P.R.China Institute of Preventive Veterinary Medicine Sichuan Agricultural UniversityChengdu 611130P.R.China Key Laboratory of Animal Disease and Human Health of Sichuan Province Chengdu 611130P.R.China

The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid （Opt-Cap） gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% （v/v） methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What＇s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles （VLPs）. These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.

关键词： capsid gene codon'optimization duck circovirus virus-like particles

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Carboxyl Terminus Truncated Human Papillomavirus Type 58 L1 Protein Maintains Its Bioactivity and Ability to Form virus-like particles

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2004年第6期24卷 537-539页

作者：李文生刘红莉郑瑾陈宏伟杨军王丽秀闫小飞王一理司履生 The Key Laboratory of Biomedical Information Enginceering of Ministry of Education and Institute for Cancer Research School of Life Science and Technology Xian Jiaotong University Xlan 710061 ChinaShan'xi Hospital School of Life Science and Techno The Key Laboratory of Biomedical Information Enginceering of Ministry of Education and Institute for Cancer Research School of Life Science and Technology Xian Jiaotong University Xian 710061 China

Summary: To prepare carboxyl terminus truncated human papillomavirus type 58 L1(HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing-HPV58L1 protein-were harvested and analyzed by SDS-PAGE and Western blot. The ProBond~TM purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.

关键词： HPV58L1 protein carboxyl terminus truncation baculovirus expression system protein purification virus-like particles

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PRODUCTION IN PICHIAPASTORISAND CHARACTERIZATION OF GENETIC ENGINEERED CHIMERIC HBV/HEV virus-like particles

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Chinese Medical Sciences Journal 2004年第2期19卷 78-83页

作者： Hong-zhaoLi Hong-yingGang Qiang-mingSun XiaoLiu Yan-bingMa Mao-shengSun Chang-baiDai InstituteofMedicalBiology ChineseAcademyofMedicalSciences＆PekingUnionMedicalCollegeKunming650118

Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichiapastorisunder the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualiza-tion. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoriswith an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immuno-genicity on surface-displayed HEnAg. Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

关键词： hepatitis E virus neutralization epitope virus-like particles

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Synthesis of Mucin-1 Glycopeptides Conjugates with virus-like particles as Promising Anticancer Vaccines

Synthesis of Mucin-1 Glycopeptides Conjugates with Virus-Lik...

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第十一届全国化学生物学学术会议

作者： Xuanjun Wu Xuefei Huang National Glycoengineering Research Center Shandong University Department of Chemistry Michigan State University

Successful vaccines targeting mucin-1（MUC1） provide an appealing approach for cancer immunotherapy. Herein, MUC1 glycopeptides, have been conjugated to bacteriophage Qb virus-like particles and the immune responses to Qβ-MUC1 conjugates have been evaluated in both wildtype（Wt） mice and human mimic immunotolerant MUC1 transgenic（***） mice. The first generation of Qβ-MUC1 conjugates produced super high titers of antibodies in Wt mice with strong binding to MUC1-expressing tumor cells. However, in *** mice, the induced antibodies exhibited very weak binding to tumor cells. To create MUC1 vaccines that can function well in *** mice, a new strategy has been developed for discovery of protective epitopes. A new generation Qβ-MUC1 conjugates focusing on the key protective MUC1 epitopes has been designed, which induced superior Ig G antibodies with strong tumor binding, high tumor killing activities and significant protection in both metastasis and solid tumor models. The antibodies exhibited remarkable selectivity towards human breast cancer tissues over normal tissues, suggesting the high translational potential of Qβ-MUC1 conjugates.

关键词： mucin-1 virus-like particles anti-cancer vaccine immunotolerant mice

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Synthesis of Mucin-1 Glycopeptides Conjugates with virus-like particles as Promising Anticancer Vaccines

Synthesis of Mucin-1 Glycopeptides Conjugates with Virus-Lik...

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第十一届全国化学生物学学术会议

作者：吴选俊 Xuefei Huang 山东大学 Department of Chemistry Michigan State University

Successful vaccines targeting mucin-1（MUC1） provide an appealing approach for cancer ***,MUC1 glycopeptides,have been conjugated to bacteriophage Qβ virus-like particles and the immune responses to Qβ-MUC1 conjugates have been evaluated in both wildtype（Wt） mice and human mimic immunotolerant MUC1 transgenic（***） *** first generation of Qβ-MUC1 conjugates produced super high titers of antibodies in Wt mice with strong binding to MUC1-expressing tumor ***,in *** mice,the induced antibodies exhibited very weak binding to tumor *** create MUC1 vaccines that can function well in *** mice,a new strategy has been developed for discovery of protective epitopes.A new generation Qβ-MUC1 conjugates focusing on the key protective MUC1 epitopes has been designed,which induced superior IgG antibodies with strong tumor binding,high tumor killing activities and significant protection in both metastasis and solid tumor *** antibodies exhibited remarkable selectivity towards human breast cancer tissues over normal tissues,suggesting the high translational potential of Qβ-MUC1 conjugates.

关键词： mucin-1 virus-like particles anti-cancer vaccine immunotolerant mice

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Minor deletions in loops of the capsids on Porcine parvovirus VP2 for the assembly of virus-like particles

Minor deletions in loops of the capsids on Porcine parvoviru...

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中国畜牧兽医学会动物传染病学分会第十二次人兽共患病学术研讨会暨第六届第十四次教学专业委员会

作者： Qunxing Pan Kongwang He Yongshan Wang Xiaoli Wang Wei Ouyang Libing Wen Rongli Guo Institute of Veterinary Medicine Jiangsu Academy of Agricultural Sciences.Key Laboratory of Veterinary Biological Engineering and TechnologyMinistry of Agriculture.National Center for Engineering Research of Veterinary Bio-products

virus-like particles(VLPs) constitute a new genetation of non-replicative vectors for delivery of heterologous epitopes and induction of immune *** is no previous information for deletions in different loops of PPV VP2 on correct assembly of *** this report,we have constructed deletion mutants in the surface of PPV capsids by adenoviruses expression *** of recombinant adenoviruses expressing PPV:AVP2(Loop2/4) were studied by IFA and Western blotting *** microscopy analysis confirmed the presence of abundant VLPs in loop2 deletion mutant, with the expected size and appropriate *** the other hand,loop4 deletions produced a lower number of particles and the morphology of the particles was not well *** study showed that rAd-△VP2(Loop2) could induce specific antibody response to PPV as strong as PPV inactivated vaccine,rAd-△VP2(Loop4) could induce specific antibody titers lower than rAd-△VP2(Loop2)(pparticles opens the way to the development of highly efficient vaccines,able to stimulate at the same time the different branches of the immune system.

关键词： virus-like particles loops deletion mutants PPV

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Disturbed gut virome with potent interferonogenic property in systemic lupus erythematosus

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Science Bulletin 2023年第3期68卷 295-304,M0004页

作者： Beidi Chen Jiabao Cao Wei Liu Yuqing Zhang Yudong Liu Min Wang Fei Xiao Jie Ma Jun Wang Xuan Zhang Department of Rheumatology Beijing HospitalNational Center of GerontologyInstitute of Geriatric MedicineChinese Academy of Medical SciencesBeijing 100730China Division of Rheumatology and Immunology Peking University Third HospitalBeijing 100191China CAS Key Laboratory of Pathogenic Microbiology and Immunology Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100101China The Key Laboratory of Geriatrics Beijing HospitalNational Center of GerontologyNational Health CommissionInstitute of Geriatric MedicineBeijing Institute of GeriatricsChinese Academy of Medical SciencesBeijing 100730China

Accumulating evidence suggests an essential role of disturbed gut microbiota in the etiopathogenesis of systemic lupus erythematosus(SLE),but it remains unclear as to gut *** this study,fecal virus-like particles(VLPs)isolated from 76 non-treated SLE patients and 75 healthy controls were subjected to gut virome *** proportion of bacteriophages was significantly elevated in the SLE gut,and the altered viral taxa were correlated with clinical *** virome and bacteriome were closely associated with each other in SLE *** combination of gut viral and bacterial markers displayed better performance in distinguishing SLE patients from healthy ***,VLPs from non-treated SLE patients promoted interferon-a production in an epithelial cell line and human immune ***,the interferon-stimulatory capacity diminished in VLPs from post-treated SLE *** findings may shed novel insights into SLE *** in-depth understanding of gut virome might help develop future biomarkers and therapeutics for SLE patients.

关键词： Systemic lupus erythematosus Gut virome virus-like particles Interferon-a

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Safety,immunogenicity,and protection provided by unadjuvanted and adjuvanted formulations of a recombinant plant-derived virus-like particle vaccine candidate for COVID-19 in nonhuman primates

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Cellular & Molecular Immunology 2022年第2期19卷 222-233页

作者： Stéphane Pillet Prabhu SArunachalam Guadalupe Andreani Nadia Golden Jane Fontenot Pyone Pyone Aye Katharina Röltgen Gabrielle Lehmicke Philipe Gobeil Charlotte Dubé Sonia Trépanier Nathalie Charland Marc-AndréD’Aoust Kasi Russell-Lodrigue Christopher Monjure Robert V.Blair Scott D.Boyd Rudolf P.Bohm Jay Rappaport François Villinger Nathalie Landry Bali Pulendran Brian J.Ward Medicago Inc. QuébecQCCanada. Institute for Immunity Transplantation and InfectionStanford University School of MedicineStanford UniversityStanfordCAUSA. Tulane National Primate Research Center CovingtonLAUSA. New Iberia Research Center University of Louisiana at LafayetteNew IberiaLAUSA. Department of Pathology Stanford University School of MedicineStanford UniversityStanfordCAUSA. Department of Microbiology and Immunology Tulane University School of MedicineNew OrleansLAUSA. Department of Microbiology and Immunology Stanford University School of MedicineStanford UniversityStanfordCAUSA. Institute for Immunity Transplantation&InfectionStanford University School of MedicineStanford UniversityStanfordCAUSA Research Institute of the McGill University Health Centre MontrealQCCanada.

Although antivirals are important tools to control severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection,effective vaccines are essential to control the current coronavirus disease 2019(COVID-19)***-derived virus-like particle(VLP)vaccine candidates have previously demonstrated immunogenicity and efficacy against ***,we report the immunogenicity and protection induced in rhesus macaques by intramuscular injections of a VLP bearing a SARS-CoV-2 spike protein(CoVLP)vaccine candidate formulated with or without Adjuvant System 03(AS03)or cytidine-phospho-guanosine(CpG)*** a single dose of the unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses,booster immunization(at 28 days after priming)and adjuvant administration significantly improved both responses,with higher immunogenicity and protection provided by the AS03-adjuvanted *** micrograms of CoVLP adjuvanted with AS03 induced a polyfunctional interleukin-2(IL-2)-driven response and IL-4 expression in CD4 T *** were challenged by multiple routes(i.e.,intratracheal,intranasal,and ocular)with a total viral dose of 106 plaque-forming units of *** viral replication in nasal swabs and bronchoalveolar lavage fluid(BALF)as well as fewer SARS-CoV-2-infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of proinflammatory cytokines and chemotactic factors in the BALF were observed in animals immunized with the CoVLP adjuvanted with *** clinical,pathologic,or virologic evidence of vaccineassociated enhanced disease was observed in vaccinated *** CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.

关键词： SARS-CoV-2 virus-like particles Non-humane primates AS03 CpG1018

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Quantitative functionalization of biosynthetic caged protein materials

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Quantitative Biology 2023年第1期11卷 1-14页

作者： Quan Cheng Xuan Wang Xian-En Zhang Chengchen Xu Feng Li College of Life Sciences and Health Wuhan University of Science and TechnologyWuhan 430065China State Key Laboratory of Virology Wuhan Institute of VirologyCenter for Biosafety Mega-ScienceChinese Academy of SciencesWuhan 430071China Faculty of Synthetic Biology Shenzhen Institute of Advanced TechnologyChinese Academy of SciencesShenzhen 518055China National Laboratory of Biomacromolecules Institute of BiophysicsChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China

Background:As one of the representative protein materials,protein nanocages(PNCs)are self-assembled supramolecular structures with multiple advantages,such as good monodispersity,biocompatibility,structural addressability,and facile *** quantitative functionalization is essential to the construction of PNCs with designed ***:With three modifiable interfaces,the interior surface,outer surface,and interfaces between building blocks,PNCs can serve as an ideal platform for precise multi-functionalization studies and *** review summarizes the currently available methods for precise quantitative functionalization of PNCs and highlights the significance of precise quantitative control in fabricating PNC-based materials or *** methods can be categorized into three groups,genetic,chemical,and combined ***:This review would be constructive for those who work with biosynthetic PNCs in diverse fields.

关键词： protein nanocages virus-like particles functionalization genetic modification chemical modification

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